The cytotoxicity of medical ultrasonic couplant was tested by MTT assay and agar overlay test. By MTT assay, when the inoculum density was high, the cytotoxicity level was low, or vice versa. The cytotoxicity grade tested by agar overlay was not accord to MTT assay's too. MTT assay is suitable to test the cytotoxicity of medical ultrasonic couplant because it is quantitative and more sensitive, however, the experimental condition and the preparative method of extraction should be determined.
Animals ; Cell Line ; Colorimetry ; Cytotoxicity Tests, Immunologic ; methods ; Mice ; Ultrasonics

OBJECTIVETo establish a cell-based detection method of ciguatoxin using fluorescence assay.

METHODSMouse neuroblastoma N-2A cells were exposed to ouabain and veratridine and different concentrations of standard ciguatoxin samples (P-CTX-1) to establish the curvilinear relationship between the toxin dosage and fluorescence intensity using the sodium fluorescence probe CoroNaTM Green. The toxicity curvilinear relationship was also generated between the toxin dosage and cell survival using CCK-8 method. Based on these standard curves, the presence of ciguatoxin was detected in 33 samples of deep-sea coral fish.

RESULTSA correlation was found between the detection results of cell-based fluorescence assay and cytotoxicity assay, whose detection limit reached 103 g/ml and 1012 g/ml, respectively. The cell-based fluorescent assay sensitivity showed a higher sensitivity than cytotoxicity assay with a 2-4 h reduction of the detection time.

CONCLUSIONSThe cell-based fluorescent assay can quickly and sensitively detect ciguatoxin and may serve as a good option for preliminary screening of the toxin.

Animals ; Cell Line, Tumor ; Ciguatoxins ; toxicity ; Cytotoxicity Tests, Immunologic ; methods ; Fishes ; Fluorescent Dyes ; Mice ; Sodium

The natural killer(NK) cell activity of mononuclear cells (MNC) from peripheral blood (PB) and synovial fluid (SF) of 40 rheumatoid arthritis(RA) patients was investigated by employing 51-chromium-(51Cr) release microcytotoxicity and single cell cytotoxicity assays against K562 target cells. It has been revealed that SF-MNC from RA patients showed a significantly lower NK activity than PB-MNC from the same patients and this might be due to an impaired target binding capacity of the effector cells and not due to a deficiency of active NK cells.
Adolescent ; Adult ; Aged ; Arthritis, Rheumatoid/*immunology ; Chromium Radioisotopes/diagnostic use ; Comparative Study ; Cytotoxicity Tests, Immunologic/methods ; *Cytotoxicity, Immunologic ; Female ; Human ; In Vitro ; Killer Cells, Natural/*immunology ; Male ; Middle Age ; Support, Non-U.S. Gov't ; Synovial Fluid/immunology

OBJECTIVETo explore whether coculture of dendritic cells (DC) with cytokine-induced killer (CIK) lead to an increase of cytotoxicity against tumor cells in vitro and in vivo.

METHODSDC and CIK were prepared from human peripheral blood mononuclear cells (PBMC) by conventional methods, the DC pulsed with or without NB4 leukemia cell lyses (LCL) was cocultured with the CIK (LCL-DC + CIK and DC + CIK), CIK was used as control. Cells phenotypes were analyzed by flow cytometry, secretion of IFN-gamma was determined by ELISPOT assay, and cytotoxicity was assayed in vitro with (51)Cr-release assay. A human leukemia cell NB4-bearing nude mice model was established to test in vivo antitumor efficacy and cell homing.

RESULTSCompared with CIK, LCL-DC + CIK got a significant increasing of proliferation rate [(18.2 +/- 2.1) times vs (11.6 +/- 2.3) times, P < 0.05] and CD(3)(+)CD(56)(+) expression rate [(51.05 +/- 2.63)% vs (30.18 +/- 1.45)%, P < 0.05], and the number of IFN-gamma secreting cells was increased significantly [(13.86 +/- 3.28)/10(4) cells vs (8.74 +/- 2.53)/10(4) cells, n = 12, P < 0.05]. Meanwhile, LCL-DC + CIK led to an increase of cytotoxic activity to NB4, K562, and KG1a cells, and showed significant inhibition of the growth of transplanted tumor cells and increased tumor free survival rate of nude mice (100% vs 66.7%, P < 0.05), DiI labeled LCL-DC + CIK were detected in spleen, lymph node and tumor within a week after injection. There was no significant different in antitumor activity between LCL-DC + CIK cell and DC + CIK cell.

