The activation of the neutrophil respiratory burst by anti-neutrophil cytoplasm autoantibody (ANCA) from patients with systemic vasculitis requires tyrosine kinases and protein kinase C activation

The ability of antineutrophil cytoplasm autoantibodies (ANCA) from patients with systemic vasculitis to stimulate protein kinase C (PKC) and tyrosine kinases was examined in human neutrophils. Using the superoxide dismutase-inhibitable reduction of ferricytochrome C, the kinetics of ANCA-induced superoxide (O2−) production were characterized and subsequently manipulated by specific inhibitors of PKC and tyrosine kinases. With this approach, ANCA IgG, but not normal IgG or ANCA F(ab′)2 fragments caused a time and dose dependent release of O2− from TNF-α primed neutrophils. The kinetics of ANCA-induced O2− production showed an initial 10–15 min lag phase compared to the N-formyl-l-methionyl-l-leucyl-l-phenylalanine response, suggesting differences in the signalling pathways recruited by these two stimuli. Inhibitor studies revealed that ANCA-activation involved members of both the Ca2+-dependent and -independent PKC isoforms and also tyrosine kinases. ANCA IgG resulted in the translocation of the βII isoform of PKC at a time corresponding to the end of the lag phase of O2− production, suggesting that PKC activity may be instrumental in processes regulating the activity of the NADPH oxidase in response to ANCA. Tyrosine phosphorylation of numerous proteins also peaked 10–15 min after stimulation with ANCA but not normal IgG. These data suggest that PKC and tyrosine kinases regulate O2− production from neutrophils stimulated with autoantibodies from patients with systemic vasculitis

Focal ischemia due to traumatic contusions documented by stable xenon-CT and ultrastructural studies

✓ A traumatic cerebral contusion causes a zone of perifocal neuronal necrosis, the cause of which is not known; the surgical management of these lesions remains controversial. To determine the pathophysiological mechanisms responsible for brain damage after contusions, the authors performed cerebral blood flow (CBF) mapping studies and related these to change in local cerebral blood volume (CBV) and ultrastructure. In 11 severely head injured patients with contusion, CBF and CBV were measured in pericontusional areas using stable xenon-computerized tomography (CT). These studies demonstrated a profound reduction in perilesional CBF (mean 17.5 ± 4 ml/100 g/min), which was always accompanied by a zone of edema defined by CT density measurements. Mean CBV in these regions was 2.3 ± 0.4 ml/100 g, a reduction to approximately one-half the value of 4.8 ml/100 g found in the nonedematous regions, and to approximately 35% of the value of 6.0 ml/100 g found in normal volunteers. Ultrastructural analysis of the pericontusional tissue, taken at surgery in four patients with high intracranial pressure showed glial swelling with narrowing of the microvascular lumina due to massive podocytic process swelling. Additionally, some suggestion of vascular occlusion due to erythrocyte and leukocyte stasis was seen. These data support the conclusion that microvascular compromise by compression and/or occlusion is a major event associated with profound perilesional hypoperfusion, which is a uniform finding within edematous pericontusional tissue

Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo

The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection