Delayed-type Hypersensitivity Response to Crude and Fractionated Antigens from Fonsecaea pedrosoi CMMI 1 Grown in Different Culture Media

Chromoblastomycosis is a subcutaneous fungal disease caused by dematiaceous fungi, especially by Fonsecaea pedrosoi, regarded as its major causative agent in Brazil. In recent years there has been a decline in the use of skin testing for delayed-type hypersensitivity (DTH) in epidemiological surveys of fungal infections, mainly because of the unpredictability of positive reactions and lack of specificity of the antigens used. The aim of the present study was to assess delayed-type skin tests in guinea pigs experimentally infected with F. pedrosoi using exoantigens prepared from two culture filtrates. Sixteen adult male guinea pigs were inoculated intratesticularly with fungal cells and submitted to sensitivity assays 4 weeks after inoculation. They received an intradermal injection with crude and fractionated antigens from Alviano’s and Smith’s cultures, and were assessed 24 and 48 h thereafter. Except for one animal, all of them had positive indurations after 48 h. There were no statistical differences between the measurements at 24 and 48 h for each exoantigen used, neither among the induration measurements at 48 h when different preparations were compared. Our results suggest that a delayed-type skin test using antigens produced in synthetic media may be useful for the assessment of primary exposure to chromoblastomycosis.     

Pathological observations in experimental candida infection of sensitized guinea pigs

Guinea pigs immunized intramuscularly with heat-killed or viable Candida albicans were infected intracutaneously with C. albicans. Animals with negative delayed hypersensitivity against C. albicans antigen showed similar lesions with non-immunized controls. Delayed hypersensitivity-positive guinea pigs, which were detected in the animals immunized with heat-killed C. albicans in CFA and IFA, demonstrated a delay of the resolution of the inflammatory tissue reaction and, in the animals immunized with C. albicans in CFA, developed a granuloma.These results suggest that both humoral and cell-mediated immunities do not play a significant role for protection against candidiasis and at a late stage of infection, cell-mediated immunity may play a secondary role of the enhancement of resistance to candida infection associated with granuloma formation.     

Eimeria stiedai: delayed hypersensitivity response in rabbit coccidiosis.

The development of delayed hypersensitivity (DH) in rabbits infected with Eimeria stiedai was shown by skin testing. A particulate antigen fraction was prepared by extraction of nonsporulated E. stiedai oocysts and found to be effective in producing dermal induration similar to that seen in a tuberculin reaction. The average diameter was 9 mm (range 7–11.0 mm, n = 26) with an average thickness of 0.4–0.5 mm for infected rabbits. All skin reactions were negative in noninfected animals (0–3.0 mm diameter and 0–0.2 mm thickness). Histological examination of dermal reactions revealed mononuclear cell infiltration within 48 hr with areas of necrobiosis. Skin reactivity was passively transferred to noninfected rabbits with lymphocyte suspensions and cell-free transfer factor but not with serum from infected skin-reactive animals. Delayed hypersensitivity was detected in 11 of 28 infected rabbits at 10 days, and by 20–30 days, 53 of the 55 animals tested showed positive skin reactions.     

Study of Delayed Hypersensitivity to Myxoviruses Induced by Vaccines

The delayed hypersensitivity response of guinea pigs to Bacillus Calmette-Gúerin (BCG) and myxovirus vaccines was investigated by use of the techniques of skin testing and inhibition of macrophage migration. A serum antibody response to the injected vaccines was readily demonstrable. Parainfluenza type 2 virus consistently failed to induce a delayed hypersensitivity state in 15 animals, even with the use of a virus adjuvant emulsion. Respiratory syncytial virus occupied an intermediate position in that positive delayed type skin tests of an erythematous nature were elicited following inoculation, but only two of seven guinea pigs yielded a positive migration inhibition test. In mumps-inoculated animals, skin testing gave rise to erythematous delayed skin reactions which varied from 0 to 20 mm in size. Migration inhibition could be demonstrated in 7 of 21 animals. In almost all guinea pigs inoculated with BCG, large, indurated, erythematous skin reactions were elicited, and inhibition of macrophage migration was readily demonstrated. The degree of skin reactivity was positively correlated with inhibition of macrophage migration. If the skin reaction to a specific antigen exceeded 9 mm of erythema, that antigen also inhibited macrophage migration. Skin testing proved to be the most sensitive indicator of viral hypersensitivity. Migration inhibition was demonstrated only when a greater than 8-mm skin reaction was evoked. This cellular hypersensitivity appeared to be a qualitative phenomenon, the expression of which could be heightened by the use of adjuvant. The applicability and sensitivity of the migration inhibition technique is considered relative to its use for in vitro monitoring of effects of viral vaccine inoculations.     

