Effects of temporal IFNγ exposure on macrophage phenotype and secretory profile: exploring GMP-Compliant production of a novel subtype of regulatory macrophages (MregIFNγ0) for potential cell therapeutic applications.

BACKGROUND: Macrophages are involved in tissue homeostasis, angiogenesis and immunomodulation. Proangiogenic and anti-inflammatory macrophages (regulatory macrophages, Mreg) can be differentiated in-vitro from CD14+ monocytes by using a defined cell culture medium and a stimulus of IFNγ. AIM OF THE STUDY: To scrutinize the potential impact of temporal IFNγ exposure on macrophage differentiation as such exposure may lead to the emergence of a distinct and novel macrophage subtype. METHODS: Differentiation of human CD14+ monocytes to Mreg was performed using a GMP compliant protocol and administration of IFNγ on day 6. Monocytes from the same donor were in parallel differentiated to MregIFNγ0 using the identical protocol but with administration of IFNγ on day 0. Cell characterization was performed using brightfield microscopy, automated and metabolic cell analysis, transmission electron microscopy, flow cytometry, qPCR and secretome profiling. RESULTS: Mreg and MregIFNγ0 showed no differences in cell size and volume. However, phenotypically MregIFNγ0 exhibited fewer intracellular vesicles/vacuoles but larger pseudopodia-like extensions. MregIFNγ0 revealed reduced expression of IDO and PD-L1 (P < 0.01 for both). They were positive for CD80, CD14, CD16 and CD38 (P < 0.0001vs. Mreg for all), while the majority of MregIFNγ0 did not express CD206, CD56, and CD103 on their cell surface (P < 0.01 vs. Mreg for all). In terms of their secretomes, MregIFNγ0 differed significantly from Mreg. MregIFNγ0 media exhibited reduced levels of ENA-78, Osteopontin and Serpin E1, while the amounts of MIG (CXCL9) and IP10 were increased. CONCLUSION: Exposing CD14+ monocytes to an alternatively timed IFNγ stimulation results in a novel macrophage subtype which possess additional M1-like features (MregIFNγ0). MregIFNγ0 may therefore have the potential to serve as cellular therapeutics for clinical applications beyond those covered by M2-like Mreg, including immunomodulation and tumor treatment.

Reduction of the acquisition time needed to obtain somatosensory evoked potentials by estimation of the required averaging sweep count by an algorithm.

Somatosensory evoked potentials are frequently acquired by stimulation of the median or tibial nerves (mSEPs and tSEPs) for intraoperative monitoring of sensory pathways. Due to their low amplitudes it is common practice to average 200 or more sweeps to discern the evoked potentials from the background EEG. The aim of this study was to investigate if an algorithm designed to determine the lowest sweep count needed to obtain reproducible evoked potentials in each patient significantly reduces the median necessary sweep count to under 200. 30 patients undergoing spinal surgery at the Department of Neurosurgery were included in the study. Beginning with a sweep count of 200 an algorithm was designed to determine the lowest sweep count that yielded reproducible evoked potentials in each patient. By this algorithm the minimal sweep count was determined in 15 patients for mSEPs and in 15 patients for tSEPs. The required sweep count was below 200 in 14 of 15 patients for mSEPs (93.3%) with a mean sweep count of 56 ± 51. For tSEPs the sweep count was below 200 in 11 of 15 patients (73.3%) with a mean sweep count of 106 ± 70 (mean ± SD). The calculated mean time to average the potentials could thereby be reduced from 48.8s to 13.7s for mSEPs and from 48.8s to 25.9s for tSEPs. The proposed algorithm allowed sweep count and acquisition time reduction in roughly 90% of all patients for mSEPs and in 70% of all patients for tSEPs.

Characterization of large extracellular vesicles (L-EV) derived from human regulatory macrophages (Mreg): novel mediators in wound healing and angiogenesis?

