D. Kiers

Short-term hyperoxia does not exert immunologic effects during experimental murine and human endotoxemia

Oxygen therapy to maintain tissue oxygenation is one of the cornerstones of critical care. Therefore, hyperoxia is often encountered in critically ill patients. Epidemiologic studies have demonstrated that hyperoxia may affect outcome, although mechanisms are unclear. Immunologic effects might be involved, as hyperoxia was shown to attenuate inflammation and organ damage in preclinical models. However, it remains unclear whether these observations can be ascribed to direct immunosuppressive effects of hyperoxia or to preserved tissue oxygenation. In contrast to these putative anti-inflammatory effects, hyperoxia may elicit an inflammatory response and organ damage in itself, known as oxygen toxicity. Here, we demonstrate that, in the absence of systemic inflammation, short-term hyperoxia (100% O2 for 2.5 hours in mice and 3.5 hours in humans) does not result in increased levels of inflammatory cytokines in both mice and healthy volunteers. Furthermore, we show that, compared with room air, hyperoxia does not affect the systemic inflammatory response elicited by administration of bacterial endotoxin in mice and man. Finally, neutrophil phagocytosis and ROS generation are unaffected by short-term hyperoxia. Our results indicate that hyperoxia does not exert direct anti-inflammatory effects and temper expectations of using it as an immunomodulatory treatment strategy.

The challenge of Clostridium difficile infection: Overview of clinical manifestations, diagnostic tools and therapeutic options.

The most important infectious cause of antibiotic-associated diarrhoea and colitis is Clostridium difficile, which is a Gram-positive, anaerobic, spore-forming, toxin-producing bacillus. In this overview we will discuss the diagnostic and therapeutic management of patients presenting with suspected or proven C. difficile infection (CDI). The clinical spectrum varies from asymptomatic C. difficile carriers to fulminant colitis with multi-organ failure. The onset of symptoms is usually within 2 weeks after initiation of antibiotic treatment. Diagnosis is based on the combination of clinical symptoms and either a positive stool test for C. difficile toxins or endoscopic or histological findings of pseudomembranous colitis. There is no indication for treatment of asymptomatic carriers, but patients with proven CDI should be treated. Treatment consists of cessation of the provoking antibiotic treatment, secondary prevention by infection control strategies, and treatment with metronidazole or vancomycin. Treatment of recurring CDI, severe infection, the need for surgery, and novel alternative potential treatment strategies will be discussed. The concurrent increase in multiresistant colonisation and increasing numbers of asymptomatic carriers of C. difficile will lead to an increase of the situation in which patients with severe infections, treated with broad-spectrum antibiotics, will develop concurrent severe CDI. We will discuss possible therapy strategies for these patients.

Characterization of a model of systemic inflammation in humans in vivo elicited by continuous infusion of endotoxin

Investigating the systemic inflammatory response in patients with critical illness such as sepsis, trauma and burns is complicated due to uncertainties about the onset, duration and severity of the insult. Therefore, in vivo models of inflammation are essential to study the pathophysiology and to evaluate immunomodulatory therapies. Intravenous bolus administration of endotoxin to healthy volunteers is a well-established model of a short-lived systemic inflammatory response, characterized by increased plasma cytokine levels, flu-like symptoms and fever. In contrast, patients suffering from systemic inflammation are often exposed to inflammatory stimuli for an extended period of time. Therefore, continuous infusion of endotoxin may better reflect the kinetics of the inflammatory response encountered in these patients. Herein, we characterize a novel model of systemic inflammation elicited by a bolus infusion of 1 ng/kg, followed by a 3hr continuous infusion of 1 ng/kg/h of endotoxin in healthy volunteers, and compared it with models of bolus administrations of 1 and 2 ng/kg of endotoxin. The novel model was well-tolerated and resulted in a more pronounced increase in plasma cytokine levels with different kinetics and more prolonged symptoms and fever compared with the bolus-only models. Therefore, the continuous endotoxin infusion model provides novel insights into kinetics of the inflammatory response during continuous inflammatory stimuli and accommodates a larger time window to evaluate immunomodulating therapies.

Enteroendocrine L Cells Sense LPS after Gut Barrier Injury to Enhance GLP-1 Secretion

Glucagon-like peptide 1 (GLP-1) is a hormone released from enteroendocrine L cells. Although first described as a glucoregulatory incretin hormone, GLP-1 also suppresses inflammation and promotes mucosal integrity. Here, we demonstrate that plasma GLP-1 levels are rapidly increased by lipopolysaccharide (LPS) administration in mice via a Toll-like receptor 4 (TLR4)-dependent mechanism. Experimental manipulation of gut barrier integrity after dextran sodium sulfate treatment, or via ischemia/reperfusion experiments in mice, triggered a rapid rise in circulating GLP-1. This phenomenon was detected prior to measurable changes in inflammatory status and plasma cytokine and LPS levels. In human subjects, LPS administration also induced GLP-1 secretion. Furthermore, GLP-1 levels were rapidly increased following the induction of ischemia in the human intestine. These findings expand traditional concepts of enteroendocrine L cell biology to encompass the sensing of inflammatory stimuli and compromised mucosal integrity, linking glucagon-like peptide secretion to gut inflammation.

