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ABSTRACT: Reactive nitrogen species, in particular, peroxynitrite (ONOO(-)) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO(-) has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin--apparent in both control and EIU eyes--was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood-aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO(-) in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.
Experimental Eye Research 05/2002; 74(4):463-71. · 3.26 Impact Factor
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ABSTRACT: To evaluate the use of an intravitreal sustained-release cyclosporine (CsA) delivery device for treatment of horses with naturally occurring recurrent uveitis.
16 horses with recurrent uveitis.
Horses with frequent recurrent episodes of uveitis or with disease that was progressing despite appropriate medication were selected for this study. Additional inclusion criteria included adequate retinal function as determined by use of electroretinography, lack of severe cataract formation, and no vision-threatening ocular complications (eg, retinal detachment, severe retinal degeneration, and posterior synechia). Sustained-release CsA delivery devices (4 microg of CsA/d) were implanted into the vitreous through a sclerotomy at the pars plana. Reexaminations were performed 1, 3, 6, and 12 months after implantation, then continued annually. Ophthalmic changes, number of recurrent episodes of uveitis, and vision were recorded.
The rate of recurrent episodes after device implantation (0.36 episodes/y) was less than prior to surgery (75 episodes/y). In addition, only 3 horses developed episodes of recurrent uveitis after surgery. Vision was detected in 14 of 16 affected eyes at a mean follow-up time of 13.8 months (range, 6 to 24 months).
This intravitreal sustained-release CsA delivery device may be a safe and important tool for long-term treatment of horses with chronic recurrent uveitis.
American Journal of Veterinary Research 01/2002; 62(12):1892-6. · 1.27 Impact Factor
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ABSTRACT: The purpose of this study was to determine the effects of an intravitreal device releasing cyclosporine A (CsA) on recurrent inflammatory episodes in experimental uveitis. Nine normal horses were immunized peripherally with H37RA-mTB antigen twice, and then received 25 microg of H37RA-mTB antigen intravitreally in the right eye and an equal volume of balanced salt solution intravitreally in the left eye. Two weeks later, the animals randomly received either a CsA or a polymer implant (without CsA) in both eyes 1 week following implantation of the devices, 25 microg of H37RA-mTB antigen was reinjected into the right eye of each animal. Clinical signs of ophthalmic inflammation were graded following injections and implantation. The animals from each group were euthanized at 3, 14, and 28 days following the second injection. Aqueous and vitreous humor protein concentrations were measured. The presence, number, and type (CD4, 5 and 8) of infiltrating inflammatory cells and amount of tissue destruction were determined. Total RNA was isolated and quantitative reverse transcriptase-polymerase chain reaction was performed for equine specific interleukin (IL) 2 and 4, interferon-gamma (IFN gamma) and beta-actin. In addition, aqueous and vitreous humor and peripheral blood were collected at the termination of the experiments and analyzed for CsA concentration by HPLC. Within 4h of the first intravitreal H37RA-mTB antigen injection, each animal developed epiphora, blepharospasm, mild corneal edema, aqueous flare, myosis, and vitreous opacity. The severity of signs peaked 48 to 72 h after injection and subsequently decreased back to normal within 14 days. Following the second injection, clinical signs in the eyes with the CsA device were less severe and significantly shorter in duration than signs with the polymer only implant eyes. Aqueous and vitreous humor protein levels, infiltrating cell numbers, total number of T-lymphocytes, and levels of IL-2 and IFN gamma-mRNA were significantly less in eyes with the CsA implant compared to eyes with the polymer only. CsA implants did not completely eliminate the development of a second ('recurrent') experimental inflammatory episode in these horses. However, the duration and severity of inflammation, cellular infiltration, tissue destruction, and pro-inflammatory cytokines RNA transcript levels were significantly less in those eyes implanted with the CsA device.
