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Biomedical subjects

J F Kearney

Publications and source records attributed to J F Kearney.

At least 91 records · Page 5Linked to original sources
PubMed1987

Probing functional sites on complement protein B with monoclonal antibodies. Evidence for C3b-binding sites on Ba.

We used four mouse monoclonal antibodies (Mab) as probes of functional sites of human complement protein B. Two Mab, HA4-1B (gamma 2a kappa) and HA4-15 (gamma 2a kappa), reacted with the same or adjacent epitopes on the Bb fragment of B, while the other two, HA4-1A (gamma 1 kappa) and FD3-20 (gamma 1 kappa), reacted with distinct epitopes on Ba. All reactive epitopes were expressed on native B and only one, recognized by the anti-Ba Mab HA4-1A was more reactive on isolated Ba than on B. These binding specificities were determined by direct binding radioassays and confirmed by inhibition studies. Immunoelectron microscopy of B and Bb in complex with anti-Ba and anti-Bb revealed that the recognized epitopes are on opposite sides of the molecule and are on discrete domains. All four Mab inhibited the hemolytic activity of B, although with different efficiencies and through different mechanisms. The main effect of the two anti-Bb Mab was an increased rate of loss of hemolytic sites from preformed EC3bBb C3 convertase presumably through accelerated dissociation of Bb. On the other hand, the main effect of the two anti-Ba Mab was inhibition of binding of B to C3b. HA4-1A was more efficient, inhibiting by 50% the binding of [125I]B to EC3b at 10 micrograms/ml as IgG and at 13 micrograms/ml as Fab. The data suggest that a binding site for C3b on intact B is located on the Ba portion of the molecule.

Antibodies, Monoclonal↗
PubMed1987

Idiotypes and autoimmunity.

By the analysis of hybridomas constructed from B cells early in development we have shown that: (i) the early neonatal B cell repertoire consists of a highly autoreactive set of B cells showing extensive multispecificity and interconnectivity; (ii) many of these antibodies express anti-idiotypic activity towards autologous germline-encoded idiotypic antibodies; (iii) the anti-idiotypic activities of such B cells and/or their antibody products play a major role in establishing the clonal dominance of certain well-characterized idiotypes in the responses to phosphorylcholine (PC) and alpha 1----3 dextran; and (iv) results obtained in comparisons between antibodies to the acetylcholine receptor and alpha 1----3 dextran in humans and mice showed extensive idiotypic connectivity. Some of the anti-idiotypic specificities involved were also apparent in the neonatally derived antibodies. These results suggest that there are extensive idiotype-directed interactions between B cells early in development which appear to be essential for establishing the adult B cell repertoire and the accompanying clonal dominance of appropriate idiotypes. Similar interactions may also play a role in the development of certain autoimmune disorders.

Animals↗
PubMed1987

Assembly and secretion of heavy chains that do not associate posttranslationally with immunoglobulin heavy chain-binding protein.

Heavy chain-binding protein (BiP) associates posttranslationally with nascent Ig heavy chains in the endoplasmic reticulum (ER) and remains associated with these heavy chains until they assemble with light chains. The heavy chain-BiP complex can be precipitated by antibody reagents against either component. To identify sites on heavy chain molecules that are important for association with BiP, we have examined 30 mouse myelomas and hybridomas that synthesize Ig heavy chains with well characterized deletions. Mutant Ig heavy chains that lack the CH1 domain could not be demonstrated to associate with BiP, whereas mutant Ig heavy chains with deletions of the CH2 or CH3 domain were still able to associate with BiP. In two light chain negative cell lines that produced heavy chains with deletions of the CH1 domain, free heavy chains were secreted. When Ig assembly and secretion were examined in mutants that did not associate with BiP, and were compared with normal parental lines, it was found that the rate of Ig secretion was increased in the mutant lines and that the Ig molecules were secreted in various stages of assembly. In one mutant line (CH1-) approximately one-third of the secreted Ig molecules were incompletely assembled, whereas the Ig molecules secreted by the parental line were completely assembled. Our data show the CH1 domain to be important for association with BiP and that when this association does not occur, incompletely assembled heavy chains can be secreted. This implies a role for BiP in preventing the transport of unassembled Ig molecules from the ER.

