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DNA fragments homologous to members of the family of P-type ion-motive ATPases were identified in Trypanosoma cruzi by polymerase chain reaction (PCR) amplification. The sequence of one fragment, which closely resembled (87% identity) the tandemly linked proton pumps in Leishmania, was used to characterize the H(+)-ATPase genes in T. cruzi. The T. cruzi proton pump locus contains four tandemly repeated genes (TCH1-4) separated by 1.1 kb intergenic regions. The nucleotide sequence of one cloned gene of the tandem array contains a 2775 nt open reading frame encoding a predicted 101908-Da protein of 925 amino acids. The TCH genes are expressed as 3.8 and 4.9 kb polyadenylated transcripts in the epimastigote stage; expression of both transcripts is reduced in metacyclic trypomastigotes. Results of 5' and 3' RACE transcript mapping indicate that the 3.8 kb message is generated from within the tandemly repeated locus. The 3.8 kb TCH transcript has the T. cruzi mini-exon appended to a short (40 nt) 5' untranslated region (UTR) and has a 927 nt 3' UTR. The full peptide sequence of the T. cruzi proton pump is 80% identical to the Leishmania pump but lacks the extended carboxyl tail present in the Leishmania ATPase. An antibody that recognizes the 110-kDa Leishmania donovani proton pump cross-reacts with a 100-kDa protein in lysates of T. cruzi epimastigotes.

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A 2253-nucleotide (nt) transcript for a Trichomonas vaginalis heat-shock protein 70, TVCHSP70, has been isolated that encodes for a protein of 659 amino acids with a predicted molecular weight of 71.3 kDa. TVCHSP70 has a short (10-nt) 5' untranslated region (UTR), and the 263-nt 3' UTR is the longest reported for a Trichomonas peptide. Amino-acid sequence analysis and phylogenetic comparison identifies TVCHSP70 as a member of the heat-inducible cytoplasmic HSP70 gene family. Southern-blot data indicate that T. vaginalis contains at least four members of the cytoplasmic HSP70 gene family. Members of the TVCHSP70 family are expressed as 2.3-kb transcripts at low levels during 37 degrees C culture, and their expression is significantly up-regulated at 43 degrees C. Slot-blot analysis of seven T. vaginalis clinical isolates demonstrated a 3- to 44-fold up-regulation of TVCHSP70 under conditions of heat shock (43 degrees C) or oxidative stress (500 microm H2O2) as compared with controls (37 degrees C).

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Patients infected with Trichomonas vaginalis mount humoral and cellular immune responses that often do not protect against reinfection. The oxidative stressors produced by leukocytes may trigger a heat-shock-like response in T. vaginalis trophozoites, helping the parasite to survive host immune defenses. The antigenicity of T. vaginalis heat-shock proteins (HSPs) was examined by immunoprecipitation of T. vaginalis heat-induced proteins with sera from infected patients and controls. When T. vaginalis was heat-shocked, HSPs of 169-167 and 140-137 kDa were specifically recognized by sera from infected male and female patients. However, the majority of T. vaginalis HSPs were also immunoprecipitated by control sera; all sera recognized 72- to 71-kDa, 47- to 45-kDa, 38- to 37-kDa, 35-kDa, and 31-kDa heat-induced proteins. At least 15 proteins from non-heat-shocked T. vaginalis were immunoprecipitated by sera from infected patients and controls, indicating that natural or cross-reacting antibodies could participate in host responses to trichomoniasis. Molecules of 158, 135, 89, and 74-72 kDa were immunoprecipitated from some non-heat-shocked parasites only by patients' sera. A 38-kDa T. vaginalis protein was immunoprecipitated only by sera from infected females and may be specific for infection in women.

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D2A21, a novel peptide antibiotic has in vitro activity against a wide spectrum of sexually transmitted diseases (STD). In this study we tested the hypothesis that intravaginal D2A21 would interfere with acquisition of Trichomonas vaginalis infection in a modified mouse model. T. vaginalis infections of estrogenized young mice pretreated with Lactobacillus vaginalis or Lactobacillus rhamnosus were more frequent and persistent than those in mice pre-treated with Lactobacillus gasseri or Lactobacillus acidophilus. One hundred percent T. vaginalis infection was achieved for 2-4 days post-challenge when intravaginal L. rhamnosus pre-treatments were given to estrogenized mice 48 hr prior to a single T. vaginalis challenge. Estrogenized mice pre-treated with L. rhamnosus were pre-medicated with intravaginal placebo gel, 0.5% or 2% D2A21 gel, or 500 microg/mL metronidazole gel prior to T. vaginalis challenge. Both 2% D2A21 and metronidazole gels were significantly more efficacious (10% or none infected) than placebo gel (53% infected) in preventing vaginal T. vaginalis infections in mice.

