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Biomedical subjects

H B Tanowitz

Publications and source records attributed to H B Tanowitz.

At least 37 records · Page 2Linked to original sources
PubMed1999

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.

Animals↗
PubMed1999

Application of cardiac gated magnetic resonance imaging in murine Chagas' disease.

To evaluate the role of gated cardiac magnetic imaging resonance (MRI) in Chagas' disease, we infected mice with Trypanosoma cruzi (Brazil strain). Two models were chosen for study, the CD1 and the inducible nitric oxide synthase knockout (NOS2-/-) mice. Infection of CD1 mice was associated with a significant increase in the right ventricular inner diameter (RVID) that was reversed in some mice by verapamil. Expression of cardiac NOS2 has been associated with myocardial dysfunction. Therefore, we evaluated chagasic cardiomyopathy in NOS2-/- and syngeneic wild type (WT) mice. Infected WT mice exhibited an increase in RVID in the acute phase (< 60 days postinfection) that was more marked during chronic infection (>100 days postinfection). Chronically infected NOS2-/- mice had an increase in RVID. The RVID in infected WT mice was greater than in NOS2-/- mice. These data demonstrate that MRI is a useful tool in the serial evaluation of the heart in murine Chagas' disease. In addition, it supports the notion that the NOS2-/-/NO pathway may contribute to the pathogenesis of murine chagasic cardiomyopathy.

Analysis of Variance↗
PubMed1998

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.

Amino Acid Sequence↗
PubMed1998

Gap junction disappearance in astrocytes and leptomeningeal cells as a consequence of protozoan infection.

Trypanosoma cruzi and Toxoplasma gondii are protozoan parasites capable of causing infections of the nervous system. In order to determine effects of infection by these organisms on intercellular communication in the brain, dye coupling and connexin abundance and distribution were examined in leptomeningeal cells and astrocytes infected with T. cruzi or T. gondii. For both cell types infected with either type of protozoan parasite, intercellular diffusion of intracellularly injected Lucifer Yellow was dramatically reduced. Immunocytochemistry with antibodies specific for connexin43 (in astrocytes) or both connexin43 and connexin26 (for leptomeningeal cells) demonstrated that punctate gap junctional staining was much reduced in infected cells, although uninfected neighbors could display normal connexin abundance and distribution. Western blot analyses revealed that connexin43 abundance in both cell types infected with either parasite was similar to that in uninfected cells. Phosphorylation state of connexin43 (inferred from electrophoretic mobility of connexin43 isoforms) was not significantly affected by the infection process. Immunocytochemistry of whole brains from animals acutely infected with either parasite also showed a marked reduction in connexin43 expression. We conclude that infection of both types of brain cells with either protozoan parasite results in a loss of intercellular communication and organized gap junction plaques without affecting expression levels or posttranslational processing of gap junction proteins. Presumably, these changes in gap junction distribution result from altered targeting of the junctional protein to the plasma membrane, and/or from changes in assembly of subunits into functional channels.

Animals↗
PubMed1998

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.

Blood Transfusion↗
PubMed1998

TNP-470 is an effective antimicrosporidial agent.

Therapy for microsporidia, which cause diarrhea and a wasting syndrome in persons with AIDS, has had limited success. Fumagillin, a naturally secreted water-insoluble antibiotic, has in vitro activity against microsporidia and has been used successfully in the treatment of superficial keratitis in patients with AIDS, but systemic therapy has been limited by toxicity of the currently available fumagillin salt. TNP-470, a semisynthetic analogue of fumagillin, was studied in vitro and in the athymic nude mouse model of microsporidiosis. RK13 cells were infected with microsporidia of the family Encephalitozoonidae and treated at day 3 with TNP-470. This agent was highly effective, with an ID50 (50% inhibitory dose compared with control) of 0.001 microg/mL. TNP-470 also demonstrated in vivo activity against Encephalitozoon cuniculi, with prolonged survival and the prevention of the development of ascites in infected athymic mice. These data suggest that the fumagillin derivative TNP-470 is a promising agent for the treatment of microsporidiosis.

Animals↗
PubMed1998

Bradyzoite development in Toxoplasma gondii and the hsp70 stress response.

