George M. Matuschak

Therapeutic manipulation of peroxynitrite attenuates the development of opiate-induced antinociceptive tolerance in mice

Severe pain syndromes reduce quality of life in patients with inflammatory and neoplastic diseases, often because chronic opiate therapy results in reduced analgesic effectiveness, or tolerance, leading to escalating doses and distressing side effects. The mechanisms leading to tolerance are poorly understood. Our studies revealed that development of antinociceptive tolerance to repeated doses of morphine in mice was consistently associated with the appearance of several tyrosine-nitrated proteins in the dorsal horn of the spinal cord, including the mitochondrial isoform of superoxide (O2-) dismutase, the glutamate transporter GLT-1, and the enzyme glutamine synthase. Furthermore, antinociceptive tolerance was associated with increased formation of several proinflammatory cytokines, oxidative DNA damage, and activation of the nuclear factor poly(ADP-ribose) polymerase. Inhibition of NO synthesis or removal of O2- blocked these biochemical changes and inhibited the development of tolerance, pointing to peroxynitrite (ONOO-), the product of the interaction between O2- and NO, as a signaling mediator in this setting. Indeed, coadministration of morphine with the ONOO- decomposition catalyst, Fe(III) 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin, blocked protein nitration, attenuated the observed biochemical changes, and prevented the development of tolerance in a dose-dependent manner. Collectively, these data suggest a causal role for ONOO- in pathways culminating in antinociceptive tolerance to opiates. Peroxynitrite (ONOO-) decomposition catalysts may have therapeutic potential as adjuncts to opiates in relieving suffering from chronic pain.

A Role for Nitric Oxide-Mediated Peroxynitrite Formation in a Model of Endotoxin-Induced Shock

The aim of the present study was to assess the relative contributions of peroxynitrite formation following induction of nitric-oxide synthase (iNOS) in the pathophysiology of endotoxin-induced shock in the rat. To this end, we used a selective inhibitor of iNOS, N-(3-(aminomethyl)benzyl)acetamidine (1400W), and a peroxynitrite decomposition catalyst, 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron III chloride (FeTTPs). Intravenous (i.v.) administration of Escherichia coli lipopolysaccharide (LPS; 4 mg/kg) elicited a time-dependent fall in mean arterial pressure as well as liver, renal, and pancreatic tissue damage. 1400W (3-10 mg/kg i.v.) administered 30 min before LPS delayed the development of hypotension but did not improve survival. On the other hand, FeTTPs administered (10-100 mg/kg i.v.) inhibited in a dose-dependent manner LPS-induced hypotension, tissue injury, and improved mortality rate. In separate experiments, rats were treated with LPS (4 mg/kg) or saline for control, and their aortas were isolated and placed in organ baths 2 h later. Tissues from LPS-treated rats had significant inhibition of contractile activity to phenylephrine as well as a significantly impaired relaxation response to acetylcholine. FeTPPs, when administered (100 mg/kg i.v.) 1 h before LPS, prevented the LPS-induced aortic contractile and endothelial dysfunction. These results demonstrate that nitric oxide-derived peroxynitrite formation plays an important role in this model of endotoxemia. Our results also suggest that use of an iNOS inhibitor in this setting has little beneficial effect in part because, in the presence of a failing eNOS system, some NO is needed to maintain adequate organ function.

Magnesium in critical illness: metabolism, assessment, and treatment.

Magnesium is the second most abundant intracellular cation and the fourth most common cation in the body [1]. Its importance as an essential nutrient has been recognized since 1932, when Kruse et al. [2] reported the effects of acute Mg deficiency in rats. Even recently Mg was considered the “forgotten cation” in clinical practice [3]; however, this is no longer the case [4]. Estimates of Mg deficiency range from 20% to 61% [5, 6, 7], while a recent study found that reductions in total serum Mg on admission are associated with increased mortality [8].

