Mitsuru Sakakibara

Reduced Numbers and Impaired Ability of Myeloid and Plasmacytoid Dendritic Cells to Polarize T Helper Cells in Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Involvement of the p38 mitogen-activated protein kinase cascade in hepatocellular carcinoma

The mitogen‐activated protein kinase (MAPK) cascade is activated in response to various extracellular stimuli. The authors investigated the involvement of the p38 MAPK, a member of the MAPK superfamily, cascade in hepatoma cell lines and in human hepatocellular carcinoma (HCC) tissue specimens.

TIE2-expressing monocytes as a diagnostic marker for hepatocellular carcinoma correlates with angiogenesis.

Angiogenesis is a critical step in the development and progression of hepatocellular carcinoma (HCC). Myeloid lineage cells, such as macrophages and monocytes, have been reported to regulate angiogenesis in mouse tumor models. TIE2, a receptor of angiopoietins, conveys pro‐angiogenic signals and identifies a monocyte/macrophage subset with pro‐angiogenic activity. Here, we analyzed the occurrence and kinetics of TIE2‐expressing monocytes/macrophages (TEMs) in HCC patients. This study enrolled 168 HCV‐infected patients including 89 with HCC. We examined the frequency of TEMs, as defined as CD14+CD16+TIE2+ cells, in the peripheral blood and liver. The localization of TEMs in the liver was determined by immunofluorescence staining. Micro‐vessel density in the liver was measured by counting CD34+ vascular structures. We found that the frequency of circulating TEMs was significantly higher in HCC than non‐HCC patients, while being higher in the liver than in the blood. In patients who underwent local radio‐ablation or resection of HCC, the frequency of TEMs dynamically changed in the blood in parallel with HCC recurrence. Most TEMs were identified in the perivascular areas of tumor tissue. A significant positive correlation was observed between micro‐vessel density in HCC and frequency of TEMs in the blood or tumors, suggesting that TEMs are involved in HCC angiogenesis. Receiver operating characteristic analyses revealed the superiority of TEM frequency to AFP, PIVKA‐II and ANG‐2 serum levels as diagnostic marker for HCC. Conclusion: TEMs increase in patients with HCC and their frequency changes with the therapeutic response or recurrence. We thus suggest that TEM frequency can be used as a diagnostic marker for HCC, potentially reflecting angiogenesis in the liver. (HEPATOLOGY 2013)

Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I.

Dendritic cells utilize various sets of Toll‐like receptors (TLR) or cytosolic sensors to detect pathogens and evoke immune responses. In patients with hepatitis C virus (HCV) infection, a higher prevalence of various infectious diseases is reported; suggesting that innate immunity against pathogens is impaired. The aim of this study was to clarify whether the TLR and retinoic acid inducible gene‐I (RIG‐I) system in myeloid dendritic cells is preserved or not in chronic HCV infection. The expression of TLRs, RIG‐I and its relatives were compared in myeloid dendritic cells between 39 patients and 52 healthy volunteers. The induction of type‐I interferon (IFN) and inflammatory cytokines was examined in response to agonists for TLR2 (palmitoyl‐3‐cysteine‐serine‐lysine‐4), TLR3/RIG‐I (polyinosine–polycytidylic acid) or TLR4 (lipopolysaccharide). The relative expressions of TLR2, TLR4, RIG‐I, and LGP2 from the patients were significantly higher than those from the volunteers, whereas TLR3 and MDA‐5 expressions did not differ. In search for factors regulating TLR/RIG‐I expression, it was shown that IFN‐α, polyinosine–polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG‐I, but TNF‐α, HCV core or HCV non‐structural proteins did not. For the functional analyses, myeloid dendritic cells from the patients induced significantly less amounts of IFN‐β, TNF‐α and IL‐12p70 in response to polyinosine–polycytidylic acid or lipopolysaccharide. It is noteworthy that the expression of TRIF and TRAF6, which are essential adaptor molecules transmitting TLR3 or TLR4‐dependent signals, is reduced in the patients. Thus, innate cytokine responses in myeloid dendritic cells are impaired regardless of enhanced expressions of TLR2, TLR4, and RIG‐I in HCV infection. J. Med. Virol. 80:980–988, 2008.

