Removal of the infrapatellar fat pad and associated synovium benefits female guinea pigs in the Dunkin Hartley model of idiopathic osteoarthritis.

Background: Several tissues contribute to the onset and advancement of knee osteoarthritis (OA). One tissue type that is worthy of closer evaluation, particularly in the context of sex, is the infrapatellar fat pad (IFP). We previously demonstrated that removal of the IFP had short-term beneficial effects for a cohort of male Dunkin-Hartley guinea pigs. The present project was designed to elucidate the influence of IFP removal in females of this OA-prone strain. It was hypothesized that resection of the IFP would reduce the development of OA in knees of a rodent model predisposed to the disease. Methods: Female guinea pigs (n=16) were acquired at an age of 2.5 months. Surgical removal of the IFP and associated synovium complex (IFP/SC) was executed at 3 months of age. One knee had the IFP/SC resected; a comparable sham surgery was performed on the contralateral knee. All animals were subjected to voluntary enclosure monitoring and dynamic weight-bearing, as well as compulsory treadmill-based gait analysis monthly; baseline data was collected prior to surgery. Guinea pigs were euthanized at 7 months. Knees from eight animals were evaluated via histology, mRNA expression, and immunohistochemistry (IHC); knees from the remaining eight animals were allocated to microcomputed tomography (microCT), biomechanical analyses (whole joint testing and indentation relaxation testing), and atomic absorption spectroscopy (AAS). Results: Fibrous connective tissue (FCT) replaced the IFP/SC. Mobility/gait data indicated that unilateral IFP/SC removal did not affect bilateral hindlimb movement. MicroCT demonstrated that osteophytes were not a significant feature of OA in this sex; however, trabecular thickness (TbTh) in medial femorae decreased in knees containing the FCT. Histopathology scores were predominantly influenced by changes in the lateral tibia, which demonstrated that histologic signs of OA were increased in knees containing the native IFP/SC versus those with the FCT. Similarly, indentation testing demonstrated higher instantaneous and equilibrium moduli in the lateral tibial articular cartilage of control knees with native IFPs. AAS of multiple tissue types associated with the knee revealed that zinc was the major trace element influenced by removal of the IFP/SC. Conclusions: Our data suggest that the IFP/SC is a significant component driving knee OA in female guinea pigs and that resection of this tissue prior to disease has short-term benefits. Specifically, the formation of the FCT in place of the native tissue resulted in decreased cartilage-related OA changes, as demonstrated by reduced Osteoarthritis Research Society International (OARSI) histology scores, as well as changes in transcript, protein, and cartilage indentation analyses. Importantly, this model provides evidence that sex needs to be considered when investigating responses and associated mechanisms seen with this intervention.

TLR4 antagonism provides short-term but not long-term clinical benefit in a full-depth cartilage defect mouse model.

PURPOSE/AIM: Cartilage injury and subsequent osteoarthritis (OA) are debilitating conditions affecting millions worldwide. As there are no cures for these ailments, novel therapies are needed to suppress disease pathogenesis. Given that joint injuries are known to produce damage-associated molecular patterns (DAMPs), our central premise is that the Toll-like receptor 4 (TLR4) pathway is a principal driver in the early response to cartilage damage and subsequent pathology. We postulate that TLR4 activation is initiated/perpetuated by DAMPs released following joint damage. Thus, antagonism of the TLR4 pathway immediately after injury may suppress the development of joint surface defects. MATERIALS AND METHODS: Two groups were utilized: (1) 8-week-old, male C57BL6 mice treated systemically with a known TLR4 antagonist and (2) mice injected with vehicle control. A full-depth cartilage lesion on the midline of the patellofemoral groove was created in the right knee of each mouse. The left knee was used as a sham surgery control. Gait changes were evaluated over 4 weeks using a quantitative gait analysis system. At harvest, knee joints were processed for pathologic assessment, Nanostring® transcript expression, and immunohistochemistry (IHC). RESULTS: Short-term treatment with a TLR4 antagonist at 14-days significantly improved relevant gait parameters; improved cartilage metrics and modified Mankin scores were also seen. Additionally, mRNA expression and IHC showed reduced expression of inflammatory mediators in animals treated with the TLR4 antagonist. CONCLUSIONS: Collectively, this work demonstrates that systemic treatment with a TLR4 antagonist is protective to further cartilage damage 14-days post-injury in a murine model of induced disease.

