Inflammation and Fibrosis in Orbital Inflammatory Disease: A Histopathologic Analysis.

PURPOSE: The purpose of this study was to compare the histopathologic inflammation and fibrosis of orbital adipose tissue in orbital inflammatory disease (OID) specimens. METHODS: In this retrospective cohort study, inflammation, and fibrosis in orbital adipose tissue from patients with thyroid-associated orbitopathy (TAO), granulomatosis with polyangiitis (GPA), sarcoidosis, nonspecific orbital inflammation (NSOI), and healthy controls were scored by 2 masked ocular pathologists. Both categories were scored on a scale of 0 to 3 with scoring criteria based on the percentage of specimens containing inflammation or fibrosis, respectively. Tissue specimens were collected from oculoplastic surgeons at 8 international centers representing 4 countries. Seventy-four specimens were included: 25 with TAO, 6 with orbital GPA, 7 with orbital sarcoidosis, 24 with NSOI, and 12 healthy controls. RESULTS: The mean inflammation and fibrosis scores for healthy controls were 0.0 and 1.1, respectively. Orbital inflammatory disease groups' inflammation (I) and fibrosis (F) scores, formatted [I, F] with respective p -values when compared to controls, were: TAO [0.2, 1.4] ( p = 1, 1), GPA [1.9, 2.6] ( p = 0.003, 0.009), sarcoidosis [2.4, 1.9] ( p = 0.001, 0.023), and NSOI [1.3, 1.8] ( p ≤ 0.001, 0.018). Sarcoidosis had the highest mean inflammation score. The pairwise analysis demonstrated that sarcoidosis had a significantly higher mean inflammation score than NSOI ( p = 0.036) and TAO ( p < 0.0001), but no difference when compared to GPA. GPA had the highest mean fibrosis score, with pairwise analysis demonstrating a significantly higher mean fibrosis score than TAO ( p = 0.048). CONCLUSIONS: Mean inflammation and fibrosis scores in TAO orbital adipose tissue samples did not differ from healthy controls. In contrast, the more "intense" inflammatory diseases such as GPA, sarcoidosis, and NSOI did demonstrate higher histopathologic inflammation and fibrosis. This has implications in prognosis, therapeutic selection, and response monitoring in orbital inflammatory disease.

Axial spondyloarthritis patients have altered mucosal IgA response to oral and fecal microbiota.

Axial spondyloarthritis (axSpA) is an inflammatory arthritis involving the spine and the sacroiliac joint with extra-articular manifestations in the eye, gut, and skin. The intestinal microbiota has been implicated as a central environmental component in the pathogenesis of various types of spondyloarthritis including axSpA. Additionally, alterations in the oral microbiota have been shown in various rheumatological conditions, such as rheumatoid arthritis (RA). Therefore, the aim of this study was to investigate whether axSpA patients have an altered immunoglobulin A (IgA) response in the gut and oral microbial communities. We performed 16S rRNA gene (16S) sequencing on IgA positive (IgA+) and IgA negative (IgA-) fractions (IgA-SEQ) from feces (n=17 axSpA; n=14 healthy) and saliva (n=14 axSpA; n=12 healthy), as well as on IgA-unsorted fecal and salivary samples. PICRUSt2 was used to predict microbial metabolic potential in axSpA patients and healthy controls (HCs). IgA-SEQ analyses revealed enrichment of several microbes in the fecal (Akkermansia, Ruminococcaceae, Lachnospira) and salivary (Prevotellaceae, Actinobacillus) microbiome in axSpA patients as compared with HCs. Fecal microbiome from axSpA patients showed a tendency towards increased alpha diversity in IgA+ fraction and decreased diversity in IgA- fraction in comparison with HCs, while the salivary microbiome exhibits a significant decrease in alpha diversity in both IgA+ and IgA- fractions. Increased IgA coating of Clostridiales Family XIII in feces correlated with disease severity. Inferred metagenomic analysis suggests perturbation of metabolites and metabolic pathways for inflammation (oxidative stress, amino acid degradation) and metabolism (propanoate and butanoate) in axSpA patients. Analyses of fecal and salivary microbes from axSpA patients reveal distinct populations of immunoreactive microbes compared to HCs using the IgA-SEQ approach. These bacteria were not identified by comparing their relative abundance alone. Predictive metagenomic analysis revealed perturbation of metabolites/metabolic pathways in axSpA patients. Future studies on these immunoreactive microbes may lead to better understanding of the functional role of IgA in maintaining microbial structure and human health.

