1. Investigación

Las alergias alimentarias son cada vez más prevalentes y de mayor gravedad, y la alergia a las proteínas de la leche de vaca (APLV) es una de las más importantes en lactantes. Varios estudios han sugerido que desequilibrios en la microbiota intestinal, posiblemente influenciados por la microbiota materna, juegan un papel importante en el desarrollo de APLV. La metabolómica ofrece un enfoque prometedor, ya que examina los cambios metabólicos en patologías como las alergias alimentarias. La metabolómica fecal, en concreto, es ideal para estudiar la microbiota intestinal en la APLV. Con este objetivo, se recogieron muestras fecales de lactantes con APLV, sus madres y sus abuelas, y se compararon con los respectivos grupos control. Se recogieron cuestionarios detallados para recabar información sobre variables como la dieta, el modo de nacimiento, el uso de antibióticos y los antecedentes alérgicos. El análisis metabolómico se llevó a cabo en la cohorte final de 200 muestras mediante cromatografía de gases acoplada a espectrometría de masas con analizador de cuadrupolo-tiempo de vuelo (GC-QTOF-MS) y electroforesis capilar con inyección multisegmento acoplada a TOF-MS (MSI-CE-TOF-MS). Con todos estos datos, la tesis se dividió en dos capítulos: el Capítulo 1 presenta la integración de los datos de metabolómica fecal y metagenómica (incluyendo secuenciación del gen 16S rRNA y shotgun) en una prueba de concepto donde se compararon los tres grupos de edad, mientras que en el Capítulo 2 se presentan las diferencias en el metaboloma fecal de los lactantes debidas a la APLV, la dieta y el modo de nacimiento. En conjunto, esta tesis aporta información valiosa sobre el desarrollo del microbioma y los metabolitos asociados a lo largo de la vida. Se necesitan más estudios para arrojar más luz sobre la interacción entre la microbiota y el desarrollo de la APLV, especialmente estudios con fenotipos más graves y el seguimiento de varios puntos temporales de recogida de muestras.

Purpose of Review Allergic diseases have become a burden in industrialized societies. Among children, food allergy (FA) constitutes a major impairment of quality of life. FA is partly due to a lack or loss of tolerance to food antigens at the level of the intestinal mucosa, where the microbiota plays a crucial role. Early changes in the composition of the gut microbiota may influence the development of the immune system and can be related to the risk of allergic diseases, including FA. This review will focus on the role of sphingolipids and the major bacteria involved in their metabolism, in the development of food antigen sensitization and FA. Recent Findings Numerous studies have identified different patterns of microbial composition between individuals with and without FA, pointing to an interaction between gut microbiota, enterocytes, and immune cells. When this interaction is lost and an imbalance in the composition of the intestinal microbiota occurs, the integrity of the epithelial barrier may be altered, leading to intestinal permeability and sensitization to food antigens and the development of FA. Gram- negative bacteria, especially those of the Proteobacteria phylum, have been associated with the development of FA. Investigating the interactions between the intestinal microbiota and the immune system, their influence on intestinal barrier function, and their production of metabolites and signaling molecules may contribute to understanding the pathogenesis of FA. Summary Sphingolipids, a class of bioactive amphipathic lipids found in cell membranes, have emerged as critical regulators of inflammation. In this review, we will attempt to summarize the existing knowledge on the role of these molecules and the major bacteria involved in their metabolism in the mechanisms underlying sensitization to food antigens and the development of FA.

Allergic asthma is a prominent disease especially during childhood. Indoor allergens, in general, and particularly house dust mites (HDM) are the most prevalent sensitizers associated with allergic asthma. Available data show that 65–130 million people are mite-sensitized world-wide and as many as 50% of these are asthmatic. In fact, sensitization to HDM in the first years of life can produce devastating effects on pulmonary function leading to asthmatic syndromes that can be fatal. To date, there has been considerable research into the pathological pathways and structural changes associated with allergic asthma. However, limitations related to the disease heterogeneity and a lack of knowledge into its pathophysiology have impeded the generation of valuable data needed to appropriately phenotype patients and, subsequently, treat this disease. Here, we report a systematic and integral analysis of the disease, from airway remodelling to the immune response taking place throughout the disease stages. We present an overview of metabolomics, the management of complex multifactorial diseases through the analysis of all possible metabolites in a biological sample, obtaining a global interpretation of biological systems. Special interest is placed on the challenges to obtain biological samples and the methodological aspects to acquire relevant information, focusing on the identification of novel biomarkers associated with specific phenotypes of allergic asthma. We also present an overview of the metabolites cited in the literature, which have been related to inflammation and immune response in asthma and other allergy-related diseases.

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.

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.

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 CD11c1 and CD41 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. (J Allergy Clin Immunol 2019;143:681-90.)

