Ultrasound detection of spontaneous hepato-cellular carcinomas in X/myc bitransgenic mice.

AIMS: To evaluate trans-abdominal ultrasound for the detection of Hepatocellular carcinoma (HCC) in a bitrasgenic murine (X/myc) model using a commercially available high-frequency ultrasound unit. METHODS: Sixty-one female animals were included in this study. These animals were submitted to a single ultrasound examination of the liver under general anesthesia (isoflurane), and then euthanized. Results of ultrasound were compared with necropsy and histopathology. RESULTS: The lesions demonstrated a fairly consistent aspect (oval- or round-shaped, well-defined hypoechoic homogeneous lesions), and lesions as small as 2 mm were identified. For detection of hepatic nodules per mouse the sensitivity was 75%, the specificity was 100% and the accuracy was 88.5%. For detection of hepatic focal lesions per lesions the overall sensitivity was 60%, the specificity was 97%, and the accuracy was 75.9%. Contrast-enhanced harmonic ultrasound imaging did not improve the identification of the lesions in our experimental conditions. CONCLUSION: High-frequency ultrasound appears to be an efficient tool allowing new possibilities to use this animal model and evaluate new therapies in longitudinal studies, which are much more powerful.

Preliminary results of interferon-alpha therapy on woodchuck hepatitis virus-induced hepatocarcinogenesis: possible benefit in female transgenic mice.

BACKGROUND: C-myc activation is a potent oncogenic event in hepatocarcinogenesis. The aim of this study was to test the preventive effect of interferon-alpha (IFN-alpha) on the development of dysplasia and subsequent hepatocellular carcinoma (HCC) in transgenic (Tg) mice overexpressing c-myc in the liver. METHODS: The WHV/c-myc Tg mice recapitulating woodchuck hepatitis virus-induced hepatocarcinogenesis were treated with IFN-alpha, starting early in life until sacrifice at pre-neoplastic or neoplastic stages. Transgene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR), hepatocyte proliferation was assessed by bromodeoxyuridine incorporation and RT-PCR for proliferating cell nuclear antigen, and apoptosis was assessed by in situ nick-end-labeling of DNA. RESULTS: C-myc expression and hepatocyte proliferation were significantly reduced in treated female mice, without modification of apoptosis, correlating with a lower severity of dysplasia in 9/12 treated animals at pre-neoplastic stages. At the neoplastic stage, 2/3 treated females neither exhibited carcinoma nor dysplasia, while all 6/6 untreated mice and 3/3 treated males developed carcinomas. CONCLUSIONS: Inhibition of c-myc and hepatocyte proliferation by long-term administration of IFN-alpha was associated with a decrease, or a delay, of oncogenesis in the mouse Tg HCC model. Whether c-myc and hepatocyte proliferation down-regulation could be relevant parameters of IFN-alpha efficiency for hepatocarcinogenesis prevention in cirrhotic patients should be established.

A new hepadnavirus endemic in arctic ground squirrels in Alaska.

We present evidence for a novel member of the hepadnavirus family that is endemic in wild arctic ground squirrels (Spermophylus parryi kennicotti) in Alaska. This virus, designated arctic squirrel hepatitis virus (ASHV), was initially detected in the livers of animals bearing large hepatic nodules by nucleic acid hybridization with hepadnavirus probes and in plasma by cross-reactivity with antibodies to hepadnavirus surface and core antigens. The complete nucleotide sequence of the 3,302-bp-long ASHV genome was determined and compared with those of ground squirrel hepatitis virus (GSHV) and woodchuck hepatitis virus (WHV); all sequences were organized into four open reading frames, designated pre-C/C, pre-S/S, pol, and X. Despite roughly equivalent variability among the three rodent hepadnaviruses (around 16% base and 19% amino acid exchanges), ASHV appeared to be more closely related to GSHV than to WHV in phylogenetic analysis. Accordingly, preliminary studies of the pathology of ASHV infection suggested that ASHV may be a less efficient oncogenic agent than WHV. About one-third of aged animals maintained in captivity, including virus-infected as well as uninfected squirrels, developed large liver nodules, consisting of hepatocellular adenomas or carcinomas or nonmalignant lesions characterized by drastic microvesicular steatosis. ASHV-infected arctic ground squirrels may serve as a new model with which to analyze the contribution of hepadnavirus- and host-specific determinants to liver pathology and tumorigenesis.

Hepadna virus integration generates virus-cell cotranscripts carrying 3' truncated X genes in human and woodchuck liver tumors.

