The exact role of hepatitis B virus in the development of liver cancer is not known. The recent identification of a viral regulatory gene HBx suggests a possible direct involvement of the virus whereby the HBx protein, acting as a transcriptional transactivator of viral genes, may alter host gene expression and lead to the development of hepatocellular carcinoma. We have tested this possibility of placing the entire HBx gene under its own regulatory elements directly into the germline of mice. Transgenic animals harbouring this viral gene succumbed to progressive histopathological changes specifically in the liver, beginning with multifocal areas of altered hepatocytes, followed by the appearance of benign adenomas, and proceeding to the development of malignant carcinomas. Male mice developed disease and died much earlier than females. This transgenic animal model appears ideal for defining the molecular events that follow the expression of the viral HBx gene and are responsible for the development of liver cancer.

A possible causative role for the recently discovered hepatitis C virus (HCV) in the development of hepatocellular carcinoma (HCC) was investigated by assay of sera from HCC patients in Japan for antibodies to a recombinant HCV antigen and to hepatitis B virus (HBV) antigens. Among the 253 HCC patients examined, 156 (61.7%) had no serum markers of either a previous or a current HBV infection (group I), 46 (18.2%) were negative for HBV surface antigen but positive for anti-HBV surface and/or anti-HBV core antibody, indicating the occurrence of a previous, transient HBV infection (group II), and 51 (20.2%) were chronically infected HBV carriers as evidenced by positivity for HBV surface antigen (group Ill). The prevalence of HCV antibody in group I (68.6%) and II (58.7%) patients was significantly higher than for group m1 (3.9%) or in 148 additional patients with other (non-HCC) cancers (10.1%) (P < 0.01). Thus, there appears to be a strong association between HCV infection and the development of HCC, particularly in patients for which HBV infection cannot be implicated as a causative factor. The data also suggest an additional mode of transmission for HCV other than blood transfusion, since a history of blood transfusion was shown in only about 30% of the HCV antibodypositive HCC patients in groups I and 11. A high prevalence of HCV antibody was also shown among patients with HCC whose disease was originally thought to be due to very high ethanol consumption.The development of specific serological tests for infection by hepatitis A virus (HAV) and hepatitis B virus (HBV) has revealed that a large proportion of hepatitis cases are not caused by either ofthese agents (1-3). The resultant diagnosis of exclusion, non-A, non-B hepatitis (NANBH), now accounts for 95% of all posttransfusion hepatitis and over one-third of sporadic, acute hepatitis cases in Japan. Although symptoms in the acute phase of this disease are generally less severe than with HAV or HBV infection, NANBH is much more likely to develop into a persistent, chronic state. Over 50% of posttransfusion NANBH cases become chronically infected versus less than 10% in the case of HBV infections; typically, no chronicity results from infection by HAV. It is also clearly established that chronic NANBH can develop into hepatic cirrhosis (4-6). Accumulated serological, pathological, epidemiological, and clinical evidence suggests a significant association ofthe HBV carrier state with hepatocellular carcinoma (HCC) (7,8). In Japan, however, less than one-third ofHCC patients are also chronic HBV carriers, and the number of surgically treated HCC cases with no serological markers of prior or current HBV infection has increased steadily in Japan during the last 10 years (9). This suggests another causative factor(s). It has been hypothesized that NANBH virus(es) might be the missing causative agent in HCC development (10)(11)(12)(13)(14).The etiological agent(s) of NANBH has long been sought by many research groups (15,16), and recently a NANBH agent, ter...

An efficient method of constructing recombinant adenoviruses (Ads)

Familial adenomatous polyposis coli (FAP) is a disease characterized by the development of multiple colorectal adenomas, and affected individuals carry germline mutations in the APC gene. With the use of a conditional gene targeting system, a mouse model of FAP was created that circumvents the embryonic lethality of Apc deficiency and directs Apc inactivation specifically to the colorectal epithelium. loxP sites were inserted into the introns around Apc exon 14, and the resultant mutant allele (Apc580S) was introduced into the mouse germline. Mice homozygous for Apc580S were normal; however, upon infection of the colorectal region with an adenovirus encoding the Cre recombinase, the mice developed adenomas within 4 weeks. The adenomas showed deletion of Apc exon 14, indicating that the loss of Apc function was caused by Cre-loxP-mediated recombination.

A recombinant adenovirus (Ad) expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off-switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ads showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1, HeLa and Jurkat cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in transgenic animals. The method is also applicable to construction of recombinant Ad bearing a gene the expression of which is deleterious to propagation of recombinant Ad.

No abstract

Glial cells express a variety of neurotransmitter receptors. Notably, Bergmann glial cells in the cerebellum have Ca2+-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors (AMPARs) assembled without the GluR2 subunit. To elucidate the role of these Ca2+-permeable AMPARs, we converted them into Ca2+-impermeable receptors by adenoviral-mediated delivery of the GluR2 gene. This conversion retracted the glial processes ensheathing synapses on Purkinje cell dendritic spines and retarded the removal of synaptically released glutamate. Furthermore, it caused multiple innervation of Purkinje cells by the climbing fibers. Thus, the glial Ca2+-permeable AMPARs are indispensable for proper structural and functional relations between Bergmann glia and glutamatergic synapses.

SUMMARY:Recently, the adenovirus expression vector attracts much attention for the application to gene therapy and the method to purify and concentrate adenovirus without loss of infectivity has become very important, especially for animal experiments and gene therapy of humans.In this report, we show a simple and efficient method for purifying infectious adenovirus. The method consists of sequential centrifugation in CsCl step gradients without loss of infectivity and can be completed in one day. The method maintained the viral infectivity after 10-fold concentration and seemed to remove more than 99.9% of carried-over proteins. We showed also that the buffers for dialyzing the purified virions influenced the stability of infectivity.The buffers of 10 mM HEPES-1 mM EDTA -10% glycerol and PBS (-)-10% glycerol resulted in higher stability than did 10 mM HEPES-1 mM MgC12-10% glycerol. The method is may be useful in many applications of recombinant adenovirus.