5 cells possess the molecular machinery to both metabolize and respond to vitamin D. Of special importance is the finding that HCV infection markedly increased the levels of calcitriol in cell cultures. This was not due to increased production of calcitriol, as the level of 1α-hydroxylase was not altered, but rather to the prevention of induction of 24 hydroxylase, the enzyme responsible for the first step in the catabolism of calcitriol. Thus, HCV increases the efficacy of the vitamin D endocrine system of the hepatocyte. It is by now established

that vitamin D promotes innate immune responses associated with pathogen elimination such as macrophage phagocytic function, and TLR2/1, TLR4, and cathelicidin induction in various cell types.39, 40 Our findings that vitamin D induced interferon and synergized with it adds another facet to its activity as an enhancer of innate immunity. Our study unravels selleck chemicals llc an interplay between vitamin D and HCV: on the one hand, viral infection increases the production of the active metabolite of vitamin D and, on the other hand, this metabolite suppresses viral infection. This interplay, together with the finding that vitamin D employs the interferon system to combat

HCV, suggests learn more a physiological role for the hormone in the antiviral arm of hepatic innate immunity. It is maintained that HCV persistence is associated with its ability to evade innate immune defenses by suppressing the RIG-I and TLR3 pathways, thereby impairing interferon production in infected hepatocytes. As mentioned before, Huh7.5 cells

have similar defects in the interferon pathway. Thymidine kinase Interestingly, treatment with vitamin D restored the ability of Huh7.5 cells to produce interferon. It seems plausible that vitamin D may have a similar effect in virus-infected normal hepatocytes, thus counteracting the disruption of the interferon pathway by the virus. It is therefore tempting to assign vitamin D a role in the ongoing coevolutionary arms race between the virus and the host. “
“Transgenic mice expressing dominant-negative retinoic acid receptor (RAR) α specifically in the liver exhibit steatohepatitis, which leads to the development of liver tumors. Although the cause of steatohepatitis in these mice is unknown, diminished hepatic expression of insulin-like growth factor-1 suggests that insulin resistance may be involved. In the present study, we examined the effects of retinoids on insulin resistance in mice to gain further insight into the mechanisms responsible for this condition. Dietary administration of all-trans-retinoic acid (ATRA) significantly improved insulin sensitivity in C57BL/6J mice, which served as a model for high-fat, high-fructose diet–induced nonalcoholic fatty liver disease (NAFLD). The same effect was observed in genetically insulin-resistant KK-Ay mice, occurring in concert with activation of leptin-signaling pathway proteins, including signal transducer and activator of transcription 3 (STAT3) and Janus kinase 2.

1. Notch1 siRNAs or Fzd10 siRNAs were respectively inhibit Notch1 or Fzd10 expression, among which Notch1 siRNA2 and Fzd10 siRNA1 showed the most effective inhibitors. The check details activities of

both Notch1 signaling and Wnt/β-catenin signaling were markedly suppressed in L02/HBx-Notch1 siRNA2 cells. However, the activity of Notch1 signaling was not apparently changed in L02/HBx-Fzd10 siRNA1 cells. Furthermore, the activity of Notch1 or Wnt/β-catenin signaling was not significantly affected by transfecting with RNAi ether tageted respectively Fzd10 or Notch1 in L02 cells. Having partially blocked Wnt/β-catenin signaling in L02/HBx cells, the promotion of growth, cell cycle progression and inhibition apoptosis induced by Notch1 were substantially attenuated. Conclusion: Our study demonstrated that crosstalk between Notch1 and Wnt/β-catenin pathways did exist in L02/HBx, and Wnt/β-catenin pathway was the downstream of Notch1 signaling in L02 cell malignant induced by HBx. Key Word(s): 1. Notch; 2. Wnt/β-catenin; 3. crosstalk; 4. HCC; Presenting Author: MAN YANG

Additional Authors: XINGSHUN QI, ZIWEI LIU, YONGZHAN NIE, GUOHONG HAN, KAICHUN WU, DAIMING FAN Corresponding Author: GUOHONG HAN Affiliations: Xijing selleck screening library Hospital of Digestive Diseases; Xijing Hospital of Digestive Diseases; Xijing Hospital of Digestive Diseases Objective: To examine sorafenib-related adverse events (AEs) and their relationship to survival in patients with unresectable hepatocellular carcinoma (HCC) receiving sorafenib combined with transarterial TCL chemoembolization (TACE). Methods: From January 2010 to December 2011, we prospectively collected data from 142 consecutive HCC patients who received combination therapy with sorafenib and TACE.

