Elucidating the risk factors for chronic obstructive pulmonary disease: an umbrella review of meta-analyses.

Chronic obstructive pulmonary disease (COPD) is commonly attributed to smoking, and other potential risk factors are ignored. We aimed to critically appraise the epidemiological credibility of the risk factors for COPD that have been examined in published meta-analyses. We performed a systematic search to capture systematic reviews and meta-analyses of observational studies on environmental factors and biomarkers for risk of COPD. We applied a set of standardised methodological criteria based on the level of statistical significance, sample size, between-study heterogeneity and statistical biases. Our search yielded 11 eligible papers, including 18 meta-analyses on environmental factors or biomarkers for COPD risk, and eight eligible papers with systematic reviews only. Eleven associations achieved statistical significance at P < 0.001 and six associations at P < 1 × 10-6. Thirteen associations presented an I²  50%, while six associations had evidence of small-study effects and/or excess significance bias. History of tuberculosis or rheumatoid arthritis, exposure to biomass fuels, tobacco smoking and second hand smoking were supported by high epidemiological credibility for an increased risk of COPD. Furthermore, highly suggestive evidence was found for increased levels of serum C-reactive protein, and serum fibrinogen in COPD patients compared with healthy controls. To summarise, our approach suggests that, while a proportion of COPD patients are non-smokers, only a narrow range of risk factors not related to smoking have been studied for an association with COPD. There is also a need to decipher possible protective factors in COPD pathogenesis given that more than a half of ever-smokers do not develop COPD.

Value of adding an IGRA to the TST to screen for latent tuberculous infection in Greek health care workers.

SETTING: Ioannina University Hospital, Ioannina, Greece. OBJECTIVE: To evaluate the value of adding an interferon-gamma release assay (IGRA) to the tuberculin skin test (TST) for detecting latent tuberculous infection (LTBI) in a Greek university hospital among health care workers (HCWs) predominantly vaccinated with bacille Calmette-Guérin (BCG). DESIGN: Of 788 HCWs enrolled, 68.1% were BCG-vaccinated. A TST ⩾ 10 mm was considered positive and was followed by the QuantiFERON-TB(®) Gold In-Tube assay (QFT-GIT) in a two-step strategy. RESULTS: Of the enrolled HCWs, 36.4% were TST-positive, of whom only 14.4% were IGRA-positive. Agreement between the tests was poor (κ = 0.019; 95%CI -0.014-0.05, P = 0.355). Both TST and IGRA positivity increased with TST diameter, from 5.7% in TST 10-14 mm to 48.8% in TST ⩾20 mm. TST-positive, IGRA-negative results were most likely in younger, recently BCG-vaccinated HCWs (84.6% in those aged 20-29 years) and less likely in older HCWs (45% in those aged 50-59 years). The two-step strategy would have been more cost saving compared to the TST-only approach if adherence to LTBI treatment in our cohort had been ⩾24%. CONCLUSIONS: Poor overall agreement between TST and QFT-GIT was found. Use of IGRA as a second step in TST-positive cases offers an appropriate tool for LTBI detection among BCG-vaccinated HCWs in low-TB-incidence settings.

Interferon-gamma release assay clarifies the effect of bacille Calmette-Guérin vaccination in Greek army recruits.

OBJECTIVE: To compare the most recent commercial interferon-gamma release assay (IGRA), the QuantiFERON-TB Gold In-Tube (QFT-GIT), with the tuberculin skin test (TST) in Greek army recruits who were bacille Calmette-Guérin (BCG) vaccinated during childhood and had no history of tuberculosis (TB) exposure. METHOD: We conducted a cross-sectional comparison study of 1750 young army recruits. TST was performed on all participants, while QFT-GIT was performed in all subjects with TST > 0 mm and in 18 TST-negative controls (TST = 0 mm). RESULTS: Among the study subjects, 5.4% (96/1750) had TST indurations of >or=10 mm, and 3.4% (59/1750) had indurations of >or=15 mm. Among subjects with a positive TST, 11.4% (11/96) tested positive on QFT-GIT. All those with QFT-GIT positivity had TST indurations of >or=15 mm, and none of those with TST indurations of 10-14 mm were positive by QFT-GIT. The overall agreement between TST and QFT-GIT was poor (kappa = 0.02). CONCLUSION: We found a significant discordance between TST and QFT-GIT in BCG-vaccinated Greek army recruits consistent with previous studies showing that BCG received after infancy produces false-positive TST reactions. Our findings underline the need for a two-step approach in diagnosing latent TB infection in all BCG-vaccinated individuals: initial TST screening, followed by an IGRA to confirm TST positivity.

