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Background: BK virus (BKV) is a ubiquitous polyoma virus that lies dormant in the genitourinary tract once acquired in early childhood. In states of cellular immunodeficiency, the virus can reactivate to cause hemorrhagic cystitis and nephritis. Children with systemic lupus erythematosus (SLE) have an increased risk of developing infectious complications secondary to their immunocompromised state from the administration of several immuno-modulatory drugs. Currently, there are no data regarding the prevalence of BK viruria or viremia in children with SLE. Methods: We conducted a prospective cohort study involving children with SLE of 18 years and younger. We obtained urine and blood samples at baseline and every 3 months up to 1 year for BK virus detection by real-time, quantitative polymerase chain reaction analysis. A comprehensive review of demographic information, clinical characteristics and medication history was also obtained. Results: Thirty-two pediatric patients (26 females and 6 males) with SLE were enrolled. Median age at the time of SLE diagnosis and enrollment into study was 13.6 years and 16.0 years old, respectively. The prevalence at enrollment was 3.1% (1/32) for BK viruria and 6.2% (2/32) for BK viremia. During the study period, 3 patients had viruria, 5 had viremia and 4 had both viruria and viremia. Of the 12 patients with BKV reactivation, only one was positive for microscopic hematuria, all others were asymptomatic. A total of nine of 97(9.2%) urine samples and 10 of 96(10.4%) blood samples were positive for BK virus. The most commonly utilized biologics in this cohort group were Rituximab (90.6%), Abatacept (12.5%), and Belimumab (9.3%). The type of medication exposure and clinical characteristics did not statistically differ between the groups that did or did not have BK viruria and/or viremia. Conclusions: Our study suggests that pediatric patients with SLE have BK viremia and/or viruria at a higher rate than the general healthy population, although the significance of the reactivation and viral level is unclear. The influence of immune-modulatory drugs on BKV reactivation is still uncertain. To understand the interplay amongst BK virus, immunosuppression and dysregulated immune system in children with SLE, ongoing research in a larger population is still warranted, which may help establish proper surveillance, diagnosis and treatment for BKV infection. [ABSTRACT FROM AUTHOR]

Background: Anti-SSA/Ro60 and anti-SSB/La are essential serological biomarkers for rheumatic diseases, specifically Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE). Currently, laboratory detection technology and platforms are designed with an emphasis on high-throughput methodology; therefore, the relationship of sensitivity with specificity remains a significant area for improvement. In this study, we used single-cell antibody nanowells (SCAN) technology to directly profile individual B cells producing antibodies against specific autoantigens such as SSA/Ro60 and SSB/La., Methods: Peripheral blood mononuclear cells were isolated using Ficoll gradient. Fluorescently labeled cells were added to fabricated nanowells and imaged using a high-speed epifluorescence microscope. The microengraving process was conducted using printed slides coated with immunoglobulins. Printed slides were hybridized with fluorescence-conjugated immunoglobulin G (IgG), SSA/Ro60, and SSB/La antigens. Microarray spots were analyzed for nanowells with single live B cells that produced antigen-specific autoantibodies., Results: Our results indicate that SCAN can simultaneously detect high frequencies of anti-SSA/Ro60 and anti-SSB/La with a specific IgG isotype in peripheral blood mononuclear cells of patients, as well as measure their individual secretion levels. The data showed that patients with SS and SLE exhibited higher frequency and greater concentration of anti-SSA/Ro60- and anti-SSB/La-producing B cells in the IgG isotype. Furthermore, individual B cells of patients produced higher levels of IgG-specific anti-SSA/Ro60 autoantibody, but not IgG-specific anti-SSB/La autoantibody, compared with healthy control subjects., Conclusions: These results support the application of SCAN as a robust multiparametric analytical bioassay that can directly measure secretion of autoantibody and accurately report antigen-specific, autoantibody-producing cells.

