RESUMEN
BACKGROUND AND AIMS: Intestinal fibrosis is a common complication of Inflammatory Bowel Disease (IBD), namely Crohn's disease (CD) and ulcerative colitis (UC), but the precise mechanism by which it occurs is incompletely understood hampering the development of effective therapeutic strategies. Here, we aimed at inducing and characterizing an inflammation-mediated fibrosis in patient-derived organoids (PDOs) issued from crypts isolated from colonic mucosal biopsies of IBD pediatric patients and age matched-control subjects (CTRLs). METHODS: Inflammatory-driven fibrosis was induced by exposing CTRL-, CD- and UC-PDOs to the pro-inflammatory cytokine TNF-α for one day, followed by a co-treatment with TNF-α and TGF-ß1 for three days. Fibrotic response was proven by analyzing inflammatory and fibrotic markers by RT-qPCR and immunofluorescence. Transcriptomic changes were assessed by RNA-sequencing. RESULTS: Co-treatment with TNF-α and TGF-ß1 caused in CTRL- and IBD-PDOs morphological changes towards a mesenchymal-like phenotype and up-regulation of inflammatory, mesenchymal, and fibrotic markers. Transcriptomic profiling highlighted that in all intestinal PDOs, regardless of the disease, the co-exposure to TNF-α and TGF-ß1 regulated EMT genes and specifically increased genes involved in positive regulation of cell migration. Finally, we demonstrated that CD-PDOs display a specific response to fibrosis compared to both CTRL- and UC-PDOs, mainly characterized by upregulation of nuclear factors controlling transcription. CONCLUSIONS: This study demonstrates that intestinal PDOs may develop an inflammatory-derived fibrosis thus representing a promising tool to study fibrogenesis in IBD. Fibrotic PDOs show increased expression of EMT genes. In particular, fibrotic CD-PDOs display a specific gene expression signature compared to UC and CTRL-PDOs.
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Fibrosis , Organoides , Factor de Crecimiento Transformador beta1 , Factor de Necrosis Tumoral alfa , Humanos , Organoides/patología , Factor de Crecimiento Transformador beta1/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedades Inflamatorias del Intestino/patología , Niño , Femenino , Masculino , Mucosa Intestinal/patología , Adolescente , Enfermedad de Crohn/patología , Enfermedad de Crohn/metabolismo , Colitis Ulcerosa/patología , Intestinos/patologíaRESUMEN
Extracellular High-mobility group box 1 (HMGB1) contributes to the pathogenesis of inflammatory disorders, including inflammatory bowel diseases (IBD). Poly (ADP-ribose) polymerase 1 (PARP1) has been recently reported to promote HMGB1 acetylation and its secretion outside cells. In this study, the relationship between HMGB1 and PARP1 in controlling intestinal inflammation was explored. C57BL6/J wild type (WT) and PARP1-/- mice were treated with DSS to induce acute colitis, or with the DSS and PARP1 inhibitor, PJ34. Human intestinal organoids, which are originated from ulcerative colitis (UC) patients, were exposed to pro-inflammatory cytokines (INFγ + TNFα) to induce intestinal inflammation, or coexposed to cytokines and PJ34. Results show that PARP1-/- mice develop less severe colitis than WT mice, evidenced by a significant decrease in fecal and serum HMGB1, and, similarly, treating WT mice with PJ34 reduces the secreted HMGB1. The exposure of intestinal organoids to pro-inflammatory cytokines results in PARP1 activation and HMGB1 secretion; nevertheless, the co-exposure to PJ34, significantly reduces the release of HMGB1, improving inflammation and oxidative stress. Finally, HMGB1 release during inflammation is associated with its PARP1-induced PARylation in RAW264.7 cells. These findings offer novel evidence that PARP1 favors HMGB1 secretion in intestinal inflammation and suggest that impairing PARP1 might be a novel approach to manage IBD.
