Pharmacokinetics of amphotericin B colloidal dispersion in critically ill patients with cholestatic liver disease.

The pharmacokinetics of lipid-bound and liberated amphotericin B (AMB) was assessed in 11 critically ill patients with cholestatic liver disease (CSLD) and in 9 subjects with normal liver function treated with AMB colloidal dispersion (ABCD). Exposure to lipid-bound AMB was higher in patients with CSLD. Levels of liberated AMB were elevated by CSLD only after the first dose, whereas its pharmacokinetics was unaffected at steady state. The standard dosage of ABCD is probably adequate for patients with CSLD.

Human tissue distribution of voriconazole.

Voriconazole concentrations were determined in autopsy samples of eight patients who had been treated for a median of 7 days (interquartile range [IQR], 5 days). Voriconazole penetrates well into various tissues, with median levels of 6.26 µg/g ((interquartile range [IQR], 7.87 µg/g) in the lung, 3.41 µg/g (IQR, 16.72 µg/g) in the brain, 6.89 µg/g (IQR, 24.16 µg/g) in the liver, 6.47 µg/g (IQR, 6.19 µg/g) in the kidneys, 5.60 µg/g (IQR, 11.49 µg/g) in the spleen, and 7.55 µg/g (IQR, 16.91 µg/g) in the myocardium.

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.

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.

INTRODUCTION: Levosimendan 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. METHODS: PMN 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. RESULTS: In 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. CONCLUSIONS: Levosimendan 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.

Influence of albumin dialysis on pharmacokinetics of amphotericin B colloidal dispersion and amphotericin B lipid complex.

Albumin dialysis (AD) is a therapeutic option in severe cholestatic liver failure. However, it can significantly enhance drug elimination. Pharmacokinetic data on antimicrobial agents--in particular on antimycotics--administered under this clinical condition are very sparse. Therefore, amphotericin B (AMB) plasma concentrations were measured in two critically ill patients who were treated with AD because of severe cholestatic liver failure and were prescribed lipid formulated AMB--either AMB colloidal dispersion (ABCD) or AMB lipid complex (ABLC)--for suspected invasive fungal infection. AD was performed with the molecular adsorbent recirculating system (MARS). Lipid-associated and liberated AMB were separately quantified on and off AD. The clearance of the liberated AMB fraction was not essentially affected (ABLC) or moderately enhanced during AD by a factor of 2.5 (ABCD). The clearance of the lipid-formulated fraction was increased by a factor of 4 during AD (ABCD) or was similar (ABLC) on and off AD. Despite the fact that there was a four-fold higher clearance of the lipid-formulated fraction of ABCD, the clinically relevant area under the concentration time curve of the liberated AMB fraction was only moderately changed (by 37% in ABCD, 70% in ABLC) during AD. Thus, the effect of AD on lipid formulated AMB appears to be moderate. A daily dose of 5 mg/kg will probably lead to adequate plasma levels in patients on AD.

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-N(G)monomethyl-arginine (L-NMMA) and a cofactor-based-inhibitor, H4-amino-biopterin (4-ABH(4)) 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-ABH(4). Furthermore we detected significant basal iNOS protein expression in unstimulated HK-2 cells. NOS inhibition by 4-ABH(4) 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-ABH(4). 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.

Hyperoncotic colloids and acute kidney injury: a meta-analysis of randomized trials.

