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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 145 records · Page 8Linked to original sources
PubMed1989

Remarkable sequence conservation of the HLA-DQB2 locus (DX beta) within the highly polymorphic DQ subregion of the human MHC.

The HLA-D region of the major histocompatibility complex (MHC) is characterized by a remarkable diversity. Most of the HLA class II genes are highly polymorphic, and in addition, the number and organization of individual loci in that region varies in different haplotypes. This extensive allelic polymorphism of immune response genes has well-known functional implications. Within the HLA-D region, two loci, DQA2 and DQB2 (formerly called DX alpha and DX beta), represent a very special case: the detailed structure of these two genes is entirely compatible with expression, yet their expression has never been demonstrated in any tissue. Consequently, there exists no known corresponding protein product. Pseudogenes are known to accumulate mutations, as observed for instance in the case of HLA-DPA2,-DPB2, or -DRB2 genes. We have therefore investigated the extent of DQ2 genes' polymorphism by DNA sequence comparison and by oligonucleotide hybridization across a large number of different haplotypes, and compared it with other genes in the HLA-D region. We show here that, contrary to the adjacent DQ1 genes, DQ2 genes exhibit little and possibly no polymorphism. This conservation of DQ2 genes in many haplotypes indicates that the DQ1-DQ2 duplication event must have preceeded the extensive diversification of DQ1 genes and raises the puzzling question of why DQ2 genes have remained nonpolymorphic. This suggests that either these genes correspond to an unusually invariant region of the MHC or they are under a strong selective pressure for the conservation of the amino acid sequence of a putative DQ2 gene product. The latter would imply that the HLA-DQ2 genes are expressed into a protein product endowed with essential functional properties.

Alleles↗
PubMed1989

Combined immunodeficiency with abnormal expression of MHC class II genes.

The MHC class II CID represents an example of immunodeficiency in which the defect in expression of membrane glycoproteins leads to abnormal cell to cell interactions and thus to abnormal immune responses. It represents an interesting model which confirms the importance of MHC molecules in all immune responses to foreign antigens. It also underlines the complexity of regulatory mechanism which control the expression of MHC class II genes. To elucidate these mechanisms, it is essential to identify and characterize the genes involved in control of MHC class II expression.

Agammaglobulinemia↗
PubMed1989

A T-cell clone recognizing an MLC stimulating epitope located on the DRw11 beta 1 chain.

An alloreactive proliferative T-cell clone, 6065 WS, was obtained in an intrafamilial cell combination where the stimulator was a homozygous DRw11, DRw52, DQw3 son and the responder his haploidentical mother. Proliferation assays on eight local DRw11 families, 75 homozygous B-cell lines (Tenth Histocompatibility Workshop panel) and blocking assays with monoclonal antibodies showed that clone 6065 WS recognizes an epitope on the DRw11 beta 1 chain. Comparison of the reactivity of clone 6065 WS with cells expressing the three known DRw11 beta 1 amino acid sequences identified two unique amino acids at positions 71 and 86 which contribute to determining the specific recognition by the T-cell clone 6065 WS. Our data suggest that one or both of these amino acids can either be directly involved in the recognition by the T-cell receptor or responsible for critical conformation of the determinants on the DR molecule. Alternatively, they could affect recognition of a self peptide bound to the major histocompatibility complex class II molecule.

Amino Acid Sequence↗
PubMed1989

DNA typing of DRw6 subtypes: correlation with DRB1 and DRB3 allelic sequences by hybridization with oligonucleotide probes.

