Clarissa von Haefen

The intracellular pathways leading to mitochondrial activation and subsequent cell death in the ceramide-mediated stress response have been intensively studied in recent years. Experimental evidence has been provided that ceramide-induced apoptosis is inhibited by overexpression of antiapoptotic proteins of the Bcl-2 family. However, the direct effect of proapoptotic gene products, e.g. Bax, on ceramide-induced death signalling has not yet been studied in detail. In the present work, we show by measurement of mitochondrial permeability transition, cytochrome c release, activation of caspase-3 and DNA fragmentation that ceramide-induced apoptosis is marginal in Bax-negative DU 145 cells. Reconstitution of Bax by generation of DU 145 cells stably expressing this proapoptotic factor, clearly enhanced ceramide-induced apoptosis at all levels of the mitochondrial signalling cascade. Using the broad-range caspase inhibitor zVAD-fmk and zDEVD-fmk, an inhibitor of caspase-3-like activities, we demonstrate that the ceramide-induced mitochondrial activation in Bax-transfected DU 145 cells is caspase-independent. On the other hand, apoptotic events located downstream of the mitochondria, e.g. DNA fragmentation, were shown to be caspase-dependent. This influence of Bax on ceramide-induced apoptosis was confirmed in another cellular system: whereas Bax-positive HCT116 wild type cells were very sensitive towards induction of cell death by C(2)-ceramide, sensitivity of Bax knock-out HCT116 cells was significantly reduced. Thus, we conclude that Bax is a key activator of ceramide-mediated death pathways.

Caspase-8 is a key effector of death-receptor-triggered apoptosis. In a previous study, we demonstrated, however, that caspase-8 can also be activated in a death receptor-independent manner via the mitochondrial apoptosis pathway, downstream of caspase-3. Here, we show that caspases-3 and -8 mediate a mitochondrial amplification loop that is required for the optimal release of cytochrome c, mitochondrial permeability shift transition, and cell death during apoptosis induced by treatment with the microtubule-damaging agent paclitaxel (Taxol). In contrast, Smac release from mitochondria followed a different pattern, and therefore seems to be regulated independently from cytochrome c release. Taxol-induced cell death was inhibited by the use of synthetic, cell-permeable caspase-3- (zDEVD-fmk) or caspase-8-specific (zIETD-fmk) inhibitors. Apoptosis signaling was not affected by a dominant-negative FADD mutant (FADD-DN), thereby excluding a role of death receptor signaling in the amplification loop and drug-induced apoptosis. The inhibitor experiments were corroborated by the use of BJAB cells overexpressing the natural serpin protease inhibitor, cytokine response modifier A. These data demonstrate that the complete activation of mitochondria, release of cytochrome c, and execution of drug-induced apoptosis require a mitochondrial amplification loop that depends on caspases-3 and -8 activation. In addition, this is the first report to demonstrate death receptor-independent caspase-8 autoprocessing in vivo.

Exogenous stress like tissue damage and pathogen invasion during surgical trauma could lead to a peripheral inflammatory response and induce neuroinflammation, which can result in postoperative cognitive dysfunction (POCD). The cholinergic anti-inflammatory pathway is a neurohumoral mechanism that plays a prominent role by suppressing the inflammatory response. Treatments with acetylcholinesterase inhibitors enhance cholinergic transmission and may therefore act as a potential approach to prevent neuroinflammation. In the presence or absence of acetylcholinesterase inhibitors, adult Wistar rats underwent surgery alone or were additionally treated with lipopolysaccharide (LPS). Physostigmine, which can overcome the blood-brain barrier or neostigmine acting only peripheral, served as acetylcholinesterase inhibitors. The expression of pro- and anti-inflammatory cytokines in the cortex, hippocampus, spleen and plasma was measured after 1 h, 24 h, 3 d and 7 d using Real-Time PCR, western blot analysis or cytometric bead array (CBA). Fluoro-Jade B staining of brain slices was employed to elucidate neurodegeneration. The activity of acetylcholinesterase was estimated using a spectrofluorometric method. Surgery accompanied by LPS-treatment led to increased IL-1beta gene and protein upregulation in the cortex and hippocampus but was significantly reduced by physostigmine and neostigmine. Furthermore, surgery in combination with LPS-treatment caused increased protein expression of IL-1, TNF-alpha and IL-10 in the spleen and plasma. Physostigmine and neostigmine significantly decreased the protein expression of IL-1 and TNF-alpha. Neuronal degeneration and the activity of acetylcholinesterase were elevated after surgery with LPS-treatment and reduced by physostigmine and neostigmine. Along with LPS-treatment, acetylcholinesterase inhibitors reduce the pro-inflammatory response as well as neurodegeneration after surgery in the cortex and hippocampus. This combination may represent a tool to break the pathogenesis of POCD.

Dexmedetomidine (DEX) is a highly selective agonist of α2-receptors with sedative, anxiolytic, and analgesic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on hippocampal neurogenesis, specifically the proliferation capacity and maturation of neurons and neuronal plasticity following the induction of hyperoxia in neonatal rats. Six-day old sex-matched Wistar rats were exposed to 80% oxygen or room air for 24 h and treated with 1, 5 or 10 μg/kg of dexmedetomidine or normal saline. A single pretreatment with DEX attenuated the hyperoxia-induced injury in terms of neurogenesis and plasticity. In detail, both the proliferation capacity (PCNA+ cells) as well as the expression of neuronal markers (Nestin+, PSA-NCAM+, NeuN+ cells) and transcription factors (SOX2, Tbr1/2, Prox1) were significantly reduced under hyperoxia compared to control. Furthermore, regulators of neuronal plasticity (Nrp1, Nrg1, Syp, and Sema3a/f) were also drastically decreased. A single administration of dexmedetomidine prior to oxygen exposure resulted in a significant up-regulation of expression-profiles compared to hyperoxia. Our results suggest that dexmedetomidine may have neuroprotective effects in an acute hyperoxic model of the neonatal rat.

Dexmedetomidine is a highly selective agonist of α2-receptors with sedative, anxiolytic, analgesic, and anesthetic properties. Neuroprotective effects of dexmedetomidine have been reported in various brain injury models. In the present study, we investigated the effects of dexmedetomidine on neurodegeneration, oxidative stress markers, and inflammation following the induction of hyperoxia in neonatal rats. Six-day-old Wistar rats received different concentrations of dexmedetomidine (1, 5, or 10 µg/kg bodyweight) and were exposed to 80% oxygen for 24 h. Sex-matched littermates kept in room air and injected with normal saline or dexmedetomidine served as controls. Dexmedetomidine pretreatment significantly reduced hyperoxia-induced neurodegeneration in different brain regions of the neonatal rat. In addition, dexmedetomidine restored the reduced/oxidized glutathione ratio and attenuated the levels of malondialdehyde, a marker of lipid peroxidation, after exposure to high oxygen concentration. Moreover, administration of dexmedetomidine induced downregulation of IL-1β on mRNA and protein level in the developing rat brain. Dexmedetomidine provides protections against toxic oxygen induced neonatal brain injury which is likely associated with oxidative stress signaling and inflammatory cytokines. Our results suggest that dexmedetomidine may have a therapeutic potential since oxygen administration to neonates is sometimes inevitable.

In this study, we asked whether overexpression of caspase-3, a central downstream executioner of apoptotic pathways, might sensitize breast cancer cells with acquired drug resistance (MT1/ADR) to drug-induced apoptosis. As control, we employed caspase-3 negative and caspase-3-transfected MCF-7 cells. Whereas mock-transfected MCF-7 cells were resistent to epirubicin, etoposide and paclitaxel (taxol), the same drugs led to breakdown of nuclear DNA in caspase-3-transfected MCF-7 cells. MT1/ADR cells express low levels of wild type caspase-3 but show defective caspase activation and apoptosis upon drug exposure. These cells also display a less efficient activation of the mitochondrial permeability transition. Caspase-3-transfected MT1/ADR clones showed a 2.8-fold increase in the protein level and a 3.7-fold higher specific enzyme activity. Procaspase-3 overexpression was not toxic and did not affect background apoptosis. Interestingly, procaspase-3-transfected MT1/ADR cells were more sensitive to cytotoxic drugs as compared with vector-transfected controls and DNA fragmentation nearly reached the levels of the original drug sensitive MT1 cells. Thus, overexpression of caspase-3 enhances chemosensitivity especially in situations where activation of the mitochondrial apoptosome is disturbed.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF), which are frequently inactivated in human cancer. Whereas p16(INK4a) acts through engagement of the Rb-cdk4/6-cyclin D pathway, both the pro-apoptotic and cell cycle-regulatory functions of p14(ARF) were shown to be primarily dependent on the presence of functional p53. Recent reports have also implicated p14(ARF) in p53-independent mechanisms of cell cycle regulation and apoptosis induction, respectively. To further explore the pro-apoptotic function of p14(ARF) in relation to functional cellular p53, we constructed a replication-deficient adenoviral vector for overexpression of p14(ARF) (Ad-p14(ARF)). As expected, Ad-p14(ARF) efficiently induced apoptosis in p53/Rb wild-type U-2OS osteosarcoma cells at low multiplicities of infection. Interestingly, Ad-p14(ARF) also induced apoptosis in both p53-deleted SAOS-2 osteosarcoma cells and HCT116 colon cancer cells with a bi-allelic knock-out of p53 (HCT116-p53(-/-)). Similarly, adenovirus-mediated overexpression of p14(ARF) induced apoptosis in p53/Bax-mutated DU145 prostate cancer cells as well as in HCT116 cells devoid of functional Bax (HCT116-Bax(-/-)). Restoration of Bax expression by retroviral gene transfer in DU145 cells did not further enhance p14(ARF)-triggered cell death. Infection with Ad-p14(ARF) induced activation of mitochondrial permeability shift transition, caspase activation and apoptotic DNA fragmentation irrespective of the presence or absence of either Bax or functional cellular p53. Nevertheless, overexpression of the anti-apoptotic Bcl-2 homolog Bcl-x(L) markedly inhibited p14(ARF)-induced apoptosis. This may indicate that p14(ARF) triggers a so far unknown activator of mitochondrial apoptosis which can be inhibited by Bcl-2 but which acts either independently or downstream of Bax. Taken together, this report demonstrates the participation of signaling pathways apart from the p53/Mdm-2 rheostat and Bax in p14(ARF)-mediated apoptosis.

The death ligand TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation. Here, we show that the synergism of TRAIL and 5-fluorouracil (5-FU) and cross-sensitization between TRAIL and 5-FU for induction of apoptosis, entirely depend on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to release cytochrome c and to activate caspase-3 and -9 when exposed to TRAIL and 5-FU. In contrast, TRAIL sensitized for 5-FU-induced apoptosis and vice versa upon reconstitution of Bax expression. Isobolographic analyses of ED50 doses for 5-FU at increasing TRAIL concentrations showed a clear synergism of TRAIL and 5-FU in Bax-expressing cells. In contrast, the effect was merely additive in DU145 cells lacking Bax. Notably, both DU145 and HCT116 Bax-deficient cells still express Bak. This indicates that Bak is not sufficient to mediate cross-sensitization and synergism between 5-FU and TRAIL. Stable overexpression of Bak in DU145 sensitized for epirubicin-induced apoptosis but failed to confer synergy between TRAIL and 5-FU. Moreover, we show by the use of EGFP-tagged Bax and Bak that TRAIL and 5-FU synergistically trigger oligomerization and clustering of Bax but not Bak. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade and delineate a higher degree of specificity in signaling for cell death by multidomain Bcl-2 homologs.

