Development of a Novel Assay for the Detection of Active Neutrophil Elastase in Patients with Chronic Obstructive Pulmonary Disease

Neutrophil elastase (NE), a biomarker of infection and inflammation, correlates with the severity of several respiratory diseases including chronic obstructive pulmonary disease (COPD). However, it’s detection and quantification in biological samples is confounded by a lack of reliable and robust methodologies. Standard assays using chromogenic or fluorogenic substrates are not specific when added to complex clinical samples containing multiple proteolytic and hydrolytic enzymes which have the ability to hydrolyse the substrate, thereby resulting in an over-estimation of the target protease. Furthermore, ELISA systems measure total protease levels which can be a mixture of latent, active and protease-inhibitor complexes. Therefore, we have developed a novel immunoassay (ProteaseTag™ Active NE Immunoassay) which is selective and specific for the capture of active NE in sputum and Bronchoalveolar Lavage (BAL) in patients with COPD. The objective of this study was to clinically validate ProteaseTag™ Active NE Ultra Immunoassay for the detection of NE in sputum from COPD patients. 20 matched sputum sol samples were collected from 10 COPD patients (M=6, F=4; 73 ± 6 years) during stable and exacerbation phases. Samples were assayed for NE activity utilising both ProteaseTag™ Active NE Ultra Immunoassay and a fluorogenic substrate-based kinetic activity assay. Both assays detected elevated levels of NE in the majority of patients (n=7) during an exacerbation (mean=217.2 μg/ml ±296.6) compared to their stable phase (mean=92.37 μg/ml ±259.8). However, statistical analysis did not show this difference to be significant (p=0.07, ProteaseTag™ Active NE Ultra Immunoassay; p=0.06 kinetic assay), most likely due to the low study number. A highly significant correlation was found between the 2 assay types (p≤0.0001, r=0.996). NE as a primary efficacy endpoint in clinical trials or as a marker of inflammation within the clinic has been hampered by the lack of a robust and simple to use assay. ProteaseTag™ Active NE Immunoassay specifically measures only active NE in clinical samples, is quick and easy to use (< 3 hours) and has no dependency on a kinetic readout. ProteaseTag™ technology is currently being transferred to a lateral flow device for use at Point of Care.

On the Origin of the Right to Copy: Charting the Movement of Copyright Law in Eighteenth Century Britain (1695-1775)

Taking as its point of departure the lapse of the 1662 Licensing Act in 1695, this book examines the lead up to the passage of the Statute of Anne 1710 and charts the movement of copyright law throughout the eighteenth century, culminating in the House of Lords decision in Donaldson v Becket (1774). The established reading of copyright's development throughout this period, from the 1710 Act to the pronouncement in Donaldson, is that it was transformed from a publisher's right to an author's right; that is, legislation initially designed to regulate the marketplace of the bookseller and publisher evolved into an instrument that functioned to recognise the proprietary inevitability of an author's intellectual labour. The historical narrative which unfolds within this book presents a challenge to that accepted orthodoxy. The traditional analysis of the development of copyright in eighteenth-century Britain is revealed to exhibit the character of long-standing myth, and the centrality of the modern proprietary author as the raison d'etre of the modern copyright regime is displaced.

Controlled release of a model antibacterial drug from a novel self-lubricating silicone biomaterial

Abstract There is considerable interest in developing medical devices that provide controlled delivery of biologically active agents, for example, to reduce the incidence of device-related infection. Silicone elastomers are one of the commonest biomaterials used in medical device production. However, they have a relatively high coefficient of friction and the resulting lack of lubricity can cause pain and tissue damage on device insertion and removal. Novel silicone cross-linking agents have recently been reported that produce inherently ‘self-lubricating’ silicone elastomers with very low coefficients of friction. In this study, the model antibacterial drug metronidazole has been incorporated into these self-lubricating silicone elastomers to produce a novel bioactive biomaterial. The in vitro release characteristics of the bioactive component were evaluated as a function of cross-linker composition and drug loading. Although conventional matrix-type release kinetics were observed for metronidazole from the silicone systems, it was also observed that increasing the concentration of the cross-linking agent responsible for the lubricious character (tetra(oleyloxy)silane) relative to that of the standard non-lubricious cross-linking agent (tetrapropoxysilane) produced an increase in the metronidazole flux rate by up to 65% for a specified drug loading. The results highlight the potential for developing lubricious silicone medical devices with enhanced drug release characteristics.

The spectrum of endogenous human chromogranin A-derived peptides identified using a modified proteomic strategy.

