Cardiac anaphylaxis in isolated guinea pig hearts perfused at constant flow or constant pressure

Acute responses to antigen-antibody interactions (anaphylactic reactions) in isolated guinea pig hearts are reported to include decreases in coronary flow, increases in heart rate, prolongation of impulse propagation, development of arrhythmias, and transient increases followed by substantial decreases in ventricular contractile force. It is not clear from these studies, however, whether all of the changes are direct effects of the mediators released by the antigen-antibody reaction or whether some of them are indirect results of the severe reduction in flow evoked by coronary vasoconstriction. Therefore, the present study was designed to assess cardiac anaphylactic events in isolated hearts of guinea pigs passively sensitized with IgG antibody to ovalbumin under conditions in which coronary perfusion pressure was maintained constant and to compare the responses to those of hearts in which coronary flow was maintained at a constant rate. Our data indicate that when coronary flow decreased during anaphylaxis (constant pressure perfusion), hearts responded to antigen challenge with greater prolongation of the PR interval, duration of arrhythmias, suppression of left ventricular systolic pressure, and release of histamine and adenosine plus inosine into the venous effluent than when coronary flow was maintained during anaphylaxis (constant flow perfusion). The data suggest that maintenance of coronary flow during cardiac anaphylaxis may attenuate the severity of the functional derangement.     

A plant histaminase modulates cardiac anaphylactic response in guinea pig

The effect of a copper amine oxidase (histaminase) purified from the pea seedling as free or immobilized enzyme on the response to specific antigen was studied in isolated hearts from actively sensitized guinea pigs. In vitro challenge with the specific antigen of hearts from actively sensitized animals evokes a positive inotropic and chronotropic effect, a coronary constriction, followed by dilation and an increase in the amount of histamine and nitrites, the oxidation product of nitric oxide, in the perfusates. In the presence of both forms of histaminases, the positive inotropic and chronotropic responses as well as the coronary constriction and the release of histamine were fully blocked. The amount of nitrites, appearing in the perfusates when anaphylaxis is elicited in the presence of both forms of histaminases, is significantly increased, as well as nitric oxide synthase activity and cyclic GMP content in cardiac tissue, while cardiac calcium overload was significantly prevented. These observations demonstrate that the decrease in the anaphylactic release of histamine and the subsequent abatement of the cardiac response to antigen can be accounted for by the inactivation by histaminase of the released histamine and by a stimulation of endogenous nitric oxide production.     

O3-induced airway hyperresponsiveness to noncholinergic system and other stimuli.

The effect of O3 exposure (3 ppm, 1 h) on the in vivo and in vitro airway responsiveness, as well as the changes in cell contents in bronchoalveolar lavage (BAL) fluid, were evaluated 16-18 h after O3 exposure in sensitized and nonsensitized male guinea pigs. The sensitization procedure was performed through repeated inhalation of ovalbumin for 3 wk. Increase in pulmonary insufflation pressure produced by the excitatory nonadrenergic noncholinergic (eNANC) system, histamine, and antigen were assessed in in vivo conditions, whereas airway responsiveness to histamine and substance P was evaluated in in vitro conditions by use of tracheal chains with or without epithelium and lung parenchymal strips. We found that O3 exposure 1) increased the neutrophil content in BAL fluids in both sensitized and nonsensitized guinea pigs, 2) caused hyperresponsiveness to eNANC stimulation in nonsensitized guinea pigs (although combination of sensitization and O3 exposure paradoxically abolished the hyperresponsiveness to eNANC stimulation), 3) increased the in vivo bronchoconstrictor responses to histamine and antigen, 4) caused hyperresponsiveness to substance P in nonsensitized tracheae with or without epithelium and in sensitized tracheae with epithelium, 5) did not modify the responsiveness to histamine in tracheae with or without epithelium (and in addition, epithelium removal caused hyperresponsiveness to histamine even in those tracheae exposed to O3), and 6) produced hyperresponsiveness to histamine in lung parenchymal strips either from sensitized or nonsensitized guinea pigs.     

