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A qualitative study was conducted using semi-structured interviews leaders in community-based organizations, health care, and local public health to understand organizational perspectives of collaboration for health equity and identify opportunities to improve collaboration. Twelve leaders were interviewed from March through May 2023 in Kent County, Michigan. All leaders saw collaboration as valuable for advancing health equity. Key themes that affected collaboration and could be a facilitator or barrier to advancing health equity in the community were inclusion, power, relationships, resources, and organizational traits. Leaders articulated the following factors as those that support collaboration for health equity: authentic inclusion, shared decision-making, taking time to foster trusting relationships, adequate resources to support the infrastructure needed for collaborations, organizational flexibility, and individual commitment. Building partnerships with these facilitators in mind may result in more robust, sustainable, and resilient collaboratives., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

One of the major causes of vision impairment among elderly people in developed nations is age-related macular degeneration (AMD). The distinctive features of AMD are the accumulation of extracellular deposits called drusen and the gradual deterioration of photoreceptors and nearby tissues in the macula. AMD is a complex and multifaceted disease influenced by several factors such as aging, environmental risk factors, and a person's genetic susceptibility to the condition. The interaction among these factors leads to the initiation and advancement of AMD, where genetic predisposition plays a crucial role. With the advent of high-throughput genotyping technologies, many novel genetic loci associated with AMD have been identified, enhancing our knowledge of its genetic architecture. The common genetic variants linked to AMD are found on chromosome 1q32 (in the complement factor H gene) and 10q26 (age-related maculopathy susceptibility 2 and high-temperature requirement A serine peptidase 1 genes) loci, along with several other risk variants. This review summarizes the common genetic variants of complement pathways, lipid metabolism, and extracellular matrix proteins associated with AMD risk, highlighting the intricate pathways contributing to AMD pathogenesis. Knowledge of the genetic underpinnings of AMD will allow for the future development of personalized diagnostics and targeted therapeutic interventions, paving the way for more effective management of AMD and improved outcomes for affected individuals.

In recent years, there has been a captivating focus of interest in elucidating the intricate crosstalk between adiponectin (APN), a versatile fat-associated adipokine and ocular pathologies. Unveiling the intricate relationship between adipocytokine APN and its receptors (AdipoRs) with aging eye disorders has emerged as a fascinating frontier in medical research. This review article delves into this connection, illuminating the hidden influence of APN on retinal health. This comprehensive review critically examines the latest findings and breakthroughs that underscore the pivotal roles of APN/AdipoRs signaling in maintaining ocular homeostasis and protecting against eye ailments. Here, we meticulously explore the intriguing mechanisms by which APN protein influences retinal function and overall visual acuity. Drawing from an extensive array of cutting-edge studies, the article highlights APN's multifaceted functions, ranging from anti-inflammatory properties and oxidative stress reduction to angiogenic regulation within retinal and macula tissues. The involvement of APN/AdipoRs in mediating these effects opens up novel avenues for potential therapeutic interventions targeting prevalent aging eye conditions. Moreover, this review unravels the interplay between APN signaling pathways and age-related macular degeneration (AMD). The single-cell RNA-seq results validate the expression of both the receptor isoforms (AdipoR1/R2) in retinal cells. The transcriptomic analysis showed lower expression of AdipoR1/2 in dry AMD pathogenesis compared to healthy subjects. The inhibitory adiponectin peptide (APN1) demonstrated over 75% suppression of CNV, whereas the control peptide did not exert any inhibitory effect on choroidal neovascularization (CNV). The elucidation of these relationships fosters a deeper understanding of adipose tissue's profound influence on ocular health, presenting new prospects for personalized treatments and preventative measures. Because APN1 inhibits CNV and leakage, it can be used to treat human AMD, although the possibility to treat human AMD is in the early stage and more clinical research is needed. In conclusion, this review provides a captivating journey into the enthralling world of APN, intertwining the realms of adipose biology and ophthalmology in aging.

Purpose: Intra-Tenon or subconjunctival injection of a solution of mitomycin C (MMC) and 1% preservative-free lidocaine (as an anesthetic) has gained popularity for its use in trabeculectomy, a filtering surgery for glaucoma. To our knowledge, no studies have analyzed the impact of lidocaine on the cytotoxic effects of MMC in this setting. This study was conducted to evaluate in vitro fibroblast cytotoxicity to a solution of MMC (0.2 mg/ml) and 1% preservative-free lidocaine., Design: Experimental study., Participants: Nonhuman subject research., Methods: Cultured human conjunctival fibroblasts were incubated in phosphate-buffered saline (PBS) (control), MMC (0.2 mg/ml), a mixture of 0.2 mg/ml MMC + 1% preservative-free lidocaine, or 1% preservative-free lidocaine. Samples were taken at 2, 5, 10, 30, and 60 minutes, and a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay with photoabsorbance testing was used to assess conjunctival cell viability., Main Outcome Measure: Mean photoabsorbance., Results: Mean photoabsorbance across all time intervals was 0.680 for solutions incubated in PBS, 0.642 for MMC, 0.612 for MMC + 1% preservative-free lidocaine, and 0.605 for 1% preservative-free lidocaine. A 2-way analysis of variance analyzing solution, time, and solution-time interaction on photoabsorbance showed that PBS was least cytotoxic and an optimal control for this study. Tukey post hoc comparisons showed that MMC was more cytotoxic than PBS (P < 0.001). However, both MMC + 1% preservative-free lidocaine and 1% preservative-free lidocaine were more cytotoxic than MMC and PBS (P < 0.01 for all). No significant differences in cytotoxicity comparing lidocaine-containing solutions were observed., Conclusions: In this in vitro study, we found an increase in cytotoxicity when MMC (0.2 mg/ml) was combined with 1% preservative-free lidocaine. We note that lidocaine did not inhibit MMC cytotoxicity and exhibited a significant cytotoxic effect on its own., (Copyright © 2020 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Introduction: Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) has spread around the globe. Susceptibility has been associated with age, biological sex, and other prior existing health conditions. However, host genes are involved in viral infectivity and pathogenicity, and polymorphisms in these could be responsible for the interethnic/interindividual variability observed in infection and progression of COVID-19. Materials and Methods: Clinical exome data of 103 individuals was analyzed to identify sequence variants in five selected candidate genes: ACE2, TMPRSS2, CD209, IFITM3 , and MUC5B to assess their prevalence and role to understand the COVID-19 infectivity and progression in our population. Results: A total of 497 polymorphisms were identified in the five selected genes in the exomes analyzed. Thirty-eight polymorphisms identified in our cohort have been reported earlier in literature and have functional significance or association with health conditions. These variants were classified into three groups: protective, susceptible, and responsible for comorbidities. Discussion and Conclusion: The two polymorphisms described in literature as risk inducing are rs35705950 in MUC5B gene and TMPRSS2 haplotype (rs463727, rs34624090, rs55964536, rs734056, rs4290734, rs34783969, rs11702475, rs35899679, and rs35041537) were absent in our cohort explaining the slower infectivity of the disease in this part of India. The 38 functional variants identified can be used as a predisposition panel for the COVID-19 infectivity and progression and stratify individuals as "high or low risk," which would help in planning appropriate surveillance and management protocols. A larger study from different regions of India is warranted to validate these results., (Copyright © 2020 Iyer, Samajder, Zubeda, S, Mali, PV, Sharma, Abbas, Bora, Narravula and Hasan.)

