7A-C).8 INT-747 and INT-767 increased the size and amount of bile infarcts, as well as LW/BW ratio, in CBDL mice (Supporting Fig. 12A,B), whereas only INT-767 significantly decreased SW/BW ratio (Supporting Fig. 12C) and showed a trend to reduction of serum ALT (Supporting Fig. 13A). Although histological examination

of H&E-stained livers revealed bile infarcts in all the groups, only INT-747 increased infiltration of inflammatory cells within the portal fields (Supporting Fig. 13B). In line with serum ALT levels, INT-767-fed CBDL mice had reduced expression of proinflammatory genes Tnf-α and Il-1β and less CD-11b- and F4/80-positive cells around bile infarcts (Supporting Fig. 14A,B). However, keratin 19 (K19) and Vcam-1 gene expression remained unchanged in CBDL mice after INT-747, INT-777, and INT-767 feeding (Supporting Fig. 15). In this study, we have addressed the selleck inhibitor therapeutic mechanisms of BA receptor signaling through the nuclear BA receptor, FXR, and the G-protein-coupled membrane BA receptor, TGR5, in the Mdr2−/− mouse cholangiopathy model. We report herein that, in this model, the novel FXR/TGR5 agonist, INT-767, reduces bile toxicity by decreasing biliary BA output and inducing HCO-rich

Y-27632 manufacturer choleresis in an FXR-dependent manner. BAs are important signaling molecules with hormonal actions through dedicated nuclear and G-protein-coupled receptors, such as FXR and TGR5, respectively.8 TGR5 and FXR polymorphisms19, 20 further support the importance

of BA signaling in human cholestastic diseases, such as PSC. Liver injury in Mdr2−/− mice is considered to evolve because of detergent properties of nonmicellar-bound free biliary BAs,29 leaving many open questions for the potential role of BA signaling in modulating biliary pathophysiology. Only the dual FXR/TGR5 agonist, INT-767, 上海皓元 was hepatoprotective in the Mdr2−/− model, as reflected by reduced serum ALT, decreased hepatic inflammation, improved reactive cholangiocyte phenotype, and reduced fibrosis. We could neither observe significant direct anti-inflammatory effects of INT-767 in RAW264.7 macrophages (with very low endogenous Fxr and Tgr5 expression), BEC cholangiocytes, or HepG2 hepatocytes (both with high levels of Fxr and very low Tgr5; data not shown) nor direct antifibrotic effects in primary MFBs (with very low endogenous Fxr and Tgr5 expression) as major fibrogenic cells in the Mdr2−/− model. Absent expression of FXR and TGR59, 11 in hepatic stellate cells further indicates that FXR and TGR5 signaling may have no direct antifibrotic effects. These findings led us hypothesize that INT-767 might improve liver injury by directly impacting on bile formation and composition. Indeed, via Fxr activation, INT-767 inhibited BA synthesis (by ileal Fgf15 and hepatic Shp induction), thus resulting in decreased biliary BA output while significantly increasing bile flow and-unexpectedly-HCO output.

To study the function of Col1a1 within fibrocytes, BM from 5′StemLoop knockout mouse was transplanted into wildtype mice, which are deficient of Col1a1 expression in fibrocytes. METHODS: Inducible Col1a1-ER-Cre mice were crossed with Rosa26-YFP

Y-27632 concentration reporter mice and Rosa26-DTA mice and were used as donors for BM transplantation into wt mice to generate Fibrocyte deleted mice. Upon tamoxifen administration, fibrocytes were labeled by YFP expression in wt mice, while successful ablation of fibrocytes (>95%) was achieved in Fibrocyte deleted mice, as indicated by disappearance of YFP+ fibrocytes. To study the function of Col1a1 in fibrocytes, the BM from 5′SL knockout mice was transplanted into wt mice to generate Fibrocyte5′SL-Col1a1 mice. Mice were subjected

