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BACKGROUND: The pathological mechanism of the gastrointestinal forms of food allergies is less understood in comparison to other clinical phenotypes, such as asthma and anaphylaxis Importantly, high-IgE levels are a poor prognostic factor in gastrointestinal allergies. METHODS: This study investigated how high-IgE levels influence the development of intestinal inflammation and the metabolome in allergic enteritis (AE), using IgE knock-in (IgEki) mice expressing high levels of IgE. In addition, correlation of the altered metabolome with gut microbiome was analysed. RESULTS: Ovalbumin-sensitized and egg-white diet-fed (OVA/EW) BALB/c WT mice developed moderate AE, whereas OVA/EW IgEki mice induced more aggravated intestinal inflammation with enhanced eosinophil accumulation. Untargeted metabolomics detected the increased levels of N-tau-methylhistamine and 2,3-butanediol, and reduced levels of butyric acid in faeces and/or sera of OVA/EW IgEki mice, which was accompanied with reduced Clostridium and increased Lactobacillus at the genus level. Non-sensitized and egg-white diet-fed (NC/EW) WT mice did not exhibit any signs of AE, whereas NC/EW IgEki mice developed marginal degrees of AE. Compared to NC/EW WT mice, enhanced levels of lysophospholipids, sphinganine and sphingosine were detected in serum and faecal samples of NC/EW IgEki mice. In addition, several associations of altered metabolome with gut microbiome-for example Akkermansia with lysophosphatidylserine-were detected. CONCLUSIONS: Our results suggest that high-IgE levels alter intestinal and systemic levels of endogenous and microbiota-associated metabolites in experimental AE. This study contributes to deepening the knowledge of molecular mechanisms for the development of AE and provides clues to advance diagnostic and therapeutic strategies of allergic diseases.
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The human gut microbiome establishes and matures during infancy, and dysregulation at this stage may lead to pathologies later in life. We conducted a multi-omics study comprising three generations of family members to investigate the early development of the gut microbiota. Fecal samples from 200 individuals, including infants (0-12 months old; 55% females, 45% males) and their respective mothers and grandmothers, were analyzed using two independent metabolomics platforms and metagenomics. For metabolomics, gas chromatography and capillary electrophoresis coupled to mass spectrometry were applied. For metagenomics, both 16S rRNA gene and shotgun sequencing were performed. Here we show that infants greatly vary from their elders in fecal microbiota populations, function, and metabolome. Infants have a less diverse microbiota than adults and present differences in several metabolite classes, such as short- and branched-chain fatty acids, which are associated with shifts in bacterial populations. These findings provide innovative biochemical insights into the shaping of the gut microbiome within the same generational line that could be beneficial in improving childhood health outcomes.
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Microbioma Gastrointestinal , Lactente , Masculino , Adulto , Feminino , Humanos , Criança , Idoso , Recém-Nascido , Microbioma Gastrointestinal/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Multiômica , Metaboloma , Fezes/microbiologia , MãesRESUMO
The prevalence and severity of allergic diseases have increased over the last 30 years. Understanding the mechanisms responsible for these diseases is a major challenge in current allergology, as it is crucial for the transition towards precision medicine, which encompasses predictive, preventive, and personalized strategies. The urge to identify predictive biomarkers of allergy at early stages of life is crucial, especially in the context of major allergic diseases such as food allergy and atopic dermatitis. Identifying these biomarkers could enhance our understanding of the immature immune responses, improve allergy handling at early ages and pave the way for preventive and therapeutic approaches. This minireview aims to explore the relevance of three biomarker categories (proteome, microbiome, and metabolome) in early life. First, levels of some proteins emerge as potential indicators of mucosal health and metabolic status in certain allergic diseases. Second, bacterial taxonomy provides insight into the composition of the microbiota through high-throughput sequencing methods. Finally, metabolites, representing the end products of bacterial and host metabolic activity, serve as early indicators of changes in microbiota and host metabolism. This information could help to develop an extensive identification of biomarkers in AD and FA and their potential in translational personalized medicine in early life.