Objectives: To explore the therapeutic role of a single dose combination of montelukast (MON) and dexamethasone (DXM) through intra-peritoneal route against paraquat (PQ)-intoxicated experimental Wistar rats., Methods: In vivo the survival rate was investigated following the administration of both MON and DXM in PQ exposed rats. Lungs parameters including enhanced pause (Penh), tidal volume (TV) and breath per minute (BPM) were determined using the whole body plethysmograph (WBP). Further chest imaging and histopathological studies were conducted to evaluate the lungs injury. In vivo the antioxidant activity was carried out by determining the levels of catalase (SOD), superoxide dismutase (CAT) and glutathione peroxidase (GSH-Px). Lungs tissue concentration of different proinflammatory cytokines like IL-1β, IL-6, TGF-β1 and TNF-α was also determined. Finally, expression of NF-kB and p-NF-kB was investigated by western blot., Results: Results of survival rate and levels of lungs parameters indicated therapeutic potential of combination treatment of MON and DXM. Protective activity on lungs was reflected in chest imaging and histopathological investigations. Moreover, combination treatment exhibited significant increased levels of all anti-oxidant parameters. Significant decrease in the levels of IL-1β; IL-6; TGF-β1 and TNF-α was also observed with the combination treatment of MON and DXM. Evidence of significant down regulation of NF-kB and phospho-NF-kB was also found with the combination treatment of MON and DXM., Conclusions: Given the advantage of therapeutic synergism activity of MON and DXM, it may be used in the prophylaxis of PQ-intoxication following clinical trials.

Background: Defense personnel utilize capsaicin-based ocular sprays as non-lethal agents for law implementation during instances of mob violence. This study involves the capsaicin antagonist Capsazepine and the investigation of whether Capsazepine's antagonistic approach can be favorably utilized for defense utilization to block capsaicin-initiated pain and inflammation via the ocular pathway., Research Design and Methods: Ocular capsazepine in situ gels were prepared with polymers Pluronic F-127 and Chitosan; optimized formulation was quantified in ocular tissues chromatographically and by in vivo live ocular imaging; anti-inflammatory efficacy was determined by eye irritation testing, corneal and retinal imaging, ocular prostaglandin estimation, and by viability and proliferation testing using human ocular cell lines, etc., Results: A physicochemically stable Capsazepine in situ gel was formulated which showed little ocular irritation, considerable transcorneal permeation; was precisely quantified in ocular tissues by gas chromatography and in vivo live ocular imaging; showed anti-inflammatory properties against capsaicin by eye imaging experiments, prostaglandin declination and showed acceptable cytocompatibility when studied using human ocular cell lines., Conclusions: The fabricated in situ Capsazepine gel system might be promising for ocular delivery as it appears a pharmacologically potent and safe development, suitable for utilization in the ocular clinical therapy, provided there is additional research to substantiate it.

Purpose: Experimental autoimmune anterior uveitis (EAAU) is a clinically relevant animal model for human idiopathic anterior uveitis (IAU). The role of the immunomodulator transforming growth factor β2 (TGF-β2) in EAAU pathology is unknown. In this study, we investigated the regulatory role of TGF-β2 in EAAU., Methods: EAAU was induced in male Lewis rats by footpad injection of melanin-associated antigen (MAA). TGF-β2 was administered intravenously (iv) in MAA-sensitized rats during the induction of EAAU, or after the clinical onset of uveitis. MAA-sensitized rats injected similarly with an equal volume of PBS served as control. Animals were examined daily between days 7 and 30 post-injection for the clinical signs of uveitis using slit lamp biomicroscopy. Animals were sacrificed at various time points and eyes were harvested for histological analysis to assess the course and severity of inflammation. For histopathological analysis, paraffin sections of harvested eyes were stained with hematoxylin and eosin. Popliteal lymph nodes (LNs) were used for CD4 + CD25 + FoxP3 + T regulatory (Tregs) population analysis and for CD4 + T cell proliferation assay., Results: Administration of recombinant TGF-β2 during the early stages of EAAU prevented the induction of uveitis. Compared to PBS, the presence of TGF-β2 in the cell culture significantly (p < 0.05) inhibited the proliferation of CD4 + T cells in response to MAA. In MAA-sensitized Lewis rats, iv treatment with recombinant TGF-β2 resulted in significantly (p < 0.05) increased percentage of Tregs compared to animals treated similarly with PBS. Thus, TGF-β2 inhibited the induction of EAAU by inhibiting CD4 + T cell proliferation and increasing the number of Tregs. Injection of TGF-β2 in rats with active EAAU resulted in diminished disease activity. Unfortunately, this treatment did not lead to the early resolution of EAAU., Conclusions: TGF-β2 plays a critical role in regulation of intraocular inflammation in EAAU. Findings reported in this study improve our understanding of immunopathology of IAU and suggest that recombinant TGF-β2 may be a promising therapeutic agent for human IAU.

Objective: Melatonin and pumpkin seed oil, along with US FDA approved UV filters were incorporated into a formulation for enhancement of UV protection by exerting an antioxidant effect. The objective of this study was to assess the protective effect of this formulation against ultraviolet (UV) radiation-induced photo dermatitis in rats, which is an established model to study the aetiopathogenic mechanisms in psoriasis vulgaris, as the former exhibits the same features to those of clinical psoriasis vulgaris in humans. Materials and methods: The animals were segregated into five groups (6/group) and all received their respective formulations dermally prior to chronic UV irradiation for 28 days. The test, placebo, and standard groups; received the test, placebo, and standard formulations respectively; whereas the positive control group received only UV radiation. A normal control group was also maintained. Disease and treatment status were analyzed using various techniques by euthanizing the rats after 28 days. Results: The test formulation was able to ameliorate the UV-induced increase in skin fold, epidermal thickness, and skin edema; inhibit the reduction of hydroxyproline content and incidence of LPO within the skin tissues of exposed animals. The formulation was also able to inhibit the release of proinflammatory cytokines; IFN-γ, IL-1β, IL-6, and TNF-α; and upregulation of NF-κB and COX-2 genes caused by chronic UV exposure. Conclusion: It can be stated that melatonin included in the newly formulated sunscreen was able to inhibit the induction of photodermatitis via immunoregulation of inflammatory cytokines along with NF-κB and COX-2 genes.

