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M J Giphart

Publications and source records attributed to M J Giphart.

At least 73 records · Page 4Linked to original sources
PubMed1990

The nucleotide sequence of bovine MHC class II DQB and DRB genes.

The nucleotide sequences of most of the exons and parts of the introns of two BoLA-DQB genes and two BoLA-DRB genes have been determined. The structure of these genes is very similar to that of human major histocompatibility complex (MHC) class II genes. The two DQB genes probably represent true alleles. Based on the exons sequenced, both DQB genes and one of the DRB genes seem to be functional. The other DRB gene is a pseudogene; stopcodons are found in the exons encoding the second and transmembrane domain and, furthermore, a 2 base pair (bp) deletion has occurred in the leader exon which places the initiation start codon out of frame. Also in this pseudogene, an almost perfect inverted repeat of 200 bp is found flanking the exon encoding the first domain, which might have been the result of a duplication/inversion event. The sequences presented in this paper do not contain any repetitions. Therefore, DNA fragments containing these sequences can be used as homologous bovine probes in restriction fragment length polymorphism (RFLP) analysis to study disease associations in cattle.

Amino Acid Sequence↗
PubMed1990

Homologies between the major histocompatibility complex of man and cattle: consequences for disease resistance and susceptibility.

The major histocompatibility complex (MHC) of mammals contains a large number of mostly duplicated genes. In the HLA system (the MHC of man), which is by far the best-studied major histocompatibility system so far, roughly 20 genes have been defined and mapped. They code for three classes of proteins: HLA-A, -B and -C (Class I), HLA-DP, -DQ and -DR (Class II) and serum complement components C2, C4 and Bf (Class III). Furthermore, the region contains genes for 21-hydroxylase (21-OH) and tumor necrosis factor (TNF). The MHC thus forms a chromosomal segment containing several clusters of genes of only partially defined biological significance, but ondoubtedly playing a role in disease susceptibility. In view of the recently obtained structural information on BoLA, the MHC of cattle, it is hypothesized that susceptibility to diseases in cattle is associated with BoLA in the same way as human diseases. Finally, new technical and conceptual developments in the field of MHC research and their application to the BoLA system are discussed.

Animals↗
PubMed1990

An unexpected effect of matching for HLA-A9 in renal transplantation.

The observation of elevated levels of HLA class I molecules in sera of HLA-A9-positive individuals, and their potential role in the regulation of the immune response, motivated us to study the effect of the presence of HLA-A9 in either kidney donor or recipient on graft survival. Analysis of data from unrelated first transplants performed within the Eurotransplant area revealed that in the group of patients who were not treated with cyclosporine (n = 2051), transplants with no HLA-DR mismatches in which donors (D) and recipients (R) shared the HLA-A9 antigen (D+R+), had significantly poorer graft survival (P = 0.0001) than all other combinations, reaching a 20% difference at 5 years posttransplantation. This effect, which was not found in the CsA-treated patient group (n = 7297), was specific for HLA-A9. The implications of this findings are discussed in relation to the mechanisms of the alloimmune response.

Cyclosporins↗
PubMed1990

Applicability of HLA-DQB oligonucleotide typing for the TA10 and 2B3 specificities in routine HLA typing.

Our group recently developed oligonucleotide probes associated to the TA10 and 2B3 specificities (1). Unambiguous typings were observed in a panel of homozygous typing cells and a family, using 32P-end labeled probes and PCR-amplified DNA (DQB exon 2). To investigate whether these TA10 and 2B3 associated oligonucleotides could be used in routine HLA-typing we extended the study to a panel of healthy, unrelated individuals. When TA10 and 2B3 typings by oligonucleotides were compared with those obtained in routine serology a complete correlation was observed for TA10. A discrepancy was seen for 2B3 typing in material obtained from DQw5- (and possibly DQw4)-positive individuals which could be explained by the CYNAP (cytotoxicity-negative, absorption-positive) phenomenon, using the IIB3 monoclonal antibody in routine tissue typing. Our results suggest that HLA-DQB oligonucleotide typing for TA10 and 2B3 is an accurate and reliable method and can be used effectively in routine HLA typing.

