Role of crystallins in ocular neuroprotection and axonal regeneration.

Neuroprotection is an emerging challenge in ophthalmology due to the particularly exposed location of retinal neurons and to the steadily increasing rate of intraocular surgical and pharmacological treatments applied to various eye diseases. Within few decades neuroprotection has developed from strongly contested approaches to being recognized and introduced as a potentially clinical application. One of the groups of putative substances for neuroprotection comprises αA- and αB-crystallins, which are types of heat-shock proteins and are considered to be molecular chaperones. The ß/γ-crystallins form their own superfamily and are characterized as proteins with a distinct structure containing four Greek key motifs. Besides being abundant in the ocular lens, crystallins are also expressed in both the developing and mature retina. Crystallins are dramatically up-regulated in numerous retinal pathologies, including mechanical injury, ischemic insults, age-related macular degeneration, uveoretinitis, and diabetic retinopathy. Crystallins of the α family are thought to play a crucial role in retinal neuron survival and inflammation. Crystallins of the ß/γ superfamily are also small proteins with a possible emerging role in retinal tissue remodeling and repair. One of the typical retinal diseases associated with crystallins is the experimental glaucomatous neuropathy that is characterized by their expression. Another typical retinal disease is the atrophy that occurs after mechanical injury to the optic nerve, which is associated with the need to regrow retinal axons. We have shown in regenerative models in vivo and in vitro that ßB2-crystallin actively supports the regenerative growth of cut retinal axons, thereby offering targets for neuroprotective and regenerative treatments. In this review we discuss the discovery that ßB2-crystallin is clearly up-regulated in the regenerating retina in vitro. ßB2-Crystallin is produced and secreted during axon elongation, while ß/γ-crystallins promote axon growth both in vivo and in vitro by acting either directly by uptake into cells, or indirectly by enhancing the production of ciliary neurotrophic factor from astrocytes to synergistically promote axon regrowth. We also discuss methods to induce the continuous production of crystallins at the site of injury and repair based on the use of transfected neural progenitor cells. This review ultimately leads to the conclusion that the postinjury fate of neurons cannot be seen merely as inevitable, but instead should be regarded as a challenge to shaping the neuroprotective and regenerative conditions that promote cell survival and axon repair.

Development of an intravitreal peptide (BQ123) sustained release system based on poly(2-hydroxyoctanoic acid) aiming at a retinal vasodilator response.

PURPOSE: Development of a novel formulation for intravitreal administration, in which the endothelinA receptor antagonist BQ123 is incorporated in a biodegradable and injectable polymer drug delivery system, poly(2-hydroxyoctanoic acid), aiming at a prolonged retinal vasodilator response. METHODS: BQ123 was incorporated in poly(2-hydroxyoctanoic acid), leading to an easily injectable, homogenous mixture. In vitro release profiles were obtained in porcine vitreous humor (n=6). The ex vivo biocompatibility was studied by placing the formulation in contact with porcine retinal tissues and performing histology. In a pilot in vivo study, the change in retinal vessel diameter of mini pigs (n=2) was followed over 3 h after an intravitreal injection of the formulation, as well as the release of BQ123 from the polymer system for approximately 7 days (n=6). RESULTS: In vitro, a constant release profile was obtained, releasing approximately 91% of BQ123 within 7 days. Histology on the porcine retinal tissues showed good ex vivo biocompatibility. In vivo, a vasodilative response was observed, with a retinal vessel diameter increase from 14% after 15 min, for approximately 39% after 3 h. At t=3 h, the BQ123 concentration in the vitreous humor was 0.7±0.2 µg/mL, followed by 1.5±1.0 and 1.1±0.8 µg/mL after 3 and 7 days, respectively. 39.9%±6.0% of BQ123 was still present in the polymer depot at t=7 days. CONCLUSIONS: The results show that an intravitreal injection of this drug delivery system leads to a prolonged vasodilative response and a BQ123 release over 7 days, suggesting its therapeutic potential in the management of retinal ischemic conditions.