The presence of 40-50% more UV radiation in high altitude areas renders the plethora of sunscreen products available in the market virtually ineffective. In this light of event, four US FDA approved UV filters were combined with melatonin and pumpkin seed oil to produce a broad spectrum sunscreen cream, which is envisaged to provide optimum sunprotection along with enhanced antioxidant activity. The objective of this study is to evaluate the protective effect of the sunscreen cream against UV radiation-induced skin photoaging in adult Wistar albino rats and identify its possible underlying mechanism. Wistar rats were exposed to broad spectrum UV radiation for 28 days. The test group received the sunscreen formulation dermally every day prior to UV radiation. The effects of the formulation against UV induced symptoms; viz. skin thickness and edema, in vivo antioxidant activities, inflammatory cytokines, collagen content, histopathological examination and expression of specific genes established the protective activity of the formulation. The test formulation was able to mitigate the harmful effects of UV radiation by increasing in vivo SOD, GSH-Px, CAT and collagen levels; decreasing skin edema, skin thickness and cytokines like IL-6, IL-1β, TNF-α and TGF-β1. UV radiation induced changes in histological architecture and arrangement of collagen and elastin fibers were also prevented by the test formulation. Finally, the formulation was able to regulate the expression of COL3A1, COX-2, bFGF, VEGF-C, Smad2, Smad4, Smad7 genes which induced significant photoprotective activity. The sunscreen formulation ameliorated UV induced photoaging by preventing oxidative collagen degradation and augmentation of TGF-β-Smad-mediated collagen production., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Graphene in nano form has proven to be one of the most remarkable materials. It has a single atom thick molecular structure and it possesses exceptional physical strength, electrical and electronic properties. Applications of the Graphene Family of Nanomaterials (GFNs) in different fields of therapy have emerged, including for targeted drug delivery in cancer, gene delivery, antimicrobial therapy, tissue engineering and more recently in more diseases including HIV. This review seeks to analyze current advances of potential applications of graphene and its family of nano-materials for drug delivery and other major biomedical purposes. Moreover, safety and toxicity are the major roadblocks preventing the use of GFNs in therapeutics. This review intends to analyze the safety and biocompatibility of GFNs along with the discussion on the latest techniques developed for toxicity reduction and biocompatibility enhancement of GFNs. This review seeks to evaluate how GFNs in future will serve as biocompatible and useful biomaterials in therapeutics., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Solar ultraviolet (UV) radiation exposure is known to cause inevitable damage to human skin via different mechanisms which include disruption of genetic material and generation of free radicals. In the ever emerging field of photoprotective agents, there have been constant endeavors to uphold the standards for optimum protection from solar UV-induced damages which include alarming conditions ranging from severe keratosis to malignant transformation of skin cells. Out of the various methods available for photoprotection, chemical photoprotective agents are most popular due to its ease of applicability, availability, and efficacy. However, the benevolences of chemophotoprotective agents are not excluded from the fact that all chemical agents are bound to suffer predestined consequences of toxicity and unwanted side effects. The present article focuses on the basic knowledge pertaining to achieve adequate sun protection and also on the beneficial and risk factors of using chemical agents as photoprotective formulations. The article highlights the US Food and Drug Administration (FDA) approved and unapproved UV filters; and also sheds light on the overall measures to protect an individual from UV radiation exposure, dispel misconceptions and present the newer technologies that are available in the market to accomplish ideal sun protection.

Gramine is a natural indole alkaloid that has been isolated from different raw plants occurring mainly in Avena sativa, etc. The study was aimed to investigate the possible in vitro antioxidant, in vitro mutagenic, in vitro antimutagenic, and in vivo genotoxic activity of gramine using ferric reducing ability of plasma (FRAP) assay, Metal chelating, Ames bacterial reverse mutation test, and the mouse bone marrow micronucleus assay as well as chromosomal aberration. Four concentrations of gramine viz. 250, 500, 1000, and 2000 μg/mL were evaluated for its antioxidant activity in FRAP Assay and Metal Chelating Test. Four concentrations of gramine (1250 μg/plate, 2500 μg/plate, 5000 μg/plate, and 10 000 μg/plate) were employed in Salmonella typhimurium strains to study the mutagenicity in the presence and absence of standard mutagens, 2-aminofluorene (2-AF), sodium azide (SA), and 2-nitrofluorene (2-NF). Three doses, i.e. 0.1, 0.2, and 0.3 × the LD 50 of gramine (i.e. 50 mg/kg, 100 mg/kg, and 150 mg/kg) were administered orally to either sex of Swiss albino mice for 48 h to study the genotoxic activity in micronucleus assay as well as chromosomal aberration. Gramine showed potent antioxidant activity in both the assay. Gramine at the given dose lacks mutagenicity as well as found to possess antimutagenic efficacy. Interestingly, S9 enzymes increase the antimutagenic activity in a dose-dependent manner. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes (MNPCEs), as well as no significant difference in the percentage of chromosomal aberrations was observed between the gramine groups and the negative groups but percentage of polychromatic erythrocytes (PCEs) is found to be higher in all the gramine groups. These results indicate significant antioxidant, non-mutagenic as well as non-genotoxic activity of gramine in vitro and in vivo in the given doses.

Compromised stability of pharmaceutical formulations loaded with volatiles is a serious problem associated with devices designed to deliver volatile compounds. The present study has been focused to evaluate the stability potential of matrix-type polymeric patches composed of volatile ethyl anthranilate for prophylaxis against vector-borne diseases. Ethyl anthranilate-loaded matrix-type polymeric patches were fabricated by solvent evaporation method on an impermeable backing membrane and attached to temporary release liners. Stability testing of the polymeric patches was performed as per the International Conference on Harmonization (ICH) guidelines for 6 months under accelerated conditions. In addition, the quantification of residual solvents was also performed as per the ICH guidelines. After conducting the stability studies for 6 months, the optimized patches showed the best possible results with respect to uniformity of drug content, physical appearance, and other analytical parameters. Furthermore, the amount of residual solvent was found well below the accepted limit. Thus, the present report outlined the analytical parameters to be evaluated to ensure the stability of a certain devices consisting of volatile compounds., (Copyright © 2016. Published by Elsevier B.V.)

Ultraviolet (UV) radiation exposure has been known to cause irreparable damages to human skin. The daunting risk of UV radiation exposure faced by military personnel led to the development of a sunscreen formulation which has superior sun protection factor combined with the ability to counteract reactive oxygen species. The present work deals with the preclinical safety evaluation of the sunscreen formulation comprising of four US FDA approved UV filters; namely avobenzone, octinoxate, oxybenzone, titanium dioxide along with melatonin and pumpkin seed oil, via OECD protocols of assessing acute oral and dermal toxicity; skin sensitizing; skin irritating; ocular irritating and genotoxic potential. Both oral and dermal LD 50 values were found to be ˃2000 mg/kg body weight in adult Wistar albino rats using acute dermal and oral toxicity tests. The sunscreen formulation was found to be non-sensitizing to the skin of guinea pigs and non-irritating to both skin and eyes of rabbits. The sunscreen formulation was also found to be non-mutagenic which was affirmed by a battery of genotoxicity and muagenicity assays. The results obtained from this preclinical study indicated that the sunscreen formulation is non toxic and safe in animal models. This study along with additional preclinical evaluations may serve as a basis for considering the formulation as a potential candidate for further trials to establish its efficacy, tolerability and applicability., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: To characterize the effect of aspirin (ASA) in mouse models of choroidal neovascularization (CNV) and retinal degeneration., Methods: In vivo: Male C57BL/6 mice were given ASA in food or regular rodent diet. CNV was induced by argon laser photocoagulation. Subretinal injections of polyethylene glycol 400 (PEG-400) were administered to induce retinal degeneration. CNV size, laser spot area and mean intensity of VEGF in the laser injured zones were measured. In the PEG injected eyes the thickness of retinal pigment epithelium (RPE) and choroid was measured. In vitro: Human ARPE-19 cells were treated with 0.5 or 2.0 mM/L of ASA for 72 h. ELISA was used to measure the concentration of VEGF and CCL-2 in the supernatants. Additionally, damaged RPE monolayer was treated with ASA (0.5 or 2.0 mM/L) and vehicle separately. Size of damaged area was measured. ELISA was used to measure secretion of VEGF-A and CCL-2 by damaged cells after 24 h., Results: No statistically significant effect of ASA on CNV size, laser spot size or VEGF expression was noted in CNV model. In the PEG model, ASA did not have any effect on RPE and choroid thickness; however, a significant increase in RPE atrophy was observed (P = 0.02 + 38%). In addition, ASA had a significant effect on the ability of the RPE cells to regenerate and become confluent after mechanical damage., Conclusions: ASA at doses consumed clinically for various medical causes may not worsen CNV in human subjects. However, ASA may increase RPE atrophy when consumed over long periods of time., (© 2016 Royal Australian and New Zealand College of Ophthalmologists.)

