EDUARDO MILTON RAMOS SANCHEZ
Projetos de Pesquisa
Unidades Organizacionais
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
7 resultados
Resultados de Busca
Agora exibindo 1 - 7 de 7
- ATP6V(0)d2 controls Leishmania parasitophorous vacuole biogenesis via cholesterol homeostasis(2019) PESSOA, Carina Carraro; REIS, Luiza Campos; RAMOS-SANCHEZ, Eduardo Milton; ORIKAZA, Cristina Mary; CORTEZ, Cristian; LEVATTI, Erica Valadares de Castro; BADARO, Ana Carolina Benites; YAMAMOTO, Joyce Umbelino da Silva; D'ALMEIDA, Vania; GOTO, Hiro; MORTARA, Renato Arruda; REAL, FernandoV-ATPases are part of the membrane components of pathogen-containing vacuoles, although their function in intracellular infection remains elusive. In addition to organelle acidification, V-ATPases are alternatively implicated in membrane fusion and anti-inflammatory functions controlled by ATP6V(0)d2, the d subunit variant of the V-ATPase complex. Therefore, we evaluated the role of ATP6V(0)d2 in the biogenesis of pathogen-containing vacuoles using ATP6V(0)d2 knock-down macrophages infected with the protozoan parasite Leishmania amazonensis. These parasites survive within IFN gamma/LPS-activated inflammatory macrophages, multiplying in large/fusogenic parasitophorous vacuoles (PVs) and inducing ATP6V(0)d2 upregulation. ATP6V(0)d2 knock-down decreased macrophage cholesterol levels and inhibited PV enlargement without interfering with parasite multiplication. However, parasites required ATP6V(0)d2 to resist the influx of oxidized low-density lipoprotein (ox-LDL)-derived cholesterol, which restored PV enlargement in ATP6V(0)d2 knock-down macrophages by replenishing macrophage cholesterol pools. Thus, we reveal parasite-mediated subversion of host V-ATPase function toward cholesterol retention, which is required for establishing an inflammation-resistant intracellular parasite niche. Author summary V-ATPases control acidification and other processes at intracellular vesicles that bacteria and parasites exploit as compartments for replication and immune evasion. We report that the protozoan intracellular parasite Leishmania amazonensis resists inflammatory macrophage immune responses and upregulates an alternative isoform of subunit d of V-ATPase (ATP6V(0)d2). Leishmania are still sequestered within acidified parasitophorous vacuoles (PVs) in cells lacking ATP6V(0)d2, but these PVs do not enlarge in volume, a distinguishing feature of intracellular infection by these parasites. Cholesterol levels in ATP6V(0)d2-deficient cells are reduced and exogenous cholesterol repletion can restore vacuole size, leading to enhanced parasite killing. This study demonstrates the ATP6V(0)d2-mediated interplay of macrophage cholesterol retention and control of the biogenesis of large pathogen-containing vacuoles. The study provides grounds for the development of new therapeutic strategies for diseases caused by intracellular pathogens sheltered in host cell compartments.
- R-Phycoerythrin-labeled Mannheimia haemolytica for the simultaneous measurement of phagocytosis and intracellular reactive oxygen species production in bovine blood and bronchoalveolar lavage cells(2018) BATISTA, Camila F.; SOUZA, Fernando N.; SANTOS, Kamila R.; SANCHEZ, Eduardo M. Ramos; REIS, Luiza Campos; BERTAGNON, Heloisa G.; BLAGITZ, Maiara G.; GOMES, Renata C.; LAGE, Andrey P.; HEINEMANN, Marcos B.; LIBERA, Alice M. M. P. DellaThe present study aimed to validate the use of R-phycoerythrin (R-PE)-labeled Mannheimia haemolytica to simultaneously stimulate phagocytosis and intracellular production of reactive oxygen species (ROS) by blood phagocytes in bronchoalveolar lavage (BAL) fluid. Initially, R-PE-labeled M. haemolytica was inactivated using a water bath at 60 degrees C for 60 min. Afterwards, R-PE labelling of bacteria was confirmed by flow cytometry. The geometric mean fluorescence intensity of R-PE-labeled bacteria (FL2 detector, 585 +/- 42 nm) was analyzed by flow cytometry and was 41.5-fold higher than the respective unlabeled controls, confirming the success of bacterial conjugation to R-PE. Phagocytosis and intracellular production of ROS by blood neutrophils and monocytes, and by BAL CD14 macrophages, in 12 healthy 6-month-old male calves were then performed using R-PE-labeled bacteria and 2',7'-dichlorofluoresceindiacetate (DCFH-DA) as probes. Confocal microscopy was used to confirm phagocytosis of R-PE-labeled M. haemolytica by phagocytes and to exclude erroneous measurements of bacteria adhering to the leukocyte membrane. The present study showed that there is no difference in the ROS production without stimulus and in the presence of M. haemolytica by peripheral blood neutrophils and monocytes, in contrast to the increased ROS production by local alveolar macrophages upon stimulation by M. haemolytica. This emphasizes the importance of alveolar macrophages in the maintenance of homeostasis and health of the respiratory system, which can be supported during the inflammatory process by the rapid recruitment of neutrophils with high microbicidal and phagocytic capacity. The method described here provides an easy and feasible tool to measure phagocytosis and intracellular ROS production by phagocytes, especially when commonly used probes for intracellular ROS production were used, such as DCFH-DA and dihydrorhodamine 123.
