Amino-acid levels in subretinal and vitreous fluid of patients with retinal detachment.

PURPOSE: To compare the concentration of amino acids in subretinal and vitreous fluid of patients with primary rhegmatogenous retinal detachment to that of control vitreous. METHODS: This prospective, observational study measured amino-acid levels in subretinal fluid of patients undergoing scleral buckle placement (n=20) and vitreous fluid in patients undergoing pars plana vitrectomy (n=5) for primary retinal detachment. Vitreous fluid from patients undergoing vitrectomy for macular hole (n=7) or epiretinal membrane (n=3) served as a control. Subretinal fluid and control vitreous were analysed using high-pressure liquid chromatography. Retinal detachment vitreous was analysed using capillary electrophoresis-laser-induced fluorescence. RESULTS: Mean levels of glutamate (27.0+/-1.7 microM), aspartate (4.1+/-4.0 microM), and glycine (44.1+/-31.0 microM) in subretinal fluid and glutamate (13.4+/-11.9 microM) in the vitreous were significantly elevated in retinal detachment compared to control vitreous. A significant, positive association was observed between levels of aspartate and glutamate in subretinal fluid (Spearman's correlation coefficient: 0.74, P<0.01). Mean arginine levels did not differ significantly between subretinal fluid and control vitreous. Levels of alanine, tyrosine, valine, isoleucine, leucine, and phenylalanine were significantly lower in subretinal fluid compared to control vitreous (all P<0.01). CONCLUSIONS: Glutamate levels in subretinal fluid and vitreous of patients with primary retinal detachment is significantly elevated in comparison to control vitreous. This finding lends further support to the hypothesis that elevated glutamate levels may result from ischaemia of the outer retina secondary to retinal detachment.

Outcomes following 25-gauge vitrectomies.

PURPOSE: Twenty-five gauge vitrectomy surgery offers potential advantages over standard 20-gauge vitrectomy surgery, but the short- and long-term post-operative complications, such as cataract formation, are still being evaluated. This study quantifies the outcomes seen following 25-gauge vitrectomies. METHODS: This is a retrospective, consecutive, non-comparative case series of 25-gauge vitrectomies performed between January 2002 and August 2004. Cases without at least 3 months of follow-up and previous vitrectomies were excluded. Analyses were performed with t-test and Kaplan-Meier curves. RESULTS: Seventy-one cases met inclusion criteria. The mean age of the patients was 65 years old (SD 11 years). A variety of surgical indications were included. A statistically significant difference was seen between the mean preoperative visual acuity (20/100) and the mean visual acuity at the 3-month post-operative visit (20/60; P<0.0001). A Kaplan-Meier curve established that for all cases 63.4% of eyes required cataract extraction at 1 year. Total mean follow-up time was 8.6+/-5.5 months. CONCLUSIONS: Statistically significant improvement was seen in mean vision by 3 months following 25-gauge vitrectomy. Cataract formation after 25-gauge vitrectomies remains an important consideration.

A new model system for lipid interactions in stratum corneum vesicles: effects of lipid composition, calcium, and pH.

We prepared large unilamellar vesicles (LUVs) with three different stratum corneum lipid compositions: constant amounts of ceramides (55 wt %) and fatty acids (15%) with varying amounts of cholesterol sulfate (0-15%) and cholesterol (15-30%). One of the compositions served as a model for normal stratum corneum, while the second one served as a model for recessive X-linked ichthyosis stratum corneum. The third composition consisted of no cholesterol sulfate. Intervesicle lipid interactions in these LUVs were monitored by fluorescence methods for content leakage, and contents mixing at pH 9, in the absence and presence of Ca2+, and at pH 6. Since the content leakage and contents mixing assays were originally developed for phospholipid vesicles, we characterized the probe binding and the probe quenching properties for stratum corneum LUV systems, and modified the assays slightly accordingly. The time-dependent fluorescence intensity changes in the probe-containing LUVs at pH 9 and 6 and in response to the addition of calcium were monitored. Our results demonstrated that all three types of LUVs were relatively stable at pH 9. Addition of Ca2+ or decreasing the pH to 6 activated intervesicle lipid mixing followed by vesicle fusion and lysis. We found that the LUVs with no cholesterol sulfate and 30% cholesterol exhibited a more extensive Ca2+- or low-pH-activated intervesicle lipid interaction than LUVs with either 5% cholesterol sulfate and 25% cholesterol or 15% cholesterol sulfate and 15% cholesterol. These results suggest that fusogenic agents such as Ca2+ and H+ act to neutralize the fatty acids in the lipid bilayer of stratum corneum vesicles. The inclusion of 5-15% cholesterol sulfate helps to prevent the collapse of fused vesicles into other structures.

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.

Molecular properties of a stratum corneum model lipid system: large unilamellar vesicles.

