Ketamine, an NMDA receptor (NMDA-R) antagonist, produces psychotomimetic effects when administered in sub-anesthetic dosages. While previous research suggests that Ketamine alters the excitation/inhibition (E/I)-balance in cortical microcircuits, the precise neural mechanisms by which Ketamine produces these effects are not well understood. We analyzed resting-state MEG data from n = 12 participants who were administered Ketamine to assess changes in gamma-band (30-90 Hz) power and the slope of the aperiodic power spectrum compared to placebo. In addition, correlations of these effects with gene-expression of GABAergic interneurons and NMDA-Rs subunits were analyzed. Finally, we compared Ketamine-induced spectral changes to the effects of systematically changing NMDA-R levels on pyramidal cells, and parvalbumin-, somatostatin- and vasoactive intestinal peptide-expressing interneurons in a computational model of cortical layer-2/3 to identify crucial sites of Ketamine action. Ketamine resulted in a flatter aperiodic slope and increased gamma-band power across brain regions, with pronounced effects in prefrontal and central areas. These effects were correlated with the spatial distribution of parvalbumin and GluN2D gene expression. Computational modeling revealed that reduced NMDA-R activity in parvalbumin or somatostatin interneurons could reproduce increased gamma-band power by increasing pyramidal neuron firing rate, but did not account for changes in the aperiodic slope. The results suggest that parvalbumin and somatostatin interneurons may underlie increased gamma-band power following Ketamine administration in healthy volunteers, while changes in the aperiodic component could not be recreated. These findings have implications for current models of E/I-balance, as well as for understanding the mechanisms underlying the circuit effects of Ketamine.

Die akzidentelle Wachheit während der Allgemeinanästhesie ist eine seltene Komplikation, die jedoch zu schweren psychologischen Folgeerscheinungen führen kann. Dieser Fortbildungsbeitrag beleuchtet die Inzidenz der Komplikation, beschreibt das klinische Bild, die möglichen Ursachen, einige Diagnoseverfahren, das postoperative Management und Möglichkeiten der Prävention.

Accidental awareness during general anaesthesia (AAGA) is a rare but severe complication. The reported incidence of AAGA may depend on the assessment of intraoperative awareness with explicit recall and there are substantial variations between subspecialties and groups of patients. The majority of prospective studies using structured interviews reported an incidence of AAGA at 0.1-0.2% during general anaesthesia, however, higher values were observed in paediatric (0.2-1.2%) and obstetric patients (0.47%). Risk factors that predispose to AAGA are patient conditions, ASA status, female gender, patient age, history of AAGA, surgical procedure, anaesthetic drug type, muscle relaxation, dosages of hypnotic or analgesic drugs, monitoring and malfunction of anaesthesia systems. Preventive strategies include careful assessment of risk factors, avoidance of underdosages of hypnotics and analgetics during general anaesthesia and monitoring of depth of anaesthesia in risk patients. The health-related consequences can be serious and psychopharmacological and psychotherapeutic interventions are indicated in patients who have experienced AAGA.

Propofol belongs to a class of molecules that are known to block learning and memory in mammals, including rodents and humans. Interestingly, learning and memory are not tied to the presence of a nervous system. There are several lines of evidence indicating that single-celled organisms also have the capacity for learning and memory which may be considered as basal intelligence. Here, we introduce a new experimental model for testing the learning ability of Physarum polycephalum, a model organism frequently used to study single-celled "intelligence". In this study, the impact of propofol on Physarum's "intelligence" was tested. The model consists of a labyrinth of subsequent bifurcations in which food (oat flakes soaked with coconut oil-derived medium chain triglycerides [MCT] and soybean oil-derived long chain triglycerides [LCT]) or propofol in MCT/LCT) is placed in one of each Y-branch. In this setting, it was tested whether Physarum memorized the rewarding branch. We saw that Physarum was a quick learner when capturing the first bifurcations of the maze; thereafter, the effect decreased, perhaps due to reaching a state of satiety. In contrast, when oat flakes were soaked with propofol, Physarum's preference for oat flakes declined significantly. Several possible actions, including the blocking of gamma-aminobutyric acid (GABA) receptor signaling, are suggested to account for this behavior, many of which can be tested in our new model.

