Normal adult rat hepatocytes remained viable and functional for at least 43 days when plated on collagen-coated dishes and fed chemically defined medium supplemented with dimethyl sulfoxide (Me2SO). Hepatocytes isolated by collagenase perfusion and cultured in the presence or absence of Me2SO were (i) examined by light and electron microscopy for morphological changes; (it) analyzed for the production of albumin and other plasma proteins; and (iiM) tested by autoradiography for DNA synthesis. Me2SO-treated cells continued to produce specific plasma proteins during the entire culture period; albumin production was consistently high (11)(12)(13)(14)(15)(16)(17)(18)(19) ,.g/ml of culture medium per 24 hr) from day 2 to at least day 43 after plating. Ultrastructural analyses demonstrated that Me2SO-treated hepatocytes resembled those from intact liver in organization of cytoplasmic organelles and cellular junctions. The optimal concentration for observing the morphological and biochemical effects of Me2SO was 2% (vol/vol). We conclude that supplementation of chemically dermed medium with Me2SO enables maintenance ofdifferentiated hepatocytes in culture for extended periods of time.Dimethyl sulfoxide (Me2SO) is a dipolar aprotic solvent that is active in biological systems (1). Addition of 1-2% (vol/vol) Me2SO to the culture medium of Friend virus-induced murine erythroleukemia (MEL) cells for 4-5 days causes 90% of the cells to express characteristics associated with normal erythroid differentiation, including alterations in morphology (2), induction of a-and P-globin synthesis (3, 4), and loss of the capacity for cell division (5). Me2SO-induced differentiation has also been observed in a human leukemia cell line (6) and in cultured fibrosarcoma (7), neuroblastoma (8), human colon carcinoma (9), human lung cancer (10), and murine embryonal carcinoma (11, 12) cell lines.Past efforts to achieve long-term culture of differentiated normal adult hepatocytes have not been successful. Limited proliferation and maintenance of adult hepatocytes can be achieved by supplementing culture medium with serum from partially hepatectomized animals (13) or by plating hepatocytes on liver extracellular matrix and maintaining them in serum-free hormonally defined medium (14). Proliferation also can be achieved by culturing hepatocytes at low cell density in the presence of insulin and epidermal growth factor (EGF), but maintenance of hepatocyte-specific characteristics requires high density or supplementation with hepatic plasma membrane material (15,16).In the present study, we employed a collagen-coated surface and supplemented the culture medium with Me2SO in an attempt to extend the time in vitro that hepatocytes remain biochemically and morphologically differentiated. The addition of Me2SO had a dramatic effect; hepatocytes retaining morphological and biochemical characteristics of normal liver could be maintained in culture for as long as 43 days. Note that Me2SO, used previously to induce differentiation in tumor cells ...
Hepatitis B virus (HBV) is a small, double-stranded DNA virus and is the prototype of the hepadnavirus family. HBV is a human pathogen capable of causing both acute and chronic hepatitis. The World Health Organization currently estimates that 350 million people are chronically infected with HBV. Persistent HBV infection is also associated with an increased risk of cirrhosis and hepatocellular carcinoma. 1 Although a tremendous amount is known about HBV, our knowledge of the virus is by no means complete. Historically, major obstacles in the study of HBV have been the inability of the virus to infect cells in vitro, and the lack of animal model systems due to a strict virus-host range. Thus, many aspects of HBV biology have been unraveled by studying related hepadnaviruses, such as the duck hepatitis virus which is capable of in vitro infection, 2 and the woodchuck hepatitis virus which allows for the in vivo study in an animal model system. 3 The duck hepatitis virus and woodchuck hepatitis virus systems were instrumental in developing an understanding of the hepadnavirus lifecycle and remain valuable models for HBV infection. However, many significant differences exist between animal hepadnaviruses and HBV. For example, avian hepatitis viruses do not encode the X gene, 4 and major transcriptional differences between woodchuck hepatitis virus and HBV have been reported. 