HIRO GOTO
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Medicina Preventiva, Faculdade de Medicina - Docente
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina - Líder
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina - Líder
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- Validation of ELISA with recombinant antigens in serological diagnosis of canine Leishmania infantum infection(2021) FUJIMORI, Mahyumi; ALMEIDA, Arleana do Bom Parto Ferreira de; BARROUIN-MELO, Stella Maria; CORTEZ, Luiz Ricardo Paes de Barros; DUTHIE, Malcolm Scott; HIRAMOTO, Roberto Mitsuyoshi; PINHO, Flaviane Alves de; REED, Steven Gregory; SOUSA, Valeria Regia Franco; SOUZA, Nazare Fonseca; SOARES, Rodrigo Martins; TOLEZANO, Jose Eduardo; SANCHEZ, Maria Carmen Arroyo; GOTO, HiroBACKGROUND Dogs are the main peridomiciliary reservoir of Leishmania infantum thus the correct diagnosis of infection is essential for the control of the transmission and treatment as well. However, the diagnosis is based on serological assays that are not fully effective. OBJECTIVE We aimed to establish an effective serological assay for the diagnosis of L. infantum infected dogs using Leishmania-derived recombinant antigens. METHODS Leishmania derived rK39-, rK28-, rKR95-based enzyme-linked immunosorbent assay (ELISA) was standardized using symptomatic and asymptomatic L. infantum-infected dogs. Then 2,530 samples from inquiry in endemic areas for VL were evaluated and the results compared with recommended assays by the Brazilian Ministry of Health (MH algorithm). Further samples from a cohort of 30 dogs were searched. FINDINGS For rK39-, rK28- and rKR95-ELISA the sensitivity was around 97% and specificity 100%. The positivity of these three ELISA in the inquiry samples was 27-28%, around 10% higher than the assays currently in use. When cohort samples were searched, we observed likely false-negative results (> 65%) with supposedly negative samples that turned positive six months later with the assays in use (MH algorithm). MAIN CONCLUSIONS For the diagnosis of L. infantum-infected dogs, rK39-based ELISA showed better diagnostic performance than other assays in use in Brazil and worldwide.
- Staphylococcus aureus Protection-Related Type 3 Cell-Mediated Immune Response Elicited by Recombinant Proteins and GM-CSF DNA Vaccine(2021) SANTOS, Kamila R.; SOUZA, Fernando N.; RAMOS-SANCHEZ, Eduardo M.; BATISTA, Camila F.; REIS, Luiza C.; FOTORAN, Wesley F.; HEINEMANN, Marcos B.; GOTO, Hiro; GIDLUND, Magnus; CUNHA, Adriano F.; FARIA, Angelica Rosa; ANDRADE, Helida M.; LAGE, Andrey P.; CERQUEIRA, Monica M. O. P.; LIBERA, Alice M. M. P. DellaStaphylococcus aureus mastitis remains a major challenge for dairy farming. Here, 24 mice were immunized and divided into four groups: G1: control; G2: Granulocyte Macrophage Colony-Stimulating Factor (GM-CSF) DNA vaccine; G3: F0F1 ATP synthase subunit alpha (SAS), succinyldiaminopimelate (SDD), and cysteinyl-tRNA synthetase (CTS) recombinant proteins; and G4: SAS+SDD+CTS plus GM-CSF DNA vaccine. The lymphocyte subpopulations, and the intracellular interleukin-17A (IL-17A) and interferon-gamma production in the draining lymph node cells were immunophenotyped by flow cytometry. The immunophenotyping and lymphocyte proliferation was determined in spleen cells cultured with and without S. aureus stimulus. Immunization with S. aureus recombinant proteins generated memory cells in draining lymph nodes. Immunization with the three recombinant proteins plus GM-CSF DNA led to an increase in the percentage of IL-17A(+) cells among overall CD44(+) (memory), T CD4(+), CD4(+) T CD44(+) CD27(-), gamma delta TCR, gamma delta TCR+ CD44(+) CD27(+), and TCRV gamma 4(+) cells. Vaccination with S. aureus recombinant proteins associated with GM-CSF DNA vaccine downregulated T(H)2 immunity. Immunization with the three recombinant proteins plus the GM-CSF DNA led to a proliferation of overall memory T, CD4(+), and CD4(+) TEM cells upon S. aureus stimulus. This approach fostered type 3 immunity, suggesting the development of a protective immune response against S. aureus.
