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Biomedical subjects

D B Hill

Publications and source records attributed to D B Hill.

At least 19 recordsLinked to original sources
PubMed2004

Fast vesicle transport in PC12 neurites: velocities and forces.

Although the mechanical behavior of single-motor protein molecules such as kinesin has been carefully studied in buffer, the mechanical behavior of motor-driven vesicles in cells is much less understood. We have tracked single vesicles in neurites of PC12 cells with a spatial precision of +/-30 nm and a time resolution of 120 ms. Because the neurites are thin, long, straight, and attached to the surface of planar cover glasses, the velocity of individual vesicles could be measured for times as long as 15 s and distances as long as 15 mum. The velocity of anterograde vesicles was in most cases constant for periods of 1-2 s, then changed in a step-like fashion to a new constant velocity. The viscoelastic modulus felt by the vesicles within live PC12 cells was determined from the Brownian motion, using Mason's generalization of the Stokes-Einstein equation. From Stokes' law, the drag force at the smallest sustained velocity was 4.2+/-0.6 pN for vesicles of radius 0.30-0.40 mum, about half the maximum force which conventional kinesin can develop during bead assays in buffer. We interpret the observed velocity steps as changes of +/-1 or occasionally +/-2 in the number of active motor proteins dragging that vesicle along a microtubule. Assuming that the motor is conventional kinesin, which hydrolyzes one ATP per 8 nm step along the microtubule, the motor protein efficiency in PC12 neurites is approximately 35%.

Animals↗
PubMed2001

Inhibition of caspases in vivo protects the rat liver against alcohol-induced sensitization to bacterial lipopolysaccharide.

BACKGROUND: The mechanisms of liver sensitization by alcohol to Gram-negative bacterial lipopolysaccharide (LPS) remain elusive. The purpose of this study was two-fold: (1) to test the hypothesis that alcohol-enhanced liver apoptosis may be a sensitizing mechanism for LPS and (2) to further characterize the liver apoptotic response to alcohol. METHODS: Rats were fed a high-fat, alcohol-containing liquid diet for 14 weeks, treated with LPS (1.0 mg/kg of body weight, intravenously) or saline, followed by injection of a pan-caspase inhibitor IDN1965; N-[(1,3-dimethylindole-2-carbonyl)-valinyl]-3-amino-4-oxo-5-fluoropentanoic acid; 10 mg/kg of body weight, intraperitoneally or vehicle, and killed. The following parameters were assessed: plasma aspartate: 2-oxoglutarate aminotransferase activity (AST); liver histology and terminal deoxyribonucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) response; caspase-3, -8, and -9 activity; and mRNA and protein expression for two apoptosis-signaling molecules: Fas receptor and Fas ligand; and three apoptosis adaptors: Bax, Bcl-XL, and Bcl-2. RESULTS: Alcohol-feeding-induced liver steatosis, slightly increased caspases' activity, the number of TUNEL-positive nuclei, and facilitated the LPS necrotic effect without affecting mRNA expression of apoptosis signals and adaptors. LPS induced a significant increase in AST and the number of TUNEL-positive nuclei, both effects being more pronounced in alcohol-treated rats. LPS produced hepatic necrosis only in alcohol-treated rats. LPS effects were associated with up-regulation of mRNA expression for both apoptosis adaptors and signaling molecules. IDN1965 administration 3 hr after LPS injection strongly inhibited caspases' activity, particularly that of caspase-3. IDN1965 also abolished the increase in TUNEL-positive nuclei, reversed the effect of LPS on plasma AST in alcohol-treated rats, and prevented LPS-induced necrosis. CONCLUSIONS: (1) Alcohol-enhanced liver apoptosis may not involve regulatory steps at the transcriptional level. LPS-induced liver apoptosis seems to involve transcriptional regulation of several apoptosis adaptors. Therefore, alcohol and LPS may enhance liver apoptosis through different mechanisms. (2) Alcohol-enhanced liver apoptosis precedes and may facilitate the hepatic effects of LPS. LPS superimposed on alcohol further elevates the rate of apoptosis in the liver. This may exceed the phagocytosing capacity of the liver so that all the apoptotic cells are not phagocytosed, but rather die of necrosis.

