MicroRNAs in liver cancer: a model for investigating pathogenesis and novel therapeutic approaches.

MicroRNAs (miRNAs) constitute a large class of short RNAs (e.g., 20-24 nucleotides in length), whose main function is to posttranscriptionally regulate the expression of protein-coding genes. Their importance in tumorigenesis has been demonstrated over the past decade, and correspondingly, they have emerged as potential therapeutic molecules and targets. Liver cancer is one of the most common neoplastic diseases worldwide, and it currently has a poor prognosis owing to largely ineffective therapeutic options. Liver cancer is also an excellent model for testing miRNA-based therapy approaches as it can be easily targeted with the systemic delivery of oligonucleotides. In recent years, the role of miRNAs in hepatocellular carcinoma (HCC) has been established with molecular studies and the development of animal models. These studies have also provided the basis for evaluating the therapeutic potential of miRNAs, or anti-miRNAs. In general, the safety of miRNAs has been proven and antitumor activity has been observed. Moreover, because of the absence or presence of mild side effects, the prophylactic use of miRNA-based approaches may be foreseen.

MicroRNA and hepatocellular carcinoma: biology and prognostic significance.

Hepatocellular carcinoma (HCC) is the most common primary liver cancer and the third most common cause of cancer-related death worldwide. In 90% of cases, HCC arises on a background of cirrhosis which, in turns, results from hepatitis (HBV and HCV) infections, alcohol abuse, metabolic disorders including NASH, and genetic metabolic diseases, autoimmune hepatitis, primary biliary cirrhosis and exposure to environmental carcinogens. The molecular mechanisms underlying HCC development are still only partially known. Despite a high molecular variability, the deregulation of definite oncogenic pathways has been confirmed as a common finding in HCC. Among these, the molecular ways controlling proliferation, apoptosis and migration play a major role. In recent years, a new class of regulatory RNAs, the microRNAs, has been discovered and their deregulated expression has been linked to the molecular pathogenesis of many cancers because of their ability to strongly impact on the expression of crucial messenger RNAs. This review focuses on some of the most relevant evidence concerning the contribution of microRNA aberrant expression to HCC development.

MiR-221 controls CDKN1C/p57 and CDKN1B/p27 expression in human hepatocellular carcinoma.

The identification of target mRNAs is a key step for assessing the role of aberrantly expressed microRNAs in human cancer. MiR-221 is upregulated in human hepatocellular carcinoma (HCC) as well as in other malignancies. One proven target of miR-221 is CDKN1B/p27, whose downregulation affects HCC prognosis. Here, we proved that the cyclin-dependent kinase inhibitor (CDKI) CDKN1C/p57 is also a direct target of miR-221. Indeed, downregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to miR-221 transfection into HCC-derived cells and a significant upregulation of both CDKN1B/p27 and CDKN1C/p57 occurs in response to antimiR-221 transfection. A direct interaction of miR-221 with a target site on the 3' UTR of CDKN1C/p57 mRNA was also demonstrated. By controlling these two CDKIs, upregulation of miR-221 can promote growth of HCC cells by increasing the number of cells in S-phase. To assess the relevance of these studies in primary tumors, matched HCC and cirrhosis samples were assayed for miR-221, for CDKN1B/p27 and CDKN1C/p57 expression. MiR-221 was upregulated in 71% of HCCs, whereas CDKN1B/p27 and CDKN1C/p57 proteins were downregulated in 77% of cases. A significant inverse correlation between miR-221 and both CDKN1B/p27 and CDKN1C/p57 was found in HCCs. In conclusion, we suggest that miR-221 has an oncogenic function in hepatocarcinogenesis by targeting CDKN1B/p27 and CDKN1C/p57, hence promoting proliferation by controlling cell-cycle inhibitors. These findings establish a basis toward the development of therapeutic strategies aimed at blocking miR-221 in HCC.

Different haemodynamic effects of a single dose of long-acting isosorbide-5-mononitrate in healthy subjects and patients with cirrhotic portal hypertension.

