Vascular relaxation of canine visceral arteries after ischemia by means of supraceliac aortic cross-clamping followed by reperfusion

<p>Abstract</p> <p>Background</p> <p>The supraceliac aortic cross-clamping can be an option to save patients with hipovolemic shock due to abdominal trauma. However, this maneuver is associated with ischemia/reperfusion (I/R) injury strongly related to oxidative stress and reduction of nitric oxide bioavailability. Moreover, several studies demonstrated impairment in relaxation after I/R, but the time course of I/R necessary to induce vascular dysfunction is still controversial. We investigated whether 60 minutes of ischemia followed by 30 minutes of reperfusion do not change the relaxation of visceral arteries nor the plasma and renal levels of malondialdehyde (MDA) and nitrite plus nitrate (NOx).</p> <p>Methods</p> <p>Male mongrel dogs (n = 27) were randomly allocated in one of the three groups: sham (no clamping, n = 9), ischemia (supraceliac aortic cross-clamping for 60 minutes, n = 9), and I/R (60 minutes of ischemia followed by reperfusion for 30 minutes, n = 9). Relaxation of visceral arteries (celiac trunk, renal and superior mesenteric arteries) was studied in organ chambers. MDA and NOx concentrations were determined using a commercially available kit and an ozone-based chemiluminescence assay, respectively.</p> <p>Results</p> <p>Both acetylcholine and calcium ionophore caused relaxation in endothelium-intact rings and no statistical differences were observed among the three groups. Sodium nitroprusside promoted relaxation in endothelium-denuded rings, and there were no inter-group statistical differences. Both plasma and renal concentrations of MDA and NOx showed no significant difference among the groups.</p> <p>Conclusion</p> <p>Supraceliac aortic cross-clamping for 60 minutes alone and followed by 30 minutes of reperfusion did not impair relaxation of canine visceral arteries nor evoke biochemical alterations in plasma or renal tissue.</p

Effects of tocilizumab on neutrophil function and kinetics

Background Decreases in circulating neutrophils (polymorphonuclear leukocytes, PMNs) have been reported in patients treated with the anti-interleukin-6 receptor (IL-6R) antibody tocilizumab (TCZ); the mechanism for this is unclear. We hypothesize that TCZ reduces circulating neutrophils by affecting margination and / or bone marrow trafficking without affecting neutrophil function or apoptosis. Materials and methods 18 healthy subjects were randomized to single intravenous dose of TCZ 8 mg/kg (n = 12) or placebo (n = 6) on day 0. On day 4, each subject had autologous indium-111-labeled neutrophils re-injected, and their kinetics quantified with longitudinal profiling in a whole body gamma-counter. TCZ-treated subjects were divided into two groups according to the extent of reduction in neutrophil count. Results Mean day 4 neutrophil counts, as % baseline, were 101.9%, 68.3% and 44.2% in the placebo, TCZ-PMN-’high’ and TCZ-PMN-’low’ groups, respectively (p < 0.001). Following TCZ, neutrophil function, activation and apoptosis ex vivo were all unaffected. In vivo, there were no differences in early blood recovery or margination to liver / spleen and bone marrow; however, later neutrophil re-distribution to bone marrow was markedly reduced in the TCZ-PMN-low group (peak pelvic count as % day 4 count on: day 5, 188% placebo vs 127% TCZ-PMN-low, p < 0.001; day 10, 180% placebo vs 132% TCZ-PMN-low, p < 0.01), with a trend towards higher liver / spleen neutrophil retention. Conclusions We have demonstrated for the first time in humans that IL-6R blockade affects neutrophil trafficking to the bone marrow without influencing neutrophil functional capacity.This work was supported by grants from F Hoffman La Roche Ltd. and the Evelyn Trust (L.S.C.L.). F.D. and B.P-B. are employees of F Hoffman La Roche Ltd. E.R.C. has received fees from F Hoffman La Roche Ltd

Role of P-selectin and nitric oxide in leukocyte-endothelial interaction in the rat mesenteric microcirculation

