Novel Associations between Related Proteins and Cellular Effects of High-Density Lipoprotein

BACKGROUND AND OBJECTIVES: Recent studies have examined the structure-function relationship of high-density lipoprotein (HDL). This study aimed to identify and rank HDL-associated proteins involved in several biological function of HDL.METHODS: HDLs isolated from 48 participants were analyzed. Cholesterol efflux capacity, effect of HDL on nitric oxide production, and vascular cell adhesion molecule-1 expression were assessed. The relative abundance of identified proteins in the highest vs. lowest quartile was expressed using the normalized spectral abundance factor ratio.RESULTS: After adjustment by multiple testing, six proteins, thyroxine-binding globulin, alpha-1B-glycoprotein, plasma serine protease inhibitor, vitronectin, angiotensinogen, and serum amyloid A-4, were more abundant (relative abundance ratio ≥2) in HDLs with the highest cholesterol efflux capacity. In contrast, three proteins, complement C4-A, alpha-2-macroglobulin, and immunoglobulin mu chain C region, were less abundant (relative abundance ratio <0.5). In terms of nitric oxide production and vascular cell adhesion molecule-1 expression, no proteins showed abundance ratios ≥2 or <0.5 after adjustment. Proteins correlated with the functional parameters of HDL belonged to diverse biological categories.CONCLUSIONS: In summary, this study ranked proteins showing higher or lower abundance in HDLs with high functional capacities and newly identified multiple proteins linked to cholesterol efflux capacity.

Lipid Raft Integrity Is Required for Survival of Triple Negative Breast Cancer Cells / 한국유방암학회지

PURPOSE: Lipid rafts are cholesterol enriched microdomains that colocalize signaling pathways involved in cell proliferation, metastasis, and angiogenesis. We examined the effect of methyl-β-cyclodextrin (MβCD)-mediated cholesterol extraction on the proliferation, adhesion, invasion, and angiogenesis of triple negative breast cancer (TNBC) cells. METHODS: We measured cholesterol and estimated cell toxicity. Detergent resistant membrane (DRM) and non-DRM fractions were separated using the OptiPrep gradient method. Cell cycles stages were analyzed by flow cytometry, apoptosis was assessed using the TdT-mediated dUTP nick end-labeling assay, and metastasis was determined using a Matrigel invasion assay. Neo-vessel pattern and levels of angiogenic modulators were determined using an in vitro angiogenesis assay and an angiogenesis array, respectively. RESULTS: The present study found that the cholesterol-depleting agent MβCD, efficiently depleted membrane cholesterol and caused concentration dependent (0.1–0.5 mM) cytotoxicity compared to nystatin and filipin III in TNBC cell lines, MDA-MB 231 and MDA-MB 468. A reduced proportion of caveolin-1 found in DRM fractions indicated a cholesterol extraction-induced disruption of lipid raft integrity. MβCD inhibited 52% of MDA-MB 231 cell adhesion on fibronectin and 56% of MDA-MB 468 cell adhesion on vitronectin, while invasiveness of these cells was decreased by 48% and 52% respectively, following MβCD treatment (48 hours). MβCD also caused cell cycle arrest at the G2M phase and apoptosis in MDA-MB 231 cells (25% and 58% cells, respectively) and in MDA-MB 468 cells (30% and 38% cells, respectively). We found that MβCD treated cells caused a 52% and 58% depletion of neovessel formation in both MDA-MB 231 and MDA-MB 468 cell lines, respectively. This study also demonstrated that MβCD treatment caused a respective 2.6- and 2.5-fold depletion of tyrosine protein kinase receptor (TEK) receptor tyrosine kinase levels in both TNBC cell lines. CONCLUSION: MβCD-induced cholesterol removal enhances alterations in lipid raft integrity, which reduces TNBC cell survival.

