Impaired CFTR function in mild cystic fibrosis associated with the S977F/T5TG12complex allele in trans with F508del mutation

AbstractBackgroundThe S977F mutation (c.2930C>T) in the CFTR gene (CFTR/ABCC7) is extremely rare. We describe the case of an adult patient carrying the complex allele S977F/T5TG12 in trans with the F508del mutation. Mild respiratory manifestations arose in adulthood associated with azoospermia, acute pancreatitis, minor hemoptysis and Cl− levels ranging from 40 to 42mEq/L.MethodDiagnosis was confirmed by repeated NPD measurements, genetic DHPLC analysis and a recently described functional assay measuring cAMP-dependent cell depolarization in peripheral blood monocytes.ResultsNPD measurements, DHPLC and monocyte functional assay (CF index=−18). Results were consistent with a CF phenotype.ConclusionsThe combined application of DHPLC and NPD analysis in the algorithm for CF diagnosis appears useful for the management of similar cases. In addition, the novel monocyte functional assay might contribute to improve our diagnostic capability, counseling and better treatment of these challenging clinical cases

Defective CFTR Expression and Function Are Detectable in Blood Monocytes: Development of a New Blood Test for Cystic Fibrosis

BACKGROUND: Evaluation of cystic fibrosis transmembrane conductance regulator (CFTR) functional activity to assess new therapies and define diagnosis of cystic fibrosis (CF) is cumbersome. It is known that leukocytes express detectable levels of CFTR but the molecule has not been characterized in these cells. In this study we aim at setting up and validating a blood test to evaluate CFTR expression and function in leukocytes. DESCRIPTION: Western blot, PCR, immunofluorescence and cell membrane depolarization analysis by single-cell fluorescence imaging, using the potential-sensitive DiSBAC(2)(3) probe were utilized. Expression of PKA phosphorylated, cell membrane-localized CFTR was detected in non-CF monocytes, being undetectable or present in truncated form in monocytes derived from CF patients presenting with nonsense mutations. CFTR agonist administration induced membrane depolarization in monocytes isolated from non-CF donors (31 subjects) and, to a lesser extent, obligate CFTR heterozygous carriers (HTZ: 15 subjects), but it failed in monocytes from CF patients (44 subjects). We propose an index, which values in CF patients are significantly (p<0.001) lower than in the other two groups. Nasal Potential Difference, measured in selected subjects had concordant results with monocytes assay (Kappa statistic 0.93, 95%CI: 0.80-1.00). RESULTS AND SIGNIFICANCE: CFTR is detectable and is functional in human monocytes. We also showed that CFTR-associated activity can be evaluated in 5 ml of peripheral blood and devise an index potentially applicable for diagnostic purposes and both basic and translational research: from drug development to evaluation of functional outcomes in clinical trials

Relationship between low Ankle-Brachial Index and rapid renal function decline in patients with atrial fibrillation: A prospective multicentre cohort study

OBJECTIVE: To investigate the relationship between Ankle-Brachial Index (ABI) and renal function progression in patients with atrial fibrillation (AF). DESIGN: Observational prospective multicentre cohort study. SETTING:Atherothrombosis Center of I Clinica Medica of 'Sapienza' University of Rome; Department of Medical and Surgical Sciences of University Magna Græcia of Catanzaro; Atrial Fibrillation Registry for Ankle-Brachial Index Prevalence Assessment-Collaborative Italian Study. PARTICIPANTS: 897 AF patients on treatment with vitamin K antagonists. MAIN OUTCOME MEASURES: The relationship between basal ABI and renal function progression, assessed by the estimated Glomerular Filtration Rate (eGFR) calculated with the CKD-EPI formula at baseline and after 2 years of follow-up. The rapid decline in eGFR, defined as a decline in eGFR >5 mL/min/1.73 m(2)/year, and incident eGFR<60 mL/min/1.73 m(2) were primary and secondary end points, respectively. RESULTS: Mean age was 71.8±9.0 years and 41.8% were women. Low ABI (ie, ≤0.90) was present in 194 (21.6%) patients. Baseline median eGFR was 72.7 mL/min/1.73 m(2), and 28.7% patients had an eGFR60 mL/min/1.73 m(2), 153 (23.9%) had a reduction of the eGFR <60 mL/min/1.73 m(2). ABI ≤0.90 was also an independent predictor for incident eGFR<60 mL/min/1.73 m(2) (HR 1.851, 95% CI 1.205 to 2.845, p=0.005). CONCLUSIONS: In patients with AF, an ABI ≤0.90 is independently associated with a rapid decline in renal function and incident eGFR<60 mL/min/1.73 m(2). ABI measurement may help identify patients with AF at risk of renal function deterioration

