We hypothesized that progression of monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM) reflects the escape of transformed plasma cells from T-cell recognition because of impaired antigen processing-presenting machinery (APM). We studied plasma cells and CD8+ T cells from bone marrow of 20 MGUS patients, 20 MM patients, and 10 control patients. Immunofluorescence and flow cytometry revealed significantly different patterns of APM component expression in plasma cells from the 3 groups. Compared with control patients, MM samples had lower expression of proteasome subunits and peptide transporters and greater expression of chaperones, considering both percentages of stained cells and molecular equivalents of soluble fluorochrome. MGUS samples had intermediate percentages of stained cells but molecular equivalents of soluble fluorochrome similar to control patients. Real-time polymerase chain reaction documented that APM changes occurred at the transcriptional level. Cytotoxicity assays demonstrated that MGUS CD8+ T cells lysed autologous transformed plasma cells more than MM CD8+ T cells did. MGUS progression correlated directly with calnexin, calreticulin, and tapasin and indirectly with delta, LMP2, and LMP10 expression levels; MM disease status did not correlate with APM levels. APM changes may allow transformed plasma cells to elude immunesurveillance in the MGUS-MM pathogenetic sequence.

Autoantibodies to intracellular antigens form a large family of immunoglobulins directed to a variety of ubiquitously expressed intracellular molecules, including numerous enzymes, some ribonucleoproteins and double-stranded DNA. These anti-self antibodies have been found to be selectively expressed in sera of patients with several systemic (non-organ-specific) autoimmune diseases, such as systemic sclerosis (SSc), SLE, mixed connective tissue disease, Sjögren's syndrome and idiopathic myopathies. Despite their important diagnostic and prognostic value and their utility in assessing disease activity, little is known about the molecular mechanisms involved in their generation and role in autoimmune diseases nor is it known why particular autoantibodies are preferentially expressed in certain diseases. Here, we review the different lines of research which are presently being conducted to understand how these autoantibodies are generated (e.g. through apoptotic body formation, molecular mimicry and other mechanisms) and how they encounter antigen in order to cause an autoimmune disease. The recently reported mechanism of intracellular immunity mediated by Ro52 (or tripartite motif containing 21, TRIM21) in a cellular model of adenovirus infection is opening new perspectives for studying the effects of autoantibodies once they get inside cells. © 2011 Elsevier B.V. All rights reserved.

Centromere-associated protein A (CENP-A), a common autoimmune target in a subset of systemic sclerosis patients, appears to have no role to explain why its corresponding auto-antibodies are more frequently found in the limited than the diffuse form of systemic sclerosis. Therefore, we investigated the fine specificity of anti-CENP-A antibodies as a first step to understanding their role in systemic sclerosis pathology. We focused on the amino-terminal portion of CENP-A spanning amino acids 1 to 17 (Ap(1-17)), which represents, along with Ap(17-30), an immunodominant epitope of the protein. Peptide Ap(1-17) was used to purify antibodies from 8 patients with systemic sclerosis. Anti-Ap(1-17) antibodies specifically reacted with human CENP-A but did not cross-react with CENP-B or Ap(17-30). Panning of a phage display peptide library with anti-Ap(1-17) antibodies from 2 patients identified two novel, partially overlapping motifs, <(5)Rx(st)xKP(10)> and <(9)KPxxPxR(15)> as the result of the alignment of specific phage clone insert sequences. Anti-Ap(1-17) IgG from the 8 patients had different reactivities to isolated phage clone insert sequences. Scanning the Swiss-Prot database revealed a large number of different types of proteins containing the two Ap(1-17) antigenic motifs. These data show that anti-CENP-A(1-17) antibodies are generated independently from anti-CENP-B antibodies and display great heterogeneity in their specificity by recognizing different motifs within that peptide sequence. This finding, along with the widespread interspecies and human tissue distribution of the two motifs, suggests that the number of motif-expressing proteins which can be the potential target of these antibodies is markedly higher than that estimated from the peptide-based epitope spreading model.

