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The focus of this article is to review recent techniques in proteomic analysis of ocular fluids. These fluids include tears, aqueous humor, and vitreous, they will also be compared to serum analysis. Furthermore, we attempt to summarize some disease correlated biomarkers in ocular fluids that were discovered through different proteomic techniques in eye diseases like dry eye, glaucoma, age-related macular degeneration, uveitis, or diabetic retinopathy. This review is trying to point out the importance of these biomarkers for clinical applications., (Copyright © 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To show the existence of IgG antibodies against retinal antigens in aqueous humor of normal tension glaucoma patients., Methods: Forty-two patients were included in this study. Aqueous humor was collected from control subjects (CO; n = 21) and patients with normal tension glaucoma (NTG; n = 21). Western blot methods against bovine retinal antigens were used to detect the IgG antibody patterns. The complex antibody repertoires were analyzed by multivariate statistical techniques. Mass spectrometry was used to identify the most important antigens., Results: Very complex IgG antibody patterns against retinal antigens were found in all analyzed aqueous humor samples. Our multivariate approach could quantify differences in immunoreactivities, and including all peaks, the analysis of discriminance revealed a statistical significant difference between the patterns of the NTG and the CO group (p < 0.001). The antigen band at 21 kDa was identified as alpha B-crystallin, the 57-kDa antigen band as vimentin, and one at 70 kDa as heat shock protein 70., Conclusions: We could demonstrate that complex IgG antibody patterns against retina exist in aqueous humor. The significant differences in the antibody pattern of the glaucoma group compared with the nonglaucoma group in aqueous humor confirm the results of previous studies using sera of glaucoma patients. These differences in antibody patterns might be further evidence for an autoimmune involvement in the pathogenesis of some glaucoma patients.

Background: The aim of this study was to compare the IgG antibody patterns against retinal antigens in sera of patients with age-related macular degeneration (AMD) and healthy subjects to learn more about possible immunological aspects of this disease and to identify some of the most important antigens., Methods: Sera of 140 patients were analyzed: healthy volunteers (CO, n=101) and patients with "wet" age-related macular degeneration (AMD, n=39). The sera were tested against western blots of bovine retinal antigens. The IgG antibody patterns were analyzed by multivariate statistical techniques and some antigens were identified via LC-MS/MS., Results: All patients showed complex patterns of IgG antibodies against retinal antigens. The discriminant analysis revealed a statistical significant difference between the antibody profiles of the AMD and the CO group (P=0.000023). Not only up-regulations of antigen-antibody-reactivities in the AMD group at some molecular weight ranges, e.g. at 46 and 52 kDa, could be seen, but also down-regulations, e.g. at 18 and 36 kDa. The 18 kDa antigen band was identified as alphaB-crystallin, the band at 46 kDa as alpha-enolase, and one at 52 kDa as glial fibrillary acidic protein., Conclusions: We could demonstrate that both groups (wet AMD and CO) show complex IgG antibody patterns against retinal antigens, which are highly specific for each group. This provides further hints for the immunological basis of the disease. These changes in the antibody profiles in "wet" AMD could represent a secondary response to retinal damage or can play a causative role in the disease.

The current review investigates the bioactivity of different glass interfaces created on thin glass cover slips as substrates. The interfaces studied are plain glass, functionalized glass using 0.5 M and 5 M of sodium hydroxide (NaOH) for 24 hrs, and glass coated with bioactive 58S Bioglass (58S). A biomimetic method, involving the exposure of the three interfaces to 1.5 times simulated body fluid (SBF) tests the bioactivity of the interfaces via creation of layer of Hydroxyapatite (HA). Fluorinated SBF will precipitate fluorine doped HA (FHA) on a bioactive interface. Higher concentration of 1.5 times of SBF used in this study intended to accelerate the formation of HA and FHA layer over the substrate. HA and FHA is found to be precipitated on the thinly coated 58S. This paper, study also the thin film coatings of three forms of bioceramics - bioactive 58S, HA and FHA. The study, also proposes to draw a relation between the morphology of HA particles with duration of exposure to SBF, the effects of fluorine on the morphology and the cell interaction with bioactive 58S, HA and FHA interfaces using pre-differentiated osteoblastic MC3T3 cells. The analysis of cells in this study is confined to three parameters that include the attachment, proliferation and viability of cells. Tests employed for the analysis of the thin film coating of HA and FHA is restricted to qualitative X-Ray Diffraction and quantitative Field Emission Scanning Electron Microscope. Other mechanical tests such as shear test are not used to test the mechanical properties of this thin layer, due to the fact that the thin film is too thin for such analysis.

