Severe hepatocytotoxicity linked to denosumab.

OBJECTIVE: Denosumab (Prolia, Amgen, Thousand Oaks, CA, USA) is a fully human antibody to the receptor activator of nuclear factor-KB ligand (RANKL). We present a case of submassive hepatic necrosis with evidence implicating cytokine induction resulting from an immune reaction to denosumab. CASE REPORT: A 72-year-old lady presented with elevated liver enzymes. One month previously, she received a s/c administration of 60 mg of denosumab. Viral hepatitis A, B and C and human herpes viruses 6-7 were negative as were routine autoimmune serology. Transaminases reached more than 50 x ULN, and gamma-glutamyl-transpeptidase (GGT) increased to more than 30 x ULN. Serum bilirubin reached 13.8 mg/dL. The serum albumin level decreased to 2.8 g/L. Prednisone (40 mg) and ursodeoxycholic acid (900 mg) were administered. The Naranjo Adverse Drug Reaction probability score was 6, consistent with a probable adverse drug reaction. A liver biopsy revealed sub-massive hepatic necrosis consistent with drug-induced liver injury (DILI). During steroid tapering, there was a slow decline in the levels of both the transaminases and the GGT, and a concomitant increase in the serum albumin. A month after stopping prednisone and ursodeoxycholic acid, there was an acute increase in the level of the transaminases and a decrease in the serum albumin. Steroid reintroduction resulted in normalization of the liver enzymes and synthetic capacity. A lymphocyte toxicity assay to denosumab was demonstrated a hypersensitivity reaction to denosumab resulting in 31% toxicity. The control patient showed no toxicity to denosumab. Cytokine levels (pg/mL) were as follows: Interleukin (IL)1 was 1193 (normal-24.5), IL8 357 (20-60), RANKL 224 (60-80), RANTES 215 (15-50), TNF-a 850 (25-50), TGF-b 546 (20-40), VEGF 735 (25-30). Serum RANKL was markedly reduced in the presence of denosumab (16 pg/mL). The elevated markers of apoptosis ccK18(M-30)(68-132) 140 IU and K18 apoptosis+ necrosis (M65) (62-213) 322 U/L implicate necrosis. CONCLUSIONS: We suggest that RANKL inhibition can produce severe hepatic necrosis together with an increase in proinflammatory cytokines.

Pyrrolizidine alkaloids enhance alcohol-induced hepatocytotoxicity in vitro in normal human hepatocytes.

