Introduction: Cancer is the second leading cause of death from diseases in the world. Only in 2018, this disease was responsible for about 9.6 million deaths, where breast cancer is the tumor type with the highest incidence and mortality in the female population worldwide. Taking into account the adverse effects of current treatments, in addition to low efficiency and specificity, it is evident the need to develop new therapies aimed at greater specificity and less side effects.Objective: This work aims to develop cationic liposomes that carry interference RNA (siRNA) molecules for silencing target genes in breast tumors. Methodology:The selection of targets was previously outlined by our research group based on data from the interactome and transcriptome of seven tumor cell lines and one non-tumor cell line. The development of the liposome was carried out by forming a lipid film of phospholipid and cholesterol followed by hydration of the it and subsequent extrusion. The particle physicochemical characterization was evaluated by dynamic light scattering (DLS) and zeta potential; and their biological effects were assessed. First, we evaluated the effect of delivery formulations (empty cationic liposome, in the presence or absence of Polyethylene glycol -PEG) on cell viability in MCF-7 tumor cell line (human breast adenocarcinoma, Luminal A), MDA-MB-231 (human breast adenocarcinomas, triple negative) and HEP-G2 (human hepatocarcinoma). Flow cytometry methodology was used to evaluate gene silencing, where cells were treated with siRNA-carrying liposomes in different concentrations for 48 hours. Results:We performed bioassays at different times and concentrations, that shows greater cell viability in the presence of liposomes with PEG when compared in the absence of it, however, both demonstrated viability greater than 50%. Then, we proceed with the evaluation of protein silencing in flow cytometry after treatment with siRNA carrier liposomes in the presence or absence of PEG, in different concentrations for 48 hours. The silencing evaluation showed a decrease in the expression of two target proteins in cells treated with liposomes containing RNAi for these two targets. Conclusion:The liposomes were successfully obtained. The evaluation of the silencing of two target proteins revealed a decrease in their expression in relation to the control, indicating the efficiency of siRNA delivery through liposome system. Thus, our results indicate a promising profile of the target genes under study for the development of an innovative therapy in the treatment of breast cancer.
Introduction:The infection caused by the new coronavirus (SARS-CoV-2) induces a severe acute respiratory syndrome called COVID-19, leading to more than six million deaths worldwide. The ChAdOx1 nCoV-19 vaccine developed by AstraZeneca and produced by Bio-Manguinhos helps to drop mortality in Brazil and many other countries. Cellular immunology is a key aspect of overall vaccine-induced immunological memory. Additionally, lymphocyte response reflected in interferon and pro-inflammatory cytokine production are presented in the COVID-19, triggering memory protective responses.Objective: To assess the profile of cellular responses elicited by the ChAdOx1 nCoV-19 vaccine considering participants with or without previous SARS-Cov-2 infection.Methodology: Blood samples obtained from participants vaccinated with ChAdOx1 nCoV-19 were collected in a follow-up strategy: 0, 7, 15, 30, 90, and 120 days after vaccination (DAV) with first dose, with a second dose at 90 DAV. Participants tested every two weeks were clustered according to any previous SARS-Cov-2 PCR positive result (COVID-19) or otherwise . The number of IFN-γ produced cells was assessed by ELISpot and levels of IL-10, IFNL3/IL-28B, and D-Dimers were quantified in plasma by Luminex technology.Results: Considering the complete cohort the number of cells producing IFN-γ presented enhanced levels 15DAV and 120DAV. Clustering participants according to the previous infection at 15DAV noCOVID-19 presented augmented levels, without differences before (0DAV) or after complete vaccination (120DAV). Luminex analyses do not present significant differences either considering vaccination follow-up, or previous infection. Conclusion:It remains elusive the cutoffs for protective memory cellular responses to achieve disease protection. Although, our results demonstrated that ChAdOx1 nCoV-19 elicits IFN-γ cellular responses both after the first and second dose, without pro-inflammatory or prothrombotic responses. Besides, here it was observed that previous SARS-Cov-2 infection modulates cellular response kinetics, presenting a faster IFN-γ production, which does not reflect on the positive final responses after complete vaccination.
The immune response is crucial for coronavirus disease 19 (COVID-19) progression, with the participation of proinflammatory cells and cytokines, inducing lung injury and loss of respiratory function. CLEC5A expression on monocytes can be triggered by viral and bacterial infections, leading to poor outcomes. SARS-CoV-2 is able to induce neutrophil activation by CLEC5A and Toll-like receptor 2, leading to an aggressive inflammatory cascade, but little information is known about the molecular interactions between CLEC5A and SARS-CoV-2 proteins.Objectives: Here, we aimed to explore how CLEC5A expression could be affected by SARS-CoV-2 infection using immunological tools with in vitro, in vivo and in silico assays.Methodology: Molecular docking modeling was performed through the ClusPro 2.0 and Pymol 2.5 software. PBMCs were subjected to assays ex vivo immunophenotyping with commercial antibodies to characterize the monocyte subpopulations and Clec5a expression. The PBMC were isolated by Ficoll-Paque® and analyzed by flow cytometry. The samples were divided into three groups: unexposed (n=18), mild COVID-19 (n=17) and severe . Quantification of the cytokines IL-2, IFN-γ, IL-6, and IL-1β was performed using an in-house multiplex liquid microarray test. Detection of CLEC5A gene expressed in blood from hamsters was performed by RT-qPCR. Blood samples were obtained at days 3, 5, 10, and 15 through exsanguination by cardiac puncture from a 36 Syrian golden hamster (Mesocricetus auratus) at 1 year of age and 150 ± 1.4 g infected intranasally with SARS-CoV-2 strains Delta (1.0 × 106 PFU/ml) and Omicron (1.0 × 106 PFU/ml). Results:The findings revealed that high levels of CLEC5A expression were found in monocytes from severe COVID-19 patients in comparison with mild COVID-19 and unexposed subjects, but not in vaccinated subjects who developed mild COVID-19. In hamsters, we detected CLEC5A gene expression during 3-15 days of Omicron strain viral challenge. Our results also showed that CLEC5A can interact with SARS-CoV-2, promoting inflammatory cytokine production, probably through an interaction with the receptor binding domain in the N-acetylglucosamine binding site (NAG-601). The high expression of CLEC5A and high levels of proinflammatory cytokine production were reduced in vitro by a human CLEC5A monoclonal antibody. Conclusion:CLEC5A was triggered by spike glycoprotein, suggesting its involvement in COVID-19 progression; therapy with a monoclonal antibody could be a good strategy for COVID-19 treatment, but vaccines are still the best option to avoid hospitalization/deaths.
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