Effects of amelogenins on angiogenesis-associated processes of endothelial cells.

OBJECTIVE: To study the effects of an amelogenin mixture on integrin-dependent adhesion, DNA synthesis and apoptosis of cultured human dermal microvascular endothelial cells and angiogenesis in an organotypic assay. METHOD: Immobilised antibodies against specific integrins (alpha-1, alpha-2, alpha-3, alpha-4, alpha-5, alpha-v, ß1, ß2, ß3, ß4, ß6, alpha-vß3, alpha-vß5 and alpha-5ß1) were used to capture treated human dermal microvascular endothelial cells, which were detected colourimetrically. DNA synthesis of the cells was monitored by 5-bromo-2'- deoxyuridine incorporation and apoptosis by a TdT-mediated dUTP nick-end labelling technique. Tubule formation from aortic arches of 13-d-old chick embryos were followed over 48h. RESULTS: The amelogenin mixture increased microvessel outgrowth by 76% (p < 0.01, n=12) from the aortic explants. Also, amelogenins increased the adhesion (p < 0.01, n = 5) by multiple angiogenesis associated integrin subunits and alpha-vß3, alpha-vß5 and alpha-5ß1 heterodimers on human dermal microvascular endothelial cells at a non-mitogenic concentration (100 µg/ml). Conversely, amelogenins at 1,000 µg/ml decreased microvessel formation possibly due to attenuation of corresponding integrins despite increasing (p < 0.001, n = 8) DNA synthesis. No significant apoptosis was detected in human dermal microvascular endothelial cells cultured on Matrigel with and without amelogenins. CONCLUSION: Increased surface expression of integrins on endothelial cells may contribute to the proangiogenic property of amelogenins.

Evaluation of a near-senescent human dermal fibroblast cell line and effect of amelogenin.

BACKGROUND: Fibroblast senescence may delay healing of chronic wounds. OBJECTIVES: To characterize a chronic human dermal fibroblast cell line (CRL-7815) with near-senescent properties, cell proliferation and production of wound-healing modulating cytokines, and biosynthesis and remodelling of collagen were compared with normal human dermal fibroblasts. Also, the response of CRL-7815 fibroblasts to the extracellular matrix protein amelogenin that is beneficial in the treatment of stalled chronic wounds was studied. METHODS: Fibroblast proliferation was monitored by time-resolved growth curves and factors secreted into the culture medium containing 10% fetal bovine serum were measured by enzyme-linked immunosorbent assays. Fibroblast-mediated reorganization was examined in three-dimensional type I collagen matrices. RESULTS: Cell proliferation over 9 days was significantly (P < 0.01) slower for CRL-7815 than for normal fibroblasts. Amelogenin at 1 mg mL(-1) increased (P < 0.01) CRL-7815 proliferation to the level of the normal fibroblasts. The neutrophil chemoattractant interleukin (IL)-8 was low while the constitutive production of monocyte chemoattractant protein (MCP)-1 was highly elevated in medium from cultured CRL-7815 fibroblasts. Amelogenin augmented IL-8 but attenuated MCP-1 secretion in CRL-7815 fibroblasts. The elevated vascular endothelial growth factor production in CRL-7815 fibroblasts was further increased with amelogenin while increased type I collagen synthesis by CRL-7815 was reduced with 0.1 mg mL(-1) amelogenin. The dramatically impaired collagen matrix remodelling with CRL-7815 fibroblasts (P < 0.001) was slightly improved with amelogenin (P = 0.0011). CONCLUSIONS: The near-senescent cell line CRL-7815 shares functional anomalies with fibroblasts isolated from nonhealing chronic cutaneous wounds. Amelogenin has the capacity to switch chronic fibroblasts into an acute-like phenotype.

Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo are eradicated by a synthetic cationic polymer.

OBJECTIVES: Antimicrobial peptides are important effectors of innate immunity. Bacteria display multiple defence mechanisms against these peptides. For example, Pseudomonas aeruginosa releases potent proteinases that inactivate the human cathelicidin LL-37. Hence, in conditions characterized by persistent bacterial colonization, such as in P. aeruginosa-infected skin wounds, there is a need for efficient means of reducing bacterial load. Here, the effect of the cationic molecule polyhexamethylenebiguanide (PHMB) was evaluated. METHODS: Infection models in human wound fluid and human skin were established. Radial diffusion methods, bacterial growth and bactericidal assays were used for determination of effects of PHMB on bacteria in the presence of plasma, wound fluid or human skin. At the protein and tissue levels, SDS-PAGE, light microscopy and scanning electron microscopy were used to study the effects of P. aeruginosa infection before and after addition of PHMB. RESULTS: PHMB killed common ulcer-derived bacteria in the presence of human wound fluid. Furthermore, elastase-expressing P. aeruginosa completely degraded wound fluid proteins as well as human skin during infection ex vivo. The infection, and consequent protein degradation, was reversed by PHMB. CONCLUSIONS: The ex vivo infection models presented here should be helpful in the screening of novel antimicrobials and constitute a prerequisite for future clinical studies.

