EDUARDO MILTON RAMOS SANCHEZ
Projetos de Pesquisa
Unidades Organizacionais
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
10 resultados
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- Brief Research Report: Expression of PD-1 and CTLA-4 in T Lymphocytes and Their Relationship With the Periparturient Period and the Endometrial Cytology of Dairy Cows During the Postpartum Period(2022) SOUZA, Carolina Menezes Suassuna de; LIMA, Ewerton de Souza; ORDONHO, Raphael Ferreira; OLIVEIRA, Bianca Rafaella Rodrigues dos Santos; RODRIGUES, Rebeca Cordeiro; MOURA, Marquiliano Farias de; LIMA, Daniel Magalhaes; BLAGITZ, Maiara Garcia; SANCHEZ, Eduardo Milton Ramos; MEDEIROS, Isac Almeida de; SOUZA, Fernando Nogueira; FERNANDES, Artur Cezar de CarvalhoThe present study sought to evaluate the expression of PD-1 and CTLA-4 in blood T lymphocytes during the periparturient period and their relationship with uterine health in dairy cows, as determined by endometrial cytology and serum concentrations of beta-hydroxybutyrate (BHB) and non-esterified fatty acids (NEFAs), which are indicators of a negative energy balance. The second objective of this study was to investigate whether the expression of PD-1 and CTLA-4 in T lymphocytes is associated with the serum acute phase-protein haptoglobin concentration during the periparturient period. To address these objectives, 26 clinically healthy dairy cows were used. Peripheral blood was collected 14 days prepartum (T-14), at calving (T0), and 30 days postpartum (T30) to measure the expression of PD-1 and CTLA-4 in blood T lymphocytes by flow cytometry. In addition, we collected blood at T0, 10 days after parturition (T10), and T30 to obtain serum and determine the serum concentrations of NEFA, BHB, and Hp. Endometrial cytology was performed at T10, 20 days after parturition (T20), and T30. In the present study, we observed higher expression of CTLA-4 and PD-1 in T lymphocytes at parturition and in the prepartum period, which could indicate a relationship between these immune checkpoints and immunological tolerance during gestation in dairy cattle. In addition, a negative association between the expression of these immune checkpoints prepartum or at parturition and endometrial cytology at T20 and T30 was observed, indicating the negative implications of these immune response regulators in susceptibility to infections. This finding was further corroborated by the relationship between the serum concentration of haptoglobin and the expression of CTLA-4 and PD-1 by T lymphocytes. However, we did not observe a relationship between the indicators of negative energy balance, evaluated by the serum concentrations of BHB and NEFA, and the expression of the immune checkpoint markers studied. Thus, our findings represent an initial step that paves the way for the development of new therapeutic alternatives directed by the host with the objective of increasing the resistance of dairy cattle to infections in this critical period of life.
- 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.
- Milk Macrophage Function in Bovine Leukemia Virus-Infected Dairy Cows(2021) LIMA, Ewerton de Souza; BLAGITZ, Maiara Garcia; BATISTA, Camila Freitas; ALVES, Alexandre Jose; FERNANDES, Artur Cezar de Carvalho; SANCHEZ, Eduardo Milton Ramos; TORRES, Hugo Frias; DINIZ, Soraia Araujo; SILVA, Marcos Xavier; LIBERA, Alice Maria Melville Paiva Della; SOUZA, Fernando Nogueira deThe implications of bovine leukemia virus (BLV) on innate and adaptive immune responses have been widely investigated; however, the effects of BLV on mammary gland immunity require further investigation. The present study investigated the viability, phagocytic capacity, and intracellular production of reactive oxygen and nitrogen species (RONS) by macrophages in milk samples from dairy cows naturally infected with BLV with or without persistent lymphocytosis (PL). No effect of BLV infection in the overall number of macrophages per milliliter and in the percentage of viable macrophages among overall milk viable cells was found. Furthermore, BLV-infected dairy cows had a higher frequency of viable milk macrophages, while healthy animals had a tendency toward a higher percentage of apoptotic milk macrophages. The percentage of milk macrophages that phagocytosed Staphylococcus aureus in seronegative animals was higher than that in BLV-infected dairy cows. No effect of BLV infection on the intracellular RONS production and the intensity of phagocytosis by milk macrophages was observed. Thus, this study provides new insights into the implications of BLV infections in the bovine mammary gland.