Background and objective: Systemic sclerosis is a highly invalidating connective tissue disease, whereby an interplay between vasculopathies, fibroblast activation and autoimmunity leads to widespread collagen deposition and tissue fibrosis. Autoimmunity is demonstrated by the presence of anti-nuclear antibodies (ANA) which include different subsets of auto-antibodies (Abs), such as anti-CENP-A Abs. Because CENP-A does not appear to have any pathogenetic (or protective) role to explain why the corresponding auto-Abs are more frequently found in the limited rather than the diffuse form of systemic sclerosis, and to get a deeper insight into the mechanism(s) by which these Abs are generated, we investigated their fine specificity, i.e., the amino acids (motif amino acids) which are recognized by this Ab population. We focused our study on the amino terminal portion of CENP-A spanning amino acid (AA) 1 to 17 (Ap1-17). This region was selected because it represents, along with Ap17-30, an immuno-dominant epitope of CENP-A. Both regions express the motif GPxRx shared with CENP-B, which is thought to be at the basis of anti-CENP-B cross reactivity with CENP-A. Methods: A CENP-A-derived peptide spanning AA 1 to 17 (Ap1-17) was insolubilized on an AffiGel-15 column, which was used for affinity purification of anti-Ap1-17 Abs from anti-CENP-positive sera of 8 patients with systemic sclerosis. SDS-GEL was employed to assess their purity and Western blot to test the reactivity of purified anti-Ap1-17 IgG with recombinant CENP-A and CENP-B. ELISAs were used to test the reactivity of sera or purified anti-peptide Abs. A phage display peptide library (PDPL) was used to identify anti-Ap1-17 Abs-specific motif AA. Web-available programs were looked up for the motif screening against protein database. Results: Anti-Ap1-17 IgG purified from these sera reacted specifically and dose-dependently with Ap1-17, whereas no reactivity was observed with Ap17-30. Panning of PDPL with anti-Ap1-17 IgG from patients pt14 and pt4 identified the 2 partially overlapping motifs &lt;5Rx(st)xKP10.&gt; and &lt;9KPxxPxR15.&gt;. Assessment of the reactivity of isolated phage clones with IgG anti-Ap1-17 divided the 8 patients into 4 groups, in function of their IgG reactivity (or lack of reactivity) with the pt14 and pt4 IgG-specific phage clones (pcs) 14 and pcs4, as follows: group #1 included pt14 IgG reacting with pcs14 only; group#2 included pt4 IgG which reacted with pcs4 only; group #3 included pt5, pt7 and pt9 IgG reacting, though to a different extent, with pc4.22, pc4.26, pc4.33 and pc4.40; group #4 included pt1, pt8 and pt15 IgG reacting neither with Pt4 nor with pt14-specific pcs. It is unlikely that pt1, pt8 and pt15 IgG reacted with the previously defined motif GPxRx expressed also by CENP-B, because of its lack of reactivity with IgG from any of the 8 patients evaluated. Conclusions: Our data show that: a) anti-Ap1-17 are independently expressed from anti-Ap17-30 and from anti-CENP-B; b) two novel motifs can be recognized by anti-CENP-A Abs; and c) IgG anti-Ap1-17 from different patients display a unique specificity, despite the recognition of the same peptide segment.

In a randomized, phase 3 study, superior complete/near-complete response (CR/nCR) rates and extended progression-free survival were demonstrated with bortezomib-thalidomide-dexamethasone (VTD) versus thalidomide-dexamethasone (TD) as induction therapy before, and consolidation after, double autologous stem cell transplantation for newly diagnosed myeloma patients (intention-to-treat analysis; VTD, n = 236; TD, n = 238). This per-protocol analysis (VTD, n = 160; TD, n = 161) specifically assessed the efficacy and safety of consolidation with VTD or TD. Before starting consolidation, CR/nCR rates were not significantly different in the VTD (63.1%) and TD arms (54.7%). After consolidation, CR (60.6% vs 46.6%) and CR/nCR (73.1% vs 60.9%) rates were significantly higher for VTD-treated versus TD-treated patients. VTD consolidation significantly increased CR and CR/nCR rates, but TD did not (McNemar test). With a median follow-up of 30.4 months from start of consolidation, 3-year progression-free survival was significantly longer for the VTD group (60% vs 48% for TD). Grade 2 or 3 peripheral neuropathy (8.1% vs 2.4%) was more frequent with VTD (grade 3, 0.6%) versus TD consolidation. The superior efficacy of VTD versus TD as induction was retained despite readministration as consolidation therapy after double autologous transplantation. VTD consolidation therapy significantly contributed to improved clinical outcomes observed for patients randomly assigned to the VTD arm of the study. The study is registered at www.clinicaltrials.gov as #NCT01134484.

