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Objectives. To determine the efficacy of widely available subtype clinical tests to characterize evaporative dry eye disease (EDED) related to meibomian gland dysfunction (MGD) compared to normal and to validate those clinical cut points in an independent sample. Methods. A diagnostic accuracy study (52 subjects), an investigator-masked study, was followed by a larger independent sample (364 subjects) analysis to confirm efficacy in normal and EDED subjects. All subjects were 18 years of age and older and were classified using a battery of clinical tests for dry eye that included symptoms, tear meniscus height, tear stability, ocular staining, evaporative-specific tests, and the Schirmer I test. Results. Normal (nondry eye; n = 26) and EDED (n = 26) subjects completed the efficacy study. The global tests of tear breakup time, staining, and symptoms all produced AUCs ≥ 0.70, representing acceptable discrimination. EDED-specific tests of eyelid marginal signs, gland secretion quality, and gland loss did not demonstrate acceptable test efficacy or differences between normal and EDED subjects. In a larger, independent sample of normal and EDED subjects, gland secretion quality and eyelid marginal score achieved acceptable diagnostic levels: AUCs of 0.789 (CI: 0.734–0.844) and 0.729 (CI: 0.648–0.810), respectively, but not lipid interferometry grade or lower eyelid gland dropout estimated using meiboscopy. Conclusions. Meibomian gland secretion quality is an efficient and useful functional indicator in EDED and should be incorporated into core outcome sets for this dry eye subtype.

Summary: The lacrimal gland (LG) is an exocrine organ responsible for the secretion of aqueous tear film. Regenerative and stem cell therapies that target LG repair are coming to the fore, although our understanding of LG cell lineage hierarchy is still incomplete. We utilize the analysis of label-retaining cells (LRCs) and genetic lineage tracing to define LG cell lineage hierarchy. Our study suggests that embryonic LG contains unique long-lived multipotent stem cells that give rise to all postnatal epithelial cell types. Following birth, lineages become established and the fate of progenitor cell descendants becomes restricted. However, some cell lineages retain plasticity after maturation and can trans-differentiate into other cell types upon injury. The demonstration that the LG contains progenitor cells with different levels of plasticity has profound implications for our understanding of LG gland function in homeostasis and disease and will be helpful for developing stem cell-based therapies in the future.

The meibomian and sebaceous glands secrete lipids to prevent desiccation of the ocular surface and skin, respectively. Precisely how these holocrine tissues regenerate is not well understood. To address this, we characterized keratin 5+ (K5) label-retaining cells (LRCs) and the lineage tracing of keratin 14 (K14) progenitors in mouse meibomian glands. Using the tet-off H2B-GFP/K5tTA mouse, H2B-GFP fluorescence dilutes 2-fold with every division in K5+ cell nuclei after doxycycline administration. In 3D reconstructions generated over a >28-day doxycycline chase, we observed LRCs at the acinus entrance where K6+ ductal epithelium terminates. For lineage tracing, K14CreERT2-Confetti mice were injected intraperitoneally with tamoxifen and euthanized at 23 and 59 weeks later. Meibomian gland acini in these mice were either monochromatic or dual-colored, whereas the duct exhibited multiple colors. In conclusion, LRCs are likely to direct meibomian gland turnover and may exist as two distinct unipotent progenitors that renew ductal and acinar tissue separately.

The cornea is the most external layer of the eye and serves two important roles in (1) the refraction of light and (2) protection from the outside environment, both of which are highly dependent on the collagen assembly of the corneal stroma. This study sought to determine the collagen fiber arrangement of the canine corneal stroma and correlate the stromal organization with tissue stiffness in the anterior and posterior cornea. Collagen organization of the canine cornea was visualized through second-harmonic generation (SHG) imaging, and tissue stiffness of the anterior and posterior corneal stroma was determined by atomic force microscopy. Analysis of the canine anterior corneal stroma using SHG imaging documented intertwining of the collagen fibers with a high degree of fiber branching, with a more lamellar and non-branching posterior stroma. The anterior stroma had significantly higher tissue stiffness in both dogs and humans, when compared with the posterior corneal stroma (canine median: 1.3 kPa vs. 0.3 kPa; human median: 14.6 kPa vs. 2.1 kPa, respectively). There was a direct correlation between corneal collagen stromal organization and tissue stiffness in the dog, which was consistent with other mammalian species previously examined and likely reflects the need for maintenance of rigidity and corneal curvature.

