Condivisione anziché delibere: dalla formazione sul campo alla valutazione del cambiamento

È stato progettato e condotto nel 2012 un intervento formativo in presenza e sul campo, indirizzato all’appropriatezza prescrittiva di PSA, VES, PCR e degli esami prima di terapia estro-progestinica. Tutti i 430 medici di medicina generale (MMG) della ASL TO3, suddivisi in 28 equipe locali di circa 20 medici l’una, hanno partecipato a 2 incontri in plenaria, intercalati da lavoro personale e di equipe sui propri comportamenti prescrittivi. Il Controllo di Gestione della ASL ha evidenziato una diminuzione complessiva del 29% della prescrizione degli esami in oggetto, con un range per i singoli esami fra 10 e 56%, nonché una diminuzione complessiva del 15,59% di tutte le richieste di esami di laboratorio.Introduction. Following an act of Piemonte region about the appropriateness of lab test orders, a project of blended education (front and “learning on the job” education) was designed and implemented, with the aim of increasing the knowledge and awareness of General Practitioners (GP) about the appropriateness of orders of a set of lab tests (PSA, VES, PCR, tests for risk assessment before estro-progestinic medication). The main expected outcome was a change in order habits, with a decrease in the number of inappropriate orders. A “halo effect” was expected as well, showed by a decrease in orders of other lab tests than the ones addressed by the educational program. Methods. The project was designed according to the nine phases of PRECEDE-PROCEED model, particularly addressed to identify effective assessment models for interventions of improvement of health and to assist their implementation and evaluation. All 430 GPs of the local Health Trust ASL TO3 were involved and joined the project, organized in 28 local teams of about 20 members each. The educational program was held in 2012 and was based on two class general meetings (introduction of the project, guidelines for the appropriateness of orders, interim report on the activities of local teams, final report on results and suggestions), intertwined with local team meetings, in which the members of the teams reported about the audit of their behavior and about the mechanisms they identified as a cause of inappropriate ordering. Many of the teams displayed the characteristics of community of practice, with a reflective and active attitude. Results. At the end of the project the orders addressed by the educational program decreased overall of 29%, with the decrease of single exams ranging from 10 to 56%. This decrease corresponded to a decrease of € 523,471 of cost. Many feedback emerged from the local teams, pointing to the influence that the dispositions from consultants had in the process of request of lab tests. As a by-product, data from the ASL TO3 management control office showed a decrease of 15.59% of all lab tests in 2012

Human β3-Adrenoreceptor is Resistant to Agonist-Induced Desensitization in Renal Epithelial Cells

Background/Aims: We recently showed that the β3-adrenoreceptor (β3AR) is expressed in mouse kidney collecting ducts (CD) cells along with the type-2 vasopressin receptor (AVPR2). Interestingly, a single injection of a β3AR selective agonist promotes a potent antidiuretic effect in mice. Before considering the feasibility of chronic β3AR agonism to induce antidiuresis in vivo, we aimed to evaluate in vitro the signaling and desensitization profiles of human β3AR. Methods: Human β3AR desensitization was compared with that of human AVPR2 in cultured renal cells. Video imaging and FRET experiments were performed to dissect β3AR signaling under acute and chronic stimulation. Plasma membrane localization of β3AR, AVPR2 and AQP2 after agonist stimulation was studied by confocal microscopy. Receptors degradation was evaluated by Western blotting. Results: In renal cells acute stimulation with the selective β3AR agonist mirabegron, induced a dose-dependent increase in cAMP. Interestingly, chronic exposure to mirabegron promoted a significant increase of intracellular cAMP up to 12 hours. In addition, a slow and slight agonist-induced internalization and a delayed downregulation of β3AR was observed under chronic stimulation. Furthermore, chronic exposure to mirabegron promoted apical expression of AQP2 also up to 12 hours. Conversely, long-term stimulation of AVPR2 with dDAVP showed short-lasting receptor signaling, rapid internalization and downregulation and apical AQP2 expression for no longer than 3 h. Conclusions: Overall, we conclude that β3AR is less prone than AVPR2 to agonist-induced desensitization in renal collecting duct epithelial cells, showing sustained cAMP production, preserved membrane localization and delayed degradation after 12 hours agonist exposure. These results may be important for the potential use of chronic pharmacological stimulation of β3AR to promote antidiuresis overcoming in vivo renal concentrating defects caused by inactivating mutations of the AVPR2

