Cinzia Scambi

Neutrophil Functions and IL-8 in Psoriatic Arthritis and in Cutaneous Psoriasis

The aim of this study is to determine some functions of neutrophil in patients affected by psoriatic arthritis and to compare them to those of patients affected by cutaneous psoriasis and to normal controls. We used a model of experimental cutaneous inflammation allowing to separate a cluster of purified and viable PMN cells. Then we analyzed, within the three groups, the IL-8 concentration in serum and in the supernatant obtained from the inflammatory site to gather data on the possible pathogenic role played by this cytokine in psoriatic arthritis. We studied neutrophil functions in patients with cutaneous psoriasis and psoriatic arthritis, in acute phase, in comparison with healthy control subjects. We investigated in vivo neutrophil migration by Senns skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. We also measured IL-8 concentration in serum and in the supernatant obtained from the inflammatory site, artificially created through the skin window scrape. Neutrophil migration in vivo was significantly higher in both groups of patients than in controls. In the presence of fMLP, blood cells showed a burst of superoxide release, which was significantly more pronounced in patients when compared to healthy controls. Neutrophils from skin window scrape showed a much higher response to fMLP as compared to blood cells of all subject groups, but no differences were observed between patients and controls. No correlation was found between the three groups in adhesion ability under basal condition or in response to different stimuli by circulating and migrating neutrophils. Our results also show a great increase of IL-8 in the exudate from patients compared to controls. Our study shows that there is no difference in neutrophil functions between patients with psoriatic arthritis and cutaneous psoriasis; moreover we suggest that the source of high IL-8 levels are neutrophils rather than the keratinocytes.

Comparative Proteomic Analysis of Serum from Patients with Systemic Sclerosis and Sclerodermatous GVHD. Evidence of Defective Function of Factor H

Background Systemic sclerosis (SSc) is an autoimmune disease characterized by immunological and vascular abnormalities. Until now, the cause of SSc remains unclear. Sclerodermatous graft-versus-host disease (ScGVHD) is one of the most severe complications following bone marrow transplantation (BMT) for haematological disorders. Since the first cases, the similarity of ScGVHD to SSc has been reported. However, both diseases could have different etiopathogeneses. The objective of this study was to identify new serum biomarkers involved in SSc and ScGVHD. Methodology Serum was obtained from patients with SSc and ScGVHD, patients without ScGVHD who received BMT for haematological disorders and healthy controls. Bi-dimensional electrophoresis (2D) was carried out to generate maps of serum proteins from patients and controls. The 2D maps underwent image analysis and differently expressed proteins were identified. Immuno-blot analysis and ELISA assay were used to validate the proteomic data. Hemolytic assay with sheep erythrocytes was performed to evaluate the capacity of Factor H (FH) to control complement activation on the cellular surface. FH binding to endothelial cells (ECs) was also analysed in order to assess possible dysfunctions of this protein. Principal Findings Fourteen differentially expressed proteins were identified. We detected pneumococcal antibody cross-reacting with double stranded DNA in serum of all bone marrow transplanted patients with ScGVHD. We documented higher levels of FH in serum of SSc and ScGVHD patients compared healthy controls and increased sheep erythrocytes lysis after incubation with serum of diffuse SSc patients. In addition, we observed that FH binding to ECs was reduced when we used serum from these patients. Conclusions The comparative proteomic analysis of serum from SSc and ScGVHD patients highlighted proteins involved in either promoting or maintaining an inflammatory state. We also found a defective function of Factor H, possibly associated with ECs damage.

Proteome analysis of biological fluids from autoimmune-rheumatological disorders.

Autoimmune‐rheumatological diseases are worldwide distributed disorders and represent a complex array of illnesses characterized by autoreactivity (reactivity against self‐antigens) of T‐B lymphocytes and by the synthesis of autoantibodies crucial for diagnosis (biomarkers). Yet, the effects of the autoimmune chronic inflammation on the infiltrated tissues and organs generally lead to profound tissue and organ damage with loss of function (i.e., lung, kidney, joints, exocrine glands). Although progresses have been made on the knowledge of these disorders, much still remains to be investigated on their pathogenesis and identification of new biomarkers useful in clinical practice. The rationale of using proteomics in autoimmune‐rheumatological diseases has been the unmet need to collect, from biological fluids that are easily obtainable, a summary of the final biochemical events that represent the effects of the interplay between immune cells, mesenchymal cells and endothelial cells. Proteomic analysis of these fluids shows encouraging results and in this review, we addressed four major autoimmune‐rheumatological diseases investigated through proteomic techniques and provide evidence‐based data on the highlights obtained in systemic sclerosis, primary and secondary Sjogrens syndrome, systemic lupus erythematosus and rheumatoid arthritis.

