A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro.

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin-biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Simultaneous measurements of serum AFP, GPC-3 and HCCR for diagnosing hepatocellular carcinoma.

BACKGROUND/AIMS: Hepatocellular carcinoma (HCC) is a prevalent malignant tumor. Tumor markers are very useful in early diagnosis; however a single marker is rather limited. We launched a test to increase the diagnostic sensitivity through the combined detection. METHODOLOGY: Serum concentration of three tumor-markers, Glypican-3 (GPC-3), Human-Cervical-Cancer-Oncogene (HCCR) and a-fetoprotein (AFP), were determined in 189 samples: 101 cases of HCC, 40 cases of cirrhosis, 18 cases of hepatitis and 30 cases of control healthy donors. Every marker was evaluated for its diagnostic value by one-way-analysis-of-variance and receiver-operating-characteristics analysis. RESULTS: GPC-3 was the best marker with an area under the curve (AUC) of 0.892; using 26.8ng/mL as the cut-off for HCC diagnosis, GPC-3 has a sensitivity of 51.5% and maintains a specificity of 92.8%. HCCR, with an AUC of 0.831, can reach a sensitivity of 22.8% and maintain a specificity of 90.9% if the cut-off is set as 58.8mAU/mL. With an AUC of 0.827, the efficacy and sensitivity of AFP were 36.6% and 98.5% when using 199.3ng/mL as the cut-off. No significant correlation was found between these three markers. Simultaneously detecting three markers can significantly increases the sensitivity to 80.2%, much higher than AFP alone. CONCLUSIONS: GPC-3 and HCCR are useful tumor markers complementary to AFP for clinical diagnosis of HCC.

[Effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl4/ethanol].

To study the effects of Smad4 on liver fibrosis and hepatocarcinogenesis in mice treated with CCl(4)/ethanol. The wild-type mice (Smad4 +/+) and the Smad4 knockout mice (Smad4 +/-) were injected subcutaneously with carbon tetrachloride(CCl(4))/ethanol twice a week for twenty weeks. The expression of Smad4, TGFbeta1, Smad2, Smad3, Smad6, TIMP1, MMP2 and MMP9 was detected by RT-PCR. In the cirrhotic liver, the expression of Smad4 mRNA was significantly higher than that in the normal liver. Comparing with wild-type mice (Smad4 +/+), the TGFbeta1-Smad4 signaling was markedly attenuated in the Smad4 knockout mice (Smad4 +/-). After induction by CCl(4)/ethanol, the hepatic fibrosis in the Smad4 knockout mice (Smad4 +/-) was obviously alleviated compared with the wild-type mice (Smad4 +/+), and the incidence rate of hepatocarcinogenesis of the former was also lower than that of the latter(32.0% vs 41.9%). These results indicate that knocking out Smad4 can delay the progression of liver fibrosis and liver cancer.

The immunosuppressive tumor microenvironment in hepatocellular carcinoma.

Increasing evidence indicates the immunosuppressive nature of the local environment in tumor. The present study was focused on analyzing the immune status within hepatocellular carcinoma. In contrast to the increasing number of CD4(+) T cells, CD8(+), CD3(-)CD56(+), CD3(+)CD56(+), and gammadeltaT cells were all found to be under-represented in tumor infiltrating lymphocytes. Notably, the relative abundance of CD3(+)CD56(+) cells appeared to be correlated with patient survival. Functional analysis demonstrated that CD4(+) cells in the tumor tended to produce more IL-10 but less IFN-gamma, whereas CD8(+) cells showed impaired capacity for the production of both IFN-gamma and perforin. Consistent with previous reports, we observed a significant increase of Foxp3(+) cells in the tumor tissue. Intriguingly, although over 90% of CD4(+)CD25(high) cells were found to be Foxp3(+), the majority of Foxp3(+) cells were identified in the CD4(+)CD25(medium) and CD4(+)CD25(-) subsets. In support of its role as a negative regulator, CD4(+)CD25(high) cells suppressed the proliferation of CD4(+)CD25(-) cells isolated from the same tissues in an APC dependent manner. In conclusion, the tumor microenvironment of hepatocellular carcinoma is featured by the presence of multiple immunosuppressive factors.

