Endothelin-1 receptors play a minor role in the protection against acute Trypanosoma cruzi infection in mice.

Chagas' disease, caused by the protozoan Trypanosoma cruzi, is a major cause of cardiovascular disability in countries where it is endemic. Damage to the heart microvasculature has been proposed to be an important factor in the pathogenesis of heart dysfunction. Endothelin-1 (ET-1) is a potent vasoconstrictor and exerts its effects via specific ET A and ET B receptors. A few studies have suggested a role for ET-1 and its receptors in the pathogenesis of Chagas' disease. We investigated the effects of treatment with bosentan, an ET A/ET B receptor antagonist, on the course of T. cruzi infection (Y strain) in C57Bl/6 mice. Treatment with bosentan (100 mg kg-1 day-1) was given per os starting day 0 after infection until sacrifice. Bosentan significantly increased myocardial inflammation, with no effects on parasitemia. Although the total number of nests was similar, a lower number of intact amastigote nests was found in the heart of bosentan-treated animals. Bosentan failed to affect the infection-associated increase in the cardiac levels of the cytokines IFN-g and TNF-a and the chemokines CCL2/MCP-1, CCL3/MIP-1a and CCL5/RANTES. In vitro, pre-incubation with ET-1 (0.1 microM) 4 h before infection enhanced the uptake of the parasites by peritoneal macrophages, and this effect was abrogated when macrophages were pre-treated with bosentan (1 microM) 15 min before incubation with ET-1. However, ET-1 did not alter killing of intracellular parasites after 48 h of in vitro infection. Our data suggest that bosentan-treated mice have a delay in controlling parasitism which is compensated for exacerbated inflammation. Infection is eventually controlled in these animals and lethality is unchanged, demonstrating that ET-1 plays a minor role in the protection against acute murine T. cruzi infection.

Infective endocarditis caused by Streptococcus mutans. A complication of idiopathic hypertrophic subaortic stenosis.

Three patients with endocarditis caused by Streptococcus mutans were seen during a six-month period. All had clinical features of subacute bacterial endocarditis, including fever, heart murmurs, and positive blood cultures. One had underlying aortic insufficiency and two had idiopathic hypertrophic subaortic stenosis. All patients were treated with parenteral antibiotics and were cured. Streptococcus mutans is a pleomorphic, microaerophilic organism that is associated with dental caries and plaque. Differentiation of S mutans from enterococcal endocarditis is important because the former condition can be treated for a shorter period of time with penicillin alone, without the addition of aminoglycoside antibiotics.

Cell cycle molecules and diseases of the cardiovascular system.

Injury to the cardiovascular system causes an elevated expression of endothelin-1 (ET-1) and activation of several important signaling pathways including the mitogen-activated kinase (MAPK) cascade. The activation of these pathways has been implicated in the pathogenesis of cardiovascular disease caused by hypoxia, infections, and ischemia /reperfusion injury, cardiomyopathy and restenosis after balloon angioplasty. Important downstream targets of the MAPK and ET-1 pathways are the cell cycle regulatory molecules (cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors). Regulation of these molecules contributes to remodeling throughout the cardiovascular system. In addition, cell cycle molecules are important in the regulation of angiogenesis. These new data have led to the development of potential therapeutic modalities targeting these regulatory molecules in order to ameliorate various cardiovascular disease states.

Alteration of the pattern of beta-adrenergic desensitization in cultured L6E9 muscle cells infected with Trypanosoma cruzi.

