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Background/Aims Patients with suspected new-onset giant cell arteritis (GCA) require urgent specialist assessment to establish a diagnosis. Early review reduces unnecessary treatment with high dose glucocorticoids in those without GCA and enhances sensitivity of temporal artery ultrasound. Ensuring patients are rapidly triaged and diagnosed poses a logistical challenge for rheumatology departments working alongside colleagues in both primary care and ophthalmology. To address this, we established a rheumatology-led Newcastle Hospital GCA fast track clinic, a service led by four consultant rheumatologists who perform clinical assessments and temporal artery ultrasound scans. Methods Data were collected prospectively from 2017. We performed a clinical service evaluation based on a retrospective review of clinical records of patients assessed in this service between 2017 to 2020. Results A total of 626 consecutive patients were assessed, with 145 (23%) diagnosed with GCA over the four-year period. Average age was 72 (range 41-96 years) and 452 (72%) were female. The cardinal symptoms of headache, jaw claudication and visual disturbance were present in 518, 103 and 283 patients respectively. The majority of referrals came from ophthalmology (42%) and primary care (34%). 614 patients had an ultrasound scan performed, returning a positive result in 121 (19.7%), negative in 415 (67.6%) and inconclusive in 78 (12.7%). A biopsy was requested for 147 patients over the four-year period; 33 were positive, 95 negative and six were inconclusive. Within this cohort, 47 patients had a biopsy performed despite a negative ultrasound scan, of which five were found to be positive for GCA. Conclusion Our data illustrate the key clinical, imaging and histological findings within one of the largest inception cohorts of GCA in the UK and demonstrate the feasibility of a one-stop rapid access clinical service for patient benefit. Disclosure C.M. Lin: None. J. Berry: None. B. Thompson: None. J. Heaney: None. K. Houghton: None. K. Baker: None. G. Reynolds: None. A. Lorenzi: None.

Improving understanding of the underlying pathophysiology of giant cell arteritis (GCA) is transforming clinical management by identifying novel avenues for targeted therapies. One key area of concern for both clinicians and patients with GCA is glucocorticoid (GC) morbidity. The first randomised controlled trials of targeted treatment to reduce cumulative GC use in GCA have been published, with tocilizumab, an interleukin (IL)-6 receptor inhibitor, now the first ever licensed treatment for GCA. Further potential therapies are emerging owing to our enhanced understanding of the pathophysiology of the disease. Other improvements in the care of our patients are rapid access pathways and imaging techniques, such as ultrasound, which are becoming part of modern rheumatology practice in the UK, Europe and beyond. These have been highlighted in the literature to reduce delay in diagnosis and improve long-term outcomes for those investigated for GCA.

Background Tocilizumab was approved for the treatment of giant cell arteritis (GCA) by NICE in April 2018. This decision followed the GiACTA (Giant Cell Arteritis Actemra) study, a randomised control trial that demonstrated a beneficial effect of tocilizumab in reducing the frequency of disease flare and overall prednisolone requirements. However, as noted in the NICE appraisal, the extent to which the patient population in the GiACTA study reflects the population it is eventually used for in UK clinical practice is not clear. To address this, we analysed the records of all patients started on tocilizumab treatment for GCA in a single centre since its approval. Methods We performed a retrospective analysis of the clinical records of all patients started on tocilizumab for GCA at the Freeman Hospital in Newcastle. All patients are discussed in a regional connective tissue disease MDT prior to the initiation of therapy to ensure they fulfil NICE guidelines. At each subsequent visit adverse events related to both tocilizumab and corticosteroids and any flares of GCA or ischaemic events are recorded. Results In total 14 patients started tocilizumab since June 2018, with a cumulative exposure of 8 patient-years. The mean age was 74 years, slightly older than the average age in the GiACTA trial (69 years). In contrast to GiACTA where 48% of patients were newly diagnosed, all our patients had established disease. In our cohort 36% of patients had CT PET evidence of large vessel vasculitis, similar to the rate in GiACTA (40%) but higher than the national average of around 5%. Incidence of visual loss in our treated patients was higher at 29% than recorded in the general GCA population in the DC-VAS study (7.9%). In those treated for relapsing disease around half (56%) had recorded previous significant adverse events with steroids, including heart failure, hypertension and mood changes. Five patients had infections requiring antibiotics (cellulitis/ulcer in three, chest infection in one, urinary tract infections in two), with two serious infections requiring hospital admission (both urinary tract infections). No patients had further ischaemic events while on treatment. All patients were on a lower dose of prednisolone following treatment with an average of a 63% reduction in steroid dose. Conclusion The modest number of patients receiving tocilizumab for GCA suggests we are not treating everyone at first relapse. The higher rates of large vessel vasculitis, visual loss and previous steroid-induced complications suggest a preference for saving tocilizumab for a more severely affected subset of patients. In this preliminary data there were reassuringly no recorded ischaemic events following treatment and steroid doses were successfully reduced in all patients. Disclosures G. Reynolds None. B. Griffiths None. K. Houghton None. B. Thompson None. A.R. Lorenzi None. J. Heaney None.

