CONCLUSIONS: A panel of replicons containing GT1-4 NS5B sequences

derived from HCV reference viruses or plasma samples was used to assess genotype specific JQ1 cell line variation in HCV NI susceptibility. IFN susceptibility was similar within and between genotypes while NI susceptibility varied according to inhibitor and genotype. On whole, GT3 NS5B replicons exhibited reduced susceptibility to SOF compared to GT2 NS5B replicons. This observation may contribute to the differential SOF treatment responses observed among individuals with GT2 and GT3 viruses. A number of NIs that display greater activity against GT2-4 viruses compared to GT1 viruses were identified. The clinical development of new, potent and pan-genotypic NIs may contribute to further improvements in HCV treatment, including duration, tolerability and outcome. Disclosures: Wei Huang – Employment: Monogram Biosciences Christos J. Petropoulos – Employment: Monogram Biosciences, LabCorp; Management

Position: Monogram Biosciences, LabCorp; Patent Held/Filed: Monogram Biosciences, LabCorp; Stock Shareholder: LabCorp Alectinib ic50 Raymond F. Schinazi – Stock Shareholder: RFS Pharma Jacqueline D. Reeves – Employment: MONOGRAM BIOSCIENCES INC. The following people have nothing to disclose: Elizabeth D. Anton, Kristi Strom-men, Amber A. Rivera, Franck Amblard Hepatitis C virus-induced, end-stage liver disease is a major indication for liver transplantation,

but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients are frequently ineligible for direct-acting antivirals, due to toxicity, resistance, or advanced MCE liver disease. Adoptive immuno-therapy with liver graft-derived, ex-vivo activated lymphocytes was previously shown to prevent hepatitis C virus-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by ”ready for use“ suicide gene-modifed lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes could potently, dose-dependently, and time-dependently, inhibit viral replication. The effect occurrs at effector:target ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is IL-2-depen-dent, IFN-v-mediated, and importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus-thymidine kinase suicide gene.

CONCLUSIONS: A panel of replicons containing GT1-4 NS5B sequences

derived from HCV reference viruses or plasma samples was used to assess genotype specific Dabrafenib clinical trial variation in HCV NI susceptibility. IFN susceptibility was similar within and between genotypes while NI susceptibility varied according to inhibitor and genotype. On whole, GT3 NS5B replicons exhibited reduced susceptibility to SOF compared to GT2 NS5B replicons. This observation may contribute to the differential SOF treatment responses observed among individuals with GT2 and GT3 viruses. A number of NIs that display greater activity against GT2-4 viruses compared to GT1 viruses were identified. The clinical development of new, potent and pan-genotypic NIs may contribute to further improvements in HCV treatment, including duration, tolerability and outcome. Disclosures: Wei Huang – Employment: Monogram Biosciences Christos J. Petropoulos – Employment: Monogram Biosciences, LabCorp; Management

Position: Monogram Biosciences, LabCorp; Patent Held/Filed: Monogram Biosciences, LabCorp; Stock Shareholder: LabCorp selleck kinase inhibitor Raymond F. Schinazi – Stock Shareholder: RFS Pharma Jacqueline D. Reeves – Employment: MONOGRAM BIOSCIENCES INC. The following people have nothing to disclose: Elizabeth D. Anton, Kristi Strom-men, Amber A. Rivera, Franck Amblard Hepatitis C virus-induced, end-stage liver disease is a major indication for liver transplantation,

but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients are frequently ineligible for direct-acting antivirals, due to toxicity, resistance, or advanced 上海皓元 liver disease. Adoptive immuno-therapy with liver graft-derived, ex-vivo activated lymphocytes was previously shown to prevent hepatitis C virus-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by ”ready for use“ suicide gene-modifed lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes could potently, dose-dependently, and time-dependently, inhibit viral replication. The effect occurrs at effector:target ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is IL-2-depen-dent, IFN-v-mediated, and importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus-thymidine kinase suicide gene.

