Italian Children Exposure to Bisphenol A: Biomonitoring Data from the LIFE PERSUADED Project

A human biomonitoring (HBM) study on bisphenol A (BPA) in Italian children and adolescents was performed within the LIFE PERSUADED project, considering the residing areas, sex and age. The median urinary BPA level was 7.02 mu g/L, with children living in the South of Italy or in urban areas having higher levels than those residing in the North or in rural areas. Children aged 4-6 years had higher BPA levels than those aged 7-10 and 11-14 years, but no differences were detected between sexes. The exposure in Italian children was higher compared to children from other countries, but lower than the HBM guidance value (135 mu g/L). The estimated daily intake was 0.17 mu g/kg body weight (bw) per day, about 24-fold below the temporary Tolerable Daily Intake of 4 mu g/kg bw per day established by the European Food Safety Authority. However, this threshold was exceeded in 1.44% of the enrolled children, raising concern about the overall exposure of Italian young population

Searching novel therapeutic targets for scleroderma: P2X7-receptor is UP-regulated and promotes a fibrogenic phenotype in systemic sclerosis fibroblasts

Objectives: Systemic sclerosis (SSc) is a connective tissue disorder presenting fibrosis of the skin and internal organs, for which no effective treatments are currently available. Increasing evidence indicates that the P2X7 receptor (P2X7R), a nucleotide-gated ionotropic channel primarily involved in the inflammatory response, may also have a key role in the development of tissue fibrosis in different body districts. This study was aimed at investigating P2X7R expression and function in promoting a fibrogenic phenotype in dermal fibroblasts from SSc patients, also analyzing putative underlying mechanistic pathways. Methods: Fibroblasts were isolated by skin biopsy from 9 SSc patients and 8 healthy controls. P2X7R expression, and function (cytosolic free Ca2+ fluxes, α-smooth muscle actin [α-SMA] expression, cell migration, and collagen release) were studied. Moreover, the role of cytokine (interleukin-1β, interleukin-6) and connective tissue growth factor (CTGF) production, and extracellular signal-regulated kinases (ERK) activation in mediating P2X7R-dependent pro-fibrotic effects in SSc fibroblasts was evaluated. Results: P2X7R expression and Ca2+ permeability induced by the selective P2X7R agonist 2'-3'-O-(4-benzoylbenzoyl)ATP (BzATP) weremarkedly higher in SSc than control fibroblasts. Moreover, increased aSMA expression, cell migration, CTGF, and collagen release were observed in lipopolysaccharides-primed SSc fibroblasts after BzATP stimulation. While P2X7-induced cytokine changes did not affect collagen production, it was completely abrogated by inhibition of the ERK pathway. Conclusion: In SSc fibroblasts, P2X7R is overexpressed and its stimulation induces Ca2+-signaling activation and a fibrogenic phenotype characterized by increased migration and collagen production. These data point to the P2X7R as a potential, novel therapeutic target for controlling exaggerated collagen deposition and tissue fibrosis in patients with SSc

Espressione e assemblaggio delle proteine capsidiche del virus adeno-associato in Saccharomyces cerevisiae.

Il progetto in cui si inserisce il mio lavoro di tesi ha come obbiettivo finale quello di proporre il lievito S. cerevisiae come sistema modello per la produzione di vettori derivati dal virus adeno-associato (AAV) per la terapia genica. AAV è un virus difettivo, non patogeno, della famiglia dei Parvovirus che dipende dalla co-infezione di un virus helper (come adenovirus o Herpes simplex) per una sua replicazione produttiva. In assenza del virus helper, AAV stabilisce un’infezione latente attraverso un’ integrazione sito specifica nel genoma dell’ospite o resta in una forma episomale persistente. La tesi si propone di aumentare e di ottimizzare l’espressione di VP1, VP2 e VP3, le tre proteine del capside di AAV, per permettere la formazione del capside stesso nel ceppo di lievito RSY12. E’ stato visto che per l’assemblaggio corretto dei capsidi ricombinanti di AAV le tre proteine devono essere espresse secondo un rapporto di concentrazione VP1:VP2:VP3 che oscilla da 1:1:8 a 1:1:20 rispettivamente. Questo è molto importante poiché solo i capsidi assemblati in maniera corretta sono capaci di infettare le cellule umane. In laboratorio, erano già disponibili un vettore in cui l’espressione della proteina VP3 è controllata dal promotore p40 di AAV ed un altro che esprime la proteina VP1 sotto il controllo del promotore inducibile da galattosio pGAL. Il vettore contenente il gene VP3 sotto il controllo del promotore p40 è stato dimostrato essere attivo in lievito ma l’espressione della proteina non raggiunge alti livelli rispetto a VP1 controllato da pGAL. Quindi, nell'intento di aumentare il livello di espressione di VP3 rispetto a VP1 abbiamo costruito, mediante diversi steps di clonaggio, tre diversi vettori contenenti le sequenze codificanti VP2 e VP3. In due vettori VP2 e VP3 sono stati clonati sotto il controllo del promotore pADH700 che è stato dimostrato essere attivo in tutte le fasi di crescita, ottenendo il vettore pESCADH700-VP2,3 e pESCADH700-UTR-VP2,3. In quest’ultimo vettore è stata mantenuta una sequenza UTR presente nel sistema naturale tra i due diversi “start sites” che potrebbe avere un funzione regolatoria. In un terzo vettore, la sequenza VP2,3 è stata clonata sotto il controllo del pGAL, inducibile da galattosio, strettamente regolato in S. cerevisiae e fortemente represso in presenza di glucosio, ottenendo il vettore pGAL-VP2,3. L’analisi dell’espressione delle proteine dai costrutti suddetti, effettuata mediante Western blot utilizzando un anticorpo specifico per le tre proteine capsidiche, ha mostrato una maggiore quantità di VP2 e VP3 ottenuta da cellule del ceppo di lievito RSY12 trasformate con pESCADH700-VP2,3 rispetto a quelle trasformate con pESCADH700-UTR-VP2,3. Ancora migliore è il livello di espressione di queste proteine con il vettore pESCGAL10VP2,3. Ottenuto l’aumento della proteine VP2,3 è stato costruito un nuovo vettore che contiene le sequenza codificante per le tre proteine capsidiche sotto la regolazione di pGAL1 e pGAL10: pESCLEUGalVP2,3GalVP1KM. Altri due vettori che abbiamo costruito ponendo sempre le tre proteine capsidiche sotto il controllo di promotori pGAL sono: pESCGAL1VP1KM e pYESGAL1VP2,3. Dopo aver trasformato le cellule di lievito con varie combinazioni di questi vettori, siamo arrivati a capire che la presenza nella cellula di un vettore contenente pGALVP2,3 determina un maggior numero di copie di questi due geni e porta ad un più alto livello delle due proteine. Finora, i risultati ottenuti indicano un aumento della quantità di proteine e presumibilmente anche di capsidi AAV nel lievito. La formazione di questi capsidi AAV viene analizzata cercando di mettere a punto la loro purificazione ed estrazione con ultracentrifuga e cuschion di saccarosio al 40%. Questo studio ci permetterà di capire se il lievito S. cerevisiae possa essere utilizzato come sistema modello non solo per lo studio dei fattori coinvolti nell’ assemblaggio del capside virale, ma anche per il suo successivo packaging con il genoma single strand di AAV

Bleomycin in the setting of lung fibrosis induction: from biological mechanisms to counteractions.

