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
- Dual Host-Intracellular Parasite Transcriptome of Enucleated Cells Hosting Leishmania amazonensis: Control of Half-Life o Host Cell Transcripts by the Parasite(2020) ORIKAZA, Cristina M.; PESSOA, Carina C.; V, Fernanda Paladino; V, Pilar T. Florentino; BARBIERI, Clara L.; GOTO, Hiro; RAMOS-SANCHEZ, Eduardo Milton; SILVEIRA, Jose Franco; RABINOVITCH, Michel; MORTARA, Renato A.; REAL, FernandoEnucleated cells or cytoplasts (cells whose nucleus is removed in vitro) represent an unexplored biological model for intracellular infection studies due to the abrupt interruption of nuclear processing and new RNA synthesis by the host cell in response to pathogen entry. Using enucleated fibroblasts hosting the protozoan parasite Leishmania amazonensis, we demonstrate that parasite multiplication and biogenesis of large parasitophorous vacuoles in which parasites multiply are independent of the host cell nucleus. Dual RNA sequencing of both host cytoplast and intracellular parasite transcripts identified host transcripts that are more preserved or degraded upon interaction with parasites and also parasite genes that are differentially expressed when hosted by nucleated or enucleated cells. Cytoplasts are suitable host cells, which persist in culture for more than 72 h and display functional enrichment of transcripts related to mitochondrial functions and mRNA translation. Crosstalk between nucleated host de novo gene expression in response to intracellular parasitism and the parasite gene expression to counteract or benefit from these host responses induces a parasite transcriptional profile favoring parasite multiplication and aerobic respiration, and a host-parasite transcriptional landscape enriched in host cell metabolic functions related to NAD, fatty acid, and glycolytic metabolism. Conversely, interruption of host nucleus-parasite cross talk by infection of enucleated cells generates a host-parasite transcriptional landscape in which cytoplast transcripts are enriched in phagolysosome-related pathway, prosurvival, and SerpinB-mediated immunomodulation. In addition, predictive in since analyses indicated that parasite transcript products secreted within cytoplasts interact with host transcript products conserving the host V-ATPase proton translocation function and glutamine/proline metabolism. The collective evidence indicates parasite-mediated control of host cell transcripts half-life that is beneficial to parasite intracellular multiplication and escape from host immune responses. These findings will contribute to improved drug targeting and serve as database for L. arnazonensis-host cell interactions.
- Asymptomatic infections in blood donors harbouring Plasmodium: an invisible risk detected by molecular and serological tools(2018) LIMA, Giselle F. M. C.; SANCHEZ, Maria C. Arroyo; LEVI, Jose E.; FUJIMORI, Mahyumi; CARAMELO, Luiza da Cruz; SANCHEZ, Arianni Rondelli; RAMOS-SANCHEZ, Eduardo M.; INOUE, Juliana; COSTA-NASCIMENTO, Maria de Jesus; MENDRONE JUNIOR, Alfredo; SANTI, Silvia M. DiBackground. Transfusion-transmitted malaria due to asymptomatic Plasmodium infections is a challenge for blood banks. There is a lack of data on the prevalence of asymptomatic infected blood donors and the incidence of transfusion-transmitted malaria in low endemicity areas worldwide. We estimated the frequency of blood donors harbouring Plasmodium in an area in which asymptomatic infections have been reported. Material and methods. To estimate the frequency of blood donors harbouring Plasmodium we used microscopy and molecular tools. Serological tests were applied to measure the exposure of candidates to Plasmodium antigens. Venous blood was collected from 91 candidates attending the ""Pro-Sangue"" Blood Centre Foundation in Sao Paulo, who lived in the municipality of Juquitiba, Sao Paulo, Brazil, where sporadic autochthonous cases of malaria have been described. Blood samples were used for parasitological, molecular and serological studies. Results. Among the 91 samples examined, rare Plasmodium forms were observed in two donors. Genus real-time polymerase chain reaction analysis demonstrated Plasmodium amplification in three candidates and species-specific nested polymerase chain reaction identified P. malariae in two. ELISA-IgG was reactive in 42.9% of samples for P. vivax (Pv-MSP1 19) and in 6.6% for P. falciparum (Pf-Zw). ELISA-IgM was reactive in 2.2% of samples for P. vivax and in 4.4% for P. falciparum. An indirect immunofluorescence assay was reactive for P. malariae in 15.4% of cases. Discussion. Reservoirs of Plasmodium represent a challenge for blood banks, since studies have shown that high levels of submicroscopic infections can occur in low transmission areas. The risk of transfusion-transmitted malaria presented here points to the need to conduct molecular investigations of candidate donors with any positive malarial antibody test.
conferenceObject Analysis of global gene expression of Leishmania infantum under Insulin-like growth factor I stimulus(2015) RAMOS-SANCHEZ, E. M.; NERLAND, A. H.; GOTO, H.- Immunological implications of bovine leukemia virus infection(2017) BLAGITZ, M. G.; SOUZA, F. N.; BATISTA, C. F.; AZEVEDO, L. F. F.; SANCHEZ, E. M. R.; DINIZ, S. A.; SILVA, M. X.; HADDAD, J. P.; LIBERA, A. M. M. P. DellaThis study examined neutrophil and monocyte functions and the blood lymphocyte profile of naturally BLV-infected cows with or without persistent lymphocytosis (PL). The percentage of neutrophils and monocytes that phagocytosed Staphylococcus aureus was lower in BLV-infected dairy cows, particularly those with PL. The relative percentage of CD44(+) monocytes and neutrophils and CD11b expression by neutrophils was also lower in BLV-infected dairy cows with PL. A correlation between the percentage of CD11b(+) neutrophils and that produced reactive oxygen species (ROS) was found. Furthermore, the percentage of CD44(+) monocytes was positively correlated with the percentage of monocytes that phagocytosed S. aureus and the same phenomenon was observed for neutrophils. In BLV-infected dairy cows, particularly those with PL, inhibition of monocyte and neutrophil apoptosis was observed. Additionally, the percentage of neutrophils producing ROS was lower in BLV-infected cows with PL, in contrast to higher intensity of intracellular production of ROS by monocytes. The result from the lymphocyte immunophenotyping of BLV-infected cows with PL was an increase in B cells, mainly B CD5(+) CD11b(+), due to the apoptosis inhibition. In conclusion, this study provides novel insight into the implications of BLV infection for cattle, which can include the dysfunction of blood monocytes and neutrophils.
conferenceObject Altered expression of microRNAs in infections with cutaneous and visceral strains of Leishmania(2019) GOTO, H.; SOUZA, M. A.; RAMOS-SANCHEZ, E. M.; MUXEL, S. M.; BRITO, M. E. F.; PEREIRA, V. R. A.; FLOETER-WINTER, L. M.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.
- 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.
- 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.