Tropismo tecidual dos genótipos principais de Trypanosoma cruzi em camundongos BALB/c com infecções mistas, não tratados e tratados na fase aguda da infecção, avaliados pela técnica de LSSP-PCR.

A correlação entre a genética do T. cruzi e as formas clínicas da doença de Chagas não está bem estabelecida. Apesar da relação parasito x hospedeiro ser complexa e multifatorial, uma das razões para essa falta de correlação pode ser a freqüente ocorrência de infecções mistas na natureza. Nesse sentido, o principal objetivo desse estudo foi avaliar, por meio da técnica de LSSP-PCR, o impacto de infecções mistas no tropismo tecidual de clones de T. cruzi pertencentes aos genótipos principais 19, 20, 39 e 32 na fase crônica da infecção. Para tal, foram avaliados seis tecidos (coração, músculo esquelético, cólon, baço, bexiga e cérebro) de quatro camundongos BALB/c fêmeas com infecção mista por clones de T. cruzi de cada genótipo principal. Os animais foram inoculados, via IP, com 5.000 tripomastigotas sanguíneos de cada clone, combinados aos pares, totalizando 24 misturas distintas. Esses camundongos foram divididos em dois grupos: não tratados (INT) e tratados (IT) com benzonidazol (BZ) e avaliados comparativamente às respectivas infecções monoclonais (10.000 tripomastigotas sanguíneos). A técnica de PCR detectou o kDNA dos oito clones de T. cruzi em todos os seis tecidos avaliados de animais INT. Após o tratamento houve redução na positividade da PCR especialmente nos animais infectados com clones dos genótipos 32 e 39 em comparação com aqueles infectados com os genótipos 19 e 20. Não foi observada uma associação entre a distância filogenética dos clones e o tropismo tecidual do kDNA do T. cruzi. Entretanto, clones dos genótipos 19 e 20 foram os únicos que apresentaram, durante a fase crônica, positividade na PCR no cérebro e maior positividade na bexiga. A comparação entre a distribuição tecidual do kDNA de clones do T. cruzi em camundongos com infecção mista e monoclonal revelou que na maioria das misturas estudadas, exceto Cuica cl1 + SO3 cl5 (20+39) houve alteração do tropismo tecidual do kDNA nos animais INT e IT. A maioria dos tecidos de animais com infecção mista (INT e IT) apresentou PCR negativa, ao contrário do observado nos animais com infecção monoclonal. Entretanto, a PCR foi positiva em animais infectados com 18/24 misturas. A identificação dos clones presentes nesses tecidos por LSSP-PCR revelou resultados esperados, considerando as infecções monoclonais, na maioria das misturas. Curiosamente, o perfil genético dos clones remanescentes nos tecidos de animais infectados com as misturas OPS21 cl11 + P209 cl1 e OPS21 cl11 + Bug2148 cl1 e tratados demonstrou a presença do clone OPS21 cl1, 100% susceptível ao BZ. Outro resultado interessante foi obtido na caracterização dos clones remanescentes nos tecidos de animais infectados com a mistura SO3 cl5 + IVV cl4 (39+32) tratados com BZ, que revelou um perfil indeterminado não compatível com o esperado para os clones originais utilizados na inoculação. A caracterização por LSSP-PCR e microssatélites dos clones presentes nos tecidos de animais infectados com as misturas que apresentaram alteração no perfil de susceptibilidade ao BZ revelou resultados semelhantes e esperados, considerando as infecções monoclonais, na maioria das misturas. Além disso, a técnica de LSSP-PCR revelou alteração no tropismo tecidual do kDNA dos clones nesses mesmos animais, sugestiva de interação entre os parasitos componentes da mistura. A técnica de LSSP-PCR apresentou maior sensibilidade quando comparada à de microssatélites, sendo capaz de detectar uma maior porcentagem de ambos os clones nos tecidos de animais INT. A identificação por LSSP-PCR dos clones remanescentes nos tecidos de animais considerados curados (TC) e que apresentaram PCR em tecido positiva demonstrou apenas a presença de um clone e os resultados foram semelhantes àqueles encontrados nos animais tratados não curados (TNC), levantando um importante questionamento a respeito do critério de cura na doença de Chagas. Somente clones dos genótipos 19 ou 20 estavam presentes nos tecidos de animais TC e infectados com as combinações genotípicas 19+32, 19+39 e 20+39, exceto na mistura Cuica cl1 + Bug2148 cl1 (20+39), que demonstrou a presença do clone pertencente ao genótipo 39. Os resultados obtidos nesse trabalho revelaram alteração no tropismo tecidual do kDNA do T. cruzi em infecções mistas sugestiva de interações entre os clones da mistura. Os dados em conjunto sugerem que a correlação entre a genética do parasito e as diferentes formas clínicas da doença é ainda mais difícil de ser estabelecida em infecções mistas em relação às infecções monoclonais.The correlation between the T. cruzi genetics and the clinical forms of Chagas’ disease is not well established. Despite the host x parasite relationship be complex and multifactorial one reason for this may be the frequent occurrence in nature of mixed infections. The aim of this study was to evaluate by LSSP-PCR technique, the impact of mixed infections in tissue tropism of T. cruzi clonal stocks from major genotypes 19, 20, 39 and 32 in the chronic phase of infection. For this, were evaluated six tissues (heart, skeletal muscle, colon, spleen, bladder and brain) of four female BALB/c mice with mixed infection by IP route, with T. cruzi clonal stocks from each genotype (5.000 blood trypomastigotes of each) combined in pairs, in a total of 24 different mixtures. Mice were divided in two groups: not treated (INT) and treated (IT) with benznidazole (BZ) and compared to the respective monoclonal infection (10.000 blood trypomastigotes of each clone). The PCR technique detected kDNA of eight clones of T. cruzi in all six tissues of INT animals. After treatment it was observed reduction of the PCR positivity, particularly in animals infected with clones of genotypes 32 and 39 when compared with those infected with genotypes 19 and 20. No association was observed between the parasites phylogenetic distance and the tissue tropism of T. cruzi kDNA. However, clones of genotypes 19 and 20 were the only who showed during the chronic phase, PCR positivity in the brain and higher positivity in bladder. The comparison between the tissue distribution of T. cruzi kDNA of each clone in mice with monoclonal and mixed infection revealed that most of the mixtures studied, except Cuica cl1 + SO3 cl5 (20 +39) changed the kDNA tissue tropism, both in IT and INT animals. Most tissues of animals with mixed infection (INT and IT) presented negative PCR, in contrast to that verified in animals with monoclonal infection. However, the PCR was positive in animals infected with 18/24 mixtures. The identification of each clone present in these tissues by LSSP-PCR revealed the expected results considering the monoclonal infections in the majority of the mixtures. Interestingly, the genetic profile of the remaining clonal stock in tissues of animals infected with treated mixtures OPS21 cl11 + P209 cl1 and OPS21 cl11 + Bug2148 cl1 demonstrated the presence of the clone OPS21 cl1, 100% susceptible to BZ. Another interesting result was obtained in the characterization of the remaining clones in tissues of animals infected with the mixture SO3cl5 + IVV cl4 (39 +32) treated with BZ, which showed an indeterminate profile not compatible with the expected for the original clones used for inoculation. The characterization by LSSP-PCR and microsatellite of the clones present in tissues of animals infected with mixtures that showed change in the BZ susceptibility profile showed expected and similar results considering the monoclonal infections, in most of the mixtures. Moreover, the technique of LSSP-PCR revealed changes in tissue tropism of kDNA of each clone in these animals, suggestive of interaction phenomena between the parasites present in the mixture. The technique of LSSP-PCR showed higher sensitivity when compared to microsatellites being able to detect a higher percentage of both clones in tissues of INT animals. The LSSP-PCR for identification of clones remaining in tissues from animals considered cured (TC), which showed positive tissue-PCR revealed only the presence of one clone and the results were similar to those obtained in treated not cured (TNC) animals, raising an important question regarding the cure criteria in Chagas’ disease. Only clones of genotypes 19 and 20 were present in tissues of TC animals infected with and the genotype combinations 19+32, 19+39 and 20+39, except the mixture Cuica cl1 + Bug2148 cl1 (20 +39), which presented the presence of the clone belonging to genotype 39. The results obtained in this study showed changes in tissue tropism of T. cruzi kDNA in mixed infections, suggestive of interactions between the clones of the mixture. Data together suggest that the genetic correlation between the parasite genetic and the different clinical forms of the disease is much more difficult to be established in mixed infections compared to monoclonal infections