- Spatial distribution of canine Leishmania infantum infection in a municipality with endemic human leishmaniasis in Eastern Bahia, Brazil(2021) VARJAO, Bruno Milen; PINHO, Flaviane Alves de; SOLCA, Manuela da Silva; SILVESTRE, Ricardo; FUJIMORI, Mahyumi; GOTO, Hiro; VARJAO, Natasha Milen; DIAS, Roberta Costa; BARROUIN-MELO, Stella MariaEfforts to control a zoonotic disease such as visceral leishmaniasis (VL) caused by Leishmania infantum can be successful if they rely on comprehensive data on animal infection. In Bahia state, Brazil, human VL is endemic, yet some areas have no epidemiological data on canine L. infantum infection and canine leishmaniasis (CanL) to date. We aimed to perform an epidemiological study describing the spatial distribution and characterizing canine L. infantum infection in two districts of the municipality of Muritiba, where human cases have occurred. Brazilian official serodiagnostic protocol (ELISA and immunochromatographic tests), PCR and clinical examination were performed in 351 owned dogs. A seroprevalence of 15.7% (55/351) was found, and L. infantum identified in 88.8% (32/36) of PCR tested samples. Spatial distribution of positive dogs indicated infection in both urban and rural districts. There was no association between seropositivity and sex or breed, but dogs older than 2 years were 3.8 times more likely to be seropositive (95% CI 1.57 - 9.18) than younger dogs. Among seropositive dogs, 80% (44/55) had clinical manifestations of CanL: 75% (33/44) presented dermatopathy, 50% (22/44) emaciation, and 29.5% (13/44) ophthalmopathy. This is the first report on canine seroprevalence and natural L. infantum infection in Muritiba, Bahia.
- Pleiotropic Effect of Hormone Insulin-Like Growth Factor-I in Immune Response and Pathogenesis in Leishmaniases(2021) REIS, Luiza C.; RAMOS-SANCHEZ, Eduardo Milton; ARAUJO, Fernanda N.; LEAL, Ariane F.; OZAKI, Christiane Y.; SEVILLANO, Orlando R.; USCATA, Bernardina A.; GOTO, HiroLeishmaniases are diseases caused by several Leishmania species, and many factors contribute to the development of the infection. Because the adaptive immune response does not fully explain the outcome of Leishmania infection and considering that the initial events are crucial in the establishment of the infection, we investigated one of the growth factors, the insulin-like growth factor-I (IGF-I), found in circulation and produced by different cells including macrophages and present in the skin where the parasite is inoculated. Here, we review the role of IGF-I in leishmaniasis experimental models and human patients. IGF-I induces the growth of different Leishmania species in vitro and alters the disease outcome increasing the parasite load and lesion size, especially in L. major- and L. amazonensis-infected mouse leishmaniasis. IGF-I affects the parasite interacting with the IGF-I receptor present on Leishmania. During Leishmania-macrophage interaction, IGF-I acts on the arginine metabolic pathway, resulting in polyamine production both in macrophages and Leishmania. IGF-I and cytokines interact with reciprocal influences on their expression. IL-4 is a hallmark of susceptibility to L. major in murine leishmaniasis, but we observed that IGF-I operates astoundingly as an effector element of the IL-4. Approaching human leishmaniasis, patients with mucosal, disseminated, and visceral diseases presented surprisingly low IGF-I serum levels, suggesting diverse effects than parasite growth. We observed that low IGF-I levels might contribute to the inflammatory response persistence and delayed lesion healing in human cutaneous leishmaniasis and the anemia development in visceral leishmaniasis. We must highlight the complexity of infection revealed depending on the Leishmania species and the parasite's developmental stages. Because IGF-I exerts pleiotropic effects on the biology of interaction and disease pathogenesis, IGF-I turns up as an attractive tool to explore biological and pathogenic processes underlying infection development. IGF-I pleiotropic effects open further the possibility of approaching IGF-I as a therapeutical target.
