Sjögren’s syndrome (SjS) is a chronic autoimmune disease characterized initially by lymphocytic infiltration and destruction of exocrine glands, followed by systemic organ damage and B-cell lymphoma. Conventional treatment is based on management of symptoms and there is a shortage of therapies that address the underlying causes of inflammation at source exocrine tissue. The aim of this study was to test a novel protein polymer-based platform consisting of diblock copolymers composed from Elastin-like Polypeptides (ELPs) fused with FKBP12, to deliver a potent immunosuppressant with dose-limiting toxicity, rapamycin (Rapa) also known as Sirolimus, and evaluate its effects on the inflamed lacrimal gland (LG) of non-obese diabetic mouse (NOD), a classic mouse model of SjS. Both soluble and diblock copolymer ELPs were fused to FKBP12 and characterized with respect to purity, hydrodynamic radii, drug entrapment and release. Both formulations showed successful association with Rapa; however, the nanoparticle formulation, FSI, released drug with nearly a 5 fold longer terminal half-life of 62.5h. The strong interaction of FSI nanoparticles with Rapa was confirmed in vivo by a shift in the monoexponential pharmacokinetic profile for free drug to a biexponential profile for the nanoparticle formulation. When acutely administered by injection into NOD mice via the tail vein, this FSI formulation significantly suppressed lymphocytic infiltration in the LG relative to the control group while reducing toxicity. There was also a significant effect on inflammatory and mammalian target of Rapamycin (mTOR) pathway genes in the LG and surprisingly, our nanoparticle formulation was significantly better at decreasing a proposed tear biomarker of SjS, cathepsin S (CATS) compared to free drug. These findings suggest that FSI is a promising tool for delivering Rapa for treatment of SjS in a murine model and may be further explored to meet the unmet medical challenge of SjS.

Objective The diagnosis of Sjögren's Syndrome (SS) in routine practice is largely a clinical one and requires a high index of suspicion by the treating physician. This great dependence upon clinical judgment frequently leads to delayed diagnosis or misdiagnosis. Tear protein profiles have been proposed as simple and reliable biomarkers for SS diagnosis. Given that cathepsin S activity is increased in the lacrimal glands and tears of NOD mice (a murine model of SS), we explored the clinical utility of using tear cathepsin S (CTSS) activity as a biomarker for SS. Methods A method to measure CTSS activity in tears eluted from Schirmer's strips was developed and validated. Schirmer's tests and CTSS activity measurements were performed on 278 female subjects, including patients with SS (n=73), rheumatoid arthritis (n=79), systemic lupus erythematosus (n=40), blepharitis (n=10), non-specific dry eye (n=31), or other autoimmune diseases (n=12), along with 33 healthy controls. Results Median tear CTSS activity in SS patients was 4.1-fold higher than in patients with non-SS autoimmune diseases, 2.1-fold higher than in patients with non-specific dry eye, and 41.1-fold higher than in healthy controls. Tear CTSS levels were equally elevated in primary and secondary SS independent of the Schirmer's strip values or of circulating anti-SSA or anti-SSB autoantibodies. Conclusion Markedly high levels of tear CTSS activity are suggestive of SS. CTSS activity in tears can be measured in a simple, quick, economical, and non-invasive fashion and may serve as a novel biomarker and indicator of autoimmune dacryoadenitis during the workup for SS.

Expression of CATS and CATH increases in parallel with proinflammatory cytokines during the development of autoimmune inflammatory disease in the NOD mouse disease model. Tear CATS may represent a biomarker for diagnosis of dacryoadenitis in SjS.

PurposeTo evaluate the efficacy of topical rapamycin in treating autoimmune dacryoadenitis in a mouse model of Sjögren's syndrome.MethodsWe developed rapamycin in a poly(ethylene glycol)-distearoyl phosphatidylethanolamine (PEG-DSPE) micelle formulation to maintain solubility. Rapamycin or PEG-DSPE eye drops (vehicle) were administered in a well-established Sjögren's syndrome disease model, the male nonobese diabetic (NOD) mice, twice daily for 12 weeks starting at 8 weeks of age. Mouse tear fluid was collected and tear Cathepsin S, a putative tear biomarker for Sjögren's syndrome, was measured. Lacrimal glands were retrieved for histological evaluation, and quantitative real-time PCR of genes associated with Sjögren's syndrome pathogenesis. Tear secretion was measured using phenol red threads, and corneal fluorescein staining was used to assess corneal integrity.ResultsLymphocytic infiltration of lacrimal glands from rapamycin-treated mice was significantly (P = 0.0001) reduced by 3.8-fold relative to vehicle-treated mice after 12 weeks of treatment. Rapamycin, but not vehicle, treatment increased tear secretion and decreased corneal fluorescein staining after 12 weeks. In rapamycin-treated mice, Cathepsin S activity was significantly reduced by 3.75-fold in tears (P < 0.0001) and 1.68-fold in lacrimal gland lysates (P = 0.003) relative to vehicle-treated mice. Rapamycin significantly altered the expression of several genes linked to Sjögren's syndrome pathogenesis, including major histocompatibility complex II, TNF-α, IFN-γ, and IL-12a, as well as Akt3, an effector of autophagy.ConclusionsOur findings suggest that topical rapamycin reduces autoimmune-mediated lacrimal gland inflammation while improving ocular surface integrity and tear secretion, and thus has potential for treating Sjögren's syndrome–associated dry eye.

