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This article has been designed to give a brief overview of antigen-antibody reactions, especially in the form used in immunoassays. The different types of label and assay design have been described briefly. The report has not been designed as a brief text book, so that diagrams referring to assay principles have been omitted. The number of tables and figures has been reduced to a minimum, the references being sufficient to enable the reader to go deeper into his areas of interest. The problems of internal and external quality assessment of immunoassays has been dealt with. Finally, a short section describes possible future trends in which immunoassay may play a role.

This article reports on the performance of two "dry" chemistry devices, (Reflotron, Roche Diagnostics and Vitros, Johnson & Johnson) and compared them with classical "wet" chemistry analysers in four commercially produced quality assessment samples (Roche PNU and PPU and Seronorm Human and Human High Controls) sent repeatedly over a 12-month observation period. Eleven analytes (including five enzymes) were studied, eight of which had target values set by reference method procedures. The results showed that both devices gave comparative results for the same sample sent in different EQA-surveys. Statistically significant differences which occurred were due to the high precision of measurement with a minimal shift in the measured concentrations. They had no clinical relevance in interpretation of results. Comparisons between "dry" and "wet" chemistry results for the same analyte were almost always statistically significantly different and often large enough to influence the clinical interpretation of results. Examples here were glucose and uric acid measured with the Reflotron and compared with other Roche devices (Cobas, Hitachi). The Vitros showed deviant values for urea and creatinine, when compared with other measuring devices using liquid reagents. Differences seen were constant over time, but must be seen in context with the matrices of the samples sent. The results show the long term stability of both reagents and test kits, a necessary prerequisite for long-term controlling of precision and indirectly accuracy of patient measurements.

During a 12-month longitudinal study, bulk-tank milk was collected from organic (n=17) and conventional (n=19) dairy farms in the UK. Milk samples were analysed for vitamin A (retinol), vitamin E (alpha-tocopherol) and beta-carotene content. The farming system type, herd production level and nutritional factors affecting the milk fat vitamin content were investigated by use of mixed model analyses. Conventionally produced milk fat had a higher mean content of vitamin A than organically produced milk fat, although there were no significant differences in the vitamin E or beta-carotene contents between the two types of milk fat. Apart from farming system, other key factors that affected milk fat vitamin content were season, herd yield and concentrate feeding level. Milk vitamin content increased in the summer months and in association with increased concentrate feeding, whilst higher-yielding herds had a lower milk vitamin E and beta-carotene content. Thus, conventional dairy farms in the UK produced milk with a higher vitamin A content, possibly owing to increased vitamin A supplementation in concentrate feeds. However, knowledge of the effects of season, access to fresh grazing or specific silage types and herd production level may also be used by all producers and processors to enhance the vitamin content in milk.

The path from diatomic molecule spectroscopy to molecular modelling and drug discovery is described, along with aspects of the commercialisation of research. It is a history tightly coupled with the advances in computers over the past 50 years, but with a future full of opportunity.

A subjective cow cleanliness scoring system was validated and used to assess the cleanliness score of dairy cows at different times in the year. A longitudinal study followed a number of farms from summer to winter, and a larger, cross-sectional study assessed a greater number of farms during the housed winter period. The scoring system was demonstrated to be both a repeatable and practical technique to use on-farm and showed that cows become dirtier in the transition from summer grazing to winter housing. Although farming system (organic or conventional) had no effect on cow cleanliness when cows were at grass, when housed in the winter, organic cows were significantly more likely to be cleaner. There was a link between cow cleanliness scores and milk quality, with herds having lower bulk tank somatic cell counts (BTSCC) tending to have a lower (cleaner) median cow cleanliness score; with this relationship strongest for the organic herds. There was no significant link between cleanliness score and Bactoscan (BS) count or clinical mastitis incidence. No major mastitis pathogens were cultured from bulk tank milk samples from the quartile of herds with the cleanest cows in contrast to the quartile of herds with the dirtiest cows, where significant mastitis pathogens were cultured. Based on this study, all farms, especially organic systems, should attempt to keep cows clean as part of subclinical mastitis control.

Finding a set of molecules, which closely resemble a given lead molecule, from a database containing potentially billions of chemical structures is an important but daunting problem. Similar molecular shapes are particularly important, given that in biology small organic molecules frequently act by binding into a defined and complex site on a macromolecule. Here, we present a new method for molecular shape comparison, named ultrafast shape recognition (USR), capable of screening billions of compounds for similar shapes using a single computer and without the need of aligning the molecules before testing for similarity. Despite its extremely fast comparison rate, USR will be shown to be highly accurate at describing, and hence comparing, molecular shapes., ((c) 2007 Wiley Periodicals, Inc.)

Point of care testing (POCT) is evolving at an ever increasing rate. This article deals mainly with the aspect of POCT for blood glucose and the problems of external quality assessment (EQA) of point of care devices (POCD). At the present time it is only possible to control precision with EQA, independent of the matrix of the test materials (synthetic polymer-base, plasma/serum, or processed whole blood). The German Federal Medical Council guidelines for laboratory performance allow an interlaboratory imprecision of +/-16%. The majority of POCD fulfill these requirements. The long-term stability of results--tested by repeated distribution of the same materials over a 12-month period--is excellent, and the performance of POCD under routine condtions is usually excellent in terms of result-comparability. The problems of accuracy in terms of control materials have still to be mastered.

