3 research outputs found
RAID3 - An interleukin-6 receptor-binding aptamer with post-selective modification-resistant affinity
<div><p>Aptamers are an emerging class of highly specific targeting ligands. They can be selected <i>in vitro</i> for a large variety of targets, ranging from small molecules to whole cells. Most aptamers selected are nucleic acid-based, allowing chemical synthesis and easy modification. Although their properties make them interesting drug candidates for a broad spectrum of applications and an interesting alternative to antibodies or fusion proteins, they are not yet broadly used. One major drawback of aptamers is their susceptibility to abundant serum nucleases, resulting in their fast degradation in biological fluids. Using modified nucleic acids has become a common strategy to overcome these disadvantages, greatly increasing their half-life under cell culture conditions or even <i>in vivo</i>. Whereas pre-selective modifications of the initial library for aptamer selection are relatively easy to obtain, post-selective modifications of already selected aptamers are still generally very labor-intensive and often compromise the aptamers ability to bind its target molecule.</p><p>Here we report the selection, characterization and post-selective modification of a 34 nucleotide (nt) RNA aptamer for a non-dominant, novel target site (domain 3) of the interleukin-6 receptor (IL-6R). We performed structural analyses and investigated the affinity of the aptamer to the membrane-bound and soluble forms (sIL-6R) of the IL-6R. Further, we performed structural analyses of the aptamer in solution using small-angle X-ray scattering and determined its overall shape and oligomeric state. Post-selective exchange of all pyrimidines against their 2′-fluoro analogs increased the aptamers stability significantly without compromising its affinity for the target protein. The resulting modified aptamer could be shortened to its minimal binding motif without loss of affinity.</p></div
Microarray Results and Corresponding Quantitative Real-Time PCR for Differentially Regulated Genes in CD or UC Compared to Normal Controls
<p>Genes were chosen on the basis of their dysregulation in IBD and represent both known genes from functional groups discussed and genes of unknown function. Quantitative real-time PCR was carried out on individual samples from group 2 patients (14–18 normal controls, 19–33 UC, and 17–22 CD samples, depending on the availability of the patient samples at the time the plates were produced), except for IL-8 and TNF-α (not on array), which were tested in group 1 patients (11 normal controls, ten UC, and ten CD patient samples) as a proof-of-principle measure. The extended cohort of group 2 patients includes those with active disease and using anti-inflammatory drugs (but not immunosuppressants or biologicals), whereas group 1 patients had active disease and were medication-free for 6 wk. Results are summarised by a ratio of medians (CD:normal or UC:normal). Complete results, including box-plots and number of samples analysed in each assay, are included in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0020199#sg001" target="_blank">Figure S1</a>. All results were significantly differentially regulated except for marked results; single dagger indicates that array result was not significant (<i>p</i> > 0.0015 or fold-change < 1.2); asterisk indicates that real-time PCR result was not significant (<i>p</i> > 0.05). A dashed line represents the fold-change level of 1.2.</p
Immunohistochemical Localization of CEACAM1, CSNK1D, and PRKCB1 in Colonic Mucosa
<div><p>Staining of a representative mucosal tissue samples from five normal controls (N), five Crohn disease (CD), and six ulcerative colitis (UC) patients using antibodies against (A) CEACAM1, (B) CSNK1D, and (C) PRKCB1.</p>
<p>(A) CEACAM1 immunoreactivity was found in the apical epithelial lining (1), crypts of inflamed tissue (2, 4). Additional staining was detected in immune cells (3) and blood vessels (5).</p>
<p>(B) CSNK1D immunoreactivity showed a strong granular staining pattern in normal (6) and the UC group (8), which was located basolaterally. Weaker staining of the apex of the crypts could be detected in CD (7).</p>
<p>(C) For PRKCB1, weak staining could be observed in the apical epithelial layer in the normal (9) and CD (10) mucosa and, interestingly, immunoreactivity was found nearly exclusively in the marginal zone of small lymph follicles (11), whereas the lamina propria was immunonegative. In contrast, strong staining was detected in the apical epithelial layer of UC mucosa (12). Furthermore, lamina propria mononuclear cells underlying the epithelial layer were also positive in the UC mucosa (13).</p></div