The protective effect of solidagenone from Solidago chilensis Meyen in a mouse model of airway inflammation.

Solidagenone is the main active constituent present in Solidago chilensis Meyen which is used in folk medicine to treat pain and inflammatory diseases. This study aimed to evaluate the anti-inflammatory activity of solidagenone in vitro and in a model of allergic airway inflammation. In vitro studies were performed in activated macrophages and lymphocytes. BALB/c mice were sensitized and challenged with ovalbumin and treated with solidagenone orally (30 or 90 mg/kg body weight) or dexamethasone, as a positive control in our in vivo analysis. Supernatant concentrations of nitrite, TNF and IL-1ß, as well as gene expression of pro-inflammatory mediators in macrophages cultures, were reduced after solidagenone treatment, without affecting macrophages viability. Besides, solidagenone significantly decreased T cell proliferation and secretion of IFNγ and IL-2. Th2 cytokine concentrations and inflammatory cell counts, especially eosinophils, in bronchoalveolar lavage fluid were reduced in mice treated with solidagenone. Histopathological evaluation of lung tissue was performed, and morphometrical analyses demonstrated reduction of cellular infiltration and mucus hypersecretion. Altogether, solidagenone presented anti-inflammatory activity in vitro and in vivo in the OVA-induced airway inflammation model, suggesting its promising pharmacological use as an anti-inflammatory agent for allergic hypersensitivity.

Leukemia Inhibitory Factor (LIF) Overexpression Increases the Angiogenic Potential of Bone Marrow Mesenchymal Stem/Stromal Cells.

Mesenchymal stem/stromal cells (MSCs) have the ability to secrete bioactive molecules, exerting multiple biological effects, such as tissue regeneration, reduction of inflammation, and neovascularization. The therapeutic potential of MSCs can be increased by genetic modification to overexpress cytokines and growth factors. Here we produced mouse MSCs overexpressing human leukemia inhibitory factor (LIF) to assess their proangiogenic potential in vitro and in vivo. Mouse bone marrow-derived MSCs were transduced by using a second-generation lentiviral system to express human LIF. Leukemia inhibitory factor expression was confirmed by RT-qPCR and by ELISA, allowing the quantification of the transcript and secreted protein, respectively. Flow cytometry analysis and trilineage differentiation assay showed that the MSC_LIF cell line maintained the immunophenotype and a multipotency characteristic of MSCs. The immunosuppressive activity of MSC_LIF was confirmed using a lymphoproliferation assay. Moreover, gene expression analysis demonstrated upregulation of genes coding for strategic factors in the neovascularization process, such as angiogenin, IL-8, MCP-1, and VEGF, and for the perivascular cell markers αSMA, Col4a1, SM22, and NG2. To evaluate the pro-angiogenic potential of MSC_LIF, we first tested its effects on endothelial cells obtained from umbilical vein in a scratch wound healing assay. Conditioned medium (CM) from MSC_LIF promoted a significant increase in cell migration compared to CM from control MSC. Additionally, in vitro tube formation of endothelial cells was increased by the presence of MSC_LIF, as shown in microvessel sprouting in aortic ring cultures. Finally, an in vivo Matrigel plug assay was performed, showing that MSC_LIF were more potent in promoting in vivo angiogenesis and tissue vascularization than control MSCs. In conclusion, LIF overexpression is a promising strategy to increase the proangiogenic potential of MSCs and sets precedents for future investigations of their potential applications for the treatment of ischemic diseases and tissue repair.

Genetic Engineering as a Strategy to Improve the Therapeutic Efficacy of Mesenchymal Stem/Stromal Cells in Regenerative Medicine.

Mesenchymal stem/stromal cells (MSCs) have been widely studied in the field of regenerative medicine for applications in the treatment of several disease settings. The therapeutic potential of MSCs has been evaluated in studies in vitro and in vivo, especially based on their anti-inflammatory and pro-regenerative action, through the secretion of soluble mediators. In many cases, however, insufficient engraftment and limited beneficial effects of MSCs indicate the need of approaches to enhance their survival, migration and therapeutic potential. Genetic engineering emerges as a means to induce the expression of different proteins and soluble factors with a wide range of applications, such as growth factors, cytokines, chemokines, transcription factors, enzymes and microRNAs. Distinct strategies have been applied to induce genetic modifications with the goal to enhance the potential of MCSs. This review aims to contribute to the update of the different genetically engineered tools employed for MSCs modification, as well as the factors investigated in different fields in which genetically engineered MSCs have been tested.

Phenotype instability of hepatocyte-like cells produced by direct reprogramming of mesenchymal stromal cells.

BACKGROUND: Hepatocyte-like cells (iHEPs) generated by transcription factor-mediated direct reprogramming of somatic cells have been studied as potential cell sources for the development of novel therapies targeting liver diseases. The mechanisms involved in direct reprogramming, stability after long-term in vitro expansion, and safety profile of reprogrammed cells in different experimental models, however, still require further investigation. METHODS: iHEPs were generated by forced expression of Foxa2/Hnf4a in mouse mesenchymal stromal cells and characterized their phenotype stability by in vitro and in vivo analyses. RESULTS: The iHEPs expressed mixed hepatocyte and liver progenitor cell markers, were highly proliferative, and presented metabolic activities in functional assays. A progressive loss of hepatic phenotype, however, was observed after several passages, leading to an increase in alpha-SMA+ fibroblast-like cells, which could be distinguished and sorted from iHEPs by differential mitochondrial content. The resulting purified iHEPs proliferated, maintained liver progenitor cell markers, and, upon stimulation with lineage maturation media, increased expression of either biliary or hepatocyte markers. In vivo functionality was assessed in independent pre-clinical mouse models. Minimal engraftment was observed following transplantation in mice with acute acetaminophen-induced liver injury. In contrast, upon transplantation in a transgenic mouse model presenting host hepatocyte senescence, widespread engraftment and uncontrolled proliferation of iHEPs was observed, forming islands of epithelial-like cells, adipocyte-like cells, or cells presenting both morphologies. CONCLUSION: The results have significant implications for cell reprogramming, suggesting that iHEPs generated by Foxa2/Hnf4a expression have an unstable phenotype and depend on transgene expression for maintenance of hepatocyte-like characteristics, showing a tendency to return to the mesenchymal phenotype of origin and a compromised safety profile.

Betulinic Acid Derivative BA5, Attenuates Inflammation and Fibrosis in Experimental Chronic Chagas Disease Cardiomyopathy by Inducing IL-10 and M2 Polarization.

Chronic Chagas disease cardiomyopathy (CCC) is a major cause of heart disease in Latin America and treatment for this condition is unsatisfactory. Here we investigated the effects of BA5, an amide semi-synthetic derivative betulinic acid, in a model of CCC. Mice chronically infected with T. cruzi were treated orally with BA5 (10 or 1 mg/Kg), three times per week, for 2 months. BA5 treatment decreased inflammation and fibrosis in heart sections but did not improve exercise capacity or ameliorate cardiac electric disturbances in infected mice. Serum concentrations of TNF-α, IFN-γ, and IL-1ß, as well as cardiac gene expression of pro-inflammatory mediators, were reduced after BA5 treatment. In contrast, a significant increase in the anti-inflammatory cytokine IL-10 concentration was observed in BA5-treated mice in both tested doses compared to vehicle-treated mice. Moreover, polarization to anti-inflammatory/M2 macrophage phenotype was evidenced by a decrease in the expression of NOS2 and proinflammatory cytokines and the increase in M2 markers, such as Arg1 and CHI3 in mice treated with BA5. In conclusion, BA5 had a potent anti-inflammatory activity on a model of parasite-driven heart disease related to IL-10 production and a switch from M1 to M2 subset of macrophages.

