RESUMO
Pancreatic bioengineering is a potential therapeutic alternative for type 1 diabetes (T1D) in which the pancreas is decellularized, generating an acellular extracellular matrix (ECM) scaffold, which may be reconstituted by recellularization with several cell types to generate a bioartificial pancreas. No consensus for an ideal pancreatic decellularization protocol exists. Therefore, we aimed to determine the best-suited detergent by comparing sodium dodecyl sulfate (SDS), sodium deoxycholate (SDC), and Triton X-100 at different concentrations. Murine (n=12) and human pancreatic tissue from adult brain-dead donors (n=06) was harvested in accordance with Institutional Ethical Committee of the University of São Paulo Medical School (CEP-FMUSP) and decellularized under different detergent conditions. DNA content, histological analysis, and transmission and scanning electron microscopy were assessed. The most adequate condition for pancreatic decellularization was found to be 4% SDC, displaying: a) effective cell removal; b) maintenance of extracellular matrix architecture; c) proteoglycans, glycosaminoglycans (GAGs), and collagen fibers preservation. This protocol was extrapolated and successfully applied to human pancreas decellularization. The acellular ECM scaffold generated was recelullarized using human pancreatic islets primary clusters. 3D clusters were generated using 0.5×104 cells and then placed on top of acellular pancreatic slices (25 and 50 μm thickness). These clusters tended to connect to the acellular matrix, with visible cells located in the periphery of the clusters interacting with the ECM network of the bioscaffold slices and continued to produce insulin. This study provided evidence on how to improve and accelerate the pancreas decellularization process, while maintaining its architecture and extracellular structure, aiming at pancreatic bioengineering.
RESUMO
This study aimed to evaluate the modes of action of harringtonine (HT) and homoharringtonine (HHT) alkaloids in cell with wild (HepG2/C3A) and mutant p53 (HuH-7.5). We performed assays for cytotoxicity, genotoxicity, induction of apoptosis, cell cycle phase, and membrane integrity. Obtained data were compared with the relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. The relative expression of the genes showed an increase in apoptosis-inducing mRNAs, such as TNF and BBC3, as well as a reduction in BCL2 and BAK. The mRNAs of CYP2E1 and CYP2C19 xenobiotic metabolism genes increased in both lineages, while CYP3A4 increased only in the HuH-7.5 lineage. The mRNA expression of endoplasmic reticulum (ER) stress genes (ERN1 and EIF2AK3) was shown to increase in HHT and HT treatments. A similar increase was recorded in the mRNA expression of the TRAF2 gene. The changes observed in this study support the hypothesis that ER stress was more strongly associated with TNF induction, causing cell death by apoptosis in p53 mutant cells. This result with wild and mutant p53 cells may have clinical implications in the use of these compounds.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Harringtoninas/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Proteína Supressora de Tumor p53/genética , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/genética , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genéticaRESUMO
AIMS: Fructooligosaccharides (FOSs) known for their health properties and ß-(2â6)-levan-type FOSs have shown prebiotic and immunomodulatory activities that overcome those of commercial ß-(2â1)-FOSs, but costs do not favour their use. Moreover, FOSs can reach the bloodstream through the diet, and little is known about their direct effect on cells. The aim of this work was to produce high-content FOSs by Bacillus subtilis natto CCT7712 in a bioreactor using commercial sucrose and to evaluate their antiproliferative effects in OVCAR-3 cells. METHODS AND RESULTS: FOS production reached 173·60 g l-1 , 0·2 vvm aeration and uncontrolled pH. Levan-type FOSs, composed of ß-(2 â 6) links and mainly GF3 (6-nystose), were identified using RMN spectroscopy, FT-IR and ESI-MS. FOSs decreased the viability and proliferation of OVCAR-3 cells, and the effects were associated with an increased pro-inflammatory response by the induction of IL-8 and TNF-α, and the repression of ER-ß genes. The metabolic profiles showed disruption of cellular homeostasis that can be associated with a decrease in proliferation. CONCLUSIONS: The high production of levan-type FOSs from B. subtilis natto CCT7712 in a bioreactor was achieved, and they showed antiproliferative potential in OVCAR-3 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: FOS could be a good target for future therapeutic studies and commercial use.
