Specific scAbs could be obtained through biopanning from phage antibody libraries by use of antigens as target molecules. scAbs specific to thrombin were separated from mouse antibody library by the panning strategy of alternating liquid-solid phase in this paper. Thrombin was biotinylated by photobiotin at first, then avidin-coated magnetic beads were utilized to isolate specific scAbs. The eluted phages were amplified and subject to the second round panning in microtiter plate to remove the unspecific reombinant phages. 4 specific scAbs were separated from 23 phage clones after four rounds of alternative panning.

Objective To investigate the clinical effect of renal transplantation in elderly patients.Methods Retrospective analysis was held on 49 elderly patients aged over 65 years who underwent renal transplantation,and compared with 87 patients under the age of 50 years who underwent the renal transplantation during the same time.Results The survival rate at one year was 85.7% in elderly patients,and 93.1% in the non-elderly patients(P＜0.05).The graft survival rate at one year was 89.8% in elderly patients,and 90.8% in the non-elderly patients,but no significant difference was found between the two groups.The rate of acute rejection response was 10.2% in elderly patients,significantly lower than that in non-elderly patients(28.7%).The rate of complication of cardio-cerebrovascular diseases in elderly patients(51.1%)were higher than those in non-elderly patients(19.5%,P＜0.05).The dosages of immunosuppressive agent cyclosporine in 3 months,3-6 months and 6-12 months were(3.6?1.1),(3.1?0.9)mg?kg~(-1)?d~(-1) and (2.9?0.7) mg?kg~(-1)?d~(-1)in elderly group,significantly lower than those[(5.4?1.4),(4.6?1.3)mg?kg~(-1)?d~(-1)and(4.1?1.0)mg?kg~(-1)?d~(-1)]in non-elderly group(P＜0.05).Conclusions Renal transplantation in elderly patients is effective and reliable.

Objective To explore the clinical distribution and significant of 23 kinds of human papillomavir-us(HPV)genetypes in normal cells and atypical squamous cells(ASC-US),meanwhile analysis result of cervi-cal histological pathology diagnosis in cases of ASC-US.Methods A total of 1 000 women with normal cells specimens were recruited into control group,and 236 women with ASC-US were selected into the ASC-US group.Polymerase chain reaction(PCR)and gene-chips technology were utilized for the detection of 23 kinds of HPV genetypes,all cases of ASC-US diagnosis of cervical pathological histology.Results A total of 106 ca-ses of HPV infection were detected in the control group,as the total HPV infection rate was 10.6%,in which the single genotypes infection rate was 9.3% and the multiple genotypes infection rate was 1.3%.A total of 139 cases of HPV infection were detected in ASC-US group,as the total HPV infection rate was 58.9%,in which the single genotypes infection rate was 38.1%,and the multiple genotypes infection rate was 20.8%. There were significant differences on the total HPV infection rate,the infection rates of type 1 and multiple geontypes between the control group and ASC-US group(P<0.05).The top six of constituent ratio in the control group were type 43,16,58,33,52,18,42,those in the ASC-US group were type 16,18,52,58,33,51,66.Conclusion PCR combined with the gene-chip technology could be used in the HPV genotypes detection in cervical cells,which has important clinical significance on the further distribution management of ASC-US,and should be draw great attention.

OBJECTIVETo explore the underlying mechanism of mesenchymal stem cells (MSCs) transfer induced cardiac function improvement in failing hearts.

METHODSCongestive heart failure (CHF) was induced in rats by cauterization of the heart wall. MSCs were cultured from autologous bone marrow and injected into the border zone and the remote myocardium 5 days after cauterization.

RESULTSTen weeks later, cardiomyocyte nucleus mitotic index, capillary density and expression of insulin-like growth factor 1 (IGF-1), hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) were significantly increased in the border zone and significantly reduced in the remote myocardium in CHF rats (all P<0.05 vs. sham). Besides cardiac function improvement and left ventricular remodeling attenuation evidenced by hemodynamic and echocardiographic examinations, expressions of IGF-1, HGF and VEGF in the remote myocardium and in the border zone were also significantly upregulated (P<0.05 or P<0.01 vs. CHF), and cardiomyocyte nucleus mitotic index as well as capillary density were significantly increased in CHF rats with MSCs (P<0.05 or P<0.01 vs. CHF). Moreover, collagen area was significantly reduced and myocardial area was significantly increased in the border zone in these rats too.

CONCLUSIONAutologous MSC implantation upregulated expressions of growth factors enhanced cardioangiogenesis which might be the underlying mechanisms for improved cardiac function and attenuated left ventricular remodeling induced by MSCs transplantation in failing rat myocardium.

