To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunization of rodents and humans with irradiation-attenuated malaria sporozoites confers preerythrocytic stage-specific protective immunity to challenge infection. This immunity is directed against intrahepatic parasites and involves T cells and interferon y, which prevent development of exoerythrocytic stages and subsequent blood infection. The present study was undertaken to determine how protective immunity is achieved after immunization of rodent hosts with irradiated Plasmodium berghei sporozoites. We present evidence that irradiated parasites persist in hepatocytes of rats and mice for up to 6 months after immunization. A relationship between the persistence of parasites and the maintenance of protective immunity was observed. Protective immunity was abrogated in irradiated-sporozoite-immunized rats following the application of chemotherapy to remove preexisting liver parasites. Additionally, protective immunity against sporozoite challenge was established in rats vaccinated with early and late hepatic stages of irradiated parasites. These results show that irradiation-attenuated sporozoites produce persistent intrahepatic stages in vivo necessary for the induction and maintenance of protective immunity.

Plasmodium berghei sporozoites delivered by mosquito bite were more infectious to outbred CD-1 mice than were sporozoites delivered by intravenous inoculation. The route of challenge also affected vaccine efficacy. In view of these findings and the fact that mosquito bites are the natural mode of sporozoite delivery, infectious mosquito bites should be considered the challenge protocol of choice for sporozoite vaccine efficacy trials.

Despite the low susceptibility of BALB/c mice to hepatic infection by Plasmodium berghei, this animal model is routinely used to investigate the basic biology of the malaria parasite and to test vaccines and the immune response against exoerythrocytic (EE) stages derived from sporozoites. A murine model in which a large number of EE parasites are established would be useful for furthering such investigations. Therefore, we assayed six mouse strains for susceptibility to erythrocytic and hepatic infections. The administration of 50 sporozoites by intravenous inoculation was suflicient to establish erythrocytic infections in five of five C57BL/6 mice compared with 10,000 sporozoites required to infect 100%/ of BALB/c mice. To assay for hepatic infections, mice received an intravenous inoculum of 106 sporozoites, and liver sections for light microscopy and histology were obtained at 29 and 44 h postinoculation. EE parasites were visualized by immunofluorescence, using an antibody to a P.fakciparum heat shock protein. The mean number of EE parasites per 100 cm2 for C57BL/6 and A/J strains was significantly higher than that for BALB/c (2,190 260, 88 38, and 6 2, respectively). The proportion of inoculated sporozoites transforming into liver schizonts was 8.2% in C57BL/6 and <1% in C3HVHeJ, DBA/1, and Swiss CD-1/ICR mice. Nonspecific inflammatory infiltrates around EE parasites were less prevalent in liver sections from C57BL/6 mice than in those from BALB/c mice, which contributed to the decrease in developing EE stages in BALB/c mice. These data indicate that the C57BL/6-P. berghei system is preferable for investigating the biology and immunology of liver stage parasites.

The elimination of liver-stage malaria parasites by nitric oxide (NO)-producing hepatocytes is regulated by T cells. Both CD8 ؉ and CD4 ؉ T cells, which surround infected hepatocytes, are evident by 24 h after sporozoite challenge in Brown Norway rats previously immunized with irradiated Plasmodium berghei sporozoites. While the number of CD4 ؉ T cells remained the same beyond 24 h postchallenge, the number of CD8 ؉ T cells increased three-and sixfold by 31 and 44 h, respectively. This increase in the number of CD8 ؉ T cells correlated with a decrease in the number of intrahepatic parasites. In immunized rats, intrahepatic parasites were reduced in number by 31 h after sporozoite challenge and cleared from the liver by 44 h, as visualized by P. berghei-specific DNA in situ hybridization. If immunized rats were treated with aminoguanidine, a substrate inhibitor of NO synthase, at the time of challenge, liver-stage protection was blocked, as shown by the increase in parasite liver burden. Further histological examination of infected livers from immunized animals treated with aminoguanidine revealed fewer and smaller cellular infiltrates surrounding the infected hepatocytes, and the number of CD8 ؉ T cells that normally accumulate within the infiltrates was drastically reduced. Consequently, the infected hepatocytes were not cleared from the liver. We hypothesize that the early production of NO may promote the influx and/or enhance local proliferation of malaria parasite-specific CD8 ؉ T cells or a CD8 ؉ T-cell subset which is required for parasite clearance.