PSGL-1-mediated adhesion of human hematopoietic progenitors to P-selectin results in suppression of hematopoiesis.

Cellular interactions are critical for the regulation of hematopoiesis. The sialomucin PSGL-1/CD162 mediates the attachment of mature leukocytes to P-selectin. We now show that PSGL-1 also functions as the sole receptor for P-selectin on primitive human CD34+ hematopoietic progenitor cells (HPC). More importantly, ligation of PSGL-1 by immobilized or soluble ligand or anti-PSGL-1 antibody results in a profound suppression of HPC proliferation stimulated by potent combinations of early acting hematopoietic growth factors. These data demonstrate an unanticipated but extremely marked growth-inhibitory effect of P-selectin on hematopoiesis and provide direct evidence that PSGL-1, in addition to its well-documented role as an adhesion molecule on mature leukocytes, is a potent negative regulator of human hematopoietic progenitors.

The monoclonal antibody 97A6 defines a novel surface antigen expressed on human basophils and their multipotent and unipotent progenitors.

Basophils (Ba) and mast cells (MC) are important effector cells of inflammatory reactions. Both cell types derive from CD34(+) hematopoietic progenitors. However, little is known about the cell subsets that become committed to and give rise to Ba and/or MC. We have generated a monoclonal antibody (MoAb), 97A6, that specifically detects human Ba, MC (lung, skin), and their CD34(+) progenitors. Other mature hematopoietic cells (neutrophils, eosinophils, monocytes, lymphocytes, platelets) did not react with MoAb 97A6, and sorting of 97A6(+) peripheral blood (PB) and bone marrow (BM) cells resulted in an almost pure population (>98%) of Ba. Approximately 1% of CD34(+) BM and PB cells was found to be 97A6(+). Culture of sorted CD34(+)97A6(+) BM cells in semisolid medium containing phytohemagglutinin-stimulated leukocyte supernatant for 16 days (multilineage assay) resulted in the formation of pure Ba colonies (10 of 40), Ba-eosinophil colonies (7 of 40), Ba-macrophage colonies (3 of 40), and multilineage Ba-eosinophil-macrophage and/or neutrophil colonies (12 of 40). In contrast, no Ba could be cultured from CD34(+)97A6(-) cells. Liquid culture of CD34(+) PB cells in the presence of 100 ng/mL interleukin (IL)-3 (Ba progenitor assay) resulted in an increase of 97A6(+) cells, starting from 1% of day-0 cells to almost 70% (basophils) after day 7. Culture of sorted BM CD34(+)97A6(+) cells in the presence of 100 ng/mL stem cell factor (SCF) for 35 days (mast cell progenitor assay) resulted in the growth of MC (>30% on day 35). Anti-IgE-induced IgE receptor cross-linking on Ba for 15 minutes resulted in a 4-fold to 5-fold upregulation of 97A6 antigen expression. These data show that the 97A6-reactive antigen plays a role in basophil activation and is expressed on multipotent CD34(+) progenitors, MC progenitors, Ba progenitors, as well as on mature Ba and tissue MC. The lineage-specificity of MoAb 97A6 suggests that this novel marker may be a useful tool to isolate and analyze Ba/MC and their progenitors.

Activity of atovaquone against Babesia microti in the Mongolian gerbil, Meriones unguiculatus.

The hydroxynaphthoquinone, atovaquone (Wellvone, Glaxo-Wellcome Ltd.) was found to have significant activity against Babesia microti, the main cause of human babesiosis in the U.S.A. This activity compares well with that of the most effective babesicide currently available for use in animals, imidocarb dipropionate, that unlike atovaquone is not licensed for use in humans. Treatment with well tolerated doses of atovaquone results in a rapid reduction in parasitemias and an early disappearance of parasites from blood smears. However, in common with all the other babesicides tested, atovaquone did not sterilize gerbils of infection, even at very high daily doses administered for up to 10 days. A combination of atovaquone and clindamycin was more effective than atovaquone alone in the treatment of both acute and chronic infections but failed to eliminate parasites completely.

