CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) IN HUMAN LEUKOCYTES

SCOPO: 1) valutare l\u2019espressione della proteine che regola il trasporto del cloro transmembrana (CFTR) e la sua attivit\ue0 funzionale nei monociti; 2) creare delle linee cellulari immortalizzate a partire da linfociti B aventi genotipi differenti; 3) valutare linee cellulari immortalizzate. CONOSCENZE DI BASE: La Fibrosi Cistica (CF), la pi\uf9 commune e grave malattia autosomale diffusa nei Paesi Caucasici, \ue8 dovuta alla presenza di mutazioni sul gene CFTR. Sebbene la CF sia una malattia multi organo, la patologia polmonare \ue8 la principale causa di morte tra i pazienti CF. E\u2019 caratterizzata da una infiammazione cronica, conseguenza di un\u2019infezione batterica. La suscettibilit\ue0 alle infezioni batteriche non \ue8 completamente conosciuta, anche se il coinvolgimento di CFTR nelle funzioni microbicide dei macrofagi sta emergendo in questo periodo. I macrofagi differenziano in situ a partire dai monociti infiltati nel tessuto e mostrano una marcata variabilit\ue0 morfologica pur avendo comuni funzioni cellulari e molecolari. Sebbene l\u2019espressione di CFTR nei macrofagi alveolari sia stata descritta, la sua espressione nei monociti non \ue8 ancora stata riportata anche se queste cellule risultano essere pi\uf9 accessibili per studi funzionali e di espressione. La valutazione dell\u2019espressione e della funzione del CFTR nelle cellule mononucleari del sangue periferico (PBMC) \ue8 un pre-requisito per valutare il loro ruolo ed il loro potenziale utilizzo in diagnostica e nello sviluppo di nuovi farmaci con azione sul difetto molecolare del CFTR. METODI: Purificazione dei monociti e dei linfociti B da sangue intero; produzione del virus Epstein-Barr (EBV); immortalizzazione dei linfociti B mediante EBV; isolamento RNA e analisi dell\u2019mRNA mediante PCR; Real-time PCR; Western blotting; citometria di flusso; immunofluorescenza; depolarizzazione di membrana; misurazione delle differenze dei potenziali nasali; analisi dei dati. RISULTATI: Utilizzando un anticorpo anti-CFTR policlonale e due monoclonali che riconoscono differenti epitopi, abbiamo rilevato mediante western blotting tutte le forme conosciute del CFTR. La citometria di flusso e la microscopia confocale ha confermato l\u2019espressione di CFTR e la sua localizzazione su membrana. Abbiamo osservato che i monociti non-CF, dopo stimolazione con uno specifico agonista di CFTR, mostravano un aumento dell\u2019intensit\ue0 di fluorescenza, una variazione che non abbiamo rilevato nei monociti CF. Questi risultati hanno suggerito una correlazione dell\u2019attivit\ue0 di CFTR con la depolarizzazione della membrana ed i dati sono stati confermati tramite uno specifico inibitore, CFTR (inh)-172. Questo approccio \ue8 stato comparato alla misurazione dei potenziali nasali (NPD) eseguiti sugli stessi soggetti ed la sovrapposizione dei dati ha rilevato una forte corrispondenza tra le due tecniche. I linfociti B sono stati immortalizzati mediante EBV e sono stati utilizzati come potenziali modelli cellulari per valutare l\u2019attivit\ue0 del CFTR. Abbiamo rilevato la maggiore forma glicosilata di CFTR in queste linee immortalizzate utilizzando un anticorpo monoclonale anti-CFTR. Una forma a minore peso molecolare \ue8 stata anch\u2019essa evidenziata mediante questo anticorpo ed uno policlonale. La citometria di flusso e la microscopia confocale ci hanno permesso di confermare l\u2019espressione e la localizzazione su membrana del CFTR. Il test di depolarizzazione della membrana \ue8 stato applicato sulle cellule B immortalizzate ottenendo gli stessi risultati visti sui monociti. CONCLUSIONI: Abbiamo dimostrato l\u2019espressione della proteina CFTR in monociti umani identificando una variante a peso molecolare corrispondente ad un basso livello di post-trasduzione della proteina. Questo \ue8 stato confermato utilizzando monociti con genotipo omozigote per la mutazione non-sense i quali perdevano l\u2019espressione della forma di CFTR. La citometria di flusso potrebbe essere utile per valutare l\u2019espressione di CFTR. Infatti abbiamo dimostrato che pu\uf2 distinguere tra non-CF ed eterozigoti da pazienti CF mediante la marcatura CD14/Rb-AF488 dei monociti. L\u2019analisi della depolarizzazione di membrana su singola cellula ha confermato l\u2019espressione funzionale del CFTR mostrando una elevata depolarizzazione di membrana a seguito della stimolazione delle cellule con uno specifico agonista del CFTR. Questo metodo potrebbe essere eseguito entro un paio di ore dal prelievo del sangue. Inoltre, \ue8 facilmente riproducibile con un minimo disturbo e rischio per il paziente e potrebbe permettere una valutazione in corso d\u2019opera degli effetti di alcune particolari terapie sull\u2019espressione e sull\u2019attivit\ue0 del CFTR. Abbiamo creato uno specifico indice capace di discriminare tra CF e non-CF. La sovrapposizione dei dati NPD e di quelli sull\u2019attivit\ue0 funzionale del CFTR sui monociti risultata in una perfetta corrispondenza tra le due tecniche. Poich\ue9 NPD \ue8 un test diagnostico che si applica su soggetti con test del sudore dubbio o con almeno una mutazione non conosciuta, possiamo promuovere la valutazione dell\u2019attivit\ue0 del CFTR nei monociti mediante tecniche ottiche come un utile metodo per valutare l\u2019attivit\ue0 di CFTR per la ricerca, includendo lo sviluppo di nuovi farmaci e la diagnosi. Dato che le cellule primarie hanno disponibilit\ue0 limitata in termini quantitativi, abbiamo preso vantaggio dall\u2019osservazione che i linfociti esprimono CFTR. Le cellule B immortalizzate potrebbero essere utilizzate come modello cellulare per studiare l\u2019espressione e l\u2019attivit\ue0 del CFTR. Abbiamo rilevato l\u2019espressione di una forma di CFTR che probabilmente rappresenta una isoforma processata a seguito dell\u2019attivit\ue0 di una specifica calpaina nei linfociti come dimostrato in letteratura. Inoltre, l\u2019indice ottenuto mediante lo studio della depolarizzazione di membrana ci ha permesso di discriminare tra gruppi CF e non-CF come osservato nei monociti. Tutti questi risultati hanno dimostrato che il CFTR \ue8 espresso ed \ue8 attivo nei linfociti umani e nelle cellule B immortalizzate. Per tale motivo, queste cellule possono essere sfruttate per valutare la risposta di specifiche mutazioni a nuovi farmaci diretti o indiretti sul difetto di base di CF.AIM: 1) to evaluate the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and functional activity in monocytes; 2) to create immortalized cell lines from human B-lymphocyte cells characterized by different genotypes; 3) to evaluate CFTR protein expression in immortalized B cells. BACKGROUND: Cystic Fibrosis (CF), the most common autosomal severe disorder in Caucasians, is caused by mutations in the CFTR gene. Although CF is a multi-organ disease, the lung pathology is the main cause of morbidity and mortality of CF patients. It is characterized by chronic inflammation as a consequence of persistent bacterial infections by several opportunistic pathogens. Mechanisms leading to increased susceptibility to bacterial infections in CF are not completely known, although the involvement of CFTR in microbicidal functions of macrophages is emerging. Tissue macrophages differentiate in situ from infiltrating monocytes and display a remarkable variability in cell morphology although common molecular and cellular functions. Although expression of CFTR in alveolar macrophages has been described, its expression has not been reported in monocytes that are more accessible for expression studies and functional analysis tests than macrophages. Evaluation of expression and functional activity of CFTR in peripheral blood mononuclear cells (PBMC) is a pre-requisite to evaluate their role and their potential use in diagnostic and developing new drugs acting on the molecular defect of CF. METHODS: Purification of monocytes and lymphocyte B cells from whole blood; production of Epstein-Barr Virus (EBV); immortalization of Lymphocytes B cells by EBV; RNA isolation and CFTR mRNA analysis by reverse-transcription and polymerase chain reaction (PCR); quantitative real-time PCR (RT-qPCR); western Blotting; Flow cytometry assay; immunofluorescence; cell depolarization assay; Nasal Potential Differences (NPDs) assay; analysis of cell depolarization assay data. RESULTS: In this study western blotting using a polyclonal and two monoclonal anti-CFTR antibodies that recognize different epitopes detected all known forms of CFTR. Flow cytometry and confocal microscopy analysis confirmed expression of CFTR protein expression and its membrane localization. Increased fluorescence intensity, corresponding to membrane depolarization, was observed only when non-CF monocytes were stimulated with CFTR agonist, while CF monocytes did not show fluorescence variation. These results suggested a correlation between CFTR activity and membrane depolarization and data were confirmed using a specific CFTR inhibitor, CFTR (inh)-172. This approach was compared to NPD measurements performed in a subset of the same patients subjected to this analysis. Results obtained by NPD overlapped those obtained by the analysis of monocytes from non-CF donors and CF patients. B-lymphocytes were then immortalized by EBV and were tested as potential cell models for CFTR activity assays. The major glycosylated form of CFTR was detected in immortalized non-CF EVB-transformed B cell line by a monoclonal anti-CFTR antibody, but a band with minor molecular weight was also detected with this antibody and with a polyclonal anti-CFTR antibody. Flow cytometry and confocal assay allowed us to confirm CFTR expression and membrane location in these cell lines. Membrane depolarization test was applied in EBV-transformed B cells and the results confirmed a stimulus induced membrane depolarization in non- CF cells. CONCLUSION: We have demonstrated that CFTR proteins are expressed in human monocytes as a variant recognized by a specific antibody. Its molecular weight is consistent with a lower level of post-translational processing and its loss in patients carrying a homozygous non-sense mutation confirmed its presence in human monocytes. Flow cytometry could be also a useful method to evaluate CFTR expression. We demonstrated that it can distinguish between non-CF and HTZ subjects and CF patients analyzing stained CD14/Rb-AF488 monocytes. Single-cell membrane depolarization analysis confirmed that, upon stimulation with CFTR agonists, normal monocytes displayed a highly reproducible membrane depolarization activity consistent with the expression of functional CFTR. Single-cell depolarization assay could be performed within a few hours after blood collection. It is also easily repeatable with a minimal discomfort and risk for the patient and it could thus allow a time-course evaluation of effects of any particular therapy on CFTR expression or functional activity. A specific activity index was devised that appears capable to discriminate among CF and non-CF cells. Overlapping NPD data and functional activity data, we observed a perfect correspondence. Since NPD is a reference diagnostic test applied when a subject has borderline sweat test and at least one unidentified CFTR mutation, we might promote the evaluation of CFTR activity in monocytes by optical techniques as a useful tool to assess CFTR activity for basic and translational research, including drug development and diagnosis. As primary cells are available in limited amounts, we have taken advantage of the observation that CFTR-associated Cl- permeability has been demonstrated in lymphocytes. So, immortalized-B-cells could be useful as cellular model to study CFTR expression and activity. We observed a form of CFTR that likely represents a processed isoform possibly linked to specific calpain activity in lymphocyte cells as demonstrated in the literature. Furthermore, the index obtained by single-cell fluorescence imaging discriminated between non-CF and CF groups as shown in monocytes. All these results demonstrated that CFTR protein is expressed and is active in human lymphocytes and EBV-transformed B cells opening interesting perspectives in this field. Indeed, these cells can be exploited to evaluate the response of specific mutations to newly developed drugs acting directly or indirectly on the basic defect of CF

