For practitioners seeing patients with unexplained acute liver disease, comprehensive catalogs of DILI ALF agents are useful, but these Target Selective Inhibitor Library lists are only “snapshots” because prescribing practices vary geographically and temporarily.3, 24, 34 Few biologicals were implicated here, but DILI from these compounds is emerging,

including fatalities.44 Within the broad spectrum of causative agents, antimicrobials dominate.13, 16, 18, 21 Isoniazid, as monotherapy or in combination, commonly causes hepatoxicity leading to liver transplantation,17 followed by sulfur drugs, nitrofurantoin, other antibiotics, and antifungals. Amoxicillin-clavulanic and NSAIDs often cause DILI,19, 28, 45 but less commonly ALF. Perhaps the inflammation caused by the infection for which antibiotics are prescribed, predisposes the patients to develop DILI.46 Antiepileptics, antimetabolites, herbal mixtures and their derivatives, slimming potions, and illicit drugs,

have strong reputations as hepatotoxins7, 47, 48 and were well represented in our study. Statin prevalence (n ≥ 6) was unexpected, as was the occasionally long duration of exposure (median 3-6 months; range, <1 month to 36 months; see also the footnote to Table 1C). Statin hepatotoxicity is generally benign,49 but statins have been responsible for a few DILI-associated fatalities,18, 19 and atorvastatin-to-simvastatin substitution hepatitis has been reported.50 In six subjects, a statin was the only potential DILI agent—albeit sometimes with a long latency (6-36 months in three of them)—and Afatinib price this increases confidence in our provocative observation that awaits confirmation by others. The latency between drug use and DILI onset varies, but is usually up to 3 months although delays of up to 12 months are considered compatible.6, 16, 19, 25, 40, 45 Extended latency is the norm for nitrofurantoin51 and some other drugs,

like diclofenac. In the current study, when the cause of DILI ALF was certain, the median exposure was 2 months, but even here six cases had 6 to 10 months of latency. For isoniazid median latency was 5 months; 6-8 months in one-third of the cases. As anticipated,10, 15, 19, 21 DILI in ALF was mostly hepatocellular (77.8%) compared click here to cholestatic and mixed reactions (19.2%) Conventional causes of cholestatic and mixed reactions (phenothiazines, macrolides, NSAIDs, carbamazepine, and phenytoin34, 52, 53) were rare. We confirmed that many drugs can cause cholestatic and mixed hepatotoxic reactions16, 19 (Table 3). Three drugs in this study have been withdrawn (bromfenac and troglitazone because of hepatotoxicity, and cerivastatin because of rhabdomyolysis), and development of the hypoglycemic agent, TAK 559, was halted. Many drugs carry warnings of hepatotoxicity (isoniazid, rifampin, ketaconazole, diclofenac, valproic acid, telithromycin, and interferon-β).

(HEPATOLOGY 2011;) Cancer stem cells have the ability to self-renew, differentiate, and proliferate, have greater tumorigenicity and chemoresistance, and have been associated with a poor

prognosis in several human malignancies.1, 2 They have also been identified in hepatocellular carcinoma (HCC): Among the HCCs with the conventional histomorphological features, there is a recently proposed subtype characterized by the expression of “stemness”-related markers, such as keratin 19 (K19), cluster of differentiation (CD)133, and epithelial cell adhesion see more molecule (EpCAM), and is associated with a poorer prognosis, compared to usual HCCs without these markers.3 The poor prognosis of stemness-related marker expressing HCCs may partly be attributed to the relationship between this subset of HCC with invasion and metastasis-related gene expression. Up-regulation of invasion and metastasis-related genes, such as VIL2 (encoding ezrin), PLAUR (uPAR: urokinase plasminogen activator receptor), and CD44, was

