ß-hydroxybutyrate protects hepatocytes against endoplasmic reticulum stress in a sirtuin 1-independent manner.

ß-hydroxybutyrate (BHB), a major ketone body in mammals, is produced from fatty acids through mitochondrial fatty acid oxidation in hepatocytes. To elucidate the role of BHB in the hepatic endoplasmic reticulum (ER), we examined the effects of BHB on hepatic ER stress induced by tunicamycin. In mouse hepatoma Hepa1c1c7 cells, BHB treatment suppressed the protein expression of ER stress responsive genes and increased cell viability, while reducing the protein expression of apoptosis inducible genes, without causing any alterations in the protein expression of sirtuin 1 (SIRT1) or the phosphorylation of AMP-activated protein kinase. The intraperitoneal administration of BHB also reduced the protein expression of ER stress responsive genes in mouse livers. In human hepatoma HepG2 cells, the protein expression levels of ER stress responsive genes were increased by the partial inhibition of BHB production with siRNA targeting endogenous 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase, whereas they were decreased by promoting BHB production with fenofibrate. These findings revealed that BHB helps to suppress hepatic ER stress via a SIRT1-independent pathway, and it might be possible to manipulate ER stress by regulating BHB production genetically or pharmacologically.

Suppression of the TGF-ß1-induced protein expression of SNAI1 and N-cadherin by miR-199a.

MicroRNA miR-199a is clustered with miR-214 on chromosome 1 and its expression is up-regulated by various factors that are associated with epithelial-to-mesenchymal transition (EMT), such as a transcriptional repressor Twist1 and transforming growth factor (TGF)-ß. miR-199a is either up-regulated or down-regulated in a variety of cancers, although EMT is associated with the progression of cancer. We found here that miR-199a suppressed the translation of SNAI1, a transcriptional repressor that plays a role in EMT, by targeting the sequence within the 3'UTR of the SNAI1 mRNA, and reduced the protein level of SNAI1. miR-199a increased the protein level of claudin-1 in both the TGF-ß1-treated and -untreated cells at least partly by decreasing the protein level of SNAI1, a transcriptional repressor for claudin-1. In addition, miR-199a targeted the sequence within the 3'UTR of the N-cadherin mRNA and suppressed the TGF-ß1-induced increase in the protein level of N-cadherin in a manner independent of SNAI1. These results indicate that miR-199a suppresses the TGF-ß1-induced protein expression of SNAI1 and N-cadherin.

Absence of primary cilia in cell cycle-arrested human breast cancer cells.

Previous studies using cultured cells showed that primary cilia are present in quiescent cells, but are absent in proliferating cells. We studied here the relationship between the presence or absence of primary cilia and the cell cycle arrest of normal epithelial cells and cancer cells in the human normal breast and breast cancer tissues. In normal breast tissues, although most epithelial cells were nonproliferating as estimated by the immunofluorescence staining of the proliferation marker Ki-67, primary cilia were present only in 20-40% of the epithelial cells. In breast cancer tissues, primary cilia were not observed in any of the breast cancer cells. Furthermore, primary cilia were hardly observed in the nonproliferating cancer cells in the orthotopic and metastatic human breast cancer xenograft tumors in mice. These results indicate that the absence of primary cilia does not necessarily represent the proliferating phases of normal epithelial cells and cancer cells.

Reduction of the ST6 ß-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1)-catalyzed sialylation of nectin-like molecule 2/cell adhesion molecule 1 and enhancement of ErbB2/ErbB3 signaling by microRNA-199a.

