Toll-like receptors and vascular markers in ocular rosacea.

PURPOSE: To delineate signals by which the vascular abnormalities inherent to ocular rosacea arise and to correlate these signals with elements of the innate immune system. METHODS: Experimental study. Immunohistochemical staining was performed for a variety of vascular markers and for toll-like receptor-4 on eyelid biopsies taken from patients with ocular rosacea and normal controls. Statistical comparisons were then performed between the 2 groups. RESULTS: Immunohistochemical staining for CD31 and integrin-ß-3 did not demonstrate any statistically significant differences between eyelids from patients with ocular rosacea and normal controls. Cutaneous biopsies from ocular rosacea patients demonstrated statistically significant enrichments of intercellular adhesion molecule-1 and CD105 among arterioles, whereas there were no statistically significant differences in the venules between normal controls and ocular rosacea patients. The correlation between the number of toll-like receptor-4-positive cells and each vascular marker was statistically significant. CONCLUSIONS: Cutaneous biopsies of the eyelid did not demonstrate an increase in the total number of blood vessels. However, the vascular abnormalities that are typical of ocular rosacea represent activated, inflamed vessels, and these phenomena may be mediated by intercellular adhesion molecule and CD105. Furthermore, the strong correlations between toll-like receptor-4 and each vascular marker suggest that the innate immune system may govern the cutaneous effects of ocular rosacea. Intercellular adhesion molecule, CD105, and toll-like receptor-4 may represent important therapeutic targets in the management of this disease.

Central Corneal Thickness in Childhood Cataract.

PURPOSE: We explored elevated central corneal thickness (CCT) in children with cataracts as possibly reflecting preexisting corneal malformation related to specific cataract morphology. METHODS: All children consecutively seen during the study periods who had cataracts and corneal pachymetry as part of their routine care were enrolled at academic centers in large cities of Canada and the United States. Study data collected included age, sex, CCT, and cataract morphology. Differences among cataract morphology groups with respect to mean CCT measurements were evaluated and compared with a historical control thickness of 558 µm. RESULTS: A total of 96 children were enrolled in this study. The average subject age was 5.1 years, and 55 children (57%) were female. The mean CCT value for all subjects was 566.1 µm. There was little evidence to conclude that the cataract morphology groups differed from each other (P = 0.65) or from controls with respect to CCT. CONCLUSIONS: In children, CCT is likely independent of cataract morphology. This implies that factors other than preoperative malformation are more likely related to elevated CCT observed in children with aphakia and pseudophakia.

Retinal abnormalities in ß-thalassemia major.

Patients with beta (ß)-thalassemia (ß-TM: ß-thalassemia major, ß-TI: ß-thalassemia intermedia) have a variety of complications that may affect all organs, including the eye. Ocular abnormalities include retinal pigment epithelial degeneration, angioid streaks, venous tortuosity, night blindness, visual field defects, decreased visual acuity, color vision abnormalities, and acute visual loss. Patients with ß-thalassemia major are transfusion dependent and require iron chelation therapy to survive. Retinal degeneration may result from either retinal iron accumulation from transfusion-induced iron overload or retinal toxicity induced by iron chelation therapy. Some who were never treated with iron chelation therapy exhibited retinopathy, and others receiving iron chelation therapy had chelator-induced retinopathy. We will focus on retinal abnormalities present in individuals with ß-thalassemia major viewed in light of new findings on the mechanisms and manifestations of retinal iron toxicity.

Burden and Depression in Caregivers of Blind Patients in New York State.

