PTGDR gene expression and response to dexamethasone treatment in an in vitro model

[EN]Asthma is a multifactorial pathology influenced by environmental and genetic factors. Glucocorticoid treatment decreases symptoms by regulating genes involved in the inflammatory process through binding to specific DNA sequences. Polymorphisms located in the promoter region of the Prostaglandin D Receptor (PTGDR) gene have been related to asthma. We aimed to analyze the effect of PTGDR promoter haplotypes on gene expression and response to corticosteroid therapy. A549 lung epithelial cells were transfected with vectors carrying four different PTGDR haplotypes (CTCT, CCCC, CCCT and TCCT), and treated with dexamethasone. Different approaches to study the promoter activity (Dual Luciferase Reporter System), gene expression levels (qPCR) and cytokine secretion (Multiplexed Bead-based Flow Cytometric) were used. In addition, in silico analysis was also performed. Cells carrying the TCCT haplotype showed the lowest promoter activity (p-value<0.05) and mRNA expression levels in basal conditions. After dexamethasone treatment, cells carrying the wild-type variant CTCT showed the highest response, and those carrying the TCCT variant the lowest (p-value<0.05) in luciferase assays. Different transcription factor binding patterns were identified in silico. Moreover, differences in cytokine secretion were also found among different promoter haplotypes. Polymorphisms of PTGDR gene influence basal promoter activity and gene expression, as well as the cytokine secretory pattern. Furthermore, an association between these positions and response to corticoid treatment was observed

PTGDR expression is upregulated through retinoic acid receptors (RAR) mechanism in allergy

[EN] Functional studies suggest that promoter polymorphisms of the Prostaglandin D Receptor (PTGDR) gene can be involved in asthma. All-trans Retinoic acid (ATRA) has also been linked to allergic diseases. We have previously described the PTGDR promoter activation mediated by ATRA through response elements (RARE) at position -549T> C. In this study we aimed to analyze the effect of retinoic acid (RA) on the expression of PTGDR, the production of cytokines as well as to evaluate the binding of RA receptors to RA-Response Elements (RARE) sequences. A549 cells were transfected with vectors carrying different PTGDR haplotypes and treated with all-Trans Retinoic Acid (ATRA). PTGDR expression was measured by qPCR. Chromatin Immunoprecipitation assays (ChIP) were performed in ATRA stimulated KU812 cells and in PBMCs of patients carrying CTCT, CCCC or CCCT haplotypes. In addition, a broad panel of cytokines was analyzed by cytometric bead assay in A549 cells. The expression of PTGDR increased in A549 cells transfected with PTGDR-variants. The CCCC haplotype showed a significantly higher expression compared with CTCT. However, we found that RA up-regulated PTGDR expression through RARα mainly in the CTCT variant. Experiments on PBMCs from allergic patients carrying the -549T and -549C variant of the PTGDR promoter after ATRA and RAR antagonist administration confirmed the modulation of PTGDR by ATRA. The cytokine analysis showed that IL4 and IL6 levels were significantly increased in A549 cells transfected with PTGDR. In addition, ATRA treatment decreased the levels of IL4, IL6 and TNFα in A549 cells, whereas it increased IL4 and TNFα levels in PTGDR-transfected cells. We observed genetic differences in the regulation of PTGDR by ATRA that could contribute to the phenotypic differences observed in allergic patients. Our findings showed that RAR modulation by PTGDR might have an impact on Th2 responses, suggesting that RAR could be a potential therapeutic target in allergic inflammation.Instituto de Salud Carlos III (ISCIII); Fondo Europeo de Desarrollo Regional-FEDER; Consejería de Salud-Junta de Castilla y Leó

Retinoic Acid Modulates PTGDR Promoter Activity

[EN] Background and objective: Vitamin A has been linked to the development of allergic diseases although its role is not fully understood, Retinoic acid (RA), a metabolite of Vitamin A, has been previously associated with the prostaglandin pathway, and PTGDR, a receptor of PGD2, has been proposed as a candidate gene in allergy and asthma. Considering the role of PTGDR in allergy, the goal of this study was to analyze the effect of RA on the activation of the promoter region of the PTGDR gene. Methods: A549 lung epithelial cells were transfected with 4 combinations of genetic variants of the PTGDR promoter and stimulated with all-trans RA (ATRA); luciferase assays were performed using the Dual Luciferase Reporter System, and real-time quantitative polymerase chain reaction was used to measure the expression of PTGDR, CYP26A1, RARA, RARB, RARG, and RXRA in basal A549 cell cultures and after ATRA treatment. We also performed an in silico analysis. Results: After ATRA treatment increased expression of CYP26A1 (12-fold) and RARB (4-fold) was detected. ATRA activated PTGDR promoter activity in transfected cells (P<.001) and RA response element sequences were identified in silico in this promoter region. Conclusions: RA modulated PTGDR promoter activity. Differential response to RA and to new treatments based on PTGDR modulation could depend on genetic background in allergic asthmatic patients.Instituto de Salud Carlos III; European Regional Development Fund (ERDF); Junta de Castilla y León; Fundación Botín-Universidad de Salamanca; Sociedad Española de Alergología e Inmunología Clínica; Fundación Salud 200

