Hikaru Fujioka

Enhanced expression of type I interferon and toll-like receptor-3 in primary biliary cirrhosis

The pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic. In order to address this issue, we analyzed by laser capture microdissection and real-time reverse transcription-polymerase chain reaction the site-specific expression of messenger RNA (mRNA) for cytokines (interferon (IFN)-α, -β, -γ, interleukin (IL)-1β, -4, -6, -10, -12p40, -18, tumor necrosis factor-α) and toll-like receptors (TLRs) (TLR-2, -3, -4, -7, -9) in portal tract and liver parenchyma from patients with early-stage PBC. Expression of IFN-α, -β and TLR-3 proteins was also studied by immunohistochemistry. Autoimmune hepatitis (AIH) and chronic hepatitis C (CHC) served as disease controls. The expression levels of type I IFN (IFN-α, -β) and TLR-3 mRNAs, which are known to induce type I IFN, were significantly higher in portal tract and liver parenchyma as compared to AIH and CHC. A strong positive correlation between the mRNA levels of type I IFN and TLR-3 was also seen in both areas. Immunohistologically, IFN-α is present in the mononuclear cells in portal tract and sinusoidal cells. Macrophages in portal tract and hepatocytes expressed IFN-β and TLR-3. Furthermore, the level of IFN-α mRNA in the portal tract was positively correlated with serum alkaline phosphatase. In conclusion, these data indicate that TLR-3 and type I IFN signaling pathways are active in both the portal tract and liver parenchyma of early-stage PBC, and form the basis for our hypothesis that these signaling pathways are involved in the pathophysiology of PBC.

Human intrahepatic biliary epithelial cells function in innate immunity by producing IL-6 and IL-8 via the TLR4-NF-kappaB and -MAPK signaling pathways.

Abstract: Background: Human intrahepatic biliary epithelial cells (HIBECs) may play active roles in both the innate and adaptive immune responses. Little is known, however, about the role of toll‐like receptors (TLRs) on HIBECs in inflammatory cholangiopathies.

Utility of technetium-99m-labeled-galactosyl human serum albumin scintigraphy for estimating the hepatic functional reserve.

Technetium-99m-diethylenetriaminepentaacetic acid-galactosyl-human serum albumin (Tc-GSA) is a receptor binding agent, specific for asialoglycoprotein receptor, that resides exclusively on the plasma membrane of mammalian hepatocytes. The usefulness of Tc-GSA for estimating the hepatic functional reserve was retrospectively evaluated in patients undergoing a hepatic resection. Tc-GSA scintigraphy was performed in 35 patients before hepatectomy, and the hepatic uptake ratio (LHL15) was calculated. The LHL15 was then compared with the findings of conventional liver function tests, the indocyanine green retention rate in 15 minutes (ICG R15), and histologic activity index (HAI) score. Significant correlations were observed between the LHL15 and values of ICG R15, prothrombin time activity, serum levels of total bilirubin, hyaluronic acid, and values of HAI score. Ratios of LHL15 to preoperative liver volume (LHL-V) correlated well with the regenerative rates of the residual liver after major hepatectomy. In addition, patients with more than 0.76 of LHL-V value had no complications in postoperative course, whereas those with less than 0.73 had several complications due to hepatic dysfunction. Tc-GSA scintigraphy thus appears to be a useful diagnostic tool for evaluating functioning mass of the liver and the values of LHL-V seems to be able to demonstrate regenerative activity in the residual liver after hepatectomy.

