Anti-IL17A Halts the Onset of Diabetic Retinopathy in Type I and II Diabetic Mice.

There are ~463 million diabetics worldwide, and more than half have diabetic retinopathy. Yet, treatments are still lacking for non-proliferative diabetic retinopathy. We and others previously provided evidence that Interleukin-17A (IL-17A) plays a pivotal role in non-proliferative diabetic retinopathy. However, all murine studies used Type I diabetes models. Hence, it was the aim of this study to determine if IL-17A induces non-proliferative diabetic retinopathy in Type II diabetic mice, as identified for Type I diabetes. While examining the efficacy of anti-IL-17A as a potential therapeutic in a short-term Type I and a long-term Type II diabetes model; using different routes of administration of anti-IL-17A treatments. Retinal inflammation was significantly decreased (p < 0.05) after Type I-diabetic mice received 1 intravitreal injection, and Type II-diabetic mice received seven intraperitoneal injections of anti-IL-17A. Further, vascular tight junction protein Zonula Occludens-1 (ZO-1) was significantly decreased in both Type I and II diabetic mice, which was significantly increased when mice received anti-IL-17A injections (p < 0.05). Similarly, tight junction protein Occludin degradation was halted in Type II diabetic mice that received anti-IL-17A treatments. Finally, retinal capillary degeneration was halted 6 months after diabetes was confirmed in Type II-diabetic mice that received weekly intraperitoneal injections of anti-IL-17A. These findings provide evidence that IL-17A plays a pivotal role in non-proliferative diabetic retinopathy in Type II diabetic mice, and suggests that anti-IL-17A could be a good therapeutic candidate for non-proliferative diabetic retinopathy.

IL-17A Enhances Retinal Neovascularization.

Retinal neovascularization occurs in proliferative diabetic retinopathy, neovascular glaucoma, and age-related macular degeneration. This type of retinal pathology normally occurs in the later stages of these ocular diseases and is a prevalent cause of vision loss. Previously, we determined that Interleukin (IL)-17A plays a pivotal role in the onset and progression of non-proliferative diabetic retinopathy in diabetic mice. Unfortunately, none of our diabetic murine models progress to proliferative diabetic retinopathy. Hence, the role of IL-17A in vascular angiogenesis, neovascularization, and the onset of proliferative diabetic retinopathy was unclear. In the current study, we determined that diabetes-mediated IL-17A enhances vascular endothelial growth factor (VEGF) production in the retina, Muller glia, and retinal endothelial cells. Further, we determined that IL-17A can initiate retinal endothelial cell proliferation and can enhance VEGF-dependent vascular angiogenesis. Finally, by utilizing the oxygen induced retinopathy model, we determined that IL-17A enhances retinal neovascularization. Collectively, the results of this study provide evidence that IL-17A plays a pivotal role in vascular proliferation in the retina. Hence, IL-17A could be a potentially novel therapeutic target for retinal neovascularization, which can cause blindness in multiple ocular diseases.

Inhibition of CD40-TRAF6-dependent inflammatory activity halts the onset of diabetic retinopathy in streptozotocin-diabetic mice.

Diabetes initiates inflammation that can impair the retinal vasculature, and lead to diabetic retinopathy; one of the leading causes of blindness. Inflammatory pathways have been examined as potential therapeutic targets for diabetic retinopathy, but there is still a need for early-stage treatments. We hypothesized that the CD40-TNF Receptor Associated Factor 6 (TRAF6) axis plays a pivotal role in the onset of diabetic retinopathy, and that the CD40-TRAF6 axis would be a prime therapeutic target for early-stage non-proliferative diabetic retinopathy. The CD40-TRAF6 complex can initiate NFκB activation, inflammation, and tissue damage. Further, CD40 and TRAF6 are constitutively expressed on Muller glia, and upregulated in the diabetic retina. Yet the role of the CD40-TRAF6 complex in the onset of diabetic retinopathy is still unclear. In the current study, we examined the CD40-TRAF6 axis in diabetic retinopathy using a small molecule inhibitor (SMI-6877002) on streptozotocin-induced diabetic mice. When CD40-TRAF6-dependent inflammation was inhibited, retinal vascular leakage and capillary degeneration was ameliorated in diabetic mice. Collectively, these data suggest that the CD40-TRAF6 axis plays a pivotal role in the onset of diabetic retinopathy, and could be a novel therapeutic target for early diabetic retinopathy.