Animals↗
PubMed1987

Analysis with antiidiotype antibody of a patient with chronic lymphocytic leukemia and a large cell lymphoma (Richter's syndrome).

A murine monoclonal antibody made against an idiotypic determinant (Id) of surface IgM/IgD lambda molecules on chronic lymphocytic leukemia (CLL) cells of a 71-year-old woman was used for clonal analysis by two-color immunofluorescence. The anti-Id antibody identified IgM+/IgD+/lambda+ B cells as the predominant cell type of her CLL clone. In addition, substantial proportions of the IgG and IgA B cells and most of the IgM plasma cells in her bone marrow and blood were Id+. Six years after diagnosis, the patient died of respiratory failure due to infiltration of lungs by malignant cells. Autopsy revealed a dramatic change in the tumor cell morphology. The lungs, hilar nodes, and liver were infiltrated by a diffuse large cell lymphoma admixed with the leukemic cells. By immunohistologic staining these anaplastic lymphoma cells were IgM+/IgD-/lambda+ B cells expressing the same Id noted earlier on the CLL cells. The immunoglobulin gene rearrangement pattern on Southern blot analysis was also the same in leukemic blood cells and in the tissues involved by the lymphoma. Thus, the combination of antiidiotype and immunoglobulin gene analyses in this patient with Richter's syndrome revealed that a CLL clone, seemingly "frozen" in differentiation, was actually undergoing isotype switching, differentiation into plasma cells, and evolution into a rapidly growing and fetal lymphoma.

Aged↗
PubMed1987

Monoclonal IgM antibodies that inhibit primary Moloney murine sarcoma growth.

Monoclonal IgM antibodies with specificity for Moloney murine sarcoma virus (M-MuSV)-Moloney murine leukemia virus (M-MuLV) from two hybridoma clones have been isolated and characterized. The monoclonal antibodies have specificity for a cytoplasmic and cell surface Friend-Moloney-Rauscher group-specific antigen. Immunoelectron microscopy revealed antibody binding to the surface of virus-expressing cells but not to the budding virus particles. Treatment of M-MuSV-injected mice with monoclonal IgM anti-M-MuSV significantly inhibited tumor growth compared to virus-inoculated animals receiving either saline or MOPC 104E. Nude mice exhibited delayed tumor induction following treatment with the monoclonal antibodies but ultimately died from tumor growth. Virus-injected euthymic mice that were treated with monoclonal IgM anti-M-MuSV generated a potentiated spleen cell-mediated cytotoxicity against Moloney sarcoma cells compared to virus-infected treated with saline. This potentiation of cytotoxicity remained after trypsinization of the spleen cells and thus was probably not due to passively adsorbed monoclonal antibody. The antibodies alone or in the presence of complement did not neutralize M-MuLV. The IgM antibodies induced specific tumor cell cytotoxicity in vitro mediated by complement spleen cells, lymph node cells, or thymus cells. In conclusion, two monoclonal IgM anti-M-MuSV antibodies that bind to the tumor cell surface did not neutralize virus can inhibit primary M-MuSV-induced tumor growth in vivo. The regression event appeared to involve heterogeneous mechanisms. Complete regression remained thymus dependent even with passive antibody therapy, but significant tumor growth inhibition was produced independent of T-cells. In vitro these IgM antibodies induced complement and cell-mediated cytotoxicity.

Animals↗
PubMed1986

Mouse monoclonal antibodies against bromelain-treated mouse erythrocytes: reactivity with erythrocytes of various species of animals and idiotypes.

