Elisa Astorri

Evolution of Ectopic Lymphoid Neogenesis and In Situ Autoantibody Production in Autoimmune Nonobese Diabetic Mice: Cellular and Molecular Characterization of Tertiary Lymphoid Structures in Pancreatic Islets

A pivotal role for tertiary lymphoid structures (TLSs) in promoting Ag-specific humoral responses during chronic inflammation is emerging in several autoimmune conditions, including rheumatoid arthritis, Sjogren’s syndrome, and autoimmune thyroiditis. However, there is limited evidence on the cellular and molecular mechanisms underlying TLS formation and their contribution to autoimmunity in the pancreas during autoimmune insulitis. In this study, we performed a detailed and comprehensive assessment of the evolution of TLSs during autoimmune insulitis in 126 female NOD mice from 4 to 38 wk of age. We demonstrated that during progression from peri- to intrainsulitis in early diabetic mice, T and B cell infiltration follows a highly regulated process with the formation of lymphoid aggregates characterized by T/B cell segregation, follicular dendritic cell networks, and differentiation of germinal center B cells. This process is preceded by local upregulation of lymphotoxins α/β and lymphoid chemokines CXCL13 and CCL19, and is associated with infiltration of B220+/IgD+/CD23+/CD21− follicular B cells expressing CXCR5. Despite a similar incidence of insulitis, late diabetic mice displayed a significantly reduced incidence of fully organized TLSs and reduced levels of lymphotoxins/lymphoid chemokines. Upon development, TLSs were fully functional in supporting in situ autoreactive B cell differentiation, as demonstrated by the expression of activation-induced cytidine deaminase, the enzyme required for Ig affinity maturation and class switching, and the presence of CD138+ plasma cells displaying anti-insulin reactivity. Overall, our work provides direct evidence that TLSs are of critical relevance in promoting autoimmunity and chronic inflammation during autoimmune insulitis and diabetes in NOD mice.

Implication of Epstein‐Barr Virus Infection in Disease‐Specific Autoreactive B Cell Activation in Ectopic Lymphoid Structures of Sjögren's Syndrome

To examine whether the B cell tropic γ‐herpesvirus Epstein‐Barr virus (EBV) is aberrantly expressed in its latent and lytic forms within ectopic lymphoid structures (ELS) in the salivary glands of patients with Sjögrens syndrome (SS), and to investigate the relationship between EBV dysregulation, B cell activation, in situ differentiation of autoreactive plasma cells, disease‐specific autoantibody production, and cytotoxicity.

Circulating Reg1α proteins and autoantibodies to Reg1α proteins as biomarkers of β-cell regeneration and damage in type 1 diabetes.

Type 1 diabetes is an autoimmune disease where β-cells are in a constant process of death and renewal. Reg genes play a role in β-cells regeneration. Reg proteins may be target of an autoimmune response in type 1 diabetes with consequent production of autoantibodies and failure of regeneration. The objective of this work was to characterize the role of Reg1α proteins and anti-Reg1α antibodies as biomarkers of β-cell regeneration and damage. Serum levels of Reg1α protein were investigated in 87 type 1 diabetic subjects (31 newly diagnosed and 56 long standing), 63 type 2 diabetic subjects, 39 subjects with systemic lupus erythematosus (SLE), a nonpancreatic autoimmune disorder, and 64 healthy subjects. The presence of anti-Reg1α antibodies and correlation with metabolic, immune, and genetic parameters were analyzed in diabetic subjects. Increased levels of Reg1α protein were observed in newly diagnosed (p=0.002), and long standing (p=0.001) type 1 diabetes patients and type 2 diabetic subjects (p<0.001). Anti-Reg1α antibodies were found in 47% of patients with type 1 diabetes. No correlation was found with metabolic, immune, and genetic parameters. Patients with SLE showed no increase in Reg1α protein. We report here for the first time raised serum Reg1α protein in type 1 and type 2 diabetes and anti-Reg1α antibodies in type 1 diabetes. Reg1α levels appear not to be influenced by genetic or metabolic control. These findings allow considering future studies on Reg1α protein and autoantibody as new tools in the evaluation and monitoring of β-cells regeneration and autoimmunity.

Ultrasound of the salivary glands is a strong predictor of labial gland biopsy histopathology in patients with sicca symptoms

BACKGROUND The international classification criteria for Sjögrens syndrome necessitate the presence of either extractable nuclear antibody or a characteristic focal inflammatory infiltrate in a minor salivary gland. Thus, patients who are extractable nuclear antibody-negative will need to have a labial salivary gland biopsy, which is an invasive procedure associated with morbidity. The aim of this study was to evaluate the viability of ultrasound imaging of the major salivary glands as a predictor of the histology to explore whether ultrasound can help in stratifying Sjögrens patients and reduce the need for biopsy. METHODS The records of 85 patients suspected of having Sjögrens syndrome and who have had biopsy and ultrasound were analysed retrospectively. The histology and the ultrasound were reported by experts independently. The reporting was impartial as the examiners were blinded to the results of the other investigations and to the diagnosis. RESULTS Out of the 85 patients, 34 had positive ultrasound, 29 of whom also had positive histology. Fifty-one patients had negative ultrasound, of whom 49 were also negative for histological features of Sjögrens syndrome. The results show that the ultrasound had a positive predictive value of 85% and a striking negative predicative value of 96% of the histology results. The overall concordance between the ultrasound and the histology was 91% (Kappa = 0.826). CONCLUSIONS Our study shows that potentially the ultrasound has a role in stratifying patients who are extractable nuclear antibody-negative and can help to prioritize the biopsy for those who have sonographic evidence of SS.

