Suppression of established IgE antibody responses with isologous anti-idiotypic antibodies in guinea pigs

Guinea pigs of strains 2 and 13 can produce isologous anti-idiotypic (aIds) antibodies against anti-benzylpenicilloyl (anti-BPO) IgG, following immunization with affinity-purified anti-BPO antibodies of the same strain. The specificity of aId was determined by inhibition of binding of aId to Fab(t) in ELISA. The results showed that the reaction of strain 2 (anti-BPO)aId can be inhibited with syngeneic anti-BPO Fab(t) and to a smaller degree with anti-BPO Fab(t) of strain 13. On the other hand, strain 13 (anti-BPO)aId reacted exclusively with syngeneic anti-BPO Fab(t). In both cases, binding of aId to anti-BPO Fab(t) could not be inhibited with BPO-epsilon-aminocaproic acid, indicating that these aId are not directed against the antigen-combining site. The effect of isologous aId on both short- and long-time established IgE responses was studied in guinea pigs of strain 13. In both situations, administration of isologous aId resulted in suppression of the anti-BPO IgE antibody response. The suppressive effect was antigen-specific and lasted for several weeks: in the case of an early-response IgE remained suppressed despite additional booster injections of antigen. In contrast to the IgE response, the production of anti-BPO IgG antibodies was only slightly affected.     

Effect of passive immunization with purified specific or cross-reacting immunoglobulin G antibodies against Treponema pallidum on the course of infection in guinea pigs.

Whole immune serum or highly purified immunoglobulin G (IgG) antibodies to Treponema pallidum exhaustively adsorbed with three strains of nonpathogenic treponemes (TPI-IgG) were used for passive immunization of inbred strain 2 guinea pigs before and after intradermal challenge with 3.4 x 10(7) virulent T. pallidum Nichols organisms. Before challenge, control animals received a similarly purified IgG fraction containing either a cocktail of antibodies against three nonpathogenic treponemes (NPTI-IgG) or IgG prepared from normal guinea pig serum (NGPS-IgG). The purified fractions contained both IgG1 and IgG2 isotypes. The antibody levels (detected by fluorescent treponemal antibody test and enzyme-linked immunosorbent assay) and molecular specificities (immunoblot) of sera obtained from recipient animals before infection reflected those of the purified fractions used for immunization. Three protocols of passive immunization were used. Whole immune serum containing specific and cross-reacting antibodies afforded better protection than TPI-IgG even though asymptomatic animals were not fully protected. A single intradermal injection (0.1 ml) of TPI-IgG or NPTI-IgG into one hind leg 22 h before infection at the same site provided relatively higher protection than multiple intravenous injections (total, 15 ml) of the respective individual preparations. Since purified NGPS-IgG injected in the same animals, into the opposite hind leg, failed to protect against the challenging infection, it is reasonable to assume that specific and cross-reacting antitreponemal antibodies of the IgG1 subclass, which in guinea pigs are homocytotropic, play a relevant role in local protection.     

Experimental lung injury induced by trimellitic anhydride inhalation on guinea pigs.

Trimellitic anhydride (TMA) causes lung injury by inhalation exposure in humans. In order to investigate more precisely the mechanism of lung injury by TMA, an experimental animal model of TMA-induced lung injury was established. Guinea pigs were intramuscularly injected with trimellitylated bovine serum albumin (TM BSA) with complete Freund's adjuvant (CFA). Appreciable amounts of antibodies against TM epitopes were detected in the sera of these animals. Guinea pigs that were passively sensitized with anti-TM antisera as well as the actively immunized animals developed hemorrhagic pneumonitis after TMA inhalation. It is well recognized that hyperimmune antisera of guinea pigs contain two subclasses of IgG antibodies, IgG1 and IgG2, which are known as homocytotropic and heterocytotropic antibodies, respectively. To determine the role of these antibodies in the airway injury with alveolar hemorrhages, they were isolated by gel filtration and by ion exchange column chromatography, and the guinea pigs that were sensitized with each of these antibodies were exposed to TMA inhalation. The extent of lung injury in these animals was quantitatively determined by the measurements of lung extravasation of Evans blue dye injected intravenously after TMA inhalation. Significant increases in the extravasation of dye were observed in both animal groups sensitized with IgG1 and IgG2 antibodies. In addition, results obtained with heat-treated antisera and IgG1 antibody did not significantly differ from those obtained with untreated samples. These results suggest that the lung injury resulting from TMA inhalation exposure can be induced with humoral antibodies, not only IgG1 but also IgG2, and that at least two types of allergic reactions are involved in the pathogenesis of lung injury.     