Enrichment of IGF-1R and PPARγ signalling pathways in orbital inflammatory diseases: steps toward understanding pathogenesis.

BACKGROUND: Orbital inflammatory disease (OID) encompasses a wide range of pathology including thyroid-associated orbitopathy (TAO), granulomatosis with polyangiitis (GPA), sarcoidosis and non-specific orbital inflammation (NSOI), accounting for up to 6% of orbital diseases. Understanding the underlying pathophysiology of OID can improve diagnosis and help target therapy. AIMS: To test the hypothesis that shared signalling pathways are activated in different forms of OID. METHODS: In this secondary analysis, pathway analysis was performed on the previously reported differentially expressed genes from orbital adipose tissue using patients with OID and healthy controls who were characterised by microarray. For the original publications, tissue specimens were collected from oculoplastic surgeons at 10 international centres representing four countries (USA, Canada, Australia and Saudi Arabia). Diagnoses were independently confirmed by two masked ocular pathologists (DJW, HEG). Gene expression profiling analysis was performed at the Oregon Health & Science University. Eighty-three participants were included: 25 with TAO, 6 with orbital GPA, 7 with orbital sarcoidosis, 25 with NSOI and 20 healthy controls. RESULTS: Among the 83 subjects (mean (SD) age, 52.8 (18.3) years; 70% (n=58) female), those with OID demonstrated perturbation of the downstream gene expressions of the IGF-1R (MAPK/RAS/RAF/MEK/ERK and PI3K/Akt/mTOR pathways), peroxisome proliferator-activated receptor-γ (PPARγ), adipocytokine and AMPK signalling pathways compared with healthy controls. Specifically, GPA samples differed from controls in gene expression within the insulin-like growth factor-1 receptor (IGF-1R, PI3K-Akt (p=0.001), RAS (p=0.005)), PPARγ (p=0.002), adipocytokine (p=0.004) or AMPK (p=<0.001) pathways. TAO, sarcoidosis and NSOI samples were also found to have statistically significant differential gene expression in these pathways. CONCLUSIONS: Although OID includes a heterogenous group of pathologies, TAO, GPA, sarcoidosis and NSOI share enrichment of common gene signalling pathways, namely IGF-1R, PPARγ, adipocytokine and AMPK. Pathway analyses of gene expression suggest that other forms of orbital inflammation in addition to TAO may benefit from blockade of IGF-1R signalling pathways.

Identifying RNA Biomarkers and Molecular Pathways Involved in Multiple Subtypes of Uveitis.

PURPOSE: Uveitis is a heterogeneous collection of diseases. We tested the hypothesis that despite the diversity of uveitides, there could be common mechanisms shared by multiple subtypes, and that evidence of these common mechanisms may be detected as gene expression profiles in whole blood. DESIGN: Cohort study. METHODS: Ninety subjects with uveitis including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulointerstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy controls were enrolled, predominantly at Oregon Health & Science University. RNA-Seq data generated from peripheral, whole blood identified 19,859 unique transcripts. We analyzed gene expression pathways via Kyoto Encyclopedia of Genes and Genomes and Gene Ontology (GO). We validated our list of upregulated genes by comparison to a previously published study on peripheral blood gene expression among 50 subjects with diverse forms of uveitis. RESULTS: Both the Kyoto Encyclopedia of Genes and Genomes and GO analysis identified multiple shared pathways or GO terms with a P value of <.0001. Almost all pathways related to the immune response and/or response to an infection. A total of 119 individual transcripts were upregulated by at least 1.5-fold and false discovery rate <.05, and 61 were downregulated by similar criteria. Comparing mRNA from our study with a false discovery rate <.05 and the prior report, we identified 10 common gene transcripts: ICAM1, IL15RA, IL15, IRF1, IL10RB, GSK3A, TYK2, MEF2A, MEF2B, and MEF2D. CONCLUSIONS: Many forms of uveitis share overlapping mechanisms. These data support the concept that a single therapeutic approach could benefit diverse forms of this disease.

Non-specific orbital inflammation: Current understanding and unmet needs.

