Richard Stratton

Proteomic analysis of scleroderma lesional skin reveals activated wound healing phenotype of epidermal cell layer

OBJECTIVE To identify using proteomic analysis, proteins of altered abundance in the skin of patients with SSc. METHODS 4 mm excision biopsies were obtained from the forearm involved skin of 12 diffuse SSc patients and 12 healthy controls. Two-dimensional gel electrophoresis was used to separate and define proteins in normal and SSc skin biopsy material. Proteins of altered abundance in the disease were formally identified by mass spectroscopy. Abnormalities of the epidermis were confirmed by immunohistochemistry. RESULTS Proteomic analysis revealed altered abundance of proteins involved in extracellular matrix production, myofibroblast contractility, energy metabolism and response to oxidative stress. In addition, proteins specific to the epidermis and involved in epidermal cell differentiation were altered in abundance in the disease. SSc epidermis is thickened, has an expanded nucleated cell layer, and exhibits abnormal persistence of basal marker keratin 14, delayed expression of maturation markers keratin 1/10 and the induction of keratins 6 and 16, normally absent from interfollicular skin and induced following epidermal injury. These changes closely resemble the activated phenotype seen during wound healing. CONCLUSIONS Consistent with previous models of SSc pathogenesis these data are showing increased contractility, increased extracellular matrix and response to oxidative stress in the involved skin of recent onset SSc patients. In addition, we show that SSc epidermis has an activated, wound healing phenotype. These findings are important because epidermal cells activated by injury induce and regulate local fibroblasts during wound repair.

Palmar fasciitis and polyarthritis syndrome: a sign of ovarian malignancy

Summary Highlighting the importance and recognition of palmar fasciitis and polyarthritis syndrome, a paraneoplastic syndrome related to ovarian and other malignancies.

Systemic vasculitis with multiple aneurysms complicating systemic lupus erythematosus.

In elderly diabetic patients with renal cholesterol atheroembolism, a common precipitant is manipulation of atherosclerotic vessels during vascular surgery or angiography. It can also result from thrombolytic and anticoagulant therapy, sometimes after a considerable delay1. Spontaneous cholesterol embolism is uncommon-found by Cross2 in 1.9% of serial necropsies, always in patients over 60 years of age. The clinical features are varied and make diagnosis difficult. Risk factors for renal cholesterol atheroembolism are advanced age (mean 66 years), hypertension, coronary atherosclerosis and renal impairment3. Spontaneous renal atheroembolism often leads to progressive decline in renal function, early dialysisdependence and high mortality3; however, renal function can recover4. Flash pulmonary oedema often points to underlying atheromatous renal artery occlusion, and we suspect that this was present in our patient. Regarding treatment, there is some evidence that a statin can stabilize atherosclerotic plaques, reduce the propensity for atheroembolism and thus preserve renal function5. Recurrent spontaneous cholesterol atheroembolism, characterized here by short-lived episodes of acute renal failure and pulmonary oedema, does not seem to have been described previously. This possibility should be considered in any elderly diabetic patient with established atherosclerotic disease who presents with impaired renal function and pulmonary oedema.

Wegener's granulomatosis with multiple pulmonary nodules – diagnostic difficulties

We describe the case of a 65-year-old woman with multiple pulmonary nodules on a chest radiograph where subsequent investigations led to diagnostic difficulties.

Commentary on a recent article—“A prostacyclin analogue, Iloprost, protects from bleomycin-induced fibrosis in mice” Zhu Y et al. Respir Res. 2010 Mar 20;11(1):34

Data from our laboratory show that in vitro fibroblasts are exquisitely responsive to prostacyclin and the prostacyclin derivative Iloprost, which block their activation by TGFβ. A recent article by Zhu Y et al confirm these effects in vivo showing that Iloprost, given as a single intraperitoneal injection, blocks lung fibrosis in the bleomycin model of lung injury and fibrosis. These results are important because at present no effective clinical treatments are available to treat idiopathic lung fibrosis, which progresses and leads to respiratory failure. Limiting factors for the clinical use of prostacyclin derivatives as anti-fibrotics are failure to achieve therapeutic levels in the involved fibrotic tissues, and dose limiting side effects due to vasodilatation and binding to the IP receptor on vascular cells. Possible approaches include fibroblast directed gene therapies or amelioration of the vascular side effects.