In the mouse, membrane cofactor protein (CD46), a key regulator of the alternative pathway of the complement system, is only expressed in the eye and on the inner acrosomal membrane of spermatozoa. We noted that although Cd46(-/-) mice have normal systemic alternative pathway activating ability, lack of CD46 leads to dysregulated complement activation in the eye, as evidenced by increased deposition of C5b-9 in the retinal pigment epithelium (RPE) and choroid. A knockout of CD46 induced the following cardinal features of human dry age-related macular degeneration (AMD) in 12-month-old male and female mice: accumulation of autofluorescent material in and hypertrophy of the RPE, dense deposits in and thickening of Bruch's membrane, loss of photoreceptors, cells in subretinal space, and a reduction of choroidal vessels. Collectively, our results demonstrate spontaneous age-related degenerative changes in the retina, RPE, and choroid of Cd46(-/-) mice that are consistent with human dry AMD. These findings provide the exciting possibility of using Cd46(-/-) mice as a convenient and reliable animal model for dry AMD. Having such a relatively straight-forward model for dry AMD should provide valuable insights into pathogenesis and a test model system for novel drug targets. More important, tissue-specific expression of CD46 gives the Cd46(-/-) mouse model of dry AMD a unique advantage over other mouse models using knockout strains., (Copyright © 2016 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Studies performed over the past decade in humans and experimental animals have been a major source of information and improved our understanding of how dysregulation of the complement system contributes to age-related macular degeneration (AMD) pathology. Drusen, the hall-mark of dry-type AMD are reported to be the by-product of complement mediated inflammatory processes. In wet AMD, unregulated complement activation results in increased production of angiogenic growth factors leading to choroidal neovascularization both in humans and in animal models. In this review article we have linked the complement system with modifiable and non-modifiable AMD risk factors as well as with prediction models of AMD. Understanding the association between the complement system, risk factors and prediction models will help improve our understanding of AMD pathology and management of this disease., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Garcinia lanceifolia Roxb. is an important and endemic medicinal plant of Assam which has been used by various ethnic communities of Northeast India to treat various disorders like dysentery, dyspepsia, and biliousness. The plant is considered to be containing much medicinal value and is also eaten raw or made into pickles by the local people. Our present study has been focused on the evaluation of the antibacterial activity of the methanolic extract of the bark of Garcinia lanceifolia which may lead us to a scientific evidence of the use of this plant in cases of dysentery and diarrhoea.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness. This study was done to characterize dry AMD-like changes in mouse retinal pigment epithelium (RPE) and retina after polyethylene glycol (PEG) treatment. We injected male C57BL/6 mice subretinally with PBS, 0.025, 0.25, 0.5 and 1.0 mg of PEG-400 and the animals were sacrificed on day 5. Eyes were harvested and processed for histological analysis. In all other experiments 0.5 mg PEG was injected and animals were sacrificed on days 1, 3, 5 or 14. Paraffin, 5 μm and plastic, 1 μm and 80 nm sections were used for further analysis. Subretinal injection of 0.5 mg PEG induced a 32% reduction of outer nuclear layer (ONL) thickness, 61% decrease of photoreceptor outer and inner segment length, 49% decrease of nuclear density in the ONL and 31% increase of RPE cell density by day 5 after injection. The maximum level of TUNEL positive nuclei in the ONL (6.8 + 1.99%) was detected at day 5 after PEG injection and co-localized with Casp3act. Histological signs of apoptosis were observed in the ONL by light or electron microscopy. Degeneration of RPE cells was found in PEG injected eyes. Gene expression data identified several genes reported to be involved in human AMD. C3, Cfi, Serping1, Mmp9, Htra1 and Lpl were up-regulated in PEG injected eyes compared to PBS controls. PEG leads to morphological and gene expression changes in RPE and retina consistent with dry AMD. This model will be useful to investigate dry AMD pathogenesis and treatment., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Dysregulation of the complement system is increasingly recognized as a contributing factor in age-related macular degeneration. Although the complement regulator CD46 is expressed ubiquitously in humans, in mouse it was previously thought to be expressed only on spermatozoa. We detected CD46 mRNA and protein in the posterior ocular segment (neuronal retina, retinal pigment epithelium, and choroid) of wild-type (WT) C57BL/6J mice. Cd46(-/-) knockout mice exhibited increased levels of the membrane attack complex and of vascular endothelial growth factor (VEGF) in the retina and choroid. The Cd46(-/-) mice were also more susceptible to laser-induced choroidal neovascularization (CNV). In Cd46(-/-) mice, 19% of laser spots were positive for CNV at day 2 after treatment, but no positive spots were detected in WT mice. At day 3, 42% of laser spots were positive in Cd46(-/-) mice, but only 11% in WT mice. A fully developed CNV complex was noted in both Cd46(-/-) and WT mice at day 7; however, lesion size was significantly (P < 0.05) increased in Cd46(-/-) mice. Our findings provide evidence for expression of CD46 in the mouse eye and a role for CD46 in protection against laser-induced CNV. We propose that the Cd46(-/-) mouse has a greater susceptibility to experimental CNV because of insufficient complement inhibition, which leads to increased membrane attack complex deposition and VEGF expression., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Age-related macular degeneration (AMD) is a leading cause of central blindness in the elderly population. The wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied the effects of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density were investigated by microscopy. Immunohistochemical staining (IHC) for von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: The purpose of this article is to evaluate the effect of nicotine on anti-vascular endothelial growth factor therapy in the treatment of neovascular age-related macular degeneration., Methods: One group of mice received nicotine in drinking water and the other group received water only. Choroidal neovascularization (CNV) was induced with a laser. Nicotinic acetylcholine receptor-α7 (nAChRα7) expression was evaluated by immunohistochemistry. Bevacizumab or adiponectin peptide II (APNpII) was injected intravitreally on Day 7 postlaser, and the effects were evaluated on Days 14 and 21. α-Bungarotoxin was injected intraperitoneally on Days 2 to 5, and its effect was evaluated on Day 14., Results: Expression of nAChRα7 was 2 to 7 times higher between Days 3 and 7 postlaser compared with naive mice. In water-fed mice, APNpII, bevacizumab, and α-bungarotoxin significantly reduced CNV size. In nicotine-fed mice, treatment with APNpII or bevacizumab did not significantly reduce CNV size, whereas α-bungarotoxin did have an effect. Comparing water- and nicotine-fed mice, CNV size was 61% to 86% smaller in water-fed mice except for the α-bungarotoxin group, where there was no difference. Platelet-derived growth factor and vascular endothelial growth factor expression was 1.5- to 2.5-fold higher at Day 14 in nicotine-treated mice., Conclusion: Nicotine significantly blocks the effect of anti-vascular endothelial growth factor therapy in the treatment of laser-induced neovascular age-related macular degeneration. nAChRα7 is significantly upregulated during the formation of CNV, and treatment with an nAChRα7 antagonist decreases CNV size irrespective of nicotine administration.