Correlation between disease severity and presence of ocular autoantibodies in juvenile idiopathic arthritis-associated uveitis.

PURPOSE: The pathogenesis of juvenile idiopathic arthritis-associated uveitis (JIAU) is undefined. This study intended to analyze the presence of antiocular autoantibodies in serum and their correlation with disease course. METHODS: Serum samples from children with JIAU (n = 47); JIA without uveitis (n = 67); idiopathic anterior uveitis (IAU; n = 12); and healthy controls (n = 52) were collected. The binding patterns of serum antibodies to ocular cryosections from swine eyes were analyzed by indirect immunohistochemistry, and were correlated to epidemiological, clinical, and laboratory test results. RESULTS: The patient groups differed with respect to their presence of antibody binding to the sections: JIAU (94%), JIA (75%), IAU (75%), and healthy controls (29%) to uveal and/or retinal structures. Serum antibodies of JIAU patients predominantly bound at iris (74%), and ciliary body (79%). Iris/ciliary body positive staining correlated with the presence of uveitis complications (P < 0.005) in JIAU patients, but not with positivity of serum antinuclear antibodies (ANA), rheumatoid factor (RF), or HLA-B27, and was independent from uveitis activity or type of anti-inflammatory therapy. CONCLUSIONS: In JIAU patients, antiocular serum antibodies can be detected more frequently than in control groups. Binding patterns to ocular tissue correlate with complicated uveitis course but not with uveitis activity and anti-inflammatory treatment. Antibody binding is not specific for this uveitis entity, and does not correlate with ANA positivity.

Design and in vitro assessment of L-lactic acid-based copolymers as prodrug and carrier for intravitreal sustained L-lactate release to reverse retinal arteriolar occlusions.

Ophthalmic conditions in which the retinal vasculature is obstructed generally lead to vision loss. Administration of the vasodilator L-lactate might offer a treatment strategy by restoring the blood flow, but unfortunately its effect after single intravitreal injection is short-lived. This study describes a concept in which the sustained release of L-lactic acid from a biodegradable copolymer system is investigated. The 50:50 (n/n) copolymer system, composed of L-lactic acid and L,D-2-hydroxyoctanoic acid, is a viscous injectable that will form an intravitreal drug depot. Hydrolysis of the copolymer will automatically lead to the release of L-lactic acid, which will convert to L-lactate at physiological pH, thereby providing a carrier and pro-drug in one. In vitro and ex vivo release studies demonstrate an L-lactic acid release over several weeks. Biocompatibility of the co-polymer and its degradation products is shown on a human retinal pigment epithelial cell line and on ex vivo retinal tissues. A low molecular weight copolymer (1200 g/mol) with low polydispersity has promising properties with a constant release profile, good biocompatibility and injectability.

Everolimus for the treatment of uveitis refractory to cyclosporine A: a pilot study.

BACKGROUND: This study investigated the efficacy of everolimus, a potent inhibitor of T lymphocyte proliferation, for treating noninfectious uveitis. The study design was an open-label prospective trial. METHODS: Twelve patients with severe chronic uveitis (anterior and intermediate n = 9, panuveitis n = 3) refractive to cyclosporine A (CsA) received additional everolimus. MAIN OUTCOME MEASURE: the primary outcome measure was uveitis inactivity at 3 months. Secondary outcome measures were uveitis recurrence, visual acuity (BCVA), laser flare photometry values, cystoid macular edema, and tapering of concomitant corticosteroids and/or immunosuppressive drugs in 12 months with the addition of everolimus and after withdrawing everolimus. Percentages of peripheral blood CD3(+)CD4(+)CD25(+)Foxp3(+) cells were studied. RESULTS: At month 3 with everolimus, uveitis was inactive in all patients. By 12 months, uveitis had recurred in four patients after tapering (n = 2) or withdrawing (n = 2) CsA. BCVA remained stable in all patients, mean foveal thickness (OCT) was slightly reduced from 308 µm at baseline to 255 µm (p = 0.1), and mean flare values were slightly reduced from 27.8 to 19.3 photons/msec (p = 0.1). It was possible to achieve a 50 % dose reduction of systemic prednisone (n = 8) or CsA (n = 8). After withdrawing everolimus, uveitis recurred in 50 % within 1 month; by 6 months, BCVA dropped ≥2 lines in five patients, and prednisone use increased ≥50 % in four patients. The percentage of peripheral blood CD3(+)CD4(+)CD25(+)FoxP3(+) T cells increased during the everolimus treatment, and dropped after withdrawal. CONCLUSIONS: Uveitis inactivity was achieved with the addition of everolimus in patients with chronic, CsA-refractive anterior and intermediate uveitis, or panuveitis.

