Hypersensitivity Reactions In Guinea Pigs Research Articles

Citrus limon Peel Essential Oil-Induced Type IV Hypersensitivity Reaction.

BackgroundSeveral studies have shown anti-inflammatory, anti-microbial, antifungal, and antioxidant effects from Citrus limon–peel essential oil (Cl-PEO). Cl-PEO can be developed as topical drugs for oral ulceration because of its potential active components. There have been no studies on the topical application of Cl-PEO inducing type IV hypersensitivity reaction.PurposeTo investigate the potential of Cl-PEO from Batu City to induce type IV hypersensitivity reactions based on clinical changes, lymphocytes, macrophages, IFNγ, andIL10 expression.MethodsThis study was adapted from a guinea pig maximization-test method in Indonesia and the guidance of ISO 10,993-10:2010, and conducted on 20 guinea pigs (Cavia cobaya) divided into a control group and a treatment group. The treatment group was given Cl-PEO and the control group CMC-Na. Clinical changes were observed, then tissue specimens taken for hematoxylin–eosin and immunohistochemistry staining.ResultsThere were no clinical changes after exposure. Lymphocyte and macrophage numbers and IFNγ and IL10 expression increased in the treatment group compared to the control group (p=0).ConclusionCl-PEO can induce type IV hypersensitivity reactions in guinea pigs based on cellular and molecular cytokines, but there are no clinical changes after topical application.

Molecular cloning and expression of MPT63 gene of Mycobacterium tuberculosis

The present investigation was undertaken to amplify, clone and express MPT63 gene of Mycobacterium tuberculosis to discern this secretary protein as a potent diagnostic and immunogenic antigen. Primer specific for MPT63 gene with restriction enzyme sites, viz BamHI and HindIII were designed. MPT63 gene was amplified using DNA from M. tuberculosis 198/94 (IVRI) strain, with designed primers by polymerase chain reaction (PCR). The 412 bp amplicon was purified and first cloned into pGEM-T vector. Positive clone was confirmed by colony PCR and restriction enzyme digestion. The pGEM-T released insert was ligated with BamHI and HindIII digested pET32b expression vector using T4 DNA ligase and transformed into Escherchia coli. Recombinant clones were selected by colony PCR and restriction enzyme digestion and induced with 1mM final concentration of Isopropylß- D-thiogalactopyranosidase (IPTG) for expression of the recombinant MPT63 protein. The expressed protein of 33 kDa was obtained during 2 h post induction. Western blot with Ni- NTA anti-histidine HRP conjugate and hyperimmune serum raised in rabbits confirmed the presence and purity of recombinant MPT63 protein by immuno reactivity at the unique 33 kDa region. Further confirmation of recombinant protein was done by dot blot and indirect ELISA using rabbit hyperimmune serum. The recombinant MPT63 protein was evaluated as a skin test antigen to produce delayed type hypersensitivity reaction in guinea pigs. Recombinant MPT63 protein produced skin erythema of 3 to 4 mm diameter in guinea pigs sensitized with killed M. tuberculosis and M. bovis culture. From the present study, it may be concluded that understanding of the immunogenic components of an infectious agent is essential through molecular characterization, in regard to the serological diagnosis, and the development of strategies for efficient immune protection and eradication of the disease.

Assessment of immunogenicity and protective efficacy of Microsporum canis secreted components coupled to monophosphoryl lipid-A adjuvant in a vaccine study using guinea pigs

Microsporum canis is the most common dermatophyte in pets and is of zoonotic importance but currently there is no effective vaccine available to prevent dermatophytosis. The aim of this work was to assess the immunogenicity and protective efficacy of secreted components (SC) from M. canis adjuvanted with the monophosphoryl lipid-A (MPLA), in a vaccine study using the guinea pig as an experimental model. Animals were vaccinated with either the SC adjuvanted with the MPLA, the MPLA adjuvant alone or PBS three times at two-week intervals, until 42 days prior to M. canis infection. A blind evaluation of dermatophytosis symptoms development and fungal persistence in skin was monitored weekly. The antibody response towards the SC and the levels of Interferon (IFN)γ and Interleukin-4 expressed in peripheral blood mononuclear cells were assessed along or at the end of the study period respectively. The animals that received MPLA had a significantly lower clinical score than those inoculated with PBS. However, no significant difference was observed between the guinea pigs vaccinated with the SC adjuvanted with the MPLA and those having received MPLA alone. The results also showed that vaccination induced a strong antibody response towards the SC and an increase in IFNγ mRNA level. Our results show that the MPLA adjuvant used in this vaccine study can induce per se a partial protection against a M. canis infection. Although they induce a delayed-type hypersensitivity reaction in guinea pigs, the SC do not confer a protection under the present experimental conditions.

