An Essential Role for SHARPIN in the Regulation of Caspase 1 Activity in Sepsis.

Sepsis is burdened by high mortality due to uncontrolled inflammatory response to pathogens. Increased caspase 1 activation causing maturation of IL1ß/18 remains a therapeutic challenge in sepsis. SHARPIN (shank-associated regulator of G-protein signaling homology domain-interacting protein), a component of the LUBAC (linear ubiquitin chain-assembly complex), regulates inflammation, with unknown effects on caspase 1 activation. Mice lacking Casp1, Casp11, or both in a Sharpin-deficient background were generated, exposed to lipopolysaccharide-induced endotoxemia, and injected with caspase 1 inhibitor. We monitored survival, Il1ß/18, and caspase 1/11 levels in plasma and organs and deciphered mechanisms of SHARPIN-dependent caspase 1 inhibition. A correlation between LUBAC and active caspase 1 was found in blood mononuclear cells from septic patients. SHARPIN bound caspase 1 and disrupted p20/p10 dimer formation, the last step of caspase 1 processing, thereby inhibiting enzyme activation and maturation of IL1ß/18 in a LUBAC-independent manner. In septic patients, LUBAC-independent decline in SHARPIN correlated with enhancement of active caspase 1 in circulating mononuclear cells. Septic Sharpin-deficient mice displayed enrichment in mature Il1ß/18 and active caspase 1, and shortened survival. Inhibition of caspase 1 reduced levels of Il1ß/18 and splenic cell death, and prolonged survival in septic Sharpin-deficient mice. Our findings identify SHARPIN as a potent in vivo caspase 1 inhibitor and propose the caspase 1-SHARPIN interaction as a target in sepsis.

Attenuation of myocardial injury by HMGB1 blockade during ischemia/reperfusion is toll-like receptor 2-dependent.

Genetic or pharmacological ablation of toll-like receptor 2 (TLR2) protects against myocardial ischemia/reperfusion injury (MI/R). However, the endogenous ligand responsible for TLR2 activation has not yet been detected. The objective of this study was to identify HMGB1 as an activator of TLR2 signalling during MI/R. C57BL/6 wild-type (WT) or TLR2(-/-)-mice were injected with vehicle, HMGB1, or HMGB1 BoxA one hour before myocardial ischemia (30 min) and reperfusion (24 hrs). Infarct size, cardiac troponin T, leukocyte infiltration, HMGB1 release, TLR4-, TLR9-, and RAGE-expression were quantified. HMGB1 plasma levels were measured in patients undergoing coronary artery bypass graft (CABG) surgery. HMGB1 antagonist BoxA reduced cardiomyocyte necrosis during MI/R in WT mice, accompanied by reduced leukocyte infiltration. Injection of HMGB1 did, however, not increase infarct size in WT animals. In TLR2(-/-)-hearts, neither BoxA nor HMGB1 affected infarct size. No differences in RAGE and TLR9 expression could be detected, while TLR2(-/-)-mice display increased TLR4 and HMGB1 expression. Plasma levels of HMGB1 were increased MI/R in TLR2(-/-)-mice after CABG surgery in patients carrying a TLR2 polymorphism (Arg753Gln). We here provide evidence that absence of TLR2 signalling abrogates infarct-sparing effects of HMGB1 blockade.

In vivo electrophysiological characterization of TASK-1 deficient mice.