P-selectin, a member of the selectin family of adhesion molecules, is believed to play a significant role in mediating leukocyte-endothelial interaction during the inflammation response. To examine the role of P-selectin in the rat mesenteric microcirculation following splanchnic arterial occlusion/reperfusion (SAO/R), pentobarbital anesthetized rats were subjected to 60 minutes of celiac and superior mesenteric artery occlusion followed by 120 minutes of reperfusion. SAO/R resulted in a significant increase in P-selectin expression on the venular endothelium as determined immunohistochemically. This increase in P-selectin expression correlated to a significant increase in leukocyte rolling along and adherence to the venular endothelium as observed via intravital microscopy. Administration of the P-selectin neutralizing monoclonal antibody, PB1.3, 10 minutes prior to reperfusion significantly attenuated leukocyte rolling and leukocyte adherence following splanchnic ischemia-reperfusion, thus indicating a significant role for P-selectin in splanchnic ischemia-reperfusion induced leukocyte-endothelial interaction in the rat. Blocking leukocyte-endothelial interaction in this manner, significantly decreased leukocyte accumulation in the post-ischemic tissue as quantified by tissue myeloperoxidase activity, and decreased tissue injury as evidenced by a decreased plasma free amino-nitrogen concentration. Similarly, administration of the potent nitric oxide donor S-nitroso-N-acetylpenicillamine (SNAP) just prior to reperfusion significantly attenuated the ischemia-reperfusion induced increases in leukocyte-endothelial interaction. Immunohistochemical localization of P-selectin following SAO/R in SNAP treated rats revealed a significant decrease in ischemia-reperfusion induced P-selectin expression, thus indicating a regulatory role for nitric oxide in P-selectin expression. To confirm the relationship between nitric oxide and endothelial expression of P-selectin, endothelial NO was directly inhibited by superfusion of the rat mesentery with the NO synthase inhibitor, N\sp{\rm G}-nitro-L-arginine methyl ester (L-NAME). Inhibition of endothelial NO resulted in a significant increase in P-selectin expression on the venular endothelial surface and a significant increase in P-selectin mediated leukocyte-endothelial interaction. The increased P-selectin expression observed following L-NAME superfusion was effectively blocked by simultaneous infusion of L-arginine, the substrate for NO synthase, thereby demonstrating that the effects of L-NAME were the result of NO inhibition. Thus, the data presented in this thesis indicate an important role for P-selectin in leukocyte-endothelial interaction following splanchnic arterial occlusion/reperfusion in the rat, and demonstrate a novel role for nitric oxide in regulating the expression of this adhesion molecule

Lipopolysaccharide-Induced Leukocyte Rolling and Adhesion in the Rat Mesenteric Microcirculation: Regulation by Glucocorticoids and Role of Cytokines

AbstractA common side effect of high dose glucocorticoid therapy is increased susceptibility to bacterial infection, an effect that is in part mediated through inhibition of leukocyte recruitment to infected areas. However, the sites at which glucocorticoids act to prevent the multistep process of leukocyte recruitment have not been fully established. In this study, the effects of the glucocorticoid dexamethasone (DEX) on leukocyte-endothelial interactions, in response to bacterial LPS, were examined utilizing a model of rat mesenteric intravital microscopy. Pretreatment of rats with DEX (0.5 mg/kg) for 18 h or 30 min before stimulation with LPS significantly inhibited LPS-induced leukocyte rolling and adhesion in mesenteric postcapillary venules. Pretreatment with DEX also inhibited LPS-induced changes in expression of L-selectin and a shared epitope of CD11b/c on circulating neutrophils. These effects of DEX may be due to DEX inhibition of IL-1, TNF, and cytokine-induced neutrophil chemoattractant-1 (CINC-1) generation, since antagonists to these mediators were able to mimic DEX effects on leukocyte-endothelial interactions and circulating leukocyte phenotype. These data indicate that inhibition of cytokine- and chemokine-induced leukocyte-endothelial interactions may be a primary mechanism by which glucocorticoids inhibit leukocyte recruitment to bacterial agents and thus increase susceptibility to infection.</jats:p

Identification of candidate substrates for ectodomain shedding by the metalloprotease-disintegrin ADAM8.

ADAM proteases are type I transmembrane proteins with extracellular metalloprotease domains. As for most ADAM family members, ADAM8 (CD156a, MS2) is involved in ectodomain shedding of membrane proteins and is linked to inflammation and neurodegeneration. To identify potential substrates released under these pathologic conditions, we screened 10-mer peptides representing amino acid sequences from extracellular domains of various membrane proteins using the ProteaseSpot™ system. A soluble ADAM8 protease containing a pro- and metalloprotease domain was expressed in E. coli and purified as active protease owing to autocatalytic prodomain removal. From 34 peptides tested in the peptide cleavage assay, significant cleavage by soluble ADAM8 was observed for 14 peptides representing membrane proteins with functions in inflammation and neurodegeneration, among them the β-amyloid precursor protein (APP). The in vivo relevance of the ProteaseSpot™ method was confirmed by cleavage of full-length APP with ADAM8 in human embryonic kidney 293 cells expressing tagged APP. ADAM8 cleaved APP with similar efficiency as ADAM10, whereas the inactive ADAM8 mutant did not. Exchanging amino acids at defined positions in the cleavage sequence of myelin basic protein (MBP) revealed sequence criteria for ADAM8 cleavage. Taken together, the results allowed us to identify novel candidate substrates that could be cleaved by ADAM8 in vivo under pathologic conditions