Farrah Monibi

Effects of opioids on phagocytic function, oxidative burst capacity, cytokine production and apoptosis in canine leukocytes.

Opioids alter immune and apoptotic pathways in several species. They are commonly used in companion animals affected with infectious and/or inflammatory disease, but the immunomodulatory and apoptotic effects of these drugs in dogs are relatively unknown. The aim of the present study was to evaluate the effects of morphine, buprenorphine and fentanyl on canine phagocyte function, oxidative burst capacity, leukocyte cytokine production, and lymphocyte apoptosis. Blood from six healthy adult dogs was incubated in the presence or absence of morphine (200 ng/mL), buprenorphine (10 ng/mL) or fentanyl (10 ng/mL) for 3 h (phagocytic function; cytokine production) or 8 h (apoptosis). Neutrophil phagocytosis of opsonized Escherichia coli, respiratory burst capacity after stimulation with opsonized E. coli or phorbol 12-myristate 13-acetate (PMA), and Annexin V-FITC staining of apoptotic lymphocytes were evaluated using flow cytometry. Leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-10 was assessed after incubation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or peptidoglycan. Morphine promoted a more intense respiratory burst. Morphine, buprenorphine and fentanyl all promoted LPS- or LTA-induced TNF-α and IL-10 production. However, the opioids tested did not alter TNF-α:IL-10 ratios. Morphine, buprenorphine and fentanyl all inhibited neutrophil apoptosis, an effect that was not concentration dependent in nature. These data indicate that opioids alter immune and apoptotic pathways in dogs. The possible effects of opioids on immune and cellular responses should be considered when designing studies and interpreting outcomes of studies involving administration of opioids in dogs.

Identification of Synovial Fluid Biomarkers for Knee Osteoarthritis and Correlation with Radiographic Assessment.

Osteoarthritis (OA) is a costly and debilitating condition that is typically not diagnosed early enough to prevent progression of disease. The purpose of this study was to evaluate synovial fluid from knees with and without OA for potential markers of joint inflammation and degradation and to correlate these findings with radiographic severity of disease. With Institutional Review Board approval, synovial fluid samples were collected before the patient undergoing total knee arthroplasty. Control knees (n = 3) were patients younger than 30 years of age with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury, and no surgical history for either knee. Weight-bearing, anterior-posterior radiographic views were used to determine radiographic OA severity using the modified Kellgren and Lawrence scale. Synovial fluid samples from 18 patients (21 knees) were analyzed using a multiplex assay. Matrix metalloproteinase (MMP)-1 (p < 0.001), interleukin (IL)-6 (p < 0.013), IL-8 (p < 0.024), and Chemokine (C-C motif) ligand 5 (CCL5) (p < 0.006) were significantly higher in the synovial fluid of OA patients compared with normal patients. The radiographic score was significantly higher in patients with OA compared with normal knees (p < 0.002). MMP-1 had a moderate positive correlation with MMP-2, IL-6, IL-8, and CCL5. IL-6 had a strong positive correlation with IL-8 and a moderate positive correlation with MMP-2. Monocyte chemotactic protein 1 had a moderate positive correlation with IL-6 and a strong positive correlation with IL-8. Radiographic scores had a strong positive correlation with IL-6 and IL-8 and a moderate positive correlation with MCP-1. These data provide novel and clinically relevant information for the investigation of synovial fluid biomarkers for knee OA.

Characterization of knee meniscal pathology: correlation of gross, histologic, biochemical, molecular, and radiographic measures of disease.