5 Within the last decade, several HBV expressing cell lines have been established by transfecting viral DNA into liverderived human cell lines and by selecting novel cell lines containing stably integrated HBV genomes. [6][7][8] The most widely used are the HepG2 2.2.15 cell line (2.2.15) derived from the HepG2 hepatoblastoma cell line and HB611 derived from the HuH6 hepatoma cell line. These and other cell lines have led to considerable progress in the study of HBV in vitro. However, there are some inherent drawbacks which preclude the use of these cell lines in studying some aspects of HBV biology. (1) Many HBV expressing cell lines were created using constructs containing strong heterologous promoters proximal to the HBV genome. The effect those promoters have on HBV transcription and replication is unclear but could differ substantially from what occurs in a natural infection in vivo in which HBV gene expression is driven solely by endogenous HBV promoters. (2) Cell lines commonly used to study HBV contain multiple copies of integrated HBV DNA. Unlike retroviruses, which integrate viral DNA into the host genome, hepadnavirus genomes are not integrated routinely but, instead, are maintained in the nucleus of infected cells in vivo as a pool of episomal covalently closed circular (CCC) DNA molecules. 9 Although the integration of HBV DNA in human liver has been reported, 10 it is not an obligatory part of the HBV lifecycle. HBV does not encode any machinery for integration into the host genome, and integration is not required for HBV replication. In addition, when integrated HBV DNA is found,
Hepatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB) is frequently caused by a mutation (G1896A) in the hepatitis B virus (HBV) precore (PC) reading frame that creates a stop codon, causing premature termination of the PC protein. During lamivudine treatment, drug resistance develops at a similar rate in HBeAg positive and HBeAg negative CHB. Lamivudine-resistant HBV mutants have been shown to replicate inefficiently in vitro in the absence of PC mutations, but it is unknown whether the presence of PC mutations affects replication efficiency or antiviral sensitivity. This study utilized the recombinant HBV baculovirus system to address these issues. HBV baculoviruses encoding the G1896A PC stop codon mutation were generated in wild-type (WT) and lamivudine-resistant (rtM204I and rtL180M ؉ rtM204V) backgrounds, resulting in a panel of 6 related recombinant baculoviruses. In vitro assays were performed to compare the sensitivities of the PC mutant viruses with lamivudine and adefovir and to compare relative replication yields. The PC mutation did not significantly affect sensitivities to either adefovir or lamivudine. WT HBV and PC mutant HBV showed similar replication yields, whereas the replication yields of the lamivudine-resistant mutants were greatly reduced in HBeAg positive HBVs, confirming previous observations. However, the presence of the PC mutation was found to compensate for the replication deficiency in each of the lamivudine-resistant mutants, increasing the replication yields of each virus. In conclusion, the PC stop codon mutation appears to increase the replication efficacy of lamivudine-resistant virus but does not affect in vitro drug sensitivity. (HEPATOLOGY 2003;37:27-35.) H epatitis B e antigen (HBeAg) negative chronic hepatitis B (CHB), a phase in the natural history of CHB, is marked by the selection of hepatitis B viruses (HBV) unable to secrete HBeAg and has become the major form of disease presentation in many parts of the world. 1 The most common of several mutations that can cause HBeAg negativity is a guanine to adenine transition at nucleotide position 1,896 (G1896A), which creates a TAG stop codon at codon 28 of the precore (PC) protein. [1][2][3][4][5] Interferon alfa and lamivudine are the only therapeutic agents approved for treatment of CHB. 6 Lamivudine is regarded as safe and as efficacious as interferon alfa, but the percentage of patients with HBeAg positive CHB who undergo HBeAg seroconversion increases with the duration of treatment. 7 Seroconversion rates between 11% and 15% per year have been reported over treatment periods up to 3 years. [7][8][9][10] Unfortunately, the frequency of antiviral drug resistance increases with the duration of therapy, rising to as high as 66% after 3 years. 10,11 The most common mutations conferring lamivudine resistance affect the active site YMDD (tyrosine-methionine-aspartate-aspartate) motif in the C domain of the HBV polymerase protein, causing the methionine (M) residue (amino acid 204) to be replaced with either isole...
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