Fig 2: Effects of taurine treatment on microglial cells in brain tissue. Representative immunohistochemical staining images of Iba-1 (microglia stained in brown) showing microglial activation with quantitative analyses of activated microglia in the hipppocampus (A, top row, bar = 100 µm; bottom row, bar = 20 µm, enlarged from the dashed line box) and cortex (B, top row, bar = 100 µm; bottom row, bar = 20 µm, enlarged from the dashed line box). Nuclei were counterstained with hematoxylin. Quantification of the immunohistochemistry data are expressed as the means ± SD of 3–4 hippocampal fields from three to four SAMP8 mice and 3–4 specific cortex areas from three to four SAMP8 mice of each group. Representative immunohistochemical staining images of TNF-alpha positive area in hippocampus (C, top row, bar = 200 µm; bottom row, bar = 100 µm, enlarged from the dashed line box) and TNF-alpha positive cells in cortex (D, top row, bar = 100 µm; bottom row, bar = 50 µm, enlarged from the dashed line box). Nuclei were counterstained with hematoxylin. Quantification of the immunohistochemistry data are expressed as the means ± SD of 4 hippocampal fields from four SAMP8 mice and 4 specific cortex areas from four SAMP8 mice of each group. *p < 0.05 and **p < 0.01 by Student’s t-test.

Fig 3: Colocalization of inflammatory molecules with neuronal and glial markers in CA3 region of 3xTg-AD mice. Double immunofluorescence staining for (A–C) TNF, (D–F) COX-2, and (G–I) iNOS with MAP2, GFAP, and IBA1 in the hippocampal CA3 region in 3xTg-AD mice. The results demonstrated that TNF-α, COX-2, and iNOS were detected in the cytoplasm of neurons (labeled with MAP2), astrocytes (labeled with GFAP), and microglial cells (labeled with IBA1), as indicated by the white arrowheads. TNF-α, COX-2 and iNOS (red); MAP2, GFAP, and IBA1 (green); and DAPI (blue) (nuclei). Bar = 50 μm.

Fig 4: Increased immunoglobulin production and antibody diversity in HS skin and complement activation.Box-and-whisker plots of BCR CDR3 expressions (A). The y axis shows normalized log-transformed BCR CDR3 expression. The x axis represents patient group. In all cases there were more BCR CDR3 sequences detected in HS skin compared with control healthy skin. Box-and-whisker plots of BCR gene segment expression. The y axis shows normalized log-transformed BCR gene segment expression. The x axis represents patient group. The Shannon diversity index for BCR CDR3 gene segment is plotted on the y axis. The x axis represents patient group. HS skin had a significantly more diverse BCR repertoire (B). Beta diversity–based principal coordinates analysis (PCoA) of BCR CDR3 sequences. Sample matrix was generated using Jaccard dissimilarities, and respective profiles were compared by PCoA. Each color represents 1 patient group, HS (red) and control (blue). This analysis revealed clear separation for κ and λ light chains but not Ig heavy chain (C) Hierarchical clustering of expressed TCR V/J gene segment expression. Heatmaps by clonal abundance across sample sets. Note good separation of HS from controls based upon clonal abundances in BCR κ and λ repertoires. Components of the complement pathway (C1q) and breakdown products of activated complement components (C3b, C4d) were increased in HS skin, particularly in the deeper layers of the skin (n = 3) (scale bar: 100 μm) (D). Complement receptors, CR1 and CR2, were increased in the deeper layers of HS, along with IgG1 immune complex deposition (n = 3) (scale bar: 100 μm) (E). Immunofluorescence of B cells (CD20) and plasma cells (CD138) showed primary localization of TNF to the plasma cell population in HS skin (n = 3) (scale bar: 50 μm) (F). For A and B, the bold vertical line represents the median, and the upper and lower limits of the box represent the interquartile range (IQR). The whiskers represent 1.5× IQR.

Fig 5: PDGF-BB as a factor for smooth muscle cells to migrate, dedifferentiate and express pro-inflammatory genes. (A) Expression of PDGF-BB or Amphiregulin (AREG) in endothelial cells of intracranial aneurysm (IA) lesions. On the 14th day after IA induction, IA lesions at the right anterior cerebral-olfactory artery bifurcation were harvested and subjected to the immunohistochemical analyses. The representative images of immunohistochemistry for PDGF-BB or AREG (green), Cadherin 5, a marker for endothelial cells, (red), nuclear staining by DAPI (blue) and merged images are shown. The magnified images corresponding to the square in the upper panels are shown in the lower panels. Bars, 10 µm. (B) Chemotactic activity of PDGF-BB or AREG on smooth muscle cells (SMCs). The migration of primary culture of SMCs across matrigel-coated pores via 100 ng/ml PGDF-BB or 100 ng/ml AREG was assessed by a transwell system. The representative images of SMCs migrated are shown. Bars, 10 µm. The number of migrated cells is shown in the lower graph. Data represents the mean ± SEM (n = 4). Statistical analysis was done by a Kruskal–Wallis test. *p < 0.05. (C) Dedifferentiation of SMCs by PDGF-BB. Primary culture of SMCs were stimulated with 100 ng/ml PGDF-BB or 100 ng/ml AREG for 72 h and expression of SMA was assessed by western blot analysis using α-tubulin as an internal control. The representative images are shown. (D,E) Induction of PTGS2 (COX-2) or IL6 by PDGF-BB in cultured SMCs. Primary culture of SMCs were stimulated with vehicle (V), 100 ng/ml PGDF-BB (P) or 100 ng/ml AREG (A) (D) or each dose of PDGF-BB (E) for 1 h and expression of TNF, CCL2, PTGS2 or IL6 was examined by quantitative RT-PCR analysis. Data represents the mean ± SEM (n = 4). Statistical analysis was done by a Kruskal–Wallis test. *p < 0.05.