CX3CL1 and CX3CR1 expression in tertiary lymphoid structures in salivary gland infiltrates: fractalkine contribution to lymphoid neogenesis in Sjögren’s syndrome

OBJECTIVES Primary SS is an autoimmune disease characterized by chronic lymphocytic inflammation and ectopic germinal centre (GC) formation within salivary glands. Fractalkine (CX3CL1), associated with the pathogenesis of RA, is the sole member of the CX3C chemokine (CK) family and acts as an adhesion and chemotactic molecule. The objectives of this work are to determine to what extent CX3CL1 and its receptor CX3CR1 expression might be altered in salivary glands obtained from patients and to establish whether these CKs might be involved in SS ectopic lymphoneogenesis. METHODS We assessed the presence of CX3CL1 protein in sera by ELISA in 21 patients with primary SS, 11 patients with Sicca syndrome (Sicca), 20 RA patients and 10 blood donors. Histological evaluation was performed on sequential sections of salivary gland tissue. Using TaqMan RT-PCR we studied CX3CL1 and CX3CR1 mRNA expression in salivary gland tissues from a molecular point of view. RESULTS Increased serum levels of CX3CL1 protein were observed in SS patients compared with controls (P < 0.0001) and in RA patients compared with controls (P < 0.0001), but no difference was found between Sicca patients and controls (P = 0.22). We identified histologically the cells expressing CX3CL1 and CX3CR1 in salivary glands of SS patients and we localized the molecule within tertiary lymphoid structures. Finally, the mRNA levels of the CK and its receptor were up-regulated in SS salivary glands. CONCLUSION We believe that our findings point to the need for future studies on CX3CL1 and CX3CR1 proteins as contributors to the formation of ectopic GCs and possibly as a new tool in the evaluation and diagnosis of SS.

Blue eyes as a risk factor for type 1 diabetes

A high frequency of blue eyes and fair skin are reported in northern European Caucasians with type 1 diabetes (T1D). Also there is an inverse relationship between latitude and T1D incidence. We determined whether iris colour and skin pigmentation are risk factors in a Caucasian population living in two Mediterranean regions located at the same latitude with higher ultraviolet B irradiance, but with different T1D incidence.

The British Society for Rheumatology guideline for the management of adults with primary Sjögren’s Syndrome

Primary Sjogren’s Syndrome (pSS) is a classic, immunemediated, condition of unknown aetiology characterized by focal lymphocytic infiltration of exocrine glands [1]. Patients characteristically complain of drying of the eyes and mucosal surfaces along with fatigue and arthralgia. There is an association with autoimmune thyroid disease, coeliac disease and primary biliary cirrhosis. Systemic features include inflammatory arthritis, scLE, immune thrombocytopenia (ITP), vasculitis with purpura, salivary gland inflammation, neuropathies, interstitial lung disease (ILD) and a 5 10% lifetime risk of B cell lymphoma [2, 3]. This guideline reviews the treatment of the glandular and systemic features of pSS The management of the glandular features includes conserving, replacing and stimulating secretions. Systemic features may require system-specific therapy and immunomodulatory treatment. Holistic

SAT0378 Autophagy is Up-Regulated in the Salivary Glands of Primary Sjogren's Syndrome Patients and Correlates with the Focus Score and Disease Activity

Background Autophagy is now considered as a major regulator in trafficking events that activates innate and adaptive immunity and consistent evidence supports its role in autoimmunity (1). Primary Sjogrens syndrome (pSS) is a systemic autoimmune disease characterized by infiltration of exocrine glands by T and B cells that, producing chemokines and cytokines, coordinate the chronic inflammatory process. No data on the role of autophagy in pSS are available in humans, although studies in mice demonstrated its involvement in the salivary and lacrimal gland homeostasis (2,3). Objectives We investigated the autophagy process in salivary gland tissue and in peripheral T lymphocytes from pSS patients to evaluate its possible implication in the pathogenesis of the disease. Methods 30 patients with pSS, 20 patients with sicca syndrome or non-specific-chronic-sialoadenitis and 30 healthy donors were studied. Peripheral T lymphocytes were isolated by standard procedures. Salivary gland biopsies were evaluated by i) H&E to assess histological pattern, the severity of inflammatory infiltrate and the presence of germinal centers, ii) RT-PCR for the expression of autophagy-related genes and IL-23p19 and IL-21 mRNA. Autophagy-related proteins (LC3, Atg5, p62/SQSTM1) were detected in peripheral T lymphocytes by western blot and in salivary gland by immunohistochemistry and immunofluorescence. IL-21 and IL-23p19 serum levels were measured by ELISA. Results Autophagy is up-regulated in T cells from the salivary glands, but not from the peripheral blood, of pSS patients and it is correlated with disease activity and damage indexes. Autophagy is also correlated with the local expression of the pro-inflammatory cytokines IL-21 and IL-23p19, but not with serum levels of these cytokines. Conclusions Our data show that, in pSS, T cells present high levels of autophagy, which may up-regulate the expression of pro-inflammatory cytokines, providing evidence for a role of this process in the pathogenesis of pSS and identifying a possible therapeutic target. References Pierdominici M, Vomero M, Barbati C et al. FASEB J. 2012; 26: 1400-1412. Morgan-Bathke M, Lin HH, Chibly AM et al. J Dent Res. 2013; 92: 911-917. Seo Y, Ji YW, Lee SM, et al. Cell Death Dis. 2014; 5: e1309. Disclosure of Interest None declared

