Parkinson's disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of alpha-synuclein protein (alpha-syn) inside brain cells. It is likely that the formation of alpha-syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding alpha-syn have been found in inherited forms of PD. alpha-Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of alpha-syn in conditioned culture media from untransfected and alpha-syn-transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti-alpha-syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, alpha-syn was identified as a single 15 kDa band, which co-migrated with a recombinant form of the protein and reacted with five different antibodies to alpha-syn. Our findings suggest that cells normally secrete alpha-syn into their surrounding media, both in vitro and in vivo. The detection of extracellular alpha-syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.

To test the hypothesis that tolerating some subretinal fluid (SRF) in patients with neovascular agerelated macular degeneration (nAMD) treated with ranibizumab using a treat-and-extend (T&E) regimen can achieve similar visual acuity (VA) outcomes as treatment aimed at resolving all SRF.Design: Multicenter, randomized, 24-month, phase 4, single-masked, noninferiority clinical trial.Participants: Participants with treatment-naïve active subfoveal choroidal neovascularization (CNV). Methods: Participants were randomized to receive ranibizumab 0.5 mg monthly until either complete resolution of SRF and intraretinal fluid (IRF; intensive arm: SRF intolerant) or resolution of all IRF only (relaxed arm: SRF tolerant except for SRF >200 mm at the foveal center) before extending treatment intervals. A 5-letter noninferiority margin was applied to the primary outcome.Main Outcome Measures: Mean change in best-corrected VA (BCVA), and central subfield thickness and number of injections from baseline to month 24.Results: Of the 349 participants randomized (intensive arm, n ¼ 174; relaxed arm, n ¼ 175), 279 (79.9%) completed the month 24. The mean change in BCVA from baseline to month 24 was 3.0 letters (standard deviation, 16.3 letters) in the intensive group and 2.6 letters (standard deviation, 16.3 letters) in the relaxed group, demonstrating noninferiority of the relaxed compared with the intensive treatment (P ¼ 0.99). Similar proportions of both groups achieved 20/40 or better VA (53.5% and 56.6%, respectively; P ¼ 0.92) and 20/200 or worse VA (8.7% and 8.1%, respectively; P ¼ 0.52). Participants in the relaxed group received fewer ranibizumab injections over 24 months (mean, 15.8 [standard deviation, 5.9]) than those in the intensive group (mean, 17 [standard deviation, 6.5]; P ¼ 0.001). Significantly more participants in the intensive group never extended beyond 4-week treatment intervals (13.5%) than in the relaxed group (2.8%; P ¼ 0.003), and significantly more participants in the relaxed group extended to and maintained 12-week treatment intervals (29.6%) than the intensive group (15.0%; P ¼ 0.005).Conclusions: Patients treated with a ranibizumab T&E protocol who tolerated some SRF achieved VA that is comparable, with fewer injections, with that achieved when treatment aimed to resolve all SRF completely.

Cultures of oral epithelial cells can be generated to confluence on AM expanded ex vivo from biopsy-derived oral mucosal tissue. Autologous transplantation was performed with these cultivated oral epithelial cells onto the ocular surfaces of keratectomized rabbit eyes. Autologous transplantation of cultivated oral epithelium is a feasible method for ocular surface reconstruction. The long-term outcome of such transplantation is not yet clear, and its feasibility in clinical use should be evaluated further.

