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J F Kearney

Publications and source records attributed to J F Kearney.

At least 109 records · Page 6Linked to original sources
PubMed1985

Induction of germ-line anti-alpha 1-3 dextran antibody responses in mice by members of the Enterobacteriaceae family.

A large panel of enteric organisms was screened for agglutination with a panel of lambda monoclonal antibodies of different heavy chain isotypes specific for alpha 1-3 dextran (DEX). Two strains were initially isolated that were bound by most of the anti-DEX antibodies. One organism, Enterobacter cloacae strain MK7, which was characterized in detail, induced a typical lambda anti-DEX response in Igh-Ca mice that had a fine idiotope profile comparable with that induced by purified B1355S dextran containing alpha 1-3 glucosidic linkages (alpha 1-3-DEX). The determinant on the bacterial surface was shown by binding inhibition with nigerotriose to contain alpha 1-3 linkages. Hyperimmunization with these organisms of normal, athymic (nu/nu), or germ-free mice induced large amounts of IgM antibodies but very little IgG. This is the first description of an organism isolated from the normal gut flora of mice that can be shown directly to be bound by alpha 1-3-DEX antibodies and to induce the typical germ-line response of the DEX family of antibodies.

Agglutination Tests↗
PubMed1984

Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

Animals↗
PubMed1984

Quantitation of complement factor D in human serum by a solid-phase radioimmunoassay.

A sensitive solid-phase radioimmunoassay is described which quantitates human D to 1-2 ng/ml. The assay was used to measure the concentration of D in normal and acute-phase sera and sera from individuals with systemic lupus erythematosus. All 3 groups of sera had comparable levels of D with mean values of 1.8, 2.3 and 2.5 micrograms/ml, respectively. Also tested were sera decomplemented in vitro by activators of the classical and alternative pathways. The results indicated that D is not depleted by alternative or classical pathway activation. However, heat inactivation (56 degrees C, 30 min) of serum resulted in almost complete loss of antigenic D.

C-Reactive Protein↗
PubMed1984

Isotype specificity of helper T cell clones. Peyer's patch Th cells preferentially collaborate with mature IgA B cells for IgA responses.

The nature of the IgA B cell precursors that receive preferential help from selected clones of T helper cells from mouse Peyer's patches (PP Th A) were studied. Activation of the PP Th A clones required the presence of antigen, sheep erythrocytes (SRBC), in a culture system supporting development of antibody-secreting plasma cells. Two types of PP Th A cells were used. Both gave vigorous IgA responses; the first also supported low IgM, and the second low IgM and IgG subclass antibody responses. Removal of sIgA+ B cells from either splenic or PP B cell cultures selectively depleted precursors of IgA antibody producers. Cultures of purified sIgA+ B cells, cloned PP Th A cells and SRBC, selectively yielded IgA antibody producers. Finally, PP Th A cells did not support IgA responses in B cell cultures derived from spleens of young mice (days 1-25), and full IgA responses were not seen until the donor mice were 6-7 wk of age. These results suggest that cloned T helper cells can recognize and collaborate with mature, IgA committed B cells.

Animals↗
PubMed1984

Cellular expression of idiotopes defined by monoclonal antibodies.

Monoclonal anti-idiotype (Id) antibodies with specificities for determinants related to the antigen-binding sites of 3 BALB/c myeloma proteins, MOPC-460, HOPC-8 and J558, were used to study Id expression on murine lymphocytes. The monoclonal antibodies were shown to react only with Id structures associated with immunoglobulin on B cells. None of these 3 Id nor a VH Id, detected by a monoclonal antibody made against HOPC-8 heavy chain, were found on T cells. These Id were detected on splenic B cells in neonatal mice; the frequencies in normal, nude and germ-free mice were similar: MOPC-460 Id+: 1.05 +/- 1.7/10(4) spleen cells, HOPC-8 Id+: 1.45 +/- 1.2/10(4) and J558: 0.35 +/- 0.6/10(4). Almost all Id+ cells bore surface IgM, a few expressed surface IgG. MOPC-460 Id+ IgG+ cells were mainly gamma 2a+ or gamma 2b+, whereas J558 and HOPC-8 Id+ IgG+ cells were gamma 3+.

Aging↗
PubMed1984

Analysis of the reactivity of four anti-mouse IgM allotype antibodies with mu+ B lineage cells at various stages of differentiation.