L6E9 myoblasts infected with Trypanosoma cruzi undergo desensitization to beta-adrenergic catecholamines in a manner distinct from uninfected control myoblasts. Following incubation of intact cells with isoproterenol for 2 h, homogenates prepared from differentiated, high density uninfected L6E9 cells retain isoproterenol-dependent adenylate cyclase activity. In addition, previous exposure to isoproterenol is accompanied by a decrease in the number of beta-adrenergic receptors. Homogenates of high density L6E9 cells infected with T. cruzi retain their adenylate cyclase responsivity to isoproterenol but demonstrate a marked decrease in beta-adrenergic receptors. Following desensitization infected cell homogenates lose their responsiveness to isoproterenol and demonstrate a more marked decrease in beta-receptors. There does not appear to be any effect of T. cruzi infection on affinity of beta-adrenergic agonists for the beta-receptor or on changes in agonist affinity associated with desensitization. Infection of low density undifferentiated cells results in no apparent change in adenylate cyclase activity or in beta-receptors. Their behavior in the setting of desensitization--decreased whole cell cyclic AMP, decreased adenylate cyclase, unchanged beta-receptors--is also not affected by infection. The pattern of desensitization to beta-adrenergic agonists in high density infected cells shares several properties with the pattern of desensitization in low density uninfected cells, suggesting that infection may be associated with part of the more primitive cellular response pattern.

The molecular characterization of the major polar tube protein gene from Encephalitozoon hellem, a microsporidian parasite of humans.

The microsporidia are obligate intracellular protozoan parasites of increasing importance as human pathogens, which are characterized by a small resistant spore with a single polar filament that coils around the sporoplasm. When stimulated, the polar filament rapidly everts out of the spore to form a hollow polar tube through which the sporoplasm passes, thus serving as a unique mechanism of transmission. A genomic library of the human microsporidium Encephalitozoon hellem was screened using a polyclonal rabbit antibody (anti-PTP Eh55) produced to the major HPLC purified polar tube protein (PTP) of E. hellem. This antibody localized to intrasporal polar filaments and extrasporal polar tubes of E. hellem by immunogold electron microscopy confirming the polar tube specificity of the antibody. A total of 14 anti-PTP Eh55 reactive genomic clones were identified and purified. A PTP gene was identified consisting of 1362 bp coding for 453 amino acids. The N-terminus of the translated protein consists of aputative N-terminal signal sequence of 22 amino acids, which when cleaved results in a mature protein of 431 amino acids with a predicted molecular mass of 43 kDa. The protein has a high proline content (14.6%) and contains a central domain of six alternating tandem repeats of 20 amino acids. After ligation of the gene into a glutathione S-transferase (GST) expression vector, a fusion protein was produced that reacted by immunoblotting with the polar tube specific anti-PTP Eh55. The gene was present as a single copy in the genome and there was no homology with other known genes. As the polar tube is a critical structure for the transmission of this organism to a new host cell, further study of PTPs may lead to the development of new therapeutic strategies and diagnostic tests.

Localization and activity of nitric oxide synthases in the gastrointestinal tract of Trypanosoma cruzi-infected mice.

Chagas' disease, caused by the protozoan parasite, Trypanosoma cruzi, is associated with gastrointestinal abnormalities. Since nitric oxide (NO) has been shown to be a factor influencing intestinal function we evaluated the distributions and activities of the NO synthase (NOS) isoforms, in the gut of mice infected with T. cruzi. Ca2+-dependent (NOS1/NOS3) activity was decreased, whereas Ca2+-independent (NOS2) activity was increased in infected mice. NOS2-immunoreactivity was demonstrated in cells within the muscle layers and epithelium in infected mice and NOS1 immunoreactivity was seen in nerve structures. These data indicate that alterations in the NO-system may be important in the pathogenesis of the gastrointestinal manifestations in Chagas' disease.

The role of endothelin in the pathogenesis of Chagas' disease.

