Biomimetic coating of organic polymers with a protein-functionalized layer of calcium phosphate: the surface properties of the carrier influence neither the coating characteristics nor the incorporation mechanism or release kinetics of the protein

Polymers that are used in clinical practice as bone-defect-filling materials possess many essential qualities, such as moldability, mechanical strength and biodegradability, but they are neither osteoconductive nor osteoinductive. Osteoconductivity can be conferred by coating the material with a layer of calcium phosphate, which can be rendered osteoinductive by functionalizing it with an osteogenic agent. We wished to ascertain whether the morphological and physicochemical characteristics of unfunctionalized and bovine-serum-albumin (BSA)-functionalized calcium-phosphate coatings were influenced by the surface properties of polymeric carriers. The release kinetics of the protein were also investigated. Two sponge-like materials (Helistat® and Polyactive®) and two fibrous ones (Ethisorb and poly[lactic-co-glycolic acid]) were tested. The coating characteristics were evaluated using state-of-the-art methodologies. The release kinetics of BSA were monitored spectrophotometrically. The characteristics of the amorphous and the crystalline phases of the coatings were not influenced by either the surface chemistry or the surface geometry of the underlying polymer. The mechanism whereby BSA was incorporated into the crystalline layer and the rate of release of the truly incorporated depot were likewise unaffected by the nature of the polymeric carrier. Our biomimetic coating technique could be applied to either spongy or fibrous bone-defect-filling organic polymers, with a view to rendering them osteoconductive and osteoinductive.

Cerium oxide nanoparticle uptake kinetics from the gas-phase into lung cells in vitro is transport limited

Nowadays, aerosol processes are widely used for the manufacture of nanoparticles (NPs), creating an increased occupational exposure risk of workers, laboratory personnel and scientists to airborne particles. There is evidence that possible adverse effects are linked with the accumulation of NPs in target cells, pointing out the importance of understanding the kinetics of particle internalization. In this context, the uptake kinetics of representative airborne NPs over 30 min and their internalization after 24 h post-exposure were investigated by the use of a recently established exposure system. This system combines the production of aerosolized cerium oxide (CeO(2)) NPs by flame spray synthesis with its simultaneous particle deposition from the gas-phase onto A549 lung cells, cultivated at the air-liquid interface. Particle uptake was quantified by mass spectrometry after several exposure times (0, 5, 10, 20 and 30 min). Over 35% of the deposited mass was found internalized after 10 min exposure, a value that increased to 60% after 30 min exposure. Following an additional 24 h post-incubation, a time span, after which adverse biological effects were observed in previous experiments, over 80% of total CeO(2) could be detected intracellularly. On the ultrastructural level, focal cerium aggregates were present on the apical surface of A549 cells and could also be localized intracellularly in vesicular structures. The uptake behaviour of aerosolized CeO(2) is in line with observations on cerium suspensions, where particle mass transport was identified as the rate-limiting factor for NP internalization.

Self-assembly of focal point oligo-catechol ethylene glycol dendrons on titanium oxide surfaces: adsorption kinetics, surface characterization, and nonfouling properties

This work covers the synthesis of second-generation, ethylene glycol dendrons covalently linked to a surface anchor that contains two, three, or four catechol groups, the molecular assembly in aqueous buffer on titanium oxide surfaces, and the evaluation of the resistance of the monomolecular adlayers against nonspecific protein adsorption in contact with full blood serum. The results were compared to those of a linear poly(ethylene glycol) (PEG) analogue with the same molecular weight. The adsorption kinetics as well as resulting surface coverages were monitored by ex situ spectroscopic ellipsometry (VASE), in situ optical waveguide lightmode spectroscopy (OWLS), and quartz crystal microbalance with dissipation (QCM-D) investigations. The expected compositions of the macromolecular films were verified by X-ray photoelectron spectroscopy (XPS). The results of the adsorption study, performed in a high ionic strength ("cloud-point") buffer at room temperature, demonstrate that the adsorption kinetics increase with increasing number of catechol binding moieties and exceed the values found for the linear PEG analogue. This is attributed to the comparatively smaller and more confined molecular volume of the dendritic macromolecules in solution, the improved presentation of the catechol anchor, and/or their much lower cloud-point in the chosen buffer (close to room temperature). Interestingly, in terms of mechanistic aspects of "nonfouling" surface properties, the dendron films were found to be much stiffer and considerably less hydrated in comparison to the linear PEG brush surface, closer in their physicochemical properties to oligo(ethylene glycol) alkanethiol self-assembled monolayers than to conventional brush surfaces. Despite these differences, both types of polymer architectures at saturation coverage proved to be highly resistant toward protein adsorption. Although associated with higher synthesis costs, dendritic macromolecules are considered to be an attractive alternative to linear polymers for surface (bio)functionalization in view of their spontaneous formation of ultrathin, confluent, and nonfouling monolayers at room temperature and their outstanding ability to present functional ligands (coupled to the termini of the dendritic structure) at high surface densities.

