SignificanceHitherto, both the autonomic nervous system and innate immune system were regarded as systems that cannot be voluntarily influenced. The present study demonstrates that, through practicing techniques learned in a short-term training program, the sympathetic nervous system and immune system can indeed be voluntarily influenced. Healthy volunteers practicing the learned techniques exhibited profound increases in the release of epinephrine, which in turn led to increased production of anti-inflammatory mediators and subsequent dampening of the proinflammatory cytokine response elicited by intravenous administration of bacterial endotoxin. This study could have important implications for the treatment of a variety of conditions associated with excessive or persistent inflammation, especially autoimmune diseases in which therapies that antagonize proinflammatory cytokines have shown great benefit.

The endothelium plays a central role in pathophysiology of sepsis. Systemic inflammation results in endothelial dysfunction, contributing to the development of shock and multiple organ dysfunction. However, an easy to obtain, early, and clinically applicable marker of endothelial dysfunction is currently not available. Such a marker could guide early and targeted therapy such as selective iNOS inhibition. Endocan is a soluble proteoglycan secreted by endothelial cells in response to pro-inflammatory cytokines, bacterial endotoxins, and angiogenic factors, and enhances microvascular permeability. Plasma endocan levels are increased in septic patients, and have been shown to correlate with sepsis severity and mortality. However, the direct relationship between endocan and endothelial function has not yet been investigated in humans in vivo.

In a variety of conditions excessive and/or persistent activation of the innate immune system has detrimental effects. In animals, electrical vagus nerve stimulation (VNS) inhibits the innate immune response in models of endotoxemia (administration of lipopolysaccharide [LPS]), sepsis, trauma, and hemorrhagic shock, via the so-called cholinergic anti-inflammatory pathway. However, human in vivo evidence is lacking. Up till now, VNS was possible through implantation of a cuff electrode wrapped around the nerve, which limits its use in acute inflammatory situations frequently encountered on the ICU. A novel, less invasive VNS method is transvenous VNS (tVNS).

Abstract Abstract 3452 Objective Anemia of chronic disease (ACD) is generally attributed to increased hepcidin production. The first-in-class hepcidin antagonist NOX-H94, a PEGylated anti-hepcidin L-RNA oligonucleotide, is in development for targeted treatment of ACD. We investigated whether NOX-H94 prevents the inflammation-induced serum iron decrease during experimental human endotoxemia. Effects on the innate immune response were studied as secondary endpoints. Materials and Methods The study protocol (ClinicalTrials.gov identifier NCT01522794) was approved by the local ethics committee of the Radboud University Nijmegen Medical Centre and was conducted in accordance with the Declaration of Helsinki (2000) of the World Medical Association. This randomized, double-blind, placebo-controlled trial was carried out in 24 healthy young men. At T=0 hours (h), 2 ng/kg E. coli endotoxin was administered intravenously (i.v.), followed by 1.2 mg/kg NOX-H94 or placebo i.v. at T=0.5h. Blood was drawn serially for 24h and at day 3, 8 and 15 after endotoxin administration for measurements of inflammatory parameters, cytokines, NOX-H94 pharmacokinetics, total hepcidin-25 (free and NOX-H94-bound), and iron parameters. The difference of serum iron change from baseline at T=9h was defined as primary endpoint. Data are expressed as mean±SD. Results Endotoxin administration led to flu-like symptoms peaking at T=1.5h, irrespective of the treatment group. Body temperature rose by 1.9±0.5 °C in both groups. Peak CRP at T=24h was also similar in NOX-H94 and placebo treated groups (34.3±18.0 vs. 36.8±8.7 mg/L) as was the rise in leucocytes at T=6 h (12.1±2.2 vs. 12.1±2.3 ×109/L). Plasma levels of TNF-α, IL-6, IL-10, and IL-1RA peaked markedly and similarly in both treatment groups. NOX-H94 was well tolerated. Plasma concentrations peaked at 0.7±0.4 h after the start of administration, after which they declined according to a two-compartment model, with rapid initial elimination followed by a slower elimination phase with a plasma half-life of 22.5 ± 4.28h. Hepcidin-25 rose to a peak of 23.0±5.2 nM at T=5.5±0.05 h in control subjects and normalized at about T=24 h. In the NOX-H94 treated group, total hepcidin-25 concentrations, which represent the sum of free hepcidin-25 and hepcidin-25 bound to NOX-H94, rose to 559±112 nM at T=9.5±1.5 h and normalized slowly until day 15. The observed serum iron concentrations are shown in the table. In the placebo group, serum iron increased from baseline to a peak at T=3h, returned close to baseline at T=6h and decreased under the baseline concentration at T=9h reaching its lowest point at T=12h. In the NOX-H94 group, serum iron concentrations rose until T=6h and then slowly declined until T=24h. From 6 to 12 h post LPS, the serum iron concentrations in NOX-H94 treated subjects were significantly higher than in placebo treated subjects (P<0.0001, ANCOVA). At T=24 h and 48 h, serum iron was lower in NOX-H94 treated subjects than in placebo controls. Conclusion Experimental human endotoxemia induced a robust inflammatory response with hepcidin release and a subsequent decrease in serum iron. Treatment with NOX-H94 had no effect on innate immunity, but effectively prevented the inflammation-induced drop in serum iron concentrations. These findings demonstrate the clinical potential of the anti-hepcidin drug NOX-H94 for further development to treat patients with ACD. Disclosures: van Eijk: NOXXON Pharma AG: Research Funding. Swinkels:NOXXON Pharma AG: Research Funding. Schwoebel:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment. Summo:NOXXON Pharma AG: Employment. Stéphanie:NOXXON Pharma AG: Employment. Riecke:NOXXON Pharma AG: Employment. Pikkers:NOXXON Pharma AG: Research Funding.

