Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells.

OBJECTIVES: Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-ß]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. METHODS: Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. RESULTS: aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-ß gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. CONCLUSIONS AND RELEVANCE: The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.

Identification of serum microRNAs with differential expression between dogs with splenic masses and healthy dogs with histologically normal spleens.

OBJECTIVE: To identify differential microRNA (miRNA) expression in dogs with splenic hemangiosarcoma, splenic hematoma, and histologically normal spleens. ANIMALS: Dogs with splenic hemangiosarcoma (n = 10), splenic hematoma (n = 5), and histologically normal spleens (n = 5). PROCEDURES: Splenic tissue and serum samples were collected from dogs with splenic masses (ie, hemangiosarcoma or hematoma samples) and healthy control dogs (ie, control samples), and total RNA was extracted. Reverse transcription quantitative real-time PCR was performed with 28 miRNAs associated with hemangiosarcoma, angiosarcoma, or associated genes. Differential expression analysis was performed. RESULTS: Control tissue and serum samples had similar miRNA expression patterns, and hemangiosarcoma tissue and serum samples did not. Hemangiosarcoma serum samples had higher expression than hemangiosarcoma tissue for 13 miRNAs and lower expression for 1 miRNA. Control tissue and hemangiosarcoma tissue had varying expressions for 12 miRNAs, with 10 more highly expressed in control samples and 2 more highly expressed in hemangiosarcoma samples. Five miRNAs (miR-214-3p, miR-452, miR-494-3p, miR-497-5p, miR-543) had significantly different expression in serum between dogs with splenic masses (ie, hemangiosarcoma or hematoma) and serum of dogs with histologically normal spleens, with higher expression in the serum of dogs with splenic masses for all 5 miRNAs. CONCLUSIONS AND CLINICAL RELEVANCE: 5 circulating miRNAs were identified that distinguished dogs with splenic hemangiosarcoma or hematoma from those with histologically normal spleens. These 5 miRNAs had higher expression in dogs with splenic masses, indicating upregulation of these circulating miRNAs occurs in these splenic disease states. These miRNAs may be useful as a noninvasive screening tool that uses serum to identify dogs with splenic masses.