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Biomedical subjects

H Tanowitz

Publications and source records attributed to H Tanowitz.

At least 19 recordsLinked to original sources
PubMed1992

Trypanosoma cruzi: alteration of cAMP metabolism following infection of human endothelial cells.

We have previously reported that Trypanosoma cruzi infection of endothelial cells results in alterations in the metabolism of Ca2+, inositol triphosphate (IP3), and prostacycline (PGI2). In this report, we demonstrate that infection also alters the metabolism of cAMP. Infection of endothelial cells does not significantly alter beta-adrenergic receptor density or affinity, adenylate cyclase activity, and whole-cell cAMP levels. However, incubation of infected endothelial cells with the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) resulted in less than a 60% increase in cell cAMP in contrast to the greater than a 100% increase observed in uninfected endothelial cells under otherwise identical reaction conditions. Infected endothelial cells demonstrated a twofold increase in phosphodiesterase activity when measured directly. Moreover, homogenates prepared from infected endothelial cells previously incubated with isoproterenol for 20 min showed little or no change in PDE activity. In contrast, homogenates prepared from uninfected endothelial cells treated under otherwise identical reaction conditions showed a 5.7-fold increase in PDE activity. In the presence of IBMX, isoproterenol-dependent stimulation of cAMP levels in infected endothelial cells reached a maximum level at 5 min of incubation, and thereafter rapidly declined. In contrast, cAMP levels in uninfected endothelial cells reached a maximum at 2 min of incubation, and thereafter remained elevated throughout the duration of the incubation. Infection-associated changes in isoproterenol dependent stimulation of cAMP accumulation appear to relate, in part, to changes in PDE activity.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1992

Diagnosis of Encephalitozoon cuniculi infection by western blot and the use of cross-reactive antigens for the possible detection of microsporidiosis in humans.

Microsporidia are very primitive, eukaryotic, obligate, intracellular, protozoan parasites. Encephalitozoon cuniculi, a microsporidian originally described from a rabbit infection, has been described in humans as well as in many species of laboratory animals. We report the detection of E. cuniculi by Western blotting in a rabbit with torticollis that was obtained from an Encephalitozoon-free colony. Cross-reactivity of this serum was observed with antigens prepared from several genera of microsporidia. Identical Western blotting patterns were obtained with sera obtained from a rabbit immunized with E. cuniculi that was purified from tissue culture cells. In addition, we were able to demonstrate cross-reactivity between E. cuniculi rabbit antisera and Enterocytozoon bieneusi antigens by indirect immunofluorescent assay techniques in human intestinal biopsy samples. These cross-reactions between microsporidia may be useful in developing diagnostic tests for non-cultivatable microsporidia such as Enterocytozoon bieneusi.

Animals↗
PubMed1992

Detection of HIV-1 protein and nucleic acid in enterochromaffin cells of HIV-1-seropositive patients.

Diarrhea contributes significantly to the morbidity and mortality of patients with the acquired immunodeficiency syndrome (AIDS). Up to 50% of AIDS patients have diarrhea, and an etiologic agent for this cannot be identified in all of them. Recent evidence suggests that enterochromaffin cells may be infected by the human immunodeficiency virus type 1 (HIV-1) and may contribute to the unexplained diarrhea. To test this hypothesis further, endoscopic biopsies of duodena from 22 HIV-1 seropositive patients [17 with diarrhea (> 500 g/day and > 3 bowel movements/day), five without diarrhea] and from 15 normal controls (no HIV risk factors) without diarrhea were studied. Formalin-fixed and paraffin-embedded 5-microns sections were examined by immunocytochemistry, using a monoclonal antibody to the HIV-1 gp41 protein, and by in situ hybridization with a full-length biotinylated HIV-1 DNA probe. Positive staining for gp41 was detected in crypt cells, consistent with the location, size, and morphology of enterochromaffin cells, in 11 of 17 HIV-1-seropositive patients with diarrhea, and in none of five without diarrhea. Nucleic acid hybridization staining was performed in five of the 11 patients who had positive gp41 staining; all showed HIV nucleic acid sequences in similar cells. All three of the five patients with positive staining for HIV nucleic acid sequences had diarrhea for which no etiologic agent for diarrhea could be found, and one each had cryptosporidia or microsporidia. No staining was observed in any of the samples from normal control tissues. These results suggest that HIV-1 may infect enterochromaffin cells and possibly alter their function. This, in turn, may contribute to the diarrhea associated with AIDS.

