Coinfection with Different Trypanosoma cruzi Strains Interferes with the Host Immune Response to Infection

A century after the discovery of Trypanosoma cruzi in a child living in Lassance, Minas Gerais, Brazil in 1909, many uncertainties remain with respect to factors determining the pathogenesis of Chagas disease (CD). Herein, we simultaneously investigate the contribution of both host and parasite factors during acute phase of infection in BALB/c mice infected with the JG and/or CL Brener T. cruzi strains. JG single infected mice presented reduced parasitemia and heart parasitism, no mortality, levels of pro-inflammatory mediators (TNF-α, CCL2, IL-6 and IFN-γ) similar to those found among naïve animals and no clinical manifestations of disease. On the other hand, CL Brener single infected mice presented higher parasitemia and heart parasitism, as well as an increased systemic release of pro-inflammatory mediators and higher mortality probably due to a toxic shock-like systemic inflammatory response. Interestingly, coinfection with JG and CL Brener strains resulted in intermediate parasitemia, heart parasitism and mortality. This was accompanied by an increase in the systemic release of IL-10 with a parallel increase in the number of MAC-3+ and CD4+ T spleen cells expressing IL-10. Therefore, the endogenous production of IL-10 elicited by coinfection seems to be crucial to counterregulate the potentially lethal effects triggered by systemic release of pro-inflammatory mediators induced by CL Brener single infection. In conclusion, our results suggest that the composition of the infecting parasite population plays a role in the host response to T. cruzi in determining the severity of the disease in experimentally infected BALB/c mice. The combination of JG and CL Brener was able to trigger both protective inflammatory immunity and regulatory immune mechanisms that attenuate damage caused by inflammation and disease severity in BALB/c mice

Análises populacionais em Trypanosoma cruzi baseadas em microssatélites polimórficos de DNA