Bleomycin (BLM) is a drug used to treat different types of neoplasms. BLM’s most severe adverse effect is lung toxicity, which induces remodeling of lung architecture and loss of pulmonary function, rapidly leading to death. While its clinical role as an anticancer agent is limited, its use in experimental settings is widespread since BLM is one of the most widely used drugs for inducing lung fibrosis in animals, due to its ability to provoke a histologic lung pattern similar to that described in patients undergoing chemotherapy. This pattern is characterized by patchy parenchymal inflammation, epithelial cell injury with reactive hyperplasia, epithelial–mesenchymal transition, activation and differentiation of fibroblasts to myofibroblasts, basement membrane and alveolar epithelium injuries. Several studies have demonstrated that BLM damage is mediated by DNA strand scission producing single- or double-strand breaks that lead to increased production of free radicals. Up to now, the mechanisms involved in the development of pulmonary fibrosis have not been fully understood; several studies have analyzed various potential biological molecular factors, such as transforming growth factor beta 1, tumor necrosis factor alpha, components of the extracellular matrix, chaperones, interleukins and chemokines. The aim of this paper is to review the specific characteristics of BLM-induced lung fibrosis in different animal models and to summarize modalities and timing of in vivo drug administration. Understanding the mechanisms of BLM-induced lung fibrosis and of commonly used therapies for counteracting fibrosis provides an opportunity for translating potential molecular targets from animal models to the clinical arena

Phthalates exposure as determinant of albuminuria in type 2 diabetes subjects: a cross-sectional study

CONTEXT: Recent epidemiological observations have reported an association among phthalates exposure and insulin resistance, obesity, and diabetes but have not related exposure to these environmental pollutants with microvascular complications of diabetes. OBJECTIVE: To explore the relationship between phthalates and renal function in subjects with diabetes. DESIGN: Cross-sectional, case-only study. Concentrations of three urinary metabolites of di-2-ethylhexylphthalate were quantified in a spot morning urine sample, normalized for creatinine urinary excretion, and related to clinical parameters and phenotype, adjusting for confounders. PATIENTS: Two hundred and nine patients with diabetes consecutively referred to our clinic. MAIN OUTCOME MEASURES: Relationship between phthalates and renal function [evaluated with estimated glomerular filtration rate (eGFR) and albuminuria]. RESULTS: Creatinine-adjusted urinary concentrations of mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate (MEOHP), and mono-2-ethyl-5-hydroxyhexyl phthalate were 7.53 µg/g (range, 4.84 to 12.60), 3.04 µg/g (range, 1.03 to 5.14), and 10.70 µg/g (7.02 to 17.40), respectively. Age, sex, body mass index, diabetes duration, smoking, blood pressure, glycated Hb, and eGFR did not influence their levels. Exposure to MEHP and MEOHP was greater in individuals with microalbuminuria/macroalbuminuria (MEHP, P = 0.0173; MEOHP, P = 0.0306). The fourth vs first quartile showed a greater risk of albuminuria (MEHP: OR, 4.83; 95% CI, 1.45 to 16.06; P = 0.0297; MEOHP: OR, 3.29; 95% CI, 1.08 to 10.04); P = 0.0352). MEOHP was greater (P = 0.034) in subjects with cardiovascular events; MEHP showed a positive trend (P = 0.061). CONCLUSION: Our findings have revealed an association between exposure to di-2-ethylhexylphthalate metabolites and the degree of albuminuria in subjects with diabetes; the lack of a relationship with eGFR suggests their urinary levels are independent of renal function

Oropharyngeal meningococcal carriage in children and adolescents, a single center study in Buenos Aires, Argentina.

BackgroundNeisseria meningitidis (Nm) pharyngeal carriage is a necessary condition for invasive disease. We present the first carriage study in children in Buenos Aires, Argentina, considering 2017 as a transition year. Aims: to assess the rate of Nm carriage, to determine genogroup, clonal complex and outer membrane protein distribution, to determine carriage risk factors by age.MethodsCross-sectional study including children 1-17 yrs, at Ricardo Gutiérrez Children's Hospital in Buenos Aires 2017. Oro-pharyngeal swabs were taken and cultured within a short time after collection. Genogroup was determined by PCR and clonal complex by MLST. Categorical variables were analyzed.ResultsA total of 1,751 children were included. Group 1: 943 children 1-9 yrs, 38 Nm were isolated; overall carriage 4.0%. Genogroup distribution: B 26.3%, W 5.3%, Y 2.6%, Z 5.3%, other groups 7.9% and capsule null (cnl) 52.6%. Participating in extracurricular activities was the only independent predictor of Nm carriage. Group 2: 808 children 10-17 yrs, 76 Nm were isolated; overall carriage 9.4%. Genogroup distribution: B 19.7%, C 5.3%, W 7.9%, Y 9.2%, Z 5.3%, other groups 7.9% and cnl 44.7%. Independent predictors of carriage: attending pubs/night clubs and passive smoking (adjusted OR: 0.55, 95%CI = 0.32-0.93; p = 0.025).ConclusionsOverall carriage was higher in 10-17 yrs. The isolates presenting the cnl locus were prevalent in both age groups and genogroup B was the second most frequent