Adult↗
PubMed1991

Myocardial beta-adrenergic adenylate cyclase complex in a canine model of chagasic cardiomyopathy.

Infection of beagles with an opossum-derived strain of Trypanosoma cruzi (Tc-O) results in features of early and chronic chagasic cardiomyopathy, that is, increases in PR interval, atrioventricular block, and frequent ventricular premature contractions, ventricular tachycardia, and decreased left ventricular ejection fraction. These signs are not observed in animals infected with a canine strain of T. cruzi (Tc-D). To understand the biochemical basis for these early cardiac effects, we examined the beta-adrenergic adenylate cyclase complex in myocardial membranes prepared from animals infected with either of the two strains. In animals infected with Tc-O (symptomatic), the maximum velocity (Vmax) decreased and concentration of agonist resulting in 50% of Vmax (Kact) increased for isoproterenol-dependent adenylate cyclase activity; in animals infected with Tc-D (asymptomatic), Vmax and Kact for isoproterenol were unchanged from control, uninfected animals. beta-Receptor density decreased by 20% in symptomatic animals with no change in affinity, whereas no differences were observed between uninfected and infected asymptomatic animals. A complex pattern of changes was apparent in the guanine nucleotide binding protein, Gs, in the setting of infection. Alterations in cholera toxin-dependent ADP-ribosylation patterns as well as immunochemical detection with anti-G alpha s antisera suggested a change in the biochemical nature of the Gs species and not necessarily a physical loss of this protein. Reconstitution of adenylate cyclase activity in cyc- membranes demonstrated a decrease in hormone-sensitive Gs activity in membranes prepared from symptomatic animals without a change in activity demonstrable in the presence of Gpp(NH)p. Collectively, the results suggest that the depression in beta-adrenergic adenylate cyclase activity associated with symptomatic infection of beagles with T. cruzi occurs primarily as a result of changes in the Gs protein complex, most likely resulting in an uncoupling of the beta-adrenergic receptor from the Gs protein.

Adenosine Diphosphate Ribose↗
PubMed1989

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.

Adenylyl Cyclases↗
PubMed1988

Isospora belli infection: treatment with pyrimethamine.

Isosporiasis is an uncommon but important diarrheal disease of humans that, like cryptosporidiosis, is life-threatening in patients with the acquired immunodeficiency syndrome (AIDS). Isospora belli infection responds rapidly to therapy with trimethoprim-sulfamethoxazole, but patients with AIDS have a high rate of adverse reactions to this therapy. The cases of two patients with AIDS, sulfonamide allergy, and I. belli infection are reported. They were treated successfully with pyrimethamine alone, 75 mg/d, and recurrence was prevented with daily pyrimethamine therapy, 25 mg/d. In patients with AIDS with sulfonamide allergy or intolerance, pyrimethamine alone seems to be a reasonable alternative therapy for I. belli infection.

Adult↗
PubMed1988

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.

Angiotensin II↗
PubMed1988

Western blot analysis of the antibody response of patients with AIDS and toxoplasma encephalitis: antigenic diversity among Toxoplasma strains.