to CCl4 (6w), livers were analyzed for development of liver fibrosis. The transcriptome of fibrocytes Vemurafenib price were assessed by RNA-seq. RESULTS: Deletion of fibrocytes in mice resulted in attenuation of CCl4-induced liver fibrosis by 50%, as shown by reduced Sirius Red, and Col1a1, alpha-SMA, TIMP1 and TGFbeta1 mRNA expression. We determined that in fibrotic liver fibrocyte give rise to 10% of myofibroblasts. Meanwhile, majority of fibrocytes contributes to hepatic myeloid cells, which serve as a significant source of TGFb1, and inflammatory cytokines such as IL-6 and IL1b1, and also suppress anti-fibrogenic (M2) macrophages. We next hypothesized that upregulation of Col1a1

may be important for fibrocyte functions. Indeed, development of liver fibrosis was reduced in Fibrocyte5′SL-Col1a1 mice by 30%, and was associated with impaired activation and migration of HSCs and reduced TGFβ1 and CCL5 in liver. CONCLUSION: Fibrocyte contribute to myofibroblast population, but most importantly, they mediate differentiation of proinflammatory macrophage and secret profibrogenic cytokine TGFβ1. Furthermore, proper collagen expression is required for fibrocyte function. Disclosures: The following people have nothing to disclose: Jun Xu, Tae Jun Park, Min Cong, Xiao Liu, David A. Brenner, Tatiana Kisseleva Background: To accomplish cell therapy with higher efficiencies, insights into transplanted cell fates is critical. The ability of mature hepatocytes as well as LSEC to engraft and MCE proliferate in liver was of therapeutic benefit. However, early clearance of transplanted cells was a major restriction in both cases. Clearance of transplanted hepatocytes was largely due to secondary release of endothelin-1 (ET1) or of chemokines/cytokines/ receptors, e.g., TNF-a, from Kupffer cells and neutrophils, whereas endothelial disruption and release of hepatoprotective factors from hepatic stellate cells (HSC) benefited cell engraftment. Based on these considerations, we defined mechanisms for engrafting transplanted LSEC in liver.

One milliliter of the culture contained

50 000 MNC, α-modified Eagle’s medium, 1.2% 1500-centipoise methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% fetal bovine serum, 1% BSA, 1 × 10−4 m 2-mercaptoethanol (Wako Pure Chemical Industries, Osaka, Japan), and cytokines including 100 ng/mL recombinant human (rh) stem cell factor, 10 ng/mL rh interleukin-3, 10 ng/mL rh granulocyte macrophage colony-stimulating factor, 10 ng/mL rh granulocyte colony-stimulating factor and 2 U/mL rh erythropoietin. All cytokines were gifts from Kirin Brewery (Tokyo, Japan). The dishes were then incubated in a humidified atmosphere with 5% CO2 in air. On day 14 of culture, colonies consisting of 40 cells or more were scored on an Olympus CK40 inverted microscope (Olympus, Tokyo, Japan). Plasma SDF-1α was measured using a human CXCL12/SDF-1α Angiogenesis inhibitor Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA). The intra- and inter-assay see more coefficients of variations were 3.5% and 10.3%, respectively. SDF-1α concentrations were measured in duplicate for each sample and standard solution. Formalin-fixed, paraffin-embedded

5-μm tissue sections of spleen specimens obtained from three splectomyzed LC patients were used for immunohistochemical staining for SDF-1α. Deparaffinized sections were heated for 5 min at 100°C in a pressure cooker to reactivate the antigen and treated with 0.3% H2O2 in methanol for 30 min to abolish endogenous peroxidase activity. Sections were blocked with 1% goat serum in PBS, covered with rabbit anti-SDF-1α polyclonal antibody (PeproTech, Rocky Hill, NJ, USA; dilution 1:200) overnight at 4°C, washed, covered with a second-step biotinylated medchemexpress antibody for 30 min,

and incubated with peroxidase-labeled streptavidin for 30 min. After washing, sections were incubated with 0.05% 3,3′-diaminobenzidine tetrahydrochloride and 0.15% H2O2, and counterstained with 10% hematoxylin (Wako Pure Chemical Industries). Controls were performed by omitting the primary SDF-1α antibody. Immunofluorescence studies were performed with similar methods using rabbit anti-human CD34 and mouse anti-human CD45 (both Abcam, Cambridge, MA, USA; dilution 1:200). The following secondary antibodies were used: FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG F(ab’)2 (both Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:400). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Molecular Probes Invitrogen, Eugene, OR, USA). Staining with appropriate FITC- or PE-conjugated isotype controls was performed as negative controls. All sections were examined under a fluorescence microscope (BZ-X700; Keyence, Osaka Japan). Statistically significant differences between groups were analyzed by Student’s t-test and the Mann–Whitney U-test. Correlations were determined by the linear regression test. Differences were considered significant at P < 0.05.