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Subtyping isolates of Trichomonas vaginalis is an essential tool for understanding the epidemiology of this common sexually-transmitted disease. Restriction fragment length polymorphism (RFLP) analysis employing a probe from the heat-inducible cytoplasmic HSP70 gene family hybridized with EcoR I-digested genomic DNA was used in the molecular typing of Trichomonas isolates. Analysis of five American Type Culture Collection (ATCC) reference strains and 31 Jackson, Mississippi, isolates from six male and 21 female patients, revealed 10 distinct RFLP pattern subtypes of Trichomonas. The subtypes were temporally stable and cosmopolitan. The RFLP profiles seen in Maryland, Ohio, Massachusetts, and New York ATCC strains were identical to those of some Mississippi isolates, even though the samples were isolated 10-35 years apart. There was no correlation between metronidazole resistance and RFLP subtype with resistant isolates from eight patients distributed among six different subtypes.

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DNA fragments homologous to P-type cation translocating ATPase genes were identified in Trichomonas vaginalis by polymerase chain reaction (PCR) amplification. The genomic locus corresponding to one PCR fragment, TVCA1, contains a 3,055 base-pair open reading frame encoding a 108,162 dalton protein composed of 981 amino acids. TVCA1 lacks introns, is present in a single copy, and is expressed as a 3.1 kb transcript with short 5' and 3' untranslated regions. Separate primer extension experiments map the 5' end of the TVCA1 transcript to 12 and 16 nucleotide bases (nt) upstream of the methionine initiation codon. The message polyadenylation site is located 62 nt downstream of the protein termination codon at a CA dinucleotide. The TVCA1 protein sequence shares 57-58% similarity with rabbit, schistosome, trypanosome and malarial sarcoplasmic-endoplasmic reticulum calcium (SERCA) pumps, and significantly lower similarity with plasma membrane calcium pumps and cation translocating ATPases of other ion specificities. Structural and functional domains identified in P-type ATPases as well as 61/68 residues specifically implicated in SERCA pump activity are conserved in TVCA1. However, TVCA1 lacks binding sites for phospholamban regulation, thapsigargin inhibition and the calmodulin dependent protein kinase site phosphorylation present in other SERCA pumps.

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Hamycin, a polyene antibiotic related to amphotericin B, has been used topically to treat fungal and protozoan infections in India. We assessed the cytotoxic activity of hamycin on nine metronidazole resistant or susceptible Trichomonas vaginalis strains isolated from symptomatic or asymptomatic patients. Cytotoxic activity of hamycin against two mammalian cell lines, BHK-21 and HeLa, was also determined. Tritiated thymidine pulse labeling after drug-washout and a recovery period was used to distinguish death of target cells from temporary static effects of the drug. Liposomal hamycin and hamycin formulated with dimethyl sulphoxide, deoxycholate or glycerin were compared. Although all hamycin preparations were trichomonacidal at approximately 1 mg/L, hamycin-dimethyl sulphoxide was stable for only 24 h, and hamycin glycerin was incompletely solubilized. Hamycin-deoxycholate remained a stable gel for 2 months at room temperature, but its activity was reduced four-fold when compared to the fresh preparation. The mammalian tissue culture cell lines HeLa and BHK-21 were killed by trichomonacidal concentrations of hamycin-deoxycholate. This cytotoxicity of hamycin for mammalian cells is a concern, but new forms of the drug are under development that may allow more widespread use of this drug.

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Fluconazole concentrations in the serum and prostate of human volunteers undergoing transurethral resection for benign prostatic hypertrophy were measured. There was a high correlation (r = 0.783) between serum (mean = 6.6 micrograms/ml) and tissue (mean = 1.9 micrograms/g) fluconazole concentrations, and these data were used to construct a model for local tissue concentrations.

