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.     

Trypanosoma cruzi: pattern of RNA synthesis following infection of vertebrate cells

The pattern of RNA synthesis of intracellular Trypanosoma cruzi amastigotes, immediately following infection of Lesch-Nyhan human fibroblasts, was studied by autoradiography. Amastigote RNA synthesis, determined by [3H]guanine incorporation, was not detected until 2 h after infection. At 8 h postinfection more than 90% of intracellular amastigotes were labeled. It was verified that extracellular trypomastigotes also synthesized RNA. Therefore it was concluded that, if RNA is required for trypomastigote-to-amastigote transformation, this nucleic acid is already present in the trypomastigotes before infection of the vertebrate cell. It is probable that the RNA synthesized by amastigotes during the prereplicative lag period (the period between initial infection and the onset of DNA synthesis) is required for intracellular growth and reproduction.     

Histamine stimulates inositol phosphate accumulation via the H1-receptor in cultured human endothelial cells

The effects of histamine on [3H]inositol phosphate ([3H]IP) accumulation was examined in the presence of lithium in [3H]inositol-prelabelled human umbilical vein endothelial cells. Histamine stimulated total [3H]IP formation in a dose-dependent manner with a half-maximal value (EC50) of around 1-2 X 10(-6) M. Mepyramine, but not cimetidine, completely abolished the histamine response indicating that activation of phosphoinositide hydrolysis is mediated via H1-receptors. These data are the first to suggest that activation of inositol lipid hydrolysis is the underlying transmembrane signalling mechanism histamine H1-receptors employ in mediating various endothelial cell functions.     

Trypanosoma cruzi: in vitro interactions between cultured amastigotes and human skin-muscle cells.

Established cultures of human skin-muscle cells were used for determining the parasite—host cell relationship of Trypanosoma cruzi amastigotes (12–16 passages) cultured in a cell-free medium (F-69) at 37 C. The medium used for this experiment was tissue culture fluid M-199 enriched with 10% fetal bovine serum and relatively high concentrations of ATP, ADP and AMP. Amastigotes entered skin-muscle cells incubated at 32 or 35 C, multiplied and completed their intracellular life cycle in about 7 days. At 35 C, 23.6% of cells became infected in 7 days and at 32 C, 43.6% were infected in 5 days. The higher infection rate of cultured cells at 32 C was probably due to more frequent and prolonged cell-parasite contact, as amastigotes multiplied in the tissue culture medium and remained viable for a longer period at the lower temperature. As a control, epimastigotes were used to infect skinmuscle cells. Epimastigotes transformed into metacyclic trypomastigotes before entering host cells, multiplied, and completed the intracellular life cycle. We conclude that the amastigotes cultured in F-69 at 37 C are biologically similar to intracellular amastigotes from the vertebrate host, in that both can multiply and complete the life cycle intracellulary.     

Trypanosoma cruzi: selective isolation of pure trypomastigotes from cultured muscle cells

Trypomastigote or trypomastigote-amastigote populations of Trypanosoma cruzi (Y strain, MERC 2C) entirely free of epimastigotes were obtained from infected muscle cell cultures, and separated from host-cell debris by passage through a DEAE-cellulose column. Approximately 75% of the parasites were recovered and mouse infectivity titrations with postcolumn trypomastigotes showed only a slight reduction in infectivity compared to starting material. Light and electron microscopic examination of material showed a high degree of purity had been achieved by the column procedure. No host-cell debris could be identified in the eluate and parasites were morphologically intact. Enumeration of trypomastigotes and amastigotes in mixed populations before and after column purification showed that there was no preferential loss of either stage and both had the same residence time. This procedure may be used to obtain clean, minimally altered, parasite material free of the invertebrate epimastigote stage and host-cell debris for studies which are sensitive to these contaminants.     

Trypanosoma cruzi alters adherens junctions in cardiomyocytes

We analyzed the distribution and expression of cadherin and beta-catenin during Trypanosoma cruzi-cardiomyocyte interaction. Confocal microscopy revealed cadherin associated with beta-catenin at the cell-cell contacts. After 24h of infection, the spatial distribution and expression of both adherens junction (AJ) proteins remained unaltered. In contrast, loss of N-cadherin-catenin complex was visualized in highly infected cardiomyocytes. Immunoblotting assays corroborated the spatial disorder, showing a 46% reduction in both N-cadherin and beta-catenin expression at later infection (72h of infection). Our data demonstrate that T. cruzi infection disturbs AJs, which can result in loss of cardiac tension and may contribute to the cardiac dysfunctions present in T. cruzi infection.     

