Indoleamine 2,3-dioxygenase (IDO) is known as an endogenous immunosuppressive enzyme which plays a significant role in the process of tumor.IDO is not only found in tumor cells but also detected in dendritic cell (DC) in tumor microenvironment,which participates in the formation of tumor immune tolerance through expressing IDO enzyme.Signal transducer and activator of transcription (STAT) is the main signal protein family which participates in the IDO transcriptional regulation of DC.It is necessary to detail the signaling pathway in regulating IDO expression,which will help us develop high specific and more active IDO inhibitors and provide new options for anti-cancer targeted therapy.

There exists a population of myeloid-origin cells that are associated with tumor immune escape in cancer patients,commonly termed as myeloid derived suppressor cells(MDSCs).M DSCs accumulate in the blood,lymph nodes,bone marrow,and inhibit both adptive and innate immunity.Different suppressive mechanisms are used by MDSCs to block tumor immunity.Reducing the numbers of MDSCs or inhibiting the suppressive pathway conducted by MDSCs will bring new highlight to biotherapy in tumor treatment.

Objective:To test the role of tumor necrosis factor-?(TNF-?)in acute lung injury and study new treatment.Method:The rabbit acid aspiration models were induced with 0.1N hydrochloric acid (HCI). Twenty rabbits were divided randomly into HCl group,verapamil and vitamin C groups.The plasma levels of TNF-? and malondialdehyde (MDA) were measured,and polymorphonuclear neutrophils (PMN)were counted in lung tissue of all animals. Result:There were increases in TNF-?,MDA and PMN after acid aspiration,which can be inhibited by verapamil and vitamin C. Conclusion:PMN can be induced by TNF-?.Vitamin C and verapamil provide a obvious protection on pulmonary tissue after acid aspiration.

Objective:To investigate the expression of indoleamine 2,3-dioxygenase and the distribution of Treg cells in breast cancer and tumor draining lymph nodes (TDLNs) and to explore the relationship between them.Methods:26 cases of breast cancer and 10 cases of breast benign diseases were collected from Tianjin medical university cancer hospital.RT-PCR was used to detect the mRNA of IDO in breast cancer,TDLNs,benign diseases and normal breast tissues.Immunohistochemistry was used to detect the expression of IDO and Foxp3 proteins in the same tissues. Results:The mRNA and the percentage of IDO~+ cells [(19.59±7.65)%] in TDLNs were higher than in breast cancer [(13.16±7.82)%] (P<0.05),while in the breast cancer were higher than in benign diseases [(3.24±1.30)%] and normal breast tissue [(2.70±1.53)%] (P<0.05).Expression of IDO protein in breast cancer was associated with tumor clinical stage and lymph nodes metastasis while had no relationship with tumor diameter,ER,PR and Her2 status.The percentage of Foxp3~+ cells in breast cancer [(3.50±1.04)%] was higher than in benign diseases [(0.71±0.42)%] (P<0.05) and normal breast tissue [(0.55±0.34)%],and that of the TDLNs [(6.13±2.31)%] was higher than in breast cancer [(3.50±1.04)%] (P<0.05).The percentage of IDO~+ cells was positive correlated with the distribution of Treg cells in breast cancer(r~2=0.449,P<0.05)and TDLNs (r~2=0.454,P<0.05).Conclusion:Expression of IDO in breast cancer is upregulated.The high level expression of IDO is accompanied by increasing Treg cells in breast cancer and TDLNs,which suggests that breast cancer can recruit Treg cells by expressing IDO to participate the immune tolerance.

Objective:To observe ability of DC vaccine transfected with tumor cell total RNA to induce specific antitumor immunity in lung cancer patients in vitro.Methods:DCs were generated from lung cancer patients' peripheral blood mononuclear cells(PBMC).Total RNA was isolated from lung cancer tumor cell line Calu-6 by Trizol.Autologous DCs transfected with Calu-6 total RNA by liposome were used to induce specific CTL proliferation.Specific cytotoxicity and IFN-? secretion were measured by LDH assay and ELISA method.Results:Transfected DCs exhibited dramatically increased expression of specific membrane markers and function-associated molecules,and were more potent in stimulating allogenous or autologous T cell proliferation than that of control DCs.Specific CTLs induced by transfected DCs showed higher cytotoxicity than LAK against Calu-6 antigens positive target cells.When sensitized lymphocytes were restimulated by transfected DCs again,IFN-? secretion enhanced significantly.Conclusion:Lung cancer patient's autologous DCs transfected with tumor total RNA are effective vaccines in stimulating specific antitumor T-cell immunity in vitro.

10.3969/j.issn.1000-8179.2013.12.001

Objective:To study the distribution pattern of CIK cells re-infused by different manner.Methods:Isotope 32P-? dATP and fluorescence dye CM-DiI were used individually to label CIK cells. CIK cells labeled by the two methods in vitro were inoculated to nude mice by intraperitoneal injection or tail vein injection. Radioactivity quantitative measurement and fluorescence microscopy were used to analysis dynamic distribution of CIK cells among organs of mice.Results:The CIK cells were quickly distributed to organs such as liver, spleen, kidney, lung, stomach and intestine after inoculation into nude mice. Among those organs, the liver, spleen and kidney showed highest distribution concentration of CIK cells. Early stage after infusion, concentration of CIK cells in lung above all reached peak via tail vein, and by means of intraperitoneal injection, distribution of CIK cells in intraperitoneal organs firstly got to max. CIK cells remained alive in liver and spleen for more than 2 weeks.Conclusion:The extensive distribution pattern of CIK cells among organs shows that CIK cells can be used as drugs against various malignant tumors in organism. Infusion of CIK cells via blood vessel maybe suit for tumor of organs with rich blood supply, and application by means of body-cavity way should suit for malignant effusions and limited lesion in it.

