An open-source computational tool to automatically quantify immunolabeled retinal ganglion cells.

A fully automated and robust method was developed to quantify β-III-tubulin-stained retinal ganglion cells, combining computational recognition of individual cells by CellProfiler and a machine-learning tool to teach phenotypic classification of the retinal ganglion cells by CellProfiler Analyst. In animal models of glaucoma, quantification of immunolabeled retinal ganglion cells is currently performed manually and remains time-consuming. Using this automated method, quantifications of retinal ganglion cell images were accelerated tenfold: 1800 images were counted in 3 h using our automated method, while manual counting of the same images took 72 h.

The Role of the Immune System in Glaucoma: Bridging the Divide Between Immune Mechanisms in Experimental Glaucoma and the Human Disease.

Glaucoma is one of the leading causes of visual impairment worldwide. Classically, clinicians have evaluated patients through a full ophthalmological examination including gonioscopy, measurement of intraocular pressure (IOP), and assessment of the optic nerve. New imaging modalities have further enhanced our ability to evaluate glaucoma; however, our treatments have not evolved as much.

Effects of High Estrogen Levels on Monocyte Chemoattractant Protein-1 and Wound Healing.

Herein, we tested the effects of high levels of supplemental estrogen treatment on cutaneous wound healing. Female mice were implanted with a 17β-estradiol (E) secreting pellet or placebo before receiving a full-thickness dermal excisional wound. Mice receiving the E pellet attained hormone levels that are comparable to those achieved during pregnancy.

ABCB5 is a limbal stem cell gene required for corneal development and repair.

Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far.

Increased resistance to Staphylococcus aureus endophthalmitis in BALB/c mice: Fas ligand is required for resolution of inflammation but not for bacterial clearance.

FasL was recently shown be required for bacterial clearance in C57BL/6 mice that express the FasL.1 allotype. The FasL.

Soluble guanylate cyclase α1-deficient mice: a novel murine model for primary open angle glaucoma.

Primary open angle glaucoma (POAG) is a leading cause of blindness worldwide. The molecular signaling involved in the pathogenesis of POAG remains unknown. Here, we report that mice lacking the α1 subunit of the nitric oxide receptor soluble guanylate cyclase represent a novel and translatable animal model of POAG, characterized by thinning of the retinal nerve fiber layer and loss of optic nerve axons in the context of an open iridocorneal angle.

NLRP3 inflammasome activation in retinal pigment epithelial cells by lysosomal destabilization: implications for age-related macular degeneration.

Purpose: To evaluate the effect of lysosomal destabilization on NLRP3 inflammasome activation in RPE cells and to investigate the mechanisms by which inflammasome activation may contribute to the pathogenesis of age-related macular degeneration (AMD).

Methods: Human ocular tissue sections from patients with geographic atrophy or neovascular AMD were stained for NLRP3 and compared to tissues from age-matched controls. Expression of the IL-1β precursor, pro-IL-1β, was induced in ARPE-19 cells by IL-1α treatment.

Opposing roles for membrane bound and soluble Fas ligand in glaucoma-associated retinal ganglion cell death.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity.

Spontaneous bacterial keratitis in CD36 knockout mice.

Purpose: CD36 is a Class B scavenger receptor that is constitutively expressed in the corneal epithelium and has been implicated in many homeostatic functions, including the homeostasis of the epidermal barrier. The aim of this study is to determine (1) whether CD36 is required for the maintenance of the corneal epithelial barrier to infection, and (2) whether CD36-deficient mice present with an increased susceptibility to bacterial keratitis.

Methods: The corneas of CD36(-/-), TSP1(-/-), TLR2(-/-), and C57BL/6 WT mice were screened via slit lamp microscopy or ex vivo analysis.

alphaB-crystallin protects retinal tissue during Staphylococcus aureus-induced endophthalmitis.

Bacterial infections of the eye highlight a dilemma that is central to all immune-privileged sites. On the one hand, immune privilege limits inflammation to prevent bystander destruction of normal tissue and loss of vision. On the other hand, bacterial infections require a robust inflammatory response for rapid clearance of the pathogen.

Control of ocular tumor growth and metastatic spread by soluble and membrane Fas ligand.

Fas ligand (FasL) can be either membrane bound, or cleaved by metalloproteinases (MMP) to produce a soluble protein. The two different forms of FasL are reported to have opposite functions-membrane-bound FasL (mFasL) is proinflammatory and soluble FasL (sFasL) is antiinflammatory. We previously showed that, within the immune-privileged eye, tumors expressing high levels of mFasL overcame the suppressive ocular environment, triggered an inflammatory response, and were subsequently rejected.

A novel treatment for ocular tumors using membrane FasL vesicles to activate innate immunity and terminate immune privilege.

Purpose: Ocular immune privilege promotes tumor growth by hindering the development of innate and adaptive immunity. A prior study showed that ocular tumors expressing the membrane-only form of Fas ligand (FasL) terminate immune privilege, induce vigorous inflammation, undergo rejection, and induce systemic protective immunity. In these previous experiments the tumor cells used were genetically engineered to express membrane FasL.

Effect of acute ethanol exposure on the dermal inflammatory response after burn injury.

Background: More than 100,000 people each year are admitted to U.S. hospitals for severe burn injury.

Elevated monocyte chemoattractant protein-1 levels following thermal injury precede monocyte recruitment to the wound site and are controlled, in part, by tumor necrosis factor-alpha.

In previous studies, mice given a full-thickness scald injury had an influx of neutrophils into the skin that followed a local increase in a neutrophil chemoattractant. Because macrophages are known to infiltrate the wound area after neutrophils and are essential for normal wound repair, studies were designed to characterize the time course of macrophage accumulation in the wound and to identify the factor(s) responsible for this influx. A macrophage infiltrate into the wound was observed at 4 days post-injury and persisted through at least 10 days.

Membrane Fas ligand activates innate immunity and terminates ocular immune privilege.

It has been proposed that the constitutive expression of Fas ligand (FasL) in the eye maintains immune privilege, in part through inducing apoptosis of infiltrating Fas(+) T cells. However, the role of FasL in immune privilege remains controversial due to studies that indicate FasL is both pro- and anti-inflammatory. To elucidate the mechanism(s) by which FasL regulates immune privilege, we used an ocular tumor model and examined the individual roles of the membrane-bound and soluble form of FasL in regulating ocular inflammation.

Estrogen regulation of immune responses after injury.

There is a naturally occurring gender difference in immune responses which persists after traumatic injury. Physiological levels of 17beta-estradiol (E(2)) are immunostimulatory, whereas high pregnancy and superphysiological levels are immunosuppressive. In contrast, at all concentrations, testosterone suppresses immune responses.

Survival and cell mediated immunity after burn injury in aged mice.

The elderly are less able to survive burn injury than young healthy individuals. Regardless of age, burn victims often succumb to secondary infections rather than the primary injury. Since immune responses diminish with age, it is likely that aged individuals are predisposed to a poor outcome by virtue of their weak immune system.