O silenciamento da ecto-5'-nucleotidase/CD73 altera a progressão de gliomas

O glioblastoma multiforme (GBM) é um tipo de tumor bastante agressivo, que apresenta uma elevada taxa de proliferação e um comportamento altamente invasivo. A ecto-5’-nucleotidase (e5NT/CD73) é uma enzima expressa de forma bastante ampla, a qual age na hidrólise do AMP extracelular, com consequente formação de adenosina, molécula com diversas funções celulares. Além da função enzimática, foi mostrado que a e5NT/CD73 também exerce importante papel na interação célula-célula e célula-matriz. Muitas evidências apontam para funções de promoção tumoral desempenhados pela e5NT/CD73, a qual se apresenta expressa em altos níveis em diferentes tumores. No presente estudo caracterizamos o papel desempenhado pela e5NT/CD73 no crescimento de GBM in vitro e in vivo através do silenciamento de sua expressão e atividade em células de glioma de rato C6. A e5NT/CD73 foi silenciada por RNA de interferência estável através de lentivírus e o silenciamento foi caracterizado por métodos de imunofluorescência e atividade enzimática. Além disso, investigamos parâmetros de malignidade in vitro, como adesão e migração celular. Para uma melhor análise do papel da proteína no crescimento tumoral, implantamos as células C6 silenciadas em um modelo de glioma em ratos. Análises patológicas e imunohistoquímicas usando o marcador nestina foram realizados. Foram obtidos dois clones silenciados de forma estável, C6-c54 e C6-c56, os quais tiveram uma significativa redução na expressão e atividade da e5NT/CD73. Foi observada uma significativa alteração no parâmetro de adesão in vitro de ambos clones silenciados, e esse efeito deve-se provavelmente à função de adesão exercida pela proteína e não da sua atividade enzimática, uma vez que o uso do inibidor APCP não foi capaz de diminuir a capacidade de adesão das células wt. Além disso, o implante das células silenciadas não foi capaz de gerar uma massa tumoral in vivo, o que foi confirmado posteriormente por análise imunohistoquímica. Não obstante, todos tumores C6 wt cresceram com todas características histopatológicas de GBM. O crescimento dos tumores C6 não foi bloqueado pela co-injeção com o inibidor enzimático APCP. Desse modo, nossos resultados corroboram outros dados da literatura que indicam um papel crucial da sinalização purinérgica na progressão de GBM. Estudos complementares são necessários para o estabelecimento de novas e mais eficazes terapias contra esses tumores, utilizando a e5NT/CD73 como alvo.Glioblastoma (GBM) is a very aggressive tumor that presents a high proliferative and invasive behavior. Ecto-5’-nucleotidase (e5NT/CD73) is a widely expressed enzyme that hydrolyses extracellular AMP, producing adenosine, an important molecule in several cell functions. Besides that, e5NT/CD73 has been shown to exert an important role in cell-cell and cell-matrix interactions. Several evidences suggest that e5NT/CD73 has tumor-promotion functions, being upregulated in several tumors. In this study we aimed to better characterize the role of e5NT/CD73 in glioma progression by silencing its expression in a rat C6 glioma cells and analyzing the in vitro and in vivo growth of GBM. Knockdown (KD) of e5NT/CD73 was obtained through stable RNA interference mediated by lentivirus and KD cells were analyzed through immunofluorescence and activity methods such as HPLC and malaquite-green. We also assessed parameters of malignancy in vitro, such as cell adhesion and migration. For a deeper investigation, we implanted KD C6 cells in rat striatum and performed pathological and immunohistochemical analyses to assess tumor size and malignancy. Two stable e5NT/CD73 KD clones of C6 glioma cells were obtained, named C6-c54 and C6- c56, and these clones presented a large reduction in expression and AMPase activity of e5NT/CD73. A significant alteration in adhesion parameter of silenced cells was observed in vitro possibly due to the loss of the adherence function of e5NT/CD73 per se. In addition, e5NT/CD73 KD cells were not able to develop a tumor mass in the striatum of rats, which was confirmed by immunohistochemistry analyses, whereas all rats injected with C6 wt cells developed tumors with all histopathological features of GBM. Tumor growth of C6 wt cells was not blocked by the co-injection with the enzymatic inhibitor APCP. Taken together these results reinforce the crucial involvement of purinergic signaling in the GBM progression. Further studies are necessary to establish new and powerful therapies against these tumors using e5NT/CD73 as target

