Corneal involvement in rheumatoid arthritis: an vivo confocal study

PURPOSE. To analyze the in vivo morphology of corneal cells and nerves in patients with rheumatoid arthritis (RA), with or without secondary Sj\uf6gren\u2019s syndrome (SSII), and to investigate the correlations between corneal alterations and RA activity. METHODS. Fifty patients with RA and 30 age- and gender-matched control subjects were studied. SSII was diagnosed according to the American-European Consensus Group criteria, and RA activity was evaluated by the Lansbury index (LI). Confocal microscopy was used to investigate corneal thickness, the number of epithelial and stromal cells, and keratocyte hyperreflectivity. In addition, the sub-basal plexus was assessed for the number, tortuosity, and reflectivity of the nerve fibers and the presence of beadlike formations. RESULTS. Sixteen percent of patients with RA also had SSII. Between the SSII and non-SSII groups, no significant differences were found in the LI or in the clinical and confocal variables. Significant differences were present between patients with RA and control subjects for all the variables studied except nerve reflectivity. In patients with RA with and without SSII, LI correlated significantly with the number of beadlike formations and the number of hyperreflective, activated keratocytes. CONCLUSIONS. Confocal microscopy of patients with RA showed several changes in corneal cells and nerves. The number of beadlike formations and the number of activated keratocytes could be interpreted as confocal signs of ocular surface disease activity. These correlations with the index of systemic disease activity, LI, may provide insight regarding the pathogenic mechanisms of dry eye in patients with RA

Taking care of systemic sclerosis patients during COVID-19 pandemic : rethink the clinical activity

COVID-19 outbreak has quickly spread worldwide, causing a high pressure on the health-care system. In Italy, from March 8, 2020, all the deferrable clinical activities have been suspended to increase the health care offer for COVID-19 patients. The hospital organization has been modified also in order to assure non-COVID-19 patients assistance. The Scleroderma Unit of ASST Pini-CTO Hospital, in Milan, in the region mostly hit by SARS-CoV-2 in Italy, follows more than 600 patients affected by systemic sclerosis (SSc). Patients with SSc need a close follow-up with a regular screening of organ involvement and frequent intravenous treatments. All SSc patients have been educated about ministerial directives to limit COVID-19 spread. The organization of our Scleroderma Unit has been quickly rethought to assure SSc patients assistance in safety for them and for health-care workers during urgent visits or infusion therapies. Using electronic way of communication with frequent virtual contact and guarantying home deliveries of some therapies, we allowed a continuity of care also outside the Hospital

AB0125 EXPRESSION OF INTERFERON TYPE I- AND TYPE II-INDUCED GENES IN PATIENTS WITH SJÖGREN'S SYNDROME WITH AND WITHOUT EXTRAGLANDULAR INVOLVEMENT

