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PubMed1995

Effects of surface modification of materials on human neutrophil activation.

The properties of a polymer surface affect the cellular functions and morphology of cells in contact with the polymer. In this paper, we will demonstrate the effects of surface modification of materials on various neutrophil markers of activation. The sulfonation of a polystyrene surface caused increases in its negative charge and hydrophilicity. The sulfonation did not affect the number of adhered neutrophils, but the shape of the neutrophils adhered on the material was different; a round shape on highly sulfonated polystyrene and a spread shape on weakly sulfonated or non-sulfonated polystyrene. Expression of the adhesion molecule, CD11b, on neutrophils was also affected by the properties of the polymer surface. CD11b was expressed in neutrophils adhered on polystyrene and the expression decreased with increasing sulfonation of the surface. The expression of CD11b on the neutrophils on highly sulfonated polystyrene was the same as that on non-adhered neutrophils. In contrast, the expression of CD11a was not affected by the properties of the material surface. The F-actin content of activated neutrophils and the production of active oxygen groups detected by means of luminol-dependent chemiluminescence were also dependent on the sulfo-group content of the material surface. Finally, the translocation of protein kinase C (PKC) was determined in neutrophils adhered to these materials. Compared to non-adhered cells, the ratio of membrane bound to cytosolic PKC increased in adhered cells, but the increase was suppressed by sulfonation of the material surface. These data suggest that activation of neutrophils on polystyrene is suppressed by surface modification with increasing negative charge and/or hydrophilicity.

Actins↗
PubMed2004

Arrest of human lung fibroblasts in G2 phase after irradiation is regulated by converging phosphatidylinositol-3 kinase and beta1-integrin signaling in vitro.

PURPOSE: Cell-matrix interactions might confer cellular radioresistance in vitro. As a function of radiation, the impact of fibronectin (FN) and phosphatidylinositol-3 kinase (PI3K)-related signaling on survival, the cell cycle, and the beta1-integrin signaling kinases integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt), and glycogen synthase kinase-3beta (GSK-3beta) was examined in normal lung fibroblasts in vitro. METHODS AND MATERIALS: Normal human CCD32 lung fibroblasts grown on polystyrene, FN, or poly-L-lysine were irradiated with 0-8 Gy. Colony forming assays, flow cytometric DNA analysis, immunoblotting (Chk1, Chk2, Cdc25C, Cdk1, 14-3-3, p53, p21), and protein kinase assays (ILK, PKBalpha/Akt, GSK-3beta) were performed with or without PI3K inhibition using LY294002 or wortmannin. RESULTS: FN significantly elevated clonogenic survival of CCD32 cells after irradiation compared with polystyrene or poly-L-lysine. FN improved accumulation of irradiated cells in G(2)/M (60%) compared with polystyrene (43%). LY294002 prevented radiation-dependent G(2) blockage on polystyrene; on FN, G(2) arrest was only slightly reduced. Radiation- and PI3K inhibition-related changes in expression and phosphorylation of the various cell cycle proteins tested correlated with the cell cycle data acquired. The kinase activities of ILK, PKBalpha/Akt, and GSK-3beta were strongly induced by irradiation on polystyrene, but not on FN, which was a result of a FN-mediated increase of basal kinase activities. In contrast to polystyrene, FN enabled radiation-dependent induction of ILK and GSK-3beta in a PI3K-independent manner. CONCLUSION: The data indicate a tight convergence of cell-matrix and cell-growth factor interactions that seem to optimize the cellular responsiveness to ionizing radiation in terms of survival and G(2) arrest. ILK, PKBalpha/Akt, and GSK-3beta involved in integrin signaling were uncovered as new molecular factors within the cellular radiation response. Our findings might also provide insight into normal tissue effects and cellular radioresistance.

Androstadienes↗
PubMed2003

The effects of natural biofilms on the reattachment of young adult zebra mussels to artificial substrata.