Alleles↗
PubMed1989

Unambiguous typing for HLA-DQ TA10 and 2B3 specificities using specific oligonucleotide probes.

Oligonucleotide probes specific for the serologically defined TA10 and 2B3 specificities were selected based on a comparison of the available HLA-DQ beta sequences. Panel and family segregation studies confirm a complete correlation between the reactivities of the selected probes and the TA10/IIB3 antibodies. The Glu residue at position 45 of the HLA-DQ beta chain is specific for the TA10 determinants, and a DQ beta Gly-Val-Tyr sequence is found at position 45-47 for all 2B3-positive DQ beta chains.

Amino Acid Sequence↗
PubMed1989

Cloning of the bovine major histocompatibility complex class II genes.

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.

Animals↗
PubMed1989

Acquired Zwa antigen on Zwa negative platelets demonstrated by western blotting.

Zwa-negative platelets that had been incubated in random donor AB plasma for several hours and subsequently cryopreserved, were found to be Zwa-positive when tested 3 years later by Western blotting. Following this serendipitous observation, we demonstrated that the Zwa antigen could be passively adsorbed onto the membrane of Zwa-negative platelets incubating for 4 h in the plasma from Zwa-positive donors. This observation supports the hypothesis that passive acquirement of Zwa antigens is a pathogenic mechanism for inducing post-tranfusion purpura, and suggests that Zwa-negative platelets should not be stored in Zwa-positive plasma before transfusion.

Adult↗
PubMed1989

Molecular analysis of the HLA-DR5 haplotype.

A panel of eleven HLA-DR5 homozygous lymphoblastoid cell lines was investigated for structural heterogeneity on the product level. HLA class II antigens were isolated by immunoprecipitation with different anti-class II monoclonal antibodies and separated by two-dimensional (2-D) gel electrophoresis. As a result, three distinct DRB1, one commonly expressed DRB3, and two distinct DQ gene products could be identified that combined to four different haplotypes associated with HLA-DR5. A hitherto serologically undetected split of HLA-DRw11 was presented by three cell lines. HLA-DRw11 and HLA-DRw12 were found to be related allospecifities that differ only in their DRB1 locus products, but are closely associated with the supertypic DRB3 allele HLA-DRw52b and with HLA-DQw7. The DRB3 alleles HLA-DRw52a and DRw52c were not detected in our cell line panel, indicating that these supertypic determinants are in negative linkage disequilibrium with HLA-DR5. Our data suggest that intra HLA-DR/DQ crossing-over events contribute to the development of the HLA class II polymorphism. Evidence is presented that the T cell defined HLA-D allospecifities are commonly determined by DRB1 and DQ gene products.

Amino Acids↗
PubMed1989

A family with an apparent HLA-DR triplet: evidence for exchange of functional HLA-DR beta-genes between different haplotypes.

Serological and protein analysis showed that HLA-DR1 and DR2 short (s) segregated together as one haplotype, resulting in a HLA-DR triplet as found in a family study. Both DR1 and DR2 molecules were coordinately expressed and were shown to function as restriction elements in antigen presentation assays. This unique HLA-DR1, 2s haplotype was further studied by Southern blot analysis. Based upon well-known restriction fragment length polymorphisms for the involved gene sets, i.e. DR1 and DR2 along with the DR type from the other haplotype, the genes as identified by restriction fragment length polymorphism of the triplet could be established. Pseudogenes, which are included in the previously described gene sets of HLA-DR1 and DR2 are apparently lacking in the triplet. We therefore postulate that during an unequal crossing-over event the DR beta-pseudogene of DR2 could be exchanged by a functional DR1 beta-gene.

Crossing Over, Genetic↗
PubMed1988

Biochemical complexity of serum HLA class I molecules.

