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Biomedical subjects

P Parham

Publications and source records attributed to P Parham.

At least 325 records · Page 18Linked to original sources
PubMed1983

Binding of monoclonal antibodies inhibits dissociation but not exchange of HLA and beta 2-microglobulin.

Hyafil and Strominger (Proc Nat Acad Sci USA 76:5834, 1979) showed that the complex of papain-solubilized HLA heavy chain and beta 2-microglobulin (beta 2-m) will measurably dissociate and exchange with free beta 2-m at 37 degrees C but not at 4 degrees C. We have investigated the affects of monoclonal antibodies on these processes. The dissociation reaction at 37 degrees C is inhibited by the presence of HLA-A,B,C specific monoclonal antibodies. Monomorphic antibodies such as BB7.7 against combinatorial determinants are most potent though antibodies against many different determinants of the HLA heavy chain are effective. This suggests that dissociation of beta 2-m from HLA involves a conformational change of the HLA heavy chain which is prevented by antibody binding. BB7.7 also increased the rate of association of HLA heavy chain and beta 2-m. This affect was significantly greater at 37 degrees C and 25 degrees C than at 4 degrees C. Monoclonal antibodies that inhibited dissociation did not inhibit the beta 2-m exchange reaction. If beta 2-m exchange proceeds through a simple dissociation/association reaction then the affect of antibody inhibition on dissociation is almost exactly compensated by its enhancement of association. Alternatively B 2-m exchange may not proceed via a simple dissociation/association reaction.

Antibodies, Monoclonal↗
PubMed1983

Recognition by xenogeneic cytotoxic T lymphocytes of cells expressing HLA-A2 or HLA-B7 after DNA-mediated gene transfer.

Murine L cells expressing HLA-A2 or -B7 antigens were isolated after cotransformation of thymidine kinase-negative cells with the herpes simplex virus thymidine kinase gene and the genomic clones containing either the HLA-A2 or -B7 genes. Monoclonal antibody binding analyses demonstrated the stable cell surface expression of HLA antigens by these cells at levels of up to 40% of the amount expressed by the human B lymphoblastoid cell line, JY. The HLA-A2 and -B7 antigens expressed by the L cells retained all of the antibody-defined, heavy-chain-associated antigenic determinants but lacked those determinants associated with human beta 2-microglobulin. These HLA transformants were capable of functioning as targets for monoclonal cytotoxic T lymphocytes (CTL) that specifically recognize the HLA-B7 or -A2 antigens expressed by JY cells. However, the efficiency of lysis, relative to the JY cell line, was 50-99% for individual CTL. In addition, not all of these CTL were capable of lysing the appropriate transformants. Because the antigens appear by serological criteria to be structurally intact and expressed at high levels, these results suggest that the complementation of the HLA heavy chains with mouse, rather than human, beta 2-microglobulin may alter the antigenic determinants that are important for CTL recognition.

Animals↗
PubMed1983

Location and distribution of the light chains in clathrin trimers.

Mouse monoclonal antibodies have been made that are specific for one of the light chains (LC-A) of calf brain clathrin. The determinant they recognize has been mapped by immunoelectron microscopy and shown to lie near the center of a clathrin trimer. Quantitative immunoprecipitation experiments with one of the monoclonal antibodies suggests that, despite the overall 1:2 stoichiometry, the light chain composition of an individual clathrin trimer is nearly random.

Animals↗
PubMed1983

Tropomyosin-like properties of clathrin light chains allow a rapid, high-yield purification.

The light chains (LCa and LCb) of bovine brain clathrin are resistant to heat denaturation by boiling, a property shared by tropomyosin (Bailey, K., 1948, Biochem. J., 43:271-281). Light chains were partially purified by boiling and centrifugation of a Tris-extract of crude membranes prepared from bovine brains (Keen, J. H., M. C. Willingham, and I. H. Pastan, 1979, Cell., 16:303-312). Contaminant polypeptides were then removed by size-exclusion high-pressure liquid chromatography. The purified light chains were separated from each other by using an immunoaffinity column prepared from a monoclonal antibody CVC.7 specific for LCa and not LCb.

Animals↗
PubMed1983

The antigenic structure of HLA-A2: an analysis with competitive binding assays and monoclonal antibodies.