Exportado OPUSMade available in DSpace on 2019-08-14T02:02:02Z (GMT). No. of bitstreams: 1 tese_helder_magno_silva_valadares.pdf: 3940062 bytes, checksum: 96eda46b0a184a502eb37cdf0eef3410 (MD5) Previous issue date: 14A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, apresenta diferentes manifestações clínicas, resultantes de uma interação complexa entre fatores genéticos e ambientais, tanto do hospedeiro quanto do parasito. Com relação aos fatores genéticos do parasito, uma variedade de estudos biológicos e moleculares revelou uma substancial variabilidade genética entre as cepas de T. cruzi. A análise de perfis de microssatélites é uma técnica poderosa para acessar a variabilidade genética em eucariotos e pode ser utilizada para distinguir os indivíduos ou populações de diferentes espécies. Estudos anteriores, usando oito loci de microssatélites compostos de dinucleotídeos, trouxeram informações valiosas sobre a estrutura das populações de T. cruzi. Entretanto, devido à metodologia empregada no isolamento desses loci, o tamanho das seqüências flanqueadoras das repetições é muito reduzido, o que dificulta o desenho de novos iniciadores que permitam bons níveis de amplificação por PCR. Além disso, loci de dinucleotídeos, em geral, são mais susceptíveis ao fenômeno da derrapagem da DNA polimerase, o que certamente dificulta as análises. No presente trabalho, nós descrevemos novos loci de microssatélites adequados para a caracterização de T. cruzi em amostras biológicas que usualmente apresentam quantidades limitantes do DNA do parasito. Para isto, e aproveitando os dados obtidos do projeto genoma de T. cruzi, nós avaliamos a abundância, composição e distribuição de microssatélites perfeitos constituídos por motivos de di, tri e tetranucleotídeos no genoma de T. cruzi. Em geral, os loci de microssatélites foram cerca de 12 vezes menos freqüentes em regiões codificantes quando comparados com as regiões não codificantes do genoma de T. cruzi, onde as repetições de dinucleotídeos foram mais abundantes (47,6%). Entretanto, as repetições de trinucleotídeos foram os microssatélites mais comuns dentro das regiões codificantes. Usando o programa Tandem Repeats Finder, foi possível identificar e caracterizar inicialmente sete novos loci de microssatélites seis compostos por repetições de trinucleotídeos(TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 e TcCAA10) e um composto por repetições de tetranucleotídeos (TcAAAT6). Todos eles, exceto o locus TcCAA10, foram fisicamente mapeados em distintas regiões intergênicas do cromossomo III de CL Brener. Constratando, o locus TcCAA10 foi localizado dentro de uma região codificante para uma proteína hipotética no genoma de T. cruzi. Todos estes loci de microssatélites foram polimórficos e, portanto, úteis para estudos de variabilidade genética de T. cruzi. Resultados notáveis foram obtidos com as análises dos tamanhos dos alelos amplificados para o locus TcTAC15. Com a utilização deste locus foi possível separar as cepas pertencentes à linhagem T. cruzi I daquelas pertencentes às linhagens T. cruzi II, T. cruzi III e híbridos. Para estabelecer um ensaio de PCR mais sensível, nós desenhamos pares extras de iniciadores para cada locus, de forma a permitir o uso de protocolos de full nested PCR. Demonstramos que, utilizando esse tipo de estratégia, foi possível detectar e diferenciar cepas presentes em tecidos humanos e de camundongos infectados. Além disso, foi possível também amplificar DNA do parasito diretamente em células únicas separadas através do FACS Cell Sorter. Para realizar isto, o procedimento para a separação de células únicas pelo FACS Cell Sorter foi otimizado e uma PCR multiplex foi desenvolvida. Até o momento, duas populações artificialmente(JG+Col1.7G2 e Silvio X10 cl1+Esmeraldo cl3) e duas naturalmente (Berenice-78 25B e A316A R7) mistas foram analisadas após a separação em células únicas. Em todos os casos, nós pudemos separar e identificar as subpopulações presentes nas populações policlonais. O isolado Be-78 25B mostrou-se constituído por no mínimodois clones: um similar à cepa Berenice-62 e pertencente à linhagem T. cruzi II (DTU IIb) e o outro pertencente ao grupo das cepas híbridas (DTU IId). O isolado A316A R7 foi também constituído por dois clones: um pertencente à linhagem T. cruzi I (DTU I) e o outro pertencente à linhagem T. cruzi II (DTU IIb). Finalmente, no presente trabalho, nós analisamos também casos de transmissão congênita da doença de Chagas de três regiões endêmicas distintas (Minas Gerais e Rio Grande do Sul, Brasil, e Buenos Aires, Argentina). As análises dos perfis de microssatélites mostraram uma perfeita identidade genotípica entre os pares mães-filhos analisados, corroborando com o diagnóstico de infecção congênita para estes casos. As análises de microssatélites associadas a outros marcadores evolutivamente mais conservados em T. cruzi (rDNA 24S, COII e mini-exon) mostraram que no estado de Minas Gerais, os parasitos isolados de todos os casos congênitos pertencem à linhagem T. cruzi II (DTU IIb), enquanto que os parasitos isolados dos casos congênitos do estado do Rio Grande do Sul e Argentina pertencem ao grupo das cepas híbridas (DTU IId). É interessante que a prevalência de casos congênitos da doença de Chagas na parte sul do Brasil e Argentina parece ser muito mais elevada do que em Minas Gerais. Então, nós pressupomos que a alta freqüência de casos congênitos da doença de Chagas numa área endêmica está associada com uma maior predominância de populações híbridas de T. cruzi (especialmente do DTU IId) circulantes nesta região.Chagas disease caused by the protozoan Trypanosoma cruzi exhibits distinct clinical manifestations resulting from complex interactions among environmental and genetic factors from both host and parasite. Concerning the parasite genetic factors, a broad range of biological and molecular studies has revealed substantial genetic variability among T. cruzi strains. Microsatellite profiles analysis is a powerful technique to access genetic variability in eukaryotes, and can be used to distinguish individuals or populations of different species. Previous studies usingeight dinucleotide microsatellite loci have provided valuable information about the structure of T. cruzi populations. However, because of the methodology used to isolate these loci, the sequences flanking of the repeats are very short, which makes the design of adequate primers to achieve good amplifications by PCR a difficult task. In addition, dinucleotide loci are especially susceptible to DNA polymerase slippages, which complicate the amplicon analyses. In the present work, we describe new microsatellite loci suitable to T. cruzi characterizationdirectly in biological samples that usually present very little parasite DNA. For that, and taking advantages from the T. cruzi genome sequencing project, we evaluate the abundance, composition and distribution of perfect microsatellites constituted of di, tri and tetranucleotide motifs in the T. cruzi genome. In general, the microsatellite loci were 12 times less frequently in coding regions compared to noncoding regions of the T. cruzi genome, where the dinucleotide repeats were the most abundant (47,6%). However, the trinucleotide repeats were the most common microsatellite within the coding regions. Using the software TandemRepeats Finder it was possible to identify and characterize seven new microsatellite loci six composed by trinucleotide (TcTAC15, TcTAT20, TcAAT8, TcATT14, TcGAG10 and TcCAA10) and one composed by tetranucleotide motifs (TcAAAT6). All, except the TcCAA10 locus, were physically mapped on distinct intergenic regions of the chromosome III of the CL Brener. Contrasting, the TcCAA10 locus was localized within a hypothetical protein gene in T. cruzi genome. All microsatellites were polymorphic and useful for T. cruzi genetic variability studies. Remarkable results were obtained by analyzing the allele sizes of the amplicons for the TcTAC15 locus. By using this locus it was possible to separate the strains belonging to T. cruzi I lineage from those belonging to T. cruzi II, T. cruzi III and hybrid group. To establish a more sensitive PCR assay, we designed extra pairs of primers for each microsatellite locus and used them in Full Nested PCR protocols. By using this strategy we could detect and differentiate T. cruzi strains directly in human and animal infected tissues. Besides, it was also possible to amplify the parasite DNA directly in single cells separated by FACS Cell Sorter. To achieve that, the current procedure for single cells separation by FACS Cell Sorter was optimizedand a multiplex PCR assay was described. Up to now two artificially (JG+Col1.7G2 and SilvioX10 cl1+Esmeraldo cl3) and two naturally (Berenice-78 25B and A316A R7) mixed populations were analyzed after single cell sorting. In all cases we could separate and identify the subpopulations present in the polyclonal populations. The Be-78 25B isolate was composed by at least two clones: one similar to the Berenice-62 strain and belonging to T. cruzi II major lineage (DTU IIb) and another belonging to the hybrid group (DTU IId). The isolate A316A R7 was also constituted by two clones: one belonging to T. cruzi I (DTU I) and another belonging to T. cruzi II lineage (DTU IIb). Finally, in the present work, we analyzed cases of congenital Chagas disease from three distinct endemic regions (Minas Gerais and Rio Grande do Sul, Brazil, and Buenos Aires, Argentine). The microsatellite profiles showed a perfect genotypic identity between the mothers-offspring pairs analyzed, in agreement with the diagnose of congenital infection for these cases. The microsatellite analyses associated to other markers evolutionarily more conserved in T. cruzi (rDNA 24S, COII and mini-exon) showed that in the Minas Gerais state the isolated parasites of all congenital cases belong to T. cruzi II lineage (DTU IIb), whereas the isolated parasites from congenital cases from the Rio Grande do Sul state and Argentine belong to hybrid strains group (DTU IId). It is interesting that the prevalence of congenital cases of Chagas disease in south part of Brazil and Argentine seems to be much higher than in Minas Gerais. Thus, we hypothezed thatthe high frequency of congenital cases of Chagas disease in an endemic area is associated with the higher predominance of T. cruzi hybrid populations (especially from the DTU IId) circulating in this region

