Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: Regenerative endodontic procedures are an effective treatment option for immature teeth with infected necrotic pulps to allow for healing and potential continued root development, yet challenges to ideal treatment outcomes remain. Consistent development of root length and width of dentin remains a challenge, as does development of the pulp-dentin complex. Previous in vitro studies have assessed the role of different growth factors and bioactive molecules in combination with scaffolds to potentially facilitate continued development of the pulp-dentin complex using dental pulp stem cells (DPSCs). The proline-rich tyrosine kinase 2 (Pyk2) is linked with osteoblast activity and the regulation of bone mass. Further, the Pyk2 inhibitor PF-4618433 (PF-46) has been shown in previous studies to enhance osteoblast activity and mineral deposition in vitro. However, whether Pyk2 targeting promotes the osteogenic differentiation of DPSCs remains unknown. Objective: The purpose of this study was to investigate the effect of a Pyk2 inhibitor, PF-46, on the proliferation, differentiation, and mineralization of human DPSCs. Materials and Methods: Human DPSCs were cultured in 24-well plates with α-MEM with 10% FBS, and containing 0 μM (vehicle control) or 0.1 μM, 0.3 μM, or 0.6 μM PF-46. Fresh media and treatments were replaced every 2-3 days. After 1 day incubation, cytotoxic effects were evaluated by using an MTS proliferation assay. After 4 days of treatment, direct cell counting was performed. To induce osteogenic differentiation, ascorbic acid and β-glycerol phosphate were added to the culture media and the DPSCs were cultured with PF-46 for 14 days. Then, an alkaline phosphatase (ALP) assay and mineral deposition assay were performed. Differences between treatment groups were analyzed by a one-way ANOVA followed by pair-wise tests conducted using Tukey’s multiple comparisons procedure with a 5% significance level. Results: The 0.6 μM PF-46 group had a significantly higher cell count, ALP activity and mineral deposition when compared to 0 μM PF-46. The 0.1 and 0.3 μM PF-46 groups also had significantly higher ALP activity compared to the 0 μM PF-46 group after 14 days of incubation. There was a general trend of increased differentiation and mineral deposition as the concentration of PF-46 increased from 0.1 μM to 0.6 μM. Conclusion: There was a general concentration-dependent increase in cell count, differentiation, and mineral deposition by human DPSCs as the concentration of PF-46 increased from 0 μM up to 0.6 μM, with the highest activity observed with 0.6 μM PF-46. Although further research is needed, these results suggest that strategies that target Pyk2 may potentially be used to improve the osteogenic differentiation of DPSCs to aid endodontic regeneration.

INTRODUÇÃO: Embora os corticosteróides sejam considerados tratamento padrão-ouro na asma, pacientes com asma grave não são totalmente controlados com este tratamento. Estudos prévios com inibidores da Rho quinase sugeriram uma influência benéfica destas drogas na asma, atuando como uma possível alternativa anti-inflamatória. No entanto, não há estudos anteriores avaliando os efeitos destes inibidores, associados ou não com corticosteróides, na modulação da mecânica do sistema respiratório e oscilatória do tecido pulmonar distal, assim como nas alterações histopatológicas, em modelo animal de inflamação pulmonar crônica. OBJETIVOS: Avaliar se o tratamento com o inibidor específico da Rho (Y- 27632), associado ou não com a dexametasona modula a resposta de mecânica pulmonar, inflamatória, de remodelamento da matriz extracelular e ativação do estresse oxidativo em cobaias com inflamação alérgica crônica. MÉTODOS: As cobaias receberam sete inalações de ovoalbumina (1-5mg/ml; grupo OVA) durante 4 semanas. A partir da quinta inalação, os animais do grupo da Rho quinase receberam inalação de Y-27632 (1mM) (grupo OVA-RHO) e ou dexametasona (2 mg.kg-1) associada ou não a Y-27632 (grupos OVA-C ou grupos ORC), 10 minutos antes de cada inalação com OVA. Setenta e duas horas depois da sétima inalação, os animais foram anestesiados, exsanguinados, o óxido nítrico exalado foi coletado e a mecânica do sistema respiratório (Ers e Rrs) e oscilatória do tecido pulmonar distal (Et e Rt) foram realizadas em condições basais e após desafio com OVA (0,1%). Após, as fatias de pulmão foram removidas e submetidas à avaliação histopatológica. RESULTADOS: Houve um aumento no óxido nítrico exalado, nas respostas máximas de Ers, Rrs, Rt e Et após desafio antigênico, nos eosinófilos, nas células positivas