INTRODUCTION: In a subset of patients with limited cutaneous (lc) systemic sclerosis (SSc), anti-CENP-A antibodies (Ab) cross-react with a peptide (FOXE3p53-62) that presents striking homology with one of the 2 immunodominant epitopes of CENP-A (Ap17-30). We searched for clinical correlates of anti-FOXE3p53-62 Ab by measuring their levels along with those of Ab to Ap17-30 and to the second immunodominant epitope of CENP-A, namely Ap1-17. METHODS: Serum samples were obtained from 121 patients with SSc, 46 patients with systemic lupus erythematosus (SLE) and 25 healthy blood donors (HBD). The reactivity of serum IgG to Ap1-17, Ap17-30 and FOXE3p53-62 was measured by ELISA. The corresponding anti-peptide Ab were affinity-purified from pooled SSc sera and used to establish standard curves for quantifying these Ab in patients and HBD. Receiver operating characteristics (ROC) analysis, comparing SSc patients who were positive for anti-CENP Ab (ACA+) to those who were negative, was used to find cut-off points for dichotomizing the anti-peptide Ab levels into positive and negative. Clinical records were reviewed to extract demographic data and information about organ involvement and disease activity. RESULTS: Of 121 SSc sera, 75 were ACA+; 88.0% of these samples reacted with Ap1-17, 82.6% with Ap17-30 and 53.3% with FOXE3p53-62. Among the 46 ACA- SSc sera, 2.2% reacted with Ap1-17, 4.3% with Ap17-30 and 11% with FOXE3p53-62. The levels of these Ab were low in ACA-, SLE and HBD groups and not significantly different among them. When ACA+ SSc patients were divided into subgroups positive or negative for anti-FOXE3p53-62 Ab, the only variables that were significantly different between groups were the levels of anti-Ap17-30 Ab and disease activity index (DAI). There was a significant association between negativity for anti-FOXE3p53-62 Ab and active disease defined as either DAI [greater than or equal to]3 (Fisher exact test, p=0.045) or less restrictive DAI[greater than or equal to]2.5 (p=0.009). CONCLUSIONS: ACA+-Anti-FOXE3p53-62+Ab identifies a subgroup of patients with lcSSc who are less likely to develop active disease. In lcSSc patients at presentation, anti-FOXE3p53-62+ can be a marker with prognostic significance.

Human leucocyte antigen (HLA)-G has a tolerogenic function and could play a role in the pathogenesis of immune-mediated diseases, including systemic sclerosis (SSc). The aim of this study was to evaluate HLA-G serum expression (sHLA-G) and the HLA-G gene 14 base pairs (bp) insertion/deletion (del(-)/del(+)) polymorphism in patients with Ssc, to search for possible associations with clinical and laboratory variables. sHLA-G was measured by enzyme-linked immunosorbent assay (ELISA) in sera from 77 patients with SSc and 32 healthy donors (HD); the 14 bp del(-)/del(+) polymorphism was evaluated by polymerase chain reaction (PCR) amplification of peripheral blood mononuclear cells (PBMC) genomic DNA. Receiver operating characteristics (ROC) analysis identified the HLA-G cut-off that best discriminated dichotomized clinical and serological variables, that was subsequently employed to subdivide SSc patients into HLA-G high (HLA-G(+)) and low (HLA-G(-)) profile groups. sHLA-G were not statistically different between SSc patients and HD, nor between distinct SSc autoantibody subsets. Subdividing SSc patients by HLA-G positivity or negativity yielded significant differences for the modified Rodnan skin score (mRss) (P = 0.032), 'general' (P = 0.031) and 'kidney' (P = 0.028) Medsger severity scores (MSS) and disease activity index, and especially Δ heart/lung (P = 0.005). A worse 'general' MSS (P = 0.002) and Δ heart/lung (P = 0.011) were more frequent in the low sHLA-G group. These two variables and mRss were associated with sHLA-G levels at logistic regression analysis. Treatment had no influence on sHLA-G. Moreover, a higher frequency of scleredema was detected in the del(+)/del(+) than the del(-)/del(+) group (P = 0.04). These data suggest modulatory effects of sHLA-G on SSc. Prospective studies are needed to investigate a role in predicting the disease course. © 2015 British Society for Immunology.

BACKGROUND. Auto-Abs to the histone H3-like centromeric protein-A (CENP-A) are frequently detected in systemic sclerosis (SSc) with limited cutaneous involvement. The long-range goal of this study is to define the origin of CENP-A Abs through the identification of proteins other than CENP-A, which can be their target or induce them. We found that Abs to an immunodominant region of the CENP-A (spanning amino acids 17 to 30; Ap17-30) recognized two distinct, though overlapping, epitopes. One of these is defined by the motif PTPxxGPXXR.OBJECTIVE: 1) to identify proteins expressing the anti-CENP-A-specific motif, 2) to analyze their homology to CENP-A; 3) to screen anti-CENP-A Ab positive sera against peptide-bearing the motif.METHODS: A Swiss-Prot database search was performed to identify proteins expressing anti-CENP-A-specific motifs. Anti-Ap17-30 IgGs were purified from sera by affinity chromatography on an insolubilized peptide column. ELISA was used to test the specificity of serum or Abs against peptides in binding and inhibition assays. Microscopy immunofluorescence assay was performed on fixed-permeabilized HeLa cells, using purified IgG anti-Ap17-30 cells and FITC-conjugated anti-human IgG as detecting reagents. Immunization of BALB/c mice with peptides was performed by intra-peritoneal injections of 10 µg-peptide on day 0, 7, 14 and 70. Sera were drawn weekly until the 13th week and stored at -80°C until used.RESULTS: A database search identified 4 proteins expressing the motif PTPxxGPxxR. Of these, the forkhead box E3 (FOXE3)-derived peptide FOXE3p displayed the highest homology with CENP-A Ap17-30 (identity: 80%), while condensing-2 complex subunit D3-derived peptide (CD3p), protein Jumonji-derived peptide (JMJp) and microtubule-associated protein 1A-derived peptide (MAP1p) displayed 60% homology to Ap17-30. These peptides' antigenic homology with Ap17-30 was evaluated, using IgG anti-Ap17-30 affinity-purified from the sera of 8 anti-CENP-A+ SSc patients (IgG pt1 to pt8). As compared to Ap17-30, Pt1 to pt8 IgG displayed the highest reactivity with FOXE3p, while a lower or no reactivity was observed with the remaining peptides. Furthermore, mouse anti-Ap17-30 and anti-FOXE3 Abs cross-reacted to each other, while Abs elicited with MAP1A partially reacted with CD3. Neither CD3 nor JMJ were found to be immunogenic. Kinetic studies of the reactivity of anti-FOXE3 Abs with Ap17-30 indicated that FOXE3 booster injection on the day 70 markedly increased their relative avidity to Ap17-30. Furthermore mouse anti-FOXE3 sera recognize CENP-A and FOXE3 in western blot, while mouse anti-Ap17-30 recognize CENP-A only. The testing of 69 human anti-CENP-A/Ap17-30+ sera with FOXE3p identified its reactivity with 50.7 % of them.CONCLUSION: 1) FOXE3 displays an antigenic and immunogenic homology with CENP-A-derived Ap17-30; 2) FOXE3p can identify a subset of anti-CENP-A Ab-positive patients. Ongoing studies are expected to establish whether any correlation between FOXE3 reactive Ab-patients and a clinical profile exists.

The safety of four different adjuvants was assessed in lupus-prone New Zealand black/New Zealand white (BW)F1 mice. Four groups of mice were injected intraperitoneally with incomplete Freund's adjuvant (IFA), complete Freund's adjuvant (CFA), squalene (SQU) or aluminium hydroxide (ALU). An additional group received plain phosphate-buffered saline (PBS) (UNT group). Mice were primed at week 9 and boosted every other week up to week 15. Proteinuria became detectable at weeks 17 (IFA group), 24 (CFA group), 28 (SQU and ALU groups) and 32 (UNT group). Different mean values were obtained among the groups from weeks 17 to 21 [week 17: one-way analysis of variance (anova) P = 0·016; weeks 18 and 19: P = 0·048; weeks 20 and 21: P = 0·013] being higher in the IFA group than the others [Tukey's honestly significant difference (HSD) post-test P < 0·05]. No differences in anti-DNA antibody levels were observed among groups. Anti-RNP/Sm antibody developed at week 19 in only one CFA-treated mouse. Mean mouse weight at week 18 was lower in the ALU group than the IFA (Tukey's HSD post-test P = 0·04), CFA (P = 0·01) and SQU (P < 0·0001) groups, while the mean weight in the SQU group was higher than in the IFA (P = 0·009), CFA (P = 0·013) and UNT (P = 0·005) groups. The ALU group weight decreased by almost half between weeks 29 and 31, indicating some toxic effect of ALU in the late post-immunization period. Thus, SQU was the least toxic adjuvant as it did not (i) accelerate proteinuria onset compared to IFA; (ii) induce toxicity compared to ALU or (iii) elicit anti-RNP/Sm autoantibody, as occurred in the CFA group.

Background: The process of epithelial_mesenchymal transition (EMT) has been regarded in systemic sclerosis (SSc) as one of the possible mechanisms favouring tissue accumulation of monocyte_derived fibrocytes or myofibroblasts, which contribute to tissue fibrosis [1]. Forkhead box E3 (FOXE3) is a transcription factor involved in EMT of lens epithelial cells (LEC). Its expression progressively decreases with the migration of LEC from the anterior to the equatorial region. FOXE3 expression cessation marks initiation of fiber differentiation, suggesting that the loss of FOXE3 expression favors a pro_fibrotic phenotype [2]. No data are available on mRNA FOXE3 expression in sites other than LEC. Objectives: In this study, we investigated the FOXE3 mRNA expression in unstimulated and TGF_ß_ or IL_4_stimulated monocytes from SSc patients and healthy blood donors (HBD), to established whether i) FOXE3 is constitutively expressed in human monocytes; ii) FOXE3 expression can be modulated in vitro by cytokines involved in SSc profibrotic process; iii) there is any association between FOXE3 expression and a particular SSc serological profile. Methods: PBMC were isolated from heparinized peripheral blood of 9 patients with SSc (5 Scl70 + ; 4 Scl70 – ), and 3 HBD by Ficoll_Hypaque density gradient centrifugation. Monocytes (CD14+) were isolated by positive selection using microbeads. Cells (1x10 6 cells/ml) were stimulated TGF_ß (10 ng/ml) and IL_4 (40 ng/ml) for 14 days. mRNA was extracted and semi_quantitative PCR was performed to assess FOXE3 expression. GM_CSF stimulation (50ng/ml) was used as positive control. The levels of FOXE3 mRNA were quantified by normalizing its expression against that of GAPDH. Expression was measured as mean relative expression level (MREL). Variation of expression was measured as mean fold change (MFC). Results: Similar baseline levels of FOXE3 mRNA was observed in unstimulated CD14 + cells from SSc patients and HBD (MREL SSc=0.32; HBD=0.26). As expected, GM_CSF stimulation of CD14 + cells from SSc patients and HBD markedly up_regulated FOXE3 expression (SSc: MFC=3.24; HBD: MFC=1.84). TGF_ß and IL_4 behaved similarly to GM_CSF in enhancing FOXE3 expression in CD14 + cells from all HBD (MFC TGF_ß =1.35; MFC IL_4 =1.59) and from 3 out 4 Scl70 – patients (MFC TGF_ß =2.36; MFC IL_4 =2.9), being the expression unchanged in the remaining Scl70 – patient. By contrast, in the 4 Scl70 + patients, CD14 + FOXE3 expression markedly decreased following these cytokines stimulation (MFC TGF_ ß =0.28; MFC IL_4 =0.31) Conclusions: This is the first study to demonstrate FOXE3 mRNA expression in monocytes from HBD and SSc patients, and its differential expression following TGF_ß and IL_4 stimulation, correlating with the serological profile of SSc patients. The data suggest that the down_regulation of FOXE3 induced by TGF_ß and IL_4 may direct monocytes toward a more profibrotic phenotype in Scl70 + as compared to Scl70 – patients. The relationship of this finding with the anti_FOXE3 antibodies recently detected in SSc sera [3], remains to be determined. References: Postlethwaite AE et al. Curr Opin Rheumatol 16:733_738, 2004. Landgren H et al. Invest Ophthalmol Vis Sci 49:4269_4277, 2008. Perosa F et al. Arthritis Res Ther 15:R72, 2013. Acknowledgements: This work was supported by a 2013 grant from the Italian Group for Systemic Sclerosis (GILS), Milan, Italy. Disclosure of Interest: None declared DOI: 10.1136/annrheumdis_2014_eular.4130 Citation: Ann Rheum Dis 2014;73(Suppl2)

