Novel application of Influenza A virus-inoculated chorioallantoic membrane to characterize a NP-specific monoclonal antibody for immunohistochemistry assaying

Monoclonal antibodies (MAbs) are widely applied in disease diagnoses. Herein, we report a MAb, WF-4, against Influenza A virus nucleoprotein (NP), its broad response with Influenza A virus, and its application in an immunohistochemistry (IHC) assay. WF-4 was screened by immunofluorescence assay (IFA). The results showed that its reactivity with baculovirus-expressed full-length recombinant NP (rNP) in Western blot (WB), indicating its IHC applicability. Fifteen Influenza A virus (reference subtypes H1 to H15) infected chicken embryonated chorioallantoic membranes (CAM), fixed by formalin, were all detectable in the WF-4-based IHC assay. Also, the reactivity of the IHC test with NP from experimentally inoculated H6N1 and from all recent outbreaks of H5 subtype avian Influenza A virus (AIV) field cases in Taiwan showed positive results. Our data indicate that CAM, a by-product of Influenza A virus preparation, is helpful for Influenza A virus-specific MAb characterization, and that the WF-4 MAb recognizes conserved and linear epitopes of Influenza A virus NP. Therefore, WF-4 is capable of detecting NP antigens via IHC and may be suitable for developing various tests for diagnosis of Influenza A virus and, especially, AIV infection.

Clinical study on the effect of warfarin on coagulation and hemorheology in patients with atrial fibrillation / 国际检验医学杂志

Objective To investigate the effect of warfarin on coagulation and hemorheology in patients with atrial fibrillation.Methods 106 cases of admitted in our hospital from October 2014 to October 2016 were randomly divided into observation group(53 cases)and control group(53 cases).The control group re-ceived routine treatment,the observation group received routine and warfarin treatment.The two groups were treated for 4 weeks.The therapeutic effects were compared between the two groups.Before and after treat-ment,indicators of heart function,blood coagulation and hemorheology were observed.Results The total effi-ciency of observation group(92.45%)was higher than that of the control group(73.58%,P<0.05).After treatment,the TT and APTT of the two groups both increased(P<0.05);The TT and APTT of the observa-tion group were higher than that of the control group(P<0.05);The plasma viscosity,erythrocyte aggrega-tion index,and fibrinogen of the two groups decreased(P<0.05);The plasma viscosity,erythrocyte aggrega-tion index,fibrinogen of the observation group were lower than that of the control group(P<0.05).Conclu-sion Warfarin has obvious curative effect on patients with atrial fibrillation,and can improve the function of blood coagulation and hemorheology.

In vitro study of regulation of shear stress on antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel / 生物医学工程学杂志

This study was designed to investigate the changes of prostaglandin I2 (PGI2) and nitric oxide (NO) secreted by endothelialized polyurethane small diameter artificial blood vessel. The peripheral blood mononuclear cells of healthy adult were separated and induced into endothelial progenitor cells (EPCs), which were identified by the methods of discrepancy microphage and fluorescent immunology labeling. After the induced cells being seeded on the polyurethane small-diameter artificial vessels, the endothelialized polyurethane small diameter artificial blood vessels were divided into four different experimental groups, including stationary group, low-flow shear stress group (5 dynes/cm2), medium-flow shearstress group (15 dynes/cm2), and high-flow shear stress group (25 dynes/cm2). Then, the levels of 6-ketoprostaglandin F1alpha (6-keto-PGF1alpha) and NO of different time were measured by enzyme-linked immunosorbent assay and nitrate reductase methods. The peripheral blood mononuclear cells differentiated into EPCs. They presented typical "spindie-shaped" appearance, and they were positively labeled by fluorescent acetylated-LDL, lectin, FLK-1 and vWF. Shear stress enhanced the production of NO and 6-keto-PGF1alpha by EPCs in a dose-dependent manner. Therefore, shear stress increases the secretion of NO and PGI2 by EPC, which suggests that shear stress can improve the antithrombogenic potentials of endothelialized polyurethane small diameter artificial blood vessel.