Increased expression of Toll-like receptor 3 in intrahepatic biliary epithelial cells at sites of ductular reaction in diseased livers

Background Toll-like receptors (TLRs) may play active roles in both innate and adaptive immune responses in human intrahepatic biliary epithelial cells (HIBECs). The role of TLR3 expressed by HIBECs, however, remains unclear. Methods We determined the in vivo expression of TLRs in biopsy specimens derived from diseased livers immunohistochemically using a panel of monoclonal antibodies against human TLRs. We then examined the response of cultured HIBECs to a TLR3 ligand, polyinosinic–polycytidylic acid (polyI:C). Using siRNAs specific for Toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1) and mitochondrial antiviral signaling protein (MAVS), we studied signaling pathways inducing IFN-β expression. Results The expression of TLR3 was markedly increased in biliary epithelial cells at sites of ductular reaction in diseased livers, including primary biliary cirrhosis (PBC), autoimmune hepatitis (AIH), and chronic viral hepatitis (CH) as compared to nondiseased livers. Although cultured HIBECs constitutively expressed TLR3 at both the protein and mRNA levels in vitro, the addition of polyI:C to culture media induced only minimal increases in IFN-β mRNA. In contrast, transfection of HIBECs with polyI:C induced a marked increase in mRNAs encoding a variety of chemokines/cytokines, including IFN-β, IL-6, and TNF-α. The induction of IFN-β mRNA was efficiently inhibited by an siRNA against MAVS but not against TICAM-1, indicating that the main signaling pathway for IFN-β induction following polyI:C transfection is via retinoic acid-inducible gene I (RIG-I)/melanoma differentiation-associated gene 5 (MDA5) in HIBECs. Conclusions TLR3 expression by biliary epithelial cells increased at sites of ductular reaction in diseased livers; further study will be necessary to characterize it’s in vivo physiological role.

Mode of Infiltrative Growth of Colorectal Liver Metastases Is a Useful Predictor of Recurrence after Hepatic Resection

AbstractMany studies have been conducted to determine prognosis on the basis of the characteristics of metastatic liver tumor from colorectal cancer. The present study was carried out to determine whether the pathological mode of infiltrative growth (INF) of a metastatic liver nodule is useful in predicting recurrence in the remnant liver after hepatic resection. A total of 42 curative hepatic resections were performed for 37 patients with isolated liver metastases from colorectal cancer. Multivariate analysis (n = 42) showed that number, INF type, and size of liver metastases were statistically significant as independent risk factors. Of these, 28 resected liver metastases (smaller than 6 cm in size or containing fewer than 4 nodules) were classified pathologically into INF alpha or beta types (INF a b; n = 14) and gamma type (INFg; n = 14). Disease-free survival at 5 years was 64% for patients with INF a b type, and 14% for those with the INF g type of liver metastases. Of these, recurrent disease of the liver after hepatic resection was found in 2 (14%) and 11 (79%) patients with INF a b and INF g types, respectively. From these observations, we concluded that pathological infiltrative growth of liver metastases is an informative predictor of disease-free survival and especially of recurrence in the remnant liver.

Vitamin D Binding Protein-Macrophage Activating Factor Inhibits HCC in SCID Mice

BACKGROUND A high incidence of recurrence after treatment is the most serious problem in hepatocellular carcinoma (HCC). Therefore, a new strategy for the treatment of the disease is needed. The aim of the present study was to investigate whether vitamin D binding protein-macrophage activating factor (DBP-maf) is able to inhibit the growth of HCC. METHODS The effects of DBP-maf on endothelial cells and macrophage were evaluated by WST-1 assay and phagocytosis assay, respectively. Human HCC cells (HepG2) were implanted into the dorsum of severe combined immunodeficiency (SCID) mice. These mice were divided into control and DBP-maf treatment groups (n = 10/group). The mice in the treatment group received 40 ng/kg/d of DBP-maf for 21 d. RESULTS DBP-maf showed anti-proliferative activity against endothelial cells and also activated phagocytosis by macrophages. DBP-maf inhibited the growth of HCC cells (treatment group: 126 ± 18mm(3), untreated group: 1691.5 ± 546.9mm(3), P = 0.0077). Histologic examinations of the tumors revealed the microvessel density was reduced and more macrophage infiltration was demonstrated in the tumor of mice in the treatment group. CONCLUSION DBP-maf has at least two novel functions, namely, an anti-angiogenic activity and tumor killing activity through the activation of macrophages. DBP-maf may therefore represent a new strategy for the treatment of HCC.