We have used two rat monoclonal antibodies (Mab) (Bet-1 and 331.12), a mouse Mab (AF6-78.25), and an alloantiserum (SJA anti-BAB/14) in two-color immunofluorescence analysis and enzyme-linked immunosorbent assays to examine the expression of IgM allotypes at various stages of mouse B cell development, especially at the pre-B cell stage. In agreement with findings previously reported by others, these antibodies displayed the patterns of allotypic reactivity with IgM+ B cells and plasma cells from the appropriate strains of mice: Bet-1 antibody is specific for a allotype, AF6-78.25 and SJA anti-BAB/14 for b allotype, and 331.12 for both a and b allotypes but not e allotype. These antibodies, however, did not react with isolated mu-heavy chains prepared from secreted molecules or synthesized in situ by normal pre-B cells or pre-B cell-derived hybridomas. The requirement of light chain participation in the expression of IgM allotypic determinant(s) was additionally suggested by analysis of a fetal liver-derived hybridoma (F1-26-11) that has been shown to secrete IgM heteromolecules composed of C57BL/6-derived mu-chain and BALB/c-derived kappa-chains. In contrast to the nonreactivity with C57BL/6 mu-only pre-B cell-derived hybridomas, all three antibodies with specificity for the b allotype (AF6-78.25, SJA anti-BAB/14, and 331.12) reacted with this particular hybridoma. After treating surface IgM molecules on B lymphocytes with papain to cleave Fab fragments, the b allotype reactivity of AF6-78.25 mouse Mab, but not 331.12 rat Mab, disappeared, whereas the a allotype reactivities of two rat Mab (Bet-1, 331.12) were not altered. These results suggest that the IgM allotypic epitopes defined by these antibodies appear to be sterically dependent on the assembly of the whole IgM molecules, and rat Mab 331.12 and mouse Mab AF6-78.25 define two separate b allotype specificities, one on the Fc portion and the other on the Fab portion of the IgM molecules, respectively.

Animals↗
PubMed1984

The use of monoclonal antibodies as probes of the three-dimensional structure of human complement factor D.

The monoclonal antibodies (MAb) were studied for their binding specificities and their effects on the hemolytic and proteolytic activities of human D. One Mab, FD10-1, was obtained from mice immunized with native D; the other, JA4-2, was similarly obtained from mice immunized with BSA coupled to a synthetic nonapeptide (Arg-Ile-Leu-Gly-Gly-Arg-Glu-Ala-Tyr) containing the seven NH2-terminal amino acids of D. By using a PEG-precipitation assay system, lactoperoxidase-iodinated D was precipitated by FD10-1 cell culture supernatant as well as by the purified MAb. Conversely, with the use of the same assay system, neither the JA4-2 cell culture supernatant nor the purified MAb precipitated any significant amount of 125I-D. In contrast, by using a solid-phase binding assay, radiolabeled JA4-2 as well as FD10-1 bound to D-coated wells. In addition, the binding of purified JA4-2 to D-coated wells could be completely inhibited by the nonapeptide, whereas the binding of purified FD10-1 at equivalent concentrations was unaffected. These binding studies correlated with the results of functional assays. FD10-1 was found to inhibit the hemolytic activity of the alternative complement pathway in human serum as well as the cleavage of radiolabeled B by D in the presence of cobra venom factor, whereas JA4-2 had no significant inhibitory effect in either of these assays. These data suggest that the NH2-terminus of native D, like that of other active serine proteases, is buried inside the molecule where it is inaccessible to the bulky JA4-2 MAb.

Animals↗
PubMed1984

Identification and characterization of an apparent germline set of auto-anti-idiotypic regulatory B lymphocytes.

By fusing BALB/c splenic lymphocytes from mice immunized with phosphorylcholine (PC) to an immunoglobulin nonproducing plasmacytoma cell line, a B cell hybridoma was isolated (MM-60) that has been shown by multiple criteria to produce a bona fide auto-anti-(anti-T15 idiotype) antibody. In vivo administration of MM-60 antibody suppressed T15+ anti-PC antibody production in an idiotope-specific manner by activation of an intervening set of anti-T15 B cells. These T15-specific B cells i) appeared to express germline-encoded variable region gene products, ii) developed in parallel to, but independent of, T15+ B cells, and iii) suppressed the anti-PC response in a T cell-independent fashion. Variants of T15+ anti-PC B cells possessing aberrant immunoglobulin heavy chain D region structure escaped from the suppression imposed by this anti-T15 B cell set, suggesting that a function of the heavy chain D region may be to contribute to the formation of molecular target sites for idiotype-directed regulatory cells and/or antibodies. The indigenous nature of these particular populations of anti-idiotypic and anti-(anti-idiotypic) B cells and the ability of their immunoglobulin products to regulate antigen-specific B cells in vivo provides strong supportive evidence for the significant role idiotype-directed network interactions play in regulating specific antibody production during a normal immune response.