BACKGROUND: The action pathways of nitrates are hypothesised to be deranged in cirrhosis. AIM: In order to confirm it, the acute haemodynamic effects of isosorbide-5-mononitrate in cirrhotic patients and controls was investigated. PATIENT: Nine cirrhotics and nine healthy controls. METHODS: Evaluation in the fasting state, 90 min after isosorbide-5-mononitrate or placebo (double-blind on two different days) and then 30 and 120 min after eating a standard meal. Various systemic and splanchnic haemodynamic parameters, including arterial impedance, assessed as Doppler pulsatility index, were measured. RESULTS: isosorbide-5-mononitrate reduced arterial pressure and increased heart rate and mesenteric pulsatility index both in controls and in cirrhotics, whereas the following parameters behaved differently in the two groups (P < 0.05): hepatic pulsatility index decreased (-9%) and the portal velocity increased (+13%) in controls, whereas hepatic pulsatility increased (+18%) and portal velocity decreased (-18%) in cirrhotics. The two groups presented a similar pattern of changes in most variables under placebo after a meal. In controls, the administration of isosorbide-5-mononitrate blunted the postprandial mesenteric vasodilation and related changes in splanchnic and systemic circulation, expected at 30 min, in comparison to those observed under placebo. In cirrhotics, instead, the postprandial pattern was similar under placebo and isosorbide-5-mononitrate. CONCLUSIONS: The acute administration of isosorbide-5-mononitrate produces different haemodynamic effects in healthy and diseased livers, both in the fasting state and after a meal, consistent with the hypothesis of a deranged response of the intrahepatic microcirculation to nitrates in cirrhosis.

Value of splanchnic Doppler ultrasound in the diagnosis of portal hypertension.

The accuracy of various Doppler parameters of portal circulation in the diagnosis of relevant portal hypertension (presence of gastroesophageal varices) was prospectively validated. The following parameters were compared in 51 patients with chronic liver disease (40 with cirrhosis and 11 with chronic hepatitis): portal vein flow velocity and congestion index, hepatic and splenic arteries resistance indexes (RI), modified liver vascular index (portal flow velocity/hepatic artery RI) and portal hypertension index, a new index calculated as: [(hepatic artery RI x 0.69) x (splenic artery RI x 0.87)]/portal vein flow velocity. Highest accuracy was achieved by the splenic artery RI and the portal hypertension index (both around 75%) at cut-offs, respectively, of 0.60 and 12 cm/s(-1), which appeared to be, therefore, the most favorable parameters for the clinical practice. Their use may limit the need for endoscopy to search for varices.

Influence of liver fibrosis on hepatic artery Doppler resistance index in chronic hepatitis of viral origin.

BACKGROUND: Hepatic arterial Doppler sonography is increasingly being used in liver diagnostics. The determinants of the elevation of hepatic artery impedance indexes in chronic liver disease, however, have still not been fully clarified. The aim of the present study was to investigate the relationship between histological alterations and liver circulation in chronic hepatitis. METHODS: Hepatic artery resistance index and portal flow velocity were measured using Doppler sonography in 47 patients with chronic hepatitis of viral origin diagnosed at histopathology. The patients were divided into two groups, those with mild and those with severe alterations, in accordance with the various histological parameters of the Knodell scoring system. RESULTS: Hepatic artery resistance index and age were higher in patients with more severe liver fibrosis (respectively 0.638 +/- 0.084 and 39.0 +/- 10.9 (years) in mild fibrosis versus 0.687 +/- 0.060 and 49.4 +/- 14.4 (years) in severe fibrosis; P < 0.05 for both), whereas no difference between the two groups was found for the other histological features (degeneration, inflammation and necrosis), nor for portal flow velocity. CONCLUSIONS: The increase in hepatic artery resistance index appears to be influenced by the extent of fibrous tissue deposition in the liver, determined by chronic inflammation and repair and, secondly, by ageing.

Serum xanthine oxidase in human liver disease.

