Nicolas A. Parejo

Relationship between plasma levels of lipopolysaccharide (LPS) and LPS- binding protein in patients with severe sepsis and septic shock

Plasma endotoxin and lipopolysaccharide-binding protein (LBP) levels were measured in a group of 253 patients at the onset of severe sepsis and/or septic shock. Endotoxin levels were significantly greater than control levels (n=33; mean +/- SD, 5.1+/-7.3 pg/mL) in 78.3% of patients. Median endotoxin levels in patients with sepsis were 300 pg/mL (25%-75% interquartile range, 110-726 pg/mL). LBP levels were elevated in 97% of patients compared with normal control values of 4.1+/-1.65 microgram/mL. Median LBP levels in patients with sepsis were 31.2 microgram/mL (interquartile range, 22.5-47.7 microgram/mL). Median endotoxin levels at study entry were more highly elevated (515 vs. 230 pg/mL; P<.01), and LBP levels were less highly elevated (28.0 vs. 33.2 microgram/mL; P<.05) in nonsurvivors than survivors over the 28-day study period. No correlation was found between endotoxin and LBP levels. The quantitative level of both endotoxin and LBP may have prognostic significance in patients with severe sepsis.

WAY-202196, a selective estrogen receptor-beta agonist, protects against death in experimental septic shock.

Objective:To determine the effect of an estrogen receptor-β selective agent in experimental models of systemic infection and sepsis. Design:WAY-202196, a nonsteroidal selective estrogen receptor-β agonist, was tested in the murine listeriosis model, the neutropenic rat Pseudomonas aeruginosa infection, and the mouse cecal ligation and puncture sepsis models. Setting:University-affiliated biomedical research laboratory. Subjects:BALB/c mice and Sprague-Dawley rats. Interventions:WAY-202196 or control (vehicle) was administered orally in doses ranging from 1.5 to 50 mg/kg at various time points in the three experimental model systems. Measurements and Main Results:Susceptibility of mice treated with a single oral dose of up to 50 mg/kg WAY-202196 did not differ from those treated with vehicle alone after systemic challenge by Listeria monocytogenes, suggesting a lack of generalized immunosuppression. In the neutropenic rat model, daily administration of WAY-202196 (50 mg/kg) significantly increased survival against an otherwise lethal challenge of P. aeruginosa 12.4.4 compared with the control group (83% vs. 25% survival; p < 0.05). Preservation of intestinal mucosal weight and prevention of histopathologic changes were also observed with the administration of WAY-202196. Similar results were obtained in a cecal ligation and puncture model, in which multiple oral doses of WAY-202196 (50 mg/kg) improved survival (83% vs. 0%; p < 0.05), preserved intestinal epithelial integrity, and significantly reduced systemic bacteremia and peritoneal interleukin-6 and tumor necrosis factor levels. The estrogen receptor-β agonist provided a comparable level of protection in both male and female animals. Conclusion:These results indicate that oral administration of WAY-202196 preserved gastrointestinal barrier function and improved outcome in experimental models of systemic infection and inflammation. WAY-202196 and similar agents may prove useful clinically as a novel treatment strategy for the treatment or prevention of severe sepsis.

Differential Antibiotic-Induced Endotoxin Release in Severe Melioidosis

Severe melioidosis is a life-threatening, systemic bacterial infection caused by Burkholderia pseudomallei. A prospective, randomized treatment trial was conducted in northeast Thailand to compare ceftazidime (a penicillin-binding protein [PBP]-3-specific agent that causes release of large amounts of endotoxin in vitro) and imipenem (a PBP-2-specific agent that kills B. pseudomallei more rapidly but releases low amounts of endotoxin) in severe melioidosis over a 6-h time course after the first dose of antibiotic. Despite similar clinical, microbiological, endotoxin, and cytokine measures at study entry, ceftazidime-treated patients (n=34) had significantly greater systemic endotoxin (P<.001) than patients treated with imipenem (n=34) after the first dose of antibiotic. No overall difference in mortality was observed (35% in both groups [95% confidence interval, 20%-50%]). Differential antibiotic-induced endotoxin release is demonstrable in severe melioidosis. These differences in endotoxin release did not appear to have a significant impact on survival in this group of patients.

