The horizontal transfer of Trypanosoma cruzi mitochondrial minicircle DNA to the genomes of naturally infected humans may play an important role in the pathogenesis of Chagas disease. Minicircle integrations within LINE-1 elements create the potential for foreign DNA mobility within the host genome via the machinery associated with this retrotransposon. Here we document integration of minicircle DNA fragments in clonal human macrophage cell lines and their mobilization over time. The movement of an integration event in a clonal transfected cell line was tracked at three months and three years post-infection. The minicircle sequence integrated into a LINE-1 retrotransposon; one such foreign fragment subsequently relocated to another genomic location in association with associated LINE-1 elements. The p15 locus was altered at three years as a direct effect of minicircle/LINE-1 acquisition, resulting in elimination of p15 mRNA. Here we show for the first time a molecular pathology stemming from mobilization of a kDNA/LINE-1 mutation. These genomic changes and detected transcript variations are consistent with our hypothesis that minicircle integration is a causal component of parasite-independent, autoimmune-driven lesions seen in the heart and other target tissues associated with Chagas disease.

As infecções pelo Trypanosoma cruzi e a doença de Chagas são endêmicas em babuínos (Papio hamadryas) reproduzidos em cativeiro na Southwest Foundation for Biomedical Research, em Santo Antonio, Texas. Nós obtivemos produtos de amplificação por PCR do DNA extraído de piolhos colhidos do cabelo e da pele de babuínos chagásicos, com primers aneladores específicos para DNAs nuclear e de cinetoplasto do protozoário. Esses produtos foram hibridizados com suas respectivas seqüências internas complementares. Seqüências selecionadas foram clonadas e o sequenciamento demonstrou a presença de DNA nuclear de T. cruzi, e de minicírculo de kDNA. A PCR competitiva com primers de kDNA determinou a quantidade de aproximadamente 23.9 ± 18.2 T. cruzi por piolho. Este achado sugere que o piolho pode ser um vetor contribuindo para a disseminação de T. cruzi na colônia de babuínos.

Trypanosoma cruzi (Schyzotrypanum, Chagas, 1909), and Chagas disease are endemic in captive-reared baboons at the Southwest Foundation for Biomedical Research, San Antonio, Texas. We obtained PCR amplification products from DNA extracted from sucking lice collected from the hair and skin of T. cruzi-infected baboons, with specific nested sets of primers for the protozoan kinetoplast DNA, and nuclear DNA. These products were hybridized to their complementary internal sequences. Selected sequences were cloned and sequencing established the presence of T. cruzi nuclear DNA, and minicircle kDNA. Competitive PCR with a kDNA set of primers determined the quantity of approximately 23.9 ± 18.2 T. cruzi per louse. This finding suggests that the louse may be a vector incidentally contributing to the dissemination of T. cruzi infection in the baboon colony.

Usamos um método molecular e demonstramos que o tratamento de infecções crônicas pelo Trypanosoma cruzi com nitroderivados, que se mostraram eficientes em diminuir parasitemias nas infecções agudas, não produziu cura parasitológica. Trinta e quatro chagásicos crônicos, com pelo menos dois entre três testes sorológicos positivos para a infecção, e 17 controles com testes sorológicos negativos formaram os grupos de estudo. Os testes de PCR com primers de DNA de T. cruzi monitoraram a eficácia do tratamento. Foram obtidos produtos de PCR a partir de DNA de pacientes chagásicos tratados e não-tratados, com primers de DNA nuclear de T. cruzi. Os produtos amplificados hibridizaram com suas sequências internas e complementares. A técnica de PCR competitiva foi usada para quantificar o número de parasitos no sangue e revelou < 1 a 75 T. cruzi/ml em chagásicos não-tratados (média 25,83 ± 26,32) e < 1 a 36 T. cruzi/ml em chagásicos tratados (média 6,45 ± 9,28). A diferença entre as médias não foi estatisticamente significativa. O resultado mostra que o tratamento da doença de Chagas crônica com drogas nitroderivadas é insatisfatório.

We used a molecular method and demonstrated that treatment of the chronic human Trypanosoma cruzi infections with nitroderivatives did not lead to parasitological cure. Seventeen treated and 17 untreated chronic Chagas' disease patients, with at least two out of three positive serologic assays for the infection, and 17 control subjects formed the study groups. PCR assays with nested sets of T. cruzi DNA primers monitored the efficacy of treatment. The amplification products were hybridized to their complementary internal sequences. Untreated and treated Chagas' disease patients yielded PCR amplification products with T. cruzi nuclear DNA primers. Competitive PCR was conducted to determine the quantity of parasites in the blood and revealed < 1 to 75 T. cruzi/ml in untreated (means 25.83 ± 26.32) and < 1 to 36 T. cruzi/ml in treated (means 6.45 ± 9.28) Chagas' disease patients. The difference between the means was not statistically significant. These findings reveal a need for precise definition of the role of treatment of chronic Chagas' disease patients with nitrofuran and nitroimidazole compounds.

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.

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.