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Primary open angle (POAG) and pseudoexfoliative glaucoma (PXG) are the most common primary and secondary forms of glaucoma, respectively. Even though the patho-physiology, aqueous humor composition, risk factors, clinical features, therapy and drug induced ocular surface changes in POAG and PXG have been widely studied, to date information concerning tear protein characterization is lacking. Tears are a source of nourishment for ocular surface tissues and a vehicle to remove local waste products, metabolized drugs and inflammatory mediators produced in several ophthalmic diseases. In glaucoma, the proteomic definition of tears may provide insights concerning patho-physiology of the disease and ocular surface modifications induced by topical therapy. Our study aimed at characterizing protein patterns in tears of patients with medically controlled POAG and PXG. A comparative tears proteomic analysis by label-free LC-MS(E) highlighted differences in the expression of several proteins in the two glaucoma sub-types and control subjects, highlighting inflammation pathways expressed in both diseases. Results were independently reconfirmed by SDS-PAGE and linear MALDI-TOF MS, validating altered levels of Lysozyme C, Lipocalin-1, Protein S100, Immunoglobulins and Prolactin Inducible Protein. Moreover, we found a differential pattern of phosphorylated Cystatin-S that distinguishes the two pathologies. The most relevant results suggest that in both pathologies there may be active inflammation pathways related to the disease and/or induced by therapy. We show, for the first time, tear protein patterns expressed under controlled intraocular pressure conditions in POAG and PXG subjects. These findings could help in the understanding of molecular machinery underlying these ophthalmologic diseases, resulting in early diagnosis and more specific therapy.

Background Carbonylation is an irreversible modification caused by the introduction into proteins of carbonyl derivatives (aldehydes and ketones), which can alter protein structure and function and lead to cellular dysfunction and tissue damage. Chronic uremia may be associated with an increased carbonyl overload ("carbonyl stress"), though carbonyl formation has been proposed so far for major plasma proteins only. In this study we looked for evidence and for the targets of plasma protein carbonylation in patients on hemodialysis. We also examined the effect of in vitro carbonylated albumin on mRNA levels of endothelial cell adhesion molecules involved in early atherogenesis. Methods Carbonylated proteins in uremic plasma were detected by a covalent hydrazine bait strategy and identified by combining electrophoretic separation with mass spectrometry analysis of tryptic digests. Some plasma samples were first depleted of albumin and immunoglobulins to improve detection of lower abundance proteins. The functional impact of carbonylation was assessed in human vein endothelial cells by studying models of modified human serum albumin. Results Post-dialysis plasma carbonylated protein levels were significantly increased compared to pre-dialysis levels. Susceptibility to carbonyl formation was described on a open-platform investigation for a number of plasma proteins, albumin being the main scavenger of carbonyl reactive species. Incubation of endothelial cells with low doses of carbonylated albumin caused a significant increase in intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 mRNA levels. Conclusions Chronic uremia appears as a state of "carbonyl stress" targeting several different plasma proteins. Carbonylated albumin displayed biological effects that may be relevant to uremic atherosclerosis.

The exposure of the aminophospholipid phosphatidylserine on the external leaflet of red blood cell plasma membrane can have several pathophysiological consequences with particular regard to the processes of cell phagocytosis, haemostasis and cell-cell interaction. A significant increase in phosphatidylserine-exposing erythrocytes has been reported in chronic haemodialysis patients and found to be strongly influenced by the uraemic milieu. To identify uraemic compound(s) enhancing phosphatidylserine externalization in erythrocytes, we fractionated by chromatographic methods the ultrafiltrate obtained during dialysis, and examined by flow cytometry the effect of the resulting fractions on phosphatidylserine exposure in human red cells. Chromatographic procedures disclosed a homogeneous fraction able to increase erythrocyte phosphatidylserine exposure. The inducer of such externalization was identified by monodimensional gel electrophoresis and mass spectrometry investigations as beta2-microglobulin. To confirm the beta2-microglobulin effect and to examine the influence of protein glycation (as it occurs in uraemia) on phosphatidylserine erythrocyte exposure, erythrocytes from normal subjects were incubated with recombinant beta2-microglobulin (showing no glycation sites at mass analysis), commercial beta2-microglobulin (8 glycation sites), or with in vitro glycated recombinant beta2-microglobulin (showing multiple glycation sites). Elevated concentrations of beta2-microglobulin (corresponding to plasma levels reached in dialysis patients) increased slightly but significantly the protein's ability to externalize phosphatidylserine on human erythrocytes. Such an effect was markedly enhanced by glycated forms of the protein. Beta2-microglobulin is recognized as a surrogate marker of middle-molecule uraemic toxins and represents a key component of dialysis-associated amyloidosis. Our study adds further evidence to the potential pathophysiologic consequences of beta2-microglobulin accumulation in chronic uraemic patients.

