Jennifer T. Wyffels

Analysis of the proteomic profile of chronic pressure ulcers

Analysis of the proteomic profile of pressure ulcers over time is a critical step in the identification of biomarkers of healing or nonhealing in pressure ulcers. The wound fluid from 32 subjects with 42 pressure ulcers was evaluated over 6 weeks at 15 time points. Samples specific to both the interior and the periphery of the wound bed were collected. Antibody screening arrays, isobaric tags for relative and absolute quantitation with mass spectrometry and multiplexed microarrays were used to characterize wound fluid and results were correlated with clinical outcome. Twenty‐one proteins were found to distinguish between healed and chronic wounds and 19 proteins were differentially expressed between the interior and periphery of wounds. Four proteins, pyruvate kinase isozymes M1/M2, profilin‐1, Ig lambda‐1 chain C regions, and Ig gamma‐1 chain C region, were present in lower levels for periphery samples when compared to interior samples and six proteins, keratin, type II cytoskeletal 6A (KRT6A), keratin, type I cytoskeletal 14, S100 calcium binding proteins A7, alpha‐1‐antitrypsin precursor, hemoglobin subunit alpha, and hemoglobin subunit beta, were present in higher levels in periphery samples when compared with interior samples. S100 calcium binding protein A6, S100 calcium binding protein A7, and soluble receptor for advanced glycation end‐products had higher levels in the periphery of chronic wounds vs. the interior in planar arrays. A significant temporal trend was noted for monokine induced by gamma interferon (MIG), synonomous with chemokine (C‐X‐C motif) ligand 9 (CXCL9), which increased as wounds healed and remained nearly constant for ulcers that were not approaching closure.

Analysis of pressure ulcer wound fluid using two‐dimensional electrophoresis

The incidence rate of pressure ulcers in the USA ranges from 0·4% to 38% in acute care settings and from 2·2% to 23·9% in long‐term care settings, and their treatment costs are in the billions of dollars yearly. The proteome of wound fluid may contain early indicators or biomarkers associated with healing in pressure ulcers that would enable treatment regimes to be optimised for each individual. Wound fluid was collected from the interior and periphery of 19 chronic pressure ulcers at 15 time points during 42 days for an analysis of protein expression. Proteins were fractionated using two‐dimensional polyacrylamide gel electrophoresis. A comparison of the spot distributions indicates a biochemical difference between the interior and the periphery of wounds. Pressure ulcers that healed show a greater number of spots for interior and peripheral locations combined over time when compared with wounds that did not heal. Using this technique, protein S100A9 was identified as a potential biomarker of wound healing. The identification of differences within the proteome of healing versus non healing pressure ulcers could have great significance in the use of current treatments, as well as the development of new therapeutic interventions.

Dexamethasone-induced apoptosis in immune cells from peripheral circulation and lymphomyeloid tissues of juvenile clearnose skates, Raja eglanteria

Juvenile clearnose skates (Raja eglanteria) were injected intramuscularly with dexamethasone-21-phosphate at 50, 75, and 100mg/kg body weight. After 24h, skates were sacrificed and lymphomyeloid tissues (thymus, spleen, Leydig organ, and epigonal organ) were removed and whole blood was sampled. Tissues were used fresh for imprints or prepared for histology by solvent fixation or freezing in liquid nitrogen. Apoptosis in fixed tissues was assessed by transmission electron microscopy. Frozen sections and cytospin preparations of peripheral blood leukocytes (PBL) were evaluated by the TUNEL reaction to detect DNA strand breaks. Dexamethasone treatment increased apoptotic activity in all lymphomyeloid tissues as well as in PBL. These studies demonstrate that immune cells of elasmobranchs have the capacity for glucocorticoid-driven apoptosis, and that programmed cell death as a mechanism to regulate immune cell production appears to have been conserved during vertebrate evolution.

Granulation tissue of chronic pressure ulcers as a predictive indicator of wound closure.

OBJECTIVE: To describe the temporal relationship between the quantity of granulation tissue in a chronic pressure ulcer (PrU) and its clinical outcome. DESIGN: Study participants were seen on days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11, 14, 21, 28, 35, and 42. On each visit, the wounds were digitally photographed with a 3-cm2 calibration target. Images were analyzed using VeV MD (version 1.1.14; VERG Inc, Winnipeg, Manitoba, Canada) and Adobe Photoshop CS3 Extended (version 10.0.1; Adobe Systems Inc, San Jose, California). Granulation tissue was selected from calibrated digital images by 1 of 2 methods: manual selection and automated selection. Granulation tissue area was expressed as a percentage of total wound area. SETTING: Academic research laboratory. PARTICIPANTS: Thirty-one chronic PrUs were observed in 27 subjects. MAIN OUTCOME MEASURES: Quantitative measure of granulation tissue area. MAIN RESULTS: There was no relationship between the amount of granulation tissue expressed as a percentage of the total PrU area and wound outcome. CONCLUSIONS: This study is the first to both quantitatively measure the amount of granulation tissue in a chronic PrU and attempt to correlate it to wound outcome. Although counterintuitive, the amount of granulation tissue was not predictive of outcome, and no temporal trends could be described.

A pilot study evaluating protein abundance in pressure ulcer fluid from people with and without spinal cord injury

Abstract Objective To determine whether the biochemistry of chronic pressure ulcers differs between patients with and without chronic spinal cord injury (SCI) through measurement and comparison of the concentration of wound fluid inflammatory mediators, growth factors, cytokines, acute phase proteins, and proteases. Design Survey. Setting Tertiary spinal cord rehabilitation center and skilled nursing facilities. Participants Twenty-nine subjects with SCI and nine subjects without SCI (>18 years) with at least one chronic pressure ulcer Stage II, III, or IV were enrolled. Outcome measures Total protein and 22 target analyte concentrations including inflammatory mediators, growth factors, cytokines, acute phase proteins, and proteases were quantified in the wound fluid and blood serum samples. Blood samples were tested for complete blood count, albumin, hemoglobin A1c, total iron binding capacity, iron, percent (%) saturation, C-reactive protein, and erythrocyte sedimentation rate. Results Wound fluid concentrations were significantly different between subjects with SCI and subjects without SCI for total protein concentration and nine analytes, MMP-9, S100A12, S100A8, S100A9, FGF2, IL-1b, TIMP-1, TIMP-2, and TGF-b1. Subjects without SCI had higher values for all significantly different analytes measured in wound fluid except FGF2, TGF-b1, and wound fluid total protein. Subject-matched circulating levels of analytes and the standardized local concentration of the same proteins in the wound fluid were weakly or not correlated. Conclusions The biochemical profile of chronic pressure ulcers is different between SCI and non-SCI populations. These differences should be considered when selecting treatment options. Systemic blood serum properties may not represent the local wound environment.