Analysis of novel Sjogren's syndrome autoantibodies in patients with dry eyes.

BACKGROUND: Dry eye is a common problem in Ophthalmology and may occur for many reasons including Sjogren's syndrome (SS). Recent studies have identified autoantibodies, anti-salivary gland protein 1 (SP1), anti-carbonic anhydrase 6 (CA6) and anti-parotid secretory protein (PSP), which occur early in the course of SS. The current studies were designed to evaluate how many patients with idiopathic dry eye and no evidence of systemic diseases from a dry eye practice have these autoantibodies. METHODS: Patients from a dry eye clinic and normal controls were assessed by Schirmer's test for tear flow. Sera were assessed for autoantibodies using ELISA assays. Statistics was performed with Prism 7 software and student's unpaired t test. RESULTS: In this study 60% of the dry eye patients expressed one of these autoantibodies. Only 30% expressed one of the autoantibodies associated with long-standing SS, which are included in the diagnostic criteria for SS, anti-Ro and anti-La. Patients with disease for less than 2 years and mild dry eyes did not express anti-Ro or anti-La, while 25% expressed anti-SP1. Similar observations, with smaller numbers, were made when patients had not only dry eye but also dry mouth. CONCLUSIONS: Antibodies to SP1, CA6 and PSP occur in some patients with idiopathic dry eyes. Further studies will be needed to determine how many of these patients go on to develop systemic manifestations of SS. Testing for these autoantibodies may allow early recognition of patients with SS. This will lead to improved management of the patients and the development of new strategies to maintain normal lacrimal and salivary gland function in patients with SS.

Different stages of primary Sjogren's syndrome involving lymphotoxin and type 1 IFN.

Primary Sjögren's syndrome (pSS) is a complex autoimmune disease starting in the salivary and lacrimal glands and continuing to involve the lungs and kidneys with the eventual development of lymphoma. Many studies have emphasized the role of type 1 IFN (IFN-α) and lymphotoxin α (LTα) in the pathogenesis of the disease. The present studies were designed to delineate the role of IFN-α in pSS using an animal model, the IL-14α (IL14αTG) transgenic mouse. IL14αTG mice lacking the type 1 IFNR (IL14αTG.IFNR(-/-)) had the same submandibular gland and lacrimal gland injury as did the IL14αTG mice, but they lacked the later parotid gland and lung injury. Development of lymphoma was delayed in IL14αTG.IFNR(-/-) mice. The switch from IgM to IgG autoantibodies as well as the increase in serum IgG2a seen is IL14αTG mice was inhibited in IL14αTG.IFNR(-/-) mice. Production of LTα was identified in both IL14αTG mice and IL14αTG.IFNR(-/-) mice at the time that salivary gland injury was occurring. These and previous studies suggest a model for pSS that separates the disease into several stages: 1) initial injury to the submandibular and lacrimal glands via an environmental insult and LTα; 2) amplification of local injury via the production of type 1 IFN; injury to the parotid glands, lungs, and kidneys is seen; 3) progression of systemic inflammation with the eventual development of large B cell lymphoma. Understanding these different stages will help to develop strategies for treatment of patients with pSS based on the status of their disease.

Investigation of novel autoantibodies in Sjogren's syndrome utilizing Sera from the Sjogren's international collaborative clinical alliance cohort.

