Activating mutations in BRAF disrupt the hypothalamo-pituitary axis leading to hypopituitarism in mice and humans

Germline mutations in BRAF and other components of the MAPK pathway are associated with the congenital syndromes collectively known as RASopathies. Here, we report the association of Septo-Optic Dysplasia (SOD) including hypopituitarism and Cardio-Facio-Cutaneous (CFC) syndrome in patients harbouring mutations in BRAF. Phosphoproteomic analyses demonstrate that these genetic variants are gain-of-function mutations leading to activation of the MAPK pathway. Activation of the MAPK pathway by conditional expression of the BrafV600E/+ allele, or the knock-in BrafQ241R/+ allele (corresponding to the most frequent human CFC-causing mutation, BRAF p.Q257R), leads to abnormal cell lineage determination and terminal differentiation of hormone-producing cells, causing hypopituitarism. Expression of the BrafV600E/+ allele in embryonic pituitary progenitors leads to an increased expression of cell cycle inhibitors, cell growth arrest and apoptosis, but not tumour formation. Our findings show a critical role of BRAF in hypothalamo-pituitary-axis development both in mouse and human and implicate mutations found in RASopathies as a cause of endocrine deficiencies in humans

Plasma levels of alpha1-antichymotrypsin and secretory leukocyte proteinase inhibitor in healthy and chronic obstructive pulmonary disease (COPD) subjects with and without severe α1-antitrypsin deficiency

BACKGROUND: Individuals with severe Z α1-antitrypsin (AAT) deficiency have a considerably increased risk of developing chronic obstructive lung disease (COPD). It has been hypothesized that compensatory increases in levels of other protease inhibitors mitigate the effects of this AAT deficiency. We analysed plasma levels of AAT, α1-antichymotrypsin (ACT) and secretory leukocyte protease inhibitor (SLPI) in healthy (asymptomatic) and COPD subjects with and without AAT deficiency. METHODS: Studied groups included: 71 asymptomatic AAT-deficient subjects (ZZ, n = 48 and SZ, n = 23, age 31 ± 0.5) identified during Swedish neonatal screening for AAT deficiency between 1972 and 1974; age-matched controls (MM, n = 57, age 30.7 ± 0.6); older asymptomatic ZZ (n = 10); healthy MM (n = 20, age 53 ± 9.6); and COPD patients (ZZ, n = 10, age 47.4 ± 11 and MM, n = 10, age 59.4 ± 6.7). Plasma levels of SLPI, AAT and ACT were analysed using ELISA and immunoelectrophoresis. RESULTS: No significant difference was found in plasma ACT and SLPI levels between the healthy MM and the ZZ or SZ subjects in the studied groups. Independent of the genetic variant, subjects with COPD (n = 19) had elevated plasma levels of SLPI and ACT relative to controls (n = 153) (49.5 ± 7.2 vs 40.7 ± 9.1 ng/ml, p < 0.001 and 0.52 ± 0.19 vs 0.40 ± 0.1 mg/ml, p < 0.05, respectively). CONCLUSION: Our findings show that plasma levels of ACT and SLPI are not elevated in subjects with genetic AAT deficiency compared MM controls and do not appear to compensate for the deficiency of plasma AAT

Effects of controlled diesel exhaust exposure on apoptosis and proliferation markers in bronchial epithelium – an in vivo bronchoscopy study on asthmatics, rhinitics and healthy subjects

BackgroundEpidemiological evidence demonstrates that exposure to traffic-derived pollution worsens respiratory symptoms in asthmatics, but controlled human exposure studies have failed to provide a mechanism for this effect. Here we investigated whether diesel exhaust (DE) would induce apoptosis or proliferation in the bronchial epithelium in vivo and thus contribute to respiratory symptoms.MethodsModerate (n?=?16) and mild (n?=?16) asthmatics, atopic non-asthmatic controls (rhinitics) (n?=?13) and healthy controls (n?=?21) were exposed to filtered air or DE (100 ?g/m 3 ) for 2 h, on two separate occasions. Bronchial biopsies were taken 18 h post-exposure and immunohistochemically analysed for pro-apoptotic and anti-apoptotic proteins (Bad, Bak, p85 PARP, Fas, Bcl-2) and a marker of proliferation (Ki67). Positive staining was assessed within the epithelium using computerized image analysis.ResultsNo evidence of epithelial apoptosis or proliferation was observed in healthy, allergic or asthmatic airways following DE challenge.ConclusionIn the present study, we investigated whether DE exposure would affect markers of proliferation and apoptosis in the bronchial epithelium of asthmatics, rhinitics and healthy controls, providing a mechanistic basis for the reported increased airway sensitivity in asthmatics to air pollutants. In this first in vivo exposure investigation, we found no evidence of diesel exhaust-induced effects on these processes in the subject groups investigated