para IL-2, IL-4, IL-5, IL-13, IFN-?, iNOS, MMP-9, TIMP-1,TGF- ß, NF?B e no conteúdo do 8-iso-PGF2?, no conteúdo de fibras elásticas, colágenas e de actina em vias aéreas e no parênquima pulmonar distal do grupo OVA comparado ao controle (P<0,05). Nos grupos OVA-RHO, OVA-C e ORC houve uma diminuição em todos os parâmetros comparados ao grupo OVA (P<0,05). A associação do Y-27632 com o tratamento com corticosteróide (grupo ORC) potencializou a atenuação do conteúdo de colágeno e IFN-? na parede das vias aéreas e IL-2, IFN-?, 8-iso-PGF2? e NF-?B no parênquima distal comparado aos grupos OVA-RHO e OVA-C (P<0,05). No grupo ORC houve uma redução nas células positivas para TIMP-1 e eosinófilos no septo alveolar comparado ao grupo OVA-C (P<0,05). CONCLUSÕES: A inibição da Rho quinase ou o tratamento com corticosteróides contribuíram para o controle da resposta de mecânica pulmonar, da resposta eosinofílica e linfocitária Th1 e Th2, do remodelamento da matriz extracelular e da ativação do estresse oxidativo nas vias aéreas e parênquima distal neste modelo animal. A associação do inibidor da Rho quinase com o corticosteróide potencializou o controle de parte da resposta de remodelamento e de inflamação nas vias aéreas e parênquima. A inibição da Rho quinase associada ou não a corticosteróides pode ser considerada uma ferramenta farmacológica futura para o tratamento de doenças pulmonares crônicas / INTRODUCTION: Although corticosteroids are considered gold standard of asthma treatment, patients with serious asthma are not totally controlled with this treatment. Previous studies with Rho-kinase inhibitors suggested a beneficial influence of these drugs on asthma, being a possible anti-inflammatory alternative. However, there are no previous studies evaluating in an animal model of chronic pulmonary inflammation the effects of such inhibitors, combined or not with corticosteroids, on the mechanics modulation of the respiratory system and oscillation of distal pulmonary tissue, as well as on histopathological alterations. OBJECTIVES: To evaluate whether treatment with the specific Rho inhibitor (Y-27632), combined or not with dexamethasone, modulates the responses of pulmonary mechanics, inflammation, extracellular matrix remodeling, and oxidative stress activation in guinea pigs with chronic allergic inflammation. METHODS: Guinea pigs received seven ovalbumin inhalations (1-5mg/ml; OVA group) during 4 weeks. After the fifth inhalation, the animals of the Rho-kinase group received inhalation of Y-27632 (1mM) (OVA-RHO group) and/or dexamethasone (2 mg.kg-1), combined or not with Y-27632 (OVA-C groups or ORC groups), 10 minutes before each inhalation with OVA. Seventy-two hours following the seventh inhalation, the animals were anesthetized, exsanguinated, the exhaled nitric oxide was collected, and mechanics of the respiratory system (Ers and Rrs) and oscillation of the distal lung tissue (Et e Rt) were performed in basal conditions and after challenge with OVA (0.1%). Afterwards, lung slices were removed and submitted to histopathological evaluation. RESULTS: There was an increase of exhaled nitric oxide, maximum responses of Ers, Rrs, Et and Rt after antigen challenge, eosinophil counts, cells positive for IL-2, IL-4, IL-5, IL-13, IFN-?, iNOS, MMP-9, TIMP-1,TGF-ß, NF?B, and in the content of 8-iso-PGF2?, elastic fibers, collagen fibers and actin in the airways in distal lung parenchyma of the OVA group, compared to the control (P<0.05). In the OVA-RHO, OVA-C and ORC groups there was a decrease in all parameters, when compared to the OVA group (P<0.05). The treatment combining Y-27632 with corticosteroid (ORC group) maximized the attenuation of the content of collagen and IFN-y in the airways walls, and of IL-2, IFN-?, 8-iso-PGF2? and NF-?B in distal parenchyma, when compared to the OVA-RHO and OVA-C groups (P<0.05). In the ORC group, there was a reduction of cells positive for TIMP-1 and eosinophils in the alveolar septum, compared to the OVA-C group (P<0.05). CONCLUSIONS: Rho kinase or treatment with corticosteroids contributed to the control of the pulmonary mechanics response, Th1 and Th2 lymphocyte and eosinophil responses, extracellular matrix remodeling, and activation of the oxidative stress in the airways and distal parenchyma of this animal model. The combined treatment with Rho kinase and corticosteroid maximized the control of part of the remodeling response and inflammation of the airways and parenchyma. Rho-kinase inhibition combined or not with corticosteroids can be considered a future pharmacological tool for treatment of chronic pulmonary diseases

No description available.

