Arnaud Lemmers

The interleukin‐17 pathway is involved in human alcoholic liver disease

Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin‐17 (IL‐17) is known to enhance neutrophil recruitment. We studied the IL‐17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL‐17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme‐linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL‐17 staining and co‐staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL‐17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL‐17 by chemotaxis assays. IL‐17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL‐17, and their CD4+ T lymphocytes disclosed an IL‐17–secreting phenotype. In the liver, IL‐17–secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL‐17+ cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL‐17+ cells infiltrates correlated to model for end‐stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL‐17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL‐17 stimulation in a dose‐dependent manner through IL‐8 and growth related oncogen α (GRO‐α) secretion in vitro. Conclusion: Human alcoholic liver disease is characterized by the activation of the IL‐17 pathway. In alcoholic hepatitis, liver infiltration with IL‐17–secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597). (HEPATOLOGY 2009;49:646–657.)

Impact of patatin‐like phospholipase‐3 (rs738409 C>G) polymorphism on fibrosis progression and steatosis in chronic hepatitis C

Only 20% of patients with chronic hepatitis C (CHC) will develop cirrhosis, and fibrosis progression remains highly unpredictable. A recent genome‐wide association study identified a genetic variant in the patatin‐like phospholipase‐3 (PNPLA3) gene (rs738409 C>G) associated with steatosis that was further demonstrated to influence severity of fibrosis in nonalcoholic fatty liver disease. The aim of this study was to assess the impact of this polymorphism on histological liver damage and response to antiviral therapy in CHC. We recruited 537 Caucasian CHC patients from three European centers (Brussels, Belgium [n = 229]; Hannover, Germany [n = 171]; Lyon, France [n = 137]); these patients were centrally genotyped for the PNPLA3 (rs738409 C>G) polymorphism. We studied the influence of rs738409 and other variants in the PNPLA3 region on steatosis and fibrosis assessed both in a cross‐sectional and longitudinal manner. Seven other variants previously associated with fibrosis progression were included. Finally, we explored the impact of rs738409 on response to standard antiviral therapy using the interferon lambda 3 (IL28B) [rs12979860 C>T] variant both as a comparator and as a positive control. After adjustment for age, sex, body mass index, alcohol consumption, and diabetes, rs738409 mutant G allele homozygote carriers remained at higher risk for steatosis (odds ratio [OR] 2.55, 95% confidence interval [CI] 1.08‐6.03, P = 0.034), fibrosis (OR 3.13, 95% CI 1.50‐6.51, P = 0.002), and fibrosis progression (OR 2.64, 95% CI 1.22‐5.67, P = 0.013). Conversely, rs738409 was not independently associated with treatment failure (OR 1.07, 95% CI 0.46‐2.49, P = 0.875) and did not influence clinical or biological variables. Conclusion: The PNPLA3 (rs738409 C>G) polymorphism favors steatosis and fibrosis progression in CHC. This polymorphism may represent a valuable genetic predictor and a potential therapeutic target in CHC liver damage. (HEPATOLOGY 2011;)

Differential liver sensitization to Toll‐like receptor pathways in mice with alcoholic fatty liver

Gut‐derived, endotoxin‐mediated hepatocellular damage has been postulated to play a crucial role in the pathogenesis of alcohol‐induced liver injury in rodents. Endotoxins induce production of tumor necrosis factor α (TNF‐α) by Kupffer cells via Toll‐like receptor (TLR) 4 and contribute to liver injury. This study addressed the contribution of other TLRs and ligands to alcoholic fatty liver. C57Bl6/J mice were fed a modified Lieber‐DeCarli diet. Serum aminotransferase measurements, histological analysis, and quantification of liver TNF‐α and TLR1‐9 messenger RNA (mRNA) were performed. The effect of TLR ligands on liver injury was assessed in vivo. Neomycin and metronidazole or diphenyleneiodonium sulfate (DPI) were administered to evaluate the role of gut bacteria and NADPH oxidase activity, respectively, in hepatic TLR expression. Enteral ethanol (EtOH) exposure induced steatosis and increased liver weight, aminotransferase levels, and expression of TLR1, 2, 4, 6, 7, 8, and 9 liver mRNA. Injection of lipoteichoic acid, peptidoglycan (PGN), lipopolysaccharide (LPS), loxoribine, and oligonucleotide containing CpG (ISS‐ODN) increased TNF‐α mRNA expression more in the livers of EtOH‐fed mice than in control mice. PGN, LPS, flagellin, and ISS‐ODN induced liver inflammatory infiltrate in EtOH‐fed mice but not control mice. Addition of antibiotics reduced the severity of alcoholic fatty liver without affecting TLR expression, whereas daily DPI injections reduced the EtOH‐mediated upregulation of TLR2, 4, 6, and 9 mRNA. In conclusion, EtOH‐fed mice exhibited an oxidative stress dependent on upregulation of multiple TLRs in the liver and are sensitive to liver inflammation induced by multiple bacterial products recognized by TLRs. (HEPATOLOGY 2006;43:989–1000.)