Intravenous injection of adipose-derived mesenchymal stromal cells benefits gait and inflammation in a spontaneous osteoarthritis model.

Osteoarthritis (OA) is a leading cause of morbidity among aging populations, yet symptom and/or disease-modification remains elusive. Adipose-derived mesenchymal stromal cells (adMSCs) have demonstrated immunomodulatory and anti-inflammatory properties that may alleviate clinical signs and interrupt disease onset and progression. Indeed, multiple manuscripts have evaluated intra-articular administration of adMSCs as a therapeutic; however, comparatively few evaluations of systemic delivery methods have been published. Therefore, the aim of this study was to evaluate the short-term impact of intravenous (IV) delivery of allogeneic adMSCs in an established model of spontaneous OA, the Hartley guinea pig. Animals with moderate OA received once weekly injections of 2 × 106 adMSCs or vehicle control for 4 weeks in peripheral veins; harvest occurred 2 weeks after the final injection. Systemic administration of adMSCs resulted in no adverse effects and was efficacious in reducing clinical signs of OA (as assessed by computer-aided gait analysis) compared to control injected animals. Further, there were significant decreases in key inflammatory mediators (including monocyte chemoattractant protein-1, tumor necrosis factor, and prostaglandin E2 ) both systemically (liver, kidney, and serum) and locally in the knee (joint tissues and synovial fluid) in animals treated with IV adMSCs relative to controls (as per enzyme-linked immunosorbent assay and/or immunohistochemistry, dictated by tissue sample). Thus, systemic administration of adMSCs by IV injection significantly improved gait parameters and reduced both systemic and intra-articular inflammatory mediators in animals with OA. These findings demonstrate the potential utility of alternative delivery approaches for cellular therapy of OA, particularly for patients with multiple affected joints.

Early removal of the infrapatellar fat pad/synovium complex beneficially alters the pathogenesis of moderate stage idiopathic knee osteoarthritis in male Dunkin Hartley guinea pigs.

BACKGROUND: The infrapatellar fat pad (IFP) is the largest adipose deposit in the knee; however, its contributions to the homeostasis of this organ remain undefined. To determine the influence of the IFP and its associated synovium (IFP/synovium complex or IFP/SC) on joint health, this study evaluated the progression of osteoarthritis (OA) following excision of this unit in a rodent model of naturally-occurring disease. METHODS: Male Dunkin-Hartley guinea pigs (n=18) received surgical removal of the IFP in one knee at 3 months of age; contralateral knees received sham surgery as matched internal controls. Mobility and gait assessments were performed prior to IFP/SC removal and monthly thereafter. Animals were harvested at 7 months of age. Ten set of these knees were processed for microcomputed tomography (microCT), histopathology, transcript expression analyses, and immunohistochemistry (IHC); 8 sets of knees were dedicated to microCT and biomechanical testing (material properties of knee joints tissues and anterior drawer laxity). RESULTS: Fibrous connective tissue (FCT) developed in place of the native adipose depot. Gait demonstrated no significant differences between IFP/SC removal and contralateral hindlimbs. MicroCT OA scores were improved in knees containing the FCT. Quantitatively, IFP/SC-containing knees had more osteophyte development and increased trabecular volume bone mineral density (vBMD) in femora and tibiae. Histopathology confirmed maintenance of articular cartilage structure, proteoglycan content, and chondrocyte cellularity in FCT-containing knees. Transcript analyses revealed decreased expression of adipose-related molecules and select inflammatory mediators in FCTs compared to IFP/SCs. This was verified via IHC for two key inflammatory agents. The medial articular cartilage in knees with native IFP/SCs showed an increase in equilibrium modulus, which correlated with increased amounts of magnesium and phosphorus. DISCUSSION/CONCLUSION: Formation of the FCT resulted in reduced OA-associated changes in both bone and cartilage. This benefit may be associated with: a decrease in inflammatory mediators at transcript and protein levels; and/or improved biomechanical properties. Thus, the IFP/SC may play a role in the pathogenesis of knee OA in this strain, with removal prior to disease onset appearing to have short-term benefits.

