Danilo Squatrito

Behçet’s syndrome pathophysiology and potential therapeutic targets

Behçet syndrome is a systemic inflammatory disorder characterized by multiorgan involvement such as oral and genital ulcers, uveitis, skin lesions as well as by less frequent, but often more severe, central nervous system and vascular manifestations. The pathogenetic mechanisms are still incompletely known; however the interaction between a specific genetic background and environmental or infectious factors certainly contributes to the immune dysregulation that characterizes this disease. The discovery of new immunological pathways in Behçet syndrome pathogenesis may help us to set up new treatments. In this review, we will focus our attention on the possible mechanisms underlying Behçet syndrome pathogenesis and their potential role as novel therapeutic targets.

Neutrophil Activation Promotes Fibrinogen Oxidation and Thrombus Formation in Behçet Disease.

Background— Behçet disease (BD) is a systemic vasculitis with a broad range of organ involvement, characterized by a multisystemic, immune-inflammatory disorder involving vessels of all sizes and often complicated by thrombosis. Systemic redox imbalance and circulating neutrophil hyperactivation have been observed in BD patients and are thought to be responsible for impaired coagulation. We here focused on the pathogenetic mechanisms potentially linking immune cell activation and thrombosis, and specifically examined whether neutrophil activation can affect fibrinogen modifications and consequently elicit thrombosis. Methods and Results— Blood samples were collected from 98 consecutive BD patients attending our dedicated Center and from 70 age- and sex-matched healthy controls; in all patients fibrinogen function and structure, fibrin susceptibility to plasmin-lysis, plasma redox status, leukocyte oxidative stress markers, and possible reactive oxygen species sources were examined. Thrombin-catalyzed fibrin formation and fibrin susceptibility to plasmin-induced lysis were significantly impaired in BD patients (P<0.001). These findings were associated with increased plasma oxidative stress markers (P<0.001) and with a marked carbonylation of fibrinogen (P<0.001), whose secondary structure appeared deeply modified. Neutrophils displayed an enhanced NADPH oxidase activity and increased reactive oxygen species production (P<0.001), which significantly correlated with fibrinogen carbonylation level (r2=0.33, P<0.0001), residual &bgr;-band intensity (r2=0.07, P<0.01), and fibrinogen clotting ability (r2=0.073, P<0.01) Conclusions— In BD patients, altered fibrinogen structure and impaired fibrinogen function are associated with neutrophil activation and enhanced reactive oxygen species production whose primary source is represented by neutrophil NADPH oxidase.

Thrombosis in vasculitis: from pathogenesis to treatment

In recent years, the relationship between inflammation and thrombosis has been deeply investigated and it is now clear that immune and coagulation systems are functionally interconnected.Inflammation-induced thrombosis is by now considered a feature not only of autoimmune rheumatic diseases, but also of systemic vasculitides such as Behçet’s syndrome, ANCA-associated vasculitis or giant cells arteritis, especially during active disease.These findings have important consequences in terms of management and treatment. Indeed, Behçet’syndrome requires immunosuppressive agents for vascular involvement rather than anticoagulation or antiplatelet therapy, and it is conceivable that also in ANCA-associated vasculitis or large vessel-vasculitis an aggressive anti-inflammatory treatment during active disease could reduce the risk of thrombotic events in early stages.In this review we discuss thrombosis in vasculitides, especially in Behçet’s syndrome, ANCA-associated vasculitis and large-vessel vasculitis, and provide pathogenetic and clinical clues for the different specialists involved in the care of these patients.

Pathogenesis and potential therapeutic targets in systemic lupus erythematosus: from bench to bedside

Systemic lupus erythematosus (SLE) is considered an autoimmune disease with multiorgan involvement. Many advances have been made during the last decade regarding inflammatory pathways, genetic and epigenetic alterations, adaptive and innate immune system mechanisms specifically involved in SLE pathogenesis. Apoptosis has been proposed as an important player in SLE pathogenesis more than a decade ago. However, only recently new key apoptotic pathways have been investigated and the link between apoptotic debris containing autoantigens, innate immunity and ongoing inflammation has been further elucidated. Better understanding of cellular mechanisms and involved cytokines contributed to the development of new biological drugs specifically addressed for SLE therapy.

Microparticles: Bridging the Gap between Autoimmunity and Thrombosis.

Microparticles (MPs) are irregularly shaped small vesicles of heterogeneous size released from the plasma membrane in a tightly controlled process, after different stimuli. MPs have been associated with proinflammatory effects and also with autoimmune processes, being a source of autoantigenic nuclear material, which can form immune complexes. In addition, recent reports have linked a large number of autoimmune disorders to an increased risk of thrombosis, and MPs seem to promote the potential for thrombotic events. A growing mass of evidence supports the idea that MPs could contribute to the generation of an inflammation-induced hypercoagulability state, having a relevant role in the pathogenesis of the thrombotic phenomena associated to autoimmune disease, such as systemic lupus erythematosus, antiphospholipid antibody syndrome, and systemic vasculitis. In this review, we focus on the procoagulant properties of circulating MPs and analyze their contribution to the pathogenesis of autoimmune diseases.

Infliximab and Cogan's syndrome.

Sir, Cogan’s syndrome (CS) is a rare inflammatory disease, with a typical form (TCS) consisting of non-syphilitic interstitial keratitis, hearing loss, and vestibular impairment and an atypical one (ACS) with different ocular lesions, variable vestibular-auditory impairment and systemic inflammation. Hearing loss is usually progressive and bilateral and leads to deafness in 37–67% of the patients. High dose steroids and immunosuppressive therapy treat the first phases, but the disease tends to recur. We describe the effectiveness of infliximab in the treatment of three cases of Cogan’s syndrome and emphasise the importance of an early treatment to prevent irreversible damages. A 30-year-old woman with acute atypical Cogan’s syndrome was treated with methylprednisolone and cyclophosphamide, but a bilateral sensorineural hearing loss and a vestibular hypofunction persisted. Therefore, infliximab was administrated at 3 mg ⁄ kg at weeks 0, 2, 6, and 8, then every 8 weeks in association with methotrexate, giving rapid improvement (Fig. 1a). The treatment was suspended during a pregnancy, completed without complications. A new Cogan’s syndrome flare was treated with infliximab with good disease control in a 5 years follow-up. A 29-year-old man developed an interstitial keratitis and symptoms resembling Meniere disease, with a bilateral, sensorineural hearing loss. After treatment with betamethasone, the patient showed a clinical worsening. Due to a diagnosis of typical Cogan’s syndrome the patient was treated with traditional immunosuppressive therapy, without stable improvement. Therefore, infliximab was administered following international protocol, giving a complete resolution of ocular symptoms and a hearing improvement (Fig. 1b). A 35-year-old man developed atypical Cogan’s syndrome characterised by left hearing loss and anterior scleritis. Despite a pulse therapy of cyclophosphamide and Fig. 2. Endoscopic view of the nasal pack in situ. C O R R E S P O N D E N C E : L E T T E R S Correspondence 441

A new cytofluorimetric approach to evaluate the circulating microparticles in subjects with antiphospholipid antibodies

INTRODUCTION Growing evidence supports the idea that microparticles (MPs) could contribute to the pathogenesis of the thrombotic phenomena associated with antiphospholipid antibody syndrome (APS), inducing a hypercoagulable state. But, to date, different approaches to evaluate circulating MPs and conflicting results have been reported. MATERIALS AND METHODS We have characterized the different circulating subpopulations of MPs in APS patients, and in asymptomatic aPL-positive subjects (carriers) by examining the correlation between the amount and phenotype of MPs and the clinical parameters. Forty-eight subjects were enrolled: 16 with primary APS, 16 aPL-positive, but without clinical criteria for APS (carriers), and 16 healthy subjects. The levels of MPs were evaluated using a new cytofluorimetric approach based on BD Horizon Violet Proliferation dye (VPD) 450. RESULTS AND CONCLUSIONS Using a new detection cytofluorimetric approach, we demonstrated that the AnnV-negative MPs, underestimated/or excluded in the previous studies, are a large subset of circulating MPs. Also, the levels of MPs in the plasma of aPL positive subjects indicate a state of cellular activation, which is much more pronounced in patients with APS compared to aPL carriers. Moreover, the preliminary data of our pilot study suggest that the evaluation of circulating MPs, in particular PMPs and EMPs, could be used as a surrogate biomarker for platelet and vascular damage monitoring and, if confirmed in a more numerous cohort of patients, it could be used as a prognostic factor to identify aPL positive subjects at higher risk of developing thrombosis.