Deglycosylation of the 45/47-kilodalton antigen complex of Mycobacterium tuberculosis decreases its capacity to elicit in vivo or in vitro cellular immune responses.

A protection against a challenge with Mycobacterium tuberculosis is induced by previous immunization with living attenuated mycobacteria, usually bacillus Calmette-Guérin (BCG). The 45/47-kDa antigen complex (Apa) present in culture filtrates of BCG of M. tuberculosis has been identified and isolated based on its ability to interact mainly with T lymphocytes and/or antibodies induced by immunization with living bacteria. The protein is glycosylated. A large batch of Apa was purified from M. tuberculosis culture filtrate to determine the extent of glycosylation and its role on the expression of the immune responses. Mass spectrometry revealed a spectrum of glycosylated molecules, with the majority of species bearing six, seven, or eight mannose residues (22, 24, and 17%, respectively), while others three, four, or five mannoses (5, 9, and 14%, respectively). Molecules with one, two, or nine mannoses were rare (1.5, 3, and 3%, respectively), as were unglycosylated species (in the range of 1%). To eliminate the mannose residues linked to the protein, the glycosylated Apa molecules were chemically or enzymatically treated. The deglycosylated antigen was 10-fold less active than native molecules in eliciting delayed-type hypersensitivity reactions in guinea pigs immunized with BCG. It was 30-fold less active than native molecules when assayed in vitro for its capacity to stimulate T lymphocytes primed in vivo. The presence of the mannose residues on the Apa protein was essential for the antigenicity of the molecules in T-cell-dependent immune responses in vitro and in vivo.

Lowering of trichosanthin immunogenicity by site-specific coupling to dextran

Trichosanthin is a type I ribosome-inactivating protein possessing a broad spectrum of biological and pharmacological activities. Therapeutic use of this compound is hampered by its immunogenicity. It was shown earlier that coupling of dextran to trichosanthin can increase plasma half-life and reduce antigenicity. However, the site where dextran attaches to trichosanthin cannot be controlled; ideally, it should be at or near the antigenic determinant. The present study attempted to couple dextran to trichosanthin at a potential antigenic site. By site-directed mutagenesis, two sites, R29 and K173, were replaced by cysteine, and dextran was coupled to the newly created cysteine residues. The dextran–trichosanthin complex retained 50% of abortifacient activity and had a mean residence time in rats 27-fold longer than natural trichosanthin. Acute hypersensitivity reaction in guinea pigs was reduced greatly after coupling of K173C (a trichosanthin mutant with lysine-173 replaced by cysteine) to dextran. Compared with natural trichosanthin, dextran-K173C had a decrease in IgG and IgE response, whereas the coupling of R29C (a trichosanthin mutant with arginine-29 replaced by cysteine) to dextran did not show significant reduction of immunogenicity. This suggests that K173 but not R29 is located at or near an antigenic determinant. This study has demonstrated an alternative approach for mapping of antigenic determinants. The information obtained is also useful in producing an improved trichosanthin derivative for therapeutic use.

Evaluation of hypersensitivity to microencapsulated ampicillin in guinea pigs.