Nectin-like molecule 2 (Necl-2)/cell adhesion molecule 1 (CADM1) is shown to be down-regulated by the promoter hypermethylation and/or loss of heterozygosity at chromosome 11q23.2 in many types of cancers, including lung and breast cancers, and is proposed to serve as a tumor suppressor. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers, and other mechanisms for the suppression of Necl-2 are presumed to be present. We previously showed that Necl-2 interacts in cis with ErbB3 and suppresses the heregulin (HRG)-induced ErbB2/ErbB3 signaling for cell movement and death. We studied here the relationship between Necl-2 and microRNA-199a (miR-199a) that is up-regulated or down-regulated in a variety of cancers. miR-199a did not directly target the Necl-2 mRNA or affect its mRNA level in human lung cancer A549 cells and human embryonic kidney HEK293 cells. Necl-2 was at least sialylated by the sialyltransferase ST6 ß-galactosamide α-2,6-sialyltransferase 1 (ST6GAL1). miR-199a targeted ST6GAL1 and reduced both the sialylation and the protein level of Necl-2. In addition, miR-199a enhanced the HRG-induced ErbB2/ErbB3 signaling. These results indicate that the suppressive role of Necl-2 in the HRG-induced ErbB2/ErbB3 signaling is regulated by miR-199a at least through the reduction of the ST6GAL1-catalyzed sialylation of Necl-2 and/or through the reduction of the protein level of Necl-2 presumably by the protein degradation.

miR-214 and hypoxia down-regulate Necl-2/CADM1 and enhance ErbB2/ErbB3 signaling.

Necl-2/CADM1 is down-regulated by the promoter hypermethylation and/or the loss of heterozygosity at chromosome 11q23.2 in many types of cancers and serves as a tumor suppressor by interacting in cis with ErbB3 and suppressing the ligand-induced ErbB2/ErbB3 signaling for cell movement and death. However, the incidence of these epigenetic and genetic abnormalities of Necl-2 is 30-60% in these cancers. We investigated here other mechanisms that down-regulate Necl-2. miR-214, that is frequently up-regulated in a variety of cancers, targeted the 3'UTR of the Necl-2 mRNA directly, suppressed the translation of Necl-2 and enhanced the ligand-induced ErbB2/ErbB3 signaling in human colon cancer Caco-2 cells. Hypoxia reduced the Necl-2 protein level in a manner independent of miR-214 or hypoxia-inducible factor-1α in Caco-2 cells. These results indicate that miR-214 and hypoxia are novel regulators that down-regulate Necl-2 and enhance ErbB2/ErbB3 signaling.

Metabolomics-based search for therapeutic agents for non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the commonest form of chronic liver disease in developed countries. Non-alcoholic steatohepatitis (NASH), which represents advanced stage NAFLD, is increasingly being recognized as a major cause of liver-related morbidity and mortality. However, no effective drugs against NASH have yet been developed. Therefore, we searched for candidate therapeutic agents based on the changes in levels of hepatic metabolites via gas chromatography mass spectrometry (GC/MS)-based metabolome analysis of livers from methionine-choline deficient (MCD) diet-fed mice, a mouse model of NASH. METHODS: The metabolites were extracted from the livers of the MCD diet-fed mice and then analyzed using GC/MS. Subsequently, the MCD diet-fed mice were supplemented with hypotaurine, and the therapeutic effects of hypotaurine against steatohepatitis were evaluated. RESULTS: Ninety-nine metabolites were detected in the livers of the MCD diet-induced steatohepatitis model mice. Among these metabolites, hypotaurine exhibited the greatest decrease in its concentration in the mice. Supplementation with 2 mmol/kgBW hypotaurine attenuated liver injuries and fat accumulation caused by the MCD diet-induced steatohepatitis. Furthermore, 10 mmol/kgBW hypotaurine supplementation ameliorated fibrosis and oxidative stress induced by the MCD diet. CONCLUSION: The present metabolome analysis-based study demonstrated that hypotaurine is a novel candidate therapeutic agent for NASH.

Current Status of Conversion Hepatectomy After Sorafenib and Lenvatinib Treatment for Unresectable Hepatocellular Carcinoma.