Among MHC class II antigens, the DRw6/Dw6 complex represents a special situation where typing on a routine basis is often troublesome, mainly because monospecific alloantisera are rare and individual subtypes numerous. We demonstrate here that the use of oligonucleotide DNA typing permits an analysis of the polymorphism within DRw6 haplotypes and provides a molecular basis for correlations with functional data. Synthetic oligonucleotide probes, most of them locus- and allele-specific, were derived from the DNA sequences of three alleles of locus DRB1 and three alleles of locus DRB3. These probes allow the positive identification of distinct DRw6 subtypes. As analyzed on a panel of 26 well-defined DRw6 cell lines, oligotyping allows a direct and absolute correlation with the DRw13 serologic specificity and with the cellularly defined Dw9,Dw16,Dw18, and Dw19 specificities. Correlations of the polymorphism at the DRB1 locus with the polymorphism at the DRB3 locus (DRw52 alleles) allow us to identify preferential allelic associations such as DRw13-Dw18-DRw52a/52b, DRw13-Dw19-DRw52c, and DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least DRw14-Dw9-DRw52b. In view of the absolute segregation of the DRw52c allele with the DRw13/Dw19 haplotype, the Dw19 cellular reactivity might involve, at least in part, epitopes on the DRw52c allele. The identification of DRw6 subtypes, as well as of other HLA class II subspecificities, by oligotyping can now complement and possibly replace serologic and cellular typing. It represents a particularly useful contribution to the optimization of class II matching in the case of bone marrow transplantation with unrelated donors.

Alleles↗
PubMed1989

Cloning of the major histocompatibility complex class II promoter binding protein affected in a hereditary defect in class II gene regulation.

The regulation of major histocompatibility complex class II gene expression is directly involved in the control of normal and abnormal immune responses. In humans, HLA-DR, -DQ, and -DP class II heterodimers are encoded by a family of alpha- and beta-chain genes clustered in the major histocompatibility complex. Their expression is developmentally controlled and normally restricted to certain cell types. This control is mediated by cis-acting sequences in class II promoters and by trans-acting regulatory factors. Several nuclear proteins bind to class II promoter sequences. In a form of hereditary immunodeficiency characterized by a defect in a trans-acting regulatory factor controlling class II gene transcription, we have observed that one of these nuclear factors (RF-X) does not bind to its target sequence (the class II X box). A cDNA encoding RF-X was isolated by screening a phage expression library with an X-box binding-site probe. The recombinant protein has the binding specificity of RF-X, including a characteristic gradient of affinity for the X boxes of HLA-DR, -DP, and -DQ promoters. RF-X mRNA is present in the regulatory mutants, indicating a defect in the synthesis of a functional form of the RF-X protein.

Base Sequence↗
PubMed1989

Inherited immunodeficiency with a defect in a major histocompatibility complex class II promoter-binding protein differs in the chromatin structure of the HLA-DRA gene.

A defect in a trans-regulatory factor which controls major histocompatibility complex class II gene expression is responsible for an inherited form of immunodeficiency with a lack of expression of human leukocyte antigen (HLA) class II antigens. We have recently described and cloned an HLA class II promoter DNA-binding protein, RF-X, present in normal B cells and absent in these class II-deficient regulatory mutants. Here we report that these in vitro results correlate with a specific change in the chromatin structure of the class II promoter: two prominent DNase I-hypersensitive sites were identified in the promoter of the HLA-DRA gene in normal B lymphocytes and found to be absent in the class II-deficient mutant cells. The same two prominent DNase I-hypersensitive sites were observed in normal fibroblastic cells induced by gamma interferon to express class II genes. Interestingly, they were also observed in the uninduced class II-negative fibroblastic cells, which have also been shown to have a normal RF-X binding pattern. We conclude that the two DNase I-hypersensitive sites in the HLA-DRA promoter reflect features in chromatin structure which correlate with the binding of the trans-acting factor RF-X and which are necessary but not sufficient for the expression of class II genes.

B-Lymphocytes↗
PubMed1989

Combined immunodeficiency with defective expression in MHC class II genes.

MHC class II deficiency is an inherited immunodeficiency disease characterized by the presence of a normal number of T and B lymphocytes and profound anomaly of cellular and humoral responses to foreign antigens. All bone-marrow-derived cells (including B lymphocytes, monocytes and activated T lymphocytes) and also enterocytes and endothelial cells do not express all HLA class II (DR, DQ and DP) molecules on their membrane. It is known that the proper recognition of foreign antigens depends on their presentation, together with HLA class II molecules, on the membrane of antigen-presenting cells. MHC class II deficient combined immunodeficiency confirms the important role of MHC gene products in immune-defence mechanisms. Patients suffer from repeated and severe infections that are frequently the cause of death. The defect in HLA class II expression is the consequence of a lack of synthesis of HLA class II alpha and beta chains in patients' cells. Studies performed at DNA and RNA levels showed that there was no gross abnormality of MHC class II genes and that mRNA for all HLA molecules was not detected in patients' cells. These results, together with segregation studies performed in several families, suggested that the defect in HLA class II gene expression involves a transacting regulatory factor. Direct transcription assays showed that the disease is characterized by an absence of HLA class II gene transcription. An analysis of the specific binding of nuclear proteins from patients' cell lines to HLA class II promotor showed that a specific protein, RF-X, which normally binds to a regulatory sequence common to HLA class II promotors, is affected in MHC class II combined immunodeficiency.

