Objective:To investigate the influence of sCD80-Linker-sCD40L fusion protein on the unspecific anti- tumor immunity in vitro.Methods:Ovarian cancer SKOV3 cells were separately transfected with recombinant adenoviral vectors containing sCD80-Linker-sCD40L fusion gene,sCD80 gene,sCD40L gene or with control adenovirus.The expres- sion of the sCD80-Linker-sCD40L fusion protein,sCD80 protein and sCD40L protein in the supernatants of SKOV3 cells was determined by ELISA.Dendritic cells(DCs)were cultured with peripheral blood mononuclear cells from a patient with ovarian carcinoma.DCs and autologous T cells were co-cuhured and were exposed to different supernatants for 48 h. The allostimulatory effects of DCs on T cells were determined by mixed lymphocyte reaction(MLR).The unspecific kill- ing activities of induced T cells against SKOV3/K562 cells were measured by LDH-releasing assay.Results:ELISA assay showed that levels of the sCD80-Linker-sCD40L fusion protein,sCD80 protein and sCD40L protein in the supernatants of transfeced SKOV3 cells were 2.791 ng/ml,1.956 ng/ml and 1.407 ng/ml,respectively.The fusion protein-exposed DCs ([0.382?0.053]vs[0.167?0.028],P

OBJECTIVETo explore the specific cytotoxic T lymphocyte (CTL) induced by dendritic cells (DC), which were transfected by the plasmid pC53-SN3 encoding p53 gene.

METHODSDC derived from HLA-A2(+) mononuclear cells of the 24-lung cancer patients was transfected with the plasmid pC53-SN3 by lipofectamine and then co-cultured with auto-unpurified T cells to induce potent CTL (T-pC53-SN3). The cytolysis of specific CTL against Calu-6, a HLA-A2(+) human lung cancer cell line, was measured by using lactate dehydrogenase (LDH) releasing assay.

RESULTSThe expression of CD(1a) and CD(83), the correlative markers of DC, increased apparently after transfected with plasmid pC53-SN3, the expression rate was (5.45 +/- 0.89)% and (3.26 +/- 0.47)% versus (52.15 +/- 11.56)% and (25.78 +/- 12.35)%. CD(14) decreased apparently, but other DC correlative markers of CD(1a), CD(40), CD(86), and HLA-DR remained almost the same as that before transfection. Compared with T-IL-2, the CTL derived from PBMNC stimulated by IL-2 (100 U/ml), the cytolytic activity of T-pC53-SN3 against Calu-6 cell line showed a significant increase, but cytolytic activity was 56.79 +/- 15.67 and 39.33 +/- 9.88, respectively, when effect cells: target cells was 10:1. The expression of the CD(8), CD(69), and CD(45)RO/CD(8) of T-pC53-SN3 cells increased significantly, but that of CD(3), CD(4), CD(86), ect, was not significantly different from those of T-pCMV-neo.

CONCLUSIONSIt showed that DC transfected by p53 gene could induce potent HLA-A(2) restrictive CTL to kill tumor cell efficiently.

Antigens, CD ; analysis ; B7-2 Antigen ; CD40 Antigens ; analysis ; Cell Line, Tumor ; immunology ; Coculture Techniques ; Cytotoxicity, Immunologic ; immunology ; Dendritic Cells ; drug effects ; immunology ; metabolism ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Membrane Glycoproteins ; analysis ; T-Lymphocytes ; immunology ; Tumor Suppressor Protein p53 ; genetics ; physiology

In order to find a method suitable for purifying large amount of CD34(+) cells, from 5 cases who accepted autologous peripheral blood stem cell transplantation, CD34(+) cells were collected and enriched by using Isolex 300i (Nexell). Phenotypes were detected by flow cytometry and the biological viability were assayed by the colony-forming experiments and cell expansion experiment in vitro. The results showed that the number of mononuclear cells first collected was about (3.5 - 6.0) x 10(10) and (0.55 - 1.2)% of cells were CD34 positive. The number of positive production was about (2.0 - 3.0) x 10(8); the CD34(+) cells purity was (75 - 85)% and the yield was (40 - 65)%. The CD34(+) cells of positive production could expand up to 2 - 3 times when cultured with SCF + IL3 + FL + TPO + EPO in vitro. The results of colony-forming experiments demonstrated that the CD34(+) cells collected has enough colony-forming ability. All results showed the enriched CD34(+) cells with biological viability. In conclusion, the CD34(+) immunomagnetic isolation apparatus Isolex300i is suitable to clinical application for a large amount of CD34(+) cell enrichment.
Antigens, CD34 ; immunology ; Colony-Forming Units Assay ; Flow Cytometry ; Hematopoietic Stem Cells ; cytology ; immunology ; Humans ; Immunomagnetic Separation ; instrumentation ; methods ; standards ; Neoplasms ; blood

OBJECTIVETo evaluate the distribution of CD4+ CD25+ regulatory T-cells (T-regs) in tumor-draining lymph nodes (TDLN) in patients with non-small cell lung caner (NSCLC), and to investigate the effect of CD4+ CD25+ T regulatory cells on the immune status of TDLN and the progression of NSCLC.

