Marco Tabone

Detection of antibodies against proteins modified by hydroxyethyl free radicals in patients with alcoholic cirrhosis

BACKGROUND/AIMS We have previously shown that hydroxyethyl free radicals produced during cytochrome P4502E1-mediated oxidation of ethanol covalently bind to microsomal proteins. The present study examined whether alkylation of proteins by hydroxyethyl radicals induces an immunologic response in alcoholic patients. METHODS A microplate enzyme-linked immunosorbent assay was developed using as antigen human serum albumin or bovine fibrinogen reacted with chemically produced hydroxyethyl radicals. RESULTS This assay showed that the sera of alcoholic cirrhotics contained both immunoglobulin (Ig) Gs and IgAs that recognized proteins modified by hydroxyethyl radicals, whereas practically no reaction was observed in the sera of healthy controls or cirrhotics without evidence of alcohol abuse. The reactivity of the sera from alcoholic patients was not influenced by the protein to which hydroxyethyl radicals were bound. The sera of alcoholic cirrhotics also contained antibodies directed against acetaldehyde-modified albumin. However, the reaction of alcoholic sera with hydroxyethyl radical epitopes was not inhibited by increasing concentrations of acetaldehyde-modified albumin produced under either reducing or nonreducing conditions. CONCLUSIONS The results indicate that a new group of antigens that do not cross-react with antibodies against acetaldehyde-derived epitopes is formed by the alkylation of protein by hydroxyethyl radicals and is involved in the development of immunologic reactions in alcoholic patients.

Interferon and amantadine in combination as initial treatment for chronic hepatitis C patients

BACKGROUND/AIMS To evaluate the efficacy and tolerance of amantadine in combination with interferon in the treatment of chronic hepatitis C. METHODS Multi-centre trial including 180 chronic hepatitis C patients without cirrhosis, randomly enrolled to receive interferon 6 MU every other day for 6 months followed by 3 MU for further 6 months (group A, 90 patients), or the same schedule plus amantadine 200 mg/day (group B, 90 patients). Primary end-point was a sustained virological and biochemical response, secondary end-points were on-treatment (third month) and end-of-treatment response rates. RESULTS The two groups had similar demographic, biochemical and virological characteristics. A sustained response after 6 months follow-up was observed in 17% of group A and 24% of group B patients (P not significant), an end-of-treatment response was observed in 37% in group A and 47% in group B (P not significant), an on-treatment response was observed in 46% in group A and 61% in group B patients (P < 0.05). No major side effects due to amantadine administration were observed. CONCLUSIONS Adding amantadine to interferon did not improve the sustained treatment efficacy. However, the rate of early response at the third month of therapy was significantly higher in the combination therapy group.

AMOUNT AND DURATION OF ALCOHOL INTAKE AS RISK FACTORS OF SYMPTOMATIC LIVER CIRRHOSIS: A CASE-CONTROL STUDY

We carried out a hospital based case-control study involving 320 patients with symptomatic liver cirrhosis (LC) and 320 pair-matched control individuals, in order to estimate the dose-response relationship between both the daily amount and the duration of alcohol intake and the risk of LC. Lifetime alcohol consumption was measured by a standardized and reproducible questionnaire, and expressed as lifetime daily alcohol intake (LDAI) and duration of alcohol consumption (DAC). The odds ratio (OR) for LC was estimated by the conditional logistic regression. It increased from 1.0 for lifetime abstainers to 4.2 for LDAI of 225 g or more. Comparing durations of alcohol consumption of < or = 10 and > or = 30 years in the model, the ORs consistently decreased for all the LDAI categories: from 4.1 to 0.6 in the 25-50 g category; from 15.1 to 0.9 in the 75-100 g category; from 67.2 to 1.5 in the 125 g or more category. Our results suggest that the dose-dependent relationship between alcohol and LC may be mediated by the degree of individual susceptibility to the detrimental effect of alcohol to the liver.

