Genetic lineage tracing reveals poor angiogenic potential of cardiac endothelial cells.

Abstract Aims Cardiac ischaemia does not elicit an efficient angiogenic response. Indeed, lack of surgical revascularization upon myocardial infarction results in cardiomyocyte death, scarring, and loss of contractile function. Clinical trials aimed at inducing therapeutic revascularization through the delivery of pro-angiogenic molecules after cardiac ischaemia have invariably failed, suggesting that endothelial cells in the heart cannot mount an efficient angiogenic response. To understand why the heart is a poorly angiogenic environment, here we compare the angiogenic response of the cardiac and skeletal muscle using a lineage tracing approach to genetically label sprouting endothelial cells. Methods and results We observed that overexpression of the vascular endothelial growth factor in the skeletal muscle potently stimulated angiogenesis, resulting in the formation of a massive number of new capillaries and arterioles. In contrast, response to the same dose of the same factor in the heart was blunted and consisted in a modest increase in the number of new arterioles. By using Apelin-CreER mice to genetically label sprouting endothelial cells we observed that different pro-angiogenic stimuli activated Apelin expression in both muscle types to a similar extent, however, only in the skeletal muscle, these cells were able to sprout, form elongated vascular tubes activating Notch signalling, and became incorporated into arteries. In the heart, Apelin-positive cells transiently persisted and failed to give rise to new vessels. When we implanted cancer cells in different organs, the abortive angiogenic response in the heart resulted in a reduced expansion of the tumour mass. Conclusion Our genetic lineage tracing indicates that cardiac endothelial cells activate Apelin expression in response to pro-angiogenic stimuli but, different from those of the skeletal muscle, fail to proliferate and form mature and structured vessels. The poor angiogenic potential of the heart is associated with reduced tumour angiogenesis and growth of cancer cells

Distribution of Exonic Variants in Glycogen Synthesis and Catabolism Genes in Late Onset Pompe Disease (LOPD)

Pompe disease (PD) is a monogenic autosomal recessive disorder caused by biallelic pathogenic variants of the GAA gene encoding lysosomal alpha-glucosidase; its loss causes glycogen storage in lysosomes, mainly in the muscular tissue. The genotype-phenotype correlation has been extensively discussed, and caution is recommended when interpreting the clinical significance of any mutation in a single patient. As there is no evidence that environmental factors can modulate the phenotype, the observed clinical variability in PD suggests that genetic variants other than pathogenic GAA mutations influence the mechanisms of muscle damage/repair and the overall clinical picture. Genes encoding proteins involved in glycogen synthesis and catabolism may represent excellent candidates as phenotypic modifiers of PD. The genes analyzed for glycogen synthesis included UGP2, glycogenin (GYG1-muscle, GYG2, and other tissues), glycogen synthase (GYS1-muscle and GYS2-liver), GBE1, EPM2A, NHLRC1, GSK3A, and GSK3B. The only enzyme involved in glycogen catabolism in lysosomes is alpha-glucosidase, which is encoded by GAA, while two cytoplasmic enzymes, phosphorylase (PYGB-brain, PGL-liver, and PYGM-muscle) and glycogen debranching (AGL) are needed to obtain glucose 1-phosphate or free glucose. Here, we report the potentially relevant variants in genes related to glycogen synthesis and catabolism, identified by whole exome sequencing in a group of 30 patients with late-onset Pompe disease (LOPD). In our exploratory analysis, we observed a reduced number of variants in the genes expressed in muscles versus the genes expressed in other tissues, but we did not find a single variant that strongly affected the phenotype. From our work, it also appears that the current clinical scores used in LOPD do not describe muscle impairment with enough qualitative/quantitative details to correlate it with genes that, even with a slightly reduced function due to genetic variants, impact the phenotype

The actin-nucleator promoting factor WASp regulates endo-lysosomal maturation and Toll-like receptor 9 signaling in Dendritic Cells.

