Hepatocyte proliferation in primary biliary cirrhosis as assessed by proliferating cell nuclear antigen and Ki-67 antigen labelling.

Expression of the proliferating cell nuclear antigen and Ki-67 antigen by hepatocytes was investigated in liver tissue specimens of 29 patients with primary biliary cirrhosis (stage I 13, stage II 6, stage III 5 and stage IV 5 patients) prior to treatment with ursodeoxycholic acid and of five control subjects using immunocytochemical methods. Proliferating cell nuclear antigen and Ki-67 expression were reevaluated in seven patients after 3 years of treatment with ursodeoxycholic acid. Proliferating cell nuclear antigen labelling indices were significantly higher in primary biliary cirrhosis (stage I, 6.4% to 32.4%, median, 10.9%; stage II, 9.6% to 21.6%, median 11.4%; stage III, 5.2% to 12.5%, median, 7.6%; stage IV, 3.8% to 8.9%, median, 5.6%) than in controls (0% to 0.5%, median, 0.1%; p < 0.005). Ki-67 antigen labelling counts were lower than proliferating cell nuclear antigen indices but elevated in all stages of primary biliary cirrhosis (stage I, 0.5% to 3.5%, median 2.0%; stage II, 1.8% to 3.6%, median 2.6%; stage III, 1.3% to 2.5%, median 1.9%; stage IV, 0.4% to 1.7%, median 1.0%) compared with controls (0% to 0.5%, median 0.3%; p < 0.005). After ursodeoxycholic acid treatment, mean proliferating cell nuclear antigen and Ki-67 labelling indices decreased from a median of 9.0% (range, 3.8% to 32.4%) to a median of 7.8% (range, 4.5% to 17.2%; p = 0.045) for proliferating cell nuclear antigen and from a median of 2.5% (range, 0.8% to 3.6%) to a median of 2.1% (range, 0.9% to 3.1%; p = 0.031) for Ki-67 antigen. It is concluded that hepatocyte proliferation is markedly increased in primary biliary cirrhosis, particularly in the early stages of the disease, and that ursodeoxycholic acid treatment reduces proliferative activity in primary biliary cirrhosis.

Serum levels of adhesion molecules and thrombomodulin as indicators of vascular injury in severe Plasmodium falciparum malaria.

Severe Plasmodium falciparum malaria is characterized by multiple organ involvement due to sequestration of infected erythrocytes in small vessels. Endothelial cell adhesion molecules play an important role in this interaction. During the course of a severe cerebral P. falciparum malaria infection we found very markedly elevated levels of the soluble adhesion molecules intercellular adhesion molecule-1, E-selectin, and vascular cell adhesion molecule-1, with a maximum increase of nine, seven, and eight times, respectively. These very high levels of soluble adhesion molecules point to an endothelial cell injury as an additional cause to physiological release or shedding due to receptor interactions. Soluble thrombomodulin (sTM) levels showed an extremely marked elevation up to 332 ng/ml (up to 13 times the normal value) as well. Malaria patients without severe organ involvement/cerebral manifestation showed only a mild elevation of sTM levels. TM is a parameter independent of the immunological system. It is regarded as a marker of vasculitis and endothelial cell destruction. Therefore, markedly elevated sTM levels document a substantial endothelial cell injury in severe malarial infection and may be of diagnostic and prognostic importance.

Hepatitis C virus, alcoholic cirrhosis, and hepatocellular carcinoma.

We undertook a retrospective study to determine the prevalence of hepatitis C virus (HCV) and hepatitis B virus (HBV) infection in 81 Caucasian patients with confirmed hepatocellular carcinoma (HCC). Besides HBV and HCV serological markers, HCV RNA and HBV DNA were detected in serum and liver tissue by polymerase chain reaction. Overall, HCV RNA was found in 20 cases (25%), HBV DNA in 21 patients (26%), and coinfection in 3 patients (3%). HCV RNA in liver tissue was not found without virus in serum, whereas HBV DNA was found in the liver tissue of one patient without viremia. In an additional analysis, 32 patients with HCC and alcoholic cirrhosis (HCC-AC) were compared to 35 cases with AC without HCC and 35 cases with alcoholic hepatitis. The prevalence of HCV RNA in HCC-AC (19%) was significantly higher than in the other groups (AC, 3%; alcoholic hepatitis, 0%). HBV DNA was present in 19% of HCC-AC as compared to 3% of AC and 0% of alcoholic hepatitis. We conclude that the form of HCC in 50% of the patients in a Western European country is related to chronic viral hepatitis. Our data obtained from a group of patients having alcoholic liver disease with or without HCC suggest that the prevalence of HCV RNA or HBV DNA in these populations increases with the severity of hepatic injury.