PURPOSE: To describe the degree of burden of care and the proportion at risk of depression among individuals caring for legally blind patients. METHODS: We performed a cross-sectional study of 486 individuals providing care to their family members who were legally blind. Best-corrected visual acuity of the better-seeing eye in patients determined group placement: Group 1, 20/200-10/200; group 2, 10/200 to light perception (LP); group 3, no light perception (NLP); group VF, visual field loss to <20 central degrees. Burden was evaluated using the Burden Index of Caregivers (BIC-11) and the prevalence at risk of depression was determined by the Center for Epidemiologic Studies Depression (CES-D) scale. RESULTS: Total mean BIC-11 scores ranged from 8.78 ± 4.82 (group 1) to 12.03 ± 5.22 (group 3; p = 0.04). Daily hours spent on close supervision, intensity of caregiving and presence of multiple chronic illnesses in caregivers were the significant covariates affecting BIC-11 scores (p < 0.05). The prevalence of caregivers at risk of depression increased with vision loss from 6.9% (group 1) to 17.9% (group 3; p < 0.05). Female caregivers had an odds ratio (OR) of 2.89 for depression (95% confidence interval, CI, 1.07-3.97; p = 0.04). Caregivers with ≥2 comorbidities had OR 4.24 (95% CI 2.41-6.11) for risk of depression (p < 0.01). CONCLUSION: Burden of care was highest among caregivers who provided greater hours of supervision. Patients with more limitations in their activities of daily living had caregivers who reported higher burden. Female caregivers and caregivers with multiple chronic illnesses were at higher risk of depression.

CD1 Mouse Retina Is Shielded From Iron Overload Caused by a High Iron Diet.

PURPOSE: High RPE iron levels have been associated with age-related macular degeneration. Mutation of the ferroxidase ceruloplasmin leads to RPE iron accumulation and degeneration in patients with aceruloplasminemia; mice lacking ceruloplasmin and its homolog hephaestin have a similar RPE degeneration. To determine whether a high iron diet (HID) could cause RPE iron accumulation, possibly contributing to RPE oxidative stress in AMD, we tested the effect of dietary iron on mouse RPE iron. METHODS: Male CD1 strain mice were fed either a standard iron diet (SID) or the same diet with extra iron added (HID) for either 3 months or 10 months. Mice were analyzed with immunofluorescence and Perls' histochemical iron stain to assess iron levels. Levels of ferritin, transferrin receptor, and oxidative stress gene mRNAs were measured by quantitative PCR (qPCR) in neural retina (NR) and isolated RPE. Morphology was assessed in plastic sections. RESULTS: Ferritin immunoreactivity demonstrated a modest increase in the RPE in 10-month HID mice. Analysis by qPCR showed changes in mRNA levels of iron-responsive genes, indicating moderately increased iron in the RPE of 10-month HID mice. However, even by age 18 months, there was no Perls' signal in the retina or RPE and no retinal degeneration. CONCLUSIONS: These findings indicate that iron absorbed from the diet can modestly increase the level of iron deposition in the wild-type mouse RPE without causing RPE or retinal degeneration. This suggests regulation of retinal iron uptake at the blood-retinal barriers.

Optic nerve morphology in normal children.

PURPOSE: To expand the normative database of pediatric eyes analyzed with the Optovue optical coherence tomography (OCT). METHODS: In a community-based, cross-sectional analysis, 77 healthy 5-year-old children were recruited from pediatric practices. No subject had any known ocular disorder. Their optic nerves were assessed using Optovue optical coherence tomography (OCT). Data were compared to the normative database obtained by Optovue for adults, ages 18-25, 40-45, and 55-60. Comparisons included thickness of retinal nerve fiber layer (RNFL) and ganglion cell complex (GCC), and optic nerve head disk area, vertical (VCD) and horizontal (HCD) cup:disk ratios, and cup area. RESULTS: In comparison to all adult age groups, the children's optic nerve heads were statistically less cupped (cup area, VCD, HCD), all with P values of <0.0003. No statistical difference was identified between the children and adults aged 18-25 and 40-45 with respect to RNFL or GCC. Both RNFL and GCC were thicker in the children compared to adults aged 55-60 (P < 0.003). Children's disk area's were marginally smaller than those of adults, the difference not reaching statistical significance. CONCLUSIONS: Our OCT results demonstrate that young children's optic nerves are statistically less cupped than those of all normal adults. The children's RNFL and GCC are statistically thicker than those of adults aged 55-60.

Fish oil improves gene targets of Down syndrome in C57BL and BALB/c mice.