The in vitro studies were performed in freshly isolated or immortalized5, 8 large cholangiocytes. The rationale for performing these studies only in large cholangiocytes is based on the fact that secretin stimulated in vivo the proliferation of only large bile ducts and that following BDL, large but not small cholangiocytes proliferate.5 Freshly isolated large cholangiocytes (≈99%

by cytokeratin-19 immunohistochemistry)5, 20 were purified by centrifugal elutriation4, 9, Small molecule library in vitro 14 followed by immunoaffinity separation by a monoclonal antibody, rat IgG2a (provided by Dr. R. Faris, Brown University, Providence, RI), against an antigen expressed by all mouse cholangiocytes.5 selleck inhibitor Our large mouse

cholangiocyte lines, which display morphological, phenotypic, and functional features similar to that of freshly isolated large cholangiocytes were cultured as described.5, 8, 9 We evaluated the expression of SR by immunohistochemistry in paraffin-embedded liver sections from the experimental groups of Table 1. Because immunohistochemistry shows that only large bile ducts from WT (but not knockout) animals express SR, we evaluated the expression of SR by way of immunofluorescence and real-time polymerase chain reaction (PCR) in freshly isolated large cholangiocytes from normal and 3- and 7-day BDL WT mice. Semiquantitative immunohistochemical analysis of SR expression in sections was performed as described.5 Light microscopy photographs of liver sections were taken by Leica Microsystems DM 4500 B Light Microscopy (Weltzlar, Germany) with a Jenoptik Prog Res C10 Plus Videocam (Jena,

Germany). Immunofluorescence for SR was also performed in large cholangiocytes from normal and 3- and 7-day BDL WT mice.5, 20 Images were visualized using an Olympus IX-71 confocal microscope. For all immunoreactions, negative controls 上海皓元医药股份有限公司 (with normal serum from the same species substituted for the primary antibody) were included. In freshly isolated large cholangiocytes from normal and BDL WT mice, messenger RNA and protein expression of SR were evaluated by way of real-time PCR23 and western blot analysis, respectively.20 For real-time PCR, RNA was extracted from cholangiocytes using the RNeasy Mini Kit (Qiagen Inc, Valencia, CA) and reverse-transcribed using the Reaction Ready First Strand cDNA synthesis kit (SuperArray, Frederick, MD). These reactions were used as templates for the PCR assays using an SYBR Green PCR master mix and specific primers designed against the mouse secretin receptor gene NM_001012322,24 and glyceraldehyde 3-phosphate dehydrogenase, the housekeeping gene (SuperArray, Frederick, MD) in the real-time thermal cycler (ABI Prism 7900HT sequence detection system). A ΔΔCt analysis was performed using normal large cholangiocytes as the control sample.

Finally, we show that miR-17-5p expression correlates well with HSP27 status in primary human HCC tissues with metastasis. Conclusion: Our findings suggest that the p38 MAPK pathway plays a crucial role in miR-17-5p-induced phosphorylation of HSP27 and, as a consequence, phosphorylated HSP27 enhances the migration of HCC cells. Our data highlight an important role of miR-17-5p in the proliferation and migration of HCC cells and support the potential application of miR-17-5p in HCC therapy. (Hepatology 2010;) MicroRNAs (miRNAs) are 21- to 25-nucleo tide RNA molecules that regulate

cellular differentiation, apoptosis, proliferation, and migration.1, 2 Many studies have shown that miRNAs are implicated in many cancers, find more and altered miRNA levels can result in the aberrant expression of gene products that may contribute to cancer biology.3, 4 Indeed, some miRNAs have been classified as tumor suppressors or oncogenes.5 Recent studies that have used hybridization-based

microarrays to investigate miRNA expression profiles in human hepatocellular carcinoma (HCC) have identified nonoverlapping signatures of a small number of miRNAs that are up-regulated and down-regulated in human HCC compared with paired peritumoral tissues.6-8 Although the roles of miRNAs in HCC have recently been postulated, their pathophysiological contributions to HCC are still largely unknown. The check details miR-17-92 cluster (composed of miR-17-5p, miR-17-3p, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92-1) first attracted attention following a series of observations linking

these miRNAs to cancer pathogenesis.9 Overexpression of the miR-17-92 locus has been identified in lung cancer,10 B cell lymphoma,9 HCC,11 and stomach solid tumors.12 Moreover, elevated expression of the miR-17-92 polycistron in hepadnavirus-associated HCC contributes to a malignant phenotype.13 The association of miR-17-92 with a broad range of cancers not only underlines the clinical significance of this locus but also suggests that miR-17-92 may regulate fundamental biological processes. miR-17-5p, an important member 上海皓元医药股份有限公司 of the miR-17-92 cluster, was found to be overexpressed in HCC.11, 13, 14 Some targets of miR-17-5p have been confirmed, such as E2F1,15 NCOA3,16 and RBL2.17 Many of these targets are known cell cycle regulators, but their identification does not sufficiently explain the oncogenic potential of miR-17-5p. The introduction of proteomics has enabled the simultaneous analysis of changes in multiple proteins. Recently, some new proteomic approaches were used to measure changes in the synthesis of several thousand proteins in response to miRNA transfection or endogenous miRNA knockdown.18, 19 These data suggest that miRNAs can directly or indirectly regulate protein synthesis of thousands of genes. These approaches are powerful means by which to identify miRNA targets.

