Reproducibility of sodium MRI measures of articular cartilage of the knee in osteoarthritis.

OBJECTIVE: To determine the stability and reproducibility of the sodium magnetic resonance imaging (MRI) signal measured in the articular cartilage of the knee in both healthy volunteers and osteoarthritis (OA) patients. DESIGN: This was a prospective Research Ethics Committee approved study that acquired sodium and proton MRI data from 15 subjects with OA (three males, age 64 ± 10) and five healthy controls age and sex matched over the group. Each subject underwent standing planar radiographs of their knees for radiological scoring as well as symptomatological assessment questionnaires. In two MRI sessions on the same day, high resolution double-echo steady state (DESS) and 3D short echo time sodium MRI images of the most diseased knee were acquired and co-registered in each session. A blinded reader (LT) manually delineated the articular cartilage into four discrete regions, and two combined regions, on the DESS images. These regions were applied to the sodium images, and a median sodium signal from each reported. Within-subject and between-subject coefficients of variation were estimated and intraclass correlation coefficients for the healthy control group, OA subject group, and all pooled subjects group were calculated. RESULTS: Within-subject variability of sodium MRI at 3T was 3.2% overall, and 2.0% in healthy age-matched volunteers compared to a reproducibility of 3.6% on OA subjects. CONCLUSIONS: The reproducibility of sodium MRI was similar in both healthy controls and OA subjects. Researchers piloting techniques in healthy controls thus may expect a similar reproducibility in a controlled trial involving subjects with American College of Rheumatology (ACR)-defined OA of the knee.

Wound debridement potential of glycosidases of the wound-healing maggot, Lucilia sericata.

The wound-healing maggot, Lucilia sericata Meigen (Diptera: Calliphoridae), degrades extracellular matrix components by releasing enzymes. The purpose of this study was to investigate the glycosylation profiles of wound slough/eschar from chronic venous leg ulcers and the complementary presence of glycosidase activities in first-instar excretions/secretions (ES1) and to define their specificities. The predominant carbohydrate moieties present in wound slough/eschar were determined by probing one-dimensional Western blots with conjugated lectins of known specificities. The presence of specific glycosidase activities in ES1 was determined using chromogenic and fluorogenic substrates. The removal of carbohydrate moieties from slough/eschar proteins by glycosidases in ES1 was determined by two-dimensional electrophoresis and Emerald 300 glycoprotein staining. α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues were detected on slough/eschar-derived proteins. Furthermore, it was demonstrated that the treatment of slough/eschar with ES1 significantly reduced uptake of the carbohydrate-specific stain. Subsequently, α-D-glucosidase, α-D-mannosidase and N-acetylglucosaminidase activities were identified in ES1. Specific chromogenic and fluorogenic substrates and gel filtration chromatography showed that these activities result from distinct enzymes. These activities were mirrored in the removal of α-D-glucosyl, α-D-mannosyl and N-acetylglucosamine residues from proteins of slough/eschar from maggot-treated wounds. These data suggest that maggot glycosidases remove sugars from slough/eschar proteins. This may contribute to debridement, which is ultimately accomplished by a suite of biochemically distinct enzymes present in ES1.

Array comparative genomic hybridisation on first polar bodies suggests that non-disjunction is not the predominant mechanism leading to aneuploidy in humans.

INTRODUCTION: Aneuploidy (the presence of extra or missing chromosomes) arises primarily through chromosome segregation errors in the oocyte at meiosis I but the details of mechanism by which such errors occur in humans are the subject of some debate. It is generally believed that aneuploidy arises primarily as a result of segregation of a whole chromosome to the same pole as its homologue (non-disjunction). Nonetheless, classical cytogenetic studies suggest that this model does not fully account for the patterns observed in human oocytes. An alternative model (precocious separation of sister chromatids) has thus been proposed, but recurring criticism of this model purports that technical issues may have led to interpretation errors. MATERIALS AND METHODS: Array comparative genomic hybridisation (aCGH) was used on 164 human first polar bodies to distinguish between whole chromosome (non-disjunction) and chromatid (precocious separation) errors. RESULTS: Single chromatid errors were over 11 times more common than whole chromosome errors, consistent with prior classical cytogenetic and fluorescence in situ hybridisation (FISH) studies. DISCUSSION: The received wisdom that non-disjunction is the primary mechanism leading to human aneuploidy should be reconsidered.

