Levamisole

B. malayi and Brugia pahangi adults extracted from the Meriones unguiculatus infection system (NIH/NIAID Filariasis Research Reagent Resource Center) (105 (link)) were maintained in daily changes of RPMI 1640 medium with l-glutamine (Sigma-Aldrich) supplemented with fetal bovine serum (FBS) (10%, vol/vol; Fisher Scientific) and penicillin-streptomycin (0.1 mg/mL; Gibco) at 37°C with 5% CO₂ unless otherwise specified. Brugia microfilariae isolated from the same system were maintained in RPMI 1640 medium supplemented with l-glutamine (Sigma-Aldrich) and penicillin-streptomycin (0.1 mg/mL; Gibco) at 37°C with 5% CO₂ unless otherwise specified.
The chemicals used in the assays included serotonin (catalog number AAB2126306; Fisher Scientific), arecoline (catalog number AC250130050; Fisher Scientific), atropine (catalog number sc-252392; Santa Cruz Biotechnology), nicotine (catalog number sc-482740; Santa Cruz Biotechnology), levamisole (catalog number TCL0231-1G; VWR), carbachol (catalog number sc-202092; Santa Cruz Biotechnology), acetylcholine (catalog number AC159170050; Fisher Scientific), oxotremorine M (catalog number sc-203656; Santa Cruz Biotechnology), and aldicarb (catalog number sc-254939; Santa Cruz Biotechnology).

Samples for blood plasma were collected from 10 animals/tank (20 animals/group) (Figure 1). The concentration of total and non-bound Ca²⁺, Cl⁻, inorganic P (Pi), pH, Zn²⁺, ALP, and bicarbonate ions were measured by the Skretting Aquaculture Innovation Laboratory (Stavanger, Norway). The non-bound Ca²⁺, Cl⁻, and Pi were determined as described in [39 (link)]. The total Ca²⁺ and pH was measured by ion selective electrode (art. #981772, #981597, respectively). Photometry at 560 nm was applied for quantitative determination of Zn²⁺ and the use of 5 Br-PAPS. Photometry at 405 nm was used for quantitative determination of bicarbonate ions and ALP levels (art. #981889, #981833, respectively). Alkaline phosphatase activity (ALP) was determined in three animals/tank (six animals/group) using p-nitrophenyl-phosphate (4-NPP) as a substrate in a 2-amino-2-methyl-1-propanol (AMP) phosphate-accepting buffer. The ALP activity was expressed as enzyme units (U/L). The activity of bone/liver/kidney ALP (b/l/k ALP) was determined with the application of 10 mmol levamisole (VWR Chemicals) to inhibit the non-intestinal form of the enzyme [62 (link),63 (link)]. The measured activity of intestinal ALP was then deducted from total plasma ALP to determine b/l/k ALP.