Background BD patients are managed equivalently by multiple medical specialties in our trust. Failure to switch from IV to s/c antiTNFalpha therapies was noted and our use of methotrexate, hydroxychloroquine and cyclophosphamide deviates from guidelines. Whether this effects outcome is unknown it but seems likely to be considered clinically useful. A national audit would determine if our management is an anomaly or in keeping with standard practice in the UK prompting review of our treatment algorithm. Methods Involved specialities provided details of BD patients in their care. Electronic case records from January 2009 to July 2019 were reviewed. Data were extracted using standard pro-forma classifying patients into 3 groups determined by their most severe manifestation: less severe (predominant musculoskeletal, mucocutaneous); ophthalmic (all eye disease) and severe disease (predominant CNS and vascular disease). Medication was assessed against guidelines for escalation and de-escalation protocols. Results 50 patients (representing approximately 7% of the national BD population) were identified from 4 specialties (Table 1). 23 were female; 45 identified as White British. 26 were regularly reviewed by 2 or more specialties, 4 had discontinued follow up. 26/50 had used antiTNFalpha therapies and 9/50 had received IV cyclophosphamide. Adherence to guidelines was determined by specialty and disease severity (Table 1). The EULAR guideline was followed least well by Rheumatology; low rates of adherence to the national guideline reflected frequent use of methotrexate and hydroxychloroquine in the less severe group and failure to switch to from IV to sub-cutaneous antiTNFalpha therapies. Deviations from national and EULAR guidelines in the ‘severe’ category related to the first line use of IV cyclophosphamide for acute organ/sight threatening disease. Conclusion BD patients are managed equivalently by multiple medical specialties in our Trust. Failure to switch from IV to s/c antiTNFalpha therapies was noted and our use of methotrexate, hydroxychloroquine and cyclophosphamide deviates from guidelines. Whether this effects outcome is unknown it but seems likely to be considered clinically useful. A national audit would determine if our management is an anomaly or in keeping with standard practice in the UK prompting review of our treatment algorithm. Disclosures J. Weightman None. B. Griffiths None. R. Pandit None. C. Stroud None. A.D. Price None. A.R. Lorenzi None.

