Ryo Nakagawa

Novel chemoimmunotherapeutic strategy for hepatocellular carcinoma based on a genome-wide association study

Pharmacotherapeutic options are limited for hepatocellular carcinoma (HCC). Recently, we identified the anti-tumor ligand MHC class I polypeptide-related sequence A (MICA) gene as a susceptibility gene for hepatitis C virus-induced HCC in a genome-wide association study (GWAS). To prove the concept of HCC immunotherapy based on the results of a GWAS, in the present study, we searched for drugs that could restore MICA expression. A screen of the FDA-approved drug library identified the anti-cancer agent vorinostat as the strongest hit, suggesting histone deacetylase inhibitors (HDACis) as potent candidates. Indeed, the HDACi-induced expression of MICA specific to HCC cells enhanced natural killer (NK) cell-mediated cytotoxicity in co-culture, which was further reinforced by treatment with an inhibitor of MICA sheddase. Similarly augmented anti-tumor activity of NK cells via NK group 2D was observed in vivo. Metabolomics analysis revealed HDACi-mediated alterations in energy supply and stresses for MICA induction and HCC inhibition, providing a mechanism for the chemoimmunotherapeutic actions. These data are indicative of promising strategies for selective HCC innate immunotherapy.

miR-425 regulates inflammatory cytokine production in CD4+ T cells via N-Ras upregulation in primary biliary cholangitis

BACKGROUND & AIMS Primary biliary cholangitis (PBC) is an autoimmune liver disease of unknown pathogenesis. Consequently, therapeutic targets for PBC have yet to be identified. CD4+ T cells play a pivotal role in immunological dysfunction observed in PBC, and therefore, microRNA (miRNA) and mRNA expression were analysed in CD4+ T cells, to investigate PBC pathogenesis and identify novel therapeutic targets. METHODS Integral miRNA and mRNA analysis of 14 PBC patients and ten healthy controls was carried out using microarray and quantitative real-time polymerase chain reaction (qRT-PCR), with gene set enrichment analysis. The functional analyses of miRNA were then assessed using reporter and miRNA-overexpression assays. RESULTS The integral analysis of miRNA and mRNA identified four significantly downregulated miRNAs (miR-181a, -181b, -374b, and -425) related to the T cell receptor (TCR) signalling pathway in CD4+ T cells of PBC. N-Ras, a regulator of the TCR signalling pathway, was found to be targeted by all four identified miRNAs. In addition, in vitro assays confirmed that decreased miR-425 strongly induced inflammatory cytokines (interleukin [IL]-2 and interferon [IFN]-γ) via N-Ras upregulation in the TCR signalling pathway. CONCLUSION The decreased expression of four miRNAs that dysregulate TCR signalling in PBC CD4+ T cells was identified. miR-425 was demonstrated as an inflammatory regulator of PBC via N-Ras upregulation. Therefore, the restoration of decreased miR-425 or the suppression of N-Ras may be a promising immunotherapeutic strategy against PBC. LAY SUMMARY Primary biliary cholangitis (PBC) is an autoimmune liver disease, but the causes are unknown. MicroRNAs are molecules known to regulate biological signals. In this study, four microRNAs were identified as being decreased in PBC patients, leading to activation of T cell receptor signalling pathways, involved in inflammation. One particular target, N-Ras, could be an attractive and novel immunotherapeutic option for PBC. TRANSCRIPT PROFILING Microarray data are deposited in GEO (GEO accession: GSE93172).

Clinical usefulness of ursodeoxycholic acid for Japanese patients with autoimmune hepatitis

AIM To evaluate the therapeutic effects of ursodeoxycholic acid (UDCA) on autoimmune hepatitis (AIH). METHODS A total 136 patients who were diagnosed with AIH were included in our study. All of the patients underwent a liver biopsy, and had at least a probable diagnosis on the basis of either the revised scoring system or the simplified scores. Initial treatment included UDCA monotherapy (Group U, n = 48) and prednisolone (PSL) monotherapy (Group P, n = 88). Group U was further classified into two subgroups according to the effect of UDCA: Patients who had achieved remission induction with UDCA monotherapy and showed no sign of relapse (Subgroup U1, n = 34) and patients who additionally received PSL during follow-up (Subgroup U2, n = 14). We compared the clinical and histological findings between each groups, and investigated factors contributing to the response to UDCA monotherapy. RESULTS In Group U, 34 patients (71%) achieved and maintained remission over 49 (range: 8-90) mo (Subgroup U1) and 14 patients (29%) additionally received PSL (Subgroup U2) during follow-up. Two patients in Subgroup U2 achieved remission induction once but additionally required PSL administration because of relapse (15 and 35 mo after the start of treatment). The remaining 12 patients in Subgroup U2 failed to achieve remission induction during follow-up, and PSL was added during 7 (range: 2-18) mo. Compared with Subgroup U2, Subgroup U1 had significantly lower alanine aminotransferase (ALT) levels at onset (124 IU/L vs 262 IU/L, P = 0.023) and a significantly higher proportion of patients with mild inflammation (A1) on histological examination (70.6% vs 35.7%, P = 0.025). When multivariate analysis was performed to identify factors contributing to the response to UDCA monotherapy, only a serum ALT level of 200 IU/L or lower was found to be associated with a significant difference (P = 0.013). CONCLUSION To prevent adverse events related to corticosteroids, UDCA monotherapy for AIH needs to be considered in patients with a serum ALT level of 200 IU/L or lower.