Meniscal pathology is an extremely prevalent problem, which inevitably leads to osteoarthritis and associated pain, swelling, and disability. Relatively little data are available regarding the molecular, biochemical, and histologic aspects of meniscal disease. This study characterizes meniscal pathology in the presence of symptomatic osteoarthritis and correlates clinical and basic science data in an attempt to delineate clinically relevant mechanisms of disease. Twenty-seven knees from 23 patients who underwent total knee arthroplasty comprised the affected group and 6 aged nonsymptomatic knees were used as controls. All meniscal tissues were harvested and subjectively scored for gross and histologic pathology. Biochemical analyses were performed to determine glycosaminoglycan (GAG) content, collagen (hydroxyproline) content, and water content. Real-time polymerase chain reaction analysis was conducted for genes involved in synthesis (collagens [col] 1, 2, 3, and 6), degradation (matrix metalloproteinases [MMP-1, -2, -3, -13]), and angiogenesis (vascular endothelial growth factor). Weight-bearing, anterior-posterior radiographic views were used to determine joint space measurements for lateral and medial compartments, and were subjectively scored for osteoarthritic changes. Data were compared for statistically significant differences and to determine the presence and strength of correlations among variables assessed. Affected menisci had significantly higher gross and histologic pathology scores compared with control menisci. Affected menisci had significantly higher water, proteoglycan, and collagen content compared with control menisci. Col 1, 3, and 6 gene expression levels for the affected group were significantly increased compared with controls. MMP-13 expression was significantly increased for the affected group. MMP-2 and -3 expression levels were significantly lower in the affected group compared with controls. The affected group had significantly more joint space narrowing and higher radiographic scores for medial compared with lateral compartments. Several strong and moderately strong correlations were present between variables. These data suggest that in vitro measures of meniscal pathology have potential value for understanding disease mechanisms and predicting clinical disease.

Meniscal biology in health and disease

ABSTRACT The knee is a fascinating yet complex joint. Researchers and clinicians agree that the joint is an organ comprised of highly specialized intrinsic and extrinsic tissues contributing to both health and disease. Key to the function and movement of the knee are the menisci, exquisite fibrocartilage structures that are critical structures for maintaining biological and biomechanical integrity of the joint. The biological/physiological functions of the menisci must be understood at the tissue, cellular and even molecular levels in order to determine clinically relevant methods for assessing it and influencing it. By investigating normal and pathological functions at the basic science level, we can begin to translate data to patients. The objective of this article is to provide an overview of this translational pathway so that progression toward improved diagnostic, preventative, and therapeutic strategies can be effectively pursued. We have thoroughly examined the pathobiological, biomarker, and imaging aspects of meniscus research. This translational approach can be effective toward optimal diagnosis, prevention, and treatment for the millions of patients who suffer from meniscal disorders each year.

Development of a Novel Canine Model for Posttraumatic Osteoarthritis of the Knee.

Translational models of posttraumatic osteoarthritis (PTOA) that accurately represent clinical pathology need to be developed. This study assessed a novel canine model for PTOA using impact injury. Impacts were delivered to the medial femoral condyle of dogs using a custom-designed impactor at 20, 40, or 60 MPa. Functional assessments and magnetic resonance imaging (MRI) were performed at 2 and 12 weeks, and arthroscopic and histologic assessments were performed at 12 weeks after injury. At 2 and 12 weeks, dogs had observable lameness, knee pain, effusion, loss in range of motion (ROM) and dysfunction in both hindlimbs with severity correlated strongly (r > 0.77) to impact level. At 12 weeks, function, pain, effusion, and ROM were significantly (p < 0.049) worse in knees impacted at 40 and 60 MPa compared with 20 MPa. MRI showed consistent cartilage and subchondral bone marrow lesions, and arthroscopy revealed synovitis and cartilage destruction in impacted knees, with increased severity for 40 and 60 MPa impacts. Histopathology was significantly (p = 0.049) more severe in 40 and 60 MPa and strongly correlated (r = 0.93) to impact level. This novel translational model appears to be valid for investigation of PTOA, including determination of temporal mechanisms of disease and preclinical testing for preventative and therapeutic strategies.

Characterization of Meniscal Pathology Using Molecular and Proteomic Analyses.

