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At least 19 recordsLinked to original sources
PubMed1975

Stimulation and inhibition of anti-hapten responses in guinea pigs immunized with hybrid liposomes.

Guinea pigs were immunized with liposomal model membranes containing phosphatidylethanolamine (PE) or glycerophosphorylethanolamine (GPE) derivatives in which the amino function was substituted with either dinitrophenylaminocaproyl (Dnp-Cap) or mono(p-azobenzenearsonic acid)tyrosyl (ABA-Tyr) residues. Previous studies have demonstrated that hapten-specific antibodies are elicited by DNP-Cap-PE or ABA-Tyr-PE sensitized liposomes and that cell-mediated immunity is induced by ABA-Tyr-PE (but not Dnp-Cap-PE) sensitized liposomes. These liposomes differ from conventional immunogens in which haptens are covalently attached to immunogenic carriers. This investigation describes two new aspects of liposomal immunogenicity in animals immunized with hybrid liposomes containing both Dnp-Cap-PE and ABA-Tyr-PE. (1) Stimulation of the anti-Dnp response by incorporation of increasing amounts of ABA-Tyr-PE; (2) inhibition of anti-ABA antibody formation by incorporation of increasing amounts of DNnp-Cap-PE. The two phenomena are dependent on the presence of each determinant in the same lipid bilayer. Thus, entrapment of the water-soluble deacylated derivative of ABA-Tyr-PE (i.e., ABA-Tyr-GPE) in a aqueous compartments of Dnp-Cap-PE sensitized liposomes does not enhance anti-Dnp antibody production. Similarly, entrapment of the non-amphipathic derivative of DNP-Cap-PE (i.e., Dnp-Cap-GPE) within ABA-Tyr-PE sensitized liposomes does not suppress anti-ABA antibody formation. Furthermore, mixtures of Dnp-Cap-PE sensitized liposomes and ABA-Tyr-PE sensitized liposomes neither stimulated nor inhibited the anti-hapten responses. These results indicate that preparation of hybrid liposomes with different N-substituted PE derivatives provides an extremely convenient method for controlling hapten and/or immunologic carrier determinant density.

Aminocaproates↗
PubMed1976

Comparative immunogenic properties of N-substituted phosphatidylethanolamine derivatives and liposomal model membranes.

This study describes some of the parameters that quantitatively or qualitatively influence the immunogenicity in guinea pigs of synthetic lipid antigens: phosphatidylethanolamine (PE) derivatives in which the amino (N) group has been substituted with either dinitrophenyl (DNP), dinitrophenylaminocaproyl (DNP-Cap), fluoresceinthiocarbamyl (Fl), or mono (p-azobenzenearsonic acid) throsyl (ABA-Tyr) residues. Previous experiments have shown that the non-covalent insertion of DNP-Cap-PE and ABA-Tyr-PE into the same lipid bilayers of sphingomyelincholesterol-dicetylphosphate liposomes markedly enhanced anti-DNP-Cap antibody formation over that produced by liposomes sensitized with only DNP-Cap-PE. The humoral response to Fl-PE and CNP-PE-sensitized liposomes is also augmented by the simultaneous incorporation of ABA-Tyr-PE. Moreover, micelles containing both DNP-Cap-PE and ABA-Tyr-PE induce more antibodies to the DNP-Cap deteminant than do micelles of DNP-Cap-PE alone, or a mixture of DNP-Cap-PE and ABA-Tyr-PE micelles. Nevertheless, in regard to a humoral response, liposomes were more potent immunogens than were their micellar counterparts. Of all the N-substituted derivatives examined so far, ABA-Tyr-PE is unique in that it can elicit cell-mediated immunity in addition to antibodies. The cellular response to ABA-Tyr-PE is not, however, stimulated by incorporation into liposomal bilayers and requires administration of either micelles or liposomes in complete Freund's adjuvant. In contrast, the ability of ABA-Tyr-PE to enhance a humoral response to another N-substituted PE derivative present in the same immunogen is also observed when the latter are given with incomplete Freund's adjuvant. The relationship of these findings to the immunogenicity of naturally occurring lipid antigens, as well as conventional immunogens having at least one determinant covalently attached to a protein carrier is discussed.

Adjuvants, Immunologic↗
PubMed1977

Two str1cturally similar haptens each induce a different inherited idiotype.

