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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. However, some acute inflammation is necessary to allow efficient bacterial and fungal clearance and prevent corneal infection. This inflammation can be triggered by microbial components that activate the innate immune system through toll-like receptor (TLR) pathways. In particular, TLR2 and TLR4 activation leads to pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) activation. Similarly, endogenous molecules released from disrupted cells, known as damage-associated molecular patterns (DAMPs), can also activate TLR2, TLR4 and NFκB, with the resultant inflammation worsening the outcome of corneal wound healing. In sterile keratitis without infection, inflammation can occur though TLRs to impact corneal wound healing and reduce corneal transparency. This review demonstrates the need for acute inflammation to prevent pathogenic infiltration, while supporting the idea that a reduction in chronic and/or excessive inflammation will allow for improved wound healing.

Purpose: 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.

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. TPMD-induced calcium increases (Ca++i) were measured in Ca++-containing and Ca++-free solutions containing thapsigargin, ryanodine, BAPTA-AM, 18-α-glycyrrhetinic acid (18α-GA), apyrase, BCTC, AMG 9810, or AMTB. Fluorescence intensity was recorded as the number of cells responding and the area under the fluorescence versus time curve. The maximum distance of responding neighboring cells in ex vivo human corneas was measured. Connexin 43 protein in HCSC and MCSC was examined using immunofluorescence staining, and corneal rubbing was applied to confirm whether TPMDs occur following mechanical manipulation. Our results demonstrate that single cell TPMDs result in Ca++ waves in neighboring keratocytes both in culture and within ex vivo corneas. The source of Ca++ is both intra-and extra-cellular, and the signal can be mediated by ATP and/or gap junctions, and is species dependent. Stromal rubbing confirmed that TPMDs do occur following mechanical manipulation. Keratocyte TPMDs and their associated signaling events are likely common occurrences following minor or major corneal trauma.

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. Here, we show in vitro that DOPG inhibits TLR2 activation induced in response to HSPB4, as well as DAMPs that are elevated in diabetes, a disease that also slows corneal wound healing. Further, we show that the co-receptor, cluster of differentiation-14 (CD14), is necessary for PAMP/DAMP-induced activation of TLR2, as well as of TLR4. Finally, we simulated the high-glucose environment of diabetes to show that elevated glucose levels enhance TLR4 activation by a DAMP known to be upregulated in diabetes. Together, our results demonstrate the anti-inflammatory actions of DOPG and support further investigation into its development as a possible therapy for corneal injury, especially in diabetic patients at high risk of vision-threatening complications.

The relationship between vitamin D and glaucoma is controversial. The objective of this study was to examine women from the Women's Health Initiative (WHI) to determine if there is an association between vitamin D and incident glaucoma in postmenopausal women. We examined the association between dietary vitamin D intake, vitamin D supplements and serum 25 hydroxyvitamin D (25(OH)D) levels and the risk of developing glaucoma. 143,389 postmenopausal women from the WHI including a subset with serum 25(OH) D measurements were examined to determine the association of dietary, supplemental and serum levels of vitamin D to the development of glaucoma. Dietary intakes of vitamin D, use of vitamin D supplements and serum levels of 25(OH) D were predictors examined for the main outcome of incident glaucoma. In multivariable models adjusted for demographic, clinical variables and medication use, dietary vitamin D, vitamin D supplements, total vitamin D intake (diet plus supplements) and serum 25 (OH) D measurements were not significantly associated with incident glaucoma. In the CaD placebo-controlled intervention clinical trial, there was also no association in the active intervention arm with glaucoma. We conclude that dietary vitamin D intake, supplements and serum levels are not significantly related to the risk of developing glaucoma in postmenopausal women.

Purpose In contact with the external environment, the cornea can easily be injured. Although corneal wounds generally heal rapidly, the pain and increased risk of infection associated with a damaged cornea, as well as the impaired healing observed in some individuals, emphasize the need for novel treatments to accelerate corneal healing. We previously demonstrated in epidermal keratinocytes that the glycerol channel aquaporin-3 (AQP3) interacts with phospholipase D2 (PLD2) to produce the signaling phospholipid phosphatidylglycerol (PG), which has been shown to accelerate skin wound healing in vivo. We hypothesized that the same signaling pathway might be operational in corneal epithelial cells. Methods We used co-immunoprecipitation, immunohistochemistry, scratch wound healing assays in vitro, and corneal epithelial wound healing assays in vivo to determine the role of the AQP3/PLD2/PG signaling pathway in corneal epithelium. Results AQP3 was present in human corneas in situ, and AQP3 and PLD2 were co-immunoprecipitated from corneal epithelial cell lysates. The two proteins could also be co-immunoprecipitated from insect cells simultaneously infected with AQP3- and PLD2-expressing baculoviruses, suggesting a likely direct interaction. A particular PG, dioleoylphosphatidylglycerol (DOPG), enhanced scratch wound healing of a corneal epithelial monolayer in vitro. DOPG also accelerated corneal epithelial wound healing in vivo, both in wild-type mice and in a mouse model exhibiting impaired corneal wound healing (AQP3 knockout mice). Conclusions These results indicate the importance of the AQP3/PLD2/PG signaling pathway in corneal epithelial cells and suggest the possibility of developing DOPG as a pharmacologic therapy to enhance corneal wound healing in patients.

Keratoconus (KC) is the most common corneal ectatic disorder affecting >300,000 people in the US. KC normally has its onset in adolescence, progressively worsening through the third to fourth decades of life. KC patients report significant impaired vision-related quality of life. Genetic factors play an important role in KC pathogenesis. To identify novel genes in familial KC patients, we performed whole exome and genome sequencing in a four-generation family. We identified potential variants in the PPIP5K2 and PCSK1 genes. Using in vitro cellular model and in vivo gene-trap mouse model, we found critical evidence to support the role of PPIP5K2 in normal corneal function and KC pathogenesis. The gene-trap mouse showed irregular corneal surfaces and pathological corneal thinning resembling KC. For the first time, we have integrated corneal tomography and pachymetry mapping into characterization of mouse corneal phenotypes which could be widely implemented in basic and translational research for KC diagnosis and therapy in the future.

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. Western blotting and immunochemistry were used to detect proteins in cultured cells exposed to 1,25(OH)2D3 and 24R,25(OH)2D3. Results VDR KO resulted in decreased corneal desmosomal desmoglein 1 (DSG1) and desmocollin 2 (DSC2) mRNA, and hemidesmosomal plectin mRNA. DSG1 and plectin protein expression were reduced in VDR KO corneas. DSG1 protein expression increased in VDR wild types (VDR WT) and VDR KO mouse primary epithelial cells (MPCEC) treated with 1,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 treatment resulted in increased plectin and integrin β4 levels in VDR WT MPCEC, and decreased levels in VDR KO MPCEC. Treatment of human corneal epithelial cells (HCEC) with 1,25(OH)2D3 and 24R,25(OH)2D3 resulted in increased DSC2 and DSG1 protein expression. Plectin and integrin β4 were only increased in 24R,25(OH)2D3 treated HCEC. Conclusions VDR KO results in reduced desmosomal and hemidesmosomal mRNA and protein levels. 1,25(OH)2D3 and 24R,25(OH)2D3 increased DSG1 protein in all cells tested. For hemidesmosome proteins, 24R,25(OH)2D3 increased plectin and integrin β4 protein expression in VDR WT and HCEC, with decreased expression in VDR KO MPCEC. Thus, vitamin D3 is involved in desmosome and hemidesmosome junction formation/regulation, and their decreased expression likely contributes to the loosely adherent corneal epithelium in VDR KO mice. Our data indicate the presence of a VDR-independent pathway.

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)(2)D3 and 24R,25(OH)(2)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). The purpose of this study was to specifically determine the effects of 1,25(OH)(2)D3 and 24R,25(OH)(2)D3 on corneal epithelial cell gap junction proteins. Connexin (Cx) 26, 30 and 43 protein expression was detected in a human corneal epithelial cell line (HCEC), wild type and vitamin D receptor knockout (VDR (−/−)) mouse corneas, and cultured mouse primary epithelial cells (MPCEC). In vitro gap junction function was assessed using the scrape loading/dye transfer assay. HCEC and MPCEC were treated with 1,25(OH)(2)D3 or 24R,25(OH)(2)D3. Western blotting was used to detect gap junction proteins. Vitamin D3 effects on epithelial intracellular Ca(++) (Ca(i)(++)) were determined using the dye Cal-520. Cx26 and Cx43 protein levels were significantly increased in HCEC and MPCEC treated with both 1,25(OH)(2)D3 and 24R,25(OH)(2)D3. Cx30 and Cx43 protein levels were also significantly increased in VDR (−/−) MPCEC. In vitro gap junction connectivity was significanlty enhanced in HCEC and MPCEC cultured with 24R,25(OH)(2)D3 and 1,25(OH)(2)D3. Ca(i)(++) was not affected by 1,25(OH)(2)D3 or 24R,25(OH)(2)D3 in HCEC or MPCEC. We conclude that both 1,25(OH)(2)D3 and 24R,25(OH)(2)D3 are positive regulators of connexin proteins and gap junction communication in the corneal epithelium. These vitamin D metabolites appear to signal through both VDR-dependent and -independent pathways. The effects of vitamin D on corneal epithelial gap junctions do not seem to be dependent on Ca(i)(++).

This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.

