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Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems.

ABSTRACT: Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis.

SUBMITTER: Huang J

PROVIDER: S-EPMC6608033 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems.

Huang Jiansheng J Milton Amber A Arnold Robert D RD Huang Hui H Smith Forrest F Panizzi Jennifer R JR Panizzi Peter P

Journal of leukocyte biology 20160216 4

Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to targe ...[more]

PMID: 26884610

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Project description:BackgroundEnzymatic activity measurements of the highly oxidative enzyme myeloperoxidase (MPO), which is implicated in many diseases, are widely used in the literature, but often suffer from nonspecificity and lack of uniformity. Thus, validation and standardization are needed to establish a robust method that is highly specific, sensitive, and reproducible for assaying MPO activity in biological samples.Principal findingsWe found conflicting results between in vivo molecular MR imaging of MPO, which measures extracellular activity, and commonly used in vitro MPO activity assays. Thus, we established and validated a protocol to obtain extra- and intracellular MPO from murine organs. To validate the MPO activity assays, three different classes of MPO activity assays were used in spike and recovery experiments. However, these assay methods yielded inconsistent results, likely because of interfering substances and other peroxidases present in tissue extracts. To circumvent this, we first captured MPO with an antibody. The MPO activity of the resultant samples was assessed by ADHP and validated against samples from MPO-knockout mice in murine disease models of multiple sclerosis, steatohepatitis, and myocardial infarction. We found the measurements performed using this protocol to be highly specific and reproducible, and when performed using ADHP, to be highly sensitive over a broad range. In addition, we found that intracellular MPO activity correlated well with tissue neutrophil content, and can be used as a marker to assess neutrophil infiltration in the tissue.ConclusionWe validated a highly specific and sensitive assay protocol that should be used as the standard method for all MPO activity assays in biological samples. We also established a method to obtain extra- and intracellular MPO from murine organs. Extracellular MPO activity gives an estimate of the oxidative stress in inflammatory diseases, while intracellular MPO activity correlates well with tissue neutrophil content. A detailed step-by-step protocol is provided.

Project description:BACKGROUND:There has been an increasing interest in understanding the usefulness of wrist-based accelerometer data for physical activity (PA) assessment due to the ease of use and higher user compliance than other body placements. PA assessment studies have relied on machine learning methods which take accelerometer data in forms of variables, or feature vectors. METHODS:In this work, we introduce automated shape feature derivation methods to transform epochs of accelerometer data into feature vectors. As the first step, recurring patterns in the collected data are identified and placed in a codebook. Similarities between epochs of accelerometer data and codebook's patterns are the basis of feature calculations. In this paper, we demonstrate supervised and unsupervised approaches to learn codebooks. We evaluated these methods and compared them with the standard statistical measures for PA assessment. The experiments were performed on 146 participants who wore an ActiGraph GT3X+ accelerometer on the right wrist and performed 33 activities of daily living. RESULTS:Our evaluations show that the shape feature derivation methods were able to perform comparably with the standard wrist model (F1-score: 0.89) for identifying sedentary PAs (F1-scores of 0.86 and 0.85 for supervised and unsupervised methods, respectively). This was also observed for identifying locomotion activities (F1-scores: 0.87, 0.83, and 0.81 for the standard wrist, supervised, unsupervised models, respectively). All the wrist models were able to estimate energy expenditure required for PAs with low error (rMSE: 0.90, 0.93, and 0.90 for the standard wrist, supervised, and unsupervised models, respectively). CONCLUSION:The automated shape feature derivation methods offer insights into the performed activities by providing a summary of repeating patterns in the accelerometer data. Furthermore, they could be used as efficient alternatives (or additions) for manually engineered features, especially important for cases where the latter fail to provide sufficient information to machine learning methods for PA assessment.