Gene Expression Regulation↗
PubMed1989

[The frequency of detection of Mycoplasma meleagridis in breeding turkeys depending on the laying age].

Coating of air sacs was recorded from day-old chicks from parents with Mycoplasma (M.) meleagridis infection, with positive findings being obtained from 9.52% of all animals early in the laying period and from 34.09% up to the 8th laying week. M. meleagridis was isolated from palatine and cloacal swabs taken of laying hens and insemination cocks, with positive findings being 50-60% prior to the laying period (28th week of age), 100% at start of laying, and 80% in the 14th laying week. M. meleagridis was identified in 50% of all embryonated eggs as of the 1st laying week and in 100% as of the 4th week. M. meleagridis was cultured from 30.67% of all sperm samples tested, between the 30th and 46th week of production. Differences were found to exist between individual cocks, with 4 cocks being without M. meleagridis at all. There was usually agreement between positive M. meleagridis findings from sperm and cloacal swabs. M. meleagridis was eliminated from cock sperm by spectinomycin (0.6 mg/ml diluting medium), but M. iowae was not.

Age Factors↗
PubMed1988

Differential effects of in vitro mycoplasma infection on interleukin-1 alpha and beta mRNA expression in U937 and A431 cells.

Cultured cells depend on cytokine mediators for sustained growth and maintenance and are routinely employed in bioassays to detect and measure minute changes in biological mediators, e.g. the interferons and interleukins. We evaluated the effects of mycoplasma infection on the steady-state mRNA levels of two cytokines IL-1 alpha and beta. Noninfected human squamous carcinoma cell line A431 expressed constitutively IL-1 alpha and beta mRNA. In contrast freshly isolated peripheral blood mononuclear cells and the monocytic cell line U937 expressed abundant IL-1 mRNA only after the appropriate stimulation. Peripheral blood mononuclear cells and U937 steady-state IL-1 beta mRNA levels were considerably greater than IL-1 alpha mRNA levels, whereas nearly equivalent high levels of IL-1 alpha and beta mRNA were detected in A431 cells. Mycoplasma infection of cultured A431 cells reduced the steady-state levels of IL-1 alpha and beta mRNA. This effect was nonspecific for A431 cells as actin mRNA steady-state levels showed similar decreases to mycoplasma contamination. However, this response was cell specific since mycoplasma-free and contaminated U937 cells differed little in IL-1 mRNA expression. These results show that the response to mycoplasma infection is at least partly cell-type dependent.

Cell Line↗
PubMed1988

Identification of HIV-infected seronegative individuals by a direct diagnostic test based on hybridisation to amplified viral DNA.

There is a need for direct detection of the virus in people infected with human immunodeficiency virus (HIV), independently of a serological response. In this study, after enzymic amplification of a specific segment of the HIV genome, a simple slot-blot hybridisation procedure allowed unequivocal identification of HIV DNA in all seropositive subjects tested. More importantly, the hybridisation test allowed the detection of HIV DNA in several seronegative subjects from very high risk groups. This new direct approach towards the diagnosis of HIV infection, which can easily be carried out on a large scale, is therefore capable of identifying HIV-infected individuals before the development of antibodies.

DNA, Viral↗
PubMed1988

Congenital immunodeficiency with a regulatory defect in MHC class II gene expression lacks a specific HLA-DR promoter binding protein, RF-X.