The purpose of this study was to determine if the sustained release of ampicillin from a biodegradable drug-delivery system (microencapsulated ampicillin anhydrate (MEAA)) will increase or decrease the intensity of a hypersensitivity reaction compared with that observed with free drug. Ovalbumin, which is known to elicit a marked hypersensitivity reaction in guinea pigs, and microencapsulated ovalbumin (MOVA) were tested in parallel with ampicillin and MEAA. Guinea pigs were sensitized biweekly by subcutaneous and intramuscular injections of ampicillin, MEAA, ovalbumin, MOVA or placebo microspheres (test articles), each mixed with Freund's adjuvant, and challenged 2 weeks later, intradermally, with the free compounds. In a separate set of experiments, guinea pigs were sensitized by implantation of the same agents in the caudal thigh of anaesthetized animals. Skin allergic reactions were tested at 1 and 3 weeks following local implantation of the test articles. Sera of sensitized guinea pigs were tested for specific IgG antibodies by enzyme-linked immunosorbent assay, and skin samples from the site of the inflammatory reaction were fixed, stained and evaluated histologically. Guinea pigs sensitized systemically with MEAA or MOVA showed smaller, but not statistically different skin allergic response than animals given corresponding free compounds. However, guinea pigs sensitized by local implantation of MEAA showed a significantly lower inflammatory response (P < 0.0001) than those given an equivalent dose of the free drug. Guinea pigs sensitized with placebo microspheres showed a low inflammatory skin reaction which was similar to those sensitized with all doses of MEAA. There was no significant difference in specific IgG antibody response in the sera of guinea pigs sensitized locally with either free or microencapsulated ampicillin or ovalbumin. Histology of skin revealed a milder inflammatory reaction with MEAA or MOVA than with ampicillin or ovalbumin, respectively. We conclude that the encapsulated ampicillin or ovalbumin and subsequent release of each agent will elicit a reduced hypersensitivity reaction in guinea pigs than will the free agent.

Endotoxin induces severe inflammatory reactions with necrosis at sites primed with delayed-type hypersensitivity reactions in guinea pigs.

Guinea pigs immunized with Freund's complete adjuvant received challenge injection of the purified protein derivative of Mycobacterium tuberculosis in the flanks and the corneas to prepare delayed-type hypersensitivity (DTH) reactions. The animals were injected subcutaneously with lipopolysaccharide (LPS) or a synthetic lipid A (LA-15-PP). At the skin site primed with DTH reaction, increased swelling and hemorrhagic reaction followed by a definite necrotic reaction occurred. Severe corneal reactions were also observed in the animals. These findings indicate that bacterial endotoxin modulates DTH reactions and induces severe inflammatory reactions.

Antigen-Specific IgG1-Mediated Epidermal Cell Injury: A Component of Contact Hypersensitivity Reactions in Guinea Pigs, Measurable In Vitro in Full-Thickness Skin Explants

Guinea pigs were sensitized by the topical application of either dinitrochlorobenzene (DNCB) or oxazolone on days 1, 2, 3, and 10. Seventeen days after the first treatment with the sensitizer, full-thickness 1.0-cm2 explants of untreated areas of skin were topically exposed in vitro to these contactants. Compared to the response of skin from control guinea pigs, skin from specifically sensitized animals showed a dose-related increase in the number of epidermal cells containing vacuoles. A specific increase in epidermal microblistering paralleled the increase in epidermal vacuolization. In addition, skin explants from sensitized animals (exposed to the contactant) showed a specific decrease in the incorporation of [14C]leucine. Full-thickness skin explants from unsensitized guinea pigs were sensitized in vitro by the intradermal injection of serum IgG1 fraction from oxazolone-sensitized guinea pigs. In such passively sensitized explants, the specific contactant produced an increase in the number of epidermal vacuoles, an increase in the amount of microblistering, and a decrease in the number of mast cells detectable by Giemsa staining. To elicit this specific response, the concentration of the specific contactant had to be mildly injurious, as well as antigenic. This requirement for nonspecific injury could be met by topically exposing skin explants to a nonspecific irritant followed by a sub-threshold concentration of the specific contactant. In contrast to vacuole formation and blistering, contactant-specific degranulation of mast cells (measured by the decrease in their number) did not require irritant levels of the contactant. These studies show that several components of contact sensitivity reactions can be reproduced in vitro by the passive transfer of sera containing antigen-specific immunoglobulins. Banks of such sera might, therefore, be useful in identifying (in human populations) many pre-existing sensitivities to chemical compounds.

