Ketavest

Two mice (age, 8–12 weeks) per gender were selected for each treatment group. LPS [Lipopolysaccharides from Escherichia coli 0111:B4, dissolved in phosphate-buffered saline (PBS)] from Sigma-Aldrich (St. Louis, MO, USA) was injected once intratracheally (10 mg/kg) in WT and KO mice. The control group was comprised of KO and WT mice injected with the same volume of sterile PBS. Bronchoalveolar lavage fluid (BALF) was collected. Briefly, following anesthesia by intraperitoneal injection with a cocktail of xylazine (Rompun 2%; Bayer AG, Leverkusen, Germany) and ketamine (Ketavest; 100 mg/ml; Pfizer, Inc., New York, NY, USA), the trachea was exposed and the lungs were lavaged three times with 0.2 ml sterile saline per wash. The BALF was stored at 4°C and samples of lung tissue were harvested for RNA, protein and histological studies 12 h after treatment, and refrigerated at −80°C.
WT mice were randomly divided into four treatment groups. Two vitamin D treatment groups (one PBS group and one LPS group) were treated with the vitamin D analog paricalcitol (Sigma-Aldrich) dissolved in propylene (glycol:ethanol, 90:10; 0.5 µg/kg body weight) through intra-peritoneal injection 30 min prior to LPS or PBS treatment. The two vehicle groups received the dissolvent only prior to LPS or PBS treatment.

Mice were divided randomly into each group. Chemically synthesized hookworm peptides, Acan1 and Nak1, were administered once via the i.p. route in sterile phosphate buffered saline at indicated doses (Fig. S9). Four hours later, mice were anesthetized with xylazine (5 mg kg⁻¹, Rompun 2% Bayer) and ketamine (50 mg kg⁻¹, Ketavest; Pfizer Inc). TNBS was prepared by dissolving 2.5 mg in 50% ethanol. Once unresponsive, mice received an enema with a 125-mg kg⁻¹ dose of TNBS using a lubricated 20-G soft catheter as described (48 , 49 (link)). Animals were monitored daily for weight loss and general well-being over 3 days. Mice were weighed daily and their overall appearance (piloerection), mobility, and fecal consistency/bleeding were quantified. On day 4 following colitis induction, colons (from cecum to rectum) were collected, quantified, and assessed macroscopically. Clinical changes were scored as described (22 (link)). Briefly, clinical pathology including adhesion, edema, wall thickening, and ulceration was scored from 0 to 3, with 3 corresponding to most severe disease. This was for a maximum total score of 12 as described (7 (link)). Mouse colons were also assessed histologically for inflammation and gene expression as described (6 ).