Establishing PAX6 as a biomarker to detect early loss of ocular phenotype in human patients with Sjögren's syndrome.

PURPOSE: Sjögren's syndrome (SS) is a common autoimmune disease that can cause aqueous-deficient dry eye and the aberrant differentiation of ocular mucosal epithelial cells toward a lineage that is pathologically keratinized and skin-like. PAX6 is the master regulator of corneal lineage commitment. Recently, we showed a functional role for PAX6 in preventing ocular surface damage induced by the proinflammatory cytokine, IL-1ß, in a mouse model of SS. Here, we examine PAX6's potential as a clinical biomarker that predicts ocular surface disease in SS patients. METHODS: Impression cytology specimens isolated from the bulbar conjunctiva of control (n = 43) and SS patients (n = 43) were used to evaluate the relative abundance of PAX6, IL-1ß, and pathologic keratinization marker, small proline-rich protein (SPRR1B) by TaqMan qPCR. Transcript expression was examined relative to clinical data, including the ocular staining score (OSS), tear breakup time (TBUT), Schirmer tear test, serum autoantibody results, and the labial salivary gland focus score. RESULTS: PAX6 expression was significantly reduced in SS patients (P = 0.010, Wilcoxon rank sum test), and highly correlated with OSS (Spearman ρ = 0.239, 95% CI 0.02-0.43; P = 0.027). The extent to which PAX6 predicted SPRR1B was largely dependent on IL-1ß expression (R(2) = 0.28, P < 0.01) and elevated IL-1ß predicted reduced TBUT (R(2) = 0.24, P = 0.035), low tear secretion (R(2) = 0.30, P = 0.011), and focus score (R(2) = 0.21, P = 0.002). CONCLUSIONS: Downregulation of PAX6 in SS patients was highly associated with ocular surface damage and largely dependent on the level of inflammation. Restoration of PAX6 may provide a clinical approach to manage dry eye in SS patients.

Autoimmunity in the pathogenesis and treatment of keratoconjunctivitis sicca.

Dry eye is a chronic corneal disease that impacts the quality of life of many older adults. Keratoconjunctivitis sicca (KCS), a form of aqueous-deficient dry eye, is frequently associated with Sjögren's syndrome and mechanisms of autoimmunity. For KCS and other forms of dry eye, current treatments are limited, with many medications providing only symptomatic relief rather than targeting the pathophysiology of disease. Here, we review proposed mechanisms in the pathogenesis of autoimmune-based KCS: genetic susceptibility and disruptions in antigen recognition, immune response, and immune regulation. By understanding the mechanisms of immune dysfunction through basic science and translational research, potential drug targets can be identified. Finally, we discuss current dry eye therapies as well as promising new treatment options and drug therapy targets.

Quantification of conjunctival TNF-α in aqueous-deficient dry eye.

PURPOSE: This study aimed to quantify and compare conjunctival epithelial tumor necrosis factor (NF) α mRNA expression in Sjögren syndrome (SS), non-Sjögren syndrome aqueous-deficient dry eye (non-SS DE), and non-dry eye (NDE) control subjects. METHODS: A total of 76 subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 non-SS DE (confirmed by symptoms and Schirmer scores ≤ 10 mm), and 26 NDE. Superior and temporal bulbar conjunctival epithelial cells were collected via impression cytology. Epithelial RNA was extracted, and TNF-α mRNA expression was quantified by real-time quantitative polymerase chain reaction. RESULTS: The expression of TNF-α mRNA was found to be significantly higher in the SS group (2.48 ± 1.79) compared to both non-SS DE (0.95 ± 1.18; p < 0.05) and NDE (0.84 ± 0.51; p < 0.05) groups. No difference in TNF-α mRNA expression was found between the non-SS DE and NDE groups (p = 0.67). CONCLUSIONS: These results demonstrate that SS-associated aqueous-deficient dry eye is associated with a significant upregulation of conjunctival epithelial TNF-α mRNA relative to both non-SS DE and control groups. The degree to which TNF-α mRNA is upregulated in SS may contribute to the severe ocular surface damage observed in these patients.

Lymphotoxin-beta receptor blockade reduces CXCL13 in lacrimal glands and improves corneal integrity in the NOD model of Sjögren's syndrome.

