Project description:Chronic spontaneous urticaria (CSU), also traditionally called Chronic Idiopathic Urticaria (CIU), is a common dermatosis. Its estimated point prevalence is 0.6 to 1%. CSU is defined by spontaneously occurring short-lived and itching wheal, angioedema or both. The current nomenclature of urticaria endorses the use of a clinical rather than an aetiological classification to recognise that a percentage of patients with CSU have an autoimmune rather than idiopathic aetiology. As it happens with other IgE-associated diseases, a better knowledge of urticaria patients requires defining phenotypes. The present knowledge about CSU phenotypes is based on the clinical characteristics, associated comorbidities, the course of the disease and its response to the available effective drugs. But a phenotype is defined as the visible characteristics or traits that are a consequence of the expression of genes as well as the influence of environmental factors. The phenotype is part a consequence of the genotype. To study which is the genotype expressed by the patients suffering of CSU can be interesting. The mechanisms involved in the mast cell activation and the role of the released mast cell mediators are well known allowing explaining of how the wheals appear and disappear. But very little is known about the genetic susceptibility and genes involved in the development of wheals in CSU patients. By means of this work, enough case series was collected to draw reliable conclusions, based on the genetic variations found. In order to assess associations between genetic polymorphisms and the different phenotypes of CSU, large case series of well-documented patients with a relevant history are needed. The primary purpose of this study was to identify the most relevant genes expressed in the tissue and the blood samples from patients with moderate and severe CSU. Objective: The primary purpose of this study was to identify the most relevant genes expressed in the tissue and the blood samples from patients with moderate and severe CSU. The knowledge of the gene profiling differences comparing wheal versus no-lesion skin in selected patients is a real and accurate approach to identify the genes expressed in the wheal developed in patients with CSU. Patients and methods: A total of 20 patients suffering of moderate and severe CSU/CIU and 10 healthy control subjects were included. The setting of the study was the Department of Dermatology and the Department of Pharmacology Clinic at the Hospital del Mar, Institut Mar d'Investigacions Mediques, Barcelona. The sample size is adequate to the exploratory character of the study. Only patients showing active moderate to severe CSU during at least 3 months were included in the study. Patients were selected prospectively. An Urticaria Activity Score (UAS) 7 defined moderate to severe CSU, this mean an UAS7 â?¥16 (UAS 7 range from 0 to 42), where the symptom itch showed a value â?¥8. All the patients included remained symptomatic in spite of the treatment with antihistamines. The diagnosis of CSU was based on clinical history data and physical examination. Any active treatment was supplied at the patient at the time where the samples were obtained. Healthy controls were selected from a pool of volunteers. A detailed anamnesis, clinical exploration and basal blood analysis was carried out in order to guarantee that any active disease nor cutaneous nor systemic was present. The CSU patients and the healthy volunteers were submitted to some interventions in order to obtain cutaneous and venous blood samples. Cutaneous biopsies were obtained after local anesthesia using punch biopsy of 4 mm in diameter. Two cutaneous biopsies including skin from an active wheal and one biopsy from non-lesion skin were obtained. The active wheal chosen for biopsy was obtained from the trunk and clinically showed erythema and edema, with a time of evolution of at least of one hour. One of the biopsies taken from the wheal was fixed in formaldehyde and after paraffin inclusion was stained with haematoxilin -eosin, Giemsa and with c-Kit as mast cell marker with the objective to characterize the diagnosis of wheal and describe accurately the characteristics of the inflammatory infiltrate. The second biopsy was immediately prepared for storage prior to be submitted for gene array. Fat and other undesired components that do not belong to the sample were removed before quick-freezing the tissue in liquid nitrogen. Cryopreserved tissue samples were stored at -80Â°C prior to the gene microarray study. The punch taken over no-lesion skin guarantee that no wheal was present in the previous 72 hours and no other skin alteration was present i.e., desquamation or pigmentation. Venous blood analysis was obtained from the basilic mediana vein in the antecubital fossae and prepared for its storage for gene array analysis. Quality control of RNA samples was performed with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Good quality samples with an RNA Integrity Number (RIN) value >6, and also some mildly degraded samples with a RIN value between 4 and 6, were processed following specific protocols. The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Based on the results provided from the microarray comparative analysis, we selected a set of genes to be confirmed by Q-PCR. The criteria followed to perform such selection were based in technical aspects that would guarantee gene confirmation. These recommendations include an adjusted P value of 0.05 and a Log fold change of 1 (plus or minor to 1). The second criteria was based in an accurate analysis of the genes up or down regulated and its possible involvement in mast cell biology, wheal pathogenesis, urticaria or allergy. The data base included shows the raw data of the gene-array performed in the available samples of the blood and the tissue of the patients with CSU and the healthy controls included in this study. 61 samples were analyzed from 30 individuals, 20 patients and 10 healthy controls. This Series includes the samples from tissue.