Animals ; Disease Models, Animal ; Heart Failure ; metabolism ; therapy ; Hepatocyte Growth Factor ; metabolism ; Insulin-Like Growth Factor I ; metabolism ; Male ; Mesenchymal Stem Cell Transplantation ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transplantation, Autologous ; Vascular Endothelial Growth Factor A ; metabolism ; Ventricular Remodeling

The prokaryotic expression plasmid pQE30-HN of hemagglutinin-neuraminidase (HN) protein gene of bovine parainfluenza virus type 3 (BPIV3) strain HJ-1 was expressed by IPTG induction in E. coli XL1Blue. The recombinant HN protein(rHN) was purified by electroeluting method, and used as coated antigen. An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect the antibody valence of BPIV3. The best working conditions of ELISA were as follows: the antigen concentration was 6 microg/mL; the serum dilution was 1:50; the blocking reagent was 5% skimmed milk; the blocking time was 60 min at 37 degrees C; the second antibody concentration was 1:10 000; The cut-off value was 0.30. The method revealed a good specificity, no cross-reaction to the positive sera of BCV, IBRV or BRSV was observed. We applied the method to detect 323 serum samples of dairy cow in Heilongjiang Province, the seropositivity rate of BPIV3 was about 58%. The indirect ELISA established provided a technological basis for the development of ELISA kit.
Animals ; Antibodies, Viral ; blood ; Blotting, Western ; Cattle ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; Female ; HN Protein ; genetics ; Parainfluenza Virus 3, Bovine ; genetics ; immunology

The purpose of this study was to investigate the vasorelaxing effect and mechanism of idoxifene (a new estrogen receptor modulator) on human internal mammary artery (HIMA). HIMA segments were harvested from men during coronary artery bypass grafting surgery. Patients with diabetes mellitus, hypercholesterolemia, hypertension, or smoking habit were excluded. The vasorelaxing effect of idoxifene on artery rings from HIMA with and without endothelium was measured by means of perfusion in vitro. Cumulative dose-response to idoxifene in the range of 0.01-10 micromol/L was observed in the presence and absence of NO synthase inhibitor L-NAME. It was also studied whether the vasodilation effect of idoxifene on HIMA was blocked by methylene blue (MB), an inhibitor of guanylate cyclase (GC). The results obtained from idoxifene were compared with those from 17beta-estradiol (E(2)). It was found that idoxifene caused a concentration-dependent relaxation on HIMA. The dose range was from 0.03 micromol/L (minimal vasodilatory concentration) to 3 mmol/L (maximal vasodilatory concentration). It was also found that the vasorelaxation effect of idoxifene on HIMA was dependent on endothelium. E(2) (0.1-100 micromol/L) also resulted in an endothelium-dependent vasorelaxation, but the vessels were 15-fold less sensitive to E(2) than to idoxifene in their vasorelaxation responses. The EC(50) for E(2) was 4.65+/-0.34 micromol/L, compared with 0.32+/-0.02 micromol/L for idoxifene. The mean maximal vasodilatory value of E(2) was 88.3+/-5.7%, compared with 88.6+/-7.2% for idoxifene. Pretreatment with L-NAME (100micromol/L) abolished idoxifene-induced vasodilation virtually by blocking nitric oxide production. The vasorelaxing effect of idoxifene disappeared in the presence of MB (10 micromol/L). These findings demonstrate that idoxifene results in an endothelium-dependent vasorelaxation of HIMA, like estrogen. The effect of idoxifene is more potent than that of traditional estrogen, and is possibly mediated by NO-GC-cGMP pathway.
Estrogen Antagonists ; pharmacology ; Humans ; Mammary Arteries ; drug effects ; physiology ; Tamoxifen ; analogs & derivatives ; pharmacology ; Vasodilation ; drug effects

OBJECTIVE: To investigate the changes of phospho-JNK (p-JNK) during the incised wound healing of the skin in mice and to explore the rule of the time-dependent change of p-JNK in wound age determination. METHODS: The changes of p-JNK expression in incised skin wound were detected by immunohistochemistry and Western blot. RESULTS: There was a minimal baseline staining of p-JNK in control mouse skin. Changes of p-JNK expression were mainly detectable in neutrophils in the wound specimens from 3 hours to 12 hours after injury. Afterwards, the p-JNK positive cells were mostly mononuclear cells and fibroblasts between post-injury day 1 and day 5, whereas the p-JNK positive cells were mostly fibroblasts between post-injury day 7 and day 14. Morphometrically, the ratio of the p-JNK positive cells to the total increased gradually in the wound specimens from 3 hours to day 1, and maximized at day 1 with a slight decrease from post-injury day 3 to day 5. The ratio showed a second peak in the specimens of day 7, and then decreased gradually from post-injury day 10 to day 14. The changes of p-JNK expression were observed throughout the wound healing stages by Western blot as well, with a peak expression occurring between 12 hour and day 3 after injury. CONCLUSION p-JNK may play a pivotal role in inducing apoptosis of neutrophils, mononuclear cells, and fibroblasts during skin wound healing and meanwhile, p-JNK may be a potentially useful marker for wound age determination.
Animals ; Biomarkers ; Female ; Forensic Medicine ; JNK Mitogen-Activated Protein Kinases/metabolism* ; Male ; Mice ; Phosphorylation ; Random Allocation ; Skin/injuries* ; Time Factors ; Wound Healing ; Wounds, Penetrating/enzymology*

OBJECTIVETo evaluate the prognosis effect of Chinese herbal medicines (CHMs) for benefiting qi and activating blood circulation adjunctive to conventional treatment in patients with acute coronary syndrome (ACS) after percutaneous coronary intervention (PCI).

