Mucopolysaccharidosis type II: preliminary data on glycosaminoglycan levels and structure in mice at baseline and after 6 weeks treatment with ERT

Mucopolysaccharidosis type II is a lysosomal storage disease due to the deficit of the enzyme iduronate 2-sulfatase (IDS) and to the consequent accumulation of heparan- (HS) and dermatan-sulfate (DS), with multi-organ involvement. In this study we characterized glycosaminoglycan (GAG) levels and structure in the brain and liver of the Ids knock-out mouse model, at 12 weeks of age and after 6 weeks treatment with human IDS (hIDS) enzyme, by using the capillary electrophoresis-laser induced fluorescence (CE-LIF) technique. As expected, Ids-ko mice showed a heavy accumulation of HS, about 15 times higher than wild-type (wt) in the brain and up to 240 times in the liver. The overall HS charge density rose by 1.5 times only in the liver, but the sulfation pattern changed in both organs. We also observed an increased chondroitin-sulfate (CS)+DS levels of about 2 times in the brain and 5 times in the liver, but an increased CS/DS ratio of about 22 times only in the liver. On the contrary, the hyaluronic acid (HA) levels did not change in both organs. We also conducted the same analysis in Ids-ko mice treated with 1 mg/kg of hIDS, once a week. As expected, we observed in the liver a huge reduction of HS (20 times vs untreated mice) and also of CS+DS and CS/DS. Instead, we did not observe a reduction of the different GAGs in the brain, confirming the enzyme inability to cross BBB. In this district a slight increase of CS/DS ratio, CS+DS and HA levels, and an about 40% increase of HS level, vs untreated ko mice, was observed. On the opposite, the overall HS charge density is decreased 2.5X vs untreated ko and wt mice. This preliminary data underline how by using a more sensitive technique of analysis, a clear separation of the GAGs pattern between wt and Ids-ko mice can be observed. This results particularly important for the brain, where application of common biochemical techniques detects very low GAG levels in both animal types, thus limiting the use of GAG analysis as possible biomarker of therapeutic efficacy in the brain district. The application of CE-LIF analysis is therefore proposed for a detailed evaluation of GAG pattern for potential monitoring of therapeutic efficacy

Cholesterol-lowering action of a novel nutraceutical combination in uremic rats: Insights into the molecular mechanism in a hepatoma cell line

Appropriate nutraceutical combinations may represent a valid approach to prevent vascular calcification associated with chronic kidney disease (CKD). In the present study, we tested the effect of a new nutraceutical combination named RenaTris®, containing MK-7, magnesium carbonate, and Sucrosomial® Iron, on vascular calcification in uremic rats. Rats were randomly divided into three groups, i.e. control (high-phosphate diet), uremic (high-phosphate diet containing 0.5% adenine), and supplemented uremic diet (0.5% adenine, MK-7, magnesium carbonate, and Sucrosomial® Iron). After six weeks, sera and vascular calcification were examined. The uremic diet increased creatinine and phosphate levels and induced extensive vascular calcification. The uremic condition also induced a mild hypercholesterolemic condition (+52% of total cholesterol; p < 0.05). The supplemented uremic diet did not reduce creatinine, phosphate levels, or vascular calcification, however, we observed a significant hypocholesterolemic effect (−18.9% in supplemental uremic vs. uremic diet; p < 0.05). Similar to simvastatin, incubation of cultured human hepatoma cells (Huh7) with MK-7 significantly reduced cholesterol biosynthesis (−38%) and induced 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and low-density lipoprotein receptor (LDLR) at both mRNA and protein levels. The effect of MK-7 on LDLR was counteracted by the co-incubation with squalene. Unlike simvastatin, MK-7 reduced PCSK9 in Huh7. These results indicated that the new nutraceutical combination significantly impacts cholesterol metabolism and its supplementation may help to control mild hypercholesterolemic conditions in CKD patients