Integration of the human and woodchuck hepatitis B viruses (HBV and WHV) in host chromosomes has been implicated in the development of hepatocellular carcinoma by different cis- and trans-acting mechanisms. The structure and coding capacity of abundant HBV and WHV transcripts of abnormal sizes produced from integrated viral sequences in one human and two woodchuck liver tumors were examined. Analysis of cDNA clones revealed in all cases hybrid virus-cell transcripts containing sequences of the viral surface gene, the viral enhancer, and different truncated versions of the viral X transactivator. Cotranscribed cellular sequences showing no significant coding function provided the signals for transcription termination. In two transcripts, the HBX and WHX genes truncated at carboxy terminal positions conserved transcriptional trans-acting capacity in transient transfection assays. These results lend support to the hypothesis that the integrated hepadnavirus X transactivator might participate in the development of woodchuck as well as human liver tumors by a common trans-acting mechanism.

Liver-specific expression and high oncogenic efficiency of a c-myc transgene activated by woodchuck hepatitis virus insertion.

The high oncogenic efficiency of woodchuck hepatitis virus (WHV) has been correlated with the ability of this virus to provoke insertional activation of myc family genes. To assess the impact of viral integration on liver cell transformation, we have generated transgenic mice carrying the mutated c-myc gene and adjacent viral DNA from a woodchuck tumor, in original configuration. Virtually all mice from two different strains developed hepatocellular carcinoma with a mean latency period of 8-12 months. The c-myc transgene was expressed transiently in neonatal livers, and re-expressed at preneoplastic and neoplastic stages in adult livers. Woodchuck c-myc mRNA driven by the normal P1 and P2 promoters and WHV-specific transcripts encoding viral surface antigens were produced in a strictly co-regulated fashion during development and tumorigenesis, indicating a predominant regulatory influence of the viral enhancer. Furthermore, the activity of the viral enhancer in response to various biological stimuli was apparently modulated by glucose uptake and glucagon/insulin balance in differentiated hepatocytes. In this model, a viral integration event selected from a naturally occurring tumor proved to be determinant for induction of hepatocarcinogenesis, although enforced, liver-specific expression of c-myc was limited to a particular developmental stage.

Recognition of the N-terminal, C-terminal, and interior portions of HBx by sera from patients with hepatitis B.

We have cloned and expressed in Escherichia coli three different parts of the HBx open reading frame, the N- and C-termini and the interior or central portion, using two vector systems. The sera of 43 hepatitis B virus patients representing three clinical categories--asymptomatic carriers, chronic active hepatitis, and hepatitis B patients with cirrhosis--known to be anti-HBx positive, were tested for reactivity against these constructs by Western blotting. The great majority of sera, regardless of the clinical categories, clearly recognise all three parts of HBx, strongly suggesting that the normal mechanism of biosynthesis of the HBx gene product is a straight-forward translation of the open reading frame starting from the first ATG. However, asymptomatic carriers show a marked, often almost exclusive, preference for recognition of the central portion of HBx, while patients with chronic hepatitis and patients with cirrhosis generally recognise all three parts of HBx to a similar extent.

Early and frequent detection of HBxAg and/or anti-HBx in hepatitis B virus infection.

To clarify the significance of the X gene of hepatitis B virus, we have tested for anti-HBx in the serum and HBxAg in the liver at different stages of the natural history of hepatitis B virus infection. Sera were screened by enzyme-linked immunosorbent assay and positive results confirmed by immunoblot. Purified recombinant MS2 Pol-HBx fusion protein was used as target for both assays. Among serial sera of patients with nonfulminant acute hepatitis, 24 of 64 patients (37.5%) were positive for anti-HBx. In fulminant cases, 15 of 36 patients (42%) had anti-HBx. In chronic hepatitis patients with high rates of hepatitis B virus replication, we found a significantly (p less than 0.01) higher prevalence of anti-HBx, 14 of 25 patients (56%), than in those with low replication, 14 of 66 patients (21%), or among asymptomatic HBsAg carrier blood donors (20 of 126 = 16%) without detectable hepatitis B virus replication (p less than 0.0001). The highest prevalence of anti-HBx was found in HBsAg carriers with cirrhosis (41 of 54 patients = 76%) and/or with hepatocellular carcinoma (18 of 33 patients = 54%). The findings suggest that anti-HBx appears as a common and early marker of hepatitis B virus infection, transient in self-limited hepatitis but persisting with progression to chronicity. In chronic hepatitis, the prevalence of anti-HBx correlated with the intensity and duration of hepatitis B virus replication but neither with the severity of the liver disease nor with malignant transformation per se.(ABSTRACT TRUNCATED AT 250 WORDS)