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BACKGROUND: Atherosclerosis is a chronic inflammatory disease. We previously reported that a diet high in alpha-linolenic acid (ALA) reduces lipid and inflammatory cardiovascular disease risk factors in hypercholesterolemic subjects. OBJECTIVE: The objective was to evaluate the effects of a diet high in ALA on serum proinflammatory cytokine concentrations and cytokine production by cultured peripheral blood mononuclear cells (PBMCs) from subjects fed the experimental diets. DESIGN: A randomized, controlled, 3-diet, 3-period crossover study design was used. Hypercholesterolemic subjects (n = 23) were assigned to 3 experimental diets: a diet high in ALA (ALA diet; 6.5% of energy), a diet high in linoleic acid (LA diet; 12.6% of energy), and an average American diet (AAD) for 6 wk. Serum interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNF-alpha) concentrations and the production of IL-6, IL-1beta, and TNF-alpha by PBMCs were measured. RESULTS: IL-6, IL-1beta, and TNF-alpha production by PBMCs and serum TNF-alpha concentrations were lower (P < 0.05 and P < 0.08, respectively) with the ALA diet than with the LA diet or AAD. PBMC production of TNF-alpha was inversely correlated with ALA (r = -0.402, P = 0.07) and with eicosapentaenoic acid (r = -0.476, P = 0.03) concentrations in PBMC lipids with the ALA diet. Changes in serum ALA were inversely correlated with changes in TNF-alpha produced by PBMCs (r = -0.423, P < 0.05). CONCLUSIONS: Increased intakes of dietary ALA elicit antiinflammatory effects by inhibiting IL-6, IL-1beta, and TNF-alpha production in cultured PBMCs. Changes in PBMC ALA and eicosapentaenoic acid (derived from dietary ALA) are associated with beneficial changes in TNF-alpha release. Thus, the cardioprotective effects of ALA are mediated in part by a reduction in the production of inflammatory cytokines.

Cancer results from an undesirable imbalance between cellular proliferation and apoptosis. Both processes may be modulated at the level of gene expression, viz., p53 and c-Ha-ras, by dietary bioactive components such as resveratrol. We tested the time-dependent effect of resveratrol on gene and protein expression in WR-21 cells containing a mutated human c-Ha-ras oncogene. We demonstrate cyclic resveratrol-mediated expression of p53, mdm2, p21(cip/waf), Rb, and cyclin G at both the RNA and the protein level at8 h. However, ras was not differentially expressed at either the RNA or the protein level. p53 was upregulated followed by p21cip/waf, then mdm2, and cyclin G, all downstream p53-activated targets. RNA transcription increased at8 h for all genes except p53, but protein levels did not suggest uncoupling of transcription and translation. At 24 h, both p53 and Rb expression returned to baseline, suggesting collapse of DNA structure and spindle assembly checkpoints characteristic of mitotic catastrophe. In summary, resveratrol at8 h induced p53-mediated effects, including apoptosis and cell-cycle arrest (G2/M). However, later, it induced cell-cycle checkpoint dysfunction, indicative of mitotic catastrophe. Thus, future studies should better elucidate the temporal mechanism of the dietary bioactive agent resveratrol on cancer cells.

The effects of linoleic acid (LA), alpha-linolenic acid (ALA), and docosahexaenoic acid (DHA) were compared to that of palmitic acid (PA), on inflammatory responses in human monocytic THP-1 cells. When cells were pre-incubated with fatty acids for 2-h and then stimulated with lipopolysaccharide for 24-h in the presence of fatty acids, secretion of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha (TNFalpha) was significantly decreased after treatment with LA, ALA, and DHA versus PA (P < 0.01 for all); ALA and DHA elicited more favorable effects. These effects were comparable to those for 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and were dose-dependent. In addition, LA, ALA, and DHA decreased IL-6, IL-1beta, and TNFalpha gene expression (P < 0.05 for all) and nuclear factor (NF)-kappaB DNA-binding activity, whereas peroxisome proliferator-activated receptor-gamma (PPARgamma) DNA-binding activity was increased. The results indicate that the anti-inflammatory effects of polyunsaturated fatty acids may be, in part, due to the inhibition of NF-kappaB activation via activation of PPARgamma.

An accumulating body of evidence suggests that resveratrol can inhibit carcinogenesis through antiproliferative and apoptotic effects. One proposed mechanism for this is the modulation of genes, for example, Ras and p53, frequently associated with human cancer. To test the effect of resveratrol on gene expression, we used the WR-21 cell line because it contains a mutated human c-Ha-ras gene. Cells at > or =70% confluency were incubated with media alone or with increasing concentrations of trans-resveratrol (0.1-1000 microM) for 24 h. Resveratrol (30-100 microM) decreased cellular proliferation by 80% (bromodeoxyuridine incorporation) and increased apoptosis by 60% (TUNEL). Cells were then treated with media alone or with 50-microM resveratrol for 24 h. RNA was isolated for nylon-based macroarray analyses and protein for immunoblotting. Resveratrol increased (+) and decreased (-) gene expression associated with apoptosis (Birc5+, Cash+, Mcl-1+, Mdm2+, Rpa-like+), cellular proliferation (Ctsd+, Mdm2+, Egr1+, ODC+) and cell cycle (cyclin D+, cyclin g+, Gadd45a-, Mad2l-, Mdm2+). Resveratrol consistently increased by > or =6-fold Mdm2 expression and other downstream p53 effectors, but not p53 itself at 24 h. Subsequent cell cycle analysis indicated a significant accumulation of cells in G2/M, and a decrease in G1/G0 suggesting a G2/M blockade. Further RT-PCR and Western blot analyses indicated no differential changes in Ras mRNA expression or p21(ras) protein levels, respectively. These results suggest that resveratrol potently inhibits cellular proliferation, increases apoptosis, alters cell cycle dynamics and modulates associated gene expression. Furthermore, these effects appear mediated, in part, by p53 without direct modulation of mutant c-Ha-ras expression.

