Julia Hasslacher

Levosimendan inhibits release of reactive oxygen species in polymorphonuclear leukocytes in vitro and in patients with acute heart failure and septic shock: a prospective observational study

IntroductionLevosimendan is an extensively investigated inodilator showing also cardioprotective and antiinflammatory effects. The aim of our study was to explore the influence of levosimendan on polymorphonuclear leucocytes (PMN), a main source of reactive oxygen species, in vitro and in patients with acute heart failure or septic myocardial depression.MethodsPMN isolated from healthy volunteers were incubated with levosimendan in vitro. After stimulation with N-formyl-Met-Leu-Phe (fMLP) or phorbol 12-myristate 13-acetate (PMA) respiratory burst was quantified using a fluorescent dye. Apoptosis and expression of cell adhesion molecules of PMN were measured by flow cytometry. For determination of in vivo effects patients with acute heart failure (n = 16) or septic cardiac failure (n = 9) receiving levosimendan treatment were enrolled consecutively. PMN were isolated to measure respiratory burst activity before treatment as well as one and two hours after initiation of levosimendan administration. Furthermore inflammatory, hemodynamic and renal function parameters were obtained.ResultsIn vitro, levosimendan suppressed respiratory burst activity in fMLP or PMA stimulated PMN in a dose dependent manner by 30 ± 11% (P < 0.001) at 100 ng/mL and by 27 ± 17% (P < 0.001) at 1000 ng/mL respectively. Markers of apoptosis and PMN cell adhesion molecule expression remained unaffected by levosimendan treatment.In vivo, levosimendan treatment for two hours resulted in a significant reduction of PMA stimulated oxidative burst by 45% (P < 0.01) and fMLP stimulated oxidative burst by 49% (P < 0.05) in patients with acute heart failure. In patients suffering from septic shock levosimendan treatment decreased oxidative burst activity in unstimulated, fMLP and PMA stimulated PMN by 48% (P < 0.05), 46% (P < 0.01) and 43% (P < 0.01) respectively.ConclusionsLevosimendan appears to exert distinct immunomodulatory effects by decreasing oxidative burst activity of PMN. This property might contribute to the previously described cardioprotective effects of the drug.

Characterization of Microvesicles in Septic Shock Using High-Sensitivity Flow Cytometry.

Purpose: Endothelial pathology is considered to play a key role in septic shock. Since endothelial-derived microvesicles (MV) are elevated in various diseases associated with endothelial pathology, they are considered surrogate markers of the endothelial state. By analyzing the signature of circulating MV with high-sensitivity flow cytometry (hsFC), we wanted to test the hypothesis whether endothelial-derived MV are increased in septic shock. Methods: MV in blood from healthy volunteers and patients with septic shock treated in a medical intensive care unit were quantified by hsFC, which has an improved detection limit of approximately 0.3 &mgr;m. Results: Patients with septic shock (n = 30) showed 3-fold higher levels of CD31+/CD41− MV (58.5 (26.4–101.2) [median (25th–75th percentile)] vs. 19.5 (12.8–25.4) MV/&mgr;L; P <0.001) compared with healthy volunteers (n = 18). Absolute counts of CD144+, CD62E+, and CD106+ MV, specific for endothelial-derived MV, were low in all groups. The number of CD31+/CD41− MV correlated significantly with leukocyte count (rs = 0.64; P <0.001). Platelet-derived CD41+ MV were significantly elevated in the group dying within 48 h after inclusion (639.1 (321.3–969.7) vs. 221.5 (119.5–456.9) MV/&mgr;L; P = 0.037). Patients dying within 48 h had also significantly higher levels of CD31+/CD41−/AnnexinV− MV (51.9 (24.9–259.8) vs. 18.9 (9.7–31) MV/&mgr;L; P = 0.028). Conclusions: Despite an improved detection limit for MV by using hsFC, counts of endothelial-specific MV are unexpectedly low in patients with septic shock. Increased amounts of CD41+ and CD31+/CD41−/AnnexinV− MV indicate release by activated platelets and possibly leukocytes correlating with unfavorable outcome.

