Carbonylation induced by antibiotic and pesticide residues on casein increases its IgE binding and allergenicity.

This work aimed to evaluate the effect of carbonylation induced by tetracyclines, ß-lactams, fluoroquinolones, and pyrethroids in caseins of bovine origin on their immunoreactivity and allergenicity. Using a spectrophotometric method, ELISA, dot-blot, and an IgE-mediated milk allergy mouse model, we confirmed that antibiotics and pesticides at their maximum residue limit, promoted the in vitro carbonylation of caseins (among 5.0 ± 0.01 and 67.5 ± 0.70 nmol of carbonyl/mg of protein); furthermore, carbonylations greater than 19 nmol significantly increase the in vitro IgE immunoreactivity of caseins (average OD among 0.63-1.50) regarding the negative control (average OD: 0.56). On the other hand, sensitized mice exposed to oxidized caseins showed increased clinical scores (2-5), positive skin tests, and footpad swelling (0.28-0.59 mm) compared to the negative control (1-2; negative skin tests; 0.1 mm, respectively), denoting increased allergenicity. These results suggest that casein carbonylation increases their IgE immunoreactivity and allergenicity, a fact that could be explained by the resistance to the digestion promoted by carbonylation and by conformational changes in the random coil casein structure, which can expose cryptic epitopes or neoepitopes.

Volcanic ash-driven worsening of mucosal inflammation in an experimental colitis model.

Particulate matter exposure and related chemical changes in drinking water have been associated with health problems and inflammatory disorders. This study aimed to examine the effect of orally administered ash-water dilution on the gut of mice under normal and inflammatory conditions. Balb/c mice received ash-released soluble and dust-suspended components in the drinking water for 14 days. On day 7, animals were intrarectally instilled with TNBS in ethanol or flagellin from Salmonella typhimurium in PBS. At sacrifice, colon segments were collected and histologic damage, mRNA expression and cytokine levels in tissue were evaluated. In addition, these parameters were also evaluated in IL-10 null mice. We found that mice that received 5% w. fine-ash dilution in the drinking water worsened colitis signs. Weight loss, shortening of the colon, tissue edema with mucosa and submucosa cell infiltration and production of pro-inflammatory cytokines and chemokines were enhanced compared to control mice. A more pronounced inflammation was observed in IL-10 null mice. In addition, markers of NLRP3-dependent inflammasome activation were found in animals exposed to ash. In conclusion, ingestion of contaminated water with dust-suspended particulate matter enhanced the inflammatory response in the gut, probably due to alteration of the gut barrier and promoting an intense contact with the luminal content. This study critically appraises the response for fine particulate matter in uncommon illnesses reported for volcanic ash pollution. We suggest actions to enable better prediction and assessment the health impacts of volcanic eruptions.

Mucosal Immunoregulatory Properties of Tsukamurella inchonensis to Reverse Experimental Food Allergy.

The intestinal mucosa is lined by epithelial cells, which are key cells to sustain gut homeostasis. Food allergy is an immune-mediated adverse reaction to food, likely due to defective regulatory circuits. Tsukamurella inchonensis is a non-pathogenic bacterium with immunomodulatory properties. We hypothesize that the anti-inflammatory effect of dead T. inchonensis on activated epithelial cells modulates milk allergy through the restoration of tolerance in a mouse model. Epithelial cells (Caco-2 and enterocytes from mouse gut) and macrophages were stimulated with T. inchonensis and induction of luciferase under the NF-κB promoter, ROS and cytokines production were studied. Balb/c mice were mucosally sensitized with cow´s milk proteins plus cholera toxin and orally challenged with the allergen to evidence hypersensitivity symptoms. After that, mice were orally administered with heat-killed T. inchonensis as treatment and then challenged with the allergen. The therapeutic efficacy was in vivo (clinical score and cutaneous test) and in vitro (serum specific antibodies and cytokines-ELISA, and cell analysis-flow cytometry) evaluated. Heat-killed T. inchonensis modulated the induction of pro-inflammatory chemokines, with an increase in anti-inflammatory cytokines by intestinal epithelial cells and by macrophages with decreased OX40L expression. In vivo, oral administration of T. inchonensis increased the frequency of lamina propria CD4+CD25+FoxP3+ T cells, and clinical signs were lower in T. inchonensis-treated mice compared with milk-sensitized animals. In vivo depletion of Tregs (anti-CD25) abrogated T. inchonensis immunomodulation. In conclusion, these bacteria suppressed the intestinal inflammatory immune response to reverse food allergy.

A novel approach to ameliorate experimental milk allergy based on the oral administration of a short soy cross-reactive peptide.

Food allergy is on the rise, and preventive/therapeutic procedures are needed. We explored a preventive protocol for milk allergy with the oral administration of a Gly-m-Bd-30K soy-derived peptide that contains cross-reactive epitopes with bovine caseins. B/T-cross-reactive epitopes were mapped using milk-specific human sera and monoclonal antibodies on overlapping and recombinant peptides of Gly-m-Bd-30K by SPOT and cell proliferation assays. Bioinformatics tools were used to characterize epitopes on the 3D-modelled molecule, and to predict the binding to HLA alleles. The peptide was orally administrated to mice that were then IgE-sensitized to milk proteins. Immunodominant B-epitopes were mainly located on the surface of the Nt-fragment. The use of a soy-peptide-containing an immunodominant cross-reactive T-epitope, along with a single B epitope, prevents IgE-mediated milk sensitization through the induction of Th1-mediated immunity and induction of blocking IgG. The use of a safe soy-peptide may represent a promising alternative for preventing milk allergy.

