It is clear that cytokines cause metabolic disturbances that are similar to known complications of AH. TNF appears to be a proximal mediator of multiple types of experimental liver injury and TNF activity is elevated in ALD, as are the levels of certain other cytokines. On the other hand, low physiologic amounts of cytokines appear to be important for liver regeneration (and perhaps are beneficial to the organism as a whole). Goals for evaluation of anticytokine therapy in ALD will be: (1) determining the timing and type of the particular anticytokine employed (such as, immediate administration of antibody followed by an inhibitor of cytokine production), (2) appropriate monitoring of drug effects on cytokine metabolism as well as liver function and outcome, and (3) maintenance of the regenerative or positive physiologic effects of cytokines while blocking the cytolytic effects. Thus, we predict that ultimate anticytokine therapy will be directed at conserving the positive growth-enhancing effects of cytokines while attenuating their cytolytic effects.

The incidence of fractures and of osteoporosis differs between Oriental and Western Caucasian women. This may depend, at least in part, on nutritional factors, including dissimilarities in dietary intake of phytoestrogens. To investigate this possibility, 2-month-old female rats were ovariectomized (OVX) or sham-operated (SHAM), fed a casein-based diet, injected daily with subcutaneous genistein (GEN), the most abundant and best characterized phytoestrogen, or vehicle (Veh) and killed 21 days after surgery. As expected, ovariectomy resulted in loss of bone mineral density (BMD) and in uterine atrophy. However, administration of 5 micrograms GEN per gram body weight (b.w.) ameliorated the ovariectomy-induced loss of BMD (189 +/- 2 mg/cm2 in OVX and 192 +/- 2 in OVX with 5 micrograms GEN/g b.w. per day; p < 0.05). One microgram GEN per gram body weight did not affect the BMD loss and the effect of the 5 micrograms and 25 micrograms GEN per gram body weight were statistically not different. A trend toward reduced uterine atrophy (21% reduction) was noted with the 25 micrograms GEN dose, but not with the 1 microgram and 5 micrograms doses. A separate experiment with 2 x 2 factorial design was conducted to elucidate the mechanism by which GEN ameliorates ovariectomy-induced bone loss. In this experiment, histomorphometry demonstrated a dramatic reduction in trabecular bone volume after ovariectomy (7.6 +/- 0.7% of total bone volume in SHAM-Veh vs 3.3 +/- 0.2% in OVX-Veh; p < 0.01) and less bone loss in OVX rats injected with 5 micrograms GEN per gram per day (3.3 +/- 0.2% of total bone volume in OVX-Veh vs 5.2 +/- 0.4% in OVX-GEN; p < 0.01). Administration of GEN was associated with higher bone formation rate per tissue volume and with a trend toward a higher number of osteoblasts per bone perimeter. The parameters of bone resorption were not affected by GEN. The concentration of serum osteocalcin and the urinary excretion of deoxypyridinoline provided corroborating results. Since production of proinflammatory cytokines is intimately involved in the pathogenesis of postmenopausal osteoporosis, the effect of GEN on lipopolysaccharide-induced in vitro production of Tumor necrosis factor-alpha (TNF alpha) was tested in monocytic cells from the same four rat groups. Production of TNF alpha was markedly elevated in OVX-Veh as compared with the SHAM-Veh rats, but this was blocked by GEN in the OVX rats. This study shows that GEN reduces both trabecular and compact bone loss after ovariectomy and that this protective effect differs from that of estrogen, since it depends on stimulation of bone formation rather than on suppression of bone resorption. Lack of action of GEN on uterine atrophy supports the possibility that this GEN dose affects target tissues via non-estrogenic mechanisms. Modulation of cytokine production may be involved in the effect of GEN on bone.

