Interactions between Leishmania mexicana mexicana promastigotes and amastigotes and murine peritoneal macrophages in vitro

Unstimulated adherent mouse peritoneal cells were cultured in vitro and infected with equal numbers of a single strain of Leishmania m. mexicana amastigotes (AM), virulent promastigotes (VP), avirulent promastigotes (AVP) and fixed promastigotes (FP). Duplicate May-Grünwald-Giemsa stained coverslips were examined at time intervals up to 13 days. By 3 hr post infection, the number of macrophages containing parasites varied between 60.5% (VP) and 84% (AM) for macrophages exposed to living parasites, compared to 6.5% for macrophages exposed for FP. However, variable numbers of parasites showed degenerative changes by 3 hr, and the number of macrophages containing morphologically intact parasites varied significantly between cells infected with AM (84%) and those infected with VP (42%) or AVP(40%). The mean number on intacte parasites/macrophage also differed significantly between AM-infected cells and living or fixed promastigotes-infected cells. Quantitation of intact and degenerated parasites indicated parasite multiplication, as well as destruction, in VP-infected cells and parasite survival and multiplication in AM-infecte monolayers; in contrast no evidence of parasite multiplication was seen in AVP-infected cells. Changes in the mono layer itself (cell loss and macrophage vacuolization) were also evaluated. These results suggest that crucial events determining the outcome of infection occur in the host-parasite relationship during the fist 24 hours of infection. These events are apparently influenced not only by parasite or host strain but by environmentally induced variation within a given strain.

Extra-tissular Schistosoma mansoni egg granulomata in the peritoneal cavity of mice

The presence of Schistosoma mansoni eggs surrounded by inflammatory cells were detected within the peritoneal cavity of experimentally infected mice. The histological and ultrastructural analysis revealed the predominantly macrophagic composition of these structures. The presence of epithelioid cells, macrophages in different stages of activation and the architectural pattern of the cells, characterize these structures as extra-tissular true granulomas. Granulomas much similar to those observed in the peritoneal cavity of infected mice were also detected after the intraperitoneal injection of viable eggs in non-infected mice. Collagen fibers were observed in between the inflammatory cells of granulomas obtained 10 weeks after infection and 48 hours after the injection of viable eggs into the peritoneal cavity. In later times of infection or injection the amount of collagen fibers increases resulting in a typical pattern of healed schistosoma egg granulomas. The possible influence of the immune response on the genesis of the granulomatous reaction as well as the influence of the vascularized connective tissue on this process is discussed.

Jatobal virus antigenic characterization by ELISA and neutralization test using EIA as indicator, on tissue culture

A virus antigenic characterization methodology using an indirect method of antibody detection ELISA with virus-infected cultured cells as antigen and a micro virus neutralisation test using EIA (NT-EIA) as an aid to reading were used for antigenic characterization of Jatobal (BeAn 423380). Jatobal virus was characterized as a Bunyaviridae, Bunyavirus genus, Simbu serogroup virus. ELISA using infected cultured cells as antigen is a sensitive and reliable method for identification of viruses and has many advantages over conventional antibody capture ELISA's and other tests: it eliminates solid phase coating with virus and laborious antigen preparation; it permits screening of large numbers of virus antisera faster and more easily than by CF, HAI, or plaque reduction NT. ELISA and NT using EIA as an aid to reading can be applicable to viruses which do not produce cytopathogenic effect. Both techniques are applicable to identification of viruses which grow in mosquito cells.

Mechanism of action of a nitroimidazole-thiadiazole derivate upon Trypanosoma cruzi tissue culture amastigotes

Megazol (CL 64,855) a very effective drug in experimental infections by Trypanosoma cruzi, and also in in vitro assays with vertebrate forms of the parasite, had its parasite, had its activity upon macromolecule biosynthesis tested using tissue culture-derived amastigote forms. Megazol presented a drastic inhibition of [3H]-uridine incorporation, suggesting a selective activity upon protein synthesis. Comparing the three drugs, megazol was more potent than nifurtimox and benznidazole in inhibiting protein an DNA synthesis. Megazol showed a 91% of inhibition of [3H]-leucine incorporation whereas nifurtimox and benznidazole, 0% and 2%, respectively. These latter two drugs inhibited the incorporation of all the precursors tested at similar levels, but the concentration of benznidazole was always three times higher, suggesting different mechanisms of action or, more probably, a greater efficiency of the 5-nitrofuran derivate in relation to the 2-nitroimidazole. So, wes conclude that the mode of action of megazol is different from the ones of nifurtimox and benznidazole and that its primary effect is associated with an impairment of protein synthesis.

Trypanosoma cruzi: experimental Chagas' disease in Rhesus monkeys. II. Ultraestructural and cytochemical studies of peroxidase and acid phosphatase activities

Ultrastructural and cytochemical studies of peroxidase and acid phosphatase were performed in skin, lymph node and heart muscle tissue of thesus monkeys with experimental Chagas's disease. At the site of inoculation ther was a proliferative reaction with the presence of immature macrophages revealed by peroxidase technique. At the lymph node a difuse inflammatory exudate with mononuclear cells, fibroblasts and immature activated macrophages reproduces the human patrtern of acute Chagas' disease inflamatory lesions. The hearth muscle cells present different degrees of degenerative alterations and a striking increase in the number of lysosomal profiles that exhibit acid hydrolase reaction product. A strong inflammatory reaction was present due to lymphocytic infiltrate or due to eosinophil granulocytes associated to ruptured cells. The present study provides some experimental evidences that the monkey model could be used as a reliable model to characterize histopathological alterations of the human disease.