OBJECTIVES: High concentrations of serum xanthine oxidase (XO) have been reported during human liver disease and hepatocyte injury in experimental settings. However, it is unclear whether this elevation reflects hepatocyte necrosis or has a different meaning. METHODS: The serum level of XO in 64 patients with chronic liver disease (17 patients with cirrhosis, 30 with chronic hepatitis, and 17 with cholestatic disorders) and in 12 control subjects was determined by a competitive ELISA. Conventional serum markers of liver damage were assessed in all patients, and grading and staging were scored in the chronic hepatitis group according to Knodell. RESULTS: The XO serum levels were significantly higher in the patients than in the controls. The differences were also significant when controls were compared to patients with chronic hepatitis and cholestatic disorders separately, but not when compared to the cirrhosis group. Patients with cholestatic disorders had XO values higher than those of patients with cirrhosis or chronic hepatitis. XO levels did not correlate with stage and grade in chronic hepatitis group. We found a weak but significant positive correlation in patients between XO serum level and gamma-glutamyl transpeptidase (r = 0.37). This correlation was stronger when chronic hepatitis (r = 0.42) and, especially cholestatic disorders (r = 0.71), were separately tested, but was absent in the cirrhosis group. The XO values positively correlated with alkaline phosphatase in patients with cholestatic disorders. A level of serum XO >32 microg/ml specifically identified cholestatic disorders in our study population. CONCLUSIONS: A marked elevation of serum XO in patients with chronic liver disease seems to reflect the presence of cholestasis. No correlation between XO levels and histological or serum evidence of hepatocyte necrosis was found in these patients.

Surveillance programme of cirrhotic patients for early diagnosis and treatment of hepatocellular carcinoma: a cost effectiveness analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of death in cirrhotic patients. This neoplasm is associated with liver cirrhosis (LC) in more than 90% of cases. Early diagnosis and treatment of HCC are expected to improve survival of patients. AIMS: To assess the cost effectiveness of a surveillance programme of patients with LC for the early diagnosis and treatment of HCC. PATIENTS: A cohort of 313 Italian patients with LC were enrolled in the surveillance programme between March 1989 and November 1991. In the same period, 104 consecutive patients with incidentally detected HCC were referred to our centre and served as a control group. METHODS: Surveillance was based on ultrasonography (US) and alpha fetoprotein (AFP) determinations repeated at six month intervals. Risk factors for HCC were assessed by multivariate analysis (Cox model). Outcome measures analysed were: (1) number and size of tumours; (2) eligibility for treatment; and (3) survival of patients. Economic issues were: (1) overall cost of surveillance programme; (2) cost per treatable HCC; and (3) cost per year of life saved (if any). Costs were assessed according to charges for procedures at our university hospital. RESULTS: Surveillance lasted a mean of 56 (31) months (range 6-100). During the follow up, 61 patients (19.5%) developed HCC (unifocal at US in 49 cases), with an incidence of 4.1% per year of follow up. AFP, Child-Pugh classes B and C, and male sex were detected as independent risk factors for developing HCC. Only 42 (68.9%) of 61 liver tumours were treated by surgical resection, orthotopic liver transplantation, or local therapy. The cumulative survival rate of the 61 patients with liver tumours detected in the surveillance programme was significantly longer than that of controls (p=0.02) and multivariate analysis showed an association between surveillance and survival. The overall cost of the surveillance programme was US$753 226, the cost per treatable HCC was US$17 934, and the cost for year of life saved was US$112 993. CONCLUSION: Our surveillance policy of patients with LC requires a large number of resources and offers little benefit in terms of patient survival. The decision whether to adopt a surveillance policy towards HCC should rely on the prevalence of the disease in the population and on the resources of a particular country.

Left ventricular volumes in liver cirrhosis.

BACKGROUND: Patients with alcoholic cirrhosis have left ventricular dimensions similar to controls. Few data have been reported in patients with cirrhosis of viral origin. AIM: To assess left ventricular dimensions in patients with pure viral cirrhosis. PATIENTS AND METHODS: Thirty patients with virus-related cirrhosis, 23 patients with alcoholic cirrhosis and 12 healthy controls were submitted to measurement of left ventricular volumes, cardiac output, mean arterial pressure and total peripheral resistance. RESULTS: Patients with cirrhosis showed a similar increase in cardiac index and heart rate and reduction of mean arterial pressure and peripheral vascular resistance in comparison to controls, irrespective of the aetiology. Left ventricular end systolic volume index was lower (p<0.01) and ejection fraction higher (p<0.01) in virus-related cirrhotic patients [mean +/- SD, respectively 12.4+/-4.1 ml/sqm and 77.9%) in comparison both to controls (21.5+/-6.3 ml/sqm and 66.8%) and alcoholics (20.6+/-7.0 ml/sqm and 68.8%). End diastolic volume index was not significantly different between the three groups. CONCLUSIONS: Our findings indicate smaller left ventricular volumes and higher ejection fraction in pure virus-related cirrhosis than in alcoholic cirrhosis and controls. Since peripheral haemodynamics proved similar in virus- and alcohol-related cirrhosis, a subclinical alcohol cardiomyopathy may be hypothesised to account for the absence of such left ventricular pattern in alcoholic patients.