Acute intraocular pressure (IOP) elevation causes accumulation of retrogradely-transported brain derived neurotrophic factor and its receptor at the optic nerve head (ONH) in rats and monkeys. Obstruction of axonal transport may therefore be involved in glaucoma pathogenesis, but it is unknown if obstruction is specific to certain transported factors or represents a generalized failure of retrograde axonal transport. The dynein motor complex mediates retrograde axonal transport in retinal ganglion cells (RGC). Our hypothesis was that elevated IOP interferes with dynein-mediated axonal transport. We studied the distribution of dynein subunits in the retina and optic nerve after acute and chronic experimental IOP elevation in the rat. IOP was elevated unilaterally in 54 rats. Dynein subunit distribution was compared in treated and control eyes by immunohistochemistry and Western blotting at 1 day (n=12), 3 days (n=4), 1 week (n=15), 2 weeks (n=12) and 4 weeks (n=11). For immunohistochemistry, sections through the ONH were probed with an anti-dynein heavy chain (HC) antibody and graded semi-quantitatively by masked observers. Other freshly enucleated eyes were microdissected for separate Western blot quantification of dynein intermediate complex (IC) in myelinated and unmyelinated optic nerve, ONH and retina. Immunohistochemistry showed accumulation of dynein HC at the ONH in IOP elevation eyes compared to controls (P0.001, Wilcoxon paired sign-rank test, n=29). ONH dynein IC was elevated by 46.5% in chronic IOP elevation eyes compared to controls by Western blotting (P0.001, 95% CI=25.9% to 67.8%, n=17). The maximum increase in ONH dynein IC was 78.7% after 1 week (P0.05, n=5), but significant increases were also detected after 4 h and 4 weeks of IOP elevation (P0.05, n=4 rats per group). Total retinal dynein IC was increased by 8.7% in chronic IOP elevation eyes compared to controls (P0.03, 95% CI 1.4% to 16.1%, n=24). In the retina, IOP elevation particularly affected the 72 kD subunit of dynein IC, which was 100.7% higher in chronic IOP elevation eyes compared to controls (P0.00001, 95% CI 71.0% to 130.4%, n=21). Dynein IC changes in myelinated and unmyelinated optic nerve were not significant (P0.05). We conclude that dynein accumulates at the ONH with experimental IOP elevation in the rat, supporting the hypothesis that disrupted axonal transport in RGC may be involved in the pathogenesis of glaucoma. The effect of IOP elevation on other motor proteins deserves further investigation in the future.

Purpose There has been recent interest in the potential use of gene therapy techniques to treat ocular disease. In this article, we consider the optic nerve diseases that are potentially most amenable to gene therapy. Methods We discuss the recent success of gene transfer experiments in animal models of glaucoma, optic neuritis, Leber's hereditary optic neuropathy (LHON), and optic nerve transection, and we assess the possibility of using similar techniques to treat human disease in the future. Results We have achieved highly efficient transfection of retinal ganglion cells in a rat model of glaucoma following a single intravitreal injection of adeno-associated virus (AAV). In our model, we have found that AAV-mediated gene therapy with brain-derived neurotrophic factor has a significant neuroprotective effect compared to saline or control virus injections. Guy and co-workers have successfully used AAV-mediated gene therapy to replace the defective mitochondrial enzyme subunit in cells derived from human patients with LHON. Gene therapy techniques have also shown promise in animal models of optic neuritis and optic nerve trauma. Conclusions Human diseases with single-gene defects such as LHON may soon be treated successfully by gene therapy, assuming that vectors continue to improve and are well tolerated in the human eye. Other optic nerve diseases such as glaucoma that do not have a single-gene defect may also benefit from gene therapy to enhance RGC survival. In all cases, the risks of treatment will need to be balanced against the potential benefits.

We designed a novel short-term bitransgenic model to better characterize the effects of benzo(a)pyrene (BP) exposure on multi-organ carcinogenesis and to evaluate the effects of a well-recognized antioxidant, N-acetyl-L-cysteine (NAC), on neoplasia. We selected the p53 heterozygous Tg.AC (v-Ha-ras) mouse model for our studies because these mice possess a carcinogen-inducible ras oncogene and one functional p53 tumor suppressor allele. Both mutations occur frequently in human cancers. In a 2 x 2 experimental design, both female and male mice were fed basal diet alone or containing 3% NAC and administered by gavage corn oil vehicle alone or containing 20 mg BP/kg body weight given twice weekly for 10 weeks. Mice (n = 15 for each grouping and sex) were subsequently observed an additional 18 weeks followed by tissue collection for evaluation of multi-organ pathology. Benzo(a)pyrene increased neoplasia in the thymus, spleen, stomach, and hematopoietic system after 28 weeks. We observed modest NAC-associated decreases in BP-induced pathology of the liver, papilloma formation and hyperplasia in the forestomach, and the occurrence of malignant lymphoma. Benzo(a)pyrene exposure reduced survival to approximately 40% in male mice, suggesting toxicity; however, survival in control groups was approximately 60%. Survival decreased to approximately 30% for females in all groups. We noted a clear, but nonsignificant, 15% decline in body weights of male, but not female, mice fed NAC, although food intake did not differ. Collectively, the data suggested carcinogen and antioxidant-associated effects on neoplasia that appeared sex-dependent. Thus, this novel short-term bitransgenic model may potentially be useful for testing dietary modulation of carcinogenesis.