Inhaled bordetella pertussis vaccine decreases airway responsiveness in guinea pigs

Bordetella pertussis (BP) has been used as adjuvant for experimental animal immunization, but its effects on airway responsiveness are uncertain. Three groups of guinea pigs were used: animals with a single exposure to inhaled BP vaccine (strain 134, total dose 1.24 × 10¹²germs), animals submitted to a sensitization procedure through inhalation of ovalbumin plus BP and healthy control animals. Four weeks after inhalation of BP or after the beginning of sensitization, dose- or concentration-response curves to histamine were constructed in vivo and in vitro (tracheal and parenchymal preparations). We found that BP alone produced lower responses to histamine than control guinea pigs in vivo (insufflation pressure, p = 0.0003) and in tracheal tissues (p = 0.04), but not in parenchymal preparations. Sensitization did not modify the responsiveness compared with their respective controls. These results suggest that some BP component(s), probably pertussis toxin, causes a long lasting airway hyporesponsiveness in guinea pigs.     

Repeated antigen challenge induced airway hyperresponsiveness to neurokinin A and vagal non-adrenergic, non-cholinergic (NANC) stimulation in guinea pigs.

The effect of repeated weekly antigen challenges by aerosol on bronchopulmonary responses to ACh, histamine, neurokinin A or atropine-resistant (NANC) component of vagal stimulation, has been studied in guinea pigs. Bronchospastic responses were measured in anaesthetized animals, 7 days after the last challenge with antigen (or vehicle). No difference was observed between control and antigen challenged guinea pigs in their responsiveness to acetylcholine (1-300 mumol kg-1 i.v.) or histamine (1-300 mumol kg-1 i.v.). On the other hand, amplitude of bronchospasm induced by neurokinin A (1-3 mumol kg-1 i.v.) or NANC vagal stimulation (20 Hz, 1 msec, 10 V, trains of 5-20 sec) was significantly increased in guinea pigs previously challenged with antigen, as compared to controls. These results suggest that repetitive antigen exposure in sensitized guinea pigs generates an increase in the responsiveness to exogenously administered or endogenously released tachykinins, at a time when no generalized hyperresponsiveness to other spasmogens could be observed.     

In vitro and inhibition of anaphylactic reactions 1-methyl-3-isobutylxanthine]

1-methyl-3-isobutylxanthine (MIBX), a potent antiphosphodiesterase drug, inhibited the anaphylactic reactions both in vitro and in vivo: anaphylactic histamine release from the isolated mast cells of rats, anaphylactic contracture of the isolated trachea of guinea pigs, passive cutaneous anaphylaxis (PCA) in mice and anaphylactic bronchial constriction (ABC) in guinea pigs. MIBX inhibited the anaphylactic reaction of the trachea, PCA and ABC more than the corresponding reactions induced by histamine.     

Prostaglandins and cardiac anaphylaxis.

Manifestations of cardiac anaphylaxis include sinus tachycardia and arrhythmias, both of which result from histamine release. The marked decrease in coronary flow, which also occurs during cardiac anaphylaxis, cannot be attributed solely to histamine release.To indirectly assess the possible role of prostaglandins in cardiac anaphylaxis, hearts from sensitized guinea pigs were challenged $\text{in}$$\text{vitro}$ in the presence of indomethacin. This resulted in a marked increase in histamine release, which caused a greater tachycardia and an increase in the incidence of arrhythmias. Moreover, for the same degree of histamine release sinus rate increments were larger in the presence of indomethacin. However, despite the enhanced cardiac dysfunction, coronary flow rate did not decrease.The results suggest that, during cardiac anaphylaxis, prostaglandins modulate histamine release and the effects of released histamine. Furthermore, since we have found that PGF_2α is released from the anaphylactic heart, we tentatively ascribe the anaphylactic reduction in coronary flow to the elaboration of PGF_2α.     

A study with BN 52021 demonstrates the involvement of PAF-acether in IgE-dependent anaphylactic bronchoconstriction

The involvement of platelet activating factor (PAF) in anaphylaxis was examined by recording the pulmonary responses of anesthetized passively sensitized guinea-pigs to the aerosolization of ovalbumin. Animals were tested with and without BN 52021 (a ginkgolide B, PAF receptor antagonist) pretreatment. Aerosolization of ovalbumin produced a bronchoconstriction (BC) which could be made refractory to additional challenges with the antigen. In animals desensitized to ovalbumin, aerosolization of PAF produced an unattenuated BC. Guinea pigs desensitized by repeated aerosolizations of PAF subsequently showed reduced responses to aerosolized antigen suggesting that PAF may be involved in the BC. Animals pretreated with BN 52021, were protected against the effects of systemically administered PAF and also showed reduced responses to aerosolized antigen. Aerosolization of the leukotriene antagonist, FPL 55712, was ineffective against anaphylactic BC under conditions where catecholamine and histamine release were blocked.     