Plasma kinetics and biliary excretion of colchicine in patients with chronic liver disease after oral administration of a single dose and after long-term treatment.

Plasma kinetics and biliary excretion of colchicine in patients with chronic liver disease were evaluated after oral administration of a single dose and after long-term treatment. A single oral dose of 1 mg colchicine led to a mean peak concentration of 3.60 +/- 1.04 ng/ml at a peak time of 2.16 +/- 0.34 h and a mean area under the plasma concentration time curve, extrapolated from time 0 to infinity, of 24.90 +/- 8.47 ng.h/ml. Comparable values were obtained after repeated administration. Distribution half-life was 2.83 +/- 0.74 h, and terminal plasma half-life was 9.81 +/- 2.08 h; the mean apparent volume of distribution and the mean apparent plasma clearance were 1448 +/- 4061 and 175.3 +/- 47.6 1/h, respectively. Colchicine concentrations in bile (2025 +/- 1368 ng/ml) were clearly higher than in plasma. Long-term treatment with colchicine (1 mg/day) in patients with various stages of primary biliary cirrhosis (PBC) was associated with colchicine concentrations varying from < 0.15 to 2.0 ng/ml, with a slight tendency to higher concentrations in PBC stages III-IV than I-II. Although about 20% of colchicine is excreted in bile within 24 h, accumulation of colchicine may appear only in patients with advanced liver disease and cholestasis.

Prevalence and serological manifestation of hepatitis C virus infection in patients with hepatitis non-A, non-B: a follow-up study.

OBJECTIVES: Sera of patients with acute hepatitis non-A, non-B prospectively followed for a mean of 11.4 years (range 9 to 15 years) were assayed for anti-hepatitis C virus (HCV) by first- and second-generation enzyme-linked immunosorbent assay (ELISA) and second-generation recombinant immunoblot assay (RIBA) to study the patterns of antibody response in relation to the outcome of disease. METHODS: From 1974 through 1981, 112 patients with acute hepatitis non-A, non-B were enrolled in a prospective study. Sera of 45 patients taken during the acute phase of hepatitis, as well as taken during follow-up in 1983 and 1990 were still available for evaluation. Sera were assayed by first- (ELISA-1) and second-generation (ELISA-2) anti-HCV ELISA, second-generation RIBA (RIBA-2) and HCV RNA by RT-polymerase chain reaction (PCR). RESULTS: Based on anti-HCV seropositivity by RIBA-2 in acute hepatitis and/or during follow-up, a total of 22/45 patients with HNANB (48.8%) were considered to have confirmed HCV infection. By ELISA-1, 11/22-patients with HCV infection (50%) were positive for anti-HCV within 6 weeks of the onset of illness, 18/22 sera (82%) were reactive by ELISA-2. Confirmation by RIBA-2 was obtained in 12 (55%) cases, while one additional patient was RIBA-2 indeterminate. In 1983, a disappearance of anti-HCV tested by RIBA-2 was observed in 7/12 resolved patients but in none of the patients with chronic hepatitis (p = 0.0018) while loss of anti-HCV was observed in 2/12 cases with resolved hepatitis and none with chronic hepatitis by ELISA (not significant). In 1990, all patients with chronic hepatitis were still positive by either ELISA or RIBA-2 but 9/12 (75%), 6/12 (50%) and 10/11 (91%) patients with resolved hepatitis had lost anti-HCV seropositivity tested by ELISA-1 (p = 0.0004), ELISA-2 (p = 0.0124) or RIBA-2 (p < 0.0001), respectively. Mostly, RIBA-2 reactivity was lost prior to ELISA-2 reactivity during follow-up. Rates of antibody loss as detected by ELISA-1, ELISA-2 and RIBA-2 were 4.1, 2.9 and 5.8 per 100 person years, respectively. CONCLUSIONS: This study shows that the continuing presence of HCV is necessary to maintain anti-HCV seropositivity. In contrast, anti-HCV reactivity is progressively lost over time in resolved patients. In addition, albeit more specific, the RIBA-2 is less sensitive than the ELISA and RIBA seroconversion to negative seems to be the earliest serological marker of a resolved HCV infection.

