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Key Clinical Message Autologous formalin-fixed tumor vaccine (AFTV) suppressed re-recurrence for more than 32 months of multiple-recurrent hepatocellular carcinoma based on hepatitis C virus-induced liver cirrhosis in a case with previous recurrence interval, 51-, 28-, 12-, and 4-months. We detected glypican-3-specific cytotoxic T lymphocytes in the peripheral blood at 12 months after AFTV.

症例は18歳女性．生来健康だったが，ウコン，女性ホルモン剤を内服していたところ黄疸，肝障害を認めた．肝生検では形質細胞浸潤，ロゼット形成を伴う肝硬変像を呈し，実質には著明な出血壊死を認めた．腹部CTでは脾腫，胃腎シャントを認め，内視鏡では胃・食道静脈瘤を認めた．自己免疫性肝炎（AIH）の国際診断基準スコアは治療前で+15であり，背景肝としてAIHが考えられた．大循環シャントが形成されており，ある期間をかけてAIHから肝硬変に進展した後に急性肝障害が加わったと推測された．

長期にラミブジン (LAM) 投与がなされたB型慢性肝炎症例を対象として, LAM耐性株の出現に伴う肝炎再燃の予測および投与中止時期決定のため, B型肝炎ウイルス (HBV) DNAの推移を高感度定量法を用いて検討した. HBV DNA陽性のB型慢性活動性肝炎で, 24カ月以上LAM投与が継続された8症例 (35～59歳, 男性が7例, F3が4例, HBe抗原陽性が3例, ゲノタイプは全例C型, コアプロモーターは全例変異型) を対象とした. HBV DNA定量法としてTMA-HPA法, アンプリコアHBVモニター法に加え, 超高感度検出系のHBV RTD-PCR direct (DIRECT) 法を導入した. 検討の結果, 4例において長期経過後にDNA量の再上昇およびYMDD変異株の出現が認められたが, 4例ともLAM投与経過中にDNA量の軽度上昇がみられていた. 1例においては経過中のDNA量の上昇をDIRECT法においてのみ捉えられており, LAM投与症例においてHBV DNAの推移を観察していく上でDIRECT法の有用性が示唆された.

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor-associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte-derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA-derived CEA and MUC1 antigens and DC-derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4+ and CD8+ T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN-γ. More importantly, coculture of fusion cells with patient-derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen-specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1-positive and HLA partially matched target cells. Antigen-specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA- and MUC1-specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen-specific CTL responses. Our report demonstrates the simultaneous induction of CRCA-specific CTL responses restricted by HLA-A2 and -A24. © 2005 Wiley-Liss, Inc.

症例は60歳女性. 黄疸を主訴に入院. 肝機能障害を認め, 肝炎ウイルスマーカー陰性で, 飲酒歴, 薬物歴なく, 抗核抗体陽性等より自己免疫性肝炎 (autoimmune hepatitis, AIH) と診断した. 肝組織所見は慢性活動性肝炎で, 肝機能障害はプレドニゾロン投与により改善し, 治療後のAIHスコアーは24点で確診例であった. さらに橋本病, 特発性副甲状腺機能低下症, 多発性筋炎の合併を認め, 多腺性自己免疫症候群 (autoimmune polyglandular syndrome, APS) を合併したAIHと診断した. AIHとAPSの合併例の報告は, わが国では少なく貴重な症例と考えられた.

症例は51歳女性, 看護婦. 主訴は全身倦怠感. 平成8年3月22日, C型慢性肝炎患者に使用した注射針にて左足背部を刺傷. 同年4月5日頃より, 全身倦怠感出現し, 4月13日の血液検査で肝機能障害を指摘され入院となった. 入院時の肝炎ウイルスマーカーはHBs抗体を除き全て陰性であったが, DNA probe法によるHCV-RNAの定量では40Meq/ml以上と高値を呈していた. そのため, インターフェロン (IFN) -βの投与を施行し, 効果が認められた. しかし, 終了後19日目に再燃がみられ, IFN-α2bの追加投与を行い著効が得られた. また, 汚染血と被汚染血中のHCV-RNAのgenotypeは1b型で一致していた. さらに, direct sequence法によるNS5A領域内の40アミノ酸の解析でも, 両者ともに2カ所に同一の変異がみられ一致していた.