INTRODUCTION: In Sjögren's syndrome, keratoconjunctivitis sicca (dry eye) is associated with infiltration of lacrimal glands by leukocytes and consequent losses of tear-fluid production and the integrity of the ocular surface. We investigated the effect of blockade of the lymphotoxin-beta receptor (LTBR) pathway on lacrimal-gland pathology in the NOD mouse model of Sjögren's syndrome. METHODS: Male NOD mice were treated for up to ten weeks with an antagonist, LTBR-Ig, or control mouse antibody MOPC-21. Extra-orbital lacrimal glands were analyzed by immunohistochemistry for high endothelial venules (HEV), by Affymetrix gene-array analysis and real-time PCR for differential gene expression, and by ELISA for CXCL13 protein. Leukocytes from lacrimal glands were analyzed by flow-cytometry. Tear-fluid secretion-rates were measured and the integrity of the ocular surface was scored using slit-lamp microscopy and fluorescein isothiocyanate (FITC) staining. The chemokine CXCL13 was measured by ELISA in sera from Sjögren's syndrome patients (n = 27) and healthy controls (n = 30). Statistical analysis was by the two-tailed, unpaired T-test, or the Mann-Whitney-test for ocular integrity scores. RESULTS: LTBR blockade for eight weeks reduced B-cell accumulation (approximately 5-fold), eliminated HEV in lacrimal glands, and reduced the entry rate of lymphocytes into lacrimal glands. Affymetrix-chip analysis revealed numerous changes in mRNA expression due to LTBR blockade, including reduction of homeostatic chemokine expression. The reduction of CXCL13, CCL21, CCL19 mRNA and the HEV-associated gene GLYCAM-1 was confirmed by PCR analysis. CXCL13 protein increased with disease progression in lacrimal-gland homogenates, but after LTBR blockade for 8 weeks, CXCL13 was reduced approximately 6-fold to 8.4 pg/mg (+/- 2.7) from 51 pg/mg (+/-5.3) in lacrimal glands of 16 week old control mice. Mice given LTBR blockade exhibited an approximately two-fold greater tear-fluid secretion than control mice (P = 0.001), and had a significantly improved ocular surface integrity score (P = 0.005). The mean CXCL13 concentration in sera from Sjögren's patients (n = 27) was 170 pg/ml, compared to 92.0 pg/ml for sera from (n = 30) healthy controls (P = 0.01). CONCLUSIONS: Blockade of LTBR pathways may have therapeutic potential for treatment of Sjögren's syndrome.

MUC1 expression in Sjogren's syndrome, KCS, and control subjects.

PURPOSE: To quantify and compare human mucin 1 (MUC1) protein and mRNA expression in tears and conjunctival epithelial cells collected from Sjogren's syndrome (SS), non-Sjogren's keratoconjunctivitus sicca (KCS) and non-dry eyed (NDE) control subjects. METHODS: Seventy-six subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 KCS (confirmed by symptoms and Schirmer scores < or = 10 mm) and 26 NDE. Tears were collected using an eye-wash technique. Impression cytology was used to gather protein and mRNA from conjunctival epithelial cells. Soluble and membrane bound MUC1 were quantified via western blotting and MUC1 mRNA was quantified by real time qPCR. RESULTS: The SS group demonstrated significantly higher concentrations of soluble MUC1 (0.12 +/- 0.11 [SS]; 0.013 +/- 0.02 [KCS; p=0.001]; 0.0023 +/- 0.0024 [NDE; p<0.001]) and MUC1 mRNA (3.18 +/- 1.44 [SS]; 1.79 +/- 1.18 [KCS; p<0.05]; 1.60 +/- 0.74 [NDE; p<0.05]) compared to both KCS and NDE groups. Soluble MUC1 expression was also higher in the KCS group compared to the NDE group (p=0.02), where as MUC1 mRNA expression was similar in both KCS and NDE groups. Membrane bound MUC1 expression differed only between the SS and NDE groups (0.005 +/- -0.003 [SS]; 0.003 +/- 0.002 [NDE; p=0.002]). CONCLUSIONS: These results demonstrate that SS subjects express greater quantities of MUC1 protein and mRNA compared to both KCS and control subjects. Increased soluble MUC1 expression was also found in KCS subjects compared to controls. Membrane bound MUC1 was present in higher concentration in SS versus NDE only. These significant changes in MUC1 expression may represent compensatory or protective responses to chronic insult to the ocular surface.