When mice of the A/J strain were immunized with azobenzenearsonate (ABA) directly coupled to a protein, they produced antibodies that share an inherited cross-reactive idiotype (CRI). In the antigenic determinant that induces CRI, ABA is very probably coupled to tyrosine, and the structure that protrudes from the polypeptide backbone has two benzene rings and a molecular weight of 419. When this same structure is separated from the polypeptide backbone by a spacer of 99 daltons, it induces a different inherited idiotype (ABA-HOP-e) in the same strain of mice. Our data suggest that antibodies with the idiotype CRI recognize the terminal benzene ring and the azo group, but do not fit closely around the second ring structure. Antibodies with the idiotype ABA-HOP-e have fine specificity for both benzene rings. Both idiotypes are inherited and both are linked to genes controlling constant regions of the heavy chains.

Animals↗
PubMed1978

T-lymphocyte activation by immunogenic determinants.

Synthetic antigens have been of great value in elucidating the relationships between antigen structure and lymphocyte activation. The compound RAT behaves as a monofunctional antigen in guinea pigs and mice, inducing T-lymphocyte responses without appreciable circulating antibody, although the ABA-specific B cell population is expanded by immunization with the monovalent molecule. On the other hand, bifunctional antigens composed of one RAT moiety serving as a carrier and a second chemical group, either identical to or different from RAT, serving as a hapten, induced antibody responses. In such responses, T cell specificity was always directed against the RAT component. Using symmetrical bifunctional antigens with rigid or flexible spacers between the two determinants, marked differences in structural requirements for cell triggering, assessed by antigen-induced lymphocyte proliferation, and for cell cooperation, determined by antibody formation, were found. Rigidly spaced bifunctional antigens serve admirably for cooperation but poorly for T cell activation, underscoring the advantage of two-point binding for the latter.

Animals↗
PubMed1979

Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III).

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

Amino Acids↗
PubMed1975

Intramolecular arsanilazotyrosine-248-Zn complex of carboxypeptidase A: a monitor of catalytic events.

The intensely chromophoric intramolecular coordination complex formed between arsanilazotyrosine-248 and the active site zinc atom of azocarboxypeptidase A (Johansen, J. T. & Vallee, B. L. (1971) Proc. Nat. Acad. Sci. USA 68, 2532-2535) is a spectrokinetic probe of catalytic events. The interconversion of the azoTyr-248-Zn complex and its constituents is measured by stopped-flow pH and temperature-jump methods. The rate of interconversion, 64,000 sec-1, is orders of magnitude faster than that of the catalytic step itself (about 0.01-100 sec-1). Rapidly turned over peptide and ester substrates disrupt the azoTyr-248-Zn complex before hydrolysis occurs. As a consequence, formation of azoTyr-248, substrate binding, and catalysis can all be monitored while catalysis is actually in progress. The results of these dynamic studies specify a course of catalytic events, different from those postulated based on x-ray structure analysis. If azoTyr-248 is displaced, the direction is opposite to the inward movement postulated on the basis of x-ray studies and is not unique to induction by substrates, since rapid changes in pH also result in analogous spectral changes. AzoTyr-248 carboxypeptidase has all the features which are essential for mechanistic studies: (1) It is enzymatically active; (2) the spectra of the metal complex differ characteristically from those of its constituents; (3) it responds dynamically to environmental factors; and (4) the response time of the probe itself is much more rapid than is required for the measurement of the catalytic step. These combined kinetic and spectral properties of the metal complex render it a powerful spectrokinetic probe to visualize and discern microscopic details of the catalytic process.

Arsenicals↗
PubMed1977

Binding to idiotypic determinants of large proportions of thymus-derived lymphocytes in idiotypically suppressed mice.

All A/J mice immunized with a conjugate of p-azophenylarsonate groups to keyhole limpet hemocyanin produce antibodies against azophenylarsonate, some of which share a crossreactive idiotype. The appearance of the idiotype can be suppressed, without reducing the response against azophenylarsonate, by injecting rabbit anti-idiotypic antibodies prior to immunization. We have now observed that mice suppressed in this way, or by adoptive transfer of leukocytes from other suppressed mice, and then immunized with the hemocyanin-azophenylarsonate conjugate, possess high proportions (up to 14%) of lymphocytes that form rosettes with A/J erythrocytes coated with Fab fragments possessing the idiotype. Idiotypic specificity was demonstrated by various experiments. Most of all of the rosette-forming lymphocytes appear to be thymus-derived lymphocytes (T cells). Treatment of T cells with trypsin eliminated the capacity to form rosettes, which was restored on standing overnight in medium. Thus, the receptors are synthesized by the cells and are not passively adsorbed. Treatment of mice with anti-idiotypic antiserum without antigenic stimulation did not elicit substantial numbers of rosette-forming cells. The requirement for antigen suggests that antigen-idiotype complexes may be a stimulatory agent. A prolonged rest period after immunization of suppressed mice was required for the induction of high percentages of rosette-forming cells. Rosette formation provides a convenient method for studying factors that induce the formation of idiotype-specific T cells.