We used immunoblotting to ascertain if toxoplasma encephalitis in disease caused by the human immunodeficiency virus (HIV) could be diagnosed by the appearance of characteristic antibodies recognizing specific Toxoplasma antigens. The profile of antibodies to Toxoplasma was examined in human serum and cerebrospinal fluid from patients with chronic and acute toxoplasmosis with or without HIV infection. Many Toxoplasma antigens were recognized by all sera; the majority were presumably surface proteins, as determined by 125I labeling. All sera recognized antigens at 38, 35, 28, and 26 kilodaltons. No specific antibody or pattern of antibodies distinguished between groups of patients. A 120-kilodalton antigen recognized by sera from Atlanta was not, however, seen in most sera from New York. Study of the recognition of the antigens of different strains of Toxoplasma gondii (RH, C56, T100) by the same human sera demonstrated strain-specific antigenic differences. These strain variations may account for the antibody diversity among the patients studied.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Myocardial adenylate cyclase activity in acute murine Chagas' disease.

We have studied the influence of myocardial infection with Trypanosoma cruzi on the beta-adrenergic adenylate cyclase complex in mouse myocardial membranes. The maximal rate of cAMP generation (Vmax) and the concentration of agonist associated with 50% of the maximal activity (apparent Kact) were determined for a series of agents. Six days after infection, the Vmax for isoproterenol significantly declines without a change in the apparent Kact. After 21 days of infection, both the Vmax and apparent Kact for isoproterenol are reduced. At 6 and 21 days of infection, the affinity of the beta-receptor for [125I]iodocyanopindolol declines from 0.84 to 3.6 and 3 nM, respectively, while the receptor density increases with the duration of infection from 33 to 57 and 82 fmol/mg protein, respectively. The Vmax (but not the apparent Kact) for forskolin and Mg2+- and Mn2+-associated activities declines also after 21 days. Another adenylate cyclase activity, which was stimulated by the nonhydrolyzable guanine nucleotide Gpp(NH)p, declines in relation to the duration of infection. Inhibitors of adenylate cyclase activity were also studied. Inhibition of adenylate cyclase activity by adenosine and by Gpp(NH)p (in the presence of forskolin) declines after 21 days of infection. The results suggested that the coupling proteins Ns and Ni, which mediate stimulatory or inhibitory control of receptors to adenylate cyclase activity, might be altered by infection. As monitored by cholera toxin- and pertussis toxin-dependent ADP ribosylation of their respective substrates, which include Ns and Ni proteins, respectively, there are declines in the availability of both substrates as a result of T. cruzi infection. For infected membranes, the addition of NADP enhances the magnitude of cholera toxin-dependent ADP ribosylation and renders the magnitude of pertussis toxin-dependent ADP ribosylation equal to that observed in uninfected membranes. The results support the hypothesis that infection with T. cruzi results in profound generalized alterations of the adenylate cyclase complex at several different sites.

Acute Disease↗
PubMed1987

Infection of L6E9 myoblasts with Trypanosoma cruzi alters adenylate cyclase activity and guanine nucleotide binding proteins.

We studied the consequences of infection of L6E9 myoblasts with T. cruzi on the adenylate cyclase complex to test the hypothesis that infection alters the functional properties of the guanine nucleotide regulatory proteins, Ns and Ni. Stimulating activities of adenylate cyclase due to isoproterenol, isoproterenol plus Gpp(NH)p, or forskolin (activities mediated by Ns) are not altered by infection. However, inhibitory activities mediated by Ni [Gpp(NH)p, acetylcholine, and adenosine inhibition of forskolin-dependent adenylate cyclase activity] are compromised by infection. The reduction in adenosine's inhibition of forskolin-dependent adenylate cyclase activity is seen throughout the effective concentration range of adenosine. Pertussis toxin does not change basal or stimulated adenylate cyclase activity in infected cells compared with normal uninfected cells, nor does it alter the inhibiting action of adenosine. To evaluate the coupling proteins (Ns and Ni) involved in the stimulation and inhibition of adenylate cyclase more directly, cholera- and pertussis-toxin-dependent ADP ribosylation studies were performed. The incorporation of [32P]ADP ribose in the presence (specific) or absence (nonspecific) of the toxins was markedly decreased in membranes prepared from infected cells. However, in membranes prepared from infected or uninfected cells previously treated with pertussis toxin, there was a significant reduction in specific pertussis-toxin dependent ADP ribosylation. The infection-associated diminution in toxin-dependent ADP ribosylation complements the impaired inhibition of adenylate cyclase data. Collectively, the data further substantiate an infection-associated alteration in the adenylate cyclase complex, probably at the level of the guanine nucleotide binding proteins.

