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Previously it was reported that combining antibiotics with L-97-1, an adenosine A1 receptor antagonist, significantly improves survival and blocks acute lung injury induced by Yersinia pestis CO 99 in a rat model of pneumonic plague. In the current studies using a conscious rat model of cecal ligation and puncture (CLP) sepsis, L-97-1 was administered in daily intravenous infusions in combination with antibiotics to simulate the use of L-97-1 as an anti-sepsis therapeutic in the clinical setting. In these studies, when administered at 12 hours (h) following CLP, in combination with broad spectrum antibiotics, ceftriaxone and clindamycin, L-97-1 improves 7 day (d) survival [25%, 35%, and 75%, respectively for L-97-1 (10 mg/kg/h, 12.5 mg/kg/h, and 15 mg/kg/h) versus (vs.) 25% for antibiotics alone] in a dose-dependent manner. The addition of L-97-1, 15 mg/kg/h to antibiotics significantly increased 7 d survival following CLP compared to therapy with either antibiotics alone (P = 0.002) or L-97-1 at 15 mg/kg/h alone (P < 0.001) and was not significantly different than survival in sham CLP animals (Log-rank (Mantel-Cox) test with Bonferroni’s correction for multiple comparisons). Moreover, in these studies, in combination with antibiotics L-97-1 dose-dependently protects the kidney, significantly improving renal function at 24 h post CLP at 10 mg/kg/h (P < 0.001), 12.5 mg/kg/h (P < 0.0001), and 15 mg/kg/h (P < 0.0001) vs. antibiotics alone (ANOVA followed by Tukey’s post-hoc test for pair-wise comparisons). The results of these studies support efficacy for L-97-1 as an anti-sepsis therapeutic.

Yersinia pestis, a Gram-negative bacillus causing plague and Centers for Disease Control and Prevention (CDC) classified Category A pathogen, has high potential as a bioweapon. Lipopolysaccharide, a virulence factor for Y. pestis, binds to and activates A1 adenosine receptor (AR)s and, in animals, A1AR antagonists block induced acute lung injury (ALI) and increase survival following cecal ligation and perforation. In this study, rats were infected intratracheally with viable Y. pestis [CO99 (pCD1+/Δpgm) 1 × 108 CFU/animal] and treated daily for 3 d with ciprofloxacin (cipro), the A1AR antagonist L-97-1, or cipro plus L-97-1 starting at 0, 6, 24, 48, or 72 h post- Y. pestis. At 72 h post- Y. pestis, cipro plus L-97-1 significantly improved 6-d survival to 60–70% vs 28% for cipro plus H2O and 33% for untreated Y. pestis controls ( P = 0.02, logrank test). Lung edema, hemorrhage and leukocyte infiltration index (LII) were evaluated histologically to produce ALI scores. Cipro plus L-97-1 significantly reduced lung edema, as well as aggregate lung injury scores vs controls or cipro plus H2O, and LII vs controls ( P

Pneumonic plague resulting from Yersinia pestis induces swiftly lethal sepsis and is a major concern as a weapon of bioterrorism. However, the role of specific plasmid-encoded vs. chromosomal Y. pestis virulence factors in the pathogenesis of acute lung injury, shock, and nonpulmonary dysfunction is unclear. We hypothesized that the pathophysiology of pneumonic plague resulting from expression of proteins encoded by the thermally regulated pCD1 plasmid differs from cardiopulmonary and inflammatory changes attributable to the chromosomal pgm gene locus.Prospective, experimental study.Research laboratory at a university medical center.Conscious, chronically catheterized male Sprague-Dawley rats (total n=104).Rats were intratracheally infected with 109 colony-forming units of Y. pestis attenuated strains CO99 (pCD1+/DeltaApgm) or KIM6+ (pCD1-/pgm+) and evaluated over 6 days. Serial evaluations of vital signs, cardiorespiratory parameters, blood cultures, inflammatory biomarkers, and organ function were obtained, as well as organ histopathology and cytokine production.Despite equivalent endotoxin contents between the inocula, CO99-infected animals had a median survival of 3 days with greater nonpulmonary organ injury, microbial growth, serum alanine aminotransferase, and liver microvascular permeability vs. KIM6+-infected animals (p.05). Parallel differences occurred in serum tumor necrosis factor-alpha levels. Notably, 75% of CO99 rats developed fatal hypotension after developing nonpulmonary organ damage.These results suggest that progression to lethal sepsis with augmented liver injury during plague pneumonia requires factors encoded by the pCD1 plasmid but not chromosomal proteins present within the pgm gene locus.

Reductions in alveolar oxygenation during lung hypoxia/reoxygenation (H/R) injury are common after gram-negative endotoxemia. However, the effects of H/R on endotoxin-stimulated cytokine production by alveolar macrophages are unclear and may depend upon thresholds for hypoxic oxyradical generation in situ. Here TNF-alpha and IL-1beta production were determined in rat alveolar macrophages stimulated with Escherichia coli lipopolysaccharide (LPS, serotype O55:B5) while exposed to either normoxia for up to 24h, to brief normocarbic hypoxia (1.5h at an atmospheric PO(2)=10+/-2mm Hg), or to combined H/R. LPS-induced TNF-alpha and IL-1beta were reduced at the peak of hypoxia and by reoxygenation in LPS+H/R cells (P0.01) compared with normoxic controls despite no changes in reduced glutathione (GSH) or in PGE2 production. Both TNF-alpha mRNA and NF-kappaB activation were reduced by hypoxia that suppressed superoxide anion generation. Thus, dynamic reductions in the ambient PO(2) of alveolar macrophages that do not deplete GSH suppress LPS-induced TNF-alpha expression, IL-1beta production, and NF-kappaB activation even as oxyradical production is decreased.

To systematically review the literature and present data on the safety and efficacy of induced moderate hypothermia combined with ICP monitoring in critically ill patients with acute liver failure. We conducted a retrospective observational search of MEDLINE database using both OVID and PubMed with the following MeSH terms, “Hypothermia, Induced,” “Brain Edema,” “Intracranial Hypertension” (ICH), “Liver failure, Acute” and “Liver Failure, Fulminant.” We limited our search to case series involving at least three human subjects and all other clinical trials. Baseline ICP, cerebral perfusion pressure (CPP) and cerebral blood flow (CBF) as well as the response of these variables to hypothermia were recorded when available. Additional clinical and demographic data were also recorded. Five case series were identified. Pre-existing coagulopathy from liver failure was reversed by various modalities in all studies prior to insertion of ICP monitors. Induction of moderate hypothermia combined with ICP monitoring consistently improved ICP, CPP and CBF in four trials; one trial demonstrated the feasibility and effectiveness of moderate induced hypothermia as part of a protocolized strategy for the management of ICH. Limited data exist concerning the safety and efficacy of moderate hypothermia and ICP monitoring for the treatment of ICH in acute liver failure. The available evidence shows that induction of moderate hypothermia in this clinical setting is feasible and possibly efficacious. Well-designed prospective clinical trials are warranted in this challenging context, given the potential of providing a bridge to liver transplantation or even clinical recovery.