Animals↗
PubMed2000

Increased monocyte nuclear factor-kappaB activation and tumor necrosis factor production in alcoholic hepatitis.

Increased tumor necrosis factor-a activity has been reported in patients with alcoholic hepatitis and is implicated in its pathogenesis. The aim of this study was to investigate potential mechanisms of increased tumor necrosis factor-a activity in alcoholic hepatitis. Monocyte nuclear factor-kB activity was assessed by electrophoretic mobility shift assay, monocyte tumor necrosis factor-a mRNA was semi-quantitatively assessed by reverse transcriptase polymerase chain reaction, and tumor necrosis factor-a in monocyte culture supernatants was measured. There was significantly greater spontaneous nuclear factor-kB activity in the monocytes of 6 patients with alcoholic hepatitis as compared with that in the monocytes of control subjects. There was spontaneous tumor necrosis factor-a mRNA and tumor necrosis factor-a release from the monocytes of patients with alcoholic hepatitis but not from the monocytes of normal subjects. Endotoxin increased nuclear factor-kB activity and induced tumor necrosis factor-a mRNA and tumor necrosis factor-a release from normal subjects' monocytes. Endotoxin further increased nuclear factor-kB activity, tumor necrosis factor-a mRNA, and tumor necrosis factor-a release from the monocytes of patients with alcoholic hepatitis. Supershift assays indicate that the monocyte nuclear factor-kB activation involves the p50 and p65 subunits. Dysregulated tumor necrosis factor-a metabolism in alcoholic hepatitis monocytes is associated with increased nuclear factor-kB activity and tumor necrosis factor-a mRNA expression.

Adult↗
PubMed2000

Does the use of electronic medical records improve surrogate patient outcomes in outpatient settings?

BACKGROUND: We reviewed the evidence regarding the effectiveness of electronic medical records (EMRs) as tools for improving surrogate patient outcomes in the outpatient primary care setting. METHODS: We searched the MEDLINE database (1966-1999) to find relevant articles for inclusion in the systematic review. Reference lists of retrieved publications were also searched for relevant citations. We included original published reports of all prospective studies evaluating the use of hybrid or complete EMR systems as a method of improving surrogate patient outcomes in the outpatient primary care setting. Criteria for evaluation included the use of a random study group assignment, appropriateness of control group, blinded assessment of outcomes, number and reasons for withdrawal of subjects, and attempts to minimize confounding interventions. RESULTS: Seven prospective trials of complete EMRs and 9 prospective trials of hybrid EMRs were located. Most evaluated the impact of EMR-generated reminders on provider and patient compliance with health maintenance interventions. Findings were equally positive for both complete and hybrid EMRs, and all but 1 trial reported positive results. However, the methodologic quality of the trials was modest. Design problems included lack of concurrent control groups, non-blinded outcome assessment, and the presence of potentially confounding concurrent interventions. CONCLUSIONS: Evidence from published trials suggests that utilization of either complete or hybrid EMRs can improve some surrogate outpatient care outcomes. However, rigorous trials that evaluate their impact on morbidity and mortality, and employ current technologies are required before widespread adoption of EMRs can be confidently recommended.

Ambulatory Care↗
PubMed2000

Apoptosis and dysregulated ceramide metabolism in a murine model of alcohol-enhanced lipopolysaccharide hepatotoxicity.

BACKGROUND: The role of apoptosis in EtOH-induced liver injury has not been investigated much. Therefore, the question whether apoptosis is a contributory factor to alcoholic liver disease remains to be answered. The purpose of this study was to characterize the liver apoptotic response in a murine model of alcohol-enhanced lipopolysaccharide (LPS) hepatotoxicity. METHODS: Mice were fed an alcohol-containing liquid diet for 49 days followed by an acute LPS challenge. The liver state was judged on the basis of histological appearance, plasma liver enzyme activity (alanine:2-oxoglutarate and aspartate:2-oxoglutarate aminotransferases, as markers of hepatocytolysis), and plasma hyaluronan levels (as a marker of the sinusoidal endothelial cell scavenging function). The liver apoptotic response was assessed by DNA fragmentation (TUNEL procedure), and caspases-3 and -8 activity. To determine if ceramide played a role in the liver apoptotic response, the activity of acidic sphingomyelinase and tissue content of ceramide were also quantified. RESULTS: Alcohol exposure induced fat accumulation and sensitized the liver to LPS injurious effects. Plasma liver enzyme activity was elevated by alcohol and this effect was potentiated by LPS. Liver apoptosis was augmented by both alcohol and LPS treatment as reflected by high frequency of positive TUNEL staining nuclei and by an increased activity of caspase-3 and -8. Acidic sphingomyelinase activity was also increased and it was associated with an elevated tissue content of ceramide. In addition, LPS also increased plasma TNF-alpha levels. These changes were accompanied by elevated plasma hyaluronan, reflecting an impaired sinusoidal endothelial cell scavenging function. CONCLUSIONS: These results provide a more complete description of the liver apoptotic response to both alcohol and LPS and may constitute the basis for further mechanistic studies on a possible role apoptosis may play in alcoholic liver injury.