Animals↗
PubMed1978

Purification of functional, determinant-specific, idiotype-bearing murine T cells.

Strain A/J mice immunized with azobenzenearsonate (ABA)-mouse IgG conjugates develop suppression for anti-trinitrophenyl(TNP) responses to doubly conjugated (ABA,TNP) proteins. This suppression is specific for the ABA epitope and is mediated by T cells in cell transfer experiments. ABA-binding T cells from suppressed animals were purified by a two-stage procedure in which B cells were removed from spleen cell populations by adherence to plastic surfaces coated with anti-mouse Ig antibody, followed by binding the nonadherent population (more 95 percent Thy-1-positive) to surfaces coated with ABA-protein conjugates. Approximately 90 percent of the cells recovered by temperature-dependent elution from the ABA plates (similar to 2 percent of the spleen cells) bound antigen immediately afterward, and up to 50 percent of the cells bound anti-cross-reactive idiotype antibody. On the other hand, the nonadherent T-cell population was completely negative in the antigen- binding and idiotype assays. Another distinguishing feature of the two T-cell populations was that 78 percent of the adherent cells, but only 2 percent of the nonadherent cells, were Ia positive, although the specific I-region marker(s) expressed on the cells was not identified. The biological function of the antigen-binding T cells was investigated using a standard cell transfer protocol. Suppressor cells were enriched in the adherent population by a factor of at least 25, establishing that functional, epitope-specific, idiotype-bearing T cells can be significantly purified by this procedure. Note Added in Proof. We have recently isolated two types of ABA-binding molecules biosynthetically labeled with (35)S-methionine from NP-40 lysates of purified antigen-specific T cells. The molecules were purified by adsorption onto an ABA-Sepharose immunoadsorbent followed by elution with 9 M urea. Autoradiograms of SDS-PAGE of the eluates revealed components with tool wt of approximately 60,000 and 33,000 dahons. These molecules were not present in eluates from a bovine IgG-Sepharose control immunoadsorbent and thus represent specific ABA-binding products synthesized by T cells.

Animals↗
PubMed1979

Mechanisms of regulation of cell-mediated immunity. IV. Azobenzenearsonate-specific suppressor factor(s) bear cross-reactive idiotypic determinants the expression of which is linked to the heavy-chain allotype linkage group of genes.

T-cell derived suppressor factor(s) (SF) specific for azobenzenearsonate (ABA) were prepared by the mechanical disruption of suppressor cells. Such suppressor factors were adsorbed to and recovered from immunoadsorbents prepared from the F(ab')2 fragments of rabbit immunoglobulin directed against the cross-reactive idiotype of A/J anti-ABA antibodies. These ABA-suppressor factors were not retained on Sepharose 4B immunoadsorbent columns which had been coupled with F(ab')2 fragments or normal rabbit immunoglobulins prepared from prebleeds of rabbits used to make anti-idiotypic antiserum. The specificity of the F(ab')2 rabbit anti-idiotypic serum was established by direct idiotypic-binding assays and by affinity purification over an immunoadsorbent consisting of CRI+ anti-ABA immunoglobulin from A/J mice. ABA-suppressor factors were shown to be specifically absorbed and eluted from F(ab')2 anti-idiotypic columns. Futhermore, the eluted suppressor factor can be specifically reabsorbed and recovered from a second anti-idiotypic immunoadsorbent. The concordance between antigen-binding specificity and the presence of idiotypic determinants was demonstrated by adsorbing ABA SF to antigen columns and then fractionating the ABA-specific factor on anti-idiotypic immunoadsorbents. ABA-suppressor factors were shown to be specifically retained on immunoadsorbents directed against major histocompatibility complex (MHC) determinants. Factor eluted from anti-MHC columns could then be specifically adsorbed to anti-idiotypic immunoadsorbents. This suggests that the same molecular complex that is recognized by the H-2 alloantiserum is specifically adsorbed to an anti-idiotypic immunoadsorbent. Genetic analysis of the expression of CRI+ suppressor factor was performed using the C.AL-20 mouse strain which has the AL/N allotype and produces CRI+ anti-ABA immunoglobulins. The implication of these findings to the nature of T-cell-derived regulatory molecules is discussed.

Animals↗
PubMed1979

Suppression of idiotype and generation of suppressor T cells with idiotype-conjugated thymocytes.

