The current studies used a high trans-fat, high fructose diet to promote murine NAFLD with fibrosis,18 revealing that L-Fabp−/− mice not only exhibit attenuated steatosis with decreased LD accumulation but are also protected against steatosis-associated fibrogenesis. Several elements of these findings merit additional discussion. Little is known about the expression of genes related to FA uptake and metabolic channeling during HSC activation. Earlier studies in freshly isolated rat HSCs revealed expression

of mRNAs encoding Brain-Fabp (B-Fabp, Fabp7), L-Fabp, as well as retinol binding protein Epigenetics inhibitor (Rbp), with decreased expression upon culture in vitro.23 The current findings in murine HSCs confirm some but not all of those findings (specifically, B-Fabp was undetectable in our hands) but also demonstrate that L-Fabp depletion temporally accompanies LD depletion from cultured WT murine HSCs and that HSCs isolated from L-Fabp−/− mice AG-014699 in vitro contain

fewer LDs. Importantly, Ad-L-Fabp expression both increased the accumulation of FA and neutral lipid and also suppressed the expression of profibrogenic genes in passaged HSCs. These findings imply that the expression of L-Fabp both promotes LD accumulation and also inhibits HSC activation in vitro. The underlying mechanisms and pathways remain to be defined, but we speculate that L-Fabp aminophylline regulates the uptake and retention of lipid mediators and signaling molecules in HSCs, analogous to functions described for L-Fabp in liganding PPARα in isolated hepatocytes.24 Other studies have established a role for L-Fabp in the metabolic channeling of FA in enterocytes for complex lipid assembly.25 The findings in TFF-fed mice revealed a striking shift in LD accumulation in L-Fabp−/− mice with decreased expression of several LD-associated genes

including Plin4, Plin5, and Cidec, each of which has been shown to be modulated as downstream targets of either Pparα26 or Pparγ27, 28 in murine liver. Our a priori hypothesis, based on the role of L-Fabp in HSC activation in vitro, was that L-Fabp−/− mice would display enhanced susceptibility to high-fat diet-induced liver injury and fibrosis. Instead we found that L-Fabp−/− mice exhibited reduced fibrogenesis, which correlated with decreased hepatic steatosis. This discrepancy may reflect the complex intracellular crosstalk and lipid signaling that occurs in vivo between hepatocytes and HSCs and highlights the importance of in vivo models in understanding complex systems. Moreover, since germline deletion of L-Fabp has been shown to alter intestinal FA trafficking,12, 14, 15 it is unclear whether the absence of L-Fabp in the intestinal mucosa may also alter the progression of experimental NAFLD.

Antiviral agents such as entecavir (ETV) and lamivudine (LVD) are thought to reduce HCC incidence, but the associations of those drugs with suppression of HCC development have not been clear. Methods: Among 1203 CHB patients who visited Okayama university hospital or the related hospitals between 2011 and 2012. The incidence rates of HCC were compared among different patient groups of age, HBV DNA, HBe antigen, and treatment. The cumulative HCC incidences were analyzed with Kaplan-Meier method and log rank test. Results: Among the 686 patients of age >= 35 years at diagnosis,

HCC were observed in 115 patients with the mean observation period of 1687 days. Among the patients with HBV DNA >=4 log copies/mL and positive HBe antigen at diagnosis (n=184; 120 GS-1101 manufacturer with ETV, 37 with LVD, and 27 with none, respectively), the HCC incidence rates were 8.4% in 5 years among those treated with ETV, 21.8% PLX-4720 order among those with LVD, and 26.4% among those without drugs, respectively. The cumulative HCC incidence was significantly reduced in ETV treated patients compared with those treated with LVD or none (p = 0.013). Among the patients with HBV DNA >=4 log copies/mL and negative