There is an ongoing controversy regarding the relevance of apoptosis induction by ionizing irradiation as compared with other end points including transient or permanent cell cycle arrest of damaged cells. Here, we show that such permanent cell cycle arrest and apoptosis represent two sides of the same coin. MCF-7 cells fail to express procaspase-3, which results in resistance to apoptosis induced by anticancer drugs. Conversely, restoration of procaspase-3 sensitizes MCF-7 cells to chemotherapeutics including epirubicine, etoposide and taxol. In contrast, irradiation does not trigger apoptotic cell death but results in prolonged arrest in the G2 phase of the cell division cycle regardless of procaspase-3 expression. This suggested that the propensity of MCF-7 cells to arrest at the G2 checkpoint results in resistance to apoptosis upon gamma-irradiation. This G2 arrest was associated with upregulation of p21CIP/WAF-1. Inhibition of DNA-damage-induced stress kinases and p21CIP/WAF-1 expression by caffeine abrogated G2 arrest and induced apoptosis of the irradiated cells in a caspase-3-dependent manner. Inhibition of cell cycle progression by adenoviral expression of the cyclin dependent kinase inhibitor p21CIP/WAF-1 prevented apoptosis upon caffeine treatment indicating that cell cycle progression, that is, G2-release, is required for induction of apoptosis. Likewise, cells homozygously deleted for p21CIP/WAF-1 (HCT116 p21-/-) display enhanced irradiation-induced apoptosis via a caspase-3-dependent mechanism. These data indicate that the disruption of G2 checkpoint control overcomes cell cycle arrest and resistance to gamma-irradiation-induced cell death. Thus, DNA damage may trigger a permanent G2 arrest as an initial inactivation step of tumor cells where the phenomenon of apoptosis is hidden unless cell cycle arrest is overcome. The efficient induction of apoptosis upon G2 release thereby depends on the propensity to activate the key executioner caspase-3. This finding is of crucial importance for the understanding of molecular steps underlying the efficacy of ionizing radiation to delete tumor cells.

Background: Chronic alcohol consumption is a major cause of liver injury.However, the molecular mechanisms by which alcohol impairs hepatocellular function and induces cell death remain unclear.Macroautophagy (hereafter called 'autophagy') is a degradation pathway involved in the survival or death of cells during conditions of cellular stress.This study examines the effect of chronic alcohol consumption on hepatocellular autophagy in an animal model.Methods: During a 12-week period male Wistar rats were fed a Lieber-DeCarli diet containing 5% alcohol (EtOH group; n=10), or an isocaloric diet (control group; n=10).Hepatic expression of key regulatory autophagy proteins (e.g.Beclin-1, ATG-3, ATG-5, p62/SQSTM1 and LC3) were detected by real-time polymerase chain reaction and Western blot analysis.Markers of cellular stress and apoptotic cell death (e.g.HO-1, caspase-3, PARP-1 and Bcl-2) were determined, and levels of reduced and oxidized glutathione were measured.Results: Chronic alcohol consumption caused cellular and oxidative stress in the liver.Transcriptional and translational expression of Beclin-1 and ATG-5 was significantly impaired.The protein expression of LC3-I and LC3-II was significantly increased, while the ratio of LC3I/II remained unchanged in the EtOH group compared with controls.Hepatocellular expression of p62/SQSTM1 and markers of apoptotic cell death (such as cleaved caspase-3 and cleaved PARP-1) were significantly increased in the EtOH group indicating a disrupted autophagic flux and increased rate of apoptosis in the liver.Conclusions: In this model, chronic alcohol consumption impaired hepatocellular autophagy and induced apoptotic cell death.It appears that changes in autophagy might contribute to alcohol-induced structural and functional hepatocellular injury.

Abstract Background Despite the intensive efforts to improve the diagnosis and therapy of sepsis over the last decade, the mortality of septic shock remains high and causes substantial socioeconomical burden of disease. The function of immune cells is time-of-day-dependent and is regulated by several circadian clock genes. This study aims to investigate whether the rhythmicity of clock gene expression is altered in patients with septic shock. Methods This prospective pilot study was performed at the university hospital Charité–Universitätsmedizin Berlin, Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK). We included 20 patients with septic shock between May 2014 and January 2018, from whom blood was drawn every 4 h over a 24-h period to isolate CD14-positive monocytes and to measure the expression of 17 clock and clock-associated genes. Of these patients, 3 whose samples expressed fewer than 8 clock genes were excluded from the final analysis. A rhythmicity score S P was calculated, which comprises values between -1 (arrhythmic) and 1 (rhythmic), and expression data were compared to data of a healthy study population additionally. Results 77% of the measured clock genes showed inconclusive rhythms, i.e., neither rhythmic nor arrhythmic. The clock genes NR1D1 , NR1D2 and CRY2 were the most rhythmic, while CLOCK and ARNTL were the least rhythmic. Overall, the rhythmicity scores for septic shock patients were significantly ( p < 0.0001) lower (0.23 ± 0.26) compared to the control group (12 healthy young men, 0.70 ± 0.18). In addition, the expression of clock genes CRY1 , NR1D1 , NR1D2 , DBP , and PER2 was suppressed in septic shock patients and CRY2 was significantly upregulated compared to controls. Conclusion Molecular rhythms in immune cells of septic shock patients were substantially altered and decreased compared to healthy young men. The decrease in rhythmicity was clock gene-dependent. The loss of rhythmicity and down-regulation of clock gene expression might be caused by sepsis and might further deteriorate immune responses and organ injury, but further studies are necessary to understand underlying pathophysiological mechanisms. Trail registration Clinical trial registered with www.ClinicalTrials.gov (NCT02044575) on 24 January 2014.

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.

We previously demonstrated that the forced expression of pro-caspase-3 can revert acquired chemoresistance in MT1-Adr breast cancer cells which show a defective activation of the mitochondrial pathway of apoptosis. We now asked whether the manipulation of mitochondrial apoptosis signaling can revert different types of drug resistance, i.e. the resistance due to impaired mitochondrial activation in the MT1-Adr cells and the resistance in MT3-Adr cells which is caused by increased expression of the Mdr-1/p-glycoprotein ABC transporter. Here we show that Bcl-2 overexpression is the underlying cause for the resistant phenotype in the MT1-Adr cells. Overexpression of the apoptosis-promoting Bax homologue Bak or the BH3 only protein Nbk/Bik reverts, as expected, acquired drug resistance in the MT1-Adr cells as recently demonstrated for pro-caspase-3. Moreover, we show that both apoptosis-promoters, Nbk/Bik and Bak, antagonize acquired chemoresistance for epirubicin-mediated apoptosis in MT3-Adr breast cancer cells. Neither drug uptake nor drug efflux were influenced by Bak or Nbk/Bik. Thus, our data show that manipulation of the downstream apoptosis signaling cascade by Bak and Nbk/Bik can overcome not only drug resistance due to mitochondrial apoptosis deficiency (in the MT1-Adr cells) but also classical, i.e. efflux-mediated, resistance for drug-induced cell death in the MT3-Adr cell line. Nbk/Bik and Bak could therefore be target genes to increase chemosensitivity and overcome different types of drug resistance.

Oxygen toxicity contributes to the pathogenesis of adverse neurological outcome in survivors of preterm birth in clinical studies. In infant rodent brains, hyperoxia triggers widespread apoptotic neurodegeneration, induces pro-inflammatory cytokines and inhibits growth factor signaling cascades. Since a tissue-protective effect has been observed for recombinant erythropoietin (rEpo), we hypothesized that rEpo would influence hyperoxia-induced oxidative stress in the developing rat brain. The aim of this study was to investigate the level of glutathione (reduced and oxidized), lipid peroxidation and the expression of heme oxygenase-1 (HO-1) and acetylcholinesterase (AChE) after hyperoxia and rEpo treatment. Six-day-old Wistar rats were exposed to 80% oxygen for 2–48 h and received 20,000 IU/kg rEpo intraperitoneally (i.p.). Sex-matched littermates kept under room air and injected with normal saline or rEpo served as controls. Treatment with rEpo significantly reduced hyperoxia-induced upregulation of oxidized glutathione (GSSG) and malondialdehyde, a product of lipid breakdown, whereas reduced glutathione (GSH) was upregulated by rEpo. In parallel, hyperoxia-treated immature rat brains revealed rEpo-suppressible upregulation of synaptic AChE-S as well as of the stress-inducible AChE-R variant, together predicting rEpo-protected cholinergic signaling and restrained inflammatory reactions. Furthermore, treatment with rEpo induced upregulation of HO-1 on mRNA, protein and activity level in the developing rat brain. Our results suggest that rEpo generates its protective effect against oxygen toxicity by a reduction of diverse oxidative stress parameters and by limiting the stressor-inducible changes in both HO-1 and cholinergic functions.

Autophagy is a self-degradative process that involves turnover and recycling of cytoplasmic components in healthy and diseased tissue. Autophagy has been shown to be protective at the early stages of programmed cell death but it can also promote apoptosis under certain conditions. Earlier we demonstrated that oxygen contributes to the pathogenesis of neonatal brain damage, which can be ameliorated by intervention with recombinant human erythropoietin (rhEpo). Extrinsic- and intrinsic apoptotic pathways are involved in oxygen induced neurotoxicity but the role of autophagy in this model is unclear. We analyzed the expression of autophagy activity markers in the immature rodent brain after exposure to elevated oxygen concentrations. We observed a hyperoxia-exposure dependent regulation of autophagy-related gene (Atg) proteins Atg3, 5, 12, Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), LC3A-II, and LC3B-II which are all key autophagy activity proteins. Interestingly, a single injection with rhEpo at the onset of hyperoxia counteracted these oxygen-mediated effects. Our results indicate that rhEpo generates its protective effect by modifying the key autophagy activity proteins.

Within the last decade, it became clear that oxygen contributes to the pathogenesis of neonatal brain damage, leading to neurocognitive impairment of prematurely born infants in later life. Recently, we have identified a critical role for receptor-mediated neuronal apoptosis in the immature rodent brain. However, the contribution of the intrinsic apoptotic pathway accompanied by activation of caspase-2 under hyperoxic conditions in the neonatal brain still remains elusive. Inhibition of caspases appears a promising strategy for neuroprotection. In order to assess the influence of specific caspases on the developing brain, we applied a recently developed pentapeptide-based group II caspase inhibitor (5-(2,6-difluoro-phenoxy)-3(R,S)-(2(S)-(2(S)-(3-methoxycarbonyl-2(S)-(3-methyl-2(S)-((quinoline-2-carbonyl)-amino)-butyrylamino)propionylamino)3-methylbutyrylamino)propionylamino)-4-oxo-pentanoic acid methyl ester; TRP601). Here, we report that elevated oxygen (hyperoxia) triggers a marked increase in active caspase-2 expression, resulting in an initiation of the intrinsic apoptotic pathway with upregulation of key proteins, namely, cytochrome c, apoptosis protease-activating factor-1, and the caspase-independent protein apoptosis-inducing factor, whereas BH3-interacting domain death agonist and the anti-apoptotic protein B-cell lymphoma-2 are downregulated. These results coincide with an upregulation of caspase-3 activity and marked neurodegeneration. However, single treatment with TRP601 at the beginning of hyperoxia reversed the detrimental effects in this model. Hyperoxia-mediated neurodegeneration is supported by intrinsic apoptosis, suggesting that the development of highly selective caspase inhibitors will represent a potential useful therapeutic strategy in prematurely born infants.