Targeted APC Activation in Cancer Immunotherapy to Enhance the Abscopal Effect

In oncology, the “abscopal effect” refers to the therapeutic effect on a distant tumor resulting from the treatment of local tumor (e. g., ablation, injection, or radiation). Typically associated with radiation, the abscopal effect is thought to be mediated by a systemic antitumor immune response that is induced by two concurrent changes at the treated tumor: (1) the release of tumor antigens and (2) the exposure of damage-associated molecular patterns. Therapies that produce these changes are associated with immunogenic cell death (ICD). Some interventions have been shown to cause an abscopal effect without inducing the release of tumor antigens, suggesting that release of tumor antigens at baseline plays a significant role in mediating the abscopal effect. With tumor antigens already present, therapies that target activation of APCs alone may be sufficient to enhance the abscopal effect. Here, we discuss two therapies targeted at APC activation, TLR9 and CD40 agonists, and their use in the clinic to enhance the abscopal effect

Differential macrophage activation alters the expression profile of NTPDase and Ecto-5´-nucleotidase

Macrophages are key elements in the inflammatory process, whereas depending on the micro-environmental stimulation they exhibit a pro-inflammatory (classical/M1) or an anti-inflammatory/reparatory (alternative/M2) phenotype. Extracellular ATP can act as a danger signal whereas adenosine generally serves as a negative feedback mechanism to limit inflammation. The local increase in nucleotides communication is controlled by ectonucleotidases, such as members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family and ecto-59-nucleotidase/CD73 (ecto-59-NT). In the present work we evaluated the presence of these enzymes in resident mice M1 (macrophages stimulated with LPS), and M2 (macrophages stimulated with IL-4) macrophages. Macrophages were collected by a lavage of the mice (6–8 weeks) peritoneal cavity and treated for 24 h with IL-4 (10 ng/mL) or LPS (10 ng/mL). Nitrite concentrations were measured using the Greiss reaction. Supernatants were harvested to determine cytokines and the ATPase, ADPase and AMPase activities were determined by the malachite green method and HPLC analysis. The expression of selected surface proteins was evaluated by flow cytometry. The results reveal that M1 macrophages presented a decreased ATP and AMP hydrolysis in agreement with a decrease in NTPDase1, -3 and ecto-59-nucleotidase expression compared to M2. In contrast, M2 macrophages showed a higher ATP and AMP hydrolysis and increased NTPDase1, -3 and ecto-59-nucleotidase expression compared to M1 macrophages. Therefore, macrophages of the M1 phenotype lead to an accumulation of ATP while macrophages of the M2 phenotype may rapidly convert ATP to adenosine. The results also showed that P1 and P2 purinoreceptors present the same mRNA profile in both phenotypes. In addition, M2 macrophages, which have a higher ATPase activity, were less sensitive to cell death. In conclusion, these changes in ectoenzyme activities might allow macrophages to adjust the outcome of the extracellular purinergic cascade in order to fine-tune their functions during the inflammatory set