Impaired Function of Dendritic Cells Circulating in Patients Infected with Hepatitis C Virus Who Have Persistently Normal Alanine Aminotransferase Levels

Hepatitis C virus (HCV) induces chronic liver disease in hosts which can eventually progresses to liver cirrhosis and hepatocellular carcinoma. However, progression of liver disease is slower in patients with persistently normal levels of alanine aminotransferase (ALT) than in those with active hepatitis. Although distinct immune responses against HCV have been proposed in asymptomatic infection, the role of circulating dendritic cells (DC) in the pathogenesis of these patients remains obscure. To address this issue, we compared the number and function of myeloid DC (MDC) and plasmacytoid DC (PDC) between uninfected individuals and HCV-infected patients with or without elevated ALT levels. Numbers of DC and DC progenitors were significantly lower in patients with chronic active hepatitis than in control subjects. However, no differences were found in the number of DC between normal controls and HCV-infected patients with persistently normal ALT levels. MDC from patients with active hepatitis were less able to polarize naive CD4 T cells into the Th1 phenotype, while their MDC and PDC primed more CD4 T cells producing IL-10 than those from normal controls. Such dysfunction of DC was also observed in patients with persistently normal ALT levels. In conclusion, circulating DC decrease in number predominantly in HCV-infected patients with active hepatitis, and the function of DC is impaired even in those with normal ALT levels.

Enhanced ability of regulatory T cells in chronic hepatitis C patients with persistently normal alanine aminotransferase levels than those with active hepatitis

Summary.  In hepatitis C virus (HCV) infection, the Th1‐type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV‐infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally‐occurring Treg (N‐Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real‐time reverse transcriptase‐polymerase chain reaction. Bulk or CD25‐depleted CD4+ T cells cultured with HCV‐NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127–CD25–FOXP3+ cells as distinct subsets other than CD25+ N‐Treg. The frequencies of N‐Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV‐specific T cell responses, showing that co‐existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127–CD25–FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV‐infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis.

Association of enhanced activity of indoleamine 2,3-dioxygenase in dendritic cells with the induction of regulatory T cells in chronic hepatitis C infection

BackgroundAltered functions of dendritic cells (DCs) and/or increases of regulatory T cells (Tregs) are involved in the pathogenesis of chronic hepatitis C virus (HCV) infection. A tryptophan-catabolizing enzyme, indoleamine 2,3-dioxygenase (IDO), is reported to be an inducer of immune tolerance. Our aim was to clarify whether or not IDO is activated in chronic hepatitis C patients and its role in immune responses.MethodsThis study enrolled 176 patients with chronic HCV infection and 37 healthy volunteers. Serum kynurenine concentration was evaluated by high-performance liquid chromatography, and its correlation with clinical parameters was examined. Monocyte-derived DCs were prepared from the subjects and subsequently stimulated with a combination of lipopolysaccharide and interferon-gamma to induce functional IDO (defined as IDO-DCs). The phenotypes, kynurenine or cytokine production, and T-cell responses with IDO-DCs were compared between the patients and healthy volunteers.ResultsThe serum kynurenine level in the patients was significantly higher than that in the healthy volunteers, and the level of serum kynurenine was positively correlated with the histological activity or fibrosis score. IDO activity in IDO-DCs from the patients was significantly higher than that in IDO-DCs from the volunteers. Furthermore, IDO-DCs from the patients induced more Tregs in vitro compared with those from the volunteers, and the frequency of induced Tregs by IDO-DCs was decreased with an IDO-specific inhibitor.ConclusionsSystemic IDO activity is enhanced in chronic hepatitis C patients in correlation with the degree of liver inflammation and fibrosis. In response to inflammatory stimuli, DCs from the patients tend to induce Tregs, with some of this action being dependent on IDO.

Clinical Trial of the Intratumoral Administration of Labeled DC Combined With Systemic Chemotherapy for Esophageal Cancer