How the andrological sector suffered from the dramatic Covid 19 outbreak in Italy: supportive initiatives of the Italian Association of Andrology (SIA)

The possible strategies for the remodulation of the andrological activity were discussed and shared within a team of national experts belonging to the Italian Society of Andrology (SIA). Initiatives for andrologists. With the various provisions of the Prime Minister that have followed all the national and local scientific meetings were canceled. In order to compensate for this abrupt lack of opportunities for scientific meetings, a cycle of live reports was activated on YOUTUBE by recognized experts, to cover many different andrological topics. The YOUTUBE channel was chosen as it can be easily followed by each member without having to download any streaming program, with the possibility to consult the contents without any time limitation. Initiatives for patients In this new context, non-urgent outpatient activities (such as Andrology) have been suspended throughout the national territory

The role of flower pollen extract in managing patients affected by chronic prostatitis/chronic pelvic pain syndrome. a comprehensive analysis of all published clinical trials

Background: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still a challenge to manage for all physicians. We feel that a summary of the current literature and a systematic review to evaluate the therapeutic efficacy of flower pollen extract would be helpful for physicians who are considering a phytotherapeutic approach to treating patients with CP/CPPS. Methods: A comprehensive search of the PubMed and Embase databases up to June 2016 was performed. This comprehensive analysis included both pre-clinical and clinical trials on the role of flower pollen extract in CP/CPPS patients. Moreover, a meta-analysis of available randomized controlled trials (RCTs) was performed. The NIH Chronic Prostatitis Symptom Index (NIH-CPSI) and Quality of Life related questionnaires (QoL) were the most commonly used tools to evaluate the therapeutic efficacy of pollen extract. Results: Pre-clinical studies demonstrated the anti-inflammatory and anti-proliferative role of pollen extract. 6 clinical, non-controlled studies including 206 patients, and 4 RCTs including 384 patients were conducted. The mean response rate in non-controlled studies was 83.6% (62.2%-96.0%). The meta-analysis revealed that flower pollen extract could significantly improve patients’ quality of life [OR 0.52 (0.34-.0.81); p = 0.02]. No significant adverse events were reported. Conclusion: Most of these studies presented encouraging results in terms of variations in NIH-CPSI and QoL scores. These studies suggest that the use of flower pollen extract for the management of CP/CPPS patients is beneficial. Future publications of robust evidence from additional RCTs and longer-term follow-up would provide more support encouraging the use of flower pollen extracts for CP/CPPS patients

numerical evaluation of the applicability of steady test bench swirl ratios to diesel engine dynamic conditions