Induced Pluripotent Stem Cell for the Study and Treatment of Sickle Cell Anemia

Sickle cell anemia (SCA) is a monogenic disease of high mortality, affecting millions of people worldwide. There is no broad, effective, and safe definitive treatment for SCA, so the palliative treatments are the most used. The establishment of an in vitro model allows better understanding of how the disease occurs, besides allowing the development of more effective tests and treatments. In this context, iPSC technology is a powerful tool for basic research and disease modeling, and a promise for finding and screening more effective and safe drugs, besides the possibility of use in regenerative medicine. This work obtained a model for study and treatment of SCA using iPSC. Then, episomal vectors were used for reprogramming peripheral blood mononuclear cells to obtain integration-free iPSC. Cells were collected from patients treated with hydroxyurea and without treatment. The iPSCP Bscd lines were characterized for pluripotent and differentiation potential. The iPSC lines were differentiated into HSC, so that we obtained a dynamic and efficient protocol of CD34+CD45+ cells production. We offer a valuable tool for a better understanding of how SCA occurs, in addition to making possible the development of more effective drugs and treatments and providing better understanding of widely used treatments, such as hydroxyurea

Persistence of PCR-positive tissue in benznidazole-treated mice with negative blood parasitological and serological tests in dual infections with Trypanosoma cruzi stocks from different genotypes.

Objectives: To assess different methodologies to better define an early post-therapeutic cure criterion after benznidazole treatment in BALB/c mice following mixed infection with dual Trypanosoma cruzi genotypes. Methods: According to the classical cure criteria, animals were classified as treated not cured (TNC 5 76.4%), treated cured (TC 5 12.5%) and dissociated (DIS 5 11.1%) using parasitological [fresh blood examination (FBE), blood culture (BC) and blood PCR] and serological methods [conventional serology (CS-ELISA) and non-conventional serology (NCS-FC-ALTA)]. Tissues were also evaluated by PCR. Results: FBE was able to detect patent parasitaemia in only 18.1% of TNC and therapeutic failure was detected in 79.1% and 97.2% of TNC by BC and blood PCR, respectively. CS-ELISA should not be used before 3 months after treatment since it may lead to false-negative results. At 3 months after treatment with benznidazole, NCS-FC-ALTA was more efficient for categorizing the groups of treated mice. In the TNC group, although a decreased frequency of PCR-positive tissue was observed in several host tissues, increased positivity was also observed, despite the T. cruzi genotype combination. All TC animals presented at least two positive tissue-PCR results. Conclusions: Our results confirm that NSC-FC-ALTA and blood PCR are the most suitable methods to early detect therapeutic failure in acute murine T. cruzi infection. Additionally, our data show that BC positivity is highly dependent upon the T. cruzi genotype combination. Moreover, our findings demonstrated that PCR tests performed on tissues from animals considered cured after benznidazole treatment still detected T. cruzi DNA, most probably indicating residual infection