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Biomedical subjects

M Wittner

Publications and source records attributed to M Wittner.

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

Expression of cardiac cytokines and inducible form of nitric oxide synthase (NOS2) in Trypanosoma cruzi-infected mice.

Expression of Cardiac Cytokines and Inducible Form of Nitric Oxide Synthase (NOS2) in Trypanosoma cruzi-infected Mice. Journal of Molecular and Cellular Cardiology (1999) 31, 75-88. Both cardiac cytokine and inducible nitric oxide synthase (NOS2) expression have been implicated in the cardiac dysfunction associated with myocarditis and cardiomyopathy. Chagas' disease, caused by Trypanosoma cruzi, is an important cause of cardiomyopathy. We examined the effect of T. cruzi (Brazil strain) infection with or without verapamil treatment on the expression of cytokines and NOS2 in the heart. Messenger RNA for NOS2, IL-1beta, and TNF-alpha was induced in the myocardium of infected mice, and Western blot analysis as well as immunohistochemistry demonstrated a significant increase in NOS2 protein. Verapamil treatment reduced the expression of cardiac NOS2 protein and the mRNAs for NOS2, TNF-alpha, and IL-1beta. Infection-associated increases in cardiac L-citrulline were also reduced by verapamil treatment. Verapamil-treated infected mice that survived for 80 days exhibited less inflammation and fibrosis compared to untreated mice. Gated MRI and echocardiography revealed an increased right ventricular inner diameter (RVID) in untreated but not in verapamil-treated infected CD1 mice. This suggests that the infection-associated expression of cytokines and NOS2 in the heart correlate with the severity of myocarditis and the effect of verapamil. The RVID was significantly increased in infected wild-type (WT) compared to infected syngeneic NOS2 knockout (NOS2-/-) mice. Fractional shortening was decreased and myocardial L-citrulline was increased in infected WT mice. These data suggest that NO generated from cardiac NOS2 may participate in the pathogenesis of murine chagasic heart disease.

Animals↗
PubMed1999

The Ca2+-sensing receptor in the rabbit cortical thick ascending limb (CTAL) is functionally not coupled to phospholipase C.

The recently cloned rabbit kidney Ca2+-sensing receptor (RabCaR) was functionally characterized in microperfused rabbit cortical thick ascending limb (CTAL) segments. Reverse transcriptase polymerase chain reaction (RT-PCR) confirmed that this nephron segment contains mRNAs coding for the RabCaR. Elevation of the extracellular Ca2+ concentration ([Ca2+]e) from 1 to 5 mmol l-1 induced an increase in the fluorescence emission ratio (R), thus reflecting an increase in intracellular Ca2+ activity ([Ca2+]i). This increase was inhibited by verapamil, nifedipine and SKF 96365, and potentiated by a previous application of Bay K 8644. Neither verapamil nor Bay K 8644 modified the resting [Ca2+]i. This suggests that the basolateral Ca2+ influx induced by a high [Ca2+]e occurs via verapamil- and dihydropyridine-sensitive Ca2+ channels, which are not open under resting conditions. In contrast to that evoked by antidiuretic hormone (ADH), the [Ca2+]i increase induced by a high [Ca2+]e did not result from an accumulation of inositol phosphates. Neomycin, Gd3+, Mg2+, commonly used agonists of the Ca2+-sensing receptor, did not increase the [Ca2+]i. In the presence of verapamil, ADH still produced a transient [Ca2+]i increase that was not observed in the presence of an increased [Ca2+]e. These results suggest that the RabCaR in rabbit CTAL cells is not functionally coupled to phospholipase C. In conclusion, the high [Ca2+]e-induced [Ca2+]i increase involves verapamil- and dihydropyridine-sensitive Ca2+ channels and is independent of phosphoinositide metabolism. Whether these channels are activated by the RabCaR remains to be elucidated.

Animals↗
PubMed1999

Disruption of the Toxoplasma gondii bradyzoite-specific gene BAG1 decreases in vivo cyst formation.