BACKGROUND/AIM: Sorafenib and lenvatinib have long been used as a first-line treatment for advanced hepatocellular carcinoma (HCC). Along with the development of systemic chemotherapy for HCC, the concept of conversion hepatectomy has recently become widespread. The present study aimed to assess the clinical outcomes of sorafenib and lenvatinib for HCC regarding the possibility of conversion hepatectomy in clinical practice. PATIENTS AND METHODS: A total of 295 patients with advanced HCC receiving sorafenib and lenvatinib, accounting for 306 treatments (sorafenib, n=157; lenvatinib, n=149, 11 patients received lenvatinib after sorafenib treatment) at five different institutions were enrolled. Patients were assessed for their clinical characteristics and therapeutic response using both Response Evaluation Criteria in Solid Tumors criteria (RECIST) and modified RECIST (mRECIST) criteria. Additionally, an indication of surgery after tyrosine kinase inhibitor administration was determined based on the tumor status of patients. RESULTS: The median survival times of patients treated with sorafenib and lenvatinib were 12.8 and 16.4 months, respectively, without significant difference (p=0.1645). The objective response rates (ORR) of sorafenib based on mRECIST and RECIST were 10.1% and 5.9%, respectively, and those of lenvatinib were 38.1% and 19.0%, respectively. Among the 306 treatments, two cases (sorafenib and lenvatinib, one each) underwent hepatectomy after systemic chemotherapy. CONCLUSION: Few cases with unresectable HCC were amenable to conversion hepatectomy after sorafenib and lenvatinib treatments due to the limited ORR by RECIST. Cautious approach must be taken when administering neoadjuvant chemotherapy aimed at conversion hepatectomy.

Association of adverse childhood experiences and cortical neurite density alterations with posttraumatic stress disorder symptoms in autism spectrum disorder.

Background: Posttraumatic stress disorder (PTSD) can be a source of significant social and daily distress in autism spectrum disorder (ASD). Compared to typically developed (TD) individuals, people with ASD are at an increased risk of adverse childhood experiences (ACEs), which can result in abnormal neuronal development. However, whether or how ACEs influence abnormal neural development and PTSD symptoms in ASD has not been fully elucidated. Methods: Thirty-nine TD individuals and 41 individuals with ASD underwent T1-weighted magnetic resonance imaging and neurite orientation dispersion and density imaging (NODDI), with axonal and dendritic densities assessed in terms of the orientation dispersion index and neurite density index (NDI), respectively. Voxel-based analyses were performed to explore the brain regions associated with PTSD symptoms, and the relationships between the severity of ACEs and PTSD symptoms and NODDI parameters in the extracted brain regions were examined. Results: There was a significant positive association between PTSD symptom severity and NDI in the bilateral supplementary motor area; right superior frontal, left supramarginal, and right superior temporal gyrus; and right precuneus in the ASD group, but not in the TD group. ACE severity was significantly associated with NDI in the right superior frontal and left supramarginal gyrus and right precuneus in the ASD group. Moreover, NDI in the right precuneus mainly predicted the severity of PTSD symptoms in the ASD group, but not the TD group. Conclusion: These results suggest that ACE-associated higher neurite density is of clinical importance in the pathophysiology of PTSD symptoms in ASD.

Factors associated with the response to atezolizumab/bevacizumab combination therapy for hepatocellular carcinoma.

Background and Aim: The purpose of this study was to analyze factors associated with the overall survival (OS) of atezolizumab/bevacizumab combination therapy for advanced hepatocellular carcinoma (aHCC). We also assessed the OS of patients with ineffective therapy and those who discontinued treatment owing to adverse events (AEs). Methods: This retrospective multicenter study involved 139 patients with aHCC who received atezolizumab/bevacizumab combination therapy between November 2020 and September 2022. Results: The median duration of treatment was 136.5 days, and the median observation period was 316 days. The overall response rate was 40%, and the disease control rate was 78% according to mRECIST criteria. Grade ≥2 AEs occurred in 63 patients (43%) and led to treatment discontinuation in 16 patients. Multivariate analysis revealed that treatment response and occurrence of grade ≥2 AEs after therapy, as well as low level of albumin-bilirubin (ALBI) grade and low level of des-gamma carboxy prothrombin (DCP) before therapy, were extracted as factors that contributed to OS. Log-rank tests with the Kaplan-Meier method showed significant differences in OS among these factors. The OS of patients who discontinued owing to AEs was significantly shorter than that of other patients. Conclusion: Not only factors before therapy but also treatment response and the appearance of AEs are involved in OS for atezolizumab/bevacizumab combination therapy. Although the development of AEs also contributed to OS, appropriate management of AEs is important to avoid discontinuing treatment with this combination.