This study investigated the role of complement in the protection of retinal ganglion cells (RGCs) in chronic ocular hypertension model of glaucoma. Intraocular pressure (IOP) was elevated in the right eye of Lewis rats by laser photocoagulation (two treatments, 7days apart) of episcleral and limbal veins. Left eye did not receive laser treatment and served as control. Animals were injected with cobra venom factor every fifth day starting day 7 after first laser, to deplete the complement system. Animals were sacrificed at 6-week post-laser. Levels of C3 split products and membrane attack complex (MAC) were elevated in the retina of eyes with increased IOP and complement depletion reduced the loss of Brn3a(+) RGCs accompanied by decreased expression of GFAP and reduced MAC deposition. In complement depleted rats with increased IOP, reduced TUNEL(+) cells in ganglion cell layer, and decreased levels of active caspase-8 and active caspase-9 was observed compared to PBS treated complement sufficient rats with increased IOP. Interestingly, complement depletion also resulted in reduction of calcium influx and levels of BAD in the retinal cells of the eyes with increased IOP. Together, our results provide evidence that complement mediated apoptosis plays a pivotal role in the loss of RGCs in chronic ocular hypertension model of glaucoma., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Purpose:  To investigate the effect of topically applied honey on intact corneas, surgically induced corneal abrasions and endotoxin induced keratitis., Materials and Methods: The effect of honey on the cornea was investigated by application of honey on intact corneas, wounded corneas and endotoxin-induced keratitis in Lewis rats. The corneas were wounded by creating an epithelial defect using a surgical blade, and the keratitis was induced by topically applying Pseudomonas aeruginosa endotoxin to scarified corneas. After treatment rats were sacrificed and cornea harvested in each case. Corneas were processed for paraffin embedding for histological and immuno-fluorescence staining. Corneas were also harvested and processed for total ribonucleic acid (RNA) isolation for reverse transcriptase-polymerase chain reaction (RT-PCR) analysis for various growth factors and inflammatory chemokines/cytokines)., Results:  Histological analysis revealed that no inflammation or morphological changes occurred after honey treatment in naive intact corneas. Vascular endothelial growth factor (VEGF) levels were also not altered after honey treatment. Topical application of honey to injured corneas resulted in faster epithelial healing and decreased expression of VEGF, transforming growth factor beta (TGF-β), interferon gamma (IFN-γ), interleukin 12 (IL-12) and tumor necrosis factor alpha (TNF-α) in injured corneas. Our results also established that honey treatment reduced the inflammation in endotoxin-induced keratitis by reducing the levels of angiogenic factors (VEGF and TGF-β), inflammatory cytokines (IL-12) and chemokines (CC chemokine receptor 5(CCR-5))., Conclusion:  Short term use of honey on intact corneas can be safe. Honey has anti-angiogenic and anti-inflammatory properties that can be explored in several corneal inflammatory and infectious conditions.

The present study investigated the interactions among the complement membrane attack complex (MAC), CCL2, and VEGF that occur in vivo during the development of choroidal neovascularization (CNV). We first investigated the sequential expression of MAC, CCL2, and VEGF during laser-induced CNV in C57BL/6 mice. Increased MAC deposition was detected at 1 h, CCL2 increased at 3 h, and VEGF was up-regulated at day 3 post-laser treatment. These results suggested that during laser-induced CNV, MAC, CCL2 and VEGF are formed and/or expressed in the following order: MAC → CCL2 → VEGF. To determine the cross-talk between MAC, CCL2, and VEGF during laser-induced CNV, neutralizing antibodies were injected both systemically and locally to block the bioactivity of each molecule. Blocking MAC formation inhibited CCL2 and VEGF expression and also limited CNV formation, whereas neutralization of CCL2 bioactivity did not affect MAC deposition; however, it reduced VEGF expression and CNV formation. When bioactivity of VEGF was blocked, CNV formation was significantly inhibited, but MAC deposition was not affected. Together, our results demonstrate that MAC is an upstream mediator and effect of MAC on the development of laser-induced CNV can be attributed to its direct effect on VEGF as well as its effect on VEGF that is mediated by CCL2. Understanding the interplay between immune mediators is critical to gain insight into the pathogenesis of CNV.

In this study, we describe a new method for inducing choroidal neovascularization (CNV) in C57BL/6 mice, an animal model of wet age-related macular degeneration (AMD). AMD is a disease that causes central blindness in humans. We injected PEG-8 subretinally in different doses (0.125-2 mg) to induce CNV. After PEG-8 injection, we examined CNV at several time points (days 3-42). We also used Western blotting, immunohistochemistry, and ELISA to examine the complement component C3 split products, C9, VEGF, TGF-β2, and basic FGF. As early as day 1 after treatment, we found that a single subretinal injection of 1 mg of PEG-8 increased the C3 split products and the C9, TGF-β2, and basic FGF levels in the retinal pigment epithelium-choroid tissue. By day 3 after PEG-8 injection, the intraocular activation of the complement system caused induction and progression of CNV, including new vessels penetrating the Bruch's membrane. At day 5 after PEG-8 injection, we observed a fully developed CNV and retinal degeneration. Thus, in this study, we present a new, inexpensive, and accelerated mouse model of CNV that may be useful to study AMD.

The objective of the current study was to delineate the pathway of complement activation that is crucial for the induction of experimental autoimmune anterior uveitis (EAAU). We studied the development of EAAU in melanin-associated antigen (MAA)-sensitized Lewis rats treated with antibody against C4 or factor B. Control animals received isotype IgG control. Antibody against C4 had no effect on EAAU, and all of the animals developed EAAU similar to those injected with control IgG. In contrast, EAAU was completely inhibited in all MAA-sensitized Lewis rats injected with factor B antibody. Treatment with anti-factor B antibody resulted in suppression of ocular complement activation. Adoptive transfer of T lymphocytes harvested from draining lymph nodes of donor animals treated with anti-factor B did not transfer EAAU to naïve syngenic rats. Anti-factor B antibody inhibited the ability of MAA-specific CD4(+) T cells to proliferate (in vitro) in response to MAA in a dose-dependent manner. Level of TNF-α and IFN-γ decreased in the presence of anti-factor B. Collectively, our results provide the novel finding that complement activation via the alternative pathway contributes to intraocular inflammation in EAAU, and anti-factor B-mediated inhibition of EAAU is due to diminished antigen-specific CD4(+) T cell responses to MAA. Our findings explain the interactions between the complement system and T cells that are critical for the induction of EAAU and may lead to the development of therapy for idiopathic anterior uveitis based on selective blockade of the alternative pathway.