Interleukin-33 and Mast Cells Bridge Innate and Adaptive Immunity: From the Allergologist's Perspective / 대한배뇨장애요실금학회지

Interleukin (IL) 33, a member of the IL-1 superfamily, is an "alarmin" protein and is secreted in its active form from damaged cells undergoing necrotic cell death. Mast cells are one of the main effector cell types in allergic disorders. They secrete a variety of mediators, including T helper 2 cytokines. As mast cells have high-affinity IgE receptors (FcepsilonRI) on their surface, they can capture circulating IgE. IgE-bound mast cells degranulate large amounts of histamine, heparin, and proteases when they encounter antigens. As IL-33 is an important mediator of innate immunity and mast cells play an important role in adaptive immune responses, interactions between the two could link innate and adaptive immunity. IL-33 promotes the adhesion of mast cells to laminin, fibronectin, and vitronectin. IL-33 increases the expression of adhesion molecules, such as intracellular adhesion molecule-1 and vascular cell adhesion molecule-1, in endothelial cells, thus enhancing mast cell adhesion to blood vessel walls. IL-33 stimulates mast cell proliferation by activating the ST2/Myd88 pathway; increases mast cell survival by the activation of survival proteins such as Bcl-XL; and promotes the growth, development, and maturation of mast cell progenitors. IL-33 is also involved in the activation of mature mast cells and production of different proinflammatory cytokines. The interaction of IL-33 and mast cells could have important clinical implications in the field of clinical urology. Epithelial dysfunction and mast cells could play an important role in the pathogenesis of interstitial cystitis. Urinary levels of IL-33 significantly increase in patients with interstitial cystitis. In addition, the number of mast cells significantly increase in the urinary bladders of patients with interstitial cystitis. Therefore, inhibition of mast cell activation and degranulation in response to increase in IL-33 is a potential therapeutic target in the treatment of interstitial cystitis.

Uso de plasma rico en plaquetas en la práctica clínica / Platelet rich plasma in the clinical practic

En los últimos 10 años se ha estado hablando con relación a los beneficios terapéuticos de la terapia con plasma rico en plaquetas (PRP) y su aplicación en distintas áreas de la salud como la odontología, la ortopedia, la cirugía vascular y la medicina estética entre otras (1-4). El uso del PRP como herramienta terapéutica se sustenta en la presencia de múltiples factores de crecimiento dentro de los gránulos alfa de las plaquetas, entre los que se encuentran el factor de crecimiento derivado de plaquetas, factor de crecimiento transformante beta, factor plaquetario 4, interleuquina 1, factor de angiogénesis derivado de plaquetas, factor de crecimiento vascular endotelial, factor de crecimiento epidérmico, factor de crecimiento endotelial derivado de plaquetas, factor de crecimiento epitelial, factor de crecimiento similar a la insulina, osteocalcina, osteonectina, fibrinógeno, vitronectina, fibronectina y trombospondina 1 (5).

In the last 10 years, the therapeutic benefits of platelet-rich plasma (PRP) therapy and its application in different areas of health such as dentistry, orthopedics, vascular surgery and aesthetic medicine have been discussed among others (1-4). The use of PRP as a therapeutic tool is supported by the presence of multiple growth factors within the alpha granules of platelets, among which are platelet-derived growth factor, transforming growth factor beta, platelet factor 4, interleukin 1, platelet-derived angiogenesis factor, vascular endothelial growth factor, epidermal growth factor, platelet-derived endothelial growth factor, epithelial growth factor, insulin-like growth factor, osteocalcin, osteonectin, fibrinogen, vitronectin, fibronectin and thrombospondin 1 (5).

Expression Change of Complement Regulator Genes of the Human Astrocytoma Cell Line after A beta1-42 and Interferon Gamma Administration

BACKGROUND: We determined the changes of complement regulator gene expression in the amyloid-beta1-42(A beta1-42) and interferon-gamma(IFN-gamma)-stimulated human astrocytoma cell line. METHODS: The human astrocytoma cell line, U373MG, was stimulated with IFN-gamma(62.5-1,000U/ml) in the presence or absence of aggregated A beta1-42(1-20micrometer) for 24 hours. Messenger RNA expression of C1 inhibitor(C1-INH), complement factor I(CFI), clusterin, vitronectin, decay accelerating factor(DAF), membrane cofactor protein(MCP), and CD59 was measured by quantitative real-time reverse transcriptase-PCR. RESULTS: IFN-gamma(final concentration, 500U/ml) markedly increased the expression of mRNA for C1-INH in a time dependent fashion. A beta1-42(final concentration, 2micrometer) induced a slight increase in the expression of C1-INH. Messenger RNAs for CFI and clusterin were minimally increased, but other regulators were unchanged or decreased by either A beta1-42 or IFN-gamma. IFN-gamma overrode A beta1-42-induced mRNA expression of C1-INH when the cells were treated with these two reagents together. CONCLUSION: Among the complement regulator genes in the human astrocytoma cell line, U373MG, only C1-INH was significantly up-regulated by IFN-gamma with or without A beta1-42 administration.