The Local Complement Activation on Vascular Bed of Patients with Systemic Sclerosis: A Hypothesis-Generating Study

Objective The role of complement system in the pathogenesis of systemic sclerosis (SSc) has been debated during the last decade but an evident implication in this disease has never been found. We carried out an explorative study on SSc patients to evaluate the expression of soluble and local C5b-9 complement complex and its relation with a complement regulator, the Membrane Cofactor Protein (MCP, CD46) on skin vascular bed as target distinctive of SSc disease. We also analyzed two polymorphic variants in the complement activation gene cluster involving the MCP region. Methods C5b-9 plasma levels of SSc patients and healthy subjects were analyzed by ELISA assay. Archival skin biopsies of SSc patients and controls were subjected to immunofluorescence analysis to detect C5b-9 and MCP on vascular endothelial cells. The expression of MCP was validated by immunoblot analysis with specific antibody. Polymorphic variants in the MCP gene promoter were tested by a quantitative PCR technique-based allelic discrimination method. Results Even though circulating levels of C5b-9 did not differ between SSc and controls, C5b-9 deposition was detected in skin biopsies of SSc patients but not in healthy subjects. MCP was significantly lower in skin vessels of SSc patients than in healthy controls and was associated with the over-expression of two polymorphic variants in the MCP gene promoter, which has been related to more aggressive phenotypes in other immune-mediated diseases. Conclusions Our results firsty document the local complement activation with an abnormal expression of MCP in skin vessels of SSc patients, suggesting that a subset of SSc patients might be exposed to more severe organ complications and clinical evolution due to abnormal local complement activation.

Preliminary evidence for cell membrane amelioration in children with cystic fibrosis by 5-MTHF and vitamin B12 supplementation: a single arm trial

Background Cystic fibrosis (CF) is one of the most common fatal autosomal recessive disorders in the Caucasian population caused by mutations of gene for the cystic fibrosis transmembrane conductance regulator (CFTR). New experimental therapeutic strategies for CF propose a diet supplementation to affect the plasma membrane fluidity and to modulate amplified inflammatory response. The objective of this study was to evaluate the efficacy of 5-methyltetrahydrofolate (5-MTHF) and vitamin B12 supplementation for ameliorating cell plasma membrane features in pediatric patients with cystic fibrosis. Methodology and Principal Findings A single arm trial was conducted from April 2004 to March 2006 in an Italian CF care centre. 31 children with CF aged from 3 to 8 years old were enrolled. Exclusion criteria were diabetes, chronic infections of the airways and regular antibiotics intake. Children with CF were supplemented for 24 weeks with 5-methyltetrahydrofolate (5-MTHF, 7.5 mg /day) and vitamin B12 (0.5 mg/day). Red blood cells (RBCs) were used to investigate plasma membrane, since RBCs share lipid, protein composition and organization with other cell types. We evaluated RBCs membrane lipid composition, membrane protein oxidative damage, cation content, cation transport pathways, plasma and RBCs folate levels and plasma homocysteine levels at baseline and after 24 weeks of 5-MTHF and vitamin B12 supplementation. In CF children, 5-MTHF and vitamin B12 supplementation (i) increased plasma and RBC folate levels; (ii) decreased plasma homocysteine levels; (iii) modified RBC membrane phospholipid fatty acid composition; (iv) increased RBC K+ content; (v) reduced RBC membrane oxidative damage and HSP70 membrane association. Conclusion and Significance 5-MTHF and vitamin B12 supplementation might ameliorate RBC membrane features of children with CF. Trial Registration ClinicalTrials.gov NCT00730509

Scleroderma Lung Fibrosis and Biologic Drugs

Biologic agents have revolutionized the management of some inflammatory diseases like rheumatoid arthritis, seronegative spondyloarthropathies and Crohn’s disease; the selective inhibition of mediators involved in systemic chronic processes have allowed to expand the knowledge among the complex pathogenic pathways that are involved in inflammation and damage. The off-label use of biologic drugs is expanding. This review highlights the theoretical basis that may support the use of biologic drugs in the treatment of Systemic Sclerosis (SSc), a disabling and sometimes devastating disease whose treatment remains largely unsatisfactory. Some progresses have been made concerning the vascular complications of the disease, but the skin and visceral fibrosis, which is the hallmark of the disease, is still orphan of effective therapy. Moreover the results of the limited clinical experience about biologic agent administration in SSc and SSc-interstitial lung disease are presented.

