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Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma.

Healey GD, Evans N, Hopkin JM, Davies G, Walker W - J. Cell. Mol. Med. (2013)

Bottom Line: STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment.In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell-line monolayer cultures.Furthermore, targeting of STAT6 with siRNA attenuated IL-13-driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, Institute of Life Science, Swansea University, Swansea, UK. g.d.healey@swansea.ac.uk

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Representative micrographs stained with haematoxylin & eosin to show (A) sheets of differentiated columnar epithelium obtained by Rhinoprobe™ curettage sampling (40×), and (B) the presence of beating cilia (indicated by arrow) which were readily discernible upon placement in culture (100×). RT-PCR analysis of NEC's was carried out at the time of sampling to compare (C) STAT6, (D) CCL26 and (E) MUC5AC mRNA expression between non-asthmatic (closed bars) and asthmatic (open bars) donor groups. Values presented are the mean fold-change, relative to the mean δCt value for the non-asthmatic group ± SD (non-asthmatic control: n = 4, asthmatic donors: n = 19). Data were analysed by Mann–Whitney U-test and values differ from non-asthmatic: *P < 0.05.
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fig01: Representative micrographs stained with haematoxylin & eosin to show (A) sheets of differentiated columnar epithelium obtained by Rhinoprobe™ curettage sampling (40×), and (B) the presence of beating cilia (indicated by arrow) which were readily discernible upon placement in culture (100×). RT-PCR analysis of NEC's was carried out at the time of sampling to compare (C) STAT6, (D) CCL26 and (E) MUC5AC mRNA expression between non-asthmatic (closed bars) and asthmatic (open bars) donor groups. Values presented are the mean fold-change, relative to the mean δCt value for the non-asthmatic group ± SD (non-asthmatic control: n = 4, asthmatic donors: n = 19). Data were analysed by Mann–Whitney U-test and values differ from non-asthmatic: *P < 0.05.

Mentions: To test whether sampling the nasal epithelium of asthmatic donors would provide an improved method for preclinical evaluation, we obtained primary NEC's from asthmatic (n = 19) and non-asthmatic (n = 4) control donors by sampling the inferior turbinate with a Rhinoprobe™ curette, patient details and baseline data for mild, moderate and severe asthma patients are provided in Table 1. Irrespective of donor status, combined curettage of the right and left nasal turbinate produced similar total epithelial cell yields (up to 1 × 105 cells, viability >70%). Attempts to culture cells obtained by curettage were initially hampered by high contamination rates. Subsequent cultures were therefore carried out in the presence of antibiotics, as described in the Materials and Methods section, to overcome this problem. Samples typically consisted of sheets of differentiated epithelium (Fig. 1A) with beating ciliated columnar epithelial cells readily discernible on microscopic examination (Fig. 1B). No evidence of contaminating cells was apparent from microscopic examination and gene expression analysis for the T cell marker CD3 was negative (data not shown). RT-PCR analysis of cells at the time of sampling showed that STAT6 mRNA expression was similar in nasal samples derived from either asthmatic (fold change, μ = 1.13 ± 0.61) or non-asthmatic (fold change, μ = 0.75 ± 0.12) donors (Fig. 1C). In contrast, CCL26 mRNA expression was significantly elevated in samples from asthmatic (fold change, μ = 31.6 ± 52.19) donors compared with the non-asthmatic (fold change, μ = 1.48 ± 1.3) controls (Fig. 1D). In addition, although not significant, a trend suggesting elevated expression of MUC5AC mRNA was also noted in the asthmatic group (fold change, μ = 169.37 ± 461.9) compared with the non-asthmatic (fold change, μ = 1.45 ±1.59) controls (Fig. 1E). No significant differences were noted between asthma patients with differing severities of disease, and no significant difference in expression levels was observed between CHI3L1 and PROM1 (data not shown). Therefore, NEC's derived from patients with bronchial asthma appear to exhibit phenotypic characteristics consistent with pro-asthmatic inflammatory events.


Evaluation of nasal epithelium sampling as a tool in the preclinical development of siRNA-based therapeutics for asthma.