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)2VitD], 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. A second generation of reagents used large fixed charge groups that increased sensitivity at the cost of a deterioration in chromatographic separation of the vitamin D derivatives. This precludes a survey of numerous vitamin D metabolites without redesigning the chromatographic system used. 2-Nitrosopyridine (PyrNO) demonstrates that one can improve ionization and gain higher sensitivity over PTAD. The resulting vitamin D derivatives facilitate high-resolution chromatographic separation of the major metabolites. Additionally, a liquid-liquid extraction followed by solid-phase extraction (LLE-SPE) was developed to selectively extract 1α,25(OH)2VitD, while reducing 2- to 4-fold ion suppression compared with SPE alone. LLE-SPE followed by PyrNO derivatization and LC/MS/MS analysis is a promising new method for quantifying vitamin D metabolites in a smaller sample volume (100 µL of serum) than previously reported methods. The PyrNO derivatization method is based on the Diels-Alder reaction and thus is generally applicable to a variety diene analytes.

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).

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.Rabbits were fed a control or vitamin D3 supplemented diet. Pilocarpine-stimulated tear fluid was collected and aqueous and vitreous humor were drawn from enucleated eyes. Plasma vitamin D was also measured. To test for epithelial vitamin D synthesis, a human corneal limbal epithelial cell line was irradiated with two doses of UV-B (10 and 20 mJ/cm(2)/day for 3 days) and vitamin D was measured in control or 7-dehydrocholesterol treated culture medium. Measurements were made using mass spectroscopy.25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3 increased significantly following D3 supplementation in all samples except vitreous humor. Tear fluid and aqueous humor had small but detectable 1,25(OH)(2)-vitamin D3 levels. Vitamin D2 metabolites were observed in all samples. Vitamin D3 levels were below the detection limit for all samples. Minimal vitamin D3 metabolites were observed in control and UV-B-irradiated epithelial culture medium except following 7-dehydrocholesterol treatment, which resulted in a UV-B-dose dependent increase in vitamin D3, 25(OH)-vitamin D3 and 24,25(OH)(2)-vitamin D3.There are measurable concentrations of vitamin D metabolites in tear fluid and aqueous and vitreous humor, and oral vitamin D supplementation affects vitamin D metabolite concentrations in the anterior segment of the eye. In addition, the UV exposure results lead us to conclude that corneal epithelial cells are likely capable of synthesizing vitamin D3 metabolites in the presence of 7-dehydrocholesterol following UV-B exposure.

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). The molecular species compositions of LPA and LPC in fresh and incubated AH were determined by liquid chromatography–tandem mass spectrometry. A high, but similar activity of lysoPLD in the samples from both control and freeze-wounded eyes was detected. Its enzymatic properties resemble those of plasma lysoPLD, identified as autotaxin. Levels of LPCs, predominant substrates of lysoPLD in AH, were several times higher in the AH samples from injured eyes than those from the control eyes. Our results suggest that lysoPLD is constitutively released from corneal tissues and/or ciliary body into the AH, with no injury-induced increase in release following freeze-wounding. They also suggest that wound-induced increases in LPA-like biological activity are due to linoleoyl species-rich molecular composition in AH from wounded eyes. A possible mechanism of the altered molecular composition is an increase in the AH concentrations of LPCs, linoleoyl species of which are preferentially converted to corresponding unsaturated LPA by the constitutively active lysoPLD.

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 (ICI LPA ) 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. Methods. Skin biopsies were taken from involved and uninvolved skin of SSc patients and controls. Whole-cell perforated patch-clamping was used to measure ICI LPA activity in fibroblasts isolated and cultured from these biopsies. Western blotting was used to measure α-smooth muscle actin (α-SMA). Proliferation was measured using a colorimetric assay. Results. Fibroblasts cultured from SSc skin show significantly increased ICI LPA activity following LPA exposure compared with control skin fibroblasts. α-SMA protein was significantly increased in cultured SSc skin fibroblasts vs controls. No significant differences in proliferation rates were found. Conclusions. Elevated ICI LPA activity is a hallmark of SSc skin fibroblasts. Blocking ICI LPA activation may be a new therapeutic approach for treating SSc-associated fibrosis.

Vitamin D deficiency may have implications for cardiovascular health. The purpose of this study was to determine the relationship of 25-hydroxyvitamin D (25[OH]D) to cholesterol and lipoprotein particles and to determine whether increasing 25(OH)D through ultraviolet (UV) irradiation impacted on these parameters in healthy young men and women. This was a randomized trial of 51 adults exposed to suberythemal doses of whole-body irradiation using UV lamps that emitted UV-A and UV-B radiation, compared with a control group, twice weekly for 12 weeks. 25-Hydroxyvitamin D, cholesterol, and lipoprotein subfractions were measured at baseline and after 12 weeks. There was a significant (P < .03) positive association between 25(OH)D and apolipoprotein A-I (Apo A-I) and lipoprotein A-I (Lp A-I). The ratio of low-density lipoprotein to high-density lipoprotein was significantly (P < or = .044) negatively correlated with 25(OH)D levels. The levels of 25(OH)D increased significantly in the treated compared with control group (P < .05). Overall, there were no significant differences between the treated and control groups in any lipoproteins or apolipoproteins after administration of UV irradiation. Subgroup analysis for Apo A-II confined to those with 25(OH)D insufficiency (25[OH]D

We successfully fabricated transparent, robust hydrogels as corneal substitutes from concentrated recombinant human type I and type III collagen solutions crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). White light transmission through these gels is comparable or superior to that of human corneas. Hydrogels from both type I and type III collagens supported in vitro epithelium and nerve over-growth. While both these biocompatible hydrogels have adequate tensile strength and elasticity for surgical manipulation, type III collagen hydrogels tended to be mechanically superior. Twelve-month post-implantation results of type I recombinant collagen-based corneal substitutes into mini-pigs showed retention of optical clarity, along with regeneration of corneal cells, nerves and tear film. For clinical use, implants based on fully characterized, recombinant human collagen eliminate the risk of pathogen transfer or xenogeneic immuno-responses posed by animal collagens.