Superoxide potentiates NF-κB activation and modulates endotoxin-induced cytokine production in alveolar macrophages

Gram-negative bacterial infection predisposes to the development of shock and acute lung injury with multiple organ dysfunction in the critically ill. Although overexpression of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6, IL-8, and other mediators is causally implicated in the pathogenesis of shock and lung injury, the underlying mechanisms following cellular exposure to gram-negative endotoxin remain unclear. De novo generation of reactive oxygen species (ROS) by monocytes/macrophages in particular has been proposed as a pivotal regulatory mechanism by which enhanced transactivation of redox-sensitive genes culminates in augmented cytokine expression within the lower respiratory tract. Here we sought to characterize the mechanism of action of a synthetic, nonpeptide, low-molecular-weight, Mn-containing superoxide dismutase mimetic (SODm), M40403, in modulating E. coli lipopolysaccharide serotype 0111:B4 (LPS)-induced cytokine production by cultured rat alveolar macrophages. Intracellular superoxide (O2.−) ion generation was measured using hydroethidine (HE) dye, and the dose-dependent effects of M40403 on TNF-α and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-κB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IκB-α were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-α, and IL-6 in LPS-stimulated alveolar macrophages, suggesting a key role for superoxide in endotoxin-induced cytokine production in the distal air spaces. In addition, M40403 decreased E. coli LPS-induced activation of NF-κB, and this effect was associated with modest suppression of cytoplasmic IκB-α degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-α and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-κB transactivation or signaling.

Cyclooxygenases 1 and 2 contribute to peroxynitrite-mediated inflammatory pain hypersensitivity

Peroxynitrite (ONOO−), the reaction product of the interaction between superoxide (O2·−) and nitric oxide (·NO), is a potent proinflammatory and cytotoxic nitrooxidative species. Its role as a mediator of hyperalgesia (clinically defined as an augmented sensitivity to painful stimuli) is not known. In light of the known proinflammatory properties of ONOO−, our study addressed its potential involvement in the development of hyperalgesia associated with tissue damage and inflammation. Intraplantar injection in rats of the ONOO− precursor O2·− (1 εM) led to the development of thermal hyperalgesia associated with a profound localized inflammatory response. Both events were blocked by L‐NAME (NG ‐nitro‐L‐arginine methyl ester, 3–30 mg/kg), a nitric oxide synthase inhibitor, or by FeTM‐4‐PyP+ [Fe(III)5,10,15,20‐tetrakis(N‐meth‐ylpyridinium‐4‐yl)porphyrin, 3–30 mg/kg], an ONOO−decomposition catalyst. These results suggested that locally synthesized ONOO− produced in situ by O2·−and ·NO is key in the development of inflammatory hyperalgesia. The direct link between ONOO− and hyperalgesia was further supported by demonstrating that intraplantar injection of soluble ONOO− itself (1 εM) similarly led to inflammatory hyperalgesia. ONOO− generated by the interaction between exogenous administration of O2·− and endogenous ·NO, or provided by direct injection of ONOO−, activated the transcription factor NF‐κB in paw tissues, enhancing expression of the inducible but not the constitutive cyclooxygenase enzyme (COX‐2 and COX‐1, respectively). ONOO−‐mediated hyperalgesia was blocked in a dose‐dependent manner by intraperitoneal injections of indomethacin (10 mg/kg), a nonselective COX‐1/ COX‐2 inhibitor, or NS398 [N‐(2‐cyclohexyloxy‐4‐nitro‐phenyl)methanesulfonamide; 10 mg/kg] a selective COX‐2 inhibitor, as well as by an anti‐prostaglandin (PG) E2 antibody (200 pg). In another established model of inflammation‐related hyperalgesia by intraplantar injection of carrageenan in rats, inhibition of ONOO− with FeTM‐4‐PyP5+ (3–30 mg/kg) inhibited the development of hyperalgesia and the release of PGE2 in paw tissue exudates. Furthermore, FeTM‐4‐PyP5+ synergized with indomethacin and NS397 (1–10 mg/kg) to block both hyperalgesia and edema. Taken together, these data show for the first time that ONOO− is a potent mediator of inflammation‐derived hyperalgesia operating via the COX‐to‐PGE2 pathway. These results provide a pharmacological rationale for the development of inhibitors of peroxynitrite biosynthesis as novel nonnarcotic analgesics. The broad implications of our study are that dual inhibition of both ONOO− formation and COX activity may provide an alternative therapeutic approach to the management of pain: effective analgesia with reduced side‐effects typically associated with the use of COX inhibitors.—Ndengele, M. N., Cuzzocrea, S., Esposito, E., Mazzon, E., Di Paola, R., Matuschak, G. M., Salvemini, D. Cyclooxygenases 1 and 2 contribute to peroxynitritemediated inflammatory pain hypersensitivity. FASEB J. 22, 3154–3164 (2008)

Spinal ceramide modulates the development of morphine antinociceptive tolerance via peroxynitrite-mediated nitroxidative stress and neuroimmune activation.