BACKGROUND: Sjogren's syndrome (SS) is a chronic autoimmune disease mainly affecting salivary and lacrimal glands. Current diagnostic criteria for SS utilize anti-Ro and anti-La as serological markers. Animal models for SS have identified novel autoantibodies, anti-salivary gland protein 1 (SP1), anti-carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP). These novel antibodies are seen in the animals at an earlier stage of SS than anti-Ro and anti-La. The current studies were designed to evaluate these novel autoantibodies in the sera of well-characterized patients with dry eyes and dry mouth and lip biopsies from the Sjogren's International Collaborative Clinical Alliance (SICCA) to determine if they indeed identify SS with less severe disease than patients expressing anti-Ro and anti-La. METHODS: Sera were obtained from SICCA registry in patients for whom lymphocytic foci per 4 mm(2) on the lip biopsies was either 0 (F = 0), <1 (F <1) or > 3 (F >3). ELISA assays were utilized to evaluate these sera for anti-Ro, anti-La, anti-SP1, anti-CA6, and anti-PSP. RESULTS: In patients with dry eyes and dry mouth but F = 0, increased expression of anti- CA6 was noted compared to the F <1 group (p = .032) or the F > 3 group (p = .006). Neither anti-PSP nor anti-SP1 reached statistical significance because of the small numbers in the F0 group, although there was a trend for their expression to be higher in the F0 group. On the other hand, the expression of anti-Ro was significantly reduced in the F0 group compared to the F <1 (p = .0021) and F > 3 (p = .0003) groups. The reduced expression of anti-La in the F0 group compared to the F <1 and F > 3 groups did not quite reach statistical significance. CONCLUSIONS: Anti-Ro and anti-La identify patients with SS and more severe disease than anti-SP1, anti-CA6, and anti-PSP. More studies are needed to identify the timing in the course of SS when these different autoantibodies are expressed and/or whether they are expressed in patients with different clinical manifestations.

Evaluation of salivary gland protein 1 antibodies in patients with primary and secondary Sjogren's syndrome.

Sjogren's syndrome (SS) has been associated with the expression of anti-Ro and anti-La antibodies. Anti-salivary gland protein 1 (SP1) antibodies have recently been identified in patients with SS. The current work involved a cross sectional study to determine whether anti-SP1 antibodies were identified in particular subgroups of patients with SS. The results of this study revealed that anti-SP1 antibodies were present in the sera of 52% of SS patients while anti-Ro/anti-La was present in 63% of patients. 19% of patients had anti-SP1 without anti-Ro/anti-La. Patients with SS and lymphoma expressed anti-Ro, anti-La and anti-SP1 together. In SS associated with RA, 50% had antibodies anti-SP1 while 40% had anti-Ro/anti-La. In conclusion, anti-SP1 antibodies are commonly seen in both primary and secondary SS and rarely in normal controls. Future studies are needed to determine the roles and timing of expression of anti-SP1 antibodies in Sjogren's syndrome.

Chromatin dynamics in living cells: identification of oscillatory motion.

Genomic DNA in mammalian cells is organized into ~1 Mbp chromatin domains (ChrD) which represent the basic structural units for DNA compaction, replication, and transcription. Remarkably, ChrD are highly dynamic and undergo both translational movement and configurational changes. In this study, we introduce an automated motion tracking analysis to measure, both in 2D and 3D, the linear displacement of early, mid and late S-phase replicated ChrD over short time periods (<1 sec). We conclude that previously identified large-scale transitions in the spatial position and configuration of chromatin, originate from asymmetric oscillations of the ChrD detectable in fractions of a second. The rapid oscillatory motion correlates with the replication timing of the ChrD with early S replicated ChrD showing the highest levels of motion and late S-phase chromatin the lowest. Virtually identical levels of oscillatory motion were detected when ChrD were measured during active DNA replication or during inhibition of transcription with DRB or α-amanitin. While this motion is energy independent, the oscillations of early S and mid S, but not late S replicated chromatin, are reduced by cell permeabilization. This suggests involvement of soluble factors in the regulation of chromatin dynamics. The DNA intercalating agent actinomycin D also significantly inhibits early S-labeled chromatin oscillation. We propose that rapid asymmetric oscillations of <1 sec are the basis for translational movements and configurational changes in ChrD previously detected over time spans of minutes-hours, and are the result of both the stochastic collisions of macromolecules and specific molecular interactions.

Temporal histological changes in lacrimal and major salivary glands in mouse models of Sjogren's syndrome.

