Search PubMed⌕ Search

Biomedical subjects

D Charron

Publications and source records attributed to D Charron.

At least 145 records · Page 8Linked to original sources
PubMed1996

HLA class II signaling mediates cellular activation and programmed cell death.

The class II human leucocyte antigens (HLA class II) are principally peptide presentation molecules. Signal transduction by these molecules has also been shown to transmit activation signals in both B and T lymphocytes by a pathway including protein tyrosine kinase activation, an intracellular calcium flux, and both the activation and transcriptional regulation of protein kinase C (PKC) isoforms. Apoptosis can also result from human leukocyte antigen class II stimulation. Inhibitors of gene transcription were used to inhibit activation and, therefore, to distinguish the signal transduction pathways important for apoptosis. This approach provided evidence that cellular activation and apoptosis undertook separate signaling pathways, and that PKC and intracellular calcium were shared between the two pathways, while tyrosine kinase activity was essential for cell activation. Further studies using cord blood B cells showed that these cells were incapable of generating a calcium flux after HLA class II ligation and were not subject to cell death. The importance of sustained levels of calcium for programmed cell death (PCD) was underlined since the restoration of a calcium flux enabled PCD of cord blood B cells via HLA class II. These results demonstrate that HLA class II stimulation initiates two distinct signal transduction pathways.

Adult↗
PubMed1995

HLA-Cw allele analysis by PCR-restriction fragment length polymorphism: study of known and additional alleles.

We describe a technique for HLA-Cw genotyping by digestion of PCR-amplified genes with restriction endonucleases. Locus-specific primers selectively amplified HLA-Cw sequences from exon 2 in a single PCR that avoided coamplification of other classical and nonclassical class I genes. Amplified DNAs were digested with selected enzymes. Sixty-three homozygous cell lines from International Histocompatibility Workshop X and 113 unrelated individual cells were genotypes for HLA-Cw and compared with serology. The present protocol can distinguish 23 alleles corresponding to the known HLA-Cw sequences. Genotyping of serologically undetectable alleles (HLA-Cw Blank) and of heterozygous cells was made possible by using this method. Six additional HLA-Cw alleles were identified by unusual restriction patterns and confirmed by sequencing; this observation suggests the presence of another family of allele-sharing clusters in the HLA-B locus. This PCR-restriction endonuclease method provides a simple and convenient approach for HLA-Cw DNA typing, allowing the definition of serologically undetectable alleles, and will contribute to the evaluation of the biological role of the HLA-C locus.

Alleles↗
PubMed1995

Comparison of two HLA-DRB high resolution microtiter plate reverse hybridization typing methods: advantage of a codon-86 valine or glycine PCR segregation.

Two rapid, nonisotopic, high-resolution HLA-DRB typing methods have been developed for DRB1, DRB3, DRB4 and DRB5 alleles. These methods are based on a single procedure consisting of the reverse hybridization of biotinylated amplicons to oligonucleotide probes that are covalently attached to a microtiter plate. Detection is by an enzymatic reaction with a fluorescent substrate. The 1 Generic Amplification (1GA) method amplifies all HLA-DRB alleles in the same reaction mix. The 2 Allelic Subset Amplification (2SA) method uses two distinct amplification reactions that distributes all DRB alleles into two equal-size subsets, according to the codon 86 Gly or Val polymorphism; this adds an extra discrimination level to the typing. 108 samples were typed using the 1GA and the 2SA methods and no discrepancies were found. Typing indeterminations due to overlapping probe combinations were compared; it was found that the 2SA method, with the extra discrimination level at the PCR step, greatly improved resolution.

Alleles↗
PubMed1995

An evaluation of a multicenter study on HLA-DPB1 typing using solid-phase Taq-cycle sequencing chemistry.