Laboratory signs of acute or recent cytomegalovirus infection are common in cirrhosis of the liver.

Human cytomegalovirus (CMV) is an ubiquitous pathogen that can cause severe and often fatal infections in immunocompromised patients. Patients with cirrhosis often show various degrees of impaired cellular immunity that could lead to acute CMV reactivation. The aim of the present study was to determine whether laboratory findings of active CMV infections are common in patients with cirrhosis. Fifty-five patients with cirrhosis were studied for acute CMV infection by virological (antigenemia and quantitative polymerase chain reaction in polymorphonuclear leukocytes) and serological (detection of anti-CMV IgM by immunoblot) methods. The same tests were carried out on 50 blood donors and on 20 chronic hepatitis patients, considered as control populations. Acute or recent CMV infection had occurred in 31 (56%) of 55 patients with cirrhosis, whereas only 1 out of 20 (5%) patients with chronic non-cirrhotic liver disease and none of the 50 blood donors had laboratory signs of active CMV infection. The difference between patients with cirrhosis and the control groups was significant (P < 0.001, chi(2) test). CMV in patients with cirrhosis was not related to age, gender, hepatitis C virus infection or hepatocellular carcinoma. There was no significant correlation between impairment of liver function and the presence of active CMV infection. Patients with cirrhosis should be considered at risk for CMV infection, that seems to be mild and asymptomatic.

Gain of imprinting at chromosome 11p15: A pathogenetic mechanism identified in human hepatocarcinomas.

Genomic imprinting is a reversible condition that causes parental-specific silencing of maternally or paternally inherited genes. Analysis of DNA and RNA from 52 human hepatocarcinoma samples revealed abnormal imprinting of genes located at chromosome 11p15 in 51% of 37 informative samples. The most frequently detected abnormality was gain of imprinting, which led to loss of expression of genes present on the maternal chromosome. As compared with matched normal liver tissue, hepatocellular carcinomas showed extinction or significant reduction of expression of one of the alleles of the CDKN1C, SLC22A1L, and IGF2 genes. Loss of maternal-specific methylation at the KvDMR1 locus in hepatocarcinoma correlated with abnormal expression of CDKN1C and IGF2, suggesting a function for KvDMR1 as a long-range imprinting center active in adult tissues. These results point to the role of epigenetic mechanisms leading to loss of expression of imprinted genes at chromosome region 11p15 in human tumors.

Allelic imbalance on 16q in small, unifocal hepatocellular carcinoma: correlation with HBV and HCV infections and cellular proliferation rate.

In advanced hepatocellular carcinoma (HCC), allelic loss on chromosome 16q may occur. To better define the frequency of this alteration in small HCC and to more closely identify the affected region for further positional cloning of the putative tumor suppressor gene contained in this region, microsatellite polymorphism analysis was conducted on small, unifocal HCC, without signs of intrahepatic or systemic spread. We also tried to assess its possible correlation with hepatitis virus infections (HBV and HCV) and cellular proliferation rate. DNA from 35 small (<4 cm), unifocal HCC and from the corresponding nontumorous surrounding tissue was analyzed by 10 sets of microsatellite polymorphic markers. Serologic markers for hepatitis virus B and C infections were investigated in all cases. AgNOR protein quantity was assessed by image analysis on cryostatic sections stained with silver. The percentage of tumours with allelic imbalance ranged from 11.1 to 37%. The minimal involved region was assessed at 16q24.3, corresponding to the D16S413 marker, which was also the most commonly affected locus (10 of 27 informative cases, 37%). Allelic imbalance on chromosome 16q was significantly associated with HBV infection: 8 of 10 cases showed an actual or previous HBV infection in the group showing allelic imbalance, versus 6 with a previous HBV infection out of 25 in the control group (P < 0.01). No difference was found as far as HCV infection is concerned. The mean (+/-SE) AgNOR protein value in six cases showing allelic imbalance was 8.36 +/- 1.2 microm2, compared to 6.45 +/- 0.68 microm2 in 13 cases retaining both the alleles at 16q but the difference proved not statistically significant. In conclusion, in this series of small, unifocal HCC the minimal region of allelic imbalance on 16q was restricted to 16q24.3. It was found to be associated with HBV infection but not with increased cellular proliferation rate.