demonstrated in HCCs with progenitor cell features (i.e., “hepatoblast”-like subtype) in a gene-expression profiling study,3 and up-regulated invasion and epithelial-mesenchymal GDC-0068 ic50 transition (EMT)-related genes was demonstrated in CD133-expressing HCCs.4 Moreover, an association between high expression levels of stemness-related markers in HCCs and tumor angiogenesis was recently reported.5 EMT is a critical part of the tumor-cell invasion process and results in the loss of epithelial characteristics click here and the acquisition of mesenchymal features, which include the expression of vimentin, N-cadherin, S100A4, metalloproteinases, snail, and twist.6 Loss of normal epithelial intercellular contact is an important feature and is demonstrated by the loss of membranous E-cadherin expression in the tumor cells. The expression of E-cadherin is controlled by transcription factors, such as snail and twist, which bind to consensus E-box sequences in the E-cadherin gene promoter.7 In addition,

E-cadherin may also undergo cleavage by matrix metalloproteinases (MMPs), resulting in the down-regulation of E-cadherin-mediated intercellular adhesion. MMPs have been shown to play important roles in tumor invasion, and MMP2 overexpression in HCC has been associated with a poor prognosis.8 VIL2 and its protein, ezrin, interact with E-cadherin and have been implicated in the invasiveness and metastasis of HCCs.9 Ezrin overexpression has been associated with K19 expression10 and poor prognosis.10, 11 An association between uPAR and EMT has also been demonstrated in breast cancer cells,12 and interactions between urokinase plasminogen activator (uPA) and uPAR have been observed in the invasion and metastasis of various tumors, including HCCs.

ATRA, in combination with sorafenib

decreased the activity of the metabolic pathways of HCC cells, and contributes to the increased sensitivity to apoptosis. Combination of anti-cancer drugs with ATRA will be useful for anti-tumor therapy for HCC by regulating cancer cell metabolism. Disclosures: The following people have nothing to disclose: Goshi Shiota, Keita Kanki Introduction: Sorafenib is standard of care for advanced hepatocellular carcinoma (HCC). however, response is often transient with development of resistance. We have recently found that sorafenib GSK2118436 treatment increased hypoxia and induced SDF1α and CXCR4 expression in HCCs – leading to tumor desmoplasia and Gr1+ myeloid suppressor cell recruitment. Anti-mouse

PD1 antibody (αPD1) treatment has been shown to boost immune response in other malignancies that are characterized by PD1/PD1L upregulation. Methods: We used an orthotopic murine HCC model – HCA1 tumor grafts in syngeneic C3H mice. HCC tumor growth was monitored by ultrasound. HCC tumor growth was monitored by small animal ultrasound. When tumor volume reached approximately 14mm3 (3×3×3mm), the mice were randomized to 4 groups of treatment (n=6): (1) control (CTRL); (2) sorafenib, 50mg/ kg/daily gavage (SOR); (3) sorafenib plus the CXCR4 inhibitor AMD3100 (10mg/kg/s.c. minipump) (SOR+AMD); and (4) SOR+AMD plus aPD1 (5 × 100μg i.p. injections every 3 days) (SOR/AMD/PD1). Palbociclib cell line Tumor growth was evaluated for 28 days. Results: At the endpoint, tumor volume was significantly smaller in the SOR/AMD and SOR/AMD/PD1 groups versus CTRL (CTRL: 240mm3 vs. SOR: 151mm3 vs. SOR/AMD: 116mm3 vs. SOR/AMD/αPD1: 79mm3). Sorafenib treatment alone resulted in accumulation of intratumoral T-regulatory cells and M2-type macrophages. Inhibition of CXCR4 by AMD3100 prevented these effects and led to inhibition of tumor metastasis and primary tumor growth. However, combination treatment of SOR/AMD did not increase the number of CD8+ T-cells. Addition of aPD1 synergized with SOR/AMD in delaying tumor growth and reducing