Separation of amastigotes and trypomastigotes of Trypanosoma cruzi from cultured cells

A method is described for the isolation and purification of trypomastigotes and amastigotes of Trypanosoma cruzi from cell cultures. L-A9, a transformed fibroblast cell line, and J774G8, a macrophage-like cell line of tumor origin, were used. Both cell lines were infected with bloodstream trypomastigotes of T. cruzi, which once within host cells transform into dividing amastigotes. After 6--8 days infection the host cells ruptured, spontaneously liberating parasites into the culture medium. L-A9 cells liberated mainly trypomastigotes while J774G8 cells liberated amastigotes. The parasites were collected and purified by centrifugation in a gradient of metrizamide. The purity of the preparation as well as the morphology of the parasites and the host cells were analysed by electron microscopy.     

The pentose phosphate pathway in Trypanosoma cruzi

The pentose phosphate pathway has been studied in Trypanosoma cruzi, Clone CL Brener. Functioning of the pathway was demonstrated in epimastigotes by measuring the evolution of (14)CO(2) from [1-(14)C] or [6-(14)C]D-glucose. Glucose consumption through the PPP increased from 9.9% to 20.4% in the presence of methylene blue, which mimics oxidative stress. All the enzymes of the PPP are present in the four major developmental stages of the parasite. Subcellular localisation experiments suggested that the PPP enzymes have a cytosolic component, predominant in most cases, although all of them also seem to have organellar localisation(s).     

Experimental Trypanosoma cruzi infection alters the shaping of the central and peripheral T-cell repertoire

We investigated the thymic and peripheral T-lymphocyte subsets in BALB/c mice undergoing acute or chronic Trypanosoma cruzi infection, in terms of expression of particular Vbeta rearrangements of the T-cell receptor. We first confirmed the severe depletion of CD4(+)CD8(+) thymocytes following acute T. cruzi infection. By contrast, the numbers of CD4(+)CD8(+) cells in subcutaneous lymph nodes increased up to 16 times. In subcutaneous lymph nodes, we found CD4(+)CD8(+) cells that expressed prohibited segments TCRVbeta5 and TCRVbeta12 (which are physiologically deleted in the thymus of BALB/c mice), as did some mature single-positive cells (CD4(+)CD8(-) and CD4(-)CD8(+)). In the thymus of infected animals, although higher numbers of immature cells bearing such Vbeta segments were seen, they were no longer detected in the mature single-positive stage, suggesting that negative selection occurs normally. We also found increased numbers of cells bearing the potentially autoreactive phenotype TCRVbeta5(+) and TCRVbeta12(+) in T-lymphocyte subsets from subcutaneous lymph nodes of T. cruzi chronically infected mice. In conclusion, our data indicate that immature T lymphocytes bearing prohibited TCRVbeta segments leave the thymus and gain the lymph nodes, where they further differentiate into mature CD4(+) or CD8(+) cells. Conjointly, these findings show changes in the shaping of the central and peripheral T-cell repertoire in both acute and chronic phases of murine T. cruzi infection. The release of potentially autoreactive T cells in the periphery of the immune system may contribute to the autoimmune process found in both murine and human Chagas' disease.     

Prevalence of anti-R-13 antibodies in human Trypanosoma cruzi infection

Abstract Infection with Trypanosoma cruzi develops in three phases: acute, indeterminate or asymptomatic, and chronic phase (with cardiac or digestive manifestations). Moreover, transmission may occur from infected mothers to newborn, the so-called congenital form. In the present study, humoral responses against T. cruzi total extract and against the 13 amino acid peptide named R-13 derived from the parasite ribosomal P protein, previously described as a possible marker of chronic Chagas heart disease, were determined pateints and in blood bank donors from endemic areas. While in sera from acute phase, only IgM anti- T.cruzi response was observed, both IgM and IgG anti- T. cruzi antibodies were detected in sera from congenitally infected newborns. The percentage of positive response in sera from blood bank donors was relatively high in endemic regions. Antibodies against the R-13 peptide were present in a large proportion of cardiac chagasic patients but were totally lacking in patients with digestive form of Chagas disease. Furthermore, anti-R-13 positive responses were detected in congenitally infected newborns.     