Gadolinium diethylenetriaminepentaacetate enhancement kinetics in the menisci of asymptomatic subjects: a first step towards a dedicated dGEMRIC (delayed gadolinium-enhanced MRI of cartilage)-like protocol for biochemical imaging of the menisci

It was our aim to investigate the gadolinium diethylenetriaminepentaacetate (Gd-DTPA(2-) ) enhancement kinetics in the menisci of the knee joint over a prolonged period of time. Six asymptomatic volunteers (four men and two women; mean age, 25 ± 2.4 years) were enrolled. Sagittal, T(1) -weighted, spin-echo MR sequences of the right knee joint were obtained at 3 T. Imaging was performed before (baseline), 1 h after and in half-hour intervals up to 9 h after the intravenous administration of 0.2 mmol/kg of Gd-DTPA(2-) . To measure the rates of contrast enhancement relative to the baseline, regions of interest that covered the anterior and posterior horns of the medial and lateral meniscus were defined on each of two adjacent sections, and enhancement curves were constructed. An enhancement peak between 2.5 and 4.5 h after Gd-DTPA(2-) administration was observed, and analysis of variance also revealed no significant difference (p=0.94), in terms of enhancement, within this time interval. Pair-wise, post hoc testing also revealed no significant differences between 2.5 and 3, 3 and 3.5, 3.5 and 4, and 4 and 4.5 h post Gd-DTPA(2-) application. Our preliminary data therefore suggest that the time window suitable for a dGEMRIC (delayed gadolinium-enhanced MRI of cartilage)-like T(1) mapping of the menisci is relatively short, and lies between 2.5 and 4.5 h after Gd-DTPA(2-) injection.

Kinetics of ocular and systemic antigen-specific T-cell responses elicited during murine cytomegalovirus retinitis

Cytomegalovirus (CMV) reactivation in the retina of immunocompromized patients is a cause of significant morbidity as it can lead to blindness. The adaptive immune response is critical in controlling murine CMV (MCMV) infection in MCMV-susceptible mouse strains. CD8(+) T cells limit systemic viral replication in the acute phase of infection and are essential to contain latent virus. In this study, we provide the first evaluation of the kinetics of anti-viral T-cell responses after subretinal infection with MCMV. The acute response was characterized by a rapid expansion phase, with infiltration of CD8(+) T cells into the infected retina, followed by a contraction phase. MCMV-specific T cells displayed biphasic kinetics with a first peak at day 12 and contraction by day 18 followed by sustained recruitment of these cells into the retina at later time points post-infection. MCMV-specific CD8(+) T cells were also observed in the draining cervical lymph nodes and the spleen. Presentation of viral epitopes and activation of CD8(+) T cells was widespread and could be detected in the spleen and the draining lymph nodes, but not in the retina or iris. Moreover, after intraocular infection, antigen-specific cytotoxic activity was detectable and exhibited kinetics equivalent to those observed after intraperitoneal infection with the same viral dose. These data provide novel insights of how and where immune responses are initiated when viral antigen is present in the subretinal space.