Age-related macular degeneration (AMD) is the commonest cause of blindness in the developed world, and is strongly associated with the Y402H polymorphism in the complement (C) factor H (CFH) gene. As liver is the primary site of C production, our hypothesis was that modification of liver CFH Y402H genotype through liver transplantation would influence the development of AMD. We recruited 223 Western European patients at least 55 years old, who had undergone LT at least 5 years previously. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype by previously described ELISA utilising variant-specific monoclonal antibodies. This approach was verified by genotyping donor tissue from a subgroup of patients (n = 49). We found that recipient AMD status was associated with recipient CFH Y402H genotype (p = 0.036) but not with donor CFH Y402H genotype (p = 0.626), after controlling for age, gender, smoking status, and body mass index. Compared to previously reported prevalence figures there was an increased prevalence of both AMD and recipient CFH Y402H risk allele frequency in LT patients. Plasma C protein levels (including C3 and C4, activation products C3a, C4a, C5a, TCC, and regulators C1 inhibitor and CFH) were similar in LT patients with and without AMD. We also compared LT patients with 30 randomly selected age matched healthy controls, without AMD or a history of LT. Plasma total CFH was higher in LT patients compared to controls, otherwise levels of all other C proteins were similar. AMD was associated with recipient CFH Y402H genotype, implying that local rather than systemic CFH protein or gene therapy may be more relevant for pathogenesis and a better target for therapy in AMD. The increased prevalence of AMD in LT patients may be explained by the observed increased frequency of the CFH 402H sequence variant

Adeno-associated virus (AAV) vectors provide a useful way to deliver genes to the eye. They have a number of important properties which make them suitable for this purpose, not least their lack of significant pathogenicity and the potential for long-term transfection of retinal cells. The optimal methods for AAV-mediated gene delivery are determined by the location and characteristics of the target cell type. Efficient gene delivery to photoreceptors and pigment epithelial cells following subretinal injection of AAV has been achieved in various animal models. AAV-mediated gene therapy has been shown to slow photoreceptor loss in rodent models of primary photoreceptor diseases and in dogs with a naturally occurring disease similar to human Leber's congenital amaurosis (LCA). Efficient gene delivery to other cell types such as retinal ganglion cells (RGCs), however, has been more problematic. In this article, we review the potential uses of AAV-mediated gene delivery to the eye. We describe the selection of an appropriate AAV vector for ocular gene transfer studies and discuss the techniques used to deliver the virus to the eye and to assess ocular transfection. We emphasize our techniques for successful gene transfer to retinal ganglion cells, which have often proven challenging to transfect with high efficiency. Using a modified AAV incorporating a chicken beta-actin (CBA) promoter and the woodchuck hepatitis posttranscriptional regulatory element, we describe how our techniques allow approximately 85% of rat retinal ganglion cells to be transfected within 2 weeks of a single intravitreal virus injection. Our techniques facilitate the study of the pathogenesis of RGC diseases such as glaucoma and the development of novel new treatments based on gene therapy.

To investigate whether the levels of free amino acids and protein in the vitreous of rat eyes are altered with chronic intraocular pressure (IOP) elevation or after optic nerve transection.The concentrations of 20 amino acids in the vitreous humor were measured by high-performance liquid chromatography in both eyes of 41 rats with unilateral IOP elevation induced by translimbal photocoagulation. Eyes were studied 1 day and 1, 2, 4, and 9 weeks after initial IOP elevation. The same amino acids were measured in 41 rats 1 day and 2, 4, and 9 weeks after unilateral transection of the orbital optic nerve. The intravitreal protein level was assayed in additional 22 rats with IOP elevation and 12 rats after nerve transection. Two masked observers evaluated the amount of optic nerve damage with a semiquantitative, light-microscopic technique.In rats with experimental glaucoma, amino acid concentrations were unchanged 1 day after treatment. At 1 week, 4 of 20 amino acids (aspartate, proline, alanine, and lysine) were higher than in control eyes (or = 0.01), but this difference was nonsignificant after Bonferroni correction for multiple simultaneous amino acid comparisons (none achieved0.0025). No amino acid was significantly different from control in the nerve transection groups (all0.05). Vitreous protein level was significantly higher in glaucomatous eyes than their paired controls at 1 day (0.0001) and 1 week (0.002). One day and 1 week after optic nerve transection, vitreal proteins were significantly elevated compared with control eyes from untreated animals (0.0020 and0.0022, respectively), though not compared with their fellow eyes ( = 0.25 and 0.10).Chronic experimental glaucoma and transection of the optic nerve increase the amount of protein in the rat vitreous above control levels. In the vitreous of rats with experimental glaucoma, a number of free amino acids were transiently elevated to a modest degree, but no significant difference in vitreous glutamate concentration was detected (0.01).

ROS are diverse and abundant in biological systems. While excessive ROS production clearly damages DNA, low levels of ROS affect cell signaling particularly at the level of redox modulation. Moreover, the specific contributions of ROS to apoptosis and mitogenesis in maintenance of cell number homeostasis remains to be elucidated. ROS dose is a critical parameter in determining the ultimate cellular response; however the shape of the dose response curve is unpredictable. When cells are stimulated with ROS, cell-signaling cascades are activated. It appears that the cellular redox potential is an important determinant of cell function and interruption of redox balance may adversely affect cell function. As a result, compounds such as antioxidants may intercept critical ROS signaling molecules and both protect cells and foster pathogenesis. As a result, further study is needed to unravel the role of ROS in redox regulation and the potential outcome of antioxidant administration on cellular responses.

