Chiarimenti sulle modalità di applicazione dei CAM ristorazione collettiva (nota a TAR Veneto, Sez. I, 4 gennaio 2021, n. 11)

Il presente contributo propone l’analisi della sentenza del Tribunale amministrativo regionale per il Veneto, Sez. I, 4 gennaio 2021, n. 11, che esclude l’applicazione dei CAM ristorazione collettiva per gruppi aggregati di prodotti, riferendola ai singoli alimenti. Una differente lettura, secondo il Collegio, lascerebbe eccessivi margini di libertà all’aggiudicataria nell’operare compensazioni tra alimenti del tutto eterogenei, con il rischio che alcune importanti categorie di prodotti biologici rimangano assenti dalla fornitura. Il TAR Veneto, inoltre, seppure incidentalmente, prende posizione sul meccanismo di operatività del vincolo previsto dall’art. 34 del d.lgs. n. 50 del 2016 – che obbliga le amministrazioni al rispetto dei CAM – limitandolo all’ipotesi in cui la lex specialis espressamente ne effettui il richiamo. Un simile orientamento risulta di interesse soprattutto se confrontato con quanto espresso da questa stessa Sezione con la pronuncia del 18 marzo 2019, n. 329, dove, per la prima volta, veniva sostenuta la vincolatività dei requisiti ambientali, indipendentemente dal loro effettivo richiamo nella legge di gara, ponendo alla base di questa lettura il principio di eterointegrazione normativa, di cui all’art. 1374 c.c. Si era in tal modo provveduto, ragionevolmente, a intensificare la portata applicativa dei CAM. La pronuncia in esame offre all’Autrice l’occasione di delineare un breve quadro della disciplina di riferimento e dell’evoluzione normativa in materia di criteri ambientali minimi, sottolineando il ruolo fondamentale del green public procurement nel raggiungimento degli obiettivi di sviluppo sostenibile

Oltre il dogmatismo: la nuova dimensione dell'inefficacia pubblicistica

Il presente saggio intende analizzare la categoria dell’inefficacia pubblicistica, così come venutasi a delineare in conseguenza, prima, dell’adozione degli artt. 120 e ss. del codice del processo amministrativo, con riferimento al destino del contratto pubblico a seguito dell’annullamento della relativa aggiudicazione e, più recentemente, delle modifiche apportate alla legge sul procedimento a opera del c.d. Decreto Semplificazioni. Si tratta di interventi che incidono su istituti e problematiche molto differenti, ma che appaiono accomunati dal peculiare utilizzo della figura dell’inefficacia: questa, come quasi alla stregua di passe-partout, viene accolta, plasmata e trasformata per il raggiungimento di precisi obiettivi. Si procederà prima a una sintetica definizione del dibattito dottrinale che ha preceduto e seguito tali evoluzioni, per poi indagare l’incidenza degli interventi legislativi e la più significativa giurisprudenza sul punto, prestando maggiore attenzione, considerata la novità della disciplina, all’inefficacia di cui all’art. 2, comma 8 bis della l. n. 241/1990. Scopo di questa analisi vuole essere quello di dare atto di come l’inefficacia sia oggi caratterizzata da una struttura elastica, capace di andare oltre le severe categorie dogmatiche e di adattarsi, in un’ottica fortemente pragmatica, alle molteplici necessità che vengono a crearsi nell’esercizio dell’attività amministrativa

PDE5 inhibitors in type 2 diabetes cardiovascular complications

Pharmacological inhibition of Phosphodiesterase type 5 (PDE5) proved its efficacy treating several pathological conditions, such as erectile dysfunction and pulmonary hypertension. Nowadays, its benefits on cardiovascular diseases are well documented, particularly in the treatment of type 2 diabetes (T2DM)-related cardiovascular complications. In this context, treatment of T2DM with PDE5 inhibitors, such as sildenafil, tadalafil or vardenafil ameliorates endothelial dysfunction both in patients and animal models through an augmented flow mediated dilation rate and an up-regulation of endothelial markers; it also reduces the inflammatory state by down-regulating inflammatory cytokines expression and improves diabetic cardiomyopathy and ischemia-reperfusion injury mainly through the activation of NO-cGMP-PKG pathway. The present review summarizes the state of art on PDE5 inhibition in the treatment of cardiovascular complications in T2DM

Identification of murine phosphodiesterase 5A isoforms and their functional characterization in HL-1 cardiac cell line