Introduction: Sjögren's syndrome (SjS) is a systemic autoimmune disease characterized by decreased salivary and lacrimal gland secretions, resulting in severe dry mouth and dry eyes. Recent studies have suggested that TH17 cells and its signature cytokine IL-17 are involved in the underlying pathogenic mechanisms leading to destructive inflammation and autoimmunity. In the present study, we examined whether IL-27, a natural inhibitor of TH17 activity, could down-regulate or reverse SjS in C57BL/6.NOD-Aec1Aec2 mice, a model of primary-SjS., Methods: Recombinant serotype 2 adeno-associated viral (AAV2) vectors expressing either IL-27 (rAAV2-IL27) or LacZ (rAAV2-LacZ) were injected into 6 or 14 week-old C57BL/6.NOD-Aec1Aec2 mice. Changes in IL-27, IL-17, and IL-10 cytokine levels in peripheral blood were determined by ELISAs, while flow cytometry analyses were used to quantify cytokine-positive splenocytes. Histological assessment of salivary glands, anti-nuclear autoantibody (ANA) staining, and stimulated saliva flow rates were used to profile SjS disease severity., Results: Mice systemically treated with intravenous rAAV2-IL27 injections at either 6 or 14 weeks of age exhibited long-term elevated levels of serum IL-27 with concomitantly reduced levels of IL-17 compared with sera from mice injected with rAAV2-LacZ or saline out to 20 weeks post-inoculation. Most importantly, disease profiles revealed that rAAV2-IL27 treatment had little effect on lymphocytic focus (LF) scores, but resulted in structural changes in LF, lower titers of ANAs with changes in staining patterns, and a less severe clinical disease as determined by saliva flow rates., Conclusions: These data support the concept that IL-27, when provided exogenously, can induce a suppressive effect on SjS development and thus may be an effective therapeutic agent for regulating TH17 pro-inflammatory activity in autoimmune diseases where the TH17 system has been shown to play an important role in their pathogenesis.

Introduction: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a key negative costimulatory molecule that displays a wide range of anti-inflammatory properties and is currently approved to treat rheumatoid arthritis as a recombinant fusion protein (CTLA4IgG). To better understand the role of CTLA4IgG in primary Sjögren's syndrome (pSS), we generated a recombinant adeno-associated virus vector serotype 2 (AAV2) expressing a chimera of mouse CTLA-4 fused with a human immunoglobulin (AAV2-CTLA4IgG) and observed the effect of this molecule in C57BL/6.NOD-Aec1Aec2 mice, an animal model of pSS., Methods: A recombinant adeno-associated virus-2 (AAV-2) vector was constructed encoding a CTLA4IgG fusion protein. The AAV2-CTLA4IgG vector and an AAV2 control vector encoding beta galactosidase (LacZ) were administered by retrograde cannulation of the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice. Protein expression was measured by ELISA and salivary glands were assessed for inflammation and activity., Results: Recombinant CTLA4IgG blocked B7 expression on macrophages in vitro. In vivo, localized expression of CTLA4IgG in the salivary glands of C57BL/6.NOD-Aec1Aec2 mice inhibited the loss of salivary gland activity and decreased T and B cell infiltration as well as dendritic cells and macrophages in the glands compared with control mice. In addition a decrease in several proinflammatory cytokines and an increase in transforming growth factor beta-1 (TGF-β1) expression were also observed., Conclusions: These data suggest expression of CTLA4IgG in the salivary gland can decrease the inflammation and improve the xerostomia reported in these mice.

Introduction: Sjögren's syndrome (SS) involves a chronic, progressive inflammation primarily of the salivary and lacrimal glands leading to decreased levels of saliva and tears resulting in dry mouth and dry eye diseases. Seminal findings regarding TH17 cell populations that secrete predominantly interleukin (IL)-17A have been shown to play an important role in an increasing number of autoimmune diseases, including SS. In the present study, we investigated the function of IL-17A on the development and onset of SS., Methods: Adenovirus serotype 5 (Ad5) vectors expressing either IL-17A or LacZ were infused via retrograde cannulation into the salivary glands of C57BL/6J mice between 6 and 8 weeks of age or between 15 and 17 weeks of age. The mice were characterized for SS phenotypes., Results: Disease profiling indicated that SS-non-susceptible C57BL/6J mice whose salivary glands received the Ad5-IL17A vector developed a SS-like disease profile, including the appearance of lymphocytic foci, increased cytokine levels, changes in antinuclear antibody profiles, and temporal loss of saliva flow., Conclusions: Induction of SS pathology by IL-17A in SS-non-susceptible mice strongly suggests that IL-17A is an important inflammatory cytokine in salivary gland dysfunction. Thus, localized anti-IL17 therapy may be effective in preventing glandular dysfunction.

