Contact lens interactions with the tear film

Biochemical changes brought about by the influence of the contact lens on the tear film are conveniently split into two categories. Firstly, the lens can remove or reduce the levels of specific components in the tear film, and secondly, the lens can augment the tear film, by stimulating the influx of new components or increasing the level of existing components. The most obvious tear film components for study in this context are lipids, proteins, mucins and electrolytes. The interactions are affected by the properties of the lens, the characteristics of the individual wearer and the wear schedule. An additional complicating factor is the fact that the lens is many times thicker than the tear film and any immobilised tear components will be more extensively exposed to oxygen and UV radiation than is the case in the absence of a lens. It is arguably the lipoidal components that are most markedly affected by lens wear, since their immobilisation on the lens surface markedly increases their susceptibility to autoxidative degradation. The limited information that is available highlights the importance of subject specificity and suggests that lipid oxidation phenomena are potentially important in contributing to the 'end of day' discomfort of symptomatic contact lens patients. It is clear that tear lipids, although regarded as relatively inert for many years, are now seen as a reactive and potentially important family of compounds in the search for understanding of contact lens-induced discomfort. The influence of the lens on tear proteins shows the greatest range of complexity. Deposition and denaturation can stimulate immune response, lower molecular weight proteins can be extensively absorbed into the lens matrix and the lens can stimulate cascade or upregulation processes leading either to the generation of additional proteins and peptides or an increase in concentration of existing components. Added to this is the stimulating influence of the lens on vascular leakage leading to the influx of plasma proteins such as albumin. The evidence from studies of mucin expression in tears is not consistent and conclusive. This is in part because sample sources, lens materials and methods of analysis vary considerably, and in some cases the study population numbers are low. Expression levels show mucin and material specificity but clear patterns of behaviour are elusive. The electrolyte composition of tears is significantly different from that of other body fluids. Sodium and potassium dominate but potassium ion concentrations in tears are much higher than in serum levels. Calcium and magnesium concentrations in tears are lower than in serum but closer to interstitial fluids. The contact lens provides the potential for increased osmolarity through enhanced evaporation and differential electrolyte concentrations between the anterior and posterior tear films. Since the changes in ocular biochemistry consequent upon contact lens wear are known to be subject-dependent - as indeed is wearer response to the lens - pre-characterisation of individual participant tear chemistry in clinical studies would enhance understanding of these complex effects. © 2013 Elsevier Ltd.

Synthesis, self-assembly and (absence of) protein interactions of poly(glycerol methacrylate)-silicone macro-amphiphiles

The present study focuses on the synthesis of amphiphilic block copolymers containing poly(glycerol monomethacrylate) (PGMMA), showing the advantages of a protection/deprotection strategy based on silyl groups. PGMMA blocks were synthesized via ATRP started by a double functional poly(dimethyl siloxane) (PDMS) macroinitiator of molecular weight ≈7000 g mol-1. The resulting triblock copolymers were characterized by low polydispersity (generally ≤1.1) and their aggregation concentration in water was essentially dominated by the PDMS block length (critical aggregation concentration substantially invariant for GMMA degree of polymerization ≥30). For GMMA blocks with DP > 50, the self-assembly in water produced 35-50 nm spherical micelles, while shorter hydrophilic chains produced larger aggregates apparently displaying worm-like morphologies. Block copolymers with long GMMA chains (DP ≈ 200) produced particularly stable micellar aggregates, which were then selected for a preliminary assessment of the possibility of adsorption of plasma proteins (albumin and fibrinogen); using diffusion NMR as an analytical technique, no significant adsorption was recorded both on micelles and on soluble PGMMA employed as a control, indicating the possibility of a "stealth" behaviour. This journal is © 2013 The Royal Society of Chemistry.

Protein lipoxidation:detection strategies and challenges

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets.