Numerous studies have been published describing the nonclassical actions of vitamin D. Our lab discovered that the cornea is capable of activating and metabolizing vitamin D (Fig. 1).1 This ability of the cornea to metabolize vitamin D was recently confirmed.2 We have also shown that vitamin D metabolites are present in tear fluid, probably serving a role in corneal maintenance, although the source of tear fluid vitamin D is unknown.1 Figure 1 Vitamin D metabolism pathway. Inactive vitamin D is either synthesized from the skin or obtained through the diet. The inactive vitamin D is converted in the anterior segment of the eye to 25(OH)D by 25-hydroxylase, and then either to 1,25(OH)2D or 24,25(OH)2D ... The classic pathway of vitamin D synthesis begins with UV light exposure of 7-dehydrocholesterol in the skin to produce previtamin D. This previtamin D3 is hydroxylated to 25 hydroxyvitamin D3 (25(OH)D3) in the liver and finally converted 1α, 25 dihydroxyvitamin D3 (1α,25(OH)D3) in the kidney. In order for this last step to take place, 25(OH)D3 needs to be reabsorbed at the proximal tubule of the kidney by the endocytic dual receptor complex megalin and cubilin.3 Megalin is a member of the low lipoprotein receptor superfamily4 and it is expressed in the plasma membrane and endocytic apparatus of many epithelial tissues including RPE and nonpigmented ciliary body epithelium,5 small intestine,6,7 renal proximal tubule,8,9 visceral yolk sac,10,11 thyroid gland,12 lungs,12 and parathyroid gland,13 among others. This receptor is known as a scavenger receptor because of its multiligand properties, which allows for nonspecific cellular uptake of proteins. Vitamin D binding protein (DBP) is one of the ligands for megalin.14 Cubilin is also an endocytic receptor that has been shown to be coexpressed with megalin in several absorptive epithelia and also has the potential for binding DBP.3 The purpose of this study was to determine the source(s) of vitamin D in tear fluid. This is particularly relevant given a recent study demonstrating that high serum vitamin D levels have a small but favorable effect on dry eye syndrome.15 In addition, because megalin and cubilin are involved in vitamin D transport and because megalin expression can be stimulated by vitamin D, we examined the expression of these proteins in the lacrimal and Harderian gland of wild-type and Vitamin D Receptor (VDR) knockout mice.16 We hypothesized that the lacrimal gland excretes vitamin D metabolites into the lacrimal fluid and that megalin and cubilin are involved in the lacrimal gland vitamin D secretory pathway. We also hypothesized that feeding the VDR knockout mice a special diet rich in calcium, phosphate, and lactose that has been shown to reverse some of the phenotypical features in these mice through normalization of their calcium and mineral metabolism,17–19 would also reverse phenotypical changes observed in the mice fed a normal diet.

Summary A single infusion of pamidronate was given to patients with systemic sclerosis (scleroderma, SSc) to assess effects on cytokine production by peripheral blood mononuclear cells (PBMC) and lymphocyte subsets. Eighteen patients with SSc received a single intravenous dose of 60 mg of pamidronate and were followed for 6 months. Assessment of cytokine production [interferon (IFN)-γ, interleukin (IL)-10, transforming growth factor (TGF)-β1, tumour necrosis factor (TNF)-α and IL-4] by PBMC and lymphocyte subsets by flow cytometry was carried out before and after the pamidronate infusion. Unstimulated PBMC produced increased amounts of IFN-γ and TNF-α and reduced levels of TGF-β1 for up to 24 weeks after the infusion. γδ T cells from patients with SSc were activated in vitro and produced increased IFN-γ. The effects of pamidronate on modulation of cytokine profiles in patients with SSc may merit future study.

Porcine and recombinant human atelocollagen I solutions were cross-linked with a water soluble carbodiimide at various stoichiometries and collagen concentrations (5-20 w/w %). The resulting hydrogels were clear and, when used as cell growth matrices, allowed cell and nerve visualization in vitro and in vivo. We have previously reported that, after six months of implantation in pigs' and rabbits' corneas, these robust hydrogels allowed regeneration of host cells and nerves to give optically clear corneas with no detected loss in thickness, indicating stable engraftment. Here, the biocompatible hydrogel formulations leading to this novel in vivo performance were characterized for amine consumption, gel hydration, thermal properties, optical clarity, refractive index, nutrient diffusion, biodegradation, tensile measurements, and average pore diameters. Gels with excellent in vitro (epithelial overgrowth, neurite penetration) and in vivo performance (clarity, touch sensitivity regeneration) had 4-11 nm pores, yet had glucose and albumin diffusive coefficients similar to mammalian corneas and allowed neurite extension through the gels.