Primary items included the incidence, severity, onset and length of sorafenib-related AEs, as well as overall survival. Results: During a median follow up of 7.9 months (interquartile range, 3.8–14.2 months), 120 (84%) patients experienced sorafenib-related AEs. Common types of sorafenib-related AEs included hand-foot skin reaction (HFSR) (62%), alopecia (52%), rash (50%), diarrhea (58%), fatigue (57%) and anorexia and/or nausea (24%). These usually occurred within 13–35 days after sorafenib and lasted for 0.7–5 months. Ten patients required dose reductions due to drug-related AEs. Fourteen patients had a transitory interruption in treatment due to AEs. Drug-related AEs leading to permanent treatment discontinuation occurred in 8 patients. The presence of sorafenib-related AEs was an independent predictor of overall survival (hazard ratio: 0.465; 95% confidence interval: 0.261–0.829). The occurrences of HFSR, rash, alopecia, diarrhea and hoarseness were significantly associated with better survival. Additionally, the survival benefit was more significant if rash and HFSR occurred within 4 weeks of starting treatment or if the severity of these AEs was increased.

It is not, however, possible to infer from these data whether this mutation was a primary event that resulted in loss of phototrophy or if it occurred secondarily. However, this appears to be the first time in a dinoflagellate morphospecies that a native phenotype (reduced chloroplasts, loss of phototrophy) has been linked to a naturally occurring genetic mutation sufficient to cause that phenotype. The failure to amplify psbA from the achlorophyllous Esoptrodinium isolate HP using methods successful for all other Esoptrodinium

isolates (and a cryptophyte) may indicate the presence of even more extensive mutations in its plastid genome, if present. Although nonphotosynthetic, the Adriamycin in vitro reduced plastids apparent in “colorless”

Esoptrodinium isolates may still function in a variety of metabolic roles as has been found in other protists such as the check details apicomplexan Plasmodium falciparum and the nonphotosynthetic chlorophyte Prototheca wickerhamii (Waller et al. 1998, Sato and Wilson 2002, Borza et al. 2005). Additional research on Esoptrodinium could shed more light on the general evolution and potential metabolic role of reduced plastids in dinoflagellates, especially through comparative analyses of isolates that appear to be in different stages of independent plastid reduction. Esoptrodinium has been found in shallow sidewalk runoff from a leaking water pipe (Calado et al. 2006) and other small temporary urban pools (C.F. Delwiche, personal communication). We have also found Esoptrodinium in high abundance in greenhouse pools and temporary rain puddles in a grassy field, far distant from any pond. Esoptrodinium can be recovered from desiccated sediment (Calado et al. 2006) and is heat tolerant, surviving (as cysts) an incubator cooling failure in our laboratory that resulted in temperatures >45°C for 2 days. Collectively, these observations Arachidonate 15-lipoxygenase have led us to believe Esoptrodinium may exist as a “soil dinoflagellate,” in the same sense that some ciliates, euglenoids, etc. are considered “soil protists,” even though they are also found in ponds (Metting

1981, Foissner 1998). If so, this would be a unique niche for a dinoflagellate with implications for its ecology and potential biogeography. In addition, we have observed that Esoptrodinium appears to induce contact-mediated lysis of C. ovata, an interesting apparent prey capture strategy that requires further investigation. The apparently degenerate plastids of some Esoptrodinium isolates could make them a new model species for genetic or other investigation of plastid loss in dinoflagellates. Finally, systematic revision is required to clarify the potentially unreliable taxonomic separation between Esoptrodinium and Bernardinium, and to determine if any species-level distinctions can be characterized in these apparently diverse dinoflagellates.