Assuntos
Bacillus subtilis/metabolismo , Proliferação de Células/efeitos dos fármacos , Oligossacarídeos/metabolismo , Oligossacarídeos/farmacologia , Reatores Biológicos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Frutanos/química , Frutanos/metabolismo , Frutanos/farmacologia , Humanos , Oligossacarídeos/química , Sacarose/metabolismoRESUMO
Pemphigus foliaceus (PF) is the most common autoimmune skin disease in dogs. It is characterized by pustules, erosions, and crusts which occur due to the presence of autoantibodies that target intercellular adhesion. Histopathological examination is considered the gold standard pattern in the diagnosis, but may sometimes be inconclusive, especially when the characteristic findings are not identified. New diagnostic tests are continuously being developed and immunofluorescence assays, could be a valuable alternative diagnostic tool. This study aimed to evaluate the applicability of direct and indirect immunofluorescence (DIF and IIF) tests for the diagnosis of canine PF. Twenty eight dogs were divided into two groups: Group I with 14 dogs with PF and Group II (control) with 14 dogs with Superficial pyoderma (differential diagnoses of PF). All animals were submitted to skin biopsy to histopathological and DIF. Blood samples were collected to assess IIF. Comparing the DIF results against the histopathology test, there was an agreement of 75% (9/12) with a Kappa index of 0.77 (P<0.001). Considering IIF, the agreement was 100% (14/14), with a Kappa index of 1.0 (P<0.001). We conclude that DIF and IIF are highly effective and were useful and effective complementary examination tests for an improvement in the diagnosis of canine PF.(AU)
O pênfigo foliáceo (PF) é considerado uma das doenças tegumentares autoimunes mais frequentes em cães. Clinicamente, caracteriza-se pela presença de pústulas, erosões e crostas. O exame histopatológico é considerado o teste diagnóstico de eleição, porém pode se mostrar inconclusivo, sobretudo quando os achados característicos da doença não são observados. Novas ferramentas diagnósticas têm sido desenvolvidas e os testes de imunofluorecência são uma valiosa alternativa. Este estudo teve como objetivo avaliar a aplicabilidade das reações de imunofluorescência direta (IFD) e indireta (IFI) para o diagnóstico do PF canino. Vinte e oito cães foram divididos em dois grupos: grupo I com 14 cães com PF e grupo II (controle) com 14 cães com piodermite superficial (um dos principais diagnósticos diferenciais do PF). Todos os animais foram submetidos à biópsia cutânea, seguida de exame histopatológico e IFD. Amostras de sangue foram coletadas para realização da IFI. Comparando-se os valores de IFD com o histopatológico, obtiveram-se valores de concordância de 75% (9/12), com índice Kappa de 0,77 (P<0,001). Já na IFI, a concordância foi de 100% (14/14), com índice Kappa de 1,0 (P<0,001). Concluiu-se, então, que a IFD e a IFI apresentaram excelentes resultados e podem ser consideradas novas alternativas diagnósticas do PF canino.(AU)
Assuntos
Animais , Cães , Cães/anormalidades , Imunofluorescência , Imunofluorescência/veterinária , Penfigoide Bolhoso/diagnósticoRESUMO
Pemphigus foliaceus (PF) is the most common autoimmune skin disease in dogs. It is characterized by pustules, erosions, and crusts which occur due to the presence of autoantibodies that target intercellular adhesion. Histopathological examination is considered the gold standard pattern in the diagnosis, but may sometimes be inconclusive, especially when the characteristic findings are not identified. New diagnostic tests are continuously being developed and immunofluorescence assays, could be a valuable alternative diagnostic tool. This study aimed to evaluate the applicability of direct and indirect immunofluorescence (DIF and IIF) tests for the diagnosis of canine PF. Twenty eight dogs were divided into two groups: Group I with 14 dogs with PF and Group II (control) with 14 dogs with Superficial pyoderma (differential diagnoses of PF). All animals were submitted to skin biopsy to histopathological and DIF. Blood samples were collected to assess IIF. Comparing the DIF results against the histopathology test, there was an agreement of 75% (9/12) with a Kappa index of 0.77 (P<0.001). Considering IIF, the agreement was 100% (14/14), with a Kappa index of 1.0 (P<0.001). We conclude that DIF and IIF are highly effective and were useful and effective complementary examination tests for an improvement in the diagnosis of canine PF.(AU)