Retinal Inflammation, Oxidative Stress, and Vascular Impairment Is Ablated in Diabetic Mice Receiving XMD8-92 Treatment.

The global number of diabetics continues to rise annually. As diabetes progresses, almost all of Type I and more than half of Type II diabetics develop diabetic retinopathy. Diabetic retinopathy is a microvascular disease of the retina, and is the leading cause of blindness in the working-age population worldwide. With such a significant health impact, new drugs are required to halt the blinding threat posed by this visual disorder. The cause of diabetic retinopathy is multifactorial, and an optimal therapeutic would halt inflammation, cease photoreceptor cell dysfunction, and ablate vascular impairment. XMD8-92 is a small molecule inhibitor that blocks inflammatory activity downstream of ERK5 (extracellular signal-related kinase 5) and BRD4 (bromodomain 4). ERK5 elicits inflammation, is increased in Type II diabetics, and plays a pathologic role in diabetic nephropathy, while BRD4 induces retinal inflammation and plays a role in retinal degeneration. Further, we provide evidence that suggests both pERK5 and BRD4 expression are increased in the retinas of our STZ (streptozotocin)-induced diabetic mice. Taken together, we hypothesized that XMD8-92 would be a good therapeutic candidate for diabetic retinopathy, and tested XMD8-92 in a murine model of diabetic retinopathy. In the current study, we developed an in vivo treatment regimen by administering one 100 µL subcutaneous injection of saline containing 20 µM of XMD8-92 weekly, to STZ-induced diabetic mice. XMD8-92 treatments significantly decreased diabetes-mediated retinal inflammation, VEGF production, and oxidative stress. Further, XMD8-92 halted the degradation of ZO-1 (zonula occludens-1), which is a tight junction protein associated with vascular permeability in the retina. Finally, XMD8-92 treatment ablated diabetes-mediated vascular leakage and capillary degeneration, which are the clinical hallmarks of non-proliferative diabetic retinopathy. Taken together, this study provides strong evidence that XMD8-92 could be a potentially novel therapeutic for diabetic retinopathy.

Aryl Hydrocarbon Receptor Agonist VAF347 Impedes Retinal Pathogenesis in Diabetic Mice.

Diabetic retinopathy is the leading cause of blindness in the working-age population worldwide. Although the cause of diabetic retinopathy is multifactorial, IL-17A is a prevalent inflammatory cytokine involved in the promotion of diabetes-mediated retinal inflammation and the progression of diabetic retinopathy. The primary source of IL-17A is Th17 cells, which are T helper cells that have been differentiated by dendritic cells in a proinflammatory cytokine environment. Aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that can manipulate dendritic cell maturation, halt the production of IL-6 (a proinflammatory cytokine), and suppress Th17 cell differentiation. In the current study, we examined the efficacy of an AhR agonist, VAF347, as a potential therapeutic for the onset of non-proliferative diabetic retinopathy in streptozotocin (STZ)-induced diabetic C57BL/6 mice. We determined that diabetes-mediated leukostasis, oxidative stress, and inflammation in the retina of STZ-diabetic mice were all significantly lower when treated with the AhR agonist VAF347. Furthermore, when VAF347 was subcutaneously injected into STZ-diabetic mice, retinal capillary degeneration was ameliorated, which is the hallmark of non-proliferative diabetic retinopathy in this diabetes murine model. Collectively, these findings provide evidence that the AhR agonist VAF347 could be a potentially novel therapeutic for non-proliferative diabetic retinopathy.

RORγt Inhibitor-SR1001 Halts Retinal Inflammation, Capillary Degeneration, and the Progression of Diabetic Retinopathy.

Diabetic retinopathy is a diabetes-mediated retinal microvascular disease that is the leading cause of blindness in the working-age population worldwide. Interleukin (IL)-17A is an inflammatory cytokine that has been previously shown to play a pivotal role in the promotion and progression of diabetic retinopathy. Retinoic acid-related orphan receptor gammaT (RORγt) is a ligand-dependent transcription factor that mediates IL-17A production. However, the role of RORγt in diabetes-mediated retinal inflammation and capillary degeneration, as well as its potential therapeutic attributes for diabetic retinopathy has not yet been determined. In the current study, we examined retinal inflammation and vascular pathology in streptozotocin-induced diabetic mice. We found RORγt expressing cells in the retinal vasculature of diabetic mice. Further, diabetes-mediated retinal inflammation, oxidative stress, and retinal endothelial cell death were all significantly lower in RORγt-/- mice. Finally, when a RORγt small molecule inhibitor (SR1001) was subcutaneously injected into diabetic mice, retinal inflammation and capillary degeneration were ameliorated. These findings establish a pathologic role for RORγt in the onset of diabetic retinopathy and identify a potentially novel therapeutic for this blinding disease.

MicroRNA-processing Enzymes Are Essential for Survival and Function of Mature Retinal Pigmented Epithelial Cells in Mice.

Age-related macular degeneration (AMD) is a major cause of irreversible vision loss. The neovascular or "wet" form of AMD can be treated to varying degrees with anti-angiogenic drugs, but geographic atrophy (GA) is an advanced stage of the more prevalent "dry" form of AMD for which there is no effective treatment. Development of GA has been linked to loss of the microRNA (miRNA)-processing enzyme DICER1 in the mature retinal pigmented epithelium (RPE). This loss results in the accumulation of toxic transcripts of Alu transposable elements, which activate the NLRP3 inflammasome and additional downstream pathways that compromise the integrity and function of the RPE. However, it remains unclear whether the loss of miRNA processing and subsequent gene regulation in the RPE due to DICER1 deficiency also contributes to RPE cell death. To clarify the role of miRNAs in RPE cells, we used two different mature RPE cell-specific Cre recombinase drivers to inactivate either Dicer1 or DiGeorge syndrome critical region 8 (Dgcr8), thus removing RPE miRNA regulatory activity in mice by disrupting two independent and essential steps of miRNA biogenesis. In contrast with prior studies, we found that the loss of each factor independently led to strikingly similar defects in the survival and function of the RPE and retina. These results suggest that the loss of miRNAs also contributes to RPE cell death and loss of visual function and could affect the pathology of dry AMD.

Electroporation of DNA into Physarum polycephalum Mitochondria: Effects on Transcription and RNA Editing in Isolated Organelles.

Mitochondrial RNAs in the acellular slime mold Physarum polycephalum contain nucleotides that are not encoded in the mitochondrial genes from which they are transcribed. These site-specific changes are quite extensive, comprising ~4% of the residues within mRNAs and ~2% of rRNAs and tRNAs. These "extra" nucleotides are added co-transcriptionally, but the means by which this is accomplished have not been elucidated. The cox1 mRNA also contains four sites of C to U changes, which occur post-transcriptionally, most likely via targeted deamination. The currently available in vitro systems for studying P. polycephalum editing are limited in that the template is the entire ~63,000 bp mitochondrial genome. This presents a significant challenge when trying to define the signals that specify editing sites. In an attempt to overcome this issue, a method for introducing DNA into isolated P. polycephalum mitochondria via electroporation has been developed. Exogenous DNA is expressed, but the transcripts synthesized from these templates are not edited under the conditions tested. However, transcripts derived from the mitochondrial genome are accurately edited after electroporation, indicating that the editing machinery is still functional. These findings suggest that this method may ultimately provide a feasible approach to elucidating editing signals.

