Open-label efficacy and safety trial of 42 days of 566C80 for Pneumocystis carinii pneumonia in AIDS patients.

Pneumocystis carinii pneumonia continues to be a cause of morbidity and mortality in AIDS patients. Current therapies have a high rate of toxicity and failure. Compound 566C80 is a 1-4,hydroxynaphthoquinone with potent antiprotozoal activity which shows good efficacy and safety in 21-day treatment trials of P. carinii pneumonia (PCP) in AIDS patients. Because there is a generally high recurrence rate after treatment of PCP and there may be a possible advantage in decreasing the P. carinii burden in the lung with extended anti-Pneumocystis therapy, we performed an open label-trial of the safety and efficacy of 42-day therapy with 566C80 for PCP in AIDS patients. Ten patients were enrolled and one was lost to follow-up. Eight of the remaining nine patients successfully completed 42 days of therapy with minimal toxicity. This trial suggests that 566C80 for 42 days can be an effective, safe, and well-tolerated oral therapy for PCP in AIDS patients.     

A radiometric method for objectively screening large numbers of compounds against Pneumocystis carinii in vitro.

A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.     

Composition of Pneumocystis carinii Neutral Lipids and Identification of Coenzyme Q10 as the Major Ubiquinone Homolog

The lipids of purified preparations of Pneumocystis carinii carinii freshly isolated from infected rats were analyzed and compared with those of whole lungs from normal and methylprednisolone-immunosuppressed uninfected rats. In this study, the neutral lipid fraction was examined in detail; the relative concentrations of individual classes making up this fraction were quantified. Of particular interest was the nature of the organism's ubiquinone (coenzyme Q, CoQ) fraction because atovaquone, a hydroxynaphtho-quinone (566C80) analog of ubiquinone, is efficacious in the treatment of P. carinii pneumonia. The ubiquinone concentration in both P. carinii and lung tissues was relatively low compared to that present in rat heart and liver tissues. Two homologs were identified in the organism: CoQ₁₀ was the predominant homolog with lesser amounts of CoQ₉ present. In contrast, the lungs of normal and immunosuppressed uninfected rats had CoQ₉ and lesser amounts of CoQ₈, but no detectable CoQ₁₀. Furthermore, radiolabeled mevalonic acid was incorporated in vitro into the ubiquinone fraction of P. carinii indicating that the organism has the de novo branch of the isoprenoid biosynthetic pathway leading to polyprenyl formation. Hence, it was concluded that CoQ₁₀ (if not both CoQ1₁₀ and CoQ₉) in P. carinii as not scavenged from the host but was synthesized by the organism. Although lung tissues contained substantial free fatty acids, the organism was enriched in these lipids. The high concentration of free fatty acids and relatively low level of triglycerides in P. carinii suggest that fatty acids may represent major carbon sources for ATP production by the organism.     

Anti-Pneumocystis carinii and antiplasmodial activities of primaquine-derived imidazolidin-4-ones

A series of primaquine-derived imidazolidin-4-ones were screened for their in vitro activity against Pneumocystis carinii and Plasmodium falciparum W2 strain. Most compounds were active against P. carinii above 10 microg/mL and displayed slight to marked activity. The imidazolidin-4-ones most active against P. carinii were also those most active antiplasmodial agents, in the muM range. One of the tested imidazolidin-4-ones was slightly more active than the parent primaquine and may represent a lead compound for the development of novel anti-P. carinii 8-aminoquinolines with increased stability and resistance to metabolic inactivation.     

Indicators of Pneumocystis carinii viability in short-term cell culture.

Growth of P. carinii in culture has been difficult to document in the absence of reliable methods for distinguishing live from dead organisms. We studied three markers of cell function in P. carinii during the course of short-term cell culture, and correlated these with the number of P. carinii present in culture supernatants. The markers were glucan synthase activity, esterase activity and cell membrane integrity. The last two were assessed by double staining with fluorescein diacetate and propidium iodide followed by analysis of fluorescence using flow cytometry. The rise in P. carinii number after 5 to 7 days in culture was associated with increased glucan synthase activity. Flow cytometry analysis of day-6 P. carinii cultures confirmed that over 80% of the organisms catalyzed the conversion of fluorescein diacetate to fluorescein and excluded propidium iodide. The demonstration of three indices of metabolic activity in an expanding P. carinii population has confirmed the efficacy of a culture system as a means of sustaining the continued activity, albeit short-lived, of viable P. carinii.     