Objectives Postoperative delirium (POD) is a common complication after elective cardiac surgery. Recent evidence indicates that a disruption in the normal activity of the cholinergic system may be associated with delirium. Design Prospective observational study. Setting Single-centre at a European academic hospital. Primary and secondary outcome measures In our study the enzyme activities of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) were determined preoperatively as well as on the first and second postoperative day. The confusion assessment method for the intensive care unit was used to screen patients for the presence of POD. Results A total of 114 patients were included in the study. POD was associated with a decrease in BChE activity on postoperative day 1 (p=0.03). In addition, patients who developed POD, had significantly lower preoperative AChE activity than patients without POD (p<0.01). Multivariate analysis identified a preoperatively decreased AChE activity (OR 3.1; 95% CI 1.14 to 8.46), anticholinergic treatment (OR 5.09; 95% CI 1.51 to 17.23), elevated European System for Cardiac Operative Risk Evaluation (OR 3.68; 95% CI 1.04 to 12.99) and age (OR 3.02; 95% CI 1.06 to 8.62) to be independently associated with the development of POD. Conclusions We conclude that a reduction in the acetylcholine hydrolysing enzyme activity in patients undergoing cardiac surgery may correlate with the development of POD.

Early and adequate restoration of endothelial and tubular renal function is a substantial step during regeneration after ischemia reperfusion (IR) injury, occurring, e.g., in kidney transplantation, renal surgery, and sepsis. While tubular epithelial cell injury has long been of central importance, recent perception includes the renal vascular endothelium. In this regard, the fibrin cleavage product fibrinopeptide Bβ15-42 mitigate IR injury by stabilizing interendothelial junctions through its affinity to VE-cadherin. Therefore, this study focused on the effect of Bβ15-42 on post-acute physiological renal regeneration. For this, adult male C57BL/6 mice were exposed to a 30 min bilateral renal ischemia and reperfusion for 24 h or 48 h. Animals were randomized in a non-operative control group, two operative groups each treated with i.v. administration of either saline or Bβ15-42 (2.4 mg/kg) immediately prior to reperfusion. Endothelial activation and inflammatory response was attenuated in renal tissue homogenates by single application of Bβ15-42. Meanwhile, Bβ15-42 did not affect acute kidney injury markers. Regarding the angiogenetic players VEGF-A, Angiopoietin-1, Angiopoietin-2, however, we observed significant higher expressions at mRNA and trend to higher protein level in Bβ15-42 treated mice, compared to saline treated mice after 48 h of IR, thus pointing toward an increased angiogenetic activity. Similar dynamics were observed for the intermediate filament vimentin, the cytoprotective protein klotho, stathmin and the proliferation cellular nuclear antigen, which were significantly up-regulated at the same points in time. These results suggest a beneficial effect of anatomical contiguously located endothelial cells on tubular regeneration through stabilization of endothelial integrity. Therefore, it seems that Bβ15-42 represents a novel pharmacological approach in the targeted therapy of acute renal failure in everyday clinical practice.