One milliliter of the culture contained

50 000 MNC, α-modified Eagle’s medium, 1.2% 1500-centipoise methylcellulose (Shinetsu Chemical, Tokyo, Japan), 30% fetal bovine serum, 1% BSA, 1 × 10−4 m 2-mercaptoethanol (Wako Pure Chemical Industries, Osaka, Japan), and cytokines including 100 ng/mL recombinant human (rh) stem cell factor, 10 ng/mL rh interleukin-3, 10 ng/mL rh granulocyte macrophage colony-stimulating factor, 10 ng/mL rh granulocyte colony-stimulating factor and 2 U/mL rh erythropoietin. All cytokines were gifts from Kirin Brewery (Tokyo, Japan). The dishes were then incubated in a humidified atmosphere with 5% CO2 in air. On day 14 of culture, colonies consisting of 40 cells or more were scored on an Olympus CK40 inverted microscope (Olympus, Tokyo, Japan). Plasma SDF-1α was measured using a human CXCL12/SDF-1α Trichostatin A concentration Quantikine enzyme-linked immunosorbent assay kit (R&D Systems, Minneapolis, MN, USA). The intra- and inter-assay Metformin coefficients of variations were 3.5% and 10.3%, respectively. SDF-1α concentrations were measured in duplicate for each sample and standard solution. Formalin-fixed, paraffin-embedded

5-μm tissue sections of spleen specimens obtained from three splectomyzed LC patients were used for immunohistochemical staining for SDF-1α. Deparaffinized sections were heated for 5 min at 100°C in a pressure cooker to reactivate the antigen and treated with 0.3% H2O2 in methanol for 30 min to abolish endogenous peroxidase activity. Sections were blocked with 1% goat serum in PBS, covered with rabbit anti-SDF-1α polyclonal antibody (PeproTech, Rocky Hill, NJ, USA; dilution 1:200) overnight at 4°C, washed, covered with a second-step biotinylated MCE公司 antibody for 30 min,

and incubated with peroxidase-labeled streptavidin for 30 min. After washing, sections were incubated with 0.05% 3,3′-diaminobenzidine tetrahydrochloride and 0.15% H2O2, and counterstained with 10% hematoxylin (Wako Pure Chemical Industries). Controls were performed by omitting the primary SDF-1α antibody. Immunofluorescence studies were performed with similar methods using rabbit anti-human CD34 and mouse anti-human CD45 (both Abcam, Cambridge, MA, USA; dilution 1:200). The following secondary antibodies were used: FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG F(ab’)2 (both Santa Cruz Biotechnology, Santa Cruz, CA, USA; dilution 1:400). Nuclei were stained with 4′,6-diamidino-2-phenylindole (Molecular Probes Invitrogen, Eugene, OR, USA). Staining with appropriate FITC- or PE-conjugated isotype controls was performed as negative controls. All sections were examined under a fluorescence microscope (BZ-X700; Keyence, Osaka Japan). Statistically significant differences between groups were analyzed by Student’s t-test and the Mann–Whitney U-test. Correlations were determined by the linear regression test. Differences were considered significant at P < 0.05.

