Evangelos M. Fragkakis

OBJECTIVE: Group 3 innate lymphoid cells (ILC3s) play a pivotal role in barrier tissues such as the gut and the skin, two important sites of disease in spondyloarthritis (SpA). This study was undertaken to investigate whether normal or injured human enthesis, a key target tissue in early SpA, harbors ILC3s in entheseal soft tissue and adjacent perientheseal bone. METHODS: Interspinous ligament and spinous process bone from donors with no systemic inflammatory disease were collected, enzymatically digested, and immunophenotyped. The immunologic profile of entheseal cells was examined, and the transcriptional profile of sorted ILC3s was compared to that of ILC3s isolated from SpA synovial fluid (SF). To assess the ability of entheseal tissue to produce interleukin-17 (IL-17) and IL-22, entheseal digests were stimulated with IL-23 and IL-1β. Osteoarthritic and ruptured Achilles tendon tissue was examined histologically. RESULTS: The proportion of ILCs in human entheseal soft tissue was higher than that in peripheral blood (P = 0.008); entheseal soft tissue and perientheseal bone both had a higher proportion of NKp44+ ILC3s (P = 0.001 and P = 0.043, respectively). Studies of retinoic acid receptor-related orphan nuclear receptor γt (RORγt), STAT3, and IL-23 receptor transcript expression validated the entheseal ILC3 phenotype. Cytokine transcript expression was similar in ILC3s isolated from enthesis and from SpA SF. Stimulation of normal entheseal digests with IL-23/IL-1β led to up-regulation of IL-17A transcript, and histologic examination of injured/damaged entheses revealed the presence of RORγt-expressing cells. CONCLUSION: This work shows that human enthesis harbors a resident population of ILC3s, with the potential to participate in the pathogenesis of SpA.

OBJECTIVES: Murine models of interleukin (IL)-23-driven spondyloarthritis (SpA) have demonstrated entheseal accumulation of γδT-cells which were responsible for the majority of local IL-17A production. However, IL-23 blockers are ineffective in axial inflammation in man. This study investigated γδT-cell subsets in the normal human enthesis to explore the biology of the IL-23/17 axis. METHODS: Human spinous processes entheseal soft tissue (EST) and peri-entheseal bone (PEB) were harvested during elective orthopaedic procedures. Entheseal γδT-cells were evaluated using immunohistochemistry and isolated and characterised using flow cytometry. RNA was isolated from γδT-cell subsets and analysed by qPCR. Entheseal γδT-cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, anti-CD3/28 or IL-23 and IL-17A production was measured by high-sensitivity ELISA and qPCR. RESULTS: Entheseal γδT-cells were confirmed immunohistochemically with Vδ1 and Vδ2 subsets that are cytometrically defined. Transcript profiles of both cell populations suggested tissue residency and immunomodulatory status. Entheseal Vδ2 cells expressed high relative abundance of IL-23/17-associated transcripts including IL-23R, RORC and CCR6, whereas the Vδ1 subset almost completely lacked detectable IL-23R transcript. Following PMA stimulation IL-17A was detectable in both Vδ1 and Vδ2 subsets, and following CD3/CD28 stimulation both subsets showed IL-17A and IL-17F transcripts with neither transcript being detectable in the Vδ1 subset following IL-23 stimulation. CONCLUSION: Spinal entheseal Vδ1 and Vδ2 subsets are tissue resident cells with inducible IL-17A production with evidence that the Vδ1 subset does so independently of IL-23R expression.

BACKGROUND: This study aims to determine the incidence of pulmonary embolism (PE) in trauma and orthopedic patients within a regional tertiary referral center and its association with the pattern of injury, type of treatment, co-morbidities, thromboprophylaxis and mortality. METHODS: All patients admitted to our institution between January 2010 and December 2011, for acute trauma or elective orthopedic procedures, were eligible to participate in this study. Our cohort was formed by identifying all patients with clinical features of PE who underwent Computed Tomography-Pulmonary Angiogram (CT-PA) to confirm or exclude the clinical suspicion of PE, within six months after the injury or the surgical procedure.Case notes and electronic databases were reviewed retrospectively to identify each patient's venous thromboembolism (VTE) risk factors, type of treatment, thromboprophylaxis and mortality. RESULTS: Out of 18,151 patients admitted during the study period only 85 (0.47%) patients developed PE (positive CT-PA) (24 underwent elective surgery and 61 sustained acute trauma). Of these, only 76% of the patients received thromboprophylaxis. Hypertension, obesity and cardiovascular disease were the most commonly identifiable risk factors. In 39% of the cases, PE was diagnosed during the in-hospital stay. The median time of PE diagnosis, from the date of injury or the surgical intervention was 23 days (range 1 to 312). The overall mortality rate was 0.07% (13/18,151), but for those who developed PE it was 15.29% (13/85). Concomitant deep venous thrombosis (DVT) was identified in 33.3% of patients. The presence of two or more co-morbidities was significantly associated with the incidence of mortality (unadjusted odds ratio (OR) = 3.52, 95% confidence interval (CI) (1.34, 18.99), P = 0.034). Although there was also a similar clinical effect size for polytrauma injury on mortality (unadjusted OR = 1.90 (0.38, 9.54), P = 0.218), evidence was not statistically significant for this factor. CONCLUSIONS: The incidence of VTE was comparable to previously reported rates, whereas the mortality rate was lower. Our local protocols that comply with the National Institute for Health and Clinical Excellence (NICE) guidelines in the UK appear to be effective in preventing VTE and reducing mortality in trauma and orthopedic patients.