Citations

Immunization Procedures:

Peptide emulsions were prepared in complete Freunds adjuvant (CFA) containing Mycobacterium tuberculosis H37RA extract (Difco Laboratories, Detroit, MI, USA) to a final concentration of 5 mg/ml. To induce disease, animals were immunized twice s.c., in inguinal and sternal regions on days 0 and 7, and all animals received pertussis toxin (PT, List Biological Laboratories, Campbell, CA, USA; 100 ng/mouse) i.p., on days 0 and 2 after the first immunization …

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)

For T reg cell depletion by DT, Foxp3EGFPDTR+ mice were treated i.p. with 50 g/kg DT (List Biological Laboratory).

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

Cell culture and transfection:

The normal epithelial breast cell line MCF-10A was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and cultured in MEBM basal medium (Lonza, Basel, Switzerland) supplemented with the MEGM Single Quot Kit (Lonza) and cholera toxin (List Biological Labs, Campbell, CA) under a humid environment with 5% CO₂ at 37°C. …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae

… SC injections were centered on segments C2-3 and C6-7 in different rats. Tracers used were cholera toxin B subunit (CTB; 1% in saline, List Biological, Campbell, CA, product #103B, lot #10327A) …

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

Induction and evaluation of EAE:

Male C57BL/6 mice (6-8 weeks old), originally obtained from the Jackson Laboratory, were injected s.c. in both flanks with 100 µg of the MOG₃₅₅₅ peptide (MEVGWYRSPFSRVVHLYRNGK) that was dissolved in PBS, emulsified in an equal volume of CFA (Difco), and supplemented with 5 mg/ml heat-killed mycobacterium tuberculosis H37Ra (Difco). The mice were also injected twice i.p. with 200 ng of pertussis toxin (List Biological Laboratories), once on the day of immunization and once 24 h later. …

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)

EAE induction, treatment and DNA microarray:

… EAE was induced in 8 weeks old female non-obese diabetic (NOD)/ShiJcl mice (CLEA Japan, Inc.). EAE was induced using a modified method we previously reported [11]. In brief, NOD mice were subcutaneously immunized into both flanks with 100 µg MOG ₃₅₅₅ (myelin oligodendrocyte glycoprotein, MEVGWYRSPFSRVVHLYRNGK) emulsified in CFA (200 l) containing Mycobacterium tuberculosis H37Ra (200 µg, Difco Laboratories), followed by intraperitoneal injections of 200 ng pertussis toxin (List Laboratories) on days 1 and 2. …

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. Reagents and cells

PTx (List Biological Laboratories, Campbell CA, stock solution 50 µg/mL) was prepared by reconstitution with 0.1 M sodium phosphate (pH 7.0), 0.5 M sodium chloride, and 50% glycerol and was stored at −20 °C.

2.3. Preparation of PTx and PTx-spiked vaccine samples:

PTx solutions for assay use were prepared immediately before use by diluting PTx stock solution in diluent, 0.2% gelatin in physiologically buffered saline …

2.4. HIST assay:

Briefly, on Day 0, groups of 20 mice each were injected by the intraperitoneal route (IP) with 0.5mL of either PBS-G diluent, PTx, vaccine, or vaccine spiked with toxin.

2.5. CHO cell assay:

The samples tested in the CHO cell assay (both PTx and vaccine samples) were the same ones that were tested in the HIST and were prepared as described above. The CHO cell assay for PTx activity in vaccine samples that was used in this study is the Direct Method assay that was performed essentially as previously described [11]. Briefly, PTx or pre-diluted (1:20) vaccine samples (100L) were two-fold serially diluted with cell culture medium in 96-well plates to yield a seven-point dilution curve.

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)

Stimulation of monocytes with LPS:

… experiments were conducted with a predictable LPS-stimulatory effect. For this purpose, monocytes from six horses were incubated in 24 well plates with RPMI-1640 containing 10% DHS and E. coli 0111:B4 LPS (List Biologicals Inc., Campbell, CA) at either 1, 50 or 100 ng/ml. …

The effect of ePL on LPS-stimulated monocytes:

In the second series of experiments (summarized in Table 1) monocytes obtained from 6 horses were stimulated with 50 ng/ml E. coli LPS 0111:B4 and incubated with either RPMI-1640 containing 10% DHS or 10% ePL. Equine monocytes incubated in RPMI-1640 supplemented with 10% DHS or pooled ePL without LPS served as control groups.

To more accurately define the potential anti-inflammatory effect of ePL we included experimental groups containing an escalating concentration of DHS. Specifically, equine monocytes from the same six horses were incubated with RPMI-1640 containing 1, 5 or 10% of DHS and 0111:B4 LPS at 50 ng/ml plus 10% pooled ePL. …

• Product #201 – LPS from Escherichia coli O111:B4

… (St. Louis, MO). Smooth-type LPS from E. coli O55:B5 (List Biological Laboratories, CA) was used as an endotoxin. …

NOTE:  At the time this paper was written, Product #203 (5mg – LPS from Escherichia coli O55:B5) was utilized.

Product #203 (5 mg – LPS from Escherichia coli O55:B5) is no longer sold.

Product #203A (2.5 mg – LPS from Escherichia coli O55:B5) is available for purchase.

• Product #203A – LPS from Escherichia coli O55:B5

Drugs used were … CT (12.5 µg/ml, List Biologicals, Campbell, CA), ), …

• Product #100B – Cholera Toxin (AZIDE-FREE) from Vibrio cholerae