This study was initiated to explore the effect of recombinant rat Crry linked to the Fc portion of rat IgG2a (Crry-Ig) on the induction of experimental autoimmune anterior uveitis (EAAU) and on established disease. EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen (MAA). MAA sensitized animals received Crry-Ig, rat IgG2a (isotype control) or PBS separately before the onset of EAAU or after the onset of clinical disease. Administration of Crry-Ig suppressed the induction of EAAU while all animals injected with IgG2a or PBS developed the normal course of EAAU. Treatment with Crry-Ig resulted in the suppression of ocular complement activation as well as the functional activity of complement in the peripheral blood. At the peak of EAAU, levels of IFN-γ, IP-10, ICAM-1 and LECAM-1 were significantly reduced within the eyes of Crry-Ig treated Lewis rats. Importantly, administration of Crry-Ig even after the onset of EAAU resulted in a sharp decline in the disease activity and early resolution of EAAU. Collectively, the evidence presented here demonstrate that inhibition of complement by Crry-Ig results in low levels of inflammatory molecules-C3 activation products, MAC, cytokines, chemokines and adhesion molecules in the eye. Down-regulation of these molecules affects the infiltration and recruitment of inflammatory cells to the eye resulting in the inhibition of EAAU., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

This study was designed to explore the effect of recombinant, membrane-targeted CD59 (rCD59-APT542) on the growth and size of fully developed neovascular complex using the murine model of laser-induced choroidal neovascularization (CNV). CNV was induced by laser photocoagulation in C57BL/6 mice using an argon laser, and the animals received rCD59-APT542 via intravitreal (ivt) route. Western blot analysis, immunohistochemistry, and total complement hemolytic assay demonstrated that exogenously administered rCD59-APT542 was incorporated as well as retained in RPE and choroid and was functionally active in vivo. Single ivt injection during the growth of the CNV (i.e. at day 3 post-laser) resulted in ∼79% inhibition of the further growth of neovascular complex. The size of the CNV complex was significantly (p < 0.05) reduced by the administration of rCD59-APT542 after the CNV complex has fully developed (i.e. at day 7 post-laser). Treatment with rCD59-APT542 blocked the formation of membrane attack complex (MAC), increased apoptosis and decreased cell proliferation in the neovascular complex. On the basis of results presented here we conclude that recombinant membrane targeted CD59 inhibited the growth of the CNV complex and reduced the size of fully developed CNV in the laser-induced mouse model. We propose that a combination of two mechanisms: increased apoptosis and decreased cell proliferation, both resulting from local inhibition of MAC, may be responsible for inhibition of CNV by rCD59-APT542.

The objective of this study was to explore the relationship between local (ie, ocular) complement factor H (CFH) and choroidal neovascularization (CNV) associated with wet age-related macular degeneration (AMD), a leading cause of irreversible blindness, in laser-treated C57BL/6 mice. Immunohistochemical and RT-PCR analysis of retinal pigmented epithelium (RPE)-choroid sclera revealed that the expression of CFH was down-regulated on day 1 with a dramatic increase on days 5 and 7 postlaser injury. Flat mount and Western blot analysis further revealed that membrane attack complex (MAC) expression was up-regulated on days 1 and 3 postlaser injury; however, MAC was down-regulated on days 5 and 7 postinjury but was still higher than in non-injured mice. Similar patterns for CFH and MAC were observed for RPE cells when serial paraffin sections of the laser spots were analyzed. Subretinal injection of siRNA directed against CFH resulted in a threefold suppression of CFH in the RPE and choroid without affecting either CFH levels in the liver or the functional activity of the alternative pathway in the peripheral blood. Ocular knock-down of CFH resulted in increased MAC deposition, which leads to the early onset as well as exacerbation of laser-induced CNV. In conclusion, our findings provide evidence that CFH present on RPE and choroid regulates local MAC formation that is critical for the development of laser-induced CNV.

The objective of this study was to inhibit experimental autoimmune anterior uveitis (EAAU) by establishing antigen-specific immune tolerance in animals pre-sensitized with melanin-associated antigen (MAA). Intravenous administration of MAA on days 6, 7, 8 and 9 post-immunization induced tolerance and inhibited EAAU in all Lewis rats. The number of cells (total T cells, CD4(+) T cells and CD8(+) T cells) undergoing apoptosis dramatically increased in the popliteal lymph nodes (LNs) of the tolerized animals compared with non-tolerized animals. In addition, Fas ligand (FasL), TNF receptor 1 (TNFR1) and caspase-8 were upregulated in tolerized rats. Proliferation of total lymphocytes, CD4(+)T cells and CD8(+) T cells (harvested from the popliteal LNs) in response to antigenic stimulation was drastically reduced in the state of tolerance compared with the cells from non-tolerized animals. The level of interferon (IFN)-gamma and IL-2 decreased, whereas TGF-beta2 was elevated in the state of tolerance. Furthermore, the number of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) increased in the popliteal LNs of tolerized animals compared with non-tolerized animals. In conclusion, our results suggest that deletion of antigen-specific T cells by apoptosis and active suppression mediated by Tregs has an important role in the induction of antigen specific immune tolerance in animals with an established immune response against MAA.

This study was initiated to induce experimental autoimmune anterior uveitis (EAAU) in Lewis rats by melanin-associated antigen (MAA; 22-kDa fragment of type I collagen alpha2 chain) derived from rat iris and ciliary body (CB), to localize MAA within the eye, and to investigate the possible mechanism of MAA generation in vivo. The EAAU model replicates idiopathic human anterior uveitis. Lewis rats sensitized to rat MAA developed anterior uveitis, and EAAU induced by rat MAA can be adoptively transferred to naive syngenic rats by MAA-primed T cells. Animals immunized with rat MAA developed cellular immunity to the antigen. MAA was detected only in the iris and CB of the eye. Iris and CB were the major source of matrix metalloproteinase-1 (MMP-1) in the naive eye, and ocular expression of MMP-1 was up-regulated, whereas expression of tissue inhibitor of metalloproteinase 1 decreased before the onset of EAAU. These results demonstrated that EAAU can be induced by autologous MAA. Uveitogenic antigen is present only in the iris and CB of the eye, and the imbalance between MMP-1 and tissue inhibitor of metalloproteinase 1 may play a role in the generation of MAA in vivo. Collectively, the evidence presented here suggests that MAA is an autoantigen in EAAU. These observations may extend to idiopathic human anterior uveitis and facilitate the development of antigen-specific therapy.

We have investigated the effect of adiponectin (APN) peptide II on new vessel growth in mouse model of choroidal neovascularization (CNV) or wet type age-related macular degeneration (AMD). Mice were injected intraperitoneally with APN peptide II, control peptide, or PBS on day 1-7 or day 5-14. APN, AdipoR1, PCNA, and VEGF localization was investigated using confocal microscopy, immunohistochemistry, and RT-PCR. APN peptide II decreased the relative area of FITC-dextran perfused vessels by 4-fold, PCNA expression by 3-fold, and the number of PCNA stained HUVEC and MAVEC cells by 38 and 46%, respectively. We concluded that APN peptide II inhibits CNV size on days 7 and 14 by inhibiting the proliferation of endothelial cells in vivo and in vitro. APN peptide II may have therapeutic potential to inhibit CNV or wet AMD.

