Eosinophils in Delayed Hypersensitivity Skin Reaction Sites

Guinea pigs immunized with egg albumin or an azobenzene arsanate conjugate of acetyl-tyrosine (ABA-tyr) in complete or incomplete Freund's adjuvant can develop eosinophil-containing delayed hypersensitivity skin reactions when challenged with specific antigen. Retest, achieved by the re-injection of antigen at the site of a prior skin test, leads to an accelerated reaction which may contain increased numbers of eosinophils. ABA-tyr which induces delayed hypersensitivity without antibody formation, dissociates the two components of the retest reaction; that antigen leads to an accelerated reaction, but one with only small numbers of eosinophils.     

Oral induction of the secretory antibody response by soluble and particulate antigens

The present experiments have been performed in order to study the immunogenicity of antigen taken up by peritoneal macrophages using the hapten-carrier model and to investigate the role of macrophages in the antigenic competition between hapten and carrier moieties of the antigen molecule we have previously described. Guinea pigs were immunized with peritoneal cells collected from guinea pigs previously injected intraperitoneally with soluble or glutaraldehyde-polymerized hapten-carrier conjugates in Freund's incomplete adjuvant. Delayed hypersensitivity reactions to the carrier and anaphylactic reactions to both the hapten and the carrier were studied 14 and 16 days after immunization. After immunization with macrophage-associated hapten-carrier conjugate, delayed hypersensitivity reactions to the carrier were first detected in the absence of anaphylactic reactions which appeared later. The capabilities of macrophage-ingested antigen to induce delayed hypersensitivity reactions, but not anaphylaxis, decreased when increasing the incubation time of macrophages with antigen. The antigenic competition between hapten and carrier was confirmed to be a transient phenomenon occurring in the macrophage.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Immune response to a thymus-independent antigen (DNP-Ficoll) in the guinea pig

Guinea pigs immunized with DNP30-Ficoll produced IgM antibody only. No IgG1, IgG2, IgE antibodies or delayed hypersensitivity were detected in these animals. However, Arthus reactions, induced by the hapten coupled to a foreign carrier or the whole antigen, were found. The time course of the IgM response was limited and the response to reinjection of the antigen reduced. Cyclophosphamide (CY), given 3 days before primary immunization, prolonged the IgM response. Given on the day of immunization or 3 days after CY reduced this response. CY given on days +3 or +7 after primary immunization completely suppressed the response to antigen reinjected 42 days later. Arthus reactions were totally suppressed by CY given on the day of immunization, or 3 or 7 days later.     

Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen.     

Antigen-induced modulation of autonomic and sensory neurons in vitro

We have addressed the hypothesis that the excitability of peripheral neurons is affected during immediate hypersensitivity reactions. Guinea pigs were actively sensitized to ovalbumin. The electrical membrane properties of neurons within the superior cervical ganglion, bronchial parasympathetic ganglion and nodose ganglion were evaluated before, during and after antigen challenge. In all preparations, antigen stimulation induced the release of histamine and arachidonic acid metabolites. Our results support the hypothesis that the excitability of sympathetic, parasympathetic and sensory C-type neurons may be increased during immediate hypersensitivity reactions.     

Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis.

We have studied the capacity of a selected fraction of Mycobacterium tuberculosis extracellular proteins (EP) released into broth culture by mid-logarithmic-growth-phase organisms to induce cell-mediated immune responses and protective immunity in a guinea pig model of pulmonary tuberculosis. Guinea pigs infected with M. tuberculosis by aerosol but not uninfected control guinea pigs exhibit strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. Guinea pigs immunized subcutaneously with EP but not sham-immunized control guinea pigs also develop strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. EP is nonlethal and nontoxic to guinea pigs upon subcutaneous immunization. Guinea pigs immunized with EP and then challenged with aerosolized M. tuberculosis exhibit protective immunity. In five independent experiments, EP-immunized guinea pigs were consistently protected against clinical illness, including weight loss. Compared with EP-immunized guinea pigs, sham-immunized control guinea pigs lost 12.9 +/- 2.0% (mean +/- SE) of their total weight. EP-immunized guinea pigs also had a 10-fold reduction in viable M. tuberculosis bacilli in their lungs and spleens (P = 0.004 and 0.001, respectively) compared with sham-immunized control animals. In the two experiments in which some guinea pigs died after aerosol challenge, EP-immunized animals were protected from death. Whereas all 12 (100%) EP-immunized guinea pigs survived challenge with aerosolized M. tuberculosis, only 6 of 12 (50%) sham-immunized control guinea pigs survived challenge (P = 0.007, Fisher exact test). This study demonstrates that actively growing M. tuberculosis cells release immunoprotective molecules extracellularly, that a subunit vaccine against tuberculosis is feasible, and that extracellular molecules of M. tuberculosis are potential candidates for a subunit vaccine.     