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Since metronidazole is a mutagen in vitro, there is concern about the widespread systemic use of this drug in women with trichomoniasis, particularly those who are pregnant. A randomized, double-blind, placebo-controlled trial compared a single 2-g intravaginal dose of metronidazole cream with a single 2-g oral dose of metronidazole in patients with a culture positive for Trichomonas organisms. Of the 302 preenrollment cultures completed, 94 (31%) were positive. Sixty-one patients were enrolled in the study. Each received either oral placebo and intravaginal metronidazole or intravaginal placebo and oral metronidazole. Follow-up cultures were done on posttreatment day 3-5. Of the 53 evaluatable patients, 14 (50%) of 28 in the intravaginal group and 22 (88%) of 25 in the oral group were microbiologically cured (P = .0037). Single-dose intravaginal metronidazole is inferior to single-dose oral metronidazole and cannot be relied on as an alternative therapy.

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Nucleoside salvage pathways are vital to the parasitic protozoan Trypanosoma cruzi, and have become important targets in the development of new chemotherapeutic agents against this organism. We produced a mutant T. cruzi clone with a defect in the uptake of the adenosine analogue tubercidin which allowed us to hypothesize that there are at least two distinct nucleoside transport pathways in this parasite. The mutant shows a marked defect in the uptake of tubercidin and thymidine, whereas the uptake of adenosine and inosine are normal. Inhibition and metabolic studies suggest that the defect is related to transport and that there are two transport processes relatively specific for purines and pyrimidines, respectively, although tubercidin is transported via the latter. This is similar to the reported dual nucleoside transport pathways in Leishmania donovani and may be a common system in the Trypanosomatidae. These transport processes are markedly different from those which have been described for mammalian cells and may play an important role in the design of strategies for the chemotherapy of human infection with these pathogenic parasites.

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Multiple variables affect the tissue destruction caused by missiles, and the interaction of these variables is incompletely understood. The recently developed technology of computerized solid modeling now makes it possible to analyze these parameters in three dimensions. A technique for creating solid models of organic structures is described. The tissues within the boundaries thus defined are ascribed physical attributes by means of finite element analysis with data derived from empirical studies. An interactive user-friendly program is being developed combining this modeling with a probabilistic scheme (Monte Carlo simulation) to describe a variety of wounding scenarios. The data from these predictions will be compared with information from wound registries and the model refined until it can project consistently accurate patterns of injury.

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Utilizing the previously reported inter-clonal differences in total DNA/organism, flow cytometry was used to analyze the population dynamics of Trypanosoma cruzi clone mixtures growing in liquid medium or vertebrate cells. The growth of clone mixtures in liquid medium can be described by unique parameters reflecting exponential growth rate (r), stationary phase population density (1/k), and the interaction between the clones (h). The relative numbers of each clone in the population change rapidly with time and the results are in quantitative agreement with mathematical models of competitive population growth. The relationship between the parameters for T. cruzi is such that, in general, there is no dynamic equilibrium with coexistence of clones with different growth rates; under all culture protocols, the faster growing clone will prevail. A computer simulation of the vertebrate cell cycle of T. cruzi suggests that clone mixtures grow relatively independently; the basic attributes of the model were substantiated experimentally. Although wide fluctuations in the proportion of each clone released occurred, the faster growing clone again predominated. Finally, these results underline the importance of working with well-defined clones in the laboratory to avoid inconsistencies and paradoxical results and stress the importance of the rapid isolation of single cell clones from clinical specimens when studying the relationship of the parasite to human disease.

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An anaerobic myocardial abscess due to Bacteroides fragilis developed in a 60-year-old man when he had an acute myocardial infarction while recuperating from surgery for a paracolonic abscess. Anaerobic bacteremia is a common event and may lead to infection in areas of low oxygen tension far removed from the original portal of entry.

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The course of lethal Plasmodium berghei infection was examined in nu/+ and T cell-deficient nu/nu BALB/c mice. A rapidly fatal neurologic syndrome, including ataxia, hemiparesis, and seizures, was seen in the nu/+ mice early in the infection, whereas this syndrome was absent in the nu/nu mice. The nu/nu mice also developed anemia more slowly, had lower levels of immune complexes and total IgG, and had smaller decreases in serum C3 compared with the nu/+ mice. Histopathologic examination of the brains revealed cerebral malaria lesions, including vascular plugging and micro-hemorrhages, in the nu/+ mice but not in the nu/nu mice. Cerebral lesions similar in frequency and severity to those in nu/+ mice developed in nu/nu mice given spleen cells from normal nu/+ mice. The results suggest that an intact immune system is necessary for the expression of cerebral malaria.

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