Stratum corneum lipids are relatively complex, and there is little detailed understanding of their chemical and physical properties at the molecular level. Large unilamellar vesicles (LUVs) with lipid compositions similar to those of stratum corneum were prepared at pH 9 with commercially available lipids. This system was used as a model system for molecular studies of stratum corneum lipids. LUVs were chosen as the model system as they are comparatively more stable and can be characterized more quantitatively in terms of lipid concentration, surface area, and volume than model systems such as lipid mixture suspensions, lipid films, and small unilamellar vesicles. Results from freeze-fracture and cryo electron microscopy studies of our LUVs showed spherical vesicles. Quasi-elastic light scattering measurements revealed a narrow size distribution, centering around 119 nm. At room temperature, the LUVs were stable for several weeks at pH 9 and for more than 15 h but less than 24 h at pH 6. Differential scanning calorimetry measurements indicated broad endothermic transitions centered near 60-65 degrees C, closely matching the transition temperature reported for stratum corneum lipid extracts. Spin probes, 5-doxylstearic acid and 12-doxylstearic acid, were used for electron paramagnetic resonance (EPR) studies of the molecular dynamics of the lipids. EPR results indicated more restricted motion near the polar headgroup region than near the center of the alkyl chain region. Motional profiles of the spin labels near the polar headgroup and within the alkyl chain region in the LUVs were obtained as a function of temperature, ranging from 25 to 90 degrees C. We also found that the partitioning between the lipid and aqueous phases for each spin probe was temperature dependent and was generally correlated with phase transitions observed by differential scanning calorimetry and with alkyl chain mobility observed by EPR. Thus, this LUV system is well suited for additional molecular studies under different experimental conditions.

Influence of the route of infection of Pasteurella anatipestifer on the clinical and immune responses of white Pekin ducks.

The clinical, pathological and immunological responses were compared in ducklings infected by the intramuscular, oral and intranasal routes with virulent Pasteurella anatipestifer. Intramuscular challenge resulted in clinical signs of infection and caused 100 per cent mortality within three days. No disease signs or death were observed in the orally challenged ducks. Whereas intranasal inoculation caused no deaths, signs of infection were observed in two of 12 birds four days later. In the orally challenged group, low concentrations of antibodies (0.17 log2 to 4.5 log2) were detected in the tracheal washes of five of nine birds examined using an enzyme-linked immunosorbent assay. Humoral antibodies were detected in only one of these birds. In the intranasally infected group, serum antibody levels ranging in titre from 0.62 log2 to 6.2 log2 were found in four of nine birds examined over seven to 14 days following infection. Nine of the birds in this group were shown to have low concentrations of antibodies (0.50 log2 to 6.33 log2) in the tracheal washings. The demonstration of antibodies in the tracheal washings, but not in the serum of nine birds examined, suggested that a local immune response had occurred. However, these studies have shown that antibodies present on the tracheal surface can also be derived from antibodies given intraperitoneally.

Development of an enzyme-linked immunosorbent assay for the detection of humoral antibody to Pasteurella anatipestifer.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to Pasteurella anatipestifer in duck sera is described. As part of the initial assay development, micro-titration plates from different manufacturers were assayed for their suitability to bind P. anatipestifer antigen. The Nunc Immunopiate II was chosen, on account of its overall reproducibility (5.5% coefficient of variation) and the absence of an edge effect. Optimum concentrations of reagents were determined and the inclusion of 1.0M NaCl in the wash buffer was found to reduce non-specific binding and increase assay sensitivity. The assay is specific in that antibodies were detected only in those ducks either exposed to or following vaccination with P. anatipestifer; sera from ducks immunised with other heterologous bacterial antigens, and having agglutinating antibodies to them, gave no detectable response in the ELISA. Between-assay coefficients of variation for the quality control serum pools representing high, medium and low levels of antibodies to P. anatipestifer were 6.8%, 8.3% and 8.6% respectively. A precision-dose profile was derived. A graph of absorbance versus log(2) serum end point titre showed a linear relationship (r = 0.99) over the range investigated. The derived regression line (P<0.001) was used to transform the absorbance measurement obtained for a single 1:100 dilution of serum into a log(2) titre value. It was demonstrated that the ELISA is a much superior method to rapid slide agglutination and agar gel precipitin tests in measuring antibody responses to exposure against P. anatipestifer type 2.

An investigation into the association between liver damage and metastatic uveal melanoma.

We examined the effect of liver damage induced by carbon tetrachloride on the incidence of hepatic metastasis in the Greene hamster melanoma. Nine of 17 animals with liver damage had melanoma growth in the liver after the intraportal vein injection of tumor cells, whereas tumor growth occurred in four of 27 control animals (P less than .01). These results suggest that, in this model, liver damage predisposes to metastatic involvement by melanoma.