Cloning and expression of the gene for the Avi-3 antigen of Mycobacterium avium and mapping of its epitopes.

The Avi-3 antigen, which is found only in Mycobacterium avium culture sonic extracts, is species specific and results in strong skin test activity in guinea pigs sensitized with heat-killed M. avium. Its gene was cloned by using a previously developed single-probe method and was sequenced. The gene encoded a 194-amino-acid polypeptide with a molecular weight of 21,500. A recombinant Avi-3 antigen expressed in Escherichia coli reacted with monoclonal and polyclonal antibodies raised against the native Avi-3 antigen. To identify epitopes on this protein for immunodiagnostic purposes, various parts of the Avi-3 antigen were expressed as beta-galactosidase fusion proteins, using pUR and pURS expression vectors. The clones screened by both antibody reactivity and T-cell proliferative activity defined fragments with coexisting B- and T-cell epitopes. A B-cell epitope (Asn-176 to Ala-186) and two T-cell epitopes (Glu-75 to Ile-86 and Arg-155 to Leu-164) were thus defined. The synthetic polymerized peptides of the T-cell epitopes were proven to elicit a delayed cutaneous hypersensitivity reaction in guinea pigs. This mapping method would be useful in the development of a subunit vaccine consisting of an immunodominant B-cell epitope linked to a T-cell epitope in the vicinity.

A major T cell antigen of Mycobacterium leprae is a 10-kD heat-shock cognate protein.

Several mycobacterial antigens, identified by monoclonal antibodies and patient sera, have been found to be homologous to stress or heat-shock proteins (hsp) defined in Escherichia coli and yeast. A major antigen recognized by most Mycobacterium leprae-reactive human T cell lines and cell wall-reactive T cell clones is a 10-kD protein that has now been cloned and sequenced. The predicted amino acid sequence of this protein is 44% homologous to the hsp 10 (GroES) of E. coli. The purified native and recombinant 10-kD protein was found to be a stronger stimulator of peripheral blood T cell proliferation than other native and recombinant M. leprae proteins tested. The degree of reactivity paralleled the response to intact M. leprae throughout the spectrum of leprosy. Limiting-dilution analysis of peripheral blood lymphocytes from a patient contact and a tuberculoid patient indicated that approximately one third of M. leprae-reactive T cell precursors responded to the 10-kD antigen. T cell lines derived from lepromin skin tests were strongly responsive to the 10-kD protein. T cell clones reactive to both the purified native and recombinant 10-kD antigens recognized M. leprae-specific epitopes as well as epitopes crossreactive with the cognate antigen of M. tuberculosis. Further, the purified hsp 10 elicited strong delayed-type hypersensitivity reactions in guinea pigs sensitized to M. leprae. The strong T cell responses against the M. leprae 10-kD protein suggest a role for this heat-shock cognate protein in the protective/resistant responses to infection.

Comparison of cross-reactivities of imipenem and other beta-lactam antibiotics by delayed-type hypersensitivity reaction in guinea pigs.

The cross-reactivity of imipenem (IPM) in a delayed-type hypersensitivity (DTH) reaction was investigated by intradermal skin reaction, macrophage migration inhibition tests (MIT) and lymphocyte stimulation tests (LST) in guinea pigs. The animals were immunized with IPM, ampicillin (ABPC) or cephalexin (CEX) using Freund's complete adjuvant. Three sensitized-drugs exhibited the same immunogenicity in the skin reaction. The cross-reactivities in skin reaction among IPM, sulbactam, aztreonam, ABPC and 6-aminopenicillanic acid as penam, and CEX, ceftizoxime, 7-aminocephalosporanic acid as cephem were examined. IPM did not show cross-reactions with any of the tested drugs in three sensitized groups, indicating that the drugs possessed no cross-reactivity with tested beta-lactams in DTH. In MIT, 4 or 5 of 6 animals reacted to be sensitized drugs in each group. The cross-reactivity of IPM- and ABPC-sensitized groups in MIT was similar to that of a skin reaction, however, the CEX-sensitized animals showed broad cross-reactions. Using a kind of test drug, one or two animals in the ABPC-sensitized group showed migration enhancement, but IPM- or CEX-sensitized animals did not exhibit migration enhancement. In LST, most of the animals tested exhibited lymphocyte prolifration by sensitized drugs but a cross-reaction was scarcely observed. The above results suggest that among beta-lactams, IPM has a very low cross-reactivity. LST is considered to be more a useful assay than MIT for in vitro identification of causative drugs in animal models.

Immunological properties of cephalexin-induced delayed type hypersensitivity reaction in guinea pigs.

Immunological properties of delayed-type hypersensitivity (DTH) reaction induced by cephalexin (CEX) in guinea pigs were investigated. The animals were immunized with CEX using Freund's complete adjuvant. The time course of CEX-induced erythema showed some differences compared with that of classical DTH reaction. The erythema appeared at 6 h after intradermal administration of CEX, reached maximum size at 12 to 24 h and to be visible until 72 h. By enzyme-linked immunosorbent assay, anti-CEX antibody was detected in only one of 15 animals tested. Normal animals (recipients) which had received immune sera from CEX-sensitized animals (donors) showed no skin reaction to CEX. In contrast, reaction to CEX was observed in recipient animals which had received a local transfer of lymphocytes or T cells from CEX sensitized animals. In immunopharmacological study, cyclosporin A suppressed the skin reaction but cyclophosphamide did not. Administration of carrageenan, an inhibitor of macrophage function, had no effect on expression of the reaction. Post administration (1 or 15 h) of clemastine, an anti-histamine drug, did not affect the reaction. By histological examination, the infiltrating cell-types at the reaction site were mainly composed of mononuclear cells and neutrophils, but no basophils, indicating that CEX-induced DTH reaction is tuberculin-type DTH and not a cutaneous basophil hypersensitivity reaction.

Mycobacteria in pond waters as a source of non-specific reactions to bovine tuberculin in New Zealand

When 71 samples were collected from ponds throughout New Zealand, 35 (49.3%) were found to contain mycobacteria. The majority of these strains (62.9%) belonged to a homogeneous group (tentative designation H-Group, which differed from any known mycobacterial species. Mycobacteria of this H-group had also been found in sphagnum vegetation growing in the immediate vicinity of many of the ponds. H-Group mycobacteria induce sensitization in guinea pigs against bovine tuberculin. The PPD sensitin prepared from these mycobacteria gave rise to larger reactions in guinea pigs than did bovine tuberculin when used in the same concentrations (500 and 50 TU). The possible sensitization of cattle to bovine tuberculin via drinking water containing H-Group mycobacteria, is discussed. The larger size of the delayed hypersensitivity reactions in guinea pigs using the same concentrations of bovine and homologous tuberculin, suggests that comparative intradermal testing might enable this non-specific reaction to be distinguished from the specific reaction developed during bovine tuberculosis infection in cattle.

Measurement of cutaneous blood flow velocity in delayed hypersensitivity reactions in guinea pigs

The blood flow velocity in the skin of the flanks of guinea pigs can be measured precisely by laser Doppler velocimetry, provided the animal is mildly sedated. The probe holder can be attached to the shaved skin with cyanoacrylate glue and can be detached after completion of blood flow velocity measurements by application of acetone without harming the animal. The technique is pain-free and non-invasive: it can be used to make serial measurements on the same animal. The system has demonstrated hyperaemia in delayed-type responses to tuberculin PPD, ovalbumin and Keyhole Limpet Haemocyanin with immunological specificity. The time course of evolution and resolution of the vasomotor response accords with previous experience. The method can detect hyperaemia-inducing mediators in the supernatant of antigen or mitogen stimulated lymphocyte cultures and could be used for their bioassay.

Relationship between basophils and eosinophils in cutaneous basophil hypersensitivity reactions in guinea pigs and susceptibility to Trichostrongylus colubriformis infection

Guinea pigs with genetically determined resistance to primary infection with the nematode parasite Trichostrongylus colubriformis had more basophil and eosinophil leucocytes in cutaneous basophil hypersensitivity (CBH) reactions to keyhole limpet haemocyanin (KLH) than T. colubriformis susceptible guinea pigs. In outbred guinea pigs there was a significant correlation between the number of eosinophils in CBH reactions to KLH after 24 h and resistance to primary infection with T. colubriformis. These results suggest that examination of cutaneous hypersensitivity reactions to non-parasite antigens might be useful in predicting inherent resistance or susceptibility to parasite infection.