BACKGROUND: Evidence in imaging studies suggests that there may be differences in glandular involvement in Sjogren's syndrome (SS) depending on the stage of the disease. No detailed histological studies are available to show if there are any such difference in glandular involvement at various time periods and stages of SS. This cross sectional study examines the inflammatory changes in mouse models of SS at various ages. METHODS: The histological changes in major salivary and lacrimal glands were studied at ages of 3, 6, 9, 12, 15 and 18 months in both sexes in well characterized mouse models of SS, non-obese diabetes mouse and Interleukin-14 alpha-transgenic mice. RESULTS: Our results indicate that early inflammation concurrently occur in submandibular and lacrimal glands around the age of 6 weeks. Parotid glands are involved much later in the course of SS with less severe inflammation. Sublingual glands are rarely involved. CONCLUSIONS: Our conclusions are that SS may be an organ specific disease with early inflammation occurring in submandibular and lacrimal glands, followed by the parotid. Non organ specific events occur in later courses of the disease. The understanding of the disease progression is important in tailoring early local therapeutic interventions before complete destruction of salivary and lacrimal glands.

Novel autoantibodies in Sjogren's syndrome.

Sjogren's syndrome (SS) is defined by autoantibodies to Ro and La. The current studies identified additional autoantibodies in SS to salivary gland protein 1 (SP-1), carbonic anhydrase 6 (CA6) and parotid secretory protein (PSP). These autoantibodies were present in two animal models for SS and occurred earlier in the course of the disease than antibodies to Ro or La. Patients with SS also produced antibodies to SP-1, CA6 and PSP. These antibodies were found in 45% of patients meeting the criteria for SS who lacked antibodies to Ro or La. Furthermore, in patients with idiopathic xerostomia and xerophthalmia for less than 2 years, 76% had antibodies to SP-1 and/or CA6 while only 31% had antibodies to Ro or La. Antibodies to SP-1, CA6 and PSP may be useful markers for identifying patients with SS at early stages of the disease or those that lack antibodies to either Ro or La.

Profiling of Myositis Specific Antibodies and Composite Scores as an Aid in the Differential Diagnosis of Autoimmune Myopathies.

(1) Background: Myositis specific antibodies (MSA) represent important diagnostic and stratification tools in idiopathic inflammatory myositis (IIM) patients. Here we aimed to evaluate the clinical performance of MSA profiled by a novel particle based multi-analyte technology (PMAT) in IIM and subsets thereof. (2) Methods: 264 IIM patients and 200 controls were tested for MSA using PMAT (Inova Diagnostics, research use only). Diagnostic performance was analyzed and composite scores were generated. (3) Results: The sensitivity/specificity of the individual MSA were: 19.7%/100% (Jo-1), 7.2%/100.0% (Mi-2), 3.0%/99.0% (NXP2), 3.8%/100.0% (SAE), 2.7%/100.0% (PL-7), 1.9%/99.5 (PL-12), 1.1%/100.0% (EJ), 15.5%/99.5% (TIF1γ), 8.3%/98.5% (MDA5), 6.1%/99.0% (HMGCR) and 1.9%/98.5% (SRP). Of all IIM patients, 180/264 tested positive for at least one of the MSAs. In the individual control group, 12/200 (6.0%) tested positive for at least one MSA, most of which had levels close to the cut-off (except one SRP and one PL-12). Only 6/264 (2.3%) IIM patients were positive for more than one antibody (MDA5/HMGCR, EJ/PL-7, 2 x MDA5/TIF1γ, EJ/SAE, SAE/TIF1γ). The overall sensitivity was 68.2% paired with a specificity of 94.0%, leading to an odds ratio of 33.8. The composite scores showed good discrimination between subgroups (e.g., anti-synthetase syndrome). (4) Conclusion: MSA, especially when combined in composite scores (here measured by PMAT), provide value in stratification of patients with IIM.

The search for an autoimmune origin of psychotic disorders: Prevalence of autoantibodies against hippocampus antigens, glutamic acid decarboxylase and nuclear antigens.