Sir, We are grateful for the opportunity to respond to the correspondence from Dr He, and we thank the author for his interest in our recent article, which demonstrated that platelet-derived growth factor (PDGF) is secreted by bone marrow-derived mesenchymal stem cells (MSCs) and confers neuroprotection to retinal ganglion cells in organotypic retinal explant culture and in an in vivo model of ocular hypertensive glaucoma (Johnson et al. , 2013). Based on our previously reported observation that intravitreal MSC transplantation confers optic nerve neuroprotection (Johnson et al. , 2010), we recently analysed the human MSC secretome and compared it to that of human fibroblasts using xMAP antibody-based arrays. As Dr He noted, we demonstrated that PDGF-AA secretion from MSCs was >500-fold greater than from fibroblasts, …

Epidemiologic studies support the protective role of dietary antioxidants in preventing cancer. However, emerging evidence from clinical trials and laboratory data suggest that in some cases individual antioxidant supplements may actually exacerbate carcinogenesis. Our goal was to explore these paradoxical activities in a rodent model that possesses genotypic characteristics of human cancers. We selected the p53 haploinsufficient Tg.AC (v-Ha-ras) mouse as a model, because it contains an activated, carcinogeninducible ras oncogene and an inactivated p53 tumor suppressor gene, which are frequent genetic alterations in human cancers. These mice develop chemically induced benign and malignant skin tumors rapidly which can easily be quantified. Mice were fed basal diets with or without 3% N-acetyl-L-cysteine (NAC), a well-recognized antioxidant, prior to, during and after topical application of the carcinogen benzo[a]pyrene (64 microg/mouse) applied twice per week for 7 weeks. Tumor incidence exceeded 90% for both groups, and NAC did not reduce tumor latency. Mice fed NAC displayed a 43% reduction (P < 0.05) in tumor multiplicity and delayed the appearance of lesions (P < 0.05). Dietary NAC also significantly (P < 0.05) improved group survival by 5 weeks. Total tumor yields were reduced in both dietary groups but malignant spindle cell tumors (SCT) increased by 25% in NAC-fed mice. The v-Ha-ras oncogene and p53 protein products were clearly co-expressed in both benign and malignant lesions from both dietary groups. In summary, dietary supplementation with NAC was chemopreventive, but the marginal increase in SCT suggests a paradoxical effect.

Editor,—Sympathetic ophthalmia with deafness has been reported rarely.12 We describe one such case and explore a hypothesis whereby genetic susceptibility may be associated with cross reactivity of antigens derived from common neural crest tissue. ### CASE REPORT A 72 year old woman who had previously undergone a left trabeculectomy had a fall in which she sustained a Colles fracture and a left total hyphaema. By 14 days the blood had cleared sufficiently to reveal that she had expelled the entire left iris into the trabeculectomy bleb, with extensive dispersion of uveal pigment into the subconjunctival space. She was also found to have dislocated the lens, and had sustained a choroidal detachment (confirmed by ultrasonography). A non-granulomatous anterior uveitis was noted in the left eye at this time. The initial visual acuity was light perception in the left eye and 6/12 in the right eye. On the 25th day after the original injury, she experienced pain and blurring of vision in her right eye, with the simultaneous onset of …

Vitamins are compounds that are required in small quantities for growth and maintenance of normal cell and organ functions; they are nutrients required primarily by vertebrates and not by lower organisms. Although vitamins may be grouped by their source or function, the most commonly used categorization relates to their chemical solubility, namely, hydrophobicity or hydrophilicity, which is directly associated with absorption processes.

Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease (CVD). One proposed mechanism for the beneficial effect of carotenoids is through functional modulation of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell (HAEC) cultures (passages 4-8) for 24 h with each of the five most prevalent carotenoids in human plasma, which are alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 micromol/l. Carotenoids were solubilized in 0.7% (v/v) tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate solubilities in aqueous medium, final concentrations of alpha-carotene, beta-carotene, beta-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 micromol/l and monolayers accumulated 647, 158, 7, 113, and 9 pmol/mg protein, respectively. Monolayers were then stimulated with IL-1beta (5 ng/ml) for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay (ELISA). To assess endothelial cell adhesion to monocytes, IL-1beta-stimulated monolayers were incubated for 10 min with 51Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with beta-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13%, respectively. Pre-incubation with beta-carotene and lutein significantly reduced E-selectin expression by 38 and 34%, respectively. Pre-treatment with beta-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18%, respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1beta-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25%, respectively. Thus, among the carotenoids, lycopene appears to be most effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface.

Alpha-tocophorel (T) is the most common form of vitamin E inplasma and tissues. Alpha-T is also believed to be superior to its homologues beta-T, gamma-T, and delta-T in antioxidant activity. Biological activity of alpha-T has been intensively studied in a number of bodily systems. In contrast, the other homologues have received little attention beyond the evaluation of their relative antioxidant activity. We as well as others have previously shown that alpha-T can enhance cell- mediated immune function of aged animals and humans. Gamma-T is a principal form of vitamin E in the American diet and some cooking oils contain substantial amount of beta-T and delta-T. Thus it is of public health interest to compare their biological effects with than of alpha-t in various systems. In this study, we used an in vitro supplementation protocol to determine immunologic effects of these T homologues on murine splenocytes. The results showed that all four T homologues enhance both spontaneous and mitogen-stimulated lymphocyte proliferation (LP) and the maximal enhancement produced by them was of the same magnitude. The dose range to produce maximal enhancement varied with different homologues. The efficiency was in the order of beta-T approximately delta-T > alpha-T. Interestingly, at 50 (optimal for alpha-T) and 150 micromol/L, while alpha-T enhanced LP, all the other homologues inhibited LP. This inhibition was found to be due to their cytotoxicity at these levels. T homologues had a differential effect on interleukin (IL)-2 and prostaglandin (PG)E(2) production. IL-2 production by mouse splenocytes was not affected by alpha-T or beta-T, but was increased by gamma-T and delta-T. All T homologues, except for beta-T, inhibited PGE(2) alpha-T. Thus, all the T homologues enhance LP. However, the dose required to reach maximal enhancement varies among the homologues. On the other hand, they have a differential effect on IL-2 and PGE(2) production. The difference in nature and magnitude of the effect on immune function does not correlate with their reported relative antioxidant activity and might be due to minor differences in their structure important to their other biological activities.