Diurnal changes of fibrinolysis in patients with liver cirrhosis and esophageal varices.

Variceal bleeding, whose triggering mechanisms are largely unknown, occurs with a circadian rhythmicity, with 2 peaks, one greater, in the evening, and one smaller, in the early morning. We assessed some clotting and hemodynamic parameters, possibly involved in variceal hemorrhage, over a 24-hour period, at 4-hour intervals, in 16 patients with cirrhosis and esophageal varices and in 9 controls. At each time interval, tissue plasminogen activator (tPA) and tPA inhibitor-1 (PAI-1) antigens and activities and total euglobulin fibrinolytic activity were determined and portal-vein flow velocity, volume, and congestion index were measured by duplex-Doppler. Significant circadian rhythms were searched for by least-squares and cosinor methods. tPA activity showed a circadian rhythm in cirrhosis, with a peak of 2.85 times the trough value, calculated at 18:42, and remained over 2.5-fold until shortly after 22:00. Total fibrinolytic activity showed a similar pattern, which was statistically significant also in controls. tPA and PAI antigens also showed significant circadian rhythm both in controls and cirrhotics, with higher values in the morning. Among the portal hemodynamic parameters only the congestion index showed significant rhythmic changes and only in cirrhosis, with the highest values in the late evening, but with limited diurnal excursion (+/- 5.5%). In conclusion, we showed the existence of a circadian rhythm of fibrinolysis in cirrhosis, whose temporal distribution might suggest a role of fibrinolysis in variceal hemorrhage on the basis of the comparison to the known chronorisk of variceal bleeding.

Assessment of vascular patterns of small liver mass lesions: value and limitation of the different Doppler ultrasound modalities.

OBJECTIVES: This study aimed to investigate the value and limitation of the different Doppler ultrasound modalities (spectral analysis, color, and power Doppler imaging) in the differential diagnosis of small liver tumors to identify the optimal diagnostic approach with the presently available Doppler technology. METHODS: Presence and distribution of color and power Doppler signals, Doppler peak frequency, resistive index, and systolic acceleration time were examined in 133 liver nodules (< or = 4 cm). RESULTS: Color and power Doppler did not identify specific diagnostic vascular patterns. By discriminant analysis, peak frequency (cut-off 1320 Hz) differentiates small hematocellular carcinoma (< or = 2 cm) from macroregenerative nodules and hemangiomas (accuracy 92.6%); resistive index (cut-off 0.65) differentiates malignancies from benign lesions (accuracy 83.8%); and systolic acceleration time (cut-off 105 ms) differentiates hepatocellular carcinoma from metastases (accuracy 80.9%). CONCLUSIONS: Power Doppler imaging is able to assess vascularity in the majority of small liver nodules, but the pattern distribution of tumoral vascular signals does not provide reliable differential diagnostic criteria. Using conventional Doppler technology, power Doppler should be used to detect vascular signals and spectral analysis, and subsequently to measure quantitative parameters such as high peak frequency and resistive index (which identify malignancy) and prolonged systolic acceleration time (which identifies primary from metastatic liver tumors).

Aberrant fragile histidine triad gene transcripts in primary hepatocellular carcinoma and liver cirrhosis.