- Emergence of livestock-associated Mammaliicoccus sciuri ST71 co-harbouring mecA and mecC genes in Brazil(2023) MOURA, Guilherme S. de; CARVALHO, Eneas de; SANCHEZ, Eduardo M. Ramos; SELLERA, Fabio P.; MARQUES, Michele F. S.; HEINEMANN, Marcos B.; VLIEGHER, Sarne De; SOUZA, Fernando N.; MOTA, Rinaldo A.The discovery and tracking of antimicrobial resistance genes are essential for understanding the evolution of bacterial resistance and restraining its dispersion. Mammaliicoccus sciuri (formerly Staphylococcus sciuri) is the most probable evolutionary repository of the mecA gene, that later disseminated to S. aureus. In this study, we describe the first double mecA/mecC homologue-positive non-aureus staphylococci and mammaliicocci (NASM) from the American continent, also representing the first report of mecC-positive NASM in Brazil. Two clonally related methicillin-resistant M. sciuri strains co-carrying mecA and mecC genes were isolated from the teat skin swab and milk sample collected from an ewe's left udder half. Both M. sciuri strains belonged to the sequence type (ST) 71. Besides mecA and mecC genes, the M. sciuri strains carried broad resistomes for clinically important antimicrobial agents, including & beta;-lactams, tetracyclines, lincosamide, streptogramin, streptomycin, and aminoglycosides. Virulome analysis showed the presence of the clumping factor B (clfB), ATP-dependent protease ClpP (ClpP) and serine-aspartate repeat proteins (sdrC and sdrE) virulence-associated genes. Phylogenomic analysis revealed that these M. sciuri strains are part of a globally disseminated branch, associated with farm and companion animals and even with food. Our findings suggest that M. sciuri is likely to emerge as a pathogen of global interest, carrying a broad repertoire of antimicrobial resistance genes with a remarkable co-presence of mecA and mecC genes. Finally, we strongly encourage to monitor M. sciuri under the One Health umbrella since this bacterial species is spreading at the human-animal-environment interface.
- The bovine leukemia virus infection prolongs immunosuppression in dairy cows during the periparturient period by sustaining higher expression of immunological checkpoints in T cells(2023) NASCIMENTO, Alice Maria Melo do; SOUZA, Carolina Menezes Suassuna de; OLIVEIRA, Ana Claudia Dumont; BLAGITZ, Maiara Garcia; SANCHEZ, Eduardo Milton Ramos; LIBERA, Alice Maria Melville Paiva Della; LEITE, Ricardo de Miranda Henriques; FERNANDES, Artur Cezar de Carvalho; SOUZA, Fernando NogueiraBovine leukemia virus (BLV) is caused by a deltaretrovirus and has been associated with immunosuppression as well as comorbidities such as bovine mastitis, the costliest disease in the dairy sector. However, no previous study has explored at the synergistic immunosuppressive effect of the peripartum period with an immunosuppressive viral disease such as BLV. Thus, our study explored the effect of BLV infection in the periparturient period on the expression of PD-1 and CTLA-4 in blood T lymphocytes, and the impact of BLV infection on the rate of new intramammary infections during the early lactation. Here, we found that BLV-infected dairy cows always had a statistically significant higher expression of CTLA-4 and PD-1 in blood T cells. Furthermore, our findings indi-cated that BLV infection prolongs immunosuppression in dairy cows during the periparturient period by sus-taining higher expression of immunological checkpoints in T cells. In addition, BLV-infected dairy cows have a higher rate of new intramammary infections during early lactation. Thus, our study provides new insights of the immunosuppressive effect of BLV on the most critical period of the cows' life with marked detrimental effect on protective T-cell immunity and comorbidities, such as bovine mastitis.
- 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.
- Mathematical Modelling Using Predictive Biomarkers for the Outcome of Canine Leishmaniasis upon Chemotherapy(2020) GONCALVES, Rafaela de Sousa; PINHO, Flaviane Alves de; DINIS-OLIVEIRA, Ricardo Jorge; AZEVEDO, Rui; GAIFEM, Joana; LARANGEIRA, Daniela Farias; RAMOS-SANCHEZ, Eduardo Milton; GOTO, Hiro; SILVESTRE, Ricardo; BARROUIN-MELO, Stella MariaPrediction parameters of possible outcomes of canine leishmaniasis (CanL) therapy might help with therapeutic decisions and animal health care. Here, we aimed to develop a diagnostic method with predictive value by analyzing two groups of dogs with CanL, those that exhibited a decrease in parasite load upon antiparasitic treatment (group: responders) and those that maintained high parasite load despite the treatment (group: non-responders). The parameters analyzed were parasitic load determined by q-PCR, hemogram, serum biochemistry and immune system-related gene expression signature. A mathematical model was applied to the analysis of these parameters to predict how efficient their response to therapy would be. Responder dogs restored hematological and biochemical parameters to the reference values and exhibited a Th1 cell activation profile with a linear tendency to reach mild clinical alteration stages. Differently, non-responders developed a mixed Th1/Th2 response and exhibited markers of liver and kidney injury. Erythrocyte counts and serum phosphorus were identified as predictive markers of therapeutic response at an early period of assessment of CanL. The results presented in this study are highly encouraging and may represent a new paradigm for future assistance to clinicians to interfere precociously in the therapeutic approach, with a more precise definition in the patient's prognosis.