During surgery or infection, peripheral inflammation can lead to neuroinflammation, which is associated with cognitive impairment, neurodegeneration, and several neurodegenerative diseases. Dexmedetomidine, an α-2-adrenoceptor agonist, is known to exert anti-inflammatory and neuroprotective properties and reduces the incidence of postoperative cognitive impairments. However, on the whole the molecular mechanisms are poorly understood. This study aims to explore whether dexmedetomidine influences microRNAs (miRNAs) in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation. Adult Wistar rats were injected with 1 mg/kg LPS intraperitoneal (i.p.) in the presence or absence of 5 µg/kg dexmedetomidine. After 6 h, 24 h, and 7 days, gene expressions of interleukin 1-β (IL1-β), tumor necrosis factor-α (TNF-α), and microRNA expressions of miR 124, 132, 134, and 155 were measured in the hippocampus, cortex, and plasma. Dexmedetomidine decreased the LPS-induced neuroinflammation in the hippocampus and cortex via significant reduction of the IL1-β and TNF-α gene expressions after 24 h. Moreover, the LPS-mediated increased expressions of miR 124, 132, 134, and 155 were significantly decreased after dexmedetomidine treatment in both brain regions. In plasma, dexmedetomidine significantly reduced LPS-induced miR 155 after 6 h. Furthermore, there is evidence that miR 132 and 134 may be suitable as potential biomarkers for the detection of neuroinflammation.

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.

Alcohol induces cellular stress and promotes cell death in immune cells. Molecular mechanisms by which ethanol impairs the function of immune cells are largely unknown. Autophagy is a degradation pathway, acting either as a pro-survival or pro-death mechanism activated during stress conditions. We examined whether ethanol influences autophagy in monocytic human U937, CD4 Jurkat, and MCF-7 cells.Effects of ethanol during starvation-induced autophagy were investigated, treating cells with ethanol alone and in combination with activation of autophagy by rapamycin or inhibition by wortmannin. Apoptotic and necrotic cell death features such as the breakdown of the mitochondrial membrane potential, DNA fragmentation, and cell permeability were assessed using FACS analyses. Expression level of Beclin-1, LC3-II, Bcl-2, and the activation of caspase-3, and PARP-1 were determined using Western blot analyses. Influence of ethanol on formation of LC3-II complexes was assessed using fluorescence microscopy in MCF-7 cells stable transfected with a GFP-LC3-II-expression vector.Ethanol down regulated autophagy proteins such as Beclin-1 and LC3-II. Apoptosis was enhanced as shown by breakdown of mitochondrial potential, up-regulation of cleaved caspase-3 and PARP-1 and down-regulation of anti-apoptotic protein Bcl-2. Formation of LC3-II complexes was inhibited by ethanol in caspase-3 deficient MCF-7 cells. Stimulation of autophagy by rapamycin prevented ethanol-induced apoptotic cell death. Inhibition of autophagy by wortmannin aggravated ethanol-mediated necrotic cell death.Inhibition of autophagy via ethanol enhances susceptibility to cell death.

The cholinergic anti-inflammatory pathway is a neural mechanism that suppresses the innate inflammatory response and controls inflammation employing acetylcholine as the key endogenous mediator. In this study, we investigated the effects of the cholinergic agonists, physostigmine and donepezil, on neurodegeneration, inflammation and oxidative stress during oxygen toxicity in the developing rat brain. The aim of this study was to investigate the level of neurodegeneration, expression of proinflammatory cytokines, glutathione and lipid peroxidation after hyperoxia and treatment with the acetylcholinesterase (AChE) inhibitors, physostigmine and donepezil in the brain of neonatal rats. Six-day-old Wistar rats were exposed to 80% oxygen for 12-24 h and received 100 μg/kg physostigmine or 200 μg/kg donepezil intraperitoneally. Sex-matched littermates kept in room air and injected with normal saline, physostigmine or donepezil served as controls. Treatment with both inhibitors significantly reduced hyperoxia-triggered activity of AChE, neural cell death and the upregulation of the proinflammatory cytokines IL-1β and TNF-α in the immature rat brain on the mRNA and protein level. In parallel, hyperoxia-induced oxidative stress was reduced by concomitant physostigmine and donepezil administration, as shown by an increased reduced/oxidized glutathione ratio and attenuated malondialdehyde levels, as a sign of lipid peroxidation. Our results suggest that a single treatment with AChE inhibitors at the beginning of hyperoxia attenuated the detrimental effects of oxygen toxicity in the developing brain and may pave the way for AChE inhibitors, which are currently used for the treatment of Alzheimer's disease, as potential candidates for adjunctive neuroprotective therapies to the immature brain.

Surgical patients are at high risk for developing infectious complications and postoperative delirium. Prolonged infections and delirium result in worse outcome. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and influenza vaccination are known to increase HLA-DR on monocytes and improve immune reactivity. This study aimed to investigate whether GM-CSF or vaccination reverses monocyte deactivation. Secondary aims were whether it decreases infection and delirium days after esophageal or pancreatic resection over time.In this prospective, randomized, placebo-controlled, double-blind, double dummy trial setting on an interdisciplinary ICU of a university hospital 61 patients with immunosuppression (monocytic HLA-DR [mHLA-DR] <10,000 monoclonal antibodies [mAb] per cell) on the first day after esophageal or pancreatic resection were treated with either GM-CSF (250 μg/m2/d), influenza vaccination (Mutagrip 0.5 ml/d) or placebo for a maximum of 3 consecutive days if mHLA-DR remained below 10,000 mAb per cell. HLA-DR on monocytes was measured daily until day 5 after surgery. Infections and delirium were followed up for 9 days after surgery. Primary outcome was HLA-DR on monocytes, and secondary outcomes were duration of infection and delirium.mHLA-DR was significantly increased compared to placebo (p < 0.001) and influenza vaccination (p < 0.001) on the second postoperative day. Compared with placebo, GM-CSF-treated patients revealed shorter duration of infection (p < 0.001); the duration of delirium was increased after vaccination (p = 0.003).Treatment with GM-CSF in patients with postoperative immune suppression was safe and effective in restoring monocytic immune competence. Furthermore, therapy with GM-CSF reduced duration of infection in immune compromised patients. However, influenza vaccination increased duration of delirium after major surgery.www.controlled-trials.com ISRCTN27114642.

Purpose: Although the role of the angiotensin II type 2 (AT2) receptor in acute lung injury is not yet completely understood, a protective role of this receptor subtype has been suggested. We hypothesized that, in a rodent model of acute lung injury, stimulation of the AT2 receptor with the direct agonist Compound 21 (C21) might have a beneficial effect on pulmonary inflammation and might improve pulmonary gas exchange. Materials and methods: Male adult rats were divided into a treatment group that received pulmonary lavage followed by mechanical ventilation (LAV, n=9), a group receiving pulmonary lavage, mechanical ventilation, and direct stimulation of the AT2 receptor with C21 (LAV+C21, n=9), and a control group that received mechanical ventilation only (control, n=9). Arterial blood gas analysis was performed every 30 min throughout the 240-min observation period. Lung tissue and plasma samples were obtained at 240 min after the start of mechanical ventilation. Protein content and surface activity of bronchoalveolar lavage fluid were assessed and the wet/dry-weight ratio of lungs was determined. Transcriptional and translational regulation of pro- and antiinflammatory cytokines IL-1β, tumor necrosis factor-alpha, IL-6, IL-10, and IL-4 was determined in lungs and in plasma. Results: Pulmonary lavage led to a significant impairment of gas exchange, the formation of lung edema, and the induction of pulmonary inflammation. Protein content of lavage fluid was increased and contained washed-out surfactant. Direct AT2 receptor stimulation with C21 led to a significant inhibition of tumor necrosis factor-alpha and IL-6 expressions in the lungs, whereas the expressions of IL-1, IL-10, and IL-4 remained unchanged. During the 240-min observation period, AT2 receptor stimulation did not improve pulmonary gas exchange or lung edema. Conclusion: In this rodent model of acute lung injury after repeated pulmonary lavage, AT2 receptor stimulation attenuates pulmonary inflammation but does not improve gas exchange. Keywords: AT2 receptor, lung failure, ARDS, acute lung injury, Compound 21 (C21)

Reactive oxygen species (ROS) and intrinsic antioxidant defense systems play an important role in both physiological cell signaling processes and many pathological states, including neurodegenerative disorders and oxygen-toxicity. Here we report that short exposures to non-physiologic oxygen levels change the balance of the ROS-dependent thioredoxin/peroxiredoxin system in the developing rat brain. The aim of this study was to evaluate the expression of peroxiredoxins, thioredoxin 1, sulfiredoxin 1, and DJ-1 on gene and protein level under hyperoxic conditions. Six-days old Wistar rats were exposed to 80% oxygen for 6–48 h while sex-matched littermates were kept in room-air and served as controls. Oxygen-toxicity significantly induced upregulation of peroxiredoxins 1 and 2, peroxiredoxin sulfonic form, thioredoxin 1, and sulfiredoxin 1 in the brains of infant rats. Additionally, hyperoxia reduced the level of DJ-1, a hydroperoxide-responsive protein in the developing rat brain. The pathology of hyperoxia-mediated injury to the developing brain is still elusive and oxygen administration to neonates is often inevitable. These findings may provide evidence for the development of targeted therapeutic strategies to enhance the antioxidative defense of the immature brain.

Background: Postoperative immune suppression, particularly a loss of cell-mediated immunity, is commonly seen after surgery and is associated with worse outcome, i.e. delayed wound healing, infections, sepsis, multiple-organ failure and cancer recurrence. However, the recovery of immune cells focusing on differences between innate and acquired immunity during severe postoperative immunosuppression is not investigated. Methods: In this retrospective randomized controlled trial (RCT) subgroup analysis, 10 postoperatively immune suppressed patients after esophageal or pancreatic resection were analyzed. Innate and acquired immune cells, the expression of human leukocyte antigen-D related on monocytes (mHLA-DR), lipopolysaccharide (LPS)-induced monocytic TNF-α and IL-10 secretion ex vivo, Concanavalin A (Con A)-induced IFN-γ, TNF-α, IL-2, IL-4, IL-5 and IL-10 release were measured preoperatively (od) until day 5 after surgery (pod5). Recovery of immune cells was defined by a significant decrease respectively increase after a significant postoperative alteration. Statistical analyses were performed using nonparametric statistical procedures. Results: Postoperative alterations of innate immune cells recovered on pod2 (eosinophils), pod3 (neutrophils) and pod5 (mHLA-DR, monocytic TNF-α and IL-10 secretion), whereas alterations of acquired immune cells (lymphocytes, T cells, T helper cells, and cytotoxic T cells) did not recover until pod5. Peripheral blood T cells showed an impaired production of the T helper (Th) 1 cytokine IFN-γ upon Con A stimulation on pod1, while Th2 specific cytokine release did not change until pod5.Conclusions: Innate immunity recovered earlier than acquired immunity during severe postoperative immunosuppression. Furthermore, we found a more anti- than pro-inflammatory T cell function on the first day after surgery, while T cell counts decreased.