External quality assessment surveys for thyroid-related antibodies have been offered by INSTAND for 20 years. During this time, some problems have remained, especially those between the similarity of samples sent and routine patient samples. Here the questions of "matrix effects" and "commutability of results" are topics discussed at most EQA-meetings. This short communication deals with the effects of lyophilization on results for thyroid-associated antibodies in an EQA-survey carried out by over 230 participants in October 2005 by INSTAND in Düsseldorf, Germany. The results show that there are small but statistically significant differences (at the level p < 0.05) in results from liquid and lyophilized samples from the same source, the tendency to higher results in the lyophilized samples for anti-TPO and anti-Tg and to lower results for TRAb. The precision in measurement was significantly better for anti-TPO and TRAb in the lyophilized samples, there being no significant difference for anti-Tg. The differences in results between liquid and lyophilized samples were minimal when compared with the numerical results for anti-TPO and anti-Tg, despite the fact that all kits were calibrated with the NIBSC reference materials, although the latter are now both 40 years old.

This report was written in response to the article by Wood published recently in this journal. It describes a practical solution to the problems of controlling the pre-analytical phase in the clinical diagnostic laboratory. As an indicator of quality in the pre-analytical phase of sample processing, a target analyte was chosen which is sensitive to delay in centrifugation and/or analysis. The results of analyses of the samples sent by satellite medical practitioners were compared with those from an on-site hospital laboratory with a controllable optimized pre-analytical phase. The aim of the comparison was: (a) to identify those medical practices whose mean/median sample values significantly deviate from those of the control situation in the hospital laboratory due to the possible problems in the pre-analytical phase; (b) to aid these laboratories in the process of rectifying these problems. A Microsoft Excel-based Pre-Analytical Survey tool (PAS tool) has been developed which addresses the above mentioned problems. It has been tested on serum potassium which is known to be sensitive to delay and/or irregularities in sample treatment. The PAS tool has been shown to be one possibility for improving the quality of the analyses by identifying the sources of problems within the pre-analytical phase, thus allowing them to be rectified. Additionally, the PAS tool has an educational value and can also be adopted for use in other decentralized laboratories.

Eosinophils are observed to localize to cholinergic nerves in a variety of inflammatory conditions such as asthma, rhinitis, eosinophilic gastroenteritis, and inflammatory bowel disease, where they are also responsible for the induction of cell signaling. We hypothesized that a consequence of eosinophil localization to cholinergic nerves would involve a neural remodeling process. Eosinophil co-culture with cholinergic IMR32 cells led to increased expression of the M2 muscarinic receptor, with this induction being mediated via an adhesion-dependent release of eosinophil proteins, including major basic protein and nerve growth factor. Studies on the promoter sequence of the M2 receptor indicated that this induction was initiated at a transcription start site 145 kb upstream of the gene-coding region. This promoter site contains binding sites for a variety of transcription factors including SP1, AP1, and AP2. Eosinophils also induced the expression of several cholinergic genes involved in the synthesis, storage, and metabolism of acetylcholine, including the enzymes choline acetyltransferase, vesicular acetylcholine transferase, and acetylcholinesterase. The observed eosinophil-induced changes in enzyme content were associated with a reduction in intracellular neural acetylcholine but an increase in choline content, suggesting increased acetylcholine turnover and a reduction in acetylcholinesterase activity, in turn suggesting reduced catabolism of acetylcholine. Together these data suggest that eosinophil localization to cholinergic nerves induces neural remodeling, promoting a cholinergic phenotype.

The Schools Malaria Project (http://emalaria.soton.ac.uk/) brings together school students with university researchers in the hunt for a new antimalaria drug. The design challenge being offered to students is to use a distributed drug search and selection system to design potential antimalaria drugs. The system is accessed via a Web interface. This e-science project displays the results of the trials in an accessible manner, giving students an opportunity for discussion and debate both with peers and with the university contacts. The project has been implemented by using distributed computing techniques, spreading computer load over a network of machines that cross institutional boundaries, forming a grid. This provides access to greater computing power and allows a much more complex and detailed formulation of the drug design problem to be tackled for research, teaching, and learning.

Exposure to non-nutritional food additives during the critical development window has been implicated in the induction and severity of behavioral disorders such as attention deficit hyperactivity disorder (ADHD). Although the use of single food additives at their regulated concentrations is believed to be relatively safe in terms of neuronal development, their combined effects remain unclear. We therefore examined the neurotoxic effects of four common food additives in combinations of two (Brilliant Blue and L-glutamic acid, Quinoline Yellow and aspartame) to assess potential interactions. Mouse NB2a neuroblastoma cells were induced to differentiate and grow neurites in the presence of additives. After 24 h, cells were fixed and stained and neurite length measured by light microscopy with computerized image analysis. Neurotoxicity was measured as an inhibition of neurite outgrowth. Two independent models were used to analyze combination effects: effect additivity and dose additivity. Significant synergy was observed between combinations of Brilliant Blue with L-glutamic acid, and Quinoline Yellow with aspartame, in both models. Involvement of N-methyl-D-aspartate (NMDA) receptors in food additive-induced neurite inhibition was assessed with a NMDA antagonist, CNS-1102. L-glutamic acid- and aspartame-induced neurotoxicity was reduced in the presence of CNS-1102; however, the antagonist did not prevent food color-induced neurotoxicity. Theoretical exposure to additives was calculated based on analysis of content in foodstuff, and estimated percentage absorption from the gut. Inhibition of neurite outgrowth was found at concentrations of additives theoretically achievable in plasma by ingestion of a typical snack and drink. In addition, Trypan Blue dye exclusion was used to evaluate the cellular toxicity of food additives on cell viability of NB2a cells; both combinations had a straightforward additive effect on cytotoxicity. These data have implications for the cellular effects of common chemical entities ingested individually and in combination.

In this study a new principle of measurement in LC-MS/MS (liquid chromatography mass spectrometry) for determination of the immunosuppressive drugs sirolimus, everolimus, tacrolimus, and cyclosporin A has been introduced by using the Cs(+) ion as the product ion in the multiple reaction monitoring mode (MRM). Separation of the immunosuppressive agents was achieved using a phenyl-hexyl-RP column together with a ternary gradient elution profile, consisting of water, methanol and acetonitrile combined with 0.1% v/v formic acid and 0.1 mmol/l Cs+. Quantification was performed using cyclosporin D, ascomycin and 32-desmethoxy-rapamycin as internal standards. The inter-run precision of this new method, expressed as the coefficient of variation, was 2.57% for sirolimus, 2.11% for everolimus, 2.31% for tacrolimus and 2.11% for cyclosporin A.

This short article describes the results obtained in both internal and external quality assessment of point of care devices (POCD) for the monitoring of blood glucose. The results show that the use of synthetic, serum and whole blood matrices for the samples do not markedly change the inaccuracy of measurement. It is only possible to check precision of POC devices for glucose in external quality assessment (EQA) surveys for POC devices for blood glucose. The majority of devices used in point of care testing for blood glucose had acceptable (im)precision. The applications and limitations of the use of POC devices for blood glucose must be clearly defined before allowing their use in patient care programmes. Finally, the results confirm that the decision of the German Federal Medical Council (Bundesärztekammer) to exclude POC devices from accuracy checks against a reference method procedure in national EQA surveys was correct, at least at the present time.

This article describes an essential improvement of the published candidate reference measurement procedure for digoxin and digitoxin and compares it with the original method. The novelty of the method lies in the measurement of the caesium (Cs+) ion as product ion in the multiple reaction monitoring mode (MRM) with potentially improved analytical specificity whilst retaining a comparable accuracy and precision at therapeutic levels. The original measurement procedure used the single-ion mode (SIM). The dissociation of the Cs+ adducts in MRM leads to the formation of Cs+ ions as main charged product in high yield. The present method results in a product ion signal intensity in MRM for digoxin and digitoxin of up to 80% of the precursor ion signal intensity in SIM. The precision, expressed as the coefficient of variation of the new method for digoxin was 3.18% (SIM) and 2.28% (MRM) at a concentration of 0.66 microg/l and 1.26% (SIM) or 1.65% (MRM) at 2.0 microg/l. The corresponding data for digitoxin were 1.21% (SIM) and 1.62% (MRM) at 24 microg/l and 1.46% (SIM) and 1.13% (MRM) at 42 microg/l.

Activated eosinophils release potentially toxic cationic granular proteins, including the major basic proteins (MBP) and eosinophil-derived neurotoxin (EDN). However, in inflammatory conditions including asthma and inflammatory bowel disease, localization of eosinophils to nerves is associated with nerve plasticity, specifically remodeling. In previous in vitro studies, we have shown that eosinophil adhesion to IMR-32 nerve cells, via nerve cell intercellular adhesion molecule-1, results in an adhesion-dependent release of granule proteins. We hypothesized that released eosinophil granule proteins may affect nerve cell signaling and survival, leading to nerve cell remodeling. Culture in serum-deprived media induced apoptosis in IMR-32 cells that was dose-dependently abolished by inclusion of MBP1 but not by EDN. Both MBP1 and EDN induced phosphorylation of Akt, but with divergent time courses and intensities, and survival was independent of Akt. MBP1 induced activation of neural nuclear factor (NF)-kappaB, from 10 min to 12 h, declining by 24 h, whereas EDN induced a short-lived activation of NF-kappaB. MBP1-induced protection was dependent on phosphorylation of ERK 1/2 and was related to a phospho-ERK-dependent upregulation of the NF-kappaB-activated anti-apoptotic gene, Bfl-1. This signaling pathway was not activated by EDN. Thus, MBP1 released from eosinophils at inflammatory sites may regulate peripheral nerve plasticity by inhibiting apoptosis.

The stable tautomeric forms of two representative arene-substituted diketoacid HIV-1 integrase inhibitors, 5-ClTEP and L-731,988, were investigated by B3LYP with 6-31G*, 6-31G(d,p), and 6-31+G(d,p) basis sets. Optimization with MP2/6-31G* was also performed for 5-ClTEP. The solvation effect was considered using a conductor-like screening model. With the density functional theory method, the trans diketo conformations are more stable than the cis conformers. The difference is 14 kJ mol(-1) for 5-ClTEP and 33 kJ mol(-1) for L-731,988. Two trans diketo structures were obtained. The difference between these two trans diketo structures is less than 4 kJ mol(-1) calculated at the B3LYP/6-311+G(3df,2p) level. Two enol forms prevail over the diketo tautomers and are calculated to have the same free energy. Because there is no barrier observed between these two enol forms, they can interchange easily such that a delocalized transition state is suggested to be the observed form. Contradictory to the results of the MP2 method that predicts a preference for the trans diketo forms, the B3LYP method predicts a preference for the enol tautomers, which is in agreement with the experimental results.

This review shows the current analytical quality for the following analytes used as tumour markers in the external quality assessment (EQA)-programmes of Instand e.V., a national EQA-organiser in Germany: Corticotropin (ACTH), growth hormone (GH, hGH), prolactin (PRL), chorionic gonadotropin (CG, hCG), calcitonin (CT, hCT), thyroglobulin (Tg), carcinoembryonic antigen (CEA), CA-Antigens 125, 72-4, 15-3 and 19-9, alpha foetoprotein (AFP) and prostate-specific antigen (PSA). The results from the participants show a large variation in the precision of the methods used as well as in the comparability of results between methods for the same analyte. In general, the hormones used as tumour markers show better performance than the "CA-markers", which are often inadequately standardised and defined. In the case of one CA-marker (CA 72-4/TAG 72-4), the differences between the lowest kit median concentration and highest kit median concentration for one sample pair were 440% and 580%. The corresponding figures for ACTH were 123% and 156% and for CEA 180% and 184%. The classical tumour markers such as carcinoembryonic antigen (CEA) and alpha foetoprotein (AFP) performed markedly better than the CA-markers and PSA with regards to both inter- and intra-method comparability. The inter-laboratory precision for a given kit and marker was acceptable in many cases. The results show that only results from the same kit/method for each tumour marker can be used for cumulative or time-dependent comparison of results - for example pre-operative and post-operative follow up. In the case of prostate specific antigen (PSA), the kits used for free and total PSA must come from the same producer, if the generally accepted ratios are to have any diagnostic value. The need for kit- and laboratory-specific reference ranges and cut-off values for setting diagnostic specificity and sensitivity is highlighted from the EQA-results. The situation for inter-method comparability for the CA-Markers has not improved over the past decade. With the exception of calcitonin for detecting medullary thyroid carcinoma, chorionic gonadotropin in germ-cell tumours in men and thyroglobulin after total thyroidectomy, none of the remaining analytes appear to be suitable for screening purposes.

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.

This article describes practical problems in carrying out witness audits in terms of the international normal ISO 15189, with special regard to the situation in medical diagnostic laboratories in Germany. The presence of central laboratories--for example in hospitals--and decentralised laboratories--usually in the private sector--present different problems, especially in terms of the preanalytical phase and its control during witness audits for laboratories accredited according to ISO 15189. Whereas the preanalytical phase can be--at least in theory--controlled completely in and by a centralised hospital laboratory, for example by the use of "sample-collection teams", the problems in preparation and transporting of samples from a peripheral practice to a decentralised analytical laboratory cannot be fully controlled. Other aspects of the preanalytical phase including biological variability, wrong specimen additives, wrong patient preparation and the ADR 2005 / UN Packaging regulations are briefly discussed together with the aims of an optimised pre-analytical phase. The paragraphs of the normal ISO 15189 which cannot be fully controlled during a witness audit are listed, together with compromise solutions.

The present article presents a critical review of the current guidelines of the Federal Medical Council (Richtlinie der Bundesärztekammer--[RiliBAK]) in Germany, both for internal and external quality control. Examples have been chosen for analytes which present problems. These include thyrotropin (TSH) and human chorionic gonadotropin (hCG). Data are presented for the difference in analyte concentrations found between methods/kits and longitudinally for external quality assessment (EQA-) surveys for analytes using reference method values as target values. These include: calcium, aspartate aminotransferase (ASAT; GOT), aldosterone, cortisol, 17beta-oestradiol and total thyroxine (TT4). Furthermore, internal data from participating laboratories concerning laboratory internal precision and accuracy have been analysed to show the state of the art with regard to both parameters. Data analysis shows the need for a reintroduction of concentration-dependent assessment of performance (crossover system) for certain analytes, where clinically relevant concentrations are measured at the extremes of the calibration curve. The current guideline is at present being restructured and should include considerations made in this review of the state-of-the-art of clinical laboratory analysis.