Healey GD, Evans N, Hopkin JM, Davies G, Walker W - J. Cell. Mol. Med. (2013)

Representative micrographs stained with haematoxylin & eosin to show (A) sheets of differentiated columnar epithelium obtained by Rhinoprobe™ curettage sampling (40×), and (B) the presence of beating cilia (indicated by arrow) which were readily discernible upon placement in culture (100×). RT-PCR analysis of NEC's was carried out at the time of sampling to compare (C) STAT6, (D) CCL26 and (E) MUC5AC mRNA expression between non-asthmatic (closed bars) and asthmatic (open bars) donor groups. Values presented are the mean fold-change, relative to the mean δCt value for the non-asthmatic group ± SD (non-asthmatic control: n = 4, asthmatic donors: n = 19). Data were analysed by Mann–Whitney U-test and values differ from non-asthmatic: *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3823017&req=5

fig01: Representative micrographs stained with haematoxylin & eosin to show (A) sheets of differentiated columnar epithelium obtained by Rhinoprobe™ curettage sampling (40×), and (B) the presence of beating cilia (indicated by arrow) which were readily discernible upon placement in culture (100×). RT-PCR analysis of NEC's was carried out at the time of sampling to compare (C) STAT6, (D) CCL26 and (E) MUC5AC mRNA expression between non-asthmatic (closed bars) and asthmatic (open bars) donor groups. Values presented are the mean fold-change, relative to the mean δCt value for the non-asthmatic group ± SD (non-asthmatic control: n = 4, asthmatic donors: n = 19). Data were analysed by Mann–Whitney U-test and values differ from non-asthmatic: *P < 0.05.
Mentions: To test whether sampling the nasal epithelium of asthmatic donors would provide an improved method for preclinical evaluation, we obtained primary NEC's from asthmatic (n = 19) and non-asthmatic (n = 4) control donors by sampling the inferior turbinate with a Rhinoprobe™ curette, patient details and baseline data for mild, moderate and severe asthma patients are provided in Table 1. Irrespective of donor status, combined curettage of the right and left nasal turbinate produced similar total epithelial cell yields (up to 1 × 105 cells, viability >70%). Attempts to culture cells obtained by curettage were initially hampered by high contamination rates. Subsequent cultures were therefore carried out in the presence of antibiotics, as described in the Materials and Methods section, to overcome this problem. Samples typically consisted of sheets of differentiated epithelium (Fig. 1A) with beating ciliated columnar epithelial cells readily discernible on microscopic examination (Fig. 1B). No evidence of contaminating cells was apparent from microscopic examination and gene expression analysis for the T cell marker CD3 was negative (data not shown). RT-PCR analysis of cells at the time of sampling showed that STAT6 mRNA expression was similar in nasal samples derived from either asthmatic (fold change, μ = 1.13 ± 0.61) or non-asthmatic (fold change, μ = 0.75 ± 0.12) donors (Fig. 1C). In contrast, CCL26 mRNA expression was significantly elevated in samples from asthmatic (fold change, μ = 31.6 ± 52.19) donors compared with the non-asthmatic (fold change, μ = 1.48 ± 1.3) controls (Fig. 1D). In addition, although not significant, a trend suggesting elevated expression of MUC5AC mRNA was also noted in the asthmatic group (fold change, μ = 169.37 ± 461.9) compared with the non-asthmatic (fold change, μ = 1.45 ±1.59) controls (Fig. 1E). No significant differences were noted between asthma patients with differing severities of disease, and no significant difference in expression levels was observed between CHI3L1 and PROM1 (data not shown). Therefore, NEC's derived from patients with bronchial asthma appear to exhibit phenotypic characteristics consistent with pro-asthmatic inflammatory events.

Bottom Line: STAT6 expression was invariant in both donor groups and amenable to suppression by siRNA treatment.In addition, STAT6 mRNA was also suppressible by apically delivered siRNA treatment in comparative differentiated nasal epithelial cell-line monolayer cultures.Furthermore, targeting of STAT6 with siRNA attenuated IL-13-driven CCL26 expression in these cells, pointing to the utility of this approach in preclinical testing.

View Article: PubMed Central - PubMed

Affiliation: College of Medicine, Institute of Life Science, Swansea University, Swansea, UK. g.d.healey@swansea.ac.uk

Show MeSH
Related in: MedlinePlus