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B Mach

Publications and source records attributed to B Mach.

At least 73 records · Page 4Linked to original sources
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↗
PubMed1987

A urine inhibitor of interleukin 1 activity affects both interleukin 1 alpha and 1 beta but not tumor necrosis factor alpha.

Urine from monocytic leukemia and other febrile patients contains an inhibitor of interleukin 1 (IL-1), as measured by prostaglandin E2 and collagenase production by human fibroblasts and synovial cells. With the use of recombinant IL-1, the IL-1 inhibitor was partially purified by using ammonium sulfate precipitation, anion-exchange, and gel filtration chromatographies. IL-1 inhibitory activity elutes with an 18,000 to 25,000 apparent molecular size. The same fractions also inhibit IL-1 assayed by the proliferation of murine thymocytes and human fibroblasts. Both forms of human recombinant IL-1, IL-1 alpha and IL-1 beta, which show only 26% homology, but nevertheless bind to the same receptor, are affected by this natural inhibitor to the same extent. In contrast, human recombinant tumor necrosis factor, which shares some of the biologic activities of IL-1, is not inhibited by the urinary IL-1 inhibitor. This study shows that the various biologic activities of both forms of human recombinant IL-1 are inhibited by a partially purified natural urine-derived factor.

Cell Division↗
PubMed1987

Constitutive and induced expression of the individual HLA-DR beta and alpha chain loci in different cell types.

The HLA-DR subregion of the human major histocompatibility complex encodes molecules involved in the regulation of the immune response. These HLA class II molecules are transmembrane heterodimers composed of an alpha and a beta chain. The polymorphic beta chains are encoded by multiple, highly homologous loci, whereas the alpha chain is encoded by a single, nonpolymorphic locus. HLA-DR is expressed constitutively on B lymphocytes and on activated T lymphocytes. It can also be induced by interferon-gamma on most nonlymphoid cells. In a quantitative study of the expression of the individual DR beta chain loci, we have investigated: the levels of mRNA transcripts of the two functional DR beta loci (beta I and beta III) in B cells of various haplotypes; whether both beta chain loci are expressed in activated T cells and, if so, the level of expression of each; whether both loci are expressed in interferon-gamma-induced nonlymphoid cells. This analysis relied on locus-specific DR beta chain oligonucleotide probes. Expression of both the beta I and the beta III loci was observed in all cell types and in all haplotypes tested. In every case the amount of beta I mRNA was about 5 times higher than that of beta III mRNA. This indicates a controlled and coordinated regulation of the mRNA levels of these two HLA-DR loci under all conditions of major histocompatibility complex class II gene expression.

B-Lymphocytes↗
PubMed1987

Characterization of N-linked oligosaccharides of an HLA-DR molecule expressed in different cell lines.

In order to determine the factors that influence the glycosylation of an integral membrane protein, we investigated the N-glycosylation of a molecule of the human major histocompatibility complex (MHC) class II, the HLA-DR antigen. This glycoprotein was studied in a human Epstein-Barr-virus-transformed B cell line and in a mouse fibroblastic cell line co-transfected with DR alpha and DR beta genes. We observed that the HLA-DR-antigen glycosylation pattern depends on the cell line in which processing takes place and is closely related to the glycosylation pattern of the overall cellular glycoproteins. Furthermore, when comparing the glycosylation of the separated alpha- and beta-chains, differences were noticed within the same molecule, showing the importance of the individual peptide backbone for the glycosylation process.

Cell Line↗
PubMed1987

Molecular cloning of glucokinase cDNA. Developmental and dietary regulation of glucokinase mRNA in rat liver.

A rat liver cDNA library enriched for glucokinase sequences was constructed using the phage expression vector lambda gt11 and screened with an antiserum to glucokinase. A positive phage clone termed lambda-GK223 was isolated by several rounds of plaque purification. When introduced in the high frequency lysogenization strain Y1089, the phage was shown to encode a fusion protein containing epitopes specific to rat liver glucokinase. The 1800-base pair cDNA insert of lambda-GK223 was subcloned in a pUC plasmid, and a resulting recombinant termed pUC-GK1 was used for hybrid selection of mRNA. The selected mRNA directed the synthesis in a cell-free translation system of a protein identified as glucokinase by electrophoresis and immunoprecipitation. The cloned cDNA was then used as a probe to measure the amount of glucokinase mRNA in rat liver during postnatal development. Glucokinase mRNA, 2.4 kilobases in length, was first detectable at day 14 after birth and increased 40-fold in amount from this age to day 31, in parallel with the emergence of glucokinase enzyme activity. In the adult rat, glucokinase mRNA was low during fasting and increased more than 50-fold above the fasting level within 6 h of an oral glucose load. However, maximal accumulation of glucokinase mRNA was short-lived and the mRNA level returned toward basal values by 18 h of refeeding. These data point to rapid and massive effects on the expression of the glucokinase gene at the transcriptional or post-transcriptional levels during ontogenic development and dietary changes in the adult animal.

