Transient plasma membrane disruption induced calcium waves in mouse and human corneal epithelial cells.

The purpose of this study was to examine transient plasma membrane disruptions (TPMDs) and TPMD-induced Ca++ waves (TPMD Ca++ Wvs) in human and mouse corneal epithelium (HCEC and MCEC). A multi-photon microscope was used to create laser-induced TPMDs in single cultured cells and in intact ex vivo and in vivo MCECs and ex vivo human cornea rim HCECs. Eye rubbing-induced TPMDs were studied by gentle rubbing with a cotton tipped applicator over a closed eyelid in ex vivo and in vivo MCECs.

Effects of 1,25-Vitamin D3 and 24,25-Vitamin D3 on Corneal Nerve Regeneration in Diabetic Mice.

Corneal nerve homeostasis is essential for the functional integrity of the ocular surface. Vitamin D deficiency (VDD) and vitamin D receptor knockout (VDR KO) have been found to reduce corneal nerve density in diabetic mice. This is the first study to comprehensively examine the influence of vitamin D on nerve regeneration following corneal epithelial injury in diabetic mice.

Topical Spironolactone in the Treatment of Ocular Graft-Versus-Host Disease.

Introduction: This two-part study aimed to investigate the therapeutic potential of topical spironolactone in ocular graft-versus-host disease (oGVHD). While off-label use of topical spironolactone has been described in dry eye, its efficacy in managing signs and symptoms of oGVHD remains unstudied. Preclinically, we tested the hypothesis that spironolactone induces corneal lipid synthesis in a mouse model.

A Method for Eliminating Fibroblast Contamination in Mouse and Human Primary Corneal Epithelial Cell Cultures.

Purpose: This study was designed to determine if previous approaches to eliminate fibroblast contamination in different cells types would be successful in eliminating fibroblast contamination from human and mouse primary corneal epithelial cell cultures, with the primary goal being to describe a simple, easy, and effective method to culture fibroblast-free primary mouse and human corneal epithelial cell cultures.

Methods: Primary human and mouse corneal stromal cells and epithelial cells were isolated and cultured from human corneal rims and mouse corneas, respectively. Several approaches previously used in other tissue types were evaluated using corneal epithelial cells and mixtures of fibroblasts and epithelial cells to determine the most effective purification method.

Dioleoylphosphatidylglycerol Inhibits Heat Shock Protein B4 (HSPB4)-Induced Inflammatory Pathways In Vitro.

Our previous work shows that dioleoylphosphatidylglycerol (DOPG) accelerates corneal epithelial healing in vitro and in vivo by unknown mechanisms. Prior data demonstrate that DOPG inhibits toll-like receptor (TLR) activation and inflammation induced by microbial components (pathogen-associated molecular patterns, PAMPs) and by endogenous molecules upregulated in psoriatic skin, which act as danger-associated molecular patterns (DAMPs) to activate TLRs and promote inflammation. In the injured cornea, sterile inflammation can result from the release of the DAMP molecule, heat shock protein B4 (HSPB4), to contribute to delayed wound healing.

Innate Immune System Activation, Inflammation and Corneal Wound Healing.

Corneal wounds resulting from injury, surgeries, or other intrusions not only cause pain, but also can predispose an individual to infection. While some inflammation may be beneficial to protect against microbial infection of wounds, the inflammatory process, if excessive, may delay corneal wound healing. An examination of the literature on the effect of inflammation on corneal wound healing suggests that manipulations that result in reductions in severe or chronic inflammation lead to better outcomes in terms of corneal clarity, thickness, and healing.

Effects of 1,25 and 24,25 Vitamin D on Corneal Fibroblast VDR and Vitamin D Metabolizing and Catabolizing Enzymes.

: To investigate the effects of 1,25-Vit D3 and 24,25-Vit D3 on corneal fibroblast expression of the vitamin D-associated enzymes CYP27B1 and CYP24A1 and the roles of the vitamin D receptor (VDR) and protein disulfide isomerase, family A, member 3 (Pdia3) in these cells.: CYP24A1, CYP27B1, VDR, and Pdia3 expression in corneas was detected using immunohistochemistry. Western blotting was used to measure protein expression in human and mouse fibroblasts, including VDR KO mouse cells, treated with 1,25-Vit D3 (20 nM) and 24,25-Vit D3 (100 nM).

Effects of Vitamin D Receptor Knockout and Vitamin D Deficiency on Corneal Epithelial Wound Healing and Nerve Density in Diabetic Mice.

