BACKGROUNDThis study was to evaluate whether anergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro and prolong cardiac allograft survival after adoptive transfer.

METHODSAnergic cells were induced in vitro by the addition of anti-CD154 and anti-CD80 monoclonal antibodies (mAbs) to primary MLR (mixed lymphocyte reaction) consisting of BALB/c as responder and C3H as stimulator. Anergic cells were added to a newly formed MLR in assessing the regulatory capacity and antigen specificity of anergic cells. The ability of anergic cells to respond to antigen and/or exogenous recombinant mouse interleukin-2 (rmIL-2) was tested. For in vivo studies, anergic cells were intravenously injected into 3.0-Gy gamma-irradiated BALB/c mice immediately after heterotopic abdominal cardiac transplantation. To prolong allograft survival, recipient mice injected with anergic cells received rapamycin therapy [1 mg.day(-1).kg(-1)].

RESULTSAnergic cells strongly suppressed the proliferation of naicaron;ve BALB/c splenocytes against the original (C3H) stimulator in a dose-dependent manner, but they failed to suppress the proliferation of naicaron;ve BALB/c splenocytes against the third-party (C57BL/6J) stimulator. The anergic state was reversed by both original (C3H) stimulator and additional exogenous IL-2. In in vivo studies, untreated irradiated BALB/c mice rejected C3H cardiac allografts with a mean survival time of (8.6 +/- 1.1) days, whereas those injected with the anergic cells rejected the allografts with a mean survival time of (11.8 +/- 1.9) days, which was slightly longer than that of the untreated mice. The protocol based on anergic cells injection plus rapamycin therapy could prolong allograft survival significantly [(29.6 +/- 4.4) days].

CONCLUSIONSAnergic cells induced by the blockade of CD40-CD154 and CD28-B7 costimulatory pathways can act as potent immunoregulatory cells in vitro, and prolong cardiac allograft survival after adoptive transfer in the presence of rapamycin therapy. This procedure might be clinically useful for prolonging allograft survival if optimal protocols are developed.

Animals ; Antibodies, Monoclonal ; pharmacology ; B7-1 Antigen ; physiology ; CD28 Antigens ; physiology ; CD40 Antigens ; physiology ; CD40 Ligand ; physiology ; Graft Survival ; Heart Transplantation ; Lymphocyte Activation ; Lymphocyte Culture Test, Mixed ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C3H ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; immunology ; Transplantation, Homologous

OBJECTIVETo establish a method for the determination of gossypol in cotton root bark.

METHODSamples were extracted with acetone, and determined on a C18 column with the mobile phase (Acetonitrile-0.2% phosphoric acid, 85:15) and UV-235 nm detector.

RESULTThe recovery rate was 97.6%, RSD 1.59% (n = 5).

CONCLUSIONThis method can be used for the determination of gossypol in cotton root bark and the content of gossypol in three different species has been determined.

Chromatography, High Pressure Liquid ; Gossypium ; chemistry ; Gossypol ; analysis ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry

Objective To study the clinical value of serum cytokeratin 19 fragment (CYFRA21-1) as a biomarker in evaluating its prognosis,monitoring and follow-up of the postoperative patients with non- small cell lung cancer (NSCLC).Methods Serum CYFRA21-1 was determined by radioimmunoassay for 207 patients with NSCLC before and after surgical operation at Tongji Hospital,Shanghai.Relationship between serum CYFRA21-1 and the prognosis,recurrence and metastasis of lung cancer was analyzed retrospectively.Results The patients were followed-up for 37 months in average.Preoperative serum CYFRA21-1 was positive in 42.0% (87/207) of all the cases,48.8% (60/123) of squamous cell earcinama and 34.6% (27/78) of adenocarcinoma,with statistical significance (X~2=3.901,P

OBJECTIVETo study the pathogen and characteristics of viral diarrhea in children in Changchun area.

METHODS460 stools specimens were collected from children with acute diarrhea cured in the childrens, hospital of Changchun in 2010. Rotavirus were detected by ELISA, caliceverus and astrovirus were detected by reverse transcription-polymerase chain reactions (RT-PCR), adenovirus were detected by polymerase chain reactions (PCR).

RESULTSA total of 460 specimens were detected. The detection rate of rotavirus, caliceverus, astrovious, adenovious respectively is 35.22%, 20.43%, 9.78%, 3.70%, the detectablerate of mixed infection is 7.61%, children under 2 years old were the major patient. The main genotypes of the virus: rotavirus (G3P[8]), caliceverus (GII-4), astrovious (type I), adenovious (Ad41).

CONCLUSIONRotavirus is the main pathogen in Changchun. Followed by caliceverus, astrovious, adenovious.

Adenoviruses, Human ; isolation & purification ; Caliciviridae ; isolation & purification ; Child, Preschool ; China ; epidemiology ; Diarrhea ; epidemiology ; etiology ; Female ; Humans ; Infant ; Male ; Mamastrovirus ; isolation & purification ; Rotavirus ; isolation & purification ; Virus Diseases ; epidemiology ; etiology

OBJECTIVETo explore the mechanism of acupoint sticking of "Hua yutie" in improving ischemic stroke.

