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BACKGROUND: The combination of senescence triggers with senolytic drugs is considered a promising new approach to cancer therapy. Here, we studied the efficacy of the genotoxic agent etoposide (Eto) and irradiation in inducing senescence of Panc02 pancreatic cancer cells, and the capability of the Bcl-2 inhibitor navitoclax (ABT-263; Nav) to trigger senolysis. METHODS: Panc02 cells were treated with Eto or irradiated with 5-20 Gy before exposure to Nav. Cell survival, proliferation, and senescence were assessed by trypan blue staining, quantification of DNA synthesis, and staining of senescence-associated ß-galactosidase (SA-ß-Gal)-positive cells, respectively. Levels of mRNA were determined by real-time polymerase chain reaction, and protein expression was analyzed by immunoblotting. Panc02 cells were also grown as pancreatic tumors in mice, which were subsequently treated with Eto and Nav. RESULTS: Eto and irradiation had an antiproliferative effect on Panc02 cells that was significantly or tendentially enhanced by Nav. In vivo, Eto and Nav together, but not Eto alone, significantly reduced the proportion of proliferating cells. The expression of the senescence marker γH2AX and tumor infiltration with T-cells were not affected by the treatment. In vitro, almost all Eto-exposed cells and a significant proportion of cells irradiated with 20 Gy were SA-ß-Gal-positive. Application of Nav reduced the percentage of SA-ß-Gal-positive cells after irradiation but not after pretreatment with Eto. In response to triggers of senescence, cultured Panc02 cells showed increased protein levels of γH2AX and the autophagy marker LC3B-II, and higher mRNA levels of Cdkn1a, Mdm2, and PAI-1, while the effects of Nav were variable. CONCLUSIONS: In vitro and in vivo, the combination of senescence triggers with Nav inhibited tumor cell growth more effectively than the triggers alone. Our data also provide some evidence for senolytic effects of Nav in vitro.
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BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a disease of the elderly mostly because its development from preneoplastic lesions depends on the accumulation of gene mutations and epigenetic alterations over time. How aging of non-cancerous tissues of the host affects tumor progression, however, remains largely unknown. METHODS: We took advantage of a model of accelerated aging, uncoupling protein 2-deficient (Ucp2 knockout, Ucp2 KO) mice, to investigate the growth of orthotopically transplanted Ucp2 wild-type (WT) PDAC cells (cell lines Panc02 and 6606PDA) in vivo and to study strain-dependent differences of the PDAC microenvironment. RESULTS: Measurements of tumor weights and quantification of proliferating cells indicated a significant growth advantage of Panc02 and 6606PDA cells in WT mice compared to Ucp2 KO mice. In tumors in the knockout strain, higher levels of interferon-γ mRNA despite similar numbers of tumor-infiltrating T cells were observed. 6606PDA cells triggered a stronger stromal reaction in Ucp2 KO mice than in WT animals. Accordingly, pancreatic stellate cells from Ucp2 KO mice proliferated at a higher rate than cells of the WT strain when they were incubated with conditioned media from PDAC cells. CONCLUSIONS: Ucp2 modulates PDAC microenvironment in a way that favors tumor progression and implicates an altered stromal response as one of the underlying mechanisms.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ratones , Animales , Proteína Desacopladora 2/genética , Proteína Desacopladora 2/metabolismo , Ratones Endogámicos C57BL , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Ratones Noqueados , Microambiente Tumoral , Neoplasias PancreáticasRESUMEN
INTRODUCTION: Cholestatic liver disease (CLD) is associated with intestinal barrier dysfunction. The peptide hormone ghrelin may exert both hepatoprotective and barrier-strengthening effects. Here, we have evaluated these effects under the conditions of experimental cholestasis. METHODS: C57BL/6J mice with bile duct ligation (BDL) or sham surgery were treated with ghrelin or solvent for 9 days. Liver injury was assessed by histological and laboratory analyses. Paracellular macromolecule permeability and transmural electrical resistance (TMER) of colonic tissues were measured using a Ussing chamber. Expression of tight junction (TJ) genes was quantified by real-time PCR. Amplicon metagenomic sequencing was employed to analyze bacterial 16S rRNA from colonic stool samples. RESULTS: Mice with BDL exhibited weight loss and signs of severe liver injury. These changes were unaffected by ghrelin treatment. FITC-4-kDa-dextran flux was increased and TMER decreased after BDL. Treatment with ghrelin tended to reduce these effects. Furthermore, application of ghrelin was associated with higher mRNA levels of claudin-4, occludin, and ZO-1 in colonic tissues of mice with BDL. Reduced alpha-diversity of the microbiome was observed in solvent-treated mice with BDL but not in ghrelin-treated animals. CONCLUSION: Ghrelin treatment did not improve weight loss and liver damage but increased gene expression of colonic TJ proteins and restored the alpha-diversity of the microbiome. Since protective effects of ghrelin might be masked by the severity of the model, we suggest follow-up studies in models of milder CLD.
