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1.
AAPS J ; 20(5): 86, 2018 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-30039346

RESUMO

Drug-induced kidney injury is often observed in the clinics and can lead to long-term organ failure. In this work, we evaluated a novel in vitro system that aims at detecting whether compounds can cause renal proximal tubule damage in man. For this, we implemented organotypic cultures of human conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1) in a three-channel OrganoPlate under microfluidic conditions. Cells were exposed to four known nephrotoxicants (cisplatin, tenofovir, cyclosporine A, and tobramycin). The effect on cell viability and NAG release into the medium was determined. A novel panel of four miRNAs (mir-21, mir-29a, mir-34a, and mir-192) was selected as potential biomarkers of proximal tubule damage. After nephrotoxicant treatment, miRNA levels in culture medium were earlier indicators than cell viability (WST-8 assay) and outperformed NAG for proximal tubule damage. In particular, mir-29a, mir-34a, and mir-192 were highly reproducible between experiments and across compounds, whereas mir-21 showed more variability. Moreover, similar data were obtained in two different laboratories, underlining the reproducibility and technical transferability of the results, a key requirement for the implementation of novel biomarkers. In conclusion, the selected miRNAs behaved like sensitive biomarkers of damage to tubular epithelial cells caused by several nephrotoxicity mechanisms. This biomarker panel, in combination with the 3D cultures of ciPTEC-OAT1 in the OrganoPlate, represents a novel tool for in vitro nephrotoxicity detection. These results pave the way for the application of miRNAs in longitudinal, time-course in vitro toxicity studies.


Assuntos
Células Epiteliais/efeitos dos fármacos , Nefropatias/induzido quimicamente , Túbulos Renais Proximais/efeitos dos fármacos , MicroRNAs/genética , Técnicas Analíticas Microfluídicas , Linhagem Celular Transformada , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Marcadores Genéticos , Humanos , Nefropatias/genética , Nefropatias/metabolismo , Nefropatias/patologia , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologia , MicroRNAs/metabolismo , Estudo de Prova de Conceito , Reprodutibilidade dos Testes , Medição de Risco , Fatores de Tempo
2.
Clin Exp Immunol ; 181(2): 343-56, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25845418

RESUMO

Exaggerated activation of lymphocytes contributes to the pathogenesis of inflammatory bowel disease (IBD). Medical therapies are linked to the BCL-2 family-mediated apoptosis. Imbalance in BCL-2 family proteins may cause failure in therapeutic responses. We investigated the role of BCL-2 inhibitor ABT-737 for lymphocyte apoptosis in mice under inflammatory conditions. B.6129P2-interleukin (IL)-10(tm1Cgn) /J (IL-10(-/-) ) weighing 25-30 g with ongoing colitis were used. Fifty mg/kg/day ABT-737 was injected intraperitoneally (i.p.). Haematological analyses were performed with an ADVIA 2120 flow cytometer and mass cytometry with a CyTOF 2. Following i.p. administration, ABT-737 was detected in both spontaneous and acute colitis in peripheral blood (PBL) and colon tissue. Treatment led to lymphopenia. CD4(+) CD44(+) CD62L(+) central memory and CD8(+) , CD44(+) CD62L(-) central memory T cells were decreased in PBL upon ABT-737 compared to vehicle-receiving controls. Increased apoptosis upon ABT-737 was determined in blood lymphocytes, splenocytes and Peyer's patches and was accompanied by a decrease in TNF and IL-1B. ABT-737 positively altered the colonic mucosa and ameliorated inflammation, as shown by colonoscopy, histology and colon length. A decreased BIM/BCL-2 ratio or absence of BIM in both Bim(-) (/) (-) and Il10(-) (/) (-) × Bim(-) (/) (-) impeded the protective effect of ABT-737. The BIM/BCL-2 ratio decreased with age and during the course of treatment. Thus, long-term treatment resulted in adapted TNF levels and macroscopic mucosal damage. ABT-737 was efficacious in diminishing lymphocytes and ameliorating colitis in a BIM-dependent manner. Regulation of inappropriate survival of lymphocytes by ABT-737 may provide a therapeutic strategy in IBD.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Compostos de Bifenilo/farmacologia , Colite/tratamento farmacológico , Proteínas de Membrana/genética , Nitrofenóis/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas/genética , Sulfonamidas/farmacologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/patologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Colite/induzido quimicamente , Colite/genética , Colite/patologia , Sulfato de Dextrana , Feminino , Expressão Gênica , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Injeções Intraperitoneais , Interleucina-10/genética , Interleucina-10/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Selectina L/genética , Selectina L/metabolismo , Linfopenia/induzido quimicamente , Linfopenia/genética , Linfopenia/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
3.
Lab Anim ; 47(4): 274-83, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23828853