The effective treatment of pain is typically limited by a decrease in the pain-relieving action of morphine that follows its chronic administration (tolerance). Therefore, restoring opioid efficacy is of great clinical importance. In a murine model of opioid antinociceptive tolerance, repeated administration of morphine significantly stimulated the enzymatic activities of spinal cord serine palmitoyltransferase, ceramide synthase, and acid sphingomyelinase (enzymes involved in the de novo and sphingomyelinase pathways of ceramide biosynthesis, respectively) and led to peroxynitrite-derive nitroxidative stress and neuroimmune activation [activation of spinal glial cells and increase formation of tumor necrosis factor-α, interleukin (IL)-1β, and IL-6]. Inhibition of ceramide biosynthesis with various pharmacological inhibitors significantly attenuated the increase in spinal ceramide production, nitroxidative stress, and neuroimmune activation. These events culminated in a significant inhibition of the development of morphine antinociceptive tolerance at doses devoid of behavioral side effects. Our findings implicate ceramide as a key upstream signaling molecule in the development of morphine antinociceptive tolerance and provide the rationale for development of inhibitors of ceramide biosynthesis as adjuncts to opiates for the management of chronic pain.

Ceramide: A Key Signaling Molecule in a Guinea Pig Model of Allergic Asthmatic Response and Airway Inflammation

Although mechanisms involved in the pathogenesis of asthma remain unclear, roles for oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation have been documented. Ceramide is a sphingolipid with potent proinflammatory and proapoptotic properties. This study aimed at determining whether increased formation of ceramide contributes to the development of airway inflammation and hyper-responsiveness, using a well characterized in vivo model of allergic asthmatic response and airway inflammation in ovalbumin-sensitized guinea pigs. Aerosol administration of ovalbumin increased ceramide levels and ceramide synthase activity in the airway epithelium associated with respiratory abnormalities, such as cough, dyspnea, and severe bronchoconstriction. These abnormalities correlated with nitrotyrosine formation in the airway epithelium and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation evident by the infiltration of neutrophils and eosinophils in lung tissues, mast cell degranulation, and release of prostaglandin D2 and proinflammatory cytokines. Inhibition of de novo ceramide synthesis with the competitive and reversible inhibitor of ceramide synthase fumonisin B1 (0.25, 0.5 and 1 mg/kg b.wt.), given i.p. daily for 4 days before allergen challenge, attenuated nitrotyrosine formation and oxidative/nitrosative stress, epithelial cell apoptosis, and airway inflammation while improving the respiratory and histopathological abnormalities. These results implicate ceramide in the development of allergic asthmatic response and airway inflammation. Strategies aimed at reducing the levels of ceramide and downstream events should yield promising novel anti-asthmatic agents.

Multicenter evaluation of a human monoclonal antibody to Enterobacteriaceae common antigen in patients with Gram-negative sepsis.

ObjectiveTo evaluate in Gram-negative sepsis patients the human monoclonal immunoglobulin M antibody (MAB-T88) directed at the enterobacterial common antigen which is a specific surface antigen closely linked to lipopolysaccharide and shared by all members of the Enterobacteriaceae family of Gram-negative bacteria. DesignProspective, randomized, double-blinded, placebo-controlled, multicenter trial. SettingThirty-three academic medical centers in the United States. PatientsPatients were entered with a clinical diagnosis of sepsis, the presence of either shock or multiple organ dysfunction, and presumptive evidence for Gram-negative infection. InterventionsPatients received a single intravenous infusion, over 30 mins, of either 300 mg of MAB-T88 formulated in albumin, or placebo (albumin). Measurements and Main ResultsThe primary analysis group was prospectively identified as those patients with documented evidence of an infection with bacteria of the family Enterobacteriaceae at any site. The primary end point was survival within the first 28 days. A total of 826 patients were enrolled with 55% (n = 455) in the primary analysis group. There were no significant differences between the intervention and control primary analysis group study groups for sites of infection, severity of illness, underlying medical conditions, adequacy of antibiotic or surgical treatment, or other baseline variables except for a higher frequency of chronic renal failure in the MAB-T88 group (4.4% vs. 1.3%, p = .051). The average Acute Physiology and Chronic Health Evaluation II scores were 26.8 ± 8.6 (mean ± sd) in the MAB-T88-treated group and 26.5 ± 8.3 in the placebo-treated group (p = .72). There was no significant difference between MAB-T88- and placebo-treated groups during the first 28-day all-cause mortality in the primary analysis group (34.2% vs. 30.8%, p = .44) or in all 826 patients enrolled (37.0% vs. 34.0%, p = .36). On subset analysis, the use of MAB-T88 was not associated with significant mortality trends. More adverse events were seen with the use of MAB-T88 in the bacteremic enterobacterial common antigen group (p < .05). ConclusionsUse of the human monoclonal antibody, MAB-T88, did not improve the mortality in patients with presumed Gram-negative sepsis or in those patients with proven enterobacterial common antigen infections. No subset trends were identified that would support further investigation of this agent in sepsis.

