Association of Delay in Appendectomy With Perforation in Children With Appendicitis.

OBJECTIVE: The aim of this study was to assess whether increased time from emergency department (ED) triage to appendectomy is associated with a greater risk of children developing appendiceal perforation. METHODS: We performed a multicenter retrospective cohort study of children younger than 18 years hospitalized with appendicitis. To avoid enrolling patients who had perforated prior to ED arrival, we included only children who had a computed tomography (CT) scan demonstrating nonperforated appendicitis. Time to appendectomy was measured as time from ED triage to incision. The main outcome was appendiceal perforation as documented in the surgical report. Variables associated with perforation in bivariate analysis (P < 0.05) were adjusted for using logistic regression. RESULTS: Overall, 857 patients had a CT scan that demonstrated nonperforated appendicitis. The median age was 12 years (interquartile range, 9-15 years), and 500 (58%) were male. The median time to appendectomy was 11 hours (interquartile range, 8-15 hours). In total, 111 patients (13%) had perforated appendicitis at operation. Children who developed perforation were more likely to require additional CT scans and return to the ED and had a significantly longer length of stay. After adjusting for potential confounders, every hour increase in the time from ED triage to incision was independently associated with a 2% increase in the odds of perforation (P = 0.03; adjusted odds ratio, 1.02; 95% confidence interval, 1.00-1.04). CONCLUSIONS: Delays in appendectomy were associated with an increase in the odds of perforation. These results suggest that prolonged delays to appendectomy might be harmful for children with appendicitis and should be minimized to prevent associated morbidity.

Mapping local matrix remodeling induced by a migrating tumor cell using three-dimensional multiple-particle tracking.

Mesenchymal cell migration through a three-dimensional (3D) matrix typically involves major matrix remodeling. The direction of matrix deformation occurs locally in all three dimensions, which cannot be measured by current techniques. To probe the local, 3D, real-time deformation of a collagen matrix during tumor cell migration, we developed an assay whereby matrix-embedded beads are tracked simultaneously in all three directions with high resolution. To establish a proof of principle, we investigated patterns of collagen I matrix deformation near fibrosarcoma cells in the absence and presence of inhibitors of matrix metalloproteinases and acto-myosin contractility. Our results indicate that migrating cells show patterns of local matrix deformation toward the cell that are symmetric in magnitude with respect to the axis of cell movement. In contrast, patterns of matrix release from the cell are asymmetric: the matrix is typically relaxed first at the back of the cell, allowing forward motion, and then at the cell's leading edge. Matrix deformation in regions of the matrix near the cell's leading edge is elastic and mostly reversible, but induces irreversible matrix rupture events near the trailing edge. Our results also indicate that matrix remodeling spatially correlates with protrusive activity. This correlation is mediated by myosin II and Rac1, and eliminated after inhibition of pericellular proteolysis or ROCK. We have developed an assay based on high-resolution 3D multiple-particle tracking that allows us to probe local matrix remodeling during mesenchymal cell migration through a 3D matrix and simultaneously monitor protrusion dynamics.

Fibronectin fibrillogenesis regulates three-dimensional neovessel formation.

During vasculogenesis and angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. While 3D-embedded endothelial cells establish adhesive interactions with surrounding ligands to optimally respond to soluble or matrix-bound agonists, the manner in which a randomly ordered ECM with diverse physico-mechanical properties is remodeled to support blood vessel formation has remained undefined. Herein, we demonstrate that endothelial cells initiate neovascularization by unfolding soluble fibronectin (Fn) and depositing a pericellular network of fibrils that serve to support cytoskeletal organization, actomyosin-dependent tension, and the viscoelastic properties of the embedded cells in a 3D-specific fashion. These results advance a new model wherein Fn polymerization serves as a structural scaffolding that displays adhesive ligands on a mechanically ideal substratum for promoting neovessel development.

Nuclear lamin A/C deficiency induces defects in cell mechanics, polarization, and migration.

Lamin A/C is a major constituent of the nuclear lamina, a thin filamentous protein layer that lies beneath the nuclear envelope. Here we show that lamin A/C deficiency in mouse embryonic fibroblasts (Lmna(-/-) MEFs) diminishes the ability of these cells to polarize at the edge of a wound and significantly reduces cell migration speed into the wound. Moreover, lamin A/C deficiency induces significant separation of the microtubule organizing center (MTOC) from the nuclear envelope. Investigations using ballistic intracellular nanorheology reveal that lamin A/C deficiency also dramatically affects the micromechanical properties of the cytoplasm. Both the elasticity (stretchiness) and the viscosity (propensity of a material to flow) of the cytoplasm in Lmna(-/-) MEFs are significantly reduced. Disassembly of either the actin filament or microtubule networks in Lmna(+/+) MEFs results in decrease of cytoplasmic elasticity and viscosity down to levels found in Lmna(-/-) MEFs. Together these results show that both the mechanical properties of the cytoskeleton and cytoskeleton-based processes, including cell motility, coupled MTOC and nucleus dynamics, and cell polarization, depend critically on the integrity of the nuclear lamina, which suggest the existence of a functional mechanical connection between the nucleus and the cytoskeleton. These results also suggest that cell polarization during cell migration requires tight mechanical coupling between MTOC and nucleus, which is mediated by lamin A/C.

Probing intercellular interactions between vascular endothelial cadherin pairs at single-molecule resolution and in living cells.

Vascular endothelial (VE) cadherin is the surface glycoprotein cadherin specific to the endothelium that mediates cell-cell adhesion and plays a major role in the remodeling, gating, and maturation of vascular vessels. To investigate the contribution of individual VE-cadherins to endothelial cell-cell interactions and investigate whether different classical cadherins display different kinetics and micromechanical properties, we characterize the binding properties of VE-cadherin/VE-cadherin bonds at single-molecule resolution and in living human umbilical vein endothelial cells (HUVECs). Our single-molecule force spectroscopy measurements reveal that type II VE-cadherin molecules form bonds that are less prone to rupture and display a higher tensile strength than bonds formed by classical type I neuronal (N) cadherin and epithelial (E) cadherin. The equilibrium lifetime of the VE-cadherin/VE-cadherin bond is significantly longer than formed by N-cadherin/N-cadherin bonds and E-cadherin/E-cadherin bonds. These results indicate that VE-cadherins form bonds that have kinetics and mechanical properties that are significantly different from those formed by classical type I cadherins, properties that are particularly well adapted to the barrier and adhesive functions of VE-cadherin in endothelial cell-cell junctions.