Objective To develop a convenient method efficiently expands the frequency of specific CTLS.Methods We used different concentrations of CMV-speeific epitope peptides pp65 to stimulate PBMCs for expansion of CMV-specific CTLs.CMV-specifie CTLs were doubly labeled by tetramers-PE and CD_8-FITC for FACS analysis.Results The method expands CMV-speeific CTLs efficiently.CMV-specific CTLs were expanded from 1% to 20% of PBMCs quickly(namely 40% of CD_8~+ T cells).The method provided a large number of cells with tetramer staining of CD_8~+ T cells for FACS analysis from a single blood sampling.Conclusions Peptides stimulation methods are convenient,easy to operate and expanded CMV- specific CTLs efficiently.The increased frequencies of CMV-specific CTLs allowed the data of different individuals to be easily compared and sequentially evaluated.The methods lay the base for adoptive immunotherapy to prevent CMV disease.

Objective To explore the role of tissue inhibitor of metalloproteinase-1(TIMP-1) during renal senescence by using human TIMP-1 transgenic mice.Methods Renal histological changes of wild type mice and transgenic mice at the age of 3,12,24 months were observed by periodic acid-schiff(PAS)staining of paraffin sections.The numbers of F4/80 positive cells were detected by immunofluoreseence.The protein expressions of TIMP-1,TIMP-2,matrix metalloproteinase(MMP)-9,MMP-2,intercellular adhesion molecule-1(ICAM-1),transforming growth factor?1(TGF-?1),collagenⅢand collagenⅣwere detected by Western blot.The activities of gelatinases and TIMP-1 were examined by gelatin zymography and reverse zymography respectively.Results Focal renal fibrosis was found in two genotypes with aging.At the age of 24 months,compared with wild type,in kidneys of transgenic type,the expressions and activities of gelatinases were dowregulated (MMP-2:2.08?0.20 vs.3.39?0.43;MMP-9:4.02?0.82 vs.6.72?1.40,all P＜0.05);the expressions of collagenⅢ,collagenⅣ,ICAM-1,and TGF-?1 were upragulated(0.72+0.11 vs.0.57?0.09;0.84?0.13 vs.0.6?0.11,0.72?0.12 vs.0.53?0.07; 0.69?0.12 vs.0.45?0.09,all P＜0.05),and the numbers of F4/80 positive cells were increased (18.8?4.4 vs.12.7?3.6,P＜0.05)with the upregulated expression and activity of TIMP-1(1.10?0.18 vs.0.62?0.09;50.75?7.25 vs.20.64?3.50,P＜0.05).Conclusions TIMP-1 could promote age-related renal fibrosis through enhancing inflammation reaction by ICAM-1 upregulation.

Objective To explore the effect of Shenfu Decoction combined with western medicine treatment of elderly patients with multiple organ dysfunction syndrome (MOF) clinical effect. Methods 65 cases of elderly patients with MOF as the research object, were randomly divided into observation group (n=35) and control group (n=30). The two groups were given conventional standard western medicine treatment, the observation group on the based on the combination of Shenfu Decoction treatment. Results After treatment, two groups of blood pressure and urine volume were significantly increased (P<0.05), and the observation group was significantly higher than the control group (P<0.05); the two groups of patients with respiratory and heart rate were significantly decreased (P<0.05), and the observation group was significantly lower than the control group (P<0.05); two groups of Scr, BUN, UA were significantly decreased (P<0.05), and the observation group was significantly lower than the control group (P<0.05); the two groups after treatment IL-6, CRP, TNF-α levels decreased significantly (P<0.05), and the observation group was significantly lower than the control group (P<0.05). Conclusion Shenfu Decoction combined with western standard clinical effect in treatment of elderly MOF is considerable.