OBJECTIVE: Herbal remedies containing pyrrilidozine alkaloids (PA)s can induce liver damage, including hepato-sinusoidal obstruction syndrome (HSOS) or veno-occlusive liver disease (VOD). Some individuals misusing alcohol consume also teas and/or herbal remedies containing PA. The interaction or additive toxicity of alcohol to PA toxicity needs to be addressed. The objectives of this study are 1) to review the scientific literature on the PA-induced liver toxicity; 2) identify possible mechanism(s) involved in PA-induced hepatocytotoxicity in the presence or absence of ethanol (EtOH) in vitro in normal human hepatocytes (NHH) in primary culture. To respond to the first objective, we systematically search all the literature engines (PubMed, Google Scholar) for liver induced damage due to PAs and summarize the results in an introductory systematic review. ORIGINAL ARTICLE EXPERIMENTAL DESIGN AND METHODS: Cells were exposed to one dose of 100 mmol/L EtOH for 24 hrs and to 2 doses of 100 mmol/L EtOH for consecutive 24 hrs periods, in the presence or absence of PAs (10 mg/mL), or the caspase-3 inhibitor IDN-1965 (50 µmol/L). Cells were analyzed for apoptosis by light microscopy, immuno-histochemistry, measuring cytokeratin-18 fragmentation, and transmission electron microscopy (TEM) (6000 cells/treatment). Cytotoxicity was determined using succinate dehydrogenase (SDH) activity, an enzyme specific to the mitochondria. RESULTS: In NHH cells, a 100 mmol/L dose of Et-OH resulted in 22±2.5 apoptosis (p<0.001 vs. control). Two consecutive doses of 100 mmol/L Et-OH for 24 hrs each caused 36±3.0% apoptosis (p<0.001 vs. control and p<0.05 vs. one dose Et-OH). Pre-treatment with 50 µmol/L caspase inhibitor significantly reduced Et-OH-induced apoptosis [12±1.5% in 100 mmol/L (p<0.05) and 20±4.0% in 2×100 mmol/L (p<0.001)]. In addition, pre-treatment with 50 µmol caspase inhibitor in cells treated with PA + EtOH reduced apoptosis significantly (vs. non-exposed to caspase-inhibitor): Δ -22±3.0 % (p<0.05). HPC significantly decreased apoptosis compared to conditions lacking this supplementation in cells treated with EtOH-exposed cells present ballooning, Mallory bodies, changes in mitochondrial cristae and apoptosis by TEM. Pre-treatment with 50 µmol caspase inhibitor significantly reduced 100 mmol/L EtOH-induced (one dose) in NHH by 14±0.5% (p<0.05) compared to cells not exposed to the caspase-inhibitor. In cells treated concomitantly with PA and EtOH 100 mM Mallory-bodies and apo-necrotic cells have been observed. Pre-treatment with 50 µmol caspase inhibitor reduced the mitochondrial damage. A significant depletion in glutathione (GSH) was observed in Et-OH treated cells after 1 and 2 treatments (p<0.001 vs. control). Treatment with Et-OH enhanced PA-induced GSH-depletion and resulted in a significant increase in PA-induced cytotoxicity (p<0.001 vs. Et-untreated cells). Exposure to EtOH increased the cell culture media levels of the pro-inflammatory cytokine TNF. PA + EtOH-treated cells increased TNF-α levels in media compared to EtOH alone [86±8 vs. 53±5 pg/mL in cells exposed to 100 mmol/L EtOH (p<0.05) and 218±14 vs. 179±8 pg/mL in cells exposed to 2×100 mmol/L EtOH (p<0.05)]. CONCLUSIONS: PA up-regulates EtOH-induced hepatocytotoxicity by inducing the inflammatory cytokines and enhancing the apoptotic effects of ethanol. There is a need for monitoring herbal medicine in order to optimize traditional medicine use and maximize the clinical benefits. Additionally, there is necessary to communicate to physicians the possible negative results of herbal remedies use. Also, the interactions between herbal remedies and drugs of misuse should be communicated to consumers.

Circulating soluble cytochrome c in liver disease as a marker of apoptosis.

OBJECTIVES: To measure levels of soluble cytochrome c, a clinical marker of apoptosis in patients with liver disease; determine whether soluble cytochrome c is derived from the liver; and correlate soluble cytochrome c level with histology and disease activity. DESIGN: Laboratory research study with comparison group. SETTING: Liver Institute, at the Rabin Medical Center, Israel, and In Vitro Toxicology Laboratory, Canada. SUBJECTS: A total of 108 patients with liver disease and 30 healthy controls. INTERVENTIONS: Paired hepatic and portal vein samples were taken via the transjugular vein in patients after liver biopsy and transjugular intrahepatic portacaval shunt, and bile from patients with external biliary drainage. Soluble cytochrome c was measured with an enzyme-linked immunosorbent assay in peripheral blood. Apoptotic cells in liver tissue were identified by morphological criteria and quantitated with the dUTP nick-end-labelling (TUNEL) assay. MAIN OUTCOME MEASURES: Soluble cytochrome c level by type of liver disease by clinical and histological findings. RESULTS: Soluble cytochrome c concentration (mean 187.1 +/- 219.5 ng x mL(-1)) was significantly higher in patients with liver disease than in controls (39.8 +/- 35.1 ng x mL(-1); P = 0.0001), with highest levels in the primary sclerosing cholangitis group (mean 1041.0 +/- 2844.8 ng x mL(-1); P = 0.001). Cytochrome c levels were correlated with serum bilirubin, alkaline phosphatase, creatinine levels, necroinflammatory score and apoptotic index, but not with serum alanine aminotransferase and synthetic liver function tests. In the 16 paired samples, soluble cytochrome c level was higher in the hepatic (mean 267.9 +/- 297.0 ng x mL(-1)) than the portal vein (mean 169.2 +/- 143.3 ng x mL(-1)), and it was highly detectable in bile (mean 2288.0 +/-4596.0 ng x mL(-1)) (P = 0.001). Untreated patients with chronic viral hepatitis (B and C) had significantly higher levels (mean 282.8 +/-304.3 ng x mL(-1)) than treated patients (77.9 +/- 35.8 ng x mL(-1); P = 0.001). CONCLUSIONS: Soluble cytochrome c levels are increased in different types of liver disease. Soluble cytochrome c is probably derived from the liver and secreted into the bile. Levels correlate with the apoptotic index and are affected by antiviral treatment. Soluble cytochrome c may serve as a serum marker of apoptosis.

