Myeloperoxidase released after neutrophil and monocyte activation can generate reactive oxygen species, leading to host tissue damage. Extracellular glomerular myeloperoxidase deposition, seen in ...ANCA-associated vasculitis, may enhance crescentic GN through antigen-specific T and B cell activation. Myeloperoxidase-deficient animals have attenuated GN early on, but augmented T cell responses. We investigated the effect of myeloperoxidase inhibition, using the myeloperoxidase inhibitor AZM198, to understand its potential role in treating crescentic GN.
We evaluated renal biopsy samples from patients with various forms of crescentic GN for myeloperoxidase and neutrophils, measured serum myeloperoxidase concentration in patients with ANCA-associated vasculitis and controls, and assessed neutrophil extracellular trap formation, reactive oxygen species production, and neutrophil degranulation in ANCA-stimulated neutrophils in the absence and presence of AZM198. We also tested the effect of AZM198 on ANCA-stimulated neutrophil-mediated endothelial cell damage
, as well as on crescentic GN severity and antigen-specific T cell reactivity in the murine model of nephrotoxic nephritis.
All biopsy specimens with crescentic GN had extracellular glomerular myeloperoxidase deposition that correlated significantly with eGFR and crescent formation.
, AZM198 led to a significant reduction in neutrophil extracellular trap formation, reactive oxygen species production, and released human neutrophil peptide levels, and attenuated neutrophil-mediated endothelial cell damage.
, delayed AZM198 treatment significantly reduced proteinuria, glomerular thrombosis, serum creatinine, and glomerular macrophage infiltration, without increasing adaptive T cell responses.
Myeloperoxidase inhibition reduced neutrophil degranulation and neutrophil-mediated endothelial cell damage in patients with ANCA-associated vasculitis. In preclinical crescentic GN, delayed myeloperoxidase inhibition suppressed kidney damage without augmenting adaptive immune responses, suggesting it might offer a novel adjunctive therapeutic approach in crescentic GN.
Rheumatoid arthritis (RA) is a common autoimmune disease that causes significant disability and reduced life expectancy. The folate antagonist methotrexate (MTX) is first-line therapy for RA when ...used weekly at low doses (5-25 mg). However, the true rate of adherence to MTX is uncertain. This is in part due to the different methods of measurement of adherence employed with no biochemical test currently available to determine adherence to low dose MTX. Common methods of MTX measurement include immunoassays in patients with high dose therapy, but these assays cross-react with MTX metabolites and lack the sensitivity required to measure adherence to low dose MTX. HPLC-SRM-MS (selected reaction monitoring-mass spectrometry) has several theoretical advantages over immunoassays with improved specificity, minimal cross-reaction and higher sensitivity. The aim of this study was to develop an assay to measure MTX and its major metabolite 7-OH-MTX in urine as a tool to monitor adherence to low dose MTX in clinic. As a proof of concept, urine samples from 4 participants with RA were measured after directly observed therapy. The assay showed improved sensitivity compared to that reported by immunoassays, with low carryover and high within-run precision. In participant samples, MTX was measurable in the urine for up to 105 hours after administration and 7-OH-MTX was detectable up to 98 hours after administration, suggesting that this assay is suitable for the measurement of adherence to therapy. The assay requires minimal sample preparation and can be adopted by other laboratories with minimal study set up.
Objective
With increasing age, skin is subject to alterations in its organization, which impact on its function as well as having clinical consequences. Proteomics is a useful tool for non‐targeted, ...semi‐quantitative simultaneous investigation of high numbers of proteins. In the current study, we utilize proteomics to characterize and contrast age‐associated differences in photoexposed and photoprotected skin, with a focus on the epidermis, dermal–epidermal junction and papillary dermis.
Methods
Skin biopsies from buttock (photoprotected) and forearm (photoexposed) of healthy volunteers (aged 18–30 or ≥65 years) were transversely sectioned from the stratum corneum to a depth of 250 μm. Following SDS‐PAGE, each sample lane was segmented prior to analysis by liquid chromatography‐mass spectrometry/mass spectrometry. Pathway analysis was carried out using Ingenuity IPA.