(Fig. 1A; Supporting Fig. S1). We examined whether HCV modulates the expression of either miR-27 isoform. Huh7.5 cells were transfected with subgenomic replicon (HCV-SGR) from the Con1 isolate (genotype 1b; Fig. 1B). Relative miR-27 GW-572016 chemical structure expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). HCV-SGR induced a 2-fold up-regulation of miR-27a expression and 5-fold up-regulation in miR-27b expression (Fig. 1C). Transfection of replication-deficient HCV-SGR ΔNS5B maintained a 2-fold up-regulation of miR-27a (Fig. 1C); however, miR-27b levels did not increase (Fig. 1C). These observations indicate that viral replication

is required for miR-27b up-regulation but HCV translation is sufficient to activate miR-27a expression. Next we examined miR-27 expression during HCV infection. We performed qRT-PCR analysis on Huh7.5 cells infected with JFH-1T, a cell-culture adapted high-titer strain of JFH-1 (genotype 2a).[25] Up-regulation of both miR-27a (2.6-fold; Fig. 1D) and miR-27b levels (1.2-fold; Fig. 1E) was observed. These results confirm that HCV infection induces miR-27

expression, and this induction is conserved across HCV genotypes. To probe the molecular mechanism by which HCV regulates miR-27, we used an miR-27 sensor plasmid containing a dual-luciferase reporter bearing two fully complementary miR-27b binding sites in the 3′-untranslated region (UTR) of the Renilla luciferase gene. Since miR-27a and miR-27b differ by only one nucleotide, both isoforms regulate http://www.selleckchem.com/products/Methazolastone.html luciferase activity. Huh7 cells were cotransfected with HCV proteins and the miR-27 sensor plasmid. HCV core and NS4B expression independently induced a decrease in luciferase signal relative to the controls (Fig. 1F). This down-regulation was reversed

MCE upon mutation of the miR-27 binding sites, demonstrating miR-27-specific activity. qRT-PCR confirmed that both core and NS4B overexpression resulted in increased miR-27a/b levels (Supporting Fig. S2). miR-27b expression can be activated in a PI3K pathway-dependent manner.[26] Since both NS4B and core have previously been shown to activate SREBP by way of the PI3K/Akt pathway,[27, 28] we hypothesized that these proteins may regulate miR-27b expression similarly. Huh7 cells were cotransfected with NS4B and core and miR-27 sensor plasmid and then treated with a PI3K inhibitor, LY294002. The results showed LY294002 impaired HCV proteins’ ability to induce miR-27-mediated gene silencing (Supporting Fig. S3), suggesting that HCV activates miR-27 expression in a PI3K-dependent fashion. We next examined whether miR-27 plays a regulatory role for lipid metabolism in Huh7 cells by transfecting with control or miR-27 mimics and inhibitors and measuring the effects. The activity of miR-27b mimics and inhibitors was confirmed using the sensor plasmid (Supporting Fig. S4).

(Fig. 1A; Supporting Fig. S1). We examined whether HCV modulates the expression of either miR-27 isoform. Huh7.5 cells were transfected with subgenomic replicon (HCV-SGR) from the Con1 isolate (genotype 1b; Fig. 1B). Relative miR-27 selleckchem expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). HCV-SGR induced a 2-fold up-regulation of miR-27a expression and 5-fold up-regulation in miR-27b expression (Fig. 1C). Transfection of replication-deficient HCV-SGR ΔNS5B maintained a 2-fold up-regulation of miR-27a (Fig. 1C); however, miR-27b levels did not increase (Fig. 1C). These observations indicate that viral replication

is required for miR-27b up-regulation but HCV translation is sufficient to activate miR-27a expression. Next we examined miR-27 expression during HCV infection. We performed qRT-PCR analysis on Huh7.5 cells infected with JFH-1T, a cell-culture adapted high-titer strain of JFH-1 (genotype 2a).[25] Up-regulation of both miR-27a (2.6-fold; Fig. 1D) and miR-27b levels (1.2-fold; Fig. 1E) was observed. These results confirm that HCV infection induces miR-27

expression, and this induction is conserved across HCV genotypes. To probe the molecular mechanism by which HCV regulates miR-27, we used an miR-27 sensor plasmid containing a dual-luciferase reporter bearing two fully complementary miR-27b binding sites in the 3′-untranslated region (UTR) of the Renilla luciferase gene. Since miR-27a and miR-27b differ by only one nucleotide, both isoforms regulate INCB024360 luciferase activity. Huh7 cells were cotransfected with HCV proteins and the miR-27 sensor plasmid. HCV core and NS4B expression independently induced a decrease in luciferase signal relative to the controls (Fig. 1F). This down-regulation was reversed

