Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted by In Vitro Neutralization and Epitope Modeling

Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic.     

Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory distress syndrome

The acute respiratory distress syndrome (ARDS) provokes three pathologic processes: unchecked inflammation, interstitial/alveolar protein accumulation, and destruction of pulmonary epithelial cells. The highly conserved heat shock protein HSP-70 can limit all three responses but is not appropriately expressed in the lungs after cecal ligation and double puncture (2CLP), a clinically relevant model of ARDS. We hypothesize that restoring expression of HSP-70 using adenovirus-mediated gene therapy will limit pulmonary pathology following 2CLP. We administered a vector containing the porcine HSP-70 cDNA driven by a CMV promoter (AdHSP) into the lungs of rats subjected to 2CLP or sham operation. Administration of AdHSP after either sham operation or 2CLP increased HSP-70 protein expression in lung tissue, as determined by immunohistochemistry and Western blot hybridization. Administration of AdHSP significantly attenuated interstitial and alveolar edema and protein exudation and dramatically decreased neutrophil accumulation, relative to a control adenovirus. CLP-associated mortality at 48 hours was reduced by half. Modulation of HSP-70 production reduces pathologic changes and may improve outcome in experimental ARDS.     

Competitive and noncompetitive inhibition of myocardial cytochrome C oxidase in sepsis

Sepsis is the most common cause of death in intensive care units worldwide. The basic pathophysiologic defect in sepsis, causing functional abnormalities in many organ systems, remains elusive. One potential cause is disruption of oxidative phosphorylation in mitochondria. Here, we report that oxidation of cytochrome c by myocardial cytochrome c oxidase, the terminal oxidase in the electron transport chain, is competitively inhibited early in experimental sepsis (cecal ligation with single or double 23-gauge puncture) in mice. In severe sepsis (cecal ligation and double puncture, 75% mortality at 48 h), inhibition becomes noncompetitive by 48 h. The development of noncompetitive inhibition is associated with a decrease in heme a,a3 content, which is the key active site in the functional subunit (I) and catalyzes the reduction of molecular oxygen. In addition, there are persistently decreased steady-state levels of subunit I mRNA and protein after cecal ligation and double puncture. Both loss of heme and loss of subunit I could explain the observed irreversible inhibition of cytochrome c oxidase. Noncompetitive inhibition of cytochrome c oxidase may interrupt oxidative phosphorylation, leading to sepsis-associated cardiac depression. Importantly, this abnormality may underlie sepsis-associated dysfunction in other organ systems.     

Sepsis-induced cholestasis, steatosis, hepatocellular injury, and impaired hepatocellular regeneration are enhanced in interleukin-6 -/- mice

OBJECTIVE: Hepatic dysfunction is an important but poorly understood component of sepsis. In severe sepsis, liver dysfunction is characterized by cholestasis, steatosis, hepatocellular injury, impaired regeneration, a decreased response to the cytokine interleukin-6, and high mortality. To determine whether loss of interleukin-6 activity caused hepatic dysfunction and mortality, we induced sepsis in wild-type (interleukin-6 +/+) and interleukin-6 knockout (interleukin-6 -/-) mice. We hypothesized that sepsis in interleukin-6 -/- mice would increase cholestasis, steatosis, hepatocellular injury, and mortality and impair hepatocyte regeneration. DESIGN: Randomized prospective experimental study. SETTING: University medical laboratory. SUBJECTS: Male adolescent C57Bl6 interleukin-6 +/+ and interleukin-6 -/- mice. INTERVENTIONS: Mild sepsis was induced using cecal ligation and single puncture (CLP). Severe, lethal sepsis was induced using cecal ligation and double puncture (2CLP). Some mice received recombinant human interleukin-6 at the time of CLP/2CLP. All animals were fluid resuscitated at the time of surgery and every 24 hrs thereafter. In survival cohorts, mortality at 16, 24, 48, and 72 hrs was recorded. In separate cohorts, surviving animals were killed at 24 and 48 hrs, and liver tissue was harvested. A separate cohort of mice received bromodeoxyuridine for detection of regeneration. MEASUREMENTS AND MAIN RESULTS: 2CLP was 100% fatal within the first 12 hrs in interleukin-6 -/- mice. Mortality from 2CLP in interleukin-6 +/+ mice before 24 hrs was nil but was 90% by 72 hrs. At 72 hrs, CLP was 40% fatal in interleukin-6 +/+ mice but 90% in interleukin-6 -/- mice. CLP induced cholestasis, steatosis, and hepatocellular injury in interleukin-6 -/-, but not interleukin-6 +/+, mice. Regeneration was absent following CLP in interleukin-6 -/- animals but occurred in interleukin-6 +/+ mice. Early administration of recombinant human interleukin-6 did not reverse abnormalities in interleukin-6 -/- mice. CONCLUSIONS: The absence of interleukin-6 is an important determinant of hepatic dysfunction and mortality in sepsis.     