Maggot chymotrypsin I from Lucilia sericata is resistant to endogenous wound protease inhibitors.

BACKGROUND: A chymotrypsin found in the secretions of Lucilia sericata and manufactured as a recombinant enzyme degrades chronic wound eschar ex vivo. OBJECTIVES: To characterize the inhibition profile of the L. sericata recombinant chymotrypsin I. METHODS: Activity of recombinant chymotrypsin I and its sensitivity to endogenous inhibitors were determined enzymatically using the fluorogenic substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-aminomethyl coumarin. RESULTS: We report the presence of high concentrations of two endogenous inhibitors, α1-antichymotrypsin and α1-antitrypsin, in wound eschar and a trace of a third, α2-macroglobulin, with the potential to inhibit this debridement process. However, the addition of a soluble and inhibitor-containing extract of chronic wound eschar to chymotrypsin I did not affect activity of the enzyme, neither did the addition of purified native α1-antichymotrypsin or α1-antitrypsin, although chymotrypsin I was inhibited by α2-macroglobulin. Conversely, the mammalian equivalent, α-chymotrypsin, was inhibited by the purified native α1-antichymotrypsin, α1-antitrypsin and α2-macroglobulin and by the soluble extract of wound eschar. CONCLUSIONS: The data suggest that the maggot-derived chymotrypsin I is biochemically distinct from human α-chymotrypsin and the lack of inhibition by wound eschar suggests a means by which chymotrypsin I activity survives within the wound to contribute towards debridement during maggot biotherapy.

A major house dust mite allergen disrupts the immunoglobulin E network by selectively cleaving CD23: innate protection by antiproteases.

Asthma is a chronic life-threatening disease of worldwide importance. Although allergic asthma and related atopic conditions correlate strongly with immune sensitization to house dust mites, it is unclear why antigens from mites provoke such powerful allergic immune responses. We have characterized the protease activity of Der p I, the group I protease allergen of the house dust mite Dermatophagoides pteronyssinus, and here report that it cleaves the low-affinity immunoglobulin (Ig) E Fc receptor (CD23) from the surface of human B lymphocytes. Der p I selectively cleaves CD23 and has no effect on the expression of any other B cell surface molecules tested. We speculate that this loss of cell surface CD23 from IgE-secreting B cells may promote and enhance IgE immune responses by ablating an important feedback inhibitory mechanism that normally limits IgE synthesis. Furthermore, since soluble CD23 is reported to promote IgE production, fragments of CD23 released by Der p I may directly enhance the synthesis of IgE. alpha 1-Antiprotease, a pulmonary antiprotease, is also shown to inhibit the cleavage of CD23 by Der p I. This may be significant in the etiopathogenesis of asthma, because other indoor pollutants associated with asthma are known to potently inhibit this antiprotease. These data suggest that the proteolytic activity of Der p I, the group I allergen of the house dust mite D. pteronyssinus, is mechanistically linked to the potent allergenicity of house dust mites. Furthermore, inhibition of Der p I by alpha 1-antiprotease suggests a mechanism by which confounding factors, such as tobacco smoke, may act as a risk factor for allergic asthma.

Experimental hookworm infection: a randomized placebo-controlled trial in asthma.

BACKGROUND: Epidemiological studies suggest that hookworm infection protects against asthma, and therefore that hookworm infection may have a direct or an indirect therapeutic potential in this disease. We now report the first clinical trial of experimental hookworm infection in people with allergic asthma. OBJECTIVES: To determine the effects of experimental hookworm infection in asthma. METHODS: Thirty-two individuals with asthma and measurable airway responsiveness to adenosine monophosphate (AMP) were randomized and double blinded to cutaneous administration of either ten Necator americanus larvae, or histamine solution (placebo), and followed for 16 weeks. The primary outcome was the change in provocation dose of inhaled AMP required to reduce forced expiratory volume in 1 s by 20% (PD(20)AMP) from baseline to week 16. Secondary outcomes included change in several measures of asthma control and allergen skin sensitivity and the occurrence of adverse effects. RESULTS: Mean PD(20)AMP improved in both groups, more in the hookworm [1.49 doubling doses (DD)] than the placebo group (0.98 DD), but the difference between groups was not significant (0.51 DD; 95% confidence interval: -1.79 to 2.80; P=0.65). There were no significant differences between the two groups for other measures of asthma control or allergen skin sensitization. Infection was generally well tolerated. CONCLUSIONS: Experimental infection with ten hookworm larvae in asthma did not result in significant improvement in bronchial responsiveness or other measures of asthma control in this study. However, infection was well tolerated and resulted in a non-significant improvement in airway responsiveness, indicating that further studies that mimic more closely natural infection are feasible and should be undertaken.