Zeta-potential of Micro- and/or Nano-bubbles in Water Produced by Some Kinds of Gases

Abstract Several reports in the literature show the acceleration in the physiological activity in plants and shellfishes when water containing micro- and nano-bubbles was used. However, the mechanisms of the phenomenon relating to aqua and agricultural fields and the stability of micro- and nano-bubbles in water have not been explained scientifically yet. The reason is the lack of knowledge about the characteristics of water containing micro- and nano-bubbles. In order to understand these characteristics, ξ-potential measurements were done in water after the generation of micro- and nano-bubbles using different kinds of gases. The values of ξ-potential were all negative and the absolute values were in the range between 34-45 mV (oxygen), 17-20 mV (air), 29-35 mV (nitrogen), 20-27 mV (carbon dioxide), and 11-22 mV (xenon). The different absolute values of ξ-potential may indicate different stability of the nano-bubbles according to the type of gas, as well as different structures at the gas-liquid interface. Furthermore, the bubbling time seemed to affect the ξ-potential, but more studies are needed to understand the phenomena during the production of micro- and nano-bubbles.

Predominance of regorafenib over sorafenib: restoration of membrane-bound MICA in hepatocellular carcinoma cells

The multi‐kinase inhibitor regorafenib (REG) was recently demonstrated to be effective in patients with sorafenib (SOR)‐resistant hepatocellular carcinoma (HCC). Interestingly, SOR is known to enhance the accumulation of membrane‐bound MHC class I polypeptide‐related sequence A (mMICA) in HCC cells and to block the production of soluble MICA (sMICA), an immunological decoy. In addition, MICA is associated with HCC in patients with chronic hepatitis C. We have now compared the impact of REG and SOR on MICA in HCC cells, as well as the immunotherapeutic implications thereof.

Enzymatic inhibition of MICA sheddase ADAM17 by lomofungin in hepatocellular carcinoma cells: Lomofungin suppresses ADAM17 in hepatocellular carcinoma cells

In our previous study on hepatocellular carcinoma (HCC) susceptibility genes in chronic hepatitis patients, we identified the MHC class I polypeptide‐related sequence A (MICA). Natural killer cells eliminate various cancer cells, including HCC, by suppressing MICA shedding. Therefore, we investigated MICA sheddases and inhibitors for HCC immunotherapy. In this study, HepG2, PLC/PRF/5, and Hep3B were treated with the siRNA of a disintegrin and metalloproteases (ADAMs) and matrix metalloproteases to measure the concentration of soluble MICA (sMICA) by ELISA to detect the therapeutic target. Furthermore, an FDA‐approved drug library was tested for the enzymatic inhibition of the targeted enzyme in an in vitro drug screening assay system. ADAM17 knockdown reduced sMICA levels and increased membrane‐bound MICA (mMICA) expression in HCC cells. In an in vitro drug screen using an FDA‐approved drug library, lomofungin, an antifungal drug, was found to strongly decrease ADAM17 activity. In HCC cells, mMICA expression was induced and sMICA production was inhibited in a dose‐dependent manner. These effects were cancelled upon ADAM17 knockdown, suggesting that lomofungin targeted ADAM17. Analysis of lomofungin analogs revealed the responsible functional groups. In summary, we suggest lomofungin to be an attractive agent for the immunological control of HCC, via the suppression of ADAM17.

Tu1830 Specifically Expressed MicroRNAs in CD4+ T Cells Participate With the Pathogenesis of Primary Biliary Cirrhosis to Regulate T Cell Signaling Pathway

A S L D A b st ra ct s 1.Circulating Th17 cells were elevated in PBC compared to HC(P<0.0001), and higher than those in chronic hepatic B(CHB) (P<0.0001). IL-17-positive cells that infiltrated the liver were higher in PBC compared to HC. 2.The progenitor of Th17 cells, CD4+CD161+ cells is increased in PBC(P<0.0001). And stimulated with IL-23 and IL-1 β, they expressed more IL-17(P=0.0556).The serum IL-1β and IL-23 were higher in PBC(P=0.023). 3.Early PBC presented with more Th17 cells in periphery blood than advanced PBC(P=0.0351). Whereas, IL-17 was higher in the liver in advanced PBC. The serum CCL20 levels were higher in PBC patients(P=0.001), especially in advanced disease(P=0.001). The CCL20 levels were more obvious in liver in PBC. Whereas, CD4+CCR6+ cells were not statistically different between PBC and HC(P=0.975). 4.IL-17 can promote the proliferation of HSCs in a dose dependent way(P<0.001), and increase the IL-8 expression in both dose and time dependent way(P<0.001). However, α-SMA production may not be influenced by IL-17(P=0.0847). Anti-IL-17 neutralizes above reactions. Conclusions: 1.Compared to HC and CHB, the levels of circulating Th17 cells and IL-17-positive cells infiltrated liver are higher in PBC. 2.CD4+CD161+ cells produce more IL-17 after stimulated with IL-23 and IL-1 βin PBC, therefore CD4+CD161+ cells are a source of increased Th17 cells in PBC. 3.The increased Th17 population is greater in circulation and less in liver in early PBC patients. Enhanced serum and hepatic CCL20 induce Th17 cells to migrate to liver, therefore Th17 cells are lower in circulation and enhanced in liver in advanced patients. 4.IL-17 can promote proliferation of HSCs in a dose dependent way and increase the IL-8 expression in both dose and time dependent way. Therefore, anti-IL-17 is a promising therapy for PBC fibrosis.