Background:It is well known that Sjögren's syndrome (SjS) is characterized by an upregulation of interferon (IFN)-induced genes. Namely, IFN type I signature has been reported in peripheral blood mononuclear cells (PBMCs) and in salivary glands of patients with this disease. However, few data are available on possible variability of IFN-induced gene upregulation in different clinical phenotypes of SjS.Objectives:To verify whether upregulation of IFN-induced genes is comparable in patients with SjS characterized by different clinical phenotypes, i.e., patients with systemic extraglandular manifestations (EGMs) versus patients with a disease limited to glandular features (GFs) and with widespread pain (WP).Methods:The study population was composed by 11 patients with SjS and EGMs (1 male, age range 18-78 years), and 10 patients with only GFs and WP (all females, age range 46-81 years), all classified according to ACR-EULAR criteria. The prevalence of anti-SSA(Ro) antibodies was 11/11 and 8/10, respectively. Lip biopsy was positive in all cases. Six healthy normal subjects were also included in the study as control population.Four IFN type I- and 5 IFN type II-induced genes were chosen for the study on the basis of previous literature data. Total RNA from each patient and control was isolated from purified PBMCs, followed by cDNA preparation and real time quantitative-PCR (RQ-PCR) analysis, using specific primer/probe sets. For calculation of relative expression, all samples were normalised against expression of a household gene (beta actin). A further normalization was performed against the mean value of relative expression obtained in the normal controls. Final fold change values were determined from the double-normalised values using the 2−ΔΔCT method (Applied Biosystems).Results:Fold change values of gene expression of both IFN type I- and type II-induced genes in PBMCs were different in the two clinical phenotypes of SjS. Fold change values of IFN type I-induced genes appeared strongly higher in patients with EGM, and some of them only moderately increased in those with only GF and WP. The expression of some of IFN type II-induced genes were slightly increased in patients belonging to both clinical phenotypes. Results are detailed in the table.Table.Fold change values of gene expression in patients with SjS plus EGMs, in patients with disease limited to GF and WP, and in controls.GeneMX1IFIT1IFT3IFI44IDO1GRP1MIGIP-10P2RY14SjS-EGMs85.938.524.440.425.18.34.51.55.5SJS-GF-WP4.21.72.04.84.11.20.60.31.3Controls2.11.61.11.51.41.31.51.31.4Legend. IFN type I-induced genes: MIX, IFN-induced GTP binding protein 1; IFIT1, IFN-induced protein with tetratricopeptide repeats 1; IFIT3, IFN-induced protein with tetratricopeptide repeats 3; IFI44, IFN-induced protein 44.IFN type II-induced genes: IDO1, indolamine-deoxygenase 1; GBP1, guanylate binding protein 1; MIG, C-X-C chemokine 9 (CXCL9); IP-10, C-X-C chemokine 10 (CXCL10); P2RY14, purinergic receptor 14.Conclusion:The present data indicate that IFN type I- and, to a lesser degree, type II-induced genes are upregulated in patients with SjS, but this phenomenon is consistently stronger in patients with systemic EGMs. In patients with only GFs IFN-induced gene upregulation is milder in PBMCs, and then probably more restricted to the exocrine target tissues.Disclosure of Interests:Nicoletta Del Papa: None declared, Claudio Vitali: None declared, Maurizio Lorini: None declared, Vincenzo Carbonelli: None declared, Wanda Maglione: None declared, Francesca Pignataro: None declared, Antonina Minniti: None declared, Nicola Montano: None declared, Roberto Caporali Consultant of: AbbVie; Gilead Sciences, Inc.; Lilly; Merck Sharp & Dohme; Celgene; Bristol-Myers Squibb; Pfizer; UCB, Speakers bureau: Abbvie; Bristol-Myers Squibb; Celgene; Lilly; Gilead Sciences, Inc; MSD; Pfizer; Roche; UC

Peripheral Microangiopathy Changes in Pulmonary Arterial Hypertension Related to Systemic Sclerosis: Data From a Multicenter Observational Study