Degradation of eschar from venous leg ulcers using a recombinant chymotrypsin from Lucilia sericata.

BACKGROUND: Larvae of the greenbottle Lucilia sericata are used to debride nonhealing wounds and stimulate the production of fresh granulation tissue. Previous publications have shown that secretions from L. sericata contain a number of proteolytic activities including a chymotrypsin that degrades a number of extracellular matrix components such as fibronectin, laminin and collagen. OBJECTIVES: To produce a recombinant L. sericata chymotrypsin (chymotrypsin I) and determine its effects on the degradation of patient wound eschar. METHODS: An active recombinant chymotrypsin I from L. sericata was cloned and expressed in Sf9 cells and its subsequent effects ex vivo on eschar from venous leg ulcers were determined by two-dimensional electrophoresis. RESULTS: The recombinant enzyme had the attributes of a chymotrypsin, possessing sequence homology with other chymotrypsins and demonstrating attributes of the native enzyme including cleavage of the chymotrypsin substrate succinyl-alanyl-alanyl-prolyl-phenylalanyl-7-amino-4-methyl coumarin, inhibition by phenylmethylsulphonyl fluoride and lack of inhibition by amidinophenylmethylsulphonyl fluoride. Importantly, the recombinant chymotrypsin cleaved the majority of proteins from slough/eschar from venous leg ulcers in a superior manner to chymotrypsins from human and bovine sources. CONCLUSIONS: The ex vivo degradation of eschar from venous leg ulcers indicates the potential value of recombinant chymotrypsin I as a novel, stand-alone debridement agent.

Population Data Centre Profiles: Centre for Data Linkage.

The Centre for Data Linkage (CDL) was established at Curtin University, Western Australia, to develop infrastructure to enable cross-jurisdictional record linkage in Australia. The CDL's operating model makes use of the 'separation principle', with content data typically provided to researchers directly by the data custodian; jurisdictional linkage where available are used within the linkage process. Along with conducting record linkage, the team has also invested in establishing a research programme in record linkage methodology and in developing modern record linkage software which can handle the size and complexity of today's workloads. The Centre has been instrumental in the development of practical methods for privacy-preserving record linkage, with this methodology now regularly used for real-world linkages. While the promise of a nation-wide linkage system in Australia has yet to be met, distributed models provide a potential solution.

Confounding experimental considerations in nanogenotoxicology.

The development of novel nanomaterials with unique physico-chemical properties is increasing at a rapid rate, with potential applications across a broad range of manufacturing industries and consumer products. Nanomaterial safety is therefore becoming an increasingly contentious issue that has intensified over the past 4 years, and in response, a steady stream of studies focusing on nanotoxicology are emerging. However, it is becoming increasingly evident that nanomaterials cannot be treated in the same manner as chemical compounds with regards to their safety assessment, as their unique physico-chemical properties are also responsible for unexpected interactions with experimental components that generate misleading data-sets. In this report, we focus on nanomaterial interactions with colorimetric and fluorometric dyes, components of cell culture growth medium and genotoxicity assay components, and the resultant consequences on test systems are demonstrated. Thus, highlighting some of the potential confounding factors that need to be considered in order to ensure that in vitro genotoxicity assays report true biological impacts in response to nanomaterial exposure.

Privacy preserving linkage using multiple match-keys.