Toxoplasma gondii is a well-described ubiquitous Apicomplexan protozoan parasite that is an important opportunistic pathogen. The factors affecting the transition of tachyzoites to the latent bradyzoite stage remain to be defined. The induction of bradyzoite development in vitro has been linked to temperature, pH, mitochondrial inhibitors, sodium arsenite, and many of the other stressors associated with heat shock protein (hsp) induction. There is evidence for other organisms that hsps are developmentally regulated. Therefore, we examined whether hsp induction is an early event in bradyzoite differentiation. Extracellular and intracellular T. gondii cells, after exposure to pH 8.1 or 7.1, were analyzed for the expression of inducible hsp70 by using monoclonal antibody C92F3A-5 (specific to hsp70). Western blotting demonstrated that a 72-kDa protein reactive with C92F3A-5 (hsp70), which we believe is part of the hsp70 family, is induced during bradyzoite development. By immunofluorescence and immunoelectron microscopy, we were able to demonstrate that hsp70 staining colocalized to T. gondii expressing bradyzoite-specific antigens and the presence of hsp70 in bradyzoites isolated from mouse brain. Quercetin, a bioflavonoid which inhibits the synthesis of hsp90, hsp70, and hsp27, suppresses the induction of bradyzoite development in vitro. Reverse transcription-PCR with conserved hsp70 primers demonstrated an increase in hsp70 in T. gondii on exposure to conditions which induce bradyzoite formation. A T. gondii hsp70 was subsequently cloned and sequenced by using this amplified fragment. We believe our evidence suggests that hsps are important in the process of bradyzoite differentiation.

Amino Acid Sequence↗
PubMed1997

Myocardial G proteins in murine Chagas' disease.

G protein alpha subunits and their corresponding mRNA levels were determined in hearts obtained from mice infected with Trypanosoma cruzi, the etiologic agent of Chagas' disease. Protein and mRNA levels of the inhibitory G proteins G alpha i2 and G alpha i3 were increased at 21 and 30 days postinfection (PI). After 60 days, the abundance of protein and corresponding mRNA for G alpha i2 and G alpha i3 were no longer significantly different from uninfected mice. Twenty-one days after infection, G alpha s protein levels decreased markedly, but mRNA for the stimulatory protein did not change. Similar to the Gi proteins, by 60 days differences in G alpha s protein between infected and uninfected mice were no longer evident. There was an increase in the magnitude of G beta subunit protein 21 and 30 days PI as compared with uninfected mice. However, 60 days PI the G beta subunit protein decreased to control levels. The close relationship between the infection-associated increase in G alpha i protein and mRNA suggests that control of protein expression is likely to be exerted at the transcription level. In contrast, control of infection-associated decrease in Gs appears to be at the translational level.

Animals↗
PubMed1996

Cardioprotective actions of verapamil on the beta-adrenergic receptor complex in acute canine Chagas' disease.

UNLABELLED: The effect of verapamil treatment on the myocardial beta-adrenergic adenylyl cyclase complex in acute canine Chagas' disease was investigated. Relative to uninfected animals, 30 days of infection with T. cruzi reduced myocardial adenylyl cyclase activity by over 75%. With continuous verapamil treatment, the infection-associated reduction in adenylyl cyclase activity was less than 50%. The individual components of the beta-adrenergic receptor complex were characterized. INFECTION: (1) increased right ventricular (RV) beta-adrenergic receptor (beta AR) density five-fold; (2) decreased left ventricle beta AR density by 20%; (3) reduced the proportion of high-affinity beta AR receptors to the same extent in both left and right ventricles; (4) reduced alpha s by 50% as determined by Western blot analysis, increased alpha 11-3 but did not change alpha 0; and (5) decreased the magnitude of pertussis-toxin-dependent [32P]ADP ribosylation by 60% as well as the proportion of [32P]ADP-ribose incorporated in alpha 0. Verapamil treatment of infected animals restored RV beta AR receptor density, alpha s and alpha i1-3 to control levels but had no influence on any aspect of pertussis-toxin-dependent [32P]ADP-ribosylation. Verapamil treatment of uninfected animals also: (1) increased beta-adrenergic adenylyl cyclase activity; (2) increased beta AR density in the RV but not the LV; (3) reduced high- to low-affinity beta-adrenergic receptors; and (4) affected only alpha i2 (50% decrease). The results indicate that the major actions of verapamil on the beta-adrenergic adenylyl cyclase complex in acute canine Chagas' disease may help to account for its cardioprotective effects.