Nasal mucosal hypersensitivity in guinea pigs intermittently exposed to cold.

To develop an animal model for experimental nasal hypersensitivity and hyperreactivity, guinea pigs were subjected to intermittent exposure to cold temperature (intermittent cold stress, SART stress) for 5 consecutive days. In SART-stressed guinea pigs, nasal mucosal hypersensitivity to histamine evoking sneeze response and nasal hypersecretion in response to methacholine were observed. The hypersensitivity remained for further 7 days after being released from SART stress. On the other hand, such nasal mucosal hypersensitivity was not caused by a continuous cold stress alone, suggesting that intermittent exposure to cold may be of importance for the appearance of nasal mucosal hypersensitivity. In passively sensitized SART-stressed guinea pigs, the quantity of nasal secretion induced by an allergen was significantly increased compared with that of a group of normal animals. The expression of muscarinic acetylcholine receptor (m-ACh.R) became higher in SART-stressed guinea pigs. Thus, hypersensitivity and hyperreactivity in this system were found to be associated with an increase in density of m-ACh.R. SART-stressed guinea pigs will serve as an animal model for hypersensitivity in nasal mucosa, which would be useful for the study of nasal allergy.     

Influenza in ferrets and guinea pigs: effect on cell-mediated immunity.

Two different animal models were studied to determine whether localized upper respiratory tract viral infection was associated with suppression of systemic cell-mediated immunity. During influenza infection in ferrets, there was no significant decrease in lymphocyte responsiveness to phytohemagglutinin (PHA). Guinea pigs given influenza showed no significant change in their response to PHA or to picryl human serum albumin (picHSA), to which they had been immunized previously. Delayed hypersensitivity skin test responses to picHSA in guinea pigs remained intact during infection. No change in the percentage of circulating T lymphocytes was detected during influenza infection. Transfer of immunity to nonsensitized recipient guinea pigs from picHSA-sensitized guinea pigs was accomplished during influenza infection. Lack of a suppressive effect on systemic cell-mediated immunity after influenza challenge in these two animal models of mild influenza confirmed previous findings in humans with mild influenza infection.     

The effect of disodium 4-chloro-2-iminodibenzoate (CCA) on IgE levels and anaphylactic shock

The effect of disodium 4-chloro-2,2-iminodibenzoate (CCA) on IgE antibody response was examined in C₃H/A and (BALB/c×C57BL/6J) F1 hybrid mice immunized with low doses of ovalbumin (OA) adsorbed on aluminium hydroxide gel. CCA administered orally at the doses of 5 and 50 mg/kg/day reduced IgE antibody production in these mice as determined by PCA test. High doses of CCA (100 mg/kg/day) given from day 7 before immunization of C57BL mice and during 1 week after immunization of mice with OA and Bordetella Pertussis Vaccine reduced the mortality of these mice subjected to anaphylactic shock on day 7 of immunization. CCA treatment was ineffective in anaphylactic shock of C57BL mice immunized with very high dose of OA, known to elicit little or no IgE antibody production but high IgG antibody response.The treatment of OA-immunized Guinea pigs with one oral dose of CCA (100 mg/kg) did not reduce mortality in protracted anaphylactic shock. Our results demonstrate that CCA inhibits IgE production as well as IgE mediated hypersensitivity reactions in mice.     

Basophil hypersensitivity response in rabbits.

A cutaneous basophil hypersensitivity response has been observed in rabbits immunized with bovine serum albumin and challenged intradermally with this antigen 7 days later. The cellular response appears to be similar to cutaneous basophil hypersensitivity reported in guinea pigs and humans. A basophil response was also observed in rabbits immunized with Staphylococcus aureus and challenged with viable staphylococcal cells 7 days later. A method of observing basophil infiltration in rabbits by means of connective tissue spreads obtained from the subcutaneous connective tissue is described. The rabbit should serve as an excellent model for the study of basophil responses as these animals have a significant basophil component with few if any tissue mast cells which may be confused both morphologically and functionally with the basophil.     

Culture of C. burnetii from the dental pulp of experimentally infected guinea pigs

An experimental model of Q fever in Guinea pigs was studied. Coxiella burnetii was cultured from the dental pulp of infected animals following bacteremia.     

Effects of contact sensitization and delayed hypersensitivity reactions on immune responses to non-related antigens. Modulation of Immune responses.