This laboratory study examined the effects of natural biofilms on the reattachment of young adult zebra mussels, Dreissena polymorpha, in Petri dishes. Natural biofilms were developed in glass and polystyrene Petri dishes using water samples collected at various times of the year. Biofilms were developed over 1, 3, 8, and 14 d. Controls were clean glass and polystyrene Petri dishes. Zebra mussels collected from the field (< or = 10 mm, ventral length) were placed in the dishes and their reattachment by byssal threads was recorded after 1 d. Zebra mussels reattached to the dish surface or the shells of other mussels in the dish, or remained unattached. The data indicate that reattachment to clean glass was greater than to clean polystyrene (p < or = 0.05, ANOVA), but there were no consistent differences between reattachment to filmed polystyrene and filmed glass dish surfaces. Zebra mussels in control and filmed glass dishes reattached in higher percentages to the dish surface compared to the shells of other mussels (p < or = 0.05, ANOVA). There was no difference in mussel of reattachment between the dish surface and the shells of other mussels in most control polystyrene dishes (p > 0.05, ANOVA), whereas in filmed polystyrene the percentage of reattachment to the dish surface was greater than to the shells of other mussels (p < or = 0.05, ANOVA). These results indicate that substratum wettability and the presence of biofilms on some types of substrata can be factors in the reattachment of young adult zebra mussels.

Adhesiveness↗
PubMed1993

Glycosphingolipid inhibition of the adhesion of thrombin-activated platelets to surfaces is potentiated by albumin.

Previous studies have shown that exogenous glycosphingolipids (GSLs) inhibit the adhesion of thrombin-activated platelets (TAP) to polystyrene plates coated with various RGD-ligands (where RGD is the peptide sequence Arg-Gly-Asp), suggesting that GSLs can modulate the platelet integrin receptor glycoprotein IIb-IIIa. However, albumin was always used as a plastic surface-blocking agent in these studies. In order to evaluate the role of albumin in these experiments, we studied the effect of various GSLs and albumin on the interaction between TAP and hydrophobic surfaces in a solid-phase assay using indium-111-labelled platelets and polystyrene plates. TAP (10(8) platelets/ml) adhered to polystyrene (half-saturation time 40 +/- 3 min) with a maximal adhesion density of 56 +/- 1 x 10(3) platelets/mm2. Platelet adhesion was only slightly affected (< 11% inhibition) by immobilized bovine serum albumin, immobilized mixed bovine brain gangliosides (MBG) or fluid-phase MBG. In contrast, fluid-phase MBG was an effective inhibitor of platelet adhesion to polystyrene (> 46% inhibition), but only after albumin was first immobilized to the plate. Covering albumin-coated polystyrene with MBG, followed by washing, was as effective as fluid-phase MBG at inhibiting platelet adhesion, thus indicating that a ganglioside-albumin interaction at the polystyrene surface was responsible for effective inhibition. When purified GSLs were substituted for MBG, it was found that all those tested (GT1b, GD1a, GM1, asialo GM1 and globoside) had similar inhibitory activity.

Blood Platelets↗
PubMed2002

A study of the initial attachment and subsequent behavior of rat oral epithelial cells cultured on titanium.

BACKGROUND: The seal between an implant and the oral epithelium is an important factor for successful implant therapy. The purpose of this study was to evaluate the initial attachment and subsequent behavior of rat oral epithelial (OE) cells on pure titanium (Ti) used for dental implants. METHODS: OE cells derived from 4-day-old rats were cultured on Ti, polystyrene culture dishes, and glass coverslips. The number of attached cells, cell areas, number of colonies, and proliferation rates were measured. Additionally, immunostaining of vinculin and laminin-5 (LN5) was performed, and LN5-immunoreactive areas were measured. RESULTS: After 24 hours of culture, there were fewer cells attached to the Ti than to the polystyrene dishes or glass coverslips, and the area of cells was greater on the polystyrene than on the Ti or glass. OE cells reached their maximum proliferation rate after 48 hours of culture on the polystyrene and glass, and after 72 hours on Ti. LN5 was deposited behind cells as they migrated, and the LN5-immunoreactive area was smaller on Ti than on polystyrene after 96 hours of culture. CONCLUSIONS: The initial attachment of OE cells to Ti was inferior to that on polystyrene or glass, and the OE cell migration area indicated by the deposition of LN5 was smaller on Ti than on the other materials. Therefore, this study suggests that further improvement of Ti surface properties is needed for rapid attachment and spreading of oral epithelium to dental implants.