CYP7A1 represents the key enzyme catalyzing BA biosynthesis. In one human study, investigating patients with obstructive cholestasis, hepatic CYP7A1 mRNA tended to be increased, similar to the results in our fatty liver patients.32 High serum cholestenone levels confirm the increased CYP7A1 mRNA expression in our study at the functional level. SHP also represses hepatocyte BA uptake by transcriptional repression of NTCP.33 In experimental obstructive cholestasis in rodents, NTCP is down-regulated by activation of FXR and subsequently induction of the repressor SHP. This mechanism prevents excessive BA transport from portal blood and uptake ICG-001 datasheet into the hepatocytes.10

Similar mechanisms are in action for human NTCP where SHP has been shown to suppress the glucocorticoid receptor-mediated transactivation of the human NTCP promoter.34 In human fatty livers, we observed an increase in NTCP expression, which in contrast to Cyp7A1 decreased with progression to NASH. The observed attenuation of baseline SHP activity under steatotic conditions was confirmed in our in vitro experiments. FFA treatment led Mitomycin C price to a significant up-regulation

of NTCP and Cyp7A1. Similar to our findings in NASH regarding NTCP expression, BA (CDCA) treatment attenuated these effects, most likely by way of FXR-SHP activation. This is consistent with recent data from ob/ob mice where SHP-induction and down-regulation of NTCP is absent despite retention of BAs.35 In a recent study, Wanninger et al.36 observed an up-regulation of hepatic CYP7A1 mRNA expression, in line with elevated serum triglyceride levels in adiponectin knockout

mice. We also observed lower adiponectin levels with advanced stages 上海皓元医药股份有限公司 of NAFLD and our in vitro data also confirm a negative regulation of Cyp7A1 by adiponectin. As Aranha et al.6 have shown, BAs within the liver are elevated in patients with steatohepatitis. In agreement with our data, this would suggest an increased BA uptake and production. Elevated intrahepatic BA amounts would lead to activation of FXR and subsequently enhanced BSEP expression, which we also observed.29 On the other hand, impaired adaptation of BSEP expression may lead to increased BA accumulation in hepatocytes and enhanced cell death. Interestingly, steatotic hepatocytes were shown to be more susceptible to BA-induced apoptosis.37 Even low elevations of BAs normally not considered harmful could enhance hepatocyte apoptosis in patients with NAFLD. However, in our in vitro studies we did not observe alterations in viability upon FFA and CDCA cotreatment. An altered intestinal FGF19 production and/or altered hepatic responsiveness to FGF19 may accordingly contribute to the dysregulation of BA homeostasis observed in our patients with NAFLD, as has previously been shown that a rather moderate increase in BA flux may superimpose an influence of FGF15.

02 mg dexametha-sone/kg) or vehicle (PBS). At termination of the study, the liver condition was evaluated by liver weight, NAFLD Activity Score (NAS), fibrosis, and paraclinical liver parameters. Result: The fructose-rich NVP-LDE225 ic50 diet induced marked signs of NASH (increased liver weight, hepatocyte

balloning, inflammation, pericellular and perivenular fibrosis and glycogen deposition). The NAFLD Activity Score (NAS) was strongly reduced in the group of rats treated with anti-CD163-dexamethasone conjugate compared to the vehicle group and the dexamethasone group, respectively (Table 1). Further, aspartate aminotransferase levels were significantly lower in rats treated with anti-CD163-dexametha-sone compared to the vehicle group. Conclusion: Targeting of macrophages by the anti-CD163-dexamethasone conjugate significantly reduced NASH changes compared to free dexa-methasone. Thus, low-dose targeting of dexamethasone to CD163-positive macrophages is a potential future macrophage specific NASH therapy without the serious and problematic side effects seen in conventional systemic glucocorticoid therapy. Disclosures: Jonas H. Graversen – Management Position: Affinicon; Stock Shareholder: Affin-icon Henning Gronbaek – Grant/Research Support: Novartis, Ipsen Holger J. Møller – Grant/Research Support:

Danish Council see more for Strategic Research; Independent Contractor: IQ-Products, NL; Patent Held/Filed: Aarhus University; Stock Shareholder: Affinicon Aps Søren K. Moestrup – Stock Shareholder: Affinicon The following people have nothing to disclose: Pia Svendsen, Hendrik V. Vilstrup Background: Emerging evidence implicates the molecular circadian clock as a critical regulator of alcoholic liver injury. Melatonin, an endogenously produced neurohormone secreted by the pineal gland, has a variety of protective effects during organ injury including promotion of cellular proliferation and differentiation. However, its effect and mechanism on the circa-dian clock medchemexpress during alcoholic liver injury remains

to be explored. The objective of this study was to evaluate the role of mela-tonin regulated microRNA-clock gene network in alcohol-induced hepatic steatosis. Methods: The miRNA expression of melatonin upstream enzymes, serotonin N-acetyltransferase (AANAT) and N-acetylserotonin O-methyltransferase (ASMT), were assessed in ethanol and LPS treated human hepatocytes (N-Heps) and cholangiocytes (H69), as well as in 5-week chronic alcohol fed mouse liver specimens with anti-miRNA vivo-morpholino treatment and control liver tissue by real-time PCR assay. The secretion of melatonin was verified by ELISA assay. Cell proliferation and survival was measured by MTS assay. The hepatic expressions of the clock circadian genes including PER1, Clock and CRY1 were also determined by real-time PCR assay.

Thus, 5 to 20% of NAFL patients progress to NASH. It is still not well understood why some patients develop NASH AZD3965 manufacturer and others remain with NAFL. Higher frequencies of Th17 cells were observed in livers of a NASH mouse model and higher IL-17 and IL-21 gene expression was described

in human livers of NASH patients. We hypothesized that the phenotype of peripheral CD4+ T cells might be predictive for the degree and quality of hepatic T cell infiltration and histopathology. Aims: To analyse differences in the hepatic and peripheral immune phenotype in patients with NAFL and NAFLD with a focus on conventional CD4+ effector T cell subsets and CD4+ regulatory T cells (Tregs). Methods: 40 patients with NAFL, 17 patients with NASH and 44 healthy controls (HC) were included in this study. Multi-colour FACS analysis was performed of PBMCs and intrahepatic lymphocytes. CD4+ T cells were stimulated with PMA and ionomycin for intracellular detection of cytokine production (IL-17, IL-4, INF-g, IL-21). Results: Patients were older, had a higher BMI and a higher CK-18 serum level in comparison to HC. In peripheral blood, NAFLD and NASH patients had higher frequencies of HLA-DR+, i.e. activated, effector memory, IFN-g+, IL-17+, IL-21+ and IL-4+ cells and lower frequencies of CD45RA+ CD25+ resting Tregs among CD4+ T cells than HC. Closer analysis of Th1 cells among PBMCs revealed that T-bet expression

was lower among CXCR3+ cells of NASH than NAFLD patients. The CD4+ T cells infiltrating the liver of NAFLD and NASH patients consisted to more than 80% of CXCR3+ effector memory cells with more than 50% Apoptosis inhibitor recently activated, i.e. HLA-DR+, cells and frequencies of cells producing the named cytokines elevated at least five- to ten-fold in comparison to PBMCs. Higher frequencies of IL-17+ cells among intrahepatic CD4+ T cells and a higher Th17/ resting Treg ratio distinguished NAFLD from NASH patients. Conclusions: Our data indicate that NAFL patients show a “prehepatitic” immune cell profile very similar to that seen in NASH. The progression from NAFLD to NASH is marked by increased frequency of IL-17+ cells among intrahepatic CD4+ T cells

and a higher Th17/Treg 上海皓元 ratio. Disclosures: The following people have nothing to disclose: Monika Rau, Anne-Kristin Schilling, Ilona Hering, Jan Meertens, Theodor Kudlich, Christian Jurowich, Niklas Beyersdorf, Andreas Geier Background: NASH is increasingly recognized as a disease of lipotoxicity induced by diet and lifestyle. The sequence of events that lead to hepatic lipid accumulation, metabolic changes and mitochondrial dysfunction in NASH are not known. We investigated the chronology of these phenomena in a widely adopted diet-induced animal model of NASH. Methods: Male C57Bl/6 mice were randomly assigned to a fast food (high fat, high cholesterol) or a standard chow (SC) diet and reared up to 36 weeks. Fructose was provided in the drinking water.