BACKGROUND/AIMS: TASK-1 is a potassium channel predominantly expressed in heart and brain. We have previously shown that anesthetized TASK-1(-/-)mice have prolonged QT intervals in surface electrocardiograms (ECGs). In addition, heart rate variability quantified by time and frequency domain parameters was significantly altered in TASK-1(-/-) mice with a sympathetic preponderance. Aims of the present study were the analysis of QT intervals by telemetric ECGs, to determine potential influences of anesthesia and ß-adrenergic stimulation on repolarization in surface ECGs, to investigate in vivo electrophysiological parameters by intracardiac electrical stimulation and to quantify heart rate turbulence after ischemia/reperfusion or ventricular pacing in TASK-1(+/+) and TASK-1(-/-) mice. METHODS: Rate corrected QT intervals (QTc) were recorded in conscious mice by telemetry and in surface ECGs following administration of various anesthetics (tribromoethanol (Avertin(®)), pentobarbital and isoflurane). TASK-1(+/+) and TASK-1(-/-) mice were characterized by programmed electrical stimulation using an intracardiac octapolar catheter. The baroreceptor reflex was analyzed by heart rate turbulence (turbulence onset and slope) after ischemia/reperfusion and by stimulated premature ventricular contractions. RESULTS: Telemetric and surface ECGs in mice sedated with Avertin(®) and pentobarbital, showed a significantly lengthened rate corrected QT interval in TASK-1(-/-) mice (telemetry: TASK-1(+/+) 43±3ms vs. TASK-1(-/-) 49±5ms, n=6, p<0.05; Avertin(®): TASK-1(+/+) 36±8ms vs. TASK-1(-/-) 48±4ms, n=13/16, p<0.0001). The prolongation of the QT interval was most pronounced at lower heart rates. Isoflurane, known for its stimulatory effects on the TASK channel family, attenuated the rate corrected QT interval prolongation in TASK-1(-/-)mice. Intracardiac electrical stimulation revealed normal values for electrical conduction and refractoriness. No significant arrhythmias after atrial and ventricular burst stimulation were induced before and after adrenergic challenge in both genotypes. Turbulence onset after premature ventricular contraction was significantly altered in TASK-1(-/-) mice. CONCLUSION: TASK-1(-/-) mice exhibit a phenotype of QT prolongation, which distinct relation to heart rate. TASK-1 deficiency does neither alter key electrophysiological parameters nor increases atrial/ventricular vulnerability after electrical stimulation. The heart rate response after premature ventricular contractions is significantly abolished indicating a diminished baroreceptor reflex in TASK-1(-/-) mice.

Systemic inflammation after aortic cross clamping is influenced by Toll-like receptor 2 preconditioning and deficiency.

BACKGROUND: The perioperative morbidity and mortality of abdominal aortic aneurysm repair is linked to systemic inflammation. Important triggers of the latter are Toll-like receptors (TLRs), which play a central role in innate immunity. Ischemia/reperfusion (I/R) injury can be influenced by either TLR stimulation before I/R (preconditioning) or TLR dysfunction (deficiency or polymorphism). The influence of TLR2 stimulation or deficiency on systemic cytokine release and organ damage after aortic cross clamping has not been evaluated yet. METHODS: Wild type (WT) and TLR2-deficient mice were subjected to 1 h ischemia and 2 or 4 h reperfusion of the infrarenal aorta. One group of WT mice was preconditioned with the synthetic TLR2 agonist Pam(3)Cys-Ser-Lys(4) (Pam(3)CSK(4)). Sham-operated animals without I/R served as controls. Plasma levels of interleukin (IL)-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, granulocyte macrophage-colony stimulating factor, tumor necrosis factor-α, alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH), and creatinine were measured. RESULTS: I/R injury caused by transient clamping of the infrarenal aorta led to a time-dependent increase of all measured cytokines except IL-4, IL-5, and granulocyte macrophage-colony stimulating factor. This was accompanied by elevated markers of organ damage (ALT, AST, and LDH) except creatinine. Preconditioning with Pam(3)CSK(4) led to lower plasma concentrations of all cytokines, with the exception of anti-inflammatory IL-10 which was significantly upregulated. Furthermore, ALT, AST, and LDH plasma concentrations were lower in the preconditioning group. TLR2-deficient mice similarly showed reduced signs of systemic inflammation and organ damage, although less distinctive. IL-2, IL-6, IL-12, ALT, and LDH were time dependently lower compared with WT mice. IL-10 concentration was higher in TLR2-deficient mice compared with WT. CONCLUSIONS: Both, preconditioning via TLR2 and TLR2 deficiency, ameliorate systemic inflammation and organ damage during I/R injury caused by clamping of the infrarenal aorta. Compared with untreated WT animals, Pam(3)CSK(4) preconditioned and TLR2-deficient mice showed lower concentrations of pro-inflammatory cytokines, whereas anti-inflammatory IL-10 was elevated in both groups. This was accompanied by reduced organ dysfunction parameters.

Evaluation of a Clinical Decision Support System for the most evidence-based approach to managing perioperative anticoagulation.