Effects of systemic and intravitreal TNF-α inhibition in experimental autoimmune uveoretinitis.

PURPOSE: To investigate the effect of systemic or local TNF-α inhibition with etanercept on experimental autoimmune uveoretinitis (EAU). METHODS: EAU was induced by immunizing B10.RIII mice with IRBPp161-180 or by adoptively transferring uveitogenic splenocytes. Mice received systemic or local treatment with etanercept in the afferent or efferent phase. For systemic treatment, mice were injected intraperitoneally. For local treatment, etanercept was injected intravitreally or subconjunctivally. Control mice received PBS. EAU scores were determined histologically. Splenic cells were assessed for [(3)H]thymidine incorporation. ELISA was performed to measure levels of cytokines produced by splenocytes. Vitreous cavity-associated immune deviation (VCAID) was induced by intravitreally injecting ovalbumin and evaluated by measuring DTH reaction. RESULTS: After systemic treatment with etanercept in the afferent phase, EAU disease scores, IRBP-specific cell proliferation, and production of Th1, Th2, and Th17 cytokines were reduced. EAU also improved after intravitreal etanercept treatment in the afferent phase, with unaltered IRBP-specific proliferation, reduced IFN-γ, but increased IL-6 and IL-10 secretion. VCAID induction was impaired after intravitreal etanercept treatment. No amelioration of EAU or reduction in IRBP-specific cell response was found after systemic or intravitreal treatment in the efferent phase or after subconjunctival treatment. After adoptive transfer, etanercept- and PBS-treated recipients showed similar disease severity and antigen-specific proliferation of splenocytes. CONCLUSIONS: It can be concluded that TNF-α participates mainly in the immunopathology in the induction phase of EAU. The mechanism of action underlying EAU improvement may be different for local and systemic etanercept treatment.

New biologic drugs: anti-interleukin therapy.

Interleukins (ILs) are cytokines which are defined by their capability to convey information between leukocytes, in this way directing proliferation, activation, and migration and also regulation of the cells. Data from anti-IL treatments in systemic autoimmune diseases have shown these drugs to be beneficial and to have a satisfactory safety profile and tolerance. Recent publications of small case series suggest that several anti-IL drugs have considerable efficacy in treating otherwise refractory uveitis. Anti-IL therapy, therefore, might constitute an option for the treatment of uveitis resistant to corticosteroids, classical immunosuppressives, or tumor necrosis factor-α inhibitors. However, due to high costs and possible long-term risks, anti-IL agents should currently be reserved to selected uveitis patients and be administered only under close interdisciplinary monitoring.

Complement and UV-irradiated photoreceptor outer segments increase the cytokine secretion by retinal pigment epithelial cells.

