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.
Animals ; Antibodies, Monoclonal ; Blotting, Western ; Chickens ; Chorioallantoic Membrane ; Diagnosis ; Disease Outbreaks ; Epitopes ; Fluorescent Antibody Technique ; Formaldehyde ; Immunohistochemistry ; Influenza A virus ; Influenza in Birds ; Influenza, Human ; Nucleoproteins ; Taiwan

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 10(8.7)TCID₅₀/mL (TCID₅₀, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity.
Adenoviridae ; Amino Acids ; Chickens ; Chorioallantoic Membrane ; Eggs ; Fowl adenovirus A ; Liver ; Malaysia ; Mortality ; Ovum ; Poultry ; Serogroup ; Specific Pathogen-Free Organisms ; Viral Load

Objective: To determine the antiangiogenic activity of Telescopium telescopium (Horn snail) extract using in ovo chorioallantoic membrane (CAM) assay. Methods Methanolic extract of Telescopium telescopium was subjected to modified Kupchan partitioning. Four treatment groups – negative control, positive control (quercetin), test samples, and blanks – were used for the in ovo chorioallantoic membrane (CAM) assay. ImageJ software was used to measure average vessel diameter (DV) and total length (LT) to determine the degree of vascularization, percent inhibition, and antiangiogenic activity. Biochemical screening was done for the crude extract and the fraction with the highest percent inhibition.
Chorioallantoic Membrane ; Gastropoda

OBJECTIVETo investigate the inhibitory effect of von Willebrand factor-cleaving protease, a disintegrin-like and metalloprotease with thrombospondin-1 repeats (ADAMTS13)on angiogenesis induced by vascular endothelial growth factor (VEGF)in vitro and in vivo.

METHODSCell proliferation assay, differentiation (tube formation)assay and wound migration assay were performed by using human umbilical vein endothelial cells (HUVECs)to explore the effect of ADAMTS13 on angiogenesis in vitro. Cells were treated with different concentrations of ADAMTS13 (1, 5, 25, 50 and 100 nmol/L)and the number of cells was counted via MTT assay. In addition, effect of ADAMTS13 on differentiation was assessed by measuring the length of capillary-like tube structures formed by HUVECS in matrigel. Effect of ADAMTS13 on HUVEC migration was assessed via calculation of wound healing distance after 8 hrs culture with VEGF or ADAMTS13. Chick embryo chorioallantoic membrane (CAM) assay and Matrigel plug assay were performed to investigate the effect of ADAMTS13 on angiogenesis in vivo.

RESULTSADAMTS13 significantly inhibited the proliferation of HUVECs induced by VEGF in a dose-dependent manner. Migration distance of HUVECs was (79 ± 22) μm in control group, (250 ± 8)μm in VEGF-treated group and (170 ± 23)μm in VEGF and ADAMTS13 cotreated-group after 8 hrs, respectively. The tube length is (450.6 ± 16.6)% in VEGF-treated group and (235.3 ± 19.0)% in VEGF and ADAMTS13 cotreated-group of that of control group after HUVECs cultured in matrigel for 16 hrs. The number of blood vessels decreased after treatment with ADAMTS13 in CAM assay. The number of blood vessels was (228.2 ± 10.8)%, (69.2 ± 21.1)%, (184.6 ± 15.2)% in VEGF treated-group, ADAMTS13 treated-group and VEGF and ADAMTS13 cotreated-group of that of control group, respectively. Formation of capillary-like network in matrigel plugs containing VEGF was reduced to 43.5% by ADAMTS13 in matrigel plug assay in mouse model.

CONCLUSIONADAMTS13 inhibits the HUVECs proliferation, differentiation and migration in vitro. ADAMTS13 inhibits chick embryos vascularization and formation of capillary-like network in vivo.

ADAM Proteins ; pharmacology ; ADAMTS13 Protein ; Animals ; Cell Movement ; Cell Proliferation ; Chick Embryo ; Chorioallantoic Membrane ; Collagen ; Drug Combinations ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Laminin ; Mice ; Neovascularization, Physiologic ; Proteoglycans ; Vascular Endothelial Growth Factor A

OBJECTIVETo investigate the inhibitory effects of icariin(ICA),an active ingredient of Herb Epimedii,on angiogenesis.