STUDY OBJECTIVE: We explored the feasibility of a Clinical Decision Support System (CDSS) to guide evidence-based perioperative anticoagulation. DESIGN: Prospective randomised clinical management simulation multicentre study. SETTING: Five University and 11 general hospitals in Germany. PARTICIPANTS: We enrolled physicians (anaesthesiologist (n = 73), trauma surgeons (n = 2), unknown (n = 1)) with different professional experience. INTERVENTIONS: A CDSS based on a multiple-choice test was developed and validated at the University Hospital of Frankfurt (phase-I). The CDSS comprised European guidelines for the management of anticoagulation in cardiology, cardio-thoracic, non-cardio-thoracic surgery and anaesthesiology. Phase-II compared the efficiency of physicians in identifying evidence-based approach of managing perioperative anticoagulation. In total 168 physicians were randomised to CDSS (PERI-KOAG) or CONTROL. MEASUREMENTS: Overall mean score and association of processing time and professional experience were analysed. The multiple-choice test consists of 11 cases and two correct answers per question were required to gain 100% success rate (=22 points). MAIN RESULTS: In total 76 physicians completed the questionnaire (n = 42 PERI-KOAG; n = 34 CONTROL; attrition rate 54%). Overall mean score (max. 100% = 22 points) was significantly higher in PERI-KOAG compared to CONTROL (82 ± 15% vs. 70 ± 10%; 18 ± 3 vs. 15 ± 2 points; P = 0.0003). A longer processing time is associated with significantly increased overall mean scores in PERI-KOAG (≥33 min. 89 ± 10% (20 ± 2 points) vs. <33 min. 73 ± 15% (16 ± 3 points), P = 0.0005) but not in CONTROL (≥33 min. 74 ± 13% (16 ± 3 points) vs. <33 min. 69 ± 9% (15 ± 2 points), P = 0.11). Within PERI-KOAG, there is a tendency towards higher results within the more experienced group (>5 years), but no significant difference to less (≤5 years) experienced colleagues (87 ± 10% (19 ± 2 points) vs. 78 ± 17% (17 ± 4 points), P = 0.08). However, an association between professional experience and success rate in CONTROL has not been shown (71 ± 8% vs. 70 ± 13%, 16 ± 2 vs. 15 ± 3 points; P = 0.66). CONCLUSIONS: CDSS significantly improved the identification of evidence-based treatment approaches. A precise usage of CDSS is mandatory to maximise efficiency.

Left ventricular dilation in toll-like receptor 2 deficient mice after myocardial ischemia/reperfusion through defective scar formation.

Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors (TLRs). TLR2(-/-)-mice show short-term advantages upon reperfusion injury as compared with WT controls. Accordingly, it has been shown that transient TLR2-blockade prior to reperfusion is associated with improved left-ventricular performance after myocardial scar formation. We present here adverse myocardial remodeling due to a chronic lack of TLR2 expression. Myocardial ischemia/reperfusion (MI/R) was surgically induced in C3HeN-mice by ligation of the left anterior descending coronary artery for 20 min, followed by 24 h or 28 days of reperfusion. TLR2(-/-)-mice and TLR2-Ab treated (T2.5) WT-mice displayed a reduction of infarct size, plasma troponin T concentrations, and leukocyte infiltration as compared with untreated controls after 24 h of reperfusion. After 28 days, however, magnetic resonance imaging revealed a marked left ventricular dilation in TLR2(-/-)-animals, which was associated with pronounced matrix remodeling characterized by reduced collagen and decorin density in the infarct scar. Our data show adverse effects on myocardial remodeling in TLR2(-/-)-mice. Although interception with TLR2 signaling is a promising concept for the prevention of reperfusion injury after myocardial ischemia, these data give cause for serious concern with respect to the time-point and duration of the potential treatment.

Functional role of TASK-1 in the heart: studies in TASK-1-deficient mice show prolonged cardiac repolarization and reduced heart rate variability.

