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.

Production of polyclonal antibodies against the S and preS2 regions of woodchuck hepatitis virus: lack of detectable low glycosylated preS2 protein (GP33) in sera from infected animals.

Polyclonal antibodies directed against the preS2 and S domains of the woodchuck hepatitis virus (WHV) envelope proteins were prepared using synthetic peptides and fusion polypeptides as immunogens. They were tested by immunoblotting and immunoprecipitation of infected woodchuck sera and lysates of a eukaryotic cell line expressing WHV envelope proteins. Only one anti-peptide serum directed against the preS2 domain was reactive with WHV envelope proteins, recognizing the preS2 and preS1 proteins by their preS2 epitopes. With recombinant fusion proteins we generated several anti-S sera, which recognized all envelope proteins, and anti-preS2 antisera, which recognized the preS proteins. Results obtained with our antisera showed that sera of infected woodchucks lack the low glycosylated form (GP33) of the preS2 protein, unlike human hepatitis B virus.

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)

Nucleotide sequence and structural organization of the human FMS proto-oncogene.

The human proto-oncogene c-fms [FMS] on chromosome 5q33.3 encodes a transmembrane glycoprotein with tyrosine kinase activity that functions as the cell surface receptor for the macrophage colony stimulating factor (CSF-1 or M-CSF). Overlapping bacteriophage clones that included 35 kb of the FMS locus and contained the complete coding sequence of the CSF-1 receptor were subjected to nucleotide sequencing analysis. Comparison with the cDNA sequence of the human c-fms gene indicated that at least one 5' noncoding exon is located far upstream (ca. 26 kb) from sequences encoding the CSF-1 receptor. The FMS coding sequence consists of 21 small exons and heterogeneously sized introns, ranging from 6.3 kb to less than 0.1 kb in complexity.