Cytokine and Growth Factor Concentrations in Canine Autologous Conditioned Serum

Abstract

(ACADEMIC)The object of this study was to compare growth factor and cytokine profiles in canine autologous conditioned serum (ACS) to canine plasma. Blood collected from 16 medium to large breed dogs was used to produce ACS (Orthokine ® vet irap 10 syringes) and citrated plasma (control). Canine-specific ELISA assays were run per manufacturers' instructions for interleukin (IL)-10, IL-4, tumor necrosis factor (TNF)-α, insulin-like growth factor (IGF)-1, fibroblast growth factor (FGF)-2, transforming growth factor (TGF)-β1, IL-1β, and interleukin-1 receptor antagonist (IL-1ra). Serum, in addition to plasma and ACS, was collected from an additional 6 dogs for TNF-α, IL-1β, and IL-1ra analysis (total of 22 dogs). Data were analyzed for differences in cytokine concentrations between ACS, plasma, and serum using the Wilcoxon signed-rank test with significance set at P<.05.There was a large variability in growth factor and cytokine concentrations between individual dogs in both plasma and ACS. There were no significant differences in IL-10, TNF-α, IGF-1, FGF-2, and TGF-β1 concentrations between ACS, plasma, or serum. ACS concentrations of IL-1β (median, range; 46.3 pg/mL, 0-828.8) and IL-4 (0.0 pg/mL, 0-244.1) were significantly increased compared to plasma (36.6 pg/mL, 0-657.1 and 0.0 pg/mL, 0-0, respectively). IL-1ra concentrations in ACS (median, range; 3458.9 pg/mL, 1,243.1-12,089.0) were significantly higher than plasma (692.3 pg/mL, 422.5-1,475.6), as was the IL-1ra:IL-1β ratio (39.9 and 7.2, respectively).iii Cytokine and Growth Factor Profiles in Canine Autologous Conditioned SerumDominique Marie Sawyere ABSTRACT (PUBLIC)Osteoarthritis (OA) is a common cause of pain and suffering in dogs and is challenging to treat effectively Current treatment for OA merely mask the signs of pain and do not promote regeneration of the damaged cartilage or alter the course of the disease. Many treatments also cause harmful side effects. Autologous conditioned serum (ACS) is a biological therapy that controls inflammation within the joint by specifically blocking the inflammatory cytokine interleukin (IL)-1β. In humans and horses ACS relieves the pain from OA and promotes cartilage regeneration. The purpose of this study was to compare levels of pro-and anti-inflammatory cytokines in canine ACS to that of normal canine plasma with the hypothesis that levels of anti-inflammatory cytokines would be higher in ACS compared to controls and that levels of pro-inflammatory cytokines would remain unchanged. Paired blood samples were collected from 22 large breed healthy dogs and processed in either irap®10 syringes (ACS) or in anti-coagulant as a control. For the last 6 dogs an additional serum sample was collected. Growth factor and cytokine levels were determined using canine-specific ELISAs. One pro-inflammatory cytokine (IL-1β) and two anti-inflammatory cytokines (IL-1ra and IL-4) were significantly higher in the ACS compared to plasma and IL-1ra levels were also higher in serum compared with plasma samples. Other levels w...

Cell-Based Therapies for Joint Disease in Veterinary Medicine: What We Have Learned and What We Need to Know

“…Both techniques increase IL-1Ra and IL-10 levels as well as growth factors IGF-1 and TGF-β1 in equine blood ( 16 ). In canine blood, IRAP I™ ( 20 ) and IRAP II™ ( 19 ) cause significantly increased IL-1ra at levels comparable to equine and human products, but no significant differences were found for other growth factors and anti-inflammatory cytokines when investigated for IRAP I™ ( 20 ). The main limitation of such studies to-date is that absolute levels, or ratios of anti-inflammatory and inflammatory cytokines have not been linked to biological or therapeutic efficacy.…”

“…Despite current clinical use of media additives from platelet lysate, to autologous serum, to serum-free culture, the effects of media additives on MSC function are largely unknown for veterinary species. Given large variation in cytokines and growth factors from equine and canine blood-derived products, it is likely that additives have varying effects on MSCs with unknown effects on their eventual therapeutic efficacy ( 14 – 20 ).…”

“…The main limitation of such studies to-date is that absolute levels, or ratios of anti-inflammatory and inflammatory cytokines have not been linked to biological or therapeutic efficacy. When equine and canine studies found that pro-inflammatory cytokines IL-1β and TNFα were also increased with ACS preparation in horses ( 15 , 16 ) and dogs ( 20 ), it was suggested that the ratio of anti-inflammatory to pro-inflammatory cytokines may be important for therapeutic effect ( 16 ). However, this has not been directly shown in veterinary species.…”

“…Short (<72 h) clearance time of ACS cytokines in vivo , coupled with minimal effects of ACS-derived TNFα on cartilage metabolism in humans may support the counter argument that cytokine profiles of ACS are of little therapeutic consequence ( 15 ). Despite lack of evidence as to the therapeutic consequence of cytokine composition, it is clear that preparation technique ( 15 – 17 , 20 ) and individual variation ( 18 , 19 ) alter the bioactive composition of equine and canine ACS. IRAP II™ has a higher IL-1Ra:IL-1 ratio than IRAP I™ in horses ( 16 ).…”

“…Due to funding constraints, many studies have a low number of animals, a lack of control groups or have not progressed beyond pilot data. In addition, variability derived from factors including individual donor and processing method challenges our ability to draw meaningful conclusions ( 14 – 20 ).…”