Alanine Transaminase↗
PubMed1999

A role for Helicobacter pylori in the gastrointestinal complaints of eating disorder patients?

UNLABELLED: Eating disorder patients frequently present with gastrointestinal complaints. Helicobacter pylori is an etiologic factor in type B gastritis, gastric and duodenal ulcers, and may cause nausea and anorexia. OBJECTIVE: To determine whether or not there is an increased prevalence of H. pylori infection in patients with eating disorders. METHOD: Serum H. pylori IgG antibody and gastrointestinal symptoms were assessed in 32 patients admitted for inpatient treatment of anorexia nervosa and/or bulimia nervosa. RESULTS: Eating disorder patients did not have an increased rate of detectable serum H. pylori IgG antibody. DISCUSSION: There is not an increased prevalence of H. pylori infection in eating disorder patients. Thus, the increased frequency of gastrointestinal complaints in eating disorder patients cannot be attributed to H. pylori infection.

Adolescent↗
PubMed1999

Increased monocyte MCP-1 production in acute alcoholic hepatitis.

Monocyte chemoattractant protein-1 (MCP-1) is a potent mononuclear cell-specific chemotactic protein. MCP-1 is a candidate chemoattractant for activation and hepatic infiltration of mononuclear cells in alcoholic hepatitis (AH). Blood was collected from 15 patients with AH (mean bilirubin 17.6+/-3.5 mg/dl; normal 0. 2-1.0 mg/dl) on admission and at time points for up to 6 months. Peripheral blood monocytes were isolated and MCP-1 production assessed by measuring MCP-1 concentrations in monocyte culture supernatants after overnight (20 h) incubation. Monocytes from normal subjects did not product detectable MCP-1 unless stimulated with endotoxin (LPS;5 microg/ml). The mean level of constitutive MCP-1 from AH patient monocytes was 4694+/-2432 pg/ml 20 h on admission. The mean MCP-1 level for LPS-treated monocytes was 4903+/-1540 pg/ml 20 h for normal subjects and was significantly elevated in AH patients to 11589+/-3266 pg/ml/20 h. AH patient monocyte MCP-1 production was decreased in vitro when monocytes were treated with N-acetylcysteine (5 mM) and also decreased over the 6-month study as the patients improved clinically. MCP-1 plasma levels were below the detection limits of the assay used in both AH patients and normal subjects. Thus, monocytes from AH patients not only constitutively product MCP-1, but also produce higher levels of MCP-1 with endotoxin stimulation. Further studies are needed to clarify the role of MCP-1 in the activation and hepatic infiltration of mononuclear cells in alcoholic liver disease.

Acetylcysteine↗
PubMed1999

Antioxidants attenuate nuclear factor-kappa B activation and tumor necrosis factor-alpha production in alcoholic hepatitis patient monocytes and rat Kupffer cells, in vitro.

UNLABELLED: There is increased tumor necrosis factor-alpha (TNF) activity in alcoholic hepatitis (AH). OBJECTIVES: To examine the effects of antioxidants and glutathione enhancing agents on NF-kappaB activation and TNF production in Kupffer cells and monocytes. DESIGN AND METHODS: Isolated rat Kupffer cells and peripheral blood monocytes from AH patients were treated in vitro. NF-kappaB activation was assessed by electrophoretic mobility shift assay and TNF was measured in cell culture supernatants. RESULTS: Monocytes from AH patients had greater TNF production compared to normal volunteers. Pretreatment with antioxidants or gluathione enhancing agents inhibited TNF production and NF-kappaB activation in both monocytes from normal and AH patients as well as in rat Kupffer cells. CONCLUSIONS: There may be a therapeutic role for antioxidants or glutathione enhancing agents in disease states with increased TNF activity such as AH.