Antinuclear autoantibody profile in systemic sclerosis patients who are negative for anticentromere and anti-topoisomerase I specificities.

Joint Bone Spine - In Press.Proof corrected by the author Available online since jeudi 25 decembre 2014

AB0125 The crosstalk between the complement system and the coagulation cascade in the antiphospholipid syndrome. preliminary data from basic research.

Background The association between antiphospholipid antibodies (aPL) and thrombophilic state in antiphospholipid syndrome (APS) is well recognized[1], but the underlying pathophysiology remains incompletely elucidated. Several findings suggest the role of complement system (CS) in the pathogenesis of antiphospholipid syndrome (APS)[2]. The importance of CS in APS is understandable since complement-derived inflammatory mediators increase vascular permeability, activate platelets and promote release of cytokines from monocytesthat favor systemic inflammation and coagulation[3]. It has been demonstrated in a mouse model of aPL-induced pregnancy loss that complement activation can amplify the fetal injury[4,5]. CS activation has been also documented in patients with APS[6], but there are far fewer clinical data. Objectives To evaluate the relation between CS and coagulation cascade in APS. Methods We used plasma samples from healthy women [H] (pregnant: n=5; non pregnant: n=5) and from patients with APS who were non pregnant (n=6), pregnant (n=10), in catastrophic phase [CAT] (n=4) or in a quiescent phase but with the same autoantibody profile of CAT (triple positivity for lupus anticoagulant, anticardiolipin and anti β2-glycoptrotein I antibodies) [SPECIAL] (n=4). The levels of activated complement components (C5a and C5b-9) and Tissue factor (TF) were analyzed using specific ELISA assays. Results During this preliminary study, main differences on C5a plasma levels were observed between H and patients with APS (mean H non pregnant: 4.28; APS non pregnant: 10.52), especially in subjects during CAT and in SPECIAL ones (mean 17.38 and 20.49, respectively). No marked differences were observed between pregnant H and with APS (mean APS: 7.97, H: 7.96). Similar data were obtained form C5b-9 analysis. Plasma TF values in samples from the same women described before were investigated. Among the two subgroups of H women, we didn’t observed significant differences in TF values. Conversely, we found interesting differences between non pregnant H controls and patients with APS (mean H: 88.46; APS: 212.21). High values were also documented in APS patients with CAT despite the anticoagulant treatment (mean 168.51), while lower TF values had been observed in pregnant women with APS and in SPECIAL ones (mean 138.17 and 104.65, respectively). We point out that pregnant women with APS and SPECIAL ones received anticoagulant drug to prevent the thrombi formation. Conclusions These preliminary data, together with other already reported, support the hypothesis of an involvement of CS in the modulation of coagulation activity in APS, especially in patients with triple positivity. References de Groot PG et al. Curr Opin Hematol 2004;11:165-9. Oku K et al. Ann Rheum Dis. 2009;68:1030-5. Cavazzana I et al. J Autoimmun 2007;28:160-164. Girardi G et al. J Clin Invest 2003;112:1644-54. Holers VM et al. J Exp Med 2002;1995:211-20. Lim W. Curr Opin Hemat 2011;18:361-365. Disclosure of Interest None Declared

Progress in Understanding the Pathogenesis of Systemic Sclerosis with Lung Involvement: The Contribution of Proteomic Studies

Systemic sclerosis (SSc) is a chronic inflammatory disease involving the skin and various internal organs. SSc is characterized by microvascular dysfunction, activation of the immune system and tissue fibrosis. Endothelial cell damage seems to be the initiating factor, but the precise triggering events that underlie the development of the disease remain unclear. Lung involvement is a frequent complication, which causes increased morbidity and mortality in patients with SSc. In particular, pulmonary arterial hypertension and interstitial lung disease are the two major clinical diseases that affect SSc patients, but the current treatments appear to have no satisfying effects on these pulmonary complications, until yet. Recently researches using innovative technologies have highlighted several peptides that might contribute to better understand the underlying pathogenic mechanisms of pulmonary injury and could promote more effective therapeutic strategies for SSc patients. Here, we focus on the major proteomic studies on biological fluid from patients with SSc.