METHODSThe chick chorioallantoic membrane(CAM)assay was adopted to evaluate the effects of various doses of the ICA on the angiogenesis. The cell growth inhibitory effect of ICA on human umbilical vein endothelial cells(HUVEC)was measured by MTT assay. Cell cycle arrest and the induction of apoptosis were evaluated by flow cytometry. The effect of ICA on the migration of HUVEC cells was measured on Transwell model.

RESULTSICA remarkably inhibited angiogenesis in CAM in a concentration-dependent manner. The proliferation of HUVEC cells was inhibited by ICA, and the effect was time-and concentration-dependent. ICA-treated HUVEC cells showed cell cycle arrest;180 μg/ml of ICA decreased the percentage of migrating HUVEC cells by 78.0%.

CONCLUSIONICA can effectively suppress angiogenesis;however,its in vivo inhibitory effect on angiogenesis warrants further investigations.

Angiogenesis Inhibitors ; Animals ; Apoptosis ; Cell Cycle ; Cell Line ; Cell Proliferation ; Chickens ; Chorioallantoic Membrane ; Flavonoids ; Human Umbilical Vein Endothelial Cells ; Humans ; Neovascularization, Physiologic

To study the in vitro anti-angiogenesis effect of three curcumin pigments (curcumin, demethoxycurcumin, bisdemethoxycurcumin). In the study, the inhibitory effect of the three curcumin pigments on proliferation of HUVEC cells induced by OX-LDL and the effect on migration of HUVEC cells were detected. The effect on neovascularization was observed by chorioallantoic membrane (CAM) test. The effect on cell adhesion factors ICAM-1 and VCAM-1 of HUVECs were tested by Real-time RT-PCR. It was found that the three curcumins could inhibit the proliferation of HUVEC cells induced by OX-LDL within the dosage range 4, 8, 16 mg x L(-1), with a dose-dependence. The proliferative effect of curcumins on HUVECs was greater than the other two derivatives (P < 0.01). All of the three curcumin pigments inhibited the migration of HUVEC cells and the angiogenesis of chick chorioallantoic membrane (CAM). The migration inhibition rate of curcumins at middle and high concentrations was greater than the other two (P < 0.01). All of the three curcumin could down-regulate the expression of VEGF and ICAM-1, and curcumins showed more obvious effect in down-regulating VEGF than demethoxycurcumin and bisdemethoxycurcumin(P < 0.01); Bisdemethoxycurcumin showed the most significant effect in down-regulating ICAM-1 (P < 0.01). All of the three showed no remarkable effect on expression of VCAM-1, and only bisdemethoxycurcumin showed the down-regulating effect (P < 0.05). According to the findings, all of the three curcumin pigments could resist angiogenesis by inhibiting proliferation and migration of endothelial cells and down-regulating the expression of VEGF and adhesion molecules ICAM-1.
Angiogenesis Inhibitors ; pharmacology ; Animals ; Cell Movement ; drug effects ; Cells, Cultured ; Chorioallantoic Membrane ; drug effects ; Curcumin ; analogs & derivatives ; pharmacology ; Humans ; Intercellular Adhesion Molecule-1 ; genetics ; RNA, Messenger ; analysis ; Vascular Cell Adhesion Molecule-1 ; genetics ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo evaluate the effect of bear bile powder (BBP) on angiogenesis, and investigate the underlying molecular mechanisms.

METHODSA chick embryo chorioallantoic membrane (CAM) assay was used to evaluate the angiogensis in vivo. Human umbilical vein endothelial cells (HUVECs) were treated with 0, 0.25, 0.5, 0.75, and 1.0 mg/mL of BBP for 24, 48 and 72 h, respectively. The 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to determine the viability of HUVECs. Cell cycle progression of HUVECs was examined by fluorescence-activated cell sorting (FACS) analysis with propidium iodide staining. HUVEC migration was determined by wound healing method. An ECMatrix gel system was used to evaluate the tube formation of HUVECs. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in both HUVECs and HepG2 human cells were examined by reverse transcription-polymerase chain reaction and enzyme linked immunosorbent assay, respectively.