Protein carbonylation is an irreversible and not reparable reaction which is caused by the introduction into proteins of carbonyl derivatives such as ketones and aldehydes, generated from direct oxidation processes or from secondary protein reaction with reactive carbonyl compounds. Several studies have demonstrated significantly increased levels of reactive carbonyl compounds, a general increase in plasma protein carbonyls and carbonyl formation on major plasma proteins in blood from uremic patients, particularly those undergoing chronic haemodialysis.In the present preliminary study, we first assessed by an in vitro filtration apparatus the possible effects of different materials used for haemodialysis membranes on protein retention and carbonylation. We employed hollow fiber minidialyzers of identical structural characteristics composed of either polymethylmethacrylate, ethylenevinyl alcohol, or cellulose diacetate materials. Protein Western Blot and SDS-PAGE coupled to mass spectrometry analysis were applied to highlight the carbonylated protein-binding characteristics of the different materials. We also investigated in vivo protein carbonylation and carboxy methyl lisine-modification in plasma obtained before and after a haemodialysis session.Our data underline a different capability on protein adsorption associated with the different properties of the filter materials, highlighting the central buffering and protective role of serum albumin. In particular, polymethylmethacrylate and cellulose diacetate showed, in vitro, the highest capacity of binding plasma proteins on the surface of the hollow fiber minidialyzers.The present study suggests that biomaterials used for fabrication of haemodialysis membrane may affect the carbonyl balance in chronic uremic patients.

Protein-adsorptive properties are a key feature of membranes used for haemodialysis treatment. Protein adsorption is vital to the biocompatibility of a membrane material and influences membrane's performance. The object of the present study is to investigate membrane biocompatibility by correlating the adsorbed proteome repertoire with chemical feature of the membrane surfaces. Dialyzers composed of either cellulose triacetate (Sureflux 50 L, effective surface area 0.5 m(2); Nipro Corporation, Japan) or the polysulfone-based helixone (FX40, effective surface area 0.4 m(2); Fresenius Medical Care AG, Germany) materials were employed to develop an ex vivo apparatus to study protein adsorption. Adsorbed proteins were eluted by a strong chaotropic buffer condition and investigated by a proteomic approach. The profiling strategy was based on 2D-electrophoresis separation of desorbed protein coupled to MALDI-TOF/TOF analysis. The total protein adsorption was not significantly different between the two materials. An average of 179 protein spots was visualised for helixone membranes while a map of retained proteins of cellulose triacetate membranes was made up of 239 protein spots. The cellulose triacetate material showed a higher binding capacity for albumin and apolipoprotein. In fact, a number of different protein spots belonging to the gene transcript of albumin were visible in the cellulose triacetate map. In contrast, helixone bound only a small proportion of albumin, while proved to be particularly active in retaining protein associated with the coagulation cascade, such as the fibrinogen isoforms. Our data indicate that proteomic techniques are a useful approach for the investigation of proteins surface-adsorbed onto haemodialysis membranes, and may provide a molecular base for the interpretation of the efficacy and safety of anticoagulation treatment during renal replacement therapy.