CONCLUSIONCoculture of CIK with DCs can promote the effect of CIK against tumor in vitro and in vivo. DC-CIK is promising as an immuno-therapeutic strategy for patients with leukemia.

Animals ; Cell Line, Tumor ; Coculture Techniques ; Cytokines ; pharmacology ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; drug effects ; immunology ; Dendritic Cells ; cytology ; immunology ; Female ; Humans ; Immunization, Passive ; methods ; K562 Cells ; Killer Cells, Natural ; cytology ; drug effects ; immunology ; Leukemia, Experimental ; immunology ; therapy ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Xenograft Model Antitumor Assays

The clinical significance of positive B-cell complement-dependent cytotoxicity crossmatching (B-CDC) in renal transplant recipients remains unclear. We reviewed 20 recipients with isolated B-CDC positivity at the time of transplantation. We compared the clinical characteristics, acute rejection and long-term graft survival between positive and negative B-CDC patients (n = 602). The number of retransplant recipients and positivity for T- and B-flowcytometric crossmatch was greater in positive B-CDC patients than in negative B-CDC patients. The overall acute rejection rate of positive B-CDC patients was significantly higher (P < 0.001), and Banff grade II or III cellular rejection was more frequently observed in positive B-CDC patients (P = 0.037). Compared with negative B-CDC patients, acute cellular rejection as a cause of graft loss was more prevalent (P = 0.020) and rescue rejection therapy was more frequently needed in positive B-CDC patients (P = 0.007). The allograft survival rate of positive B-CDC patients was significantly lower than that of negative B-CDC patients (P < 0.001), and B-CDC positivity independently increased the risk of allograft failure 2.31-fold (95% CI 1.15-4.67; P = 0.019) according to multivariate analysis. In conclusion, isolated B-CDC positivity is an independent long-term prognostic factor for allograft survival.
Acute Disease ; Adult ; Aged ; B-Lymphocytes/*immunology ; Complement Activation ; Cytotoxicity Tests, Immunologic ; Female ; Graft Survival/*immunology ; Histocompatibility Testing/*methods ; Humans ; *Kidney Transplantation/immunology ; Male ; Middle Aged ; Prognosis ; Retrospective Studies ; Risk Factors ; Survival Analysis ; T-Lymphocytes/immunology ; Transplantation, Homologous

TNF-related apoptosis-inducing ligand (TRAIL) is a member of factor TNF family, which could be potentially developed as novel antitumor agent due to its selective and efficient induction of apoptosis in tumor cells. Gene recombinant expression is an important tool for production of pharmaceutical protein. In this paper, the gene encoding human soluble TRAIL (114-281aa fragment) was cloned by PCR and then inserted into the Pichia Pastoris expression vector pPIC9K. The transformants were double-screened on plates containing neomycin G418 and many clones with high levels of G418-resistance were selected for further studies on protein expression. The recombinant human soluble TRAIL was secreted into the BMMY media under the condition of 3% methanol. And the recombinant protein was purified to homogeneity (-80% purity) by using Ni-agarose affinity chromatography. The yield of this protein is about 1-2 mg per liter culture. Cell viability assays demonstrated that human soluble TRAIL was cytotoxic in both leukemia cells Jurkat and lung cancer cells A549. After treatment with 0.05 microg/ml TRAIL, the survival rate of Jurkat cells was about 10%. The expressed TRAIL showed dose-dependent cytotoxicity in A549 cells within the range of 0.1-1 microg/ml. When the protein concentration reached 1 microg/ml, the survival rates of A549 cells were about 30%. However, the recombinant human soluble TRAIL did not show obvious cytotoxicity in human skin fibroblast cells (HSF) at concentrations tested. There results demonstrate that human soluble TRAIL is selectively cytotoxic in tumor cells. The expression system constructed in this experiment might contribute to further production of soluble TRAIL and TRAIL-based novel fusion proteins in large quantities.
Antineoplastic Agents ; pharmacology ; Cell Line, Tumor ; Cloning, Molecular ; Cytotoxicity Tests, Immunologic ; methods ; Genetic Vectors ; genetics ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; biosynthesis ; genetics

OBJECTIVETo construct a recombined phage vaccine and to evaluate the efficiency of this phage vaccine against EGFR-positive tumors.