RESULTSCompared with the untreated group, BBP inhibited angiogenesis in vivo in the CAM model (P< 0.01). In addition, treatment with 0.25-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability by 14%-27%, 29%-69% and 33%-91%, compared with the untreated control cells (P< 0.01). Additionally, BBP inhibited the proliferation of HUVECs via blocking the cell cycle G to S progression, compared with the S phase of untreated cells 48.05%± 5.00%, 0.25-0.75 mg/mL BBP reduced S phase to 40.38%± 5.30%, 36.54± 4.50% and 32.13± 3.50%, respectively (Pglt; 0.05). Moreover, BBP inhibited the migration and tube formation of HUVECs, compared with the tube length of untreated cells 100%± 12%, 0.25-0.75 mg/mL BBP reduced the tube length to 62%± 9%, 43%± 5% and 17%± 3%, respectively (p< 0.01). Furthermore, BBP treatment down-regulated the mRNA and protein expression levels of VEGF-A in both HepG2 cells and HUVECs.

CONCLUSIONBBP could inhibit the angiogenesis by reducing VEGF-A expression, which may, in part, explain its anti-tumor activity.

Animals ; Bile ; chemistry ; Cell Cycle ; Cell Movement ; Cell Proliferation ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; Gene Expression Regulation ; Hep G2 Cells ; Human Umbilical Vein Endothelial Cells ; cytology ; Humans ; Neovascularization, Physiologic ; Powders ; RNA, Messenger ; genetics ; metabolism ; Ursidae ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

BACKGROUND/OBJECTIVES: Abundant consumption of seaweeds in the diet is epidemiologically linked to the reduction in risk of developing cancer. In larger cases, however, identification of particular seaweeds that are accountable for these effects is still lacking, hindering the recognition of competent dietary-based chemo preventive approaches. The aim of this research was to establish the antiproliferative potency and angiosuppressive mode of action of Stoechospermum marginatum seaweed methanolic extract using various experimental models. MATERIALS/METHODS: Among the 15 seaweeds screened for antiproliferative activity against Ehrlich ascites tumor (EAT) cell line, Stoechospermum marginatum extract (SME) was found to be the most promising. Therefore, it was further investigated for its anti-proliferative activity in-vitro against choriocarcinoma (BeWo) and non-transformed Human embryonic kidney (HEK 293) cells, and for its anti-migratory/tube formation activity against HUVEC cells in-vitro. Subsequently, the angiosuppressive activity of S. marginatum was established by inhibition of angiogenesis in in-vivo (peritoneal angiogenesis and chorioallantoic membrane assay) and ex-vivo (rat cornea assay) models. RESULTS: Most brown seaweed extracts inhibited the proliferation of EAT cells, while green and red seaweed extracts were much less effective. According to the results, SME selectively inhibited proliferation of BeWo cells in-vitro in a dose-dependent manner, but had a lesser effect on HEK 293 cells. SME also suppressed the migration and tube formation of HUVEC cells in-vitro. In addition, SME was able to suppress VEGF-induced angiogenesis in the chorio allantoic membrane, rat cornea, and tumor induced angiogenesis in the peritoneum of EAT bearing mice. A decrease in the microvessel density count and CD31 antigen staining of treated mice peritoneum provided further evidence of its angiosuppressive activity. CONCLUSIONS: Altogether, the data underline that VEGF mediated angiogenesis is the target for the angiosuppressive action of SME and could potentially be useful in cancer prevention or treatment involving stimulated angiogenesis.
Allantois ; Animals ; Antigens, CD31 ; Carcinoma, Ehrlich Tumor ; Cell Line ; Chorioallantoic Membrane ; Choriocarcinoma ; Cornea ; Diet ; Female ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Kidney ; Methanol ; Mice ; Microvessels ; Models, Theoretical* ; Peritoneum ; Pregnancy ; Rats ; Seaweed ; Vascular Endothelial Growth Factor A

