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Transforming growth factor‐β (TGF‐β) becomes rapidly activated in the infarcted heart. Hence, TGF‐β‐mediated persistent activation of cardiac fibroblasts (CFs) and exaggerated fibrotic responses may result in adverse cardiac remodelling and heart failure. Additionally, peptidylarginine deiminase 4 (PAD4) was described to be implicated in organ fibrosis. Here, we investigated the impact of PAD4 on CF function and myofibroblast transdifferentiation in vitro. The expression of fibrosis‐related genes was largely similar in cultured WT and PAD4‐/‐ CFs of passage 3, although collagen III was reduced in PAD4‐/‐ CFs. Exposure to TGF‐β inhibited proliferation and increased contractile activity and migration of WT CFs, but not of PAD4‐/‐ CFs. However, under baseline conditions, PAD4−/− CFs showed comparable functional characteristics as TGF‐β‐stimulated WT CFs. Although the SMAD‐dependent TGF‐β pathway was not disturbed in PAD4‐/‐ CFs, TGF‐β failed to activate protein kinase B (Akt) and signal transducer and activator of transcription 3 (STAT3) in these cells. Similar results were obtained in WT CFs treated with the PAD4 inhibitor Cl‐amidine. Abrogated Akt activation was associated with diminished levels of phosphorylated, inactive glycogen synthase kinase‐3β (GSK‐3β). Consequently, PAD4‐/‐ CFs did not upregulate collagen I and α‐smooth muscle actin (α‐SMA) expression after TGF‐β treatment. Thus, PAD4 is substantially involved in the regulation of non‐canonical TGF‐β signalling and may represent a therapeutic target for the treatment of adverse cardiac remodelling.

Neutrophil extracellular traps (NETs) are web-like structures, which are released upon neutrophil activation. It has previously been demonstrated that NETs are present in atherosclerotic lesions of both humans and animal models thus playing a decisive role in atherosclerosis. Besides, macrophages have a crucial role in disease progression, whereby classically activated M1 macrophages sustain inflammation and alternatively activated M2 macrophages display anti-inflammatory effects. Although NETs and macrophages were found to colocalize in atherosclerotic lesions, the impact of NETs on macrophage function is not fully understood. In the present study, we aimed to investigate the effect of NETs on human and murine macrophages in respect to the expression of pro-inflammatory cytokines, matrix metalloproteinases (MMPs) and uptake of oxidized LDL (oxLDL) in vitro. Human THP-1 and murine bone marrow-derived macrophages were cultured under M1 (LPS + IFN-γ)- and M2a (IL-4)-polarizing culture conditions and treated with NETs. To mimic intraplaque regions, cells were additionally cultured under hypoxic conditions. NETs significantly increased the expression of IL-1β, TNF-α and IL-6 in THP-M1 macrophages under normoxia but suppressed their expression in murine M1 macrophages under hypoxic conditions. Notably, NETs increased the number of oxLDL-positive M1 and M2 human and murine macrophages under normoxia, but did not influence formation of murine foam cells under hypoxia. However, oxLDL uptake did not strongly correlate with the expression of the LDL receptor CD36. Besides, upregulated MMP-9 expression and secretion by macrophages was detected in the presence of NETs. Again, hypoxic culture conditions dampened NETs effects. These results suggest that NETs may favor foam cell formation and plaque vulnerability, but exert opposite effects in respect to the inflammatory response of human and murine M1 macrophages. Moreover, effects of NETs on macrophages’ phenotype are altered under hypoxia.

Myocardial hypertrophy is present in many heart diseases, representing a strong predictor of adverse cardiovascular outcomes. Regarding therapeutic intervention, mesenchymal stem cells (MSCs) have been suggested to significantly reduce cardiac hypertrophy and progression to heart failure. Preconditioning of MSCs was previously demonstrated to highly improve their paracrine activity resulting in modulation of immune responses and the progression of diseases. Here, we studied the effects of bone marrow-derived preconditioned MSCs on hypertrophied induced pluripotent stem cell-derived cardiomyocytes (iPS-CM) and also sought to identify MSC-derived antihypertrophic molecules. Phenylephrine (PE) was used to induce hypertrophy in murine iPS-CM, and markers of hypertrophy were identified by microarray analysis. Murine MSCs were treated with IFN-γ and IL-1β to enhance their paracrine activity, and transcriptional profiling was performed by microarray analysis. Hypertrophied iPS-CM were subsequently cocultured with preconditioned MSCs or MSC-conditioned medium (CM), respectively. Effects on hypertrophied iPS-CM were studied by cell area quantification, real-time PCR, and western blot. In some experiments, cells were incubated with fractions of MSC-CM obtained by ultrafiltration or by MSC-CM supplemented with inhibitory antibodies. Intracellular and extracellular levels of vascular endothelial growth factor (VEGF) were evaluated by western blot and ELISA. PE-induced hypertrophy in iPS-CM was associated with an upregulation of neuron-derived orphan receptor (Nor1) expression, activation of Akt, and inhibition of both strongly prevented hypertrophy induction in iPS-CM. VEGF secreted by preconditioned MSCs provoked hypertrophy regression in iPS-CM, and a negative correlation between Nor1 expression and hypertrophic growth could be evidenced. Our results demonstrate that Nor1 expression strongly supports hypertrophy in iPS-CM. Moreover, the secretome of preconditioned MSCs triggered regression of hypertrophy in iPS-CM in a VEGF-dependent manner. We suggest that the delivery of the MSC-derived secretome may represent a therapeutic strategy to limit cardiac hypertrophy. However, additional in vivo studies are needed to prove this hypothesis.

Cardiopulmonary bypass (CPB) provokes inflammation culminating in organ dysfunction and increased mortality. Recently, neutrophil extracellular traps (NETs) have been found to be involved in a variety of cardiovascular diseases promoting tissue and organ injury. Here, we aimed to elaborate the proinflammatory potential of circulating cell-free (cf)DNA in patients undergoing cardiac surgery with CPB. Plasma was collected pre- and postoperatively as well as at d1, d3, d5 and d8 after surgery. At d1, we found circulating cfDNA levels to be significantly increased in patients with prolonged CPB duration (>100 min) when compared to those with shorter CPB times (CPB 100 min induced NETs release by neutrophils from healthy donors which was not suppressed by inhibitors of intracellular toll-like receptor (TLR)9. DNA binding to neutrophils’ surface (s)TLR9 has been evidenced. Altogether, we demonstrate that elevated plasma cfDNA might be useful to assess CPB-mediated detrimental effects, including endothelial damage, in cardiac surgical patients with prolonged CPB duration. cfDNA-triggered NETosis is independent of classical TLR9 signaling.

The use of cardiopulmonary bypass (CPB) results in the activation of leukocytes, release of neutrophil extracellular traps (NETs) and severe inflammation. We hypothesize that targeting of circulating cell-free DNA (cfDNA) by DNases might represent a feasible therapeutic strategy to limit CPB-associated side effects. Male Wistar rats (n = 24) underwent CPB with deep hypothermic circulatory arrest (DHCA) and were divided into 3 groups: control (group 1), one i.v. bolus DNase I before CPB start (group 2) and a second DNase I dose before reperfusion (group 3). We found a positive correlation between plasma cfDNA/NETs levels and compromised endothelial vasorelaxation after CPB. DNase I administration significantly diminished plasma cfDNA/NETs levels. Further, a dose-dependent improvement in endothelial function accompanied by significant reduction of circulating intercellular adhesion molecule (ICAM)-1 was observed. Rats of group 3 had significantly reduced plasma IL-6 levels and downregulated expression of adhesion molecules resulting in impaired leukocyte extravasation and reduced MPO activity in lungs. Mechanistically, digestion of NETs by DNase I significantly diminished NETs-dependent upregulation of adhesion molecules in human endothelial cells. Altogether, systemic DNase I administration during CPB efficiently reduced cfDNA/NETs-mediated endothelial dysfunction and inflammation and might represents a promising therapeutic strategy for clinical practice.