Phosphodiesterase 5A (PDE5A) specifically degrades the ubiquitous second messenger cGMP and experimental and clinical data highlight its important role in cardiac diseases. To address PDE5A role in cardiac physiology, three splice variants of the PDE5A were cloned for the first time from mouse cDNA library (mPde5a1, mPde5a2 and mPde5a3). The predicted amino acidic sequences of the three murine isoforms are different in the N-terminal regulatory domain. mPDE5A isoforms were transfected in HEK293T cells and they showed high affinity for cGMP and similar sensitivity to sildenafil inhibition. RT-PCR analysis showed that mPde5a1, mPde5a2 and mPde5a3 had differential tissue distribution. In the adult heart, mPde5a1 and mPde5a2 were expressed at different levels whereas mPde5a3 was undetectable. Overexpression of mPDE5As induced an increase of HL-1 number cells which progress into cell cycle. mPDE5A1 and mPDE5A3 overexpression increased the number of polyploid and binucleated cells, mPDE5A3 widened HL-1 areas and modulated hypertrophic markers more efficiently respect to the other mPDE5A isoforms. Moreover, mPDE5A isoforms had differential subcellular localization: mPDE5A1 was mainly localized in the cytoplasm, mPDE5A2 and mPDE5A3 were also nuclear localized. These results demonstrate for the first time the existence of three PDE5A isoforms in mouse and highlight their potential role in the induction of hypertrophy. This article is protected by copyright. All rights reserved

FGF9 – Nodal signaling negatively control meiotic entry of postnatal male germ cells

Fibroblast growth factor 9 (FGF9) produced by the somatic cells of the testis acts directly on germ cells to inhibit meiosis by upregulating levels of the RNA binding protein Nanos2, both in fetal and postnatal gonad [1]. Several studies have recently demonstrated that the anti-meiotic effect of FGF9 in the fetal testis is mediated by Nodal, a member of the TGF-Beta morphogen family. In fetal male germ cells FGF9 upregulates Cripto, which is a Nodal co-receptor, making these cells unable to enter meiosis [2]. However it is currently unknown how FGF9 acts postnatally to inhibit entry into meiosis of male mitotic germ cells. We first investigated which was the signal transduction pathway activated by FGF9 on postnatal mouse spermatogonia. We found that FGF9 signaling is dependent on ERK but not on AKT activation, which is instead triggered by Kit ligand (an inducer of meiotic entry that cooperates with retinoic acid). By qRT-PCR we found that Nodal and Cripto are expressed in postnatal spermatogonia and that FGF9 (but not Kit ligand) treatment promotes their upregulation. By western blot we found that phospho-Smad2, an indicator of active Nodal signaling, is increased in spermatogonia upon FGF9 treatment, suggesting that Nodal mediates FGF9 action also in postnatal male germ cells

Carcinoid Syndrome. Preclinical Models and Future Therapeutic Strategies

Carcinoid syndrome represents a debilitating paraneoplastic disease, caused by the se- cretion of several substances, occurring in about 10–40% of patients with well-differentiated neu- roendocrine tumors (NETs). The main signs and symptoms associated with carcinoid syndrome are flushing, diarrhea, hypotension, tachycardia, bronchoconstriction, venous telangiectasia, dyspnea and fibrotic complications (mesenteric and retroperitoneal fibrosis, and carcinoid heart disease). Although there are several drugs available for the treatment of carcinoid syndrome, the lack of therapeutic response, poor tolerance or resistance to drugs are often reported. Preclinical models are indispensable tools for investigating the pathogenesis, mechanisms for tumor progression and new therapeutic approaches for cancer. This paper provides a state-of-the-art overview of in vitro and in vivo models in NETs with carcinoid syndrome, highlighting the future developments and therapeutic approaches in this field

Characterization of three PDE5 isoforms in murine cardiomyocytes

Phosphodiesterase 5 (PDE5A) is responsible for hydrolysis of cGMP, a second messenger regulating many physiological functions in cardiac myocytes. PDE5A involvement in cardiac hypertrophy has been reported and the use of its inhibitor, sildenafil, has reverted the pathological increase of cardiac size in humans and in animal models (Nagendran et al., 2007). In humans, a single PDE5 gene encodes for three isoforms (PDE5A1, A2 and A3), which differ in their N-terminus being translated from alternative initiation sites (Lin et al., 2000). The isoforms exhibit specific tissue expression patterns and different sensitivities to pharmacological inhibitors. However, little is known about their specific biological roles. The existence of three murine PDE5A isoforms was predicted through human gene homology and confirmed by RT-PCR. Tissue expression pattern of each variant was uncovered by RT-PCR and western blot analysis. In adult heart, transcripts encoding for the three isoforms were detected. In cardiomyocytes primary cultures and cell lines PDE5A isoforms localization was revealed by fluorescence microscopy analysis and subcellular fractioning. Their phosphodiesterasic activities and sildenafil sensibilities were measured by radioactive assays. Finally, post-translational modifications were explored. Hypertrophic stimuli resulted in Ser 92 phosphorylation of PDE5A isoforms, possibly through by Protein Kinase A. In summary, the understanding of PDE5A isoforms localization and differential activation and activity might be an important step toward the improvement of the diagnostic, prognostic, and predictive values of PDE5A in hypertrophy treatment