Enhanced heat shock protein 70 expression alters proteasomal degradation of IkappaB kinase in experimental acute respiratory distress syndrome

OBJECTIVES: Acute respiratory distress syndrome is a common and highly lethal inflammatory lung syndrome. We previously have shown that an adenoviral vector expressing the heat shock protein (Hsp)70 (AdHSP) protects against experimental sepsis-induced acute respiratory distress syndrome in part by limiting neutrophil accumulation in the lung. Neutrophil accumulation and activation is modulated, in part, by the nuclear factor-kappaB (NF-kappaB) signal transduction pathway. NF-kappaB activation requires dissociation/degradation of a bound inhibitor, IkappaBalpha. IkappaBalpha degradation requires phosphorylation by IkappaB kinase, ubiquitination by the SCFbeta-TrCP (Skp1/Cullin1/Fbox beta-transducing repeat-containing protein) ubiquitin ligase, and degradation by the 26S proteasome. We tested the hypothesis that Hsp70 attenuates NF-kappaB activation at multiple points in the IkappaBalpha degradative pathway. DESIGN: Laboratory investigation. SETTING: University medical center research laboratory. SUBJECTS: Adolescent (200 g) Sprague-Dawley rats and murine lung epithelial-12 cells in culture. INTERVENTIONS: Lung injury was induced in rats via cecal ligation and double puncture. Thereafter, animals were treated with intratracheal injection of 1) phosphate buffer saline, 2) AdHSP, or 3) an adenovirus expressing green fluorescent protein. Murine lung epithelial-12 cells were stimulated with tumor necrosis factor-alpha and transfected. NF-kappaB was examined using molecular biological tools. MEASUREMENTS AND MAIN RESULTS: Intratracheal administration of AdHSP to rats with cecal ligation and double puncture limited nuclear translocation of NF-kappaB and attenuated phosphorylation of IkappaBalpha. AdHSP treatment reduced, but did not eliminate, phosphorylation of the beta-subunit of IkappaB kinase. In vitro kinase activity assays and gel filtration chromatography revealed that treatment of sepsis-induced lung injury with AdHSP induced fragmentation of the IkappaB kinase signalosome. This stabilized intermediary complexes containing IkappaB kinase components, IkappaBalpha, and NF-kappaB. Cellular studies indicate that although ubiquitination of IkappaBalpha was maintained, proteasomal degradation was impaired by an indirect mechanism. CONCLUSIONS: Treatment of sepsis-induced lung injury with AdHSP limits NF-kappaB activation. This results from stabilization of intermediary NF-kappaB/IkappaBalpha/IkappaB kinase complexes in a way that impairs proteasomal degradation of IkappaBalpha. This novel mechanism by which Hsp70 attenuates an intracellular process may be of therapeutic value.     

Adenoviral Vector Transfection into the Pulmonary Epithelium after Cecal Ligation and Puncture in Rats

Background: Adenoviral-targeted gene delivery to respiratory epithelium can augment production of specific proteins. Therefore, it may be valuable in treating the acute respiratory distress syndrome. The authors tested the hypothesis that adenoviral vector uptake after cecal ligation and double puncture in rats, an animal model of the acute respiratory distress syndrome, is higher than that observed in controls that did not undergo operation ("nonoperated") or those that underwent a sham operation ("sham-operated").

Methods: Adenoviruses expressing green fluorescent protein or Lac-Z were delivered into the lungs of anesthetized rats via tracheal catheter. Animals were killed 24 or 48 h later. Histopathology and green fluorescent protein expression were examined using light of fluorescence microscopy. Cellular localization of Lac-Z was determined with electron microscopy or semithin sectioning. Viral receptor density and localization were determined using immunoblotting and immunohistochemistry.

Results: After cecal ligation and double puncture, rats were hypoxic and tachypneic. Alveoli were segmentally consolidated, contained proteinaceous debris and neutrophils, and had thickened septa. Administration of adenoviruses to rats that were sham-operated or underwent cecal ligation and double puncture resulted in high levels of marker protein expression in cells lining alveoli. Use of 3 x 1011 plaque-forming units instead of 3 x 1012 plaque-forming units resulted in similar levels of green fluorescent protein expression with negligible viral-mediated lymphocytic infiltration. Semithin section and electron microscopy revealed expression primarily localized to type II alveolar cells. Abundance of [alpha]v[beta]3 integrins and human coxsackie-adenovirus receptor (receptors that modulate viral attachment and internalization) was increased after cecal ligation and double puncture, predominantly in type II pneumocytes.