Mechanical ventilation can cause ventilator-induced brain injury via afferent vagal signaling and hippocampal neurotransmitter imbalances. The triggering mechanisms for vagal signaling during mechanical ventilation are unknown. The objective of this study was to assess whether pulmonary transient receptor potential vanilloid type-4 (TRPV4) mechanoreceptors and vagal afferent purinergic receptors (P2X) act as triggers of ventilator-induced brain injury.Controlled, human in vitro and ex vivo studies, as well as murine in vivo laboratory studies.Research laboratory.Wild-type, TRPV4-deficient C57BL/6J mice, 8-10 weeks old. Human postmortem lung tissue and human lung epithelial cell line BEAS-2B.Mice subjected to mechanical ventilation were studied using functional MRI to assess hippocampal activity. The effects of lidocaine (a nonselective ion-channel inhibitor), P2X-purinoceptor antagonist (iso-PPADS), or genetic TRPV4 deficiency on hippocampal dopamine-dependent pro-apoptotic signaling were studied in mechanically ventilated mice. Human lung epithelial cells (BEAS-2B) were used to study the effects of mechanical stretch on TRPV4 and P2X expression and activation. TRPV4 levels were measured in postmortem lung tissue from ventilated and nonventilated patients.Hippocampus functional MRI analysis revealed considerable changes in response to the increase in tidal volume during mechanical ventilation. Intratracheal lidocaine, iso-PPADS, and TRPV4 genetic deficiency protected mice against ventilationinduced hippocampal pro-apoptotic signaling. Mechanical stretch in both, BEAS-2B cells and ventilated wild-type mice, resulted in TRPV4 activation and reduced Trpv4 and P2x expression. Intratracheal replenishment of adenosine triphosphate in Trpv4 mice abrogated the protective effect of TRPV4 deficiency. Autopsy lung tissue from ventilated patients showed decreased lung TRPV4 levels compared with nonventilated CONCLUSIONS:: TRPV4 mechanosensors and purinergic receptors are involved in the mechanisms of ventilator-induced brain injury. Inhibition of this neural signaling, either using nonspecific or specific inhibitors targeting the TRPV4/adenosine triphosphate/P2X signaling axis, may represent a novel strategy to prevent or treat ventilator-induced brain injury.

Infections and perioperative stress can lead to neuroinflammation, which in turn is linked to cognitive impairments such as postoperative delirium or postoperative cognitive dysfunctions. The α2-adrenoceptor agonist dexmedetomidine (DEX) prevents cognitive impairments and has organo-protective and anti-inflammatory properties. Macroautophagy (autophagy) regulates many biological processes, but its role in DEX-mediated anti-inflammation and the underlying mechanism of DEX remains largely unclear. We were interested how a pretreatment with DEX protects against lipopolysaccharide (LPS)-induced inflammation in adult male Wistar rats. We used Western blot and activity assays to study how DEX modulated autophagy- and apoptosis-associated proteins as well as molecules of the cholinergic anti-inflammatory pathway, and qPCR to analyse the expression of autophagy and inflammation-associated microRNAs (miRNA) in the spleen, cortex and hippocampus at different time points (6 h, 24 h, 7 d). We showed that a DEX pretreatment prevents LPS-induced impairments in autophagic flux and attenuates the LPS-induced increase in the apoptosis-associated protein cleaved poly(ADP-ribose)-polymerase (PARP) in the spleen. Both, DEX and LPS altered miRNA expression and molecules of the cholinergic anti-inflammatory pathway in the spleen and brain. While only a certain set of miRNAs was up- and/or downregulated by LPS in each tissue, which was prevented or attenuated by a DEX pretreatment in the spleen and hippocampus, all miRNAs were up- and/or downregulated by DEX itself - independent of whether or not they were altered by LPS. Our results indicate that the organo-protective effect of DEX may be mediated by autophagy, possibly by acting on associated miRNAs, and the cholinergic anti-inflammatory pathway. Preventive effects of DEX on LPS-induced inflammation. DEX restores the LPS-induced impairments in autophagic flux, attenuates PARP cleavage and alters molecules of the cholinergic system in the spleen. Furthermore, DEX alters and prevents LPS-induced miRNA expression changes in the spleen and brain along with LPS.

Impaired cerebellar development of premature infants and the associated impairment of cerebellar functions in cognitive development could be crucial factors for neurodevelopmental disorders. Anesthetic- and hyperoxia-induced neurotoxicity of the immature brain can lead to learning and behavioral disorders. Dexmedetomidine (DEX), which is associated with neuroprotective properties, is increasingly being studied for off-label use in the NICU. For this purpose, six-day-old Wistar rats (P6) were exposed to hyperoxia (80% O2) or normoxia (21% O2) for 24 h after DEX (5 µg/kg, i.p.) or vehicle (0.9% NaCl) application. An initial detection in the immature rat cerebellum was performed after the termination of hyperoxia at P7 and then after recovery in room air at P9, P11, and P14. Hyperoxia reduced the proportion of Calb1+-Purkinje cells and affected the dendrite length at P7 and/or P9/P11. Proliferating Pax6+-granule progenitors remained reduced after hyperoxia and until P14. The expression of neurotrophins and neuronal transcription factors/markers of proliferation, migration, and survival were also reduced by oxidative stress in different manners. DEX demonstrated protective effects on hyperoxia-injured Purkinje cells, and DEX without hyperoxia modulated neuronal transcription in the short term without any effects at the cellular level. DEX protects hyperoxia-damaged Purkinje cells and appears to differentially affect cerebellar granular cell neurogenesis following oxidative stress.

The risk of oxidative stress is unavoidable in preterm infants and increases the risk of neonatal morbidities. Premature infants often require sedation and analgesia, and the commonly used opioids and benzodiazepines are associated with adverse effects. Impairment of cerebellar functions during cognitive development could be a crucial factor in neurodevelopmental disorders of prematurity. Recent studies have focused on dexmedetomidine (DEX), which has been associated with potential neuroprotective properties and is used as an off-label application in neonatal units. Wistar rats (P6) were exposed to 80% hyperoxia for 24 h and received as pretreatment a single dose of DEX (5µg/kg, i.p.). Analyses in the immature rat cerebellum immediately after hyperoxia (P7) and after recovery to room air (P9, P11, and P14) included examinations for cell death and inflammatory and oxidative responses. Acute exposure to high oxygen concentrations caused a significant oxidative stress response, with a return to normal levels by P14. A marked reduction of hyperoxia-mediated damage was demonstrated after DEX pretreatment. DEX produced a much earlier recovery than in controls, confirming a neuroprotective effect of DEX on alterations elicited by oxygen stress on the developing cerebellum.

Preterm birth is a risk factor for cardiometabolic disease. The preterm heart before terminal differentiation is in a phase that is crucial for the number and structure of cardiomyocytes in further development, with adverse effects of hypoxic and hyperoxic events. Pharmacological intervention could attenuate the negative effects of oxygen. Dexmedetomidine (DEX) is an α2-adrenoceptor agonist and has been mentioned in connection with cardio-protective benefits. In this study, H9c2 myocytes and primary fetal rat cardiomyocytes (NRCM) were cultured for 24 h under hypoxic condition (5% O2), corresponding to fetal physioxia (pO2 32-45 mmHg), ambient oxygen (21% O2, pO2 ~150 mmHg), or hyperoxic conditions (80% O2, pO2 ~300 mmHg). Subsequently, the effects of DEX preconditioning (0.1 µM, 1 µM, 10 µM) were analyzed. Modulated oxygen tension reduced both proliferating cardiomyocytes and transcripts (CycD2). High-oxygen tension induced hypertrophy in H9c2 cells. Cell-death-associated transcripts for caspase-dependent apoptosis (Casp3/8) increased, whereas caspase-independent transcripts (AIF) increased in H9c2 cells and decreased in NRCMs. Autophagy-related mediators (Atg5/12) were induced in H9c2 under both oxygen conditions, whereas they were downregulated in NRCMs. DEX preconditioning protected H9c2 and NRCMs from oxidative stress through inhibition of transcription of the oxidative stress marker GCLC, and inhibited the transcription of both the redox-sensitive transcription factors Nrf2 under hyperoxia and Hif1α under hypoxia. In addition, DEX normalized the gene expression of Hippo-pathway mediators (YAP1, Tead1, Lats2, Cul7) that exhibited abnormalities due to differential oxygen tensions compared with normoxia, suggesting that DEX modulates the activation of the Hippo pathway. This, in the context of the protective impact of redox-sensitive factors, may provide a possible rationale for the cardio-protective effects of DEX in oxygen-modulated requirements on survival-promoting transcripts of immortalized and fetal cardiomyocytes.

Past studies have observed that brain atrophy may accelerate after surgical procedures. Furthermore, an association of systemic inflammation with neurodegeneration has been described. We hypothesize that postoperative interleukin (IL) levels in circulation as well as the perioperative change in interleukin levels are associated with increased postoperative atrophy in the Nucleus basalis magnocellularis (of Meynert, NBM) which is the major source of cortical acetylcholine. We analyzed data from the BioCog cohort which included patients ≥ 65 years presenting for elective major surgery (≥ 60min). Blood samples were taken before surgery and on the first postoperative day. Magnetic resonance imaging of the brain and neuropsychological assessments were conducted before surgery and after three months follow-up. We used linear regression analysis to determine the association of three interleukins (IL6, IL8 and IL18) with NBM atrophy (in % volume change from baseline before surgery to follow-up), as well as to examine the associations of NBM atrophy and volume with postoperative cognitive ability and perioperative cognitive change. Receiver-operating curves were used to determine the prognostic value of preoperative interleukin levels. For IL8 (N = 97) and IL18 (N = 217), but not IL6 (N = 240), we observed significant associations of higher postoperative IL levels at the first postoperative day with higher NBM atrophy at three months after surgery. Subsequent analyses suggested that in both IL8 and IL18, this association was driven by a more general association of chronically elevated IL levels and NBM atrophy, reflected by preoperative IL concentrations, rather than IL response to surgery, measured as the difference between pre- and postoperative IL concentrations. At follow-up, NBM volume was positively associated with the level of cognitive performance, but NBM atrophy was not significantly related to perioperative cognitive change. Prognostic value of preoperative IL concentrations for NBM atrophy was low. Our results suggest that an association of postoperative interleukin levels with NBM atrophy is driven by preoperatively elevated interleukins due to pre-existing inflammation, rather than perioperative change in interleukin levels in response to surgery and anesthesia. The BioCog study has been registered at clinicaltrials.gov on Oct 15, 2014 (NCT02265263).

Background: Acute ethanol intoxication has the potential to alter immune reactivity by various pathways. The aim of this study was to investigate T-helper cell subsets transcription factors and cytokines in human peripheral blood mononuclear cells (PBMCs) following a single dose of lipopolysaccharide (LPS) with or without ethanol exposure. Methods: Human PBMCs were cultured in the presence of 100 mM ethanol and/or 100 ng/ml LPS for various time periods (1, 3, 8, and 24 hours) and analyzed for the kinetics of gene expression by quantitative real-time PCR of selected transcription factors (T-bet, GATA3, Foxp3, and RORγt) and cytokines (TNF-α, IL-6, IL-10, and IFN-γ). The proportion of Th17 and Treg cells was identified 24 hours after treatment with ethanol and LPS by multiparameter flow cytometry. Viability and amount of dead cells were analyzed after 24 and 48 hours by MTT assay and flow cytometry. Results: Following LPS challenge, gene expression of Foxp3 increased, whereas RORγt decreased after 3 hours, GATA3 decreased within 1 hour, whereas expression of T-bet did not change at any time. Gene expression of TNF-α, interferon-γ (IFN-γ), and IL-6 peaked after 3 hours, expression of IL-10 peaked after 8 hours. Ethanol suppressed the LPS-induced gene expression of Foxp3, RORγt, and T-bet after 8 hours, expression of TNF-α and IFN-γ was also suppressed after 3 and 8 hours. Markers of inflammation including TNF-α and IL-1β in supernatant of PBMCs were significantly decreased, while levels of IL-10 and IL-6 remained unchanged following ethanol exposure. Furthermore, ethanol-treated cells alone or in combination with LPS had significantly fewer IL-17- and IFN-γ-secreting CD4+ T cells but constant proportion of Treg cells when compared to control cells. Proliferation and viability of the cells were not influenced under these conditions. Conclusions: Alcohol interferes with the kinetics of Foxp3, RORγt, and T-bet gene expression and the production of TNF-α and IL-1ß and influences the balance of Treg/Th17 cells following LPS exposure.

