IMPACT OF CALCIFIC MITRAL STENOSIS ON SURVIVAL IN PATIENTS UNDERGOING TRANSCATHETER AORTIC VALVE REPLACEMENT

Objectives. This study was performed to investigate the prevalence and impact on survival of baseline calcific mitral stenosis (MS) in patients undergoing transcatheter aortic valve replacement (TAVR) due to the presence of severe symptomatic aortic stenosis (AS). Methods. This retrospective study included 928 consecutive patients with severe, symptomatic AS undergoing TAVR in two institutions, from January 2012 to August 2016. Mean follow-up was 40.8±13.9 months. Results. Based on mean mitral gradients (MMG) at baseline, 3 groups were identified: normal-mild, MMG<5 mmHg (n=737, 81.7%); moderate MMG5 and <10 mmHg (n=147, 16.3%); severe MMG10 mmHg (n=17, 1.9%). These latter were more frequently women, with a smaller body surface area, a higher prevalence of atrial fibrillation, chronic obstructive pulmonary disease and previous history of CABG/PCI. At baseline, patients with MMG10 mmHg compared with 5 and <10 mmHg and normal patients exhibited a lower mitral valve area (2.4±0.94 vs 2.1±0.86 vs 1.5±0.44 cm2) a lower prevalence of MR2+ (5.9% vs 28.6% and 15.6%, p<0.0001), a higher prevalence of severe mitral annular calcium (70.6% vs 45.6% and 13.0%, p<0.0001) and a higher systolic pulmonary arterial pressure (50.6±12.1 vs 47.2±14.5 and 41.6±14.4, p<0.0001). Despite the low prevalence of MMG10 mmHg, these patients experienced higher 5-year mortality compared to the other groups (adjusted Hazard Ratio: 2.91, 95% Confidence Interval [CI]:1.17-7.20, p=0.02). Conclusions. Severe calcific MS is uncommon in patients undergoing TAVR. However, its presence is associated with higher long-term mortality whereas moderate MS is not. The presence of severe calcific MS might identify a subgroup of patients in whom a double valve intervention should be considered

Valutazione dell’induzione di risposta adattativa a mutageni in colture cellulari di mammifero in seguito ad esposizioni a campi elettromagnetici non ionizzanti