An autoimmune response to odorant binding protein 1a is associated with dry eye in the Aire-deficient mouse.

Sjögren's Syndrome (SS) is a human autoimmune disease characterized by immune-mediated destruction of the lacrimal and salivary glands. In this study, we show that the Aire-deficient mouse represents a new tool to investigate autoimmune dacryoadenitis and keratoconjunctivitis sicca, features of SS. Previous work in the Aire-deficient mouse suggested a role for alpha-fodrin, a ubiquitous Ag, in the disease process. Using an unbiased biochemical approach, however, we have identified a novel lacrimal gland autoantigen, odorant binding protein 1a, targeted by the autoimmune response. This novel autoantigen is expressed in the thymus in an Aire-dependent manner. The results from our study suggest that defects in central tolerance may contribute to SS and provide a new and clinically relevant model to investigate the pathogenic mechanisms in lacrimal gland autoimmunity and associated ocular surface sequelae.

[Pathogenesis and diagnosis of Sjögren's syndrome]. / Pathogenese und Diagnostik des Sjögren-Syndroms.

Sjögren's syndrome is a common autoimmune disorder. Several genetic risk factors such as STAT-4, ILT6 and the haplotype HLA-B8/DR3 have been identified. In addition, there are environmental risk factors, possibly chronic viral infections. In the pathophysiology of Sjögren's syndrome T and B cells infiltrate the salivary and lacrimal glands. As a consequence of the destruction of glandular cells by cytotoxic T cells, production of cytokines and autoantibodies inhibiting glandular function, the production of saliva and tears is decreased. The feeling of dry eyes and mouth is frequently not noticed by the patients. Therefore, Sjögren's syndrome should also be considered when extraglandular manifestations such as vasculitis, polyneuropathy or arthritis occur, even when the patients do not complain of dry eyes and mouth. Establishing the diagnosis of Sjögren's syndrome requires verification of reduced glandular function, for example using Schirmer's test and the Saxon test. The confirmation of Sjögren's syndrome as a cause of sicca syndrome is subsequently performed by the detection of autoantibodies against Ro (SS-A) and La (SS-B) and/or by a salivary gland biopsy.

MUC16 expression in Sjogren's syndrome, KCS, and control subjects.

PURPOSE: To investigate the expression of MUC16 protein in tears and conjunctival cell membranes and MUC16 mRNA in conjunctival cells of Sjogren's syndrome (SS), keratoconjunctivitus sicca (KCS) and non-dry eyed (NDE) subjects. The relationship of tear flow and soluble MUC16 concentration was also measured. METHODS: Seventy-six subjects were recruited for this study: 25 SS (confirmed via American-European Consensus Criteria 2002), 25 KCS (confirmed by symptoms and Schirmer scores < or =10 mm) and 26 NDE. Tear flow was measured by the Schirmer test without anesthesia for 5 min. Tears were collected using an eye-wash technique. Protein and mRNA were isolated from conjunctival epithelial cells collected via impression cytology. Soluble and membrane bound MUC16 were quantified via western blotting and MUC16 mRNA was quantified by real time qPCR. RESULTS: The SS group demonstrated significantly higher concentrations of soluble MUC16 (7.28 [SS] +/- 3.97 versus 3.35 [KCS] +/- 4.54 [p=0.004] and versus 1.61 [NDE] +/- 1.22 [p<0.001]) and MUC16 mRNA (4.66 [SS] +/- 5.06 versus 1.84 [KCS] +/- 2.26 [p=0.01] and 1.52 [NDE] +/- 1.04 [p=0.003]) compared to both KCS and NDE groups, respectively. No differences in soluble MUC16 or MUC16 mRNA were found between the KCS and NDE groups. Membrane bound MUC16 was similar in all three groups. No significant correlation was found between mean Schirmer values and any measure of MUC16 expression. CONCLUSIONS: These results demonstrate that SS subjects display a significant increase in both soluble MUC16 and MUC16 mRNA concentrations compared to other forms of aqueous deficient dry eye and non dry-eyed individuals. There was no correlation between tear flow and soluble MUC16 concentration.