INTRODUÇÃO: Embora os corticosteróides sejam considerados tratamento padrão-ouro na asma, pacientes com asma grave não são totalmente controlados com este tratamento. Estudos prévios com inibidores da Rho quinase sugeriram uma influência benéfica destas drogas na asma, atuando como uma possível alternativa anti-inflamatória. No entanto, não há estudos anteriores avaliando os efeitos destes inibidores, associados ou não com corticosteróides, na modulação da mecânica do sistema respiratório e oscilatória do tecido pulmonar distal, assim como nas alterações histopatológicas, em modelo animal de inflamação pulmonar crônica. OBJETIVOS: Avaliar se o tratamento com o inibidor específico da Rho (Y- 27632), associado ou não com a dexametasona modula a resposta de mecânica pulmonar, inflamatória, de remodelamento da matriz extracelular e ativação do estresse oxidativo em cobaias com inflamação alérgica crônica. MÉTODOS: As cobaias receberam sete inalações de ovoalbumina (1-5mg/ml; grupo OVA) durante 4 semanas. A partir da quinta inalação, os animais do grupo da Rho quinase receberam inalação de Y-27632 (1mM) (grupo OVA-RHO) e ou dexametasona (2 mg.kg-1) associada ou não a Y-27632 (grupos OVA-C ou grupos ORC), 10 minutos antes de cada inalação com OVA. Setenta e duas horas depois da sétima inalação, os animais foram anestesiados, exsanguinados, o óxido nítrico exalado foi coletado e a mecânica do sistema respiratório (Ers e Rrs) e oscilatória do tecido pulmonar distal (Et e Rt) foram realizadas em condições basais e após desafio com OVA (0,1%). Após, as fatias de pulmão foram removidas e submetidas à avaliação histopatológica. RESULTADOS: Houve um aumento no óxido nítrico exalado, nas respostas máximas de Ers, Rrs, Rt e Et após desafio antigênico, nos eosinófilos, nas células positivas para IL-2, IL-4, IL-5, IL-13, IFN-?, iNOS, MMP-9, TIMP-1,TGF- ß, NF?B e no conteúdo do 8-iso-PGF2?, no conteúdo de fibras elásticas, colágenas e de actina em vias aéreas e no parênquima pulmonar distal do grupo OVA comparado ao controle (P<0,05). Nos grupos OVA-RHO, OVA-C e ORC houve uma diminuição em todos os parâmetros comparados ao grupo OVA (P<0,05). A associação do Y-27632 com o tratamento com corticosteróide (grupo ORC) potencializou a atenuação do conteúdo de colágeno e IFN-? na parede das vias aéreas e IL-2, IFN-?, 8-iso-PGF2? e NF-?B no parênquima distal comparado aos grupos OVA-RHO e OVA-C (P<0,05). No grupo ORC houve uma redução nas células positivas para TIMP-1 e eosinófilos no septo alveolar comparado ao grupo OVA-C (P<0,05). CONCLUSÕES: A inibição da Rho quinase ou o tratamento com corticosteróides contribuíram para o controle da resposta de mecânica pulmonar, da resposta eosinofílica e linfocitária Th1 e Th2, do remodelamento da matriz extracelular e da ativação do estresse oxidativo nas vias aéreas e parênquima distal neste modelo animal. A associação do inibidor da Rho quinase com o corticosteróide potencializou o controle de parte da resposta de remodelamento e de inflamação nas vias aéreas e parênquima. A inibição da Rho quinase associada ou não a corticosteróides pode ser considerada uma ferramenta farmacológica futura para o tratamento de doenças pulmonares crônicas / INTRODUCTION: Although corticosteroids are considered gold standard of asthma treatment, patients with serious asthma are not totally controlled with this treatment. Previous studies with Rho-kinase inhibitors suggested a beneficial influence of these drugs on asthma, being a possible anti-inflammatory alternative. However, there are no previous studies evaluating in an animal model of chronic pulmonary inflammation the effects of such inhibitors, combined or not with corticosteroids, on the mechanics modulation of the respiratory system and oscillation of distal pulmonary tissue, as well as on histopathological alterations. OBJECTIVES: To evaluate whether treatment with the specific Rho inhibitor (Y-27632), combined or not with dexamethasone, modulates