Twelve mouse monoclonal antibodies against HLA-A2 were analyzed by competitive binding assays on cells. The A2 molecule was found to have two topologically distinct regions of highly specific alloantigenic determinants. One region encompasses determinants shared with HLA-A28 and the other contains determinants shared with HLA-B17 and a variant, A28*, of HLA-A28. Each of these two alloantigenic regions is more closely associated with a different broadly polymorphic determinant shared by many HLA gene products. The topographic relationship between the two alloantigenic regions, four monomorphic determinants, and the carbohydrate moiety of the HLA heavy chain was also assessed. All the antigenic determinants preferentially associate with one or other of the two alloantigenic regions. An analysis of these results with reference to the amino acid sequences of A2 and A28 suggests a working hypothesis whereby the A2,A28*,B17 region primarily involves residues of the NH2-terminal or first domain, and that the A2,A28 region primarily involves residues of the second domain of the A2 heavy chain.

Amino Acid Sequence↗
PubMed1983

On the fragmentation of monoclonal IgG1, IgG2a, and IgG2b from BALB/c mice.

Methods for the production and purification of F(ab')2 fragments from BALB/c monoclonal IgG1, IgG2a, and IgG2b with pepsin and other proteases were examined. The overall susceptibility to degradation is IgG2b greater than IgG2a greater than IgG1. Stable F(ab')2 can be produced in good yield from IgG1 with pepsin at pH 3.5 to 4.0 and can be made directly by pepsin treatment of ascites fluids or cell culture supernatants containing IgG1. IgG2a is cleaved in two steps by pepsin, first to F(ab')2 and then to Fab'. With carefully chosen conditions, F(ab')2 can be obtained in acceptable yield. The primary cleavage for the IgG2a heavy chain appears to be on the COOH terminal side of the interheavy chain disulfides, and secondary cleavage is on the NH2-terminal side. For IgG2b the reverse is true, and F(ab')2 has not been obtained in useful amounts; however, the primary cleavage of IgG2b appears to be assymetric with respect to the two heavy chains, and Fab/c fragments that have one Fab plus Fc can be made. Digestion with elastase resulted in the best yield of Fab/c. This finding may provide a method for retaining cytotoxicity in monoclonal antibodies against cell surface antigens while eliminating their capacity to modulate. The cleavage patterns of the three classes of IgG are rationalized in terms of the structure of their hinge regions.

Animals↗
PubMed1982

Chromosome localization and gene synteny of the major histocompatibility complex in the owl monkey, Aotus.

Rodent cells were hybridized with owl monkey (Aotus) cells of karyotypes II, III, V, and VI. Aotus-rodent somatic hybrid lines preferentially segregating Aotus chromosomes were selected to determine the chromosomal location of the major histocompatibility complex and other genes with which it is synthetic in man. Based on correlation between concordant segregation of the chromosome as visualized by G-banding and expression of the Aotus antigens or enzymes in independent Aotus-rodent hybrid clones, we have assigned Aotus gene loci for the MHC, GLO, ME1, SOD2, and PGM3 to Aotus chromosome 9 of karyotype VI (2n = 49/50), chromosome 10 of karyotype V (2n = 46), and chromosome 7 of karyotype II and III (2n = 54 and 53). On the basis of banding patterns we previously postulated that these chromosomes of the different karyotypes were homologous. The gene assignments reported here provide independent evidence for that hypothesis. Aotus chromosomes 9 (K-VI), 10 (K-V), and 7 (K-II, III) are homologous to human chromosome 6 in that they all code for the MHC, GLO, ME1, SOD2, and PGM3. The structural differences between these homologous chromosomes probably resulted from a pericentric inversion.

Animals↗
PubMed1982

Evolution of HLA antigenic determinants: species cross-reactions of monoclonal antibodies.

The reactions of mouse monoclonal HLA-A-, -B-, -C-specific antibodies on cells from 50 species were analyzed by binding assay. Antibodies which are monomorphic in humans generally show monomorphic behavior in other species and many significant cross-reactions were seen. Antibodies which recognize highly specific polymorphic determinants gave few cross-reactions with other species. Antibodies which recognize broad polymorphic determinants in humans cross-reacted in a polymorphic manner with many other species. The patterns of cross-reaction for both monomorphic and broadly polymorphic determinants correlated roughly with the current picture of phylogenetic relationships. These results show there are two fundamentally different classes of polymorphic determinants, one which appears relatively conserved in evolution and one which is the product of recent change. They probably correlate with the public and private antigens of mouse H-2 antigens. A simple genetic model of HLA-A, -B, -C genes, whereby all specificities at a locus are true alleles, is sufficient to explain these patterns of cross-reaction. It seems likely that cross-reactions previously seen with alloantisera were due to broadly polymorphic antibodies rather than to allele-specific antibodies. If this were the case, then no experimental basis for Bodmer's model of MHC polymorphism being due to control of expression of a tandem array of genes exists.

