Suppression of tumour necrosis factor production from mononuclear cells by a novel synthetic compound, CLX-090717.

OBJECTIVES: To evaluate the clinical efficacy of a novel synthetic peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonist, CLX-090717, in several in vitro cell culture systems and murine CIA, an experimental model of RA. METHODS: Peripheral blood monocytes purified by elutriation, and rheumatoid synovial cells isolated from clinical tissue were cultured with CLX-090717 and TNF-alpha release was measured. Molecular mechanism of action was analysed by western blotting and electrophoretic mobility shift assay. Thioglycollate-elicited murine peritoneal macrophages were cultured with CLX-090717 and lipopolysaccharide (LPS)-induced TNF-alpha release was assayed. Therapeutic studies were done in mice with established arthritis by evaluating clinical parameters and histology. In addition, type II collagen response of lymphocytes from mice with CIA was examined. RESULTS: CLX-090717 significantly inhibited spontaneous TNF-alpha release by RA synovial membrane cells, as well as LPS-induced TNF-alpha release from human and murine monocytic cells. Inhibition of TNF-alpha in monocytes was mediated partially through a nuclear factor-kappaB (NF-kappaB)-dependent pathway, as judged by sustained levels of IkappaBalpha in cytosolic extracts and a reduced level of LPS-induced NF-kappaB activity in nuclear extracts. CLX-090717 reduced clinical signs of arthritis and damage to joint architecture when administered therapeutically to arthritic mice. Mechanisms of action in CIA involved the reduction in proliferation of arthritic lymphocytes to antigen in vitro as well as reduced TNF-alpha release. CONCLUSIONS: Our data suggest that the synthetic compound CLX-090717 has potential as a small molecular weight anti-inflammatory therapeutic for chronic inflammatory conditions.

Urban legend versus rural reality: patients' experience of attendance at accident and emergency departments in west Wales.

OBJECTIVES: To investigate why and how patients decide to attend accident and emergency (A&E) departments, and to assess their satisfaction with the experience, in a predominantly rural west Wales population. METHODS: This was a semi-structured follow up telephone interview of patients who walked in to A&E in one of four general hospitals in west Wales and were triaged as Manchester Triage score 4 or 5. Patients were recruited by nurses during the period July-November 2002. The study sample consisted of 176 male and 145 female patients, mean (SD) age 36.6 (20.0) years. The main outcome measure was a quantitative and qualitative description of the recalled experiences of A&E attenders, the circumstances of their attendance, and their satisfaction with the experience. RESULTS: Of the study sample, 78% attended with injury or illnesses of recent origin, and 50% with actual or presumed musculoskeletal injury, 73% of which were sustained within 10 miles of home. Travel to hospital was by private transport for 86%, average distance 7.4 miles. The majority (90%) were registered with a local GP, but 32% felt A&E was the obvious choice, and a further 44% considered their GP inaccessible to their needs. Patients' reasons for seeking health care at A&E were similar to those described in an English urban study. Waiting times were rarely excessive; 80% left within 2 hours, and patient satisfaction was generally high. Among the 87 patients (27%) who reported a less satisfactory experience, 48 (55%) of these complained of dismissive attitudes of doctors. CONCLUSIONS: Anecdotal accounts of abuse of A&E services and unreasonable patient expectations gain the status of "urban legends" within the medical profession. Among the predominantly settled rural population in west Wales, there is little evidence of unreasonable patient expectations, and most patients report high satisfaction levels. Patients' bad experiences most frequently arise from a dismissive attitude on the part of medical staff. These attitudes are often consequent on an A&E culture that views some patients' attendances as less appropriate than others.

Processes affecting the remediation of chromium-contaminated sites.

The remediation of chromium-contaminated sites requires knowledge of the processes that control the migration and transformation of chromium. Advection, dispersion, and diffusion are physical processes affecting the rate at which contaminants can migrate in the subsurface. Heterogeneity is an important factor that affects the contribution of each of these mechanisms to the migration of chromium-laden waters. Redox reactions, chemical speciation, adsorption/desorption phenomena, and precipitation/dissolution reactions control the transformation and mobility of chromium. The reduction of CrVI to CrIII can occur in the presence of ferrous iron in solution or in mineral phases, reduced sulfur compounds, or soil organic matter. At neutral to alkaline pH, the CrIII precipitates as amorphous hydroxides or forms complexes with organic matter. CrIII is oxidized by manganese dioxide, a common mineral found in many soils. Solid-phase precipitates of hexavalent chromium such as barium chromate can serve either as sources or sinks for CrVI. Adsorption of CrVI in soils increases with decreasing chromium concentration, making it more difficult to remove the chromium as the concentration decreases during pump-and-treat remediation. Knowledge of these chemical and physical processes is important in developing and selecting effective, cost-efficient remediation designs for chromium-contaminated sites.

Reduced activity of tryptophan 2,3-dioxygenase in the liver of rats treated with chlorinated dibenzo-p-dioxins (CDDs): dose-responses and structure-activity relationship.

The activity of tryptophan 2,3-dioxygenase (TdO) was measured in the livers of male Sprague-Dawley rats after acutely toxic doses (LD20-LD80) of chlorinated dibenzo-p-dioxins (CDDs) with 4 of the up to 7 chlorine substituents occupying the 2,3,7,8-positions. Treatment with toxic doses of CDDs results in voluntary feed refusal of rats. A corresponding involuntary reduction of feed intake in naive animals (pair-feeding) causes elevated levels of TdO activity. In the CDD treated rats, however, TdO activities were dose-dependently reduced. An LD80 reduced TdO activity to about 50% of the level found in the corresponding pair-fed animals. This decrease of TdO activity explains the dose-dependent increase of serum tryptophan, which in turn is the likely cause of voluntary feed refusal observed in CDD-treated rats. The activity of another enzyme which is regulated in a fashion very similar to that of TdO, viz., tyrosine aminotransferase (TAT), was consistently, but not dose-dependently, affected by treatment with CDDs.

Running economy is impaired following a single bout of resistance exercise.

The purpose of this study was to determine whether a low-volume high-intensity resistance training session influenced running economy during a subsequent aerobic treadmill run. Nine well trained distance runners (mean +/- SD; VO2max, 66.6 +/- 10.2 ml x kg(-1) x min(-1); weight, 65.8 +/- 10.2 kg; height, 173.4 +/- 7.8 cm; age 20 +/- 1.1 years) with resistance training experience performed treadmill running at two different speeds (0.56 m x sec(-1) and 0.20 m x sec(-1) below speed corresponding to lactate equilibrium) either rested or 1, 8 or 24 hours after a 50-minute whole body resistance training session. Running economy was assessed using open circuit spirometry while heart rate was recorded telemetrically. The contractile properties of the quadriceps femoris were also determined following each resistance training session and prior to each treadmill run using percutaneous electrical stimulation. Submaximal oxygen consumption was significantly increased one hour (2.6 +/- 2.3%, p= 0.007), and eight hours (1.6 +/- 2.5%, p= 0.032), but not 24 hours after resistance training. No significant differences were found in exercising heart rate, ventilation, respiratory exchange ratio, ratings of perceived exertion, or running mechanics. Peak twitch torque, time to peak torque, and half relaxation time of the quadriceps femoris were significantly reduced immediately following resistance training while peak twitch torque was also lower one hour following resistance training. Running economy following a resistance training session is impaired for up to 8 hours. This change was not paralleled by a concomitant change in exercising heart rate. The mechanism responsible for increased oxygen consumption following resistance training may be related to impairment of the force generating capacity of skeletal muscle, as there was a significant decrement in the contractile properties of the quadriceps femoris following resistance training.

Certification of Total Arsenic in Blood and Urine Standard Reference Materials by Radiochemical Neutron Activation Analysis and Inductively Coupled Plasma - Mass Spectrometry.

A newly developed procedure for determination of arsenic by radiochemical neutron activation analysis (RNAA) was used to measure arsenic at four levels in SRM 955c Toxic Elements in Caprine Blood and at two levels in SRM 2668 Toxic Elements in Frozen Human Urine for the purpose of providing mass concentration values for certification. Samples were freeze-dried prior to analysis followed by neutron irradiation for 3 h at a fluence rate of 1×1014cm-2s-1. After sample dissolution in perchloric and nitric acids, arsenic was separated from the matrix by extraction into zinc diethyldithiocarbamate in chloroform, and 76As quantified by gamma-ray spectroscopy. Differences in chemical yield and counting geometry between samples and standards were monitored by measuring the count rate of a 77As tracer added before sample dissolution. RNAA results were combined with inductively coupled plasma - mass spectrometry (ICP-MS) values from NIST and collaborating laboratories to provide certified values of (10.81 ± 0.54) µg/kg and (213.1 ± 0.73) µg/kg for SRM 2668 Levels I and II, and certified values of (21.66 ± 0.73) µg/kg, (52.7 ± 1.1) µg/kg, and (78.8 ± 4.9) µg/kg for SRM 955c Levels 2, 3, and 4 respectively. Because of discrepancies between values obtained by different methods for SRM 955c Level 1, an information value of < 5 µg/kg was assigned for this material.

Simultaneous detection of selenium by atomic fluorescence and sulfur by molecular emission by flow-injection hydride generation with on-line reduction for the determination of selenate, sulfate and sulfite.

An inductively coupled plasma atomic fluorescence spectrometry (ICP-AFS) instrument, was modified so that it was capable of monitoring transient chromatographic or flow-injection profiles and that sulfur molecular emission and selenium atomic fluorescence could be monitored simultaneously in an argon-hydrogen diffusion flame on a glass burner. The analytes were introduced as hydrogen selenide and hydrogen sulfide, generated on a flow-injection manifold. Selenate was reduced to hydride-forming selenite by microwave-assisted on-line reaction with hydrochloric acid, and sulfate, or sulfite, was reduced to hydride-forming sulfide by a mixture of hydriodic acid, acetic acid and sodium hypophosphite. The effects of the nature of reducing agent, flow rate, microwave power and coil length were studied. The limit of detection (3s) for selenium was 10microgL(-1), and for sulfide was 70microgL(-1) (200-microL injection volume). The calibration was linear for selenium up to 2mgL(-1) and to 10mgL(-1) for sulfide. The throughput was 180h(-1). The three sulfur species could be differentiated on the basis of reactivity at various microwave powers.

Sleep latency and lifestyle in Oxfordshire villages.

Sleep latency, the usual delay in falling asleep, was estimated in 689 male and 757 female residents of a group of Oxfordshire villages by means of a sleep questionnaire. Sleep latency is analysed in relation to 59 descriptive elements of lifestyle and reported feelings. The pattern of association is broadly similar in the two sexes, and multivariate analysis identifies boredom and insufficient to do, mild morbidity, and use of sleeping tablets and tranquillizers as important correlates of long sleep latency in both sexes. These data are compared with a similar analysis of the correlates of sleep duration and sleep quality. It is concluded that sleep latency represents an important measure of well-being.

Association between smoking and drinking and sleep duration.

In a study of lifestyles and health of the adult population of some Oxfordshire villages, data were collected upon the usual sleep duration and quality, smoking and drinking habits of 725 men and 759 women. A strong negative association has been found between cigarette smoking and sleep duration on both sexes, and between alcohol consumption and sleep duration in men. There is no equivalent association between drinking or smoking and reported poor quality sleep. While these findings do not prove a causal relationship, the absence of complaints of poor quality sleep among the smokers and drinkers suggests that these habits are not simply the correlates of underlying psychological problems leading to insomnia.

Comparison of deoxyribonucleic acid uptake and marker integration in bacilli and protoplasts of Bacillus subtilis.

trp(+)his(-) donor deoxyribonucleic acid (DNA) was added to highly competent trp(-)his(+) recipient bacilli and to protoplasts prepared from these bacilli, and the cell-DNA complexes were incubated for 30 min. The complexes were then washed and lysed, and their DNA was analyzed on a trp(-)his(-) strain for the donor marker trp(+), the resident marker his(+), and for the recombinant trp(+)his(+) combination. The extracts of the bacillary complexes contained a normal percentage of donor markers (0.1-0.2%), and the number of trp(+)his(+) doubles (20% of all trp(+) transformants) indicated that the donor DNA had become integrated into the resident genomes. The protoplast complexes contained 10 to 1,000 times fewer donor markers and almost no recombinants. This indicated that, in protoplasts, marker uptake was minimal and recombination was absent. Uptake was also measured with (3)H-labeled DNA. On the average, protoplasts took up one-fiftieth as much DNA as bacilli. It was concluded that, probably, protoplasts took up no DNA at all, that there were no DNA affinity sites on the surface of the protoplasts, and that the residual marker and radioactivity uptake was due to imperfections in the experimental system. The data and conclusions differed sharply from earlier ones of Hirokawa and Ikeda despite the fact that the techniques of these authors were followed in repeat experiments.

Anaesthetic management of a child with Burkitt's lymphoma of the larynx.

An eight-year-old boy with a Burkitt's lymphoma of the upper airway is described. The use of sevoflurane for induction of anaesthesia in patients with airway obstruction is discussed. The logistical problems of upper airway surgery and anaesthesia in this type of patient are considered.

Urinary hormone levels: a population study of associations between steroid and catecholamine excretion rates.

Urinary levels of steroid metabolites (17-hydroxycorticosteroids and 17-oxosteroids) were measured in 408 males at two times during the normal working day. Hormone levels are expressed both as rates of excretion and as creatinine ratios and are compared with the values for adrenaline and noradrenaline previously measured in the same samples. Strong associations are found between catecholamines and steroids. Generally the greatest correlation is with hormones of the same biochemical class, although the 17-hydroxycorticosteroid level is more strongly correlated with the adrenaline level than is the level of noradrenaline.

Similarities between husbands and wives in rates of catecholamine excretion.

Husband-wife correlations for catecholamine excretion rates are presented. Highly significant positive correlations are found in adrenaline excretion in samples collected in the early morning and at around noon, and in noon noradrenaline excretion.

Catecholamine excretion rates in relation to life-styles in the male population of Otmoor, Oxfordshire.

The paper gives the results of the number of analyses of aspects of life-style and dietary patterns of members of the Otmoor population, in relation to their catecholamine excretion rates. The data reported here are restricted to males. Feelings of boredom were associated with low adrenaline excretion rates. Reported physical tiredness was associated with low adrenaline levels, while mental tiredness seems to be related to high adrenaline levels. People who regarded themselves as having a competitive personality, as being faced by a large number of life challenges, as having to meet self-set deadlines, as choosing to focus on more than one task at the same time, or as being under time pressure had high rates. Cigarette smoking and coffee consumption were related to high adrenaline excretion rates. Taken together these variables can explain 16-20% of variance in adrenaline excretion. Smoking and coffee consumption are of primary importance. The results of similar analyses of noradrenaline are reported.

Identification of residues critical for enzymatic activity in the domain encoded by exons 8 and 9 of the human inducible nitric oxide synthase.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of several diseases including airway inflammation of asthma. iNOS is active only as a homodimer. We previously demonstrated that the region encoded by exons 8 and 9 is critical for dimerization. In this study, alanine-scanning mutagenesis was used to identify critical amino acids in that region by expression of mutant proteins in human embryonic kidney 293 cells. All iNOS mutants yielded iNOS protein as detected by Western analysis. Four iNOS mutants with alanine replacing Trp260, Asn261, Tyr267, or Asp280 did not generate NO. Dimer formation was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis at 4 degrees C, followed by immunoblotting. Wild-type iNOS migrated both as monomers and dimers. iNOS mutants with alanine replacing Trp260, Asn261, or Tyr267, however, migrated only as monomers, suggesting that their inability to produce NO is related to a defect in dimer formation. Interestingly, the Asp280 mutant retained the ability to dimerize, indicating that it represents an inactive form of an iNOS dimer. These data identify four amino acids in exons 8 and 9 critical for iNOS activity, three of which also influence dimerization. These residues are strictly conserved among all NOS isforms and across species. Thus all NOS isoforms share general structural similarities, including specific amino acids critical for dimerization and catalytic activity. These data increase our understanding of the structural elements critical for NO synthesis and lay the groundwork for future studies aimed at downregulation of iNOS activity.

Use of diclofenac in children with asthma.

This study investigated the effect of diclofenac on the lung function of 70 children aged 6-15 years with a diagnosis of asthma, recruited from a hospital respiratory clinic. Peak flow and a forced expiratory flow-volume loop were measured and the patients were then given 1-1.5 mg.kg-1 effervescent diclofenac orally. Spirometry was repeated at 10, 20 and 30 min, a 15% decrease in results being considered a significant reduction in lung function. No patient demonstrated a consistent reduction in lung function of > 15% during the study and there were no reports of wheezing or increased bronchodilator use after completion of the spirometry. In conclusion, we studied a group of genuine asthmatics and found no clinically significant incidence of bronchospasm with the use of a single therapeutic dose of diclofenac.

Cloning and characterization of human inducible nitric oxide synthase splice variants: a domain, encoded by exons 8 and 9, is critical for dimerization.

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242-335 of the oxygenase domain. In this study, iNOS8(-)9(-) and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8(-)9(-) in 293 cell line resulted in generation of iNOS8(-)9(-) mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8(-)9(-) did not form dimers. Similar to iNOSFL, iNOS8(-)9(-) exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two "control" iNOS deletion mutants were synthesized, lacking either residues 45-52 of the oxygenase domain or residues 1131-1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.