Dimerization of GPCRs:Novel insight into the role of FLNA and SSAs regulating SST₂ and SST₅ homo- and hetero-dimer formation

The process of GPCR dimerization can have profound effects on GPCR activation, signaling, and intracellular trafficking. Somatostatin receptors (SSTs) are class A GPCRs abundantly expressed in pituitary tumors where they represent the main pharmacological targets of somatostatin analogs (SSAs), thanks to their antisecretory and antiproliferative actions. The cytoskeletal protein filamin A (FLNA) directly interacts with both somatostatin receptor type 2 (SST2) and 5 (SST5) and regulates their expression and signaling in pituitary tumoral cells. So far, the existence and physiological relevance of SSTs homo- and hetero-dimerization in the pituitary have not been explored. Moreover, whether octreotide or pasireotide may play modulatory effects and whether FLNA may participate to this level of receptor organization have remained elusive. Here, we used a proximity ligation assay (PLA)-based approach for the in situ visualization and quantification of SST2/SST5 dimerization in rat GH3 as well as in human melanoma cells either expressing (A7) or lacking (M2) FLNA. First, we observed the formation of endogenous SST5 homo-dimers in GH3, A7, and M2 cells. Using the PLA approach combined with epitope tagging, we detected homo-dimers of human SST2 in GH3, A7, and M2 cells transiently co-expressing HA- and SNAP-tagged SST2. SST2 and SST5 can also form endogenous hetero-dimers in these cells. Interestingly, FLNA absence reduced the basal number of hetero-dimers (-36.8 ± 6.3% reduction of PLA events in M2, P &lt; 0.05 vs. A7), and octreotide but not pasireotide promoted hetero-dimerization in both A7 and M2 (+20.0 ± 11.8% and +44.1 ± 16.3% increase of PLA events in A7 and M2, respectively, P &lt; 0.05 vs. basal). Finally, immunofluorescence data showed that SST2 and SST5 recruitment at the plasma membrane and internalization are similarly induced by octreotide and pasireotide in GH3 and A7 cells. On the contrary, in M2 cells, octreotide failed to internalize both receptors whereas pasireotide promoted robust receptor internalization at shorter times than in A7 cells. In conclusion, we demonstrated that in GH3 cells SST2 and SST5 can form both homo- and hetero-dimers and that FLNA plays a role in the formation of SST2/SST5 hetero-dimers. Moreover, we showed that FLNA regulatesSST2 and SST5 intracellular trafficking induced by octreotide and pasireotide. </p

Low dose rate brachytherapy (LDR-BT) as monotherapy for early stage prostate cancer in Italy: practice and outcome analysis in a series of 2237 patients from 11 institutions

OBJECTIVE: Low-dose-rate brachytherapy (LDR-BT) in localized prostate cancer is available since 15 years in Italy. We realized the first national multicentre and multidisciplinary data collection to evaluate LDR-BT practice, given as monotherapy, and outcome in terms of biochemical failure. METHODS: Between May 1998 and December 2011, 2237 patients with early-stage prostate cancer from 11 Italian community and academic hospitals were treated with iodine-125 ((125)I) or palladium-103 LDR-BT as monotherapy and followed up for at least 2 years. (125)I seeds were implanted in 97.7% of the patients: the mean dose received by 90% of target volume was 145 Gy; the mean target volume receiving 100% of prescribed dose (V100) was 91.1%. Biochemical failure-free survival (BFFS), disease-specific survival (DSS) and overall survival (OS) were estimated using Kaplan-Meier method. Log-rank test and multivariable Cox regression were used to evaluate the relationship of covariates with outcomes. RESULTS: Median follow-up time was 65 months. 5- and 7-year DSS, OS and BFFS were 99 and 98%, 94 and 89%, and 92 and 88%, respectively. At multivariate analysis, the National Comprehensive Cancer Network score (p < 0.0001) and V100 (p = 0.09) were correlated with BFFS, with V100 effect significantly different between patients at low risk and those at intermediate/high risk (p = 0.04). Short follow-up and lack of toxicity data represent the main limitations for a global evaluation of LDR-BT. CONCLUSION: This first multicentre Italian report confirms LDR-BT as an excellent curative modality for low-/intermediate-risk prostate cancer. ADVANCES IN KNOWLEDGE: Multidisciplinary teams may help to select adequately patients to be treated with brachytherapy, with a direct impact on the implant quality and, possibly, on outcome