metastasis. aPD1 treatment significantly selleck inhibitor increased intratumoral infiltration of cytotoxic CD8+ T cells and their activation (as demonstrated by elevated levels of IL-2, TNF-α and IFN-γ expression). CD8+ T-cells co-localized with caspase-3 positive apoptotic HCC cells. Conclusion: Modulation and activation of immune responses by combining AMD3100 and aPD1 may be a novel approach to inhibit local and distant tumor evasion from sorafenib treatment in HCC. Disclosures: Thomas Reiberger – Grant/Research Support: Roche, Gilead, MSD, Phenex; Speaking and Teaching: Roche, Gilead, MSD Rakesh K. Jain – Board Membership: XTuit, H&Q Healthcare Investors, H&Q Life Sciences Investors; Consulting: Enlight Biosciences, Noxxon, Zyngenia; Grant/ Research Support: Dyax, MedImmune, Roche; Stock Shareholder: Enlight Biosciences, SynDevRx, XTuit Dan G.

The infection was carried out at 500 pfu/cells in α minimum essential medium supplemented with 0.2% BSA for 6 hours at 33°C. The virus-containing high throughput screening medium was then removed, and cells were incubated for 24 hours in α minimum essential medium supplemented with 4% fetal bovine serum before being differentiated and treated as described. Timp3−/− mice on a C57/BL6 background

have been described,16 as have metabolic testing procedures10–12 (see also Supporting Information). Animal studies were approved by the University of Tor Vergata Animal Care and Use Committee. All animals received human care according to the criteria outlined in the “Guide for the Care and Use of Laboratory Animals” prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, selleck compound revised

1985). For the extraction of matrix-bound Timp3, tissues were treated as described.10 Histology was perfomed as described.12 (See Supporting Information for details.) Total RNA was isolated from wild-type (WT) and Timp3−/− mice and from SV40-transformed hepatocytes using Trizol reagent (Invitrogen Corp, Carlsbad, CA). Two micrograms of total RNA were reverse-transcribed into complementary DNA using the High Capacity cDNA Archive kit (Applied Biosystems, Foster City, CA). Quantitative real-time polymerase chain reaction was performed using an ABI PRISM 7700 System and TaqMan reagents (Applied Biosystems). Each reaction was performed in triplicate using standard reaction conditions. The Applied Biosystems primers used are listed in the Supporting Methods. Shotgun proteomics and ingenuity pathways analysis were performed as described17, 18 and are reported in an extended version in the Supporting Information. Assays for S-adenosylmethionine and S-adenosylhomocysteine in liver and cells—methionine and homocysteine in serum—were

click here performed as described19 and are reported in an extended version in the Supporting Methods. Results of the experimental studies are expressed as the mean ± standard deviation as indicated. Statistical analysis was performed using one-way analysis of variance, two-way analysis of variance, or an unpaired Student t test as appropriate. Values of p < 0.05 were considered statistically significant. We have recently described that regulation of TNF-α release from plasma membrane through its ectodomain shedding by TACE has a role in accelerating liver inflammation and steatosis when coupled with an insulin-resistant environmental and genetic background.10–12 Because TACE haploinsufficiency protects from lipotoxicity and glucotoxicity in an in vivo model, we analyzed three different in vitro cell culture models—3T3-F442A adipocytes, C2C12 myocytes, and SV40-tranformed hepatocytes—to study mechanisms that link metabolic dysfunction to TACE activation. TACE activity was significantly increased by treatment with a free fatty acid, palmitic acid (0.