In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volume-activated Cl− current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1–5, with an apparent increase in EDG-3, -4, and -5 following brief SDS application to cell lines, and EDG receptors 2–5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.

The phospholipid growth factors (PLGFs), including lysophosphatidic acid (LPA), have been implicated in corneal wound healing. PLGF concentrations and activities are elevated after corneal injury. Using real-time PCR, we quantified receptor mRNA levels in the healing rabbit cornea. In intact corneas, transcripts for S1P1, LPA1, and LPA3receptor subtypes were detected, as was lipid phosphate phosphatase 1 (LPP1). After wounding, the trend for endothelium and keratocytes was for significant decreases in transcript numbers for the three receptor subtypes, whereas epithelial cells showed increased transcript numbers, except for an S1P1decrease in healing cells. LPP1 transcript numbers were decreased in keratocytes and endothelium, although LPP-specific activity was unchanged. LPA-elicited Ca2+transients were significantly reduced in the healing endothelium. Consistent with reduced LPA3receptor numbers, dioctylglycerol pyrophosphate, a selective antagonist, reduced LPA-induced Ca2+transients 2.7-fold in nonwounded epithelium but only 1.5-fold in wound-healing endothelium. These data for the first time establish physiologically relevant differential changes in the expression of PLGF receptor subtypes and provide evidence for the changing role of LPA3receptors in endothelial cells.

Gap junctional communication in the epithelium and endothelium of human corneas was studied. The influence of corneal storage on endothelial gap junctions was also examined.Donor human corneal cells were injected with carboxyfluorescein while surrounding cells were monitored for traces of fluorescence. Dye-spread coefficients were measured in corneal endothelial cells. Western blot and immunohistochemical analysis of the endothelium and epithelium was employed to determine if connexin 43 was present.Dye coupling occurs in both the epithelium and endothelium of the human cornea. Epithelial dye coupling was extensive in the basal layers but less apparent in the superficial layers. Endothelial dye coupling was similar to that reported for rabbit corneas. Western blot and immunohistochemical analyses demonstrated the presence of connexin 43 in both cell types.Gap junctional communication occurs in the endothelium and epithelium of human corneas, and both cell types express connexin 43. These results are similar to previous rabbit studies, thereby strengthening the use of the rabbit cornea as a gap junction model.

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. Previous gap junction studies in cornea epithelium and keratocytes were performed using cultured cells or ex vivo invasive techniques. These invasive techniques were unable to measure diffusion coefficients and likely were disruptive to normal cell physiology. METHODS Corneas from VDR knockout and control mice were stained with 5(6)-carboxyfluorescein diacetate (CFDA). Gap junction diffusion coefficients of the corneal epithelium phenotypes and of keratocytes, residing in intact corneas, were detected using FRAP. RESULTS Diffusion coefficients equaled 18.7, 9.8, 5.6, and 4.2 μm(2)/s for superficial squamous cells, middle wing cells, basal cells, and keratocytes, respectively. Corneal thickness, superficial cell size, and the superficial squamous cell diffusion coefficient of 10-week-old VDR knockout mice were significantly lower than those of control mice (P < 0.01). The superficial cell diffusion coefficient of heterozygous mice was significantly lower than control mice (P < 0.05). CONCLUSIONS Our results demonstrate differences in gap junction dye spread among the epithelial cell phenotypes, mirroring the epithelial developmental axis. The VDR knockout influences previously unreported cell-to-cell communication in superficial epithelium.

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).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).Age and tanner score were inversely associated with all biomarkers (BALP, OC, NTX/Cr) (P 0.001). Males had higher BALP and OC than females (P 0.001). Body mass index (BMI) was inversely associated with OC and NTX/Cr (P 0.001). There was no significant association of biomarkers with lifestyle related factors, ALL treatment-related factors, dietary calcium, vitamin D, or LS-BMD Z-score.In this population of long-term survivors of ALL, bone turnover was significantly associated with age, gender, tanner stage, and BMI. ALL-related treatments did not influence bone turnover and bone turnover was not predictive of volumetric LS-BMD Z-score.