Hypofunction of the N-methyl-D-aspartate receptor (NMDAR) has been implicated as a possible mechanism underlying cognitive deficits and aberrant neuronal dynamics in schizophrenia. To test this hypothesis, we first administered a sub-anaesthetic dose of S-ketamine (0.006 mg/kg/min) or saline in a single-blind crossover design in 14 participants while magnetoencephalographic data were recorded during a visual task. In addition, magnetoencephalographic data were obtained in a sample of unmedicated first-episode psychosis patients (n = 10) and in patients with chronic schizophrenia (n = 16) to allow for comparisons of neuronal dynamics in clinical populations versus NMDAR hypofunctioning. Magnetoencephalographic data were analysed at source-level in the 1-90 Hz frequency range in occipital and thalamic regions of interest. In addition, directed functional connectivity analysis was performed using Granger causality and feedback and feedforward activity was investigated using a directed asymmetry index. Psychopathology was assessed with the Positive and Negative Syndrome Scale. Acute ketamine administration in healthy volunteers led to similar effects on cognition and psychopathology as observed in first-episode and chronic schizophrenia patients. However, the effects of ketamine on high-frequency oscillations and their connectivity profile were not consistent with these observations. Ketamine increased amplitude and frequency of gamma-power (63-80 Hz) in occipital regions and upregulated low frequency (5-28 Hz) activity. Moreover, ketamine disrupted feedforward and feedback signalling at high and low frequencies leading to hypo-and hyper-connectivity in thalamo-cortical networks. In contrast, first-episode and chronic schizophrenia patients showed a different pattern of magnetoencephalographic activity, characterized by decreased task-induced high-gamma band oscillations and predominantly increased feedforward/feedback-mediated Granger causality connectivity. Accordingly, the current data have implications for theories of cognitive dysfunctions and circuit impairments in the disorder, suggesting that acute NMDAR hypofunction does not recreate alterations in neural oscillations during visual processing observed in schizophrenia.

Abstract Background Hypofunction of the N-methyl-D-aspartate receptor (NMDA-R) has been implicated as a possible mechanism underlying cognitive deficits and aberrant neuronal dynamics in schizophrenia (ScZ). Methods In a single-blind cross-over design, 14 participants received either a subanaesthetic dose of S-ketamine (0.006 mg/Kg) or saline while Magnetoencephalographic (MEG) data were recorded during a visual task. In addition, MEG-data were obtained in a sample of unmedicated first-episode psychosis (FEP) patients (n = 10) and in patients with chronic ScZ (n = 16). MEG-data were analyzed at source-level in the 1–90 Hz frequency range in occipital and thalamic regions of interest (ROIs). In addition, directed functional connectivity analysis was performed using Granger Causality (GC). Psychopathology was assessed by the Positive and Negative Syndrome Scale (PANSS). Results Behavioral impairments were accompanied by increased amplitude and frequency of gamma-power (63–80 Hz) in occipital regions during Ketamine administration, while low-frequency (~5–30 Hz) activity was upregulated. Moreover, Ketamine disrupted feedforward (FF) and feedback (FB) signaling at high and low frequencies leading to hypoconnectivity in thalamo-cortical (TC) networks. In contrast, FEP and chronic ScZ patients showed a different pattern of MEG-activity, characterized by decreased task-induced high-gamma band oscillations and increased FF/FB-mediated GC-connectivity. Discussion The effects of Ketamine on high-frequency oscillations and their connectivity profile are not consistent with observations in FEP and chronic ScZ-patients. Accordingly, the current data have implications for theories of cognitive dysfunctions and circuit impairments in the disorder, suggesting that the effects of acute NMDA-R hypofunction are not consistent with impairments in visuo-perceptual oscillations in ScZ-patients.

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.