Animals↗
PubMed1982

Expression of HLA antigens by human thymic epithelial cells.

Human thymuses were examined by tissue section staining with antibodies specific for monomorphic and polymorphic HLA-A, B, C, and DR determinants. The principal cell type expressing high levels of HLA antigens has the distribution of epithelial cells. Immunoelectron microscopy confirmed their epithelial nature. As in the mouse, both medullary and cortical epithelial cells express high levels of class II (DR) antigens, a finding that is remarkable in that these antigens were originally thought to be restricted to lymphoid and accessory cells. Class I (A, B, and C) antigens are also present on thymic epithelial cells. They are easily detectable on medullary epithelial cells, but two distinct patterns of cortical staining were observed. One group of antibodies produced intense dendritic staining throughout the cortex; the other group produced only faint or no cortical dendritic staining at all. These different staining patterns do not correlate with known properties of the antibodies and thus appear to be due to intrinsic properties of the different A, B, and C antigens.

Adult↗
PubMed1982

Cell surface properties of HLA antigens on Epstein-Barr virus-transformed cell lines.

A number of monoclonal antibodies have been used to investigate the distributions and rates of lateral motion of the HLA-A,B, and-DR antigens on several Epstein--Barr virus-transformed B-cell lines. The lateral diffusion coefficients (D) of fluorescein conjugates of the monoclonal antibodies bound to the cell surface were determined by fluorescence recovery after pattern photobleaching. Ds of HLA-A and-B were found to be comparable and of the order of 10(-9) to 10(-10) cm2/sec for each of the seven monoclonal antibodies and four cell lines examined. The HLA antigens appear to be monomeric on the cell surface based on experiments using mixtures of arsanilic acid-conjugated and fluorescein-conjugated antibodies. Four monoclonal antibodies against DR antigens were examined. Two of these, Genox 3.53 and L243, labeled the cell surface uniformly and gave Ds comparable to those obtained for the HLA-A and -B antigens. The other two, DA2 and 2.06, rapidly patched on the cell surface and were immobile. The DA2, L243, and Genox 3.53 antibodies bound outside of the caps formed with the arsanilic acid-conjugated 2.06 antibody and a second-step rhodamine-conjugated rabbit anti-arsanilate antibody. This is consistent with recent biochemical evidence that there are multiple distinct antigens coded for by the HLA-DR region.

Antibodies, Monoclonal↗
PubMed1982

HLA antigen structural gene mutants selected with an allospecific monoclonal antibody.

The HLA-A2 antigen-specific monoclonal antibody BB7.2 and complement were used to immunoselect mutants from an ethyl methanesulfonate-mutagenized human B lymphoid cell line, T5-1. Surviving colonies were screened by radioimmune binding with BB7.2 and with a monospecific HLA-A2 alloantiserum, Stewart, and HLA antigens of selected clones were immunoprecipitated and studied by isoelectric focusing. Several classes of mutants could be distinguished: mutants that expressed no HLA-A2 heavy chain; mutants that expressed an HLA-A2 heavy chain that was unable to associate with beta 2-microglobulin (beta 2m) and was not expressed at the cell surface; mutants with reduced HLA-A2 heavy chain-beta 2m association and cell surface expression of HLA-A2 dimer with or without heavy chain charge alterations; mutants with normal HLA-A2 heavy chain-beta 2m association and normal quantitative cell surface HLA-A2 expression but with HLA-A2 heavy chain charge alterations; and mutants with as yet incompletely defined lesions. Mutants with altered cell surface HLA antigens were not found in previous selections with alloantisera and should be useful for epitope mapping and structure-function studies of HLA molecules.

Antibodies, Monoclonal↗
PubMed1982

Monomorphic anti-HLA-A,B,C monoclonal antibodies detecting molecular subunits and combinatorial determinants.