The etiology of psychotic disorders is still unknown, but in a subgroup of patients symptoms might be caused by an autoimmune reaction. In this study, we tested patterns of autoimmune reactivity against potentially novel hippocampal antigens. Serum of a cohort of 621 individuals with psychotic disorders and 257 controls were first tested for reactivity on neuropil of rat brain sections. Brain reactive sera (67 diseased, 27 healthy) were further tested for antibody binding to glutamic acid decarboxylase (GAD) isotype 65 and 67 by cell-based assay (CBA). A sub-cohort of 199 individuals with psychotic disorders and 152 controls was tested for the prevalence of anti-nuclear antibodies (ANA) on HEp2-substrate as well as for reactivity to double-stranded DNA, ribosomal P (RPP), and cardiolipin (CL). Incubation of rat brain with serum resulted in unidentified hippocampal binding patterns in both diseased and control groups. Upon screening with GAD CBA, one of these patterns was identified as GAD65 in one individual with schizophrenia and also in one healthy individual. Two diseased and two healthy individuals had low antibody levels targeting GAD67 by CBA. Antibody reactivity on HEp-2-substrate was increased in patients with schizoaffective disorder, but only in 3 patients did antibody testing hint at a possible diagnosis of systemic lupus erythematosus. Although reactivity of serum to intracellular antigens might be increased in patients with psychotic disorder, no specific targets could be identified. GAD antibodies are very rare and do not seem increased in serum of patients with psychotic disorders.

Chromatin dynamics is correlated with replication timing.

Discrete chromatin domains (ChrD), containing an average of approximately 1 Mbp DNA, represent the basic structural units for the regulation of DNA organization and replication in situ. In this study, a bio-computational approach is employed to simultaneously measure the translational motion of large populations of ChrD in the cell nucleus of living cells. Both movement and configurational changes are strikingly higher in early S-phase replicating ChrD compared to those that replicate in mid and late S-phase. The chromatin dynamics was not sensitive to transcription inhibition by alpha-amanitin but was significantly reduced by actinomycin D treatment. Since a majority of active genes replicate in early S-phase, our results suggest a correlation between levels of chromatin dynamics and chromatin poised for active transcription. Analysis of ChrD colocalization with transcription sites and cDNA with ChrD and transcription sites further supports this proposal.

Organization of the amplified type I interferon gene cluster and associated chromosome regions in the interphase nucleus of human osteosarcoma cells.

The organization of the amplified type I interferon (IFN) gene cluster and surrounding chromosomal regions was studied in the interphase cell nucleus of the human osteosarcoma cell line MG63. Rather than being arranged in a linear ladder-like array as in mitotic chromosomes, a cluster of approximately 15 foci was detected that was preferentially associated along the periphery of both the cell nucleus and a chromosome territory containing components of chromosomes 4, 8, and 9. Interspersed within the IFN gene foci were corresponding foci derived from amplified centromere 4 and 9 sequences. Other copies of chromosomes 4 and 8 were frequently detected in pairs or higher-order arrays lacking discrete borders between the chromosomes. In contrast, while chromosomes 4 and 8 in normal WI38 human fibroblast and osteoblast cells were occasionally found to associate closely, discrete boundaries were always detected between the two. DNA replication timing of the IFN gene cluster in early- to mid-S phase of WI38 cells was conserved in the amplified IFN gene cluster of MG63. Quantitative RT-PCR demonstrated a approximately 3-fold increase in IFN beta transcripts in MG63 compared with WI38 and RNA/DNA FISH experiments revealed 1-5 foci of IFN beta transcripts per cell with only approximately 5% of the cells showing foci within the highly amplified IFN gene cluster.

Matrin 3: chromosomal distribution and protein interactions.

Matrin 3 (matr3), an abundant protein of the internal nuclear matrix, has been linked to a variety of functional events. As a step toward defining its multifunctional nature, we have studied the association of matr3 with chromosome territories and identified potential interacting proteins. A similar staining pattern of matr3 was observed in fixed WI38 fibroblast cells and in live HeLa cells using a matr3-GFP construct. Matr3 was detected throughout autosomal and the active X chromosome territories. Conversely, matr3 was strikingly excluded from the inactive X chromosome as well as within both the perinuclear and perinucleolar heterochromatin. Yeast two hybrid analysis identified matr3 interactions with 33 unique nuclear localized proteins and also revealed its propensity for self association. A majority of these proteins are involved in RNA metabolism and chromatin remodeling while others function in protein translation, DNA replication/repair and apoptosis. Further analysis of a selection of these proteins and scaffold attachment factor A (SAFA) by co-localization and co-immunoprecipitation experiments using HeLa cells confirmed their interactions with matr3.

Spatio-temporal dynamics of replication and transcription sites in the mammalian cell nucleus.