Objective To explore the different apoptotic gene expressions and apoptotic signaling transduction of human rhabdomyosarcoma (RD) cells infected by enterovirus 71 (EV71) in different stage.Methods The survival of EV71-infected RD cells was observed by trypan blue staining.The apoptotic morphology and rates of RD cells were surveyed and detected by Annexin V-FITC/PI staining and flow cytometry,respectively.PCR array was employed to analyze 88 apoptotic gene expressions from EV71-infected RD cells at 8 h and 20 h postinfection (p.i),respectively.Results After RD cells were infected with EV71 (MOI =5) at 8 h p.i,the viability was significantly decreased.Flow cytometry data demonstrated that the apoptotic rates of EV71-infected RD cells had increased to 18.0％ and 19.1％ at 20 h p.i in early and later stage respectively.RT-PCR array studies revealed significant variations in the expression of apoptotic genes.Among 88 genes,only the expression of IFN-β1 was upregulated 5.22 folds,whereas 47 genes including ACIN1,Akt,Apaf1,caspase and CIDEB were found to be downregulated that were lower than 2 folds at 8 h p.i.However,28 genes including FasL,CD40L,TNF,caspase-10 and caspase-3 were induced more than 2 folds after EV71 infection at 20 h.Conclusion The downregulation of apoptosis-related genes is associated with viral apoptosis-suppressing effect in RD cells in the early stage of EV71 infection.The death receptor signaling pathways including Fas/FasL and TNF/TNFR are activated to induce cell apoptosis in the late stage of EV71 infection.Moreover,host cell can also inhibit apoptosis by regulating signal pathway of CD40/CD40L,NF-κB/RelA and PI3K/Akt activation.

Objective To investigate the effects of dexmedetomidine pretreatment on rat lung oxidative stress injury induced by ischemia-reperfusion (I/R).Methods The model of lung I/R injury was established by clamping the hilum of left lung for 45 min followed by reperfusion.Twenty-four SD rats were randomly divided into sham group,model group,and experiment group.Each group had eight rats.Rats in the sham group were underwent left thoracotomies with no I/R.Rats were received normal saline or dexmedetomidine at a rate of 3.0 μg · kg-1 · h-1 for 60 min in the model group or experiment group before ischemia,respectively.After reperfusion of 120 min,blood sample from left pulmonary vein was collected for blood gas analysis.HE staining was used to evaluate lung injury score (LIS).Xanthine oxidase method and thiobarbituric acid method were used to detected superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in the left lung tissues,respectively.Western Blot was used to detected nuclear factor E2-related factor 2 (Nrf2) protein expression of left lung tissue.Results Compared with the sham group of LIS 3.5 ± 0.6,MDA content (1.6 ± 0.3) nmol · mg-1,SOD activity (65.1 ± 4.8) U · mg-1,the LIS 8.5 ± 1.3 and MDA content (5.8 ± 2.2) nmol · mg-1 in model group increased significantly;while the SOD activity (30.3 ± 4.1) U · mg-1 in the model group decreased significantly (P ＜ 0.05).Compared with the model group,LIS (5.0 ± 1.2) and MDA content (2.7 ± 0.3) nmol · mg-1 in experiment group reduced significantly(P ＜ 0.05),while SOD activity (61.4 ± 1.9) U · mg-1 in experiment group increased significantly also (P ＜ 0.05).Compared with the sham group on nuclear Nrf2 protein expression of 0.25 ± 0.04,nuclear Nrf2 protein expression in the model group decreased with 0.12 ± 0.02,which had significant difference (P ＜ 0.05).Compared with the model group,the nuclear Nrf2 protein expression in the experiment group increased of 0.53 ± 0.05,which showed significant difference (P ＜ 0.05).Conclusion Dexmedetomidine pretreatment decreased the MDA content and increased the SOD activity via activating Nrf2 pathway,and ameliorated lung oxidative stress induced by I/R and improved lung function.