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Colitis , Proteína HMGB1 , Enfermedades Inflamatorias del Intestino , Poli(ADP-Ribosa) Polimerasa-1 , Animales , Humanos , Ratones , Colitis/inducido químicamente , Citocinas , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamación , Organoides , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
Pelvic radiation disease (PRD), a frequent side effect in patients with abdominal/pelvic cancers treated with radiotherapy, remains an unmet medical need. Currently available preclinical models have limited applications for the investigation of PRD pathogenesis and possible therapeutic strategies. In order to select the most effective irradiation protocol for PRD induction in mice, we evaluated the efficacy of three different locally and fractionated X-ray exposures. Using the selected protocol (10 Gy/day × 4 days), we assessed PRD through tissue (number and length of colon crypts) and molecular (expression of genes involved in oxidative stress, cell damage, inflammation, and stem cell markers) analyses at short (3 h or 3 days after X-ray) and long (38 days after X-rays) post-irradiation times. The results show that a primary damage response in term of apoptosis, inflammation, and surrogate markers of oxidative stress was found, thus determining a consequent impairment of cell crypts differentiation and proliferation as well as a local inflammation and a bacterial translocation to mesenteric lymph nodes after several weeks post-irradiation. Changes were also found in microbiota composition, particularly in the relative abundance of dominant phyla, related families, and in alpha diversity indices, as an indication of dysbiotic conditions induced by irradiation. Fecal markers of intestinal inflammation, measured during the experimental timeline, identified lactoferrin, along with elastase, as useful non-invasive tools to monitor disease progression. Thus, our preclinical model may be useful to develop new therapeutic strategies for PRD treatment.
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Traumatismos por Radiación , Ratones , Animales , Rayos X , Modelos Animales de Enfermedad , Apoptosis/efectos de la radiación , InflamaciónRESUMEN
Cisplatin [cis-diamminedichloroplatinum(II) (cis-DDP)] is one of the most successful anticancer agents effective against a wide range of solid tumors. However, its use is restricted by side effects and/or by intrinsic or acquired drug resistance. Here, we probed the role of glutathione transferase (GST) P1-1, an antiapoptotic protein often overexpressed in drug-resistant tumors, as a cis-DDP-binding protein. Our results show that cis-DDP is not a substrate for the glutathione (GSH) transferase activity of GST P1-1. Instead, GST P1-1 sequesters and inactivates cisplatin with the aid of 2 solvent-accessible cysteines, resulting in protein subunits cross-linking, while maintaining its GSH-conjugation activity. Furthermore, it is well known that GST P1-1 binding to the c-Jun N-terminal kinase (JNK) inhibits JNK phosphorylation, which is required for downstream apoptosis signaling. Thus, in turn, GST P1-1 overexpression and Pt-induced subunit cross-linking could modulate JNK apoptotic signaling, further confirming the role of GST P1-1 as an antiapoptotic protein.
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Cisplatino/química , Gutatión-S-Transferasa pi/química , Proteínas Quinasas JNK Activadas por Mitógenos/química , Neoplasias/tratamiento farmacológico , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Cisplatino/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Glutatión/química , Gutatión-S-Transferasa pi/genética , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Neoplasias/genética , Fosforilación , Unión Proteica/efectos de los fármacos , Conformación Proteica , Transducción de Señal/efectos de los fármacosRESUMEN
Crohn's disease (CD) and ulcerative colitis (UC) are chronic inflammatory disorders of the gastrointestinal tract. Chronic inflammation is the main factor leading to intestinal fibrosis, resulting in recurrent stenosis, especially in CD patients. Currently, the underlying molecular mechanisms of fibrosis are still unclear. ZNF281 is a zinc-finger transcriptional regulator that has been characterized as an epithelial-to-mesenchymal transition (EMT)-inducing transcription factor, suggesting its involvement in the regulation of pluripotency, stemness, and cancer. The aim of this study is to investigate in vivo and in vitro the role of ZNF281 in intestinal fibrogenesis. Intestinal fibrosis was studied in vivo in C57BL/6J mice with chronic colitis induced by two or three cycles of administration of dextran sulfate sodium (DSS). The contribution of ZNF281 to gut fibrosis was studied in vitro in the human colon fibroblast cell line CCD-18Co, activated by the pro-fibrotic cytokine TGFß1. ZNF281 was downregulated by siRNA transfection, and RNA-sequencing was performed to identify genes regulated by TGFß1 in activated colon fibroblasts via ZNF281. Results showed a marked increase of ZNF281 in in vivo murine fibrotic colon as well as in in vitro human colon fibroblasts activated by TGFß1. Moreover, abrogation of ZNF281 in TGFß1-treated fibroblasts affected the expression of genes belonging to specific pathways linked to fibroblast activation and differentiation into myofibroblasts. We demonstrated that ZNF281 is a key regulator of colon fibroblast activation and myofibroblast differentiation upon fibrotic stimuli by transcriptionally controlling extracellular matrix (ECM) composition, remodeling, and cell contraction, highlighting a new role in the onset and progression of gut fibrosis.
