11 research outputs found

    LTβR dependent growth suppressive activity also affects fully developed splenic tissue.

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    <p>(A) WT recipients without prior splenectomy were either sham operated or received WT or LTβR<sup>-/-</sup> splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous WT spleen. Indicated are means and standard deviation (n = 4–11, * = p < 0.05, ** = p < 0.01). (B) LTβR<sup>-/-</sup> recipients without prior splenectomy were either sham operated or received WT or LTβR<sup>-/-</sup> splenic implants. Eight weeks later weight (left side) and cell number (right side) of the endogenous spleen was determined. Only WT splenic regenerates significantly reduced weight and cell number of the endogenous LTβR<sup>-/-</sup> deficient spleen. Indicated are means and standard deviation (n = 5–8, ** = p < 0.01). These experiments were 2 times independently performed.</p

    LTβR expression by non-splenic tissues suppresses growth of regenerating splenic tissue.

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    <p>(A) Macroscopic appearance of splenic regenerates 8 weeks after implantation of wild-type splenic tissue (WT) into wild-type recipients (WT), LTβR deficient splenic tissue (LTβR<sup>-/-</sup>) into WT recipients, and WT splenic tissue into LTβR deficient recipients (LTβR<sup>-/-</sup>). (B) Weight (left side) and cell number (right side) of splenic regenerates. Indicated are means and standard deviation (n = 4–9, ** = p < 0.01). (C) Microscopic appearance of splenic regenerates. Cryostat sections were stained by immunohistochemistry for T cells (brown; TCRβ<sup>+</sup>) and B cells (blue; B220<sup>+</sup>). Red pulp (RP), T-cell zone (T), and B-cell zone (B) are well developed except in the LTβR<sup>-/-</sup> into WT combination where T and B cells are intermixed and only the red pulp (RP) is clearly recognizable (bar: 100 μm). This experiment was 3 times independently performed.</p

    Identification of LTβR regulated proteins by two-dimensional differential gel electrophoresis and mass spectrometry.

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    <p>(A) 2D-DIGE experiment performed with splenic stroma of LTβR<sup>-/-</sup> (Cy3, green) and WT (Cy5, red) mice. The Cy2 channel is masked (representative gel of three replicas). The encircled area is shown in (B). (B) Gel section showing four spots with an increase in volume ratio when LTβR<sup>-/-</sup> (left side) and WT (right side) splenic stroma was compared. Mass spectrometry identified the four spots as four isoforms of MFAP4 (representative gel of three replicas). (C) Indicated is the MFAP4 abundance in splenic stroma of LTβR<sup>-/-</sup> spleen, LTβR<sup>-/-</sup> spleen from a donor which received a WT splenic implant 8 weeks earlier (LTβR<sup>-/-</sup> + WT), and WT spleen. The different 2D-DIGE experiments were compared quantitatively by summing up the volume ratios of the four isoforms of MFAP4 and relating it to the abundance of MFAP4 in LTβR<sup>-/-</sup> splenic stroma which was set to one. The abundance of MFAP4 increased from LTβR<sup>-/-</sup> splenic stroma to LTβR<sup>-/-</sup> splenic stroma obtained from mice with a WT splenic regenerate and further to WT splenic stroma. Indicated are means and standard deviation (n = 3–6). This experiment was 2 times independently performed. (D) Western blot analysis of MFAP4 from splenic stroma lysates of LTβR<sup>-/-</sup> mice, LTβR<sup>-/-</sup> mice which received a WT splenic implant 8 weeks earlier (LTβR<sup>-/-</sup> + WT), and WT mice. Three different sets of experiments are shown.</p

    Architecture and cellular structure of WT spleen and WT into LTβR<sup>-/-</sup> splenic regenerates are comparable.

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    <p>(A) The compartment sizes of normal WT spleens were analyzed and compared to that of splenic regenerates (WT→LTβR<sup>-/-</sup>) 8 weeks after implantation. Indicated is the size of individual splenic zones as percent of total section area (n = 6–7 animals per group; RP: red pulp, MZ: marginal zone, WP: white pulp, T: T-cell zone, B: B-cell zone, T/B: mixed T/B-cell zone). (B) Frequency of leukocyte subsets in normal WT spleens and splenic regenerates (WT→LTβR<sup>-/-</sup>) as determined by flow cytometry (n = 5–8 animals per group; B: B cells, IgD<sup>-</sup> B: memory B cells, T: T cells; CD8: CD8<sup>+</sup> T cells, CD4: CD4<sup>+</sup> T cells, CD25: CD25<sup>+</sup>CD4<sup>+</sup> -regulatory- T cells). (C) Expression of cytokines and chemokines in normal WT spleens and splenic regenerates (WT→ LTβR<sup>-/-</sup>). Shown is the relative expression level normalized to the housekeeping gene (MLN 51) and relative to WT (fold expression change). The bars represent the mean values ± SD. Mann-Whitney <i>U</i> test was used to indicate a significant difference between WT spleens and splenic regenerates (*p < 0.05; ***p < 0.001).</p

    MFAP4 does not suppress the regeneration of splenic implants.

