Guenter Huhle

Prognostic Value of Plasma N-Terminal Pro-Brain Natriuretic Peptide in Patients With Severe Sepsis

Background—Increased plasma levels of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) have been identified as predictors of cardiac dysfunction and prognosis in congestive heart failure and ischemic heart disease. In severe sepsis patients, however, no information is available yet about the prognostic value of natriuretic peptides. Therefore, the aim of the present study was to determine the role of the N-terminal prohormone forms of ANP (NT-proANP) and BNP (NT-proBNP) in the context of outcome of septic patients. Furthermore, the effect of treatment with recombinant human activated protein C [drotrecogin alfa (activated)] on plasma levels of natriuretic peptides in severe sepsis was evaluated. Methods and Results—Fifty-seven patients with severe sepsis were included. Levels of NT-proANP and NT-proBNP were measured on the second day of sepsis by ELISA. Septic patients with NT-proBNP levels >1400 pmol/L were 3.9 times more likely (relative risk [RR], 3.9; 95% CI, 1.6 to 9.7) to die from sepsis than patients with lower NT-proBNP values (P<0.01). NT-proANP levels, however, were not predictive of survival in our patient population. A highly significant correlation was found between troponin I levels and plasma concentrations of NT-proBNP in septic patients (r=0.68, P<0.0001). In addition, troponin I significantly accounted for the variation in NT-proBNP levels (P<0.0001), suggesting an important role for NT-proBNP in the context of cardiac injury and dysfunction in septic patients. Twenty-three septic patients who received treatment with drotrecogin alfa (activated) presented with significantly lower concentrations of NT-proANP, NT-proBNP, and troponin I compared with patients not receiving drotrecogin alfa (activated). Conclusions—NT-proBNP may serve as useful laboratory marker to predict survival in patients presenting with severe sepsis.

Recombinant human activated protein C upregulates cyclooxygenase- 2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

Prostacyclin (PGI(2)) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI(2) in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI(2) release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500 ng/ml-20 microg/ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3-9 h and caused a time- and dose-dependent release of 6-keto PGF(1 alpha), the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-alpha (TNF-alpha) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostaglandin 12 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6-keto PGF(1 alpha)-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA upregulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

Time course of systemic markers of inflammation in patients presenting with acute coronary syndromes.

Abstract Inflammation within coronary plaques may cause an acute coronary syndrome by promoting rupture and erosion. It was the aim of this study to examine whether markers of inflammation derive from a cardiac or extracardiac source and how their levels develop over time. Blood samples were taken from patients with acute coronary syndromes (ACS) with proven atherosclerotic lesion(s) of the left coronary artery (n = 13) and from control patients without coronary artery disease (n = 13). Blood was taken from the femoral vein and the coronary sinus vein before and after coronary angioplasty (day 0) and on days 1 and 120. Levels of tumor necrosis factor-α (TNF-α), interleukin- 6 (IL-6), interleukin-1-receptor antagonist (IL-1 ra) and soluble CD40 ligand (sCD40L) were higher in ACS patients as compared to controls and remained elevated up to day 120. In the long-term time course these markers of inflammation and plaque remodeling slightly decreased in ACS patients. There were no statistically significant differences detectable in the levels of TNF-α, IL-6, IL-1 β, IL-10, IL-1 ra, sCD40L and monocyte chemoattractant protein-1 (MCP-1) in the blood of ACS patients taken from a cardiac source as compared to an extracardiac source (coronary sinus vs. femoral vein). This study demonstrates the importance of a systemic inflammatory condition in patients with ACS, in whom markers of inflammation are increased as compared to controls. During long-term follow-up the pro-inflammatory activity remains elevated in ACS patients, supporting the concept of a systemic rather than a local vascular inflammation contributing to the development of atherosclerosis.

Determination of heparin-induced IgG antibody by fluorescence-linked immunofiltration assay (FLIFA)

A fluorescence-linked immunofiltration assay (FLIFA) was developed for the determination of heparin-induced IgG in heparin-induced thrombocytopenia (HIT) type II patients. Protein A was immobilized on a nitrocellulose membrane to bind heparin-induced IgG of HIT type II patients. Fluorescein-5-isothiocynate (FITC)-heparin was added to platelet factor 4 present in normal serum to form the neo-antigen which was captured by heparin-induced IgG. The heparin-induced IgG was quantified by the relative fluorescence intensity (RFI) of bound FITC-heparin. Values were expressed as a RFI ratio (RFI patient / RFI normal) and were 1.965+/-0.413 in HIT type II patients (n = 36) and 1.064+/-0.162 in healthy controls (n = 50, p<0.0001). The intra- and inter-assay coefficients of variation were 4.9 and 10.4%, respectively. The heparin-induced IgG FLIFA will be useful in individual and epidemiological studies in patients during treatment with heparin. The FLIFA technique offers an alternative, rapid and sensitive methodological approach for studies on the interaction between antigen-antibody or ligand-receptor.

