Biomedical subjects

J D Capra

Publications and source records attributed to J D Capra.

At least 181 records · Page 10Linked to original sources

PubMed1984

Use of JH4 joining segment gene by an anti-arsonate antibody that bears the major A-strain cross-reactive idiotype but displays diminished antigen binding.

One of the antibody families utilized by the A/J mouse in its response to p-azophenylarsonate (Ars) is characterized by the expression of the major anti-arsonate cross-reactive idiotype (CRI) of the A strain. This family has been termed the Ars-A family. A hybridoma antibody (HP 101F11 ) obtained after immunization of an A/J mouse with Ars was identified initially as displaying the CRI, but was subsequently found to bind antigen at a level much lower than most members of the Ars-A family. The results of binding studies suggested that HP 101F11 possesses reduced avidity for antigen. When isolated light and heavy chains were allowed to recombine with the heavy and light chains of a strongly antigen-binding, strongly CRI-positive antibody of the Ars-A family (HP 93G7 ), the low level of antigen binding by HP 101F11 was found to be due to a structurally variant heavy chain. Whereas antibodies of the Ars-A family with normal avidity for antigen had been shown to use the JH2 joining segment gene, amino acid sequence analysis of HP 101F11 revealed that this antibody has a JH segment with a sequence identical to that encoded by a portion of a different JH gene, JH4 . The implication that 101F11 uses the JH4 gene instead of JH2 was supported by the observation that the productively rearranged gene is associated with an Eco R1 restriction fragment 0.95 Kb smaller than the corresponding fragments of Ars-A hybridomas with normal avidity for antigen. The size difference of 0.95 Kb corresponds exactly to the known distance between the JH2 and JH4 genes in BALB/c germline DNA. In addition to the structural differences immediately attributable to the use of JH4 , HP 101F11 has shown an amino acid interchange in the DH segment, and a single amino acid deletion at the DH-JH boundary. These results show that variation among members of the Ars-A family in the DH and/or JH segments provides alternative structural forms of Ars-A antibodies upon which selective processes can operate during the course of an immune response.

Amino Acid Sequence↗

PubMed1984

Primary structural variation among serologically indistinguishable DS antigens: the MB3-bearing molecule in DR4 cells differs from the MB3-bearing molecule in DR5 cells.

HLA-DS molecules bearing the MB3 supertypic specificity have been isolated from two DR4 and two DR5 homozygous cell lines by using the monoclonal antibody IVD12 . Limited amino-terminal amino acid sequence analysis of these molecules demonstrates polymorphism of the HLA-DS subregion. Although the distribution of amino-terminal tyrosine residues in the alpha-chains of all IVD12 -reactive molecules was identical, amino-terminal amino acid sequence differences existed between DS beta-chains isolated from these two groups of cell lines bearing different DR specificities. These studies indicate that two DS molecules bearing the same serologic determinant ( MB3 ), although similar to one another, may be structurally distinct.

Amino Acid Sequence↗

PubMed1984

Expression of HLA-DR, MB, MT and SB antigens on human mononuclear cells: identification of two phenotypically distinct monocyte populations.

The expression of HLA-DR, SB, MB, and MT antigens in different populations of human mononuclear cells was investigated with the use of monoclonal antibodies that recognize distinct human Ia-like antigens. Our results indicate that in man, as previously reported in other species, two phenotypically distinct populations of monocytes or macrophages can be identified on the basis of expression of Class II MHC antigens. Virtually all circulating monocytes displayed determinants associated with HLA-DR, SB, and MT. In addition, a subpopulation of human monocytes expressed MB/DS-associated antigens, as detected with monoclonal antibodies specific for MB1, MB3, and DS-framework determinants. Most B lymphocytes expressed antigens associated with HLA-DR, and the specificities SB2, SB3, MB1, MB3, MT2, and MT3 were also present. Resting T lymphocytes were unreactive with antibodies that recognize all of the Class II MHC antigens tested. T lymphocytes activated by soluble antigen or alloantigens, and expanded in culture, expressed DR, SB, MB, and MT. The majority of the MB/DS+ cells present in the adherent population were monocytes, because they were phagocytic and had the monocyte-specific marker 63D3. The rest of the cells were not identified. They are likely to include mostly B lymphocytes. The presence of other cells, such as dendritic cells, in this subset needs to be determined.

Antibodies, Monoclonal↗

PubMed1984

Junctional diversity is essential to antibody activity.