Study of anaphylectic hypersensitivity manifestations to blood protein in guinea pigs with using the neutrophil damage test in vitro]

The paper is devoted to the study of hypersensitivity of anaphylactic type to horse serum in guinea pigs by means of neutrophil injury index in vitro. Simultaneously, for control, the same method was applied to the study of delayed hypersensitivity manifestations; for this purpose allergy to hemolytic staphylococcus was reproduced in these animals. The state of hypersensitivity of "anaphylactic type" against the foreign serum in guinea pigs was not accompanied by any enhanced blood neutrophil reactions, whereas high values of the test under study were characteristic of the process of guinea pigs sensitization with staphylococcus culture.     

Delayed type hypersensitivity in guinea pig infected subcutaneously with Naegleria fowleri Carter.

A soluble fraction, derived from Naegleria fowleri trophozoites disrupted by freeze-thawing, was tested for antigenic properties. Intradermal injections of this preparation were administered to guinea pigs previously infected subcutaneously with viable N. fowleri. Delayed hypersensitivity to the antigen and loss of weight, the diagnostic symptom of visceral naegleriasis, were observed in the surviving animals. Fifty percent of the guinea pigs, however, did not lose weight and had a reduced reaction to the antigen. The apparent differences in the immunocompetence of guinea pigs inoculated subcutaneously and intranasally with N. fowleri are compared.     

Detection of hypersensitivity toLegionella pneumophila in guinea pigs by skin test

Cross-reacting antigens of three serogroups ofLegionella pneumophila differed serologically and immunologically from the serogroup-specific antigens. Intradermal injection of cross-reacting antigens into sensitized guinea pigs evoked skin hypersensitivity. Animals were sensitized either by injection of inactivatedL. pneumophila in adjuvant or by infection with live organisms. Skin reactions were measurable about 2–4 h after injection and continued to increase in intensity for the first 24 h, followed by a gradual decline over the next 48 h. Histological examination of skin reactions taken from test sites at 48 h revealed infiltration of mononuclear cells in and about the small subcutaneous blood vessels and throughout the dermis, compatible with a delayed-type reaction. The overall appearance and time course of the reaction resembled a combination of immediate and delayed types of hypersensitivity. Each cross-reacting antigen of the three serogroups evoked skin reactions in animals which had been sensitized to any of those serogroups, but was not reactive in nonsensitized animals. These observations indicate the possibility of detecting present or past infection ofL. pneumophila by skin tests.     

Relation between Delayed Skin Reactivity and Macrophage Migration Inhibition or Lymphocyte Transformation in Tuberculin-Type Hypersensitivity and Jones-Mote Hypersensitivity