OP0048 IL-31 Is An Inflammatory Pro-Fibrotic Factor Elevated in A Subset of Scleroderma Patients with Severe Pruritus

Background Systemic sclerosis (SSc) is an autoimmune rheumatic disease associated with fibroblast activation in the skin and visceral organs. In SSc, refractory pruritus is a common symptom in a subgroup of patients. IL-31 is a Th2 cell derived cytokine implicated in severe itch in other conditions including atopic dermatitis and T cell lymphoma. T-lymphocyte-derived factors provide a possible link between autoimmune inflammation and fibrosis reported in SSc. In this study we measured IL-31 levels in SSc tissue fluid and plasma, and sought correlation with itch severity and clinical parameters. Recombinant IL-31 was found to influence fibroblast and fibroblast precursor activity. Methods IL-31 was measured by ELISA of dermal tissue fluid samples obtained by a suction blister analysis method from the involved skin of SSc patients or matched site of healthy controls (SSc n=28, controls n=15), and matched plasma samples. IL-31 receptor mRNA expression was assayed by qPCR of SSc and control fibroblast lysates, as well as epithelial tissue biopsy samples. Normal skin fibroblasts as well as fat derived mesenchymal stem cells (MSCs) were treated with IL-31 (50 ng/ml) and migratory and fibrotic responses were assayed. Results IL-31 levels were increased in dermal interstitial fluid in SSc patients compared to controls (mean of 99.4 pg/ml vs 2.3 pg/ml in controls, P<0.0003) and in plasma samples (mean plasma IL-31 1370 vs 196 pg/ml, P<0.01). Dermal fluid IL-31 levels correlated strongly with itch severity in these patients R=0.72, P<0.0038). Raised blister fluid IL-31 was characteristic of a subgroup of SSc patients with severe pruritus, which included individuals from both limited and diffuse SSc clinical subsets. Both normal and disease fibroblast extracts contained IL-31 receptor mRNA (qPCR relative copy number 6.4 SSc vs 1.4 controls, p<0.01) which was also seen in epidermal tissue extracts (qPCR 18.4 SSc vs 8.2 in control, p NS). Treatment of normal dermal fibroblasts with IL-31 led to induction of type I collagen but not CTGF, and promoted fibroblast and MSC migration dependent on ERK and PI3kinase pathways. Conclusions This is the first analysis of IL-31 in systemic sclerosis and we have shown increased expression in the skin and plasma of a subgroup of SSc patients, correlating with severe itch. IL-31 protein induced fibroblasts and MSCs and may link T-cell autoimmune responses to fibroblast and precursor cell activation in SSc. Blocking IL-31 therapeutically may provide effective treatment in a subgroup of SSc patients identified clinically by severe pruritus. Disclosure of Interest None declared

