Pneumocystis causes pneumonia in immunodeficient hosts but also likely causes infection in healthy hosts. To characterize infection in healthy mice, we developed and validated a real-time polymerase chain reaction assay for quantitation of Pneumocystis carinii f. sp. muris. In healthy mice exposed to Pneumocystis-infected animals, organisms were first detected at 2-3 weeks, peaked at 5-6 weeks, and were cleared by 7-9 weeks. The peak organism load in healthy animals was 2-3 logs lower than that in immunodeficient animals. This approach should facilitate studies of anti-Pneumocystis immune mechanisms in healthy hosts and provide insights into the development of Pneumocystis pneumonia in immunodeficient hosts.
A method for reliable quantification of Pneumocystis carinii in research models of P. carinii pneumonia (PCP) that is more convenient and reproducible than microscopic enumeration of organisms would greatly facilitate investigations of this organism. We developed a rapid quantitative touchdown (QTD) PCR assay for detecting P. carinii f. sp. carinii, the subspecies of P. carinii commonly used in research models of PCP. The assay was based on the single-copy dihydrofolate reductase gene and was able to detect <5 copies of a plasmid standard per tube. It was reproducibly quantitative (r ؍ 0.99) over 6 log values for standards containing >5 copies/tube. Application of the assay to a series of 10-fold dilutions of P. carinii organisms isolated from rat lung demonstrated that it was reproducibly quantitative over 5 log values (r ؍ 0.99). The assay was applied to a recently reported in vitro axenic cultivation system for P. carinii and confirmed our microscopy findings that no organism multiplication had occurred during culture. For all cultures analyzed, QTD PCR assays showed a decrease in P. carinii DNA that exceeded the expected decrease due to dilution of the inoculum upon transfer. In conclusion, a rapid, sensitive, and reproducible quantitative PCR assay for P. carinii f. sp. carinii has been developed and is applicable to in vivo as well as in vitro systems. The assay should prove useful for conducting studies in which quantification of organism burden or growth assessment is critical, such as in vitro antimicrobic susceptibility testing or in vivo immunopathological experiments.
Because thymidine metabolism is a potential target for therapy of Pneumocystis pneumonia, it was investigated whether Pneumocystis organisms have a salvage pathway for thymidine by administering 5-bromo-29-deoxyuridine (BrdU) to mice and rats with Pneumocystis pneumonia. Although BrdU incorporation was detected in host cells, no incorporation was seen in Pneumocystis organisms infecting either rats or mice. This suggests that Pneumocystis organisms do not have a salvage pathway for thymidine, and that inhibitors of de novo synthesis, such as thymidylate synthase inhibitors, may be effective drugs for treating Pneumocystis pneumonia. INTRODUCTIONPneumocystis jirovecii is an atypical fungus that infects patients suffering from a variety of immunosuppressive diseases, such as AIDS and cancer (Kovacs et al., 2001a;Roblot et al., 2002). Although there has been a decrease in morbidity among AIDS patients due to the availability of potent anti-retroviral regimens, Pneumocystis pneumonia (PCP) continues to be the most common life-threatening opportunistic infection among HIV-infected patients. Therapy with the combination of trimethoprim and sulfamethoxazole (TMP/SMX) is the preferred approach for primary prophylaxis in susceptible patients (i.e. for AIDS patients with CD4 + count levels <200 mm 23 ) as well as for treatment of active PCP (Kovacs & Masur, 2000;Phair et al., 1990). These drugs target the enzymes dihydrofolate reductase and dihydropteroate synthase (DHPS), respectively. Other drugs including dapsone, atovaquone and pentamidine are alternatives to TMP/SMX, but they are either more toxic or less active against P. jirovecii (Hughes Hughes, 1998;Kovacs et al., 2001a;Sattler et al., 1988; Vasconcelles et al., 2000). Unfortunately, recent studies have identified mutations in the gene encoding DHPS suggesting the emergence of resistance in P. jirovecii to sulfa drugs (dapsone and SMX) (Helweg-Larsen et al., 1999;Kazanjian et al., 2000;Ma et al., 1999;Navin et al., 2001). Development of new drugs to treat or prevent PCP thus remains an important priority for research.P. jirovecii is closely related to Pneumocystis species that infect rats (Pneumocystis carinii) and mice (P. carinii f. sp. muris). A better understanding of the metabolic pathways of Pneumocystis species may lead to the identification of novel therapeutic targets. Thymidine metabolism has been partially characterized in Pneumocystis: thymidylate synthase has been identified, isolated, cloned and crystallized (Anderson et al., 2000; Edman et al., 1989;Kovacs et al., 1990;Santi et al., 1991). Thus, Pneumocystis organisms possess de novo synthetic pathways for thymidine, a target of chemotherapy in other infections. Therapies that target de novo thymidylate synthesis can potentially be bypassed using thymidine salvage pathways. To see if Pneumocystis organisms possessed a salvage pathway for thymidine, 5-bromo-29-deoxyuridine (BrdU) was administered for 14 days to Pneumocystis-infected rats and mice. BrdU is a thymidine analogue which can be incorpora...
X-linked Hyper IgM Syndrome (HIM) is a rare congenital immunodeficiency recently demonstrated to be caused by a mutation in the gene encoding CD40 ligand. These patients are susceptible to Pneumocystis carinii pneumonia, which implies an important role for CD40L in host defense against P. carinii. In this study we undertook to investigate whether treatment of P. carinii infected scid mice with murine recombinant CD40 ligand trimer (muCD40L) for 21 days would facilitate clearance of the organisms. We found no significant difference in organism burden in treated compared to control animals. Therefore in this model treatment with muCD40L alone is ineffective in clearing P. carinii infection.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.