Trypanosoma cruzi: The immunological consequences of infection

Trypanosoma cruzi, the causative agent of Chagas' disease, infects an estimated 12 million people in Latin America and may induce cardiopathy and megaformation of the oesophagus and colon. During the early, acute stage of the infection, parasite-induced inflammatory infiltrates may cause transitory disease which terminates with the emergence of an immune response sufficient to reduce the parasite to insignificant levels. Even so, severe disease may develop many years after the original infection. It has been suggested that this might result from an autoimmune process triggered by the parasite and mediated either (1) by the adsorption of parasite antigens to host cells, thus rendering these cells susceptible to the host's own antiparasite immune response, or (2) via cross-reactive antigens shared by the host and parasite. In common with many parasitic diseases, there is an urgent need for studies on the T-cell response to T cruzi infection, as this might not only hold the key to the immunopathology but also serve as a means of clearing this lifelong infection which survives by sequestering into an intracellular site.     

Isolation of cardiac mast cells in experimental Trypanosoma cruzi infection

Mast cells (MC) secrete diverse pre-stored chemical mediators that are pivotal in inflammatory and fibrotic etiologies, such as Trypanosoma cruzi-induced myocardiopathy. However, due to reduced number of cardiac MC, in situ and in vitro identification, and difficult tissue isolation, these cells are rarely addressed. In this work we optimized the identification of cardiac and peritoneal MC and developed an enzymatic method for MC isolation using control and T. cruzi-infected mice. MC were identified by: toluidine blue (TB); alcian blue (AB)/safranin (S); AB or a mixed solution composed by AB/S/TB. Previous evaluations of cardiac MC in T. cruzi infection were based on TB staining and our results using AB/S/TB solution showed an increase in, at least, five times the detection of MC. This mixed solution may improve the identification of MC populations also from skin, mucosa and tissues that are infected by other pathogens or under the influence of chronic inflammation, leading to more precise results. Furthermore, the appropriate combination of samples (frozen/unfixed/thick slices) and staining protocols can assure the best evaluation of MC. We have also isolated cardiac MC using collagenase and developed a highly efficient 60%/70% Percoll-graded protocol that enriched in, at least, 95% the population of cardiac MC.     

Isolation and purification of amastigotes of Trypanosoma cruzi from cultured vero cells

A method is described for the isolation and purification of the intracellular amastigotes of Trypanosoma cruzi from cultured Vero cells. Host cells were infected with metacyclic forms obtained in Grace's medium. Six days after infection, the cells wer subjected to treatment with trypsin to obtain the intracellular forms. The parasites were collected and purified by Percoll discontinuous gradient centrifugation.     

Bioluminescent imaging of Trypanosoma cruzi infection

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi, is a major public health problem in Central and South America. The pathogenesis of Chagas disease is complex and the natural course of infection is not completely understood. The recent development of bioluminescence imaging technology has facilitated studies of a number of infectious and non-infectious diseases. We developed luminescent T. cruzi to facilitate similar studies of Chagas disease pathogenesis. Luminescent T. cruzi trypomastigotes and amastigotes were imaged in infections of rat myoblast cultures, which demonstrated a clear correlation of photon emission signal strength to the number of parasites used. This was also observed in mice infected with different numbers of luminescent parasites, where a stringent correlation of photon emission to parasite number was observed early at the site of inoculation, followed by dissemination of parasites to different sites over the course of a 25-day infection. Whole animal imaging from ventral, dorsal and lateral perspectives provided clear evidence of parasite dissemination. The tissue distribution of T. cruzi was further determined by imaging heart, spleen, skeletal muscle, lungs, kidneys, liver and intestines ex vivo. These results illustrate the natural dissemination of T. cruzi during infection and unveil a new tool for studying a number of aspects of Chagas disease, including rapid in vitro screening of potential therapeutical agents, roles of parasite and host factors in the outcome of infection, and analysis of differential tissue tropism in various parasite-host strain combinations.     