2010 - 2011Nell’ambito del progetto di ricerca dal titolo “Valutazione dell’induzione di risposta adattativa a mutageni in colture cellulari di mammifero in seguito ad esposizioni a campi elettromagnetici non ionizzanti” è stato caratterizzato l’effetto protettivo (risposta adattativa) dell’esposizione a radiofrequenza (RF) dal danno indotto in colture cellulari da agenti a nota azione genotossica. La sperimentazione è stata eseguita in colture cellulari primarie (linfociti umani da 27 donatori sani) e in colture stabilizzate di roditore (fibroblasti di polmone di criceto, V79). In una fase iniziale, l’attività di ricerca ha riguardato l’approfondimento di osservazioni precedenti, dove si era riscontrato che linfociti umani da sangue periferico, pre-esposti ad un campo elettromagnetico alla frequenza di 900 MHz, segnale GSM, e trattati con Mitomicina C (MMC) mostravano un danno cromosomico ridotto rispetto ai trattamenti con sola MMC (Sannino et al., 2009). Applicando il test del micronucleo (MN) col blocco della citodieresi, è stato infatti, dimostrato che la pre-esposizione a RF è in grado di proteggere dal danno al DNA solo quando viene effettuata nella fase S del ciclo cellulare, inducendo risposta adattativa (RA), ma non ha alcun effetto in fase G0 o G1. Successivamente, l’attenzione è stata focalizzata su un segnale di telefonia mobile di terza generazione quale il segnale UMTS alla frequenza di 1950 MHz per valutare il ruolo a) dei parametri dell’esposizione (frequenza, modulazione e tasso di assorbimento specifico, SAR), b) del mutageno impiegato e c) del modello cellulare nella RA indotta da RF. La sperimentazione su colture cellulari di linfociti da sangue periferico ha evidenziato che anche pre-esposizioni a 1950 MHz sono in grado di evocare risposta adattativa, ma il grado di protezione dal danno indotto da MMC è strettamente dipendente dal SAR applicato. Inoltre, le stesse condizioni di esposizione si sono mostrate efficaci anche nella protezione di danno cromosomico indotto da trattamenti con raggi X, evidenziando che pre-esposizioni a RF sono in grado di ridurre il danno al DNA indipendentemente dalla natura e dal meccanismo di azione del mutageno impiegato. Infatti, mentre la MMC è un agente alchilante che induce cross-link nella molecola di DNA, i raggi X sono un agente clastogeno che induce rotture del singolo e doppio filamento. Risultati analoghi sono stati ottenuti quando sono state impiegate le V79 come modello cellulare, mostrando che il fenomeno dell’adattamento da RF non è limitato a cellule primarie quali i linfociti umani ma si riscontra anche in linee cellulari stabilizzate, sebbene in quest’ultimo caso siano richieste condizioni sperimentali più spinte, sia in termini di pre-trattamento (SAR) che di dosi di MMC. L’ultima parte del lavoro sperimentale ha riguardato la valutazione dei possibili meccanismi di azione alla base della RA indotta da RF, sulla base delle indicazioni riportate in letteratura sulla RA indotta da radiazioni ionizzanti. A tale scopo, nelle condizioni sperimentali che davano adattamento nei due tipi cellulari studiati, sono stati valutati effetti sulla vitalità cellulare (test di esclusione del tripan blue), sulla progressione del ciclo cellulare (test citofluorimetrico di incorporazione dello ioduro di propidio) e sul sistema di riparo del DNA (inibizione degli enzimi di riparazione mediante trattamento con 3-Aminobenzamide, 3AB). Nel caso dei linfociti umani è stata anche valutata l’apoptosi mediante il test citofluorimetrico dell’annessina V-FITC/ioduro di propidio. I risultati ottenuti sia con i linfociti umani sia con le V79 indicano che i meccanismi di azione alla base della risposta adattativa indotta da RF non coinvolgono la vitalità, il ciclo cellulare e l’apoptosi. Invece, è stato evidenziato un possibile ruolo degli enzimi di riparo del DNA nell’induzione del fenomeno. Infatti, in colture trattate con 3AB, che inibisce il legame della poli(ADP-ribosio) polimerasi alla cromatina, non si osserva adattamento. Riassunto E’ interessante sottolineare che, a conferma delle osservazioni riportate in questo progetto di ricerca, nel periodo di svolgimento del presente dottorato altri gruppi hanno riscontrato la capacità della RF ad indurre RA sia in vitro in colture di HL-60 (Jin et al., 2012) sia in vivo in topi e ratti (Cao et al., 2010, 2011; Jiang et al., 2012; Mortazavi et al., 2011, 2012) valutando differenti target biologici. [a cura dell'autore]X n.s

Genotoxicity of radiofrequency electromagnetic fields: Protocol for a systematic review of in vitro studies.

Abstract Background Exposure to radiofrequency electromagnetic fields (RF-EMF, 100 kHz – 300 GHz) emitted by wireless communication technologies is pervasive and ubiquitous. Concern has been raised about possible adverse effects to human health. In 2011 the International Agency for Research on Cancer has classified RF-EMF as possibly carcinogenic to humans, highlighting that the evidence is weak and far from conclusive. Updated systematic reviews of the scientific literature on this topic are lacking, especially for mechanistic studies. Objectives To develop a protocol for a systematic review of experimental studies investigating genotoxic effects induced by RF-EMF in in vitro cellular models. Genotoxicity is one of the key-biological indicators of carcinogenicity, and the most common characteristics of established carcinogens. The predefined procedures for conducting the systematic review are outlined below. Methods We will follow the guidelines developed by the National Toxicology Program-Office of Health Assessment and Translation (NTP-OHAT), adapted to the evaluation of in vitro studies. Eligibility criteria We will include experimental in vitro studies addressing the relationship between controlled exposures to RF-EMF and genotoxicity in mammalian cells only. Eligibility for inclusion will be further restricted to peer reviewed articles reporting findings from primary studies. Information sources We will search the scientific literature databases NCBI PubMed, Web of Science, and EMF-Portal. No filter on publication date will be applied. Only studies published in English will be considered. The reference lists of the included papers and available reviews will be screened for unidentified relevant papers. References will be managed through Endnote X9 software. Data extraction and synthesis of results Data from included papers will be extracted according to predefined forms. Heterogeneity within the available evidence will determine the type of evidence synthesis that is appropriate. Findings will be summarized in tables, graphical displays and in a narrative synthesis of the available evidences. A meta-analysis will be carried out if subgroups of studies homogeneous in terms of exposure characteristics, endpoint, and cell types will be identified. Risk of bias The internal validity of included studies will be assessed using the NTP-OHAT Risk of Bias Rating Tool for animal studies, adapted to in vitro studies. This stage of the process will be managed through the Health Assessment Workspace Collaborative (HAWC). Evidence appraisal To rate confidence in the body of evidence, we will use the OHAT GRADE-based approach for animal studies. Framework and funding This protocol concerns one of the evidence streams considered in a larger systematic review of the scientific literature on the potential carcinogenicity of RF-EMF, performed by scientists from several Italian public research agencies. The project is supported by the Italian Workers' Compensation Authority (INAIL) in the framework of the CRA with the Istituto Superiore di Sanita "BRiC 2018/06 – Scientific evidence on the carcinogenicity of radiofrequency electromagnetic fields"