Expression of CIITA-related MHCII molecules in tumors linked to prognosis in hepatocellular carcinoma.

The prognosis of hepatocellular carcinoma (HCC) after surgery is poor due to its high recurrence rate. In order to unfold the mechanism of different recurrent-free survival (RFS) times following resection, expression profiling of tumor tissues from 32 HCC patients with different RFS time were used to identify differential expression of individual genes and signaling pathway components correlated with RFS time. Quantitative RT-PCR, Western blotting, and immunohistochemistry were used to validate the expression of selected genes. Up-regulation of several immune related genes and pathways, especially HLA II-related antigen presenting pathways, significantly correlated with longer RFS time. The expression of MHCII molecules were found to be mainly located in either CD68+ cells or CD45+ cells, and their expression significantly correlated with the expression of CIITA (HLA II genes transactivator) in the tumor. The results suggest that the high expression level of CIITA and MHCII molecules in hepatocellular carcinoma tissue is an effective prognostic marker for longer RFS time in HCC.

[Effects of galectin-3 on proliferation and apoptosis of hepatic stellate cells].

OBJECTIVE: To observe the effects of galectin-3 on proliferation and apoptosis of hepatic stellate cells. METHODS: RT-PCR and Western blot were used to detect the expression of galectin-3 in hepatic stellate cells. Short hairpin DNA targeting galectin-3 of rat was was ligated into the recombinant vector pGCsilencer U6/Neo/GFP/shRNA plasmid. Then the plasmid was transfected into rat hepatic stellate cells. RT-PCR and Western blot were used to detect the interfering efficiency. Cell proliferation level was observed by CCK8 method at 24, 48 and 72 hours after transfection. Cell apoptosis was measured by Annexin V/PI-labeled flow cytometric analysis. RESULTS: Expression of galectin-3 in HSC was verified by both RT-PCR and Western blot. The recombinant vector was successfully constructed and verified, and was transfected into rat hepatic stellate cells. Western Blot and RT-PCR results demonstrated that the expression level of Galectin-3 was significantly down-regulated in galectin-3 shRNA transfected cells compared to control vector transferred cells. CCK8 assay indicated that proliferation of Galectin-3 knockdown cells was lower than that of control cells 48 and 72 hours post-transfection. Apoptotic cells in shRNA-interfering group were higher than those in control group both in early stage and advanced stage. CONCLUSION: Hepatic stellate cells can express galectin-3. Inhibition of galectin-3 using RNAi technique can suppress proliferation and induce apoptosis in HSC.

Plac1 is a tumor-specific antigen capable of eliciting spontaneous antibody responses in human cancer patients.

Immunoselection and tumor evasion constitutes one of the major obstacles in cancer immunotherapy. A potential solution to this problem is the development of polyvalent vaccines, and the identification of more tumor-specific antigens is a prerequisite for the development of cancer vaccines. To identify novel tumor-specific antigens, suppression subtractive hybridization (SSH) was performed to isolate genes differentially expressed in human hepatocellular cancer (HCC) tissues. PLAC1 (PLACenta-specific 1) was one of the genes identified highly expressed in HCC tissues but not in paired noncancerous tissues. Further analyses revealed its expression in several other types of cancer tissues as well as tumor cell lines, but not in normal tissues except for placenta. Among HCC samples tested, 32% (22/69) showed PLAC1 mRNA expression while the protein was detected in 23.3% (7/30). A serological survey revealed that 3.8% (4/101) of HCC patients had anti-PLAC1 antibody response, suggesting the immunogenicity of PLAC1 in HCC patients. PLAC1 represents a new class of tumor associated antigen with restricted expression in placenta and cancer tissues, that may serve as a target for cancer vaccination.

Identification of new cytotoxic T-lymphocyte epitopes from cancer testis antigen HCA587.