Guinea pigs were immunized intracutaneously into the ears with sheep red blood cells (SRBC). Application of a sensitizing dose of the contact allergen dinitrochlorobenzene (DNCB) onto the same ears was shown to suppress or enhance the humoral response to SRBC depending on the time of application. When guinea pigs were sensitized to a contact allergen, application of a sensitizing dose of a non-related allergen on the same ears either had no effect or caused a clear enhancement of the development of delayed type hypersensitivity (DTH). Strongest enhancement was found when both sensitizations were performed on the same day. Further experiments on the effects of a concomitant DTH reaction elicited at the site of application of a contact allergen showed a strong potentiation of DTH when B-cell suppression was minimized by pretreatment with cyclophosphamide (CY). It was considered that CY-DTH-immunopotentiation might be a useful tool for achieving a higher level of sensitivity after epicutaneous sensitization.     

Immunotherapy of a guinea pig hepatoma with mycobacterial vaccines: comparison of BCG cell walls and cell wall skeletons.

BCG cell wall skeletons (SK) derived from BCG cell walls (CW) by treatment with proteolytic enzymes and organic solvents were tested for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basic, SK were as effective as CW in causing tumor regression, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with SK. On a weight basis, CW were more active than SK in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with SK. In unimmunized animals the inflammatory response to intradermally administered CW was greater than that evoked by SK. CW and SK provoked delayed cutaneous hypersensitivity reactions of similar strength in animals immunized with living BCG. This study provided no compelling reasons for using SK instead of CW in clinical trials of cancer treatment by mycobacterial vaccines.     

In vivo and in virto cellular responses to cytoplasmic and cell wall antigens of Histoplasma capsulatum in artificially immunized or infected guinea pigs.

Guinea pigs were infected with different doses of yeasts of Histoplasma capsulatum or artifically immunized with several concentrations of unextracted yeast cell walls, and then tested in vivo and in vitro for cell-mediated responses to various subcellular fractions of the fungus. Three types of cell-mediated responses were measured, viz., skin test activity, production of migration inhibition factor, and lymphocyte transformation. Positive cutaneous reactions were elicited in animals immunized with 100 or 1,000 mug of cell walls when such animals were skin-tested with cell wall glycoprotein of soluble cytoplasmic substances, whereas animals immunized with 2,000 mug of cell walls did not react significantly greater than unsensitized animals when skin-tested with the same antigens. Histoplasmin did not elicit cutaneous sensitivity in guinea pigs infected with the smallest inoculum, 6 X 10(5) yeast cells, or in animals immunized with cell walls, regardless of the concentration of cell walls used as immunogen. However, hypersensitivity to H. capsulatum could be detected with cytoplasmic substances in animals infected with 6X 10(5). In guinea pigs infected with larger doses, i.e., 10 X 10(7), 15 X10(7), or 20 X 10(7), hypersensitivity could be detected with histoplasmin, cell wall glycoprotein, a ribosome-rich fraction, and soluble cytoplasmic substances. Both cell wall glycoprotein and soluble cytoplasmic substances were functional in migration inhibition factor assays with peritoneal exudate cells from animals immunized with 100 or 1,000 mug of cell walls. The transformation of lymphocytes from infected and artificially immunized guinea pigs in the presence of cell wall glycoprotein and soluble cytoplasmic substances was variable and unpredictable, the lymphocytes from some animls within a given group transforming and those from other animals showing no evidence of stimulation. Moreover, the level of stimulation could not be correlated with the degree of dermal hypersensitivity. These findings suggest that cell wall glycoprotein, and the fractions containing ribosomes and soluble cytoplasmic substances, could be useful antigens in assays for cellular immunity, and warrant further investigation with respect to specificity and active components.     

Comparative adjuvant activities of Legionella pneumophila and Mycobacterium tuberculosis

Adjuvant activity of heat-killed Legionella pneumophila was demonstrated and compared with that of inactivated Mycobacterium tuberculosis H37Rv. The two species of bacteria were suspended separately in oil and Arlacel A. Bovine serum albumin (BSA) in saline was then emulsified within the respective adjuvants and injected intradermally into guinea pigs. Antibodies to the BSA antigen in the sera of the animals were quantitated with the kinetic-dependent enzyme-linked immunosorbent assay (k-Elisa). Guinea pigs immunized with BSA in adjuvant with killed L. pneumophila produced high titers of anti-BSA antibody, which, on the average, were nearly as high as in those immunized with BSA in complete Freund's adjuvant with M. tuberculosis H37Rv, and which were much greater than in others immunized with incomplete adjuvant, lacking bacteria. Moreover, with a polypeptide hapten, the L. pneumophila evoked as much or more antibody in rabbits as the mycobacterium adjuvant. The effect of the legionella adjuvant upon the cellular immune response was examined using skin tests. For this purpose guinea pigs were immunized with picryl-guinea pig albumin in these adjuvants. 6 weeks later, they were skin-tested with that antigen. They showed reactions which appeared to have immediate as well as delayed components when examined grossly and histologically. Others, immunized with incomplete adjuvant, did not exhibit delayed reactions. Accordingly, heat-killed L. pneumophila acts as a potent adjuvant. Under the circumstances of these experiments, it was as effective as heat-killed M. tuberculosis.     