The purpose of this study is to examine the hydrolytic degradation of electron beam irradiated ring-opening polymerized (ROP) poly(l-lactide) (PLLA-ir) and non-irradiated melt polycondensation polymerized poly(l-lactic acid) (PLLA-pc). It was observed that irradiation increases the hydrolytic degradation rate constant for ROP PLLA. This was due to a more hydrophilic PLLA-ir, as a result of irradiation. The degradation rate constants (k) of PLLA-ir samples were also found to be similar, regardless of the radiation dose, and an empirically formulated equation relating hydrolytic degradation time span to radiation dose was derived. The k value for PLLA-pc was observed to be lower than that for PLLA-ir, though the latter had a higher molecular weight. This was due to the difference in degradation mechanism, in which PLLA-ir undergoes end group scission, through a back- biting mechanism, during hydrolysis and thus a faster hydrolysis rate. Electron beam irradiation, though accelerates the degradation of PLLA, has been shown to be useful in accurately controlling the hydrolytic time span of PLLA. This method of controlling the hydrolytic degradation time was by far an easier task than through melt polycondensation polymerization. This would allow PLLA to be used for drug delivery purposes or as a temporary implant that requires a moderate time span (3-6 months).

Purpose: Glaucoma is characterized by a progressive loss of retinal ganglion cells that results in a characteristic optic neuropathy associated with visual field loss. In previous studies, changes in the antibody profiles have been shown in the sera of patients with glaucoma, and these findings suggest a role for autoimmune involvement in the pathogenesis of glaucoma in some patients. The purpose of this study was to compare the antibody profiles against optic nerve antigens in patients with glaucoma in two different study populations from Germany and the United States., Methods: One hundred twenty patients were included in the study, 60 from Germany and 60 from the United States: a control group (CTRL, n = 20), a group of patients with primary open-angle glaucoma (POAG, n = 20), and one group of patients with normal-pressure glaucoma (NPG, n = 20) from each country. Western blot analyses against bovine optic nerve antigens were used to detect the IgG antibody patterns present in the patients' sera. The complex antibody profiles were analyzed by multivariate statistical techniques., Results: Complex IgG autoantibody repertoires were present in all patients with glaucoma as well as healthy subjects from both the German and the United States study population. A large similarity between all antibody profiles in both study populations was demonstrated in the number and frequency of both up- and downregulation of antibody reactivities in patients with glaucoma of both national cohorts. The multivariate analysis of discriminance found a significant difference between the glaucoma groups and healthy subjects against optic nerve antigens. As in previous studies, the NPG group revealed the highest variance from the control group (P < 0.01). Furthermore, a newly described antibody biomarker in both study populations was identified as alpha-fodrin. Western blot results revealed that there was an increased frequency and enhanced immunoreactivity to alpha-fodrin (120 kDa) in the sera of patients with NPG. The presence of alpha-fodrin autoantibodies were confirmed by ELISA, in which a highly elevated anti-alpha-fodrin titer in patients with NPG was found to be significantly greater than in the control subjects (P < 0.01) or age-matched patients with POAG (P < 0.04)., Conclusions: Complex IgG antibody patterns against optic nerve antigens can be reproducibly identified in the serum of study populations from the United States and Germany. In both cohorts, patients with glaucoma have characteristic differences in serum autoantibody repertoires from those in control subjects. A newly described autoantibody to alpha-fodrin found in other neurodegenerative diseases such as Alzheimer's, further implicate a role for autoimmunity and the neurodegenerative processes in glaucoma. The high correspondence of the autoantibody patterns found in the study populations from different continents provides further evidence that serum autoantibody patterns may be useful biomarkers for glaucoma detection or for determining prognosis in future studies by means of pattern-matching algorithms.