Digestive Disease Week 2000 conference report: New treatments for chronic hepatitis C.

At the Digestive Disease Week (DDW) conference and 101st Meeting of the American Gastroenterological Association in San Diego, California, May 22 to 24, 2000, over 400 abstracts on hepatitis C were submitted for posters or oral presentations to the American Association for the Study of Liver Diseases. A substantial portion of the program discussed the treatment of chronic hepatitis C, focusing on interferon and ribavirin combination therapy, substitution of amantadine for ribavirin, and the use of pegylated interferons. In randomized, clinical trials, combination therapy with interferon- alpha-2B and ribavirin results in a greater sustained virilogical response than treatment with interferon alone. Combination therapy is generally safe and well tolerated, but there is a need to monitor patients throughout treatment for hemolytic side effects, depression, and weight and lipid profiles. In the present review some background information on chronic hepatitis C is given and some of the more relevant abstracts presented on this subject at the DDW conference are highlighted.

Immunopathogenesis of hepatitis C viral infection: Th1/Th2 responses and the role of cytokines.

Hepatitis C virus is a common cause of hepatocellular injury that is associated with complex and vigorous immunologic mechanisms. Both humoral and cell-mediated immune responses participate in the host defense against hepatitis C viral infection, but there is increasing recognition of the roles played by the cell-mediated response, and in particular the cytokine system, in the immunopathogenesis of chronic hepatitis C. The cell-mediated response depends on cytotoxic and helper T-cell activity, and functions through the actions of cytokines to regulate macrophages, natural killer cells, and antiviral cellular proteins. Cytokines produced in the liver are essential in defending the host against hepatitis C invasion, but they have also been implicated in the hepatocellular injury seen in the majority of chronically infected patients. Cytokines are thought to be involved in the pathogenesis of hepatitis C under conditions where the virus can mutate effectively and evade T-cell immune defense mechanisms. Persistent infection upsets the balance between immunostimulatory and inhibitory cytokines which can prolong inflammation and lead to necrosis, fibrosis, and chronic liver disease.

Cytokines as predictors for sustained response and as markers for immunomodulation in patients with chronic hepatitis C.

OBJECTIVES: (i) To characterize serum cytokine levels of tumor necrosis factor alpha (TNF alpha), interleukin 6 (IL 6), IL 8 and IL 12 in non-cirrhotic patients with chronic hepatitis C, (ii) to correlate the levels of these cytokines with the degree of the disease at the basal level, (iii) to correlate these levels with the response to therapy, (iv) to compare profiles of cytokines in monotherapy (MT) versus combination therapy (CT), and (v) to compare the immunomodulatory effects of MT versus CT. DESIGN AND METHODS: 47 patients were enrolled in the study. The controls were 120 volunteers (recruited from students and staff) that did not present HCV RNA positive and were not known to suffer any other metabolic disease. Thirty patients formed the other group of controls, with alcoholic liver disease (ALD). Serum cytokine levels were assessed using enzyme-linked immunosorbent assay (ELISA). RESULTS: The sustained responders (SRs) have basal values much lower than relapsed responders (RRs) and non-responders (NRs) regardless of the therapy. CONCLUSIONS: Cytokines can be used as non-invasive markers for sustained response and as monitors for the outcome of therapy.