Atovaquone and proguanil hydrochloride: a review of nonclinical studies.

BACKGROUND: Safe and effective antimalarial drugs are needed for treatment and prophylaxis of malaria. The combination of atovaquone and proguanil hydrochloride is a new antimalarial drug combination that has recently become available in many countries. METHODS: Data were reviewed from nonclinical studies evaluating the microbiology, secondary pharmacology, pharmacokinetics, and toxicology of atovaquone and proguanil hydrochloride. RESULTS: Atovaquone is highly active against asexual erythrocytic stages of Plasmodium falciparum in vitro (IC50 0.7-6 nM) and in animal models. Proguanil per se has only weak antimalarial activity in vitro (IC50 2.4-19 microM), and its effectiveness depends on the active metabolite cycloguanil (IC50 0.5-2.5 nM). The combination of atovaquone and proguanil is synergistic in vitro. Both drugs also have activity against gametocytes and pre-erythrocytic (hepatic) stages of malaria parasites. Atovaquone is a ubiquinone antagonist that inhibits mitochondrial electron transport and collapses mitochondrial membrane potential. The proguanil metabolite cycloguanil is a dihydrofolate reductase inhibitor, but the mode of action of proguanil is unknown. In screening evaluations of secondary pharmacology, neither atovaquone nor proguanil had activity that adversely affected gastrointestinal, cardiovascular, or central or autonomic nervous system functions at clinically relevant concentrations. After oral administration, atovaquone exposure is extensive in rats but limited in dogs, while proguanil and cycloguanil exposure is extensive in dogs but limited in rats. In both species, toxicity was related to proguanil exposure, the principal manifestations being salivation, emesis, and loss of body weight. Neither atovaquone nor proguanil was teratogenic or mutagenic. An increased incidence of hepatic adenomas and adenocarcinomas was seen in mice, but not rats, after lifetime exposure to atovaquone, and appears to be related to species-specific differences in hepatic enzymatic activity. No additional toxicity was evident in animals treated with the combination of atovaquone and proguanil hydrochloride compared to those treated with either drug alone. CONCLUSION: Nonclinical studies of atovaquone and proguanil hydrochloride supported the clinical development of this combination for treatment and prophylaxis of malaria.

Isolation and molecular characterization of the bifunctional hydroxymethyldihydropterin pyrophosphokinase-dihydropteroate synthase gene from Toxoplasma gondii.

Toxoplasma gondii is an important cause of AIDS-related opportunistic infection, manifest as toxoplasmic encephalitis. The clinical treatment of choice is the synergistic combination of antifolate agents, pyrimethamine and sulphadiazine, of which the latter targets the parasite's dihydropteroate synthase (DHPS) activity. Here, we describe the isolation of the gene encoding this activity in T. gondii. The nucleotide sequence contains an open reading frame interrupted by five introns, which encodes a protein of 664 amino acids with an M(r) of 72991. Sequence analysis revealed that, in addition to DHPS, the predicted protein contains a second enzyme function, hydroxymethyldihydropterin pyrophosphokinase (PPPK). This enzyme immediately precedes DHPS in the folate biosynthetic pathway. The bifunctional arrangement of the T. gondii pppk-dhps gene is the same as that observed in the related protozoan parasite, Plasmodium falciparum, and confirms previous biochemical data that these activities were inseparable. Recently, specific mutations within conserved motifs of the DHPS gene of P. falciparum have been identified which give rise to sulphonamide drug resistance. Analysis of seven clinical isolates of T. gondii did not reveal any similar mutations in this limited sample of organisms that had been subjected to drug pressure.

Therapeutic efficacy of atovaquone against the bovine intraerythrocytic parasite, Babesia divergens.