Introduction: Recently, we reported the development of the C57BL/6.NOD-Aec1Aec2 mouse that carries two genetic intervals derived from the non-obese diabetic (NOD) mouse capable of conferring Sjögren's syndrome (SjS)-like disease in SjS-non-susceptible C57BL/6 mice. In an attempt to define the molecular bases underlying the onset of stomatitis sicca (xerostomia) in this C57BL/6.NOD-Aec1Aec2 mouse model, we have carried out a study using genomic microarray technology., Methods: By means of oligonucleotide microarrays, gene expression profiles of salivary glands at 4, 8, 12, 16, and 20 weeks of age were generated for C57BL/6.NOD-Aec1Aec2 male mice. Using Linear Models for Microarray Analysis and B-statistics software, 480 genes were identified as being differentially expressed (P < 0.01 and Q < 0.0001) during the development of SjS-like disease in the salivary glands., Results: The 480 genes could be arranged into four clusters, with each cluster defining a unique pattern of temporal expression, while the individual genes within each cluster could be grouped according to related biological functions. By means of pair-wise analysis, temporal changes in transcript expressions provided profiles indicating that many additional genes are differentially expressed at specific time points during the development of disease. Multiple genes reportedly showing an association with autoimmunity and/or SjS, in either humans or mouse models, were found to exhibit differential expressions, both quantitatively and temporally. Selecting various families of genes associated with specific functions (for example, antibody production, complement, and chemokines), we noted that only a limited number of family members showed differential expressions and these correlated with specific phases of disease., Conclusions: Taking advantage of known functions of these genes, investigators can construct interactive gene pathways, leading to modeling of possible underlying events inducing salivary gland dysfunction. Thus, these different approaches to analyzing microarray data permit the identification of multiple sets of genes of interest whose expressions and expression profiles may correlate with molecular mechanisms, signaling pathways, and/or immunological processes involved in the development and onset of SjS.

Introduction: Sjögren syndrome (SjS) is a systemic autoimmune disease in which an immunological attack primarily against the salivary and lacrimal glands results in the loss of acinar cell tissue and function, leading to stomatitis sicca and keratoconjunctivitis sicca. In recent years, two genetic regions, one on chromosome 1 (designated autoimmune exocrinopathy 2 or Aec2) and the second on chromosome 3 (designated autoimmune exocrinopathy 1 or Aec1) derived from nonobese diabetic (NOD) mice, have been shown to be necessary and sufficient to replicate SjS-like disease in nonsusceptible C57BL/6 mice., Methods: Starting with the SjS-susceptible C57BL/6-derived mouse, referred to as C57BL/6.NOD-Aec1Aec2, we generated a large set of recombinant inbred (RI) lines containing portions of Aec2 as a means of identifying more precisely the genetic elements of chromosome 1 responsible for disease development., Results: Disease profiling of these RI lines has revealed that the SjS susceptibility genes of Aec2 lie within a region located at approximately 79 +/- 5 cM distal to the centromere, as defined by microsatellite markers. This chromosomal region contains several sets of genes known to correlate with various immunopathological features of SjS as well as disease susceptibility genes for both type 1 diabetes and systemic lupus erythematosus in mice. One gene in particular, tumor necrosis factor (ligand) superfamily member 4 (or Ox40 ligand), encoding a product whose biological functions correlate with both physiological homeostasis and immune regulations, could be a potential candidate SjS susceptibility gene., Conclusions: These new RI lines represent the first step not only in fine mapping SjS susceptibility loci but also in identifying potential candidate SjS susceptibility genes. Identification of possible candidate genes permits construction of models describing underlying molecular pathogenic mechanisms in this model of SjS and establishes a basis for construction of specific gene knockout mice.