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Cow's milk allergy (CMA) is one of the most prevalent food allergies in children. Several studies have demonstrated that gut microbiota influences the acquisition of oral tolerance to food antigens at initial stages of life. Changes in the gut microbiota composition and/or functionality (i.e., dysbiosis) have been linked to inadequate immune system regulation and the emergence of pathologies. Moreover, omic sciences have become an essential tool for the analysis of the gut microbiota. On the other hand, the use of fecal biomarkers for the diagnosis of CMA has recently been reviewed, with fecal calprotectin, α-1 antitrypsin, and lactoferrin being the most relevant. This study aimed at evaluating functional changes in the gut microbiota in the feces of cow's milk allergic infants (AI) compared to control infants (CI) by metagenomic shotgun sequencing and at correlating these findings with the levels of fecal biomarkers (α-1 antitrypsin, lactoferrin, and calprotectin) by an integrative approach. We have observed differences between AI and CI groups in terms of fecal protein levels and metagenomic analysis. Our findings suggest that AI have altered glycerophospholipid metabolism as well as higher levels of lactoferrin and calprotectin that could be explained by their allergic status.
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Microbioma Gastrointestinal , Hipersensibilidade a Leite , Feminino , Animais , Bovinos , Leite/química , Lactoferrina/metabolismo , Hipersensibilidade a Leite/diagnóstico , Fezes/química , Biomarcadores/análiseRESUMO
Non-IgE-mediated gastrointestinal food allergy (non-IgE-GI-FA) is the name given to a series of pathologies whose main entities are food protein-induced allergic proctocolitis (FPIAP), food protein-induced enteropathy (FPE), and food protein-induced enterocolitis syndrome (FPIES). These are more uncommon than IgE-mediated food allergies, their mechanisms remain largely unknown, and their diagnosis is mainly done by clinical history, due to the lack of specific biomarkers. In this review, we present the latest advances found in the literature about clinical aspects, the current diagnosis, and treatment options of non-IgE-GI-FAs. We discuss the use of animal models, the analysis of gut microbiota, omics techniques, and fecal proteins with a focus on understanding the pathophysiological mechanisms of these pathologies and obtaining possible diagnostic and/or prognostic biomarkers. Finally, we discuss the unmet needs that researchers should tackle to advance in the knowledge of these barely explored pathologies.
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PURPOSE: To mimic the perioperative microenvironment where bacterial products get in contact with colorectal cancer (CRC) cells and study its impact on protein release, we exposed six CRC cell lines to lipopolysaccharide (LPS) and investigated the effect on the secretome using in-depth mass spectrometry-based proteomics. EXPERIMENTAL DESIGN: Cancer cell secretome was harvested in bio-duplicate after LPS treatment, and separated in EV and soluble secretome (SS) fractions. Gel-fractionated proteins were analysed by label-free nano-liquid chromatography coupled to tandem mass spectrometry. NF-κB activation, triggered upon LPS treatment, was evaluated. RESULTS: We report a CRC secretome dataset of 5601 proteins. Comparison of all LPS-treated cells with controls revealed 37 proteins with altered abundance in the SS, including RPS25; and 13 in EVs, including HMGB1. Comparing controls and LPS-treated samples per cell line, revealed 564 significant differential proteins with fold-change >3. The LPS-induced release of RPS25 was validated by western blot. CONCLUSIONS AND CLINICAL RELEVANCE: Bacterial endotoxin has minor impact on the global CRC cell line secretome, yet it may alter protein release in a cell line-specific manner. This modulation might play a role in orchestrating the development of a permissive environment for CRC liver metastasis, especially through EV-communication.
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LipopolissacarídeosRESUMO
The connection between gut microbiota and human health is becoming increasingly relevant and the number of groups working in this field is constantly growing. In this context, from high-throughput gene sequencing to metabolomics analysis, the omics technologies have contributed enormously to unveil the secret crosstalk between us and our microbes. All the omics technologies produce a great amount of information, and processing this information is time-consuming and expensive. For this reason, a correct experimental design and a careful pre-analytical planning are crucial. To study the human gut microbiota, faeces are the sample of choice. Faecal material is complex, and procedures for collecting and preserving faeces are not well-established. Furthermore, increasing evidence suggests that multiple confounding factors, such as antibiotics consumption, mode of delivery, diet, aging and several diseases and disorders can alter the composition and functionality of the microbiota. This review is focused on the discussion of critical general issues during the pre-analytical planning, from patient handling to faeces sampling, including collection procedures, transport, storage conditions and possible pre-treatments, which are critical for a successful research in omics with a special attention to metabolomics and gene sequencing. We also point out that the adoption of standard operating procedures in the field is needed to guarantee accuracy and reproducibility of results.
