Objective To investigate whether the Notch-1 signaling pathway is involved in the acquisition of the epithelial-mesenchymal transition (EMT) phenotype of gefitinib-acquired resistant lung cancer cells.Methods The PC9 cell line (harboring EGFR exon 19 deletion) and PC9/AB2 cells (gefitinibacquired resistant PC9 cells) were used.siRNA targeting Notch-1 eukaryotic expression vector (siNotch-1) was constructed and PC9/AB2 cells were transfected with siNotch-1.The protein expression of EGFR,Akt,Erk,Notch receptors and ligands,TGF-β receptors,E-cadherin,Vimentin,and Snail were detected by Western blot assay.DNA sequencing and fluorescence in situ hybridization (FISH) analysis were processed to detect mutation of EGFR exon 20 and MET amplification,respectively.For cytotoxicity assay,cell viability was assessed by Cell Counting Kit 8.Results Gefitinib resistant cell line PC9/AB2 had no evidence of MET amplification or EGFR T790M mutation.The expression of Notch-1 was upregulated in gefitinib resistant PC9/AB2 cells compared with that in gefitinib-sensitive PC9 cells.There were no significant protein expression differences of other Notch receptors,Notch ligands or TGF-β receptors between both paired cell lines.Western blot results showed that protein expression of E-cadherin was greatly reduced in PC9/AB2 cells,while elevated levels of Vimentin and Snail were observed.A significant reduction of the expression of Snail and Vimentin in Notch-1 siRNA transfected PC9/AB2 cells with increased E-cadherin expression was found by Western blot assay.PC9/AB2 cells displayed a round like cell morphology after Notch-1 siRNA transfection.Silence of Notch-1 decreased colony-formation ability but enhanced sensitivity of gefitinib on PC9/AB2 cells.Conclusion Notch-1 might play a novel role in acquired resistance to gefitinib,which could be reversed by inhibiting Notch-l.

To enhance the specificity of anti-TNF-α single chain Fv antibody (TNF-scFv) to inflamed site, we constructed a bispecific antibody BsDb that targets TNF-α and ED-B-containing fibronectin (B-FN) by covalently linking TNF-scFv and the anti-ED-B scFv L19 at the gene level via a flexible peptide linker deriving from human serum albumin. BsDb was successfully secreted from Pichia pastoris as functional protein, identified by immunoblotting, and purified to homogeneity with affinity chromatography. BsDb retained the immunoreactivity of its original antibodies TNF-scFv and L19, and showed a marked gain in antigen-binding affinity and in TNF-α-neutralizing ability, when compared to TNF-scFv and L19 that were produced in Escherichia coli. In the adjuvant-induced arthritis (AIA) mice model, BsDb showed selective accumulation and retention in the inflamed paws but rapid clearance from blood, resulting in high arthritic paw to blood ratios. These data indicate that BsDb is endowed with high specificity to inflamed site and low toxicity to normal tissues and holds great potential for in vivo application for the targeted therapy of RA and other chronic inflammatory diseases.
Animals ; Antibodies, Bispecific ; biosynthesis ; immunology ; Antibodies, Neutralizing ; biosynthesis ; immunology ; Escherichia coli ; Fibronectins ; chemistry ; immunology ; Humans ; Mice ; Single-Chain Antibodies ; biosynthesis ; immunology ; Tumor Necrosis Factor-alpha ; immunology

Objective To study the protective effect of different doses of rosiglitazone (RSG)against focal cerebral ischemia-reperfusion injury in rats．Methods Forty male sprague Dawley rats were randomly divided into the following groups：sham-operation，control，lowdose RSG(1 mg/kg·d)，moderate-dose(2 mg/kg·d)，high-dose(4 mg/kg·d)goups(n=8 in each grow)．A rat model of middle cerebral artery occlusion for 2 hours and reperfusion for 22 hours were induced by the intraluminal suture method．An automatie biochemical analyzer was used to detect the blood glucose and lipid levels．A solid phase sandwich enzyme-linked immunosorbent assay was used to determine serum insulin levels，and the insulin sensitivity indexes were calculated．The effect of rosiglitazone on infarct volume and behavioristics was observed．Results Compared with the control group，the infarct volume was significantly reduced，and the neurological function scores were improved significantly in each RSG group(P<0．05 or P<0．01)．The insulin sensitivity was significantly increased(P<0．05 or P<0．01)，and hyperglycemia was reduced significantly after cerebral ischemia(P<0．05)and showed a dose-dependent manner.Conclusions RSG has obvious neuroprotective effect against cerebral ischemia／repeffusion injury in rats．Its mechanism may be associated with the increase of insulin sensitivity．

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.

