Impairment of the growth of Plasmodium falciparum in HbEE erythrocytes.

HbE is a beta-chain mutant frequently found among inhabitants of Southeast Asia and surrounding territories. We find that Plasmodium falciparum multiplies more slowly in erythrocytes from individuals homozygous for HbE than in cells from HbA individuals. In contrast, this parasite grows normally in erythrocytes heterozygous for HbE. This is the first direct evidence that suggests what has been suspected on the basis of circumstantial data, that HbE-containing erythrocytes might be advantageous to the carrier in regions with endemic malaria.

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.

Verapamil ameliorates clinical, pathologic and biochemical manifestations of experimental chagasic cardiomyopathy in mice.

The influence of long-term verapamil administration on the consequences of Trypanosoma cruzi infection in mice was studied with regard to animal mortality, morbidity, myocardial pathologic features and myocardial beta-adrenergic adenylate cyclase activity. Verapamil administration dramatically decreased the mortality rate from 60% to 6% during the 70 day period of infection. Three clinical stages of infection were evident. In the acute stage (17 days after infection with maximal parasitemia), verapamil treatment not only decreased the incidence of myocardial disease (fibrosis and inflammation), but also protected myocardial beta-adrenergic adenylate cyclase activity. In addition, there was no increase in total body weight, which was regarded as an index of right-sided heart failure. In the subacute stage (30 to 60 days after infection), administration of verapamil continued to decrease myocardial disease and preserve beta-adrenergic adenylate cyclase activity. In addition, verapamil ameliorated the morbidity and mortality associated with this stage of infection. The chronic stage of infection was characterized by a decrease in myocardial disease and in beta-adrenergic adenylate cyclase activity. Thus, independent of the state of infection, long-term verapamil treatment enhanced beta-adrenergic adenylate cyclase activity. In addition, verapamil ameliorated the morbidity associated with infection. Although the relation among these various effects of verapamil in the setting of T. cruzi infection remains to be determined, collectively the results suggested that verapamil administration attenuated the consequences of 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.

Release of guanosine triphosphate binding protein alpha subunits from mouse myocardial membranes: basic properties and their alterations in acute murine Chagas disease.

OBJECTIVE: The aim was to investigate the arrangement of heterotrimeric alpha beta gamma G proteins in myocardial membranes using GTP gamma S dependent release characteristics of their alpha subunits in acute murine Chagas disease. METHODS: The properties of GTP gamma S dependent alpha subunit release were monitored immunochemically as well as by cholera toxin and pertussis toxin catalysed [32P]ADP ribosylation. RESULTS: GTP gamma S, as opposed to other nucleotides, caused optimal and virtually instantaneous release of soluble 40 kDa [32P]ADP ribosylated protein in pertussis toxin treated membranes. When determined immunochemically, infection decreased both the sensitivity to GTP gamma S dependent release of alpha i subunits and appeared to facilitate the appearance of GTP gamma S dependent release of alpha i3. GTP gamma S also caused the release of soluble 45 and 40 kDa proteins as detected by cholera toxin-[32P]ADP ribosylated membranes and immunochemical analysis. With regard to cholera toxin-[32P]ADP ribosylated Gs substrates sensitive to GTP gamma S dependent release, infection (1) decreased the amount of 45 kDa alpha s protein, (2) increased the amount of 40 kDa protein, and (3) enhanced sensitivity to GTP gamma S. In contrast, there was no effect of infection on the magnitude or sensitivity to GTP gamma S dependent release of immunochemical alpha s. CONCLUSIONS: The diverse characteristics of GTP gamma S dependent release of the very similar alpha subunits from myocardial membranes and their unique sensitivity to infection with T cruzi suggest that these very similar proteins are arranged within the plasma membrane in such a manner as to modify their biochemical behaviour.

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.

Alterations in intracellular calcium following infection of human endothelial cells with Trypanosoma cruzi.

Trypanosoma cruzi infection in cultured human umbilical vein endothelial cells increased basal cellular calcium levels from 55 to 110 nM, as monitored with the fluorescent probe, fura-2. It also influenced intracellular calcium such that consistently higher total levels were observed in response to bradykinin, angiotensin II and norepinephrine, as compared to similarly treated uninfected cells. However, bradykinin and angiotensin II-dependent increases in calcium, when considered as the absolute increment or fold elevation over basal, were significantly lower in infected endothelial cells. Infection also influenced changes in calcium levels due to agents that operate independently of plasma membrane receptors. In the presence of ionomycin, the magnitude and rate of rise of intracellular calcium were decreased; additionally the calcium peak was delayed and the subsequent decline slowed. Similar to the results with bradykinin and angiotensin II, infection decreased both the increment in and fold stimulation of intracellular calcium in response to ionomycin. In contrast, infection altered only the total calcium stimulated in response to oligomycin; neither the fold stimulation of, nor increment in intracellular calcium was affected. These results indicate that (1) infection by T. cruzi alters calcium homeostasis in endothelial cells under basal and stimulated conditions; (2) both receptor-dependent and receptor-independent mechanisms are affected by infection. The possible contribution of altered calcium homeostasis induced by T. cruzi in the pathogenesis of chagasic cardiomyopathy is considered.

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.

Studies of in vitro infection by Trypanosoma cruzi. I. Ultrastructural studies on the invasion of macrophages and L-cells.

The interactions of Trypanosoma cruzi with L-cells, and with normal and activated macrophages in vitro were studied by ultrastructural techniques. T. cruzi actively invades cultured L-cells and uniformly destroys them. Normal macrophages could control a 1:1 (parasite to host cell) infection, but were destroyed by a 10:1 infection. BCG-activated macrophages, however, controlled a 10:1 infection but not one at a ratio of 100:1. It appears that parasites that survive within host cells do so outside cytoplasmic vacuoles, whereas when they are relegated to host cell phagosomes they are destroyed. Culture forms of T. cruzi have several means of access into host cells. Marcrophages are better able to survive infection than are non-phagocytic cells. Finally, it is suggested that control of an experimental infection in vitro is dependent upon numbers of parasites to macrophages as well as the state of the macrophages.

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.

Abnormalities of the coronary microcirculation in acute murine Chagas' disease.

Chronic Chagasic heart disease has many features characteristic of other congestive cardiomyopathies, including ventricular and atrial chamber enlargement, hypertrophy, focal scarring, and mural thrombi. Histologically, there is often lymphocytic inflammation, spotty necrosis, and few parasites. Although immunologic mechanisms have been invoked to explain the development of myocardial degeneration, there have been suggestions that the focal alterations in the heart are secondary to abnormalities of the coronary microcirculation. Based on work from our laboratories which has demonstrated microvascular hyperreactivity in several other models of congestive cardiomyopathy, we investigated whether the cardiac microcirculation of mice acutely infected with Trypanosoma cruzi was also abnormal. We perfused animals at 15-17 days post-infection with silicone rubber which fills the arterioles, capillaries, and venules of the beating heart. After clearing the tissue, we observed numerous areas of focal vascular constriction, microaneurysm formation, dilatation, and proliferation of microvessels which were not present in control animals. These lesions were similar to those we have observed in other congestive cardiomyopathies. Since at this stage of infection there is minimal cardiac degeneration or fibrosis, the presence of these vascular lesions even early in Chagas' disease, may be significant for the pathogenesis of focal myocardial damage. These observations during acute infection provide additional support for the suggestions of others that the myocardial microcirculation is abnormal in Chagas' disease.