Analysis of Variance↗
PubMed1990

Novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG in human serum using recombinant gag-env hybrid protein as antigen.

A novel and sensitive enzyme immunoassay (immune-complex-transfer enzyme immunoassay) for antihuman T cell leukemia virus type 1 IgG (anti-HTLV-1 IgG) in human serum using recombinant gag(14-139)-env-(197-295) hybrid protein is described. Anti-HTLV-1 IgG in test serum was reacted with dinitrophenyl biotinyl bovine serum albumin-recombinant gag-env hybrid protein conjugate. The complex formed was trapped onto polystyrene balls coated with affinity-purified antidinitrophenyl group IgG. After washing to eliminate nonspecific IgG in the test serum, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with streptavidin. After washing, anti-HTLV-1 IgG in the complex trapped onto the streptavidin-coated polystyrene balls was reacted with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Peroxidase activity bound to the streptavidin-coated polystyrene balls was assayed by fluorometry. By transfer of the complex, the nonspecific binding of nonspecific human IgG was considerably reduced, and the detection limit of anti-HTLV-1 IgG in serum was lowered 30-300-fold compared with that by Western blotting, gelatin particle agglutination, and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with test serum and, after washing, with antihuman IgG gamma-chain Fab'-peroxidase conjugate. Usefulness of the immune-complex-transfer enzyme immunoassay was demonstrated using 271 serum samples.

Blotting, Western↗
PubMed1991

Sensitive detection of anti-human T-cell leukemia virus type I IgG in human serum by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag-env hybrid protein as antigen.

Anti-human T-cell leukemia virus type I IgG (anti-HTLV-I IgG) in human serum was detected with high sensitivity by a novel enzyme immunoassay (immune complex transfer enzyme immunoassay) using recombinant gag(14-139)-env(197-295) hybrid protein. Anti-HTLV-I IgG in test serum was reacted simultaneously with dinitrophenyl bovine serum albumin-recombinant gag-env hybrid protein conjugate and recombinant gag-env hybrid protein-horseradish peroxidase conjugate. The complex formed of the three components was trapped onto polystyrene balls coated with affinity-purified anti-dinitrophenyl group IgG. After washing the polystyrene balls to eliminate nonspecific IgG in the test serum and excess of the peroxidase conjugate, the complex was eluted from the polystyrene balls with dinitrophenyl-L-lysine and transferred to polystyrene balls coated with affinity-purified anti-human IgG gamma-chain IgG. Peroxidase activity bound to the polystyrene balls was remarkably reduced by transfer of the complex and the detection limit of anti-HTLV-I IgG in serum was lowered 300 to 3000-fold compared with that by Western blotting and the conventional enzyme immunoassay, in which a recombinant gag-env hybrid protein-coated polystyrene ball was incubated with the test serum and, after washing, with anti-human IgG gamma-chain Fab'-peroxidase conjugate. The immune complex transfer enzyme immunoassay may overcome some difficulties with currently used methods.

Gene Products, env↗
PubMed1989

Measurement of anti-thyroglobulin IgG in serum by novel and sensitive immune complex transfer enzyme immunoassay.

Anti-thyroglobulin IgG in human serum was measured by a novel and sensitive immune complex transfer enzyme immunoassay. Anti-thyroglobulin IgG in human serum was reacted with dinitrophenyl thyroglobulin, and the complex formed between human anti-thyroglobulin IgG and dinitrophenyl thyroglobulin was trapped onto an affinity-purified rabbit (anti-dinitrophenyl bovine serum albumin) IgG-coated polystyrene ball. The polystyrene ball was washed to eliminate most nonspecific IgG in test serum, and the complex was eluted from the polystyrene ball, to which nonspecific IgG had been adsorbed, with dinitrophenyl-L-lysine and transferred to a clean polystyrene ball coated with rabbit anti-thyroglobulin IgG. The amount of human anti-thyroglobulin IgG in the complex on the rabbit anti-thyroglobulin IgG-coated polystyrene ball was estimated using rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. The present enzyme immunoassay was 1,000 to 3,000-fold more sensitive than the conventional enzyme immunoassay, in which a human thyroglobulin-coated polystyrene ball was incubated with serum containing human anti-thyroglobulin IgG and, after washing, with rabbit (anti-human IgG gamma-chain) Fab'-horseradish peroxidase conjugate. By the present enzyme immunoassay, anti-thyroglobulin IgG was demonstrated in about 10% of healthy subjects and in all patients with Graves' disease and chronic thyroiditis. The principle of the present method may make it possible to sensitively measure other autoantibodies including anti-thyroid peroxidase and anti-thyrotropin receptor antibodies to aid diagnosis of thyroid diseases.