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Colestasis , Microbiota , Ratones , Animales , Ghrelina/farmacología , Ghrelina/uso terapéutico , Ratones Endogámicos C57BL , ARN Ribosómico 16S/genética , Conductos Biliares/cirugía , Colestasis/microbiología , Colestasis/patología , Hígado/patología , Pérdida de Peso , Solventes , Modelos Animales de EnfermedadRESUMEN
BACKGROUND: Pancreatic stellate cells (PSCs) foster the progression of pancreatic adenocarcinoma and chronic pancreatitis (CP) by producing a dense fibrotic stroma. However, the incomplete knowledge of PSCs biology hampers the exploration of antifibrotic therapies. Here, we explored the role of the Hippo pathway in the context of PSCs activation and experimental CP. METHODS: CP model was created in rats with the tail vein injection of dibutyltin dichloride (DBTC). The expression of Yes-associated protein (YAP) in CP tissue was assessed. Primary and immortalized rats PSCs were treated with the YAP-inhibitor verteporfin. Furthermore, YAP siRNA was employed. Subsequently, DNA synthesis, cell survival, levels of α-smooth muscle actin (α-SMA) protein, presence of lipid droplets and PSCs gene expression were evaluated. Upstream regulators of YAP signaling were studied by reporter gene assays. RESULTS: In DBTC-induced CP, pronounced expression of YAP in areas of tubular structures and periductal fibrosis was observed. Verteporfin diminished DNA replication in PSCs in a dose-dependent fashion. Knockdown of YAP reduced cell proliferation. Primary cultures of PSCs were characterized by a decrease of lipid droplets and increased synthesis of α-SMA protein. Both processes were not affected by verteporfin. At the non-cytotoxic concentration of 100 nmol/L, verteporfin significantly reduced mRNA levels of transforming growth factor-ß1 (Tgf-ß1) and Ccn family member 1 (Ccn1). YAP signaling was activated by TGF-ß1, but repressed by interferon-γ. CONCLUSIONS: Activated YAP enhanced PSCs proliferation. The antifibrotic potential of Hippo pathway inhibitors warrants further investigation.
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Adenocarcinoma , Neoplasias Pancreáticas , Pancreatitis Crónica , Animales , Ratas , Adenocarcinoma/patología , Fibrosis , Páncreas/patología , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/metabolismo , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/genética , Factor de Crecimiento Transformador beta1/metabolismo , Verteporfina/farmacologíaRESUMEN
BACKGROUND AND AIM: Malnutrition is a frequent complication of chronic pancreatitis. Adequate nutritional support is imperative, but there is still uncertainty about the optimal nutritional treatment. This work systematically compiles evidence from randomized controlled trials investigating dietary interventions in chronic pancreatitis and, in a further step, contrasts those findings with existing dietary recommendations. METHODS: The literature search (PubMed and Cochrane Central Register of Controlled Trials) included English and German full-text articles, which had been published in peer-reviewed journals. Two independent reviewers identified and selected studies. For meta-analysis, forest plots with 95% confidence intervals were generated using a random-effects model. RESULTS: Eleven randomized controlled trials fulfilled all selection criteria. In these trials, the following dietary interventions were tested: antioxidant treatment (n = 6), vitamin D supplementation (n = 3), supplementation with oral nutritional supplements (n = 1), and symbiotics supplementation (n = 1). Studies were of good methodological quality (mean Jadad score of 3.6) but heterogeneous in terms of interventions and study populations. Only for vitamin D, there was convincing evidence for efficacy of supplementation. We found no effect for antioxidant treatment on pain relief (standardized mean difference = -0.12; 95% confidence interval -0.73 to 0.48) and limited generalizability for interventions with oral nutritional supplements and symbiotics. CONCLUSIONS: Nutritional management in chronic pancreatitis remains challenging. As well-designed randomized controlled trials are scarce, in large part, recommendations can only be based on low-level evidence studies or expert opinion. For now, consumption of a balanced diet remains the cornerstone recommendation for prevention, whereas more goal-directed interventions are indicated for specific nutrient deficiencies.
