32 research outputs found

    Image_3_Plasma Cell Alloantigen 1 and IL-10 Secretion Define Two Distinct Peritoneal B1a B Cell Subsets With Opposite Functions, PC1high Cells Being Protective and PC1low Cells Harmful for the Growing Fetus.jpg

    No full text
    <p>B cells possess various immuno regulatory functions. However, research about their participation in tolerance induction toward the fetus is just emerging. Accumulating evidence supports the idea that B cells can play seemingly conflicting roles during pregnancy, either protecting or harming the fetus. Previous findings indicated the presence of two different peritoneal B cell subsets, defined by the expression of the plasma cell alloantigen 1 (PC1) and with distinct immune modulatory functions. Here, we aimed to study the participation of these two B cell subsets, on pregnancy outcome in a murine model of disturbed fetal tolerance. The frequencies and cell numbers of peritoneal and splenic CD19<sup>+</sup>IL-10<sup>+</sup> and CD19<sup>+</sup>CD5<sup>+</sup>IL-10<sup>+</sup>PC1<sup>+</sup> cells were assessed in virgin as well as normal pregnant (NP) and abortion-prone (AP) females during the course of gestation. Peritoneal PC1<sup>low</sup> or PC1<sup>high</sup> B1a B cells were sorted, analyzed for their ability to secrete IL-10 and adoptively transferred into NP or AP females. On gestation day (gd) 12, the abortion rate as well as the frequencies and cell numbers of regulatory T cells, TH1 and TH17 cells were determined in spleens and decidua. In addition, mRNA expression of IL-10, TGF-β, IFN-γ, and TNF-α was analyzed in decidual tissue. Peritoneal CD19<sup>+</sup>IL-10<sup>+</sup> and CD19<sup>+</sup>CD5<sup>+</sup>IL-10<sup>+</sup>PC1<sup>+</sup> frequencies fluctuated during the progression of normal pregnancies while no significant changes were observed in spleen. AP females showed significantly reduced frequencies of both B cell populations and exhibited an altered peritoneal PC1<sup>high</sup>/PC1<sup>low</sup> ratio at gd10. Adoptive transfers of PC1<sup>low</sup> B1a B cells into NP females increased the abortion rate in association with a reduced splenic regulatory T/TH17 ratio. By contrast, the transfer of PC1<sup>high</sup> B1a B cells into AP females significantly diminished the fetal rejection rate and significantly reduced the numbers of splenic TH17 cells. Our results suggest that the peritoneum harbors two distinct B1a B cell subsets that can be distinguished by their PC1 expression. Whereas PC1<sup>high</sup> B1a B cells seem to support fetal survival, PC1<sup>low</sup> cells B1a B cells may compromise fetal well-being.</p

    Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

    No full text
    <div><p>The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [<sup>3</sup>H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.</p></div

    Flow cytometry analysis of γH2AX-stained DNA double-strand breaks.

    No full text
    <p>Mean γH2AX intensity was assessed in PBMCs immediately, 1 h and 20 h after indicated exposure conditions. (A) Representative overlay histogram of γH2AX-intensity 1 h after indicated exposure (black line) and of corresponding control (gray line). (B) Difference of mean fluorescence intensity (MFI) of γH2AX and IgG-isotype control staining from 16 independent experiments at three different time points after exposure as mean ± SEM (***: P ≤ 0.001; ns: P > 0.05).</p

    Effect of glucose and lactate on cellular ATP content in IPEC-1 cells after 1 h treatment.

    No full text
    <p>IPEC-1 cells were grown to confluence (7 d) and treated for 1 h with HBSS-based solutions. Supernatant was removed and ATP content was measured by a luminescence based assay. Statistical analysis was done by Kruskal-Wallis and Dunn's post-hoc test. Different superscripts indicate statistically significant (p<0.05) differences.</p

    AGE accumulation in MCF-7 and TamR cells.

