Mutation affecting the proximal promoter of Endoglin as the origin of hereditary hemorrhagic telangiectasia type 1.

BACKGROUND: Hereditary hemorrhagic telangiectasia (HHT) is a vascular multi-organ system disorder. Its diagnostic criteria include epistaxis, telangiectases in mucocutaneous sites, arteriovenous malformations (AVMs), and familial inheritance. HHT is transmitted as an autosomal dominant condition, caused in 85% of cases by mutations in either Endoglin (ENG) or Activin receptor-like kinase (ACVRL1/ACVRL1/ALK1) genes. Pathogenic mutations have been described in exons, splice junctions and, in a few cases with ENG mutations, in the proximal promoter, which creates a new ATG start site. However, no mutations affecting transcription regulation have been described to date in HHT, and this type of mutation is rarely identified in the literature on rare diseases. METHODS: Sequencing data from a family with HHT lead to single nucleotide change, c.-58G > A. The functionality and pathogenicity of this change was analyzed by in vitro mutagenesis, quantitative PCR and Gel shift assay. Student t test was used for statistical significance. RESULTS: A single nucleotide change, c.-58G > A, in the proximal ENG promoter co-segregated with HHT clinical features in an HHT family. This mutation was present in the proband and in 2 other symptomatic members, whereas 2 asymptomatic relatives did not harbor the mutation. Analysis of RNA from activated monocytes from the probands and the healthy brother revealed reduced ENG mRNA expression in the HHT patient (p = 0.005). Site-directed mutagenesis of the ENG promoter resulted in a three-fold decrease in luciferase activity of the mutant c.-58A allele compared to wild type (p = 0.005). Finally, gel shift assay identified a DNA-protein specific complex. CONCLUSIONS: The novel ENG c.-58G > A substitution in the ENG promoter co-segregates with HHT symptoms in a family and appears to affect the transcriptional regulation of the gene, resulting in reduced ENG expression. ENG c.-58G > A may therefore be a pathogenic HHT mutation leading to haploinsufficiency of Endoglin and HHT symptoms. To the best of our knowledge, this is the first report of a pathogenic mutation in HHT involving the binding site for a transcription factor in the promoter of ENG.

SOCS3 silencing attenuates eosinophil functions in asthma patients.

Eosinophils are one of the key inflammatory cells in asthma. Eosinophils can exert a wide variety of actions through expression and secretion of multiple molecules. Previously, we have demonstrated that eosinophils purified from peripheral blood from asthma patients express high levels of suppressor of cytokine signaling 3 (SOCS3). In this article, SOCS3 gene silencing in eosinophils from asthmatics has been carried out to achieve a better understanding of the suppressor function in eosinophils. SOCS3 siRNA treatment drastically reduced SOCS3 expression in eosinophils, leading to an inhibition of the regulatory transcription factors GATA-3 and FoxP3, also interleukin (IL)-10; in turn, an increased STAT3 phosphorilation was observed. Moreover, SOCS3 abrogation in eosinophils produced impaired migration, adhesion and degranulation. Therefore, SOCS3 might be regarded as an important regulator implicated in eosinophil mobilization from the bone marrow to the lungs during the asthmatic process.

Simulated gastrointestinal digestion reduces the allergic reactivity of shrimp extract proteins and tropomyosin.

Shrimp are highly allergenic foods. Current management are limited to the avoidance of foods. Therefore, there is an unmet need for a safe and effective therapy using modified allergens. This study focuses on assessing the potential for modification of the allergenicity of shrimp proteins following heat treatment or simulated gastric digestion. Shrimp proteins do not reduce their IgE reactivity after heat treatment but it is reduced by simulated gastric digestion in a time- and dose-dependent manner. Tropomyosin in shrimp extract is worse digested than purified tropomyosin. After 60 min of 10 U/µg pepsin digestion, a strong inhibition was produced in the in vivo skin reactivity of shrimp extracts and in activation of basophils from allergic patients. Immunisation experiments performed in rabbits demonstrated that digested boiled shrimp extract is able to induce IgG antibodies that block the IgE binding to the untreated boiled shrimp extract in shrimp-allergic patients. Building on our observations, digestion treatment could be an effective method for reducing shrimp allergenicity while maintaining the immunogenicity.

Gene silencing of SOCS3 by siRNA intranasal delivery inhibits asthma phenotype in mice.

Suppresors of cytokine signaling (SOCS) proteins regulate cytokine responses and control immune balance. Several studies have confirmed that SOCS3 is increased in asthmatic patients, and SOCS3 expression is correlated with disease severity. The objective of this study was to evaluate if delivering of SOCS3 short interfering RNA (siRNA) intranasally in lungs could be a good therapeutic approach in an asthma chronic mouse model. Our results showed that intranasal treatment with SOCS3-siRNA led to an improvement in the eosinophil count and the normalization of hyperresponsiveness to methacholine. Concomitantly, this treatment resulted in an improvement in mucus secretion, a reduction in lung collagen, which are prominent features of airway remodeling. The mechanism implies JAK/STAT and RhoA/Rho-kinase signaling pathway, because we found a decreasing in STAT3 phosphorylation status and down regulation of RhoA/Rho-kinase protein expression. These results might lead to a new therapy for the treatment of chronic asthma.

Misregulation of suppressors of cytokine signaling in eosinophilic esophagitis.

BACKGROUND: Several findings suggest that eosinophilic esophagitis (EoE) is strongly associated with atopy and allergen-driven, Th2-type immune responses, indicating the association of EoE with immune dysregulation. The objective of this study is to ascertain the molecular mechanism involved in EoE disease development a Th2 condition. METHODS: 25 patients with diagnosis of EoE and 17 non-EoE controls were recruited by the gastroenterology and allergy departments from three different hospitals. Transcription analysis of suppressors of cytokine signaling 1, 3, 5 (SOCS), interleukin-5 (IL), IL-13, eotaxin (CCL26), eoataxin receptor (CCR3), and mitogen-activated protein kinase 1 (MAPK1) was performed in esophageal biopsies by real time PCR. Western blot of ERK esophageal protein and additional measures of IL-5 and VEGF levels in serum were performed. RESULTS: The esophagus of EoE patients expresses and synthesizes high levels of SOCS1 and SOCS3 proteins (P < 0.05), and these expression correlated with levels of IL-5, IL-13, CCL26, CCR3, and MAPK1 genes. In addition, we demonstrate the implication of the ERK pathway (P < 0.001). CONCLUSIONS: SOCS proteins probably contribute to EoE pathogenesis by directly or indirectly inducing the Th2 profile, as well as by promoting the production of Th2 cytokines. All these findings further enhance our understanding of the mechanism of EoE, and accumulating evidence suggests that EoE pathogenesis is likely to be due to misregulation of immunological pathways.