Abundant surface and cytoplasmatic FcεRI α-chain expression only in subconfluent human intestinal tumor cell lines.

<p>Immunofluoresence staining for (A–H) FcεRIα is performed in (A, B) subconfluent and (C, D) confluent Caco2/TC7 as well as in (E, F) subconfluent and (G, H) confluent HCT-8. Triton-X-100 permeabilized (A, C, E, G) and untreated cells (B, D, F, H) were compared. (I–L) Representative control staining in subconfluent Caco2/TC7 with the (I, J) anti-lamin A/C or (K, L) unspecific murine IgG2b, (I, K) permeabilized or (J, L) untreated. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×40.</p

Expression of FcεRI α- and γ-chain but not β-chain mRNA in human intestinal epithelial cells.

<p>The expression pattern of the FcεRI complex was analyzed by real-time PCR analysis using specific primers for detection of (A) FcεRI α-, β- (not shown), and (B) γ-chain. Target gene expression levels were normalized to the average of housekeeping genes and are depicted relative to the value of subconfluent Caco-2/TC7 cells. The values are presented as means +/− SD (n = 3) from one experiment. The results are representative of two independent experiments.</p

Co-expression of FcεRI α- and γ-chain, but not FcεRI α- and β-chain is observed in epithelial cells of intestinal tissue.

<p>In sections from cancer patient No. 23 FcεRI γ-chain (green) is found to be co-expressed in FcεRIα (red) positive epithelial cells of (A) the small intestine, as well as in (B) colon tissue and (C) tumor sample. In addition, sections from CD patient No. 8 are double-positive for FcεRI α- and γ-chain in (D) the small intestinal, (E) colon and in (F) lesional tissue. (G) Only in the subepithelial tissue FcεRI β-chain (green) positive cells are detected as shown here in the crypts of small intestinal tissue. (H) Negative control with mouse IgG2b and goat IgG isotype control antibodies. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×64.</p

FcεRI β- and γ-chain staining results in FcεRI α-chain expressing intestinal tissue.

<p>n.a., not applicable;</p

Positive staining for FcεRI α-chain and defensin-5 is observed in serial sections from intestinal tissue.

<p>(A) FcεRI α-chain is detected on the membrane, as well as in the cytoplasm of epithelial cells in small intestine of cancer patient No. 16. FcεRI α-chain positive cells are also found in (B) the colon and (C) a tumor sample from the same patient. (D) Staining with anti-defensin-5 antibodies confirmed that FcεRIα expressing cells at the basis of the small intestinal crypts are Paneth cells. Defensin-5 is expressed also in (E) colon and (F) tumor sample in same areas, but to a lesser extend when compared with FcεRI α-chain staining. Similar staining pattern are observed also for the CD patient No. 8, as FcεRI α-chain positive cells are detected in (G) the epithelium of small intestinal tissue, (H) colon and (I) lesional region. Defensin-5 positive cells are located at (J) the crypt basis of small intestine, (K) along the colon crypt, as well as in (L) the lesional region. Original magnification ×10, inset ×40.</p

Patients' characteristics and staining results.

<p>n.d., not determined; n.a., not applicable; Pc, Paneth cell staining; M, Membrane staining; sV, single cell staining in villi; V, all villus epithelium.</p

Western blot analysis reveals FcεRI in human intestinal tumor cell lines.

<p>(A) FcεRI α-chain and (B) FcεRI γ-chain expression is investigated in Caco2/TC7 and HCT8 subconfluent and confluent cell lines. In all experiments RBL cells transfected with the human FcεRI receptor served as positive controls and the protein expression signal was normalized to the expression of house-keeping protein actin.</p

Abundant FcεRI γ-chain expression and IgE binding in subconfluent human intestinal tumor cell lines.

<p>Immunofluorescence staining of (A, D, G, J) FcεRI β- and (B, E, H, K) FcεRI γ-chain are performed in (A–C) subconfluent and (D–F) confluent Caco2/TC7 and in (G–I) subconfluent and (J–L) confluent HCT-8. (C, F, I, L) According to the expression pattern of FcεRI in the subconfluent cells, IgE binding is observed exclusively in subconfluent, non-mature intestinal cells. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×40.</p

FcεRI α-chain is expressed in epithelial cells.

<p>Co-staining with anti- FcεRI α-chain (red) and anti-keratin 8 antibodies (green) verifies the epithelial expression of FcεRI in (A) the crypts of small intestinal section, where FcεRIα is found primarily in the supranuclear region, in (B) colon tissue and in (C) tumor sample of cancer patient No. 16. (D) Negative control with mouse IgG2b and rabbit IgG. The blue fluorescence DAPI staining indicates the nuclei. Original magnification ×40.</p

Nitration of β-Lactoglobulin but Not of Ovomucoid Enhances Anaphylactic Responses in Food Allergic Mice

<div><p>Background</p><p>We revealed in previous studies that nitration of food proteins reduces the risk of <i>de novo</i> sensitization in a murine food allergy model. In contrast, in situations with preformed specific IgE antibodies, <i>in vitro</i> experiments suggested an increased capacity of effector cell activation by nitrated food proteins.</p><p>Objective</p><p>The aim of this study was to investigate the influence of protein nitration on the effector phase of food allergy.</p><p>Design</p><p>BALB/c mice were immunized intraperitoneally (i.p.) with the milk allergen β-lactoglobulin (BLG) or the egg allergen ovomucoid (OVM), followed by intragastric (i.g.) gavages to induce a strong local inflammatory response and allergen-specific antibodies. Subsequently, naïve and allergic mice were intravenously (i.v.) challenged with untreated, sham-nitrated or nitrated BLG or OVM. Anaphylaxis was monitored by measuring core body temperature and determination of mouse mast cell protease-1 (mMCP-1) levels in blood.</p><p>Results</p><p>A significant drop of body temperature accompanied with significantly elevated concentrations of the anaphylaxis marker mMCP-1 were only observed in BLG allergic animals challenged with nitrated BLG and not in OVM allergic mice challenged with nitrated OVM. SDS-PAGE and circular dichroism analysis of the differentially modified allergens revealed an effect of nitration on the secondary protein structure exclusively for BLG together with enhanced protein aggregation.</p><p>Conclusion</p><p>Our data suggest that nitration affects differently the food allergens BLG and OVM. In the case of BLG, structural changes favored dimerization possibly explaining the increased anaphylactic reactivity in BLG allergic animals.</p></div