Veterinary Immunology and Immunopathology 11/2000; 76(3-4):239-55. · 2.08 Impact Factor
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ABSTRACT: OBJECTIVE: To determine the long-term toxicity of an intravitreal device releasing continuous cyclosporinee A (CsA) in normal eyes of horses by evaluating clinical signs, electroretinography, and histopathology. Animals Studied Ten adult horses with normal ophthalmic examinations were used in this study Procedure(s) Four horses had one eye implanted with a CsA device, and six horses had the right eye implanted with a CsA-containing device (10 eyes with CsA in total) and the left eye (six eyes in total) with the device without drug (control). The implants were placed in the vitreous of the eyes through a sclerotomy 1 cm posterior to the limbus in the dorso-temporal quadrant of the eye. Scotopic electroretinograms were performed prior to implantation and at 1 week, and at 1, 3, 6, 9, and 12 months postimplantation. Two of the unilaterally implanted horses were euthanized at 1 weeks postimplantation, and two at 6 weeks postimplantation. Two of the bilaterally implanted horses were euthanized at 6 months, two at 9 months, and two at 12 months postimplantation. At euthanasia, the eyes were removed, aqueous and vitreous humor aspirated, and tissues fixed in 10% buffered formalin and processed for histopathology. CsA concentrations were measured by high pressure liquid chromatography in the aqueous and vitreous humors, and in peripheral blood. RESULTS: The devices were tolerated well in 14 of 16 eyes. There was minimal postoperative inflammation in most eyes, with a normal appearance within 7 days. In two eyes implanted with the CsA device, severe inflammation resulted in phthisis bulbi by 28 days. One of these eyes exhibited suspected bacterial endophthalmitis, and one had a sterile endophthalmitis and cataract presumably from trauma to the lens during implantation. In the other 14 eyes, no change was observed in the scotopic electroretinograms (ERG) from preoperative results, and no significant differences between the right (CsA) and left (control device) eyes were observed. CsA levels in the aqueous and vitreous humor, and peripheral blood were below the detection limit of the HPLC. Histologic findings revealed only a mild lymphoplasmacytic cellular infiltrate in the ciliary body and pars plana near the implantation site. CONCLUSIONS: The CsA devices were well tolerated with no long-term complications from the implants themselves. However, complications may occur from inadvertent implantation trauma or contamination during surgery. The long-term safety of the device may make it useful for delivery of CsA in the control of equine recurrent uveitis.
Veterinary Ophthalmology 02/2000; 3(2-3):105-110. · 0.75 Impact Factor
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ABSTRACT: Equine recurrent uveitis (ERU), a chronic, recurrent inflammation primarily of the anterior uveal tract, is the most common cause of blindness in horses. Recently, T-lymphocytes have been found to be the most numerous cell type to infiltrate the anterior uveal of horses with ERU. In the present study, we characterized the T-lymphocyte population in the anterior uveal tract of eyes of horses with chronic ERU by evaluating the microscopic appearance (histopathologic features), the T-lymphocyte subsets, and the relative levels and amounts of T-lymphocyte cytokine mRNA in the anterior uvea. Seven inflamed eyes (from six horses with chronic ERU) and 5 normal eyes (from five horses with nonocular problems) were studied. After clinical examination, the eyes were removed, ocular fluids were aspirated, and anterior uveal tissues (iris and ciliary body) were processed for histologic and molecular (RNA isolation) analyses. Histologic examination by hematoxylin and eosin (H and E) staining and immunohistochemistry evaluating T-lymphocyte subsets (anti-CD4, CD8, CD5) were performed for each sample. RNA samples were analyzed for levels of messenger (m) RNA specific for interleukin (IL)-2, 4, and interferon-gamma (IFNgamma) by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR). Eyes with ERU exhibited characteristic clinical signs, including corneal edema, aqueous flare, posterior synechia, corpora nigra degeneration, and cataract formation. Histologically, infiltration of the uveal tract with lymphocytes, plasma cells, and macrophages was most evident in the ciliary body and base of the iris. Loss of tissue structure (destruction) was most evident in the ciliary processes. Infiltrating lymphocytes were predominantly CD4+ T-cells (e.g. 48% CD4+ and 18% CD8+ in the ciliary body stroma), as determined by immunohistochemistry. Few inflammatory cells were observed in the normal eyes. The QRT-PCR results revealed increased transcription of IL-2 and IFNgamma and low IL-4 mRNA expression in eyes with chronic ERU compared to normal eyes, demonstrating a Thelper (Th) 1-like inflammatory response in eyes with ERU.