Use of cardiopulmonary bypass in cardiac surgery triggers systemic inflammation by neutrophil activation leading to neutrophil extracellular traps (NETs) release. Hence, nuclear DNA released by necrotic and apoptotic cells might contribute to an increase in circulating cell-free DNA (cfDNA). cfDNA/NETs might induce endothelial damage and organ dysfunction. This study focuses on the accuracy of cfDNA to predict acute kidney injury (AKI) after on-pump surgery. 58 cardiac patients undergoing on-pump surgery were prospectively enrolled. Blood samples were taken preoperatively, immediately after surgery, at day 1, 2, 3 and 5 from patients with (n = 21) or without (n = 37) postoperative AKI development. Levels of cfDNA, neutrophil gelatinase-associated lipocalin (NGAL) and creatinine in patients’ plasma were quantified. ROC curves were used to assess the predictive value of the biomarkers for AKI. Further baseline characteristics and perioperative variables were analyzed.cfDNA and NGAL levels highly increased in AKI patients and significant intergroup differences (vs. non-AKI) were found until day 3 and day 5 after surgery, respectively. cfDNA levels were significantly elevated in patients who developed late AKI (>24 hours), but not in those with AKI development during the first 24 hours (early AKI). NGAL and creatinine, which were highest in patients with early AKI, accurately predicted during the first 24 postoperative hours (early AKI). At day 3, at a threshold of 260.53 ng/ml cfDNA was the best predictor for AKI (AUC = 0.804) compared to NGAL (AUC = 0.699) and creatinine (AUC = 0.688). NGAL, but not cfDNA, was strongly associated with AKI stages and mortality. Monitoring of cfDNA levels from the first postoperative day might represent a valuable tool to predict late AKI after on-pump surgery.

The demand to develop convergent technology platforms, such as bio-functionalized medical devices, is rapidly increasing. However, the loss of biological function of the effector molecules during sterilization represents a significant and general problem. Therefore, we have developed and characterized a nano-coating (NC) formulation capable of maintaining the functionality of proteins on biological-device combination products. As a proof of concept, the NC preserved the structural and functional integrity of an otherwise highly fragile antibody immobilized on polyurethane during deleterious sterilizing irradiation (≥ 25 kGy). The NC procedure enables straight-forward terminal sterilization of bio-functionalized materials while preserving optimal conditioning of the bioactive surface.

Vascular ischemic diseases, hypertension, and other systemic hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). NO but also its active derivates like nitrite or nitroso compounds are important effector and signal molecules with vasodilating properties. Our previous findings point to a therapeutical potential of cutaneous administration of NO in the treatment of systemic hemodynamic disorders. Unfortunately, no reliable data are available on the mechanisms, kinetics and biological responses of dermal application of nitric oxide in humans in vivo. The aim of the study was to close this gap and to explore the therapeutical potential of dermal nitric oxide application. We characterized with human skin in vitro and in vivo the capacity of NO, applied in a NO-releasing acidified form of nitrite-containing liniments, to penetrate the epidermis and to influence local as well as systemic hemodynamic parameters. We found that dermal application of NO led to a very rapid and significant transepidermal translocation of NO into the underlying tissue. Depending on the size of treated skin area, this translocation manifests itself through a significant systemic increase of the NO derivates nitrite and nitroso compounds, respectively. In parallel, this translocation was accompanied by an increased systemic vasodilatation and blood flow as well as reduced blood pressure. We here give evidence that in humans dermal application of NO has a therapeutic potential for systemic hemodynamic disorders that might arise from local or systemic insufficient availability of NO or its bio-active NO derivates, respectively.