PURPOSE: Age-related macular degeneration (AMD) is accompanied by increased complement activation, and by lipofuscin accumulation in retinal pigment epithelial (RPE) cells due to incomplete degradation of photoreceptor outer segments (POS). The influence of POS, ultraviolet (UV)-irradiated POS and human complement sera (HCS) on cytokine secretion from RPE cells was therefore examined. METHODS: RPE cells were incubated with POS or UV-POS every other day for 1 week. The autofluorescence (AF) was measured photometrically and by flow cytometry. Senescence-associated genes were analyzed by RT-PCR. Internalization and degradation of POS were determined using phagocytosis and degradation assays, and lysosomal function by neutral red uptake. RPE cells in polycarbonate cell culture inserts were incubated apically with POS or UV-POS and afterward basally with HCS. C7-deficient HCS was used as control. The integrity of the cell monolayer was assessed by measuring the transepithelial electrical resistance (TER) and the permeability. Interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, and vascular endothelial growth factor were quantified by ELISA. RESULTS: POS treatment led to an increased AF and senescence marker expression, which were further elevated in response to UV-POS. UV-POS were preferentially accumulated over POS and the lysosomal function was impaired due to UV-POS. HCS intensified the cytokine production compared with controls. POS had no effect, though UV-POS combined with HCS induced a significant increase in all cytokines. CONCLUSIONS: RPE cultivation with UV-POS might serve as a model to investigate the accumulation of lipofuscin-like structures. The enhanced cytokine secretion due to UV-POS with HCS may account for an increased susceptibility for lipofuscin-loaded cells to complement, inducing a proinflammatory environment as observed in AMD.

Amniotic membrane induces peroxisome proliferator-activated receptor-γ positive alternatively activated macrophages.

PURPOSE: Amniotic membrane transplantation (AMT) reportedly improves herpetic stromal keratitis (HSK). Here we studied the role of the amniotic membrane (AM) on macrophages. METHODS: BALB/c mice with necrotizing HSK received an AMT or tarsorrhaphy (TAR) as control. Apoptosis of F4/80+ cells was determined using the annexinV/7-AAD system. Macrophage invasion was determined using a cornea invasion assay. Cytokine secretion was quantified by ELISA. Arginase activity was measured by bioassay. Expression of nuclear factor (NF)-κB or peroxisome proliferator-activated receptor (PPAR)-γ related proteins was detected by Western blot analysis, and the expression of costimulatory surface molecules or PPAR-γ by flow cytometry. Lipid accumulation was observed by Oil red O and Sudan B staining. RESULTS: After AMT apoptotic features of corneal macrophages, but also macrophage invasion increased. IL-6, IL-10, IL-12, TNF-α, and NF-κB content in HSK corneas had decreased with AMT. AMT increased expression of PPAR-γ, arginase 1 and 2, and arginase activity in AM-treated HSK corneas. In vitro, NF-κB, cytokine production, costimulatory molecules (CD80, CD86, CD40), phagocytic capacity, proliferation, viability, and accessory function to herpes simplex virus (HSV)-1 specific draining lymph node (DLN) cells were reduced in bone marrow derived macrophages (BM) cocultured with AM, while CD206, CD204, CD163, and CD68, lipid accumulation in the cytoplasm, PPAR-γ expression, and arginase activity was increased. An increase in viability and proliferation was observed in the presence of AM combined with apoptotic cells, compared with AM alone. CONCLUSIONS: Based on these results it can be concluded that the action mechanism of AM is associated with modulation of classically activated macrophages into alternatively activated macrophages or macrophage cell death, probably by engaging lipid metabolism and activating the PPAR-γ pathway, consequently curtailing effector T cell functions. Apoptotic cells induced in the environment with AM support the presence and survival of such macrophages.

Intravitreal treatment with antisense oligonucleotides targeting tumor necrosis factor-α in murine herpes simplex virus type 1 retinitis.

