The use of an indirect fluorescent antibody test for detecting Pneumocystis carinii.

Pneumocystis carinii pneumonia is a major cause of morbidity and mortality in immunocompromised patients. An indirect fluorescent antibody (IFA) test has been developed using monoclonal antibodies specific for antigens on the surface of P carinii. We tested the sensitivity and specificity of this IFA test for detecting P carinii in respiratory specimens of immunocompromised patients with pulmonary symptoms undergoing bronchoscopy. Both the bronchial wash and bronchoalveolar lavage specimens of patients with and without P carinii pneumonia were studied. The bronchoalveolar lavage and bronchial wash specimens were examined using modified Wright-Giemsa and methenamine silver stains. In addition, aliquots of the specimen were fixed and stained with IFA and read with a fluorescent microscope. Fifty-nine patients were found to have P carinii organisms. The bronchial wash specimen has been shown to be less sensitive than the bronchoalveolar lavage specimen for detecting the presence of P carinii. In the bronchial wash specimen from these 59 patients, only 60% had positive modified Wright-Giemsa stains, and 70% had positive methenamine silver stains. The IFA stain was positive in 93% of the specimens tested (significantly higher than the other two stains). There was only one false-positive IFA test result among the 54 patients tested with negative results. We found the IFA stain to be superior to conventional stains when examining less-than-adequate specimens, such as those from bronchial washes.

Visceral leishmaniasis in congenic mice of susceptible and resistant phenotypes: immunosuppression by adherent spleen cells.

Visceral leishmaniasis is one of several parasitic diseases of humans characterized by immune suppression. A murine model of disseminated leishmaniasis utilizing inbred strains of specific genetic constitution was used to study the mechanisms of immunosuppression elicited during the course of infection. Resistant (Lshr) and susceptible (Lshs) strains of mice were challenged with amastigotes of Leishmania donovani and evaluated as to immune status at intervals between 2 and 40 weeks after challenge. The proliferative responses of splenic lymphocytes to T-cell mitogens, a B-cell mitogen, and parasite antigens were measured to evaluate the relative immune status of parasitized mice and noninfected control mice. Lymphocytes from resistant C3Heb/FeJ (C3H) mice responded normally to concanavalin A and phytohemagglutinin throughout the course of infection. Parasite antigen responses appeared 2 weeks after challenge of C3H mice and remained vigorous for periods up to 6 months. In contrast, immune suppression during infection was profound in both the curing (C57B1/10) and noncuring (B10.D2) phenotypes of Lshs congenic mice. Both Lshs strains developed severe infection as evidenced by high parasite burdens in the liver and spleen 4 to 5 weeks after challenge; splenic lymphocytes taken from these mice between 2 and 8 weeks became increasingly unresponsive to the T-cell mitogens as well as to parasite antigens. The noncuring B10.D2 mice which suffered chronic infection continued to be suppressed for as long as 40 weeks. C57B1/10 (curing) mice, in contrast, cleared infection between 12 and 16 weeks. After spontaneous recovery or elimination of parasites by antimonial drug therapy, the response of spleen cells to T-cell mitogens or parasite antigens were restored to normal. The spleen cells from the Lshs strains of mice obtained during the height of infection suppressed the proliferative responses of spleen cells from their uninfected counterparts upon cocultivation in vitro. Removal of adherent cells from the suppressive spleen cell populations restored normal mitogen responses. On the basis of adherence characteristics, phagocytosis, and morphology, the suppressor was identified as a macrophage population which appears to be responsible for a nonspecific immunosuppression of Lshs mice with significant parasite burdens of L. donovani.

Visceral leishmaniasis in congenic mice of susceptible and resistant phenotypes: T-lymphocyte-mediated immunosuppression.

This paper continues a comparative study (A. D. Nickol and P. F. Bonventre, Infect. Immun. 50:160-168, 1985) describing immune responses exhibited by congenic, Lshs mouse strains C57B1/10 (cure) and B10.D2 (noncure) during the course of disseminated leishmaniasis. We report that sublethal whole-body irradiation of B10.D2 mice before challenge with Leishmania donovani converted the noncuring mice to a curing phenotype. Splenic lymphocytes from L. donovani-infected B10.D2 mice failed to proliferate in response to parasite antigen stimulation in vitro. Splenic lymphocytes from irradiated, cured B10.D2 mice regained the capacity to respond to the parasite antigen stimulus. Transfer of T cells but not B cells from L. donovani-infected B10.D2 mice prevented the acquisition of immunity and recovery from infection in X-irradiated mice. In addition, a splenic T-cell population from L. donovani-infected B10.D2 mice suppressed the proliferation in vitro of parasite antigen-stimulated lymphocytes of irradiation-cured B10.D2 mice. Suppressor T cells were not demonstrable in the spleens of spontaneously cured C57B1/10 mice. Splenic lymphocytes from infected B10.D2 mice were deficient in the production of macrophage-activating factor (MAF) upon stimulation by L. donovani antigens in vitro. Deficient MAF production was specific for parasite antigen stimulation, because MAF production subsequent to concanavalin A stimulation of splenic lymphocytes from infected B10.D2 mice was not suppressed. The data suggest that a genetically based immunological defect in B10.D2 mice prevents the acquisition of effective cell-mediated immunity and subsequent elimination of L. donovani from tissue macrophages. The immunological deficit, not apparent in the curing C57B1/10, appears to be caused by the development of parasite antigen-specific suppressor T cells during the course of the disseminated leishmaniasis.

Immunosuppression associated with visceral leishmaniasis of hamsters.

Immunosuppression was demonstrated during the course of Leishmania donovani infection of outbred and inbred hamsters. Proliferative responses of splenic lymphocytes to the mitogen concanavalin A (Con A) and to promastigote antigens were used as indicators of immune responsiveness. Although splenic lymphocyte proliferative responses to parasite antigens were demonstrable 3 weeks after challenge, antigen specific lymphocyte responses diminished as the infection progressed. Two types of immunosuppression were demonstrable. The first was a non-specific anergy of splenic lymphocytes to Con A stimulation. Thus, spleen cells from infected animals did not actively suppress the Con A responses of normal lymphocytes in mixed cultures A second immunosuppression mechanism, specific for leishmania antigens was mediated by a nylon wool non-adherent cell population. The suppressor, tentatively identified as a T cell population, inhibited the proliferation of parasite antigen sensitized responder lymphocytes in mixed culture. Elimination of the parasite burden by glucantime therapy restored responsiveness of lymphocytes to parasite antigens. Con A responses, however, remained suppressed 1 week after drug cure.

Development of protective immunity against bacterial and viral infections in tumor-bearing mice coincident with suppression of tumor immunity.

This report addresses the question whether Meth A (methylcholanthrene-induced fibrosarcoma) tumor bearing Balb/c mice are able to develop specific antimicrobial immunity. Although specific suppressor T lymphocytes appeared during tumor growth which prevented expression of antitumor immunity, the development of protective immunity to L monocytogenes, S. pneumoniae or ectromelia virus infections was unimpaired. The Meth A tumor produced a soluble immunosuppressive factor which inhibited lymphocyte and macrophage functions in vitro. Tumor growth failed to inhibit the formation of immunoglobulin essential to antipneumococcal immunity, or the development of a specific acquired cellular resistance of primary importance in immunity to listeria and ectromelia virus infections. That tumor growth did not interfere with the development of cell mediated immunity was demonstrated by the effective transfer of antilisteria immunity by immune spleen from tumor-bearing mice.

Acquired immunity against mouse typhoid: genetic restriction and comparative efficacy of ribosomal and conventional vaccines.

Strains of mice immunised with a ribosomal preparation of Salmonella typhimurium varied in their ability to survive an intraperitoneal challenge of virulent S. typhimurium. Immunised nude (nu/nu), heterozygous (nu/+) mice, strain C57Bl/6J and strain CBA/J succumbed to lethal infection whereas strains C3D2F1/J, B6D2F1/J and A/J, and Swiss mice were fully protected. Strains DBA/2J and C3H/HeJ were partially protected. Enumeration of the systemic bacterial population after challenge with S. typhimurium indicated that all immunised mouse strains were able to reduce the infectious load. S. typhimurium was rapidly inactivated in the peritoneal cavity of immunised mice, effectively reducing the challenge and thereby limiting the number of organisms available to seed the systemic circulation. This response was also obtained in immunised athymic mice and was therefore attributed to a T-cell independent antibody response. Organisms that escaped destruction in the peritoneal cavity multiplied rapidly in the reticuloendothelial organs. Only mice from strains genetically capable of developing an effective cell-mediated immune response to the antigenic stimulus provided by the challenge organism itself survived infection. The efficacy of ribosomal immunisation was compared with immunisation by heat-killed bacteria, viable attenuated and viable virulent organisms by enumeration of the systemic bacterial population after intravenous challenge with S. typhimurium. Vaccination with ribosomal preparations or heat-killed organisms provided limited protection whereas immunity provided by viable organisms was far superior.

Impaired resistance to bacterial infection after tumor implant is traced to lactic dehydrogenase virus.

A BALB/c mouse-passaged methylcholanthrene-induced fibrosarcoma tumor caused severe impairment of resistance to systemic listeriosis. Depressed resistance expressed immediately after tumor implantation was traced to inadvertent association of tumor with lactic dehydrogenase virus. Tumor cured of virus was totally inactive.

Anomalous high native resistance to athymic mice to bacterial pathogens.

Congenitally athymic (nude) mice exhibited an anomalous high resistance against infections with the facultative intracellular parasite Listeria monocytogenes and other bacterial pathogens. Protection against lethal infection was demonstrated to result from the presence of naturally occurring activated macrophages in the reticuloendothelial organs of the nude mice. This was exemplified after intravenous challenge by enhanced bacterial clearance from the blood and augmented bacterial killing in the spleens and livers of nude mice as compared with immunologically competent control mice. Resident peritoneal macrophages of nude mice were not activated in terms of phagocytic, bactericidal, or tumoricidal potential. The development of activated fixed tissue macrophages appears to arise as a result of the T-lymphocyte deficiency since thymus implantation abrogated the enhanced resistance of nude mice. Antibiotic elimination of intestinal bacteria also modified resistance to bacterial infection, indicating a role of environmental factors on macrophage activation. Several possible mechanisms leading to macrophage activation and heightened resistance to infection in nude mice are offered.