Scoring was made on a 4+ scale for the amount of perivascular mononuclear cell infiltration: 0 = none, 1+ = sparse, 2+ = some, 3+ = modest, and 4+ = abundant. Scores of ≥2+ were considered consistent with a DTH reaction, similar to earlier reports whereby DTH reactions consisted of perivascular mononuclear cell infiltrate layers ≥5× “thicker” than normal

or negative control monkey skin [23]. The histological scores were summarized by group as a general indication of DTH reactivity. Heparinized blood samples were collected 7 days after the last booster. PBMC were isolated by Ficoll gradient. Cells were maintained at a density of 1 × 106 cells/mL in RPMI-1640 medium supplemented with 10% FBS (PHA).

PBMC from each monkey were loaded or not with the immunization antigen (no adjuvant) for 24 h, and later LY2157299 concentration labeled with CFSE (target cells) as described [24]. Subsequently these cells were mixed with autologous PBMC (effectors cells) at a 2:1 ratio and direct cytolysis was evaluated by FACS as the percent reduction of Obeticholic Acid purchase the CFSE labeled population [25] and [26]. A week after the sixth immunization each group of animals was conditioned to receive acute controlled full-thickness skin wounds on the dorsal area under sodium pentobarbital (30 mg/kg of body weight) anesthesia. Four symmetric ulcers were inflicted in the dorsum of each rat using disposable 8 mm diameter punch biotomes (Biopunch®, Fray). Following hemostasia, the wound contours were traced upon transparent plastic sheets for planimetric analysis. This served as the original wounded area. Wound closure dynamics were studied using the standard cutaneous round ulcer model as described previously [27]. During this period, no immunization procedures were conducted. While in the case of the weekly schedules the wounds were allowed to completely heal, for biweekly schedules the animals were sacrificed 12 days after the ulcer induction and the extent of the healing

process was to confirmed by histopathological evaluation. The effects of the vaccine administration on skin healing were evaluated using the 4 mm punch biopsies done for DTH histological assessment. Following hemostasis, the wound contours were traced upon transparent plastic sheets for planimetric analysis and closure dynamics determined using the standard cutaneous round ulcer model [27]. Wound closure dynamics was studied at days 0, 6, 12 and 21 and the percent of wound healing was calculated as the percent of wound area reduction as compared to the initial values. Wound healing measurements and histopathological characterization of both, monkeys and rat lesions were performed by technical personnel unaware of the animal’s treatment. Furthermore, the final processing of the data was also performed in a blinded fashion by a skilled professional.