Self-expandable metal stents for the treatment of benign upper GI leaks and perforations.

BACKGROUND Self-expandable metal stents (SEMSs) have been suggested for the treatment of benign upper GI leaks and perforations. Nevertheless, uncomplicated removal remains difficult. Placement of a self-expandable plastic stent (SEPS) into an SEMS can facilitate retrieval. OBJECTIVES This study reviews our experience with sequential SEMS/SEPS placement in patients with benign upper GI leaks or perforations. DESIGN A retrospective review of the chart of each patient who underwent SEMS placement for benign upper GI leaks or perforations, including (1) fistula after bariatric surgery, (2) other postoperative fistulae, (3) Boerhaave syndrome, (4) iatrogenic perforations, and (5) other perforations. SETTING Single, tertiary center. PATIENTS Eighty-eight patients (37 male, average age 51.6 years, range 18-89 years). INTERVENTIONS SEMS placement and removal, with or without SEPS placement. MAIN OUTCOME MEASUREMENTS Feasibility of SEMS removal and successful treatment of lesions and short-term and long-term complications. RESULTS A total of 153 SEMSs were placed in 88 patients; all placements were successful. Six patients died (not SEMS-related deaths) and 6 patients were lost to follow-up with SEMSs still in place. Seventy-three of the remaining 76 patients had successful SEMS removal (96.1%). The rate of successful SEMS removal per stent was 97.8% (132/135). Resolution of leaks and perforations was achieved in 59 patients (77.6%) with standard endoscopic treatment, and in 64 patients (84.2%) after prolonged, repeated endoscopic treatment. Spontaneous migration occurred in 11.1% of stents, and there were minor complications (dysphagia, hyperplasia, rupture of coating) in 20.9% and major complications (bleeding, perforation, tracheal compression) in 5.9%. LIMITATIONS Retrospective design and highly selected patient population. CONCLUSIONS Use of SEMSs for the treatment of benign upper GI leaks and perforations is feasible, relatively safe, and effective, and SEMSs can be easily removed 1 to 3 weeks after SEPS insertion. Leaks and perforations were closed in 77.6% of cases.

Common polymorphism in the PNPLA3/adiponutrin gene confers higher risk of cirrhosis and liver damage in alcoholic liver disease

BACKGROUND & AIMS A recent genome-wide association study identified genetic polymorphism (rs738409 C>G) in the PNPLA3/adiponutrin gene associated with liver steatosis. This variant has also been linked to increased risk of alcoholic liver disease (ALD) and cirrhosis in Mestizo Mexicans with excessive alcohol intake. Our aim was to study the influence of this polymorphism on European Caucasian patients with histologically suggestive ALD. METHODS Three-hundred-and-twenty-eight healthy controls and 330 ALD patients, among whom 265 had cirrhosis, were genotyped for the rs738409 polymorphism. We studied the impact of rs738409 on clinical and biological parameters, together with histological staging of steatosis and fibrosis. PNPLA3 messenger RNA (mRNA) levels were measured by quantitative real-time PCR according to the patients phenotype. RESULTS The G-allele was significantly more frequent in ALD patients than in controls (odds ratio [OR] = 1.54, 95% confidence interval [CI] = 1.12-2.11 p = 0.008) and was, among ALD patients, significantly associated with steatosis (p = 0.048), fibrosis (p = 0.001), and greater risk of cirrhosis (p = 0.001). In multivariate analysis, rs738409 remained the strongest independent factor associated with risk of cirrhosis (OR = 2.08; 95% CI = 1.15-3.77; p = 0.02). Furthermore, the PNPLA3 mRNA liver expression level was significantly lower in patients with more advanced fibrosis (p = 0.03) and negatively correlated with the hepatic venous pressure gradient (r = -0.41, p = 0.006). CONCLUSIONS In European Caucasians, the rs738409 variant is associated with increased risk of ALD, liver damage, and cirrhosis. Further prospective studies are required to confirm these results and to evaluate the potential of PNPLA3 as both a predictor and a therapeutic target in ALD.

Enteral nutrition with or without N-acetylcysteine in the treatment of severe acute alcoholic hepatitis: a randomized multicenter controlled trial.