Hyperglycemia in hospitalized patients is associated with increased morbidity and mortality. Postoperative infections are particularly common in the setting of perioperative hyperglycemia; however, the relationship between perioperative glucose levels and the innate immune system remains unclear.Immune cells, monocytic respectively T cell function and T cell subspecies of 32 patients after esophageal or pancreatic resection were analyzed preoperatively and on the first day after surgery (POD 1). Perioperative blood glucose was measured hourly via arterial blood gas analyses. Groups were classified by maximum perioperative glucose levels: <180 mg/dL versus at least one episode of ≥180 mg/dL. The suppression of immune cells and cytokines was defined as the difference between pre- and postoperative values.In perioperative hyperglycemic patients, preoperative CD4+/CD8+ ratio (P=0.039), count of CD4+ T cells (P=0.039) and release of IFN-γ (P=0.013) and TNF-α (P=0.045) after ex-vivo T cell stimulation of whole blood were significantly higher. Furthermore, the postoperative count of basophils was significantly lower (P=0.011), HLA-DR expressing CD8- T cells were tendentially lower (P=0.058) and more suppressed (P=0.035). The suppression of IFN-γ (P=0.003) and TNF-α (P=0.006) was significantly higher in these patients after ex-vivo T cell stimulation but absolute values were similar between the groups. IL-10 release of lipopolysaccharide-stimulated whole blood was tendentially more suppressed after perioperative hyperglycemia (P=0.084).Severe perioperative hyperglycemia attenuated postoperative basophil count, T cell activation and monocytic function. These patients were also at preoperative higher immune activation.

Exogenous stress like tissue damage and pathogen invasion during surgical trauma could lead to a peripheral inflammatory response and induce neuroinflammation, which can result in postoperative cognitive dysfunction (POCD).The cholinergic anti-inflammatory pathway is a neurohumoral mechanism that plays a prominent role by suppressing the inflammatory response.Treatments with acetylcholinesterase inhibitors enhance cholinergic transmission and may therefore act as a potential approach to prevent neuroinflammation.In the presence or absence of acetylcholinesterase inhibitors, adult Wistar rats underwent surgery alone or were additionally treated with lipopolysaccharide (LPS).Physostigmine, which can overcome the blood-brain barrier or neostigmine acting only peripheral, served as acetylcholinesterase inhibitors.The expression of pro-and anti-inflammatory cytokines in the cortex, hippocampus, spleen and plasma was measured after 1 h, 24 h, 3 d and 7 d using Real-Time PCR, western blot analysis or cytometric bead array (CBA).Fluoro-Jade B staining of brain slices was employed to elucidate neurodegeneration.The activity of acetylcholinesterase was estimated using a spectrofluorometric method.Surgery accompanied by LPS-treatment led to increased IL-1beta gene and protein upregulation in the cortex and hippocampus but was significantly reduced by physostigmine and neostigmine.Furthermore, surgery in combination with LPS-treatment caused increased protein expression of IL-1, TNF-alpha and IL-10 in the spleen and plasma.Physostigmine and neostigmine significantly decreased the protein expression of IL-1 and TNFalpha.Neuronal degeneration and the activity of acetylcholinesterase were elevated after surgery with LPS-treatment and reduced by physostigmine and neostigmine.Along with LPS-treatment, acetylcholinesterase inhibitors reduce the proinflammatory response as well as neurodegeneration after surgery in the cortex and hippocampus.This combination may represent a tool to break the pathogenesis of POCD.

Molecular mechanisms accompanying ethanol-induced cytotoxicity remain to be defined. The renin–angiotensin system with its respective receptors, the angiotensin AT1 and AT2 receptor (AT1R and AT2R), has been implicated in these processes. The AT2R seems to counteract the pro-inflammatory, pro-hypertrophic, and pro-fibrotic actions of the AT1R and is involved in cellular differentiation and tissue repair. Recently, we identified poly(ADP-ribose) polymerase-1 (PARP-1) as a novel negative transcriptional regulator of the AT2R. However, the complex interactions between ethanol, PARP-1, and the AT2R are largely unknown. In this in vitro study, we aimed to clarify whether acute ethanol treatment modifies AT2R promoter activity or AT2R mRNA and protein levels and whether PARP-1 is involved in ethanol-mediated regulation of the AT2R. Murine fibroblasts of the R3T3 and MEF line (murine embryonic fibroblasts) were exposed to ethanol for 24 h. AT2R promoter activity, mRNA and protein levels were analyzed with and without PARP-1 inhibition and in PARP-1 knockout MEF cells. Expression of PARP-1 was analyzed over course of time, and cell viability and DNA fragmentation were measured on single-cell level by flow cytometry. Ethanol exposition induced substantial downregulation of the AT2R on promoter, mRNA and protein levels in a dose-dependent manner. Pharmacological inhibition or ablation of PARP-1 completely abolished this effect. Ethanol treatment did not have any effect on AT1R mRNA and protein levels in MEF cells. Further, acute ethanol treatment promoted DNA fragmentation and caused transcriptional induction of PARP-1. Our findings reveal that PARP-1 is an upstream transcriptional regulator of the AT2 receptor in the context of ethanol exposure and represses the AT2R gene in fibroblasts in vitro. Variations in expression of the potentially tissue-protective AT2R might contribute to ethanol-mediated pathology.

Monocytic human leukocyte antigen D related (mHLA-DR) is essential for antigen-presentation. Downregulation of mHLA-DR emerged as a general biomarker of impaired immunity seen in patients with sepsis and pneumonia and after major surgery. Influencing factors of mHLA-DR such as age, overweight, diabetes, smoking, and gender remain unclear.We analyzed 20 patients after esophageal or pancreatic resection of a prospective, randomized, placebo-controlled, double-blind trial (placebo group). mHLA-DR was determined from day of surgery (od) until postoperative day (pod) 5. Statistical analyses were performed using multivariate generalized estimating equation analyses (GEE), nonparametric multivariate analysis of longitudinal data, and univariate post hoc nonparametric Mann-Whitney tests.In GEE, smoking and gender were confirmed as significant influencing factors over time. Univariate analyses of mHLA-DR between smokers and nonsmokers showed lower preoperative levels (p = 0.010) and a trend towards lower levels on pod5 (p = 0.056) in smokers. Lower mHLA-DR was seen in men on pod3 (p = 0.038) and on pod5 (p = 0.026). Overweight patients (BMI > 25 kg/m2) had lower levels of mHLA-DR on pod3 (p = 0.039) and pod4 (p = 0.047).Smoking is an important influencing factor on pre- and postoperative immune function while postoperative immune function was influenced by gender and overweight. Clinical trial registered with ISRCTN27114642.

Dexmedetomidine is an alpha-2 adrenoreceptor agonist with anti-inflammatory and anti-delirogenic properties. Pathogenesis of postoperative delirium (POD) includes cholinergic dysfunction and deregulated inflammatory response to surgical trauma. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) are discussed as biomarkers for both POD and severity in acute inflammation. To show whether there is a link between blood cholinesterase activities and dexmedetomidine, we performed a secondary analysis of a randomised, double-blind, placebo-controlled trial that recently showed a lower incidence of POD in the dexmedetomidine group. Abdominal or cardiac surgical patients aged ≥ 60 years were randomised to receive dexmedetomidine or placebo intra- and postoperatively in addition to standard general anaesthesia. We analysed the course of perioperative cholinesterase activities of 56 patients, measured preoperatively and twice postoperatively. Dexmedetomidine resulted in no change in AChE activity and caused a rapid recovery of BChE activity after an initial decrease, while placebo showed a significant decrease in both cholinesterase activities. There were no significant between-group differences at any point in time. From these data it can be assumed that dexmedetomidine could alleviate POD via altering the cholinergic anti-inflammatory pathway (CAIP). We advocate for further investigations to show the direct connection between dexmedetomidine and cholinesterase activity.

Patients undergoing high-risk surgery show haemodynamic instability and an increased risk of morbidity. However, most of the available data concentrate on the intraoperative period. This study aims to characterise patients with advanced haemodynamic monitoring throughout the whole perioperative period using electrical cardiometry.In a prospective, observational, monocentric pilot study, electrical cardiometry measurements were obtained using an Osypka ICON™ monitor before surgery, during surgery, and repeatedly throughout the hospital stay for 30 patients with primary ovarian cancer undergoing multivisceral cytoreductive surgery. Severe postoperative complications according to the Clavien-Dindo classification were used as a grouping criterion.The relative change from the baseline to the first intraoperative timepoint showed a reduced heart rate (HR, median - 19 [25-quartile - 26%; 75-quartile - 10%]%, p < 0.0001), stroke volume index (SVI, - 9.5 [- 15.3; 3.2]%, p = 0.0038), cardiac index (CI, - 24.5 [- 32; - 13]%, p < 0.0001) and index of contractility (- 17.5 [- 35.3; - 0.8]%, p < 0.0001). Throughout the perioperative course, patients had intraoperatively a reduced HR and CI (both p < 0.0001) and postoperatively an increased HR (p < 0.0001) and CI (p = 0.016), whereas SVI was unchanged. Thoracic fluid volume increased continuously versus preoperative values and did not normalise up to the day of discharge. Patients having postoperative complications showed a lower index of contractility (p = 0.0435) and a higher systolic time ratio (p = 0.0008) over the perioperative course in comparison to patients without complications, whereas the CI (p = 0.3337) was comparable between groups. One patient had to be excluded from data analysis for not receiving the planned surgery.Substantial decreases in HR, SVI, CI, and index of contractility occurred from the day before surgery to the first intraoperative timepoint. HR and CI were altered throughout the perioperative course. Patients with postoperative complications differed from patients without complications in the markers of cardiac function, a lower index of contractility and a lower SVI. The analyses of trends over the whole perioperative time course by using non-invasive technologies like EC seem to be useful to identify patients with altered haemodynamic parameters and therefore at an increased risk for postoperative complications after major surgery.