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Colitis , Enfermedad de Crohn , Proteínas Represoras/metabolismo , Animales , Colitis/inducido químicamente , Colitis/genética , Colitis/metabolismo , Colon/patología , Enfermedad de Crohn/metabolismo , Sulfato de Dextran , Fibroblastos/metabolismo , Fibrosis , Humanos , Ratones , Ratones Endogámicos C57BL , ARN Interferente Pequeño/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Zinc/metabolismoRESUMEN
OBJECTIVES: The gut-liver axis has been recently investigated in depth in relation to intestinal and hepatic diseases. Key actors are bile acid (BA) receptors, as farnesoid-X-receptor (FXR), pregnane-X-receptor (PXR), and G-protein-coupled-receptor (GPCR; TGR5), that control a broad range of metabolic processes as well as inflammation and fibrosis. The present study aims to investigate the impact of intestinal inflammation on liver health with a focus on FXR, PXR, and TGR5 expression. The strategy to improve liver health by reducing gut inflammation is also considered. Modulation of BA receptors in the inflamed colonic tissues of inflammatory bowel disease (IBD) pediatric patients is analyzed. METHODS: A dextran sodium sulphate (DSS) colitis animal model was built. Co-cultures with Caco2 and HepG2 cell lines were set up. Modulation of BA receptors in biopsies of IBD pediatric patients was assessed by real-time PCR and immunohistochemistry. RESULTS: Histology showed inflammatory cell infiltration in the liver of DSS mice, where FXR and PXR were significantly decreased and oxidative stress was increased. Exposure of Caco2 to inflammatory stimuli resulted in the reduction of BA receptor expression in HepG2. Caco2 treatment with dipotassium glycyrrhizate (DPG) reduced these effects on liver cells. Inflamed colon of patients showed altered FXR, PXR, and TGR5 expression. CONCLUSIONS: This study strongly suggests that gut inflammation affects hepatic cells by altering BA receptor levels as well as increasing the production of pro-inflammatory cytokines and oxidative stress. Hence, reducing gut inflammation is needed not only to improve the intestinal disease but also to protect the liver.
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Hepatopatías , Animales , Ácidos y Sales Biliares , Células CACO-2 , Niño , Humanos , Inflamación , Ratones , Ratones Endogámicos C57BLRESUMEN
OBJECTIVE: The importance of autophagy in mechanisms underlying inflammation has been highlighted. Downstream effects of the bacterial sensor NOD2 include autophagy induction. Recently, a relationship between defects in autophagy and adherent/invasive Escherichia coli (AIEC) persistence has emerged. The present study aims at investigating the interplay between autophagy, NOD2 and AIEC bacteria and assessing the expression level of autophagic proteins in intestinal biopsies of pediatric patients with inflammatory bowel disease (IBD). METHODS: A human epithelial colorectal adenocarcinoma (Caco2) cell line stably over-expressing NOD2 was produced (Caco2NOD2). ATG16L1, LC3 and NOD2 levels were analysed in the Caco2 cell line and Caco2NOD2 after exposure to AIEC strains, by western blot and immunofluorescence. AIEC survival inside cells and TNFα, IL-8 and IL-1ßmRNA expression were analysed by gentamicin protection assay and real time PCR. ATG16L1 and LC3 expression was analyzed in the inflamed ileum and colon of 28 patients with Crohn's disease (CD), 14 with ulcerative colitis (UC) and 23 controls by western blot. RESULTS: AIEC infection increased ATG16L1 and LC3 in Caco2 cells. Exposure to AIEC strains increased LC3 and ATG16L1 in Caco2 overexpressing NOD2, more than in Caco2 wild type, while a decrease of AIEC survival rate and cytokine expression was observed in the same cell line. LC3 expression was increased in the inflamed colon of CD and UC children. CONCLUSIONS: The NOD2-mediated autophagy induction is crucial to hold the intramucosal bacterial burden, especially towards AIEC, and to limit the resulting inflammatory response. Autophagy is active in inflamed colonic tissues of IBD pediatric patients.