Animals↗
PubMed1999

Effects of exogenous superoxide anion and nitric oxide on the scavenging function and electron microscopic appearance of the sinusoidal endothelium in the isolated, perfused rat liver.

BACKGROUND/AIMS: Functional and morphological alterations of the hepatic sinusoidal endothelial cell occur in several models of experimental liver injury and in clinical settings. The causes of these alterations are multiple. The aim of this study was to test the hypothesis that the early functional impairment and morphological alterations of the sinusoidal endothelial cell and hepatic sinusoid associated with liver injury are mediated by free radical species, such as superoxide anion and nitric oxide. METHODS: Isolated rat livers were perfused by recirculation with hemoglobin-free, Krebs-Henseleit bicarbonate buffer and presented with a source of superoxide anion (xanthine oxidase+hypoxanthine) or nitric oxide (S-nitroso-N-acetyl penicillamine). Hyaluronan uptake (an index of sinusoidal endothelial cell scavenging function), thiobarbituric acid-reactive substances content of the tissue (a marker of lipid peroxidation), reduced and oxidized glutathione (a marker of the thiol system oxidation/reduction state), lactate dehydrogenase and alanine aminotransferase activities (markers of cytolysis), as well as scanning and transmission electron microscopic appearance of the sinusoid were evaluated. RESULTS: At the high concentrations used, both free radical generating systems suppressed hyaluronan uptake, increased malondialdehyde content of the tissue, enhanced the release of both liver enzymes, decreased the total glutathione content of the liver, and altered the ratio of reduced/oxidized glutathione. Both free radical species induced dose-dependent morphological alterations of the sinusoid, consisting of the appearance of large gaps replacing the sieve-plated fenestration. CONCLUSIONS: The free radical species-induced functional impairment and morphological alterations of the liver sinusoid, presented in this study, closely resemble the early in vivo changes associated with liver injury under a variety of conditions, such as preservation and reperfusion, or administration of hepatotoxicants such as D-galactosamine, Gram-negative bacterial lipopolysaccharides, acetaminophen, alcohol and others. Therefore, we suggest that early liver sinusoid injury, observed under these conditions, can be attributed to the action of free radicals, such as superoxide anion and nitric oxide.

Alanine Transaminase↗
PubMed1999

Increased urinary F2-isoprostane excretion in alcoholic liver disease.

Free radical-induced lipid peroxidation (LP) is thought to be important in alcoholic liver disease (ALD), however, direct demonstration of increased LP in patients with ALD has been difficult. Quantification of F2-isoprostanes (F2-isoP), prostanoids produced by peroxidation of arachidonic acid, in plasma and urine are sensitive and specific indices of LP in vivo. To determine if LP is increased in ALD, 24-h urinary excretion of F2-isoPs were measured in 10 patients hospitalized because of ALD. The mean urinary excretion of the F2-isoP in the ALD patients' urine was 9.6+/-3.5 ng/mg creatinine, which was significantly elevated compared to controls' urinary excretion, which was 1.7+/-0.2 ng/mg creatinine (p<.01). The urinary excretion of F2-isoP decreased to 3.6+/-1.1 ng/mg creatinine as the patients improved clinically with abstinence over the 1-month period. These data suggest that lipid peroxidation, as assessed by this noninvasive method, is increased in patients with acute ALD and decreases with time as the patients improve clinically with abstinence.

Alanine Transaminase↗
PubMed1999

Modulation of caspase-3 activity and Fas ligand mRNA expression in rat liver cells in vivo by alcohol and lipopolysaccharide.

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.

Alanine Transaminase↗
PubMed1998

Tumor necrosis factor and alcoholic liver disease.