The hypothesis that chromogranin A (CgA), a protein of neuroendocrine cell secretory granules, may be a precursor of biologically active peptides, rests on observed activities of peptide fragments largely produced by exogenous protease digestion of the bovine protein. Here we have adopted a modified proteomic strategy to isolate and characterise human CgA-derived peptides produced by endogenous prohormone convertases. Initial focus was on an insulinoma as previous studies have shown that CgA is rapidly processed in pancreatic beta cells and that tumours arising from these express appropriate prohormone convertases. Eleven novel peptides were identified arising from processing at both monobasic and dibasic sites and processing was most evident in the C-terminal domain of the protein. Some of these peptides were identified in endocrine tumours, such as mid-gut carcinoid and phaeochromocytoma, which arise from endocrine cells of different phenotype and in different anatomical sites. Two of the most interesting peptides, GR-44 and ER-37, representing the C-terminal region of CgA, were found to be amidated. These data would imply that the intact protein is C-terminally amidated and that these peptides are probably biologically active. The spectrum of novel CgA-derived peptides, described in the present study, should provide a basis for biological evaluation of authentic entities.

An investigation into the structure and bioavailability of a-tocopherol dispersions in Gelucire 44/14

In this investigation we describe the preparation, physical characterisation and in vivo behaviour of solid dispersions of a liquid nutraceutical, ±-tocopherol, in Gelucire 44/14 with a view to establishing whether dispersion in this matrix may provide a means of formulating a liquid drug in a solid dosage form while also improving the oral bioavailability. Using Vitamin E Preparation USP as the source of ±-tocopherol, dispersions were prepared using a melt-fusion method with active loadings up to 50% (w/w) and characterised using differential scanning calorimetry and optical microscopy. Capsules containing 300 IU ±-tocopherol were manufactured and the absorption profiles compared to a commercial soft gelatin capsule preparation in healthy human volunteers. Confocal laser scanning microscopy (CLSM) studies were performed in order to elucidate the mechanism by which drug release may be occurring. Differential scanning calorimetry studies indicated that the presence of the active had a negligible effect on the melting profile of the carrier, indicating limited miscibility between the two components, a conclusion supported by the microscopy studies. Similarly, the dispersions were shown to exhibit a glass transition corresponding to the incorporated drug, indicating molecular cooperativity and hence phase separation from the lipid base. Despite the phase separation, it was noted that capsules stored for 18 months under ambient conditions showed no evidence of leakage. Bioavailability studies in six healthy male volunteers indicated that the Gelucire 44/14 formulation showed an approximately two-fold increase in total ±-tocopherol absorption compared to the commercial preparation. Confocal laser scanning microscopy studies indicated that, on contact with water, the dispersions formed two interfacial layers, from which the Gelucire 44/14 disperses in the liquid medium as small particles. Furthermore, evidence was obtained for the dispersed material becoming incorporated into the hydrated lipid. In conclusion, the dispersion of the liquid drug in Gelucire 44/14 appears to allow the dual advantages of the preparation of a solid formulation and improved bioavailability of this material.

Sexual behaviour of young people in Northern Ireland: First sexual experience

Two National Surveys of Sexual Attitudes and Lifestyles in Britain (Natsal) were conducted, one in 1990 and one in 2000. Northern Ireland was excluded from both studies. Now, for the first time, comparable data about sexual attitudes and lifestyles of young people (14- to 25-year-olds) in Northern Ireland are available. Data were collected through self-administered questionnaires, one-to-one interviews and focus-group discussions. As in Natsal 1990 and 2000, young people were asked about their sexual attitudes towards sex, experiences of sex education, knowledge of sexually transmitted infections (STIs) and, if sexually active, about the circumstances in which sexual intercourse occurred. A total of 1013 young people in the target age group completed the self-administered questionnaire. Young people in Northern Ireland do not differ significantly from their counterparts in Britain in terms of sexual lifestyles and attitudes. Some 53.3% of all respondents reported that they had had sexual intercourse. Condom use at first sex was reported by 64% of sexually active respondents; 27.4% said they used no contraception; 26.7% of all respondents said they had sex before age 16. Respondents who first had sex when they were 15 or 16 years were more likely than other respondents to say that 'being drunk' was the main reason why intercourse occurred. Peer pressure to engage in sex was more prevalent among males than females. Young people in Northern Ireland regard friends as their most important source of sex education. School is the second most important source but most respondents wanted more sex education in school. It is important that it is needs focused and includes potentially sensitive and contentious information.

Drug Delivery of Aminolevulinic Acid from Topical Formulations Intended for Photodynamic Therapy

Topical photodynamic therapy is used for a variety of malignant and pre-malignant skin disorders, including Bowen's Disease and Superficial Basal Cell Carcinoma. A haem precursor, typically 5-aminolevulinic acid (ALA), acting as a prodrug, is absorbed and converted by the haem biosynthetic pathway to photoactive protoprophyrin IX (PpIX), which accumulates preferentially in rapidly dividing
cells. Cell destruction occurs when PpIx is activated by an intense light source of appropriate wavelength. Topical delivery of ALA avoids the prolonged photosensitivity reactions associated with systemic administration of photosensitisers but its clinical utility is influenced by the tissue penetration characteristics of the drug, its ease of application and the stability of the active agent in the applied dose. This review, therefore, focuses on drug delivery applications for topical, ALA-based PDT. Issues considered in detail include physical and chemical enhancement strategies for tissue penetration of ALA and subsequent intracellular accumulation of PpIX, together with formulation strategies and drug delivery design solutions appropriate to various clinical applications. The fundamental aspects of drug diffusion in
relation to the physicochemical properties of ALA are reviewed and specific consideration is given to the degradation pathways of ALA in formulated systems that, in turn, influence the design of stable topical formulations.

A comparison of theoretical Si VIII emission line ratios with observations from SERTS

Recent R-matrix calculations of electron impact excitation rates in N-like Si VIII are used to derive theoretical emission line intensity ratios involving 2s(2)2p(3)-2s2p(4) transitions in the 216 -320 Angstrom wavelength range. A comparison of these with an extensive dataset of solar active region, quiet- Sun, sub-flare and off-limb observations, obtained during rocket flights of the Solar EUV Research Telescope and Spectrograph (SERTS), indicates that the ratio R-1 = I(216.94 Angstrom)/I(319.84 Angstrom) may provide a usable electron density diagnostic for coronal plasmas. The ratio involves two lines of comparable intensity, and varies by a factor of about 5 over the useful density range of 10(8)-10(11) cm(-3). However R-2 = I(276.85 Angstrom)/I(319.84 Angstrom) and R-3 = I(277.05 Angstrom)/I(319.84 Angstrom) show very poor agreement between theory and observation, due to the severe blending of the 276.85 and 277.05 Angstrom lines with Si VII and Mg VII transitions, respectively, making the ratios unsuitable as density diagnostics. The 314.35 Angstrom feature of Si VIII also appears to be blended, with the other species contributing around 20% to the total line flux.

Multi-ionization of helium and lithium using the independent electron and independent event models with intrinsic CDW

Cross sections for the multi-ionization of He and Li are presented for impact energies in the range of 50 to 1000 keV/amu. These are calculated using the eikonal initial state approximation to represent the input and exit channels of the active electrons. The ionization process is simulated in a variety of ways, most notably an attempt to account for the effects of electron correlation via the inclusion of a continuum density of states (CDS) term. Inadequacies, of the CDW formulation at small impact parameters, and of the models themselves, are discussed and conclusions are drawn on what repercussions this has for the cross sections calculated.

Biotransformation of maximakinin, a bradykinin-related nonadecapeptide from toad venom, by mammalian kallikrein and salivary proteases

Maximakinin is an N-terminally extended bradykinin (DLPKINRKGPRPPGFSPFR) from the venom of a Chinese toad (Bombina maxima) that displays highly selective activity at mammalian arterial smooth muscle receptors. In this study, we report that incubation of maximakinin with either kallikrein or human saliva generates catabolites with enhanced bioactivity that retain the tissue selective effects of the parent molecule. In addition, we have observed that kallikrein rapidly cleaves the C-terminal arginyl residue of both maximakinin and bradykinin – a cleavage hitherto considered to be performed by a carboxypeptidase that facilitates selective bradykinin receptor targeting. Maximakinin has thus evolved as a `smart' defensive weapon in the toad with inherent resistance to the signal-terminating protease hardware in the potential predator. Thus, natural selection of amphibian skin peptides for antipredator defence, through interspecies delivery by an exogenous secretory mode, produces subtle structural stabilization modifications that can potentially provide new insights for the design of orally active and selectively targeted peptide therapeutics.

Novel frenatins from the skin of the Australasian Giant White-lipped tree frog, Litoria infrafrenata: cloning of precursor cDNAs and identification in defensive skin secretion.