Background Chronic alcohol consumption leads to oxidative stress in a variety of cells, especially in brain cells because they have a reduced oxidative metabolism of alcohol. Uncoupling proteins ( UCP s) are anion channels of the inner mitochondrial membrane, which can decouple internal respiration. “Mild uncoupling” of the mitochondrial respiratory chain leads to a reduced production of free radicals (reactive oxygen species) and a reduction in oxidative cell stress. The extent to which chronic alcohol consumption regulates UCP‐2 and ‐4 in the brain is still unknown. Methods We examined the effects of a 12‐week 5% alcohol diet in the brain of male W istar rats ( n = 34). Cerebral gene and protein expression of UCP ‐2, ‐4, as well as B cl‐2, and the release of cytochrome c out of the mitochondria were detected by real‐time polymerase chain reaction and W estern blot analysis. The percentage of degenerated cells was determined by F luoro– J ade B staining of brain slices. Results Brains of rats with a chronic alcohol diet showed an increased gene and protein expression of UCP ‐2 and ‐4. The expression of the antiapoptotic protein B cl‐2 in the brain of the alcohol‐treated animals was decreased significantly, whereas cytochrome c release from mitochondria was increased. In addition increased neurodegeneration could be demonstrated in the alcohol‐treated animals. Conclusions Chronic alcohol consumption leads to a cerebral induction of UCP ‐2 and ‐4 with a simultaneous decrease in the antiapoptotic protein B cl‐2, cytochrome c release from mitochondria and increased neurodegeneration. This study reveals a compensatory effect of UCP ‐2 and ‐4 in the brain during chronic alcohol consumption.

Synthetic alkylphospholipids (APLs), exhibit similarity to the platelet-activating factor (PAF). These compounds have antiproliferative effects on tumour cells and can therefore be regarded as a new class of drugs. Unlike classic cytostatic agents, synthetic alkylphospholipids do not interfere with the DNA or the mitotic spindle apparatus. Instead, due to their aliphatic character, alkylphospholipids accumulate in cell membranes, where they have an impact on lipid metabolism and lipid-dependent signalling pathways which leads to inhibition of proliferation and induction of apoptosis in malignant cells. Normal cells remain unaffected by these compounds. Glycosidated phospholipids, are a novel class of alkylphospholipids, in which carbohydrates or carbohydrate-related molecules are introduced in the chemical lead of PAF. These hybrid alkylphospholipids also exhibit anti-proliferative capacity. Furthermore, members of this subfamily also modulate cell adhesion, differentiation, apoptosis and migration of tumour cells. Among the members of this group, Inositol-C2-platelet-activating factor (Ino-C2-PAF) is the most effective compound developed so far. Recently, we also showed that Ino-C2-PAF exhibited the strongest impact on the gene expression levels of immortalised keratinocytes in comparison to edelfosine and another glycosidated alkylphospholipid, Glucose-platelet-activating factor (Glc-PAF). Furthermore, Ino-C2-PAF reduced the expression of genes encoding proteins associated with inflammation and the innate and acquired immune responses. Keywords: Apoptosis, cell proliferation, differentiation, gene expression, glycosidated phospholipids, migration.

BACKGROUND: Granulocyte macrophage colony-stimulating factor (GM-CSF) can be used as a potent stimulator for immune suppressed patients as defined by a decrease of human leukocyte antigen-D related expression on monocytes (mHLA-DR) after surgery.However, the exact role of GM-CSF on monocytic and T cell function is unclear.METHODS: In this retrospective randomized controlled trial (RCT) subgroup analysis, monocytic respectively T cell function and T cell subspecies of 20 immune suppressed (i.e.mHLA-DR levels below 10,000 monoclonal antibodies (mAb) per cell at the first day after surgery) patients after esophageal or pancreatic resection were analyzed.Each 10 patients received either GM-CSF (250 μg/m²/d) or placebo for a maximum of three consecutive days if mHLA-DR levels remained below 10,000 mAb per cell.mHLA-DR and further parameters of immune function were measured preoperatively (od) until day 5 after surgery (pod5).Statistical analyses were performed using nonparametric statistical procedures.RESULTS: In multivariate analysis, mHLA-DR significantly differed between the groups (p < 0.001).mHLA-DR was increased on pod2 (p < 0.001) and pod3 (p = 0.002) after GM-CSF application.Tumor necrosis factor-α (TNF-α) release of lipopolysaccharide (LPS) stimulated monocytes multivariately significantly differed between the groups (p < 0.008) and was increased in the GM-CSF group on pod2 (p < 0.001) and pod3 (p = 0.046).Th17/regulatory T (Treg) cell ratio was higher after GM-CSF treatment on pod2 (p = 0.041).No differences were seen in lymphocytes and T helper cell (Th)1/Th2 specific cytokine production after T cell stimulation with Concanavalin (Con) A between the groups.CONCLUSIONS: Postoperative application of GM-CSF significantly enhanced qualitative monocytic function by increased mHLA-DR and TNF-α release after LPS stimulation and apparently enhanced Th17/Treg ratio.Clinical trial registered with www.controlled-trials.com(ISRCTN27114642) 05 December 2008.

Haemophagocytic lymphohistiocytosis (HLH) in adults is characterised by toxic immune activation and a sepsis-like syndrome, leading to high numbers of undiagnosed cases and mortality rates of up to 68%. Early diagnosis and specific immune suppressive treatment are mandatory to avoid fatal outcome, but the diagnostic criteria (HLH-2004) are adopted from paediatric HLH and have not been validated in adults. Experimental studies suggest biomarkers to sufficiently diagnose HLH. However, biomarkers for the diagnosis of adult HLH have not yet been investigated.The HEMICU (Diagnostic biomarkers for adult haemophagocytic lymphohistiocytosis in critically ill patients) study aims to estimate the incidence rate of adult HLH among suspected adult patients in intensive care units (ICUs). Screening for HLH will be performed in 16 ICUs of Charité - Universitätsmedizin Berlin. The inclusion criteria are bicytopaenia, hyperferritinaemia (≥500 µg/L), fever or when HLH is suspected by the clinician. Over a period of 2 years, we expect inclusion of about 100 patients with suspected HLH. HLH will be diagnosed if at least five of the HLH-2004 criteria are fulfilled, together with an expert review; all other included patients will serve as controls. Second, a panel of potential biomarker candidates will be explored. DNA, plasma and serum will be stored in a biobank. The primary endpoint of the study is the incidence rate of adult HLH among suspected adult patients during ICU stay. Out of a variety of measured biomarkers, this study furthermore aims to find highly potential biomarkers for the diagnosis of adult HLH in ICU. The results of this study will contribute to improved recognition and patient outcome of adult HLH in clinical routine.The institutional ethics committee approved this study on 1 August 2018 (Ethics Committee of Charité - Universitätsmedizin Berlin, EA4/006/18). The results of the study will be disseminated in an international peer-reviewed journal and presented at international conferences.NCT03510650.

Exposure to N-methyl-d-aspartate (NMDA) receptor antagonists has been demonstrated to induce neurodegeneration in newborn rats. However, in clinical practice the use of NMDA receptor antagonists as anesthetics and sedatives cannot always be avoided. The present study investigated the effect of the indirect cholinergic agonist physostigmine on neurotrophin expression and the extracellular matrix during NMDA receptor antagonist induced injury to the immature rat brain. The aim was to investigate matrix metalloproteinase (MMP)-2 activity, as well as expression of tissue inhibitor of metalloproteinase (TIMP)-2 and brain-derived neurotrophic factor (BDNF) after co-administration of the non-competitive NMDA receptor antagonist MK801 (dizocilpine) and the acetylcholinesterase (AChE) inhibitor physostigmine. The AChE inhibitor physostigmine ameliorated the MK801-induced reduction of BDNF mRNA and protein levels, reduced MK801-triggered MMP-2 activity and prevented decreased TIMP-2 mRNA expression. Our results indicate that AChE inhibition may prevent newborn rats from MK801-mediated brain damage by enhancing neurotrophin-associated signaling pathways and by modulating the extracellular matrix.

Uncoupling proteins (UCPs) are anion channels that can decouple the mitochondrial respiratory chain. "Mild uncoupling" of internal respiration reduces free radical production and oxidative cell stress. Chronic alcohol consumption is a potent inducer of oxidative stress in multiple tissues and regulates UCP-2 and -4 expression in the brain. To analyse the impact of chronic alcohol intake on UCP-2 expression in tissues with high endogenous UCP-2 contents, male Wistar rats (n=34) were treated with a 12-week 5% alcohol diet. In the lungs and the spleen of rats with a chronic alcohol diet cytochrome c release from mitochondria was significantly increased. Both organs did not show any altered gene and protein expression of UCP-2. Different to cerebral tissue chronic alcohol consumption has no regulatory effect on UCP-2 gene and protein expression in organs with a high endogenous UCP-2 content. Therefore, chronic alcohol consumption leads to a tissue specific expression of UCP-2.

Abstract Objective To assess the effects of epidural anesthesia (EA) on patients who underwent liver resection. Design Secondary analysis of a prospective randomized controlled trial. Setting This single-center study was conducted at an academic medical center. Methods A subset of 110 1:1 propensity score–matched patients who underwent liver resection with and without EA were analyzed. Outcome measures were pain intensity ≥5 on a numeric rating scale (NRS) at rest and during movement on postoperative days 1–5, analyzed with logistic mixed-effects models, and postoperative complications according to the Clavien-Dindo classification, length of hospital stay (LOS), and one-year survival. One-year survival in the matched cohorts was compared using a frailty model. Results EA patients were less likely to experience NRS ≥5 at rest (odds ratio = 0.06, 95% confidence interval [CI] = 0.01 to 0.28, P &amp;lt; 0.001). These findings were independent of age, sex, Charlson comorbidity index, baseline NRS, and surgical approach (open vs laparoscopic). The number and severity of postoperative complications and LOS were comparable between groups (P = 0.258, P &amp;gt; 0.999, and P = 0.467, respectively). Reduced mortality rates were seen in the EA group one year after surgery (9.1% vs 30.9%, hazard ratio = 0.32, 95% CI = 0.11 to 0.90, P = 0.031). No EA-related adverse events occurred. Earlier recovery of bowel function was seen in EA patients. Conclusions Patients with EA had better postoperative pain control and required fewer systemic opioids. Postoperative complications and LOS did not differ, although one-year survival was significantly improved in patients with EA. EA applied in liver surgery was effective and safe.

Repeated pulmonary lavage allows to reliably reproduce failure of gas exchange and major histological findings of acute lung injury (ALI). However, because the capacity of pulmonary lavage to induce pulmonary inflammation is not well established in rodents, this study aims to characterize the induction of pulmonary inflammation in a rat model of ALI.Male adult rats were divided into a treatment group (n = 9) that received pulmonary lavage with consecutive mechanical ventilation, and a control group that received mechanical ventilation only (n = 9). Arterial blood gas analyses were performed every 30 min throughout the study. Pressure-volume curves, and lung tissue and plasma samples, were obtained at 240 min after the start of mechanical ventilation. Protein content and surface activity of bronchoalveolar lavage fluid was assessed. Transcriptional and translational regulation of pro- and anti-inflammatory cytokines IL-1β, TNF-α, IL-6, and IL-10 was determined in lungs and plasma. Markers of cellular stress were measured in lung tissue.Pulmonary lavage significantly decreased lung compliance, induced hypoxia and hypercapnia, and mediated respiratory acidosis. Protein content of lavage fluid was significantly increased and contained washed out surfactant. Expression of IL-1β, TNF-α, and IL-6 mRNA and protein expression of IL-1β and TNF-α was significantly induced in lavaged lungs, without spillover into the systemic circulation. Markers of cellular stress were significantly upregulated in lavaged lungs.This model of ALI applied in rats can induce pulmonary inflammation. The model might be used to develop therapeutic strategies that target pulmonary inflammation in ALI.