THU0008 A Negative High-Resolution Salivary Gland Ultrasound is Highly Predictive of Negative Labial Gland Biopsy in Patients with SICCA Symptoms

Background Sjögrens syndrome (SS) is a chronic autoimmune disease that affects salivary and lacrimal glands and results in xerostomia and xerophtalmia. Current criteria require the presence of either anti-Ro/La antibodies (ENA) or a positive focus score at labial salivary gland biopsy (LSGB) for SS classification [1]. Salivary gland biopsy is a highly specific marker for the diagnosis of SS but as an invasive procedure it is often indicated only in ENA- patients with sicca symptoms. Objectives Here we investigated the capacity of high-resolution salivary gland ultrasound (US) to predict the result of a LSGB biopsy in a consecutive cohort of patient with sicca. Methods Eighty-five consecutive patients attending the Combined Oral Medicine/Rheumatology SS Clinic at Barts and The London NHS Trust who underwent US and LSGB were recruited in the study. All patients displayed subjective and objective symptoms and signs of sicca. US imaging was scored as described by Salaffi et al. [2] while LSGB were scored following immunohistology, including CD3/CD20/CD138/CD21 markers [3]. US and LSGB were scored blindly with the radiologist and the pathologist unaware of the clinical data. Results Of the 85 patients recruited, 36 fulfilled SS criteria (15 LSGB+ENA+, 16 LSGB+ENA−, 5 LSGB−ENA+) while 49 were classified as sicca (LSGB−ENA−). Within the whole cohort, abnormal US findings were observed in 34 patients, of these 29 displayed a positive LSGB. Concordance between US and LSGB was 91.76% (Kappa=0.826). Irrespectively of diagnosis and ENA status, the positive predictive value of having a positive LSGB with abnormal US findings was 85.29% whilst a negative US gave a negative LSGB predictive value of 96.08%. Conclusions Our results demonstrate that high-resolution salivary gland US has a high concordance with LSGB. In particular, a negative US result is highly predictive of a negative labial gland biopsy in patients with sicca symptoms. These data suggests that, particularly in ENA− patients, the role of US is to guide whether or not LSGB is indicated, thus avoiding an invasive procedure in patients with an extremely low chance of a positive result. Thus, we propose that US should be considered in the SS diagnostic algorithm as a screening test before LSGB is performed. References Vitali C, Bombardieri S, Jonsson R, Moutsopoulos HM, Alexander EL, Carsons SE, et al. Classification criteria for Sjogrens syndrome: a revised version of the European criteria proposed by the American-European Consensus Group. Ann Rheum Dis. 2002 Salaffi F, Carotti M, Iagnocco A, Luccioli F, Ramonda R, Sabatini E, et al. Ultrasonography of salivary glands in primary Sjogrens syndrome: a comparison with contrast sialography and scintigraphy. Rheumatology (Oxford). 2008 Barone F, Bombardieri M, Rosado MM, Morgan PR, Challacombe SJ, De Vita S, et al. CXCL13, CCL21, and CXCL12 expression in salivary glands of patients with Sjogrens syndrome and MALT lymphoma: association with reactive and malignant areas of lymphoid organization. J Immunol. 2008 Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.3114

Diabetes-Related Autoantibodies in Children With Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common subtype of leukemia in children. Although ALL and type 1 diabetes appear to be biologically unrelated, there are common threads in both epidemiology and etiology. Rising incidence rates of both ALL (1) and type 1 diabetes (2) observed over recent decades in many Western countries seem to support common etiological factors (3). In the current study, we report on diabetes-related autoantibodies (Abs) in a group of patients with ALL. Thirty-four consecutive children (19 males and 15 females, mean age 6.2 ± 4.6 years) were referred to our institution in 2004 for newly diagnosed ALL. Patients were tested for Abs to islet and thyroid antigens. After the initial investigation and treatment, 31/34 (91%) patients (3 died in the mean time) were followedup for 6 years to evaluate the evolution of the autoimmune markers and progression toward type 1 diabetes. Glutamic acid decarboxylase (GAD) Abs by direct …