Browsing by Subject "Cornea"
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Item Destruction of Corneal Nerves Promotes Corneal Allograft Rejection(2014-06-11) Paunicka, Kathryn Joy; Street, Nancy E.; Niederkorn, Jerry Y.; Gruchalla, Rebecca S.; Gill, Michelle A.The human corneal endothelium has very little regenerative capacities and cannot fully heal in response to infection or trauma. Evolutionarily, the eye developed a mechanism to retain visual acuity by protecting the eye from inflammatory damage, referred to as immune privilege. The mechanisms that protect the eye from inflammation-induced injury are 1.) the presence of immunosuppressive molecules within the aqueous humor; 2.) the expression of pro-apoptotic factors on corneal cells ; and 3.) the induction of a form of immune tolerance called anterior chamber-associated immune deviation (ACAID). Immune privilege contributes to the 90% success rate of corneal allografts without the need for histocompatibility matching and use of systemic immunosuppressive therapy. However, when one or more parameters that contribute to immune privilege are broken, the cornel allograft becomes vulnerable to the immune system, resulting in corneal allograft failure. Patients that elect to have a corneal allograft replaced are the population at the highest risk of immune rejection and have only a 70% success rate with the second corneal allograft, and the rate of rejection continues to increase with each successive graft. Why subsequent corneal allografts have an increased incidence of rejection is unknown. Due to the high success rate of corneal allografts, the donor tissues are not tissue matched to the recipients. With the limited documentation on the histocompatibility gene loci expressed by the corneal tissue donors, it is unknown if the rejection of the initial corneal allograft sensitizes the corneal allograft recipient. This study provides evidence that the sensitization of the corneal transplant recipient towards alloantigens also expressed on the subsequent corneal allografts is not a requisite for the high incidence of graft rejection. Furthermore, the enhanced incidence of graft rejection is an immune response directed towards alloantigens expressed on the subsequent corneal transplant. The aim of this study is to determine why the corneal transplantation procedure enhances the rejection of subsequent corneal allografts in both eyes. Experimental evidence demonstrates that the destruction of the corneal nerves in one eye fundamentally alters the expression of the immunoregulatory neuropeptides in the contralateral eye. The altered expression of these neuropeptides inhibits both the induction and maintenance of immune privilege. The alteration in the microenvironment mediated a quick and prolonged loss of immune privilege, which could be reversed by blocking the activity of the pro-inflammatory neuropeptide substance P (SP). The survival of the corneal allograft requires the participation of antigen-specific T regulatory cells. Neuropeptides within the ocular environment are important for immune privilege through the induction of tolerance. Our results demonstrate the destruction of the corneal nerves and the release of the pro-inflammatory neuropeptide SP inhibits both the generation and function of T regulatory cells, which ultimately leads to corneal allograft rejection.Item Effect of ULK1 Inhibition on Corneal Epithelial Cells During Pseudomonas aeruginosa Infection(2024-01-30) Abdallah, Joelle; Ayilam Ramachandran, Rajalakshmy; Robertson, Danielle M.INTRODUCTION: Pseudomonas aeruginosa (PA) keratitis is a severe infection of the cornea that can lead to blindness. Studies in our lab have shown that PA exploits autophagy, a major cellular degradation process, in corneal epithelial cells (hTCEpi cells) to promote intracellular survival. We have further shown that the inhibition of autophagy by the Unc 51-like kinase (Ulk1), an enzyme that mediates formation of the autophagosome, reduces intracellular levels of PA. More recently, we have demonstrated that PA infection negatively impacts host mitochondria. ULK1/2 has been reported to translocate to mitochondria to mediate mitophagy however, a role for ULK1/2 in mitochondrial homeostasis during infection has not yet been explored. In this study, we investigated the effects of the inhibition of Ulk1 during PA infection on host mitochondria. METHODS: Telomerase-immortalized human corneal epithelial (hTCEpi) cells were used for this study. Cells were cultured in serum-free defined keratinocyte media with growth supplements. Cells were inoculated with 106 CFU/ml of PA in log growth phase with or without treatment with 1 ï�M of the Ulk1/2 inhibitor MRT68921. Intracellular levels