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BACKGROUND: Nitric oxide (NO) produced in macrophages plays a pivotal role as a leishmanicidal agent. A previous study has demonstrated that 20% of the L. (V.) braziliensis isolated from initial cutaneous lesions of patients from the endemic area of Corte de Pedra, Bahia, Brazil, were NO resistant. Additionally, 5 to 11% of the patients did not respond to three or more antimony treatments" (refractory patients). The aim of this study is to investigate if there is an association between the resistance of L. (V.) braziliensis to NO and nonresponsiveness to antimony therapy and cytokine production. METHODS: We evaluated the in vitro toxicity of NO against the promastigotes stages of L. (V.) braziliensis isolated from responsive and refractory patients, and the infectivity of the amastigote forms of these isolates against human macrophages. The supernatants from Leishmania infected macrophage were used to measure TNF-α and IL-10 levels. RESULTS: Using NaNO2 (pH 5.0) as the NO source, L. (V.) braziliensis isolated from refractory patients were more NO resistant (IC50 = 5.8 ± 4.8) than L. (V.) braziliensis isolated from responsive patients (IC50 = 2.0 ± 1.4). Four isolates were selected to infect human macrophages: NO-susceptible and NO-resistant L. (V.) braziliensis isolated from responsive and refractory patients. NO-resistant L. (V.) braziliensis isolated from refractory patients infected more macrophages stimulated with LPS and IFN-γ at 120 hours than NO-susceptible L. (V.) braziliensis isolated from refractory patients. Also, lower levels of TNF-α were detected in supernatants of macrophages infected with NO-resistant L. (V.) braziliensis as compared to macrophages infected with NO-susceptible L. (V.) braziliensis (p < 0.05 at 2, 24 and 120 hours), while no differences were detected in IL-10 levels. CONCLUSION: These data suggest that NO resistance could be related to the nonresponsiveness to antimony therapy seen in American Tegumentary Leishmaniasis.

BACKGROUND: L. braziliensis causes cutaneous (CL) and mucosal (ML) leishmaniasis. Wound healing neutrophil (PMN) and macrophage responses made following the bite of the vector sand fly contribute to disease progression in mice. To look at the interplay between PMN and macrophages in disease progression in humans we asked whether polymorphisms at genes that regulate their infiltration or function are associated with different clinical phenotypes. Specifically, CXCR1 (IL8RA) and CXCR2 (IL8RB) are receptors for chemokines that attract PMN to inflammatory sites. They lie 30-260 kb upstream of SLC11A1, a gene known primarily for its role in regulating macrophage activation, resistance to leishmaniasis, and wound healing responses in mice, but also known to be expressed in PMN, macrophages and dendritic cells. METHODS Polymorphic variants at CXCR1, CXCR2 and SLC11A1 were analysed using Taqman or ABI fragment separation technologies in cases (60 CL; 60 ML), unrelated controls (n = 120), and multicase families (104 nuclear families; 88 ML, 250 CL cases) from Brazil. Logistic regression analysis, family-based association testing (FBAT) and haplotype analysis (TRANSMIT) were performed. RESULTS: Case-control analysis showed association between the common C allele (OR 2.38; 95% CI 1.23-4.57; P = 0.009) of CXCR1_rs2854386 and CL, supported by family-based (FBAT; Z score 2.002; P = 0.045) analysis (104 nuclear families; 88 ML, 250 CL cases). ML associated with the rarer G allele (Z score 1.999; P = 0.046). CL associated with a 3' insertion/deletion polymorphism at SLC11A1 (Z score 2.549; P = 0.011). CONCLUSIONS: The study supports roles for CXCR1 and SLC11A1 in the outcome of L. braziliensis infection in humans. Slc11a1 does not influence cutaneous lesion development following needle injection of Leishmania in mice, suggesting that its role here might relate to the action of PMN, macrophage and/or dendritic cells in the wound healing response to the sand fly bite. Together with the CXCR1 association, the data are consistent with hypotheses relating to the possible role of PMN in initiation of a lesion following the delivery of parasites via the sand fly bite. Association of ML with the rare derived G allele suggests that PMN also have an important positive role to play in preventing this form of the disease.

