MiR-155 modulates the inflammatory phenotype of intestinal myofibroblasts by targeting SOCS1 in ulcerative colitis

Abnormal levels of microRNA (miR)-155, which regulate inflammation and immune responses, have been demonstrated in the colonic mucosa of patients with inflammatory bowel diseases (IBD), although its role in disease pathophysiology is unknown. We investigated the role of miR-155 in the acquisition and maintenance of an activated phenotype by intestinal myofibroblasts (IMF), a key cell population contributing to mucosal damage in IBD. IMF were isolated from colonic biopsies of healthy controls, ulcerative colitis (UC) and Crohn's disease (CD) patients. MiR-155 in IMF was quantified by quantitative reverse transcription-PCR in basal condition and following exposure to TNF-alpha, interleukin (IL)-1 beta, lipopolysaccharide (LPS) or TGF-beta 1. The effects of miR-155 mimic or inhibitor transfection on cytokine release and suppressor of cytokine signaling 1 (SOCS1) expression were assessed by enzyme-linked immunosorbent assay and western blot, respectively. Regulation of the target gene SOCS1 expression by miR-155 was assessed using luciferase reporter construct. We found that miR-155 was significantly upregulated in UC as compared with control-and CD-derived IMF. Moreover, TNF-alpha and LPS, but not TGF-beta 1 and IL-1 beta, significantly increased miR-155 expression in IMF. Ectopic expression of miR-155 in control IMF augmented cytokines release, whereas it downregulated SOCS1 expression. MiR-155 knockdown in UC-IMF reduced cytokine production and enhanced SOCS1 expression. Luciferase reporter assay demonstrated that miR-155 directly targets SOCS1. Moreover, silencing of SOCS1 in control IMF significantly increased IL-6 and IL-8 release. In all, our data suggest that inflammatory mediators induce miR-155 expression in IMF of patients with UC. By downregulating the expression of SOCS1, miR-155 wires IMF inflammatory phenotype

Aberrant MET activation impairs perinuclear actin cap organization with YAP1 cytosolic relocation

: Little is known about the signaling network responsible for the organization of the perinuclear actin cap, a recently identified structure holding unique roles in the regulation of nuclear shape and cell directionality. In cancer cells expressing a constitutively active MET, we show a rearrangement of the actin cap filaments, which crash into perinuclear patches associated with spherical nuclei, meandering cell motility and inactivation of the mechano-transducer YAP1. MET ablation is sufficient to reactivate YAP1 and restore the cap, leading to enhanced directionality and flattened nuclei. Consistently, the introduction of a hyperactive MET in normal epithelial cells, enhances nuclear height and alters the cap organization, as also confirmed by TEM analysis. Finally, the constitutively active YAP1 mutant YAP5SA is able to overcome the effects of oncogenic MET. Overall, our work describes a signaling axis empowering MET-mediated YAP1 dampening and actin cap misalignment, with implications for nuclear shape and cell motility

The autocrine loop of ALK receptor and ALKAL2 ligand is an actionable target in consensus molecular subtype 1 colon cancer

In the last years, several efforts have been made to classify colorectal cancer (CRC) into well-defined molecular subgroups, representing the intrinsic inter-patient heterogeneity, known as Consensus Molecular Subtypes (CMSs)

Serum Albumin Is Inversely Associated With Portal Vein Thrombosis in Cirrhosis

We analyzed whether serum albumin is independently associated with portal vein thrombosis (PVT) in liver cirrhosis (LC) and if a biologic plausibility exists. This study was divided into three parts. In part 1 (retrospective analysis), 753 consecutive patients with LC with ultrasound-detected PVT were retrospectively analyzed. In part 2, 112 patients with LC and 56 matched controls were entered in the cross-sectional study. In part 3, 5 patients with cirrhosis were entered in the in vivo study and 4 healthy subjects (HSs) were entered in the in vitro study to explore if albumin may affect platelet activation by modulating oxidative stress. In the 753 patients with LC, the prevalence of PVT was 16.7%; logistic analysis showed that only age (odds ratio [OR], 1.024; P = 0.012) and serum albumin (OR, -0.422; P = 0.0001) significantly predicted patients with PVT. Analyzing the 112 patients with LC and controls, soluble clusters of differentiation (CD)40-ligand (P = 0.0238), soluble Nox2-derived peptide (sNox2-dp; P < 0.0001), and urinary excretion of isoprostanes (P = 0.0078) were higher in patients with LC. In LC, albumin was correlated with sCD4OL (Spearman's rank correlation coefficient [r(s)], -0.33; P < 0.001), sNox2-dp (r(s), -0.57; P < 0.0001), and urinary excretion of isoprostanes (r(s), -0.48; P < 0.0001) levels. The in vivo study showed a progressive decrease in platelet aggregation, sNox2-dp, and urinary 8-iso prostaglandin F2 alpha-III formation 2 hours and 3 days after albumin infusion. Finally, platelet aggregation, sNox2-dp, and isoprostane formation significantly decreased in platelets from HSs incubated with scalar concentrations of albumin. Conclusion: Low serum albumin in LC is associated with PVT, suggesting that albumin could be a modulator of the hemostatic system through interference with mechanisms regulating platelet activation