In vitro experimental infection of primary human hepatocytes with hepatitis B virus.

BACKGROUND/AIMS: Studies on the interaction of hepatitis B virus (HBV) with its host cell require a suitable tissue culture system. This study used primary adult hepatocytes from healthy human liver tissue to establish productive infection in vitro. METHODS: Hepatocytes were inoculated overnight with HBV. Production of viral proteins was assessed by radioimmunoassay and by [35S]methionine labeling, and production of viral DNA was assessed by Southern blotting and endogenous polymerase assay. RESULTS: Secretion of high levels of hepatitis B surface antigen (HBsAg) and low levels of hepatitis B virus e antigen (HBeAg) into the medium was detectable 6 days after infection and reached maximum values after 12 days. Metabolic labeling showed production of viral proteins to be a result of de novo synthesis. The appearance of single-stranded HBV DNA in the cytoplasm of infected cells, typically present in immature cores, indicated viral replication. HBV DNA containing particles possessing an active viral DNA polymerase could be immunoprecipitated from the medium 12 days after infection. An antiserum specific for the preS1 region of the viral envelope was capable to block infection. Presence of dimethyl sulfoxide in the medium greatly improved the yield of viral proteins. CONCLUSIONS: Primary adult human liver cells are competent for infection with HBV.

The humoral immune response to p53 in patients with hepatocellular carcinoma is specific for malignancy and independent of the alpha-fetoprotein status.

Recently, p53 gene aberrations have been recognized as a relevant factor in hepatocarcinogenesis, in tumors from both high-risk and low-risk areas. Because p53 gene mutations typically result in increased p53 levels in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, we have analyzed sera from 80 European patients with hepatocellular carcinoma for the presence of p53 antibodies. For this purpose we developed an immunoblot assay using recombinant p53 as antigen. Sixty-seven sera from patients with different acute and chronic liver diseases were used as controls. In addition, serum alpha-fetoprotein assays were performed. Circulating antibodies against p53 were found in 25% (20 of 80) of the sera from patients with hepatocellular carcinoma but not in various nonmalignant liver diseases. The association of p53 antibodies with malignancy was highly significant (p < 0.00003). In 73.8% (59 of 80) of the hepatocellular carcinoma sera the alpha-fetoprotein levels were elevated. Among the 21 alpha-fetoprotein-negative hepatocellular carcinoma sera, 5 were found to contain p53 antibodies (23.8%). In conclusion, an antibody response against p53 developed in a significant proportion of patients with hepatocellular carcinoma but not in those with nonmalignant liver diseases. Serological testing for p53 antibodies gives the opportunity to identify a subgroup of patients with hepatocellular carcinoma not detected by conventional tests for serum alpha-fetoprotein.

Visceral leishmaniasis (kala-azar) after a visit to the Mediterranean region.

The case of a 17-year-old patient is presented who became ill 10 months after a holiday visit to Malta. Symptoms included fever peaking daily at 40 degrees C, pancytopenia, and splenomegaly. There was no evidence of bacterial or virological involvement, and probatory treatment with antibiotics followed by corticosteroids was without success. Examination of bone marrow led to the diagnosis of visceral leishmaniasis (kala-azar). A therapy with pentavalent antimony brought rapid improvement in clinical symptoms and led to complete recovery. A short review is presented of the epidemiology, diagnosis, and therapy of visceral leishmaniasis. The aim of this presentation is to remind the attendant physician of the clinical symptoms involved with the possible case of visceral leishmaniasis.

[Antineutrophil cytoplasmic antibodies in chronic inflammatory bowel diseases]. / Antineutrophile zytoplasmatische Antikörper bei chronisch-entzündlichen Darmerkrankungen.

Serum samples from 83 patients (42 women, 41 men, mean age 41 [19-85] years) with chronic inflammatory bowel diseases (ulcerative colitis: n = 41, Crohn's disease: n = 42) of differing degrees of activity were tested for antineutrophil cytoplasmic antibodies (ANCA) by immunofluorescence microscopy and various ELISA techniques. Seven patients with ulcerative colitis and one with Crohn's disease were suffering from associated primary sclerosing cholangitis. ANCA were detected in 18 sera, 13 from patients with ulcerative colitis (31.7%) and five from patients with Crohn's disease (11.9%). Six of the eight patients with primary sclerosing cholangitis were ANCA-positive. Nine sera showed a cytoplasmic (c-ANCA-) pattern and 9 others showed a partially atypical perinuclear (p-ANCA-) pattern. Among the ANCA-positive sera, ELISA techniques showed that two had antibodies against serine proteinase 3, two against lactoferrin, two against elastase and one against myeloperoxidase. There was no correlation between the anatomical pattern or activity of the disease and the presence of ANCA. The antineutrophil cytoplasm antibodies demonstrable in chronic inflammatory bowel disease appear to be directed against so far unknown antigens. They are particularly frequent in patients with associated primary sclerosing cholangitis.