Infection with Trypanosoma cruzi causes a generalised vasculitis of several vascular beds. This vasculopathy is manifested by vasospasm, reduced blood flow, focal ischaemia, platelet thrombi, increased platelet aggregation and elevated plasma levels of thromboxane A(2) and endothelin-1. In the myocardium of infected mice, myonecrosis and a vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium are observed. Immunohistochemistry studies employing anti-endothelin-1 antibody revealed increased expression of endothelin-1, most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for prepro endothelin-1, endothelin converting enzyme and endothelin-1 were observed in the infected myocardium. When T. cruzi-infected mice were treated with phosphoramidon, an inhibitor of endothelin converting enzyme, there was a decrease in heart size and severity of pathology. Mitogen-activated protein kinases and the transcription factor activator-protein-1 regulate the expression of endothelin-1. Therefore, we examined the activation of mitogen-activated protein kinases in the myocardium by T. cruzi. Western blot demonstrated an extracellular signal regulated kinase. In addition, the activator-protein-1 DNA binding activity, as determined by electrophoretic mobility shift assay, was increased. Increased expression of cyclins A and cyclin D1 was observed in the myocardium, and immunohistochemistry studies revealed that interstitial cells and vascular and endocardial endothelial cells stained intensely with antibodies to these cyclins. These data demonstrate that T. cruzi infection of the myocardium activates extracellular signal regulated kinase, activator-protein-1, endothelin-1, and cyclins. The activation of these pathways is likely to contribute to the pathogenesis of chagasic heart disease. These experimental observations suggest that the vasculature plays a role in the pathogenesis of chagasic cardiomyopathy. Additionally, the identification of these pathways provides possible targets for therapeutic interventions to ameliorate or prevent the development of cardiomyopathy during T. cruzi infection.

The putative mechanistic basis for the modulatory role of endothelin-1 in the altered vascular tone induced by Trypanosoma cruzi.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of heart disease in Latin America. T. cruzi-induced microvascular compromise, in turn, is thought to play a major role in chagasic heart disease. Previous in vitro studies have implicated endothelin-1 (ET-1) as a potentially important vasomodulator present in increased levels in the supernatant of T. cruzi infected cultured human umbilical vein endothelial cells (HUVEC). Thus, the goal of the present investigation was to further evaluate the potentially important contribution of ET-1 to T. cruzi-induced alterations in vascular tone in vitro. Bioassay studies once again documented that exposure of isolated rat aortic rings to infected HUVEC supernatants elicited contractile responses whose steady-state magnitude was significantly greater than contractile responses elicited by exposure of aortic rings to uninfected HUVEC supernatants. Furthermore, the increased aortic contractility was significantly attenuated by the presence of the ET(A) subtype selective antagonists BMS-182,874 or BQ-123. Additionally, incubation of HUVEC with either verapamil or phosphoramidon prior to infection was also associated with reduced aortic contractility, upon exposure to the supernatant. Phosphoramidon, but not verapamil, produced a significant decrease in the measured ET-1 levels in the HUVEC supernatant. Consistent with the bioassay results, preincubation of Fura-2-loaded cultured rat aortic vascular smooth muscle cells with verapamil resulted in a near complete ablation of ET-1-induced transmembrane Ca2+ flux. Taken together, these data are consistent with the hypothesis that ET-1-induced vasoconstriction may play an important modulatory role in the vascular compromise characteristic of T. cruzi infection.

Chagas' disease: Microvascular and interstitial matrix abnormalities characteristic of congestive cardiomyopathy of diverse etiology.

Chagas' disease, due to a chronic and persistent infection with the parasite Trypanosoma cruzi, is the cause of one of the most prevalent forms of congestive cardiomyopathy in South and Central America. In common with other congestive cardiomyopathies due to acquired and hereditary etiologies seen worldwide, chagasic cardiomyopathy is characterized by cardiomegaly, ventricular chamber remodeling, myocellular necrosis, and multifocal areas of interstitial and replacement fibrosis. The microscopic presence of the parasite in the late stages of the disease is rare. This review examines some of the evidence for the role of a dysfunctional microvasculature as a pathophysiological mechanism for myocardial damage in chagasic cardiomyopathy. Perturbations of the interstitial connective tissue matrix in Chagas' disease also are described in regard to the remodeling characteristic of the affected ventricle. Similar abnormalities of the microvasculature and matrix have been reported in other congestive cardiomyopathies, thereby suggesting that common pathophysiologic mechanisms may lead to ventricular damage even when the initiating etiologic agent is different. Preventive treatment or palliation of Chagas' cardiomyopathy and other congestive cardiomyopathies may result from a better understanding of these secondary pathogenetic events.

Myocardial expression of endothelin-1 in murine Trypanosoma cruzi infection.