Thirteen monoclonal antibodies that react with monomorphic determinants on the HLA-A,B,C-beta 2-microglobulin (beta 2m) molecule were characterized. Analysis of antibody activity included inhibition by papain-solubilized HLA antigens and free beta 2m, antibody binding to mouse-human somatic cell hybrids containing human chromosome 6 or 15, and antibody cross-reactivity with lymphocytes from nonhuman species. Two criteria for monomorphism were established: 1) equal inhibition or absorption of antibody activity by all papain-solubilized HLA antigens or cell lines of different HLA specificities tested; and 2) nonpolymorphic cross-reactivity within another species or subspecies. On the basis of soluble antigen inhibition and binding to somatic cell hybrids, 3 classes of antibodies were detected: anti-beta 2m, anti-heavy chain, and anti-complex (against a combinatorial determinant formed by heavy chain and beta 2m). Antibody cross-reaction patterns in nonhuman species were suggestive that these monomorphic antibodies detect a limited number of determinants, minimally one on each chain and 2 combinatorial determinants. Examination of the known primary sequences for HLA-A2, HLA-B7, H-2Kb, and mouse, rabbit and human beta 2m provides a molecular explanation for this limited mouse anti-HLA monomorphic antibody activity.

Animals↗
PubMed1981

Monoclonal antibodies against two separate alloantigenic sites of HLA-B40.

Two monoclonal antibodies (MB40.2 and MB40.3) which are highly specific for HLA-B40 and HLA-B7 were made. They appear to be directed against two separate alloantigenic sites of these HLA molecules. Semiquantitative analysis of the kinetics of antibody binding show that MB40.2 recognizes a site which shows a degree of cross-reactivity with B7 and is specific to some B40 molecules. This antibody also distinguishes between different molecules types as B40. In contrast, MB40.3 recognizes an antigenic determinant which is less variable between B7 and B40 and more closely approximates a public antigen or common antigenic site. This study suggests that the introduction of monoclonal antibodies into MHC serology not only permits but demands a quantitative analysis of these complex systems of homologous but highly polymorphic molecules.

Antibodies, Monoclonal↗
PubMed1981

Of cows and men: a comparative study of histocompatibility antigens.

Bovine histocompatibility antigens cross-react highly with HLA antigens in comparison to their homologues in other nonprimate species. A panel of 34 anti-HLA monoclonal antibodies was tested for binding activity with lymphocytes from five breeds of cattle. Eleven antibodies demonstrated extensive cross-reactivity. Lymphocytes from several species of ungulate and subungulate were then tested with the same panel of antibodies. The buffalo and hyrax also displayed some cross-reactivity, though not as extensive as seen with the cattle lymphocytes. Other ungulates showed essentially no cross-reactivity. The significance of these results is discussed in terms of the structure and evolution of histocompatibility antigens.

Animals↗
PubMed1981

Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLA-A28.

The purification and properties of a cytotoxic mouse monoclonal anti-HLA-A2 antibody are described. This antibody, BB7.2, can be used as an HLA-A2 typing reagent with little modification of current typing techniques. It also recognizes a low frequency variant of HLA-A28. BB7.2 provides an example of an antibody which requires bivalent attachment to a polymeric antigen, e.g., a cell, to produce a readily detectable complex. This is due to a high rate of dissociation of the complex formed between a single BB7.2 combining sites and HLA-A2. The consequences of this property were investigated and some complications for potential uses of such monoclonal antibodies are discussed.

Animals↗
PubMed1980

Influenza virus-specific cytotoxic T lymphocytes recognize HLA-molecules. Blocking by monoclonal anti-HLA antibodies.

The effect of monoclonal anti-HLA antibodies on lysis of influenza virus-infected target cells by sensitized cytotoxic T lymphocytes was measured. Lysis was blocked, provided the antibody was present in a concentration sufficient to saturate the HLA determinants on the target cells. Antibodies to monomorphic HLA determinants and to beta 2-microglobulin inhibited. Antibody to HLA A2 and B17 blocked lysis of target cells that shared these antigens with the effector cell. It was concluded that the restriction elements for influenza virus-specific cytotoxic T cells are the HLA molecules themselves.

Adult↗
PubMed1980

Molecular characterization of HLA-A, B homologues in owl monkeys and other nonhuman primates.

Mouse anti-HLA-A, B monoclonal antibodies have been used to study the homologues of HLA-A, B antigens in other primate species. Immunoprecipitates of primate histocompatibility antigens from extracts of radioactively labeled lymphocytes were analyzed by SDS-polyacrylamide electrophoresis. Primate histocompatibility antigens appear to have similar molecular structure to human HLA antigens. Owl monkeys, which react polymorphically with some monomorphic anti-HLA antibodies, showed biochemical differences which correlated with the serological polymorphism. An antibody (W6/32) which only reacts with the HLA/beta 2-microglobulin complex in humans and not with the free HLA heavy chain has the reverse specificity in some owl monkeys.

Animals↗