Objective To prepare Exosomes secreted by mouse hepatoma cell (H_(22)) and heat stressed Exosomes (HS-Exo) derived from heat stress-treated mouse hepatoma cell (H_(22)), in order to study the possible anti-tumor immune mechanism. Methods Exosomes and HS-Exo were purified by serial ultracentrifugation and sucrose density gradiant centrifugation, and were observed and identified by electron microscope. The components and production of the protein and the effects of the host immune response against hepatocellular carcinoma of HS-Exo were observed by using Exosomes as the control. Their immunological factors were detected by Western blot. Lymphocyte proliferation and specific cytotoxic activity of mouse splenic cells were determined by MTT. CD4~+ and CD8~+ lymphocytes infiltration in mouse tumor tissues immunized by both were analysed by immunohistochemical staining. Results HS-Exo was similar in morphology to the Exosomes, the important immune-ralated protein expressed in HS-Exo was increased (P<0.05). HS-Exo immunized mouse group showed more effective inhibition of tumor growth, better-induced lymphocyte proliferation,more significantly enhanced the cytotoxic activity of spleen lymphocytes, as well as a more prominent role in tumor therapy than Exosomes immunized mouse control group (P<0.05). Conclusions Heat stress treatment method for the preparation of HS-Exo was feasible. HS-Exo had a stronger role in the immuneactivity and tumor treatment than control Exosomes.

OBJECTIVEThis study was designed to explore the incidence rate of occult HBV infection in patients with anti-HBc positive alone and analyze the possible reasons of occult infection.

METHODSSera of 183 patients carrying anti-HBc alone(A < or = 0.1) were collected and real-time PCR was used to select samples with HBV DNA positive. HBV pre-S/S amplification products were obtained by PCR, and clonal sequencing were then used for these samples with HBV DNA positive.

RESULTSDNA quantitative results of three samples were greater than 10(3) copies/ml in 183 samples, with a fraction of 1.6%. Pre-S/S sequencing results of two samples from these three samples were obtained. Point mutations within "a" determinant with Q129R/P mutations and co-existence of the mutant type and wild type were found in the two samples.

CONCLUSIONSOccult HBV infection existed in samples with anti-HBc alone. Factors contributing to the loss of HBsAg detection by immunoassays include S gene mutations and low levels of circulating antigen which are below the assay limit of detection. Occult HBV infection not only can lead to a false clinical diagnosis, but also can result in hematological pollution due to such occult infection of blood donors.

Base Sequence ; Blood Donors ; DNA, Viral ; analysis ; Genotype ; Hepatitis B ; diagnosis ; immunology ; Hepatitis B Antibodies ; Hepatitis B Core Antigens ; isolation & purification ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B virus ; genetics ; immunology ; Humans ; Polymerase Chain Reaction ; Protein Precursors ; genetics

OBJECTIVETo investigate the effects of polysaccharide fraction of Cordyceps sinensis (PSCS) on triptolide (TPL)-induced apoptosis in the HL-60 cells and the involved molecular mechanism.

METHODSThe cultured leukemia HL-60 cells were divided into three groups: control group, TPL group (cells were treated with 5 ng/ml TPL only), and PSCS+TPL cells group (cells treated with 5 ng/ml TPL and 100 microg/ml or 200 microg/ml PSCS for 18 h). Cell viability was tested by MTT assay and apoptotic cells were quantitatively measured by flow cytometry with Annexin V/PI double stain.The expressions of Caspase-3, 6, 7, 9 and NF-kappa B proteins were tested by Western blot.

RESULTMTT assay showed that different concentrations of PSCS inhibited the cell viability. Flow cytometry indicated that TPL markedly increased the apoptosis rate of the HL-60 cells, and PSCS enhanced the apoptosis in a dose-dependent manner. Western blot showed that TPL did not inhibit the expression of the Caspase-3, 6, 7, 9 and NF-kappa B proteins, and when cells were treated with PSCS, the expression of proteins decreased with the PSCS concentration rising.

CONCLUSIONPSCS can enhance TPL-induced apoptosis in HL-60 cells and inhibit the expression of NF-kappa B and Caspase 3,6,7,9,which might be the possible signaling pathway of inducing apoptosis.

Apoptosis ; drug effects ; Caspases ; metabolism ; Cordyceps ; chemistry ; Diterpenes ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Epoxy Compounds ; pharmacology ; HL-60 Cells ; Humans ; NF-kappa B ; metabolism ; Phenanthrenes ; pharmacology ; Polysaccharides ; isolation & purification ; pharmacology

OBJECTIVETo investigate the expression of angiopoietin (Ang)-1 and Ang-2 in colorectal tumors and its relations to microvessel density (MVD) in tumor tissue.

METHODSAng-1, Ang-2 and factor VIII-related antigen were stained immunohistochemically in 91 cases of primary colorectal adenocarcinoma, 20 cases of colorectal adenoma and 24 cases of normal colorectal mucosal tissue, and MVD was also assayed in above tissue specimens.

RESULT(1) A significantly higher Ang-1 (7.07+/-2.00) was observed in normal tissue compared with 1.75 +/-1.98 in the adenoma and 1.40 +/- 1.22 in the adenocarcinoma (P<0.01). (2) Ang-2 protein positive rate in adenocarcinoma was significantly higher than that in normal tissue and adenoma (P<0.01). The expression of Ang-2 in adenocarcinoma was closely associated with poor differentiation and vessel invasion. (3) There were significant correlations between Ang-1 and Ang-2 (r=-0.338, P<0.01), Ang-1 and MVD (r=-0.388, P<0.01), Ang-2 and MVD (r=0.594, P<0.01) in the 135 cases.

CONCLUSIONThe overexpression of Ang-2 may play an important role in angiogenesis of colorectal adenocarcinoma. It can be regarded as an index for malignancy and prognosis in colorectal adenocarcinoma.

Adenocarcinoma ; blood supply ; metabolism ; Adult ; Aged ; Aged, 80 and over ; Angiopoietin-1 ; biosynthesis ; genetics ; Angiopoietin-2 ; biosynthesis ; genetics ; Biomarkers, Tumor ; Capillaries ; pathology ; Colorectal Neoplasms ; blood supply ; metabolism ; Female ; Humans ; Male ; Middle Aged ; Prognosis ; von Willebrand Factor ; biosynthesis ; genetics

AIMTo provide experimental basis for clinical use of cryopreserved dendritic cells (DCs) by investigating the biological properties of cryopreserved DCs derived from cord blood.

METHODSThe biological discrepancies between unfrozen DCs (UFDC) derived from unfrozen cord blood (UFCB) and DCs from cryopreserved cord blood (CPCB) or cryopreserved DCs (CPDCs) were explored by morphological observation and immunophenotype analysis. Moreover, the stimulating index (SI) in mixed lymphocyte culture and cytotoxic eliminating rate (ER) were measured by MTT.

RESULTSThe TBR of CPCB and CPDCs were 95.8% and 88.7% respectively. Compared to UFCB, CPCB differentiated toward DCs in a delayed and decreased mode, displayed lower expression of CD1a, CD83 and HLA-DR, and had reduced SI and ER. Similarly, the CPDCs became adherent DCs later and grew less in number than UFDCs. After culture, the expression of CD1a, CD83 and HLA-DR as well as SI and ER were lower in CPDCs than those in UFDCs.

CONCLUSIONThough the biological properties of CPCB and CPDCs were injured after cryopreservation, they still had relatively complete basic function. Their recoveries rate were > 85%.

Cell Proliferation ; Cells, Cultured ; Cryopreservation ; Dendritic Cells ; cytology ; Fetal Blood ; cytology ; Humans

AIMTo observe the basical properties of adherent stromal cells in culture derived from cryopreserved bone marrow cells (BMCs), and to provide laboratory evidences for clinical application of cryopreserved stromal cells.

METHODSFresh BMCs and adherent stromal cells cultured for 14 days in Dexter long-term culture system (fresh stromal cells) plus 5% DMSO-6% HES cryopreservatives were frozen in -80 degrees C refrigerator, and cryopreserved in - 196 degrees C liquid nitrogen for 2 weeks (the former is called cryopreserved BMCs, the latter called cryopreserved stromal cells). These cells were cultured in Dexter long-term culture system after they were thawed. We have examined the growth features, constituents and stimulating functions of the culture of these cells.

RESULTSGrowth features: Cryopreserved BMCs produced adherent stromal cells, cell clusters and cell layer 1-2 days later than fresh BMCs. Cryopreserved stromal cells formed cell layer in 2nd day of culture, and were 12-18 h later than fresh stromal cells. Cryopreserved BMCs and stromal cells proliferated significantly less than fresh BMCs and stromal cells. Constituents: The ratio of fibrocytes and endothelial cells were lower, and the ratio of macrophages and fat cells were higher in culture of cryopreserved BMCs than that in fresh BMCs. The measurements in culture of cryopreserved stromal cells were much more significant compared with that in fresh stromal cells. Numbers of cells containing apoptic bodies in culture of cryopreserved BMCs and stromal cells were more than that in fresh BMCs and stromal cells. TBRR of cryopreserved BMCs and stromal cells were 92.5% and 89.5% respectively. The expression rates of CD14 and HLA-DR in culture of cryopreserved BMCs and stromal cells were higher than that in fresh BMCs and stromal cells, and just in contrast with the expression rates of CD45 and CD33. Stimulating functions: CAFA and LTC-IC on the stromal cell layer derived from cryopreserved BMCs, cryopreserved stromal cells, fresh BMCs and fresh stromal cells were all growing well, and there were no significant differences among these groups.

CONCLUSIONBiological properties of adherent stromal cells derived from BMCs and stromal cells were injured slightly and still maintained completely after cryopreservation with 5% DMSO-6% HES cryopreservatives.

Bone Marrow Cells ; cytology ; Cell Survival ; Cells, Cultured ; Cryopreservation ; Humans ; Stromal Cells ; cytology

OBJECTIVETo compare the behavioral effects of psychoactive drugs between two strains of mice.

METHODSThe Kunming (KM) and ICR mice were injected intraperitoneally with caffeine (3, 10, 30, 100 mg/kg), ephedrine (3, 10, 30, 100 mg/kg), diazepam (1, 3,1 0 mg/kg) and chloral hydrate (10, 30, 100 mg/kg), respectively. Ten min after injection, the locomotor activity in the open field was recorded for 2 h. The total distance, the distance ratio to total distance and the time in central region were analyzed for each drugs. Thirty min after injection, the latent time in the passive avoidance test was measured in a shuttle box.

RESULTSCaffeine and diazepam prolonged the latent time, and ephedrine and chloral hydrate decreased the latent time, but there were no differences between the two strains. The two strains of mice exhibited significant differences in the total distance after injection of ephedrine 10 mg/kg, diazepam 3 mg/kg and chloral hydrate 100 mg/kg. Compared to KM mice, ICR mice exhibited an increase in the distance ratio and the time in central region after injection of ephedrine 10-100 mg/kg, but a decrease after diazepam 3-10 mg/kg.

CONCLUSIONKM and ICR mice show no differences in latent time, but significant differences in the total distance, the distance ratio and the time in central region in the locomotor activity. Therefore, selection of mouse strains is important in the study of psychoactive drugs.

Animals ; Caffeine ; pharmacology ; Central Nervous System Agents ; pharmacology ; Chloral Hydrate ; pharmacology ; Diazepam ; pharmacology ; Dose-Response Relationship, Drug ; Ephedrine ; pharmacology ; Mice ; Mice, Inbred ICR ; Motor Activity ; drug effects

Three cyclotides were isolated from the whole plant of Viola yedoensis in this study. The two, vary peptide E and cycloviolacin Y5, were previously reported, and a novel cycloviolacin VY1 was characterized according to the interpretation of MS/MS fragmentation of peptides which were produced from the reduced and alkylated parent peptide with the digestion of Endo Lys-C, trypsin and chymotrypsin, separately. The stability of remarkable resistance to proteolytic degradation by trypsin and chymotrypsin, and that of thermal denaturation was confirmed again. Besides, the IC50 value of cycloviolacin VY1 against influenza A H1N1 virus was (2.27 +/- 0.20) microg x mL(-1). It is the first cyclotide reported with anti-influenza A H1N1 virus activity in vitro assay.
Antiviral Agents ; isolation & purification ; pharmacology ; Cyclotides ; pharmacology ; Influenza A Virus, H1N1 Subtype ; drug effects ; Tandem Mass Spectrometry ; Viola ; chemistry

OBJECTIVETo explore the possible factors causing the increase of rabies cases and to provide references for the development of related prevention and control strategy in Hunan.

METHODSData was collected and analyzed on epidemic situation of rabies in the past ten years, and studies were carried in some counties.

RESULTSFrom 1994 to 1999, the annual cases sustained between 17 and 78. However, the number of cases has continued to increase since 2000. The annual reported cases in 2001 and 2002 were 311 and 313, and accounted for 34.8% and 30.0% of the total cases in the whole country respectively. The epidemic areas were mainly located in the south and middle parts of Hunan. Furthermore, the epidemic areas had been expanding. In 1997, human rabies cases were only reported in 7 cities but increased to 12 cities in 2004.

CONCLUSIONFactors as increasing number but low inoculating rate to dogs, and incorrect treatment on the wounds etc. might have served important roles for the recurrence of rabies. Hence, it is necessary to take comprehensive preventive measures to control and prevent the epidemics of rabies in Hunan.

Adolescent ; Adult ; Aged ; Child ; Child, Preschool ; China ; epidemiology ; Female ; Humans ; Incidence ; Male ; Middle Aged ; Rabies ; epidemiology

AIMTo explore proper cryopreservative systems for hematopoietic stem cells.

METHODSPeripheral blood mononuclear cells from 20 persons were mixed with different cryopreservative agent, dimethyl suflfoxide (DMSO) or combination of DMSO and hydroxyethyl starch (HES), then cooled in -80 degrees C low temperature refrigerator (Refr) or autocontrolled programmed cryogenic system (PCS), preserved in Refr or in liquid nitrogen. GM-CFU, LTC-IC, CD34+ cells and typeran blue resistance (TBR) were assayed after different period of cryopreservation.

RESULTSThe recovery rates of CFU-GM, LTC-IC, CD34+ cells and TBR in peripheral blood mononuclear cells which were cooled and preserved in Refr with 5% DMSO-6% HES were 82.2% +/- 14.7%, 83.0% +/- 12.2%, 94.2% +/- 4.3% and 97.7% +/- 3.9% respectively, significantly higher than that in Refr with 10% DMSO (P < 0.05). When cells were cryopreservated with the same cryopreservatives, there was no significantly difference of recovery rate in group of Refr and group of Refr with PCS. Meanwhile, there was not significantly difference of recovery rate among all three groups, preserved in Refr ahead of liquid nitrogen, in Refr merely, in liquid nitrogen with PCS within one year (p > 0.05). However, the recovery rate of CFU-GM, LTC- IC, CD34+ cells and TBR decreased dramatically if cells were cooled and preserved in Refr for two years. After cells were thawed, the cell activity declined gradually at room temperature if the cryopreservatives were not removed or diluted. The cell activity of 10% DMSO group was affected more than that of 5% DMSO-6% HES group.

CONCLUSION5% DMSO-6% HES is better than 10% DMSO as cryopreservatives for hematopoietic stem cells. Refr cryopreservation is a simple and effective method if cells would be cryopreserved for less than one year. If cells would be cryopreserved for more than one year, liquid nitrogen cryopreservation should be recommended. The cryopreservatives should be diluted or removed immediately after cells were thawed.

Blood Preservation ; methods ; Cell Survival ; drug effects ; Cryopreservation ; methods ; Cryoprotective Agents ; pharmacology ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; drug effects ; Humans