ObjectiveTo investigate the effect of isoflurane anesthesia on plasma cortisol,and brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in hippocampus in rots.MethodsThirty-six adult male SD rots,aged 10 weeks,weighing 250-280 g,were randomly assigned into 6 groups:control group (group C,n =6) and O2 group (group O,n =6),isoflurane group (group Ⅰ,n =24).The rats were exposed to 2％ isoflurane for 2 h (FGF 3 L/min) in group Ⅰ.While the rats were only exposed to the pure oxygen in group O.Six rats in each group were chosen to perform Morris water maze test after inhalation of pure oxygen was stopped in group O,and at 2 h,and at 1,7 and 14 days after the end of administration in group Ⅰ.The escape latency and swimming distance in place navigation test,and the number of crossing the platform and swimming distance in spatial probe test were recorded.After water maze test was terminated at each time point,blood samples were taken from the fossa orbitalis to determine the plasma cortisol concentration and the hippocampal tissue was obtained for measurement of the contents of BDNF and NGF.ResultsCompared with group C,the number of crossing the platform was significantly decreased,the swimming distance was significantly shortened,and the plasma cortisol concentration was significantly decreased in spatial probe test in group O,and the escape latency and swimming distance were significantly prolonged at 1 day after the end of administration in plaee navigation test,and the number of crossing the platform and the content of BDNF in the hippocampal tissue were significantly decreased,and the swimming distance was significantly shortened in spatial probe test in group Ⅰ (P ＜ 0.05 or 0.01 ).Conclusion lsoflurane anesthesia exerts a transient inhibitory effect on cognitive function in the short term,and promotion of the cortisol release and synthesis of BDNF is involved in the mechanism,but not the synthesis of NGF in hippocampus in rats.

In order to enhance the specificity of TNF-α monoclonal antibody to inflamed site, a bispecific antibody BsDb that targets TNF-α and the extra-domain B (ED-B) of fibronectin (FN) was constructed by covalently linking the anti-TNF-α single chain Fv antibody (TNF-scFv) and the anti-ED-B scFv L19 via a flexible peptide linker deriving from human serum albumin (HSA). ED-B is an antigen specifically expressed at the inflamed site. BsDb is expressed in E. coli, identified by immunoblot, and purified with affinity chromatography. This was followed by further examination of its bioactivities and pharmacokinetics. We demonstrated that BsDb retained the immunoreactivity of its original antibodies as it could simultaneously bind to TNF-α and ED-B and neutralize the biological action of TNF-α. In the collagen-induced arthritis mice model, BsDb selectively accumulate in the inflamed joint with a maximal uptake of (12.2 ± 1.50)% ID/g in a single inflamed paw and retain in the inflamed paw for at least 72 h. In contrast, BsDb showed a short serum half-life of (0.50 ± 0.05) h and a rapid clearance from normal tissues. The findings reported herein indicate that BsDb has good specificity to the inflamed site and low toxicity to normal tissues. BsDb is therefore likely to have greater clinical applications in the treatment of rheumatoid arthritis and other autoimmune diseases. This laid a stable basis for its preclinical study.
Animals ; Antibodies, Bispecific ; chemistry ; Antibodies, Monoclonal ; chemistry ; Arthritis, Experimental ; Escherichia coli ; Fibronectins ; chemistry ; Half-Life ; Humans ; Mice ; Single-Chain Antibodies ; chemistry ; Tumor Necrosis Factor-alpha ; chemistry

BACKGROUND AND OBJECTIVEExtraskeletal Ewing's sarcoma (EES) is a rare, rapidly growing, round-cell, malignant tumor that can develop in the soft tissues at any location. This study was to analyze the clinical features, diagnosis and treatment of EES.

METHODSClinical data of 18 patients with EES, treated at between Cancer Center of Sun Yat-sen University between 1995 and 2007, were analyzed.

RESULTSOf the 18 patients, 13 were male and 8 were female, aged from 8 months to 60 years. Twelve (66.7%) patients were between 5-25 years of age. Eight (44.4%) patients had tumors originated from low extremities.Sixteen patients had masses at their first visit. Sixteen patients were treated by the combined modality therapy, and 2 patients were treated by the single modality therapy. The 1-, 3- and 5- year actuarial survival rates were 82.4%, 64.2% and 32.1%, respectively. The presence of metastatic disease at the time of diagnosis and the mode of treatment were prognostic factors.

CONCLUSIONSEES is common in adolescent. It often manifests as a localized mass. The combined modality therapy is recommended for this disease. The presence of metastatic disease at the time of diagnosis and the mode of treatment are prognostic factors.