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Microbioma Gastrointestinal , Microbiota , Fezes , Microbioma Gastrointestinal/genética , Humanos , Metabolômica , Reprodutibilidade dos TestesRESUMO
BACKGROUND: In areas of high exposure to grass pollen, allergic patients are frequently sensitized to profilin, and some experience severe profilin-mediated food-induced reactions. This specific population of patients is ideal to study the relationship between respiratory and food allergies. OBJECTIVE: We sought to determine the role of oral mucosal epithelial barrier integrity in profilin-mediated allergic reactions. METHODS: Thirty-eight patients with profilin allergy stratified into mild or severe according to their clinical history and response to a profilin challenge test and 6 nonallergic subjects were recruited. Oral mucosal biopsies were used for measurement of CD11c, CD3, CD4, tryptase, claudin-1, occludin, E-cadherin, and vascular endothelial growth factor A levels; Masson trichrome staining; and POSTN, IL33, TPSAB, TPSB, and CMA gene expression analysis by using quantitative RT-PCR. Blood samples were used for basophil activation tests. RESULTS: Distinct features of the group with severe allergy included the following: (1) impaired epithelial integrity with reduced expression of claudin-1, occludin, and E-cadherin and decreased numbers of epithelial cells, which is indicative of acanthosis, higher collagen deposition, and angiogenesis; (2) inflammatory immune response in the mucosa, with an increased number of CD11c+ and CD4+ infiltrates and increased expression of the cytokine genes POSTN and IL33; and (3) a 10-fold increased sensitivity of basophils to profilin. CONCLUSIONS: Patients with profilin allergy present with significant damage to the oral mucosal epithelial barrier, which might allow profilin penetration into the oral mucosa and induction of local inflammation. Additionally, severely allergic patients presented with increased sensitivity of effector cells.
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Basófilos/imunologia , Hipersensibilidade Alimentar/imunologia , Mucosa Bucal/patologia , Hipersensibilidade Respiratória/imunologia , Junções Íntimas/patologia , Adulto , Alérgenos/imunologia , Claudina-1/genética , Claudina-1/metabolismo , Reações Cruzadas , Feminino , Humanos , Imunoglobulina E/metabolismo , Masculino , Pessoa de Meia-Idade , Poaceae/imunologia , Pólen/imunologia , Profilinas/imunologia , Adulto JovemRESUMO
Allergic diseases, such as respiratory, cutaneous, and food allergy, have dramatically increased in prevalence over the last few decades. Recent research points to a central role of the microbiome, which is highly influenced by multiple environmental and dietary factors. It is well established that the microbiome can modulate the immune response, from cellular development to organ and tissue formation exerting its effects through multiple interactions with both the innate and acquired branches of the immune system. It has been described at some extent changes in environment and nutrition produce dysbiosis in the gut but also in the skin, and lung microbiome, inducing qualitative and quantitative changes in composition and metabolic activity. Here, we review the potential role of the skin, respiratory, and gastrointestinal tract (GIT) microbiomes in allergic diseases. In the GIT, the microbiome has been proven to be important in developing either effector or tolerant responses to different antigens by balancing the activities of Th1 and Th2 cells. In the lung, the microbiome may play a role in driving asthma endotype polarization, by adjusting the balance between Th2 and Th17 patterns. Bacterial dysbiosis is associated with chronic inflammatory disorders of the skin, such as atopic dermatitis and psoriasis. Thus, the microbiome can be considered a therapeutical target for treating inflammatory diseases, such as allergy. Despite some limitations, interventions with probiotics, prebiotics, and/or synbiotics seem promising for the development of a preventive therapy by restoring altered microbiome functionality, or as an adjuvant in specific immunotherapy.
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The development of techniques and methods for allergen purification is essential for diagnosis and the development of safe immunotherapeutic agents. The most common purification techniques include chromatographic methodologies. In this chapter, we review and describe the details of the methodologies of using ion-exchange, gel-filtration, and affinity chromatography to purify two well-known panallergens, profilin and parvalbumin.