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Suplementos Dietéticos , Terapia Nutricional/métodos , Pancreatitis Crónica/dietoterapia , Ensayos Clínicos Controlados Aleatorios como Asunto , Antioxidantes/administración & dosificación , Humanos , Vitamina D/administración & dosificaciónRESUMEN
The impact of environmental factors, such as diet, and the genetic basis of autoimmune pancreatitis (AIP) are largely unknown. Here, we used an experimental murine AIP model to identify the contribution of diet to AIP development, as well as to fine-map AIP-associated genes in outbred mice prone to develop the disease. For this purpose, we fed mice of an autoimmune-prone intercross line (AIL) three different diets (control, calorie-reduced and western diet) for 6 months, at which point the mice were genotyped and phenotyped for AIP. Overall, 269 out of 734 mice (36.6%) developed AIP with signs of parenchymal destruction, equally affecting mice of both sexes. AIP prevalence and severity were reduced by approximately 50% in mice held under caloric restriction compared to those fed control or western diet. We identified a quantitative trait locus (QTL) on chromosome 4 to be associated with AIP, which is located within a previously reported QTL. This association does not change when considering diet or sex as an additional variable for the mapping. Using whole-genome sequences of the AIL founder strains, we resolved this QTL to a single candidate gene, namely Map3k7. Expression of Map3k7 was largely restricted to islet cells as well as lymphocytes found in the exocrine pancreas of mice with AIP. Our studies suggest a major impact of diet on AIP. Furthermore, we identify Map3k7 as a novel susceptibility gene for experimental AIP. Both findings warrant clinical translation.
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Pancreatitis Autoinmune/etiología , Dieta/efectos adversos , Susceptibilidad a Enfermedades , Predisposición Genética a la Enfermedad , Alelos , Animales , Pancreatitis Autoinmune/diagnóstico , Pancreatitis Autoinmune/metabolismo , Biomarcadores , Mapeo Cromosómico , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Interacción Gen-Ambiente , Genotipo , Inmunohistoquímica , Quinasas Quinasa Quinasa PAM/genética , Quinasas Quinasa Quinasa PAM/metabolismo , Masculino , Ratones , Sitios de Carácter Cuantitativo , Índice de Severidad de la EnfermedadRESUMEN
Muscle wasting represents a constant pathological feature of common chronic gastrointestinal diseases, including liver cirrhosis (LC), inflammatory bowel diseases (IBD), chronic pancreatitis (CP) and pancreatic cancer (PC), and is associated with increased morbidity and mortality. Recent clinical and experimental studies point to the existence of a gut-skeletal muscle axis that is constituted by specific gut-derived mediators which activate pro- and anti-sarcopenic signalling pathways in skeletal muscle cells. A pathophysiological link between both organs is also provided by low-grade systemic inflammation. Animal models of LC, IBD, CP and PC represent an important resource for mechanistic and preclinical studies on disease-associated muscle wasting. They are also required to test and validate specific anti-sarcopenic therapies prior to clinical application. In this article, we review frequently used rodent models of muscle wasting in the context of chronic gastrointestinal diseases, survey their specific advantages and limitations and discuss possibilities for further research activities in the field. We conclude that animal models of LC-, IBD- and PC-associated sarcopenia are an essential supplement to clinical studies because they may provide additional mechanistic insights and help to identify molecular targets for therapeutic interventions in humans.
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Enfermedades Gastrointestinales/patología , Músculo Esquelético/patología , Atrofia Muscular/patología , Animales , Enfermedad Crónica , Humanos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: In short bowel syndrome, epithelial surface loss results in impaired nutrient absorption and may lead to intestinal insufficiency or intestinal failure. Nucleotide oligomerization domain 2 (Nod2) dysfunction predisposes to the development of intestinal failure after intestinal resection and is associated with intestinal barrier defects. Epithelial barrier function is crucial for intestinal absorption and for intestinal adaptation in the short bowel situation. AIMS: The aim of the study was to characterize the effects of the GLP-2 analogue Teduglutide in the small intestine in the presence and absence of Nod2 in a mouse model of short bowel syndrome. METHODS: Mice underwent 40% ICR and were thereafter treated with Teduglutide versus vehicle injections. Survival, body weight, stool water, and sodium content and plasma aldosterone concentrations were determined. Intestinal and kidney tissue was examined with light and fluorescence microscopy, Ussing chamber studies and quantitative PCR in wild type and transgenic mice. RESULTS: Teduglutide reduced intestinal failure incidence in Nod2 k.o. mice. In wt mice, Teduglutide attenuated intestinal insufficiency as indicated by reduced body weight loss and lower plasma aldosterone concentrations, lower stool water content, and lower stool sodium losses. Teduglutide treatment was associated with enhanced epithelial paracellular pore function and enhanced claudin-10 expression in tight junctions in the villus tips, where it colocalized with sodium-glucose cotransporter 1 (SGLT-1), which mediates Na-coupled glucose transport. CONCLUSIONS: In the SBS situation, Teduglutide not only maximizes small intestinal mucosal hypertrophy but also partially restores small intestinal epithelial function through an altered distribution of claudin-10, facilitating sodium recirculation for Na-coupled glucose transport and water absorption.