BACKGROUND: Tumor necrosis factor alpha (TNF-α) is a proinflammatory cytokine known to participate in intraocular inflammatory disease. This study investigated whether treatment with intravitreal antisense-oligonucleotides (ASON) targeting TNF-α mRNA affects the progression of herpes simplex virus 1 (HSV-1) retinitis in mice. METHODS: The in vivo uptake of the oligonucleotid after intravitreal injection was determined with FITC-labeled TNF-α ASON. HSV-retinitis was induced on day 0 by the injection of HSV-1 (KOS strain) into the anterior chamber (AC) of the right eyes of BALB/c mice (von Szily model). The left contralateral eyes were injected intravitreally on day 7 with TNF-α ASON, sequence-unspecific control ASON (CON), or buffer. The clinical course of retinitis, ocular inflammatory cell-infiltration, TNF-α expression in the eye by ELISA, delayed-type hypersensitivity (DTH) reaction, virus-neutralizing antibody titers in the serum, uptake of [3H]thymidine from regional lymph node (rln) cells, and viral content in the eyes were determined. RESULTS: In vivo, strong fluorescence of FITC- TNF-α ASON was detected in the choroid and retina up to 3 days after intravitreal injection, but none in the rln. After treatment of eyes with ASON, decreased expression of TNF-α in the eye, and reduced incidence and severity of retinitis on day 10 after infection (P < 0.05) could be found. The other parameters were not significantly influenced after TNF-α ASON treatment. CONCLUSIONS: TNF-α participates in the pathology of HSV-1 retinitis. Local inhibition of TNF-α mRNA by intraocular TNF-α ASON injection did not influence the systemic HSV-specific immune response or the antiviral response in the eye, but reduced ocular inflammatory bystander damage.

Anti-inflammatory treatment of uveitis with biologicals: new treatment options that reflect pathogenetic knowledge of the disease.

BACKGROUND: Endogenous uveitis is a sight-threatening disease. In addition to corticosteroids, immunosuppressive agents are commonly used to treat patients with severe course. Immunosuppressive drugs act nonspecifically, rather than providing a specific interaction with the critical pathogenetic pathways of uveitis. Better knowledge of the basic mechanisms underlying uveitis and of the molecules that are important for regulating inflammation has helped to create new and more specific treatment approaches. Biological therapy for inflammatory diseases employs substances that interfere with specific molecules or pathways induced in the body during the inflammatory process. METHODS: This review gives an overview on molecules that play a critical role in the pathogenetic process of uveitis, as has been observed in patients or the respective animal models, and summarizes the current experience with biologicals for the treatment of uveitis refractive to conventional immunosuppressives.

Amniotic membrane transplantation induces apoptosis in T lymphocytes in murine corneas with experimental herpetic stromal keratitis.

PURPOSE: To investigate the effect of human amniotic membrane transplantation (AMT) on T-cell immune response in murine corneas with herpetic stromal keratitis (HSK). METHODS: Herpes simplex virus (HSV)-1-infected BALB/c mice with necrotizing HSK were treated with AMT. CD3(+) cell apoptosis was determined in treated corneas and in vitro by flow cytometric analysis using the annexin V/7-AAD system. The effect of interleukin (IL)-2, cyclosporine, rapamycin, or Fas on T-cell survival was measured. Activation phenotype was measured by (3)H-thymidine uptake and flow cytometry (CD25, CD69, major histocompatibility complex class II). Cytokine/chemokine secretion from amniotic membrane (AM)-treated corneas or draining lymph node cells was measured. The immune-modulating capacity of long-term AMT treatment and adoptive transfer of AM-treated splenocytes was tested. RESULTS: After AMT, HSK and corneal inflammatory cell infiltration improved, and T-lymphocyte apoptosis occurred. T-cell apoptosis was also induced in vitro, independently of rIL-2, cyclosporine, rapamycin, or Fas. AMT-treated corneas and cultured lymphocytes had reduced IL-2, IL-10, IL-12, CRG-2, and CCL-2 content. Long-term AMT treatment decreased the proliferative response and type 1 helper T-cell cytokine level in draining lymph node cells. The improvement in HSK did not persist. Delayed-type hypersensitivity or HSV-1-specific cytotoxicity was not altered CONCLUSIONS: The results suggest that murine HSK improves after AMT through reduced local T-helper cell immune responses by inducing apoptosis in T lymphocytes, independently of passive apoptosis or activation-induced cell death. AM also reduces local T-helper cytokine and chemokine levels but does not result in immune deviation. Immunologic memory against HSV-1 is not affected by AMT, and long-term protection or tolerance is not induced.

Subconjunctival antisense oligonucleotides targeting TNF-alpha influence immunopathology and viral replication in murine HSV-1 retinitis.