Aim: Secretion of proteins into basal tears of Parkinson’s disease (PD) patients may be altered by changes in nerve function. Materials & methods: Oligomeric α-SynOligo and total α-SynTotal, CCL-2, DJ-1, LF and MMP-9 were measured in basal tears from 93 PD patients and 82 age- and sex-equivalent healthy controls. Results: α-SynTotal was decreased (p = 0.0043), whereas α-SynOligo (p < 0.0001) and the ratio of α-SynOligo/α-SynTotal (p < 0.0001) were increased in basal tears from PD patients compared with healthy controls. Area under receiver-operating curves of α-SynOligo and α-SynOligo/α-SynTotal contents were 0.70 (95% confidence limits: 0.621–0.774) and 0.72 (95% confidence limits: 0.642–0.792). Conclusion: PD patient basal tears may contain biomarkers that can be assayed noninvasively and inexpensively.

Cathepsin S (CTSS) activity is elevated in Sjögren’s Syndrome (SS) patient tears. Here we tested whether protease inhibition and cystatin C (Cys C) levels are reduced in SS tears, which could lead to enhanced CTSS-driven degradation of tear proteins. CTSS activity against Cys C, LF and sIgA was tested in SS or healthy control tears. Tears from 156 female subjects (33, SS; 33, rheumatoid arthritis; 31, other autoimmune diseases; 35, non-autoimmune dry eye (DE); 24, healthy controls) were analyzed for CTSS activity and Cys C, LF, and sIgA levels. Cys C and LF showed enhanced degradation in SS tears supplemented with recombinant CTSS, but not supplemented healthy control tears. CTSS activity was significantly increased, while Cys C, LF and sIgA levels were significantly decreased, in SS tears compared to other groups. While tear CTSS activity remained the strongest discriminator of SS in autoimmune populations, combining LF and CTSS improved discrimination of SS beyond CTSS in DE patients. Reductions in Cys C and other endogenous proteases may enhance CTSS activity in SS tears. Tear CTSS activity is reconfirmed as a putative biomarker of SS in an independent patient cohort while combined LF and CTSS measurements may distinguish SS from DE patients.

The purpose of the present study was to test the potential of mouse bone marrow-derived mesenchymal stem cells (BD-MSCs) in improving tear production in a mouse model of Sjögren's syndrome dry eye and to investigate the underlying mechanisms involved. NOD mice (n = 20) were randomized to receive i.p. injection of sterile phosphate buffered saline (PBS, control) or murine BD-MSCs (1 × 106 cells). Tears production was measured at baseline and once a week after treatment using phenol red impregnated threads. Cathepsin S activity in the tears was measured at the end of treatment. After 4 weeks, animals were sacrificed and the lacrimal glands were excised and processed for histopathology, immunohistochemistry, and RNA analysis. Following BD-MSC injection, tears production increased over time when compared to both baseline and PBS injected mice. Although the number of lymphocytic foci in the lacrimal glands of treated animals did not change, the size of the foci decreased by 40.5% when compared to control animals. The mRNA level of the water channel aquaporin 5 was significantly increased following delivery of BD-MSCs. We conclude that treatment with BD-MSCs increases tear production in the NOD mouse model of Sjögren's syndrome. This is likely due to decreased inflammation and increased expression of aquaporin 5.

The purpose of the present studies was to investigate the impact of chronic inflammation of the lacrimal gland, as occurs in Sjögren’s syndrome, on the morphology and function of myoepithelial cells (MECs). In spite of the importance of MECs for lacrimal gland function, the effect of inflammation on MECs has not been well defined. We studied changes in MEC structure and function in two animal models of aqueous deficient dry eye, NOD and MRL/lpr mice. We found a statistically significant reduction in the size of MECs in diseased compared to control lacrimal glands. We also found that oxytocin receptor was highly expressed in MECs of mouse and human lacrimal glands and that its expression was strongly reduced in diseased glands. Furthermore, we found a significant decrease in the amount of two MEC contractile proteins, α-smooth muscle actin (SMA) and calponin. Finally, oxytocin-mediated contraction was impaired in lacrimal gland acini from diseased glands. We conclude that chronic inflammation of the lacrimal gland leads to a substantial thinning of MECs, down-regulation of contractile proteins and oxytocin receptor expression, and therefore impaired acini contraction. This is the first study highlighting the role of oxytocin mediated MEC contraction on lacrimal gland function.