The bradyzoite stage of the Apicomplexan protozoan parasite Toxoplasma gondii plays a critical role in maintenance of latent infection. We reported previously the cloning of a bradyzoite-specific gene BAG1/hsp30 (previously referred to as BAG5) encoding a cytoplasmic antigen related to small heat shock proteins. We have now disrupted BAG1 in the T. gondii PLK strain by homologous recombination. H7, a cloned null mutant, and Y8, a control positive for both cat and BAG1, were chosen for further characterization. Immunofluorescence and Western blot analysis of bradyzoites with BAG1 antisera demonstrated expression of BAG1 in the Y8 and the PLK strain but no expression in H7. All three strains expressed a 116 kDa bradyzoite cyst wall antigen, a 29 kDa matrix antigen and the 65 kDa matrix reactive antigen MAG1. Mice inoculated with H7 parasites formed significantly fewer cysts than those inoculated with the Y8 and the PLK strains. H7 parasites were complemented with BAG1 using phleomycin selection. Cyst formation in vivo for the BAG1-complemented H7 parasites was similar to wild-type parasites. We therefore conclude that BAG1 is not essential for cyst formation, but facilitates formation of cysts in vivo.

Animals↗
PubMed1999

Infection of endothelial cells with Trypanosoma cruzi activates NF-kappaB and induces vascular adhesion molecule expression.

Transcriptional activation of vascular adhesion molecule expression, a major component of an inflammatory response, is regulated, in part, by the nuclear factor-kappaB/Rel (NF-kappaB) family of transcription factors. We therefore determined whether Trypanosoma cruzi infection of endothelial cells resulted in the activation of NF-kappaB and the induction or increased expression of adhesion molecules. Human umbilical vein endothelial cells (HUVEC) were infected with trypomastigotes of the Tulahuen strain of T. cruzi. Electrophoretic mobility shift assays with an NF-kappaB-specific oligonucleotide and nuclear extracts from T. cruzi-infected HUVEC (6 to 48 h postinfection) detected two major shifted complexes. Pretreatment with 50x cold NF-kappaB consensus sequence abolished both gel-shifted complexes while excess SP-1 consensus sequence had no effect. These data indicate that nuclear extracts from T. cruzi-infected HUVEC specifically bound to the NF-kappaB consensus DNA sequence. Supershift analysis revealed that the gel-shifted complexes were comprised of p65 (RelA) and p50 (NF-kappaB1). Northern blot analyses demonstrated both the induction of vascular cell adhesion molecule 1 and E-selectin and the upregulation of intercellular adhesion molecule 1 mRNA in HUVEC infected with T. cruzi. Immunocytochemical staining confirmed adhesion molecule expression in response to T. cruzi infection. These findings are consistent with the hypothesis that the activation of the NF-kappaB pathway in endothelial cells associated with T. cruzi infection may be an important factor in the inflammatory response and subsequent vascular injury and endothelial dysfunction that lead to chronic cardiomyopathy.

Animals↗
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↗
PubMed1998

A Ba(2+)-insensitive K+ conductance in the basolateral membrane of rabbit cortical thick ascending limb cells.

The nature of the K+ exit across the basolateral membrane of microperfused rabbit cortical thick ascending limbs (cTALs) was investigated using the transepithelial and transmembrane potential difference (PDte, PDbl) and conductance measurements. An increase in bath K+ concentration from 4 to 10, 25, 50 mmol/l depolarized the basolateral membrane in a concentration-dependent manner, accompanied by a decrease in the fractional resistance of the basolateral membrane (FRbl). The Cl- channel blocker, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid (NPPB), did not prevent these effects. The effect of Ba2+ on PDbl was bimodally distributed: paradoxically, in the tubules in which Ba2+ largely depolarized, the effects on PDbl of the bath K+ concentration increases were not inhibited by extracellular Ba2+, in tubules in which Ba2+ moderately depolarized, Ba2+ partially inhibited the K+ concentration increase-induced depolarization of the basolateral membrane. However, the parallel decrease in FRbl was Ba2+ insensitive, indicating that the K+ channel of the basolateral membrane was not modified by extracellular Ba2+. The Ba(2+)-induced depolarizations were prevented by furosemide suggesting that Ba2+ acts by inhibiting basolateral KCl extrusion. Finally, the K+ concentration increase-induced depolarizations were insensitive to tetraethylammonium, charybdotoxin, apamin and verapamil. In conclusion, the present study provides evidence that, in addition to a Ba(2+)-sensitive KCl cotransport system, the basolateral membrane of rabbit cTAL cells possesses a K+ conductance which is insensitive to extracellular Ba2+.