Engine coherent flow structures such as swirl and tumble motions are key factors for the combustion process due to their capability to rise turbulence levels and enhance mixing which, in turns, severely influence both fuel efficiency and pollutant emissions. Automotive industry has therefore put great efforts over the last decades in evaluating air flow during induction stroke and air flow within the cylinder. Nowadays swirl and tumble motion characterizing a specific cylinder head are evaluated experimentally at design stage mainly using stationary flow benches. Such tests allow characterizing each head prototype using non-dimensional parameters like swirl and tumble ratios and, finally, to compare the different designs. In the present work the authors focused their attention on the swirl ratio characterization, firstly reviewing the two main methodologies for evaluating such parameter and more precisely the AVL and the Ricardo ones. A numerical method is then proposed in order to reproduce the stationary test bench with the final goal to develop a fast and accurate virtual test bench for cylinder head design. Simulations have been carried out on different VM Motori engine heads for which experimental data were available. The comparison between computational and experimental swirl ratios allowed to evaluate the suitability of using a virtual test bench as alternative or complementary to experiments. These results widened the understanding of the swirl fluid-dynamics and suggested that care must be taken when comparing duct designs having no geometrical similarity. Finally dynamic simulations have been performed for the head prototypes in order to compute the engine swirl in realistic conditions and to compare it with the steady bench results. This allowed evaluating the capability of the two different "static" swirl ratio definition (AVL/Ricardo) in correctly estimating real engine swirl. © 2015 The Authors. Published by Elsevier Ltd

Temperature profile of ex-vivo organs during radio frequency thermal ablation by fiber Bragg gratings.

We report on the integration of fiber optic sensors with commercial medical instrumentation for temperature monitoring during radio frequency ablation for tumor treatment. A suitable configuration with five fiber Bragg grating sensors bonded to a bipolar radio frequency (RF) probe has been developed to monitor the area under treatment. A series of experiments were conducted on ex-vivo animal kidney and liver and the results confirm that we were able to make a multipoint measurement and to develop a real-time temperature profile of the area, with a temperature resolution of 0.1°C and a spatial resolution of 5 mm during a series of different and consecutive RF discharges

Serenoa repens associated with selenium and lycopene extract and bromelain and methylsulfonylmethane extract are able to improve the efficacy of levofloxacin in chronic bacterial prostatitis patients

Objective: To date, the management of patients with chronic bacterial prostatitis (CBP) is not satisfactory, especially in terms of symptoms relief. Here, we evaluated the efficacy and the safety of a combination of serenoa repens, selenium and lycopene extract + bromelain and methylsulfonylmethane extract associated with levofloxacin in patients with CBP. Materials and methods: All patients with clinical and instrumental diagnosis of CBP, admitted to a single Urological Institution from March to June 2015 were enrolled in this phase III study. All enrolled patients were randomized into two groups: Group A received levofloxacin 500 mg o.d. for 14 days associated with lycopene and methylsulfonylmethane; Group B received levofloxacin (500 mg o.d. for 14 days) only. Clinical and microbiological analyses were carried out at the time of admission (T0) and during the followups at 1 month (T1) and 6 months (T2) from the end of the treatment. NIH Chronic Prostatitis Symptom Index (CPSI), International Prostatic Symptom Score (IPSS) and Quality of Well-Being (QoL) questionnaires were used. The main outcome measures were the rate of microbiological cure and the improvement in questionnaire results from baseline at the end of the follow-ups period. Results: Forty patients were enrolled in Group A and 39 in Group B. During the follow-up (T1), we recorded a significant changes in terms of NIH-CPSI and IPSS in Group A (mean difference: 17.6 ± 2.65; 12.2 ± 2.33; p < 0.01; p < 0.05, respectively) and versus Group B at the intergroup analysis (mean difference: -9 ± 1.82; -8.33 ± 1.71; p < 0.05; p < 0.05, respectively). No differences were reported in terms of microbiological findings between the two groups. At the second follow-up visit (T2), questionnaire results demonstrated statistically significant differences between groups (p < 0.001). One patient in Group A (2.5%) and 7 patients (17.9%) in Group B showed a symptomatic and microbiological recurrence (p = 0.02). Conclusions: The combination of serenoa repens, selenium, lycopene + bromelain and methylsulfonylmethane extracts improved the clinical efficacy of levofloxacin in patients affected by CBP without the development of side effects