Human serum was found to contain a variety of class I-like molecules by Western blotting with anti-class I heavy chain reagents: major bands usually are observed around Mr 44,000, 40,000, and 35,000-37,000. HLA-A24-positive individuals are distinguished by higher serum levels of Mr 44,000 and 40,000 class I-like molecules than those found in HLA-A24-negative individuals. The Mr 44,000 serum molecules are probably intact class I molecules that have been shed from the cell membrane, because they contain both a transmembrane segment (TM), as deduced from detergent-binding experiments, and a cytoplasmic tail (CT), as inferred from reactivity with an antipeptide serum specific for the cytoplasmic domain of class I antigens (RaCT). The Mr 35,000 and 37,000 molecules contain neither a TM nor a CT region and therefore are probably proteolytic breakdown products of cellular and/or serum Mr 44,000 molecules, although the existence of Q10-like molecules in man cannot be ruled out. The Mr 40,000 molecules do not contain a TM region. Mr 40,000 molecules reactive with the RaCT serum were found in the minority (2/13) of sera tested. We conclude that alternative splicing resulting in a precise excision of the TM exon plays a minor role in the generation of serum HLA class I antigens.

Animals↗
PubMed1988

Polymorphism and complexity of HLA-DR: evidence for intra-HLA-DR region crossing-over events.

HLA-DR molecules were isolated from HLA-DR3, -5, and -w6 positive homozygous B-cell lines by immunoprecipitation with monoclonal antibodies and analyzed by gel electrophoretic techniques. DNA isolated from the same cell lines was digested with the restriction enzyme Taq I and hybridized with a DR beta full-length cDNA probe. We demonstrated that certain DR beta I alleles are found in combination with different DR beta III alleles as defined by Southern blotting, protein chemistry, a functional assay using purified protein derivative-specific T-cell lines, and, in one case, also alloreactive T-cell reagents. Our results indicate that within the family of HLA-DRw52-associated haplotypes DR beta chain genes may have been transferred from one haplotype to another. The implications of these findings are discussed.

Alleles↗
PubMed1988

An HLA-DQ alpha allele identified at DNA and protein level is strongly associated with celiac disease.

An HLA-DQ alpha cDNA probe showed upon hybridization a highly significant discrepancy between the RFLP of celiac disease patients and healthy controls. The 4.0-kb Bgl II restriction fragment was present in 97% of celiac disease patients (n = 30), compared to 56% in a healthy control population (n = 72) (RR = 14.9; p less than 0.0005). At the product level all celiac disease patients tested to date have one DQ alpha chain in common, designated HLA-DQ alpha 2.3, which is associated with the 4.0-kb Bgl II fragment. This HLA-DQ alpha allele identified at the DNA level and product level seems to be a better marker for genetic susceptibility to develop celiac disease than those available to date.

Alleles↗
PubMed1988

Molecular analysis of HLA-DP specificities HLA-DPw1, -DPw2 and -DPw4: DP beta chain heterogeneity correlates with PLT subtyping.

HLA-DP molecules were isolated from Epstein-Barr virus transformed B cell lines by immunoprecipitation with monoclonal antibody B7/21.2 and subsequently analysed by two-dimensional gel electrophoresis. The results obtained demonstrate that the HLA-DP molecules that can be isolated from cells positive for the HLA-DP specificities HLA-DPw1, -DPw2 and -DPw4 display DP beta chain isoelectric point differences, whereas no DP alpha chain polymorphism was observed. These results suggest that the PLT defined HLA-DP specificities (HLA-DPw1, -DPw2 and -DPw4) are probably DP beta chain structures.

Cell Line↗
PubMed1987

Molecular diversity of HLA-DQ. DQ alpha and beta chain isoelectric point differences and their relation to serologically defined HLA-DQ allospecificities.