Novel ageing-biomarker discovery using data-intensive technologies

Ageing is accompanied by many visible characteristics. Other biological and physiological markers are also well-described e.g. loss of circulating sex hormones and increased inflammatory cytokines. Biomarkers for healthy ageing studies are presently predicated on existing knowledge of ageing traits. The increasing availability of data-intensive methods enables deep-analysis of biological samples for novel biomarkers. We have adopted two discrete approaches in MARK-AGE Work Package 7 for biomarker discovery; (1) microarray analyses and/or proteomics in cell systems e.g. endothelial progenitor cells or T cell ageing including a stress model; and (2) investigation of cellular material and plasma directly from tightly-defined proband subsets of different ages using proteomic, transcriptomic and miR array. The first approach provided longitudinal insight into endothelial progenitor and T cell ageing.This review describes the strategy and use of hypothesis-free, data-intensive approaches to explore cellular proteins, miR, mRNA and plasma proteins as healthy ageing biomarkers, using ageing models and directly within samples from adults of different ages. It considers the challenges associated with integrating multiple models and pilot studies as rational biomarkers for a large cohort study. From this approach, a number of high-throughput methods were developed to evaluate novel, putative biomarkers of ageing in the MARK-AGE cohort.

Protein modification and phospholipid oxidation

Phospholipid oxidation can generate reactive and electrophilic products that are capable of modifying proteins, especially at cysteine, lysine and histidine residues. Such lipoxidation reactions are known to alter protein structure and function, both with gain of function and loss of activity effects. As well as potential importance in the redox regulation of cell behaviour, lipoxidation products in plasma could also be useful biomarkers for stress conditions. Although studies with antibodies suggested the occurrence of lipoxidation adducts on ApoB-100, these products had not previously been characterized at a molecular level. We have developed new mass spectrometry-based approaches to detect and locate adducts of oxidized phospholipids in plasma proteins, as well as direct oxidation modifications of proteins, which avoid some of the problems typically encountered with database search engines leading to erroneous identifications of oxidative PTMs. This approach uses accurate mass extracted ion chromatograms (XICs) of fragment ions from peptides containing oxPTMs, and allows multiple modifications to be examined regardless of the protein that contains them. For example, a reporter ion at 184.074 Da/e corresponding to phosphocholine indicated the presence of oxidized phosphatidylcholine adducts, while 2 reporter ions at 100.078 and 82.025 Da/e were selective for allysine. ApoB-100-oxidized phospholipid adducts were detected even in healthy human samples, as well as LDL from patients with inflammatory disease. Lipidomic studies showed that more than 350 different species of lipid were present in LDL, and were altered in disease conditions. LDL clearly represents a very complex carrier system and one that offers a rich source of information about systemic conditions, with potential as indicators of oxidative damage in ageing or inflammatory diseases.

The lipid- and lipoprotein- [LDL-Lp(a)] apheresis techniques. Updating

Therapeutic plasmapheresis allows the extracorporeal removal of plasmatic lipoproteins (Lipid-apheresis) (LA). It can be non selective (non specific), semi - selective or selective low density lipoprotein-lipoprotein(a) (specific [LDL- Lp(a)] apheresis) (Lipoprotein apheresis, LDLa). The LDL removal rate is a perfect parameter to assess the system efficiency. Plasma-Exchange (PEX) cannot be considered either specific nor, selective. In PEX the whole blood is separated into plasma and its corpuscular components usually through centrifugation or rather filtration. The corpuscular components mixed with albumin solution plus saline (NaCl 0.9%) solution at 20%-25%, are then reinfused to the patient, to substitute the plasma formerly removed. PEX eliminates atherogenic lipoproteins, but also other essential plasma proteins, such as albumin, immunoglobulins, and hemocoagulatory mediators. Cascade filtration (CF) is a method based on plasma separation and removal of plasma proteins through double filtration. During the CF two hollow–fiber filters with pores of different diameter are used to eliminate the plasma components of different weight and molecular diameter. A CF system uses a first polypropylene filter with 0.55 µm diameter pores and a second one of diacetate of cellulose with 0.02 µm pores. The first filter separates the whole blood, and the plasma is then perfused through a second filter which allows the recovery of molecules with a diameter lower than 0.02 µm, and the removal of molecules larger in diameter as apoB100–containing lipoproteins. Since both albumin and immunoglobulins are not removed, or to a negligible extent, plasma-expanders, substitution fluids, and in particular albumin, as occurs in PEX are not needed. CF however, is characterized by lower selectivity since removes also high density lipoprotein (HDL) particles which have an antiatherogenic activity. In the 80’s, a variation of Lipid-apheresis has been developed which allows the LDL-cholesterol (LDLC) (-61%) and Lp(a) (-60%) removal from plasma through processing 3 liters of filtered plasma by means of lipid-specific thermofiltration, LDL immunoadsorption, heparin-induced LDL precipitation, LDL adsorption through dextran sulphate. More recently (90’s) the DALI®, and the Liposorber D® hemoperfusion systems, effective for apoB100- containing lipoproteins removal have been developed. All the above mentioned systems are established LDL-apheresis techniques referable to the generic definition of LDLa. However, this last definition cannot describe in an appropriate manner the removal of another highly atherogenic lipoprotein particle: the Lp(a). Thus it would be better to refer the above mentioned techniques to the wider scientific and technical concept of lipoprotein apheresis. Lipid apheresis - Lipoprotein apheresis - LDL-apheresis - Severe Dyslipidemia.