Many mechanisms of antibody diversification have been shown to exist, including combinatorial pairings of heavy and light chains, the use of multiple gene segments (combinatorial diversity), and the imprecise joining of these gene segments (junctional diversity). The contributions of each of these mechanisms to functional antibody activity has not been fully explored, especially in the case of junctional diversity. A chain recombination experiment between an anti-arsonate monoclonal antibody and an anti-oxazolone molecule in which light chains differ essentially only at the V/J junctional position show that junctional diversity may play an important role in antigen binding.

Animals↗

PubMed1983

Amino acid sequence diversity within the family of antibodies bearing the major antiarsonate cross-reactive idiotype of the A strain mouse.

VH region amino acid sequences are described for five A/J anti-p-azophenylarsonate (anti-Ars) hybridoma antibodies for which the VL region sequences have previously been determined, thus completing the V domain sequences of these molecules. These antibodies all belong to the family designated Ars-A which bears the major anti-arsonate cross-reactive idiotype (CRI) of the A strain mouse. However, they differ in the degree to which they express the CRI in standard competition radioimmunoassays. Although the sequences are closely related, all are different from each other. Replacements are distributed throughout the VH region and occur in positions of the chain encoded by all three gene segments, VH, DH, and JH. It is likely that somatic diversification processes play a dominant role in producing the sequence variability in each of these segments. The number of differences from the sequence encoded by the germline is smallest for antibodies that express the CRI most strongly, suggesting that somatic diversification is responsible for loss of the CRI in members of the Ars-A antibody family. There is an unusual degree of clustering of differences in both CDR2 and CDR3 and many of the substitutions are located in "hot spots" of variation. The large number of differences between the chains prohibits the unambiguous identification of positions at which alterations play a major role in reducing the expression of the CRI. However, the data suggest that the loss of the CRI is associated with a definable repertoire of somatic changes at a restricted number of highly variable sites.

Amino Acid Sequence↗

PubMed1983

Structural analysis of a human I-A homologue using a monoclonal antibody that recognizes an MB3-like specificity.

Monoclonal antibody IVD12 was used to isolate and characterize a human Ia molecule present on B cells that generally display DR4 or DR5 phenotypes. The specificity of binding of IVD12 to human peripheral blood B cells from 75 normal individuals and 19 homozygous human lymphoblastoid B cell lines was identical to the supertypic specificity MB3 previously defined. Furthermore, IVD12-reactivity was shown to segregate with HLA in three informative families. In each family, individuals positive for IVD12 binding were also positive for DR4 or DR5. Using IVD12, a molecule has been isolated from the homozygous cell line PRIESS (DR4/4) and has been shown by amino acid sequence analysis to be homologous to the murine I-A and human HLA-DS molecules. These findings suggest that the MB3 specificity is found on a molecule encoded by loci distinct from those loci which encode HLA-DR molecules. This molecule represents the third family of HLA-D region molecules isolated from the cell line PRIESS. Both HLA-DR and HLA-SB molecules from this cell line were previously shown by amino acid sequence analysis to be I-E-like but distinct from one another. Collectively, these data provide evidence that the HLA-D region contains at least six loci encoding distinct alpha and beta chains for the HLA-SB, HLA-DR, and HLA-DS molecules.

Amino Acid Sequence↗

PubMed1983

Structural analysis of monoclonal anti-arsonate antibodies: idiotypic specificities are determined by the heavy chain.

Hybrid immunoglobulin molecules were constructed from the isolated heavy and light chains of monoclonal anti-arsonate antibodies which differed in their expression of a cross-reactive idiotype. The reconstructed molecules were tested for public and private idiotypic determinants associated with the A-strain response to the hapten p-azophenylarsonate and it was found that both an appropriate heavy chain and an appropriate light chain were required for expression of idiotypic determinants. While appropriate heavy chains could be derived only from idiotype-positive antibodies, appropriate light chains could be derived not only from idiotype positive antibodies, but also from certain idiotype-negative molecules. Similarly, recombinant (hybrid) molecules constructed from two idiotype-positive immunoglobulins differing quantitatively in the degree of idiotypic character, expressed the character at a level which correlated with that of the heavy chain donor. Thus, while the serological expression of idiotypic determinants occurs only in the context of the appropriate VHVL combination, these data demonstrate that the determinants comprising the major cross-reactive idiotype of the anti-arsonate response of A/J mice have their bases in heavy chain structures.

Animals↗

PubMed1983

A murine anti-I-Ak monoclonal antibody recognizes human DR (I-E-like) antigens.

A monoclonal antibody. H39-49.5, originally raised against the murine I-Ak antigen, recognizes a nonpolymorphic determinant on HLA-DR-like molecules from human cells. These HLA-DR-like molecules have been characterized by amino acid sequencing and have been found to be homologous to the murine I-E antigens rather than the murine I-A antigens. These results suggest a common origin for the murine I-E, I-A, and HLA-DR antigens and caution that serological cross-reactivity alone cannot be used to reliably establish the presence of structural homologues of I-E- or I-A-like molecules in other species.

