Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.     

Experimental Trypanosoma cruzi infection in platelet-activating factor receptor-deficient mice

The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR(-/-) mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR(-/-) mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-gamma and TNF-alpha in heart tissue of infected animals. Moreover, serum concentrations of TNF-alpha, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR(-/-) animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B(4) or MCP-1 and produced less nitric oxide when infected and activated with IFN-gamma. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.     

Activation of potassium and chloride channels by tumor necrosis factor alpha. Role in liver cell death

Despite abundant evidence for changes in mitochondrial membrane permeability in tumor necrosis factor (TNF)-mediated cell death, the role of plasma membrane ion channels in this process remains unclear. These studies examine the influence of TNF on ion channel opening and death in a model rat liver cell line (HTC). TNF (25 ng/ml) elicited a 2- and 5-fold increase in K(+) and Cl(-) currents, respectively, in HTC cells. These increases occurred within 5-10 min after TNF exposure and were inhibited either by K(+) or Cl(-) substitution or by K(+) channel blockers (Ba(2+), quinine, 0.1 mm each) or Cl(-) channel blockers (10 microm 5-nitro-2-(3-phenylpropylamino)benzoic acid and 0.1 mm N-phenylanthranilic acid), respectively. TNF-mediated increases in K(+) and Cl(-) currents were each inhibited by intracellular Ca(2+) chelation (5 mm EGTA), ATP depletion (4 units/ml apyrase), and the protein kinase C (PKC) inhibitors chelerythrine (10 micrometer) or PKC 19-36 peptide (1 micrometer). In contrast, currents were not attenuated by the calmodulin kinase II 281-309 peptide (10 micrometer), an inhibitor of calmodulin kinase II. In the presence of actinomycin D (1 micrometer), each of the above ion channel blockers significantly delayed the progression to TNF-mediated cell death. Collectively, these data suggest that activation of K(+) and Cl(-) channels is an early response to TNF signaling and that channel opening is Ca(2+)- and PKC-dependent. Our findings further suggest that K(+) and Cl(-) channels participate in pathways leading to TNF-mediated cell death and thus represent potential therapeutic targets to attenuate liver injury from TNF.     

Different types of tea products attenuate inflammation induced in Trypanosoma brucei infected mice

An in vivo study was carried out to determine the effect of different types of Kenyan tea extracts on male Swiss albino mice infected with Trypanosoma brucei brucei isolate KETRI 2710. The isolate produced a similar clinical picture after a pre-patent period of 5 days post-infection (DPI). Parasitemia levels in the untreated mice and those given different teas developed exponentially at similar rates reaching similar densities at the peak of parasitemia 8 DPI. Between 9 and 13 DPI parasitemia decreased more rapidly in tea treated compared to the untreated mice which indicated that tea lowered parasitemia level. Anaemia indicated by a fall in erythrocyte packed cell volume (PCV) occurred within 4 DPI and remained below the normal levels until the terminal stages of the disease. A significant difference (P<0.05) was observed 11 DPI between the tea treated and the untreated mice indicating that tea enhanced resistance to erythrocyte destruction. Mice treated with tea exhibited significantly (P<0.01) reduced parasite-induced hypoalbuminemia as compared to the untreated. Since albumin is a negative acute phase protein, it shows a decrease during inflammatory conditions and therefore its elevation in the mice given tea in this study clearly demonstrated that tea ameliorated inflammation induced by T. b. brucei. Although green and white teas were superior in most of these characteristics, black tea, which is the principle tea product from Kenya, displayed remarkable properties some even comparable to those of green tea. Interestingly, tea was more efficacious than dexamethasone an established anti-inflammatory drug, demonstrating its therapeutic potential.     

Extracellular ATP induces cell death in CD4+/CD8+ double-positive thymocytes in mice infected with Trypanosoma cruzi

In the acute phase of Trypanosoma cruzi infection, there is dramatic atrophy of the thymus. However, the pathways involved in this change have not yet been identified. This event is mainly characterized by a massive loss of cortical CD4+/CD8+ double-positive cells, but also by other structural and functional alterations in the organ. A number of molecules, including extracellular ATP, have been suggested to play a role in the selective processes that take place in the thymus. ATP and analogues trigger many different cellular responses in thymocytes and other cell types, such as the opening of plasma membrane cation channels and a pore that may induce cell death. Herein, we investigated the possible involvement of extracellular ATP in thymus atrophy induced by infection with T. cruzi. We observed that ATP induces an increase in plasma membrane permeabilization and cellular death in CD4+/CD8+ double-positive thymocytes collected from infected mice during the atrophy phase. No differences were observed prior to the atrophy phase or during the chronic phase. Our results indicate that P2Z/P2X7 receptors may play a central role in thymus atrophy during T. cruzi infection.     

