Ketaject

Mice were anesthetized by injection with Ketamine (Ketaject, Phoenix Pharmaceuticals Inc., St. Joseph, MO) and Xylazine (XYLA-JECT, Phoenix Pharmaceuticals Inc., St. Joseph, MO) prior to administration of either vehicle or an aliquot of vehicle containing SP-A1. The SP-A1 was isolated from stably transfected CHO cells and purified using mannose affinity chromatography as described previously^{48 (link)}. SP-A1 preparations were made with the SP-A1 6A² variant. This SP-A1 variant occurs in the general population with the greatest frequency^{8 (link),49 (link)}. The exogenous SP-A1 treatment contained SP-A1 (10 μg) in 50 μl of sterile saline with 1 mM CaCl₂. We have used similar doses of exogenous SP-A in previous rescue studies^{22 ,24 (link),29 (link),35 (link),37 (link),38} . Control animals received 50 μl of vehicle (saline and 1 mM CaCl₂) alone. We suspended the anesthetized mice by their maxillary incisors, placed the bolus containing SP-A1 or vehicle in the pharynx, and briefly blocked the nostrils, resulting in the aspiration of the instilled bolus. After recovering from anesthesia, the mice were returned to their cages until it was time to harvest alveolar macrophages. In previous studies²² we have found this method of introducing SP-A and other substances to the lungs to be very consistent and reproducible.

These studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and using protocols 2016–17 and 2017–62 approved by the Food and Drug’s Animal Care and Use Committee (ACUC) and conducted in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care’s (AAALAC) guidelines. The animals were housed in approved facilities and monitored daily by veterinarians and facility personnel. The macaques were kept in double housing (paired), fed a complete diet that included fresh fruits and vegetables. The cages were arranged in large rooms to allow the monkeys visual, olfactory and auditory interactions with each other. Food and water were available ad libitum and vitamins were provided. The animals were also provided with environmental enrichment, such as toys designed especially for monkeys, to promote psychological well-being. All inoculations, measurements and biopsies were performed under anesthesia using Ketaject; 5–7 mg/kg of body weight (Phoenix Pharmaceuticals), anesthesia was reversed with atipamezole hydrochloride (Antisedan, 100 μg/kg of body weight; Zoetis Services).

Male rhesus macaques (Macaca mulatta) were obtained from the National Institutes of Health (NIH) colony in South Carolina [38 (link)], housed in approved facilities, and monitored daily by veterinarians and facility personnel. Animals were 2 years of age, and their average weight was 3.4 kg (range 2.6-4kg). Treatment groups were balanced for weight prior to starting the study. Animals were grouped into five groups based on the following treatments, Saline, Sb^V 20 mg/ml, Sb^V 5 mg/ml, Sb^V 2.5 mg/kg, Sb^V 0 mg/ml. Following 3 doses of Sb^V given every other day IM, the animals in groups Sb^V 20, 5, 2.5, 0 mg/kg were given a single dose of D35 1 mg/kg SC at a distant site. Blood and 4 mm skin biopsies (Miltex, Inc) were taken distant from the sites were drugs were previously administered. Skin biopsies were placed immediately into Trizol and kept on ice until samples could be processed. Macaques were anesthetized with ketamine for all procedures (Ketaject; 5–10 mg/kg of body weight; Phoenix Pharmaceuticals), following the procedure the anesthetic was reversed with atipamezole hydrochloride (Antisedan, 100 μg/kg of body weight; Zoetis Services). The animals showed no sign of itching or pain related to the lesions or the biopsies and efforts were made to minimize their suffering.