YRNAs overexpression and potential implications in allergy

[EN] Background Small non-coding RNAs (snRNAs) develop important functions related to epigenetic regulation. YRNAs are snRNAs involved in the initiation of DNA replication and RNA stability that regulate gene expression. They have been related to autoimmune, cancer and inflammatory diseases but never before to allergy. In this work we described for the first time in allergic patients the differential expression profile of YRNAs, their regulatory mechanisms and their potential as new diagnostic and therapeutic targets. Methods From a previous whole RNAseq study in B cells of allergic patients, differential expression profiles of coding and non-coding transcripts were obtained. To select the most differentially expressed non coding transcripts, fold change and p-values were analyzed. A validation of the expression differences detected was developed in an independent cohort of 304 individuals, 208 allergic patients and 96 controls by using qPCR. Potential binding and retrotransponibility capacity were characterized by in silico structural analysis. Using a novel bioinformatics approach, RNA targets identification, functional enrichment and network analyses were performed. Results We found that almost 70% of overexpressed non-coding transcripts in allergic patients corresponded to YRNAs. From the three more differentially overexpressed candidates, increased expression was independently confirmed in the peripheral blood of allergic patients. Structural analysis suggested a protein binding capacity decrease and an increase in retrotransponibility. Studies of RNA targets allowed the identification of sequences related to the immune mechanisms underlying allergy. Conclusions Overexpression of YRNAs is observed for the first time in allergic patients. Structural and functional information points to their implication on regulatory mechanisms of the disease.Instituto de Salud Carlos III; European Regional Development Fund; Junta de Castilla y León; Consolidated Research Unit of Castilla y León; Ramón y Cajal program; FEDER/Ministerio de Ciencia, Innovación y Universidades - Agencia Estatal de Investigació

Increased TPSAB1 Copy Number in a Family With Elevated Basal Serum Levels of Tryptase

[EN]Background: Some recent familial studies have described a pattern of autosomal dominant inheritance for increased basal serum tryptase (BST), but no correlation with mRNA expression and gene dose have been reported. Objective: We analyzed TPSAB1 mRNA expression and gene dose in a four-member family with high BST and in two control subjects. Methods: Blood samples were collected from the family and control subjects. Complete morphologic, immunophenotypical, and molecular bone marrow mast cell (MC) studies were performed. mRNA gene expression and gene dose were performed in a LightCycler 480 instrument. Genotype and CNV were performed by quantitative real-time digital PCR (qdPCR). Results: CNV analysis revealed a hereditary copy number gain genotype (3β2α) present in all the family members studied. The elevated total BST in the family members correlated with a significant increase in tryptase gene expression and dose. Conclusions and Clinical Relevance: We present a family with hereditary α-tryptasemia and elevated BST which correlated with a high expression of tryptase genes and an increased gene dose. The family members presented with atypical MC-mediator release symptoms or were even asymptomatic. Clinicians should be aware that elevated BST does not always mean an MC disorder.Spanish Foundation of the Spanish Association of Allergy and Clinical Immunology (Sociedad Española de Alergologia e Inmunologia Clínica); the Council of Castilla y León; European Social Fund, the Immunoallergic Association of Salamanca; Asthma, Allergic, and Adverse Reactions networks for Cooperative Research in Health (ARADyAL); Instituto de Salud Carlos III; European Regional Development Fund (ERDF)

Application of a Pharmacogenetics-Based Precision Medicine Model (5SPM) to Psychotic Patients That Presented Poor Response to Neuroleptic Therapy

[EN] Antipsychotics are the keystone of the treatment of severe and prolonged mental disorders. However, there are many risks associated with these drugs and not all patients undergo full therapeutic profit from them. The application of the 5 Step Precision Medicine model(5SPM), based on the analysis of the pharmacogenetic profile of each patient, could be a helpful tool to solve many of the problematics traditionally associated with the neuroleptic treatment. In order to solve this question, a cohort of psychotic patients that showed poor clinical evolution was analyzed. After evaluating the relationship between the prescribed treatment and pharmacogenetic profile of each patient, a great number of pharmacological interactions and pharmacogenetical conflicts were found. After reconsidering the treatment of the conflictive cases, patients showed a substantial reduction on mean daily doses and polytherapy cases, which may cause less risk of adverse effects, greater adherence, and a reduction on economic costs