Suppressive effects of transdermal clonidine administration on contact hypersensitivity reactions in guinea pigs

In the present study, immunopharmacological effects of clonidine-TTS on allergic contact dermatitis (ACD) to non-related, established contact sensitizers were investigated in guinea pigs. First, to evaluate the hypotensive effect of clonidine-TTS in guinea pigs, intra-arterial blood pressure was recorded. After 4 day of treatment with one (or two) TTS per animal, a reduction of arterial blood pressure from 71 ± 1 to 51 ± 2 mm Hg was observed. We subsequently assessed the effects of clonidine-TTS on contact hypersensitivity reactions to 2,4-dinitrochlorobenzene (DNCB) and 4-ethoxymethylene-2-phenyl-oxazolone (Ox). This study indicates that clonidine-TTS suppressed the elicitation of contact hypersensitivity reactions. The observed immunosuppressive effect of clonidine may account for the relatively weak hypersensitivity reactions to this drug in experimental animal studies. Further studies are needed to determine whether such findings are of relevance to the clinical use of clonidine in patient populations.

Retained T-cell reactivity of rye grass pollen extract following cleavage with cyanogen bromide and nitrothiocyanobenzoic acid.

Rye grass pollen extract was fragmented by sequential treatments with cyanogen bromide and 2-nitro-5-thiocyanobenzoic acid, and a fraction containing fragments of molecular weight greater than 10,000 Mr was isolated. The in vitro reactivity of the extract with specific IgE was extensively reduced by fragmentation. Less reduction in activity was shown either by skin testing or by inhibition of an extract-specific IgG-binding assay. Reactivity with, and ability to induce, extract-specific mouse T cells were retained by the fragment preparation, and the ability to cause transformation of lymphocytes from atopic donors was unchanged. Fragments did not induce extract-specific IgG antibody in mice, were unable to stimulate the production of T-helper cells which could collaborate in an adoptive cell-transfer system, and did not induce delayed hypersensitivity reactions in guinea pigs. The possibility that such T-cell-reactive modified allergens (T'allergoids) might be used to stimulate selectively T-cell subsets and, therefore, could be used to advantage in immunotherapy is discussed.

Purification, characterization and identification of a 32 kDa protein antigen of Mycobacterium bovis BCG

An immunogenic protein called P32 has been purified from Sauton zinc deficient culture filtrate of Mycobacterium bovis BCG using successively hydrophobic chromatography on Phenyl-Sepharose, ion exchange on DEAE-Sephacel and molecular sieving on Sephadex G-100. The final preparation was found to be homogeneous as based on several analyses. This P32 protein was a constituent of BCG cells grown in normal conditions. It represented about 3% of the soluble fraction of a cellular extract, and appeared as the major protein released in normal Sauton culture filtrate. This protein was found to have a molecular weight of 32000 by SDS-polyacrylamide gel electrophoresis and in molecular sieving. Its amino acid composition showed an abundance of acidic amino acids (or their amides). The NH2-terminal amino acid sequence (6 amino acids) was determined. Purified P32 was tested by various crossed immunoelectrophoresis techniques, and was shown to belong to the antigen 85 complex in the reference system for BCG antigens. It was more precisely identified as antigen 85A. The protein antigen elicited a weak delayed hypersensitivity reaction in guinea pigs sensitized with heat-killed or living BCG. No delayed hypersensitivity reaction was observed in living BCG sensitized mice, however, it induced significant amounts of gamma Interferon in cultured spleen cells from BCG-sensitized mice. Moreover, P32 either pure or as part of BCG soluble extract promoted substantial antibody levels when injected in rabbits.