Apoptosis and cytokine release in human monocytes cultured on polystyrene and fibrinogen-coated polystyrene surfaces.

The effects of polystyrene (PS) material surface preadsorption with fibrinogen (3 mg/ml) and a low concentration of lipopolysaccharide (LPS; 10 ng/ml) and polystyrene particles (PS; 10(5)/ml) on human monocyte adhesion, viability and cytokine release were studied during 24h culture in vitro. LPS caused an upregulation of CD14 in adherent cells. In comparison with unstimulated cells on uncoated polystyrene surfaces, LPS did not alter the number of adherent cells but caused a markedly increased release of the proinflammatory cytokines (IL-1alpha and TNF-alpha) and the down-regulating IL-10. The expression of indicators of various stages of cell death, TdT, annexin-V, propidium iodide (PI) and lactate dehydrogenase (LDH), were unaltered, decreased, decreased and increased, respectively, after LPS stimulation. PS particles (3 microm psi) caused an increased DNA fragmentation but had a reduced proportion of annexin-V and PI positive cells in comparison with unstimulated cells on uncoated PS. In contrast, 1microm psi particles had a similar proportion of TdT, annexin-V and PI expressing cells as unstimulated controls. Cultures stimulated with particles (irrespective of size), had a similar concentration of proinflammatory cytokines as unstimulated controls, whereas a higher level of IL-10 was detected. Precoating of PS with fibrinogen revealed an enhanced cell adhesion and a concomitant reduction of CD14 expression. irrespective of stimulation with various agonists. The proportions of TdT, annexin-V and PI positive cells were unaltered or reduced on fibrinogen-coated PS in both unstimulated and agonist-challenged cultures. However, depending on the presence and type of agonist, fibrinogen mediated either a markedly increased (LPS) or equivalent (particles and unstimulated) IL-1alpha and TNFalpha release. Further, in comparison with uncoated substrates, fibrinogen was associated with a reduction of IL-10 release, irrespective of the type of stimuli. These observations, using low concentrations of bacterial and material products, indicate that fibrinogen modulates cell material interactions and up- and down-regulates specific events depending on the nature/ type of immediate stimuli.

In vitro study of monocyte viability during the initial adhesion to albumin- and fibrinogen-coated surfaces.

Surface adherent monocytes and macrophages play a central role in the inflammatory response to biomaterials. In the present study the adhesion, viability and apoptotic changes in material surface adherent monocytes during the first hours of cell-surface interactions in vitro were studied, using tissue culture polystyrene surfaces coated with human albumin and fibrinogen. Human peripheral blood monocytes were enriched by a two-step gradient centrifugation and resuspended (1 x 10(6)/ml) in RPMI with 10% fetal bovine serum. The cells were added to polystyrene surfaces coated with human fibrinogen or albumin and incubated in 37 degrees C (5% CO2, 100% humidity) for 30 min, 1, 2, 3 and 24 h. The adherent cells were stained for early apoptotic changes (exposed phosphatidylserine) and cell death using Annexin-V-fluorescein and propidium iodide staining, respectively. A bi-phasic adhesion was observed on the fibrinogen coated surface, having the highest number of adherent cells after 30 min and 24 h, while the cell number was markedly reduced after 1-3 h. The number of adherent cells on albumin was relatively low after all short time incubations but had reached a high level after 24 h. The number of adherent dead cells was highest after I h on both albumin (approximately 30%) and fibrinogen (approximately 15%). In the 24 h cultures, the viability of adherent cells was high on both surfaces (95-100%). Viable cells staining positive for early apoptotic changes could only be clearly observed on the albumin coated surface, after 30 min of cell-material surface interaction. Cell death, including apoptotic death, thus seems to play an important role during the initial interactions between monocytes and a foreign surface.

Intracellular Ca(2+) mobilization and kinase activity during acylated homoserine lactone-dependent quorum sensing in Serratia liquefaciens.