Animals↗
PubMed1998

Calcium-sensing receptor: regulation of electrolyte transport in the thick ascending limb of Henle's loop.

A calcium-sensing receptor (CaR) has functionally been described in the cortical thick ascending limb of Henle's loop (CTAL) of rat and mouse. This G protein-coupled receptor activates phospholipase C and increases the intracellular Ca2+ concentration. We observed that in the mouse CTAL cAMP formation, induced by 10(-8) mol/l AVP, was inhibited by more than 90% when the extracellular Ca2+ concentration ([Ca2+]e) was increased from 0.5 to 3 mmol/l. Measurements of transepithelial potential difference (PDte) in rat and mouse CTAL and medullary thick ascending limb (mTAL) segments and of transepithelial ion net fluxes in the mouse CTAL (isotonic perfusion conditions: 150 mmol/l NaCl in the lumen and bath) showed that an increase in the [Ca2+]e had no effect on basal and arginine vasopressin (AVP, 10(-10) mol/l)-stimulated transepithelial PDte, NaCl and Mg2+ transport. However, Ca2+ reabsorption was strongly inhibited by increased [Ca2+]e. Addition of AVP reversed this inhibitory effect of increased [Ca2+]e. Under hypotonic perfusion conditions (lumen 50 mmol/l NaCl; bath 150 mmol/l NaCl), a high [Ca2+]e induced a 50% decrease in Mg2+ reabsorption which was restored by AVP. Under these conditions, the effects on Ca2+ transport described above were still observed. In conclusion, activation of the CaR in the mouse TAL has no effect on basal and AVP-stimulated transepithelial NaCl reabsorption despite its large inhibitory effect on cAMP synthesis. The CaR, however, could play a role in the regulation of transepithelial Ca2+ and Mg2+ reabsorption.

Absorption↗
PubMed1997

Alpha2-adrenoreceptor agonist, dexmedetomidine, alters acute gene expression after global ischemia in gerbils.

The effect of dexmedetomidine, a selective alpha2-adrenoreceptor agonist and neuroprotective agent on the expression of immediate early genes and heat shock protein hsp70, was studied using quantitative in situ hybridization in a global ischemia model. At the dose previously shown to be neuroprotective dexmedetomidine inhibited the expression of c-fos and hsp70 mRNA, did not affect jun-B mRNA, and enhanced the induction of NGFI-A mRNA in the postischemic gerbil hippocampus. The reduced gene expression of c-fos and hsp70 was detected in the CA1 pyramidal cells which are prone to ischemic degeneration, whereas the increased gene expression of NGFI-A was measured from the CA3 and dentate gyrus, areas relatively resistant to ischemia. These alterations in early gene expression possibly reflect the mechanisms mediating the neuroprotective effects of alpha2-adrenoreceptor agonists.

Adrenergic alpha-2 Receptor Agonists↗
PubMed1997

Calcium and magnesium transport in the cortical thick ascending limb of Henle's loop: influence of age and gender.

Previous studies from our laboratory have shown that Ca2+ and Mg2+ absorption in the mouse cortical thick ascending limb of Henle's loop (cTAL) is a passive, paracellular process driven by the transepithelial voltage. The passive permeability of the epithelium is enhanced by peptide hormones. The present study investigated whether divalent cation absorption in the cTAL is influenced by cell maturation and/or gender. For this purpose, mouse cTAL segments were microdissected from kidneys of female and male animals aged 4 and 8 weeks. The microdissected tubules were perfused in vitro at a luminal flow rate of 1.5 to 2.5 nl/min. Transepithelial Na+, Cl-, Ca2+ and Mg2+ net fluxes (JX, pmol.min-1.mm-1) were measured using electron microprobe analysis, and the transepithelial potential difference (PDte) was measured continuously. No differences were found in the PDte, JNa and JCl of the various animal groups but the transepithelial Ca2+ and Mg2+ transport capacity of the cTAL was higher in adults (8 weeks) than in young animals (4 weeks). Furthermore, irrespective of age, transepithelial Ca2+ net absorption was greater in male than in female animals. In contrast, the NaCl transport was maximal at 4 weeks in both genders. We conclude therefore that transepithelial divalent cation absorption in the mouse cTAL is an inductive process influenced by cell maturation and gender. The molecular basis of these inductions remains to be elucidated.

Absorption↗
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↗