Spleen and peritoneal cells from unimmunized BALB/c mice were cultured in the presence of LPS for 24 hr and fused to produce hybridomas secreting antibodies against bromelain-treated mouse erythrocytes (BrMRBC). Three clones from spleen cells and eight clones from peritoneal cells were isolated and characterized further. All the monoclonal antibodies had IgMK isotype. Their reactivities against untreated and bromelain-treated erythrocytes from various species were assessed by hemolysis and indirect radioimmunoassay; all the clones had similar antigen specificities. On the isoelectric focussing patterns of light chains, they were separated into two groups, two and nine clones, and all the light chains in each group showed identical patterns. The two groups shared no common idiotope detectable by anti-idiotype antibodies prepared by immunization of rabbits with the monoclonal antibodies, but all the antibodies in each group shared common idiotopes. In each group, one antibody had a unique idiotope different from any other antibody, but eight antibodies in a group shared another identical idiotope. These findings suggest the restricted heterogeneity of anti-BrMRBC antibodies in the mouse.

Animals↗
PubMed1986

Idiotypic network connectivity and a possible cause of myasthenia gravis.

Extensive idiotypic connectivity has been discovered between the antibodies composing the immune responses against the acetylcholine receptor (AChR) and alpha-1,3-dextran. The idiotypic connections form an elaborate network linking these disparate antigen systems, and there is an hierarchical organization of the antibodies in this network. The key anti-Ids that interconnect these two responses are more crossreactive, lower-affinity antibodies. Interestingly, 15% of patients with MG, which is caused by autoantibodies against the AChR, have serum antibodies against DEX. Control sera are negative for anti-DEX antibodies. Certain anti-DEX antibodies also bind to anti-AChR antibodies via idiotypic interactions. These findings suggest a model for the initiation of autoimmunity in MG. Antibodies made in response to DEX epitopes on the surface of certain bacteria would elicit the production of anti-Ids. However, some of these anti-Ids would also be autoantibodies against the AChR. Thus, is some circumstances, autoimmunity may develop as a consequence of the normal operation of regulatory idiotypic networks.

Animals↗
PubMed1986

Rearrangement of antigen receptor genes is defective in mice with severe combined immune deficiency.

A process unique to lymphocyte differentiation is the rearrangement of genes encoding antigen-specific receptors on B and T cells. A mouse mutant (C.B-17scid) with severe combined immune deficiency, i.e., that lacks functional B and T cells, shows no evidence of such gene rearrangements. However, rearrangements were detected in Abelson murine leukemia virus-transformed bone marrow cells and in spontaneous thymic lymphomas from C.B-17scid mice. Most of these rearrangements were abnormal: approximately 80% of Igh rearrangements deleted the entire Jh region, and approximately 60% of TCR beta rearrangements deleted the entire J beta 2 region. The deletions appeared to result from faulty D-to-J recombination. No such abnormal rearrangements were detected in transformed tissues from control mice. The scid mutation may adversely affect the recombinase system catalyzing the assembly of antigen receptor genes in developing B and T lymphocytes.

Abelson murine leukemia virus↗
PubMed1986

Functional characterization of monoclonal auto-anti-idiotype antibodies isolated from the early B cell repertoire of BALB/c mice.

A large number of hybridomas were constructed by fusion of B cells from perinatal liver and spleen. Many of these showed multispecificity, high interconnectivity and anti-idiotype (Id) activity. Several of these were subjected to a detailed analysis to evaluate their influence on the developing immune system. A hybridoma BD2 (mu,kappa), derived from 2-day-old liver, was shown to have anti-T15 and anti-J558 activity by inhibition enzyme-linked immunosorbent assay and by in vivo administration. BD2 reduced primary T15 and J558 Id in adult BALB/c by 50%. In contrast, timed administration of this antibody during neonatal periods resulted in enhancement of responses to phosphorylcholine (PC) and alpha (1----3)-linked dextran (Dex) when these mice were challenged as adults. Another hybridoma DB3 (mu,kappa), derived from a lipopolysaccharide-stimulated fetal liver, reacts with GB4-10 (anti-T15) and not with PC. It also reacts with BD2. It is thus anti-anti-Id with respect to T15 and J558. Early administration of this antibody also led to an enhancement of anti-PC and anti-Dex responses, apparently via expansion of a set of intermediate anti-Id BD2-like B cells. In adult mice it suppressed responses to both antigens. A third hybridoma FC4 (mu,kappa), derived from 3-day-old spleen, reacts with GB4-10 as well as EB3-7 (anti-J558). Introduction of this antibody into neonatal mice enhanced anti-Dex responses while in adults it caused suppression of T15 Id. The results presented here suggest a possible role for neonatal anti-Id B cells in the primary activation of antigen-reactive B cells by Id selection.