上海皓元医药股份有限公司 upon mutation of the miR-27 binding sites, demonstrating miR-27-specific activity. qRT-PCR confirmed that both core and NS4B overexpression resulted in increased miR-27a/b levels (Supporting Fig. S2). miR-27b expression can be activated in a PI3K pathway-dependent manner.[26] Since both NS4B and core have previously been shown to activate SREBP by way of the PI3K/Akt pathway,[27, 28] we hypothesized that these proteins may regulate miR-27b expression similarly. Huh7 cells were cotransfected with NS4B and core and miR-27 sensor plasmid and then treated with a PI3K inhibitor, LY294002. The results showed LY294002 impaired HCV proteins’ ability to induce miR-27-mediated gene silencing (Supporting Fig. S3), suggesting that HCV activates miR-27 expression in a PI3K-dependent fashion. We next examined whether miR-27 plays a regulatory role for lipid metabolism in Huh7 cells by transfecting with control or miR-27 mimics and inhibitors and measuring the effects. The activity of miR-27b mimics and inhibitors was confirmed using the sensor plasmid (Supporting Fig. S4).

(Fig. 1A; Supporting Fig. S1). We examined whether HCV modulates the expression of either miR-27 isoform. Huh7.5 cells were transfected with subgenomic replicon (HCV-SGR) from the Con1 isolate (genotype 1b; Fig. 1B). Relative miR-27 www.selleckchem.com/products/pf-562271.html expression was analyzed by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). HCV-SGR induced a 2-fold up-regulation of miR-27a expression and 5-fold up-regulation in miR-27b expression (Fig. 1C). Transfection of replication-deficient HCV-SGR ΔNS5B maintained a 2-fold up-regulation of miR-27a (Fig. 1C); however, miR-27b levels did not increase (Fig. 1C). These observations indicate that viral replication

is required for miR-27b up-regulation but HCV translation is sufficient to activate miR-27a expression. Next we examined miR-27 expression during HCV infection. We performed qRT-PCR analysis on Huh7.5 cells infected with JFH-1T, a cell-culture adapted high-titer strain of JFH-1 (genotype 2a).[25] Up-regulation of both miR-27a (2.6-fold; Fig. 1D) and miR-27b levels (1.2-fold; Fig. 1E) was observed. These results confirm that HCV infection induces miR-27

expression, and this induction is conserved across HCV genotypes. To probe the molecular mechanism by which HCV regulates miR-27, we used an miR-27 sensor plasmid containing a dual-luciferase reporter bearing two fully complementary miR-27b binding sites in the 3′-untranslated region (UTR) of the Renilla luciferase gene. Since miR-27a and miR-27b differ by only one nucleotide, both isoforms regulate Selleck Doramapimod luciferase activity. Huh7 cells were cotransfected with HCV proteins and the miR-27 sensor plasmid. HCV core and NS4B expression independently induced a decrease in luciferase signal relative to the controls (Fig. 1F). This down-regulation was reversed

上海皓元医药股份有限公司 upon mutation of the miR-27 binding sites, demonstrating miR-27-specific activity. qRT-PCR confirmed that both core and NS4B overexpression resulted in increased miR-27a/b levels (Supporting Fig. S2). miR-27b expression can be activated in a PI3K pathway-dependent manner.[26] Since both NS4B and core have previously been shown to activate SREBP by way of the PI3K/Akt pathway,[27, 28] we hypothesized that these proteins may regulate miR-27b expression similarly. Huh7 cells were cotransfected with NS4B and core and miR-27 sensor plasmid and then treated with a PI3K inhibitor, LY294002. The results showed LY294002 impaired HCV proteins’ ability to induce miR-27-mediated gene silencing (Supporting Fig. S3), suggesting that HCV activates miR-27 expression in a PI3K-dependent fashion. We next examined whether miR-27 plays a regulatory role for lipid metabolism in Huh7 cells by transfecting with control or miR-27 mimics and inhibitors and measuring the effects. The activity of miR-27b mimics and inhibitors was confirmed using the sensor plasmid (Supporting Fig. S4).