conferenceObject CHOLESTEROL ESTER TRANSFER PROTEIN (CETP) EXPRESSION IN MICE PROMOTES CONTROL OF EXPERIMENTAL CUTANEOUS LEISHMANIASIS(2021) OZAKI, C. Y.; DANTAS, F. E.; SANTANA, K. G.; TAFURI, W.; SOTTO, M. N.; GOTO, H.; CAZITA, P. M.- Improved Performance of ELISA and Immunochromatographic Tests Using a New Chimeric A2-Based Protein for Human Visceral Leishmaniasis Diagnosis(2021) FIGUEIREDO, Maria Marta; SANTOS, Anna R. R. dos; GODOI, Lara C.; CASTRO, Natalia S. de; ANDRADE, Bruno C. de; SERGIO, Sarah A. R.; JERONIMO, Selma M. B.; OLIVEIRA, Edward J. de; VALENCIA-PORTILLO, Ruth T.; BEZERRA, Lucilandia M.; GOTO, Hiro; SANCHEZ, Maria C. A.; JUNQUEIRA, Caroline; TEIXEIRA, Santuza M. R.; FONSECA, Flavio G. da; GAZZINELLI, Ricardo T.; FERNANDES, Ana PaulaHuman visceral leishmaniasis (VL) is a major public health problem worldwide, leading to significant mortality rates if not properly treated and controlled. Precise identification of infected patients is essential to establish treatment and control measures. Although several VL serological diagnosis advances have been accomplished lately, mainly using recombinant antigens and immunochromatographic tests (ICTs), improvements may still be achieved using multiepitope chimeric proteins in different test platforms. Here, we reported on the evaluation of ELISA and an ICT developed with a new chimeric protein, named DTL-4, based on repetitive antigenic sequences, including those present in the A2 protein. Methods. A total of 1028 sera samples were used for the development and validation of ELISA (321 samples from L. infantum-infected patients, 62 samples from VL/AIDS coinfected patients, 236 samples from patients infected with other diseases, and 409 samples from healthy donors). A total of 520 sera samples were used to develop and validate ICT (249 samples from L. infantum-infected patients, 46 samples from VL/AIDS coinfected patients, 40 samples from patients infected with other diseases, and 185 samples from healthy donors). Findings. Using the validation sera panels, DTL-4-based ELISA displayed an overall sensitivity of 94.61% (95% CI: 89.94-97.28), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 97.02% (95% CI: 94.61-98.38), while for ICT, sensitivity, specificity, and accuracy values corresponded to 91.98% (95% CI: 86.65-95.39), 100.00% (95% CI: 96.30-100.00), and 95.14% (95% CI: 91.62-97.15), respectively. When testing sera samples from VL/AIDS coinfected patients, DTL-4-ELISA displayed a sensitivity of 77.42% (95% CI: 65.48-86.16), a specificity of 99.41% (95% CI: 96.39-99.99), and an accuracy of 93.51% (95% CI: 89.49%-96.10%), while for DTL-4-ICT, sensitivity was 73.91% (95% CI: 59.74-84.40), specificity was 90.63% (95% CI: 81.02-95.63), and accuracy was 82.00% (95% CI: 73.63-90.91). Conclusion. DTL-4 is a promising candidate antigen for serodiagnosis of VL patients, including those with VL/AIDS coinfection, when incorporated into ELISA or ICT test formats.