METHODSRegional tumor-draining lymph nodes of 53 NSCLC patients were resected during the operation. The percentage of CD4+ CD25+ T-regs as a subset of CD4+ T cells and CD8+ T cells were detected by immunofluorescence and regular immunohistochemistry, respectively. The level of cytokines TGF-beta1 and IL-10 was detected by real time quantitative RT-PCR.

RESULTSCD4+ CD25+ T-regs in tumor-infiltrating lymph nodes from the patients with NSCLC accounted for 28.80% +/- 8.06% of total CD4+ T cells, and were significantly increased comparing with that (15.48% +/- 4.66%) in the tumor-free lymph nodes (P < 0.01). The percentage of CD4+ CD25+ T-regs in TDLN of NSCLC patients was negatively correlated with the amount of CD8+ T cells within the lymph nodes (r = -0. 756, P < 0.001), but positively correlated with the level of TGF-beta1 (r = 0.645, P < 0.001) and IL-10 (r = 0.769, P < 0.001). It also increased as NSCLC getting progressed, which was 30.42% +/- 7.47% in stage III versus 16.22% +/- 4.88% in stage I and III; 32.58% +/- 7.52% in N2 versus 22.76% +/- 4.67% in N1, with a significant difference between the two groups, respectively (P < 0.01).

CONCLUSIONThe population of CD4+ CD25+ T regulatory cells in tumor-draining lymph nodes in patients with non-small cell lung caner is positively correlated with the progression and infiltration of lung cancer, which might provide new immunologic method to evaluate the progression and prognosis of non-small cell lung caner. The outcomes of biotherapy for NSCLC may be improved in the future through regulating the CD4+ CD25+ T regulatory cells.

Aged ; CD4 Lymphocyte Count ; CD8-Positive T-Lymphocytes ; pathology ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Female ; Humans ; Interleukin-10 ; metabolism ; Lung Neoplasms ; metabolism ; pathology ; Lymph Nodes ; immunology ; metabolism ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Staging ; T-Lymphocytes, Regulatory ; pathology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo evaluate the changes of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer.

METHODSSixty four patients with breast cancer, 15 patients with benign breast tumors and 9 healthy volunteers were included in this study. The proportion of CD(4)(+)CD(25)(+) T cells population in total T cells was evaluated by flow cytometric analysis. The cytokine production (TGF-beta1) was measured by ELISA.

RESULTSThe population of CD(4)(+)CD(25)(+) T cells in peripheral blood from patients with breast cancer accounted for (5.1 +/- 2.9)% of the total amount of T lymphocytes, and was significantly higher in comparison with that in patients with benign tumors and in healthy volunteers (P < 0.05). The CD(4)(+)CD(25)(+) T cells population in breast cancer patients was positively correlated with the cancer size and with TGF-beta1 level (r = 0.511 and r = 0.253, respectively), and negatively correlated with CD(8)(+)CD(28)(+) T cells and NK cells (r = -0.243 and r = -0.301, respectively).

CONCLUSIONThe CD(4)(+)CD(25)(+) regulatory T cells in peripheral blood of patients with breast cancer is significantly increased in comparison with that in patients with benign breast tumor and in healthy subjects. It may be responsible for immune suppression in breast cancer patients.

Adult ; Aged ; Breast Neoplasms ; immunology ; CD4-Positive T-Lymphocytes ; immunology ; Cell Count ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Interleukin-2 Receptor alpha Subunit ; immunology ; Middle Aged ; T-Lymphocyte Subsets ; immunology ; Transforming Growth Factor beta ; biosynthesis ; genetics

OBJECTIVETo investigate the influence of mutual interactions between FasL expressed by colon carcinoma cells and endogenous cytokines interleukin-18 on liver metastasis and invasion of human colon cancer cells.