Objectives: Sepsis and septic shock are commonly present in the ICU and accompanied by significant morbidity, mortality, and cost. The frequency of secondary adrenal insufficiency in sepsis remains open to debate and a challenge to identify and treat appropriately. Animal models of sepsis using genetic or surgical initiation of adrenal insufficiency resulted in increased mortality, but the mechanisms are still unclear. The present study investigates the impact of adrenal inflammation in septic mice challenged with cecal ligation and puncture.Design: Prospective experimental study.Setting: University laboratory.Subjects: C57BL/6N wild-type mice.Interventions: Sepsis, induced by cecal ligation and puncture for 24 and 48 hours.Measurements and Main Results: Both septic and control mice were carefully monitored (every 30 min) for up to 48 hours and divided into survivors and nonsurvivors. We observed a significant and massive increase of interleukin-6, interleukin-1, and tumor necrosis factor- in adrenal protein extracts of nonsurvivors compared with sham animals and survivors. This pattern was partly reflected in liver and lung but not in plasma samples. Notably, a significant increase in nonsurvivors compared with survivors was only found for lung interleukin-6. In line with these findings, we detected a higher degree of leukocyte infiltration and hemorrhage in the adrenal glands of deceased mice. Evaluation of the hypothalamic-pituitary-adrenal axis response in these animals revealed an increase of adrenocorticotropic hormone, which was only partly reflected in the corticosterone level. Notably, using the adrenocorticotropic hormone stimulation test, we found an impaired adrenocorticotropic hormone response, particularly in nonsurvivors, which significantly correlated with the number of infiltrated leukocytes.Conclusions: Cecal ligation and puncture-induced murine sepsis induces a strong inflammatory response in the adrenal glands, which is accompanied by cell death and hemorrhage. Our data suggest that mortality and adrenal incapacitation are associated with the degree of adrenal inflammation, thereby underscoring the importance of adrenal function on survival.

The disruption of endothelial integrity is a crucial step for the development of vascular leakage and consequently ischemia-reperfusion injury (IRI). Regarding the molecular cell–cell interaction, the fibrinopeptide Bβ15–42 prevents vascular leakage by stabilizing the inter-endothelial junctions via association with the vascular endothelial-cadherin. In a previous study we showed that a renoprotective effect in early IRI may be achieved by intravenous administration of Bβ15–42 at the time of reperfusion. We now aimed to investigate whether additional pre-ischemic application of Bβ15–42 could enhance this effect. Therefore C57BL/6 mice were subjected to 0.5 h bilateral renal ischemia followed by reperfusion. The animals were randomized into 6 groups (n = 6): two control groups treated with i.v. administration of NaCl at reperfusion for 0.5 h (NaCl 1 h) and 2.5 h (NaCl 3 h), two groups with Bβ15–42 at reperfusion for 0.5 h (Bβrep 1 h) and 2.5 h (Bβrep 3 h), and two groups with administration of Bβ15–42 immediately pre-ischemic as well as at reperfusion for 0.5 h (Bβperi 1 h) and 2.5 h (Bβperi 3 h). We found that both Bβrep and Bβperi mice displayed reduced early renal damage compared with NaCl treated mice. However, there was no further reduction of the IR damage through added pre-ischemic application of Bβ15–42. Overall, we detected significantly reduced endothelial activation, lower tissue infiltration of neutrophils as well as lower tissue levels of neutrophil gelatinase-associated lipocalin (NGAL) in all mice treated with Bβ15–42 compared to mice treated with NaCl. Our data confirm the renoprotective effect of Bβ15–42 in the early therapeutic treatment of acute kidney injury due to ischemia and reperfusion. However, a combined pre-and post-ischemic administration of Bβ15–42 appears to provide no additional benefit compared with a sole administration at reperfusion.

BACKGROUNDIntestinal fatty acid binding protein (iFABP) is elevated in plasma by intestinal injury. We investigated the influence of surgical trauma and severe sepsis caused by abdominal and pulmonary infection on plasma iFABP concentrations.METHODSSeventy-nine patients were included in this prospective observational study: 31 patients before elective major abdominal surgery (EMS), 33 patients with severe sepsis on admission to the intensive care unit (ICU), and 15 healthy volunteers who served as controls. Blood samples were taken before and after surgery for a period up to 5 d.RESULTSPrior to surgery, EMS patients had increased iFABP concentrations in those patients with intestinal cancer compared with patients without intestinal cancer (217 pg/mL, interquartile range [IQR] I-III 100-369 pg/mL versus 79 pg/mL, IQR I-III: 0-182 pg/mL; p<0.01) and with controls (114 pg/mL, IQR I-III: 103-124 pg/mL; p<0.01). Surgical trauma increased iFABP levels in patients without intestinal cancer (240 pg/mL, IQR I-III 111-305 pg/mL; p<0.01). Within 24 h after surgery, iFABP levels decreased to normal values. Patients with severe sepsis of abdominal origin had elevated concentrations compared with controls (324 pg/mL [IQR I-III 0-649 pg/mL]; p=0.05); in patients with pneumonia, iFABP levels were not significantly increased. Discrimination between intestinal- and pulmonary-induced sepsis was low (area under the curve [AUC] 0.693; 95% confidence interval 0.512-0.874).CONCLUSIONSSurgical trauma and severe sepsis lead to elevated iFABP concentrations. However, intestinal malignant disease and in some patients severe sepsis caused by pneumonia also resulted in elevated iFABP concentrations. The results support the idea that epithelial injury of many causes leads to elevated concentrations of iFABP. The value of iFABP for differentiating pulmonary from intestinal sepsis is limited.

