RESUMEN
Transglutaminase (TGase)-catalyzed crosslinking has gained substantial traction as a novel strategy for reducing allergenic risk in food proteins, particularly within the realm of hypoallergenic food production. This study explored the impact of TGase crosslinking on conformational changes in a binary protein system composed of soy protein isolate (SPI) and sodium caseinate (SC) at varying mass ratios (10:0, 7:3, 5:5, 3:7 (w/w)). Specifically, the immunoglobulin E (IgE) binding capacity of soy proteins within this system was examined. Prolonged TGase crosslinking (ranging from 0 h to 15 h) resulted in a gradual reduction in IgE reactivity across all SPI-SC ratios, with the order of IgE-binding capability as follows: SPI > SPI5-SC5 > SPI7-SC3 > SPI3-SC7. These alterations in protein conformation following TGase crosslinking, as demonstrated by variable intrinsic fluorescence, altered surface hydrophobicity, increased ultraviolet absorption and reduced free sulfhydryl content, were identified as the underlying causes. Additionally, ionic bonds were found to play a significant role in maintaining the structure of the dual-protein system after crosslinking, with hydrophobic forces and hydrogen bonds serving as supplementary forces. Generally, the dual-protein system may exhibit enhanced efficacy in reducing the allergenicity of soy protein.
Asunto(s)
Inmunoglobulina E , Conformación Proteica , Proteínas de Soja , Transglutaminasas , Transglutaminasas/química , Transglutaminasas/metabolismo , Proteínas de Soja/química , Proteínas de Soja/inmunología , Inmunoglobulina E/inmunología , Inmunoglobulina E/metabolismo , Reactivos de Enlaces Cruzados/química , Interacciones Hidrofóbicas e Hidrofílicas , Humanos , Caseínas/química , Caseínas/metabolismo , Caseínas/inmunologíaRESUMEN
Casein, the major allergen in cow's milk, presents a significant challenge in providing nutritional support for children with allergies. To address this issue, we investigated a composite enzyme, comprising papain and chymotrypsin, to reduce the allergenicity of casein. Enzymatic hydrolysis induced substantial structural changes in casein, diminishing its affinity for specific IgE and IgG antibodies. Additionally, in a BALB/c mouse model, casein hydrolysate alleviated allergic symptoms, evidenced by lower serum IgE and IgG levels, reduced plasma histamine, and decreased Th2 cytokine release during cell co-culture. Peptidomic analysis revealed a 52.38% and 60% reduction in peptides containing IgE epitopes in casein hydrolyzed by the composite enzyme compared to papain and chymotrypsin, respectively, along with a notable absence of previously reported T cell epitopes. These results demonstrate the potential of enzyme combinations to enhance the efficiency of epitope destruction in allergenic proteins, providing valuable insights into the development of hypoallergenic dairy products.
Asunto(s)
Alérgenos , Caseínas , Quimotripsina , Hipersensibilidad a la Leche , Papaína , Animales , Bovinos , Femenino , Humanos , Ratones , Alérgenos/inmunología , Alérgenos/química , Caseínas/inmunología , Caseínas/química , Quimotripsina/química , Quimotripsina/inmunología , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/química , Inmunoglobulina E/inmunología , Ratones Endogámicos BALB C , Leche/química , Leche/inmunología , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/prevención & control , Papaína/inmunología , Papaína/químicaRESUMEN
BACKGROUND: The Trial to Reduce IDDM in the Genetically at Risk (TRIGR) (NCT00179777) found no difference type 1 diabetes risk between hydrolyzed and regular infant formula. However, cow milk consumption during childhood is consistently linked to type 1 diabetes risk in prospective cohort studies. OBJECTIVES: Our primary aim was to study whether humoral immune responses to cow milk and cow milk consumption are associated with type 1 diabetes in TRIGR children. METHODS: TRIGR comprised 2159 children with genetic susceptibility to type 1 diabetes born between 2002 and 2007 in 15 countries. Children were randomly assigned into groups receiving extensively hydrolyzed casein or a regular cow milk formula and followed up until age 10 y. Type 1 diabetes-related autoantibodies and antibodies to cow milk proteins were analyzed. Infant formula intake was measured by structured dietary interviews and milk consumption with a food frequency questionnaire. Associations of milk antibodies and milk consumption with risk to develop type 1 diabetes were analyzed using Cox survival model. RESULTS: Cow milk antibody concentrations both in cord blood [hazards ratio (HR) for islet autoimmunity: 1.30; 95% CI: 1.05, 1.61; HR for type 1 diabetes: 1.32; 95% CI: 1.02, 1.71] and longitudinally from birth to 3 years (HR for islet autoimmunity: 1.39; 95% CI: 1.07, 1.81; HR for type 1 diabetes: 1.43; 95% CI: 1.04, 1.96) were associated with increased risk of developing type 1 diabetes. The amount of regular infant formula was associated with reduced islet autoimmunity risk in the regular infant formula group (HR: 0.92; 95% CI: 0.85, 0.99). Furthermore, frequent liquid milk consumption after infancy was associated with increased risk of islet autoimmunity or type 1 diabetes. CONCLUSIONS: Elevated cow milk antibody concentrations and high consumption of liquid milk after infancy are related to type 1 diabetes development in children with an increased genetic susceptibility to type 1 diabetes. Enhanced antibody concentrations to cow milk may provide a biomarker of immune system prone to develop islet autoimmunity. This trial was registered at clinicaltrials.gov as NCT00179777.