BACKGROUND & AIMS Severe acute alcoholic hepatitis is associated with a high mortality rate. Oxidative stress is involved in the pathogenesis of acute alcoholic hepatitis. Previous findings had also suggested that enteral nutritional support might increase survival in patients with severe acute alcoholic hepatitis. Therefore, the aim of the present study was to evaluate the efficacy of N-acetylcysteine in combination with adequate nutritional support in patients with severe acute alcoholic hepatitis. METHODS Patients with biopsy-proven acute alcoholic hepatitis and mDF ≥32 were randomized to receive N-acetylcysteine intravenously or a placebo perfusion along with adequate nutritional support for 14 days. The primary endpoint was 6-month survival; secondary endpoints were biological parameter evolution and infection rate. RESULTS Fifty-two patients were randomized in the study (28 into the N-acetylcysteine arm, 24 into the control arm), and among them, five were excluded from the analysis for protocol violation. The two groups did not differ in baseline characteristics. Survival rates at 1 and 6 months in N-acetylcysteine and control groups were 70.2 vs. 83.8% (p=0.26) and 62.4 vs. 67.1% (p=0.60), respectively. Early biological changes, documented infection rate at 1 month, and incidence of hepatorenal syndrome did not differ between the two groups. CONCLUSIONS In this study, high doses of intravenous N-acetylcysteine therapy for 14 days conferred neither survival benefits nor early biological improvement in severe acute alcoholic hepatitis patients with adequate nutritional support. However, these results must be viewed with caution, since the study suffered from a lack of power.

Profile of soluble cytokine receptors in Crohn’s disease

Introduction: Soluble cytokine receptors (sCRs) modulate the in vivo activity of cytokines. Deficient sCR production could participate in the pathogenesis and course of Crohn’s disease (CD). The aim of the study was to examine the profile of sCRs in CD patients and their modulation by infliximab and corticosteroids. Methods: We prospectively examined active CD patients (aCD) treated with either infliximab (n = 21) or corticosteroids (n = 9), CD patients in clinical remission (rCD, n = 20), ulcerative colitis patients (UC, n = 24), and healthy subjects (HS, n = 15). Cultures of colonic biopsies were also examined from CD inflamed (n = 8), CD non-inflamed (n = 7), and healthy mucosa (n = 8). Levels of tumour necrosis factor α (TNF-α), soluble TNF receptor I (sTNFRI), soluble TNF receptor II (sTNFRII), interleukin 1β (IL-1β), soluble IL-1 receptor I (sIL-1RI), soluble IL-1 receptor II (sIL-1RII), IL-6, soluble IL-6 receptor (sIL-6R), and sgp130 were measured using ELISA. Results: Higher levels of sTNFRI (p<0.05, p<0.01), sTNFRII (p<0.01, p<0.01), sIL-1RI (p<0.05, NS), IL-6 (p<0.01, p<0.01), and sIL-6R (p<0.05, NS) were observed in aCD compared with rCD and HS. Interestingly, sIL-1RII (p<0.05, p<0.01) and sgp130 (p<0.01, p<0.01) were profoundly decreased in aCD compared with rCD and HS, and were negatively correlated with CRP. Deficient production of sIL-1RII was specific to CD (not observed in ulcerative colitis), and was further confirmed at the mucosal level. Infliximab decreased sTNFRII at one and four weeks (p<0.05) and enhanced sIL-6R levels at one week (p<0.05). Corticosteroids increased sIL-1RII levels at one week (p<0.05). Conclusion: CD is associated with dysregulated production of sCRs. Deficiency in sIL-1RII and sgp130 may be essential to CD pathogenesis. Their replacement through the use of fusion proteins could represent future alternative therapeutic strategies for CD.

Galectin-3 and galectin-3-binding site expression in human adult astrocytic tumours and related angiogenesis.

Using computer‐assisted microscopy, the present work aimed to quantitatively characterize the level of the histochemically detectable expression of galectin‐3 and galectin‐3‐binding sites in sections of a series of 84 astrocytic tumours (including 22 grade II, 21 grade III and 41 grade IV specimens) and seven non‐tumoural specimens used as controls. The presence of galectin‐3 and reactive sites for this lectin were monitored by means of a specific polyclonal anti‐galectin‐3 antibody (aGal3) and biotinylated galectin‐3 (Gal3), respectively. The pattern of expression of galectin‐3‐binding sites is compared to the pattern of expression of laminin (a potential galectin‐3 ligand) revealed using a biotinylated anti‐laminin antibody (aLam). Three variables quantitatively characterizing histochemical staining reactions were evaluated by means of computer‐assisted microscopy for each of the 3 probes under study (aGal3, Gal3 and aLam). The labelling index (LI) is the percentage of tissue area specifically stained by a histochemical probe. The mean optical density (MOD) denotes staining intensity. The concentration heterogeneity (CH) feature expresses the concentrational spread of individual fields. The data obtained in the present study show that: (i) white matter of a non‐tumoural brain expresses galectin‐3 (and also galectin‐3‐binding sites); (ii) the level of galectin‐3 expression significantly decreases in the majority of tumour astrocytes from low to high grade astrocytic tumours; while (iii) some tumour cell clones expressing high amounts of galectin‐3 emerged with increasing levels of malignancy; and (iv) the level of accessible galectin‐3‐binding sites was apparently not heavily modified in the course of malignancy progression. In conclusion, the results obtained in the present study show that human astrocytic tumours are very heterogenous in their galectin‐3 levels of expression. If high levels of galectin‐3 determine the invasiveness potential of a tumour cell, then within a heterogenous tumour the presence of even a small, but actively proliferating number of tumour cell clones expressing high levels of galectin‐3 can be expected to lead to tumour invasiveness.

