Although NFkappaB binding activity is induced during liver regeneration after partial hepatectomy, the physiological consequence of this induction is unknown. We have assessed the role of NFkappaB during liver regeneration by delivering to the liver a superrepressor of NFkappaB activity using an adenoviral vector expressing a mutated form of IkappaBalpha. This adenovirus (Ad5IkappaB) was almost exclusively expressed in the liver and inhibited NFkappaB DNA binding activity and transcriptional activity in cultured cells as well as in the liver in vivo. After partial hepatectomy, infection with Ad5IkappaB, but not a control adenovirus (Ad5LacZ), resulted in the induction of massive apoptosis and hepatocytes as demonstrated by histological staining and TUNEL analysis. In addition, infection with Ad5IkappaB but not Ad5LacZ decreased the mitotic index after partial hepatectomy. These two phenomena, increased apoptosis and failure to progress through the cell cycle, were associated with liver dysfunction in animals infected with the Ad5IkappaB but not Ad5LacZ, as demonstrated by elevated serum bilirubin and ammonia levels. Thus, the induction of NFkappaB during liver regeneration after partial hepatectomy appears to be a required event to prevent apoptosis and to allow for normal cell cycle progression.

The pattern of lysyl hydroxylation in the nontriple helical domains of collagen is critical in determining the cross-linking pathways that are tissue specific. We hypothesized that the tissue specificity of type I collagen cross-linking is, in part, due to the differential expression of lysyl hydroxylase genes (Procollagen-lysine,2-oxyglutarate,5-dioxygenase 1, 2, and 3 [PLOD1, PLOD2, and PLOD3]). In this study, we have examined the expression patterns of these three genes during the course of in vitro differentiation of human osteoprogenitor cells (bone marrow stromal cells [BMSCs]) and normal skin fibroblasts (NSFs). In addition, using the medium and cell layer/matrix fractions in these cultures, lysine hydroxylation of type I collagen ␣ chains and collagen cross-linking chemistries have been characterized. High levels of PLOD1 and PLOD3 genes were expressed in both BMSCs and NSFs, and the expression levels did not change in the course of differentiation. In contrast to the PLOD1 and PLOD3 genes, both cell types showed low PLOD2 gene expression in undifferentiated and early differentiated conditions. However, fully differentiated BMSCs, but not NSFs, exhibited a significantly elevated level (6-fold increase) of PLOD2 mRNA. This increase coincided with the onset of matrix mineralization and with the increase in lysyl hydroxylation in the nontriple helical domains of ␣ chains of type I collagen molecule. Furthermore, the collagen cross-links that are derived from the nontriple helical hydroxylysine-aldehyde were found only in fully differentiated BMSC cultures. The data suggests that PLOD2 expression is associated with lysine hydroxylation in the nontriple helical domains of collagen and, thus, could be partially responsible for the tissue-specific collagen cross

Destruction of Kupffer cells with gadolinium chloride (GdCl(3)) and intestinal sterilization with antibiotics diminished ethanol-induced steatosis in the enteral ethanol feeding model. However, mechanisms of ethanol-induced fatty liver remain unclear. Accordingly, the role of Kupffer cells in ethanol-induced fat accumulation was studied. Rats were given ethanol (5 g/kg body wt) intragastrically, and tissue triglycerides were measured enzymatically. Kupffer cells were isolated 0-24 h after ethanol, and PGE(2) production was measured by ELISA, whereas inducible cyclooxygenase (COX-2) mRNA was detected by RT-PCR. As expected, ethanol increased liver triglycerides about threefold. This increase was blunted by antibiotics, GdCl(3), the dihydropyridine-type Ca(2+) channel blocker nimodipine, and the COX inhibitor indomethacin. Ethanol also increased PGE(2) production by Kupffer cells about threefold. This increase was also blunted significantly by antibiotics, nimodipine, and indomethacin. Furthermore, tissue triglycerides were increased about threefold by PGE(2) treatment in vivo as well as by a PGE(2) EP(2)/EP(4) receptor agonist, whereas an EP(1)/EP(3) agonist had no effect. Moreover, permeable cAMP analogs also increased triglyceride content in the liver significantly. We conclude that PGE(2) derived from Kupffer cells, which are activated by ethanol, interacts with prostanoid receptors on hepatocytes to increase cAMP, which causes triglyceride accumulation in the liver. This mechanism is one of many involved in fatty liver caused by ethanol.

The continuous intragastric in vivo enteral feeding model in the rat developed by Tsukamoto and French has been very useful; however, it requires surgical expertise. Recently, we found that Kupffer cells isolated from rats treated only once with ethanol were sensitized to endotoxin 24 hours later. Accordingly, these experiments were designed to determine if a new, simple animal model of ethanol hepatotoxicity could be developed based on Kupffer cell sensitization. Female Wistar rats were given ethanol (5 g/kg body weight) once every 24 hours intragastrically. Livers were stained with hematoxylin-eosin to assess steatosis, inflammation, and necrosis, and tissue triglycerides, serum transaminases, and plasma endotoxin were measured. Kupffer cells were isolated 0 to 24 hours after one intragastric dose of ethanol daily, and intracellular Ca 2؉ ([Ca 2؉ ] i ) was measured using fura-2, while tumor necrosis factor ␣ (TNF-␣) was measured by enzyme-linked immunosorbent assay. CD14 was evaluated by Western and Northern analysis. Ethanol caused steatosis, necrosis, and inflammation in only a few weeks, and after 8 weeks, serum aspartate transaminase (AST) levels were doubled. Values were similar to levels achieved in the enteral feeding model. Triglycerides were also increased significantly by ethanol as expected, and endotoxin levels were increased to 70 to 80 pg/mL. This latter increase was prevented (F20 pg/mL) by antibiotics implicating endotoxin. In isolated Kupffer cells from untreated control rats, [Ca 2؉ ] i increased to 82 ؎ 7 nmol/L after addition of lipopolysaccharide (LPS) (100 ng/mL), and levels were elevated about twofold by ethanol given 24 hours earlier ( Although it is well known that alcoholic liver disease results from the dose-and time-dependent consumption of ethanol, mechanisms remain unclear. For a long time, a significant drawback for research in this area was the lack of appropriate animal models. For example, feeding ethanol chronically with the diet causes only fatty liver. 1 The establishment of a continuous intragastric in vivo enteral feeding model in the rat by Tsukamoto and French represented a major development in this area. 2,3 With this model, it has been shown that inactivation of Kupffer cells with gadolinium chloride (GdCl 3 ) prevented early alcohol-induced liver injury. 4 Moreover, intestinal sterilization with antibiotics (polymyxin B and neomycin) 5 or decreasing endotoxin with lactobacillus 6 prevented alcohol-induced liver injury. These experiments pointed to a role for activation of Kupffer cells by gut-derived endotoxin as a primary event in mechanisms of alcohol-induced liver injury. Although this chronic enteral ethanol model has been very useful, it is both timeconsuming and expensive, and requires surgical expertise.Recently, we found that Kupffer cells isolated from rats treated once with ethanol 24 hours earlier exhibited enhanced sensitivity to lipopolysaccharide (LPS) as a result of up-regulation of the endotoxin receptor, CD14. 7 Furthermore, Su et al. 8 showed that ...

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The levels of induced TGF-beta1 and TGF-beta1 m-RNA after L burn injury are higher and peak earlier than after M burn injury. Elevated TGF-beta1 may be associated with cell death in hepatocytes. The TGF-beta1 rise may be associated with hepatocyte injury and systemic response to massive burn.

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