Background Giant cell arteritis (GCA) is the commonest large-vessel vasculitis. Until recently high dose corticosteroids had been the mainstay of treatment. Significant concern about steroid related co-morbidities have driven interest in developing new treatment strategies, and the potential to reduce costs associated with managing these complications was pivotal in securing funding for tocilizumab. Despite this little is known about the at-risk population, comprising both those diagnosed with GCA and those suspected of GCA, as both groups undergo the same steroid exposure. We therefore sought to determine if patients referred to our GCA fast track pathway (GCA-FTP) were more or less at risk than the general age matched population of developing corticosteroid related side effects. Methods Data from 356 consecutively referred patients to our GCA-FTP between January 1st 2017 and December 31st 2018 were reviewed. Co-morbidity data is routinely collected as part of our GCA-FTP rolling audit. We determined the frequency of pre-existing co-morbidities most often associated with steroid related side effects and compared these with published background prevalence estimates for a similar age group in the UK. Results In general rates of co-morbidities were lower than published estimates in over-70s, as we did not actively pursue their identification beyond what was readily available at referral (Table 1). Interestingly, however, whilst the prevalence of diabetes mellitus in our cohort (n = 356) was comparable to that in the general population (23.6%), it was significantly lower in those diagnosed with GCA (n = 83) at 11% (p = 0.0017). Despite this difference, background rates of cardiovascular and peripheral vascular disease were similar in each group. Conclusion In our cohort, diabetes mellitus seems to be a protective factor against the development of GCA. Similar results were reported in 2015 from a meta-analysis of inception cohorts involving 903 patients with GCA and 1064 controls. The interaction between diabetes and the onset of GCA merits further investigation. Disclosures L. Spray None. G. Reynolds None. J. Heaney None. K. Houghton None. B. Hargreaves None. B. Thompson None. A. Lorenzi None.

A 75-year-old hypertensive female with stable idiopathic intermediate uveitis presented with bilateral sequential optic neuropathy with optic disc swelling. The optic neuropathy in the first affected eye (right) was thought to be due to non-arteritic anterior ischaemic optic neuropathy (NAION). Asymptomatic left optic disc swelling was found at routine review 2 months later, and a diagnosis of giant cell arteritis (GCA) was sought. Temporal artery duplex ultrasound showed the “halo sign,” but a subsequent temporal artery biopsy showed light-chain (AL) amyloidosis with no signs of giant cell arteritis. In this case, bilateral sequential ischaemic optic neuropathy mimicking non-arteritic anterior ischaemic optic neuropathy was the presenting sign of systemic amyloidosis involving the temporal arteries.

Background:Giant cell arteritis (GCA) is an idiopathic vasculitis affecting large and medium-sized vessels. The pattern of arterial involvement is heterogeneous with two overlapping categories recognised: classical cranial GCA and extra-cranial GCA (or large vessel vasculitis – LVV) that predominantly affects the aorta and its proximal branches. Although LVV is present in around 80% of patients with cranial GCA, and around one third will develop large vessel complications, there are no guidelines for which patients should be screened for it (1). We sought to investigate whether clinical and laboratory features were a useful guide to the severity of LVV on FDG PET-CT.Objectives:To retrospectively analyse whether baseline patient characteristics are able to predict the extent of large vessel vasculitis on PET-CT.Methods:Clinical data for 65 patients referred for a PET-CT scan by Rheumatology at the Freeman Hospital, Newcastle between January 2015 and May 2018 were retrospectively analysed. The most recent full blood count and inflammatory markers prior to the scan were used. Scans were reviewed by a consultant radiologist and trainee. The arterial network was split in to ten potentially involved territories (aortic arch, thoracic aorta, abdominal aorta, iliac vessels, axillary, brachiocephalic, subclavian, carotid, vertebral and femoral arteries. Both the value of highest standardised uptake value (SUV max) and the territory affected was recorded for each positive scan.Results:In the period analysed 65 PET-CT scans were requested, mostly (77%) as baseline investigations for symptoms with LVV in the differential diagnosis. Of these 22 (34%) were positive for LVV and in that group the majority of patients (64%) were female. In those with a negative scan, 47.5% were on concurrent steroid treatment compared to 9% with a positive scan. Regression analysis suggested that the number of systemic features (weight loss, pyrexia, polymyalgia) was weakly correlated with the number of affected territories (p=0.04). In contrast there was no correlation between laboratory tests ((CRP (p=0.91), ESR (p=0.46), Hb (p=0.44), platelets (p=0.74)) and the number of territories affected. The aortic arch (47%) was most commonly the territory with the highest degree of FDG uptake (SUV max) followed by the abdominal aorta (21%) and thoracic (10%) and femoral arteries (10%). There was no correlation between SUV max and laboratory tests ((CRP (p=0.55), ESR (p=0.89), Hb (p=0.82), platelets (p=0.17)) or the number of systemic features (p=0.7). There was no significant difference in the number of territories affected between those on steroid treatment at the time of the scan and steroid-naïve patients, albeit the number of positive scans in those on steroid treatment was low (n=5).Conclusion:These results suggest that clinical and laboratory features are a poor guide to predicting the maximal severity and extent of disease on FDG PET-CT.References:[1]Koster MJ, Matteson EL, Warrington KJ. Large-vessel giant cell arteritis: diagnosis, monitoring and management. Rheumatology (Oxford). 2018;57(suppl_2):ii32-ii42.Disclosure of Interests:None declared

Background The recently published UK guidelines for the management of SLE recommend biologic therapy for severe or refractory disease. The British Isles Lupus Assessment Group Biologics Register (BILAG-BR) has shown rituximab (RTX) to be safe, effective and corticosteroid [CS] sparing when used to treat refractory SLE. In 2013 NHS England published an interim clinical commissioning policy statement with criteria determining when RTX can be used to treat SLE. Objectives We evaluated our centre’s RTX retreatment strategy in patients with SLE and the consequent outcomes (disease activity and CS dose). Methods Records for the first 50 patients receiving RTX for refractory SLE who consented to join BILAG-BR from our centre between December 2013 and January 2016 were analysed (data cut off July 2016). Demographics, disease activityBILAG 2004/SLEDAI-2K), change in CS dose, retreatment schedules and adverse events were analysed. Results Median(IQR) age and disease duration were 42.8 (33–53) years and 9.5 (4–15.8) years respectively. Male: female ratio was 1:25. 80% were Caucasian, 6% Asian, 4% Caribbean and 10% other. All patients met SLICC/ACR classification criteria for SLE. The median(IQR) SLEDAI-2K scores and BILAG 2004 scores reduced from 6 (4–8) to 4 (0–4) (p 30/50 patients received their 1 st course of RTX at BILAG-BR baseline visit. 23 met criteria for active disease (at least 1A or 2Bs), 6 were taking an unacceptably high maintenance CS dose, and 1 was planning pregnancy. Median(IQR) CS dose in this group at baseline was 10 mg(1.5–20), and 5 mg(3–9) at 6 months. 70.8% demonstrated complete response at 6 months. 17 (57%) went on to have retreatment due to active disease, of which 11 (64.7%) had responded at 6 months post retreatment. Median(IQR) time to retreatment was 8 (6–12) months. 20/50 patients had received retreatment at predetermined intervals prior to their baseline BILAG-BR visit. Median(IQR) numbers of previous RTX courses were 3 (2–6). Median(IQR) CS dose was 8.75 mg(0–11.3) at baseline BILAG-BR visit and 5.5 mg(0–10) at 6 months. Median(IQR) time from baseline to retreatment was 6 (6–9.5) months. Median(IQR) sustained response was 18 (13.5–18) months. Conclusions Historically, our centre used time from 1 st treatment to flare as a guide to a patient’s future RTX retreatment schedule; patients were, on average, treated 2 months earlier than those treated under the current commissioning policy. Findings suggest that earlier retreatment led to sustained disease control and reduction of CS dose with no increase in adverse events. 11/16 incomplete responders responded following retreatment. Early retreatment may be associated with better outcomes for the patient and further research is needed in this area. Disclosure of Interest None declared

Lymphocyte depleting therapies have been used to treat refractory autoimmune disease, including RA, but treatment may be associated with long-term lymphopenia. It is unclear whether delayed reconstitution preferentially affects lymphocyte subsets, how this modulates immune challenges and whether thymic function influences the outcome. These questions are now addressed in a detailed analysis of RA patients 12 years after alemtuzumab (anti-CD52) treatment.Blood was obtained from 20 RA patients 12 years after alemtuzumab treatment. Lymphocyte subsets were enumerated by flow cytometry. T-cell receptor excision circles (TRECs)/ml were determined to quantify thymic function, and serological responses to neoantigens and recall antigens were assessed.RA patients remained lymphopenic 12 years after their first dose of alemtuzumab. CD5(+) B cells, which may be associated with autoantibody production, were significantly reduced in alemtuzumab-treated patients compared with age-matched disease controls. In addition, naïve and memory CD4(+) T-cell subsets were present in altered proportions in patients who had received alemtuzumab, with increased effector memory CD4(+) T cells, and decreased naïve and central memory CD4(+) T cells. TRECs were detectable in alemtuzumab-treated patients and correlated with CD4(+) lymphocyte counts. Vaccine responses to neoantigens and recall antigens fell within the normal range for an ageing population.Alemtuzumab therapy resulted in long-term alterations in lymphocyte subsets. The significance of these changes remains uncertain but patients respond normally to antigenic challenges. Thymic function remains an important determinant of T-cell reconstitution even several years after lymphocytotoxic therapy.

Genome integrity is continuously challenged by the DNA damage that arises during normal cell metabolism. Biallelic mutations in the genes encoding the genome surveillance enzyme ribonuclease H2 (RNase H2) cause Aicardi-Goutières syndrome (AGS), a pediatric disorder that shares features with the autoimmune disease systemic lupus erythematosus (SLE). Here we determined that heterozygous parents of AGS patients exhibit an intermediate autoimmune phenotype and demonstrated a genetic association between rare RNASEH2 sequence variants and SLE. Evaluation of patient cells revealed that SLE- and AGS-associated mutations impair RNase H2 function and result in accumulation of ribonucleotides in genomic DNA. The ensuing chronic low level of DNA damage triggered a DNA damage response characterized by constitutive p53 phosphorylation and senescence. Patient fibroblasts exhibited constitutive upregulation of IFN-stimulated genes and an enhanced type I IFN response to the immunostimulatory nucleic acid polyinosinic:polycytidylic acid and UV light irradiation, linking RNase H2 deficiency to potentiation of innate immune signaling. Moreover, UV-induced cyclobutane pyrimidine dimer formation was markedly enhanced in ribonucleotide-containing DNA, providing a mechanism for photosensitivity in RNase H2-associated SLE. Collectively, our findings implicate RNase H2 in the pathogenesis of SLE and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity., La lista completa de autores que integran el documento puede consultarse en el archivo, Instituto de Física de Líquidos y Sistemas Biológicos

Objective. To determine whether low-dose ciclosporin was a more effective corticosteroid-sparing agent than AZA in patients with SLE. Methods. Patients with SLE requiring a change or initiation of a corticosteroid-sparing agent and who were taking >/=15 mg of prednisolone/day were randomized to receive either ciclosporin or AZA during this 12-month open-label multi-centre trial. There were strict guidelines for the reduction of prednisolone. The primary outcome was the absolute mean change in prednisolone. Results. Eighty-nine patients were randomized. Using an intention-to-treat analysis, the absolute mean change in prednisolone dose between baseline and 12 months, adjusted for baseline prednisolone dose, was 9.0 mg for ciclosporin (95% CI 7.2, 10.8) and 10.7 mg for AZA (95% CI 8.8, 12.7). The difference in the change between treatment groups was -1.7 mg (95% CI -4.4, 0.9; P = 0.2). No significant differences were detected for the secondary outcomes: change in disease activity [classic British Isles Lupus Assessment Group (BILAG) index], number of flares, development of new damage or change in quality of life. A similar number of patients in each arm stopped the study drugs due to adverse events and ineffectiveness. No patient developed severe hypertension or a persistent rise in creatinine. One patient in the ciclosporin arm developed a significant increase in proteinuria due to disease activity. Conclusions. Both drugs were effective corticosteroid-sparing agents. Ciclosporin was not a more effective corticosteroid-sparing agent. Ciclosporin may be considered in patients who are unable to tolerate AZA. Patients on ciclosporin require close monitoring of blood pressure and creatinine. Trial registration. Current Controlled Trials, http://www.controlled-trials.com/, ISRCTN35919612. (Less)

The authors regret that an error occurred in Section 2.4. Real time quantitative PCR, on the fifth sentence. The corrected sentence is 'Each reaction contained 200 ng DNA.'