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Proteína Adaptadora de Señalización NOD2/metabolismo , Péptidos/farmacología , Síndrome del Intestino Corto/metabolismo , Animales , Modelos Animales de Enfermedad , Fármacos Gastrointestinales/farmacología , Péptido 2 Similar al Glucagón/metabolismo , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos ICR , Uniones Estrechas/metabolismoRESUMEN
BACKGROUND: Current animal models of chronic pancreatitis (CP) often provide only limited pathophysiological insights since they incompletely reflect the human disease. CP induced by injection of dibutyltin dichloride (DBTC-pancreatitis) shares with human CP the important feature of extended fibrosis and would be an even more attractive model if it could be transferred from rats to mice, as recently suggested in the context of combined ethanol and DBTC application. This study aimed to evaluate the effects of DBTC in pancreas and liver of C57BL/6 mice, a strain commonly used to engineer genetic mouse models. METHODS: C57BL/6 mice and Lewis rats were exposed to variable doses of DBTC. After an investigation period of up to 4 weeks, laboratory findings and histopathological changes of pancreas and liver were evaluated. RESULTS: Chronic DBTC-pancreatitis in rats was characterized by acinar cell damage, ductal changes, fibrosis, and inflammatory cell infiltrates. Mice treated with DBTC at 6-8 mg/kg body weight, the standard doses in rats, showed transient increases of lipase activities but no morphological signs of chronic DBTC-pancreatitis 4 weeks after injection of the drug. Increased doses of 10-12 mg/kg DBTC were intolerable due to their high toxicity. In contrast, mice and rats presented with a similar histopathology of the liver that can be characterized as a chronic-proliferative DBTC-cholangitis with predominating damage and proliferation of the small bile ducts as well as secondary portal inflammatory cell infiltrates and a beginning portal fibrosis. CONCLUSIONS: The DBTC-model cannot be transferred from rats to C57BL/6 mice with respect to chronic DBTC-pancreatitis, but might be of interest to study DBTC-cholangitis in both species.
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Colangitis/patología , Modelos Animales de Enfermedad , Hígado/patología , Páncreas/patología , Pancreatitis/patología , Animales , Colangitis/inducido químicamente , Enfermedad Crónica , Fibrosis , Lipasa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Compuestos Orgánicos de Estaño , Pancreatitis/inducido químicamente , Pancreatitis/enzimología , Ratas , Ratas Endogámicas LewRESUMEN
Liver cirrhosis is frequently accompanied by disease-related malnutrition (DRM) and sarcopenia, defined as loss of skeletal muscle mass and function. DRM and sarcopenia often coexist in cirrhotic patients and are associated with increased morbidity and mortality. The clinical manifestation of both comorbidities are triggered by multifactorial mechanisms including reduced nutrient and energy intake caused by dietary restrictions, anorexia, neuroendocrine deregulation, olfactory and gustatory deficits. Maldigestion and malabsorption due to small intestinal bacterial overgrowth, pancreatic insufficiency or cholestasis may also contribute to DRM and sarcopenia. Decreased protein synthesis and increased protein degradation is the cornerstone mechanism to muscle loss, among others mediated by disease- and inflammation-mediated metabolic changes, hyperammonemia, increased myostatin and reduced human growth hormone. The concise pathophysiological mechanisms and interactions of DRM and sarcopenia in liver cirrhosis are not completely understood. Furthermore, most knowledge in this field are based on experimental models, but only few data in humans exist. This review summarizes known and proposed molecular mechanisms contributing to malnutrition and sarcopenia in liver cirrhosis and highlights remaining knowledge gaps. Since, in the prevention and treatment of DRM and sarcopenia in cirrhotic patients, more research is needed to identify potential biomarkers for diagnosis and development of targeted therapeutic strategies.
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Cirrosis Hepática , Desnutrición , Músculo Esquelético , Sarcopenia , Humanos , Cirrosis Hepática/complicaciones , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Desnutrición/etiología , Desnutrición/metabolismo , Desnutrición/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Sarcopenia/etiología , Sarcopenia/metabolismo , Sarcopenia/patologíaRESUMEN
Nucleotide-binding oligomerization domain-containing protein 2 (NOD2) gene mutations are a risk factor for Crohn's disease and also associated with worse outcome in short bowel syndrome (SBS) patients independent of the underlying disease. The aim of this study was to analyze the effect of Nod2 deficiency on barrier function and stool microbiome after extensive ileocecal resection in mice. Male C57BL6/J wild-type (WT) and Nod2-knockout (KO) mice underwent 40% ileocecal resection. Sham control mice received simple transection of the ileum. Clinical outcome was monitored daily. Barrier function was measured with Ussing chambers using FITC-4-kDa-Dextran flux, transmucosal electrical resistance, and dilution potentials. Immunofluorescence of claudin-2 was studied. Composition of the stool microbiome was assessed by 16S rRNA gene sequencing. Resected Nod2-KO mice had impaired clinical outcome compared with resected WT mice. This was accompanied by increased stool water contents and increased plasma aldosterone. Histomorphological adaptation was independent of Nod2. Barrier function studies revealed impaired sodium to chloride permeability and altered claudin-2 localization in the absence of Nod2. Resection induced decreases of bacterial diversity and a shift of bacteriodetes-to-firmicutes ratios. Ileum and cecum resection-induced increase in proteobacteria was absent in Nod2-deficient mice. Verrucomicrobia were temporarily increased in Nod2-KO mice. Nod2 deficiency functionally impairs adaptation to short bowel syndrome via a lesser increase of epithelial sodium pore permeability, altered epithelial barrier function, and the microbiome.NEW & NOTEWORTHYNOD2 gene mutations are associated with the development of severe short bowel syndrome and intestinal failure. The influence of Nod2 mutations on intestinal adaptation in experimental short bowel syndrome has not been studied yet. Here, we provide data that Nod2 deficiency worsens clinical outcome and functional adaptation under SBS conditions in mice, indicating that NOD2 is required for successful adaptation after ileocecal resection.