It has been widely accepted that development of porto-pulmonary hypertension (POPH) is independent of the cause of portal hypertension. The degree of hepatic damage and liver function do not correlate with predisposition to POPH or its severity. However, portal hypertension has been confirmed as a prerequisite for developing pulmonary hypertension. Transthoracic echocardiography is the best screening test for the presence of POPH, but a diagnosis of POPH can be established only by right heart catheterization. Randomized controlled trials comparing the efficacy and safety of different pharmacologic strategies are lacking in patients with POPH. The general management includes diuretics and oxygen supplementation. Notably, moderate to severe POPH predisposes candidates for orthotopic liver transplantation to a higher risk of perioperative mortality. Vasomodulating pharmacologic agents are used in patients with moderate to severe POPH to decrease pulmonary arterial hypertension, thereby permitting liver transplantation to be performed safely. Epo-prostenol is the best-studied medication, and bosentan appears promising.

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.

Our previous studies have indicated that hypoxia-induced mitogenic factor (HIMF) has angiogenic properties in an in vivo matrigel plug model and HIMF upregulates expression of vascular endothelial growth factor (VEGF) in mouse lungs and cultured lung epithelial cells. However, whether HIMF exerts angiogenic effects through modulating endothelial cell function remains unknown. In this study, mouse aortic rings cultured with recombinant HIMF protein resulted in enhanced vascular sprouting and increased endothelial cell spreading as confirmed by Dil-Ac-LDL uptake, von Willebrand factor and CD31 staining. In cultured mouse endothelial cell line SVEC 4–10, HIMF dose-dependently enhanced cell proliferation, in vitro migration and tubulogenesis, which was not attenuated by SU1498, a VEGFR2/Flk-1 receptor tyrosine kinase inhibitor. Moreover, HIMF stimulation resulted in phosphorylation of Akt, p38 and ERK1/2 kinases in SVEC 4–10 cells. Treatment of mouse aortic rings and SVEC 4–10 cells with LY294002, but not SB203580, PD098059 or U0126, abolished HIMF-induced vascular sprouting and angiogenic responses. In addition, transfection of a dominant-negative mutant of phosphatidylinositol 3-kinase (PI-3K), Δp85, blocked HIMF-induced phosphorylation of Akt, endothelial activation and tubulogenesis. These results indicate that HIMF enhances angiogenesis by promoting proliferation and migration of endothelial cells via activation of the PI-3K/Akt pathways.

Transactivation of the DNA-binding proteins nuclear factor-κB (NF-κB) and activator protein (AP)-1 by de novo oxyradical generation is a stereotypic redox-sensitive process during hypoxic stress of the liver. Systemic trauma is associated with splanchnic hypoxia-reoxygenation (H/R) followed by intraportal gram-negative bacteremia, which collectively have been implicated in posttraumatic liver dysfunction and multiple organ damage. We hypothesized that hypoxic stress of the liver before stimulation by Escherichia coli serotype O55:B5 (EC) amplifies oxyradical-mediated transactivation of NF-κB and AP-1 as well as cytokine production compared with noninfectious H/R or gram-negative sepsis without prior hypoxia. Livers from Sprague-Dawley rats underwent perfusion for 180 min with or without 0.5 h of hypoxia (perfusate Po2, 40 ± 5 mmHg) followed by reoxygenation and infection with 109EC or 0.9% NaCl infusion. In H/R + EC livers, nuclear translocation of NF-κB and AP-1 was unexpectedly reduced in gel shift assays vs. normoxic EC controls, as were perfusate TNF-α and IL-1β levels. Preceding hypoxic stress paradoxically increased postbacteremic reduced-to-oxidized glutathione ratios plus nuclear localization of IκBα and phospho-IκBα, but not JunB/FosB profiles. Notably, xanthine oxidase inhibition increased transactivation as well as cytokine production in H/R + EC livers. Thus brief hypoxic stress of the liver before intraportal gram-negative bacteremia potently suppresses activation of canonical redox-sensitive transcription factors and production of inflammatory cytokines by mechanisms including xanthine oxidase-induced oxyradicals functioning in an anti-inflammatory signaling role. These results suggest a novel multifunctionality of oxyradicals in decoupling hepatic transcriptional activity and cytokine biosynthesis early in the posttraumatic milieu.

We hypothesized that the sympathomimetic cocaine may alter cardiovascular and inflammatory responses and enhance susceptibility to endotoxemia due to innate differences in patterns of sympathetic and cardiovascular responsiveness.Prospective study.Experimental animal laboratory.Fifty-six conscious, instrumented albino rats.Rats were instrumented for determination of arterial pressure and intravenous drug administration and, in some rats, for cardiac output. After recovery, rats were given cocaine (5 mg/kg i.v., twice daily with 4-6 trials) to identify one of two hemodynamic response patterns: a) an increase in systemic vascular resistance with cardiac depression (vascular responders) or b) smaller increases in systemic vascular resistance and no change or an increase in cardiac output (mixed responders). At least 1 month after characterizing response patterns to cocaine, animals were pretreated with cocaine (5 mg/kg i.v.) or an equivalent bolus of vehicle (0.9% saline) while recording hemodynamics. Five minutes later, Escherichia coli lipopolysaccharide (serotype O55:B5, 20 mg/kg i.v.) was administered for 15 mins.Hemodynamic responses, pupillary diameter, and serum cytokines were determined at several time points. Lipopolysaccharide administration (5-40 mg/kg) without cocaine produced dose-dependent depressor responses with recovery typically within 2 hrs. Although 87% of rats survived a single 20 mg/kg dose of lipopolysaccharide when given alone, pretreatment of vascular responders with cocaine before lipopolysaccharide resulted in greater increases in systemic vascular resistance and pupillary mydriasis and lethality in five of six vascular responders, whereas only one of six mixed responders died. Pretreatment with the alpha1-adrenoceptor antagonist prazosin (0.1 mg/kg i.v.) before cocaine and lipopolysaccharide attenuated hemodynamic responses and improved survival among vascular responders. Serum interleukin-6 and interleukin-10 were elevated in rats treated with cocaine and lipopolysaccharide compared with rats treated with lipopolysaccharide alone, whereas serum tumor necrosis factor-alpha was reduced by cocaine pretreatment. Moreover, serum interleukin-1beta, tumor necrosis factor-alpha, and interleukin-6 were elevated in nonsurvivors compared with survivors after cocaine and lipopolysaccharide administration.We conclude that cocaine enhances susceptibility and worsens outcome from endotoxic shock by augmenting sympathetic activity, particularly in vascular responders, and that alpha-adrenoceptors mediate the altered inflammatory responses.

Exacerbations of cystic fibrosis (CF) lung dis- ease are characterized by increased inspissation of abnor- mally viscid pulmonary secretions with resultant plugging of small airways, worsened ventilation/perfusion mismatch, and increased physiological deadspace. In this circumstance, hypoxic respiratory failure necessitating mechanical venti- lation can be life-threatening. We present such a case of CF lung disease poorly responsive to conventional mechanical ventilatory strategies, in which high-frequency percussive ventilation (HFPV) using volumetric diffusive respiration mobilized copious amounts of inspissated pulmonary secre- tions and improved refractory hypoxia. Subsequent transient hypercarbia necessitated titrating ventilator parameters to return the PaCO2 to baseline; the voluminous clearance of secretions and improvement in oxygenation were sustained. HFPV appears unique in its ability to function as a meth- odological continuum from noninvasive percussion to invasive percussive ventilation for airway clearance, a fun- damental tenet of the CF treatment paradigm.

Episodes of tissue hypoxia and reoxygenation frequently occur during gram-negative bacteremia that progresses to septic shock. However, few studies have evaluated modulation by hypoxia and reoxygenation of the proinflammatory cytokine gene expression that is normally induced by gram-negative bacteremia or endotoxemia. In buffer-perfused organs, hypoxia downregulates Escherichia coli-induced expression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the liver but upregulates these cytokines in the lungs. To identify molecular mechanisms underlying these events, we investigated the effects of brief (1.5-h) hypoxia on TNF-alpha and IL-1beta expression in cultured RAW 264.7 cells during their continuous exposure to lipopolysaccharide (LPS) endotoxin derived from E. coli (serotype 055:B5) for up to 24 h. IL-1beta and TNF-alpha concentrations in cell lysates and culture supernatants were measured by ELISA, and steady-state mRNA was measured by Northern analysis. LPS-induced IL-1beta synthesis was downregulated by hypoxia at both the protein and mRNA levels despite no change in cellular redox status as measured by levels of GSH. In contrast, LPS-induced TNF-alpha production was unaffected by hypoxia as assessed by cell lysate mRNA and lysate and supernatant protein levels. Nuclear runoff analysis showed that downregulation of IL-1beta gene expression by hypoxia occurred transcriptionally. Allopurinol or catalase treatment did not alter modulation of LPS-induced IL-1beta expression by hypoxia, suggesting that this suppression was not caused by reactive oxygen species. Cycloheximide pretreatment suggested that hypoxia-induced downregulation of IL-1beta expression did not require de novo protein synthesis.

We report a unique case of progressive tracheobronchial stenosis in a 52-year-old woman who presented to us with stridor and dyspnea at rest. Her initial symptoms began 20 years earlier, at which time subglottic stenosis of ill-defined etiology necessitated tracheal resection with end-to-end anastomosis. Tracheal biopsy at the time revealed nonspecific inflammation without granulomas, vasculitis, infection, amyloidosis, or malignancy. Over subsequent years, she underwent multiple endobronchial laser resections of the trachea for recurrent disease. On presentation to us, flexible bronchoscopy showed inflammatory stenoses of the left mainstem bronchus and bronchus intermedius. Bronchial biopsy showed acute and chronic stromal inflammation with scattered plasma cells and myofibroblasts against a background of dense fibrosis. Review of the initial tracheal resection specimens and subsequent bronchial specimens revealed areas of high collagenous content with a relatively scant overall myofibroblastic cellular infiltrate; stains for S-100 and anaplastic lymphoma kinase were negative. A diagnosis of idiopathic tracheal stenosis was made with unusual accompanying bronchial involvement, that is, idiopathic tracheobronchial stenosis. Inflammatory airway bronchostenoses were stabilized by high-dose steroids followed by weekly methotrexate therapy, as evidenced by serial flexible bronchoscopies and sequential chest computed tomography with 3-dimensional reconstruction imaging. To our knowledge, this is the first reported case of combined idiopathic tracheal and bronchial stenosis stabilized with anti-inflammatory treatment.

This study describes an unusual patient with X-linked Alport syndrome (XLAS) in whom diffuse alveolar hemorrhage (DAH) developed as a complication of alemtuzumab therapy following renal transplantation. A 26-year-old man with XLAS underwent retransplantation with a cadaveric renal allograft. He received alemtuzumab therapy as a part of an immunosuppressive induction protocol, and dyspnea and hemoptysis developed. A chest CT scan showed diffuse alveolar opacities. Bronchoscopy was performed to determine the cause of hemoptysis and hypoxia. BAL showed a characteristic increasingly bloody return in the sequential aliquots. There was no growth of pathogenic bacteria or evidence of opportunistic infection. Clinical improvement occurred with the initiation of steroids, and the patient required short-term mechanical ventilation for acute respiratory failure. To our knowledge, this is the first reported case of DAH associated with use of alemtuzumab therapy, although other pulmonary toxicities have been described. The prevalence of this form of pulmonary toxicity is unclear and requires further systematic study.

Introduction. Muramlytripeptide phosphatidylethanolamine (MTP) stimulates synthesis of cytokines by hepatic Kupffer cells. We have shown in a perfused rat liver model that secondary ischemia/reperfusion (I/R) downregulates tumor necrosis factor alpha (TNF-α) expression after Escherichia coli (EC) bacteremia. Here, we tested the hypothesis that pretreatment with MTP restores cytokine response after sequential bacteremia and I/R. Methods. Thirty-eight livers were studied in eight groups after intraportal injection of 10 9 live EC or normal saline (NS): (1) normoxic EC; (2) EC + I/R (ischemia began 30 min after EC followed by 2 h of reperfusion); (3) normoxic NS controls; and (4) NS + I/R. Four groups of rats received 300 μg/kg of MTP i.v. 24 h prior to liver harvesting: (5) MTP + EC; (6) MTP + EC + I/R; (7) MTP + NS; and (8) MTP + NS + I/R. Bioactive and antigenic TNF-α, PGE 2 and bacterial clearance were assessed. Results. MTP increased bioactive TNF-α response to EC (MTP + EC vs EC controls: 685 ± 255 U/ml vs 250 ± 180 U/ml, P < 0.02). I/R did not downregulate TNF-α in animals treated with MTP (MTP + NS vs MTP + NS + I/R, P = 0.83). Pretreatment with MTP restored TNF« after I/R MTP + EC + I/R vs EC + I/R: 671 ± 215 U/ ml vs 27 ± 14 U/ml, P < 0.001) to levels similar to those found in the MTP + EC group (MTP + EC + I/R vs MTP + EC: 671 ± 215 U/ml vs 685 ± 255 U/ml, P = 0.75). Finally, bacterial clearance was increased in groups which received MTP. Conclusion. In vivo administration of MTP increases hepatic TNF-α response to intraportal EC bacteremia by a PGE 2 independent mechanism. This response is maintained even after subsequent ischemia and reperfusion.

We employed a bile duct ligation (BDL) model of cholestatic liver injury to test the hypothesis that this form of preexisting hepatic dysfunction alters the kinetics of circulating TNF-alpha and IL-6 after Escherichia coli endotoxemia, thereby augmenting mortality and lung injury by a TNF-alpha:leukotriene (LT) axis of inflammation. Male rats were catheterized 13 d after BDL or sham surgery and studied while awake 18 to 24 h later. Cholestasis after BDL was confirmed by baseline serum bilirubin (BDL = 7.34 +/- 0.72 mg/dl, mean +/- SEM, n = 17 versus Sham = 0.25 +/- 0.07, n = 20; p < 0.005) and histopathology. Sham and BDL animals received E. coli lipopolysaccharide serotype O55:B5 (LPS, 5 mg/kg i.v.) or 0.9% NaCl (NS) ending at t = 0 and were monitored over 24 h for vital signs and hemodynamics. In parallel studies, lipoxygenase inhibition was performed using diethylcarbamazine or the 5-lipoxygenase activating-protein inhibitor MK-886. Blood was collected at baseline and at t = 1.5, 3.5, and 24 h for formed elements and for serum endotoxin, TNF-alpha, IL-6, bilirubin, and alanine aminotransferase (ALT). Organs were evaluated at 24 h for histopathology, including neutrophil (PMN) densities and wet/dry weight (W/D) ratios. Cholestasis reduced survival after otherwise nonlethal endotoxemia, with seven of 11 BDL + LPS rats dying within 24 h versus no deaths in BDL + NS (n = 6), Sham + LPS (n = 14), or Sham + NS (n = 6) animals (p < 0.01). Despite equivalent serum endotoxin between groups, circulating TNF-alpha was 8-fold higher in BDL + LPS than in Sham + LPS rats at 1.5 and 3.5 h (p < 0.001), whereas serum TNF-alpha did not differ between BDL + NS and Sham + NS rats. IL-6 likewise was increased differentially by 1.5 h in BDL + LPS animals (11.98 +/- 2.42 ng/ml) versus Sham + LPS rats (3.05 +/- 0.58 ng/ml, p < 0.05). Hypothermia, bradycardic hypotension, and leukopenia were most severe and prolonged in BDL + LPS rats, which also had significantly higher ALT values, W/D ratios, and organ PMN counts. LT inhibition failed to reduce BDL-related differences in serum cytokines or survival after endotoxemia. Thus, cholestasis augments inflammatory responses to gram-negative endotoxemia, sensitizing the host to enhanced fluid flux in multiple organs and to mortality by a LT-independent mechanism.

Decreases in the alveolar O2 tension commonly follow gram-negative bacteremic shock that progresses to the acute respiratory distress syndrome (ARDS). To examine the effects of alveolar hypoxia and reoxygenation (H/R) on postbacteremic pulmonary cytokine expression, lungs from Sprague-Dawley rats (n = 43) were perfused over 180 min after hematogenous infection with 10(9) live Escherichia coli serotype O55:B5 (EC) or infusion of 0.9% NaCl (NS). Compared with normoxic EC and NS controls, EC + H/R and NS + H/R lungs received 90 min of constant-flow hypoxia followed by 60 min of reoxygenation. Perfusates were cultured and analyzed for TNF-alpha, IL-1alpha, IL-1beta, and PGE2 while monitoring pulmonary artery pressure (Ppa). Changes in the filtration coefficient (Kf) were evaluated at 180 min when cytokine mRNA levels were assessed in lung homogenates. Transcripts of the anti-inflammatory cytokine TGF-beta1 and of inducible cyclooxygenase (COX-2) were similarly analyzed. For equivalent EC clearance, Ppa, and Kf as in normoxic EC, postbacteremic H/R increased TNF-alpha gene expression and doubled the export of TNF-alpha from the lungs, an effect not blocked by allopurinol. IL-1alpha transcripts were also increased in EC + H/R versus EC lungs, in contrast to the lack of change in IL-1beta, TGF-beta, or COX-2 mRNA levels, or in cell-associated or circulating IL-1beta and PGE2. Thus, gram-negative bacteremic lung infection and secondary alveolar H/R upregulate the expression of specific inflammatory cytokines compared with pulmonary infection under normoxic conditions, independently of xanthine oxidase-induced O2 radicals. These findings identify the alveolar PO2 as a potent immunomodulatory signal whose reductions early after gram-negative sepsis may enhance lung inflammation in ARDS.

Purpose: During gram-negative bacteremia (GNB), tumor necrosis factor-α (TNF-α) is a critical early mediator of host defense, whose overexpression can initiate acute lung injury, multiple organ failure, and death. In this study we evaluated the ability of a chimeric fusion protein containing two extracellular domains of the human p80 TNF-α receptor and the Fc region of human IgG 1 (TNFR:Fc) to reduce circulating TNF-α, and to ameliorate organ injury and improve survival in a rodent model of GNB during immunosuppression-related neutropenia. Materials and Methods: Conscious catheterized male rats ( n = 37) with stable cyclophosphamide-induced neutropenia were infected intravenously (IV) with 5 × 10 9 live Escherichia coli (EC, serotype 055:135) ending at t = 0. All animals received antibiotics (penicillin/ amikacin sulfate) at t = 0.5 and t = 8 hours, and 0.9% sodium chloride (normal saline solution (NS), 1 mL/h) from t = 0 to 8 hours. Subgroups were post-treated at t = 0.5 hours with a 1.0 mL IV dose of TNFR:Fc (60, 600, or 1,200 μg; Immunex), 600 μg of human IgG1-κ or IgG1-λ (Sigma), or NS alone (controls). A separate TNFR:Fc pretreatment subgroup received 600 μg/rat of the fusion protein 5 minutes before starting EC infusion. Hemodynamics were monitored continuously through t = 24 hours, and arterial samples were collected at baseline and at t = 1.5,4.5,8, and 24 hours after EC were analyzed for blood gases, quantitative culture, serum endotoxin, bioactive and antigenic TNF-α, and formed elements. Postmortem tissues were examined for histopathologic changes. Results: Compared with antibiotic-treated and fluid-supported controls, TNFR:Fc dose-dependently reduced bioactive but not antigenic TNF-α without altering bacterial clearance, serum endotoxin, or 24-hour survival. Of note, 600 pg of IgGl-κ or IgG1-λ attenuated peak bioactive TNF-α to a similar degree as 1,200 μg TNFR:Fc, and also significantly reduced serum endotoxin levels. Nevertheless, by t = 8 hours all bacteremic rats were hypothermic with tachypnea-related hypocarbia and hyperoxemia and were thrombocytopenic. At death, all subgroups showed similar hepatic glycogen depletion and pulmonary congestion with perivascular edema and alveolar hemorrhage. Conclusions: Although TNFR:Fc and its idiotypic control IgG1 reduced circulating bioactive TNF-α, neither treatment prevented progression of lethal shock with attendant organ injury in this conscious, antibiotic-treated and fluid-resuscitated model of immunosuppression-related GNB.

Lactoferrin is a glycoprotein with anti-infective and anti-inflammatory properties found in secretions and immune cells. Talactoferrin alfa, a recombinant form of human lactoferrin, has similar properties and plays an important role in maintaining the gastrointestinal mucosal barrier integrity. In experimental animal models, administration of talactoferrin reduces translocation of bacteria from the gut into the systemic circulation and mortality from sepsis. Our objective was to determine if talactoferrin could reduce 28-day all-cause mortality in patients with severe sepsis and to assess its safety.Prospective, randomized, double-blind, placebo-controlled, multicenter phase 2 trial.Adult ICUs and emergency departments in the United States.One hundred ninety-four adults within 24 hrs of the onset of severe sepsis.Enterally administered talactoferrin 1.5g or placebo every 8 hrs for up to 28 days or until discharge from the ICU.Modified intention-to-treat analysis was used to assess the primary (28-day all-cause mortality) and secondary endpoints. The all-cause mortality at 28 days was 26.9% in the placebo group and 14.4% in the talactoferrin group (two-sided p = 0.052), representing a 12.5% absolute and a 46.5% relative reduction in mortality, meeting the protocol-specified primary endpoint. Reduction in all cause mortality was sustained at 6 months (p = 0.039). These reductions in mortality were observed across a wide spectrum of subgroups. The drug was well tolerated with a safety profile similar to that of placebo.Enteral administration of talactoferrin reduced 28-day all-cause mortality in patients with severe sepsis. This reduction in mortality was sustained at 6 months. Talactoferrin was very well tolerated.