Asunto(s)
Autoinmunidad , Diabetes Mellitus Tipo 1 , Inmunoglobulina G , Fórmulas Infantiles , Islotes Pancreáticos , Proteínas de la Leche , Leche , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Humanos , Animales , Lactante , Femenino , Masculino , Inmunoglobulina G/sangre , Islotes Pancreáticos/inmunología , Bovinos , Proteínas de la Leche/inmunología , Preescolar , Autoanticuerpos/sangre , Predisposición Genética a la Enfermedad , Factores de Riesgo , Niño , Dieta , Estudios de Seguimiento , Caseínas/inmunologíaRESUMEN
BACKGROUND: Milk oral immunotherapy is the riskiest and most unpredictable form of oral immunotherapy. We aimed to produce a low allergenic product than conventional once baked-cake/muffin, to develop indirect in-house ELISA to check the tolerance status with milk products and evaluate IgE reactivity of patients' sera via western blotting (WB) and indirect in-house ELISA. METHOD: A low allergenic product named biscotti-twice baked-cake was developed, and the total protein concentration was determined. The protein content was studied by SDS-PAGE and proteomics. Milk-specific IgE (sIgE) binding assays were performed by WB and indirect in-house ELISA by using patients' sera. RESULTS: Casein band intensity was observed to be lower in the biscotti-twice baked-cake than in the once baked-cake (p = .014). Proteomics analysis and αS1-casein measurement showed that the lowest intensity of casein was found in biscotti. The low binding capacity of milk sIgE to biscotti compared with once baked-cake was shown by WB (p = .0012) and by indirect in-house ELISA (p = .0001). In the ROC analysis, the area under the curve (AUC) of the in-house ELISA IgE was comparable with Uni-CAP milk and casein sIgE. The AUC of the in-house ELISA IgE for cake (0.96) and biscotti (1) was slightly better than Uni-CAP milk sIgE (0.94; 0.97) and casein sIgE (0.96; 0.97), respectively. CONCLUSION: The low allergenicity of the newly developed low allergenic product "biscotti-twice baked-cake" has been demonstrated by in vitro experiments. Biscotti could be a safe treatment option than once baked-cake/muffin in patients who are reactive to once baked-milk products.
Asunto(s)
Alérgenos , Desensibilización Inmunológica , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina E , Hipersensibilidad a la Leche , Humanos , Inmunoglobulina E/sangre , Inmunoglobulina E/inmunología , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/diagnóstico , Hipersensibilidad a la Leche/sangre , Alérgenos/inmunología , Femenino , Masculino , Preescolar , Niño , Desensibilización Inmunológica/métodos , Animales , Leche/inmunología , Leche/efectos adversos , Lactante , Caseínas/inmunología , Proteómica/métodos , Western Blotting , Administración Oral , AdolescenteRESUMEN
BACKGROUND: The basophil activation test is an emerging clinical tool in the diagnosis of cow's milk allergy (CMA). The aim was to assess the association between the basophil allergen threshold sensitivity to the major milk protein casein (casein-specific CD-sens), the levels of milk- and casein-specific Immunoglobulin E antibodies (IgE-ab), and the severity of allergic reactions at milk challenges. METHODS: We enrolled 34 patients aged 5-15 (median 9) years who underwent a double-blind placebo-controlled milk-challenge (DBPCMC) as screening before inclusion in an oral immunotherapy study for CMA. The severity of the allergic reaction at the DBPCMC was graded using Sampson's severity score. Venous blood was drawn before the DBPCMC. Milk- and casein-specific IgE-ab were analyzed. Following in vitro stimulation of basophils with casein, casein-specific CD-sens, was determined. RESULTS: Thirty-three patients completed the DBPCMC. There were strong correlations between casein-specific CD-sens and IgE-ab to milk (rs = 0.682, p < .001), and between casein-specific CD-sens and IgE-ab to casein (rs = 0.823, p < .001). There was a correlation between the severity of the allergic reaction and casein-specific CD-sens level (rs = 0.395, p = .041) and an inverse correlation between casein-specific CD-sens level and the cumulative dose of milk protein to which the patient reacted at the DBPCMC (rs = -0.418, p = .027). Among the 30 patients with an allergic reaction at the DBPCMC, 67% had positive casein-specific CD-sens, 23% had negative casein-specific CD-sens, and 10% were declared non-responders. CONCLUSION: Two thirds of those reacting at the DBPMC had positive casein-specific CD-sens, but reactions also occurred despite negative casein-specific CD-sens. The association between casein-specific CD-sens and the severity of the allergic reaction and cumulative dose of milk protein, respectively, was moderate.
