Antibody-mediated CD31 ‘immobilization’ enhances activated T cell chemokinesis.

<p>Naïve (a) and activated (b) WT T cells were incubated overnight in RPMI 0.5% FCS. Some T cells were pre-incubated with an anti-CD31 mAb at saturating concentrations (5 µg/ml) for 30 minutes at RT. Migration in response to CXCL10 or medium through a transwell was assessed over 6 hours. Percentage migration was calculated by dividing the number of cells harvested from the bottom chamber following 6 hours incubation at 37°C by the original number of cells plated onto the transwell. The average percentage migration from three independent experiments is shown. Error bars indicate SD (*p<0.05).</p

Dose and time dependency of BaP1 effects on type IV collagen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with different amounts of BaP1 (10, 30 and 100 μg) for either 5 or 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for type IV collagen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type: (A) arterioles, (B) capillaries, and (C) PCV per cremaster (n = 4). Below each graph, representative three-dimensional images of each vessel type immunostained for type IV collagen are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions) for BaP1 (30 μg) and control at 15 min. The images show a decrease in fluorescence intensity for type IV collagen in BM of capillaries of treated tissues as compared to control, whereas no significant reduction was observed in arterioles and PCV. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; Col IV: type IV collagen; PCV: post-capillary venules.</p

Effect of BaP1 and CsH1 on size and density of gaps between adjacent smooth muscle and pericytes on mouse cremaster muscle vasculature.

<p>Isolated cremaster muscles were incubated with either 30 μg of BaP1 or 15 μg of CsH1 (model without blood flow). In another experiment, anesthetized mice were injected by intrascrotal route with either 60 μg of BaP1 or 30 μg of CsH1 (model with blood flow). Controls were incubated or injected with PBS. After 15 min of exposition to toxin in each model, whole cremaster muscles were fixed and immunostained for observation by confocal microscopy and analysis of the gaps between adjacent smooth muscle and pericytes. Results are expressed as the mean ± SEM of the (A, B) gap size and (C, D) gap density (number of gaps per vessel area) of at least five images of arterioles and PCV per cremaster (n = 4). (E) Representative three-dimensional images of each vessel type immunostained for actin α smooth muscle are shown for BaP1 30 μg and control in the model without blood flow. Notice the increase in the gap size (arrows) in arterioles and PCV of treated tissues as compared to control. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. C: control; PCV: post-capillary venules.</p

Effect of BaP1 on laminin and nidogen from vascular BM on isolated mouse cremaster muscle.

<p>Isolated cremaster muscles were incubated with 30 μg of BaP1 for 15 min (model without blood flow). Control tissues were incubated with PBS. Whole tissues were fixed and immunostained for observation by confocal microscopy and analysis of total fluorescence intensity for (A) laminin and (B) nidogen. Results are expressed as the mean ± SEM of the percentage of intensity related to control of at least five images of each vessel type per cremaster (n = 4). (C) Representative three-dimensional images of each vessel type immunostained for laminin are shown with a gray color coding spectrum (black as low fluorescence intensity regions and white as high fluorescence intensity regions). The images show a decrease in fluorescence intensity for laminin in BM of capillaries and PCV of treated tissues as compared to control, whereas no reduction in the fluorescence intensity was observed for nidogen. Scale bar represents 30 μm. *p < 0.05, **p < 0.001 as compared to control. Lam: laminin; Nid: nidogen; PCV: post-capillary venules.</p

Design of an Activity-Based Probe for Human Neutrophil Elastase: Implementation of the Lossen Rearrangement To Induce Förster Resonance Energy Transfers

Human neutrophil elastase is an important regulator of the immune response and plays a role in host defense mechanisms and further physiological processes. The uncontrolled activity of this serine protease may cause severe tissue alterations and impair inflammatory states. The design of an activity-based probe for human neutrophil elastase reported herein relies on a sulfonyloxyphthalimide moiety as a new type of warhead that is linker-connected to a coumarin fluorophore. The inhibitory potency of the activity-based probe was assessed against several serine and cysteine proteases, and the selectivity for human neutrophil elastase (<i>K</i>_i = 6.85 nM) was determined. The adequate fluorescent tag of the probe allowed for the in-gel fluorescence detection of human neutrophil elastase in the low nanomolar range. The coumarin moiety and the anthranilic acid function of the probe, produced in the course of a Lossen rearrangement, were part of two different Förster resonance energy transfers