Effects of ethanol and ethanol metabolites on intrinsic function of mesenteric resistance arteries.

Evidence suggests that ethanol-induced hypertension is associated with increased cardiovascular responsiveness to vasopressors in vivo and enhanced reactivity of isolated arteries to vasopressors ex vivo. The underlying mechanisms are not well understood and the contribution of ethanol metabolites to vascular effects induced by ethanol consumption are unclear. Mesenteric resistance arteries were harvested from Sprague-Dawley rats. Pressure myography was utilized to test effects of ethanol, acetaldehyde and phosphatidylethanol on myogenic tone and on vasoconstriction induced by phenylephrine, arginine vasopressin (aVP), endothelin-1 and KCl. Ethanol, acetaldehyde and phosphatidylethanol concentrations were monitored during the experiments. Ethanol concentrations in the vessel bath decreased with a half-life of 25min; acetaldehyde and phosphatidylethanol concentrations remained constant. Pretreatment with ethanol dose-dependently increased the potency of phenylephrine to induce vasoconstriction 4-fold (p<0.01). These effects were comparable when arteries were pre-treated with a single dose of ethanol for 30min and when ethanol concentrations were kept constant during 30min and 60min of pretreatment. While ethanol also dose-dependently increased the potency of aVP to induce vasoconstriction 1.7-fold (p<0.05), it did not affect vasoconstriction induced by endothelin-1 or KCl. Acetaldehyde pre-treatment (30 min) dose-dependently increased the potency of phenylephrine to induce vasoconstriction 2.7-fold (p<0.01) but did not affect other vasoconstrictor responses. Phosphatidylethanol did not affect any vasoconstrictor responses. Ethanol and its metabolites did not affect myogenic tone. These data suggest that ethanol and acetaldehyde selectively sensitize intrinsic constrictor responses upon activation of vascular α1-adrenergic and/or vasopressin receptors at clinically relevant concentrations. Our findings support the concept that enhanced vasoreactivity to vasoactive hormones contributes to the development of hypertension induced by ethanol consumption. Ex vivo exposure of resistance arteries to ethanol and acetaldehyde resembles effects of chronic ethanol consumption on intrinsic vascular function, and thus could serve as test platform to evaluate interventions aimed to mitigate vascular effects associated with ethanol consumption

Extracellular Ubiquitin Increases in Packed Red Blood Cell Units During Storage

Ubiquitin (Ub) is involved in intracellular protein metabolism, but may also have extracellular roles in host defense and immunomodulation. Erythrocytes contain high amounts of Ub and hemolysis is one potential source of extracellular Ub in vivo. Since hemolysis also occurs with storage of packed RBC units (pRBCs) in vitro, we hypothesized that Ub is released during storage and that it correlates with immunological properties of pRBCs. Daily aliquots were drawn from pRBCs ( n = 3) for 42 days and plasma was isolated. Ub was measured by ELISA. Immunomodulatory properties of plasma were assessed by measuring endotoxin-stimulated cytokine (TNF-α, IL-6, IL-8) production of normal whole blood, and cell proliferation in phytohemagglutinin-stimulated peripheral blood mononuclear cells. Plasma Ub linearly increased (49 ± 2 ng/mL/day; r 2 = 0.82, P < 0.001) 20-fold to 2170 ± 268 ng/mL on day 42. Plasma inhibited TNF-α production but stimulated IL-8 production of normal whole blood, which correlated with time-dependent Ub release (TNFα: r spearman = −0.626, P < 0.001; IL-8: r spearman = 0.427, P = 0.004). Addition of exogenous Ub (equaling day 42 concentration) to day 0–4 plasma inhibited TNF-α production by one-third of the effect detected for day 42 plasma, but also inhibited IL-8 production by 40%. IL-6 production and cell proliferation was unchanged between day 0–4 plasma with or without Ub supplementation and day 42 plasma. Extracellular Ub release in pRBCs correlates with in vitro immunomodulatory effects and may partially contribute to transfusion-related immune modulation. Additionally, the linear kinetics of the ubiquitin release during pRBC storage suggest Ub is a suitable in vitro quality control parameter