the responses of pulmonary mechanics, inflammation, extracellular matrix remodeling, and oxidative stress activation in guinea pigs with chronic allergic inflammation. METHODS: Guinea pigs received seven ovalbumin inhalations (1-5mg/ml; OVA group) during 4 weeks. After the fifth inhalation, the animals of the Rho-kinase group received inhalation of Y-27632 (1mM) (OVA-RHO group) and/or dexamethasone (2 mg.kg-1), combined or not with Y-27632 (OVA-C groups or ORC groups), 10 minutes before each inhalation with OVA. Seventy-two hours following the seventh inhalation, the animals were anesthetized, exsanguinated, the exhaled nitric oxide was collected, and mechanics of the respiratory system (Ers and Rrs) and oscillation of the distal lung tissue (Et e Rt) were performed in basal conditions and after challenge with OVA (0.1%). Afterwards, lung slices were removed and submitted to histopathological evaluation. RESULTS: There was an increase of exhaled nitric oxide, maximum responses of Ers, Rrs, Et and Rt after antigen challenge, eosinophil counts, cells positive for IL-2, IL-4, IL-5, IL-13, IFN-?, iNOS, MMP-9, TIMP-1,TGF-ß, NF?B, and in the content of 8-iso-PGF2?, elastic fibers, collagen fibers and actin in the airways in distal lung parenchyma of the OVA group, compared to the control (P<0.05). In the OVA-RHO, OVA-C and ORC groups there was a decrease in all parameters, when compared to the OVA group (P<0.05). The treatment combining Y-27632 with corticosteroid (ORC group) maximized the attenuation of the content of collagen and IFN-y in the airways walls, and of IL-2, IFN-?, 8-iso-PGF2? and NF-?B in distal parenchyma, when compared to the OVA-RHO and OVA-C groups (P<0.05). In the ORC group, there was a reduction of cells positive for TIMP-1 and eosinophils in the alveolar septum, compared to the OVA-C group (P<0.05). CONCLUSIONS: Rho kinase or treatment with corticosteroids contributed to the control of the pulmonary mechanics response, Th1 and Th2 lymphocyte and eosinophil responses, extracellular matrix remodeling, and activation of the oxidative stress in the airways and distal parenchyma of this animal model. The combined treatment with Rho kinase and corticosteroid maximized the control of part of the remodeling response and inflammation of the airways and parenchyma. Rho-kinase inhibition combined or not with corticosteroids can be considered a future pharmacological tool for treatment of chronic pulmonary diseases

Radiotherapy is a major treatment modality used to treat muscle-invasive bladder cancer, with patient outcomes similar to surgery. However, radioresistance is a significant factor in treatment failure. Cell-free extracts of muscle-invasive bladder tumors are defective in nonhomologous end-joining (NHEJ), and this phenotype may be used clinically by combining radiotherapy with a radiosensitizing drug that targets homologous recombination, thereby sparing normal tissues with intact NHEJ. The response of the homologous recombination protein RAD51 to radiation is inhibited by the small-molecule tyrosine kinase inhibitor imatinib. Stable RT112 bladder cancer Ku knockdown (Ku80KD) cells were generated using short hairpin RNA technology to mimic the invasive tumor phenotype and also RAD51 knockdown (RAD51KD) cells to show imatinib's pathway selectivity. Ku80KD, RAD51KD, nonsilencing vector control, and parental RT112 cells were treated with radiation in combination with either imatinib or lapatinib, which inhibits NHEJ and cell survival assessed by clonogenic assay. Drug doses were chosen at approximately IC40 and IC10 (nontoxic) levels. Imatinib radiosensitized Ku80KD cells to a greater extent than RAD51KD or RT112 cells. In contrast, lapatinib radiosensitized RAD51KD and RT112 cells but not Ku80KD cells. Taken together, our findings suggest a new application for imatinib in concurrent use with radiotherapy to treat muscle-invasive bladder cancer. Cancer Res; 73(5); 1611-20. (c)2012 AACR.