Toll like receptor signaling in "inflammaging": microRNA as new players.

none7nopubblicazione scientificaopenOlivieri F; Rippo MR; Prattichizzo F; Babini L; Graciotti L; Recchioni R; Procopio ADOlivieri, Fabiola; Rippo, Maria Rita; Prattichizzo, Francesco; Babini, Lucia; Graciotti, Laura; Recchioni, R; Procopio, Antonio Domenic

Human mesenchymal stem cells from chorionic villi and amniotic fluid are not susceptible to transformation after extensive in vitro expansion.

Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. Increasing evidence suggests that MSCs isolated from fetal tissues are more plastic and grow faster than adult MSCs. In this study, we characterized human mesenchymal progenitor cells from chorionic villi (CV) and amniotic fluid (AF) isolated during the first and second trimesters, respectively, and compared them with adult bone marrow-derived MSCs (BM). We evaluated 10 CV, 10 AF, and 6 BM samples expanded until the MSCs reached senescence. We used discarded cells from prenatal analyses for all the experiments. To evaluate the replicative stability of these cells, we studied the telomerase activity, hTERT gene transcription, and telomere length in these cells. Spontaneous chromosomal alterations were excluded by cytogenetic analysis. We studied the expression of c-myc and p53, tumor-associated genes, at different passage in culture and the capacity of these cells to grow in an anchorage-independent manner by using soft agar assay. We isolated homogeneous populations of spindle-shaped CV, AF, and BM cells expressing mesenchymal immunophenotypic markers throughout the period of expansion. CV cells achieved 14 ± 0.9 logs of expansion in 118 days and AF cells achieved 21 ± 0.9 logs in 118 days, while BM cells achieved 11 × 0.4 logs in 84 days. Despite their high proliferation capacity, fetal MSCs showed no telomerase activity, no hTERT and c-myc transcriptions, and maintained long, stable telomeres. A constant expression level of p53 and a normal karyotype were preserved throughout long-term expansion, suggesting the safety of fetal MSCs. In conclusion, our results indicate that fetal MSCs could be an alternative, more accessible resource for cell therapy and regenerative medicine

Hormone replacement therapy enhances IGF-1 signaling in skeletal muscle by diminishing miR-182 and miR-223 expressions : a study on postmenopausal monozygotic twin pairs

MiRNAs are fine-tuning modifiers of skeletal muscle regulation, but knowledge of their hormonal control is lacking. We used a co-twin case-control study design, that is, monozygotic postmenopausal twin pairs discordant for estrogen-based hormone replacement therapy (HRT) to explore estrogen-dependent skeletal muscle regulation via miRNAs. MiRNA profiles were determined from vastus lateralis muscle of nine healthy 54-62-years-old monozygotic female twin pairs discordant for HRT (median 7 years). MCF-7 cells, human myoblast cultures and mouse muscle experiments were used to confirm estrogen's causal role on the expression of specific miRNAs, their target mRNAs and proteins and finally the activation of related signaling pathway. Of the 230 miRNAs expressed at detectable levels in muscle samples, qPCR confirmed significantly lower miR-182, miR-223 and miR-142-3p expressions in HRT using than in their nonusing co-twins. Insulin/IGF-1 signaling emerged one common pathway targeted by these miRNAs. IGF-1R and FOXO3A mRNA and protein were more abundantly expressed in muscle samples of HRT users than nonusers. In vitro assays confirmed effective targeting of miR-182 and miR-223 on IGF-1R and FOXO3A mRNA as well as a dose-dependent miR-182 and miR-223 down-regulations concomitantly with up-regulation of FOXO3A and IGF-1R expression. Novel finding is the postmenopausal HRT-reduced miRs-182, miR-223 and miR-142-3p expression in female skeletal muscle. The observed miRNA-mediated enhancement of the target genes' IGF-1R and FOXO3A expression as well as the activation of insulin/IGF-1 pathway signaling via phosphorylation of AKT and mTOR is an important mechanism for positive estrogen impact on skeletal muscle of postmenopausal women.Peer reviewe

Ruolo del sistema FASL/FAS nella biologia delle cellule mesenchimali staminali

Le cellule staminali mesemchimali (MSC) sono una popolazione eterogenea di cellule stromali multipotenti, possono essere isolate da diversi tessuti adulti e possono dare origine a diversi tipi cellulari(21). Nel midollo osseo (BM) differenziano principalmente in osteoblasti e adipociti, e l’ interazione con questo microambiente influenza da vicino il loro programma differenziativo. FasL è una citochina molto conosciuta per il suo ruolo pro-apoptotico, in associazione con il suo recettore specifico Fas, tuttavia diversi studi propongono che sia coinvolta anche in altri processi biologici. Questo lavoro dimostra per la prima volta che FasL può essere coinvolto nella proliferazione e nella differenziazione delle BM-MSC. Le BM-MSC trattate con una bassa dose di FasL (0.5 ng/ml) proliferano più rapidamente delle cellule non trattate, senza andare incontro ad apoptosi o a processi differenziativi, mentre dosi più alte (25 ng/ml) inducono il processo apoptotico nel 20% delle cellule, selezionando una popolazione con un fenotipo ancora staminale. A livello molecolare il trattamento con FasL 0.5 ng/ml induce la fosforilazione di ERK 1/2 e l’aumento dell’espressione della survivina mentre FasL 25 ng/ml determina l’attivazione delle caspasi 8 e 3. Inoltre FasL 25 ng/ml attraverso la modulazione di PPARγ e FABP4/aP2 inibisce reversibilmente la differenziazione delle BM-MSC in adipociti. I dati di inibizione dell’adipogenesi in vitro sono stati confermati su topi Fas lpr, mutati per Fas, che hanno mostrato una maggiore espressione dell’mRNA e delle proteine PPARγ e FABP4/aP2 rispetto ai controlli. I nostri dati suggeriscono che il sistema FasL/Fas è coivolto nella biologia delle BM-MSC, e può regolarne sia la proliferazione che il differenziamento adipogenico. Questi risultati possono avere una rilevanza clinica, chiarendo i meccanismi che determinano l’aumento dell’adipogenesi nel midollo durante l’invecchiamento o in alcune condizioni patologiche come l’osteoporosi

Exploitation of a polyphasic PCR-DGGE approach forthe investigation of the microbiota of traditional Italian raw milk cheeses.