INTRODUCTION: It has been hypothesized that hyperoncotic colloids might contribute to acute kidney injury (AKI). However, the validity of this hypothesis remains unclear. METHODS: A meta-analysis was conducted of randomized controlled trials evaluating AKI after infusion of hyperoncotic albumin and hydroxyethyl starch (HES) solutions. Mortality was a secondary endpoint. Eligible trials were sought by multiple methods, and the pooled odds ratios (OR) for AKI and death and 95% confidence intervals (CI) were computed under a random effects model. RESULTS: Eleven randomized trials with a total of 1220 patients were included: 7 evaluating hyperoncotic albumin and 4 hyperoncotic HES. Clinical indications were ascites, surgery, sepsis and spontaneous bacterial peritonitis. Hyperoncotic albumin decreased the odds of AKI by 76% (OR, 0.24; CI, 0.12-0.48; P < 0.0001), while hyperoncotic HES increased those odds by 92% (OR, 1.92; CI, 1.31-2.81; P = 0.0008). Parallel effects on mortality were observed, with hyperoncotic albumin reducing the odds of death by 48% (OR, 0.52; CI, 0.28-0.95; P = 0.035) and hyperoncotic HES raising those odds by 41% (OR, 1.41; CI, 1.01-1.96; P = 0.043). CONCLUSIONS: This meta-analysis does not support the hypothesis that hyperoncotic colloid solutions per se injure the kidney. Renal effects appear instead to be colloid-specific, with albumin displaying renoprotection and HES showing nephrotoxicity.

Pulmonary epithelial lining fluid concentrations after use of systemic amphotericin B lipid formulations.

Amphotericin B (AMB) concentrations were determined in pulmonary epithelial lining fluid (ELF) of 44 critically ill patients, who were receiving treatment with liposomal AMB (LAMB) (n = 11), AMB colloidal dispersion (ABCD) (n = 28), or AMB lipid complex (ABLC) (n = 5). Mean AMB levels (+/- standard errors of the means) in ELF amounted to 1.60 +/- 0.58, 0.38 +/- 0.07, and 1.29 +/- 0.71 microg/ml in LAMB-, ABCD-, and ABLC-treated patients, respectively (differences are not significant).

Flow-dependent regulation of endothelial Toll-like receptor 2 expression through inhibition of SP1 activity.

Innate immune system activation is associated with atherosclerotic lesion development. The specific sites of lesion development are believed to be defined by the shear stress of blood flow. Consequently, we investigated the responsiveness of human coronary artery endothelial cells (HCAECs) to Toll-like receptor (TLR) 2 and 4 agonists in an in vitro model of chronic laminar flow. HCAECs under chronic laminar flow were found to be normally responsive to lipopolysaccharide (and tumor necrosis factor) in terms of E-selectin expression but were found to be hyporesponsive to stimulation with the specific TLR2 ligands macrophage activating lipopeptide-2, PAM2-Cys, and Lip19; this was observed to be attributable to downregulation of TLR2 transcription and protein expression. We found that laminar flow induced SP1 serine phosphorylation by protein kinase CK2 and thereby blocked SP1 binding to the TLR2 promoter, which is required for TLR2 expression. This regulatory mechanism also blocked lipopolysaccharide- and tumor necrosis factor-induced TLR2 upregulation in HCAECs and could be important for suppression of other flow-sensitive endothelial proteins. These results extend the role of flow in controlling endothelial responsiveness. Given the current evidence that TLRs are proatherogenic, flow suppression of TLR2 expression may be atheroprotective.

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 µm. 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/µL; 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/µL; 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/µL; 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.

Neurokinin-1 receptor interacts with PrP(106-126)-induced dendritic cell migration and maturation.

Circulating monocytic cells may mediate neuroinvasion in transmissible spongiform encephalopathies by transporting prion protein (PrP) from sites of entry to the nervous system. Factors regulating monocyte-derived dendritic cell (DC) functions in neuronal tissue include neurogenic mediators, but their interactions with prion-infected DCs are unknown. Here, we report that neuropeptides regulate the interaction of DCs exposed to PrP(106-126). Inhibition of neurokinin-1-receptors activation with neurokinin-1-receptor antagonist or antibody attenuates substance P-induced enhancement of DC migration induced by PrP(106-126) and prion-protein-induced DC maturation. Other neuropeptides arrest chemotaxis. Data support a priming role of the neuropeptide substance P in PrP(106-126)-induced migration of DCs involving neurokinin-1-receptors.