Interaction of nectin-like molecule 2 with integrin alpha6beta4 and inhibition of disassembly of integrin alpha6beta4 from hemidesmosomes.

In normal epithelial cells, integrin α(6)ß(4) is abundantly expressed and forms hemidesmosomes, which is a cellular structure that mediates cell-extracellular matrix binding. In many types of cancer cells, integrin α(6)ß(4) is up-regulated, laminin is cleaved, and hemidesmosomes are disrupted, eventually causing an enhancement of cancer cell movement and facilitation of their invasion. We previously showed that the immunoglobulin-like cell adhesion molecule Necl-2 (Nectin-like molecule 2), known as a tumor suppressor, inhibits cancer cell movement by suppressing the ErbB3/ErbB2 signaling. We show here that Necl-2 interacts in cis with integrin α(6)ß(4). The binding of Necl-2 with integrin ß(4) was mediated by its extracellular region. In human colorectal adenocarcinoma Caco-2 cells, integrin α(6)ß(4) was localized at hemidesmosomes. Small interfering RNA-mediated suppression of Necl-2 expression enhanced the phorbol ester-induced disruption of the integrin α(6)ß(4) complex at hemidesmosomes, whereas expression of Necl-2 suppressed the disruption of this structure. These results indicate that tumor-suppressive functions of Necl-2 are mediated by the stabilization of the hemidesmosome structure in addition to the inhibition of the ErbB3/ErbB2 signaling.

Significance of post-progression therapy after tyrosine kinase inhibitors for advanced hepatocellular carcinoma.

Background and Aim: Molecular-targeted therapies such as sorafenib and lenvatinib have long been used as first-line treatment for advanced hepatocellular carcinoma (aHCC). However, adverse events or limited therapeutic effects may necessitate the change to another therapeutic option, known as post-progression therapy. To investigate the significance of post-progression therapy, we analyzed the outcomes of aHCC patients following first-line molecular-targeted therapy in a real-world study. Methods: This retrospective, multicenter study involved patients with aHCC who received sorafenib or lenvatinib as first-line therapy between January 2011 and September 2021. Results: In total, 513 patients were analyzed: 309 treated with sorafenib and 204 with lenvatinib. The overall response and disease control rates were 15 and 50%, respectively, in the sorafenib group and 30 and 75%, respectively, in the lenvatinib group (P < 0.001). Kaplan-Meier analysis revealed no significant differences in progression-free survival and overall survival (OS) between the two treatments. Multivariate analysis revealed that fibrosis-4 index, disease control rate, post-progression therapy, and use of an immune checkpoint inhibitor (ICI) were significantly associated with OS. OS was significantly longer in patients who received post-progression therapy than in those who did not (log-rank P < 0.001). Most patients who received an ICI as post-progression therapy had previously received lenvatinib. Among lenvatinib-treated patients, OS was significantly longer in patients who received an ICI than in patients received another or no post-progression therapy (P = 0.004). Conclusion: The introduction of newer drugs for post-progression therapy is expected to prolong survival. ICI-based regimens appear to be effective after lenvatinib.

Localization of nectin-free afadin at the leading edge and its involvement in directional cell movement induced by platelet-derived growth factor.

Afadin is an actin-filament-binding protein that binds to nectin, an immunoglobulin-like cell-cell adhesion molecule, and plays an important role in the formation of adherens junctions. Here, we show that afadin, which did not bind to nectin and was localized at the leading edge of moving cells, has another role: enhancement of the directional, but not random, cell movement. When NIH3T3 cells were stimulated with platelet-derived growth factor (PDGF), afadin colocalized with PDGF receptor, alphavbeta3 integrin and nectin-like molecule-5 at the leading edge and facilitated the formation of leading-edge structures and directional cell movement in the direction of PDGF stimulation. However, these phenotypes were markedly perturbed by knockdown of afadin, and were dependent on the binding of afadin to active Rap1. Binding of Rap1 to afadin was necessary for the recruitment of afadin and the tyrosine phosphatase SHP-2 to the leading edge. SHP-2 was previously reported to tightly regulate the activation of PDGF receptor and its downstream signaling pathway for the formation of the leading edge. These results indicate that afadin has a novel role in PDGF-induced directional cell movement, presumably in cooperation with active Rap1 and SHP-2.