Atorvastatin Inhibits Functional Expression of Proatherogenic TLR2 in Arterial Endothelial Cells

Background: There is growing evidence that TLR2 plays a role in the pathogenesis of atherosclerosis. It is highly expressed in endothelial cells in areas of disturbed blood flow, like plaques or vessel bifurcations, but laminar blood flow suppresses endothelial TLR2 expression and is therefore thought to be atheroprotective. We sought for means to also protect lesion prone sites from TLR2 over-expression and subsequent endothelial activation. Methods: Human coronary artery endothelial cells (HCAEC) were treated with atorvastatin (ATV) and TLR2 surface expression was determined by FACS analyses. Western blot analyses were used to explore the phosphorylation status of SP1. Results: ATV profoundly inhibited basal and stimulated endothelial TLR2 expression in a time- and dose-dependent manner. It also inhibited HCAEC activation by MALP-2. TLR2 surface expression was inversely correlated to SP1 serine phosphorylation and was casein kinase 2 dependent. Conclusion: We demonstrate that ATV can control over-expression of proinflammatory endothelial TLR2 protein and TLR2-mediated endothelial activation. The mechanism involves casein kinase 2 and SP1 phosphorylation. ATV effects on endothelial cell TLR2 are comparable to those of laminar blood flow and might therefore also be atheroprotective.

Outcome prediction and temperature dependency of MR-proANP and Copeptin in comatose resuscitated patients.

OBJECTIVE To evaluate the prognostic potential of serum C-terminal provasopressin (CT-proAVP or Copeptin) and midregional pro-A-type natriuretic peptide (MR-proANP) to predict neurological outcome following resuscitation from cardiac arrest. METHODS In this prospective observational study, we employed novel ultra sensitive immunoassay technology to examine serial serum samples from 134 cardiac arrest patients. Patients were either allocated to mild therapeutic hypothermia using an endovascular device or normothermia. Serial blood samples were obtained from resuscitated cardiac arrest survivors during their first 7 days in an intensive care unit, and serum Copeptin and MR-proANP were measured. Cerebral function assessments were made using cerebral performance categorization (CPC) at discharge from hospital. Copeptin and MR-proANP data were analyzed using dichotomized CPC scores (1-2 versus 3-5). RESULTS Sixty-nine patients (51%) had a poor outcome (CPC 3-5) at hospital discharge. MR-proANP and Copeptin peaked on day 1 (i.e. 0-24h) with the medians being 249.3pmol/L and 77.2pmol/L, respectively. In the first 48h maximum levels of MR-proANP and Copeptin showed an AUC in the ROC of 0.743 (95% CI: 0.658-0.828) and 0.677 (95% CI: 0.583-0.771). Binary logistic regression revealed MR-proANP and Copeptin within 48h after ROSC being significantly associated with functional outcome (p<0.05). Copeptin within 48h was also associated with outcome in the hypothermia group (p<0.05). CONCLUSION Systemic levels of MR-proANP and Copeptin peak early in cardiac arrest patients in the 48h post-resuscitation period. MR-proANP and Copeptin were highly predictive for poor outcome in comatose resuscitated patients.

Insufficient performance of serum cystatin C as a biomarker for acute kidney injury of postrenal etiology.

Dear Editor, Royakkers et al. [1] recently reported that serum and urinary cystatin C appear to be poor biomarkers for the early detection of acute kidney injury (AKI) in critically ill patients and cannot predict the need for renal replacement therapy (RRT). In this context we present the case of a 65-year-old man with AKI showing nearly normal serum cystatin C levels despite excessive creatinine levels due to AKI. The patient, who had a history of type II diabetes, hypertension, and chronic kidney disease (CKD, stage 3), was admitted to the hospital with severe shortness of breath and bilateral pleural effusions. The right kidney had been removed because of tuberculosis several years before. During the 2 weeks before admission the patient had noticed gross hematuria. The initial CT scan revealed moderate hydronephrosis as well as a minor infiltration in the right middle lobe of the lung. The patient’s condition deteriorated rapidly, requiring intubation for acute respiratory failure as well as vasopressor therapy and volume resuscitation for vasodilatory shock. Inflammatory markers were significantly increased [C-reactive protein (CRP) 1.22 mg/dl, normal 0–0.7; procalcitonin (PCT) 6 lg/l, normal 0–0.5; white blood cell (WBC) 16.7 G/l). The sepsis-related organ failure assessment (SOFA) and acute physiology, age, chronic health evaluation (APACHE) II scores at admission were 15 and 43, respectively. Placement of a urinary catheter resulted in 200 ml of urine with gross hematuria and leukocytes. A diagnosis of urosepsis due to postrenal obstruction was made. Because of persistent oliguria, acidosis (pH 6.9), and hyperkalemia (potassium 7.44 mmol/l) and severe azotemia (creatinine 17.23 mg/dl, normal 0.67–1.17; urea 298.4 mg/dl, normal 18–55) continuous venovenous hemodialysis (CVVHD) was initiated using regional citrate anticoagulation (MultiFiltrate Ci-Ca, Fresenius Medical Care, Bad Homburg, Germany) with an Ultraflux AV 1000 S filter. Blood flow was 100 ml/min and dialysate rate 2,000 ml/h. Despite pronounced azotemia serum cystatin C was only slightly elevated (1.43 mg/l, normal range 0.47–1.09). Within 12 h of CVVHD serum creatinine decreased to 7.68 mg/dl and serum cystatin C to 1.14 mg/l (Fig. 1). During the following days of treatment serum creatinine and urea constantly declined, whereas serum cystatin C increased for another 7 days until the maximum level of 2.33 mg/l was reached. After stabilization of the patient a diagnostic urethrocystoscopy was performed and revealed a multilocalized, infiltrating carcinoma of the bladder obstructing the urethra—presumably the cause of postrenal failure and urosepsis. After 15 days CVVHD was discontinued and the patient was switched to intermittent hemodialysis. Cystatin C is a 13.3-kDa protein that is produced at a constant rate by nucleated cells. It is freely filtered by glomeruli, reabsorbed, and almost completely metabolized by the proximal renal tubule. Serum cystatin C has been shown to respond promptly to early reductions of (glomerular filtration rate) GFR in CKD [2]. In cases of a sudden drop of GFR characteristic of AKI, serum cystatin C was found to increase similarly to creatinine but more rapidly [3]. In discrepancy to this concept our patient with underlying CKD had only slightly elevated serum cystatin C levels in the presence of severe azotemia due to postrenal obstruction on admission. A likely explanation for this finding may be that during the acute phase of obstructive AKI some glomerular filtration is still