Animals↗
PubMed1987

DNA typing of HLA-DR beta chain genes can discriminate between undetected alleles and real homozygotes.

The polymorphism of HLA-DR antigens has been studied by Southern blot hybridization under conditions specific for the detection of the DR beta chain genes. Haplotype-specific patterns were defined with DNA from DR1, 2, 3, 4, 7, w8, w11, w12, and W13 homozygous typing cells, with restriction enzymes Eco RI, Bgl I, and Pvu II. Certain serological specificities, such as DR2, DR3, and DR7, can be encoded by distinct allelic forms of DR beta chain genes. The procedure of "DNA typing" was applied to family analysis of individuals expressing only a single DR specificity upon serological typing. Three cases are described here: (1) in family GR, phenotypic DR 7 homozygotes correspond to genomic heterozygotes, and a novel DR7 allele is described: (2) in family RU, the genes corresponding to a serologically undetected (blank) DR allele were identified by restriction fragment length polymorphism (RFLP); this novel DR haplotype has an RFLP pattern similar to those of the DRw52 family, even though this specificity was not expressed on the DR-blank lymphocytes; (3) in family RG, there is no blank allele, but a homozygote RFLP situation at the DR subregion.

Alleles↗
PubMed1987

Oligonucleotide genotyping shows that alleles at the HLA-DR beta III locus of the DRw52 supertypic group segregate independently of known DR or Dw specificities.

Using locus- and allele-specific oligonucleotide probes, we have studied the polymorphism of the HLA-DR beta III locus within the haplotypes of the DRw52 supertypic group. DNA from a number of homozygous typing cells typed for both Dw and DR was used. The DR beta III polymorphisms, DRw52a and DRw52b, do not segregate with Dw typing, or with DR typing, indicating that the determinants responsible for Dw-defined T-cell response and for DR haplotypic recognition are not encoded by the DR beta III locus. Hence, we can conclude that these DR specificities are encoded by the other functional DR locus, DR beta I, while the DR beta III locus encodes only the supertypic product.

Base Sequence↗
PubMed1987

Structural comparison of the genes of two HLA-DR supertypic groups: the loci encoding DRw52 and DRw53 are not truly allelic.

The organization and sequence of the HLA-DR beta chain genes are compared in the two supertypic groups, DRw52 and DRw53, which together account for more than 80% of HLA-DR alleles. From the structural data, we conclude that these two groups represent distinct lineages which have followed different patterns of evolution. The fine structure of the beta chain locus encoding the DRw53 specificity corresponds most closely to the DR beta II pseudogene in the DRw52 haplotypes. Concomitantly, the DR beta I locus in DRw53 haplotypes is more closely related to both of the two expressed DR beta loci of the DRw52 haplotypes (DR beta I and DR beta III). These two loci are the result of a recent duplication. This leads to the proposal that both expressed DR beta chain genes in the DRw52 haplotypes (DR beta I and DR beta III) are derived from a single precursor locus, while the two loci expressed in the DRw53 haplotypes are derived from distinct ancestral loci. The genes encoding DRw52 and DRw53 are therefore not true alleles of the same original locus. A scheme is proposed that accounts for the evolution of DR specificities within the DRw52 and DRw53 groups of haplotypes. It is evident that the different HLA-DR alleles are not structurally equidistant and that one must take into consideration different degrees of heterozygosity or mismatch among the DR alleles.

Alleles↗
PubMed1987

Characterization of an HLA-DR beta pseudogene in the DRw52 supertypic group.

The nature of the DR beta II pseudogene in a haplotype of the DRw52 supertypic group was investigated by nucleotide sequence analysis. It revealed several deleterious mutations in the signal sequence and second domain regions in addition to the complete absence of the first domain and adjacent sequences. No expression of DR beta II pseudogene mRNA can be detected. The same DR beta II pseudogene is probably present in other members of the DRw52 supertypic group. The pattern of mutations in this DR beta II pseudogene is different from that observed in the DR beta pseudogene of the DRw53 supertypic group, indicating a distinct evolutionary pathway for these two groups of DR haplotypes.

Amino Acid Sequence↗
PubMed1987

HLA-DR alpha, -DX alpha, and DR beta III gene association studies in DR3 individuals.

In this study we have examined the results of probing with synthetic oligomers at the DR beta III locus, together with restriction fragment length polymorphisms defined by BglII digestion and a cDNA DR alpha probe, and Taq 1 digestion and a genomic DQ alpha probe. We have demonstrated heterogeneity of the human leukocyte antigen DR3 and close association of the DR alpha, DR beta III, and DX alpha genes. Two DR3-related preferential allelic associations have been identified, which may prove useful in family analysis as well as for investigations of DR3-related diseases.