The cancer testis (CT) antigen HCA587 is highly expressed in human hepatocellular carcinoma (HCC) and induces specific T-cell responses in a significant proportion of HCC patients. To explore its potential in cancer immunotherapy, a reverse immunology approach was adopted to identify HCA587-derived HLA-A( *)0201-restricted epitopes. Multiple peptides with a top ranking in various prediction programs were thus synthesized and three of them-p248-256, p140-149 and p144-152-were found to bind to HLA-A(*)0201 molecules with a high affinity and effectively induced a recall response of CD8+ T cells, which were either primed in vitro with the HCA587 antigen or directly isolated from HCC patients bearing HCA587+ tumors. Notably, these peptide-specific CD8+ T cells exhibited potent cytotoxic activity over HCA587+ tumor cells. Taken together, the present study has identified three new HLA-A(*)0201-restricted cytotoxic T cell epitopes in the CT antigen HCA587, which may serve as targets for peptide-based immunotherapy for HCC patients.

[NY-ESO-1 and cancer immunotherapy].

NY-ESO-1 is an important member of cancer-testis antigen family and is widely distributed among many cancer types. As a tumor-specific antigen with the strongest immunogenicity so far identified, it can induce spontaneous antibody and T-cell responses in patients with NY-ESO-1-positive tumors. Therefore, it has been a good vaccine candidate in the immunotherapy against many malignancies. This article reviews the recent research advances in NY-ESO-1 and its relevant vaccines.

Long-limb length difference had no effect on outcomes of laparoscopic Roux-en-Y gastric bypass surgery for obese Chinese patients with type 2 diabetes mellitus: A CONSORT compliant article.

Bariatric surgery is effective in treating different components of metabolic syndrome including obesity, type 2 diabetes mellitus (T2DM), and hyperlipidemia. But there is no consensus on the ideal biliopancreatic and Roux limb length. This study aimed to explore the effect of biliopancreatic limb and Roux limb lengths during laparoscopic Roux-en-Y gastric bypass (LRYGB) procedures on weight loss and T2DM control.We studied the clinical records of 58 patients with metabolic syndrome, T2DM, and body mass index (BMI) 32 to 50 kg/m who underwent LRYGB in our hospital. The short limb group (Group A) underwent LRYGB with a limb length of 160 to 200 cm (n = 31) and the long limb group (Group B) underwent LRYGB with a limb length of 210 to 240 cm (n = 27) were compared.The occurrence of acute or chronic internal hernia in Group B was higher than that in Group A (P = .026). Twelve months after surgery, patients from the 2 groups were also observed with reduction in BMI, percent excess weight loss (EWL), preoperative FPG, and HbA1c as compared with these indicators before surgery. However, the differences of these indicators between 2 groups were not significant at the time point of before and 3, 6, 12 months after surgery.LRYGB had significant effects on weight loss and diabetes control in obese T2DM patients. However, there was no significant difference in the short term on weight loss and diabetes control in the patients receiving different limb lengths.

[Identification of a naturally presented MAGE-A3 epitope on the surface of HLE cell line by mass spectrometry].

OBJECTIVE: To identify a naturally presented HLA-A2-restricted epitope of MAGE-A3 antigen, FLWGPRALV (MAGE-A3(271 - 279)), on the surface of a human hepatocellular carcinoma (HCC) cell line HLE. METHODS: Synthetic peptide FLWGPRALV, served as positive control target, was analyzed by HPLC and HPLC-ESI-TOF-MSMS, in order to determine its HPLC elution time, mass-spectrometric characteristics and the lowest detection limitation by the two approaches. 3 x 10(9) HLE cells were collected, peptides naturally presented by major histocompatibility complex (MHC) molecules on the cell surface were isolated by mild acid elution, and concentrated by lyophilization, then the mixtures of peptides were fractioned by HPLC. The ingredient ranged from 2 min before the elution time determined by the synthetic peptide to 2 min after that was collected, concentrated by lyophilization, and analyzed by HPLC-ESI-TOF-MSMS, to identify the existence of the MAGE-A3(271 - 279) peptide. RESULTS: The HPLC-ESI-TOF-MSMS detection provided an evidence for the existence of a doubly charged ion of (m/z)(2) 529.9, which was further analyzed by collision induced dissociation. The doubly charged ion was ultimately identified as the MAGE-A3(271 - 279) peptide, its amino sequence was FLWGPRALV and its molecular weight was 1058.4 Da. CONCLUSIONS: MAGE-A3(271 - 279) epitope could be naturally presented by HLA-A2 molecules to the surface of HCC cell line and MAGE-A3(271 - 279) peptide may have potential immunotherapeutic value in HCC patients.

