RESUMO
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.
Assuntos
Fibrose , Organoides , Fator de Crescimento Transformador beta1 , Fator de Necrose Tumoral alfa , Humanos , Organoides/patologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Doenças Inflamatórias Intestinais/patologia , Criança , Feminino , Masculino , Mucosa Intestinal/patologia , Adolescente , Doença de Crohn/patologia , Doença de Crohn/metabolismo , Colite Ulcerativa/patologia , Intestinos/patologiaRESUMO
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.
Assuntos
Colite , Proteína HMGB1 , Doenças Inflamatórias Intestinais , Poli(ADP-Ribose) Polimerase-1 , Animais , Humanos , Camundongos , Colite/induzido quimicamente , Citocinas , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Inflamação , Organoides , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
A tight relationship between gut-liver diseases and brain functions has recently emerged. Bile acid (BA) receptors, bacterial-derived molecules and the blood-brain barrier (BBB) play key roles in this association. This study was aimed to evaluate how non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) impact the BA receptors Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) expression in the brain and to correlate these effects with circulating BAs composition, BBB integrity and neuroinflammation. A mouse model of NAFLD was set up by a high-fat and sugar diet, and NASH was induced with the supplementation of dextran-sulfate-sodium (DSS) in drinking water. FXR, TGR5 and ionized calcium-binding adaptor molecule 1 (Iba-1) expression in the brain was detected by immunohistochemistry, while Zonula occludens (ZO)-1, Occludin and Plasmalemmal Vesicle Associated Protein-1 (PV-1) were analyzed by immunofluorescence. Biochemical analyses investigated serum BA composition, lipopolysaccharide-binding protein (LBP) and S100ß protein (S100ß) levels. Results showed a down-regulation of FXR in NASH and an up-regulation of TGR5 and Iba-1 in the cortex and hippocampus in both treated groups as compared to the control group. The BA composition was altered in the serum of both treated groups, and LBP and S100ß were significantly augmented in NASH. ZO-1 and Occludin were attenuated in the brain capillary endothelial cells of both treated groups versus the control group. We demonstrated that NAFLD and NASH provoke different grades of brain dysfunction, which are characterized by the altered expression of BA receptors, FXR and TGR5, and activation of microglia. These effects are somewhat promoted by a modification of circulating BAs composition and by an increase in LBP that concur to damage BBB, thus favoring neuroinflammation.
Assuntos
Ácidos e Sais Biliares , Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Barreira Hematoencefálica/metabolismo , Ocludina/metabolismo , Células Endoteliais/metabolismo , Doenças Neuroinflamatórias , Encéfalo/metabolismoRESUMO
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.
Assuntos
Colite , Doença de Crohn , Proteínas Repressoras/metabolismo , Animais , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Colo/patologia , Doença de Crohn/metabolismo , Sulfato de Dextrana , Fibroblastos/metabolismo , Fibrose , Humanos , Camundongos , Camundongos Endogâmicos C57BL , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Zinco/metabolismoRESUMO
In the last decade, the widespread application of shotgun metagenomics provided extensive characterization of the bacterial "dark matter" of the gut microbiome, propelling the development of dedicated, standardized bioinformatic pipelines and the systematic collection of metagenomic data into comprehensive databases. The advent of next-generation sequencing also unravels a previously underestimated viral population (virome) present in the human gut. Despite extensive efforts to characterize the human gut virome, to date, little is known about the childhood gut virome. However, alterations of the gut virome in children have been linked to pathological conditions such as inflammatory bowel disease, type 1 diabetes, malnutrition, diarrhea and celiac disease.
Assuntos
Doença Celíaca/virologia , Diabetes Mellitus Tipo 1/virologia , Diarreia/virologia , Doenças Inflamatórias Intestinais/virologia , Mucosa Intestinal/virologia , Viroma , Doença Celíaca/microbiologia , Criança , Diabetes Mellitus Tipo 1/microbiologia , Diarreia/microbiologia , Humanos , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/microbiologia , MetagenomaRESUMO
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.