TASK-1, a member of the recently identified K2P channel family, is mainly expressed in the heart and the nervous system. TASK-1 is regulated by several physiological and pathological conditions and functions as a background potassium channel. However, there are limited data concerning the significance of TASK-1 in cardiac physiology. We studied the functional role of TASK-1 in the heart by cardiac phenotyping the TASK-1-deficient mouse (TASK-1(-/-)). TASK-1 was predominantly expressed in the ventricles of control animals. Real-time PCR and immunoblot demonstrated that the expression of seven other K2P channels was unchanged in TASK-1(-/-) mice. No structural or functional abnormalities were found by histology and echocardiography. Electrophysiological studies recording monophasic action potentials (MAPs) showed a significant prolongation of action potential duration in spontaneously beating and atrially paced hearts, respectively. Surface ECGs of TASK-1(-/-) mice revealed a significant prolongation of the rate corrected QT interval. Telemetric ECG recordings for 24 h, during physical and pharmacological stress testing and after ischemia/reperfusion injury did not result in a higher incidence of arrhythmias. Infarct size was comparable in both genotypes. However, TASK-1(-/-) mice had a higher mean heart rate and significantly reduced heart rate variability (HRV). Time and frequency domain measurements as well as baroreceptor reflex testing revealed a sympathovagal imbalance with a shift to an increase in sympathetic influence in TASK-1(-/-) mice. In conclusion, TASK-1 plays a functional role in the repolarization of the cardiac action potential in vivo and contributes to the maintenance of HRV.

Cerebrospinal Fluid Concentrations of Meropenem and Vancomycin in Ventriculitis Patients Obtained by TDM-Guided Continuous Infusion.

Effective antibiotic therapy of cerebral infections such as meningitis or ventriculitis is hindered by low penetration into the cerebrospinal fluid (CSF). Because continuous infusion of meropenem and vancomycin and routine therapeutic drug monitoring (TDM) have been proposed to optimize antimicrobial exposure in ventriculitis patients, an individualized dosing strategy was implemented in our department. We present a retrospective analysis of meropenem and vancomycin concentrations in serum and CSF in the first nine ventriculitis patients treated with continuous infusion and TDM-guided dose optimization aiming at 20-30 mg/L. Median initial dosing was 8.8 g/24 h meropenem and 4.25 g/24 h vancomycin, respectively, resulting in median serum concentrations of 21.3 mg/L for meropenem and 24.5 mg/L for vancomycin and CSF concentrations of 3.4 mg/L for meropenem and 1.7 mg/L for vancomycin. Median CSF penetration was 15% for meropenem and 7% for vancomycin. With initial dosing, all but one patient achieved CSF concentrations above 1 mg/L. Dose adjustment according to TDM ensured sufficient CSF concentrations in all patients within 48 h of treatment. Given the limited penetration, continuous infusion of meropenem and vancomycin based on renal function and TDM-guided dose optimization appears a reasonable approach to attain sufficient CSF concentrations in ventriculitis patients.

Toll-like receptor 2 signaling triggers fatal arrhythmias upon myocardial ischemia-reperfusion.

OBJECTIVE: Restoration of myocardial blood flow after ischemia triggers an inflammatory response involving toll-like receptors. Toll-like receptor 2 deficiency is associated with a reduced infarct size after myocardial ischemia and reperfusion. Because a marked mortality was observed in C3HeN wild-type mice, which was absent in TLR2 mice, we tested whether cardiac arrhythmias are the underlying pathology and aimed to elucidate how toll-like receptor 2 ligation might prevent lethal arrhythmias. DESIGN: Experimental animal model. SETTING: University hospital research laboratory. SUBJECTS: Male C3HeN mice. INTERVENTIONS: Myocardial ischemia and reperfusion was surgically induced by ligation of the left anterior descending coronary artery for 20 mins followed by 24 hrs of reperfusion. Electrocardiography was continuously recorded during the observation period through an implantable telemetry transmitter to detect cardiac arrhythmias during reperfusion. MEASUREMENTS AND MAIN RESULTS: Toll-like receptor 2 expression was associated with a 51% mortality rate (23 of 45 mice died) after myocardial ischemia and reperfusion. Absence of toll-like receptor 2 improved survival toward 100% (17 of 17 mice survived). Electrocardiography diagnostics in conscious animals and histologic analysis revealed that absence of toll-like receptor 2 signaling prevented the formation of pathologic heart rate turbulence after myocardial ischemia and reperfusion and modulated the density of connexin 43-positive gap junctions in the ischemic area compared with wild-type hearts, indicating arrhythmia as the cause underlying the observed mortality. CONCLUSIONS: The results presented here indicate toll-like receptor 2 as a novel target for the prevention of lethal arrhythmic complications after myocardial ischemia and reperfusion.