    No full text
    <p>MCF-7-Md and TamR-Md cells were grown under standard conditions and harvested in the logarithmic phase. Cells were fractionated by a detergent based protocol and AGEs detected by Western blotting. Based on protein determination and Coomassie staining (A), equal amounts of protein were loaded for both cell lines and the AGEs MG-AGE (B) and CML (C) detected by Western analysis. Differences in the pattern of AGE-modified proteins are indicated by arrows.</p

    Image_2_Plasma Cell Alloantigen 1 and IL-10 Secretion Define Two Distinct Peritoneal B1a B Cell Subsets With Opposite Functions, PC1high Cells Being Protective and PC1low Cells Harmful for the Growing Fetus.jpg

    No full text
    <p>B cells possess various immuno regulatory functions. However, research about their participation in tolerance induction toward the fetus is just emerging. Accumulating evidence supports the idea that B cells can play seemingly conflicting roles during pregnancy, either protecting or harming the fetus. Previous findings indicated the presence of two different peritoneal B cell subsets, defined by the expression of the plasma cell alloantigen 1 (PC1) and with distinct immune modulatory functions. Here, we aimed to study the participation of these two B cell subsets, on pregnancy outcome in a murine model of disturbed fetal tolerance. The frequencies and cell numbers of peritoneal and splenic CD19<sup>+</sup>IL-10<sup>+</sup> and CD19<sup>+</sup>CD5<sup>+</sup>IL-10<sup>+</sup>PC1<sup>+</sup> cells were assessed in virgin as well as normal pregnant (NP) and abortion-prone (AP) females during the course of gestation. Peritoneal PC1<sup>low</sup> or PC1<sup>high</sup> B1a B cells were sorted, analyzed for their ability to secrete IL-10 and adoptively transferred into NP or AP females. On gestation day (gd) 12, the abortion rate as well as the frequencies and cell numbers of regulatory T cells, TH1 and TH17 cells were determined in spleens and decidua. In addition, mRNA expression of IL-10, TGF-β, IFN-γ, and TNF-α was analyzed in decidual tissue. Peritoneal CD19<sup>+</sup>IL-10<sup>+</sup> and CD19<sup>+</sup>CD5<sup>+</sup>IL-10<sup>+</sup>PC1<sup>+</sup> frequencies fluctuated during the progression of normal pregnancies while no significant changes were observed in spleen. AP females showed significantly reduced frequencies of both B cell populations and exhibited an altered peritoneal PC1<sup>high</sup>/PC1<sup>low</sup> ratio at gd10. Adoptive transfers of PC1<sup>low</sup> B1a B cells into NP females increased the abortion rate in association with a reduced splenic regulatory T/TH17 ratio. By contrast, the transfer of PC1<sup>high</sup> B1a B cells into AP females significantly diminished the fetal rejection rate and significantly reduced the numbers of splenic TH17 cells. Our results suggest that the peritoneum harbors two distinct B1a B cell subsets that can be distinguished by their PC1 expression. Whereas PC1<sup>high</sup> B1a B cells seem to support fetal survival, PC1<sup>low</sup> cells B1a B cells may compromise fetal well-being.</p

    Cell viability analysis of unstimulated PBMCs by CellTiter-Blue assay.

    No full text
    <p>Metabolic activity was measured 24 h, 48 h and 84 h after indicated exposure conditions. Diagrams display mean ± SEM of 16 independent experiments (***: P ≤ 0.001; **: P ≤ 0.01; ns: P > 0.05).</p

    Different Ca<sup>2+</sup> responses of CB and adult CD31<sup>+</sup> naive T cells.