The meniscus is a complex tissue and is integral to knee joint health and function. Although the meniscus has been studied for years, a relatively large amount of basic science data on meniscal health and disease are unavailable. Genomic and proteomic analyses of meniscal pathology could greatly improve our understanding of etiopathogenesis and the progression of meniscal disease, yet these analyses are lacking in the current literature. Therefore, the objective of this study was to use microarray and proteomic analyses to compare aged-normal and pathologic meniscal tissues. Meniscal tissue was collected from the knees of five patient groups (n = 3/group). Cohorts included patients undergoing meniscectomy with or without articular cartilage pathology, patients undergoing total knee arthroplasty with mild or moderate-severe osteoarthritis, and aged-normal controls from organ donors. Tissue sections were collected from the white/white and white/red zones of posterior medial menisci. Expression levels were compared between pathologic and control menisci to identify genes of interest (at least a ×1.5 fold change in expression levels between two or more groups) using microarray analysis. Proteomics analysis was performed using mass spectrometry to identify proteins of interest (those with possible trends identified between the aged-normal and pathologic groups). The microarray identified 157 genes of interest. Genes were categorized into the following subgroups: (1) synthesis, (2) vascularity, (3) degradation and antidegradation, and (4) signaling pathways. Mass spectrometry identified 173 proteins of interest. Proteins were further divided into the following categories: (1) extracellular matrix (ECM) proteins; (2) proteins associated with vascularity; (3) degradation and antidegradation proteins; (4) cytoskeleton proteins; (5) glycolysis pathway proteins; and (6) signaling proteins. These data provide novel molecular and biochemical information for the investigation of meniscal pathology. Further evaluation of these disease indicators will help researchers develop algorithms for diagnostic, therapeutic, and prognostic strategies related to meniscal disorders.

Morphine and buprenorphine do not alter leukocyte cytokine production capacity, early apoptosis, or neutrophil phagocytic function in healthy dogs.

Opioids have immunomodulatory properties in many species, but there is little information pertaining to these properties in dogs. Our objective was to compare the in vivo effects of morphine, buprenorphine, and control solution on innate immune system function and apoptosis in healthy dogs. Six adult dogs received a 24-hour infusion of morphine, buprenorphine, or control solution (saline) in a randomized, controlled, crossover block design. Leukocyte apoptosis, phagocytosis, and oxidative burst were evaluated using flow cytometry. Lipopolysaccharide, lipoteichoic acid, and peptidoglycan-stimulated leukocyte production of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 were determined using canine specific multiplex assays. No significant treatment effects were detected among groups. These data suggest that healthy dogs could be less sensitive to the immunomodulatory effects of acute opioid administration compared with other species. Larger investigations in healthy and immunologically challenged dogs are recommended prior to application of these results in clinical patients.

Effects of tramadol and o-desmethyltramadol on canine innate immune system function

OBJECTIVE Tramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10. STUDY DESIGN In vitro pharmacodynamic study. ANIMALS Six healthy dogs. METHODS Blood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay. RESULTS No differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production. CONCLUSIONS Tramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift. CLINICAL RELEVANCE These data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.

Identification of Novel Synovial Fluid Biomarkers Associated with Meniscal Pathology

The menisci are integral components within the knee for ensuring optimal joint function. The overall goal of this study was to identify proteomic markers of meniscal disease within synovial fluid samples obtained from control knees versus knees affected with varying degrees of meniscal injury and osteoarthritis. Joint fluid samples were collected before the patient underwent an arthroscopic knee procedure or total knee arthroplasty. Normal controls included patients younger than 30 years with no history of anterior cruciate ligament, posterior cruciate ligament, or meniscal injury. A total of 21 joint fluid aspirates were analyzed using mass spectrometry, and a total of 296 proteins were identified. Among these, 50 proteins were determined to be of interest as potential biomarkers based on initial analysis and known functions in articular metabolism. Further statistical analysis comparing protein concentrations among clinical groups identified 13 proteins with significant differences between at least two of the patient cohorts. These data provide novel information for the investigation of synovial fluid biomarkers and treatment strategies for meniscal pathology.