A new quantitative cytometric technique, termed the ArrayScan™, is described and used to measure NF-B nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-␣ (TNF␣). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-B was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4 -5 min slower than that of IB␣ degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNF␣ and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNF␣ stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNF␣ stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent IB␣ degradation and subsequent NF-B translocation could also be discriminated; LeuLeu-Leu aldehyde was only a partial inhibitor with an IC 50 of 2 M, while clastolactacystin ␤-lactone was a complete inhibitor with an IC 50 of 10 M. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNF␣ stimulation in both cell types with an IC 50 of 0.4 M. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNF␣ concentration measured after 17 h of stimulation. These results suggest that the ArrayScan™ technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-B. IL-11 and TNF␣ are two master cytokines that induce an almost identical proinflammatory response, including the production of chemotactic cytokines, adhesion molecules, and enzymes such as cyclooxygenase, nitric-oxide synthetase, and matrix metalloproteinases (1, 2). Many of these effects are a result of the activation by both IL-1 and TNF␣ of the NF-B transcription factor pathway, which is associated with the activation of many cellular defense genes (3, 4). Composed of p65 (RelA) and p50 proteins, NF-B is normally present in the cytoplasm in an inactive state in a complex with members of the IB inhibitor protein family, chiefly the 37-kDa IB␣ form. In this complexed form, a nuclear localization sequence found on NF-B is masked by the IB␣, preventing nuclear translocation of NF-B, DNA binding, and subsequent transcriptional activation (5-12). IL-1 or TNF␣ receptor activation induces within several minutes the specific phosphorylation of Ser 32 and Ser 36 on IB␣, the destruction of the phosphorylated IB␣ protein by proteasomes, and the translocation of NF-B to the nucleus (13-1...

S-adenosylmethionine (Adomet) is a substrate for de novo synthesis of choline. Adomet deficiency occurs in certain types of liver injury, and the injury is attenuated by exogenous Adomet. Tumor necrosis factor-α (TNF-α) is also a mediator of these models of hepatotoxicity. We investigated the role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats made deficient in both Adomet and choline. Rats were maintained on either a methionine-restricted and choline-deficient (MCD) diet or a diet containing sufficient amounts of all nutrients [methionine and choline sufficient (MCS)] and then administered either LPS or saline. MCS-LPS rats had normal liver histology and no change in serum transaminases compared with the MCS-saline control group. MCD-saline rats had hepatosteatosis but no necrosis, and a five- to sevenfold increase in transaminases vs. the MCS-saline group. MCD-LPS rats additionally had hepatonecrosis and a 30- to 50-fold increase in transaminases. Exogenous Adomet administration to MCD-LPS rats corrected the hepatic deficiency of Adomet but not of choline, prevented necrosis but not steatosis, and attenuated transaminases. Serum TNF-α was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-α in MCD-LPS rats.

The purpose of this study was to determine if exacerbation of apoptosis precedes liver injury during chronic exposure of rats to alcohol. After 7 weeks of feeding an alcohol- or dextrin-containing liquid diet, the animals were treated with gram-negative bacterial lipopolysaccharide (1 mg x kg(-1) body weight, intravenously) or sterile saline and sacrificed 3 hr after the treatment. Alanine:2-oxoglutarate aminotransferase (ALT) and lactate:NAD oxidoreductase [lactate dehydrogenase (LDH)] were measured in plasma. The caudate lobe of the liver was resected for histology, while the rest of the organ was perfused with collagenase to isolate hepatocytes, Kupffer cells (KCs), and sinusoidal endothelial cells (SECs) by centrifugal elutriation. Hepatocyte mitochondria were isolated by differential centrifugation of the cell homogenate. Reduced and oxidized glutathione (GSH and GSSG) in isolated hepatocytes and hepatocyte mitochondria, and malondialdehyde in hepatocytes were assayed. Caspase-3 activity and Fas ligand mRNA expression were determined in hepatocytes, KCs, and SECs. Plasma ALT and LDH activity, liver histology, GSH, GSSG and their ratio, and malondialdehyde content were not affected by alcohol treatment Caspase-3 activity was significantly increased in alcohol-treated rats in all three cell types, with the lowest response observed in hepatocytes and the highest in KCs. Fas ligand mRNA expression, which had the highest level in SECs, followed by KCs and hepatocytes, was not affected by alcohol administration. Lipopolysaccharide had the following effects: an increase in ALT in both pair- and alcohol-fed rats, and LDH only in alcohol-fed rats, a decrease in GSH + GSSG levels in both mitochondria and hepatocytes, an elevation of malondialdehyde content in hepatocytes, a raise in caspase-3 activity in all groups and cell types, and an augmentation of Fas ligand expression in hepatocytes and KCs, but not in SECs. These data suggest that, during chronic alcohol consumption, an exacerbated apoptosis precedes alcohol-induced liver injury.