A study with BN 52021 demonstrates the involvement of PAF-acether in IgE-dependent anaphylactic bronchoconstriction

The involvement of platelet activating factor (PAF) in anaphylaxis was examined by recording the pulmonary responses of anesthetized passively sensitized guinea-pigs to the aerosolization of ovalbumin. Animals were tested with and without BN 52021 (a ginkgolide B, PAF receptor antagonist) pretreatment. Aerosolization of ovalbumin produced a bronchoconstriction (BC) which could be made refractory to additional challenges with the antigen. In animals desensitized to ovalbumin, aerosolization of PAF produced an unattenuated BC. Guinea pigs desensitized by repeated aerosolizations of PAF subsequently showed reduced responses to aerosolized antigen suggesting that PAF may be involved in the BC. Animals pretreated with BN 52021, were protected against the effects of systematically administered PAF and also showed reduced responses to aerosolized antigen. Aerosolization of the leukotriene antagonist, FPL 55712, was ineffective against anaphylactic BC under conditions where catecholamine and histamine release were blocked.     

Characterization and genetic control of the immune response to synthetic polypeptide antigens of defined geometry.

Both humoral and cell-mediated immune responses to the synthetic helical hapten-carrier conjugate poly-Glu-Tyr-Lys(TNP)-(Glu-Tyr-Ala)5 were found to be linked to the major histocompatibility locus in mice and guinea pigs. The responder mouse strains (H-2d haplotype) showed a primary IgM response with an IgG component appearing after the secondary immunization. The antibody response was accompanied by a positive DTH reaction in responder strains. Nonresponder mice (H-2b or H-2k haplotypes) showed neither IgM nor IgG antibodies and the DTH reaction was negative. Administration of the antigen as a complex with an immunogenic carrier was not effective in inducing a response in nonresponder mice. In guinea pig studies, it was found that strain 2 animals were able to mount an antibody response against the TNP-hapten and a DTH response against the polypeptide backbone. Strain 13 animals gave no anti-TNP antibodies at the lower dose levels and DTH activity was entirely negative for all doses of immunizing antigen. Replacement of the TNP hapten by the arsanilazo dipeptide derivative, BOC-gly-ARA-tyrosine, converted the nonresponder strain 13 guinea pigs into complete responders showing antibody and DTH reactions to both the hapten and the polypeptide backbone.     

A comparison of autoimmunity and experimental allergic thyroiditis in strain 2 and Hartley strain guinea pigs

The response of Strain 2 guinea pigs to immunization with thyroid extract in complete adjuvant was compared with the response of the Hartley strain. The Strain 2 did not develop thyroiditis as frequently or in as great a degree as the Hartley strain. This difference in degree of thyroiditis between these two strains occurred consistently even when the animals were immunized with a wide range of doses of thyroid extract and of mycobacteria in adjuvant, and was independent of the strain origin of the antigen used for immunization. Although the Strain 2 made less thyroiditis than the Hartley strain, it made as much agglutinating antibody, and often as much delayed sensitivity as the Hartley strain. The genetic controls regulating autoimminity to the thyroid and production of thyroiditis appear to differ from those regulating production of experimental allergic encephalomyelitis.     

Involvement of platelet-activating factor and leukotrienes in anaphylactic segmental venoconstriction in ovalbumin sensitized guinea pig livers