4%) were on therapy. Conclusions: Based on this small sample of 14 clinics, adoption of the CASL Guidelines for the management of CHB has been poor at the primary care level in Canada. Physicians are frequently not screening for GHB, not testing patients, nor assessing viral replication adequately. DAPT ic50 When viral replication was assessed, two-thirds of patients who might require treatment were not being treated. Screening for HGG was also not well done. Education of PGPs in the management of GHB

is urgently needed, and communication between PGPs and specialists can be improved to ensure better patient management. Disclosures: Morris Sherman – Advisory Committees or Review Panels: Merck, ABT-888 ic50 Tibotec, Roche, Gilead, Celsion, Janssen; Speaking and Teaching: Gilead, Bristol Myers Squibb, Bayer Phuong Nguyen – Advisory Committees or Review Panels:

GILEAD, BMS, MERCK, PFIZER, ASTRA ZENECA, GSK, TAKEDA, BI, ELI LILLY, AMGEN, GALDERMA, NOVARTIS, ASTELLAS, ABBOTT Jean Palmart – Independent Contractor: Gilead Sciences Canada Background: Currently 4 million persons in the US have active hepatitis C infection and world- wide there may be as many as 170 million people with this infection. Most patients have never been treated and newer therapies herald the potential for wider uptake and acceptance of treatment. Primary care providers will be needed to help expand access to care, but few models of collaborative primary care hepatitis C practices exist. Methods: Retrospective

analysis of collaborative primary care clinic for evaluation and treatment of patients with chronic hepatitis C at a single VA medical center. A single half-day clinic was organized with 4 primary care MDs, two nurse practitioners, one nurse case manager, and 1-2 hepatologists. A co-located psychiatrist and one pharmacist were integrated into the clinic, and bi-monthly noon meetings were held to discuss treatment issues. Clinic productivity and outcomes related to the number of patients who initiated and completed treatment with Carnitine dehydrogenase direct acting antivirals (DAA) and pegylated interferon and ribavirin from July 2011 through December 2012. Results: This clinic had 1890 confirmed HGV registry patients and a total of 1690 clinic visits during this 18 month time period. Clinic capacity included 215 patient slots per month. Same week appointment access was provided. During this time 74 patients with HGV genotype 1 initiated DAA antiviral therapy. Primary care providers treated 47 patients (32% cirrhotic) vs. 27 patients treated by hepatologists (48% cirrhotic). The percentage of patients that completed 0-19 weeks, 20-28 weeks, 29-36 weeks, and greater than 36 weeks of antiviral treatment 25. 9%, 36. 2%, 10.3%, and 27. 6, respectively. Final SVR rate was 46% (33. 3% cirrhotics vs 55. 2% noncirrhotics).

As open synovectomy has

been effective in controlling synovitis and recurrent bleeding, it requires a large incision, prolonged VX-809 order hospitalization, protocolized rehabilitation, and large amount of factor replacement, and has been associated with a infection rate and reduced range of motion [10,11,12]. Long-term results on joint bleeding control and on overall joint function were satisfactory with these traditional techniques. Arthroscopic synovectomy represents a less invasive approach, with a low complication rate [13], and it has become the preferred alternative to the open technique. The use of arthroscopic synovectomy in persons with haemophilia was first attempted on knees and reported in 1983 [14,15], producing satisfactory results even after a prolonged follow-up with a significant reduction of joint bleeding recurrence and preservation of joint mobility [16]. This procedure, Selleck Tyrosine Kinase Inhibitor Library mainly performed in children or adolescents, allowed a significant reduction of bleeding rate and pain relief, suggesting that the beneficial effects of this surgery are greater if it is performed before the onset of severe radiological changes [17]. Additional data has been published suggesting that arthroscopic synovectomy is a cost-effective

means of addressing target joint bleeding [18]. Associated musculoskeletal disorders (i.e. flat foot, axial deviation of lower limbs) have to be treated as soon as possible in order to reduce the likelihood of secondary joint disease. Orthopaedic surgeons have to deal with two challenges:

the management of haemophilic paediatric patients coming from countries where factor replacement therapy is not available and patients with inhibitors. Cases of severe arthropathy with severe joint involvement that we commonly encountered many years ago are now seen Pomalidomide supplier only in such patients. Patients with inhibitors have more severe and incapacitating degrees of arthropathy than those without [19], with fixed knee flexion deformity as a common problem. If conservative treatment fails, surgical procedures have to be considered, including supracondylar femoral extension osteotomy, joint distraction, posterior capsulotomy and arthroscopic release. Potential complications of these procedures are fractures, neurovascular lesions, knee instability, and recurrent deformity with continued growth [20]. In patients with an immature skeleton, anterior femoral stapling is a less invasive method to treat fixed knee flexion deformity, is well tolerated, and provides an excellent alternative to osteotomy by allowing gradual correction through growth manipulation [21]. Joint replacement surgery, as last resort, could be performed in such patients when marked joint destruction is present and pain or deformity compromises function. Relief of pain, reduction of the deformity, and dramatic improvement in functional status and quality of life can be achieved in most patients.