Epidemiological and clinical studies indicate that vitamin E may reduce the risk of cardiovascular disease (CVD). Modulation of adhesion molecule expression and chemokine production by vitamin E may contribute to its beneficial effect. In this study we found that the enrichment of confluent human aortic endothelial cells (HAEC) or U937 monocytic cells with increasing doses of vitamin E (d-alpha-tocopherol, 20, 40, and 60 micromol/l for 20 h) inhibited their adhesion when either or both cell types were stimulated with interleukin (IL)-1beta. Enrichment of HAEC with the same doses of vitamin E suppressed IL-1beta-stimulated expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin). Supplementation with increasing doses of vitamin E up to 60 micromol/l was not effective in preventing spontaneous production of monocyte chemoattractant protein-1 (MCP-1), but supplementation with vitamin E at 60 micromol/l reduced IL-8 production significantly. However, IL-1beta-induced productions of both MCP-1 and IL-8 were dose-dependently suppressed by enrichment of cells with vitamin E. Vitamin E, at the doses used, did not significantly change the spontaneous production but dose-dependently inhibited the IL-1beta-induced production of inflammatory cytokine IL-6. We concluded that vitamin E could inhibit production of chemokines and inflammatory cytokines, in addition to inhibiting adhesion of HAEC to monocytes by reducing expression of adhesion molecules when cells were activated with an inflammatory cytokine. These mediators are actively involved in the pathogenesis of atherosclerosis. Therefore, their inhibition by vitamin E may contribute to vitamin E's reported reduction in risk of CVD.

We report the case of young man with a 4-month history of recurrent, transient left monocular visual loss due to intermittent ocular hypertension and a prepapillary vascular loop. Examination showed narrow iridocorneal angles and a prepapillary vascular loop in the left eye. The working diagnosis was of intermittent transient occlusion of the prepapillary vascular loop due to transient ocular hypertension related to intermittent angle occlusion. He underwent bilateral peripheral iridotomies and was started on G Pilocarpine 0.1% and G Latanaprost to the left eye. One year later, he remained asymptomatic with no further episodes of visual disturbance. A 22-year-old man presented with three episodes in 4 months of transient left monocular visual loss. During each episode, his vision became “grey and washed out” and cleared within 5–30 min. This was not associated with posture …

Recent evidence supports the contention that unaccustomed and strenuous exercise may lead to disruption of the delicate oxidant/antioxidant balance, resulting in oxidative stress within the human body. It is generally accepted that many instances of cellular and tissue dysfunction observed after exercise may be attributed to oxidative stress. Vitamin E is the most prevalent and potent lipid soluble antioxidant in the human body and may have an important role in decreasing oxidative stress associated with exercise by improving the oxidant/antioxidant balance. Concentrations of vitamin E can be significantly increased in muscle tissues within a relatively short period of time by dietary intake and/or supplementation. Concentrations of vitamin E may also vary among the muscle fibre types. Although rarely observed, vitamin E deficiency may induce muscle fibre degradation, affect skeletal muscle fibre type distribution, promote deposition of lipid granules, and result in other increased indications of oxidative stress. Oxygen consumption increases markedly during strenuous exercise and is believed to contribute to the increase in oxygen radical formation during exercise. Acute exercise and exercise training increase antioxidant enzyme levels in skeletal muscle of animals. Since exercise reduces vitamin E content of skeletal muscle, the requirement for vitamin E may be substantially above the current recommended dietary allowance (RDA), particularly for individuals who regularly exercise. Data suggesting a protective effect of vitamin E supplementation on human performance are limited; however, the preponderance of evidence indicates beneficial effects from long-term vitamin E supplementation in decreasing exercise-induced oxidative stress and associated risk factors.

Numerous epidemiological studies support a strong inverse relationship between consumption of carotenoid-rich fruits and vegetables and the incidence of some degenerative diseases. One proposed mechanism of protection by carotenoids centers on their putative antioxidant activity, although direct evidence in support of this contention is limited at the cellular level. The antioxidant potential of beta-carotene (BC) and lutein (LUT), carotenoids with or without provitamin A activity, respectively, was evaluated using the human liver cell line HepG2. Pilot studies showed that a 90-min exposure of confluent cultures to 500 mumol/L tert-butylhydroperoxide (TBHP) at 37 degrees C significantly (P0.05) increased lipid peroxidation and cellular leakage of lactate dehydrogenase (LDH), and decreased the uptake of 3H-alpha-aminoisobutyric acid and 3H-2-deoxyglucose. Protein synthesis, mitochondrial activity and glucose oxidation were not affected by TBHP treatment, suggesting that the plasma membrane was the primary site of TBHP-induced damage. Overnight incubation of cultures withor = 1 mumol/L dl-alpha-tocopherol protected cells against oxidant-induced changes. In parallel studies, overnight incubation of HepG2 in medium containing micelles with either BC or LUT (final concentrations of 1.1 and 10.9 mumol/L, respectively), the cell content of the carotenoids increased from0.04 to 0.32 and 3.39 nmol/mg protein, respectively. Carotenoid-loaded cells were partially or completely protected against oxidant-induced changes in lipid peroxidation, LDH release and amino acid and deoxyglucose transport. These data demonstrate that BC and LUT or their metabolites protect HepG2 cells against oxidant-induced damage and that the protective effect is independent of provitamin A activity.