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Autofagia , Colitis Ulcerosa/inmunología , Enfermedad de Crohn/inmunología , Infecciones por Escherichia coli/inmunología , Proteína Adaptadora de Señalización NOD2/inmunología , Adolescente , Proteínas Relacionadas con la Autofagia/inmunología , Células CACO-2 , Niño , Preescolar , Citocinas/genética , Células Epiteliales/microbiología , Femenino , Humanos , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Intestinos/citología , Masculino , Proteínas Asociadas a Microtúbulos/inmunologíaAsunto(s)
Aorta Torácica/efectos de los fármacos , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Ácido Glicirrínico/farmacología , Animales , Aorta Torácica/metabolismo , Aorta Torácica/patología , Enfermedades de la Aorta/etiología , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Aterosclerosis/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Proteína HMGB1/metabolismo , Mediadores de Inflamación/metabolismo , Lípidos/sangre , Ratones Noqueados para ApoE , Placa Aterosclerótica , Factores SexualesRESUMEN
BACKGROUND: Faecal biomarkers have emerged as important tools in managing of inflammatory bowel disease [IBD], which includes Crohn's disease [CD] and ulcerative colitis [UC]. AIM: To identify new biomarkers of gut inflammation in the stools of IBD patients using a proteomic approach. METHODS: Proteomic analysis of stools was performed in patients with both active CD and CD in remission and in controls by 2-DIGE and MALDI-TOF/TOF MS. An ELISA was used to confirm results in a second cohort of IBD patients and controls. RESULTS: 2-DIGE analysis detected 70 spots in the stools of patients with active CD or patients in remission CD and in controls. MALDI-TOF/TOF MS analysis identified 21 proteins with Chymotrypsin C, Gelsolin and Rho GDP-dissociation inhibitor 2 [RhoGDI2] best correlating with the levels of intestinal inflammation. Results were confirmed in a second cohort of IBD patients and controls [57 CD, 60 UC, 31 controls]. The identified faecal markers significantly correlated with the severity of intestinal inflammation in IBD patients [SES-CD in CD, Mayo endoscopic subscore in UC] [CD; Chymotrypsin-C: r = 0.64, p < 0.001; Gelsolin: r = 0.82, p < 0.001; RhoGDI2: r = 0.64, p < 0.001; UC; Chymotrypsin-C: r = 0.76, p < 0.001; Gelsolin: r = 0.75, p < 0.001; RhoGDI2: r = 0.63, p < 0.001]. Moreover, ROC analysis showed that Gelsolin [p < 0.0002] and RhoGDI2 [p < 0.0001] in CD, and RhoGDI2 [p = 0.0004] in UC, have higher sensitivity and specificity than faecal calprotectin in discriminating between patients and controls. CONCLUSIONS: We show for the first time that 2-DIGE is a reliable method to detect proteins in human stools. Three novel faecal biomarkers of gut inflammation have been identified that display good specificity and sensitivity for identifying IBD and significantly correlate with IBD severity.
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Colitis Ulcerosa , Enfermedad de Crohn , Enfermedades Inflamatorias del Intestino , Humanos , Quimotripsina/metabolismo , Gelsolina/metabolismo , Proteómica , Inhibidor beta de Disociación del Nucleótido Guanina rho/metabolismo , Mucosa Intestinal/metabolismo , Enfermedades Inflamatorias del Intestino/complicaciones , Enfermedades Inflamatorias del Intestino/diagnóstico , Enfermedades Inflamatorias del Intestino/metabolismo , Colitis Ulcerosa/diagnóstico , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/diagnóstico , Enfermedad de Crohn/metabolismo , Biomarcadores/análisis , Inflamación/metabolismo , Complejo de Antígeno L1 de Leucocito/análisis , Heces/química , Índice de Severidad de la EnfermedadRESUMEN
BACKGROUND: The incidence of non-alcoholic fatty liver disease (NAFLD) and its more severe and progressive form, non-alcoholic steatohepatitis (NASH) is increasing worldwide. Gut inflammation seems to concur to the pathogenesis of NASH. No drugs are currently approved for NASH treatment. AIMS: To investigate if inflamed gut directly contributes to the progression of NASH through gut epithelial and vascular barrier impairment and to evaluate the efficacy of dipotassium glycyrrhizate (DPG) to improve the liver disease. METHODS: A NASH model was set up by feeding mice, for 8 and 13 weeks, with high fat diet with high fructose and glucose (HFD-FG) supplemented periodically with dextran sulfate sodium (DSS) in drinking water. A group was also treated with DPG by gavage. Histological, immunohistochemical and molecular analysis were performed. RESULTS: DSS-induced colitis increased steatosis, inflammatory (IL-6, TNFα, NLRP3, MCP-1) as well as fibrotic (TGF-ß, α-SMA) mediator expression in HFD-FG mice. Beneficial effect of DPG was associated with restoration of intestinal epithelial and vascular barriers, evaluated respectively by ZO-1 and PV-1 expression, that are known to limit bacterial translocation. CONCLUSION: Colonic inflammation strongly contributes to the progression of NASH, likely by favouring bacterial translocation. DPG treatment could represent a novel strategy to reduce liver injury.