Non-specific orbital inflammation (NSOI) is a noninfectious inflammatory condition of the orbit. Although it is generally considered the most common diagnosis derived from an orbital biopsy, it is a diagnosis of exclusion, meaning that the diagnosis requires exclusion of a systemic process or another identifiable etiology of orbital inflammation. The clinical diagnosis of NSOI is ill-defined, but it is typically characterized by acute orbital signs and symptoms, including pain, proptosis, periorbital edema, chemosis, diplopia, and less commonly visual disturbance. NSOI poses a diagnostic and therapeutic challenge: The clinical presentations and histological findings are heterogeneous, and there are no specific diagnostic criteria or treatment guidelines. The etiology and pathogenesis of NSOI are poorly understood. Here we recapitulate our current clinical understanding of NSOI, with an emphasis on the most recent findings on clinical characteristics, imaging findings, and treatment outcomes. Furthermore, gene expression profiling of NSOI and its implications are presented and discussed.

Revising the Diagnosis of Idiopathic Uveitis by Peripheral Blood Transcriptomics.

PURPOSE: To test the hypothesis that idiopathic uveitis can be categorized into subtypes based on gene expression from blood. DESIGN: Case control study. METHODS: We applied RNA-Seq to peripheral blood from patients with uveitis associated with 1 of 4 systemic diseases, including axial spondyloarthritis (n = 17), sarcoidosis (n = 13), inflammatory bowel disease (n = 12), tubulo-interstitial nephritis with uveitis (n = 10), or idiopathic uveitis (n = 38) as well as 18 healthy control subjects evaluated predominantly at Oregon Health and Science University. A high-dimensional negative binomial regression model implemented in the edgeR R package compared each disease group with the control subjects. The 20 most distinctive genes for each diagnosis were extracted. Of 80 genes, there were 75 unique genes. A classification algorithm was developed by fitting a gradient boosting tree with 5-fold cross-validation. Messenger RNA from subjects with idiopathic uveitis were analyzed to see if any fit clinically and by gene expression pattern with one of the diagnosable entities. RESULTS: For uveitis associated with a diagnosable systemic disease, gene expression profiling achieved an overall accuracy of 85% (balanced average of sensitivity plus specificity, P < .001). Although most patients with idiopathic uveitis presumably have none of these 4 associated systemic diseases, gene expression profiles helped to reclassify 11 of 38 subjects. CONCLUSIONS: Peripheral blood gene expression profiling is a potential adjunct in accurate differential diagnosis of the cause of uveitis. Validation of these results and characterization of the gene expression profile from additional discrete diagnoses could enhance the value of these observations.

Radiologic imaging shows variable accuracy in diagnosing orbital inflammatory disease and assessing its activity.

Radiologic orbital imaging provides important information in the diagnosis and management of orbital inflammation. However, the diagnostic value of orbital imaging is not well elucidated. This study aimed to investigate the diagnostic accuracy of orbital imaging to diagnose orbital inflammatory diseases and its ability to detect active inflammation. We collected 75 scans of 52 patients (49 computed tomography (CT) scans; 26 magnetic resonance (MR) imaging scans). Clinical diagnoses included thyroid eye disease (TED) (41 scans, 31 patients), non-specific orbital inflammation (NSOI) (22 scans, 14 patients), sarcoidosis (4 scans, 3 patients), IgG4-related ophthalmic disease (IgG4-ROD) (5 scans, 3 patients), and granulomatosis with polyangiitis (GPA) (3 scans, 1 patient). Two experienced neuroradiologists interpreted the scans, offered a most likely diagnosis, and assessed the activity of inflammation, blinded to clinical findings. The accuracy rate of radiological diagnosis compared to each clinical diagnosis was evaluated. Sensitivity and specificity in detecting active inflammation were analyzed for TED and NSOI. The accuracy rate of radiologic diagnosis was 80.0% for IgG4-ROD, 77.3% for NSOI, and 73.2% for TED. Orbital imaging could not diagnose sarcoidosis. Orbital CT had a sensitivity of 50.0% and a specificity of 75.0% to predict active TED using clinical assessment as the gold standard. The sensitivity/specificity of orbital MR was 83.3/16.7% for the detection of active NSOI. In conclusion, orbital imaging is accurate for the diagnosis of IgG4, NSOI, and TED. Further studies with a large number of cases are needed to confirm this finding, especially with regard to uncommon diseases. Orbital CT showed moderate sensitivity and good specificity for identifying active TED.