Background We have previously shown that the slow Wallerian degeneration mutation, whilst delaying axonal degeneration after optic nerve crush, does not protect retinal ganglion cell (RGC) bodies in adult rats. To test the effects of a combination approach protecting both axons and cell bodies we performed combined optic nerve crush and lens injury, which results in both enhanced RGC survival as well as axon regeneration past the lesion site in wildtype animals. Results As previously reported we found that the Wld S mutation does not protect RGC bodies after optic nerve crush alone. Surprisingly, we found that Wld S transgenic rats did not exhibit the enhanced RGC survival response after combined optic nerve crush and lens injury that was observed in wildtype rats. RGC axon regeneration past the optic nerve lesion site was, however, similar in Wld S and wildtypes. Furthermore, activation of retinal glia, previously shown to be associated with enhanced RGC survival and axon regeneration after optic nerve crush and lens injury, was unaffected in Wld S transgenic rats. Conclusions RGC axon regeneration is similar between Wld S transgenic and wildtype rats, but Wld S transgenic rats do not exhibit enhanced RGC survival after combined optic nerve crush and lens injury suggesting that the neuroprotective effects of lens injury on RGC survival may be limited by the Wld S protein.

The effects of tau hyperphosphorylation and aggregation on axonal transport were investigated in the optic nerve of mice transgenic for human mutant P301S tau. Transport was examined using cholera toxin B tracing. Retrograde transport was reduced in transgenic mice at 3 and 5 months of age, when compared to C57/Bl6 control mice. Anterograde axonal transport was also reduced in 3-month-old transgenic mice. Mild excitotoxic injury of retinal ganglion cells resulted in greater nerve cell loss in retinas from 3- and 5-month old P301S transgenic mice, when compared to controls. In conjunction with the detection of abnormal tau in the optic nerve in human and experimental glaucoma, the present findings suggest that tau hyperphosphorylation and aggregation may constitute targets for neuroprotective therapies in glaucoma as well as tauopathies.

PURPOSE In vivo imaging of the retina is becoming an increasingly important research method. General anesthesia rapidly compromises the corneal surface, which increases scattering. In addition, wavefront aberrations limit the maximum imaging resolution. Three common methods of stabilizing the air-cornea interface and reducing scattering are the use of a contact lens, a microscopy slide coverslip, or mineral oil. These methods have not yet been analyzed regarding their impact on scattering and wavefront aberrations. METHODS Nineteen eyes of 19 rats were analyzed with a custom-made Hartmann-Shack (HS) wavefront sensor. The amount of scattering was determined by analysis of the HS spot width, and the wavefront was reconstructed for the naked eye and each scattering-reducing method. Their effect on optical quality was determined by calculating the modulation transfer function (MTF). RESULTS The three methods applied significantly reduced scattering but were differentially effective, with the coverslip performing the best and the mineral oil the worst. The root mean square (RMS) of the wavefront aberration, as well as the intereye variability of the RMS, was significantly smaller with the contact lens than with the coverslip. The MTF was best for the contact lens and worst for the coverslip, which was also illustrated by image simulations. CONCLUSIONS The coverslip, contact lens, and mineral oil, when applied to the cornea, all reduced scattering. The best-performing method, the coverslip, increased wavefront aberrations. Overall, the contact lens had the best influence on image quality, and it appears to be the method of choice for high-resolution retinal imaging in rats.

The potential use of stem or progenitor cell transplantation to treat neurodegenerative diseases is a subject of intensive research. The eye is a good candidate for therapeutic cell transplantation, which could benefit the retina by neuronal replacement or neuroprotection. As examples of neuronal replacement, both neonatal photoreceptor precursors1 and human embryonic stem cell–derived retinal progenitors2 can functionally replace photoreceptors when transplanted into animals with retinal dystrophy. However, so far only a very small proportion of grafted cells have been shown to integrate into the host retina. Replacement of retinal ganglion cells (RGCs) presents an even greater challenge. RGCs are selectively and progressively lost in glaucoma, which is the leading cause of irreversible blindness worldwide.3 In contrast to photoreceptors, RGCs have complex afferent retinal connections and long axons that project to precise brain targets. Thus, functional replacement of RGCs necessitates overcoming numerous fundamental barriers. Neuroprotective strategies, in which transplanted cells protect endogenous neural tissue, have also shown promising results in animal models of retinal disease. Cell transplantation slows the loss of neurons and/or preserves vision in models of inherited photoreceptor degeneration4,5 and retinal ischemia,6 and has also been investigated in models of glaucoma.7–9 The neuroprotective mechanism of action appears to include trophic factor secretion and/or modulation of inflammatory processes.8 In addition, cell transplantation may activate endogenous repair mechanisms by modulating inhibitory signals to promote axonal regrowth and neuritic sprouting.10,11 Although retinal stem cell therapy seems promising, multiple fundamental problems must be resolved before it can be applied clinically. Arguably, the most basic of these obstacles is the inhibitory barrier that prevents migration of grafted cells from the transplantation site into the retina. Overcoming this barrier is a necessary prerequisite to the long-term efficacy of retinal stem cell therapy, regardless of whether the ultimate goal is regenerative or protective in nature. Much like the rest of the mature central nervous system (CNS), the retina is implastic and relatively inhibitory to cellular migration. We and others have reported that only ∼1% of intraocularly transplanted cells commonly migrate into the retina, whereas most remain as a bolus outside of the neural tissue.1,7,12–17 This limitation appears to be common to a variety of stem cell types, including those of neural and mesenchymal lineage. Intravitreally grafted cells line the surface of the inner limiting membrane (ILM) without penetrating the host retina.15 Endogenous extracellular matrix (ECM) molecules contribute partially to the blockade of subretinal transplants, and the targeted disruption of these molecules can modestly enhance retinal integration.16–18 The contribution of basement membrane-associated proteins to the inhibitory environment has not been investigated. Moreover, the activity of nonneuronal retinal cell types may also inhibit graft migration.13,17,19 However, the relative importance of each component is largely unknown. In this study, we systematically assessed the contributions of the inner basal lamina ECM and the retinal glial cell population to the blockade of graft migration after intravitreal transplantation. We demonstrated in vitro that, although mechanical ILM peeling facilitated graft migration, enzymatic degradation of the inner basal lamina alone, without concurrent Muller cell trauma, did not recapitulate the effect. However, treatment with a glia-specific toxin dramatically improved cell integration even when the ILM was fully intact. Furthermore, we confirmed that this latter effect was preserved in vivo, suggesting that suppression of glial reactivity may be a necessary component of future retinal stem cell transplantation therapies.

