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  • The macrophage marker translocator protein (TSPO) is down-regulated on pro-inflammatory 'M1' human macrophages.

    3 October 2018

    The translocator protein (TSPO) is a mitochondrial membrane protein, of as yet uncertain function. Its purported high expression on activated macrophages, has lent utility to TSPO targeted molecular imaging in the form of positron emission tomography (PET), as a means to detect and quantify inflammation in vivo. However, existing literature regarding TSPO expression on human activated macrophages is lacking, mostly deriving from brain tissue studies, including studies of brain malignancy, and inflammatory diseases such as multiple sclerosis. Here, we utilized three human sources of monocyte derived macrophages (MDM), from THP-1 monocytes, healthy peripheral blood monocytes and synovial fluid monocytes from patients with rheumatoid arthritis, to undertake a detailed investigation of TSPO expression in activated macrophages. In this work, we demonstrate a consistent down-regulation of TSPO mRNA and protein in macrophages activated to a pro-inflammatory, or 'M1' phenotype. Conversely, stimulation of macrophages to an M2 phenotype with IL-4, dexamethasone or TGF-β1 did not alter TSPO expression, regardless of MDM source. The reasons for this are uncertain, but our study findings add some supporting evidence for recent investigations concluding that TSPO may be involved in negative regulation of inflammatory responses in macrophages.

  • Beneficial effects of autologous bone marrow-derived mesenchymal stem cells in naturally occurring tendinopathy.

    3 October 2018

    Tendon injuries are a common age-related degenerative condition where current treatment strategies fail to restore functionality and normal quality of life. This disease also occurs naturally in horses, with many similarities to human tendinopathy making it an ideal large animal model for human disease. Regenerative approaches are increasingly used to improve outcome involving mesenchymal stem cells (MSCs), supported by clinical data where injection of autologous bone marrow derived MSCs (BM-MSCs) suspended in marrow supernatant into injured tendons has halved the re-injury rate in racehorses. We hypothesized that stem cell therapy induces a matrix more closely resembling normal tendon than the fibrous scar tissue formed by natural repair. Twelve horses with career-ending naturally-occurring superficial digital flexor tendon injury were allocated randomly to treatment and control groups. 1X10(7) autologous BM-MSCs suspended in 2 ml of marrow supernatant were implanted into the damaged tendon of the treated group. The control group received the same volume of saline. Following a 6 month exercise programme horses were euthanized and tendons assessed for structural stiffness by non-destructive mechanical testing and for morphological and molecular composition. BM-MSC treated tendons exhibited statistically significant improvements in key parameters compared to saline-injected control tendons towards that of normal tendons and those in the contralateral limbs. Specifically, treated tendons had lower structural stiffness (p<0.05) although no significant difference in calculated modulus of elasticity, lower (improved) histological scoring of organisation (p<0.003) and crimp pattern (p<0.05), lower cellularity (p<0.007), DNA content (p<0.05), vascularity (p<0.03), water content (p<0.05), GAG content (p<0.05), and MMP-13 activity (p<0.02). Treatment with autologous MSCs in marrow supernatant therefore provides significant benefits compared to untreated tendon repair in enhancing normalisation of biomechanical, morphological, and compositional parameters. These data in natural disease, with no adverse findings, support the use of this treatment for human tendon injuries.

  • Macrophage sub-populations and the lipoxin A4 receptor implicate active inflammation during equine tendon repair.

    3 October 2018

    Macrophages (Mφ) orchestrate inflammatory and reparatory processes in injured connective tissues but their role during different phases of tendon healing is not known. We investigated the contribution of different Mφ subsets in an equine model of naturally occurring tendon injury. Post mortem tissues were harvested from normal (uninjured), sub-acute (3-6 weeks post injury) and chronically injured (>3 months post injury) superficial digital flexor tendons. To determine if inflammation was present in injured tendons, Mφ sub-populations were quantified based on surface antigen expression of CD172a (pan Mφ), CD14(high)CD206(low) (pro-inflammatory M1Mφ), and CD206(high) (anti-inflammatory M2Mφ) to assess potential polarised phenotypes. In addition, the Lipoxin A(4) receptor (FPR2/ALX) was used as marker for resolving inflammation. Normal tendons were negative for both Mφ and FPR2/ALX. In contrast, M1Mφ predominated in sub-acute injury, whereas a potential phenotype-switch to M2Mφ polarity was seen in chronic injury. Furthermore, FPR2/ALX expression by tenocytes was significantly upregulated in sub-acute but not chronic injury. Expression of the FPR2/ALX ligand Annexin A1 was also significantly increased in sub-acute and chronic injuries in contrast to low level expression in normal tendons. The combination of reduced FPR2/ALX expression and persistence of the M2Mφ phenotype in chronic injury suggests a potential mechanism for incomplete resolution of inflammation after tendon injury. To investigate the effect of pro-inflammatory mediators on lipoxin A(4) (LXA(4)) production and FPR2/ALX expression in vitro, normal tendon explants were stimulated with interleukin-1 beta and prostaglandin E(2). Stimulation with either mediator induced LXA(4) release and maximal upregulation of FPR2/ALX expression after 72 hours. Taken together, our data suggests that although tenocytes are capable of mounting a protective mechanism to counteract inflammatory stimuli, this appears to be of insufficient duration and magnitude in natural tendon injury, which may potentiate chronic inflammation and fibrotic repair, as indicated by the presence of M2Mφ.