To determine whether transcriptional alterations of the fragile histidine triad (FHIT) gene play a role in the development and progression of human hepatocellular carcinoma (HCC) we used reverse transcription-PCR to examine mRNA FHIT expression in 28 paired samples of HCC (24 in cirrhotic and 4 in noncirrhotic livers) and matched noncancerous tissue and in 10 normal livers. We also assessed loss of heterozygosity of the polymorphic D3S1300 microsatellite marker in the intron between exons 5 and 6 of the FHIT gene. Abnormal FHIT transcripts were detected in 13 cases (46.4%): 10 in the cancerous tissue only, 1 with the same pattern in both cancerous and matched noncancerous tissue, and 2 in the noncancerous tissue only. The four HCCs that arose in noncirrhotic liver all showed abnormal FHIT transcripts. No alterations were found in normal livers. Sequence analysis of abnormally sized transcripts revealed that they were generated by the fusion of exons 3 or 4 with exons 8 or 9. Among the cancerous specimens, one case showed only an abnormal sized transcript derived from the fusion of exons 4 and 9 in the absence of any normal-sized transcript, and another case showed deletion of a sequence comprised between nucleotides -35 and 399 resulting in an exon 4-9 fusion not respecting the exons' bounds. Loss of heterozygosity was found in two cases with abnormal FHIT transcripts and in only one case with normal transcript. Patients with aberrant FHIT transcripts showed a significantly higher relapse rate and shorter recurrence time (P = 0.001). This could be related to a primary genomic instability affecting particularly susceptible regions like FRA3B and could be associated with an increasing risk of recurrence without involving a causative role.

Systemic and splanchnic hemodynamic changes after liver transplantation for cirrhosis: a long-term prospective study.

The effect of orthotopic liver transplantation (OLT) on the systemic and splanchnic hemodynamic alterations of cirrhosis is still largely unknown. The aim of this study was to prospectively investigate the long-term changes induced by OLT on several hemodynamic parameters. In 28 patients undergoing OLT for cirrhosis, the following parameters were measured before surgery and subsequently at 6-month intervals (mean follow-up period, 17 months): cardiac index, mean arterial pressure (MAP), heart rate, total peripheral resistance (TPR), portal vein flow velocity and flow volume, spleen size, and Doppler ultrasound resistance or pulsatility indexes (RI or PI) in the: 1) interlobular renal, 2) superior mesenteric, 3) splenic, and 4) hepatic arteries. The same parameters were measured in 10 healthy controls. After OLT, cardiac index and heart rate significantly decreased (P <.01), while MAP and TPR increased (P <.001), so that any significant difference from controls disappeared. Renal RI progressively decreased, achieving a significant reduction (P <.05) to normal values at the 12th month of follow-up. Portal flow velocity and hepatic and splenic RI returned to values not significantly different from controls. Portal flow volume increased over normal values after OLT (P <.001), and SMA PI, lower than normal before OLT, did not show any statistically significant increase thereafter. Spleen size decreased significantly, but persisted to be larger than in controls. In conclusion, systemic, renal, and most, but interestingly not all, splanchnic circulatory alterations of cirrhosis are restored to normal after OLT.

Pilot study of triple antiviral therapy for chronic hepatitis C in interferon alpha non-responders.

BACKGROUND: No effective therapy exists for interferon non-responding chronic hepatitis C patients. AIMS: Pilot study evaluating the potential efficacy and safety of triple antiviral therapy in interferon-alpha non-responders. PATIENTS AND METHODS: Twenty consecutive adult patients with chronic hepatitis C who had failed to respond to a 6-month course of interferon alpha were randomly assigned to receive a combination of interferon alpha + oral ribavirin (double therapy), or the same combination + oral amantadine (triple therapy), for 6 months. RESULTS: By the end of therapy, normal alanine transaminase (biochemical response) was obtained in 2 out of 10 patients on double therapy but in 7 out of 10 on triple therapy (p < 0.05), and negative serum hepatitis C virus (HCV) RNA (virological response) occurred in 1 out of 10 patients on double therapy but in 7 out of 10 patients on triple therapy (p < 0.01). Six months after therapy, biochemical response was sustained in 1 (double therapy) and 4 patients (triple therapy), respectively, and the virological response was sustained in no patient on double therapy but in 3 patients on triple therapy. CONCLUSIONS: Triple antiviral therapy seems to be able to induce biochemical and virological responses in interferon alpha non-responders with chronic hepatitis C.

Determination of xanthine oxidase in human serum by a competitive enzyme-linked immunosorbent assay (ELISA).

Xanthine oxidase was purified from human milk and used to immunise rabbits. A competitive immunoenzymatic assay with purified enzyme and rabbit antiserum was optimised to measure xanthine oxidase in human serum, the lowest detectable amount being 0.03 pmol of enzymatic protein. Thus, the test (i) is sensitive enough to determine xanthine oxidase in human serum, being more sensitive than the spectrophotometric method, (ii) it is more convenient for clinical laboratories than other sensitive tests and (iii) it has the advantage over the enzyme activity-based assays of also detecting inactive enzyme molecules. A competitive enzyme-linked immunosorbent assay (ELISA) was used to measure the serum xanthine oxidase level in healthy donors and in patients with liver diseases, and it was found that any concentration below 1 mg/L is in the normal range.

Imbalance of IL-1 beta and IL-1 receptor antagonist mRNA in liver tissue from hepatitis C virus (HCV)-related chronic hepatitis.

Increased levels of IL-1 beta and IL-1 receptor antagonist (IL-1Ra) have been found in serum of patients with chronic liver diseases, although their expression in liver tissue has not been extensively investigated. The aim of this study was therefore to examine the relationship between IL-1 beta and IL-1Ra at tissue level in patients with HCV-related chronic active hepatitis (CAH) of varying degrees of severity. IL-1 beta and IL-1Ra mRNA expression was investigated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 31 patients with CAH of varying severity (classified as minimal/mild in 13 cases and moderate/severe activity in 18 cases) and in 12 control subjects. Quantitative evaluation of IL-1 beta and IL-1Ra corresponding bands was performed by densitometric image analysis, and expressed in arbitrary units. The 12 controls expressed a similar pattern with a mean IL-1 beta/IL-1Ra ratio of 1.03 (1.03 +/- 0.15 (mean +/- s.e.m.), median 0.92, range 0.71-1.45). Minimal/mild activity CAH showed a prevalence of IL-1Ra mRNA expression (1.14 +/- 0.64, median 0.43, range 0-8.75) when compared with controls (0.27 +/- 0.04, median 0.23, range 0.11-0.45) and with moderate/severe activity CAH (0.20 +/- 0.04, median 0.12, range 0-0.67; P = 0.01). Since IL-1 beta expression was similar in the three groups, a significantly different IL-1 beta/IL-1Ra ratio emerged between controls, patients with moderate/severe CAH (2.22 +/- 0.48, median 2.76, range 0-6.12) and those with minimal/mild activity CAH (0.62 +/- 0.15, median 0.5, range 0-1.58, P = 0.005). Patients with higher grades of fibrosis showed a higher IL-1 beta/IL-1Ra ratio (2.49 +/- 0.56, median 2.15, range 0.35-6.12) in comparison with lower grade fibrosis (1.06 +/- 0.30, median 0.59, range 0.03-4.50) and control patients (P = 0.01). These results suggest that an imbalance between IL-1 beta and IL-1Ra, at the tissue level, may contribute to the pathogenesis and the activity of chronic active hepatitis C.

Superior mesenteric artery impedance in chronic liver diseases: relationship with disease severity and portal circulation.

OBJECTIVE: The increase of splanchnic blood flow volume in liver cirrhosis is attributed to decreased arterial resistance. The aim of this study was to noninvasively investigate superior mesenteric artery impedance in patients with chronic liver diseases and to assess its relationship with portal hemodynamics and with clinical parameters. METHODS: Superior mesenteric artery (SMA) pulsatility (SMA-PI) and resistance (SMA-RI) indices and portal vein flow parameters (velocity, volume, and congestion index) were measured by duplex-Doppler ultrasound in 14 patients with chronic hepatitis, in 73 cirrhotics, in 30 liver transplant recipients, and in 31 control subjects. RESULTS: SMA-PI significantly differed among the five groups (p < 0.0001), being lower in cirrhotics (2.55+/-0.70) and transplanted patients (2.77+/-0.69) than in chronic hepatitis (3.28+/-0.57) and control subjects (3.42+/-0.92). SMA-PI was lower in ascitic cirrhosis (2.40+/-0.71) than in compensated cirrhosis (2.71+/-0.70) (p < 0.01) and in cirrhotics with large varices (2.30+/-0.67) than in those without varices (2.75+/-0.65) (p < 0.05). Moreover SMA-PI correlated with numeric Child-Pugh score (r=-0.28) and portal vein congestion index (r=-0.36). CONCLUSION: Hyperdynamic splanchnic circulation, noninvasively assessed by a decrease of mesenteric artery impedance, occurs in cirrhosis since the early stage of the disease and tends to worsen in relation to liver failure and the severity of portal hypertension. Furthermore, the persistent SMA-PI decrease in transplant recipients suggests a consistent contribution to this circulatory alteration from a patent portosystemic collateral circulation.