Posttrauma apoptosis resistance of neutrophils (PMN) is related to overshooting immune responses, systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF). Recently, we have shown that the apoptosis resistance in circulating PMN from severely injured patients which is known to be mediated by high serum levels of pro-inflammatory cytokines can be overcome by the activation of Fas death receptor. Here, we aimed to study whether stimulation of surface Fas leads to the inactivation of hyperactivated PMN from critically ill patients with SIRS. PMN from 23 multiple trauma patients (mean injury severity score (ISS) 34±1.9) were isolated at day 1 after admission to the trauma center. PMN from 17 volunteer blood donors served as controls. Neutrophil activity has been determined after ex vivo short (1 h) and long-term (4 h) stimulation of freshly isolated PMN with immobilized agonistic anti-Fas antibodies. We found neutrophil chemotactic migration in response to IL-8, phagocytosis and oxidative burst to be significantly inhibited in control cells already after short-term (1 h) Fas stimulation. In contrast, inactivation of trauma PMN by agonistic anti-Fas antibodies was found to be efficient only after long-term (4 h) incubation of cells with agonistic antibodies. Thus, in trauma PMN down-regulation of neutrophil activity seems to be delayed when compared to cells isolated from healthy controls, suggesting impaired susceptibility for Fas stimulation in these cells. Interestingly, whereas Fas-mediated inhibition of phagocytosis and oxidative burst could be prevented by the broad range caspase inhibitor t-butoxycarbonyl-aspartyl(O-methyl)-fluoromethyl ketone (BocD-fmk), the chemotactic activity in response to IL-8 was unaffected. In conclusion, we demonstrate that stimulation of neutrophil Fas does not only initiate apoptosis but also induces inhibition of neutrophil functions, partially by non-apoptotic signaling.

The systemic inflammatory response syndrome and subsequent organ failure are mainly driven by activated neutrophils with prolonged life span, which is believed to be due to apoptosis resistance. However, detailed underlying mechanisms leading to neutrophil apoptosis resistance are largely unknown, and possible therapeutic options to overcome this resistance do not exist. Here we report that activated neutrophils from severely injured patients exhibit cell death resistance due to impaired activation of the intrinsic apoptosis pathway, as evidenced by limited staurosporine-induced mitochondrial membrane depolarization and decreased caspase-9 activity. Moreover, we found that these neutrophils express high levels of antiapoptotic Mcl-1 and low levels of proapoptotic Bax protein. Mcl-1 up-regulation was dependent on elevated concentrations of GM-CSF in patient serum. Accordingly, increased Mcl-1 protein stability and GM-CSF serum concentrations were shown to correlate with staurosporine-induced apoptosis resistance. However, cross-linking of neutrophil Fas by immobilized agonistic anti-Fas IgM resulted in caspase-dependent mitochondrial membrane depolarization and apoptosis induction. In conclusion, the observed impairment of the intrinsic pathway and the resulting apoptosis resistance may be overcome by immobilized agonistic anti-Fas IgM. Targeting of neutrophil Fas by immobilized agonistic effector molecules may represent a new therapeutic tool to limit neutrophil hyperactivation and its sequelae in patients with severe immune disorders.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil-derived circulating free DNA (cf-DNA) and neutrophil cytoplasm-derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf-DNA/NETs for identification of septic arthritis. Forty-two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf-DNA/NETs (j-cf-DNA) levels were directly quantified. Diagnostic value of j-cf-DNA was compared with white blood cells (WBC), synovial white blood cells (j-WBC), C-reactive protein (CRP), j-IL-6, j-TNF alpha, j-IL-1 beta, and myeloperoxidase (j-MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC-curves for each parameter were calculated. Synovial fluid cf-DNA/NETs values from patients with septic arthritis (3,286 +/- 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 +/- 208 ng/ml, n = 17) or osteoarthritis (278 +/- 34 ng/ml, n = 16, p < 0.01). In conjunction with j-cf-DNA, j-IL-6 and j-IL-1 beta were significantly elevated (p < 0.01), but WBC, CRP, and j-WBC were not. At a cut-off of 300 ng/ml, j-cf-DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf-DNA/NETs (0.933) and j-IL-6 (0.951). cf-DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial.