Nitric oxide is considered an important contributory factor in the pathogenesis of septic shock. The sepsis-associated elevated expression of inducible nitric oxide-synthase and subsequent increase in nitric oxide largely accounts for the down-regulation of endothelial nitric oxide-synthase, resulting in an impaired vasodilatory response to substances such as substance P, bradykinin and acetylcholine. Given the overwhelming evidence in favour of endothelial dysfunction during sepsis, the finding of Kienbaum et al. [1] that acetylcholine-induced vasodilation is preserved in septic shock patients is surprising. This paper represents the first investigation of vascular reactivity in septic patients, for which we congratulate the authors, though we feel it is important to make some comments. In a previous study using the perfused forearm, conducted prior to and during experimental endotoxaemia in humans it was found that an infusion of acetylcholine comparable to that used by Kienbaum resulted in a 40% reduction in the vasodilatory response during endotoxemia compared to baseline response [2, 3]. Endothelial dysfunction was found 4–6 h after lipopolysaccharide administration, at the time that circulating cytokines are at their highest. In Kienbaum’s study, it is not clear in what phase of sepsis the patients were and it appears plausible that during later phases of septic shock, endothelial function may recover. In addition, in septic shock patients, paired observations are not possible and with a small sample size inter-individual differences may become important. Acetylcholine is highly unstable in blood and vasodilator responses to acetylcholine are dependent on aceylcholinesterase activity, sex, basal blood flow and forearm length [4]. Furthermore, it appears that Kienbaum et al. measured forearm blood flow in the infused arm only. In our opinion, and in previous studies, measurement of forearm blood flow in both forearms would be a better method. Measurements of the flow in the non-infused arm can be used as a contemporaneous time-control for the infused arm. In this way systemic effects can be observed and vasoactive effects can be expressed as a quotient of the infused and the non-infused arm [2, 5]. Kienbaum et al. report that the acetylcholine-induced decrease in forearm vascular resistance (forearm blood flow/mean arterial pressure) was similar between septic patients and controls, however, since septic patients had lower vascular resistance initially, decrease in vascular resistance may be less. In addition, since it was only feasible to infuse two acetylcholine concentrations, it cannot be determined at which part of the acetylcholine dose-response curve the measurements were made. It is conceivable that with higher infusion rates of acetylcholine or other endothelium-dependent agonists, like substance P and bradykinin, it would be possible to demonstrate endothelial dysfunction during early sepsis. Finally, the lack of a vasopressor response after intra-arterial infusion of the nitric oxide-synthase inhibitor L-N monomethylarginine in the controls is probably caused by the much lower infusion rates used compared to previous studies [2, 5]. In conclusion, in contrast to prior experimental and clinical work, endothelial dysfunction during systemic inflammation was not confirmed by Kienbaum et al. Although the ‘perfused forearm model’ enables us to investigate direct vascular effects of various pharmacological agents, allowing us to determine direct vasoactivity and endothelial function in various groups of patients, caution should be taken in the interpretation of the results obtained. In view of the above issues, additional studies are necessary before we can conclude that endothelial dysfunction is not present in septic patients.