Asunto(s)
Alérgenos , Basófilos , Caseínas , Inmunoglobulina E , Hipersensibilidad a la Leche , Humanos , Basófilos/inmunología , Basófilos/metabolismo , Caseínas/inmunología , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/diagnóstico , Hipersensibilidad a la Leche/sangre , Inmunoglobulina E/inmunología , Inmunoglobulina E/sangre , Femenino , Masculino , Niño , Adolescente , Preescolar , Alérgenos/inmunología , Animales , Leche/inmunología , Leche/efectos adversos , Método Doble CiegoRESUMEN
Casein (CN) is the primary allergenic protein in cow's milk, contributing to the worldwide escalating prevalence of food allergies. However, there remains limited knowledge regarding the effect of structural modifications on CN allergenicity. Herein, we prepared three modified CNs (mCN), including sodium dodecyl sulfate and dithiothreitol-induced linear CN (LCN), transglutaminase-cross-linked CN (TCN), and glucose-glycated CN (GCN). The electrophoresis results indicated widespread protein aggregation among mCN, causing variations in their molecular weights. The unique internal and external structural characteristics of mCN were substantiated by disparities in surface microstructure, alterations in the secondary structure, variations in free amino acid contents, and modifications in functional molecular groups. Despite the lower digestibility of TCN and GCN compared to LCN, they significantly suppressed IL-8 production in Caco-2 cells without significantly promoting their proliferation. Moreover, GCN showed the weakest capacity to induce LAD2 cell degranulation. Despite the therapeutic effect of TCN, GCN-treated mice displayed the most prominent attenuation of allergic reactions and a remarkably restored Th1/Th2 imbalance, while LCN administration resulted in severe allergic phenotypes and endotypes in both cellular and murine models. This study highlighted the detrimental effect of linear modifications and underscored the significance of glycation in relation to CN allergenicity.
Asunto(s)
Alérgenos , Caseínas , Ratones Endogámicos BALB C , Células TH1 , Células Th2 , Animales , Humanos , Ratones , Células Th2/inmunología , Caseínas/inmunología , Caseínas/química , Células TH1/inmunología , Alérgenos/inmunología , Alérgenos/química , Células CACO-2 , Femenino , Glicosilación , Bovinos , Homeostasis , Hipersensibilidad a los Alimentos/inmunologíaRESUMEN
Bovine casein is a major allergen present in cow milk to induce anaphylaxis. In this study, the potential allergenicity of enzymatically hydrolyzed casein (HC) was evaluated based on in vitro and in vivo. The results showed that Alcalase and Protamex treatment (AT, PT) reduced the potential allergenicity of CN, with the greatest reductions of 68.25% and 50.75%, respectively. In addition, in vivo results showed that HC effectively alleviated allergic response symptoms of Balb/c mice; a significant tendency toward decreased serum IgG1 and mast cell tryptase levels was observed, accompanied by a decrease of Th2-associated IL-4, IL-5, and IL-13 and an increase of IFN-γ levels in spleen. Moreover, the inflammation of the lung, jejunum, and ileum was remarkably ameliorated. The findings indicated that HC induced a shift toward Th1 response and maintained the Th1/Th2 immune balance. Importantly, our results provide the basis for the production of hypoallergenic dairy products.
Asunto(s)
Alérgenos , Caseínas , Ratones Endogámicos BALB C , Células Th2 , Animales , Ratones , Caseínas/inmunología , Alérgenos/inmunología , Femenino , Células Th2/inmunología , Hidrólisis , Inmunoglobulina G/sangre , Modelos Animales de Enfermedad , Bovinos , Bazo/inmunología , Hipersensibilidad a la Leche/inmunología , Interferón gamma/metabolismo , Células TH1/inmunología , Interleucina-4/metabolismo , Triptasas/metabolismo , Citocinas/metabolismo , Yeyuno/inmunología , Leche/inmunología , Leche/química , Interleucina-13/inmunología , Interleucina-13/metabolismo , Anafilaxia/inmunología , Anafilaxia/inducido químicamente , Anafilaxia/prevención & control , Interleucina-5/inmunologíaRESUMEN
Multiple sclerosis (MS) is a neuroinflammatory demyelinating disease of the central nervous system (CNS) with a high socioeconomic relevance. The pathophysiology of MS, which is both complex and incompletely understood, is believed to be influenced by various environmental determinants, including diet. Since the 1990s, a correlation between the consumption of bovine milk products and MS prevalence has been debated. Here, we show that C57BL/6 mice immunized with bovine casein developed severe spinal cord pathology, in particular, demyelination, which was associated with the deposition of immunoglobulin G. Furthermore, we observed binding of serum from casein-immunized mice to mouse oligodendrocytes in CNS tissue sections and in culture where casein-specific antibodies induced complement-dependent pathology. We subsequently identified myelin-associated glycoprotein (MAG) as a cross-reactive antigenic target. The results obtained from the mouse model were complemented by clinical data showing that serum samples from patients with MS contained significantly higher B cell and antibody reactivity to bovine casein than those from patients with other neurologic diseases. This reactivity correlated with the B cell response to a mixture of CNS antigens and could again be attributed to MAG reactivity. While we acknowledge disease heterogeneity among individuals with MS, we believe that consumption of cow's milk in a subset of patients with MS who have experienced a previous loss of tolerance to bovine casein may aggravate the disease. Our data suggest that patients with antibodies to bovine casein might benefit from restricting dairy products from their diet.
Asunto(s)
Anticuerpos/inmunología , Caseínas/inmunología , Reacciones Cruzadas , Enfermedades Desmielinizantes/inmunología , Esclerosis Múltiple/inmunología , Glicoproteína Asociada a Mielina/inmunología , Animales , Especificidad de Anticuerpos , Humanos , Ratones , Ratones Endogámicos C57BL , Leche/inmunologíaRESUMEN
Background: Oral immunotherapy (OIT) is an emerging treatment for cow's milk protein (CMP) allergy in children. The mechanisms driving tolerance following OIT are not well understood. Regulatory T cells (TREG) cells are key inhibitors of allergic responses and promoters of allergen-specific tolerance. In an exploratory study, we sought to detect induction of allergen-specific TREG in a cohort of subjects undergoing OIT. Methods: Pediatric patients with a history of allergic reaction to cow's milk and a positive Skin Pick Test (SPT) and/or CMP-specific IgE >0.35 kU, as well as a positive oral challenge to CMP underwent OIT with escalating doses of milk and were followed for up to 6 months. At specific milestones during the dose escalation and maintenance phases, casein-specific CD4+ T cells were expanded from patient blood by culturing unfractionated PBMCs with casein in vitro. The CD4+ T cell phenotypes were quantified by flow cytometry. Results: Our culture system induced activated casein-specific FOXP3+Helios+ TREG cells and FOXP3- TEFF cells, discriminated by expression of CD137 (4-1BB) and CD154 (CD40L) respectively. The frequency of casein-specific TREG cells increased significantly with escalating doses of milk during OIT while casein-specific TEFF cell frequencies remained constant. Moreover, expanded casein-specific TREG cells expressed higher levels of FOXP3 compared to polyclonal TREG cells, suggesting a more robust TREG phenotype. The induction of casein-specific TREG cells increased with successful CMP desensitization and correlated with increased frequencies of casein-specific Th1 cells among OIT subjects. The level of casein-specific TREG cells negatively correlated with the time required to reach the maintenance phase of desensitization. Conclusions: Overall, effective CMP-OIT successfully promoted the expansion of casein-specific, functionally-stable FOXP3+ TREG cells while mitigating Th2 responses in children receiving OIT. Our exploratory study proposes that an in vitro TREG response to casein may correlate with the time to reach maintenance in CMP-OIT.
Asunto(s)
Caseínas/inmunología , Desensibilización Inmunológica/métodos , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/terapia , Linfocitos T Reguladores/inmunología , Administración Oral , Adolescente , Alérgenos/administración & dosificación , Animales , Ligando de CD40/sangre , Bovinos , Niño , Estudios de Cohortes , Femenino , Factores de Transcripción Forkhead/sangre , Humanos , Técnicas In Vitro , Masculino , Hipersensibilidad a la Leche/sangre , Linfocitos T Reguladores/clasificación , Células Th2/inmunología , Factores de Tiempo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/sangreRESUMEN
The burden of infections on an individual and public health is profound. Many observational studies have shown a link between infections and the pathogenesis of disease; however a greater understanding of the role of host genetics is essential. Children from the longitudinal birth cohort, the Avon Longitudinal Study of Parents and Children, had 14 antibodies measured in plasma at age 7: Alpha-casein protein, beta-casein protein, cytomegalovirus, Epstein-Barr virus, feline herpes virus, Helicobacter pylori, herpes simplex virus 1, influenza virus subtype H1N1, influenza virus subtype H3N2, measles virus, Saccharomyces cerevisiae, Theiler's virus, Toxoplasma gondii, and SAG1 protein domain, a surface antigen of Toxoplasma gondii measured for greater precision. We performed genome-wide association analyses of antibody levels against these 14 infections (N = 357 - 5010) and identified three genome-wide signals (P < 5×10-8), two associated with measles virus antibodies and one with Toxoplasma gondii antibodies. In an association analysis focused on the human leukocyte antigen (HLA) region of the genome, we further detected 15 HLA alleles at a two-digit resolution and 23 HLA alleles at a four-digit resolution associated with five antibodies, with eight HLA alleles associated with Epstein-Barr virus antibodies showing strong evidence of replication in UK Biobank. We discuss how our findings from antibody levels complement other studies using self-reported phenotypes in understanding the architecture of host genetics related to infections.