Sjögren's syndrome-like disease of C57BL/6.NOD-Aec1 Aec2 mice: gender differences in keratoconjunctivitis sicca defined by a cross-over in the chromosome 3 Aec1 locus.

Sjögren's syndrome (SjS) is a systemic autoimmune disease in which an immunological attack primarily against the salivary and lacrimal glands results in loss of acinar cell tissue and function leading to stomatitis sicca and keratoconjunctivitis sicca. In recent years, the NOD mouse has become an accepted model of SjS, exhibiting a spontaneously developing disease that strongly mimics the human condition. Two genetic regions, one on chromosome 1 (designated Aec2) and the second on chromosome 3 (designated Aec1) of NOD mice, have been shown to be necessary and sufficient to recapitulate SjS-like disease in non-susceptible C57BL/6 mice. Here we describe a newly derived strain, C57BL/6.NOD-Aec1R1Aec2, in which a recombination in Aec1 has resulted in reducing this genetic region to less than 20 cM from 48.5 cM. Profiling of this recombinant inbred strain has revealed that male mice maintain a full SjS-like disease, whereas female mice exhibit stomatitis sicca in the absence of detectable keratoconjunctivitis sicca. These data suggest SjS-like disease in the NOD mouse shows gender-specific regulation determined by autosomal genes.

[Psoriasis and hypogonadism in chronic blepharokeratoconjunctivitis. A case report]. / Psoriasis und Hypogonadismus bei chronischer Blepharokeratokonjunktivitis. Ein Fallbericht.

BACKGROUND: Ocular symptoms occur in approximately 10% of patients with psoriasis vulgaris. PATIENT: We report the clinical course of a 35-year-old male patient with obstructive meibomian gland dysfunction, keratoconjunctivitis and reduced reflex secretion of both eyes. Psoriasis vulgaris and hypothalamic hypogonadism were also present. Genetic testing (cytogenetic and DNA analysis) was performed because of additional facial dysmorphia, brachydactylia and obesity. No chromosomal anomaly was found and no genetic syndrome has yet been diagnosed. The therapeutic regimen included preservative-free artificial tears, occlusion of the puncta and a systemic dose of doxycycline. Dermatological symptoms were treated topically and the hypogonadism was treated with intramuscular injections of testosterone. CONCLUSION: Lacrimal and meibomian glands are influenced by androgens. Therefore hormonal dysfunction can also have contributed to the blepharokeratoconjunctivitis in this patient.

[Congenital familial cornea plana with ptosis, peripheral sclerocornea and conjunctival xerosis]. / Kongenitale familiäre Cornea plana mit Ptosis, peripherer Sklerokornea und Bindehaut-Xerose.

BACKGROUND: Cornea plana is an extremely rare, congenital hereditary malformation of the corneo-scleral shape. The curvatures of cornea and sclera are nearly equal with an indistinct limbus. In addition to the flatness, there is a peripheral sclerocornea that produces a pseudomicrocornea. The low corneal refraction and the short anterior segment often result in hyperopia. Myopia is also described. Usually the posterior segment is not involved. MATERIALS AND METHODS: A young man of 22 years, his three- and five-year-old sons, and his newborn daughter showed this hereditary abnormality of the cornea. In this uncommon anomaly we measured corneal curvature, refraction, diameter and in three of the four patients echographical length of the bulbi. RESULTS: The family showed an autosomal dominant inheritance of the cornea plana. The corneal refraction was less than 32 diopters. The scleral encroachment caused an oval cornea measuring horizontally between 5 and 6.5 mm, vertically 4 to 5 mm. Additionally a pseudoblepharoptosis and a conjunctival xerosis of the father and his sons was observed, which is not regularly found. A-scan measuring of the bulbi revealed age-related normal values. CONCLUSIONS: There is no evidence for progression of this anomaly during life. No therapeutical consequences are necessary. To preserve a satisfactory function a conscientious orthoptical maintainance should be guaranteed.