O silenciamento da ecto-5'-nucleotidase/CD73 altera a progressão de gliomas

O glioblastoma multiforme (GBM) é um tipo de tumor bastante agressivo, que apresenta uma elevada taxa de proliferação e um comportamento altamente invasivo. A ecto-5’-nucleotidase (e5NT/CD73) é uma enzima expressa de forma bastante ampla, a qual age na hidrólise do AMP extracelular, com consequente formação de adenosina, molécula com diversas funções celulares. Além da função enzimática, foi mostrado que a e5NT/CD73 também exerce importante papel na interação célula-célula e célula-matriz. Muitas evidências apontam para funções de promoção tumoral desempenhados pela e5NT/CD73, a qual se apresenta expressa em altos níveis em diferentes tumores. No presente estudo caracterizamos o papel desempenhado pela e5NT/CD73 no crescimento de GBM in vitro e in vivo através do silenciamento de sua expressão e atividade em células de glioma de rato C6. A e5NT/CD73 foi silenciada por RNA de interferência estável através de lentivírus e o silenciamento foi caracterizado por métodos de imunofluorescência e atividade enzimática. Além disso, investigamos parâmetros de malignidade in vitro, como adesão e migração celular. Para uma melhor análise do papel da proteína no crescimento tumoral, implantamos as células C6 silenciadas em um modelo de glioma em ratos. Análises patológicas e imunohistoquímicas usando o marcador nestina foram realizados. Foram obtidos dois clones silenciados de forma estável, C6-c54 e C6-c56, os quais tiveram uma significativa redução na expressão e atividade da e5NT/CD73. Foi observada uma significativa alteração no parâmetro de adesão in vitro de ambos clones silenciados, e esse efeito deve-se provavelmente à função de adesão exercida pela proteína e não da sua atividade enzimática, uma vez que o uso do inibidor APCP não foi capaz de diminuir a capacidade de adesão das células wt. Além disso, o implante das células silenciadas não foi capaz de gerar uma massa tumoral in vivo, o que foi confirmado posteriormente por análise imunohistoquímica. Não obstante, todos tumores C6 wt cresceram com todas características histopatológicas de GBM. O crescimento dos tumores C6 não foi bloqueado pela co-injeção com o inibidor enzimático APCP. Desse modo, nossos resultados corroboram outros dados da literatura que indicam um papel crucial da sinalização purinérgica na progressão de GBM. Estudos complementares são necessários para o estabelecimento de novas e mais eficazes terapias contra esses tumores, utilizando a e5NT/CD73 como alvo.Glioblastoma (GBM) is a very aggressive tumor that presents a high proliferative and invasive behavior. Ecto-5’-nucleotidase (e5NT/CD73) is a widely expressed enzyme that hydrolyses extracellular AMP, producing adenosine, an important molecule in several cell functions. Besides that, e5NT/CD73 has been shown to exert an important role in cell-cell and cell-matrix interactions. Several evidences suggest that e5NT/CD73 has tumor-promotion functions, being upregulated in several tumors. In this study we aimed to better characterize the role of e5NT/CD73 in glioma progression by silencing its expression in a rat C6 glioma cells and analyzing the in vitro and in vivo growth of GBM. Knockdown (KD) of e5NT/CD73 was obtained through stable RNA interference mediated by lentivirus and KD cells were analyzed through immunofluorescence and activity methods such as HPLC and malaquite-green. We also assessed parameters of malignancy in vitro, such as cell adhesion and migration. For a deeper investigation, we implanted KD C6 cells in rat striatum and performed pathological and immunohistochemical analyses to assess tumor size and malignancy. Two stable e5NT/CD73 KD clones of C6 glioma cells were obtained, named C6-c54 and C6- c56, and these clones presented a large reduction in expression and AMPase activity of e5NT/CD73. A significant alteration in adhesion parameter of silenced cells was observed in vitro possibly due to the loss of the adherence function of e5NT/CD73 per se. In addition, e5NT/CD73 KD cells were not able to develop a tumor mass in the striatum of rats, which was confirmed by immunohistochemistry analyses, whereas all rats injected with C6 wt cells developed tumors with all histopathological features of GBM. Tumor growth of C6 wt cells was not blocked by the co-injection with the enzymatic inhibitor APCP. Taken together these results reinforce the crucial involvement of purinergic signaling in the GBM progression. Further studies are necessary to establish new and powerful therapies against these tumors using e5NT/CD73 as target

Comparação entre métodos de extração de DNA para determinação de carga viral de PCV2

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Comparação entre métodos de extração de DNA para determinação de carga viral de PCV2

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In situ vaccination with defined factors overcomes T cell exhaustion in distant tumors

Irreversible T cell exhaustion limits the efficacy of programmed cell death 1 (PD-1) blockade. We observed that dual CD40-TLR4 stimulation within a single tumor restored PD-1 sensitivity and that this regimen triggered a systemic tumor-specific CD8+ T cell response. This approach effectively treated established tumors in diverse syngeneic cancer models, and the systemic effect was dependent on the injected tumor, indicating that treated tumors were converted into necessary components of this therapy. Strikingly, this approach was associated with the absence of exhausted PD-1hi T cells in treated and distant tumors, while sparing the intervening draining lymph node and spleen. Furthermore, patients with transcription changes like those induced by this therapy experienced improved progression-free survival with anti–PD-1 treatment. Dual CD40-TLR4 activation within a single tumor is thus an approach for overcoming resistance to PD-1 blockade that is unique in its ability to cause the loss of exhausted T cells within tumors while sparing nonmalignant tissues