A machine-learning based bio-psycho-social model for the prediction of non-obstructive and obstructive coronary artery disease

Background: Mechanisms of myocardial ischemia in obstructive and non-obstructive coronary artery disease (CAD), and the interplay between clinical, functional, biological and psycho-social features, are still far to be fully elucidated. Objectives: To develop a machine-learning (ML) model for the supervised prediction of obstructive versus non-obstructive CAD. Methods: From the EVA study, we analysed adults hospitalized for IHD undergoing conventional coronary angiography (CCA). Non-obstructive CAD was defined by a stenosis &lt; 50% in one or more vessels. Baseline clinical and psycho-socio-cultural characteristics were used for computing a Rockwood and Mitnitski frailty index, and a gender score according to GENESIS-PRAXY methodology. Serum concentration of inflammatory cytokines was measured with a multiplex flow cytometry assay. Through an XGBoost classifier combined with an explainable artificial intelligence tool (SHAP), we identified the most influential features in discriminating obstructive versus non-obstructive CAD. Results: Among the overall EVA cohort (n = 509), 311 individuals (mean age 67 ± 11&nbsp;years, 38% females; 67% obstructive CAD) with complete data were analysed. The ML-based model (83% accuracy and 87% precision) showed that while obstructive CAD was associated with higher frailty index, older age and a cytokine signature characterized by IL-1β, IL-12p70 and IL-33, non-obstructive CAD was associated with a higher gender score (i.e., social characteristics traditionally ascribed to women) and with a cytokine signature characterized by IL-18, IL-8, IL-23. Conclusions: Integrating clinical, biological, and psycho-social features, we have optimized a sex- and gender-unbiased model that discriminates obstructive and non-obstructive CAD. Further mechanistic studies will shed light on the biological plausibility of these associations. Clinical trial registration: NCT02737982

Frequency of left ventricular hypertrophy in non-valvular atrial fibrillation

Left ventricular hypertrophy (LVH) is significantly related to adverse clinical outcomes in patients at high risk of cardiovascular events. In patients with atrial fibrillation (AF), data on LVH, that is, prevalence and determinants, are inconsistent mainly because of different definitions and heterogeneity of study populations. We determined echocardiographic-based LVH prevalence and clinical factors independently associated with its development in a prospective cohort of patients with non-valvular (NV) AF. From the "Atrial Fibrillation Registry for Ankle-brachial Index Prevalence Assessment: Collaborative Italian Study" (ARAPACIS) population, 1,184 patients with NVAF (mean age 72 \ub1 11 years; 56% men) with complete data to define LVH were selected. ARAPACIS is a multicenter, observational, prospective, longitudinal on-going study designed to estimate prevalence of peripheral artery disease in patients with NVAF. We found a high prevalence of LVH (52%) in patients with NVAF. Compared to those without LVH, patients with AF with LVH were older and had a higher prevalence of hypertension, diabetes, and previous myocardial infarction (MI). A higher prevalence of ankle-brachial index 640.90 was seen in patients with LVH (22 vs 17%, p = 0.0392). Patients with LVH were at significantly higher thromboembolic risk, with CHA2DS2-VASc 652 seen in 93% of LVH and in 73% of patients without LVH (p &lt;0.05). Women with LVH had a higher prevalence of concentric hypertrophy than men (46% vs 29%, p = 0.0003). Logistic regression analysis demonstrated that female gender (odds ratio [OR] 2.80, p &lt;0.0001), age (OR 1.03 per year, p &lt;0.001), hypertension (OR 2.30, p &lt;0.001), diabetes (OR 1.62, p = 0.004), and previous MI (OR 1.96, p = 0.001) were independently associated with LVH. In conclusion, patients with NVAF have a high prevalence of LVH, which is related to female gender, older age, hypertension, and previous MI. These patients are at high thromboembolic risk and deserve a holistic approach to cardiovascular prevention

Caratterizzazione elettrofisiologica e morfologica di sinapsi glutammatergiche nel muscolo scheletrico di mammifero