BACKGROUND: Large extracellular vesicles (L-EV) with a diameter between 1 and 10 µm are released by various cell types. L-EV contain and transport active molecules which are crucially involved in cell to cell communication. We have shown that secretory products of human regulatory macrophages (Mreg) bear pro-angiogenic potential in-vitro and our recent findings show that Mreg cultures also contain numerous large vesicular structures similar to L-EV with so far unknown characteristics and function. AIM OF THIS STUDY: To characterize the nature of Mreg-derived L-EV (L-EVMreg) and to gain insights into their role in wound healing and angiogenesis. METHODS: Mreg were differentiated using blood monocytes from healthy donors (N = 9) and L-EVMreg were isolated from culture supernatants by differential centrifugation. Characterization of L-EVMreg was performed by cell/vesicle analysis, brightfield/transmission electron microscopy (TEM), flow cytometry and proteome profiling arrays. The impact of L-EVMreg on wound healing and angiogenesis was evaluated by means of scratch and in-vitro tube formation assays. RESULTS: Mreg and L-EVMreg show an average diameter of 13.73 ± 1.33 µm (volume: 1.45 ± 0.44 pl) and 7.47 ± 0.75 µm (volume: 0.22 ± 0.06 pl) respectively. Flow cytometry analyses revealed similarities between Mreg and L-EVMreg regarding their surface marker composition. However, compared to Mreg fewer L-EVMreg were positive for CD31 (P < 0.01), CD206 (P < 0.05), CD103 (P < 0.01) and CD45 (P < 0.05). Proteome profiling suggested that L-EVMreg contain abundant amounts of pro-angiogenic proteins (i.e. interleukin-8, platelet factor 4 and serpin E1). From a functional point of view L-EVMreg positively influenced in-vitro wound healing (P < 0.05) and several pro-angiogenic parameters in tube formation assays (all segment associated parameters, P < 0.05; number of meshes, P < 0.05). CONCLUSION: L-EVMreg with regenerative and pro-angiogenic potential can be reproducibly isolated from in-vitro cultured human regulatory macrophages. We propose that L-EVMreg could represent a putative therapeutic option for the treatment of chronic wounds and ischemia-associated diseases.

Effects of remote ischemic preconditioning (RIPC) and chronic remote ischemic preconditioning (cRIPC) on levels of plasma cytokines, cell surface characteristics of monocytes and in-vitro angiogenesis: a pilot study.

Remote ischemic preconditioning (RIPC) protects the heart against myocardial ischemia/reperfusion (I/R) injury and recent work also suggested chronic remote ischemic conditioning (cRIPC) for cardiovascular protection. Based on current knowledge that systemic immunomodulatory effects of RIPC and the anti-inflammatory capacity of monocytes might be involved in cardiovascular protection, the aim of our study was to evaluate whether RIPC/cRIPC blood plasma is able to induce in-vitro angiogenesis, identify responsible factors and evaluate the effects of RIPC/cRIPC on cell surface characteristics of circulating monocytes. Eleven healthy volunteers were subjected to RIPC/cRIPC using a blood pressure cuff inflated to > 200 mmHg for 3 × 5 min on the upper arm. Plasma and peripheral blood monocytes were isolated before RIPC (Control), after 1 × RIPC (RIPC) and at the end of 1 week of daily RIPC (cRIPC) treatment. Plasma concentrations of potentially pro-angiogenic humoral factors (CXCL5, Growth hormone, IGFBP3, IL-1α, IL-6, Angiopoietin 2, VEGF, PECAM-1, sTie-2, IL-8, MCSF) were measured using custom made multiplex ELISA systems. Tube formation assays for evaluation of in-vitro angiogenesis were performed with donor plasma, monocyte conditioned culture media as well as IL-1α, CXCL5 and Growth hormone. The presence of CD14, CD16, Tie-2 and CCR2 was analyzed on monocytes by flow cytometry. Employing in-vitro tube formation assays, several parameters of angiogenesis were significantly increased by cRIPC plasma (number of nodes, P < 0.05; number of master junctions, P < 0.05; number of segments, P < 0.05) but were not influenced by culture medium from RIPC/cRIPC treated monocytes. While RIPC/cRIPC treatment did not lead to significant changes of the median plasma concentrations of any of the selected potentially pro-angiogenic humoral factors, in-depth analysis of the individual subjects revealed differences in plasma levels of IL-1α, CXCL5 and Growth hormone after RIPC/cRIPC treatment in some of the volunteers. Nevertheless, the positive effects of RIPC/cRIPC plasma on in-vitro angiogenesis could not be mimicked by the addition of the respective humoral factors alone or in combination. While monocyte conditioned culture media did not affect in-vitro tube formation, flow cytometry analyses of circulating monocytes revealed a significant increase in the number of Tie-2 positive and a decrease of CCR2 positive monocytes after RIPC/cRIPC (Tie-2: cRIPC, P < 0.05; CCR2: RIPC P < 0.01). Cardiovascular protection may be mediated by RIPC and cRIPC via a regulation of plasma cytokines as well as changes in cell surface characteristics of monocytes (e.g. Tie-2). Our results suggest that a combination of humoral and cellular factors could be responsible for the RIPC/cRIPC mediated effects and that interindividual variations seem to play a considerable part in the RIPC/cRIPC associated mechanisms.

Hypoxia directed migration of human naïve monocytes is associated with an attenuation of cytokine release: indications for a key role of CCL26.