BACKGROUND: Disseminated leishmaniasis is an emerging infectious disease, mostly due to L. braziliensis, which has clinical and histopathological features distinct from cutaneous leishmaniasis. METHODS: In the current study we evaluated the in vitro production of the cytokines IFN-γ, TNF-α, IL-5 and IL-10 by peripheral blood mononuclear cells (PBMC) from 15 disseminated leishmaniasis and 24 cutaneous leishmaniasis patients upon stimulation with L. braziliensis antigens genotyped as disseminated leishmaniasis or cutaneous leishmaniasis isolates. RESULTS: Regardless of the source of L. braziliensis antigens, PBMC from cutaneous leishmaniasis patients produced significantly higher IFN-γ than PBMC from disseminated leishmaniasis patients. Levels of TNF-α by PBMC from cutaneous leishmaniasis patients were significantly higher than disseminated leishmaniasis patients only when stimulated by genotyped cutaneous leishmaniasis antigens. The levels of IL-5 and IL-10 production by PBMC were very low and similar in PBMCs from both disseminated leishmaniasis and cutaneous leishmaniasis patients. The immune response of each patient evaluated by the two L. braziliensis antigens was assessed in a paired analysis in which we showed that L. braziliensis genotyped as disseminated leishmaniasis isolate was more potent than L. braziliensis genotyped as cutaneous leishmaniasis isolate in triggering IFN-γ and TNF-α production in both diseases and IL-5 only in cutaneous leishmaniasis patients. CONCLUSION: This study provides evidence that antigens prepared from genotypically distinct strains of L. braziliensis induce different degrees of immune response. It also indicates that both parasite and host play a role in the outcome of L. braziliensis infection.

p. 508–514 Submitted by Ana Valéria de Jesus Moura (anavaleria_131@hotmail.com) on 2011-12-11T17:03:46Z No. of bitstreams: 1 1077.pdf: 1159272 bytes, checksum: 42eaf0f40906bf9b35739de038ee64f2 (MD5) Made available in DSpace on 2011-12-11T17:03:46Z (GMT). No. of bitstreams: 1 1077.pdf: 1159272 bytes, checksum: 42eaf0f40906bf9b35739de038ee64f2 (MD5) Previous issue date: 2004-01 In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP’s geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.

Submitted by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2019-11-29T14:36:13Z No. of bitstreams: 1 Dissert_ICS Camila Sampaio Ribeiro.pdf: 3022458 bytes, checksum: 951dd5ed9a1594bc1b24fbbf9292fad2 (MD5) Made available in DSpace on 2019-11-29T14:36:13Z (GMT). No. of bitstreams: 1 Dissert_ICS Camila Sampaio Ribeiro.pdf: 3022458 bytes, checksum: 951dd5ed9a1594bc1b24fbbf9292fad2 (MD5) Fundação do Amparo à Pesquisa de São Paulo (FAPESP) e Sociedade Brasileira de Dermatologia – Funaderm Objetivos: Avaliar a eficácia da terapia combinada de antimônio pentavalente (Sbv) com tamoxifeno, oral (TO) ou tópico (TT), em comparação com Sbv em monoterapia para o tratamento da Leishmaniose cutânea (LC). Determinar a expressão de CD8, IL17 e outros marcadores imunes na lesão por meio da imuno-histoquímica (IHQ) e correlacionar com desfecho terapêutico. Métodos: Ensaio clínico de fase II randomizado com três grupos. Estudo realizado no posto de saúde de Corte de PedraBahia. Adultos com LC apresentando 01-05 úlceras variando entre 01-05 cm foram incluídos. Foi realizada uma biópsia pré-tratamento para estudo in situ. Resultados: 38 pacientes foram alocados em três grupos: Sbv (n = 15); Sbv + TT (n = 11) e Sbv + TO (n = 12). Na análise por intenção de tratar (D90), a taxa de cura foi de de 53% (8/15), 45% (5/11) e 67% (8/12), respectivamente (p = 0,58). Na análise por protocolo, a taxa de cura foi de 57% (8/14), 50% (5/10), 80% (8/10), respectivamente (p = 0,35). Não houve diferença significativa na frequência de efeitos colaterais entre os grupos. A expressão in situ de CD8 ou de IL-17 entre pacientes que falharam e que curaram não apresentou diferença significativa. Conclusões: A terapia combinada de Sbv com Tamoxifeno é um tratamento seguro para LC e o grupo Sbv+TO apresentou maior taxa de cura (p=ns). A expressão tecidual de CD8 e IL-17 não se correlacionou com o desfecho terapêutico.

