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.

Food-sensitized pediatric patients show colonic cow's milk protein-specific Th2 cells.

Food allergies have become a health concern worldwide. Around 6% to 10% of children are allergic to cow's milk proteins. We have previously characterized colorectal polyps in patients sensitized to food allergens. These polyps are classified as inflammatory and present a type 2 environment, with elevated interleukin (IL)-13 and IL-4, and are a site of immunoglobulin E synthesis. In this study, we characterized and isolated cow's milk protein-specific T cell lines and T cell clones from the lamina propria of polyps from patients sensitized to these proteins. Isolated T cells responded to cow's milk proteins similarly to peripheral blood T cells, showing antigen-specific cell proliferation and Th2 cytokines release in vitro. T cell clones obtained were all CD4+ T cells and expressed the membrane TCRαß receptor and secreted higher IL-4, IL-5, and IL-13 amounts than unstimulated cells, whereas interferon γ secretion remained unchanged. Remarkably, the gut homing chemokine receptor CCR9 was augmented in cow's milk-specific peripheral and lamina propria T cells, and CCL25 was found to be expressed in the inflammatory polyp tissue and not in the adjacent mucosa. In conclusion, we isolated and characterized cow's milk-specific lamina propria CD4+ Th2 cells from colonic inflammatory polyps. CCR9 expression on these cells, along with increase secretion of CCL25 in the polyp, favors recruitment and cow's milk-specific allergic response within the inflammatory polyp tissue. Our findings may be critical to understand the underlying mechanism that promotes immunoglobulin E synthesis in the colon of cow's milk proteins allergic patients, contributing to the development of novel T cell-targeted immunotherapies.

Characterization of protein cargo of Echinococcus granulosus extracellular vesicles in drug response and its influence on immune response.

BACKGROUND: The Echinococcus granulosus sensu lato species complex causes cystic echinococcosis, a zoonotic disease of medical importance. Parasite-derived small extracellular vesicles (sEVs) are involved in the interaction with hosts intervening in signal transduction related to parasite proliferation and disease pathogenesis. Although the characteristics of sEVs from E. granulosus protoscoleces and their interaction with host dendritic cells (DCs) have been described, the effect of sEVs recovered during parasite pharmacological treatment on the immune response remains unexplored. METHODS: Here, we isolated and characterized sEVs from control and drug-treated protoscoleces by ultracentrifugation, transmission electron microscopy, dynamic light scattering, and proteomic analysis. In addition, we evaluated the cytokine response profile induced in murine bone marrow-derived dendritic cells (BMDCs) by qPCR. RESULTS: The isolated sEVs, with conventional size between 50 and 200 nm, regardless of drug treatment, showed more than 500 cargo proteins and, importantly, 20 known antigens and 70 potential antigenic proteins, and several integral-transmembrane and soluble proteins mainly associated with signal transduction, immunomodulation, scaffolding factors, extracellular matrix-anchoring, and lipid transport. The identity and abundance of proteins in the sEV-cargo from metformin- and albendazole sulfoxide (ABZSO)-treated parasites were determined by proteomic analysis, detecting 107 and eight exclusive proteins, respectively, which include proteins related to the mechanisms of drug action. We also determined that the interaction of murine BMDCs with sEVs derived from control parasites and those treated with ABZSO and metformin increased the expression of pro-inflammatory cytokines such as IL-12 compared to control cells. Additionally, protoscolex-derived vesicles from metformin treatments induced the production of IL-6, TNF-α, and IL-10. However, the expression of IL-23 and TGF-ß was downregulated. CONCLUSIONS: We demonstrated that sEV-cargo derived from drug-treated E. granulosus protoscoleces have immunomodulatory functions, as they enhance DC activation towards a type 1 pro-inflammatory profile against the parasite, and therefore support the proposal of a new approach for the prevention and treatment of secondary echinococcosis.

Case report: De novo SAMD9L truncation causes neonatal-onset autoinflammatory syndrome which was successfully treated with hematopoietic stem cell transplantation.

During recent years, the identification of monogenic mutations that cause sterile inflammation has expanded the spectrum of autoinflammatory diseases, clinical disorders characterized by uncontrolled systemic and organ-specific inflammation that, in some cases, can mirror infectious conditions. Early studies support the concept of innate immune dysregulation with a predominance of myeloid effector cell dysregulation, particularly neutrophils and macrophages, in causing tissue inflammation. However, recent discoveries have shown a complex overlap of features of autoinflammation and/or immunodeficiency contributing to severe disease phenotypes. Here, we describe the first Argentine patient with a newly described frameshift mutation in SAMD9L c.2666delT/p.F889Sfs*2 presenting with a complex phenotypic overlap of CANDLE-like features and severe infection-induced cytopenia and immunodeficiency. The patient underwent a fully matched unrelated HSCT and has since been in inflammatory remission 5 years post-HSCT.

Lidocaine reinforces the anti-inflammatory action of dexamethasone on myeloid and epithelial cells activated by inflammatory cytokines or SARS-CoV-2 infection.

BACKGROUND: Severe cases of Coronavirus Disease 2019 (COVID-19) that require admission to the Intensive Care Unit (ICU) and mechanical ventilation assistance show a high mortality rate with currently few therapeutic options available. Severe COVID-19 is characterized by a systemic inflammatory condition, also called "cytokine storm", which can lead to various multi-organ complications and ultimately death. Lidocaine, a safe local anesthetic that given intravenously is used to treat arrhythmias, has long been reported to have an anti-inflammatory and pro-homeostatic activity. METHODS: We studied the capacity of lidocaine to modulate cytokine secretion of mouse and human myeloid cell lines activated by different cytokines or Toll Like Receptor (TLR) ligands (flagellin (FliC), Lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (Poly I:C) and N-Palmitoyl-S- [2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteinyl-(S)-seryl-(S)-lysyl-(S)-lysyl-(S)-lysyl-(S)-lysine x 3HCl (Pam3Cys-SKKKK)) or by Severe acute respiratory syndromecoronavirus 2 (SARS-CoV-2) infection to epithelial cells. Reporter cell lines were used to study modulation of lidocaine of specific signaling pathways. RESULTS: Lidocaine used in combination with dexamethasone, had an additive effect in the modulation of cellular inflammatory response triggered by Tumoral Necrosis Factor alpha (TNFα), Interleukin 1 beta (IL-1ß) as well as different TLR ligands. We also found that lidocaine in combination with dexamethasone modulates the Nuclear factor kappa B (NF-κB) pathway, inflammasome activation as well as interferon gamma receptor (IFNγR) signaling without affecting the type I interferons (Type I IFNs) pathway. Furthermore, we showed that lidocaine and dexamethasone treatment of epithelial cells infected with SARS-CoV-2 modulated the expression of chemokines that contribute to pro-inflammatory effects in severe COVID. CONCLUSIONS: We reported for the first time in vitro anti-inflammatory capacity of lidocaine on SARS-CoV-2 triggered immune pathways. These results indicated the potential of lidocaine to treat COVID-19 patients and add tools to the therapeutic options available for these concerning cases.

Type-2 Cytokines Promote the Secretion of the Eosinophil-Attractant CCL26 by Intestinal Epithelial Cells in Food-Sensitized Patients.

Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at inflammation sites. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. In this work, we studied the colorectal polyp-confined tissue containing eosinophils and we hypothesized that intestinal epithelial cells are the cell source of eotaxin-3 or CCL26, a potent chemoattractant for eosinophils. We analyzed colorectal polyps (n=50) from pediatric patients with rectal bleeding by H&E staining and eosin staining, and different pro-inflammatory cytokines were assessed by RT-qPCR and ELISA. IgE and CCL26 were investigated by RT-qPCR, ELISA and confocal microscopy. Finally, the intracellular signaling pathway that mediates the CCL26 production was analyzed using a kinase array and immunoblotting in human intestinal Caco-2 cell line. We found a dense cell agglomeration within the polyps, with a significantly higher frequency of eosinophils than in control adjacent tissue. IL-4 and IL-13 were significantly up-regulated in polyps and CCL26 was elevated in the epithelial compartment. Experiments with Caco-2 cells showed that the type-2 cytokine IL-13 increased STAT3 and STAT6 phosphorylation and eotaxin-3 secretion. The addition of the blocking antibody Dupilumab or the inhibitor Ruxolitinib to the cytokine-stimulated Caco-2 cells diminished the CCL26 secretion to basal levels in a dose-dependent manner. In conclusion, our findings demonstrate a high frequency of eosinophils, and elevated levels of type-2 cytokines and eotaxin-3 in the inflammatory stroma of colorectal polyps from pediatric patients. Polyp epithelial cells showed to be the main cell source of CCL26, and IL-13 was the main trigger of this chemokine through the activation of the STAT3/STAT6/JAK1-2 pathway. We suggest that the epithelial compartment actively participates in the recruitment of eosinophils to the colonic polyp-confined inflammatory environment.

Federation of Clinical Immunology Societies Goes South 2021: advanced course on molecular and cellular translational immunology.

The Federation of Clinical Immunology Societies (FOCIS) regularly organizes scientific meetings to foster advances in immunology. A new event of this type is FOCIS Goes South, a course and workshop organized by FOCIS Centers of Excellence (FCEs) from across Latin America, which consists of a course on advanced immunology, a flow cytometry workshop and seminars on cutting-edge research in autoimmunity, tolerance, cancer, infectious diseases and vaccines. Due to the COVID-19 pandemic, the second version of FOCIS Goes South, hosted by the Millennium Institute on Immunology and Immunotherapy in Chile, took place virtually from 15 to 18 November 2021, with more than 950 registered participants. The present article summarizes the key findings and insights discussed at FOCIS Goes South 2021.

Spatiotemporal regulation of galectin-1-induced T-cell death in lamina propria from Crohn's disease and ulcerative colitis patients.

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is characterized by chronic, relapsing intestinal inflammation. Galectin-1 (Gal-1) is an endogenous lectin with key pro-resolving roles, including induction of T-cell apoptosis and secretion of immunosuppressive cytokines. Despite considerable progress, the relevance of Gal-1-induced T-cell death in inflamed tissue from human IBD patients has not been ascertained. Intestinal biopsies and surgical specimens from control patients (n = 52) and patients with active or inactive IBD (n = 97) were studied. Gal-1 expression was studied by RT-qPCR, immunoblotting, ELISA and immunohistochemistry. Gal-1-specific ligands and Gal-1-induced apoptosis of lamina propria (LP) T-cells were determined by TUNEL and flow cytometry. We found a transient expression of asialo core 1-O-glycans in LP T-cells from inflamed areas (p < 0.05) as revealed by flow cytometry using peanut agglutinin (PNA) binding and assessing dysregulation of the core-2 ß 1-6-N-acetylglucosaminyltransferase 1 (C2GNT1), an enzyme responsible for elongation of core 2 O-glycans. Consequently, Gal-1 binding was attenuated in CD3+CD4+ and CD3+CD8+ LP T-cells isolated from inflamed sites (p < 0.05). Incubation with recombinant Gal-1 induced apoptosis of LP CD3+ T-cells isolated from control subjects and non-inflamed areas of IBD patients (p < 0.05), but not from inflamed areas. In conclusion, our findings showed that transient regulation of the O-glycan profile during inflammation modulates Gal-1 binding and LP T-cell survival in IBD patients.

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.

Las vacunas para COVID-19 están mostrando seguridad y una elevada eficacia en la protección / Vaccines for COVID-19 are showing safety and high efficacy in protection

Las vacunas para COVID han mostrado parámetros aceptables de seguridad y una elevada eficacia de protección para contraer COVID-19 en los estudios de fase 3 en individuos voluntarios. Definir estrategias de vacunación y administrarlas en la mayor proporción de la población permitirá alcanzar la inmunidad de rebaño que restrinja la circulación del SARS-CoV-2. Sólo de esta manera se podrán evitar nuevas olas epidémicas y retomar hábitos de vida normales (AU)

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 Role of Cytokines in the Fibrotic Responses in Crohn's Disease.

Crohn's disease is an idiopathic disorder of the gut thought to be caused by a combination of environmental and genetic factors in susceptible individuals. It is characterized by chronic transmural inflammation of the terminal ileum and colon, with typical transmural lesions. Complications, including fibrosis, mean that between 40 and 70% of patients require surgery in the first 10 years after diagnosis. Presently, there is no evidence that the current therapies which dampen inflammation modulate or reverse intestinal fibrosis. In this review, we focus on cytokines that may lead to fibrosis and stenosis and the contribution of experimental models for understanding and treatment of gut fibrosis.

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.

Avances en las inmunoterapias para alergias alimentarias / New insights in immunotherapies for food allergies / Novas perspectivas em imunoterapias para alergias alimentares

Las enfermedades alérgicas son las inmunopatologías que con mayor prevalencia se presentan en el mundo. Pueden o no estar mediadas por anticuerpos IgE, sin embargo estas últimas son las que más intensamente se han estudiado por el riesgo que presentan para la vida del paciente. Si bien el único tratamiento que logra revertir estos mecanismos es la no exposición al alergeno, esto no siempre es posible. Por esta razón, y a partir del mayor conocimiento alcanzado del sistema inmune de mucosas junto al desarrollo de modelos animales de alergia, existe un marcado interés en la especialidad para el desarrollo de inmunoterapias que controlen y reviertan el estado de alergia. A partir de los ensayos pre-clínicos en animales y la aplicación de protocolos terapéuticos en ensayos clínicos, se han desarrollado terapias mucosales que logran inducir mecanismos de tolerancia específicos del alergeno, los cuales son capaces de revertir la sensibilización alérgica. Dado que el principal escollo siguen siendo las reacciones adversas inducidas durante el tratamiento, se requiere profundizar los estudios para desarrollar protocolos terapéuticos más seguros. En este punto la medicina traslacional encuentra un campo próspero para fortalecer las interacciones entre la ciencia básica, la aplicada y la clínica.

Allergic diseases are the most prevalent immunopathologies worldwide. Although different mechanisms -IgE-independent or IgE-dependent- can be involved in the immunopathogenesis, the latter are the most studied reactions since they can be life-threatening. Nowadays, allergen avoidance is the unique effective treatment for allergic patients. However, this is rather difficult to implement. For this reason, and based on the new insights into the mucosal immune system and the development of animal models of allergy, there is an increasing interest in developing novel therapies to control or reverse allergic disorders. Pre-clinical studies and clinical trials have been successful to prove that immunotherapies may accomplish mucosal mechanisms of allergen-specific tolerance, which are able to revoke the allergic sensitization. Since the main obstacle in these therapies still has adverse reactions induced during treatment, further studies are required to explore safe and effective therapeutic protocols. At this point, translational medicine is a flourishing field in the areas of basic science, applied science, and clinical research.

As doenças alérgicas são as imunopatologias mais prevalentes em todo o mundo. Embora possam estar mediadas ou não por anticorpos IgE, estas últimas são as reacções mais intensamente estudadas, devido ao risco que apresentam para a vida do paciente. Ainda que o único tratamento eficaz para reverter este mecanismos seja a não exposição dos pacientes ao alergeno, isto nem sempre é possível. Por este motivo, e com base nas novas perspectivas sobre o sistema imune de mucosas, junto com o desenvolvimento de modelos e para o animais de alergia, existe um interesse crescente na especialidade para o desenvolvimento de imunoterapias que controlem e revertam o estado de alergia. A partir de estudos pré-clínicos em animais e a aplicação de protocolos terapêuticos em ensaios clínico, foram desenvolvidas terapias mucosas que conseguem induzir mecanismos de tolerância específicos do alergeno, que são capazes de reverter a sensibilização alérgica. Devido a que o principal obstáculo nestas terapias continuam sendo as reações adversas induzidas durante o tratamento, é necessário realizar mais estudos para desenvolver protocolos terapêuticos mais seguros. Neste ponto, medicina translacional é um campo próspero para fortalecer as interações entre a ciência básica, a aplicada e a clínica.

A galectin-specific signature in the gut delineates Crohn's disease and ulcerative colitis from other human inflammatory intestinal disorders.

Inflammatory bowel diseases (IBD) are chronic and relapsing inflammatory conditions of the gastrointestinal tract including Crohn's disease (CD) and ulcerative colitis (UC). Galectins, defined by shared consensus amino acid sequence and affinity for ß-galactosides, are critical modulators of the inflammatory response. However, the relevance of the galectin network in the pathogenesis of human IBD has not yet been explored. Here, we analyzed the expression of relevant members of the galectin family in intestinal biopsies, and identified their contribution as novel mucosal markers in IBD. Colonic biopsies were obtained from 59 IBD patients (22 CD and 37 UC), 9 patients with gut rejection after transplantation, 8 adult celiac patients, and 32 non-IBD donors. Galectin mRNA expression was analyzed by RT-PCR and qPCR using specific primers for individual galectins. A linear discriminant analysis (LDA) was used to analyze galectin expression in individual intestinal samples. Expression of common mucosal-associated galectins (Gal-1, -3, -4, -9) is dysregulated in inflamed tissues of IBD patients compared with non-inflamed IBD or control samples. LDA discriminated between different inflammation grades in active IBD and showed that remission IBD samples were clusterized with control samples. Galectin profiling could not distinguish CD and UC. Furthermore, inflamed IBD was discriminated from inflamed tissue of rejected gut in transplanted patients and duodenum of celiac patients, which could not be distinguished from control duodenum samples. The integrative analysis of galectins discriminated IBD from other intestinal inflammatory conditions and could be used as potential mucosal biomarker.

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.

Métodos electroforético e inmunoquímicos para detectar proteínas de leche, soja y huevo en premezclas comerciales / Electrophoretic and immunochemical methods for the detection of milk, soy and egg proteins in commercial mix products

The presence of milk, egg and soy proteins was evaluated in thirteen commercial products, which were premixes to make cream caramel, cake, soy "milanesas", "tortas fritas", pizza, gnocchi and complementary foods. Samples were analysed using SDS-PAGE, immunoblotting and ELISA methods. The electrophoretic method and immunoblotting were useful to confirm the presence of milk, egg and soy proteins in four samples that declared them as ingredients. In other samples, both methods showed negative results for some of these proteins, although they were also declared as ingredients. This suggests that those proteins were not added as ingredients in these products. The ELISA kits detected very low concentration of the allergenic proteins in four products with precautionary phrases and also in five samples that made no reference to them in their labels. ELISA methods are useful to detect cross contamination due to their high sensitivity. The food industry should be responsible for the declaration of milk, egg and soy proteins in their food labels.

Se evaluó la detección de proteínas de leche, huevo y soja en trece productos comerciales, correspondientes a premezclas para preparar flanes, bizcochuelo, milanesas de soja, tortas fritas, pizza, ñoquis y productos en polvo a base de harinas para niños pequeños. Las muestras fueron analizadas por SDS-PAGE, inmunoblotting y métodos de ELISA. El método electroforético e inmunoblotting resultaron útiles para confirmar la presencia de las proteínas alergénicas en algunas muestras que las declaraban como ingredientes. En cuatro muestras que también declaraban como ingredientes alguna de estas proteínas, tanto con SDS-PAGE como con Inmunoblotting, los resultados fueron negativos, sugiriendo que dichas proteínas no fueron agregadas como ingredientes en dichos alimentos. Los kits de ELISA permitieron la detección de concentraciones muy bajas de estos alérgenos en cuatro productos que presentaban frases de advertencia e incluso en otros cinco que no tenían ninguna declaración de alérgenos. Los métodos de ELISA son útiles para detectar contaminaciones cruzadas, dada su elevada sensibilidad. Se concluye que resulta necesario una declaración responsable de estas proteínas alergénicas, en los rótulos de este tipo de alimentos por parte de los fabricantes.

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.