Project description:Chronic spontaneous urticaria (CSU), also traditionally called Chronic Idiopathic Urticaria (CIU), is a common dermatosis. Its estimated point prevalence is 0.6 to 1%. CSU is defined by spontaneously occurring short-lived and itching wheal, angioedema or both. The current nomenclature of urticaria endorses the use of a clinical rather than an aetiological classification to recognise that a percentage of patients with CSU have an autoimmune rather than idiopathic aetiology. As it happens with other IgE-associated diseases, a better knowledge of urticaria patients requires defining phenotypes. The present knowledge about CSU phenotypes is based on the clinical characteristics, associated comorbidities, the course of the disease and its response to the available effective drugs. But a phenotype is defined as the visible characteristics or traits that are a consequence of the expression of genes as well as the influence of environmental factors. The phenotype is part a consequence of the genotype. To study which is the genotype expressed by the patients suffering of CSU can be interesting. The mechanisms involved in the mast cell activation and the role of the released mast cell mediators are well known allowing explaining of how the wheals appear and disappear. But very little is known about the genetic susceptibility and genes involved in the development of wheals in CSU patients. By means of this work, enough case series was collected to draw reliable conclusions, based on the genetic variations found. In order to assess associations between genetic polymorphisms and the different phenotypes of CSU, large case series of well-documented patients with a relevant history are needed. The primary purpose of this study was to identify the most relevant genes expressed in the tissue and the blood samples from patients with moderate and severe CSU. Objective: The primary purpose of this study was to identify the most relevant genes expressed in the tissue and the blood samples from patients with moderate and severe CSU. The knowledge of the gene profiling differences comparing wheal versus no-lesion skin in selected patients is a real and accurate approach to identify the genes expressed in the wheal developed in patients with CSU. Patients and methods: A total of 20 patients suffering of moderate and severe CSU/CIU and 10 healthy control subjects were included. The setting of the study was the Department of Dermatology and the Department of Pharmacology Clinic at the Hospital del Mar, Institut Mar d'Investigacions Mediques, Barcelona. The sample size is adequate to the exploratory character of the study. Only patients showing active moderate to severe CSU during at least 3 months were included in the study. Patients were selected prospectively. An Urticaria Activity Score (UAS) 7 defined moderate to severe CSU, this mean an UAS7 â?¥16 (UAS 7 range from 0 to 42), where the symptom itch showed a value â?¥8. All the patients included remained symptomatic in spite of the treatment with antihistamines. The diagnosis of CSU was based on clinical history data and physical examination. Any active treatment was supplied at the patient at the time where the samples were obtained. Healthy controls were selected from a pool of volunteers. A detailed anamnesis, clinical exploration and basal blood analysis was carried out in order to guarantee that any active disease nor cutaneous nor systemic was present. The CSU patients and the healthy volunteers were submitted to some interventions in order to obtain cutaneous and venous blood samples. Cutaneous biopsies were obtained after local anesthesia using punch biopsy of 4 mm in diameter. Two cutaneous biopsies including skin from an active wheal and one biopsy from non-lesion skin were obtained. The active wheal chosen for biopsy was obtained from the trunk and clinically showed erythema and edema, with a time of evolution of at least of one hour. One of the biopsies taken from the wheal was fixed in formaldehyde and after paraffin inclusion was stained with haematoxilin -eosin, Giemsa and with c-Kit as mast cell marker with the objective to characterize the diagnosis of wheal and describe accurately the characteristics of the inflammatory infiltrate. The second biopsy was immediately prepared for storage prior to be submitted for gene array. Fat and other undesired components that do not belong to the sample were removed before quick-freezing the tissue in liquid nitrogen. Cryopreserved tissue samples were stored at -80Â°C prior to the gene microarray study. The punch taken over no-lesion skin guarantee that no wheal was present in the previous 72 hours and no other skin alteration was present i.e., desquamation or pigmentation. Venous blood analysis was obtained from the basilic mediana vein in the antecubital fossae and prepared for its storage for gene array analysis. Quality control of RNA samples was performed with the Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA). Good quality samples with an RNA Integrity Number (RIN) value >6, and also some mildly degraded samples with a RIN value between 4 and 6, were processed following specific protocols. The hybridization procedure was performed according to the Agilent 60-mer oligo microarray processing protocol using the Agilent Gene Expression Hybridization Kit (Agilent Technologies). Based on the results provided from the microarray comparative analysis, we selected a set of genes to be confirmed by Q-PCR. The criteria followed to perform such selection were based in technical aspects that would guarantee gene confirmation. These recommendations include an adjusted P value of 0.05 and a Log fold change of 1 (plus or minor to 1). The second criteria was based in an accurate analysis of the genes up or down regulated and its possible involvement in mast cell biology, wheal pathogenesis, urticaria or allergy. The data base included shows the raw data of the gene-array performed in the available samples of the blood and the tissue of the patients with CSU and the healthy controls included in this study. 61 samples were analyzed from 30 individuals, 20 patients and 10 healthy controls. This Series includes the samples from blood.