Systemic sclerosis (SSc) is a connective tissue disease characterized by immune-system alterations, fibrosis involving the skin and internal organs and diffuse microangiopathy. Pulmonary arterial hypertension (PAH) is a severe complication of SSc affecting about 10-15% of the patients and it is a leading cause of mortality. Due to the devastating nature of SSc-PAH, there is a clear need to systematically adopt appropriate screening programs. Nail fold videocapillaroscopy (NVC) studies have shown a more severe peripheral microvascular dysfunction in SSc patients with PAH suggesting that abnormalities in peripheral microcirculation may correlate with pulmonary microangiopathy. This is a cross-sectional study involving four tertiary University Rheumatology Units in the Center-North of Italy. Seventy patients, 35 adults with SSc and PAH confirmed by RHC (F/M 34/1; median age 65.2 ± 8.9 SD yrs), and 35 SSc patients without PAH were enrolled (F/M 3471; median age 63.3 ± 10.3 SD yrs). Clinical, laboratoristic and instrumental data were collected and NVC was performed in all patient. Specific NVC parameters were evaluated and a semi-quantitative rating scale was adopted to score these changes. Finally, patients were distributed into the suitable NVC pattern belonging to the scleroderma pattern. Our aim was to compare the peripheral microangiopathy changes in SSc patients with and without PAH, and to investigate the relationship between NVC findings and the main hemodynamic parameters of pulmonary vasculopathy. Patients with SSc-PAH+ showed a significant higher frequency of interstitial lung disease (ILD). No significant differences regarding clinical and laboratoristic parameters were observed. NVC abnormalities, avascular areas were more frequent in SSc patients with PAH, respect to those without (p = 0.03), and capillary density was significantly lower when considering grade 3 (p = 0.02). A higher NVC semiquantitative mean was found in SSc-PAH+ patients and a greater rate of the "late" pattern was detected in SSc-PAH+ subjects in respect to PAH- (57.1% vs. 25.7%) (p = 0.03). A significant correlations between pulmonary pressure values (sPAP by TTE and mPAP by RHC) and the capillary density (Spearman's rho 0.35, p = 0.04 for both). Our findings provide additional evidence to the literature data, confirming that a higher degree of peripheral nailfold microangiopathy is more common in SSc-PAH patients, and further strengthening the concept that NVC changes may run parallel with similar abnormalities inside pulmonary microcirculation

POS0424 DETERMINING CIRCULATING ENDOTHELIAL CELLS USING CELLSEARCH SYSTEM IN SYSTEMIC SCLEROSIS PATIENTS

Background:Endothelial damage and fibroproliferative vasculopathy of small vessels are pathological hallmarks of Systemic Sclerosis (SSc). Detection and analysis of circulating endothelial cells (CECs) detached from affected blood vessels may be an informative tool to study vascular dysfunction and could be considered a novel biomarker of scleroderma vasculopathy. Our group first showed the presence of CECs in SSc by fluorescence-activated cell sorting (FACS), demonstrating that a raised counts of active CECs may represent direct evidence of active vascular disease in SSc. Despite these interesting data, issues related to difficulties in CEC counting through FACS analysis, due their very low concentration in peripheral blood, prevented further investigations in this field. Recently, a specific kit for the detection of CECs has been developed through the CellSearch System (CS), a semi-automated device for the standardized analysis of rare cells, such as CECs, in peripheral blood.Objectives:To assess the counts of CECs determined by the CS in SSc patients and to evaluate their clinical implication and potential as vascular biomarker in SSc.Methods:10mL of blood samples were collected from 29 subjects (19 SSc patients and 10 healthy donors - HDs) and stored in tubes containing a specific preservative, to allow the analysis of 4mL of blood within 72 hours, according to manufacturer instructions. Out of 19 SSc patients, 18 were female, 10 had the limited form and 9 the diffuse cutaneous variant of SSc. CS uses a proprietary kit containing a ferrofluid-based reagent, that target CD146 to magnetically capture CECs, and the immunofluorescent reagents to stain the CECs, defined as CD146+, CD105-PE+, DAPI+ and CD45-APC-. Clinical, laboratoristic and demographic data were also collected.Results:The mean number of CECs in patients with SSc was significantly higher in comparison to HDs (554/4mL vs. 53.5/4 mL, p=0.0042). When analyzed according to disease subset, both lcSSc and dcSSc showed significantly increased levels of CECs in comparison with HDs (p=0.003 and p=0.005, respectively). No statistical difference was observed in the mean number of CECs in patients with lcSSc compared to those with dcSSc. Regarding vascular involvement, the CECs counts strictly correlated with the presence of digital ulcers (DUs) (p=0.0001) showing a median of 863cells/4mL for the SSc patients with DUs versus a median of 276.2/4mL for the SSc patients without DUs. No statistical correlation was found between CECs and serological autoantibody pattern, skin parameters, or joint and muscle involvement. Patients with active disease, according to the EUSTAR Activity Index, showed a higher CECs value than those with inactive disease (p=0.0012).Conclusion:The amount of CECs detectable in peripheral blood has been recently proposed as a marker of endothelial damage in different vascular diseases, including SSc. However, currently no standardized method is available to determine CEC counts, which makes reported data on CECs reliable and suitable. The CS system is a commercially available semi-automated system that enables standardized determination of CECs. Thus, we examined clinical utility of CECs count by this system in SSc patients. Our results confirm that baseline CEC counts, evaluated by a new standardized method, may represent direct evidence of endothelial damage in SSc and could be a promising tool for monitoring active disease and evaluating therapeutic responses to vascular and immunosuppressive treatments.References:[1]Del Papa N, Pignataro F. Front Immunol. 2018 Jun 18;9:1383[2]De Simone C et al. J Eur Acad Dermatol Venereol. 2014 May;28(5):590-6[3]Del Papa N et al. Arthritis Rheum. 2004 Apr;50(4):1296-304Disclosure of Interests:Francesca Pignataro: None declared, Laura Zorzino: None declared, Wanda Maglione: None declared, Antonina Minniti: None declared, Giulia Clericuzio: None declared, Marco Picozzi: None declared, Cecilia Simonelli Employee of: Menarini Silicon Biosystems, Francesco Picardo Employee of: Menarini Silicon Biosystems, Roberto Caporali: None declared, Nicoletta Del Papa: None declare