The hepatic anaphylactic venoconstriction is partly involved in anaphylactic hypotension, and is characterized by significant post-sinusoidal constriction and liver congestion in guinea pigs. We determined what chemical mediators are involved in anaphylaxis-induced segmental venoconstriction and liver congestion in perfused livers isolated from ovalbumin sensitized guinea pigs. Livers were perfused portally and recirculatingly at constant flow with diluted blood. The sinusoidal pressure was measured by the double occlusion pressure (Pdo), and was used to determine the pre-sinusoidal (Rpre) and post-sinusoidal (Rpost) resistances. An antigen injection increased both the portal vein pressure and Pdo, resulting in 4.1- and 2.3-fold increases in Rpre and Rpost, respectively. Hepatic congestion was observed as reflected by liver weight gain. Pretreatment with TCV-309 (10microM, platelet-activating factor (PAF) receptor antagonist) or ONO-1078 (100microM, human cysteinyl-leukotriene (Cys-LT) receptor 1 antagonist), but not indomethacin (10microM, cyclooxygenase inhibitor), ketanserin (10microM, serotonin receptor antagonist), or diphenhydramine (100microM, histamine H1 antagonist), significantly attenuated this anaphylactic hepatic venoconstriction. Anaphylaxis-induced increases in Rpre and Rpost were significantly inhibited by TCV-309 (by 48%) and ONO-1078 (by 36%), respectively. Combined TCV-309 and ONO-1078 pretreatment exerted additive inhibitory effects on anaphylactic hepatic venoconstriction. Anaphylactic hepatic weight gain was converted to weight loss when post-sinusoidal constriction was attenuated. It is concluded that anaphylaxis-induced pre-sinusoidal constriction is mainly caused by PAF and the post-sinusoidal constriction by Cys-LTs in guinea pig livers.     

Determinant specific suppression of antigen-induced T cell proliferation in the guinea pig. II. Determinant specific suppression of in vitro T cell responsiveness parallels a selective suppression of anti-hapten but not anti-carrier antibody responses

Using an antigen of defined physical structure with precisely mapped immunogenic sites, we asked whether those molecular sites previously shown to be critical for immune response gene-mediated initiation of T cell proliferation and T help correspond to the same molecular regions capable of inducing antigen-specific suppression of T cell proliferation and antibody production. Inbred strain 2, 13, and 2 x 13 F1 hybrid guinea pigs were immunized with various species variants or fragments of insulin adjuvant before subsequent immunization with antigen in complete Freund's adjuvant. Analysis of the patterns of depressed T cell responsiveness showed a striking correspondence to the Ir gene-dependent mechanism that controls the recognition of discrete regions within the insulin molecule observed in T cell help in antibody production. In addition, suppression of carrier-specific T cells parallels suppression of anti-hapten antibody responses when hapten is presented on the suppressed carrier without a concomitant suppression of the anti-carrier antibody response.     

Muscarinic-receptor functioning in tracheas from normal and ovalbumin-sensitive guinea pigs

Responses to bilateral vagal nerve stimulation, to field stimulation, and to exogenous methacholine and histamine were compared in tracheas isolated from (a) saline injected (i.p.) and saline-aerosol exposed guinea pigs (control), (b) ovalbumin-sensitized and saline-aerosol exposed (sensitized) guinea pigs, and (c) ovalbumin-sensitized and 2% ovalbumin-aerosol exposed (challenged) guinea pigs. Tracheal pressor responses (cmH2O; 1 cmH2O = 98.1 Pa) to nerve and field stimulation, and maximal responses to methacholine and histamine were significantly increased in animals from group c compared with groups a and b. Dose-response lines in response to the two agonists, expressed as percent maximal contraction, did not differ among the groups. The M1 antagonist pirenzepine (0.1-10 nM) selectively reduced responses to nerve stimulation in all three groups. The M2 antagonist gallamine potentiated responses to nerve or field stimulation in all three groups. We conclude that M1, M2, and M3 muscarinic receptor functioning is similar in control and ovalbumin-sensitized guinea pigs. Changes in post-receptor transduction mechanisms may mediate the increased responsiveness noted in animals from group c.     

Lack of adrenomedullin on antigen-induced bronchoconstriction in guinea pigs in vivo

Antigen challenge can provoke acute bronchoconstriction, recognized as immediate asthmatic response (IAR), but the evolving events in this reaction are not well defined. Recently, a novel peptide, designated adrenomedullin, was isolated from human pheochromocytoma, and has been shown to have potent systemic and pulmonary vasodilator activity.The purpose of this study was to elucidate the influence of adrenomedullin in the development of IAR. Passively sensitized guinea pigs were anesthetized and treated with diphenhydramine hydrochloride, and then artificially ventilated. Ovalbumin was inhaled after an intravenous administration of adrenomedullin. Other studies were performed in naive guinea pigs to investigate the airway responses to inhaled methacholine or histamine after an intravenous administration of adrenomedullin. Antigen challenge caused bronchoconstriction in sensitized guinea pigs. Adrenomedullin did not inhibit the antigen-induced bronchoconstriction in sensitized guinea pigs or the dose-dependent responses to inhaled methacholine or histamine in naive animals in spite of its vasodilating effect. We conclude that an intravenous administration of adrenomedullin does not influence antigen-induced bronchoconstriction or bronchial responsiveness to inhaled methacholine or histamine in vivo.     