A strong negative association between alcohol consumption and the risk of hepatocellular carcinoma in cirrhotic patients - A case-control study

We carried out a hospital-based, case-control study to assess the association of both the Hepatitis B Virus (HBV) infection and the lifetime daily alcohol intake with the risk of developing hepatocellular carcinoma (HCC) in patients with liver cirrhosis (LC). Cases were 62 consecutive inpatients of a Gastroenterology Division in whom a first diagnosis of HCC superimposed on LC was made. Two control groups were used: 310 patients without liver disease, matched 1:5 with cases and randomly selected from inpatients of the same hospital, and 97 consecutive asymptomatic inpatients in whom the first diagnosis of LC was made. Alcohol intake was quantified in all subjects by a standardized questionnaire. HBV infection was associated with HCC development in cirrhotics (odds ratio =6.8; 95% confidence interval =1.4−32.3), whereas we observed a trend towards a decreased HCC risk at increased alcohol intake values (odds ratio from 1 for lifetime abstainers to 0.2 for drinkers of 175 g/day or more). Our results suggest that alcohol intake is not a direct determinant of HCC, but its role is mediated by LC. Cirrhotics with high alcohol intake do not usually survive long enough to develop HCC.

Re-treatment with interferon-beta of patients with chronic hepatitis C virus infection

Objective To evaluate the efficacy of interferon-beta (IFN-β) in the re-treatment of patients with chronic hepatitis C who did not respond to IFN-α monotherapy. Patients and methods Thirty patients (24 men and six women; mean age, 41 ± 13 (SD) years; range, 23–62 years), with chronic hepatitis C that was non-responsive to a standard course of IFN-α therapy, were re-treated with recombinant human IFN-β-1a. All patients received IFN-β, 12 MIU subcutaneously, three times weekly for 3 months, after which time patients’ responses were evaluated. Responders (normal alanine aminotransferase, and negative for serum hepatitis C virus RNA) continued to receive IFN-β, 12 MIU, for a further 3 months. Non-responders had their dose increased to 18 MIU for the remaining 3 months of treatment. After 6 months of treatment, therapy was stopped and patients were followed-up for a further 6 months. Results Overall, six (20%) of the 30 patients exhibited a response at the end of treatment. One patient (3.3%) maintained a sustained virological response at the end of post-treatment follow-up. Conclusions Treatment with recombinant IFN-β, at doses of up to 18 MIU for 6 months, is safe and well tolerated. However, the results of the trial do not support the use of IFN-β monotherapy in patients with chronic hepatitis C that is resistant to IFN-α.

Serum interferon gamma in primary biliary cirrhosis: Effect of ursodeoxycholic acid and prednisone therapy alone and in combination

Background Interferon‐&ggr; may have immunopathogenic importance in primary biliary cirrhosis, stimulating aberrant expression on biliary epithelium of class II major histocompatibility molecules and inter‐cellular adhesion molecule‐1. Liver transcripts for interferon‐&ggr; are found in primary biliary cirrhosis. Its serum level is increased in pretransplantation stages and decreases after transplantation. Objectives (1) To verify whether serum interferon‐&ggr; levels are increased in non‐cirrhotic stages of primary biliary cirrhosis. (2) To evaluate the effect of ursodeoxycholic acid and prednisone alone and in combination on serum levels of interferon‐&ggr; and soluble inter‐cellular adhesion molecule‐1. Methods Nine non‐cirrhotic, anicteric patients with primary biliary cirrhosis (patient test group), 14 healthy, negative controls and 14 positive controls, with chronic hepatitis related to hepatitis C virus were studied in basal condition. Primary biliary cirrhosis patients were treated with ursodeoxycholic acid, prednisone and the association of the two drugs for three 4‐week periods, each period separated by a 4‐week wash‐out. Interferon‐&ggr; and soluble inter‐cellular adhesion molecule‐1 were measured in serum by commercially available immuno‐enzymatic kits. Results Median interferon‐&ggr; levels were increased in patients with primary biliary cirrhosis compared with healthy controls (44 vs 19 pg/ml; P < 0.01) but similar to those in chronic hepatitis patients (47 pg/ml). Serum soluble inter‐cellular adhesion molecule‐1 was significantly reduced by ursodeoxycholic acid, and an even greater reduction was obtained on addition of prednisone. No treatment affected interferon‐&ggr; levels. Conclusion Serum interferon‐&ggr; is increased in non‐cirrhotic patients with primary biliary cirrhosis, but this is not disease‐specific. Neither ursodeoxycholic acid, nor prednisone, nor the combination of the two drugs influenced this immunological pathway of primary biliary cirrhosis. Eur J Gastroenterol Hepatol 12:463‐468