Clinical Relevance of Baseline TCP in Transcatheter Aortic Valve Replacement

AIMS: To investigate the influence of baseline thrombocytopenia (TCP) on short-term and long-term outcomes after transcatheter aortic valve replacement (TAVR). METHODS AND RESULTS: A total of 732 consecutive patients with severe, symptomatic aortic stenosis undergoing TAVR from January 2012 to December 2015 were included. Primary outcomes of interest were the relationship of baseline TCP with 30-day and 1-year all-cause mortality. Secondary outcomes of interest were procedural complications and in-hospital mortality in the same subgroups. The prevalence of TCP (defined as platelet count <150 × 109/L) at baseline was 21.9%, of whom 4.0% had moderate/severe TCP (defined as platelet count <100 × 109/L). Compared to no or mild TCP, moderate/severe TCP at baseline was associated with a significantly higher 30-day mortality (23.3% vs 2.3% and 3.1%, respectively; P<.001) and 1-year mortality (40.0% vs 8.3% and 13.4%, respectively; P<.001). In Cox regression analysis, moderate/severe baseline TCP was an independent predictor of 30-day and 1-year mortality (hazard ratio [HR], 13.18; 95% confidence interval [CI], 4.49-38.64; P<.001 and HR, 5.90; 95% CI, 2.68-13.02; P<.001, respectively). CONCLUSIONS: In conclusion, baseline TCP is a strong predictor of mortality in TAVR patients, possibly identifying a specific subgroup of frail patients; therefore, it should be taken into account when addressing TAVR risk

Design of Antibody-Functionalized Polymeric Membranes for the Immunoisolation of Pancreatic Islets

none8noAn immunoencapsulation strategy for pancreatic islets aimed to reduce the risk of rejection in transplanted patients due to the immune response of the host organism is proposed. In this sense, a polyethylene glycol (PEG) hydrogel functionalized with an immunosuppressive antibody (Ab), such as Cytotoxic T-lymphocyte antigen-4 Ig (CTLA4-Ig), would act as both passive and active barrier to the host immune response. To demonstrate the feasibility of this approach, a photopolymerizable-PEG was conjugated to the selected antibody and the PEG-Ab complex was used to coat the islets. Moreover, to preserve the antigen-recognition site of the antibody during the conjugation process, a controlled immobilization method was setup through the attachment of the His-tagged antigen to a solid support. In detail, a gold-coated silicon wafer functionalized with 11-Mercaptoundecanoic acid was used as a substrate for further modification, leading to a nickel(II)-terminated ligand surface. Then, the immobilized antigen was recognized by the corresponding antibody that was conjugated to the PEG. The antibody-PEG complex was detached from the support prior to be photopolymerized around the islets. First, this immobilization method has been demonstrated for the green fluorescent protein (GFP)–anti-green fluorescent protein (Anti-GFP) antigen-antibody pair, as proof of principle. Then, the approach was extended to the immunorelevant B7-1 CTLA-4-Ig antigen-antibody pair, followed by the binding of Acryl-PEG to the immobilized constant region of the antibody. In both cases, after using an elution protocol, only a partial recovery of the antibody-PEG complex was obtained. Nevertheless, the viability and the functional activity of the encapsulated islets, as determined by the glucose-stimulated insulin secretion (GSIS) assay, showed the good compatibility of this approach.openAnna Cavallo; Ugo Masullo; Alessandra Quarta; Alessandro Sannino; Amilcare Barca; Tiziano Verri; Marta Madaghiele; Laura BlasiCavallo, Anna; Masullo, Ugo; Quarta, Alessandra; Sannino, Alessandro; Barca, Amilcare; Verri, Tiziano; Madaghiele, Marta; Blasi, Laur