HBe antigen at diagnosis (n=237; 128 with ETV, 19 with LVD, and 90 with none, respectively), the cumulative HCC incidences were 14.1% in 5 years among those treated with ETV, while 26.4% among those without drugs. The cumulative HCC incidence was comparable between the groups. Among the patients with HBV DNA <4 log copies/mL at diagnosis (n=265; 38 with ETV, 2 with LVD, and 225 with

none, respectively), HCC were observed only in 7 patients (2 treated with LVD and 5 without drug therapy, respectively). The cumulative incidence rates Mirabegron of HCC were 2.5% at year 5 in the non-treated patients. Similar analyses were done for the patients with age <35 years. There were no significant differences in HCC incidence among the different patient groups during the follow-up periods. Conclusions: In CHB patients with age >= 35 years, HBV DNA >= 4 log copies/mL and positive HBe antigen at diagnosis, ETV treatment is recommended for suppression of HCC development. 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, Akinobu Takaki, Tomonori Seno, Koichi Takaguchi Introduction Cases of tubular dysfunction have been reported in both HBV and HIV-infected patients receiving TDF. However, little is known about the impact on renal tubular function in CHB patients under long-term use of ETV and TDF. The aim of this study is to evaluate markers of renal tubular function and bone turnover in CHB patients treated with ETV or TDF for at least two years.

After 3 h, the cells were washed using fresh LB media, mixed with l-arabinose at the final concentration of 100 mM and incubated for another 2 h at 37 °C. Tenfold dilutions were made and plated on LB-Km and LB-Strep and incubated overnight at 37 °C. Colonies grown in LB-Km plates were considered to be unresolved and so were discarded, while those grown on LB-Strep plates were considered to be the required recombinants. The loss of the rpsL-neo

marker was confirmed by colony-PCR following the above-mentioned protocol. The ability of APEC1 click here wild type and its isogenic mutant APEC1LoxP to utilize ferric iron as the only source of iron was tested. Bacteria were cultured overnight at 37 °C in LB containing 200 μM of iron chelator 2, 2′-dipyridyl (DIP) (Sigma). The bacteria were then diluted 1 : 100 in LB containing 200 μM DIP and incubated for 3 h followed by washing in PBS. Approximately 1 × 105 CFU were mixed with 3 mL soft agar and plated onto LB plates supplemented with 375 μM DIP. Wells (5 mm diameter) were cut in the agar and filled with 100 μL of iron source (100 μM ferric chloride) or sterile double distilled water (negative control). Growth around wells was assessed

visually after an overnight incubation at 37 °C. For growth curves, strains were iron-limited overnight by culturing in LB containing 200 μM DIP. Prior to inoculation, strains were washed in PBS and approximately 105 CFU sdfd inoculated into liquid LB alone, LB containing 300 μM DIP, and 50 μM ferric chloride Y-27632 datasheet or no additional iron source and put into a 96-well plate. The general scheme of deletion is shown in Fig. 1a. PCR was performed buy Docetaxel using two primer sets HT51 and HT53, each containing 70 bp at the 5′ ends that are complimentary to regions upstream and downstream of the fiu gene and intergenic region 2051–52, followed by 34 bp for loxP site and 24 bp at the 3′ ends to amplify a cassette containing a rpsL-neo marker. The regions chosen for the deletion were designed based on the complete genome sequence of APEC O1 available

(Accession Number: NC_008563.1). The regions are the fiu gene, one of the 12 iron receptor genes in APEC (Ons et al., 2007) that encodes an iron-regulated outer membrane protein known as ferric iron uptake protein (Curtis et al., 1988; Newman & Shapiro, 1999) and an intergenic region 2051–52 between two divergently expressed genes APEC O1_2051 and APEC O1_2052 (Fig. 1b) (Johnson et al., 2007). The results from colony PCR demonstrated that the KmR recombinants analyzed contained the desired integration of LoxP cassette (Fig. 2) in respective regions and sequencing confirmed the integration and unidirectional orientation of loxP sites (data not shown). These results confirmed the generation of strains APEC1LoxP1 (Table 1) (Fig. 2a) and APEC1LoxP2 (Table 1) (Fig. 2b). The unidirectional orientation of the loxP sites is required for the deletion of the DNA segment (Nagy, 2000).