Apoptosis resistance in activated neutrophils is known to be associated with collateral damage of surrounding tissue, as well as immune and organ dysfunction. Thus, the safe removal of neutrophils by apoptosis induction represents a prerequisite for the resolution of inflammation. Here, we report that intrinsic apoptosis resistance in human neutrophils, isolated from severely injured patients, is based on enhanced stabilization of antiapoptotic myeloid cell leukemia 1 and subsequent impairment of downstream apoptotic pathways. Whereas extrinsic apoptosis induction by the activation of Fas death receptor on inflammatory neutrophils was accompanied by caspase- and proteasome-mediated myeloid cell leukemia 1 degradation, intrinsic apoptosis induction by staurosporine led to a significant stabilization of myeloid cell leukemia 1 protein, which impeded on truncated forms of B cell lymphoma 2-associated X protein and B cell lymphoma 2 homology domain 3-interacting domain death translocation and subsequent cytochrome c release from the mitochondria. We show further that profound inhibition of myeloid cell leukemia 1 degradation is based on the inhibition of caspases and sustained activation of kinases involved in cell survival, such as Akt. Accordingly, impeded myeloid cell leukemia 1 phosphorylation on Ser159 by glycogen synthase kinase 3 and protein ubiquitination has been demonstrated. Inhibition of myeloid cell leukemia 1 activity markedly increased sensitivity to staurosporine-induced cell death. Altogether, these results provide new insights into the mechanisms underlying myeloid cell leukemia 1-mediated apoptosis resistance to staurosporine under inflammatory situations and should be considered for the development of novel therapeutic strategies.

The life span of neutrophilic granulocytes has a determining impact on the intensity and duration of neutrophil driven lung inflammation. Based on the compatible solute ectoine, we aimed to prevent anti-apoptotic reactions in neutrophils triggered by the inflammatory microenvironment in the lung. Neutrophils from chronic obstructive pulmonary disease patients and control individuals were exposed to inflammatory mediators and xenobiotics in the presence or absence of ectoine. The in vivo relevance of this approach was tested in xenobiotic-induced lung inflammation in rats. The reduction of apoptosis rates of ex vivo-exposed neutrophils from all study groups was significantly restored in the presence of ectoine. However, natural apoptosis rates not altered by inflammatory stimuli were not changed by ectoine. Mechanistic analyses demonstrated the preventive effect of ectoine on the induction of anti-apoptotic signalling. Neutrophilic lung inflammation induced by single or multiple expositions of animals to environmental particles was reduced after the therapeutic intervention with ectoine. Analyses of neutrophils from bronchoalveolar lavage indicate that the in vivo effect is due to the restoration of neutrophil apoptosis. Ectoine, a compound of the highly compliant group of compatible solutes, demonstrates a reproducible and robust effect on the resolution of lung inflammation.

Trauma represents the leading cause of death among young people in western countries. Among the beneficial role of neutrophils in host defence, excessive priming and activation of neutrophils after major trauma lead to an overwhelming inflammatory response and secondary host tissue injury due to the release of toxic metabolites and enzymes. Hyperbaric oxygen (HBO) therapy has been proposed to possess antiinflammatory effects and might represent an appropriate therapeutic option to lower inflammation in a broad range of patients. Here, we studied the effects of HBO on the activity of neutrophils isolated from severely injured patients (days 1-2 after trauma), in fact on the production of reactive oxygen species (ROS) and release of neutrophil extracellular traps (NETs). We found exposure to HBO therapy to significantly diminish phorbol-12-myristate-13-acetate (PMA)-induced ROS production in neutrophils isolated from patients and healthy volunteers. At the same time, marked decrease in NETs release was found in control cells and a less pronounced reduction in patient neutrophils. Impaired ability to produce ROS following exposure to HBO was demonstrated to be linked to a strong downregulation of the activity of p38 MAPK. Only slight suppression of ERK activity could be found. In addition, HBO did not influence neutrophil chemotaxis or apoptosis, respectively. Collectively, this study shows for the first time that HBO therapy suppresses ROS production in inflammatory human neutrophils, and thus might impair ROS-dependent pathways, e.g. kinases activation and NETs release. Thus, HBO might represent a feasible therapy for patients suffering from systemic inflammation, including those with multiple trauma.

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics, or biological responses relating to cutaneous exposure to NO in humans in vivo. Here we show that, owing to its excellent diffusion capacity, cutaneously applied NO rapidly penetrates the epidermal barrier in significant amounts, strongly enriching skin tissue and blood plasma with its vasoactive derivates. In parallel, it significantly increased vasodilatation and blood flow and reduced thrombocyte aggregation capacity. Data presented here for the first time show that, in humans, dermal application of NO has strong potential for use in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bioactive derivates. Clinical Pharmacology & Therapeutics (2012); 91 6, 1074–1082. doi:10.1038/clpt.2011.366

Introduction. Neutrophil extracellular traps (NET) consist of a DNA scaffold that can be destroyed by Deoxyribonuclease (DNase). Thus DNases are potential prerequisites for natural counter regulation of NETs formation. In the present study, we determined the relationship of NETs and DNase after major trauma. Methods. Thirty-nine major trauma patients, 14 with and 25 without sepsis development were enrolled in this prospective study. Levels of cell-free (cf)-DNA/NETs and DNase were quantified daily from admission until day 9 after admission. Results. Levels of cf-DNA/NETs in patients who developed sepsis were significantly increased after trauma. In the early septic phase, DNase values in septic patients were significantly increased compared to patients without sepsis ( ). cf-DNA/NETs values correlated to values of DNase in all trauma patients and patients with uneventful recovery ( ) but not in septic patients. Recombinant DNase efficiently degraded NETs released by stimulated neutrophils in a concentration-dependent manner in vitro. Conclusions. DNase degrades NETs in a concentration-dependent manner and therefore could have a potential regulatory effect on NET formation in neutrophils. This may inhibit the antibacterial effects of NETs or protect the tissue from autodestruction in inadequate NETs release in septic patients.

Delayed neutrophil apoptosis and overshooting neutrophil activity contribute to organ dysfunction and subsequent organ failure in sepsis. Here, we investigated apoptotic signaling pathways that are involved in the inhibition of spontaneous apoptosis in neutrophils isolated from major trauma patients with uneventful outcome as well as in those with sepsis development. DNA fragmentation in peripheral blood neutrophils showed an inverse correlation with the organ dysfunction at d 10 after trauma in all patients, supporting the important role of neutrophil apoptosis regulation for patient’s outcome. The expression of the antiapoptotic Bcl-2 protein members A1 and Mcl-1 were found to be diminished in the septic patients at d 5 and d 10 after trauma. This decrease was also linked to an impaired intrinsic apoptosis resistance, which has been previously shown to occur in neutrophils during systemic inflammation. In patients with sepsis development, delayed neutrophil apoptosis was found to be associated with a disturbed extrinsic pathway, as demonstrated by reduced caspase-8 activity and Bid truncation. Notably, the expression of Dad1 protein, which is involved in protein N-glycosylation, was significantly increased in septic patients at d 10 after trauma. Taken together, our data demonstrate that neutrophil apoptosis is regulated by both the intrinsic and extrinsic pathway, depending on patient’s outcome. These findings might provide a molecular basis for new strategies targeting cell death pathways in apoptosis-resistant neutrophils during systemic inflammation.

Rationale: Human skin contains photolabile nitric oxide derivates like nitrite and S -nitroso thiols, which after UVA irradiation, decompose and lead to the formation of vasoactive NO. Objective: Here, we investigated whether whole body UVA irradiation influences the blood pressure of healthy volunteers because of cutaneous nonenzymatic NO formation. Methods and Results: As detected by chemoluminescence detection or by electron paramagnetic resonance spectroscopy in vitro with human skin specimens, UVA illumination (25 J/cm 2 ) significantly increased the intradermal levels of free NO. In addition, UVA enhanced dermal S -nitrosothiols 2.3-fold, and the subfraction of dermal S -nitrosoalbumin 2.9-fold. In vivo, in healthy volunteers creamed with a skin cream containing isotopically labeled 15 N-nitrite, whole body UVA irradiation (20 J/cm 2 ) induced significant levels of 15 N-labeled S -nitrosothiols in the blood plasma of light exposed subjects, as detected by cavity leak out spectroscopy. Furthermore, whole body UVA irradiation caused a rapid, significant decrease, lasting up to 60 minutes, in systolic and diastolic blood pressure of healthy volunteers by 11±2% at 30 minutes after UVA exposure. The decrease in blood pressure strongly correlated ( R 2 =0.74) with enhanced plasma concentration of nitrosated species, as detected by a chemiluminescence assay, with increased forearm blood flow (+26±7%), with increased flow mediated vasodilation of the brachial artery (+68±22%), and with decreased forearm vascular resistance (−28±7%). Conclusions: UVA irradiation of human skin caused a significant drop in blood pressure even at moderate UVA doses. The effects were attributed to UVA induced release of NO from cutaneous photolabile NO derivates.

