Monocyte-derived dendritic cells: a potential target for therapy in multiple sclerosis (MS)

Monocytes can differentiate into dendritic cells (DC), cells with a pivotal role in both protective immunity and tolerance. Defects in the maturation or function of DC may be important in the development of autoimmune disease. We sought to establish if there were differences in the cytokine (granulocyte-macrophage colony-stimulating factor and IL-4)-driven maturation of monocytes to DC in patients with MS and whether drugs used to treat MS affected this process in vitro. We have demonstrated that there is no defect in the ability of magnetic activated cell sorting (MACS)-purified monocytes from patients with MS to differentiate to DC, but equally they show no tendency to acquire a DC phenotype without exogenous cytokines. Interferon-beta1a prevents the acquisition of a full DC phenotype as determined by light and electron microscopy and by flow cytometry. Methylprednisolone not only prevents the development of monocyte-derived DC but totally redirects monocyte differentiation towards a macrophage phenotype. Evidence is evolving for a role for DC in central nervous system immunity, either within the brain or in cervical lymph nodes. The demonstrated effect of both drugs on monocyte differentiation may represent an important site for immune therapy in MS.

Detection of synthetic glucocorticoid residues in cattle tissue and hair samples after a single dose administration using LC-MS/MS

A sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay for the detection of several synthetic glucocorticoids in kidney, muscle and hair samples of cattle after a single intramuscular injection is described. After a dichloromethane wash of the hair samples, analytes were released from the hair matrix by enzymatic digestion. Muscle samples were also digested enzymatically using proteinase, while kidney samples were deconjugated by Helix pomatia juice. These preliminary steps were followed by a methanol extraction and a solid phase extraction (SPE) clean up step for all matrices. Chromatographic separation was achieved on a Hypersil Hypercarb column and MS/MS data were obtained in the multiple reaction monitoring mode using negative electrospray ionization. The developed protocols were evaluated by assessing residue concentrations in muscle, kidney and hair samples of thirteen calves, treated with a particular intramuscular injection of glucocorticoid. The lowest residue levels were found in muscle samples (approximately 5% of the residue levels in kidney), while high residue levels were obtained in hair samples. Hair is an interesting matrix since the sampling is non-invasive and the drugs may stay incorporated for a longer period of time. (C) 2004 Elsevier B.V. All rights reserved.

Development and validation of a dried blood spot-LC-APCI-MS assay for estimation of canrenone in paediatric samples

A selective and sensitive liquid chromatography (LC)-atmospheric pressure chemical ionisation (APCI)-mass spectroscopic (MS) assay of canrenone has been developed and validated employing Dried Blood Spots (DBS) as the sample collection medium. DBS samples were prepared by applying 30 mu l of spiked whole blood onto Guthrie cards. A 6 mm disc was punched from the each DBS and extracted with 2 ml of methanolic solution of 17 alpha-methyltestosterone (Internal Standard). The methanolic extract was evaporated to dryness and reconstituted in acetonitrile:water (1:9, v/v). The reconstituted solution was further subjected to solid phase extraction using HLB cartridges. Chromatographic separation was achieved using Waters Sunfire C18 reversed-phase column using isocratic elution, followed by a high organic wash to clear late eluting/highly retained components. The mobile phase consisted of methanol:water (60:40, v/v) pumped at a flow rate of 0.3 ml/min. LC-APCI-MS detection was performed in the selected-ion monitoring (SIM) mode using target ions at m/z 341.1 and 303.3 for canrenone and internal standard respectively. The selectivity of the method was established by analysing DBS samples from 6 different sources (individuals). The calibration curve for canrenone was found to be linear over 25-1000 ng/ml (r >0.994). Accuracy (% RE) and precision (% CV) values for within and between day were

Nigrocin-2 peptides from Chinese Odorrana frogs – integration of UPLC/MS/MS with molecular cloning in amphibian skin peptidome analysis

Peptidomics is a powerful set of tools for the identification, structural elucidation and discovery of novel regulatory peptides and for monitoring the degradation pathways of structurally and catalytically important proteins. Amphibian skin secretions, arising from specialized granular glands, often contain complex peptidomes containing many components of entirely novel structure and unique site-substituted analogues of known peptide families. Following the discovery that the granular gland transcriptome is present in such secretions in a PCR-amenable form, we designed a strategy for peptide structural characterization involving the integration of ‘shotgun’ cloning of cDNAs encoding peptide precursors, deduction of putative bioactive peptide structures, and confirmation of these structures using tandem MS/MS sequencing. Here, we illustrate this strategy by means of elucidation of the primary structures of nigrocin-2 homologues from the defensive skin secretions of four species of Chinese Odorrana frogs, O. schmackeri, O. livida, O. hejiangensis and O. versabilis. Synthetic replicates of the peptides were found to possess antimicrobial activity. Nigrocin-2 peptides occur widely in the skin secretions of Asian ranid frogs and in those of the Odorrana group, and are particularly well-represented and of diverse structure in some species. Integration of the molecular analytical technologies described provides a means for rapid structural characterization of novel peptides from complex natural libraries in the absence of systematic online database information.

Investigating the mechanism of the H 2-assisted selective catalytic reduction (SCR) of NOx with octane using fast cycling transient in situ DRIFTS-MS analysis

A mechanistic study of the H-2-assisted Selective Catalytic Reduction (SCR) of NOx with octane as reductant over a Ag/Al2O3 catalyst was carried out using a modified DRIFTS cell coupled to a mass spectrometer Using fast transient cycling switching of H-2 with a time resolution of a few seconds It was possible to differentiate potential reaction intermediates from other moieties that are clearly spectator species Using such a periodic operation mode effects were uncovered that are normally hidden in conventional transient studies which typically consist of a single transient In experiments based on a single transient addition of H-2 to or removal of H-2 from the SCR feed it was found that the changes in the concentrations of gaseous species (products and reactants) were not matched by changes at comparable timescales of the concentration of surface species observed by IR This observation indicates that the majority of sur face species observed by DRIFTS under steady-state reaction conditions are spectators In contrast under fast cycling experimental conditions It was found that a surface isocyanate species had a temporal response that matched that of N-15(2) This suggests that some of the isocyanate species observed by infrared spectroscopy could be important intermediates in the hydrogen-assisted SCR reaction although it is emphasised that this may be dependent on the way in which the infrared spectra are obtained It is concluded that the use of fast transient cycling switching techniques may provide useful mechanistic information under certain circumstances.

Development and validation of a dried blood spot LC-MS/MS assay to quantify ranitidine in paediatric samples.

A novel approach has been developed to determine ranitidine in paediatric samples using dried blood spots (DBS) on Guthrie cards (Whatman 903). A selective and sensitive HPLC-MS/MS assay has been developed and validated using small volumes of blood (30µl). A 6mm disc was punched from each DBS and extracted with methanolic solution of the internal standard (IS) nizatidine. This was further subjected to solid phase extraction (SPE), followed by reversed phase HPLC separation, using a XBridge™ C18 column and mobile phase 10mM ammonium acetate/methanol (98:2 v/v) with a flow rate of 0.3mL/min. This was combined with multiple reaction monitoring (MRM) mass detection using electrospray ionisation (ESI). The calibration curve for ranitidine was found linear over the range 10-500ng/mL (r=0.996). The limit of quantification (LOQ) of the method was validated at 10ng/mL. Accuracy and precision values for within and between days were

Fabrication and Ms Conduct: Scrutinising Practice Through Feminist Theory

This paper describes a collaborative practice, between an architect (the author) and a textile designer; its outcomes and the critical theoretical and feminist contexts from which the practice evolved and to which it still responds. The practice advocates the interweaving of more than the yarns, material and cultures on which it is physically based, but also the intertwining of theory and technology as a means to advance architectural practice. This is done in response to Ahrentzen’s charge to feminist scholars and practitioners to ‘embrace not only the abstract conceptual nature of much postmodernist theorizing but also that derived from the serious “hanging out”, looking at, listening to, scrutinising and theorizing lived experiences of the everyday’, in this instance the everyday practice of combining concrete and textiles.

Development and validation of a method for the confirmation of nicarbazin in chicken liver and eggs using LC-electrospray MS-MS according to the revised EU criteria for veterinary drug residue analysis

A method is described for the quantitative confirmation of 4,4'-dinitrocarbanilide (DNC), the marker residue for nicarbazin in chicken liver and eggs. The method is based on LC coupled to negative ion electrospray MS-MS of tissue extracts prepared by liquid-liquid extraction. The [M-H](-) ion at m/z 301 is monitored along with two transition ions at m/z 137 and 107 for DNC and the [M-H](-) ion at m/z 309 for the internal standard, d(8)-DNC. The method has been validated according to the new EU criteria for the analysis of veterinary drug residues at 100, 200 and 300 mug kg(-1) in liver and at 10, 30 and 100 mug kg(-1) in eggs. Difficulties concerning the application of the new analytical limits, namely the decision limit (CC) and the detection capability (CC) to the determination of DNC in both liver and eggs are discussed.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400 mg kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.