Lymphoid organ alterations enhanced by sub-lethal doses of coronaviruses in experimentally induced Trypanosoma cruzi infection in mice

The effect of sub-lethal doses of coronaviruses on the course of disease in CBA mice experimentally infected with a mildly pathogenic strain of Trypanosoma cruzi was investigated. Mice were inoculated with either T. cruzi, 0.1 median lethal dose (LD50) of coronavirus (mouse hepatitis virus [MHV-3] or virus X), or both pathogens. Levels of parasitemia, mortality, and the extent of pathologic alterations in lymphoid organs were determined. Mice inoculated with T. cruzi had mild alterations in their lymphoid organs and survived infection. In contrast, mice inoculated with both pathogens died, and had significantly higher levels of parasitemia and profound alterations in lymphoid organs. These results indicate that the pathologic profile of T. cruzi infection can be profoundly altered by subclinical infection with coronaviruses.     

Tumor necrosis factor alpha and inflammation disrupt the polarity complex in intestinal epithelial cells by a posttranslational mechanism

Inflammatory processes disrupt the barrier function in epithelia. Increased permeability often leads to chronic of inflammation. Important among other cytokines, tumor necrosis factor alpha (TNF-α) initiates an NF-κB-mediated response that leads to upregulation of myosin light chain kinase (MLCK), a hallmark of the pathogenesis of inflammatory bowel disease. Here, we found that two components of the evolutionarily conserved organizer of tight junctions and polarity, the polarity complex (atypical protein kinase C [aPKC]-PAR6-PAR3) were downregulated by TNF-α signaling in intestinal epithelial cells and also in vivo during intestinal inflammation. Decreases in aPKC levels were due to decreased chaperoning activity of Hsp70 proteins, with failure of the aPKC rescue machinery, and these effects were rescued by NF-κB inhibition. Comparable downregulation of aPKC shRNA phenocopied effects of TNF-α signaling, including apical nonmuscle myosin II accumulation and myosin light chain phosphorylation. These effects, including ZO-1 downregulation, were rescued by overexpression of constitutively active aPKC. We conclude that this novel mechanism is a complementary effector pathway for TNF-α signaling.     

Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis

Microbial lung infections are the major cause of morbidity and mortality in the hereditary metabolic disorder cystic fibrosis, yet the molecular mechanisms leading from the mutation of cystic fibrosis transmembrane conductance regulator (CFTR) to lung infection are still unclear. Here, we show that ceramide age-dependently accumulates in the respiratory tract of uninfected Cftr-deficient mice owing to an alkalinization of intracellular vesicles in Cftr-deficient cells. This change in pH results in an imbalance between acid sphingomyelinase (Asm) cleavage of sphingomyelin to ceramide and acid ceramidase consumption of ceramide, resulting in the higher levels of ceramide. The accumulation of ceramide causes Cftr-deficient mice to suffer from constitutive age-dependent pulmonary inflammation, death of respiratory epithelial cells, deposits of DNA in bronchi and high susceptibility to severe Pseudomonas aeruginosa infections. Partial genetic deficiency of Asm in Cftr(-/-)Smpd1(+/-) mice or pharmacological treatment of Cftr-deficient mice with the Asm blocker amitriptyline normalizes pulmonary ceramide and prevents all pathological findings, including susceptibility to infection. These data suggest inhibition of Asm as a new treatment strategy for cystic fibrosis.     

Exacerbation of experimental Trypanosoma cruzi infection in mice by concomitant malaria.

The effect of malaria on the chronic phase of Chagas' disease was investigated in mice. The animals were given Plasmodium bergheri-infected red blood cells 2 to 12 months after their initial inoculation with trypomastigotes of 3 different strains of Trypanosoma cruzi (Y. CL and Gilmar). in all the experiments carried out with one of the strains (CL), a somewhat variable but always considerable percentage of mice (average 39%) relapsed in to the acute phase of Chagas' disease. This relapse was characterized by a significant increase in the number of circulating trypomastigotes. Recrudescence was observed also with a 2nd strain of T. cruzi (Gilmar), which is similar in many aspects to the CL strain, e.g. the morphology of blood stages, curved of parasitemia and susceptibility to antibodies in vitro. In mice whose chronic phase was induced by trypomastigotes of the Y strain, malaria infections did not induce a typical acute phas with high parasitemia by T. cruzi. Bloodstream forms of Y parasites differ from those of CL and Gilmar strains morphologically as well as immunologically, i.e. only the Y strain is easily agglutinated and partly inactivated by specific immune serum. In light of this and other known characteristics of the strains used in the present work, the author speculates on mechanisms which allow malaria infections selectively to suppress acquired host resistance to certain strains of T. cruzi.     

