Murray Wittner

The microsporidia and microsporidiosis

Historic Perspective on the Microsporidia: Expanding Horizons Structure of the Microsporidia Development Morphology and Life Cycles of the Microsporidia Molecular Biology, Molecular Phylogeny, and Molecular Diagnostic Approaches to the Microsporidia Microsporidian Biochemistry and Physiology The Structure, Function, and Composition of the Microsporidian Polar Tube Host-Parasite Relationships in Microsporidiosis: Animal Models and Physiology Clinical Syndromes Associated with Microsporidiosis Ocular Microsporidiosis Laboratory Diagnosis of Microsporidiosis Culture and Propagation of Microsporidia Microsporidia in Higher Vertebrates Fish Microsporidia Microsporidia in Insects Epidemiology of Microsporidiosis

Trypanosoma cruzi: role of the immune response in the natural resistance of inbred strains of mice.

Abstract Nine inbred strains of mice were challenged with 104 or 105 trypomastigotes of the Brazil strain of Trypanosoma cruzi. A spectrum of resistance was evident ranging from highly susceptible strains, e.g., C3H, which developed high parasitemias and died within 3 to 4 weeks, to resistant strains, e.g., C57BL/10, which developed low parasitemias and survived. Impairment of the immune system in resistant C57BL/10 mice by X-irradiation, splenectomy, or treatment with silica led to high, often fatal parasitemias. Athymic nude mice, in particular, attained exceptionally high parasitemias before dying. The immune response appears to be necessary for survival and to play a role in the natural resistance of some mouse strains by effectively eliminating parasites and minimizing parasitemia. Using congenic strains of mice, it was shown that the principal genetic determinant of resistance is not associated with their H-2 haplotype.

A Cell Culture System for Study of the Development of Toxoplasma gondii Bradyzoites

ABSTRACT. Toxoplasma gondii is a ubiquitous apicomplexan parasite and a major opportunistic pathogen under AIDS‐induced conditions, where it causes encephalitis when the bradyzoite (cyst) stage is reactivated. A bradyzoite‐specific Mab, 74.1.8, reacting with a 28 kDa antigen, was used to study bradyzoite development in vitro by immuno‐electron microscopy and immunofluorescence in human fibroblasts infected with ME49 strain T. gondii. Bradyzoites were detected in tissue culture within 3 days of infection. Free floating cyst‐like structures were also identified. Western blotting demonstrated the expression of bradyzoite antigens in these free‐floating cysts as well as in the monolayer. Bradyzoite development was increased by using media adjusted to pH 6.8 or 8.2. The addition of γ‐interferon at day 3 of culture while decreasing the total number of cysts formed prevented tachyzoite overgrowth and enabled study of in vitro bradyzoites for up to 25 days. The addition of IL‐6 increased the number of cysts released into the medium and increased the number of cysts formed at pH 7.2. Confirmation of bradyzoite development in vitro was provided by electron microscopy. It is possible that the induction of an acute phase response in the host cell may be important for bradyzoite differentiation. This system should allow further studies on the effect of various agents on the development of bradyzoites.

Successful Chemotherapy of Transfusion Babesiosis

We describe babesiosis transmitted by transfusion. The infected blood donor was identified and a minimum period of infectivity of the donors blood was established. We report a new modality for chemotherapy consisting of quinine plus clindamycin, and a new endemic focus for this zoonosis on Fire Island, New York. There are insufficient data to establish a reasonably safe period after which visitors and residents of Babesia-endemic foci can become blood donors. Screening of such persons by a rapid serologic test, such as the ELISA or immunofluorescent antibody tests, is suggested.

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.

Initial Characterization of CST1, a Toxoplasma gondii Cyst Wall Glycoprotein

ABSTRACT Toxoplasma gondii is an important protozoan pathogen of humans that can cause encephalitis in immunocompromised individuals such as those with AIDS. This encephalitis is due to reactivation of latent infection in T. gondii-seropositive patients. Latent organisms survive within tissue cysts, which are specialized parasitophorous vacuoles containing bradyzoites. The cyst wall of this structure is produced by modification of the parasitophorous vacuole by the parasite and is important in cyst survival. The components of the cyst wall have been poorly characterized. By using immunofluorescence and immunoelectron microscopy, we have identified a monoclonal antibody (MAb 93.18) that reacts with the cyst wall. This antibody recognizes a 116-kDa glycoprotein, which we have termed CST1, containing sugar residues that bind Dolichos biflorans lectin (DBA). CST1 is distinct from T. gondii antigen labeled with succinylTriticum vulgare lectin (S-WGA) and represents the major DBA-binding component in T. gondii. The carbohydrate components of the tissue cyst, such as CST1, are probably important in both providing stability and facilitating persistence in its host. As is seen in the carbohydrate capsules of fungi, glycoproteins in theT. gondii cyst wall may protect cysts from the immune response of the host. Further characterization of the formation of the cyst wall and its components should lead to insights into the mechanism of tissue cyst persistence and may suggest novel therapeutic approaches to eliminate tissue cysts of this organism.