Reactive Oxygen Species Production, Expression of Complement Regulator Genes and Phagocytosis in the Murine Microglial Cell after Administration of Beta-Amyloid(A beta1-42) Protein

BACKGROUND: Microglia is a primary cellular component of neuritic plaques in Alzheimer's disease. Beta amyloid deposits attract microglia and activate them to produce inflammatory mediators. The objectives of this study were to characterize activation of the microglia; production of reactive oxygen species (ROS), constitutive and upregulated expression of complement regulators, and intracellular localization of amyloid by phagocytosis. METHODS: BV-2 cells, mouse microglia cell line, were incubated for 3~18 hours with a single dose of 20 micro M of aggregated A beta1-42. ROS measurement was done with FACScan. Messenger RNA expressions of C1-INH, vitronectin, CD59, clusterin, factor H, and superoxide dismutase (SOD) were detected by RT-PCR. The intensity of bands from 6% polyacrylamide electrophoretic gel was analyzed by a bioimage analyzer. The intracellular localization of A beta in the phagocytosed microglia was observed by transmission electron microscope. RESULTS: A beta1-42 activates microglia with an increase of ROS production. Expression of mRNA for SOD was also increased. Messenger RNA for C1-INH and vitronectin were upregulated. A beta fibrils were located in the phagosome of microglia. CONCLUSIONS: A beta activated microglia are playing dual roles as effector and scavenger cell, which as a result, may contribute to the chronic neuroinflammation of Alzheimer's disease.

Envolvimento da proteína prion celular e seus ligantes nos mecanismos de plasticidade neuronal e neuroproteção / Involvement of cellular prion protein and its ligands in the mechanisms of neuronal plasticity and neuroprotection

Atualmente muitos grupos de pesquisa estão empenhados em desvendar a função da forma celular da proteína prion, PrPc. PrPc é uma isoforma normal da proteína prion infecciosa (PrPsc-proteína prion scrapie) relacionada às Encefalopatias Espongiformes Transmissíveis (TSEs), genericamente designadas por doenças priônicas. Uma das maneiras de esclarecer o papel de PrPc é investigar moléculas ligantes e associá-las a fenômenos biológicos. As funções propostas para PrPc vão desde atividade semelhante a superóxido dismutase, proteção contra estresse oxidativo, diferenciação neuronal à sinalização. Em 1997, descrevemos um receptor/ligante para PrPc utilizando o princípio da hidropaticidade complementar. No presente trabalho, apresentamos o isolamento e identificação deste ligante de PrPc como sendo STI1 (?Stress inducible protein 1?), uma co-chaperonina. In vitro, a STI1 interage com PrPc de maneira específica, saturável e com alta afinidade (Kd=10-7M). Ex vivo, a interação entre PrPc e STI1, possui efeito neuroprotetor ao ativar a via de PKA (Proteína quinase dependente de AMPc), além disso, promove crescimento neurítico através da via de MAPK (Proteína quinase ativada por mitógeno) em neurônios do sistema nervoso central. Paralelamente, mostramos que PrPc atua como um ligante de proteínas de matriz extracelular: vitronectina e laminina. A interação entre vitronectina e PrPc leva ao crescimento axonal de células do sistema nervoso periférico. Ao interagir com laminina, PrPc induz formação e manutenção de neuritos e se mostra importante para os mecanimos de consolidação da memória de curta e longa duração, sendo que este processo requer a ativação de vias clássicas de sinalização (PKA/MAPK). Assim, a caracterização das interações PrPc-STI1, PrPc-Vn e PrPc-Ln representa contribuições importantes para a elucidação do papel biológico de PrPc...(AU)