Veterinary Immunology and Immunopathology 11/1999; 71(1):17-28. · 2.08 Impact Factor
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ABSTRACT: To determine whether nuclear transcription factor-kappaB (NF-kappaB) is activated in human retinal pigment epithelial (hRPE) cells in response to interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha), or interferon-gamma (IFN-gamma) alone or in combination and if so, whether expression of proinflammatory genes induced by these agents can be blocked by a proteasome inhibitor, MG-132, which inhibits the degradation of I kappaB, an NF-kappaB inhibitor, thereby preventing nuclear translocation of NF-kappaB.
Cultured hRPE were pretreated for 60 minutes with medium alone or medium containing the proteasome inhibitor MG-132 (20 microM) and then exposed to TNF-alpha (1.1 x 10(3) U/ml), IL-1beta (5 U/ml), or IFN-gamma (7.5 x 10(3) U/ml) alone or in combination (TII). Nuclear translocation of NF-kappaB was determined by fluorescence staining of the NF-kappaB Rel A (p65) subunit. Cytoplasmic I kappaB protein was measured by western blot analysis. Nuclear extract binding to kappaB DNA motifs was measured by electrophoretic mobility shift assay and antibody supershift assay. Steady state mRNA expression of the chemokines melanoma growth stimulating activity (MGSA)/gro-alpha, regulated on activation normal T-cell expression and secreted (RANTES), and monocyte chemoattractant protein (MCP-1), the cytokines IL-1beta and macrophage colony stimulating factor (M-CSF) and intercellular adhesion molecule-1 (ICAM-1) was evaluated by semiquantitative reverse transcription-polymerase chain reaction. Chemokine and cytokine protein secretion was measured by enzyme-linked immunosorbent assay. Cell-surface ICAM-1 expression was determined by flow cytometry.
TNF-alpha, IL-1beta, and TII but not IFN-gamma alone caused degradation of I kappaB, Rel A nuclear translocation, and increased NF-kappaB DNA binding activity, effects that were blocked by pretreatment with MG-132. MG-132 suppressed MGSA/gro-alpha, RANTES, MCP-1, IL-1beta, M-CSF, and ICAM-1 mRNA expression and secreted RANTES, MCP-1, and M-CSF protein, and cell-surface ICAM-1 that were induced by IL-1beta, TNF-alpha, and TII.
TNF-alpha, IL-1beta, and TII induce expression of proinflammatory cytokines and ICAM-1 in hRPE cells through an NF-kappaB-dependent signal transduction pathway. This effect is blocked by MG-132, a proteasome inhibitor that prevents I kappaB degradation. Inhibition of NF-kappaB may be a useful strategy to treat proliferative vitreoretinopathy and uveitis, ocular diseases initiated and perpetuated by cytokine activation.
Investigative Ophthalmology & Visual Science 03/1999; 40(2):477-86. · 3.60 Impact Factor
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ABSTRACT: Recent studies have implicated nitric oxide and peroxynitrite in the pathogenesis of many diseases, such as septic shock, arthritis, lung disease, and atherosclerosis. Nitric oxide (.NO) exerts many diverse effects on vascular tone, affecting neurotransmission and cellular cytotoxicity/communication. Our laboratory and others have documented a proinflammatory role for .NO in ocular inflammation. Uveitis, which is an inflammation of the highly vascular uveal tract in the eye, is a debilitating condition that can lead to visual impairment and blindness. It is characterized by acute, recurrent, or persistent inflammation with disruption of the blood-aqueous barrier and is accompanied by protein leakage and leukocyte infiltration into the aqueous humor and anterior chamber. Systemic injection of endotoxin into mice and rats, or intraocular injection of endotoxin into mice, rats, and rabbits induces acute uveitis, which clinically and histologically resembles acute anterior uveitis in humans. These models facilitate the study of pathogenic mechanisms that contribute to ocular inflammation. In addition to .NO, superoxide anion radicals (O2.-), and peroxynitrite (ONOO-), the products of the reaction between .NO and O2.-, are also implicated in uveitis. The role of peroxynitrite in ocular inflammation is still largely unknown. Characterization of the roles of these important uveitic mediators in the ocular inflammatory response will provide information critical to the understanding of the pathogenesis of intraocular inflammation so that more effective therapeutic intervention(s) can be developed.
