Dose-controlled exposure of A549 epithelial cells at the air-liquid interface to airborne ultrafine carbonaceous particles.

The geometry of commercially available perfusion chambers designed for harbouring three membrane-based cell cultures was modified for reliable and dose-controlled air-liquid interface (ALI) exposures. Confluent A549 epithelial cells grown on membranes were integrated in the chamber system and supplied with medium from the chamber bottom. Cell viability was not impaired by the conditions of ALI exposure without particles. Expression of the inflammatory cytokines interleukin 6 and interleukin 8 by A549 cells during ALI exposure to filtered air for 6h and subsequent stimulation with tumor necrosis factor was not altered compared to submersed controls, indicating that the cells maintained their functional integrity. Ultrafine carbonaceous model particles with a count median mobility diameter of about 95+/-5 nm were produced by spark discharge at a stable concentration of about 2 x 10(6) cm(-3) and continuously monitored for accurate determination of the exposure dose. Delivery to the ALI exposure system yielded a homogeneous particle deposition over the membranes with a deposition efficiency of 2%. Mid dose exposure of A549 cells to this aerosol for 6h yielded a total particle deposition of (2.6+/-0.4) x 10(8) cm(-2) corresponding to (87+/-23) ng cm(-2). The 2.7-fold (p < or = 0.05) increased transcription of heme oxygenase-1 indicated a sensitive antioxidant and stress response, while cell viability did not reveal a toxic mechanism.

Experimental analysis of flow calculations based on HRCT imaging of individual bifurcations.

Flow simulations in airways and arteries allow the non-invasive study of transport and deposition processes in these vessel systems. Individual vessel geometries as input for such simulations are highly desirable. Computed tomography (CT) permits the acquisition of binary data to reconstruct such geometries. To prove the suitability of this reconstruction method, we compared measured with simulated velocities within model bifurcations. Particle image velocimetry was applied to measure flow velocities. Numerical simulations of these velocities were carried out by using the CT data of the same models as input to flow calculations (CFD). Within the resolution limits good agreement between measured and simulated velocities was found. For the smallest bifurcation (tube diameter: 2 mm) the agreement was less, indicating a methodical limitation by the actual resolution of the CT-scan technique. The study showed that a combination of CT and CFD can be considered as an appropriate step towards realistic simulations of particle transportation and deposition in individual geometries of the respiratory or cardiovascular systems.

Viscoelastic properties of canine tracheal mucus: effects of structural inhomogeneities and storage.

Tracheal mucus is an inhomogeneous material and thus its viscoelastic properties may vary substantially within a mucus sample. In this study on canine tracheal mucus, we investigated this variation of mucus viscoelasticity and addressed the question whether or not the viscoelasticity is affected by storage of the mucus at low temperatures. Mucus samples were collected from 16 beagle dogs kept under clean air conditions. By means of magnetic rheometry, dynamic viscosity- and elasticity were determined either in the fresh state of these samples or after storage at -80 degrees C. Owing to inhomogeneities within the gel-structure of the mucus, local values of the viscoelastic parameters differed by more than an order of magnitude. Short storage periods (5 d) were found to decrease, long periods (100 d) to increase the viscosity as well as elasticity parameters of the mucus material. These results emphasize the fact that measured viscoelastic properties of mucus are average values depending on the measuring protocol. In addition, these values are modified by storage of the mucus at low temperatures.

Shear dependence of the fibrin coagulation kinetics in vitro.

Fibrin thrombus formation, in vivo and in vitro, preferentially occurs in regions of retarded, recirculating flow which promote local variations of the distribution of blood components, e.g. thrombin, and shear rates. To better understand the effects of shear forces on the thrombin induced fibrin coagulation process the time course of fibrin formation in a fibrinogen/thrombin solution was studied for different shear rates gamma (0 s-1 < or = gamma < or = 500 s-1) and thrombin concentrations cthr (0.1 units/ml < or = cthr < or = 1.0 units/ml). The clotting curves at zero shear and the shear induced alterations of these curves could essentially be described in terms of a reaction kinetics defined by two rate coefficients k1, k2 which can be attributed to fibrinogen cleavage by thrombin and fibrin polymerisation, respectively. For cthr > approximately 0.5 units/ml and gamma > approximately 15 s-1 an additional mechanism, presumably fibrin breakage, had to be assumed. The rate coefficient k2 was markedly more affected by cthr and shear forces then was k1. The results fit well to the growth kinetics of fibrin thrombi formed in glass models of an arterial branching.

[Are pyrethroids harmless? Evaluation of experimental data]. / Sind Pyrethroide unbedenklich? Zur Bewertung experimenteller Befunde.

Pyrethroids are synthetic insecticides which world-wide increasingly replace DDT, Lindane, Organophosphate-pesticides etc. in fields like agriculture, hygienics, household, wear- and building industry. Being lipophilic and strongly neurotoxic substances, a considerable health risk has been predicted in regard of pyrethroid applications. On the other hand the published experimental data are often interpreted as proof of a small, calculable and, therefore, controllable risk. The present paper attempts to specify the reasons for these contrary judgements with regard to the pyrethroid effects. Since the arguments of both sides are based on the same experimental data, first the scientifically unquestioned evidence about effects and stability of the pyrethroids is acquired and critically reviewed. The different arguments are in turn discussed in details by means of this evidence. It was demonstrated that the scientific understanding of the pyrethroid effects is incomplete, as far as that the health risk cannot be considered as calculable. Impairments of health caused by pyrethroid effects, particularly known from scientific investigations carried out in China but also occurring with increasing incidence in Germany, can, therefore, not be excluded anymore. As practical consequences, moreover, an obligation to complete publication of pyrethroid intoxications and an extensive health education of the public about the risks associated with pyrethroid applications are demanded.