Cyclophosphamide-induced neutropenia exacerbates septic shock and multiple organ injury in conscious rats during Escherichia coli (EC) bacteremia despite antibiotics and fluid administration. We hypothesized that such shock and inflammatory organ injury would be mitigated by rBPI23's microbicidal activity and/or binding of EC endotoxins. Four days after 100 mg cyclophosphamide/kg, catheterized rats with < 300 PMNs/microL were pretreated with rBPI23 or the irrelevant 22 kDa protein thaumatin [3.3-6.6 mg/kg, i.v. in 0.9% NaCl (NS)] 5 min before graded i.v. infection with 5 x 10(9) or 1 x 10(10) cfu of EC serotype 055:B5 ending at t = 0. Posttreatment with each protein continued (3.3-6.6 mg/kg in 1 mL NS/h) through 8 h, in addition to penicillin plus amikacin sulfate at t = 1.5 and 8 h. Arterial samples were obtained before pretreatment and at t = 1.5, 4.5, 8, and 24 h when animals were necropsied. One of eight thaumatin + 5 x 10(9) EC rats and none of six thaumatin + 10(10) EC rats survived 24 h. In contrast, rBPI23 significantly reduced mortality after either inoculum, improved bacterial clearance, and led to renormalization of early EC-induced hypotension, hypothermia, tachypnea, hyperoxemia, and hypocarbia. Compared with thaumatin, however, rBPI23 did not reduce circulating endotoxin or bioactive and antigenic tumor necrosis factor-alpha. Sepsis-induced severe neutropenia (< 50 PMNs/microL) evident in all EC rats by t = 1.5 h was reversed with rBPI23 by t = 8 h, but thrombocytopenia (< 5 x 10(4) platelets/microL) evident in all groups by t = 4.5 h was not altered.(ABSTRACT TRUNCATED AT 250 WORDS)

We tested the hypothesis that reducing the hepatic O2 supply by 30 min of constant-flow hypoxia (PO2, approximately 45 Torr) following gram-negative bacteremia downregulates tumor necrosis factor-alpha (TNF-alpha) in buffer-perfused rat lives (total n = 44). Eight groups were studied after intraportal 10(9) viable E. coli serotype 055:B5 (EC) or 0.9% NaCl (NS) at t = 0:1) normoxic EC; 2) normoxic NS controls; 3) EC+hypoxia (H)-reoxygenation (R) in which H began 30 min after EC followed by 120 min of R; and 4) NS+H/R. To assess the role of cyclooxygenase vs. xanthine oxidase activation, the effects of 10(-5) M indomethacin (Indo) in 5) Indo+EC+H/R and 6) Indo+NS+H/R were compared with allopurinol (Allo) in 7) Allo+EC+H/R and 8) Allo+NS+H/R groups. Bacterial clearance, bioactive and antigenic TNF-alpha, and hepatic O2 uptake and performance were serially assessed, as was prostaglandin (PG) E2 at baseline and peak hypoxia in EC-challenged groups. Intrahepatic bacterial killing and TNF-alpha mRNA were determined at t = 180 min. Bioactive venous TNF-alpha did not increase in normoxic NS controls (6 +/- 3 U/ml at t = 180 min; mean +/- SE), whereas levels rose in NS4H/R by 180 min (111 +/- 34 U/ml; P < 0.01) without increases in TNF-alpha mRNA. In contrast, EC-induced increases in TNF-alpha transcripts during normoxia were attenuated in EC+H/R, as were protein levels (57 +/- 20 U/ml; P < 0.05), despite similar bacterial clearance. Neither Indo-mediated reductions in PGE2 nor allopurinol increased TNF-alpha after EC+H/R.(ABSTRACT TRUNCATED AT 250 WORDS)

Endobronchial leiomyomas are rare benign tumors of the lung, arising from the smooth muscle of the bronchial tree. Symptomatology is based on the degree of endoluminal bronchial obstruction, and surgical resection has generally been the mainstay of treatment. We describe a mechanically ventilated patient with recurrent atelectasis and a postobstructive pneumonia caused by an occlusive endobronchial leiomyoma who was successfully weaned off the ventilator after treatment with argon plasma coagulation delivered via flexible bronchoscopy. We also briefly review the literature.

Cyclophosphamide (CY)-induced neutropenia exacerbates septic shock and acute lung injury during Candida albicans (CA) fungemia in conscious rats. We hypothesized that treatment of such animals with recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) improves host defense during disseminated candidiasis by increasing peripheral neutrophils (PMNs) and enhancing endogenous production of antifungal cytokines including tumor necrosis factor-alpha (TNF). Naive (neutrophil-replete) or neutropenic rats were infected with 10(7) yeast-phase CA; subgroups received GM-CSF (25 micrograms/kg sc) or sterile 0.9% NaCl (NS) twice a day beginning 3 days before CA infection. Arterial hemodynamics, formed blood elements, bioactive TNF in serum and bronchoalveolar lavage fluid (BALF), and lung histopathology were monitored for up to 72 h after infection. All naive animals receiving GM-CSF (n = 5) and 78% of naive rats given NS (n = 9) remained normotensive through 72 h with no lung injury, differing principally in baseline PMNs before CA infection (8.8 +/- 1.8 x 10(3)/microliters, mean +/- SE, vs. 3.7 +/- 0.4 x 10(3)/microliters, respectively, P < 0.01). Neutropenic rats given NS (baseline PMN = 41 +/- 10/microliters, n = 7) were sensitized to CA, and 100% died of hypothermic shock with severe respiratory distress within 56 h of infection. Pulmonary periarterial and alveolar hemorrhage were prominent. Although GM-CSF did not increase baseline PMNs in CY animals by the outset of infection (162 +/- 58/microliters, n = 8), 62% of these rats remained normotensive and eupneic through 72 h (P < 0.01), and their lungs showed no perivascular hemorrhage, alveolar disruption, or fungi.(ABSTRACT TRUNCATED AT 250 WORDS)

The liver modulates host responses to endotoxemia by production and clearance of tumor necrosis factor alpha (TNF-alpha) and eicosanoid lipoxygenation products. Reductions in liver blood flow (QL) are common during endotoxemia, but it is unknown whether the kinetics of TNF-alpha and leukotrienes (LTs) are thereby altered to amplify lung inflammation. To test this hypothesis, reductions in QL were modeled by an end-to-side portacaval shunt (PCS) in Sprague-Dawley rats. Conscious animals received 2.5 mg/kg of intravenous E. coli lipopolysaccharide (LPS) serotype 055:B5 (PCS + LPS; n = 17) or saline (n = 5). Responses were compared with those in sham-operated rats (sham + LPS; n = 13) and NSS-challenged control rats (n = 5). Cardiopulmonary changes, serum TNF-alpha, and formed elements were determined at t = 0, 1.5, 3.5, and 24 h, when organ wet/dry ratios (W/D) were measured with TNF-alpha, LTB4, and polymorphonuclear neutrophils (PMN) in bronchoalveolar lavage fluid (BALF). In PCS + LPS rats, mortality was 59% and serum TNF-alpha peaked at 1.5 h (2,784 +/- 658 U/ml, mean +/- SEM) coincident with the onset of hypotension. Despite equivalent endotoxemia and liver- and lung-associated TNF-alpha in sham + LPS rats at 1.5 h, peak serum TNF-alpha was 38% less and mortality was 15% (p0.05). Cardiac, hepatic, and cecal W/D were likewise increased in PCS + LPS versus sham + LPS rats, as were BALF PMNs (p0.05). In parallel studies, the disappearance kinetics of infused rTNF-alpha were not altered in nonendotoxemic PCS animals, implicating enhanced lung uptake of LPS and systemic export of TNF-alpha in PCS + LPS rats.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Candida albicans (CA) increasingly causes septic shock, acute lung injury, and multiple organ damage during immunosuppression-related neutropenia. However, the effects of neutrophil (PMN) depletion on induction of tumor necrosis factor-alpha (TNF) by CA and its potential mediation of Candida septic shock are unknown. We hypothesized that reduced CA uptake by circulating PMNs during cyclophosphamide (CY)-related neutropenia sensitizes to TNF-mediated shock from enhanced cytokine production after phagocytosis by tissue macrophages. Absolute or relative neutropenia (PMNs < or = 500/microliters or 2,500/microliters) was modeled in rats by intraperitoneal CY 4-8 days before 10(9) yeast-phase CA (acute studies < or = 24 h, n = 81 animals) or 10(6) CA (subacute studies < or = 72 h, n = 25). Compared with neutrophil-sufficient rats, absolute neutropenia accelerated hemodynamic collapse and respiratory distress after 10(9) CA, and pulmonary microvascular permeability was amplified. These changes evolved without increased candidemia or elevations in bioactive or antigenic serum TNF, which remained low even at death (42.3 +/- 14.8 U/ml vs. 12.6 +/- 2.9 U/ml for CY + saline, means +/- SE, P = NS). In contrast, significant TNF in lung tissue and bronchoalveolar lavage fluid (BALF) was evident within 6 h in CY + 10(9) CA rats. Electron microscopy confirmed hyphal proliferation into alveoli from yeast within mononuclear cells in lung capillaries. Subacute disseminated candidiasis after 10(6) CA was not associated with elevated serum, lung, or BALF TNF. We conclude that differential systemic and intrapulmonary TNF production occur in CA septic shock during preexisting neutropenia, with compartmentalized TNF production in the lower respiratory tract accompanying yeast-mycelial transformation. Thus TNF is not an obligate mediator of acute candidemic shock or subacute disseminated candidiasis during CY-induced immunosuppression but may initiate pulmonary injury accompanying high-grade candidemia.

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.

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-alpha (TNF-alpha) and interleukin (IL)-1beta, 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-alpha and IL-6 biosynthesis by ELISAs. Upstream redox-sensitive signaling events involving the pleiotropic transcription factor NF-kappaB were determined in nuclear extracts by electrophoretic mobility shift assays (EMSAs) and p65 subunit Western blot. The levels of the cytosolic inhibitory protein IkappaB-alpha were also assessed by Western analysis. We found that M40403 potently suppressed the production of superoxide, TNF-alpha, 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-kappaB, and this effect was associated with modest suppression of cytoplasmic IkappaB-alpha degradation. Together, these results suggest that removal of superoxide by M40403 inhibits endotoxin-induced production of TNF-alpha and IL-6 in alveolar macrophages by a mechanism involving suppression of redox-sensitive NF-kappaB transactivation or signaling.

Objective: To 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. Design: Prospective, randomized, double-blinded, placebocontrolled, multicenter trial. Setting: Thirty-three academic medical centers in the United States. Patients: Patients were entered with a clinical diagnosis of sepsis, the presence of either shock or multiple organ dysfunction, and presumptive evidence for Gram-negative infection. Interventions: Patients received a single intravenous infusion, over 30 mins, of either 300 mg of MAB-T88 formulated in albumin, or placebo (albumin). Measurements and Main Results: The 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). Conclusions: Use of the human monoclonal antibody, MABT88, 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. (Crit Care Med 2003; 31:419 ‐427)

Objectives We have shown previously that inactivation of catecholamines by superoxide anions contributes to the loss of vascular reactivity to norepinephrine and the subsequent hypotension that develops in Gram-negative endotoxic shock. In addition to their vasopressor actions, catecholamines, via beta-adrenoceptor activation, are important regulators of cytokine production. Here we examined if maintenance of serum catecholamine levels by the superoxide dismutase mimetic, M40401, modulates serum cytokine levels and arterial hypotension in an Escherichia coli-infected conscious rat model of septic shock. Design Controlled laboratory animal study. Setting University animal research laboratory. Subjects Pathogen-free male Sprague-Dawley rats (n = 51). Interventions Conscious, antibiotic-treated animals with chronic in-dwelling carotid arterial and jugular venous catheters were intravenously infected with 10(10) live E. coli bacteria (O55:B5, n = 51) over 30 mins, ending at time = 0 hrs. At 0.5 or 3 hrs, infected rats were administered an intravenous infusion of either M40401 (n = 33) or 0.9% saline (n = 18) for 6 hrs at a rate of 1 mL/h. In additional experiments, anesthetized animals with catheterized left femoral arteries and veins were administered a dose-range of norepinephrine (0.1-1 microg/kg) as bolus intravenous injections. Thereafter, E. coli lipopolysaccharide (4 mg/kg, n = 6) was administered as a 0.3-mL slow bolus intravenous injection. One hour later, the norepinephrine protocol was repeated, after which the rats were administered an intravenous infusion of either M40401 or 0.9% saline for 15 mins. At 2 hrs, the dose response to norepinephrine was repeated. Measurements and main results Rats infected with live E. coli exhibited a biphasic fall in mean arterial pressure, with mortality reaching 83% by 24 hrs. Rats treated with M40401 (0.25, 2.5, or 25 microg x kg-1 x hr-1 ) 3 hrs after bacteremic sepsis maintained a normal mean arterial pressure, and mortality was dose-dependently reduced to 44, 33, and 22%, respectively, at 24 hrs. Furthermore, serum catecholamine levels were diminished in E. coli-infected rats treated with saline compared with rats treated with M40401. In separate experiments, E. coli-infected rats were administered M40401 (25 microg x kg-1 x hr-1 ) 0.5 hr after bacterial challenge. Blood samples taken at 0, 1.5, 3.5, and 6 hrs were analyzed for tumor necrosis factor-alpha, interleukin (IL)-1 beta, IL-6, and IL-10 and for norepinephrine and epinephrine. Serum levels of tumor necrosis factor-alpha and IL-1 beta were significantly depressed in M40401-treated septic rats, whereas IL-10 was elevated. Moreover, serum catecholamine levels were greater in M40401-treated septic rats at the same time points. IL-6 levels were unaffected by M40401 treatment. Finally we examined whether treatment with M40401 could reverse the hyporeactivity to norepinephrine typifying early septic shock. Using the E. coli lipopolysaccharide (4 mg/kg) challenged anesthetized rat model of shock, we demonstrated that the vasoconstrictor ability of norepinephrine was indeed restored after M40401 treatment (25 microg/kg). Conclusion Postinfection treatment with the superoxide dismutase mimetic M40401 protects against hypotension, vascular hyporeactivity to catecholamines, and mortality associated with septic shock. Such beneficial effects correlate with both reduced oxidative inactivation of serum catecholamines and a reduction in canonical cytokine mediators of inflammation.