The release of "neutrophil extracellular traps" (NETs) has been identified as a novel immune response in innate immunity. NETs are composed of neutrophil-derived circulating free DNA (cf-DNA) and neutrophil cytoplasm-derived proteins such as proteases. In this study, we analyzed the putative diagnostic value of synovial cf-DNA/NETs for identification of septic arthritis. Forty-two patients with a joint effusion who had undergone arthrocentesis were included. From synovial fluid, cf-DNA/NETs (j-cf-DNA) levels were directly quantified. Diagnostic value of j-cf-DNA was compared with white blood cells (WBC), synovial white blood cells (j-WBC), C-reactive protein (CRP), j-IL-6, j-TNF alpha, j-IL-1 beta, and myeloperoxidase (j-MPO). Sensitivity, specificity, positive and negative predictive value, as well as ROC-curves for each parameter were calculated. Synovial fluid cf-DNA/NETs values from patients with septic arthritis (3,286 +/- 386 ng/ml, n = 9) were significantly increased compared to patients with noninfectious joint inflammation (1,040 +/- 208 ng/ml, n = 17) or osteoarthritis (278 +/- 34 ng/ml, n = 16, p0.01). In conjunction with j-cf-DNA, j-IL-6 and j-IL-1 beta were significantly elevated (p0.01), but WBC, CRP, and j-WBC were not. At a cut-off of 300 ng/ml, j-cf-DNA had a sensitivity of 0.89, a specificity of 1.0, a positive predictive value of 1.0, and a negative predictive value of 0.97. Receiver operation curves revealed largest areas under the curve for cf-DNA/NETs (0.933) and j-IL-6 (0.951). cf-DNA/NETs seem to be a valuable additional marker for the diagnosis of septic arthritis or periprosthetic infections. However, this result should be confirmed in a large clinical trial.

The inhalation of combustion-derived nanoparticles leads to adverse health effects in the airways. In this context the induction of membrane-coupled signalling is considered as causative for changes in tissue homeostasis and pro-inflammatory reactions. The identification of these molecular cell reactions allowed to seek for strategies which interfere with these adverse effects. In the current study, we investigated the structurally different compatible solutes mannosylglycerate (firoin) from thermophilic bacteria and ectoine from halophilic bacteria for their capability to reduce signalling pathways triggered by carbon nanoparticles in target cells in the lung. The pre-treatment of lung epithelial cells with both substances decreased the particle-specific activation of mitogen-activated protein kinases and also the endpoints proliferation and apoptosis. Firoin applied into the lungs of animals, like ectoine, led to a significant reduction of the neutrophilic lung inflammation induced by particle exposure. The pro-inflammatory effect of carbon nanoparticles on human neutrophil granulocytes ex vivo was significantly reduced by both substances via the reduction of the anti-apoptotic membrane-dependent signalling. The data of this study together with earlier studies demonstrate that two structurally non-related compatible solutes are able to prevent pathogenic reactions of the airways to carbon nanoparticles by interfering with signalling events. The findings highlight the preventive or therapeutic potential of compatible solutes for adverse health effects caused by particle exposure of the airways.

Many local hemodynamic and vascular disorders may be the result of impaired bioavailability of nitric oxide (NO). Previous findings point to a therapeutic potential of dermal NO application in the treatment of hemodynamic disorders, but no reliable data are available on the mechanisms, kinetics and biological responses of cutaneous NO exposure in humans in vivo. Here we show that due to its excellent diffusion capacity, cutaneously applied NO rapidly and in significant amounts penetrates the epidermal barrier, strongly enriched in skin tissue or blood plasma in the form of its vasoactive derivates, and in parallel significantly increased vasodilatation and blood flow, and reduced the thrombocyte aggregation capacity. Data presented for the first time give evidence that in humans dermal NO application has a strong potential in the therapy of local hemodynamic disorders arising from insufficient availability of NO or its bio-active derivates, respectively.

