Validation and characterization of the inverted <i>in vitro</i> model of human FAE.

<p>(a) Schematic representation of the successive steps leading to the establishment of the M-like cell-containing Caco-2 cell monolayer. Molecular and cellular partners added as a function of the experimental setting are depicted. D, day. (b) Slight decrease of transepithelial electrical resistance (TEER) was observed during M cell conversion of polarized Caco-2 cell monolayer. (c) Localization by immunofluorescence of ZO-1 and CA19.9 in the mono- and co-culture models shows tight intercellular junctions around CA19.9⁺ M cells (top view). (d1) Identification of M-like cells by transmission electron microscopy (TEM). In co-culture, M-like cells were identified by the effacement of microvilli at the apical surface and the presence of enfolded lymphocytes (side view). Mono-cultures present a columnar shape as well as a brush border. (d2) Scanning electron microscopy (SEM) confirmed the presence of M-like cells and the lack of microvilli at their apical surface (top view). (e1) Identification and localization of M-like cells and enterocyte cells by immunofluorescence staining with anti-CA19.9-PE mAb and lectin UEA-1-FITC, respectively (top view). (e2) Co-localization evaluated by immunofluorescence microscopy of CA19.9-PE and IgA2-GFP indicates binding of the Ab to M-like cells (top view). (f) Increased apical-to-basolateral transport of 0.2 µm yellow-green-conjugated NPs across the co-cultures as compared to mono-cultures. Absence of transport at 4°C is indicative of an active membrane trafficking process. Each set of experiments was repeated at least twice.</p

Influence of glycosylation and sialylation on the uptake of m-IgA2 by M-like cells.

<p>(a) Mono- and co-cultures were incubated for 90 min at 37°C with various hypoglycosylated, or enzymatically deglycosylated, IgA2 constructs, and transported recombinant IgA (m-IgA2) was evaluated by associated luminescence (<i>n</i> = 4). PNGase, Peptide N:glycosidase; Neura, neuraminidase. (b) Same set of experiments performed with recombinant anti-CD20 m-IgA2, human colostrum SIgA, and plasma-purified IgA; transported IgA was measured by ELISA. (c) IgA deglycosylation and desialylation was monitored by Western blot analysis using detection with HRP-conjugated lectins Ulex europaeus-1 and wheat germ agglutinin, respectively. Lane content: 1, m-IgA2 G0; 2, m-IgA2 G2; 3, m-IgA2; 4, colostrum SIgA+PNGase; 5, colostrum SIgA.</p

IgA2 interacts with Dectin-1 and Siglec-5–expressing cells.

<p>(a) Recognition by m-IgA2 of recombinant Dectin-1 and Siglec-5 used as coating molecules (<i>n</i> = 6), as measured by ELISA. (b) Determination by immunofluorescence of the binding of m-IgA2 to Dectin-1 and Siglec-5 receptors expressed by double-transfected HEK cells. Co-localization resulted in the appearance of yellow areas in the cell periphery. In control experiments, no binding of IgA1 was detected, and nontransfected HEK cells did not stain (<i>n</i> = 2). (c) In flow cytometry analysis, cells were plotted according to the FSC and SSC profiles and gated to include only HEK cells. A second selection was performed to include only those cells positive for Dectin-1 and Siglec-5.</p

Dectin-1 on M cells is essential for SIgA uptake and elicitation of Ab responses against an associated Ag.

<p>(a) Incubation of p24-SIgA (red, indicated by arrowheads) for 90 min in a ligated intestinal loop containing a PP resulted in the targeting of the complex to Dectin-1⁺ M cells in the FAE, as indicated by the appearance of numerous yellow spots. V, villi. (b) Oral immunization of wt mice and chimeric-wt:KO (wt mice reconstituted with bone marrow cells from Dectin-1 KO mice) with HIVp24-SIgA resulted in the production of antigen-specific seric Abs, whereas Dectin-1 KO mice and chimeric-KO:wt mice (Dectin-1 KO mice reconstituted with bone marrow cells from wt mice) did not respond. (c) An identical pattern of Ag-specific Ab production was observed in the feces of immunized mice. Samples were collected 1 wk after the last immunization and Ab production was measured by ELISA. Horizontal bars show the mean value ± SEM (*<i>p</i><0.05).</p

Antibody production.