Pre-treatment levels of anti-HCV core IgM antibodies are closely associated with response to alpha interferon therapy in chronic hepatitis C patients

Objectives: To investigate the relationship between pre‐treatment levels of antihepatitis C virus (HCV) immunoglobulin M (IgM) antibodies and the outcome of interferon therapy, and also the relationship with genotypes and quantitative viraemia. Patients: One hundred and four patients with biopsy‐proven chronic hepatitis C without cirrhosis, consecutively enrolled in three general hospitals in Turin, Italy, and treated according to the same interferon schedule (3 MU of recombinant &agr;‐2b interferon three times a week for 6 months). Anti‐HCV IgM were measured by a secondgeneration enzyme‐linked immunoassay and results expressed as sample‐cutoff ratio. In 30 patients, determination of viraemia by branched DNA (bDNA) and genotyping were performed and the correlation with anti‐HCV IgM ratios was assessed. Results: According to univariate analysis, anti‐HCV IgM ratios, age, serum gammaglutamyltranspeptidase (&ggr;‐GT) and ferritin levels were significantly associated with sustained response to therapy. A log‐linear model, testing the effect of these variables on response to therapy, showed that anti‐HCV IgM ratio was the only independently associated variable (P= 0.00057). Anti‐HCV IgM were associated with viraemia levels (r=0.57), but not with genotype distribution. Patients with anti‐HCV IgM ratio less than 1 were sustained responders to the ‘standard therapy’ in 65% of cases. By contrast, among patients with a ratio greater than 3, sustained response was achieved in only one patient (3%), while 73% were non‐responders; the majority of relapsers were found among patients with a ratio between 1 and 3. Conclusion: Anti‐HCV IgM antibodies provide an easily accessible and cheap serological marker of active viral replication, and are significantly related to the outcome of interferon therapy in patients with chronic hepatitis C.

Serum levels of anti-hepatitis C virus IgM core antibodies may predict the response to interferon-α therapy in chronic hepatitis C

SUMMARY. We measured the optical densities (OD) of serum anti‐hepatitis C virus IgM core antibodies in 40 HCV‐positive patients (24 males and 16 females) with histologically proven chronic active hepatitis but without cirrhosis. All patients were treated with i.m. injections of 3 MU thrice weekly of interferon‐α (IFN‐α) for 6 months and followed‐up monthly. Optical densities were evaluated in thawed sera before beginning treatment and 6 months after completion, and in fresh sera obtained at the end of an 8–12‐month follow‐up period. Patients were grouped into three categories according to the OD obtained: <0.3 (negative test): 0.3‐0.6 (intermediate positivity): >0.6 (high positivity). According to the response to treatment during the follow‐up period, patients were further divided into three classes: sustained responders: relapsers or partial responders: non‐responders. In each patient, the OD values were similar in the three determinations before, after therapy and at the end of the follow‐up period. All patients with an intermediate positive test for anti‐HCV IgM core antibodies were relapsers or partial responders, and all patients with high OD values were non‐responders. Conversely, 71% of the patients with a negative test were sustained responders. We conclude that this cheap and easily performed test may be useful in predicting the response to IFN therapy.

