Comparison of 4-HNE expression in the placental beds between the control and IUGR/PE groups.

<p>Parallel sections of placental beds of normal pregnancy (C) and IUGR/PE (D) stained with cytokeratin 7 antibody, 4-HNE antibody (A, B) revealed that 4-HNE immunopositivity (red staining) is strongly expressed in extravillous trophoblast in IUGR/PE (B), and only very weak in healthy placental beds (A), Original magnification 200×, bar represents 100 µm. Other cells such as endothelial cells showed strong immunopositivity for 4-HNE (A, B arrows) but were dropped from the scoring. The IRS score showed a significant increase in the 4-HNE expression in the extra villous trophoblast in IUGR/PE cases when compared with the control groups. **p<0.005 when compared to the controls.</p

Schematic representation of the invasion route of interstitial and endovascular trophoblast in human pregnancy.

<p>Blue: fetal tissues, including interstitial trophoblast and its intravasating derivatives. Brown: maternal tissues. Endovascular trophoblast is derived from a side route of interstitial trophoblast (Kaufmann et al. 2003, modified).</p

Expression of VEGF in the placental beds of women suffering from IUGR/PE or controls and the Immunoreactive Score (IRS) of this staining in the EVT.

<p>Interstitial trophoblast in controls display a stronger immunopositivity for VEGF (A), which are positive for cytokeratin 7 (C) than in early onset IUGR/PE samples (B). In control pregnancies decidual cells showed also a higher VEGF activity (A, arrows) but these cells were not analyzed. Original magnification 200×(bar = 100 µm). (E) The figure represent the mean score + (SEM) of VEGF immunolocalisation to endovascular and interstitial trophoblast cells in both control and early onset IUGR/PE groups. *p<0.05 between the two groups using Student’s t test.</p

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb

Additional file 4: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Exogenous application of CRAMP reduced NFκB translocation in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT (A) or KO (B) mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 30 min, 1 or 2 h. After incubation cells were fixed and immunolabeled using anti-NFκB p65 antibody (red) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 19383 kb

Invasion of extravillous trophoblast.

<p>Immunohistochemistry using an antibody directed against cytokeratin 7 revealed the invasive depth and the numerical density of invasive cells in control cases (A, B) and early onset IUGR/PE (C). Original magnification 400× (B, C). (A) In order to allow publication of a survey picture illustrating the complete invasive pathway, immunoreactivity of cytokeratin 7-positive cells was enhanced by image analysis. Figure A represents the placental, endometrial and myometrial parts of the sample Original magnification 100×.</p

Expression of Nrf2 and cytokeratin 7 in the placental beds from patients with early onset IUGR/PE and controls with Immunoreactive Score (IRS) of staining for Nrf2.

<p>In placental beds of IUGR/PE cases, cytokeratin 7-positive cells (D) surround a spiral artery, reveals a strong cytoplasmic immunopositivity for Nrf2 (B). In contrast, very weak immunostaining is seen (A) in the same cytokeratin 7-positive cells (C) in control placental beds. Blue haematoxylin counterstain was performed. Original magnification 200×(bar = 100 µm). Mean score + (SEM) of Nrf2 immunolocalisation to endovascular and interstitial trophoblast cells in both control and early onset IUGR/PE groups (E) confirms the previously results. ***p<0.0001 versus the control group using Student’s t test. The arrows in (B) represent the leukocyte populations in decidua and myometrium, which were also positive for Nrf2 but they were excluded from the scoring.</p

Clinical characteristics of control and pathological pregnancies.

<p>Values were presented as mean ± SD or n (%).</p>***<p>P<0.005 when compared with normal pregnancy.</p>#<p>P<0.001 when compared with normal pregnancy.</p

Additional file 3: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Proliferation and apoptosis induction after bacterial stimulation in CRAMP-WT or CRAMP-KO microglial cells. Microglial cells from CRAMP-knockout (KO) or wild-type (WT) mice were incubated with bacterial supernatants of Gram-positive bacterium Streptococcus pneumoniae (SP) or Gram-negative bacterium Neisseria meningitidis (NM) and bacterial cell wall components lipopolysaccharide (LPS) or peptidoglycan (PGN) for 24 h. After incubation, glial cells were fixed and immunolabeled using the proliferation marker Ki67 (red), TUNEL reaction mixture for apoptosis and DAPI for nuclear counterstaining (blue). (A) Representative results from one of three independent experiments. (B) Ki67 proliferation index was calculated by the number of positive cells expressing Ki67 divided by the total number of cells in each field. These results were calculated for at least 20 separate cells. Scale bar = 20 μm. (TIFF 6059 kb

Additional file 5: of CRAMP deficiency leads to a pro-inflammatory phenotype and impaired phagocytosis after exposure to bacterial meningitis pathogens

Co-stimulation of exogenous CRAMP and bacterial supernatant NM induced increase of HO-1 immunofluorescence in CRAMP-KO microglial cells. Microglial cells from CRAMP-WT or KO mice were incubated with 1, 2 or 10 μM mouse CRAMP with or without supernatant of NM for 6 h. After incubation cells were fixed and immunolabeled using anti-HO-1 antibody (green) and nuclear counterstaining DAPI (blue) and examined with fluorescence microscopy. The figure shows representative results from three independent experiments. Scale bar = 20 μm. (TIFF 3834 kb