- Dual Role of Insulin-Like Growth Factor (IGF)-I in American Tegumentary Leishmaniasis(2021) MENDES-AGUIAR, Carolina; LOPES-SIQUEIRA, Camilla; PETTITO-ASSIS, Fabricio; PEREIRA-OLIVEIRA, Marcia; OLIVEIRA-NETO, Manoel Paes de; PIRMEZ, Claude; DA-CRUZ, Alda Maria; GOTO, HiroBackground. Cytokines and growth factors involved in the tissue inflammatory process influence the outcome of Leishmania infection. Insulin-like growth factor (IGF-I) constitutively present in the skin may participate in the inflammatory process and parasite-host interaction. Previous work has shown that preincubation of Leishmania (Leishmania) amazonensis with recombinant IGF-I induces accelerated lesion development. However, in human cutaneous leishmaniasis (CL) pathogenesis, it is more relevant to the persistent inflammatory process than progressive parasite proliferation. In this context, we aimed to investigate whether IGF-I was present in the CL lesions and if this factor may influence the lesions' development acting on parasite growth and/or on the inflammatory/healing process. Methodology. Fifty-one CL patients' skin lesion samples from endemic area of L. (Viannia) braziliensis infection were submitted to histopathological analysis and searched for Leishmania and IGF-I expression by immunohistochemistry. Results. In human CL lesions, IGF-I was observed preferentially in the late lesion (more than 90 days), and the percentage of positive area for IGF-I was positively correlated with duration of illness (r=0.42, P<0.05). IGF-I was highly expressed in the inflammatory infiltrate of CL lesions from patients evolving with good response to therapy (2.8%+/- 2.1%; median=2.1%; n=18) than poor responders (1.3%+/- 1.1%; median: 1.05%; n=6; P<0.05). Conclusions. It is the first time that IGF-I was detected in lesions of infectious cutaneous disease, specifically in American tegumentary leishmaniasis. IGF-I was related to chronicity and good response to treatment. We may relate this finding to the efficient anti-inflammatory response and the known action of IGF-I in wound repair. The present data highlight the importance of searching nonspecific factors besides adaptive immune elements in the study of leishmaniasis' pathogenesis.
- Early reduction in PD-L1 expression predicts faster treatment response in human cutaneous leishmaniasis(2021) DEY, Nidhi S.; SENARATNE, Sujai; SOMARATNE, Vijani; MADARASINGHE, Nayani P.; SENEVIRATNE, Bimalka; FORRESTER, Sarah; OCA, Marcela Montes de; REIS, Luiza Campos; MOULIK, Srija; WALRAD, Pegine B.; CHATTERJEE, Mitali; GOTO, Hiro; WICKREMASINGHE, Renu; LAGOS, Dimitris; KAYE, Paul M.; RANASINGHE, ShalindraCutaneous leishmaniasis (CL) is caused by Leishmania donovani in Sri Lanka. Pentavalent antimonials (e.g., sodium stibogluconate [SSG]) remain first-line drugs for CL with no new effective treatments emerging. We studied whole blood and lesion transcriptomes from Sri Lankan patients with CL at presentation and during SSG treatment. From lesions but not whole blood, we identified differential expression of immune-related genes, including immune checkpoint molecules, after onset of treatment. Using spatial profiling and RNA-FISH, we confirmed reduced expression of programmed death-ligand 1 (PD-L1) and indoleamine 2,3-dioxygenase 1 (IDO1) proteins on treatment in lesions of a second validation cohort and further demonstrated significantly higher expression of these checkpoint molecules on parasite-infected compared with noninfected lesional CD68+ monocytes and macrophages. Crucially, early reduction in PD-L1 but not IDO1 expression was predictive of rate of clinical cure (HR = 4.88) and occurred in parallel with reduction in parasite load. Our data support a model whereby the initial anti-leishmanial activity of antimonial drugs alleviates checkpoint inhibition on T cells, facilitating immune-drug synergism and clinical cure. Our findings demonstrate that PD-L1 expression can be used as a predictor of rapidity of clinical response to SSG treatment in Sri Lanka and support further evaluation of PD-L1 as a host-directed therapeutic in leishmaniasis.