Betulinic acid derivative BA5, a dual NF-kB/calcineurin inhibitor, alleviates experimental shock and delayed hypersensitivity.

Betulinic acid (BA) is a naturally occurring triterpenoid with several biological properties already described, including immunomodulatory activity. Here we investigated the immunomodulatory activity of eight semi-synthetic amide derivatives of betulinic acid. Screening of derivatives BA1-BA8 led to the identification of compounds with superior immunomodulatory activity than BA on activated macrophages and lymphocytes. BA5, the most potent derivative, inhibited nitric oxide and TNFα production in a concentration-dependent manner, and decreased NF-κB activation in Raw 264.7 cells. Additionally, BA5 inhibited the proliferation of activated lymphocytes and the secretion of IL-2, IL-4 IL-6, IL-10, IL-17A and IFNÉ£, in a concentration-dependent manner. Flow cytometry analysis in lymphocyte cultures showed that treatment with BA5 induces cell cycle arrest in pre-G1 phase followed by cell death by apoptosis. Moreover, BA5 also inhibited the activity of calcineurin, an enzyme that plays a critical role in the progression of cell cycle and T lymphocyte activation. BA5 has a synergistic inhibitory effect with dexamethasone on lymphoproliferation, showing a promising profile for drug combination. Finally, we observed immunosuppressive effects of BA5 in vivo in mouse models of lethal endotoxemia and delayed type hypersensitivity. Our results reinforce the potential use of betulinic acid and its derivatives in the search for potent immunomodulatory drugs.

Mesenchymal Stem cells in the context of canine atopic dermatitis: A Review / Células-tronco mesenquimais no contexto da dermatite atópica canina: uma revisão

Canine atopic dermatitis (CAD) is a chronic inflammatory skin disease and has a high frequency among dermatological diseases. The interaction of genetic factors, skin and environmental conditions affect the expression of the disease, developing a complex pathology. Current multimodal treatment has numerous adverse effects and variations in its efficacy and safety, demonstrating the need to develop safe and effective therapeutic resources for patients with CAD. Mesenchymal stem cells (MSCs) are multipotent cells, with special characteristics, such as self-renewal, immunomodulatory properties, and de-differentiation, making them useful for several clinical problems. The discovery of the immunosuppressive effect of MSCs on T cells has opened the potential for new perspectives with its use as a therapeutic agent for immune diseases, such as CAD. The scarce number of research using the MSC as a treatment for CAD result in the lack of knowledge about the benefits and possible protocols to be followed for the use of this cell therapy. In this review, we highlighted the clinical studies and potential biological mechanisms of MSC-based cell therapy effects attenuating canine atopic dermatitis compared to conventional treatment, which might lead to a safe improvement of the animals clinical condition in a short period without causing adverse effects.(AU)

A dermatite atópica canina (DAC) é uma doença inflamatória crônica da pele e tem alta frequência entre as doenças dermatológicas. A interação de fatores genéticos, pele e condições ambientais afetam a expressão da doença, desenvolvendo uma patologia complexa. O tratamento multimodal atual apresenta inúmeros efeitos adversos e variações em sua eficácia e segurança, demonstrando a necessidade de desenvolver recursos terapêuticos seguros e eficazes para pacientes com DAC. As células-tronco mesenquimais (CTM) são células multipotentes, com características especiais, como auto renovação, propriedades imunomoduladoras e desdiferenciação, tornando-se úteis para diversos problemas clínicos. A descoberta do efeito imunossupressor das CTMs sobre as células T abriu o potencial para novas perspectivas com sua utilização como agente terapêutico para doenças imunológicas, como a DAC. O escasso número de pesquisas utilizando o MSC como tratamento para DAC resulta no desconhecimento dos benefícios e dos possíveis protocolos a serem seguidos para a utilização dessa terapia celular. Nesta revisão, destacamos os estudos clínicos e os potenciais mecanismos biológicos dos efeitos da terapia celular baseada em MSC que atenuam a dermatite atópica canina em comparação com o tratamento convencional, podendo levar a uma melhora segura da condição clínica do animal em um curto período, sem causar efeitos adversos.(AU)