THE A BILIT Y TO CREA TE N EW H UM A N TISSU ES in the laboratory or on the workbench is no longer the stuff of science fiction. Bioengineered skin is routinely used in skin grafting procedures, and ventricular assist devices are now being used to prolong the life of cardiac patients on the waiting list for donor hearts. Worldwide, the waiting lists for donor organs and tissues are extraordinarily long, regardless of the tissue type. The ability to construct and/or grow these tissues in the laboratory is the goal of tissue engineering. Our work and the work of our team (including among others Dr. Griffith’s colleagues at the University of Ottawa, Dr. Rosemarie Osborne at Procter & Gamble, and Dr. Charles Doillon at the Universitaire de Quebec) has focused on the development of an artificial cornea. To date, we have engineered a corneal equivalent designed to be useful as an alternative to animal (Draize) testing, as well as for drug efficacy testing and basic wound healing research (1). Our current work is focused on expanding the technology used in this corneal equivalent to construct a transplantable cornea. We had several goals in mind for the corneal equivalent when we began this project in 1995. Overall, we set out to design a corneal equivalent that had the morphological and physiologica l characteristics of a human cornea. Thus, the construct had to contain the basic cellular layers of a cornea, including a stratified epithelium, Bowman’s layer, keratocytes residing in the stroma, and an endothelial monolayer with its limiting membrane (Descemet’s membrane). We were looking to use cells derived from human corneas, which we immortalized (i.e., extend the lifespans) to obtain a continuous and homogeneous supply of cells for research purposes and also for in vitro assays of toxicity or drug safety and efficacy. Functionally , we wanted the cornea to have the capacity to swell and to be able to maintain its hydration state through a ouabain inhibitable endothelial pump mechanism. We also wanted the construct to be transparent, and to lose its transparency following toxic insults as is seen in in vivo corneas. In addition, after toxic insult, we wanted the cells of the corneas to exhibit changes in the expression of genes known to be affected during corneal wound healing. Finally, we wanted to add a vascularized sclera to the constructs. To engineer a cornea with the features stated above, two research pathways had to be followed, a cellular path and a biomaterials path. Of course, in the end, these pathways had to merge in that the cells had to live within the biopolymer matrix. The remainder of this review will focus on the cellular pathway. Figure 1 shows the morphology of the corneal equivalents as compared to cultured human eye bank corneas (A–C, E), along with the vascularized sclera (D). Figure 1F shows epithelial cells invading the stromal matrix, the result of utilizing poor cell selection criteria. Improving this selection criteria was one of the many novel approaches we took in constructing these corneal equivalents, specifically in that we used ion current categorization via patch clamping as one of our initial selection criteria. The Watsky lab has been examining corneal cell transport, ion channels and currents for more than 10 years (for example see review [2]), with the most recent work focusing on changes in ion channels during

Human corneal equivalents comprising the three main layers of the cornea (epithelium, stroma, and endothelium) were constructed. Each cellular layer was fabricated from immortalized human corneal cells that were screened for use on the basis of morphological, biochemical, and electrophysiological similarity to their natural counterparts. The resulting corneal equivalents mimicked human corneas in key physical and physiological functions, including morphology, biochemical marker expression, transparency, ion and fluid transport, and gene expression. Morphological and functional equivalents to human corneas that can be produced in vitro have immediate applications in toxicity and drug efficacy testing, and form the basis for future development of implantable tissues.

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.

Tonic immobility (TI) is an unlearned behavioral response characterized by a state of immobility and torpor. Effect of inter-trial interval on duration of tonic immobility was assessed in thirty, juvenile lemon sharks (Negaprion brevirostris). Regression analyses showed that massed trials of 12 per session increased the average duration of tonic immobility by 475 sec compared to spaced trials of 1 per session. Each experiment was composed of 24 trials. TI is stable and durations much longer in the lemon shark than for other sharks. These findings have enabled us to develop a quantitative bioassay for use in testing chemical shark repellents.

Single-channel patch-clamp techniques as well as standard and perforated-patch whole cell voltage-clamp techniques have been applied to the study of ionic channels in the corneal endothelium of several species. These studies have revealed two major K+ currents. One is due to an anion- and temperature-stimulated channel that is blocked by Cs+ but not by most other K+ channel blockers, and the other is similar to the family of A-currents found in excitable cells. The A-current is transient after a depolarizing voltage step and is blocked by both 4-aminopyridine and quinidine. These two currents are probably responsible for setting the -50 to -60 mV resting voltage reported for these cells. A Ca(2+)-activated ATP-inhibited nonselective cation channel and a tetrodotoxin-blocked Na+ channel are possible Na+ inflow pathways, but, given their gating properties, it is not certain that either channel works under physiological conditions. A large-conductance anion channel has also been identified by single-channel patch-clamp techniques. Single corneal endothelial cells have input resistances of 5-10 G omega and have steady-state K+ currents that are approximately 10 pA at the resting voltage. Pairs or monolayers of cells are electrically coupled and dye coupled through gap junctions.