Adult↗
PubMed2002

Comparative uptake studies of bioadhesive and non-bioadhesive nanoparticles in human intestinal cell lines and rats: the effect of mucus on particle adsorption and transport.

PURPOSE: The interaction of nanoparticles (NP), consisting of hydrophobic polystyrene, bioadhesive chitosan, and stealth PLA-PEG with two human intestinal cell lines, the enterocyte-like Caco-2 and mucus-secreting MTX-E12, was investigated and compared to the in vivo NP uptake in rats. METHODS: The extent and mechanism of cellular association of different NP with Caco-2 and MTX-E12 was investigated using confocal laser scanning microscopy (CLSM) and a cellular association assay. In vitro results were compared to gastrointestinal distribution of chitosan NP in rats after intra-duodenal injection. RESULTS: Cellular association of NP with Caco-2 cell monolayers showed the following rank order: polystyrene > chitosan >> PLA-PEG. Mucus (MTX-E12) significantly decreased the association of hydrophobic polystyrene NP. While no mucus binding was observed for PLA-PEG, association of chitosan NP with mucus strongly increased. Intra-duodenal administration of chitosan NP in rats confirmed these in vitro results, demonstrating that NP could be detected in both epithelial cells and Peyer's patches. Chitosan NP internalization was saturable, as well as energy- and temperature-dependent. It could be inhibited by an excess of protamine and by removal of anionic sites of the apical membrane. By contrast, polystyrene NP uptake was found to be largely independent of these factors, except for a temperature-dependency. CONCLUSIONS: In contrast to Caco-2 cells, the presence of mucus presented a major barrier for the uptake of hydrophobic polystyrene NP and showed an even more profound effect upon the uptake of chitosan NP. A correlation between the uptake in cell culture models and in vivo rat epithelial cells was confirmed for chitosan NP. Moreover, chitosan NP seemed to be taken up and transported by adsorptive transcytosis, while polystyrene NP uptake was probably mediated by non-adsorptive transcytosis.

Adhesives↗
PubMed2003

Cell adhesion to the extracellular matrix protein fibronectin modulates radiation-dependent G2 phase arrest involving integrin-linked kinase (ILK) and glycogen synthase kinase-3beta (GSK-3beta) in vitro.

Cell adhesion to extracellular matrix (ECM) is thought to confer resistance against cell-damaging agents, that is, drugs and radiation, in tumour and normal cells in vitro. The dependence of cell survival on beta1-integrin-linked kinase (ILK), protein kinase Balpha/Akt (PKBalpha/Akt) and glycogen synthase kinase-3beta (GSK-3beta) activity, which participate in beta1-integrin signalling and cell cycle progression was investigated as a function of radiation exposure. Colony-formation assays on polystyrene, fibronectin (FN), laminin (LA), bovine serum albumin (BSA) or poly-L-lysine (poly-L) (0-8 Gy), kinase assays, flow cytometric DNA and annexin-V analysis and immunoblotting were performed in nonirradiated and irradiated (2 or 6 Gy) A549 human lung cancer cells and CCD32 normal human lung fibroblasts. Cell contact to FN in contrast to polystyrene elevated basal ILK, PKBalpha/Akt and GSK-3beta kinase activities in A549 and CCD32 cells, as well as the basal amount of A549 G2 phase cells. Irradiation on FN or LA as compared to polystyrene, BSA or poly-L significantly improved cell survival. Following irradiation, kinase activities were stimulated strongly on polystyrene but showed to be less prominent on FN, which was because of the FN-related basal induction. Following irradiation, FN compared to polystyrene enlarged and prolonged G2 arrest in both the cell lines. For the analysis of phosphatidylinositol-3 kinase (PI3-K) dependence of protein kinases and cell cycle transition, the PI3-K inhibitors LY294002 and wortmannin were used showing decreased kinase activities, antiproliferative and radiation-dependent G2 accumulation-abrogating effects accompanied by downregulation of cyclin D1 and phospho-pRb in cells attached to polystyrene. Fibronectin partly abrogated these effects PI3-K-independently. These findings suggest a novel pathway that makes direct phosphorylation of GSK-3beta by ILK feasible after irradiation. Conclusively, the data indicate that ILK, PKBalpha/Akt and GSK-3beta are involved in modulations of the cell cycle after irradiation. These interactions are strictly dependent on ECM components in a cell line-specific manner. Our findings provide molecular insights into mechanisms likely to be important for ECM-dependent cell survival and cellular radioresistance as well as tumour growth.