Adenylyl Cyclases↗
PubMed1996

Gastrointestinal manifestations.

Gastrointestinal disease is a common problem in the setting of HIV-1 infection. As patients live longer and other opportunistic pathogens are suppressed, these problems are becoming even more important in the quality of life.

AIDS-Related Opportunistic Infections↗
PubMed1996

Prevalence of microsporidiosis due to Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis among patients with AIDS-related diarrhea: determination by polymerase chain reaction to the microsporidian small-subunit rRNA gene.

Microsporidia are emerging as opportunistic pathogens in patients with AIDS. Enterocytozoon bieneusi and Encephalitozoon (Septata) intestinalis have been implicated in enteric infections in AIDS patients with chronic diarrhea, a wasting syndrome, and malabsorption. We used the polymerase chain reaction (PCR) and primers that amplify the conserved regions of the small-subunit rRNA (SSU-rRNA) gene of E. bieneusi and E. intestinalis in tissue specimens from HIV-infected patients with and without diarrhea to examine the association between microsporidia and diarrhea in patients with AIDS. Tissue specimens were obtained from 68 patients with AIDS and diarrhea (mean CD4 lymphocyte count, 21/mm3) and 43 AIDS patients without diarrhea (mean CD4 lymphocyte count, 60/mm3). By means of PCR with use of the SSU-rRNA primers specific for E. bieneusi and E. intestinalis, we found that 44% of patients with diarrhea were infected with microsporidia, whereas only 2.3% of the patients without diarrhea were infected with microsporidia (P < .001). There was a clear association between the presence of microsporidia and diarrhea. In addition, the SSU-rRNA primers proved to be sensitive and specific when used in this clinical setting.

AIDS-Related Opportunistic Infections↗
PubMed1996

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.

Animals↗
PubMed1996

Compromised microcirculation in acute murine Trypanosoma cruzi infection.

Microvascular compromise has been 1 of many factors implicated in the etiology of the cardiomyopathy associated with Chagas' disease. To further assess the effect of Trypanosoma cruzi infection on the microcirculation, we examined the cremaster microvascular model in CD-1 male mice infected with the Brazil strain at 20-25 days postinfection. There was a significant decrease in red cell velocity (Vrbc) in first and third-order arterioles and venules, which was reversed by verapamil treatment. Video recordings revealed a marked inflammatory response that was confirmed by transmission electron microscopy. A marked inflammatory response was not seen in verapamil-treated infected mice. Segmental vasospasm and dilatation was evident in the microvascular bed of infected mice. This was not seen in control or verapamil-treated mice. This model provides a readily accessible method to observe directly the effects of T. cruzi infection on the microcirculatory flow in vivo. In addition, it confirms and extends our previous observations regarding T. cruzi-associated microvascular spasm and underscores a role for verapamil, a calcium-channel blocker, in the amelioration of the Chagas' disease.

Acute Disease↗
PubMed1996

Effects of verapamil on acute murine Chagas' disease.

Continuous administration of verapamil significantly reduced the mortality rate of acute murine Trypanosoma cruzi infection (P < 0.05). The mechanistic basis for these observations was investigated. Verapamil and other calcium-channel blockers did not inhibit the growth of epimastigotes in culture. Furthermore, verapamil did not inhibit the intracellular growth of amastigotes in endothelial cells as determined by the uptake of 3H-uracil. There were no significant differences in parasitemia between infected mice that were untreated and those treated with verapamil. Twenty days postinfection infected, untreated mice had a parasitemia of 5.8 x 10(6) trypomastigotes/ml (SD +/- 2 x 10(6)), whereas infected, verapamil-treated mice had a parasitemia of 2.2 x 10(6) trypomastigotes/ml (SD +/- 0.5 x 10(6)). There was no significant difference in mortality between mice administered verapamil only for the initial 10 days of murine infection compared to those treated continuously. A 3-day delay in the initiation of verapamil administration reduced the mortality rate, but a 10-day delay did not. Propranolol (beta-adrenergic blocker), prazosin (alpha 1-adrenergic blocker), and diltiazem (another calcium-channel blocker) reduced the mortality but not significantly (P = 0.07). In biochemical studies of the beta- adrenergic signal transduction complex, we determined that verapamil and propranolol reversed the infection-associated decrease in myocardial beta- adrenergic adenylyl cyclase activity. In contrast, complementary western blot analysis revealed no significant changes in the G-proteins of the beta- adrenergic receptor complex 45 days postinfection. Therefore, these results suggest that the basis of verapamil's influence on the early critical period of infection is multifactorial and independent of a direct trypanocidal effect.

