To study the chemical constituents of Rabdosia japonica var. glaucocalyx and their anti-complementary activity on the basis of preliminary studies. Target isolation guided by anti-complementary activity test, compounds in the chloroform and n-butanol fractions were isolated and purified by silica gel and Sephadex LH-20 column chromatographies, and preparative HPLC. The structures were identified by various spectroscopic data including ESI-MS, 1H-NMR and 13C-NMR data. The compounds were evaluated for anti-complementary activity in vitro. Eleven compounds were isolated from the chloroform and n-butanol soluble fractions and identified as stigmasterol (1), stigmas-9 (11) -en-3-ol (2), glaucocalyxin D (3), kamebakaurin (4), maslinic acid (5), corosolic acid (6), minheryins I (7), diosmetin (8), caffeic acid ethylene ester (9), caffeic acid (10) and vitexin (11). Isoquercetrin, rutin, quercetin, 3-methylquercetin, luteolin, 7-methylluteolin, and apigenin which were isolated from the preliminary studies together with compounds 9 and 10 showed inhibition of the complement system by the classical pathway. Compounds 2, 4, 6-9 and 11 were obtained from this plant for the first time. Caffeic acid (10) showed the strongest activity in vitro with a CH50 value of 0.041 g x L(-1).
Animals ; Antioxidants ; pharmacology ; Caffeic Acids ; pharmacology ; Chromatography ; Chromatography, High Pressure Liquid ; Complement Hemolytic Activity Assay ; methods ; Complement Inactivating Agents ; chemistry ; pharmacology ; Cricetinae ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Erythrocytes ; drug effects ; Esters ; Ethylenes ; pharmacology ; Female ; Isodon ; chemistry ; Magnetic Resonance Spectroscopy ; Male ; Plant Components, Aerial ; chemistry ; Plant Growth Regulators ; pharmacology ; Sheep ; Spectrometry, Mass, Electrospray Ionization

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 assess the immunotoxicologic effects of genetically modified drought resistant wheat T349 with GmDREB1 gene.

METHODSA total of 250 female BALB/c mice (6-8 week-old, weight 18-22 g) were divided into five large groups (50 mice for each large group) by body weight randomly. In each large group, the mice were divided into five groups (10 mice for each group) by body weight randomly, which were set as negative control group, common wheat group, parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, respectively. Mice in negative control and positive control group were fed with feedstuff AIN-93G, mice in common wheat group, non-genetically modified parental wheat group and genetically modified wheat group were fed with feedstuffs added corresponding wheat (proportion up to 76%) for 30 days, then body weight, organ coefficient of spleen and thymus, peripheral blood lymphocytes phenotyping, serum cytokine, serum immunoglobulin, antibody plaque-forming cell (PFC), serum 50% hemolytic value (HC50), mitogen-induced splenocyte proliferation, delayed-type hypersensitivity (DTH) reaction and phagocytic activities of phagocytes were detected respectively.

RESULTSAfter 30 days raise, among negative control group, common wheat group, non-genetically modified parental wheat group, genetically modified wheat group and cyclophosphamide positive control group, mice body weight were (21.0±0.3), (20.4±0.7), (21.1±1.0), (21.1±1.0), (19.4±1.0) g, respectively (F=7.47, P<0.01); organ coefficient of spleen were (0.407±0.047)%, (0.390±0.028)%, (0.402±0.042)%, (0.421±0.041)%, (0.304±0.048)%, respectively (F=12.41, P<0.01); organ coefficient of thymus were (0.234±0.032)%, (0.246±0.028)%, (0.249±0.040)%, (0.234±0.034)%, (0.185±0.039)%, respectively (F=5.58, P<0.01); the percentage of T cell in peripheral blood were (70.43±4.44)%, (68.33±5.37)%, (73.04±2.68)%, (74.42±2.86)%, (90.42±1.66)%, respectively (F=57.51, P<0.01); the percentage of B cell were (13.89±3.19)%, (15.34±4.84)%, (13.06±4.22)%, (12.93±2.36)%, (3.01±0.96)%, respectively (F=12.79, P<0.01); the percentage of Th cell were (55.87±3.80)%, (55.24±4.60)%, (57.92±3.70)%, (59.57±2.54)%, (77.37±2.31)%, respectively (F=68.58, P<0.01);the Th/Ts ratio were 4.16±0.29, 4.73±0.96, 4.19±0.78, 4.52±0.40, 6.34±0.73, respectively (F=17.57, P<0.01);the serum IgG were (1046.38±210.67), (1065.49±297.22), (1517.73±299.52), (1576.67±241.92), (1155.88±167.05) µg/ml, respectively (F=10.53, P<0.01); the serum IgM were (333.83±18.97), (327.73±27.72), (367.47±27.18), (363.42±46.14), (278.71±24.42) µg/ml, respectively (F=12.11, P<0.01); the serum IgA were (51.69±10.10), (42.40 ± 8.35), (32.11±4.22), (37.12±4.90), (41.45±8.89) µg/ml, respectively (F=8.25, P<0.01); the PFC were (29.2±14.6), (28.0±20.0), (34.8±30.9), (33.2±25.1), (4.8±5.3) per 10(6) splenocyte, respectively (F=3.33, P<0.05); the HC50 were 82.3±6.5, 79.7±4.6, 75.8±4.1, 74.9±3.6, 70.8±2.1, respectively (F=9.99, P<0.01);the LPS-induced splenocyte proliferation were 0.21±0.10, 0.21±0.14, 0.26±0.12, 0.25±0.14, 0.07±0.06, respectively (F=4.18, P<0.05).

CONCLUSIONThe genetically modified drought-resistant wheat T349 was substantially equivalent to parental wheat in the effects on immune organs and immunologic functions of mice, and it didn't show immunotoxicity.

Animals ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; Droughts ; Female ; Mice ; Mice, Inbred BALB C ; Plants, Genetically Modified ; immunology ; toxicity ; Triticum ; genetics ; immunology ; toxicity

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 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

OBJECTIVEFunctional defects in NK cells have been proposed to be responsible for the impairment of anti-tumor immune responses. However, it remained unclear whether the function of NK cells were impaired in patients with hepatocellular carcinoma. To address this issue, we analyzed the frequency and function of peripheral NK cell subsets in hepatocellular carcinoma (HCC) patients.

METHODS35 HCC patients and 24 healthy controls (HC) were enrolled in the study. Peripheral NK frequency was analyzed using flow cytometry. In addition, the capacity of NK cells to produce IFN gamma and to lyse K562 cells was evaluated.

RESULTSIn contrast with the healthy controls, the frequency of peripheral NK cells in hepatocellular carcinoma patients was decreased (12.19%+/-10.85% vs 24.01%+/-8.78%, u = 4.01, probability value less than 0.01), while the frequency of CD56(bright)CD16(neg) NK cells was increased (0.62%+/-0.39% vs 0.48%+/-0.28%, u = 1.96, probability value less than 0.05), and the frequency of CD56(dim)CD16(pos) NK cells was significantly decreased (11.59%+/-7.49% vs 22.66%+/-8.84%, u = 3.92, probability value less than 0.01). In addition, peripheral NK cells from HCC patients exhibited decreased capacity to produce IFN gamma (effective cells 13.31%) and to lyse K562 cells (mixed ratio 30:1, 10:1, 1:1, effective cells 16.72%+/-7.33% vs 26.29%+/-12.36%, u = 2.52, P less than 0.05, 8.01%+/-4.40% vs 13.09%+/-5.03%, u = 3.32, probability value less than 0.05, 3.51%+/-2.82% vs 3.42%+/-1.64%, u = 1.56, probability value more than 0.05, respectively) as compared with healthy subjects.

CONCLUSIONAnti-tumor activity of NK cells in HCC patients was impaired.

Adult ; CD56 Antigen ; immunology ; Carcinoma, Hepatocellular ; immunology ; Case-Control Studies ; Cytotoxicity Tests, Immunologic ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; metabolism ; K562 Cells ; Killer Cells, Natural ; immunology ; Liver Neoplasms ; immunology ; Lymphocyte Subsets ; immunology ; Male ; Middle Aged ; Receptors, IgG ; immunology

When some testing institutions performed biological evaluation to the disposable medical syringe piston, cytotoxicity was found. According to the biological evaluation testing Selection Guide proposed by Ministry of Health and the Comments of Sample Provider, We performed biological evaluation to one sample by using 5 tests of basic biological evaluation. Cytotoxicity was found, which was probably caused by the residue of the lotion. This research provides reference for objective evaluation of disposable medical syringe piston and safe guarantee of the product.
Cytotoxicity Tests, Immunologic ; Disposable Equipment ; Syringes ; adverse effects

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

Animals ; Carcinogenicity Tests ; Cytotoxicity Tests, Immunologic ; Humans ; Mutagenicity Tests ; Soot ; toxicity

OBJECTIVETo study comparatively the cytotoxicity induced by acid bentonite and organic bentonite.

METHODSThe cytotoxicity of two kinds of bentonite was detected using CCK8 assay, neutral red uptake (NRU) assay, lactate dehydrogenase (LDH) leakage assay, apoptosis assay and hemolysis assay. In hemolysis assay human erythrocytes served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 0.3125, 0.6250, 1.2500 and 2.5000 mg/ml for ten min. In other four assays, human B lymphoblast cells (HMy2.CIR) served as target cells and were exposed to the two kinds of bentonite at the doses of 0, 10, 20, 30, 60, 120 and 180 microg/ml for four h.

RESULTSIn hemolysis assay, the hemolysis rates induced by two kinds of bentonite at all doses were significantly higher than that of control (P<0.05); in CCK-8 assay, the cellular activities in acid bentonite group at the doses > or =30 microg/ml and in organic bentonite group at the doses > or =20 microg/ml were significantly lower than that of control (P<0.01); the similar results appeared in NRU assay and LDH assay, and the dose-effect relationship was observed in above 4 assays. In apoptosis assay, the early apoptosis cell rates in acid bentonite group at the dose of 180 microg/ml and in organic bentonite group at the doses of 120,180 microg/ml were significantly higher than that of control (P<0.05). Moreover, the results of five in vitro assays indicated the cytotoxicity induced by organic bentonite was higher than that induced by acid bentonite.

CONCLUSIONTwo kinds of bentonite could induce cytotoxicity, such as apoptosis and damage of cell membrane. The cytotoxicity of organic bentonite is higher than that of acid bentonite due to the different industrial treatment and characteristics of two kinds of bentonite particles.

Apoptosis ; drug effects ; Bentonite ; toxicity ; Cell Line ; Cytotoxicity Tests, Immunologic ; Erythrocytes ; drug effects ; pathology ; Hemolysis ; drug effects ; Humans ; Lymphocytes ; drug effects ; pathology