Paula Borralho

Necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis

Hepatocyte cell death, inflammation and oxidative stress constitute key pathogenic mechanisms underlying non-alcoholic fatty liver disease (NAFLD). We aimed to investigate the role of necroptosis in human and experimental NAFLD and its association with tumour necrosis factor α (TNF-α) and oxidative stress. Serum markers of necrosis, liver receptor-interacting protein 3 (RIP3) and phosphorylated mixed lineage kinase domain-like (MLKL) were evaluated in control individuals and patients with NAFLD. C57BL/6 wild-type (WT) or RIP3-deficient (RIP3(-/-)) mice were fed a high-fat choline-deficient (HFCD) or methionine and choline-deficient (MCD) diet, with subsequent histological and biochemical analysis of hepatic damage. In primary murine hepatocytes, necroptosis and oxidative stress were also assessed after necrostatin-1 (Nec-1) treatment or RIP3 silencing. We show that circulating markers of necrosis and TNF-α, as well as liver RIP3 and MLKL phosphorylation were increased in NAFLD. Likewise, RIP3 and MLKL protein levels and TNF-α expression were increased in the liver of HFCD and MCD diet-fed mice. Moreover, RIP3 and MLKL sequestration in the insoluble protein fraction of NASH (non-alcoholic steatohepatitis) mice liver lysates represented an early event during stetatohepatitis progression. Functional studies in primary murine hepatocytes established the association between TNF-α-induced RIP3 expression, activation of necroptosis and oxidative stress. Strikingly, RIP3 deficiency attenuated MCD diet-induced liver injury, steatosis, inflammation, fibrosis and oxidative stress. In conclusion, necroptosis is increased in the liver of NAFLD patients and in experimental models of NASH. Further, TNF-α triggers RIP3-dependent oxidative stress during hepatocyte necroptosis. As such, targeting necroptosis appears to arrest or at least impair NAFLD progression.

Rofecoxib: a possible cause of acute colitis.

Nonsteroidal antiinflammatory agents may cause relevant small bowel and colonic side effects, apart from gastroduodenal lesions. The synthesis of selective cyclooxygenase-2 (COX-2) inhibitors has been an important breakthrough in antiinflammatory therapy by decreasing the incidence of upper gastrointestinal lesions. However, there is little information available concerning their effects on gut mucosa distally to the duodenum. A case history is described of a 52-year-old woman with a temporary colostomy after resection of a sigmoid tumor and who presented with bloody diarrhea 5 days after beginning therapy with rofecoxib. The hemorrhage had its origin in the transverse colon, and the endoscopic appearance was that of actively bleeding acute hemorrhagic colitis. No other colonic lesions were detected, nor was there any evidence of related infection, bleeding diathesis, or other systemic diseases. On discontinuing rofecoxib and instituting parenteral rehydration, the bleeding and diarrhea stopped. Endoscopic follow-up revealed regenerating mucosa in the transverse colon. The time relation, the absence of other causes of hemorrhage, and the clinical evolution all strongly support the probability of a causal relation between rofecoxib and hemorrhagic colitis. This case may raise awareness concerning the possibility of colonic lesions related to the new COX-2 inhibitors, similar to what is known about nonselective antiinflammatory agents.

An unusual cause of pseudoachalasia: the Alport syndrome-diffuse leiomyomatosis association.

Alport syndrome (AS) is a hereditary disease characterized by glomerular nephropathy progressing to end-stage renal disease, frequently associated with sensorineural deafness and ocular abnormalities. Rarely, AS coexists with diffuse leiomyomatosis, a benign proliferation of smooth muscle in the gastrointestinal tract, mostly of the oesophagus, but also of the tracheobronchial tree and the female genital tract. Patients with this association have been shown to have contiguous gene deletion involving both COL4A5 and COL4A6 genes. The authors report the case of a 25-year-old man with AS and long-standing dysphagia. The patient received a renal transplant at the age of 23 because of end-stage renal disease. Clinical assessment as well as endoscopic, manometric and radiologic studies suggested the diagnosis of achalasia, which was treated by Hellers myotomy with Dor fundoplication. Postprocedure dysphagia led to an endoscopic ultrasound that showed diffuse thickening of the second layer, resulting in the hypothesis of oesophageal leiomyomatosis. The diagnosis was confirmed through histological study of endoscopic biopsies and genetic analysis.