Contribution of Genetic Background, Traditional Risk Factors, and HIV-Related Factors to Coronary Artery Disease Events in HIV-Positive Persons

We show in human immunodeficiency virus-positive persons that the coronary artery disease effect of an unfavorable genetic background is comparable to previous studies in the general population, and comparable in size to traditional risk factors and antiretroviral regimens known to increase cardiovascular ris

Epithelial ovarian cancer is infiltrated by activated effector T cells co-expressing CD39, PD-1, TIM-3, CD137 and interacting with cancer cells and myeloid cells

IntroductionDespite predicted efficacy, immunotherapy in epithelial ovarian cancer (EOC) has limited clinical benefit and the prognosis of patients remains poor. There is thus a strong need for better identifying local immune dynamics and immune-suppressive pathways limiting T-cell mediated anti-tumor immunity.MethodsIn this observational study we analyzed by immunohistochemistry, gene expression profiling and flow cytometry the antigenic landscape and immune composition of 48 EOC specimens, with a focus on tumor-infiltrating lymphocytes (TILs).ResultsActivated T cells showing features of partial exhaustion with a CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ surface profile were exclusively present in EOC specimens but not in corresponding peripheral blood or ascitic fluid, indicating that the tumor microenvironment might sustain this peculiar phenotype. Interestingly, while neoplastic cells expressed several tumor-associated antigens possibly able to stimulate tumor-specific TILs, macrophages provided both co-stimulatory and inhibitory signals and were more abundant in TILs-enriched specimens harboring the CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ signature.ConclusionThese data demonstrate that EOC is enriched in CD137+CD39+PD-1+TIM-3+CD45RA-CD62L-CD95+ T lymphocytes, a phenotype possibly modulated by antigen recognition on neoplastic cells and by a combination of inhibitory and co-stimulatory signals largely provided by infiltrating myeloid cells. Furthermore, we have identified immunosuppressive pathways potentially hampering local immunity which might be targeted by immunotherapeutic approaches

Cytoskeleton protein Filamin A is required for efficient Somatostatin receptor type 2 internalization and recycling through Rab5 and Rab4 sorting endosomes in tumor somatotroph cells

The high expression of somatostatin receptor 2 (SST2) in growth hormone (GH)-secreting tumors represents the rationale for the clinical use of somatostatin analogs (SSAs) in acromegaly. Recently, the cytoskeletal protein Filamin A (FLNA) has emerged as key modulator of the responsiveness of GH-secreting pituitary tumors to SSAs by regulating SST2 signaling and expression. The aim of this study was to explore FLNA involvement in SST2 intracellular trafficking in tumor somatotroph cells. By biotinylation assay we found that FLNA silencing abolished octreotide-mediated SST2 internalization in rat GH3 cell line (28.0±2.7% vs 4±4.3% SST2 internalization, control vs FLNA siRNA cells, respectively, P&lt;0.001) and human GH-secreting primary cultured cells (70.3±21.1% vs 24±19.2% SST2 internalization, control vs FLNA siRNA cells, respectively, P&lt;0.05). In addition, confocal imaging revealed impaired SST2 recycling to the plasma membrane in FLNA silenced GH3 cells. Co-immunoprecipitation and immunofluorescence experiments showed that FLNA, as well as β-arrestin2, is timely-dependent recruited to octreotide-stimulated SST2 receptors both in rat and human tumor somatotroph cells. Although FLNA expression knock down did not prevent the formation of β-arrestin2-SST2 complex in GH3 cells, it significantly impaired efficient SST2 loading into cytosolic vesicles positive for the early endocytic and recycling markers Rab5 and Rab4, respectively (33.7±8.9% down to 25.9±6.9%, p&lt;0.05, and 28.4±7.4% down to 17.6±5.7%, p&lt;0.01, for SST2-Rab5 and SST2-Rab4 colocalization, respectively, in control vs FLNA siRNA cells). Altogether these data support an important role for FLNA in the mediation of octreotide-induced SST2 trafficking in GH-secreting pituitary tumor cells through Rab5 and Rab4 sorting endosomes

Extracorporeal carbon dioxide removal through ventilation of acidified dialysate: An experimental study

