DOCK2 is required for chemokine-promoted human T lymphocyte adhesion under shear stress mediated by the integrin alpha4beta1

The alpha4beta1 integrin is an essential adhesion molecule for recruitment of circulating lymphocytes into lymphoid organs and peripheral sites of inflammation. Chemokines stimulate alpha4beta1 adhesive activity allowing lymphocyte arrest on endothelium and subsequent diapedesis. Activation of the GTPase Rac by the guanine-nucleotide exchange factor Vav1 promoted by CXCL12 controls T lymphocyte adhesion mediated by alpha4beta1. In this study, we investigated the role of DOCK2, a lymphocyte guanine-nucleotide exchange factor also involved in Rac activation, in CXCL12-stimulated human T lymphocyte adhesion mediated by alpha4beta1. Using T cells transfected with DOCK2 mutant forms defective in Rac activation or with DOCK2 small interfering RNA, we demonstrate that DOCK2 is needed for efficient chemokine-stimulated lymphocyte attachment to VCAM-1 under shear stress. Flow chamber, soluble binding, and cell spreading assays identified the strengthening of alpha4beta1-VCAM-1 interaction, involving high affinity alpha4beta1 conformations, as the adhesion step mainly controlled by DOCK2 activity. The comparison of DOCK2 and Vav1 involvement in CXCL12-promoted Rac activation and alpha4beta1-dependent human T cell adhesion indicated a more prominent role of Vav1 than DOCK2. These results suggest that DOCK2-mediated signaling regulates chemokine-stimulated human T lymphocyte alpha4beta1 adhesive activity, and that cooperation with Vav1 might be required to induce sufficient Rac activation for efficient adhesion. In contrast, flow chamber experiments using lymph node and spleen T cells from DOCK2(-/-) mice revealed no significant alterations in CXCL12-promoted adhesion mediated by alpha4beta1, indicating that DOCK2 activity is dispensable for triggering of this adhesion in mouse T cells, and suggesting that Rac activation plays minor roles in this process.

Shear stress modulates the expression of thrombospondin-1 and CD36 in endothelial cells in vitro and during shear stress-induced angiogenesis in vivo

Binding of thrombospondin-1 (TSP-1) to the CD36 receptor inhibits angiogenesis and induces apoptosis in endothelial cells (EC). Conversely, matrix-bound TSP-1 supports vessel formation. In this study we analyzed the shear stress-dependent expression of TSP-1 and CD36 in endothelial cells in vitro and in vivo to reveal its putative role in the blood flow-induced remodelling of vascular networks. Shear stress was applied to EC using a cone-and-plate apparatus and gene expression was analyzed by RT-PCR, Northern and Western blot. Angiogenesis in skeletal muscles of prazosin-fed (50 mg/l drinking water; 4 d) mice was assessed by measuring capillary-to-fiber (C/F) ratios. Protein expression in whole muscle homogenates (WMH) or BS-1 lectin-enriched EC fractions (ECF) was analyzed by Western blot. Shear stress downregulated TSP-1 and CD36 expression in vitro in a force- and time-dependent manner sustained for at least 72 h and reversible by restoration of no-flow conditions. In vivo, shear stress-driven increase of C/F in prazosin-fed mice was associated with reduced expression of TSP-1 and CD36 in ECF, while TSP-1 expression in WMH was increased. Down-regulation of endothelial TSP-1/CD36 by shear stress suggests a mechanism for inhibition of apoptosis in perfused vessels and pruning in the absence of flow. The increase of extra-endothelial (e.g. matrix-bound) TSP-1 could support a splitting type of vessel growth.

Thrombospondin-1 mediates platelet adhesion at high shear via glycoprotein Ib (GPIb): an alternative/backup mechanism to von Willebrand factor

Acute thrombotic arterial occlusion is the leading cause of morbidity and mortality in the Western world. Von Willebrand factor is thought to be the only indispensable adhesive substrate to promote thrombus formation in high shear environments. We found that thrombospondin-1, a glycoprotein enriched in arteriosclerotic plaques, might function as an alternative substrate for thrombus formation. Platelets adhered to thrombospondin-1 in a shear dependent manner with an optimum shear as found in stenosed arteries. Adhesion is extremely firm, with no detachment of platelets up to a shear rate of 4000 s(-1). Experiments using platelets from a patient completely lacking von Willebrand factor showed that von Willebrand factor is not involved in platelet binding to thrombospondin-1. Platelet adhesion to thrombospondin-1 is not mediated via beta3-integrins or GPIa. CD36 partially mediates the adhesion of pre-activated platelets. We identified GPIb as high shear adhesion-receptor for thrombospondin-1. Soluble GPIb, as well as antibodies against the GPIb, blocked platelet adhesion almost completely. The new discovered thrombospondin-1-GPIb adhesion axis under arterial shear conditions might be important, not only during thrombus formation but also for pathological processes where other cells bind to the endothelium or subendothelium, including arteriosclerosis, inflammation and tumor metastasis, and a promising therapeutic target.

