Keisuke Nagatsuma

Role of Transglutaminase 2 in Liver Injury via Cross-linking and Silencing of Transcription Factor Sp1

BACKGROUND & AIMS Despite high morbidity and mortality of alcoholic liver disease worldwide, the molecular mechanisms underlying alcohol-induced liver cell death are not fully understood. Transglutaminase 2 (TG2) is a cross-linking enzyme implicated in apoptosis. TG2 levels and activity are increased in association with various types of liver injury. However, how TG2 induces hepatic apoptosis is not known. METHODS Human hepatic cells or primary hepatocytes from rats or TG2+/+ and TG2-/- mice were treated with ethanol. Mice were administered anti-Fas antibody or alcohol. Liver sections were prepared from patients with alcoholic steatohepatitis. Changes in TG2 levels, Sp1 cross-linking and its activities, expression of hepatocyte growth factor receptor, c-Met, and hepatic apoptosis were measured. RESULTS Ethanol induced apoptosis in hepatic cells, enhanced activity and nuclear accumulation of TG2 as well as accumulation of cross-linked and inactivated Sp1, and reduced expression of the Sp1-responsive gene, c-Met. These effects were rescued by TG2 knockdown, restoration of functional Sp1, or addition of hepatocyte growth factor, whereas apoptosis was reproduced by Sp1 knockdown or TG2 overexpression. Compared with TG2+/+ mice, TG2-/- mice showed markedly reduced hepatocyte apoptosis and Sp1 cross-linking following ethanol or anti-Fas treatment. Treatment of TG2+/+ mice with the TG2 inhibitors putrescine or cystamine blocked anti-Fas-induced hepatic apoptosis and Sp1 silencing. Moreover, enhanced expression of cross-linked Sp1 and TG2 was evident in hepatocyte nuclei of patients with alcoholic steatohepatitis. CONCLUSIONS TG2 induces hepatocyte apoptosis via Sp1 cross-linking and inactivation, with resultant inhibition of the expression of c-Met required for hepatic cell viability.

Current Immunotherapeutic Approaches in Pancreatic Cancer

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer.

Free fatty acids induce transglutaminase 2-dependent apoptosis in hepatocytes via ER stress-stimulated PERK pathways†

Non‐alcoholic steatohepatitis (NASH), a progressive form of fatty liver, shares histological similarities with alcoholic steatohepatitis (ASH), including accumulated fat, hepatic apoptosis, and fibrous tissues in the liver, but the molecular mechanisms responsible for hepatic apoptosis remain unclear. We previously reported that transglutaminase 2 (TG2), induced in the nuclei of ethanol‐treated hepatocytes, crosslinks and inactivates the transcription factor Sp1, leading to hepatic apoptosis. In this study, we investigated whether a similar change is involved in NASH, and if so, how TG2 and crosslinked Sp1 (CLSp1) are induced. Elevated nuclear TG2 and CLSp1 formation was demonstrated in NASH patients, as well as increased activation of apoptosis inducing factor (AIF) and release of cytochrome c. In Hc human normal hepatocytes treated with free fatty acids (FFAs), biochemical analyses revealed that ethanol and FFAs provoked fat accumulation, endoplasmic reticulum (ER) stress, increased nuclear factor kappa B (NFκB), and nuclear TG2. Salubrinal, a selective inhibitor of the ER stress‐induced pancreatic ER kinase (PERK) signaling pathway, inhibited NFκB activation, nuclear TG2 expression, and apoptosis only if it was induced by FFAs, but not by ethanol. These results suggest that FFAs could increase ER stress and lead to nuclear NFκB activation and TG2 induction through PERK‐dependent pathways, resulting in TG2‐mediated apoptosis accompanying crosslinking and inactivation of Sp1, activation of AIF, and release of cytochrome c. J. Cell. Physiol. 227: 1130–1137, 2012.

Combined TLR2/4-Activated Dendritic/Tumor Cell Fusions Induce Augmented Cytotoxic T Lymphocytes

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4+ and CD8+ T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4+CD25+Foxp3+ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.

Lecithin: retinol acyltransferase protein is distributed in both hepatic stellate cells and endothelial cells of normal rodent and human liver.

Background: To determine the extent to which hepatic stellate cell (HSC) activation contributes to liver fibrosis, it was found necessary to develop an alternative structural and functional stellate cell marker for in situ studies. Although several HSC markers have been reported, none of those are associated with particular HSC functions.