Cardiovascular determinants of the hemodynamic response to acute endotoxemia in the dog

The cardiovascular changes that occur early in hyperdynamic, hypotensive septic shock are poorly understood. In a splenectomized but otherwise intact pentobarbital-anesthetized canine model (n = 11), we simultaneously characterized the determinants of ventricular and peripheral vascular function in response to a bolus infusion of E. coli endotoxin (1.0 mg/kg). Right-ventricular (RV) and left-ventricular (LV) stroke volumes were measured by electromagnetic flow probes. Instantaneous RV function and venous return curves 1,2 and steady state arterial pressure-flow (P/Q) and LV function curves were generated by incremental volume loading both before (control) and 30 minutes after (endo) endotoxin infusion. Blood volume (BV) was determined by indicator dilution. Within five minutes of endotoxin infusion, hypotension and hypoperfusion developed because of a decrease in mean systemic pressure (P ma ) with no change in ventricular function of the arterial P/Q relation. However, after 30 minutes endo, compared with control, cardiac output increased (2.4 ± .3 to 3.2 ± .3 L/min, x ± XE. P P ma decreased further (11.3 ± 0.9 to 9.5 ± 0.9 mm Hg, P ma ) was unchanged. Ventricular stroke volumes at similar filling pressures were increased ( P

Outcomes of Etomidate in Severe Sepsis and Septic Shock

BACKGROUND The use of single-dose etomidate to facilitate intubation in critically ill patients has recently been debated given its suppression of steroidogenesis with possible resultant adverse outcomes. Our objective was to assess the effects of single-dose etomidate used during rapid-sequence intubation (RSI) on various measures of outcome, such as mortality, vasopressor use, corticosteroid use, ICU length of stay (ICU-LOS), and number of ventilator days. METHODS A retrospective 18-month cohort study was performed in a multidisciplinary ICU of an academic tertiary care institution. Consecutive patients with severe sepsis or septic shock who were intubated and mechanically ventilated were identified and grouped as having received single-dose etomidate during intubation or not. Hospital mortality, ICU length of stay, number of ventilator days, corticosteroid use, vasopressor use, and demographic and clinical variables were recorded. RESULTS Two hundred twenty-four patients were identified; 113 had received etomidate. The mean Acute Physiology and Chronic Health Evaluation II scores in the etomidate and nonetomidate groups were 21.3 ± 8.1 and 21.9 ± 8.3, respectively (P = .62). The relative risks for mortality and vasopressor use were 0.92 (CI, 0.74-1.14; P = 0.51) and 1.16 (CI, 0.9-1.51; P = .31), respectively, in the etomidate group. There were no significant differences in ICU-LOS (mean, 14 vs 12 days; P = .31) or number of ventilator days (mean, 11 vs 8 days; P = .13) between the etomidate and nonetomidate groups, respectively. The relative risk for corticosteroid use in the etomidate group was 1.34 (CI, 1.11-1.61; P = .003). Multivariate analysis using logistic regression demonstrated no significant association of etomidate with mortality (OR, 0.9; CI, 0.45-1.83; P = .78). CONCLUSION Single-dose etomidate used during RSI in critically ill patients with severe sepsis and septic shock was not associated with increased mortality, vasopressor use, ICU-LOS, or number of ventilator days. Patients intubated with etomidate had an increased incidence of subsequent corticosteroid use, with no difference in outcomes.