BACKGROUND: Numerous tissue-derived factors have been postulated to be involved in tissue migration of circulating monocytes. The aim of this study was to evaluate whether a defined hypoxic gradient can induce directed migration of naïve human monocytes and to identify responsible autocrine/paracrine factors. METHODS: Monocytes were isolated from peripheral blood mononuclear cells, transferred into chemotaxis chambers and subjected to a defined oxygen gradient with or without the addition of CCL26. Cell migration was recorded and secretome analyses were performed. RESULTS: Cell migration recordings revealed directed migration of monocytes towards the source of hypoxia. Analysis of the monocyte secretome demonstrated a reduced secretion of 70% (19/27) of the analyzed cytokines under hypoxic conditions. The most down-regulated factors were CCL26 (- 99%), CCL1 (- 95%), CX3CL1 (- 95%), CCL17 (- 85%) and XCL1 (- 83%). Administration of recombinant CCL26 abolished the hypoxia-induced directed migration of human monocytes, while the addition of CCL26 under normoxic conditions resulted in a repulsion of monocytes from the source of CCL26. CONCLUSIONS: Hypoxia induces directed migration of human monocytes in-vitro. Autocrine/paracrine released CCL26 is involved in the hypoxia-mediated monocyte migration and may represent a target molecule for the modulation of monocyte migration in-vivo.

Remote ischemic preconditioning attenuates intestinal mucosal damage: insight from a rat model of ischemia-reperfusion injury.

BACKGROUND: Remote ischemic preconditioning (RIPC) is a phenomenon, whereby repeated, non-lethal episodes of ischemia to an organ or limb exert protection against ischemia-reperfusion (I/R) injury in distant organs. Despite intensive research, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms, especially in the intestine. Aim of this study was to evaluate possible protective effects RIPC on intestinal I/R injury. METHODS: Thirty rats were randomly assigned to four groups: I/R; I/R + RIPC; Sham; Sham + RIPC. Animals were anesthetized and the superior mesenteric artery was clamped for 30 min, followed by 60 min of reperfusion. RIPC-treated rats received 3 × 5 min of bilateral hindlimb I/R prior to surgery, sham groups obtained laparotomy without clamping. After I/R injury serum/tissue was analyzed for: Mucosal damage, Caspase-3/7 activity, expression of cell stress proteins, hydrogen peroxide (H2O2) and malondialdehyde (MDA) production, Hypoxia-inducible factor-1α (HIF-1α) protein expression and matrix metalloproteinase (MMP) activity. RESULTS: Intestinal I/R resulted in increased mucosal injury (P < 0.001) and elevated Caspase-3/7 activity (P < 0.001). RIPC significantly reduced the histological signs of intestinal I/R injury (P < 0.01), but did not affect Caspase-3/7 activity. Proteome profiling suggested a RIPC-mediated regulation of several cell stress proteins after I/R injury: Cytochrome C (+ 157%); Cited-2 (- 39%), ADAMTS1 (+ 74%). Serum concentrations of H2O2 and MDA remained unchanged after RIPC, while the reduced intestinal injury was associated with increased HIF-1α levels. Measurements of MMP activities in serum and intestinal tissue revealed an attenuated gelatinase activity at 130 kDa within the serum samples (P < 0.001) after RIPC, while the activity of MMPs within the intestinal tissue was not affected by I/R injury or RIPC. CONCLUSIONS: RIPC ameliorates intestinal I/R injury in rats. The underlying mechanisms may involve HIF-1α protein expression and a decreased serum activity of a 130 kDa factor with gelatinase activity.

Doxycycline protects human intestinal cells from hypoxia/reoxygenation injury: Implications from an in-vitro hypoxia model.

Intestinal ischemia/reperfusion (I/R) injury is a grave clinical emergency and associated with high morbidity and mortality rates. Based on the complex underlying mechanisms, a multimodal pharmacological approach seems necessary to prevent intestinal I/R injury. The antibiotic drug doxycycline, which exhibits a wide range of pleiotropic therapeutic properties, might be a promising candidate for also reducing I/R injury in the intestine. To investigate possible protective effects of doxycycline on intestinal I/R injury, human intestinal CaCo-2 cells were exposed to doxycycline at clinically relevant concentrations. In order to mimic I/R injury, CaCo-2 were thereafter subjected to hypoxia/reoxygenation by using our recently described two-enzyme in-vitro hypoxia model. Investigations of cell morphology, cell damage, apoptosis and hydrogen peroxide formation were performed 24h after the hypoxic insult. Hypoxia/reoxygenation injury resulted in morphological signs of cell damage, elevated LDH concentrations in the respective culture media (P<0.001) and increased protein expression of proapoptotic caspase-3 (P<0.05) in the intestinal cultures. These events were associated with increased levels hydrogen peroxide (P<0.001). Preincubation of CaCo-2 cells with different concentrations of doxycycline (5µM, 10µM, 50µM) reduced the hypoxia induced signs of cell damage and LDH release (P<0.001 for all concentrations). The reduction of cellular damage was associated with a reduced expression of caspase-3 (5µM, P<0.01; 10µM, P<0.01; 50µM, P<0.05), while hydrogen peroxide levels remained unchanged. In summary, doxycycline protects human intestinal cells from hypoxia/reoxygenation injury in-vitro. Further animal and clinical studies are required to prove the protective potential of doxycycline on intestinal I/R injury under in-vivo conditions.