Aging↗
PubMed1983

Fine idiotype analysis of B cell precursors in the T-dependent and T-independent responses to alpha 1-3 dextran in BALB/c mice.

In this study BALB/c B cell precursors responsive to the T-independent (TI) type 2 (TI-2) antigen, dextran B1355S (DEX), and the T-dependent (TD) derivative, dextran-Limulus hemocyanin (DEX-Hy) were examined for isotype and idiotope expression using the splenic focus assay. The predominant isotype detected in the TI assay was IgM, while IgA was the predominant isotype expressed in the TD assay. There was also a fourfold increase in the number of foci secreting more than one isotype in the TD assay vs. the TI assay without an overall change in anti-DEX precursor frequency, suggesting that carrier-primed T cells enhance the expression of non-IgM isotypes possibly by increasing the frequency of isotype switching by individual B cell precursors. A panel of distinct monoclonal antiidiotype antibodies (MAIDs) was then used to examine idiotope expression by antibodies secreted in splenic foci responding to DEX and DEX-Hy. This analysis revealed considerable diversity in the idiotope profiles expressed by all isotypes tested. There appeared to be no differences in idiotope diversity among the various isotypes. A similar diversity of idiotope profiles was obtained from both TI and TD splenic foci, indicating that a comparable degree of diversity was associated with the antibodies generated by TI and TD precursors. Idiotype analysis of IgM-IgA-secreting foci with a panel of monoclonal antiidiotope antibodies revealed slight idiotypic differences between the two isotypes secreted in the same focus in about half the cases. These results suggest that somatic variation occurs during the antigen-driven maturation of B cell precursors, within the 15-d time frame of the splenic focus assay, and may be associated with isotype switching.

Animals↗
PubMed1983

An enzyme-linked immunoabsorbent assay for measuring antibodies against muscle acetylcholine receptor.

An enzyme-linked immunoabsorbent assay has been developed for measuring anti-acetylcholine receptor antibodies from sera of patients with myasthenia gravis or tissue culture supernatants from hybridomas. Acetylcholine receptor from a detergent extract of muscle tissue was bound indirectly to microtiter plates via a monoclonal anti-receptor antibody already coupled to the polyvinyl plates. Myasthenic sera or antibodies in tissue culture media were then tested for binding to the acetylcholine receptor attached to the monoclonal antibody. Anti-receptor antibodies were detected in the serum of 80% of myasthenic patients when assayed by this method and the levels of antibody corresponded fairly well with antibody titers determined by an immunoprecipitation assay. Occasional myasthenic patients had serum antibodies which reacted specifically with the monoclonal antibody attached to the microtiter plate. The assay described here was far less time-consuming than immunoprecipitation assays, required only small quantities of receptor, and did not require the use of radioisotopes such as 125I-alpha-bungarotoxin.

Animals↗
PubMed1983

Analysis of idiotypic heterogeneity in the anti-alpha 1-3 dextran and anti-phosphorylcholine responses using monoclonal anti-idiotype antibodies.

Panels of monoclonal anti-idiotype antibodies (MAIDs) specific for individual (IdI) and cross-reactive (IdX) idiotopes were prepared and used to study the expression of these idiotopes on anti-DEX and anti-PC antibodies produced in response to antigenic stimulation in vivo, clonal expression of idiotopes in an in vitro splenic focus assay, and the alterations in the idiotypic profile of these responses after in vivo administration of monoclonal anti-Id antibodies. Using these panels of MAIDs, it was possible to inactivate IdI-bearing B cells both in neonates and adult mice without affecting the responsiveness of IdI- B cells. By contrast, suppression with IdX-specific antibodies resulted in greatly reduced antibody responses. By studying the idiotypic profile of anti-DEX clones in the splenic focus assay, it was shown that IgM, IgG, and IgA antibody Id were diverse and paralleled those expressed in serum. Within some clones there was evidence that idiotope-isotype associations differed, suggesting that V region variants may have been generated within the progeny of a clone following stimulation by dextran. An anti-anti-Id antibody isolated from a BALB/c mouse undergoing a normal immune response to R36A was shown to have a T-cell independent highly idiotope-specific regulatory effect on the T15+ anti-PC response, apparently affecting induction of anti-idiotypic B cells.

