Genótipo e expressão da proteína fluorescente verde melhorada em ratos da linhagem LEW-Tg (EGFP) F455.5/Rrrc

The Green fluorescent protein (GFP) was first described after being extracted from Aequorea victoria in 1987. Since then, GFP and its derivatives have been widely used in several experiments as cell and protein marker. In the present study it was verified the genotype of the offspring from crosses between heterozygote Lewis LEW-Tg (EGFP) F455.5/ Rrrc rats and analyzed the expression of the enhanced green fluorescent protein (EGFP) in different cell types and genotypes. The genotype of the offspring was assessed by PCR and analysis of EGFP expression in different cells and genotypes, including mesenchymal stem cells (MSC) derived from adipose tissue and calvarial osteoblast cells. Expression of EGFP was verified by flow cytometry, fluorescence microscopy, and immunostaining. Through these methods, it was identified the genotypes of the offspring and determined the levels of expression of EGFP in two cell types. A difference in expression between the (EGFP +/+) and (EGFP +/-) genotypes was also observed in addition to the presence of autofluorescence. Further studies on the natural fluorescence of cells with the (EGFP +/-) genotype and that induced by presence of the EGFP are necessary.A proteína fluorescente verde (GFP) foi descrita pela primeira vez após ter sido extraída de Aequorea victoria em 1987. Desde então, a GFP e seus derivados têm sido amplamente utilizados em várias experiências como marcador celular e de proteínas. O objetivo do presente estudo foi o de verificar o genótipo dos descendentes de cruzamentos entre ratos Lewis LEW-Tg (EGFP) F455.5/Rrrc heterozigotos e de analisar a expressão da proteína fluorescente verde melhorada (EGFP) em diferentes tipos celulares e genótipos. O genótipo da descendência foi avaliado por PCR e pela análise da expressão da EGFP em diferentes células e genótipos, incluindo-se as células-tronco mesenquimais (MSC) derivadas de tecido adiposo e de osteoblastos de calvária. A expressão da EGFP foi verificada por citometria de fluxo, microscopia de fluorescência e imunocoloração. Foram, identificados os genótipos da descendência e determinados os níveis de expressão de EGFP em dois tipos de células. Foi também constatada uma diferença de expressão entre os genótipos (EGFP +/+) e (EGFP +/-) além da presença de autofluorescência. Mais estudos são necessários para esclarecer a fluorescência natural de células com o genótipo (EGFP +/-) e aquela induzida pela presença da EGFP

Heating Capacity and Biocompatibility of Hybrid Nanoparticles for Magnetic Hyperthermia Treatment

Cancer is one of the deadliest diseases worldwide and has been responsible for millions of deaths. However, developing a satisfactory smart multifunctional material combining different strategies to kill cancer cells poses a challenge. This work aims at filling this gap by developing a composite material for cancer treatment through hyperthermia and drug release. With this purpose, magnetic nanoparticles were coated with a polymer matrix consisting of poly (L-co-D,L lactic acid-co-trimethylene carbonate) and a poly(ethylene oxide)–poly(propylene oxide)–poly(ethylene oxide) triblock copolymer. High-resolution transmission electron microscopy and selected area electron diffraction confirmed magnetite to be the only iron oxide in the sample. Cytotoxicity and heat release assays on the hybrid nanoparticles were performed here for the first time. The heat induction results indicate that these new magnetic hybrid nanoparticles are capable of increasing the temperature by more than 5 °C, the minimal temperature rise required for being effectively used in hyperthermia treatments. The biocompatibility assays conducted under different concentrations, in the presence and in the absence of an external alternating current magnetic field, did not reveal any cytotoxicity. Therefore, the overall results indicate that the investigated hybrid nanoparticles have a great potential to be used as carrier systems for cancer treatment by hyperthermia

SET domain-containing protein 4 (SETD4) is a newly identified cytosolic and nuclear Lysine Methyltransferase involved in breast cancer cell proliferation

Cancer is comprised of a multitude of epigenetic abnormalities, including the global loss and regional gain of DNA methylation as well as alterations in histone methylation. Here, we characterize a new methyltransferase, SET domain-containing protein 4 (SETD4), which is involved in breast carcinogenesis. Quantitative real-time PCR (qPCR) showed elevated expression levels of SETD4 in several breast cancer cell lines. SETD4 overexpression was confirmed by western blot analysis suggesting a correlation between high expression of SETD4 and a lack of the estrogen receptor (ER) in breast cancer. In addition, cell fractionation studies and confocal immunofluorescence revealed the nuclear and non-nuclear localization of this new protein. SETD4 knockdown in breast cancer cell lines significantly suppressed their proliferation and delayed the G1/S cell cycle transition without affecting apoptosis. Furthermore, western blot analysis showed that knockdown of SETD4 decreased cyclin D1 expression, revealing the involvement of SETD4 in cell cycle regulation. These data imply that SETD4 plays a crucial role in breast carcinogenesis and could be a novel molecular target for the development of new strategies for the diagnosis and treatment of breast cancer