Effects of 5-hydroxytamine and its antagonists on hepatic stellate cells.

BACKGROUND: Hepatic stellate cell (HSC) plays a key role in hepatic fibrosis. This study was undertaken to investigate the expression of 5-hydroxytamine receptors in HSC and the effect of 5-hydroxytamine on biological characteristics of HSC. METHODS: Liver ex vivo perfusion of collagenase and density gradient centrifugation were used to isolate HSCs. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to detect the expression of 5-hydroxytamine receptor subtypes 1A, 2A, 2B and 3. Western blot hybridization was used to elucidate the effect of 5-hydroxytamine and its 2A receptor antagonist ketanserin and 3 receptor antagonist ondanosetron on the expression of transforming growth factor-beta1 (TGF-beta1) and Smad4 in HSC. RESULTS: HSC expressed 5-hydroxytamine receptor subtypes 1A, 2A and 2B. 5-hydroxytamine significantly increased the expression of TGF-beta1 and Smad4 in HSC (P<0.05). This action can be antagonized by ketanserin, not by ondanosetron. CONCLUSIONS: HSC expresses 5-hydroxytamine receptors. 5-Hydroxytamine could effect the biological characteristics of HSC through its receptor mediation, and may play a role in the pathogenesis of liver cirrhosis and portal hypertension.

Identification of two naturally presented MAGE antigenic peptides from a patient with hepatocellular carcinoma by mass spectrometry.

In the absence of efficient systemic chemotherapy, immunotherapy is considered a hopeful treatment for controlling recurrence of hepatocellular carcinoma (HCC). The identification of proper antigenic peptides presented by MHC class I molecules is a critical step for the development of therapeutic vaccines against tumors. Currently, the "reverse immunology" approach is the most commonly used technique in the identification of the tumor-associated T cell epitopes. However, it is based on T cell dependent approach and cannot fully reflect the actual presentation of epitope in tumor in vivo. In the present study, we managed to identify the naturally presented MAGE epitopes of HCC directly by epitope prediction, HPLC differential analysis and MS detection. We successfully detected a naturally processed peptide FLWGPRALV (MAGE-3(271-279), HLA-A2-restricted) with an estimated number of 38-39 copies/cell in HCC. To our knowledge, this is the first evidence that the naturally processed MAGE-3(271-279) can be isolated and identified from the tumor tissue of HCC patient. Furthermore, specific CD8(+) T cell responses to this epitope were also found after tumor relapse by IFN-gamma release Cytospot and tetramer assay indicating that MAGE-3(271-279) was indeed presented by HCC cells in vivo. In addition, another new antigen peptide was found, which may be derived from MAGE-1. Our findings demonstrate the potential of the direct approach for identification of tumor-associated epitopes. This approach may become a useful tool for the development of vaccine against cancer in the future.

Expression of cancer/testis (CT) antigens in Chinese hepatocellular carcinoma and its correlation with clinical parameters.