owadays, many research groups are interested in unraveling the role of the cellular prion protein, PrPc, which is the normal isoform of protein associated with the Transmissible Spongiform Encephalopaties (TSEs), generically designated prion diseases. Several biological roles for PrPc have been proposed. In 1997, our group described a PrPc receptor/ligand based on the complementary hydropathy theory. Herein, we showed the identification of the PrPc receptor/ligand as STI1, or Stress inducible protein 1. In vitro studies demonstrated that STI1 is a specific, saturable and high affinity ligand for PrPc (Kd=10-7M). Ex vivo, PrPc-STI1 interaction promoted neurite outgrowth through MAPK (Mitogen activated protein kinase) and showed neuroprotective effects by activating PKA (cAMP-dependent protein kinase) in central nervous system neurons. We also demonstrated that PrPc act as an extracellular matrix protein receptor for vitronectin (Vn) and laminin (Ln). The interaction between Vn and PrPc led to axonal outgrowth in peripheral nervous system. Upon its interaction with Ln, PrPc induced formation and maintenance of neurites and participated in short and long term memory consolidation mechanisms by activating classical signaling pathways (PKA/MAPK). Thus, the characterization of PrPc-STI1, PrPcVn and PrPc-Ln interactions represents important contributions for the elucidation of PrPc physiological roles (AU)

Construction of CHO-IVB, A serum-independent, apoptosis-resistant cell line that can grow in adherence / 生物工程学报

Without serum to provide adherent factors, CHO-dhfr- cells grow in suspension when cultured in serum-free medium. Although this offers advantages in some applications, in most production systems adherent cell growth is preferable. Gene transfection, clonal selection and amplification can be easier for adherent cells; the density of immobilized cells is often higher than those in suspension culture, which results in a higher protein productivity; washout of cells by perfused medium during continuous fermentation can be avoided; for high-throughput microplate assays, adherent cells are preferred to facilitate medium changes and cell washing. It has been proved that purified vitronectin alone was able to mediate attachment and spreading of CHO cells in serum-free medium. So we constructed a tricistronic expression vector expressing Igf-1, Vitronectin and Bel-2 at the same time. The vector was transfected into CHO-dhfr- cells and one clone, namely CHO-IVB2, expressing high level of the three proteins was screened out by Western blot. The cell line showed similar apoptosis-resistant and serum-independent properties to CHO-IB, an engineered cell line constructed before. When cultured in IMEM protein-free medium without any components supplemented, CHO-IVB can grow adherently. The viable cell numbers and growth rate of CHO-IVB were much higher than CHO-IB, making CHO-IVB an apoptosis-resistant host for production of recombinant proteins which can grow adherently in protein-free medium.

A proteína prion celular e seus ligantes - vitronectina, STI1 e laminina - nos mecanismos de plasticidade neuronal / The cellular prion protein and its ligants - vitronectin, STI1 and laminin - in the mechanisms of neuronal plasticity

Prions são agentes etiológicos das encefalopatias espongiformes transmissíveis, doenças que acometem tanto homens quanto animais. A proteína infecciosa, PrPsc, é uma isoforma de uma proteína celular normal denominada PrPc. As funções de PrPc ainda causam controvérsia na literatura, mas já foi demonstrada a participação de PrPc em uma variedade de fenômenos biológicos, como homeostase de íons cobre, proteção contra estresse oxidativo, sinalização celular e neuritogênese entre outros. A interação de PrPc com laminina, uma proteína de matriz extracelular, leva a formação e manutenção de neuritos em neurônios hipocampais. Seguindo este caminho, demonstramos no presente trabalho a interação de PrPc com outra proteína de matriz extracelular, vitronectina (Vn)...

Heparan sulfate and control of cell division: adhesion and proliferation of mutant CHO-745 cells lacking xylosyl transferase

We have examined the role of cell surface glycosaminoglycans in cell division: adhesion and proliferation of Chinese hamster ovary (CHO) cells. We used both wild-type (CHO-K1) cells and a mutant (CHO-745) which is deficient in the synthesis of proteoglycans due to lack of activity of xylosyl transferase. Using different amounts of wild-type and mutant cells, little adhesion was observed in the presence of laminin and type I collagen. However, when fibronectin or vitronectin was used as substrate, there was an enhancement in the adhesion of wild-type and mutant cells. Only CHO-K1 cells showed a time-dependent adhesion on type IV collagen. These results suggest that the two cell lines present different adhesive profiles. Several lines of experimental evidence suggest that heparan sulfate proteoglycans play a role in cell adhesion as positive modulators of cell proliferation and as key participants in the process of cell division. Proliferation and cell cycle assays clearly demonstrate that a decrease in the amount of glycosaminoglycans does not inhibit the proliferation of mutant CHO-745 cells when compared to the wild type CHO-K1, in agreement with the findings that both CHO-K1 and CHO-745 cells take 8 h to enter the S phase