Rheumatoid arthritis (RA) is an immune-mediated disease involving chronic low-grade inflammation that may progressively lead to joint destruction, deformity, disability and even death. Despite its predominant osteoarticular and periarticular manifestations, RA is a systemic disease often associated with cutaneous and organ-specific extra-articular manifestations (EAM). Despite the fact that EAM have been studied in numerous RA cohorts, there is no uniformity in their definition or classification. This paper reviews current knowledge about EAM in terms of frequency, clinical aspects and current therapeutic approaches. In an initial attempt at a classification, we separated EAM from RA co-morbidities and from general, constitutional manifestations of systemic inflammation. Moreover, we distinguished EAM into cutaneous and visceral forms, both severe and not severe. In aggregated data from 12 large RA cohorts, patients with EAM, especially the severe forms, were found to have greater co-morbidity and mortality than patients without EAM. Understanding the complexity of EAM and their management remains a challenge for clinicians, especially since the effectiveness of drug therapy on EAM has not been systematically evaluated in randomized clinical trials. Keywords: Rheumatoid arthritis; Extra-articular manifestation; Co-morbidities; Classification; Incidence/rate; Vasculitides; Amyloidosis; Felty's syndrome

The surface presentation of peptides by major histocompatibility complex (MHC) class I molecules is critical to all CD8+ T-cell adaptive immune responses, including those against tumors. The generation of peptides and their loading on MHC class I molecules is a multistep process involving multiple molecular species that constitute the so-called antigen processing and presenting machinery (APM). The majority of class I peptides begin as proteasome degradation products of cytosolic proteins. Once transported into the endoplasmic reticulum by TAP (transporter associated with antigen processing), peptides are not bound randomly by class I molecules but are chosen by length and sequence, with peptidases editing the raw peptide pool. Aberrations in APM genes and proteins have frequently been observed in human tumors and found to correlate with relevant clinical variables, including tumor grade, tumor stage, disease recurrence, and survival. These findings support the idea that APM defects are immune escape mechanisms that disrupt the tumor cells’ ability to be recognized and killed by tumor antigen–specific cytotoxic CD8+ T cells. Detailed knowledge of APM is crucial for the optimization of T cell–based immunotherapy protocols.