Quorum sensing in Gram-negative bacteria involves acylated homoserine lactones (AHLs) and a transcription factor, activated by the AHLs. In this study, a possible involvement of intracellular Ca(2+) as second messenger and/or protein kinase activity during signal transduction is analyzed. When N-hexanoyl-l-homoserine lactone was added to a suspension of Fura-2-loaded Serratia liquefaciens, there was a decline in [Ca(2+)](i), measured as a decrease in the Fura-2 fluorescence ratio. As controls, the addition of the signal molecule N-3-oxohexanoyl-l-homoserine lactone, which is not produced by S. liquefaciens, did not induce changes in [Ca(2+)](i). Using a protein kinase activity assay on AHL-stimulated cells, an increase in kinase activity after N-butanoyl-l-homoserine lactone stimulation of S. liquefaciens cells was detected, whereas the kinase activity induced by N-3-oxohexanoyl-l-homoserine lactone was not statistically significant. The conclusion from this study is that changes in [Ca(2+)](i) are involved in quorum sensing signal transduction in the Gram-negative bacteria S. liquefaciens. We also conclude that kinase activity is induced in S. liquefaciens upon AHL stimulation. We suggest that the transient intracellular [Ca(2+)] changes and kinase activity, activated by the AHL signal, are critical for the quorum-sensing signal transduction.

Cooperativity in the antibody binding to surface-adsorbed antigen.

The binding to surface-adsorbed antigen of monoclonal mouse IgG-antibodies (mAbs), with two different affinities to dinitrophenyl (DNP), was measured by a calibrated ELISA. The concentration-dependence of antibody binding to surface-bound antigen of different epitope densities was analysed using Scatchard plots. The dissociation of bound tritium-labelled antibodies was measured in the presence of unlabelled antibodies in the bulk. At low surface concentration of bound anti-DNP, both high-affinity mAb and low-affinity mAb show a positive cooperativity in the binding reaction to antigen of high epitope density. Using antigen of lower epitope densities, the positive cooperativity is more pronounced for low-affinity clones. At higher surface concentrations of bound anti-DNP, the Scatchard plots indicate a negative cooperativity of binding, which is also implied by the increased dissociation found in the presence of antibodies in solution. The study confirms previous findings that the binding of antibodies to surface-bound antigen not only depends on intrinsic antibody affinity measured in solution. Other factors, such as self-interaction, also affect the heterogeneous binding reaction.

Effect of antibody affinity on the isotherm of antibody binding to surface-immobilized antigen.

The binding of monoclonal antibodies to surface-adsorbed antigen was studied. Mouse IgG antibodies directed against dinitrophenyl groups (DNP) and O6-ethyl-2'-deoxyguanosine with known affinity for the antigen were used. The hapten was coupled to a protein, bovine serum albumin (BSA) or keyhole limpet hemocyanin, and adsorbed to polystyrene or silicone surfaces. Four different DNP-BSA epitope densities were used. Antibodies were incubated with the antigen-coated surface overnight. The bound antibodies were detected either optically by ellipsometry or by enzyme-conjugated anti-mouse IgG antibodies in the common ELISA technique. Absorbance values from ELISA measurements were transformed to surface density through calibration by ellipsometry. The experimental data showed that the binding of a high affinity antibody (Ka = 2.0 X 10(10] was diffusion rate limited after 24 h incubation time. Identical binding isotherms were found for high and low affinity clones of anti-DNP antibodies (Ka = 4.1 X 10(7) and 3.5 X 10(5] when antigen of high epitope density was used. At low epitope density the amount of bound low affinity antibodies decreased. Electron microscopy was used for studies of the distribution of colloidal gold-antibody complexes bound to surface-immobilized antigen. The results of the experiment showed that low affinity antibodies were bound in clusters whereas high affinity antibodies bound as single particles. These findings were related to the ELISA measurements. The results indicate that the binding isotherm of antibody to surface adsorbed antigen is not merely a reflection of the intrinsic antibody affinity measured in solution. Other macromolecular properties of antibodies, e.g., lateral intermolecular interactions and phase separation, affect the heterogeneous binding reaction.

A diffusion limited reaction theory for a microtiter plate assay.

Calculations are presented describing the diffusion limited kinetics of a solid-phase immunoassay in which reactants are immobilized at the inner surface of a cylindrical well. The calculations refer to an unstirred situation and simplified expressions are presented which can be used for calibration and optimization of the assay.

Kinetics of antibody binding to solid-phase-immobilised antigen. Effect of diffusion rate limitation and steric interaction.

The binding of monoclonal antibodies to surface-immobilised antigen was studied. Antibodies against dinitrophenyl-benzene and O6-ethyl-2'-deoxyguanosine with a known affinity for the antigen were used. The amount of bound antibodies was measured by ellipsometry with an accuracy of +/- 0.15 pmol/cm2, and a sensitivity of 0.11 pmol/cm2. The binding rate of the initial antibody binding could become diffusion rate limited, and the binding rate at surface concentrations above 1 pmol/cm2 was affected by steric interaction between bound antibodies. Bound antibodies did not dissociate when rinsed with saline for up to 20 h, but dissociated in the presence of antigen (0.1 mM). The dissociation rate did not follow any identifiable rate constant. The results are discussed in relation to theoretical models of the kinetics of antigen-antibody reactions at solid-liquid interfaces.