Fluid shear stress upregulated endothelial nitric oxide synthase gene expression and nitric oxide formation in human endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>To observe the effect of fluid shear stress on the eNOS gene expression and NO production in endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The peripheral blood mononuclear cells from healthy volunteers were inducted into EPCs and divided into stationary group (0 dyn/cm(2), 1 dyn/cm(2) = 0.1 Pa), low-flow shear stress group (5 dyn/cm(2)), medium-flow shear stress group (15 dyn/cm(2)) and high-flow shear stress group (25 dyn/cm(2)). The effects of shear stress on the endothelial nitric oxide synthase (eNOS) gene expression and nitric oxide (NO) production in human EPCs were measured.</p><p><b>RESULTS</b>Typical "spindle-shaped" appearance was shown in EPCs derived from peripheral blood mononuclear cells and were positively labeled by acetylated-LDL, lectin, FLK-1 and vWF. After 4 hours treatment with various shear stresses, the ratio of eNOS/beta-actin mRNA expression by human EPCs in low, medium and high-flow shear stress group was 0.364, 0.505 and 0.548 respectively, which was significantly higher than that in stationary group (0.183, all P < 0.05) and the NO secretion in human EPCs in low, medium and high-flow shear stress group was also significantly higher than that in stationary group (all P < 0.05).</p><p><b>CONCLUSION</b>Fluid shear stress enhances the eNOS mRNA expression and NO secretion in human EPCs, therefore, shear stress could potentiate the repair efficacy of EPCs for endothelial injury.</p>

In vitro induction, differentiation and seeding of source cells for the endothelialization of small-diameter artificial blood vessel of polyurethane / 中国组织工程研究

BACKGROUND: At present, after transplantation of small diameter artificial blood vessel, long-term patency rate is low due to being lacking of endothelial cells for lining and anti-thrombus characters. In some studies,mature endothelial cells were tried to be seeded in the artificial vessel to boost up its anti-thrombus capability so as to improve the long-term patency rate, but we got unsatisfied effect due to the defects of seed cells and scaffolds. Therefore, in clinic, proper seed cells and vascular scaffolds have been searched for improving the long-term low pateney rate in transplantation of small diameter artificial blood vessel.OBJECTIVE: To investigate the feasibility that differentiation of bone marrow mononuclear cells induced in vitro into endothelial-progenitor cells (EPCs) and seed polyurethane small diameter artificial blood vessel so as to provide proper seed cells for endotheliazation of polyurethane small diameter artificial blood vessel.DESIGN: Observation experiment SETTING: Cardivascular Medical Department and Staff Room of Immunology, First Hospital Affiliated to Sun Yat-sen University MATERILAS: This experiment was carried out at the First Hospital Affiliated to Sun Yat-sen University from September 2004 to May 2005. About 10 mL of bone marrow from healthy adult volunteers (n=7) was used in this experiment.METHODS: Bone marrow mononuclear cells of healthy adult were collected and put in the fibronectin pre-coated DMEM culture medium, then induced by vascular endothelial growth factor and basic fibroblast growth factor. Induced cells were observed under fluorescence microscope and identified with immunohistochemical staining. The induced and proliferated EPCs were seeded onto the surface of polyurethane small diameter artificial blood vessel. Morphological change was observed under scanning electron microscope.MAIN OUTCOME MEASURES: ① Cellular morphological change.② Staining results of immunohistochemical VWF and CD 34 antibody . ③ Adhesive growth status of EPCs on the polyurethane small diameter artificial blood vessel RESULTS: ① In the vascular endothelial growth factor and basic fibroblast growth factor and other inducers , bone marrow mononuclear cells differentiated into EPCs , presenting typical "spindle-shaped" appearance under an inverted fluorescence microscope and became to form a monolayer that arrayed in "cobblestone-like" ② Immunohistochemical staining showed von willebrand factor(VWF) and CD34 antigen stained positive. ③ Under the scanning electron microscope, surface of polyurethane small diameter artificial blood vessel without seeded cells presented typical polyporous honeycomb-like structure , and the size of hole suited the crawling of EPCs. After seeding the cells, we observed the adhesion, crawling and spreading of the EPCs on the surface of polyurethane small diameter artificial blood vessel. Some EPCs grew into the honeycomb-like holes were seen occasionally.CONCLUSION: Bone marrow mononuclear cells can be induced and differentiated into EPCs, while induced and differentiated EPCs well grow adhesively in the polyurethane small diameter artificial vessels, suggesting that differentiation of bone marrow mononuclear cells induced in vitro into EPCS, which can be used as seed cells for endothelialization of polyurethane small diameter artificial blood vessels.