Increased consumption of fruits and vegetables is associated with a lower risk of chronic disease such as cardiovascular disease, some forms of cancer, and neurodegeneration. Pro-oxidant-induced oxidative stress contributes to the pathogenesis of numerous chronic diseases and, as such, dietary antioxidants can quench and/or retard such processes. Dietary polyphenols, ie, phenolic acids and flavonoids, are a primary source of antioxidants for humans and are derived from plants including fruits, vegetables, spices, and herbs. Based on compelling evidence regarding the health effects of polyphenol-rich foods, new dietary supplements and polyphenol-rich foods are being developed for public use. Consumption of such products can increase dietary polyphenol intake and subsequently plasma concentrations beyond expected levels associated with dietary consumption and potentially confer additional health benefits. Furthermore, bioavailability can be modified to further increase absorption and ultimately plasma concentrations of polyphenols. However, the upper limit for plasma concentrations of polyphenols before the elaboration of adverse effects is unknown for many polyphenols. Moreover, a considerable amount of evidence is accumulating which supports the hypothesis that high-dose polyphenols can mechanistically cause adverse effects through pro-oxidative action. Thus, polyphenol-rich dietary supplements can potentially confer additional benefits but high-doses may elicit toxicity thereby establishing a double-edge sword in supplement use.

The mechanisms of progression in glaucoma remain incompletely understood, but basic science research is gradually elucidating the processes involved. Axonal injury is a major event in glaucoma and the cellular mechanisms involved in axonal degeneration are different from those that lead to retinal ganglion cell body death. Thus, preventing cell body death is necessary but not sufficient to prevent progressive loss of function in glaucoma, and axonal protection is an important therapeutic goal. Basic science approaches are also suggesting new ways to detect and monitor progression. Techniques to image individual retinal ganglion cells and their axons in the living eye are improving rapidly. Cells undergoing apoptosis can be labelled and identified, and it may be possible in the future to use retinal ganglion cell apoptosis rates as a biomarker of glaucoma progression. However, slow progression may be difficult to detect using such techniques; therefore, biomarkers that identify ganglion cells that are sick but not yet committed to die are an important research goal.

Purpose To develop and characterize a retinal explant culture system to facilitate investigation of novel methods of improving retinal stem cell therapy. Methods Retinas explanted from adult rats were cultured in serum-free medium (B27/N2) or medium containing normal horse serum (NHS). Tissue viability was assessed by gross morphology, propidium iodide (PI) uptake, cell survival quantification, activated caspase-3 expression, and immunohistochemistry. Muller progenitor cells (hMIO-M1), or mesenchymal stem cells (MSC) were placed on explants, to model intravitreal cell transplantation. Explants were compared with whole eyes, with or without experimental glaucoma and/or intravitreal cell transplantation. Results Explants cultured in B27/N2 medium were viable for at least 17 days, as assessed by the aforementioned parameters. NHS medium was associated with obvious tissue degradation, greater/more diffuse PI uptake, significant cell loss over time, and temporal increase in activated caspase-3(+) cells. Explants in B27/N2 medium strongly expressed beta-III-tubulin, neurofilament, NeuN, Brn3a, Thy-1, GFAP, vimentin, nestin, and glutamine synthetase, whereas immunoreactivity was weak in NHS medium and decreased further with time. Seven and 14 days after coculture or transplantation, glial reactivity (GFAP/vimentin expression) was highly upregulated in explants and eyes, respectively. Some grafted cells migrated into the retina, but most remained outside the inner limiting membrane. Conclusions Retinal explants prepared using the described techniques and cultured in B27/N2 medium are viable for at least 2 weeks and mimic in vivo glial reactivity to transplantation while allowing few grafted cells to integrate. This system may be a useful in vitro model for investigating methods of enhancing retinal stem cell therapy.

PURPOSE. Stem cell transplantation is a potential treatment strat- egy for neurodegenerative diseases such as glaucoma. The Muller stem cell line MIO-M1 can be differentiated to produce retinal neurons and glia. The survival, migration, differentia- tion, and integration of MIO-M1 cells were investigated in a rat model of glaucoma. The effect of modulating the retinal envi- ronment with either chondroitinase ABC or erythropoietin was also studied. METHODS. Intraocular pressure was chronically increased uni- laterally by using a laser glaucoma model in adult rats. EGFP- transduced MIO-M1 cells were transplanted into the vitreous or subretinal space of glaucomatous or untreated eyes. Oral im- mune suppressants were administered to reduce xenograft rejection. Survival, migration, differentiation, and integration of grafted cells were assessed by immunohistochemistry. RESULTS. Transplanted cells survived for 2 to 3 weeks in vivo, although microglia/macrophage infiltration and a reduction in graft survival were seen by 4 weeks. Grafted cells displayed a migratory phenotype with an elongated bipolar shape often oriented toward the retina. Transplanted cells expressed mark- ers such as PSA-NCAM, GFAP, and -III-tubulin. The host retina was resistant to MIO-M1 migration, but modification of the local environment with erythropoietin or chondroitinase ABC facilitated retinal infiltration by MIO-M1 cells. CONCLUSIONS. The results demonstrate that differentiating MIO-M1 cells within the glaucomatous eye produced cells that expressed neuronal and glial cell markers. The retina was relatively resistant to transplant integration, and long-term xenograft survival was limited. However, local modulation of the retinal environment enhanced the integration of MIO-M1 cells into the glaucomatous retina. (Invest Ophthalmol Vis Sci. 2008;49:3449 -3456) DOI:10.1167/iovs.08-1770

A 23-year-old Caucasian female with known hydrocephalus and a ventriculoperitoneal shunt in situ presented with a 3-month history of intermittent blurred vision, and headaches of increasing frequen...