Induction of T and B cell immunity by anti-idiotypic antibody

A small dose of the IgG1 fraction of anti-idiotypic antibody (aId1) raised in guinea pigs against a strain A/J antibody specific for streptococcal Group A carbohydrate sensitizes A/J mice against Group A streptococci. This is opposed to the previously established suppressive function of anti-idiotypic antibody of the IgG2 class (aId2). Correspondingly, aId1 but not aId2 is eliminated from the circulation in the way typical of an immunogenic molecule. However, the stimulatory component in the IgG1 fraction is not necessarily itself IgG1 antibody. Sensitization occurs in both B and helper T lymphocytes and is specific for Group A streptococci. In the B cell compartment sensitization is restricted to precursor cells expressing the idiotype. The concomitant activation of T helper cells therefore suggests that these cells make use of receptors with a similar or identical idiotype. Efficient sensitization by aId1 of both T and B cells is also demonstrated in strain C57L/J mice which upon immunization with Group A streptococci express a partially cross-reacting idiotype as a minor component. When such animals were primed with aId1, essentially all of the anti-carbohydrate antibody carried the partially cross-reacting idiotype.     

Mucosal immunization with a novel nanoemulsion-based recombinant anthrax protective antigen vaccine protects against Bacillus anthracis spore challenge

The currently available commercial human anthrax vaccine requires multiple injections for efficacy and has side effects due to its alum adjuvant. These factors limit its utility when immunizing exposed populations in emergent situations. We evaluated a novel mucosal adjuvant that consists of a nontoxic, water-in-oil nanoemulsion (NE). This material does not contain a proinflammatory component but penetrates mucosal surfaces to load antigens into dendritic cells. Mice and guinea pigs were intranasally immunized with recombinant Bacillus anthracis protective antigen (rPA) mixed in NE as an adjuvant. rPA-NE immunization was effective in inducing both serum anti-PA immunoglobulin G (IgG) and bronchial anti-PA IgA and IgG antibodies after either one or two mucosal administrations. Serum anti-PA IgG2a and IgG2b antibodies and PA-specific cytokine induction after immunization indicate a Th1-polarized immune response. rPA-NE immunization also produced high titers of lethal-toxin-neutralizing serum antibodies in both mice and guinea pigs. Guinea pigs nasally immunized with rPA-NE vaccine were protected against an intradermal challenge with approximately 1,000 times the 50% lethal dose ( approximately 1,000x LD(50)) of B. anthracis Ames strain spores (1.38 x 10(3) spores), which killed control animals within 96 h. Nasal immunization also resulted in 70% and 40% survival rates against intranasal challenge with 10x LD(50) and 100x LD(50) (1.2 x 10(6) and 1.2 x 10(7)) Ames strain spores. Our results indicate that NE can effectively adjuvant rPA for intranasal immunization. This potentially could lead to a needle-free anthrax vaccine requiring fewer doses and having fewer side effects than the currently available human vaccine.     

Specificity of antigens in aqueous phenol extracts of skin examined by means of guinea pig and rabbit immune sera.

Human, guinea pig and rabbit skin homogenates were digested with trypsin and extracted with phenol water. Antisera were raised in guinea pigs and rabbits by immunization with extract recovered from the water phase (TPW extract). All sera showed increased titres in indirect haemagglutination tests. The results of absorption and inhibition experiments indicated antibodies against a common antigenic determinant. These antibodies also agglutinated erythrocytes sensitized with autologous antigen. In addition, serum from rabbits immunized with human or guinea pig skin extract contained antibodies against species-specific determinants. Rabbit antiserum precipitated guinea pig skin extract. The antigen involved had specificity identical with that of an antigen in the human, but not in the rabbit skin, extract. Oxidation of the human TPW extract with periodate destroyed the precipitinogen and the species-specific haemagglutinogen while the common determinant was not affected.     

