Prolonged recurrence-free survival following OK432-stimulated dendritic cell transfer into hepatocellular carcinoma during transarterial embolization.

Despite curative locoregional treatments for hepatocellular carcinoma (HCC), tumour recurrence rates remain high. The current study was designed to assess the safety and bioactivity of infusion of dendritic cells (DCs) stimulated with OK432, a streptococcus-derived anti-cancer immunotherapeutic agent, into tumour tissues following transcatheter hepatic arterial embolization (TAE) treatment in patients with HCC. DCs were derived from peripheral blood monocytes of patients with hepatitis C virus-related cirrhosis and HCC in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor and stimulated with 0·1 KE/ml OK432 for 2 days. Thirteen patients were administered with 5 × 106 of DCs through arterial catheter during the procedures of TAE treatment on day 7. The immunomodulatory effects and clinical responses were evaluated in comparison with a group of 22 historical controls treated with TAE but without DC transfer. OK432 stimulation of immature DCs promoted their maturation towards cells with activated phenotypes, high expression of a homing receptor, fairly well-preserved phagocytic capacity, greatly enhanced cytokine production and effective tumoricidal activity. Administration of OK432-stimulated DCs to patients was found to be feasible and safe. Kaplan-Meier analysis revealed prolonged recurrence-free survival of patients treated in this manner compared with the historical controls (P = 0·046, log-rank test). The bioactivity of the transferred DCs was reflected in higher serum concentrations of the cytokines IL-9, IL-15 and tumour necrosis factor-α and the chemokines CCL4 and CCL11. Collectively, this study suggests that a DC-based, active immunotherapeutic strategy in combination with locoregional treatments exerts beneficial anti-tumour effects against liver cancer.

Novel nuclear signaling pathway mediates activation of fibroblast growth factor-2 gene by type 1 and type 2 angiotensin II receptors.

In bovine adrenal medullary cells synergistically acting type 1 and type 2 angiotensin II (AII) receptors activate the fibroblast growth factor-2 (FGF-2) gene through a unique AII-responsive promoter element. Both the type 1 and type 2 AII receptors and the downstream cyclic adenosine 1',3'-monophosphate- and protein kinase C-dependent signaling pathways activate the FGF-2 promoter through a novel signal-transducing mechanism. This mechanism, which we have named integrative nuclear FGF receptor-1 signaling, involves the nuclear translocation of FGF receptor-1 and its subsequent transactivation of the AII-responsive element in the FGF-2 promoter.

Upregulation of type I interferon receptor by IFN-gamma.

Type I interferon (IFN) receptor has a multichain structure composed of at least two distinct subunits, IFNAR-1 and IFNAR-2. In the present study, we demonstrated that IFN-gamma induced the expression of mRNA for IFNAR-1 and IFNAR-2 in a human hepatoma cell line, HepG2 cells. The induction was dose and time dependent. Because of this result, we examined the effect of combined treatment with type I IFN and IFN-gamma. The intracellular 2-5A-synthetase activity induced by combined treatment was significantly higher than that by type I IFN alone. This study suggests that combined treatment with type I IFN and IFN-gamma may be more effective than that of type I IFN alone and that the upregulation of type I IFN receptor may be one of the reasons. Our findings may have some relevance to the clinical use of IFN.

Combined therapy of transcatheter hepatic arterial embolization with intratumoral dendritic cell infusion for hepatocellular carcinoma: clinical safety.

The curative treatments for hepatocellular carcinoma (HCC), including surgical resection and radiofrequency ablation (RFA), do not prevent tumour recurrence effectively. Dendritic cell (DC)-based immunotherapies are believed to contribute to the eradication of the residual and recurrent tumour cells. The current study was designed to assess the safety and bioactivity of DC infusion into tumour tissues following transcatheter hepatic arterial embolization (TAE) for patients with cirrhosis and HCC. Peripheral blood mononuclear cells (PBMCs) were differentiated into phenotypically confirmed DCs. Ten patients were administered autologous DCs through an arterial catheter during TAE treatment. Shortly thereafter, some HCC nodules were treated additionally to achieve the curative local therapeutic effects. There was no clinical or serological evidence of adverse events, including hepatic failure or autoimmune responses in any patients, in addition to those due to TAE. Following the infusion of (111)Indium-labelled DCs, DCs were detectable inside and around the HCC nodules for up to 17 days, and were associated with lymphocyte and monocyte infiltration. Interestingly, T lymphocyte responses were induced against peptides derived from the tumour antigens, Her-2/neu, MRP3, hTERT and AFP, 4 weeks after the infusion in some patients. The cumulative survival rates were not significantly changed by this strategy. These results demonstrate that transcatheter arterial DC infusion into tumour tissues following TAE treatment is feasible and safe for patients with cirrhosis and HCC. Furthermore, the antigen-non-specific, immature DC infusion may induce immune responses to unprimed tumour antigens, providing a plausible strategy to enhance tumour immunity.

Cross-reactivity between hepatitis C virus and Influenza A virus determinant-specific cytotoxic T cells.

The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8(+) T cells of HCV-infected patients is the HCV(NS3-1073) peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10(-8) M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

Expression of interferon alpha/beta receptor in the liver of chronic hepatitis C patients.