- Unusual manifestation of genital cutaneous leishmaniasis in an immunocompetent patient from Sao Paulo, Brazil: A case report(2021) REIS, Luiza Campos; LINDOSO, Jose Angelo Lauletta; CELESTE, Beatriz Julieta; BRAZ, Lucia Maria Almeida; RAMOS-SANCHEZ, Eduardo Milton; YAMASHIRO-KANASHIRO, Edite Hatsumi; GOTO, Hiro; OYAFUSO, Luiza Keiko MatsukaA 31-year-old male patient developed an ulcer on the glans penis that evolved for three months without healing. We diagnosed it as leishmaniasis using polymerase chain reaction. No immunosuppression or associated diseases were observed. The patient was treated with meglumine antimoniate that cured the lesion in a month post-treatment. Here, we report this case of cutaneous leishmaniasis lesion at the unusual location of glans penis in an immunocompetent individual. The lesion likely developed due to the bite of a vector, highlighting the need for considering cutaneous leishmaniasis among differential diagnosis of sexually transmitted diseases in areas endemic for leishmaniasis.
- miR-548d-3p Alters Parasite Growth and Inflammation in Leishmania (Viannia) braziliensis Infection(2021) SOUZA, Marina de Assis; RAMOS-SANCHEZ, Eduardo Milton; MUXEL, Sandra Marcia; LAGOS, Dimitris; REIS, Luiza Campos; PEREIRA, Valeria Rego Alves; BRITO, Maria Edileuza Felinto; ZAMPIERI, Ricardo Andrade; KAYE, Paul Martin; FLOETER-WINTER, Lucile Maria; GOTO, HiroAmerican Tegumentary Leishmaniasis (ATL) is an endemic disease in Latin America, mainly caused in Brazil by Leishmania (Viannia) braziliensis. Clinical manifestations vary from mild, localized cutaneous leishmaniasis (CL) to aggressive mucosal disease. The host immune response strongly determines the outcome of infection and pattern of disease. However, the pathogenesis of ATL is not well understood, and host microRNAs (miRNAs) may have a role in this context. In the present study, miRNAs were quantified using qPCR arrays in human monocytic THP-1 cells infected in vitro with L. (V.) braziliensis promastigotes and in plasma from patients with ATL, focusing on inflammatory response-specific miRNAs. Patients with active or self-healed cutaneous leishmaniasis patients, with confirmed parasitological or immunological diagnosis, were compared with healthy controls. Computational target prediction of significantly-altered miRNAs from in vitro L. (V.) braziliensis-infected THP-1 cells revealed predicted targets involved in diverse pathways, including chemokine signaling, inflammatory, cellular proliferation, and tissue repair processes. In plasma, we observed distinct miRNA expression in patients with self-healed and active lesions compared with healthy controls. Some miRNAs dysregulated during THP-1 in vitro infection were also found in plasma from self-healed patients, including miR-548d-3p, which was upregulated in infected THP-1 cells and in plasma from self-healed patients. As miR-548d-3p was predicted to target the chemokine pathway and inflammation is a central to the pathogenesis of ATL, we evaluated the effect of transient transfection of a miR-548d-3p inhibitor on L. (V.) braziliensis infected-THP-1 cells. Inhibition of miR-548d-3p reduced parasite growth early after infection and increased production of MCP1/CCL2, RANTES/CCL5, and IP10/CXCL10. In plasma of self-healed patients, MCP1/CCL2, RANTES/CCL5, and IL-8/CXCL8 concentrations were significantly decreased and MIG/CXCL9 and IP-10/CXCL10 increased compared to patients with active disease. These data suggest that by modulating miRNAs, L. (V.) braziliensis may interfere with chemokine production and hence the inflammatory processes underpinning lesion resolution. Our data suggest miR-548d-3p could be further evaluated as a prognostic marker for ATL and/or as a host-directed therapeutic target.