Comparison of T cell subsets in CSF and blood of HIV-infected and uninfected subject.

<p>(A) Relation of CD4+ T cells present in the CSF to those present in the blood amongst the 4 subject groups with regression line (solid line; slope  = 1.395, r² 0.7608, p<0.0001) and 95% confidence intervals (dotted lines). (B) Relation of CD8+ T cells present in the CSF to those present in the blood amongst the 4 subject groups with regression line (slope  = 0.9768, r² 0.5794, p<0.0001) and 95% confidence intervals. (C) Comparison of CSF and blood CD4:CD8 ratio between the 4 subject groups.</p

ART influences the absolute lymphocyte subset counts present in the CSF of HIV-infected subjects.

<p>Absolute number of CSF monocytes (A), CD3+ T (B), CD4+ T (C), CD8+ T (D), B (E) and NK (F) cell subsets present in CSF of the 4 patient subgroups <i>(Off, Rx Viremic, Rx VL<500</i> and <i>HIV-uninfected)</i> are shown. Median and interquartile range for each cell population are shown in the box plots. Whiskers are set at 10–90%. Mean values are indicated by (+). ANOVA for significant overall differences between the 3 HIV-infected treatment groups was conducted, with the p value depicted at the top of each panel. Post-hoc analysis using Dunn’s test comparing differences between pairs of groups, if statistically significant, are as indicated in each panel.</p

HIV-infected and uninfected subjects have distinct blood and CSF lymphocyte proportions and counts.

<p>Proportions of CD3+, CD4+ and CD8+ T cells; B cells; and NK cells present in the blood (A) and CSF (B) of HIV-uninfected (HIV-) and HIV-infected (HIV+) subjects are depicted as a percentage of total lymphocyte population. Absolute CSF cell counts of lymphocyte subsets for HIV-uninfected and HIV infected (C) subjects are shown. Median and interquartile range for each cell population are shown in the box plots. Whiskers are set at 10–90%. Mean values are indicated by (+).</p

Proportion and Absolute Count of CSF Populations.

*<p>calculated using Mann-Whitney U-test.</p

Flow cytometry gating strategy to define blood and CSF white blood cell (WBC) populations.

<p>Data was compensated and gated using FlowJo to define TruCOUNT™ beads, (i) from both blood (left panel) and CSF (right panel) data. Remaining events were displayed in a CD45 vs FSC plot (ii) to gate out debris and define a total WBC population. CD14 was used to gate monocytes (iii) from the total WBC gate, and CD14− cells were displayed on a CD45 vs SSC plot (iv) to define lymphocyte and granulocyte gates. Lymphocytes were subdivided into CD3+ T cells and CD3− lymphocytes (v), CD3− lymphocytes were displayed on a CD19 vs CD56&16 (vi) plot to define B cells and NK cells respectively. CD3+ T cells were displayed on a CD4 vs CD8 plot (vii) to define CD4+ and CD8+ T cells.</p

Background Characteristics of Study Subjects.

<p>SD = standard deviation.</p><p>IQR = interquartile range.</p><p>NA = Not applicable.</p

Additional file 1: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) The 3F4-tag in PrPC does not alter the ADAM10-mediated shedding. Western blot analysis of forebrain homogenates comparing PrPC shedding between mice expressing endogenous wild-type PrPC (PrPCWT) and knock-in mice expressing 3F4-tagged PrPC instead (PrP3F4KI). Quantification was done by referring the sPrP signal to the respective fl-PrP signal (POM2 Ab) of the re-probed blot and is shown on the right (n = 3). As in other parts of this study, forebrain homogenates of Prnp0/0, ADAM10 cKO and tga20 mice served as specificity controls. The position of air bubbles on the membrane (indicated by arrows) further supports the slight molecular weight shift between sPrP and fl-PrP described in Fig. 1b. To prove genotypes of PrPCWT and PrP3F4KI mice, in a parallel blot shown below, PrPC was first detected with an antibody directed against the 3F4 epitope and re-probed with POM2. (JPEG 225 kb

Additional file 1: Figure S1. of Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site

Antibodies 192wt and 7a6 only detect sAPP in brain soluble fractions of wildtype mice. To test the specificity of the antibodies 7A6 and 192wt we compared brain soluble fractions of wildtype and APP ko mice. Here, we could show that by using the corresponding antibodies both sAPPα and sAPPβ are detectable in brain soluble fractions of wildtype, but are lacking in those of APP ko. Since APLP2 can also be detected in APP ko brain membrane fractions, we show that the knock-out is restricted to APP and does not involve other members of the APP family. (PDF 646 kb

Additional file 4: of Structural and mechanistic aspects influencing the ADAM10-mediated shedding of the prion protein

(.jpg) Differences in the glycopattern between total and cell surface PrPC in N2a cells. (A) Western blot analysis and (B) densitometric quantification of glycoform proportions of total (cell lysates) versus cell surface fl-PrP (biotinylated samples) using POM2 antibody for detection. Absence of actin and almost exclusive expression of mature ADAM10 (with almost no premature ADAM10) in the biotinylated samples confirm technical soundness of the assay (the upwards shift of ADAM10 in gel likely results from the assay protocol). Quantification reveals that the fraction of diglycosylated PrP at the cell surface is increased compared to total PrP in cell lysates (diglycosylated: 68.0 ± 0.7% (surface PrP) vs. 55.1 ± 2.4% (total PrP); monoglycosylated: 23.5 ± 0.8% (surface PrP) vs. 29.3 ± 0.6% (total PrP); unglycosylated: 8.5 ± 0.2% (surface PrP) vs. 15.5 ± 1.9% (total PrP); n = 3; ±SD). (JPEG 610 kb

Additional file 4: Figure S4. of Generation of aggregation prone N-terminally truncated amyloid β peptides by meprin β depends on the sequence specificity at the cleavage site

Meprin β prefers cleavage of wt APP compared to APP A673T. 15 nM recombinant meprin β was incubated with synthetic APP peptides for different time intervals. HPLC analysis showed that processing kinetics of APP A376T (F-J) were decreased compared to wt APP (A-E). Occurring peaks with smaller retention times were identified as cleavage products by MALDI MS. Peak intensity has been measured at 214 nm. (PDF 1715 kb