GMP LPS

• Product #9433 – cGMP LPS, from E. coli O113

In vivo experimental human endotoxaemia model and ex vivo analyses

Eight healthy (as confirmed by medical history, physical examination and routine laboratory testing) male volunteers with ages ranging from 18 years to 35 years provided written informed consent to participate in experimental endotoxaemia experiments conducted at the research unit of the intensive care department of the Radboud University Medical Center. ...

A continuous endotoxin infusion regimen was used, as is described in detail elsewhere40. In short, participants were admitted to the research unit, and an antecubital vein and radial artery were cannulated to allow administration of fluids and endotoxin, and blood sampling and haemodynamic monitoring, respectively. A 3-lead ECG was recorded continuously throughout the experiment. After iso-osmolar pre-hydration (1.5 l NaCl 0.45% and glucose 2.5% administered intravenously in the hour before the start of endotoxin infusion), volunteers were intravenously challenged with a loading dose of 1 ng kg⁻¹ bodyweight endotoxin (Escherichia coli lipopolysaccharide (LPS) type O113, lot no. 94332B1; List Biological Laboratories), directly followed by continuous infusion of 0.5 ng kg⁻¹ h⁻¹ for 3 h. Participants were monitored for 8 h after the endotoxin loading dose after which they were discharged from the research unit. ...

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

Experimental human endotoxemia

All endotoxemia-related procedures were performed according to our standard continuous LPS-infusion protocol (12). In short, subjects were admitted to the research unit situated on the Medium Care department of the Radboud university medical center for approximately ten hours. Subjects had to refrain from consumption of alcohol and caffeine (24 h), and food and drinks (12 h) prior to LPS administration. Upon admission on the first LPS challenge day, two antebrachial venous cannulas were placed to allow administration of fluids, study medication and LPS. A radial artery catheter (BD Infusion Therapy Systems, Sandy, Utah, USA) was placed under ultrasound guidance to allow serial blood sampling and continuous hemodynamic monitoring. To reduce the risk of vasovagal responses (14), hydration fluids (2.5% glucose/0.45% sodium chloride) were administered as an initial prehydration bolus of 1.5L in the 45 min prior to LPS administration, and at a rate of 150 mL/hr for the remainder of the experiment. After prehydration, a bodyweight adjusted bolus dose of 1 ng/kg LPS (E. coli type O:113, lot no. 94332B1; List Biological Laboratories, Campbell, USA) was administered intravenously, directly followed by continuous infusion of LPS at a rate of 0.5 ng/kg/hr for three hours. Blood samples were serially obtained to construct time-concentration curves of circulating cytokines. Heart rate and intra-arterial blood pressure were measured using a 4-lead electrocardiogram (M50 Monitor, Philips, Eindhoven, the Netherlands) and a radial artery pressure transducer (Edwards Lifesciences, Irvine, California, USA). Hemodynamic parameters were recorded every 5 s using in-house developed software. Every 30 min, body temperature was measured using a tympanic thermometer (FirstTemp Genius 2, Covidien, Dublin, Ireland). Flu-like symptoms (headache, nausea, cold shivers, myalgia and back pain) were scored every 30 min using a numeric six-point Likert scale (0 = no symptoms, 5 = worst ever experienced). A composite score was then calculated as the sum of all previous elements, resulting in a total symptom score ranging from 0 to 25. Furthermore, general malaise was scored on an eleven-point scale (0 = no symptoms, 10 = worst ever experienced).

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

2.2. General session procedures

We performed all LPS-related procedures as described previously (Jansen et al., 2022). We tested three participants simultaneously in one room at the Medium Care Unit of Radboudumc. Sixty to 90 min before LPS administration, we placed a radial artery catheter and antebrachial venous cannula to allow serial blood sampling, hemodynamic monitoring, and administration of fluids and LPS, respectively and these stayed in during the remaining of the test-day. In the 45 min prior to LPS administration, we administered hydration fluids (2.5% glucose / 0.45% sodium chloride) as a 1.5L prehydration bolus to reduce the risk of vasovagal collapse(van Eijk et al., 2004), and thereafter at a rate of 150 mL/h for the remainder of the experiment. Directly after prehydration, we administered a bodyweight-adjusted bolus dose of 1 ng/kg LPS (E. Coli-derived, Type O113, lot no. 94332B1; List Biological Laboratories) intravenously. Behavioural testing took place at three timepoints: 45 min before injection (S1), 2 h post injection (S2) and 5 h post injection (S3). Timing was based on previous studies showing that sickness symptoms and cytokines peak 1.5-2 h after LPS administration and are largely normalized 5 post-LPS administration (Kox et al., 2015, Leentjens et al., 2012). At S2, participants were able to perform behavioural tasks while cytokine levels and fatigue are still high (Draper et al., 2018). Participants filled out mood questionnaires each hour after LPS administration. Furthermore, we obtained blood samples at various timepoints prior to and following LPS administration (see below). ...

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

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).

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

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.

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

Human blood samples

For volunteers treated with endotoxin, blood samples were acquired from a random 17 out of 100 volunteers who participated in the 100LPS human endotoxemia study (NL68166.091.18, CMO: 2018-4983). ...

On the day of the LPS challenge, volunteers were hospitalized at the Radboud University Medical Center and received a single intravenous administration of 1 ng/kg LPS (Escherichia coli O:113; List Biological Laboratories, Campbell, CA, USA), inducing controlled systemic inflammation. Participants were constantly monitored by a care physician for sepsis-related symptoms such as high blood pressure, high heart rate or fever, among others. ...

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

Systemic LPS challenge

After participants had undergone the IMQ skin challenge, an i.v. LPS challenge was performed on Day 7 after the start of treatment. All participants received 1 ng/kg purified Escherichia coli O113 LPS (List Labs, US, Lot #94332B4) reconstituted in 0.5 mL glucose 2.5%-sodium chloride (NaCl) 0.45% and administered as a 2- min infusion. To ensure that participants stayed adequately hydrated, glucose 2.5%-NaCl 0.45% was infused, starting 2 h prior to LPS administration and continuing until 6 h afterwards.18 Circulating inflammatory proteins (acute phase proteins, cytokines), leukocyte differentiation, and clinical parameters (pulse rate, blood pressure, and temperature) were evaluated over time, starting 0.5 h after the end of the infusion and ending up to 24 h after infusion.

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113

- 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) ...

• Product #9433 - GMP LPS Lipopolysaccharide List HPT From Escherichia Coli Type O113