4-Hydroxy-7-oxo-5-heptenoic Acid Lactone Induces Angiogenesis through Several Different Molecular Pathways.

Oxidative stress and angiogenesis have been implicated not only in normal phenomena such as tissue healing and remodeling but also in many pathological processes. However, the relationships between oxidative stress and angiogenesis still remain unclear, although oxidative stress has been convincingly demonstrated to influence the progression of angiogenesis under physiological and pathological conditions. The retina is particularly susceptible to oxidative stress because of its intensive oxygenation and high abundance of polyunsaturated fatty acyls. In particular, it has high levels of docosahexanoates, whose oxidative fragmentation produces 4-hydroxy-7-oxo-5-heptenoic acid lactone (HOHA-lactone). Previously, we found that HOHA-lactone is a major precursor of 2-(ω-carboxyethyl)pyrrole (CEP) derivatives, which are tightly linked to age-related macular degeneration (AMD). CEPs promote the pathological angiogenesis of late-stage AMD. We now report additional mechanisms by which HOHA-lactone promotes angiogenesis. Using cultured ARPE-19 cells, we observed that HOHA-lactone induces secretion of vascular endothelial growth factor (VEGF), which is correlated to increases in reactive oxygen species and decreases in intracellular glutathione (GSH). Wound healing and tube formation assays provided, for the first time, in vitro evidence that HOHA-lactone induces the release of VEGF from ARPE-19 cells, which promotes angiogenesis by human umbilical vein endothelial cells (HUVEC) in culture. Thus, HOHA-lactone can stimulate vascular growth through a VEGF-dependent pathway. In addition, results from MTT and wound healing assays as well as tube formation experiments showed that GSH-conjugated metabolites of HOHA-lactone stimulate HUVEC proliferation and promote angiogenesis in vitro. Previous studies demonstrated that HOHA-lactone, through its CEP derivatives, promotes angiogenesis in a novel Toll-like receptor 2-dependent manner that is independent of the VEGF receptor or VEGF expression. The new studies show that HOHA-lactone also participates in other angiogenic signaling pathways that include promoting the secretion of VEGF from retinal pigmented epithelial cells.

JAK/STAT regulation of Aspergillus fumigatus corneal infections and IL-6/23-stimulated neutrophil, IL-17, elastase, and MMP9 activity.

IL-6 and IL-23 (IL-6/23) induce IL-17A (IL-17) production by a subpopulation of murine and human neutrophils, resulting in autocrine IL-17 activation, enhanced production of reactive oxygen species, and increased fungal killing. As IL-6 and IL-23 receptors trigger JAK1, -3/STAT3 and JAK2/STAT3 phosphorylation, respectively, we examined the role of this pathway in a murine model of fungal keratitis and also examined neutrophil elastase and gelatinase (matrix metalloproteinase 9) activity by IL-6/23-stimulated human neutrophils in vitro. We found that STAT3 phosphorylation of neutrophils in Aspergillus fumigatus-infected corne as was inhibited by the JAK/STAT inhibitor Ruxolitinib, resulting in impaired fungal killing and decreased matrix metalloproteinase 9 activity. In vitro, we showed that fungal killing by IL-6/23-stimulated human peripheral blood neutrophils was impaired by JAK/STAT inhibitors Ruxolitinib and Stattic, and by the retinoic acid receptor-related orphan receptor γt inhibitor SR1001. This was also associated with decreased reactive oxygen species, IL-17A production, and retinoic acid receptor-related orphan receptor γt translocation to the nucleus. We also demonstrate that IL-6/23-activated neutrophils exhibit increased elastase and gelatinase (matrix metalloproteinase 9) activity, which is inhibited by Ruxolitinib and Stattic but not by SR1001. Taken together, these observations indicate that the regulation of activity of IL-17-producing neutrophils by JAK/STAT inhibitors impairs reactive oxygen species production and fungal killing activity but also blocks elastase and gelatinase activity that can cause tissue damage.

