Distinct ability to accumulate eosinophils during the inflammatory cellular response to M. bovis BCG in the mouse pleural cavity.

OBJECTIVE AND DESIGN: The host response to Mycobacteria focuses on the development of cell-mediated immunity and granuloma formation. Here, we investigated the onset of cellular responses to mycobacteria in murine pleurisy. MATERIAL: Distinct mouse strains previously described as Bcg susceptible or resistant were inoculated intrathoracically with different doses of live M. bovis BCG. METHODS: At various time intervals, cells harvested from the inflammatory site were identified and ultra-structurally analysed. RESULTS: BCG-induced pleurisy had two peaks of cellular influx at 1 and 15 days after infection. At the first half hour, macrophages were found to be heavily infected. Neutrophil arrival started after 2 h of infection and peaked at 4 h. At this time, neutrophils were found ingesting mycobacteria exclusively with a high infecting dose. BCG was potently more eosinophilotactic in Bcg susceptible mice than in the resistant ones and to other well known eosinophilia inducers: IL-5, PAF-acether or LPS. CONCLUSIONS: Mycobacterial load and mouse susceptibility seem to determine the early granulocyte dynamics in the lesion.

Ultrastructural distribution of poly (A)+ RNA during Trypanosoma cruzi-cardiomyocyte interaction in vitro: a quantitative analysis of the total mRNA content by in situ hybridization.

Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A)+ RNA remained about the same up to 24 h post-infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.

Trypanosoma cruzi infection affects actin mRNA regulation in heart muscle cells.

We have previously described alterations in the cytoskeletal organization of heart muscle cells (HMC) infected with Trypanosoma cruzi in vitro. Our aim was to investigate whether these changes also affect the regulation of the actin mRNAs during HMC differentiation. Northern blot analysis revealed that alpha-cardiac actin mRNA levels increased during cell differentiation while beta-actin mRNA levels declined. Nonmuscle cells displayed beta-actin mRNA signal localized at the cell periphery, while alpha-cardiac actin mRNA had a perinuclear distribution in myocytes. Trypanosoma cruzi-infected cells showed 50% reduction in alpha-cardiac actin mRNA expression after 72 h of infection. In contrast, beta-actin mRNA levels increased approximately 79% after 48 h of infection. In addition, in situ beta-actin mRNA was delocalized from the periphery into the perinuclear region. These observations support the hypothesis that Trypanosoma cruzi affects actin mRNA regulation and localization through its effect on the cytoskeleton of heart muscle cells.

Glycolipid and protein profiles in trypanosomatids.

A comparative study of glycolipid and protein composition in Trypanosoma cruzi and non-pathogenic trypanosomatids was carried out using Triton X-114 extraction. Protein profiles were analysed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE), and glycolipids were detected using high-performance thin-layer chromatography (HPTLC). Hydrophilic protein profiles were similar in non-pathogenic protozoa. Endotrypanum schaudinni, Crithidia luciliae and T. mega showed five characteristic protein bands ranging between 30 and 66 kDa. In the hydrophobic phase, a band of 50 kDa was present only in T. mega. Strain-specific protein distribution was detected in T. cruzi clone Dm28c and T. cruzi G and Y strains; clone Dm28c had five typical hydrophilic proteins at between 24 and 45 kDa, the G strain had two bands at 45 kDa in the hydrophilic phase and the Y strain had a major protein band at 24 kDa in both phases. T. dionisii and T. cruzi clone Dm28c showed a characteristic distribution of three hydrophilic proteins of approx. 45 kDa. Qualitative analysis of glycolipid composition showed that the T. cruzi strains and Dm28c clone and T. dionisii had four orcinol-positive spots, whereas in the other non-pathogenic trypanosomatids only three glycolipids were detected.

Myofibrillar breakdown and cytoskeletal alterations in heart muscle cells during invasion by Trypanosoma cruzi: immunological and ultrastructural study.

