Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food 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.

The inhibition of voltage-gated H+ channel (HVCN1) induces acidification of leukemic Jurkat T cells promoting cell death by apoptosis.

Cellular energetic deregulation is widely known to produce an overproduction of acidic species in cancer cells. This acid overload must be counterbalanced with a high rate of H+ extrusion to maintain cell viability. In this sense, many H+ transporters have been reported to be crucial for cell survival and proposed as antineoplastic target. By the way, voltage-gated proton channels (Hv1) mediate highly selective H+ outward currents, capable to compensate acid burden in brief periods of time. This structure is canonically described acting as NADPH oxidase counterbalance in reactive oxygen species production. In this work, we show, for the first time in a oncohematologic cell line, that inhibition of Hv1 channels by Zn2+ and the more selective blocker 2-(6-chloro-1H-benzimidazol-2-yl)guanidine (ClGBI) progressively decreases intracellular pH in resting conditions. This acidification is evident minutes after blockade and progresses under prolonged exposure (2, 17, and 48 h), and we firstly demonstrate that this is followed by cell death through apoptosis (annexin V binding). Altogether, these results contribute strong evidence that this channel might be a new therapeutic target in cancer.

Inflammation Controls Sensitivity of Human and Mouse Intestinal Epithelial Cells to Galectin-1.

Galectins play key roles in the inflammatory cascade. In this study, we aimed to analyze the effect of galectin-1 (Gal-1) in the function of intestinal epithelial cells (IECs) isolated from healthy and inflamed mucosa. IECs isolated from mice or patients with inflammatory bowel diseases (IBD) were incubated with different pro-inflammatory cytokines, and Gal-1 binding, secretion of homeostatic factors and viability were assessed. Experimental models of food allergy and colitis were used to evaluate the in vivo influence of inflammation on Gal-1 binding and modulation of IECs. We found an enhanced binding of Gal-1 to: (a) murine IECs exposed to IL-1ß, TNF, and IL-13; (b) IECs from inflamed areas in intestinal tissue from IBD patients; (c) small bowel of allergic mice; and (d) colon from mice with experimental colitis. Our results showed that low concentrations of Gal-1 favored a tolerogenic micro-environment, whereas high concentrations of this lectin modulated viability of IECs through mechanisms involving activation of caspase-9 and modulation of Bcl-2 protein family members. Our results showed that, when added in the presence of diverse pro-inflammatory cytokines such as tumor necrosis factor (TNF), IL-13 and IL-5, Gal-1 differentially promoted the secretion of growth factors including thymic stromal lymphopoietin (TSLP), epidermal growth factor (EGF), IL-10, IL-25, and transforming growth factor (TGF-ß1 ). In conclusion, we found an augmented binding of Gal-1 to IECs when exposed in vitro or in vivo to inflammatory stimuli, showing different effects depending on Gal-1 concentration. These findings highlight the importance of the inflammatory micro-environment of mucosal tissues in modulating IECs susceptibility to the immunoregulatory lectin Gal-1 and its role in epithelial cell homeostasis.

Poly(allylamine)-tripolyphosphate polymeric nanoparticle as an NLRP3-dependent systemic adjuvant for the vaccine development.

Nanotechnology plays a crucial role in vaccine development and provides the opportunity to design functional nanoparticles (Np) of different compositions, sizes, charges and surface properties for biomedical applications. The present study aims to evaluate a complex coacervate-like Np composed of poly(allylamine hydrochloride) (PAH) and tripolyphosphate (Tpp) as a safe vehicle and adjuvant for systemic vaccines. We investigated the activation of different antigen-presenting cells (APCs) with Np-PAH and its adjuvanticity in Balbc/c and different KO mice that were intraperitoneally immunized with Np-OVA. We found that Np-PAH increased the expression of CD86 and MHCII and promoted the production and secretion of interleukin-1ß (IL-1ß) and IL-18 through the inflammasome NLRP3 when macrophages and dendritic cells were co-incubated with LPS and Np-PAH. We evidenced an unconventional IL-1ß release through the autophagosome pathway. The inhibition of autophagy with 3-methyladenine reduced the LPS/Np-PAH-induced IL-1ß secretion. Additionally, our findings showed that the systemic administration of mice with Np-OVA triggered a significant induction of serum OVA-specific IgG and IgG2a, an increased secretion of IFN-γ by spleen cells, and high frequencies of LT CD4 + IFN-γ + and LT CD8 + IFN-γ + . In conclusion, our findings show that PAH-based Np promoted the inflammasome activation of innate cells with Th1-dependent adjuvant properties, making them valuable for formulating of novel preventive or therapeutic vaccines for infectious and non-infectious diseases.

Poly(allylamine)-tripolyphosphate Ionic Assemblies as Nanocarriers: Friend or Foe?