AIMS Shock wave therapy (SWT) represents a clinically widely used angiogenic and thus regenerative approach for the treatment of ischaemic heart or limb disease. Despite promising results in preclinical and clinical trials, the exact mechanism of action remains unknown. Toll-like receptor 3, which is part of the innate immunity, is activated by binding double-stranded (ds) RNA. It plays a key role in inflammation, a process that is needed also for angiogenesis. We hypothesize that SWT causes cellular cavitation without damaging the target cells, thus liberating cytoplasmic RNA that in turn activates TLR3. METHODS AND RESULTS SWT induces TLR3 and IFN-β1 gene expression as well as RNA liberation from endothelial cells in a time-dependant manner. Conditioned medium from SWT-treated HUVECs induced TLR3 signalling in reporter cells. The response was lost when the medium was treated with RNase III to abolish dsRNAs or when TLR3 was silenced using siRNAs. In a mouse hind limb ischaemia model using wt and TLR3(-/-) mice (n = 6), SWT induced angiogenesis and arteriogenesis only in wt animals. These effects were accompanied by improved blood perfusion of treated limbs. Analysis of main molecules of the TLR3 pathways confirmed TLR3 signalling in vivo following SWT. CONCLUSION Our data reveal a central role of the innate immune system, namely Toll-like receptor 3, to mediate angiogenesis upon release of cytoplasmic RNAs by mechanotransduction of SWT.

Nerve injury-induced protein (Ninjurin [Ninj]) 1 is an adhesion molecule originally identified in Schwann cells after nerve injury, whereas it is also expressed in leukocytes, epithelium, endothelium, and various organs, and is induced under inflammatory conditions. Its contribution to inflammation was so far restricted to the nervous system and exclusively attributed to its role during leukocyte migration. We hypothesized a proinflammatory role for Ninj1 also outside the nervous system. To elucidate its impact during inflammation, we analyzed expression levels and its contribution to inflammation in septic mice and studied its effect on inflammatory signaling in vitro. The effect on inflammation was analyzed by genetic (only in vitro) and pharmacologic repression in septic mice (cecal ligation and puncture) and cell culture, respectively. Repression of Ninj1 by an inhibitory peptide or small interfering RNA attenuated LPS-triggered inflammation in macrophages and endothelial cells by modulating p38 phosphorylation and activator protein-1 activation. Inhibition of Ninj1 in septic mice reduced systemic and pulmonary inflammation as well as organ damage, and ameliorated survival after 24 hours. Ninj1 is elevated under inflammatory conditions and contributes to inflammation not only by mediating leukocyte migration, but also by modulating Toll-like receptor 4-dependent expression of inflammatory mediators. We assume that, owing to both mechanisms, inhibition reduces systemic inflammation and organ damage in septic mice. Our data contribute to a better understanding of the complex inflammatory mechanisms and add a novel therapeutic target for inflammatory conditions such as sepsis.