Objective:This work aims determine the expression of the neurotensin (NTS) gene in hepatocellular carcinoma (HCC) subgrouping using immunohistochemical staining (IHC) as well as to evaluate the correlation between the activation of NTS/IL-8 pathway in HCC and inflammatory response in microenvironment and epithelial mesenchymal transition (EMT) in cancer and in the prognosis of patients. Methods:Tumor tissues and corresponding adjacent normal tissue were collected from 64 cases of HCC patients. The expression levels of NTS protein and multiple inflammation and EMT-related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin,β-Catenin, and Vimentin, were examined in 64 cases of paraffin-embedded HCC tissues using the immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) among 64 cases of HCC patients were compared. Results:We found that the frequency of NTS-expressing tissues among all HCC samples was 17.19%(11/64). Significantly increased IL-8 protein was confirmed in 90.91%of NTS+HCC samples and was positively correlated with the levels of NTS protein in cancer tissues (P=0.036), which implied the dysfunctional activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 were significantly correlated with the co-expression of NTS and IL-8 in HCC. Evident features of EMT, including decreased membrane expression of E-Cadherin and increased accumulation of cytoplasmicβ-Catemin and Vimentin, were found in NTS+IL-8+samples. The co-expression of NTS and IL-8 in cancer was significantly correlated with the clinical outcomes of patients, as the mortality rate of NTS+IL-8+HCC patients is 2.5-fold higher than that of others after surgery (P=0.022).Accordingly, the OS of NTS+IL-8+HCC patients significantly decreased (24.65±4.45 m vs. 75.79±16.32 m, P=0.013), and these patients are at a higher risk of death at an expected hazard ratio (HR) of 3.457. Conclusion:The NTS/IL-8 pathway is dysfunctionally activated in a subgroup of HCC samples. Highly expressed NTS is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.

Objective: To separate and identify the exosomes derived from MCF-7 tumor cells,and observe their function on IL-2 induced lymphocyte proliferation.Methods:The exosomes were separated by serial centrifugation,concentration and density gradient centrifugation, and then identified by transmission electron microscope.The effects of exosomes on IL-2 induced proliferation were studied by MTT method.Results:Total 20 mL exosomes from 2×10~7 MCF-7 cells with concentration of protein were(1.84±0.01) g/L. The diameters of these purified bi-layer membrane exosomes were 55-70 nm, which were consistent with the internal vesicles in multi-vesicular endosomes.The result of MTT test showed that proliferation of donor peripheral blood lymphocytes was inhibited by tumor exosomes at a dose-dependent manner in response to IL-2.Conclusion:It is feasible to separate high-purity exosomes from tumor cell culture medium supernatant by serial centrifugation and concentrate method. These tumor derived exosomes have inhibitory effect on IL-2 induced proliferation.

Objective To explore whether antisense-EGFR could enhance the radiosonsitivity of human lung cancer spc-a-1 cell line.Methods The spc-a-1 cells were transfected with antisenso.EGFR-pcDNA3 by lipofectamine 2000(pcDNA3 antiEGFR group).Two other groups were used for comparison:control group(spc-a-1 cell without transfection)and pcDNA3 group(spc-a-1 cell transfeeted with pcDNA3 which did not contain antisense EGFR).Cell clones that stable expressing antisense-EGFR wa8 selected with G41 8 and the suppression of the expression of EGFR mRNA and protein were detected by RT-PCR and Western blot.The influence of antisense-EGFR on cell cycle was testified by flow cytometry assay.The cell apoptosis was analyzed by flow cytometry after 8 Gy irradiation.Further,cells of each group were irradiated with X-rays at the dose of 0,2,4,6 and 8 Gy.Dose-survival curve of each group was established by colony-forming assay.Results The expression of EGFR mRNA and protein were significantly inhibited after antisense-EGFR-pcDNA3 transfection.The cells arrested at the G2/M phase in the pcDNA3 antiEGFR group,control group and pcDNA3 group were (29.53±1.91)%,(13.7±1.30)%and(12.40±1.34)%,respectively.The apoptosis index of spc-a-1 cells in the antisonse-EGFR combined with irradiation group was obviously higher than that of the comparable groups [(39.24±1.57)%,(13.79±0.63)%and(15.02±0.85%)].The values of D0,Dq,SF2 of pcDNA3 antiEGFR group declined obviously compared with the control group(2.11,2.49,0.84 vs 1.19,0.15,0.32).Conclusions Antisense-EGFR could induce the G2/M cell cycle arrest,promote cell apoptosis and inhibit the ability of sublethal cell damage repair induced by irradiation,80 that it could significantly improve the radiosensitivity of spc-a-1 cell in vitro.