HygR and PurR plasmid vectors for episomal transfection of Trypanosoma cruzi

This work describes the development and functional testing of two episomes for stable transfection of Trypanosoma cruzi. pHygD contained the 5'- and 3'- flanking regions of the gene encoding the cathepsin B-like protease of T. cruzi as functional trans-splicing and polyadenylation signals for the hygR ORF. Evidence is presented to support extrachromosomal maintenance and organization as tandem repeats in transfected parasites. pPac was derived from pHygD by replacement of the entire hygR ORF with a purR coding region. The ability to modify pHygD and the availability of the complete DNA sequence make these plasmids useful tools for the genetic manipulation of T. cruzi.

Current millenium biotechniques for biomedical research on parasites and host-parasite interactions

The development of biotechnology in the last three decades has generated the feeling that the newest scientific achievements will deliver high standard quality of life through abundance of food and means for successfully combating diseases. Where the new biotechnologies give access to genetic information, there is a common belief that physiological and pathological processes result from subtle modifications of gene expression. Trustfully, modern genetics has produced genetic maps, physical maps and complete nucleotide sequences from 141 viruses, 51 organelles, two eubacteria, one archeon and one eukaryote (Saccharomices cerevisiae). In addition, during the Centennial Commemoration of the Oswaldo Cruz Institute the nearly complete human genome map was proudly announced, whereas the latest Brazilian key stone contribution to science was the publication of the Shillela fastidiosa genomic sequence highlythed on a Nature cover issue. There exists a belief among the populace that further scientific accomplishments will rapidly lead to new drugs and methodological approaches to cure genetic diseases and other incurable ailments. Yet, much evidence has been accumulated, showing that a large information gap exists between the knowledge of genome sequence and our knowledge of genome function. Now that many genome maps are available, people wish to know what are we going to do with them. Certainly, all these scientific accomplishments will shed light on many more secrets of life. Nevertheless, parsimony in the weekly announcements of promising scientific achievements is necessary. We also need many more creative experimental biologists to discover new, as yet un-envisaged biotechnological approaches, and the basic resource needed for carrying out mile stone research necessary for leading us to that "promised land" often proclaimed by the mass media

Cross-reactivity of antibodies in human infections by the kinetoplastid protozoa Trypanosoma cruzi, Leishmania chagasi and Leishmania (viannia) braziliensis

We have detected antibodies, in the sera of Chagas disease, Kala-azar and Mucocutaneous leishmaniasis patients, that bind multiple antigens shared between the three causative agents. The Chagas disease sera showed 98 to 100 positive results by ELISA when the Leishmania braziliensis and Leishmania chagasi antigens were used, respectively. The Kala-azar sera showed 100 positive results with Trypanosoma cruzi or L. braziliensis antigens by immunofluorescence assays. The antibodies in the sera of Mucocutaneous leishmaniasis patients showed 100 positive results by ELISA assays with T. cruzi or L. chagasi antigens. Furthermore, the direct agglutination of L. chagasi promastigotes showed that 95 of Kala-azar and 35 of Mucocutaneous leishmaniasis sera agglutinated the parasite in dilutions above 1:512. In contrast, 15 of Chagas sera agglutinated the parasite in dilutions 1:16 and below. Western blot analysis showed that the Chagas sera that formed at least 24 bands with the T. cruzi also formed 13 bands with the L. chagasi and 17 bands with the L. braziliensis. The Kala-azar sera that recognized at least 29 bands with the homologous antigen also formed 14 bands with the T. cruzi and 10 bands with the L. braziliensis antigens. Finally, the Mucocutaneous leishmaniasis sera that formed at least 17 bands with the homologous antigen also formed 10 bands with the T. cruzi and four bands with the L. chagasi antigens. These results indicate the presence of common antigenic determinants in several protozoal proteins and, therefore, explain the serologic cross-reactions reported here.

Emprego de quatro exames imunológicos na determinaçäo da prevalência da doença de Chagas nos garis do Serviço de Limpeza Urbana do Distrito Federal / The use of four immunological tests to determine the prevalence of Chagas' disease in street cleaners of the Urban Cleaning Service of the Federal District

Seropositivity for Trypanosoma cruzi infection was studied in 368 street-sweepers of the SLU, Federal District, Brazil, with the aid of haemaglutination, immunofluorescence and, also, a delayed-type skin test to the parasite T12E antigen. It showed 32.1 per cent, 42.1 per cent and 38.6 per cent positive results, respectively for each assay. Among these, however, only 47 per cent were positive with each of three exams performed. In addition, 19.7 per cent were positive with two out of three exams performed. The remaining 33.3 per cent sera yielded one positive result out of three exams employed and were submitted to the immunoblot assay. This analysis confirmed 3 cases (37.5 per cent) positive by hemmaglutination, 3 (11.5 per cent) positive by skin test, and 1 (3.7 per cent) positive by immunofluorescence. At the end of the analysis, it was shown that 129 (35 per cent) individuals yielded at least two positive assays and, therefore, they should be considered as T. cruzi-infected individuals.

Inserçäo de DNA de Trypanosoma cruzi no genoma de célula hospedeira de mamífero por meio de infecçäo / Insertion of Trypanosoma cruzi DNA in the genome of mammal host cell through infection

Observamos a cromatina de formas amstigotad de T. cruzi associada a cromossomos de macrófagos metafásicos obtidos em diversos períodos da infecçäo aguda. O estudo imunocitogenético demonstrou que o material genético inserido naqueles cromossomos era produto do T. cruzi. Pelo teste de hibridaza ao in situ com sonda biotinilada de DNA de T. cruzi, foi confirmada a inserçäo de DNA do protozoário nos cromossomos murinos. A inserçäo seletiva de 3H-DNA do protozoário em alguns cromossomos sugere que podem ocorrer rearranjos transxenogênicos em infecçöes de mamiferos pelo T. cruzi