Hubert Louis

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.)

Multisystemic production of interleukin 10 limits the severity of acute pancreatitis in mice

Background—Interleukin 10 (IL-10) decreases the severity of experimental acute pancreatitis. The role of endogenous IL-10 in modulating the course of pancreatitis is currently unknown. Aims—To examine the systemic release of IL-10 and its messenger RNA production in the pancreas, liver, and lungs and analyse the effects of IL-10 neutralisation in caerulein induced acute pancreatitis in mice. Methods—Acute necrotising pancreatitis was induced by intraperitoneal caerulein. Serum levels of IL-10 and tumour necrosis factor (TNF), and tissue IL-10 and TNF-α gene expression were assessed. After injecting control antibody or after blocking the activity of endogenous IL-10 by a specific monoclonal antibody, the severity of acute pancreatitis was assessed in terms of serum enzyme release, histological changes, and systemic and tissue TNF production. Results—In control conditions, serum IL-10 levels increased and correlated with the course of pancreatitis, with a maximal value eight hours after induction. Both IL-10 and TNF-α messengers showed a similar course, and were identified in the pancreas, liver, and lungs. Neutralisation of endogenous IL-10 significantly increased the severity of pancreatitis and associated lung injury as well as serum TNF protein levels (+75%) and pancreatic, pulmonary, and hepatic TNF messenger expression (+33%, +29%, +43%, respectively). Conclusions—In this non-lethal model, systemic release of IL-10 correlates with the course of acute pancreatitis. This anti-inflammatory response parallels the release of TNF and both cytokines are produced multisystemically. Endogenous IL-10 controls TNF-α production and plays a protective role in the local and systemic consequences of the disease.

CCR5 deficiency exacerbates T-cell–mediated hepatitis in mice†

Experimental T‐cell–mediated hepatitis induced by concanavalin A (Con A) involves the production of different cytokines and chemokines and is characterized by leukocyte infiltration. Because the chemokine receptor CCR5 and its ligands (CCL3, CCL4, and CCL5) regulate leukocyte chemotaxis and activation, we investigated the role of CCR5 during Con A–induced liver injury. Serum levels of CCR5 ligands and their hepatic transcript levels were significantly increased after Con A injection, whereas CCR5+ liver mononuclear cells were recruited to the liver. CCR5‐deficient (CCR5−/−) mice disclosed increased mortality and liver injury following Con A administration compared with wild‐type mice. CCR5−/− mice also exhibited increased production of interleukin 4, tumor necrosis factor α, CCL3, CCL4, and CCL5, and a prominent liver mononuclear cell infiltrate, among which many cells were CCR1+. In vivo neutralization of CCR5 ligands in CCR5−/− mice afforded a protection against hepatitis only when CCL5 was neutralized. In conclusion, CCR5 deficiency exacerbates T‐cell–mediated hepatitis, and leads to increased levels of CCR5 ligands and a more pronounced liver mononuclear infiltrate, suggesting that CCR5 expression can modulate severity of immunomediated liver injury. (HEPATOLOGY 2005;42:854–862.)

n -acetylcysteine Decreases Severity of Acute Pancreatitis in Mice

Oxidative stress plays a major role in the early stage of acute pancreatitis. This study assessed the effects of N-acetylcysteine (NAC), a reduced glutathione (GSH) provider and a direct scavenger of reactive oxygen intermediates, in the course of acute pancreatitis in mice. Acute pancreatitis (AP) was induced by intraperitoneal (i.p.) injections of cerulein. Mice received NAC (1,000 mg/kg, i.p.) every 3 h, starting either 1 h before the first cerulein injection (prophylactic group) or 1 h after the first cerulein injection (therapeutic group), or i.p. saline injections for controls. Severity of AP was evaluated by histology, serum hydrolase levels, and serum and intrapancreatic levels of MCP-1 and interleukin 6 (IL-6). Pancreatic conjugated dienes and intrapancreatic and intrahepatic GSH levels were measured to assess the local and systemic oxidative processes. Acute pancreatitis was also induced with a CDE diet in controls and mice receiving either both NAC ad libidum in drinking water and 1,000 mg/kg i.p. injection once daily. The severity of pulmonary lesions was assessed by arterial blood gases (po2) and intrapulmonary myeloperoxidase (MPO content) measurements as well as the survival of mice. The severity of cerulein-induced AP was significantly decreased in the prophylactic group compared with the therapeutic and control groups. Prophylactic administration of NAC also decreased the intrapancreatic levels of conjugated dienes compared with controls. The intrapancreatic and systemic release of MCP-1 and IL-6 was also decreased in the prophylactic group 3 and 6 hours after AP induction. In addition, NAC pretreatment also reduced hepatic IL-6 production at 3 and 6 hours after starting cerulein challenge. In CDE-induced AP, the severity of lung injury (hypoxemia, MPO content) was decreased, and survival was improved by NAC. NAC administered in a prophylactic protocol limits the severity of experimental acute pancreatitis in mice, as well as its systemic complications and related mortality.