Ten Years of Experience Support Pharmacogenetic Testing to Guide Individualized Drug Therapy

[EN]Precision medicine utilizing the genetic information of genes involved in the metabolism and disposition of drugs can not only improve drug efficacy but also prevent or minimize adverse events. Polypharmacy is common among multimorbid patients and is associated with increased adverse events. One of the main objectives in health care is safe and efficacious drug therapy, which is directly correlated to the individual response to treatment. Precision medicine can increase drug safety in many scenarios, including polypharmacy. In this report, we share our experience utilizing precision medicine over the past ten years. Based on our experience using pharmacogenetic (PGx)-informed prescribing, we implemented a five-step precision medicine protocol (5SPM) that includes the assessment of the biological-clinical characteristics of the patient, current and past prescription history, and the patient's PGx test results. To illustrate our approach, we present cases highlighting the clinical relevance of precision medicine with a focus on patients with a complex history and polypharmacy.Instituto de Salud Carlos III; European Regional Development Fun

Efecto de la dexametasona sobre el mecanismo inflamatorio que subyace en el asma

[ES] La prostaglandina D2 es el principal metabolito del ácido araquidónico, producido por los mastocitos activados durante una reacción alérgica. Dicho metabolito ejerce su respuesta biológica mediante la interacción con 2 tipos de receptores transmembrana: PTGDR y CRTH2. Desde hace más de un siglo se conoce que la alergia respiratoria tiene un importante componente genético. Entre los factores genéticos asociados a la enfermedad destaca el gen del Receptor de la Prostaglandina D2 (PTGDR), en el que se han caracterizado variantes polimórficas en la región promotora significativamente asociadas a la respuesta inflamatoria que subyace en la alergia, y a la existencia de distintas características fenotípicas en los pacientes asmáticos. Además, los resultados obtenidos en modelos animales han demostrado su papel crucial en la respuesta inflamatoria de vías aéreas mediada por alérgenos. La administración de glucocorticoides constituye la base del tratamiento del asma. Ejercen su efecto a través de unos sitios específicos GRE (Elementos de Respuesta a Glucocorticoides) localizados en la región promotora de genes implicados en el proceso inflamatorio, mediante la activación o represión de la expresión de los genes efectores que acabaría influyendo en el desencadenamiento de la respuesta alérgica

Las intervenciones farmacéuticas con Valeriana officinalis o Passiflora incarnata junto con la educación en la higiene del sueño mejoran los síntomas climatéricos y los problemas del sueño en la menopausia