Systemic administration of a pharmacologic iron chelator reduces cartilage lesion development in the Dunkin-Hartley model of primary osteoarthritis.

Iron has been emerging as a key contributor to aging-associated, chronic disorders due to the propensity for generating reactive oxygen species. To date, there are a limited number of publications exploring the role of iron in the pathogenesis of primary/age-related osteoarthritis (OA). The objective of this study was to determine whether reduced iron via pharmacologic iron chelation with deferoxamine (DFO) affected the development and/or severity of cartilage lesions in a primary OA model. At 12-weeks-of-age, 15 male Dunkin-Hartley guinea pigs received either 46 mg/kg DFO (n = 8) or vehicle control (n = 7) injected subcutaneously twice daily for five days each week. Movement changes, captured via overhead enclosure monitoring, were also determined. Termination occurred at 30-weeks-of-age. Iron was quantified in serum, urine, liver, and femoral head articular cartilage. Left knees were evaluated for: structural changes using histopathology guidelines; and immunohistochemistry. Gene expression analysis was conducted on right knee articular cartilage. DFO reduced iron levels in femoral head articular cartilage (p = 0.0006) and liver (p = 0.02), and increased iron within urine (p = 0.04) and serum (p = 0.0009). Mobility of control animals declined, while the DFO group maintained activity levels similar to the first month of treatment (p = 0.05). OA-associated cartilage lesions were reduced in knees of DFO animals (p = 0.0001), with chondrocyte hypocellularity a key histologic difference between groups (p < 0.0001). DFO-receiving animals had increased immunostaining for phosphorylated adenosine monophosphate activated protein kinase alpha within knee articular cartilage; lower transcript counts of several proapoptotic genes (p = 0.04-0.0004) and matrix-degrading enzymes (p = 0.02-<0.0001), and increased expression of the anti-apoptotic gene Bcl-2 (p < 0.0001) and a tissue inhibitor of matrix-metalloproteinases (p = 0.03) were also observed. These results suggest that iron chelation delayed the progression of primary OA in an animal model and could hold potential as a translational intervention. These findings provide expanded insight into factors that may contribute to the pathogenesis of primary OA.

Treatment of an Alveolar Rhabdomyosarcoma Allograft with Recombinant Myxoma Virus and Oclacitinib.

PURPOSE: Rhabdomyosarcomas (RMS) are difficult tumors to treat with conventional therapies. Publications indicate that oncolytic virotherapy (OV) could benefit cancer patients with tumors that are refractory to conventional treatments. It is believed that the efficacy of OV can be enhanced when used in combination with other treatments. This study evaluated the response of mice with aggressive alveolar RMS (ARMS) allografts to treatment with an OV [recombinant myxoma virus (MYXVΔserp2)] in combination with a Janus kinase (JAK) inhibitor (oclacitinib). Oclacitinib is known to inhibit JAK1 and JAK2 cell signaling pathways, which should limit the antiviral Type I interferon response. However, oclacitinib does not inhibit immune pathways that promote antigen presentation, which help stimulate an anti-cancer immune response. MATERIALS AND METHODS: To determine if MYXVΔserp2 and oclacitinib could improve outcomes in animals with ARMS, nude mice were inoculated subcutaneously with murine ARMS cells to establish tumors. Immune responses, tumor growth, and clinical signs in mice treated with combination therapy were compared to mice given placebo therapy and mice treated with OV alone. RESULTS: Combination therapy was safe; no viral DNA was detected in off-target organs, only within tumors. As predicted, viral DNA was detected in tumors of mice given oclacitinib and MYXVΔserp2 for a longer time period than mice treated with OV alone. Although tumor growth rates and median survival times were not significantly different between groups, clinical signs were less severe in mice treated with OV. CONCLUSION: Our data indicate that MYXVΔserp2 treatment benefits mice with ARMS by reducing clinical signs of disease and improving quality of life.