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Adaptación Fisiológica , Absorción Intestinal , Mucosa Intestinal/metabolismo , Proteína Adaptadora de Señalización NOD2/genética , Síndrome del Intestino Corto/genética , Aldosterona/metabolismo , Animales , Cloruros/metabolismo , Conductividad Eléctrica , Microbioma Gastrointestinal , Íleon/metabolismo , Íleon/microbiología , Transporte Iónico , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína Adaptadora de Señalización NOD2/deficiencia , Síndrome del Intestino Corto/metabolismo , Síndrome del Intestino Corto/fisiopatología , Sodio/metabolismoRESUMEN
The immunopathogenesis of autoimmune pancreatitis (AIP) is poorly understood. Here, we have used MRL/MpJ mice, a model of spontaneous AIP, to address the role of cellular autoimmune processes in the initiation and progression of the disease. Therefore, different T cell subpopulations were adoptively transferred from sick to still healthy (but susceptible) MRL/MpJ mice. Unpurified splenocytes and CD3+ T cells both efficiently induced AIP, while CD4+ and CD8+ T cells alone, as well as splenocytes from healthy mice, were insufficient to trigger the disease. Strikingly, CD4+ CD44high memory T cells, although transferred at lower numbers than other T cells, also induced AIP in recipient mice. Employing a modified experimental design, we also evaluated the effects of regulatory T cells (Tregs ) on the progression of AIP in already diseased mice. Under the given experimental conditions, there was no significant suppressive effect of adoptively transferred Tregs on pancreatic histopathology. The results of our studies suggest a key role of T cell-mediated processes in murine AIP. The effects of CD4+ CD44high memory T cells are in accordance with genetic studies of our group, which had previously implicated this cell type into the pathogenesis of AIP. In follow-up studies, we will focus on the interplay of cellular and humoral autoimmunity in the context of AIP.
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Enfermedades Autoinmunes/inmunología , Receptores de Hialuranos/inmunología , Pancreatitis/inmunología , Linfocitos T Reguladores/inmunología , Animales , Enfermedades Autoinmunes/patología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Humanos , Ratones , Pancreatitis/patologíaRESUMEN
BACKGROUND: Uncoupling protein 2 (UCP2) has been suggested to inhibit mitochondrial production of reactive oxygen species (ROS) by decreasing the mitochondrial membrane potential. Experimental acute pancreatitis is associated with increased UCP2 expression, whereas UCP2 deficiency retards regeneration of aged mice from acute pancreatitis. Here, we have addressed biological and molecular functions of UCP2 in pancreatic stellate cells (PSCs), which are involved in pancreatic wound repair and fibrogenesis. METHODS: PSCs were isolated from 12 months old (aged) UCP2-/- mice and animals of the wild-type (WT) strain C57BL/6. Proliferation and cell death were assessed by employing trypan blue staining and a 5-bromo-2'-deoxyuridine incorporation assay. Intracellular fat droplets were visualized by oil red O staining. Levels of mRNA were determined by RT-PCR, while protein expression was analyzed by immunoblotting and immunofluorescence analysis. Intracellular ROS levels were measured with 2', 7'-dichlorofluorescin diacetate. Expression of senescence-associated beta-galactosidase (SA beta-Gal) was used as a surrogate marker of cellular senescence. RESULTS: PSCs derived from UCP2-/- mice proliferated at a lower rate than cells from WT mice. In agreement with this observation, the UCP2 inhibitor genipin displayed dose-dependent inhibitory effects on WT PSC growth. Interestingly, ROS levels in PSCs did not differ between the two strains, and PSCs derived from UCP2-/- mice did not senesce faster than those from corresponding WT cells. PSCs from UCP2-/- mice and WT animals were also indistinguishable with respect to the activation-dependent loss of intracellular fat droplets, expression of the activation marker alpha-smooth muscle actin, type I collagen and the autocrine/paracrine mediators interleukin-6 and transforming growth factor-beta1. CONCLUSIONS: A reduced proliferative capacity of PSC from aged UCP2-/- mice may contribute to the retarded regeneration after acute pancreatitis. Apart from their slower growth, PSC of UCP2-/- mice displayed no functional abnormalities. The antifibrotic potential of UCP2 inhibitors deserves further attention.