Introduction. Physiological and endocrine changes occur during menopause that can negatively affect the sleep-wake cycle and contribute to objective and subjective sleep problems. Objective. To assess the effectiveness of a pharmaceutic intervention with two different complementary  treatments and sleep hygiene education on climacteric symptoms and sleep domains in menopausal women  with sleep disturbance. Material and methods. A sample of 109 women (45-64 years) participated in a 3-month randomized study,  35 received sleep hygiene instructions (SHI), 36 received capsules containing Passiflora incarnata 3 times a  ay plus SHI (PI), and 38 received capsules containing Valerian officinalis 3 times a day plus SHI (VO). Participants were evaluated by a) the Menopause Quality of Life (MENQOL) instrument, b) Pittsburgh Sleep  Quality Index (PSQI), c) Insomnia Severity Index, d) Epworth Sleepiness Scale, and e) Mental component of  SF-12 health survey. Results. MENQOL scores were similar at baseline in the three groups but were reduced (improved  vasomotor domain and physical subscale) at the end of the study in the VO group when compared with PI  and SHI counterparts (both, p<0.05). The SF-12 mental function showed improvement in the VO group  (p<0.05). Global PSQI score was significantly improved by PI and VO treatments at the end of treatment  (p=0.046 and p=0.034, respectively). VO group was more effective than PI in alleviating mild insomnia.  Change in vasomotor symptoms positively and significantly correlated with changes in all items of PSQI  components, except for sleep duration and the association was strongest with sleep latency. Most  participants evaluated the pharmaceutical and educational interventions provided as satisfactory. Conclusions. The Valerian officinalis was the preferable treatment for the climateric symptoms and sleep  difficulties associated with menopause. This study provided evidence that community pharmacists can play a  crucial role in referring menopausal women with symptoms of insomnia to potential medicinal plants  therapy and sleep hygiene instructions.  Introducción. Durante la menopausia se producen cambios fisiológicos y endocrinos que pueden afectar  negativamente al ciclo sueño-vigilia y contribuir a problemas objetivos y subjetivos del sueño. Objetivos. Evaluar la efectividad de la intervención farmacéutica con dos tratamientos complementarios  diferentes y educación en la higiene del sueño sobre los síntomas climatéricos y los dominios del sueño en  mujeres menopáusicas con trastornos del sueño. Métodos. Una muestra de 109 mujeres (45-64 años) participaron en un estudio aleatorizado de 3 meses, 35  recibieron instrucciones de higiene del sueño (SHI), 36 recibieron cápsulas que contenían Passiflora incarnata  3 veces al día más SHI (PI), y 38 recibieron cápsulas que contenían Valerian officinalis 3 veces al día  más SHI (VO). Las participantes completaron los cuestionarios de a) calidad de vida especifica de la  menopausia (MENQOL), b) índice de calidad del sueño de Pittsburgh (PSQI), c) índice de gravedad del  insomnio, d) escala de somnolencia de Epworth y e) dimensión salud mental del cuestionario de salud SF-12. Resultados. Las puntuaciones del cuestionario de calidad de vida específico de la menopausia fueron  similares en todos los grupos al inicio del estudio y se redujeron (dominio vasomotor y subescala física) al  final del estudio en el grupo VO en comparación con PI y SHI (ambos, p <0.05). La función mental SF-12  mostró una mejoría en las mujeres del grupo VO (p <0.05). La puntuación global de PSQI mejoró  significativamente con PI y VO al final del tratamiento (p = 0.046 y p = 0.034, respectivamente). El grupo VO  fue más efectivo que PI para aliviar el insomnio leve. El cambio en los síntomas vasomotores mostró  correlaciones significativas positivas con todos los ítems en los componentes del PSQI, excepto en la  duración del sueño. La asociación fue más mayor con la latencia del sueño. La mayoría de las participantes  evaluaron las intervenciones farmacéuticas y educativas prestadas como satisfactorias. Conclusiones. La Valeriana officinalis asociada a la higiene del sueño fue el tratamiento preferible para los  síntomas climáticos y las dificultades de sueño en la menopausia. Este estudio proporcionó evidencia de que  los farmacéuticos comunitarios pueden desempeñar un papel importante derivando a las mujeres  menopáusicas con síntomas de insomnio a la terapia potencial de plantas medicinales e higiene del sueño.

PTGDR expression is upregulated through retinoic acid receptors (RAR) mechanism in allergy.

Functional studies suggest that promoter polymorphisms of the Prostaglandin D Receptor (PTGDR) gene can be involved in asthma. All-trans Retinoic acid (ATRA) has also been linked to allergic diseases. We have previously described the PTGDR promoter activation mediated by ATRA through response elements (RARE) at position -549T> C. In this study we aimed to analyze the effect of retinoic acid (RA) on the expression of PTGDR, the production of cytokines as well as to evaluate the binding of RA receptors to RA-Response Elements (RARE) sequences. A549 cells were transfected with vectors carrying different PTGDR haplotypes and treated with all-Trans Retinoic Acid (ATRA). PTGDR expression was measured by qPCR. Chromatin Immunoprecipitation assays (ChIP) were performed in ATRA stimulated KU812 cells and in PBMCs of patients carrying CTCT, CCCC or CCCT haplotypes. In addition, a broad panel of cytokines was analyzed by cytometric bead assay in A549 cells. The expression of PTGDR increased in A549 cells transfected with PTGDR-variants. The CCCC haplotype showed a significantly higher expression compared with CTCT. However, we found that RA up-regulated PTGDR expression through RARα mainly in the CTCT variant. Experiments on PBMCs from allergic patients carrying the -549T and -549C variant of the PTGDR promoter after ATRA and RAR antagonist administration confirmed the modulation of PTGDR by ATRA. The cytokine analysis showed that IL4 and IL6 levels were significantly increased in A549 cells transfected with PTGDR. In addition, ATRA treatment decreased the levels of IL4, IL6 and TNFα in A549 cells, whereas it increased IL4 and TNFα levels in PTGDR-transfected cells. We observed genetic differences in the regulation of PTGDR by ATRA that could contribute to the phenotypic differences observed in allergic patients. Our findings showed that RAR modulation by PTGDR might have an impact on Th2 responses, suggesting that RAR could be a potential therapeutic target in allergic inflammation