Submitted by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2019-12-02T14:28:58Z No. of bitstreams: 1 Tese_ICS_Carolina Cincurá Barreto.pdf: 2076665 bytes, checksum: eb1e24dd1cf491524b393d7418a8f0e2 (MD5) Made available in DSpace on 2019-12-02T14:28:58Z (GMT). No. of bitstreams: 1 Tese_ICS_Carolina Cincurá Barreto.pdf: 2076665 bytes, checksum: eb1e24dd1cf491524b393d7418a8f0e2 (MD5) Introdução: A leishmaniose mucosa (LM) é uma forma grave de apresentação da leishmaniose tegumentar americana (LTA) associada a uma resposta inflamatória persistente. Concentrações elevadas de TNF, IFN-γ, CXCL9 e CXCL10 são encontradas em pacientes com LM e a associação terapêutica da pentoxifilina ao antimonial tem se mostrado mais efetiva do que o uso isolado do antimonial, reduzindo o tempo de cura em pacientes com LM. O presente estudo avalia a existência de correlação entre a produção de citocinas e quimiocinas e a gravidade da LM, o efeito da associação da pentoxifilina na resposta imune e evolução clínica e se alguma dessas citocinas e quimiocinas podem funcionar como marcador de resposta terapêutica. Material e métodos: Estudo prospectivo avaliando 86 pacientes com LM provenientes de uma área endêmica para Leishmania braziliensis. Os pacientes foram classificados em estágios variando de I a V de acordo com a gravidade da doença mucosa. A produção de TNF, IFN-γ, CXCL-9 e CXCL-10 em resposta ao antígeno solúvel de Leishmania (SLA) foi determinada por ELISA em sobrenadantes de células mononucleares de sangue periférico (CMSP) antes e depois do tratamento com antimonial isolado, ou com a associação da pentoxifilina ao antimonial. Resultados: A mediana do TNF no grupo com doença leve (I-II) foi de 1064 pg/ml (142 - 3738 pg/ml), enquanto que no grupo com doença de moderada a grave (III-V) foi de 1941pg/ml (529 - 5294 pg/ml) (p= 0,008). Foi observada correlação direta entre a gravidade da LM e as concentrações de TNF (r=0,44; p=0,007). Pacientes tratados com a associação do antimonial com a pentoxifilina cicatrizaram mais rapidamente do que os pacientes que utilizaram o antimonial isolado (52 ± 27 dias versus 77 ± 48, risco relativo= 0,60; intervalo de confiança 95%= 0,37-0,96; p=0,013). Falha terapêutica foi maior no grupo que usou apenas o antimonial (25% versus 7%; p=0,041). Houve uma redução significativa das concentrações de CXCL9 após o tratamento para LM em ambos os grupos (p=0,013;p=0,043). A diferença de concentração do TNF antes e depois do tratamento foi preditora de cura (Curva ROC= área sob a curva de 0,65; IC 95%= 0,53-0,78; p=0,05). Conclusão: A produção de TNF esta associada a gravidade da doença mucosa e a associação da pentoxifilina ao antimonial reduz o tempo de cura e as taxas de falha terapêutica, devendo ser recomendada no tratamento de pacientes com LM.