Precise time-course studies on delayed skin reaction, lymphocyte transformation and macrophage migration inhibition were carried out from day 3 to 270 and from day 3 to 120, respectively, in guinea pigs immunized with bovine gamma-globulin (BGG) in complete Freund's adjuvant (CFA) and those immunized with BGG in incomplete Freund's adjuvant (IFA). a) Delayed skin reactions could be elicited for a long period of time after immunization with BGG in CFA in the presence of prominent antibody production and were accompanied by induration. b) Delayed reactions could be elicited transiently after immunization with BGG in IFA and were not accompanied by induration. c) At the peak of hypersensitivity, infiltrating cells at the reaction sites were composed largely of mononuclear cells and basophils, respectively, in the animals immunized with BGG in CFA and those immunized with BGG in IFA. d) Uptake of ³H-thymidine by lymphocytes was increased remarkably in the presence of BGG when cells were obtained at early stages after immunization by both methods. e) Macrophage migration inhibition was strongly positive in animals immunized with BGG in CFA but weakly positive in those immunized with BGG in IFA. Increased lymphocyte transformation preceded the appearance of a positive migration inhibition. f) After immunization with BGG in CFA, Jones-Mote hypersensitivity appeared to precede the development of tuberculin-type hypersensitivity.     

Effect of antilymphocyte serum on animals experimentally infected with Histoplasma capsulatum or Cryptococcus neoformans

The anti-mouse and anti-guinea pig antilymphocyte sera (ALS) prepared for this study were shown to contain cytoxic and leucoagglutinating antibodies, and were capable of producing severe lymphopenia in these animals. Guinea pigs treated weekly with ALS were more susceptible to development of fatal infection when inoculated with Histoplasma capsulatum. No fatalities occurred in guinea pigs infected with equal doses of H. capsulatum but treated with normal rabbit serum (NRS) or saline. The time necessary to reach 50% fatality in mice infected with Cryptococcus neoformans was greatly reduced by pretreatment with ALS in comparison with infected controls treated with NRS or saline. When low dosages were used (0.1 ld(50)), the effect was even more pronounced. Spleen homogenates from mice infected with equal dosages of H. capsulatum and treated with ALS or NRS were cultured. More than 150 times as many organisms were present in the spleens of the ALS-treated group. Similar results were obtained from culturing the lungs and liver. Delayed hypersensitive skin reactions were radically decreased or abrogated in H. capsulatum-infected guinea pigs inoculated intraperitoneally with ALS 12 hr before skin testing with histoplasmin. When ALS was given weekly, the influence on skin reactivity was less notable. Given intradermally, ALS was shown to inhibit the delayed reaction to histoplasmin within a radius of 40 mm.     

Allergenicity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations in Mice and Guinea Pigs

Guinea pigs were injected subcutaneously with mycobacterial ribosomal fraction incorporated in Freund's incomplete adjuvant and tested 6 and 12 weeks later by the intradermal injection of 0.5 μg (25 TU) of Purified Protein Derivative. No evidence of delayed-type hypersensitivity could be detected in these animals, although large necrotic reactions were obtained in guinea pigs sensitized with living, attenuated mycobacterial cells. Mice also were vaccinated by the intraperitoneal injection of mycobacterial ribosomal fraction or ribonucleic acid (RNA) and tested for sensitivity to tuberculin at various subsequent times. No evidence of true tuberculin hypersensitivity could be detected at any time, although what appeared to be small Arthus type reactions were seen in mice given the largest vaccinating doses. Attempts to recall tuberculin sensitivity in vaccinated mice by the intravenous injection, 4 weeks after vaccination of living cells, of either the virulent or attenuated mycobacterial strains were unsuccessful. Instead, when the virulent cells were injected, a suppression of footpad reactivity was noted in animals made sensitive to tuberculin by the previous intraperitoneal injection of viable attenuated mycobacterial cells. Both guinea pigs and mice, vaccinated as described above, were also skin tested or footpad tested, respectively, with 2 μg of the ribosomal fraction or RNA used for vaccination. No evidence of true tuberculin hypersensitivity could be obtained; instead, in guinea pig skin very small dermonecrotic areas were noted, and in mice swelling and redness of the footpad occurred to an equal extent in both vaccinated and nonvaccinated mice. The possible role of tuberculin hypersensitivity in acquired immunity to tuberculosis is discussed, and the conclusion is reached that its part, if any, is minor.     

