G. Verme

Immunofluorescence detection of new antigen-antibody system (delta/anti-delta) associated to hepatitis B virus in liver and in serum of HBsAg carriers.

A new antigen-antibody system associated with the hepatitis B virus and immunologically distinct from the HB surface, core, and e systems is reported. The new antigen, termed delta, was detected by direct immunofluorescence only in the liver cell nuclei of patients with HBsAg positive chronic liver disease. At present, the intrahepatic expression of HBcAg and delta antigen appears to be mutually exclusive. No ultrastructural aspect corresponding to the delta antigen could be identified under the electron microscope. delta antibody was found in the serum of chronic HBsAg carriers, with a higher prevalence in patients with liver damage. The nuclear fluorescence patterns of HBcAg and delta antigen were similar; it is only possible to discriminate between the two antigens by using the respective specific antisera.

A new hepatitis B virus strain in patients with severe anti-HBe positive chronic hepatitis B

In hepatitis B virus carriers who are anti-HBe positive despite ongoing viral replication (HBcAg in liver and HBV-DNA in serum) the natural course of hepatitis is severe and the response to interferon is low. We investigated whether a new hepatitis B virus (HBV) strain could be involved. A translational termination codon at the carboxyterminal end of the pre-C region responsible for the lack of HBeAg secretion was found in 18 of 19 HBV clones isolated from seven pedigreed patients with this clinical syndrome. The same findings were confirmed by direct sequencing. One of these patients underwent a liver transplant and HBV infection of the new liver resulted in high titered viremia and intrahepatic expression of HBcAg, without detectable HBeAg in serum. Another patient was superinfected by hepatitis delta virus (HDV) and developed high titres of total and IgM anti-HD. In spite of this, chronic hepatitis remained unchanged during 7 years of follow-up. These data strongly suggest that a viable precore minus mutant of hepatitis B virus is responsible for the lack of HBeAg in the serum of these patients. The HBV variant may explain the peculiar geographic distribution of anti-HBe positive hepatitis. The variations in the virus genome sequence may cause the more severe form of liver disease and modify the pathogenicity in the case of HDV superinfection.

A randomized controlled trial of interferon alfa-2b as therapy for chronic non-A, non-B hepatitis

Eighty patients with chronic non-A, non-B hepatitis completed a randomized controlled trial of the therapeutic efficacy of recombinant interferon alfa-2b. Twenty-nine received 1 million units and 26 received 3 million units of interferon subcutaneously thrice weekly for 6 months, and 25 were controls. Normalization or a significant decrease of alanine aminotransferase values was obtained in 19/29 (66%) patients treated with 1 million units, in 18/26 (69%) patients treated with 3 million units and in one control patient (4%, p less than 0.05). However, when control patients were randomized after the initial 24 weeks to receive 1 or 3 million units of interferon for 48 weeks, 12/14 (86%) patients receiving 3 million units responded to therapy versus 3/11 patients receiving 1 million units (27%, p less than 0.05). After a 1 to 6 months follow-up period post treatment, an alanine aminotransferase relapse was observed in 18/30 (60%) responders to 3 million units and in 17/22 (77%) responders to 1 million units. Cirrhotic patients responded less than patients with non-cirrhotic disease (47 vs. 78%, p less than 0.05). Only responders treated with 3 million units significantly ameliorated their histologic picture (pre-therapy Knodells index = 8.9, post-therapy = 6.0, p less than 0.05). The data confirm that treatment with interferon is of benefit in patients with chronic non-A, non-B hepatitis.

Hepatitis G virus RNA in the serum of patients with elevated gamma glutamyl transpeptidase and alkaline phosphatase: a specific liver disease