Results
Comparison of skin proteomes at buttock and forearm sites revealed differences in relative protein abundance. Ageing in skin on the photoexposed forearm resulted in 80% of the altered proteins being increased with age, in contrast to the photoprotected buttock where 74% of altered proteins with age were reduced. Functionally, age‐altered proteins in the photoexposed forearm were associated with conferring structure, energy and metabolism. In the photoprotected buttock, proteins associated with gene expression, free‐radical scavenging, protein synthesis and protein degradation were most frequently altered.
Conclusion
This study highlights the necessity of not considering photoageing as an accelerated intrinsic ageing, but as a distinct physiological process.
Résumé
Objectif
Avec l’âge, la peau est sujette à des altérations dans son organisation, et outre le fait d'avoir des conséquences cliniques cela a un impact sur sa fonction. La protéomique est un outil utile pour l’évaluation non ciblée, semi‐quantitative, simultanée d'un nombre élevé de protéines. Dans cette étude, nous utilisons la protéomique pour caractériser et comparer les différences associées à l’âge entre une peau photoexposée et une peau photoprotégée, avec une attention particulière sur l’épiderme, la jonction dermo–épidermique et le derme papillaire.
Méthodes
Des biopsies de peau de la fesse (photoprotégée) et de l'avant‐bras (photoexposée) de volontaires sains (âgés de 18 à 30 ans ou de ≥ 65 ans) ont été sectionnées transversalement depuis la couche cornée jusqu’à une profondeur de 250 μm. Suite à une électrophorèse SDS‐PAGE, chaque échantillon a été segmenté avant l'analyse par chromatographie en phase liquide couplée à la spectrométrie de masse/spectrométrie de masse. Une analyse des voies de signalisation a été réalisée à l'aide d'Ingenuity IPA.
Résultats
La comparaison des protéomes de la peau des sites des fesses et de l'avant‐bras a révélé des différences dans l'abondance relative de protéines. Le vieillissement de la peau de l'avant‐bras photoexposée montre une augmentation de 80% des protéines altérées avec l’âge, contrairement à la peau des fesses photoprotégée où une réduction de 74 % des protéines altérées avec l’âge a été mesurée. Sur le plan de la fonction, les protéines altérées par l’âge dans la peau de l'avant‐bras photoexposée étaient associées à une structure, une énergie et un métabolisme. Dans la peau des fesses photoprotégée, les protéines associées à l'expression génique, la neutralisation des radicaux‐libres, la synthèse des protéines et la dégradation des protéines étaient le plus fréquemment altérés.
Conclusion
Cette étude souligne la nécessité de ne pas considérer le photovieillissement comme un vieillissement accéléré intrinsèque, mais comme un processus physiologique distinct.
In this study, we investigated the impact of ageing and light on the proteomes of skin collected at photoexposed and photoprotected sites. Although most of the altered proteins in skin from aged individuals were higher in abundance than in skin from younger individuals, in the photoprotected buttock the majority of the altered proteins were reduced with age. Differences in the functions of altered proteins between photoprotected and photoexposed sites also suggest that the nature of photoageing in skin may be very different and perhaps distinct from non‐photoageing.
Stages of CKD are currently defined by eGFR and require measurement of serum creatinine concentrations. Previous studies have shown a good correlation between salivary and serum urea levels and the ...stage of CKD. However, quantitative salivary urea assays in current clinical use require costly and labor-intensive commercial kits, which restricts the advantage of using saliva and limits wider applicability as a quick and easy means of assessing renal function. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy has been shown to provide a potentially straightforward, reagent-free method for the identification of a range of disease-related biomarkers and is in current clinical use for analyses of the chemical composition of kidney stones. We assessed the feasibility of ATR-FTIR spectroscopy as an alternative method to measure salivary urea in patients with different stages of CKD. The ATR-FTIR spectra of dried saliva samples from six healthy controls and 20 patients with CKD (stages 1-5) were analyzed to provide their urea concentrations. The lower limit of detection of salivary urea by the ATR-FTIR spectroscopy method was 1-2 mM, at the lower end of the clinically relevant range. Statistically significant differences in salivary urea concentrations were demonstrated between healthy subjects (4.1±0.5 mM) and patients with CKD stages 3-5 (CKD stage 3, 6.8±0.7 mM; CKD stage 4, 9.1±1 mM; CKD stage 5, 14.8±1.6 mM). These salivary urea concentrations correlated well with serum urea levels in the same patients measured by an automated analyzer (Spearman rank correlation coefficient of 0.71;
<0.001). The ability of the method to detect and stage CKD was assessed from the sensitivity and specificity parameters of a receiver operating characteristics (ROC) curve analysis. This proof-of-concept study demonstrates that quantitation of salivary urea by ATR-FTIR spectroscopy could provide a viable tool for rapid and cost-effective diagnosis of stages 3-5 CKD.