Diabetic keratopathy occurs in ∼70% of all people with diabetes. This study was designed to examine the effects of vitamin D receptor knockout (VDR) and vitamin D deficiency (VDD) on corneal epithelial wound healing and nerve density in diabetic mice. Diabetes was induced using the low-dose streptozotocin method.

Transient Cell Membrane Disruptions induce Calcium Waves in Corneal Keratocytes.

The purpose of this study was to determine if transient cell membrane disruptions (TPMDs) in single keratocytes can trigger signaling events in neighboring keratocytes. Stromal cells were cultured from human corneas (HCSC) and mouse corneas (MCSC). TPMDs were produced using a multiphoton microscope in Cal-520-AM loaded cells.

Influence of Vitamin D on Corneal Epithelial Cell Desmosomes and Hemidesmosomes.

Purpose: We have observed noticably weak epithelial attachment in vitamin D receptor knockout mice (VDR KO) undergoing epithelial debridement. We hypothesized that VDR KO negatively affects corneal epithelial cell desmosomes and/or hemidesmosomes.

Methods: Transcript levels of desmosome and hemidesmosome proteins in VDR KO corneas were assessed by qPCR.

Vitamin D receptor and metabolite effects on corneal epithelial cell gap junction proteins.

Vitamin D is a fat-soluble prohormone that can be activated both systemically and within individual tissues. Our lab has previously demonstrated that the corneal epithelium can activate vitamin D and that the vitamin D metabolites 1,25(OH)D3 and 24R,25(OH)D3 can affect corneal epithelial migration, proliferation, and tight and gap junction function. These vitamin D-derived metabolites signal through the vitamin D receptor (VDR).

Effects of 1,25 and 24,25 Vitamin D on Corneal Epithelial Proliferation, Migration and Vitamin D Metabolizing and Catabolizing Enzymes.

This study investigated the effects of 1,25(OH)D3 and 24R,25(OH)D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)D3 increased CYP24A1 protein expression and decreased CYP27B1 expression.

A new sensitive LC/MS/MS analysis of vitamin D metabolites using a click derivatization reagent, 2-nitrosopyridine.

There is an increased demand for comprehensive analysis of vitamin D metabolites. This is a major challenge, especially for 1α,25-dihydroxyvitamin D [1α,25(OH)VitD], because it is biologically active at picomolar concentrations. 4-Phenyl-1,2,4-triazoline-3,5-dione (PTAD) was a revolutionary reagent in dramatically increasing sensitivity of all diene metabolites and allowing the routine analysis of the bioactive, but minor, vitamin D metabolites.

Vitamin D in Tear Fluid.

Purpose: To determine the source(s) of vitamin D in tear fluid and examine the expression of the endocytic proteins and putative vitamin D transporters megalin and cubilin in lacrimal and Harderian glands.

Methods: Wild-type, heterozygous, and vitamin D receptor (VDR) knockout C57BL/6 mice were used, with a subset of knockout mice fed a replenishment diet for some studies. Mouse lacrimal and Harderian glands from each group were used to measure megalin and cubilin by RT-PCR, Western blot, and immunohistochemistry.

Effect of vitamin D receptor knockout on cornea epithelium wound healing and tight junctions.

Purpose: Our laboratory previously determined that vitamin D3, the vitamin D receptor (VDR), and 1α hydroxylase are present and active in the eye. In this study, we examined the effects of VDR knockout on wound healing, the tight junction-associated proteins occludin and ZO-1, and tight junction numbers in mouse corneas.

Methods: Epithelial wounds (2-mm) were made with an agar brush on 4-week-old and 10-week-old wild-type, heterozygous, and VDR knockout mouse corneas.

Effects of vitamin D receptor knockout on cornea epithelium gap junctions.

Purpose: Gap junctions are present in all corneal cell types and have been shown to have a critical role in cell phenotype determination. Vitamin D has been shown to influence cell differentiation, and recent work demonstrates the presence of vitamin D in the ocular anterior segment. This study measured and compared gap junction diffusion coefficients among different cornea epithelium phenotypes and in keratocytes using a noninvasive technique, fluorescence recovery after photobleaching (FRAP), and examined the influence of vitamin D receptor (VDR) knockout on epithelial gap junction communication in intact corneas.

Bone turnover in long-term survivors of childhood acute lymphoblastic leukemia.