Toll-like receptor 4 functions intracellularly in human coronary artery endothelial cells: roles of LBP and sCD14 in mediating LPS responses.

Endothelial cells are activated by microbial agonists through Toll-like receptors (TLRs) to express inflammatory mediators; this is of significance in acute as well as chronic inflammatory states such as septic shock and atherosclerosis, respectively. We investigated mechanisms of lipopolysaccharide (LPS)-induced cell activation in human coronary artery endothelial cells (HCAEC) using a combination of FACS, confocal microscopy, RT-PCR, and functional assays. We found that TLR4, in contrast to TLR2, is not only located intracellularly but also functions intracellularly. That being the case, internalization of LPS is required for activation. We further characterized the HCAEC LPS uptake system and found that HCAEC exhibit an effective LPS uptake only in the presence of LPS binding protein (LBP). In addition to its function as a catalyst for LPS-CD14 complex formation, LBP enables HCAEC activation at low LPS concentrations by facilitating the uptake, and therefore delivery, of LPS-CD14 complexes to intracellular TLR4-MD-2. LBP-dependent uptake involves a scavenger receptor pathway. Our findings may be of pathophysiological relevance in the initial response of the organism to infection. Results further suggest that LBP levels, which vary as LBP is an acute phase reactant, could be relevant to initiating inflammatory responses in the vasculature in response to chronic or recurring low LPS.

Rho-GTPase-dependent platelet-neutrophil interaction affected by HMG-CoA reductase inhibition with altered adenosine nucleotide release and function.

Platelet activation and aggregation is considered a crucial step in the initiation and aggravation of arterial thrombosis. ADP from activated platelets is recognized as major factor in thrombus formation and is a potent stimulator of oxygen-free radical release from neutrophils. The aim of the present investigation was to determine in vitro the direct effects of statins on ATP and ADP secretion by platelets and its impact on subsequent oxidative burst activity in neutrophils. Human neutrophils and platelets were isolated from peripheral blood. Levels of platelet-derived ATP and ADP were measured by high-performance liquid chromatography, oxygen-free radical release of neutrophils was measured fluorometrically, and chemotaxis experiments were performed. Rho-GTPases were studied by Western blot analysis. Thrombin-activated platelets primed neutrophils for enhanced oxygen-free radical release on triggering with formyl-Met-Leu-Phe, reduced by cerivastatin and simvastatin treatment of platelets. The two statins decreased the amount of adenosine-derivative release in these cells. Rho-GTPases, required for the thrombin signaling in platelets and neutrophils, were decreased after coincubation with statins. Data demonstrate that inhibition of Rho-GTPases by statins inhibit platelet ADP and ATP release and the consecutive augmentation of neutrophil oxygen-free radical release. Statins affect platelet-neutrophil interactions by altering Rho-GTPase-dependent adenosine nucleotide function.

Reversal of thrombin-induced deactivation of CD39/ATPDase in endothelial cells by HMG-CoA reductase inhibition: effects on Rho-GTPase and adenosine nucleotide metabolism.

Adenosine triphosphate and diphosphate that activate platelet, leukocyte, and endothelium functions are hydrolyzed by endothelial CD39/ATPDase. Because CD39/ATPDase is downregulated in endothelial cells by inflammation and this may be affected by HMG-CoA reductase inhibitors, we examined the role of cerivastatin and simvastatin in regulation of endothelial CD39/ATPDase expression, metabolism of ATP/ADP, and function in platelets. Thrombin-stimulated endothelial cells in vitro were treated with the statins, and hydrolysis of exogenous ADP and ATP was assessed by high-performance liquid chromatography and malachite green assay. Platelet aggregation studies were performed with endothelial cell supernatants as triggers. CD39/ATPDase surface expression by endothelial cells was determined immunologically by fluorescence-activated cell sorter, mRNA expression by RT-PCR, and thrombin-induced dissociation of Rho-GTPases by Western blotting. Treatment by simvastatin or cerivastatin restored impaired metabolism of exogenous ATP and ADP in thrombin-activated endothelial cells by preventing thrombin-induced downregulation of CD39/ATPDase. In platelet aggregation studies, ATP and ADP supernatants of thrombin-activated endothelial cells were less stimulatory in the presence of statins than in their absence. Data show that statins preserve CD39/ATPDase activity in thrombin-treated endothelial cells involving alterations by statins of Rho-GTPase function and CD39/ATPDase expression. Preservation of adenine nucleotide metabolism may directly contribute to the observed anti-thrombotic and anti-inflammatory actions of statins.

