EDUARDO MILTON RAMOS SANCHEZ
Projetos de Pesquisa
Unidades Organizacionais
LIM/38 - Laboratório de Epidemiologia e Imunobiologia, Hospital das Clínicas, Faculdade de Medicina
3 resultados
Resultados de Busca
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- 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.
- 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.
- 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.