An Approach to Differential Diagnosis of Antiphospholipid Antibody Syndrome and Related Conditions

The antiphospholipid antibody syndrome is a systemic, acquired, immune-mediated disorder characterized by episodes of venous, arterial, or microcirculation thrombosis and/or pregnancy abnormalities, associated with the persistent presence of autoantibodies, confirmed at least in two occasions 12 weeks apart, directed to molecular complexes consisting of phospholipids and proteins. Antiphospholipid antibody syndrome should always be considered as a potential diagnosis especially for young patients presenting with a history of thrombotic events, in particular when they occur without any obvious external trigger or any inherited thrombophilic mutation (even if 2006 criteria do not exclude antiphospholipid antibody syndrome in patients with other inherited or acquired prothrombotic conditions), or for women with recurrent pregnancy losses or later fetal deaths. Many other disorders are able to mimic antiphospholipid antibody syndrome, so a broad range of alternative diagnoses should be investigated and ruled out during clinical workup.

Off-label use of rituximab for systemic lupus erythematosus in Europe.

Objectives Rituximab (RTX) is a biological treatment used off-label in patients with systemic lupus erythematosus (SLE). This survey aimed to investigate the off-label use of RTX in Europe and compare the characteristics of patients receiving RTX with those receiving conventional therapy. Methods Data on patients with SLE receiving RTX were taken from the International Registry for Biologics in SLE retrospective registry and complemented with data on patients with SLE treated with conventional therapy. For nationwide estimates of RTX use in patients with SLE, investigators were asked to provide data through case report forms (CRFs). Countries for which no data were submitted through CRFs, published literature and/or personal communication were used, and for European countries where no data were available, estimates were made on the assumption of similarities with neighbouring countries. Results The estimated off-label use of RTX in Europe was 0.5%–1.5% of all patients with SLE. In comparison with patients with SLE on conventional therapy, patients treated with RTX had longer disease duration, higher disease activity and were more often treated with immunosuppressives. The most frequent organ manifestations for which either RTX or conventional therapy was initiated were lupus nephritis followed by musculoskeletal and haematological. The reason for treatment was, besides disease control, corticosteroid-sparing for patients treated with conventional therapy. Conclusions RTX use for SLE in Europe is restrictive and appears to be used as a last resort in patients for whom other reasonable options have been exhausted.

Successful Treatment of Ocular Sarcoidosis with Rituximab

Sarcoidosis is a multisystem inflammatory disease of undetermined origin. Thoracic involvement is the most common, with lymphoadenopathy and a various degree of lung lesions, such as granulomatous nodules and diffuse interstitial thickening, potentially leading to lung fibrosis. Skin, joints, heart, brain, and eye, however, can also be involved with a wide spectrum of clinical manifestations. Ocular sarcoidosis occurs approximately in one-third of patients, also in apparent absence of disease manifestations in other locations and can potentially involve any element of the eye. Among ocular lesions, however, some are more suggestive of sarcoidosis, such as bilateral granulomatous uveitis. Despite the recent advances in the development of new therapeutic agents, sarcoidosis remains a great therapeutic challenge, because of the current lack of data on the efficacy of the immunosuppressive and biological treatments. A 50-year-old woman was referred to our Center for relapsing uveitis. She had been facing recurrent severe uveitis involving both the eyes for 3 years, with poor and transitory responses to topical treatments and oral steroid therapy. Uveitis recurred regularly shortly after steroid withdrawal. A complete ophthalmic evaluation revealed a bilateral granulomatous panuveitis characterized by anterior chamber Tyndall, keratic precipitates, synechiae, vitreous opacity 3þ in RE and 1þ in LE, and cystoid macular edema OU. Best-corrected visual acuity (BCVA) was 0.70 logMAR RE and 0.20 logMAR LE (Figure 1). A systemic disease was suspected because of the concomitant presence of multiple, raised, erythematous lesions on the anterior surface of the lower legs, identifiable as erythema nodosum, and neurological symptoms, consisting of left arm paresthesias. A brain nuclear magnetic resonance (NMR) was performed, revealing the presence of multiple, nonenhancing lesions of the white matter, compatible with neurosarcoidosis. Full blood count, metabolic panel, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), and the other laboratory tests gave results within normal limits. Antinuclear antibodies (ANA), anti-dsDNA, anti-extractable nuclear antigens (ENA), anti-neutrophil cytoplasm antibodies (ANCA), anti-phospholipid antibodies (aPL), and lupus anticoagulant (LAC) tests were also negative. Angiotensin converting enzyme (ACE), however, was significantly increased (106 U/L). A high-resolution CT of the lung with contrast revealed multiple nodular bilateral lung lesions with mild hilar and mediastinal lymphoadenopathies. To confirm the diagnostic hypothesis we also performed a scintigraphy that revealed abnormal gallium uptake by both parotid glands and lungs. The patient did not consent to a bronchoscopy, but the lung involvement was clinically mild, so the predictive value of such a procedure was low. We started topical therapy (dexamethasone 2% and tropicamide 1%) and oral prednisone