Background: Extracorporeal (EC) carbon dioxide (CO2) removal (ECCO2R) may be a powerful alternative to ventilation, possibly avoiding the need for mechanical ventilation and endotracheal intubation. We previously reported how an infusion of lactic acid before a membrane lung (ML) effectively enhances ECCO2R. We evaluated an innovative ECCO2R technique based on ventilation of acidified dialysate. Methods: Four swine were sedated, mechanically ventilated, and connected to a venovenous dialysis circuit (blood flow, 250 ml/min). The dialysate was recirculated in a closed loop circuit including a ML (gas flow, 10 liters/min) and then returned to the dialyzer. In each animal, 4 different dialysis flows (DF) of 200, 400, 600, and 800 ml/min were evaluated with and without lactic acid infusion (2.5 mEq/min); the sequence was completed 3 times. At the end of each step, we measured the volume of CO2R by the ML (Vco2ML) and collected blood and dialysate samples for gas analyses. Results: Acid infusion substantially increased Vco2ML, from 33 \uc2\ub1 6 ml/min to 86 \uc2\ub1 7 ml/min. Different DFs had little effect on Vco2ML, which was only slightly reduced at DF 200 ml/min. The partial pressure of CO2of blood passing through the dialysis filter changed from 60.9 \uc2\ub1 3.6 to 37.1 \uc2\ub1 4.8 mm Hg without acidification and to 32.5 \uc2\ub1 5.3 mm Hg with acidification, corresponding to a pH increase of 0.18 \uc2\ub1 0.03 and 0.03 \uc2\ub1 0.04 units, respectively. Conclusions: Ventilation of acidified dialysate efficiently increased ECCO2R of an amount corresponding to 35% to 45% of the total CO2production of an adult man from a blood flow as low as 250 ml/min. \uc2\ua9 2014 International Society for Heart and Lung Transplantation. All rights reserved

β-arrestin 2 is required for dopamine receptor type 2 inhibitory effects on AKT phosphorylation and cell proliferation in pituitary tumors.

Dopamine receptor type 2 (DRD2) agonists are the first-choice treatment for PRL-secreting pitui-tary tumors but are poorly effective in non-functioning pituitary neuroendocrine tumors (NF-PitNET). DRD2 reduces AKT phosphorylation in lactotrophs, but no data are available in NF-PitNETs. DRD2 effects on AKT are mediated by a β-arrestin2-dependent mechanism in mouse striatum. The aim of this study was to investigate DRD2 effects on AKT phosphorylation and cell proliferation in human primary cultured NF-PitNET cells and in rat tumoral lactotroph cells MMQ, and to test β-arrestin 2 involvement. We found that DRD2 agonist BIM53097 induced a reduction of p- AKT /total-AKT ratio in MMQ (-32.8±17.6%, p&lt;0.001 vs basal) and in a subset (n=15/41,36.6%) of NF-PitNETs (subgroup 1). In the remaining NF-PitNETs (subgroup 2), BIM53097 induced an increase of p-AKT. The ability of BIM53097 to reduce p-AKT correlated to its antimitotic effect, since the majority of subgroup 1 NF-PitNETs was responsive to BIM53097 and nearly all subgroup 2 NF-PitNETs were resistant. β-arrestin 2 was expressed in MMQ and in 80% of subgroup 1 NF-PitNETs, whereas it was undetectable in 77% of subgroup 2 NF-PitNETs. In MMQ, β-arrestin 2 silencing prevented DRD2 inhibitory effects on p-AKT and cell proliferation. Accordingly, β-arrestin 2 transfection in subgroup 2 NF-PitNETs conferred to BIM53097 the ability to inhibit both p-AKT and cell growth. In conclusion, we demonstrated that β-arrestin 2 is required for DRD2 inhibitory effects on AKT phosphorylation and cell proliferation in MMQ and NF-PitNETs, paving the way for a potential role of β-arrestin 2 as a biomarker predicting NF-PitNETs responsiveness to treatment with dopamine agonists

Octreotide and pasireotide effects on medullary thyroid carcinoma (MTC) cells growth, migration and invasion