Toll-like receptors (TLRs) are innate immune receptors, which play a key role in both innate and adaptive immune responses, are important to recognize pathogenic components and to trigger inflammatory responses. However, TLRs could be inappropriately activated by self and endogenous nucleic acids leading to unwarranted inflammation with dangerous outcomes, including autoimmune diseases. Intracellular TLRs compartmentalization is essential to regulate initiation and termination of signalling, thereby avoiding excessive inflammation. Therefore, studies of mechanisms and adaptors that limit unrestrained activation of innate immune cells upon TLRs triggering are of increasing interest as their potential in understanding autoimmunity. Wiskott-Aldrich Syndrome (WAS) is a X-linked primary immune deficiency, caused by mutations on WAS-protein (WASp), a hematopoietic specific actin nucleator promoting factor (NPFs). WAS is characterized by recurrent infections, and a marked predisposition to develop autoimmune phenomena. This thesis aimed to dissect the molecular mechanism that leads to excessive production of type-I interferon in the context of WAS, using a model of WASp null dendritic cells (WKO DCs). First, we developed and validated new cellular models to study WASp. We demonstrated that, after TLR9 stimulation, WKO cells produce higher amount of type-I IFN and proinflammatory cytokines, both in BM-DCs and Hoxb8-DCs models. Moreover, our dose-response assay of endogenous ligands showed that WKO display a lower TLR9 activation threshold compared to wild-type (WT) DCs. Intriguingly, our morphological analysis revealed structural alterations in endo-lysosomes of WKO DCs. Using flow cytometry and endocytic probes we observed that the lack of WASp leads to aberration in cargo recycling and degradation. We then investigated whether WKO structural and functional remodeling could influence TLR9-ligand intracellular trafficking. By blocking protein synthesis, we demonstrated that WASp expression is important to control TLR9 degradation rate. In addition, biochemical and immunofluorescence analysis revealed that exogenous and endogenous TLR9 ligands are engulfed in WKO cells. In particular, ligands traffic slowly across the first endocytic organelles and are degraded less efficiently than in WT DCs. Finally, using actin inhibitors, we observed that treated cells display impaired endo-lysosome maturation and reduced TLR9 degradation. Most importantly, TLR9 signalling is enhanced leading to higher production of type-I IFN. Taken together, these results prove that the perturbation of actin dynamics closely resembles WASp deficiency phenotype. In conclusion, our findings shed light on the mechanism underlying excessive TLR9 activation: actin nucleation mediated by WASp is required to maintain a correct endo-lysosomal organization and to control TLR9 threshold, activation and signalling in DCs

Identification of Novel Mutations by Targeted NGS Panel in Patients with Hyperferritinemia

Background. Several inherited diseases cause hyperferritinemia with or without iron overload. Differential diagnosis is complex and requires an extensive work-up. Currently, a clinical-guided approach to genetic tests is performed based on gene-by-gene sequencing. Although reasonable, this approach is expensive and time-consuming and Next Generation Sequencing (NGS) technology may provide cheaper and quicker large-scale DNA sequencing. Methods. We analysed 36 patients with non-HFE-related hyperferritinemia. Liver iron concentration was measured in 33 by magnetic resonance. A panel of 25 iron related genes was designed using SureDesign software. Custom libraries were generated and then sequenced using Ion Torrent PGM. Results. We identified six novel mutations in SLC40A1, three novel and one known mutation in TFR2, one known mutation and a de-novo deletion in HJV, and a novel mutation in HAMP in ten patients. In silico analyses supported the pathogenic role of the mutations. Conclusions. Our results support the use of an NGS-based panel in selected patients with hyperferritinemia in a tertiary center for iron metabolism disorders. However, 26 out of 36 patients did not show genetic variants that can individually explain hyperferritinemia and/or iron overload suggesting the existence of other genetic defects or gene-gene and gene-environment interactions needing further studies

Cellular Signaling Pathways Activated by Functional Graphene Nanomaterials

The paper reviews the network of cellular signaling pathways activated by Functional Graphene Nanomaterials (FGN) designed as a platform for multi-targeted therapy or scaffold in tissue engineering. Cells communicate with each other through a molecular device called signalosome. It is a transient co-cluster of signal transducers and transmembrane receptors activated following the binding of transmembrane receptors to extracellular signals. Signalosomes are thus efficient and sensitive signal-responding devices that amplify incoming signals and convert them into robust responses that can be relayed from the plasma membrane to the nucleus or other target sites within the cell. The review describes the state-of-the-art biomedical applications of FGN focusing the attention on the cell/FGN interactions and signalosome activation

Melka Kunture: tecniche digitali per l’archeologia preistorica

La musealizzazione del sito di Melka Kunture rappresenta la conclusione di 40 anni di ricerche e scavi compiuti dalla Missione Archeologica Francese diretta da Jean Chavaillon tra il 1965 e il 1995 e dalla Missione Archeologica Italiana dell’Università di Roma “La Sapienza” e del Ministero degli Affari Esteri, diretta da Marcello Piperno a partire dal 1999 e tuttora in corso. L’estensione del sito, la sua lunga sequenza culturale (1.7-0.2 milioni di anni) insieme alla molteplicità e varietà delle situazioni archeologiche presenti nelle sue diverse fasi fanno di Melka Kunture un complesso straordinario e unico, paragonabile soltanto alla Gola di Olduvai in Tanzania

Melka Kunture: tecniche digitali per l'archeologia preistorica.

8La musealizzazione del sito di Melka Kuntu-re rappresenta la conclusione di 40 anni di ricerche e scavi compiuti dalla Missione Archeologica Francese diretta da Jean Chavaillon tra il 1965 e il 1995 e dalla Missione Archeologica Italiana dell'Università di Roma "La Sapienza" e del Ministero degli Affari Esteri, diretta da Marcello Piperno a partire dal 1999 e tuttora in corso. L'estensione del sito, la sua lunga sequenza culturale (1.7-0.2 milioni di anni) insieme alla mOlteplicità e varietà delle situazioni archeologiche presenti nelle sue diverse fasi fanno di Melka Kunture un complesso straordinario e unico, paragonabile soltanto alla Gola di Olduvai in Tanzania.Speciale Archeomatica 2007reservedmixedR. SALVINI; SALVI M. C; GRUPPIONI G; CARMIGNANI L; PIPERNO M; GALLOTTI R; KIEFFER G.; BULGARELLI M.GSalvini, Riccardo; Salvi, MARIA CRISTINA; Gruppioni, G; Carmignani, Leonardo; Piperno, M; Gallotti, R; Kieffer, G.; Bulgarelli, M. G

Chemo-radiotherapy plus durvalumab for loco-regional relapse of resected NSCLC

Abstract Background tumor recurrence after NSCLC surgical resection is the most common cause of treatment failure that sharply reduces the patient’s life expectancy. The optimal treatment strategy for loco-regional recurrences developing after surgical resection in patients with non–small-cell lung cancer (NSCLC) is not established yet. This report aims to describe the pattern of relapse, PFS, and OS in patients treated with radio-chemotherapy and durvalumab for loco-regional relapse after surgery. Methods  We conducted a multicenter, retrospective study including subjects who underwent surgical resection for NSCLC and were treated with Pacific protocol after loco-regional relapse. Results Twenty-four patients met the inclusion criteria. At the time of diagnosis mean age was 65 years (range 47–78), the majority being male (58.3%). The 12-month progression-free survival rate was 68.7%, the 18-month progression-free survival rate was 45.8%, and the 24-month progression-free survival rate was 34.3%. There were three deaths: the 12-month survival rate was 91%, and the 18-month survival rate was 82.8%. Conclusions In this article, we propose a treatment strategy that might prolong post recurrence survival in patients with good performance status experiencing loco-regional relapse after surgery