- miR-548d-3p Is Up-regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages (vol 12, 826039, 2022)(2022) RAMOS-SANCHEZ, Eduardo Milton; REIS, Luiza Campos; SOUZA, Marina de Assis; MUXEL, Sandra Marcia; SANTOS, Kamila Reis; LAGOS, Dimitris; PEREIRA, Valeria Rego Alves; BRITO, Maria Edileuza Felinto de; KAYE, Paul Martin; FLOETER-WINTER, Lucile Maria; GOTO, Hiro
- miR-548d-3p Is Up-Regulated in Human Visceral Leishmaniasis and Suppresses Parasite Growth in Macrophages(2022) RAMOS-SANCHEZ, Eduardo Milton; REIS, Luiza Campos; SOUZA, Marina de Assis; MUXEL, Sandra Marcia; SANTOS, Kamila Reis; LAGOS, Dimitris; PEREIRA, Valeria Rego Alves; BRITO, Maria Edileuza Felinto de; KAYE, Paul Martin; FLOETER-WINTER, Lucile Maria; GOTO, HiroVisceral leishmaniasis caused by Leishmania (Leishmania) infantum in Latin America progress with hepatosplenomegaly, pancytopenia, hypergammaglobulinemia, and weight loss and maybe lethal mainly in untreated cases. miRNAs are important regulators of immune and inflammatory gene expression, but their mechanisms of action and their relationship to pathogenesis in leishmaniasis are not well understood. In the present study, we sought to quantify changes in miRNAs associated with immune and inflammatory pathways using the L. (L.) infantum promastigote infected- human monocytic THP-1 cell model and plasma from patients with visceral leishmaniasis. We identified differentially expressed miRNAs in infected THP-1 cells compared with non-infected cells using qPCR arrays. These miRNAs were submitted to in silico analysis, revealing targets within functional pathways associated with TGF-beta, chemokines, glucose metabolism, inflammation, apoptosis, and cell signaling. In parallel, we identified differentially expressed miRNAs in active visceral leishmaniasis patient plasma compared with endemic healthy controls. In silico analysis of these data indicated different predicted targets within the TGF-beta, TLR4, IGF-I, chemokine, and HIF1 alpha pathways. Only a small number of miRNAs were commonly identified in these two datasets, notably with miR-548d-3p being up-regulated in both conditions. To evaluate the potential biological role of miR-548d-3p, we transiently transfected a miR-548d-3p inhibitor into L. (L.) infantum infected-THP-1 cells, finding that inhibition of miR-548d-3p enhanced parasite growth, likely mediated through reduced levels of MCP-1/CCL2 and nitric oxide production. Further work will be required to determine how miR-548d-3p plays a role in vivo and whether it serves as a potential biomarker of progressive leishmaniasis.
- Orange-Emitting ZnSe:Mn2+ Quantum Dots as Nanoprobes for Macrophages(2020) KHAN, Zahid U.; UCHIYAMA, Mayara K.; KHAN, Latif U.; RAMOS-SANCHEZ, Eduardo M.; REIS, Luiza Campos; NAKAMURA, Marcelo; GOTO, Hiro; SOUZA, Ana O. De; ARAKI, Koiti; BRITO, Hermi F.; GIDLUND, MagnusThe biocompatibility, bionanointeraction, uptake efficiency, and entry pathway of luminescent nanomaterials are the key factors to understand development of an efficient bionanoprobe. The foremost objective of this work is to explore the potential of 3-mercaptopropionic acid (3-MPA) capped ZnSe:xMn(2+) (x = 5, 10, and 15 mol %) quantum dots (QDs) for the development of bionanoprobe used in future biological and clinical applications. For this purpose, highly intense orange-emitting activator Mn2+ ion doped ZnSe QDs were synthesized via a high-temperature organometallic method and rendered water-soluble by a ligand exchange approach. The morphological and physicochemical characterizations displayed the ultrasmall zinc-blend cubic crystal structure of QDs with an elliptical shape nanocrystals and average diameter of 4 nm. The luminescent nanomaterials exhibited orange emission centered at 584 nm under excitation at 385 nm. The biocompatibility, time-dependent cellular uptake, and the uptake mechanism of QDs were studied in RAW 264.7 macrophages, accomplished by various cytotoxicity assays, CytoViva hyperspectral enhanced dark-field and dual-mode fluorescence (DMF) microscopy, and transmission electron microscopy (TEM) images. The cytotoxicity study did not confirm any noticeable deleterious effect of QDs within incubation for 6 h. The fluorescence images of cells incubated with QDs showed efficient emission, which is a manifestation that QDs are photochemically stable in the intracellular environment. The cellular uptake findings demonstrated that the QDs were predominantly internalized via clathrin- and caveolae-mediated pathways. After the uptake, QDs aggregates appeared inside the vesicles in the cytoplasm, and their number and size gradually increased as a function of time. Nevertheless, the fluorescent QDs presented remarkable colloidal stability in various media, biocompatibility within the designated time, efficient time-dependent uptake, and distinct entry pathway in RAW macrophages, suggesting promising candidates to explore for the development of future bionanoprobes.
- 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.