To study when and where active genes replicated in early S phase are transcribed, a series of pulse-chase experiments are performed to label replicating chromatin domains (RS) in early S phase and subsequently transcription sites (TS) after chase periods of 0 to 24 h. Surprisingly, transcription activity throughout these chase periods did not show significant colocalization with early RS chromatin domains. Application of novel image segmentation and proximity algorithms, however, revealed close proximity of TS with the labeled chromatin domains independent of chase time. In addition, RNA polymerase II was highly proximal and showed significant colocalization with both TS and the chromatin domains. Based on these findings, we propose that chromatin activated for transcription dynamically unfolds or "loops out" of early RS chromatin domains where it can interact with RNA polymerase II and other components of the transcriptional machinery. Our results further suggest that the early RS chromatin domains are transcribing genes throughout the cell cycle and that multiple chromatin domains are organized around the same transcription factory.

Ladder-like amplification of the type I interferon gene cluster in the human osteosarcoma cell line MG63.

The organization of the type I interferon (IFN) gene cluster (9p21.3) was studied in a human osteosarcoma cell line (MG63). Array comparative genomic hybridization (aCGH) showed an amplification of approximately 6-fold which ended at both ends of the gene cluster with a deletion that extended throughout the 9p21.3 band. Spectral karyotyping (SKY) combined with fluorescence in-situ hybridization (FISH) identified an arrangement of the gene cluster in a ladder-like array of 5-7 'bands' spanning a single chromosome termed the 'IFN chromosome'. Chromosome painting revealed that the IFN chromosome is derived from components of chromosomes 4, 8 and 9. Labelling with centromeric probes demonstrated a ladder-like amplification of centromeric 4 and 9 sequences that co-localized with each other and a similar banding pattern of chromosome 4, as well as alternating with the IFN gene clusters. In contrast, centromere 8 was not detected on the IFN chromosome. One of the amplified centromeric 9 bands was identified as the functional centromere based on its location at the chromosome constriction and immunolocalization of the CENP-C protein. A model is presented for the generation of the IFN chromosome that involves breakage-fusion-bridge events.

Anti-DFS70 antibodies: an update on our current understanding and their clinical usefulness.

INTRODUCTION: Anti-DFS70 antibodies and their clinical associations remain an immunological paradox. Unlike other antinuclear antibodies (ANA), there is a growing body of evidence that anti-DFS70 antibodies, when present in high titers and in isolation (without accompanying other antibodies), are useful to aid in the exclusion of ANA associated rheumatic diseases. Areas covered: This review aims to analyze and interpret the current published knowledge and recent findings to provide guidance in the use of anti-DFS70 antibodies to analyze associations of this unique autoantibody. Expert commentary: Although the association of anti-DFS70 antibodies is still unknown, their use as a biomarker that can aid in the exclusion of ANA related diseases is well-established.

Anti-OJ autoantibodies: Rare or underdetected?

Anti-OJ autoantibodies are rare myositis-specific autoantibodies that have been described to target isoleucyl-tRNA synthetase. Routinely used multiplex assays perform poorly in detection of anti-OJ antibodies. In this manuscript, we review the existing literature on critical issues in detection of anti-OJ and the clinical features associated with anti-OJ. The challenging detection with line/blot immunoassays and ELISAs is most likely related to the characteristics of the autoantigen involved, which is part of a multi-enzyme synthetase complex. Anti-OJ autoantibodies might therefore be more aptly termed anti-OJ complex autoantibodies. Anti-OJ autoantibodies are associated with the anti-synthetase syndrome, with interstitial lung disease (ILD) frequently being the sole manifestation. Myositis, present in the majority of patients with anti-OJ antibodies, is more severe than in patients with other anti-aminoacyl-tRNA synthetases. Most patients respond to glucocorticoid therapy. As detection of anti-OJ is relevant for treatment, reliable and practical detection is needed. Meanwhile, clinicians need to be aware of the possibility of anti-OJ in patients with ILD, isolated or in combination with myositis.

Identifying functional neighborhoods within the cell nucleus: proximity analysis of early S-phase replicating chromatin domains to sites of transcription, RNA polymerase II, HP1gamma, matrin 3 and SAF-A.