SUMMARY. We tested the sera of 67 consecutive patients for hepatitis G virus (HGV) RNA by reverse transcriptase‐polymerase chain reaction (RT‐PCR). These patients (42 males and 2 5 females, median age 35 years, range 13–64 years) had liver disease of unknown aetiology and were without markers of hepatitis (A‐E) viruses or signs of genetically determined, autoimmune, alcoholic or drug‐induced liver disease. The controls in this study were 110 patients (50 females and 60 males, median age 45 years, range 9–65 years) with chronic hepatitis B virus (HBV) infection (19 patients) or hepatitis C virus (HCV) infection (91 patients). Ten of 67 (14.9%) patients with cryptogenic disease were positive for HGV RNA by at least three separate tests; HGV RNA was also detected in one of 19 (5.3%) hepatitis B surface antigen (HBsAg) carriers and in nine of 91 (16.6%) patients with antibody to HCV. These data suggest that HGV occurs as frequently in HCV infected patients as in those with cryptogenic disease. Elevated serum gamma glutamyl transpeptidase (γ‐GT) (higher than twice the normal value) and alkaline phosphatase levels were found in eight of 10 (80%) HGV RNA positive patients and in six of 57 (10.5%) HGV RNA negative patients (P < 0.0001). Five (50%) HGV RNA positive patients had non‐specific inflammatory bile duct lesions. A statistically significant difference was observed between HGV RNA positive and negative patients with chronic HBV or HCV infections (P < 0.029). Therefore, the spectrum of liver disease associated with HGV is wide, but a characteristic lesion of the bile duct leading to elevation of cholestatic enzymes might be specific for this virus.

INHIBITION OF HEPATITIS B VIRUS REPLICATION BY VIDARABINE MONOPHOSPHATE CONJUGATED WITH LACTOSAMINATED SERUM ALBUMIN

Vidarabine (ara A) produces severe dose-dependent side-effects. To examine whether its monophosphate ester (ara-AMP) can be effective in the treatment of chronic hepatitis B when given in reduced dosage as a conjugate with lactosaminated human serum albumin (L-HSA), which selectively enters hepatocytes, five patients with chronic type B hepatitis (HBsAg/HBV-DNA positive for at least 2 years) were treated with the conjugate. The daily dose of conjugate given (35 mg/kg) contains 1.5 mg ara-AMP, whereas the usual daily dose of free ara-AMP is 5-10 mg/kg. In three patients HBV-DNA fell to undetectable levels and remained negative in two; in one of them anti-HBe developed. In the other two patients HBV-DNA decreased but was detectable during treatment--one received three cycles of therapy, and became HBV-DNA negative and anti-HBe positive 45 days after the end of treatment; the other remained HBeAg/HBV-DNA positive. No adverse effects were observed, and biochemical variables (including aminotransferases) remained unchanged or decreased with viraemia. No antibodies (IgM and IgG classes) that bound the conjugate were detected. Thus L-HSA-ara-AMP inhibits HBV replication as well as free ara-AMP but at a third to a sixth of the dose.

Autoantibodies and response to α-interferon in patients with chronic viral hepatitis

One hundred and fifteen patients with chronic type B, D and non-A, non-B hepatitis treated with recombinant alpha-interferon were tested for six different autoantibodies prior to or during therapy, and the course of treatment was compared in autoantibody-positive and -negative patients. Three out of 25 (12%) hepatitis B patients, 14 out of 30 (47%) hepatitis D patients and 19 out of 60 (32%) chronic non-A, non-B hepatitis carriers had baseline or post-therapy autoantibodies. The rate of response between patients with and without autoantibodies among B, D and non-A, non-B patients was, respectively, 67 vs. 79%, 23 vs. 25%, 70 vs. 61% (p = N.S.). No adverse reaction was observed in the 36 patients who had or developed nuclear, smooth muscle, parietal cells and thyroid autoantibodies during therapy. A patient with baseline antibodies against liver and kidney microsomes developed an icteric acute hepatitis at the fourth month of therapy, but five other patients with this reactivity responded to therapy uneventfully. The presence of autoantibodies before therapy or their induction following therapy is not a contraindication to the use of interferon in patients with chronic viral hepatitis.

Liver targeting of antiviral nucleoside analogues through the asialoglycoprotein receptor.