Licorice-gypsum (gancao-shigao, GC-SG) drug pair was used as the research object, using supramolecular chemistry to explore the scientific connotation of combining herbal medicine GC with insoluble mineral medicine SG in clinical application of traditional Chinese medicine. ① The Tyndall effect, microscopic morphology and particle size of the single and co-decocted of GC and SG were observed, the paste content and conductivity were determined, and the interaction between GC and SG was detected by isothermal titration calorimetry (ITC) and infrared absorption spectroscopy (IR). ② Calcium chloride (CaCl₂), a soluble calcium salt of equal gypsum quality, was used instead of SG with GC for co-decocting to explore the effect of calcium salt content on the water decocting, and the characteristics were combined with the Tyndall effect, microscopic morphology, paste content and conductivity. ITC and IR techniques were used to detect the interaction between the two, and the interaction between them was detected by ITC and IR. The zeta potential and ultraviolet-visible spectrophotometry (UV-vis) of GC-SG and GC-CaCl₂ co-decoction were compared, and the inorganic and organic components in the co-decoction were detected by inductively coupled plasma optical emission spectrometer (ICP-OES) and high performance liquid chromatography (HPLC). The results showed: ① Compared with the liquid phase of single decoction, GC-SG co-decoction had more obvious Tyndall effect, and showed uniform spherical nanoparticles under electron microscope. Physical characterization results such as paste content and conductivity showed that co-decoction promoted the dissolution of each other's components; ITC and IR results showed that there was strong interaction between GC and SG, which preliminatively indicated that GC and SG co-decoction promoted the formation of uniform and stable supramolecular system of traditional Chinese medicine. ② When soluble calcium salt was used to substitute insoluble SG with GC for co-decocting, a stronger but astigmatic light path appeared than single decocting solution, the zeta potential was reduced, and a large number of accumulated polymers were formed. The results of paste content and conductivity showed that the dissolution of the co-decocting component was reduced than the single decocting component. ITC, UV-vis and IR results showed that there was interaction between GC with Ca²⁺ and SG. The formation of polysink indicated that a large amount of soluble calcium salt would destroy the stability of supramolecular Chinese medicine. The results of ICP-OES and HPLC showed that the glycyrrhizic acid (GA) content of the former lower than the latter, which was related to the formation of a large number of polycondensates with the increase of Ca²⁺ concentration and the decrease of the dissolution of GA and other active ingredients. This study indicates that the compatibility of GC and SG can form a uniform and stable supramolecular system of traditional Chinese medicine. Calcium salt, the main component of SG, is taken as the starting point. Excessive soluble Ca²⁺ can promote the aggregation of active ingredients such as GA, so as to reveal the scientific connotation of the compatibility of GC and SG, an insoluble mineral medicine.

OBJECTIVETo investigate the epidemiological features, genetic drift in the epitopes of hemagglutinin (HA) of the novel influenza A (H1N1) virus and oseltamivir-resistant variants characterized by H275Y and N295S mutations in children in Shanghai since the outbreak.

METHODBetween June 2009 and May 2012, a prospective surveillance study was carried out in Shanghainese children who attended the outpatient clinic of Children's Hospital of Fudan University for influenza-like illness. One-step real-time fluorescence quantitative RT-PCR was performed to detect seasonal influenza A and influenza B virus and the novel influenza A (H1N1) virus in the respiratory samples. Genetic drift from the vaccine strain in HA epitopes of the novel influenza H1N1 virus and the molecular markers associated with oseltamivir resistance in neuraminidase (NA) were analyzed.

RESULTOut of 3475 enrolled cases, the novel influenza A (H1N1) virus was confirmed virologically in 222 (6.4%) otherwise healthy children with 133 (59.9%) being boys and 89 (40.1%) girls. The median ages of children with the novel influenza A (H1N1) virus infection during the first wave from August 2009 to February 2010 and the second wave from December 2010 to February 2011 were 53.5 months and 32.0 months, respectively (Z = -4.601, P = 0.000); 119 (46.9%) had the close contact with persons suffering from fever or respiratory infection, of whom, 68 (57.1%) contacts were family members and 47 (39.5%) contacts were classmates. During the outbreak in 2009-2010 season, 66 (40.9%) were exposed to primary index cases, school students were the major exposure subjects, accounting for 50.0%. The nucleotide sequences of HA1 gene were highly homologous between the vaccine strain A/California/07/2009 and Shanghai circulating novel influenza A (H1N1) strains and only S83P mutation in epitope E of HA was detected inclusively in the circulating strains. The H275Y and N295S amino acid mutations associated with oseltamivir resistance were not found in the circulating novel influenza (H1N1) strains.