Serum levels of lycopene, beta-carotene, and retinol and their correlation with sperm DNA damage in normospermic and infertile men

Background: Oxidative stress in reproductive system leads to sperm DNA damage and sperm membrane lipid peroxidation and may play an important role in the pathogenesis of male infertility, especially in idiopathic cases. Antioxidants such as carotenoids function against free radical damages. Objective: The aim of this study was to determine the levels of lycopene, beta-carotene and retinol in serum and their relationship with sperm DNA damage and lipid peroxidation in infertile and normospermic males. Materials and Methods: Sixty two infertile men and 71 normospermic men participated in this study. Blood and semen samples were collected from all subjects. Sperm DNA damage was measured using TUNEL method. Carotenoids, retinol, and malonedildehyde in serum were also determined. Results: DNA fragmentation was higher in infertile group comparing to control group. Serum levels of lycopene, beta-carotene and, vitamin A in infertile men were significantly lower than normospermic men (p< 0.001, =0.005, and =0.003 respectively). While serum MDA was not significantly different between two groups, MDA in seminal plasma of infertile men was significantly higher than control group (p< 0.001). Conclusion: We concluded that lycopene, beta-carotene, and retinol can reduce sperm DNA fragmentation and lipid peroxidation through their antioxidant effect. Therefore the DNA fragmentation assay and determination of antioxidants factors such as lycopene, beta-carotene and retinol, along with sperm analysis can be useful in diagnosis and treatment of men with idiopathic infertility.

Longitudinal comparison between plasma and seminal HIV-1 viral loads during antiretroviral treatment

This study was designed to investigate the impact of anti-retroviral therapy on both plasma and seminal HIV-1 viral loads and the correlation between viral loads in these compartments after treatment. Viral load, CD4+ and CD8+ T-cell counts were evaluated in paired plasma and semen samples from 36 antiretroviral therapy-naïve patients at baseline and on days 45, 90, and 180 of treatment. Slopes for blood and seminal viral loads in all treated patients were similar (p = 0.21). Median HIV-1 RNA titers in plasma and semen at baseline were 4.95 log10 and 4.48 log10 copies/ml, respectively. After 180 days of therapy, the median viral load declined to 3.15 log10 copies/ml (plasma) and 3.2 log10 copies/ml (semen). At this timepoint 22 patients presented HIV-1 viral load below 400 copies/ml in either plasma or semen, but only 9 had viral loads below 400 copies/ml in both compartments.