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Enfermedad del Hígado Graso no Alcohólico , Animales , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Inflamación/complicaciones , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/patologíaRESUMEN
Microalgae are natural sources of valuable bioactive compounds, such as polyunsaturated fatty acids (PUFAs), that show antioxidant, anti-inflammatory, anticancer and antimicrobial activities. The marine microalga Isochrysis galbana (I. galbana) is extremely rich in ω3 PUFAs, mainly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Probiotics are currently suggested as adjuvant therapy in the management of diseases associated with gut dysbiosis. The Lactobacillus reuteri (L. reuteri), one of the most widely used probiotics, has been shown to produce multiple beneficial effects on host health. The present study aimed to present an innovative method for growing the probiotic L. reuteri in the raw seaweed extracts from I. galbana as an alternative to the conventional medium, under conditions of oxygen deprivation (anaerobiosis). As a result, the microalga I. galbana was shown for the first time to be an excellent culture medium for growing L. reuteri. Furthermore, the gas-chromatography mass-spectrometry analysis showed that the microalga-derived ω3 PUFAs were still available after the fermentation by L. reuteri. Accordingly, the fermented compound (FC), obtained from the growth of L. reuteri in I. galbana in anaerobiosis, was able to significantly reduce the adhesiveness and invasiveness of the harmful adherent-invasive Escherichia coli to intestinal epithelial cells, due to a cooperative effect between L. reuteri and microalgae-released ω3 PUFAs. These findings open new perspectives in the use of unicellular microalgae as growth medium for probiotics and in the production of biofunctional compounds.
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Técnicas de Cultivo Celular por Lotes/métodos , Haptophyta/microbiología , Limosilactobacillus reuteri/crecimiento & desarrollo , Medios de Cultivo/química , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Ácidos Grasos Omega-3 , Ácidos Grasos Insaturados/química , Fermentación , Haptophyta/metabolismo , Microalgas/química , Probióticos/metabolismoRESUMEN
Introduction: An early diagnosis of necrotizing enterocolitis (NEC), a major gastrointestinal emergency in preterm newborns, is crucial to improve diagnostic approach and prognosis. We evaluated whether fecal high-mobility group box protein 1 (HMGB1) may early identify preterms at risk of developing NEC. Materials and Methods: A case-control study including neonates admitted at the Neonatal Intensive Care Unit (NICU) of the Sapienza University Hospital "Umberto I" in Rome, from July 2015 to December 2016. Stool samples obtained from cases (preterm newborns with NEC) and controls (newborns without NEC) were collected at the enrolment (T0) and within 7-14 days after the first sample collection (T1). HMGB1, extracted and measured with western blot, was reported as densitometry units (DUS). Results: HMGB1 levels in 30 cases (n = 28-Bell stage 1, n = 2 Bell stage 2) were higher [T0: 21,462 DUS (95% CI, 16,370-26,553 DUS)-T1: 17,533 DUS (95% CI, 13,052-22,014 DUS)] than in 30 preterm controls [T0: 9,446 DUS (95% CI, 6,147-12,746 DUS)-T1: 9,261 DUS (95% CI, 5,126-13,396 DUS), p < 0.001). Preterm newborns showed significant higher levels of HMGB1 (15,690 DUS (95% CI, 11,929-19,451 DUS)] in comparison with 30 full-term neonates with birth weight >2,500 g [6,599 DUS (95% CI, 3,141-10,058 DUS), p = 0.003]. Multivariate analysis showed that the risk of NEC was significantly (p = 0.012) related to the HMGB1 fecal levels at T0. Conclusions: We suggest fecal HMGB1 as a reliable marker of early NEC in preterm neonates. This study supports further investigation on the role of fecal HMGB1 assessment in managing preterm newborns at risk of NEC. Further studies are advocated to evaluate diagnostic accuracy of this marker in more severe forms of the disease.
RESUMEN
Gut mucosal healing (MH) is considered a key therapeutic target and prognostic parameter in the management of inflammatory bowel disease (IBD). The dipotassium glycyrrhizate (DPG), a salt of the glycoconjugated triterpene glycyrrhizin, has been shown to inhibit the High Mobility Group Box 1 (HMGB1) protein, an allarmin strongly implicated in the pathogenesis of most inflammatory and auto-immune disorders. Here we discuss new insights on how DPG acts on MH comparing the acute phase and the recovery phase from experimental colitis in mice. We found that DPG strongly accelerates MH by differently regulating pro-inflammatory (CXCL1, CXCL3, CXCL5, PTGS2, IL-1ß, IL-6, CCL12, CCL7) and wound healing (COL3A1, MMP9, VTN, PLAUR, SERPINE, CSF3, FGF2, FGF7, PLAT, TIMP1) genes as observed only during the recovery phase of colitis. Relevant issue is the identification of extracellular matrix (ECM) remodeling genes, VTN, and PLAUR, as crucial genes to achieve MH during DPG treatment. Furthermore, a noticeable recovery of intestinal epithelial barrier structural organization, wound repair ability, and functionality is observed in two human colorectal adenocarcinoma cell lines exposed to DPG during inflammation. Thus, our study identifies DPG as a potent tool for controlling intestinal inflammation and improving MH.