Inactivation of porcine endogenous retrovirus by human serum as a function of complement activated through the classical pathway

BACKGROUND: The clinical use of organs and cells of pig donors as a source of tissue for xenotransplantation and extracorporeal therapies has been problematic due to the risk for zoonotic infection of porcine endogenous retroviruses (PERV). METHODS: The effect of human serum on PERV was evaluated using an infectivity assay and virolysis assay. Cell-free PERV infection to human 293 cells was determined by the presence of proviruses 5 days post-infection by a highly sensitive nested PCR, and the lysis of PERV virions was determined by the reverse transcriptase activities released into the supernatant. RESULTS: Treatment of PERV-PK, the culture supernatant of a pig kidney cell line containing the virus titer of 10(2.8) TCID(50) units/ml, with a quarter volume of human serum completely inactivated the infectivity. This activity was heat-labile and sensitive to an anti-complement agent, nafamostat mesilate, and a Ca(2+)-chelator, EGTA, indicating the crucial involvement of complement activated through the classical pathway. Since a synthetic galactosyl alpha1-3 galalactose (Galalpha1-3Gal) largely absorbed the activity from the serum, natural antibodies to the Galalpha1-3Gal epitopes are likely to trigger the complement activation. CONCLUSION: Cell-free PERV seems no longer be infectious in human serum. This greatly encourages the clinical application of pig tissues in particular for extracorporeal therapies such as a bioartificial liver, in which pig cells do not come in direct contact with a recipient.

Role of intrasplenic hepatocyte transplantation in improving survival and liver regeneration after hepatic resection in cirrhotic rats.

We studied the effect of preoperative hepatocyte transplantation on the prevention of liver failure in cirrhotic rats after hepatic resection. Two groups of Lewis rats were rendered cirrhotic by IP injection of 1% dimethylnitrosamine and were subjected to 33% hepatectomy. Two days before the resection, 36 rats in group I received intrasplenic hepatocyte transplantation, and 25 rats in group II were given intrasplenic injection of normal saline as a control. By the end of the third postoperative day, the rats in group I had better survival and a better biochemical profile than those in group II. The liver growth rate and the labeling index of proliferating cell nuclear antigen (PCNA-LI) showed a steady rise in group I. Compared with group II, group I had a significantly lower transforming growth factor (TGF-β1) level (p < 0.05). We conclude that preoperative intrasplenic hepatocyte transplantation improves survival and facilitates regeneration in cirrhotic rats after hepatic resection.

Expression of microsomal prostaglandin E synthase-1 in human hepatocelluar carcinoma

Background/Aims: The objective of this study was to evaluate the expression of microsomal prostaglandin E synthase‐1 (mPGES‐1) in hepatocellular carcinoma (HCC) tissues.

Pancreatic carcinogenesis: apoptosis and angiogenesis.

Abstract: Apoptosis and angiogenesis are critical biologic processes that are altered during carcinogenesis. Both apoptosis and angiogenesis may play an important role in pancreatic carcinogenesis. Despite numerous advances in the diagnosis and treatment of pancreatic cancer, its prognosis remains dismal and a new therapeutic approach is much needed. Recent research has revealed that apoptosis and angiogenesis are closely interrelated. Several reports show that a tumor suppresser gene that is expressed in pancreatic carcinoma and related to malignant potential can induce apoptosis and also inhibit angiogenesis. At present, it is generally accepted that tumor growth in cancers, including pancreatic cancer, depends on angiogenesis. We have identified 2 new angiogenesis inhibitors from a conditioned medium of human pancreatic carcinoma cell line (BxPC-3): antiangiogenic antithrombin III (aaAT-III) and vitamin D binding protein–macrophage activating factor (DBP-maf). These molecules were able to regress tumors in severe combined immunodeficiency disease (SCID) mice, demonstrating potent inhibition of endothelial cell proliferation. Moreover, the angiogenesis inhibitors induced tumor dormancy in the animal model. These results suggest that antiangiogenic therapy using angiogenesis inhibitors may become a new strategy for treatment of pancreatic cancer in the near future.