METHODSEighty rats were randomly divided into 5 groups, a model group, an acupoint sticking group, an acupuncture group, a Nimodipine group and a normal group. Middle cerebral artery occlusion (MCAO) was used for preparation of focal cerebral ischemic rat model. After modeling, any treatment was not given to the model group; for the acupoint sticking group, "Hua yutie" was applied at "Dazhui" (GV 14) ,"Qihai" (CV 6) and "Mingmen" (GV 4); for the acupuncture group, acupuncture was given at the same acupoints as those in the acupoint sticking group; the Nimodipine group received intragastric administration of Nimodipine. And the normal group did not receive any treatment. Their infarction volume, the cerebral water content, expression of vascular endothelial growth factor (VEGF) and the protein level were observed.

RESULTSThe infarction volume coincided with the dominative scope of the middle cerebral artery of the electric coagulation. There were significant differences in the cerebral water content as the various treatment groups compared with that of the model group (all P<0.05). The VEGF positive cell number and the protein level around the infarction area in the acupoint sticking group were increased as compared with those in the model group (P<0.01), with no significant difference as compared with the Nimodipine group and the acupuncture group (all P>0.05).

CONCLUSIONAcupoint sticking of "Hua yutie" alleviates the cerebral damage after ischemia possibly through enhancing the expression and protein level of VEGF.

Acupuncture Points ; Administration, Cutaneous ; Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; Cerebral Infarction ; drug therapy ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Gene Expression ; drug effects ; Humans ; Male ; Rats ; Rats, Wistar ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Dendritic cell (DC) plays a key role in antitumor immune response. However, there is a deficiency of DC function in the majority of leukemia patients. It is a novel idea that expanding DC in vitro and enhancing their antitumor immune function and DC-based tumor vaccines may be used as an efficient immune therapy for leukemia. In the project, the condition to induce DC from myeloid leukemia cell lines and its anti-leukemia response were investigated. HL-60, K562 and THP-1 cells were cultured with various combinations of cytokines for inducing DC. The morphologic features were analyzed with optical and electron microscopy. The phenotype of DC was detected by FCM with CD1a, CD40, CD80, CD86, HLA-A, B, C and HLA-DR monoclonal antibodies. The ability of DC stimulating lymphocyte proliferation was observed by allo-mixed lymphocyte reaction using (3)H-TdR incorporation. Cytotoxicity assay was measured by (51)Cr-release method. The level of IL-12 and IFN-gamma in supernatant of DC culture was measured by ELISA. It was proved that the DCs derived from K562, HL-60 and THP-1 cells showed a typical morphology of dendritic cell. The induced cells expressed the surface differentiation antigens of DC. A high expression of phenotypes was found in HL-60-DC and THP-1-DC stimulated by GM-CSF + IL-4 + TNF-gamma and K562-DC with GM-CSF + IL-4 + IL-12. The DCs from the 3 leukemia cell lines stimulated allo-MLR and CTL reaction strongly. Different contents of IL-12 were detected in the supernatants of DC culture and IFN-gamma in the coculture of DC and blood mononuclear cells. It is concluded that the myeloid leukemia cells are able to be induced DCs by cytokines in vitro. The different leukemia cells need different cytokines and cultural conditions. DCs derived from leukemia cells express phenotype of antigen-presenting cells. They have the ability of stimulating T lymphocyte proliferation and inducing CTL reaction to clear leukemia cells, and the DCs secrete IL-12 and increase secretion of IFN-gamma by T cells.
Antigens, CD ; immunology ; Antigens, CD1 ; immunology ; B7-2 Antigen ; CD40 Antigens ; immunology ; Coculture Techniques ; Cytokines ; pharmacology ; Cytotoxicity, Immunologic ; drug effects ; Dendritic Cells ; drug effects ; immunology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; HL-60 Cells ; HLA Antigens ; immunology ; Humans ; Immunophenotyping ; Interferon-gamma ; pharmacology ; Interleukin-12 ; pharmacology ; Interleukin-4 ; pharmacology ; K562 Cells ; Membrane Glycoproteins ; immunology ; Microscopy, Electron ; Time Factors ; Tumor Cells, Cultured ; drug effects ; immunology ; ultrastructure ; Tumor Necrosis Factor-alpha ; pharmacology

OBJECTIVETo investigate transient expression of fusion protein with a chimeric HBsAg-HSP70 construct in HepG2 cells.

METHODSEnkaryotic expression plasmids inserted HBsAg gene or chimeric HBsAg-HSP70 gene were prepared and transfected into HepG2 cells by means of cationic liposome. mRNA were detected by RT-PCR and proteins expressed in the cells were detected by immunocytochemistry 48 hours later. HBsAg in cultured supernatants and cell lysates were assayed by ELISA.