For investigating the expression of cancer/testis (CT) antigens in patients with hepatocellular carcinoma (HCC) in China, and evaluating the correlations between the expression of these CT antigens and clinical parameters, we collected tumors and adjacent non-cancerous tissues of 43 HCC patients from Beijing and 30 HCC patients from Guangxi province. Expression of the mRNA of 14 CT antigens was evaluated by reverse transcription PCR (RT-PCR). The correlation between CT antigen expression and clinical parameters was statistically analyzed. The mRNA expression frequencies of CT antigens in tumor tissue were: MAGE-A1, 69.9%; MAGE-A3, 47.9%; MAGE-A4, 20.0%; MAGE-A10, 36.7%; SSX-1, 67.4%; SSX-2, 35.6%; SSX-4, 48.8%; SSX-5, 30.2%; NY-ESO-1, 42.5%; MAGE-B1, 52.0%; MAGE-B2, 60.0%; MAGE-C1, 48.0%; MAGE-C2, 68.0%; and SCP-1, 33.3%. However, in adjacent tissues, no CT antigen mRNA expression was detected, except SSX-1 in 9.3% patients. In each HCC tissue, the expression of a minimum of one, two, or three CT antigens was in the range of 80-90, 70-80 or 50-70%, respectively. MAGE-A3 mRNA expression differed between the HCC patients in Beijing and Guangxi (P=0.002). The average age of the HCC patients bearing CT antigen positive tumors was higher than that of the HCC patients bearing CT antigen negative tumors. The expression of MAGE-A3, SSX-1, SSX-2, SSX-4, MAGE-B2, MAGE-C1, and MAGE-C2 correlated significantly with older age (P<0.05). Moreover, the expressions of MAGE-A4 and SCP-1 were related to alpha-fetoprotein abnormality (P<0.05), and the expression of NY-ESO-1 was related to early tumor stage (P<0.05). There was no correlation observed between the expression of CT antigens and the sex, HBV infection or tumor size.

The spontaneous CD8+ T-cell response to HLA-A2-restricted NY-ESO-1b peptide in hepatocellular carcinoma patients.

PURPOSE: Hepatocellular carcinoma (HCC) can express various cancer-testis antigens including NY-ESO-1, members of the SSX family, members of the MAGE family, SCP-1, and CTP11. Immunotherapy directed against these antigens is a potential alternative treatment for HCC. To date, it remains unclear whether HCC patients have spontaneous immune responses to these tumor antigens. The objectives of this study were to measure immune responses to NY-ESO-1, a promising cancer vaccine candidate, in HCC patients using the HLA-A2-restricted NY-ESO-1b peptide (p157-165) to measure cellular responses and whole protein to measure antibody responses. EXPERIMENTAL DESIGN: In HLA-A2(+) patients with NY-ESO-1(+) HCC, we analyzed T-cell antigen-dependent interferon (IFN)-gamma and/or Granzyme B release by enzyme-linked immunospot (ELISPOT) assay and IFN-gamma-producing intracellular cytokine flow cytometry (CytoSpot). As an assay independent of T-cell function, we performed tetramer staining. Antibodies to whole NY-ESO-1 were assayed by enzyme-linked immunosorbent assay. RESULTS: The frequency of specific CD8(+) T-cell responses to NY-ESO-1b in 28 NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients was 35.7% (10 of 28). The average magnitude of effector CD8(+) T cells was 0.3% (89 +/- 59 per 2.5 x 10(4) CD8(+) cells) and 1.2% as measured by IFN-gamma release ELISPOT and CytoSpot assays, respectively. These in vitro induced NY-ESO-1b-specific CD8(+) T cells can also recognize HepG2 cells transfected with pcDNA3.1-NY-ESO-1 in both IFN-gamma and Granzyme B ELISPOT assays. Frequencies of NY-ESO-1b-specific T cells in several patients were confirmed by tetramer staining. Nonfunctional tetramer(+)CD8(+) T cells were also present. The CD8(+) T-cell response was apparently increased in patients with late-stage HCC. A discordance between antibody and CD8(+) T-cell responses in HCC patients was observed. CONCLUSIONS: The elevated frequency of specific CD8(+) T-cell responses to NY-ESO-1b in NY-ESO-1 mRNA(+)HLA-A2(+) HCC patients suggests that NY-ESO-1 is appropriate for use in the immunotherapy of HCC patients.

Effect of ligand of peroxisome proliferator-activated receptor gamma on the biological characters of hepatic stellate cells.