Lack of Correlation between Seminal and Plasma HIV-1 Viral Loads is Associated with CD4T Cell Depletion in Therapy-naïve HIV-1+ Patients

The present study was conducted to investigate a possible correlation between plasma (PVL) and seminal viral load (SVL) on treatment-naïve HIV-1-infected patients in Vitória, ES, Brazil. We also evaluated whether the progressive immunosuppression associated with HIV disease (as evidenced by declining CD4 T cell counts) has any impact on the correlation between PVL and SVL HIV-1. Viral load on paired blood and semen samples from 56 consecutive treatment-naïve patients were evaluated and compared to CD4 cell counts. Viral load and T cell counts (cells/µl) were determined by NASBA and by flow cytometry, respectively. Overall, a strong positive correlation between PVL and SVL (rho = 0.438, p = 0.001) was observed. However, when patients were grouped according to their CD4 counts, this correlation was only significant among patients with CD4 counts > 200 cells/µl. Results presented here demonstrate the existence of a strong correlation between PVL and SVL on patients with CD4 cell counts > 200 cells/µl, suggesting that this association may correlate with disease progression.

Binding of d-methadone, l-methadone, and dl-methadone to proteins in plasma of healthy volunteers: role of the variants of alpha 1-acid glycoprotein.

The plasma concentrations of alpha 1-acid glycoprotein (AAG), albumin, triglycerides, cholesterol, and total proteins, as well as the plasma binding of racemic, d-methadone, and l-methadone were measured in 45 healthy subjects. The AAG phenotypes and the concentrations of AAG variants were also determined. The measured free fractions for racemic, d-methadone, and l-methadone were, respectively, 12.7% +/- 3.3%, 10.0% +/- 2.9%, and 14.2% +/- 3.2% (mean +/- SD). A significant correlation was obtained between the binding ratio (B/F) for dl-methadone and the total AAG concentration (r = 0.724; p less than 0.001). A multiple stepwise regression analysis showed that AAG was the main explanatory variable for the binding of the racemate. When concentrations of AAG variants were considered, a significant correlation was obtained between the binding ratio of dl-methadone and orosomucoid2 A concentration (r = 0.715; p less than 0.001), a weak correlation between dl-methadone and orosomucoid1 S concentration (r = 0.494; p less than 0.001), and no correlation between dl-methadone and orosomucoid1 F1 concentration (r = 0.049; not significant). Similar findings were obtained with the enantiomers. This study shows the importance of considering not only total AAG but also concentrations of AAG variants when measuring the binding of methadone and possibly of other drugs in plasma.

Cloning of a cDNA encoding a plasma membrane-associated, uronide binding phosphoprotein with physical properties similar to viral movement proteins.

Oligogalacturonides are structural and regulatory homopolymers from the extracellular pectic matrix of plants. In vitro micromolar concentrations of oligogalacturonates and polygalacturonates were shown previously to stimulate the phosphorylation of a small plasma membrane-associated protein in potato. Immunologically cross-reactive proteins were detected in plasma membrane-enriched fractions from all angiosperm subclasses in the Cronquist system. Polygalacturonate-enhanced phosphorylation of the protein was observed in four of the six dicotyledon subclasses but not in any of the five monocotyledon subclasses. A cDNA for the protein was cloned from potato. The deduced protein is extremely hydrophilic and has a proline-rich N terminus. The C-terminal half of the protein was predicted to be a coiled coil, suggesting that the protein interacts with other macromolecules. The recombinant protein was found to bind both simple and complex galacturonides. The behavior of the protein suggests several parallels with viral proteins involved in intercellular communication.

The role of plasma membrane STIM1 and Ca(2+)entry in platelet aggregation. STIM1 binds to novel proteins in human platelets.

Ca(2+) elevation is essential to platelet activation. STIM1 senses Ca(2+) in the endoplasmic reticulum and activates Orai channels allowing store-operated Ca(2+) entry (SOCE). STIM1 has also been reported to be present in the plasma membrane (PM) with its N-terminal region exposed to the outside medium but its role is not fully understood. We have examined the effects of the antibody GOK/STIM1, which recognises the N-terminal region of STIM1, on SOCE, agonist-stimulated Ca(2+) entry, surface exposure, in vitro thrombus formation and aggregation in human platelets. We also determined novel binding partners of STIM1 using proteomics. The dialysed GOK/STIM1 antibody failed to reduced thapsigargin- and agonist-mediated Ca(2+) entry in Fura2-labelled cells. Using flow cytometry we detect a portion of STIM1 to be surface-exposed. The dialysed GOK/STIM1 antibody reduced thrombus formation by whole blood on collagen-coated capillaries under flow and platelet aggregation induced by collagen. In immunoprecipitation experiments followed by proteomic analysis, STIM1 was found to extract a number of proteins including myosin, DOCK10, thrombospondin-1 and actin. These studies suggest that PM STIM1 may facilitate platelet activation by collagen through novel interactions at the plasma membrane while the essential Ca(2+)-sensing role of STIM1 is served by the protein in the ER.