Esophageal cancer is a highly aggressive disease, and improved modalities for its treatment are needed. We performed chemoimmunotherapy involving the intratumoral administration of 111In-labeled dendritic cells (DC) in combination with preoperative chemotherapy in 5 esophageal cancer patients. Mature DC were generated and traced by scintigraphy after their administration. No adverse events that were directly related to the intratumoral DC administration were observed. Delayed-type hypersensitivity skin tests against keyhole limpet hemocyanin, which was added to the culture medium, detected a positive response in 3 patients, and keyhole limpet hemocyanin antibody production was observed in 4 patients, suggesting that intratumorally administered DC migrate to the lymph nodes, where they function as antigen-presenting cells. However, scintigraphic images obtained after the DC administration demonstrated that the DC remained at the esophageal tumor injection sites in all cases, and no DC accumulation was observed elsewhere. The accumulation of CD83+ cells in the primary tumor was also observed in 2 out of 4 patients in an immunohistochemical analysis using surgically resected specimens. Although the induction of tumor-specific immune responses during chemoimmunotherapy was also analyzed in enzyme-linked immunosorbent assay against 28 tumor antigens, none of the antibodies against the antigens displayed enhanced titers. No changes of NY-ESO-1-specific cellular immune response was observed in a patient who displayed NY-ESO-1 antibody production before the DC administration. These results suggest that the intratumoral administration of 111In-labeled mature DC during chemotherapy does not lead to detectable DC migration from the primary tumor to the draining lymph nodes, and therefore, might not achieve an optimal clinical response.

Quick generation of fully mature dendritic cells from monocytes with OK432, low-dose prostanoid, and interferon-alpha as potent immune enhancers.

Dendritic cells (DCs) are one of the promising tools for enhancing antigen-specific immune responses in clinical settings. Many studies have been performed thus far to verify the efficacy of the DC vaccine in cancer patients; however, the responses have not always been satisfactory, partly because of DC incompetence. To obtain DCs potentially applicable for vaccination of cancer patients, our group sought to establish the strategy of DC generation mainly by modulating culture periods and maturation stimuli. Novel mature DCs that can be generated from monocytes within 3 days by using a combination of OK432 (Streptococcus pyogenes preparation), low-dose prostaglandin E2 (PGE2), and interferon-α (OPA-DCs) were developed. They strongly express CD83, CD86, and CCR7 and have potent ability to migrate to CCL21. In addition, they were able to activate natural killer and T helper 1 (TH1) cells and to induce peptide-antigen-specific cytotoxic T lymphocytes more significantly than monocyte-derived DCs stimulated with a conventional cytokine cocktail of tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and PGE2 (monocyte-conditioned medium [MCM]-mimic DCs). The profound ability of OPA-DCs to stimulate these effectors is attributable to their higher expression of IL-12p70, IL-23, and IL-27 than MCM-mimic DCs, which was supported by the findings that the neutralization of IL-12p70 and IL-23 reduced the TH1 priming ability of OPA-DCs. Even when from advanced gastric or colonic cancer patients, OPA-DCs displayed abilities of migration and TH1 induction comparable to those from healthy subjects. Therefore, OPA-DCs may serve as a feasible vaccine with the potential to enhance TH1-dominant and cytolytic immune responses against cancers.

Three-Dimensional Registration of Images Obtained Before and After Radiofrequency Ablation of Hepatocellular Carcinoma to Assess Treatment Adequacy

OBJECTIVE The aim of our study was to evaluate the value of 3D registration images reconstructed by fusion of pre- and posttreatment CT or MRI for the assessment of ablative margins after percutaneous radiofrequency ablation (RFA) of hepatocellular carcinoma (HCC). MATERIALS AND METHODS From January 2007 to May 2011, we performed RFA in 84 patients to treat 139 HCC nodules, the margins of which had been assessed by comparing pre- and postablation images side by side. The same nodules were retrospectively assessed again with 3D registration images after classification into four margin grades. We analyzed the cumulative local recurrence rate for each grade and reviewed the origin of recurrence. RESULTS Three-dimensional registration images predicted local recurrences more accurately than did the conventional side-by-side method (area under the curve, 0.678 and 0.536, respectively; p = 0.0144). The cumulative rates of local recurrence were significantly different among the margin grades assessed with 3D registration images (p = 0.0088). Three-dimensional registration images detected that the major origins of recurrence (n = 22) were residuals (n = 13) and sites of no margin (n = 6), especially proximate to blood vessels more than 3 mm in diameter. CONCLUSION Three-dimensional registration of pre- and postablation CT or MRI more accurately assesses the ablative margin than the conventional method. It can predict a proclivity for local recurrence after RFA according to margin grade. It also indicated that residuals and sites of no margin proximate to blood vessels that are more than 3 mm in diameter are high-risk locations for local recurrence after ablation.