ELISPOT multiscreen plates (Millipore, Billerica,
MA, USA) were coated with anti-mouse IgG (1 μg/mL, Southern Biotech) or precoated with poly-L-lysine (Sigma) followed by calf thymus DNA (20 μg/mL, Sigma) or highly purified recombinant nucleolin (10 μg/mL, Diarect, Freiburg, Germany) kindly provided by U. Wellmann (University Erlangen-Nuremberg, Germany). Purity of recombinant nucleolin was assessed by silver staining (Supporting Information Fig. 2B). After blocking with 2% FCS in PBS, single cell suspensions from kidney, spleen and BM from two femurs were incubated for 2 h at 37°C. Selleck R788 Plates were washed and incubated with HRP-goat antibody to mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) for 1 h at 20°C. Spots were detected by tetramethylbenzidine MEK inhibitor substrate (Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA) and analyzed using an automatic ELISPOT reader (AID Diagnostics, Strassberg, Germany) and AID ELISPOT reader software 4.0. Data were analyzed using either a non-parametric Mann–Whitney U test or a two-tailed paired T-test using GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA). Both, the Kolmogorov–Smirnov test and the Shapiro–Wilks test were applied to test for a normal distribution. Significant differences are indicated as * for p<0.05,
** for p<0.01 and *** for p<0.001. We are grateful to Daniela Graef and Miriam Reutelshöfer (Department of Pathology) for expert technical assistance. This work was supported by grants from the German Research Society (FOR 831 TP 8; VO673/3-2) and Collaborative Research Center (SFB 643; project B3), the BayImmuNet Atazanavir program of the state of Bavaria and the Interdisciplinary Center for Clinical Research Erlangen (IZKF, project number A31). Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance
to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. “
“In addition to archetypal cognitive defects, Down syndrome (DS) is characterized by altered lymphocyte development and function, including premature thymic involution and increased incidence of infections. However, the potential mechanisms for these changes have not been fully elucidated. The current study used the Ts65Dn mouse model of DS to assess deficiencies in T-cell development and possible molecular alterations. Ts65Dn mice exhibited premature thymic involution and a threefold to fourfold decrease in the number and proportion of immature, double-negative thymocyte progenitors. In addition, there were twofold fewer double-positive and CD4 single-positive thymocytes in Ts65Dn thymuses.