O pênfigo foliáceo (PF) é considerado uma das doenças tegumentares autoimunes mais frequentes em cães. Clinicamente, caracteriza-se pela presença de pústulas, erosões e crostas. O exame histopatológico é considerado o teste diagnóstico de eleição, porém pode se mostrar inconclusivo, sobretudo quando os achados característicos da doença não são observados. Novas ferramentas diagnósticas têm sido desenvolvidas e os testes de imunofluorecência são uma valiosa alternativa. Este estudo teve como objetivo avaliar a aplicabilidade das reações de imunofluorescência direta (IFD) e indireta (IFI) para o diagnóstico do PF canino. Vinte e oito cães foram divididos em dois grupos: grupo I com 14 cães com PF e grupo II (controle) com 14 cães com piodermite superficial (um dos principais diagnósticos diferenciais do PF). Todos os animais foram submetidos à biópsia cutânea, seguida de exame histopatológico e IFD. Amostras de sangue foram coletadas para realização da IFI. Comparando-se os valores de IFD com o histopatológico, obtiveram-se valores de concordância de 75% (9/12), com índice Kappa de 0,77 (P<0,001). Já na IFI, a concordância foi de 100% (14/14), com índice Kappa de 1,0 (P<0,001). Concluiu-se, então, que a IFD e a IFI apresentaram excelentes resultados e podem ser consideradas novas alternativas diagnósticas do PF canino.(AU)
Assuntos
Animais , Cães , Cães/anormalidades , Imunofluorescência/estatística & dados numéricos , Imunofluorescência/veterinária , Penfigoide Bolhoso/diagnósticoRESUMO
O objetivo deste estudo prospectivo foi analisar a função do ventrículo esquerdo de cadelas que apresentavam sepse ou não e diagnosticar a depressão miocárdica de forma precoce, por meio do feature tracking imaging bidimensional (FTI-2D).Vinte e nove cadelas diagnosticadas com piometra, patologia usada como modelo experimental para sepse, foram avaliadas. Os animais foram submetidos ao exame ecocardiográfico convencional e ao FTI-2D e divididos em três grupos: grupo I (controle), grupo II (piometra sepse) e grupo III (piometra não sepse). Em relação aos parâmetros da ecocardiografia convencional, não houve diferença estatística entre os grupos avaliados. Quanto ao strain radial, os grupos II e III apresentaram função ventricular inferior no momento do diagnóstico (22,805,80 grupo I; 18,455,96 grupo II; 18,719,45 grupo III; P=0,032) e o grupo III manteve essa redução no retorno. Já no strain rate radial, apenas o grupo III obteve função ventricular inferior no momento do diagnóstico (1,900,57 grupo I; 1,910,70 grupo II, 1,570,53 grupo III; P=0,021). Para o strain longitudinal, observou-se função ventricular mais baixa nos grupos II e III no momento do diagnóstico (11,043,38 grupo I; 8,593,97 grupo II; 8,211,99; P=0,048) e após 10 dias de pós-cirúrgico (11,043,38 grupo I; 9,002,16 grupo II; 8,122,27 grupo III; p=0,048). O FTI-2D foi capaz de detectar alterações precoces da função ventricular esquerda, que não foram observadas no exame ecocardiográfico convencional, sugerindo quadro de depressão miocárdica, até mesmo em cadelas com piometra sem quadro clínico evidente de sepse. Assim, essa modalidade pode ser útil no diagnóstico, pois pode detectar alterações cardíacas precoces, possibilitando uma intervenção terapêutica rápida.(AU)
The aim of this prospective study was to analyze the left ventricular function of dogs who had sepsis or not and diagnose myocardial depression early on through the two-dimensional feature tracking imaging (2D-FTI). Twenty-nine dogs diagnosed with pyometra, a pathology used as an experimental model for sepsis were evaluated. The animals were subjected to conventional echocardiography and 2D-FTI, and divided into three groups: Group I (control), group II (sepsis pyometra) and group III (no sepsis pyometra). Regarding the parameters of conventional echocardiography, there was no statistical difference between the groups. As for the radial Strain, group II and III had lower ventricular function at diagnosis (22.805.80 Group I; 18.455.96 Group II; 18.719.45 Group III; P = 0.032) and group III maintained this reduction in return. Regarding the radial Strain rate, only the III group had lower ventricular function at diagnosis (1.900.57 Group I; 1.910.70 Group II, 1.570.53 Group III; P = 0.021). For the Longitudinal Strain we observed lower ventricular function in groups II and III at diagnosis (11.043.38 Group I, Group II 8.593.97, 8.211.99; P = 0.048) and ten days after surgery (11.043.38 Group I; 9.002.16 Group II; 8.122.27 Group III; P = 0.048). The 2D-FTI was able to detect early changes in left ventricular function, suggesting myocardial depression frame, even in female dogs with pyometra without clear clinical picture of sepsis, which were not seen in conventional echocardiography. Thus, this model can be useful in diagnosis, because it can detect early cardiac changes, enabling a rapid therapeutic intervention.(AU)