METHODSA total of 702 patients with ACS who underwent PCI were enrolled and randomly assigned to receive conventional treatment plus CHMs for benefiting qi and activating blood circulation (treatment group, 351 cases) or conventional treatment alone (control group, 351 cases) for 6 months. Six months later, all patients received conventional treatment alone. Follow-ups were scheduled at 6th, 12th, 18th, 24th month after enrollment in April 2008, and the final follow-up visit was during September 2011 and November 2011. The primary endpoint was the composite of cardiac death, nonfatal myocardial infarction or revascularization (PCI or coronary artery bypass grafting); and the secondary endpoint was the composite of re-admission for ACS, congestive heart failure, nonfatal stroke or other thrombus events.

RESULTSA total of 621 (88.59%) patients completed 35.4±3.8 months follow-up, while 80 (11.41%) patients withdrew from the trial (41 in the treatment group and 39 in the control group). The incidence of primary endpoint was 5.7% (20 patients) in the treatment group versus 10.86% (38 patients) in the control group [relative risk (RR): 0.53; 95% confidence interval (CI): 0.30, 0.88; P=0.013; absolute risk reduction (ARR):-0.052, 95% CI: -0.06, 0.01]. The incidence of secondary endpoint was 5.98% (21 patients) in the treatment group versus 10.28% (36 patients) in control group (RR: 0.58, 95% CI: 0.33, 0.97, P=0.037; ARR: -0.043, 95% CI: 0.06, 0.01). Most of the primary and secondary endpoints were occurred in 18 months (84.50% in the treatment group versus 78.10% in the control group).

CONCLUSIONCHMs for benefiting qi and activating blood circulation adjunctive to conventional treatment improved clinical outcomes for patients with ACS after PCI in long-term follow-up.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People’s Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories’ results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample’s highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.

Objective: To investigate the current status and real performance of the detection of RUNX1-RUNX1T1 fusion transcript levels and WT1 transcript levels in China through interlaboratory comparison. Methods: Peking University People's Hospital (PKUPH) prepared the samples for comparison. That is, the fresh RUNX1-RUNX1T1 positive (+) bone morrow nucleated cells were serially diluted with RUNX1-RUNX1T1 negative (-) nucleated cells from different patients. Totally 23 sets with 14 different samples per set were prepared. TRIzol reagent was added in each tube and thoroughly mixed with cells for homogenization. Each laboratory simultaneously tested RUNX1-RUNX1T1 and WT1 transcript levels of one set of samples by real-time quantitative PCR method. All transcript levels were reported as the percentage of RUNX1-RUNX1T1 or WT1 transcript copies/ABL copies. Spearman correlation coefficient between the reported transcript levels of each participated laboratory and those of PKUPH was calculated. Results: ①RUNX1-RUNX1T1 comparison: 9 samples were (+) and 5 were (-) , the false negative and positive rates of the 20 participated laboratories were 0 (0/180) and 5% (5/100) , respectively. The reported transcript levels of all 9 positive samples were different among laboratories. The median reported transcript levels of 9 positive samples were from 0.060% to 176.7%, which covered 3.5-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.5 to 12.3 (one result which obviously deviated from other laboratories' results was not included) , 85% (17/20) of the laboratories had correlation coefficient ≥0.98. ②WT1 comparison: The median reported transcript levels of all 14 samples were from 0.17% to 67.6%, which covered 2.6-log. The ratios of each sample's highest to the lowest reported transcript levels were from 5.3-13.7, 62% (13/21) of the laboratories had correlation coefficient ≥0.98. ③ The relative relationship of the reported RUNX1-RUNX1T1 transcript levels between the participants and PKUPH was not always consistent with that of WT1 transcript levels. Both RUNX1-RUNX1T1 and WT1 transcript levels from 2 and 7 laboratories were individually lower than and higher than those of PKUPH, whereas for the rest 11 laboratories, one transcript level was higher than and the other was lower than that of PKUPH. Conclusion: The reported RUNX1-RUNX1T1 and WT1 transcript levels were different among laboratories for the same sample. Most of the participated laboratories reported highly consistent result with that of PKUPH. The relationship between laboratories of the different transcript levels may not be the same.
China ; Core Binding Factor Alpha 2 Subunit ; Humans ; Leukemia, Myeloid, Acute ; RUNX1 Translocation Partner 1 Protein ; Real-Time Polymerase Chain Reaction ; Transcription, Genetic ; WT1 Proteins