Experimental autoimmune anterior uveitis (EAAU) serves as an animal model for human idiopathic AU, the most common form of intraocular inflammation of significant morbidity whose recurrence can lead to permanent vision loss. This study was undertaken to inhibit EAAU by inducing tolerance to melanin-associated antigen (MAA) and to investigate the underlying mechanisms responsible for tolerance induction. Intravenous administration of MAA both induced tolerance and inhibited EAAU in Lewis rats. Flow cytometric analysis revealed that the proliferation of lymph node cells in response to antigenic stimulation was drastically reduced in the state of tolerance both in vivo and in vitro. Our results from co-culture experiments demonstrated that intravenous administration of MAA led to the generation of T-regulatory cells that suppress T-cell proliferative responses and induce tolerance. Expression levels of both interleukin-10 and transforming growth factor-beta2 were elevated whereas reduced levels of tumor necrosis factor-alpha, interferon-gamma, and interleukin-2 were detected in tolerance-induced animals. Tolerance was reversed by replenishing these animals with recombinant interleukin-2. Tolerance could be adoptively transferred by removing lymph node cells from tolerance-induced donors and giving them to recipient rats. Interestingly, adoptive transfer of tolerance failed when lymph nodes cells were depleted of CD4(+)CD25(+) T cells. In conclusion, T-cell nonresponsiveness because of active suppression mediated by T-regulatory cells facilitates the development of tolerance to MAA in EAAU.

The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.

Functionally active complement system and complement regulatory proteins are present in the normal human and rodent eye. Complement activation and its regulation by ocular complement regulatory proteins contribute to the pathology of various ocular diseases including keratitis, uveitis and age-related macular degeneration. Furthermore, a strong relationship between age-related macular degeneration and polymorphism in the genes of certain complement components/complement regulatory proteins is now well established. Recombinant forms of the naturally occurring complement regulatory proteins have been exploited in the animal models for treatment of these ocular diseases. It is hoped that in the future recombinant complement regulatory proteins will be used as novel therapeutic agents in the clinic for the treatment of keratitis, uveitis, and age-related macular degeneration.

Cellular oxidative stress plays an important role in retinal pigment epithelial (RPE) cell death during aging and the development of age-related macular degeneration. Early reports indicate that during phagocytosis of rod outer segments, there is an increase of RPE oxidative stress and an upregulation of PPARgamma mRNA in these cells. These studies suggest that activation of PPARgamma may modulate cellular oxidative stress. This paper presents a brief review of recent studies that investigate RPE oxidative stress under various experimental conditions. This is followed by a detailed review on those reports that examine the protective effect of the natural PPARgamma ligand, 15d-PGJ(2), against RPE oxidative stress. This agent can upregulate glutathione and prevent oxidant-induced intracellular reactive oxygen species accumulation, mitochondrial depolarization, and apoptosis. The cytoprotective effect of this agent, however, is not shared by other PPARgamma agonists. Nonetheless, this property of 15d-PGJ(2) may be useful in future development of pharmacological tools against retinal diseases caused by oxidative stress.

Purpose: Experimental autoimmune anterior uveitis (EAAU) serves as an animal model of human idiopathic anterior uveitis. This study was undertaken to investigate the role of apoptosis in the resolution of EAAU., Methods: EAAU was induced in Lewis rats by bovine melanin-associated antigen (MAA). Animals were killed at different time points during EAAU, and apoptosis of the inflammatory cells within the eye was monitored., Results: Flow cytometry, TUNEL staining, and light microscopy demonstrated that CD11b/c(+) and CD4(+) T cells undergo apoptosis during EAAU. Electron microscopic analysis demonstrated that the macrophages remove these apoptotic infiltrating cells from the eye by phagocytosis. Caspase-3 levels peaked during the resolution of EAAU, and the upregulation of caspase-8 and -9 preceded that of caspase-3, suggesting that both extrinsic and intrinsic pathways of apoptosis are involved. There was an inverse relationship between the expression of proapoptotic protein Bax and antiapoptotic protein Bcl-2 during EAAU. Cytochrome c was present in the cytoplasm of the infiltrating cells undergoing apoptosis., Conclusions: These results demonstrate that extrinsic and intrinsic pathways of apoptosis are involved in the resolution of EAAU. They further suggest that apoptosis followed by phagocytosis plays a critical role in the clearance of infiltrating cells from eyes with uveitis and leads to the resolution of EAAU.

In the normal eye, the complement system is continuously activated at low levels and both membrane-bound and soluble intraocular complement regulatory proteins tightly regulate this spontaneous complement activation. This allows protection against pathogens without causing any damage to self-tissue and vision loss. The complement system and complement regulatory proteins control the intraocular inflammation in autoimmune uveitis and play an important role in the development of corneal inflammation, age-related macular degeneration and diabetic retinopathy. The evidence derived from both animal models and patient studies support the concept that complement inhibition is a relevant therapeutic target in the treatment of various ocular diseases. Currently, several clinical trials using complement inhibitors are going on. It is possible that, in the near future, complement inhibitors might be used as therapeutic agents in eye clinics.

The aim of this study was to investigate the role of adiponectin (APN) in a mouse model of laser induced choroidal neovascularization (CNV). We have shown by immunohistochemistry that the expression of APN, adiponectin receptor 1, adiponectin receptor 2 and T cadherin gradually increased from day 1 to day 7 post-laser in laser treated mice compared to controls. Recombinant APN (rAPN) was injected intraperitoneally (i.p., 25 microg/mouse) or intravitreally (2 microg/eye) in lasered mice. Another set of lasered mice received APN peptide via i.p. (75 microg/mouse) or intravitreal (30 microg/eye) route. Control mice received a similar treatment with PBS, control protein or control peptide after laser treatment. We found that in the i.p. and intravitreal injection of rAPN resulted in 78% and 68% inhibition respectively in the size of CNV complex compared to control mice. Similar results were observed when APN peptide was injected intravitreally or i.p. Treatment with rAPN or the peptide resulted in decreased levels of vascular endothelial growth factor. Thus, APN inhibited choroidal angiogenesis and may have therapeutic implications in the treatment of wet age related macular degeneration.

We have shown that membrane attack complex (MAC) formation via the activation of the alternative pathway plays a central role in the laser-induced choroidal neovascularization (CNV). This study was undertaken to understand the role of a complement regulatory protein, CD59, which controls MAC assembly and function, in this model. CNV was induced by laser photocoagulation in C57BL/6 and Cd59a(-/-) mice using an argon laser. Animals from each group were sacrificed on day 1, 3, 5, and 7 postlaser. Retinal pigment epithelium-choroid-scleral tissue was examined to determine the incidence and size of CNV complex, and semiquantitative RT-PCR and Western blot analysis for CD59a was studied. Recombinant soluble mouse CD59a-IgG2a fusion (rsCD59a-Fc) protein was injected via i.p. or intravitreal routes 24 h before laser. Our results demonstrated that CD59a (both mRNA and protein) was down-regulated during laser-induced CNV. Cd59a(-/-) mice developed CNV complex early in the disease process. Increased MAC deposition was also observed in these Cd59a(-/-) mice. Administration of rsCD59a-Fc inhibited the development of CNV complex in the mouse model by blocking MAC formation and also inhibited expression of angiogenic growth factors. These data provide strong evidence that CD59a plays a crucial role in regulating complement activation and MAC formation essential for the release of growth factors that drive the development of laser-induced CNV in mice. Thus, our results suggest that the inhibition of complement by soluble CD59 may provide a novel therapeutic alternative to current treatment.

