Abstract Background: Propolis exhibits huge potential in the pharmaceutical industry. In the present study, its effects were investigated on dendritic cells (DCs) stimulated with a tumor antigen (MAGE-1) and retinoic acid (RA) and on T lymphocytes to observe a possible differential activation of T lymphocytes, driving preferentially to Th1 or Treg cells. Methods: Cell viability, lymphocyte proliferation, gene expression (T-bet and FoxP3), and cytokine production by DCs (TNF-α, IL-10, IL-6 and IL-1β) and lymphocytes (IFN-γ and TGF-β) were analyzed. Results: MAGE-1 and RA alone or in combination with propolis inhibited TNF-α production and induced a higher lymphoproliferation compared to control, while MAGE-1 + propolis induced IL-6 production. Propolis in combination with RA induced FoxP3 expression. MAGE-1 induced IFN-γ production while propolis inhibited it, returning to basal levels. RA inhibited TGF-β production, what was counteracted by propolis. Conclusion: Propolis affected immunological parameters inhibiting pro-inflammatory cytokines and favoring the regulatory profile, opening perspectives for the control of inflammatory conditions.

ABSTRACT Background: Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs. Methods: Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis. Results: It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation. Conclusions: FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.

▪ ABSTRACT Introduction: Aiming to obtain autogenous and injectable lipografts from resected tissues in dermolipectomies, this study proposes a new method for harvesting and processing adipose tissue through a specific fragmenting device. The main objective was to establish a comparative analysis of the quality and viability characteristics of the new lipofragmentation technique and those of the well-known liposuction technique, widely accepted as a viable source of fat grafting. In vivo and in vitro assays were designed to evaluate the biological behavior of the samples to guide new and possible human studies with clinical applications. Methods: A post-bariatric patient who underwent abdominal dermolipectomy had her surgical specimen resected, which was divided into four parts that underwent liposuction and lipofragmentation, with and without prior infiltration. All samples were centrifuged and distributed for assays with assessments involving histological analysis, immunohistochemistry, flow cytometry, cell culture, and xenograft injection on the back of 10 Wistar rats, which was evaluated after six weeks for mass, volume, and histological features. Results: The structural characteristics and biological behaviors of fragmented, dry, and infiltrated fat samples were similar to those of liposuction samples. Conclusions: Fat fragmentation transformed the subcutaneous cellular tissue of dermolipectomies into a new, viable injectable lipograft variant, with biological characteristics similar to those of traditional liposuction. Although still preliminary, our results support further investigations to optimize the technique and improve fat grafting and its possible applications in regenerative medicine.

▪ RESUMO Introdução: Com o objetivo de obter lipoenxerto autógeno e injetável de tecido ressecado em dermolipectomias, este estudo propõe um novo método para colheita e processamento do tecido adiposo, através de um dispositivo fragmentador específico. O principal objetivo foi estabelecer uma análise comparativa das características de qualidade e viabilidade do novo lipofragmentado em relação ao já conhecido lipoaspirado, amplamente aceito como fonte viável de lipoenxerto. Ensaios in vivo e in vitro foram delineados para avaliar o comportamento biológico das amostras, a fim de orientar novos e possíveis estudos em humanos com aplicações clínicas. Métodos: Uma paciente pós-bariátrica que foi submetida a dermolipectomia abdominal teve sua peça cirúrgica ressecada e dividida em quatro partes que foram submetidas a Lipoaspiração e Lipofragmentação, sem e com infiltração prévia. Todas as amostras foram submetidas a centrifugação e então distribuídas para os ensaios que envolveram avaliação histológica, imunohistoquímica, citometria de fluxo, cultura celular e ainda a injeção de xenoenxerto no dorso de 10 ratos Wistar, retirados após seis semanas para avaliação de massa, volume e características histológicas. Resultados: As amostras de gordura fragmentada, seca e infiltrada, mostraram características estruturais e comportamento biológico semelhantes aos das amostras de lipoaspirado. Conclusões: A fragmentação da gordura transformou o tecido celular subcutâneo das dermolipectomias em uma nova variante de lipoenxerto injetável e viável, com características biológicas semelhantes àquelas do lipoaspirado tradicional. Embora ainda preliminares, nossos resultados embasam a realização de novas investigações buscando otimizar a técnica com vistas ao aprimoramento da enxertia gordurosa e suas possíveis aplicações na medicina regenerativa.