METHODSUsing immunohistochemical streptavidin-biotin complex (SABC) method, the expressions of Fas receptor and Fas ligand in SW620 colon carcinoma cells and Chang liver cells were observed so as to provide morphological evidence for the functions of Fas receptor and Fas ligand. In an effort to examine the cytotoxicity of effector cells, CytoTox(96) Non-Radioactive Cytotoxicity Assay was adopted to measure the LDH releasing value after the SW620 cells were co-cultured with Chang liver cells.

RESULTSIt was shown that the Fas ligand of colon carcinoma SW620 cells was positive and the positive substances were distributed in the cell membrane and cytoplasm, and the Fas receptor of colon carcinoma SW620 cells was negative. The Fas receptor of Chang liver cells turned out to be positive and the positive substances were distributed in the cell membrane, and the Fas ligand of Chang liver cells was negative. At 6 hours after co-culture of IFN-γ-stimulated Chang liver cells with interleukin-18-stimulated (for 36 h) SW620 cells or unstimulated SW620 cells, the cytotoxicity of SW620 cells to IFN-stimulated Chang liver cells at effector-to-target ratios of 10:1, 5:1, 2.5:1 and 1.25:1 was 68.3%, 49.8%, 21.1%, 9.7% (F = 76.87, P < 0.05) and 32.7%, 21.8%, 11.1%, 6.7% (F = 7.27, P < 0.05), respectively. The non-radioactive cytotoxicity assay showed that the apoptotic rate of Chang liver cells was remarkably increased with the increase of planting concentration of SW620 after the SW620 cells were co-cultured with Chang liver cells. The cytotoxicity was significantly enhanced by interleukin-18.

CONCLUSIONThe FasL expression of human colon cancer cells may be regulated by endogenous interleukin-18 in the host microenvironment and enhance the liver colonization competence of colon cancer cells through induction of apoptosis in the Fas-expressing hepatocytes.

Apoptosis ; Cell Line, Tumor ; Cells, Cultured ; Coculture Techniques ; Colonic Neoplasms ; immunology ; metabolism ; pathology ; Cytotoxicity, Immunologic ; Fas Ligand Protein ; metabolism ; Hepatocytes ; cytology ; metabolism ; Humans ; Interferon-gamma ; pharmacology ; Interleukin-18 ; pharmacology ; L-Lactate Dehydrogenase ; metabolism ; fas Receptor ; metabolism

OBJECTIVETo investigate the anti-tumor effects and mechanism of tumor vaccines and whether chemotherapeutic agents administered prior to immunotherapy could augment the efficacy of the vaccines.

METHODSC57/BL mice inoculated with Lewis lung cancer cells were used as tumor models. Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene modified LA795 and Lewis lung cancer cell lines were administered as allogeneic and autologous tumor vaccines, respectively. After Lewis cells (1 x 10(7)) inoculation, the mice received irradiated GM-CSF secreting cancer vaccine solely or in combination with carboplatin. The survival of the mice was observed. The cytotoxicity of spleen cells or purified CD8(+) cells was analyzed by lactate dehydrogenase (LDH) assay. Serum level of IL-4 and IFN-gamma was detected using ELISA method.

RESULTSThe cytotoxicity of the spleen cells or purified CD8(+) T cells against Lewis cells in the mice immunized with cancer cell vaccine was significantly increased, relative to that of the control, untreated group (P < 0.05). Serum level of Th1-type cytokine IFN-gamma was increased after vaccination, whereas Th2-type cytokine IL-4 showed no significant change. The GM-CSF secreting cancer cell vaccine had no significant influence on the survival of the mice with established heavy tumor burden. The combination of chemotherapy and cancer vaccine could statistically prolong the survival time; whereas any method itself had no significant effect.

CONCLUSIONThe GM-CSF secreting cancer cell vaccine can induce immune responses. The chemotherapeutic agents may be beneficial to enhance the anti-tumor activity of cancer vaccine.

Animals ; Antineoplastic Agents ; therapeutic use ; Cancer Vaccines ; therapeutic use ; Carboplatin ; therapeutic use ; Carcinoma, Lewis Lung ; blood ; metabolism ; pathology ; therapy ; Cell Line, Tumor ; Female ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; metabolism ; Interferon-gamma ; blood ; Interleukin-4 ; blood ; Lung Neoplasms ; metabolism ; pathology ; Mice ; Mice, Inbred C57BL ; Transfection

