Raymond Mcmahon

Oxidative stress in chronic hepatitis C: not just a feature of late stage disease.

BACKGROUND/AIMS Chronic hepatitis C infection is a major world-wide problem, frequently progressing to cirrhosis, liver failure or hepatoma. The pathological mechanisms of disease progression are unclear but oxidant stress may play a role. METHODS Markers of lipid peroxidation, antioxidant status, hepatic fibrogenesis and liver function were measured in blood or urine from 42 chronic hepatitis C patients. Fibrosis was graded histologically in a subgroup of 33 patients. RESULTS The lipid peroxidation marker 8-isoprostane and the ratio of oxidized to reduced glutathione were significantly elevated (P<0.001, P=0.006). The antioxidants glutathione, selenium and vitamins A, C and E were significantly decreased (all P<0.001) compared to age and sex matched controls. Abnormal values were more marked in cirrhotics, but significant changes were also observed in the non-cirrhotic group. The fibrosis score correlated positively with urinary 8-isoprostane and type III procollagen peptide and negatively with vitamin A. CONCLUSIONS Oxidant stress, as reflected in blood and urine by a wide range of pro- and antioxidant markers, is a significant feature of hepatitis C infection. Although more severe in the cirrhotic group, there was clear evidence of oxidant stress in non-cirrhotic patients. Antioxidant therapy may therefore have a role in slowing disease progression to cirrhosis.

Oxidant stress is a significant feature of primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is a chronic cholestatic disorder characterised by an immunological, and often granulomatous, attack on bile ducts leading to fibrosis, cirrhosis, liver failure and death. Animal and human studies suggest that oxidant stress plays a key role in progression of other liver diseases, but no comprehensive investigation has been performed previously in PBC. A wide range of lipid peroxidation and antioxidant markers were measured in the blood and urine of 41 patients with histologically confirmed PBC. Lipid peroxidation markers were significantly elevated [plasma and urinary 8-isoprostane, P<0.001; plasma malondialdehyde (MDA), P=0.007] compared to age- and sex-matched controls. The most striking antioxidant depletion occurred with plasma total glutathione where levels were significantly reduced (30% of controls). Total serum antioxidant levels were decreased (P=0.013) and serum selenium and vitamin A were also lower (both P<0.001); vitamins C and E were normal. Most patients had early disease biochemically and were Child-Pugh grade A. Urinary 8-isoprostane correlated positively with Ludwig stage and markers of hepatic injury and cholestasis. This study clearly demonstrates that oxidant stress, as reflected in a comprehensive spectrum of lipid peroxidation and antioxidant markers, is a significant feature of early-stage PBC.

Distribution of constitutive (COX-1) and inducible (COX-2) cyclooxygenase in postviral human liver cirrhosis: a possible role for COX-2 in the pathogenesis of liver cirrhosis.

Aims: Prostaglandins produced by the action of cyclooxygenases (COX) are important mediators of systemic vasodilatation and inflammation in liver cirrhosis. The aim of this study was to investigate the distribution of COX-1 and COX–2 in postviral cirrhosis. Methods: The immunohistochemical expression of the constitutive (COX-1) and the inducible (COX-2) isoenzymes was investigated in 15 patients with cirrhosis after hepatitis B and C infection; three normal control livers were also analysed. Results: COX-2 was absent from normal liver but was highly expressed in cirrhosis, mainly in the inflammatory, sinusoidal, vascular endothelial, and biliary epithelial cells. Low amounts of COX-1 were expressed in both normal and cirrhotic livers, exclusively in sinusoidal and vascular endothelial cells, with no differences seen between normal and cirrhotic livers. Conclusions: COX-2 is overexpressed in liver cirrhosis, and possibly contributes to prostaglandin overproduction, which may be a major component of the inflammation and hyperdynamic circulation associated with cirrhosis. Because COX-2 is thought to contribute to tumour development, high COX-2 production could be a contributor to hepatocellular carcinoma development in cirrhosis. The finding of COX-2 and not COX-1 upregulation in cirrhosis could provide a possible new role for selective COX-2 inhibitors in reducing inflammation and minimising the occurrence of hepatocellular carcinoma in patients with cirrhosis.

Expression of parathyroid hormone-related protein and its receptor in bone metastases from prostate cancer

Studies of breast cancer suggest that parathyroid hormone‐related protein (PTHrP) is important in the development of bone metastases. To determine whether PTHrP expression is important in prostate cancer metastasis, immunohistochemistry and in situ hybridization were used to assess the expression of PTHrP and its receptor in primary prostate cancer and bone metastases from both prostate and non‐prostate cancers. PTHrP was expressed in more prostate primary tumours than bone metastases (p=0.003, Fishers exact test). All bone metastases from non‐prostate cancers expressed PTHrP. In contrast, PTHrP receptor was expressed in all bone metastases, but in only 19% of primary prostate tumours (p=0.001). The receptor to PTHrP was found to be highly expressed in bone metastases from prostate and other primaries, whereas PTHrP protein was found to have lower expression in the bone metastases than in the primary tumours. In conclusion, the expression of the receptor to PTHrP is increased in bone metastases from prostate cancer and may play an important role in their formation. Copyright

Expression of nitric oxide synthase isoforms in human liver cirrhosis.