Cytokine network in nonresponding chronic hepatitis C patients with genotype 1: role of triple therapy with interferon alpha, ribavirin, and ursodeoxycholate.

OBJECTIVE: (i) to characterize the profile of tumor necrosis factor alpha (TNF alpha), interleukin-6 (IL-6), IL 10, Fas-ligand and transforming growth factor beta (TGF beta), chronic hepatitis C (HCV) patients with genotype 1; (ii) to determine the influence of triple therapy (TT) with interferon alpha (IFN alpha) + ribavirin + ursodeoxycholic acid on these cytokines and (iii) to establish the relationship between the pro-inflammatory cytokines and the outcome of treatment. DESIGN AND METHODS: 22 patients infected with HCV-genotype 1 a/b and non responsive to IFN-alpha monotherapy were enrolled in the TT. The controls were 49 HCV naïve patients with genotype 1 a/b. Cytokine levels were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: The baseline TNF alpha values (pg/mL) in the sustained responders (SRs) (63+/-3) were significantly lower than non-responders (NRs) (140+/-16) (p < 0.001). Baseline Fas (ng/mL) levels were also lower in SRs (4.3+/-0.2) than NRs (5.4+/-0.4) (p < 0.05). CONCLUSIONS: Fas and TNF alpha may be used as serological markers of inflammation and effectiveness of therapy.

Accumulation of macrophages in primary sclerosing cholangitis.

OBJECTIVES: To determine what changes are occurring in patients with primary sclerosing cholangitis (PSC) by examining perisinusoidal macrophages (Kupffer cells) in liver biopsies; 2-to measure transforming growth factor beta (TGFbeta) as a marker of fibrosis in these patients. DESIGN AND METHODS: Transmission electron microscopy and immunohistochemistry of 15 PSC, 26 primary biliary cirrhosis (PBC), 30 alcoholic liver disease (ALD) and 51 with normal histology was used. Five PSC, 30 ALD and 120 normal volunteers were sampled for serum levels of TGFbeta. RESULTS: There was a three-fold increase in relative numbers of Kupffer cells in PSC compared to PBC and to patients whose livers had normal histology. In PSC there was an accumulation of perisinusoidal macrophages, which was not associated with focal necrosis or with cholestasis. The levels of TGFbeta in PSC were 54 +/- 2 in cirrhotic versus 34 +/- 5 in non-cirrhotic patients (p < 0.005). CONCLUSION: The persistent activation of these macrophages may lead to the chronic release of TGFbeta and contribute to chronic inflammation, fibrosis and cirrhosis.

CYP2E1-mediated modulation of valproic acid-induced hepatocytotoxicity.

OBJECTIVES: To determine the cytotoxicity of valproic acid (VPA) and its metabolite, 4-ene-valproic acid (4-ene-VPA) in human hepatoblastoma cells (Hep G2), and to study the modulatory effect of cytochrome P450 2E1 induction in this model. METHODS: Cells were exposed to VPA or 4-ene-VPA in the presence of either ethanol (EtOH), or EtOH combined with disulphiram (DS). Some cells were exposed to alpha-fluoro-VPA or to alpha-fluoro-4-ene-VPA in the absence of CYP2E1 inducers. Apoptosis and necrosis were measured by analyzing 6000 cells per sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. RESULTS: VPA + EtOH increased VPA cytotoxicity. 4-ene-VPA + EtOH significantly increased toxicity, while DS + EtOH significantly reduced this toxicity. Alpha-fluorinated analogues reduced cytotoxicity compared to the corresponding VPA compounds. Neither VPA nor alpha-fluorinated VPA increased the release of IL-6 or TNF-alpha in media. A significant increase in the release of TNF-alpha was observed in cells exposed to 4-ene-VPA that further increased with EtOH exposure. CONCLUSIONS: Cells exposed to 4-ene-VPA experience greater cytotoxicity than those treated with VPA. Cytochrome P450 2E1 inducers enhance toxicity in VPA-exposed cells, while alpha-fluorination of VPA diminishes cytotoxicity by directly interfering with the beta-oxidation of the 4-ene-VPA metabolite.