AIM: To study the effect of rosiglitazone, which is a ligand of peroxisome proliferator-activated receptor gamma (PPARgamma), on the expression of PPARgamma in hepatic stellate cells (HSCs) and on the biological characteristics of HSCs. METHODS: The activated HSCs were divided into three groups: control group, 3 micromol/L rosiglitazone group, and 10 micromol/L rosiglitazone group. The expression of PPARgamma, alpha-smooth muscle actin (alpha-SMA), and type I and III collagen was detected by RT-PCR, Western blot and immunocytochemical staining, respectively. Cell proliferation was determined with methylthiazolyltetrazolium (MTT) colorimetric assay. Cell apoptosis was demonstrated with flow cytometry. RESULTS: The expression of PPARgamma at mRNA and protein level markedly increased in HSCs of 10 micromol/L rosiglitazone group (t value was 10.870 and 4.627 respectively, P<0.01 in both). The proliferation of HSCs in 10 micromol/L rosiglitazone group decreased significantly (t = 5.542, P<0.01), alpha-SMA expression level and type I collagen synthesis ability were also reduced vs controls (t value = 10.256 and 14.627 respectively, P<0.01 in both). The apoptotic rate of HSCs significantly increased in 10 micromol/L rosiglitazone group vs control (chi(2) = 16.682, P<0.01). CONCLUSION: By increasing expression of PPARgamma in activated HSCs, rosiglitazone, an agonist of PPARgamma, decreases alpha-SMA expression and type I collagen synthesis, inhibits cell proliferation, and induces cell apoptosis.

Clinical analysis of surgical treatment of portal hypertension.

AIM: To review the experience in surgery for 508 patients with portal hypertension and to explore the selection of reasonable operation under different conditions. METHODS: The data of 508 patients with portal hypertension treated surgically in 1991-2001 in our centers were analyzed. Of the 508 patients, 256 were treated with portaazygous devascularization (PAD), 167 with portasystemic shunt (PSS), 62 with selective shunt (SS), 11 with combined portasystemic shunt and portaazygous devascularization (PSS+PAD), 9 with liver transplantation (LT), 3 with union operation for hepatic carcinoma and portal hypertension (HCC+PH). RESULTS: In the 167 patients treated with PSS, free portal pressure (FPP) was significantly higher in the patients with a longer diameter of the anastomotic stoma than in those with a shorter diameter before the operation (P < 0.01). After the operation, FPP in the former patients markedly decreased compared to the latter ones (P < 0.01). The incidence rate of hemorrhage in patients treated with PAD, PSS, SS, PSS+PAD, and HCC+PH was 21.09% (54/256), 13.77 (23/167), 11.29 (7/62), 36.36% (4/11), and 100% (3/3), respectively. The incidence rate of hepatic encephalopathy was 3.91% (10/256), 9.58% (16/167), 4.84% (3/62), 9.09% (1/11), and 100% (3/3), respectively while the operative mortality was 5.49% (15/256), 4.22% (7/167), 4.84% (3/62), 9.09% (1/11), and 66.67% (2/3) respectively. The operative mortality of liver transplantation was 22.22% (2/9). CONCLUSION: Five kinds of operation in surgical treatment of portal hypertension have their advantages and disadvantages. Therefore, the selection of operation should be based on the actual needs of the patients.

[Establishment of dendritomas by fusion of human dendritic cells with human hepatocellular carcinoma cell line HLE cells].

OBJECTIVE: To construct dendritomas by fusion of human dendritic cells with HLE cells, a human hepatocellular carcinoma cell line. METHODS: HLE cells were cultured in RPMI 1640 with 15% FCS. Human dendritic cells (DCs) were obtained from peripheral blood monocytes cultured in the presence of GM-CSF and IL-4 for 7 days, matured with TNF-alpha and PGE(2) for 2 days. The DCs and HLE cells were labeled with green fluorescence dye PKH67-GL and red fluorescence dye PKH26-GL, respectively, and fused in 50% polyethylene glycol (PEG) + 10% dimethyl sulfoxide (DMSO) to generate dendritomas for rapid fluorescence-activated cell sorting (FACS). RESULTS: Dendritomas with dual red-green fluorescence were constructed successfully, and FACS analysis showed the effective fusion rate was 16.8%. CONCLUSION: With fluorescence dyes PKH67-GL and PKH26-GL as fusion markers, dendritomas for rapid fluorescence-activated cell sorting are constructed, which may throw new light on immunotherapy of hepatocellular carcinoma.