RNA-Seq of human whole blood: Evaluation of globin RNA depletion on Ribo-Zero library method.

Peripheral blood is a highly accessible biofluid providing a rich source of information about human physiology and health status. However, for studies of the blood transcriptome with RNA sequencing (RNA-Seq) techniques, high levels of hemoglobin mRNAs (hgbRNA) present in blood can occupy valuable sequencing space, impacting detection and quantification of non-hgbRNAs. In this study, we evaluated two methods for preparing ribosomal RNA (rRNA)-depleted sequencing libraries for RNA-Seq of whole blood, one of which is also designed to deplete hgbRNAs. Two experiments were performed: one evaluating library performance across 6 human blood samples and the other examining library reproducibility and performance in a two-subject subset. We find that addition of hgbRNA depletion to the rRNA-depletion protocol for library preparation from blood RNA effectively reduces highly abundant hgbRNA reads; however, it does not result in a statistically significant increase in differentially expressed genes in our patient-control study. Bioinformatic removal of globin gene counts in non-hgbRNA depleted libraries provides improvement in overall performance of these libraries. We conclude that use of a standard ribosomal RNA depletion method for library preparation coupled with bioinformatic removal of globin gene counts is sufficient for reproducible and sensitive measurement of both coding and noncoding RNAs in the blood transcriptome.

Gene Expression Pathways across Multiple Tissues in Antineutrophil Cytoplasmic Antibody-associated Vasculitis Reveal Core Pathways of Disease Pathology.

OBJECTIVE: To identify commonalities in gene expression data across all antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) tissues thus far characterized. METHODS: Gene expression data were collected from the 3 AAV tissues thus far characterized (orbit, peripheral leukocytes, and sinus brushings). These data were analyzed to identify commonly expressed genes and disease pathways. The pathways data were adjusted for multiple comparisons using a combined local false discovery rate, which estimates the probability of a false discovery of a given pathway in all 3 tissues analyzed. RESULTS: Only 4 genes were upregulated in all 3 tissues - IL1RN, TLR2, SLC11A1, and MMP9. After multiple comparison adjustments, the network pathway analysis revealed 28 pathways associated with all 3 tissues. The most strongly associated pathway for all 3 tissues was the neutrophil degranulation pathway [multidimensional local false discovery (md-locfdr) = 1.05 × 10-12], followed by the osteoclast differentiation (md-locfdr = 3.8 × 10-05), cell surface interactions at the vascular wall (md-locfdr = 4.2 × 10-04), signaling by interleukins (md-locfdr = 6.1 × 10-04), and phagosome (md-locfdr = 0.003) pathways. There were no downregulated genes or pathways common to all 3 tissues. CONCLUSION: This analysis identified individual genes and pathways of disease common to all AAV tissues thus far characterized. The use of a network pathway analysis allowed us to identify pathologic mechanisms that were not readily apparent in the commonly expressed genes alone. Many of these pathways are consistent with current theories about infectious drivers and the crossroads of innate and adaptive immune mechanisms. In addition, this analysis highlights novel pathways, such as vessel wall interactions and platelet activation, which require further investigation.

Gene Expression Profiling and Heterogeneity of Nonspecific Orbital Inflammation Affecting the Lacrimal Gland.