Accuracy of Faecal Calprotectin and Neutrophil Gelatinase B-associated Lipocalin in Evaluating Subclinical Inflammation in UlceRaTIVE Colitis-the ACERTIVE study.

Background and Aims Mucosal healing and histological remission are different targets for patients with ulcerative colitis, but both rely on an invasive endoscopic procedure. This study aimed to assess faecal calprotectin and neutrophil gelatinase B-associated lipocalin as biomarkers for disease activity in asymptomatic ulcerative colitis patients. Methods This was a multicentric cross-sectional study including 371 patients, who were classified according to their endoscopic and histological scores. These results were evaluated alongside the faecal levels of both biomarkers. Results Macroscopic lesions [i.e. endoscopic Mayo score ≥1] were present in 28% of the patients, and 9% had active disease according to fht Ulcerative Colitis Endoscopic Index of Severity. Moreover, 21% presented with histological inflammation according to the Geboes index, whereas 15% and 5% presented with focal and diffuse basal plasmacytosis, respectively. The faecal levels of calprotectin and neutrophil gelatinase B-associated lipocalin were statistically higher for patients with endoscopic lesions and histological activity. A receiver operating characteristic-based analysis revealed that both biomarkers were able to indicate mucosal healing and histological remission with an acceptable probability, and cut-off levels of 150-250 μg/g for faecal calprotectin and 12 μg/g for neutrophil gelatinase B-associated lipocalin were proposed. Conclusions Faecal calprotectin and neutrophil gelatinase B-associated lipocalin levels are a valuable addition for assessment of disease activity in asymptomatic ulcerative colitis patients. Biological levels of the analysed biomarkers below the proposed thresholds can rule out the presence of macroscopic and microscopic lesions with a probability of 75-93%. However, caution should be applied whenever interpreting positive results, as these biomarkers present consistently low positive predictive values.

Unusual Cause for Smoldering Dysphagia

A 30-year-old black woman presented with heartburn and odynophagia. She had a 2-year history of Behçet’s disease and systemic lupus erythematosus and had been treated with colchicine, hydroxychloroquine, and sucralfate. Odynophagia was not related to the presence of oral ulcers as they were painless and when they were in remission the patient would still intermittently complain of substernal pain. The patient underwent upper digestive endoscopy that revealed only small mucosal irregularities in the upper third of the esophagus (Fig. 1). Biopsies of these segments showed marked acanthosis and papillomatosis of the squamous epithelium as well as intense lymphoplasmacytic infiltrate with an increased number of intraepithelial lymphocytes (IEL). There were neither granulocytes nor signs of viral infection. The endoscopic findings were then attributed to regenerative changes of the epithelium and the patient was started on a proton pump inhibitor (PPI), assuming gastroesophageal reflux disease (GERD). During the following years there were flare-ups of rheumatologic disease activity due to the patient’s lack of adherence to therapy. However, there was no correlation of the patient’s maintained (although scarce) complaints of transitory dysphagia and substernal pain. In 2008, 2 years after the first endoscopy, the symptoms progressed to persistent dysphagia for solids and the patient localized the point of obstruction to beneath the sternum. There were also episodes of self-remitting food impaction. There were no abnormal findings on physical examination or laboratory tests. The gastroesophageal contrast radiograph was negative (Fig. 2). She was referred to our GI clinic and upper endoscopy was repeated. A narrowed esophageal lumen with multiple rings was seen in the upper third of the esophagus, suggesting a feline esophagus (Fig. 3a, b). Biopsies were undertaken in the upper and lower thirds of the esophagus.