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Proliferación Celular , Células Estrelladas Pancreáticas/metabolismo , Proteína Desacopladora 2/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Genotipo , Iridoides/farmacología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Estrelladas Pancreáticas/efectos de los fármacos , Células Estrelladas Pancreáticas/patología , Fenotipo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Proteína Desacopladora 2/antagonistas & inhibidores , Proteína Desacopladora 2/deficiencia , Proteína Desacopladora 2/genéticaRESUMEN
BACKGROUND: Modulation of the stroma response is considered a promising approach for the treatment of chronic pancreatitis and pancreatic cancer. The aim of this study was to evaluate the effects of three clinically available small molecule kinase inhibitors, regorafenib, trametinib and dactolisib, on effector functions of activated pancreatic stellate cells (PSCs), which play a key role in pancreatic fibrosis. METHODS: Cultured rat PSCs were exposed to small molecule kinase inhibitors. Proliferation and cell death were assessed by measuring the incorporation of 5-bromo-2'-deoxyuridine and cytotoxicity, respectively. Levels of mRNA were determined by real-time PCR, while protein expression and phosphorylation were analyzed by immunoblotting. Interleukin-6 levels in culture supernatants were quantified by ELISA. Zymography assays were performed to monitor collagenase activity in culture supernatants. RESULTS: The MEK inhibitor trametinib and the dual phosphatidylinositol 3-kinase/mTOR inhibitor dactolisib, but not the multi-kinase inhibitor regorafenib, efficiently inhibited PSC proliferation. Trametinib as well as regorafenib suppressed the expression of two autocrine mediators of PSC activation, interleukin-6 and transforming growth factor-beta1. Dactolisib-treated cells expressed less alpha1 type I collagen and lower levels of alpha-smooth muscle actin, a marker of the myofibroblastic PSC phenotype. Simultaneous application of dactolisib and trametinib displayed additive inhibitory effects on cell growth without statistically significant cytotoxicity. Activity of matrix metalloproteinase-2 was not affected by any of the drugs. CONCLUSION: We suggest the combination of two drugs, that specifically target two key signaling pathways in PSC, Ras-Raf-MEK-ERK (trametinib) and phosphatidylinositol 3-kinase-AKT-mTOR (dactolisib), as a concept to modulate the activation state of the cells in the context of fibrosis.
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Proliferación Celular/efectos de los fármacos , Imidazoles/farmacología , Células Estrelladas Pancreáticas/efectos de los fármacos , Pancreatitis Crónica/tratamiento farmacológico , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/farmacología , Piridonas/farmacología , Pirimidinonas/farmacología , Quinolinas/farmacología , Actinas/genética , Actinas/metabolismo , Animales , Células Cultivadas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Relación Dosis-Respuesta a Droga , Fibrosis , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Estrelladas Pancreáticas/enzimología , Células Estrelladas Pancreáticas/patología , Pancreatitis Crónica/enzimología , Pancreatitis Crónica/genética , Pancreatitis Crónica/patología , Fosfatidilinositol 3-Quinasa/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Ratas Endogámicas Lew , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Factores de Tiempo , Factor de Crecimiento Transformador beta1/genética , Factor de Crecimiento Transformador beta1/metabolismoRESUMEN
BACKGROUND: Autoimmune pancreatitis (AIP) in humans invariably responds to steroid treatment, but little is known about the underlying pathogenesis and the benefits of alternative treatments. OBJECTIVE: To study the pathogenesis, and the efficacy of alternative immunosuppressant agents in the MRL/Mp mouse model of AIP. DESIGN: MRL/Mp mice were pretreated for 4 weeks with polyinosinic:polycytidylic acid to induce AIP. Pancreatic sections of mice genetically deleted for CTLA-4 were analysed. Blockage of CTLA-4 was achieved by intraperitoneal antibody treatment with 2 µg/g anti-mouse-CD152. Subsequent therapeutic studies were performed for a period of 4 weeks using cyclosporine A (40 µg/g), rapamycin (1 µg/g) or azathioprine (15 µg/g). RESULTS: Blockage of CTLA-4 in MRL/Mp mice suppressed regulatory T cell (Treg) function and raised the effector T cell (Teff) response with subsequent histomorphological organ destruction, indicating that AIP is a T cell-driven disease. Using an established histopathological score, we found that dexamethasone, cyclosporine A and rapamycin, but less so azathioprine, reduced pancreatic damage. However, the beneficial effects of cyclosporine A and rapamycin were achieved via different mechanisms: cyclosporine A inhibited Teff activation and proliferation whereas rapamycin led to selective expansion of Tregs which subsequently suppressed the Teff response. CONCLUSIONS: The calcineurin inhibitor cyclosporine A and the mammalian target of rapamycin (mTOR) inhibitor, rapamycin, improve the course of AIP in MRL/Mp mice via different mechanisms. These findings further support the concept of autoreactive T cells as key players in the pathogenesis of AIP and suggest that cyclosporine A and rapamycin should be considered for treatment of AIP in humans.
