25D₃ and cathelicidin in serum and bladder tissue of women during a three-month period of 25D₃ supplementation.

<p>Postmenopausal women were treated with 2000 units of oral 25D₃ daily for 12 weeks. Serum samples were collected before, at 6 and 12 weeks. Serum 25D₃ levels increased from a baseline median value of 68.5 nmol/L to 104.5 nmol/L at 6 weeks, <i>P</i><0.001 (Wilcoxon rank test), and 117 nmol/L at 12 weeks, <i>P</i><0.01 (Wilcoxon rank test). The dotted line denotes the recommended serum 25D₃ level of 75 nmol/L and median values are indicated (<b>A</b>). Despite treatment with 25D₃, serum cathelicidin levels did not increase (<b>B</b>). <i>E. coli</i>-infected bladder biopsies from women receiving 25D₃ supplementation responded with higher expression of <i>CAMP</i> mRNA, <i>P</i><0.05 (Mann-Whitney test) (<b>C</b>) and cathelicidin protein, <i>P</i> <0.05 (Mann-Whitney test) (<b>D</b>) compared to infected pieces prior to supplementation. Fluorescence immunohistochemistry showed increased cathelicidin protein expression in infected bladder biopsies. Tissue samples shown are from the same patient before (<b>E</b>) and after (<b>F</b>) 12 weeks of 25D₃ treatment. Cathelicidin is localized in the uroepithelium including the larger umbrella cells closest to the bladder lumen. Staining without primary antibody serves as negative control (<b>G</b>). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm. Cathelicidin is stained with AlexaFluor 594 (red), the cell nucleus with DAPI (blue).</p

Human bladder cells respond with <i>CYP24A1</i> and <i>CAMP</i> induction to 1,25D₃ and 25D₃.

<p>Normal bladder cells (HBEP, upper panel) and TERT-NHUC bladder cells (lower panel) were stimulated with 10⁻⁷ M 25D₃ or 10⁻⁸ M 1,25D₃ for 24 hours. Levels of gene-specific mRNA for <i>CYP24A1</i> (<b>A</b>, <b>B</b>), <i>CAMP</i> (<b>C</b>, <b>D</b>), <i>VDR</i> (<b>E</b>, <b>F</b>) and <i>CYP27B1</i> (<b>G</b>, <b>H</b>) in stimulated cells were determined and are presented as fold change compared to vector-treated control cells. Data presented are pooled from three independent experiments and shown as mean and standard deviation; ***<i>P</i><0.001, **<i>P</i><0.01, *<i>P</i><0.05 (ANOVA with Dunnett's post-test).</p

1,25D₃ or 25D₃ in combination with <i>E. coli</i> increased cathelicidin protein in bladder epithelial cells.

<p>Production of cathelicidin increased in TERT-NHUC bladder cells after treatment with 1,25D₃ or 25D₃, and was further increased by infection with <i>E. coli</i> CFT073 (UPEC). Images are captured at ×63 magnification and depict an area corresponding to 119×119 µm (<b>A</b>). The control image (Neg control) is shown in the right upper panel. Fluorescence intensity was quantified and is presented as mean values with standard deviation; ***<i>P</i><0.001, *<i>P</i><0.05 (ANOVA with Dunnett's post-test) (<b>B</b>). An increase in cathelicidin protein was also recorded by flow cytometry in <i>E. coli</i>-infected TERT-NHUC bladder cells treated with 1,25D₃ or 25D₃. Grey is vector +<i>E. coli</i>, red is 1,25D₃ 10⁻⁸ M+<i>E. coli</i> and black is 25D₃ 10⁻⁷ M+<i>E. coli</i> (<b>C</b>).</p