HLA-DQ molecules were isolated from a panel of HLA-DR-DQ homozygous cell lines, partially of consanguineous origin, derived by the use of monoclonal antibody SPV-L3, and subsequently analyzed by gel electrophoretic techniques. It is demonstrated that both the DQ alpha and beta chain exhibit an extensive isoelectric point polymorphism. Within a panel of 29 B-cell lines tested, at least 5 distinct alpha and 6 distinct DQ beta chain gene products were observed. Of the 30 theoretically possible DQ alpha-beta dimers, only 10 could be identified within the panel: 5 different dimers are associated with the DQw1 allospecificity; HLA-DQw2 and -DQw3 are associated with 2 types of dimers, whereas another DQ alpha-beta combination was expressed by a cell line with a so-called DQ-blank specificity. The relation between the specificities 2B3 and TA10 appeared to be complicated as far as DQ beta chain isoelectric point differences are concerned: monoclonal antibody IIB3 seems to be reactive with four distinct DQ beta chain alleles whereas monoclonal antibody TA10 only reacted with one type of DQ beta chain. These results suggest that the polymorphic DQ alpha and beta chains may both contribute to the definition of the HLA-DQ allospecificity. A particular DQ beta chain was present in two types of HLA-DQw1 molecules, as well as in one type of HLA-DQw2 and -DQw3 (2B3 positive) molecule, and formed dimers with electrophoretic distinct DQ alpha chains. On the other hand, HLA-DQw2 molecules isolated from HLA-DR3-positive cells and one type of HLA-DQw3 (TA10 positive) molecule were found to be constructed of identical alpha chains but appeared to differ in the composition of their DQ beta chain gene products. The implications of these findings will be discussed.

Alleles↗
PubMed1987

HLA class II DNA analysis by RFLP reveals novel class II polymorphism.

Polymorphism of the HLA-D/DR region has been defined by serologic and cellular methods. Additionally, protein and DNA analyses not only confirmed and refined the definition of the established polymorphisms but also revealed further polymorphisms for which no serologic or cellular correlate are known (yet). To study these in more detail, we analyzed the banding patterns obtained from Southern blot hybridizations with DR beta and DQ alpha cDNA probes. Specific fragments reflecting already defined polymorphisms could be identified. A refined HLA-D/DR definition based upon the presence of DNA subtypes could be introduced. Fragments have also been identified that are associated with the DR/Dw specificities. Moreover, individuals with different DR types may also share fragments in hybridization assays. These shared hybridizing fragments (SHFs) are, for instance, found in individuals typed as DR4, DR5, DR7, and DRw8. In total, 20 SHFs were found using two restriction enzymes and the DR beta and DQ alpha cDNA probes. Some of these SHFs correlate with antigenic determinants defined by broad reacting alloantisera, such as DRw52, but for 14 of these SHFs, no serologic equivalent has been found so far. Thus, SHFs reflect a conservation at the DNA level of the HLA class II region, which suggests that the polymorphic class II genes may be more conserved than previously thought. The possible biologic implications of conserved sequences in the HLA class II genes will be discussed.

Cell Line↗
PubMed1987

Polymorphisms within the HLA-DRw6 haplotype. III. DQ alpha and DQ beta polymorphism associated with HLA-D.

We have studied HLA-DQ encoded antigens from HLA-DRw6 homozygous cells and analyzed the DQ region at the DNA level. HLA-DQ molecules were isolated from EBV transformed B-cell lines and analyzed for DQ alpha and DQ beta polymorphism. From the same set of cells, DNA was isolated and analyzed for RFLP. Polymorphism could be detected by both techniques, i.e., on the protein level and on the DNA. The variation in pI of the DQ alpha and beta chains correlated with the polymorphism as detected by HTC typing, as did the variation in molecular weight of the bands hybridizing to DQ specific cDNA probes; identical patterns were detected for cells of one HLA-D specificity and different patterns for different HLA-D types. Additionally, DQ reactive PLT reagents were raised against DRw6 positive cells. Panel studies revealed that these DQ reactive proliferative T cells can discriminate between the polymorphic DQ antigens on cells with different HLA-D specificities.

Antibodies, Monoclonal↗
PubMed1987

Two alleles detected for HLA-DZ alpha on the basis of a polymorphic restriction site in the third exon.

The restriction enzyme ApaI has been used to define Restriction Fragment Length Polymorphism (RFLP) within the DZ alpha gene. Digestion of genomic DNA of 96 individuals with ApaI reveals the existence of two different patterns. Both homozygous and heterozygous individuals were detected. Hardy-Weinberg analysis indicates that DZ alpha is part of a di-allelic system with allele frequencies of 50%. DZ alpha polymorphism in the population studied does not correlate with serologically determined class I and HLA-DR, DQ and cellular DP typing data. A limited family study demonstrates that DZ alpha polymorphism segregates with HLA-class II phenotypes.

Alleles↗