Chiara Favero

Nutrients Intake Is Associated with DNA Methylation of Candidate Inflammatory Genes in a Population of Obese Subjects

The aim of the present study was to evaluate the potential association between dietary nutrients and alterations in DNA methylation in a set of five candidate genes, including CD14, Et-1, iNOS, HERV-w and TNFα, in a population of overweight/obese subjects. We evaluated possible associations between gene methylation and clinical blood parameters, including total cholesterol (TC), low- and high-density lipoprotein cholesterol (LDL-C and HDL-C), triglyceride and homocysteine levels. We employed validated methods to assess anthropometric, clinical and dietary data, as well as pyrosequencing to evaluate DNA methylation of the five candidate genes in 165 overweight/obese subjects. There was no association between body mass index and DNA methylation of the five candidate genes in this group of subjects. Positive associations were observed between TNFα methylation and blood levels of LDL-C (β = 0.447, p = 0.002), TC/HDL-C (β = 0.467, p = 0.001) and LDL-C/HDL-C (β = 0.445, p = 0.002), as well as between HERV-w methylation and dietary intakes of β-carotene (β = 0.088, p = 0.051) and carotenoids (β = 0.083, p = 0.029). TNFα methylation showed negative associations with dietary intakes of cholesterol (β = −0.278, p = 0.048), folic acid (β = −0.339, p = 0.012), β-carotene (β = −0.332, p = 0.045), carotenoids (β = −0.331, p = 0.015) and retinol (β = −0.360, p = 0.008). These results suggest a complex relationship among nutrient intake, oxidative stress and DNA methylation.

Histologic muscular history in steroid-treated and untreated patients with Duchenne dystrophy

Objective: Duchenne muscular dystrophy (DMD) is a lethal disease. The outcome measures used in numerous therapeutic trials include skeletal muscle biopsy. We studied the natural history of DMD from the standpoint of muscle histology with the aim of providing a reproducible tool for use in evaluating and comparing any histologic changes occurring in patients with DMD undergoing treatment and hence be able to determine how therapy modulates the histologic evolution of the disease. Methods: Three independent operators analyzed 56 muscle biopsies from 40 patients not treated with steroids, aged 1 to 10 years and 16 individuals treated with steroids, aged 7 to 10 years. We analyzed morphologic measures, normalized every measure for the average number of fibers observed for each year of age, and calculated intraclass correlation coefficients. Results: The average proportion of connective tissue in patients not treated with steroids was 16.98% from ages 1 to 6 years and 30% from ages 7 to 10 years (p < 0.0001). The average proportion in patients treated with steroids was 24.90%. Muscle fiber area mirrored that of connective tissue in both groups. Conclusions: Having provided a reproducible tool for evaluation and comparison of histologic changes occurring in patients undergoing clinical trials, it was observed that at ages 6 to 7 years, fibrotic tissue rapidly peaks to 29.85%; this is a crucial moment when muscle tissue loses its self-regeneration ability, veering toward fibrotic degeneration. These data should be considered when deciding the most suitable time to begin therapy.

Plasmatic extracellular vesicle microRNAs in malignant pleural mesothelioma and Asbestos-Exposed subjects suggest a 2-miRNA signature as potential biomarker of disease

Background Malignant Pleural Mesothelioma (MPM) is an aggressive cancer mainly caused by asbestos exposure and refractory to current therapies. Specific diagnostic markers for early MPM diagnosis are needed. Changes in miRNA expression have been implicated in several diseases and cancers, including MPM. We examined if a specific miRNA signature in plasmatic extracellular vesicles (EV) may help to discriminate between malignant pleural mesothelioma patients (MPM) and subjects with Past Asbestos Exposure (PAE). Methodology/Principal findings We investigated 23 MPM patients and 19 cancer-free subjects with past asbestos exposure (PAE). We screened 754 miRNAs in plasmatic EVs by OpenArray and found 55 differential miRNAs using logistic regression models adjusted for age, sex, BMI, and smoking. Among the top-20 differential miRNAs chosen for validation by Real time PCR, 16 were confirmed. Using receiver operating characteristic (ROC) curve analysis, the most discriminating miRNA combination was given by miR-103a-3p + miR-30e-3p, which generated an AUC of 0.942 (95% CI 0.87–1.00), with a sensitivity of 95.5% and a specificity of 80.0%. Using multivariate Cox regression analysis including gender, age, BMI and smoking we found a Hazard Ratio for miR-103a-3p above the median of 0.37 (95%CI 0.13–1.13) and of 0.51 (95%CI 0.17–1.52) for miR-30e-3p. Conclusions This study suggests EV-associated miR-103a-3p and miR-30e-3p are able to discriminate MPM from PAE subjects. Larger and prospective studies are needed to confirm these two-miRNA signature alone or in combination with other biomarkers as diagnostic tools for MPM.