Interactions between alcohol, infection, and surgery and their effect on differentiation and functionality of T helper cells are not yet completely understood. We hypothesized that alcohol and surgery disturb differentiation of T helper cells and contribute to an impaired immune response.Mice were treated with alcohol for two weeks. Saline treatment served as control. Clinical performance and weight were assessed. On day 14, a median laparotomy was performed and animals were challenged with Klebsiella pneumoniae intranasally. Bacterial load was determined in lungs and blood. T helper cell subpopulations and the released cytokines were assessed in lungs, spleens, and plasma. Key transcription factors of T cell differentiation were evaluated.Alcohol significantly impaired clinical appearance and body weight of animals with postsurgical infection (p < 0.05). Bacterial load was significantly higher after alcohol treatment (p < 0.05). T helper cell subsets and released cytokine levels were significantly altered in lung, but not in spleen. Expression of transcription factors of T helper cell lineage commitment did not translate into different counts of T helper cells.Alcohol and surgery lead to significant cellular and functional modulations of T helper cells during postsurgical infection. These effects may contribute to an impaired immune response after surgery.

p14ARF Induces G2 Cell Cycle Arrest in p53- and p21-deficient Cells by Down-regulating p34cdc2 Kinase ActivityJournal of Biological ChemistryVol. 280Issue 8PreviewThe human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16INK4a and p14ARF. Although primarily proposed to require a functional p53·Mdm-2 signaling axis, recently p14ARF has been implicated in p53-independent cell cycle regulation. Here we show that p14ARF preferentially induces a G2 arrest in tumor cells lacking functional p53 and/or p21. Expression of p14ARF impaired mitotic entry and enforced a primarily cytoplasmic localization of p34cdc2 that was associated with a decrease in p34cdc2 kinase activity and reduced p34cdc2 protein expression. Full-Text PDF Open Access This article has been withdrawn by the authors. The authors reported that in figure 3A the Ad-LacZ histogram for the HCT116-p21+/+ cells was inadvertently reused as Ad-p14ARF histogram for the HCT116-p21+/+ cells and provided an amended figure. However, the Journal analysis concluded there was an additional issue regarding a possible image reuse of the control blots corresponding to β-actin HCT116 p53-/- panel in figure 6A, and DU145 Ad-p14ARF (25, 50, 100) MOI β-actin panel in figure 6C. The issue could not be resolved due to the lack of original data more than 18 years after publication. The withdrawing authors stand by the overall findings and conclusions of the study.

Objectives Preoperative hypoalbuminaemia is associated with adverse outcome, including increased postoperative mortality in cardiovascular surgery, neurosurgery, trauma and orthopaedic surgery. However, much less is known about the association between preoperative serum albumin and clinical outcomes after liver surgery. In this study, we sought to determine whether hypoalbuminaemia before partial hepatectomy is associated with a worse postoperative outcome. Design Observational study. Setting University Medical Centre in Germany. Participants We analysed 154 patients enrolled in the perioperative PHYsostigmine prophylaxis for liver resection patients at risk for DELIrium and postOperative cognitive dysfunction (PHYDELIO) trial with a preoperative serum albumin assessment. Hypoalbuminaemia was defined as serum albumin &lt;35 g/L. Subgroups classified as hypoalbuminaemia and non-hypoalbuminaemia consisted of 32 (20.8%) and 122 (79.2%) patients, respectively. Outcome measures The outcome parameters of interest were postoperative complications according to Clavien (moderate: I, II; major: ≥III), length of intensive care unit (ICU) stay, length of hospital stay and survival rates 1 year after surgery. Results Preoperative hypoalbuminaemia was associated with the occurrence of major postoperative complications (OR 3.051 (95% CI 1.197 to 7.775); p=0.019) after adjusting for age, sex, randomisation, American Society of Anesthesiologists physical status, preoperative diagnosis and Child-Pugh class. Both ICU and hospital lengths of stay were significantly prolonged in patients with preoperative hypoalbuminaemia (OR 2.573 (95% CI 1.015 to 6.524); p=0.047 and OR 1.296 (95% CI 0.254 to 3.009); p=0.012, respectively). One-year survival was comparable between patients with and without hypoalbuminaemia. Conclusions We found that low serum albumin before surgery was associated with a worse short-term outcome after partial hepatectomy, which strengthens the prognostic value of serum albumin in the setting of liver surgery. Trial registration numbers ISRCTN18978802 and EudraCT 2008-007237-47.

Editor, We would like to report on a randomised, double-blind controlled trial we undertook in our University Hospital, Department of Anaesthesiology and Intensive Care Medicine in Berlin, Germany. The primary aim of this trial was to investigate the effects on mHLA-DR (monocytic human leucocyte antigen) expression and postoperative infection rates of preoperative vaccination in cancer patients undergoing surgery of the upper aerodigestive tract. In a previous pilot study, our research group found an increase of mHLA-DR expression after influenza vaccination compared with placebo in patients with cancer of the upper aero-digestive tract without subsequent surgery.1 Ethics approval for this study was provided by the local ethics committee (Ethics board Charité–University Medicine Berlin, Campus Mitte, chairperson: Prof. Uebelhack), on 9 March 2005 (ref: EA 1/048/04). The study evaluated consecutive patients scheduled for surgery on upper aero digestive tract cancer (neck dissection or upper oesophageal resection) between November 2005 and July 2008. Patients were excluded if less than 18 years old, pregnant or lactating, were of American Society of Anesthesiologists (ASA) classification IV or V, had any diagnosed infection in the previous 14 days, were HIV positive, had liver cirrhosis (Child B or C), had undergone organ transplantation, were immune suppressed, had been treated with corticosteroids in the previous 4 weeks, had a recent myocardial infarction (less than 8 weeks previously), had diabetes mellitus with sequelae, had undergone a splenectomy, had a hypoproteinaemia, had a known incompatibility or allergy against any component of the vaccine or had a significant psychiatric illness. After obtaining written informed consent, patients were randomised to one of the two treatment groups: vaccination group or the placebo group stratified to the type of surgery. The first vaccination was performed on the day before surgery at 07:00 p.m.; the second vaccination was performed on the day of surgery at 7 : 00 a.m. Four blood samples for analysis of mHLA-DR expression were taken from each patient: immediately before the first vaccination, immediately before the second vaccination, and on the first and third postoperative days (POD). Vaccination was performed with the haemagglutinin antigens of the influenza virus (Mutagrip; Sanofi Pasteur MSD, Lyon, France) for the northern hemisphere. The nonvaccinated group of patients received 0.5 ml 0.9% normal saline subcutaneously. Statistical analyses of metric parameters were performed by nonparametric tests such as (exact) Mann–Whitney U test (for independent samples). Nominal-scaled clinical data were analysed using (exact) Chi-square tests. Differences between treatments with reference to baseline were tested with both univariate and multivariate nonparametric analysis of covariance for repeated measures. All results for metrically scaled data are given as median and interquartile range [25 to 75%]. The intention-to-treat population consisted of 66 patients (33 per group). Baseline characteristics did not differ between the two groups except for the frequency of alcohol use disorders and known allergies: these were more frequent in the vaccination group. Both groups showed postoperative immunodepression, documented by decreased mHLA-DR expression on day 1 and 3 after surgery, but there was no significant difference between vaccinated and nonvaccinated patients. The proportion of patients with mHLA-DR expression of less than 10 000 antibodies per cell was 27% in the placebo group vs. 19% in the vaccination group (P = 0.218). Cumulative incidence of postoperative infections is shown in Fig. 1. Overall, 10 days after surgery, infection rate did not differ between the groups. However, until POD 5, there was a slight tendency for decreased infection rates in the vaccination group (P = 0.190), whereas between POD 6 and POD 10, the incidence of infections increased in vaccinated patients compared with the nonvaccinated patients (P = 0.016). The following infections occurred (according to the centers for disease control and prevention definitions for nosocomial infections): surgical wound infection, pneumonia, bloodstream infection (with sepsis) and urinary tract infection.Fig. 1: Cumulative incidence of infections, n = 33 (Mutagrip), n = 33 (Placebo), POD = post operative day.One patient in each group died. Median duration of ICU stay in the vaccination group was 2.2 [0.8 to 6.7] days vs. 3.3 [0.7 to 4.9] days in the nonvaccination group (P = 0.807). Mean hospital stay in the vaccination group was 12.5 [9.3 to 24.0] days vs. 14.0 [11.8 to 22.3] days in the nonvaccination group (P = 0.305). Our results suggest that postoperative immune suppression cannot be avoided by preoperative vaccination measured by mHLA-DR expression on monocytes. Other studies that addressed restoring monocyte function and mHLA-DR expression as well as cell-mediated immune response by influenza vaccination were performed only in nonsurgical patients.2,3 Haining et al.2 demonstrated an increased mHLA-DR expression after influenza vaccination in children after haemopoietic stem cell transplantations. The study of Holvast et al.3 revealed an increase of interferon (IFN)-gamma after influenza vaccination in patients with systemic lupus erythematosus (SLE) and healthy control individuals. These effects were not seen after surgery in our study. In addition, recent evidence looking at leucocyte gene expression in patients with trauma and burns suggest that the pro and anti-inflammatory responses occur simultaneously.4 This is in contrast to the paradigm that trauma/surgery leads to a pro-inflammatory response phase followed by an anti-inflammatory response phase on which the rationale of this trial was based. The postoperative incidence of infections did not differ between vaccinated and nonvaccinated patients from POD 0 to POD 5, which is consistent with the lack of vaccination effect on cell-mediated immune response. From POD 6 to POD 10, the cumulative incidence of infections was higher in vaccinated patients than in nonvaccinated patients. This result is in contrast to our hypothesis that preoperative vaccination can reduce the postoperative incidence of infections. It was not an issue of the safety analysis, as this result became apparent after un-blinding all patients during final data analysis. As the trial is underpowered, the findings have to be interpreted with caution. Due to the small sample size, it was not possible to consider possible different effects between the surgical procedures (i.e. neck dissection and oesophageal resection) nor was it possible to consider the effects of additional confounders such as the type of anaesthesia, duration of surgery or blood loss (although duration of surgery or blood loss did not differ between the groups). Our results suggest that the vaccination of patients before surgery does not enhance HLA-DR expression or reduce postoperative infections. Whether earlier vaccination would be effective is unknown. No serious adverse reactions were observed. Acknowledgements relating to this article Assistance with the study: we are grateful to Claudia Nitschke, Anja Baethge, Jana Kindermann, Annabell Hijazi, Pascal Kucik, Birgit Rückershausen and Sarah Keller for data and blood sample collection. Financial support and sponsorship: the trial was financially supported by inner university grants and DFG grant 432 (German Research Foundation). Conflicts of interest: none. ISRCTN Registration Number: ISRCTN16108096.

We previously demonstrated that the forced expression of pro-caspase-3 can revert acquired chemoresistance in MT1-Adr breast cancer cells which show a defective activation of the mitochondrial pathway of apoptosis. We now asked whether the manipulation of mitochondrial apoptosis signaling can revert different types of drug resistance, i.e. the resistance due to impaired mitochondrial activation in the MT1-Adr cells and the resistance in MT3-Adr cells which is caused by increased expression of the Mdr-1/p-glycoprotein ABC transporter. Here we show that Bcl-2 overexpression is the underlying cause for the resistant phenotype in the MT1-Adr cells. Overexpression of the apoptosis-promoting Bax homologue Bak or the BH3 only protein Nbk/Bik reverts, as expected, acquired drug resistance in the MT1-Adr cells as recently demonstrated for pro-caspase-3. Moreover, we show that both apoptosis-promoters, Nbk/Bik and Bak, antagonize acquired chemoresistance for epirubicin-mediated apoptosis in MT3-Adr breast cancer cells. Neither drug uptake nor drug efflux were influenced by Bak or Nbk/Bik. Thus, our data show that manipulation of the downstream apoptosis signaling cascade by Bak and Nbk/Bik can overcome not only drug resistance due to mitochondrial apoptosis deficiency (in the MT1-Adr cells) but also classical, i.e. efflux-mediated, resistance for drug-induced cell death in the MT3-Adr cell line. Nbk/Bik and Bak could therefore be target genes to increase chemosensitivity and overcome different types of drug resistance.