Assuntos
Hepatopatias , Animais , Ácidos e Sais Biliares , Células CACO-2 , Criança , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BLRESUMO
OBJECTIVE: The aim of the study was to systematically review the diagnostic utility of serum biomarkers for the diagnosis of necrotizing enterocolitis (NEC). METHODS: We conducted an electronic and manual search of the available evidence. We included studies reporting data on the diagnostic accuracy of "serum" biomarkers for the diagnosis of NEC, available until January 2016. RESULTS: We selected 22 studies from the 1296 articles retrieved. Only S100 A8/A9 protein and apolipoprotein-CII showed high sensitivity (100% and 96.4%, respectively) and specificity (90% and 95%, respectively) in the studies using Bell stage II NEC as target condition. High sensitivity and specificity were reported for interleukin-10 (100% and 90%), interleukin1-receptor antagonist (100% and 91.7%), intestinal fatty acid-binding protein (100% and 91%) and ischemia-modified albumin (94.7% and 92%), when tested to predict the evolution from definite to advanced NEC. Given the amount of uncertainty, the limited availability of data and heterogeneity among the populations in the different studies, we were unable to perform a meta-analysis. Major concerns about the applicability stemmed from the spectrum of patients enrolled and the inclusion of diseases different from Bell stage ≥2 NEC as target conditions. CONCLUSIONS: We identified only few markers with good diagnostic accuracy and found an overall low quality of the studies on serum NEC biomarkers. In conclusion, data supporting their use are insufficient.
Assuntos
Progressão da Doença , Enterocolite Necrosante/sangue , Enterocolite Necrosante/diagnóstico , Apolipoproteína C-II/sangue , Biomarcadores/sangue , Calgranulina A/sangue , Proteínas de Ligação a Ácido Graxo , Humanos , Interleucina-10/sangue , Sensibilidade e Especificidade , Albumina Sérica HumanaRESUMO
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.
Assuntos
Autofagia , Colite Ulcerativa/imunologia , Doença de Crohn/imunologia , Infecções por Escherichia coli/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Adolescente , Proteínas Relacionadas à Autofagia/imunologia , Células CACO-2 , Criança , Pré-Escolar , Citocinas/genética , Células Epiteliais/microbiologia , Feminino , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Intestinos/citologia , Masculino , Proteínas Associadas aos Microtúbulos/imunologiaRESUMO
OBJECTIVES: Recently published works showed that occupational exposure to antineoplastic drugs (ANPD) is still frequent in hospital settings, despite significant safety policy improvements. The aim of this study was to assess the current level of occupational exposure to ANPD and any potentially associated cytogenetic damages in hospital nurses routinely handling ANPD. METHODS: Occupationally ANPD-exposed (n = 71) and ANPD-unexposed (n = 77; control) nurses were recruited on a voluntary basis from five hospitals in Northern and Central Italy. Evaluation of surface contamination and dermal exposure to ANPD was assessed by determining cyclophosphamide (CP) on selected surfaces (wipes) and on exposed nurses' clothes (pads). The concentration of unmetabolized CPas a biomarker of internal dosewas measured in end-shift urine samples. Biomonitoring of genotoxic effects (i.e., biological effect monitoring) was conducted by analyzing micronuclei (MN) and chromosome aberrations (CA) in peripheral blood lymphocytes. Genetic polymorphisms for enzymes involved in metabolic detoxification (i.e., glutathione S-transferases) were analyzed as well. RESULTS: We observed a significant increase in MN frequency (5.30 ± 2.99 and 3.29 ± 1.97; mean values ± standard deviation; p < 0.0001) in exposed nurses versus controls, as well as in CA detection (3.30 ± 2.05 and 1.84 ± 1.67; p < 0.0001), exposed subjects versus controls. Our results provide evidence that, despite safety controlled conditions, ANPD handling still represents a considerable genotoxic risk for occupationally exposed personnel. CONCLUSIONS: Because both MN and CA have been described as being predictive of group-increased cancer risk, our findings point to a need for improving specific safety procedures in handling and administering ANPD.