Alzheimer disease and familial British dementia are neurodegenerative diseases that are characterized by the presence of numerous amyloid plaques in the brain. These lesions contain fibrillar deposits of the ␤-amyloid peptide (A␤) and the British dementia peptide (ABri), respectively. Both peptides are toxic to cells in culture, and there is increasing evidence that early "soluble oligomers" are the toxic entity rather than mature amyloid fibrils. The molecular mechanisms responsible for this toxicity are not clear, but in the case of A␤, one prominent hypothesis is that the peptide can induce oxidative damage via the formation of hydrogen peroxide. We have developed a reliable method, employing electron spin resonance spectroscopy in conjunction with the spin-trapping technique, to detect any hydrogen peroxide generated during the incubation of A␤ and other amyloidogenic peptides. Here, we monitored levels of hydrogen peroxide accumulation during different stages of aggregation of A␤-(1-40) and ABri and found that in both cases it was generated as a short "burst" early on in the aggregation process. Ultrastructural studies with both peptides revealed that structures resembling "soluble oligomers" or "protofibrils" were present during this early phase of hydrogen peroxide formation. Mature amyloid fibrils derived from A␤-(1-40) did not generate hydrogen peroxide. We conclude that hydrogen peroxide formation during the early stages of protein aggregation may be a common mechanism of cell death in these (and possibly other) neurodegenerative diseases.There is mounting evidence for the importance of oxidative damage to the brain in a wide range of neurodegenerative diseases based on detection of markers such as elevated levels of redox-active transition metal ions, lipid peroxidation, DNA and protein oxidation, and the introduction of carbonyl groups into proteins (reviewed, for example, in Refs. 1-6). These are hallmarks of attack by reactive oxygen species (ROS), 3 including superoxide, hydrogen peroxide, and the hydroxyl radical. The ␤-amyloid peptide (A␤), which is responsible for senile plaque formation in Alzheimer disease (AD), has been reported to generate hydrogen peroxide from molecular oxygen through electron transfer interactions involving bound redox-active metal ions (7-10). Hydrogen peroxide is readily converted into the aggressive hydroxyl radical by Fenton chemistry and these two ROS could be responsible for some of the oxidative damage seen in the brain in AD. Familial British dementia (FBD) is an inherited neurodegenerative disorder that is strikingly similar in neuropathology to AD, including the presence of extracellular amyloid plaques and intracellular neurofibrillary tangles. FBD is due to a stop codon mutation in the BRI gene, the protein product of which undergoes proteolytic cleavage to release an abnormally long peptide fragment (ABri) that rapidly aggregates in vitro into toxic oligomers (11,12). Only ABri with an intact intramolecular disulfide bond can do this, whereas the correspondi...

A model of cultivated corneal endothelial transplantation for corneal endothelial dysfunction was established in primates whose corneal endothelial cells have less proliferative capacity in vivo. Our results suggest that this is a useful model for long-term observation in advance of the future clinical application of cultivated corneal endothelial transplantation.

The prostate gland is conventionally divided into zones or regions. This morphology is of clinical significance as prostate cancer (CaP) occurs mainly in the peripheral zone (PZ). We obtained tissue sets consisting of paraffin-embedded blocks of cancer-free transition zone (TZ) and PZ and adjacent CaP from patients (n = 6) who had undergone radical retropubic prostatectomy; a seventh tissue set of snap-frozen PZ and TZ was obtained from a CaP-free gland removed after radical cystoprostatectomy. Paraffin-embedded tissue slices were sectioned (10-mum thick) and mounted on suitable windows to facilitate infrared (IR) spectra acquisition before being dewaxed and air dried; cryosections were dessicated on BaF(2) windows. Spectra were collected employing synchrotron Fourier-transform infrared (FTIR) microspectroscopy in transmission mode or attenuated total reflection-FTIR (ATR) spectroscopy. Epithelial cell and stromal IR spectra were subjected to principal component analysis to determine whether wavenumber-absorbance relationships expressed as single points in "hyperspace" might on the basis of multivariate distance reveal biophysical differences between cells in situ in different tissue regions. After spectroscopic analysis, plotted clusters and their loadings curves highlighted marked variation in the spectral region containing DNA/RNA bands ( approximately 1490-1000 cm(-1)). By interrogating the intrinsic dimensionality of IR spectra in this small cohort sample, we found that TZ epithelial cells appeared to align more closely with those of CaP while exhibiting marked structural differences compared to PZ epithelium. IR spectra of PZ stroma also suggested that these cells are structurally more different to CaP than those located in the TZ. Because the PZ exhibits a higher occurrence of CaP, other factors (e.g., hormone exposure) may modulate the growth kinetics of initiated epithelial cells in this region. The results of this pilot study surprisingly indicate that TZ epithelial cells are more likely to exhibit what may be a susceptibility-to-adenocarcinoma spectral signature. Thus, IR spectroscopy on its own may not be sufficient to identify premalignant prostate epithelial cells most likely to progress to CaP.

Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet b-cells in most cases of type-2 diabetes mellitus (T2Dm). Human amylin 'oligomers' are toxic to these cells. Using two different experimental techniques, we found that H 2 O 2 was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H 2 O 2 and did not interact with copper. We conclude that the formation of H 2 O 2 from amylin could contribute to the progressive degeneration of islet cells in T2Dm.

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