Mesenchymal Stem cells in the context of canine atopic dermatitis: A Review

ABSTRACT Canine atopic dermatitis (CAD) is a chronic inflammatory skin disease and has a high frequency among dermatological diseases. The interaction of genetic factors, skin and environmental conditions affect the expression of the disease, developing a complex pathology. Current multimodal treatment has numerous adverse effects and variations in its efficacy and safety, demonstrating the need to develop safe and effective therapeutic resources for patients with CAD. Mesenchymal stem cells (MSCs) are multipotent cells, with special characteristics, such as self-renewal, immunomodulatory properties, and de-differentiation, making them useful for several clinical problems. The discovery of the immunosuppressive effect of MSCs on T cells has opened the potential for new perspectives with its use as a therapeutic agent for immune diseases, such as CAD. The scarce number of research using the MSC as a treatment for CAD result in the lack of knowledge about the benefits and possible protocols to be followed for the use of this cell therapy. In this review, we highlighted the clinical studies and potential biological mechanisms of MSC-based cell therapy effects attenuating canine atopic dermatitis compared to conventional treatment, which might lead to a safe improvement of the animals clinical condition in a short period without causing adverse effects.

RESUMO A dermatite atópica canina (DAC) é uma doença inflamatória crônica da pele e tem alta frequência entre as doenças dermatológicas. A interação de fatores genéticos, pele e condições ambientais afetam a expressão da doença, desenvolvendo uma patologia complexa. O tratamento multimodal atual apresenta inúmeros efeitos adversos e variações em sua eficácia e segurança, demonstrando a necessidade de desenvolver recursos terapêuticos seguros e eficazes para pacientes com DAC. As células-tronco mesenquimais (CTM) são células multipotentes, com características especiais, como auto renovação, propriedades imunomoduladoras e desdiferenciação, tornando-se úteis para diversos problemas clínicos. A descoberta do efeito imunossupressor das CTMs sobre as células T abriu o potencial para novas perspectivas com sua utilização como agente terapêutico para doenças imunológicas, como a DAC. O escasso número de pesquisas utilizando o MSC como tratamento para DAC resulta no desconhecimento dos benefícios e dos possíveis protocolos a serem seguidos para a utilização dessa terapia celular. Nesta revisão, destacamos os estudos clínicos e os potenciais mecanismos biológicos dos efeitos da terapia celular baseada em MSC que atenuam a dermatite atópica canina em comparação com o tratamento convencional, podendo levar a uma melhora segura da condição clínica do animal em um curto período, sem causar efeitos adversos.

Geração de células semelhantes a hepatócitos por reprogramação direta de células-tronco mesenquimais