RESUMO

An impaired intestinal epithelial barrier is thought to be a major factor in the pathogenesis of human inflammatory bowel disease (IBD). IBD is frequently investigated by inducing a damaged barrier in murine models of colitis. This can be done by feeding mice with dextran sulfate sodium (DSS) polymers in their drinking water. Refinement measures should focus on alleviating unnecessary suffering during this probably painful condition. Appropriate parameters are needed to decide when to terminate the experiments. Our aim was to investigate whether a change in burrowing behaviour is a sensitive measure of animal welfare in murine models of colitis. Acute colitis was induced in C57BL/6 mice with 2.0% DSS over nine days. The burrowing test is based on the species-typical behaviour of mice to spontaneously displace items from tubes within their home cage. As a burrowing apparatus, a water bottle (250 mL, 150 mm length, 55 mm diameter) filled with 138-142 g of pellets of the animal's diet was used. The presence of intestinal inflammation as a result of acute DSS-induced colitis was confirmed by a decrease in body weight, colon length and an increase of murine endoscopic index of colitis severity, histological score and spleen weight in the group receiving DSS as compared with the control group. An onset of intestinal inflammation correlated with a significant decrease in burrowing behaviour (P < 0.05). Altered adrenal gland histology indicated stress as a result of acute colitis. Our findings provide evidence that changes of spontaneous burrowing behaviour correlate with the onset of inflammation in acute DSS-induced colitis.


Assuntos
Bem-Estar do Animal , Comportamento Animal , Estresse Fisiológico , Animais , Colite/induzido quimicamente , Colite/patologia , Colonoscopia/veterinária , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Dor
4.
Clin Exp Immunol ; 173(3): 493-501, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23668821

RESUMO

We investigated the role of B cell lymphoma (BCL)-2-interacting mediator of cell death (Bim) for lymphocyte homeostasis in intestinal mucosa. Lymphocytes lacking Bim are refractory to apoptosis. Chronic colitis was induced in Bim-deficient mice (Bim(-/-) ) with dextran sulphate sodium (DSS). Weight loss and colonoscopic score were increased significantly in Bim(-/-) mice compared to wild-type mice. As Bim is induced for the killing of autoreactive cells we determined the role of Bim in the regulation of lymphocyte survival at mucosal sites. Upon chronic dextran sulphate sodium (DSS)-induced colitis, Bim(-/-) animals exhibited an increased infiltrate of lymphocytes into the mucosa compared to wild-type mice. The number of autoreactive T cell receptor (TCR) Vß8(+) lymphocytes was significantly higher in Bim(-/-) mice compared to wild-type controls. Impaired removal of autoreactive lymphocytes in Bim(-/-) mice upon chronic DSS-induced colitis may therefore contribute to aggravated mucosal inflammation.


Assuntos
Proteínas Reguladoras de Apoptose/deficiência , Colite/imunologia , Colite/metabolismo , Linfócitos/imunologia , Linfócitos/metabolismo , Proteínas de Membrana/deficiência , Proteínas Proto-Oncogênicas/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Autoimunidade/genética , Proteína 11 Semelhante a Bcl-2 , Morte Celular/genética , Colite/induzido quimicamente , Citocinas/biossíntese , Sulfato de Dextrana/efeitos adversos , Inflamação/genética , Inflamação/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fenótipo , Proteínas Proto-Oncogênicas/genética , Prolapso Retal/genética
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