    No full text
    <p>(A-B) Ca<sup>2+</sup> mobilization in CD4<sup>+</sup>CD45RA<sup>+</sup>CD31<sup>+</sup> T cells of one healthy donor (representative of at least eight healthy individuals) of PBMCs (A, adult) or CB (B) were performed in response to 0.05 μg/ml anti-CD3 Ab plus 0.5 μg/ml soluble anti-CD28 Ab (red curve) or only 0.05 μg/ml of anti-CD3 Ab alone (black curve, with anti-CD28 isotype Ab) in combination with GAMIg. The blue dotted line displays the maximum Ca<sup>2+</sup> response of adult CD31<sup>+</sup> naive T cells for anti-CD3 Ab stimulation alone. (C) Box plot with scatter plots representing means and SD of Ca<sup>2+</sup> influx response normalized by maximal Ca<sup>2+</sup> influx response to ionomycin of adult (gray circle) and CB (black circle) and their dependency on anti-CD28 Ab costimulation (anti-CD3 Ab plus anti-CD28 Ab (red box) or anti-CD3 Ab with anti-CD28 Ab isotype (blue box)). Statistical significance between groups * <i>P<</i>0.05 was determined by two-tailed ANOVA with Tukey-Kramer post-hoc test. n = number of individuals. (D) Comparison of different CD4<sup>+</sup> T cell subset stimulated with anti-CD3 Ab plus anti-CD28 Ab (red box) or with anti-CD3 Ab alone (blue box). Box plot with scatter plots representing means and standard deviations of Ca<sup>2+</sup> influx response normalized by maximal Ca<sup>2+</sup> influx response to ionomycin. Statistical significance of differences between anti-CD3/anti-CD28 Ab or anti-CD3 Ab stimulation at concentration 0.05 μg/ml of anti-CD3 Ab CD31<sup>+</sup> between groups of different T cell subsets was determined by two-tailed ANOVA <i>P</i> = 0.7745, CD45RA<sup>+</sup> <i>P</i> = 0.8195, CD4<sup>+</sup> <i>P</i> = 0.9926. ns = not significant. n = number of individuals. (E) CD4<sup>+</sup>CD45RA<sup>+</sup>CD31<sup>+</sup> T cells of CB (filled line), and adults (dashed line) treated with 0.5 μg/ml soluble anti-CD3 Ab and anti-CD28 Ab cross-linked with GAMIg in the presence (red) or absence (black) of 2 mM EGTA. One representative experiment out of three comparable experiments is shown. (F) STIM1 protein expression in CD4<sup>+</sup> T cells of CB (black) and in naive CD31<sup>+</sup> T cells from adults (gray) after stimulation using anti-CD3/anti-CD28 Ab. The densitometric analyses of the ratio of STIM1/α Tubulin are shown. Results are representative of at least two experiments.</p

    Age-dependent signatory Ca<sup>2+</sup> influx and cytokine concentrations in supernatants of naive CD4<sup>+</sup> T cells.

    No full text
    <p>(A-B) CD4<sup>+</sup>CD45RA<sup>+</sup>CD31<sup>+</sup> T cells stimulated with anti-CD3 Ab as indicated either with costimulation by 0.5 μg/ml soluble anti-CD28 Ab (A, dark gray) or with the CD28 isotype control (B, light gray). Compiled data of box plots with scatter plots represent the Ca<sup>2+</sup> influx response normalized to the maximal Ca<sup>2+</sup> influx ionomycin response. Statistical significance between groups was determined by two tailed ANOVA Tukey-Kramer post-hoc test * <i>P<</i>0.05. n = number of individuals. (C) NFATc2 expression after anti-CD3 Ab plus anti-CD28 Ab engagement. The densitometric analyses of the immunoblots for the relative protein expression levels are shown as ratios of NFATc2 or pNFATc2 to αTubulin. Lysates from three different donors were pooled. Results are representative of at least two independent experiments. IFNγ (D), IL-2 (E), and TNFα (F) concentrations in the supernatants of unstimulated (white), of soluble anti-CD3 Ab plus anti-CD28 Ab stimulated (dark gray), and of TCR/CD3 stimulated alone (light gray, with anti-CD28 Ab isotype) of naive T cells using a Bio-Plex cytokine assay (Bio-Rad). The mean value and SD are indicated for five independent experiments (two tailed ANOVA Tukey-Kramer post-hoc test * <i>P<</i>0.05). n = number of individuals.</p

    Frequencies of naive CD31<sup>+</sup> T cells within the CD4<sup>+</sup>CD45RA<sup>+</sup> compartment remain constant over ages.

    No full text
    <p>(A) T cell subsets of CB or PBMCs analyzed by flow cytometry. Representative flow cytometry dot plots and histograms are shown with the percentage of CD4<sup>+</sup>CD45RA<sup>+</sup> T cell subsets for CB (top), infant (middle; 2.8 months), and adult (bottom) samples. (B) Frequencies of CD4<sup>+</sup> among lymphocytes, (C) of CD45RA<sup>+</sup> among CD4<sup>+</sup> T cells, and (D) CD31<sup>+</sup> among peripheral CD4<sup>+</sup>CD45<sup>+</sup> cells are shown (* <i>P<</i>0.05; two-sided ANOVA Tukey-Kramer post-hoc test). The mean value and standard deviations (SD) are indicated. Age of infants and children is indicated in months. n = number of individuals.</p
    corecore