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.

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.

Foram detectados anticorpos, nos soros de pacientes com doença de Chagas, Calazar e Leishnnaniose cutâneo-mucosa, que se ligam a antígenos compartilhados pelos três agentes causais. Os pacientes chagásicos mostraram 98 a 100% de soropositividade pelo ELISA quando antígenos de Leishmania braziliensis e de Leishmania chagasi foram usados, respectivamente. Os soros de Calazar mostraram resultados positivos com antígenos de Trypanosoma cruzi ou com L. braziliensis pela imunofluorescência. Ademais, os anticorpos nos soros de pacientes com Leishmaniose cutâneo-mucosa tinham 100% de resultados positivos pelo ELISA com antígenos de T. cruzi ou de L. chagasi. Ademais, a aglutinação direta de promastigotas de L. chagasi mostrou que 95% dos soros de Calazar e 35% de Leishmaniose cutâneo-mucosa algutinaram o parasito em diluições acima de 1:512. Em contraste, 15% dos soros chagásicos aglutinaram o parasito na diluição de 1:16 ou abaixo. Análises pelo Western blot mostraram que os soros chagásicos que formaram pelo menos 24 bandas com antígeno de T. cruzi também foramaram 13 bandas com antígeno de L. chagasi e 17 bandas com L. braziliensis. Os soros de Calazar que reconheceram pelo menos 29 bandas com o antígeno homólogo também formaram 14 bandas com o T. cruzi e 10 bandas com L. braziliensis. Finalmente, os soros de Leishmaniose cutâneo-mucosa que formaram pelo menos 17 bandas com o antígeno homólogo também formaram 10 bandas com os antígenos de T. cruzi e quatro com L. braziliensis. Esses resultados indicam a presença de determinantes antigênicos comuns em várias proteínas desses protozoários e, portanto, explicam as reações sorológicas cruzadas descritas aqui.

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.

A prevalência da infecção chagásica em 368 garis do Serviço de Limpeza Urbana (SLU) do Distrito Federal foi inicialmente indicada pelos exames de hemaglutinação e imunofluorescência, quando encontrou-se 32,1% e 47,6% de resultados positivos, respectivamente, para cada exame. Em vista das discrepâncias obsevadas. utilizou-se o teste cutâneo com o antígeno T12E para esclarecimento do diagnóstico em 15,5% dos que tinham apenas um teste sorológico positivo. O teste cutâneo foi positivo em 38,6% dos garis. A análise dos resultados de 183 garis mostrou 47% dos casos com três exames concordantes positivos. Mais 19, 7% tiveram dois exames concordantes positivos. Porém, 33,3% dos garis tinham apenas um dos três exames positivo. Empregou-se também o "immunoblot" na tentativa de esclarecer o diagnóstico nos 61 casos com apenas um resultado positivo. Foram encontrados 37,5% (3/8) dos casos positivos pela hemaglutinação, 11,5% (3/26) pelo teste cutâneo, e apenas 3,7% (1/27) pela imunofluorescência. Após a adição desses casos positivos pelo "immunoblot", o total de chagásicos passou a ser de 129, ou seja, 35%/ dos garis da amostra estudada.

Seropositivity for Trypanosoma cruzi infection was studied in 308 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%, 42.7% and 38.6% positive results, respectively for cach assay. Among these, however, only 47% were positive with each of three exams performed. In addition, 19.7% were positive with two out of three exams performed. The remaining 33-3% sera yielded one positive result out of three exams employed and were submitted to the immunoblot assay. This analysis confirmed 3 cases (37.5%) positive by hemmaglutination. 3 (11.5%) positive by skin test, and 1 (3-7%) positive by immunofluorescence. At the end of the analysis, it was shown that 129 (35%) individuals yielded at least two positive assays and therefore, they should be cousidered as T. cruzi-infected individuals.

Observamos a cromatina deformas amastigotas de T. cruzi associada a cromossomos de macrófagos metafásicos obtidos em diversos períodos da infecção aguda. O estudo imunocitogenético demonstrou que o material genético inserido naqueles cromossomos era produto do T. cruzi. Pelo teste de hibridizaçâo in situ com sonda biotinilada de DNA de T. cruzi, foi confirmada a inserção de DNA do protozoário nos cromossomos murinos. A inserção seletiva de ³H-DNA do protozoário em alguns cromossomos sugere que podem ocorrer rearranjos transxenogénicos em infecções de mamíferos pelo T. cruzi.

Trypanosoma cruzi chromatin was observed in macrophage chromosome spreads obtained at different periods of infections in BALB/c mice. Immunofluorescent studies showed that genetic materials associated with the chromosomes were T. cruzi products. In situ hybridization showed the protozoon DNA insertion in the host cell genome. In addition, selective ³H-DNA insertion in chromosomes 3, 6 and 11 was observed, which suggested that transxenogene rearrangement may take place in T. cruzi infections of mammals.