Acetylcholine↗
PubMed1985

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.

Aneurysm↗
PubMed1983

Growth of Trypanosoma cruzi in a cloned macrophage cell line and in a variant defective in oxygen metabolism.

A continuous cloned murine macrophage-like cell line, clone 16 derived from J774, has been found upon appropriate stimulation to be capable of oxidizing glucose by the hexose monophosphate shunt and producing O2- and H2O2. A variant in oxidative metabolism, clone C3C, was selected from this cell line which under similar conditions is unable to produce significant amounts of O2- and H2O2. When cells of the parental clone 16 were infected with epimastigotes of Trypanosoma cruzi, there was significant killing or growth inhibition of the parasites at 3 to 4 days after infection. In contrast, the parasites grew in the oxidative variant, clone C3C. Trypomastigote forms of T. cruzi were found to be only partially killed in the parental clone 16 but grew abundantly in the oxidative variant. Infection of the parental clone, but not the variant, was sufficient to stimulate oxygen metabolism as demonstrated by the increased reduction of nitro blue tetrazolium. Studies on the killing of T. cruzi epimastigotes in cell-free suspension by xanthine-xanthine oxidase indicated that 90% of the killing was catalase sensitive and due to H2O2, with at most 7 to 8% killing which could be inhibited by scavengers of . OH and singlet oxygen (1O2). In the in vitro experiment with H2O2 produced by glucose and glucose oxidase, the 50% lethal doses of epimastigotes and trypomastigotes were 6.0 and 8.7 nmol of H2O2 per min per ml, respectively, indicating that trypomastigotes were more resistant to killing by H2O2 than epimastigotes were. A reconstitution experiment of trypanocidal activity in clone C3C by ingestion of zymosan particles coupled with glucose oxidase showed that H2O2 was essential for this cytocidal process in the macrophage cell line. These results provide clear evidence for killing of an intracellular parasite by a continuous macrophage-like cell line and suggest the importance of the oxidative cytocidal mechanism in this process.

Animals↗
PubMed1982

Reconstitution of a variant macrophage cell line defective in oxygen metabolism with a H2O2-generating system.

A variant clone, C3C, derived from the cloned macrophage cell line J774.16 lacks the capacity to produce O2- or H2O2 after appropriate stimulation. When the parental and variant cell lines were infected with epimastigotes of Trypanosoma cruzi, the parasites were killed or their growth was inhibited by the parental line, but they grew readily in the variant clone C3C. It was possible to reconstitute the variant cell line with an enzyme system targeted to the lysosomal compartment capable of generating a single oxygen metabolite, H2O2. This was accomplished by allowing the cells to phagocytize zymosan particles covalently coupled with glucose oxidase (GO-Zy particles). Approximately one-third of the H2O2 theoretically expected to be produced by the ingested GO-Zy particles could be detected outside the cells by the cytochrome c peroxidase assay; this fraction may represent the efficiency of extracellular assays for H2O2 production. When T. cruzi-infected clone C3C cells were reconstituted with GO-Zy particles, upon addition of glucose, intracellular killing of the parasites occurred. It was possible to estimate the level of H2O2 production required to kill a single parasite (8.7 x 10(-7) nmol/min) by GO-Zy particles in suspension and to formulate a first approximation of the killing potency of the reconstituted cells--i.e., number of parasites expected to be killed--that correlated well with the observed growth of the parasites intracellularly.

Animals↗
PubMed1981

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.

Erythrocytes↗