Chagas' disease, caused by Trypanosoma cruzi, is an important cause of myocarditis and chronic cardiomyopathy and is accompanied by microvascular spasm and myocardial ischemia. We reported previously that infection of cultured endothelial cells with T. cruzi increased the synthesis of biologically active endothlein-1 (ET-1). In the present study, we examined the role of ET-1 in the cardiovascular system of CD1 mice infected with the Brazil strain of T. cruzi and C57BL/6 mice infected with the Tulahuen strain during acute infection. In the myocardium of infected mice myonecrosis and multiple pseudocysts were observed. There was also an intense vasculitis of the aorta, coronary artery, smaller myocardial vessels and the endocardial endothelium. Immunohistochemistry studies employing anti-ET-1 antibody revealed increased expression of ET-1 that was most intense in the endocardial and vascular endothelium. Elevated levels of mRNA for preproET-1, endothelin converting enzyme and ET-1 were observed in the same myocardial samples. Plasma ET-1 levels were significantly elevated in infected CD1 mice 10-15 days post infection. These observations suggest that increased levels of ET-1 are a consequence of the initial invasion of the cardiovascular system and provide a mechanism for infection-associated myocardial dysfunction.

Parasitic infections in AIDS patients. Cryptosporidiosis, isosporiasis, microsporidiosis, cyclosporiasis.

AIDS is characteristically associated with several intracellular enteric protozoan infections that often cause chronic and sometimes fatal intractable large-volume diarrhea. Until the AIDS epidemic, several of these parasitic infections were almost unknown as causes of human disease. This article reviews the diseases produced by cryptosporidia, isospora, cyclospora, and microsporidia in humans.

The ethnic minority traveler.

Although the ethnic minority traveler is exposed to the same risks as other travelers, there are special considerations that make them vulnerable to certain diseases. In addition, many ethnic minority travelers are traditionally underserved by the medical community and often travel without the benefit of adequate counseling and immunization. The specific disease entities covered in this article include parasitic diseases (e.g. malaria, trypanosomiasis, intestinal helminths), tuberculosis, and other respiratory diseases, dengue, and sexually transmitted diseases and HIV.

Taeniasis and cysticercosis.

This article focuses on clinical issues of taeniasis and cysticercosis, including a comprehensive review of the clinical data, standard and latest chemotherapy, modern concepts of pathogenesis, conventional and advanced diagnostic tests, current epidemiology, and effective means of control. Fundamental parasitology is covered to familiarize physicians and scientists with the latest concepts of parasites.

Pyrimethamine concentrations in serum during treatment of acute murine experimental toxoplasmosis.

Central nervous system toxoplasmosis is a major opportunistic infection in patients with acquired immunodeficiency syndrome. The standard therapy for this infection is pyrimethamine (PYR) and sulfonamides. To assess in vivo if PYR alone could adequately treat toxoplasmosis, a murine model of acute toxoplasmosis was used. The CD1 strain of mice was infected intraperitoneally with 10(4) parasites of the RH strain of Toxoplasma gondii. Pyrimethamine was administered in mouse chow at concentrations of 0, 0.03125, 0.0625, 0.125, 0.25, or 1.0 mg of PYR/g of food, which provides the following daily PYR dosages: 0, 6.25, 12.5, 25, 50, and 200 mg/kg/day. No sulfonamides were administered. Serum PYR levels proved more accurate than mg of PYR/g of food in predicting survival. Mice with serum PYR levels greater than or equal to 500 ng/ml (2 microM) survived and had no parasites present on peritoneal lavage. Mice with serum PYR levels less than 100 ng/ml (0.4 microM) had a 100% mortality rate and the average parasite count was 3 x 10(7) organisms in the lavage fluid. At a PYR level of 370 ng/ml, six of 11 mice survived and the lavage fluid contained 2.5 x 10(5) organisms. Previously, using 3H-uracil in an in vitro assay, PYR at a concentration of 500 ng/ml was shown to be as effective in inhibiting Toxoplasma growth as the combination of PYR (100 ng/ml) and sulfonamides 25 micrograms/ml). These data suggest the potential usefulness of PYR for monotherapy of toxoplasmosis and are consistent with previously described in vitro assays.

Protection against fatal murine Chagas' disease with a Leishmania braziliensis panamensis stock.