Abstract INTRODUCTION: Chronic hepatitis C is a leading cause of liver disease. Infection triggers an immediate immune response in the host that is mediated by humoral/cellular mechanisms. T cells respond to infection via secretion of cytokines, which inhibit or stimulate one another, leading to cytokine imbalance and ultimately affecting treatment. Studies using interferon (IFN) and ribavirin (RBV) showed that TCD8+ cells and cytokine levels are associated with sustainable virological response (SVR). However, studies that investigated the effects of triple therapy (TT) are limited. METHODS: The study included hepatitis C virus (HCV)+ RNA, naives, genotype 1, ≥18 years, and advanced fibrosis (F≥3) patients. Samples were collected at baseline and after 12 weeks (W12) of TT. Six cytokines were analyzed by flow cytometry. RESULTS: Of 31 patients, four were excluded (two deaths, one interrupted TT, and one F2 patient). Of the 27 remaining patients, 21 (78%) were cirrhotic. SVR was achieved in 63% of the patients. The patients had a mean age of 55.11 ± 10.03 years. Analyses at baseline showed that the chemokine CCL5/Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES) (p=0.04) and interleukin (IL)-6 (p=0.02), which was associated with SVR. RANTES (p=0.04) and IL-8 (p=0.01) levels were associated with SVR at W12. CONCLUSIONS Similar to patterns observed during double therapy, IL-6, IL-8, and RANTES levels were associated with SVR in TT, indicating the potential role of interferon in immune response to hepatitis C virus.

BACKGROUND The main clinical forms of paracoccidioidomycosis (PCM) are the acute/subacute form (AF) and the chronic form (CF), and they both display considerable clinical variability. The immune responses of PCM patients, during and after treatment, remain neglected, mainly in the case of CF patients, due to the high prevalence of pulmonary sequelae. OBJECTIVE To evaluate the distribution of whole blood T cell subsets, serum cytokines, and biomarkers of pulmonary fibrosis in PCM patients, according to the clinical form and at different time points, during the antifungal therapy. METHODS Eighty-seven PCM patients, from an endemic area in Brazil, were categorised into groups, according to the clinical form (AF or CF) and the moment of treatment. The peripheral blood T lymphocyte subsets of these patients were analysed using fluorescence-activated cell sorting. The serum levels of cytokines, basic fibroblast growth factor and surfactant protein-D (SP-D) were also analysed. FINDINGS In the CF patients, an expansion of the peripheral blood TCD4+ cells was observed during the treatment, and this persisted even after two years of antifungal treatment. In addition, these patients showed high serum levels of SP-D. CONCLUSION Our findings highlight the immunological changes CF patients undergo, during and after treatment, possibly due to the hypoxia triggered by pulmonary fibrosis and emphysema.

Abstract Purpose: To investigate the effects of cyclosporine A on renal ischemia-reperfusion injury during transient hyperglycemia in rats. Methods: In a model of ischemia-reperfusion-induced renal injury and transiently induced hyperglycemia by intraperitoneal injection of glucose, 2.5 g.kg-1, Wistar rats were anesthetized with either isoflurane or propofol and received intravenous cyclosporine A, 5 mg.kg-1, five minutes before reperfusion. Comparison groups were isoflurane and propofol sham groups and isoflurane and propofol ischemia-reperfusion-induced renal injury. Renal tubular cell viability was quantitatively assessed by flow cytometry after cell culture and classified as early apoptosis, necrotic cells, and intact cells. Results: Early apoptosis was significantly higher in isoflurane and propofol anesthetized animals subjected to renal ischemia-reperfusion injury when compared to both cyclosporine A treated and sham groups. Necrosis percentage was significantly higher in propofol-anesthetized animals subjected to renal ischemia-reperfusion injury. The percentage of intact cells was lower in both, isoflurane and propofol anesthetized animals subjected to renal ischemia-reperfusion injury. Conclusion: In a model of ischemia-reperfusion-induced renal injury, cyclosporine A, 5 m.kg-1, administered five minutes before renal reperfusion in rats with acute-induced hyperglycemia under either isoflurano or propofol anesthesia, attenuated early apoptosis and preserved viability in renal tubular cells, regardless of the anesthetic used.

Abstract Background Agaricus brasiliensis is a medicinal mushroom with immunomodulatory and antitumor activities attributed to the β-glucans presented in the polysaccharide fraction of its fruiting body. Since β-glucans enhance cellular immunoresponsiveness, in this study we aimed to evaluate the effect of an acid-treated polysaccharide-rich fraction (ATF) of A. brasiliensis on the ability of human monocytes to adhere/phagocyte C. albicans yeast cells, their expression of pattern recognition receptors and their ability to produce cytokines. Methods Adhesion/phagocytosis of FITC-labeled C. albicans was evaluated by flow cytometry. Cells were incubated with specific fluorochrome-labeled antibodies for TLR2 and 4, βGR and MR and also evaluated by flow cytometry. Monocytes were cultured with ATF, and culture supernatants were collected for analysis of in vitro cytokine production by ELISA (TNF-α, IL-1β, IL-12 and IL-10). Results ATF significantly increased the adherence/phagocytosis of C. albicans by monocytes and this was associated with enhanced expression of TLR2 and TLR4, while no effect was observed on βGR or MR. Moreover, expression of TLR4 and TLR2 was associated with higher levels of in vitro production of TNF-α and IL-1, respectively. Production of IL-10 was also increased by ATF treatment, but we found no association between its production and the expression of Toll-like receptors. Conclusion Our results provided us with evidence that A. brasiliensis polysaccharides affect human monocytes probably through the modulation of Toll-like receptors.

PURPOSE: To study the effect of isoflurane (Iso) or propofol (Prop) anesthesia on renal ischemia/reperfusion injury (IRI) during transient hyperglycemia. METHODS: Thirty six rats were randomly assigned into six groups of six animals each: PHS (Sham-Prop=1mg.kg-1.min-1 + Hyperglycemia=2.5g.kg-1 of glucose solution administered intraperitoneally); HIS (Sham-Iso + Hyperglycemia); PHI (Prop + Hyperglycemia + Ischemia); IHI (Iso + Hyperglycemia + Ischemia); PI (Prop + Ischemia), and II (Iso + Ischemia). After 30 minutes of anesthesia induction, right nephrectomy was performed (all animals) and the left renal artery was clamped during 25 minutes (ischemia). The animals were sacrificed after 24 hours and blood collection (to dose creatinine) and left kidney removal were performed for histological analysis, and flow cytometry (FCM): percentage of initial apoptosis (APTi) and viable cells (VC). RESULTS: Serum creatinine (mg/dL) was statistically different in groups PHI (3.60±0.40) and IHI (3.23±1.08), p<0.05. Histological analysis was statistically different in groups PHI (4.0[4.0;5.0]) and IHI (4.5[4.0;5.0]), p<0.05. APTi percentage was statistically different in groups PHI (73.2±7.1), and IHI (48.1±14). VC percentage was statistically different in groups PHI (25.8±6.9) and IHI (38.5±9.2), p<0.05. CONCLUSIONS: Propofol and isoflurane showed the same level of protection against ischemia/reperfusion injury in the normoglycemic groups. Transient hyperglycemia is associated with an increase in IRI.

