In vitro investigation of Dimethylsulfoxide upon human lymphocyte proliferation

Dimetilsulfóxido (DMSO) é um solvente amplamente utilizado para diluir ou incorporar componentes ou misturas em meios aquosos para ensaios biológicos in vitro. Neste trabalho, seus efeitos sobre a proliferação de linfócitos humanos foram avaliados observando-se a multiplicação celular por citometria de fluxo, análise morfológica em citocentrifugados corados e quantificação de AgNOR. Células mononucleares de sangue periférico humano foram induzidas a proliferar por fitohemaglutinina na presença de DMSO (150-300mM), por 5 dias a 37°C e 5% de CO2. Observou-se que o DMSO inibiu significativamente, proporcional à concentração, a proliferação de linfócitos. A morfologia celular mostrou predominância de linfócitos nas culturas tratadas com DMSO, enquanto baixos números de AgNORs, que são segmentos de DNA que transcrevem RNA ribossomal, foram encontrados. Estes resultados demonstram que o DMSO é um solvente inapropriado para estudos biológicos que envolvam imunomodulação de linfócitos humanos.Dimethylsulfoxide (DMSO) is a solvent widely used for diluting or including compounds or mixtures for in vitro biological assays. In this work, the effects of DMSO on human lymphocytes proliferation were evaluated through cell multiplication in a flow cytometer, morphological analyses on stained smears, and AgNOR quantification. Human peripheral blood mononuclear cells induced to proliferate by phytohemagglutinin were treated with DMSO (150- 300mM) for 5 days at 37 °C, and 5% CO2. DMSO inhibited significantly the lymphocyte proliferation in a dosedependent manner. Lymphocyte morphology predominated in DMSO-treated cultures while low numbers of Ag- NOR, which are DNA segments that transcribe ribosomal RNA, were found. The results showed that DMSO is not appropriate for using as a solvent for biological assays involving human lymphocytes immunomodulation.Colegio de Farmacéuticos de la Provincia de Buenos Aire

Multiplexed Flow Cytometric Approach for Detection of Anti-SARS-CoV-2 IgG, IgM and IgA Using Beads Covalently Coupled to the Nucleocapsid Protein

Flow cytometry has emerged as a promising technique for detection of SARS-CoV-2 antibodies. In this study, we described a new methodology to detect simultaneously IgG, IgM and IgA of SARS-CoV-2 nucleocapsid protein in human serum by flow cytometry. The Nucleocapsid protein was covalently bound on functional beads surface applying sulfo-SMCC chemistry. BUV395 anti-IgG, BB515 anti-IgM, biotinylated anti-IgA1/IgA2 and BV421 streptavidin were used as fluorophore conjugated secondary antibodies. Serum and antibodies reaction conditions were optimized for each antibody isotype detection and a multiplexed detection assay was developed. This new cell-free multiplex approach based on flow cytometry was able to efficiently discriminate COVID-19 negative and positive samples. The simultaneous detection of IgG, IgM and IgA showed a sensibility of 88.5-96.2% and specificity of 100%. The combined detection of antibody isotypes offers greater spectrum for detection and monitoring of COVID-19 vaccines and seroconversion. This novel strategy opens a new avenue for flow cytometry-based diagnosis.</p

Environmental Contaminants Modulate Breast Cancer Development and Outcome in TP53 p.R337H Carriers and Noncarriers

Two major concerns associated with cancer development in Paran&aacute; state, South Brazil, are environmental pollution and the germline TP53 p.R337H variant found in 0.27&ndash;0.30% of the population. We assessed breast cancer (BC) risk in rural (C1 and C2) and industrialized (C3) subregions, previously classified by geochemistry, agricultural productivity, and population density. C2 presents lower organochloride levels in rivers and lower agricultural outputs than C1, and lower levels of chlorine anions in rivers and lower industrial activities than C3. TP53 p.R337H status was assessed in 4658 women aged &gt;30 years from C1, C2, and C3, subsequent to a genetic screening (Group 1, longitudinal study). BC risk in this group was 4.58 times higher among TP53 p.R337H carriers. BC prevalence and risk were significantly lower in C2 compared to that in C3. Mortality rate and risk associated with BC in women aged &gt;30 years (n = 8181 deceased women; Group 2) were also lower in C2 than those in C3 and C1. These results suggest that environmental factors modulate BC risk and outcome in carriers and noncarriers