After 3 h, the cells were washed using fresh LB media, mixed with l-arabinose at the final concentration of 100 mM and incubated for another 2 h at 37 °C. Tenfold dilutions were made and plated on LB-Km and LB-Strep and incubated overnight at 37 °C. Colonies grown in LB-Km plates were considered to be unresolved and so were discarded, while those grown on LB-Strep plates were considered to be the required recombinants. The loss of the rpsL-neo

marker was confirmed by colony-PCR following the above-mentioned protocol. The ability of APEC1 Smoothened antagonist wild type and its isogenic mutant APEC1LoxP to utilize ferric iron as the only source of iron was tested. Bacteria were cultured overnight at 37 °C in LB containing 200 μM of iron chelator 2, 2′-dipyridyl (DIP) (Sigma). The bacteria were then diluted 1 : 100 in LB containing 200 μM DIP and incubated for 3 h followed by washing in PBS. Approximately 1 × 105 CFU were mixed with 3 mL soft agar and plated onto LB plates supplemented with 375 μM DIP. Wells (5 mm diameter) were cut in the agar and filled with 100 μL of iron source (100 μM ferric chloride) or sterile double distilled water (negative control). Growth around wells was assessed

visually after an overnight incubation at 37 °C. For growth curves, strains were iron-limited overnight by culturing in LB containing 200 μM DIP. Prior to inoculation, strains were washed in PBS and approximately 105 CFU sdfd inoculated into liquid LB alone, LB containing 300 μM DIP, and 50 μM ferric chloride Proteasome assay or no additional iron source and put into a 96-well plate. The general scheme of deletion is shown in Fig. 1a. PCR was performed Paclitaxel in vitro using two primer sets HT51 and HT53, each containing 70 bp at the 5′ ends that are complimentary to regions upstream and downstream of the fiu gene and intergenic region 2051–52, followed by 34 bp for loxP site and 24 bp at the 3′ ends to amplify a cassette containing a rpsL-neo marker. The regions chosen for the deletion were designed based on the complete genome sequence of APEC O1 available

(Accession Number: NC_008563.1). The regions are the fiu gene, one of the 12 iron receptor genes in APEC (Ons et al., 2007) that encodes an iron-regulated outer membrane protein known as ferric iron uptake protein (Curtis et al., 1988; Newman & Shapiro, 1999) and an intergenic region 2051–52 between two divergently expressed genes APEC O1_2051 and APEC O1_2052 (Fig. 1b) (Johnson et al., 2007). The results from colony PCR demonstrated that the KmR recombinants analyzed contained the desired integration of LoxP cassette (Fig. 2) in respective regions and sequencing confirmed the integration and unidirectional orientation of loxP sites (data not shown). These results confirmed the generation of strains APEC1LoxP1 (Table 1) (Fig. 2a) and APEC1LoxP2 (Table 1) (Fig. 2b). The unidirectional orientation of the loxP sites is required for the deletion of the DNA segment (Nagy, 2000).

Previous studies revealed that protein kinase B (Akt) played a key role in tumor progress and prognosis and it has a close correlation with tumor lymphnode metastasis. But

the role of Akt played in the lymphangiogenesis of stomach cancer is still unknown. In the current study, we analysed the relationship of the protein expression of Akt/mTOR signaling pathway with lymphangiogenic factor VEGF-C/-D and also with lymph vessel density (LVD) in situ and in vitro and eventually to clarify whether a Akt/mTOR/ VEGF-C/-D signaling pathway exist in the gastric cancer. Methods: 1. In situ gastric cancer tissue JQ1 concentration experiments: 55 fresh gastric cancer tissues with matched normal gastric mucosas from patients with disparate pathological stages were collected and fresh-frozen in liquid nitrogen after surgical resections performed at the first affiliated hospital of Nanchang University. Of these, 42 were male and 13 were female. None of the patients had received chemo-, radio- or immuno-therapy before resection. Part of each specimen were routinely processed, fixed in 10% buffered formalin, and embedded selleck in paraffin for histopathological analysis (hematoxylin and eosin