A study of association between age-related circulating endothelial progenitor cells and arterial elasticity / 中华心血管病杂志

<p><b>OBJECTIVE</b>Reduced arterial elasticity is a hallmark of aging in healthy humans independently of diseases and endothelial-cell injury and dysfunction may be responsible for this fall in arterial elasticity. We hypothesized that circulating endothelial progenitor cells (EPCs) are involved in endothelial repair and that lack of EPCs contributes to impaired arterial elasticity.</p><p><b>METHODS</b>A total of 56 healthy male volunteers were divided into young (n = 26) and elderly (n = 30) groups. Large and small artery elasticity indices were non-invasively assessed by using pulse wave analysis. Flow cytometer was used to count the number of circulating CD34(+) mononuclear cells (MNCs), which were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. EPCs were characterized as adherent cells double positive staining for DiI-acLDL uptake and lectin binding with using fluorescent microscope.</p><p><b>RESULTS</b>C(1) (large artery elasticity index) and C(2) (small artery elasticity index) were significantly reduced in the elderly group compared with those in the young group (11.73 +/- 1.45 vs 16.89 +/- 1.69 ml/mm Hg x 10, P < 0.001; 8.40 +/- 1.45 vs 10.58 +/- 1.18 ml/mm Hg x 100, P < 0.001 respectively). In parallel, the number of circulating EPCs was significantly reduced in the elderly group compared with the young group (0.13 +/- 0.02 vs 0.17 +/- 0.04%, P < 0.05). The number of circulating EPCs correlated with C(1) large and C(2) small artery elasticity indices (r = 0.47, P < 0.01; r = 0.4, P < 0.01). Fluorescent microscope was used to identify EPCs, which were double positive staining for DiI-acLDL uptake and lectin binding.</p><p><b>CONCLUSION</b>The present findings suggested that the fall in circulating EPCs with subsequently impaired endothelial-cell repair and function might contribute to reduced arterial elasticity in humans with aging. The decrease in circulating EPCs could serve as a surrogate biologic measure of vascular function and human age.</p>

Change in endothelial progenitor cells and endothelial function in patients with unstable angina pectoris / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the relationship between endothelial progenitors cells (EPCs) and endothelium-dependent vasodilation in patients with unstable angina pectoris.</p><p><b>METHODS</b>Thirty patients with unstable angina pectoris (UAP) and thirty control subjects were recruited. Flow-mediated dilation (FMD) in the brachial artery was evaluated by using ultrasound Doppler flow method. The number of circulating EPCs was analyzed by flow cytometry analysis. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. CD34 antigen of adherent cells was identified by immunohistochemical assay. EPCs were characterized as adherent cells double positive for FITC-UEA-I binding and DiI-acLDL uptake by direct fluorescent staining under inverted fluorescent microscope.</p><p><b>RESULTS</b>FMD was significantly impaired in the UAP group compared with the control group (5.93% +/- 2.67% vs 11.1% +/- 4.36%, P < 0.05). There was no significant difference in NMD between two groups (13.60% +/- 5.03% vs 14.18% +/- 4.50%, P > 0.05). The number of CD34(+) cells significantly increased in the UAP group compared with the control group (0.13% +/- 0.05% vs 0.09% +/- 0.04%, P < 0.05). There was a negative association between impaired FMD and increased CD34(+) cell (r = -0.385, P < 0.05). A positive antigen of CD34 of adhesion cells and double positive adhesion cells were found.</p><p><b>CONCLUSION</b>This study shows that the levels of peripheral CD34(+) cells in patients with UAP increase with an impaired endothelial function. The increase in EPCs may be an important compensatory response to acute coronary ischemia and impaired endothelium in patients with UAP.</p>