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis

Molecular and biological characterization of Trypanosoma cruzi strains isolated from children from Jequitinhonha Valley, State of Minas Gerais, Brazil

Introduction The biological diversity of Trypanosoma cruzi strains plays an important role in the clinical and epidemiological features of Chagas disease. Methods Eight T. cruzi strains isolated from children living in a Chagas disease vector-controlled area of Jequitinhonha Valley, State of Minas Gerais, Brazil, were genetically and biologically characterized. Results The characterizations demonstrated that all of the strains belonged to T. cruzi II, and showed high infectivity and a variable mean maximum peak of parasitemia. Six strains displayed low parasitemia, and two displayed moderate parasitemia. Later peaks of parasitemia and a predominance of intermediate and large trypomastigotes in all T. cruzi strains were observed. The mean pre-patent period was relatively short (4.2±0.25 to 13.7±3.08 days), whereas the patent period ranged from 3.3±1.08 to 34.5±3.52 days. Mortality was observed only in animals infected with strain 806 (62.5%). Histopathological analysis of the heart showed that strains 501 and 806 caused inflammation, but fibrosis was observed only in animals infected with strain 806. Conclusions The results indicate the presence of an association between the biological behavior in mice and the genetic characteristics of the parasites. The study also confirmed general data from Brazil where T. cruzi II lineage is the most prevalent in the domiciliary cycle and generally has low virulence, with some strains capable of inducing inflammatory processes and fibrosis

Biological, biochemical and molecular features of Trypanosoma cruzi strains isolated from patients infected through oral transmission during a 2005 outbreak in the state of Santa Catarina, Brazil: its correspondence with the new T. cruzi Taxonomy Consensus (2009).