Importance: Although a variety of well-characterized diseases, such as sarcoidosis and granulomatosis with polyangiitis, affect the lacrimal gland, many patients with dacryoadenitis are diagnosed as having nonspecific orbital inflammation (NSOI) on the basis of histology and systemic disease evaluation. The ability to further classify the disease in these patients should facilitate selection of effective therapies. Objective: To test the a priori hypothesis that gene expression profiles would complement clinical and histopathologic evaluations in identifying well-characterized diseases and in subdividing NSOI into clinically relevant groups. Design, Setting, and Participants: In this cohort study, gene expression levels in biopsy specimens of inflamed and control lacrimal glands were measured with microarrays. Stained sections of the same biopsy specimens were used for evaluation of histopathology. Tissue samples of patients were obtained from oculoplastic surgeons at 7 international centers representing 4 countries (United States, Saudi Arabia, Canada, and Taiwan). Gene expression analysis was done at Oregon Health & Science University. Participants were 48 patients, including 3 with granulomatosis with polyangiitis, 28 with NSOI, 7 with sarcoidosis, 4 with thyroid eye disease, and 6 healthy controls. The study dates were March 2012 to April 2017. Main Outcomes and Measures: The primary outcome was subdivision of biopsy specimens based on gene expression of a published list of approximately 40 differentially expressed transcripts in blood, lacrimal gland, and orbital adipose tissue from patients with sarcoidosis. Stained sections were evaluated for inflammation (none, mild, moderate, or marked), granulomas, nodules, or fibrosis by 2 independent ocular pathologists masked to the clinical diagnosis. Results: Among 48 patients (mean [SD] age, 41.6 [19.0] years; 32 [67%] female), the mclust algorithm segregated the biopsy specimens into 4 subsets, with the differences illustrated by a heat map and multidimensional scaling plots. Most of the sarcoidosis biopsy specimens were in subset 1, which had the highest granuloma score. Three NSOI biopsy specimens in subset 1 had no apparent granulomas. Thirty-two percent (9 of 28) of the NSOI biopsy specimens could not be distinguished from biopsy specimens of healthy controls in subset 4, while other examples of NSOI tended to group with gene expression resembling granulomatosis with polyangiitis or thyroid eye disease. The 4 subsets could also be partially differentiated by their fibrosis, granulomas, and inflammation pathology scores but not their lymphoid nodule scores. Conclusions and Relevance: Gene expression profiling discloses clear heterogeneity among patients with lacrimal inflammatory disease. Comparison of the expression profiles suggests that a subset of patients with nonspecific dacryoadenitis might have a limited form of sarcoidosis, while other patients with NSOI cannot be distinguished from healthy controls.

Molecular diagnosis: Implications for ophthalmology.

The effort to subdivide diseases and to individualize therapies based on characteristics of the patient has been labeled precision medicine. Jameson and Longo define precision medicine as "treatments targeted to the needs of individual patients on the basis of genetic, biomarker, phenotypic or psychosocial characteristics that distinguish a given patient from other patients with similar clinical presentations" (Jameson and Longo, 2015). We illustrate how molecular diagnosis can be applied to orbital inflammatory disease to achieve the goals of precision medicine.

The Role of the Immune Response in the Pathogenesis of Thyroid Eye Disease: A Reassessment.

BACKGROUND: Although thyroid eye disease is a common complication of Graves' disease, the pathogenesis of the orbital disease is poorly understood. Most authorities implicate the immune response as an important causal factor. We sought to clarify pathogenesis by using gene expression microarray. METHODS: An international consortium of ocular pathologists and orbital surgeons contributed formalin fixed orbital biopsies. RNA was extracted from orbital tissue from 20 healthy controls, 25 patients with thyroid eye disease (TED), 25 patients with nonspecific orbital inflammation (NSOI), 7 patients with sarcoidosis and 6 patients with granulomatosis with polyangiitis (GPA). Tissue was divided into a discovery set and a validation set. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays which include 54,000 probe sets. RESULTS: Principal component analysis showed that gene expression from tissue from patients with TED more closely resembled gene expression from healthy control tissue in comparison to gene expression characteristic of sarcoidosis, NSOI, or granulomatosis with polyangiitis. Unsupervised cluster dendrograms further indicated the similarity between TED and healthy controls. Heat maps based on gene expression for cytokines, chemokines, or their receptors showed that these inflammatory markers were associated with NSOI, sarcoidosis, or GPA much more frequently than with TED. CONCLUSION: This is the first study to compare gene expression in TED to gene expression associated with other causes of exophthalmos. The juxtaposition shows that inflammatory markers are far less characteristic of TED relative to other orbital inflammatory diseases.

Orbital pseudotumor can be a localized form of granulomatosis with polyangiitis as revealed by gene expression profiling.