Past attempts to immunize mice against a fatal Trypanosoma cruzi infection utilizing related hemoflagellates have been unsuccessful. In the present study, C57BL/6 mice received a footpad inoculation of 10(7) promastigotes of Leishmania braziliensis panamensis. Six and 9 weeks subsequent to this inoculation mice were infected intraperitoneally with the Tulahuen strain of T. cruzi. All immunized mice survived infection over the 6-month period of observation whereas control mice regularly died. There was an early transient T. cruzi parasitemia in the immunized mice. Culture of blood and organs as well as histopathological examination of various organs 6 months post-challenge failed to yield any evidence of T. cruzi. Heat-killed and freeze-thawed extracts of promastigotes did not confer any protection. These observations raise the possibility that certain leishmanial species might confer natural protection against a T. cruzi infection and that this information could be useful in the development of a vaccine.

Acetylcholinesterase levels in skeletal muscle of mice infected with Trypanosoma cruzi.

Acetylcholinesterase (AChE) activity was measured in skeletal muscle from susceptible (A/J) and resistant (C57BL/6) mice infected with the Brazil strain (myotropic) of Trypanosoma cruzi. There was a 60% decrease in activity in skeletal muscle obtained from A/J mice 20 days post-infection as compared to controls. There was no decrease in AChE activity in skeletal muscle obtained from infected C57BL/6 mice 20 and 150 days post-infection. Histologic examination of skeletal muscle from infected A/J mice revealed marked necrosis, pseudocysts, and minimal inflammation. Similar examinations in C57BL/6 mice revealed marked inflammation in the absence of necrosis and parasites. These data provide additional biochemical support that denervation hypersensitivity is an important concomitant of Chagas' disease and that it is already present during the acute stage. Additionally, it may support the notion that the presence of the parasite mediates these abnormalities.

Evidence for guanosine triphosphate--binding proteins in Trypanosoma cruzi.

The transformation of the parasite Trypanosoma cruzi from the blood-borne trypomastigote to the intracellular amastigote constitutes a key clinical feature in the pathophysiology of Chagas' disease. That this transition occurs without change in the integrity of the plasma membrane of the parasite suggests the presence of biochemical structures, i.e., signal transduction systems, that convey information regarding the external milieu of the host so as to facilitate this transformation. In higher eukaryotes, it has been found that a heterotrimeric GTP-binding protein (G-protein), composed of alpha beta gamma subunits, constitutes a critical component of this complex. Two closely related groups of G-proteins are substrates for cholera toxin (CT)- (Gs) and pertussis toxin (PT)- (Gi1-3 and Go) dependent ADP ribosylation. In concert, they link plasma membrane receptors to adenylate cyclase, resulting in the stimulation or inhibition, respectively, of cAMP generation. In this report, we demonstrate the presence of both groups of G-proteins. Cholera toxin-dependent ADP ribosylation of 42- and 45-kD proteins was demonstrable in amastigotes (AMAST), in the cytosol of epimastigotes (EPI), and weakly in trypomastigotes (TRYP), suggesting the presence of the stimulatory GTP-binding protein, Gs, in T. cruzi. Antisera generated against the alpha s subunit of the Gs heterotrimeric protein (anti-alpha s) bound to a 45-kD protein CT substrate in the rank order TRYP >> AMAST approximately EPI cytosol. Immunoprecipitation of CT-32P-ADP-ribosylated membranes with anti-alpha s resulted in 42- and 45-kD proteins. However, no Gs-mediated activation of adenylate cyclase was demonstrable in reconstitution studies using cyc- lymphoma cells, which lack a functional Gs but possess a beta-adrenergic receptor and adenylyl cyclase enzyme. Pertussis toxin-catalyzed ADP ribosylation was demonstrable in 39-40-kD particulate proteins of EPI, less strongly in AMAST, and least in TRYP, consistent with the presence of inhibitory (Gi) and Go GTP-binding proteins. In support of this observation, immunochemical analysis of the PT substrates identified the presence of alpha o and alpha i1-2-3 in EPI, AMAST and TRYP, although, with the exception of alpha i3, both toxin and associated immunochemical PT substrates are decreased in AMAST and TRYP relative to EPI. Although the functions of these putative G-proteins in T. cruzi are still unclear, their expression may be regulated by the state of parasite differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Atovaquone in the treatment of Babesia microti infections in hamsters.