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Células Epiteliales/efectos de los fármacos , Matriz Extracelular/efectos de los fármacos , Ácido Glicirrínico/farmacología , Mucosa Intestinal/efectos de los fármacos , Cicatrización de Heridas/efectos de los fármacos , Animales , Células CACO-2 , Línea Celular , Línea Celular Tumoral , Colitis/tratamiento farmacológico , Colitis/metabolismo , Citocinas/metabolismo , Células Epiteliales/metabolismo , Matriz Extracelular/metabolismo , Femenino , Proteína HMGB1/metabolismo , Células HT29 , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Mucosa Intestinal/metabolismo , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND AND AIMS: Recent evidence implicates gut microbiota (GM) and immune alterations in autism spectrum disorders (ASD). We assess GM profile and peripheral levels of immunological, neuronal and bacterial molecules in ASD children and controls. Alarmin HMGB1 was explored as a non-invasive biomarker to monitor gastrointestinal (GI) symptoms. METHODS: Thirty ASD children and 14 controls entered into the study. GM metagenomic analysis was performed for 16 ASD patients and 7 controls. GM functional profile was assessed by GO term analysis. Blood levels of IL-1ß, TNFα, TGFß, IL-10, INFγ, IL-8, lipopolysaccharide, Neurotensin, Sortilin1 and GSSG/GSH ratio were analyzed in all subjects by ELISA. Fecal HMGB1 was analyzed by Western blot. RESULTS: We observed a significant decrease in bacterial diversity. Furthermore, 82 GO terms underrepresented in ASD. Four of them pointed at 3,3 phenylpropionate catabolism and were imputable to Escherichia coli (E. coli) group. Serum levels of TNFα, TGFß, NT, and SORT-1 increased in ASD patients. Fecal levels of HMGB1 correlated with GI sign severity in ASD children. CONCLUSIONS: We suggest that a decrease of E. coli might affect the propionate catabolism in ASD. We report occurrence of peripheral inflammation in ASD children. We propose fecal HMGB1 as a non-invasive biomarker to detect GI symptoms.
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Trastorno del Espectro Autista/microbiología , Enfermedades Gastrointestinales/inmunología , Microbioma Gastrointestinal , Inflamación , Trastorno del Espectro Autista/inmunología , Trastorno del Espectro Autista/fisiopatología , Estudios de Casos y Controles , Niño , Desarrollo Infantil , Preescolar , Comorbilidad , Citocinas/sangre , Escherichia coli/aislamiento & purificación , Heces/microbiología , Femenino , Humanos , Masculino , Fenilpropionatos/metabolismoRESUMEN
The analysis of microbiota composition in humans, animals, and built environments is important because of emerging roles and applications in a broad range of disease and ecological phenotypes. Next Generation Sequencing is the current method of choice to characterize microbial community composition. The taxonomic profile of a microbial community can be obtained either by shotgun analysis of random DNA fragments or through 16S ribosomal RNA gene (rDNA) amplicon sequencing. It has been previously shown that the 16S rDNA amplicon sequencing approach yields quantitatively and qualitatively different results compared to shotgun metagenomics when the two techniques are used to assess microbial community composition on the same samples. However, most of such comparisons were either based on the recovery of 16S rDNA sequences in the shotgun metagenomics data or limited to a single microbiome or synthetic samples. Direct comparison of shotgun metagenomics and 16S rDNA amplicon sequencing on the same samples was performed only once in the recent literature, suggesting that the two methods yield comparable results. Here, we set out to compare the outcome of these two alternative approaches to the microbiome characterization in human gut microbiomes from stool samples. To this end, we processed six different samples with both techniques. We report here that shotgun next generation sequencing metagenomics allows much deeper characterization of the microbiome complexity, allowing identification of a larger number of species for each sample, compared to 16S rDNA amplicon sequencing. Further comparative studies in independent samples are called for.