Use of a Collagen Membrane to Enhance the Survival of Primary Intestinal Epithelial Cells.

Intestinal epithelial cell culture is important for biological, functional, and immunological studies. Since enterocytes have a short in vivo life span due to anoikis, we aimed to establish a novel and reproducible method to prolong the survival of mouse and human cells. Cells were isolated following a standard procedure, and cultured on ordered-cow's collagen membranes. A prolonged cell life span was achieved; cells covered the complete surface of bio-membranes and showed a classical enterocyte morphology with high expression of enzymes supporting the possibility of cryopreservation. Apoptosis was dramatically reduced and cultured enterocytes expressed cytokeratin and LGR5 (low frequency). Cells exposed to LPS or flagellin showed the induction of TLR4 and TLR5 expression and a functional phenotype upon exposure to the probiotic Bifidobacterium bifidum or the pathogenic Clostridium difficile. The secretion of the homeostatic (IL-25 and TSLP), inhibitory (IL-10 and TGF-ß), or pro-inflammatory mediators (IL-1ß and TNF) were induced. In conclusion, this novel protocol using cow's collagen-ordered membrane provides a simple and reproducible method to maintain intestinal epithelial cells functional for cell-microorganism interaction studies and stem cell expansion. J. Cell. Physiol. 232: 2489-2496, 2017. © 2016 Wiley Periodicals, Inc.

Targeting a cross-reactive Gly m 5 soy peptide as responsible for hypersensitivity reactions in a milk allergy mouse model.

BACKGROUND: Cross-reactivity between soybean allergens and bovine caseins has been previously reported. In this study we aimed to map epitopes of the major soybean allergen Gly m 5 that are co-recognized by casein specific antibodies, and to identify a peptide responsible for the cross-reactivity. METHODS: Cow's milk protein (CMP)-specific antibodies were used in different immunoassays (immunoblotting, ELISA, ELISA inhibition test) to evaluate the in vitro recognition of soybean proteins (SP). Recombinant Gly m 5 (α), a truncated fragment containing the C-terminal domain (α-T) and peptides of α-T were obtained and epitope mapping was performed with an overlapping peptide assay. Bioinformatics tools were used for epitope prediction by sequence alignment, and for modelling the cross-recognized soy proteins and peptides. The binding of SP to a monoclonal antibody was studied by surface Plasmon resonance (SPR). Finally, the in vivo cross-recognition of SP was assessed in a mouse model of milk allergy. RESULTS: Both α and α-T reacted with the different CMP-specific antibodies. α-T contains IgG and IgE epitopes in several peptides, particularly in the peptide named PA. Besides, we found similar values of association and dissociation constants between the α-casein specific mAb and the different milk and soy components. The food allergy mouse model showed that SP and PA contain the cross-reactive B and T epitopes, which triggered hypersensitivity reactions and a Th2-mediated response on CMP-sensitized mice. CONCLUSIONS: Gly m 5 is a cross-reactive soy allergen and the α-T portion of the molecule contains IgG and IgE immunodominant epitopes, confined to PA, a region with enough conformation to be bound by antibodies. These findings contribute to explain the intolerance to SP observed in IgE-mediated CMA patients, primarily not sensitised to SP, as well as it sets the basis to propose a mucosal immunotherapy for milk allergy using this soy peptide.

Down-regulation of NF-κB signaling by Gordonia bronchialis prevents the activation of gut epithelial cells.

The immunomodulatory power of heat-killed Gordonia bronchialis was studied on gut epithelial cells activated with pro-inflammatory stimuli (flagellin, TNF-α or IL-1ß). Light emission of luciferase-transfected epithelial cells and mRNA expression of IL-1ß, TNF-α, IL-6, CCL20, IL-8 and MCP-1 were measured. NF-κB activation was assessed by immunofluorescence and immunoblotting, and induction of reactive oxygen species (ROS) was evaluated. In vivo inhibitory properties of G. bronchialis were studied with ligated intestinal loop assay and in a mouse model of food allergy. G. bronchialis promoted the down-regulation of the expression of CCL20 and IL-1ß on activated epithelial cells in a dose-dependent manner. A concomitant blocking of nuclear p65 translocation with increased production of ROS was found. In vivo experiments confirmed the inhibition of CCL20 expression and the suppression of IgE sensitization and hypersensitivity symptoms in the food allergy mouse model. In conclusion, heat-killed G. bronchialis inhibited the activation of NF-κB pathway in human epithelial cells, and suppressed the expression of CCL20. These results indicate that G. bronchialis may be used to modulate the initial steps of innate immune activation, which further suppress the allergic sensitization. This approach may be exploited as a therapy for intestinal inflammation.