4-Hydroxy-7-oxo-5-heptenoic Acid (HOHA) Lactone is a Biologically Active Precursor for the Generation of 2-(ω-Carboxyethyl)pyrrole (CEP) Derivatives of Proteins and Ethanolamine Phospholipids.

2-(ω-Carboxyethyl)pyrrole (CEP) derivatives of proteins were previously shown to have significant pathological and physiological relevance to age-related macular degeneration, cancer and wound healing. Previously, we showed that CEPs are generated in the reaction of ε-amino groups of protein lysyl residues with 1-palmityl-2-(4-hydroxy-7-oxo-5-heptenoyl)-sn-glycero-3-phosphatidylcholine (HOHA-PC), a lipid oxidation product uniquely generated by oxidative truncation of docosahexanenate-containing phosphatidylcholine. More recently, we found that HOHA-PC rapidly releases HOHA-lactone and 2-lyso-PC (t1/2 = 30 min at 37 °C) by nonenzymatic transesterification/deacylation. Now we report that HOHA-lactone reacts with Ac-Gly-Lys-OMe or human serum albumin to form CEP derivatives in vitro. Incubation of human red blood cell ghosts with HOHA-lactone generates CEP derivatives of membrane proteins and ethanolamine phospholipids. Quantitative analysis of the products generated in the reaction HOHA-PC with Ac-Gly-Lys-OMe showed that HOHA-PC mainly forms CEP-dipeptide that is not esterified to 2-lysophosphatidycholine. Thus, the HOHA-lactone pathway predominates over the direct reaction of HOHA-PC to produce the CEP-PC-dipeptide derivative. Myleoperoxidase/H2O2/NO2(-) promoted in vitro oxidation of either 1-palmityl-2-docosahexaneoyl-sn-glycero-3-phosphatidylcholine (DHA-PC) or docosahexaenoic acid (DHA) generates HOHA-lactone in yields of 0.45% and 0.78%, respectively. Lipid oxidation in human red blood cell ghosts also releases HOHA-lactone. Oxidative injury of ARPE-19 human retinal pigmented epithelial cells by exposure to H2O2 generated CEP derivatives. Treatment of ARPE-19 cells with HOHA-lactone generated CEP-modified proteins. Low (submicromolar), but not high, concentrations of HOHA-lactone promote increased vascular endothelial growth factor (VEGF) secretion by ARPE-19 cells. Therefore, HOHA-lactone not only serves as an intermediate for the generation of CEPs but also is a biologically active oxidative truncation product from docosahexaenoate lipids.

Neutrophil IL-1ß processing induced by pneumolysin is mediated by the NLRP3/ASC inflammasome and caspase-1 activation and is dependent on K+ efflux.

Although neutrophils are the most abundant cells in acute infection and inflammation, relatively little attention has been paid to their role in inflammasome formation and IL-1ß processing. In the present study, we investigated the mechanism by which neutrophils process IL-1ß in response to Streptococcus pneumoniae. Using a murine model of S. pneumoniae corneal infection, we demonstrated a requirement for IL-1ß in bacterial clearance, and we showed that Nod-like receptor protein 3 (NLRP3), apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC), and caspase-1 are essential for IL-1ß production and bacterial killing in the cornea. Neutrophils in infected corneas had multiple specks with enzymatically active caspase-1 (YVAD-FLICA 660), and bone marrow neutrophils stimulated with heat-killed S. pneumoniae (signal 1) and pneumolysin (signal 2) exhibited multiple specks when stained for NLRP3, ASC, or Caspase-1. High-molecular mass ASC complexes were also detected, consistent with oligomer formation. Pneumolysin induced K(+) efflux in neutrophils, and blocking K(+) efflux inhibited caspase-1 activation and IL-1ß processing; however, neutrophils did not undergo pyroptosis, indicating that K(+) efflux and IL-1ß processing is not a consequence of cell death. There was also no role for lysosomal destabilization or neutrophil elastase in pneumolysin-mediated IL-1ß processing in neutrophils. Taken together, these findings demonstrate an essential role for neutrophil-derived IL-1ß in S. pneumoniae infection, and they elucidate the role of the NLRP3 inflammasome in cleavage and secretion of IL-1ß in neutrophils. Given the ubiquitous presence of neutrophils in acute bacterial and fungal infections, these findings will have implications for other microbial diseases.