Thiamine deficiency predominantly occurs in patients with ALD, but may also occur as a consequence of malnutrition in end-stage cirrhosis of any cause. The cerebral symptoms disorientation, alteration of consciousness, ataxia, and dysarthria cannot be differentiated as being the result of thiamine deficiency or hyperammonemia by clinical examination.[149] In any case of doubt, thiamine should be given IV before glucose-containing solutions. Data upon the effect of the underlying liver disease on brain function are sparse, except for alcoholism and hepatitis C. Rare, but difficult, cases may be the result of Wilson’s

disease. Even patients with alcohol disorder and no clinical disease have been shown to exhibit deficits in episodic memory,[150] working memory and executive functions,[151] visuoconstruction abilities,[152] and upper- and lower-limb motor skills.[153] The cognitive AZD1208 nmr dysfunction is more pronounced in those patients with alcohol disorder who are at risk of Wernicke’s encephalopathy as a result of malnutrition or already show signs of the problem.[154] Thus, it remains

unclear whether Cell Cycle inhibitor the disturbance of brain function in patients with ALD is the result of HE, alcohol toxicity, or thiamine deficiency. There is mounting evidence that HCV is present and replicates within the brain.[155-158] Approximately half of HCV patients suffer chronic fatigue irrespective of the grade of their liver disease,[159, 160] and even patients with only mild liver disease display cognitive dysfunction,[161,

162] involving verbal learning, attention, executive function, and memory. Likewise, patients with primary biliary cirrhosis and primary sclerosing cholangitis may have severe fatigue and impairment of attention, concentration, and psychomotor function irrespective of the grade of liver disease.[163-168] Because HE shares symptoms with all concomitant disorders and underlying diseases, it is difficult in the individual case to differentiate between the effects of HE and those resulting from other causes. In some cases, the time course and response to therapy may be the best support of HE. As mentioned, a normal blood ammonia level in a patient suspected of HE calls for consideration. None of the diagnostic measures used at present has been evaluated for their ability to differentiate medchemexpress between HE and other causes of brain dysfunction. The EEG would not be altered by DM or alcohol disorders, but may show changes similar to those with HE in cases of renal dysfunction, hyponatremia, or septic encephalopathy. Psychometric tests are able to detect functional deficits, but are unable to differentiate between different causes for these deficits. Brain imaging methods have been evaluated for their use in diagnosing HE, but the results are disappointing. Nevertheless, brain imaging should be done in every patient with CLD and unexplained alteration of brain function to exclude structural lesions.

Thiamine deficiency predominantly occurs in patients with ALD, but may also occur as a consequence of malnutrition in end-stage cirrhosis of any cause. The cerebral symptoms disorientation, alteration of consciousness, ataxia, and dysarthria cannot be differentiated as being the result of thiamine deficiency or hyperammonemia by clinical examination.[149] In any case of doubt, thiamine should be given IV before glucose-containing solutions. Data upon the effect of the underlying liver disease on brain function are sparse, except for alcoholism and hepatitis C. Rare, but difficult, cases may be the result of Wilson’s

disease. Even patients with alcohol disorder and no clinical disease have been shown to exhibit deficits in episodic memory,[150] working memory and executive functions,[151] visuoconstruction abilities,[152] and upper- and lower-limb motor skills.[153] The cognitive VX-765 mw dysfunction is more pronounced in those patients with alcohol disorder who are at risk of Wernicke’s encephalopathy as a result of malnutrition or already show signs of the problem.[154] Thus, it remains

unclear whether CH5424802 ic50 the disturbance of brain function in patients with ALD is the result of HE, alcohol toxicity, or thiamine deficiency. There is mounting evidence that HCV is present and replicates within the brain.[155-158] Approximately half of HCV patients suffer chronic fatigue irrespective of the grade of their liver disease,[159, 160] and even patients with only mild liver disease display cognitive dysfunction,[161,