In HLA Class II genes, polymorphism is mainly located in the second exon. Most DNA based typing methods are confined to the identification of specific sequence motifs in the second exon. In contrast, Sequencing Based Typing (SBT) elucidates the entire exon 2 sequence for typing. Comparison of the obtained exon 2 sequence with an allele sequence library results in allele assignment. We tested the applicability of SBT using a protocol for amplification followed by solid phase Taq-cycle sequencing for HLA-DPB1 typing. A panel of 32 samples were typed by SBT at five test sites which are participating in the Sequencing Based Typing component of the 12th International Histocompatibility Workshop. The panel represents the existing polymorphism at all known polymorphic positions of exon 2, both in homozygous and heterozygous combinations. In this multicenter study we focused on the reliability of analyzing heterozygous sequences for HLA typing. A multi-sequence analysis approach, Polall, was developed to evaluate sequences obtained. The assignment of homozygosity and heterozygosity was validated by cluster analysis of chromatographic data of all sequences. Sequence characteristics were examined and considered for appropriate assignment. Differences in sequence characteristics that occurred between the test sites are considered in detail. The evaluation of data of 5 test sites reveals that Taq-cycle sequencing can reliably be performed for HLA-DPB1 SBT.

DNA-Directed DNA Polymerase↗
PubMed1995

Determination of DQB1 alleles using PCR amplification and allele-specific primers.

Molecular genotyping of HLA class II genes is commonly carried out using polymerase chain reaction (PCR) in combination with sequence-specific oligotyping (PCR-SSO) or a combination of the PCR and restriction fragment length polymorphism methods (PCR-RFLP). However, the identification of the DQB1 type by PCR-SSO and PCR-RFLP is very time-consuming which is disadvantageous for the typing of cadaveric organ donors. We have developed a DQB1 typing method using PCR in combination with allele-specific amplification (PCR-ASA), which allows the identification of the 17 most frequent alleles in one step using seven amplification mixtures. PCR allele-specific amplification HLA-DQB1 typing is easy to perform, and the results are easy to interpret in routine clinical practice. The PCR-ASA method is therefore better suited to DQB1 typing for organ transplantation than other methods.

Alleles↗
PubMed1995

Matching for MHC haplotypes results in improved survival following unrelated bone marrow transplantation.

Unrelated bone marrow donor-recipient pairs were assessed retrospectively for matching of the HLA-B, -C region (beta-block) and HLA-DR, DQ region (delta block) of the major histocompatibility complex (MHC) using a new DNA-based method referred to as MHC-block typing. The method utilises non-HLA DNA polymorphisms in the MHC as markers of blocks of ancestral haplotypes. Kaplan-Meier analysis of recipients who were matched at both the beta- and delta-blocks revealed a 6 months survival of 54%. Survival was better than for patients who were matched only by conventional criteria, including SSO-typing for class II.

Adult↗
PubMed1994

[Human major histocompatibility complex: the HLA system].

The HLA (human leukocyte antigens) system, or human major histocompatibility complex, is the most polymorphic functional genetic entity known at present. It consists of HLA class I genes and molecules (A, B and C) which control CD8+ cell-mediated antiviral responses, and class II genes and molecules (DR, DQ and DP) which control CD4+ cell responses (anti-bacterial and anti-toxin). HLA molecules function by presenting antigenic peptides to CD8+ cells (class I) and CD4+ cells (class II). Antigen presentation depends on the intracellular location of the antigen. Antigens present in the exocytosis pathway are presented by class I molecules, while class II molecules present antigens associated with the endocytosis pathway. More than 200 alleles have been detected by means of serological testing (microlymphocytotoxicity) and biochemical methods (IEF) in the HLA class I system, and now by means of molecular biology techniques for class II molecules (PCR-SSO and PCR-RFLP). This molecular typing has revealed the amino acids in HLA molecules that confer genetic susceptibility or resistance to numerous HLA-associated diseases. This is the case for example of ankylosing spondylitis (region 45-46 of HLA-B27 molecules), juvenile diabetes (aa 57 of D beta Q) and rheumatoid arthritis (aa 65-71 of DR beta). Thus, the HLA system is a genetic tool for diagnostic and therapeutic decision-making.

Animals↗
PubMed1994

Epstein-Barr virus (EBV) latent membrane protein 1 increases HLA class II expression in an EBV-negative B cell line.