Bioelectrical impedance vector analysis in the critically ill: cool tool or just another 'toy'?

Assessment of volume and hydration status is far from easy and therefore technology such as bioelectrical impedance vector analysis (BIVA) may complement our examination techniques. This study highlights the fact that clinical assessment of volume balance and BIVA may correlate, but whether the routine use of BIVA will avoid significant volume overload in the critically ill remains unknown. Further studies are needed but at the moment appear a little way off.

Differential Effects of NO Inhibition in Renal Epithelial and Endothelial Cells in Mono-Culture vs. Co-Culture Conditions

Nitric oxide (NO) plays a critical role in the regulation of renal hemodynamics and tubular function after post-ischemic damage or sepsis. Diminished NO bioavailability contributes to endothelial dysfunction and may be caused by reduced NO synthesis due to substrate or co-factor deficiency. The aim of this study was to investigate the effects of NOS inhibition and NO depletion in a renal endo-epithelial bilayer model compared to monolayers of proximal tubular epithelial (HK-2) cells and endothelial cells of venous origin (EA.hy 926) with respect to cellular integrity, apoptosis and cytokine release. Two different NOS inhibitors have been used: an arginine-based-inhibitor, L-NGmonomethyl-arginine (L-NMMA) and a cofactor-based-inhibitor, H4-amino-biopterin (4-ABH4) showing iNOS selectivity. We found significantly higher basal NO production by epithelial than by endothelial monolayers, which was significantly reduced by both NOS-inhibitors with a stronger effect demonstrated by 4-ABH4. Furthermore we detected significant basal iNOS protein expression in unstimulated HK-2 cells. NOS inhibition by 4-ABH4 was associated with increased LDH release, apoptosis and reduced IL-6 production in epithelial but not in endothelial monolayers. These effects on epithelial cells were abolished under co-culture conditions. In contrast, endothelial cells showed higher IL-6 and IL-8 release under co-culture conditions than in monolayers, with IL-8 production being largely suppressed by L-NMMA but not by 4-ABH4. In conclusion, inhibition of basal NO production in epithelial monolayers shows detrimental effects on cell integrity and viability. Under co-culture conditions interrelation between epithelial and endothelial cells appears to counteract these potentially harmful effects of epithelial NOS inhibition.

Coagulation Disorders in Subjects Undergoing Pump-Driven Veno-Venous Ecco2-R For Severe Acute Hypercapnic Respiratory Failure - a Single Center Experience

Recent evidence suggests low-flow extracorporeal CO2 removal (ECCO2-R) systems as safe and promising adjunctive therapy to avoid endotracheal intubation and the related negative consequences in subjects with severe hypercapnic respiratory failure [1]. in high-flow extracorporeal membrane oxygenation systems heterogeneous coagulation disorders are a well-known complication. However, to date there is little evidence for the influence of pump-driven low-flow veno-venous ECCO2-R on the coagulation system.

Effects of mild therapeutic hypothermia on acute kidney injury after cardiopulmonary resuscitation

About the effect of mild therapeutic hypothermia on renal function after cardiopulmonary resuscitation little data exists. From animal and human studies it is known that renal function might be impaired, which is not reflected by serum creatinine levels if creatinine production is reduced.