Regulation of Foxo-1 and the angiopoietin-2/Tie2 system by shear stress

Transcription factor Foxo-1 can be inactivated via Akt-mediated phosphorylation. Since shear stress activates Akt, we determined whether Foxo-1 and the Foxo-1-dependent, angiogenesis-related Ang-2/Tie2-system are influenced by shear stress in endothelial cells. Expression of Foxo-1 and its target genes p27Kip1 and Ang-2 was decreased under shear stress (6dyn/cm(2), 24h), nuclear exclusion of Foxo-1 by phosphorylation increased. eNOS and Tie2 were upregulated. No effects on Ang-1 expression were detected. In conclusion, Foxo-1 and Ang-2/Tie2 are part of the molecular response to shear stress, which may regulate angiogenesis.

Confidence Bands for Convex Median Curves using Sign-Tests

Suppose that one observes pairs (x1,Y1), (x2,Y2), ..., (xn,Yn), where x1 < x2 < ... < xn are fixed numbers while Y1, Y2, ..., Yn are independent random variables with unknown distributions. The only assumption is that Median(Yi) = f(xi) for some unknown convex or concave function f. We present a confidence band for this regression function f using suitable multiscale sign tests. While the exact computation of this band seems to require O(n4) steps, good approximations can be obtained in O(n2) steps. In addition the confidence band is shown to have desirable asymptotic properties as the sample size n tends to infinity.

Effect of zirconia surface treatments on the shear strength of zirconia/veneering ceramic composites

Aim of the investigation was to assess the effect of different surface treatments on the bond strength of veneering ceramics to zirconia. In a shear test, the influences of polishing, sandblasting, and silica-coating of the zirconia surface on bonding were assessed with five different veneering ceramics. In addition the effect of liner application was examined. With one veneering ceramic, the impact of regeneration firing of zirconia was also evaluated. Statistical analysis was performed with one-way ANOVA and post hoc Scheffé's test. Failure in every case occurred in the veneering ceramic adjacent to the interface with a thin layer of ceramic remaining on the zirconia surface, indicating that bond strength was higher than the cohesive strength of the veneering ceramic. Shear strength ranged from 23.5 +/- 3.4 MPa to 33.0 +/- 6.8 MPa without explicit correlation to the respective surface treatment. Regeneration firing significantly decreased the shear strength of both polished and sandblasted surfaces. Findings of this study revealed that bonding between veneering ceramics and zirconia might be based on chemical bonds. On this note, sandblasting was not a necessary surface pretreatment to enhance bond strength and that regeneration firing was not recommended.

Shear bond strength of resin cements to human dentin

OBJECTIVES: The objectives of this in vitro study were (1) to assess the bond strength of the universal cement RelyX Unicem to dentin and to compare it with three conventional resin cements, (2) to test the influence of aging on their bonding capacity and (3) to test the influence of the operator on bonding quality by performing the same test in two different centers. METHODS: 160 third molars, divided into 80 for tests at the University of Zurich (Z) and 80 for tests at the University of Berne (B), were assigned to 2 x 8 subgroups of 10 teeth each. The specimens were prepared with the corresponding bonding agents and acrylic rods were luted either with RelyX Unicem (U), RelyX ARC (A), Multilink (M) or Panavia 21 (P). All specimens were stored in water for 24h (W) and half of the specimens were subjected to 1500 cycles of thermocycling (5 degrees C and 55 degrees C) (T). Bond strength was measured by means of a shear test. RESULTS: After water storage RelyX Unicem exhibited lowest bond strength (UWZ: 9.2+/-1.6 MPa, UWB: 9.9+/-1.2 MPa, AWZ: 15.3+/-6.0 MPa, AWB: 12.2+/-4.3 MPa, MWZ: 15.6+/-3.3 MPa, MWB: 12.4 MPa+/-2.4, PWZ: 13.4+/-2.9 MPa, PWB: 14.9+/-2.6 MPa). Thermocycling affected the bonding performance of all four cements. However, bond strength of RelyX Unicem was least influenced by thermocycling (UTZ: 9.4+/-2.9 MPa, UTB: 8.6+/-1.3 MPa, ATZ: 11.4+/-6.3 MPa, ATB: 13.3+/-3.7 MPa, MTZ: 15.4+/-3.1 MPa, MTB: 10.3+/-2.4 MPa, PTZ: 11.1+/-2.8 MPa, PTB: 11.3+/-2.8 MPa). SIGNIFICANCE: Although the bond strength of RelyX Unicem to dentin was lower in comparison to RelyX ARC, Multilink and Panavia 21, its bond strength was less sensitive to variations in handling and aging.