Preconditioning by toll-like receptor 2 agonist Pam3CSK4 reduces CXCL1-dependent leukocyte recruitment in murine myocardial ischemia/reperfusion injury.

OBJECTIVE: To test whether preconditioning with a toll-like receptor (TLR) 2 agonist protects against myocardial ischemia and reperfusion by interfering with chemokine CXCL1 release from cardiomyocytes. DESIGN: C3H mice were challenged with vehicle or synthetic TLR2 agonist Pam3Cys-Ser-Lys4 (Pam3CSK4; 1 mg/kg) 24 hrs before myocardial ischemia (20 mins) and reperfusion (2 hrs or 24 hrs). Infarct size, troponin T release, and leukocyte recruitment were quantified. In murine cardiomyocytes (HL-1), we studied the expression/activation profile of TLR2 in response to stimulation with Pam3CSK4 (0.01-1 mg/mL). Furthermore, we studied the chemokine ligand 1 (CXCL1) response to Pam3CSK4 and ischemia/reperfusion in vivo and in vitro. SETTING: University hospital research laboratory. SUBJECTS: Anesthetized male mice and murine cardiomyocytes. MEASUREMENTS AND MAIN RESULTS: Preconditioning by Pam3CSK4 reduced infarct size and troponin T release. This was accompanied by a decreased recruitment of leukocytes into the ischemic area and an improved cardiac function. In HL-1 cells, TLR2 activation amplified the expression of the receptor in a time-dependent manner and led to CXCL1 release in a concentration-dependent manner. Preconditioning by Pam3CSK4 impaired CXCL1 release in response to a second inflammatory stimulus in vivo and in vitro. CONCLUSIONS: Preconditioning by TLR2 agonist Pam3CSK4 reduces myocardial infarct size after myocardial ischemia/reperfusion. One of the mechanisms involved is a diminished chemokine release from cardiomyocytes, which subsequently limits leukocyte infiltration.

Intravenous sphingosylphosphorylcholine protects ischemic and postischemic myocardial tissue in a mouse model of myocardial ischemia/reperfusion injury.

HDL, through sphingosine-1-phosphate (S1P), exerts direct cardioprotective effects on ischemic myocardium. It remains unclear whether other HDL-associated sphingophospholipids have similar effects. We therefore examined if HDL-associated sphingosylphosphorylcholine (SPC) reduces infarct size in a mouse model of transient myocardial ischemia/reperfusion. Intravenously administered SPC dose-dependently reduced infarct size after 30 minutes of myocardial ischemia and 24 hours reperfusion compared to controls. Infarct size was also reduced by postischemic, therapeutical administration of SPC. Immunohistochemistry revealed reduced polymorphonuclear neutrophil recruitment to the infarcted area after SPC treatment, and apoptosis was attenuated as measured by TUNEL. In vitro, SPC inhibited leukocyte adhesion to TNFα-activated endothelial cells and protected rat neonatal cardiomyocytes from apoptosis. S1P3 was identified as the lysophospholipid receptor mediating the cardioprotection by SPC, since its effect was completely absent in S1P3-deficient mice. We conclude that HDL-associated SPC directly protects against myocardial reperfusion injury in vivo via the S1P3 receptor.

TLR2-Dependent Reversible Oxidation of Connexin 43 at Cys260 Modifies Electrical Coupling After Experimental Myocardial Ischemia/Reperfusion.