BACKGROUND/OBJECTIVES: Abundant consumption of seaweeds in the diet is epidemiologically linked to the reduction in risk of developing cancer. In larger cases, however, identification of particular seaweeds that are accountable for these effects is still lacking, hindering the recognition of competent dietary-based chemo preventive approaches. The aim of this research was to establish the antiproliferative potency and angiosuppressive mode of action of Stoechospermum marginatum seaweed methanolic extract using various experimental models. MATERIALS/METHODS: Among the 15 seaweeds screened for antiproliferative activity against Ehrlich ascites tumor (EAT) cell line, Stoechospermum marginatum extract (SME) was found to be the most promising. Therefore, it was further investigated for its anti-proliferative activity in-vitro against choriocarcinoma (BeWo) and non-transformed Human embryonic kidney (HEK 293) cells, and for its anti-migratory/tube formation activity against HUVEC cells in-vitro. Subsequently, the angiosuppressive activity of S. marginatum was established by inhibition of angiogenesis in in-vivo (peritoneal angiogenesis and chorioallantoic membrane assay) and ex-vivo (rat cornea assay) models. RESULTS: Most brown seaweed extracts inhibited the proliferation of EAT cells, while green and red seaweed extracts were much less effective. According to the results, SME selectively inhibited proliferation of BeWo cells in-vitro in a dose-dependent manner, but had a lesser effect on HEK 293 cells. SME also suppressed the migration and tube formation of HUVEC cells in-vitro. In addition, SME was able to suppress VEGF-induced angiogenesis in the chorio allantoic membrane, rat cornea, and tumor induced angiogenesis in the peritoneum of EAT bearing mice. A decrease in the microvessel density count and CD31 antigen staining of treated mice peritoneum provided further evidence of its angiosuppressive activity. CONCLUSIONS: Altogether, the data underline that VEGF mediated angiogenesis is the target for the angiosuppressive action of SME and could potentially be useful in cancer prevention or treatment involving stimulated angiogenesis.
Allantois ; Animals ; Antigens, CD31 ; Carcinoma, Ehrlich Tumor ; Cell Line ; Chorioallantoic Membrane ; Choriocarcinoma ; Cornea ; Diet ; Female ; HEK293 Cells ; Human Umbilical Vein Endothelial Cells ; Humans ; Kidney ; Methanol ; Mice ; Microvessels ; Models, Theoretical* ; Peritoneum ; Pregnancy ; Rats ; Seaweed ; Vascular Endothelial Growth Factor A

This work aimed to assess some pharmacological activities of P. leptostachya var. asiatica Hara. The dried roots of P. leptostachya var. asiatica Hara were extracted with 70% ethanol to generate the powdered extract, named PLE. Anti-angiogenic activity was detected using chick chorioallantoic membrane (CAM) assay. In vitro anti-inflammatory activity was evaluated via analyzing nitric oxide (NO) content, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. Antioxidant activity was determined by 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and reactive oxygen species (ROS) level in the stimulated macrophage cells. Matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) activities in the culture media were detected using zymography. PLE exhibits an anti-angiogenic activity in the CAM assay, and displays an inhibitory action on the generation of NO in the LPS-stimulated macrophage cells. In the stimulated macrophage cells, it is able to diminish the enhanced ROS level. It can potently scavenge the stable DPPH free radical. It suppresses the induction of iNOS and COX-2 and the enhanced MMP-9 activity in the stimulated macrophage cells. Both monooxygenase and oxidase activities of tyrosinase were strongly inhibited by PLE. Taken together, the dried roots of P. leptostachya var. asiatica Hara possess anti-angiogenic, anti-inflammatory, antioxidant and skin whitening activities, which might partly provide its therapeutic efficacy in traditional medicine.
Chorioallantoic Membrane ; Culture Media ; Cyclooxygenase 2 ; Ethanol ; Macrophages ; Matrix Metalloproteinase 9 ; Medicine, Traditional ; Monophenol Monooxygenase ; Nitric Oxide ; Nitric Oxide Synthase Type II ; Oxidoreductases ; Reactive Oxygen Species ; Skin*