Acute Disease↗
PubMed1994

Trypanosoma cruzi: effects of infection on receptor-mediated chronotropy and Ca2+ mobilization in rat cardiac myocytes.

Acute Trypanosoma cruzi infection is commonly associated with disorders of impulse conduction and muscle contraction in heart. In order to determine the extent to which receptor function changed in response to infection, infected neonatal rat cardiac myocytes in culture were compared with matched controls with regard to chronotropic response and Ca2+ mobilization following the application of adrenergic agonists. At 7-9 days in culture (5-7 days postinfection), spontaneous beat rates of control myocytes were four times as rapid as those in infected cells. Control cells responded to 10(-5) M isoproterenol (ISO) and 10(-6) M norepinephrine (NE) with increases in beat rate of 34 and 40%, respectively. Effects of ISO on infected cells were similar, and adenylate cyclase activity was similar in control and infected cells when measured in the presence of ISO alone or in combination with Gpp(NH)p. NE produced a more marked chronotropic response in infected cultures and altered Ca2+ mobilization. NE treatment increased Ca2+ levels in control cardiac myocytes from 51.8 +/- 4.4 to 113 +/- 16 nM (in 0 Ca2+ medium) and from 85.2 +/- 6.8 to 131.3 +/- 24.5 nM (1 mM external Ca2+). In infected cardiac myocytes, NE increased Ca2+ from 116.8 +/- 17 to 164.7 +/- 9.6 nM (in 0 Ca2+ medium) and from 132.2 +/- 13.2 to 162.5 +/- 0.3 nM (1 mM Ca2+ medium). Thus, basal and alpha-adrenergic-stimulated Ca2+ levels were higher in infected than uninfected myocytes regardless of the extracellular Ca2+ levels, although the fractional increase in infected myocytes was significantly lower than that in controls (1.4- and 1.2-fold vs 2.2- and 1.5-fold). Therefore, both chronotropic and Ca(2+)-mobilization responses to the alpha-adrenergic agonist NE are altered in T. cruzi-infected cardiac myocytes; the chronotropic response of similarly infected cells to the beta-adrenergic agonist ISO was not affected. These data indicating that T. cruzi infection may be associated with a dissociation in responses to these agonists suggest a possible mechanism to explain, in part, the cardiac dysfunction characteristic of Chagas' disease.

Animals↗
PubMed1994

Conduction defects and arrhythmias in Chagas' disease: possible role of gap junctions and humoral mechanisms.

The protozoan parasite Trypanosoma cruzi causes Chagas' disease, a major cause of cardiac dysfunction in Latin Americans. Chagas' disease exhibits both acute and chronic phases, and each may be characterized by cardiac conduction disturbances. In acutely infected cultures of rodent heart cells, synchronized spontaneous beating becomes less regular, and coupling between cells is reduced. The basis of this decreased conduction is apparently in localization of the gap junction protein (Cx43) inside infected cells. Although total Cx43 is normal in infected cells, little is recognizable at appositional membranes. Electrophysiological properties are also altered by this infection. Action potentials are shortened, resting Ca2+ levels are elevated, and response to alpha-adrenergic agonists was altered, compared to controls. Humoral factors may contribute to the conduction defects in chronic Chagas' disease. Sera from chronically infected rabbits produced ECG abnormalities in Langendorff-perfused rabbit hearts. These findings indicate that chagasic infection may modify ion channel function in the heart, and we suggest that these changes may be manifested in the conduction disturbances that characterize this disease.

Action Potentials↗
PubMed1994

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)

Adenosine Diphosphate↗