A 12-week DBPC dose-finding study with sublingual monomeric allergoid tablets in house dust mite-allergic patients.

BACKGROUND: In sublingual immunotherapy, optimal doses are a key factor for therapeutic outcomes. The aim of this study with tablets containing carbamylated monomeric house dust mite allergoids was to determine the most effective and safe dose. METHODS: In this double-blind, placebo-controlled dose-finding study, 131 patients with house dust mite-induced allergic rhinoconjunctivitis were randomized to 12-week treatments with 300 UA/day, 1000 UA/day, 2000 UA/day, 3000 UA/day or placebo. Conjunctival provocation tests (CPT) were performed before, during and after treatment. The change in mean allergic severity (primary endpoint), calculated from the severity of the CPT reaction, and the proportion of patients with an improved CPT threshold (secondary endpoint) determined the treatment effect. RESULTS: The mean allergic severity decreased in all groups, including the placebo group. It was lower in all active treatment groups (300 UA/day: 0.14, 1000 UA/day: 0.15, 2000 UA/day: 0.10, 3000 UA/day: 0.15) than in the placebo group (0.30). However, this difference was not statistically significant (P < 0.1). The percentage of patients with an improved CPT threshold was higher in the active treatment groups (300 UA/day: 73.9%; 1000 UA/day: 76.0%; 2000 UA/day: 88.5%; 3000 UA/day: 76.0%) than in the placebo group (64.3%). The difference between placebo and 2000 UA/day was statistically significant (P = 0.04). In 13 (10%) exposed patients, a total of 20 treatment-related adverse events of mild severity were observed. CONCLUSIONS: The 12-week daily treatment using 2000 UA/day monomeric allergoid sublingual tablets is well tolerated and reduces the CPT reaction in house dust mite-allergic patients.

TRPC1 channels regulate directionality of migrating cells.

Cell migration depends on the generation of structural asymmetry and on different steps: protrusion and adhesion at the front and traction and detachment at the rear part of the cell. The activity of Ca(2+) channels coordinate these steps by arranging intracellular Ca(2+) signals along the axis of movement. Here, we investigated the role of the putative mechanosensitive canonical transient receptor potential channel 1 (TRPC1) in cell migration. We analyzed its function in transformed renal epithelial (Madin-Darby canine kidney-focus) cells with variation of TRPC1 expression. As shown by time lapse video microscopy, TRPC1 knockdown cells have partially lost their polarity and the ability to persistently migrate into a given direction. This failure is linked to the suppression of a local Ca(2+) gradient at the front of migrating TRPC1 knockdown cells, whereas TRPC1 overexpression leads to steeper Ca(2+) gradients. We propose that the Ca(2+) signaling events regulated by TRPC1 within the lamellipodium determine polarity and directed cell migration.

Analysis of flow in a cone-and-plate apparatus with respect to spatial and temporal effects on endothelial cells.

Endothelial cells, covering the inner surface of vessels and the heart, are permanently exposed to fluid flow, which affects the endothelial structure and the function. The response of endothelial cells to fluid shear stress is frequently investigated in cone-plate systems. For this type of device, we performed an analytical and numerical analysis of the steady, laminar, three-dimensional flow of a Newtonian fluid at low Reynolds numbers. Unsteady oscillating and pulsating flow was studied numerically by taking the geometry of a corresponding experimental setup into account. Our investigation provides detailed information with regard to shear-stress distribution at the plate as well as secondary flow. We show that: (i) there is a region on the plate where shear stress is almost constant and an analytical approach can be applied with high accuracy; (ii) detailed information about the flow in a real cone-plate device can only be obtained by numerical simulations; (iii) the pulsating flow is quasi-stationary; and (iv) there is a time lag on the order of 10(-3) s between cone rotation and shear stress generated on the plate.

Functional role of Na+-HCO3- cotransport in migration of transformed renal epithelial cells.

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na(+)-H(+) exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na(+)- HCO(3)(-) cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH(i)) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH(i) and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 (SLCA4A gene product) at a much higher level than the isoform kNBC3 (SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.

Quantitative morphodynamics of endothelial cells within confluent cultures in response to fluid shear stress.

To evaluate shear stress-induced effects on cultured cells we have extended the mechanical setup of a multichannel in vitro rheological system and developed software allowing entire processing control and image data analysis. The values of cell motility, degree of orientation (alignment), and cell elongation were correlated as a function of time (morphodynamics). Collective and individual endothelial cells within confluent cultures displayed a shear stress-dependent characteristic phase behavior of the following time course: resting conditions (phase I), change of motility (phase II), onset of alignment (phase III), and finally cell elongation (phase IV). Especially cell motility was characterized by a randomized zigzag movement around mean trajectories (fluctuations) together with mean cell locomotion. Onset of shear stress caused a down-regulation of fluctuations of 30% within <10 min and simultaneously increased locomotion velocities preferring the flow direction (phase II). After a lag period of 10 to 20 min cells orientated in the direction of flow (phase III) without significant cell elongation, which finally occurs within hours (phase IV). These data provide first evidence that cells within confluent endothelial monolayers respond to shear stress with a characteristic phase behavior.

Endothelial barrier function under laminar fluid shear stress.

It has been suggested that increasing levels of shear stress could modify endothelial permeability. This might be critical in venous grafting and in the pathogenesis of certain vascular diseases. We present a novel setup based on impedance spectroscopy that allows online investigation of the transendothelial electrical resistance (TER) under pure laminar shear stress. Shear stress-induced change in TER was associated with changes in cell motility and cell shape as a function of time (morphodynamics) and accompanied by a reorganization of catenins that regulate endothelial adherens junctions. Confluent cultures of porcine pulmonary trunk endothelial cells typically displayed a TER between 6 and 15 ohms cm2 under both resting conditions and low shear stress levels (0.5 dyn/cm2). Raising shear stress to the range of 2 to 50 dyn/cm2 caused a transient 2% to 15% increase in TER within 15 minutes that was accompanied by a reduction in cell motility. Subsequently, TER slowly decreased to a minimum of 20% below the starting value. During this period, acceleration of shape change occurred. In the ensuing period, TER values recovered, reaching control levels within hours and associated with an entire deceleration of shape change. A heterogeneous distribution of alpha-, beta-, and gamma-catenin, main components of the endothelial adherens type junctions, was also observed, indicating a differentiated regulation of shear stress-induced junction rearrangement. Additionally, catenins were partly colocalized with beta-actin at the plasma membrane, indicating migration activity of these subcellular parts. Shear stress, even at peak levels of 50 dyn/cm2, did not cause intercellular gap formation. These data show that endothelial monolayers exposed to increased levels of laminar shear stress respond with a shear stress-dependent regulation of permeability and a reorganization of junction-associated proteins, whereas monolayer integrity remains unaffected.

Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress.

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin. Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.