Inoculation of A/J mice with syngeneic thymocytes conjugated with specifically purified A/J anti-phenylarsonate (anti-Ar) antibodies, selectively suppressed the subsequent synthesis of those anti-Ar antibodies which carry the major cross-reactive idiotype. High titers of anti-Ar antibodies were produced upon subsequent immunization but in most mice the idiotype was undetectable. Suppression similarly occurred in F1(A/J X BALB/c) and in C.AL-20 mice. Although some mice were suppressed when unconjugated antibody was injected, the suppressive effect was much more pronounced, particularly in the F1 and C.AL-20 recipients, when the antibody was coupled to thymocytes. The state of suppression could be adoptively transferred with T cells to mildly irradiated syngeneic recipients. A population enriched for B cells had little if any suppressive effect. There was no requirement for antigen in the generation of suppressors. Thymocytes conjugated with antibody did not induce idiotype-specific suppression in mice that had been recently challenged with antigen. Thymocytes from BALB/c and C57BL/10 mice were effective carriers for the anti-Ar antibodies, i.e., there was no evidence for H-2 restriction. The experiments demonstrate the feasibility of suppressing idiotype production and generating idiotype-specific suppressor T cells without the use of anti-idiotypic antibody or antigen.

Animals↗
PubMed1979

Antigen- and receptor-driven regulatory mechanisms. I. Induction of suppressor T cells with anti-idiotypic antibodies.

Delayed-type hypersensitivity (DTH) to the azobenzenearsonate (ABA) hapten can be readily induced in A/J mice injecting ABA-coupled syngeneic spleen cells subcutaneously. To further characterize this T-cell-dependent immunological phenomenon, the effect of passively administered anti-cross-reactive idiotype common to anti-ABA antibodies of A/J mice (CRI) antibodies on the development of ABA-specific DTH was investigated. Animals given daily injections (of minute amounts) of anti-CRI antibodies subsequent to immunization with ABA-coupled cells show significant reduction of ABA specific responses. This inhibition is antigen specific and requires the intact immunoglobulin molecule, as F(ab')2 treatments were ineffective in suppressing the reaction. Investigations of the mechanism of the anti-CRI-induced suppression of ABA DTH revealed that the observed suppression is a result of the activation of suppressor cells. Spleen cells taken from animals which received anti-CRI antibodies were able to adoptively transfer suppression to naive recipients. This suppression was shown to be mediated by T cells, as anti-Thy1.2 plus complement completely abrogated the transfer of suppression. In addition, animals pretreated with low doses of cyclophosphamide were not suppressed by the administration of anti-CRI antibodies. The genetic restriction of anti-CRI-induced suppression was demonstrated. Antibodies to the major cross-reactive idiotype, (CRI) associated with anti-ABA antibodies in A/J mice were unable to suppress the development of DTH to ABA in BALB/c mice (H-2d, Igh-1a). Such antibodies were, however, fully active in suppressing ABA DTH in the allotype-congenic C.AL-20 strain which has an allotype (Igh-1d) similar to that of A/J (Igh-1e) on a BALB/c background, and which produces humoral antibodies with the CRI.

Animals↗
PubMed1979

Antigen- and receptor-driven regulatory mechanisms. II. Induction of suppressor T cells with idiotype-coupled syngeneic spleen cells.

Anti-p-azobenzenearsonate (ABA) antibodies, coupled covalently to normal syngeneic spleen cells and then given intravenously to normal animals, were found to be potent tolerogens for delayed-type hypersensitivity (DTH) to ABA. The ability of the antibody-coupled cells to induce tolerance was determined to be a result of the cross-reactive idiotype (CRI+) fraction of the antibodies, because anti-ABA antibodies lacking the CRI+ components when coupled to spleen cells were unable to cause any significant inhibition. Furthermore, genetic analysis revealed that the ability of CRI-coupled cells to inhibit ABA-specific DTH is linked to Igh-1 heavy chain allotype, in as much animals which possess heavy chain allotypes similar to that of A/J were sensitive to this inhibition. Adoptive transfer experiments provided evidence that CRI-coupled cells induce suppressor cells, and spleen cells or thymocytes from animals received CRI-coupled cells were able to transfer suppression to naive recipients. In addition, treatment with anti-Thy1.2 serum plus complement completely abrogated their ability to transfer suppression. Thus, this active suppression is a T-cell-dependent phenomenon. In investigating the specificity of these suppressor T cells, it was found that they functioned in an antigen-specific manner and were unable to suppress the development of DTH to an unrelated hapten 2,4-dinitro-1-fluorobenzene.

Animals↗