- Memory CD4+ and CD8+ T lymphocyte proliferation in vaccinated dairy cows with different histories of Staphylococcus aureus mastitis(2022) SOARES, Thais C. S.; SANTOS, Kamila R.; LIMA, Daniel M.; MAIA, Raysa Brenda M.; RAMOS-SANCHEZ, Eduardo M.; REIS, Luiza C.; GIDLUND, Magnus; CUNHA, Adriano F. da; ORDINOLA-RAMIREZ, Carla M.; CERQUEIRA, Monica M. O. P.; HEINEMANN, Marcos B.; LIBERA, Alice M. M. P. Della; GOTO, Hiro; SOUZA, Fernando N.Staphylococcus aureus mastitis constitutes a serious threat to dairy cows. The reasons why available vaccines are not fully effective remain poorly understood; thus, in the present study, we investigated CD4(+) and CD8(+) T lymphocyte proliferation in dairy cows vaccinated with a polyvalent mastitis vaccine that had distinct precedent Staphylococcus aureus mastitis. We studied 17 S. aureus-infected dairy cows (11 vaccinated and six unvaccinated) and eight vaccinated healthy dairy cows with no previous S. aureus mastitis infections. Flow cytometry was used to assess lymphocyte proliferation using an anti-Ki67 antibody, and monoclonal antibodies were used to identify T cell subsets. S. aureus-infected cows exhibited reduced overall lymphocyte proliferation, including CD4(+) T lymphocyte proliferation, and memory lymphocyte proliferation in response to S. aureus isolate stimulus. Immunization did not influence the expansion of blood lymphocyte populations. Furthermore, CD8(+) T cells, memory CD8(+) T lymphocytes, and effector memory CD8(+) T lymphocytes displayed reduced proliferation 21 days after the third vaccine dose compared with before vaccination at time zero. The present data demonstrates an overall negative regulation of the T-cell response suggesting its detrimental impact leading to the persistence of S. aureus intramammary infections. Furthermore, the lack of vaccination effect on T-cell mediated immunity (e.g., proliferation) may be related to poor vaccine efficacy.
- 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.
- Bovine-associated staphylococci and mammaliicocci trigger T-lymphocyte proliferative response and cytokine production differently(2023) SOUZA, Fernando N.; SANTOS, Kamila R.; FERRONATTO, Jose A.; SANCHEZ, Eduardo M. Ramos; TOLEDO-SILVA, Bruno; HEINEMANN, Marcos B.; VLIEGHER, Sarne De; LIBERA, Alice M. M. P. DellaWe examined whether distinct staphylococcal and mammaliicoccal species and strains trigger B-and T-lymphocyte proliferation and interleukin (IL)-17A and interferon (IFN)-gamma production by peripheral blood mononuclear cells in nulliparous, primiparous, and multiparous dairy cows. Flow cytometry was used to measure lymphocyte proliferation with the Ki67 anti-body, and specific monoclonal antibodies were used to identify CD3, CD4, and CD8 T lymphocyte and CD21 B lymphocyte populations. The supernatant of the peripheral blood mononuclear cell culture was used to measure IL-17A and IFN-gamma production. Two distinct, inactivated strains of bovine-associated Staphylococcus aureus [one causing a persistent intramammary infec-tion (IMI) and the other from the nose], 2 inactivated Staphylococcus chromogenes strains [one causing an IMI and the other from a teat apex), as well as an inactivated Mammaliicoccus fleurettii strain originat-ing from sawdust from a dairy farm, and the mitogens concanavalin A and phytohemagglutinin M-form (both specifically to measure lymphocyte proliferation) were studied. In contrast to the ""commensal"" Staph. aureus strain originating from the nose, the Staph. aureus strain causing a persistent IMI triggered proliferation of CD4+ and CD8+ subpopulations of T lymphocytes. The M. fleurettii strain and the 2 Staph. chromogenes strains had no effect on T-or B-cell proliferation. Fur-thermore, both Staph. aureus and Staph. chromogenes strains causing persistent IMI significantly increased IL-17A and IFN-gamma production by peripheral blood mononuclear cells. Overall, multiparous cows tended to have a higher B-lymphocyte and a lower T-lymphocyte proliferative response than primiparous and nulliparous cows. Peripheral blood mononuclear cells of multipa-rous cows also produced significantly more IL-17A and IFN-gamma. In contrast to concanavalin A, phytohemagglu-tinin M-form selectively stimulated T-cell proliferation.