Reproducibility and intraindividual variation over days in buccal cell DNA methylation of two asthma genes, interferon γ (IFNγ) and inducible nitric oxide synthase (iNOS).

The biological mechanisms responsible for the onset and exacerbation of asthma symptoms in children may involve the epigenetic regulation of inflammatory genes after environmental exposures. Using buccal cells, we hypothesized that DNA methylation in promoter regions of two asthma genes, inducible nitric oxide synthase (iNOS) and interferon γ (IFNγ), can vary over several days. Repeat buccal samples were collected 4 to 7 days apart from 34 children participating in the Columbia Center for Children's Environmental Health (CCCEH) birth cohort study. Several field duplicates (sequential collection of two samples in the field) and replicates (one sample pyrosequenced twice) also were collected to ensure consistency with collection and laboratory procedures. DNA methylation was assessed by pyrosequencing a PCR of bisulfite-treated DNA. We found that replicate and field duplicate samples were correlated strongly (r = 0.86 to 0.99, P < 0.05), while repeat samples demonstrated low within-subject correlations (r = 0.19 to 0.56, P = 0.06 to 0.30). Our data reveal DNA methylation as a dynamic epigenetic mechanism that can be accessed safely and reproducibly in an inner city pediatric cohort using non-invasive buccal swabs and pyrosequencing technology.

Mechanism of action of imiquimod 5% cream in the treatment of anogenital warts.

Objective: The objective of this randomized, double-blind, placebo-controlled trial was to evaluate the mechanism of action of imiquimod cream in the treatment of anogenital warts, and to apply the findings to the results of previously conducted safety and efficacy trials.Methods: Imiquimod (16 patients) or placebo (3 patients) cream was applied 3 times a week for up to 16 weeks; cream remained on the skin overnight for 8 +/- 2 hours. Wart biopsies were taken at prestudy, week 6, and the end of treatment (just prior to clearance or at week 16) and analyzed using PCR for HPV/DNA and RT-PCR for mRNA to identify cytokines, cellular markers, markers of proliferation and differentiation, and viral gene products. Efficacy was assessed based on wart area regression as documented by wart area measurements and photographs.Results: All patients enrolled in the trial had HPV type 6/11. All imiquimod-treated patients experienced a >/=75% clearance in baseline/target wart area. Imiquimod treatment stimulated significant increases in mRNA for IFN-alpha, 2'5' AS and IFN-gamma. Increases in mRNA for CD4, CD8, and TNF-alpha were also observed, suggesting activation of a T-helper type-1 cell mediated response. During the trial one of the vehicle treated patients also experienced spontaneous wart clearance; comparisons of the cytokine levels for this patient were similar to those observed for the imiquimod treated patients.Conclusions: The results of this mechanism of action trial indicate that the stimulation of local cytokines and cellular infiltrates by imiquimod leads to a reduction of HPV types 6 and 11 viral load with subsequent wart regression and normalization of keratinocyte proliferation without evidence of scarring. In two previous randomized vehicle-controlled trials evaluating patients with anogenital warts, the majority of patients had HPV-DNA types 6 or 11 as assessed by in situ hybridization. These results provide additional insight into the mechanism of total clearance for these otherwise healthy patients. The Th1 response demonstrated in this trial also explains the lower total clearance rates demonstrated in HIV-positive and AIDS patients.