Purpose: The aim of this study was to analyze and compare the entire IgG autoantibody patterns against different ocular antigens (retina, optic nerve, and optic nerve head) in sera of glaucoma patients and healthy subjects., Methods: Sixty-six patients were included in this study: healthy volunteers without any ocular disorders (CO, n=30), patients with primary open-angle glaucoma (POAG, n=19), and patients with normal-tension glaucoma (NTG, n=17). The sera were tested for antibodies against retinal, optic nerve, and optic nerve head tissues. Immunodetection was performed using 4-chloro-1-naphthol staining. The autoantibody patterns were digitized and subsequently analyzed by multivariate statistical techniques., Results: All patients showed a complex repertoire of IgG antibodies against retinal, optic nerve, and optic nerve head antigens. The analysis of discriminance revealed a statistically significant differences between the patterns of all three groups. Our multivariate approach could quantify the differences in immunoreactivities of patient sera against the three antigens. The POAG group had the most significant difference against retinal antigens (P=0.0021) compared with the other antigens. In the NTG group the highest reactivity appeared against optic nerve head (P=0.00053) and optic nerve (P=0.0025)., Conclusions: All groups showed different and complex antibody patterns against the three ocular tissues. These autoantibodies are highly specific for each patient group. The analysis of these patterns could provide further information about possible autoimmune mechanisms in the pathogenesis of glaucoma.

The purpose of this study is to examine the effect of electron-beam (e-beam) radiation on the hydrolytic degradation of poly(lactide-co-glycolide) (PLGA) films. PLGA films were irradiated and observed to undergo radiation-induced degradation through chain scission, as observed from a drop in its average molecular weight with radiation dose. Irradiated (5, 10 and 20 Mrad) and non-irradiated (0 Mrad) samples of PLGA were subsequently hydrolytically degraded in phosphate-buffered saline solution at 37.0 degrees C over a span of 12 weeks. It was observed that the natural logarithmic molecular weight (lnMn) of PLGA decreases linearly with hydrolytic degradation time. The rate of water uptake is higher for samples irradiated at higher radiation dose (e.g. 20 Mrad) and subsequently causing an earlier onset of mass loss. It is postulated that the increase in water uptake is due to the presence of more hydrophilic end groups, which results in the formation of microcavities because of an increase in osmotic pressure. A relationship between radiation dose and the rate of hydrolytic degradation of PLGA films, through its molecular weight was also established. This relationship allows a more accurate and precise control of the life span of PLGA through the use of e-beam radiation.

Isothermal crystallization through annealing at 115 degrees C was conducted to increase the degree of crystallinity of poly (lactide-co-glycolide) (PLGA). The maximum increase in the degree of crystallinity (approximately 21%) was achieved after 60 min of annealing. The crystal size/perfection was observed to increase with annealing time. The annealed PLGA films were then hydrolytically degraded in phosphate buffered saline solution of pH 7.4 at 37 degrees C for up to 150 days. Minimal mass loss was observed throughout the time investigated, suggesting that the samples were still in the first phase of degradation. The increase in the degree of crystallinity of the PLGA samples annealed at 15 and 30 min was found to retard their overall rate of hydrolytic degradation, when compared to those samples with higher initial crystallinity (annealed for 45 and 60 min) that had faster degradation rates. The increased degradation rate at higher crystallinity was associated with the loss of amorphous material and the formation of voids during annealing, which decreases the glass transition temperature and increases the average water uptake in the samples annealed for longer times. Therefore, the increase in degree of crystallinity is found to retard hydrolytic degradation but only to a certain extent, beyond which the formation of voids through annealing increases the rate of hydrolytic degradation.