Properties of TAS-1D3, a Tuberculin-Active Substance from BCG,in Regard to Delayed Hypersensitivity

The properties of TAS-1D3, a tuberculin-active substance purified from the cell extract of Mycobacterium bovis BCG, were studied in vivo and in vitro. In the delayed hypersensitivity skin reaction, TAS-1D3 showed far more potent activity than tuberculin purified protein derivative (PPD) in guinea pigs sensitized with BCG vaccine. This was consistently observed from 6 to 24 weeks after sensitization. The histological findings of the skin reaction to TAS-1D3 were similar to those of the reaction to PPD. Moreover, TAS-1D3 induced well both thymidine incorporation and the production of migration inhibitory factor (MIF) by the spleen cells from guinea pigs sensitized with BCG vaccine. In contrast, TAS-1D3 showed weaker activity than PPD in guinea pigs sensitized with either heat-killed M. tuberculosis Aoyama B or heat-killed M. tuberculosis H37Ra, and it weakly stimulated the spleen cells from animals sensitized with M. tuberculosis Aoyama B to incorporate thymidine and to produce MIF.     

The immunopathological effects of intracolonic injection of Mycobacterium bovis BCG cell wall emulsions in guinea pigs

Summary The immunological and pathological responses of guinea pigs to an intramural colonic injection of emulsions containing cell wall (CW) extracts of Mycobacterium bovis BCG and mineral oil were studied from week 1 to week 36 post-inoculation. The emulsions contained variable concentrations of BCG CW attached to (lipid-phase), or separate from (aqueous-phase), the mineral oil. Delayed cutaneous hypersensitivity to PPD was present throughout the course of the study in a variable percentage of guinea pigs inoculated with either type of emulsion. PPD-induced blast transformation of peripheral blood lymphocytes, studied in guinea pigs which received lipid-phase emulsion, was also detectable throughout the course of the study, with maximal response seen 2 weeks post-inoculation. The intracolonic inoculations were well tolerated, with the exception of the most concentrated lipid-phase emulsion (3 mg/ml BCG CW and 5% oil), after which one of eight guinea pigs died due to a colonic impaction and rupture at the site of inoculation. The pathological response to either type of emulsion was a focal granulomatous colitis, which tended to be more severe as the concentration of BCG CW and oil increased. Extracolonic lesions were usually limited to a granulomatous lymphadenitis of lymph nodes draining the injection site; however, the most concentrated lipid-phase emulsion occasionally produced granulomatous inflammatory foci in the liver and lungs. In general, the lesions induced by the lipid-phase emulsions were more severe than those induced by aqueous-phase emulsions, but the intensity of both types of lesions peaked at 2 or 4 weeks post-inoculation. It was concluded that the guinea pig may serve as a useful model to study BCG immunotherapy of colonic neoplasms, since intracolonic injection of BCG CW resulted in systemic immunity toward mycobacterial antigens and a localized accumulation of macrophages without untoward complications. The abbreviations used in this paper are: BCG CW, bacillus Calmette-Guérin cell walls; PPD, purified protein derivative; PBL, peripheral blood lymphocytes; cpm, counts per minute; SI, stimulation index; DCH, delayed cutaneous hypersensitivity     

The role of B lymphocytes in cell-mediated immunity II. Delayed hypersensitivity induced by dinitrophenyl-ficoll in dinitrophenyl-keyhole limpet hemocyanin-immunized guinea pigs.

Dinitrophenyl (DNP)-Ficoll will elicit typical delayed hypersensitivity skin reactions in guinea pigs immunized with DNP-keyhole limpet hemocyanin (KLH). We observed that lymph node cells (LNC) from these animals produced the lymphokine, monocyte chemotactic factor (MNL CTX) when stimulated by DNP-Ficoll in vitro. This response was antigen and hapten specific since LNC from nonimmune guinea pigs or those immunized with nonDNP containing antigens were not stimulated by DNP-Ficoll. Lymph node cells were fractionated into T- and B-cell-enriched populations to determine the nature of the DNP-Ficoll-responsive cell. Only the B-lymphocyte-enriched population produced MNL CTX in response to DNP-Ficoll. The purity of the B-cell population was demonstrated by its failure to respond to PHA and by the fact that B cells derived from DNP-although they could no longer respond without T-cell help to the T-dependent antigen, DNP-OVA. These findings suggest that the hapten-specific response of guinea pigs to DNP-Ficoll may be a form of B-cell-mediated delayed hypersensitivity.     