Objective: Thymic function declines exponentially with age. Impaired thymic function has been associated with autoimmune disease in adults but has never been formally assessed in childhood autoimmunity. Therefore, thymic function in children with the autoimmune disease juvenile idiopathic arthritis (JIA) was determined. Methods: Thymic function was measured in 70 children and young adults with JIA (age range 2.1–30.8 (median 10.4)) and 110 healthy age-matched controls using four independent assays. T cell receptor excision circles (WBLogTREC/ml) and the proportion of CD4+ CD45RA+CD31+ T cells (representing recent thymic emigrants; %RTEs) were quantified and intrathymic proliferation measured by calculating the αTREC/ΣβTREC ratio. Lastly, regulatory T cells (TReg) of thymic origin (CD4+FOXP3+) were quantified in peripheral blood to assess the ability of the thymus in JIA to generate this T cell subset. Results: Thymic function was equivalent by all four parameters in JIA when compared with the control population. Furthermore, there was no consistent effect of JIA subtype on thymic function, although intrathymic proliferation was higher in the small rheumatoid factor (RF)+ polyarticular group. There were no significant effects of disease-modifying antirheumatic drugs (DMARDs) or oral corticosteroids on thymic function, although those with the worst prognostic ILAR (International League of Associations for Rheumatology) subtypes were also those most likely to be on a DMARD. Conclusions: It is demonstrated that children and young adults with JIA, unlike adults with autoimmune diseases, have thymic function that is comparable with that of healthy controls. The varied pathologies represented by the term “JIA” suggest this observation may not be disease specific and raises interesting questions about the aetiology of thymic impairment in adult autoimmunity.

OBJECTIVE: To assess immunologically relevant outcomes in a cohort of rheumatoid arthritis (RA) patients with prolonged therapy-induced lymphopenia. METHODS: Morbidity (infection or malignancy) and mortality were assessed in 53 RA patients who were treated with the lymphocytotoxic monoclonal antibody alemtuzumab between 1991 and 1994. Data were obtained by interview, medical record review, and Office for National Statistics mortality monitoring. Lymphocyte subsets were enumerated by flow cytometry. A retrospective, matched-cohort study of mortality was performed with 102 control subjects selected from the European League Against Rheumatism database of patients with rheumatic disorders. RESULTS: Lymphopenia persisted in the patients: median CD3+CD4+, CD3+CD8+, CD19+, and CD56+ lymphocyte counts measured at a median followup of 11.8 years from the first administration of alemtuzumab were 0.50 x 10(9)/liter, 0.26 x 10(9)/liter, 0.11 x 10(9)/liter, and 0.09 x 10(9)/liter, respectively. Twenty-seven of 51 cases and 46 of 101 controls with available data had died, yielding a mortality rate ratio of 1.20 (95% confidence interval 0.72-1.98). Causes of death were similar to those that would be expected in a hospital-based RA cohort. No opportunistic infections were noted, and only 3 infections were documented following 36 elective orthopedic procedures. CONCLUSION: Despite continued lymphopenia 11.8 years after therapy, our patient cohort did not exhibit excess mortality or unusual infection-related morbidity, and surgery was well tolerated. These data should be reassuring for clinicians and patients who are considering lymphocytotoxic or other immunomodulatory therapy for RA.