BACKGROUND: To investigate the role of tumor necrosis factor-alpha (TNF-alpha) in immunopathology and viral replication in the contralateral eye in the von Szily model of herpes simplex virus (HSV)-1 acute retinitis. METHODS: In vivo distribution was analyzed after subconjunctival injection of FITC-labeled antisense oligonucleotides (ASON). After HSV-1 (KOS) was injected in the right anterior chamber (AC) in BALB/c mice, the course of the contralateral retinitis was evaluated. The left eyes were treated with either TNF-alpha ASON, sequence-unspecific control (CON), or buffer. The ocular TNF-alpha content was quantified by ELISA. The delayed-type hypersensitivity (DTH) reaction, uptake of [3H]thymidine from regional lymph nodes (rln)- and spleen cells, serum-neutralizing antibodies, and viral titer in the eyes were evaluated. RESULTS: After subconjunctival injection, FITC-labeled ASON were found in the choroid and retina. In the TNF-alpha ASON-treated eyes, TNF-alpha expression and the incidence and severity of retinitis were reduced on day 8 postinfection (PI) (p < 0.05). On day 10 PI, higher viral titers were only seen in the eyes of the TNF-alpha ASON group (p < 0.05), and retinitis was slightly more severe on day 12 PI. While the HSV-1 specific [3H]thymidine uptake from rln cells was higher in the TNF-alpha ASON mice (p < 0.05), the [3H]thymidine uptake from spleen cells, the DTH response, and the neutralizing-antibody titers did not differ between the groups. CONCLUSIONS: After regional blockade of TNF-alpha in experimental HSV-1 retinitis TNF-alpha seems to possess an antiviral capacity against HSV-1 in the contralateral eye and participates in the immunopathology of HSV-1-induced acute retinitis.

On the influence of neutrophils in corneas with necrotizing HSV-1 keratitis following amniotic membrane transplantation.

Necrotizing herpetic stromal keratitis (HSK) in mice rapidly improved after amniotic membrane transplantation (AMT). In this study we determined the fate of polymorphonuclear neutrophils (PMN) after AMT. AMT or tarsorrhaphy (T) was performed in BALB/c mice with ulcerative HSK. After 2 days, corneas were studied histologically and by transmission electron microscopy (TEM). CD11b, Gr-1, and TUNEL-positive cells were identified. Macrophages were depleted by subconjunctival injection of dichloromethylene-diphosphonate-liposomes (Cl(2)MDP-LIP) before AMT. Corneas were studied for interleukin (IL)-1alpha, IL-2, interferon (IFN)-gamma, CXCL1, CXCL2, and tumor necrosis factor (TNF)-alpha production by ELISA. PMN-enriched cell preparations co-cultured with amniotic membrane (AM) or with AM and such recombinant (r) cytokines as rIL-1alpha, rIL-2, and rTNF-alpha or supernatants from activated lymphocytes were investigated by flow cytometry (Annexin-V/7-AAD and TUNEL), and a dimethylthiazolyl-diphenyltetrazolium-bromide (MTT)-viability assay. Corneas in the AMT mice had less inflammation, fewer PMN-like cells and fewer CD11b+, and Gr-1+ cells (P<0.01), but a higher ratio of apoptotic to viable PMN-resembling cells (P<0.01) than the T mice. Phagocytic removal of apoptotic PMN-like cells by macrophages was evident in the AMT group. After Cl(2)MDP-LIP treatment, the corneas had more cell debris and apoptotic cells with PMN-like morphology. The concentrations of IL-1alpha, IL-2, CXCL1, and TNF-alpha were reduced in corneas of the AMT group as compared to that of the T group, while the concentration of CXCL2 was increased. Apoptosis of PMN-resembling cells was detected following cocultivation with AM, even when proinflammatory cytokines were present. Resolution of corneal inflammation in mice with necrotizing HSK after AMT is associated with increased apoptosis of PMN-like cells, reduction of pro-inflammatory cytokines, an increase of CXCL2, and increased removal of apoptotic PMN-like cells by macrophages.