Discutindo a educação ambiental no cotidiano escolar: desenvolvimento de projetos na escola formação inicial e continuada de professores

A presente pesquisa buscou discutir como a Educação Ambiental (EA) vem sendo trabalhada, no Ensino Fundamental e como os docentes desta escola compreendem e vem inserindo a EA no cotidiano escolar., em uma escola estadual do município de Tangará da Serra/MT, Brasil. Para tanto, realizou-se entrevistas com os professores que fazem parte de um projeto interdisciplinar de EA na escola pesquisada. Verificou-se que o projeto da escola não vem conseguindo alcançar os objetivos propostos por: desconhecimento do mesmo, pelos professores; formação deficiente dos professores, não entendimento da EA como processo de ensino-aprendizagem, falta de recursos didáticos, planejamento inadequado das atividades. A partir dessa constatação, procurou-se debater a impossibilidade de tratar do tema fora do trabalho interdisciplinar, bem como, e principalmente, a importância de um estudo mais aprofundado de EA, vinculando teoria e prática, tanto na formação docente, como em projetos escolares, a fim de fugir do tradicional vínculo “EA e ecologia, lixo e horta”.Facultad de Humanidades y Ciencias de la Educació

Modificazione della flora intestinale e obesità : possibile ruolo di resistin-like Molecule Beta (RELM Beta)