Distal esophageal spasm treated by peroral endoscopic myotomy.

with the information of every individual trainee on progression at any period in time and not just at the endpoint. It allows us to tailor our training program to the individual needs of trainees and also to assess any intervention in an individual or group training program. Th e results of our study prove the feasibility of this approach. Our next steps have been two-fold: (1) we have developed a web-based e-portfolio including the Rotterdam Assessment Form for colono scopy (RAF-c) available for trainees in the Netherlands and (2) we have expanded the selfassessment form including questions on the second and third domain for colonoscopy as we have described above. Th is includes questions on withdrawal times, PDR, and polypectomy, and is, in our opinion, the exact answer to the questions raised by Dr Singh. It is now too early to evaluate the results aft er the last modifi cation of RAF-c. Th is is a subject for ongoing study and currently under investigation.

Hepatocyte proliferative activity in human liver cirrhosis

BACKGROUND/AIMS The objective of this study was to validate, with an independent prospective cohort of patients, our previous data indicating that the proliferating cell nuclear antigen-labeling index (PCNA-LI) reflects the liver functional reserve in human cirrhosis and might have prognostic significance for patient survival. We also examined how this proliferative index is related to the expression of transforming growth factor beta1 (TGFbeta1) as a possible correlate of hepatocyte proliferative activity. METHODS The present group (n=70 patients) was similar in composition to our previous group regarding age, sex and severity of liver cirrhosis. PCNA and TGFbeta1 immunostaining were analyzed on methanol-fixed, paraffin-embedded liver biopsies. RESULTS Our data show that PCNA-LI declined significantly with worsening Child class and was negatively correlated with the Pugh score. Twenty-five patients died and 10 underwent liver transplantation during the observation period. Liver function, hepatic venous pressure gradient and hepatocyte PCNA-LI were significantly different in survivors and non-survivors. At a mean follow-up of 356 days, the patients with a PCNA-LI higher than 4.4% (the previously determined best cut-off value) had a significantly higher probability of survival than those with a PCNA-LI < or = 4.4% (0.87 vs 0.48, p=0.0009). TGFbeta1 expression in liver parenchyma correlated negatively with PCNA-LI, suggesting that this cytokine could be involved in the impaired regeneration observed in worsened liver cirrhosis. CONCLUSIONS This prospective study strengthens our previous observation that, in cirrhosis, hepatocyte proliferative activity, as evaluated by the PCNA-LI, provides information on liver functional reserve as well as on the patients prognosis.

Endoscopic implantation of Enteryx for the treatment of gastroesophageal reflux disease: technique, pre-clinical and clinical experience

The implantation of Enteryx polymer in the LES is a fast, minimally invasive procedure with anticipated low procedural risks and limited costs. Preliminary clinical data after 6 months of follow-up show good results in more than 80% of the patients, with objective improvement of acid reflux time. Enteryx is effective in the management of GERD, as evidenced by the ability of GERD patients with a history of use of PPIs and other GERD medications to eliminate or significantly reduce use of these medications. Mechanisms of action of Enteryx implantation suggest a change in distensibility of the LES, allowing a greater competency of the cardia. LES pressure and length might be increased after polymer implantation, and further studies are needed to study the effect of Enteryx on transient relaxations of the LES, which is the prevalent mechanism of reflux in patients with mild GERD. Challenges with this procedure include refining technique to deliver adequate volume in each patient and determining if the ring-shaped implant is needed. In addition to acid reflux, another indication for use of this treatment might be biliary reflux in gastrectomized patients; preliminary data suggest that Enteryx may reduce symptoms and improve bile reflux (J. Devière, unpublished observations, 2002). Further studies are needed to definitively establish the safety of the procedure. This is a major point because gastroenterologists are not dealing with ill patients but primarily with individuals who have a good quality of life while taking PPIs. The other area to investigate is the efficacy of the treatment through a sham-controlled study. Finally, this endoscopic technique will have to be evaluated in terms of cost-effectiveness against medical and surgical therapies.