Alterations in Leukocyte Function following Surgical Trauma: Differentiation of Distinct Reaction Types and Association with Tumor Necrosis Factor Gene Polymorphisms

Endotoxin-stimulated blood cytokine responses have been widely used to describe compromised host defense mechanisms after trauma. We investigated whether blood cytokine production after endotoxin stimulation is able to define distinct trauma-induced alteration patterns and whether alteration patterns are associated with tumor necrosis factor (TNF) gene polymorphisms. In 48 patients undergoing joint replacement, the levels of TNF alpha (TNF-α), interleukin 6 (IL-6), and IL-8 production in blood after endotoxin stimulation were measured preoperatively on the day of surgery and 24 h thereafter. Patients were genotyped for the TNF-α position −308 G/A polymorphism and the TNF-β NcoI polymorphism. Postoperative alterations, i.e., increases or decreases of cytokine levels (TNF-α versus IL-6, P = 0.013; TNF-α versus IL-8, P = 0.001; IL-6 versus IL-8, P = 0.007), and relative postoperative changes, i.e., percentages of preoperative cytokine levels (TNF-α versus IL-6, r(s) = 0.491, P < 0.001; TNF-α versus IL-8, r(s) = 0.591, P < 0.001; IL-6 versus IL-8, r(s) = 0.474, P < 0.001 [where r(s) is the Spearman rank correlation coefficient]), had significant positive correlations among the cytokines. Overall enhanced postoperative alteration patterns were found in 10 patients, attenuated patterns were found in 18 patients, and mixed patterns were found in 20 patients. Preoperative cytokine production levels differed significantly between these groups (those of the overall enhanced pattern group were less than those of the mixed pattern group, which were less than those of the overall attenuated pattern group). TNF polymorphisms were not associated with overall alteration patterns, but the A*TNFB1 haplotype was associated with a postoperative increase in TNF-α production (P = 0.042). Whole-blood cytokine responses to endotoxin define the following preexisting patterns in leukocyte function: low baseline production and overall enhanced alteration patterns after trauma (type 1), intermediate baseline production and mixed alteration patterns (type 2), and high baseline production and overall attenuated alteration patterns (type 3). TNF gene polymorphisms were associated with changes in TNF-α production but do not explain the overall reaction patterns of cytokine production after trauma. The clinical correlate of these newly defined reaction types remains to be determined

Therapeutic potential of exogenous ubiquitin during resuscitation from severe trauma

Recent studies suggest that extracellular ubiquitin could have a physiologic role in immunodepression in sepsis and trauma. The therapeutic potential of exogenous ubiquitin after trauma has not been examined. To fill this gap, we designed a series of experiments in a clinically relevant trauma model. Forty minutes after femur fractures and hemorrhage, swine received 1.3 mg of ubiquitin per kilogram or bovine serum albumin intravenously followed by fluid resuscitation to maintain systemic hemodynamics. Leukocyte function and the immunomodulatory capacity of serum were assessed measuring endotoxin-evoked tumor necrosis factor-alpha (TNF alpha) production ex vivo. TNF alpha and ubiquitin were quantified with enzyme-linked immunosorbent assay. Intravenous ubiquitin had no significant hemodynamic effect in normal animals. After injury, ubiquitin significantly reduced fluid requirements by at least 60% (p < 0.05). The injury was associated with transient immunodepression, as reflected by reduced endotoxin-evoked TNF alpha production by 40% to 50%. With ubiquitin, this response remained depressed for 100 to 160 minutes (p < 0.05), but fully recovered to baseline with albumin. Ubiquitin is apparently safe and effective for reducing fluid requirements as a measure of diffuse capillary leak. This immunomodulatory property suggests a new therapeutic approach after injury in particular, and for infectious and noninfectious inflammation in general