Background: We investigated the effects of demographic, lifestyle (self-reported smoking status and physical activity levels), cancer-related treatment factors (radiation and chemotherapy), and diet (calcium and vitamin D intake) on bone turnover and the relationship of bone turnover to lumbar spine bone mineral density (BMD) Z-scores (LS-BMD Z-scores) determined by quantitative computed tomography (QCT) in 418 ≥5-year survivors of childhood acute lymphoblastic leukemia (ALL).

Procedure: Bone turnover was assessed by biomarkers including serum bone-specific alkaline phosphatase (BALP), osteocalcin (OC), and urinary N-telopeptide of type I collagen indexed to creatinine (NTX/Cr). The 215 males ranged in age from 9 to 36 years (median age 17 years).

Enhancement of vitamin D metabolites in the eye following vitamin D3 supplementation and UV-B irradiation.

Purpose: This study was designed to measure vitamin D metabolites in the aqueous and vitreous humor and in tear fluid, and to determine if dietary vitamin D3 supplementation affects these levels. We also determined if the corneal epithelium can synthesize vitamin D following UV-B exposure.

Methods: Rabbits were fed a control or vitamin D3 supplemented diet.

Lysophospholipids and lysophospholipase D in rabbit aqueous humor following corneal injury.

We previously found that lysophosphatidic acid (LPA)-like activity eliciting Cl(-) currents in Xenopus oocytes is increased in rabbit aqueous humor (AH) following corneal freeze wounds. The purpose of this study was to examine whether actual levels of LPA in AH from wounded eyes are higher than those from control eyes, and to determine the sources and enzymatic pathways of AH LPA in control and wounded conditions. Lysophospholipase D (lysoPLD) activity was measured by the enzymatic determination of choline following incubation of AH samples with exogenous lysophosphatidylcholines (LPCs).

Vitamin D enhances corneal epithelial barrier function.

Purpose: The purpose of this study was to determine whether 25-hydroxyvitamin D(3) (25(OH)D(3)) and/or its active metabolite, 1α,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), can enhance corneal epithelial barrier function. The authors also determined if corneas contain mRNA for the vitamin D receptor (VDR) and 1α-hydroxylase, the enzyme required to convert 25(OH)D(3) to 1,25(OH)(2)D(3), and measured vitamin D metabolite concentrations in aqueous and vitreous humor.

Methods: RT-PCR was used to examine mouse, rabbit, and human corneal epithelial VDR and 1α-hydroxylase mRNA.

Lysophosphatidic acid-activated Cl- current activity in human systemic sclerosis skin fibroblasts.

Objectives: SSc (scleroderma) is an often fatal disease characterized by widespread tissue fibrosis. Fibroblasts play a key role in SSc-associated fibrosis. This study was designed to determine: (i) whether fibroblasts isolated from skin of patients with SSc have increased lysophosphatidic acid-activated Cl- current (IClLPA) activity vs healthy controls; (ii) whether myofibroblast differentiation is involved in SSc skin fibrosis; and (iii) whether SSc fibroblasts have different proliferation rates vs controls.

New insights into the mechanism of fibroblast to myofibroblast transformation and associated pathologies.

Myofibroblasts are a differentiated cell type essential for wound healing, participating in tissue remodeling following insult. Myofibroblasts are typically activated fibroblasts, although they can also be derived from other cell types, including epithelial cells, endothelial cells, and mononuclear cells. In most organ systems, cell signals initiated following tissue-specific insult or during the metastatic process lead to differentiation of fibroblasts or other precursor cells to the myofibroblast phenotype.

Teriparatide is safe and effectively increases bone biomarkers in institutionalized individuals with osteoporosis.

Institutionalized adults with severe developmental disabilities have a high rate of minimal trauma and appendicular fracture. There is little information about osteoporosis treatment in this population. In this efficacy and safety study, men and women with severe developmental disabilities and osteoporosis received 20 mcg teriparatide subcutaneously daily for 18-24 months.

Synthetic neoglycopolymer-recombinant human collagen hybrids as biomimetic crosslinking agents in corneal tissue engineering.

Saturated neoglycopolymers, prepared via tandem ROMP-hydrogenation (ROMP=ring-opening metathesis polymerization) of carbohydrate-functionalized norbornenes, are investigated as novel collagen crosslinking agents in corneal tissue engineering. The neoglycopolymers were incorporated into recombinant human collagen type III (RHC III) as collagen crosslinking agents and glycosaminoglycan (GAG) mimics. The purely synthetic nature of these composites is designed to reduce susceptibility to immunological and allergic reactions, and to circumvent the transmission of animal infectious diseases.