Increased levels of hepatic and serum tumor necrosis factor (TNF) have been documented in animal models of alcoholic liver disease and in human alcoholic liver disease. This dysregulated TNF metabolism has been postulated to play a role in many of the metabolic complications and the liver injury of alcoholic liver disease. One potential therapy for alcoholic liver disease may be agents that downregulate TNF production or block TNF activity. Indeed, agents such as prostaglandins and glucocorticoids (both inhibit TNF production) have been used in both human liver disease and experimental models of liver injury, and anti-TNF antibody has recently been shown to attenuate the hepatotoxicity in an animal model of alcoholic-related liver disease. In this study, we demonstrate that a simple ex vivo system can be used to initially assess potential efficacy of anticytokine agents when administered to humans. Both prednisone and a prostaglandin analog were effective in downregulating TNF and interleukin-8 production. The liver is normally resistant to TNF cytotoxicity. Sensitivity to TNF cytotoxicity is thought to occur when there is inadequate production of hepatic protective factors. In this study, we showed that, when patients with acute alcoholic hepatitis were matched with trauma patients for serum levels of interleukin-6, they had similar depressions in the negative acute phase protein, albumin, but markedly different increases in the major acute phase protein, C reactive protein. Patients with alcoholic hepatitis had a very blunted response. We also showed that inhibiting activation of the redox sensitive transcription factor NFkappaB sensitizes to TNF-induced hepatocyte death in vitro. This transcription factor is important for the production of both cytokines and many acute phase protective factors. Several hepatic protective factors are induced by TNF. One possible mechanism for liver injury in alcoholic hepatitis may be inadequate generation of hepatic protective factors. Our future understanding of mechanisms of alcoholic liver disease will involve understanding the balance between noxious and protective factors in the liver, and this should lead to rational therapy for this disease process.

Animals↗
PubMed1998

Alcoholic liver disease. Treatment strategies for the potentially reversible stages.

Even modest alcohol ingestion can increase the risk of steatosis, and long-term, excessive consumption can lead to alcoholic hepatitis and eventually cirrhosis. Most patients with clinically significant alcoholic liver disease have histologic findings typical of all three conditions. The only clearly beneficial treatment is abstinence from alcohol. Abstinence in combination with proper nutrition and general supportive care is state of the art. Steatosis is reversible upon withdrawal of alcohol, but alcoholic hepatitis can persist even with abstinence and may progress to cirrhosis. Corticosteroid therapy may reduce short-term mortality rates in patients with moderate or severe alcoholic hepatitis who have hepatic encephalopathy but no evidence of infection or gastrointestinal bleeding. Treatment with colchicine may decrease the risk of cirrhosis; however, once cirrhosis has developed, the liver damage is irreversible. The prognosis is improved with abstinence, but complications (e.g., ascites, gastrointestinal bleeding) often occur. Liver transplantation may be considered in patients with severe complications.

Humans↗
PubMed1998

Use of transfected liver cells to evaluate potential mechanisms of alcohol-induced liver injury.

There is increased activity of the proinflammatory cytokine, tumor necrosis factor (TNF) in alcoholic liver disease (ALD). Hepatic neutrophil infiltration is a principal injurious manifestation of ALD. TNF can induce cellular oxidative injury directly, and indirectly by inducing neutrophil chemotactic factor (IL-8) production by hepatocytes. IL-8 activates and chemotactically attracts neutrophils to the liver where they release oxidizing substances. Patients with ALD also have decreased protective factors for cellular oxidative injury. Manganous superoxide dismutase (MnSOD) is an antioxidant protective factor. The objectives of these studies were to investigate mechanisms for induction of an injurious factor (IL-8) and a protective factor (MnSOD) in the HepG2 human hepatoma cell line. In the first set of experiments, IL-8 gene reporter constructs were used to transiently transfect a derivative (MVh2E1-9) of the HepG2 cell line which expresses P-4502E1 and metabolizes ethanol. Inactivation of the NF-kappaB and 3'NF-IL-6 DNA binding sites decreased IL-8 gene transcriptional activation in response to TNF while inactivation of the 5'NF-IL-6 binding site increased IL-8 gene transcriptional activity in response to TNF. This system may be useful to assess the effects of ethanol on TNF-induced hepatocyte IL-8 production. In the second set of experiments, HepG2 cells were cultured in 25 to 100 mmol concentrations of ethanol. Both TNF and ethanol increased HepG2 cell MnSOD activity in short-term (72 hr) cultures with ethanol. However, after long-term (10 weeks) culture with ethanol, there was no induction of MnSOD by ethanol and there was a diminished induction of MnSOD in response to TNF. Further studies are needed to assess the effect of this diminished induction of MnSOD with chronic ethanol culture on HepG2 cell susceptibility to TNF cytotoxicity. We conclude that transfected liver cell lines can be used to evaluate mechanisms for increased injurious factors and decreased protective factors in alcoholic liver injury.