14. A great mimicking vasculitis

Introduction: We report a case of a seemingly typical presentation of giant cell arteritis (GCA) with lack of response to steroid therapy which was, in fact, foundtobeduetounderlying infectionwithsyphiliscausinga clinical syndrome with symptoms which mimicked GCA. Treatment of the infection ledtocompleteresolutionofthesymptoms.Whilstsyphilis is knowntocauseawiderangeofmulti-systemicsymptoms, it isnotusually considered in the different diagnosis in patients presenting with symptomssuggestiveofGCA,asevidencedbythescarcityofpreviouslydocumentedcases. Case description: A 64 year old male was referred to the infectious disease teambyhisGPwith aneight-weekhistoryofdrenching, intermittent night sweats, weight loss and low-grade raised inflammatory markers (CRP 37 mg/L, ESR 44 mm/hr). Screening for HIV and viral hepatitis was negative and there was no personal or contact history of TB nor any evidence of infectious or tropical disease. Autoantibody screen had been performed and ANA was found to be weakly positive at 1/100 but ENA, rheumatoid factor, anti-CCP and ANCA were all negative. CT of chest, abdomen and pelvis was normal however the patient subsequently reported a new-onset generalised headache. Inflammatory markers remained raised. On further detailed questioning, the patient reported some recent aching of the proximal upper limbs. He was hence referred for rheumatology opinion.Threeweeks later, thepatientpresented to the emergencydepartmentwithsevereunilateral temporalheadache,asensation of burning of the scalp, hair loss and some visual blurring. Examination revealed a tender, thickened and hyperaesthetic temporal artery. Inflammatory markers remained elevated with ESR 32 mm/hr and CRP 20 mg/L. The impression was of PMR evolving into GCA and the patientwascommencedon60 mgdailyofprednisolone. Thepatientwas reviewed six days later and reported an improvement of his symptoms and a plan was made for weaning of the steroids. Temporal artery biopsy wasperformedandshowednoevidenceof temporalarteritis.Onemonth later, the patient requested an urgent review due to a relapse of all of his symptoms. On this occasion, the patient was particularly concerned about worsening alopecia and more severe bilateral shoulder aching. Inflammatory markers remained at similar levels to those previous. His prednisolone was increased again and, it was thought his steroids had beenweanedtoorapidly.Hewasalsoreferredtodermatologyforanopinion regarding his hair loss. During the same period, the patient’s wife was referred to the genitourinaryclinic for infective symptoms and the patient was subsequently requested for testing as a potential contact. It was noted in the sexual health clinic that the patient had additionally developed a mildly pruritic, faint, papular rash on both palms. Treponema pallidumparticleassay (TPPA)waspositivewitharapidplasmaregain (RPR) titre of 1: 32. Secondary syphilis was confirmed and the patient was subsequently treated with intramuscular penicillin-G. Within two weeks, the patient reported rapid resolution of all his symptoms including the rash, hair loss,headache,scalptendernessandshoulderpain.CRPwasmeasured at 4 mg/L and ESR was 14 mm/hr. At the following clinic review, the patient had regained weight and steroids were tapered. The patient remained well at subsequent follow up (CRP<1,ESR 2) andcontinued to remainasymptomaticonnosteroid therapy.Oneyearon,heremainswell withnorecurrenceofhissymptoms. Discussion: Syphilis has long been known as the great mimicker for its ability topresent inawide rangeofsymptomsaffecting multiplesystems. Secondary syphilis represents haematogenous dissemination from the initial site of infection (as sometimes represented by a primary chancre) andmostcommonlypresentswithskinrashes,alopeciaandlymphadenopathy. Late or tertiary syphilis often occurs two or more years after acquisition and causes gumma formation, aortitis and neurological involvement such as focal arteritis causing infarction and meningeal inflammation.Thiscase representsa rarecaseofsecondarysyphilispresenting initially with symptoms highly suggestive of classic giant cell arteritis. Only two previous such cases have been reported in the literature. Such was the clear constellation of symptoms, the patient was treated with high dose oral steroids as is common practice but tellingly failed to respond accordingly and, indeed, subsequently experienced a worseningofsymptoms.Theonly initialclueregardingtheatypicalnature of this case, prior to the later development of the palmar rash, was the prominent alopecia which is described in secondary syphilis. Tertiary neurosyphilis with meningeal involvement is rare but would present with additional neurological symptoms such a seizure or cranial nerve palsy. The mechanism behind secondary infection causing headache and a mimicking of giant cell arteritis is unclear and a negative temporal artery biopsy would not include or exclude the condition regardless. Whilst an extremely rare manifestation, this case is interesting in being a reminder that other systemic infections can often present similarly to inflammatory conditionsseenwithin rheumatology.Secondarysyphilis shouldbeconsideredwithin thedifferentialdiagnosisofpatientspresentingwithsymptoms suggestive of GCA, especially in atypical cases or when there is a lackofresponsetosteroidtherapy. Key Learning Points: Include multi-systemic infection within the differential for patients presenting with symptoms consistent with inflammatoryoforconnective tissuedisease.Excludeothercauseswhengiantcell arteritis remainsunresponsivetoconventionaltreatment. Disclosure: S. Black: None. C. Raine: None. D. Ivens: None. R. Stratton:None.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Advances in Vascular Medicine

Scleroderma is a severe systemic disorder affecting 240 per million of population, characterized by fibrosis of the skin and internal organs, by autoimmune phenomena, and by vascular injury (for review, see1). Scleroderma patients form a heterogeneous group with a very wide spectrum of disease severity. In the most mildly affected individuals, Raynaud’s phenomenon and barely noticeable skin thickening may be the only manifestations of the disease. At the opposite end of the disease spectrum are patients with total skin encasement and life-threatening pulmonary, cardiac, or renal complications.2