Host Nitric Oxide Disrupts Microbial Cell-to-Cell Communication to Inhibit Staphylococcal Virulence.

Staphylococcus aureus is a commensal bacterium that can asymptomatically colonize its host but also causes invasive infections. Quorum sensing regulates S. aureus virulence and the transition from a commensal to a pathogenic organism. However, little is known about how host innate immunity affects interbacterial communication. We show that nitric oxide suppresses staphylococcal virulence by targeting the Agr quorum sensing system. Nitric oxide-mediated inhibition occurs through direct modification of cysteine residues C55, C123, and C199 of the AgrA transcription factor. Cysteine modification decreases AgrA promoter occupancy as well as transcription of the agr operon and quorum sensing-activated toxin genes. In a staphylococcal pneumonia model, mice lacking inducible nitric oxide synthase develop more severe disease with heightened mortality and proinflammatory cytokine responses. In addition, staphylococcal α-toxin production increases in the absence of nitric oxide or nitric oxide-sensitive AgrA cysteine residues. Our findings demonstrate an anti-virulence mechanism for nitric oxide in innate immunity.

Hepatocellular heme oxygenase-1: a potential mechanism of erythropoietin-mediated protection after liver ischemia-reperfusion injury.

Hepatic ischemia-reperfusion (IR) results in progressive injury; initiated by oxidative stress during ischemia and compounded by cytokine-mediated inflammation during reperfusion. Recovery requires strict regulation of these events. Recombinant human erythropoietin (rhEPO) is thought to mitigate hepatocellular IR injury by altering the nonparenchymal liver microenvironment. This study sought to identify additional mechanisms whereby rhEPO is protective after liver IR injury. Mice were treated with rhEPO (4 units/g s.c.) at the onset of partial liver ischemia and assessed for transaminase and histologic injury at intervals after reperfusion. Induction of cytokines, activation of signal transducers and activators of transcription (STATs), suppressors of cytokine signaling (Socs1, Socs3, Cis), caspase-3 activation, and heme oxygenase-1 (HO-1) expression were assessed in postischemic liver. Effects of rhEPO stimulation were further characterized in whole-liver lysates from mice undergoing rhEPO injection alone and in cultured AML-12 hepatocytes. Recombinant human erythropoietin treatment at the onset of severe (90 min) hepatic IR confirmed commensurate biochemical and histological protection without affecting tissue cytokine levels. Although Socs3 and STAT5 activation were induced in normal liver after in vivo rhEPO injection, this treatment did not augment expression beyond that seen with IR alone, and neither was induced in cultured hepatocytes treated with rhEPO. Recombinant human erythropoietin inhibited caspase-3 activation in nonparenchymal cells, whereas hepatocellular HO-1 was rapidly induced both in vivo and in vitro with rhEPO treatment. These data suggest HO-1 as a potent mechanism of rhEPO-mediated protection after liver IR, which involves both direct hepatocellular and nonparenchymal mechanisms.

Focused ultrasound to displace renal calculi: threshold for tissue injury.

BACKGROUND: The global prevalence and incidence of renal calculi is reported to be increasing. Of the patients that undergo surgical intervention, nearly half experience symptomatic complications associated with stone fragments that are not passed and require follow-up surgical intervention. In a clinical simulation using a clinical prototype, ultrasonic propulsion was proven effective at repositioning kidney stones in pigs. The use of ultrasound to reposition smaller stones or stone fragments to a location that facilitates spontaneous clearance could therefore improve stone-free rates. The goal of this study was to determine an injury threshold under which stones could be safely repositioned. METHODS: Kidneys of 28 domestic swine were treated with exposures that ranged in duty cycle from 0%-100% and spatial peak pulse average intensities up to 30 kW/cm(2) for a total duration of 10 min. The kidneys were processed for morphological analysis and evaluated for injury by experts blinded to the exposure conditions. RESULTS: At a duty cycle of 3.3%, a spatial peak intensity threshold of 16,620 W/cm(2) was needed before a statistically significant portion of the samples showed injury. This is nearly seven times the 2,400-W/cm(2) maximum output of the clinical prototype used to move the stones effectively in pigs. CONCLUSIONS: The data obtained from this study show that exposure of kidneys to ultrasonic propulsion for displacing renal calculi is well below the threshold for tissue injury.

A non-coding cationic lipid DNA complex produces lasting anti-leukemic effects.

Cationic lipid DNA complex (CLDC) is an immunostimulatory preparation that has significant anti-leukemic effects in multiple murine models of leukemia: BCR-ABL(+) myelogenous leukemia in C3H/HeJ animals and myelomonocytic leukemia in BALB/c mice. Following leukemic challenge, CLDC treatment inhibits tumor cell growth in vivo and extends survival, sometimes resulting in apparent eradication of tumor cells. CLDC induces multiple cytokines including interferon-gamma (IFNγ), and intravenous treatment results in a more rapid and robust response than subcutaneous treatment. IFNγ is induced in a dose-dependent manner, and tachyphylaxis results from repeated doses of CLDC. Tachyphylaxis of therapeutic effects is exacerbated at higher doses, thus the optimal survival benefits are seen at intermediate doses. Animals whose leukemia has been successfully treated with CLDC exhibit a survival advantage when faced with a secondary leukemic challenge, suggesting the existence of an adaptive anti-leukemic response. This work demonstrates the effectiveness of CLDC in multiple experimental leukemias and is consistent with a stimulation of a lasting TH(1) anti-leukemic immune response.

Suppressor of cytokine signaling expression with increasing severity of murine hepatic ischemia-reperfusion injury.

BACKGROUND/AIMS: Preservation of function requires tight regulation of the cellular events initiated when hepatic ischemia is followed by reperfusion (IR). One important mechanism modulating the cytokine-directed response to injury is Suppressors of Cytokine Signaling. SOCS1 and SOCS3 ensure appropriate intensity and duration of cytokine signaling through negative feedback on JAK-STAT signaling. The contribution of SOCS1 and SOCS3-mediated regulation to the evolution of hepatic IR injury is unknown. METHODS: C57Blk6 mice were subjected to mild (20 min) or severe (90 min) hepatic ischemia. Liver was analyzed for cytokine and SOCS1/3 induction as well as JAK-STAT activation at intervals after reperfusion. RESULTS: Tnf, Il-1beta, and Il-6 expression paralleled increasing injury severity. Despite early phosphorylation of both STAT1 and STAT3 after severe injury, only nuclear translocation of activated STAT3, suggesting that the induction of target genes through JAK-STAT after IR is predominantly via STAT3. Socs3 was expressed across the injury spectrum while Socs1 was induced only in the face of severe IR injury. Severe IR in Il-6 deficient mice confirmed that Il-6, acting via STAT3, serves as a primary inducer of both regulatory mechanisms. CONCLUSIONS: Under the influence of IL-6-mediated STAT3 signaling, Socs1 serves as a complimentary regulatory mechanism when Socs3 is insufficient to limit cytokine-mediated inflammation after hepatic IR.