Annexin A5↗
PubMed1998

Optimal conditions of immune complex transfer enzyme immunoassay for p24 antigen of HIV-1.

In the immune complex transfer enzyme immunoassay for HIV-1 p24 antigen, different preparations of anti-p24 Fab'-beta-D-galactosidase conjugate, various periods of time for immunoreactions involved, and shaking for incubations with polystyrene beads were tested. On the basis of the results of these experiments, p24 antigen was measured as follows. The antigen was reacted simultaneously with 2,4-dinitrophenyl-biotinyl-bovine serum albumin-affinity-purified rabbit anti-p24 Fab' conjugate and highly polymerized monoclonal mouse anti-p24 Fab'-beta-D-galactosidase conjugate at 37 degrees C for 2 hr. The immune complex formed comprising the three components was trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG for 1.5 hr and was transferred to white polystyrene beads coated with streptavidin in the presence of epsilon N-2,4-dinitrophenyl-L-lysine for 1.5 hr. The incubations with polystyrene beads were performed at room temperature with shaking. beta-D-Galactosidase activity bound to the white polystyrene beads was assayed by fluorometry at 30 degrees C for 2 hr. The detection limit of p24 antigen (0.1 amol/tube and 10 amol (0.24 pg)/ml of serum) was equal to that obtained when the formation, trapping, and transferring of the immune complex were performed for 4, 16, and 3 hr, respectively, by incubation without shaking. Namely, the period of time required for the immune complex transfer enzyme immunoassay of p24 antigen was markedly shortened (25.5-7 hr) without loss of the sensitivity. By the improved immune complex transfer enzyme immunoassay, p24 antigen was detected 12-20 days earlier than the detection of antibodies to HIV-1, i.e., seroconversion by the conventional ELISA.

2,4-Dinitrophenol↗
PubMed1998

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection.

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.

2,4-Dinitrophenol↗
PubMed2000

Capillary zone electrophoresis of sub-microm-sized particles in electrolyte solutions of various ionic strengths: size-dependent electrophoretic migration and separation efficiency.

To gain insight into the mechanisms of size-dependent separation of microparticles in capillary zone electrophoresis (CZE), sulfated polystyrene latex microspheres of 139, 189, 268, and 381 nm radius were subjected to CZE in Tris-borate buffers of various ionic strengths ranging from 0.0003 to 0.005, at electric field strengths of 100-500 V cm(-1). Size-dependent electrophoretic migration of polystyrene particles in CZE was shown to be an explicit function of kappaR, where kappa(-1) and rare the thickness of electric double layer (which can be derived from the ionic strength of the buffer) and particle radius, respectively. Particle mobility depends on kappaR in a manner consistent with that expected from the Overbeek-Booth electrokinetic theory, though a charged hairy layer on the surface of polystyrene latex particles complicates the quantitative prediction and optimization of size-dependent separation of such particles in CZE. However, the Overbeek-Booth theory remains a useful general guide for size-dependent separation of microparticles in CZE. In accordance with it, it could be shown that, for a given pair of polystyrene particles of different sizes, there exists an ionic strength which provides the optimal separation selectivity. Peak spreading was promoted by both an increasing electric field strength and a decreasing ionic strength. When the capillary is efficiently thermostated, the electrophoretic heterogeneity of polystyrene microspheres appears to be the major contributor to peak spreading. Yet, at both elevated electric field strengths (500 V/cm) and the highest ionic strength used (0.005), thermal effects in a capillary appear to contribute significantly to peak spreading or can even dominate it.