Phosphorylation governs the activity of many proteins. Insight into molecular mechanisms in biology would be immensely improved by robust, sensitive methods for identifying precisely sites of ...phosphate addition. An approach to selective mapping of protein phosphorylation sites on a specific target protein of interest using LC-MS is described here. In this approach multiple reaction monitoring is used as an extremely sensitive MS survey scan for potential phosphopeptides from a known protein. This is automatically followed by peptide sequencing and subsequent location of the phosphorylation site; both of these steps occur in a single LC-MS run, providing greater efficiency of sample use. The method is capable of detecting and sequencing phosphopeptides at low femtomole levels with high selectivity. As proof of the value of this approach in an experimental setting, a key Schizosaccharomyces pombe cell cycle regulatory protein, Cyclin B, was purified, and associated proteins were identified. Phosphorylation sites on these proteins were located. The technique, which we have called multiple reaction monitoring-initiated detection and sequencing (MIDAS), is shown to be a highly sensitive approach to the determination of protein phosphorylation.
Aim: To determine knowledge and practice of foot care in people with diabetes.
Methods: A questionnaire was completed by patients in Middlesbrough, South Tees, UK. A knowledge score was calculated ...and current practice determined. Practices that put patients at risk of developing foot ulcers and barriers to good practice were identified. Patients at high risk of ulceration were compared to those at low risk.
Results: The mean knowledge score was 6.5 (S.D. 2.1) out of a possible 11. There was a positive correlation between the score and having received advice on foot care (6.9 versus 5.4,
P=0.001). Deficiencies in knowledge included the inability to sense minor injury to the feet (47.3%), proneness to ulceration (52.4%) and effect of smoking on the circulation (44.5%). 24.6% (20.1–29.2) never visited a chiropodist, 18.5% (14.2–22.7) failed to inspect their feet and 83% (79.1–86.9) did not have their feet measured when they last purchased shoes. Practices that put patients at risk included use of direct forms of heat on the feet and walking barefoot. Barriers to practice of foot care were mainly due to co-morbidity. Those with high risk feet showed a higher (6.8) but not significant knowledge score compared to those at low risk (6.5) and their foot care practise was better.
Conclusion: The results highlight areas where efforts to improve knowledge and practice may contribute to the prevention of foot ulcers and amputation.
Mass spectrometry (MS)-based proteomics is a powerful tool to explore pathogenic changes of a disease in an unbiased manner and has been used extensively in Alzheimer disease (AD) research. Here, by ...performing a meta-analysis of high-quality proteomic studies, we address which pathological changes are observed consistently and therefore most likely are of great importance for AD pathogenesis. We retrieved datasets, comprising a total of 21,588 distinct proteins identified across 857 postmortem human samples, from ten studies using labeled or label-free MS approaches. Our meta-analysis findings showed significant alterations of 757 and 1,195 proteins in AD in the labeled and label-free datasets, respectively. Only 33 proteins, some of which were associated with synaptic signaling, had the same directional change across the individual studies. However, despite alterations in individual proteins being different between the labeled and the label-free datasets, several pathways related to synaptic signaling, oxidative phosphorylation, immune response and extracellular matrix were commonly dysregulated in AD. These pathways represent robust changes in the human AD brain and warrant further investigation.