Hypofunctioning of the N-methyl-D-aspartate receptor (NMDA-R) has been prominently implicated in the pathophysiology of schizophrenia (ScZ). The current study tested the effects of ketamine, a dissociative anesthetic and NMDA-R antagonist, on resting-state activity recorded with magnetoencephalography (MEG) in healthy volunteers. In a single-blind cross-over design, each participant (n = 12) received, on 2 different sessions, a subanesthetic dose of S-ketamine (0.006 mg/Kg) and saline injection. MEG-data were analyzed at sensor-and source-level in the beta (13-30 Hz) and gamma (30-90 Hz) frequency ranges. In addition, connectivity analysis at source-level was performed using transfer entropy (TE). Ketamine increased gamma-power while beta-band activity was decreased. Specifically, elevated 30-90 Hz activity was pronounced in subcortical (thalamus and hippocampus) and cortical (frontal and temporal cortex) regions, whilst reductions in beta-band power were localized to the precuneus, cerebellum, anterior cingulate, temporal and visual cortex. TE analysis demonstrated increased information transfer in a thalamo-cortical network after ketamine administration. The findings are consistent with the pronounced dysregulation of high-frequency oscillations following the inhibition of NMDA-R in animal models of ScZ as well as with evidence from electroencephalogram-data in ScZ-patients and increased functional connectivity during early illness stages. Moreover, our data highlight the potential contribution of thalamo-cortical connectivity patterns towards ketamine-induced neuronal dysregulation, which may be relevant for the understanding of ScZ as a disorder of disinhibition of neural circuits.

Background Ischemia-reperfusion edema is a common early complication after lung transplantation where the hypoxia-induced vascular endothelial growth factor (VEGF)-A plays a pivotal role. It remains unclear whether a VEGF blockade is beneficial in lung transplantation.Methods VEGF-A blockade was investigated in an orthotopic rat model of lung transplantation. VEGF-A antibody was added into the preservation solution alone (-VEGF D/-), in the preservation solution and systemically to the recipient before reperfusion (-VEGF D/R), or applied to the recipient alone before reperfusion (-VEGF -/R). Forty-eight hours after lung transplantation, left lungs were collected and wet-to-dry ratio, Western blotting, RT-PCR, and immunohistology were performed.Results VEGF-A blockade in -VEGF D/-, -VEGF D/R, and -VEGF -/R resulted in neutralization of tissue VEGF-A. Reperfusion edema was only reduced in -VEGF D/R and -VEGF D/- groups versus Perfadex controls. Some -VEGF -/R rats showed a hyperinflammation leading to increased pro-inflammatory cytokine expressions as well as increased edema. Whereas generally the -VEGF D/- group showed decreased inflammation, the combination with anti-VEGF treatment to the recipient resulted in a pro-inflammatory and a pro-apoptotic phenotype. Short-term survival, however, was not significantly different in all groups as compared to the controls. In the -VEGF (D/R) or (D/-) groups, animals mainly died from arterial thromboembolisms and in the -VEGF (-/R) group, hyperinflammation was the main cause of death.Conclusion VEGF-A directly contributes to the formation of a reperfusion edema, which might be reduced by its blockade. However, the -VEGF effect on the endothelial integrity might also favor arterial thrombosis formation.