[Inducing hepatocellular carcinoma-specific cytotoxic T lymphocyte response using formed by fusion of FastDCs and allogeneic human hepatocellular carcinoma cells].

OBJECTIVE: To fuse human hepatocellular carcinoma (HCC) cells with mature monocyte-derived dendritic cells (FastDC) and to observe in vitro the function of the fused cells in stimulating autologous T cells proliferation and inducing HCC-specific cytotoxic T lymphocyte (CTL) response. METHODS: CD14(+) cells were isolated and purified from the peripheral blood of a healthy HLA-A2 blood donor and cultured in fresh dendritic cell complete medium for 24 h, then proinflammatory mediators were supplemented for another 24 h, thus generating mature dendritic cell (FastDCs). The FastDCs were fused with human HCC cells of the line HCCLM3 to generate novel dendritoma. T cells were isolated from selected CD14(-) cells and then divided into 4 groups to be stimulated with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells respectively for 96 hours. 18 hours before the end of cultivation (3)H-TdR was added into the culture fluid. Scintillation counter was used to measure the cpm values. CD8(+)T cells were isolated from CD14(-) cells, and added with different stimulating cells radiated by (60)Co and IL-2, IL-6, and IL-7. The values of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells was measured. HCCLM3, K562, HLE, self monocytes labeled with Na(2)(51)CrO(4) were added with effector cells, gamma-scintillation counter was used to measure the cpm value so as to calculate the killing ability of CTL. RESULTS: The CTLs activated by dendritoma cells specifically killed the HCCLM3 cells in the context of MHC class I and acted less vigorously against the control target cells. The CTLs activated by dendritoma cells were stronger in killing HCCLM3 cells than DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells (all P < 0.05). The killing activity was decreased on the HCCLM3 cells incubated with anti-HLA-ABC antibody. Three, five, and seven days after co-cultivation the value of IFN-gamma in the supernatants of the culture fluid of CD8(+)T cells with fused cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells increased gradually, especially in the supernatants of the culture fluid of CD8(+)T cells with dendritoma cells (400 pg/ml +/- 60 pg/ml 3 days after, 1030 pg/ml +/- 160 pg/ml 5 days after, and 1260 pg/L +/- 180 pg/L 7 days after). CONCLUSION: The novel dendritomas formed with HCCLM3 cells and mature FastDCs from healthy human peripheral blood CD14(+) monocytes are potent stimulators for CD8(+)T cells in inducing HCCLM3 cell-specific lysis. With shorter time required for in vitro DC development, the rapid method of generation of dendritoma is more economic and may represent a new strategy for immunotherapy of hepatocellular carcinoma.

[Regulation of hepatic stellate cell activation by interleukin-10/platelet derived growth factor/mitogen-activated protein kinase pathway].

OBJECTIVE: To investigate the regulatory effect of interleukin-10 (IL10) on the activation of hepatic stellate cells (HSC) through platelet derived growth factor (PDGF) and mitogen-activated protein kinase (MAPK) pathways. METHODS: HSC were divided randomly into 4 groups. Group 1 served as a control. HSC were incubated with 1 ng/ml, 5 ng/ml, and 25 ng/ml IL-10 in groups 2, 3 and 4. RT-PCR and western blot were used to detect the expression of PDGF and MAPK protein ERK and p38 and alpha-SMA. RESULTS: Compared with the control group, expressions of ERK, p38 and alpha-SMA of groups 2, 3 and 4 were significantly lower (F values were 240.47, 21.39, 28.86 respectively. IL-10 inhibited PDGF and MAPK protein ERK and p38 and alpha-SMA expression in a dose-dependent way. CONCLUSION: IL-10 inhibits activation of HSC through the PDGF/MAPK pathway.