Alleles↗
PubMed1987

Analysis of HLA-D micropolymorphism by a simple procedure: RNA oligonucleotide hybridization.

Recent progress in the molecular genetics of HLA class II antigens has revealed the existence of multiple loci and of a large degree of polymorphism, with more individual alleles than was expected. An accurate detection and analysis of this extensive polymorphism is essential for optimal HLA typing for transplantation and for a reevaluation of HLA-disease association. Because of the limitations of the current typing methods, including restriction fragment length polymorphisms, we have proposed a DNA typing procedure based on hybridization with loci- and allele-specific oligonucleotides. Here we present a much simpler way of analyzing class II micropolymorphism down to the level of single nucleotide differences. RNA oligonucleotide typing (ROT) relies on RNA dot blots and requires 10-20 ml of blood. It is shown that with appropriate oligonucleotide probes, ROT can reliably and unambiguously identify any polymorphism at any of the HLA loci, including new alleles, not identified with previous methods. This illustrates the importance of oligonucleotide typing to optimize HLA matching, in particular for transplantation involving unrelated donors.

Alleles↗
PubMed1987

Expression of human IL 1 alpha and beta messenger RNAs and IL 1 activity in human peripheral blood mononuclear cells.

The macrophage-derived lymphokine interleukin 1 (IL 1) plays a critical role in modulating immune (cellular and humoral) and nonimmune responses. For example, the relative expression of IL 1 alpha and beta under various states may be crucial to the success of the immune system in response to infection. Until recently, a comparative study of IL 1 mRNA expression and IL 1 biological activity was not possible. We have cloned both IL 1 alpha and beta cDNAs and employed them as probes in Northern blot analysis to determine in mitogen-stimulated peripheral blood mononuclear cells the steady-state expression of their cognate mRNAs with respect to IL 1 activity. IL 1 was determined by the lymphocyte-activating factor (IL 1/LAF) and the mononuclear cell factor (IL 1/MCF) activities. In lectin-stimulated PBMC, maximum cell-associated activities whereas detected at 12 and 24 hr after stimulation whereas maximum extracellular activities appeared between 24-48 hr. In the same cultures, the kinetics of IL 1 mRNA steady-state expression were determined by Northern gel blot analysis with IL 1 alpha and beta cDNA probes. IL 1 mRNAs were undetectable in noncultured freshly isolated PBMC (time zero). Both IL 1 mRNAs appeared as early as 4 hr after lectin stimulation as did IL 1 beta mRNA in unstimulated cultures. Both IL 1 alpha and beta mRNA steady-state levels were barely detectable by 48 hr. At all time points, IL 1 mRNA levels were considerably lower in unstimulated cultures. IL 1 beta mRNA was always considerably more abundant than IL 1 alpha mRNA. The less abundant IL 1 alpha mRNA showed a decrease in its stead-state levels prior to the reduction in the levels of IL 1 beta mRNA. TNF alpha activity and mRNA were not detected under these culture conditions. Poly(A) + RNA injected into Xenopus oocytes revealed that the Northern blot detected IL 1 mRNAs were biologically active. To understand the precise nature of IL 1 in immune and nonimmune events, we felt it necessary to first study the kinetics of IL 1 mRNA steady-state levels with respect to its cell-associated and extracellular biological activities. The data presented here may allow for a better understanding of the etiology of various immune and nonimmune responses that are modulated through the expression of IL 1.

Animals↗
PubMed1986

Alternative splicing and alternative initiation of translation explain the four forms of the Ia antigen-associated invariant chain.

The Ia antigen-associated invariant chain (In) exists in humans as four related polypeptides, p33, p35, p41 and p43, all associated with HLA-class II antigens. As described previously, two of these forms of In chain, p33 and p35, result from the use of two in-phase initiation AUG codons on the unique In p33 mRNA. In addition to cDNA clones derived from In p33 mRNA, we have isolated a new cDNA clone, called p41-1, which differs from p33-1 by an additional segment in the coding region. The DNA sequence encoding the segment unique to p41-1 was identified in the genomic sequence in the intron between exon 6 and 7, and we refer to it as exon 6b. Cells transfected with a full length p41 cDNA clone in an expression vector synthesize the two larger forms of the In chain, p41 and p43. We propose that the larger mRNA, encoding p41, results from alternative splicing of exon 6b, and that p41 and p43 result from the use of the two functional initiation AUG codons identified in p33 mRNA. Alternative splicing, together with alternative initiation of translation, allows therefore the synthesis of four related In chain polypeptides from a single gene.

B-Lymphocytes↗