Several factors including ITPA polymorphism influence ribavirin-induced anemia in chronic hepatitis C

AIM To construct formulae for predicting the likelihood of ribavirin-induced anemia in pegylated interferon α plus ribavirin for chronic hepatitis C. METHODS Five hundred and sixty-one Japanese patients with hepatitis C virus genotype 1b who had received combination treatment were enrolled and assigned randomly to the derivation and confirmatory groups. Single nucleotide polymorphisms at or nearby ITPA were genotyped by real-time detection polymerase chain reaction. Factors influencing significant anemia (hemoglobin concentration < 10.0 g/dL at week 4 of treatment) and significant hemoglobin decline (declining concentrations > 3.0 g/dL at week 4) were analyzed using multiple regression analyses. Prediction formulae were constructed by significantly independent factors. RESULTS Multivariate analysis for the derivation group identified four independent factors associated with significant hemoglobin decline: hemoglobin decline at week 2 [P = 3.29 × 10(-17), odds ratio (OR) = 7.54 (g/dL)], estimated glomerular filtration rate [P = 2.16 × 10(-4), OR = 0.962 (mL/min/1.73 m(2))], rs1127354 (P = 5.75 × 10(-4), OR = 10.94) and baseline hemoglobin [P = 7.86 × 10(-4), OR = 1.50 (g/dL)]. Using the model constructed by these factors, positive and negative predictive values and predictive accuracy were 79.8%, 88.8% and 86.2%, respectively. For the confirmatory group, they were 83.3%, 91.0% and 88.3%. These factors were closely correlated with significant anemia. However, the model could not be constructed, because no patients with rs1127354 minor genotype CA/AA had significant anemia. CONCLUSION Reliable formulae for predicting the likelihood of ribavirin-induced anemia were constructed. Such modeling may be useful in developing individual tailoring and optimization of ribavirin dosage.

Impact of IL28B polymorphisms on 24‐week telaprevir‐based combination therapy for Asian chronic hepatitis C patients with hepatitis C virus genotype 1b

The aim of this study was to clarify which or how factors could influence the probability of sustained virological response (SVR) in 24‐week telaprevir‐based triple combination therapy for East Asian chronic hepatitis C patients infected with hepatitis C virus genotype 1b.

LAP degradation product reflects plasma kallikrein-dependent TGF-β activation in patients with hepatic fibrosis

Byproducts of cytokine activation are sometimes useful as surrogate biomarkers for monitoring cytokine generation in patients. Transforming growth factor (TGF)-β plays a pivotal role in pathogenesis of hepatic fibrosis. TGF-β is produced as part of an inactive latent complex, in which the cytokine is trapped by its propeptide, the latency-associated protein (LAP). Therefore, to exert its biological activity, TGF-β must be released from the latent complex. Several proteases activate latent TGF-β by cutting LAP. We previously reported that Camostat Mesilate, a broad spectrum protease inhibitor, which is especially potent at inhibiting plasma kallikrein (PLK), prevented liver fibrosis in the porcine serum-induced liver fibrosis model in rats. We suggested that PLK may work as an activator of latent TGF-β during the pathogenesis of liver diseases in the animal models. However, it remained to be elucidated whether this activation mechanism also functions in fibrotic liver in patients.Here, we report that PLK cleaves LAP between R58 and L59 residues. We have produced monoclonal antibodies against two degradation products of LAP (LAP-DP) by PLK, and we have used these specific antibodies to immunostain LAP-DP in liver tissues from both fibrotic animals and patients.The N-terminal side LAP-DP ending at R58 (R58 LAP-DP) was detected in liver tissues, while the C-terminal side LAP-DP beginning at L59 (L59 LAP-DP) was not detectable. The R58 LAP-DP was seen mostly in α-smooth muscle actin-positive activated stellate cells.These data suggest for the first time that the occurrence of a PLK-dependent TGF-β activation reaction in patients and indicates that the LAP-DP may be useful as a surrogate marker reflecting PLK-dependent TGF-β activation in fibrotic liver both in animal models and in patients.

Transplantation of Liver Organoids in the Omentum and Kidney

Liver organoids were reconstructed by mouse-immortalized hepatocytes and nonparenchymal cells (sinusoidal endothelial cells and hepatic stellate cells) in a radial-flow bioreactor (RFB). A biodegradable apatite-fiber scaffold (AFS) was used as a scaffold packed in the RFB, which enables three-dimensional cell cultures. The organoids cocultured in the RFB showed a liver-like structure with high-density layers of hepatocytes and the formation of vessel-like structures. A liver organoid consisting of three cocultured cells was transplanted under the kidney capsule (kidney group) or into the omentum (omentum group) using BALB/c nude mice. Transplanted liver organoids survived in the kidney or omentum. The expression of mRNAs of albumin, connexin 26 and 32, hepatocyte nuclear factor 4α, and glucose-6-phosphatase was increased in both groups at 8 weeks after transplantation in comparison to the pretransplant status. Tyrosine aminotransferase appeared only in the omentum group. The results suggested that the functions of liver organoids differed depending on the transplanted site in the recipient animals.

Immunologic Monitoring of Cellular Responses by Dendritic/Tumor Cell Fusion Vaccines

Although dendritic cell (DC)- based cancer vaccines induce effective antitumor activities in murine models, only limited therapeutic results have been obtained in clinical trials. As cancer vaccines induce antitumor activities by eliciting or modifying immune responses in patients with cancer, the Response Evaluation Criteria in Solid Tumors (RECIST) and WHO criteria, designed to detect early effects of cytotoxic chemotherapy in solid tumors, may not provide a complete assessment of cancer vaccines. The problem may, in part, be resolved by carrying out immunologic cellular monitoring, which is one prerequisite for rational development of cancer vaccines. In this review, we will discuss immunologic monitoring of cellular responses for the evaluation of cancer vaccines including fusions of DC and whole tumor cell.