A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Fracture liaison services for the evaluation and management of patients with osteoporotic fracture: a cost-effectiveness evaluation based on data collected over 8 years of service provision.

SUMMARY: The cost-effectiveness of Fracture Liaison Services (FLSs) for prevention of secondary fracture in osteoporosis patients in the United Kingdom (UK), and the cost associated with their widespread adoption, were evaluated. An estimated 18 fractures were prevented and £21,000 saved per 1,000 patients. Setup across the UK would cost an estimated £9.7 million. INTRODUCTION: Only 11% to 28% of patients with a fragility fracture receive osteoporosis treatment in the UK. FLSs provide an efficient means to identify patients and are endorsed by the Department of Health but have not been widely adopted. The objective of this study was to evaluate the cost-effectiveness of FLSs in the UK and the cost associated with their widespread adoption. METHODS: A cost-effectiveness and budget-impact model was developed, utilising detailed audit data collected by the West Glasgow FLS. RESULTS: For a hypothetical cohort of 1,000 fragility-fracture patients (740 requiring treatment), 686 received treatment in the FLS compared with 193 in usual care. Assessments and osteoporosis treatments cost an additional £83,598 and £206,544, respectively, in the FLS; 18 fractures (including 11 hip fractures) were prevented, giving an overall saving of £21,000. Setup costs for widespread adoption of FLSs across the UK were estimated at £9.7 million. CONCLUSIONS: FLSs are cost-effective for the prevention of further fractures in fragility-fracture patients. The cost of widespread adoption of FLS across the UK is small in comparison with other service provision and would be expected to result in important benefits in fractures avoided and reduced hospital bed occupancy.

High copper concentration reduces biofilm formation in Acidithiobacillus ferrooxidans by decreasing production of extracellular polymeric substances and its adherence to elemental sulfur.

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms. SIGNIFICANCE: Copper is a metal frequently found in high concentrations at mining environments inhabitated by acidophilic microorganisms. Copper resistance determinants of A. ferrooxidans have been previously studied in planktonic cells. Although biofilms are recurrent in these types of environments, the effect of copper on their formation has not been studied so far. The results obtained indicate that high concentrations of copper reduce the capacity of A. ferrooxidans ATCC 23270 to form biofilms on sulfur. These findings may be relevant to consider for a bacterium widely used in copper bioleaching processes.

What Is Unfair about Unequal Brute Luck? An Intergenerational Puzzle.

According to Luck egalitarians, fairness requires us to bring it about that nobody is worse off than others where this results from brute bad luck, but not where they choose or deserve to be so. In this paper, I consider one type of brute bad luck that appears paradigmatic of what a Luck Egalitarian ought to be most concerned about, namely that suffered by people who are born to badly off parents and are less well off as a result. However, when we consider what is supposedly unfair about this kind of unequal brute luck, luck egalitarians face a dilemma. According to the standard account of luck egalitarianism, differential brute luck is unfair because of its effects on the distribution of goods. Yet, where some parents are worse off because they have chosen to be imprudent, it may be impossible to neutralize these effects without creating a distribution that seems at least as unfair. This, I argue, is problematic for luck egalitarianism. I, therefore, explore two alternative views that can avoid this problem. On the first of these, proposed by Shlomi Segall, the distributional effects of unequal brute luck are unfair only when they make a situation more unequal, but not when they make it more equal. On the second, it is the unequal brute luck itself, rather than its distributional effects, that is unfair. I conclude with some considerations in favour of this second view, while accepting that both are valid responses to the problem I describe.

Detection of peroxisome proliferators using a reporter construct derived from the rat acyl-CoA oxidase promoter in the rat liver cell line H-4-II-E.

Peroxisome proliferators are nongenotoxic carcinogens capable of causing rapid transcriptional activation of genes comprising the rodent beta-oxidation pathway. Numerous compounds, such as hypolipidemic drugs, herbicides, plasticizers, and analgesics have been identified as peroxisome proliferators in rodents. We have developed a whole-cell in vitro assay to detect peroxisome proliferators in approximately 48 h. A promoter::chloramphenicol acetyltransferase (CAT) fusion construct for rat acyl-CoA oxidase (ACOX), the rate-limiting enzyme in the peroxisomal beta-oxidation pathway, was stably transfected into the rat liver cell line H-4-II-E. Treatment of the recombinant cell line (ACOX::CAT) with peroxisome proliferators, WY 14,643, clofibrate, di(2-ethylhexyl) phtalhate, and acetylsalicylic acid resulted in differential increases of CAT protein 48 h after exposure. Nonsteroidal anti-inflammatory drugs including ibuprofen, fenbupen, naproxen, and acetaminophen also up-regulated ACOX::CAT. Phorbol 12-myristate 13-acetate, a nongenotoxic carcinogen that is not classified as a peroxisome proliferator, also resulted in a slight induction of ACOX::CAT, consistent with the role of cell proliferation in tumor progression. The carcinogenic compounds 4-nitroquinoline N-oxide, ethyl methanesulfonate, diethylstilbestrol, and 2-aminoanthracene did not induce ACOX::CAT. Although the significance of peroxisome proliferators and their impact on humans is still unknown, the ability to identify them is of interest to the pharmaceutical and chemical industries. This assay was able to detect known peroxisome proliferators tested in approximately 48 h of exposure and to distinguish them from genotoxic carcinogens.

Economic evaluation in chronic pain: a systematic review and de novo flexible economic model.

There is unmet need in patients suffering from chronic pain, yet innovation may be impeded by the difficulty of justifying economic value in a field beset by data limitations and methodological variability. A systematic review was conducted to identify and summarise the key areas of variability and limitations in modelling approaches in the economic evaluation of treatments for chronic pain. The results of the literature review were then used to support the development of a fully flexible open-source economic model structure, designed to test structural and data assumptions and act as a reference for future modelling practice. The key model design themes identified from the systematic review included: time horizon; titration and stabilisation; number of treatment lines; choice/ordering of treatment; and the impact of parameter uncertainty (given reliance on expert opinion). Exploratory analyses using the model to compare a hypothetical novel therapy versus morphine as first-line treatments showed cost-effectiveness results to be sensitive to structural and data assumptions. Assumptions about the treatment pathway and choice of time horizon were key model drivers. Our results suggest structural model design and data assumptions may have driven previous cost-effectiveness results and ultimately decisions based on economic value. We therefore conclude that it is vital that future economic models in chronic pain are designed to be fully transparent and hope our open-source code is useful in order to aspire to a common approach to modelling pain that includes robust sensitivity analyses to test structural and parameter uncertainty.

The stress-response proteins poly(ADP-ribose) polymerase and NF-kappaB protect against bile salt-induced apoptosis.

Bile salts induce apoptosis and are implicated as promoters of colon cancer. The mechanisms by which bile salts produce these effects are poorly understood. We report that the cytotoxic bile salt, sodium deoxycholate (NaDOC), activates the key stress response proteins, NF-kappaB and poly(ADP-ribose) polymerase (PARP). The activation of NF-kappaB and PARP, respectively, indicates that bile salts induce oxidative stress and DNA damage. The pre-treatment of cells with specific inhibitors of these proteins [pyrrolidine dithiocarbamate (NF-kappaB inhibitor) and 3-aminobenzamide (PARP inhibitor)] sensitizes cells to the induction of apoptosis by NaDOC, indicating that these stress response pathways are protective in nature. Colon cancer risk has been reported to be associated with resistance to apoptosis. We found an increase in activated NF-kappaB at the base of human colon crypts that exhibit apoptosis resistance. This provides a link between an increased stress response and colon cancer risk. The implications of these findings with respect to apoptosis and to colon carcinogenesis are discussed.

Septicemia caused by Capnocytophaga in a granulocytopenic patient with glossitis.

A patient receiving immunosuppressive treatment for multiple myeloma became granulocytopenic, and acute glossitis developed. Blood cultures were positive for Capnocytophaga, a fastidious Gram-negative bacillus that is known to be part of the normal oral flora and a pathogen for periodontitis. The infection responded to treatment with antibiotics, including penicillin G, to which the organism was sensitive. This is one of the first reports of Capnocytophaga septicemia, and suggests that this organism may be an important pathogen in immunosuppressed patients with oral mucosal lesions.