All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis.

The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated × protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661 W cells, cultured neural retinas and a retinal degeneration model, Abca4(-/-)Rdh8(-/-) mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis.

Activation of neutrophils by autocrine IL-17A-IL-17RC interactions during fungal infection is regulated by IL-6, IL-23, RORγt and dectin-2.

Here we identified a population of bone marrow neutrophils that constitutively expressed the transcription factor RORγt and produced and responded to interleukin 17A (IL-17A (IL-17)). IL-6, IL-23 and RORγt, but not T cells or natural killer (NK) cells, were required for IL-17 production in neutrophils. IL-6 and IL-23 induced expression of the receptors IL-17RC and dectin-2 on neutrophils, and IL-17RC expression was augmented by activation of dectin-2. Autocrine activity of IL-17A and its receptor induced the production of reactive oxygen species (ROS), and increased fungal killing in vitro and in a model of Aspergillus-induced keratitis. Human neutrophils also expressed RORγt and induced the expression of IL-17A, IL-17RC and dectin-2 following stimulation with IL-6 and IL-23. Our findings identify a population of human and mouse neutrophils with autocrine IL-17 activity that probably contribute to the etiology of microbial and inflammatory diseases.

Degeneration of retinal ganglion cells in diabetic dogs and mice: relationship to glycemic control and retinal capillary degeneration.

PURPOSE: The purpose of this study was to investigate (i) the effect of diabetes on retinal ganglion cell death in diabetic dogs and mice, (ii) the effect of prolonged glycemic control on diabetes-induced death of retinal ganglion cells, (iii) whether retinal ganglion cell death in diabetes is associated with degeneration of retinal capillaries, and (iv) the effect of diet on diabetes-induced degeneration of retinal ganglion cells in mice. METHODS: Diabetes was induced in dogs using streptozotocin, and levels of glycemic control (good, moderate, and poor) were maintained for 5 years. Diabetes was studied in two mouse models (diabetes induced in C57Bl/6J mice using streptozotocin and spontaneously diabetic Ins2Akita mice). Retinal ganglion cell death was investigated by counting the number of axons from the ganglion cells in the optic nerve and with terminal transferase deoxyuridine triphosphate nick-end labeling and annexin V staining in mice. RESULTS: As reported previously, the development and severity of vascular lesions of diabetic retinopathy in diabetic dogs were strongly associated with glycemic control. Loss of retinal ganglion cells was extensive in dogs kept in poor glycemic control, and was essentially prevented in diabetic dogs kept in good glycemic control for the 5 years of study. In contrast, "moderate" glycemic control (intermediate between poor and good glycemic control) caused a significant increase in vascular pathology, but did not cause loss of retinal axons in the optic nerve. Using this validated optic nerve axon counting method, the two mouse models of diabetic retinopathy were studied to assess ganglion cell death. Despite 10 months of diabetes (a duration that has been shown to cause retinal capillary degeneration in both models), neither mouse model showed loss of optic nerve axons (thus suggesting no loss of retinal ganglion cells). Likewise, other parameters of cell death (terminal transferase deoxyuridine triphosphate nick-end labeling and annexin V labeling) did not suggest ganglion cell death in diabetic C57Bl/6J mice, and ganglion cell death was not increased by a different commercial diet. CONCLUSIONS: Retinal ganglion cell death in diabetic dogs is significantly inhibited by good or even moderate glycemic control. The finding that diabetic dogs in moderate glycemic control had appreciable vascular disease without apparent retinal ganglion cell degeneration does not support the postulate that neural degeneration causes the vascular pathology. Studies of diabetic mice in our colony again fail to find evidence of ganglion cell death due to prolonged diabetes in this species.

