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Biomedical subjects

B Mach

Publications and source records attributed to B Mach.

At least 55 records · Page 3Linked to original sources
PubMed1990

Interferon gamma drastically modifies the regulation of interleukin 1 genes by endotoxin in U937 cells.

IL-1 alpha, IL-1 beta, and tumor necrosis factor alpha (TNF-alpha) gene expression is induced by LPS (endotoxin) in monocytes/macrophages and in some monocytic cell lines. IFN gamma and 1 alpha,25-dihydroxyvitamin D3 (1,25[OH]2D3) are important macrophage-activating factors. They induce changes in the human monocyte cell line U937 that reflect cellular differentiation. We have studied the effect of IFN-gamma and of 1,25(OH)2D3 on the expression of IL-1 and TNF-alpha messenger RNA in response to LPS. The induction of these genes by LPS is immediate and transient, with a maximum in 3 h. Preincubation of the cells with IFN-gamma or with 1,25(OH)2D3 increases these mRNA responses to LPS about fourfold. More importantly, cells exposed to IFN-gamma for 72 h exhibit a drastically different and unexpected pattern of IL-1 alpha and IL-1 beta gene response to LPS. Instead of the normal transient response, one then observes a sustained increase in IL-1 alpha and IL-1 beta gene expression over at least 16 h after LPS stimulation. This was measured both at the level of mRNA and by direct transcription assays (run-off). This striking effect of IFN-gamma on the kinetics of IL-1 gene response does not apply to the TNF-alpha gene. Interestingly, 1,25(OH)2D3, which shares with IFN-gamma a number of important effects on monocytes/macrophages, does not affect the kinetics of IL-1 gene response to LPS. In view of the biological relevance of endotoxin as a macrophage activator, the potential clinical implication of this prolonged induction of IL-1 gene expression is discussed.

Blotting, Northern↗
PubMed1989

[Current approach to HLA typing for bone marrow transplantation: oligonucleotide typing by hybridization on DNA amplified by polymerase chain reaction].

The extensive polymorphism of major histocompatibility complex HLA class II antigens plays a crucial role in transplantation immunology. The molecular biology of the HLA-D region has revealed that the polymorphism at the HLA-DR, -DQ and -DP loci is much greater than was expected from serology, and thus requires accurate typing technology. We have shown that HLA class II polymorphism can be analyzed directly at the DNA level by hybridization with locus- and allele-specific oligonucleotide probes (oligotyping) derived from the variable first domain exon sequences of DR, DQ and DP genes. The same HLA typing procedure can be performed by direct hybridization on DNA previously amplified by the polymerase chain reaction (PCR). Here we show that oligotyping can complement and/or replace serological as well as cellular (Dw) typing and serves to predict a positive mixed lymphocyte culture. It is now operational (a) to replace serology when class II expression is absent or aberrant (e.g. leukemic patients, class II deficiencies) and (b) to improve, by the analysis of HLA-DR and -DQ micropolymorphism, the speed and reliability of the selection of optimally-matched unrelated donors for bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1989

Correlation of structure with T cell responses of the three members of the HLA-DRw52 allelic series.

A third allele at the DRB3 locus, DRw52c, represents an intermediate sequence between DRw52a and DRw52b and may have arisen by a gene conversion-like event. The recognition of cells bearing these molecules by a number of alloreactive and antigen-specific DR-restricted T cell clones was analyzed. On the basis of a theoretical model of HLA class II structure, distinct amino acid clusters have been identified as motifs controlling TCR recognition. These are located both in the cleft and in the alpha-helical edge of the MHC class II recognition platform. Motifs shared between two alleles may restrict public T cell clones.

Alleles↗
PubMed1989

Induction of HLA class II genes by IFN-gamma is transcriptional and requires a trans-acting protein.

HLA class II Ag are encoded by a family of related genes clustered in the HLA-D region of the MHC. The expression of this multi-gene family is highly regulated and this regulation is essential for the control of the immune response. Class II gene expression is constitutive in a limited number of cell types and can be induced by IFN-gamma in a number of class II negative cells. In this study, we have clarified two essential aspects of the regulation of HLA class II genes by IFN-gamma. 1) The induction mechanism operates at the level of transcription and there is a long lag phase in the signal transduction process. 2) The induction of class II genes requires the de novo synthesis of a new protein(s). On this basis, we propose that IFN-gamma regulates the transcription of HLA class II genes via the de novo synthesis of a trans-acting activator protein.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1989

Characterization of three functional sites in alpha beta 1 DR of DRw13. All three sites are potentially involved in major histocompatibility complex-peptide interaction.

An HLA-DR product encoded by the HLA-DRw13/Dw19 haplotype has been identified as the HLA class II molecule involved in antigen presentation to several influenza-specific helper T cell clones. Three different functional sites were identified on this molecule by comparing the structure of HLA-DR products of known sequences and their ability to efficiently present foreign antigen to the T cell clones. These functional sites were mapped on the recently proposed three-dimensional structure of HLA class II molecules. From their position, these sites are all potentially involved in HLA-peptide interaction and capable of affecting the binding and/or the conformation of the foreign peptide. This suggests that polymorphic residues essential in major histocompatibility complex restriction are mostly involved in peptide binding.

Amino Acid Sequence↗
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↗