RESULTSFusion protein (HBsAg-HSP70) transient expression in HepG2 cells were confirmed by RT-PCR, immunocytochemistry or ELISA, but fusion protein was not assayed in cell cultured supernatants by ELISA.

CONCLUSIONSTransfection of HepG2 cells with a chimeric HBsAg-HSP70 construct leads to express fusion protein, but it does not secrete into cell cultured supernatants.

Bacterial Proteins ; Enzyme-Linked Immunosorbent Assay ; HSP70 Heat-Shock Proteins ; genetics ; Hepatitis B Surface Antigens ; genetics ; Hepatitis B Vaccines ; immunology ; Humans ; Immunohistochemistry ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Vaccines, DNA ; immunology

OBJECTIVETo investigate whether Candida albicans-native phospholipomannan (PLM) induce an inflammation response through Toll-like receptor(TLRé2 in human acute monocytic leukemia cell line (THP-1) cells.

METHODSHuman THP-1 monocytes were challenged with PLM in vitro. The mRNA expressions of TLR2, TLR4, proinflammatory cytokine [interleukin(IL)-6], and chemokine (IL-8) were assayed by real time reverse transcription polymerase chain reaction. The secretions of IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay. The expression of TLR2 was analyzed with Western blot.

RESULTSPLM increased the mRNA expressions and secretions of proinflammatory cytokines (IL-6) and chemokines (IL-8) in THP-1 cells (all P=0.0000). PLM up-regulated the mRNA and protein levels of TLR2 (P=0.0000), whereas the mRNA level of TLR4 was not altered. PLM hydrolyzed with β-D-mannoside manno hydrolase failed to induce gene and protein expressions of TLR2, IL-6, and IL-8. Anti-TLRS-neutralizing antibody blocked the PLM-induced secretions of IL-6 and IL-8 in THP-1 cells (P = 0.0003, P = 0.0010).

CONCLUSIONCanidada albicans-native PLM may contribute to the inflammatory responses during Candida infection in a TLR2-dependent manner.

Candida albicans ; chemistry ; Cells, Cultured ; Glycolipids ; pharmacology ; Humans ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Monocytes ; drug effects ; immunology ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVERegarding the strong antigen-presenting abilities of dendritic cells (DC), this study was carried out based on the induction and proliferation of DC derived from human umbilical cord blood; the anti-HBV effect of cytotoxicity T lymphocytes (CTL) activated by those DC pulsed with HBsAg was also carried out to explore a new way to activate the HBsAg-specific CTL.

METHODSCord blood was collected from the cord veins of normal placentae after Cesarean sections, from which cord blood mononuclear cells (CBMC) were separated through density gradient centrifugation. The CBMC were cultured in RPMI 1640 with a cytokine cocktail. Pulsed with HBsAg, the DC were prepared to activate the HBsAg-specific CTL among the CBMC. The cytotoxic effect of CBMCs activated by the DC primed with HBsAg was assayed through the killing of those HepG2-S target cells.

RESULTSTypical DC could be induced from CMBC cultured with a cytokine cocktail. DC pulsed by HBsAg activated HBsAg-specific CTL, which killed the target HepG2-S cells to some extent.

CONCLUSIONDC can be induced from CMBC with the cytokine cocktail and they show a strong antigen-presenting ability. DC produced in this way and pulsed by HBsAg can activate HBsAg-specific CTL in vitro. This might mean that it could be a new way to break the tolerance to HBV in chronic HBV-infected patients.

Cell Culture Techniques ; Cells, Cultured ; Dendritic Cells ; cytology ; immunology ; Fetal Blood ; cytology ; Hep G2 Cells ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; immunology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology

Abstract: Our previous work revealed berberine can significantly enhance the susceptibility of fluconazole against fluconazole-resistant Candida albicans, which suggested that berberine has synergistic antifungal activity with fluconazole. Preliminary SAR of berberine needs to be studied for the possibility of investigating its target and SAR, improving its drug-likeness, and exploring new scaffold. In this work, 13-substitutited benzyl berberine derivatives and N-benzyl isoquinoline analogues were synthesized and characterized by 1H NMR and MS. Their synergetic activity with fluconazole against fluconazole-resistant Candida albicans was evaluated in vitro. The 13-substitutited benzyl berberine derivatives 1a-1e exhibited comparable activity to berberine, which suggested that the introduction of functional groups to C-13 can maintain its activity. The N-benzyl isoquinolines, which were designed as analogues of berberine with its D ring opened, exhibited lower activity than berberine. However, compound 2b, 2c, and 4b showed moderate activity, which indicated that berberine may be deconstructed to new scaffold with synergistic antifungal activity with fluconazole. The results of our research may be helpful to the SAR studies on its other biological activities.
Antifungal Agents ; pharmacology ; Berberine ; pharmacology ; Candida albicans ; drug effects ; Drug Resistance, Fungal ; Drug Synergism ; Fluconazole ; pharmacology ; Isoquinolines ; pharmacology ; Microbial Sensitivity Tests