Automation of shear-wave splitting parameter determination of local earthquakes at Yellowstone : application as indicator of crustal stress and temporal variation

Shear-wave splitting can be a useful technique for determining crustal stress fields in volcanic settings and temporal variations associated with activity. Splitting parameters were determined for a subset of local earthquakes recorded from 2000-2010 at Yellowstone. Analysis was automated using an unsupervised cluster analysis technique to determine optimum splitting parameters from 270 analysis windows for each event. Six stations clearly exhibit preferential fast polarization values sub-orthogonal to the direction of minimum horizontal compression. Yellowstone deformation results in a local crustal stress field differing from the regional field dominated by NE-SW extension, and fast directions reflect this difference rotating around the caldera maintaining perpendicularity to the rim. One station exhibits temporal variations concordant with identified periods of caldera subsidence and uplift. From splitting measurements, we calculated a crustal anisotropy of ~17-23% and crack density ~0.12-0.17 possibly resulting from stress-aligned fluid filled microcracks in the upper crust and an active hydrothermal system.

Experimental results on gravity driven fully condensing flows in vertical tubes, their agreement with theory, and their differences with shear driven flows' boundary-condition sensitivities

This doctoral thesis presents the experimental results along with a suitable synthesis with computational/theoretical results towards development of a reliable heat transfer correlation for a specific annular condensation flow regime inside a vertical tube. For fully condensing flows of pure vapor (FC-72) inside a vertical cylindrical tube of 6.6 mm diameter and 0.7 m length, the experimental measurements are shown to yield values of average heat transfer co-efficient, and approximate length of full condensation. The experimental conditions cover: mass flux G over a range of 2.9 kg/m2-s ≤ G ≤ 87.7 kg/m2-s, temperature difference ∆T (saturation temperature at the inlet pressure minus the mean condensing surface temperature) of 5 ºC to 45 ºC, and cases for which the length of full condensation xFC is in the range of 0 < xFC < 0.7 m. The range of flow conditions over which there is good agreement (within 15%) with the theory and its modeling assumptions has been identified. Additionally, the ranges of flow conditions for which there are significant discrepancies (between 15 -30% and greater than 30%) with theory have also been identified. The paper also refers to a brief set of key experimental results with regard to sensitivity of the flow to time-varying or quasi-steady (i.e. steady in the mean) impositions of pressure at both the inlet and the outlet. The experimental results support the updated theoretical/computational results that gravity dominated condensing flows do not allow such elliptic impositions.

Biomechanical analysis of torsion and shear forces in lumbar and lumbosacral spine segments of nonchondrodystrophic dogs

OBJECTIVE: To determine stiffness and load-displacement curves as a biomechanical response to applied torsion and shear forces in cadaveric canine lumbar and lumbosacral specimens. STUDY DESIGN: Biomechanical study. ANIMALS: Caudal lumbar and lumbosacral functional spine units (FSU) of nonchondrodystrophic large-breed dogs (n=31) with radiographically normal spines. METHODS: FSU from dogs without musculoskeletal disease were tested in torsion in a custom-built spine loading simulator with 6 degrees of freedom, which uses orthogonally mounted electric motors to apply pure axial rotation. For shear tests, specimens were mounted to a custom-made shear-testing device, driven by a servo hydraulic testing machine. Load-displacement curves were recorded for torsion and shear. RESULTS: Left and right torsion stiffness was not different within each FSU level; however, torsional stiffness of L7-S1 was significantly smaller compared with lumbar FSU (L4-5-L6-7). Ventral/dorsal stiffness was significantly different from lateral stiffness within an individual FSU level for L5-6, L6-7, and L7-S1 but not for L4-5. When the data from 4 tested shear directions from the same specimen were pooled, level L5-6 was significantly stiffer than L7-S1. CONCLUSIONS: Increased range of motion of the lumbosacral joint is reflected by an overall decreased shear and rotational stiffness at the lumbosacral FSU. CLINICAL RELEVANCE: Data from dogs with disc degeneration have to be collected, analyzed, and compared with results from our chondrodystrophic large-breed dogs with radiographically normal spines.