METHODST7 phage display system was used to display five fragments of the extracellular domain of chicken EGFR. The EGFR was expressed as a fused protein on the surface of the T7 phage 10B capsid protein. The EGFR expression of the phage vaccine was verified by Western-blot analysis. Anti-EGFR antibody was detected by ELISA. Splenic lymphocytes of the immunized mice were separated and used to determine the immunotoxic effect against A431 cells. The phage vaccines were injected into C57 mice 4 times before Lewis lung cancer cells inoculation. Tumor volume was recorded to evaluate the anti-tumor effect of each vaccine.

RESULTSFive phage vaccines inserted with the chicken EGFR gene were successfully constructed. Western blot assay showed that the extracellular domain of chicken EGFR proteins were displayed on the surface of the phage. Specific antibody was induced in the immunized mice, compared with the control group. Splenic lymphocytes of the immunized mice were shown to be immunotoxic against A431 cells. The killing rates of the experimental groups were higher than that of control group (P < 0.001, t-Student test). The highest killing rate was (45.74 +/- 7.21)%. The tumor growth was inhibited in the experimental groups compared with those of control groups (P < 0.05 in C1, C2, C3, C4 groups, P > 0.05 in C5 group).

CONCLUSIONOur results demonstrated that recombined EGFR phage vaccines may be used to induce therapeutic anti-tumor immunity against EGFR-positive tumors.

Animals ; Bacteriophage T7 ; genetics ; Blotting, Western ; Cancer Vaccines ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; genetics ; metabolism ; Carcinoma, Lewis Lung ; immunology ; pathology ; therapy ; Cell Line, Tumor ; Chickens ; Cytotoxicity Tests, Immunologic ; Immunotherapy ; methods ; Male ; Mice ; Mice, Inbred C57BL ; Neoplasm Transplantation ; Random Allocation ; Receptor, Epidermal Growth Factor ; genetics ; immunology ; metabolism ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo investigate the enhanced effect of bone marrow-derived dendritic cells (DC) pulsed with SVYDFFVWL, a MHC class I peptide located in 180-188 amino acid residues of human melanoma-associated antigen tyrosinase- related protein 2 ( hTRP2) on the immunity against melanomas elicited by adenovirus encoding hTRP2 (Ad hTRP2).

METHODSThe mice were intradermally immunized with Ad hTRP2, and three weeks later with Ad hTRP2 or DC/SVYDFFVWL once more. Analysis of CTL killing activity and IFN-gamma-producing CD8 + T cells in the total CD8 + T cells of spleen were made using in vivo CTL and intracellular staining of IFN-gamma, respectively. Additionally, the survival of mice was checked after the subcutaneous inoculation with mouse melanoma B16. F10 cells.

RESULTSThe 6 h CTL killing and IFN-gamma producing CD8 +T cells in the total CD8 ' T cells of spleens were 68. 40%+/-5. 50% and 0. 67%+/-0.16% in Ad hTRP2 (priming)-Ad hTRP2 (boosting) group,28. 50%+/-6.40% and 0.22%+/-0.07% in DC/SVYDFFVWL (priming)-DC/ SVYDFFVWL (boosting) group,and 98. 90%+/-0.90% and 1.05%+/-0.21% in Ad hTRP2 (priming)-DC/ SVYDFFVWI, (boosting) group, respectively. In the tumor-bearing model, none of mice survived in DC/SVYDFFVWL (priming)-DC/SVYDFFVWL (boosting) group, and just only 40% of mice were tumor-free in Ad hTRP2 (priming) -Ad hTRP2 (boosting) group, whereas 100% of mice survived in Ad hTRP2 (priming)-DC/SVYDFFVWL (boosting) group.

CONCLUSIONBoosting with DC/ SVYDFFVWL can significantly enhance the immunity against melanomas elicited by priming with Ad hTRP2, indicating that first priming with Ad hTRP2 and then boosting with DC/SVYDFFVWL is a potentially effective regimen for overcoming the disadvantage that anti-tumor immune response can not be significantly increased by readministration of adenovirus.

Adenoviridae ; genetics ; Animals ; CD8-Positive T-Lymphocytes ; immunology ; Cancer Vaccines ; immunology ; Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity Tests, Immunologic ; Dendritic Cells ; cytology ; immunology ; Female ; Genetic Vectors ; genetics ; Humans ; Immunization, Secondary ; methods ; Intramolecular Oxidoreductases ; immunology ; Melanoma, Experimental ; immunology ; pathology ; therapy ; Membrane Proteins ; immunology ; Mice ; Mice, Inbred C57BL ; Peptide Fragments ; immunology ; Survival Analysis ; T-Lymphocytes, Cytotoxic ; immunology