Assuntos
Animais , Feminino , Cães , Miocárdio/patologia , Sepse/veterinária , Disfunção Ventricular Esquerda/diagnóstico por imagem , Cardiomiopatias/veterinária , Ecocardiografia/veterinária , Piometra/veterináriaRESUMO
O objetivo deste estudo prospectivo foi analisar a função do ventrículo esquerdo de cadelas que apresentavam sepse ou não e diagnosticar a depressão miocárdica de forma precoce, por meio do feature tracking imaging bidimensional (FTI-2D).Vinte e nove cadelas diagnosticadas com piometra, patologia usada como modelo experimental para sepse, foram avaliadas. Os animais foram submetidos ao exame ecocardiográfico convencional e ao FTI-2D e divididos em três grupos: grupo I (controle), grupo II (piometra sepse) e grupo III (piometra não sepse). Em relação aos parâmetros da ecocardiografia convencional, não houve diferença estatística entre os grupos avaliados. Quanto ao strain radial, os grupos II e III apresentaram função ventricular inferior no momento do diagnóstico (22,805,80 grupo I; 18,455,96 grupo II; 18,719,45 grupo III; P=0,032) e o grupo III manteve essa redução no retorno. Já no strain rate radial, apenas o grupo III obteve função ventricular inferior no momento do diagnóstico (1,900,57 grupo I; 1,910,70 grupo II, 1,570,53 grupo III; P=0,021). Para o strain longitudinal, observou-se função ventricular mais baixa nos grupos II e III no momento do diagnóstico (11,043,38 grupo I; 8,593,97 grupo II; 8,211,99; P=0,048) e após 10 dias de pós-cirúrgico (11,043,38 grupo I; 9,002,16 grupo II; 8,122,27 grupo III; p=0,048). O FTI-2D foi capaz de detectar alterações precoces da função ventricular esquerda, que não foram observadas no exame ecocardiográfico convencional, sugerindo quadro de depressão miocárdica, até mesmo em cadelas com piometra sem quadro clínico evidente de sepse. Assim, essa modalidade pode ser útil no diagnóstico, pois pode detectar alterações cardíacas precoces, possibilitando uma intervenção terapêutica rápida.(AU)
The aim of this prospective study was to analyze the left ventricular function of dogs who had sepsis or not and diagnose myocardial depression early on through the two-dimensional feature tracking imaging (2D-FTI). Twenty-nine dogs diagnosed with pyometra, a pathology used as an experimental model for sepsis were evaluated. The animals were subjected to conventional echocardiography and 2D-FTI, and divided into three groups: Group I (control), group II (sepsis pyometra) and group III (no sepsis pyometra). Regarding the parameters of conventional echocardiography, there was no statistical difference between the groups. As for the radial Strain, group II and III had lower ventricular function at diagnosis (22.805.80 Group I; 18.455.96 Group II; 18.719.45 Group III; P = 0.032) and group III maintained this reduction in return. Regarding the radial Strain rate, only the III group had lower ventricular function at diagnosis (1.900.57 Group I; 1.910.70 Group II, 1.570.53 Group III; P = 0.021). For the Longitudinal Strain we observed lower ventricular function in groups II and III at diagnosis (11.043.38 Group I, Group II 8.593.97, 8.211.99; P = 0.048) and ten days after surgery (11.043.38 Group I; 9.002.16 Group II; 8.122.27 Group III; P = 0.048). The 2D-FTI was able to detect early changes in left ventricular function, suggesting myocardial depression frame, even in female dogs with pyometra without clear clinical picture of sepsis, which were not seen in conventional echocardiography. Thus, this model can be useful in diagnosis, because it can detect early cardiac changes, enabling a rapid therapeutic intervention.(AU)
Assuntos
Animais , Feminino , Sepse/veterinária , Disfunção Ventricular Esquerda , Cardiomiopatias/veterinária , Piometra/veterinária , Ecocardiografia/veterináriaRESUMO