A method for quantitative determination of flow induced human platelet adhesion and aggregation.

Adhesivity and aggregability of blood platelets may reflect normal and diseased states. A quantitative assessment of such reactivity should thus provide further insight into disease mechanism. The well defined conditions of stagnation point flow using platelet-rich-plasma (PRP) as fluid permit quantitative treatment of the formation of platelet microthrombi on the stagnation plate. By measuring the growth kinetics (photometry) and corresponding morphology (light- and electron microscopy) of the microthrombus, platelet adhesion as well as aggregation could be analyzed separately by means of a quantitative procedure. Parameters are evaluated for PRP samples obtained from patients exhibiting deep venous thrombosis, arterial angiopathy or myocardial infarction, respectively. The results obtained demonstrate the usefulness of the proposed method for quantifying platelet reactivity as reflected by type and degree of flow induced platelet deposition. This, in turn, provides a basis for correlating the in vitro findings with the disease state.

Action of tocainide and mexiletine on the excitation threshold of myelinated nerves.

The antiarrhythmic drugs tocainide and mexiletine increase the excitation threshold and decrease the Na permeability (PNa) in nerves. Hodgkin-Huxley model calculations suggest that the PNa decrease is not sufficient to explain the threshold increase at low concentrations (less than 0.7 mM tocainide; less than 0.1 mM mexiletine). On the other hand, a drug action on the membrane surface potential would be consistent with results of recent experiments. To clarify this point electrophysiological experiments on single nerve fibres (Rana esculenta) were carried out. Changes of the threshold interval (difference between membrane and threshold potential; Vm-Vs) were measured and compared with the corresponding changes of Vm and Vs (Vs was measured by means of the transition voltage VTr). The quantitatively good agreement between changes of Vm-Vs and those of VTr suggests that at low concentrations tocainide and mexiletine decrease nerve excitability by acting mainly on the membrane surface charge. This possibility was supported by an independent electrophoresis experiment with myelin vesicles from the same nerve material.

Electrophoretic measurements about the relation between transition voltage and zeta-potential of biological membranes.

The effects of inorganic cations, n-hexanol, saccharose and 2H2O on the electrophoretic mobility and zeta-potential of membrane vesicles from nerve myelin were measured and the results compared with the corresponding effects of the same reagents on the transition voltage, VTr, of the nerve axon membrane. Different cation concentrations and 2H2O affect both potentials, the zeta-potential and VTr, in a parallel way. Saccharose and n-hexanol, however, shift VTr but leave the electrophoretic mobility of the myelin vesicles unchanged. These results suggest that VTr shifts are not necessarily linked to changes in the membrane surface charge density but may also be caused by an interaction between the reagent and non-polar groups of the membrane interior.

The effect of n-alkanols on the stationary current voltage behavior and action potential of myelinated nerve.

Stationary current voltage characteristics and the action potential of single myelinated nerve fibres were measured to examine the effect of n-alkanols (methanol to octanol) on the electrophysiological function of the axon membrane. K+-depolarized membranes show alkanol-dependent shifts of VTr, the membrane transition voltage, whereas in veratridine-depolarized membranes such VTr-shifts are not observed. In the latter case, n-alkanols reduce both the stationary Na+ current and the conductivity step between the high- and low-ohmic conductivity state of the membrane. Action potential amplitude, however, is less affected by the alkanols as is the stationary Na+ current. The results are compared with the alkanol-dependent changes of the thermotropic phase transition in phospholipid bilayers.

On reversible changes in the steady state ion-conductance of the Ranvier node membrane under low frequency sound fields.

The influence of sound waves on the stationary membrane conductivity of the axon membrane in myelinated nerve was investigated. Sound fields with frequencies within the auditory limit shift the transition voltage Vtr and correspondingly the threshold voltage of the action potential in polarisation direction. The results indicate that these changes of the electrical membrane behavior are due to a direct mechanical interaction between low frequency sound fields and membrane structure.

Elemental distribution of Na, P, Cl and K in different structures of myelinated nerve of Rana esculenta.

Measurements of the distribution of Na, P, Cl and K were performed in different structures of the myelinated nerve. Whereas the axon shows a typical intracellular distribution pattern for Na, Cl and K, the interstitial space and the myelin sheath show a typical extracellular pattern. These measurements have demonstrated that Na is present in the myelin sheath close to the node of Ranvier.

[About the importance of paranodal structures of the Ranvier node for the impulse regeneration (author's transl)]. / Uber die tbedeutung paranodaler Strukturen für die Impulsregeneration am Ranvier'schen Schnürring

The site of the pulse regeneration in myelinated nerve is generally assumed to be the unmyelinated part of the axon membrane in thr Ranvier node. To check this, a micro-irradiation technique using laser pulses (lambda equals 347 nm, t equals 20 ns) was used to produce morphological lesions of about 1 mum diameter in various regions of the Ranvier node. The electro-physiological functions were monitored parallel to the irradiation. Depending on the localizing of the lesions two types of changes in these functions were observed: 1. If a definite site in the paranodal myelin sheath was damaged without affecting the axon, an action potential could no longer be elicited, although the resting potential as well as the stationary current-voltage behaviour remained unchanged. 2. A damage of the axon resulted in a break down of membrane potential and resistance. In most of the cases the excitability recovered after spontaneous or current induced restitution of the membrane resting potential and resistance. These observations indicate, that structures in the paranodal region are vital for the Na+-activation and inactivation. The membrane potential and stationary current-voltage behavior can be attributed to the axon membrane. A new hypothesis concerning the mechanism of the Na+-activation-inactivation process is suggested.