Aldehyde dehydrogenase 1A1 (ALDH1A1) and ALDH3A1 are corneal crystallins. They protect inner ocular tissues from ultraviolet radiation (UVR)-induced oxidative damage through catalytic and non-catalytic mechanisms. Additionally, ALDH3A1 has been postulated to play a regulatory role in the corneal epithelium based on several studies that report an inverse association between ALDH3A1 expression and corneal cell proliferation. The underlying molecular mechanisms and the physiological significance of such association remain poorly understood. In the current study, we established Tet-On human corneal epithelial cell (hTCEpi) lines, which express tetracycline-inducible wild-type (wt) or catalytically-inactive (mu) ALDH3A1. Utilizing this cellular model system, we confirmed that human ALDH3A1 decreases corneal cell proliferation; importantly, this effect appears to be partially mediated by its enzymatic activity. Mechanistically, wt-ALDH3A1, but not mu-ALDH3A1, promotes sequestering of tumor suppressor p53 in the nucleus. In the mouse cornea, however, augmented cell proliferation is noted only in Aldh1a1(-/-)/3a1(-/-) double knockout (DKO) mice, indicating in vivo the anti-proliferation effect of ALDH3A1 can be rescued by the presence of ALDH1A1. Interestingly, the hyper-proliferative epithelium of the DKO corneas display nearly complete loss of p53 expression, implying that p53 may be involved in ALDH3A1/1A1-mediated effect. In hTCEpi cells grown in high calcium concentration, mRNA levels of a panel of corneal differentiation markers were altered by ALDH3A1 expression and modulated by its enzyme activity. In conclusion, we show for the first time that: (i) ALDH3A1 decreases corneal epithelial proliferation through both non-enzymatic and enzymatic properties; (ii) ALDH1A1 contributes to the regulation of corneal cellular proliferation in vivo; and (iii) ALDH3A1 modulates corneal epithelial differentiation. Collectively, our studies indicate a functional role of ALDH3A1 in the maintenance of corneal epithelial homeostasis by simultaneously modulating proliferation and differentiation through both enzymatic and non-enzymatic mechanisms.

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

Current immunofluorescence protocols are limited as they do not provide reliable antibody staining within large tissue volumes (mm(3)) and cannot localise and quantify multiple antigens or cell populations in the same tissue at high resolution. To address this limitation, we have developed an approach to three-dimensionally visualise large tissue volumes (mm(3)) at high resolution (

Keratoplasty is the most effective treatment for corneal blindness, but suboptimal medical conditions and lack of qualified medical personnel and donated cornea often prevent the performance of corneal transplantation in developing countries. Our study aims to develop alternative treatment regimens for congenital corneal diseases of genetic mutation.Human mesenchymal stem cells isolated from neonatal umbilical cords were transplanted to treat thin and cloudy corneas of lumican null mice. Transplantation of umbilical mesenchymal stem cells significantly improved corneal transparency and increased stromal thickness of lumican null mice, but human umbilical hematopoietic stem cells failed to do the same. Further studies revealed that collagen lamellae were re-organized in corneal stroma of lumican null mice after mesenchymal stem cell transplantation. Transplanted umbilical mesenchymal stem cells survived in the mouse corneal stroma for more than 3 months with little or no graft rejection. In addition, these cells assumed a keratocyte phenotype, e.g., dendritic morphology, quiescence, expression of keratocyte unique keratan sulfated keratocan and lumican, and CD34. Moreover, umbilical mesenchymal stem cell transplantation improved host keratocyte functions, which was verified by enhanced expression of keratocan and aldehyde dehydrogenase class 3A1 in lumican null mice.Umbilical mesenchymal stem cell transplantation is a promising treatment for congenital corneal diseases involving keratocyte dysfunction. Unlike donated corneas, umbilical mesenchymal stem cells are easily isolated, expanded, stored, and can be quickly recovered from liquid nitrogen when a patient is in urgent need.

Mucopolysaccharidosis type I (MPS I) is an inherited α-L-iduronidase (IDUA, I) deficiency in which glycosaminoglycan (GAG) accumulation causes progressive multisystem organ dysfunction, neurological impairment, and death. Current MPS I mouse models, based on a NOD/SCID (NS) background, are short-lived, providing a very narrow window to assess the long-term efficacy of therapeutic interventions. They also develop thymic lymphomas, making the assessment of potential tumorigenicity of human stem cell transplantation problematic. We therefore developed a new MPS I model based on a NOD/SCID/Il2rγ (NSG) background. This model lives longer than 1 year and is tumor-free during that time. NSG MPS I (NSGI) mice exhibit the typical phenotypic features of MPS I including coarsened fur and facial features, reduced/abnormal gait, kyphosis, and corneal clouding. IDUA is undetectable in all tissues examined while GAG levels are dramatically higher in most tissues. NSGI brain shows a significant inflammatory response and prominent gliosis. Neurological MPS I manifestations are evidenced by impaired performance in behavioral tests. Human neural and hematopoietic stem cells were found to readily engraft, with human cells detectable for at least 1 year posttransplantation. This new MPS I model is thus suitable for preclinical testing of novel pluripotent stem cell-based therapy approaches.