  • Inflamm-aging and arachadonic acid metabolite differences with stage of tendon disease.

    3 October 2018

    The contribution of inflammation to the pathogenesis of tendinopathy and high prevalence of re-injury is not well established, although recent evidence suggests involvement of prostaglandins. We investigated the roles of prostaglandins and inflammation-resolving mediators in naturally occurring equine tendon injury with disease stage and age. Levels of prostaglandins E(2) (PGE(2)), F(2α) (PGF(2α)), lipoxin A(4) (LXA(4)) and its receptor FPR2/ALX were analysed in extracts of normal, sub-acute and chronic injured tendons. To assess whether potential changes were associated with altered PGE(2) metabolism, microsomal prostaglandin E synthase-1 (mPGES-1), prostaglandin dehydrogenase (PGDH), COX-2 and EP(4) receptor expression were investigated. The ability of tendons to resolve inflammation was determined by assessing FPR2/ALX expression in natural injury and IL-1β stimulated tendon explants.Alterations in the profile of lipid mediators during sub-acute injury included low PGE(2) and elevated LXA(4) levels compared to normal and chronic injuries. In contrast, PGF(2α) levels remained unchanged and were three-fold lower than PGE(2). The synthetic capacity of PGE(2) as measured by the ratio of mPGES-1:PGDH was elevated in sub-acute injury, suggesting aberrations in tendon prostaglandin metabolism, whilst COX-2 and EP(4) receptor were unchanged. Paradoxically low tendon PGE(2) levels in early injury may be attributed to increased local clearance via PGDH or the class switching of lipid mediators from the prostaglandin to the lipoxin axis. PGE(2) is therefore implicated in the development of tendon inflammation and its ensuing resolution. Whilst there was no relationship between age and tendon LXA(4) levels, there was an age-associated decline in FPR2/ALX receptor expression with concurrent increased PGE(2) levels in injury. Furthermore, uninjured tendon explants from younger (<10 years) but not older horses (≥10 years) treated with IL-1β responded by increasing FPR2/ALX suggesting aged individuals exhibit a reduced capacity to resolve inflammation via FPR2/ALX, which may present a potential mechanism for development of chronic tendinopathy and re-injury.

  • Osseous abnormalities associated with collateral desmopathy of the distal interphalangeal joint: part 1.

    3 October 2018

    REASONS FOR PERFORMING STUDY: Osseous abnormalities associated with collateral ligament (CL) injury of the distal interphalangeal (DIP) joint have been documented using magnetic resonance imaging (MRI) but there is currently limited information about the frequency of osseous pathology associated with CL injury. OBJECTIVES: To determine the frequency of occurrence of osseous abnormality coexistent with CL injury of the DIP joint and describe the distribution and character of osseous lesions; and to establish if there was an association between osseous abnormality and increased radiopharmaceutical uptake (IRU). HYPOTHESES: There would be a higher incidence of osseous abnormality at the insertion of an injured CL than at the origin; and a relationship between the presence of osseous abnormality and duration of lameness. MATERIALS AND METHODS: Magnetic resonance images of 313 feet of 289 horses with foot pain and a definitive diagnosis of collateral desmopathy of the DIP joint were analysed retrospectively for presence and type of osseous abnormality in the middle and distal phalanges. Scintigraphic images were examined and the presence of IRU in the middle or distal phalanges recorded. RESULTS: Osseous abnormalities were detected in 143 (45.7%) feet, 27 (18.8%) of which had osseous and CL injury alone, while the remaining 116 had CL related osseous injury and multiple injuries within the hoof capsule. Entheseous new bone and endosteal irregularity of the middle and distal phalanges were the most frequent types of osseous abnormality. There was a higher incidence of osseous abnormalities medially than laterally and at the ligament insertion than at the origin. There was a significant association between presence of IRU and osseous injury. CONCLUSIONS: A variety of osseous lesions of differing severity are associated with CL injury. Normal radiopharmaceutical uptake does not preclude significant osseous pathology associated with CL injury. CLINICAL RELEVANCE: Further studies are necessary in order to determine if osseous abnormalities associated with CL injury influence prognosis for return to performance.