This study demonstrates the activity of the hydroxynaphthoquinone (HNQ), atovaquone, against Babesia divergens, the cause of a rare but lethal form of human babesiosis. In vitro studies showed that unlike other anti-malarial drugs, the HNQs studied have a high level of anti-babesial activity and atovaquone was more active than imidocarb, the most effective compound used so far for human B. divergens babesiosis and also used routinely for the treatment of bovine babesiosis. Atovaquone also proved to be extremely active against B. divergens in gerbils (Meriones unguiculatus). Acute fulminating infections were effectively treated with as little as 1.0 mg/kg with increasing effectiveness up to 10 mg/kg, which compares well with the activity of imidocarb. Although immunosuppression with dexamethasone slowed the decline of parasitemias after treatment with atovaquone, gerbil survival was unaffected. Pretreatment of gerbils with 4 daily low doses of atovaquone did not have any effect on the development of subsequent infections. However, if treatment was continued after infection, daily doses as low as 0.5 mg/kg effectively suppressed the parasites.

The effect of atovaquone (566C80) on the maturation and viability of Plasmodium falciparum gametocytes in vitro.

Atovaquone (566C80), a hydroxynaphthoquinone, was investigated for activity against Plasmodium falciparum gametocytes (NF54 strain) in vitro. After 96 h of continuous exposure to the drug at 1.4 x 10(-7) M (a concentration achievable in humans 14 d after administration of a therapeutic dose of 10 mg/kg) reductions of 75%, 54% and 20% in the number of gametocyte stages 2, 3 and 4, respectively, were achieved. A small increase (14%) in stage 5 gametocytes was seen. At the same concentration, atovaquone showed greater activity against the asexual stages of P. falciparum, reductions of 93%, 96% and 43% in the number of rings, schizonts and trophozoites, respectively, being achieved. These data are consistent with inhibition of maturation of trophozoites. The observed effect on maturation of gametocytes is similarly consistent with blockade of gametocyte recruitment from merozoites produced by the preceding schizogony, or to stasis of intraerythrocytic sexual development before the formation of stage 2 gametocytes.

Inhibitory activity of the anti-malarial atovaquone (566C80) against ookinetes, oocysts, and sporozoites of Plasmodium berghei.

Ookinete formation from mature Plasmodium berghei gametocytes in vitro was partially inhibited by 0.05-0.1 microM atovaquone and almost totally blocked at a concentration of 0.25 microM. Microgametocyte exflagellation was not affected by atovaquone at concentrations up to 300 microM. Ookinete formation was also inhibited in culture when addition of 0.20 microM atovaquone was delayed by 4 hr, by which time DNA replication was likely to have been completed. Inhibition of ookinete formation by atovaquone was not reversed by orotic acid. Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal 4, 7, 14, and 20 days postinfection (p.i.) from mice that had been treated with atovaquone or control diluent 8 hr previously. Atovaquone blood feeds on day 4 reduced oocyst numbers on days 6-12, although sporozoite numbers in the thorax and abdomen on day 20 were not significantly reduced. Blood feeds on day 7 slowed oocyst growth, blood feeds on day 14 did not significantly reduce sporozoite numbers, and feeds to mosquitoes on day 20 p.i. had no effect on transmission to naive mice. Sporozoite invasion of human hepatoma cells was unaffected by the presence of atovaquone.

Interactions of atovaquone with other antimalarial drugs against Plasmodium falciparum in vitro.

A series of Plasmodium falciparum in vitro drug sensitivity studies were conducted in order to evaluate atovaquone in combination with other antimalarial drugs and thus to identify a potential partner for a fixed combination. The derived isobolograms indicated that drug interactions ranged from antagonism through addition to synergy. Of particular note were the quinolines and artemisinin analogues, which were all antagonistic, and the biguanides and tetracycline, which showed synergy. Proguanil emerged as the most promising of the current antimalarials as a partner for atovaquone in a fixed combination, with tetracycline as back-up.

Inhibitory action of the anti-malarial compound atovaquone (566C80) against Plasmodium berghei ANKA in the mosquito, Anopheles stephensi.