Clearance of Pneumocystis carinii in mice is dependent on B cells but not on P carinii-specific antibody

Both CD4(+) T cells and B cells are critical for defense against Pneumocystis carinii infection; however, the mechanism by which B cells mediate protection is unknown. We show that P. carinii-specific IgM is not sufficient to mediate clearance of P. carinii from the lungs since CD40-deficient mice produced normal levels of specific IgM, but were unable to clear the organisms. Using chimeric mice in which the B cells were deficient in CD40 (CD40KO chimeras) we found that clearance of P. carinii infection is delayed compared with wild-type controls. These CD40KO chimeric mice produced normal levels of P. carinii-specific IgM, but did not produce class-switched IgG or IgA. Similarly, clearance of P. carinii was delayed in mice deficient in FcgammaRI and III (FcgammaRKO), indicating that P. carinii-specific IgG partially mediates opsonization and clearance of P. carinii. Opsonization of organisms by complement did not compensate for the lack of specific IgG or FcgammaR, since C3-deficient and C3-depleted FcgammaRKO mice were still able to clear P. carinii. Finally, micro MT and CD40KO chimeric mice had reduced numbers of activated CD4(+) T cells in the lungs and lymph nodes compared with wild-type mice, suggesting that B cells are important for activation of T cells in response to P. carinii. Together these data indicate that P. carinii-specific IgG plays an important, but not critical, role in defense against P. carinii. Moreover, these data suggest that B cells also mediate host defense against P. carinii by facilitating CD4(+) T cell activation or expansion.     

Studies of potential filaricides: Part 14--Activity of 1-iso-butoxycarbonyl-4-methylpiperazine against experimental filariasis

The synthesis and filaricidal activity of 1-iso-butoxycarbonyl-4-methylpiperazine against Litomosoides carinii in Sigmodon hispidus and Dipetalonema viteae in Mastomys natalensis is reported. At an intraperitoneal or oral dose of 3 mg/kg given for 6 days, the compound removed 91% of the circulating microfilariae but had no effect on adult L. carinii. However, it killed all microfilariae and adults of D. viteae at a subcutaneous dose of 50 mg/kg given for 6 days. The compound also possessed chemoprophylactic activity against the larvae of L. carinii and D. viteae at a dose of 30 and 50 mg/kg respectively.     

Inhibitory potential of prodomain of Plasmodium falciparum protease serine repeat antigen 5 for asexual blood stages of parasite

Plasmodium falciparum serine repeat antigen 5 (SERA5) is a target for both drug and vaccine intervention against malaria. SERA5 is secreted in the parasitophorous vacuole where it is proteolytically processed before schizont rupture. Among the processed products is a 50.8-kDa central domain of the protease, which possesses chymotrypsin-like activity and consists of a 28.9-kDa catalytic domain with a 21.9-kDa N-terminal prodomain, which remain attached together. Because SERA5 has been implicated in merozoite egress from host erythrocytes, the effect of the prodomain and a heptapeptide derived from its C-terminus spanning from D(560) to F(566) (DNSDNMF) on parasite growth was studied. When E. coli-expressed prodomain was incubated with parasite culture, a significant delay in transition from schizont to ring stages was observed up to nanomolar concentrations. The peptide, DNSDNMF also showed similar effects but at nearly 1000-fold higher concentrations. The peptide was also found to interact with the catalytic domain. These data demonstrate the crucial role of SERA5 prodomain for the egress process. Given the inhibitory potential of the prodomain for the parasite, we suggest that peptidomimetic inhibitors based on SERA5 prodomain sequences can be developed as future therapeutics against malaria.     

Low malaria morbidity in a cohort of Senegalese children with free access to health structures

We report the impact of the free access to health facilities on malaria morbidity in children from two to 15 years old, during a malaria transmission season in Niakhar, Senegal. Between July and December 2002, 227 malaria attacks occured in 566 children. Only one case of severe malaria was observed and no death has been reported. Our results demonstrate furthermore that easier access to health facilities and to early treatment is playing a key role in malaria control.     