Immune response gene control of antibody specificity

The expression of the histocompatibility-linked PLL Ir gene was investigated in guinea pig B cells. Strain 2 and F₁ (2 × 13) guinea pigs, immunized with the αDnp-Lys₉, produce both T cells and antibody which are equally discriminatory for αDnp-Lys₉. In contrast strain 13 (PLL Ir gene negative) guinea pigs immunized with αDnp-Lys₉ do not develop specific T-cell responses and the antibody produced while restricted in heterogeneity cannot differentiate the immunizing antigen from Dnp-OH. However, if in a F₁ (2 × 13) environment, PLL Ir gene-negative B cells are provided with F₁ (2 × 13) T cells they express the ability to make antibody as specific and discriminatory as the antibody produced by PLL Ir gene-positive B cells. These findings strongly suggest that in the guinea pigs the PLL Ir gene defect is localized to the T cells and that the repertoire of specificity of B cells is similar if not identical in both responder and nonresponder animals. In addition these observations support the notion that the cellular locus for the PLL Ir gene expression in the guinea pigs is limited to T cells and not to macrophages and B lymphocytes.     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.     

Major histocompatibility complex restriction of T-cell responses to varicella-zoster virus in guinea pigs.

Varicella-zoster virus (VZV), adapted to grow in guinea pig fibroblasts, was injected subcutaneously into Hartley, strain 2, and strain 13 guinea pigs. Serum immunoglobulin G antibodies were detected 2 weeks later, and T-cell proliferative responses by blood lymphocytes were found 3 weeks after injection. The proliferating cells bound the 155 antibody, which defines a CD4-like subset of guinea pig T lymphocytes. VZV-infected fibroblasts of human, Hartley, and strain 13 origin elicited equivalent amounts of proliferation, which was quantitatively greater than that obtained with an extracted VZV antigen. Uninfected (control) human or guinea pig fibroblasts did not elicit T-cell proliferation. The proliferative response to VZV required the presence of autologous (strain 2 or 13) antigen-presenting cells and was blocked by the addition of an anti-class II major histocompatibility complex antibody. Effector cells obtained from in vitro cultures mediated class II-restricted cytotoxicity to L2C cells incubated with VZV. Class I-restricted responses were obtained only by cross-priming strain 2 animals with strain 13 peritoneal exudate cells which had been preincubated with VZV. The data indicate that guinea pigs resemble humans in that class II-restricted T cells with specificity for VZV are more readily cultured from blood than are class I-restricted cells.     

Antigen-induced suppression of mitogen responses and resistance to experimental autoimmune encephalomyelitis

Random-bred Hartley and inbred Strain 2 and Strain 13 guinea pigs were inoculated for acute experimental autoimmune encephalomyelitis (EAE). Sixty-six percent (69/103) of the Hartleys developed signs of EAE while the remaining 34% (34/103) were resistant. No Strain 2 and all Strain 13 guinea pigs developed EAE. Histologic examination of nervous tissue revealed that susceptible Hartleys and Strain 13 and Strain 2 animals had lesions characteristic of EAE. Tissue from resistant Hartleys showed fewer and less severe changes. Lymphocyte-transformation assays with EAE-inducing and noninducing antigens and T-cell mitogens revealed three different sets of responses in vitro: (i) lymphocytes from all animals responded to mitogens; (ii) lymphocytes from susceptible animals responded to EAE-inducing antigens; and (iii) lymphocytes from resistant Hartleys were suppressed from responding to the mitogens solely by EAE-inducing antigens. Plasmas from all EAE-sensitized animals had equivalent anti-myelin basic proteins (MBP) antibody titers and skin tests of EAE-inoculated Hartleys were all positive for MBP sensitization. Therefore, resistance and reduced histologic changes characteristic of EAE correlated with a disease-specific antigen-induced suppression of lymphocyte responses to T-cell mitogens. This suggests that clinical resistance to EAE in Hartley guinea pigs is mediated by an immunologic suppressor mechanism.     

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.