This study was aimed to study the potential effects of alloreactive NK cells (allo-NKs) in therapy of relapsed lung cancer after haploidentical hematopoietic stem cell transplantation using donor lymphocyte infusion (DLI). The F1 donors derived-NK cells were purified with MACS magnetic separation system, in which the proportion of the alloreactive Ly49A(+) cells was detected by flowcytometry and alloreactivity was measured by LDH method. The relapse model of lung cancer after haploidentical-HSCT was established. The distribution kinetic of infused donor lymphocytes in vivo was analyzed. The inhibition of relapse tumor, infiltration of lymphocytes in situ and fluctuation of 22 kinds of cytokines in serum after DLI were compared among different groups. The results showed that the infused donor cells of allo-NK group were accumulated mostly in lung, spleen and kidney for more than 48 hours with considerable higher levels according to the distribution kinetic curve. The sizes of relapse tumors between chemotherapy + PBS group and chemotherapy + DLI group showed no difference. However, the relapsed tumors in allo-NK + DLI group were significantly smaller than that in chemotherapy + DLI group or allo-NK + PBS group, in which increased infiltration of lymphocytes were defined in situ. The levels of cytokines such as MCP-1, IL-17, IL-12 and MCP-5 in serum of allo-NK + DLI group ascended compared with control group, though the level of IL-10 declined simultaneously. It is concluded that allo-NKs prolong the survival time of infused donor lymphocytes in vivo, promote the secretion of inflammatory cytokines and Th1-type of cytokines, and further improve the antitumor effects of DLI against relapse after transplantation.
Animals ; Cytokines ; blood ; Hematopoietic Stem Cell Transplantation ; methods ; Killer Cells, Natural ; cytology ; Lung Neoplasms ; therapy ; Lymphocyte Transfusion ; methods ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Neoplasm Recurrence, Local ; therapy ; Transplantation Conditioning ; methods

Two strategies, direct ligation after enzyme digestion and over-lap PCR technology, were adopted to construct a fusion gene which was composed of the antimelanoma single chain antibody gene and the staphylococcal enterotoxin A gene without N-terminal signal sequence. The fusion gene was subcloned into pET28-a vector and transformed into E. coli BL21(DE3). Ni-NTA system was selected to separate and purify the expresstd products. The inhibition ratio of the fusion protein was tested by MTT method. It is shown that the 6His-ScFv-SEA fusion protein can be expressed stably in E. coli BL21 (DE3). The quantity of the fusion protein was shown up to 30% of the total protein of the bacteria and mainly in inclusion body. By activation the effective cells, the fution protein can inhibit the melanoma cell whith expressed corresponding antigen.
Cell Line, Tumor ; Cell Survival ; drug effects ; Cells, Cultured ; Electrophoresis, Polyacrylamide Gel ; Enterotoxins ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Humans ; Inclusion Bodies ; genetics ; metabolism ; Melanoma ; drug therapy ; immunology ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology ; therapeutic use ; Single-Chain Antibodies ; genetics ; metabolism

OBJECTIVETo observe the supplementary analgesic effect of electroacupuncture and its influence on the maintenance of anesthesia and the speed of recovery of patients undergoing craniotomy.

METHODSEighty cases of supratentorial tumor resection were randomly divided into group A and group S, 40 cases in each group. All the patients were anesthetized with 2% Sevoflurane. The patients in group A received electroacupuncture at Hegu (LI 4) and Waiguan (TE 5), Jinmen (BL 63) and Taichong (LR 3), Zusanli (ST 36) and Qiuxu (GB 40) from anesthesia beginning to the end of operation, and in group S without electroacupuncture. The end-tidal Sevoflurane concentration, minimum alveolar concentration (MAC), bispectral index (BIS) and the information during anesthesia recovery stage were recorded, respectively.

RESULTSThe end-tidal concentration and MAC of Sevoflurane in group A at all times were significant lower than those in group S (P<0.05, P<0.01) with a Sevoflurane saving of 9.62% on average. The BIS in group A during a few phases were higher than that in group S (all P<0.05). During anesthesia recovery stage, the time of each phase in group A was significantly shorter than that in group S (all P<0.01). No dysphoria and one case with nausea and vomiting were shown in group A, but in group S, 2 patients had dysphoria and 3 patients had nausea and vomiting.

CONCLUSIONElectroacupuncture combined with Sevoflurane anesthesia can decrease the dosage of Sevoflurane, shorten the recovery time of anesthesia and improve the quality of anesthesia recovery of the patients undergoing resection of supratentorial tumor.

Acupuncture Analgesia ; Adolescent ; Adult ; Anesthesia Recovery Period ; Electroacupuncture ; Female ; Humans ; Male ; Methyl Ethers ; administration & dosage ; adverse effects ; Middle Aged ; Supratentorial Neoplasms ; drug therapy ; surgery ; therapy ; Young Adult