Necl-5/PVR enhances PDGF-induced attraction of growing microtubules to the plasma membrane of the leading edge of moving NIH3T3 cells.

Microtubules (MTs) search for and grow toward the leading edge of moving cells, followed by their stabilization at a specific structure at the rear site of the leading edge. This dynamic re-orientation of MTs is critical to directional cell movement. We previously showed that Necl-5/poliovirus receptor (PVR) interacts with platelet-derived growth factor (PDGF) receptor and integrin α(v) ß(3) at the leading edge of moving NIH3T3 cells, resulting in an enhancement of their directional movement. We studied here the role of Necl-5 in the PDGF-induced attraction of growing MTs to the leading edge of NIH3T3 cells. Necl-5 enhanced the PDGF-induced growth of MTs and attracted them near to the plasma membrane of the leading edge of NIH3T3 cells in an integrin α(v) ß(3) -dependent manner. Furthermore, Necl-5 enhanced the PDGF-induced attraction of the plus-end-tracking proteins (+TIPs), including EB1, CLIP170, an intermediate chain subunit of cytoplasmic dynein, and p150(Glued) , a subunit of dynactin, near to the plasma membrane of the leading edge. Thus, Necl-5 plays a role in the attraction of growing MTs to the plasma membrane of the leading edge of moving cells.

[A case of primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype: comparison of hemodynamics and histopathology].

A 58-year-old man was followed up for HBV-associated chronic hepatitis. A low echoic hepatic nodule 1.6cm in diameter developed in segment 8 of the liver. The tumor was hypervascular and showed enhancement on CV during hepatic arteriography (CTHA) and a defect on CT during arterial portography (CTAP). Strong enhancement, which lasted for 30 seconds, was observed at the margin of the tumor on single-level dynamic CTHA. The resected tumor was whitish, had no capsule, and consisted mainly of intermediate immature cells together with HCC-like and CCC-like tumor cells. These findings led to the diagnosis of primary liver carcinoma of intermediate (hepatocyte-cholangiocyte) phenotype. Cytokeratin (CK) 7, CK8, CK19, EMA and vimentin were positive and HP-1 and c-kit tests were negative on immunohistochemical staining. Staining with CD34+alphaSMA showed more muscular arterial vessels and sinusoid-like vessels in the peripheral zone of the tumor than in the central zone. Six months after the resection of the tumor, swollen abdominal lymph nodes were observed on US and CT, which aspiration needle biopsy showed to be metastasis of a hepatic tumor.

Quasispecies variant of pre-S/S gene in HBV-related hepatocellular carcinoma with HBs antigen positive and occult infection.

BACKGROUND: Hepatocellular carcinoma (HCC) can develop in patients who are negative for the hepatitis B surface antigen (HBsAg) in serum but positive for hepatitis B virus (HBV) DNA in the liver, referred to as occult HBV infection (OBI). Previous reports showed that HBV variants in OBI-related HCC are different from those in HBsAg-positive HCC. In the present study, HBV quasispecies based on the pre-S/S gene in OBI-related HCC patients were examined by high throughput sequencing and compared with those in HBsAg-positive HCC. METHODS: Nineteen tissue samples (9 OBI-related and 10 HBsAg-positive non-cancerous tissues) were collected at the time of surgery at Kobe University Hospital. The quasispecies with more than 1% variation in the pre-S/S region were isolated and analysed by ultra-deep sequencing. RESULTS: There were no significant differences in the major HBV populations, which exhibit more than 20% variation within the entire pre-S/S region, between OBI-related HCC and HBsAg-positive HCC. However, the prevalences of major populations with pre-S2 region mutations and of minor populations with polymerized human serum albumin-binding domain mutations were significantly higher in OBI-related HCC than in HBsAg-positive HCC. Moreover, the major variant populations associated with the B-cell epitope, located within the pre-S1 region, and the a determinant domain, located in the S region, were detected frequently in HBsAg-positive HCC. The minor populations of variants harbouring the W4R, L30S, Q118R/Stop, N123D and S124F/P mutations in the pre-S region and the L21F/S and L42F/S mutations in the S region were detected more frequently in OBI-related HCC than in HBsAg-positive HCC. CONCLUSIONS: Ultra-deep sequencing revealed that the B-cell epitope domain in the pre-S1 region and alpha determinant domain in the S region were variable in HBsAg-positive HCC, although the quasispecies associated with the pre-S2 region were highly prevalent in OBI-related HCC. TRIAL REGISTRATION: Ref: R000034382/UMIN000030113; Retrospectively registered 25 November 2017.