Effects of propofol on wound closure and barrier function of cultured endothelial cells: An in vitro experimental study.

BACKGROUND: Propofol is widely used in routine clinical practice for the induction and maintenance of anaesthesia. Although propofol is regarded as a well tolerated anaesthetic, its effect on intact or damaged endothelial cells has not yet been elucidated. OBJECTIVE: The aim of this study was to investigate the effects of different concentrations of propofol on cell damage, metabolic activity, barrier function and wound healing capacity of human endothelial cells. DESIGN: An in vitro investigation. SETTING: Research Laboratory of the Department of Anaesthesiology and Intensive Care Medicine, University Hospital Schleswig-Holstein, Kiel, Germany. MATERIALS: In vitro cultures of primary human umbilical vein endothelial cells (HUVECs). INTERVENTIONS: Intact HUVEC or wounded HUVEC monolayers were incubated with or without different concentrations of propofol (10, 30 and 100 µmol l). MAIN OUTCOME MEASURES: Cell damage, metabolic activity, monolayer permeability, wound healing capacity, protein phosphorylation. RESULTS: Propofol did not alter the morphology, induce cell damage or influence metabolic activity of intact HUVEC cells. Permeability of a HUVEC monolayer was increased by propofol 100 µmol l (P < 0.05). Wound closure was inhibited by the addition of propofol 30 and 100 µmol l (P < 0.05 and P < 0.01). This effect was associated with increased phosphorylation of extracellular signal regulated kinases (Erk) 1/2 (30 and 100 µmol l; both P < 0.05) and decreased phosphorylation of Rho kinase (Rock) (100 µmol l; P < 0.05). CONCLUSION: Propofol does not damage intact endothelial cells, but increases permeability of an endothelial cell monolayer at high concentrations and inhibits wound closure in vitro. Further experimental and clinical in vivo research should be performed to clarify the influence of propofol on endothelial wound healing.

Effect of Xenon Anesthesia Compared to Sevoflurane and Total Intravenous Anesthesia for Coronary Artery Bypass Graft Surgery on Postoperative Cardiac Troponin Release: An International, Multicenter, Phase 3, Single-blinded, Randomized Noninferiority Trial.

BACKGROUND: Ischemic myocardial damage accompanying coronary artery bypass graft surgery remains a clinical challenge. We investigated whether xenon anesthesia could limit myocardial damage in coronary artery bypass graft surgery patients, as has been reported for animal ischemia models. METHODS: In 17 university hospitals in France, Germany, Italy, and The Netherlands, low-risk elective, on-pump coronary artery bypass graft surgery patients were randomized to receive xenon, sevoflurane, or propofol-based total intravenous anesthesia for anesthesia maintenance. The primary outcome was the cardiac troponin I concentration in the blood 24 h postsurgery. The noninferiority margin for the mean difference in cardiac troponin I release between the xenon and sevoflurane groups was less than 0.15 ng/ml. Secondary outcomes were the safety and feasibility of xenon anesthesia. RESULTS: The first patient included at each center received xenon anesthesia for practical reasons. For all other patients, anesthesia maintenance was randomized (intention-to-treat: n = 492; per-protocol/without major protocol deviation: n = 446). Median 24-h postoperative cardiac troponin I concentrations (ng/ml [interquartile range]) were 1.14 [0.76 to 2.10] with xenon, 1.30 [0.78 to 2.67] with sevoflurane, and 1.48 [0.94 to 2.78] with total intravenous anesthesia [per-protocol]). The mean difference in cardiac troponin I release between xenon and sevoflurane was -0.09 ng/ml (95% CI, -0.30 to 0.11; per-protocol: P = 0.02). Postoperative cardiac troponin I release was significantly less with xenon than with total intravenous anesthesia (intention-to-treat: P = 0.05; per-protocol: P = 0.02). Perioperative variables and postoperative outcomes were comparable across all groups, with no safety concerns. CONCLUSIONS: In postoperative cardiac troponin I release, xenon was noninferior to sevoflurane in low-risk, on-pump coronary artery bypass graft surgery patients. Only with xenon was cardiac troponin I release less than with total intravenous anesthesia. Xenon anesthesia appeared safe and feasible.

Adverse effects of hydroxyethyl starch (HES 130/0.4) on intestinal barrier integrity and metabolic function are abrogated by supplementation with Albumin.

BACKGROUND: Volume resuscitation with hydroxyethyl starch (HES) is controversially discussed and we recently showed that HES perfusion impairs endothelial and epithelial intestinal barrier integrity. Here we investigated whether Albumin containing HES solutions are superior to HES alone in maintaining intestinal barrier function. METHODS: An isolated perfused model of the mouse small intestine was used to investigate the effects of: (i) 3 % Albumin (Alb), (ii) 3 % HES or (iii) 1.5 % HES/1.5 % Albumin (HES/Alb). Intestinal morphology, cell damage, metabolic functions, fluid shifts and endothelial/epithelial barrier permeability were evaluated. Potentially involved signaling mechanisms (Erk1/2, Akt and Stat5 phosphorylation) were screened. RESULTS: HES induced histomorphological damage (p < 0.01 vs. Alb), by trend elevated the amount of luminal intestinal fatty acid binding protein and reduced galactose uptake (p < 0.001 vs. Alb). Luminal and lymphatic flow rates were increased (p < 0.001 vs. Alb), while vascular flow was decreased (p < 0.001 vs. Alb) during HES perfusion. HES also increased the vascular to luminal FITC-dextran transfer (p < 0.001 vs. Alb), pointing towards a fluid shift from the vascular to the luminal and lymphatic compartments during HES perfusion. Addition of Alb (HES/Alb) reversed all adverse effects of HES (p < 0.05 vs. HES), restored barrier integrity (p < 0.05 vs. HES) and improved metabolic function of the intestine (p < 0.001 vs. HES; p < 0.05 vs. Alb). Mechanistically, HES/Alb perfusion resulted in an increased phosphorylation of Erk1/2 and Akt kinases (p < 0.001 vs. HES), while Stat5 remained unchanged. CONCLUSIONS: Albumin supplementation abrogates the adverse effects of HES in the intestine and underlying mechanism may function via phosphorylation of Erk1/2 and Akt. Albumin containing HES solutions are superior to HES alone and may improve the suitability of HES in the clinic.

Dynamic Variables Fail to Predict Fluid Responsiveness in an Animal Model With Pericardial Effusion.

OBJECTIVES: The reliability of dynamic and volumetric variables of fluid responsiveness in the presence of pericardial effusion is still elusive. The aim of the present study was to investigate their predictive power in a porcine model with hemodynamic relevant pericardial effusion. DESIGN: A single-center animal investigation. PARTICIPANTS: Twelve German domestic pigs. INTERVENTIONS: Pigs were studied before and during pericardial effusion. Instrumentation included a pulmonary artery catheter and a transpulmonary thermodilution catheter in the femoral artery. Hemodynamic variables like cardiac output (COPAC) and stroke volume (SVPAC) derived from pulmonary artery catheter, global end-diastolic volume (GEDV), stroke volume variation (SVV), and pulse-pressure variation (PPV) were obtained. MEASUREMENTS AND MAIN RESULTS: At baseline, SVV, PPV, GEDV, COPAC, and SVPAC reliably predicted fluid responsiveness (area under the curve 0.81 [p = 0.02], 0.82 [p = 0.02], 0.74 [p = 0.07], 0.74 [p = 0.07], 0.82 [p = 0.02]). After establishment of pericardial effusion the predictive power of dynamic variables was impaired and only COPAC and SVPAC and GEDV allowed significant prediction of fluid responsiveness (area under the curve 0.77 [p = 0.04], 0.76 [p = 0.05], 0.83 [p = 0.01]) with clinically relevant changes in threshold values. CONCLUSIONS: In this porcine model, hemodynamic relevant pericardial effusion abolished the ability of dynamic variables to predict fluid responsiveness. COPAC, SVPAC, and GEDV enabled prediction, but their threshold values were significantly changed.

Profiling of cell stress protein expression in cardiac tissue of cardiosurgical patients undergoing remote ischemic preconditioning: implications for thioredoxin in cardioprotection.