IgG2 antibodies block IgE antibody-induced asthma in guinea pigs

In order to examine the blocking activity of IgG2 antibodies to guinea pig for IgE antibodies-induced guinea pig asthma, experiments were carried out as follows. Guinea pigs were passively sensitized intravenously with guinea pig serum containing IgE antibodies to ovalbumin (OA). 8 days after sensitization, IgG2 purified from guinea pigs hyperimmunized with OA was intravenously injected. One hour later, the guinea pigs were challenged by inhalation of OA solution. Asthma attacks were not observed in the guinea pigs, whereas the attacks were observed in guinea pigs passively sensitized with the IgE antibodies but injected IgG2 fraction from normal guinea pigs 1 h before inhalation. These observations suggested that IgG antibodies that increased after immunotherapy might block asthma caused by inhalation of allergens in humans.     

Anterior hypothalamic lesions decrease anaphylactic contractions in guinea pig trachea in vitro by reducing histamine and LTC4 reactivity

Discrete lesions in the anterior hypothalamus (AHA) of the guinea pig brain reduce the anaphylactic contraction of the trachea in vitro after active in vivo sensitization by 40%. This difference in anaphylactic contraction does not correlate with a difference in homocytotropic antibodies but coincides with a decreased smooth muscle response to the anaphylactic mediators histamine and leukotriene C4. No difference in the beta-adrenoceptor function of the tracheal preparations can be found. The results suggest that AHA lesions afford protection against anaphylaxis in actively sensitized guinea pigs at least in part through a reduced smooth muscle response to anaphylactic mediators.     

Studies on the heterogeneity and idiotypic specificity of anti-dinitrophenyl antibodies from inbred guinea pigs

The heterogeneity of antibodies raised to 2,4-dinitrophenyl keyhole limpet hemocyanin (DNP-KLH) in inbred guinea pigs was investigated by isoelectric focusing (IEF) techniques. Some, but not all, of immunized strain 2 (Heston) and strain 13 guinea pigs produced antibodies giving restricted banding patterns, detectable by protein staining, in the basic region of IEF gels. In all cases studied, these antibodies were directed against the DNP group. Their banding patterns resembled those given by myeloma proteins from mice or electrophoretically restricted antibodies from rabbits. Antibodies in at least four pairs of sera from strain 2 animals gave very similar banding patterns on IEF gels. Immunoglobulin fractions with anti-DNP activity, which gave indistinguishable, restricted banding patterns, were isolated from two individual animals, and used to prepare anti-idiotypic antisera in rabbits. The fractions were shown to be idiotypically cross-reactive, but not identical, by passive hemagglutination and hemagglutination inhibition techniques. Purified anti-DNP antibodies from some, but not all strain 2 guinea pigs, and antibodies in sera from strain 2 and strain 13 guinea pigs immunized with DNP-KLH, were shown to be idiotypically cross-reactive in the hemagglutination inhibition system.     

In vivo and in vitro immunological cross-reactions between basic encephalitogen and synthetic basic polypeptides capable of suppressing experimental allergic encephalomyelitis

A significant extent of immunological cross-reactivity has been demonstrated between the basic encephalitogenic protein of bovine origin and several synthetic amino acid copolymers which have suppressive effect on experimental allergic encephalomyelitis (EAE). This cross-reactivity has been conclusively established on the cellular level, both in vivo by means of delayed hypersensitivity skin tests and in vitro using transformation of sensitized lymphocytes, as measured by incorporation of radioactive thymidine. The in vitro experiments have been conducted with lymph node cells from guinea pigs of both random bred and inbred strains, and on spleen and lymph node cells from rabbits. Definite cross-reactivity was observed between the basic encephalitogen and all the synthetic copolymers which were previously shown effective in suppression of EAE, whereas ineffective copolymers or unrelated proteins did not show any cross-reactivity. In the case of strain 2 guinea pigs and rabbit lymph node cells the cross-reactivity in vitro was manifested by direct cross-stimulation of the lymphocytes, whereas in random-bred or strain 13 guinea pigs and rabbit spleen cells, the cross-reaction was detected only by means of specific inhibition of the homologous stimulation by the heterologous antigen. A limited extent of cross-reactivity was observed on the humoral level as well; antibodies provoked in guinea pigs against the synthetic copolymer Cop 1 cross-reacted in the passive cutaneous anaphylaxis assay with the bovine basic encephalitogen.     