Inflammatory biomarker, neopterin, suppresses B lymphopoiesis for possible facilitation of granulocyte responses, which is severely altered in age-related stromal-cell-impaired mice, SCI/SAM.

Neopterin is produced by monocytes and is a useful biomarker of inflammatory activation. We found that neopterin enhanced in vivo and in vitro granulopoiesis triggered by the stromal-cell production of cytokines in mice. The effects of neopterin on B lymphopoiesis during the enhancement of granulopoiesis were determined using the mouse model of senescent stromal-cell impairment (SCI), a subline of senescence-accelerated mice (SAM). In non-SCI mice (a less senescent stage of SCI mice), treatment with neopterin decreased the number of colonies, on a semisolid medium, of colony-forming units of pre-B-cell progenitors (CFU-preB) from unfractionated bone marrow (BM) cells, but not that from a population rich in pro-B and pre-B cells without stromal cells. Neopterin upregulated the expression of genes for the negative regulators of B lymphopoiesis such as tumor necrosis factor-alpha (TNF-alpha ), interleukin-6 (IL-6), and transforming growth factor-beta (TGF-beta) in cultured stromal cells, implying that neopterin suppressed the CFU-preB colony formation by inducing negative regulators from stromal cells. The intraperitoneal injection of neopterin into non-SCI mice resulted in a marked decrease in the number of femoral CFU-preB within 1 day, along with increases in TNF-alpha and IL-6 expression levels. However, in SCI mice, in vivo and in vitro responses to B lymphopoiesis and the upregulation of cytokines after neopterin treatment were less marked than those in non-SCI mice. These results suggest that neopterin predominantly suppressed lymphopoiesis by inducing the production of negative regulators of B lymphopoiesis by stromal cells, resulting in the selective suppression of in vivo B lymphopoiesis. These results also suggest that neopterin facilitated granulopoiesis in BM by suppressing B lymphopoiesis, thereby contributing to the potentiation of the inflammatory process; interestingly, such neopterin function became impaired during senescence because of attenuated stromal-cell function, resulting in the downmodulation of the host-defense mechanism in the aged.

Factors associated with the decrease in hepatitis B surface antigen titers following interferon therapy in patients with chronic hepatitis B: Is interferon and adefovir combination therapy effective?