Several mediators of systemic vasodilatation in liver cirrhosis have been reported. Among these is nitric oxide (NO), which has been proposed as one of the main mediators. In this study, sera and liver biopsies were analysed from 15 patients with clinically and pathologically diagnosed liver cirrhosis. In addition, sera from seven and liver biopsies from three healthy controls were used. Serum levels of nitrite (the end product of NO) were measured using the Griess reaction and the expression of the inducible nitric oxide synthase (iNOS) and constitutive nitric oxide synthase (ecNOS) proteins was investigated using immunohistochemistry. This study shows that serum nitrite levels (94 ± 9.8 µmol/l) in cirrhotic patients were significantly (p < 0.05) increased in comparison with the controls (36.6 ± 11.03 µmol/l). iNOS was completely absent from the control group but was highly expressed in the livers from the cirrhotic group. iNOS was seen mainly in the inflammatory cells infiltrating the portal tracts, blood monocyte‐like cells, hepatocytes, sinusoidal cells, and endothelial cells. However, expression of ecNOS was only seen in the vascular endothelial cells of both the control and the cirrhotic groups, but was much higher in the latter. It is therefore clear that NO is augmented in cirrhotic patients and it is mainly produced by induction of iNOS. Moreover, NO up‐regulation is dependent on the inflammatory stage of liver cirrhosis. ecNOS production could be a normal chronic adaptation mechanism of the endothelium to the chronically increased splanchnic blood flow secondary to portal hypertension. In the near future, the appropriate inhibition of NO activity by using NOS‐active agents may provide a novel strategy for the treatment of patients with liver cirrhosis. Copyright

Epithelioid angiosarcoma arising in an arteriovenous fistula

Vascular tumours with an epithelioid endothelial morphology are now well recognized. In contrast to nonepithelioid endothelial vascular tumours, which only express endothelial markers, epithelioid tumours also express cytokeratin. The occurrence of sarcomas at sites of altered vasculature (arteriovenous fistula/Dacron graft)’ has been reported, but that of an epithelioid angiosarcoma in an arteriovenous haemodialysis fistula appears unique.

Dopexamine reduces the incidence of acute inflammation in the gut mucosa after abdominal surgery in high-risk patients.

OBJECTIVE To evaluate the effect of dopexamine on the incidence of acute inflammation in the stomach/duodenum in patients undergoing abdominal surgery > or =1.5 hrs with a minimum of one high-risk criterion. DESIGN Prospective, randomized, double-blind, placebo-controlled study. This study was conducted as a side arm to a multicenter, multinational study. SETTING University hospital in an adult intensive care unit. PATIENTS Thirty-eight patients. INTERVENTIONS Patients were stabilized with fluid, blood products, and supplementary oxygen to achieve predetermined goals: cardiac index > 2.5 L/min/m2, mean arterial blood pressure of 70 mm Hg, pulmonary arterial occlusion pressure of 10 mm Hg, hemoglobin of 100 g/L, and arterial saturation of 94%. After stabilization, the study drug (either placebo [group A], dopexamine 0.5 microg/kg/min [group B], or dopexamine 2.0 microg/kg/min [group C]) was commenced. The study drug infusion was started 2 to 12 hrs before surgery and infused for 24 hrs after surgery. Estimation of upper gut blood flow was assessed using a gastric tonometer, and gastroscopy with biopsy was performed before surgery (after induction of anesthesia) and 72 hrs after surgery. Comparisons were made between endoscopic findings and histologic proof of acute inflammatory changes. In addition, biopsies were assessed for the presence in the mucosa of mast cells, myeloperoxidase activity, and inducible nitric oxide synthase. MEASUREMENTS AND MAIN RESULTS Intramucosal pH decreased significantly with time in all three groups (p < .001), reaching the lowest point at the end of surgery. There was no difference among the groups. Endoscopy visualized acute inflammatory changes in 58.3% of group A patients, 46.2% of group B patients, and 53.90% of group C patients after hemodynamic optimization. At 72 hrs, dopexamine-treated patients compared with placebo-treated patients had a significantly lower incidence of gastric and duodenal acute inflammatory changes, as defined by myeloperoxidase activity (37.5% in groups B and C vs. 86% in group A; p < .05). CONCLUSION Dopexamine in doses of 0.5 and 2.0 microg/kg/min affords significant histologic protection to the upper gastrointestinal tract mucosa 72 hrs after operation in high-risk surgical patients undergoing abdominal surgery.