Subclinical increased anterior stromal reflectivity with topical taprenepag isopropyl.

PURPOSE: To assess the effect of topical taprenepag isopropyl on each layer of the cornea by confocal microscopy. METHODS: Thirty-two ocular hypertensive or glaucoma patients were randomized into a 2-period, crossover study of 14 days of 0.1% taprenepag alone and in unfixed combination with 0.005% latanoprost (combination therapy). Baseline and sequential slit-lamp biomicroscopy, fluorescein staining, central ultrasonic pachymetry, and confocal microscopy were performed. Confocal images were analyzed for the density of the central superficial and basal epithelium, midstromal keratocytes, and endothelium, as well as endothelial coefficient of variation and percentage of hexagonal cells, and reflectivity of anterior stromal and midstromal layers. RESULTS: Corneal staining increased from baseline, reaching a peak at day 13 (69% and 63% of subjects treated with monotherapy and combination therapy, respectively), which resolved by day 35. A statistically significant increase in mean corneal thickness for both eyes and both treatments occurred on days 7 and 13 (range, 20-27 µm; P < 0.001) but recovered (≤ 6 µm) by day 35. No statistically significant changes were observed in the basal epithelial, midstromal, or endothelial cells. Mean ratio of average reflectivity of anterior stroma to midstroma increased on days 13 and 35 in period 1 for each treatment (range, 1.2-1.9; P < 0.001), and this increase persisted during period 2. CONCLUSIONS: Anterior stromal reflectivity may remain increased even when biomicroscopic and confocal images of corneal layers remain normal or have recovered after topical taprenepag. This subclinical measure may be useful to detect a persistent adverse effect of a topical agent on the cornea.

Cutaneous penetration of the topically applied photosensitizer Pc 4 as detected by intravital 2-photon laser scanning microscopy.

The fundamental mechanism of photodynamic therapy (PDT)-induced cell death has been characterized, but early critical PDT events in vivo remain incompletely defined. With the recent development in advanced fluorescence imaging modalities, such as intravital 2-photon laser scanning microscopy (2P-LSM), researchers are now able to investigate and visualize biological processes with high resolution in real time. This powerful imaging technology allows deep tissue visualization with single-cell resolution, thus providing dynamic information on the 3-dimensional architectural makeup of the tissue. The main goal of this study was to determine the cutaneous penetration of a topically applied photosensitizer, the silicon phthalocyanine Pc 4, into the skin of live animals and to assess the effective absorption of Pc 4 through the skin barrier. Our 2P-LSM images indicate that Pc 4 penetrates to the epidermal/dermal junction of mouse skin. The data also indicate that the degree of Pc 4 absorption is dose dependent. These findings represent initial steps that may help in improving the clinical utilization of topical Pc 4-PDT.

Monocular visual deprivation in macaque monkeys: a profile in the gene expression of lateral geniculate nucleus by laser capture microdissection.

