Influence of Micropatterned Grill Lines on Entamoeba histolytica Trophozoites Morphology and Migration.

Entamoeba histolytica, the causal agent of human amoebiasis, has two morphologically different phases: a resistant cyst and a trophozoite responsible for the invasion of the host tissues such as the colonic mucosa and the intestinal epithelium. During in vitro migration, trophozoites usually produce protuberances such as pseudopods and rarely filopodia, structures that have been observed in the interaction of trophozoites with human colonic epithelial tissue. To study the different membrane projections produced by the trophozoites, including pseudopods, filopodia, uropods, blebs, and others, we designed an induction system using erythrocyte extract or fibronectin (FN) in micropatterned grill lines (each micro-line containing multiple micro-portions of FN or erythrocyte extract) on which the trophozoites were placed in culture for migration assays. Using light, confocal, and scanning electron microscopy, we established that E. histolytica trophozoites frequently produce short and long filopodia, large retractile uropods in the rear, pseudopods, blebs, and others structures, also showing continuous migration periods. The present study provides a simple migration method to induce trophozoites to generate abundant membrane protrusion structures that are rarely obtained in normal or induced cultures, such as long filopodia; this method will allow a-better understanding of the interactions of trophozoites with FN and cell debris. E. histolytica trophozoites motility plays an important role in invasive amoebiasis. It has been proposed that both physical forces and chemical signals are involved in the trophozoite motility and migration. However, the in vivo molecules that drive the chemotactic migration remain to be determined. We propose the present assay to study host molecules that guide chemotactic behavior because the method is highly reproducible, and a live image of cell movement and migration can be quantified.

Kinetoplast DNA-binding protein profile in the epimastigote form of Trypanosoma cruzi.

BACKGROUND: The Trypanosomatidae family possesses one of the most unusual DNAs found in nature: the kinetoplast genome. It consists of a few dozen maxicircles that encode for some subunits of mitochondrial enzymes and rRNAs in a cryptic pattern and thousands of minicircles that encode for the guide RNAs (gRNAs), all catenated and constituting a dense network. The complexity of kinetoplast genome based on its intricate DNA structure is well known; however, only a small number of proteins associated with kinetoplast DNA (kDNA) have been described, and the majority are related with the replication process. METHODS: We describe the protein profile obtained using formaldehyde as a cross-linking agent to obtain the kinetoplast DNA-protein complex, and Southwestern assay to identify the kDNA binding proteins present in the complex. RESULTS: We identified seven proteins eluted from the kDNA complex fixed by formaldehyde. Polyclonal antiserum developed against the kDNA-protein complex recognized only four proteins in crude extracts of epimastigote stage, suggesting immunogenic differences among these proteins and/or their availability in the kinetoplast genome. Southwestern assay using minicircle fragments showed nine kDNA binding proteins in crude extracts of Trypanosoma cruzi epimastigote. CONCLUSIONS: We describe several proteins associated with the kDNA. Some could be involved in the essential process for parasite life and also could be a good target for drug or vaccine development. The results contribute to understanding the organization of the kinetoplast genome.

American trypanosomiasis (Chagas'Disease) and blood banking in Mexico City: seroprevalance and its potential transfusional transmission risk

Background. American trypanosomiasis (Chagas' disease), an anthropozoonosis fairly common in rural Latin American, has become an urban disease due to continuous migration, intra- and internationally. Blood transfusion, the second important pathway for transmission, increases its impact. Recognition of seropositive subjects among blood donors is now recommended, and clinical and serological screening enforced. Maneuvers to inactivate or remove Trypanosoma cruzi present in collected blood are recommended. Methods. We surveyed voluntary donors at the National Institute of Cardiology in Mexico City in of anti-T. cruzi by indirect immunofluorescence, ELISA, and Western blot analysis. Seropositive donors were identified and tested for immunoglobulin. We used types and fractions of donated blood to extract DNA and perform the PCR technique using kinetoplast primers seeking parasite DNA in blood. Results. After 3,300 donors were screened, we identified 10 seropositive subjects (0.3 percent). These subjects were considered as indeterminate chagasic patients, came mainly from rural areas, and had IgG (100 percent) and IgA (30 percent) antibodies aginst a crude extract as well as a recombinant T. cruzi antigen. Identification of parasite DNA in red cell and platelet fraction was achieved from eight blood units. Conclusions. The present data provide evidence that blood donors at an urban hospital are seropositive for T. cruzi and at least 50 percent of donors carry the parasite potentially able to transmit T. cruzi in their cellular blood products. Serological screening should be included in routine blood-banking. It is also necessary to adopt measures to inactivate or eliminate organisms in donated blood