Electrolytes↗
PubMed1981

The influence of haemoperfusion on haemostasis and cellular constituents of the blood in the treatment of intoxications: a comparative study of three types of columns (Haemocol, Amberlite XAD-4, Gambro Adsorba 300 C).

The influence of haemoperfusion on blood-coagulation and cellular constituents of the blood was studied in three groups of patients. In four patients haemoperfusion was performed using a column containing acrylic-hydrogel coated activated charcoal (Haemocol), in five patients with a column containing uncoated XAD-4 nonionic polystyrene resin (Amberlite) and in five patients with a column containing cellulose coated activated charcoal (Gambro Adsorba 300 C). Perfusion was performed during 4 h with a flow of 300 ml/min. Before the start, 2 h after the start, at the end and 2 h after the end of the perfusion the haemoglobin concentration, haematocrit, leucocyte number, differential white cell count, thrombocyte number and heparin concentration were measured. Before the start and 2 h after the end prothrombin time, thrombin time, partial thromboplastin time, reptilase time, fibrinogen, prothrombin, factors V, VII, X, antithrombin III, bleeding time (Ivy), ethanol gelation test, fibrin split products and plasminogen were measured. The following conclusions can be drawn: haemoperfusion per se causes haemodilution; polystyrene resin causes in some patients a temporary reduction of the leucocyte number during haemoperfusion; polystyrene resin causes a significant reduction of thrombocyte number compared to coated activated charcoal; polystyrene resin and to a lesser extent acrylic-hydrogel-coated activated charcoal causes in some patients a prolongation of bleeding time probably by inducing alteration of thrombocyte function caused by release; polystyrene resin and probably also acrylic-hydrogel-coated activated charcoal causes an increased fibrinolytic activity without signs of disseminated intravascular coagulation.

Adult↗
PubMed2006

A new time-resolved fluorometric microarray detection system using core-shell-type fluorescent nanosphere and its application to allergen microarray.

We have developed a new time-resolved fluorometric (TRF) microarray detection system consisting of fluorescent NH2 nanosphere, TRF microarray detector and gamma-irradiated polystyrene chip. Using the TRF microarray detector, we detected 500 particles of the fluorescent nanosphere in one channel. Cross-talk fluorescence from the adjacent channels was little observed in the TRF microarray detector (<0.0004 %). The TRF microarray detection system was further applied for serum allergen-specific immunoglobulin E (IgE) multi-analyses. As a labeled tag antibody, an anti-human IgE Fab' fragment-conjugated fluorescent nanosphere (Fab' nanosphere) was prepared as described previously. As a chip surface appropriate for allergen immobilization, the polystyrene chip surface was modified by gamma irradiation. The immunoassay reactivity using the gamma-irradiated polystyrene chip was approximately 2.5-times improved compared with that of the non-treated polystyrene chip. Non-specific adsorption of the Fab' nanosphere onto the gamma-irradiated polystyrene chip surface was very low level (<0.0009 %). In only 20 mul of serum, six allergen-specific IgEs could be simultaneously determined in one reaction well in fewer than 90 min. Good correlation curves were obtained between the microarray immunoassay and the CAP RAST fluoro-enzyme immunoassay (CAP/RAST FEIA) method (r > 0.961). Reproducibility (CVs) of the microarray immunoassay was 8.6 % to 19.0 % (n = 5).

Allergens↗
PubMed1986

Pinocytosis and phagocytosis: the effect of size of a particulate substrate on its mode of capture by rat peritoneal macrophages cultured in vitro.