Os primeiros estudos demonstrando o potencial de trandiferenciação neural das células-tronco mesenquimais (CTMs) provenientes da medula óssea (MO) foram conduzidos em camundogos e humanos no início da década de 2000. Após esse período, o número de pesquisas e publicações com o mesmo propósito tem aumentado, mas com raros ou escassos estudos na espécie equina. Nesse sentindo, o objetivo desse trabalho foi avaliar o potencial in vitro da transdiferenciação neural das CTMs provenientes da MO de equinos utilizando-se dois protocolos: P1 (forksolin e ácido retinóico) e P2 (2-βmecarptoetanol). Após a confirmação das linhagens mesenquimais, pela positividade para o marcador CD90 (X=97,94%), negatividade para o marcador CD34 e resposta positiva a diferenciação osteogênica, as CTMs foram submetidas a transdiferenciação neural (P1 e P2) para avaliação morfológica e expressão dos marcadores neurais GFAP e β3 tubulina por citometria de fluxo. Os resultados revelaram mudanças morfológicas em graus variados entre os protocolos testados. No protocolo 1, vinte quatro horas após a incubação com o meio de diferenciação neural, grande proporção de células (>80%) apresentaram morfologia semelhante a células neurais, caracterizadas por retração do corpo celular e grande número de projeções protoplasmáticas (filopodia). Por outro lado, de forma comparativa, já nos primeiros 30 minutos após a exposição ao antioxidante β-mercaptoetanol (P2) as CTMs apresentaram rápida mudança morfológica caracterizada principalmente por retração do corpo celular e menor número de projeções protoplasmáticas. Também ficou evidenciado com o uso deste protocolo, menor aderência das células após tempo de exposição ao meio de diferenciação, quando comparado ao P1. Com relação a análise imunofenotípica foi observado uma maior (P<0,001) expressão dos marcadores GFAP e β3 tubulina ao término do P2 quando comparado ao P1. A habilidade das CTMs em gerar tipos celulares relacionados a linhagem neural é complexa e multifatorial, dependendo não só dos agentes indutores, mas também do ambiente no qual estas células são cultivadas. Desta forma um maior número de estudos é necessário para o melhor entendimento do processo de transdiferenciação neural a partir de CTMs de equinos.

The first studies showing the potential of neural transdifferentiation of mesenchymal stem cells (MSCs) from bone marrow (BM) were conducted in camundogos and humans in the early 2000s. After this period, the number of research and publications with the same purpose increased, but with rare or scarce studies in horses. The aim of this study was to evaluate in vitro neuronal transdifferentiation potential of MSCs from equine BM using two protocols: P1 (forksolin and retinoic acid) and P2 (2-βmecarptoetanol). After confirming the mesenchymal lineages, by positivity for the marker CD90 (X=97.94%), negative for the marker CD34 and positive response for osteogenic differentiation, MSCs were subjected to neural transdifferentiation (P1 and P2) for morphological analysis and expression of neural markers GFAP and β3 tubulin by flow cytometry. The results revealed morphological changes in varying degrees between the tested protocols. In protocol 1, twenty four hours after incubation with the media of neural differentiation, a large proportion of cells (>80%) had similar morphology to neural cells, characterized by retraction of cellular body and a large number of cytoplasmic extension (filopodia). However, comparatively, within the first 30 minutes after exposure to the antioxidant β-mercaptoethanol (P2) MSCs showed rapid morphological changes characterized mainly by retraction of cellular body and less cytoplasmic extension. It was also evidenced with the use of this protocol, lower cellular adhesion after exposure to media when compared to P1. Regarding the immunophenotyping analysis it was observed a higher (P<0.001) expression of the markers GFAP and β3 tubulin at the end of P2 compared to P1. The ability of MSCs to generate cell types related to neural lineage is complex and multifactorial, depending not only of inducing agents, but also the environment in which these cells will be cultivated. Thus a greater number of studies are necessary to better understand the process of neural transdifferentiation of MSCs from equine.