CONCLUSIONTwo major waves of the novel influenza A (H1N1) outbreaks occurred in Shanghainese children during 2009-2011. Institutional children were the major affected individuals during the 2009 pandemic wave. Households and schools were the main sites of transmission among children during influenza pandemic. Influenza vaccination should be enhanced in children and their close family contacts. The novel influenza A (H1N1) virus in Shanghai has not undergone significant genetic changes. Oseltamivir is effective for the treatment of the novel influenza A (H1N1) virus.

Adolescent ; Amino Acid Sequence ; Antiviral Agents ; pharmacology ; Child ; Child, Preschool ; China ; epidemiology ; Drug Resistance, Viral ; Female ; Hemagglutinins, Viral ; genetics ; Humans ; Infant ; Influenza A Virus, H1N1 Subtype ; classification ; genetics ; isolation & purification ; Influenza, Human ; drug therapy ; epidemiology ; pathology ; virology ; Male ; Molecular Epidemiology ; Molecular Sequence Data ; Neuraminidase ; genetics ; Oseltamivir ; pharmacology ; Pandemics ; Viral Vaccines ; genetics ; immunology

To prepare cyclosporine A (CyA) loaded block copolymer micelles and observe its release behaviors in vitro and pharmacokinetics in rats, methoxylpoly (ethylene glycol)-poly (D, L-lactide-co-glycolide) (mPEG-PLGA) was synthesized by ring-opening copolymerization of lactide and glycolide in the presence of methoxylpoly (ethylene glycol) (mPEG) as initiator. The structure of the mPEG-PLGA copolymer was confirmed with 1H NMR and FT-IR. The cyclosporine A loaded micelles (CyA-PM) were prepared by solvent evaporation method and their morphology was observed by the transmission electron microscope (TEM). The mean size and size distribution were determined by dynamic light scattering (DLS). The release behaviors in vitro and pharmacokinetics in rats were investigated by HPLC method using cyclosporine A injection commercial agent, sandimmune, as the reference. The obtained CyA-PM showed spherical shape with the core-shell structure, the mean particle sizes are in the range of 136.1-141.9 nm. The drug loading amount and entrapment efficiency were increased and the particle size became smaller with decreasing the ratio of acetone to water. With the increasing of the amount of cyclosporine A fed the drug loading increased, entrapment efficiency decreased and the particle size had no change. CyA-PM showed significant sustained release behave in vitro compared with sandimmune and only 9.7% of encapsulated cyclosporine A was released after 12 hours, the release characteristics was well fitted with Higuchi equation (r = 0.999). The Pharmacokinetics study at equal administration dosage (5 mg x kg(-1)) in rats showed the half-life (t1/2) of CyA-PM extended and the area under concentration-time curve (AUC) increased compared to sandimmune. The results also showed that cyclosporine A concentration-time data were all in accord with two compartment model. Cyclosporine A loaded mPEG-PLGA micelles showed obviously solubility enhancement, sustained release and overcome the side effect and toxicity of sandimmune resulted from solubiling agent-polyoxyethylene castor oil (Cremophor EL) and might be developed as a novel dosage form of cyclosporine A.
Animals ; Area Under Curve ; Cyclosporine ; administration & dosage ; pharmacokinetics ; Delayed-Action Preparations ; Drug Carriers ; administration & dosage ; pharmacokinetics ; Drug Compounding ; Half-Life ; Immunosuppressive Agents ; administration & dosage ; pharmacokinetics ; Male ; Micelles ; Particle Size ; Polyesters ; administration & dosage ; chemical synthesis ; pharmacokinetics ; Polyethylene Glycols ; administration & dosage ; chemical synthesis ; pharmacokinetics ; Polymers ; administration & dosage ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