Liver transplantation for acute or chronic liver failure by hepatitis non-A, non-B and hepatitis C viral infections: a postoperative follow-up.

Orthotopic liver transplantation (OLT) was performed for liver failure related to hepatitis non-A, non-B (HNANB) or hepatitis C (HCV) infections in 12 patients. Of those, 8 patients had chronic and 4 acute hepatic failure. To determine the incidence of recurrent infection, the clinical course, histological findings and serological HCV markers (HCV-RNA and detection of anti HCV antibodies, respectively) were comparatively studied in these patients. Recurrent infection was apparent in 5 of 6 patients transplanted for liver cirrhosis attributable to chronic HCV infection and with HCV-RNA detectable in serum. The clinical course of infection after OLT varied considerably. Chronic active hepatitis, progressing to liver cirrhosis 13 months postoperatively and an acute hepatitis, resolving spontaneously were seen in one case each. Recurrent infection led to chronic persistent hepatitis in the remainder. None of the patients with acute liver failure experienced recurrent infection. HCV-RNA was detectable in all the patients after OLT, with HCV-RNA present pretransplant, however the presence of HCV-RNA in serum was not necessarily associated with clinical illness.

[Combined ursodeoxycholic acid plus colchicine--treatment of primary biliary cirrhosis: results of a placebo-controlled double-blind study]. / Kombinierte Ursodeoxycholsäure plus Colchizin--Behandlung bei primär biliärer Zirrhose: Ergebnisse einer Placebo-kontrollierten Doppelblindstudie.

In the present pilot study we investigated the effects of urso treatment alone in comparison to a combined treatment with urso plus colchicine in PBC. 22 patients with PBC in the histological stages 1-3 entered the study. All patients were pretreated with urso alone (10-12 mg/kg) for 12 months. Thereafter treatment was continued in a double-blind randomized fashion with urso plus placebo or urso plus colchicine (1 mg/day) for another 12 months. During the initial 12 months urso treatment liver function tests improved significantly in all patients, pruritus improved in 60% of patients. After randomization to the different treatment groups the biochemical parameters stabilized at the lower level and no significant differences could be found between urso plus placebo and urso plus colchicine treatment concerning aminotransferases, alkaline phosphatase, bilirubin, cholinesterase, albumin or cholesterol. The results of this pilot study suggest that the addition of colchicine to an initial urso treatment does not lead to further improvement of aminotransferases, alkaline phosphatase, bilirubin or clinical symptoms like pruritus.

Immunogenicity, reactogenicity and consistency of a new, inactivated hepatitis A vaccine--a randomized multicentre study with three consecutive vaccine lots.

We investigated immunogenicity, reactogenicity and consistency of three consecutive lots of an inactivated hepatitis A vaccine in 204 seronegative volunteers. Each volunteer received a total of three doses of vaccine (720 EIU) according to a 0, 1 month primary vaccination schedule with a booster dose given at month 6. Mild, moderate and mostly local side effects were reported in 49.7% after the first and in less than 30% after the third injection. Seroconversion rate after one vaccine dose was as high as 91%. All subjects but one had already seroconverted by 1 month after the second injection, corresponding to a seroconversion rate of 99%. The geometric mean titres (GMT) increased with each dose of vaccine administered. Our results show that this inactivated hepatitis A vaccine is highly immunogenic, safe and well tolerated. Furthermore, there were no significant differences between the three vaccine lots in respect to seroconversion rate, size of antibody titre or reactogenicity.

Follow-up of patients with hepatitis non-A, non-B: incidence and persistence of anti-HCV depend on route of transmission.

Of 32 patients with non-A, non-B hepatitis, 10 (31%) were still anti-HCV-positive 12.8 years after the acute phase of the disease. Seven of the patients (21.9%) still had elevated ALT levels, and among these, 5 out of 5 patients who had been subject to parenteral risk were anti-HCV-positive. In contrast, none of the patients who had not been subject to parenteral risks were positive.