Effect of the host specific treatment in the phagocytosis of Trypanosoma cruzi blood forms by mouse peritoneal macrophages

Single doses of drugs active aginst Trypanosoma cruzi (megazol, nifurtimox and benznidazole) induce a rapid clearence of the blood parasites in experimentally infected mice. Furthermore, the in vitro phagocytosis and intracellular destruction by mouse peritoneal macrophage of blood forms collected from the treatment animals is strongly enhanced as compared with parasites from untreated controls. The uptake of the blood forms by macrophages is significantly higher with megazol than with benznidazole and nifurtimox, a finding that concurs with data showing that megazol is also the most active compound in the living host. The possibility that macrophages participate in a synergic effect between the host immune response and chemotherapeutic effect is discussed.

The distribution of agglutinins and lytic activity against Trypanosoma rangeli and erythrocytes in Rhodnius prolixus and Triatoma infestans tissue extracts and haemolymph

Haemolymph, heads, salivary glands, crops, midguts, hindguts, and Malpighian tubules from Rhodnius prolixus and Triatoma infestans were extracted in phosphate or Tris buffer saline with calcium, and tested for agglutination and lytic activities by microtitration against both vertebrateerythrocytes and cultured epimatigote forms of Trypanosoma rangeli. Haemagglutination activity against rabbit erythrocytes was found in the crop, midgut and hindgut extracts of T. infestans but only in the haemolymph of R. prolixus. Higher titres of parasite agglutinins were found in R. prolixus haemolymph than T. infestans, whilst the converse occurred for the tissue extracts. In addition, the extracts of T. infestans salivary glands, but not those of R. prolixus, showed a trypanolytic activity that was heat-inactivated and was not abolished by pre-incubation with any of the sugars or glycoproteins tested. T. infestans, which is refractory to infection by T. rangeli, thus appears to contain a much wider distribution of agglutinating and trypanolytic factors in its tissues than the more susceptible species, R. prolixus

Hyperplasia of elastic tissue in hepatic schistosomal fibrosis

Elastic tissue hyperplasia, revealed by means of histological, immunocytochemical and ultrastructural methods, appeared as a prominent change in surgical liver biopsies taken from 61 patients with schistosomal periportal and septal fibrosis. Such hyperplasia was absent in ecperimental murine schistosomiasis, including mice with "pipe-stem" fibrosis. Displaced connective tissue cells in periportal areas, such as smooth muscle cells, more frequently observed in human material, could be the site of excessive elastin synthesis, and could explain the differences observed in human and experimental materials. Elastic tissue, sometimes represented by its microfibrillar components, also appeared to be more condensed in areas of matrix (collagen) degradation, suggesting a participation of this tissue in the remodelling of the extracellular matrix. By its rectratile properties elastic tissue hyperplasia in hepatic schistosomiasis can cause vascular narrowing and thus play a role in the pathogenesis of portal hypeertension.

Plant tissue culture techniques

Plant cell and tissue culture in a simple fashion refers to techniques which utilize either single plant cells, groups of unorganized cells (callus) or organized tissues or organs put in culture, under controlled sterile conditions.

Trypanosoma cruzi recognition by macrophages and muscle cells: perspectives after a 15-year study

Macrophages and muscle cells are the main targets for invasion of Trypanosoma cruzi. Ultrastructural studies of this phenomenon in vitro showed that invasion occurs by endocytosis, with attachment and internalization being mediated by different components capable of recognizing epi-or trypomastigotes (TRY). A parasitophorus vacuole was formed in both cell types, thereafter fusing with lysosomes. Then, the mechanism of T. cruzi invasion of host cells (HC) is essentially similar (during a primary infection in the abscence of a specific immune response), regardless of wether the target cell is a professional or a non-professional phagocytic cell. Using sugars, lectins, glycosidases, proteinases and proteinase inhibitors, we observed that the relative balance between exposed sialic acid and galactose/N-acetyl galactosamine (GAL) residues on the TRY surface, determines the parasite's capacity to invade HC, and that lectin-mediated phagocytosis with GAL specificity is important for internalization of T. cruzi into macrophages. On the other hand, GAL on the surface to heart muscle cells participate on TRY adhesion. TRY need to process proteolytically both the HC and their own surface, to expose the necessary ligands and receptors that allow binding to, and internalization in the host cell. The diverse range of molecular mechanisms which the parasite could use to invade the host cell may correspond to differences in the available "receptors"on the surface of each specific cell type. Acute phase components, with lectin or proteinase inhibitory activities (a-macroglobulins), may also be involved in T. cruzi-host cell interaction.

Host tissue destruction by Entamoeba histolytica: molecules mediating adhesion, cytolysis, and proteolysis

Entamoeba histolytica, the protozoan parasite causing human amoebisis, has recently been found to comprise two genetically distinct forms, potentially pathogenic and constitutively nonpathogenic ones. Host tissue destruction by pathogenic forms is belived to result from cell functions mediaed by a lectin-type adherence receptor, a pore-forming peptide involved in host cell lysis, and abundant expression of cysteine proteinase(s). Isolation and molecular cloning of these amoeba products have provided the tools for structural analyses and manipulations of cell functions including comparisons between pathogenic and nonpathogenic forms.