Animals↗
PubMed1998

Hyperhyaluronanemia in alcoholic hepatitis is associated with increased levels of circulating soluble intercellular adhesion molecule-1.

The purpose of this study was to evaluate the role of the sinusoidal endothelial cell (SEC) during the clinical course of alcoholic hepatitis. Twenty consenting patients (mean age: 49.4 +/- 11.0 years) with moderate or severe hepatitis were studied. The patients were selected and characterized according to their history of drinking and laboratory profile, including serum aminotransferases, bilirubin, total white blood cell and neutrophil count, and prothrombin times. C-reactive protein and interleukin-6 were also measured as markers of the hepatic acute phase response. A marker of the SEC functional state, the circulating level of hyaluronan, was measured in parallel with the circulating levels of soluble intercellular adhesion molecule (sICAM)-1 over a 6-month observation period. All patients were hospitalized for the first month and encouraged to abstain from drinking for the duration of the study. The initial increased levels of both hyaluronan (542 +/- 32 ng x ml(-1) serum) and sICAM-1 (488 +/- 70 ng x ml(-1) serum), gradually fell during the 6-month observation period, eventually reaching values close to those seen in healthy subjects. A positive correlation was obtained between changes in these two markers of SEC function/activation on the one hand, and between these two tests and bilirubin, on the other hand. These data indicate that abnormalities of SEC function/activation, as reflected by serum hyaluronan and siCAM-1, are prominent in alcoholic hepatitis, and these alterations improve within relatively short periods of time after cessation of alcohol consumption.

Adult↗
PubMed1997

Cytokines and alcoholic liver disease.

Chemical messengers called cytokines play an important role during the body's initial response to infection (i.e., acute inflammation). Cytokines attract and activate components of the immune system, promote blood clotting, and facilitate the release of additional chemical messengers. In addition, cytokines induce the liver to shift its physiological function, emphasizing inflammatory and immune responses at the expense of normal metabolism. Alcohol consumption may cause excessive cytokine production in the liver, leading to inflammatory liver disease. Researchers are seeking ways to moderate the toxic effects of cytokines while sparing their protective functions.

Animals↗
PubMed1995

In vitro tumor necrosis factor cytotoxicity in Hep G2 liver cells.

Tumor necrosis factor-alpha (TNF-alpha) is a mediator of liver injury. The objective of this study was to develop an in vitro model of TNF-mediated liver cell injury using the Hep G2 cell line. Hep G2 cells normally are insensitive to TNF cytotoxicity, but they were rendered susceptible, or sensitized, to TNF cytotoxicity by inhibitors of RNA and protein synthesis. The concentration of TNF required to kill 50% of Hep G2 cells sensitized with 0.8 mumol/L actinomycin D (Act D) was 35 pmol/L compared with 5 pmol/L for LM fibroblasts, a classic target cell used in TNF cytotoxicity bioassays. Similarly, TNF cytotoxicity occurred in Hep G2 cells sensitized with cycloheximide (CHX), and cytotoxicity to both inhibitors was dose dependent. Both protein and RNA synthesis were inhibited in Hep G2 cells by the concentrations of CHX and Act D associated with TNF cytotoxicity. Hep G2 cells pretreated with TNF alone and later exposed to normally toxic concentrations of TNF with DACT did not develop cytotoxicity. Thus, in vitro tolerance to TNF was induced. Cytotoxicity also was more severe at modestly increased temperatures (39 degrees C versus 37 degrees C), which may have clinical relevance to hepatic decompensation during febrile episodes. We suggest that the Hep G2 cell line sensitized by inhibiting RNA and protein synthesis is a useful in vitro model for evaluating mechanism(s) of TNF-mediated liver cell injury.

Cell Survival↗