ESOPE-Equivalent Pulsing Protocols for Calcium Electroporation: An In Vitro Optimization Study on 2 Cancer Cell Models

Reversible electroporation is used to increase the uptake of chemotherapeutic drugs in local tumor treatment (electrochemotherapy) by applying the pulsing protocol (8 rectangular pulses, 1000 V/cm, 100 µs) standardized in the framework of the European Standard Operating Procedure on Electrochemotherapy multicenter trial. Currently, new electrochemotherapy strategies are under development to extend its applicability to tumors with different histology. Electrical parameters and drug type are critical factors. A possible approach is to test pulse parameters different from European Standard Operating Procedure on Electrochemotherapy but with comparable electroporation yield (European Standard Operating Procedure on Electrochemotherapy-equivalent protocols). Moreover, the use of non-toxic drugs combined with electroporation represents the new frontier for electrochemotherapy applications; calcium electroporation has been recently proposed as a simple tool for anticancer therapy. In vitro investigations facilitate the optimization of electrical parameters and drugs for in vivo and clinical testing. In this optimization study, new pulsing protocols have been tested by increasing the pulse number and reducing the electric field with respect to the standard. European Standard Operating Procedure on Electrochemotherapy-equivalent protocols have been identified in HL-60 and A431 cancer cell models, and a higher sensitivity in terms of electroporation yield has been recorded in HL-60 cells. Moreover, cell killing efficacy of European Standard Operating Procedure on Electrochemotherapy-equivalent protocols has been demonstrated in the presence of increasing calcium concentrations on both cell lines. Equivalent European Standard Operating Procedure on Electrochemotherapy protocols can be used to optimize the therapeutic effects in the clinic, where different regions of the same cancer tissue, with different electrical properties, might result in a differential electroporation yield of the standard protocol over the same tissue, or, eventually, in an override of the operational limits of the instrument. Moreover, using calcium can help overcome the drawbacks of standard drugs (side effects, high costs, difficult handling, preparation, and storage procedures). These results support the possibility of new treatment options in both standard electrochemotherapy and calcium electroporation, with clear advantages in the clinic

Assessment of the Severity of Paravalvular Regurgitation and its Role on Survival After Transcatheter Aortic Valve Replacement

Background: To evaluate the impact of various measurements of paravalvular regurgitation (PVR) on survival after transcatheter aortic valve replacement (TAVR). PVR can be difficult to grade and both its incidence and impact on survival may be decreasing as TAVR evolves. Methods: This retrospective study included 911 patients undergoing TAVR in two institutions. PVR was graded according to the 3-grade scheme proposed by the guidelines (PVR grade), and subsequently grade 2 and 3, and grade 0 and 1 were lumped together. PVR was also graded as a composite score (PVR score), based on 6 commonly used metrics. PVR grade, PVR score and its six individual components were tested against the risk of both 1-year and longer term mortality after TAVR. Results: Patients with moderate/severe PVR had a higher Society of Thoracic Sugeons (STS) score, higher levels of serum creatinine and larger left atria compared to patients with none/mild PVR. Moderate/severe PVR was more frequent with self-expandable and larger valves. After adjusting for American College of Cardiology (ACC) TAVR risk score, neither PVR grade, PVR score nor its six components were associated with an increased risk of mortality at 1-year (severe PVR adjusted HR: 0.75, 95% Confidence Interval [CI]: 0.19, 3.01, p = 0.50). However, intervention for clinically severe PVR increased the risk of mortality by more than 7-fold (adjusted hazard ratio [HR]: 7.6, 95% CI: 2.4, 23.5, p < 0.0001). Conclusions: In the contemporary era, moderate-severe PVR is uncommon. However, re-intervention for PVR portends a poor prognosis. This highlights the crucial importance of clinical judgment over imaging alone