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.

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.

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.

10.3969/j.issn.1000-8179.2013.12.001

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.

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: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 study tumor immune escape in a gastric carcinoma cell line expressing human indoleamine 2,3-dioxygenase(IDO).Methods Human IDO gene was cloned by RT-PCR and the vector for pIRES_2-EGFP-IDO was constructed.BGC-823 cells were transfected with the plasmid using eleetroporation.The integrated INDO genes were detected by RT-PCR and Western blot.The enzyme activity of IDO were measured.T cells from gastric cancer patients were cecuhured with BGC-823 transfected with IDO or added with 1-MT circumstance,T cell-mediated cytotoxicity and proliferation were detected.Results Higher level expression of IDO mRNA and IDO protein Was detected in tumor cells transfected with IDO gene.The level of kynurenic acid was higher in transfected cells compared with no-transfeeted group (4.84±0.11)mg/L vs.(1.83±0.10)mg/L,P=0.000.The cytotoxicity ratio of the IDO transfected group and transfected group with 1-MT circumstance (1-MT group) was lower than control group (P<0.05).The inhibition rate of transfected group with 1-MT group Was higher than control group(P<0.05).Conclusion Gastric cancer cell lines encoded with IDO inhibits T cell-mediated cytotoxicity and proliferation.

Objective To study the effect of feto-matemal microchimerism in the treatment of activated human leukocyte antigen (HLA) haploidentical mobilized peripheral blood cells against solid tumors. Methods Genomic DNA samples of 25 pairs of HLA haploidentical donors and recipients were extracted. The donor-derived HLA-DRB loci were detected with nested PCR-sequence specific primer(SSP) typing. The mixed lymphocyte proliferation action between the patients and respective donors, the engraftment of donor's cells and the serum levels of Th1/Th2 type of cytokines were measured with MTT,FISH and EIJSA method respectively. The survival time of patients with or without feto-matemal microchimerism were compared as well. Results Using nested PCR-SSP typing, the positive rates of feto-maternal microchimerism in the 25 pairs of HLA haploidentical donors and recipients were 40% in the maternal/children pairs and 0 in the paternal/children pairs. The chimerism positive patients showed less proliferation activity when cocultured with respective donors as compared with unrelated ones (P=0.03).Only one chimerism positive patient experienced the engraft of donor's cell 3 months after treatment as the donor derived XX chromosome was identified with FISH. When the data of chimerism positive patients were deleted, the serum levels of IFNγ 1 month after treatment dropped dramatically from 171.4 (26. 3～258.4) ng/L to 29. 4(1.2～39.9)ng/L. The survival time in chimerism positive patients of the maternal/children pairs was significantly longer than that in chimerism negative patients, which was (31.2±4. 3) months and (11.1±3.3) months, respectively (P=0.036). Conclusion Feto-maternal microchimerism might induce anergy in the HLA haploidentical donors, favor the engraftment of donor's progenitors and maintenance of positive microenvironment and prolong the survival time.

Objective: To evaluate the anti-tumor and side effects of activated-HLA haploidentical peripheral blood tem cells (haplo PBSCs) in the treatment of advanced refractory solid tumor patients. Methods: Forty-two patients with advanced refractory tumor, who were diagnosed in our hospital from Oct. 2004 to Oct. 2007, were enrolled in this study (all patients signed informed consent), including 12 with ovarian cancer, 9 with renal cancer, 8 with lung cancer, 8 with breast cancer, 2 with colon cancer, 2 with gastric cancer, and 1 with melanoma. The donors were healthy direct relatives of the patients; the donors' haplo-PBSCs were mobilized, collected, and activated by rhIL-2 in vitro. The clinical efficacy and side effects of haplo-PBSCs therapy were assessed by CT/PET-CT scanning, RESIST standard, KPS score, and clinical response rates, etc. Results: All 42 patients received one episode of haplo-PBSCs treatment. The progression-free survivals (PFS) were 6 months and the clinical beneficial rate (CR+PR+SD) was 73.8%. The beneficial rate of life quality was 76.2% and the KPS increased by 20 (0-30) points on average after haplo-PBSCs treatment. The patients with KIR unmatched in GVH direction had better outcomes than those with KIR matched or KIR unmatched in HVG direction (P<0.05), and the clinical beneficial rate, PFS and total beneficial rate were 94.1% vs 60.0%, (13.4±1.3) vs (8.0±0.9) months, and 89.5% vs 65.2%, respectively (all P<0.05). The donor/recipient relation as the mother/child had a better outcome than that as the father/child (P<0.05). Patients with renal cancer or ovarian cancer had better outcomes than those with other cancers, with clinical beneficial rates being 90.0% and 81.8%, respectively. Conclusion: Activated haplo-PBSCs therapy can induce non-specific anti-tumor effect, and improve the clinical symptom and life quality of advanced tumor patients.