Purified Protoplasmic Peptides of Mycobacteria: In Vivo and In Vitro Comparison of the Species Specificity of Purified Protoplasmic Peptides and Purified Protein Derivatives of Mycobacterial Culture Filtrates

The specificity of purified protein derivatives (PPD) prepared from the culture filtrates of Mycobacterium tuberculosis (PPD), M. kansasii (PPD-Y), M. intracellulare (PPD-B), and M. scrofulaceum (PPD-G) were compared to comparable protoplasmic extracts (PPP) of the same organisms by gel diffusion and delayed hypersensitivity reactions in sensitized guinea pigs. PPD and, to a lesser degree, PPD-Y demonstrated specificities sufficient to enable identification of homologously sensitized guinea pigs in the above group of four mycobacteria. PPD-B and PPD-G did not always elicit the largest reaction in homologously sensitized animals. The PPP sensitins from M. tuberculosis and M. kansasii produced as good skin reactions at 24 and at 48 hr as did their PPD counterparts. The PPP from M. scrofulaceum and M. intracellulare were more specific and more reactive than corresponding PPD, regardless of the time of comparison. Although based on different immunological mechanisms, the specificity of these two groups of sensitins, as demonstrated by delayed hypersensitivity, correlated well with serological comparisons in the gel diffusion test. The low degree of specificity of PPD-B and PPD-G in contrast to that of corresponding PPP was reflected in the precipitin bands in agar gel.     

IgE antibody-mediated cutaneous basophil hypersensitivity reactions in guinea pigs

Cutaneous basophil hypersensitivity (CBH) reactions are a heterogeneous group of delayed time course basophil-rich immune responses that can be mediated in the guinea pig by T cells, B cells, or IgG1 antibody. This study examined whether guinea pig IgE antibody could also mediate CBH reactions. IgE antibody to picryl or oxazolone determinants was induced by immunizing Hartley strain guinea pigs pretreated with cyclophosphamide. Hyperimmune serum from these animals was passed through a heavy chain-specific anti-IgG1 affinity column. The presence of IgE anti-hapten antibody in the filtrate fraction was verified by passive cutaneous anaphylaxis (PCA) testing with a 7-day period of local passive sensitization and by the heat lability (56 degrees C, 4 hr) of PCA activity. This IgE-rich fraction and the IgG1 fraction eluted from the column with base (0.2 M Na2CO3, pH 11.3) were transferred i.v. to separate groups of normal guinea pigs. Both fractions mediated delayed time course reactions that contained basophils. Macroscopic and microscopic reactions mediated by the IgE-rich fraction were abolished with heat (56 degrees C, 4 hr). Thus, two antigen-specific factors in guinea pig serum can mediate delayed time course basophil-containing reactions: IgG1 and IgE antibodies. IgE-mediated CBH reactions are similar to the late-phase reaction that follows IgE-dependent wheal-and-flare reactions in humans. The finding that guinea pig IgE can mediate a late reaction that contains basophils makes this a possible model for the human late-phase response, and suggests that some forms of CBH may play a role in human allergic disease.     

Role of mediators of cellular hypersensitivity in the stimulation of the antibody formation to unrelated antigen

The effect of a concurrent delayed hypersensitivity reaction on the antibody response to sheep red cells was assessed by a plaque assay. Guinea pigs with delayed hypersensitivity to tuberculin purified protein derivative (PPD) or egg albumin showed an increased antibody response to sheep red cells when the cells were injected intravenously at the same time as PPD or egg albumin. This effect was transferred to normal guinea pigs by serum from guinea pigs with delayed hypersensitivity to PPD or egg albumin taken 24 hr after injecting the corresponding antigen. Supernatants containing migratory inhibitory factor were prepared by incubating lymphocytes from sensitized rabbits with antigen. These supernatants were injected with sheep red cells and gave rise to an enhanced plaque response. Similar results were obtained with supernatants from normal rabbit thymus cells. The role of mediators of delayed hypersensitivity in enhancing antibody formation and in T cell/B cell cooperation is discussed.     

