Environment, interactions between Trypanosoma cruzi and its host, and health: [review] / Meio-ambiente, interações entre Trypanosoma cruzi e seu hospedeiro e saúde humana: [revisão]

An epidemiological chain involving Trypanosoma cruzi is discussed at the environmental level, and in terms of fine molecular interactions in invertebrate and vertebrate hosts dwelling in different ecosystems. This protozoan has a complex, genetically controlled plasticity, which confers adaptation to approximately 40 blood-sucking triatomine species and to over 1,000 mammalian species, fulfilling diverse metabolic requirements in its complex life-cycle. The Tr. cruzi infections are deeply embedded in countless ecotypes, where they are difficult to defeat using the control methods that are currently available. Many more field and laboratory studies are required to obtain data and information that may be used for the control and prevention of Tr. cruzi infections and their various disease manifestations. Emphasis should be placed on those sensitive interactions at cellular and environmental levels that could become selected targets for disease prevention. In the short term, new technologies for social mobilization should be used by people and organizations working for justice and equality through health information and promotion. A mass media directed program could deliver education, information and communication to protect the inhabitants at risk of contracting Tr. cruzi infections.

Uma rede epidemiológica envolvendo o Trypanosoma cruzi foi discutida nos níveis ambientais e de interações moleculares nos hospedeiros que habitam em 19 diferentes ecossistemas. O protozoário tem uma enorme plasticidade controlada geneticamente que confere sua adaptação a cerca de quarenta espécies de triatomíneos e mais de mil espécies de mamíferos. Essas infecções estão profundamente embutidas em inúmeros ecótopos, onde elas estão inacessíveis aos métodos de controle utilizados. Muito mais estudos de campo e de laboratório são necessários à obtenção de dados e informação pertinentes ao controle e prevenção das infecções pelo Tr. cruzi e as várias manifestações da doença. Ênfase deve ser dada àquelas interações que ocorrem nos níveis celulares e ambientais que se poderiam tomar como alvos seletivos para prevenção da doença. Novas tecnologias para mobilização social devem ser disponibilizadas para os que trabalham pela justiça e pela igualdade, mediante informação para a promoção da saúde. Um programa direcionado de educação de massa pode prover informação e comunicação necessárias para proteger os habitantes atualmente expostos ao risco de contrair as infecções pelo Tr. cruzi.

Hitchhiking Trypanosoma cruzi minicircle DNA affects gene expression in human host cells via LINE-1 retrotransposon

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.

Evolution and pathology in Chagas disease: a review

Trypanosoma cruzi acute infections often go unperceived, but one third of chronically infected individuals die of Chagas disease, showing diverse manifestations affecting the heart, intestines, and nervous systems. A common denominator of pathology in Chagas disease is the minimal rejection unit, whereby parasite-free target host cells are destroyed by immune system mononuclear effectors cells infiltrates. Another key feature stemming from T. cruzi infection is the integration of kDNA minicircles into the vertebrate host genome; horizontal transfer of the parasite DNA can undergo vertical transmission to the progeny of mammals and birds. kDNA integration-induced mutations can enter multiple loci in diverse chromosomes, generating new genes, pseudo genes and knock-outs, and resulting in genomic shuffling and remodeling over time. As a result of the juxtaposition of kDNA insertions with host open reading frames, novel chimeric products may be generated. Germ line transmission of kDNA-mutations determined the appearance of lesions in birds that are indistinguishable from those seen in Chagas disease patients. The production of tissue lesions showing typical minimal rejection units in birds' refractory to T. cruzi infection is consistent with the hypothesis that autoimmunity, likely triggered by integration-induced phenotypic alterations, plays a major role in the pathogenesis of Chagas disease.

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.

Blood-sucking lice may disseminate Trypanosoma cruzi infection in baboons

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

Persistent infections in chronic Chagas'disease patients treated with anti-Trypanosoma cruzi nitroderivatives

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.

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

Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

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.

Attitudes towards Chagas' disease in an endemic Brazilian community

A doença de Chagas persiste como importante problema de saúde pública em grande parte da América Latina. No Brasil, vários segmentos da populaçäo apresentam taxas de infecçäo pelo Trypanosoma cruzi que chegam até 65 por cento, sugerindo que os programas de controle ainda säo necessários. Há poucos dados disponíveis sobre as crenças populares relativas à doença de Chagas entre comunidades com altas taxas de infecçäo. Tais crenças podem ter um impacto significativo sobre a eficácia das intervençöes. Avalia-se as crenças relativas à doença de Chagas numa populaçäo com altas taxas de infecçäo pelo agente etiológico da doença. A populaçäo-alvo do estudo foi composta de moradores da Posse, uma comunidade rural no Estado de Goiás. Os resultados indicam que a maioria da populaçäo tinha um alto grau de conhecimento quanto à doença de Chagas e seu vetor. Os achados apontam para a eficácia dos componentes educativos dos programas de controle implementados pelo Ministério da Saúde.

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.