O transplante hepático permanece como o único tratamento eficaz disponível para hepatopatias graves. Entretanto, fatores como a escassez de órgãos viáveis e a rejeição imunológica limitam significativamente este tipo de terapia. O transplante de hepatócitos é uma opção terapêutica em estudo, contudo, a perda de funções metabólicas após técnicas convencionais de cultivo e a inabilidade de proliferar in vitro impedem a reprodução em larga escala deste tipo de procedimento. Devido a estes fatores, uma fonte extra e autóloga de hepatócitos poderia suprir essa carência. Estudos recentes apresentam as células-tronco mesenquimais (CTM) como uma fonte promissora de células hepáticas a partir de reprogramação direta com superexpressão de fatores de transcrição envolvidos no desenvolvimento embrionário hepático. OBJETIVO: O presente estudo visou estabelecer um protocolo para geração de células semelhantes a hepatócitos, do ponto de vista morfológico,fenotípico e funcional, através da reprogramação direta de CTMs. MÉTODOS: A reprogramação hepática foi induzida a partir da superexpressão dos fatores de transcrição FOXA2 e HNF4α através de sistema lentiviral de segunda geração e cultivo das CTM modificadas em meio de indução da diferenciação hepática contendo fatores como HGF, FGF-4 e EGF. As células reprogramadas foram caracterizadas fenotipicamente, ultra-estruturalmente e seu perfil de expressão gênica e propriedades funcionais foram avaliados. Protocolos utilizando cell sorting e pequenas moléculas foram estabelecidos para purificação e maturação da linhagem celular reprogramada. RESULTADOS: Após o cultivo em meio de indução da diferenciação hepática, foi observada a presença de colônias com morfologia epitelial característica de células que expressam marcadores de hepatócitos adultos, tais como albumina,tirosina aminotransferase (TAT) e alfa-1-antitripsina (AAT). A linhagem também apresentou características funcionais como capacidade de estocar glicogênio, lipídios, captação e depuração de indocianina verde, assim como expressão e atividade de enzimas do complexo citocromo P450. A análise ultraestrutural demonstrou características citoplasmáticas semelhantes às de hepatócitos primários como por exemplo o montante de conteúdo mitocondrial. A diferença de conteúdo mitocondrial entre células reprogramadas e CTM foi utilizada para purificação da linhagem por cell sorting. Após a etapa de maturação, foi observada o surgimento de células com aspecto de hepatócitos primários e de células com perfil de colangiócitos. CONCLUSÃO: O protocolo de reprogramação hepática direta desenvolvido mostrou-se eficiente e robusto, gerando células com propriedades hepáticas funcionais que podem ser utilizadas em plataformas de testes de toxicidade de drogas, estudos do desenvolvimento hepático, além de abrir perspectivas de novas alternativas terapêuticas para hepatopatias crônicas e agudas.

Avaliação farmacológica da tiossemicarbazona triapina na infectividade do T. cruzi e na proliferação espontânea em células leucêmicas infectadas pelo HTLV-1

A Triapina é um quelante de ferro que inibe a proliferação de diversas linhagens tumorais. Doenças infecciosas como a doença de Chagas e o HTLV-1 permanecem sem um tratamento adequado, tornando necessária a busca de novos medicamentos. No presente trabalho, investigamos a atividade anti-T. cruzi e o potencial inibitório da proliferação de células leucêmicas infectadas pelo HTLV-1 da triapina. O efeito tripanocida foi avaliado inicialmente através da determinação de valores de CI50 para formas tripomastigota de T. cruzi (cepa Y). A eficácia da triapina na infecção aguda por T. cruzi foi avaliada utilizando camundongos BALB/c infectados com T. cruzi e tratados com diferentes doses da triapina. A eficácia do tratamento foi avaliada pelo monitoramento da parasitemia. Através de ensaios de microscopia eletrônica, foi possível identificar as principais alterações induzidas no parasito pela triapina. Por último, o potencial inibitório da proliferação de células leucêmicas infectadas pelo HTLV-1 foi determinado em células da linhagem MT2. Observamos uma potente atividade tripanocida da triapina frente às formas tripomastigotas do T. cruzi, onde a mesma apresentou um valor de CI50 de 1,7 µM. Sobre as mesmas condições, a droga de referência, o benzonidazol, apresentou um valor de CI50 de 11,4 µM. Também observamos uma potente atividade da triapina nos ensaios in vivo, evidenciada por uma redução significativa (P < 0.05) da parasitemia nos animais tratados com a triapina. A microscopia eletrônica de transmissão revelou que o tratamento de tripomastigotas com a triapina resultou em alterações na mitocôndria, cinetoplasto, complexo de Golgi e o aparecimento de vacúolos atípicos. Para atividade antiproliferativa frente a células infectadas com o HTLV-1, a triapina apresentou um valor de CI50 de 1,0 µM, enquanto a ciclosporina A apresentou um valor de CI50 de 0,9 µM. Os resultados demonstram que a triapina possui uma potente atividade antiproliferativa e antiparasitária