Anti-hcv antibodies in patients with chronic liver disease and different amounts of alcohol intake: a multivariate analysis

Objective To investigate the association between the mean daily alcohol intake and positivity for serum anti-hepatitis C virus antibodies in patients with chronic liver disease of varying severity. To test the hypothesis that alcohol intake and hepatitis C virus infection have an independent role in determining chronic liver disease. Design Retrospective study using a multivariate analysis model. Methods In 212 consecutive patients with chronic liver disease, serum anti-hepatitis C virus antibodies were detected using a second-generation test and recombinant immunoblotting assay. The lifetime mean daily alcohol intake was measured by a standardized questionnaire. Patients were subsequently divided according to the histological presence or absence of liver cirrhosis and stratified into progressive categories of alcohol intake. The dose-effect relationship between anti-hepatitis C virus status and mean daily alcohol intake was assessed by a model of unconditional logistic regression, where age, gender, degree of severity of liver disease and chronic hepatitis B virus infection were considered as covariates. Results There was a strong negative association between the mean daily alcohol intake and the presence of anti-hepatitis C virus antibodies, with odds ratio decreasing down to 0.01 for the category of highest alcohol intake; no association was found for the other variables considered. Conclusion Our data support the hypothesis that alcohol intake and infection with hepatitis viruses are independent determinants of chronic liver disease.

Adjuvant iodine‐131‐labeled lipiodol for prevention of intrahepatic recurrence of hepatocellular carcinoma: Which is the best treatment schedule?

We read with interest the paper by Boucher et al.1 on the improvement of disease-free survival (DFS) rate in patients with cirrhosis treated with adjuvant intra-arterial injection of iodine-131-labeled lipiodol (131-I-Lip) after resection for hepatocellular carcinoma (HCC). Since January 2002, we have treated with this adjuvant treatment 10 consecutive male patients with hepatitis C virus–related cirrhosis after radical liver resection for primary HCC. In contrast to other centers, according to the Italian radioprotection regulations, our protocol involved injection of a single dose of 1100 MBq, which allowed us to discharge patients after 5 days, when their radioactivity fell below 600 MBq. 131-I-Lip was injected into the hepatic artery within the sixth postoperative month. Patients’ ages were 67 8 years, 30% of them had a major liver resection, tumor size was 5.4 1.8 cm, and microscopic vascular invasion was present in 50% and capsular invasion in 60%. The DFS rate of our 10 patients was compared with that of 20 patients who underwent liver resection before 2002 and were matched for sex, age, tumor size, and presence of vascular or capsular invasion. The KaplanMeier DFS rate was plotted and compared using the log-rank test. Figure 1 shows that our DFS rate is similar to that reported by Boucher et al., confirming the efficacy of this adjuvant therapy. When our experience is compared with other published data, we believe there are some unclear aspects which should be further investigated; the timing and dose of the injected radioactivity are still empirical and probably not yet optimal. In the pilot study by Lau et al.,2 1,850 MBq were injected within 6 weeks; in the study by Boucher et al., 2,400 MBq were injected within 3 months, and a third study by Partensky et al.,3 1,100 MBq were injected within 6 months and then repeated after 2 years in disease-free patients. Despite these differences in study protocols, it seems that the results of DFS are similar, suggesting the existence of a variable that has not been taken into account. It is conceivable this similarity may be due to the target volume of the remnant liver. It is likely the volume differed greatly between the various series of patients. Lau et al. report that 50% of patients underwent a major liver resection; such was the case in only 21% of the patients treated by Boucher et al., who used the highest injected activity, and conversely 75% of major resections were performed by Partensky et al., who used the lowest injected radioactivity. The delivered radiation dose (Gy) is known to be directly associated with the administered activity and inversely related to the volume of the target organ. Boucher et al. suggest that 2,400 MBq injected into the liver correspond to an estimated radiation dose of 5,000 cGy, but it is possible that the tumoricidal dose may be lower than this value. Thus, we suggest that the measure of the volume of the remnant liver targeted by the I-131-Lip should be included as a variable for results, tailoring the optimal dose for such an adjuvant therapy.