Hipersensibilidade tardia a antígeno de Trypanosoma cruzi: I- Estudo experimental em coelhos / Delayed hypersensitivity to Trypanosoma cruzi antigen: I- Experimental study in rabbits

Reaçoes de hipersensibilidade cutânea tardia em coelhos infectados com Trypanosoma cruzi homólogo ou heterólogo, foram obtidas pela injeçao de 50 mug de proteína do antígeno Tl2E, derivado de um clone do parasito. A especificidade do teste foi indicada pela ausência de reaçao em coelhos controles normais que receberam a mesma quantidade de antígeno na pele. A injeçao intradérmica do antígeno, em cinco ocasioes, nao induziu reaçao cutânea positiva ou soro conversao dos exames de hemaglutinaçao e aglutinaçao, em coelhos controles normais. Por outro lado, coelhos chagásicos submetidos a série de cinco testes cutâneos, com intervalos de uma semana, nao exibiram alteraçao da intensidade das reaçoes de hipersensibilidade ou dos perfís dos títulos de anticorpos séricos. Os registros eletrocardiográficos também nao foram alterados pelos testes cutâneos em coelhos normais e chagásicos. A inocuidade do antígeno Tl2E foi confirmada em experimentos realizados em 36 coelhos chagásicos e 19 coelhos controles normais. Esse estudo mostra que o teste cutâneo com o antígeno Tl2E pode ser útil no diagnóstico, além de servir como indicador de morbidade da doença.

Hipersensibilidade tardia a antígeno de Trypanosoma cruzi: II- Emprego do teste cutâneo com antígeno T12E para diagnóstico da doença de Chagas / Delayed hypersensitivity to trypanosoma cruzi antigen: II- Use of the skin test with T!@E antigen for the diagnosis of Chagas disease

Reaçoes cutâneas de hipersensibilidade tardia ao antígeno Tl2E foram identificadas em 35,7 por cento da amostra de 842 indivíduos do município de Mambaí, Goiás. Suas especificidade e sensibilidade foram comparadas com aquelas dos exames sorológicos. Em 94 pacientes chagásicos comprovados pelo xenodiagnóstico, o teste cutâneo foi positivo em 98,7 por cento dos casos, a imunofluorescência em 97,8 por cento e a fixaçao do complemento em 80,6 por cento. A hemaglutinaçao foi positiva em todos esses casos. O índice de 0,897 mostrou a estreita relaçao entre os percentuais positivos dos exames de hemaglutinaçao e de ímunofluorescência com o teste cutâneo, nos chagásicos sem comprovaçao parasitológica. Esse dado indica que em aproximadamente 90 por cento dos casos os resultados desses três exames sao concordantes. A quantidade de 50mug do antígeno Tl2E empregada no teste cutâneo nao apresentou efeitos colaterais e nao produziu conversao das provas imunológicas, mesmo quando foi repetido cinco vezes em voluntários sadios, em intervalos de 15 dias. A potência do antígeno permaneceu inalterada após a estocagem a -1O graus Celsius, durante 24 meses.

Hipersensibilidade tardia a antígeno de Trypanosoma cruzi: III- sensibilidade do teste cutâneo com antígeno T12E no diagnóstico de doença de Chagas em pacientes hospitalizados / Delayed hypersensitivity to trypanosoma cruzi antigen: III- Sensitivity of the skin test with T12E antigen in the diagnosis of Chagas disease in hospitalized patients

Foi feita avaliaçao de três métodos imunológicos para diagnóstico de doença de Chagas, em l20 pacientes hospitalizados. O teste cutâneo e a imunofluorescência foram positivos em lO por cento dos casos. A hemaglutinaçao foi positiva em 14,l por cento dos pacientes. A co-positividade do teste cutâneo com a hemaglutinaçao e dessa com a imunofluorescência foi de 7,5 por cento. Apenas 5 por cento dos pacientes estudados tinham os três exames concordantes positivos. Todavia, 19,1 por cento dos pacientes tinham pelo menos um dos três exames positivos. Neste estudo a especificidade do teste cutâneo foi semelhante a da imunofluorescência. A sensibilidade desses testes, entretanto, foi menor que a da hemaglutinaçao indireta. Estes dados mostram que o teste cutâneo com o antígeno Tl2E faz o diagnóstico da doença de Chagas por uma simples reaçao de hipersensibilidade cutânea tardia de fácil execuçao.

Autoimmunity in Chagas' disease

This paper reports on research that has been carried out over the last 25 years, aiming at the understanding of basic mechanisms associated with Chagas'disease lesions. Early papers that showed evidence for a role of autoimmunity in the pathogenesis of the disease have been followed by an increasing number of publications in this field, but several intricate immunologic features remains to be understood. In fact, the multidisciplinary research work that has been done in several laboratories sheds light on some molecular features of very intricate immunological mechanisms in this disease. However, the Typanosoma cruzi-induced, clinically recognized immunologic manifestations still require further research in the fields of parasitology, biochemistry, molecular biology and immunology in order to unravel their pathogenetic mechanisms.

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