INTRODUCTION: Available and practical methods for privacy preserving linkage have shortcomings: methods utilising anonymous linkage codes provide limited accuracy while methods based on Bloom filters have proven vulnerable to frequency-based attacks. OBJECTIVES: In this paper, we present and evaluate a novel protocol that aims to meld both the accuracy of the Bloom filter method with the privacy achievable through the anonymous linkage code methodology. METHODS: The protocol involves creating multiple match-keys for each record, with the composition of each match-key depending on attributes of the underlying datasets being compared. The protocol was evaluated through de-duplication of four administrative datasets and two synthetic datasets; the 'answers' outlining which records belonged to the same individual were known for each dataset. The results were compared against results achieved with un-encoded linkage and other privacy preserving techniques on the same datasets. RESULTS: The multiple match-key protocol presented here achieved high quality across all datasets, performing better than record-level Bloom filters and the SLK, but worse than field-level Bloom filters. CONCLUSION: The presented method provides high linkage quality while avoiding the frequency based attacks that have been demonstrated against the Bloom filter approach. The method appears promising for real world use.

Evaluation of approximate comparison methods on Bloom filters for probabilistic linkage.

INTRODUCTION: The need for increased privacy protection in data linkage has driven the development of privacy-preserving record linkage (PPRL) techniques. A popular technique using Bloom filters with cryptographic analyses, modifications, and hashing variations to optimise privacy has been the focus of much research in this area. With few applications of Bloom filters within a probabilistic framework, there is limited information on whether approximate matches between Bloom filtered fields can improve linkage quality. OBJECTIVES: In this study, we evaluate the effectiveness of three approximate comparison methods for Bloom filters within the context of the Fellegi-Sunter model of recording linkage: Sørensen-Dice coefficient, Jaccard similarity and Hamming distance. METHODS: Using synthetic datasets with introduced errors to simulate datasets with a range of data quality and a large real-world administrative health dataset, the research estimated partial weight curves for converting similarity scores (for each approximate comparison method) to partial weights at both field and dataset level. Deduplication linkages were run on each dataset using these partial weight curves. This was to compare the resulting quality of the approximate comparison techniques with linkages using simple cut-off similarity values and only exact matching. RESULTS: Linkages using approximate comparisons produced significantly better quality results than those using exact comparisons only. Field level partial weight curves for a specific dataset produced the best quality results. The Sørensen-Dice coefficient and Jaccard similarity produced the most consistent results across a spectrum of synthetic and real-world datasets. CONCLUSION: The use of Bloom filter similarity comparisons for probabilistic record linkage can produce linkage quality results which are comparable to Jaro-Winkler string similarities with unencrypted linkages. Probabilistic linkages using Bloom filters benefit significantly from the use of similarity comparisons, with partial weight curves producing the best results, even when not optimised for that particular dataset.

Beam-induced oxidation of mixed-valent Fe (oxyhydr)oxides (green rust) monitored by STEM-EELS.

Analytical transmission electron microscopy (TEM) is often used to investigate morphologies, crystal structures, chemical compositions and oxidation states of highly reactive mixed-valent mineral phases. Of prime interest, due to its potential role in toxic metal remediation, is green rust sulphate (GRSO4) an FeII-FeIII layered double hydroxide. In this study, we quantified the effects that TEM analysis has on GRSO4 in order to ensure the measured material properties are a result of synthesis and reaction kinetics, and not due to sample preparation and analysis technique. To do this, we compared two sample preparation techniques (anoxic drop-cast with drying, and frozen-hydrated cryogenic) and exposed samples to the electron beam for several minutes, acquiring fluence series between ca. 40 e- Å-2 and 10,000 e- Å-2. TEM imaging and electron diffraction showed that the hexagonal plate-like morphology and crystal structure of GRSO4 were largely unaffected by sample preparation and analysis technique. However, quantitative analysis of a series of monochromated Fe L3,2-edge electron energy loss spectra (EELS) showed that electron irradiation induces oxidation. We measured an Fe(II)/Fe(III) ratio of 1.94 (as expected for GRSO4) at 50 e- Å-2. However, above this fluence, the ratio logarithmically decreased and dropped to ca. 0.5 after 1000 e- Å-2. This trend was approximately the same for both sample preparation techniques implying that it is the beam alone which causes valence state changes, and not exposure to oxygen during transfer into the TEM or the vacuum of the TEM column. Ultimately this work demonstrates that GR valence can be quantified by EELS provided that the sample is not over exposed to electrons. This also opens the possibility of quantifying the effect of redox-sensitive toxic metals (e.g., As, Cr, Se) on Fe oxidation state in GR phases (relevant to the treatment of contaminated soils and water) with a higher spatial resolution than other techniques (e.g., Mössbauer spectroscopy).