We have shown previously that during myocardial ischemia/reperfusion (MI/R), toll-like receptor 2 (TLR2) signaling regulates connexin 43 (Cx43) subcellular localization and function and dampens arrhythmia formation. We aimed to identify sites capable of TLR2-dependent redox modification within Cx43. Post-ischemic TLR2-/- or wild-type (WT) mouse hearts were analyzed by OxICAT. Cx43 was mutated to exclude redox modification and transfected into HL-1 cardiomyocytes (CM) that were challenged with a TLR2 agonist. We identified Cys260 of Cx43 to be susceptible to reversible oxidation MI/R; TLR2-/- leads to reduced H2O2 production in post-ischemic isolated mitochondria and subsequently reduced oxidation of Cx43 at Cys260. Cx43 was dephosphorylated in WT, while phosphorylation was preserved in TLR2-/-. Mutation of Cx43 (C260A) and lentiviral transfection in HL-1 CM accelerated pacemaker activity and reduced activity after TLR2 ligand stimulation. We here provide evidence for TLR2-dependent reversible oxidation of Cx43 at Cys260, which led to decreased Cx43 phosphorylation and affected CM pacemaker frequency and intercellular communication.

Rosiglitazone is cardioprotective in a murine model of myocardial I/R.

The peroxisome proliferator-activated receptor gamma (PPAR-gamma) is a regulator of anti-inflammatory genes. One of its agonists, rosiglitazone-widely used in the treatment of type 2 diabetes mellitus-has recently been reported to increase the risk for myocardial infarction. In contrast, various studies provide evidence for a rosiglitazone-induced cardioprotection in different models of acute myocardial I/R. Here, we report that this protection can still be observed after 28 days of reperfusion in a murine model even when treatment commenced after the period of ischemia (reperfusion therapy). In vitro, cells from the rat cardiomyoblast cell line H9c2(2-1) are protected against oxidative stress by incubation with rosiglitazone, which can be abrogated by dexamethasone or cycloheximide. The antioxidant enzyme heme oxygenase 1 is up-regulated in these cells after rosiglitazone treatment. Our data provide further evidence that rosiglitazone exerts protective effects during myocardial I/R and might contribute to the reevaluation of the approved drug rosiglitazone.

Caspase inhibitor zVAD.fmk reduces infarct size after myocardial ischaemia and reperfusion in rats but not in mice.

OBJECTIVE: Apoptosis of cardiomyocytes has been suggested to contribute to outcome following myocardial ischaemia and reperfusion (MI/R). Caspase inhibitors were developed as potential therapeutics for MI/R. However, various reports using the broad-spectrum caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD.fmk) in the latter setting present conflicting results. Therefore, it is still unclear whether inhibition of apoptosis by caspase inhibitors promotes cardioprotection. MATERIALS AND METHODS: This study evaluated whether zVAD.fmk or novel caspase inhibitor quinoline-Val-Asp(Ome)-CH2-O-phenoxy (Q-VD-OPh) reduce myocardial infarct size in mice. Secondly, we tested zVAD.fmk's potential infarct-sparing effects in rats and whether these are accompanied by improved left ventricular function. RESULTS: In mice neither zVAD.fmk nor Q-VD-OPh reduced infarct size. In rats, however, zVAD.fmk reduced infarct size following ischaemia (25min) and reperfusion (7 days) by approximately 53%. This was, however, accompanied by an increase in left ventricular end-diastolic pressure. CONCLUSION: This study provides further evidence that abrogation of apoptosis via caspase inhibition might not be sufficient to effectively limit infarct size following MI/R.

Lipoteichoic acid induces delayed myocardial protection in isolated rat hearts: a comparison with endotoxin.

OBJECTIVE: Preconditioning with bacterial wall fragments lipopolysaccharide (LPS) or lipoteichoic acid (LTA) reduce myocardial infarct size after ischaemia and reperfusion (I/R) in rats. Preconditioning with LTA reduces neutrophil accumulation during reperfusion and thereby ameliorates one of myocardial reperfusion injury's most important mechanisms. In this study, we use an ex vivo model of regional myocardial I/R to investigate LTA versus LPS induced preconditioning in a system devoid of leukocytes. METHODS: Rats were subjected to LTA or LPS with or without dexamethasone pre-treatment. Twenty-four hours after LTA or LPS challenge, hearts were removed and retrogradely perfused in a Langendorff set-up. Hearts underwent 20 min of regional ischaemia followed by 2 h of reperfusion. Ischaemic preconditioning (IPC) was performed as a positive control. Myocardial infarct size was determined as the primary end-point. RESULTS: LTA and LPS preconditioning both lead to a marked reduction in infarct size similar to IPC, however, no significant differences were found between LTA and LPS. The reduction in infarct size was abrogated by dexamethasone pre-treatment. CONCLUSION: We conclude that preconditioning with LTA and likewise with LPS confers myocardial protection in an ex vivo setting devoid of leukocytes. Dexamethasone inhibits preconditioning, suggesting that the underlying mechanism is dependent upon induction of a systemic inflammatory response to a LTA or LPS stimulus.