Evaluation of mesenchymal cells and dapsone for the treatment of dermonecrotic wounds caused by Loxosceles laeta venom in rabbits

This study aimed to evaluate the efficacy of mesenchymal stem cells (MSC), alone or associated with dapsone (DAP), in treating dermonecrotic wounds caused by Loxosceles laeta venom. Twenty-five male rabbits were distributed into five groups. Negative control received ultrapure water (C-), whilst all other groups were injected with 20 μg of L. laeta venom. After 4 hours, each group received one of the following treatments: PBS (C+), DAP, MSC, and DAP+MSC. Animals were evaluated daily and photographic records made for analysis of wound area. Twelve days after, animals were euthanized and skin samples removed for histological analysis. We observed that DAP showed the best percentage of wound contraction at day 3. In the treatments using MSCs, a negative value of wound contraction was observed for the isolated MSCs, as well as a lower contraction value for the association of the MSC + DAP when compared to PBS, probably, by the increase in initial inflammation after the application of stem cells, due to the fact that MSCs secrete a broad spectrum of bioactive molecules such as cytokines and growth factors that favor regeneration. Histologically, it was observed that animals of C+ showed extensive areas of necrosis, ulcers, neutrophilic infiltrate, and mineralization. Collagen deposition showed increase in MSC+DAP treatment, however vascularization remained unchanged. This is the first report using MSC and MSC+DAP as a treatment for cutaneous loxoscelism and more studies are needed to determine its use as an alternative therapy for dermonecrotic lesions caused by Loxosceles spider. Key words: loxoscelism; MSC; stem cells; spider venom; wound repair

Role of immune complexes from pacients with different clinical forms of Schistosomiasis in the modulation of in vitro granuloma reaction.

Schistosomiasis is a disease whose pathology is strongly related to the granulomatous reaction formed around parasite eggs trapped in host tissues. Studies have shown that the chronic intestinal form (INT) of this infection is associated with a variety of immunoregulatory mechanisms which lead to a diminished granulomatous reaction. Using an in vitro model of granuloma reaction, we show that immune complexes (IC) isolated from sera of INT patients are able to reduce granulomatous reaction developed by peripheral blood mononuclear cells (PBMC) from acute (AC), INT and hepatosplenic (HE) patients to soluble egg antigen (SEA)-conjugated polyacrylamide beads (PB-SEA). This inhibitory activity is also observed in cell proliferation assay of PBMC from INT and HE patients stimulated with SEA and adult worm antigen (SWAP). Furthermore, IC isolated from sera of patients with different clinical forms of the disease are also able to suppress INT patients PBMC reactivity. Therefore, our results show that circulating IC present in sera of patients with different clinical forms of schistosomiasis may downregulate PBMC reactivity to parasite antigens resulting in a diminished granuloma reaction to parasite eggs

Nitric oxide-mediated immune complex-induced prostaglandin E2 production by peripheral blood mononuclear cells of humans infected with Schistosoma mansoni.

Granuloma reaction around Schistosoma mansoni eggs is the prominent lesion in human schistosomiasis. Studies have suggested the involvement of a series of suppressive mechanisms in the control of this reaction. Using an in vitro model of granuloma formation, we have shown that immune complexes (IC) isolated from sera of chronic intestinal schistosomiasis patients were able to reduce granulomatous reaction developed against soluble egg antigen-conjugated polyacrylamide beads. In this system, the role of the Larginine– nitric oxide (NO) pathway in the formation of prostaglandin E2 (PGE2) by human peripheral blood mononuclear cells (PBMC) of patients infected with schistosomiasis was investigated using IC. Preincubation of PBMC with IC produced a significant increase of both nitrite and PGE2 levels in the cell supernatant. This effect was inhibited by coincubation of cells with Nv-nitro-L-arginine methyl ester (L-NAME), a NO synthase inhibitor, showing that the release of PGE2 subsequent to IC stimulation was driven by NO. The inhibitory effect of L NAME on PGE2 release by ICtreated PBMC was reversed by sodium nitroprusside, a known NO donor. Our results indicate that NO could be an important second signal for the stimulation of PGE2 production induced by IC in PBMC from human schistosomiasis patients