BACKGROUND: Transient episodes of ischemia in a remote organ (remote ischemic preconditioning, RIPC) can attenuate myocardial ischemia/reperfusion injury but the underlying mechanisms of RIPC in the target organ are still poorly understood. Recent animal studies suggested that the small redox protein thioredoxin may be a potential candidate for preconditioning-induced organprotection. Here we employed a human proteome profiler array to investigate the RIPC regulated expression of cell stress proteins and particularly of thioredoxin in heart tissue of cardiosurgical patients with cardiopulmonary bypass (CPB). METHODS: RIPC was induced by four 5 minute cycles of transient upper limb ischemia/reperfusion using a blood pressure cuff. Right atrial tissue was obtained from patients receiving RIPC (N = 19) and control patients (N = 19) before and after CPB. Cell stress proteome profiler arrays as well as Westernblotting and ELISA experiments for thioredoxin (Thio-1) were performed employing the respective tissue samples. RESULTS: Protein arrays revealed an up-regulation of 26.9% (7/26; CA IX, Cyt C, HSP-60, HSP-70, pJNK, SOD2, Thio-1) of cell stress associated proteins in RIPC tissue obtained before CPB, while 3.8% (1/26; SIRT2) of the proteins were down-regulated. Array results for thioredoxin were verified by semi-quantitative Westernblotting studies which showed a significant up-regulation of thioredoxin protein levels in cardiac tissue samples of RIPC patients taken before CPB (RIPC: 5.36 ± 0.85 a.u.; control: 3.23 ± 0.39 a.u.; P < 0.05). Quantification of thioredoxin levels in tissue of RIPC and control patients by ELISA experiments further confirmed the Westernblotting results (RIPC: 0.30 ± 0.02 ng/mg protein; control: 0.24 ± 0.02 ng/mg protein; P < 0.05). CONCLUSION: We provide evidence for thioredoxin as a RIPC-induced factor in heart tissue of cardiosurgical patients and identified several cell stress associated proteins that are regulated by RIPC and may play a role in RIPC-mediated cardioprotection.

Culture media from hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury.

Remote ischemic preconditioning (RIPC) is a phenomenon, whereby short episodes of non-lethal ischemia to an organ or tissue exert protection against ischemia/reperfusion injury in a distant organ. However, there is still an apparent lack of knowledge concerning the RIPC-mediated mechanisms within the target organ and the released factors. Here we established a human cell culture model to investigate cellular and molecular effects of RIPC and to identify factors responsible for RIPC-mediated intestinal protection. Human umbilical vein cells (HUVEC) were exposed to repeated episodes of hypoxia (3 × 15 min) and conditioned culture media (CM) were collected after 24h. Human intestinal cells (CaCo-2) were cultured with or without CM and subjected to 90 min of hypoxia/reoxygenation injury. Reverse transcription-polymerase chain reaction, Western blotting, gelatin zymography, hydrogen peroxide measurements and lactate dehydrogenase (LDH) assays were performed. In HUVEC cultures hypoxic conditioning did not influence the profile of secreted proteins but led to an increased gelatinase activity (P<0.05) in CM. In CaCo-2 cultures 90 min of hypoxia/reoxygenation resulted in morphological signs of cell damage, increased LDH levels (P<0.001) and elevated levels of hydrogen peroxide (P<0.01). Incubation of CaCo-2 cells with CM reduced the hypoxia-induced signs of cell damage and LDH release (P<0.01) and abrogated the hypoxia-induced increase of hydrogen peroxide. These events were associated with an enhanced phosphorylation status of the prosurvival kinase Erk1/2 (P<0.05) but not Akt and STAT-5. Taken together, CM of hypoxia conditioned endothelial cells protect human intestinal cells from hypoxia/reoxygenation injury. The established culture model may help to unravel RIPC-mediated cellular events and to identify molecules released by RIPC.

Accuracy of an autocalibrated pulse contour analysis in cardiac surgery patients: a bi-center clinical trial.

BACKGROUND: Less-invasive and easy to install monitoring systems for continuous estimation of cardiac index (CI) have gained increasing interest, especially in cardiac surgery patients who often exhibit abrupt haemodynamic changes. The aim of the present study was to compare the accuracy of CI by a new semi-invasive monitoring system with transpulmonary thermodilution before and after cardiopulmonary bypass (CPB). METHODS: Sixty-five patients (41 Germany, 24 Spain) scheduled for elective coronary surgery were studied before and after CPB, respectively. Measurements included CI obtained by transpulmonary thermodilution (CITPTD) and autocalibrated semi-invasive pulse contour analysis (CIPFX). Percentage changes of CI were also calculated. RESULTS: There was only a poor correlation between CITPTD and CIPFX both before (r (2) = 0.34, p < 0.0001) and after (r (2) = 0.31, p < 0.0001) CPB, with a percentage error (PE) of 62 and 49 %, respectively. Four quadrant plots revealed a concordance rate over 90 % indicating an acceptable correlation of trends between CITPTD and CIPFX before (concordance: 93 %) and after (concordance: 94 %) CPB. In contrast, polar plot analysis showed poor trending before and an acceptable trending ability of changes in CI after CPB. CONCLUSIONS: Semi-invasive CI by autocalibrated pulse contour analysis showed a poor ability to estimate CI compared with transpulmonary thermodilution. Furthermore, the new semi-invasive device revealed an acceptable trending ability for haemodynamic changes only after CPB. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02312505 Date: 12.03.2012.