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Alérgenos/química , Alérgenos/isolamento & purificação , Produtos Biológicos/química , Cromatografia de Afinidade/métodos , Cromatografia em Gel/métodos , Cromatografia por Troca Iônica/métodos , HumanosRESUMO
Because lipids are hydrophobic, the development of efficient bioconversions in aqueous media free of organic solvents is particularly challenging for green oleochemistry. Within this aim, enzymes exhibiting various abilities to catalyze acyltransfer reaction in water/lipid systems have been identified. Among these, CpLIP2 from Candida parapsilosis has been characterized as a lipase/acyltransferase, able to catalyze acyltransfer reactions preferentially to hydrolysis in the presence of particularly low acyl acceptor concentration and high thermodynamic activity of water (aw>0.9). Lipase/acyltransferases are thus of great interest, being able to produce new esters at concentrations above the thermodynamic equilibrium of hydrolysis/esterification with limited to no release of free fatty acids. Here, we present a 3D model of CpLIP2 based on homologies with crystallographic structures of Pseudozyma antarctica lipase A. Indeed, the two enzymes have 31% of identity in their primary sequence, yielding a same general structure, but different catalytic properties. The quality of the calculated CpLIP2 model was confirmed by several methods. Limited proteolysis confirmed the location of some loops at the surface of the protein 3D model. Directed mutagenesis also supported the structural model constructed on CAL-A template: the functional properties of various mutants were consistent with their structure-based putative involvement in the oxyanion hole, substrate specificity, acyltransfer or hydrolysis catalysis and structural stability. The CpLIP2 3D model, in comparison with CAL-A 3D structure, brings insights for the elucidation and improvement of the structural determinants involved in the exceptional acyltransferase properties of this promising biocatalyst and of homologous enzymes of the same family.
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Aciltransferases/química , Candida/química , Proteínas Fúngicas/química , Lipase/química , Ácidos Palmíticos/química , Aciltransferases/genética , Aciltransferases/metabolismo , Sequência de Aminoácidos , Candida/enzimologia , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Hidrólise , Lipase/genética , Lipase/metabolismo , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Mutação , Pichia/genética , Pichia/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Conformação Proteica , Engenharia de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Mustard is a condiment added to a variety of foodstuffs and a frequent cause of food allergy. A new strategy for the detection of mustard allergen in food products is presented. The methodology is based on liquid chromatography analysis coupled to mass spectrometry. Mustard allergen Sin a 1 was purified from yellow mustard seeds. Sin a 1 was detected with a total of five peptides showing a linear response (lowest LOD was 5ng). Sin a 1 was detected in mustard sauces and salty biscuit (19±3mg/kg) where mustard content is not specified. Sin a 1, used as an internal standard, allowed quantification of this mustard allergen in foods. A novel LC/MS/MS SRM-based method has been developed to detect and quantify the presence of mustard. This method could help to detect mustard allergen Sin a 1 in processed foods and protect mustard-allergic consumers.
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Antígenos de Plantas/análise , Cromatografia Líquida/métodos , Análise de Alimentos/métodos , Espectrometria de Massas/métodos , Mostardeira/química , Proteínas de Plantas/análise , Antígenos de Plantas/imunologia , Hipersensibilidade Alimentar/imunologia , Mostardeira/imunologia , Proteínas de Plantas/imunologiaRESUMO
SCOPE: IgE-epitope mapping of allergens reveal important information about antigen components involved in allergic reactions. The peptide-based microarray immunoassay has been used to map epitopes of some food allergens. We developed a peptide microarray immunoassay to map allergenic epitopes in parvalbumin from Atlantic cod (Gad m 1), the most consumed cod species in Spain. METHODS AND RESULTS: Sera from 13 fish-allergic patients with specific IgE to cod parvalbumin were used. A library of overlapping peptides was synthesized, representing the primary sequence of Gad m 1. Peptides were used to analyze allergen-specific IgE antibodies in patient sera. 100% of the patients recognized one antigenic region of 15 amino acids in length in Gad m 1. This region only partially correlated with one of the three antigenic determinants of Gad c 1 (Allergen M), parvalbumin from Baltic cod (Gadus callarias). In the 3D model of the protein, this region was located on the surface of the protein. CONCLUSION: We have identified a relevant antigenic region in Gad m 1. This epitope could be considered as a severity marker and provides additional information to improve fish allergy diagnosis and the design of safe immunotherapeutic tools.