1C). Together, these results indicate that

AIB1 is important for CCA cell proliferation. To explore the mechanism by which knockdown of AIB1 inhibits proliferation of CCA cells, cell cycle analysis was performed to examine whether AIB1-knockdown cells are arrested in a specific phase of the cell cycle. Cells were synchronized by serum starvation for 24 hours, then the cell cycle was measured by flow cytometric analysis after serum addition for 24 hours. As shown in Fig. 2A, AIB1 knockdown induced G2/M arrest in QBC939 cells. To Ibrutinib better understand the role of AIB1 in cell cycle progression, cells were synchronized to the G1/S boundary by the double thymidine block method or arrested at G2/M phase by thymidine/nocodazole, then the cell cycle was measured by flow cytometric analysis after drug withdrawal. The results in Supporting Fig. 2 confirmed the conclusion that down-regulation of AIB1 resulted in G2/M arrest. In addition, the subG1 population of AIB1-knockdown cells, which represents apoptotic cells, was significantly larger than that of control cells, indicating that AIB1 protein is required for both proliferation and survival of CCA cells. To address the molecular mechanism underlying the crucial role of AIB1 in control of the CCA cell cycle, we examined

Nutlin-3a manufacturer the effect of knocking down AIB1 on key G2/M regulatory genes expression by western blotting. The expression of G2/M regulatory proteins such as Cyclin A, Cyclin B, Cdk1, and Cdc25 was detected at different time after serum addition following synchronization of cells by serum starvation for 24 hours. The results showed that the expression of mitosis-promoting factors including Cyclin A, Cyclin B, and Cdk1 recovered more slowly and less in AIB1-knockdown QBC939 cells than those in control cells, whereas the expression of G1-related factor Cyclin D1 was comparable in both cells (Fig. 2B). In addition, down-regulation of AIB1 markedly inhibited the expression of phosphorylated Akt (Fig. 2B). Because activation of the Akt

signaling pathway can lead to the promotion of cell cycle progression at the G2/M phase,7, 8 we investigated whether reduced Akt signaling pathway is involved in G2/M arrest by check details using the PI3K/Akt inhibitor LY294002 to treat QBC939 cells. As shown in Fig. 2C, LY294002 treatment reduced the phosphorylation of Akt and inhibited the recovery of Cyclin A, Cyclin B, and Cdk1 after serum addition. These results indicate that down-regulation of AIB1 inhibits the cell cycle progression at least in part through reducing the activity of Akt signaling pathway. Resistance of CCA to chemotherapeutic drugs is a major problem that limits the effectiveness of chemotherapies used to treat this cancer. Better understanding drug sensitivity and the mechanism of drug resistance of this cancer is essential.

1C). Together, these results indicate that

AIB1 is important for CCA cell proliferation. To explore the mechanism by which knockdown of AIB1 inhibits proliferation of CCA cells, cell cycle analysis was performed to examine whether AIB1-knockdown cells are arrested in a specific phase of the cell cycle. Cells were synchronized by serum starvation for 24 hours, then the cell cycle was measured by flow cytometric analysis after serum addition for 24 hours. As shown in Fig. 2A, AIB1 knockdown induced G2/M arrest in QBC939 cells. To Depsipeptide manufacturer better understand the role of AIB1 in cell cycle progression, cells were synchronized to the G1/S boundary by the double thymidine block method or arrested at G2/M phase by thymidine/nocodazole, then the cell cycle was measured by flow cytometric analysis after drug withdrawal. The results in Supporting Fig. 2 confirmed the conclusion that down-regulation of AIB1 resulted in G2/M arrest. In addition, the subG1 population of AIB1-knockdown cells, which represents apoptotic cells, was significantly larger than that of control cells, indicating that AIB1 protein is required for both proliferation and survival of CCA cells. To address the molecular mechanism underlying the crucial role of AIB1 in control of the CCA cell cycle, we examined

