The mRNA level of Charcot-Leyden crystal protein/galectin-10 is a marker for CRTH2 activation in human whole blood in vitro.

CRTH2 is one of the prostaglandin D2 receptors and plays a proinflammatory role in allergic diseases. Gene expression markers in whole blood induced by CRTH2 activation have not previously been reported. Using microarray analyses of 54 675 genes, we revealed modest gene expression changes in human whole blood stimulated in vitro by a selective CRTH2 agonist, DK-PGD2. Five genes were found to exhibit 1.5- to 2.6-fold changes in expression. The expression of Charcot-Leyden crystal protein/galectin-10 (CLC/Gal-10) in particular was consistently enhanced in human whole blood stimulated by DK-PGD2, as confirmed by quantitative real-time polymerase chain reaction analyses. DK-PGD(2)-induced increases in blood CLC/Gal-10 mRNA levels were largely attenuated by the CRTH2 antagonist CAY10471.Thus, the DK-PGD2-induced CLC/Gal-10 mRNA level can serve as a potential marker for monitoring pharmacodynamic effects of blood exposure to CRTH2 modulating agents.

A re-evaluation of the final step of vanillin biosynthesis in the orchid Vanilla planifolia.

A recent publication describes an enzyme from the vanilla orchid Vanilla planifolia with the ability to convert ferulic acid directly to vanillin. The authors propose that this represents the final step in the biosynthesis of vanillin, which is then converted to its storage form, glucovanillin, by glycosylation. The existence of such a "vanillin synthase" could enable biotechnological production of vanillin from ferulic acid using a "natural" vanilla enzyme. The proposed vanillin synthase exhibits high identity to cysteine proteases, and is identical at the protein sequence level to a protein identified in 2003 as being associated with the conversion of 4-coumaric acid to 4-hydroxybenzaldehyde. We here demonstrate that the recombinant cysteine protease-like protein, whether expressed in an in vitro transcription-translation system, E. coli, yeast, or plants, is unable to convert ferulic acid to vanillin. Rather, the protein is a component of an enzyme complex that preferentially converts 4-coumaric acid to 4-hydroxybenzaldehyde, as demonstrated by the purification of this complex and peptide sequencing. Furthermore, RNA sequencing provides evidence that this protein is expressed in many tissues of V. planifolia irrespective of whether or not they produce vanillin. On the basis of our results, V. planifolia does not appear to contain a cysteine protease-like "vanillin synthase" that can, by itself, directly convert ferulic acid to vanillin. The pathway to vanillin in V. planifolia is yet to be conclusively determined.

Use of transdermal fentanyl without prior opioid stabilization in patients with cancer pain.

OBJECTIVE: To determine the safety and efficacy of transdermal fentanyl for pain relief in cancer patients and to compare the effects on patients according to whether they had previously received strong opioids, weak opioids or non-opioid analgesia. METHODS: Cancer patients requiring strong analgesia were recruited into an open-label, multicentre study, conducted in eight countries. Patients received transdermal fentanyl treatment for 28 days. Pain severity, overall satisfaction with pain control, convenience of use of patches and treatment preferences were recorded daily. RESULTS: Of the 292 participants, 135 had previously received a strong opioid, 84 had previously received a weak opioid and 73 had received no regular opioids. Thirty-eight patients did not complete the study, mainly due to adverse events. For all groups the proportion of patients with 'good to excellent' pain control increased after transdermal fentanyl treatment. Transdermal fentanyl was well tolerated, with the most common treatment-related adverse events being nausea, vomiting and constipation. The percentage of strong-opioid-tolerant patients with constipation decreased following transdermal fentanyl treatment and increased slightly in the strong-opioid-naïve groups. Most patients rated the convenience of the patches as 'good to excellent', and most preferred transdermal fentanyl to their previous therapy. CONCLUSIONS: Transdermal fentanyl is an effective and well-tolerated treatment for cancer-related pain for patients regardless of whether they have previously received opioids. Previous guidelines have often advocated initial dose finding with short-acting opioids but this study demonstrates that such a complex titration and conversion schedule may not be necessary,and that treatment may be initiated directly with long-acting formulations such as transdermal fentanyl when previous analgesic therapy fails to provide adequate relief.

Unusual 4-hydroxybenzaldehyde synthase activity from tissue cultures of the vanilla orchid Vanilla planifolia.

Tissue cultures of the vanilla orchid, Vanilla planifolia, produce the flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde) and vanillin precursors such as 4-hydroxybenzaldehyde. A constitutively expressed enzyme activity catalyzing chain shortening of a hydroxycinnamic acid, believed to be the first reaction specific for formation of vanilla flavor compounds, was identified in these cultures. The enzyme converts 4-coumaric acid non-oxidatively to 4-hydroxybenzaldehyde in the presence of a thiol reagent but with no co-factor requirement. Several forms of this 4-hydroxybenzaldehyde synthase (4HBS) were resolved and partially purified by a combination of hydrophobic interaction, ion exchange and gel filtration chromatography. These forms appear to be interconvertible. The unusual properties of the 4HBS, and its appearance in different protein fractions, raise questions as to its physiological role in vanillin biosynthesis in vivo.

Systemic biomarkers of neutrophilic inflammation, tissue injury and repair in COPD patients with differing levels of disease severity.

The identification and validation of biomarkers to support the assessment of novel therapeutics for COPD continues to be an important area of research. The aim of the current study was to identify systemic protein biomarkers correlated with measures of COPD severity, as well as specific protein signatures associated with comorbidities such as metabolic syndrome. 142 protein analytes were measured in serum of 140 patients with stable COPD, 15 smokers without COPD and 30 non-smoking controls. Seven analytes (sRAGE, EN-RAGE, NGAL, Fibrinogen, MPO, TGF-α and HB-EGF) showed significant differences between severe/very severe COPD, mild/moderate COPD, smoking and non-smoking control groups. Within the COPD subjects, univariate and multivariate analyses identified analytes significantly associated with FEV(1), FEV(1)/FVC and DLCO. Most notably, a set of 5 analytes (HB-EGF, Fibrinogen, MCP-4, sRAGE and Sortilin) predicted 21% of the variability in DLCO values. To determine common functions/pathways, analytes were clustered in a correlation network by similarity of expression profile. While analytes related to neutrophil function (EN-RAGE, NGAL, MPO) grouped together to form a cluster associated with FEV(1) related parameters, analytes related to the EGFR pathway (HB-EGF, TGF-α) formed another cluster associated with both DLCO and FEV(1) related parameters. Associations of Fibrinogen with DLCO and MPO with FEV(1)/FVC were stronger in patients without metabolic syndrome (r  =  -0.52, p  =  0.005 and r  =  -0.61, p =  0.023, respectively) compared to patients with coexisting metabolic syndrome (r  =  -0.25, p  =  0.47 and r  =  -0.15, p  =  0.96, respectively), and may be driving overall associations in the general cohort. In summary, our study has identified known and novel serum protein biomarkers and has demonstrated specific associations with COPD disease severity, FEV(1), FEV(1)/FVC and DLCO. These data highlight systemic inflammatory pathways, neutrophil activation and epithelial tissue injury/repair processes as key pathways associated with COPD.

Cellular and molecular characterization of ozone-induced pulmonary inflammation in the Cynomolgus monkey.

We investigated the cellular and molecular effects of ozone exposure in Cynomolgus monkeys. Thirty-six Cynomolgus monkeys were exposed to single or repeat ozone challenge. Pulmonary inflammation was assessed using bronchoalveolar lavage fluid (BAL) and histology. Gene expression profiling in lung and blood was performed. Ozone challenge evoked BAL cellular inflammation and increases in total protein, alkaline phosphatase and cytokines. Lung histology revealed cellular inflammation and epithelial necrosis. Gene expression profiling identified oxidative phosphorylation, immune response and cell adhesion pathways altered in response to ozone, with common and unique profiles in lung and blood. Lipocalin 2, CD177, the FK-506 and S100A8 binding proteins and ST-2 represent novel peripheral biomarkers of ozone toxicity. Repeat ozone challenge evoked reproducible inflammation but attenuated cell damage. These studies provide data on the molecular mechanisms and biomarker identification of ozone-evoked toxicity, and support the use of the Cynomolgus monkey as a model of human ozone challenge.