Citations

Experimental Human Endotoxemia (IV LPS Administration)

All experimental endotoxemia-related procedures were performed as described previously and were identical on both LPS challenge days (days 0 and 7) [19, 22]. In short, subjects were admitted to the research unit of the Radboud University Medical Center for 8 h. Subjects had to refrain from alcohol and caffeine (24 h) and food and drinks (12 h) prior to LPS administration. A radial artery catheter (BD Infusion Therapy Systems, Sandy, UT, USA) and antebrachial venous cannula were placed to allow serial blood sampling, hemodynamic monitoring, and administration of fluids and LPS, respectively. In the 45 min prior to LPS administration, hydration fluids (2.5% glucose/0.45% sodium chloride) were administered as a 1.5-L prehydration bolus to reduce the risk of vasovagal collapse [23] and thereafter at a rate of 150 mL/h for the remainder of the experiment. Directly after prehydration, a bodyweight-adjusted bolus dose of 1 ng/kg LPS (Escherichia coli-derived, Type O113, lot No. 94332B1; List Biological Laboratories, Campbell, CA, USA) was administered. Blood samples were serially obtained to construct time-concentration curves of circulating cytokines.

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… At T=0, 1ng/kg bodyweight LPS (E. coli type O113, Lot no. 94332B1; List Biological Laboratories, Campbell, USA) was administered intravenously to elicit a transient systemic inflammatory response …

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IN VIVO EXPERIMENTAL HUMAN ENDOTOXEMIA

Human endotoxemia experiments were conducted at the research unit of the Intensive
Care department of the Radboud university medical center according to our standard
protocol (21). Subjects refrained from caffeine and alcohol in the 24 h before the
experiment and from food and drinks 10 h before the experiment. One venous canulae
was placed in the antecubital vein, for fluid infusion and endotoxin administration.
Subjects were pre-hydrated in the hour before LPS administration (1.5 L 2.5%
glucose/0.45% saline) followed by 150 mL/h for 6 h (until the end of the experiment).
Blood pressure monitoring and blood withdrawals were performed using an arterial
cannula placed in the radial artery. In addition, ECG was continuously monitored and
vital signs were recorded every 5 seconds on a Phillips MP50 patient monitor using an
in-house developed data capturing system. Body temperature was measured every 30
mins with infrared tympanic measurements (FirstTemp Genius 2, Covidien Geldermalsen,
the Netherlands). At T=0, 1 ng/kg bodyweight LPS (E. coli type O113, Lot no. 94332B1;
List Biological Laboratories, Campbell, USA) was administered intravenously to elicit a
transient systemic inflammatory response.

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2.3. Experimental human endotoxemia

Subjects were admitted twice to the intensive care research unit of the Radboud university medical center in Nijmegen, The Netherlands, to receive both LPS challenges. All subjects underwent the same procedures on both hospitalization days which were 7 days apart. One day prior to both admissions, subjects needed to refrain from alcohol and caffeine, and no food or drinks were allowed from 10 hours before start of the challenge days. After admission, an intravenous cannula was inserted in the antebrachial vein to administrate fluids and LPS. A radial artery catheter was placed to withdraw blood and monitor blood pressure continuously. One hour before the challenge, 1.5 L prehydration fluid (2.5% Glucose/0.45% NaCl) was administered. Subsequently, a bolus of 2 ng/kg LPS (E. coli type O113, Lot no. 94332B1; List Biological Laboratories, Campbell, USA) was administered intravenously and similar hydration fluid was continued at an infusion rate of 150 mL/h for 8 hours. Heart rate was monitored using a 4-lead electrocardiogram (M50 Monitor, Philips, Eindhoven, The Netherlands) and intra-arterial blood pressure was measured with a pressure transducer (Edward Lifesciences, Irvine, USA). Every 30 minutes, we measured core temperature with a tympanic thermometer (FirstTemp Genius 2, Covidien, Dublin, Ireland) and LPS-induced symptoms (headache, nausea, cold shivers, muscle- and back pain) were scored using a numeric six-point scale (0 = no symptoms, 5 = worst symptoms experienced ever). The sum of these scores in addition of 3 points in case of vomiting, resulted in a total symptom score ranging from 0 to 28. Blood was withdrawn before prehydration (T = -60 minutes), just before LPS administration (T = 0 minutes), and respectively 60, 90, 120, 180, 240, 360 and 480 minutes after the LPS bolus. Plasma cytokine concentrations (tumour necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-8, IL-10, macrophage inflammatory protein (MIP)-1α, MIP-1β, monocyte chemoattractant protein (MCP)-1 and IL-1RA (receptor antagonist)) were determined batchwise using a simultaneous luminex assay (Milliplex, Millipore, Billerica, USA).

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… Clinical center reference endotoxin (GMP endotoxin prepared from E. coli O113 by List Biological Laboratories, Inc., Campbell, CA) was administered after informed consent to healthy subjects enrolled in an IRB-approved protocol (Pro00070829 at Duke University) …

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… Intravenous LPS challenge. Intravenous LPS challenges were only performed on occasion 2 and 3. Subjects received 1 ng/kg (cohort 1) or 2 ng/kg (cohort 2) E. Coli-purified LPS (GMP-grade from Lot#94332B4, List Biological Laboratories Inc …

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Experimental Human Endotoxemia:

Purified LPS (continuous infusion study: Escherichia coli O:113, Lot no. 94332B4, List Biological Laboratories, Campbell, USA; bolus administration study: US Standard Reference Endotoxin Escherichia coli O:113, Pharmaceutical Development Section of the National Institutes of Health, Bethesda, USA) was supplied as a lyophilized powder and dissolved in normal saline 0.9% as described previously [23,24]. For the continuous infusion study (c-LPS), a total of 4 ng/kg LPS was administered as a intravenous loading bolus of 1 ng/kg body weight at T = 0, followed by an intravenous infusion of 1 ng/kg/h for a period of 3 h [24]. For the bolus administration study (b-LPS), LPS was administered, as described previously, as an intravenous bolus injection at a dose of 2 ng/kg body weight in 1 min at T = 0 [23].

All subjects received 1.5 L of 2.5% glucose/0.45% saline solution in the hour before initiation of LPS administration, followed by 150 mL/h during the first 6 h after LPS administration and 75 mL/h until the end of the experiment, according to our protocol [24].

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Endotoxin:

All endotoxin lots were derived from an E. coli Type O113:H10:K-negative strain of bacteria. Endotoxin was stored at the investigational site in a dedicated fridge at 28C, and reconstituted prior to administration, both according to the manufacturers instructions. The temperature of the fridge was constantly monitored and logged using an automated system. Lot #1188844 was derived from bulk material produced by the National Institute of Allergy and Infectious Diseases and FDA in 1967 and vialled in 2006 under GMP conditions by the Pharmaceutical Development Section of the NIH (Bethesda, MD, USA). The lots #94332B1 and #94332B4 were both derived from the same new GMP-grade bulk material and vialled under GMP conditions by List Biological Laboratories, Inc (Campbell, California, USA). This GMP-grade endotoxin has previously shown to be safe for administration in humans.

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… 2.2. Dose regimens. Currently, the only commercially available LPS for use in humans is the GMP-grade E. Coli Type O113 (Lot no. 94332) produced by List Biological Laboratories in Campbell, USA. …

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– One hour prior to study drug administration, systemic inflammation was induced by bolus administration of 1 ng/kg Escherichia coli type O113 lipopolysaccharide (LPS; List Biological Laboratories Inc. Campbell, California, USA) …

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