We have considered a novel gene targeting approach for treating pathologies and conditions whose genetic bases are defined using diet and nutrition. One such condition is Down syndrome, which is linked to overexpression of RCAN1 on human chromosome 21 for some phenotypes. We hypothesize that a decrease in RCAN1 expression with dietary supplements in individuals with Down syndrome represents a potential treatment. Toward this, we used in vivo studies and bioinformatic analysis to identify potential healthy dietary RCAN1 expression modulators. We observed Rcan1 isoform 1 (Rcan1-1) protein reduction in mice pup hippocampus after a 4-week curcumin and fish oil supplementation, with only fish oil reduction being statistically significant. Focusing on fish oil, we observed a 17% Rcan1-1 messenger RNA (mRNA) and 19% Rcan1-1 protein reduction in BALB/c mice after 5 weeks of fish oil supplementation. Fish oil supplementation starting at conception and in a different mouse strain (C57BL) led to a 27% reduction in hippocampal Rcan1-1 mRNA and a 34% reduction in spleen Rcan1-1 mRNA at 6 weeks of age. Hippocampal protein results revealed a modest 11% reduction in RCAN1-1, suggesting translational compensation. Bioinformatic mining of human fish oil studies also revealed reduced RCAN1 mRNA expression, consistent with the above studies. These results suggest the potential use of fish oil in treating Down syndrome and support our strategy of using select healthy dietary agents to treat genetically defined pathologies, an approach that we believe is simple, healthy, and cost-effective.

Overexpression of RCAN1 isoform 4 in mouse neurons leads to a moderate behavioral impairment.

OBJECTIVES: Recent evidence supports the involvement of RCAN1 in Down syndrome and Alzheimer's disease. To better assess this, we generated and analyzed transgenic mice overexpressing human RCAN1 isoform 4 in neurons. METHODS: Cognitive behavioral (Morris water maze, open field, zero maze, elevated plus maze assays); cognitive-associated proteins (CREB, ERK and Tau Western immunoblotting); motor coordination (Rotarod assay); structural abnormalities (immunohistological analyses), and proinflammatory cytokines (cytometric bead assay) were measured in young (2 month) and old (18 month) transgenics and compared with wild type controls. RESULTS: In old mice, male but not female transgenics exhibited a significant decrease in anxiety as compared with wild type controls, whereas female but not male transgenic mice exhibited significantly less motor coordination. No differences were observed in the Morris water maze (spatial learning). pERK levels were reduced in transgenic males but not females, while no differences were observed between genotypes for pCREB and pTau. In young mice, a modest learning and exploratory behavior was observed in transgenic mice using a limited number of mice, and at higher N values, pCREB and pERK (but not pTau) levels were reduced in transgenics. No macro- and micro-scopic structural abnormalities or proinflammatory cytokine level differences were observed. DISCUSSION: These results indicate that elevated RCAN1 isoform 4 in neurons leads to a modest cognition-related impairment that is overall stronger at 2 months, suggesting a compensatory adaptation over time. These RCAN1 isoform 4 effects may contribute to at least some of the observed phenotypes in individuals with Down syndrome and Alzheimer's.

Degraded mitochondrial DNA is a newly identified subtype of the damage associated molecular pattern (DAMP) family and possible trigger of neurodegeneration.