Signaling for ethanol-induced apoptosis and repair in vitro.

OBJECTIVES: To evaluate whether caspases are involved in ethanol (EtOH)-induced apoptosis and if polyenylphosphatidylcholine (PPC) affects apoptosis, in vitro in Hep G2 cells. METHODS: Cells were treated with 100 mmol/L EtOH for 24 h and with 2 doses of 100 mmol/L EtOH (1/24 h) in the presence of absence of 20 mmol/L of PPC or 50 micromol/L caspase 3 inhibitor (IDN). Cells were analyzed for apoptosis by transmission electron microscopy (TEM) 6000 cells/treatment, DNA fragmentation by ELISA and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (T dt-mediated d-UTP) nick-end-labeling, TUNEL. RESULTS: 100 mmol/L dose of EtOH resulted in 22 +/- 2.5% (p < 0.001) apoptosis (vs. control). Two consecutive doses of 100 mmol/L EtOH for 24 h each caused 36 +/- 3.0% (p < 0.001 vs. control and p < 0.05 vs. one dose). PPC significantly reduced apoptosis (vs. non exposed to PPC): 100 mmol/L -12 +/- 1.5% (p < 0.05) and 2 x 10(-)(0) mmol/L -20 +/- 2.0% (p < 0.001). Pretreatment with 50 micromol caspase inhibitor reduced EtOH-induced apoptosis in a similar proportion. CONCLUSIONS: PPC downregulates EtOH-apoptosis by a mechanism similar to caspase inhibition.

The toxicity of Callilepis laureola, a South African traditional herbal medicine.

OBJECTIVES: To review the literature on the toxicity of Callilepis laureola, and to assess the cytotoxicity of C. laureola in human hepatoblastoma Hep G2 cells in vitro. DESIGN AND METHODS: Cells were incubated for up to 48 h in the presence of increasing concentrations of an aqueous extract of C. laureola (0.3-13.3 mg/mL). Cytotoxicity was quantitated spectrophotometrically by the metabolism of the tetrazolium dye MTT. Cytoviability of the control cells was considered to be 100%. RESULTS: C. laureola produced cytotoxicity in a concentration-dependent manner. Cytotoxicity was significant at all concentrations tested (0.3-2.5 mg/mL, p < 0.05 vs. controls and 3.3-13.3 mg/mL, p < 0.0001 vs. controls). After 6 h, 100% toxicity was observed at a concentration of 6.7 mg/mL. CONCLUSION: C. laureola causes significant cytotoxicity in Hep G2 cells in vitro. These findings are in accordance with the observed hepatotoxicity in clinical cases of C. laureola poisoning.

Mechanisms of alcoholic liver disease: cytokines.

This article represents the proceedings of a workshop at the 2000 ISBRA Meeting in Yokohama, Japan. The chair was Manuela G. Neuman. The presentations were (1) New aspects of hepatic fibrosis, by D. A. Brenner; (2) Cellular immune response in hepatitis C models, by B. Rehermann; (3) The role of interleukin-10 in acute alcoholic hepatitis, by J. Taieb, S. Chollet-Martin, M. Cohard, J. J. Garaud, and T. Poynard; (4) Cytokine-mediated apoptosis in vitro, by M. G. Neuman; (5) Signaling for apoptosis and repair in vitro, by G. G. Katz, R. G. Cameron, N. H. Shear, and M. G. Neuman; (6) Interferons activate the P42/44 mitogen-activated protein kinase and Janus Kinase signal transducers and activation of transcription (JAK-STAT) signaling pathways in hepatocytes: Differential regulation by acute ethanol via a protein kinase C-dependent mechanism, by B. Gao; (7) Genetic polymorphisms of interleukin-1 in association with the development of Japanese alcoholic liver disease, by M. Takamatsu, M. Yamauchi, M. Ohata, S. Saito, S. Maeyama, T. Uchikoshi, and G. Toda; and (8) Increased levels of macrophage migration inhibitory factor in sera from patients with alcoholic liver diseases, by T. Kumagi, S. M. F. Akbar, M. Abe, K. Michitaka, N. Horiike, and M. Onji.