Apoptosis of Alveolar Cells in Pneumocystis Carinii Pneumonia: Application of Electron Microscopic Terminal Deoxynucleotidyl Transferase-Mediated dUTP-Biotin Nick End Labeling Method

BACKGROUND: Pneumocystis carinii (P. carinii) attaches to alveolar cells and causes injury to the epithelial cells by direct toxic effects or inhibition of epithelial growth and replication. Although respiratory cell damage or death is a common feature in P. carinii pneumonia, there has been little reports about expression of apoptosis of the lung tissue in the literatures. METHODS: We examined expression of fibronectin and vitronectin in the interaction between P. carinii and alveolar cells, and in situ terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end-labeling (TUNEL) expression of apoptosis in the respiratory cells by immunohistochemistry and pre-embedding immunoelectron microscopy. RESULTS: Light microscopic (LM) and electron microscopic (EM) immunohistochemical stains for the fibronectin and vitronectin showed strong expressions on the pellicles and tubular extensions of P. carinii and weak expression along the surfaces of type I alveolar cells. LM and EM TUNEL stains showed positive expression in the nuclei of alveolar cells, apoptotic bodies in the cytoplasm of alveolar macrophages and cellular debris in alveolar spaces. CONCLUSIONS: P. carinii induces injury and apoptosis of alveolar cells after attachment of the organisms to host cells, and alveolar macrophages enhance the clearance of apoptotic bodies of alveolar cells as well as phagocytosis and degradation of P. carinii.

Inhibitory Effect of Antibody to alphavbeta5 in Corneal Angiogenesis

PURPOSE: This study investigated the importance of alphavbeta5 function during vascular endothelial growth factor (VEGF) induced corneal angiogenesis by examining the effects of antibody to alphavbeta5 that blocks alphav 5-mediated cell adhesion to vitronectin. METHODS: A hydrogel disk containing 500 ng of VEGF was implanted into the superior corneal stroma of each of sixteen New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent to the first, randomized to contain either 40 g of antibody to alphavbeta5 (n=8) or phosphate-buffered saline (PBS)(n=8). Both disks were positioned 1.2 mm apart from the superior limbus. Eyes were examined daily under a stereomicroscope by two observers and assigned an angiogenesis score based on number and length of new blood vessels. RESULTS: On days 3 through 7 postimplantation, angiogenesis scores were significantly lower in eyes treated with antibody to alphavbeta5 (averaged score=16.33) as compared to eyes treated with PBS (averaged score=26.52)(P<0.05, Wilcoxon signed rank test). CONCLUSIONS: In a rabbit corneal micropocket assay, antibody to alphavbeta5 inhibits corneal angiogenesis induced by VEGF. Substances that target the integrin alphavbeta5 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.

Expression of Fibronectin, Vitronectin, Surfactant-A and D in Interaction of Pneumocystis carinii and Alveolar Epithelial Cells in Pneumocystis carinii Pneumonia

Both fibronectin and vitronectin bind to Pneumocystis carinii (P. carinii) and mediate the attachment of the organisms to respiratory epithelial cells. Surfactant A and D play a role in the interaction between P. carinii and host cells. In this study we examined the expression of fibronectin, vitronectin, surfactant-A and D in the interaction between P. carinii and alveolar epithelial cells by immunohistochemistry and pre-embedding immunoelectron microscopy. The experimental rat model of P. carinii pneumonia was induced by administration of low protein diet (8%) and drinking water containing dexamethasone (2 mg/liter) for 6 to 8 weeks. The primary antibodies for light and electron microscopic immunohistochemistries were monoclonal antibodies including fibronectin (1:100) and vitronectin (1:100), and polyclonal antibodies including surfactant A (1:50) and D (1:50), respectively. Light microscopic immunohistochemistry for the fibronectin, vitronectin, surfactant-A and D showed strong expressions on the P. carinii and surface linings of type I alveolar epithelial cells. The electron microscopic immunohistochemistry of the fibronectin and vitronectin showed a strong immunoexpression along the surface pellicles and tubular extensions of P. carinii trophozoites, and surface membranes of the type I epithelial cells. The surfactant-A and D proteins showed a strong expression on the pellicles of P. carinii and surface membranes of the type I epithelial cells, but a weak expression on the free-floating surfactant materials. In conclusions, the trophozoites of P. carinii were mostly attached to type I epithelial cells. The fibronectin, vitronectin, surfactant-A and D were strongly expressed, and played an enhancing role in the binding between the P. carinii organisms and the type I alveolar epithelial cells.