The expression of MHC class II genes is tightly regulated. One form of congenital severe combined immunodeficiency (SCID) is characterized by a regulatory defect that precludes expression of HLA class II genes. B lymphocyte cell lines from such SCID patients provide a tool for identifying putative regulatory proteins that bind to class II gene promoters. We have identified three proteins binding to specific segments of the HLA-DRA promoter, two of which interact to form the predominant DNA-protein complex observed. One of these proteins, defined as an X box binding protein (RF-X), is specifically missing in cells from class II deficient SCID patients. We propose that the molecular defect in this congenital HLA class II regulatory deficiency is a lack of RF-X and that this factor plays an important role in the normal regulation of MHC class II gene expression.

B-Lymphocytes↗
PubMed1988

Regulation of genes for HLA class II antigens in cell lines from patients with severe combined immunodeficiency.

HLA Class II-negative severe combined immunodeficiency (SCID) results from a congenital defect characterized by an absence of HLA Class II antigens. Patients with the disorder have no HLA-DR, DQ, or DP antigens or mRNAs in their peripheral-blood lymphocytes. The affected gene is a recessive, transacting regulatory gene that controls the expression of Class II genes. We studied the regulation of HLA Class II gene expression with the use of established Epstein-Barr virus-transformed B-cell lines and skin fibroblast lines from a group of patients with SCID. Lymphoblastoid B-cell lines from the patients contained no mRNA for HLA-DR, DQ, and DP alpha and beta polypeptides, but did express mRNA for the HLA-associated invariant chain, which is normally coregulated with HLA Class II antigens. In the B-cell line from one patient, a very low amount of DR mRNA could be detected, indicating some heterogeneity in SCID. The lymphokine gamma-interferon, a strong inducer of Class II genes in a variety of normal cells, did not restore Class II gene expression in any of the SCID B-cell lines. More important, gamma-interferon was unable to induce any Class II mRNA in fibroblast lines from patients with SCID, in contrast to the efficient induction observed in normal fibroblasts. The invariant-chain gene, however, was induced in the SCID fibroblasts, confirming a unique uncoupling in the regulation of invariant and Class II genes. Thus, the genetic defect in patients with SCID affects not only the B-cell lineage but also the inducible expression of HLA Class II genes that is normally observed in Class II-negative cells, such as fibroblasts. This unresponsiveness to gamma-interferon in vitro indicates that patients with SCID will not respond to treatment with this lymphokine. Our data also increase understanding of the normal mechanisms regulating the genes for the HLA Class II cell-surface glycoproteins.

Cell Line↗
PubMed1988

Functional polymorphism of each of the two HLA-DR beta chain loci demonstrated with antigen-specific DR3- and DRw52-restricted T cell clones.

HLA-DR3- and HLA-DRw52-associated functional polymorphism was investigated with selected tetanus toxoid (TT)-specific T cell clones. We have shown earlier that HLA-DR antigens are encoded by two distinct loci, DR beta I and DR beta III. The alloantigenic determinant(s) defined by the serological HLA-DR3 specificity map to the former, while the supratypic HLA-DRw52 determinants map to DR beta III. Furthermore, we have recently recognized by DNA sequencing three alleles of HLA-DRw52 at locus DR beta III, referred to as 52 a, b, and c. Our objective was to correlate the pattern of T cell restriction with the gene products of individual DR beta chain loci and with the three newly described alleles of locus DR beta III. Among the selected T cell clones, 5 reacted exclusively when TT was presented by HLA-DR3+ APCs (TT-DR3-APC). In contrast, two T cell clones were stimulated by TT-DRw52-APC. More specifically, these two T cell clones (Clones 10 and 16) were stimulated by different subsets of TT-DRw52-APC. Clone 16 responded to some DR3 and TT-DRw6-APC, while clone 10 was stimulated by other TT-DR3 and TT-DRw6, and all TT-DR5-APC. This same pattern of DRw52 restriction was found in panel, as well as in family studies. Because this suggested a correlation with the pattern of DRw52 polymorphism observed earlier by DNA sequencing and oligonucleotide hybridization, the APC used in these experiments were typed for the 52 a, b, and c alleles of locus DR beta III by allele-specific oligonucleotide probes. This distribution overlapped exactly with the stimulation pattern defined by the T cell clones. Clone 16 responded to TT-52a-APC, clone 10 to TT-52b-APC, and both clones to a TT-52c-APC. The response of the T cell clones was inhibited differentially by mAbs to DR. Raising TT concentration, or increasing HLA-class II expression with INF-gamma both affected the magnitude of response of the TT-specific clones but did not modify their specificities. These results demonstrate that a restriction specificity can be attributed to the DR beta III locus and illustrate the functional relevance of the polymorphism observed at this locus. This is of special interest in view of the striking difference in the pattern of structural diversity among alleles of DR beta I and DR beta III.

