Facultad de Medicina

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.

Omics strategies have triggered a revolution in the understanding of the microorganisms that reside in our body, and their implications in health and disease. For diagnosis and therapeutics, metabolomic fingerprinting is the most powerful approach, since the metabolites represent the actual interplay between humans and microbes. Studying the metabolome requires several new high-throughput analytical techniques and innovative computational methodologies. Herein, we will focus on the metabolomics workflow for gut microbiota analysis, including sampling, laboratory procedures, and available analytical techniques, paying special attention to microbiota isolation and multiplatform complementarity. Finally, we will summarize some applications and implications of gut microbiota metabolites in biomarkers discovery and several therapeutic strategies, such as fecal microbiota transplantation and the usage of prebiotics and probiotics.

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.)

Background: The use of contact lenses has increased in recent years as has the incidence of Dry Eye Syndrome, partly due to their use. Artificial tears are the most common treatment option. Since these changes can facilitate Acanthamoeba infection, the present study has been designed to evaluate the effect of three artificial tears treatments in the viability of Acanthamoeba genotype T4 trophozoites. Optava Fusion™, Oculotect®, and Artelac® Splash were selected due to their formulation. Methods: Viability was assessed using two staining methods, Trypan Blue stain and CTC stain at different time intervals (2, 4, 6, 8 and 24 h). Trypan Blue viability was obtained by manual count with light microscopy while the CTC stain was determined using flow cytometry. Results: Trypan Blue staining results demonstrated a decrease in viability for Optava Fusion™ and Artelac® Splash during the first 4 h of incubation. After, this effect seems to lose strength. In the case of Oculotect®, complete cell death was observed after 2 h. Using flow cytometry analysis, Optava Fusion™ and Oculotect® exhibited the same effect observed with Trypan Blue staining. However, Artelac® Splash revealed decreasing cell respiratory activity after four hours, with no damage to the cell membrane. Conclusions: The present study uses, for the first time, CTC stain analyzed by flow cytometry to establish Acanthamoeba viability demonstrating its usefulness and complementarity with the traditional stain, Trypan Blue. Artelac® Splash, with no preservatives, and Optava Fusion TM, with Purite®, have not shown any useful amoebicidal activity. On the contrary, promising results presented by Ocultect®, with BAK, open up a new possibility for Acanthamoeba keratitis prophylaxis and treatment although in vivo studies should be carried out.

Purpose: To assess variability in the use of Tomita and modified Bauer scores in spine metastases. Materials and methods: Clinical data and imaging from 90 patients with biopsy-proven spinal metastases, were provided to 83 specialists from 44 hospitals. Spinal levels involved and the Tomita and modified Bauer scores for each case were determined twice by each clinician, with a minimum of 6-week interval. Clinicians were blinded to every evaluation. Kappa statistic was used to assess intra and inter-observer agreement. Subgroup analyses were performed according to clinicians' specialty (medical oncology, neurosurgery, radiology, orthopedic surgery and radiation oncology), years of experience (⩽7, 8-13, ⩾14), and type of hospital (four levels). Results: For metastases identification, intra-observer agreement was "substantial" (0.600.80) at the other levels. Inter-observer agreement was "almost perfect" at lumbar spine, and "substantial" at the other levels. Intra-observer agreement for the Tomita and Bauer scores was almost perfect. Inter-observer agreement was almost perfect for the Tomita score and substantial for the Bauer one. Results were similar across specialties, years of experience and type of hospital. Conclusion: Agreement in the assessment of metastatic spine disease is high. These scoring systems can improve communication among clinicians involved in oncology care.

Objectives: Calcifying pseudoneoplasms of the neuraxis (CAPNON) are rare lesions occurring anywhere in the central nervous system (CNS). Since their description, only 55 cases have been reported. We present the largest series reviewing their imaging features, histology and potential origins. Patients and methods: four patients with histopathologically verified CAPNON are presented. Subsequently, we review all reports published with respect to study type, number of patients, clinical presentation, anatomical area (intracranial, spinal, or both), radiological features, therapy, histopathologic features, duration of follow-up, complications, and outcome. Moreover, current management of CNS CAPNON are discussed. Autopsy patients were excluded. Results: Four patients with histopathologically verified diagnosis of CAPNON are presented between 46-73 years-old. Three of them were located in the spinal cord (levels C3, D2, and L2) and one intracranial (left atrium). The spine ones were diagnosed due to radicular pain, paraparesis and numbness in lower limb, the intracranial because of intense headache. The differential diagnosis included cavernous malformation, in the case of the lumbar CAPNON this suspicion put back the surgery six months. All cases were surgically treated with complete resection. No recurrence showed at the 12-month follow-up. A total of retrospective 30 articles were selected: 10 case series (33.33%) and 20 reports of single cases (66.66%). The 30 articles and our additional cases added up to a total of 27 patients with spinal CAPNON and 32 patients with intracranial CAPNON. All patients were treated surgically. A follow-up, conducted in 48 patients, showed no signs of recurrence in 46 of the 48. Conclusions: Calcifying pseudoneoplasms are rare benign lesions of yet unknown origin. They should be taken into consideration in the differential diagnosis of calcified lesions because an inaccurate diagnosis can result in potentially harmful and unnecessary therapies, as prognosis for these lesions is generally favorable.

Pediatric Central Nervous System (CNS) tumors are the most fatal cancer diseases in childhood. Due to their localization and infiltrative nature, some tumor resections or biopsies are not feasible. In those cases, the use of minimally invasive methods as diagnostic, molecular marker detection, prognostic or monitoring therapies are emerging. The analysis of liquid biopsies which contain genetic information from the tumor has been much more widely explored in adults than in children. We compare the detection of BRAF V600E targetable mutation by digital-PCR from cell-free-DNA and EV-derived DNA (ctDNA) in serum, plasma and cerebrospinal fluid (CSF) isolated from a cohort of 29 CNS pediatric patients. Here we demonstrate that ctDNA isolated from serum and plasma could be successfully analyzed to obtain tumor genetic information which could be used to guide critical treatment decisions.

The aim of this work was to study if early overnutrition in rats is associated with cardiovascular insulin resistance in adulthood. For that purpose we used the experimental model of litter reduction. At birth, rats were organized either in litters of 12 pups/mother (L12-controls) or in litters of 3 pups/mother (L3-Overfed). After weaning rats were fed ad libitum with a standard chow and sacrificed at the age of 6 months. After sacrifice, hearts were set into a Langendorff system whereby increasing insulin doses were administered and coronary perfusion pressure, heart rate and heart contractility were recorded. Likewise, 2mm rings of aorta were mounted in an organ bath whereby changes in vascular tension in response to increasing insulin concentrations were recorded. To assess the activation of the two main pathways involved in insulin intracellular signalling, total proteins were obtained from myocardial and arterial tissues and the MAPK/Akt expression and activation in response to insulin were analyzed. Myocardial contractility in response to insulin was significantly decreased in hearts from overfed rats due to a decreased activation of the PI3K/Akt. On the contrary, in the vascular reactivity experiments insulin induced a higher vasodilation in aorta segments from L3 rats that was not mediated by the activation of the PI3K/Akt pathway and the subsequent release of nitric oxide. In conclusion, overfeeding during lactation in rats induces alterations in vascular function in response to circulating hormones like insulin. This fact could be related with the cardiovascular alterations reported in this experimental mode,