Pilot study on the effects of intravesical oxybutynin hydrochloride instillations on the validity of doping control urine samples

According to class M2.1 of the World Anti-Doping Agency (WADA) Prohibited List, the manipulation of doping control urine samples to alter their integrity and validity is prohibited both in- and out-of-competition. However, some paraplegic athletes with an overactive bladder need to be regularly treated with anti-cholinergic and anti-spasmodic drugs such as oxybutynin, which are often administered intravesically to reduce the substantial side effects observed after oral application. So far, it remains unclear whether such bladder instillations have a negative impact on analytical procedures and thus represent an anti-doping rule violation. Within this pilot study, urine samples were collected from five paraplegic athletes before and after an intravesical oxybutynin hydrochloride instillation. The samples were routinely tested for the presence of performance-enhancing drugs and afterwards fortified with 25 model compounds representing different classes of doping agents (anabolic agents, cannabinoids, diuretics, glucocorticoids, hormone and metabolic modulators, and stimulants) at low and medium concentrations. Additionally, the pH value and specific gravity were measured and the presence of oxybutynin was qualitatively determined by gas chromatography-mass spectrometry (GC-MS). In initial testing procedures, all samples were tested negative. Oxybutynin was present in most of the samples but found to have no significant effect on the detectability of the 25 model compounds subsequently added to each urine specimen. Therefore, it can be concluded that intravesical instillations with oxybutynin hydrochloride do not alter the integrity and validity of doping control urine samples

Differential Induction of Ly6G and Ly6C Positive Myeloid Derived Suppressor Cells in Chronic Kidney and Liver Inflammation and Fibrosis

<div><p>CD11b⁺Gr1⁺ myeloid derived suppressor cells (MDSC) are known to be very potent suppressors of T cell immunity and can be further stratified into granulocytic MDSC and monocytic MDSC in mice based on expression of Ly6G or Ly6C, respectively. Here, using these markers and functional assays, we aimed to identify whether MDSC are induced during chronic inflammation leading to fibrosis in both kidney and liver and whether additional markers could more specifically identify these MDSC subsets. In an adenine-induced model of kidney inflammation/fibrosis suppressive Ly6G^pos MDSC were induced. The suppressive function within the Ly6G⁺ MDSC population was exclusively present in IFNγRβ expressing cells. In contrast, in chronic inflammation in the liver induced by bile duct ligation, suppressive capacity was exclusively present in the Ly6C^pos MDSC subset. Gene expression analyses confirmed the differential origins and regulation of those MDSC subsets. Additionally, depletion of MDSC in either kidney or liver fibrosis enhanced fibrosis markers, indicating a protective role for MDSC in organ fibrosis. Thus, our data demonstrate that during liver inflammation and kidney fibrosis MDSC with similar function arise bearing a distinct marker profile and arising from different cell populations.</p></div

Differential distribution of monocytic and granulocytic myeloid derived suppressor cells within Gr-1 positive cells in liver and kidney inflammation and fibrosis.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet, or were injected i.v. with LPS/IFNγ. Furthermore, BM-MDSC were generated <i>in vitro</i> by culture of bone marrow cells with CSF2 (GM-CSF) (A, B). After the indicated times liver, kidney, spleen or bone marrow cells were isolated and analysed by flow cytometry. Histograms depict viable (Hoechst negative), non-parenchymal cells stained with CD11b and Gr-1 (A) or viable, CD11b^pos cells stained for Ly6G and Ly6C (B). Representative (A, B) and cumulative (C) data of 4 (liver, spleen) or 3 (kidney, bone marrow) independent experiments are shown (n>9). Absolute numbers of CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ meyloid cells in the liver (D) and kidney (E) at the indicated time-points after BDL or adenine-feeding, respectively. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Suppressive capacity of monocytic and granulocytic MDSC subsets.

<p>C57BL/6 mice were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Gating strategy for sorting (A). At the indicated times myeloid subsets from liver (B), kidney (C), spleen (D) or in vitro bone marrow culture (E) were isolated and CD11b⁺ cells were sorted on the basis of their Ly6C or Ly6G expression and additional expression of IFNγRβ (A), yielding 4 separate subsets of CD11b⁺ myeloid cells. Naïve CFSE-labelled CD8 T cells were stimulated using αCD3/αCD28 coated beads and the different subsets of sorted myeloid cells were added at a 3:1 ratio (B-E). After 72h T cell proliferation was analysed by flow cytometry and the percentage of proliferated T cells is depicted (B-E). Cumulative data from 2 independent experiments are shown. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001. ND = not detectable.</p

Fibrosis markers in the liver and kidney after all-trans-retinoic acid treatment.

<p>C57BL/6 mice underwent bile-duct ligation, were fed an adenine-enriched diet or as a control were left untreated. After 7 days mice were treated with 1g/L all-trans-retinoic acid (ATRA) in their drinking water (BDL: n = 7, Adenine: n = 4) or not (BDL: n = 8, adenine: n = 4)) for the remaining time until analysis. At day 14 (BDL and adenine feeding), total liver and kidney RNA was isolated for real-time PCR of fibrosis markers. Shown are mRNA expression levels for α-SMA, collagen IV, TGF-β and vimentin relative to the levels in non-treated mice (n = 3), which was set to 1. Data are depicted as mean +/- SEM. Significance was calculated by ANOVA. *p≤0.05, **p≤0.01, ***p≤0.001.</p

Surface marker expression on suppressive and non-suppressive myeloid subsets.

<p>C57BL/6 mice were treated and cells were isolated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119662#pone.0119662.g001" target="_blank">Fig. 1</a>. Myeloid cells from non-treated mice served as controls (steady state). Flow cytometric analysis of surface markers associated with MDSC induction/function on CD11b⁺Ly6C⁺ and CD11b⁺Ly6G⁺ myeloid cells are depicted in the histograms. Specific staining: black lines. Isotype controls: filled grey. Representative data of 3 independent experiments is shown.</p