Compensatory Expression of Nur77 and Nurr1 Regulates NF-κB-Dependent Inflammatory Signaling in Astrocytes.

Inflammatory activation of glial cells promotes loss of dopaminergic neurons in Parkinson disease. The transcription factor nuclear factor κB (NF-κB) regulates the expression of multiple neuroinflammatory cytokines and chemokines in activated glial cells that are damaging to neurons. Thus, inhibition of NF-κB signaling in glial cells could be a promising therapeutic strategy for the prevention of neuroinflammatory injury. Nuclear orphan receptors in the NR4A family, including NR4A1 (Nur77) and NR4A2 (Nurr1), can inhibit the inflammatory effects of NF-κB, but no approved drugs target these receptors. Therefore, we postulated that a recently developed NR4A receptor ligand, 1,1bis (3'indolyl) 1(pmethoxyphenyl) methane (C-DIM5), would suppress NF-κB-dependent inflammatory gene expression in astrocytes after treatment with 1-methyl-4-phenyl 1, 2, 3, 6-tetrahydropyridine (MPTP) and the inflammatory cytokines interferon γ and tumor necrosis factor α C-DIM5 increased expression of Nur77 mRNA and suppressed expression of multiple neuroinflammatory genes. C-DIM5 also inhibited the expression of NFκB-regulated inflammatory and apoptotic genes in quantitative polymerase chain reaction array studies and effected p65 binding to unique genes in chromatin immunoprecipitation next-generation sequencing experiments but did not prevent p65 translocation to the nucleus, suggesting a nuclear-specific mechanism. C-DIM5 prevented nuclear export of Nur77 in astrocytes induced by MPTP treatment and simultaneously recruited Nurr1 to the nucleus, consistent with known transrepressive properties of this receptor. Combined RNAi knockdown of Nur77 and Nurr1 inhibited the anti-inflammatory activity of C-DIM5, demonstrating that C-DIM5 requires these receptors to inhibit NF-κB. Collectively, these data demonstrate that NR4A1/Nur77 and NR4A2/Nurr1 dynamically regulated inflammatory gene expression in glia by modulating the transcriptional activity of NF-κB.

A novel diindolylmethane analog, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane, inhibits the tumor necrosis factor-induced inflammatory response in primary murine synovial fibroblasts through a Nurr1-dependent mechanism.

The progression of rheumatoid arthritis involves the thickening of the synovial lining due to the proliferation of fibroblast-like synoviocytes (FLS) and infiltration by inflammatory cells. Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine involved in progression of the disease. Under rheumatoid conditions, FLS express the tumor necrosis factor (TNF)-recognition complex (TNFR1, TNFR2, VCAM-1 and ICAM-1), which induces local macrophage activation and leads to downstream nuclear factor κB (NF-κB) signaling. The NF-κB-regulated inflammatory gene, cyclooxygenase (COX), increases synthesis of prostaglandins that contribute to the propagation of inflammatory damage within the joint. Because the nuclear orphan receptor, NR4A2 (Nurr1), can negatively regulate NF-κB-dependent inflammatory gene expression in macrophages, we postulated that activation of this receptor by the Nurr1 ligand 1,1-bis(3'-indolyl)-1-(p-chlorophenyl) methane (C-DIM12) would modulate inflammatory gene expression in synovial fibroblasts by inhibiting NF-κB. Treatment with C-DIM12 suppressed TNFα-induced expression of adhesion molecules and NF-κB regulated genes in primary synovial fibroblasts including vascular adhesion molecule 1 (VCAM-1), PGE2 and COX-2. Immunofluorescence studies indicated that C-DIM12 did not prevent translocation of p65 and stabilized nuclear localization of Nurr1 in synovial fibroblasts. Knockdown of Nurr1 expression by RNA interference prevented the inhibitory effects of C-DIM12 on inflammatory gene expression, indicating that the anti-inflammatory effects of this compound are Nurr1-dependent. Collectively, these data suggest that this receptor may be a viable therapeutic target in RA.

Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity.

BACKGROUND: As the primary immune response cell in the central nervous system, microglia constantly monitor the microenvironment and respond rapidly to stress, infection, and injury, making them important modulators of neuroinflammatory responses. In diseases such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, and human immunodeficiency virus-induced dementia, activation of microglia precedes astrogliosis and overt neuronal loss. Although microgliosis is implicated in manganese (Mn) neurotoxicity, the role of microglia and glial crosstalk in Mn-induced neurodegeneration is poorly understood. METHODS: Experiments utilized immunopurified murine microglia and astrocytes using column-free magnetic separation. The effect of Mn on microglia was investigated using gene expression analysis, Mn uptake measurements, protein production, and changes in morphology. Additionally, gene expression analysis was used to determine the effect Mn-treated microglia had on inflammatory responses in Mn-exposed astrocytes. RESULTS: Immunofluorescence and flow cytometric analysis of immunopurified microglia and astrocytes indicated cultures were 97 and 90% pure, respectively. Mn treatment in microglia resulted in a dose-dependent increase in pro-inflammatory gene expression, transition to a mixed M1/M2 phenotype, and a de-ramified morphology. Conditioned media from Mn-exposed microglia (MCM) dramatically enhanced expression of mRNA for Tnf, Il-1ß, Il-6, Ccl2, and Ccl5 in astrocytes, as did exposure to Mn in the presence of co-cultured microglia. MCM had increased levels of cytokines and chemokines including IL-6, TNF, CCL2, and CCL5. Pharmacological inhibition of NF-κB in microglia using Bay 11-7082 completely blocked microglial-induced astrocyte activation, whereas siRNA knockdown of Tnf in primary microglia only partially inhibited neuroinflammatory responses in astrocytes. CONCLUSIONS: These results provide evidence that NF-κB signaling in microglia plays an essential role in inflammatory responses in Mn toxicity by regulating cytokines and chemokines that amplify the activation of astrocytes.

Significance of ERα, HER2, and CAV1 expression and molecular subtype classification to canine mammary gland tumor.

Canine mammary gland tumor (CMT) and human breast cancer (HBC) share many similarities regarding their risk factors, histological features, and behavior. Despite the increasing evidence of molecular marker expression as a prognostic indicator for HBC, few studies have applied this approach to CMT. The aim of the present study is to evaluate the significance of the expression of estrogen receptor-alpha (ERα), human epidermal growth factor receptor 2 (HER2), and caveolin-1 (CAV1) to the behavior and the clinical outcome of CMT. Additionally, the correlation between subtype classification (luminal A, luminal B, HER2-overexpressing, basal-like, and normal-like) and tumor behavior prognosis were assessed. Canine mammary gland tissues were immunohistochemically stained for ERα, HER2, and CAV1 and evaluated and classified into 5 subtypes on the basis of immunoreactivity. Although there were no statistically significant differences in the molecular marker immunoreactivity of different subtypes, the degree of positive staining for ERα, extranuclear ERα, HER2, and CAV1 showed significant correlations (P < 0.05) with the behavior and prognosis of the tumor. The current study indicates the prognostic value of immunohistochemical staining status of ERα, HER2, and CAV1 for CMT. In addition, some trends were seen in subtype classification on the prognosis of the tumor, implying that, although further analysis is needed, there is potential clinical application of 5-subtype classification for CMT.