Purpose: Glaucoma is one of the leading causes for blindness in the world. It is characterized by a progressive loss of retinal ganglion cells. An elevated intraocular pressure cannot explain the disease in all subjects. Autoimmune mechanisms maybe involvemed in the pathogenesis of the disease. The aim of our study was to analyze the IgG autoantibody repertoires in sera of glaucomatous subjects against optic nerve antigens., Methods: Ninety-four subjects were included in this comparative cross-sectional study of healthy (CTRL), primary-open-angle-glaucoma (POAG), ocular-hypertension (OHT), and normal-tension-glaucoma (NTG) volunteers. Sera of subjects were tested against western blots of optic nerve antigens. For each western blot, a densitograph was built by digital image analysis and subsequently a multivariate analysis of discriminance was performed., Results: Complex IgG autoantibody repertoires could be found in all subjects, even in healthy subjects. The multivariate analysis of discriminance can test for statistical differences between the groups using the whole complex staining pattern for the calculation. A significant difference between all groups against optic nerve antigens was found. The NTG group had the highest variance from controls (p<0.01)., Conclusions: This study demonstrates immunological effects in POAG and NTG and may provide further evidence for an involvement of autoantibodies in the pathogenesis of both NTG and POAG. Using the techniques presented in this study, the differences in the complex autoantibody repertoires were assessed by means of statistical analysis. Further studies are needed to determine whether these changes in autoantibodies could be helpful in the diagnosis of glaucoma.

Purpose: Glaucoma is the second cause of blindness worldwide. It is usually considered a neurodegenerative disease. There is evidence that an autoimmune mechanism is involved in the development of glaucoma in some patients. The aim of this study was to analyze the IgG autoantibody repertoires in sera of glaucoma patients and healthy subjects., Methods: A total of 82 patients were divided into four groups: healthy volunteers without any ocular disorders (CO, n = 30), patients with primary open-angle glaucoma (POAG, n = 19), ocular hypertension (OHT, n = 16), and normal tension glaucoma (NTG, n = 17). All groups were matched for age and gender. The sera of these patients were tested against Western blots of retinal antigens. Immunodetection was done using 4-chloro-1-naphthol staining. The autoantibody patterns were digitized and subsequently analyzed by multivariate statistical techniques., Results: All patients showed different, complex staining patterns of autoantibodies against retinal antigens. There was an increase in the number of peaks in sera of patients with primary open-angle glaucoma (POAG) compared to healthy subjects (CO). Including all peaks the analysis of discriminance revealed a statistically significant difference between the patterns of POAG compared to all other groups (p < 0.01). Sera of normal tension glaucoma (NTG) had no statistically different autoantibody pattern compared to those of control subjects., Conclusions: In this study, we demonstrated a difference in the IgG autoantibody patterns of primary open-angle glaucoma patients compared to healthy subjects. However, the patterns were not significantly different in normal tension glaucoma compared to control subjects.

Normal sera contain a large number of naturally occurring autoantibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because of its ability to simultaneous screening for a wide spectrum of different antigens. In previous studies we have shown the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF MS (surface-enhanced laser desorption/ionization-time of flight mass spectrometry) technology for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The microscale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biologically activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n = 12). After washing, the chips were incubated with a complex solution of autoantigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were to be detected by mass spectrometry. Previous studies using monoclonal antibodies have demonstrated that the detection limit is in the attomole level. Furthermore, all sera were analyzed by conventional Western blotting for direct comparison. In the present study, we have shown complex on-chip antibody-antigen reactions. At higher molecular weights (> 30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular mass, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time-consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody distributions for diagnostic purposes and early detection of autoimmune diseases might be possible.