The cytoskeletal organization of normal and Trypanosoma cruzi-infected mouse embryo heart muscle cells (HMC) in primary culture was investigated using immunofluorescence and transmission electron microscopy. Fluorescent probes revealed that in the early stages of infection, up to 24 h, the HMC cytoskeleton appeared to accommodate the intracellular parasites perinuclearly, with only a few cells displaying a slight disturbance in the distribution of filaments. However, as the infection progressed (48 to 72 h), microtubules and desmin filaments were disrupted. Breakdown of myofibrils occurred in regions where the parasites were present, followed by formation of actin polygons. Using Triton X-100 treated whole cell mount, we obtained a striking preservation of the three-dimensional architecture of the cytoskeleton. Combining electron spectroscopic imaging (ESI) with contrast tuning, we detected a highly interconnected cytoskeletal network in normal cells, and a loose network in infected cells. Bundles of filaments running under and over the parasites were also observed. Our results demonstrate that T. cruzi infection induces myofibrillar breakdown and destruction of several cytoskeleton filaments in heart muscle cells.

Glycolipid and protein profiles of normal and Trypanosoma cruzi infected heart muscle cells.

Using Triton X-114, glycolipids and proteins were extracted from heart muscle cells (HMC) infected with Trypanosoma cruzi clone Dm28c and from uninfected HMC, and analysed by SDS-PAGE and high-performance thin-layer chromatography (HPTLC). Two major differences were observed: (a) two proteins with a molecular mass of 92 kDa and 69 kDa were present in the uninfected cells but absent from the infected cells and (b) a 70-90 kDa protein band was detected only in parasitized cells. These differences would seem to constitute alterations taking place during the process of cell recognition and/or parasite interiorization. No differences were observed in the respective glycolipid compositions, of control and infected cells analysed by HPTLC. A glycolipid with the same mobility as the neutral glycolipid isolated from epimastigotes of T. cruzi was detected in the uninfected cells. This finding may lend support to the previously described hypothesis that molecular mimicry is implicated in the cardioneuropathology of Chagas' disease.

Immunoelectron localization of mitochondrial F1 factor in cryosections of heart muscle cells.

Immunocytochemistry was used to investigate the localization of F1 ATPase in mitochondria of cryosections of adult mouse heart muscle cells. The initial aldehyde fixation was the only denaturation step for antigens. The fine structure was preserved with contrast enhancement as the sections were maintained hydrated, with the advantage that the entire procedure is completed in one working day. The reaction was highly specific, and entire mitochondria were labeled with the Protein A-gold complex. A new analytical technique, electron spectroscopic imaging (ESI), contributed to a better visualization of the localization of the F1 factor.

Interaction of Trypanosoma cruzi with macrophages in vitro: dissociation of the attachment and internalization phases by low temperature and cytochalasin B.

Chicken macrophages, obtained by cultivation of blood monocytes, were infected with epimastigote and bloodstream trypomastigote forms of Trypanosoma cruzi strain Y. The percentage of macrophages containing parasites within parasitophorous vacuoles and of flagellates attached to cell surfaces was determined. By incubation of the macrophages at 4 degrees C or in the presence of cytochalasin B it was possible to dissociate the attachment from the internalization phases in the process of infection of macrophages. Both treatments had a marked effect on the internalization of epimastigote and trypomastigote forms. Cytochalasin B treatment and placement of the macrophages at 4 degrees C before infection inhibited this process by about 99 and 96%, respectively. These results suggest that endocytosis is the principal mechanism of internalization of T. cruzi by macrophages. They show also that epimastigote and trypomastigote forms of T. cruzi have a different rate of adhesion to the macrophage surface.

Interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi with chicken macrophages in vitro.

Chicken macrophages, obtained by cultivation of blood monocytes, were infected with epimastigote and bloodstream trypomastigotes of the Y and the CL strains of Trypanosoma cruzi. The percentage of infected cells and the mean number of parasites per cell were determined after 2, 6, 12 and 24 h of parasite-cell contact. After 6 h of contact about 80% and 40% of the macrophages were infected by trypomastigotes of the Y and CL strains respectively. After longer periods of contact almost all macrophages were infected by Y trypomastigotes while only about 60% were infected by those of the CL strain. After 2 h of contact almost all macrophages were infected by CL epimastigotes while only about 50% were infected by Y epimastigotes. After 6 h of contact almost all macrophages were infected by epimastigotes of both strains. These results are discussed taking into consideration differences between parasites of the two strains and between the two developmental stages of the T. cruzi lifecycle.