Project description:Introduction: Systems vaccinology is a novel approach to predict immune response in vaccines. We have used a systems biology approach to identify early gene ‘signatures’ that predicted viral load control after analytical therapy interruption (ATI) in HIV-1 infected patients vaccinated with a dendritic cell-based (DC) HIV-1 vaccine. Methods: Frozen post-vaccination PBMC samples from participants of a previously published DC vaccine (DCV2) clinical trial were used for the study. mRNA and miRNA were extracted and gene expression was determined by microarray method. Differential gene expression analysis was performed on both mRNA and miRNA between responders (> 1 log10 copies/mL drop of VL after 12 weeks of ATI) and non-responders (<= 1 log10 copies/mL drop in VL at week 12 of ATI). Gene set enrichment analysis (GSEA) was carried out with the hallmark gene sets of the Broad Institute on the mRNA data. After the stand-alone analyses of mRNA and miRNA we performed an additional GSEA with gene sets defined by the genes regulated by significantly differentially expressed miRNAs. Statistical analysis was done using R and the GSEA software of the Broad Institute. Results: There were 15 responders and 20 non-responders. No differentially expressed mRNAs were observed between responders and non-responders. As compared with non-responders, responders showed an up-regulation of gene sets corresponding to TNF- alpha signaling via the NFkB pathway, inflammatory response, coagulation, the complement system, Il6 and Il2 JAK-STAT signaling, or reactive oxygen-species pathways were up-regulated, and a down-regulation of gene sets corresponding to E2F targets, oxidative phosphorylation, or interferon alpha response. We found 9 differentially expressed miRNAs between responders and non-responders: miR-32-3p, miR-185-3p, miR-223-3p, miR-500b-3p, miR-550a-3p, miR-1183, miR-1184, miR-4455, and miR-8063. Twelve Broad hallmark gene sets that were significantly deregulated in the GSEA showed significant overlap with genes regulated by one or more of these miRNAs, 10 of them with genes regulated by miR-223-3p. We also observed that the expression of genes regulated by miR-223-3p, miR-1183 and miR-8063 was significantly down-regulated in responders as compared with non-responders. Conclusions: Deregulation of certain gene sets related to inflammatory processes seems fundamental in viral control during ATI. miR-223-3p may be one of the miRNAs that fine tune part of these processes.

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.

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