Suppression of collagen arthritis in rats by heterologous anti-idiotypic antisera against anticollagen antibodies

Affinity-purified rat anti-type II collagen antibodies were used to prepare anti-idiotypic antibodies in rabbits. It has been demonstrated that such anti-idiotypic antibodies are capable of binding to anti-type II collagen antibodies in vitro. Intravenous administration of heterologous anti-idiotypic antisera at the time of immunization with type II collagen resulted in a significant suppression of anti-type II collagen antibody formation and the development of arthritis, although delayed-type hypersensitivity skin test response to type II collagen was not affected. However, treatment of rats with heterologous anti-idiotypic antisera at Day 7 after immunization was ineffective in altering disease expression. On the other hand, treatment with heterologous anti-idiotypic antisera had no significant suppressive effect on the incidence or severity of adjuvant arthritis. These results indicate that the effect of heterologous anti-idiotypic antisera directed toward anti-type II collagen antibodies is disease specific and is restricted to collagen arthritis.     

The use of hapten-polysaccharide conjugates for the induction of B-cell tolerance involving IgE responses. III. Specific tolerance induced by sulphonamide-substituted levan in the guinea pig.

Inbred strain 2 and (strain 2 x strain 13)F1 guinea pigs were made allergic by intraperitoneal injections of sulphonamide-chicken gamma-globulin (CGG) conjugates. Two sulphonamides were used: 4-sulphanilamide benzoic acid (4-SABA) and sulphamethoxazole (SMX). 4-SABA was coupled to CGG through its carboxylic group and SMX was coupled following diazotization of its sulphanilamide group. The anti-4-SABA and anti-SMX reaginic antibodies formed did not show any cross-reactivity with each other. Injection of 4-SABA coupled to native levan effectively suppressed the allergic responses of these guinea pigs when given either prior to or after immunization with 4-SABA-CGG. This treatment is specific as it did not affect anti-SMX or anti-CGG reaginic responses. Guinea pigs seem more sensitive to the regime used for tolerance induction than correspondingly sensitized mice in that less tolerogen is required on a body weight basis.     

Histocompatibility antigens and genetic control of the immune response in guinea pigs. II. Specific inhibition of antigen-induced lymphocyte proliferation by anti-receptor alloantisera

The in vitro recognition of acetylsalicilyl ovalbumin, penicilloylated bovine IgG (BPO-BGG) and the multichain copolymer poly-L-(Tyr, Glu)-poly-D L-Alapoly-L-Lys [(T, G)-A-L] by primed (2 x 13) F1 cells could be markedly and specifically blocked by alloantisera directed against "receptors" (R) or recognition structures for these antigens. The antisera were raised against lymphoid cells from various F2 hybrid guinea pigs of (2 x 13) histocompatibility type in which responsiveness to low doses of acetylsalicylic acid anhydride, BPO-BGG and (T, G)-A-L segregated independently of each other. One of the genes required for the expression of high responsiveness appears to be linked to the 13 MHC genes. The anti-R antisera did not contain antibodies capable of interacting directly with the stimulating antigen since passage of the sera through antigen-immunoadsorbent columns did not remove antibodies capable of suppressing in vitro recognition. Furthermore, the antisera did not appear to contain cytotoxic antibodies directed against specific antigen-sensitive cells. The activity of the anti-R sera was highly specific: their inhibitory activity could only be absorbed by lymphoid cells bearing the specific recognition structures for the appropriate antigen. Preliminary experiments yield no indication that the anti-R antisera are specific for immunoglobulin idiotypes.     

Modulation of immune response to Lol p I by pretreatment with anti-idiotypic antibody is not restricted to the idiotypic expression.