Active Immunization with a Detoxified Escherichia coli J5 Lipopolysaccharide Group B Meningococcal Outer Membrane Protein Complex Vaccine Protects Animals from Experimental Sepsis

The passive infusion of antibodies elicited in rabbits with a detoxified J5 lipopolysaccharide (LPS)/group B meningococcal outer membrane protein complex vaccine protected neutropenic rats from heterologous lethal gram-negative bacterial infection. In this study, active immunization was studied in neutropenic rats infected with Pseudomonas aeruginosa, in the presence or absence of ceftazidime therapy, and with Klebsiella pneumoniae. This vaccine elicited a > 200-fold increase in anti-J5 LPS antibody, which remained elevated throughout the duration of cyclophosphamide-induced neutropenia and for < or = 3 months. There was improved survival among immunized versus control animals: 48% (13/28) versus 7% (2/29) in Pseudomonas-challenged rats; 61% (11/18) versus 0% (0/10) in Pseudomonas- and ceftazidime-treated rats; and 64% (9/14) versus 13% (2/15) in Klebsiella-challenged rats (P < 0.01 for each comparison). Immunized animals had lower levels of bacteria in organs and lower levels of circulating endotoxin at the onset of fever. In conclusion, active immunization with an anti-endotoxin vaccine improved survival after infection with > or = 2 heterologous, clinically relevant bacterial species in immunocompromised animals. Active immunization with this vaccine merits further investigation.

A monoclonal antibody against RAGE alters gene expression and is protective in experimental models of sepsis and pneumococcal pneumonia.

The RAGE (receptor for advanced glycation end products) is believed to play a role in sepsis by perpetuating inflammation. The interaction of RAGE with a variety of host-derived ligands that accumulate during stress and inflammation further induces the expression of RAGE. It was previously shown that a rat anti-RAGE monoclonal antibody protected mice from lethality in a cecal ligation and puncture model. We studied the effects of a humanized anti-RAGE monoclonal antibody in the murine pneumococcal pneumonia model of sepsis. Moreover, a gene expression analysis was performed in lung tissue of animals that underwent cecal ligation and puncture and treated with the rat anti-RAGE monoclonal antibody, compared with controls. Administration of humanized anti-RAGE mAb 6 h after intratracheal infection with Streptococcus pneumoniae improved mortality in BALB/c mice whether a 7.5 mg/kg (P < 0.01) or a 15 mg/kg dose (P < 0.01) was administered in combination with antibiotics. Gene expression analysis showed that many of the genes modulated by treatment with the anti-RAGE antibody were those that play an important role in regulating inflammation. Anti-RAGE monoclonal antibody offered a survival advantage to septic mice. This protective role in treated animals is supported by the observed gene expression profile changes of genes involved in sepsis and inflammation.

Recombinant Human Interleukin-11 in Experimental Pseudomonas aeruginosa Sepsis in Immunocompromised Animals

The therapeutic potential of recombinant human interleukin-11 (rhIL-11) was tested in a neutropenic rat model that mimics the clinical consequences of myelosuppressive chemotherapy complicated by Pseudomonas aeruginosa sepsis. rhIL-11-treated animals (150 micrograms/kg intravenously every 24 h for 3 days) had reduced endotoxin levels (P < .05) and less pulmonary edema fluid (P < .001) and were protected (P < .01) against thinning and necrosis of the intestinal mucosa compared with the control group. The survival rate in rhIL-11-treated animals was 40% (19/47), whereas it was 0 (0 of 19) in the control group (P < .01). The addition of ciprofloxacin (10 mg/kg every 12 h) resulted in a survival rate of 9 (60%) of 15, while the combination of rhIL-11 and ciprofloxacin resulted in 100% survival (15/15; P < .05). These results indicate that rhIL-11 supports mucous membrane integrity of the alimentary tract and decreases the systemic inflammatory response to experimental gram-negative infection in immunocompromised animals.

Chloroquine Enhances Survival in Bacillus anthracis Intoxication

The intentional release of anthrax in the United States in 2001 resulted in 11 cases of inhalational disease, with an attendant mortality rate of 45%. Current therapeutic options for anthrax are limited; antimicrobials target only replicating organisms, thus allowing bacterial toxins to cause unchecked, devastating physiological derangements in the host. Novel approaches that target the cytotoxic effects of anthrax exotoxins are needed. Chloroquine (CQ), a commonly used antimalarial agent, endows anthrax-intoxicated murine peritoneal macrophages with a 50% and 35% marginal survival advantage at 2 and 4 h, respectively, over that of untreated control cells. The cell rescue is dose dependent and, at lower concentrations, results in delayed cell death. We subsequently studied the effect of CQ in BALB/c mice challenged with anthrax lethal toxin. CQ-treated mice demonstrated reduced tissue injury, as assessed by histopathological examination of the spleen and by peripheral blood differential cell count ratios. CQ significantly enhanced survival and may augment current treatment and prophylaxis options for this otherwise lethal infection.

Effect of anti-CD14 monoclonal antibody on clearance of Escherichia coli bacteremia and endotoxemia.