ABSTRACT To gain insight in the pathogenesis of increased vascular permeability during sepsis, we studied the effect of plasma obtained during human experimental endotoxemia on the permeability of cultured endothelial monolayers. Eight healthy subjects received an i.v. dose of 2 ng/kg Escherichia coli O:113 lipopolysaccharide (LPS). The concentration of various plasma mediators that supposedly induce vascular permeability was measured over time. Plasmas that were obtained before, and 2 and 4 h after the administration of LPS were added to human umbilical venular endothelial cells that were cultured on semipermeable membranes.The permeability of the endothelial monolayers to fluorescein isothiocyanate-labeled bovine serum albumin was determined and expressed as the relative concentration of fluorescein isothiocyanate-bovine serum albumin when compared with that measured across empty Transwell-COL (Corning Life Sciences B.V., Schiphol-Rijk, The Netherlands) membranes (i.e., without endothelial monolayers). The permeability levels were correlated with the concentrations of various mediators.Experimental endotoxemia resulted in elevated levels of tumor necrosis factor &agr;, interleukin (IL) 1&bgr;, IL-6, IL-8, IL-10, and vascular endothelial growth factor and a moderate increase of IL-12 and IFN-&ggr; (all P values < 0.01). Incubation of human umbilical venular endothelial cells with plasma obtained 2 and 4 h after the administration of LPS increased the relative permeability from a baseline level (median) of 17% (range, 14% - 31%) to 23% (range, 12% - 39%; P = not significant) and 28% (range, 11% - 40%; P < 0.05), respectively. Plasma levels of vascular endothelial growth factor and IL-10, but not TNF-&agr; or any other mediators, significantly correlated with the increase in endothelial permeability (r = 0.47, P = 0.038; r = 0.43, P = 0.038, respectively).The data presented here demonstrate that plasmas obtained from experimental human endotoxemia increase endothelial albumin permeability in vitro. Thus, cultured human endothelial monolayers provide a model to study sepsis-associated vascular changes.

Clinical experience suggests that the administration of fluids in human endotoxemia reduces symptoms. In the present study, the effects of a standardised fluid protocol on symptoms, inflammatory and hemodynamic parameters in human endotoxemia are determined. With approval of the local ethics committee, 16 healthy volunteers received 2 ng/kg of Escherichia coli endotoxin (O:113). After an overnight fast, nine subjects received 1.5 l of 2.5% glucose/0.45% NaCl the hour prior to the endotoxin administration and 150 ml/h during the course of the experiment ('prehydrated group'). Seven subjects only received a continuous infusion of 75 ml/h during the experiment ('non-prehydrated group'). The course of inflammatory parameters and symptoms were determined and mean arterial pressure, heart rate and forearm blood flow were measured. In the prehydrated group, TNF-alpha increased to 522 +/- 63 pg/ml (mean +/- SEM) while the maximum in the non-prehydrated group was 927 +/- 187 pg/ml (P < 0.04). IL-10 increased similarly in both groups (non-prehydrated 117 +/- 18 pg/ml and prehydrated 99 +/- 18 pg/ml; P = NS). The prehydrated group had a significantly lower (P < 0.004) symptom score and recovered sooner (P = 0.004). Endotoxin-induced changes in hemodynamics revealed no significant differences between groups. We demonstrate that prehydration in experimental human endotoxemia significantly shifts the cytokine balance towards a more anti-inflammatory pattern. This effect is associated with a reduction in symptoms, whereas the changes in hemodynamic parameters are not influenced by prehydration.