Animals↗
PubMed1986

In vivo suppression of perinatal multispecific B cells results in a distortion of the adult B cell repertoire.

The isolation of multispecific B cell hybridomas with a variety of anti-idiotype (anti-Id) activities from the lymphoid organs of fetal and neonatal BALB/c mice suggested that the development of the immune system may depend on Id interactions among autologous B cells. In vitro analysis of antibodies secreted by these hybridomas showed extensive sharing of an idiotope defined by the monoclonal antibody FD5-1. Early and timed administration of this antibody during the perinatal period results in a distortion of the phosphorylcholine (PC) and alpha (1----3)dextran (Dex)-specific B cell precursor compartment of the developing repertoire and is reflected by a drastic reduction of antibody responses to these antigens when challenged as adults. These observations provide strong evidence for the involvement of the early appearing multispecific B cells in Id interactions that bring about the uniform development of the normal adult B cell repertoire. Interference with these interactions at critical stages of developmental results in permanent deficiencies in the adult B cell repertoire.

Animals↗
PubMed1986

Analysis of the hepatobiliary transport of IgA with monoclonal anti-idiotype and anti-allotype antibodies.

The processing and fate of mixed immune complexes is influenced by the antibody isotypes present. The hepatobiliary transport of mixed immune complexes containing the mouse IgA myeloma protein J558 and corresponding monoclonal IgG or IgM anti-J558 idiotype or monoclonal IgG anti-mouse IgA allotype antibodies has been studied. The anti-idiotype or anti-allotype antibodies were radiolabeled and injected into mice with or without mouse polymeric IgA (J558). IgG anti-idiotype antibodies to J558 IgA were selectively transported into bile by J558 IgA. This process occurred with a radiolabeled Fab preparation of the IgG anti-idiotype and was inhibitable with IgA of an irrelevant antigenic specificity. Thus, polymeric IgA influenced the fate of IgA-IgG idiotype-anti-idiotype serum immune complexes. A monoclonal anti-idiotype antibody of the IgM isotype (D8-3) was not selectively transported into bile by itself or as an IgA-IgM complex. A monoclonal IgG antibody (CB5-6) to a mouse allotype determinant in the Fc portion of IgA was not selectively transported into bile. This anti-allotype monoclonal antibody inhibited the hepatobiliary transport of 125I-polymeric J558 IgA and therefore appeared to directly or indirectly block the site in the Fc region of IgA recognized by the hepatic receptor.

Animals↗
PubMed1986

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas.

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.

Animals↗
PubMed1986

Idiotype-directed interactions during ontogeny play a major role in the establishment of the adult B cell repertoire.

IgM hybridomas derived from perinatal B cells show a high degree of auto-reactivity and many had demonstrable anti-idiotypic reactivities by binding studies. Selected multispecific antibodies were also shown to have potent idiotype-specific biological activities and if administered at appropriate stages of development could dramatically alter the responses of these mice when challenged with appropriate antigens in adult life. The results obtained suggest that idiotype-directed interactions between neonatal B cells play an important role in the early establishment of the B cell repertoire which is subsequently expressed in adult mice.

Age Factors↗
PubMed1986

Clonotypic analysis of the fetal B cell repertoire: evidence for an early and predominant expression of idiotypes associated with the VH 36-60 family.