A concentração, a composição e a qualidade do plasma seminal na preservação do sêmen eqüino a +4ºC.

O presente trabalho constou de três experimentos. O primeiro objetivou verificar a influência de diferentes concentrações de plasma seminal e de dois diluentes na motilidade e na integridade e funcionalidade da membrana plasmática de espermatozóides eqüinos resfriados. Para tanto, foram utilizados 4 garanhões, comprovadamente férteis e em atividade sexual. Imediatamente após a coleta, o sêmen foi avaliado, diluído 1:2 com EDTA-glicose, dividido em oito alíquotas e centrifugado a 600g, por 10 minutos, para remoção do plasma seminal. O pellet de cada alíquota foi ressuspendido com um determinado volume do plasma seminal, previamente removido e acrescido de um determinado volume de um dos dois diluente (leite desnatado UHT ou leite desnatado-glicose) até atingir uma concentração final entre 40 e 50x106 espermatozóides/ml, contendo as seguintes concentrações finais de plasma seminal: 0%, 2,5%, 5% e 10%. Imediatamente após a diluição, o sêmen foi avaliado quanto à motilidade progressiva e total e funcionalidade e integridade da membrana plasmática. A seguir, os oito frascos contendo o sêmen, com um volume aproximado de 12 ml cada, foram resfriados em câmara a +4ºC a uma taxa de resfriamento de 0,3º C/min, sendo o sêmen novamente avaliado às 24, 48 e 72 horas.