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Enfermedades Autoinmunes/tratamiento farmacológico , Ciclosporina/uso terapéutico , Inmunosupresores/uso terapéutico , Páncreas/inmunología , Pancreatitis Crónica/tratamiento farmacológico , Sirolimus/uso terapéutico , Subgrupos de Linfocitos T/metabolismo , Animales , Enfermedades Autoinmunes/inducido químicamente , Enfermedades Autoinmunes/inmunología , Enfermedades Autoinmunes/patología , Azatioprina/uso terapéutico , Biomarcadores/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Proliferación Celular/efectos de los fármacos , Ciclosporina/farmacología , Dexametasona/uso terapéutico , Esquema de Medicación , Femenino , Citometría de Flujo , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos , Páncreas/efectos de los fármacos , Páncreas/patología , Pancreatitis Crónica/inducido químicamente , Pancreatitis Crónica/inmunología , Pancreatitis Crónica/patología , Poli I-C , Distribución Aleatoria , Sirolimus/farmacología , Subgrupos de Linfocitos T/efectos de los fármacos , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVES: The gut-liver axis is discussed to play an important role in hepatic cirrhosis. Decompensated liver cirrhosis is associated with portal hypertension, which can lead to a variety of complications. Transjugular intrahepatic portosystemic shunt (TIPS) is an established treatment option for the complications of portal hypertension. In this study we focused on the effect of TIPS on intestinal microbial composition in cirrhotic patients. METHODS: Thirty patients with liver cirrhosis were compared to 18 healthy adults. Seventeen patients with cirrhosis and portal hypertension received a TIPS. Clinical characteristics, including age, sex, and liver function measured with a Child-Pugh score and model for end-stage liver disease score, were obtained. Intestinal microbial composition was assessed via 16S rRNA gene amplicon sequencing from stool probes before and after TIPS. RESULTS: TIPS led to a reduction of hepatic venous pressure gradient. However, TIPS did not cause a shift in the intestinal bacterial communities. Independent from the application of TIPS, antibiotic therapy was associated with a significant difference in the intestinal bacterial microbiota and also a reduced α-diversity. In addition, a significant difference was observed in the intestinal bacterial composition between patients with liver cirrhosis and healthy controls. CONCLUSION: The presence of liver cirrhosis and the use of antibiotic therapy, but not the application of TIPS, were associated with a significant shift of the intestinal bacterial communities, showing a high impact on the microbiota of patients with liver cirrhosis.
Asunto(s)
Antibacterianos , Microbioma Gastrointestinal , Hipertensión Portal , Cirrosis Hepática , Derivación Portosistémica Intrahepática Transyugular , Humanos , Cirrosis Hepática/microbiología , Cirrosis Hepática/complicaciones , Femenino , Masculino , Microbioma Gastrointestinal/fisiología , Persona de Mediana Edad , Estudios Prospectivos , Antibacterianos/uso terapéutico , Hipertensión Portal/etiología , Anciano , Adulto , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/análisis , Estudios de Casos y Controles , Heces/microbiologíaRESUMEN
Animal models are crucial to preclinical oncological research and drug development. Animal experiments must be performed in accordance with the 3R principles of replacement and reduction, if possible, and refinement where these procedures remain crucial. In addition, European Union legislations demand a continuous refinement approach, as well as pro- and retrospective severity assessment. In this study, an objective databased severity assessment was performed in murine models for pancreatic cancer induced by orthotopic, subcutaneous, or intravenous injection of Panc02 cells. Parameters such as body weight change, distress score, perianal temperature, mouse grimace scale, burrowing, nesting behavior, and the concentration of corticosterone in plasma and its metabolites in feces were monitored during tumor progression. The most important parameters were combined into a score and mapped against a reference data set by the Relative Severity Assessment procedure (RELSA) to obtain the maximum achieved severity for each animal (RELSAmax). This scoring revealed a significantly higher RELSAmax for the orthotopic model than for the subcutaneous and intravenous models. However, compared to animal models such as pancreatitis and bile duct ligation, the pancreatic cancer models are shown to be less severe. Data-based animal welfare assessment proved to be a valuable tool for comparing the severity of differently induced cancer models.