Tumor necrosis factor alpha enhances the cytotoxicity induced by nitric oxide in cultured cerebral endothelial cells

It has been shown that independent sources of nitric oxide (NO) and the inflammatory cytokine tumor necrosis factor alpha (TNFalpha) contribute to the breakdown of the blood-brain barrier (BBB) in the pathogenesis of a number of brain disorders. However, the interaction of NO and TNFalpha has not been elucidated. The present study was designed to determine whether the toxicity induced by NO is altered by TNFalpha in brain capillary endothelial cells (BCECs), and if so, whether it is related to the generation of superoxide. TNFalpha (50-400 U/ml) did not produce toxicity until at a concentration of 800 U/ml. This toxic effect was completely blocked by copper-zinc superoxide dismutase (SOD)/catalase or N(omega)-nitro-L-arginine methyl ester (L-NAME) or oxyhemoglobin (HbO2). Sodium nitroprusside (SNP) reduced with 0.4 mM ascorbate (SNP/Vc) significantly increased Lactate dehydrogenase (LDH) efflux in a concentration-dependent manner. This cytotoxicity of SNP/Vc was also completely inhibited by SOD/catalase or HbO2. When SNP/Vc used in combination with 400 U/ml TNFalpha, a more remarkable LDH efflux was induced than SNP/Vc alone, even as little as 0.01 mM SNP/Vc was toxic, although a dose of 400 U/ml TNFalpha alone had no effect on LDH efflux. In addition, either 0.4 mM SNP/Vc and 800 U/ml TNFalpha alone or 0.4 mM SNP/Vc and 400 U/ml TNFalpha in combination significantly increased malondialdehyde (MDA) content, but nitric oxide synthase (NOS) activity was inhibited only by SNP/Vc and TNF in combination. These results suggest that TNFalpha enhances the toxicity of NO in BCECs and that at least part of this enhancement involves the generation of superoxide.     

Trypanosoma cruzi: alpha-2-macroglobulin regulates host cell apoptosis induced by the parasite infection in vitro

A2M is a broad spectrum proteinase inhibitor and cytokine carrier, besides presenting anti-apoptotic activity through the binding to its receptor, LRP. During Trypanosoma cruzi infection, apoptosis of host cells and intracellular parasites is commonly observed both in vivo and in vitro. Since plasma as well as tissue A2M levels are increased in both murine and human acute T. cruzi infection, we evaluated the possible role of A2M (its methylamine transformed Fast form-A2M-F) in regulating apoptotic events in peritoneal macrophages and cardiomyocytes during in vitro interaction with the parasite. Our data showed that DNA fragmentation (a hallmark of apoptosis) of both host cells and parasites was inhibited by A2M-F. Impaired apoptosis was also noted when A2M-F was added to the cultures maintained under serum deprivation. In addition, macrophages from C57/BL6 mice, known to display higher LRP levels as compared to those of C3H lineage, displayed higher reduction in the apoptotic levels during the A2M-F treatment.     

Trypanosoma cruzi: quantification in tissues of experimentally infected mice by limiting dilution analysis

A limiting dilution assay (LDA) was developed for the quantification of Trypanosoma cruzi in the heart and blood of infected mice. Three groups of swiss mice were injected ip with "CL", "Colombiana," and "Y" strains. At 1-day intervals after infection, blood and the heart were removed. Serial blood dilutions in LIT medium were performed and distributed in four groups of 24 microplate wells. The growth of parasite was visually checked in an inverted microscope. It was found that curves of parasitemia obtained by parasite counting in a hemocytometer or estimated by LDA were similar. A similar method was used to quantify parasites in the heart of mice. The heart was cut, washed, dried, and its weight was determined. The heart pieces were disrupted by passage through a mesh stainless-steel screen into LIT. Serial dilutions of the heart homogenate were made in LIT and added to at least 24 replicate microplate wells. Parasites were detectable earlier in the heart of mouse infected with Y strain when compared to CL and Colombiana strains. Parasites were detected in the heart of mice of all strains by 6 days after infection. This LDA for quantification of T. cruzi permits a more precise evaluation of the number of living parasites in infected tissues.