Exogenous mineralization of hard tissues using photo-absorptive minerals and femto-second lasers; the case of dental enamel.

A radical new methodology for the exogenous mineralization of hard tissues is demonstrated in the context of laser-biomaterials interaction. The proposed approach is based on the use of femtosecond pulsed lasers (fs) and Fe3+-doped calcium phosphate minerals (specifically in this work fluorapatite powder containing Fe2O3 nanoparticles (NP)). A layer of the synthetic powder is applied to the surface of eroded bovine enamel and is irradiated with a fs laser (1040 nm wavelength, 1 GHz repetition rate, 150 fs pulse duration and 0.4 W average power). The Fe2O3 NPs absorb the light and may act as thermal antennae, dissipating energy to the vicinal mineral phase. Such a photothermal process triggers the sintering and densification of the surrounding calcium phosphate crystals thereby forming a new, dense layer of typically ∼20 µm in thickness, which is bonded to the underlying surface of the natural enamel. The dispersed iron oxide NPs, ensure the localization of temperature excursion, minimizing collateral thermal damage to the surrounding natural tissue during laser irradiation. Simulated brushing trials (pH cycle and mechanical force) on the synthetic layer show that the sintered material is more acid resistant than the natural mineral of enamel. Furthermore, nano-indentation confirms that the hardness and Young's modulus of the new layers are significantly more closely matched to enamel than current restorative materials used in clinical dentistry. Although the results presented herein are exemplified in the context of bovine enamel restoration, the methodology may be more widely applicable to human enamel and other hard-tissue regenerative engineering. STATEMENT OF SIGNIFICANCE: In this work we provide a new methodology for the mineralisation of dental hard tissues using femtosecond lasers and iron doped biomaterials. In particular, we demonstrate selective laser sintering of an iron doped fluorapatite on the surface of eroded enamel under low average power and mid-IR wavelength and the formation of a new layer to substitute the removed material. The new layer is evaluated through simulated brushing trials and nano-indentation. From the results we can conclude that is more acid resistant than natural enamel while, its mechanical properties are superior to that of current restorative materials. To the best of our knowledge this is the first time that someone demonstrated, laser sintering and bonding of calcium phosphate biomaterials on hard tissues. Although we here we discuss the case of dental enamel, similar approach can be adopted for other hard tissues, leading to new strategies for the fixation of bone/tooth defects.

ß-pyrophosphate: A potential biomaterial for dental applications.

Tooth hypersensitivity is a growing problem affecting both the young and ageing population worldwide. Since an effective and permanent solution is not yet available, we propose a new methodology for the restoration of dental enamel using femtosecond lasers and novel calcium phosphate biomaterials. During this procedure the irradiated mineral transforms into a densified layer of acid resistant iron doped ß-pyrophosphate, bonded with the surface of eroded enamel. Our aim therefore is to evaluate this densified mineral as a potential replacement material for dental hard tissue. To this end, we have tested the hardness of ß-pyrophosphate pellets (sintered at 1000°C) and its mineral precursor (brushite), the wear rate during simulated tooth-brushing trials and the cytocompatibility of these minerals in powder form. It was found that the hardness of the ß-pyrophosphate pellets is comparable with that of dental enamel and significantly higher than dentine while, the brushing trials prove that the wear rate of ß-pyrophosphate is much slower than that of natural enamel. Finally, cytotoxicity and genotoxicity tests suggest that iron doped ß-pyrophosphate is cytocompatible and therefore could be used in dental applications. Taken together and with the previously reported results on laser irradiation of these materials we conclude that iron doped ß-pyrophosphate may be a promising material for restoring acid eroded and worn enamel.

HREM of the [111] surfaces of iron oxide nanoparticles.