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Microbioma Gastrointestinal , Metagenoma , Metagenómica , ARN Ribosómico 16S , Adolescente , Niño , Biología Computacional/métodos , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Metagenómica/métodos , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Celiac disease (CD) is a gluten-related immunological disorder resulting in inflammatory enteropathy. AIMS: We assessed a stool marker of intestinal inflammation, the HMGB1 protein, in children with CD on a gluten free diet (GFD) at baseline and at follow up (FU). METHODS: Thirty-nine children were investigated at diagnosis and at FU. Traditional serum markers of CD (anti-transglutaminase and anti-endomysial antibodies) and faecal HMGB1 (by enzyme-linked immunosorbent assay and immunoblotting) were tested. RESULTS: There was a marked increase at baseline in both serum anti-transglutaminase IgA (anti-tTGAs) and faecal HMGB1; the latter being undetectable in controls. A strong correlation occurred between the two markers. At 12-month FU in 24 patients on GFD, HMGB1 decreased in all subjects, yet still being detectable in six children: high anti-tTGAs where evident in three, while the three with normal anti-tTGAs were complaining of intestinal symptoms and reported a low GFD adherence. CONCLUSIONS: Faecal HMGB1 is a valuable marker of intestinal inflammation and may have a role in complementing serology in the management of CD children. Future studies including larger patient cohorts and small bowel mucosa histology will be designed to assess the relationship between faecal HMGB1 levels and duodeno-jejunal histopathology.
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Enfermedad Celíaca/dietoterapia , Dieta Sin Gluten , Heces/química , Proteína HMGB1/análisis , Adolescente , Autoanticuerpos/sangre , Biomarcadores/análisis , Estudios de Casos y Controles , Enfermedad Celíaca/metabolismo , Niño , Preescolar , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Proyectos Piloto , Transglutaminasas/sangre , Cumplimiento y Adherencia al TratamientoRESUMEN
BACKGROUND: Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). AIMS: The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis to intestinal inflammation. METHODS: In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overexpressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory bowel disease (IBD) (20 with Crohn's disease; 20 with ulcerative colitis) and from 20 controls. RESULTS: RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8, IL-1ß, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects membrane permeability by altering cell-cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targeting necroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in cultured intestinal explants from IBD. CONCLUSION: We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affects intestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and altering epithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects. These data strongly support the view that targeting necroptosis may represent a promising new option for the treatment of inflammatory enteropathies.
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Apoptosis , Permeabilidad de la Membrana Celular , Células Epiteliales/fisiología , Inflamación/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Adolescente , Clorometilcetonas de Aminoácidos/farmacología , Cadherinas/metabolismo , Caspasa 1/metabolismo , Adhesión Celular , Supervivencia Celular/efectos de los fármacos , Niño , Preescolar , Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Células HCT116 , Proteína HMGB1/metabolismo , Humanos , Imidazoles/farmacología , Indoles/farmacología , Inflamasomas/metabolismo , Interleucina-1beta/metabolismo , Interleucina-8/metabolismo , Mucosa Intestinal/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Necrosis , Fosforilación , Proteínas Quinasas/genética , Transporte de Proteínas/efectos de los fármacos , ARN Mensajero , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
BACKGROUND: Fecal high mobility group box 1 (HMGB1) has been suggested to be a novel noninvasive biomarker of gut inflammation. We aimed to assess the reliability of fecal HMGB1, compared with fecal calprotectin (FC), in detecting intestinal inflammation in pediatric and adult patients with inflammatory bowel disease (IBD) and to evaluate the accuracy of HMGB1 in identifying patients with IBD in clinical and endoscopic remission who still have histologic features of inflammation. METHODS: Stool samples from 85 children with IBD (49 Crohn's disease [CD] and 36 ulcerative colitis [UC] and 119 adults [57 Crohn's disease and 62 ulcerative colitis]) were analyzed for the study. Age-matched healthy subjects were used as controls. Fecal HMGB1 and fecal calprotectin were detected through western blot and ELISA, respectively. RESULTS: Fecal HMGB1 expression was significantly increased in pediatric and adult patients with Crohn's disease and ulcerative colitis and strongly correlated with the disease severity. Fecal calprotectin and HMGB1 significantly correlated in pediatric (r: 0.60, P < 0.001) and adult (r: 0.72, P < 0.001) IBD patients. Moreover, in patients with clinical and endoscopic remission only fecal HMGB1 showed a strong match with the degree of histological scores of inflammation (CGHAS/IGHAS for Crohn's disease and Geboes Score for ulcerative colitis). CONCLUSIONS: Fecal HMGB1 is confirmed to be a reliable biomarker of intestinal inflammation; indeed, it significantly correlates with fecal calprotectin in pediatric and adult IBD patients. Moreover, only fecal HMGB1 identifies histologic inflammation in subjects with IBD in clinical and endoscopic remission.