Designing effective drug nanocarriers that are easy to synthesize, robust, and nontoxic is a significant challenge in nanomedicine. Polyamine-multivalent molecule nanocomplexes are promising drug carriers due to their simple and all-aqueous manufacturing process. However, these systems can present issues of colloidal instability over time and cellular toxicity due to the cationic polymer. In this study, we finely modulate the formation parameters of poly(allylamine-tripolyphosphate) complexes to jointly optimize the robustness and safety. Polyallylamine was ionically assembled with tripolyphosphate anions to form liquid-like nanocomplexes with a size of around 200 nm and a zeta potential of -30 mV. We found that nanocomplexes exhibit tremendous long-term stability (9 months of storage) in colloidal dispersion and that they are suitable as protein-loading agents. Moreover, the formation of nanocomplexes induced by tripolyphosphate anions produces a switch-off in the toxicity of the system by altering the overall charge from positive to negative. In addition, we demonstrate that nanocomplexes can be internalized by bone-marrow-derived macrophage cells. Altogether, these nanocomplexes have attractive and promising properties as delivery nanoplatforms for potential therapies based on the immune system activation.

In vivo evidence of cross-reactivity between cow's milk and soybean proteins in a mouse model of food allergy.

BACKGROUND: Cow's milk allergy (CMA) is an important problem worldwide and the development of an in vivo system to study new immunotherapeutic strategies is of interest. Intolerance to soybean formula has been described in CMA patients, but it is not fully understood. In this work, we used a food allergy model in BALB/c mice to study the cross-reactivity between cow's milk protein (CMP) and soy proteins (SP). METHODS: Mice were orally sensitized with cholera toxin and CMP, and then challenged with CMP or SP to induce allergy. Elicited symptoms, plasma histamine, humoral and cellular immune response were analyzed. Th1- and Th2-associated cytokines and transcription factors were assessed at mucosal sites and in splenocytes. Cutaneous tests were also performed. RESULTS: We found that the immediate symptoms elicited in CMP-sensitized mice orally challenged with SP were consistent with a plasma histamine increase. The serum levels of CMP-specific IgE and IgG1 antibodies were increased. These antibodies also recognized soy proteins. Splenocytes and mesenteric lymph node cells incubated with CMP or SP secreted IL-5 and IL-13. mRNA expression of Th2-associated genes (IL-5, IL-13, and GATA-3) was upregulated in mucosal samples. In addition, sensitized animals exhibited positive cutaneous tests after the injection of CMP or SP. CONCLUSIONS: We demonstrate that CMP-sensitized mice, without previous exposure to soy proteins, elicited hypersensitivity signs immediately after the oral administration of SP, suggesting that the immunochemical cross-reactivity might be clinically relevant. This model may provide an approach to further characterize cross-allergenicity phenomena and develop new immunotherapeutic treatments for allergic patients.

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.

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.

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.

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.

Skin expression of IL-23 drives the development of psoriasis and psoriatic arthritis in mice.

Psoriasis (PS) is a chronic skin inflammation. Up to 30% of the patients with PS develop psoriatic arthritis (PsA), a condition characterized by inflammatory arthritis that affects joints or entheses. Although there is mounting evidence for a critical role of interleukin-23 (IL-23) signaling in the pathogenesis of both PS and PsA, it remains unclear whether IL-23-induced skin inflammation drives joint disease. Here, we show that mice expressing increased levels of IL-23 in the skin (K23 mice) develop a PS-like disease that is characterized by acanthosis, parakeratosis, hyperkeratosis, and inflammatory infiltrates in the dermis. Skin disease preceded development of PsA, including enthesitis, dactylitis, and bone destruction. The development of enthesitis and dactylitis was not due to high circulating levels of IL-23, as transgenic animals and controls had similar levels of this cytokine in circulation. IL-22, a downstream cytokine of IL-23, was highly increased in the serum of K23 mice. Although IL-22 deficiency did not affect skin disease development, IL-22 deficiency aggravated the PsA-like disease in K23 mice. Our results demonstrate a central role for skin expressed IL-23 in the initiation of PS and on pathogenic processes leading to PsA.

Diphenhydramine inhibits voltage-gated proton channels (Hv1) and induces acidification in leukemic Jurkat T cells- New insights into the pro-apoptotic effects of antihistaminic drugs.

An established characteristic of neoplastic cells is their metabolic reprogramming, known as the Warburg effect, with greater reliance on energetically less efficient pathways (such as glycolysis and pentose phosphate shunt) compared with oxidative phosphorylation. This results in an overproduction of acidic species that must be extruded to maintain intracellular homeostasis. We recently described that blocking the proton currents in leukemic cells mediated by Hv1 ion channels triggers a marked intracellular acidification and apoptosis induction. Moreover, histamine H1-receptor antagonists were found to induce apoptosis in tumoral cells but the mechanism is still unclear. By using Jurkat T cells, we now show how diphenhydramine inhibits Hv1 mediated currents, inducing a drop in intracellular pH and cellular viability. This provides evidence of a new target structure responsible of the known pro-apoptotic action of antihistaminic drugs.

The Major Soybean Allergen Gly m Bd 28K Induces Hypersensitivity Reactions in Mice Sensitized to Cow's Milk Proteins.

Reactions to soy have been reported in a proportion of patients with IgE-mediated cow's milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics' tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP.

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions.

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-ß were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-ß-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-ß-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

Cross-reactivity between the soybean protein p34 and bovine caseins.

PURPOSE: Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity. METHODS: In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model. RESULTS: P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-γ remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34. CONCLUSIONS: Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34.

Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy.

Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow's milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.

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.

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.