Uveitis is a general term for inflammatory disorders of the uveal tract and encompasses a wide range of underlying etiologies. It may be idiopathic, associated with systemic diseases or result from a variety of infectious agents. Uveitis is responsible for over 2.8% of blindness in the United States. Each year, 17.6% of active uveitis patients experience a transient or permanent loss of vision. Anterior uveitis (AU), which refers to inflammation within the anterior segment of the eye, is the most common form of uveitis. Experimental autoimmune AU (EAAU) is an organ-specific autoimmune disease of the eye, which serves as an animal model of idiopathic human AU. Recently, type I collagen was identified as the target autoantigen in EAAU. Thus, human AU may be an e xample of autoimmunity to local ocular collagen.

The complement system is a major component of innate immunity. During an inflammatory reaction, the eye is potentially threatened by homologous complement attack, and unregulated complement activation could lead to tissue damage and vision loss. The complement system is continuously activated at low levels in the normal eye, and intraocular complement-regulatory proteins (CRPs) tightly regulate this spontaneous complement activation so that there is elimination of potential pathogens without the induction of destructive intraocular inflammation. The presence of a complement activation product (iC3b) during the early phase of antigen and antigen-presenting cell contact is essential for the induction of systemic tolerance to antigen injected into the anterior chamber of the eye and the establishment of ocular immune privilege. The complement system and complement-regulatory proteins control intraocular inflammation in autoimmune anterior uveitis and may play an important role in the development of age-related macular degeneration. Thus, in the eye, complement functions as a double-edged sword - on one hand it provides innate immunity against pathogens while simultaneously instructing the adaptive immune response to develop tolerance to such pathogens to avoid inadvertent tissue damage in a critical organ.

The objective of this study was to explore the role of classical, lectin, and alternative pathways of complement activation in laser-induced choroidal neovascularization (CNV). The classical and alternative pathways were blocked in C57BL/6 mice by small interfering RNAs (siRNA) directed against C1q and factor B, respectively. C4(-/-) mice developed CNV similar to their wild-type controls and inhibition of C1q by siRNA had no effect on the development of CNV. In contrast, CNV was significantly inhibited (p < 0.001) in C5(-/-) mice and C57BL/6 mice treated with factor B siRNA. Inhibition of the alternative pathway by factor B siRNA resulted in decreased levels of membrane attack complex and angiogenic factors-vascular endothelial growth factor and TGF-beta2. Furthermore, factor B was up-regulated in complement sufficient C57BL/6 mice at day 1 postlaser and remained elevated at day 7. Significantly reduced levels of factor H were observed at day 3 in these animals. In conclusion, our results demonstrate that activation of the factor B-dependent alternative pathway, but not the classical or lectin pathways, was essential for the development of CNV in mouse model of laser-induced CNV. Thus, specific blockade of the alternative pathway may represent a therapeutically relevant strategy for the inhibition of CNV.

This study was undertaken to explore the role of complement regulatory proteins (CRPs) in experimental autoimmune anterior uveitis (EAAU). We observed that the levels of CRPs, Crry and CD59, in the eyes of Lewis rats increased during EAAU and remained elevated when the disease resolved. The in vivo role of these CRPs in EAAU was explored using neutralizing mAbs, antisense oligodeoxynucleotides (AS-ODNs), and small interfering RNAs against rat Crry and CD59. Suppression of Crry in vivo at days 9, 14, or 19 by neutralizing mAb or AS-ODNs resulted in the early onset of disease, the exacerbation of intraocular inflammation, and delayed resolution. Suppression of CD59 was only effective when the Abs and ODNs were given before the onset of disease. The most profound effect on the disease was observed when a mixture of Crry and CD59 mAbs or AS-ODNs was administered. A similar effect was observed with a combination of Crry and CD59 small interfering RNA. There was no permanent histologic damage to ocular tissue after the inflammation cleared in these animals. Increased complement activation as determined by increased deposition of C3, C3 activation fragments, and membrane attack complex was observed in the eyes of Lewis rats when the function and/or expression of Crry and CD59 was suppressed. Thus, our results suggest that various ocular tissues up-regulate the expression of Crry and CD59 to avoid self-injury during autoimmune uveitis and that these CRPs play an active role in the resolution of EAAU by down-regulating complement activation in vivo.

One of the pathologic complications of exudative (i.e. wet-type) age-related macular degeneration (AMD) is choroidal neovascularization (CNV). The aim of this study was to investigate whether chronic and heavy alcohol consumption influenced the development of CNV in a rat model. The oxidative metabolism of alcohol is minimal or absent in the eye, so that ethanol is metabolized via a nonoxidative pathway to form fatty acid ethyl esters (FAEE). Fatty acid ethyl ester synthase (FAEES) was purified from the choroid of Brown Norway (BN) rats. The purified protein was 60 kDa in size and the antibody raised against this protein showed a single band on western blot. BN rats on a regular diet were fed alcohol for 10 weeks. Control rats were fed water with a regular diet and pair-fed control rats were fed regular diet, water and glucose. We found that FAEES activity was increased 4.0-fold in the choroid of alcohol-treated rats compared with controls. The amount of ethyl esters produced in the choroid of 10 week alcohol-fed rats was 7.4-fold more than rats fed alcohol for 1 week. The increased accumulation of ethyl esters was associated with a 3.0-fold increased expression of cyclin E and cyclin E/CDK2; however, the level of the cyclin kinase inhibitor, p27Kip, did not change. The increased accumulation of ethyl esters was also associated with 3.0-fold decreased expression of APN in the choroid. We also found that the size of CNV increased by 28% in alcohol-fed rats. Thus, our study showed that chronic, heavy alcohol intake was associated with both an increased accumulation of ethyl esters in the choroid and an exacerbation of the CNV induced by laser treatment. These results may provide insight into the link between heavy alcohol consumption and exudative AMD.

Purpose: The role of complement in ocular autoimmunity was explored in a experimental autoimmune anterior uveitis (EAAU) animal model., Methods: EAAU was induced in Lewis rats by immunization with bovine melanin-associated antigen. Complement activation in the eye was monitored by Western blot for iC3b. The importance of complement to the development of EAAU was studied by comparing the course of intraocular inflammation in normal Lewis rats (complement-sufficient) with cobra venom factor-treated rats (complement-depleted). Eyes were harvested from both complement-sufficient and complement-depleted rats for mRNA and protein analysis for IFN-gamma, IL-10, and interferon-inducible protein (IP)-10. Intracellular adhesion molecule (ICAM)-1 and leukocyte-endothelial cell adhesion molecule (LECAM)-1 were detected by immunofluorescent staining. OX-42 was used to investigate the importance of iC3b and CR3 interaction in EAAU., Results: There was a correlation between ocular complement activation and disease progression in EAAU. The incidence, duration, and severity of disease were dramatically reduced after active immunization in complement-depleted rats. Complement depletion also completely suppressed adoptive transfer EAAU. The presence of complement was critical for local production of cytokines (IFN-gamma and IL-10), chemokines (IP-10), and adhesion molecules (ICAM-1 and LECAM-1) during EAAU. Furthermore, intraocular complement activation, specifically iC3b production and engagement of complement receptor 3 (CR3), had a significant impact on disease activity in EAAU., Conclusions: The study provided the novel finding that complement activation plays a central role in the pathogenesis of ocular autoimmunity and may serve as a potential target for therapeutic intervention.