Alleles↗
PubMed1988

Serological recognition of HLA-DR allodeterminant corresponding to DNA sequence involved in gene conversion.

HLA class II molecules were isolated from mouse L cells transfected with a DR alpha gene and an allele, 52a, of locus DR beta III from an HLA-homozygous cell line, AVL, of the DR3 haplotype. The isolated molecules were found to possess a new allospecificity, named TR81. This specificity behaved allelic to the previously described specificity TR22 encoded by another allele, 52b, of the DR beta III locus. The TR81 specificity was also present on the DR beta I gene product of the DR3 haplotype. The nucleotide sequence of the gene encoding TR81 differs from TR81-negative DR beta genes of the DRw52 family in only two codons, both located in the regions known to be involved in a gene conversion event. Consequently, the following conclusions can be formulated. (a) TR81 is a bi-locus specificity and allelic to TR22 only in its DR beta III locus localization. (b) The TR81 specificity is the phenotypic counterpart of the gene conversion event which led to the generation of the DR beta I gene of the DR3 haplotype. (c) One or both individual amino acid substitutions in the first domain of the DR beta chain are responsible for the TR81 allospecificity. (d) Since TR81 is expressed on the DR beta I chain of the DR3 haplotype, it is possible that TR81 and DR3 represent the same serological specificity.

Alleles↗
PubMed1988

The single DR beta gene of the DRw8 haplotype is closely related to the DR beta 3III gene encoding DRw52.

In most individuals two HLA-DR beta genes are expressed from each chromosome. One of these genes encodes one of the classical DR specificities, while the other encodes either of the supertypic DRw52/DRw53 specificities. In addition to these genes usually one or two DR beta pseudogenes are present. In contrast, the DRw8 chromosomal region only contains a single DR beta gene. To determine the relationship of this single gene to the multiple DR beta genes of other DR specificities, comparisons of Southern genomic blots were carried out. In this analysis genomic clones for each individual DR beta chain locus were included. The DR beta w8 gene was indistinguishable from the DR beta III gene of DR3 cells (encoding DRw52), suggesting that it is closely related to the latter gene. The functional implications of this finding are discussed.

Genes, MHC Class II↗
PubMed1988

Identification and distribution of three serologically undetected alleles of HLA-DR by oligonucleotide.DNA typing analysis.

Recent progress in the molecular biology of human major histocompatibility complex class II genes (HLA-DP, -DQ, -DR) have shown that the genetic complexity and allelic polymorphism are greater than expected. In the case of HLA-DR, three DR beta-chain loci have been identified and linked, two of which (DR beta I and DR beta III, now assigned names HLA-DRIB and HLA-DR3B) are functional. We have shown that the HLA micropolymorphism detected at the DNA sequence level can easily be analyzed by hybridization with allele-specific oligonucleotides (HLA "oligotyping"). In the case of the HLA DRw52 supertypic specificity, which includes the DR3, DR5, DRw6, and DRw8 haplotypes, three alleles, referred to as DRw52a, DRw52b, and DRw52c, have recently been identified at the HLA-DR3B locus by DNA sequencing. Hybridization with locus- and allele-specific oligonucleotide probes (designated 52a, 52b, and 52c) has been performed on DNA from normal individuals forming a panel of 82 haplotypes to establish the distribution of these three alleles. Individuals of the DR3 haplotype had either the DRw52a or DRw52b allele, and individuals of extended haplotype HLA-A1,B8,DR3 had only the DRw52a allele. DR5 individuals all had the DRw52b allele, while individuals of DRw6 haplotype had the DRw52a, -52b, or -52c allele. None of these three alelles are found in DRw8 individuals. Analysis of this micropolymorphism, undetectable by common typing procedures, is therefore now operational for more accurate HLA matching for transplantation and for improving correlations between HLA and disease susceptibility.

Alleles↗