Amino Acid Sequence↗

PubMed1983

Use of antipeptide antibodies to demonstrate external orientation of the NH2-terminus of the low density lipoprotein receptor in the plasma membrane of fibroblasts.

The low density lipoprotein (LDL) receptor is a member of a class of receptors that bind macromolecules at the cell surface and facilitate their cellular uptake by receptor-mediated endocytosis. The orientation of the LDL receptor in the plasma membrane is unknown. In the current studies the sequence of amino acids at the NH2-terminus of the bovine adrenal LDL receptor was determined, and a synthetic peptide corresponding to amino acids 1-16 was prepared. Antibodies against this peptide were raised in rabbits and were shown by immunoblotting analysis to react specifically with the bovine LDL receptor. The anti-receptor peptide antibodies also bound to the LDL receptor on the outer surface of the plasma membrane of intact human fibroblasts, as visualized by indirect immunofluorescence. Specificity of this binding reaction was confirmed by the observation that the anti-receptor peptide antibodies did not bind to mutant fibroblasts from a patient with homozygous familial hypercholesterolemia that lack LDL receptors. These data demonstrate that the LDL receptor is oriented in the plasma membrane with its NH2-terminus facing the extracellular surface.

Adrenal Cortex↗

PubMed1983

Association of susceptibility to certain hematopoietic malignancies with the presence of Ia allodeterminants distinct from the DR series; utility of monoclonal antibody reagents.

The Ia antigen allospecificities of individuals with either B type chronic lymphocytic leukemia or hairy cell leukemia resembled one another and differed significantly from those of a control population. In contrast, the Ia alloantigens of individuals with non-Hodgkin's lymphoma were distinctly different from those of the leukemic group but differed little from the control group. A monoclonal antibody, IVD12, directed to an MB3-like determinant reacted with the greatest proportion of the leukemic individuals and yielded the highest positive relative risk. A lower degree of positive association was found with the presence of the MT2 determinant. In contrast, the low observed frequency of the MT1/MB1 determinant among leukemic individuals was associated with the most significant negative relative risk. The relative risk associated with the presence of DR5 was positive, while among patients with chronic lymphocytic leukemia the relative risk associated with DR2 was negative. Among patients with Hodgkin's Disease the relative risk associated with the presence of DR5 was significantly increased.

Antibodies, Monoclonal↗

PubMed1982

Alpha and beta chains of SB and DR antigens are structurally distinct.

Limited amino-terminal amino acid sequences of the HLA-controlled SB and DR antigens from the cell line PREISS (DR4/4, SB3/4) show differences in both the alpha and beta chain sequences of the two molecules. SB antigens, like DR antigens, appear to be homologues of the murine I-E antigens.

Amino Acid Sequence↗

PubMed1982

Somatic mutation in genes for the variable portion of the immunoglobulin heavy chain.

The size of the gene pool potentially encoding antibodies to p-azophenyl arsonate has been examined. A heavy chain-specific full-length complementary DNA clone has been constructed with the use of messenger RNA from a hybridoma that produces antibodies to the arsonate hapten and bears nearly a full complement of the determinants comprising the cross-reactive idiotype (CRI). The sequences of both the complementary DNA clone and the corresponding immunoglobulin heavy chain have been independently determined. A probe for the variable region gene was prepared from the original heavy chain complementary DNA clone and used to analyze, by Southern filter hybridization, genomic DNA from both A/J (CRI positive) and BALB/c (CRI negative) mice. Approximately 20 to 25 restriction fragments containing "germline" variable region gene segments were detected in both strains, and many are shared by both, Since 35 CRI-positive heavy chains have been partially sequenced thus far and 31 are different, the results of the hybridization analysis suggest that somatic mutation events involving the variable region gene segments of the heavy chain play a role in the origin of the amino acid sequence diversity seen in this system.

Amino Acid Sequence↗

PubMed1982

Ubiquitous nonimmunoglobulin p-azobenzenearsonate-binding molecules from lymphoid cells.

A ubiquitous nonimmunoglobulin molecule that binds p-azobenzenearsonate (ABA) has been detected in the cytoplasm of several murine cell lines, including T cell hybridomas as well as in normal liver and spleen. Similar to many recently described antigen-specific T cell factors, this ABA-binding protein has a 62,000 mol wt, and, when analyzed by direct binding, the molecule reacts with several different rabbit anti-idiotypic antisera specific to the ABA system. The presence of this antigen-specific, "idiotype positive" molecule in many different cells indicates that it is not an important immunoregulatory molecule.