Antibacterial and antiinflammatory kinetics of curcumin as a potential antimucositis agent in cancer patients

The antiinflammatory agent curcumin (diferuloylmethane) has a potential to mitigate cancer therapy-induced mucositis. We assessed the in vitro extent of its bactericidal activity and determined the kinetics of its antiinflammatory effect on pharyngeal cells. Bactericidal activity was assessed using the LIVE/DEAD® Kit after 4 h of exposure to curcumin (50-200 μM) in 18 oropharyngeal species commonly associated with bacteremia in febrile neutropenia. Moraxella catarrhalis or its outer membrane vesicles were used to determine the inhibitory effect of curcumin on bacteria-induced proinflammatory activity as determined by cytokine release into the supernatant of Detroit 562 pharyngeal cells using the Luminex® xMAP® technology. Curcumin exerted a concentration-dependent bactericidal effect on all 18 species tested. After 4 h at 200 μM, 12 species tested were completely killed. Preincubation of Detroit cells with 200 μM curcumin for 5 to 60 min resulted in complete suppression of the release of tumor necrosis factor-α, interleukin (IL)-6, IL-8, monocyte chemoattractant protein 1, granulocyte macrophage-colony stimulating factor, and vascular endothelial growth factor. Fibroblast growth factor-2 and interferon-γ were not affected. Repetitive exposure to curcumin resulted in repetitive suppression of cytokine/chemokine expression lasting from 4 to 6 h. Through reduction of oral microbial density as well as suppression of inflammation cascades curcumin may prevent cancer therapy-induced oral mucositis, e.g., when applied as multiple daily mouth washes.

Capillary electrophoretic investigation of the enantioselective metabolism of propafenone by human cytochrome P-450 SUPERSOMES: Evidence for atypical kinetics by CYP2D6 and CYP3A4

An enantioselective CE method was used to identify the ability of CYP450 enzymes and their stereoselectivity in catalyzing the transformation of propafenone (PPF) to 5-hydroxy-propafenone (5OH-PPF) and N-despropyl-propafenone (NOR-PPF). Using in vitro incubations with single CYP450 enzymes (SUPERSOMES), 5OH-PPF is shown to be selectively produced by CYP2D6 and N-dealkylation is demonstrated to be mediated by CYP2D6, CYP3A4, CYP1A2, and CYP1A1. For the elucidation of kinetic aspects of the metabolism with CYP2D6 and CYP3A4, incubations with individual PPF enantiomers and racemic PPF were investigated. With the exception of the dealkylation in presence of R-PPF only, which can be described by the Michaelis-Menten model, all CYP2D6-induced reactions were found to follow autoactivation kinetics. For CYP3A4, all NOR-PPF enantiomer formation rates as function of PPF enantiomer concentration were determined to follow substrate inhibition kinetics. The formation of NOR-PPF by the different enzymes is stereoselective and is reduced significantly when racemic PPF is incubated. Clearance values obtained for CYP3A4 dealkylation are stereoselective whereas those of CYP2D6 hydroxylation are not. This paper reports the first investigation of the PPF hydroxylation and dealkylation kinetics by the CYP2D6 enzyme and represents the first report in which enantioselective CE data provide the complete in vitro kinetics of metabolic steps of a drug.

Structure and binding kinetics of three different human CD1d-alpha-galactosylceramide-specific T cell receptors

Invariant human TCR Valpha24-Jalpha18+/Vbeta11+ NKT cells (iNKT) are restricted by CD1d-alpha-glycosylceramides. We analyzed crystal structures and binding characteristics for an iNKT TCR plus two CD1d-alpha-GalCer-specific Vbeta11+ TCRs that use different TCR Valpha chains. The results were similar to those previously reported for MHC-peptide-specific TCRs, illustrating the versatility of the TCR platform. Docking TCR and CD1d-alpha-GalCer structures provided plausible insights into their interaction. The model supports a diagonal orientation of TCR on CD1d and suggests that complementarity determining region (CDR)3alpha, CDR3beta, and CDR1beta interact with ligands presented by CD1d, whereas CDR2beta binds to the CD1d alpha1 helix. This docking provides an explanation for the dominant usage of Vbeta11 and Vbeta8.2 chains by human and mouse iNKT cells, respectively, for recognition of CD1d-alpha-GalCer.