Akt inhibitor the effect of knocking down AIB1 on key G2/M regulatory genes expression by western blotting. The expression of G2/M regulatory proteins such as Cyclin A, Cyclin B, Cdk1, and Cdc25 was detected at different time after serum addition following synchronization of cells by serum starvation for 24 hours. The results showed that the expression of mitosis-promoting factors including Cyclin A, Cyclin B, and Cdk1 recovered more slowly and less in AIB1-knockdown QBC939 cells than those in control cells, whereas the expression of G1-related factor Cyclin D1 was comparable in both cells (Fig. 2B). In addition, down-regulation of AIB1 markedly inhibited the expression of phosphorylated Akt (Fig. 2B). Because activation of the Akt

signaling pathway can lead to the promotion of cell cycle progression at the G2/M phase,7, 8 we investigated whether reduced Akt signaling pathway is involved in G2/M arrest by selleck chemicals llc using the PI3K/Akt inhibitor LY294002 to treat QBC939 cells. As shown in Fig. 2C, LY294002 treatment reduced the phosphorylation of Akt and inhibited the recovery of Cyclin A, Cyclin B, and Cdk1 after serum addition. These results indicate that down-regulation of AIB1 inhibits the cell cycle progression at least in part through reducing the activity of Akt signaling pathway. Resistance of CCA to chemotherapeutic drugs is a major problem that limits the effectiveness of chemotherapies used to treat this cancer. Better understanding drug sensitivity and the mechanism of drug resistance of this cancer is essential.

This indicates that the HCV particles released from infected-HepaRG cells (HCV-RG) are indeed infectious. To determine the buoyant density distribution of HCV RNA, E1E2 and core antigens, the viral preparations from media collected at days 28 and 42 PD-0332991 purchase p.p. (Fig. 1A,b) were pooled and subjected to iodixanol gradient density centrifugation. Figure 1D shows that the HCV-RG particles had a relatively homogeneous distribution between 1.06 and 1.12 g/mL. They expressed E1E2 envelope proteins and contained RNA and core antigen. In addition, the positive fractions reacted with polyclonal antibodies against apoE (++, P/N ratio = 5-6) and

apoB (+, P/N ratio = 3-4), suggesting that host lipoproteins could be associated with these particles mimicking circulating HCV.14 Immunohistochemistry experiments were performed to investigate intracellular expression of HCV E1E2 and core antigens (Ag) in infected-HepaRG cells at 28 and 56 JQ1 days p.p. (infection 1). Figure 1E shows that the HCVsp-infected HepaRG cells at D28 p.p. exhibited a very strong staining of cytoplasm and perinuclear regions for E1E2 Ag (a). Fifty to sixty percent of cells were positive. Core Ag staining (b) appeared

also in the cytoplasm possibly around lipid droplets. Some cells were labeled both in the cytosol and the nucleus. Control HCV(−) uninfected HepaRG cells were clearly negative in the presence of D32.10 (a) or C7.50 (b), as well as HCV-infected cells in the presence of a control IgG1 antibody (not shown). Positively stained infected cells exhibited morphological features of hepatocytes.5 Altogether, these results indicate that the human selleck HepaRG cells can be infected with HCVsp when proliferated and do produce de novo infectious lipoprotein-associated enveloped complete HCV particles for up to 6 weeks when differentiated. To investigate whether the unique E1E2-specific D32.10 mAb inhibits HCV infection, the infection experiment (infection 3) was performed after preincubation of HCVsp with D32.10 at a 0.5 μg/mL concentration. Figure 2 shows that the D32.10 mAb completely inhibited HCV RNA production in HepaRG culture supernatants.

The total amount of HCV RNA remained at very low levels throughout the follow-up of the infection from day 1 to day 21 in the presence of D32.10 with a mean inhibition of 80.5 ± 11.6% (Fig. 2A). When HCV RNA was quantified by qPCR, 5log10 copies/mL were detected at day 21 after control infection. The preincubation of the inoculum with D32.10 reduced by ≈97% the extracellular HCV RNA (−2 log10, Fig. 2B). To further support that control-infected HepaRG cells produced viral particles, iodixanol density gradient analysis was performed from HCV RNA-associated particles present in the culture media collected at days 14 and 21 (Fig. 2C). As seen previously (infection 1), both HCV RNA, E1E2, and core antigens were recovered as a major peak between 1.