ObjectiveTo determine the effects of an anti-CD14 monoclonal antibody on the clearance of a bacteremic Escherichia coli challenge in the presence or absence of antimicrobial agents. DesignProspective randomized animal study. SettingUniversity-affiliated research laboratory. SubjectsNew Zealand White rabbits weighing 1.5–2.5 kg. InterventionsAnimals were pretreated with either an anti-lapine CD14 monoclonal antibody (immunoglobulin G2a, 5 mg/kg intravenously) or an isotype control monoclonal antibody. The animals then were challenged with 1 × 106E. coli 018:K1 in the presence or absence of ceftazidime (50 mg/kg intravenously). There were four groups of six animals randomized to receive either anti-CD14 monoclonal antibody without ceftazidime, isotype control monoclonal antibody without ceftazidime, anti-CD14 monoclonal antibody with ceftazidime, or isotype control antibody with ceftazidime. Measurements and Main ResultsSerial measurement of quantitative bacteremia and endotoxemia was performed over 24 hrs after the administration of the bacterial challenge. Animals also underwent necropsy with quantitative bacterial cultures from multiple organ tissue samples. The anti-lapine CD14 monoclonal antibody significantly impaired the bloodstream clearance of E. coli (p < .01) and increased quantitative counts of E. coli in tissue culture samples when compared with isotype control antibody in the absence of simultaneous administration of ceftazidime. No differences in quantitative bacteremia, endotoxemia, or organ tissue counts were found after anti-CD14 antibody and control antibody-treated animals in the presence of ceftazidime treatment. ConclusionsAnti-CD14 monoclonal antibody has the capacity to interfere with the innate immune response and systemic microbial clearance in experimental animals with E. coli bacteremia. The concomitant administration of effective antimicrobial therapy eliminated differences in the rate of microbial clearance between the control antibody and the CD14 monoclonal antibody. These results indicate that care should be taken in clinical trials with anti-CD14 monoclonal antibodies to ensure that adequate antimicrobial therapy is administered in the presence of systemic bacterial infection.

Estrogen Receptor β Agonism Increases Survival in Experimentally Induced Sepsis and Ameliorates the Genomic Sepsis Signature: A Pharmacogenomic Study

BACKGROUND Nonsteroidal agonists have been developed that selectively bind to and activate estrogen receptor beta (ERbeta) rather than estrogen receptor alpha (ERalpha). ERbeta is expressed equally in both male and female mammals in multiple extragonadal tissues. Work reported elsewhere has demonstrated that ERbeta agonists have beneficial effects in multiple (but not all) models of inflammatory diseases and also increase survival in experimentally induced sepsis. METHODS In these experiments, ERbeta agonists (ERB-041 or WAY-202196) were compared with vehicle control in the murine cecal ligation and puncture (CLP) model and in the pneumococcal pneumonia model of sepsis. The effect of WAY-202196 on the gene expression profile in the CLP model was further studied by transcriptome analysis of lung and small intestine tissue samples. RESULTS ERbeta agonists provided a significant survival benefit in both experimental models of bacterial sepsis. This survival advantage was accompanied by reduced histologic evidence of tissue damage, reduced transcription of multiple proinflammatory proteins by transcriptome analysis and was not associated with increased bacterial outgrowth. CONCLUSIONS ERbeta agonist administration provided a survival advantage in septic animals and appears to be a promising therapeutic modality in sepsis.

Orally Administered Recombinant Human Interleukin-11 Is Protective in Experimental Neutropenic Sepsis

Recombinant human interleukin (IL)-11 is a multifunctional cytokine with hematopoietic, immunomodulatory, and epithelial cell protective activities. IL-11alpha receptors are expressed on the luminal surface of intestinal epithelial cells. It was hypothesized that orally administered IL-11 would prevent mucosal damage and protect against microbial invasion in a neutropenic rat model of gram-negative sepsis. IL-11 was administered daily by enteric, coated multiparticle pellets over the course of chemotherapy-induced neutropenia. Compared with the placebo group, IL-11-treated rats retained mucosal mass and had prolonged survival time, reduced pathologic changes, and reduced systemic levels of bacterial endotoxin and concentrations of Pseudomonas aeruginosa in target tissues. Enterocyte messenger RNA levels for tumor necrosis factor-alpha and interferon-gamma revealed that oral IL-11 reduced but did not prevent increased expression of these cytokine genes. These results indicate that orally administered IL-11 may preserve epithelial cell integrity in the presence of cytoreductive chemotherapy. This may represent a new treatment strategy for the prevention of infection in neutropenic hosts.