Approccio globale alle mucopolisaccaridosi: creazione piattaforma web-based

Scopo dello studio: Lo scopo di questo studio è progettare una banca dati accessibile via web in grado di contenere numerosi dati biochimici e clinici di soggetti arruolati all’interno di uno studio nazionale multicentrico PRIN2012. Lo studio prevede, inoltre, la raccolta e la valutazione di campioni provenienti dai modelli murini per la MPSI e MPSII, in trattamento con ERT o Genisteina. Questo progetto si basa sull’applicazione di procedure analitiche ad alta risoluzione per la valutazione qualitativa e quantitativa dei GAG per la diagnosi neonatale di MPS e la verifica dell’efficacia terapeutica in pazienti da MPS e in modelli animali. Nei tre anni di studio si prevede l’arruolamento di circa 60 pazienti MPS in trattamento e non e di 600 neonati. Per la parte preclinica è prevista la valutazione di circa 500 topi. Di ogni categoria verranno raccolte diverse tipologie di campioni biologici in diversi tempi sui quali verranno effettuati differenti tipi di analisi dei GAG. Contemporaneamente verrà effettuata la valutazione clinica dei pazienti MPS. L’obiettivo della creazione di una banca dati è di facilitare l’inserimento, la gestione e la condivisione dei dati, nonché favorire l’interoperabilità tra gli specialisti clinici e biochimici. Metodi utilizzati: E` stata sviluppata una piattaforma software web-based con tecnologia Sharepoint 2010, ospitata su Windows Server 2008. Tale piattaforma collaborativa consta di due macro-moduli, uno per la gestione dei dati, un Relational Database Management System sviluppato in SQL Server 2008 R2 , e uno per la gestione del flusso dei campioni spediti tra le diverse unità di ricerca, un Workflow Management System capace di organizzare, pianificare ed attuare non solo le attività svolte ma anche le informazioni trasferite e poi immagazzinate nel Database condiviso . Risultati: Le cinque unità operative hanno accessi differenziati. L’inserimento dei dati avviene in modo guidato a seconda della classe considerata (neonato, patologico, topo), dell’eventuale patologia e della tipologia di dato, clinico o biochimico. La lettura dati è facilitata da filtri (soggetto, campioni ricevuti, classe di appartenenza, patologia). I soggetti dello studio e tutti i campioni ad esso riferiti vengono codificati mediante un applicativo che consente la creazione di un codice ID dato da una stringa alfanumerica generata in base a delle regole stabilite a priori e analoghe per ogni unità di ricerca in modo che si generi un codice univoco che consenta di collegare tutti i dati relativi ad un determinato soggetto. Infine è possibile esportare i dati raccolti in un formato compatibile con l’importazione in SPSS con cui poter eseguire analisi statistiche. Conclusioni: I dati biochimici e clinici raccolti secondo protocolli definiti, verranno integrati nel database condiviso, indispensabile strumento per ottimizzare la valenza dei dati stessi e promuoverne una più efficace analisi. Tutti questi aspetti contribuiranno ad arricchire la comprensione clinica delle MPS, la variabile presentazione fenotipica e la progressione della malattia. Consentiranno, inoltre una valutazione dell’efficacia terapeutica ottenuta, sulla base dell’analisi dei dati clinici e dei dati biochimici ottenuti con procedura high-throughput. Ricerca finanziata con fondi Progetto PRIN 2012

In vitro and in vivo anticancer activity of tridentate thiosemicarbazone copper complexes: Unravelling an unexplored pharmacological target

Certain metal complexes can have a great antitumor activity, as the use of cisplatin in therapy has been demonstrating for the past fifty years. Copper complexes, in particular, have attracted much attention as an example of anticancer compounds based on an endogenous metal. In this paper we present the synthesis and the activity of a series of copper(II) complexes with variously substituted salicylaldehyde thiosemicarbazone ligands. The in vitro activity of both ligands and copper complexes was assessed on a panel of cell lines (HCT-15, LoVo and LoVo oxaliplatin resistant colon carcinoma, A375 melanoma, BxPC3 and PSN1 pancreatic adenocarcinoma, BCPAP thyroid carcinoma, 2008 ovarian carcinoma, HEK293 non-transformed embryonic kidney), highlighting remarkable activity of the metal complexes, in some cases in the low nanomolar range. The copper(II) complexes were also screened, with good results, against 3D spheroids of colon (HCT-15) and pancreatic (PSN1) cancer cells. Detailed investigations on the mechanism of action of the copper(II) complexes are also reported: they are able to potently inhibit Protein Disulfide Isomerase, a copper-binding protein, that is recently emerging as a new therapeutic target for cancer treatment. Good preliminary results obtained in C57BL mice indicate that this series of metal-based compounds could be a very promising weapon in the fight against cancer