Ten patients who suffered from acute hepatitis with various clinical forms due to hepatitis B virus (HBV) were studied. HBV variants with a mutation in the precore region were dominant in two patients with fulminant hepatitis and in a patient with the most severe acute hepatitis. However, these mutant viruses were not detected in a patient who had the fulminant form of acute HBV infection on chronic liver damage or in most patients who had severe acute hepatitis. Furthermore, mutant viruses were also not detected in a patient with complicating myopathy and in one who had an atypical clinical course with three transaminase peaks. These results suggest that precore mutants may be involved in the pathogenesis of some cases of severe acute hepatitis, the same as for fulminant hepatitis, but not in other clinical forms of acute hepatitis.

To construct formulae for predicting the likelihood of ribavirin-induced anemia in pegylated interferon α plus ribavirin for chronic hepatitis C.Five hundred and sixty-one Japanese patients with hepatitis C virus genotype 1b who had received combination treatment were enrolled and assigned randomly to the derivation and confirmatory groups. Single nucleotide polymorphisms at or nearby ITPA were genotyped by real-time detection polymerase chain reaction. Factors influencing significant anemia (hemoglobin concentration10.0 g/dL at week 4 of treatment) and significant hemoglobin decline (declining concentrations3.0 g/dL at week 4) were analyzed using multiple regression analyses. Prediction formulae were constructed by significantly independent factors.Multivariate analysis for the derivation group identified four independent factors associated with significant hemoglobin decline: hemoglobin decline at week 2 [P = 3.29 × 10(-17), odds ratio (OR) = 7.54 (g/dL)], estimated glomerular filtration rate [P = 2.16 × 10(-4), OR = 0.962 (mL/min/1.73 m(2))], rs1127354 (P = 5.75 × 10(-4), OR = 10.94) and baseline hemoglobin [P = 7.86 × 10(-4), OR = 1.50 (g/dL)]. Using the model constructed by these factors, positive and negative predictive values and predictive accuracy were 79.8%, 88.8% and 86.2%, respectively. For the confirmatory group, they were 83.3%, 91.0% and 88.3%. These factors were closely correlated with significant anemia. However, the model could not be constructed, because no patients with rs1127354 minor genotype CA/AA had significant anemia.Reliable formulae for predicting the likelihood of ribavirin-induced anemia were constructed. Such modeling may be useful in developing individual tailoring and optimization of ribavirin dosage.

We investigated the immunological factors concerning the backgrounds of non- and low-responders who could not respond well to the hepatitis B (HB) vaccine. We injected 10 micrograms of the recombinant HB vaccine to the medical staffs of our hospital intramuscularly three times, 199 subjects of whom could receive the full course of immunization with the HB surface (HBs) antigen. We found that 14 subjects were non-responders whose titer of HBs antibody was under the 1.9 cut off index (C.I.) examined by radioimmunoassay (RIA) and 47 ones were low-responders whose titers were between 2.0 to 9.9 C.I. and the frequency of non- and low-responders was higher in males after the full course of HB vaccination. We chose 46 subjects and divided them into 4 groups according to the titer of HBs antibody at 8 and 28 weeks. We compared those groups to each other according to the amounts of total protein, gamma-globulin, IgG, IgA and IgM, numbers of peripheral white blood cells and lymphocytes, and percentages of peripheral lymphocyte and subsets of T cell including the examination by two-color flow cytometry using monoclonal antibodies. However we could not get any significant difference in any of those immunological factors by T assay. From these findings we suggest that the response to the HB vaccine does not depend on the quantity of the immunological factors before vaccination but on specific reactivity to HBs antigen after vaccination.

There are cases of hepatitis involving occult hepatitis B virus (HBV) infection in which, even though the HB surface antigen (HBsAg) is negative, HBV-DNA is detected by a polymerase chain reaction (PCR). We conducted a sequence analysis of the entire HBV region in a case of non-B non-C chronic hepatitis in a 46-year-old female. A diagnosis of non-B non-C chronic hepatitis was made. Although HBV markers, such as HBs antibody (anti-HBs), anti-HBc, HBeAg and anti-HBe, were negative, HBV-DNA was positive. Nested PCR was performed to amplify the precore region of HBV-DNA and all remaining regions by long nested PCR. Sequence analysis of the two obtained bands was conducted by direct sequencing. Compared with the control strains, the ATG (Methionine) start codon in the X region had mutated to GTG (Valine). It is assumed that a mutation at the start codon in the X region may be the reason why HBV markers are negative in some cases of hepatitis that involve occult HBV infection.