355, P<0.0001) were predictors of achieving qHB-sAg level ≧2 log 10IU/mL during treatment in HBeAg-negative

patients. Conclusion: Baseline qHBsAg and ALT levels are predictors of HBeAg loss during ETV therapy in HBeAg-positive patients. Baseline qHBsAg levels and on-treatment qHBsAg decline from baseline are predictors of achieving qHBsAg level ≧2 log10IU/mL during ETV therapy in both HBeAg-positive and -negative patients. Disclosures: The following people have nothing to disclose: Cheng-Yuan Peng, Hsueh-Chou Lai, Wen- Pang Su, Chia-Hsin Lin, Po-Heng Chuang, learn more Sheng-Hung Chen Background & Aims: Chronic hepatitis B virus (HBV) infection leads to cirrhosis and hepatocellular carcinoma (HCC). Antiviral agents are thought to reduce the risk of hepatic failure and HCC development. Methods: We compared the incidence of HCC in 332 nucleoside analogue (NA) treated patients

(NA group) and 494 non-treated HBV patients (control group). In the NA group, patients were initially prescribed LAM or entecavir (ETV). The drug Romidepsin ic50 mutation resistance was treated with LAM and adefovir (ADV) or ETV and ADV. Patient characteristics of the NA group and the control group differed significantly in age, gender, genotype, baseline HBV DNA level. Propensity score matching was used to eliminate the baseline above, resulting in a sample size of 137 patients per cohort. Results: The cumulative incidence rates of HCC in the NA groups were 1.6% at year 2, 3.5% at year 3, 4.5% at year 5, and 上海皓元医药股份有限公司 1 0.5% at year 10, while 0.7%, 2.3%, 3.2%, and 7.4%, respectively in the matched control group. Cox proportional hazard regression analysis showed that the NA group had similar risks of HCC development as the control group (hazard ratio 1.42, P = 0.54). In the patients treated with NA, serum HBV DNA decreased from 6.9 log IU/mL to 2.6 log IU/mL, albumin increased from 4.0 g/dL to 4.3 g/dL and ALT decreased from 82 IU/L to 23 IU/L, after 48 weeks treatment (p<0.001, <0.001, and <0.001, respectively). Conclusions: Even with long-term NA treatment,

the incidence of HCC development was not reduced significantly in HBV-infected patients. Though long-term NA treatment can bring back hepatic reserve effectively, careful observation with periodical HCC screening is recommended. Disclosures: Kazuhide Yamamoto – Advisory Committees or Review Panels: Shionogi Pharmaceutical Co; Grant/Research Support: Tanabe Mitsubishi Co, MSD, Chugai Pharmaceutical Co, Esai Co The following people have nothing to disclose: Tetsuya Yasunaka, Fusao Ikeda, Nozomu Wada, Yuuki Morimoto, Kenji Kuwaki, Akinobu Takaki Background: It has already been reported that adefovir dip-ivoxil (ADV) causes renal impairment at a certain frequency but few studies have analyzed serum ADV concentrations. Methods: The study subjects were 89 lamivudine (LAM)-resistant patients who were co-administered ADV in our hospital. LAM and ADV doses were 100 mg/day and 10 mg/day, respectively.

“
“(Headache 2010;50:761-768) CH5424802 cell line Objective.— To study the relationship between childhood physical abuse and migraine in adolescents. Background.— Childhood maltreatment might lead to an increased probability of migraine among adults. Nevertheless, the relationship between migraine and childhood

abuse is unknown in adolescents. Methods.— We enrolled 3955 students, ages 13-15, from 3 middle schools. Each participant completed a valided headache questionnaire for headache diagnosis and the Adolescent Depression Inventory (ADI). A classification of physical maltreatment was given to students who reported they had been beaten by parents or elder family members. Results.— A total of 926 (23.4%) students were diagnosed with migraine or probable migraine occurring within the 3 months prior to the survey. Physical maltreatment was reported by 945 Adriamycin concentration (23.9%) students, including a frequency of “rarely” in 762 (19.3%) students and “sometimes or often” in 183 (4.6%). The students reporting physical maltreatment were more likely to suffer migraine or probable migraine compared with those who reported no physical maltreatment (30.3% vs 21.3%, odds ratios = 1.6, 95%, CI: 1.4-1.9, P < .001). A higher frequency of physical maltreatment was associated with a higher likelihood of migraine diagnosis (21.3%