The activity of atovaquone against Plasmodium berghei ANKA during sporogonic development has been examined. Anopheles stephensi mosquitoes were fed on gametocyte infected mice which had been treated 8 h previously with atovaquone or diluent alone. Mosquito midguts were examined for oocysts, and salivary gland infections were estimated using an ELISA for the circumsporozoite protein (CSP). The number of oocysts per midgut fell by at least 97% when mosquitoes were fed on mice dosed with 0.1-10 mg atovaquone/kg body weight. This was paralleled by a decrease in the prevalence of oocyst-infected mosquitoes from 70-90% in controls to 40% or 10% respectively. No oocysts were observed at a dose of 100 mg/kg. CSP ELISA results indicated that mosquitoes fed on atovaquone failed to produce sporozoites. Mosquitoes which fed on gametocytaemic, atovaquone-treated mice (0.1-100 mg/kg) did not transmit malaria to naive mice. These results demonstrate that atovaquone has a highly potent inhibitory activity against the mosquito stages of P. berghei.

In vitro and in vivo activities of the hydroxynaphthoquinone atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, or minocycline against Toxoplasma gondii.

The efficacy of atovaquone alone or combined with pyrimethamine, sulfadiazine, clarithromycin, and minocycline was examined in vitro and in a murine model of acute toxoplasmosis. In vitro studies were performed with MRC5 fibroblast tissue cultures, with quantification of Toxoplasma growth by an enzyme-linked immunosorbent assay. For in vivo studies, mice were acutely infected intraperitoneally with 10(4) tachyzoites of the virulent RH strain and then treated perorally for 10 days from day 1 postinfection. The following drug regimens were investigated: atovaquone at 100 and 50 mg/kg of body weight per day and the combinations of atovaquone at 50 mg/kg with sulfadiazine at 200 mg/kg, pyrimethamine at 12.5 mg/kg, clarithromycin at 200 mg/kg, or minocycline at 50 mg/kg. Efficacy was assessed by determination of survival rates and sequential determination of parasite burdens in blood, brain, and lungs. In vitro, atovaquone inhibited Toxoplasma growth at a concentration of > or = 0.02 mg/liter; the 50% inhibitory concentration was estimated to be 0.023 mg/liter. No synergistic effect was observed when it was combined with sulfadiazine, clarithromycin, or minocycline, whereas a significant antagonistic effect was noted for the combination of atovaquone with pyrimethamine. In vivo, administration of atovaquone at 100 or 50 mg/kg/day for 10 days resulted in prolonged survival compared with that in untreated mice; this survival was associated with a reduction of parasite burdens in blood and tissues during the course of treatment. The combinations of atovaquone with pyrimethamine, clarithromycin, or sulfadiazine were more efficient than each drug administered alone, in terms of survival, but parasite burdens in blood and organs were not reduced compared with those in mice treated with any of the agents alone. These experimental results confirmed the activity of atovaquone against Toxoplasma gondii, but no marked improvement in efficacy was observed in vitro and in vivo when this drug was combined with pyrimethamine, sulfadiazine, minocycline, or clarithromycin.

The novel hydroxynaphthoquinone 566C80 inhibits the development of liver stages of Plasmodium berghei cultured in vitro.

The causal prophylactic activity of the novel hydroxynaphthoquinone, 566C80, was assessed against the exo-erythrocytic (EE) stages of Plasmodium berghei cultured in the human hepatoma cell line, HepG2. 566C80 was found to be highly active as an inhibitor of EE development and was more active than the established causal prophylactic pyrimethamine. A 566C80 concentration of 1.85 x 10(-9) M, added 3 h after sporozoite invasion, reduced the numbers of EE forms visible at 48 h by 50 degrees o, while the equivalent concentration of pyrimethamine was 1.95 x 10(-8) M.

Site of action of the antimalarial hydroxynaphthoquinone, 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80).