The influence of serum, lysophosphatidic acid (LPA), and hyposmotic stress on the ion channel activity of normal and cryo-injured rabbit corneal keratocytes was investigated. Whole cell currents were examined using the amphotericin perforated-patch technique. In cells from wounded corneas, fetal bovine serum activated large, holding voltage-insensitive, fast-activating, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-, flufenamic acid-, and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB)-blockable outward currents showing inactivation at depolarized voltages. LPA activated identical currents, also only in cells from wounded corneas. Blocker and reversal potential experiments characterized the current as a Cl- currents (Icl). Lysophosphatidylcholine (10 microM) failed to activate the current. An identical current was activated by hyposmotic stimulation in cells from control and wounded corneas. Hyposmotic stimulation also activated Icl in cells from wounded corneas that were unresponsive to LPA. We conclude that serum, LPA, and hypotonic stress activate Icl in keratocytes from wounded corneas. We also conclude that LPA is a serum factor that can activate Icl and that hyposmotic activation may work through a signaling pathway separate from that of LPA.

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. In addition to their beneficial and necessary role in wound healing, myofibroblasts also contribute to a number of pathologies, primarily fibrotic processes and tumor invasiveness. This review explores both traditional and nontraditional concepts of myofibroblast differentiation in the cornea, skin, heart, and other tissues, as well as some of the pathologies associated with myofibroblast activities.

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. In addition to their beneficial and necessary role in wound healing, myofibroblasts also contribute to a number of pathologies, primarily fibrotic processes and tumor invasiveness. This review explores both traditional and nontraditional concepts of myofibroblast differentiation in the cornea, skin, heart, and other tissues, as well as some of the pathologies associated with myofibroblast activities.

Using the perforated patch version of whole-cell recording, we have measurd currents from isolated frog lens epithelial cells. Three types of currents were seen. A time-independent outwardly rectifying potassium current was identified that sets the resting voltage. This potassium current differs significantly from any of the potassium currents recorded with the whole-cell technique in mammalian lens epithelial cells. In addition to the potassium current, the two other currents present were both outwardly rectifying: one was time-independent while the other showed distinct activation.

The effects of ouabain and extracellular calcium removal on gap junctional coupling of isolated rabbit corneal endothelium was examined using a modified dye-spread technique. This technique is a modification of a microelectrode procedure that now utilizes patch electrodes connected to a current-clamp circuit for dye iontophoresis and a shuttering system in the excitation light path to reduce phototoxic effects in the monolayer. It was found that a significant degree of junctional uncoupling occurred after 45 min of exposure to ouabain, quantified as a reduction in the effective diffusion coefficient of injected Lucifer yellow CH: 1.74 x 10(-7) cm2/s (control) versus 0.43 x 10(-7) cm2/s (ouabain-treated). It was also determined that no gap junctional uncoupling occurs after extended exposure (up to 3.5 h) to a calcium-free extracellular environment.

The perforated patch technique was utilized to obtain whole-cell currents from freshly dissociated rabbit corneal keratocytes. We describe and provide the initial characterization of two distinct whole cell currents in rabbit keratocytes: a K(+)-selective delayed rectifier and a voltage-sensitive, tetrodotoxin blockable Na+ current. The voltage-sensitive Na+ current is of sufficient magnitude to allow us to initiate action potentials when current-clamping the cells. This is the first detailed electrophysiological study of corneal keratocytes.

Systemic sclerosis (SSc) is an often fatal disease characterized by autoimmunity and inflammation, leading to widespread vasculopathy and fibrosis. Lysophosphatidic acid (LPA), a bioactive phospholipid in serum, is generated from lysophospholipids secreted from activated platelets in part by the action of lysophospholipase D (lysoPLD). Sphingosine 1-phosphate (S1P), a member of the bioactive lysophospholipid family, is also released from activated platelets. Because activated platelets are a hallmark of SSc, we wanted to determine whether subjects with SSc have altered serum lysophospholipid levels or lysoPLD activity. Lysophospholipid levels were measured using mass spectrometric analysis. LysoPLD activity was determined by quantifying choline released from exogenous lysophosphatidylcholine (LPC). The major results were that serum levels of arachidonoyl (20:4)-LPA and S1P were significantly higher in SSc subjects versus controls. Furthermore, serum LPA:LPC ratios of two different polyunsaturated phospholipid molecular species, and also the ratio of all species combined, were significantly higher in SSc subjects versus controls. No significant differences were found between other lysophospholipid levels or lysoPLD activities. Elevated 20:4 LPA, S1P levels and polyunsaturated LPA:LPC ratios may be markers for and/or play a significant role in the etiology of SSc and may be future pharmacological targets for SSc treatment.

The field of tissue engineering is multidisciplinary, seeking to fabricate artificial organs or substitutes in order to replace failing or damaged organs. These engineered tissues and organs are mostly targeted as substitutes for human donor tissues and as such are developed to avoid the complications associated with donor matching and immune rejection [1]. Examples include substitutes of organs including liver, heart, kidney, skin, teeth, and cornea [2].