Abstract HCC is the third leading cause of cancer-related mortality worldwide. Metabolic syndrome diseases are shown to facilitate the progression of HCC in industrial countries. Recent studies unraveled pivotal roles of long non-coding RNAs (lncRNAs) in carcinogenesis and cancer metastasis. The controversy that the non-translated H19 mRNA harbors pro- as well as anti-tumorigenic potential shows that its function is poorly characterized. TLR3 plays a key role in the recognition of exogenous pathogen-associated dsRNA and even more importantly TLR3 is activated by endogenous injury-associated ligands. There is increasing evidence that the implication of TLR3 in HCC development might be associated with tumor size, aggressiveness and survival. To study the relationship between the H19 mRNA and TLR3 we first looked at the TLR3 signaling in livers from TLR3-/- mice in comparison to WT mice. By performing real-time RT-PCR experiments, we could demonstrate that TLR3-/- mice showed a significantly enhanced H19 mRNA expression. In order to find out whether H19 mRNA acts as a regulator of TLR3 signaling, we used RNA interference (RNAi) and added the synthetic TLR3 ligand poly I:C on the human hepatoma cell line HepG2. TLR3 knockdown was followed by a significant increase in H19 mRNA expression levels, whereas poly I:C treatment resulted in significantly decreased H19 levels. Vice versa, H19 mRNA knockdown verified a significant reduction in TLR3 mRNA expression. Determination of free RNA in the cell culture supernatants revealed a significant increase of free RNA only after hypoxic incubation whereas no changes could be detected after poly I:C nor RNAi application. Treatment based on H19 inhibition may be a specific approach to target tumor cells, as it is already known that silencing of H19 significantly reduces cell viability. Due to the implication of TLR3 in mRNA stabilization, we addressed to question of a reciprocal relation between both genes by actinomycin D treatment. Indeed, H19 expression was shown to depend on TLR3 expression and a reduced H19 stability was detected after RNAi of TLR3. Since caspase-3 is a key executor of apoptosis and due to the fact that TLRs are known to be involved in the regulation of the apoptotic machinery, we performed caspase-3-like activity assay. Whereas no significant changes in basal caspase-3 like activity could be detected, a strong increase in caspase-3 like activity was seen after siRNA-mediated TLR3 knockdown under normoxic and hypoxic conditions. This indicates that TLR3 may play a crucial role in mediating the signaling through non-coding RNAs, which function is so far poorly understood. We show for the first time the direct relation between H19 and TLR3. Changes in expressions of either of these molecules results in an altered cell response. The knowledge of this interplay might lead to novel strategies for treating HCC. Citation Format: Patrick Paulus, Anja Urbschat, Christin Reissig, Kai Zacharowski, Stefan Dröse, Bertram Scheller, Elisabeth Tybl. Toll-like receptor (TLR3) signaling in human hepatocellular carcinoma (HCC) depends on the tumor-associated long-noncoding RNA H19. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2014-537

Disruption of the renal endothelial integrity is pivotal for the development of a vascular leak, tissue edema and consequently acute kidney injury. Kidney ischemia amplifies endothelial activation and up-regulation of pro-inflammatory mechanisms. After restoring a sufficient blood flow, the kidney is damaged through complex pathomechanisms that are classically referred to as ischemia and reperfusion injury, where the disruption of the inter-endothelial connections seems to be a crucial step in this pathomechanism. Focusing on the molecular cell-cell interaction, the fibrinopeptide Bβ15-42 prevents vascular leakage by stabilizing these inter-endothelial junctions. The peptide associates with vascular endothelial-cadherin, thus preventing early kidney dysfunction by preserving blood perfusion efficacy, edema formation and thus organ dysfunction. We intended to demonstrate the early therapeutic benefit of intravenously administered Bβ15-42 in a mouse model of renal ischemia and reperfusion. After 30 minutes of ischemia, the fibrinopeptide Bβ15-42 was administered intravenously before reperfusion was commenced for 1 and 3 hours. We show that Bβ15-42 alleviates early functional and morphological kidney damage as soon as 1 h and 3 h after ischemia and reperfusion. Mice treated with Bβ15-42 displayed a significantly reduced loss of VE-cadherin, indicating a conserved endothelial barrier leading to less neutrophil infiltration which in turn resulted in significantly reduced structural renal damage. The significant reduction in tissue and serum neutrophil gelatinase-associated lipocalin levels reinforced our findings. Moreover, renal perfusion analysis by color duplex sonography revealed that Bβ15-42 treatment preserved resistive indices and even improved blood velocity. Our data demonstrate the efficacy of early therapeutic intervention using the fibrinopeptide Bβ15-42 in the treatment of acute kidney injury resulting from ischemia and reperfusion. In this context Bβ15-42 may act as a potent renoprotective agent by preserving the endothelial and vascular integrity.