Inhibition of Corneal Angiogenesis by Antibody to Integrin b3 Subunit

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.

Inhibition of Corneal Angiogenesis by Antibody to Integrin b3 Subunit

Vascular endothelial cell expression of avb3, and integrin receptor that binds von Willebrand factor, vitronectin, and fibrinogen, increases in response to angiogenic stimuli such as basic fibroblast growth factor(bFGF) and during capillary proliferation in vivo. We investigated the importance of avb3 function during bFGF-induced corneal angiogenesis by examining the effects of 9D491, a monoclnal natibody against b3 that blocks avb3-mediated cell adhesion to vitronectin and fibrinogen in vitro. A hydrogel disk containing 500ng of bFGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the firtt, randomized to contain either 3ug of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and counted an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 post-implantation, angiogenesis scores were significantly lower in eyes treated with avb3 mAb(averaged score=3.3) as compared to eyes treated with 6E10(averaged score=8.0)(p<0.002, Wilcoxon rank sum test). In a rabbit corneal pocket model of angiogenesis, neutralizing monoclonal antibody to b3 inhibits corneal angiogenesis induced by bFGF. Substances that target the integrin b3 subunit may have therapeutic potential in disorders characterized by ocular neovascularization.

Inhibition of Vascular Endothelial Growth Factor Induced Corneal Angiogenesis by Antibody to Intergrin beta3 Subunit

Vascular cells respond to multiple cytokines, they also express a variety of integrin adhesion receptor. A number of the vascular cell integrins are functionally and structurally homologous, suggesting some level of biologic redundancy. We investigated the importance of alphavbeta3 function during vascular endothelial growth factor(VEGF) induced corneal angiogenesis by examining the effects of 9D491, a monoclonal antibody against beta3 that blocks alphavbeta3-mediated cell adhesion to vitronectin and fibrinogen. A hydrogel disk containing 500ng of VEGF was implanted into the superior corneal stroma of each of twelve New Zealand white rabbit eyes. Each eye also received a second hydrogel disk placed adjacent of the first, randomized to contain either 2.6microgram of 9D491 mAb(n=6) or 6E10, an irrelevant antibody of the same isotype, (n=6). Both disks were positioned 1.2mm from the superior limbus. Eyes were examined daily under a streomicroscope by two masked observers and assigned an angiogenesis score based on number and length of new blood vessels. On days 5 through 7 postimplantation, angiogenesis scores were not significantly lower in eyes treated with anti-alphavbeta3 mAb (averaged score=21.6) as compared to eyes treated with 6E10 (averaged score=24.0) (p<0.2, Wilcoxon rank sum test). In a rabbit corneal pocket assay, monoclonal antibody to beta3 could not inhibit corneal angiogenesis induced by VEGF.

Analysis of Platelet Membrane Glycoprotein Iib-IIIa Complex in Whole Blood of Glanzmann's Thrombasthenia by Flow Cytometry

Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder characterized by prolonged bleeding time, ad deficient or absent clot retraction in the presence of normal platelet count. The major underlying abnormality in this disease is grossly defective first-phase aggregation of platelet, which are unresponsive to ADP or other platelet agonists such as epinephrine, collagen, thrombin in any concentration. This disability is caused by a decrease or absence of the platelet membrans glycoprotein IIb-IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix fibronectin, and vitronectin On the development of surface labeling technique, a variety of biochemical techniques such as radioimmunoassay, crossed immunoelectrophoresis and SDS-PAGE have been used to study the structure and the function of platelet membrane glycoproteins, and to detect the platelet functional defect. But all of these techniques demand a relatively large amount of homogeneous paletelet population that requires manipulation through isolation and washing procedures before analysis. In order to eliminaste such an intricate procedure, we have applied method for analyzing platelet surface components in whole blood using monoclonal antibody and flow cytometry to recognize the absence of severe reduction of platelet membrane glycoprotien llb-llla complex. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as well as for carriers of this disease. Fow cytometry technique provides a sensitive tool for investigating platelet functional defects caused by altered expression or deficiency of platelet surface proteins.