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. Markers of bone formation [procollagen type 1 intact N-terminal propeptide (P1NP)] and resorption [C-telopeptide (CTx)] were measured at three-month intervals. Serum calcium was measured at two-week intervals for 12 weeks and thereafter at three-month intervals. Twenty-seven individuals received at least one injection. The incidence of hypercalcemia was 11.1% but was persistent and led to medication discontinuation in only one participant. Biomarkers of bone formation increased rapidly, doubling by three months. At 12 months, P1NP and CTx remained elevated from baseline; P1NP had risen from 66.95 +/- 83.71 microg/l (mean +/- SD) to 142.42 +/- 113.85 microg/l (P = 0.05), and CTx had increased from 0.377 +/- 0.253 to 1.016 +/- 1.048 ng/ml (P = 0.01). The majority of participants had an increase in P1NP of over 10 microg/l. In conclusion, teriparatide is safe and effective in developmentally disabled institutionalized adults. Serial calcium measurements are warranted, particularly during the first three months of therapy.

A biointeractive collagen-phospholipid corneal Substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed ail overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and, regeneration (despite MPCs known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea.In addition, the glucose and albumin permeability were comparable to those Of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be Substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal Source materials.

PURPOSE. To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS. Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS. Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS. Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

To determine the effects of chloride channel 3 (ClC-3) knockdown and overexpression on lysophosphatidic acid (LPA)- and volume-regulated anion channel Cl−currents ( ICl,LPAand ICl,VRAC, respectively), cell differentiation, and cell volume regulation, a short hairpin RNA (shRNA) expression system based on a mouse U6 promoter was used to knock down ClC-3 in human corneal keratocytes and human fetal lung fibroblasts. ClC-3 overexpression was achieved by electroporating full-length ClC-3, within a pcDNA3.1 vector, into these two cell lines. RT-PCR and Western blot analysis were used to detect ClC-3 mRNA and protein levels. Whole cell perforated patch-clamp recording was used to measure ICl,LPAand ICl,VRACcurrents, and fluorescence-activated cell sorting analysis was used to measure cell volume regulation. ClC-3 knockdown significantly decreased ICl,LPAand ICl,VRACactivity in the presence of transforming growth factor-β1(TGF-β1) compared with controls, whereas ClC-3 overexpression resulted in increased ICl,LPAactivity in the absence of TGF-β1. ClC-3 knockdown also resulted in a reduction of α-smooth muscle actin (α-SMA) protein levels in the presence of TGF-β1, whereas ClC-3 overexpression increased α-SMA protein expression in the absence of TGF-β1. In addition, keratocytes transfected with ClC-3 shRNA had a significantly blunted regulatory volume decrease response following hyposmotic stimulation compared with controls. These data confirm that ClC-3 is important in VRAC function and cell volume regulation, is associated with the ICl,LPAcurrent activity, and participates in the fibroblast-to-myofibroblast transition.

To determine bone mineral content (BMC), bone mineral density (BMD), Z scores, and markers of bone turnover in African American children with juvenile rheumatoid arthritis (JRA).Eight children with JRA with no prior exposure to corticosteroids were evaluated. Lumbar spine (L1-L4) and total body and total hip BMC and BMD were determined using dual x-ray absorptiometry (DXA), and Z scores (BMD) were calculated. Serum samples of markers of bone turnover including pyridinoline (PYR), N-terminal propeptide of type I procollagen (P1NP), osteocalcin (OC), and bone-specific alkaline phosphatase (BSAP) were measured.The mean Z score (BMD) at the lumbar spine (L1-L4) in patients with JRA was -1.2+/-0.8. Z scores for total body and total hip were within 1 standard deviation of normal compared with healthy historical controls matched for age, sex, and race.BMD was normal for chronological age (defined as Z scoreor= 2.0) in African American children with JRA who had not previously been treated with corticosteroids. Further studies are needed on the effects of JRA on skeletal health in African American children.

PURPOSE. To develop a simple corneal substitute from crosslinked collagen. METHODS. Porcine type I collagen (10%; pH 5), was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and Nhydroxysuccinimide (NHS). The final homogenous solution was molded to corneal dimensions, cured, and then implanted into rabbits and minipigs by lamellar keratoplasty. The implants were followed for up to 6 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, topography and esthesiometry for nerve function. Histopathologic examinations were also performed on rabbit corneas harvested after 6 months. RESULTS. Cross-linked collagen (refractive index, 1.35) had optical clarity superior to human corneas. Implanted into rabbit and porcine corneas, only 1 of 24 of the surgical corneas showed a slight haze at 6 months after surgery. All other implants showed no adverse reactions and remained optically clear. Topography showed a smooth surface and a profile similar to that of the contralateral nonsurgical eye. The implanted matrices promoted regeneration of corneal cells, tear film, and nerves. Touch sensitivity was restored, indicating some restoration of function. The corneas with implants showed no significant loss of thickness and demonstrated stable host‐graft integration. CONCLUSIONS. Collagen can be adequately stabilized, using water soluble carbodiimides as protein cross-linking reagents, in the fabrication of corneal matrix substitutes for implantation. The simple cross-linking methodology would allow for easy fabrication of matrices for transplantation in centers where there is a shortage of corneas, or where there is need for temporary patches to repair perforations in emergency situations. (Invest Ophthalmol Vis Sci. 2006;47:1869‐1875)