Determining the mechanisms by which B cells develop that culminates in the generation of a diverse repertoire is critical to our understanding of how the immune response is regulated. The B cell specificity repertoire appears to be developmentally acquired in a predetermined, temporally ordered fashion. Thus, in the Balb/c mouse, the ontogenetic appearance of functional B cells specific for various hapten probes occurs in the order of dinitrophenol (DNP), fluorescein (Fl), and phosphorylcholine (PC). In addition, when the influenza virus hemagglutinin molecule was used as an antigen probe, similar conclusions were drawn regarding a patterned acquisition of the specificity repertoire. More recently, we have used a fetal organ culture system to show that hapten-responsive B cells appear in the same predictable, temporal order in vitro. The importance of these studies was that the effects of environmental influences were minimized in the absence of circulation and cell migration, and therefore the results indicated that the patterned emergence of the specificity repertoire observed was largely the result of genetic regulatory processes. Recent molecular findings may relate to such genetic regulatory processes. The VH genes in Balb/c mice have been grouped into eight families based on sequence homology, and have been mapped relative to the constant region genes. Yancopoulos et al. and Perlmutter et al. have shown that the VH gene segments closest to the JH cluster, the VH1B 7183 family, are preferentially utilized in transformed fetal pre-B cell lines and in fetal B cell hybridomas. This led to the hypothesis that the developmental control of the expression of VH gene segments is related to chromosomal organization. A logical extension of these findings is that the programmed appearance of particular clonotypes in ontogeny may be explained, in part, by the preferential use of particular VH gene segments. However, to what extent the transformed B cell lines represent members of the functional expressed repertoire could not be evaluated. In the studies described herein, the fetal organ culture system was used to assess the early expressed repertoire at the clonotypic level using idiotypic analysis. Anti-DNP secreting clones were derived from fetal organ cultures and tested for the presence of two idiotypes, 36 and MOPC 460 (460). The 36 idiotype is a predominant DNP clonotype of the neonatal repertoire, while the 460 idiotype is a major cross-reactive idiotype of the adult DNP response.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1986

In vitro effect of monoclonal anti-idiotype antibodies (anti-M104E) on MOPC 104E myeloma cells.

We investigated the effect of three monoclonal anti-idiotype antibodies (anti-M104E) on various functions of MOPC 104E myeloma cells in vitro. The antibodies used were N-20-2 [immunoglobulin M (IgM), BALB/c], SJL18-1 [IgM, BALB/c X SJL F1], and CD3-2 [immunoglobulin G1 (IgG1), BALB/c X A/J F1]. The two IgM antibodies were very efficient in blocking surface M104E IgM as shown by rosette inhibition, whereas the IgG1 isotype was not very effective. The reexpression of surface M104E IgM was different from antibody to antibody. The secretion of M104E IgM by MOPC 104E cells was partially blocked by the two IgM antibodies, but the IgG1 antibody had no effect. All three anti-idiotype antibodies inhibited the stem cell renewal activity of MOPC 104E cells assayed by colony formation assay. On the other hand, in suspension culture, the two IgM antibodies inhibited the growth of MOPC 104E cells in the absence of complement or effector cells of antibody-dependent cellular cytotoxicity, but IgG1 antibody had no effect. The starting tumor inoculum size was critical in the observations of the effects seen on both the growth and the colony-forming activity of MOPC 104E cells. The results of this study show the functional differences between various monoclonal anti-idiotype antibodies and also indicate that some anti-idiotype antibodies can inhibit the growth of MOPC 104E myeloma cells directly without any help of complement or effector cells of antibody-dependent cellular cytotoxicity.

Animals↗
PubMed1985

Developmentally controlled expression of immunoglobulin VH genes.

Although antibody diversity arises mainly from apparently random combinatorial and somatic mutational mechanisms acting upon a limited number of germline antibody genes, the antibody repertoire develops in an ordered fashion during mammalian ontogeny. A series of early pre-B and B-lymphocyte cell lines were examined to determine whether an ordered rearrangement of gene families of the variable region of immunoglobulin heavy chains (VH) may be the basis for the programmed development of the antibody response. The results indicated that the VH repertoire of fetal B-lineage cells is largely restricted to the VH 7183 gene family and that subsequent recruitment of additional VH gene families occurs during neonatal development. These results have important implications in understanding the ontogeny of immune function.

Aging↗