Animals↗
PubMed1983

Ten percent of normal B cells and plasma cells share A VH determinant(s) (J606-GAC) with a distinct subset of murine VHIII plasmacytomas.

Our findings indicate that a subset of VHIII antibodies, which we refer to as J606-GAC, contains a determinant(s) that is present on 5 to 15% of normal splenic B cells and plasma cells as detected by immunofluorescence. This subpopulation is detected by purified antibody, 0-1, which was prepared against a murine anti-group A carbohydrate (anti-GAC) hybridoma antibody. The J606-GAC subset includes the beta 2, 1 fructosan myelomas J606, EPC109, W3082, ABPC4, and UPC61, as well as 13 anti-GAC hybridomas. The 0-1 antiserum failed to react with hybridoma and myeloma immunoglobulins from murine VH groups I and II or other VHIII antibodies. By Western blot analysis, it was observed to react with isolated heavy, but not light, chains of J606-GAC-bearing antibodies. 0-1 failed to react with myelomas XRPC44 and J539, which have the same J region as J606 but a very different VH region. These observations indicate that 0-1 is detecting a VH region determinant. The J606-GAC marker recognized by 0-1 was expressed as early as 4 days after birth and was expressed at similar frequencies in germfree and conventional mice. Immunoprecipitation of both surface and biosynthetically labeled proteins from spleen cells or J606-GAC-positive hybridoma cell lines, respectively, confirmed that 0-1 was recognizing an immunoglobulin determinant.

Animals↗
PubMed1983

Analysis of the anti-alpha 1 leads to 3 dextran response with monoclonal anti-idiotype antibodies.

The antibody response to alpha 1 leads to 3 dextran (DEX) in BALB/c mice consists of a family of closely related yet highly heterogeneous molecules. Although these antibodies have been previously characterized both idiotypically and structurally, detailed analysis of responding clones has not been possible using conventional anti-idiotype antibodies. Monoclonal syngeneic and allogeneic anti-idiotype antibodies (MAIDs) specific for anti-DEX antibodies were used in this study to dissect the serum antibody response to DEX in BALB/c mice. The constructed MAIDs showed considerable heterogeneity by isoelectric focusing and by their binding characteristics to a series of DEX specific myeloma and hybridoma proteins. The predominant heavy chain isotype of these MAIDs was gamma 1. These antibodies were used to identify individual idiotypic structures (IdI) on J558, or M104E as well as cross-reactive determinants common to both (IdX). Although both IdX and IdI MAIDs were obtained, IdI specific antibodies were obtained more frequently. BALB/c mice immunized with DEX produced antibodies expressing both IdI but in highly variable amounts. A large percentage of, but not all DEX specific antibody, could be accounted for by IdX bearing antibodies. Suppression of adult and neonatal mice by IdI specific MAIDs was effective with precise elimination of only those clones expressing IdI determinants leaving the total lambda bearing anti-DEX response intact. Suppression of adults and neonates by an IdX specific MAID resulted in a temporary and partial suppression of the total lambda bearing anti-DEX response along with total suppression of the IdX portion of the response. Unlike other systems these monoclonal antibodies produce only suppression, and under a variety of conditions enhancement of anti-DEX responses has not been observed.

Animals↗
PubMed1983

Localization of the phosphocholine-binding sites on C-reactive protein by immunoelectron microscopy.

C-reactive protein (CRP) was reacted with monoclonal IgG antibody or Fab antibody fragments directed against the phosphocholine- (PC) binding site or a second unrelated site. The resulting immune complexes were viewed by a negative stain immunoelectron microscopy technique. Monoclonal anti-PC-binding site antibody bound to a single epitope on each of the five CRP subunits. The orientation of the PC-binding sites was determined to be slightly medial to one of the planar faces (A-face) of the molecule. The second monoclonal antibody, which was not PC-binding site related, bound to epitopes (one per CRP subunit) that were located slightly lateral to the other planar face (B-face) of the CRP molecule, i.e., opposite of the PC-binding site. Thus, the PC-binding site and the non-PC-binding site are oriented nearly perpendicular but on opposite sides with respect to the plane of the CRP molecule. The functional significance of this configuration is discussed.

Animals↗
PubMed1983

Nonrandom escape of tumor cells from immune lysis due to intraclonal fluctuations in antigen expression.