Both phagocytosis (of particles) and pinocytosis (of solutes) occur in macrophages. It is not known, however, whether particles, if they are small enough, can enter by pinocytosis, nor whether there is a minimum size of particle capable of triggering phagocytic uptake. These questions have been investigated by studying, in vitro, the uptake by rat peritoneal macrophages of particles ranging in diameter from 30 nm to 1100 nm. Percoll (30 nm diameter) and polystyrene beads (100, 300, 600, 800 or 1100 nm diameter) were 125I-iodinated and their uptake by macrophages was measured in the absence or presence of metabolic and cytoskeletal inhibitors. Since uptake, expressed as an Endocytic Index (microliter/10(6) cells per h), increased steadily with the duration of incubation and was inhibited by low temperature or metabolic inhibitors, it was concluded that true endocytosis, and not a superficial cell-association, was being measured. Rates of clearance increased with increasing particle diameter. The rate of uptake of Percoll was 10-times, and of 100 nm polystyrene beads 100-times, the rate of fluid-phase pinocytosis, as measured by the uptake of 125I-labelled polyvinylpyrrolidone. Polystyrene beads of 1100 nm diameter were captured at 700-times this rate. The differential effects of colchicine and cytochalasin B on the uptake of 125I-labelled polyvinylpyrrolidone and of 1100 nm polystyrene beads were taken as indicators of their effects on pinocytosis and phagocytosis respectively. It is concluded that Percoll, although particulate, is captured by pinocytosis. The pattern of inhibition of uptake of polystyrene particles suggests that there is no radical discontinuity between pinocytic and phagocytic uptake, but that the contribution of phagocytosis steadily increases with increasing particle diameter. The results are discussed.

2,4-Dinitrophenol↗
PubMed1999

Migration of lymphocytes on fibronectin-coated surfaces: temporal evolution of migratory parameters.

Lymphocytes typically interact with implanted biomaterials through adsorbed exogenous proteins. To provide a more complete characterization of these interactions, analysis of lymphocyte migration on adsorbed extracellular matrix proteins must accompany the commonly performed adhesion studies. We report here a comparison of the migratory and adhesion behavior of Jurkat cells (a T lymphoblastoid cell line) on tissue culture treated and untreated polystyrene surfaces coated with various concentrations of fibronectin. The average speed of cell locomotion showed a biphasic response to substrate adhesiveness for cells migrating on untreated polystyrene and a monotonic decrease for cells migrating on tissue culture-treated polystyrene. A modified approach to the persistent random walk model was implemented to determine the time dependence of cell migration parameters. The random motility coefficient showed significant increases with time when cells migrated on tissue culture-treated polystyrene surfaces, while it remained relatively constant for experiments with untreated polystyrene plates. Finally, a cell migration computer model was developed to verify our modified persistent random walk analysis. Simulation results suggest that our experimental data were consistent with temporally increasing random motility coefficients.

Adsorption↗
PubMed1999

Characterization of patocytosis: endocytosis into macrophage surface-connected compartments.

Previously, we described a unique macrophage endocytosis pathway in which aggregated low density lipoproteins and microcrystalline cholesterol induce and enter a labyrinth of membrane-bound compartments that remain connected to the cell surface. We now show that certain types of non-lipid particles such as polystyrene microspheres and colloidal gold also induce and enter macrophage surface-connected compartments (SCC), a process we call patocytosis. A common property among particles that stimulate patocytosis is their hydrophobic nature. Both aggregated LDL and microcrystalline cholesterol that we showed previously to stimulate patocytosis are hydrophobic. We now show that hydrophobic polystyrene microspheres and gold particles but not their hydrophilic counterparts triggered patocytosis. Uptake by patocytosis was limited to hydrophobic polystyrene microsphere particles less than 0.5 micron in diameter. Hydrophobic polystyrene microspheres greater than this size entered macrophages by phagocytosis. Actin-independent capping of hydrophobic polystyrene microspheres on the plasma membrane preceded actin-dependent uptake of the microspheres into SCC. Sequential rounds of microsphere uptake into SCC over two successive days could occur. There was some mixing of initial and subsequently accumulated microspheres in SCC. SCC formed from plasma membrane invaginations that connected with spaces created by unfolding of stacks of internal microvilli. Microsphere transport from plasma membrane invaginations into these spaces was inhibited by primaquine. Patocytosis is a unique endocytic process in macrophages triggered by small hydrophobic particles that provides a mechanism to sequester large amounts of these materials within a labyrinth of SCC.

Actins↗