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    <p>(A) WT and MFAP4 deficient recipients were splenectomized and then received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. Indicated are means and standard deviation (n = 6). (B) WT and MFAP4 deficient recipients without prior splenectomy received a WT splenic implant. Eight weeks later the weight of the splenic regenerate was determined. No difference was seen between WT and MFAP4 deficient recipients. In addition, the suppressive activity of the endogenous spleen was similar in both groups (compare weights to the weights under A). Indicated are means and standard deviation (n = 6). These experiments were 2 times independently performed.</p

    The splenic transplantation model.

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    <p>After removal of the endogenous spleen the recipient (WT or LTβR<sup>-/-</sup>) receives a splenic implant which is composed of two pieces representing 50% of a WT or LTβR deficient spleen. The implanted splenic tissue first becomes necrotic because the blood supply is lacking. Then, formation of splenic tissue starts and within eight weeks splenic regeneration is completed.</p

    Activation markers in lymphocytes from thymus and subcutaneous lymph nodes of <i>T. cruzi</i> infected mice.

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    <p>Mice were infected with <i>T. cruzi</i> and 15 days later lymphocytes were isolated from thymus and subcutaneous lymph nodes. <b>(a)</b> Thymocytes were stained with CD4-PE, CD8-APC and marker-specific fluorescein isothiocyanate-labeled antibodies prior to flow cytometry analysis. The histograms represent the expression of the CD44, CD69 and CD62L markers in total thymocytes (upper panel) and CD4<sup>+</sup>CD8<sup>+</sup> T cells (lower panel) from chagasic and normal mice. <b>(b)</b> Comparison of the CD62L expression level from normal or chagasic CD4<sup>+</sup>CD8<sup>+</sup> T cells <i>versus</i> normal single-positive CD4<sup>+</sup> and CD8<sup>+</sup> T cells undergoing intrathymic maturation. Panel <b>(c)</b> depicts representative histograms of CD4<sup>+</sup>CD8<sup>+</sup> from <i>T. cruzi</i> infected subcutaneous lymph nodes (solid lines), compared with naïve CD4<sup>+</sup> and CD8<sup>+</sup> T cells obtained from non-infected mice. <b>(d)</b> Representative histograms of CD44 and CD69 expression in CD4<sup>+</sup>CD8<sup>+</sup> T cells, activated single-positive CD4<sup>+</sup> and CD8<sup>+</sup> T cells from subcutaneous lymph nodes obtained from <i>T. cruzi</i> infected mice. Animals were sacrificed on day 15 post-infection. The data are representative of three independent experiments. The values on the upper right corner indicate the mean fluorescent intensity of the expression of the markers from each histogram. Differences between chagasic DP and activated T cells <i>versus</i> naïve T cells are significant (<i>P</i><0.05). These data are representative of three independent experiments using five mice per group.</p

    Intrathymic expression of Bim in the course of acute <i>T. cruzi</i> infection.

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    <p><b>(a)</b> Thymuses were collected from normal or <i>T cruzi</i> infected BALB/c mice (day 15 post-infection). All numbers are given in 10<sup>6</sup>, and each value represents the mean of total thymocyte numbers in 4-8 mice per group. <b>(b)</b> From individual groups of the same experiments, DP cells isolated by cell sorting from thymus of infected mice at the indicated time-points or normal mice as controls were lysed and proteins were solubilized by detergent lysis and immunoprecipitated with anti-Bim antibody for Western blotting analysis probing with the same antibody. The upper blot shows kinetics of the Bim expression from day 10 to 16 post-infection, during the thymic atrophy, whereas the lower panel reveals a blot was probed with actin, applied as a loading control. <b>(c)</b> Optical densitometry of the western blots using NIH Image software, where Bim expression was normalized with the actin expression. The data are representative of three independent experiments. *Differences between control <i>versus</i> infected mice are significant (<i>P</i><0.05). These data are representative of two independent experiments using five mice per group.</p

    Intrathymic tissue-restricted antigen expression levels in atrophic thymus during <i>T cruzi</i> infection.

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    <p><b>(a)</b> Thymuses were collected from normal or <i>T. cruzi</i> infected BALB/c (day 15 post-infection) and expression levels of Aire and TRA genes were analyzed by real-time PCR. The expression of Aire as well as TRA genes was comparable in infected and uninfected thymus. Data are mean ± standard error. of triplicate measurements in one of two representative experiments using five mice per group. <b>(b)</b> Thymuses were stained with anti-Aire antibody and analyzed by immunofluorescence. The histological profiles show the presence of Aire-positive cells (arrows) in <b>(upper left)</b> normal thymus and <b>(upper right)</b> atrophic thymus at day 15 post-infection. The respective staining controls without the primary antibodies are represented in <b>(lower left)</b> and <b>(lower right)</b>. Inserts represent higher magnifications of Aire-positive cells (brown).</p

    Thymic atrophy in BALB/c acutely infected with <i>T. cruzi</i>.

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    <p>The histological profiles show the <b>(upper left)</b> normal thymic architecture (C, cortex; M, medulla) and <b>(upper right)</b> marked cortical and medullary atrophy in the thymus of an acutely-infected mouse. <b>(lower left)</b> The numerous metallophilic macrophages are present in the cortico-medullary zone (arrows) of normal thymus. However, following <i>T. cruzi</i> acute infection <b>(lower right)</b> the number and distribution of metallophilic macrophages are changed with the cells dispersed throughout not only the cortico-medullar area but also the cortical region. Infected mice were evaluated herein at day 15 post-infection. These data are representative of two independent experiments using four mice per group.</p
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