PubMed Health⌕ Search

Biomedical subjects

M Giphart

Publications and source records attributed to M Giphart.

At least 19 recordsLinked to original sources
PubMed1999

Matching for TNF microsatellites is strongly associated with matching for other non-HLA MHC sequences in unrelated bone marrow donor-recipient pairs.

The use of unrelated donors for bone marrow transplantation is associated with an increased morbidity and mortality when compared with HLA identical siblings. We have demonstrated previously that matching of unrelated donors and recipients for TNFa microsatellites is correlated with lower CTLp frequencies. Matching of unrelated donors and recipients for other non-HLA sequences in the major histocompatibility complex has been reported to result in less graft-versus-host disease and improved survival. It has been argued that matching for non-HLA sequences in the MHC in addition to the HLA genes themselves results in matching for the entire MHC and is therefore the equivalent of providing an HLA identical sibling donor. In order to test this hypothesis we have examined TNFa microsatellites of unrelated donor recipient pairs in whom matching for HLA loci, non-HLA sequences near HLA B (beta-block markers) and non-HLA sequences near DRB1 (delta-block markers) had been determined. All 17 patients who were matched for HLA and non-HLA markers were also matched for TNF microsatellites. This data supports the idea that matching for HLA genes and non-HLA markers results in matching at all other loci in the MHC.

Bone Marrow Transplantation↗
PubMed1996

HLA-DRB1*04 high resolution typing.

High resolution typing for HLA-DR4 is required to identify the individual subtypes. In this study a panel of DR4-positive samples was typed by both sequencing-based typing (SBT) and oligohybridization (PCR sequence-specific oligonucleotide; PCR-SSO). SBT reveals the highest resolution; moreover, ambiguous DRB1*04 allelic combinations can be resolved by a selective amplification of the individual alleles and subsequent sequencing. An extended DR4-specific PCR-SSO makes high resolution typing possible; however, an additional protocol is required to resolve ambiguities.

Alleles↗
PubMed1993

Organization and evolution of C4 and CYP21 genes in primates: importance of genomic segments.

The evolutionary relationship between two central major histocompatibility complex (MHC) genes, C4 and CYP21, was investigated by employing pulsed field gel electrophoresis (PFGE) and conventional restriction fragment length polymorphism (RFLP) analyses in human and nonhuman primates. Using Taq I in conjunction with C4 and CYP21 probes, it has been found that there are four major types of C4 genes [defined by 7.0, 6.4, 6.0, and 5.4 kilobases (kb) Taq I fragments] and two major types of CYP21 genes (3.7 and 3.2 kb fragments) in human and nonhuman primates including chimpanzee, gorilla, and orangutan. All of the eight possible combinations of C4 and CYP21 genes can be identified on one or more human ancestral haplotypes (AH). It is concluded that each of the major types of C4 and CYP21 (and each of the combinations between these) predated human speciation. PFGE analysis with Mlu I and Pvu I suggested that each C4+CYP21 segment has a specific length of 30-50 kb and that each AH carries one, two, three, or even more segments. In the case of C4, it is important to note that there is no simple relationship between the RFLP and the protein classifications. Thus, at least some of the expressed polymorphisms could be relatively recent in that they are carried by the same or different gene types. These findings are consistent with the hypothesis that MHC AHs have been formed from a large pool of specific genomic segments and that further haplospecific polymorphism has developed subsequently.

Animals↗
PubMed1991

Major histocompatibility complex ancestral haplotypes in the chimpanzee: identification using C4 allotyping.

In humans, certain major histocompatibility complex (MHC) supratypes mark unique DNA segments which have been conserved from a common but remote ancestor. In order to determine whether these ancestral haplotypes (AHs) exist in nonhuman primates, C4 allotyping was undertaken on 71 chimpanzees. Four large pedigrees were available. There are at least seven codominant C4 alleles at two loci. Null alleles are also present. It was possible to assign class I, class II, and C4 alleles to 37 unrelated haplotypes; several supratypes occurred two or more times. These putative AHs included some with alleles which resemble those carried by certain human AHs. These data provide evidence that similar MHC AHs are present in the chimpanzee and human. The present approach provides a basis for comparative studies examining the evolutionary and functional significance of the MHC.