Asunto(s)
Infecciones Bacterianas/genética , Toxoplasmosis/genética , Virosis/genética , Adolescente , Anticuerpos Antibacterianos/sangre , Anticuerpos Antiprotozoarios/sangre , Anticuerpos Antivirales/sangre , Antígenos de Protozoos/genética , Antígenos de Protozoos/inmunología , Bacterias/inmunología , Infecciones Bacterianas/sangre , Infecciones Bacterianas/inmunología , Caseínas/genética , Caseínas/inmunología , Niño , Estudio de Asociación del Genoma Completo , Antígenos HLA/genética , Humanos , Estudios Longitudinales , Polimorfismo de Nucleótido Simple , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Toxoplasmosis/sangre , Toxoplasmosis/inmunología , Virosis/sangre , Virosis/inmunología , Virus/inmunologíaRESUMEN
Currently, there is a need to explore the effects of different types of protein-anthocyanin complexations, as well as the possible changes in the nutrition and allergenicity of the formed complexes. Here, we systematically investigated the covalent and non-covalent interactions between cyanidin-3-O-glucoside (C3G) and two major milk proteins, α-casein (α-CN) and ß-lactoglobulin (ß-LG). Fluorescence quenching data showed that, under non-covalent conditions, C3G quenched the fluorescence of the two proteins via a static process, with the interaction forces being revealed; for covalent products, decreased fluorescence intensities were observed with red shifts in the λmax. Multiple spectroscopic analyses implied that C3G-addition induced protein structural unfolding through transitions between the random coil and ordered secondary components. With a two-stage simulated gastrointestinal (GI) digestion model, it was seen that covalent complexes, not their non-covalent counterparts, showed reduced protein digestibility, ascribed to structural changes resulting in the unavailability of enzyme cleaving sites. The GI digests displayed prominent 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation-scavenging abilities (3.8-11.1 mM Trolox equivalents per mL digest), in contrast to the markedly reduced 1,1-diphenyl-2-picrylhydrazyl radical-scavenging capacities. Additionally, covalent protein-C3G complexes, but not their non-covalent counterparts, showed lower IgE-binding levels in comparison to the native control. This study provides new understanding for the development of anthocyanin-milk protein systems as functional ingredients with health-beneficial properties.
Asunto(s)
Alérgenos/inmunología , Antocianinas/química , Caseínas/química , Lactoglobulinas/química , Animales , Antocianinas/inmunología , Antocianinas/metabolismo , Caseínas/inmunología , Caseínas/metabolismo , Digestión , Dispersión Dinámica de Luz/métodos , Humanos , Inmunoglobulina E/química , Inmunoglobulina E/inmunología , Lactoglobulinas/inmunología , Lactoglobulinas/metabolismo , Proteínas de la Leche/química , Proteínas de la Leche/inmunología , Proteínas de la Leche/metabolismo , Tamaño de la Partícula , Unión Proteica , Conformación Proteica , Espectrometría de Fluorescencia/métodosRESUMEN
The detection of allergen-specific IgE is of value for the diagnosis of children''s milk allergy. However, its accuracy will interfere with the presence of high levels of specific IgG in the serum of children with milk allergy. To solve this problem, we established a light-initiated chemiluminescent assay (LICA) based on nanomicrospheres, which neutralized the interference of specific IgG by increasing the amount of antigen coated on the microspheres. The ability of this method to resist IgG interference was confirmed by adding extra specific IgG to the serum of allergic patients. Finally, the positive rate of allergen-specific IgE was increased to 85%, which was better than the indirect ELISA (70%), indicating that this method has certain advantages for the detection of specific IgE in children with milk allergy.
Asunto(s)
Caseínas/inmunología , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina E/sangre , Inmunoglobulina G/sangre , Hipersensibilidad a la Leche/diagnóstico , Biomarcadores/sangre , Estudios de Casos y Controles , Humanos , Luz , Mediciones Luminiscentes , Hipersensibilidad a la Leche/sangre , Hipersensibilidad a la Leche/inmunología , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
Thermal treatment of food products leads to the formation of dietary advanced glycation endproducts (dAGEs). It was previously shown that dAGEs induce TNF-α secretion in human macrophage-like cells. To what extent gastrointestinal digestion of dAGEs influences these pro-inflammatory effects and what the implications of these pro-inflammatory characteristics further down the human gastrointestinal tract are, are currently unknown. In one of our previous studies, dAGEs were digested using the TNO gastroIntestinal Model and analysed for dAGE quantity after digestion. In the current study both digested and undigested dAGEs were used to expose human macrophage-like cells, which were subsequently analysed for TNF-α secretion. In addition, the obtained digests were fractionated, and human macrophage-like cells were exposed to the different fractions to determine whether specific fractions induce TNF-α secretion. The results show that digested dAGEs have an increased pro-inflammatory effect on human macrophage-like cells compared to undigested dAGEs. This paper therefore shows that the digestion of food-components, and specifically dAGEs, plays an important role in determining their biological activity.
Asunto(s)
Digestión/fisiología , Tracto Gastrointestinal/metabolismo , Productos Finales de Glicación Avanzada/inmunología , Productos Finales de Glicación Avanzada/metabolismo , Caseínas/inmunología , Caseínas/metabolismo , Línea Celular , Humanos , Macrófagos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: Inclusion of baked-milk products to the diet appears to markedly accelerate tolerance to unheated milk compared to a strict avoidance diet. OBJECTIVE: : The present study aims to investigate the predictors of baked-milk tolerance in children with Immunoglobulin E (IgE)-mediated cow's milk (CM) allergy. METHODS: The study included 80 patients diagnosed with IgE-mediated CM allergy upon oral food challenge (OFC) testing at our clinic. Patients who developed and did not develop reactions during OFC with baked milk were compared considering clinical and laboratory parameters. RESULTS: Eighty patients with CM allergy comprised 48 male and 32 female infants with an average age of 7.25 ± 2.45 (3-13) months. We found that 62.5% of them showed tolerance to baked milk in the OFC test performed with cakes containing 2.6-g milk protein. When the patients who tolerated and could not tolerate baked-milk products were compared for test results, we detected a statistically significant intergroup difference regarding diameter of wheal in skin prick test (SPT) performed with muffin slurry, levels of specific Immunoglobulin E (sIgE) in CM, sheep's milk (SM), goat's milk (GM), casein, and the amount of unheated milk consumed until a reaction developed in the OFC test performed with unheated milk (P < 0.05). CONCLUSION: We defined novel decision points based on CM, SM, GM, casein sIgE levels, wheal diameter in SPT with muffin slurry, and the amount of milk ingested during OFC performed with unheated milk that may be useful in predicting outcomes of baked-milk ingestion.
Asunto(s)
Alérgenos/administración & dosificación , Culinaria , Inmunoglobulina E/sangre , Hipersensibilidad a la Leche/diagnóstico , Leche/inmunología , Administración Oral , Alérgenos/efectos adversos , Alérgenos/inmunología , Animales , Caseínas/administración & dosificación , Caseínas/efectos adversos , Caseínas/inmunología , Femenino , Estudios de Seguimiento , Cabras , Humanos , Inmunoglobulina E/inmunología , Lactante , Masculino , Leche/efectos adversos , Leche/química , Hipersensibilidad a la Leche/sangre , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/prevención & control , Estudios Prospectivos , Ovinos , Pruebas CutáneasRESUMEN
The presence of various proteins, including modified ones, in food which exhibit diverse immunogenic and sensitizing properties increases the difficulty of predicting host immune responses. Still, there is a lack of sufficiently reliable and comparable data and research models describing allergens in dietary matrices. The aim of the study was to estimate the immunomodulatory effects of ß-lactoglobulin (ß-lg) in comparison to those elicited by κ-casein (κ-CN), in vivo and ex vivo, using naïve splenocytes and a mouse sensitization model. Our results revealed that the humoral and cellular responses triggered by ß-lg and κ-CN were of diverse magnitudes and showed different dynamics in the induction of control mechanisms. ß-Lg turned out to be more immunogenic and induced a more dominant Th1 response than κ-CN, which triggered a significantly higher IgE response. For both proteins, CD4+ lymphocyte profiles correlated with CD4+CD25+ and CD4+CD25+Foxp3+ T cells induction and interleukin 10 secretion, but ß-lg induced more CD4+CD25+Foxp3- Tregs. Moreover, ex vivo studies showed the risk of interaction of immune responses to different milk proteins, which may exacerbate allergy, especially the one caused by ß-lg. In conclusion, the applied model of in vivo and ex vivo exposure to ß-lg and κ-CN showed significant differences in immunoreactivity of the tested proteins (κ-CN demonstrated stronger allergenic potential than ß-lg), and may be useful for the estimation of allergenic potential of various food proteins, including those modified in technological processes.
Asunto(s)
Caseínas/inmunología , Lactoglobulinas/inmunología , Hipersensibilidad a la Leche/inmunología , Linfocitos T/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
Casein, ß-lactoglobulin and α-lactalbumin are major milk protein allergens. In the present study, the structural modifications and antigenic response of these bovine milk allergens as induced by non-thermal treatment by atmospheric cold plasma were investigated. Spark discharge (SD) and glow discharge (GD), as previously characterized cold plasma systems, were used for protein treatments. Casein, ß-lactoglobulin and α-lactalbumin were analyzed before and after plasma treatment using SDS-PAGE, FTIR, UPLC-MS/MS and ELISA. SDS-PAGE results revealed a reduction in the casein and α-lactalbumin intensity bands after SD or GD treatments; however, the ß-lactoglobulin intensity band remained unchanged. FTIR studies revealed alterations in protein secondary structure induced by plasma, particularly contents of ß-sheet and ß-turn. The UPLC-MS/MS results showed that the amino acid compositions decreased after plasma treatments. ELISA of casein and α-lactalbumin showed a decrease in antigenicity post plasma treatment, whereas ELISA of ß-lactoglobulin showed an increase in antigenicity. The study indicates that atmospheric cold plasma can be tailored to mitigate the risk of bovine milk allergens in the dairy processing and ingredients sectors.