[40, 50-52] In line with previous results,[53, 54] the presence of steatosis, which was observed in 62% of patients, was independently associated with older age, increased BMI, and hyperglycemia, but not with viral features, such as HBeAg status, and viral load, thus suggesting that metabolic alterations

are click here the leading cause of steatosis in CHB, as in the general population and in CHC,[55] whereas differently from hepatitis C, the virus itself does not play a role.[54] The high prevalence of steatosis in the present series[54] can be explained by the high prevalence of metabolic risk factors and the inclusion criteria (e.g., allowance of excessive alcohol consumption). The major finding of the present

study is the I148M polymorphism representing a genetic determinant of steatosis susceptibility in CHB. Similarly to what was observed in CHC,[40, 50] the 148M allele was an independent predictor of steatosis of any degree, but it was even more strongly associated, together with increased BMI, with the presence of severe steatosis, Selleck KPT330 increasing the risk by approximately 6-fold. Interestingly, the effect was particularly evident in the 35% of patients with acquired cofactors, such as a positive history of alcohol intake and/or severe overweight, whereas it was negligible in low-risk teetotalers with normal weight, which is consistent with the hypothesis that severe steatosis HAS1 results from the interaction of different predisposing conditions, including

the 148M PNPLA3 allele.[41] Recently, an interaction between the PNPLA3 I148M polymorphism and tea drinking in the pathogenesis of steatosis have been reported in an epidemiological study conducted in Asia.[56] Although a limitation of the present study is that tea and coffee drinking was not quantitatively assessed, tea drinking was not frequent in Italian patients, and both coffee and tea consumption were not associated with steatosis (not shown). Of note, increased BMI and active alcohol consumption were also independently associated with advanced fibrosis, and a nonsignificant trend for an association between advanced disease and severe steatosis (or the NAS) was also observed, thus leaving open the possibility that altered hepatic lipid metabolism is a risk factor for fibrosis progression also in CHB,[15, 17] although prospective studies are required for confirmation. As a result of the many confounders influencing disease history, the PNPLA3 I148M polymorphism was not associated with fibrosis severity, but, despite the relatively large number of well-characterized biopsied patients included, the power of the study was not sufficient to formally test the interaction between genetic and acquired risk factors in the pathogenesis of liver fibrosis.

HO-1 has been shown to decrease proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α), in vitro5, 6 as well as in vivo.7, 8 We could show previously that induction or overexpression of HO-1 reduces liver damage in mouse models of acute inflammation.7, 8 Here, we investigated

the effects of HO-1 induction on chronic inflammation and fibrosis of the liver. Chronic hepatitis, induced by, for example, viral infections or chronic exposure to toxins represents a severe health problem, because it bears a risk of progression to hepatocellular carcinoma (HCC), which represents the fifth most common cancer worldwide.9, 10 To investigate HO-1 effects on chronic hepatitis, we used an animal model selleckchem of chronic portal inflammation and bile duct proliferation with progression to liver fibrosis and HCC (multidrug resistance transporter 2 knockout [Mdr2ko] mouse; FVB.129P2-Abcb4tm1Bor).11 Mdr2ko mice are lacking the Mdr2 P-glycoprotein, a phosphatidylcholine transporter, resulting in dysfunctional phospholipid secretion.12 Signs of inflammation (e.g. intense hepatic leukocyte infiltrations), which appear within the

first weeks of age, are accompanied by an increase in plasma Vadimezan solubility dmso transaminase levels Hydroxychloroquine nmr and followed by enhanced connective tissue storage and progression to fibrosis.13 Fibrogenesis is a dynamic process associated with activation of hepatic stellate cells (HSCs), which is characterized by collagen

and alpha smooth muscle actin (α-SMA) expression as well as chemokine secretion and activation of matrix metalloproteinases (MMPs) and their inhibitors, tissue inhibitors of matrix metalloproteinases (TIMPs).14 As a consequence of chronic inflammation and progressing fibrosis, Mdr2ko mice have been shown to develop HCC within 12-15 months of age.15 We investigated the effect of HO-1 induction on chronic inflammation and fibrogenesis in mice with mild or established fibrosis. Our results show that HO-1 induction decreased chronic inflammation by regulating immune cell infiltration or proliferation, TNF receptor (TNFR) expression, and subsequent events in proinflammatory cytokine signaling, such as extracellular signal-related kinase (ERK) phosphorylation. Liver damage was significantly ameliorated for at least 8 weeks beyond treatment. HO-1 induction improved lobular fibrosis as well as portal inflammation to a state observed before the onset of treatment. In conclusion, induction of HO-1 interfered with chronic inflammation and fibrosis formation.