Marked 25-hydroxyvitamin D deficiency is associated with poor prognosis in patients with alcoholic liver disease.

BACKGROUND & AIMS Vitamin D deficiency has been frequently reported in advanced liver disease. However, its influence on alcoholic liver disease (ALD) has been poorly elucidated. We investigated the association of vitamin D with clinical, biological, and histological parameters and survival in ALD patients. Furthermore, we explored the effect of vitamin D treatment on ALD patient peripheral blood mononuclear cells (PBMCs), and in a murine experimental model of ALD. METHODS Serum levels of 25-hydroxyvitamin D [25(OH)D] were determined in 324 Caucasian ALD patients and 201 healthy controls. In vitro experiments on vitamin D pre-treated PBMCs evaluated TNFα production by ELISA in culture supernatants. Mice were submitted to an ethanol-fed diet and some of them were orally supplemented three times per week with 1,25(OH)2D. RESULTS Severe deficiency in 25(OH)D (<10 ng/ml) was significantly associated with higher aspartate aminotransferase levels (p=1.00 × 10(-3)), increased hepatic venous pressure gradient (p=5.80 × 10(-6)), MELD (p=2.50 × 10(-4)), and Child-Pugh scores (p=8.50 × 10(-7)). Furthermore, in multivariable analysis, a low 25(OH)D concentration was associated with cirrhosis (OR=2.13, 95% CI=1.18-3.84, p=0.013) and mortality (HR=4.33, 95% CI=1.47-12.78, p=7.94 × 10(-3)) at one year. In addition, in vitro, 1,25(OH)2D pretreatment decreased TNFα production by stimulated PBMCs of ALD patients (p=3.00 × 10(-3)), while in vivo, it decreased hepatic TNFα expression in ethanol-fed mice (p=0.04). CONCLUSIONS Low 25(OH)D levels are associated with increased liver damage and mortality in ALD. Our results suggest that vitamin D might be both a biomarker of severity and a potential therapeutic target in ALD.

Role of a cirrhosis risk score for the early prediction of fibrosis progression in hepatitis C patients with minimal liver disease.

BACKGROUND & AIMS Fibrosis progression in patients with chronic hepatitis C (CHC) is highly variable. A Cirrhosis Risk Score (CRS) based on seven genetic variants has been recently developed for identifying patients at risk for cirrhosis. The objective of this study was to assess the role of the CRS for the early prediction of fibrosis progression in CHC patients with mild liver fibrosis. In addition, we evaluated the potential benefit, for prediction accuracy, of a recently described non-invasive fibrosis staging assay, the Enhanced Liver Fibrosis (ELF) test. METHODS Two separate cohorts of HCV patients (Brussels, Belgium/Hannover, Germany) were retrospectively analyzed. Only patients with a fibrosis Ishak or METAVIR score of F0-F1 at baseline were included. Patients were classified as progressors if they showed an increase ≥2 fibrosis stages at the second histological evaluation after a follow-up ≥5years. The CRS was calculated locally. Genotyping was performed by PCR and oligonucleotide ligation with the resulting signal detected with a Luminex® 200TM and computer analysis. RESULTS In Brussels, 12/25 patients progressed (48%); similarly in Hannover, 16/31 (52%) patients progressed. In both sample sets, the CRS was significantly associated with fibrosis progression (p=0.050 in Brussels; p=0.018 in Hannover). The ELF test was only a significant predictor in Hannover (p=0.015). In multivariate analysis the CRS remained the only variable associated with fibrosis progression (odds-ratio=2.23, 95%CI 1.21-4.11 p=0.01). CONCLUSIONS Although conducted on a limited number of patients, this study in two independent centres confirms that the CRS predicts fibrosis progression in initially mild CHC.