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].

We hypothesized that ischemia/reperfusion (I/R) injury of the liver during normotensive gram-negative bacteremic sepsis alters the kinetics of circulating endotoxin, tumor necrosis factor-alpha (TNF-alpha), and coinduced mediators, thereby exacerbating sepsis-induced lung inflammation. Liver and lung dysfunction were studied after hematogenous infection of Sprague-Dawley rats with 10(9) Escherichia coli serotype O55:B5 (EC) and 90 min of secondary hepatic ischemia in EC + I/R and saline-infused (normal saline NS) x I/R rats, followed by brief (1 h) or longer reperfusion (24 h). TNF- alpha:leukotriene interactions in this model were examined using the 5-lipoxygenase-activating protein inhibitor MK-886. Compared with sham-operated EC + Sham animals, peak serum endotoxin, TNF-alpha, alanine aminotransferase, interleukin-6 (IL-6), and hepatic neutrophil (PMN) influx were higher in EC + I/R rats through 24 h (p0.05) despite comparable arterial pressure. Lung PMN influx and wet/dry weight ratios were likewise enhanced in EC + I/R versus EC + Sham or NS + I/R rats. MK-886 attenuated TNF-alpha concentrations and ischemic liver injury but not mortality. Thus, focal hepatic I/R augments circulating endotoxin, TNF-alpha, and postbacteremic lung inflammation early after normotensive E. coli bacteremic sepsis.

Reductions in hepatic O2delivery are common early after gram-negative bacteremic sepsis owing to cardiopulmonary dysfunction and derangements in sinusoidal perfusion. Although gram-negative endotoxin and cellular hypoxia independently enhance activation of nuclear factor-κB (NF-κB) via generation of reactive O2species (ROS), the combination of these stimuli downregulates hepatic TNF-α gene expression. Here we tested the hypothesis that hypoxic suppression of postbacteremic TNF-α gene expression is transcriptionally mediated by reduced activation of NF-κB. Buffer-perfused rat livers ( n = 52) were studied over 180 min after intraportal infection at t = 0 with 109live Escherichia coli (EC), serotype O55:B5, or 0.9% NaCl controls under normoxic conditions, compared with 0.5 h of constant-flow hypoxia (Po2∼41 ± 7 Torr) beginning at t = 30 min, followed by 120 min of reoxygenation. In parallel studies, tissue was obtained at peak hypoxia ( t = 60 min). To determine the role of xanthine oxidase (XO)-induced ROS in modulating NF-κB activity after hypoxia/reoxygenation (H/R), livers were pretreated with the XO inhibitor allopurinol, with results confirmed in organs of tungstate-fed animals. Electrophoretic mobility shift assays were performed on nuclear extracts of whole liver lysates using32P-labeled oligonucleotides specific for NF-κB. Compared with normoxic EC controls, hypoxia reduced postbacteremic NF-κB nuclear translocation and TNF-α bioactivity, independent of reoxygenation, tissue levels of reduced glutathione, or posthypoxic O2consumption. XO inhibition reversed the hypoxic suppression of NF-κB nuclear translocation and ameliorated decreases in cell-associated TNF-α. Thus decreases in hepatic O2delivery reduce postbacteremic nuclear translocation of NF-κB and hepatic TNF-α biosynthesis by signaling mechanisms involving low-level generation of XO-mediated ROS.

Dimorphic Candida albicans spp. increasingly cause lethal septic shock and disseminated infection in the critically ill. Following candidemia, production of specific fungal exotoxins coincident with the yeast to hyphal phenotypic transition is believed to be important in the pathogenesis of Candida septic shock. However, overexpression of the pleiotropic cytokine tumor necrosis factor (TNF)-alpha by the host following hyphal germination is also thought to be a mechanism of Candida-related cardiopulmonary dysfunction, as well as of bacteremic shock. In this study, we hypothesized that increases in circulating TNF-alpha coinciding with the yeast to hyphal transition modulate the onset and progression of shock with multiple organ injury early after hematogenous candidiasis.Prospective, controlled laboratory animal study.University hospital animal research facility.Pathogen-free, male Sprague-Dawley rats (n = 26).Conscious, antibiotic-treated animals with chronic indwelling carotid arterial and jugular venous catheters were intravenously infected with 10(9) viable blastoconidia of the C. albicans clinical pathogen, CA-MEN (n = 10), over 30 mins and ending at t = 0 hr, compared with an equivalent inoculum of its viable agerminative mutant, CA-MM2002 n = 11), or an intravenous infusion of 0.9% sodium chloride (n = 5).Mean arterial pressure (MAP), pulse rate, respiratory frequency, rectal temperature, acid-base status, quantitative blood cultures, circulating alanine aminotransferase (ALT), and bioactive TNF-alpha were serially measured in all three groups over 24 hrs or until death. Organ cultures, wet/dry weight ratios, and histopathologic changes in the lungs, heart, liver, and kidneys were determined in Candida-infected and 0.9% sodium chloride (normal saline)-infused subgroups at 6 and 24 hrs. Animals hematogenously infected with the C. albicans clinical isolate developed lethal nonendotoxemic shock inor = 6 hrs (MAP 49 +/- 7 mm Hg [SEM]; p.05 vs. t = 0 hr), and at death (7.0 +/- 0.3 hrs) were acidotic, hypocapnic, and hypothermic (rectal temperature 33.2 +/- 0.7 degrees C). Despite similar peak concentrations of circulating fungal colony-forming units (cfu) and kinetics of vascular clearance in both Candida-infected groups, survival and MAP in rats challenged with the agerminative C. albicans mutant were unchanged for8 hrs, as were pH, Pco2, and rectal temperature. No germination of the agerminative fungal strain occurred in vivo over 6 hrs. Serum TNF was nearly undetectable at t = 0 hr in all three groups. Although shock developed soon after fungemia with the C. albicans clinical isolate, TNF-alpha concentrations did not increase above normal saline values in either candidemic group at t = 1.5, 4.5, or 6 hrs (17 +/- 7 vs. 14 +/- 1 U/mL in the parent C. albicans organism vs. its agerminative mutant at t = 6 hrs). Greater numbers of agerminative C. albicans than its dimorphic parent strain were recovered from the lungs (5.41 +/- 1.0 vs. 2.02 +/- 0.38 x 10(7) cfu/g, respectively; p.05) and kidneys (p.01). By 24 hrs, modest germination of the mutant Candida strain was observed in the tissues. However, lung wet/dry ratios, intrapulmonary hyphal proliferation, and alveolar hemorrhage were all greater after infection with the parent fungal isolate. Likewise, myocardial necrosis and hepatic glycogen depletion with vacuolization were more severe after infection with the C. albicans clinical isolate vs. candidemia with its agerminative mutant, although serum ALT values did not differ between these groups.Lethal C. albicans sepsis with lung injury and multiple organ damage are temporally associated with the in vivo yeast to hyphal transition in this model. However, this candidemic septic shock syndrome is modulated by circulating fungal virulence factors or host mediators other than TNF-alpha, a cytokine considered essen

