Bacterial Toxin Research Citations

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 Blood was withdrawn before prehydration (T = -60 minutes), just before LPS administration (T = 0 minutes), and respectively 60, 90, 120, 180, 240, 360 and 480 minutes after the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

C. difficile Toxin B (TcdB) List Biological Laboratories Cat#155A

Exposure Chemical Solutions

… Stocks of ultrapure LPS (Escherichia coli O111:B4; #421; Lot #4217A1; 2.6 EU/ng; List Biologicals) were sonicated in PBS, adenosine 5′-triphosphate disodium salt hydrate (ATP; 100 mM, A46419; Sigma-Aldridge), suspended in sterile dH2O, and stored at −20°C. …

• Product #421 – ULTRA PURE LPS from Escherichia coli O111:B4

2.2. Immunization and clinical scoring

Active immunization of 10-week-old mice was performed following standard protocols for each strain (Cruz-Orengo et al., 2011, 2014) (Fig. 1). Briefly, EAE was induced by subcutaneous injection of myelin emulsion containing Complete Freund’s Adjuvant (CFA). B6 mice received 50 ​μg msMOGp35-55 (GenScript, Piscataway, NJ) with 500 ​μg Mycobacterium tuberculosis (Mtb) H37Ra peptide (Difco Laboratories, Detroit, MI), and SJL mice were immunized with 150 ​μg msPLPp139-151 (GenScript, Piscataway, NJ) with 200 ​μg Mtb. Total emulsion volume per mouse was 100 ​μl, equally distributed at four injection sites at the shoulder blades and flanks, ~25 ​μl per site. On the day of immunization, and again 2 days post-immunization (dpi), mice received intraperitoneal (i.p.) injections of pertussis toxin (PTX, List Biological Laboratories, Inc, Campbell CA) in 0.9% NaCl injectable saline; 300 ​ng and 200 ​ng for B6 and SJL, respectively. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

Preparation of C. difficile toxins

Purified C. difficile TcdA (#153) and TcdB (#154A), as well as the components of CDT, CDTa (#157A), and CDTb (#158A), were procured from List Biological Laboratories, Inc. …

Author provided incorrect List Labs product #’s; refer to the following corrected product #’s:

• Product #152C – Toxin A from Clostridium difficile
• Product #155 – Toxin B from Clostridium difficile
• Product #753A – Anti-Clostridium difficile Toxin A (Chicken lgY)
• Product #754A – Anti-Clostridium difficile Toxin B (Chicken lgY)

… Additional intratracheal treatments included lipopolysaccharide (LPS, List Labs), …

Author did not specify which List Labs LPS product was utilized in their research.
List Labs provides the following LPS products: https://listlabs.com/product-information/lipopolysaccharides/

… Seven days before perfusion cholera toxin B-fragment (CTB; 1%, 10µl; List Biologicals) was bilaterally injected into the extensor carpi radialis muscle, using a Hamilton syringe, to anterogradely label PA axons in the spinal gray matter as well as retrogradely label motoneurons in the ventral horn. CTB was tinted with Evans Blue …

• Product #104 – Cholera Toxin B Subunit (Choleragenoid) from Vibrio cholerae in Low Salt

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

2.5 Experimental animals and the establishment ofthe EAE model

… Each mouse was injected with antigen emulsion to induce EAE in the dorsal lumbosacral bulking region. The day of immunization was recorded as day 0 post-immunization (p.i.). Each mouse was intraperitoneally injected with 300ng pertussis toxin (PTX, List Labs, USA) on day 0 and day 2 p.i. …

Author did not specify which List Labs Pertussis Toxin was utilized. List Labs provides the following Pertussis Toxin products:
• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer
• Product #181 – Pertussis Toxin from B. pertussis, Lyophilized (Salt-Free)
• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)

… Pertussis Toxin List Biologicals Cat#179A …

G-protein live-cell BRET measurements

…For endpoint measurements shown in Figure 1—figure supplement 1, data is presented as raw BRET ratios (535 nm luminescence/460 nm luminescence) of unstimulated cells. For Figure 2—figure supplement 1, PTX treatments consisted of overnight incubations with 0.2 µg/ml of the toxin. …

• Product #179A – Pertussis Toxin from B. pertussis (in Glycerol)