Apoptosis in diseases of the liver.

Apoptosis, or programmed cell death, and the elimination of apoptotic cells are crucial factors in the maintenance of liver health Apoptosis allows hepatocytes to die without provoking a potentially harmful inflammatory response In contrast to necrosis, apoptosis is tightly controlled and regulated via several mechanisms, including Fas/Fas ligand interactions, the effects of cytokines such as tumor necrosis factor alpha (TNF-alpha) and transforming growth factor beta (TGF-beta), and the influence of pro- and antiapoptotic mitochondria-associated proteins of the B-cell lymphoma-2 (Bcl-2) family. Efficient elimination of apoptotic cells in the liver relies on Kupffer cells and endothelial cells and is thought to be regulated by the expression of certain cell surface receptors. Liver disease is often associated with enhanced hepatocyte apoptosis, which is the case in viral and autoimmune hepatitis, cholestatic diseases, and metabolic disorders. Disruption of apoptosis is responsible for other diseases, for example, hepatocellular carcinoma. Use and abuse of certain drugs, especially alcohol, chemotherapeutic agents, and acetaminophen, have been associated with increased apoptosis and liver damage. Apoptosis also plays a role in transplantation-associated liver damage, both in ischemia/reperfusion injury and graft rejection. The role of apoptosis in various liver diseases and the mechanisms by which apoptosis occurs in the liver may provide insight into these diseases and suggest possible treatments.

A novel lymphocyte toxicity assay to assess drug hypersensitivity syndromes.

OBJECTIVES: To validate the novel lymphocyte toxicity assay (LTA) based on the mitochondrial succinate dehydrogenase (SDH) activity vs. the LTA by using trypan blue exclusion and to determine the utility of the assay to confirm drug hypersensitivity syndrome (DHS) to sulphonamides (SMX) and aromatic anticonvulsants. METHODS: Incubation of patient lymphocytes, with or without murine hepatic microsomes with anticonvulsants or SMX. The viability of lymphocytes was based on SDH activity that can be measured spectrophotometrically. The percentage of cells displaying cytotoxicity compared to controls (cells treated only with drug) was calculated. Seventy-two immunocompetent and 16 immunocompromised (HIV) patients with DHS to SMX were sampled. The results were validated vs. 26 controls that had not experienced DHS to SMX. Sixty-two patients who had DHS to anticonvulsants were compared with 24 controls that did not have any DHS to the same anticonvulsants. RESULTS: The results showed a very good percentage of sensitivity 98 and specificity 96 with a kappa-score of 0.96. LTA higher than 13.5% was considered positive for the immunocompetent population and LTA higher than 22% was positive for the immunocompromised population. In two of the 26 controls, LTA was positive. CONCLUSION: The high quantitative kappa-value 0.96 emphasizes that the novel LTA is at least as good as the trypan blue assay. The latter is labor intensive, time consuming, and prone to human error. The new assay is objective, faster, and has been reproducible in assessing cytotoxicity.

Inducers of cytochrome P450 2E1 enhance methotrexate-induced hepatocytoxicity.