of PA were quantified using a gentamicin survival assay. Oxygen consumption and mitochondrial polarization were assessed using Seahorse metabolic flux analysis and tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1), respectively. Levels of pro-inflammatory cytokines were assessed using ELISA. Untargeted metabolomics was performed using mass spectrometry. Cellular changes were further evaluated using transmission electron microscopy (TEM). RESULTS: PA infection induced robust mitochondrial depolarization. There was a corresponding increase in secretion of IL-6 and IL-8. Treatment with MRT68921 restored mitochondrial polarization and reduced IL-6, but had no effect on IL-8. MRT68921 also reduced intracellular levels of PA. TEM demonstrated robust mitochondrial fission during PA infection. Treatment with MRT68921 preserved mitochondrial structure and polarization during PA infection. CONCLUSIONS: Taken together, these data suggest that Ulk1/2 modulates the host mitochondrial response to PA infection. Further studies are needed to determine the mechanism by which MRT68921 preserves mitochondrial function and its potential use as an adjunct therapeutic for PA-mediated keratitis.Item Interactive Effects of Obstructive Sleep Apnea and Type 2 Diabetes Mellitus on Corneal Nerves: Preliminary Findings(2017-01-17) Stuard, Whitney; Gallerson, Bryan; Robertson, Danielle M.INTRODUCTION: In vivo corneal confocal microscopy (IVCM) is increasingly being used to evaluate the corneal subbasal nerve plexus (SBNP) in patients with Type 2 Diabetes Mellitus (T2DM). Current data suggests that loss of the SBNP is a surrogate marker for peripheral diabetic neuropathy (DPN). Obstructive Sleep Apnea (OSA) is an under diagnosed condition that is highly prevalent in patients with T2DM. The purpose of this study is to examine the impact of OSA on SBNP changes in patients with T2DM. METHODS: A total of 184 patients will be recruited across four groups: A) T2DM and OSA, 2) OSA, 3) T2DM, and 4) healthy controls. A physician diagnosis of T2DM and/or OSA is required for inclusion in each study group. Groups are matched for sex, age, and obesity status. Each patient undergoes testing for the following: serology for Hemoglobin A1c and high sensitivity C-reactive protein, an eye exam with dry eye testing and dilated fundus examination, anthropometric measurements, tear collection, IVCM, measurement of corneal sensitivity, and optical coherence tomography to assess the retinal nerve fiber layer (rNFL) and macula. Patients are also asked to complete three questionnaires including the Stop-Bang questionnaire (to assess risk of OSA), the ocular surface disease index questionnaire (to assess dry eye disease), and a CPAP compliance survey. IVCM images are analyzed for determination of nerve fiber length and density, tortuosity (short term and long term), and basal corneal epithelial cell and dendritic cell density. Human tears are analyzed for tear levels of IGFBP3 using ELISA and extracellular tear DNA is quantified using a Qubit Fluorometer. Anthropometric measurements are used to calculate body mass index and waist to height ratio. RESULTS: Preliminary data confirms that Hemoglobin A1c is highest in the two groups with T2DM patients; STOP BANG scores and male neck circumference are highest in the two groups with OSA. Nerve fiber length is shortest in patients with T2DM. Tortuosity and morphological differences have been found amongst the four groups. A slight decrease has been found in the rNFL for patients with OSA and T2DM. DISCUSSION: A recent study in the UK analyzed the risk and severity of DPN in patients with OSA. Among 266 patients with T2DM, those with OSA had a higher prevalence of DPN and the severity of neuropathy was related to the severity of OSA and not the duration of T2DM. The results of the present study will be the first to show a definitive relationship in changes in the SBNP and rNFL between patients with OSA and T2DM.Item Reconstitution of a Multi-layered, Differentiated Cornea by HTERT-Immortalized Corneal Epithelial Cells Transduced with Thymidine Kinase Transplanted onto Denuded Mouse Corneas(2011-10-25) Kalangara, Jerry P.; Cavanagh, H. DwightOBJECTIVE: To develop an animal model for the implementation of human telomerase enzyme reverse transcriptase (hTERT)-immortalized human corneal epithelial cell line (hTCEpi) transduced with the hygromycin-thymidine kinase gene (HyTK) as a viable cell source for the reconstruction of the corneal surface. METHODS: HyTK cells were cultured in KGM-2 serum-free culture media under hygromycin B selection. Limbal stem cell deficiency (LSCD) was established in athymic nude mice (n=75) using ethylenediaminetetraacetic acid (EDTA) and mitomycin C treatment