stain) and for immunohistochemical staining. The expression status of p-Akt, p-mTOR, D2-40, VEGF-C and – D was detected by immunohistochemistry. 2. In vitro experiments: SGC7901 stomach cancer cells were divided into LY294002 intervention group and Rapamycin intervention group. MTT method was used to determine the effects of these two drugs on SGC7901 stomach cancer cells surviving. Western blot was used to detected the expression level of Akt, p-Akt, mTOR, p-mTOR, VEGF-C and -D after the above two drugs intervention. Results:  1. Immunohistochemical expression and relationship of p-Akt, p-mTOR, VEGF-C, VEGF-D in gastric cancer tissue. The positive expression rates of p-Akt, p-mTOR, VEGF-C, VEGF-D in gastric caner were 74.5%, 85.45%, 72.73% and 58.18%, respectively. The expression level of p-Akt was correlated with p-mTOR, VEGF-C and -D expression

levels, respectively (P < 0.05 or 0.01). At the meanwhile, the expression level of p-mTORwas also correlated with VEGF-C and – D (P < 0.05 or 0.01). 2.  Immunohistochemical expression of p-Akt, p-mTOR, VEGF-C, VEGF-D in gastric cancer tissue and their Docetaxel cell line relationships with LVD. The LVD was deteceted by immunostain of D2-40. The results revealed that the mean LVD in gastric cancer was 94.18±72.965, ranged from 0-263.The mean LVD was 23.31±21.569 in normal gastric tissue, ranged from 0-95, which has a significant difference when compared with that of cancer tissue (P < 0.001). A closely relationship was found between LVD and p-Akt, p-mTOR, VEGF-C, VEGF-D, respetively(P < 0.05 or 0.01). We also found that the expression level of p-mTOR, VEGF-C and VEGF-D were different among the p-Akt’s negative group, positive group,and strongly positive group (P < 0.05).

A major unanswered question in PBC is that although all nucleated cells have mitochondria, the damage is limited to small biliary epithelial cells (BECs).27, 28 In this regard, there have been a number of studies that have focused on identifying the unique properties of BECs,

as compared with epithelial cells from other tissues. One such finding has been the unique process of Pifithrin-�� clinical trial apoptosis in BECs after exposure of PDC-E2 to the effector processes of the immune system. The data presented herein adds significance to the concept of a role for unique pathways involved in the apoptosis of BECs in PBC. Thus, BECs express CD40 and are exquisitely sensitive to CD40L-mediated apoptosis29; indeed, after stimulation with CD40L, there is a sustained up-regulation of Fas ligand, and induction of apoptosis is accompanied by the activation of the activator protein 1 (c-Fos/c-Jun) and phosphorylated

signal transducer and activator of transcription 3 signaling pathways.30, 31 It is important to note that inadequate glutathiolation has been reasoned to lead to the exposure of PDC-E2 by biliary cells, making the BECs a potential source of neoantigens responsible for the activation of autoreactive T lymphocytes.32, 33 We extended this work and demonstrated that in contrast to learn more other epithelial cells, PDC-E2 remains immunologically intact within the apoptotic bleb when BECs undergo apoptosis.34 We also demonstrated that there was a marked increase in inflammatory cytokine production in the presence

of the unique triad of normal BEC blebs, PBC monocyte-derived macrophages, and AMA.35 We interpret these data to suggest that the presence of intact immunologically active PDC-E2 within the blebs of BECs gives rise to a local proinflammatory milieu. Importantly, it has also been suggested that macrophages can directly kill BECs via CD40-CD40L interaction.36 This insight into innate immunity provides one explanation for our understanding of BEC destruction and the key role of CD40-CD40L axis in this process. In a larger context, it has an implication in our understanding of the tissue specificity of many autoimmune diseases. Finally, check details high levels of CD40L expression in PBC patients appear to be related to elevated levels of serum IgM, a common, distinct feature of PBC. Little is known about the mechanism of hyper-IgM in PBC. CD40L has a crucial role in Ig class switching in B cells and mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome.5 An early study by Higuchi et al. investigated the presence of mutations in the CD40L gene in PBC patients by single-strand conformational polymorphism. However, the results of these studies led to a failure to identify any differences between patients and controls.