OBJECTIVES: To evaluate endomyocardial and epimyocardial left ventricular circumferential and longitudinal peak systolic strain and strain rate in dogs with myxomatous mitral valve disease using two-dimensional feature tracking imaging echocardiography. MATERIALS AND METHODS: Epimyocardial and endomyocardial global and regional myocardial peak systolic strain and strain rate using two-dimensional feature tracking imaging were evaluated in healthy dogs and those in stages B1, B2 and C of myxomatous mitral valve disease. Strain and strain rate in circumferential and longitudinal aspect were evaluated in 48 small- and medium-sized dogs. RESULTS: Global endomyocardial circumferential strain and global epimyocardial circumferential strain systolic peak were lower in stage C than in stage B2 (P = 0 · 04 and P = 0 · 02) and similar to healthy dogs. Endomyocardial circumferential strain rate in septal and inferior segments were lower in stage C compared to B2 (P = 0 · 0007 and P = 0 · 0056), but not different from healthy dogs. There were no statistical differences in the epimyocardial circumferential strain rate, longitudinal strain and strain rate between healthy and affected dogs. CLINICAL SIGNIFICANCE: Two-dimensional feature tracking imaging determination of myocardial deformation in epimyocardial and endomyocardial layers allows detection of increased compensatory circumferential left ventricular myocardial systolic performance due to volume overload and absence of this response as disease advances to congestive heart failure.
Assuntos
Doenças do Cão/diagnóstico por imagem , Prolapso da Valva Mitral/veterinária , Ultrassonografia Doppler em Cores/veterinária , Animais , Estudos de Casos e Controles , Cães , Feminino , Masculino , Prolapso da Valva Mitral/diagnóstico por imagem , Valor Preditivo dos Testes , Sístole , Disfunção Ventricular Esquerda/fisiopatologiaRESUMO
The compounds 6-dimethylaminopurine (6-DMAP) and cyclohexamide (CHX) are currently used to stimulate the development of embryos produced by nuclear transfer in the production of cloned mammals with a great deal success. This study investigated the effects of 6-DMAP and CHX in vivo using biological assays to evaluate reproductive performance in females, teratogenesis, and mutagenesis. The results of this study demonstrated that the activating agents of oocyte cytoplasm, 6-DMAP and CHX, altered the reproductive performance of the experimental animals, as well as increased the rate malformations. In addition to these adverse effects, the administration of these compounds in pregnant females resulted in genotoxic and mutagenic toxicity, as determined by comet and micronucleus assays carried out in peripheral blood samples, respectively. Based on these findings and that alterations in DNA are important, morpho-functional teratogenesis and diminished embryonic viability, suggesting that 6-DMAP and CHX, which are utilized during the cloning of mammals, are responsible for the fact that embryos produced by nuclear transfer show low viability after implantation in utero or after birth because of congenital malformations and/or alterations in their DNA.
Assuntos
Adenina/análogos & derivados , Clonagem de Organismos , Cicloeximida/efeitos adversos , Mutagênicos/efeitos adversos , Reprodução/efeitos dos fármacos , Adenina/efeitos adversos , Animais , Transferência Embrionária , Feminino , Camundongos , Gravidez , Reprodução/genética , Teratogênese/efeitos dos fármacosRESUMO
Polyphenolic compounds present in rosemary were found to have antioxidant properties, anticarcinogenic activity, and to increase the detoxification of pro-carcinogens. The aim of the study was to determine the effect the aqueous extract of rosemary (AER) on mutagenicity induced by methylmethane sulfonate in meristematic cells of Allium cepa, as well as to describe its mode of action. Anti-mutagenicity experiments were carried out with 3 different concentrations of AER, which alone showed no mutagenic effects. In antimutagenicity experiments, AER showed chemopreventive activity in cultured meristematic cells of A. cepa against exposure to methylmethane sulfonate. Additionally, post-treatment and simultaneous treatment using pre-incubation protocols were the most effective. Evaluation of different protocols and the percent reduction in DNA indicated bioantimutagenic as well desmutagenic modes of action for AER. AER may be chemopreventive and antimutagenic.