Vaccines are essential public health tools with a favorable safety profile and prophylactic effectiveness that have historically played significant roles in reducing infectious disease burden in populations, when the majority of individuals are vaccinated. The COVID-19 vaccines are expected to have similar positive impacts on health across the globe. While serious allergic reactions to vaccines are rare, their underlying mechanisms and implications for clinical management should be considered to provide individuals with the safest care possible. In this review, we provide an overview of different types of allergic adverse reactions that can potentially occur after vaccination and individual vaccine components capable of causing the allergic adverse reactions. We present the incidence of allergic adverse reactions during clinical studies and through post-authorization and post-marketing surveillance and provide plausible causes of these reactions based on potential allergenic components present in several common vaccines. Additionally, we review implications for individual diagnosis and management and vaccine manufacturing overall. Finally, we suggest areas for future research.

El asma puede presentar múltiples fenotipos, como el asma alérgico o no alérgico. Su tratamiento es complejo, existiendo pacientes graves que no responden a las medicaciones actualmente disponibles, sufren exacerbaciones frecuentes y presentan comorbilidades como la poliposis nasosinusal. La estratificación de pacientes mediante el uso de biomarcadores permitiría mejorar el tratamiento y descubrir nuevas dianas terapéuticas. Para identificar biomarcadores de estratificación por gravedad, se estudiaron pacientes asmáticos alérgicos estratificados por gravedad utilizando metabolómica y proteómica. Los pacientes con asma grave no controlado presentaron una activación característica de las rutas del ácido araquidónico, la fosfolipasa A2, y la respuesta Th2. Además, para evaluar la contribución del fenotipo alérgico al asma, se realizó un análisis metabolómico de pacientes graves no controlados con y sin alergia. Los pacientes asmáticos alérgicos mostraron una activación de la ruta de la fosfolipasa A2 y una alteración en el perfil de ácidos biliares. Por último, para conocer el papel sistémico y local de la alergia en la poliposis nasal, se estudiaron pacientes con poliposis con y sin alergia utilizando metabolómica de suero y tejido y análisis histológicos. Los pacientes alérgicos presentaron niveles reducidos de lisofosfolípidos, bilirrubina y cortisol; y un mayor número de eosinófilos en sus pólipos.

Background: Mechanisms causing the onset and perpetuation of inflammation in severe allergic patients remain unknown. Our previous studies suggested that severe allergic inflammation is linked to platelet dysfunction. Methods: Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) samples were obtained by platelet-apheresis from severe (n = 7) and mild (n = 10) allergic patients and nonallergic subjects (n = 9) to perform platelet lipidomics by liquid chromatography coupled to mass spectrometry (LC–MS) and RNA-seq analysis. Significant metabolites and transcripts were used to identify compromised biological pathways in the severe phenotype. Platelet and inflammation-related proteins were quantified by Luminex. Results: Platelets from severe allergic patients were characterized by high levels of ceramides, phosphoinositols, phosphocholines, and sphingomyelins. In contrast, they showed a decrease in eicosanoid precursor levels. Biological pathway analysis performed with the significant lipids revealed the alteration of phospholipases, calcium-dependent events, and linolenic metabolism. RNAseq confirmed mRNA overexpression of genes related to platelet activation and arachidonic acid metabolism in the severe phenotypes. Pathway analysis indicated the alteration of NOD, MAPK, TLR, TNF, and IL-17 pathways in the severe phenotype. P-Selectin and IL-17AF proteins were increased in the severe phenotype. Conclusions: This study demonstrates that platelet lipid, mRNA, and protein content is different according to allergy severity. These findings suggest that platelet load is a potential source of biomarkers and a new chance for therapeutic targets in severe inflammatory pathologies.

La alergia es una enfermedad crónica en la que el sistema inmune reacciona de una manera desmesurada contra una sustancia externa del ambiente. A pesar de que recientemente se han hecho muchos descubrimientos en la fisiopatología alérgica, los mecanismos moleculares que la controlan no son del todo conocidos. Previamente descubrimos que pacientes alérgicos graves presentaban manifestaciones clínicas no descritas con anterioridad incluyendo un extenso remodelado en la mucosa oral y cambios metabólicos a nivel sistémico que implicaban a los linfocitos T. Debido a ello, hipotetizamos que los cambios observado en la mucosa y el metabolismo en pacientes alérgicos graves no solo están asociados con la fisiopatología alérgica si no que dictan la respuesta alérgica. Para comprobarlo, primero analizamos si los cambios en la barrera epitelial respiratoria tienen asociados cambios metabólicos a su vez que puedan ser utilizados potencialmente como biomarcadores de su estado. Además, comprobamos si los cambios metabólicos sistémicos observados previamente tienen algún efecto sobre la función de los linfocitos T y el desarrollo de la enfermedad. En esta tesis se concluyó que los cambios metabólicos en mucosa respiratoria y células T puedan están estrechamente relacionados en la patofisiología alérgica.