OBJECTIVES: To study the effect of cytochrome P450 2E1-inducers on methotrexate (MTX)-induced cytotoxicity in human hepatocytes, and investigate the role of silymarin in preventing this toxicity. DESIGN AND METHODS: Cells were exposed to MTX in the presence of either ethanol (EtOH) or acetaminophen (APAP), or either combined with silymarin (S). Apoptosis and necrosis were measured by analyzing 6000 cells/sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. Cytokine expression was measured by RT-PCR. Gluthatione (GSH) content was determined in cytosolic (c) and mitochondrial (m) fractions. RESULTS: MTX+EtOH and MTX+APAP increased MTX cytotoxicity 2.9-fold and 1.9-fold, respectively. S abolished this toxicity. MTX + EtOH increased the release of IL 6, IL 8 and TNF alpha by 1.0, 1.2, and 1.1 times, respectively. Cytokine expression was upregulated versus control for IL 6 (22%), IL 8 (38%), and TNF alpha (29%). Addition of 0.5 mmol/L S downregulated TNF alpha expression and reduced cytokine release. TNF alpha increased cytotoxicity by 22%, while anti-TNFalpha antibody eradicated it. MTX+EtOH depleted 45% mGSH (0 < 0.001) while S replenished it to 87% (p < 0.001), when both were compared to control levels. CONCLUSIONS: Cytochrome P450 2E1-inducers contribute to increase oxidative stress in MTX-exposed cells by increasing TNF alpha and depleting both cGSH and mGSH. This enhances MTX-cytotoxicity and promotes apoptosis.

Predictors of sustained response to alpha interferon therapy in chronic hepatitis C.

OBJECTIVES: To utilize cytokine levels to predict sustained response (SR) to alpha interferon (IFN alpha) therapy in chronic hepatitis C patients, and to determine the relationship between serum tumor necrosis factor alpha (TNF alpha), interleukin (IL) IL 6, IL 8, IL 12, transforming growth factor beta (TGF beta 1) and the degree of liver damage as reflected by traditional markers. DESIGN AND METHODS: Serum cytokine levels were assessed using ELISA in 18 patients included in a controlled clinical trial of IFN alpha. RESULTS: Of the 18 patients, 27% were sustained responders (SR), 27% were response and relapse responders (RR), and 46% were non-responders (NR). Multivariate analysis showed that a low serum TNF alpha level and high serum IL 8 levels were independent factors associated with SR to IFN alpha therapy. Serum TNF alpha level highly correlated with viral load and genotype predictive values (p < 0.001). Therapy lowered the IL 6 and IL 12 profile. TGF beta 1 levels in serum are positively correlated with fibrinogenesis. CONCLUSIONS: IFN alpha therapy modulates immune response to hepatitis C virus, contributing to sustained response.

Ethanol-induced apoptosis in vitro.

OBJECTIVES: The aim is to study the apoptotic process in a human hepatocyte model for ethanol (EtOH)-induced apoptosis. DESIGN AND METHODS: Normal human primary hepatocytes (HPH) and Hep G2 cells were exposed to increasing EtOH. 6000 cells/ sample were analyzed by transmission electron microscopy. RESULTS: Apoptotic cells were observed (mmol/L EtOH): 40: 6 +/-0.5%, 60:13 +/- 2% (p < 0.05), 80: 26 +/- 1% (p < 0.001) (vs. control). Two consecutive doses of 80 mmol/L for 24 h each additionally increased apoptosis 55 +/- 3% (p < 0.0001 vs. control and p < 0.001 vs. single dose). In response to this exposure, there is a stronger apoptotic activity in HPH when compared to Hep G2 (p < 0.05). CONCLUSIONS: In vitro, EtOH-induced apoptosis is regulated by dose level and the frequency of exposure.

Novel morphologic findings in alcoholic liver disease.

OBJECTIVES: To study light and electron microscopic changes in alcohol-liver disease (ALD) patients, and characterize the expression pattern of Kupffer and stellate cells, correlating changes with serum cytokine levels. DESIGN AND METHODS: Liver biopsies studied in 35 ALD patients were compared to 51 normal histology patients. Quantitation was done using immunochemistry for Kupffer cells and morphometry, electron microscopy for stellate cells. ELISA was used to measure serum cytokines in 80 controls and ALD patients. RESULTS: Biopsies of ALD patients confirmed increased number of perisinusoidal, multivesicular, and stellate cells compared to controls. There was a significant increase in the number and activity of multivesicular stellate cells in ALD patients (p < 0.001). These changes were associated with significant increase in the degree of perisinusoidal collagenisation (p < 0.001). The number of Kupffer cells, serum tumor necrosis factor (TNFalpha), interleukins-6 (IL-6) and IL-12 levels, were also significantly higher in ALD patients than controls. CONCLUSIONS: In non-cirrhotic ALD, stellate cells may be involved in lipid transport and a cytokine network may influence liver inflammation.