Rosiglitazone promotes AQP2 plasma membrane expression in renal cells via a Ca-dependent/cAMP-independent mechanism.

Background/Aims: Thiazolidinediones are highly beneficial in the treatment of type II diabetes. However, they are also associated with edema and increased risk of congestive heart failure. Several studies demonstrated that rosiglitazone (RGZ) increases the abundance of aquaporin-2 (AQP2) at the plasma membrane of renal cells. The aim of this study was to investigate whether RGZ might activate a transduction pathway facilitating AQP2 membrane accumulation in renal cells. Methods: We analyzed the effect of RGZ on renal AQP2 intracellular trafficking in MCD4 renal cells by confocal microscopy and apical surface biotinylation. Cytosolic Ca2+ dynamics were measured by a video-imaging approach in single cell. Transient Receptor Potential (TRP) channels expression was determined by RT-PCR. Results: We showed that in MCD4 cells, short-term exposure to RGZ dramatically increases the amount of apically expressed AQP2 independently on cAMP production, PKA activation and AQP2 phosphorylation. RGZ elicited a cytosolic Ca2+ transient due to Ca2+ influx prevented by ruthenium red, suggesting the involvement of TRP plasma membrane channels. We identified TRPV6 as the possible candidate mediating this effect. Conclusions: Taken together these results provide a possible molecular mechanism explaining the increased AQP2 membrane expression under RGZ treatment: in renal cells RGZ elicits Ca2+ transients facilitating AQP2 exposure at the apical plasma membrane, thus increasing collecting duct water permeability. Importantly, this effect suggests an unexplored application of RGZ in the treatment of pathological states characterized by impaired AQP2 trafficking at the plasma membrane

Congenital rhabdomyosarcoma: a different clinical presentation in two cases

Background: Rhabdomyosarcoma (RMS), one of the most common soft tissue sarcomas of childhood, is very rare in the neonatal period (0.4-2% of cases). In order to gain a deeper understanding of this disease at such age, patient and tumor features, as well as treatment modality and outcome need to be reported. Case presentation: We describe two cases with congenital RMS treated at Bambino Gesù Children's Hospital between 2000 and 2016. They represent only 2.24% of all RMS patients diagnosed during that period in our Institution; this data is in agreement with the incidence reported in the literature. They reflect the two different clinical forms in which the disease may manifest itself. One patient, with the alveolar subtype (positive for specific PAX3-FOXO1 fusion transcript) and disseminated disease, had a fatal outcome with central nervous system (CNS) progression despite conventional and high dose chemotherapy. The other child, with the localized embryonal subtype, was treated successfully with conservative surgery and conventional chemotherapy, including prolonged maintenance therapy. He is disease free at 7 years of follow-up. Conclusions: RMS can also be diagnosed during the neonatal period. Given the young age, disease management is often challenging, and especially for the alveolar subtype, the outcome is dismal despite intensified multimodality therapy. In fact, it characteristically manifests with multiple subcutaneous nodules and progression most commonly occurs in the CNS (Rodriguez-Galindo et al., Cancer 92(6):1613-20, 2001). In this context, CNS prophylaxis could play a role in preventing leptomeningeal dissemination, and molecular studies can allow a deeper tumor characterization, treatment stratification and identification of new potential therapeutic targets

Current evidence of antifungal prophylaxis and therapy in pediatric patients

Invasive fungal infections (IFI) are an important complication in pediatric haematological and oncological patients who undergo intensive chemotherapy for leukemia, solid tumour at advanced stage or relapsed, and hematopoietic stem cell transplantation. The incidence of IFI is lower than bacterial infection but mortality rate remains high. This review is designed to help paediatric oncologists in choosing the appropriate anti-fungal strategy and agents for prophylaxis, empirical, pre-emptive and specific therapy on the basis of published evidence

Healthcare Professional Communication on Sexual Health: A Report from the Italian Working Group on Adolescents and Young Adults with Cancer

Background: Sexual function is an important concern for adolescent and young adult (AYA) with cancer. The aim of this study was to explore the attitude of Italian health care professionals who deal with AYA patients with cancer toward sexual health communication. Materials and methods: A 11-question survey was developed by the AIOM (Associazione Italiana di Oncologia Medica) and AIEOP (Associazione Italiana Ematologia Oncologia Pediatrica) AYA workgroup and sent to AIOM and AIEOP members. Results: The sample comprised 360 respondents, 54.2% AIEOP and 45.8% AIOM members. Eighty percent were physicians, 14.5% nurses, 4.7% psychologists, and 0.8% other professionals. Medical oncologists are more used to investigate about AYA sexual health than pediatric oncologists (58.2% vs. 46.2%), even if pediatrics more frequently refer patients to specific and shared protocol (40% vs. 26.1%). Both AIOM and AIEOP participants mostly talk about sexual health only on request or occasionally (78.8% and 79%, respectively). Clinician-reported barriers to communication identified in this study are lack of preparation and embarrassment for both the categories, plus the presence/interference of parents for pediatrics and lack of time for medical oncologists. Overall, less than 5% of clinicians in our survey received specific training on potential sexual health issues in AYA patients with cancer and only 2% felt adequately prepared to speak about it. Conclusion: Sexual health is a key component of comprehensive care for AYA with cancer during treatments. This study highlighted the need of Italian providers for specific training and guidelines on sex-related health issues encountered by AYA patients

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression

Drug resistance; Soft tissue sarcoma; Targeted therapyResistencia a las drogas; Sarcoma de tejido blando; Terapia dirigidaResistència als fàrmacs; Sarcoma dels teixits tous; Teràpia dirigidaRhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.This research was funded by Associazione Italiana per la Ricerca sul Cancro (AIRC) #15312 to RR and #24696 to FM; Italian Ministry of Health (Ricerca Corrente) to BDA, CQ, and RR; AIRC 5xmille #9962 to FL; Italian Ministry of Health (Fondi 5xmille 2021-2022) to RR; Alleanza Contro il Cancro (ACC) Italian Network-Working Group Sarcomas to BDA, RM, and RR; Fondi Ateneo 2019 to FM; MIUR-Italy: Grant to Department of Science, Roma Tre University (Dipartimento di Eccellenza, ARTICOLO 1, COMMI 314—337 LEGGE 232/2016) to MCe and PM

Activation of the Thiazide-Sensitive Sodium-Chloride Cotransporter by Beta3-Adrenoreceptor in the Distal Convoluted Tubule

We previously showed that the beta-3 adrenergic receptor (BAR3) is expressed in most segments of the nephron where its agonism promotes a potent antidiuretic effect. We localized BAR3 in distal convoluted tubule (DCT) cells expressing the thiazide-sensitive sodium-chloride cotransporter (NCC). Aim of this study is to investigate the possible functional role of BAR3 on NCC modulation in DCT cells. Here, we found that, in mice, the knockout of BAR3 was paralleled by a significant attenuation of NCC phosphorylation, paralleled by reduced expression and activation of STE-20/SPS1-related proline-alanine-rich kinase (SPAK) and WNKs the main kinases involved in NCC activation. Conversely, in BAR1/2 knockout mice, we found reduced NCC abundance with no changes in the phosphorylation state of NCC. Moreover, selective BAR3 agonism promotes both SPAK and NCC activation in wild-type mouse kidney slices. In conclusion, our findings suggest a novel role for BAR3 in the regulation of NCC in DCT