followed by mechanical debridement of corneal and limbal epithelium. Immunofluorescence (IF) using Anti-Laminin and Propidium iodide (PI) was used to assess presence of basement membrane after epithelial removal. Cultured HyTK cells were stained with Cell Tracker Green CMFDA (5-chloromethylfluorescein diacetate) and transplanted onto the right eye after epithelial removal; the left eye served as a control. Transplanted cells were evaluated at 4 hours, 1 day, and 7 days post-transplantation using laser scanning confocal microscopy (LSCM). At 4 hours and 1 day, corneas were imaged for the presence of CMFDA. At day 7, corneas were stained using antibodies to Keratin 3, Ki-67, and the fluorescent probes – PI, and Phalloidin. Cytotoxicity of ganciclovir was assessed at concentrations of 0.1, 0.5, 1.0, 5.0, and 10.0 μM using Live-Dead Assay. RESULTS: IF confirmed an intact corneal basement membrane following epithelial removal. At 4 hours and 1 day post-transplantation, CMFDA staining demonstrated that transplanted cells were dispersed throughout the corneal surface. After 7 days, HyTK cells showed stratification and IF confirmed a differentiated corneal epithelial phenotype. Incubation in ganciclovir induced a cytotoxic effect on HyTK cells in vitro and had no significant effect on the hTCEpi control cell line. This effect was significant at 0.5 µM (p < 0.05). CONCLUSIONS: These studies demonstrate the first reconstitution of a multi-layered, differentiated corneal epithelium by HyTK cells in the nude mouse model; further, proliferating HyTK cells can be killed with ganciclovir treatment in vitro, which may reduce the potential for risk of oncogenic transformation in vivo.Item The Role of Insulin-Like Growth Factor Binding Protein 3 in Mitochondrial Homeostasis(2022-05) Stuard, Whitney Leigh; Sieber, Matthew H.; Mootha, Vinod; Robertson, Danielle M.; DeBerardinis, Ralph J.The insulin-like growth factor (IGF) family includes three extracellular ligands and their cognate receptors, along with six IGF-binding proteins (IGFBPs 1-6). Of these, Insulin-like growth factor binding protein-3 (IGFBP-3) is expressed in corneal epithelial cells (CECs) and is present in human tear fluid. IGFBP-3 is a highly glycosylated secretory protein with known roles in growth and survival. In circulation, IGFBP-3 binds IGF-1 to inhibit activation of the IGF-1 receptor, thereby extending the half-life of IGF-1. IGFBP-3 also plays key roles in apoptosis, DNA repair, cell cycle control, the induction of autophagy, angiogenesis, hypoxia, and insulin resistance. However, the exact function of IGFBP-3 is cell and context-dependent. Prior studies in our lab have demonstrated that expression of IGFBP-3 is increased in response to hyperglycemia and hypoxia, suggesting a potential role for IGFBP-3 in the corneal epithelial stress response. Using a combination of contemporary cellular, molecular, and biochemical approaches, we now provide new data showing that IGFBP-3 stabilizes mitochondria in CECs through alterations in mitochondrial morphology and mitophagy. This occurs through activation of the mechanistic target of the rapamycin (mTOR) signaling pathway. Taken together, these data highlight a new role for IGFBP-3 in the regulation of mitochondrial and metabolic homeostasis in the corneal epithelium. We next explored the pathophysiological role of IGFBP-3 in mitochondrial respiration, homeostasis, and mitophagy in CECs subject to hyperosmolar stress, as seen in Dry Eye Disease. Unlike hyperglycemia, hyperosmolar stress due to an increase in levels of salt downregulates IGFBP-3 in vitro. The loss of IGFBP-3 in response to hyperosmolar stress is associated with a shift towards a more respiratory phenotype and an increase in mitochondrial fission. In contrast, co-treatment with recombinant human IGFBP-3 induced robust mitochondrial fusion and maintained metabolic activity. The decrease in intracellular IGFBP-3 in response to hyperosmolarity was further confirmed in the mouse corneal epithelium in vivo using an aqueous-deficient dry eye model. These data confirm a novel role for IGFBP-3 in metabolic and mitochondrial homeostasis.Item [Southwestern News](1998-04-17) Abila, ReyesItem Subbasal Nerve Plexus Changes in Type 2 Diabetes Mellitus Correlate with Tear Levels of IGFBP-3(2018-01-23) Stuard, Whitney L.; Titone, Rossella; Robertson, Danielle M.INTRODUCTION: Changes in the corneal subbasal nerve plexus have been reported in patients with Type 2 Diabetes Mellitus (T2DM) and suggest that these changes may provide an early, surrogate marker for the onset of peripheral neuropathy. Increasing studies are investigating the use of tear film biomarkers that correlate with corneal nerve changes in diabetic disease. Our prior