Biopsies and ANCA testing for limited forms of granulomatosis with polyangiitis (GPA) are frequently non-diagnostic. We characterized gene expression in GPA and other causes of orbital inflammation. We tested the hypothesis that a sub-set of patients with non-specific orbital inflammation (NSOI, also known as pseudotumor) mimics a limited form of GPA. Formalin-fixed, paraffin-embedded orbital biopsies were obtained from controls (n=20) and patients with GPA (n=6), NSOI (n=25), sarcoidosis (n=7), or thyroid eye disease (TED) (n=20) and were divided into discovery and validation sets. Transcripts in the tissues were quantified using Affymetrix U133 Plus 2.0 microarrays. Distinct gene expression profiles for controls and subjects with GPA, TED, or sarcoidosis were evident by principal coordinate analyses. Compared with healthy controls, 285 probe sets had elevated signals in subjects with GPA and 1472 were decreased (>1.5-fold difference, false discovery rate adjusted p<0.05). The immunoglobulin family of genes had the most dramatic increase in expression. Although gene expression in GPA could be readily distinguished from gene expression in TED, sarcoidosis, or controls, a comparison of gene expression in GPA versus NSOI found no statistically significant differences. Thus, forms of orbital inflammation can be distinguished based on gene expression. NSOI/pseudotumor is heterogeneous but often may be an unrecognized, localized form of GPA.

Fibrosis, gene expression and orbital inflammatory disease.

BACKGROUND/AIMS: To clarify the pathogenesis of fibrosis in inflammatory orbital diseases, we analysed the gene expression in orbital biopsies and compared our results with those reported for idiopathic pulmonary fibrosis. METHODS: We collected 140 biopsies from 138 patients (58 lacrimal glands; 82 orbital fat). Diagnoses included healthy controls (n=27), non-specific orbital inflammation (NSOI) (n=61), thyroid eye disease (TED) (n=29), sarcoidosis (n=14) and granulomatosis with polyangiitis (GPA) (n=7). Fibrosis was scored on a 0-3 scale by two experts, ophthalmic pathologists. Gene expression was quantified using Affymetrix U133 plus 2.0 microarray. RESULTS: Within orbital fat, fibrosis was greatest among subjects with GPA (2.75±0.46) and significantly increased in tissue from subjects with GPA, NSOI or sarcoidosis (p<0.01), but not for TED, compared with healthy controls (1.13±0.69). For lacrimal gland, the average score among controls (1.36±0.48) did not differ statistically from any of the four disease groups. Seventy-three probe sets identified transcripts correlating with fibrosis in orbital fat (false discovery rate <0.05) after accounting for batch effects, disease type, age and sex. Transcripts with increased expression included fibronectin, lumican, thrombospondin and collagen types I and VIII, each of which has been reported upregulated in pulmonary fibrosis. CONCLUSIONS: A pathologist's recognition of fibrosis in orbital tissue correlates well with increased expression of transcripts that are considered essential in fibrosis. Many transcripts implicated in orbital fibrosis have been previously implicated in pulmonary fibrosis. TED differs from other causes of orbital fat inflammation because fibrosis is not a major component. Marked fibrosis is less common in the lacrimal gland compared with orbital adipose tissue.

Parallel Gene Expression Changes in Sarcoidosis Involving the Lacrimal Gland, Orbital Tissue, or Blood.

IMPORTANCE: Sarcoidosis is a major cause of ocular or periocular inflammation. The pathogenesis of sarcoidosis is incompletely understood and diagnosis often requires a biopsy. OBJECTIVE: To determine how gene expression in either orbital adipose tissue or the lacrimal gland affected by sarcoidosis compares with gene expression in other causes of orbital disease and how gene expression in tissue affected by sarcoidosis compares with gene expression in peripheral blood samples obtained from patients with sarcoidosis. DESIGN, SETTING, AND PARTICIPANTS: In a multicenter, international, observational study, gene expression profiling of formalin-fixed biopsy specimens, using GeneChipp U133 Plus 2 microarrays (Affymetrix), was conducted between October 2012 and January 2014 on tissues biopsied from January 2000 through June 2013. Participants included 12 patients with orbital sarcoidosis (7 in adipose tissue; 5 affecting the lacrimal gland) as well as comparable tissue from 6 healthy individuals serving as controls or patients with thyroid eye disease, nonspecific orbital inflammation, or granulomatosis with polyangiitis. In addition, results were compared with gene expression in peripheral blood samples obtained from 12 historical individuals with sarcoidosis. MAIN OUTCOMES AND MEASURES: Significantly differentially expressed transcripts defined as a minimum of a 1.5-fold increase or a comparable decrease and a false discovery rate of P < .05. RESULTS: Signals from 2449 probe sets (transcripts from approximately 1522 genes) were significantly increased in the orbital adipose tissue from patients with sarcoidosis. Signals from 4050 probe sets (approximately 2619 genes) were significantly decreased. Signals from 3069 probe sets (approximately 2001 genes) were significantly higher and 3320 (approximately 2283 genes) were significantly lower in the lacrimal gland for patients with sarcoidosis. Ninety-two probe sets (approximately 69 genes) had significantly elevated signals and 67 probe sets (approximately 56 genes) had significantly lower signals in both orbital tissues and in peripheral blood from patients with sarcoidosis. The transcription factors, interferon-response factor 1, interferon-response factor 2, and nuclear factor κB, were strongly implicated in the expression of messenger RNA upregulated in common in the 3 tissues. CONCLUSIONS AND RELEVANCE: Gene expression in sarcoidosis involving the orbit or lacrimal gland can be distinguished from gene expression patterns in control tissue and overlaps with many transcripts upregulated or downregulated in the peripheral blood of patients with sarcoidosis. These observations suggest that common pathogenic mechanisms contribute to sarcoidosis in different sites. The observations support the hypothesis that a pattern of gene expression profiles could provide diagnostic information in patients with sarcoidosis.