Regulation of inositol phosphate levels by prostaglandins in cultured endometrial cells

Stimulation of cultured rabbit endometrial cells by one of the rabbit endometrial cell culture proliferation factors, prostaglandin F_2α (PGF_2α), resulted in a very rapid increase in the intracellular levels of [³H]-inositol triphosphate (IP₃), [³H]-inositol biphosphate (IP₂), and [³H]-inositol monophosphate (IP₁) in cells prelabeled with [³H]-inositol. These increases in inositol phosphate levels were detected in periods of stimulation as short as 30 seconds, reached a maximum by 1 1/2?2 min and declined to control levels by 6–10 min. The stimulation was dose-dependent with maximal increases observed near 10^?6 M PGF_2α. The cholinergic agent, carbachol, also led to time and dose-independent increases in IP₃. Lithium, cadmium, silver, copper, and zinc ions had no effect either on the breakdown of IP₃ or on the accumulation of IP₁. In contrast, vanadate at 10^?6 or 10^?5 M did lead to a decrease in the breakdown of IP₁ and a concomitant increase in IP₁, IP₂, and IP₃. PGF_2α was found previously to induce an increase in rabbit endometrial cell DNA synthesis which was inhibited by concomitant or prior addition of prostaglandin E₁ (PGE₁). PGE₁, in a dose-dependent manner, was found to inhibit the observed IP₃ increase by PGF_2α at 1 1/2 min of stimulation. PGF_2α treated and control cultures did not differ in cAMP or cGMP levels, cellular ⁴⁵Ca uptake, nor cellular ²²Na uptake. We propose that IP₃ may be one of the intracellular messenger(s) synthesized following the treatment of rabbit endometrial cell cultures with the proliferation agent PGF_2α and that it may play a crucial role with cAMP in growth regulation.     

Regulation of histamine-mediated prostacyclin synthesis in cultured human vascular endothelial cells

Histamine stimulates the production of prostacyclin (PGI₂) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI₂ synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI₂ from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI₂ when treated with histamine. Endothelial cells, however, produced similar amounts of PGI₂ in response to histamine and arachidonic acid. Thrombin also stimulates PGI₂ production in endothelial cells. Histamine and thrombin yielded an additive production of PGI₂ when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI₂ produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI₂ biosynthetic response rapidly returned to normal by $\text{4}\text{1}\text{2}$ hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI₂ from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI₂ production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI₂ synthesis in response to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.     

Bradykinin-induced changes in phosphoinositides, inositol phosphate production and intracellular free calcium in cultured bovine aortic endothelial cells

Acute hydrolysis of phosphoinositides has been demonstrated in bovine aortic endothelial cells (BAEC) treated with bradykinin (BK) (10(-7)M). The first phosphoinositide to decrease was phosphatidylinositol-4,5-bisphosphate (PIP2) indicating this to be the initial substrate of phospholipase action. Other lipid changes associated with the stimulation of BAEC were an increase in diacylglycerol (DAG) and arachidonic acid (AA) with a sustained production of phosphatidic acid (PA). The changes in cell phospholipids were accompanied by the release of inositol phosphates. Inositol-1,4,5-trisphosphate (Ins-1,4,5-P3) was produced within 10 s of stimulation with BK. There was no evidence for the production of inositol-1,3,4-trisphosphate. The release of ionic calcium (Ca2+) intracellularly was demonstrated. The timecourse of the rise in intracellular Ca2+ was consistent with the timecourse of production of IP3. Intracellular Ca2+ rose from 127 +/- 21 nM to 462 +/- 27 nM. The Ca2+ peak was at 7.0 +/- 0.4 s and took 3 min to reach a steady state which remained above the basal level. When extracellular Ca2+ was depleted in the extracellular medium a spike of intracellular Ca2+ release was measured with an immediate return to basal. Entry of extracellular Ca2+ into the cell after ionophore A23187 treatment does not induce inositol phosphate release, indicating that phosphoinositide hydrolysis is likely to be the cause rather than consequence of the elevation in cytosolic Ca2+. These data indicate action of phospholipase C (PLC) on PIP2 after BK stimulation of BAEC with the subsequent production of InsP3 causing the resulting intracellular Ca2+ release.(ABSTRACT TRUNCATED AT 250 WORDS)