Recombinant human activated protein C upregulates the release of soluble fractalkine from human endothelial cells

Fractalkine is a unique endothelial cell‐derived chemokine that functions both as a chemoattractant and as an adhesion molecule. Recent findings suggest that fractalkine plays an important role in inflammatory diseases by modulating leucocyte endothelial cell interactions. A modulating effect on the immune system in severe sepsis has been suggested for recombinant human activated protein C (rhAPC). However, a little is known about the effect of rhAPC on the endothelial release of soluble fractalkine. The effect of rhAPC (50 ng/ml to 10 μg/ml) and protein C (in equimolar concentrations) on the synthesis of fraktalkine‐mRNA and release of soluble protein in human umbilical vein endothelial cells (HUVEC) was determined by reverse transcription‐polymerase chain reaction and by an enzyme‐linked immunosorbent assay. rhAPC at supra‐pharmacological concentrations (1–10 μg/ml) stimulated fractalkine‐messenger RNA‐gene transcription and release of soluble fractalkine in a time‐ and dose‐dependent manner, whereas the zymogen protein C was ineffective. As shown by experiments using monoclonal antibodies against the thrombin receptor, protease‐activated receptor‐1 (PAR‐1), PAR‐2 and against the endothelial protein C receptor (EPCR), the effect of rhAPC on fractalkine upregulation was mediated by binding to the EPCR‐receptor and signalling via PAR‐1. These in vitro data demonstrate that induction of fractalkine release is an important response of HUVEC to stimulation with rhAPC and may lead to a better understanding of the molecular pathways involved in the mode of action of rhAPC. Further clinical trials are needed to confirm the in vivo relevance of these data.

Prognostic value of platelet-derived growth factor in patients with severe sepsis.

Primary objective: Platelet-derived growth factor-BB (PDGF-BB) has been shown to promote the structural integrity of the vessel wall and to increase wound healing capacity. Aim of the present study was to determine the role of PDGF-BB in the context of outcome of septic patients. Furthermore, the effect of treatment with recombinant human activated protein C (rhAPC) on plasma levels of PDGF-BB in severe sepsis was evaluated as well as the in vitro effect of rhAPC on PDGF-BB-release from human endothelial cells (HUVEC). Research design, methods and procedures: PDGF-BB levels were measured in 46 patients on day 3 of severe sepsis. Twenty-one of these patients received treatment with rhAPC. The in vitro effect of rhAPC on PDGF-BB-messenger RNA synthesis and release of PDGF-BB into supernatants was measured by reverse transcriptase-polymerase chain reaction and ELISA-methods. Main outcomes and results: Survivors of severe sepsis presented with higher PDGF-BB levels than non-survivors (p < 0.05). Septic patients with PDGF-BB levels below 200 pg/ml were 7.3 times more likely (RR = 7.3, 95% CI: 1.4–44.5; p < 0.05) to die from sepsis than patients with higher PDGF-BB values. RhAPC (1–10 μg/ml) stimulated endothelial PDGF-BB-messenger RNA transcription and PDGF-BB-release in vitro. Plasma levels of PDGF-BB in patients receiving rhAPC were significantly (p < 0.01) higher (median 277.7; 25–75th percentiles: 150.5–414.4 pg/ml) than in patients not treated with rhAPC (median: 125.6; 25–75th percentiles: 55.3–344.7 pg/ml). Conclusions: The ability of rhAPC to upregulate endothelial PDGF-BB production may represent a new molecular mechanism by which rhAPC controls vessel wall homeostasis and increases tissue healing capacity in severe sepsis. PDGF-BB may serve as useful laboratory marker to predict survival in patients presenting with severe sepsis.

N-terminal pro-atrial natriuretic peptide as a biochemical marker of long-term interventional success after radiofrequency catheter ablation of paroxysmal supraventricular tachyarrhythmias

Abstract Radiofrequency (RF) catheter ablation has been shown to be highly effective in the treatment of supraventricular tachycardias. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (B-type natriuretic peptide; BNP) are secreted by the heart mainly in response to myocardial stretch induced by volume load. The aim of the present study was to determine the time course of the N-terminal prohormone forms of ANP (NT-proANP) and BNP (NT-proBNP) in patients undergoing radiofrequency (RF) catheter ablation for paroxysmal supraventricular tachycardias. Serial blood samples were taken from 13 patients with symptomatic paroxysmal supraventricular tachycardias undergoing RF ablation and from 13 age- and gender-matched healthy controls. Blood was taken before ablation (day 0, baseline), and at day one and day 120 after ablation. Levels of NT-proANP were significantly higher before RF ablation (4862 ± 726 pmol/l) as compared to day one (2021 ± 220 pmol/l) and day 120 after RF ablation (2470 ± 349 pmol/l) (with p < 0.01 on day one and p < 0.05 on day 120; n = 13). The size of the left atrium decreased from 41.0 ± 5.5 mm before ablation to 34.9 ± 5.9 mm (n = 13; p < 0.05) on day 120 as measured by M-mode echocardiography. Levels of NT-proBNP showed comparable values before and on day one and day 120 after ablation and were not significantly elevated as compared to healthy controls. NT-proANP levels are increased in patients presenting with paroxysmal supraventricular tachycardias and decrease one day after radiofrequency catheter ablation, possibly reflecting a transient reduction of ANP secretion from injured myocardial cells. Lower NT-proANP levels in the long-term time course may result from reduction of atrial volume load and reconstitution of atrial architecture after successful treatment of supraventricular tachycardias. NT-proANP may serve as a useful laboratory marker to describe the long-term interventional success after RF ablation.