A Novel ELISA to Detect Methionine Sulfoxide−Containing Apolipoprotein A−I

Atherosclerosis manifests a state of increased oxidative stress characterized by comparable lipid and protein oxidation in the affected arterial wall. While oxidative modification of low density lipoprotein (LDL) has been extensively studied, increasing attention has been focused recently on oxidation of high-density lipoproteins (HDL) and its functional consequences in relation to atherosclerosis. Oxidative modification is thought to generate “dysfunctional” HDL that has lost anti-atherosclerotic activities, including the ability to remove cholesterol from lipid-laden cells. Therefore, there has been much interest in the detection of oxidized HDL. Unfortunately, available methods to detect oxidized HDL are limited at present, in part because oxidative modification of HDL is a complex process and ‘oxidized HDL’ is not a chemically defined entity. What is known however is that conversion of methionine (Met) residues of apolipoprotein (apo) A-I to methionine sulfoxide (MetO) is a process that occurs commonly as HDL undergoes oxidative modification. For example, human apoA-I+16 (containing MetO86 or MetO112) and apoA-I+32 (MetO86 plus MetO112) are generated when apoA-I reacts with lipid hydroperoxides formed as a consequence of the lipoprotein being exposed to 1e−oxidants. The formation of MetO in apoA−I induced by 2e−oxidants (i.e., hydrogen peroxide, hypochlorous acid or myeloperoxidase/hydrogen peroxide/chloride system) is associated with an impaired ability of the apolipoprotein to facilitate reactions relevant to reverse cholesterol transport. In addition, a previous study has suggested the plasma content of apoA-I+32 to be increased in certain subjects that have an increased risk to develop cardiovascular disease (CVD). Moreover, the MetO content in circulating, HDL−associated apoA−I is elevated in type 1 diabetes, a disorder commonly associated with increased oxidative stress and a risk factor for atherosclerosis. Therefore, in the present study, an existing HPLC method was applied to HDL samples from the Fletcher−Challenge study, a nested case control study, to test the potential usefulness of MetO-containing apoA-I as a marker of oxidative stress and/or CVD in a general population. Plasma samples whose HDL contained detectable apoA-I+16 and/or apoA-I+32 had significantly elevated levels of F2-isoprostanes, a marker of in vivo lipid oxidation, consistent with MetO-containing apoA-I being a useful marker of in vivo protein oxidation. Despite this however, there was no significant difference between controls and cases in their concentrations of HDL apoA-I+16 and apoA-I+32 or F2-isoprostanes, suggesting that markers of protein and lipid oxidation are not associated with the risk of coronary heart disease (CHD) in this general population. A limitation of the Fletcher−Challenge study was that only 22% of the 534 HDL samples analyzed contained apoA-I+16 and/or apoA-I+32. In addition, the HPLC−based method used is expensive and time−consuming and may lack the sensitivity needed for apolipoproteins to clinical studies. Thus, a mouse monoclonal anti-human apoA-I+32 antibody (MOA−1) was raised using HPLC−purified apoA-I+32 as immunogen. A sensitive ELISA was then developed using a commercial anti-human apoA-I monoclonal antibody as capture and biotinylated MOA−1 as detection antibody, respectively. The assay detected lipid−free HPLC−purified human apoA-I+32 in a concentration-dependent manner and with a significantly lower limit of detection (i.e., 3 ng/mL) than the HPLC method (1 μg/mL). The ELISA also detected lipid-free apoA-I modified by 2e-oxidants (hydrogen peroxide, hypochlorous acid, peroxynitrite), and HDL oxidized by 1e- or 2e-oxidants and present in buffer or human plasma. Moreover, the extent of recognition of MetO by MOA−1 increased with increasing numbers of MetO in apoA−I, as assessed by the experiments with H2O2−oxidized forms of apoA−I mutants, in which one, two or three Met residues were replaced with Leu. Their detection was concentration-dependent, reproducible, and exhibited a linear response over a physiologically plausible range of concentrations of oxidized HDL. In contrast, MOA-I failed to recognize native apoA-I, native apoA-II, apoA-I modified by hydroxyl radicals or metal ions, or LDL modified by 2e-oxidants. Furthermore, MOA−1 did not detect other Met−containing proteins oxidized by either hypochlorous acid or hydrogen peroxide. Taken together, the results showed that recognition of oxidized proteins by MOA−1 is limited to MetO contained in apoA−I. Finally, in a pilot study, plasma samples obtained from subjects with coronary artery disease (CAD) proven by angiography, and samples from CAD patients undergoing percutaneous coronary intervention (PCI) were analyzed by the ELISA. The preliminary data obtained showed elevated levels of MetO-containing apoA-I in plasma samples of CAD patients compared to those of corresponding control subjects. Unexpectedly, levels of MetOcontaining apoA-I decreased PCI compared to before PCI. A possible explanation for these results is that HDL−associated apoA−I become displaced by acute phase proteins, such as serum amyloid A, in response to PCI. In summary, the ELISA developed here specifically detects apoA-I containing MetO in HDL and human plasma. As such it may provide a useful tool for investigating the relationship between oxidized HDL and CAD