miRNA-21 ablation protects against liver injury and necroptosis in cholestasis

Inhibition of microRNA-21 (miR-21) prevents necroptosis in the mouse pancreas. Necroptosis contributes to hepatic necro-inflammation in the common bile duct ligation (BDL) murine model. We aimed to evaluate the role of miR-21 in mediating deleterious processes associated with cholestasis. Mechanistic studies established a functional link between miR-21 and necroptosis through cyclin-dependent kinase 2-associated protein 1 (CDK2AP1). miR-21 expression increased in the liver of primary biliary cholangitis (PBC) patients and BDL wild-type (WT) mice at both 3 and 14 days. Notably, under BDL, miR-21−/− mice displayed decreased liver injury markers in serum compared with WT mice, accompanied by reduced hepatocellular degeneration, oxidative stress and fibrosis. Hallmarks of necroptosis were decreased in the liver of BDL miR-21−/− mice, via relieved repression of CDK2AP1. Further, miR-21−/− mice displayed improved adaptive response of bile acid homeostasis. In conclusion, miR-21 ablation ameliorates liver damage and necroptosis in BDL mice. Inhibition of miR-21 should arise as a promising approach to treat cholestasis.

Microbiota Modulation With Synbiotic Decreases Liver Fibrosis in a High Fat Choline Deficient Diet Mice Model of Non-Alcoholic Steatohepatitis (NASH)

Background Gut microbiota may play a role in non-alcoholic steatohepatitis (NASH). Previous studies showed that prebiotics and probiotics might halt the progression of steatohepatitis. Aim To clarify the potential effect of Synbiotic 2000®Forte (Synb) in preventing or ameliorating diet induced steatohepatitis, particularly in fibrosis progression and how this intervention correlates with gut microbiota composition and endotoxinemia. Methods Twenty-seven C57BL/6 mice were divided into three groups: chow diet (CD, n = 7); high-fat choline deficient diet (HFCD, n = 10) and HFCD diet supplemented with Synbiotic 2000®Forte (four probiotic strains and four prebiotics mixture) (HFCD + Synb, n = 10). At 6 and 18 weeks, blood samples (lipopolysaccharides assay – LPS), cecal feaces (gut microbiota) and liver tissue (histology) were collected for analysis. Results Both HCFD diet mice developed steatohepatitis with ballooning at 6 and 18 weeks, opposite to CD. Comparison of histological scores in HFCD and HFCD + Synb, at 6 and 18 weeks showed no significant difference regarding steatosis, inflammation, or ballooning. Evaluating fibrosis with Sirius Red, and degree of smooth-muscle cell activation, HFCD mice had significantly more fibrosis; addition of Synb significantly reduced fibrosis at 6 weeks and 18 weeks. Serum endotoxin levels were similarly increased in HFCD and HFCD + Synb at week 6; however at week 18 HFCD + Synb had significantly lower endotoxin levels than HFCD. Gut microbiota of HFCD vs CD, showed no significant differences regarding the phyla Firmicutes and Bacteroidetes, either at 6 or 18 weeks; Proteobacteria increased at 6 week (3.3) and 18 week (7.5), while the addition of Synb resulted in a decrease at week 18 (−3.90). Fusobacteria markedly increase at week 18 (10.0), but less so with the addition of Synb (5.2). Conclusion Synbiotic 2000®Forte is able to modulate the mouse gut microbiota reducing the degree of fibrosis while simultaneously decreasing endotoxemia.

Aberrant gastric apomucin expression in ulcerative colitis and associated neoplasia