Animals↗
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

Differential expression of DRw52-like determinants detected by monoclonal antibodies.

The reactivity of three monoclonal antibodies (MoAb) directed against DRw52-like determinants was studied in relation to the reactivity of an anti-DR MoAb using fluorescence-activated cell sorter (FACS) analysis. The MCS-7 MoAb reacted with all DRw52+ cells and in addition with DR2+, DR4+, and a DR7+ cell. Both the I-LR2 and the 7.3.19.1 MoAb reacted with DR3+, DR5+, and DRw6+ cells only. However, whereas the 1-LR2 MoAb reacted strongly with all those cells compared with the anti-DR MoAb, the 7.3.19.1 MoAb reacted strongly with DR3+ cells only, and somewhat less with DR5+ and DRw6+ cells. The implications of this for the location of DRw52-like determinants on DR beta chains is discussed.

Antibodies, Monoclonal↗
PubMed1986

Quantitative and qualitative differences in HLA-DR molecules correlated with antigen-presentation capacity.

The monoclonal antibodies 7.3.19.1 (anti-DRw52-like) and B8.11.2 (anti-DR framework) were used for the isolation and characterization of HLA class II molecules expressed by HLA-DR3 and DR5 homozygous B cell lines. Sequential immunoprecipitation studies demonstrated that from these cells class II molecules can be isolated which are characterized by the presence or absence of DR framework (DR) and DRw52-like (DRw62) determinants: (DR+, DRw52+), (DR+, DRw52-) and (DR-, DRw52+). The DR3 donor cells appeared to express only the (DR+, DRw52+) and (DR-, DRw52+) class II molecules whereas DR5-positive cells express only the (DR+, DRw52+) and (DR+, DRw52-) class II molecules. Besides qualitative differences some of the above-mentioned molecules appeared to differ in their levels of expression. To investigate whether this might have functional implications, cells with the HLA-DR3 and -5 haplotypes were used to present antigen purified protein derivative of tuberculin (PPD) to PPD-specific T cell lines and the blocking capacity of the two monoclonal antibodies 7.3.19.1 and B8.11.2 was determined. A remarkable correlation was observed between the type of class II molecule blocked by these monoclonal antibodies and its quantitative expression. However, (DR-, DRw52+) molecules, clearly expressed by DR3 cells, were not involved in the presentation of PPD. This indicates that not only quantitative but also qualitative aspects may play a role in the selection of the type of class II molecule that will be involved in antigen presentation.

Antibodies, Monoclonal↗
PubMed1986

T90/44 (9.3 antigen). A cell surface molecule with a function in human T cell activation.

T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb). It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits. The function of T90/44 was investigated in a series of T cell function assays. 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M. leprae antigens. The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration. The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface. The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M. tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of [3H]-thymidine incorporation. After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10. High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes. The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb. The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb. In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44.

Antibodies, Monoclonal↗
PubMed1986

Polymorphisms within the HLA-DR3 haplotypes. I. HLA-DR polymorphisms detected at the protein and DNA levels are reflected by T-cell recognition.

HLA-DR molecules were isolated from eight different HLA-DR3 homozygous B-cell lines by immunoprecipitation with monoclonal antibodies, and they were subsequently analyzed by two-dimensional gel electrophoresis. We found that HLA-DR3 homozygous B-cell lines of consanguineous origin express two types of HLA-DR molecules. One type of HLA-DR molecule was present in all the cell lines tested, whereas the second DR molecule appears to be polymorphic. DNA isolated from the different HLA-DR3 homozygous cell lines was studied by Southern blot analysis to determine whether any DR beta restriction fragment length polymorphism could be observed. Polymorphisms detected at both the product and genomic level have been compared to each other, and their relations to the serological (HLA-DR) and cellular (HLA-D and LB-Q1) typing data will be discussed.