Development and validation of an experimental model for the assessment of radiofrequency ablation of pancreatic parenchyma

Objectives: The aim of this study was to develop and validate an ex vivo model for the assessment of radiofrequency (RF) ablation of the pancreas. Methods: Porcine pancreata were used within 1 hour of sacrifice. RF was delivered to a premarked area in the center of the pancreatic head using a thermocouple-tipped multiprobe array. Four temperature presets were evaluated: 70°-100°C in 10° increments. Immediately after ablation serial sections of the pancreatic head were cut to incorporate duodenum, portal vein, and bile duct. For each experiment, a portion of pancreatic tail was sampled as nonablated control. Hematoxylin and eosin (H&E) slides together with nicotinamide adenine dinucleotide (NADH) stained preparations were made. The NADH staining was quantified using computerized digital image recognition techniques. Results: Control sections (n = 20) demonstrated normal pancreatic architecture on H&E and strong NADH staining indicating preserved tissue oxidative metabolism. RF produced a temperature-dependent destruction of parenchymal architecture (H&E) with a corresponding loss of NADH activity. There was no evidence of thermal injury to the duodenum. Quantification of NADH staining demonstrated a median positive staining of 69.26% (55.87-97.28) for control tissue compared with 1.40% (0-7.77) for ablated pancreas (P < 0.001; Mann-Whitney U test). Conclusion: This study describes the development of a relatively simple, reliable, and reproducible model for evaluation of RF ablation of pancreatic parenchyma.

Antioxidant properties of colchicine in acute carbon tetrachloride induced rat liver injury and its role in the resolution of established cirrhosis

Antioxidant and antifibrotic properties of colchicine were investigated in the carbon tetrachloride (CCl(4)) rat model. (1) The protective effect of colchicine pretreatment on CCl(4) induced oxidant stress was examined in rats subsequently receiving a single lethal dose of CCl(4). Urinary 8-isoprostane, kidney and liver malondialdehyde and kidney glutathione levels increased following CCl(4) treatment, but only the rise in kidney malondialdehyde was significantly inhibited by colchicine pretreatment. Serum total antioxidant levels were significantly higher in the colchicine pretreatment group. (2) The long term effects of colchicine treatment on CCl(4) induced liver damage were investigated using liver histology and biochemical markers (hydroxyproline and type III procollagen peptide). Co-administration of colchicine with sub-lethal doses of CCl(4) over 10 weeks did not prevent progression to cirrhosis. However, rats made cirrhotic with repeated CCl(4) challenge and subsequently treated with colchicine for 12 months, all showed histological regression of cirrhosis. (3) The antioxidant effect of colchicine in vitro was evident only at very high concentrations compared to other plasma antioxidants. In summary, colchicine has only weak antioxidant properties, but does afford some protection against oxidative stress; more importantly, long term treatment with this drug may be of value in producing regression of established cirrhosis.

Tumour MLH1 promoter region methylation testing is an effective prescreen for Lynch Syndrome (HNPCC)

Background and aims Lynch syndrome (LS) patients have DNA mismatch repair deficiency and up to 80% lifetime risk of colorectal cancer (CRC). Screening of mutation carriers reduces CRC incidence and mortality. Selection for constitutional mutation testing relies on family history (Amsterdam and Bethesda Guidelines) and tumour-derived biomarkers. Initial biomarker analysis uses mismatch repair protein immunohistochemistry and microsatellite instability. Abnormalities in either identify mismatch repair deficiency but do not differentiate sporadic epigenetic defects, due to MLH1 promoter region methylation (13% of CRCs) from LS (4% of CRCs). A diagnostic biomarker capable of making this distinction would be valuable. This study compared two biomarkers in tumours with mismatch repair deficiency; quantification of methylation of the MLH1 promoter region using a novel assay and BRAF c.1799T>A, p.(Val600Glu) mutation status in the identification of constitutional mutations. Methods Tumour DNA was extracted (formalin fixed, paraffin embedded, FFPE tissue) and pyrosequencing used to test for MLH1 promoter methylation and presence of the BRAF c.1799T>A, p.(Val600Glu) mutation 71 CRCs from individuals with pathogenic MLH1 mutations and 73 CRCs with sporadic MLH1 loss. Specificity and sensitivity was compared. Findingss Unmethylated MLH1 promoter: sensitivity 94.4% (95% CI 86.2% to 98.4%), specificity 87.7% (95% CI 77.9% to 94.2%), Wild-type BRAF (codon 600): sensitivity 65.8% (95% CI 53.7% to 76.5%), specificity 98.6% (95% CI 92.4% to 100.0%) for the identification of those with pathogenic MLH1 mutations. Conclusions Quantitative MLH1 promoter region methylation using pyrosequencing is superior to BRAF codon 600 mutation status in identifying constitutional mutations in mismatch repair deficient tumours.