Achyranthes bidentata polysaccharides (ABPS) was extracted from the root of A. bidentata. Dendritic cells (DC), which were stimulated with ABPS and/or tumor antigen SW480, were co-cultured with cytokine induced killer cells (CIK) to test the cytotoxic effect on colon cancer cell line SW480. Peripheral blood mononuclear cells (PBMNCs) which were separated from human peripheral blood were cultured to DC and CIK separately. (1) DC were divided into four groups: pure DC served as control group; ABPS (50 mg x L(-1)) stimulated DC served as experimental group; SW480 tumor antigen stimulated DC served as the second experimental group; ABPS (50 mg x L(-1)) and SW480 tumor antigen co-stimulated DC served as the third experimental group. Flow cytometry was used to detect the difference of the positive rate of molecules in the cell surface of DC, include CD80, CD86, CD1c, CD40, HLA-DR (6 samples for each group). (2) The four DC groups were mixed with CIK at the ratio 1:5 and acted as effect cells (DC + CIK groups), and the colon cancer cell line SW480 acted as target cells. The effect cells and the target cells were mixed together at the ratio 30: 1, 20:1 and 10:1 separately, and the CCK-8 kit was used to test the cytotoxic effect on colon cancer cell line SW480. (3) At the mixing ratio 30:1 of effect cells and target cells, ELISA was used to test the level of cytokines secretion, including IL-2, IL-12p70, IL-17 and TNF-alpha, in the liquid supernatant of every test group (3 duplication per sample). The results showed as following: (1) The positive rates of CD80, CD11c, HLA-DR, in the cell surface of DC which was co-stimulated by ABPS (50 mg x L(-1)) and SW480 tumor antigen, were obviously higher than the other DC groups (P < 0.05), and the positive rates of CD86, CD40 were obviously higher than the pure DC group (P < 0.05), and there was no remarkable difference with the other two DC groups. (2) At the mixing ratio 30:1, 20:1 and 10:1 of the effect cells and the target cells, the cytotoxic effect of ABPS stimulated DC + CIK group and SW480 tumor antigen stimulated DC + CIK group was obviously higher than DC + CIK group (P < 0.05), the cytotoxic effect of ABPS and SW480 tumor antigen co-stimulated DC + CIK group was obviously higher than all the other groups. (3) At the mixing ratio 30:1 of the effect cells and the target cells, the secretion levels of IL-12p70 and TNF-alpha in the liquid supernatant of the ABPS and SW480 tumor antigen co-stimulated DC + CIK group were obviously higher than all the other groups (P < 0.05), the secretion levels of IL-2 and IL-17 in the liquid supernatant of every test group have no remarkable difference. The cytotoxic effect of ABPS stimulated DC + CIK on SW480 was obviously increased. The cytotoxic effect of ABPS and SW480 tumor antigen co-stimulated DC + CIK group was obviously higher than all the other.
Achyranthes ; chemistry ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cells, Cultured ; Colonic Neoplasms ; drug therapy ; immunology ; physiopathology ; Cytokine-Induced Killer Cells ; drug effects ; immunology ; Cytotoxicity, Immunologic ; drug effects ; Dendritic Cells ; drug effects ; immunology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Interferon-gamma ; immunology ; Interleukin-2 ; immunology ; Polysaccharides ; pharmacology

OBJECTIVETo improve the anti-tumor effect of dendritic cytoma vaccine (DCV) for finding an effective anti-tumor biotherapy.

METHODSDC vaccine prepared by transfection of adenovirus mediated melanoma-associated antigen gene (gp100) into bone marrow-derived dendritic cell (DC) was used to study the immuno-therapeutic effect and the mechanism of lentinan (LNT) in different dosages, used alone or combined with gp100-DC for treatment of B16 melanoma bearing mice.

RESULTSAfter being treated with LNT combining gp100-DC, the growth of malignant melanoma was inhibited with the tumor-free survival in the experimental animals being 66.7%. The treatment could also significantly enhance the activity of cytotoxicity T lymphocyte (CTL) and natural killer (NK) cells, elevate the levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) in splenocytes, and histological examination showed that a large amount of inflammatory cells infiltrated inside and around the tumor, and obvious necrosis of tumor cells was found.

CONCLUSIONBy combined use with LNT the anti-tumor immuno-reaction of DCV vaccine could be enhanced effectively.

Adjuvants, Immunologic ; pharmacology ; therapeutic use ; Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; therapeutic use ; Cancer Vaccines ; administration & dosage ; immunology ; Cell Line ; Cell Line, Tumor ; Combined Modality Therapy ; Dendritic Cells ; cytology ; immunology ; Female ; Humans ; Lentinan ; pharmacology ; therapeutic use ; Melanoma, Experimental ; immunology ; pathology ; therapy ; Membrane Glycoproteins ; genetics ; immunology ; Mice ; Mice, Inbred C57BL ; Random Allocation ; Shiitake Mushrooms ; chemistry ; Survival Analysis ; Transfection ; gp100 Melanoma Antigen

Objective To establish a RP-HPLC method for determining indigo and indirubin in Baphicacanthus cusia from different producing areas and medicinal parts. Methods The separation was achieved by an Agilent TC-C18 Column (4.6 mm×250 mm, 5 μm) at 25 ℃ using methanol-water (75??25) as mobile phase at a flow rate of 1 mL??min-1.The detection wavelength was 290 nm. Results Indigo had a good linear relationship with peak area at range of 0. 051 3-0.820 8 μg (r=0.999 3).The recovery rate was 99.00% and RSD was 1.30% (n=6).Indirubin had a good linear relationship with peak area at range of 0.049 5-0.792 0 μg (r=0.999 9).The recovery rate was 98.88% and RSD was 1.51% (n=6). Conclusion The contents of the two components are obviously different in Baphicacanthus cusia because of different places or medicinal parts. The proposed method is simple, rapid and reliable. This method for determination of indigo and indirubin in Baphicacanthus cusia by RP-HPLC provides a basis for quality control of Baphicacanthus cusia.

ObjectiveTo prepare a novel etimicin-encapsuled chitosan/hydroxyapatite nano-scaffolds and offer assistances for bone defect or osteomylitis.MethodsDrug-carried chitosan nanoparticles which was prepared by ionotropic gelation were combined with nano-hydroxyapatite.The mixture were shaped in molds and then prepared into porous scaffolds by freeze-dry.The surface of one scaffold was scanned.The grinded,particles of the scaffold were detected by field emission scanning electron microscope; X-ray diffraction was used to analyze components of the scaffold and total porosity.Staphylococcus aureus was choosed as the experimental bacteria,we studied lasting antibacterial property of drug-carried bone scaffold by antibacterial experiments,long-term drug releasing experiments and accumulation drug releasing experiments.Bone mesenchymal stem cells were used to detect the histocompatibility and inductivity of etimicin-carried scaffold.ResultsFreeze-dried porous scaffold has a surface with proper pore distribution (total porosity 70.68％) and the grinded scaffold has a globular and coliformed microstructure known after scanned by electron microscope.The drug-carried scaffold has a typical wave of hydroxyapatite under X-ray diffraction.The lasting antibacterial property study indicated that the drug-carried bone scaffold had maintained an inhibition zone for more than 7 days.The long-term drug releasing experiments and accumulation drug releasing experiments show that the fictional drug-carried bone scaffold released above the bacteriostasis concentration after one week and the accumulative amount within the safety scale.The scaffold had not an inhibitory effect on bone mesenchymal stem cells.ConclusionThe etimicin-encapsuled chitosar/ hydroxyapatite nano-scaffolds has similar microstructure and components of bone tissue.It is promising in bone tissue engineering applications because of its slow-release,antibacterial properties and satisfactory histocompatibility.

OBJECTIVETo investigate the interaction between myeloid-derived suppressor cells (MDSCs) and nature killer cells during acute fulminate hepatitis.

METHODSAcute fulminate hepatitis were induced by i.p. co-injection of LPS and D-GalN in mice, and the ratio of MDSCs,NK cells and the activation of NK cells in different tissues were analyzed by FACS at 0 h,1.5 h,3 h and 6 h.

RESULTSThe percentage of MDSCs and NKG2D+NK cells in different tissues increased as acute fulminate hepatitis progressed, with the increased NK cells in liver tissue. The mean fluorescence intensity of NKG2D on NK cells in different tissues were also enhanced.

CONCLUSIONMDSCs induce the proliferation and activation of NK cells in mice with acute fulminate hepatitis.

Acute Disease ; Animals ; CD8-Positive T-Lymphocytes ; immunology ; Female ; Hepatitis ; etiology ; immunology ; Killer Cells, Natural ; immunology ; Lipopolysaccharides ; Liver Failure, Acute ; chemically induced ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Myeloid Cells ; immunology ; T-Lymphocytes, Regulatory ; immunology

Objective To investigate the liver injury and pathological changes of rat and patients with Clonorchis sinensis(C, sinensis) infection, and to clarify the role of apoptosis in the injury induced by C. sinensis.Methods Wistar rats were divided into two group: 60 in infection group and 20 in control. The rats in infection group were infected with C. sinensis via oral feeding encysted cercaria;rats in control group were fed with normal saline. The rats were sacrificed 4, 6, 8 and 12 weeks after infection, respectively. Liver tissue specimens of the patients infected with C. sinensis were collected. The pathological changes of liver tissue were observed by light microscopy and the apoptofic rate of hepatocyte was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) assay. Results Parasites and eggs could he seen around the bile duct, and the duct was associated with mucosa and adenoma papillary hyperplasia, wall thickening, inflammatory cell infiltration, a small amount of fibrous tissue hyperplasia, and periportal liver cells surrounded by a number of nuclear condensation, all these changes meant morphological characteristics of apoptosis. Apoptotic rates of liver cells in infection group 4, 6,8 and 12 weeks after infection were (7.15 ± 1.50)%,(11.61 ± 3.09)%,(13.21 ± 3.47)% and (11.26 ± 4.06)%,respectively, which was significantly higher than that in control group [(2.57 ± 0.72)%, (3.17 + 0.77)%, (3.67 ±0.96)% and (2.84 ± 0.87)%, t values were 4.45, 5.49, 5.95 and 4.74, respectively, all P < 0.01]. Conclusions These findings indicate that C, sinensis can stimulate both hepatoeytic apoptosis and degeneration which may he related to clinical manifestations and liver lesions in patients with clonorchiasis.

Autophagy is an evolutionarily well conserved cellular catabolic process . Cellular proteins and organelles are engulfed to autophagosomes and eventually delivered to lysosomes for degradation .Autophagy is closely associated with a variety of human diseases especially cancer .At present , it has been widely accepted that autophagy is a“double-edged sword” in cancer: it blocks tumorigenesis at the early stage , while it facilitates tumor development at the promotion and progression stage . Moreover , autophagy can be induced by chemotherapy and radiotherapy , which is generally play a pro-survival function .Thus, autophagy is an important target for cancer therapy , and suppression of autophagy to enhance the efficacy of cancer therapeutic agents is a novel strategy in cancer therapy under active clinical trials .

AIMTo investigate the fragmentation behavior of triazolobenzodiazepines and to develop a specific, sensitive and rapid LC/MSn assay for simultaneous determination of estazolam, alprazolam and triazolam in human urine.

METHODSAfter oral administration of a single 4 mg dose of the drugs to each of three healthy volunteers, urine samples were purified by solid-phase extraction, and then injected into an ODS column (150 mm x 4.6 mm) with a mobile phase of methanol-water (8:2) for LC/MSn analysis. The structures of estazolam, alprazolam and triazolam in human urine were identified by direct comparison of the observed mass spectra and the chromatographic retention time with those of the reference substance. The mass spectrometer (Finnigan LCQ) was operated in positive mode and in two scan modes including SIM and full scan MS/MS mode. The obtained mass spectra was analyzed assisted with the software Mass Frontier 1.0 for their fragmentation pathways.

RESULTSThe full scan MS/MS spectra of each compound gave characteristic fragment ions of [M + H - N2]+ and [M + H - Cl]+. The detection limit was below 0.5 ng.mL-1 for estazolam, alprazolam and triazolam in human urine.

CONCLUSIONThe method is useful in forensic and clinical toxicology in which unequivocal identification of eatazolam, alprazolam and triazolam is desired.

Alprazolam ; urine ; Anti-Anxiety Agents ; urine ; Chromatography, Liquid ; Estazolam ; urine ; Humans ; Male ; Spectrometry, Mass, Electrospray Ionization ; Triazolam ; urine

OBJECTIVETo develop and optimize real-time fluorescence quantitative PCR (FQ-PCR) with the housekeeping gene RAG2 as cell number control to quantify T-cell receptor excision circle (TREC) in the peripheral blood.

METHODSThe real-time PCR system for amplifying TREC and RAG2 genes was established on the basis of ABI 7000 apparatus using Golden Taq system, designed primers, TaqMan-MGB probes and optimized buffer. PCR conditions were optimized with standard samples of TREC plasmid.

RESULTSThe amplification with the primer pair T(3) and T(4) was more efficient than that with T(1) and T(2). More specific and efficient amplification in FQ-PCR was achieved using TaqMan-MGB probes as compared with general Taq-Man probes. Golden Taq was more effective than general Taq in improving the specificity and decreasing the artifact, and 95 degrees C; for 10 min, 95 degrees C; for 5 s, and 53 degrees C; for 30 s for a total of 40 cycles using ABI7000 was found as the optimized thermal parameter setting.

CONCLUSIONAn optimized real-time PCR protocol for detecting TREC in peripheral blood mononuclear cells is established.

Adolescent ; Adult ; DNA-Binding Proteins ; genetics ; Female ; Gene Rearrangement, T-Lymphocyte ; genetics ; Humans ; Leukocytes, Mononuclear ; metabolism ; Male ; Middle Aged ; Nuclear Proteins ; genetics ; Polymerase Chain Reaction ; methods ; Receptors, Antigen, T-Cell ; genetics ; Reproducibility of Results

OBJECTIVETo investigate the preventive effect of interleukin-18 (IL-18) gene modified mature dendritic cells (mDC) vaccine on airway inflammation in mouse asthma model.

METHODSThe asthma model was induced by injection of ovalbumin (OVA) in BALB/c mice. IL-18 gene modified mouse mature dendritic cells (mDC) were detected by flow cytometry and its capacity of inducing allogeneic T cell responses was examined by mixed lymphocyte reaction (MLR). The OVA-induced asthmatic mice were randomly divided into 6 groups: PBS group, DXM group, mDC group, Ad-LacZ-mDC group, Ad-IL-18-mDC group and control group. The pathological changes in lung tissues were assayed by HE and AB-PAS staining. The numbers of inflammatory cells and percentage of eosinophils (EOS) in bronchoalveolar lavage fluid (BALF) were counted. The levels of IFN-γ IL-4 and IL-13 in culture supernatant of splenocytes were measured by ELISA method. The percentage of CD4(+)CD25(+)Foxp3(+) Treg was assessed by flow cytometry analysis.

RESULTThe vaccine was effective in decreasing the infiltration of EOS and accumulation of airway goblet cells in lung tissues, the numbers of inflammatory cells and percentage of EOS in BALF, and the levels of IL-4 and IL-13 in culture supernatant of splenocytes, and in increasing the levels of IFN-γ in culture supernatant of splenocytes and the percentage of CD4(+)CD25(+)foxP3(+) reg.

CONCLUSIONIL-18 gene modified mDC vaccine has a preventive effect on airway inflammation in OVA-induced asthmatic mice.

Animals ; Asthma ; immunology ; pathology ; prevention & control ; Dendritic Cells ; immunology ; Disease Models, Animal ; Genetic Therapy ; Interleukin-18 ; genetics ; Lung ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Ovalbumin ; immunology