Environmental Health Perspectives 11/1998; 106 Suppl 5:1145-9. · 7.04 Impact Factor
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ABSTRACT: Transforming growth factor-beta 2 (TGF-beta 2) is a pluripotent cytokine which has been suggested to play a number of roles in ocular physiologic and pathologic states. Intraocular fluid (i.o.f.) levels of TGF-beta 2 are quite high. Although the sources of ocular TGF-beta are not completely defined, the retinal pigment epithelium, the epithelium of the ciliary body and trabecular meshwork cells all secrete it. In this study we utilized canine lens and rabbit ciliary pigmented epithelial cell cultures to quantitate the in vitro secretion of TGF-beta 2. In addition, the effects of aphakia or the presence of cataractous lenses on IOF TGF-beta 2 levels were determined.
Lens and ciliary body epithelial cell culture supernatants and aqueous humors were assayed for total TGF-beta 2 levels by ELISA and bioassay.
TGF-beta 2 accumulated in the media bathing lens epithelial cell cultures (0.7 +/- 0.03 ng/ml at day 2) and ciliary pigmented epithelial cell cultures (0.8 +/- 0.06 ng/ml at day 2) in a time-dependent manner. Surprisingly, aqueous humor from aphakic rabbit eyes contained significantly higher levels of TGF-beta 2 than their contralateral phakic controls. Furthermore, aqueous humor from canine eyes with cataracts also contained significantly higher levels of TGF-beta 2 than normal eyes.
These results suggest that the lens secretes TGF-beta 2 and that the presence and status of the lens may influence IOF TGF-beta 2 levels.
Albrecht von Graæes Archiv für Ophthalmologie 05/1998; 236(4):305-11. · 2.17 Impact Factor
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Veterinary Ophthalmology 02/1998; 1(4):181-187. · 0.75 Impact Factor
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ABSTRACT: Nitric oxide (NO) is a highly reactive radical which plays an integral role in physiological and pathophysiological processes. NO is produced endogenously in small amounts by a constitutive NO synthase (cNOS) as a regulator of vascular tone and neurotransmission. NO can also be produced in large amounts by an inducible NOS (iNOS) in response to endotoxin and cytokines, and has been reported to be a mediator of lipopolysaccharide (LPS)-induced uveitis in rats. The purpose of the present study was to investigate the effects of NOS inhibitors with different NOS isoform specificities in the rabbit model of endotoxin-induced ocular inflammation. LPS and/or inhibitors of NOS. NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG), were injected intravitreally and the eyes observed by slit lamp for 24 hr. Coinjection of LPS with L-NAME inhibited anterior inflammation in rabbits. Iridal hyperemia (IH) and aqueous flare (AF) were completely abolished in eight out of nine rabbits in a dose-dependent manner. In addition, total cell counts were significantly suppressed (7393 +/- 697 vs. 325 +/- 188, P < 0.05) and aqueous protein levels were reduced to near control levels (25 +/- 0.75 vs. 1.72 +/- 0.36, P < 0.05). Similar suppression was seen with AG (cell counts = 351 +/- 246 and proteins = 3.1 +/- 1.2). Administration of L-NAME 0.5 hr after LPS injection suppressed inflammation to a lesser extent than coinjection. In contrast, administration of L-NAME 6 hr after LPS injection was not inhibitory, and in fact significantly increased cellular infiltration. However, AG given 6 hr after LPS had a remarkably different effect, since it significantly decreased both protein extravasation and cellular infiltration into the aqueous humor. In fact, our results suggest that cNOS may play a greater role in the earlier stages of this developing inflammatory response. These results extend others' observations that NO is a key mediator in uveitis, that induction of iNOS plays a critical role in experimental uveitis, and suggest that NO has a complex role in the ocular inflammatory process. Inhibitors of NOS can abort the LPS-induced inflammatory response if administered early enough, but could potentially exacerbate an established inflammatory episode.