Assuntos
Antineoplásicos/efeitos adversos , Aberrações Cromossômicas/induzido quimicamente , Micronúcleos com Defeito Cromossômico/induzido quimicamente , Recursos Humanos de Enfermagem Hospitalar , Exposição Ocupacional/efeitos adversos , Adulto , Antineoplásicos/urina , Biomarcadores/urina , Ciclofosfamida/análise , Dano ao DNA , Monitoramento Ambiental/métodos , Feminino , Humanos , Itália , Linfócitos/efeitos dos fármacos , Exposição Ocupacional/análise , Enfermagem OncológicaRESUMO
Intestinal epithelial cells (IECs) form a physiochemical barrier that separates the intestinal lumen from the host's internal milieu and is critical for electrolyte passage, nutrient absorption, and interaction with commensal microbiota. Moreover, IECs are strongly involved in the intestinal mucosal inflammatory response as well as in mucosal innate and adaptive immune responses. Cell death in the intestinal barrier is finely controlled, since alterations may lead to severe disorders, including inflammatory diseases. The emerging picture indicates that intestinal epithelial cell death is strictly related to the maintenance of tissue homeostasis. This review is focused on previous reports on different forms of cell death in intestinal epithelium.
Assuntos
Mucosa Intestinal/metabolismo , Intestinos/citologia , Necrose/metabolismo , Animais , Apoptose/fisiologia , Homeostase/fisiologia , Humanos , Imunidade Inata/fisiologia , Mucosa Intestinal/citologiaRESUMO
OBJECTIVES: A new caspase-independent mode of programmed cell death, termed necroptosis, has recently been identified. Altered expression of molecules involved in the necroptosis pathway has been shown to trigger intestinal inflammation. The initiation of necroptosis is principally mediated by the release of receptor interacting protein 3 (RIP3) from suppression by caspase-8. Furthermore, it has been suggested that the mixed lineage kinase domain-like (MLKL) factor is an interacting target of RIP3 in active necroptosis. This study aims at investigating the occurrence of necroptosis in children with inflammatory bowel disease (IBD) and its contribution to human intestinal inflammation. METHODS: Biopsy samples were collected from the ileum and colon of 33 children with Crohn's disease, 30 with ulcerative colitis, and 20 healthy controls. Ten children with allergic colitis (AC) were used as non-IBD comparators. RIP3, caspase-8, and MLKL protein expression levels were evaluated by western blotting. The adenocarcinoma cell line HT29 was used for in vitro experiments. RESULTS: RIP3 and MLKL increased (P<0.01) in inflamed tissues of IBD and AC patients, whereas caspase-8 was reduced. No variations were observed in uninflamed tissues of patients. The relationship between RIP3 increase, active necroptosis, and intestinal inflammation was confirmed by in vitro analyses. CONCLUSIONS: We show for the first time that necroptosis is strongly associated with intestinal inflammation in children with IBD and contributes to strengthen the inflammatory process. We believe that RIP3 and MLKL could represent attractive targets for the management of human IBD.
Assuntos
Caspase 8/metabolismo , Morte Celular/genética , Doenças Inflamatórias Intestinais/genética , Proteínas Quinases/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Adolescente , Fatores Etários , Biópsia por Agulha , Western Blotting , Sobrevivência Celular , Criança , Pré-Escolar , Estudos de Coortes , Colite Ulcerativa/genética , Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Colo/patologia , Doença de Crohn/genética , Doença de Crohn/patologia , Doença de Crohn/fisiopatologia , Progressão da Doença , Feminino , Regulação da Expressão Gênica , Humanos , Íleo/patologia , Imuno-Histoquímica , Doenças Inflamatórias Intestinais/patologia , Doenças Inflamatórias Intestinais/fisiopatologia , Masculino , Estudos Prospectivos , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco , Índice de Gravidade de Doença , Transdução de Sinais , Estatísticas não ParamétricasAssuntos