Transformation of normal resting B cells by the Epstein-Barr virus (EBV) leads to the establishment of permanent lymphoblastoid cell lines (LCL) which express high levels of HLA antigens and which are highly efficient in antigen presentation. Certain features of the LCL phenotype can be reproduced by transfecting EBV-negative B lymphoma (BL) cell lines with individual EBV latent genes under heterologous promoters. In this work we have analyzed a series of subclones derived from the EBV-negative cell line Louckes, stably transfected with constructs encoding EBV latent genes for their expression of HLA class II molecules. Louckes parental cells and control transfectants expressed detectable levels of HLA-DR, DQ and DP antigens on the cellular surface by cytofluorometry, but these levels were significantly increased in transfectants expressing the virus-coded latent membrane protein 1 (LMP-1). Northern blotting for the individual alpha and beta chain mRNA at each of the three HLA class II loci indicated correspondingly increased levels of HLA class II transcripts in the LMP-1 transfectants. Transfectants expressing the virus-coded nuclear antigens EBNA-1, EBNA-2 or EBNA-LP showed no significant changes in these parameters. These observations indicate that up-regulation of HLA-class II molecules can be a part of the changes induced by LMP-1 in B cells.

Antigens, Viral↗
PubMed1994

Differential regulation of mRNAs encoding protein kinase C isoenzymes in activated human B cells.

Stimulation of human B cells via HLA class II antigens leads to an increase of PKC activity as a consequence of a transcriptional upregulation of the PKC. Extending previous data, other known B-cell activators, which include anti-IgM, SAC, and TSST1, are shown here to increase the cytosolic PKC activity significantly. Human B cells express significant mRNA levels of the PKC alpha, beta, delta, epsilon, and zeta species while the gamma species is consistently absent. The levels of PKC alpha and epsilon mRNA are increased by exposure to a nonmitogenic anti-IgM antibody in a lymphoblastoid B-cell line while PKC beta and delta mRNA are instead downregulated by this agent. An anti-HLA class II antibody (D1.12) induced an increase of PKC alpha, beta, and delta mRNA. A time study of PKC mRNA levels in anti-IgM-treated cells showed that the accumulation of the PKC alpha mRNA precedes the increase of PKC enzymatic activity. Moreover, PKC beta mRNA decreased following treatment with SAC while, on the contrary, it increased following TPA, anti-HLA class II (1.35) mAb, or mitogenic anti-IgM treatment. Our results underline the complexity of signal transduction via the PKC pathway by revealing that the PKC isoforms are differentially regulated and are in keeping with the idea that they may have distinct physiologic roles in human B cells.

B-Lymphocytes↗
PubMed1994

Influence of human leukocyte antigen genes on TCR V gene segment frequencies.

Human leukocyte antigen (HLA)-dependent selection mechanisms exerted during thymic maturation are supposed to be main contributing factors to the genetic predetermination of the TCR repertoire and may have a detectable effect on adult peripheral blood lymphocyte V segment frequencies. Here, we analyzed whether polymorphic or non-polymorphic HLA determinants are associated with selected expression of some V gene segment specificities. We first examined the reactivity of 17 V segment specific mAb on purified CD4+ and CD8+ cell fractions in 10 unrelated people. We found a significant overexpression of only three V segment products (V beta 2, V beta 5.1 and V beta 6.7) in CD4+ and none in CD8+ cell fractions in most individuals. Skewing of certain V beta segments by non-polymorphic HLA determinants (i.e. class II for CD4+ and class I for CD8+ cells) is therefore more limited (3/17) than previously thought. Considering the effects of polymorphic HLA determinants, we compared TCR V segment frequencies in HLA-identical siblings to sibling pairs who differ at one or both HLA haplotypes, using 13 V beta specific mAb. In pairwise comparisons, we found that the HLA complex had no detectable effect on TCR repertoire in five large families with multiple siblings. Together, these observations suggest that HLA-predicted selection mechanisms exerted during thymic maturation might not have a predominant influence shaping the TCR repertoire of normal adults.

Antibodies, Monoclonal↗