AIM To evaluated the presence of gastric metaplasia in colonic mucosa of patients with ulcerative colitis and its relationship with dysplasia/neoplasia. MATERIAL AND METHODS Ninety patients with UC were selected. The duration and the extent of disease were registered in all the cases. Biopsies were histologically and immunohistochemically assessed. Crypt distortion, goblet cell depletion, Paneth cell metaplasia and inflammatory activity were graded, as well as dysplasia and invasive neoplasia (absent or present). Monoclonal antibodies against the gastric apomucins MUC5AC (foveolar) and MUC6 (mucopeptic) were used. RESULTS Neoplasia was observed in 16 patients, 8 non-invasive (dysplasia) and 8 invasive (adenocarcinoma). MUC5AC and MUC6 were detected in 63 and 16 out the 90 cases, 70.0% and 17.8%, respectively. The staining was patchy for both antibodies, affecting groups of cells more often than isolated cells. The presence of MUC5AC correlated positively with inflammatory activity and goblet cell depletion (R=0.231, p=0.03 and R=0.211, p=0.048, respectively). The expression of MUC6 correlated positively with age (R=0.297, p=0.005), duration of disease (R=0.287, p=0.008), extent of disease (R=0.342, p=0.001), crypt distortion (R=0.276, p=0.01) and the presence of neoplasia (R=0.483, p<0.00). There was no correlation between Paneth cell metaplasia and apomucin expression. CONCLUSIONS Our study demonstrates the aberrant expression of gastric apomucins in UC and suggests that MUC5AC is associated with inflammation while MUC6 is related to the presence of neoplasia. The demonstration of metaplastic cell lineages preceding dysplasia supports the biological link between inflammation and neoplasia, MUC6 emerging as a putative biomarker of dysplasia in ulcerative colitis patients.

Comparison of different histological indexes in the assessment of UC activity and their accuracy regarding endoscopic outcomes and faecal calprotectin levels

Objective Histological remission is being increasingly acknowledged as a therapeutic endpoint in patients with UC. The work hereafter described aimed to evaluate the concordance between three histological classification systems—Geboes Score (GS), Nancy Index (NI) and RobartsHistopathologyIndex (RHI), as well as to evaluate their association with the endoscopic outcomes and the faecal calprotectin (FC) levels. Design Biopsy samples from 377 patients with UC were blindly evaluated using GS, NI and RHI. The results were compared with the patients’ Mayo Endoscopic Score and FC levels. Result GS, NI and RHI have a good concordance concerning the distinction between patients in histological remission or activity. RHI was particularly close to NI, with 100% of all patients classified as being in remission with NI being identified as such with RHI and 100% of all patients classified as having activity with RHI being identified as such with NI. These scores could also predict the Mayo Endoscopic Score and the FC levels, with their sensitivity and specificity levels depending on the chosen cut-offs. Moreover, higher FC levels were statistically associated with the presence of neutrophils in the epithelium, as well as with ulceration or erosion of the intestinal mucosa. Conclusions GS, NI and RHI histopathological scoring systems are comparable in what concerns patients’ stratification into histological remission/activity. Additionally, FC levels are increased when neutrophils are present in the epithelium and the intestinal mucosa has erosions or ulcers. The presence of neutrophils in the epithelium is, indeed, the main marker of histological activity.

Farnesoid X Receptor Expression in Microscopic Colitis: A Potential Role in Disease Etiopathogenesis

Introduction: Microscopic colitis (MC) is a chronic inflammatory bowel disease with unclear etiology. Bile acid (BA) malabsorption has been described in MC patients. Farnesoid X receptor (FXR) is the main BA receptor; FXR-mediated mechanisms prevent the noxious effects of BA accumulation, preserving the integrity of the intestinal epithelial barrier and preventing intestinal inflammation. Aim: Our aim was to describe the expression of FXR in patients with MC. Methods: Archival formalin-fixed paraffin-embedded samples from the terminal ileum, right and left colon were obtained from patients with MC and matched controls. Immunohistochemistry was performed and nuclear FXR expression scored in a semi-quantitative way. Results: 169 formalin-fixed paraffin-embedded samples from 35 patients with MC and 31 controls were retrieved. There was a significant reduction of FXR expression in patients with MC versus controls both in the right colon (moderate-strong FXR expression: 21.1 vs. 64.3%; p = 0.003) and left colon (moderate-strong FXR expression: 8.3 vs. 38.7%; p = 0.027). No significant differences in FXR expression were observed in the ileum of patients with MC (moderate-strong FXR expression: 76.9 vs. 90.9%; p = 0.5). We found no difference in FXR expression between the two types of MC. No association between the degree of lymphocyte infiltration or the thickness of collagen band and FXR expression was found. Conclusions: Patients with MC present a significantly lower expression of FXR in the colon. This could render colonic epithelial cells more susceptible to the deleterious effects of BA, contributing to disease pathogenesis and symptoms in MC.