FlowJo (Tree Star) was used for gating analysis. Luciferase Immunoprecipitation Systems (LIPS) Assay LIPS assay was performed as previously described . of with a pSS-like transcript profile. Moreover, UM B cell gene expression analysis identified 187 differentially expressed genes between pSS and HCs. Conclusion A decrease in UM B cells is usually characteristic of established pSS as well as with serologic hyperactivity thereby suggesting their value as biomarkers of future disease progression and in understanding disease pathogenesis. Overall, the mRNA transcript analysis of UM B cells suggests their activation in pSS through innate immune pathways in the context of attenuated antigen-mediated adaptive signaling. Thus, our findings provide important insight into the mechanisms and potential consequences of decreased UM B cell in pSS. Primary Sj?grens Syndrome (pSS) is a systemic autoimmune disease characterized by abnormal lymphocytic infiltration in the lacrimal and the salivary glands. The most prevalent and distinctive features of this disease are keratoconjunctivitis (dry eyes) and xerostomia (dry mouth) – symptoms. pSS patients can also suffer from extra-glandular manifestations that may either precede full-blown disease or present late in the course of the disease . Nevertheless, symptoms can be present in Dihydroartemisinin the general population frequently accompanied by immunological abnormalities but in the absence of obvious autoimmune disease. Thus, in absence of definitive diagnostic assessments, early diagnosis of pSS is usually difficult to make . Accordingly, classification criteria have been proposed to assess disease activity and provide a more homogeneous case classification for research studies . However, these classification criteria often fail to capture patients early in the course of the disease C before underlying immunological mechanisms lead to destructive pathology. A role for B cells in the pathogenesis of pSS is usually strongly indicated by multiple lines of evidence, including elevated levels of total serum immunoglobulin, high levels of several autoantibodies, and greatly increased levels of B cell survival and differentiation factors like BAFF (B cell Activating Factor) and IL-21 [4C6]. Additionally, pSS patients have major disturbances of peripheral-blood B cell homeostasis [7C9] and have lymphocytic infiltrates in the salivary glands that frequently include the presence of ectopic germinal-center reactions . The pathogenic significance of B cells is also supported by promising results obtained by B cell-targeting therapies [11, 12]. The precise contribution of B cells to pSS pathology remains to be fully understood, as is the potential diagnostic value of the observed B cell abnormalities. Studies of B cell profiling in pSS typically concentrate on univariate analysis of B cell populations in patients with definitive diagnosis. However, given disease heterogeneity and the multiple, often opposing functions of B cell populations [13, 14], it is important to understand the global B cell profile of autoimmune diseases. In this work, we examined the B cell memory phenotypic and gene expression profile of patients with a wide spectrum of disease. We found the loss of Dihydroartemisinin unswitched (IgDpos/CD27pos) memory B cells was associated with clinical disease indicators in pSS and that this loss was present in a subset of patients lacking a conclusive pSS diagnosis. Furthermore, gene expression studies demonstrate unswitched memory B cells from pSS patients had an altered profile characterized by lower expression of cell signaling genes necessary for adaptive immunity. These findings may provide a model for eventual advanced diagnostics and rational design of B cell targeted therapies. Patients and Methods Study Subjects This study was approved by the University of Rochester Research Subject Review Board and all subjects provided informed consent. Blood was drawn Dihydroartemisinin from 26 pSS patients meeting AECG classification criteria  (pSS), 27 patients and 22 healthy control donors. patients comprised the following subgroups: patients with Sirt7 symptoms in the context of a clinical diagnosis of pSS established by the managing rheumatologist but without fulfilling AECG criteria (non-AECG pSS; n=4); and patients with symptoms but neither clinical nor AECG-based diagnosis of pSS (patients included 27 females, and healthy controls included 22 females. Patient characteristics are detailed in Table 1. For microarray analysis, whole blood was acquired from 5 pSS patients defined by AECG classification criteria, 5 patients, and 3-5 healthy control donors per experiment. Table 1 Patient Characteristics symptoms (meanSD)27.5 117 6.01NSAIDS (%)1511Corticosteroids (%)87Hydroxychloroquine (%)3115Methotrexate (%)40 Open in a separate window RF = rheumatoid factor; ANA = antinuclear antibody WBC = white blood cell; ESR = erythrocyte sedimentation rate Sample Preparation Peripheral blood mononuclear.