Tumor necrosis factor-alpha induces release of endothelial microparticles from human endothelial cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>The present study was designed to investigate whether Tumor necrosis factor (TNF)-alpha stimulates release of endothelial microparticles (EMPs) by human endothelial cells, and whether EMPs may serve as a promising marker for endothelial injury and dysfunction.</p><p><b>METHODS</b>Human umbilical venous endothelial cells (HUVEC) were incubated with or without TNF-alpha for 24 hours at 37 degrees C. EMPs generated on the surface of HUVEC were observed with a scanning electron microscopy. The CD31 and CD51 positive EMPs in culture supernatants were measured by flow cytometer.</p><p><b>RESULTS</b>Fewer vesicles were observed on cell surface of control group, in TNF-alpha-stimulated one, however, cells manifested a blebby surface (eruption phenomenon) and more vesicles on surface were observed. The levels of EMPs were significantly increased in TNF-alpha stimulated cells compared with controls [CD31 + EMP, (164 +/- 63)/1000 cells vs. (42 +/- 10)/1000 cells, P < 0.05; CD51 + EMP, (260 +/- 108)/1000 cells vs. (19 +/- 4)/1000 cells, P < 0.05].</p><p><b>CONCLUSION</b>TNF-alpha can stimulate HUVEC to release EMPs which may serve as a surrogate marker for endothelial injury and dysfunction.</p>

Impaired endothelium-dependent vasodilation and arterial elasticity in patients with coronary artery disease / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the change in endothelium-dependent vasodilation and arterial elasticity and the association between them in patients with coronary artery disease (CAD).</p><p><b>METHODS</b>Thirty patients with CAD and thirty control subjects were recruited for this study. Flow-mediated dilation (FMD) in the brachial artery was evaluated by ultrasound Doppler flow method. They also underwent a non-invasive assessment of C(1) large artery and C(2) small artery indices by using pulse wave analysis.</p><p><b>RESULTS</b>FMD was significantly reduced in CAD group compared with that in control group [(5.17 +/- 2.13)% vs (11.1 +/- 4.36)%, P < 0.05], C(1) large artery elasticity index was similar between the two groups [(11.59 +/- 4.56) ml/mm Hg x 10 vs (12.11 +/- 3.82) ml/mm Hg x 10, P > 0.05]. However, C(2) small artery elasticity index was significantly reduced in CAD group compared with that in control group [(4.20 +/- 1.80) ml/mm Hg x 100 vs (6.26 +/- 2.36) ml/mm Hg x 100, P < 0.05]. There was a positive association between reduced C(2) small artery elasticity index and impaired FMD (r = 0.53, P < 0.05).</p><p><b>CONCLUSIONS</b>There were impaired endothelium-dependent vasodilation and reduced C(2) small artery elasticity index in the patients with CAD, which were closely correlated with each other. The present study suggested that the measurement of C(2) small artery elasticity might be used as a novel index for the determination of endothelial function.</p>

Fluid shear stress regulates secretion of tissue-type plasminogen activator in human endothelial progenitor cells / 中华心血管病杂志

<p><b>OBJECTIVE</b>In order to investigate the role of shear stress in the regulation of endothelial function, we assessed here effects of shear stress on tissue-type plasminogen activator in human endothelial progenitor cells (EPCs).</p><p><b>METHODS</b>The peripheral blood mononuclear cells were separated from healthy adult and inducted into EPCs, which were identified by double staining for the fluorescent labeled acetylated-LDL and lectin. EPCs were seeded on the small diameter artificial vessels, and then divided into four different experimental groups including stationary group, low-flow shear stress group (5 dyn/cm(2)), medium-flow shear stress group (15 dyn/cm(2)) and high-flow shear stress group (25 dyn/cm(2)). The levels of t-PA in EPC culture medium at 0 hour, 5 hours, 10 hours, 15 hours, 20 hours and 25 hours after culture were measured by enzyme-linked immunosorbent assay.</p><p><b>RESULTS</b>The peripheral blood mononuclear cells differentiated into EPCs after induction, which were positively labeled by fluorescent acetylated-LDL and lectin. Shear stress enhanced production of the t-PA by EPCs, which was paralleled to levels and times of shear stress.</p><p><b>CONCLUSIONS</b>Shear stress increases t-PA secretion by human EPCs, suggesting that shear stress not only regulates vascular endothelial function but also participates in the pathogenesis of arteriosclerosis.</p>