Introduction Deregulated apoptosis and overshooting neutrophil functions contribute to immune and organ dysfunction in sepsis and multiple organ failure (MOF). In the present study, we determined the role of soluble Fas (sFas) in the regulation of posttraumatic neutrophil extrinsic apoptosis and the development of sepsis. Methods Forty-seven major trauma patients, 18 with and 29 without sepsis development during the first 10 days after trauma, were enrolled in this prospective study. Seventeen healthy volunteers served as controls. Blood samples from severely injured patients were analyzed at day 1, day 5 and day 9 after major trauma. sFas levels, plasma levels of neutrophil elastase (PMNE) and levels of interleukin (IL)-6 were quantified by enzyme-linked immunosorbent assay and related to patients' Sequential Organ Failure Assessment (SOFA) score and Multiple Organ Dysfunction Score (MODS). Neutrophil apoptosis was determined by propidium iodide staining of fragmented DNA and flow cytometry. sFas-mediated effects on neutrophil apoptosis were investigated in cells cultured with agonistic anti-Fas antibodies in the presence of recombinant sFas, sFas-depleted serum or untreated serum from septic patients. Results Serum levels of sFas in patients who later developed sepsis were significantly increased at day 5 (P < 0.01) and day 9 (P < 0.05) after trauma compared with patients with uneventful recovery. Apoptosis of patient neutrophils was significantly decreased during the observation period compared with control cells. Moreover, Fas-mediated apoptosis of control neutrophils was efficiently inhibited by recombinant sFas and serum from septic patients. Depletion of sFas from septic patient sera diminished the antiapoptotic effects. In septic patients, sFas levels were positively correlated with SOFA at day 1 (r = 0.7, P < 0.001), day 5 (r = 0.62, P < 0.01) and day 9 (r = 0.58, P < 0.01) and with PMNE and leukocyte counts (r = 0.49, P < 0.05 for both) as well as MODS at day 5 (r = 0.56, P < 0.01) after trauma. Conclusions Increased sFas in patients with sepsis development impairs neutrophil extrinsic apoptosis and shows a positive correlation with the organ dysfunction scores and PMNE. Therefore, sFas might be a therapeutic target to prevent posttrauma hyperinflammation and sepsis.

Introduction Although the formation of neutrophil (PMN) extracellular traps (NETs) has been detected during infection and sepsis, their role in vivo is still unclear. This study was performed in order to evaluate the influence of NETs depletion by administration of recombinant human (rh)DNase on bacterial spreading, PMN tissue infiltration and inflammatory response in a mouse model of polymicrobial sepsis. Methods In a prospective controlled double-armed animal trial, polymicrobial sepsis was induced by cecal ligation and puncture (CLP). After CLP, mice were treated with rhDNase or phosphate buffered saline, respectively. Survival, colony forming unit (CFU) counts in the peritoneal cavity, lung, liver and blood were determined. PMN and platelet counts, IL-6 and circulating free (cf)-DNA/NETs levels were monitored. PMN infiltration, as well as organ damage, was analyzed histologically in the lungs and liver. Capability and capacity of PMN to form NETs were determined over time. Results cf-DNA/NETs were found to be significantly increased 6, 24, and 48 hours after CLP when compared to the levels determined in sham and naïve mice. Peak levels after 24 hours were correlated to enhanced capacity of bone marrow-derived PMN to form NETs after ex vivo stimulation with phorbol-12-myristate-13-acetate at the same time. rhDNase treatment of mice resulted in a significant reduction of cf-DNA/NETs levels 24 hours after CLP (P < 0.001). Although overall survival was not affected by rhDNase treatment, median survival after 24 hours was significantly lower when compared with the CLP group (P < 0.01). In mice receiving rhDNase treatment, CFU counts in the lung (P < 0.001) and peritoneal cavity (P < 0.05), as well as serum IL-6 levels (P < 0.001), were found to be already increased six hours after CLP. Additionally, enhanced PMN infiltration and tissue damage in the lungs and liver were found after 24 hours. In contrast, CFU counts in mice without rhDNase treatment increased later but more strongly 24 hours after CLP (P < 0.001). Similarly, serum IL-6 levels peaked after 24 hours (P < 0.01). Conclusions This study shows, for the first time, that depletion of NETs by rhDNase administration impedes the early immune response and aggravates the pathology that follows polymicrobial sepsis in vivo.