Comparison of Platelet Rich Plasma and Bone Marrow Aspirate Concentrate for Osteoprogenitor Cell Retention and Osteoinductive Potential for Osteochondral Allograft

Objectives: Osteochondral allograft (OCA) transplantation is effective for treatment of large articular defects in the knee, hip, ankle, and shoulder of athletes. While success after OCA transplantation is good, one mechanism of failure involves inintegration of OCA bone into recipient bone. Because the OCA bone is devoid of viable cells and blood supply at implantation, and is allogeneic, integration is dependent upon cellular repopulation and neovascularization via creeping substitution. Enhancing this process using bone marrow aspirate concentrate (BMAC) or platelet rich plasma (PRP) could minimize graft failures and improve patient outcomes. Therefore, this study was designed to test the hypothesis that BMAC would be associated with superior viable osteoprogenitor cell repopulation of OCAs and osteoinductive protein production compared to PRP and saline using an ex vivo model. Methods: With ACUC approval, BMAC and leukoreduced PRP were processed from BMA (proximal humerus) and whole blood (jugular vein), respectively, of adult dogs using a commercially available system. Femoral condyles were harvested from adult dogs (n=3) immediately after euthanasia for unrelated reasons and preserved using tissue bank protocol. On day 21 of preservation, cylindrical OCAs (8 mm diam x 8 mm depth) were created (n=36; 12/dog), and randomly assigned to treatments: (1) NEG - bone portion of OCA lavaged with 10 ml saline (2) BMAC - bone portion of OCA lavaged, dried, and then saturated with 0.5 ml BMAC (3) PRP - bone portion of OCA lavaged, dried, and then saturated with 0.5 ml PRP. OCAs were cultured for 7 or 14 days (n=6/group/day), media were changed and collected on days 3, 7, and 14 for biomarker analysis. On days 7 and 14, OCAs were evaluated for viable cell colonization and infiltration using Calcein AM staining. To determine if cells were osteoprogenitors, colony forming unit (CFU) analysis was performed using crystal violet staining to determine CFUs/ml for each BMAC and PRP sample. OCA culture media were assessed for alkaline phosphatase (ALP), dickkopf-related protein (DKK), osteoprotegerin (OPG), osteopontin (OPN), adrenocorticotropic hormone (ACTH), bone morphogenic protein-2 (BMP-2), and bone morphogenic protein-7 (BMP-7) using commercially available assays. Data were compared for statistically significance (p≤0.05) differences. Results: For all BMAC OCAs, viable cells were present on the surface and deep areas of the bone at days 7 and 14. Viable cells were not observed in any part of the bone of PRP or NEG OCAs at either time point (Fig). BMAC samples had a significantly higher (p=0.029) CFU/ml compared to PRP. Concentrations of OPG were significantly higher in BMAC and PRP compared to NEG at days 3 (p<0.001) and 7 (p≤0.004). The concentration of DKK was significantly (p=0.038) higher in BMAC compared to NEG at day 3. Concentrations of BMP-2 were significantly higher in BMAC at days 3 (p<0.001) and 7 (p=0.017) and PRP at day 3 (p=0.009) compared to NEG. The concentration of ALP was significantly lower in PRP compared to NEG at day 3 (p=0.03). Concentrations of BMP-7 and OPN were below detectable limits of the assay for all groups and time points. Conclusion: BMAC showed superior viable osteoprogenitor cell repopulation of OCAs and osteoinductive protein production compared to PRP and the current standard-of-care (saline). BMAC has potential to enhance integration of osteochondral allograft bone and to improve graft survivorship and patient outcomes. Figure Viable (green staining) osteoprogenitor cells on OCAs treated with BMAC, PRP or saline on days 7 and 14 of culture