Role of Nuclear Lamin A/C in the Regulation of Nav1.5 Channel and Microtubules: Lesson From the Pathogenic Lamin A/C Variant Q517X

In this work, we studied an lmna nonsense mutation encoding for the C-terminally truncated Lamin A/C (LMNA) variant Q517X, which was described in patients affected by a severe arrhythmogenic cardiomyopathy with history of sudden death. We found that LMNA Q517X stably expressed in HL-1 cardiomyocytes abnormally aggregates at the nuclear envelope and within the nucleoplasm. Whole-cell patch clamp experiments showed that LMNA Q517X-expressing cardiomyocytes generated action potentials with reduced amplitude, overshoot, upstroke velocity and diastolic potential compared with LMNA WT-expressing cardiomyocytes. Moreover, the unique features of these cardiomyocytes were 1) hyper-polymerized tubulin network, 2) upregulated acetylated α-tubulin, and 3) cell surface Nav1.5 downregulation. These findings pointed the light on the role of tubulin and Nav1.5 channel in the abnormal electrical properties of LMNA Q517X-expressing cardiomyocytes. When expressed in HEK293 with Nav1.5 and its β1 subunit, LMNA Q517X reduced the peak Na+ current (INa) up to 63% with a shift toward positive potentials in the activation curve of the channel. Of note, both AP properties in cardiomyocytes and Nav1.5 kinetics in HEK293 cells were rescued in LMNA Q517X-expressing cells upon treatment with colchicine, an FDA-approved inhibitor of tubulin assembly. In conclusion, LMNA Q517X expression is associated with hyper-polymerization and hyper-acetylation of tubulin network with concomitant downregulation of Nav1.5 cell expression and activity, thus revealing 1) new mechanisms by which LMNA may regulate channels at the cell surface in cardiomyocytes and 2) new pathomechanisms and therapeutic targets in cardiac laminopathies

Functional characterization of a novel truncating mutation in Lamin A/C gene in a family with a severe cardiomyopathy with conduction defects

Background/Aims: Truncating LMNA gene mutations occur in many inherited cardiomyopathy cases, but the molecular mechanisms involved in the disease they cause have not yet been systematically investigated. Here, we studied a novel frameshift LMNA variant (p.D243Gfs*4) identified in three members of an Italian family co-segregating with a severe form of cardiomyopathy with conduction defects. Methods: HEK293 cells and HL-1 cardiomyocytes were transiently transfected with either Lamin A or D243Gfs*4 tagged with GFP (or mCherry). D243Gfs*4 expression, cellular localization and its effects on diverse cellular mechanisms were evaluated with western blotting, laser-scanning confocal microscopy and video-imaging analysis in single cells. Results: When expressed in HEK293 cells, GFP- (or mCherry)-tagged LMNA D243Gfs*4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfs*4 did not significantly induce ER stress response, abnormal Ca2+ handling and apoptosis when compared with HEK293 cells expressing another truncated mutant of LMNA (R321X) which similarly accumulates within the ER. Of note, HEK293-LMNA D243Gfs*4 cells showed a significant reduction of connexin 43 (CX43) expression level, which was completely rescued by activation of the WNT/β-catenin signaling pathway. When expressed in HL-1 cardiomyocytes, D243Gfs*4 significantly impaired the spontaneous Ca2+ oscillations recorded in these cells as result of propagation of the depolarizing waves through the gap junctions between non-transfected cells surrounding a cell harboring the mutation. Furthermore, mCh-D243Gfs*4 HL-1 cardiomyocytes showed reduced CX43-dependent Lucifer Yellow (LY) loading and propagation. Of note, activation of β-catenin rescued both LY loading and LMNA D243Gfs*4 -HL-1 cells spontaneous activity propagation. Conclusion: Overall, the present results clearly indicate the involvement of the aberrant CX43 expression/activity as a pathogenic mechanism for the conduction defects associated to this LMNA truncating alteration