Renal cell carcinoma (RCC) is the tenth most common cancer although the incidence is increasing. The main clinical problems stem from the relatively late presentation of many patients due to the ...often asymptomatic nature of the illness, and the relative insensitivity of metastatic disease to conventional chemotherapy and radiotherapy. Despite increasing knowledge of some of the genetic changes underlying sporadic renal cancer such as those involving the Von Hippel Lindau (VHL) gene, many of the underlying pathophysiological changes are ill‐defined and there remains a need for the identification of disease markers for use in diagnosis and prognosis or as potential therapeutic targets. This study has used a proteomic approach, based on two‐dimensional gel electrophoresis and mass spectrometry, to compare the protein profiles of conventional RCC tissue with patient‐matched normal kidney cortex. Sequencing of 32 protein spots with significantly increased expression in RCC samples (≥ 4/6 patients) and 41 proteins whose levels decreased (6/6 patients) confirmed several previously known RCC‐associated changes such as increases in Mn‐superoxide dismutase, lactate dehydrogenase‐A, aldolase A and C, pyruvate kinase M2, and thymidine phosphorylase. Additionally, several previously unknown changes were identified, including increased expression of three members of the annexin family and increased levels of the actin depolymerisation factor cofilin. The Warburg effect was also demonstrated with the identification of increases in proteins involved in the majority of steps in the glycolytic pathway and decreases in the gluconeogenic reactions, together with a parallel decrease in several mitochondrial enzymes. A number of the alterations seen were further confirmed in additional samples by immunohistochemistry, Western blotting, and laser capture microdissection.
Isobaric tags for relative and absolute quantitation, an approach to concurrent, relative quantification of proteins present in four cell preparations, have recently been described. To validate this ...approach using complex mammalian cell samples that show subtle differences in protein levels, a model stem cell-like cell line (FDCP-mix) in the presence or absence of the leukemogenic oncogene TEL/PDGFRbeta has been studied. Cell lysates were proteolytically digested, and peptides within each sample were labeled with one of four isobaric, isotope-coded tags via their N-terminal and/or lysine side chains. The four labeled samples are mixed and peptides separated by two-dimensional liquid chromatography online to a mass spectrometer (LC-MS). Upon peptide fragmentation, each tag releases a distinct mass reporter ion; the ratio of the four reporters therefore gives relative abundances of the given peptide. Relative quantification of proteins is derived using summed data from a number of peptides. TEL/PDGFRbeta leukemic oncogene-mediated changes in protein levels were compared with those seen in microarray analysis of control and transfected FDCP-mix cells. Changes at the protein level in most cases reflected those seen at the transcriptome level. Nonetheless, novel differences in protein expression were found that indicate potential mechanisms for effects of this oncogene.
Theory for the chronoamperometric positive feedback mode of the scanning electrochemical microscope (SECM) is extended to include the situation where the oxidised and reduced forms of the redox ...mediator couple have arbitrary diffusion coefficients. Under typical positive feedback conditions, the solution initially contains a redox-active species, R, along with excess supporting electrolyte. The potential of the tip ultramicroelectrode (UME), positioned close to an interface of interest, is adjusted to a value where R is electrolysed to produce species O at a diffusion-controlled rate. O diffuses away from the tip towards the interface, where the reverse redox reaction occurs leading to the production, and diffusional feedback of R for electrolysis at the tip electrode. When positive feedback measurements are carried out under diffusion-controlled chronoamperometric conditions, the form of the normalised current–time behaviour, at a particular tip to interface distance, is found to be sensitive to the ratio of the diffusion coefficients of the O/R couple. As a steady-state is established, the normalised current becomes independent of the diffusion coefficient ratio and depends only on the tip to interface distance. Experimental measurements on the chronoamperometric oxidation of ferrocene (Fc) in acetonitrile solution at a Pt tip UME positioned close to a Pt substrate electrode, provide support for the theoretical predictions and demonstrate that the diffusion coefficient ratio can readily be determined from SECM chronoamperometry. Although Fc and Fc
+ are often assumed to have the same diffusion coefficients, steady-state and chronoamperometric measurements at a conventional UME yield a value of 2.0×10
−5 cm
2 s
−1 for the diffusion coefficient of Fc, while SECM chronoamperometry indicates that the diffusion coefficient of Fc
+ is 1.6×10
−5 cm
2 s
−1.
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