The thymus contributes naïve, self MHC reactive, self tolerant T cells to the peripheral immune system throughout life, albeit with a log-linear decline with age. Quantification of thymic function is clinically relevant in the setting of lymphoablation, but a phenotypic marker distinguishing recent thymic emigrants from long lived naïve T cells remains elusive. T cell receptor excision circles (TREC) are present in thymocytes exiting the thymus and quantification of the most frequent of these, the deltarec-psiJalpha rearrangement has been widely used as a measure of recent thymic function. However, interpretation of results presented as TREC per cell has been criticised on the basis that extra-thymic cellular proliferation impacts on peripherally determined TREC numbers. TREC/ml is now considered to be more representative of thymic function than TREC/cell, especially where significant cellular proliferation occurs (e.g. during reconstitution following stem cell transplantation). Here we describe the validation of a novel variation to the established assay, directly quantifying TREC/ml from 300 microl whole blood. We show the assay to be reproducible, robust and stable longitudinally and we show equivalence of performance when compared with more standard assays. This assay particularly lends itself to the measurement of thymic function in children and where monitoring clinical variables is limited by tissue availability.

In 1998 Douek and colleagues described a novel approach to measuring thymic output using T-cell receptor excision circles (TRECs) measured in CD4+ and CD8+ T cells. TRECs are byproducts of T-cell receptor gene re-arrangements, whereby sections of excised DNA re-form as stable episomes that do not replicate with mitosis. TRECs present in T cells arising from the thymus are thus 'diluted out' upon T-cell division and can be used as a surrogate marker of thymic output. However, the interpretation of TREC number in lymphocyte subsets is confounded by the influences of T-cell proliferation, intracellular TREC degradation and T-cell death, and it has been criticised as a measurement of thymic function. Here, we describe a method for quantifying TRECs in whole blood. This revised assay provides a representation of 'total peripheral TRECs'. The effects of T-cell proliferation and cell death on the determined absolute TREC level are minimised, providing us with an improved estimation of thymic function over time. In a series of validation experiments our assay has been shown to have excellent intra-assay and inter-assay reproducibility and to generate results consistent with previously published work in healthy controls. We are now using this assay to determine thymic function in the autoimmune disease systemic lupus erythematosus to test our hypothesis that a defect in thymic function contributes to lupus pathogenesis. To measure the absolute TREC number per unit volume of blood (as a representation of 'total peripheral TRECs'), the amount of DNA extracted from a volume of whole blood must be representative of 'total' DNA in that volume of blood. We have compared four methods of DNA extraction from whole blood for consistency of yield and purity. Figure ​Figure1a1a shows the mean DNA yield from parallel extractions (n = 10) performed after (a) 1 hour, (b) 6 hours and (c) 24 hours from collection for a single sample. The overall mean yield was 32.23 μg/ml ± 2.3 standard deviations (SDs). Two further, separate, samples were similarly prepared in parallel to confirm consistency – (d) 26.8 μg ± 1.97 SD and (e) 26.9 μg ± 2.23 SD (n = 10). To measure TRECs in samples we have used a quantitative real-time PCR (TAQMAN™) assay to amplify a known proportion of the extracted DNA. The total TREC number per sample is determined absolutely from a standard curve (derived from a plasmid containing TREC sequence) and final values are presented as TRECs per millilitre of whole blood. Figure ​Figure1a1a (top axis) shows that the TREC number is reproducibly calculated from parallel extractions (n = 5). The intra-assay coefficient of variation is 1.4% (range 0.8–2.5%) while the inter-assay co-efficient of variation is 2.4% (range 0.6–4.4%). The coefficient of variation is negatively correlated with TREC number (r2 = 0.83 and 0.66, respectively). Using this assay we have also shown that TRECs are reliably detectable in healthy controls (Fig. ​(Fig.1b)1b) and show an age-related decline in keeping with the known reduction in thymic volume over time. TRECs are not detectable in RAJI (human B-cell lymphoma line) or SW48 (human colonic carcinoma cell line) (data not shown). We are now using this assay to determine the TREC number in the autoimmune disease systemic lupus erythematosus to test our hypothesis that a defect in thymic function contributes to lupus pathogenesis. Figure 1 (a) Lower axis: serial DNA extractions (see text). Bars represent mean of 10 ± standard deviation. Upper axis: T-cell receptor excision circles (TRECs) per millitre of blood from the same samples (mean ± deviation). (b) TRECs per millitre ... While measurement of TRECs in T cells and their subsets provides a representation of thymic function in the steady state, in the context of autoimmunity, where abundant auto-antigen provides a constant T-cell-activating stimulus, interpretation of the TREC number within lymphocyte subsets is confounded. By reliably and reproducibly estimating total peripheral TRECs we aim to minimise the impact of T-cell turnover on TREC number in order to gain a better understanding of thymic function in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease in which environmental exposures like virus infection and UV-irradiation trigger activation of the innate and adaptive immune system in genetically predisposed individuals. Heterozygous mutations of the 3' repair exonuclease 1 (TREX1) are associated with SLE. Biallelic mutations in TREX1 and the three subunits of ribonuclease H2 (RNASEH2A-C) cause Aicardi-Goutieres syndrome, an inflammatory encephalopathy with clinical overlap with SLE. We therefore investigated the role of RNase H2 in SLE pathogenesis. RNase H2 is responsible for the removal of misincorporated ribonucleotides from DNA and is indispensable for genome integrity. We demonstrated a genetic association for rare RNase H2 sequence variants with SLE. RNase H2-deficient fibroblasts of AGS and SLE patients accumulated ribonucleotides in genomic DNA resulting in chronic low-level DNA damage, constitutive p53 phosphorylation and senescence. Patient fibroblasts proliferated slower than fibroblasts from healthy individuals and showed impairment of cell cycle progression. In addition, patient fibroblasts exhibited constitutive up-regulation of interferon-stimulated genes and an enhanced type I interferon response to the nucleic acid poly(I:C) and UV-irradiation. UV-irradiation induced enhanced cyclobutane pyrimidine dimer formation in ribonucleotide-containing DNA. This suggests that innate immune activation may be caused by immune recognition of DNA metabolites of DNA damage repair and may also explain photosensitivity in SLE patients with RNase H2 mutation. In summary, our findings implicate RNase H2 in the pathogenesis of SLE, and suggest a role of DNA damage-associated pathways in the initiation of autoimmunity.