Summary. In order to reduce the extrahepatic side‐effects of antiviral nucleoside analogues in the treatment of chronic viral hepatitis, these drugs are conjugated with galactosyl‐terminating macromolecules. The conjugates selectively enter hepatocytes after interaction of the carrier galactose residues with the asialoglycoprotein receptor present in large amounts and high affinity only on these cells. Within hepatocytes the conjugates are delivered to lysosomes where enzymes split the bond between the carrier and the drug, allowing the latter to become concentrated in the liver. The validity of this chemotherapeutic strategy has been endorsed by a clinical study. Adenine arabinoside monophosphate (ara‐AMP), conjugated with lactosaminated human serum albumin (L‐HSA) and administered to hepatitis B virus (HBV)‐infected patients for 28 days, exerted an antiviral activity to the same extent as the free drug without producing any clinical side‐effects, including the severe neurotoxicity caused by the free drug. Preclinical studies are now underway with conjugates obtained using lactosaminated poly‐L‐lysine (Lacpoly(Lys)) as the hepatotropic carrier. These new conjugates have some advantages over those prepared with L‐HSA: they can be administered by the intramuscular route; they are obtained entirely by chemical synthesis, thus eliminating the problems involved in the use of haemoderivatives; they have a heavy drug load, which permits administration of smaller quantities of conjugate that are more easily digested in lysosomes; and they enable higher quantities of drug to be introduced into hepatocytes. The results of the experiments with two Lac‐poly(Lys) conjugates, one with ara‐AMP and one with ribavirin, are reported in this review.

‘e’ Antigen defective hepatitis B virus and course of chronic infection

We studied the relations between HBV heterogeneity and different phases of HBV infection and disease in 145 HBsAg-positive carriers followed-up for 28 months (range 24-60 months). Viraemia was characterized for the relative prevalence of wild-type and HBeAg minus HBVs after HBV-DNA amplification by PCR using an oligonucleotide hybridization assay. HBeAg minus HBV was detected in 27% of immunotolerant HBV carriers, in 67% of patients with chronic hepatitis B (immunoelimination phase) and in 17% of HBsAg carriers with latent infection. Serum HBV-DNA and IgM anti-HBc became undetectable and ALT levels normalized, either spontaneously or after interferon therapy in 12 (36.3%) of 33 patients with an exclusive wild-type viraemia, but only in two (5.7%) of 35 patients with homogeneous HBeAg minus HBV (p = 0.005). An HBeAg minus viraemia higher than 20% was associated, in both HBeAg- and anti-HBe-positive patients, with HBV-induced liver disease and an unfavourable outcome of hepatitis. These findings suggest that surgence of HBeAg defective HBV is a virus strategy to survive under peculiar conditions dictated by the interplay between HBV and the hosts immune system. The HBeAg/anti-HBe serological status is determined not only by the extent of virus replication and integration of HBV-DNA into cellular DNA but also by heterogeneity of HBV. The study of HBV heterogeneity in baseline sera of patients undergoing antiviral therapy appears to have a predictive value of the outcome of HBV infection in the single patient.

Quantitative detection of hepatitis C virus RNA in the serum of patients with chronic hepatitis C treated with interferon: A pilot study

BACKGROUNDnIt is not known whether the measurement of serum hepatitis C virus (HCV) RNA by reverse transcription polymerase chain reaction (RT-PCR) could improve the management of patients with chronic hepatitis C being treated with interferon.nnnOBJECTIVESnWe analysed, in a pilot study, the relations between the variations of HCV-RNA and alanine aminotransferase (ALT) serum levels in 18 anti-HCV positive patients treated with interferon.nnnSTUDY DESIGNnSerum HCV-RNA was measured, using a non competitive coamplification assay (Amplicor HCV Monitor), before (at 3, 2 and 1 months and baseline), during (first, third and sixth month) and after treatment for at least 8 months (range 8-17 months). HCV-RNA levels fluctuations were correlated with those of ALT and treatment outcome. According to the ALT pattern, four patients were non responders, seven partial responders, four relapsers and two long term responders.nnnRESULTSnThe median and mean baseline HCV-RNA levels were significantly different in patients infected by HCV type 1, 2 and 3, being 248,449, 235,506; 4170, 17,866 and 22,315, 79,273 molecules per ml, respectively (P < 0.0001). We did not find any significant difference between median and mean baseline viremia of responders and non responders. After 1 month of treatment viremia was below the sensitivity levels of the assay in 77.7% (14/18) of the patients who normalized ALT, at least temporarily. On the contrary, HCV-RNA remained detectable in non responders.nnnCONCLUSIONSnOur data suggest that HCV-RNA detection using Amplicor Monitor at the first month of treatment can be useful to identify non responders, avoiding three additional months of treatment as would be required by ALT monitoring alone. During the post-treatment follow-up, persistence of undetectable HCV-RNA and normal ALT levels helps to identify long term responders from patients with the risk of relapse in spite of biochemical remission.