Animals↗

PubMed1982

Monoclonal antibodies to immunoglobulin G4 induce histamine release from human basophils in vitro.

Monoclonal murine antibodies to human IgE, IgG1, IgG2, IgG3, IgG4, IgM, and IgA1 were prepared and their abilities to stimulate histamine release from human peripheral blood leukocytes were investigated. As expected, anti-IgE caused 8% to 76% histamine release from the leukocytes of each of 14 nonallergic donors. In one subject anti-IgE alone had no effect, but a 15% histamine release did occur after the subsequent addition of a goat anti-mouse IgG antiserum to crosslink cell-bound anti-IgE molecules. Anti-IgG4 alone caused 5% to 59% histamine release from the cells of four of 14 donors. Subsequent challenge with goat anti-mouse IgG caused an additional release of up to 15% of the basophil histamine, but only from leukocytes of donors responsive to anti-IgG4 alone. No histamine release was detected after stimulation by monoclonal antibodies to IgG1, IgG2, IgG3, IgM, or IgA1. The results of this study indicate that monoclonal antibodies to IgG4 as well as to IgE can stimulate human peripheral blood leukocytes to release histamine. These observations support the theory that antigen-IgG4 interactions can stimulate histamine release from human basophils.

Animals↗

PubMed1982

Amino acid sequence of the CH2 domain from various lagomorph IgGs.

The complete amino acid sequence of a CNBr fragment (C3) corresponding to the CH2 domain of hare IgG was determined. The results when compared to analogous sequences for the domestic rabbit reveal extensive homology as expected. Similar comparisons were made for sequences of tryptic peptides isolated from the same region of the Fc from several other hares, the cottontail rabbit and pika.

Amino Acid Sequence↗

PubMed1982

Amino acid sequence homologies and glycosylation differences between the fourth component of murine complement and sex-limited protein.

Limited primary sequence data have been obtained for all three subunits of the fourth component of murine complement (C4) and its related homologue, the sex-limited protein (Slp). These data show a high degree of NH2-terminal homology between C4 and Slp: four of the six residues identified for the alpha chain, seven of eight for the beta chain, and four of four for the gamma chain. This suggests that apparent molecular weight differences between C4 and Slp subunits are not, as previously suggested, due to a shift in the proteolytic processing sites in the pro-Slp polypeptide molecule. Chemical deglycosylation (apparently complete) of the C4 and Slp alpha chains with trifluoromethanesulfonic acid removes the molecular weight difference between them, suggesting that acquisition of extra glycosylation sites in the latter is responsible for this difference.

Amino Acid Sequence↗

PubMed1982

Monoclonal antibodies against human monocytes. II. Recognition of two distinct cell surface molecules.

Three monoclonal antibodies (63D3, 63D2, and 61D3) with reactivity against human monocytes have been studied. They reacted with most adherent mononuclear cells but not with pure populations of peripheral blood B and T lymphocytes. Two of these antibodies, 63D3 and 63D2, competed in fluorescence inhibition experiments, recognized a monocyte surface antigen of about 200,000 daltons, and were idiotypically related. The third antibody, 61D3, did not compete with the others in fluorescence inhibition experiments, recognized a different surface molecule, and was idiotypically distinct from 63D3 and 63D2. Whereas 63D3 and 63D2 reacted very weakly with some granulocytes, 61D3 did not, suggesting that it is specific for monocytes alone.

Animals↗

PubMed1982

Antiarsonate antibody response: a model for studying antibody diversity.

The use of two polyclonal activators, dextran sulfate (DxS) and lipopolysaccharide (LPS), with or without the presence of additional antigen, is presented here as a system for exploring the antibody response of normal (naive) amd primed B cells. This system expands populations of cells not normally observed under in vivo regulation. By fusing such unnaturally activated B cells, anti-p-azophenylarsonate hybrids were produced that secrete different isotypes of antibodies. The frequencies of isotopes expressed by these hybrids may correspond to the chromosomal order of the heavy chain genes because greater numbers of IgM- and IgG3-secreting hybrids were produced than IgG2a hybrids. Only one IgA hybrid was observed. When DxS and LPS were used to stimulate antigen-primed B cells, hybrids were generated that simultaneously secrete two isotopes of antibody. These hybrids may represent a model of the antigen-stimulated maturational class-switch step observed in normal B cells that involves the expression of IgM and IgG isotypes by the same cell. Such hybrids offer an opportunity to study antibody regulation and diversity by examining the rearrangement of genes during the Ig switch, by exploring the nature of the necessary transitions of mRNA transcription and translation to produce functional antibodies, and by probing the structure and specificity of such antibodies.

Animals↗