A major unanswered question in PBC is that although all nucleated cells have mitochondria, the damage is limited to small biliary epithelial cells (BECs).27, 28 In this regard, there have been a number of studies that have focused on identifying the unique properties of BECs,

as compared with epithelial cells from other tissues. One such finding has been the unique process of buy Selinexor apoptosis in BECs after exposure of PDC-E2 to the effector processes of the immune system. The data presented herein adds significance to the concept of a role for unique pathways involved in the apoptosis of BECs in PBC. Thus, BECs express CD40 and are exquisitely sensitive to CD40L-mediated apoptosis29; indeed, after stimulation with CD40L, there is a sustained up-regulation of Fas ligand, and induction of apoptosis is accompanied by the activation of the activator protein 1 (c-Fos/c-Jun) and phosphorylated

signal transducer and activator of transcription 3 signaling pathways.30, 31 It is important to note that inadequate glutathiolation has been reasoned to lead to the exposure of PDC-E2 by biliary cells, making the BECs a potential source of neoantigens responsible for the activation of autoreactive T lymphocytes.32, 33 We extended this work and demonstrated that in contrast to high throughput screening compounds other epithelial cells, PDC-E2 remains immunologically intact within the apoptotic bleb when BECs undergo apoptosis.34 We also demonstrated that there was a marked increase in inflammatory cytokine production in the presence

of the unique triad of normal BEC blebs, PBC monocyte-derived macrophages, and AMA.35 We interpret these data to suggest that the presence of intact immunologically active PDC-E2 within the blebs of BECs gives rise to a local proinflammatory milieu. Importantly, it has also been suggested that macrophages can directly kill BECs via CD40-CD40L interaction.36 This insight into innate immunity provides one explanation for our understanding of BEC destruction and the key role of CD40-CD40L axis in this process. In a larger context, it has an implication in our understanding of the tissue specificity of many autoimmune diseases. Finally, Fossariinae high levels of CD40L expression in PBC patients appear to be related to elevated levels of serum IgM, a common, distinct feature of PBC. Little is known about the mechanism of hyper-IgM in PBC. CD40L has a crucial role in Ig class switching in B cells and mutations in the gene encoding CD40L are known to induce X-linked hyper-IgM syndrome.5 An early study by Higuchi et al. investigated the presence of mutations in the CD40L gene in PBC patients by single-strand conformational polymorphism. However, the results of these studies led to a failure to identify any differences between patients and controls.

Although the precise etiological mechanism of DIAIH has not been elucidated yet, we can speculate that the variations in their developing patterns are due to the different metabolic activity and immunological reactions. We think that a wider range of drugs has the potential to cause AIH, and incidence of AIH with a drug-related Everolimus etiology is more frequent than we have previously thought. In cases of DILI, careful follow-up will be needed, keeping in mind that AIH can develop even after normalization of

liver enzymes. Furthermore, establishment of the diagnostic criteria and therapeutic strategy for DIAIH will be needed. Kazushi Sugimoto M.D., Ph.D.*, Takeshi Ito M.D., Ph.D.*, Norihiko Yamamoto M.D., Ph.D.*, Katsuya

Shiraki M.D., Ph.D.*, * Department of Gastroenterology and Hepatology, Mie University School of Medicine, Mie, Japan. “
“Liver fibrogenesis is associated with the transition of quiescent hepatocytes and I BET 762 hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl4. To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive Phosphoprotein phosphatase CcnE1−/− and CcnE2−/− knock-out mice with CCl4 to induce liver fibrosis. Interestingly, CcnE1−/− mice were protected against CCl4-mediated liver

fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2−/− mice showed accelerated fibrogenesis after CCl4 treatment. We isolated primary HSCs from WT, CcnE1−/−, and CcnE2−/− mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1−/− HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis.