Systemic TLR2 Antibody Application in Renal Ischaemia and Reperfusion Injury Decreases AKT Phosphorylation and Increases Apoptosis in the Mouse Kidney.

Acute kidney injury remains an important cause of renal dysfunction. In this context, Toll-like receptors have been demonstrated to play a critical role in the induction of innate and inflammatory responses. Among these, Toll-like receptor 2 (TLR2) is constitutively expressed in tubular epithelial cells (TECs) of the kidney and is also known to mediate ischaemia reperfusion (IR) injury. Adult male C57BL/6JRj mice were randomized into seven groups (n = 8): a non-operative control group (CTRL) and six interventional groups in which mice were subjected to a 30 min. bilateral renal ischaemia. Immediately before reperfusion, mice were treated either with saline or with TLR2 antibody (clone T2.5) and harvested after ischaemia and reperfusion for 3, 24 and 48 hr. Analysed kidney homogenates of TLR2 antibody-treated mice displayed significantly decreased levels of TLR2 protein after 3 hr of IR compared to saline-treated mice. Accordingly, the degree of AKT phosphorylation was significantly decreased after 3 hr of IR compared to saline-treated animals. TUNEL staining revealed significantly higher apoptosis rates in TLR2 antibody-treated animals compared to saline-treated mice after 3 and 24 hr of IR. Further, a positive correlation between TLR2 protein expression and phosphorylation of AKT as well as a negative correlation with the number of TUNEL-positive cells could be observed. Inhibition of TLR2 and its signalling pathway by a single application of TLR2 antibody results in reduced phosphorylation of AKT and consecutively increased apoptosis.

High-density lipoproteins and their constituent, sphingosine-1-phosphate, directly protect the heart against ischemia/reperfusion injury in vivo via the S1P3 lysophospholipid receptor.

BACKGROUND: All treatments of acute myocardial infarction are aimed at rapid revascularization of the occluded vessel; however, no clinical strategies are currently available to protect the heart from ischemia/reperfusion injury after restitution of blood flow. We hypothesized that some of the cholesterol transport-independent biological properties of high-density lipoprotein (HDL) implied in atheroprotection may also be beneficial in settings of acute myocardial reperfusion injury. METHODS AND RESULTS: In an in vivo mouse model of myocardial ischemia/reperfusion, we observed that HDL and its sphingolipid component, sphingosine-1-phosphate (S1P), dramatically attenuated infarction size by approximately 20% and 40%, respectively. The underlying mechanism was an inhibition of inflammatory neutrophil recruitment and cardiomyocyte apoptosis in the infarcted area. In vitro, HDL and S1P potently suppressed leukocyte adhesion to activated endothelium under flow and protected rat neonatal cardiomyocytes against apoptosis. In vivo, HDL- and S1P-mediated cardioprotection was dependent on nitric oxide (NO) and the S1P3 lysophospholipid receptor, because it was abolished by pharmacological NO synthase inhibition and was completely absent in S1P3-deficient mice. CONCLUSIONS: Our data demonstrate that HDL and its constituent, S1P, acutely protect the heart against ischemia/reperfusion injury in vivo via an S1P3-mediated and NO-dependent pathway. A rapid therapeutic elevation of S1P-containing HDL plasma levels may be beneficial in patients at high risk of acute myocardial ischemia.

CpG oligonucleotide activates Toll-like receptor 9 and causes lung inflammation in vivo.