A randomised trial on the effect of anti-platelet therapy on the systemic inflammatory response in human endotoxaemia

The use of acetylsalicylic acid (ASA) is associated with improved outcome in patients with sepsis, and P2Y12 inhibitors have been suggested to also have immunomodulatory effects. Therefore, we evaluated the effects of clinically relevant combinations of antiplatelet therapy on the immune response in experimental endotoxaemia in humans in vivo. Forty healthy subjects were randomised to seven days of placebo, placebo with ASA, ticagrelor and ASA, or clopidogrel and ASA treatment. Systemic inflammation was elicited at day seven by intravenous administration of Escherichia coli endotoxin. ASA treatment profoundly augmented the plasma concentration of pro-inflammatory cytokines, but did not affect anti-inflammatory cytokines. Addition of either P2Y12 antagonist to ASA did not affect any of the circulating cytokines, except for an attenuation of the ASA-induced increase in TNFα by ticagrelor. Systemic inflammation increased plasma adenosine, without differences between groups, and although P2Y12 inhibition impaired platelet reactivity, there was no correlation with cytokine responses.

Short-Term Hypoxia Dampens Inflammation in vivo via Enhanced Adenosine Release and Adenosine 2B Receptor Stimulation

Hypoxia and inflammation are closely intertwined phenomena. Critically ill patients often suffer from systemic inflammatory conditions and concurrently experience short-lived hypoxia. We evaluated the effects of short-term hypoxia on systemic inflammation, and show that it potently attenuates pro-inflammatory cytokine responses during murine endotoxemia. These effects are independent of hypoxia-inducible factors (HIFs), but involve augmented adenosine levels, in turn resulting in an adenosine 2B receptor-mediated post-transcriptional increase of interleukin (IL)-10 production. We translated our findings to humans using the experimental endotoxemia model, where short-term hypoxia resulted in enhanced plasma concentrations of adenosine, augmentation of endotoxin-induced circulating IL-10 levels, and concurrent attenuation of the pro-inflammatory cytokine response. Again, HIFs were shown not to be involved. Taken together, we demonstrate that short-term hypoxia dampens the systemic pro-inflammatory cytokine response through enhanced purinergic signaling in mice and men. These effects may contribute to outcome and provide leads for immunomodulatory treatment strategies for critically ill patients.

LPS-Induced ex vivo Cytokine Production is Not Augmented in Patients with Von Hippel-Lindau Disease

Dear Editor, Patients with Von Hippel-Lindau disease (VHLd) have a monoallelic germline mutation in the Von HippelLindau (VHL) gene. The Von Hippel-Lindau protein (pVHL) facilitates degradation of the transcription factor Hypoxia Inducible Factor (HIF)-1a [1]. When a second hit affects the only well-functioning VHL gene left, HIF-1a is no longer degraded and its transcriptional activity can contribute to the development of highly vascularized benign and malignant tumours in VHLd [1]. HIF-1a appears to play an important role in innate immunity. For instance, mice with a myeloid HIF-1a deficiency show an attenuated in vivo cytokine responses upon administration of lipopolysaccharide (LPS) [2]. These observations have led us and others [3] to hypothesize that VHL haploinsufficiency may result in a more pro-inflammatory phenotype of myeloid cells. Such a pro-inflammatory phenotype may contribute to tumorigenesis, as an inflammatory environment created by tumour-associated macrophages promotes tumour growth [4]. Herein, we evaluated whether ex vivo LPSinduced cytokine production is altered in patients with VHLd. All procedures were approved by the local ethics committee (CMO 2010/104) and in accordance with the declaration of Helsinki. We included VHLd patients and age-matched healthy subjects in a 1:2 ratio. PMBCs were isolated and stimulated with 10 ng/ml LPS (E. Coli, serotype 0111:B4, Sigma-Aldrich, St Louis, MI, USA) or

Hypoxia and hypoxia-mimetics attenuate the inflammatory response during murine endotoxemia

Hypoxia has been shown to exert immunomodulatory effects1. As oxygenation is daily practice in critical care, and the majority of critically ill patients suffer from inflammatory-related conditions, permissive hypoxia might be a novel therapeutic strategy. In addition, there are pharmacologic hypoxia-mimetics available that can replicate the hypoxia-effects without the potential drawbacks of systemic hypoxia. The hypoxic immunomodulatory effects are thought to be mediated through a group of transcription factors called hypoxia-inducible factors (HIFs)2. However, in vitro studies have demonstrated that, depending on the cell-type, these effects can be both pro- and anti-inflammatory. The net effects of hypoxia during systemic inflammation in vivo are therefore unknown.