To study the role of anti-idiotypic antibodies in the regulation of the immune response to Lol p I (the major allergenic component of rye grass pollen), we have recently generated a panel of three MoAbs directed against distinct epitopes of Lolp I and an anti-idiotypic MoAb directed against the idiotype borne by one of the anti-Lol p I MoAbs (290A-167). The effects of pretreatment with this anti-idiotypic MoAb in BALB/c mice before immunization with the antigen have been examined. The anti-idiotypic MoAb or unrelated MoAb were given weekly for 8 weeks intraperitoneally. Mice then received the antigen (2 micrograms) adsorbed with alum (2 mg) at weeks 9, 11 and 13. Serum anti-Lol p I antibodies (IgG or IgE) and specific idiotypic responses were measured. Anti-Lol p I IgG antibodies could be detected before immunization with Lol p I only in mice pretreated with anti-idiotypic MoAb. Immunization with Lol p I induced an anti-Lol p I IgG response in both groups, but this response was higher in mice that received anti-idiotypic MoAb. Similar profiles were seen for specific IgE antibodies and idiotypic responses. Surprisingly, idiotypes borne by other anti-Lol p I MoAbs (539A-6 and 348A-6) had also been enhanced after pretreatment with the anti-290A-167 MoAb. These observations suggested that the pretreatment with this anti-idiotypic MoAb modulates not only the expression of the respective idiotype, but also affects other idiotype responses.     

Biological and serological comparison of syngeneic and allogeneic anti-idiotypic antibodies

The majority of BALB/c antibodies with specificity for the (1→3)-glucosidic linkage of dextran B1355, fraction S (-dextran), share an idiotype crossreactive with the -dextran-binding BALB/c myeloma proteins J558 and MOPC104E. This crossreactive idiotype (IdX558) is denned by an allogeneic antiidiotypic antiserum raised in mice of strain A/HeJ against J558 myeloma protein (Carson & Weigert, 1973). Allogeneic anti-IdX558 antisera induce a long lasting complete suppression of the total anti--dextran immune response when given neonatally to BALB/c mice. Mice which recover from suppression fail to express the IdX558. In contrast, when BALB/c mice are immunized by use of J558 myeloma protein, no measurable effect on the subsequent total anti--dextran immune response or on the expression of IdX558 is found in mice making anti-J558 themselves nor in those mice which received the syngeneic anti-J558 idiotype antibody neonatally. In addition, no significant alteration of the anti--dextran response can be detected in BALB/c mice which were immunized against MOPC104E protein prior to -dextran immunization. This different suppressive capacity may be explained by different specificities of the allogeneic and syngeneic antiidiotype antisera. Hemagglutination and isoelectric focusing studies of the antisera show that BALB/c mice fail to produce antibodies against the crossreactive idiotype whereas in the allogeneic situation the anti-IdX558 is the overwhelming one. Syngeneic anti-idiotypic antibodies react only with the corresponding myeloma protein used for immunization, thus defining individual idiotypes (IdI558 and IdI104). IdI558 and IdI104 are expressed by every BALB/c mouse in response to -dextran but as a minor component of their idiotypic spectrum.     

Production and isolation of guinea pig IgE antibody

Immunoglobulins of the IgE and IgG classes have been causally associated with hypersensitivity reactions in man and in numerous animal species including mice, rats and guinea pigs. The use of the guinea pig as an animal model for both pulmonary and dermal hypersensitivity reactions, and the recent recognition of the importance of IgE antibodies in both early- and late-onset hypersensitivity responses, has heightened interest in production, separation, and isolation of this immunoglobulin class from the guinea pig. IgE antibodies were produced by treatment of strain 13 guinea pigs with cyclophosphamide followed by injection with S. aureus enterotoxin. Serum was obtained and the globulin fraction isolated by addition of caprylic acid then ammonium sulfate. Immunoglobulins were separated into classes using fast protein liquid chromatography (FPLC) employing a Mono Q column and a linear gradient of 0.01-0.3 M Na,K phosphate buffer, pH 7.5 (buffer B). IgG eluted in two major peaks. IgG2 was not retained on the column and emerged with the starting buffer; IgG1 was eluted with 15-20% buffer B. IgE, detected as heat labile homocytotropic antibody, was found in the fraction eluting with 30-35% buffer B. The elution profile of the guinea pig immunoglobulins was predicted from the pattern obtained with immunoglobulin classes from other species. This chromatographic procedure enabled rapid isolation of immunoglobulin classes from guinea pig sera and effectively separated IgG1 from IgE, the two classes associated with hypersensitivity reactions.     