Effects of potentised substances on growth kinetics of Saccharomyces cerevisiae and Schizosaccharomyces pombe

BACKGROUND: Homeopathic potencies are used as specific remedies in complementary medicine. Since the mode of action is unknown, the presumed specificity is discussed controversially. OBJECTIVE: This study investigated the effects of potentised substances on two yeast species, Saccharomyces cerevisiae and Schizosaccharomyces pombe, in a stable and reliable test system with systematic negative controls. MATERIALS AND METHODS: Yeast cells were cultivated in either potentised substances or water controls in microplates and their growth kinetics were measured photometrically. Water control runs were performed repeatedly to investigate the stability of the experimental set-up (systematic negative controls). RESULTS: 4 out of 14 screened substances seem to have affected the growth curve parameters slope or yield. Out of these substances, azoxystrobin and phosphorus were chosen for 8 further replication experiments, which partly confirmed the results of the screening. On the average of all experiments, azoxystrobin affected the slope of the growth curve of Saccharomyces cerevisiae (p < 0.05), and phosphorus affected the slope of the growth curve of Schizosaccharomyces pombe (p < 0.05). No effects were seen in the water control runs. In addition, significant interactions between treatment with potentised substances and experiment number were observed in all experiments with potentised substances (p < 0.01), but not in the water control runs. CONCLUSIONS: Both yeast species reacted to certain potentised substances by changing their growth kinetics. However, the interactions found point to additional factors of still unknown nature, that modulate the effects of potentised substances. This stable test system with yeasts may be suitable for further studies regarding the efficacy of homeopathic potencies.

Kinetics of the interaction of desAABB-fibrin monomer with immobilized fibrinogen

The soluble and stable fibrin monomer-fibrinogen complex (SF) is well known to be present in the circulating blood of healthy individuals and of patients with thrombotic diseases. However, its physiological role is not yet fully understood. To deepen our knowledge about this complex, a method for the quantitative analysis of interaction between soluble fibrin monomers and surface-immobilized fibrinogen has been established by means of resonant mirror (IAsys) and surface plasmon resonance (BIAcore) biosensors. The protocols have been optimized and validated by choosing appropriate immobilization procedures with regeneration steps and suitable fibrin concentrations. The highly specific binding of fibrin monomers to immobilized fibrin(ogen), or vice versa, was characterized by an affinity constant of approximately 10(-8)M, which accords better with the direct dissociation of fibrin triads (KD approximately 10(-8) -10(-9) M) (J. R. Shainoff and B. N. Dardik, Annals of the New York Academy of Science, 1983, Vol. 27, pp. 254-268) than with earlier estimations of the KD for the fibrin-fibrinogen complex (KD approximately 10(-6) M) (J. L. Usero, C. Izquierdo, F. J. Burguillo, M. G. Roig, A. del Arco, and M. A. Herraez, International Journal of Biochemistry, 1981, Vol. 13, pp. 1191-1196).

Modeling of transfer kinetics at the serum-cerebrospinal fluid barrier in rabbits with experimental meningitis: application to grepafloxacin

The goals of the present study were to model the population kinetics of in vivo influx and efflux processes of grepafloxacin at the serum-cerebrospinal fluid (CSF) barrier and to propose a simulation-based approach to optimize the design of dose-finding trials in the meningitis rabbit model. Twenty-nine rabbits with pneumococcal meningitis receiving grepafloxacin at 15 mg/kg of body weight (intravenous administration at 0 h), 30 mg/kg (at 0 h), or 50 mg/kg twice (at 0 and 4 h) were studied. A three-compartment population pharmacokinetic model was fit to the data with the program NONMEM (Nonlinear Mixed Effects Modeling). Passive diffusion clearance (CL(diff)) and active efflux clearance (CL(active)) are transfer kinetic modeling parameters. Influx clearance is assumed to be equal to CL(diff), and efflux clearance is the sum of CL(diff), CL(active), and bulk flow clearance (CL(bulk)). The average influx clearance for the population was 0.0055 ml/min (interindividual variability, 17%). Passive diffusion clearance was greater in rabbits receiving grepafloxacin at 15 mg/kg than in those treated with higher doses (0.0088 versus 0.0034 ml/min). Assuming a CL(bulk) of 0.01 ml/min, CL(active) was estimated to be 0.017 ml/min (11%), and clearance by total efflux was estimated to be 0.032 ml/min. The population kinetic model allows not only to quantify in vivo efflux and influx mechanisms at the serum-CSF barrier but also to analyze the effects of different dose regimens on transfer kinetic parameters in the rabbit meningitis model. The modeling-based approach also provides a tool for the simulation and prediction of various outcomes in which researchers might be interested, which is of great potential in designing dose-finding trials.