Antibodies, Monoclonal↗
PubMed1986

Divergent and invariant HLA class II beta chain isoelectric points.

Class II molecules were isolated from consanguineous HTCs (DR1-DRw8) by sequential immunoprecipitation with the monoclonal antibodies 7.3.19.1 (anti-DRw52-like), B8.11.2 (anti-DR backbone), and 7.5.10.1 (anti-HLA class II backbone). Depending upon the DR-serotype of the cell line used, two or three class II antigen families, distinct in molecular weight, could be isolated (see Hum Immunol 9:221, 1984). Immunoprecipitated class II molecules were treated with NaNase and then analyzed on 1D-IEF gels. Each HLA class II antigen family contained two alpha chains conserved in pI. Furthermore, the various haplotypes show distinct electrophoretic beta chain patterns. The number of beta chain charge configurations detected varies from 2 to 5, depending upon the antigen family or haplotype studied. Some of these chains have a pI which is specific for a given class II serotype whereas other beta chain pIs are invariant and shared among more antigen families or haplotypes.

Antibodies, Monoclonal↗
PubMed1986

DNATYPE--a personal computer program for HLA-DR typing based upon restriction fragment length polymorphisms.

The use of specific cDNA probes and selected combinations of restriction endonucleases makes it possible to characterize specific antigens by the molecular weights of the hybridized restriction enzyme digests of DNA. We have developed a simple program suitable for personal computers which utilizes such results to deduce HLA-DR phenotypes of any nucleated cell from which DNA may be extracted.

Computers↗
PubMed1986

Reduced complexity of RFLP for HLA-DR typing by the use of a DR beta 3' cDNA probe.

The polymorphism of the HLA system has been defined by alloantisera, monoclonal antibodies, MLC reactivity, protein chemistry and RFLP patterns in DNA analysis. Typing for the alleles of HLA-DR at the DNA level as an additional typing technique is useful since any nucleated cell can be used. Moreover, it is not known whether the additional polymorphism found at the DNA level in an unambiguous serotype is of functional importance and thus needs to be included in HLA-DR typing. A main problem in DNA typing is the interpretation of the complex patterns in Southern blot analysis, especially in heterozygous individuals. Therefore we constructed subprobes from full length DR beta, DQ alpha and DQ beta cDNA to reduce the number of hybridizing fragments while retaining the discriminating capacity. The clearest differences among DR alleles have been found using the restriction enzyme PvuII and the subprobe containing the 3' untranslated region of the DR beta probe. Although further characterization is necessary to be able to type at the DNA level, the simplified patterns facilitate DNA typing in heterozygous individuals for a number of haplotypes. Interestingly, the number of fragments thus obtained corresponds with the number of genes described for DR1 to DRw8 haplotypes. Based upon the finding of common hybridizing patterns in DR3, DR5 and DRw6 it may be concluded that DR3, DR5 and DRw6 have been evolved from a common ancestor. For the same reason DR4, DR7 and DRw9 may have evolved in an identical way.

Cloning, Molecular↗
PubMed1985

Typing for HLA class II at the product level.

Class II antigens were isolated from consanguineous homozygous typing cells by sequential immunoprecipitation with the MoAbs: 7.3.19.1 (anti-DRw52-like), B.8.11.2 (anti-DR backbone) and 7.5.10.1 (anti-HLA class II backbone). Depending on the DR serotype of the cell line used, two or three families of class II antigens could be isolated [1]. For each homozygous typing cell the different families of class II antigens were analysed on 1D-IEF gels. Charge heterogeneity showed that the different haplotypes are distinct in electrophoretic beta chain patterns. For each homozygous typing cell at least one beta chain was observed that possessed a haplotype unique pI. This means that typing for HLA class II at the product level is possible.