Ogni azione effettuata dal sistema nervoso come la percezione dei segnali sensoriali il controllo motorio, le funzioni cognitive come l\u2019apprendimento e la memoria, dipende da precise connessioni a livello dei neuroni. Queste specifiche connessioni sono realizzate durante la fase di sviluppo embrionale e post-natale. Lo studio dei meccanismi che sono alla base della formazione dei complessi circuiti neuronali ha da sempre affascinato ed interessato un ampio numero di scienziati. Negli ultimi decenni, gli avanzamenti delle tecniche di microscopia e di biologia molecolare hanno contribuito ad aumentare le nostre conoscenze sui principali meccanismi alla base della formazione delle sinapsi e sulle molecole responsabili della specificit\ue0 delle connessioni sinaptiche. Nonostante l\u2019enorme progresso delle nostre conoscenze nel campo restano ancora molti quesiti senza risposta, come ad esempio, non sono ancora noti i meccanismi che determinano il tipo di neurotrasmettitore nei neuroni. E\u2019 noto che la giunzione neuromuscolare di mammifero utilizza come neurotrasmettitore l\u2019acetilcolina, ma recentemente, \ue8 stato osservato che in particolari condizioni neuroni glutammatergici di mammifero sono in grado di formare sinapsi funzionali con fibre muscolari. Infatti in uno studio eseguito su ratto da un gruppo di ricercatori dell\u2019Universit\ue0 di Brescia, \ue8 stato osservato che, con l\u2019innesto di un nervo periferico tra il midollo spinale e un muscolo addominale, \ue8 possibile sostituire la sinapsi colinergica della giunzione neuromuscolare, con una nuova connessione glutammatergica proveniente da neuroni sopraspinali. Inoltre, \ue8 stato osservato che i mioblasti dello Xenopus durante lo sviluppo esprimono una grande variet\ue0 di recettori per vari tipi di neurotrasmettitori e che a seconda del fenotipo neurotrasmettitoriale, espresso dal terminale presinaptico, le cellule muscolari mantengono l\u2019appropriato recettore, suggerendo cos\uec, che la scelta del tipo di recettore dipende dal neurotrasmettitore liberato. Lo scopo della mia tesi \ue8 quello di approfondire i meccanismi alla base della formazione delle insolite sinapsi glutammatergiche muscolari nel mammifero. Nella prima parte del lavoro ho cercato di studiare mediante tecniche di elettrofisiologia intracellulare le sinapsi glutammatergiche muscolari che si formano in vivo tra vie discendenti e fibre muscolari nel ratto. Da questa prima parte non ho ottenuto delle registrazioni intracellulari in quanto gli animali operati hanno presentato tutti innervazione di tipo colinergico. Nella seconda parte del lavoro ho caratterizzato, con tecniche morfologiche, elettrofisiologiche e di biologia molecolare la formazione in vitro di sinapsi glutammatergica tra neuroni corticali e cellule di muscolo scheletrico di mammifero. Studi di immunofluorescenza hanno evidenziato la presenza di recettori AMPA (AMPAR) su cellule muscolari in cocoltura con neuroni corticali. In colture di soli miotubi il AMPAR non \ue8 espresso. In studi di imaging del calcio ho osservato che in seguito a stimolazione dei neuriti che contattano i miotubi si ha un incremento della concentrazione di calcio intracellulare. La stimolazione dei neuriti ha indotto contrazione dei miotubi. La somministrazione del curaro, un antagonista dei recettori colinergici, non ha modificato la contrazione della cellula muscolare, mentre la somministrazione di un antagonista dei recettori per il glutammato ha immediatamente bloccato la contrazione dei miotubi. Infine studi di immunoprecipitazione ed immunoblotting eseguiti su cocolture di neuroni corticali e cellule muscolari hanno dimostrato che il recettore del glutammato di tipo AMPA \ue8 espresso solamente dalle cellule muscolari in cocoltura e che l\u2019espressione del recettore \ue8 accompagnata dalla presenza di proteine che solitamente costituiscono la struttura postsinaptica di sinapsi centrali. Quindi i risultati del mio lavoro hanno dimostrato, per la prima volta, che \ue8 possibile indurre la formazione in vitro di una sinapsi glutammatergica nelle fibre muscolari di mammifero.Each of the behavioral tasks performed by the nervous system, from the perception of sensory input and the control of motor output to cognitive functions such as learning and memory, depends on precise interconnections of many millions of neurons. These connections are developed during embryonic and post natal development. The study of the mechanisms that underlie synaptic formation has always interested a great number of scientists. In the last ten years, microscopy and molecular biology techniques have supplied a great number of knowledges about the mechanisms of synaptic formation. Despite the amount of information about synaptogenesis there are still many unknown processes behind the development of a synapse, such as the mechanisms that induce the correct expression of neurotransmitters (and their receptors). It is well known that acetylcholine is the only neurotransmitter in mammalian neuromuscular junction, but recently it has been demonstrated that glutamatergic neurons are able to form functional synapses with mammalian muscle under particular conditions. At the University of Brescia it has been observed that a new glutamatergic innervation of skeletal muscle replaces the original cholinergic one when the transversus abdominal muscle is denervated and a distal nerve stump is inserted in the white lateral matter of spinal cord in rat. Moreover, Xenopus muscle cells express several classes of transmitter receptors in addition to those for acetylcholine in early embryonic development. During normal differentiation and innervation of muscle, the other classes of receptors disappear. Changing the expression of transmitters by altering calcium spike activity leads to retention of the classes of cognate, noncholinergic receptors. This indicates that the expression of appropriate receptors is due to the neurotransmitter The aim of this work was to study the mechanisms that underlie the formation of glutamatergic muscular synapses observed at the University of Brescia. Muscles, to which descending glutamatergic fibres had been diverted from the spinal cord by means of a peripheral nerve graft, were studied by intracellular electrophysiological techniques in the first part of the project. Surgical procedures were performed at Brescia University. Unfortunately, all analyzed muscles showed cholinergic innervation. In the second part of the project, murine co-cultures with cortical neurons and skeletal muscle cells were developed. Electrophysiological, biochemical and morphological analysis showed that, under these conditions, cortical neurons develop functional glutamatergic synapses with muscle cells. Immunofluorescence studies also have demonstrated the presence of AMPA receptors (AMPARs) on muscular cells in the cocultures. Calcium imaging studies showed that electrical stimulation of cortical neurites, reaching myotubes, induced an increase of intracellular calcium concentration in the muscular cells. Electrical stimulation of neurites also produced contraction of myotubes. The contractions were resistant to curare, a common blocker of acetylcholine receptors, but they were sensitive to the glutamate AMPAR antagonist (GYKI 52466). Finally, coimmunoprecipitation and immunoblotting analysis demonstrated that only myotubes cocultured with cortical neurons express AMPARs and moreover it was seen that muscle cells express proteins of central post synaptic density (PSD). These data show, for the first time, that it is possible to induce the formation of a glutamatergic synapse on mammalian muscle cell in vitro