Objective The aim of this study was to investigate the outcome of overlap/switch to adefovir dipivoxil (ADV) monotherapy for chronic hepatitis B (CHB) patients with lamivudine (LAM)-resistant HBV, who responded to LAM plus ADV combination therapy. Methods In 29 of 35 LAM-resistant CHB patients, serum HBV-DNA levels decreased to

AIM: To assess the efficacy and advantages of 4-wk pegylated interferon α-2a (peg-IFN-α2a) monotherapy for chronic hepatitis C patients with strong predictors of sustained virologic response (SVR). METHODS: Patients (n = 33) with genotype 2 and low viral load (< 100 KIU/mL), who became HCV RNA negative after 1 wk of IFN treatment, were randomly allocated to receive a 4- or 12-wk treatment course at a ratio of 2:1, respectively, with a subsequent 24-wk follow-up period. Peg-IFN-α2a was administered subcutaneously at a dose of 180 μg or 90 μg once weekly. SVR was defined as absence of serum HCV RNA at the end of the follow-up period. RESULTS: All patients completed the treatment schedule, and more than half were symptom-free during the treatment. In the 4-wk treatment group, 20 of 22 (91%) patients achieved SVR. Two patients relapsed, but achieved SVR following re-treatment with peg-IFN-α2a alone. In the 12-wk treatment group, 11 of 11 (100%) patients attained SVR. CONCLUSION: Our results show that a 4-wk course of peg-IFN-α2a monotherapy can achieve a high SVR rate in “IFN-sensitive” patients, without negatively affecting outcome.

Two young man patients with refractory post-bulbar duodenal ulcer (post-bulbar ulcer) were encountered. They had a single punched-out ulcer in the absence of an underlying disease. Patient 1 was Helicobacter pylori (Hp)-positive, and did not respond to Hp eradication therapy. The ulcer scarred after the long-term administration of a proton pump inhibitor (PPI), but recurred after a reduction in the dose. Patient 2 was Hp-negative. His ulcer did not scar even after long-term PPI administration, but it formed a fistula into the gallbladder, and the fistula was surgically closed. In both patients, laboratory and imaging studies excluded Zollinger-Ellison syndrome, but suggested a hyperacidic tendency. Unlike duodenal bulb ulcer (bulbar ulcer), the post-bulbar ulcer in Patient 1 did not heal with Hp eradication therapy, suggesting that post-bulbar ulcer differs etiologically from bulbar ulcer. We speculate that the possible causes of the refractoriness to treatment in both patients were ulcer penetration, callosity formation, and insufficient inhibition of gastric acid secretion due to the impaired passage of PPI into the deep portion of the duodenum as a result of luminal narrowing.

The patient was a 63-year-old woman with attacks of fever and abdominal pain, starting from the age of 53 years and recurring every month. Despite various examinations at another hospital, the etiology remained unclear. She was under symptomatic treatment, and was referred to our department for further evaluation. Although she had onset in middle age, the clinical symptoms and examination findings suggested familial Mediterranean fever, and administration of colchitine inhibited the attacks completely. Therefore, the patient was diagnosed as having the disease. We were not able to analyze the entire MEFV gene, but detected only a heterozygous M694I mutation. Amyloidosis did not develop as a complication. The disease is rare in Japan, and its onset in the fifties is extremely rare in the world.

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2−/HLA-24−), carcinoembryonic antigen (CEA)+, and MUC1+ as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2+ and/or HLA-A24+) and allogeneic COLM-6 resulted in MHC class I– and MHC class II–restricted proliferation of CD4+ and CD8+ T cells, high levels of IFN-γ production in both CD4+ and CD8+ T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

We evaluated the effect of a "tailor-made" chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailor-made approach, we first selected gefitinib (epidermal growth factor receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric fibroblasts, NF22, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between fibroblasts and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n = 8), 11.9; treated (n = 8), 17.3; p = 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p = 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p = 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, 22; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination.

To develop a new sensitive and inexpensive hepatitis C virus (HCV) combination test (HCV Guideline test) that enables the determination of HCV genotypes 1, 2 and 3, and simultaneous determination of HCV viral load using commercial Amplicor GT HCV MONITOR test v2.0 (microwell version).The HCV Guideline test used the PCR product generated in commercial Amplicor GT HCV Monitor test v2.0 for viral load measurement using microwell plate version of Amplicor HCV Monitor and also captured on separate plates containing capture probes and competitive oligonucleotide probes specific for HCV genotypes 1, 2 and 3, The HCV genotype was subsequently determined using the biotin-labeled PCR product and five biotin-labeled HCV-specific probes.The sensitivity of the HCV Guideline test was 0.5 KIU/mL. Specificity of the HCV Guideline test was confirmed by direct sequencing of HCV core region and molecular evolutionary analyses based on a panel of 31 samples. The comparison of the HCV Guideline test and an in-house HCV core genotyping assay using 252 samples from chronic hepatitis C patients indicated concordant results for 97.2% of samples (59.5% genotype 1, 33.7% genotype 2, 6.0% genotype 3, and 0.8% mixed genotypes). Similarly, the HCV Guideline test showed concordance with a serological test, and the serological test failed to assign any serotype in 12.7% of the samples, indicating a better sensitivity of the HCV Guideline test.Clinically, both viral load and genotypes (1, 2 and 3) have been found to be major predictors of antiviral therapy outcome regarding chronic hepatitis C based on guidelines and they are, in normal circumstances, performed as separate stand-alone assays. The HCV Guideline test is a useful method for screening large cohorts in a routine clinical setting for determining the treatment regimen and for predicting the outcome of antiviral therapy of chronic hepatitis C.