Molecular diagnosis of orbital inflammatory disease.

Orbital inflammatory diseases include thyroid eye disease (TED), granulomatosis with polyangiitis (GPA), sarcoidosis, and nonspecific orbital inflammation (NSOI). Histopathological diagnosis usually relies on the clinical context and is not always definitive. Gene expression profiling provides diagnostic and therapeutic information in several malignancies, but its role in evaluating nonmalignant disease is relatively untested. We hypothesized that gene expression profiling could provide diagnostic information for NSOI. We collected formalin-fixed, paraffin-embedded orbital biopsies from 10 institutions and 83 subjects including 25 with thyroid eye disease, 25 nonspecific orbital inflammation, 20 healthy controls, 6 with granulomatosis with polyangiitis, and 7 with sarcoidosis. Tissues were divided into discovery and validation sets. Gene expression was quantified using Affymetrix U133 Plus 2.0 microarrays. A random forest statistical algorithm based on data from 39 probe sets identified controls, GPA, or TED with an average accuracy of 76% (p=0.02). Random forest analysis indicated that 52% of tissues from patients with nonspecific inflammation were consistent with a diagnosis of GPA. Molecular diagnosis by gene expression profiling will augment clinical data and histopathology in differentiating forms of orbital inflammatory disease.

IgG4 immunostaining and its implications in orbital inflammatory disease.

OBJECTIVE: IgG4-related disease is an emerging clinical entity which frequently involves tissue within the orbit. In order to appreciate the implications of IgG4 immunostaining, we analyzed gene expression and the prevalence of IgG4- immunostaining among subjects with orbital inflammatory diseases. METHODS: We organized an international consortium to collect orbital biopsies from 108 subjects including 22 with no known orbital disease, 42 with nonspecific orbital inflammatory disease (NSOI), 26 with thyroid eye disease (TED), 12 with sarcoidosis, and 6 with granulomatosis with polyangiitis (GPA). Lacrimal gland and orbital adipose tissue biopsies were immunostained for IgG4 or IgG secreting plasma cells. RNA transcripts were quantified by Affymetrix arrays. RESULTS: None of the healthy controls or subjects with TED had substantial IgG4 staining. Among the 63 others, the prevalence of significant IgG4-immunostaining ranged from 11 to 39% depending on the definition for significant. IgG4 staining was detectable in the majority of tissues from subjects with GPA and less commonly in tissue from subjects with sarcoidosis or NSOI. The detection of IgG4+ cells correlated with inflammation in the lacrimal gland based on histology. IgG4 staining tissue expressed an increase in transcripts associated with inflammation, especially B cell-related genes. Functional annotation analysis confirmed this. CONCLUSION: IgG4+ plasma cells are common in orbital tissue from patients with sarcoidosis, GPA, or NSOI. Even using the low threshold of 10 IgG4+ cells/high powered field, IgG4 staining correlates with increased inflammation in the lacrimal gland based on histology and gene expression.

Impact of IL-1 signalling on experimental uveitis and arthritis.

