CD25 knock-out (CD25KO) mice spontaneously develop Sjögren Syndrome (SS)-like inflammation. We investigated the role of commensal bacteria by comparing CD25KO mice housed in conventional or germ-free conditions. Germ-free CD25KO mice have greater corneal barrier dysfunction, lower goblet cell density, increased total lymphocytic infiltration score, increased expression of IFN-γ, IL-12 and higher a frequency of CD4IFN-γ cells than conventional mice. CD4 T cells isolated from female germ-free CD25KO mice adoptively transferred to naive immunodeficient RAG1KO recipients caused more severe Sjögren-like disease than CD4 T cells transferred from conventional CD25KO mice. Fecal transplant in germ-free CD25KO mice reversed the spontaneous dry eye phenotype and decreased the generation of pathogenic CD4IFN-γ cells. Our studies indicate that lack of commensal bacteria accelerates the onset and severity of dacryoadenitis and generates autoreactive CD4T cells with greater pathogenicity in the CD25KO model, suggesting that the commensal bacteria or their metabolites products have immunoregulatory properties that protect exocrine glands in the CD25KO SS model.

PurposeToll-like receptor 4 (TLR4) alerts cells to the presence of bacteria by initiating an inflammatory response. We hypothesize that disruption of the ocular surface barrier in dry eye enhances TLR4 signaling. This study determined whether dry eye enhances expression of inflammatory mediators in response to topically applied TLR4 ligand.MethodsA single dose of lipopolysaccharide (LPS) or vehicle (endotoxin-free water) was applied to the cornea of nonstressed (NS) mice or mice subjected to 5 days of desiccating stress (DS). After 4 hours, corneal epithelium and conjunctiva were extracted to analyze expression of inflammatory mediators via PCR. Protein expression was confirmed by immunobead assay and immunostaining.ResultsTopically applied LPS increased expression of inflammatory mediators IL-1β, CXCL10, IL-12a, and IFN-γ in the conjunctiva, and IL-1β and CXCL10 in the cornea of NS mice compared to that in untreated controls. LPS in DS mice produced 3-fold increased expression of IL-1β in cornea and 2-fold increased expression in IL-12a in conjunctiva compared to that in LPS-treated control mice.ConclusionsLPS increased expression of inflammatory cytokines on the ocular surface. This expression was further increased in dry eye, which suggests that epithelial barrier disruption enhances exposure of LPS to TLR4+ cells and that the inflammatory response to endotoxin-producing commensal or pathogenic bacteria may be more severe in dry eye disease.

The purpose of this study was to evaluate the effectiveness of a novel fluorocarbon-based sevoflurane emulsion in dogs previously shown to produce short-term rodent anesthesia. On the basis of an unexpected allergic-type clinical reaction, we also tested the hypothesis that this type of formulation causes histamine release and complement activation. Physiological parameters, plasma histamine levels (radioimmunoassay), and complement activation (enzyme immunoassay) were quantified in response to emulsion components, including F13M5 (the emulsion’s fluorocarbon-based polymer) and methoxy poly(ethylene glycol) 5000 (the polymer’s hydrophilic block). Although the emulsion produced general anesthesia in dogs, they also experienced hypotension and clinical signs suggestive of an allergic-like response (i.e., vasodilation, urticaria, and pruritus upon recovery). Emulsions lacking sevoflurane failed to induce anesthesia but did elicit the allergic response. Plasma histamine levels were significantly increased following injection of micellar solutions of F13M5. Direct complement activation by the emulsion or its components was weak or absent. An allergic response leading to histamine release, likely initiated by the F13M5 component via an immunoglobulin pathway, is associated with an intravenous fluorocarbon-based emulsion of sevoflurane. Subsequently, its usefulness in medicine in its present formulation is limited.

Purpose: Age-related macular degeneration (AMD) is a common disease trending towards epidemic proportions and is a leading cause of irreversible vision loss in people over the age of 65. A pathomechanism of AMD is death and/or dysfunction of retinal pigment epithelial (RPE) cells; RPE loss invariably results in photoreceptor atrophy. Treatment options for AMD are very limited, and include vitamin supplements and lifestyle changes. An exciting potential therapy currently being tested in clinical trials is transplantation of stem cell-derived RPE. Methods: We developed a NIH-registered embryonic stem line (CR-4), and in this study set out to determine if CR4-RPE are tolerated in normal mice and in murine models of retinal degeneration by injecting a bolus of CR4-RPE cells in the subretinal space of immunosuppressed wild-type, Mer mutant (Merkd), and Elovl4 deficient mice. Results: Mice with CR-RPE grafts were monitored daily, were examined routinely using OCT, and histology was prepared and examined at terminal end-points. Based on the parameters of the study, none of the animals with CR-RPE grafts (n=36) experienced any obvious adverse reactions. Conclusions: We conclude that transplanted CR-4 hES-derived RPE cells are well tolerated in immunosuppressed healthy and dystrophic murine retinas.

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Nanoparticles have shown promise in the field of drug delivery. Many have the potential to improve the solubility and safety‐profile of our current drugs. Polyethylene glycol is commonly used as the hydrophilic segment in many polymer‐based nanoparticle systems. There have been reports of hypersensitivities to PEGylated particles. In order for these novel drug delivery systems to reach clinical use, it is essential to investigate the mechanism behind their allergic‐like responses. This study examines hypersensitivities to PEGylated particles in a canine model. Dogs are a very sensitive model for this type of allergy, which is thought to be caused through either direct complement activation or an antibody‐based mechanism. We have tested a number of PEG‐fluorocarbon polymers using this model and have performed blood analysis pre and post injection. Current results indicate an antibody‐mediated pathway is likely responsible for this type of hypersensitivity.This project was supported by the UW Institute for Clinical and Translational Research funded through a National Institutes of Health Clinical and Translational Science Award, 1UL1 RR025011, and the STEP program administered by the Wisconsin Alumni Research Foundation.

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