Changes of titers of acquired hepatitis B surface (HBs) antibodies against HB vaccine were investigated by measuring them again after a long lapse of time. Ten micrograms of the recombinant HB vaccine was intramuscularly injected respectively to the staff of the authors' hospital three times. Four weeks after a full course of the vaccination 185 persons could acquire antibodies, whose titers were 2.0 or more in cut off index (C.I.) by radioimmunoassay (RIA). Of these members titers of antibodies of 48 subjects could be remeasured for the first time 38 months after the measurement of the titers in the 4th week after a full course of the vaccination. Four weeks after the last vaccination 7 persons were high responders whose titers of antibodies were 50 or more in C.I. 29 were medium responders with their titers from 49 to 10, and 12 were low responders having titers of less than 10. However, 38 months after the course the titers of those responders decreased so much that nobody remained highly responders, 22 were low responders, and the remaining 22 turned to be negative again with titers less than 2.0. Supposing that the titer of antibody enough to protect the infection of HB virus is 10 or more in C.I., 32 of 36 persons needed booster shots 38 months later because their titers dwindled down to less than 10.(ABSTRACT TRUNCATED AT 250 WORDS)

Pegylated interferon α (IFNα) in combination with ribavirin is currently recommended as a standard-of-care treatment for chronic hepatitis C virus (HCV) infection. This combination therapy has drastically improved the rate of sustained virological response, specifically in difficult-to-treat patients. Recently, individualized treatment, such as response-guided therapy, is being developed based on host-, HCV- and treatment-related factors. Furthermore, modified regimens with currently available medications, novel modified IFNα and ribavirin or combinations with specifically targeted antiviral therapy for HCV agents, are currently being investigated. The purpose of this review is to address some issues and epoch-making topics in the treatment of chronic HCV infection, and to discuss more optimal and highly individualized therapeutic strategies for HCV-infected patients.

AIM: To investigate the roles of Bax and p53 proteins in photosensitivity of human colon cancer cells by using lysosome-localizing photosensitizer, ATX-S10Na (II). METHODS: HCT116 human colon cancer cells and Bax-null or p53-null isogenic derivatives were irradiated with a diode laser. Early apoptosis and cell death in response to photodynamic therapy were determined by MTT assays, annexin V assays, transmission electron microscopy assays, caspase assays and western blotting. RESULTS: Induction of early apoptosis and cell death was Bax- and p53-dependent. Bax and p53 were required for caspase-dependent apoptosis. The levels of anti-apoptotic Bcl-2 family proteins, Bcl-2 and Bcl-xL, were decreased in Bax- and p53-independent manner. CONCLUSION: Our results indicate that early apoptosis and cell death of human colon cancer cells induced by photodynamic therapy with lysosome-localizing photosensitizer ATX-S10Na (II) are mediated by p53-Bax network and low levels of Bcl-2 and Bcl-xL proteins. Our results might help in formulating new therapeutic approaches in photodynamic therapy.

著者らは抗癌剤の併用による温熱の効果を,著者らの教室にて樹立しえたアルブミン高産生性のヒト肝細胞癌JHH-4株を用い,in vitroにおいて検討を行った.ペトリ皿に付着増殖した肝癌細胞を,抗癌剤として0～20μg/mlのAdriamycinを含む培養液にて,従来のコロニー形成法とは異なり,温度勾配培養装置を用い,37～43℃で2日間培養を行い,生細胞数の算定のみならず機能的,形態的にも判定を行った.温度の上昇に伴い生細胞数の減少,培養上清中のアルブミン濃度の低下,3Hラベルのサイミジン,ウリジン,ロイシンの取込みの低下,付着細胞の形態的変化がみられた.温熱単独でみられた肝癌細胞に対するin vitroにおけるこれらの効果は,Adriamycinを併用することにより増強が認められた.