Ethanol-modulated cytokine production and expression in skin cells exposed to methotrexate.

Evidence suggests a link between alcohol consumption, psoriasis and the response of psoriatic patients to methotrexate (MTX) therapy. Ethanol (EtOH) may play a role in the pathogenesis of psoriasis by upregulating the expression and inducing the local secretion of proinflammatory cytokines, e.g. interleukins IL-1alpha, IL-6, chemokine IL-8 and tumor necrosis factor alpha (TNF-alpha). We investigated whether EtOH or MTX or their combination influence the secretion of these cytokines using normal human primary skin cells (NHPSC) and epidermoid cell line A431. The objectives of this study were: (1) to quantify the differences in cellular changes induced by MTX, (2) to measure the effect of EtOH on MTX toxicity and (3) to determine the relationship between MTX and EtOH exposure and production of proinflammatory cytokines. NHPSC and A431 were incubated with 0-10 mM MTX or alpha-MEM (control) in the presence or absence of 40 mM EtOH. A formazan 3-(4,5-dimethylthiazole-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay was used as a marker for cell viability (control was 100%). Significance was calculated by ANOVA. Cytokine release into media was quantitated by ELISA. After 24 h of MTX exposure, the release of IL-1alpha was unchanged. IL-6 increased 1.7 times in both cultures, and IL-8 increased 1.7 times in NHPSC and 2.1 times in A431. TNF-alpha release increased twice in A431 but not in NHPSC. Human recombinant IL-1alpha and IL-6 for 24 h had no effect, while TNF-alpha reduced cytoviability by 30% in NHPSC and 22% in A431. Anti-TNF-alpha reversed the effect produced by TNF-alpha in NHPSC and reduced it in A431 (11.8%, p < 0.05). We concluded that in vitro in normal human primary keratinocytes, toxicity and inflammatory responses are enhanced by EtOH.

Role of cytokines in ethanol-induced cytotoxicity in vitro in Hep G2 cells.

BACKGROUND & AIMS: As shown previously by us, ethanol (EtOH) causes time- and concentration-dependent reduction in cytoviability. Tauroursodeoxycholic acid (TUDCA) and ursodeoxycholic acid (UDCA) were shown to reduce cytotoxicity. Long-term EtOH exposure leads to immunoregulatory and detoxification impairment. This study aimed to determine the relationship between cytokine (interleukin [IL]-1 alpha and IL-6 and tumor necrosis factor [TNF]-alpha) production and expression, glutathione (GSH) status, and EtOH-induced cytotoxicity on Hep G2 cells. METHODS: Cells were incubated with 80 mmol/L EtOH or alpha-minimal essential medium (control) in the presence or absence of 50 mumol/L TUDCA or UDCA. Cytokine release was quantitated by enzyme-linked immunosorbent assay. Cytokine expression was measured by reverse-transcription polymerase chain reaction. GSH content was determined in both the cytosolic and mitochondrial fractions. RESULTS: After 24 hours of EtOH exposure, the release of IL-1 alpha doubled, that of IL-6 increased 10 times, and that of TNF-alpha increased 3.5 times. Cytokine expression was up-regulated compared with control for IL-1 alpha (42%), IL-6 (26%), and TNF-alpha (52%). Addition of 50 mumol/L TUDCA or UDCA reduced cytokine release and expression. TNF-alpha increased cytotoxicity by 18%. Anti-TNF-alpha antibody almost abolished it. EtOH depleted mGSH levels by 55% (P < 0.001). TUDCA replenished them by 88%. CONCLUSIONS: EtOH up-regulated expression of cytokines in Hep G2 cells is down-regulated by bile acids. Increased amounts of TNF-alpha and depletion in both cytosolic and mitochondrial GSH contribute to EtOH cytotoxicity. Bile acids prevent toxicity.