Ch.H. Heusser

Dipetalonema viteae: Phosphorylcholine and non-phosphorylcholine antigenic determinants in infective larvae and adult worms

The humoral immune response of Balb/c mice to live infective larvae or adult worm extract of Dipetalonema viteae is composed of two antibody populations either with or without specificity for phosphorylcholine. Absorption of immune serum on phosphorylcholine-Sepharose and separation of the antibody population demonstrated that anti-larvae serum contains a larger ratio of phosphorylcholine versus non-phosphorylcholine antibodies as compared to anti-adult serum. Immunofluorescence on crossections of female worms revealed that antigen expressing phosphorylcholine determinants were mainly found on certain internal structures, like egg, uterine, and intestinal membranes, but not on the cuticle. Immunoblotting using an adult worm extract demonstrated that protein bands reacted with either one or both populations of antibodies. The patterns were heterogeneous and moreover differed between the anti-larvae serum and the anti-adult serum.

Monoclonal Antibodies to Human Renin: Properties and Applications

A series of 11 different monoclonal antibodies generated against human kidney renin have been characterised. Their binding affinity, inhibition of renin activity, epitope distribution, crossreactivity with related enzymes and finally in vivo pharmacological effects were analysed. All antibodies were found to be specific for primate renin recognising 6 independent antigenic structures on the renin molecule. They expressed different effects on renin activity namely (1) no inhibition, (2) only partial, or (3) complete inhibition. Partially inhibiting antibodies demonstrated specific degrees of inhibition (30, 60 or 80%). One antibody, R-36-16, demonstrated an IC 50 of 1.3 X 10(-11) M/L and, when injected into marmosets, induced complete inhibition of plasma renin activity and reduction of blood pressure. Using a selected pair of antibodies a radioimmunoassay has been established providing a fast and highly reproducible determination of human and marmoset immunoreactive renin, detecting both active and inactive renin down to concentrations of 10 pg/ml (1.25 X 10(-17) moles of renin per 50 microliter sample).

Mechanism of inhibition of human renin by monoclonal antibodies.

The mechanism by which monoclonal antibodies directed against human renin (R3-36-16 and R3-47-10) inhibit renin activity was investigated using various substrates. Both antibodies acted as potent inhibitors of human renin activity when human angiotensinogen was used as a substrate. However, their effects differed clearly in the presence of synthetic tetradecapeptide. When low concentrations of tetradecapeptide were used as substrate, renin activity was only partially inhibited by R3-47-10, whereas it was stimulated by R3-36-16. At higher synthetic substrate concentrations, both antibodies stimulated angiotensin I production. This effect was independent of the pH. Both antibodies exerted their effects in the presence of CGP 29287, a peptidic transition-state competitive renin inhibitor, indicating that their binding sites differed from that of CGP 29287. In combination, the stimulatory effect of R3-36-16 was not blocked by R3-47-10, but the inhibition produced by R3-47-10 was reversed by R3-36-16. Both antibodies may prevent the large natural substrate angiotensinogen from entering the enzymatic cleft by steric hindrance. At a low substrate concentration, R3-47-10 may also partially hinder the access of synthetic tetradecapeptide into the active cleft by steric hindrance. In contrast, the stimulating effect of both antibodies may be due to a conformational change in the renin molecule, allowing an increased access of tetradecapeptide or a more rapid release of the product from the enzymatic cleft.

New concepts of IgE regulation.

B cell switch to IgE expression is mediated by IL-4 and is regarded as a T helper cell-related phenomenon. In this overview we describe that IgE switch can also be induced by mast cell/basophil like cells (from splenic non-B, non-T cells), activated by IgE receptor cross-linking and/or IL-3 which results in IL-4 production by these cells. Furthermore, activated mast cells produce their own growth factors, IL-3 and GM-CSF. Thus, activation of mast cells can provoke an ongoing local allergic reaction as long as antigen confrontation is maintained, a process which is sustained by further IgE production as well as renewal of mast cells. It is furthermore demonstrated that in certain established immune situations the IgE response may become independent of IL-4, namely in the spontaneous in vitro IgE expression of cells from atopic individuals as well as in an in vitro antigen-induced secondary IgE response of spleen cells derived from previously immunized mice. Thus, IgE-switched B cells may persist in vivo and may represent a pool of potentially IgE-producing cells. Finally, a selective inhibition of the IgE response is described in vitro and in vivo by the use of so-called non-anaphylactic monoclonal anti-IgE antibodies. Such antibodies bind to surface IgE+ B cells, but not to IgE-sensitized mast cells, and thereby inhibit IgE responses. Non-anaphylactic antibodies blocked the binding of allergen-specific IgE to mast cells by competing with the Fc epsilon on these cells. As a consequence they do not induce but rather prevent allergen-induced mediator release by mast cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Establishment of a memory in vitro murine IgE response to benzylpenicillin and its resistance to suppression by anti-IL-4 antibody.

Regulation of a memory IgE antibody response may be different from the induction of a primary response and may, therefore, be more relevant to the study of allergic diseases and the therapeutic manipulation of IgE antibody formation. In this paper a murine hapten-specific in vitro memory IgE antibody response to benzylpenicilloyl(BPO)-KLH is described. The response was analyzed by determining the number of antibody-producing cells (APC) in an ELISA spot assay. Of the total number of BPO-specific APC (10,000 APC/10(6) cultured spleen cells), about 1% were IgE-producing cells (100/10(6) cultured cells), as detected on day 6 of culture. The level of the antibody response is antigen dose-dependent, and the detected APC are BPO specific. The memory IgE response is not inhibited by the addition of anti-IL-4 antibody (11B11), even at a high excess. In the presence of the mitogen lipopolysaccharide, it has been shown that switch of B cells to IgE is induced by IL-4, a process which can be inhibited by anti-IL-4 antibody. Because the antigen-induced IgE response cannot be inhibited by anti-IL-4 antibody, in vitro responding cells derived from BPO-KLH-preimmunized mice may, therefore, have already switched in vivo to IgE. On the other hand, B cells switching to IgE in a situation of cognate T-B cell interaction might receive IL-4 in a transsynaptical way from T cells which might not be accessible to inhibition by anti-IL-4 antibody. The identification of the two possibilities in situations of established allergic disorders will be decisive for determining whether pharmacological inhibition of IL-4 (or IL-4-induced switch)--e.g., by putative low molecular weight compounds--will ever be a meaningful approach to suppress allergic diseases.