collagenase灌流法および低速遠心法により単離したラット肝実質細胞をPercollを使用した新しい直線密度勾配遠心法により亜分画した.摂食および絶食ラットの単離肝実質細胞は死細胞を除き3亜分画に,70%肝部分切除3日後のそれは1亜分画,7日後は2亜分画に明瞭に分離した.摂食ラットの3亜分画細胞の小葉内分布を決定するためFluorescein diacetateの灌流および透過電顕,走査電顕により機能的・形態的に検討をした.その結果,低密度肝実質細胞は小葉中心域肝細胞,高密度肝実質細胞は小葉周辺域の肝細胞に一致した.中間密度肝実質細胞は形態学的には高密度肝実質細胞に近似していた.また亜分画細胞の可溶性蛋白量とglucose-6-phosphatase (G6Pase)を測定した.その結果,可溶性蛋白量は亜分画により著しい差を認めたが,G6Paseは若干の差を認めるのみであった.さらに,亜分画細胞は5日間初代単層培養され,形態学的特徴が5日間持続することを認めた.

Reuber H-35 hepatoma cells were examined for their ability to synthesize protein in vitro, especially to produce alpha-fetoprotein (AFP). The presence of AFP in the culture supernatant solution was determined immunologically by the micro-Ouchterlony method. Charge heterogeneity of AFP was examined electrophoretically in continuous gradient polyacrylamide microgels. With regard to the duration of culture, there was no remarkable change in the ratio of two peaks of AFP, and which came out as a major combined peak and a similar peak by PAS staining on the condition of added SDS. These findings indicated that Reuber H-35 hepatoma cells had potential to produce two charge variants of AFP in vitro.

Ubiquitin, which can conjugate with cellular proteins, is classified into two forms: free ubiquitin and multiubiquitin chains. The latter is active as a signal for degradation of the targeted proteins. We found both forms in human serum and, using two immunoassays, quantitated them in sera from healthy subjects and patients with some diseases. Because of putative leakage of erythrocyte ubiquitin, hemolytic serum and serum obtained after long incubation (>1–2 h) of blood at room temperature were excluded. Serum concentrations of multiubiquitin chains and free ubiquitin were substantially higher in rheumatoid arthritis and hemodialysis patients, respectively, than healthy subjects. Additionally, in acute viral hepatitis, serum multiubiquitin chain concentrations were increased in the acute phase, decreased in the recovery phase, and correlated with alanine and aspartate aminotransferase activities (r = 0.676 and 0.610, P

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- γ at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+IFN-γ+CD8+ T cells and CD154+ IFN-γ+CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.

Background Human hepatocellular carcinoma (HCC) cells express WT1 and/or carcinoembryonic antigen (CEA) as potential targets for the induction of antitumor immunity. In this study, generation of cytotoxic T lymphocytes (CTL) and regulatory T cells (Treg) by fusions of dendritic cells (DCs) and HCC cells was examined. Methods HCC cells were fused to DCs either from healthy donors or the HCC patient and investigated whether supernatants derived from the HCC cell culture (HCCsp) influenced on the function of DCs/HCC fusion cells (FCs) and generation of CTL and Treg. Results FCs coexpressed the HCC cells-derived WT1 and CEA antigens and DCs-derived MHC class II and costimulatory molecules. In addition, FCs were effective in activating CD4+ and CD8+ T cells able to produce IFN-γ and inducing cytolysis of autologous tumor or semiallogeneic targets by a MHC class I-restricted mechanism. However, HCCsp induced functional impairment of DCs as demonstrated by the down-regulation of MHC class I and II, CD80, CD86, and CD83 molecules. Moreover, the HCCsp-exposed DCs failed to undergo full maturation upon stimulation with the Toll-like receptor 4 agonist penicillin-inactivated Streptococcus pyogenes. Interestingly, fusions of immature DCs generated in the presence of HCCsp and allogeneic HCC cells promoted the generation of CD4+ CD25high Foxp3+ Treg and inhibited CTL induction in the presence of HCCsp. Importantly, up-regulation of MHC class II, CD80, and CD83 on DCs was observed in the patient with advanced HCC after vaccination with autologous FCs. In addition, the FCs induced WT1- and CEA-specific CTL that were able to produce high levels of IFN-γ. Conclusion The current study is one of the first demonstrating the induction of antigen-specific CTL and the generation of Treg by fusions of DCs and HCC cells. The local tumor-related factors may favor the generation of Treg through the inhibition of DCs maturation; however, fusion cell vaccination results in recovery of the DCs function and induction of antigen-specific CTL responses in vitro. The present study may shed new light about the mechanisms responsible for the generation of CTL and Treg by FCs.