INTRODUCTION:Septic shock is associated with increased microvascular permeability. As a model for study of the pathophysiology of sepsis, endotoxin administration to humans has facilitated research into inflammation, coagulation and cardiovascular effects. The present study was undertaken to determine whether endotoxin administration to human volunteers can be used as a model to study the sepsis-associated increase in microvascular permeability.METHODS:In an open intervention study conducted in a university medical centre, 16 healthy volunteers were evaluated in the research unit of the intensive care unit. Eight were administered endotoxin intravenously (2 ng/kg Escherichia coli O113) and eight served as control individuals. Microvascular permeability was assessed before and 5 hours after the administration of endotoxin (n = 8) or placebo (n = 8) by three different methods: transcapillary escape rate of I(125)-albumin; venous occlusion strain-gauge plethysmography to determine the filtration capacity; and bioelectrical impedance analysis to determine the extracellular and total body water.RESULTS:Administration of endotoxin resulted in the expected increases in proinflammatory cytokines, temperature, flu-like symptoms and cardiovascular changes. All changes were significantly different from those in the control group. In the endotoxin group all microvascular permeability parameters remained unchanged from baseline: transcapillary escape rate of I(125)-albumin changed from 7.2 +/- 0.6 to 7.7 +/- 0.9%/hour; filtration capacity changed from 5.0 +/- 0.3 to 4.2 +/- 0.4 ml/min per 100 ml mmHg x 10(-3); and extracellular/total body water changed from 0.42 +/- 0.01 to 0.40 +/- 0.01 l/l (all differences not significant).CONCLUSION:Although experimental human endotoxaemia is frequently used as a model to study sepsis-associated pathophysiology, an endotoxin-induced increase in microvascular permeability in vivo could not be detected using three different methods. Endotoxin administration to human volunteers is not suitable as a model in which to study changes in microvascular permeability.

Meningococcal septic shock is an important cause of morbidity and mortality in children and young adults worldwide and is the prototypical gram-negative septic shock. One of the key factors in the development of shock is increased microvascular permeability. Vascular endothelial growth factor (VEGF) is a central factor in angiogenesis and is an important mediator of vascular permeability. Thirteen patients with meningococcal infection (eight presenting with shock) were investigated in the early phase of invasive meningococcal disease. Cytokines, complement activation, and VEGF plasma concentrations were measured during the first 48 h on the pediatric intensive care unit. Increased cytokine concentrations and activation of the complement system were observed. VEGF plasma concentrations were increased (median 193 pg/mL, range 71-1082) and were highest in the presence of shock (208 pg/mL, 169-1082) compared with patients presenting without shock (92 pg/mL range 71-299). VEGF concentration at admission correlated with the severity of disease (pediatric risk of mortality score, R = 0.90 [Spearman], P = 0.0001) and the amount of fluids administered within the first 24 h (R = 0.90, P < 0.0001). In all patients, a decrease in VEGF was associated with a decrease in fluid intake during t = 24 to 48 h. The results suggest that apart from correlation with IL-1β, −10, −12, and complement activation, microvascular permeability in sepsis is also closely linked to the plasma concentration of VEGF. The role of VEGF in sepsis-associated increased microvascular permeability needs further exploration and may represent a new therapeutic target.

Objective To review the prenatal assessment of associated renal pathology, non-renal pathology and renal biometry, fetal outcome and postnatal urological management in the presence of unilateral fetal multicystic dysplastic kidney.Methods A total of 38 singleton pregnancies with fetal unilateral multicystic dysplastic kidney was studied over a 13-year period. Prenatally, fetal biometry, including head and abdominal circumferences and largest longitudinal diameter of the affected and contralateral kidneys, was performed. The amount of amniotic fluid was assessed. Fetal karyotyping was offered in cases of contralateral renal or non-renal pathology. A MAG 3 scan and voiding cystogram was performed approximately 4 weeks after delivery to establish renal function and to exclude urinary reflux.Results Unilateral fetal multicystic dysplastic kidney, was left-sided in 53% and right-sided in 47% of cases. The fetus was male in 63% and female in 37% of cases. Associated renal and non-renal pathology existed in 21% and 5% of cases, respectively. The fetal karyotype in these subsets was always normal. The longitudinal diameter of the multicystic dysplastic kidney was above the 95th centile in 87%. There was polyhydramnios in three cases and oligohydramnios in two cases. The prematurity rate was 16%. Postnatal examination revealed a non-functional multicystic kidney in 87% (33138) of cases. Following surgical removal of the affected kidney, these infants progressed normally. Of the remaining five infants, four died because of associated anomalies and one infant developed normally, without surgery.Conclusions Fetal outcome is determined by associated renal and/or non-renal structural pathology, and not by the size/location of the unilateral multicystic dysplastic kidney or amniotic fluid volume.