The traditional therapy for the treatment of human Babesia microti infections has been the combination of clindamycin and quinine. However, in recent years, it has become apparent that some patients have not responded to this regimen. We became involved in the treatment of several cases of babesiosis in which atovaquone was used to treat this infection. Therefore, using the hamster model, we determined the efficacy of atovaquone alone as well as atovaquone plus azithromycin for the treatment of experimental babesiosis. Atovaquone (100 mg/kg/day) and atovaquone (100 mg/kg/day) with azithromycin (150 mg/kg/day) were effective agents for the treatment of experimental babesiosis in hamsters. When atovaquone was used as monotherapy recrudescences occurred. Organisms obtained from recrudescent animals, when inoculated into uninfected animals, proved to be unresponsive to atovaquone therapy, suggesting the emergence of drug resistance. Resistant organisms did not emerge in hamsters treated with the combination of atovaquone and azithromycin. Atovaquone should be considered in the therapeutic regimen of patients with babesiosis who have either failed standard therapy or have become intolerant to such therapy.

Infection of organotypic cultures of spinal cord and dorsal root ganglia with Trypanosoma cruzi.

Although the involvement of the nervous system in Chagas' disease is well described, the mechanism of the neuronal destruction is unclear. Immunologic, toxic mechanisms and direct invasion have been advocated. Organotypic cultures of spinal cord and dorsal root ganglion derived from Swiss outbred mice were infected with the Brazil strain of Trypanosoma cruzi. Light microscopic and ultrastructural studies were performed at regular intervals. It was found that trypomastigotes were rapidly taken up by glial and other supporting cells. Neurons were rarely parasitized and demyelination was not evident. Loss of several cytoskeletal components was seen. Dendrites were swollen and axons lost their normal filamentous structures but synaptic membranes remained intact. Mitochondrial swelling was evident even in nonparasitized neurons from infected cultures. By 7-10 days of infection the majority of neurons lost their typical morphology and were eventually destroyed by mechanisms other than direct parasite invasion. Organotypic cultures exposed to T. cruzi-conditioned medium exhibited no change in morphology. Since neurons were found only rarely to be parasitized, it is suggested that neuronal destruction is an indirect result of the parasitism of supporting cells such as glial cells and macrophages.

Effect of verapamil on the development of chronic experimental Chagas' disease.

In previous work, we have shown that the chronic administration of verapamil, a calcium channel blocker, ameliorated the mortality, pathology, and biochemical alterations associated with acute murine Chagas' disease. To extend these studies to an established chronic model, C3H/Hej mice were infected with the Sylvio X10/4 clone. This clone does not cause symptomatic acute disease but does induce cardiac pathology incorporating several pathological features of human chagasic cardiomyopathy. Cardiac pathology was assessed at 60, 90, and 180 days postinfection. There was a significant decrease in the degree of inflammation and fibrosis in the group infected and treated with verapamil. Myocardial beta-adrenergic adenylate cyclase (AC) activity was determined 180 days postinfection. In the infected group not treated with verapamil, there was no significant change in the maximum rate of conversion of ATP to cAMP (Vmax) or in the concentration of agonist giving 50% of Vmax (apparent Kact) for isoproterenol (ISPN)-dependent AC activation. The increase in Vmax for ISPN determined in the presence of 5'-guanylylimidodiphosphate (Gpp[NH]p) was consistently lower in infected than in uninfected mice, suggesting that infection altered the potential synergistic activation of AC by the guanine nucleotide. In the infected group treated with verapamil, there was a slight increase in the Vmax for ISPN. However, there was a marked enhancement of the synergistic contribution of Gpp(NH)p. These observations suggest that verapamil had preserved that aspect of the AC complex mediating guanine nucleotide sensitive activation of AC. Collectively, the observations in the acute and chronic models of murine Chagas' disease suggest that verapamil may be a useful adjunct in treatment.