Antibodies, Monoclonal↗
PubMed1984

HLA-DC antigens can serve as recognition elements for human cytotoxic T lymphocytes.

The specificity of four cytotoxic T lymphocyte (CTL) clones which recognize class II major histocompatibility complex (MHC) antigens was analyzed. All clones recognized antigens associated with the serologically defined HLA-DRw6 specificity. The activity of two of these clones, JR-2-2 and JR-2-10, could be inhibited by a monoclonal antibody Q 5/13 specific for a monomorphic determinant present on HLA-DR. In contrast, the activity of the two other CTL clones, JR-2-19 and JR-2-26, was not blocked by Q 5/13, but by a new monoclonal reagent, SPV-L3. This latter monoclonal antibody precipitated a two-chain structure of 28 kDa and 33 kDa and reacts with a monomorphic determinant. The molecular weight of the polypeptides precipitated with SPV-L3 was slightly less than those precipitated with a HLA-DR-specific monoclonal reagent. In addition two-dimensional gel electrophoresis showed that the antigen precipitated by SPV-L3 differed in charge from those precipitated with the anti-HLA-DR antibody. These results indicate that SPV-L3 recognizes a class II MHC product different from HLA-DR. This observation was confirmed by partial amino acid sequence analysis of the two chains which revealed that the molecule precipitated by SPV-L3 is homologous to HLA-DC/DS molecules. Therefore this report provides the first evidence that human cytotoxic T cells can recognize HLA-DC/DS antigens.

Amino Acid Sequence↗
PubMed1984

Analysis of the functional epitopes on different HLA-A2 molecules.

Recent studies show that the serologically defined HLA-A2 molecule can be subdivided according to functional and biochemical characteristics. By the use of various HLA-A2-specific cytotoxic T lymphocytes (CTLs) and isoelectric focusing, the serologically homogeneous HLA-A2 molecule can be divided into four subtypes. The polymorphism of the serologically defined HLA-A2 molecule has also been demonstrated by the use of HLA-A2-restricted CTLs. This study was designed to analyze the functional epitopes on different HLA-A2 molecules with special regard to the recognition patterns of different types of HLA-A2-restricted CTLs directed against minor histocompatibility (minor H) antigens. Fifteen so-called HLA-A2 variants belonging to distinct HLA-A2 subtypes were tested as target cells in the cell-mediated lympholysis (CML) assay against (1) HLA-A2-restricted antiminor H-Y CTLs, (2) HLA-A2 and -B7-restricted antiminor H-Y CTLs, and (3) HLA-A2, -Bw62 and -B27-restricted antiminor "HA" CTLs. We found that those three CTLs recognized only one of those HLA-A2 variants. Furthermore, positive reactions by the antiminor H CTLs were only observed on those variant cells which carried, in addition to the HLA-A2 variant, either another "normal" HLA-A2 molecule or another required restricting class I molecule necessary for associative recognition. These results indicate that the absence of HLA-A2 normal allotypic target determinant(s) leads to the loss of epitope(s) necessary for recognition of minor H-Y and minor "HA" transplantation antigens by HLA-restricted CTLs. We can conclude from the present study that HLA-A2-restricted antiminor H CTLs use, in general, the same epitope (or cluster of epitopes) for cellular recognition as alloimmune HLA-A2-specific CTLs.

Cytotoxicity, Immunologic↗
PubMed1984

A mouse monoclonal antibody detecting a DR-related MT2-like specificity: serology and biochemistry.

A mouse monoclonal antibody (7.3.19.1) was produced which reacts with class II molecules on B cells and monocytes of DR3, DR5, and/or DRw6 positive donors only. Using this moab and two others, three different groups of class II molecules could be identified. Furthermore, a differential precipitation pattern was found which correlates with a DR-related variable expression of the MT2-like polymorphic determinants on the cell surface. Addition of 7.3.19.1 to MLCs did not result in significant inhibition in controls to the two other moabs tested. Normal CTL activity was found in such a stimulated responder population.

Animals↗