Project description:Background: Exosomes are nanovesicles of endocytic origin believed to be involved in communication between cells. Recently, it has been shown that mast cell exosomes contain RNA named "exosomal shuttle RNA". The aim of this study was to evaluate whether exosomal shuttle RNA could play a role in the communication between human mast cells and between human mast cells and human CD34 positive progenitor cells. Results: Exosomes from the human mast cell line HMC-1 contain RNA. The exosomes contain no or very little ribosomal RNA compared to their donor cells. The mRNA and microRNA content in exosomes and their donor cells was examined using microarray analyses. We found 116 microRNA in the exosomes and 134 microRNA in the cells, from which some were expressed at different level. DNA microarray experiments revealed the presence of approximately 1800 mRNAs in the exosomes, which represent 15% of the donor cell mRNA content. Transfer experiments revealed that exosomes and their RNA can transfer to other HMC-1 cells and to CD34 positive progenitors. Conclusions: To conclude, HMC-1 exosomes contain mRNA and microRNA that can be transferred to other mast cells and to CD34 progenitors. This shuttle of exosomal RNA may represent a powerful mode of communication between cells where cells send genetic information to other cells over a distance via exosomes. [miRNA profiling] Identification of microRNA was performed by Exiqon (www.exiqon.com). Briefly, the quality of the total RNA was verified by an Agilent 2100 Bioanalyzer. Total RNA from the exosome and the HMC-1 cell samples were labelled with Hy3 and Hy5 fluorescent stain, respectively, using the miRCURY Hy3/Hy5 power labelling kit. The Hy3-labelled exosome samples and a Hy5-labeled mast cells were mixed pair-wise and hybridized to the miRCURY? LNA array (v9.2). The hybridization was performed according to the miRCURY? LNA array manual using a Tecan HS4800 hybridization station (Tecan Systems, Inc. San Jose, CA). The miRCURY? LNA array microarray slides were scanned by a ScanArray 4000 XL scanner (Packard Biochip Technologies, Billerica, MA ,USA) and the image analysis was carried out using the ImaGene 6.1.0 software (BioDiscovery, Inc, El Segundo, CA USA). The quantified signals were normalized using the global Lowess (LOcally WEighted Scatterplot Smoothing) regression algorithm. MicroRNA with signals equal to or below the background signal in 2 or more of the 4 replicate measurements were identified as absent in that slide. The limit for a miRNA to be listed as detectable was set to signal intensities higher than 3 x background (3 x median Hy3 or Hy5 for the total slide). In addition, where signals were detected for <3 of the slides, they were considered unreliable and excluded from sets of detected miRNAs. The experiment was performed in triplicate samples. The signal was calculated as the mean value of the log2MeanRatio Hy3/Hy5 for the triplicates M-BM-1 SD. [mRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol . The microarray experiments were performed by SweGene (www.swegene.org/) according to Affymetrix microarray DNA chip analysis (n=4). p0739_E1, p0739_ E2, p0739_E3 and p0739_E4 for the exosomes samples and p0739_C1, p0739_C2, p0739_C3, and p0739_C4 for the HMC-1 cells. [miRNA profiling] Exosomes were prepared from the supernatant of HMC-1 cells by differential centrifugations and filtration. RNA was isolated from the exosomes and their parental cells using Trizol followed by RNeasy clean-up. The microarray experiments were performed by Exiqon.

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Plasma-Derived Exosomes in Chronic Spontaneous Urticaria Induce the Release of Inflammatory Mediators from Mast cell through miRNA-619-5p/SOCS4 Pathway

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