Mixed phase Fe3O4-gamma-Fe2O3 (magnetite-maghemite) iron oxide nanoparticles have been fabricated by colloidal routes. HRTEM/HRSTEM images of the nanoparticles show the presence of [111] facets that terminate with enhanced contrast, which is shown to be caused by the presence of additional cations at the edges of the nanoparticles. HRTEM images were taken on a FEI CM200 FEGTEM, a JEOL 3100 with a LaB6 source, and a double aberration corrected JEOL-JEM 2200FS FEGTEM. The enhanced contrast effect was observed on the [111] surface atomic layers resolved using each machine. HRSTEM images, taken on an aberration corrected STEM, resolved enhanced contrast at specific surface sites. Exit wave reconstruction was also carried out on focal series taken on a double aberration corrected JEOL-JEM 2200FS and showed similar highly resolved enhanced contrast at specific surface cation sites. It is apparent that additional cations are occupying the [111] terminating layers of these nanoparticle surfaces. The use of different microscopes and techniques in this paper provides strong evidence that the enhanced contrast is a real effect and not an effect caused by microscope aberrations.

Malignant melanoma arising in sebaceous naevus (of Jadassohn): a case report.

Sebaceous naevus are associated with malignant transformation. They commonly occur in the head and neck region and are associated with malignant transformation into basal cell and squamous cell carcinomas. This case report describes a case of a malignant melanoma arising from a longstanding sebaceous naevus.

Proton therapy for carcinoma of the nasopharynx: a study in comparative treatment planning.

Radiation therapy for nasopharyngeal carcinoma is technically difficult because of the complexity of the regional anatomy and the natural history of the disease. The results of a study are presented showing how detailed diagnostic information available from MRI is helpful in defining the target volume to be irradiated and the critical normal structures. By using 3-dimensional planning techniques, an assessment was made of the relative merits of proton beam therapy and of X ray treatment for patients with early stage and locally advanced carcinoma of the nasopharynx. For both types of patient, the study suggests that the use of protons for the major part of treatment results in a more even distribution of dose to the tumor and an increase of approximately 5 Gy in median tumor dose with substantial reductions in doses to adjacent normal tissues. The superior dose distributions possible with protons should translate into improved local control and reduced morbidity. The difficulties of proton treatment planning for this site are addressed.

Three-dimensional photon treatment planning for carcinoma of the nasopharynx.

The role of 3-D treatment planning for carcinoma of the nasopharynx was assessed in a four institution study. Two patients were worked up and had an extensive number of CT scans on which target volumes and normal tissues were defined. Treatment planning was then performed using state of the art dose planning systems for these patients to assess the value of the new technology. In general, it was demonstrated that multi-field conformal plans could achieve good tumor dose coverage, while at the same time reducing normal tissue doses, compared to standard treatment planning techniques. The role of inhomogeneity corrections, beam energy, and the use of CT vs. simulation films for defining target volumes were also discussed. In addition, techniques to evaluate 3-D plans for the nasopharynx were considered, and some analysis of this problem is presented in this paper.

Three-dimensional photon treatment planning for Hodgkin's disease.

A multi-institutional study was undertaken using computerized planning systems to develop three-dimensional (3-D) radiotherapy plans for Hodgkin's disease (H.D.). Two patients, the first afflicted with bulky stage II disease and another one with early stage I H.D., were studied. Three main categories of plan were produced for each patient: a) a traditional plan which modelled a conventional mantle treatment on the 3-D system, b) a 3-D standard plan where anterior and posterior fields were designed to cover 3-D target volumes, and c) a 3-D unconstrained plan where innovational techniques were employed. Three-dimensional planning provides information about the dose distribution throughout the large volume irradiated in patients with H.D. that is not available with conventional mantle planning. The use of 3-D techniques resulted in improved tumor coverage, but by allowing for uncertainties such as motion, the doses to normal tissues tended to be higher. The use of unorthodox beam arrangements introduced added complexities, and further increased the lung doses. The most even dose distributions were obtained by incorporating compensating filters into anterior fields. Clinicians showed wide variations in their assessment of the plans, possible reasons for which are addressed in this paper. In addition, calculated probabilities from models of tumor control and normal tissue damage are also presented.