PURPOSE. To develop a modified adenoassociated viral (AAV) vector capable of efficient transfection of retinal ganglion cells (RGCs) and to test the hypothesis that use of this vector to express brain-derived neurotrophic factor (BDNF) could be protective in experimental glaucoma. METHODS. Ninety-three rats received one unilateral, intravitreal injection of either normal saline (n 30), AAV-BDNF-woodchuck hepatitis posttranscriptional regulatory element (WPRE; n 30), or AAV-green fluorescent protein (GFP)-WPRE (n 33). Two weeks later, experimental glaucoma was induced in the injected eye by laser application to the trabecular meshwork. Survival of RGCs was estimated by counting axons in optic nerve cross sections after 4 weeks of glaucoma. Transgene expression was assessed by immunohistochemistry, Western blot analysis, and direct visualization of GFP. RESULTS. The density of GFP-positive cells in retinal wholemounts was 1,828 299 cells/mm 2 (72,273 11,814 cells/ retina). Exposure to elevated intraocular pressure was similar in all groups. Four weeks after initial laser treatment, axon loss was 52.3% 27.1% in the saline-treated group (n 25) and 52.3% 24.2% in the AAV-GFP-WPRE group (n 30), but only 32.3% 23.0% in the AAV-BDNF-WPRE group (n 27). Survival in AAV-BDNF-WPRE animals increased markedly and the difference was significant compared with those receiving either AAV-GFP-WPRE (P 0.002, t-test) or saline (P 0.006, t-test). CONCLUSIONS. Overexpression of the BDNF gene protects RGC as estimated by axon counts in a rat glaucoma model, further supporting the potential feasibility of neurotrophic therapy as a complement to the lowering of IOP in the treatment of glaucoma. (Invest Ophthalmol Vis Sci. 2003;44:4357‐ 4365)

Purpose To use a rat model of optic nerve injury to differentiate primary and secondary retinal ganglion cell (RGC) injury. Methods Under general anesthesia, a modified diamond knife was used to transect the superior one third of the orbital optic nerve in albino Wistar rats. The number of surviving RGC was quantified by counting both the number of cells retrogradely filled with fluorescent gold dye injected into the superior colliculus 1 week before nerve injury and the number of axons in optic nerve cross sections. RGCs were counted in 56 rats, with 24 regions examined in each retinal wholemount. Rats were studied at 4 days, 8 days, 4 weeks, and 9 weeks after transection. The interocular difference in RGCs was also compared in five control rats that underwent no surgery and in five rats who underwent a unilateral sham operation. It was confirmed histologically that only the upper optic nerve had been directly injured. Results At 4 and 8 days after injury, superior RGCs showed a mean difference from their fellow eyes of -30.3% and -62.8%, respectively (P = 0.02 and 0.001, t-test, n = 8 rats/group), whereas sham-operation eyes had no significant loss (mean difference between eyes = 1.7%, P = 0.74, t-test). At 8 days, inferior RGCs were unchanged from control, fellow eyes (mean interocular difference = -4.8%, P = 0.16, t-test). Nine weeks after transection, inferior RGC had 34.5% fewer RGCs than their fellow eyes, compared with 41.2% fewer RGCs in the superior zones of the injured eyes compared with fellow eyes. Detailed, serial section studies of the topography of RGC axons in the optic nerve showed an orderly arrangement of fibers that were segregated in relation to the position of their cell bodies in the retina. Conclusions A model of partial optic nerve transection in rats showed rapid loss of directly injured RGCs in the superior retina and delayed, but significant secondary loss of RGCs in the inferior retina, whose axons were not severed. The findings confirm similar results in monkey eyes and provide a rodent model in which pharmacologic interventions against secondary degeneration can be tested.

Background Consumption of edible mushrooms has been suggested to improve health. A number of isolated mushroom constituents have been shown to modulate immunity. Five commonly consumed edible mushrooms were tested to determine whether whole mushrooms stimulate the immune system in vitro and in vivo. Results The white button (WB) extracts readily stimulated macrophage production of TNF-α. The crimini, maitake, oyster and shiitake extracts also stimulated TNF-α production in macrophage but the levels were lower than from WB stimulation. Primary cultures of murine macrophage and ovalbumin (OVA) specific T cells showed that whole mushroom extracts alone had no effect on cytokine production but co-stimulation with either lipopolysacharide or OVA (respectively) induced TNF-α, IFN-γ, and IL-1β while decreasing IL-10. Feeding mice diets that contained 2% WB mushrooms for 4 weeks had no effect on the ex vivo immune responsiveness or associated toxicity (changes in weight or pathology of liver, kidney and gastrointestinal tract). Dextran sodium sulfate (DSS) stimulation of mice that were fed 1% WB mushrooms were protected from DSS induced weight loss. In addition, 2% WB feeding protected the mice from transient DSS induced colonic injury. The TNF-α response in the colon and serum of the DSS challenged and 2% WB fed mice was higher than controls. Conclusion The data support a model whereby edible mushrooms regulate immunity in vitro. The in vivo effects of edible mushrooms required a challenge with DSS to detect small changes in TNF-α and transient protection from colonic injury. There are modest effects of in vivo consumption of edible mushrooms on induced inflammatory responses. The result is not surprising since it would certainly be harmful to strongly induce or suppress immune function following ingestion of a commonly consumed food.