Obesity is due to complex interactions between genetic background and environmental factors, such as diet and lifestyle. Experimental studies suggest that gut microbiota of obese subjects differs from lean ones and that changes in gut microbiota (prevalence of phylum Firmicutes in obese condition) are associated to body weight gain and fat accumulation. Conventional mice have significantly more body fat than germ-free counterparts, and germ-free mice are protected from diet-induced obesity. New evidences in literature show the important role of Resistine-like molecule β ( Relmβ ) played in microbiota composition and in induction of insuline resistence. Moreover the intestinal bacterial omeostasis is regulated from antimicrobical peptides secreted from Paneth cells. In the gut of α Defensin5 KO mice there is a predominant presence of bacteria from phylum Firmicutes. In order to investigate the relationship among intestinal microbiota changes, innate immunity and pathogenesis of obesity, male C57Bl/6 WT and Tlr2-/- mice were randomly allocated to receive either regular chow food (RCF) or a very high fat diet (VHFD) for 10 weeks (wks). Body weight was recorded weekly, oral glucose tolerance test, steatosis, liver inflammation, antimicrobical peptides and Relmβ expression were assessed in ileum of animals sacrificed following 2, 4, 6 and 10 weeks of diet. In addition, faecal samples were collected every two weeks and the faecal flora studied by PCRDGGE with universal primers. In order to investigate the potential effects of probiotics in balancing of gut microbiota and improve metabolic syndrome, male C57Bl/6 mice 4 weeks old were fed VHFD and daily supplemented with 108 cfu of Lactobacillus crispatus M247 for 10 weeks. Gut microbiota of diet-induced obese mice resulted in an increased presence of phylum Firmicutes compared to mice fed a normocaloric diet. These changes in Gram positive microbiota composition were showed after 4 weeks on VHFD and these modifications were stably maintained over the period of VHFD administration. WT and TLR2-/- mice on RCF showed comparable body weight gain whereas TLR2-/- mice on VHFD were more prone to body weight gain than WT mice (32.05±0.5 g and 27.5±0.49 g, respectively, at 10 wks, p<0.02). In WT mice on VHFD Relm β mRNA level (p=0,02 vs mice on RCF) is increased whilst expression of intestinal antimicrobical peptides (RegIIIβ, RegIIIγ) mRNAs is downregulated ( p=0,01vs mise on RCF ). Indeed, hRELMβ caused a significant upregulation of βDefensins and αDefensins ( p=0,03 and p=0,02 respectively ) in an vitro model of intestinal epithelial cells ( CaCo2 ). Interestingly, we observed limited changes of gut microbiota in mice receiving a VHF and oral supplementation with L. crispatus M247 (e.g. the presence of Firmitutes is not predominant like mice on VHFD without supplementation of probiotic strain). Indeed, mRNA level of Relm β in ileum was down-regulated (p=0,01 vs mice on VHFD) whilst the expression of intestinal antimicrobical peptides was up-regulated ( p=0,05 vs mise on VHFD). Strikingly, L. crispatus M247 significantly inhibited stearic acid induced Relm β up-regulation in Caco2 cells in vitro. This data is extremely interesting since L. crispatus M247 supplementation significantly improved glucose tolerance test (AUC: 591±94 mg/dl2 in VHFD mice vs 397±37 mg/dl2 in VHFD mice plus L. crispatus M247, p<0.02) although mice receiving probiotic supplementation did not show significant differences in body weight gain as compared to mice receiving only VHFD. In summary we suggest that microbiota modifications preceed the development of a diet induced obese phenotype and these changes can be triggered by changes in the mucosal production of RELMβ, a key factor involved in glucose and gut microbiota homeostasis. Probiotic supplementation, affecting RELMβ production might favorably affect gut microbiota and metabolic syndrome severityL’obesità è dovuta alla complessa interazione tra componenti genetiche e fattori ambientali, come la dieta e lo stile di vita. Studi sperimentali condotti in vivo dimostrano come la composizione della flora intestinale dei soggetti obesi differisca dai soggetti non obesi e come questi cambiamenti siano associati all’aumento di peso. I topi germ-free presentano meno accumulo di grasso rispetto ai topi normali e sono meno predisposti all’obesità non genetica ( obesità indotta dalla dieta ). Studi recenti hanno messo in evidenza l’importanza del ruolo svolto dalla Resistin-like Molecul β ( Relm β ) nella composizione della flora intestinale e sull’insulino-resistenza. L’omeostasi batterica, tuttavia, viene regolata dalla produzione a livello intestinale da peptidi antimicrobici secreti dalla cellule del Paneth: le defensine. In topi mancanti del gene che codifica per la αDefensina 6, infatti, si verifica la prevalenza del phylum batterico dei Firmicutes. Allo scopo di chiarire la relazione tra la modificazione della flora intestinale, immunità innata e obesità, topi C57Bl/6 WT e Tlr2-/- (deficienti del gene che codifica per il recettore TLR2 dell’immunità innata) sono stati posti per 10 settimane a dieta normocalorica e iperlipidica (VHF Very High Fat diet). Il peso corporeo è stato registrato settimanalmente, mentre il test di tolleranza al glucosio, la steatosi, l’infiammazione, la valutazione dell’espressione di Relmβ e dei peptidi antimicrobici nell’ileo degli animali trattati, sono state effettuate a 2, 4, 6, 10 settimane. Contemporaneamente campioni fecali sono stati raccolti ogni due settimane e lo studio sulla composizione della flora batterica è stato condotto tramite PCR-DGGE con primer universali amplificanti la regione 16S ribosomiale batterica. Allo scopo di investigare l’effetto di un microrganismo probiotico sulla composizione della flora intestinale e sulla sindrome metabolica, topi C57Bl/6 di 4 settimane di vita sono stati alimentati con dieta normocalorica e VHF supplementata e non con Lactobacillus crispatus M247 per 10 settimane. I topi WT C57Bl/6 tenuti a dieta VHF presentano una flora intestinale costituita prevalentemente da Firmicutes rispetto ai topi WT tenuti a dieta normocalorica. Questi cambiamenti sono stati osservati dopo 4 settimane di dieta grassa e queste modificazioni sono persistite per la restante parte del periodo di somministrazione della dieta. I topi WT e TLR2-/- tenuti a dieta normocalorica hanno avuto un incremento di peso paragonabile, mentre nel gruppo VHF i i topi TLR2-/-hanno raggiunto un peso maggiore rispetto ai topi WT ( 32.05±0.5 g and 27.5±0.49 g, a dieci settimane rispettivamente p<0.02). Nei topi WT a dieta VHF i livelli di mRNA di Relm β nell’ileo risultano aumentati rispetto ai topi tenuti a dieta normocalorica (p=0,02 vs topi a dieta normocalorica ), mentre l’espressione dei peptidi antimicrobici risulta diminuita (p=0,01vs topi a dieta normocalorica ). Inoltre, hRELMβ causa una significativa down-regulation di βDefensine ed αDefensina (p=0,03 and p=0,02 rispettivamente) in un modello in vitro di cellule epiteliali intestinali ( CaCo2 ). Sorprendentemente è stato osservato un limitato cambiamento nella composizione del microbiota in topi che hanno ricevuto una dieta VHF supplementata con Lactobacillus crispatus M247. ( la presenza di Firmicutes non è così predominante come nei topi a dieta VHF senza supplementazione con il probiotico ). Inoltre i livelli di mRNA di Relm β nell’ileo risulta down-regolata (p=0,01 vs topi VHF), mentre l’espressione di peptidi antimicrobici risulta up-regolata (p=0,05 vs topi VHF). Inoltre, L. crispatus M247 inibisce l’up-regolazione indotta da Acido Stearico di Relm β nelle cellule Caco2 in vitro. Questi dati sembrano molto interessanti se si pensa che la supplementazione di L. crispatus M247 migliora la tolleranza al glucosio (AUC: 591±94 mg/dl2 in topi VHF vs 397±37 mg/dl2 in topi VHF+L. crispatus M247, p<0.02), sebbene i topi che hanno ricevuto la supplementazione non mostrano differenza significative nell’aumento di peso rispetto ai topi tenuti a dieta VHF senza supplementazione. Riassumento si può concludere che: le modificazioni della flora intestinale precede l’aumento di peso nel modello di obesità non genetica utilizzato e che, questi cambiamenti potrebbero essere legati ai cambiamenti di produzione mucosale di RELMβ, fattore chiave nell’omeostasi del glucosio e del microbiota intestinale. Inoltre la supplementazione dietetica, attraverso una down-regolazione dell’espressione di RELMβ favorisce l’attenuarsi della severità della sindrome metabolic

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: Involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells