Kinetics of TH2 biomarkers in sputum of asthmatics following inhaled allergen.

BACKGROUND: Allergen-induced late airway response offers important pharmacodynamic targets, including T helper 2 (TH2) biomarkers. However, detection of inflammatory markers has been limited in dithiothreitol-processed sputum. OBJECTIVES: To test whether allergen-induced TH2 inflammatory markers can be reproducibly quantified by sensitive detection techniques in ultracentrifuged sputum and the effect of fluticasone (FP) on these endpoints. METHODS: Thirteen allergic asthmatics with dual allergen-induced airway responses, documented during a single-blind placebo run-in period, participated in a double-blind, two-period crossover study. Each period consisted of three consecutive days, separated by ≥3 weeks. Following randomization, subjects inhaled FP (500 µg bid, five doses total) or placebo. On Day 2 in each study period, allergen challenge was performed and airway response measured by forced expiratory volume in 1 sec (FEV1) until 7 h post-challenge. Sputum was induced 24 h pre-allergen and 7 and 24 h post-allergen. Sputum samples were split into two portions: TH2 biomarkers were quantified by Meso Scale multiplex platform following ultracentrifugation, and cell differentials were counted on Giemsa-May-Grünwald-stained cytospins. Allergen-induced changes in inflammatory endpoints were compared between FP and placebo using a mixed model ANCOVA. RESULTS: Inhaled allergen induced dual airway responses in all subjects during both placebo periods with reproducible late asthmatic response (LAR) and increased sputum inflammatory biomarkers (IL-2, IL-4, IL-13, and eotaxin-1) and eosinophil counts. FP effectively blunted both the LAR and the inflammatory biomarkers. CONCLUSIONS: Combining novel, sensitive quantification methods with ultracentrifugation allows reproducible quantification of sputum biomarkers following allergen challenge, reversed by FP. This approach allows non-invasive identification of pharmacodynamic targets for anti-asthma therapies.

High-throughput simultaneous analysis of RNA, protein, and lipid biomarkers in heterogeneous tissue samples.

BACKGROUND: With expanding biomarker discovery efforts and increasing costs of drug development, it is critical to maximize the value of mass-limited clinical samples. The main limitation of available methods is the inability to isolate and analyze, from a single sample, molecules requiring incompatible extraction methods. Thus, we developed a novel semiautomated method for tissue processing and tissue milling and division (TMAD). METHODS: We used a SilverHawk atherectomy catheter to collect atherosclerotic plaques from patients requiring peripheral atherectomy. Tissue preservation by flash freezing was compared with immersion in RNAlater®, and tissue grinding by traditional mortar and pestle was compared with TMAD. Comparators were protein, RNA, and lipid yield and quality. Reproducibility of analyte yield from aliquots of the same tissue sample processed by TMAD was also measured. RESULTS: The quantity and quality of biomarkers extracted from tissue prepared by TMAD was at least as good as that extracted from tissue stored and prepared by traditional means. TMAD enabled parallel analysis of gene expression (quantitative reverse-transcription PCR, microarray), protein composition (ELISA), and lipid content (biochemical assay) from as little as 20 mg of tissue. The mean correlation was r = 0.97 in molecular composition (RNA, protein, or lipid) between aliquots of individual samples generated by TMAD. We also demonstrated that it is feasible to use TMAD in a large-scale clinical study setting. CONCLUSIONS: The TMAD methodology described here enables semiautomated, high-throughput sampling of small amounts of heterogeneous tissue specimens by multiple analytical techniques with generally improved quality of recovered biomolecules.

Distinct properties and advantages of a novel peroxisome proliferator-activated protein [gamma] selective modulator.

Antidiabetic thiazolidinediones (TZDs) and non-TZD compounds have been shown to serve as agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma). Here, we report the identification and characterization of a novel non-TZD selective PPARgamma modulator (nTZDpa). nTZDpa bound potently to PPARgamma with high selectivity vs. PPARalpha or PPARdelta. In cell-based assays for transcriptional activation, nTZDpa served as a selective, potent PPARgamma partial agonist and was able to antagonize the activity of PPARgamma full agonists. nTZDpa also displayed partial agonist effects when its ability to promote adipogenesis in 3T3-L1 cells was evaluated. Assessment of protein conformation using protease protection or solution nuclear magnetic resonance spectroscopy methods showed that nTZDpa produced altered PPARgamma conformational stability vs. full agonists, thereby establishing a physical basis for its observed partial agonism. DNA microarray analysis of RNA from 3T3-L1 adipocytes treated with nTZDpa or several structurally diverse PPARgamma full agonists demonstrated qualitative differences in the affected gene expression profile for nTZDpa. Chronic treatment of fat-fed, C57BL/6J mice with nTZDpa or a TZD full agonist ameliorated hyperglycemia and hyperinsulinemia. However, unlike the TZD, nTZDpa caused reductions in weight gain and adipose depot size. Feed efficiency was also substantially diminished. Unlike TZDs, nTZDpa did not cause cardiac hypertrophy in mice. When a panel of PPARgamma target genes was examined in white adipose tissue, nTZDpa produced a different in vivo expression pattern vs. the full agonist. These findings establish that novel selective PPARgamma modulators can produce altered receptor conformational stability leading to distinctive gene expression profiles, reduced adipogenic cellular effects, and potentially improved in vivo biological responses. Such compounds may lead to preferred therapies for diabetes, obesity, or metabolic syndrome.