The Australasian anuran amphibian genus Litoria, contains many phenotypically-diverse species as a result of radial evolution of an ancestral species into different biotopes much in the manner of the indigenous marsupial mammals. In common with members of the Central/South American genus Phyllomedusa, their specialized skin granular glands are factories for the production of a plethora of biologically-active peptides. Here we report a more detailed study of those present in the defensive skin secretion of the Australasian giant white-lipped tree frog, Litoria infrafrenata, and, for the first time, we have identified three novel frenatins by deduction of primary structures from cDNAs that were cloned from a library constructed from lyophilized skin secretion using a recently-developed technique. All open-reading frames consisted of a putative signal peptide and an acidic pro-region followed by a single copy of a frenatin peptide. Processed peptides corresponding in molecular mass to the deduced molecular masses of frenatins (named 1.1, 3, 3.1 and 4.1) were identified in the same secretion sample using HPLC and mass spectroscopy. The application of this technique thus permits parallel peptidomic and transcriptomic analyzes on the same lyophilized skin secretion sample circumventing sacrifice of specimens from endangered herpetofauna.

Identification of three novel Phyllomedusa sauvagei dermaseptins (sVI-sVIII) by cloning from a skin secretion-derived cDNA library

The defensive skin secretions of many amphibians contain a wide spectrum of biologically active compounds, particularly antimicrobial peptides that act as a first line of defence against bacterial infection. Here we describe for the first time the identification of three novel dermaseptin-related peptides (dermaseptins sVI–sVIII) whose primary structures were deduced from cDNAs cloned from a library constructed from lyophilised skin secretion of the South American hylid frog, Phyllomedusa sauvagei. The molecular masses of each were subsequently confirmed by interrogation of archived LC/MS files of fractionated skin secretion followed by automated Edman degradation sequencing. The heterogeneity of primary structures encountered in amphibian skin antimicrobial peptides may in part be explained by individual variation—a factor essential for selective functional molecular evolution and perhaps, ultimately in speciation.

Carboxypeptidase-mediated metabolism of calcitonin gene-related peptide in human gingival crevicular fluid - a role in periodontal inflammation?

Background: Metabolism by peptidases plays an important role in modulating the levels of biologically-active neuropeptides. The metabolism of the anti-inflammatory neuropeptide calcitonin gene-related peptide (GCRP), but not the pro-inflammatory neuropeptides substance P (SP) and neurokinin A (NKA) by components of the gingival crevicular fluid (GCF), could potentiate the inflammatory process in periodontitis.

The production and characterisation of dinitrocarbanilide antibodies raised using antigen mimics.

Polyclonal antibodies were produced to detect the coccidiostat nicarbazin. Due to structural constraints of the active component of nicarbazin, dinitrocarbanilide (DNC), three different compounds that shared a common substructure with DNC were used as antigen mimics. The compounds (N-suceinyl-L-alanyl-L-alanyl-L-alanine 4-nitroanilide (SAN), L-glutamic acid gamma-(p-nitroanilide) (GAN) and p-nitrosuccinanilic acid (NSA)) were conjugated to a carrier protein and used in the immunisation of rabbits. Five different polyclonal sera were produced and consequently characterised. The antibodies exhibited an IC50 range of 2.3-7.6 ng/ml using a competitive ELISA procedure, Serum from one rabbit, R555, exhibited an IC50 of 2.9 ng/ml for DNC and cross-reactivity studies showed that this serum was specific for DNC and did not cross-react with other coccidiostats such as halofuginone, toltrazuril or ronidazole. (C) 2002 Elsevier Science B.V. All rights reserved.

Robustness of Damping Control Implemented by Energy Storage Systems Installed in Power Systems

An energy storage system (ESS) installed in a power system can effectively damp power system oscillations through controlling exchange of either active or reactive power between the ESS and power system. This paper investigates the robustness of damping control implemented by the ESS to the variations of power system operating conditions. It proposes a new analytical method based on the well-known equal-area criterion and small-signal stability analysis. By using the proposed method, it is concluded in the paper that damping control implemented by the ESS through controlling its active power exchange with the power system is robust to the changes of power system operating conditions. While if the ESS damping control is realized by controlling its reactive power exchange with the power system, effectiveness of damping control changes with variations of power system operating condition. In the paper, an example power system installed with a battery ESS (BESS) is presented. Simulation results confirm the analytical conclusions made in the paper about the robustness of ESS damping control. Laboratory experiment of a physical power system installed with a 35kJ/7kW SMES (Superconducting Magnetic Energy Storage) was carried out to evaluate theoretical study. Results are given in the paper, which demonstrate that effectiveness of SMES damping control realized through regulating active power is robust to changes of load conditions of the physical power system.