162] involving verbal learning, attention, executive function, and memory. Likewise, patients with primary biliary cirrhosis and primary sclerosing cholangitis may have severe fatigue and impairment of attention, concentration, and psychomotor function irrespective of the grade of liver disease.[163-168] Because HE shares symptoms with all concomitant disorders and underlying diseases, it is difficult in the individual case to differentiate between the effects of HE and those resulting from other causes. In some cases, the time course and response to therapy may be the best support of HE. As mentioned, a normal blood ammonia level in a patient suspected of HE calls for consideration. None of the diagnostic measures used at present has been evaluated for their ability to differentiate MCE between HE and other causes of brain dysfunction. The EEG would not be altered by DM or alcohol disorders, but may show changes similar to those with HE in cases of renal dysfunction, hyponatremia, or septic encephalopathy. Psychometric tests are able to detect functional deficits, but are unable to differentiate between different causes for these deficits. Brain imaging methods have been evaluated for their use in diagnosing HE, but the results are disappointing. Nevertheless, brain imaging should be done in every patient with CLD and unexplained alteration of brain function to exclude structural lesions.

The following Abs were used in this study: anti-I-Ab FITC, anti-H-2Kb FITC, anti-CD11c PE, anti-CD8a

PE, anti-CD19 PE, anti-CD1d PE, anti-CD4 PerCP/Cy5.5, anti-CD80 PerCP/Cy5.5, anti-CD11c PerCP/Cy5.5, (Biolegend, San Diego, CA), anti-CD3 FITC, anti-CD86 PE, anti-CD40 PE, Wnt drug anti-NK1.1 APC (eBioscience, San Diego, CA). For intracellular staining, liver mononuclear cells were incubated with brefeldin A (10 μg/mL) (BD Biosciences, San Diego, CA) at 37°C for 1 hour, then incubated with anti-CD16/32 Abs, followed by staining with PerCP/Cy5.5-conjugated CD3 and PE-conjugated PBS57 loaded CD1d tetramer (originally produced by the NIH tetramer facility, and supplied through Dr. David Serreze), permeabilized with Cytofix/Cytoperm reagent (BD Biosciences), and stained with Alexa Fluor 488-conjugated anti-IFN-γ (clone XMG1.2), Alexa Fluor 488-conjugated anti-IL-4 (clone 11B11), or rat IgG1 isotype control (clone R3-34) (BD Biosciences). Stained cells were assessed on a FACSCalibur (BD Biosciences) using FlowJo softwares (Tree Star, Ashland, OR). Portions of the liver were excised and immediately fixed with 10% buffered formalin solution for 2 days at room temperature. Paraffin-embedded tissue sections

were then cut into 4-μm slices for routine hematoxylin and eosin (H&E), silver, and Azan staining. Scoring of liver inflammation was performed on coded H&E-, silver-, or Azan-stained sections of liver using a set of indices by a “blinded” pathologist (K.T.); these indices Pexidartinib molecular weight quantitated the degree of portal inflammation, parenchymal inflammation, bile duct damage, granulomas, and fibrosis. Each section was scored as either 0 = no significant change, 1 = minimal, 2 = mild, 3 = moderate, 上海皓元 and 4 = severe pathology. Details of this scoring system have been described.21 Finally, to detect the presence of alpha-smooth muscle actin (α-SMA)-positive cells, an immunochemical analysis was performed with

a well-characterized monoclonal antibody (mAb) for α-SMA.22 Results are expressed as the mean ± standard error of the mean (SEM). All graphing and statistical analyses were performed using the Prism graphing program (GraphPad Software, San Diego, CA). P-values were calculated using a two-tailed unpaired Mann-Whitney test except in Table 1; the frequency of liver damage in Table 1 was evaluated using Fisher’s exact test. Significance levels were set at P = 0.05. First, to confirm the activity of α-GalCer, a nested substudy was performed in which we intravenously injected α-GalCer to naive mice and analyzed IFN-γ and IL-4 production in serum and in iNKT cells of mice. As shown in Fig. 1A, both IFN-γ and IL-4 were increased in mice injected with α-GalCer. Serum IFN-γ was detectable at 2 hours, peaked at approximately 6 hours, and was maintained until 24 hours after α-GalCer injection, whereas IL-4 peaked at 2 hours and became undetectable after 6 hours (Fig. 1A).