RESUMEN
Gene targeting experiments have shown that the cytokine erythropoietin (EPO), its cognate erythropoietin receptor (EPO-R), and associated Janus tyrosine kinase, JAK2, are all essential for erythropoiesis. Structural-functional and murine knock-in experiments have suggested that EPO-R Tyr-343 is important in EPO-mediated mitogenesis. Although Stat5 binds to EPO-R phosphotyrosine 343, the initial Stat5-deficient mice did not have profound erythroid abnormalities suggesting that additional Src homology 2 (SH2) domain-containing effectors may bind to EPO-R Tyr-343 and couple to downstream signaling pathways. We have utilized cloning of ligand target (COLT) screening to demonstrate that EPO-R Tyr(P)-343 and Tyr(P)-401 bind to the SH2 domain-containing adaptor protein SH2B1ß. Immunoprecipitation and in vitro mixing experiments reveal that EPO-R binds to SH2B1 in an SH2 domain-dependent manner and that the sequence that confers SH2B1 binding to the EPO-R is pYXXL. Previous studies have shown that SH2B1 binds directly to JAK2, but we show that in hematopoietic cells, SH2B1ß preferentially associates with the EPO-R. SH2B1 is capable of constitutive association with EPO-R, which is necessary for its optimal SH2-dependent recruitment to EPO-R-Tyr(P)-343/Tyr(P)-401. We also demonstrate that SH2B1 is responsive to EPO stimulation and becomes phosphorylated, most likely on serines/threonines, in an EPO dose- and time-dependent manner. In the absence of SH2B1, we observe enhanced activation of signaling pathways downstream of the EPO-R, indicating that SH2B1 is a negative regulator of EPO signaling.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Receptores de Eritropoyetina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/aislamiento & purificación , Animales , Línea Celular , Eritroblastos/metabolismo , Eritropoyetina/fisiología , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Fragmentos de Péptidos/metabolismo , Fosforilación , Cultivo Primario de Células , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Receptores de Eritropoyetina/química , Receptores de Eritropoyetina/aislamiento & purificación , Transducción de SeñalRESUMEN
BACKGROUND/OBJECTIVES: Completion of pancreatic wound healing requires termination of pancreatic stellate cell (PSC) activation to prevent fibrosis. Besides induction of apoptosis and return to a quiescent phenotype, senescence of PSC followed by immune cell-mediated cytolysis represents a potential mechanism. Here, we have studied if the cell cycle inhibitor cyclin-dependent kinase inhibitor 1A (Cdkn1a, p21/Waf1), expression of which is increased in senescent rat PSC, plays a causative role in the senescence process. METHODS: Senescence was induced by doxorubicin treatment. The functions of Cdkn1a were analyzed using two approaches, treatment of primary rat PSC with siRNA and tetracycline-regulated overexpression of Cdkn1a in immortalized rat cells. Expression of senescence-associated ß-galactosidase (SA ß-Gal) was used as a surrogate marker of senescence. RESULTS: The knockdown of Cdkn1a significantly attenuated the growth-inhibitory effect of doxorubicin and strongly diminished the portion of SA ß-Gal-positive cells. Overexpression of Cdkn1a enhanced both the antiproliferative effect of doxorubicin and induction of senescence. In primary PSC, doxorubicin treatment was associated with increased expression of interleukin-6 (IL-6) and matrix metalloproteinase (MMP)-9, while expression of the activation marker α-smooth muscle actin (α-SMA), p53, Cdk1 and Rad54 was diminished. The application of Cdkn1a siRNA specifically antagonized the effects of doxorubicin on the expression of p53, Cdk1 and Rad54 but not IL-6 and α-SMA, while MMP-9 expression and also activity were even enhanced. CONCLUSIONS: Cdkn1a plays a direct role in the process of rat PSC senescence. Additional Cdkn1a-independent pathways may contribute to the partial maintenance of a gene expression profile typical of senescent PSC.
Asunto(s)
Senescencia Celular/fisiología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Células Estrelladas Pancreáticas/fisiología , Animales , Línea Celular , Senescencia Celular/efectos de los fármacos , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/farmacología , Células Estrelladas Pancreáticas/efectos de los fármacos , ARN Interferente Pequeño/farmacología , Ratas , beta-Galactosidasa/biosíntesisRESUMEN
As research institutions prepare roadmaps for "systems medicine," we ask how this differs from applications of systems biology approaches in medicine and what we (should) have learned from about one decade of funding in systems biology. After surveying the area, we conclude that systems medicine is the logical next step and necessary extension of systems biology, and we focus on clinically relevant applications. We specifically discuss three related notions. First, more interdisciplinary collaborations are needed to face the challenges of integrating basic research and clinical practice: integration, analysis, and interpretation of clinical and nonclinical data for diagnosis, prognosis, and therapy require advanced statistical, computational, and mathematical tools. Second, strategies are required to (i) develop and maintain computational platforms for the integration of clinical and nonclinical data, (ii) further develop technologies for quantitative and time-resolved tracking of changes in gene expression, cell signaling, and metabolism in relation to environmental and lifestyle influences, and (iii) develop methodologies for mathematical and statistical analyses of integrated data sets and multilevel models. Third, interdisciplinary collaborations represent a major challenge and are difficult to implement. For an efficient and successful initiation of interdisciplinary systems medicine programs, we argue that epistemological, ontological, and sociological aspects require attention.