Modulation of the immune response by passive antibodies. I. Anti-hapten antibodies enhanced delayed hypersensitivity to the carrier and depressed antibody synthesis to the hapten.

The modulating effects of passive antibodies on both delayed hypersensitivity to the carrier and antibody synthesis to carrier and hapten determinants were studied in guinea pigs. Animals were injected with antibodies directed against either the carrier or the hapten prior to immunization with the hapten-carrier conjugate in Freund's complete adjuvant. Anti-hapten antibodies have been shown to have an enhancing effect on delayed hypersensitivity to the carrier and a suppressive effect on antibody synthesis to the hapten. In this experiment, anti-carrier anti-bodies seemed to have had no effect on delayed hypersensitivity to the carrier and on antibody synthesis to the hapten.     

Estimation of guinea pig antigen-specific and non-specific suppressor cell activity

A functional assay for the quantitative estimation of suppressor cell (SC) activity in guinea pigs has been developed. Cultures of antigen-stimulated peripheral blood lymphocytes (PBL) from sensitized guinea pigs develop SC activity. The suppression of proliferation can be demonstrated in antigen-stimulated autologous co-cultures of precultured and freshly isolated PBL. The extent of suppression is dependent on the preculture antigen concentration but not the preculture period and it consists, as demonstrated with PBL from doubly sensitized guinea pigs, of an antigen-specific and a non-specific component. The observed SC activities were not due to an alteration of the kinetics of the co-cultures. The estimates of suppression are highly dependent on corrections for the values of the control cultures. The present method may prove useful in immunological studies of mycobacterial infections in guinea pigs.     

Neonatal colonization of rats induces immunological tolerance to bacterial antigens

We wanted to investigate the immunological events occurring in rats intestinally colonized from birth (neonatally) or at adult age with an ovalbumin (OVA)-producing Escherichia coli O6K13 strain, carrying type 1 pili. The neonatally colonized animals responded with lower delayed type hypersensitivity (DTH) against OVA and lower levels of IgG antibodies against OVA, O6 lipopolysaccharide (LPS) and type 1 pili compared to age-matched controls. The IgG antibody response against the bystander antigen, human serum albumin (HSA), was lower in the neonatally colonized animals than in the controls co-immunized with HSA and E. coli, indicating a release of suppressive factors induced by the bacterial antigens. The adult colonized animals showed an increased DTH and antibody response against OVA after immunization. They also had high pre-immunization levels of IgG anti-O6 LPS antibodies compared to controls. However, the relative increase in IgG anti-O6 LPS antibody levels after the immunization with dead E. coli was much lower in the adult colonized animals. The present results suggest that neonatal animals develop tolerance against antigen on bacterial colonizers of the intestine. In addition, this tolerance contains components of suppression.     

Elicitation of homologous passive cutaneous anaphylactic reactions by a benzylpenicillin preparation

Four out of five commercially available benzylpenicillin preparations elicited homologous passive cutaneous anaphylactic (PCA) reaction in sensitized guinea pigs with anti-benzylpenicilloyl (anti-BPO) reaginic sera. The same preparations could not evoke PCA reaction in sensitized guinea pigs with anti-BPO γ₁ homocytotropic antibodies. The PCA reactions were completely inhibited by a prior injection of BPO--aminocaproic acid (BPO-EACA). Chromatographic analysis of one of the benzylpenicillin preparations on Sephadex G 10 revealed that the reagin-mediated PCA reaction was not evoked with the fractions from the main peak of the benzylpenicillin but with fractions eluted earlier. None of the fractions gave positive γ₁-mediated PCA reactions. These results indicated that some commercial benzylpenicillin preparations contained minute amounts of the impurities that could elicit the homologous PCA reaction in guinea pigs sensitized with anti-BPO reaginic sera. It was also indicated that the PCA elicitation activity of the benzylpenicillin preparation in the system of reagin-mediated PCA differed from that of γ₁-mediated PCA.