Promoter optimisation of lentiviral vectors for efficient insulin gene expression in canine mesenchymal stromal cells: potential surrogate beta cells.

BACKGROUND: The lack of an ideal cell type that can be easily acquired, modified to produce insulin, and re-implanted has been a limitation for ex vivo insulin gene therapy. Canine diabetes is currently treated with human insulin and is a good model for human diabetes. Mesenchymal stromal cells (MSCs) are a promising candidate cell type for gene therapy. In the present study, we optimised insulin production using lentiviral transduced canine MSCs (cMSCs), aiming to evaluate their ability for use as surrogate beta cells. METHODS: Canine MSCs were derived from bone marrow and validated by measuring the expression of MSC lineage specific markers. Lentivirus vectors encoding the proinsulin gene (with or without a Kozak sequence) under the control of spleen focus forming virus, cytomegalovirus, elongation factor 1α and simian virus 40 promotors were generated and used to transduce primary cMSCs and a hepatocyte cell line. The insulin-producing capacity of transduced primary cMSCs was assessed by measuring the concentration of C-peptide produced. RESULTS: Primary cMSC could be readily expanded in culture and efficiently transduced using lentiviral vectors encoding proinsulin. Increasing the multiplicity of infection from 3 to 20 led to an increase in C-peptide secretion (from 1700 to 4000 pmol/l). The spleen focus forming virus promoter conferred the strongest transcriptional ability. CONCLUSIONS: The results of the present study suggest that optimised lentiviral transduction of the insulin gene into primary cMSCs renders these cells capable of secreting insulin over both the short- and long-term, in sufficient quantities in vitro to support their potential use in insulin gene therapy.

Unilateral squamous cell carcinoma of the renal pelvis with hydronephrosis in a cat.

A 4-year-old female neutered domestic shorthair cat was presented for evaluation of gradual onset of lethargy and anorexia. Physical examination revealed moderate abdominal distension. Investigations performed included complete blood count, serum biochemistry, urinalysis, pyelocentesis, abdominal fluid analysis, abdominal ultrasonography and exploratory celiotomy. Nephrectomy was performed on the hydronephrotic kidney and a sample of the omentum was also taken, as it was grossly abnormal. No other abnormalities were found in the remainder of the abdominal organs. Findings were consistent with unilateral hydronephrosis and squamous cell carcinoma of the renal pelvis with abdominal carcinomatosis. The patient was given supportive treatment while the results of the biopsies from the renal tissue and the omentum were pending. The patient deteriorated a short time after surgical intervention and was euthanased. This is the first report of a squamous cell carcinoma arising from the renal pelvis in a cat. A comparison with the disease presentation in humans is also discussed.

Trichobezoar obstruction after stapled jejunal anastomosis in a dog.

OBJECTIVE: To describe an unusual long-term complication of circular end-to-end anastomosis (CEEA) stapling in a dog. STUDY DESIGN: Clinical case report. ANIMAL: An 11-year-old, female neutered, Labrador Retriever. METHODS: The dog was referred for clinical signs of bowel obstruction. An enterectomy was performed 2 years before presentation using a CEEA stapling device. Palpation, plain radiographs, and ultrasound of the abdomen confirmed the presence of a mass in the bowel, causing obstruction, and requiring surgical approach. RESULTS: An exploratory celiotomy revealed a 5 cm mass in the jejunum, involving the site of the previous surgery. The mass was removed by enterectomy. Dissection of the mass revealed the presence of many staples at the previous enterectomy site, and a trichobezoar entangled in the exposed parts of the staples. CONCLUSIONS: An enterectomy was required to treat an intestinal obstruction caused by a trichobezoar entangled in a CEEA-stapled anastomosis. CLINICAL RELEVANCE: Development of trichobezoar and subsequent bowel obstruction should be considered an unusual but potential long-term complication of CEEA-stapled anastomosis.