INTRODUCTION Since the last decade there has been an increasing demand for artisan cheeses manufactured with raw milk and traditional procedures. In raw milk cheeses, fermentation and ripening are carried out by a heterogeneous microbial community, which includes starter lactic acid bacteria (LAB), able to rapidly convert lactose into lactic acid, and non starter LAB (NSLAB) mainly contributing to the development of cheese flavour and aroma. Materials & methods Four traditional cheeses manufactured with local raw milk (Marche region, central Italy) were analysed for physico-chemical (pH, aw), compositional (NaCl, raw protein, fat content) and microbiological traits (occurrence of pathogens and their toxins, and viable counts of lactobacilli, lactococci and thermophilic streptococci). The composition of the LAB population was investigated with a poorly exploited polyphasic PCR-DGGE approach relying on the analysis of the bacterial DNA extracted either from the cheeses or the bulk cells harvested from all the dilution plates used for LAB viable counting. Results & discussion The results of traditional microbiological analyses fully complied with the European legislation [Regulation EC No. 2073/2005]. A high cheese diversity was revealed by physico-chemical, microbiological and PCR-DGGE analyses. As the fingerprints obtained with the two approaches were compared, the analysis of the cultivable communities allowed a significantly higher diversity than the direct approach to be disclosed, although in some cases, species not detected in the bulk cells were identified by analysing the DNA extracted directly from the cheeses. CONCLUSIONS The overall PCR-DGGE results clearly demonstrated that although either approach, taken singly, gives an incomplete picture of the real microbial diversity by introducing bias, their combination allows a greater pool of information to be collected

Monitoraggio terapeutico dei farmaci immunosoppressori: confronto tra metodiche

BACKGROUND: Therapeutic drug monitoring (TDM) of Immunosuppressive drugs (ISD) is crucial to the success of organ transplantation. Considering this background, the analytical performance of the assays for the ISD dosing performed on Dimension EXL 200 platform and on the currently in use analyzers for ISD were compared. We evaluated the possible performance improvement due to pre-treatment standardization, consolidation of TDM on a single platform and TAT reduction. METHODS: We evaluated our conventional assays i.e., Abbott CMIA™ cyclosporine, tacrolimus and sirolimus (on EDTA-anticoagulated whole blood) performed on Architect i1000SR platform, ThermoFisher Scientific QMS™ everolimus (K2EDTA-whole blood) on CDX90 analyzer and ThermoFisher Scientific CEDIA™ mycophenolic acid (on plasma/serum) on ILab Taurus, in comparison with the equivalent Siemens ACMIA™ (cyclosporine, tacrolimus, sirolimus, everolimus) and Petinia™ (mycophenolic acid) Dimension EXL 200 assays. For the comparison, a total of 450 consecutive samples were processed and results were statistically compared. Within-run and within-laboratory precision on Dimension were evaluated using quality control materials. RESULTS: Within-run and within-laboratory CVs% are ≤10% for all the Siemens assays, according to the IATDMCT guidelines for ISD monitoring. The regression analysis of tacrolimus, cyclosporine, sirolimus and mycophenolic acid data show good linear correlation (r≥0.96). Bland-Altman plots reveal a positive percent bias for cyclosporine (+19.9%), sirolimus (+22.8%) and mycophenolic acid (+9.65%) while tacrolimus assay shows a slight negative absolute bias (-0.23 ng/mL). Everolimus assay shows the weakest correlation between methods (r=0.95) and Bland-Altman plots show an increasing absolute bias (+3.1 ng/mL) in association with a positive 48.8% bias which tends to decrease with increasing everolimus concentrations. CONCLUSIONS: Our findings suggest that the automated assays lead to workflow improvement giving an edge to Dimension analyzer as compared to other platforms. Tacrolimus, cyclosporine, sirolimus and mycophenolic acid assays demonstrated good precision and good correlation between methods and we also quite clearly managed to identify the bias type for these assays. Everolimus assay shows the weakest correlation between methods and the simultaneous presence of two different types of bias

BDNF modulates heart contraction force and long-term homeostasis through truncated TrkB.T1 receptor activation

Brain-derived neurotrophic factor (BDNF) is critical for mammalian development and plasticity of neuronal circuitries affecting memory, mood, anxiety, pain sensitivity, and energy homeostasis. Here we report a novel unexpected role of BDNF in regulating the cardiac contraction force independent of the nervous system innervation. This function is mediated by the truncated TrkB.T1 receptor expressed in cardiomyocytes. Loss of TrkB.T1 in these cells impairs calcium signaling and causes cardiomyopathy. TrkB.T1 is activated by BDNF produced by cardiomyocytes, suggesting an autocrine/paracrine loop. These findings unveil a novel signaling mechanism in the heart that is activated by BDNF and provide evidence for a global role of this neurotrophin in the homeostasis of the organism by signaling through different TrkB receptor isoforms