Download C1-esterase inhibitor attenuates the inflammatory

Detailed information on C1INH-concentrate:
Cetor® consists of dried C1INH prepared from human plasma. The product is known to be
>90% pure. After being dissolved in the prescribed volume of water for injections the product
contains 100U of C1INH/ ml. Analysis of the batch, used in the current study, demonstrated a
protein amount of 13.7g/l and a specific activity of 5.9 U/ml.
Detailed information on the measurements of the hemodynamic and clinical response:
After admission to our research intensive care unit, the radial artery was cannulated with a 20
gauge arterial catheter (Angiomat, Deseret Medical, Becton Dickinson, Sandy, UT) under
local anaesthesia (lidocaine HCL 20 mg/ml). This catheter was connected to an arterial
pressure monitoring set (Edwards Lifesciences LLC, Irvine, CA) and a Philips IntelliVue
MP70 monitor (Philips Medical Systems, Eindhoven, The Netherlands). The mean arterial
pressure (MAP) was determined using the electronically integrated area under the arterial
pulse wave curve. Heart rate and respiratory rate monitoring were performed during the entire
experiment using a 5-lead ECG. A cannula was placed in an antecubital vein to permit
infusion of prehydration fluid (1.5 L 2.5% glucose/0.45% saline in 1 hr), endotoxin, C1INH
or placebo and a continuous infusion of 150 ml/hr 2.5% glucose/0.45% saline during the rest
of the experiment until discharge.
Detailed information on the assays performed:
All blood samples (anticoagulated with EDTA) were drawn from the arterial line, except for
samples obtained at t=24 h when blood was sampled using regular vena puncture. For
cytokine measurements, plasma was obtained immediately after blood sampling by
centrifugation at 2000xg for 15 minutes at a temperature of 4°C. Hereafter, plasma was stored
at -80°C until analysis. The concentrations of circulating cytokines TNF-α, IL-10, IL-6,
Monocyte Chemotactic Protein (MCP)-1, IL1-β and IL-1RA, as well as concentrations of
soluble vascular cell and inter-cellular adhesion molecules (VCAM-1 and ICAM-1), von
Willebrand Factor (VWF) and platelet (P)-selectin were determined in EDTA plasma
obtained immediately before endotoxin administration and serially thereafter. Measurements
of all cytokines and of the soluble adhesion molecules VCAM-1 and ICAM-1 were performed
by a multiplex assay according to the manufacturer’s instructions (Bio-plex cytokine assay,
BioRad, Hercules, CA, USA at Luminex 100, Luminex Corporation, Oosterhout,
The Netherlands). Coefficients of variation were less than 10% for all cytokines and for
VCAM-1 and ICAM- analysis. The lower limits of detection in this assay amounted 6 pg/ml
for IL-6 and IL-10, 7 pg/ml for MCP-1, 10 pg/ml for IL-1β, 20 pg/ml for TNF-α, 72 pg/ml for
IL1-RA, 581 pg/ml for VCAM-1 and 3633 pg/ml for ICAM-1.
VWF and P-selectin were determined by ELISA (Progen biotechnik, Heidelberg, Germany
and R&D systems, Minneapolis, MN). Activity of C1INH, C1INH antigen and complement
factor 4 were determined just before and serially after endotoxin administration on plasma
obtained immediately after sampling by centrifugation at 2000xg for 15 minutes at 20°C. The
determination of C1INH activity was performed using the Berichrom C1INH kit (Siemens
healthcare diagnostics, Deerfield, IL, USA, previously Dade Behring) and a Tecan Freedom
EVO 150 robot (Tecan Group, Männedorf, Switzerland). The quantitative determination of
C1INH antigen and complement factor 4 was achieved using a Behring Nefelometer (BNII)
(Siemens healthcare diagnostics, Deerfield, IL, previously Dade Behring).
C-reactive protein (CRP) levels were measured just before and at 8 and 24 hrs after endotoxin
administration in lithium heparin plasma using immunologic detection according to the
instructions of the manufacturer (Aeroset, Abbott Laboratories, Abbott Park, IL, U.S.A.).
Hematologic values were determined in EDTA anti-coagulated blood using flow-cytometry
(Sysmex XE-2100, Goffin Meyvis, Etten- Leur, The Netherlands) just before and serially
after endotoxin infusion.