Experimental Eye Research 01/1996; 62(1):21-8. · 3.26 Impact Factor
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ABSTRACT: Transforming growth factor-beta (TGF-beta), a multifunctional cytokine which has been identified in normal and inflamed ocular fluids, may play a role in the evolution of inflammatory ocular lesions. In this study we utilized a rabbit model of LPS-induced uveitis to determine if exogenous TGF-beta 2 could alter its course. Recombinant TGF-beta 2 (1-2000 ng), LPS (10 or 20 ng), or TGF-beta 2 (100 ng) plus LPS (10 ng) were injected intravitreally in one eye of a New Zealand white rabbit and the contralateral eye served as a paired control which received an equal volume of vehicle. The uveitic response was assessed by biomicroscopic examination of the anterior uvea and analysis of protein and cells in the aqueous humor. Ocular tissues were processed for histologic, immunohistochemical and in situ hybridization analyses. Rabbits injected with doses of TGF-beta 2 > or = 500 ng developed a mild uveitic response, compared to LPS alone, accompanied by expression of IL-1 beta mRNA and protein in the anterior uvea. Interestingly, rabbits coinjected with LPS (10 ng) and a nonuveitic dose (100 ng) of TGF-beta 2 exhibited a similar increase in ocular vascular permeability, but a decrease in inflammatory cell infiltration into the anterior uvea and aqueous humor (1185 +/- 117 versus 2465 +/- 176; p < 0.05). No evidence of inflammation was observed in eyes injected with 100 ng TGF-beta 2 alone. Similar to other models of inflammation, TGF-beta may interrupt the cascade of events leading to ocular inflammation, thereby suggesting therapeutic potential.
Current Eye Research 01/1996; 15(1):95-103. · 1.28 Impact Factor
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ABSTRACT: Reactive nitrogen species, in particular, peroxynitrite (ONOO−) have been proposed to play an important role in the pathogenesis of endotoxin-induced uveitis (EIU). Tyrosine nitration by ONOO− has been shown in other model systems to inhibit the activity of the superoxide anion quenching enyzme, manganese superoxide dismutase (MnSOD), perhaps contributing to progression of disease. In this study, it is confirmed through immunoanalysis that nitrated proteins are produced during EIU, and furthermore, that MnSOD is a target of nitration during the inflammatory response. In addition, through microsequencing analyses, nitrated albumin—apparent in both control and EIU eyes—was identified. Positive immunostaining of nitrated proteins was seen in the ciliary epithelium, inflammatory cells, and protein exudate of eyes from rats injected with endotoxin. Incubation of nitrotyrosine immunoprecipitates from the iris and ciliary body (ICB) with a polyclonal antibody against MnSOD revealed that nitrated MnSOD was present only in the ICB of EIU rats. When the total activity of the enzyme was examined, it was observed that despite the presence of nitrated MnSOD, activity was increased relative to control. Analysis of MnSOD mRNA and protein from the ICB of both groups demonstrated an increase in mRNA expression and consequently a three- to five-fold increase in MnSOD protein in EIU rats as compared to control rats. Further examination of MnSOD protein expression through immunohistochemistry noted enhanced immunostaining in the ciliary epithelium of eyes of EIU rats. Additional investigation of a 70 kDa band apparent in nitrotyrosine immunoprecipitates from the ICB of control and EIU rats revealed that the plasma protein albumin is nitrated as well. This protein is present as a result of the breakdown of the blood–aqueous barrier during inflammation. In summary, two endogenous nitration targets, albumin and MnSOD, were identified. Nitrated MnSOD appears to be specifically targeted to the ICB during inflammation, underscoring the importance of the interface in EIU. Furthermore, the expression and activity of the enzyme is increased in the ICB during EIU, perhaps regulating reactive nitrogen species produced within the cells. This study implicates ONOO− in the pathogenesis of EIU and imparts the putative role MnSOD plays in disease resolution.
Experimental Eye Research.