Considerable heterogeneity in the amount of surface antigen can regularly be demonstrated by cytofluorometric analysis among genetically identical cells in a tumor clone. We have used monoclonal idiotype-specific antibodies to investigate the patterns of change in amounts of an idiotypic tumor antigen and how such changes affect the immune escape of malignant B cells expressing this antigen. By the use of fluorescence-activated cell sorting, we separated cells expressing either very large or very small amounts of the idiotypic target antigen and then analyzed these subpopulations of tumor cells at various times after isolation for expression of idiotype. We found that the differences in amount of antigen expression were not heritable, and that over a period of about 7 days of continuous growth in vitro, the fluorescence-activated cell sorted populations gradually came to express normal amounts of idiotype. The mechanisms regulating the quantity of surface idiotype were independent of those affecting the amounts of other membrane-associated molecules such as H-2 antigen. Furthermore, these nonheritable intraclonal differences in amounts of antigen expression were unrelated to stages of the cell cycle but clearly did affect the susceptibility of the cells from immune lysis. Thus, tumor cells expressing the lowest amount of surface idiotype were much more resistant to the lytic effects of antiidiotypic antibody and complement but had the same cell cycle distribution as did unseparated cells. These results demonstrate that nonheritable, non-cell cycle-related heterogeneity in amount of tumor antigen expression can significantly determine which cells in a cloned malignant cell population preferentially escape monoclonal tumor-specific antibody therapy.

Animals↗
PubMed1982

Immunoglobulin VH determinants defined by monoclonal antibodies.

Hybridoma clones secreting antibodies against common VH determinants were readily produced by fusion of cells from mice immunized with isolated V mu fragments of human immunoglobulins (Ig), but not with intact Ig molecules or isolated heavy chains. Four monoclonal antibodies to the V mu fragments of different IgM paraproteins were selected for analysis: MH-44 (mu kappa), GB-24 (mu kappa), NF-11 (gamma 1 kappa), and SA-44 (gamma 1 kappa). Each antibody reacted with the homologous V mu fragment, homologous mu chain, and normal gamma chains, but not with the intact IgM molecules, intact IgG, or isolated light chains, as determined by radioimmunoassay. The VH reaction spectra with a panel of myeloma heavy chains showed overlapping but distinctive patterns for the four antibodies. Each of the four monoclonal anti-VH antibodies appeared to react with a different "hidden" VH determinant that is not exposed on undenatured, intact Ig molecules and differs from conventional VH subgroup determinants. In immunofluorescence studies, the monoclonal anti-VH antibodies did not bind to surface Ig on viable B lymphocytes, but visibly stained subpopulations of fixed B lymphocytes, pre-B cells, and normal plasma cells. The mean frequencies of VH+ plasma cells were 30% (MH-44), 17% (GB-24), 13% (NF-11), and 3% (SA-44), and similar frequencies were obtained for the VH+ B cell subpopulations. While subpopulations of B cells could be identified at all stages in differentiation by immunofluorescence with the anti-VH antibodies, neither resting nor activated T cells expressed these VH determinants in detectable amounts.

Animals↗
PubMed1982

Murine Peyer's patch T cell clones. Characterization of antigen-specific helper T cells for immunoglobulin A responses.

We successfully cloned antigen-specific T cells from murine gut-associated lymphoreticular tissue, i.e., Peyer's patches, which are dependent upon T cell growth factor and independent of antigen for continuous growth. These clones exhibit helper activity for IgA responses to sheep erythrocytes (SRBC) and have been designated T helper (Th) A. Two broad categories of Th A clones have been maintained in continuous culture. The first group supports IgM and largely IgA anti-SRBC plaque-forming cell (PFC) responses in both normal and SRBC-primed splenic B cell cultures, whereas the second group supports low IgM, IgG1, and IgG2 and high IgA PFC responses. Subclones derived from single cells maintain the parent helper properties when propagated in culture for long periods (greater than 7 mo). Cloned Th A cells are antigen specific and do not support polyclonal or immune responses to other thymus dependent antigens in normal B cell cultures. Th A cells require full histocompatibility for helper functions because addition of cloned Th A cells to B cell cultures from other H-2 types does not result in IgA responses. Cloned Th A cells are Thy-1.2+ and Lyt-1+ and Lyt-2-, Ig-, and I-A-. Th A cells bear Fc receptors for IgA and do not possess receptors for IgM or IgG isotypes. Thus, T cells that primarily promote IgA isotype responses have been isolated in high frequency from murine PP, an anatomical site of major importance for induction and regulation of the IgA response.

Animals↗