Bacterial Toxin Research Citations

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.

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2.2. Experimental design

… The two experimental sessions were held between two and four weeks apart to mitigate potential carry-over effects from LPS-mediated immune activation. …

The LPS infusion was prepared on-site immediately before administration, following manufacturer protocols for reconstitution and dilution under sterile conditions. In short, the product was reconstituted and initially diluted with sterile water, then diluted to the final dose in 30 mL sterile saline within 15 min of administration. For each participant, a single fresh vial of Clinical Center Reference lyophilized endotoxin (lipopolysaccharide/LPS; lot 94332B1) was used, prepared from Escherichia coli O113:H10:K by List Biological Laboratories, Inc. The infusion was administered at a slow rate of 1 mL/min over 30 min using an infusion pump (Taudorf et al., 2007). In the first session, LPS was administered at a very low dosage of 0.1 ng/kg of participant body weight, and in the second session at a low dosage of 0.4 ng/kg. …

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Methods
In a monocentric, randomized, double-blind, placebo-controlled, parallel-group, phase I trial, 57 healthy, smoking subjects were treated for 28 days with either oral BI 1026706 100 mg bid or placebo. At day 21, turbo-inversion recovery magnitude magnetic resonance imaging (TIRM MRI) was performed. On the last day of treatment, pre-challenge bronchoalveolar lavage fluid (BAL) and biopsies were sampled, followed by segmental LPS challenge (40 endotoxin units/kg body weight) and saline control instillation in different lung lobes. Twenty-four hours later, TIRM MRI was performed, then BAL and biopsies were collected from the challenged segments. In BAL samples, cells were differentiated for neutrophil numbers as the primary endpoint. Other endpoints included assessment of safety, biomarkers in BAL (e.g. interleukin-8 [IL-8], albumin and total protein), B1R expression in lung biopsies and TIRM score by MRI as a measure for the extent of pulmonary oedema.

… Next, Lipopolysaccharide (Endotoxin from E. coli Type O113 provided by National Institutes of Health Clinical Center, Bethesda, Maryland, USA and produced by List Biological Laboratories Inc., Campbell, California, USA) at a dose of 40 endotoxin units (E.U.)/kg …

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2.3.2. Lipopolysaccharide preparation and administration

LPS doses were prepared in the UAB CRU using an aseptic, no-touch technique. LPS (E. coli Type 0113 Clinical Center Reference Endotoxin) was manufactured by List Biological Laboratories, Inc. The Lot #94332B1 vials containing 1.0 mcg endotoxin were reconstituted to 1.0mcg/1.0 mL, using sterile water. 0.5 mL was drawn and diluted with 9.5 mL of sterile water to produce a 50 ng/mL solution for dosing. Dosages were drawn at 0.4 ng/kg (FM N = 7; HC N = 7) or 0.3 ng/kg (FM N = 5; HC N = 6) body weight. Finally, the dose was diluted in 30 mL sterile saline for administration.

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Experimental Human Endotoxemia (IV LPS Administration)

… Following that, it was continuously given at a rate of 150mL/h for the remaining duration of the experiment. Promptly after prehydration, a bodyweight-adjusted bolus dose of 1ng/kg LPS (Escherichia coli-derived, Type O0113, lot no.94332B1; List Biological Laboratories, Campbell, CA, USA) was administered. Blood samples were serially obtained to construct time-concentration curves of circuiting cytokines (Jansen et al., 2022). Temperature and cardiovascular monitoring (blood pressure and heart rate) and sickness symptoms (headache, muscle pain, back pain, nausea, shivers and vomiting were recorded throughout the whole experiment with intervals of 30 minutes. …

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2.5. Experimental human endotoxemia

On days 0 and 7, experimental endotoxemia was performed according to our standardized protocol8. Briefly, subjects were admitted to the research unit of the Radboud university medical center for eight hours, where a radial artery catheter (BD Infusion Therapy Systems) and antebrachial venous cannula were placed to allow serial blood sampling, hemodynamic monitoring and administration of fluids and LPS, respectively. In the 45 minutes prior to LPS administration, hydration fluids (2.5% glucose/0.45% sodium chloride) were administered as a 1.5L prehydration bolus to reduce the risk of vasovagal collapse13, 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 (E. Coli-derived, Type O113, lot no. 94332B1; List Biological Laboratories) was administered.

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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. …

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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). …

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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. …

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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).

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