13 research outputs found
Heatmap of glycolipid complex binding in GBS and control sera.
<p>Clustered heatmap for the 35 antigens significantly associated with GBS (arranged on the x-axis), comparing the 181 GBS sera and 161 control sera (on the y-axis). Each row represents a single serum. The log transformed arbitrary binding intensity for each antigen is represented by the colour scale, from red (log transformed arbitrary intensity of 18, most intense) to the green background (log transformed arbitrary intensity of 0, no binding), as shown at the top of the figure. Each heatmap panel represents a separate group, as labelled. (GBS, Guillain-Barré syndrome; MS, Multiple Sclerosis; ONND, Other Non-Inflammatory Neurological Diseases; HC, Healthy Controls). Blue boxes delineate clusters of GM1 and sulfatide-complex antigens. Arrows mark sulfatide complexes which cluster separately. </p
GM1 complex antigens.
<div><p>(<b>A</b>) Individual value plots for selected antigens.</p>
<p>(<b>B</b>) Association between sulfatide:GM1 and GM1 binding intensities in individual sera. Points on the y-axis are GM1 negative, sulfatide:GM1 positive. Sera close to the diagonal blue line (joining points of identical binding intensity for both antigens) bind both GM1 and sulfatide:GM1 complex with similar intensity. </p>
<p>(<b>C</b>) Association between phosphatidylserine:GM1 and GM1 binding intensities in individual sera. Points on the y-axis are GM1 negative, PS:GM1 positive. Sera on the x-axis are GM1 positive, PS:GM1 negative. </p>
<p>(<b>D</b>) Association between phosphatidylserine:GM1 and sulfatide:GM1 binding intensities in individual sera.</p></div
Complex enhanced binding.
<div><p>(<b>A</b>) Receiver operator curves for single ganglioside antigens, single glycolipids, glycolipid complexes, and for all 181 antigens. Complex antigens (dashed green line) afford the best trade-off between sensitivity and specificity.</p>
<p>(<b>B</b>) Individual value plots for GBS sera binding intensity to single sulfatide (sul), single CTH, the sum of these single intensities (sulfatide+CTH), and CTH:sulfatide complexes.</p>
<p>(<b>C</b>) Comparison between the sum of single sulfatide and CTH binding intensities (sul+CTH) and binding intensity to CTH:sulfatide complexes in individual sera. Each line links the respective values in a single serum. Lines sloping upwards from left to right indicate complex enhanced binding with a single serum.</p>
<p>(<b>D</b>) Overview heatmap of corrected complex binding intensities. The intensity value for each complex has been corrected by subtracting the binding intensities of each component glycolipid. Any residual intensity indicates complex enhanced binding. As before, GBS sera lie above and healthy control (HC) sera below the horizontal white line. The same colour scale as Figure 1 has been used. A more detailed, zoomable version of this heatmap, allowing resolution of results for each complex antigen and each serum, is available online (Figure S3).</p></div
Both sialylated and non-sialylated <i>C. jejuni</i> are scavenged by macrophages in the splenic marginal zone.
<p>Mice were injected with CFSE-labelled GB2 wt (A and C) or Cst-II mutant (B) bacteria and spleens were isolated after 1 h. Spleen sections were stained for sialoadhesin (A and B) or CD209b (C and D) and counterstained for B220 (purple). Insets show colocalisation of GB2 wt and ko bacteria with sialoadhesin and CD209b positive macrophages.</p
Sialylation of <i>C. jejuni</i> increases <i>in vivo</i> phagocytosis by splenic DC but not macrophages.
<p>Mice were sacrificed 1 h or 3 h after i.v. injection of PBS or CFSE-labelled GB2 (wt or Cst-II mutant). Bacterial deposition in spleen sections, stained for sialoadhesin, CD209b and B220, was quantified as detailed in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034416#s4" target="_blank">Materials and Methods</a> section. The CFSE-positive area of digital images was similar between GB2 wt and Cst-II mutant injected animals (A). No differences were observed in the CFSE-positive area within MOMA1 (B) or CD209b-positive cells (C). For each mouse, 6 pictures from 2 spleen sections were used for analysis (n = 4). Using Facs-analysis, the number of CFSE-positive metallophilic macrophages (Sialoadhesin+), red pulp macrophages (F4/80+), DC (CD11c+) and neutrophils (Ly-6G/C+) was determined per spleen (n = 3; D). Shown are means ± sd. * p<0.05; t-test with Welch's corrections.</p
Sialylated GB11 LOS induces higher cytokine levels by myeloid cells.
<p>BM-MΦ (A, C and E) and BM-DC (B, D and F) were incubated overnight (for IL-6 and IL-10) or for 4 h (IFN-β) with purified LOS derived from GB11 <i>C. jejuni</i> or Cst-II mutant <i>C. jejuni</i> and cytokine production was measured by ELISA. Sialylated LOS induces more IL-10 (A and B), IL-6 (C and D) and IFN-β (E and F) than non-sialylated LOS. Graphs represent pooled data as mean ± standard error from 3 (IL-10 and IFN-β) or 4 (IL-6) independent experiments. * p<0.05; paired t-test, n.d.: not detectable.</p
Increased type I interferon production in the spleens of GB2 wildtype injected animals.
<p>Mice were injected with PBS, GB2 wildtype or Cst-II mutant bacteria and spleens were isolated after 1 h. Spleen sections were homogenised for RNA extraction and cytokine expression was analysed by real-time PCR. Data is expressed as fold increase (mean ± sd; n = 4) compared to PBS-injected control animals. * p<0.05, Mann-Whitney U test.</p
Increased phagocytosis of sialylated <i>C. jejuni</i> by BM-MΦ.
<p>(A) BM-MΦ or (C) BM-DC were incubated with CFSE-labelled wt or Cst-II mutant GB11 for 60 min at 37°C (open histograms) or 4°C (filled histograms). Phagocytosis of <i>C. jejuni</i> by F4/80+ cells or CD11c<sup>+</sup> cells was quantified as fold increase in fluorescence intensity using background fluorescence of 4°C, NaN<sub>3</sub> cultured cells as a reference. Phagocytosis of sialylated wt GB11 and unsialylated GB11 for 0, 15, 30 and 60 min shows that sialylation increases <i>C. jejuni</i> phagocytosis by BM-MΦ (B) but not DM-DC (D). One representative experiment out of 3 is shown (B and D; means ± sd of triplicates). * p<0.05; t-test.</p
Serum samples from patients with GBS and from controls with other neurological disease (OND) and family controls (FC) were tested for IgG activity to lipo-oligosaccharides (LOS) from 6 <i>C. jejuni</i>.
<p>Subcharts represent serum IgG activity to LOS from (<b>A</b>) <i>C. jejuni</i> BD-07 isolated from a GBS patient (<b>B</b>) <i>C. jejuni</i> BD-39 isolated from a GBS patient, (<b>C</b>) <i>C. jejuni</i> BD-10 isolated from a GBS/MFS patient, (<b>D</b>) <i>C. jejuni</i> BD-67 isolated from a GBS/MFS patient, (<b>E</b>) <i>C. jejuni</i> BD-94 isolated from a GBS patient (<b>F</b>) <i>C. jejuni</i> Penner HS:03 serostrain (CCUG 10937), lacking ganglioside mimics, as a control. *p<0.001. Lines () indicate cut-off value. GBS, Guillain-Barré syndrome; OND, other neurological disease; FC, family controls; LOS, lipo-oligosaccharides.</p
Binding of mouse monoclonal antibodies against LOS from <i>C. jejuni</i> BD-07 and BD-39.
<p>DG-1 (binding to GM1), TBG-3 (binding to GD1a), EG-7 (binding to GD1b), EG-3 (binding to GQ1b) and EG-1 (binding to GQ1b/GT1a).</p