LONGITUDINAL STUDY OF RPE65-ASSOCIATED INHERITED RETINAL DEGENERATIONS

PURPOSE: To study the disease course of RPE65-associated inherited retinal degenerations (IRDs) as a function of the genotype, define a critical age for blindness, and identify potential modifiers. METHODS: Forty-five patients with IRD from 33 families with biallelic RPE65 mutations, 28 stemming from a genetic isolate. We collected retrospective data from medical charts. Coexisting variants in 108 IRD-associated genes were identified with Molecular Inversion Probe analysis. RESULTS: Most patients were diagnosed within the first years of life. Daytime visual function ranged from near-normal to blindness in the first four decades and met WHO criteria for blindness for visual acuity and visual field in the fifth decade. p.(Thr368His) was the most common variant (54%). Intrafamilial variability and interfamilial variability in disease severity and progression were observed. Molecular Inversion Probe analysis confirmed all RPE65 variants and identified one additional variant in LRAT and one in EYS in two separate patients. CONCLUSION: All patients with RPE65-associated IRDs developed symptoms within the first year of life. Visual function in childhood and adolescence varied but deteriorated inevitably toward blindness after age 40. In this study, genotype was not predictive of clinical course. The variance in severity of disease could not be explained by double hits in other IRD genes

Training shortens search times in children with visual impairment accompanied by nystagmus.

Contains fulltext : 139609.pdf (publisher's version ) (Open Access)Perceptual learning (PL) can improve near visual acuity (NVA) in 4-9 year old children with visual impairment (VI). However, the mechanisms underlying improved NVA are unknown. The present study compares feature search and oculomotor measures in 4-9 year old children with VI accompanied by nystagmus (VI+nys [n = 33]) and children with normal vision (NV [n = 29]). Children in the Vl+nys group were divided into three training groups: an experimental PL group, a control PL group, and a magnifier group. They were seen before (baseline) and after 6 weeks of training. Children with NV were only seen at baseline. The feature search task entailed finding a target E among distractor E's (pointing right) with element spacing varied in four steps: 0.04, 0.5, 1, and 2. At baseline, children with Vl+nys showed longer search times, shorter fixation durations, and larger saccade amplitudes than children with NV. After training, all training groups showed shorter search times. Only the experimental PL group showed prolonged fixation duration after training at 0.5 and 2 spacing, p's respectively 0.033 and 0.021. Prolonged fixation duration was associated with reduced crowding and improved crowded NVA. One of the mechanisms underlying improved crowded NVA after PL in children with Vl+nys seems to be prolonged fixation duration

Training shortens search times in children with visual impairment accompanied by nystagmus

Contains fulltext : 131483.pdf (publisher's version ) (Open Access)Perceptual learning (PL) can improve near visual acuity (NVA) in 4-9 year old children with visual impairment (VI). However, the mechanisms underlying improved NVA are unknown. The present study compares feature search and oculomotor measures in 4-9 year old children with VI accompanied by nystagmus (VI+nys [n = 33]) and children with normal vision (NV [n = 29]). Children in the Vl+nys group were divided into three training groups: an experimental PL group, a control PL group, and a magnifier group. They were seen before (baseline) and after 6 weeks of training. Children with NV were only seen at baseline. The feature search task entailed finding a target E among distractor E's (pointing right) with element spacing varied in four steps: 0.04, 0.5, 1, and 2. At baseline, children with Vl+nys showed longer search times, shorter fixation durations, and larger saccade amplitudes than children with NV. After training, all training groups showed shorter search times. Only the experimental PL group showed prolonged fixation duration after training at 0.5 and 2 spacing, p's respectively 0.033 and 0.021. Prolonged fixation duration was associated with reduced crowding and improved crowded NVA. One of the mechanisms underlying improved crowded NVA after PL in children with Vl+nys seems to be prolonged fixation duration.11 p

Saccade latencies during a preferential looking task and objective scoring of grating acuity in children with and without visual impairments

Contains fulltext : 207064.pdf (publisher's version ) (Open Access

Monocular and binocular development in children with albinism, infantile nystagmus syndrome, and normal vision

Item does not contain fulltextAbstract Background/aims: To compare interocular acuity differences, crowding ratios, and binocular summation ratios in 4- to 8-year-old children with albinism (n = 16), children with infantile nystagmus syndrome (n = 10), and children with normal vision (n = 72). Methods: Interocular acuity differences and binocular summation ratios were compared between groups. Crowding ratios were calculated by dividing the single Landolt C decimal acuity with the crowded Landolt C decimal acuity mono- and binocularly. A linear regression analysis was conducted to investigate the contribution of 5 predictors to the monocular and binocular crowding ratio: nystagmus amplitude, nystagmus frequency, strabismus, astigmatism, and anisometropia. Results: Crowding ratios were higher under mono- and binocular viewing conditions for children with infantile nystagmus syndrome than for children with normal vision. Children with albinism showed higher crowding ratios in their poorer eye and under binocular viewing conditions than children with normal vision. Children with albinism and children with infantile nystagmus syndrome showed larger interocular acuity differences than children with normal vision (0.1 logMAR in our clinical groups and 0.0 logMAR in children with normal vision). Binocular summation ratios did not differ between groups. Strabismus and nystagmus amplitude predicted the crowding ratio in the poorer eye (p = 0.015 and p = 0.005, respectively). The crowding ratio in the better eye showed a marginally significant relation with nystagmus frequency and depth of anisometropia (p = 0.082 and p = 0.070, respectively). The binocular crowding ratio was not predicted by any of the variables. Conclusions: Children with albinism and children with infantile nystagmus syndrome show larger interocular acuity differences than children with normal vision. Strabismus and nystagmus amplitude are significant predictors of the crowding ratio in the poorer eye

Noise patterns in 6- to 8-year-old children’s eye movements during crowded visual search

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Low vision aids in young visually impaired children: prospects from motor control

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Specificity and retention of visual perceptual learning in young children with low vision

Contains fulltext : 220263.pdf (publisher's version ) (Open Access