Background and aims: Exposure to N-Methyl-D-Aspartate (NMDA) receptor antagonists has been demonstrated to induce neurodegeneration in newborn rats. However, in clinical practice the use of NMDA receptor antagonists as anesthetics and sedatives cannot always be avoided. This study investigated the effect of the acetylcholinesterase (AChE) inhibitor physostigmine on neurotrophin expression and the extracellular matrix during NMDA receptor antagonist-induced injury to the immature rat brain. Aims: The aim was to investigate matrix metalloproteinase (MMP)-2 activity, as well as expression of tissue inhibitor of metalloproteinase (TIMP)-2 and brain-derived neurotrophic factor (BDNF) after co-administration of the non-competitive NMDA receptor antagonist MK801 (dizocilpine) and the AChE inhibitor physostigmine. Methods: Six-day-old Wistar rat pups received intraperitoneal injections of dizocilpine ((+)MK801; 0,5 mg/kg 0, 8, 16 h) or vehicle in combination with physostigmine at 0 h and were sacrificed at defined timepoints (6, 12, 24 h) following treatment. All procedures were approved by the local state authorities for animal welfare (G0182/09) and followed institutional guidelines. Brains were processed for molecular analysis (real-time PCR, Western blotting and gelatin zymography) to measure BDNF, MMP-2 and TIMP-2. Results: The AChE inhibitor physostigmine ameliorated the MK801-induced reduction of BDNF mRNA and protein levels, reduced MK801-triggered MMP-2 activity and prevented decreased TIMP-2 mRNA expression. Conclusions: The present study suggests that AChE may work as a therapeutic target to limit the toxicity resulting from NMDA receptor antagonist-induced injury in the developing brain. Our results show that a single application of the AChE inhibitor physostigmine modulates NMDA receptor blockade-induced changes in the levels of BDNF, MMP-2 and TIMP-2.

<h3>Background and Aim</h3> As demonstrated previously, oxygen contributes to the pathogenesis of neonatal brain damage and leading to neurocognitive impairment of prematurely born infants in later life. Reactive oxygen species (ROS) and intrinsic antioxidant defense systems play an important role in both physiological cell signaling processes and many pathological conditions, including neurodegenerative disorders and oxygen-toxicity. Beside the glutathione-system several other redox-modulating proteins are known to be involved in redox-homeostases. The aim of this study was to evaluate potential alterations within the thioredoxin/peroxiredoxin system after exposures to nonphysiologic high oxygen levels in the developing rat brain. <h3>Methods</h3> Six-days old Wistar rats were exposed to 80% oxygen for 6, 12, 24 or 48 hours and littermates kept in room air served as controls (n=6–8). Brains (excluding cerebellum) were evaluated after perfusion with PBS and dissection of both hemispheres for RNA and protein analyses. <h3>Results</h3> We demonstrate that elevated oxygen concentrations change the balance of the ROS-dependent thioredoxin/peroxiredoxin system. Oxygen-toxicity significantly induced upregulation of peroxiredoxins in infant rat brain. In parallel, hyperoxia reduced the level of DJ-1, a hydroperoxide-responsive protein. <h3>Discussion</h3> These findings are highly relevant from a clinical aspect because oxygen administration to neonates is often inevitable, and we recommend that every effort should be made in neonatal medicine to limit exposure of these immature babies to high oxygen concentrations. These results may also contribute to receive optimal therapeutical approaches to ameliorate oxygen toxicity.

Background and aims: Experimental studies show that oxygen, which is widely used in neonatal medicine for resuscitation and treatment of pulmonary hypertension, triggers a disruption of the maintenance of intracellular redox homeostasis. This disturbance can lead to oxidative stress and furthermore to neuronal apoptosis in the developing brain. The role of caspase-2 in apoptosis is poorly defined. Many in vitro studies of caspase-2 knockdown in cultured cells have implicated this caspase in cell death in response to different signaling pathways. To elucidate mechanisms of the caspase-2 inhibitor TRP601 and its mode of functioning in the developing brain in the context of hyperoxia, we investigated its impact on the levels of APAF-1, AIF, cytochrome c, caspase-9 and -3. Methods: Six-day old rats were exposed together with their mothers to 80% oxygen in the presence or absence of the caspase-2 inhibitor TRP601 (1 mg/kg) and were sacrificed after 12 or 24 hrs of hyperoxia following treatment. Dissected brains were either examined histologically to visualize degenerating cells or were subjected to protein studies. Results: Oxygen exposure triggered cell death at 12 to 24 hrs, which was attenuated by TRP601 treatment. Our protein studies demonstrated an upregulation of APAF-1, AIF, cytochrome c, caspase-9 and -3 in the cytosolic fraction of brain homogenates after hyperoxia, which reached control levels following TRP601 treatment. Conclusion: These findings suggest a protective role for the caspase-2 inhibitor TRP601 in the prevention of neonatal oxygen-induced apoptotic brain damage. Supported by the European Commission (Sixth Framework Program, contract no LSHM- CT-2006-036534).

<h3>Introduction/Background</h3> High-risk surgery frequently is associated with haemodynamic instability leading to the administration of high amounts of intravenous fluids and catecholamines to stabilize cardiovascular function. The haemodynamic instability is commonly attributed to the inflammatory response related to surgical trauma. In this study, we investigated also, how the induction of anaesthesia and the opening of the abdominal cavity impact haemodynamics. <h3>Methodology</h3> In a prospective, observational, monocentric study, haemodynamic measurements were obtained by the non-invasive methodology of thoracic electrical cardiometry (EC) in 29 patients with primary ovarian cancer undergoing multivisceral cytoreductive surgery. The change of haemodynamic parameters was analyzed from the day prior to surgery to the first intraoperative measurement by non-parametric longitudinal data analysis in a two-factorial experiment (dependent factor time). <h3>Results</h3> Median age of patients was 59 [25-quartile 50; 75-quartile 61] years. 8 (28%) patients had a diagnosis of arterial hypertension, otherwise, no cardiovascular diseases were shown. The FIGO stages were in 22 (75%) patients above stage IIIc and 9 (31%) patients had more than 500 ml of ascites. The relative change from the baseline to the first intraoperative timepoint showed a reduced heart rate (HR, median -19 [25-quartile -26%; 75-quartile -10%]%, p&lt;0.0001), stroke volume index (SVI, -9.5 [-15.3;3.2]%, p=0.0038), cardiac index (CI, -24.5 [-32;-13]%, p&lt;0.0001) and the inotropic marker index of contractility (ICON, -17.5 [-35.3;-0.8]%, p&lt;0.0001). <h3>Conclusion</h3> Substantial changes in HR, SVI, CI, and ICON occurred from the day prior to surgery to the first intraoperative measurement, indicating that patients without relevant cardiovascular morbidity showed reduced cardiocirculatory flow and cardiac function. Furthermore, these data indicate that pharmacological modulation might optimize haemodynamic care during high-risk gynaecological surgery.

<h3>Introduction/Background</h3> Patients undergoing high-risk surgery show an increased haemodynamic instability and have an increased risk of morbidity and mortality. However, the available haemodynamic data concentrate only on the intraoperative period. The aim of this study is to characterize patients hemodynamically throughout the whole intra- and postoperative period non-invasively by thoracic electrical cardiometry (EC) for advanced cardiovascular assessment. <h3>Methodology</h3> In a prospective, observational, monocentric study, EC measurements were obtained before surgery, during surgery, and repeatedly throughout the hospital stay in 30 patients with primary ovarian cancer undergoing multivisceral cytoreductive surgery. The inflammatory markers interleukine-6 (IL-6) and inter-cellular-adhesion-molecule-1 (ICAM-1) were analyzed perioperatively. Severe postoperative complications were classified according to the Clavien-Dindo classification and used as a binary grouping criterion (≥3 and &lt;2 or no complications). <h3>Results</h3> Throughout the perioperative course, patients showed a longitudinally reduced cardiac index (CI, p&lt;0.0001), while the stroke volume index (SVI, p=0.0528) remained unchanged. Patients suffering from postoperative complications differed over the longitudinal perioperative course in the index of contractility (ICON, p=0.0435) and the systolic time ratio (p=0.0008), without showing differences in CI (p=0.3337). Also, the groups differed during the longitudinal perioperative course in IL-6 (p = 0.0343) and ICAM-1 (p=0.0398). <h3>Conclusion</h3> Longitudinally over the perioperative time course CI showed a relevant decrease. Patients with postoperative complications differed from patients without complications in the markers of cardiac function, ICON and STR, as well as showing a lower SVI. These data show haemodynamic alterations during surgery in all patients and especially the association of alterations to complications during the postoperative course. Therefore, they are a first approach to improve noninvasive haemodynamic patient assessments and interdisciplinary perioperative care in the future.

Abstract Background: Although intensive efforts to improve diagnosis and therapy of sepsis over the last decade, the mortality of septic shock remains high and causes substantial socioeconomical burden of disease. The function of immune cells is time-of-day-dependent and is regulated by several circadian clock genes. This study aims to investigate whether the rhythmicity of clock gene expression is altered in patients with septic shock. Methods: This prospective pilot study was performed at the university hospital Charité – Universitätsmedizin Berlin, Department of Anesthesiology and Operative Intensive Care Medicine (CCM, CVK). We included 20 patients with septic shock between May 2014 and January 2018, from whom blood was drawn every 4 hours over a 24-hour period to isolate CD14-positive monocytes and to measure expression of 17 clock and clock-associated genes. Of these patients, 3 whose samples expressed fewer than 8 clock genes were excluded from the final analysis. Expression data were compared to data of a healthy study population and a rhythmicity score S P was calculated, which comprises values between -1 (arrhythmic) and 1 (rhythmic). Results: Overall, the rhythmicity scores for septic shock patients were significantly (p &lt; 0.0001) lower (0.23 ± 0.26) compared to the control group (12 healthy young men, 0.70 ± 0.18). 77% of the measured clock genes were classified as having inconclusive rhythms, i.e. neither rhythmic nor arrhythmic. The clock genes NR1D1 , NR1D2 and CRY2 were the most rhythmic, while CLOCK and ARNTL were the least rhythmic. In addition, the expression of clock genes CRY1 , NR1D1 , NR1D2 , DBP , and PER2 was suppressed in septic shock patients and CRY2 was significantly upregulated compared to controls. Conclusion: Compared to young healthy men, molecular rhythms in immune cells of septic shock patients were substantially decreased. The decrease in rhythmicity was clock gene-dependent. The loss of rhythmicity and downregulation of clock gene expression might be caused by sepsis and might further deteriorate immune responses and organ injury, but further studies are necessary to understand underlying pathophysiological mechanisms.Clinical trial registered with www.ClinicalTrials.gov (NCT02044575) on 24 January 2014.

Additional file 1: Table S1. Confidence intervals per clock gene and subject, representing the range of circadian amplitudes of gene expression that are consistent with the measurements at the 95% level (i.e., p