The site of action of the antimalarial compound 2-[trans-4-(4'-chlorophenyl) cyclohexyl]-3-hydroxy-1,4-naphthoquinone (566C80), would appear to be the mitochondrial respiratory chain. Studies reported herein have demonstrated 566C80 to be a potent and selective mitochondrial inhibitor with mitochondria isolated from Plasmodium falciparum and P. yoelii. Selective assay of individual respiratory chain complexes has shown the primary site of action of 566C80 to be the cytochrome bc1 complex (Complex III): supportive evidence from difference spectroscopy indicates the site of inhibition to lie between cytochromes b and c1 of this complex. Using [14C]566C80, evidence is presented which suggests that 566C80 may become irreversibly bound to a polypeptide with an approximate molecular mass of 11,500 Da.

566C80: a potent broad spectrum anti-infective agent with activity against malaria and opportunistic infections in AIDS patients.

566C80 is a novel hydroxynaphthoquinone with broad-spectrum anti-parasitic properties. In vitro the compound was more potent against Plasmodium falciparum than any of the established anti-malarial drugs. It had good activity against the pathogen in Aotus monkeys and was also effective in rodents infected with various drug-resistant strains of P. yoelii and P. berghei. In mice the compound showed significant activity against Toxoplasma gondii. Evaluation of the metabolic stability of 566C80 to NADPH-mediated oxidative metabolism was made using microsome preparations from a number of species including man. Unlike other quinones examined, 566C80 was shown to be inert in these assays. In Phase 1 clinical studies up to 750mg of compound were given as a single oral dose to fasted healthy male adults. This was well tolerated and the plasma drug elimination half-life was approximately 70h. In these subjects a 450mg dose gave plasma concentrations of 0.1-0.3 micrograms/ml which were achieved 1 h post-dosing and remained so for at least 7 days. Volunteers ingesting food prior to drug administration had quinone plasma levels which were significantly higher. Phase II trials are now underway to assess 566C80 for use against malaria and opportunistic infections in AIDS patients.

Efficacy of a hydroxynaphthoquinone, 566C80, in experimental Pneumocystis carinii pneumonitis.

The efficacy of a new class of drugs for Pneumocystis carinii pneumonitis was demonstrated. 566C80, a hydroxynaphthoquinone, administered orally in a dose of greater than or equal to 100 mg/kg of body weight per day prophylactically prevented P. carinii pneumonitis in 90% or more of rats, while all untreated control animals developed pneumonitis. When 566C80 (100 mg/kg per day) was administered for 3 weeks after P. carinii pneumonitis was established, therapy was totally effective and all of the untreated controls had progressive P. carinii pneumonitis. A dose of 566C80 of between 25 and 50 mg/kg per day protected 50% of the rats from P. carinii pneumonitis, and a dose of between 50 and 100 mg/kg per day cured 50% of those treated for P. carinii pneumonitis. Both prophylaxis and treatment with 566C80 were at least as effective as with trimethoprim-sulfamethoxazole. Animals maintained on immunosuppression after completion of treatment remained free of P. carinii, suggesting a killing effect. Clearance of P. carinii was associated with levels of 60 micrograms or more of 566C80 per ml of plasma. This hydroxynaphthoquinone offers promise as an anti-P. carinii drug.

The peculiarities of Blastocrithidia triatomae.

Blastocrithidia triatomae parasitizes triatomine bugs, the vectors of Chagas disease. Co-cultivation with a host-derived cell line permits continuous culture but the host cells are destroyed. Removal of the reduviid cells induces the formation of drought-resistant cysts, but the factors that induce encystment are unknown. Excystment is triggered after the onset of blood digestion in the insect host, a transition that is associated with unusual ultrastructural alterations. Günter Schaub, Dagmar Reduth and Mary Pudney believe that B. triatomae is a good candidate for the biological control of Chagas disease, not least because of its capacity to form highly resistant cysts in vitro.

Effect of mitochondrial inhibitors on adenosinetriphosphate levels in Plasmodium falciparum.

