Bacterial Toxin Research Citations

Active EAE model

EAE was induced by immunizing C57BL/6J mice with myelin oligodendrocyte glycoprotein (MOG)35-55 peptide (100 μg per mouse, ANASPEC, 60130-5) and complete Freund’s adjuvant (CFA) containing 4 mg ml−1 (0.4 mg per mouse) of heat-killed Mycobacterium tuberculosis (Chondrex, 7001)24. Mice were intraperitoneally injected with pertussis toxin (PTX, 250 ng/mouse, List Biological Laboratories, 180) on days 0 and 2. PBS or ITA (50 mg kg−1) was intraperitoneally injected every other day from day 0 to day 14. An ITA solution was adjusted to pH 7.4 with 1 N NaOH at 37 °C. …

• Product #180 – Pertussis Toxin from B. pertussis, Lyophilized in Buffer

4.5. EAE Induction and Clinical Evaluation of Experimental Mice

Mice were divided in a random manner into four groups (six mice in each group), including: group 1: naïve, untreated control mice; group 2: MCP-immunized EAE mice; group 3: MCP was immunized and BEY was administered from day 3 to day 27; group 4: BEY orally administered to mice from day 3 to day 27 of experiment. Mice were immunized with MOG35–55 (100 μg; Peptide International) emulsified in complete Freund’s adjuvant (CFA; Sigma-Aldrich, St Louis, MO, USA) containing 10 mg/mL heat-killed Mycobacterium tuberculosis H37Ra (Difco Laboratories, Detroit, MI). The mice were injected intraperitoneally with pertussis toxin (PTX) (List Biological Laboratories, Detroit, MI; 500 ng) on days 0 and 2. A total of 200 µL of emulsion (MCP) was administered subcutaneously on mice’s flanks at four locations. …

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)

2.2. Surgery

Under isoflurane anesthesia, 1.0 µl of a 1 % solution of the retrograde neuronal tract tracer cholera toxin B subunit (CTb; Lot #10331A1; List Biological Laboratories, Inc., Campbell, CA) was unilaterally deposited into the basal forebrain (n = 6–8/group) using the following stereotaxic coordinates, relative to Bregma [16]: anterior (AP) −0.8 mm, lateral (L) +/- 2.5 mm, ventral (DV) –8.0 mm (from skull surface). Left and right hemispheres were equally represented. CTb was pressure injected over a 10 min interval (0.1 µl/min) and the needle remained in place for an additional 10 min post-infusion to allow diffusion away from the needle tract. Animals whose CTb deposits were not centered in the VP/SI, based on post-mortem immunohistological labeling, were excluded from analysis. Prophylactic buprenorphine (0.02 mg/kg, s.c.; Med-Vet International, Mettawa, IL) was given at the conclusion of surgery to minimize post-operative pain during emergence from anesthesia. Animals were allowed one week recovery from surgery, while handling, habituation, and food intake measurement continued.

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

… Upon immunization and 2-day post immunization, mice were also intravenously injected with 300ng/mouse pertussis toxin (List Biological Laboratories) …

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)

… LPS from Salmonella minnesota Re 595 (List Biological Laboratories, Campbell, CA), …

• Product #304 – LPS from Salmonella minnesota R595 (Re)

3. Experimental

3.1. Assays

3.1.1. General
All biochemical analyses and cell assays were performed as previously reported by Lin et al. [22] and Turner, Nielsen et al. [14] with the following modifications.

3.1.2. SNAPtide Enzyme Activity Assay
The assay was conducted as previously described [22]. The final assay concentrations are as follows: SNAPtide substrate #523 = 4 µM; BoNT/A LC = 10 nM; assay buffer = 40 mM HEPES pH 7.4, 0.01% Triton X-100, 1% DMSO.

3.1.3. Screening
Initial compound screening was performed at room temperature with SNAPtide substrate #521 (List Labs, Campbell, CA, USA) at a compound concentration of 40 µM.

3.1.4. Endpoint Assay
In total, 500 nM BoNT/A LC was incubated with 5 µM of each compound for 30 min at 37 °C. At the indicated timepoints, 2 µL aliquots were quenched in 48 µL of SNAPtide #523 substrate (25-fold dilution) to give a SNAPtide assay final concentration of 20 nM BoNT/A LC and 0.2 µM compound.

• Product #521 – SNAPtide® Peptide Substrate (FITC/DABCYL) for C. botulinum Type A Neurotoxin
• Product #523 – SNAPtide® Peptide Substrate flP6(DABCYL/5-IAF) for C. botulinum Type A Neurotoxin

Tregs depletion and agent administration

To deplete Tregs, DEREG mice were injected intraperitoneally with 1 μg DT (List Biological Laboratories, inc, USA) 24 h before LPC injection, then 0.5 μg DT every third day until 10 days after LPC injection. Control mice were treated with PBS injection. Flow cytometry in spleens and blood were used to confirm Tregs depletion. …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… Diphtheria toxin, Unnicked, from Corynebacterium diphtheriae List Biological Laboratories Cat# 150 …

• Product #150 – Diphtheria Toxin, Unnicked, from Corynebacterium diphtheriae

… Biotinylated recombinant PA83 (List Biological Labs, Campbell, CA) was bound …

• Product #171E – Anthrax Protective Antigen (PA), Recombinant from B. anthracis

… Briefly, MOG/CFA emulsion was subcutaneously injected into the axilla of mouse (10 mg kg-1), followed by i.p. injection of pertussis toxin (… List Biological Laboratories, Inc., Campbell, CA) on the day of immunization and 48 h post immunization. Body weight and EAE scores of mice were monitored every day. EAE scoring criteria were as follows …

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)