No abstract

We investigated the sequence of expression of osteoblast gene markers during bone formation in vivo by in situ hybridization. Cylindrical lesions were induced in the femora of sheep with titanium analytic bone implants that allow removal of serial core samples to study bone formation. At 2 weeks (2W), granulation tissue made up of spindle-shaped cells had partially replaced the blood clot. Islands of osseous tissue, first noted in the periphery of the ingrowing tissue at 3W, became the predominant tissue by 6W. The surfaces of newly forming bone at 3W were apposed by cuboidal cells, which in some areas were several layers thick. By 6W, most of the cells lining bone trabeculae had assumed a flattened morphology. The temporal and spatial distribution of osteoblast gene markers was examined by in situ hybridization with nonradioactive digoxigenin probes for alpha 1(I) procollagen, alkaline phosphatase (ALP), osteopontin (OP), and bone Gla protein (BGP). The spindle-shaped cells in the granulation tissue expressed mRNA for alpha 1(I) procollagen, ALP, and OP but not BGP, suggesting that they may be osteoblast precursor cells. alpha 1(I) procollagen mRNA was strongly expressed by all cells on the surface of bone, with a peak intensity at 3W and then reducing sharply by 6W. Initially, only pockets of cuboidal cells on bone surfaces expressed ALP mRNA, with a peak intensity at 5W. Similarly, only a proportion of cuboidal cells expressed OP mRNA early in bone formation, but the number of cells expressing OP mRNA increased with time. Clumps of cuboidal cells expressed BGP mRNA only when bone was present, and the degree of expression increased with the amount of bone formed. This model allows the study of temporal and spatial sequence of gene expression in cells participating in osteogenesis. The temporal sequence is similar to that shown in vitro in other models of mineralization. The geographic localization of cells expressing mRNA for alpha 1(I) procollagen, ALP, OP, and BGP implies subspecialization of osteoblasts in bone formation.

No abstract

ogy, genomic organization, and replication by means of reHepatitis B virus (HBV) has been demonstrated in bile verse transcription. 1,3 duct epithelial cells (BDEC) during chronic infection.The replication of human HBV has been extensively studThe persistence of virus in BDEC may play an important ied in hepatocytes, but comparatively less is known about role in disease pathogenesis, and may be at least partly HBV infection of nonparenchymal cells within the liver. Hepresponsible for the relapse phenomenon observed in adnaviral antigens have been shown in bile duct epithelial antiviral treatments using nucleoside analogues. The cells (BDEC) in both human 7,8 and animal 9,10 livers, but the aims of this study were to examine the morphological significance of these findings has not been fully determined. changes within the liver in the duck hepatitis B modelThe HBV life cycle is complex, and there is evidence that following bile duct ligation (BDL), and to assess the efit may vary depending on the cell type, such as lymphoid fect of biliary hyperplasia upon viral DNA and proteins.cells compared with hepatocytes. 11-15 Also, HBV replication Seven-day-old ducklings, congenitally infected with the appears to be cell-cycle dependent with enhanced viral repliduck hepatitis B virus (DHBV), were subject to BDL. The cation occurring in quiescent compared with proliferating hepathological and virological changes were then followed patocytes. 16 Recent studies have shown that HBV replication at 5, 10, 15, and 20 days after ligation. All results were is inversely correlated with cellular DNA synthesis with noncompared with age-matched unligated control birds cytolytic cell arrest resulting in increased viral expression. 1.3 days. The proliferated BDEC displayed immunohistoTherefore, the interaction of HBV with BDEC warrants more chemical features identical to resting BDEC. More than detailed investigation. The aim of the present study was to 50% of BDEC in unligated controls, and more than 46% characterize DHBV infection in BDEC of congenitally inof proliferated BDEC in ligated animals were positive fected animals, and to examine the effect of cell division on for DHBV DNA and structural proteins. The intensity of hepadnaviral proteins and DNA in proliferating BDEC using immunohistochemical staining and in situ hybridization immunohistochemistry and in situ hybridization, respecsignal in the BDEC was consistently greater than that tively. of the hepatocytes, both before and after BDL. BDL induces biliary hyperplasia in the duck model, and BDEC MATERIALS AND METHODS division does not reduce the viral burden in infected cells. (HEPATOLOGY 1997;25:463-469.)Animals and Experimental Strategy. One-day-old Pekin-Aylesbury ducklings, congenitally infected with an Australian strain of DHBV, were obtained from a commercial supplier.17 Viraemia was monitored by dot blot hybridization as previously described.9,18 Thirty-eight conInfection of Pekin-Aylesbury ducks with the duck hepatitis genitally infected ducklings aged between 7 and 9 ...