Percoll e plasma seminal na preservação do sêmen eqüino a +4º C

O presente estudo visou verificar o efeito sobre alguns parâmetros da seleção por gradiente de Percoll® e da adição de plasma seminal do sêmen eqüino preservado a +4oC. O primeiro experimento avaliou a taxa de recuperação de espermatozóides após seleção por Percoll® em diferentes protocolos de centrifugação. Foram realizadas 5 coletas de sêmen de um garanhão. Imediatamente após a coleta, o sêmen foi avaliado quanto à motilidade, vigor e concentração. Foram retiradas duas amostras de 4 mL, diluídas em leite desnatado UHT com concentrações de 50 e 100 x 106 espermatozóides por mL cada. Cada uma destas amostras foi dividida em 4 alíquotas de 1 mL, que foram então colocadas sobre Percoll® e submetidas a diferentes tempos e velocidades de centrifugação. V1 - 200 g (5 min) + 800 g (10 min); V2 - 800 g (10 min); V3 - 800 g (15 min); V4 - 800 g (20 min). Após esse processo, o sobrenadante foi desprezado e o pellet de cada alíquota ressuspendido com 0,5 mL de leite UHT. As 8 amostras foram novamente avaliadas para concentração, motilidade e vigor. O segundo experimento estudou o efeito da adição de plasma seminal de diferentes qualidades ao sêmen eqüino selecionado por gradiente de Percoll® e resfriado a +4°C por até 72 horas. Foram utilizados 40 ejaculados de 4 garanhões, sendo dois com boa qualidade de sêmen e dois com baixa qualidade de sêmen. Imediatamente após a coleta, o sêmen foi avaliado quanto à motilidade, vigor e concentração e preparadas cinco frações de 100x106 espermatozóides, diluídas 1:1 (v/v) em EDTA-Glicose. Quatro delas, constituídas por 1mL a 2mL, foram depositadas sobre Percoll®. A fração restante foi centrifugada em tubo de vidro de 10 mL, sob as mesmas condições de tempo e velocidade das demais amostras. Foi realizada centrifugação por 5 minutos em 200 g, seguida de 10 minutos em 800 g. O sobrenadante foi descartado e o pellet ressuspendido com leite UHT desnatado compondo os seguintes tratamentos: Sp: 1,5 mL de leite UHT desnatado sem adição de plasma seminal; Hp: 1,425 mL de leite UHT desnatado acrescido de 75 L de plasma seminal homólogo; Ap: 1,425 mL de leite UHT desnatado acrescido de 75 L de plasma seminal do pool de alta qualidade; Bp: 1,425mL de leite UHT desnatado acrescido de 75 L plasma seminal do pool de baixa qualidade; Cc: foi centrifugada sem seleção por Percoll®, teve seu sobrenadante descartado e ressuspendida com 2 mL de leite UHT; C : uma amostra de sêmen diluído em leite UHT foi mantida como controle no processo de armazenamento. As amostras foram resfriadas a +4°C e examinadas a cada 24h até as 72 horas em relação à motilidade, funcionalidade de membrana (teste hiposmótico) e integridade de membrana (CFDA/PI). A seleção por gradiente descontínuo de Percoll® 90/45% mostrou-se efetiva na recuperação de espermatozóides com motilidades progressiva e total. A adição de 5% de plasma seminal ou a ausência de plasma não influenciaram os valores da motilidade das amostras selecionadas por gradiente de Percoll®. A seleção por Percoll não influenciou na percentagem de células com membrana plasmática funcional. Concentrações superiores a 2% de plasma seminal resultaram em decréscimo do número de células com membrana funcional, enquanto que as amostras sem plasma seminal apresentaram os melhores resultados e o grupo controle, que apresentava a maior percentagem de plasma, os piores. A utilização de Percoll® não separou células com alteração de membrana. A percentagem de células com membrana completamente íntegra foi significativamente maior nas amostras que sofreram processo de centrifugação, independentemente da seleção. O processo de seleção por Percoll® foi efetivo na recuperação de espermatozóides de eqüino com motilidade progressiva, mas não selecionou espermatozóides quanto à funcionalidade nem à integridade de membrana. Concentrações inferiores a 2% de plasma seminal melhoraram a funcionalidade de membrana. A ausência de plasma seminal melhorou os resultados de integridade das membranas plasmática e acrossomal; e a adição de plasma de alta qualidade não melhorou a motilidade de espermatozóides selecionados por Percoll®.

Análise comparativa do perfil lipídico do plasma seminal associado ao estresse oxidativo

O estresse oxidativo é um dos fatores mais importantes na diminuição da qualidade do sêmen, pois leva a perda da integridade da membrana dos espermatozóides e de danos estruturais ao DNA através da cascata de lipoperoxidação. Os danos funcionais relacionados ao estresse oxidativo como a diminuição da motilidade e da viabilidade do espermatozóide são algumas das principais causas de infertilidade masculina. Ainda, os lipídios que compõe a membrana plasmática são macromoléculas que, além de estarem envolvidas em complexos sistemas biológicos e processos metabólicos da célula, são altamente susceptíveis ao processo de lipoperoxidação desencadeado pelo estresse oxidativo. Neste contexto, este projeto propôs o estudo das alterações no perfil lipídico do plasma seminal que pudessem estar relacionadas ao estresse oxidativo e posterior comparação destes perfis em busca de biomarcadores de infertilidade. Para isso, foram coletadas amostras de sêmen de 116 pacientes que procuraram o setor de Reprodução Humana da Universidade Federal de São Paulo. Estas amostras foram submetidas a técnica de TBARS para quantificação dos produtos finais do estresse oxidativo e separação dos grupos, e em seguida, ao protocolo de extração de lipídios para obtenção dos espectros de massa através da técnica de MALDI (Matrix Assisted Laser Disorption Ionization). Com esta análise foi possível a identificação de 31 lipídeos super representados nos diferentes grupos e que, futuramente, poderão vir a ser utilizados na avaliação da qualidade seminal