Aorta Torácica/efeitos dos fármacos , Doenças da Aorta/prevenção & controle , Aterosclerose/prevenção & controle , Ácido Glicirrízico/farmacologia , Animais , Aorta Torácica/metabolismo , Aorta Torácica/patologia , Doenças da Aorta/etiologia , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Aterosclerose/etiologia , Aterosclerose/metabolismo , Aterosclerose/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Proteína HMGB1/metabolismo , Mediadores da Inflamação/metabolismo , Lipídeos/sangue , Camundongos Knockout para ApoE , Placa Aterosclerótica , Fatores SexuaisRESUMO
OBJECTIVES: The aim of this study was to evaluate in patients with cystic fibrosis (CF) the effect of Lactobacillus reuteri (LR) on the rate of respiratory exacerbations and of the infections of both upper respiratory and gastrointestinal tracts. METHODS: Prospective randomized, double-blind, placebo-controlled study enrolling 61 patients with CF with mild-to-moderate lung disease at the Regional Center for CF of the Department of Pediatrics, University of Rome "La Sapienza." All of the patients were not hospital inpatients at the time of the enrollment. Inclusion criteria were forced expiratory volume in the first second (FEV1) >70% predicted; no inhaled or systemic steroids, no anti-inflammatory drugs, antileukotrienes, and mast cell membrane stabilizers; and no serious organ involvement. Exclusion criteria were a history of pulmonary exacerbation or upper respiratory infection in the previous 2 months; changes in medications in the last 2 months; a history of hemoptysis in the last 2 months; and colonization with Burkholderia cepacia or mycobacteria. Patients were randomly assigned to receive LR (30 patients) in 5 drops per day (10(10) colony-forming units) or placebo (31 patients) for 6 months. Main outcomes were number of episodes of pulmonary exacerbations and hospital admissions for pulmonary exacerbations, number of gastrointestinal and upper respiratory tract infections. FEV1, fecal calprotectin, and cytokine profile in induced sputum and plasma were assessed at baseline and at the end of the trial. RESULTS: Pulmonary exacerbations were significantly reduced in the LR group compared with the placebo group (P<0.01; odds ratio 0.06 [95% confidence interval {CI} 0-0.40]; number needed to treat 3 [95% CI 2-7]). Similarly, the number of upper respiratory tract infections (in our series only otitis) was significantly reduced in the LR group compared with the placebo group (P<0.05; odds ratio 0.14 [95% CI 0-0.96]; number needed to treat 6 [95% CI 3-102]). The 2 groups did not differ statistically in the mean number and duration of hospitalizations for pulmonary exacerbations and gastrointestinal infections. There was no significant statistical difference in the mean delta value of FEV1, fecal calprotectin concentration, and tested cytokines (tumor necrosis factor-α and interleukin-8) between the 2 groups. CONCLUSIONS: LR reduces pulmonary exacerbations and upper respiratory tract infections in patients with CF with mild-to-moderate lung disease. LR administration may have a beneficial effect on the disease course of CF.
Assuntos
Fibrose Cística/tratamento farmacológico , Gastroenteropatias/prevenção & controle , Limosilactobacillus reuteri , Pulmão , Probióticos/uso terapêutico , Infecções Respiratórias/prevenção & controle , Adolescente , Adulto , Criança , Fibrose Cística/complicações , Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/metabolismo , Método Duplo-Cego , Feminino , Volume Expiratório Forçado , Hospitalização , Humanos , Interleucina-8/metabolismo , Limosilactobacillus reuteri/classificação , Complexo Antígeno L1 Leucocitário/metabolismo , Pulmão/metabolismo , Pulmão/microbiologia , Pulmão/patologia , Pneumopatias/prevenção & controle , Masculino , Proteínas Nucleares/metabolismo , Números Necessários para Tratar , Estudos Prospectivos , Fatores de Transcrição , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
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.