1. The effects of mitochondrial inhibitors on the ATP levels of intraerythrocytic Plasmodium falciparum have been studied. 2. Changes in parasite ATP or ADP levels with time in response to various mitochondrial inhibitors appear quite complex; ATP levels may be initially depressed and then elevated above normal, but the nature of the response depends upon the stage in the intraerythrocytic cycle and in some cases upon the concentration of the inhibitor used. 3. After ca 2 hr incubation of cultures with inhibitors ATP levels appear to be stabilized and are similar to those of untreated parasites. However, ADP levels of trophozoites show significant increases after a 2 hr incubation with inhibitors, particularly with oligomycin and to a lesser extent with antimycin A; increases in ADP levels however were not observed in ring-stages of the parasite. 4. Inhibition of red cell and parasite glycolysis leads to rapid decreases in parasite ATP levels which are not significantly affected by oligomycin. Incubation of in vitro cultures with oligomycin can result in a decreased, rather than increased rate of lactate production with a concomitant appearance of pyruvate in the growth medium. 5. This investigation would indicate that if there is a mitochondrial contribution to the parasite ATP pool it is relatively small, and that a short-fall in this contribution is quickly compensated for by ATP from other source(s), although this is not necessarily met by increased glycolysis.

Cultivation of Blastocrithidia triatomae (Trypanosomatidae) on a cell line of its host Triatoma infestans (Reduviidae).

Blastocrithidia triatomae parasitizes vectors of Chagas' disease and is very difficult to cultivate in conventional media. However, co-cultivation with a cell line of its host Triatoma infestans (TI-32; in Schneider's Drosophila medium supplemented with 20% foetal calf serum and 10% tryptose phosphate broth) led to vigorous growth at 24 or 28 degrees C without an adaptation phase. More than 60 primary cultures were initiated successfully without any failures. Subcultures could be started immediately at weekly intervals. The doubling time was similar in both the primary cultures (41 h) and the 33rd subculture (39 h). The importance of the reduviid cells for B. triatomae became clear after removal of the insect cells, when multiplication of epimastigotes stopped and mainly cysts were formed. Cysts produced in vitro were infective for reduviids. Scanning electron microscopy showed that B. triatomae attached to the host cells, inserted its flagellum into them and destroyed them.

The causal prophylactic activity of the novel hydroxynaphthoquinone 566C80 against Plasmodium berghei infections in rats.

The influence of the novel hydroxynaphthoquinone 566C80 on exoerythrocytic development of Plasmodium berghei was examined in Brown Norway rats. The procedure employed was designed to identify residual activity of the drug against tissue merozoites emerging into the bloodstream and to distinguish this from any observed causal prophylactic activity against the liver stages. Single oral doses of 10 and 1 mg/kg of 566C80 administered 3 hours after sporozoite-inoculation were effective in preventing the appearance of a patent parasitaemia, while a dose of 0.1 mg/kg significantly reduced the severity of the ensuing blood infection. There was a pronounced residual effect of 566C80 against the blood forms at a dose of 10 mg/kg, a slight residual effect at a dose of 1 mg/kg, but no apparent residual effect at 0.1 mg/kg. At the time when EE merozoites would normally emerge into the bloodstream, an aliquot of blood was sub-inoculated into mice from sporozoite-infected, 566C80-treated rats. This procedure confirmed that 566C80 is active against the exoerythrocytic stages of Plasmodium berghei.

Moulting and exsheathment of the infective larvae of Onchocerca lienalis (Filarioidea) in vitro.

Fourth-stage larvae (L4) of the cattle filarial parasite Onchocerca lienalis were produced from third stage forms (L3) and maintained in vitro using a variety of culture media and feeder cell layers. Up to 50% of the L3 larvae moulted to the fourth stage in the presence of two jird (Meriones unguiculatus) cell lines, a monkey kidney cell line and also with Vero cells. Moulting took place 2-5 days after of the initiation of the cultures: These L4 parasites could be maintained in a healthy condition for up to 88 days, although no significant increases in size of these larvae were observed during the culture period. The medium giving the most promising results, both in terms of moulting and survival, was 199 supplemented with heat inactivated foetal bovine serum (20%) and glucose (2 mg/ml). The feeder layer cells could be replaced by fresh jird red blood cells. Moulting of parasites occurred in medium alone but the viability was reduced under these conditions.