Equivalent Mortality in Normal and Athymic Mice Infected with Naegleria fowleri

Naegleriafowleri, a normally free-living amoeba, is the causative agent of primary amoebic meningoencephalitis (PAM), and since its first description in 1965 over 100 cases have been reported worldwide (John, 1982, Annual Review of Microbiology 36: 101-123). A perplexing aspect of PAM is that pathogenic N. fowleri are often isolated from recreational waters, yet exposed individuals only rarely develop PAM and it occurs in apparently healthy humans. Although affected individuals might have some underlying immune dysfunction it is most likely PAM is the result of environmental or genetic factors that serve to enhance virulence of amoebae. Studies have been directed at elucidating the role of the immune system in resistance to PAM. In laboratory animals, immunization by a variety of routes with Naegleria antigen can heighten resistance to a subsequent lethal infection (John et al., 1977, Infection and Immunity 16: 817820; Thong et al., 1978, The American Journal of Tropical Medicine and Hygiene 27: 238-240). Investigations of cellular immune components showed that Naegleria antigen could elicit a delayed hypersensitivity reaction in guinea pigs (Diffley et al., 1976, Zeitschrift fur Parasitenkunde 49: 133-137) and macrophage inhibition by lymphokine macrophage inhibition factor (Cursons et al., 1980, Infection and Immunity 29: 408-410). Although Naegleria antigen can evoke an immunological response which may lessen susceptibility to a lethal infection, little is known about the role of the humoral and cellular immune systems in innate resistance to PAM. Congenitally athymic (nude [nu/nu]) mice are deficient in T-cell-dependent cell-mediated immunity and in the humoral response to Tcell-dependent antigens (Wortis, 1974, Contemporary Topics in Immunobiology 3: 243-263). Investigations on the pathogenicity of N. fowleri in nude mice would serve as an useful aid for delineating the role of T-cell-dependent activities in the innate resistance against development of PAM. Providing that the T-cell-dependent arm of the immune system is a significant factor in innate resistance, it would be expected nude mice would show increased susceptibility and the course of PAM would be more rapid compared to normal mice. The purpose of this study was to determine if the development of PAM and the course of the disease was more rapid in congenitally athymic mice than in phenotypically normal mice. Mice were also infected with trophozoites of Naegleria lovaniensis, an amoeba physiologically and morphologically similar to N. fowleri, but nonpathogenic in immunocompetent mice (Stevens et al., 1980, International Journal for Parasitology 10: 51-64). The Lee strain of N. fowleri and N. lovaniensis were cultivated in tubes containing Chang's medium (De Jockheere, 1977, Applied and Environmental Microbiology 33: 751-757). Amoebae in late exponential growth were enumerated by hemacytometer counts, centrifuged (1,000 g for 15 min), and adjusted to the desired density in Chang's medium so that 25 1l delivered the appropriate number of amoebae (Fig. 1). Mice were of Swiss Webster background and congenitally athymic mice were obtained by mating homozygous (nu/nu) males with heterozygous (+/nu) females. Mice were maintained under specific pathogen free conditions in an area separate from other laboratory animals. For infection, mice of either sex and approximately 12 wk old were anesthetized with ether and 25 Al of an amoebae suspension was delivered into the nares with a micropipet. Development of PAM was evident by typical symptoms of anorexia, excitability, and ruffling of neck hair, which were followed by convulsions and death. PAM was

Induction and down-regulation of conjunctival type-I hypersensitivity reactions in guinea pigs sensitized topically with fluoresceinyl ovalbumin.

Type-I hypersensitivity reactions were induced in guinea pigs by repeated topical/conjunctival application of fluoresceinyl ovalbumin (FL-OA). The ocular reactivity in early responding animals was maximal between 16 and 25 days and decreased exponentially thereafter. Desensitized eyes responded minimally to compound 48/80 but maximally to histamine. Unilateral sensitization and challenge with FL-OA produced desensitization of the immunized eye, but the reactivity of the contralateral eye persisted. A significant reduction in conjunctival stained mast cells was found in repeatedly challenged eyes. The desensitization therefore was due to a loss of reactive mast cells. Systemic infection with Ascaris suum, after repeated topical challenge with FL-OA had led to desensitization, produced a reappearance of type-I hypersensitivity reactions toward both FL-OA and ascarid antigens.