El error de observación y su influencia en los análisis morfológicos de restos óseos humanos. Datos de variación discreta.

        RESUMEN A partir de la década de 1960 se incrementó el empleo de los rasgos no métricos del cráneo en los análisis de relaciones poblacionales. Si bien uno de los supuestos en que se bas6 el uso de estos rasgos es la facilidad de estandarizar su registro, varios trabajos sugieren que el error interobservador en los rasgos discretos es elevado. En consecuencia, los objetivos del trabajo son evaluar el error intra e interobservador en el registro de rasgos craneales no métricos y analizar el efecto del error interobservador sobre las distancias biológicas calculadas. Con este fin, se analizó una muestra arqueológica procedente del Valle inferior del Río Negro -10 con deformación pseudocircular y 10 con deformación planolámbdica-. Se seleccionaron ocho variables discretas del cráneo que fueron relevadas por cuatro observadores. El error de observación fue evaluado mediante un diseño experimental de bloques aleatorios con tres repeticiones y el empleo del índice Kappa y la prueba de McNemar. La distancia biológica entre las dos muestras se estimó mediante la Medida Media de Divergencia y un análisis de escalamiento multidimensional. Los resultados obtenidos indican que el error intraobservador disminuyó mediante la aplicación del diseño experimental, que el error interobservador se incrementó en las sucesivas series y que las diferencias entre los observadores alteraron los resultados de las medidas de distancia calculadas entre las muestras.   ABSTRACT Since the early 1960s, there was an increasing interest in the application of non-metric cranial traits to the analysis of relationships between populations. One of the assumptions for the use of these traits is based upon the simplicity to standardize the recordings. However, several papers suggest that the interobserver error on such recordings is high. Therefore, the goals of this paper are to evaluate the intra and interobserver error on the scoring of nonmetric cranial traits, as well as to analyze the effect of the interobserver error in the biological distances estimated with them. An archeological sample (n=20) from the lower stretch Valley of Rio Negro Valley (Rio Negro Province, Argentina) was analyzed. Eight discrete variables from the skull were recorded independently by four observers. The observation error was evaluated by means of a randomized complete blocks design, with three repetitions, and employing the Kappa index and the McNemar test. The biological distance between the two samples was estimated through the Mean Measure of Divergence and a multidimensional scaling test. Our results indicate that 1) the intraobserver error Diminishes with the application of observational designs; 2) the interobserver error increases in successive series and; 3) the differences among observers modify the results of calculated biodistance between samples