The purpose of antiviral therapy in chronic hepatitis B (CHB) is generally to achieve a decrease and ultimately disappearance of HBs antigen (HBsAg). Interferon (IFN) therapy of CHB appears to be less effective in Asian countries than in European countries, and the advantage of IFN and nucleotide(s) analog (NA) combination therapy has yet to be fully investigated. The present study focused on the factors associated with a decrease in HBs antigen following IFN monotherapy or IFN + NA combination therapy. A total of 35 patients with CHB who received IFN-based therapy (mean ± standard deviation age 36.7±8.5 years; 27 males and 8 females) were enrolled in this study. Of the 35 patients, 21 patients received pegylated IFN monotherapy and 14 patients received IFN and adefovir (ADV) combination therapy. We examined the factors associated with reductions in the HBsAg titer of >1.0 log IU/ml from the initial HBsAg titer to the end of treatment and to 24 weeks after treatment. Although 13 patients (37%) had a reduction in HBsAg of >1.0 IU/ml at the end of treatment, it was only maintained to 24 weeks after treatment in 7 patients (20%). The HBV core-related antigen (HBcrAg) titer before treatment was significantly higher in patients with a decrease in HBsAg at the end of treatment than in patients without a decrease in HBsAg (6.56±0.78 vs. 5.30±1.66 log IU/ml, P<0.05). Moreover, an increase in alanine aminotransferase (ALT) of >2 times from baseline occurred significantly more frequently in patients with a decrease in HBsAg (62 vs. 14%, P<0.05). The proportion of patients with a decrease in HBsAg was significantly greater in patients who received IFN monotherapy than in patients who received IFN and ADV combination therapy (43 vs. 29%, P<0.05). The present results revealed that the HBcr antigen titer before therapy and an on-treatment elevation of ALT (indicative of host instruction flare) are important factors associated with a decrease in HBsAg titers after IFN-based therapy. The efficacy of IFN and ADV combination therapy was not apparent in terms of a reduction in the HBsAg titer.

Inflammatory biomarker, neopterin, enlarges splenic mast-cell-progenitor pool: prominent impairment of responses in age-related stromal cell-impairment mouse SCI/SAM.

Neopterin is produced by monocytes and is a useful biomarker of inflammatory responses. We found that neopterin enhances granulopoiesis, but suppresses B-lymphopoiesis triggered by the positive and negative regulations of cytokines produced by stromal cells in mice. In this study, neopterin was found to regulate mast cell development, which was confirmed in the mouse model of senescent stromal-cell impairment (SCI). In non-SCI mice (=less senescent stage of SCI mice), neopterin decreased the number of colonies of IL-3-dependent mast-cell progenitor cells (CFU-mast) from unfractionated bone-marrow cells, but not that from the lineage-negative bone-marrow cell population without stromal cells in a semisolid in vitro system. Neopterin increased the gene expression and protein production of TGF-beta, a negative regulator of CFU-mast, in cultured stromal cells, indicating that neopterin suppressed CFU-mast colony formation by inducing TGF-beta in stromal cells. In contrast to this in vitro study, in vivo treatment with neopterin did not significantly up-regulate TGF-beta. The intravenous injection of neopterin into mice decreased the number of femoral CFU-mast and the expression level of the gene for stem cell factor (SCF), a positive regulator of CFU-mast, whereas the number of splenic CFU-mast and SCF gene expression level increased. In SCI mice, the in vivo and in vitro responses of mast cell development and cytokine gene expression level to neopterin treatment were less marked than those in non-SCI mice. These results suggest that, firstly, neopterin augments the splenic pool of CFU-mast by the production of SCF, and secondly, such neopterin function becomes impaired during senescence because of an impaired stromal-cell function, resulting in the down-modulation of host-defense mechanisms.

Yap reprograms glutamine metabolism to increase nucleotide biosynthesis and enable liver growth.

The Hippo pathway is an important regulator of organ size and tumorigenesis. It is unclear, however, how Hippo signalling provides the cellular building blocks required for rapid growth. Here, we demonstrate that transgenic zebrafish expressing an activated form of the Hippo pathway effector Yap1 (also known as YAP) develop enlarged livers and are prone to liver tumour formation. Transcriptomic and metabolomic profiling identify that Yap1 reprograms glutamine metabolism. Yap1 directly enhances glutamine synthetase (glul) expression and activity, elevating steady-state levels of glutamine and enhancing the relative isotopic enrichment of nitrogen during de novo purine and pyrimidine biosynthesis. Genetic or pharmacological inhibition of GLUL diminishes the isotopic enrichment of nitrogen into nucleotides, suppressing hepatomegaly and the growth of liver cancer cells. Consequently, Yap-driven liver growth is susceptible to nucleotide inhibition. Together, our findings demonstrate that Yap1 integrates the anabolic demands of tissue growth during development and tumorigenesis by reprogramming nitrogen metabolism to stimulate nucleotide biosynthesis.