Asunto(s)
Atmósfera/química , Caseínas/química , Caseínas/inmunología , Leche/química , Gases em Plasma/química , Proteína de Suero de Leche/química , Proteína de Suero de Leche/inmunología , Alérgenos/química , Alérgenos/inmunología , Animales , Bovinos , Leche/inmunologíaRESUMEN
BACKGROUND: Patients are commonly challenged with foods containing baked milk, for example muffins, yet little is known about the specific allergen content of muffins used in milk challenges or of the effect that baking has on allergenicity. OBJECTIVE: Our objective was to compare the levels of major milk allergens in uncooked and baked muffins using monoclonal immunoassays and IgE antibody binding before and after baking. METHODS: Uncooked and baked muffins were prepared using recipes from Mount Sinai and Imperial College. Allergen levels were compared by ELISA for Bos d 5 (ß-lactoglobulin) and Bos d 11 (ß-casein). IgE reactivity was assessed using sera from milk-sensitized donors in direct binding and inhibition ELISA. RESULTS: Bos d 5 was reduced from 680 µg/g in uncooked muffin mix to 0.17 µg/g in baked muffins, representing a >99% decrease after baking. Conversely, Bos d 11 levels in baked muffin remained high and only decreased by 30% from a mean of 4249 µg/g in uncooked muffin mix to 2961 µg/g when baked (~181 mg Bos d 11 per muffin). Baked muffins retained ~70% of the IgE binding to uncooked muffin mix. Baked muffin extract inhibited IgE binding to uncooked muffin mix by up to 80%, demonstrating retention of in vitro IgE reactivity. CONCLUSIONS AND CLINICAL RELEVANCE: High levels of Bos d 11 in baked muffins pose a risk for adverse reactions, especially in patients who have high anti-casein IgE antibodies.
Asunto(s)
Alérgenos/inmunología , Caseínas/inmunología , Calor , Inmunoglobulina E/inmunología , Lipocalinas/inmunología , Hipersensibilidad a la Leche/inmunología , Desnaturalización Proteica , Culinaria , Ensayo de Inmunoadsorción Enzimática , HumanosRESUMEN
There is no effective therapy for milk allergy. The role of lactic acid bacteria (LAB) and probiotics in protection against allergy-related outcomes is still under investigation. The aim of the study was to evaluate the immunomodulative and therapeutic potential of yogurt drinks in cow's milk allergy (CMA) management. We compared immunoreactivity of α-casein (α-CN), ß-casein (ß-CN), κ-casein (κ-CN), α-lactalbumin (α-LA), and ß-lactoglobulin (ß-LG) in 27 yogurt drinks fermented with different basic yogurt cultures, or yogurt cultures enriched with Lactobacillus plantarum and/or Bifidobacterium lactis strains, by competitive ELISA assay. Drinks with the lowest antigenic potential were used as allergoids for CMA therapy. BALB/c mice were sensitized via intraperitoneal injection of α-CN + ß-LG mixture with aluminum adjuvant, and gavaged with increasing doses of selected low-immunogenic drinks (YM-basic, or YM-LB-enriched with L. plantarum and B. lactis) to induce tolerance. Milk- or phosphate-buffered saline (PBS)-dosed mice served as controls. Compared to milk, the immunoreactivity of proteins in drinks increased or decreased, depending on the bacterial sets applied for fermentation. Only a few sets acted synergistically in reducing immunoreactivity. The selected low-immunogenic drinks stimulated allergic mice for profiling Th2 to Th1 response and acquire tolerance, and the effect was greater with YM-LB drink, which during long-lasting interventional feeding strongly increased the secretion of regulatory cytokines, i.e., IL-10 and TGF-ß, and IgA and decreased IL-4, IgE, and anti-(α-CN + ß-LG) IgG1. The studies revealed variations in the potency of yogurt bacteria to change allergenicity of milk proteins and the need for their strict selection to obtain a safe product for allergy sufferers. The YM-LB drink with reduced antigenic potential may be a source of allergoids used in the immunotherapy of IgE mediated CMA, but further clinical or volunteer studies are required.
Asunto(s)
Bacterias/inmunología , Tolerancia Inmunológica , Proteínas de la Leche/inmunología , Leche/microbiología , Probióticos/farmacología , Yogur/microbiología , Animales , Peso Corporal , Caseínas/inmunología , Ciego/microbiología , Citocinas/metabolismo , Conducta Alimentaria , Femenino , Fermentación , Microbioma Gastrointestinal , Inmunidad Humoral , Ratones Endogámicos BALB C , Ganglios Linfáticos Agregados/patología , Bazo/patología , Proteína de Suero de Leche/inmunologíaRESUMEN
Background: Early nutrition may influence the development of food allergies later in life. In the absence of breastfeeding, hydrolysates from cow's milk proteins (CMP) were indicated as a prevention strategy in at risk infants, but their proof of effectiveness in clinical and pre-clinical studies is still insufficient. Thanks to a validated mouse model, we then assessed specific and nonspecific preventive effects of administration of extensive hydrolysates from caseins (eHC) on the development of food allergy to CMP. The additional nonspecific effect of the probiotic Lactobacillus GG (LGG), commonly used in infant formula, was also assessed. Methods: Groups of young BALB/cByJ female mice were pretreated by repeated gavage either with PBS (control mice), or with PBS solution containing non-hydrolyzed milk protein isolate (MPI), eHC or eHC+LGG (eq. of 10 mg of protein/gavage). All mice were then experimentally sensitized to CMP by gavage with whole CM mixed with the Th2 mucosal adjuvant Cholera toxin. All mice were further chronically exposed to cow's milk. A group of mice was kept naïve. Sensitization to both caseins and to the non-related whey protein ß-lactoglobulin (BLG) was evaluated by measuring specific antibodies in plasma and specific ex vivo Th2/Th1/Th17 cytokine secretion. Elicitation of the allergic reaction was assessed by measuring mMCP1 in plasma obtained after oral food challenge (OFC) with CMP. Th/Treg cell frequencies in gut-associated lymphoid tissue and spleen were analyzed by flow cytometry at the end of the protocol. Robust statistical procedure combining non-supervised and supervised multivariate analyses and univariate analyses, was conducted to reveal any effect of the pretreatments. Results: PBS pretreated mice were efficiently sensitized and demonstrated elicitation of allergic reaction after OFC, whereas mice pretreated with MPI were durably protected from allergy to CMP. eHC+/-LGG pretreatments had no protective effect on sensitization to casein (specific) or BLG (non-specific), nor on CMP-induced allergic reactions. Surprisingly, eHC+LGG mice demonstrated significantly enhanced humoral and cellular immune responses after sensitization with CMP. Only some subtle changes were evidenced by flow cytometry. Conclusion: Neither specific nor nonspecific preventive effects of administration of casein-derived peptides on the development of CMP food allergy were evidenced in our experimental setup. Further studies should be conducted to delineate the mechanisms involved in the immunostimulatory potential of LGG and to clarify its significance in clinical use.
Asunto(s)
Caseínas/administración & dosificación , Lacticaseibacillus rhamnosus/fisiología , Hipersensibilidad a la Leche/prevención & control , Probióticos/administración & dosificación , Animales , Anticuerpos/sangre , Caseínas/inmunología , Células Cultivadas , Citocinas/sangre , Modelos Animales de Enfermedad , Femenino , Tracto Gastrointestinal/inmunología , Tracto Gastrointestinal/metabolismo , Tracto Gastrointestinal/microbiología , Inmunidad Celular , Inmunidad Humoral , Lacticaseibacillus rhamnosus/inmunología , Ratones Endogámicos C57BL , Hipersensibilidad a la Leche/sangre , Hipersensibilidad a la Leche/inmunología , Hipersensibilidad a la Leche/microbiología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Colaboradores-Inductores/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismoRESUMEN
Proteolytic starter cultures with intrinsic immunomodulatory activities are desirably features for the development of functional foods, which would significantly reduce the cost of their production (one-strain starter) having an additional beneficial effect on the host. In this work, Lactobacillus delbrueckii strains were selected according to their ability to efficiently hydrolyse ß-casein and to modulate the immune system. Among 36 strains evaluated, the highest proteolytic activities were found for L. delbrueckii subsp. lactis CRL581 and L. delbrueckii subsp. bulgaricus CRL656. The immunomodulatory effect of both strains and their ß-casein hydrolysates (CRL581 and CRL656 hydrolysates, respectively) were studied in a murine model. Balb/c mice were fed lactobacilli or their hydrolysates for three days. One day after the last lactobacilli or hydrolysate treatments, mice were challenged with the Toll-like receptor 3 (TLR3) agonist poly(I:C) by intraperitoneal injection. Before and after poly(I:C) challenge the phagocytic and microbicidal activity of peritoneal macrophages, intestinal immunoglobulin A (IgA), cytokine profile, and histological analysis of the intestine were analysed. L. delbrueckii subsp. lactis CRL581 significantly increased the activation of peritoneal macrophages as well as the levels of intestinal IgA, interleukin (IL)-10 and interferon (IFN)-γ when compared to untreated controls. In addition, the CRL581 strain was able to significantly reduce the intestinal inflammatory damage triggered by TLR3 activation. L. delbrueckii CRL581 increased the levels of IL-10, IFN-γ and IFN-ß, and reduced tumour necrosis factor alpha and IL-6 concentrations in the intestine of poly(I:C)-challenged mice. No immunomodulatory effects were observed for the CRL656 strain or for the CRL581 or CRL656 hydrolysates. The results of this work show that the technologically relevant and high proteolytic strain L. delbrueckii CRL581 is able to beneficially modulate the intestinal innate antiviral immune response. Although further studies with the CRL581 strain are required to corroborate and deepen its immunological effects, this bacterium is an interesting alternative for the development of new functional foods with antiviral capabilities.