Eosinophils release a number of mediators that are potentially toxic to nerve cells. However, in a number of inflammatory conditions, such as asthma and inflammatory bowel disease, it has been shown that eosinophils localize to nerves, and this is associated with enhanced nerve activity. In in vitro studies, we have shown that eosinophil adhesion via neuronal ICAM-1 leads to activation of neuronal NF-kappaB via an ERK1/2-dependent pathway. In this study, we tested the hypothesis that eosinophil adhesion to nerves promotes neural survival by protection from inflammation-associated apoptosis. Exposure of differentiated IMR-32 cholinergic nerve cells to IL-1beta, TNF-alpha, and IFN-gamma, or culture in serum-deprived medium, induced neuronal apoptosis, as detected by annexin V staining, caspase-3 activation, and DNA laddering. Addition of human eosinophils to IMR-32 nerve cells completely prevented all these features of apoptosis. The mechanism of protection by eosinophils was by an adhesion-dependent activation of ERK1/2, which led to the induced expression of the antiapoptotic gene bfl-1. Adhesion to nerve cells did not influence the expression of the related genes bax and bad. Thus, prevention of apoptosis by eosinophils may be a mechanism by which these cells regulate neural plasticity in the peripheral nervous system.

The deacylation step of serine protease catalysis is studied using DFT and ab initio QM/MM calculations combined with MD/umbrella sampling calculations. Free energies of the entire reaction are calculated in the gas phase, in a continuum solvent, and in the enzyme elastase. The calculations show that a concerted mechanism in the gas phase is replaced by a stepwise mechanism when solvent effects or an acetate ion are added to the reference system, with the tetrahedral intermediate being a shallow minimum on the free energy surface. In the enzyme, the tetrahedral intermediate is a relatively stable species ( approximately 7 kcal/mol lower in energy than the transition state), mainly due to the electrostatic effects of the oxyanion hole and Asp102. It is formed in the first step of the reaction, as a result of a proton transfer from the nucleophilic water to His57 and of an attack of the remaining hydroxyl on the ester carbonyl. This is the rate-determining step of the reaction, which requires approximately 22 kcal/mol for activation, approximately 5 kcal/mol less than the reference reaction in water. In the second stage of the reaction, only small energy barriers are detected to facilitate the proton transfer from His57 to Ser195 and the breakdown of the tetrahedral intermediate. Those are attributed mainly to a movement of Ser195 and to a rotation of the His57 side chain. During the rotation, the imidazolium ion is stabilized by a strong H-bond with Asp102, and the C(epsilon)(1)-H...O H-bond with Ser214 is replaced by one with Thr213, suggesting that a "ring-flip mechanism" is not necessary as a driving force for the reaction. The movements of His57 and Ser195 are highly correlated with rearrangements of the binding site, suggesting that product release may be implicated in the deacylation process.

Using personal computers in a grid is permitting the in silico screening of millions of molecules to seek out potential inhibitors of agents that pose bioterror threats. Current projects are targeting anthrax and smallpox, but the approach has many attractions for investigating any known protein target and its inhibition.

Protein similarity estimations can be achieved using reduced dimensional representations and we describe a new application for the generation of two-dimensional maps from the three-dimensional structure. The code for the dimensionality reduction is based on the concept of pseudo-random generation of two-dimensional coordinates and Monte Carlo-like acceptance criteria for the generated coordinates. A new method for calculating protein similarity is developed by introducing a distance-dependent similarity field. Similarity of two proteins is derived from similarity field indices between amino acids based on various criteria such as hydrophobicity, residue replacement factors and conformational similarity, each showing a one factor Gaussian dependence. Results on comparisons of misfolded protein models with data sets of correctly folded structures show that discrimination between correctly folded and misfolded structures is possible. Tests were carried out on five different proteins, comparing a misfolded protein structure with members of the same topology, architecture, family and domain according to the CATH classification.

This mortality study extends the period of observation of an article published in 1988 of 5414 workers in Vermont granite sheds and quarries to assess whether previously reported reductions in silicosis and tuberculosis mortality were maintained. The relationship between lung cancer and quartz exposure is also examined by comparing mortality in workers hired before and after 1940, when dust controls were introduced and exposures were reduced by 80% to 90%. Before 1940, general stone shed air contained 20 million particles/cubic foot (mppcf) (approximately equivalent to 0.2 mg/m of quartz), and pneumatic chisel workers were exposed on average to 60 mppcf (approximately equivalent to 0.6 mg/m of quartz). Other workers had variable exposures. After 1940, a period of decline occurred in dust levels and then stabilized in approximately 1955, when average dust levels were 5 to 6 mppcf (equivalent to 0.05-.06 mg/m of quartz). Dust exposures in the Vermont industry is considered to be free of confounding occupational substances such as arsenic, although cigarette smoking was common. By the end of 1996, 2539 workers, or 46.9% of the cohort, had died. There were no silicosis deaths in workers hired after 1940 who were exposed only in the Vermont granite industry, illustrating the effect of lowering quartz exposures. Tuberculosis caused 2 deaths in those hired after 1940 (standardized mortality ratio [SMR] = 0.52; not significant). Overall lung cancer mortality was elevated in shed workers who had been exposed both to high levels of quartz before 1940 and to the lower levels prevailing after 1940 (SMR = 1.32; P < 0.01). Quarry workers did not show an excess of lung cancer (SMR = 0.73; not significant). When shed workers with high and low exposure histories (before and after 1940) but with comparable latency and tenure were contrasted, lung cancer mortality was similar. Differing levels of quartz exposure, which resulted in large differences in the mortality experience from silicosis, did not result in differences in lung cancer mortality. The results do not support the hypothesis that granite dust exposure has a causal association with lung cancer.

Pegylated interferon alpha-a (40 kDa) has recently been shown to be superior to conventional interferon. Furthermore, in a pilot study, pegylated interferon alpha-2b together with lamivudine was superior to lamivudine monotherapy. Therefore, the pegylated interferons alone or in combination with antiviral agents are likely to be the treatment of the future. Selecting patients for treatment and knowing when to treat them is as important as knowing which medicine to treat them with.

Eosinophil localization to cholinergic nerves occurs in a variety of inflammatory conditions, including asthma. This localization is mediated by interactions between eosinophil integrins and neuronal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Eosinophil-nerve cell interactions lead to generation of neuronal reactive oxygen species and release of eosinophil proteins. The effects of eosinophil adhesion on neuronal intracellular signaling pathways were investigated. Eosinophil adhesion to IMR32 cholinergic nerves led to a rapid and sustained activation of the nuclear transcription factors nuclear factor (NF)-kappaB and activator protein (AP)-1 in the nerve cells. Eosinophil binding to neuronal ICAM-1 led to a rapid activation of ERK1/2 in nerve cells. Inhibition of ERK1/2 prevented NF-kappaB activation. Eosinophil adhesion to VCAM-1 resulted in AP-1 activation, mediated partially by rapid activation of the p38 mitogen-activated protein kinase. These data show that adhesion of eosinophils induces mitogen-activated protein kinase-dependent activation of the transcription factors NF-kappaB and AP-1 in nerve cells, indicating that eosinophil adhesion may control nerve growth and phenotype.

Four thyroid antibodies (antibodies to microsomes [MAb], thyroid peroxidase [anti-TPO], thyroglobulin [anti-Tg] and TSH-receptor [TRAB, THYBIA]) have been followed up over a 10-year period in a national external quality assessment scheme (EQAS) organised by the Institute for Standardisation and Documentation in the Medical Laboratory (INSTAND e.V.). The following points were observed: I. The introduction of samples with properties similar to patient serum (filtered, recalcified defibrinated plasma without stripping) improved performance and inter-method comparability for thyroid antibodies. II. Regular statistical analysis of EQAS data allows adjustment of target ranges to be made when necessary. III. There are large inter-method variations in reporting, both on qualitative and quantitative results. IV. The samples often gave rise to different constellations of antibodies, which were kit-dependent. V. Despite use of international reference preparations, there was no numerical comparability between quantitative methods for the same analyte. In general, the performance in EQAS for thyroid antibodies has improved over the past decade. There is still a real need for standardisation in the field of thyroid antibody analysis.

Studies of 4 to 6 months of treatment with interferon for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B virus (HBV) infection have shown clearance of HBeAg to be higher in treated patients than it is in controls by approximately 25%. These results are considerably better than those with antiviral agents. Therefore, the recent European Association for the Study of the Liver (EASL) Consensus Committee recommended the use of interferon alpha for this condition. Treatment with pegylated interferons in several trials has shown better results still. Lamivudine in combination with interferon, however, did not improve the results at 6 months after the end of therapy. In HBeAg-negative chronic HBV infection, pegylated interferon alpha is superior to lamivudine, and, again, combination with lamivudine does not improve the results. Side effects in all studies have been tolerable. Thus, these observations in chronic HBV infection, whether HBeAg-positive or HBeAg-negative, suggest an important, even primary, role for pegylated interferon therapy.

The change from measuring enzyme catalytic activity concentrations from 25 degrees C to 37 degrees C in the German Federal Republic has led to the need for new reference ranges for defined patient groups and for healthy individuals. Up to now, these are only present as tentative values and are incomplete, especially for children. This article describes a method for deriving reference ranges from results obtained from measurement at 25 degrees C and 37 degrees C and the use of percentiles to establish values for 37 degrees C. A total of 1,111,378 data from 507,305 patients were used to establish reference ranges for the following 11 enzymes at 37 degrees C using the test kits from Roche Diagnostics measured on the Modular analyser: acid phosphatase, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, cholinesterase, creatine kinase, creatine kinase - MB subunit, gamma glutamyl transpeptidase, glutamate dehydrogenase, lactate dehydrogenase and lactate dehydrogenase - isoenzyme 1. The computed reference ranges from the data used gave rise to reference ranges, some of which were in agreement with the data from the producer, some of which, however, showed deviations from the values given by the producer. Ranges for newborns, children and adolescents could be computed with the prerequisite that ranges for 25 degrees C were available and that these had been established and validated. This method of establishing reference ranges for catalytic enzyme activities can be used for all producers, providing the number of data used is sufficient to allow for valid statistical analysis.

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 +/- 2% of control at 10(-7) M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 +/- 23% of control at 10(-5) M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 +/- 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 +/- 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.

Conventional interferon therapy has been used for the treatment of chronic hepatitis B (CHB) for many decades. However, the use of interferon has been limited by its short half-life and high incidence of dose-related side effects. A meta-analysis investigating the short- and long-term consequences of interferon therapy showed that, whilst interferon therapy was beneficial in the short term, resulting in normalization of alanine aminotransferase (ALT) levels, loss of HBeAg, 'e' seroconversion and suppression of hepatitis B virus (HBV) DNA, the long-term benefits were less substantial. Pegylation of interferon (peginterferon alpha-2a [40 kDa]) led to improved pharmacokinetic and pharmacodynamic profiles, which translated to superior efficacy compared with conventional, nonpegylated interferon, in the treatment of chronic hepatitis C. A phase II study investigated the safety and efficacy of peginterferon alpha-2a (40 kDa) in the treatment of chronic hepatitis B. The results demonstrated a rapid and dramatic reduction in HBV DNA levels, HBeAg clearance and normalization of ALT with peginterferon alpha-2a (40 kDa) compared with conventional interferon. Furthermore, peginterferon alpha-2a (40 kDa) conferred a notably improved treatment response in patients with 'difficult-to-treat' hepatitis B infection. In conclusion, peginterferon alpha-2a (40 kDa) is a promising emerging therapy for CHB.

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.

High surface area alumina coatings were prepared on surface acoustic wave (SAW) mass balances. These coatings were fabricated by anodic etching of evaporated aluminum films. The coatings consisted of roughly collinear pores penetrating through the monolithic alumina film. The nanoporous (NP) coatings were characterized by scanning electron microscopy, and the pore number density and diameter were found to be (3.8 +/- 0.5) x 10(3) pores/microm2 and 6.8 +/- 4.8 nm, respectively. The mass of volatile organic compounds that adsorbed onto naked and chemically modified NP alumina coatings was measured using SAW mass balances and compared to the mass absorbed onto SAW devices having planar aluminum coatings. Thirty-four times more heptane adsorbed to the naked NP coating than to the planar coating. The mass loading response was also measured after modification with organic thin films (3-12 nm thick) that spanned the pores of the NP coating. These organic thin films were composed of sixth-generation, amine-terminated poly(amido amine) dendrimers and poly(maleic anhydride)-c-poly(methyl vinyl ether) (Gantrez). The key result of this study is that these organic thin films modulate adsorption of VOCs onto the pore walls of the NP alumina. Specifically, a single 3-nm-thick monolayer of the dendrimer reduces permeability of the VOCs by approximately 17%, whereas a 12-nm-thick G6-NH2/Gantrez composite reduces permeability by 100%. Thus, the polymer composite acts as a nonselective gate that controls access of VOCs to the underlying surface area of the pores.

This article describes a method of high analytical sensitivity, reproducibility and trueness for the determination of digoxin and digitoxin in serum or plasma at therapeutic levels using a combination of high-pressure liquid chromatography (HPLC), isotope-dilution mass spectrometry (IDMS) and caesium-adduct formation. A method for threefold deuterium substitution in the glycosides was developed, which could be performed within 24 hours without distillation giving yields > 98% of the theoretical value. Extraction from a serum or plasma matrix was performed using a liquid-phase extraction with ammonium acetate buffer/tertiary butylmethyl ether/ethyl acetate at pH 9.5. The HPLC-separation used a 10 x 2 mm LiChrospher RP-18 5 microm guard column in combination with a 125 x 2 mm main column of the same material and a gradient containing methanol, caesium ions and formic acid. Quantification of digoxin and digitoxin was made with IDMS using deuterated internal standards and the system run in single ion monitoring (SIM) mode. The methods had a lower limit of determination of 0.25 microg/l for digoxin and digitoxin, a trueness between 97.5 and 104% for digoxin and between 98 and 101% for digitoxin, respectively and had a coefficient of variation of less than 3% in the therapeutic range for both glycosides. Maximally 1 ml serum or plasma was needed for the procedure. The method is used to set target values for materials used in external quality assessment surveys (EQAS) run by INSTAND as part of a national EQAS-programme.)

This article describes the preparation and internal and external evaluation of materials, critical issues in the external quality assessment (EQA) of point-of-care testing (POCT) devices for measuring blood glucose. A comparison was made between different materials, both of natural and synthetic origin and with and without stabilisers. The aims were to produce a material which was compatible with as many POCT-devices as possible and so reduce the number of materials sent out in each campaign as well as to optimise the precision and comparability of results between methods and devices. Although the use of near natural material--sterile-filtered plasma spiked with glucose--survived internal testing, this material proved to be unsuitable for EQA surveys. The study resulted in the reduction of materials for each survey to stabilised whole blood for one device, stabilised plasma for two devices and a synthetic material based on a polyethylene glycol matrix for all other devices. Samples were sent as pairs six times annually. The POCT-devices tested measured precisely but inaccurately in the synthetic material, when compared with the reference method (gas-chromatography coupled with isotope-dilution mass-spectrometry; GC-IDMS), so that the devices could only be evaluated for precision. The construction of ratios between the concentrations measured on the two samples distributed allowed an indirect assessment of accuracy. The need for surveillance of POCT devices is stressed in this publication, which combines theory and practice in setting up and running an EQA programme for blood glucose.

By reducing protein structures to two-dimensional representations, it is possible to speed up the alignment of the structures and hence calculate similarity indices faster that using three-dimensional representations. Using amino acid based representations gives much better discrimination between proteins and faster calculations. Taking into account the relative similarity of the amino acids involved allowed improved accuracy at very little time cost.

In vivo, eosinophils localise to airway nerves in patients with asthma as well as in animal models of hyperreactivity. In both, in vivo and in vitro studies, we have shown that this localisation changes both cholinergic nerve and eosinophil function. In particular, it leads to an increase in acetylcholine release due to loss of function of a neuronal autoreceptor, the M(2) muscarinic receptor. This loss of M(2) receptor function occurs because eosinophils become activated and degranulate as a result of interactions that occur via specific adhesion molecules expressed on nerves that are recognised by counter ligands on eosinophils.

Ab initio QM/MM dynamics simulation is employed to examine the stability of the tetrahedral intermediate during the deacylation step in elastase-catalyzed hydrolysis of a simple peptide. An extended quantum region includes the catalytic triad, the tetrahedral structure, and the oxyanion hole. The calculations indicate that the tetrahedral intermediate of serine proteases is a stable species on the picosecond time scale. On the basis of geometrical and dynamical properties, and in agreement with many experimental and theoretical studies, it is suggested that the crucial hydrogen bonds involved in stabilizing this intermediate are between Asp-102 and His-57 and between the charged oxygen of the intermediate and the backbone N-H group of Gly-193 in the oxyanion hole. The mobility of the imidazolium ring between O(w) and O(gamma), two of the oxygens of the tetrahedral structure, shows how the intermediate could proceed toward the product state without a "ring-flip mechanism", proposed earlier on the basis of NMR data. In addition to the proposed C(epsilon)(1)-H.O hydrogen bond between the imidazolium ring and the backbone carbonyl of Ser-214, we observe an alternative C(epsilon)(1)-H.O hydrogen bond with the backbone carbonyl of Thr-213, that can stabilize the intermediate during the imidazolium movement. Proton hopping occurs between Asp-102 and His-57 during the simulation. The proton is, however, largely localized on the nitrogen, and hence it does not participate in a low-barrier hydrogen bond. The study also suggests factors that may be implicated in product release: breaking the hydrogen bond of the charged oxygen with the backbone of Ser-195 in the oxyanion hole and a loop opening between residues 216-225 that enables the breaking of a hydrogen bond in subsite S(3).

A feature of Peter Kollman's research was his exploitation of the latest computational techniques to devise novel applications of the free energy perturbation method. He would certainly have seized upon the opportunities offered by massively distributed computing. Here we describe the use of over a million personal computers to perform virtual screening of 3.5 billion druglike molecules against protein targets by pharmacophore pattern matching, together with other applications of pattern recognition such as docking ligands without any a priori knowledge about the binding site location., (Copyright 2002 Wiley Periodicals, Inc.)

Structural genomics will yield an immense number of protein three-dimensional structures in the near future. Automated theoretical methodologies are needed to exploit this information and are likely to play a pivotal role in drug discovery. Here, we present a fully automated, efficient docking methodology that does not require any a priori knowledge about the location of the binding site or function of the protein. The method relies on a multiscale concept where we deal with a hierarchy of models generated for the potential ligand. The models are created using the k-means clustering algorithm. The method was tested on seven protein-ligand complexes. In the largest complex, human immunodeficiency virus reverse transcriptase/nevirapin, the root mean square deviation value when comparing our results to the crystal structure was 0.29 A. We demonstrate on an additional 25 protein-ligand complexes that the methodology may be applicable to high throughput docking. This work reveals three striking results. First, a ligand can be docked using a very small number of feature points. Second, when using a multiscale concept, the number of conformers that require to be generated can be significantly reduced. Third, fully flexible ligands can be treated as a small set of rigid k-means clusters.

The CD8 coreceptor of cytotoxic T lymphocytes binds to a conserved region of major histocompatibility complex class I molecules during recognition of peptide-major histocompatibility complex (MHC) class I antigens on the surface of target cells. This event is central to the activation of cytotoxic T lymphocyte (CTL) effector functions. The contribution of the MHC complex class I light chain, beta(2)-microglobulin, to CD8alphaalpha binding is relatively small and is mediated mainly through the lysine residue at position 58. Despite this, using molecular modeling, we predict that its mutation should have a dramatic effect on CD8alphaalpha binding. The predictions are confirmed using surface plasmon resonance binding studies and human CTL activation assays. Surprisingly, the charge-reversing mutation, Lys(58) --> Glu, enhances beta(2)m-MHC class I heavy chain interactions. This mutation also significantly reduces CD8alphaalpha binding and is a potent antagonist of CTL activation. These results suggest a novel approach to CTL-specific therapeutic immunosuppression.