Cellular localization of interleukin 13 receptor alpha2 in human primary bronchial epithelial cells and fibroblasts.

BACKGROUND: Interleukin (IL) 13 is a key cytokine in asthma, regulating fibrosis, airway remodeling, induction of immunoglobulin E synthesis by B cells, bronchial hyperresponsiveness, and mucus production. IL-13 signals through the type II IL-4 receptor (IL-4R), which is composed of the IL-4Ralpha and the IL-13Ralpha1 chains. Another IL-13 binding chain, IL-13Ralpha2, binds IL-13 with high affinity but has no known signaling capability and is thought to serve as a decoy receptor providing tight regulation of IL-13 responses. METHODS: In this study, we investigated the cellular localization of IL-13Ralpha2 in human primary bronchial epithelial cells and fibroblasts using flow cytometry and confocal microscopy, as well as the in vivo expression of IL-13Ralpha2 in the human bronchial mucosa by means of immunohistochemistry. RESULTS: IL-13Ralpha2 is predominantly an intracellular rather than a membrane-bound molecule in both human primary bronchial epithelial cells and fibroblasts and displays a diffuse granular cytoplasmic distribution in both cell types. IL-13Ralpha2 protein is expressed in vivo in the human bronchial mucosa with its expression being higher in bronchial epithelial cells than bronchial fibroblasts both in vivo and in vitro. CONCLUSIONS: IL-13Ralpha2 is expressed by both human primary bronchial epithelial cells and fibroblasts as an intracellular protein with a diffuse cytoplasmic distribution. In vivo, IL-13Ralpha2 is expressed in the human airway mucosa mainly by bronchial epithelial cells.

Genetic association studies of interleukin-13 receptor alpha1 subunit gene polymorphisms in asthma and atopy.

Interleukin (IL)-13 plays a central role in asthma pathogenesis by binding to the IL-13 receptor, which is a heterodimer composed of the IL-13 receptor alpha1 subunit (IL-13Ralpha1) and IL-4Ralpha. The genetic diversity at the IL-13Ralpha1 gene (IL13RA1) locus on chromosome Xq24 was characterised and the association of identified polymorphisms with asthma and atopy phenotypes examined. The promoter and coding region of IL13RA1 were screened for common genetic variants, and polymorphisms found were genotyped in a large cohort of 341 asthmatic Caucasian families (each containing at least two asthmatic siblings) and 182 nonasthmatic control subjects. Genetic association was determined using case-control and transmission disequilibrium test analyses. Two common polymorphisms were identified, a newly found thymidine (T) to guanine (G) transition of nucleotide -281 (-281T>G) single nucleotide polymorphism in the IL13RA1 promoter and the previously described 1365A>G variant in the IL13RA1 proximal 3' untranslated region. No significant association of either -281T>G or 1365A>G with risk of asthma or atopy phenotypes was found, apart from a suggestive association between the IL13RA1 -281T/1365A haplotype and raised total serum immunoglobulin E levels in adult female asthmatics. These findings indicate that the interleukin-13 receptor alpha1 subunit gene -281T>G and 1365A>G polymorphisms do not contribute to asthma susceptibility or severity, although the interleukin-13 receptor alpha1 subunit gene locus might be involved in the control of immunoglobulin E production.

Identification, substrate specificity, and inhibition of the Streptococcus pneumoniae beta-ketoacyl-acyl carrier protein synthase III (FabH).

In the bacterial type II fatty acid synthase system, beta-ketoacyl-acyl carrier protein (ACP) synthase III (FabH) catalyzes the condensation of acetyl-CoA with malonyl-ACP. We have identified, expressed, and characterized the Streptococcus pneumoniae homologue of Escherichia coli FabH. S. pneumoniae FabH is approximately 41, 39, and 38% identical in amino acid sequence to Bacillus subtilis, E. coli, and Hemophilus influenzae FabH, respectively. The His-Asn-Cys catalytic triad present in other FabH molecules is conserved in S. pneumoniae FabH. The apparent K(m) values for acetyl-CoA and malonyl-ACP were determined to be 40.3 and 18.6 microm, respectively. Purified S. pneumoniae FabH preferentially utilized straight short-chain CoA primers. Similar to E. coli FabH, S. pneumoniae FabH was weakly inhibited by thiolactomycin. In contrast, inhibition of S. pneumoniae FabH by the newly developed compound SB418011 was very potent, with an IC(50) value of 0.016 microm. SB418011 also inhibited E. coli and H. influenzae FabH with IC(50) values of 1.2 and 0.59 microm, respectively. The availability of purified and characterized S. pneumoniae FabH will greatly aid in structural studies of this class of essential bacterial enzymes and facilitate the identification of small molecule inhibitors of type II fatty acid synthase with the potential to be novel and potent antibacterial agents active against pathogenic bacteria.

Refined structures of beta-ketoacyl-acyl carrier protein synthase III.

beta-Ketoacyl-acyl carrier protein synthase III (FabH) is a condensing enzyme that plays central roles in fatty acid biosynthesis. Three-dimensional structures of E. coli FabH in the presence and absence of ligands have been refined to 1.46 A resolution. The structures of improved accuracy revealed detailed interactions involved in ligand binding. These structures also provided new insights into the FabH mechanism, e.g. the possible role of a water or hydroxyl anion in Cys112 deprotonation. A structure of the apo enzyme uncovered large conformational changes in the active site, exemplified by the disordering of four essential loops (84-86, 146-152, 185-217 and 305-307) and the movement of catalytic residues (Cys112 and His244). The disordering of the loops leads to greater than 50 % reduction in the FabH dimer interface, suggesting a dynamic nature for an unusually large portion of the dimer interface. The existence of a large solvent-accessible channel in the dimer interface as well as two cis-peptides (cis-Pro88 and cis-Phe308) in two of the disordered loops may explain the observed structural instabilities.

Crystallization of Escherichia coli beta-ketoacyl-ACP synthase III and the use of a dry flash-cooling technique for data collection.

beta-Ketoacyl-acyl carrier protein (ACP) synthase III (FabH) is a condensing enzyme active in the fatty-acid biosynthesis pathway of bacteria. The enzymes of this pathway provide a set of targets for the discovery of previously unknown antibiotics. FabH from Escherichia coli has been crystallized in two crystal forms using the sitting-drop vapor-diffusion technique. The first form crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 63.1, b = 65.1, c = 166.5 A; the second form crystallized in the tetragonal space group P4(1)2(1)2, with unit-cell parameters a = b = 72.7, c = 99.8 A. A flash-cooling technique using no cryoprotectant was utilized in obtaining data from the second type of crystals.

Expression, purification, and crystallization of the Escherichia coli selenomethionyl beta-ketoacyl-acyl carrier protein synthase III.

Bacterial beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III, also called FabH) catalyzes the condensation and transacylation of acetyl-CoA with malonyl-ACP. In order to understand the mode of enzyme/substrate interaction and design small molecule inhibitors, we have expressed, purified, and crystallized a selenomethionyl-derivative of E. coli KAS III. Several lines of evidence confirmed that purified selenomethionyl KAS III was homogenous, stably folded, and enzymatically active. Dynamic light scattering, size exclusion chromatography, and mass spectrometry results indicated that selenomethionyl KAS III is a noncovalent homodimer. Diffraction quality crystals of selenomethionyl KAS III/acetyl-CoA complex, which grew overnight to a size of 0.2 mm(3), belonged to the tetragonal space group P4(1)2(1)2.

Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis.

Beta-ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys(112) proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His(244) and Asn(274). The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.

Purification, characterization, and kinetic mechanism of cyclin D1. CDK4, a major target for cell cycle regulation.

The cyclin D1.CDK4-pRb (retinoblastoma protein) pathway plays a central role in the cell cycle, and its deregulation is correlated with many types of cancers. As a major drug target, we purified dimeric cyclin D1.CDK4 complex to near-homogeneity by a four-step procedure from a recombinant baculovirus-infected insect culture. We optimized the kinase activity and stability and developed a reproducible assay. We examined several catalytic and kinetic properties of the complex and, via steady-state kinetics, derived a kinetic mechanism with a peptide (RbING) and subsequently investigated the mechanistic implications with a physiologically relevant protein (Rb21) as the phosphoacceptor. The complex bound ATP 130-fold tighter when Rb21 instead of RbING was used as the phosphoacceptor. By using staurosporine and ADP as inhibitors, the kinetic mechanism of the complex appeared to be a "single displacement or Bi-Bi" with Mg2+.ATP as the leading substrate and phosphorylated RbING as the last product released. In addition, we purified a cyclin D1-CDK4 fusion protein to homogeneity by a three-step protocol from another recombinant baculovirus culture and observed similar kinetic properties and mechanisms as those from the complex. We attempted to model staurosporine in the ATP-binding site of CDK4 according to our kinetic data. Our biochemical and modeling data provide validation of both the complex and fusion protein as highly active kinases and their usefulness in antiproliferative inhibitor discovery.

The isolation and characterization of cDNA encoding the mouse bifunctional ATP sulfurylase-adenosine 5'-phosphosulfate kinase.

Biosynthesis of the activated sulfate donor, adenosine 3'-phosphate 5'-phosphosulfate, involves the sequential action of two enzyme activities: ATP sulfurylase, which catalyzes the formation of adenosine 5'-phosphosulfate (APS) from ATP and free sulfate, and APS kinase, which subsequently phosphorylates APS to produce adenosine 3'-phosphate 5'-phosphosulfate. Oligonucleotide primers were derived from a human infant brain-expressed sequence tag putatively encoding a portion of APS kinase. Using these primers, reverse transcriptase-polymerase chain reaction was performed on mRNA from neonatal normal mice resulting in amplification of a 127-bp DNA fragment. This fragment was subsequently used to screen a mouse brain lambda gt11 cDNA library, yielding a 2.2-kb clone. Primers were designed from the 5'-end of the 2.2-kb clone, and 5'-rapid amplification of cDNA ends was used to obtain the translation start site. Sequence from the overlapping clones was assembled into a 2475-bp composite sequence, which contains a single open reading frame that translates into a 624-deduced amino acid sequence. Northern blots of total RNA from neonatal mice yielded a single message species at approximately 3.3 kb. Southern blot of genomic DNA digested with several restriction enzymes suggested the gene is present as a single copy. Comparison against sequence data bases suggested the composite sequence was a fused sulfurylase-kinase product, since the deduced amino acid sequence showed extensive homology to known separate sequences of both ATP sulfurylase and APS kinase from several sources. The first 199 amino acids corresponded to APS kinase sequence, followed by 37 distinct amino acids, which did not match any known sequence, followed by 388 amino acids that are highly homologous to known ATP sulfurylase sequences. Finally, recombinant enzyme expressed in COS-1 cells exhibited both ATP sulfurylase and APS kinase activity.

Hydrolyses of alpha- and beta-cellobiosyl fluorides by cellobiohydrolases of Trichoderma reesei.

Cellobiohydrolase II hydrolyses alpha- and beta-D-cellobiosyl fluorides to alpha-cellobiose at comparable rates, according to Michaelis-Menten kinetics. The stereochemistry, absence of transfer products and strict hyperbolic kinetics of the hydrolysis of alpha-cellobiosyl fluoride suggest that the mechanism for the alpha-fluoride may be the enzymic counterpart of the SNi reaction observed in the trifluoroethanolysis of alpha-glucopyranosyl fluoride [Sinnott and Jencks (1980) J. Am. Chem. Soc. 102, 2026-2032]. The absolute factors by which this enzyme accelerates fluoride ion release are small and greater for the alpha-fluoride than for the beta, suggesting that its biological function may not be just glycoside hydrolysis. Cellobiohydrolase I hydrolyses only beta-cellobiosyl fluoride, which is, however, an approx. 1-3% contaminant in alpha-cellobiosyl fluoride as prepared and purified by conventional methods. Instrumental assays for the various components of the cellulase complex are discussed.