We previously showed a preferential degradation and down-regulation of mitochondrial DNA and RNA in hamster fibroblasts in response to hydrogen peroxide. Subsequent studies by others demonstrated that mitochondrial DNA can stimulate immune cells as a DAMP (damage associated molecular patterns) family member. However, the actual physical structure of this mitochondrial DNA DAMP and its importance in non-immune cell types are poorly understood. Here we report that transfected oxidant-initiated degraded mitochondrial polynucleotides, which we term "DeMPs", strongly induce the proinflammatory cytokines interleukin 6, monocyte chemotactic protein-1, and tumor necrosis factor α in mouse primary astrocytes. Additionally, proinflammatory IL1ß was induced, implicating DeMPs in inflammasome activation. Furthermore, human cerebrospinal fluid (CSF) and plasma were found to contain detectable DeMP signal. Finally, significant degradation of mitochondrial DNA was observed in response to either a bolus or steady state hydrogen peroxide. Combined, these studies demonstrate, all for the first time, that a pathophysiologically relevant form of mitochondrial DNA (degraded) can elicit a proinflammatory cytokine induction; that a brain cell type (astrocytes) elicits a proinflammatory cytokine induction in response to these DeMPs; that this induction includes the inflammasome; that astrocytes are capable of inflammasome activation by DeMPs; that DeMPs are detectable in CSF and plasma; and that hydrogen peroxide can stimulate an early stage cellular degradation of mitochondrial DNA. These results provide new insights and are supportive of our hypothesis that DeMPs are a newly identified trigger of neurodegenerative diseases such as Alzheimer's disease, which are known to be associated with early stage inflammation and oxidation.

The calcineurin inhibitor RCAN1 is involved in cultured macrophage and in vivo immune response.

Studies on the role of regulator of calcineurin 1 (RCAN1) in immunity are limited, but have demonstrated an involvement in T-lymphocyte function. Here, we expand these studies to macrophages and in vivo infection. The treatment of RAW and primary mouse macrophages with lipopolysaccharide from Escherichia coli strongly induced RCAN1 isoform 4 (RCAN1-4), but not isoform 1. RCAN1-4 induction involved calcium, calcineurin, and reactive oxygen species. Subsequent analysis with whole bacteria including gram-negative E. coli and gram-positive Staphylococcus aureus revealed strong RCAN1-4 inductions by both, and where tested, dependence on calcium. Staphylococcus aureus cell wall components peptidoglycan and lipoteichoic acid also strongly induced RCAN1-4. In vivo, a significant induction in the proinflammatory cytokines monocyte chemotactic protein-1, interleukin-6, interferon-γ, and tumor necrosis factor-α was observed in knockout (KO) lung vs. wild-type (WT) mice 7 days after nasal infection with Fransicella tularensis. This induction was not accompanied by a significant increase in F. tularensis burden in the KO lung. Additionally, a modest increase in respiratory burst activity in KO vs. WT macrophages was observed. Combined, these studies indicate that RCAN1 is involved in macrophage and the overall in vivo immune response, and provide additional evidence that RCAN1 plays an important role in cell immunity and infectious disease.

Select phytochemicals suppress human T-lymphocytes and mouse splenocytes suggesting their use in autoimmunity and transplantation.

We have considered a novel "rational" gene targeting approach for treating pathologies whose genetic bases are defined using select phytochemicals. We reason that one such potential application of this approach would be conditions requiring immunosuppression such as autoimmune disease and transplantation, where the genetic target is clearly defined; i.e., interleukin-2 and associated T-cell activation. Therefore, we hypothesized that select phytochemicals can suppress T-lymphocyte proliferation both in vitro and in vivo. The immunosuppressive effects of berry extract, curcumin, quercetin, sulforaphane, epigallocatechin gallate (EGCG), resveratrol, alpha-tocopherol, vitamin C and sucrose were tested on anti-CD3 plus anti-CD28-activated primary human T-lymphocytes in culture. Curcumin, sulforaphane, quercetin, berry extract and EGCG all significantly inhibited T-cell proliferation, and this effect was not due to toxicity. IL-2 production was also reduced by these agents, implicating this important T-cell cytokine in proliferation suppression. Except for berry extract, these same agents also inhibited mouse splenic T-cell proliferation and IL-2 production. Subsequent in vivo studies revealed that quercetin (but not sulforaphane) modestly suppressed mouse splenocyte proliferation following supplementation of BALB/c mice diets. This effect was especially prominent if corrected for the loss of supplement "recall" as observed in cultured T-cells. These results suggest the potential use of these select phytochemicals for treating autoimmune and transplant patients, and support our strategy of using select phytochemicals to treat genetically-defined pathologies, an approach that we believe is simple, healthy, and cost-effective.