Reliability of the surgical Pleth index for assessment of postoperative pain: a pilot study.

BACKGROUND: Postoperative pain control is essential and may have a beneficial effect on postoperative outcome and morbidity. Analgesia quality is controlled using tools such as a Numerical Rating Scale (NRS). These tools require cooperation and often fail in the presence of reduced awareness. The Surgical Pleth index (SPI) has been introduced as a monitoring tool for intraoperative pain under general anaesthesia. OBJECTIVE: We investigated the correlation between SPI and pain intensity, analgesic consumption and fitness for discharge in the postanaesthesia care unit. DESIGN: An observational study. SETTING: The central postanaesthesia care unit of our tertiary care hospital. PATIENTS: Written informed consent was obtained from 100 patients scheduled for elective surgery under general anaesthesia. Patients below the age of 18 years and those with an abnormal cardiac rhythm were excluded from the study. INTERVENTION: Patients were interviewed every 10 min for 2 h. MAIN OUTCOME MEASURES: Pain intensity measured by NRS, discomfort and Aldrete and Post-Anaesthetic Discharge Scoring System (PADSS) scores were noted. SPI and total dose of opioids administered were recorded. RESULTS: A total of 1300 pain measurements were recorded; 482 (37%) reflected no or mild pain (NRS 0 to 3), 532 (41%) moderate pain (NRS 4 to 6) and 286 (22%) severe pain (NRS 7 to 10). Both NRS (r = 0.62, P < 0.001) and SPI (r = 0.38, P < 0.001) correlated significantly with total opioid consumption. SPI showed a moderate correlation with NRS (r = 0.49, P < 0.001). Receiver operating characteristic analysis showed moderate sensitivity and specificity for discrimination between low and moderate pain (NRS ≤3) (sensitivity 67%, specificity 69% for SPI ≤45), and between moderate and severe pain (NRS >6) (sensitivity 72%, specificity 72% for SPI ≥57). SPI and NRS showed weak negative correlations with Aldrete and PADSS scores. CONCLUSION: Sensitivity and specificity of SPI to discriminate between low, moderate and severe pain levels was moderate. Both NRS and SPI correlated significantly with total opioid consumption.

Comparison of a continuous noninvasive arterial pressure device with invasive measurements in cardiovascular postsurgical intensive care patients: a prospective observational study.

BACKGROUND: Arterial pressure monitoring using the a continuous noninvasive arterial pressure (CNAP) device during general anaesthesia is known to be interchangeable with continuous invasive arterial pressure (CIAP) monitoring. Agreement with invasive measurements in cardiovascular postsurgical intensive care patients has not been assessed. OBJECTIVE: The objective of this study is to assess the agreement and interchangeability of CNAP with CIAP in cardiovascular postsurgical patients and to determine the effects of cardiac arrhythmia, catecholamine dosage, respiratory weaning and calibration intervals on agreement. DESIGN: A prospective observational study. SETTING: German university hospital cardiovascular ICU. Data were collected from April 2010 to December 2011. PATIENTS: From 110 enrolled patients, 104 were included. Inclusion criteria were American Society of Anaesthesiologists (ASA) physical status III or IV patients undergoing controlled ventilation. Exclusion criteria included emergencies, complete heart block and marked arterial pressure differences greater than 10 mmHg in the two arms. MAIN OUTCOME MEASURES: Bland-Altman plots, bias, precision, 95% limits of agreement, percentage error and agreement : tolerability indexes (ATIs) were estimated to determine clinical agreement. RESULTS: From 11 222 arterial pressure readings, biases (SD) for CIAP-CNAP for systolic arterial pressure (SAP), diastolic arterial pressure (DAP) and mean arterial pressure (MAP) for all patients were 4.3 (11.6), -9.4 (8) and -6 (7.6) mmHg, respectively. Cardiac arrhythmia (4.1 (13.1), -14.4 (8.3), -9.5 (8.9) mmHg) and long interval to last calibration [4.5 (15), -9.8 (9.5), -6.4 (9.1) mmHg] impaired the accuracy of CNAP with failed interchangeability criteria defined by the percentage error. In contrast, use of catecholamines (epinephrine or norepinephrine infusions >0.1 µg kg min), short calibration intervals and weaning conditions did not affect accuracy, interchangeability and agreement, especially of MAP. Agreement was defined as acceptable for MAP for all data and subgroups (ATI 0.8 to 1.0) and at worst, marginal for SAP and DAP (ATI 0.9 to 1.6). CONCLUSION: CNAP showed acceptable agreement defined by the ATI with invasive measurements for MAP and partially for DAP, but there was considerable variability for SAP. MAP should be preferred for clinical decision making. Cardiac arrhythmia, in contrast to catecholamine dosage or weaning procedures, impaired the accuracy, agreement and interchangeability of CNAP. TRIAL REGISTRATION: Clinical trials.gov identifier NCT01003665.