<p>(a) Schematic representation of recombinant IgA2 constructs bearing GFP or Luciferase (Luc) at the N terminus. Numbers in brackets correspond to the lanes of gels depicted in (b) and (c). Glycosylation sites on asparagine residues (N) are indicated in rectangles connected to alpha chain domains. (b) Recombinant Igs separated by SDS-PAGE under reducing conditions and stained with Coomassie blue. (c) Analysis of a selection of recombinant Igs by SDS-PAGE under nonreducing conditions and stained with Coomassie blue. The identification of the various assembled forms was determined based on immunodetection with Abs specific to the alpha and kappa chains, respectively (not shown). 1, human m-IgA2 (monomer); 2, human d-IgA2 (dimer carrying the J chain); 3, murine m-IgA (monomer); 4, human IgE; 5, human IgG; 6, human m-IgA1 (monomer); 7, human mI-gA2 (monomer); 8, human m-IgA2 G0 (no glycosylation – monomer); 9, human m-IgA2 G1 (1 glycosylation site (Asn²⁶³) – monomer); 10, human m-IgA2 G2 (2 glycosylations sites (Asn²⁶³ and Asn⁴⁶⁹) – monomer); 11, human m-IgA2 (monomer); 12, human m-IgA2 dPB (monomer lacking the basal part); 13, human m-IgA2 dCα3 (monomer lacking the basal part and Cα3); 14, human m-IgA2 dCα2/3 (monomer lacking the basal part, and domains Cα3 and Cα2); 15, human m-IgA2 Cα1 G0 (Cα1 domain only, no glycosylation); 16, human m-IgA2 Cα1 (Cα1domain only).</p

Relative frequencies of tumour biopsies in the main diagnostic groups for CNS tumours as compared to their frequencies in the whole of Sweden.

<p>*according to the “classification scheme for childhood cancer” <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008239#pone.0008239-Birch1" target="_blank">[47]</a>.</p><p>**from the Swedish Childhood CNS Tumour Working Group (VCTB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008239#pone.0008239-Lannering1" target="_blank">[44]</a>.</p>§<p>Patients with tumours that can be due to inheritable diseases.</p>1<p>3/11 patients had Giant cell astrocytoma with Tuberous Sclerosis, and 1/4 had Pilocytic astrocytoma with Neurofibromatosis type I.</p>2<p>1/2 patients had Leptomeningeal oligodendromatosis.</p>3<p>this patient had Lumbar Schwannoma with Neurofibromatosis type II.</p

Structural mapping of sugars affecting transport of IgA2.

<p>(a) Mono- and co-cultures were pre-incubated apically with 5 mg of different mono/polysaccharides and sialic acid, prior to addition of various m-IgA2 constructs for 90 min at 37°C. The concentration of transported IgA was evaluated by associated luminescence (<i>n</i> = 4). (b) Similar experiments performed with 10 µg of blocking Abs directed against potential receptors (<i>n</i> = 4). Detection of Dectin-1 and Siglec-5 on M-like cells <i>in vitro</i> assessed by (c) immunofluorescence (top view), (d) flow cytometry, and (e) Western blot analysis using specific mAbs. Only cells or lysates recovered from co-cultures stained positive.</p

Specific transport of IgA2-Luc across the <i>in vitro</i> model of human FAE and in a mouse ligated loop containing a PP.

<p>(a) Mono- and co-cultures were incubated for 90 min at 37°C with various IgA2 constructs, and transported IgA was evaluated by associated luminescence (<i>n</i> = 4). The transport of m-IgA2, d-IgA2, d-IgA2+SC, m-IgA2 Ca1, and m-IgA2 dCα2/3 was significantly promoted in the co-cultures compared with the mono-cultures. (b) Only murine SIgA-Cy3, but not IgG2a-PE, incubated for 60 min in a mouse ligated intestinal loop is efficiently transported in underlying lymphoid tissues. Dotted lines delineate the interface between the intestinal lumen and tissue (FAE, side view). SIgA or IgG2a positive cells were quantified from three individual PPs.</p

Characteristics of patients and their CNS tumours.

1<p>Age at diagnosis/age at operation (* relapsing tumour).</p>2<p>Multiple nodules.</p

Specific uptake and transport of SIgA across murine and human FAE.

<p>(a) Mouse SIgA-Cy3 incubated for 60 min in a ligated intestinal loop containing a PP was taken up by M cells. Co-localization was observed with both UEA-1-FITC (a1) and GP2-FITC (a2) (<i>n</i> = 3). (b) Colocalization of SIgA-Cy3 with the Dectin-1 receptor stained in green with a specific mAb in the FAE overlying the PP (<i>n</i> = 2). (c) No entry of SIgA-Cy3 occurred in Dectin-1 KO mice (<i>n</i> = 2). (d–f) Images obtained from patient biopsy samples taken from the distal duodenum. V, villi. Biopsies were immunolabeled with human GFP-IgA2 and Dectin-1-PE (d) or Siglec-5-PE (e) at room temperature for 2 h. Conspicuous co-localization between GFP-IgA2 and Dectin-1-PE or Siglec-5-PE was observed (<i>n</i> = 2). (f) Negative controls were stained with secondary Abs alone (<i>n</i> = 2). On all pictures, dotted lines delineate the FAE separating the intestinal lumen and the lymphoid tissue (side view).</p