  • Osseous abnormalities associated with collateral desmopathy of the distal interphalangeal joint. Part 2: treatment and outcome.

    3 October 2018

    REASONS FOR PERFORMING STUDY: There are currently few long-term follow-up data relating to recovery from injury of a collateral ligament (CL) of the distal interphalangeal (DIP) joint and limited information about the effect of associated osseous injury on prognosis. OBJECTIVES: To describe long-term follow-up results for horses with CL injury, with and without associated osseous injury; and to determine the effect of extracorporeal shock wave therapy (ECSWT) or radial pressure wave therapy (RPWT) on outcome. HYPOTHESES: Prognosis for return to performance for horses with CL-related osseous injury would be worse than for horses with CL injury alone. METHODS: Magnetic resonance images from 313 feet of 289 horses with foot pain and a definitive diagnosis of collateral desmopathy of the DIP joint were analysed retrospectively for presence of osseous abnormality associated with the ligament origin or insertion and the middle and distal phalanges. Horses were assigned to groups according to the combination of their injuries. Type of treatment was recorded and follow-up information obtained. Thirty-two horses with additional sources of lameness were excluded from analysis of outcome. RESULTS: Follow-up data were available for 182 horses, 55 of which had follow-up information for up to 2 years after presentation. Twenty-seven percent of horses with CL injury alone and 34% of horses with CL related osseous injury returned to their previous performance level. Prognosis for a combination of injuries to multiple soft tissue and osseous structures within the hoof capsule was substantially worse. There was no effect of ECSWT or RPWT on outcome. CONCLUSIONS: The presence of mild to moderate CL related osseous injury does not appear to influence prognosis compared with CL injury alone. CLINICAL RELEVANCE: Further studies of a larger number of horses are necessary in order to ascertain if specific types of osseous pathology influence return to performance levels.

  • Translocator Protein as an Imaging Marker of Macrophage and Stromal Activation in Rheumatoid Arthritis Pannus.

    3 October 2018

    PET radioligands targeted to translocator protein (TSPO) offer a highly sensitive and specific means of imaging joint inflammation in rheumatoid arthritis (RA). Through high expression of TSPO on activated macrophages, TSPO PET has been widely reported in several studies of RA as a means of imaging synovial macrophages in vivo. However, this premise does not take into account the ubiquitous expression of TSPO. This study aimed to investigate TSPO expression in major cellular constituents of RA pannus-monocytes, macrophages, fibroblastlike synoviocytes (FLS cells), and CD4-positive (CD4+) T lymphocytes (T cells)-to more accurately interpret TSPO PET signal from RA synovium. Methods: Three RA patients and 3 healthy volunteers underwent PET of both knees using the TSPO radioligand 11C-PBR28. Through 3H-PBR28 autoradiography and immunostaining of synovial tissue in 6 RA patients and 6 healthy volunteers, cellular expression of TSPO in synovial tissue was evaluated. TSPO messenger RNA expression and 3H-PBR28 radioligand binding was assessed using in vitro monocytes, macrophages, FLS cells, and CD4+ T cells. Results:11C-PBR28 PET signal was significantly higher in RA joints than in healthy joints (average SUV, 0.82 ± 0.12 vs. 0.03 ± 0.004; P < 0.01). Further, 3H-PBR28-specific binding in synovial tissue was approximately 10-fold higher in RA patients than in healthy controls. Immunofluorescence revealed TSPO expression on macrophages, FLS cells, and CD4+ T cells. The in vitro study demonstrated the highest TSPO messenger RNA expression and 3H-PBR28-specific binding in activated FLS cells, nonactivated M0 macrophages, and activated M2 reparative macrophages, with the least TSPO expression being in activated and nonactivated CD4+ T cells. Conclusion: To our knowledge, this study was the first evaluation of cellular TSPO expression in synovium, with the highest TSPO expression and PBR28 binding being found on activated synovial FLS cells and M2 macrophages. TSPO-targeted PET may therefore have a unique sensitivity in detecting FLS cells and macrophage-predominant inflammation in RA, with potential utility for assessing treatment response in trials using novel FLS-cell-targeted therapies.