Higher order chromatin organization in concert with epigenetic regulation is a key process that determines gene expression at the global level. The organization of dynamic chromatin domains and their associated protein factors is intertwined with nuclear function to create higher levels of functional zones within the cell nucleus. As a step towards elucidating the organization and dynamics of these functional zones, we have investigated the spatial proximities among a constellation of functionally related sites that are found within euchromatic regions of the cell nucleus including: HP1gamma, nascent transcript sites (TS), active DNA replicating sites in early S-phase (PCNA) and RNA polymerase II sites. We report close associations among these different sites with proximity values specific for each combination. Analysis of matrin 3 and SAF-A sites demonstrates that these nuclear matrix proteins are highly proximal with the functionally related sites as well as to each other and display closely aligned and overlapping regions following application of the minimal spanning tree (MST) algorithm to visualize higher order network-like patterns. Our findings suggest that multiple factors within the nuclear microenvironment collectively form higher order combinatorial arrays of function. We propose a model for the organization of these functional neighborhoods which takes into account the proximity values of the individual sites and their spatial organization within the nuclear architecture.

Simultaneous Distinction of Monospecific and Mixed DFS70 Patterns During ANA Screening with a Novel HEp-2 ELITE/DFS70 Knockout Substrate.

Systemic autoimmune connective tissue disorders are characterized by circulating antinuclear antibodies (ANA). Although there are several technologies available for ANA screening, indirect immunofluorescence (IIF) using Human epithelial cells-2 (HEp-2) substrate remains the primary and recommended method because of its superior sensitivity. HEp-2 substrates can detect a multitude of patterns resulting from autoantibody binding to various protein and nucleic acid autoantigens distributed throughout the nucleus and cytoplasm of the cells. The great diversity of monospecific and mixed patterns resulting from positive reactions on HEp-2 substrate also complicate the interpretation and accuracy of reporting. One specific example which received utmost attention recently is the dense fine speckled 70 (DFS70) pattern resulting from autoantibodies that specifically bind to a protein called lens epithelium derived growth factor (LEDGF). Lack of clear association with a specific systemic autoimmune disease and high prevalence in healthy populations have made accurate interpretation of DFS70 pattern important. Accurate distinction of DFS70 pattern from disease-associated patterns using conventional HEp-2 substrate is challenging. Moreover, frequent co-occurrence of DFS70 pattern along with disease-associated patterns such as homogeneous, speckled, and mixed homogeneous-speckled patterns complicate the IIF interpretation. The goal of this paper is to demonstrate the utility of a novel engineered HEp-2 IIF substrate that retains all advantages of conventional HEp-2 substrate while simultaneously providing the ability to distinguish DFS70 pattern with high confidence in both monospecific and mixed ANA positive examples. The new substrate is further able to unmask disease-associated ANA patterns previously concealed by DFS70 pattern.

Analysis of DFS70 pattern and impact on ANA screening using a novel HEp-2 ELITE/DFS70 knockout substrate.

Indirect immunofluorescence (IIF) using human epithelial cell (HEp-2) substrate is a widely used and the recommended method for screening of antinuclear antibodies (ANA). Dense fine speckled (DFS70) pattern on HEp-2 has been widely reported in various healthy and disease groups. Interpretation of DFS70 pattern can be challenging on a conventional HEp-2 substrate due to its similarity to some of the disease associated patterns. The high prevalence of DFS70 autoantibodies in normal population, lack of association with a particular disease group and a general negative association with systemic and ANA associated autoimmune rheumatic diseases (SARD/AARD) necessitates the confirmation of DFS70 pattern. Results using available commercial assays for confirmation of DFS70 autoantibodies do not always agree with IIF screening results further complicating the lab work flow and ANA algorithms. In this review, we discuss the prevalence of DFS70 antibodies and factors affecting the performance of IIF and DFS70 specific confirmatory assays. Factors that contribute to disagreement between DFS70 suspicion by IIF and confirmatory assays will also be discussed. In addition, we also describe a novel IIF HEp-2 substrate, and its positive impact on DFS70 reporting and ANA screening-confirmation algorithm.