BACKGROUND: Bacterial DNA containing motifs of unmethylated CpG dinucleotides (CpG-ODN) initiate an innate immune response mediated by the pattern recognition receptor Toll-like receptor 9 (TLR9). This leads in particular to the expression of proinflammatory mediators such as tumor necrosis factor (TNF-alpha) and interleukin-1beta (IL-1beta). TLR9 is expressed in human and murine pulmonary tissue and induction of proinflammatory mediators has been linked to the development of acute lung injury. Therefore, the hypothesis was tested whether CpG-ODN administration induces an inflammatory response in the lung via TLR9 in vivo. METHODS: Wild-type (WT) and TLR9-deficient (TLR9-D) mice received CpG-ODN intraperitoneally (1668-Thioat, 1 nmol/g BW) and were observed for up to 6 hrs. Lung tissue and plasma samples were taken and various inflammatory markers were measured. RESULTS: In WT mice, CpG-ODN induced a strong activation of pulmonary NFkappaB as well as a significant increase in pulmonary TNF-alpha and IL-1beta mRNA/protein. In addition, cytokine serum levels were significantly elevated in WT mice. Increased pulmonary content of lung myeloperoxidase (MPO) was documented in WT mice following application of CpG-ODN. Bronchoalveolar lavage (BAL) revealed that CpG-ODN stimulation significantly increased total cell number as well as neutrophil count in WT animals. In contrast, the CpG-ODN-induced inflammatory response was abolished in TLR9-D mice. CONCLUSION: This study suggests that bacterial CpG-ODN causes lung inflammation via TLR9.

Takotsubo Cardiomyopathy Triggered by Venous Air Embolism During Craniotomy in the Sitting Position.

BACKGROUND: We present a case of stress-induced cardiomyopathy (Takotsubo cardiomyopathy) caused by a venous air embolism during a craniotomy performed in the sitting position. CASE DESCRIPTION: A 69-year-old woman was admitted to the neurosurgical department and scheduled for elective resection of a cerebellar metastasis in the sitting position. After craniotomy and opening of the posterior fossa, a venous air embolism was detected via transesophageal echocardiography. The patient immediately presented with cardiac decompensation with signs of takotsubo or stress-induced cardiomyopathy. CONCLUSIONS: Intensivists and anesthesiologists in the operating room and in intensive care units need to be aware of stress-induced cardiomyopathy as a probably underdiagnosed disease entity, especially as management differs significantly from other forms of cardiogenic shock. Diagnosis can be accomplished quickly by bedside echocardiography, emphasizing the need for availability of this tool and the integration of stress-induced cardiomyopathy in diagnostic algorithms in the intensive care unit.

The Role of ABO Blood Group in Cerebral Vasospasm, Associated Intracranial Hemorrhage, and Delayed Cerebral Ischemia in 470 Patients with Subarachnoid Hemorrhage.

OBJECTIVE: Rupture of an intracranial aneurysm usually presents with an acute onset and requires multidisciplinary intensive care treatment and the overall death and disability rates are high. The ABO blood type is known to play an important role in hemostasis, thrombosis, and vascular NO response. The aspect of ABO blood type in onset, clinical progress, and outcome after subarachnoid hemorrhage (SAH) is largely unexplored. We conducted this study to elucidate the association of ABO blood type with the occurrence and outcome of aneurysmal SAH. METHODS: In our retrospective study, 470 patients with aneurysmal SAH treated at our institution were included. We performed a χ2 test for comparison between blood types and World Federation of Neurosurgical Societies admission status, cerebral vasospasm, delayed infarction, associated intracerebral hemorrhage and Fisher grade for analysis for their association with SAH. RESULTS: No significant difference between blood type and the reviewed variables for SAH outcome were identified: World Federation of Neurosurgical Societies admission status (odds ratio, 1.12; 95% confidence interval [CI], 0.7-1.6; P = 0.56); SAH-associated intracerebral hemorrhage (odds ratio, 0.81; 95% CI, 0.5-1.3; P = 0.36); cerebral vasospasm (odds ratio, 1.08; 95% CI, 0.7-1.6; P = 0.71); DCI (odds ratio, 1.23; 95% CI, 0.8-1.8; P = 0.30); Fisher grade (odds ratio, 1.13; 95% CI, 0.7-1.6; P = 0.19). CONCLUSIONS: Although a possible relationship between the ABO blood group and the clinical course of patients with SAH was hypothesized, our study showed no significant influence of patient's ABO blood type on cerebral vasospasm onset, SAH-associated intracerebral hemorrhage, or delayed infarction.