The culture conditions found to result in stable proliferation of normal rat hepatocytes are: (i) subconfluent cell densities; (ii) serum-free medium; (Wi) hormonally defined medium containing epidermal growth factor, insulin, glucagon, prolactin, and other growth factors; and (iv) substrata of liver extracellular matrix depleted of growth inhibitors. Serum was found deleterious to parenchymal cells: it was inhibitory to the expression of liver-specific functions, cytostatic to parenchymal cells at all seeding densities, and cytotoxic to them at low seeding densities. These studies emphasize the relevance of synergies in the influences of hormones and extracellular matrix in regulating hepatocellular physiology.Past efforts to produce long-term proliferation of adult hepatocytes in culture have met with limited success (1-8 Substrata. Cells were plated on one of the following substrata: (i) Tissue culture plastic. Sixty-millimeter tissue culture dishes (Falcon). (ii) Type I collagen gels. Sixty-millimeter tissue culture dishes (Falcon) were coated with type I collagen gels prepared from rat tail tendons (10). (ifi) Normal rat liver biomatrix. Livers from normal Sprague-Dawley rats were utilized to prepare biomatrix by the methods of Reid (12). The biomatrix was pulverized into a fine powder with a Spex-Mill liquid nitrogen pulverizer (Spex-Mills, Metuchen, NJ), thickly smeared onto 60-mm Petri dishes (Falcon) and then sterilized by 'y irradiation (cesium-135) at 10,000 rads (100 grays). Just before use, the plates were rinsed with serum-free RPMI 1640 medium supplemented with penicillin at 200 units/ml and streptomycin at 200 pug/ml. (iv) Rat regenerating liver biomatrix. Five days after partial hepatectomy (18), livers were used for biomatrix preparation (12). (v) Normal rat liver biomatrix prepared by low-salt extraction. Biomatrix from normal rat liver was prepared by extracting the tissue with 1.0 M NaCl and omitting the initial wash in distilled water, prepared as described (11).Morphological Studies. Cultures of hepatocytes were plated under various conditions and maintained for 1-2 weeks. They were examined by using a Nikon inverted phase-contrast microscope. Cultures were evaluated by a number of investigators independently. Representative cultures were selected and photographed with phase-contrast microscopy.[3H]Thymidine Incorporation. At 1411The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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We studied 67 HBsAg‐negative Israeli patients (36 negative for all HBV serological markers as group 1 and 31 positive for antibodies to HBs and HBc as group 2) with chronic liver disease and cirrhosis of unknown origin using a rapid, sensitive and specific assay for the detection of low levels of hepatitis B virus in serum. This technique uses a high‐affinity monoclonal antibody to HBs against an a domain epitope of HBsAg to capture the virion, followed by hepatitis B virus DNA amplification with the polymerase chain reaction. In addition, 55 subjects without liver disease served as controls: Group 3 (n = 32) was negative for all hepatitis B virus markers; group 4 (n = 23) was positive for antibodies to HBs and HBc. We found 11 individuals in group 1 (31%) and 10 in group 2 (29%) harboring low levels of hepatitis B virus DNA in serum. In contrast, no one in group 3 or group 4 was positive by this technique (p < 0.0001). Using polymerase chain reaction primers spanning other regions of the hepatitis B virus genome and a method of restriction‐fragment analysis of polymerase chain reaction–amplified sequences, we detected significant DNA sequence heterogeneity, suggesting infection with distinct hepatitis B virus strains. DNA extracted from paraffin‐embedded liver biopsy specimens of 42 patients from groups 1 and 2 was shown to contain hepatitis B virus DNA by polymerase chain reaction in 11 of 12 patients with circulating virion DNA. More important, 18 additional patients whose sera were negative by HBs‐antibody capture/polymerase chain reaction amplification had hepatitis B virus DNA sequences in their livers. Hepatitis C virus antibodies were found in 71% of group 1, in 65% of group 2, in 3% of group 3 and in 4% of group 4 (p < 0.0001). Coexistence of hepatitis B virus infection and hepatitis C virus antibodies were common (>30%). We conclude that infection with hepatitis B virus undetectable by conventional assays and with hepatitis C virus may represent important unrecognized causes of idiopathic chronic liver disease in Israel, accounting for the possible origin in more than 90% of patients. (HEPATOLOGY 1991;13:1044–1051.)

The recently characterized GH-binding protein (GH-BP) has an amino acid sequence identical to the extracellular domain of the GH receptor. Serum GH-BP reflects the amount of GH receptors, and the liver seems to be their main source. To evaluate the effect of liver disease on GH-BP, 52 patients with liver cirrhosis were studied. Serum GH-BP was measured by a binding assay with dextran-coated charcoal separation. Levels of GH-BP were correlated against the clinical state, assessed by Pugh's score. The GH-BP of 31 Pugh's class A patients was 9.7 +/- 0.5%/50 microL serum, and that of 21 Pugh's class B and C patients was 7.2 +/- 0.5%/50 microL serum compared to 11.3 +/- 0.5%/50 microL serum in age-matched controls. GH-BP correlated negatively with Pugh's score and serum bilirubin, and positively with serum albumin. It did not correlate with serum liver enzymes or serum insulin-like growth factor-I. Scatchard analysis of GH binding to the GH-BP revealed similar binding affinities in Pugh's A, B, and C patients and controls. The binding capacity in cirrhosis was significantly lower than that in controls. We conclude that serum GH-BP is controlled mainly by the liver and can provide an additional measure of disease severity in liver cirrhosis.