vs 28.3%, vs 38.3%, “never” vs “rarely” vs “sometimes or often maltreated,” respectively, P < .001). In addition, among the students diagnosed with migraine, those reporting physical maltreatment had higher mean ADI scores, a higher frequency of headaches, and a greater proportion of severe headaches. Conclusions.— The results suggest that physical maltreatment is associated

with migraine in adolescents and that physical maltreatment may be MCE公司 related to an increase in the frequency and intensity of headaches in adolescents with migraines. A history of physical maltreatment may be helpful in the treatment of adolescents suffering from migraine. “
“The neuro-ophthalmology examination is critical to anyone who sees patients with the common symptom of headache. By examining the visual acuity, pupils, visual fields, motility, and fundus, clues to both secondary causes of headache and primary headaches exist. In this review, we discuss how to do the neuro-ophthalmology examination and we review cases of primary and secondary headache where key features of the examination assisted in making the correct diagnosis. “
“Many neurologists and headache specialists are befuddled by inside the Beltway wheelings and dealings as they follow health care politics. A few of us join lobbying efforts, and even fewer become strangers in a strange land. “
“Background.— Although diagnostic rates for migraine have increased over the past 5 years, the proportion of migraine sufferers using triptans has remained essentially stable. Objectives.— To assess the rate of onset of new triptan prescriptions among persons with migraine and the predictors of initiating therapy. Methods.

36 Therefore, static adhesion assays were repeated after coculturing immature ductular cells with a myofibroblastic cell line (8B) in a transwell system.38 Coculture with stellate cells increased static adhesion of NKT cells to ductular cells; this was also markedly attenuated when Hh-neutralizing antibodies were selleck compound added to the medium (Fig. 2E). Therefore, Hh-pathway activation during NASH promotes the hepatic recruitment and retention of NKT cells. Fibrotic

livers with NASH also expressed higher levels of CD1d (Fig. 3A) and IL15 (Fig. 3B), both of which can promote NKT cell survival. Consistent with evidence that MCD diet-induced NASH results in a nurturing microenvironment for NKT cells, the livers from MCD diet-treated mice were significantly enriched in CD1d tetramer-reactive NKT cells (Fig. 3C,D). Immunostaining for CD57, another NKT cell marker, confirmed that

NASH liver parenchyma harbored about 2× more CD57 (+) cells than control livers (Fig. 3E,F). Ptc+/− mice (which exhibit sustained Hh-signaling after pathway activation25) develop more liver fibrosis than wildtype mice when fed MCD diets.39 Therefore, we fed Ptc+/− mice MCD or control diets for 8 weeks, isolated LMNCs, and performed FACS to determine if overactivating the Hh-pathway influenced NKT cell accumulation. NKT cells comprised ∼10% of the LMNC in chow-fed Ptc+/− mice (Fig. 4A), and 8 weeks of MCD diet feeding resulted in ∼4-fold enrichment of NKT cells (Fig. 4B,C). Thus, although LMNC from Ptc+/− and WT C57Bl6 mice contained medchemexpress similar proportions of NKT cells before injury-related Dorsomorphin in vivo activation of hepatic Hh signaling, the degree of both Hh-pathway activation and NKT cell enrichment was greater in Ptc+/− mice during NASH. The findings may be relevant because Ptc+/− mice are also known to develop more severe liver fibrosis than WT mice during MCD diet-induced NASH. To more directly evaluate the significance of hepatic NKT accumulation for NASH-related fibrogenesis, MCD diet feeding was repeated in CD1d-deficient mice which lack NKT cells26 and littermate controls (n = 3/group). Livers were harvested after 8 weeks for assessment of collagen gene expression

and hepatic hydroxyproline content. Both assays demonstrated significant attenuation of fibrogenesis in the NKT cell-deficient mice (Fig. 4D,E). Primary hepatic NKT cells produce Shh ligand, and Hh ligands stimulate them to produce fibrogenic factors, including IL4 and IL13,29 suggesting that soluble factors from NKT cells promote liver fibrosis. To investigate this more directly, primary LMNC were isolated from healthy mice and incubated with αGalCer,40 which specifically activates NKT cells; cell-conditioned medium was then added to primary cultures of mouse stellate cells. One day later, cultures were harvested to obtain RNA for PCR analysis. Parallel studies were done with conditioned medium from LMNC that were treated with vehicle.