This indicates that the HCV particles released from infected-HepaRG cells (HCV-RG) are indeed infectious. To determine the buoyant density distribution of HCV RNA, E1E2 and core antigens, the viral preparations from media collected at days 28 and 42 buy Palbociclib p.p. (Fig. 1A,b) were pooled and subjected to iodixanol gradient density centrifugation. Figure 1D shows that the HCV-RG particles had a relatively homogeneous distribution between 1.06 and 1.12 g/mL. They expressed E1E2 envelope proteins and contained RNA and core antigen. In addition, the positive fractions reacted with polyclonal antibodies against apoE (++, P/N ratio = 5-6) and

apoB (+, P/N ratio = 3-4), suggesting that host lipoproteins could be associated with these particles mimicking circulating HCV.14 Immunohistochemistry experiments were performed to investigate intracellular expression of HCV E1E2 and core antigens (Ag) in infected-HepaRG cells at 28 and 56 see more days p.p. (infection 1). Figure 1E shows that the HCVsp-infected HepaRG cells at D28 p.p. exhibited a very strong staining of cytoplasm and perinuclear regions for E1E2 Ag (a). Fifty to sixty percent of cells were positive. Core Ag staining (b) appeared

also in the cytoplasm possibly around lipid droplets. Some cells were labeled both in the cytosol and the nucleus. Control HCV(−) uninfected HepaRG cells were clearly negative in the presence of D32.10 (a) or C7.50 (b), as well as HCV-infected cells in the presence of a control IgG1 antibody (not shown). Positively stained infected cells exhibited morphological features of hepatocytes.5 Altogether, these results indicate that the human check details HepaRG cells can be infected with HCVsp when proliferated and do produce de novo infectious lipoprotein-associated enveloped complete HCV particles for up to 6 weeks when differentiated. To investigate whether the unique E1E2-specific D32.10 mAb inhibits HCV infection, the infection experiment (infection 3) was performed after preincubation of HCVsp with D32.10 at a 0.5 μg/mL concentration. Figure 2 shows that the D32.10 mAb completely inhibited HCV RNA production in HepaRG culture supernatants.

The total amount of HCV RNA remained at very low levels throughout the follow-up of the infection from day 1 to day 21 in the presence of D32.10 with a mean inhibition of 80.5 ± 11.6% (Fig. 2A). When HCV RNA was quantified by qPCR, 5log10 copies/mL were detected at day 21 after control infection. The preincubation of the inoculum with D32.10 reduced by ≈97% the extracellular HCV RNA (−2 log10, Fig. 2B). To further support that control-infected HepaRG cells produced viral particles, iodixanol density gradient analysis was performed from HCV RNA-associated particles present in the culture media collected at days 14 and 21 (Fig. 2C). As seen previously (infection 1), both HCV RNA, E1E2, and core antigens were recovered as a major peak between 1.

These criteria were chosen because sustained virological response to anti-HCV therapy improves the outcomes of these patients. The study was approved by appropriate regulatory bodies at all centers, and written informed consent was obtained from all patients for participation in medical research. NAFLD was diagnosed based on the following: (1) elevated aminotransferases for at least 6 months; (2)

click here liver biopsy showing changes consistent with advanced fibrotic NAFLD (detailed below); and (3) exclusion of other etiologies, including viral, autoimmune, cholestatic, genetic, metabolic, alcoholic, or drug-induced liver diseases. These other etiologies were excluded using specific biochemical, clinical, radiological, and/or histological criteria.