Interferon (IFN) demonstrates antiviral activity by binding to receptors on the cell surface. Expression of the IFN receptor in hepatocytes may be directly associated with a hepatitis C virus (HCV) infection and the response to IFN therapy. A competitive PCR method was developed to measure IFN alpha/beta (alphabeta) receptor mRNA in liver samples obtained by needle biopsy. Thirty-one patients with chronic hepatitis C (21 without cirrhosis, 10 with cirrhosis) and six normal subjects were used. Eighteen of the 21 patients without cirrhosis received the IFN therapy. Competitive PCR was carried out using IFN alphabeta receptor gene-specific primers and a specific competitor. Expression of the receptor was detected in all liver samples. There was no association between the expression level and serum alanine aminotransferase level, serum (2'-5') oligo (A) synthetase level, amount of serum HCV RNA, or HCV genotype. The expression level in patients with chronic hepatitis was significantly higher than that in normal livers (P < 0.05) and in cirrhotic livers (P< 0.01). Seven of the 18 patients treated with IFN demonstrated a sustained response to IFN (sustained responders), and the remaining 11 did not (nonsustained responders). The expression level of IFN alphabeta receptor mRNA in the sustained responders was significantly higher than that in the nonsustained responders (P< 0.01). Thus, the expression of IFN alphabeta receptor mRNA may be one of the host factors influencing the response to IFN therapy.

Serum levels of soluble interferon Alfa/Beta receptor as an inhibitory factor of interferon in the patients with chronic hepatitis C.

Human serum contains a soluble form of interferon alfa/beta (sIFN alpha/beta) receptors, the functional and clinical significance of which has not been investigated in patients with chronic hepatitis C. In the present study, serum levels of sIFN alpha/beta receptor were assessed in 81 patients with chronic hepatitis C and correlated with the effectiveness of IFN therapy in these patients. Serum levels of sIFN alpha/beta receptor were significantly higher in patients with chronic hepatitis C than in healthy control patients (P <.0001). In these patients, serum levels of sIFN alpha/beta receptor were correlated with those of alanine transaminase (ALT) (P <.05), (2'-5')serum oligo(A) synthetase (2-5AS) (P <.0001), and pathological stages of liver fibrosis (P <.01). In 55 patients with chronic hepatitis C who underwent IFN therapy, there was an inverse correlation between the pretherapeutic serum levels of sIFN alpha/beta receptor and the rate of increase in serum levels of 2-5AS after the start of IFN (P <.01). Pretherapeutic serum levels of sIFN alpha/beta receptor were significantly lower in patients who showed sustained response to IFN therapy compared with those who did not respond to the therapy (P <.05). Multivariate analysis showed that low levels of serum sIFN alpha/beta receptor (

Cell-cycle dependent tyrosine phosphorylation on mortalin regulates its interaction with fibroblast growth factor-1.

We previously reported that endogenously expressed, intracellularly localized fibroblast growth factor (FGF)-1 interacts with mortalin. Here we report that FGF-1 added to the culture medium of quiescent BALB/c3T3 cells is taken up by the cells and interacts with mortalin in the cells in a regulated manner. Although both the internalized FGF-1 and mortalin were present at high levels throughout the FGF-1-initiated cell cycle, their interaction became apparent only in late G1 phase. Interestingly, mortalin was preferentially tyrosine phosphorylated at the same time, and when its normally weak phosphorylation in early G1 phase was augmented by treating the cells with vanadate, a strong interaction between mortalin and FGF-1 was established. Conversely, when phosphorylated mortalin was treated with tyrosine phosphatase, its interaction with FGF-1 was abrogated. These results indicate that FGF-1 taken up by cells preferentially interacts with mortalin in late G1 phase of the cell cycle, and that tyrosine phosphorylation of mortalin regulates this interaction.

Glutathione S-transferase Pi is a dopamine-inducible suppressor of dopamine-induced apoptosis in PC12 cells.

The finding that the neurotransmitter dopamine induces apoptosis in neurons implies the existence of a cellular mechanism by which dopaminergic neurons protect themselves from dopamine-induced apoptosis. By profiling the expression of a number of genes in differentiating PC12 cells which exhibit elevated levels of dopamine biosynthesis, we found that expression of glutathione S-transferase class Pi (GSTp) mRNA was selectively up-regulated. Interestingly, dopamine added to the culture medium of PC12 cells also augmented their expression of GSTp mRNA. Suppression of GSTp expression by transfection of its antisense expression vector augmented dopamine-induced apoptosis of PC12 cells. Conversely, overexpression of GSTp made the resultant PC12 transfectants highly resistant to dopamine-induced apoptosis. Transfection of the antisense or sense GSTp expression vectors also resulted in corresponding augmentation or suppression of dopamine-induced activation of cell-associated Jun-N-terminal kinase (JNK), which has been suggested to mediate dopamine-induced apoptosis in neuronal cells. These results indicate that GSTp is a dopamine-inducible suppressor of dopamine-induced apoptosis in PC12 cells, and suggest that this activity is exerted through inhibition of JNK activity.

Fibroblast growth factor-1 interacts with the glucose-regulated protein GRP75/mortalin.

Fibroblast growth factor-1 (FGF-1), which lacks a signal peptide and is intracellularly localized as a result of endogenous expression or endocytosis, is thought to be involved in regulating cell growth and differentiation. In the study reported here, we purified proteins that bind intracellular FGF-1. Affinity adsorption was used to purify FGF-1-binding proteins from rat L6 cells expressing FGF-1. One of the isolated proteins was identified as the glucose-regulated protein GRP75/mortalin/PBP-74/mthsp70, a member of the hsp70 family of heat-shock proteins known to be involved in regulating glucose responses, antigen processing and cell mortality. The interaction of FGF-1 and GRP75/mortalin in vivo was confirmed by co-immunoprecipitation, immunohistochemical co-localization in Rat-1 fibroblasts and by using the yeast two-hybrid system. Moreover, a binding assay in vitro with the use of recombinant FGF-1 and mortalin demonstrated a direct physical interaction between the two proteins. These results reveal that GRP75/mortalin is an intracellular FGF-1-binding protein in cells and suggest that GRP75/mortalin is involved in the trafficking of and/or signalling by FGF-1.