The genomic sequence of a contemporary wild-type mumps virus strain.

Vaccination has the potential to eradicate mumps, and 82 countries now include a live attenuated mumps vaccine as part of their childhood vaccination programme. Although, monotypic, genetic variants of mumps virus (MuV) have been described based on comparison of the SH gene sequences, and at least seven genotypes have been identified. We now report the entire sequence of a recently isolated wild type MuV strain, Glouc1/UK96 (Glouc1) by direct sequencing of the cDNA obtained from cell culture fluid. The genome of this recent isolate was 15384 nucleotides in length. There were 579 nucleotide differences (3.8%) and 71 amino acid differences (1.5%) between Glouc1, a genotype G strain and Ur-AM9, a genotype B strain. Other MuV strains with available sequences were also compared with this pathological strain. The sequence of the contemporary strain reported here provides a picture of the variability of MuV over its entire genome (GenBank accession no. AF280799).

Transcriptional activity of quinone methides derived from the tumor promoter butylated hydroxytoluene in HepG2 cells.

Butylated hydroxytoluene (BHT) is a pulmonary toxin and tumor promoter in mice presumably due to the formation of two quinone methides (QMs) that alkylate cellular nucleophiles. The activation of stress genes by these electrophilic metabolites was investigated with an assay system consisting of 14 recombinant cell lines derived from the human hepatoma line HepG2, each carrying a unique promoter or response element construct fused to the reporter gene for chloramphenicol acetyl transferase (CAT). The largest responses to QMs occurred in cells containing either the metallothionein IIA, glutathione S-transferase Ya, or 70 kDa heat shock protein promoter, or the xenobiotic response element. The other cell lines exhibited only small or no effects. These results are consistent with transcriptional activities reported for several other electrophiles known to undergo covalent interactions with proteins.

Successful treatment of parvovirus B19 infection and red cell aplasia occurring after an allogeneic bone marrow transplant.

Chronic parvovirus B19 infection in the immunocompromised host may cause severe anaemia secondary to failure of erythropoiesis. This has been previously documented in patients with the Acquired Immune Deficiency Syndrome (AIDS), congenital immunodeficiencies and in children with acute lymphoblastic leukaemia during maintenance chemotherapy. We describe persistent parvovirus infection in a 14-year-old boy after HLA-matched sibling allogeneic bone marrow transplantation for acute lymphoblastic leukaemia in second remission.

Inhibition of the cytochrome bd-terminated NADH oxidase system in Escherichia coli K-12 by divalent metal cations.

Co(II), Zn(II) and Cd(II) ions inhibited NADH oxidase activity in membranes prepared from two cytochrome bo'-deficient mutants of Escherichia coli K-12 with the following order of potency: Zn(II) > Cd(II) >> Co(II). The degree of inhibition exhibited by these metal ions was not diminished in membranes which contained elevated levels of the cytochrome bd complex, suggesting that the most sensitive site precedes this complex in the aerobic respiratory chain. For each of the metal ions studied, inhibition was determined to be of the non-competitive type. Based upon the efficacy with which EDTA alleviated inhibition, Co(II), Zn(II) and Cd(II) ions are proposed to inhibit NADH oxidase activity by binding to at least two sites in the respiratory chain with significantly different affinities.

Evidence for the transport of zinc(II) ions via the pit inorganic phosphate transport system in Escherichia coli.

A locus involved in zinc(II) uptake in Escherichia coli K-12 was identified through the generation of a zinc(II)-resistant mutant by transposon (Tn10dCam) mutagenesis. The mutation was located within the pitA gene, which encodes the low-affinity inorganic phosphate transport system (Pit). The pitA mutant accumulated reduced amounts of zinc(II) when exposed to 0.5-2.0 mM ZnSO(4) during growth in Luria-Bertani medium.

Metal ion-catalysed hydrolysis of ampicillin in microbiological growth media.

Anodic stripping voltammetry of bacterial growth medium containing copper(II) and ampicillin shows that Cu(II) is complexed by the antibiotic and that this complex decomposes to give Cu(II) complexes with ligands derived from ampicillin. At pH 7, substantial decomposition of ampicillin occurs over a few minutes, and even the very low levels of Cu(II) in Chelex-extracted medium are able effectively to catalyse the decomposition. The significance of this observation was shown during the screening of an Escherichia coli cosmid library for clones exhibiting increased resistance to Zn(II), Co(II) or Cd(II); the unexpected growth of the ampicillin-sensitive host E. coli strain on Luria-Bertani plates containing ampicillin and any of these metals was attributed to metal ion-catalysed decomposition of ampicillin. The instability of ampicillin (and other beta-lactam antibiotics) to metal ion-catalysed hydrolysis means that great care must be taken to ensure that such reactions do not occur in growth media. Furthermore, it is clear that double selection for resistance to ampicillin and metals such as Cu(II), Zn(II), Co(II) and Cd(II) is impossible.

Blood donor screening for parvovirus B19.

A programme of blood donor screening for parvovirus B19 was conducted from January to May 1990. The main aim of the study was to identify a B19 positive donation that could be used as a source of viral antigen for diagnostic serology. Out of 24,000 donors tested one was positive for B19 antigen by counter current immunoelectrophoresis and over 100 ml of undiluted B19 containing material was obtained. However, much of the positive donation was incorporated in a plasma pool of 28 donations. An acid dissociation technique was used to recover B19 antigen from immune complexes formed in the plasma pool.

Treatments for spasticity and pain in multiple sclerosis: a systematic review.

OBJECTIVES: To identify the drug treatments currently available for the management of spasticity and pain in multiple sclerosis (MS), and to evaluate their clinical and cost-effectiveness. DATA SOURCES: Electronic bibliographic databases, National Research Register, MRC Clinical Trials Register and the US National Institutes of Health Clinical Trials Register. REVIEW METHODS: Systematic searches identified 15 interventions for the treatment of spasticity and 15 interventions for treatment of pain. The quality and outcomes of the studies were evaluated. Reviews of the treatment of spasticity and pain when due to other aetiologies were also sought. RESULTS: There is limited evidence of the effectiveness of four oral drugs for spasticity: baclofen, dantrolene, diazepam and tizanidine. Tizanidine appears to be no more effective than comparator drugs such as baclofen and has a slightly different side-effects profile. Despite claims that it causes less muscle weakness, there was very little evidence that tizanidine performed any better in this respect than other drugs, although it is more expensive. The findings of this review are consistent with reviews of the same treatments for spasticity derived from other aetiologies. There is good evidence that both botulinum toxin (BT) and intrathecal baclofen are effective in reducing spasticity, and both are associated with functional benefit. However, they are invasive, and substantially more expensive. None of the studies included in the review of pain were designed specifically to evaluate the alleviation of pain in patients with MS and there was no consistency regarding the use of validated outcome measures. It was suggested that, although expensive, the use of intrathecal baclofen may be associated with significant savings in hospitalisation costs in relation to bed-bound patients who are at risk of developing pressure sores, thus enhancing its cost-effectiveness. No studies of cost-effectiveness were identified in the review of pain. There is evidence, albeit limited, of the clinical effectiveness of baclofen, dantrolene, diazepam, tizanidine, intrathecal baclofen and BT and of the potential cost-effectiveness of intrathecal baclofen in the treatment of spasticity in MS. CONCLUSIONS: Many of the interventions identified are not licensed for the alleviation of pain or spasticity in MS and the lack of evidence relating to their effectiveness may also limit their widespread use. Indeed, forthcoming information relating to the use of cannabinoids in MS may result in there being better evidence of the effectiveness of new treatments than of any of the currently used drugs. It may therefore be of value to carry out double-blind randomised controlled trials of interventions used in current practice, where outcomes could include functional benefit and impact on quality of life. Further research into the development and validation of outcomes measures for pain and spasticity may also be useful, as perhaps would cost-utility studies.

Activation of the promoters of genes associated with DNA damage, oxidative stress, ER stress and protein malfolding by the bile salt, deoxycholate.

Toxic bile salts, retained within the liver because of impaired biliary excretion, are considered to play a major role in liver injury during cholestasis. Bile salts cause cellular stresses that may result in apoptosis. To better understand such cellular stresses, the effect of the bile salt sodium deoxycholate (NaDOC) on activation of 13 specific gene promoters or response elements associated with different cellular stresses was measured in the transformed human hepatoma line, HepG2. NaDOC was found to activate transcription factors and induce or activate the promoters of genes that respond to protein malfolding (grp78 and hsp70), DNA damage (gadd153, hsp70 and c-fos), oxidative stress (NF-kappaB, c-fos, hsp70 and gadd153), ER stress (grp78) and Ca++ imbalance (grp78).

Stress responses to DNA damaging agents in the human colon carcinoma cell line, RKO.

DNA damage results from a wide variety of external agents such as chemicals and radiation. The consequences of exposure to agents that damage DNA have been traditionally studied from the perspective of cell survival and mutagenesis. Mutations are late endpoints of DNA damage. Cells respond to the earlier stages of DNA damage by inducing the expression of several genes, including those specific of the nature of the lesion. These early transcriptional responses are likely to predetermine the later fate of the damaged cell. Genes activated during this early response include those involved in DNA repair, replication, and growth control. We are interested in the transcriptional mechanisms by which cells respond to DNA damaging agents. To facilitate the measurement of gene induction, we used seven different reporter constructs integrated stably into the RKO cell line derived from a human colon carcinoma. These constructs were derived from promoters and/or response elements isolated from genes associated with DNA damage responses in human cells, and were fused to the bacterial reporter gene, choramphenicol acetyl transferase (CAT). The cell lines generated in this manner contain the promoters and/or response elements representing DNA polymerase beta, p53, gadd (growth arrest and DNA damage) 45 and 153, c-fos, TPA response element, and tissue-type plasminogen activator. These recombinant cell lines were assembled in a 96-well microtiter plate permitting their simultaneous exposure to compounds and subsequent CAT protein measurement. This assembly has been designated the CAT-Tox (D) assay. These cell lines were exposed to different classes of DNA damaging agents including those which covalently join bases to form dimers (e.g., UVC irradiation), generate DNA adducts by alkylation (e.g., methylmethane sulfonate [MMS], ethylmethane sulfonate [EMS], N-methyl-N-nitro-N-nitrosoguanine [MNNG], dimethylnitrosamine [DMN]), cross-link DNA (e.g., mitomycin C), and inhibit DNA replication by intercalative (e.g., actinomycin D) and nonintercalative (e.g., hydroxyurea) mechanisms. The transcriptional responses were measured as a function of the accumulation of CAT protein using antibodies against CAT protein in a standard ELISA. Endogenous cellular responses were evaluated for a number of the genes represented in the assay at both the mRNA and protein levels by Northern and Western blot analysis, respectively. These data corroborate the stress-induced responses measured by CAT ELISA in the CAT-Tox (D) assay, demonstrating the usefulness of this assay as a rapid and sensitive method for detection of DNA damaging agents in human cells.

Office-based arthroscopic synovectomy of the wrist in rheumatoid arthritis.

We present an office-based technique for performing arthroscopic synovectomy of the wrist in patients with rheumatoid arthritis. Intra-articular anesthesia as well as subcutaneous portal anesthesia are used. Standard portals are used in the radial carpal and midcarpal joints. Standard instrumentation is used and the synovectomy is accomplished using a motorized shaver. We performed 30 procedures in 21 patients: 15 complete synovectomies, 3 radioulnar carpal synovectomies because of only limited disease, and 12 limited synovectomies because these patients were participants in a clinical trial and required only limited synovectomy for investigational purposes. There were no complications. Office-based arthroscopic synovectomy of the wrist in patients with refractory rheumatoid arthritis can be performed safety and effectively. This technique is useful in both a clinical as well as a research setting.