Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Dimethylsulfoxide (DMSO) is an amphipathic molecule composed of a polar domain characterized by the sulfinyl and two nonpolar methyl groups, for this reason it is able to solubilize polar and nonpolar substances and transpose hydrophobic barriers. DMSO is widely used to solubilize drugs of therapeutic applications and studies indicated that 10% v/v concentration did not modify culture viability when used to treat human peripheral blood mononuclear cells (PBMC). However, some DMSO concentrations could influence lymphocyte activation and present anti-inflammatory effects. Therefore, the objective of this study was to evaluate the effect of DMSO on lymphocyte activation parameters. Cell viability analysis, proliferation, and cytokine production were performed on PBMC from six healthy subjects by flow cytometry. The results indicated that 2.5% v/v DMSO concentrations did not modify lymphocytes viability. DMSO at 1% and 2% v/v concentrations reduced the relative proliferation index of lymphocytes and at 5% and 10% v/v concentrations reduced the percentage of total lymphocytes, cluster of differentiation 4+ (CD4+) T lymphocytes and CD8+ T lymphocytes interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2) producers. Thus, it was concluded that DMSO has an in vitro anti-inflammatory effect by reducing lymphocyte activation demonstrated with proliferation reduction and the decrease of cytokine production.

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Control of Chagas disease is based on insecticide spraying in domiciles in order to exterminate triatomine populations. However, since the vectors differ in susceptibility to currently used insecticides, the screening of the toxic potential of Brazilian flora may identify new molecules lethal to triatomines. This study evaluated the toxicity of ethanolic extract of Ageratum fastigiatum (Gardner) R.M. King & H. Rob., Asteraceae, on Rhodnius nasutus, a known vector of Chagas disease. Ethanolic extracts of the aerial parts of A. fastigiatum were prepared at 25 and 50 mg/mL concentrations, and 5 µL was applied to fifth-instar nymphs of R. nasutus (n=30). Controls included nymphs that were treated with 5 µL ethanol (n=30) or left untreated (n=30). The percentage of dead insects in each group was observed at 24, 48, 72, 96 and 120 h after application. The extracts of A. fastigiatum showed a mortality rate of about 37% and 77% after 120 h, at concentrations of 25 and 50 mg/mL, respectively. In control groups, the mortality rate remained under 7%. The extract of A. fastigiatum contains a coumarin, a molecule with recognized toxicity in insects, and which may be responsible for killing the triatomines

Agareratum fastigiatum is a Brazilian medicinal plant used as anti-inflammaroty and for wound healing by the folk medicine. In vitro and in vivo studies involving A. fastigiatum essential oil (EOAF) showed indications of antiinflammatory activity, however, its effect on membrane integrins involved on cell migration is still unclear. Hence, it was evaluated in the present study the effect of EOAF on CD18 frequency on human lymphocytes. By using gas chromatography/mass spectrometry it was identified 9 compounds on EOAF: αpinene; β-pinene; β-myrcene; d-limonene; β-ocimene; sesquiterpenes; α-copaene; 4,8-β-epóxi-caryophyllene; germacrene and bicyclogermacrene. On in vitro tests, 6.25 x 10 -3 and 12.5 x 10 -3 µL/mL EOAF reduced CD18 frequency on phorbol-12-myristate-13-acetate (PMA)-stimulated lymphocytes. Such cells were obtained from peripheral blood of healthy volunteers, and were treated or not with EOAF. They were stained with fluorescent anti-CD18 monoclonal antibodies, after 24 hours incubation. Our data corroborates previous findings, indicating a possible anti-inflammatory activity of EOAF.

Pseudobrickellia brasiliensis (Asteraceae) is a plant commonly known as arnica-do-campo and belongs to the native flora of the Brazilian Cerrado. The alcoholic extract of the plant has been used as an anti-inflammatory agent in folk medicine, but the biological mechanism of action has not been elucidated. The present study evaluated the composition of P. brasiliensis aqueous extract and its effects on pro-inflammatory cytokine production and lymphocyte proliferation. The extracts were prepared by sequential maceration of P. brasiliensis leaves in ethanol, ethyl acetate, and water. Extract cytotoxicity was evaluated by trypan blue exclusion assay, and apoptosis and necrosis were measured by staining with annexin V-FITC and propidium iodide. The ethanolic (ETA) and acetate (ACE) extracts showed cytotoxic effects. The aqueous extract (AQU) was not cytotoxic. Peripheral blood mononuclear cells stimulated with phorbol myristate acetate and ionomycin and treated with AQU (100 μg/mL) showed reduced interferon (IFN)-γ and tumor necrosis factor (TNF)-α expression. AQU also inhibited lymphocyte proliferative response after nonspecific stimulation with phytohemagglutinin. The aqueous extract was analyzed by liquid chromatography coupled with photodiode array detection and mass spectrometry. Quinic acid and its derivatives 5-caffeoylquinic acid and 3,5-dicaffeoylquinic acid, as well as the flavonoids luteolin and luteolin dihexoside, were detected. All these compounds are known to exhibit anti-inflammatory activity. Taken together, these findings demonstrate that P. brasiliensis aqueous extract can inhibit the pro-inflammatory cytokine production and proliferative response of lymphocytes. These effects may be related to the presence of chemical substances with anti-inflammatory actions previously reported in scientific literature.

O uso de plantas medicinais é praticado na medicina popular, sobretudo, nos países em desenvolvimento. As plantas medicinais são elementos que constituem parte da biodiversidade e são largamente utilizadas desde os primórdios da civilização por vários povos e de diversas maneiras. Estas plantas têm se mostrado de importante relevância como medicamentos caseiros, visto que em suas composições possuem constituintes químicos eficazes para o tratamento de certas doenças. Neste estudo, o objetivo foi realizar uma revisão bibliográfica acerca das plantas medicinais utilizadas no município de Diamantina (MG) e seus entornos, a fim de compilar as informações obtidas para auxiliar nos avanços dos estudos. Utilizando os descritores Plantas medicinais, Diamantina e fitoterápicos foram encontrados 10 artigos, sendo as plantas mais citadas as espécies: Ageratum fastigiatum e Euphorbia tirucalli, as quais apresentaram as atividades sobre células do sistema imune em estudos in vitro. Novas pesquisas e estudos ainda são necessários para comprovação de propriedades farmacológicas e padronização do uso, portanto, ainda há poucas informações sobre a segurança e a confiabilidade das plantas medicinais e novos estudos devem ser apurados acerca do assunto.Palavras-chave: Plantas Medicinais. Vale do Jequitinhonha. Diamantina.AbstractThe use of medicinal plants is practiced by many populations, especially in developing countries. Medicinal plants are elements that constitute part of biodiversity and have been widely used since the dawn of civilization by various peoples and in various ways. They have been shown to be of significant relevance as home remedies, since in their compositions they have effectice chemical constituents for the treatment of certain diseases. In this work the objective was to carry out a bibliographical review about the medicinal plants used in the city of Diamantina (MG) and its surroundings, in order to compile the obtained information to support in the studies advances. Using the descriptors Medicinal plants, Diamantina and phytorerapics, 10 articles were found, the most cited being the species Ageratum fastigiatum and Euphorbia tirucalli, which presented the activities on the cells of the immune system in vitro studies. New research and studies are still necessary to prove pharmacological properties and standardization of the use, therefore, there is still little information about the safety and reliability of medicinal plants and new studies should be determined on the subject.Keywords: Medicinal Plants. Jequitinhonha Valley. Diamantina.