Although the precise etiological mechanism of DIAIH has not been elucidated yet, we can speculate that the variations in their developing patterns are due to the different metabolic activity and immunological reactions. We think that a wider range of drugs has the potential to cause AIH, and incidence of AIH with a drug-related PLX4032 in vitro etiology is more frequent than we have previously thought. In cases of DILI, careful follow-up will be needed, keeping in mind that AIH can develop even after normalization of

liver enzymes. Furthermore, establishment of the diagnostic criteria and therapeutic strategy for DIAIH will be needed. Kazushi Sugimoto M.D., Ph.D.*, Takeshi Ito M.D., Ph.D.*, Norihiko Yamamoto M.D., Ph.D.*, Katsuya

Shiraki M.D., Ph.D.*, * Department of Gastroenterology and Hepatology, Mie University School of Medicine, Mie, Japan. “
“Liver fibrogenesis is associated with the transition of quiescent hepatocytes and TSA HDAC manufacturer hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl4. To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive these CcnE1−/− and CcnE2−/− knock-out mice with CCl4 to induce liver fibrosis. Interestingly, CcnE1−/− mice were protected against CCl4-mediated liver

fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2−/− mice showed accelerated fibrogenesis after CCl4 treatment. We isolated primary HSCs from WT, CcnE1−/−, and CcnE2−/− mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1−/− HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis.

Briefly, 1 x 106 MNCs were incubated with antimouse CD3, antimouse check details CD4, antimouse CD25, antimouse CD8, antimouse CD19, antimouse NK1.1, antimouse CD11c, antimouse F4/80, and antimouse CD206 conjugated with fluorescein isothiocyanate (FITC; BD Biosciences, Franklin Lakes, NJ), phycoerythrin

(PE; BD Biosciences), peridinin chlorophyll protein (BD Biosciences), or allophycocyanin (APC; BD Biosciences). MNCs derived from livers were stained for different markers of cell subsets (i.e., CD4, CD8, NK1.1, CD11c, and F4/80) and concomitantly for the intracellular content of TNFα, IFNγ, and IL-17, -10, and -4. To this end, monoclonal antibodies were used as follows: antimouse TNFα and antimouse IL-10 conjugated with APC (BD Bioscience), antimouse IL-4, IFNγ, and IL-17 conjugated with PE. Intracellular staining for forkhead box protein 3 (Foxp3) was performed using the BD Biosciences fixation/permeabilization buffer kit. Stained cells were counted using a BD Biosciences FACSCalibur, and the results were analyzed with WinMDI software. For the detection Sotrastaurin supplier of apoptosis, the Annexin V– binding capacity of liver MNCs and splenocytes was examined by flow cytometry using the Annexin V FITC Detection Kit (BD Pharmingen, San Jose, CA), as previously described.16 Individual mouse serum was collected, and serum levels of IFNγ, TNFα, and IL-17, -4, and -10 were measured by enzyme-linked immunosorbent

assay (ELISA) using ELISA kits (R&D Systems, Minneapolis, MN). Individual spleens of Con A–untreated WT and Gal-3−/− mice were collected. The single-cell suspension of splenocytes was cultured in 24-well plates at 4 x 106 cells per well and was stimulated with 5 μg/mL of Con A (Sigma-Aldrich). After 24 hours, supernatants

were collected and cytokine concentrations were measured by ELISA kits (R&D Systems). All statistics were carried out using SPSS 13.0 for Windows software (SPSS, Inc., Chicago, IL). Results were analyzed using the Student t test. All data in nearly this study were expressed as the mean ± standard error of the mean (SEM). Values of P < 0.05 were considered as statistically significant. First, we investigated whether acute liver injury in humans would affect Gal-3 expression in the liver. Human liver tissue sections were obtained from patients suffering from acute liver disease induced by isoniazid or hepatitis B virus (HBV) and were compared to healthy controls. Compared to healthy controls (Supporting Fig. 1A), Gal-3 was strongly expressed in lining cells of hepatic sinuses both in patients with isoniazid-induced (Supporting Fig. 1B) and HBV-induced (Supporting Fig. 1C) fulminant hepatitis, suggesting a possible role of Gal-3 in liver inflammation. Next, to investigate the role of Gal-3 in experimental fulminant hepatitis, we injected Con A into WT and C57Bl/6 mice with the targeted disruption of Gal-3 gene (Gal-3−/− mice).