Activities of cardiac tissue matrix metalloproteinases 2 and 9 are reduced by remote ischemic preconditioning in cardiosurgical patients with cardiopulmonary bypass.

BACKGROUND: Transient episodes of ischemia in a remote organ or tissue (remote ischemic preconditioning, RIPC) can attenuate myocardial injury. Myocardial damage is associated with tissue remodeling and the matrix metalloproteinases 2 and 9 (MMP-2/9) are crucially involved in these events. Here we investigated the effects of RIPC on the activities of heart tissue MMP-2/9 and their correlation with serum concentrations of cardiac troponin T (cTnT), a marker for myocardial damage. METHODS: In cardiosurgical patients with cardiopulmonary bypass (CPB) RIPC was induced by four 5 minute cycles of upper limb ischemia/reperfusion. Cardiac tissue was obtained before as well as after CPB and serum cTnT concentrations were measured. Tissue derived from control patients (N = 17) with high cTnT concentrations (≥0.32 ng/ml) and RIPC patients (N = 18) with low cTnT (≤0.32 ng/ml) was subjected to gelatin zymography to quantify MMP-2/9 activities. RESULTS: In cardiac biopsies obtained before CPB, activities of MMP-2/9 were attenuated in the RIPC group (MMP-2: Control, 1.13 ± 0.13 a.u.; RIPC, 0.71 ± 0.12 a.u.; P < 0.05. MMP-9: Control, 1.50 ± 0.16 a.u.; RIPC, 0.87 ± 0.14 a.u.; P < 0.01), while activities of the pro-MMPs were not altered (P > 0.05). In cardiac biopsies taken after CPB activities of pro- and active MMP-2/9 were not different between the groups (P > 0.05). Spearman's rank tests showed that MMP-2/9 activities in cardiac tissue obtained before CPB were positively correlated with postoperative cTnT serum levels (MMP-2, P = 0.016; MMP-9, P = 0.015). CONCLUSIONS: Activities of MMP-2/9 in cardiac tissue obtained before CPB are attenuated by RIPC and are positively correlated with serum concentrations of cTnT. MMPs may represent potential targets for RIPC mediated cardioprotection. TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT00877305.

The incidence and prevention of hypothermia in newborn bonding after cesarean delivery: a randomized controlled trial.

BACKGROUND: Little is known about thermoregulation of the newborn while bonding on the mother's chest immediately after cesarean delivery. Newborn hypothermia is associated with serious complications and should be avoided. Therefore, we evaluated whether newborns develop hypothermia during intraoperative bonding while positioned on their mothers' chests and investigated the effects of active cutaneous warming of the mothers and babies during a 20-minute intraoperative bonding period. METHODS: We enrolled 40 parturients scheduled for elective cesarean delivery under spinal anesthesia. Mothers and their newborns were randomized to receive either passive insulation or forced-air skin-surface warming during the surgical procedure and bonding period. The primary outcome was neonatal core temperature at the end of the bonding period. Core temperatures of the newborns were measured with a rectal probe. Body temperatures of the mothers were assessed by sublingual measurements. Skin temperatures, thermal comfort of the mothers, and perioperative shivering were evaluated. RESULTS: Without active warming from the beginning of the surgical procedure until the end of the bonding period, the mean (SD) neonatal core temperature decreased to 35.9 (0.6)°C. Seventeen of 21 (81%) newborns became hypothermic (defined as a core temperature below 36.5°C). Active skin-surface warming from the beginning of the surgical procedure until the end of the bonding period resulted in a neonatal core temperature of 37.0 (0.2)°C and a decreased incidence of hypothermia (1 of 19 (5%) newborns (P < 0.0001)). In addition, active warming increased the mean skin temperatures of the infants, maternal core and skin temperatures, maternal thermal comfort, and reduced perioperative shivering. CONCLUSIONS: Active forced-air warming of mothers and newborns immediately after cesarean delivery reduces the incidence of infant and maternal hypothermia and maternal shivering, and increases maternal comfort.