Asunto(s)
Colitis Ulcerosa/metabolismo , Enfermedad de Crohn/metabolismo , Heces/química , Proteína HMGB1/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Western Blotting , Estudios de Casos y Controles , Niño , Colitis Ulcerosa/patología , Colitis Ulcerosa/cirugía , Colonoscopía , Enfermedad de Crohn/patología , Enfermedad de Crohn/cirugía , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Mucosa Intestinal/patología , Complejo de Antígeno L1 de Leucocito/análisis , Masculino , Persona de Mediana Edad , Periodo Posoperatorio , Inducción de Remisión , Reproducibilidad de los Resultados , Índice de Severidad de la Enfermedad , Adulto JovenRESUMEN
BACKGROUND: Krill oil is a marine derived oil rich in phospholipids, astaxanthin and omega-3 fatty acids. Several studies have found benefits of krill oil against oxidative and inflammatory damage. AIMS: We aimed at assessing the ability of krill oil to reduce intestinal inflammation by improving epithelial barrier integrity, increasing cell survival and reducing pathogenicity of adherent-invasive Escherichia coli. METHODS: CACO2 and HT29 cells were exposed to cytomix (TNFα and IFNγ) to induce inflammation and co-exposed to cytomix and krill oil. E-cadherin, ZO-1 and F-actin levels were analyzed by immunofluorescence to assess barrier integrity. Scratch test was performed to measure wound healing. Cell survival was analyzed by flow cytometry. Adherent-invasive Escherichia coli LF82 was used for adhesion/invasion assay. RESULTS: In inflamed cells E-cadherin and ZO-1 decreased, with loss of cell-cell adhesion, and F-actin polymerization increased stress fibres; krill oil restored initial conditions and improved wound healing, reduced bacterial adhesion/invasion in epithelial cells and survival within macrophages; krill oil reduced LF82-induced mRNA expression of pro-inflammatory cytokines. CONCLUSIONS: Krill oil improves intestinal barrier integrity and epithelial restitution during inflammation and controls bacterial adhesion and invasion to epithelial cells. Thus, krill oil may represent an innovative tool to reduce intestinal inflammation.
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Adhesión Bacteriana/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Euphausiacea , Ácidos Grasos Omega-3/farmacología , Viabilidad Microbiana/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Actinas/metabolismo , Animales , Células CACO-2 , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Escherichia coli/patogenicidad , Escherichia coli/fisiología , Infecciones por Escherichia coli/inmunología , Células HT29 , Humanos , Interferón gamma/farmacología , Interleucina-8/genética , Interleucina-8/metabolismo , Ratones , Células RAW 264.7 , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Cicatrización de Heridas/efectos de los fármacos , Proteína de la Zonula Occludens-1/metabolismoRESUMEN
AIMS: Oxidative stress and inflammation are always associated. Appropriate management of oxidative mediators may represent a therapeutic strategy to reduce inflammation, and use of antioxidant can be protective against inflammatory diseases. Glycyrrhizin (GL) plays an anti-inflammatory and antioxidant effect by inhibiting high mobility group box 1 (HMGB1) or 11-ß-hydroxysteroid dehydrogenase type II (11ßHSD2) enzyme. In this study, the potential role of dipotassium glycyrrhizate (DPG), a salt of GL, to reduce oxidative stress in intestinal inflammatory condition was investigated in vivo and the mechanism of action of DPG was studied in vitro. RESULTS: In a colitis mouse model DPG affected oxidative stress reducing iNOS and COX-2 expression, as well as NO and PGE2 levels. By means of LPS-stimulated macrophages we found that DPG inhibited the expression of pro-inflammatory cytokines and reduced iNOS and COX-2 expression in a time dependent manner, through two different ways of signal. DPG reduced, at a later time, both iNOS and COX-2, through a mechanism HMGB1-dependent, and at an earlier time only COX-2, through a mechanism AMP-activated kinase (AMPK)-phosphorylation-mediated. CONCLUSION: DPG has a protective effect on colitis and inflammation through the inhibition of oxidative stress. This study clarifies the two-ways mechanism by which DPG inhibits iNOS and COX-2 during inflammation and demonstrates for the first time that AMPK is a target of DPG. Uncovering this mechanism is significant to clarify the relationship between energy homeostasis and anti-oxidative responses and suggests that DPG could play a relevant role in the development of new therapy against inflammatory diseases associated to oxidative stress.