This study is to investigate the modulation of Kudiezi (KDZ) injection on differential protein expression in cerebral cortex of rats with cerebral ischemic stroke and heat toxin syndrome established by intraperitoneal injection of carrageenan and middle cerebral artery occlusion (MCAO) methods. According to random number table rats were divided into three groups: drug group, model group and sham group. The tripheye tetrazolium chloride (TTC) staining and HE staining were used to observe brain tissue injury of rats. After therapeutic intervention with above drug for seventy-two hours, the level of differential protein expression was analyzed by two-dimensional gel electrophoresis (2-DE). The results show that there are differential protein expressions between cerebral ischemic stroke and heat toxin syndrome rats and sham rats. Furthermore, as a Chinese medicine injection with effect of clearing heat, resolving toxin and dredging collaterals, KDZ injection can decrease alleviate morphological changes of cerebral ischemia, regulate the levels of some differential proteins expression.
Animals ; Brain Ischemia ; drug therapy ; genetics ; metabolism ; pathology ; Cerebral Cortex ; drug effects ; metabolism ; pathology ; Drugs, Chinese Herbal ; administration & dosage ; Gene Expression ; drug effects ; Humans ; Male ; Rats ; Rats, Sprague-Dawley ; Stroke ; drug therapy ; genetics ; metabolism ; pathology

To study the effect of Tibetan medicine Zuotai on the activity, protein and mRNA expression of CYP1A2 and NAT2, three different doses (1.2, 3.8 and 12 mg x kg(-1)) of Zuotai were administrated orally to rats once a day or once daily for twelve days, separately. Rats were administrated orally caffeine (CF) on the second day after Zuotai administration, and the urine concentration of CF metabolite 5-acetylamino-6-formylamino-3-methyl-uracil (AFMU), 1-methyluric acid (1U), 1-methylxanthine (1X), 1, 7-dimethylxanthine (17U) at 5 h after study drug administration was determined by RP-HPLC. The activity of CYP1A2 and NAT2 was evaluated by the ratio of metabolites (AFMU+1X+1U)/17U and the ratio of AFMU/(AFMU+1X+1U), respectively. The protein and mRNA expression of CYP1A2 and NAT2 were determined by ELISA and RT-PCR method, respectively. After single administration of Zuotai 3.8 mg x kg(-1) and repeated administration of Zuotai 3.8 and 12 mg x kg(-1), the activity of CYP1A2 and NAT2 decreased significantly compared with control group and there was no significant difference between other dose group and control group. The protein expression of CYP1A2 was significant lower than that in control group after repeated administration of Zuotai 12 mg x kg(-1), and the mRNA expression of CYP1A2 decreased significantly compared with that of control group after single administration of Zuotai 3.8 mg x kg(-1) and repeated admistration of Zuotai 12 mg x kg(-1), separately. The protein expression of NAT2 decreased significantly compared with that of control group after single and repeated administration of Zuotai 3.8 mg x kg(-1), respectively, and the mRNA expression of CYP1A2 decreased significantly compared with control group after single administration of Zuotai 3.8 mg x kg(-1). This study found that Tibetan medicine Zuotai had significant effect on the activity, protein and mRNA expression of CYP1A2 and NAT2.
Administration, Oral ; Animals ; Arylamine N-Acetyltransferase ; genetics ; metabolism ; Caffeine ; metabolism ; urine ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Female ; Male ; Medicine, Tibetan Traditional ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; Theophylline ; urine ; Uracil ; analogs & derivatives ; urine ; Uric Acid ; analogs & derivatives ; urine ; Xanthines ; urine