All patients had current and past consumption of ethanol less than 20 g per day on direct questioning of both the patients and a close relative. A complete medical history and physical examination was undertaken. Body mass index (BMI) was calculated using the following formula: weight (in kilograms)/height2 (in meters). Waist circumference (to the nearest half centimeter) was measured at the midpoint between the lower border of the ribcage and the iliac crest. Serum measurements included routine liver biochemistry (alanine aminotransferase [ALT] and aspartate aminotransferase [AST] levels, total bilirubin, albumin, alkaline phosphatase, and gamma glutamyl transpeptidase), complete blood count, fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein (HDL) cholesterol, and total trigycerides, selleckchem serology PLX4032 datasheet for hepatitis B and C viruses, iron studies, autoantibodies, alpha 1 antitrypsin levels and phenotype, and ceruloplasmin levels. Components of the metabolic syndrome, including central obesity, hyperglycemia,

hypertrigylceridemia, hypertension, and low HDL cholesterol, were recorded. Liver biopsies were stained with hematoxylin and eosin, Masson’s trichrome, and special stains for iron and copper. Liver biopsies were read by a single liver pathologist in each participating center. Histological features of NAFLD, such as steatosis, inflammation, hepatocyte ballooning, and fibrosis, were scored as previously described.14, 15 Only those patients that had steatosis of at least 5% plus severe fibrosis (stage 3 [septal/bridging]) or cirrhosis (stage 4) fibrosis were included in this analysis. Other histological changes of steatohepatitis, such as inflammation and ballooning, were not required as inclusion criteria. For HCV, the degree of fibrosis was scored according to the METAVIR scale16 as follows: stage 0, no fibrosis; stage 1, enlarged portal tract without septa; stage 2, enlarged portal tract with rare septa; stage 3, numerous septa without cirrhosis; stage 4, cirrhosis. Only those patients with fibrosis stage 3 or 4 disease were included.

These criteria were chosen because sustained virological response to anti-HCV therapy improves the outcomes of these patients. The study was approved by appropriate regulatory bodies at all centers, and written informed consent was obtained from all patients for participation in medical research. NAFLD was diagnosed based on the following: (1) elevated aminotransferases for at least 6 months; (2)

ICG-001 in vivo liver biopsy showing changes consistent with advanced fibrotic NAFLD (detailed below); and (3) exclusion of other etiologies, including viral, autoimmune, cholestatic, genetic, metabolic, alcoholic, or drug-induced liver diseases. These other etiologies were excluded using specific biochemical, clinical, radiological, and/or histological criteria.

All patients had current and past consumption of ethanol less than 20 g per day on direct questioning of both the patients and a close relative. A complete medical history and physical examination was undertaken. Body mass index (BMI) was calculated using the following formula: weight (in kilograms)/height2 (in meters). Waist circumference (to the nearest half centimeter) was measured at the midpoint between the lower border of the ribcage and the iliac crest. Serum measurements included routine liver biochemistry (alanine aminotransferase [ALT] and aspartate aminotransferase [AST] levels, total bilirubin, albumin, alkaline phosphatase, and gamma glutamyl transpeptidase), complete blood count, fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein (HDL) cholesterol, and total trigycerides, see more serology MG-132 chemical structure for hepatitis B and C viruses, iron studies, autoantibodies, alpha 1 antitrypsin levels and phenotype, and ceruloplasmin levels. Components of the metabolic syndrome, including central obesity, hyperglycemia,

hypertrigylceridemia, hypertension, and low HDL cholesterol, were recorded. Liver biopsies were stained with hematoxylin and eosin, Masson’s trichrome, and special stains for iron and copper. Liver biopsies were read by a single liver pathologist in each participating center. Histological features of NAFLD, such as steatosis, inflammation, hepatocyte ballooning, and fibrosis, were scored as previously described.14, 15 Only those patients that had steatosis of at least 5% plus severe fibrosis (stage 3 [septal/bridging]) or cirrhosis (stage 4) fibrosis were included in this analysis. Other histological changes of steatohepatitis, such as inflammation and ballooning, were not required as inclusion criteria. For HCV, the degree of fibrosis was scored according to the METAVIR scale16 as follows: stage 0, no fibrosis; stage 1, enlarged portal tract without septa; stage 2, enlarged portal tract with rare septa; stage 3, numerous septa without cirrhosis; stage 4, cirrhosis. Only those patients with fibrosis stage 3 or 4 disease were included.