Facultad de Medicina

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.

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.

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,

Background and Aim: Caloric restriction (CR) improves insulin sensitivity and is one of the dietetic strategies most commonly used to enlarge life and to prevent aging-induced cardiovascular alterations. The aim of this study was to analyze the possible beneficial effects of caloric restriction (CR) preventing the aging-induced insulin resistance in the heart of male Wistar rats. Methods and results: Three experimental groups were used: 3 months old rats (3m), 24 months old rats (24m) and 24 months old rats subjected to 20% CR during their three last months of life (24m-CR). After sacrifice hearts were mounted in a perfusion system (Langendorff) and heart function in basal conditions and in response to accumulative doses of insulin (10 9-10 7 M), in the presence or absence of Wortmannin (10 6 M), was recorded. CR did not attenuate the aging-induced decrease in coronary artery vasodilation in response to insulin administration, but it prevented the aging-induced downregulation of cardiac contractility (dp/dt) through activation of the PI3K/Akt intracellular pathway. Insulin stimulated in a greater extent the PI3K/Akt pathway vs the activation of the MAPK pathway and increased the protein expression of IR, GLUT-4 and eNOS in the hearts of 3m and 24m-CR rats, but not in the hearts of 24m rats. Furthermore, CR prevented the aging induced increase in endothelin-1 protein expression in myocardial tissue. Conclusion: In conclusion CR partially improves cardiac insulin sensitivity and prevents the aging induced decrease in myocardial contractility in response to insulin administration through activation of PI3K/Akt pathway.