Models of chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a major global health problem and is predicted to become the third most common cause of death by 2020. Apart from the important preventive steps of smoking cessation, there are no other specific treatments for COPD that are as effective in reversing the condition, and therefore there is a need to understand the pathophysiological mechanisms that could lead to new therapeutic strategies. The development of experimental models will help to dissect these mechanisms at the cellular and molecular level. COPD is a disease characterized by progressive airflow obstruction of the peripheral airways, associated with lung inflammation, emphysema and mucus hypersecretion. Different approaches to mimic COPD have been developed but are limited in comparison to models of allergic asthma. COPD models usually do not mimic the major features of human COPD and are commonly based on the induction of COPD-like lesions in the lungs and airways using noxious inhalants such as tobacco smoke, nitrogen dioxide, or sulfur dioxide. Depending on the duration and intensity of exposure, these noxious stimuli induce signs of chronic inflammation and airway remodelling. Emphysema can be achieved by combining such exposure with instillation of tissue-degrading enzymes. Other approaches are based on genetically-targeted mice which develop COPD-like lesions with emphysema, and such mice provide deep insights into pathophysiological mechanisms. Future approaches should aim to mimic irreversible airflow obstruction, associated with cough and sputum production, with the possibility of inducing exacerbations

EAACI position paper on occupational rhinitis

The present document is the result of a consensus reached by a panel of experts from European and non-European countries on Occupational Rhinitis (OR), a disease of emerging relevance which has received little attention in comparison to occupational asthma. The document covers the main items of OR including epidemiology, diagnosis, management, socio-economic impact, preventive strategies and medicolegal issues. An operational definition and classification of OR tailored on that of occupational asthma, as well as a diagnostic algorithm based on steps allowing for different levels of diagnostic evidence are proposed. The needs for future research are pointed out. Key messages are issued for each item

Novel FOXA2 mutation causes Hyperinsulinism, Hypopituitarism with Craniofacial and Endoderm-derived organ abnormalities.

Congenital hypopituitarism (CH) is characterized by the deficiency of one or more pituitary hormones and can present alone or in association with complex disorders. Congenital hyperinsulinism (CHI) is a disorder of unregulated insulin secretion despite hypoglycaemia that can occur in isolation or as part of a wider syndrome. Molecular diagnosis is unknown in many cases of CH and CHI. The underlying genetic etiology causing the complex phenotype of CH and CHI is unknown. In this study, we identified a de novo heterozygous mutation in the developmental transcription factor, forkhead box A2, FOXA2 (c.505T>C, p.S169P) in a child with CHI and CH with craniofacial dysmorphic features, choroidal coloboma and endoderm-derived organ malformations in liver, lung and gastrointestinal tract by whole exome sequencing. The mutation is at a highly conserved residue within the DNA binding domain. We demonstrated strong expression of Foxa2 mRNA in the developing hypothalamus, pituitary, pancreas, lungs and oesophagus of mouse embryos using in situ hybridization. Expression profiling on human embryos by immunohistochemistry showed strong expression of hFOXA2 in the neural tube, third ventricle, diencephalon and pancreas. Transient transfection of HEK293T cells with Wt (Wild type) hFOXA2 or mutant hFOXA2 showed an impairment in transcriptional reporter activity by the mutant hFOXA2. Further analyses using western blot assays showed that the FOXA2 p.(S169P) variant is pathogenic resulting in lower expression levels when compared with Wt hFOXA2. Our results show, for the first time, the causative role of FOXA2 in a complex congenital syndrome with hypopituitarism, hyperinsulinism and endoderm-derived organ abnormalities.The human embryonic and fetal material was provided by the Joint Medical Research Council (MRC)/Wellcome Trust Human Developmental Biology Resource (www.hdbr.org) (Grant 099175). M.L.V, A.G., have been funded by Action Medical Research (Grant Number GN2272); Barts and the London Charity (BTLC; Grant Number 417/2238); V.S and C.G-M are currently funded by Early Career Fellowship from the Medical College of Saint Bartholomew’s Hospital Trust and Action Medical Research. D.G is funded by a research grant from Sandoz (UK) limited through the University of Liverpool, UK (Grant number Jxg 70001