Submitted by Flávia Ferreira (flaviaccf@yahoo.com.br) on 2019-05-22T13:40:02Z No. of bitstreams: 1 Tese ICS Juliana Almeida Silva.pdf: 2339661 bytes, checksum: 5a980b1047fefb87ed7b45a62abe05d5 (MD5) Made available in DSpace on 2019-05-22T13:40:02Z (GMT). No. of bitstreams: 1 Tese ICS Juliana Almeida Silva.pdf: 2339661 bytes, checksum: 5a980b1047fefb87ed7b45a62abe05d5 (MD5) Introdução A leishmaniose tegumentar Americana (LTA) pode resultar de infecções humanas com Leishmania (Viannia) braziliensis, a qual é responsável por três formas de doença: leishmaniose cutânea (LC), mucosa (LM) e disseminada (LD). Essas três formas da doença podem ser encontradas em Corte de Pedra (CP), Bahia/Brasil. A ocorrência desses diferentes desfechos da doença nesta área endêmica, sugere que a cepa parasitária exerça um importante papel sobre a determinação do prognóstico da infecção. Objetivos Buscamos avaliar a dinâmica na constituição genotípica da população parasitária de L. (V.) braziliensis ao longo do tempo, sua associação com os desfechos clínicos de LTA em Corte de Pedra-BA, e se a carga parasitária está relacionada com a gravidade da doença. Métodos DNA total foi obtido a partir da suspensão de L. (V.) braziliensis isoladas de sujeitos com LC, LM ou LD pertencentes a duas amostras temporalmente distintas: Amostra A, 35 sujeitos recrutados entre 1992 e 2001; Amostra B, 126 sujeitos recrutados entre 2008 e 2011. Dois loci polimórficos (CHR24/3074 e CHR28/425451) dessas L. (V.) braziliensis foram amplificados por PCR, clonados, sequenciados e seus genes comparados entre os parasitas das duas amostras, para a caracterização de seus alelos. Esses loci foram explorados para avaliar a associação entre genótipos do parasita e as formas clínicas da doença. Um total de 194 amostras de DNA do parasita obtidos a partir de biópsias de lesões dos pacientes com LTA foram quantificadas por qPCR e utilizadas para avaliar a associação entre a carga parasitária e as manifestações clínicas da LTA. Resultados Identificamos no CHR24/3074 11 eventos de polimorfismos (SNP’s e Indel’s) e um total de 15 haplótipos de acordo com as nossas definições, sendo que 4 apresentaram-se mais frequentes. Já o locus CHR28/425451 apresentou 3 eventos polimórficos com um total de 7 haplótipos, dos quais 3 mantiveram-se consistentes entre as amostras A e B. Quatro haplótipos foram detectados exclusivamente na amostra mais recente, B. A avaliação da carga parasitária revelou que a alta carga pode estar associada à resposta terapêutica dos pacientes. Conclusão As avaliações entre genótipos parasitários e os fenótipos da LTA confirmaram estudos anteriores, mostrando associação entre os genótipos e desfechos clínicos da doença. Além disso, pudemos constatar como a qPCR pode ser eficaz para quantificação da carga parasitária, e esta pode exercer um papel importante na resposta terapêutica desses pacientes. Introduction American tegumentary leishmaniasis (ATL) can result from human infections with Leishmania (Viannia) braziliensis, which is responsible for three forms of disease: cutaneous leishmaniasis (CL), mucosa (ML) and disseminated (DL). These three forms of the disease can be found in Corte de Pedra (CP), Bahia /Brazil. The occurrence of these different disease outcomes in this endemic area suggests that the parasitic strain plays an important role in determining the prognosis of the infection. Objectives We sought to evaluate the dynamics in the genotypic constitution of the parasitic population of L. (V.) braziliensis over time, its association with the clinical outcomes of ATL in the of Corte de Pedra-BA, and whether the parasitic load is related to the severity of the disease. Methods Total DNA was obtained from the suspension of L. (V.) braziliensis isolated from subjects with LC, LM or LD belonging to two temporally distinct samples: Sample A, 35 subjects recruited between 1992 and 2001; Sample B, 126 subjects recruited between 2008 and 2011. Two polymorphic loci (CHR24/3074 and CHR28/425451) of these L. (V.) braziliensis were amplified by PCR, cloned, sequenced and their genes compared between the parasites of the two samples, to the characterization of their alleles. These loci were explored to evaluate the association between genotypes of the parasite and the clinical forms of the disease. A total of 194 parasite DNA samples obtained from lesions biopsies of patients with ATL were quantified by qPCR and used to evaluate the association between parasite load and clinical manifestations of ATL. Results We identified in the CHR24/3074 11 polymorphism events (SNP's and Indel's) and a total of 15 haplotypes according to our definitions, 4 of which were more frequent. The CHR28/425451 locus presented 3 polymorphic events with a total of 7 haplotypes, of which 3 remained consistent between samples A and B. Four haplotypes were detected exclusively in the most recent sample, B. The parasite load evaluation revealed that the high load may be associated with the therapeutic response of the patients. Conclusion Evaluations between parasite genotypes and ATL phenotypes have confirmed earlier studies showing association between genotypes and clinical outcomes of the disease. In addition, we have verified how qPCR can be effective for the quantification of the parasite load, and this can play an important role in the therapeutic response of these patients.