Assuntos
Colite Ulcerativa , Doença de Crohn , Doenças Inflamatórias Intestinais , Humanos , Quimotripsina/metabolismo , Gelsolina/metabolismo , Proteômica , Inibidor beta de Dissociação do Nucleotídeo Guanina rho/metabolismo , Mucosa Intestinal/metabolismo , Doenças Inflamatórias Intestinais/complicações , Doenças Inflamatórias Intestinais/diagnóstico , Doenças Inflamatórias Intestinais/metabolismo , Colite Ulcerativa/diagnóstico , Colite Ulcerativa/metabolismo , Doença de Crohn/diagnóstico , Doença de Crohn/metabolismo , Biomarcadores/análise , Inflamação/metabolismo , Complexo Antígeno L1 Leucocitário/análise , Fezes/química , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Single-balloon enteroscopy (SBE) has not been reported in pediatric Crohn's disease (CD). OBJECTIVE: To determine technical performance, yield, safety, and clinical impact of SBE in pediatric patients with suspected and established CD. DESIGN: Prospective, cohort study. SETTING: Academic tertiary-care referral center. PATIENTS: This study involved 16 patients (group A) with suspected CD and unspecific upper and lower GI endoscopy results and 14 patients (group B) with longstanding CD with previous surgery and showing signs unaccountable by conventional endoscopy. All underwent magnetic resonance imaging, and 14 patients in group A also underwent wireless capsule endoscopy. INTERVENTION: SBE. MAIN OUTCOME MEASUREMENTS: SBE diagnostic and therapeutic yield, technical performance, clinical impact, and safety. RESULTS: In group A, SBE aided diagnosis of CD in 12 patients and eosinophilic enteropathy in 2 patients, whereas no lesions were found in 2 patients. WCE was diagnostic of CD in 3 patients, suggestive of CD in 7 patients, and unspecific in the remaining patients. In group B, SBE revealed moderate-to-severe disease activity in most patients, leading to the introduction of or change in biological therapy, with a marked decrease in the pediatric Crohn's disease activity index scores. SBE allowed successful dilation of small-bowel strictures in 2 patients in group A and 3 in group B. No complications occurred. LIMITATIONS: Small sample size, no direct comparison with imaging or other endoscopic techniques. CONCLUSION: SBE is a useful and safe endoscopic procedure for evaluating the small bowel in pediatric patients with suspected or established CD. Not only does it allow a definite diagnosis of CD when the latter is uncertain, but it is also very effective in the management of small-bowel strictures, thus avoiding surgery. It may be helpful in redirecting therapy in selected CD patients.
Assuntos
Doença de Crohn/diagnóstico , Endoscopia Gastrointestinal/métodos , Intestino Delgado/patologia , Adolescente , Endoscopia por Cápsula , Cateterismo , Criança , Constrição Patológica/patologia , Constrição Patológica/terapia , Doença de Crohn/patologia , Doença de Crohn/terapia , Endoscopia Gastrointestinal/efeitos adversos , Enterite/diagnóstico , Eosinofilia/diagnóstico , Feminino , Gastrite/diagnóstico , Humanos , Imageamento por Ressonância Magnética , Masculino , Estudos Prospectivos , Índice de Gravidade de Doença , Estatísticas não ParamétricasRESUMO
Inflammatory bowel disease (IBD) is the result of an altered immune homeostasis within the intestinal mucosa against the gut microbiota, leading to chronic inflammation in genetically predisposed individuals. Under normal conditions, the immune system defends against pathogens and prevents the passage of excessive intestinal bacteria; regulatory pathways must maintain a low-grade, controlled inflammation in a healthy gut, but also induce a protective response against pathogens. The innate immune system is the first-line defense from microbes; dendritic cells, macrophages, and epithelial cells produce an initial, immediate response. The immune system constantly controls commensal bacteria and utilizes constitutive antimicrobial mechanisms to sustain immune homeostasis. The discovery that several genes linked to IBD modulate microbial recognition and innate immune pathways, such as nucleotide oligomerization domain 2 (Nod2), and genes that mediate autophagy (ie, ATG16L1, IRGM), has highlighted the critical role of host-microbe interactions in controlling intestinal immune homeostasis. Commensal microorganisms actively interact with the intestinal mucosa and influence the activity of the immune system as well as the amplitude of the immune response. In contrast, host factors can influence microbes, which in turn modulate disease susceptibility. In this paper, we focus on the mechanisms that mediate host-microbe interactions and how the disruption of this balance leads to chronic intestinal inflammation in IBD.
Assuntos
Imunidade Inata/imunologia , Doenças Inflamatórias Intestinais/imunologia , Doenças Inflamatórias Intestinais/microbiologia , Mucosa Intestinal/microbiologia , Metagenoma , Bactérias , Criança , Predisposição Genética para Doença , Humanos , Doenças Inflamatórias Intestinais/genética , Mucosa Intestinal/imunologia , Intestinos/imunologia , Intestinos/microbiologiaRESUMO
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.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica , Modelos Animais de Doenças , Inflamação/complicações , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
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.
Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Haptófitas/microbiologia , Limosilactobacillus reuteri/crescimento & desenvolvimento , Meios de Cultura/química , Ácidos Docosa-Hexaenoicos/química , Ácido Eicosapentaenoico/química , Ácidos Graxos Ômega-3 , Ácidos Graxos Insaturados/química , Fermentação , Haptófitas/metabolismo , Microalgas/química , Probióticos/metabolismoRESUMO
OBJECTIVES: High-mobility group box 1 (HMGB1) is a nuclear protein with functions in the regulation of transcription. In inflammatory conditions, HMGB1 is actively secreted from immune cells in the extracellular matrix, where it behaves as a proinflammatory cytokine. The aim of the present study was to investigate the role of HMGB1 in pediatric inflammatory bowel disease (IBD). METHODS: We analyzed the stools of 19 children with Crohn's disease (CD), 21 with ulcerative colitis (UC), and 13 controls. The gene/protein expression levels of HMGB1 were assessed in bioptic specimens of all children using real-time PCR and western blot assay. Finally, intracellular localization of the protein was analyzed by western blot, after separation of nuclear and cytoplasmic extracts, and by immunohistochemistry. RESULTS: HMGB1 protein levels were significantly increased (P<0.001) in the stools of patients, but were undetectable in the controls; fecal HMGB1 correlated well with fecal calprotectin levels (r: 0.77 in CD, r: 0.70 in UC; P<0.01); and mRNA and protein expression were unchanged in inflamed bioptic tissues compared with controls. However, by separately analyzing the nuclear and cytoplasmic fraction, we detected the cytoplasmic HMGB1 expression to be significantly enhanced (P<0.01) in the inflamed tissues of the patients. In addition, HMGB1 was significantly detected in 16 patients with inactive disease, whose endoscopic scores showed persisting inflammation, suggesting that it may be a sensitive marker of mucosal inflammation, although the disease is clinically inactive. CONCLUSIONS: It was shown for the first time in our study that HMGB1 is secreted by human inflamed intestinal tissues and abundantly found in the stools of IBD patients. Hence, it can be considered as a novel marker for intestinal inflammation. We can also suggest that the presence of HMGB1 in large amounts in the fecal stream of IBD patients is mainly due to active secretion of the protein stored in the nucleus rather than a "de novo" synthesis.
Assuntos
Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Fezes/química , Proteína HMGB1/metabolismo , Mucosa Intestinal/metabolismo , Transporte Ativo do Núcleo Celular , Adolescente , Biomarcadores/análise , Biomarcadores/metabolismo , Biópsia , Células CACO-2/imunologia , Células CACO-2/metabolismo , Núcleo Celular/metabolismo , Criança , Pré-Escolar , Colite Ulcerativa/patologia , Doença de Crohn/patologia , Feminino , Proteína HMGB1/análise , Proteína HMGB1/genética , Humanos , Lactente , Interferon-alfa/imunologia , Interferon gama/imunologia , Interleucina-8/genética , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
OBJECTIVES: Our work is aimed at identifying ex vivo new transcription factors, potentially involved in the pathogenesis of pediatric inflammatory bowel disease (IBD), by using a microarray approach. PATIENTS AND METHODS: Microarray, including 84 transcription factors, was performed in inflamed and uninflamed mucosal tissues of pediatric patients with Crohn disease (CD) and in healthy controls. Real-time polymerase chain reaction was used to confirm microarray results on a larger size of CD and patients with ulcerative colitis (UC). Protein expression was evaluated by Western blot assay. RESULTS: Microarray assay showed 40 genes differentially regulated in the inflamed mucosa and 17 in the uninflamed mucosa of patients with CD as compared with controls. Real-time polymerase chain reaction analysis revealed 10 transcripts in CD and 4 in UC, selected among those with higher differences as compared with healthy controls, significantly overexpressed in the inflamed tissues of patients. Moreover, 4 transcripts in CD and 2 in UC were found significantly upregulated in the uninvolved tissue. A further investigation evidenced an increased protein expression of activating transcription factor 3 and hypoxia-inducible transcription factor-1α in patients with CD as well as in Caco2 cell line stimulated by cytokines and hypoxia. CONCLUSIONS: The present study shows an evident upregulation of several transcription factors in the inflamed and uninflamed mucosa of children with IBD, suggesting that the inflammatory process is somehow activated at molecular levels even in the macroscopically normal mucosa of patients. A differential pattern of gene expression between CD and UC indicates distinct molecular mechanisms underlying the pathogenesis of 2 diseases. Finally, activating transcription factor 3 and hypoxia-inducible transcription factor-1α are proposed as new transcription factors potentially involved in the onset and maintenance of IBD.