The β-tubulin gene from rat and human isolates of Pneumocystis carinii

The development of new drugs for treating Pneumocystis carinii infections in AIDS patients is hampered by the lack of long-term culture systems, and by our generally limited knowledge of this organism. Recently, however, we observed significant activity of various benzimidazoles against growth of this organism in short-term cultures. Benzimidazoles inhibit microtubule polymerization; there is strong evidence that the primary target is the beta-tubulin subunit. To understand the basis for benzimidazole activity against P. carinii, and to examine the apparent relatedness of this organism to fungi, we have cloned and sequenced the single beta-tubulin gene from a rat P. carinii isolate. There was 89-91% identity at the amino acid level to beta-tubulins from filamentous fungi, but only 79-82% identity to yeast and protozoal beta-tubulins. Also, eight introns were distributed throughout the P. carinii beta-tubulin gene in a pattern characteristic of filamentous fungi. Specific residues previously implicated in benzimidazole sensitivity were conserved in P. carinii beta-tubulin. The polymerase chain reaction was used to amplify a segment of P. carinii beta-tubulin DNA from bronchoalveolar lavages obtained from two patients with AIDS. There was considerable divergence at the DNA level between the human and rat sequences, but 100% identity at the amino-acid level.     

Antimicrobial activity of polycationic peptides

The in vitro activity of six polycationic peptides, buforin II, cecropin P1, indolicidin, magainin II, nisin, and ranalexin, were evaluated against several clinical isolates of gram-positive and gram-negative aerobic bacteria, yeasts, Pneumocystis carinii and Cryptosporidium parvum, by using microbroth dilution methods. The peptides exhibited different antibacterial activities and rapid time-dependent killing. The gram-negative organisms were more susceptible to buforin II and cecropin P1, whereas buforin II and ranalexin were the most active compounds against the gram-positive strains. Similarly, ranalexin showed the highest activity against Candida spp., whereas magainin II exerted the highest anticryptococcal activity. Finally, the peptides showed high anti-Pneumocystis activity, whereas no compound had strong inhibitory effect on C. parvum.     

Survival and Infectivity of Pneumocystis carinii Outside the Mammalian Host.

SUMMARY Air blower prefilters servicing HEPA-filtered Biobubble housing Pneumocystis carinii -infected rats were stored for up to five months at -80°C to room temperature. After storage, 76% of immunosuppressed rats exposed to these prefilters developed P. carinii infections. In contrast, only 4% of control immunosuppressed rats exposed to autoclaved filters had P. carinii infections. These observations indicate that the organism has a dormant form that remains infective for at least several months outside the mammalian host.     

Deficiency of two red-cell flavin enzymes in a population in Sardinia: was glutathione reductase deficiency specifically selected for by malaria?

In two areas in Italy where malaria was endemic--in the Po delta and Maremma on the west coast--we have found a high prevalence of an inherited flavin-deficient red cell in the normal population, suggesting selection by malaria. This study in Sardinia enabled a direct comparison of red-cell activities of FAD-dependent glutathione reductase (EGR) and FMN-dependent pyridoxine phosphate (PNP) oxidase in an ethnically homogeneous population, between two coastal villages where malaria was endemic from 300 B.C. and two mountain villages with no history of malaria. Both enzyme activities were significantly lower on the coast, and it did not seem that this could be explained by possible small differences in dietary riboflavin. As was thought to be the case in Ferrara and Grosseto, it is probable that a genetically controlled flavin-deficient red cell was selected for by malaria. Low EGR apoenzyme activity was more common on the coast, usually explaining the accompanying low basic EGR activity, and may also have been selected for by malaria. This adds to evidence from others that the mechanism of defence of a flavin-deficient red cell against malaria may be through EGR deficiency. It could also play a part in the protection given by heterozygous beta-thalassemia. The multifactorial protection of the population against malaria is discussed.     

Inhibitory effect of human natural yeast killer toxin-like candidacidal antibodies on Pneumocystis carinii.

BACKGROUND: Human natural antibodies have been found that owe their candidacidal action to the mimicry of a yeast killer toxin produced by the yeast Pichia anomala (PaKT). Candidacidal human natural antibodies (KTAb) are elicited by and bind to a KT receptor (PaKTR) present on the cell surface of infectious PaKT-sensitive microorganisms. Because of the recognized susceptibility of Pneumocystis carinii organisms to PaKT upon the occurrence of specific PaKTR, we examined whether human natural KTAb could also bind to and inhibit P. carinii. MATERIALS AND METHODS: Immunoaffinity-purified KTAb from the vaginal fluid of patients affected by candidiasis were tested and compared with PaKT for their ability to inhibit rat-derived P. carinii attachment to epithelial lung cells as well as infectivity to nude rats. Immunofluorescence studies were also performed by biotinylated PaKT in competition with human KTAb to establish their specific binding to PaKTR on the surface of rat-derived and human P. carinii organisms. RESULTS: Human natural candidacidal KTAb exerted a strong, specific inhibitory activity against rat-derived P. carinii organisms that are susceptible to PaKT itself. The antimicrobial activity of human KTAb was abolished by adsorption with a specific PaKT-neutralizing mAb KT4. Immunofluorescence studies of competition with PaKT showed that human KTAb efficiently bind to the specific PaKTR on the surface of rat-derived and human P. carinii organisms. CONCLUSIONS: The results strongly suggest that human KTAb, elicited by a common transphyletic receptor of different pathogenic microorganisms during infection, may play a role in antibody-mediated cross-immunity and, if properly engineered, as functionally equivalent recombinant antibodies they could exert a therapeutic activity against pneumocystosis in vivo.     

