A 10-year-old, 12.3 kg, castrated male Shih Tzu was referred to the Veterinary Medicine Teaching Hospital (VMTH) of Seoul National University with severe pruritus and anorexia for the past 7 months. The dog had been treated with prednisolone orally twice daily (4.4 mg/kg/day) and systemic antibiotics after the diagnosis of pemphigus foliaceus was made at a local veterinary clinic. Despite the treatment, the waxing and waning lesions worsened over time.
Physical examination revealed mild depression and generalized crusting. Papules and pustules were seen on the neck, elbows, ears, abdomen, perianal area, inguinal area, axillae, and the dorsal part of the trunk. Alopecia of the forelimbs was also evident (Figure 1). Skin scrapings were performed to exclude Demodex canis and other ectoparasites. Cytology of an impression smear from a pustule revealed neutrophils and acantholytic keratinocytes. Bacterial and fungal infections had been ruled out at the previous local veterinary clinic. The blood profile (complete blood count and serum biochemistry) showed leukocytosis [white blood cells (WBCs) 58100/μL] and mild anemia [packed cell volume (PCV) 27%]. Serum biochemistry revealed an elevation of liver enzymes [alkaline phosphatase (ALP) 1828 U/L and gamma glutamyl transferase (GGT) 22 U/L], most likely caused by the long-term corticosteroid treatment. Due to malnutrition, hypoproteinemia was also present [total protein (TP) 3.3 g/dL, albumin 2.2 g/dL].
The definitive diagnosis of pemphigus foliaceus was made by histopathological examination of skin biopsies taken from the lesions on the forelimbs. Histopathology revealed a mixture of neutrophils, fibrin debris, and numerous acantholytic keratinocytes. The dermal inflammation was mild and mostly mastocytic and neutrophilic (Figure 2). The patient was treated with prednisolone (4 mg/kg/day, orally twice daily) for immunosuppression, cephalexin (60 mg/kg/day, orally twice daily) for controlling secondary infections, and with liver protectant drugs (lefotil 1 T/day, silymarin 20 mg/kg/day, and ursodeoxycholic acid 20 mg/kg/day, orally twice daily). The symptoms initially improved with a decrease in pruritus; however, recurred after one month. Cyclosporine (5 mg/kg/day, orally once daily) and azathioprine (2 mg/kg/day, orally once daily), were prescribed adjunctively but did not improve the clinical signs.
Because none of the treatment had the desired effect, ATMSCs injection therapy was decided with the consent of owner. Stem cell therapies were carried out in the Cell Therapy and Animal Cloning Clinic of VMTH with the approval of College of Veterinary Medicine, Seoul National University. Canine ATMSCs was provided by K-STEMCELL Co. Ltd. (Seoul, Korea). To increase the potency, the dog CTLA4 gene was transduced into canine ATMSCs by lentiviral expression systems as described in a previous study [15]. Canine ATMSCs and CTLA4 overexpressing ATMSCs (CTLA4-ATMSCs) positively expressed CD29, CD44, and CD90, and did not express the surface markers CD34 and CD45. The patient received CTLA4 ATMSCs (2 × 106 cells/kg) and/or ATMSCs (1 × 107 cells/kg) intravenously. Additional medicinal treatments, antibiotics (cephalexin 60 mg/kg/day, orally twice daily) and with liver protectant drugs (lefotil 1 T/day, silymarin 20 mg/kg/day, and ursodeoxycholic acid 20 mg/kg/day, orally twice daily) were prescribed continually. After the first administration of CTLA4-ATMSCs, the dog became less pruritic, and the skin lesions improved. CTLA4-ATMSCs administration was performed 6 times and naive ATMSCs administration was performed 18 times over a period of 20 months at intervals of 2 to 8 week. CTLA4-ATMSCs and naive ATMSCs were concurrently transplanted at 4th, 5th and 6th administrations. During these 20 months, the dose of prednisolone was gradually reduced and azathioprine was discontinued (Figure 3). No recurrence of the skin lesions was seen (Figure 4). After the termination of ATMSCs treatment, the skin lesions were well managed with a low dose of prednisolone (0.25 mg/kg/day, orally once daily). Furthermore, the body weight increased and the blood profile showed an improvement in the leukocytosis, anemia, and liver enzyme elevation together with an improvement in the body condition (Figure 5). The manageable state lasted for 12 months after the last ATMSCs treatment. Unfortunately, this dog died newly developed pulmonary edema. At this time, skin lesions showed mild crusting which were manageable with low dose prednisolone.