The cross-reactive immune response between infective larvae and adult worms of Acanthocheilonema (Dipetalonema) viteae is dominated by phosphorylcholine

Summary The immune response of BALB/c mice against living L3 or adult extract of Acanthocheilonema viteae induces three antibody populations, namely antibodies which cross‐react between the two forms of A. viteae, and either (i)do, or (ii) do not express specificity for phosphorylcholine (PC), and (iii) antibodies which do not cross‐react between the two stages. In the anti‐L3 serum, almost all cross‐reactive antibodies to adult antigen arc PC specific and of the IgM isotype, apart from a minor non‐PC‐reactive IgE response. On the other hand, the cross‐reactive antibodies in the anti‐adult scrum are not PC reactive and of the IgG3 isotype. The non‐cross‐reactive antibodies in the two sera were predominantly IgM and IgG2/IgG1 for the anti‐L3 and anti‐adult respectively. Immunofluorescence and immunocytochemical studies on third‐stage larvae and female worms revealed that antigens expressing PC determinants were mainly found on certain internal structures, whereas the cuticles of adults and the outer cuticle part of L3 are PC negative. Topographical analyses revealed distinct differences in labelling patterns by the two antisera, in the different areas. Thus the cross‐reactive and non‐cross‐reactive antibodies induced by BALB/c mice to the two stages of A. viteae are different in nature. Since the antibody response induced by the L3 form and cross‐reactive to the adult form is directed towards an ubiquitous antigen, namely PC, which is not present on cuticula of either the L3 or adult worms, this response may not contribute to protection against the parasite.

Regulation of immunoglobulin isotype expression in mice by antibodies in immune complexes

Fc-dependent regulation of humoral immune responses was investigated by immunization of BALB/c mice with immune complexes. These complexes were composed of DNP- and PC-conjugated KLH or Ficoll, and monoclonal T15 idiotype-positive anti-PC antibodies of different isotypes but indistinguishable V-region properties. Since the response to DNP was analyzed, effects due to masking of antigenic determinants by anti-PC antibodies are excluded. The responses to free and complexed antigens showed significant differences in the proportions of DNP-specific IgM, IgG1, IgG2 and IgG3 antibodies. Complexes containing the T-dependent carrier KLH elicited serum antibodies to DNP with significantly decreased IgM levels, irrespective of the isotype in the complex. Under these conditions, however, DNP-specific IgG classes were augmented to various extents, depending on the isotype in the complex. In the case of the T-independent carrier Ficoll, only complexes with IgM or IgG3 suppressed the IgM response. Moreover, immunization with complexes composed of IgM or IgG2a led to a significant decrease of DNP-specific IgG1. In contrast to changes induced by antibodies bound to the T-dependent antigen, immunization with T-independent complexes did not enhance the production of any of the immunoglobulin isotypes.

MODIFICATION OF THE INTERACTION OF HUMAN RENIN WITH DIFFERENT SUBSTRATES BY MONOCLONAL ANTIBODIES.

The mechanism by which anti-renin antibody inhibits renin activity was studied by following the kinetics of the reaction with angiotensinogen or a low molecular weight synthetic substrate, tetradecapeptide (TDP). Two monoclonal antibodies (70 pM) inhibited the production of angiotensin I from angiotensinogen but they differed when hog TDP was used as a substrate. R3-47-10 partially and non-competitively inhibited, whereas R3-36-16 stimulated the activity of renin. This is in contrast to the effects of the synthetic renin inhibitor, CGP 29 287, which competitively inhibits the enzyme activity with both substrates. These antibodies probably bind to the renin molecule on the flap which protects the active cleft. Angiotensinogen may be prevented from entering the cleft due to steric hindrance from bound antibody. However TDP, because of its smaller size may still be able to reach the catalytic site. In addition R3-36-16 might freeze the flap in an open position allowing a greater turnover of TDP whereas R3-47-10 may prevent the flap from fully opening and thereby hinder the reaction of TDP with the active site.

Monoclonal Antibody Secreting Mouse Hybridoma Clones Express Different Sets of Membrane Glycosphingolipids in Different Antigenic Systems

Abstract B-cell hybridomas developed for the secretion of monoclonal antibodis are also a rich source of membrane material for biochemical analyses of other than Ig,H-2 and Ia-antigens. Here, the glycosphingolipid labelling patterns of hybridomas are reported, which produced antibodies against streptococcal group A polysaccharides (ACHO), azobenzene arsonate (ABA) or phosphorylcholine (PC). While the Sp2/O tumour cell line revealed by high performance thin layer chromatography (HPTLC) only 3 ganglioside bands, hybridomas exhibited up to 50 additional GSL. In the streptococcal system at least three different GSL-patterns could be distinguished. These were independent of H-2, Ig-allotype, -class, and -subclass. Distinct GSL patterns were found for hybridomas of the ABA and PC systems without correlation to the major idiotypes. However, there is the indication that IgM and IgG antibody producing hybridomas show as a result of differentiation unique but distinct GSL patterns.