Introduction Analyses of large clinical datasets from early arthritis cohorts permit the development of algorithms that may be used for outcome prediction in individual patients. The value added by routine use of musculoskeletal ultrasound (MSUS) in an early arthritis setting, as a component of such predictive algorithms, remains to be determined. Methods The authors undertook a retrospective analysis of a large, true-to-life, observational inception cohort of early arthritis patients in Newcastle upon Tyne, UK, which included patients with inflammatory arthralgia but no clinically swollen joints. A pragmatic, 10-minute MSUS assessment protocol was developed, and applied to each of these patients at baseline. Logistic regression was used to develop two "risk metrics" that predicted the development of a persistent inflammatory arthritis (PIA), with or without the inclusion of MSUS parameters. Results A total of 379 enrolled patients were assigned definitive diagnoses after ≥12 months follow-up (median 28 months), of whom 162 (42%) developed a persistent inflammatory arthritis. A risk metric derived from 12 baseline clinical and serological parameters alone had an excellent discriminatory utility with respect to an outcome of PIA (area under receiver operator characteristic (ROC) curve 0.91; 95% CI 0.88 to 0.94). The discriminatory utility of a similar metric, which incorporated MSUS parameters, was not significantly superior (area under ROC curve 0.91; 95% CI 0.89 to 0.94). Neither did this approach identify an added value of MSUS over the use of routine clinical parameters in an algorithm for discriminating PIA patients whose outcome diagnosis was rheumatoid arthritis (RA). Conclusions MSUS use as a routine component of assessment in an early arthritis clinic did not add substantial discriminatory value to a risk metric for predicting PIA.