Three-year results of a multicenter prospective study of transoral incisionless fundoplication

Background. To date, there are no long-term data on the use of transoral incisionless fundoplication (TIF) for the treatment of chronic gastroesophageal reflux disease (GERD). We sought to prospectively evaluate the long-term safety and durability of TIF in a multi-center setting. Methods. A longitudinal per protocol (PP) and a modified intention-to-treat (mITT) analysis at 1 and 3 years consisted of symptom evaluation using the GERD health-related quality of life (GERD-HRQL) questionnaire, medication use, upper gastrointestinal endoscopy, and pH-metry. Results. Of 79 patients previously reported at 1 year, 12 were lost to follow-up, and 1 died from an unrelated cause. The remaining 66 patients were followed up and analyzed (mITT). Of 66 patients, 12 underwent revisional procedures, leaving 54 patients for PP analysis at a median of 3.1 years (range = 2.9-3.6). No adverse events related to TIF were reported at 2- or 3-year follow-up. On PP analysis, median GERD-HRQL score off proton pump inhibitors (PPIs) improved significantly to 4 (range 0-32) from both off (25 [13-38], P < .0001) and on (9 [0-22], P < .0001) PPIs. Discontinuation of daily PPIs was sustained in 61% (mITT) and 74% (PP) of patients. Of 11 patients with pH data at 3 years (PP), 9 (82%) remained normal. Based on mITT analysis, 9/23 (39%) remained normal at 3 years. Conclusions. The clinical outcomes at 3 years following TIF, patient satisfaction, healing of erosive esophagitis, and cessation of PPI medication support long-term safety and durability of the TIF procedure for those with initial treatment success. Although complete normalization of pH studies occurred in a minority of patients, successful cases showed long-term durability.

DOMINO HEPATIC TRANSPLANTATION USING THE LIVER FROM A PATIENT WITH PRIMARY HYPEROXALURIA

Background. We report a case of domino liver transplantation using the liver harvested from a patient who underwent a combined liver and kidney transplantation for primary hyperoxaluria (PH). Method. A cadaveric liver transplantation was performed in a 19-year-old man with PH. In a second step, the PH liver harvested from the first patient was transplanted in a 69-year-old man with hepatitis C-related cirrhosis, not a candidate for a classic liver graft owing to multifocal hepatocellular carcinoma. Results. At 8 months after transplantation, the domino recipient has normal hepatic function and no signs of tumoral recurrence, but he progressively developed hyperoxalemia, hyperoxaluria, and renal insufficiency. Conclusion. Regarding the favorable postoperative clinical evolution, domino liver transplantations using livers from PH patients may represent a new opportunity for marginal candidates for liver transplantation. However, the progressive renal insufficiency expected in such domino recipients should limit this procedure to selected cases.

Chronic alcohol exposure sensitizes mice to galactosamine-induced liver injury through enhanced keratinocyte chemoattractant and defective IL-10 production

BACKGROUND/AIMS Alcohol sensitizes the liver to several injuries. The mechanisms leading to this sensitization are poorly defined. In the present study, we developed a mouse model of chronic exposure to alcohol vapours that sensitize mice to galactosamine (GAL) liver injury. METHODS C57BL/6 mice were exposed to ethanol vapours for 10 days. Liver injury was induced by intraperitoneal injection of GAL (1 g/kg) and mice were killed 24 h later. RESULTS GAL challenge after ethanol pre-treatment significantly raised serum alanine aminotransaminase (ALT) levels and enhanced liver inflammation when compared with the controls (GAL alone). Serum keratinocyte chemoattractant (KC) and monocyte chemoattractant protein-1 (MCP-1) levels were significantly increased in the GAL+ethanol group. On the contrary, serum interleukin 10 (IL-10) levels were lower than in controls. Anti-KC, anti-tumour necrosis factor alpha antibodies and intestinal decontamination significantly protected mice from liver injury. In GAL+ethanol-treated mice, IL-10 treatment reduced ALT release, KC and MCP-1 serum and hepatic mRNA levels, and improved liver inflammation. CONCLUSIONS Enhancement of GAL-induced liver injury by ethanol is associated with an imbalance between proinflammatory cytokines and the anti-inflammatory cytokine IL-10 and depends on gut bacterial flora.