Brachiola vesicularum, n. g., n. sp., a new microsporidium associated with AIDS and myositis

Brachiola vesicularum, n. g., n. sp., is a new microsporidium associated with AIDS and myositis. Biopsied muscle tissue, examined by light and electron microscopy, revealed the presence of organisms developing in direct contact with muscle cell cytoplasm and fibers. No other tissue types were infected. All parasite stages contain diplokaryotic nuclei and all cell division is by binary fission. Sporogony is disporoblastic, producing 2.9 times 2 μm diplokaryotic spores containing 8‐10 coils of the polar filament arranged in one to three rows, usually two. Additionally, this microsporidium produces electron‐dense extracellular secretions and vesiculotubular appendages similar to Nosema algerae. However, the production of protoplasmic extensions which may branch and terminate in extensive vesiculotubular structures is unique to this parasite. Additionally, unlike Nosema algerae, its development occurred at warm blooded host temperature (37‐38° C) and unlike Nosema connori, which disseminates to all tissue types, B. vesicularum infected only muscle cells. Thus, a new genus and species is proposed. Because of the similarities with the genus Nosema, this new genus is placed in the family Nosematidae. Successful clearing of this infection (both clinically and histologically) resulted from treatment with albendazole and itraconozole.

Effect of Octreotide on Refractory AIDS-associated Diarrhea: A Prospective, Multicenter Clinical Trial

OBJECTIVE To determine the efficacy and safety of octreotide for treatment of refractory, profuse diarrhea in patients with the acquired immunodeficiency syndrome (AIDS). DESIGN A prospective, open-label study. SETTING Inpatient metabolic units of four university medical centers. PATIENTS Fifty-one patients infected with human immunodeficiency virus (HIV) who had uncontrolled diarrhea (greater than or equal to 500-mL liquid stool per day) despite treatment with maximally tolerable doses of antidiarrheal medications. INTERVENTION After initial baseline studies, patients received octreotide, 50 micrograms every 8 hours for 48 hours. If stool volume was not reduced to less than 250 mL/d, the dose of octreotide was increased stepwise to 100, 250, and 500 micrograms. MAIN RESULTS Fifty men and one woman (mean age, 36.3 +/- 1.1 years) entered and completed the 28-day protocol (14 days of inpatient therapy and 14 days of outpatient therapy). Stool frequency and volume decreased significantly (6.5 +/- 0.5 stools per day on day 0 compared with 3.8 +/- 0.3 stools per day on day 21 [P less than 0.001] and 1604 +/- 180 mL/d on day 0 compared with 1084 +/- 162 mL/d on day 14 [P less than 0.001], respectively). Twenty-one patients (41.2%) were considered to be partial or complete responders (reduction in daily stool volume by greater than or equal to 50% of initial collections or reduction to less than or equal to 250 mL/d). Of the 21 responders, 14 (67%) had no identifiable pathogens at initial screening compared with 9 of 30 (30%) nonresponders (P less than 0.01). CONCLUSION Patients with AIDS-associated refractory watery diarrhea, especially those without identifiable pathogens, may respond favorably to subcutaneously administered octreotide. This drug deserves further study in a randomized, placebo-controlled trial.

Role of Endothelin 1 in the Pathogenesis of Chronic Chagasic Heart Disease

ABSTRACT On the basis of previous observations, endothelin 1 (ET-1) has been suggested as contributing to the pathogenesis of Chagasic cardiomyopathy. Therefore, ET-1flox/flox;α-MHC-Cre(+) mice in which the ET-1 gene was deleted from cardiac myocytes and ET-1flox/flox;Tie 2 Cre(+) mice in which the ET-1 gene was deleted from endothelial cells were infected with Trypanosoma cruzi. Genetic controls for these cell-specific ET-1 knockout mice were used. Ninety percentage of all mice survived acute infection with the Brazil strain and were evaluated 130 days postinfection. Inflammation and fibrosis were observed in all infected mice; however, fibrosis was reduced in ET-1flox/flox;α-MHC-Cre(+) mice. Cardiac magnetic resonance imaging revealed that infection resulted in a significant increase in right ventricular internal diameter (RVID) in all mice except ET-1flox/flox;α-MHC-Cre(+) mice; i.e., RVID was not changed in infected ET-1flox/flox;α-MHC-Cre(+) mice. Echocardiography of the left ventricle demonstrated increased left ventricular end-diastolic diameter, reduced fractional shortening, and decreased relative wall thickness in infected mice. However, the magnitude of the changes was significantly less in ET-1flox/flox;α-MHC-Cre(+) mice compared to other groups. These data provide further evidence of a role for ET-1, particularly cardiac myocyte-derived ET-1, in the pathogenesis of chronic Chagasic cardiomyopathy.

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.