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Alérgenos/química , Mapeamento de Epitopos/métodos , Epitopos/química , Gadus morhua/imunologia , Imunoglobulina E/sangue , Adolescente , Adulto , Alérgenos/imunologia , Sequência de Aminoácidos , Animais , Antígenos/sangue , Antígenos/imunologia , Estudos de Casos e Controles , Criança , Epitopos/imunologia , Feminino , Hipersensibilidade Alimentar/diagnóstico , Hipersensibilidade Alimentar/imunologia , Humanos , Imunoensaio , Masculino , Dados de Sequência Molecular , Parvalbuminas/química , Parvalbuminas/imunologia , Conformação Proteica , Espanha , População Branca , Adulto JovemRESUMO
The prevalence of exotic pet allergies has been increasing over the last decade. Years ago, the main allergy-causing domestic animals were dogs and cats, although nowadays there is an increasing number of allergic diseases related to insects, rodents, amphibians, fish, and birds, among others. The current socio-economic situation, in which more and more people have to live in small apartments, might be related to this tendency. The main allergic symptoms related to exotic pets are the same as those described for dog and cat allergy: respiratory symptoms. Animal allergens are therefore, important sensitizing agents and an important risk factor for asthma. There are three main protein families implicated in these allergies, which are the lipocalin superfamily, serum albumin family, and secretoglobin superfamily. Detailed knowledge of the characteristics of allergens is crucial to improvement treatment of uncommon-pet allergies.
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BACKGROUND: IgE epitope mapping of allergens reveals important information about antigen elicitors involved in allergic reactions. The peptide-based microarray immunoassay offers an advantage of scale and parallel design over previous methods of epitope mapping. It has been used to map epitopes of some food allergens but has never been used with fish allergens. OBJECTIVE: We sought to develop a peptide microarray immunoassay to map allergenic fish epitopes of two isoforms of Atlantic salmon (Salmo salar) parvalbumin, Sal s 1 beta 1 and Sal s 1 beta 2. METHODS: Sera from 16 fish-allergic patients with specific IgE to salmon parvalbumin were used. Twelve healthy volunteers were used as negative controls. A library of overlapping peptides was synthesized commercially, representing the primary sequence of Sal s 1 beta 1 and Sal s 1 beta 2. Peptides were used to analyze allergen-specific IgE antibodies by immunolabeling with patient sera. RESULTS: Three antigenic regions, not previously described, were identified in Sal s 1 beta 1. Two of them correlated with those previously reported in Gad c 1, parvalbumin from Baltic cod (Gadus callarias). No allergenic regions were found in Sal s 1 beta 2. This could be explained by crucial amino acid substitutions between isoforms. CONCLUSIONS: We have identified three antigenic regions in Sal s 1 beta 1 using a peptide microarray immunoassay. These three sequential epitopes formed a unique antigenic determinant in the three-dimensional model of the protein. In addition, we proved that isoforms from the same protein might have a different allergenic behavior.
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Alérgenos/imunologia , Mapeamento de Epitopos/métodos , Imunoensaio , Análise em Microsséries , Peptídeos/imunologia , Salmo salar/imunologia , Adolescente , Adulto , Sequência de Aminoácidos , Animais , Criança , Epitopos/química , Epitopos/imunologia , Feminino , Humanos , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Conformação Proteica , Adulto JovemRESUMO
BACKGROUND: Plant profilins have been reported as minor allergens. They are a well-known pan-allergen family responsible for cross-reactivity between plant-derived foods and pollens. Watermelon profilin has been reported to be a major allergen in watermelon (Citrullus lanatus).The aim of this study was to characterize recombinant watermelon profilin, confirming its reactivity for diagnostic purposes and the development of immunotherapy. METHODS: Native profilin was purified from watermelon extract by affinity chromatography using poly-L-proline. Recombinant His-tagged profilin was produced in Pichia pastoris yeast using pPICZαA vector and purified by metal chelate affinity chromatography. ELISA and immunoblot were carried out with sera from 17 watermelon-allergic patients. Biological activity was tested by the basophil activation test. RESULTS: Native profilin and recombinant profilin were purified and identified by mass spectrometry. Both show similar IgE reactivity in vitro and are biologically active. CONCLUSIONS: Similarities were found in the IgE-binding patterns and biological activity of recombinant profilin and native profilin. Recombinant profilin may be a powerful tool for specific diagnosis.