Adverse effect of heparin on antithrombin action during endotoxemia: microhemodynamic and cellular mechanisms.

A recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin + AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH + AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPS-treated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p < 0.01), whereas Heparin + AT and LMWH + AT animals showed microcirculatory alterations comparable to that in controls. In additional in vitro chemotaxis assays. AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH. Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia. These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.

Inhibition of human neutrophil chemotaxis toward interleukin 8 with six clinical antithrombin concentrates in vitro.

OBJECTIVE: Antithrombin exerts direct effects on neutrophils by inhibiting chemokine-induced migration. This study examined the potency of different pharmaceutical antithrombin preparations in inhibiting neutrophil chemotaxis toward interleukin 8. METHODS: Cell migration was tested by the leading front assay in modified Boyden microchemotaxis chambers bearing nitrocellulose filters. Human neutrophils were incubated with six different antithrombin concentrates or an immunopurified antithrombin preparation at concentrations of 1 micro IUeth-5 IU/ml. RESULTS: All antithrombin concentrates irrespective of the pharmaceutical source deactivated neutrophil chemotaxis. At concentrations below 100 mIU/ml neutrophil chemotaxis toward interleukin 8 was decreased by the antithrombin preparations with varying potency, but at 1 mIU/ml no significant differences were observed. CONCLUSIONS: As the ability of antithrombin to deactivate neutrophil chemotaxis toward interleukin 8 shows differences depending on the source of commercial antithrombin, these results suggest that at equivalent WHO standard concentrations clinical antithrombin concentrates may differ in anti-inflammatory potential.

Secretoneurin as a marker for hypoxic brain injury after cardiopulmonary resuscitation.

PURPOSE: The neuropeptide secretoneurin (SN) shows widespread distribution in the brain. We evaluated whether SN is elevated after cardiopulmonary resuscitation (CPR) and could serve as a potential new biomarker for hypoxic brain injury after CPR. METHODS: This was a prospective observational clinical study. All patients admitted to a tertiary medical intensive care unit after successful CPR with expected survival of at least 24 h were consecutively enrolled from September 2008 to April 2013. Serum SN and neuron-specific enolase were determined in 24 h intervals starting with the day of CPR for 7 days. Neurological outcome was assessed with the Cerebral Performance Categories Scale (CPC) at hospital discharge. RESULTS: A total of 134 patients were included with 49 % surviving to good neurological outcome (CPC 1-2). SN serum levels peaked within the first 24 h showing on average a sixfold increase above normal. SN levels were significantly higher in patients with poor (CPC 3-5) than in patients with good neurological outcome [0-24 h: 75 (43-111) vs. 38 (23-68) fmol/ml, p < 0.001; 24-48 h: 45 (24-77) vs. 23 (16-39) fmol/ml, p < 0.001]. SN determined within the first 48 h showed a receiver operating characteristic (ROC) area under the curve (AUC) of 0.753 (0.665-0.841). NSE in the first 72 h had a ROC-AUC of 0.881 (0.815-0.946). When combining the two biomarkers an AUC of 0.925 (0.878-0.972) for outcome prediction could be reached. CONCLUSIONS: SN is a promising early biomarker for hypoxic brain injury. Further studies will be required for confirmation of these results.