Adenosine receptors expression in cardiac fibroblasts of patients with left ventricular dysfunction due to valvular disease

Context: Adenosine restores tissue homeostasis through the interaction with its membrane receptors (AR) expressed on fibroblasts, endothelial cells, smooth muscle cells and leukocytes, but their modulation is still not fully understood. Objective: To evaluate whether changes in the transcriptomic profiling of adenosine receptors (AR) occur in cardiac fibroblasts (CF) of patients (pts) with LV dysfunction due to valvular disease (V). The secondary aim was to compare in the same pts the results obtained at cardiac level with those found in circulating leukocytes. Materials and methods: Auricle fragments were excised from 13 pts during prosthetic implantation while blood samples were collected from pts (n = 9) and from healthy subjects (C, n = 7). In 7 pts cardiac biopsy and blood samples were taken simultaneously. A human CF atrial cell line (cc) was used as control. Results: AR higher levels of mRNA expression were observed with real-time PCR in Vpts compared to C, both at cardiac (overexpression A1R:98%, A2AR:63%, A2BR:87%, A3R:85%, CD39:92%, CD73:93%) and at peripheral level (A1R vs C: p = .0056; A2AR vs C: p = .0173; A2BR vs C: p = .0272; A3R vs C: p = .855; CD39 vs C: p = .0001; CD73 vs C: p = .0091). Conclusion: All AR subtypes were overexpressed in CF of Vpts. The same trends in AR expression at cardiac level was assessed on circulating leukocytes, thus opening a new road to minimally invasive studies of the adenosinergic system in cardiac patients

Big gamma-glutamyltransferase is associated with epicardial fat volume and cardiovascular outcome in the general population

Aims: Gamma-glutamyltransferase (GGT) has been recognized as a cardiovascular risk factor, and its highest molecular weight fraction [big GGT (b-GGT)] is found in vulnerable atherosclerotic plaques. We explored the relationship between b-GGT, computed tomography findings, and long-term outcomes in the general population. Methods and results: Between May 2010 and October 2011, subjects aged 45-75 years living in a Tuscan city and without known cardiac disease were screened. The primary endpoint was a composite of cardiovascular death or acute coronary syndrome requiring urgent coronary revascularization. Gamma-glutamyltransferase fractions were available in 898 subjects [median age 65 years (25th-75th percentile 55-70), 46% men]. Median plasma GGT was 20 IU (15-29), and b-GGT was 2.28 (1.28-4.17). Coronary artery calcium (CAC) score values were 0 (0-60), and the volume of pro-atherogenic epicardial fat was 155 mL (114-204). In a model including age, sex, low-density lipoprotein (LDL) cholesterol, current or previous smoking status, hypertension, diabetes, obesity, b-GGT independently predicted epicardial fat volume (EFV) (r = 0.162, P < 0.001), but not CAC (P = 0.198). Over a 10.3-year follow-up (9.6-10.8), 27 subjects (3%) experienced the primary endpoint. We evaluated couples of variables including b-GGT and a cardiovascular risk factor, CAC or EFV. Big GGT yielded independent prognostic significance from age, LDL cholesterol, current or previous smoking status, hypertension, diabetes, obesity, but not CAC or EFV. Conversely, GGT predicted the primary endpoint even independently from CAC and EFV. Conclusion: Big GGT seemed at least as predictive as the commonly available GGT assay; therefore, the need for b-GGT rather than GGT measurement should be carefully examined