Moreover, bilirubin at 50 μM buy Fostamatinib concentration significantly decreased the expression of RUNX2 (Table 4; Fig. 3A). The results observed in the experiments performed with serum from healthy subjects and patients were somewhat dissimilar, because expression of OPG decreased but RANKL increased, leading to a significant enhancement in the RANKL/OPG ratio (Table 4; Fig. 3B). In addition, no significant changes on RUNX2 expression were observed with pooled samples from patients and controls, although lower levels of mRNA

expression were observed in parallel with increasing concentrations of serum (from 2% to 20%) in the culture media (Table 4). The results of the current study, carried out using primary human osteoblasts, indicate that bilirubin has detrimental effects on cell viability, but also on osteoblast differentiation and mineralization. Thus, the presence of 50 μM unconjugated bilirubin in the culture media resulted in a decreased cell differentiation, as assessed by the alkaline phosphatase assay. Moreover, this concentration of bilirubin in the culture media decreased

cell mineralization in SAOS-2 cells as well. The detrimental effects of bilirubin are in accordance with those induced by sera samples from jaundiced patients, even though the increased bilirubin was conjugated in these patients and the potential effects of other retained substances cannot be ruled out. In

buy Compound Library these experiments, 66 μM bilirubin, which was obtained in the experiments with 20% concentration, also decreased cell differentiation and mineralization, using similar approaches. This study confirms previous data Molecular motor on the harmful effect of bilirubin on cell survival. Thus, as observed by Janes et al., the presence of sera with a high concentration of bilirubin resulted in decreased cell viability.5 Moreover, depression of proliferation of other cells of calcifying tissues by bilirubin has also been reported.21 The current study, however, adds new information, because reduced osteoblast viability was observed with sera samples from jaundiced patients, particularly in the experiments performed with the highest bilirubin concentration in culture media (66 μM). Conversely, serum from nonjaundiced patients had no detrimental effects or had lesser effects on survival. The differences in cell viability observed between the experiments performed with bilirubin in the media or with sera samples from healthy subjects and jaundiced and nonjaundiced patients may be explained, at least in part, by the presence of molecules other than bilirubin in the experiments. Among these other molecules, increased bile acid or different cytokines and growth factors released as a consequence of the pathological condition could participate in these detrimental effects on osteoblast function.

To determine the Ag persistence after Ad-HCV-NS3 infection, we analyzed the expression of FLAG-tagged

HCV-NS3 protein in the liver by IP-western blot after administration of 2 × 107, 1 × 109 or 1 × 1010 PFU of the virus. The Ag expression in the liver could be found in both core (+) and core (−) mice on 21 days after infection with 1 × 1010 PFU. When 1 × 109 PFU of Ad-HCV-NS3 was administrated, HCV NS3-protein was almost cleared from the liver of core (−) mice at day 21 post-infection, whereas the Ag expression persisted in the liver of core (+) mice until day 21 post-infection (Fig. 6). It is important to note that the loss of Ag expression in the liver of core (−) mice after infection with 1 × 109 PFU coincided with the high HCV-NS3-specific CD8 T-cell

response at 14 days post-infection (Fig. 2c), whereas Ag persistence in the liver of core (+) mice after infection with 1 × 109 PFU or the liver of core (−) and core (+) mice after infection with 1 × 1010 PFU was associated check details with strongly diminished Ag-specific CD8 T-cell response (Fig. 2c). It is likely that the expression of core protein and the high amount of Ag in the liver contributed to the functional exhaustion of HCV-NS3-specific CD8 T cells. IN THIS STUDY, we found an impaired response of HCV-NS3-specific intrahepatic CD8 T cell in a high dose setting (1 × 1010 PFU) of Ad-HCV-NS3 infection. Furthermore, higher levels of expression of regulatory molecules, Tim-3 and PD-1, by intrahepatic CD8 T cells and click here PD-L1 by intrahepatic APC were observed in HCV core Tg mice and the expression increased dependent Protein kinase N1 on infectious dose. In addition, we found a significant inverse correlation

