Sequential monolayer-suspension culture of human airway epithelial cells

AbstractIn this article, we describe a technique for culturing human airway epithelial cells, developed in Leuven, as a new tool for a most reliable diagnosis for ciliary disorders. This technique that allows to keep both structural and functional primary abnormalities of inherited ciliary abnormalities, while avoiding the secondary ones, can also be useful to a number of other studies, namely in cystic fibrosis

Lack of a-disintegrin-and-metalloproteinase ADAM10 leads to intracellular accumulation and loss of shedding of the cellular prion protein in vivo

<p>Abstract</p> <p>Background</p> <p>The cellular prion protein (PrP^C) fulfils several yet not completely understood physiological functions. Apart from these functions, it has the ability to misfold into a pathogenic scrapie form (PrP^Sc) leading to fatal transmissible spongiform encephalopathies. Proteolytic processing of PrP^Cgenerates N- and C-terminal fragments which play crucial roles both in the pathophysiology of prion diseases and in transducing physiological functions of PrP^C. A-disintegrin-and-metalloproteinase 10 (ADAM10) has been proposed by cell culture experiments to be responsible for both shedding of PrP^Cand its α-cleavage. Here, we analyzed the role of ADAM10 in the proteolytic processing of PrP^C<it>in vivo</it>.</p> <p>Results</p> <p>Using neuron-specific <it>Adam10 </it>knockout mice, we show that ADAM10 is the sheddase of PrP^Cand that its absence <it>in vivo </it>leads to increased amounts and accumulation of PrP^Cin the early secretory pathway by affecting its posttranslational processing. Elevated PrP^Clevels do not induce apoptotic signalling via p53. Furthermore, we show that ADAM10 is not responsible for the α-cleavage of PrP^C.</p> <p>Conclusion</p> <p>Our study elucidates the proteolytic processing of PrP^Cand proves a role of ADAM10 in shedding of PrP^C<it>in vivo</it>. We suggest that ADAM10 is a mediator of PrP^Chomeostasis at the plasma membrane and, thus, might be a regulator of the multiple functions discussed for PrP^C. Furthermore, identification of ADAM10 as the sheddase of PrP^Copens the avenue to devising novel approaches for therapeutic interventions against prion diseases.</p

Clinically Relevant Response to Cisplatin-5-Fluorouracyl in Intestinal-Type Sinonasal Adenocarcinoma with Loss of Vision: A Case Report

A 68-year-old man presented with rapid progressive visual loss caused by a progressive local invasive sinonasal intestinal-type adenocarcinoma (ITAC) with intracranial invasion. The local relapse of ITAC in the ethmoid sinus was previously treated with palliative radiotherapy and carboplatin-paclitaxel, without response, hence disease progression was seen. Ophthalmological examination revealed irreversible blindness of the left eye and a dramatic progressive visual loss of the right eye. Due to important visual loss caused by optic nerve invasion, a palliative treatment with cisplatin-5-fluorouracyl was started. This therapy resulted in a good clinical response with a regression of the local mass and a partial recovery of the vision

Evaluation of skin prick location on the forearm using a novel skin prick automated test device

BackgroundThe skin prick test (SPT) is the gold standard for identifying allergic sensitization in individuals suspected of having an inhalant allergy. Recently, it was demonstrated that SPT using a novel skin prick automated test (SPAT) device showed increased reproducibility and tolerability compared to the conventional SPT, among other benefits.ObjectiveThis study aimed to evaluate prick location bias using the novel SPAT device.MethodsA total of 118 volunteers were enrolled in this study and underwent SPATs with histamine (nine pricks) and glycerol control (one prick) solutions on the volar side of their forearms. Imaging of the skin reactions was performed using the SPAT device, and the physician determined the longest wheal diameter by visually inspecting the images using a web interface. Prick location bias was assessed along the medial vs. lateral and proximal vs. distal axes of the forearm.ResultsIn total, 944 histamine pricks were analyzed. Four medial and four lateral histamine pricks were grouped, and wheal sizes were compared. The longest wheal diameters were not significantly different between the medial and lateral prick locations (p = 0.41). Furthermore, the pricks were grouped by two based on their position on the proximal–distal axis of the forearm. No significant difference was observed among the four groups of analyzed prick locations (p = 0.73).ConclusionThe prick location on the volar side of the forearm did not influence wheal size in SPAT-pricked individuals

Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease

European Respiratory Society guidelines for the diagnosis of primary ciliary dyskinesia

The diagnosis of primary ciliary dyskinesia is often confirmed with standard, albeit complex and expensive, tests. In many cases, however, the diagnosis remains difficult despite the array of sophisticated diagnostic tests. There is no "gold standard" reference test. Hence, a Task Force supported by the European Respiratory Society has developed this guideline to provide evidence-based recommendations on diagnostic testing, especially in light of new developments in such tests, and the need for robust diagnoses of patients who might enter randomised controlled trials of treatments. The guideline is based on pre-defined questions relevant for clinical care, a systematic review of the literature, and assessment of the evidence using the GRADE (Grading of Recommendations, Assessment, Development and Evaluation) approach. It focuses on clinical presentation, nasal nitric oxide, analysis of ciliary beat frequency and pattern by high-speed video-microscopy analysis, transmission electron microscopy, genotyping and immunofluorescence. It then used a modified Delphi survey to develop an algorithm for the use of diagnostic tests to definitively confirm and exclude the diagnosis of primary ciliary dyskinesia; and to provide advice when the diagnosis was not conclusive. Finally, this guideline proposes a set of quality criteria for future research on the validity of diagnostic methods for primary ciliary dyskinesia

Expert curation of the human and mouse olfactory receptor gene repertoires identifies conserved coding regions split across two exons.

BACKGROUND: Olfactory receptor (OR) genes are the largest multi-gene family in the mammalian genome, with 874 in human and 1483 loci in mouse (including pseudogenes). The expansion of the OR gene repertoire has occurred through numerous duplication events followed by diversification, resulting in a large number of highly similar paralogous genes. These characteristics have made the annotation of the complete OR gene repertoire a complex task. Most OR genes have been predicted in silico and are typically annotated as intronless coding sequences. RESULTS: Here we have developed an expert curation pipeline to analyse and annotate every OR gene in the human and mouse reference genomes. By combining evidence from structural features, evolutionary conservation and experimental data, we have unified the annotation of these gene families, and have systematically determined the protein-coding potential of each locus. We have defined the non-coding regions of many OR genes, enabling us to generate full-length transcript models. We found that 13 human and 41 mouse OR loci have coding sequences that are split across two exons. These split OR genes are conserved across mammals, and are expressed at the same level as protein-coding OR genes with an intronless coding region. Our findings challenge the long-standing and widespread notion that the coding region of a vertebrate OR gene is contained within a single exon. CONCLUSIONS: This work provides the most comprehensive curation effort of the human and mouse OR gene repertoires to date. The complete annotation has been integrated into the GENCODE reference gene set, for immediate availability to the research community

Real-life assessment of chronic rhinosinusitis patients using mobile technology: the mySinusitisCoach project by EUFOREA

Background: Chronic rhinosinusitis (CRS) is a chronic inflammatory disease associated with a substantial personal and socioeconomic burden. Monitoring of patient‐reported outcomes by mobile technology offers the possibility to better understand real‐life burden of CRS. Methods: This study reports on the cross‐sectional evaluation of data of 626 users of mySinusitisCoach (mSC), a mobile application for CRS patients. Patient characteristics of mSC users were analysed as well as the level of disease control based on VAS global rhinosinusitis symptom score and adapted EPOS criteria. Results: The mSC cohort represents a heterogeneous group of CRS patients with a diverse pattern of major symptoms. Approximately half of patients reported nasal polyps. 47.3% of all CRS patients were uncontrolled based on evaluation of VAS global rhinosinusitis symptom score compared to 40.9% based on adapted EPOS criteria. The impact of CRS on sleep quality and daily life activities was significantly higher in uncontrolled versus well‐controlled patients. Half of patients had a history of FESS (functional endoscopic sinus surgery) and reported lower symptom severity compared to patients without a history of FESS, except for patients with a history of more than 3 procedures. Patients with a history of FESS reported higher VAS levels for impaired smell. Conclusion: Real‐life data confirm the high disease burden in uncontrolled CRS patients, clearly impacting quality of life. Sinus surgery improves patient‐reported outcomes, but not in patients with a history of more than 3 procedures. Mobile technology opens a new era of real‐life monitoring, supporting the evolution of care towards precision medicine