Additional file 1: of Indomethacin promotes survival of new neurons in the adult murine hippocampus accompanied by anti-inflammatory effects following MPTP-induced dopamine depletion

Figure S1. Effects of dopamine depletion and indomethacin treatment on the stages of adult hippocampal neurogenesis. Neuronal development originates from a Nestin-positive, triangular-shaped stem cell (type 1). Then, neurogenesis progresses over the stages of the putative progenitor cells (type 2a, type 2b, and type 3) and ends in the NeuN-positive mature granule cell. Neurotoxic treatment leads to a decreased number of newly generated (type 2a cells) and mature neurons, whereas indomethacin treatment afterwards promotes the development towards mature neurons. MPTP: 1-methyl-4-(2′-methylphenyl)-1,2,3,6-tetrahydropyridine hydrochloride; NeuN: neuronal nuclei. (TIF 624 kb

Summary of the changes in cytokine expression.

<p>Cytokine release was measured 24 hrs or 3 days after incubation of normal PBMCs with isu-peptide homopolymers alone or in the presence of a mitogen, respectively. The full name of the abbreviated molecules is given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Donor-dependence of IL-10 and MMP-1 release.

<p>a, PBMCs from three donors were incubated with the same batch of the isu peptide polymer or medium and IL-10 release and MMP-1 mRNA expression were measured simultaneously. The donor-dependence of IL-10 release was shown using PBMCs from more than 50 donors some are shown in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>. b, Kinetic date of IL-10 release from PBMCs from a high responder donor A (column 1, dark grey) and low responder donor B (column 3, dark grey), both treated with the isu peptide homopopymer. Untreated medium control cells from both donors (Colum 2 and 4, light grey) did not release IL-10. The p values were calculated using the Student’s t-test, n = 3, p = 0.00015 in the case of a high responder and p = 0.03 in the case of a low responder.</p

Influence of the isu peptide on cytokine release and gene expression.

<p>(a) PBMCs were incubated for 24 hrs with or without the isu peptide homopolymer. The cytokine array VI (see Material and Methods) was used. The up-regulated cytokines are circled. (b) PBMCs were treated with Con A and incubated with and without the isu peptide homopolymer for three days and the cytokine array I was used. The down-regulated cytokines are circled. Similar results were obtained with PBMCs from ten other donors. (c) Cytokine array measuring simultaneous release of ten different cytokines after incubation of PBMCs from one donor with and without the isu peptide homopolymer at different time points (6 to 24 hrs), confirming and extenting the results shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g002" target="_blank">Figure 2a</a>. Control PBMCs were incubated with medium. Compare the increase in IL-10 expression with that in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3a</a>. (d) Genes with the highest up-regulation (upper part) and down-regulation (lower part) of expression in PBMC of one donor in response to the isu peptide treatment. Using specific real-time PCRs for the up- and down-regulated genes, the changes were confirmed in PBMCs of other donors (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g003" target="_blank">Figure 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone-0055199-g004" target="_blank">4</a>, Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s002" target="_blank">Figure S2</a>). Fold changes (Fc) indicates gene expression compared to control cells incubated in medium. The full names of the genes are given in Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055199#pone.0055199.s008" target="_blank">Table S4</a>.</p

Kinetics of the modulation of cytokine release and gene expression induced by the isu peptide.

<p>a, Kinetics of the IL-10 and IL-6 release and expression of IL-10 and IL-6 mRNA. PBMCs were incubated with (gray) or without (light gray) isu peptide homopolymers and supernatants as well as mRNA were collected at different time points between 0–24 hours. The p values were calculated using the Student’s t-test, n = 3. When comparing the IL-10 release induced by the isu peptide homopolymer and that by medium alone, the p value at the peak release (15 hrs) is p = 2.16E-05, the p value for IL-10 RNA at 24 hrs is p = 0.09. All other values were accordingly. b, c, Kinetics of the expression of MMP-1, TREM-1, FCN1, CXCL9 and SEPP-1 in PBMCs incubated with (dotted line) or without (straight line) isu-peptide homopolymers. The figures show a representative result obtained with PBMCs from more than five donors. The p values were calculated using the Student’s t-test, n = 3, the p-value of sTREM-1 release at 24 hrs is p = 0.02.</p

Localisation and activity of the immunosuppressive (isu) domain of gp41 of HIV-1.

<p>(a) Functional domains in gp41 of HIV-1 (accession-nr. NCBI K03455): FP, fusion peptide; FPPR, fusion peptide proximal region; NHR, N-terminal helical region; ISU, isu domain; S-S, cystein-cystein loop; CHR, C-terminal helical region; MPER, membrane proximal external region; MSD, membrane spanning domain, 3S, domain binding to the gC1qR and inducing NKp44L expression. In the amino acid sequence of the isu domain stars (*) indicate NH₂-groups, points (.) mark COOH groups relevant for polymerisation. (b) Localisation of the isu domain after interaction of the NHR with the CHR generating a six helix bundle (only one molecule of the trimer is shown). (c) Influence of the isu-peptide on the proliferation of PHA stimulated PBMCs from a healthy donor. Cell proliferation was measured by ³H-thymidine incorporation. ³H-thymidine was added on day zero, one, two or three and cells were then harvested one the next day and the counts per minute were determined, gray - medium control, dark gray – isu peptide-BSA conjugates, added at day 0. (d) Dose dependent induction of IL-6 and Il-10 release by the isu peptide homopolymer (triangle) as measured in ELISAs. In contrast, the amidated control peptide (square) is inactive. (e) Comparative ELISA analysis of IL-10 release from PBMCs of seven donors all treated with the same amount and batch of the isu-peptide homopolymer, the IL-10 release of their PBMCs incubated with medium alone was zero. The p values were calculated using the Student's t-test, n = 3. The p value for the high responder donor 1 was p = 0.001, that of the low responder donor 7 p = 0.03.</p