Experimental model for dermal granulomatous hypersensitivity in Q fever.

Q fever has been associated with granulomatous changes in clinical biopsy material obtained from liver and bone marrow. Local reactions to skin testing have been described in previously sensitized humans, but histological studies of such reactions have not been reported. We note that delayed hypersensitivity reactions to whole-cell phase I Q fever vaccine in immunized guinea pigs have a time course of development of induration characteristic of granulomatous hypersensitivity. Histological examination of such skin reactions on day 9 after testing revealed epithelioid cell infiltration and the presence of large numbers of multinucleated giant cells. Prominent in the sections were fragments of disintegrating polymorphonuclear leukocytes having the appearance of leukocytoclasis. Electron microscopic studies confirmed the presence of epithelioid changes in cells of the mononuclear phagocyte series, as well as extensive collagen deposition. This animal system affords a readily reproducible model of dermal granulomatous hypersensitivity and an opportunity to analyze the immunological basis of this reaction.     

Oral recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing HIV-1 antigens as a freeze-dried vaccine induces long-term,HIV-specific mucosal and systemic immunity

Induction of HIV-1-specific immune responses was evaluated using a recombinant BCG (rBCG) vector-based vaccine expressing HIV-1 Env V3 peptide (rBCG-pSOV3J1). rBCG-pSOV3J1 was manufactured as a freeze-dried preparation based on good laboratory practice guidelines. Guinea pigs were immunized with the freeze-dried rBCG vaccine by oral administration to test the effectiveness of what is generally considered the most convenient and practical route for vaccination. While delayed-type hypersensitivity (DTH) skin reactions to purified protein derivative were not detected in any of the animals receiving oral rBCG-pSOV3J1, HIV-1 V3J1 antigen-specific DTH responses were detected in all of the immunized guinea pigs 1.5 years after immunization. In addition, significant proliferative responses against HIV-1 V3J1 antigen were measured in peripheral blood mononuclear cells and splenocytes from all animals receiving oral rBCG. Interestingly, intestinal intraepithelial lymphocytes from the animals also exhibited high levels of proliferative activity against HIV-1 V3J1 antigen. These results suggest that oral vaccination of guinea pigs with freeze-dried rBCG-pSOV3J1 induces high levels of functional T cells specific for HIV-1 antigens in both mucosal and systemic compartments and suggest that this approach has potential for use as a vaccine against HIV-1.     

Comparative studies with various substrains of Mycobacterium bovis BCG on the production of an antigenic protein, MPB70.

A protein, isolated and purified from the unheated culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172) and designated MPB70, elicited a delayed skin reaction in guinea pigs sensitized with viable cells of BCG but not in those sensitized with heat-killed cells. The skin reaction reached the maximum 4 to 8 weeks after the inoculation of the BCG and then decreased gradually, resulting in conversion to negative after 20 weeks, whereas the skin reaction to purified protein derivative (PPD) continued to be positive. Guinea pigs immunized with viable cells of various substrains of BCG were skin tested with MPB70 and PPD. Guinea pigs immunized with the BCG substrain Tokyo 172 and the substrain Moreau (Brazil) showed strong delayed skin reactions to both MPB70 and PPD. On the other hand, guinea pigs immunized with the Pasteur substrain 1173P2, the Glaxo substrain 1077, the Copenhagen substrain 1331, the Tice substrain, or the Beijing substrain 64-42 showed negative skin reactions to MPB70, whereas they were strongly positive to PPD. In a two-dimensional acrylamide gel electrophoretic analysis of proteins from the culture filtrates of the BCG substrains, the culture filtrates of the Tokyo and Moreau substrains showed the spot of MPB70 on the gel slabs, whereas those of the other BCG substrains did not.     

Effect of nicotinamide on the development of allergic encephalomyelitis

Daily injections of nicotinamide to guinea pigs immunized with an encephalitogenic preparation from day 1 through 16 postimmunization suppress the development of clinical manifestations of experimental allergic encephalomyelitis and reduce mortality of experimental animals. In nicotinamide-treated animals, weakened delayed-type hypersensitivity skin reactions to myelin and adhesive activity of peripheral blood leukocytes as well as enhanced cytochrome P-450-dependent monooxygenase activity are noted. Translated fromByulleten', Eksperimental'noi Biologii i Meditsiny, Vol. 125, No. 2, pp. 180–182, February, 1998