Effects of cyclophosphamide on the expression and induction of delayed hypersensitivity.

Erythematous delayed reactions without induration, presumably assigned to Jones-Mote type, were characterized by the resistance to treatment with cyclophosphamide (CY) before elicitation or immunization in guinea pigs immunized with BGG in IFA or CFA. CY-treatment before elicitation converted delayed erythematous reactions from negative to positive at late intervals after immunization with BGG in IFA. Such a treatment augmented erythematous delayed reactions in animals immunized with BGG in CFA, but abolished induration at the reaction sites. CY-treatment before elicitation or immunization reduced the numbers of basophils at the reaction sites, although erythematous delayed reactions were augmented. Effector T cells responsible for delayed erythematous reaction without induration appear to persist for a long period of time after immunization in the presence of antibody production or tuberculin hypersensitivity and the expression of their function may be inhibited by suppressive mechanisms.     

Immune responses to myelin basic protein in mycobacterial-induced suppression of experimental allergic encephalomyelitis

Pretreatment of guinea pigs with complete Freund's adjuvant (CFA) 21 days prior to injection with myelin basic protein (BP) in CFA resulted in marked attenuation of clinical and pathologic manifestations of experimental allergic encephalomyelitis (EAE). Delayed hypersensitivity skin tests to homologous BP were likewise depressed in protected animals. The protected guinea pigs also demonstrated diminished in vitro reactivity to BP as assessed by BP-induced proliferative response of peritoneal exudate cells (PEC) and BP-induced inhibition of macrophage migration. Broad-based suppression of immunologic reactivity did not occur in these animals, as manifested by larger skin tests to PPD, a greater proliferative response to old tuberculin (OT), by PEC and peripheral blood lymphocytes (PBL), as well as marked PPD-induced inhibition of macrophage migration. Diminution of the degree of cell-mediated reactivity to BP may be one of the mechanisms by which prior treatment with CFA suppresses subsequent development of EAE.     

Resistance of vaccinated mice to typical and atypical strains of Coccidioides immitis

In earlier reports, it was shown that mice and monkeys could be immunized against otherwise lethal challenge doses of Coccidioides immitis arthrospores. The vaccine was composed of Formalin-killed, in vitro grown, endosporulating spherules of C. immitis strain Silveira. In this study, mice were immunized as in the earlier work and then challenged intranasally with arthrospores from seven heterologous strains of C. immitis. Two of these strains were typical of the species, and five were atypical with respect to their cultural characteristics and morphology of microscopic structures. The vaccinated animals were well protected against challenge doses that were lethal to a majority of the control animals, regardless of the strain of fungus employed. The infection ratios among surviving vaccinated and control animals were comparable, but demonstrable lesions were generally smaller and less numerous in the vaccinated groups. It is suggested that these strains are at least immunogenically similar, although not necessarily identical, and that a vaccine prepared from a single strain of C. immitis would be practical for an immunization program.     

Blocking of delayed hypersensitivity by humoral antibody in an in vivo mouse transfer assay using sensitized guinea pig cells

Humoral antibody was shown to interfere specifically with the expression of cell-mediated immunity (delayed hypersensitivity) in an in vivo system. Mice that received peritoneal exudate cells obtained from guinea pigs sensitized to 1-chloro-2,4dinitrobenzene (DNCB) exhibited delayed hypersensitivity reactions after challenge with the sensitizing agent. While control groups that received either normal sera, saline, or anti-BSA (bovine serum albumin) in addition to peritoneal exudate cells from sensitized guinea pigs exhibited positive delayed reactons to challenge with DNCB, mice that received anti-DNP (dinitrophenyl group) in addition to the senstized cells were prevented from exhibiting a delayed reaction to DNCB.