EDUARDO MILTON RAMOS SANCHEZ
Projetos de Pesquisa
Unidades Organizacionais
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
16 resultados
Resultados de Busca
Agora exibindo 1 - 10 de 16
- 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.
- 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.
- 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.
- Insulin-Like Growth Factor-I as an Effector Element of the Cytokine IL-4 in the Development of a Leishmania major Infection(2018) REIS, Luiza C.; RAMOS-SANCHEZ, Eduardo Milton; PETITTO-ASSIS, Fabricio; NERLAND, Audun H.; HERNANDEZ-VALLADARES, Maria; SELHEIM, Frode; FLOETER-WINTER, Lucile Maria; GOTO, HiroCertain cytokines modulate the expression of insulin-like growth factor-(IGF-) I. Since IL-4 and IGF-I promote growth of the protozoan Leishmania major, we here addressed their interaction in downregulating the expression of Igf-I mRNA using small interfering RNA (siRNA) in Leishmania major-infected macrophages. Parasitism was decreased in the siRNA-treated cells compared with the nontreated cells, reversed by the addition of recombinant IGF-I (rIGF-I). In IL-4-stimulated macrophages, parasitism and the Igf-I mRNA amount were increased, and the effects were nullified upon siRNA transfection. IGF-I downregulation inhibited both parasite and macrophage arginase activation even in IL-4-stimulated cells. Searching for intracellular signaling components shared by IL-4 and IGF-I, upon siRNA transfection, phosphorylated p44, p38, and Akt proteins were decreased, affecting the phosphatidylinositol-3-kinase (PI3K)/Akt pathway. In L. major-infected C57BL6-resistant mice, the preincubation of the parasite with rIGF-I changed the infection profile to be similar to that of susceptible mice. We conclude that IGF-I constitutes an effector element of IL-4 involving the PI3K/Akt pathway during L. major infection.
conferenceObject EFFECTS OF INSULIN-LIKE GROWTH FACTOR-I AND IL-4 ON LEISHMANIA (L.) INFANTUM-INFECTED HUMAN MACROPHAGES(2018) GOTO, Hiro; SEVILLANO, Orlando; REIS, Luiza; RAMOS-SANCHEZ, Eduardo- 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
- Comparison of antibody repertories against Staphylococcus aureus in healthy and infected dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine(2020) CUNHA, A. F.; ANDRADE, H. M.; SOUZA, F. N.; FIALHO JUNIOR, L. C.; ROSA, D. L. S. O.; SANCHEZ, E. M. Ramos; GIDLUND, M.; GOTO, H.; BRITO, M. A. V. P.; GUIMARAES, A. S.; LAGE, A. P.; REIS, L. C.; LIBERA, A. M. M. P. Della; HEINEMANN, M. B.; CERQUEIRA, M. M. O. P.Staphylococcus aureus is one of the pathogens most frequently isolated from cases of mastitis worldwide. To decrease the effect of S. aureus mastitis in dairy farming, alternative strategies for controlling mastitis are needed that depend on a better knowledge of cowto-cow variations in S. aureus antibody production. The present study sought to explore the diversity of S. aureus antibodies produced by dairy cows with a distinct mastitis history and vaccinated with a polyvalent mastitis vaccine. We obtained protein extracts from S. aureus isolates derived from persistent subclinical mastitis. Proteins were fractionated using 2-dimensional gel electrophoresis and Western blotting. Then, Western blotting membranes were exposed to sera from 24 dairy cows that had been divided into the following groups: vaccinated dairy cows that were infected with S. aureus, further subdivided according to whether they (a) remained infected by S. aureus or (b) recovered from the intramammary infection; unvaccinated dairy cows infected with S. aureus; and vaccinated healthy dairy cows with no history of S. aureus mastitis. Proteins found to be reactive by Western blot were identified by mass spectrometry (MALDI/TOF-TOF). Our most important finding was that F0F1 ATP synthase subunit a, succinyl-diaminopimelate desuccinylase, and cysteinyl-tRNA synthetase were potential candidate proteins for the prevention of S. aureus mastitis. This study strengthens the notion that variations among animals should not be ignored and shows that the heterogeneity of antibody production against anti-staphylococcal antigens in animals may enable the identification of new immunotherapy targets.
conferenceObject Th2-skewed response depends on Insulin-like growth factor I in Leishmania (Leishmania) major-infection(2015) REIS, L. C.; RAMOS-SANCHEZ, E. M.; GOTO, H.- Staphylococcus aureus-Cure-Associated Antigens Elicit Type 3 Immune Memory T Cells(2022) SANTOS, Kamila R.; SOUZA, Fernando N.; RAMOS-SANCHEZ, Eduardo M.; BATISTA, Camila F.; REIS, Luiza C.; FOTORAN, Wesley L.; HEINEMANN, Marcos B.; CUNHA, Adriano F.; ROCHA, Mussya C.; FARIA, Angelica R.; ANDRADE, Helida M.; CERQUEIRA, Monica M. O. P.; GIDLUND, Magnus; GOTO, Hiro; LIBERA, Alice Maria M. P. DellaBackground: Staphylococcus aureus is one of the most frequently major mastitis pathogens that cause clinical and subclinical mastitis worldwide. Current antimicrobial treatments are usually ineffective, and the commercially available vaccines lack proven effectiveness. The immunological response elicited by the recombinant S. aureus-cure-associated proteins phosphoglycerate kinase (PGK), enolase (ENO), and elongation factor-G (EF-G) in combination with the granulocyte-macrophage colony-stimulating factor (GM-CSF) DNA vaccination was studied in this work. Methods: Here, twenty-three C57BL/6 mice were divided into four groups and vaccinated with: G1: none (control); G2: GM-CSF DNA plasmid DNA vaccine; G3: the combination of EF-G+ENO+PGK; and G4: the combinations of EF-G+ENO+PGK proteins plus GM-CSF plasmid DNA vaccine. After 44 days, spleen cells were collected for immunophenotyping and lymphocyte proliferation evaluation by flow cytometry upon S. aureus stimulus. Results: Immunization with the three S. aureus recombinant proteins alone resulted in a higher percentage of IL-17A(+) cells among CD8(+) T central memory cells, as well as the highest intensity of IL-17A production by overall lymphocytes indicating that the contribution of the combined lymphocyte populations is crucial to sustaining a type 3 cell immunity environment. Conclusion: The immunization with three S. aureus-cure-associated recombinant proteins triggered type 3 immunity, which is a highly interesting path to pursue an effective bovine S. aureus mastitis vaccine.