Lorenzo Emmi

Behçet's syndrome patients exhibit specific microbiome signature

BACKGROUND AND AIMS Behçet syndrome is a systemic inflammatory condition characterized by muco-cutaneous and ocular manifestations, with central nervous system, vascular and/or gastro-intestinal involvement. The association of microbiota with Behçet syndrome has not been shown yet. Our work was aimed to compare the gut microbiota structure and the profiles of short-chain fatty acids production in Behçet syndrome patients and healthy control relatives. METHODS Here, we compared the fecal microbiota of 22 patients with Behçet syndrome and that of 16 healthy co-habiting controls, sharing the same diet and lifestyle by pyrosequencing of the V3-V4 hypervariable regions of the 16 rDNA gene and biochemical analyses. RESULTS Our analyses showed significant differences in gut microbiota between Behçet patients and healthy cohabitants. In particular we found that Behçets patients were significantly depleted in the genera Roseburia and Subdoligranulum. Roseburia showed a relative abundance value of 10.45±6.01% in healthy relatives and 4.97±5.09% in Behçets patients, and Subdoligranulum, which reached a relative abundance of 3.28±2.20% in healthy controls, was only at 1.93±1.75% of abundance in Behçets patients. Here we report, for the first time, that a peculiar dysbiosis of the gut microbiota is present in patients with Behçet syndrome and this corresponds to specific changes in microbiome profile. A significant decrease of butyrate production (P=0.0033) in Behçets patients was demonstrated. Butyrate is able to promote differentiation of T-regulatory cells, and consequently the results obtained prompt us to speculate that a defect of butyrate production might lead to both reduced T-reg responses and activation of immuno-pathological T-effector responses. CONCLUSIONS Altogether, our results indicate that both a peculiar dysbiosis of the gut microbiota and a significant decrease of butyrate production are present in patients with Behçet syndrome.

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.

Possible Pathogenetic Role of Activated Platelets in the Primary Antiphospholipid Syndrome Involving the Central Nervous System

Neurological disorders occurring in the primary antiphospholipid syndrome (neuro-PAPS) have not yet been completely understood. Platelet activation has been suggested to play a crucial role in the pathogenesis of hemostatic disorders in the antiphospholipid syndrome, but no association with neuro-PAPS has been investigated so far. Therefore, we investigated 16 patients with PAPS by flow cytometry in the presence of circulating activated platelets as defined by the surface expression of activation-dependent glycoprotein CD62. In addition, the relationship among activated platelets and anticardiolipin antibodies (aCL) was evaluated. Compared to normal subjects CD62 was found significantly increased in these patients. Furthermore, a significantly increased percentage of CD62-positive platelets was found in the neuro-PAPS group (nine patients) compared to the non-neuro-PAPS patients (seven subjects). On the contrary, no significant difference was found between the two groups with regard to aCL IgG and platelet number. Furthermore, within the neuro-PAPS group, no difference was evidenced, in the CD62-positive platelet percentage, between the four subjects with thrombocytopenia and the five with the normal blood platelet count. Similarly, neuro-PAPS subjects with previous peripheral arterial and/or venous thrombosis did not show a significantly more elevated level of CD62-positive platelets. Finally, a linear correlation was found between the aCL IgG level and the CD62-positive platelet percentage in all the patients and, more significantly, in the neuro-PAPS group, but not within the non-neuro-PAPS patients. Our data demonstrate that circulating activated platelets are detectable by flow cytometry in the majority of PAPS patients and suggest the existence of a relationship among activated platelets, aCL, and neurological disease that patients affected by PAPS might undergo.

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.