Submitted by Ana Maria Fiscina Sampaio ([email protected]) on 2012-11-21T20:34:37Z No. of bitstreams: 1 Andrade SG Biological biochemical....pdf: 2010895 bytes, checksum: 1d866da26b9fc441d2257f34ddb9849b (MD5)Made available in DSpace on 2012-11-21T20:34:37Z (GMT). No. of bitstreams: 1 Andrade SG Biological biochemical....pdf: 2010895 bytes, checksum: 1d866da26b9fc441d2257f34ddb9849b (MD5) Previous issue date: 2011Fundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal de Santa Catarina. Departamento de Microbiologia e Parasitologia. Florianópolis, SC, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, BrasilUniversidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, BrasilFundação Oswaldo Cruz. Centro de Pesquisas Gonçalo Moniz. Salvador, BA, BrasilUniversidade Federal de Minas Gerais. Departamento de Bioquímica e Imunologia. Belo Horizonte, MG, BrasilWe examined strains of Trypanosoma cruzi isolated from patients with acute Chagas disease that had been acquired by oral transmission in the state of Santa Catarina, Brazil (2005) and two isolates that had been obtained from a marsupial (Didelphis aurita) and a vector (Triatoma tibiamaculata). These strains were characterised through their biological behaviour and isoenzymic profiles and genotyped according to the new Taxonomy Consensus (2009) based on the discrete typing unities, that is, T. cruzi genotypes I-VI. All strains exhibited the biological behaviour of biodeme type II. In six isolates, late peaks of parasitaemia, beyond the 20th day, suggested a double infection with biodemes II + III. Isoenzymes revealed Z2 or mixed Z1 and Z2 profiles. Genotyping was performed using three polymorphic genes (cytochrome oxidase II, spliced leader intergenic region and 24Sα rRNA) and the restriction fragment length polymorphism of the kDNA minicircles. Based on these markers, all but four isolates were characterised as T. cruzi II genotypes. Four mixed populations were identified: SC90, SC93 and SC97 (T. cruzi I + T. cruzi II) and SC95 (T. cruzi I + T. cruzi VI). Comparison of the results obtained by different methods was essential for the correct identification of the mixed populations and major lineages involved indicating that characterisation by different methods can provide new insights into the relationship between phenotypic and genotypic aspects of parasite behaviour

Identification of suitable reference genes for quantitative gene expression analysis in innervated and denervated adipose tissue from cafeteria diet‐fed rats

Our previous studies show that cafeteria diet increases body adiposity, plasma insulin levels, and sympathetic activity to brown adipose tissue (BAT) and white adipose tissue (WAT) of Wistar rats, leading to rapid and progressive changes in the metabolic profile. The identification of suitable reference genes that are not affected by the experimental conditions is a critical step in accurate normalization of the reverse transcription quantitative real‐time PCR (qRT‐PCR), a commonly used assay to elucidate changes in the gene expression profile. In the present study, the effects of the cafeteria diet and sympathetic innervation on the gene expression of adrenoceptor beta 3 (Adrb3) from BAT and WAT were assessed using one of the most stable and one of the least stable genes as normalizers. Rats were fed the cafeteria diet and on the 17th day, interscapular BAT or retroperitoneal WAT was denervated and, 7 days after surgery, the contralateral innervated tissue was used as control. Ten reference genes were evaluated (18S, B2m, Actb, CypA, Gapdh, Hprt1, Rpl32, Tbp, Ubc, and Ywhaz) and ranked according to their stability using the following algorithms: geNorm, NormFinder, BestKeeper, and comparative delta threshold cycle (ΔC t) method. According to the algorithms employed, the normalization of Adrb3 expression by the least stable genes produced opposite results compared with the most stable genes and literature data. In cafeteria and control diet‐fed rats, the three most stable genes were Hprt1, Tbp, and Rpl32 for interscapular BAT and Tbp, B2m, and Hprt1 for retroperitoneal WAT, while the least stable genes were 18S, Actb, and Gapdh for both tissues544231244CAPES - Coordenação de Aperfeiçoamento de Pessoal e Nível SuperiorCNPQ - Conselho Nacional de Desenvolvimento Científico e TecnológicoFAPESP – Fundação de Amparo à Pesquisa Do Estado De São PauloNão tem445764/2014‐7Não te

Trypanosoma cruzi : impact of dual-clone infections on parasite biological properties in BALB/c mice.

Herein, we have analyzed major biological properties following dual-clone Trypanosoma cruzi infections in BALB/c mice. Eight T. cruzi clonal stocks, two of each principal genotype, including genotype 19 and 20 (T. cruzi I), hybrid genotype 39 (T. cruzi) and 32 (T. cruzi II) were combined into 24 diVerent dual-clone infections. Special attention was given to characterize biological parameters assayed including: prepatent period, patent period, maximum of parasitemia, day of maximum parasitemia, area under the parasitemia curve, infectivity, mortality, and hemoculture positivity. Our Wndings clearly demonstrated that features resultant of dual-clone infections of T. cruzi clonal stocks did not display either the characteristics of the corresponding monoclonal infections or the theoretical mixture based on the respective monoclonal infections. SigniWcant changes in the expected values were observed in 4.2–79.2% of the mixtures considering the eight biological parameters studied. A lower frequency of signiWcant diVerences was found for mixtures composed by phylogenetically distant clonal stocks. Altogether, our data support our hypothesis that mixed T. cruzi infections have a great impact on the biological properties of the parasite in the host and re-emphasizes the importance of considering the possible occurrence of natural mixed infections in humans and their consequences on the biological aspects of ongoing Chagas’ disease