Mutli-layer CTS antenna array in PCB technology for SatCom applications

International audienceA low-profile antenna for satellite communications (SatCom) applications is presented here. The antenna covers the entire K/Ka frequency bands from 19 GHz to 31 GHz with a reflection coefficient better than -10 dB, corresponding to a relative bandwidth of more than 50%. A continuous transverse stub (CTS) antenna array is chosen as a radiating aperture for its very wideband performance. The radiating aperture is fed by a pillbox quasi-optical beamformer. The full antenna is realized in printed circuit board (PCB) technology by stacking several dielectric layers without any buried or blind vias in the full stack-up. Multiple horns are placed along the focal plane of the pillbox coupler to achieve beam-scanning in elevation. +/- 25 degrees. The peak value of the realized gain is 19 dBi and radiation efficiency equals 50%

Perilymphatic Gusher in stapedectomy: demonstration of a fistola of internal auditory canal.

Gusher is a very rare phenomenon, generally associated with congenital stapes fixation or otosclerosis in adult age, that may present during stapedectomy. A sudden perilymph flow occurs following platinotomy, due to congenital malformation (abnormally wide cochlear aqueduct or internal auditory canal fistula), that causes an abnormal connection between subarachnoid and perilymphatic spaces. This report deals with a case of bilateral gusher, occurring during stapedectomy, caused by an osseous fistula between bottom of internal auditory canal and the osseous labyrinth later observed at computed tomography scan. The usefulness of a radiologic examination is stressed for a correct therapeutic approach in the even contralateral ear stapedectomy

Ultra-Low-Profile Continuous Transverse Stub Array for SatCom Applications

International audienceModern K-/Ka-band satellite communications have become increasingly demanding in recent years with regard to the performance of the terminal antenna used in the link. These terminals should be high gain and broadband, as well as guarantee polarization diversity over a large field of view. A low profile is also highly desired to integrate the antenna system into a variety of moving platforms, such as aircraft and trains. An ultra-low-profile continuous transverse stub array is proposed here to tackle these challenges. The antenna array is realized in printed circuit board technology by adopting a contactless transition between the stacked-up layers without resorting to blind or buried vias. The proposed antenna is wideband (impedance matched within the 19-31 GHz band, i.e., similar to 50%) and provides multibeam radiation for polar angles as far as +/- 22.5 degrees. The peak value of the gain is 19.7 dBi, and the maximum radiation efficiency equals 50%. Furthermore, the antenna array is combined with a dual-band polarization converter. The latter provides orthogonal circular polarization (CP) in two non-adjacent bands. The overall antenna system performs right- and left-hand CPs within the SatCom downlink and uplink in the K-/Ka-band, respectively. For each polarization, the axial ratio is below 3 dB over 9.47% and 4.42% fractional bandwidths, respectively