between the percentages of IFN-γ-producing cells and expression of regulatory molecules in Ag-specific intrahepatic CD8 T cells. These results indicated that high infectious dose and the presence of HCV core gene were strongly involved in ineffective CD8 T-cell responses. Recently, a novel mechanism of T-cell dysfunction was demonstrated in a murine model of chronic LCMV infection.[24] It was found that the expression of PD-1 was upregulated on dysfunctional LCMV-specific CD8 T cells in mice.[24] In vivo blockade of PD-1/PD-L1 interaction restored the functions of LCMV-specific CD8 T cells and reduced the viral titer.[24] More recently, other inhibitory receptors such as Tim-3 have also been studied as the factors that can cause T-cell impairments in chronic viral infections.[25] These influential discoveries led to extensive investigations of inhibitory receptors in the regulation of T cells in human chronic viral infections.[25, 26] Chronic HCV infection in humans is characterized by CD8 T-cell exhaustion and dysfunction.[27] As in chronic LCMV infection, the expression of PD-1 is similarly upregulated on the virus-specific CD8 T cells in chronic HCV infection, and HCV-specific PD-1high T cells are functionally impaired.[28-30] Also, Tim-3 is overexpressed on HCV-specific dysfunctional CD8 T cells.

For primers sequences, see Supporting Table S1. Real-time quantitative PCR data represent relative changes in hepatic gene expression. Results are reported as relative differences in gene expression with GAPDH used as an internal control. Samples were homogenized in a lysis buffer (50 mM Tris·HCl, pH 7.4, containing 150 mM NaCl, find more 25

mM EDTA, 5 mM EGTA, 0.25% sodium deoxycholate, 1% Nonidet P-40, and 1 mM DTT) containing protease inhibitor cocktail (Calbiochem) with phosphatase inhibitor. Homogenates were centrifuged at 12,000g for 5 minutes at 4°C and mixed with 5× reducing electrophoresis sample buffer (50 mM Tris·HCl, pH 6.8, containing 10% glycerol, 2% sodium dodecyl sulfate [SDS], 1% β-mercaptoethanol, and 0.02% bromophenol blue) and heated for 5 minutes at 95°C. Samples containing 10-25 μg protein were then resolved by 10% SDS polyacrylamide gel electrophoresis and transferred overnight onto nitrocellulose membranes by electrophoresis. Antibodies against SAPK/JNK, p-SAPK/JNK (Thr183/Tyr185) (81E11), Bip, Doxorubicin datasheet IKKβ, eIF2α, p-eIF2α (Ser51), C/EBP homologous transcription factor (CHOP) (L63F7), were obtained from Cell Signaling Technology (Danvers, MA), ATF-6 was obtained from (Pro-Sci, CA), GADD34 (C-19), p-c-Jun (KM-1), and c-Jun(H-7a) were obtained

from Santa Cruz Biotechnology (Santa Cruz, CA), and ICAM-1 was obtained from Protein Tech Group (Chicago, IL). β-Actin antibody (Sigma Diagnostics, St. Louis, MO) was used to confirm equal protein loading among samples. Nuclear proteins were isolated from fresh liver tissue as described23 using a Nuclear Extract kit from Active Motif (Carlsbad, CA) according to the manufacturer’s Protirelin protocol. The NF-κB and AP-1 DNA binding

activity assays were performed using Trans-AM enzyme-linked immunosorbent assay (ELISA)-based kits from Active Motif (Carlsbad, CA) according to the manufacturer’s protocol. Nuclear extracts from liver tissue were incubated in a 96-well plate coated with oligonucleotide containing NF-κB or AP-1 consensus binding site. Activated transcription factors from extracts specifically bound to the respective immobilized oligonucleotide were detected using the antibodies to NF-κB p65 and p50 in NF-κB assays or c-Jun in the Ap-1 assay followed by a secondary antibody conjugated to horseradish peroxidase in an ELISA-like assay. Hepatic SAM and SAH levels were measured by high-performance liquid chromatography (HPLC) using the method of Henkel et al.24 For pharmacologic JNK inhibition experiments, cohorts of 5-10 C57BLKS mice were started on either the MCD or control diet.