BACKGROUND: Uveitis, or inflammatory eye disease, is a common extra-articular manifestation of many systemic autoinflammatory diseases involving the joints. Anakinra (recombinant interleukin (IL)-1 receptor antagonist (Ra)) is an effective therapy in several arthritic diseases; yet, few studies have investigated the extent to which IL-1 signalling or IL-1Ra influences the onset and/or severity of uveitis. OBJECTIVE: To seek possible links between arthritis and uveitis pathogenesis related to IL-1 signalling. METHODS: The eyes of IL-1Ra-deficient BALB/c mice were monitored histologically and by intravital videomicroscopy to determine if uveitis developed along with the expected spontaneous arthritis in ankles and knees. Expression levels of IL-1R and its negative regulators (IL-1Ra, IL-1RII, IL-1RAcP and single Ig IL-1R-related molecule) in eye and joint tissues were compared. Differences in uveitis induced by intraocular injection of lipopolysaccharide (LPS) in mice lacking IL-1R or IL-1Ra were assessed. RESULTS: Deficiency in IL-1Ra predisposes to spontaneous arthritis, which is exacerbated by previous systemic LPS exposure. The eye, however, does not develop inflammatory disease despite the progressive arthritis or LPS exposure. Organ-specific expression patterns for IL-1Ra and negative regulators of IL-1 activity were observed that appear to predict predisposition to inflammation in each location in IL-1Ra knockout mice. The eye is extremely sensitive to locally administered LPS, and IL-1Ra deficiency markedly exacerbates the resulting uveitis. CONCLUSION: This study demonstrates that IL-1Ra plays an important role in suppressing local responses in eyes injected with LPS and that there is discordance between murine eyes and joints in the extent to which IL-1Ra protects against spontaneous inflammation.

Differential expression of microRNA and predicted targets in pulmonary sarcoidosis.

BACKGROUND: Recent studies show that various inflammatory diseases are regulated at the level of RNA translation by small non-coding RNAs, termed microRNAs (miRNAs). We sought to determine whether sarcoidosis tissues harbor a distinct pattern of miRNA expression and then considered their potential molecular targets. METHODS AND RESULTS: Genome-wide microarray analysis of miRNA expression in lung tissue and peripheral blood mononuclear cells (PBMCs) was performed and differentially expressed (DE)-miRNAs were then validated by real-time PCR. A distinct pattern of DE-miRNA expression was identified in both lung tissue and PBMCs of sarcoidosis patients. A subgroup of DE-miRNAs common to lung and lymph node tissues were predicted to target transforming growth factor (TGFß)-regulated pathways. Likewise, the DE-miRNAs identified in PBMCs of sarcoidosis patients were predicted to target the TGFß-regulated "wingless and integrase-1" (WNT) pathway. CONCLUSIONS: This study is the first to profile miRNAs in sarcoidosis tissues and to consider their possible roles in disease pathogenesis. Our results suggest that miRNA regulate TGFß and related WNT pathways in sarcoidosis tissues, pathways previously incriminated in the pathogenesis of sarcoidosis.

The effects of corneal endothelium on graft survival in a murine model of lamellar keratoplasty.

PURPOSE: Here we investigate the role of donor endothelium on allograft rejection in a lamellar keratoplasty (LK) model using grafts with or without donor endothelium. METHODS: Corneal buttons of donor C57BL/6 mice (2.0 mm) were transplanted to lamellar recipient beds (1.5 mm) in BALB/c mice. Two variations of the LK procedure were performed: (1) standard LK (SLK) (n = 13) without donor endothelium and (2) modified LK (MLK) (n = 14) with retained donor endothelium. The graft status was assessed by slit lamp biomicroscopy and scored for stromal opacity, corneal edema, neovascularization, and anterior chamber reaction up to 46 days post-transplantation. Corneas were also observed histologically. RESULTS: The presence of a grafted corneal endothelium promoted graft rejection; 92.9% (13/14) of grafts were rejected in MLK after an average of 8.3 days, while 69.2% (9/13) of grafts were rejected in SLK on average 10.8 days after transplantation. The former's stromal opacity was significantly greater at all time points after day 14 except for day 21 (p = 0.77) and day 32 (p = 0.25). Corneal edema was significantly greater in the former at all time points after day 10 except for day 21 (p = 0.16). Neovascularization was significantly greater in the former at all time points after day 10 except for day 25 (p = 0.22). CONCLUSION: Variations of this model of LK may be useful for studies of immunological mechanisms in corneal transplantation. The donor corneal endothelium may serve as a target of the immune response which promotes inflammation, neovascularization, and graft rejection.