studies have demonstrated that the primary insulin-like growth factor (IGF)-1 binding protein, IGF-binding protein-3 (IGFBP-3), is elevated in the diabetic tear film. This study examined tear levels of IGFBP-3 in basal tears of patients with T2DM compared to age, sex, and obesity-matched controls; and assessed the relationship between tear levels of IGFBP-3 with morphological changes in the subbasal nerve plexus and corneal epithelial cells. METHODS: This study is a single visit, cross-sectional study comparing two groups: 1) T2DM and 2) healthy controls. A physician diagnosis of T2DM was required for inclusion in this test group. Groups were matched for age, sex, and obesity status. Each volunteer underwent serology testing for Hemoglobin A1c and high sensitivity C-reactive protein, completed the ocular surface disease index (OSDI) questionnaire and clinical measurements of dry eye, assessment of anthropometric parameters, tear analysis, in vivo confocal microscopy to assess corneal nerve morphology, corneal sensitivity testing, and ocular coherence tomography to assess the retinal nerve fiber layer and macula. Anthropometric measurements were used to calculate BMI and waist to height ratio. Human tears were collected for the analysis of tear levels of IGFBP-3 using an IGFBP-3 Quantikine ELISA kit (R&D Systems, Minneapolis, MN). Confocal data was analyzed using ImageJ and MetaMorph Software. RESULTS: A total of 40 participants were included in this study. There were no differences in corneal sensitivity or dry eye parameters between groups. IGFBP-3 levels in tears of T2DM patients were 3.5 times higher than controls (P<0.05). HbA1c was not correlated to IGFBP-3 (R=0.318, P=0.062). Tear levels of IGFBP-3 were correlated with nerve fiber length (R=0.522 P=0.001) and nerve branch density (R=0.481 P=0.003). IGFBP-3 was more tightly correlated with nerve changes than HbA1c. Consistent with our animal models, there was a decrease in corneal basal epithelial cell density in T2DM compared to controls (P=0.04). DISCUSSION: This study demonstrates that IGFBP-3 is higher in patients with T2DM. These studies further suggest that tear levels of IGFBP-3 may be a novel biomarker for monitoring ocular damage in diabetes. Further studies are needed to stratify tear levels of IGFBP-3 with severity of disease.Item Tear Biomarkers and Corneal Sensitivity as an Indicator of Neuropathy in Type 2 Diabetes(2020-05-01T05:00:00.000Z) Iyengar, Meera Farzana; Chang, Mary; Lingvay, Ildiko; Rajora, NilumBACKGROUND: Diabetic peripheral neuropathy (DPN) is a debilitating, progressive complication of type 2 diabetes. The high cost of management leads to amputations in approximately 6% of individuals with DPN in poor-resource settings due to medical noncompliance or lack of finances. Having an effective means of early detection of DPN is crucial for early intervention, which would have a major impact in alleviating its social, economic, and medical burden. OBJECTIVES: To explore the potential of 31 tear biomarkers involved in both corneal growth and development and inflammatory pathways in screening for diabetic peripheral neuropathy (DPN). Additionally, the utility of aesthesiometry for measuring corneal damage in DPN was assessed. METHODS: This screening test pilot study recruited 90 participants from a tertiary hospital in Lima, Peru. Participants were categorized into three groups based on diabetes and neuropathy status. Tears were collected on Schirmer strips, and proteins were measured by both ELISA and multiplex-bead assay. Corneal sensitivity was measured by aesthesiometry, and DPN was measured through vibration perception threshold testing. RESULTS: A total of 89 participants were included in the analysis. The mean age was 55.7+1.46, and 58.4% were female. After adjusting for potential confounders, MMP-9 and TGF-alpha levels showed a strong upward trend in participants with DPN when compared to those with diabetes alone, though not significant. Decreased corneal sensitivity, as measured by aesthesiometry, was negatively correlated with MMP-9 levels (p<0.01) in individuals with DPN. Aesthesiometry was significantly decreased in individuals with DPN when compared to participants with diabetes alone (p<0.01) and normal controls (p<0.01). CONCLUSIONS: Although tears are a simple and inexpensive resource with promise to help detect DPN, it is an insufficient standalone tool for detecting DPN based on the present study. Aesthesiometry is a simple, inexpensive, and accurate method to assess corneal damage associated with DPN, and its integration into screening practices has potential to improve detection of DPN in poor-resource settings.Item [UT Southwestern Medical Center News](2008-04-01) Rian, Russell