Hepatic cytokine gene expression is independently stimulated by circulating microbial products and reductions in the cellular O2 supply. Although these stimuli occur sequentially after gram-negative bacteremia, it is unknown whether their interplay augments production of interleukin (IL)-1 by the liver. We studied the effects of intraportal Escherichia coli (EC) bacteremia and secondary constant-flow hypoxia (Po2, approximately 46 Torr for 30 min) on IL-1 alpha and IL-1 beta gene expression in ex situ buffer-perfused rat livers over 180 min (n = 67). At t = 0, normoxic EC and normal saline (NS) controls received 10(9) live EC serotype 055:B5 and 0.9% NaCl, respectively; in livers subjected to EC+hypoxia-reoxygenation (H/R) and NS+H/R, hypoxia began 0.5 h after EC or NS and was followed by 120 min of reoxygenation. Portal and hepatic venous perfusates were serially analyzed for bacterial colony-forming units, O2 uptake, and aspartate aminotransferase. At 60 min (peak hypoxia) and 180 min, cDNAs for IL-1 alpha and IL-1 beta were hybridized to whole liver RNA, and IL-1 beta protein levels in venous perfusates were assessed. Intrahepatic levels of reduced glutathione (GSH) were measured as an index of oxidative stress. Compared with normoxic EC, IL-1 alpha transcripts decreased at 180 min in EC+H/R livers (P < 0.0001) as did IL-1 beta mRNA (P < 0.05), despite similar EC clearance, GSH levels, posthypoxic O2 uptake, and aspartate aminotransferase release. Hepatic secretion of IL-1 beta likewise fell in EC+H/R vs. EC controls (P < 0.005). Prostaglandin H synthase-2 (COX-2) message accumulation was not enhanced by H/R, and indomethacin did not reverse H/R-mediated suppression of IL-1 production. In contrast, H/R-related falls in EC-induced IL-1S expression were reversed by allopurinol or catalase. Thus brief hypoxic stress of the liver causing neither GSH depletion nor functional impairment downregulates postbacteremic IL-1 expression by a mechanism involving O2 radicals but not cyclooxygenase metabolites.

This article examines interactions between the lungs and the liver during microbial sepsis from four interrelated elements of host defense: (1) control of systemic endotoxemia, bacteremia, and vasoactive by-products of sepsis via hepatic mononuclear phagocytic (Kupffer's) cell clearance; (2) biosynthesis and export of cytokine and eicosanoid inflammatory mediators into hepatic venous blood; (3) metabolic inactivation and detoxification of these mediators by Kupffer's cell-hepatocyte interactions at the hepatic sinusoidal interface; and (4) cytokine-driven synthesis of acute phase proteins, including α 2 -macroglobulin, C-reactive protein, and lipopolysaccharide-binding protein, that are capable of modifying sepsis-related changes in metabolism and inflammation.

We tested the hypothesis that gram-negative bacteremia (GNB) and brief (30 min) reductions in the hepatic O2 supply by low-flow ischemia differentially modulate tumor necrosis factor-alpha (TNF-alpha) gene expression owing to sequence-specific activation of cyclooxygenase vs. complement (C) pathways. Buffer-perfused Sprague-Dawley rat livers (n = 82) were studied over 180 min after intraportal 10(9) live E. coli serotype 055:B5 (EC) or 0.9% NaCl (NS) at t = 0. Compared with EC and NS controls receiving constant-flow perfusion, sequential GNB and ischemia/reperfusion (I/R) were studied in EC + 30 I/R and NS + 30 I/R livers, in which 30 min of ischemia (I) beginning 0.5 h after EC or NS was followed by 120 min of reperfusion (R). This sequence was reversed in 30 I/R + EC and 30 I/R + NS groups. Bacterial clearance, bioactive and antigenic TNF-alpha, prostaglandin E2 (PGE2), and hepatic O2 uptake and performance were serially assessed. Venous TNF-alpha increased in EC controls to peak at 155 +/- 29 U/ml after 180 min (P < 0.001 vs. NS controls) as did hepatic TNF-alpha mRNA. Both TNF-alpha transcripts and protein levels were markedly attenuated in EC + 30 I/R (P < 0.001 vs. EC) despite equivalent EC clearance by Kupffer cells. Indomethacin (10(-5) M) decreased I/R-induced PGE2 secretion and restored TNF-alpha to control levels. In contrast, TNF-alpha levels in 30 I/R + EC perfusates exceeded those of EC + 30 I/R livers (P < 0.05) and were indistinguishable from EC controls. Allopurinol pretreatment but not heat inactivation of C or infusion of soluble human complement receptor type 1 inhibited TNF-alpha production in 30 I/R + EC organs. These results identify a novel sequence-dependent interaction whereby hepatic O2 deprivation after GNB downregulates TNF-alpha via generation of cyclooxygenase metabolites, whereas ischemia preceding GNB increases cytokine expression via reactive O2 species but not C activation.

We tested the hypothesis that regulation of tumor necrosis factor-alpha (TNF-alpha) and IL-6 by the liver differs after intraportal challenge with Candida albicans spp. vs. gram-negative or gram-positive bacteria, independent of microbial clearance kinetics or hepatic O2 consumption (VO2). Buffer-perfused rat livers were infected with equivalent inocula (10(9) colony-forming units) of viable Escherichia coli serotype 055:B5 (EC), exotoxin C-producing Staphylococcus aureus (SA), or two strains of yeast phase C. albicans (CA-1 and CA-2). Microbial clearance and circulating cytokine levels were assessed over 180 min while monitoring VO2 and functional parameters, after which organ-based microbial killing, cell-associated TNF-alpha, and cytokine mRNA levels were determined. Compared with saline controls (normal saline solution; NSS), circulating and cell-associated TNF-alpha and TNF-alpha transcripts minimally increased after CA. In contrast, large increases in perfusate TNF-alpha occurred after EC, peaking at 180 min [135 +/- 32 U/ml (mean + SE)], concomitant with rises in cell-associated cytokine and TNF-alpha transcripts (P < 0.01 vs. NSS). Circulating TNF-alpha also rose after SA but neither cell-associated nor mRNA levels exceeded NSS values. There were no pathogen-specific differences in microbial clearance or VO2. IL-6 gene expression paralleled that for TNF-alpha, but IL-6 bioactivity in perfusates was inhibited by TNF-alpha-dependent and -independent mechanisms. We conclude that hepatic TNF-alpha and IL-6 expression are differentially regulated after taxonomically diverse microbial challenges, with E. coli eliciting the strongest and Candida spp. the weakest stimulatory responses.