Role of Pten deletion and BRafV600E mutation in the generation of testicular germ cell tumors

Testicular germ cell tumors (TGCT) represent the most common solid malignancy affecting males between the ages of 15 and 35, while ovarian germ cell tumours (OGCT) are a type of ovarian neoplasm principally affecting young women. Germ cell tumors (GCTs) account for about 95 % of testicular cancer cases and for only 2-3% of ovarian cancer cases (Siegel et al., 2011). Most TGCT are potentially curable, however approximately 5% of patients with TGCT develop chemoresistance and die from the disease. PTEN deletion and mutational activation of BRAF are frequent genetic alterations found in human TGCTs, suggesting that they might be directly involved in germ cell tumorigenesis. Furthermore, BRAF mutation positively correlates with the acquisition of resistence to cisplatin, the most commonly chemotherapic agent employed for the treatment of human TGCTs. We obtained heterozygous floxed Ptenloxp/+ BRafCA Spo11Cre mice showing ovarian teratomas and testicular tumors with an incidence of about 30% at 20 days post partum (dpp) . Since Spo- 11Cre is active at around 13.5 days post coitum (dpc) in female germ cells and at around 7 dpp in male germ cells (18), these results suggest that ovarian teratomas origin from early meiotic germ cells in the fetal period whereas GCT formation in males can be a postnatal event. By histological inspection, we found that cancer cells in testes showed features reminiscent of seminoma such as a diffuse, confluent multinodular pattern. However, by immunohistochemical staining, we observed that the cells within the tumor showed heterogeneous positivity for the pluripotency markers Oct4, Sox2, Nanog, Kit and Prdm14, suggesting that they can represent a mixed form of seminoma and embryonal carcinoma cells. Our results indicate that deregulated MAP and PI3 Kinase activation can lead to postnatal male germ cells transformation

Diabetic cardiomiopathy progression is triggered by miR122-5p and involves extracellular matrix: a 5-year prospective study

The purpose of this study was to follow the long-term progression of diabetic cardiomyopathy by combining cardiac magnetic resonance (CMR) and molecular analysis

Characterization of c-Kit receptor function in cardiac regeneration by using transgenic mouse models

Background. Cardiac stem cells expressing the tyrosine kinase receptor c-kit have been recently used in in vivo and in vitro cardiac regenerative studies. However, it remains to be clarified whether the c-kit receptor itself plays a critical role in the process of cardiac regeneration. In order to clarify this point, we will explore whether c-Kit receptor affects cardiac stem cells proliferation, survival, migration and differentiation after heart injury. Methods and Results. We have generated transgenic mice in which an activatory point mutation (c-KitD814Y mice) has been introduced in the kinase domain of the c-kit gene. Initially, we have analyzed c-kit expression in tissues and organs at different stages of embryonal and post-natal development through immunohystochemical and biochemical analyses. We have found that in two transgenic lines the receptor is highly expressed and activated in heart, testis and cerebellum, compared to wild type mice. In order to follow the fate of the c-Kit transgenic stem cells we crossed c-KitD814Y mice with mice expressing GFP under c-Kit regulative sequences control. By cytofluorimetric and fluorescence microscopy analyses, we observed a 2 fold of increase in the number of c-kit positive cells on heart samples from double transgenic mice at different ages. To verify the c-kit role in cardiac regeneration we performed a necrotic heart damage in vivo and monitored cardiac repair in transgenic mice versus wild-type mice. After 9 days the wounded hearts of transgenic mice presented a larger connectival tissue area compared to wild-type mice. On the contrary, after 45 days a consistent reduction of fibrotic area was observed in transgenic mice. These preliminary results suggest a faster repair of damaged heart area that contain stem cells with an activated c-kit receptor. Further in vitro and in vivo experiments will be performed to assess whether transgenic c-kit cells directly transdifferentiate into cardiomyocytes or whether they act in a paracrine manner. In summary, the generation of transgenic mice carrying a constitutively activated c-kit in cardiac stem cells, will allow to investigate the role of the receptor and to highlight the molecular mechanism underlying heart regeneration