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine neoplasm of the parafollicular thyroid C cells. Although somatostatin receptors are expressed by MTCs, treatment with octreotide has shown poor efficacy, whereas recently pasireotide has demonstrated antiproliferative effects in persistent postoperative MTCs. Aim of this study was to test the effects of octreotide and pasireotide on MTC cells proliferation, cell cycle proteins expression, MAPK activation, apoptosis, calcitonin secretion, migration and invasion in TT cell line as well as in primary MTC cultured cells. Our results showed that both octreotide and pasireotide reduced TT cell proliferation (− 35.2 ± 12.1%, p &lt; 0.001, and − 25.3 ± 24.8%, p &lt; 0.05, at 10− 8 M, respectively), with concomitant inhibition of ERK phosphorylation and cyclin D1 expression. This cytostatic effect was accompanied by a proapoptotic action, with an increase of caspase3/7 activity of 1.5-fold. Moreover, both octreotide and pasireotide inhibited cell migration (− 50.9 ± 11.3%, p &lt; 0.01, and − 40.5 ± 17%, p &lt; 0.05, respectively) and invasion (− 61.3 ± 35.1%, p &lt; 0.05, and − 49.7 ± 18%, p &lt; 0.01, respectively). No effect was observed on calcitonin secretion. We then tried to extend these observations to primary cultures (n = 5). Octreotide and/or pasireotide were effective in reducing cells proliferation in 3 out of 5 tumors, and to induce cell apoptosis in 1 out of 3 MTCs. Both octreotide and pasireotide were able to reduce cell migration in all MTC tested. SST2, SST3 and SST5 were expressed in all MTC, with a tendency to increased expression of SST2 in RET mutated vs wild type MTCs. In agreement, inhibition of mutated RET in TT cells reduced SST2 expression. In conclusion, we demonstrated that octreotide and pasireotide inhibited cell proliferation and invasiveness in a subset of MTC, supporting their potential use in the control of tumor growth

LA GESTIONE DELLE EMERGENZE SANITARIE E SOCIO-SANITARIE NEI TERRITORI TRANSFRONTALIERI. RACCOLTA DI BEST PRACTICES DEGLI ENTI LOCALI E STRATEGIE DI SANITÀ PUBBLICA NEL FRAMEWORK DEL PROGETTO INTERREG GESTI.S.CO. COVID-19.

Introduzione. La pandemia COVID-19 ha evidenziato le difficoltà di gestione delle emergenze sanitarie, sociosanitarie ed ambientali in territori fragili o transfrontalieri con caratteristiche epidemiologiche e socio-demografiche simili. Le autorità locali si sono trovate in difficoltà e necessitano oggi di strategie e best practices per far fronte a possibili emergenze di carattere sanitario e rischi ambientali soprattutto in territori fragili, isolati o transfrontalieri. Materiali e Metodi. Lo studio descrive i risultati di un workshop di co-design svolto presso il Politecnico di Milano (Italia) con 95 referenti di autorità locali, protezione civile ed esperti di Sanità Pubblica. Durante il kickoff meeting del progetto INTERREG “GESTI.S.CO. Covid-19”, sono state state utilizzate metodologie di co-design e audience-interaction in due sessioni distinte. Lo studio presenta i dati raccolti ed i risultati preliminari del progetto. Nella prima sessione (A) di interazione con il pubblico è stato utilizzato un real time survey on-line (Sli.do) in cui ogni partecipante (n=39; response rate: 41%) poteva fornire parole chiave specifiche per descrivere le sfide affrontate durante la gestione dell’emergenza Covid-19. Nella seconda sessione (B) i partecipanti sono stati suddivisi in 5 tavoli di focus group di circa 2 ore dove, attraverso domande semi-strutturate, è stato possibile raccogliere strategie congiunte ed esperienze locali. Risultati. I partecipanti sono figure professionali prevalentemente afferenti all’ingegneria (26%), architettura (23%) e sanità pubblica (15%), oltre che Disaster Management e Psicologia, legati al mondo delle istituzioni ed enti locali (i.e. polizia locale, protezione civile, ATS, pubbliche amministrazioni). La prima sessione interattiva (A) ha evidenziato che la maggior parte delle criticità sono legate alla mancanza di pianificazione e comunicazione nelle politiche di Sanità Pubblica, soprattutto a livello locale. I tavoli di lavoro (B) hanno identificato cinque aree obiettivo e strategie di sviluppo per le autorità locali e i manager della salute pubblica: i. Rete dei servizi sociali e sanitari ii. Vaccini e campagne massive iii. Gestione delle emergenze con implicazioni sanitarie iv. Salute mentale durante le situazioni pandemiche v. La prima risposta dei governi locali alla pandemia Conclusioni. Lo studio evidenzia le esigenze dei diversi stakeholder durante, prima e dopo l'emergenza covid-19 con specifico riguardo alle sfide affrontate dagli amministratori pubblici nei territori fragili e transfrontalieri