PURPOSE: Amblyopia is the most common cause of visual impairment in children. Early detection of amblyopia and subsequent intervention are vital in preventing visual loss. Understanding the molecular pathogenesis of amblyopia would greatly facilitate development of therapeutic interventions. An animal model of amblyopia induced by monocular vision deprivation has been extensively studied in terms of anatomic and physiologic alterations that affect visual pathways. However, the molecular events underlying these changes are poorly understood. This study aimed to characterize changes of gene expression profiles in the lateral geniculate nucleus (LGN) associated with amblyopia induced by monocular visual deprivation. METHODS: Monocular vision deprivation was generated by either opaque dark contact lens or tarsorrhaphy of newborn rhesus monkeys. LGN was harvested at two or four months following induction of vision deprivation. Laser capture microdissection was used to obtain individual LGN layers for total RNA isolation. Linear T7-based in vitro RNA amplification was used to obtain sufficient RNA to conduct DNA microarray studies. The resulting Affymetrix GeneChip Expression data were analyzed using Affymetrix GeneChip Operating Software. Real-time quantitative polymerase chain reaction and in situ hybridization were used to further analyze expression of selected genes. RESULTS: Using 52,699 microarray probe sets from a Rhesus array, we identified 116 transcripts differentially expressed between deprived and nondeprived parvocellular layers: 45 genes were downregulated and 71 genes were upregulated in deprived parvocellular layers. We also observed substantial changes in deprived magnocellular laminae: 74 transcripts exhibited altered expression, 42 genes were downregulated, and 32 genes were upregulated. The genes identified in this study are involved in many diverse processes, including binding (calcium ion binding, nucleic acid binding, and nucleotide binding), catalytic activity, and signal transducer activity. CONCLUSIONS: There were significant differences in gene expression profiles between deprived and nondeprived parvocellular layers and magnocellular laminae of LGN. These alterations in gene expression may play a critical role in the molecular pathogenesis of amblyopia. The genes identified in this study may provide potential targets for therapeutic intervention of this disease.

Salicylate-based anti-inflammatory drugs inhibit the early lesion of diabetic retinopathy.

It has been previously reported that aspirin inhibited the development of diabetic retinopathy in diabetic animals, raising the possibility that anti-inflammatory drugs may have beneficial effects on diabetic retinopathy. To further explore this, we compared effects of oral consumption of three different salicylate-based drugs (aspirin, sodium salicylate, and sulfasalazine) on the development of early stages of diabetic retinopathy in rats. These three drugs differ in their ability to inhibit cyclooxygenase but share an ability to inhibit nuclear factor-kappaB (NF-kappaB). Diabetes of 9-10 months duration significantly increased the number of TUNEL (transferase-mediated dUTP nick-end labeling)-positive capillary cells and acellular (degenerate) capillaries in the retinal vasculature, and all three salicylate-based drugs inhibited this cell death and formation of acellular capillaries without altering the severity of hyperglycemia. In short-term diabetes (2-4 months), all three salicylates inhibited the diabetes-induced loss of neuronal cells from the ganglion cell layer. Oral aspirin (as a representative of the salicylate family) inhibited diabetes-induced increase in NF-kappaB DNA-binding affinity in electrophoretic mobility shift assay and transcription factor array in nuclear extract isolated from whole retina. All three salicylates inhibited the diabetes-induced translocation of p50 (a subunit of NF-kappaB) into nuclei of retinal vascular endothelial cells of the isolated retinal vasculature, as well as of p50 and p65 into nuclei of cells in the ganglion cell layer and inner nuclear layer on whole-retinal sections. Sulfasalazine (also as a representative of the salicylates) inhibited the diabetes-induced upregulation of several inflammatory gene products, which are regulated by NF-kappaB, including vascular cell adhesion molecule, intracellular adhesion molecule-1, inducible nitric oxide synthase, and cyclooxygenase-2 in whole-retinal lysate. Salicylates, in doses administrated in our experiments, inhibited NF-kappaB and perhaps other transcription factors in the retina, were well tolerated, and offered new tools to investigate and inhibit the development of diabetic retinopathy.

Antimicrobial polypeptides of the human colonic epithelium.

The lumen of the human colon is heavily colonized with microbes, but infections across its epithelial surface are infrequent. To address the hypothesis that antimicrobial polypeptides contribute to the barrier function of colonic epithelial cells, we examined cellular extracts from non-inflamed colonic mucosa using an antimicrobial assay. This approach yielded five polypeptides: three antimicrobials were previously identified as ribosomal polypeptides (L30, S19 and ubiquicidin), and two were members of the histone family (H1.5 and H2B). All exhibited bactericidal activity against Escherichia coli, and with the exception of S19, had been isolated by others based on their potent antimicrobial activity in other cells and tissues. These polypeptides normally reside inside cells and are proposed to contribute to the formation of the functional antimicrobial barrier of the colonic epithelium.