Background and objective: In a subset of systemic sclerosis (SSc) patients (pts), anti-CENP-A Ab cross reacts with a FOXE-3-derived peptide (FOXE3p), which presents a striking homology with one (Ap17-30) of the 2 immunodominant epitopes of CENP-A. Preliminary analysis, obtained by absorbance binding, showed that active disease was less likely to occur in anti- FOXE-3+ patients as compared to anti- FOXE-3- patients. Here, we have set up a titration assay using appropriate standards to quantify IgG anti-FOXE-3 and to define whether any clinical correlate exist with the presence of anti FOXE3. As control, clinical correlates with the level of antibodies to Ap17-30 and to the 2nd immunodominant epitope of CENP-A (spanning amino acids 1-to 17, Ap1-17) were also evaluated. Methods: Ap17-30, FOXE3p and Ap1-17 were synthesized at Primm peptide synthesis service (Naples, Italy). The reactivity of serum IgG to specific peptides was measured by indirect ELISAs, using BSA-conjugated peptide as coating reagents and HRP-conjugated xeno-Ab to the Fc portion of human IgG as probe. Sera from 117 SSc pts were tested for anti-centromere and anti-topoisomerase Ab (Diamedix). Calibration curves to measure the levels of Ab were settled, using IgG from sera purified by affinity column coupled to Ap17-30 and Ap1-17 peptides. Clinical records were reviewed to achieve demographic data and information about organ involvement and disease activity, previously evaluated according to a core set of 10 variables. Control sera were obtained from SLE (#47 pts) and from healthy blood donor (HBD) (#25 pts). Results: Purify IgG anti-Ap1-17 and anti-Ap17-30 were used to set-up a standard calibration curve to measure serum levels of anti-Ap1-17 (detection limit range: 2.68-6037ug/ml) and Ap17-30 (0.36-32.85 ug/ml) respectively. As anti-Ap17-30 cross-react with FOXE-3, calibration curve to measure serum levels of Abs to this peptide was achieved using purified anti-Ap17-30 IgG and FOXE-3p as coating reagents (detection limit range: 9.25-880 ug/ml). Of 117 SSc sera, 68 (58.1%) were anti-CENP positive (CENP+) and 94% (#65) of them reacted with Ap17-30 and/or Ap1-17, while no reactivity was observed on the replacement of CENP+ with CENP-, SLE, and HBD sera. The best cut-off concentration of Ab levels able to discriminate CENP+ from CENP- patients (obtained with ROC analysis) were &gt;10.4 ug/ml (sensitivity: 86.8, specificity 98%), &gt;1.2ug/ml (sens. 83%, spec. 96%) and &gt; 74.5 ug/ml (sens.&gt;52.6%, spec. 90%) for anti-Ap1-17, anti-Ap17-30 and anti-FOXE3p respectively. Following dichotomization of CENP+ patients according to their disease activity index score in group 1 (61 patients with score &lt;3), and group 2 (7 patients with score&gt; 3; active disease), a Fisher exact test showed a statistical association of FOXE3p+ sera with group 1 patients (p=0.003, OR: 39.5). Conclusion: Our data confirm the identification of a subset of patients (FOXE3+) that are less likely to have active disease.

Raynaud's phenomenon (RP) is a well defined clinical syndrome characterized by recurrent episodes of digital vasospasm triggered by exposure to physical/chemical or emotional stress. RP has been classified as primary or secondary, depending on whether it occurs as an isolated condition (pRP) or is associated to an underlying disease, mainly a connective tissue disease (CTD-RP). In both cases, it manifests with unique “triple” (pallor, cyanosis and erythema), or “double” color changes. pRP is usually a benign condition, while sRP can evolve and be complicated by acral digital ulcers and gangrene, which may require surgical treatment. The pathogenesis of RP has not yet been entirely clarified, nor is it known whether autoantibodies have a role in RP. Even so, recent advances in our understanding of the pathophysiology have highlighted novel potential therapeutic targets. The aim of this review is to discuss the etiology, epidemiology, risk factors, clinical manifestations, recently disclosed pathogenic mechanisms underlying RP and their correlation with the available therapeutic options, focusing primarily on pRP and CTD-RP.

Severe pulmonary arterial hypertension (PAH) is rarely observed as the initial manifestation of systemic lupus erythematosus (SLE), and the diagnosis is often delayed. Here we present the case of a 32-year-old woman with severe PAH as the initial manifestation of SLE, who was successfully treated with mycophenolate mofetil and cyclosporine. This case offered the opportunity to critically review the epidemiology data, predictive markers, and pathogenic pathways of SLE-associated PAH (SLE-PAH) in relation to the currently available therapeutic options and to the main clinical trials of the last 10 years focused on the treatment of SLE-PAH. Mycophenolate mofetil and cyclosporine - currently used in the maintenance phase of the disease in certain clinical settings - should be considered, as an alternative to cyclophosphamide, in future clinical trials aimed at evaluating the most effective treatment of SLE-PAH at presentation.