乳酸乳球菌乳脂亚种W56中质粒pJW566的生物学特性

从丹麦乳酪发酵启子乳酸乳球菌乳脂亚种 (Lactococcuslactissubsp .cremoris)W56中 ,分离到一个 2 2 4kb的质粒pJW566,将该质粒转化到无质粒且噬菌体敏感的L .lactisMG1 61 4、SMQ86菌株中 ,所得转化子对常见 963、c2和P335属的噬菌体具有一定抗性。经测定噬菌体以及含有pJW566的菌株所繁育的噬菌体效价 ,发现该质粒对外源DNA具有限制和修饰 (Re strictionandModification ,R M)作用。将pJW566转化到一株噬菌体敏感的乳酪工业生产菌株L .lactisCHCC2 2 81 ,在牛奶发酵中 ,表现出较强的噬菌体抗性。体外内切酶活性测定表明 ,该质粒具有的限制性内切酶需要Mg2 +和ATP ,而AdoMet(S adenosylmethionine,AdoMet)对酶活有促进作用     

Atovaquone - a novel broad-spectrum anti-infective drug

Atovoquone is a novel hydroxynaphthoquinone that is currently showing clinical promise for the treatment of malaria and the AIDS-associated diseases Pneumocystis carinii pneumonia and toxoplosmosis. The drug is the end product of half a century of research by numerous groups who have investigated the ontiporositic properties of many related compounds. Atovaquone is the only member of the series to show therapeutic activity in humans when taken orally. In this article, Alan Hudson explores the background to the discovery of this drug and reviews its mode of action, and biological and clinical profiles.     

Treatment of experimental Pneumocystis carinii pneumonia with analogs of pentamidine

Seven analogs of pentamidine were tested for their activity against an immunosuppressed rat model of Pneumocystis carinii pneumonia. Structural alterations of the pentamidine molecule included variations of the alkyl chain linking the two p-amidino phenoxy moieties and relocation of the amidine groups from the para to the meta position on the phenoxy rings. All analogs of pentamidine were active against P. carinii pneumonia when compared to a saline-treated control group. One derivative, 1, 4-di(4'-amidinophenoxy)butane, proved to be statistically more active than the parent drug.     

Treatment of Experimental Pneumocystis carinii Pneumonia with Analogs of Pentamidine

Seven analogs of pentamidine were tested for their activity against an immunosuppressed rat model of Pneumocystis carinii pneumonia. Structural alterations of the pentamidine molecule included variations of the alkyl chain linking the two p-amidino phenoxy moieties and relocation of the amidine groups from the para to the meu position on the phenoxy rings. All analogs of pentamidine were active against P. carinii pneumonia when compared to a saline-treated control group. One derivative, 1, 4-di(4'-amidinophenoxy)butanc, proved to be statistically more active than the parent drug.     

Plasminogen activator production in a rat model of Pneumocystis carinii pneumonia

Several studies have indicated that the serine protease urokinase-plasminogen-activator (uPA) is an important factor in host defense against pulmonary pathogens. To gain a better insight into the role of uPA in Pneumocystis carinii (P. carinii) pneumonia (PCP), we evaluated PA production in alveolar macrophages (AMs) obtained from rats with steroid-induced PCP. Treatment with cortisone acetate favored PCP in 91% of rats. In the bronchoalveolar lavage (BAL) samples of immunosuppressed rats both with and without PCP, we observed a decrease in uPA activity as well as a decrease in cell number. Urokinase-PA production by AMs was reduced in rats treated with cortisone alone. However, an increase in cell-associated uPA was observed in rats with PCP. This increase appears to be produced in response to P carinii infection. In fact, when AMs obtained from untreated healthy or immunosuppressed uninfected rats were challenged with P carinii, a significant increase in PA activity in cell lysates was observed, though a lower response was obtained in cortisone-treated animals. Our results suggest that healthy AMs respond to the presence of P carinii with an increase in uPA production and that this response in immunodepressed rat-AMs is partially impaired.     

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.