Project description:Histological examination of bone microstructure provides insight into extant and extinct vertebrate physiology. Fossil specimens sampled for histological examination are typically first embedded in an inexpensive polyester resin and then cut into thin sections, mounted on slides, and polished for viewing. Modern undecalcified bone is chemically processed prior to embedding in plastic resin, sectioning, mounting, and polishing. Conversely, small fossil material and modern undecalcified bone are typically embedded in higher priced epoxy resin because these specimen types require final sections near or below 100 µm thick. Anecdotal evidence suggests thin sections made of polyester resin embedded material polished thinner than 100 µm increases likelihood of sample peeling, material loss, and is unsuitable for modern tissue and small fossil material. To test this assertion, a sample of modern bones and fossil bones, teeth, and scales were embedded in either polyester resin or epoxy resin. Embedded specimens were sectioned and mounted following standard published protocol. Thin sections were ground on a lapidary wheel using decreasing grit sizes until tissue microstructure was completely discernible when viewed under a polarizing light microscope. Additionally, eight prepared thin sections (four from polyester resin embedded specimens and four from epoxy resin embedded specimens) were continuously ground on a lapidary wheel using 600 grit carbide paper until peeling occurred or material integrity was lost. Slide thickness when peeling occurred was measured for comparing slide thickness when specimen integrity was lost between the two resin types. Final slide thickness ranged from 38 µm to 247 µm when tissue was identifiable using a polarizing microscope. Finished slide thickness varied between resin types despite similar tissue visibility. However, finished slide thickness appears more dependent on hard tissue composition than resin type. Additionally, we did not find a difference of slide thickness when material was lost between resin types. The results of this preliminary study suggest that polyester resins can be used for embedding undecalcified modern hard tissues and fossilized hard tissues without loss of tissue visibility or material integrity, at least in the short term.

Project description:BackgroundEnzyme-linked immunosorbent assay (ELISA) has traditionally been used to detect myeloperoxidase (MPO) and proteinase 3 (PR3) antibodies, although it is time-consuming and physically demanding. As a novel and highly effective immunoassay, we compared chemiluminescent immunoassay (CIA) with ELISA to verify the application value of CIA in MPO and PR3 antibodies detection.MethodsBy ELISA and CIA, serum levels of anti-MPO and anti-PR3 antibodies were measured in 63 anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) patients (AAV group), including 47 microscopic polyangiitis (MPA) patients and 16 granulomatosis with polyangiitis (GPA) patients, in addition, 68 patients in interference control group (IC group), 19 healthy subjects in healthy control group (HC group). We compared MPO and PR3 antibodies levels and positive rates measured by these two methods among groups. Relationship and coincidence rate between ELISA and CIA were investigated. Diagnostic values for clinical outcomes for MPO and PR3 antibodies were assessed by receiver operator characteristic (ROC) curve.ResultsIn AAV patients, when detecting anti-MPO (r = .90) and anti-PR3 (r = .81), CIA was highly correlated with ELISA, companying with highly total (88.89%, 92.06%, respectively) and positive coincidence rates (84.78%, 77.27%, respectively). In HC group, anti-PR3 positive rate detected by both immunoassay were 0, anti-MPO almost were 0, which without statistically significant difference (P = .32). In IC group, the total (76.47%, 58.82, respectively) and positive coincidence rates (48.38%, 30.00%, respectively) of anti-MPO and anti-PR3 were the lowest, but the negative coincidence rates reached 100%. By CIA, similar to ELISA, the levels of anti-MPO were significantly higher both in AAV patients (56.00; [4.40-235.30]) and MPA patients (98.00; [27.90-324.70]) compared with either IC group (3.20; [3.20-18.55) (P < .0001) or HC group (3.20; [3.20-3.20]) (P < .0001), yielded an area under curve (AUC) of 0.76 for AAV and 0.89 for MPA, the concentration of anti-PR3 in GPA group (66.65; [24.43-150.00]) was significantly higher than that in IC group (2.3; [2.3-10.95]) (P < .0001) and HC group (2.3; [2.3-2.3]) (P < .0001), with an AUC of 0.92.ConclusionSimilar to ELISA, CIA was competent to detect MPO and PR3 antibodies in AAV patients and healthy population, thus distinguish AAV patients from IC group and HC group and effectively diagnose MPA and GPA.

Dataset Information

Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems.

Publications

Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems.

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