12E7 Antigen ; Adolescent ; Adult ; Antigens, CD ; metabolism ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Bone Neoplasms ; secondary ; Cell Adhesion Molecules ; metabolism ; Child ; Child, Preschool ; Combined Modality Therapy ; Female ; Humans ; Infant ; Lower Extremity ; Lung Neoplasms ; secondary ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm, Residual ; Radiotherapy, High-Energy ; Sarcoma, Ewing ; diagnosis ; metabolism ; pathology ; surgery ; therapy ; Soft Tissue Neoplasms ; diagnosis ; metabolism ; pathology ; surgery ; therapy ; Survival Rate ; Vimentin ; metabolism ; Young Adult

The present paper aims to investigate the relation between characteristic parameters of transesophageal photoelectric pulse wave in descending aorta and ambulatory artery blood pressure. The chests of ten adult experimental dogs were performed to take the photoelectric pulse wave of descending aorta transesophageally. The concurrent femoral artery invasive blood pressure was recorded simultaneously. Stepwise regression analysis method was used to study the correlation efficient between characteristic parameters of descending aorta pulse wave (H, h, h/H, g/H, At, s, H(1 + ts/td), k)and invasive artery blood pressure. The characteristic parameters, k and h/H (ratio: 90% and 80%) was proved that they had good correlation with systolic pressure; and k, H and s (ratio: 90%, 80% and 70%), had good correlation with diastolic pressure; while k and H (ratio: 90% for both) had good correlation with mean pressure. The mean values of multiple correlation coefficients of the selected characteristic parameters of descending aorta pulse wave with systolic pressure, diastolic pressure and mean pressure of femoral artery were 0.871, 0.900 and 0.856, respectively. The characteristic parameters of descending aorta pulse wave had specific correlation with systolic pressure, diastolic pressure and mean pressure.
Animals ; Aorta, Thoracic ; physiology ; Blood Pressure ; physiology ; Blood Pressure Monitoring, Ambulatory ; methods ; Dogs ; Electrophysiologic Techniques, Cardiac ; methods ; Female ; Femoral Artery ; physiology ; Male ; Pulse Wave Analysis ; methods ; Regression Analysis

Objective To investigate the protective mechanism of dexmedetomidine ( Dex) hydrochloride to lung ischemia/reperfusion injury ( LIRI) in mice. Methods The wild type ( WT) and Toll-like receptor 4 knockout ( TLR4-/-) C57BL/6 Balb/c female mouse randomly divided into four groups: sham group ( S group) , pulmona-ry ischemia/reperfusion group ( I/R group) , normal saline group ( NS group) and Dex group ( D group) . In S group, the chest was opened only, but in I/R group, NS group and D group, model of lung ischemia/reperfusion injury in mice was made by clamping left pulmonary hilum for 30 min, and then reperfusion for 3 h. The lung tis-sue was observed by HE staining. RT-qPCR detected the expression of TLR4 mRNA, and ELISA measured the TNF-α, IL-6 and IL-1 levels, including WT and TLR4-/-. Western blot measured the expression of NLRP3 in lung tissue in both WT and TLR4-/-. Results Dex significantly decreased the pathological damage of LIRI, re-duced the expression of levels of TLR4 mRNA and the production of inflammatory cytokines ( P＜0.01) , and also suppress production and activation of NLRP3 ( P＜0.01) in lung ischemia/reperfusion tissue in WT mice. But no cytokines was found to be inhibited in TLR4-/- mice. Conclusions Dex may decrease the release of a variety of pro-inflammatory factors and inhibit production and activation of NLRP3 inflammasome by TLR4, thereby protect lung against LIRI.

OBJECTIVETo investigate the effects of ulinastatin on gut mucosal apoptosis and bacterium translocation in a rat model of sepsis.

METHODSFifty rats were randomly assigned into 4 groups, namely the control (n=5, no operation or drugs), ulinastatin pretreatment (n=15, treated with 25,000 U/kg ulinastatin 2 h before operation), ulinastatin treatment (n=15, treated with 25,000 U/kg ulinastatin 2 h after operation) and sepsis model (n=15, without drug treatment) groups. The rats in the later 3 groups were subjected to cecal ligation and puncture (CLP). At 3, 6 and 12 h after CLP, the rats were sacrificed and the ileum was removed to examine the pathology and apoptosis of the mucosa. The DNA of Bacillus coli in the whole blood was detected using PCR.

RESULTSSepsis caused of epithelial cell loss in the ileal villi, ulceration and blebbing of the lamina propria. Ulinastatin treatment administered before and after the operation both significantly alleviated these morphological anomalies. The sepsis rats showed significantly increased intestinal mucosal apoptotic index as compared with the other 3 groups (P<0.05). Ulinastatin pretreatment, in comparison ulinastatin treatment 12 h after CLP, significantly increased the intestinal mucosal apoptotic index (P<0.05). Bacillus coli DNA was positive in sepsis and postoperative ulinastatin treatment groups but negative in the control and pretreated groups.

CONCLUSIONIncreased intestinal musocal apoptosis and gut bacterial translocation occur in rats following sepsis, and ulinastatin can effectively decrease intestinal mucosal apoptosis and inhibit bacterial translocation.

Animals ; Apoptosis ; drug effects ; Bacterial Translocation ; drug effects ; Female ; Glycoproteins ; pharmacology ; therapeutic use ; Ileum ; drug effects ; microbiology ; pathology ; Intestinal Mucosa ; drug effects ; microbiology ; pathology ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Sepsis ; drug therapy ; Trypsin Inhibitors ; pharmacology ; therapeutic use