Blood DNA methylation, nevi number, and the risk of melanoma

Germline mutations determining increased cutaneous malignant melanoma (CMM) risk have been identified in familial and sporadic CMM cases, but they account only for a small proportion of CMM cases. Recent evidence suggests that germline epimutations (e.g. DNA methylation alterations), which can be inherited similarly to genomic mutations and can be detected in normal body cells (including blood), might increase susceptibility to cancer. The aim of the study was to identify germline epimutations of genes that were found to be mutated in familial CMM (p16INK4a, p14ARF, CDK4, MC1R, hTERT), immune and inflammatory genes (ICAM-1, TNF&agr;), DNA mismatch repair gene (MLH1), and repetitive elements (ALU, LINE-1, HERV-w). We measured DNA methylation using bisulfite pyrosequencing in peripheral blood mononuclear cells from 167 CMM cases and 164 sex-matched and age-matched controls. We used multivariable logistic regression models to evaluate the association between methylation levels and CMM status or presence of dysplastic nevi. We found an association between the risk of CMM and peripheral blood mononuclear cell methylation levels of TNF&agr; [odds ratio (OR)=1.11, 95% confidence interval (CI)=1.03–1.18], CDK4 (OR=0.76, 95% CI=0.64–0.91), and MLH1 (OR=1.12, 95% CI=1.02–1.22). In control participants, the risk of developing dysplastic nevi was associated with methylation levels of TNF&agr; (OR=0.81, 95% CI=0.69–0.95), hTERT (OR=0.90, 95% CI=0.82–0.99), and ALU (OR=1.56, 95% CI=1.02–2.39). Epimutations in CMM susceptibility genes and in genes involved in response to oxidative damage are associated with the risk of developing CMM or dysplastic nevi. Further studies measuring methylation levels of these genes in prospectively collected samples are warranted to further elucidate their role in the development and progression of CMM.

Short-term particulate matter exposure induces extracellular vesicle release in overweight subjects

Background: Extracellular vesicles (EVs) represent a plausible molecular mechanism linking particulate matter (PM) inhalation to its systemic effects. Microvesicles (MVs) are released from many cell types in response to various stimuli. Increased body mass index (BMI) could modify the response to PM exposure due to enhanced PM uptake and/or an underlying pro‐oxidative state. We investigated the relationship between EV release and PM10/PM2.5 exposure in a cohort of 51 volunteers. Subjects were stratified based on their BMI to evaluate whether overweight BMI is a determinant of hypersusceptibility to PM effects. Results: Exposure to PM10/PM2.5 was assessed with a personal sampler worn for 24 hours before plasma collection and confirmed with monitoring station data. Size and cellular origin of plasma EVs were characterized by Nanosight analysis and flow cytometry, respectively. Multivariate regression models were run after log‐transformation, stratifying subjects based on BMI (≥ or <25 kg/m2). PM exposure resulted in increased release of EVs, with the maximum observed effect for endothelial MVs. For PM10 and PM2.5, the adjusted geometric mean ratio and 95% confidence interval were 3.47 (1.30, 9.27) and 3.14 (1.23, 8.02), respectively. Compared to those in normal subjects, PM‐induced EV alterations in overweight subjects were more pronounced, with visibly effect in all MV subtypes, particularly endothelial MVs. Conclusions: Our findings emphasize the role of EV release after PM exposure and the susceptibility of overweight subjects. Larger studies with accurate exposure assessment and complete EVs characterization/content analysis, could further clarify the molecular mechanism responsible for PM effects and of hypersusceptibility of overweight subjects. HighlightsWe investigated the association between EV release and personal exposure to PM.Increased PM exposure was associated with increased release of EVs.The maximum effect was found for endothelial EVs.PM‐induced EV alterations were more pronounced in overweight subjects.

MicroRNAs are associated with blood-pressure effects of exposure to particulate matter: Results from a mediated moderation analysis.

Aims Exposure to particulate air pollution is associated with increased blood pressure (BP), a well-established risk factor for cardiovascular disease. To elucidate the mechanisms underlying this relationship, we investigated whether the effects of particulate matter of less than 10 μm in aerodynamic diameter (PM10) on BP are mediated by microRNAs. Methods and results We recruited 90 obese individuals and we assessed their PM10 exposure 24 and 48 h before the recruitment day. We performed multivariate linear regression models to investigate the effects of PM10 on BP. Using the TaqMan® Low-Density Array, we experimentally evaluated and technically validated the expression levels of 377 human miRNAs in peripheral blood. We developed a mediated moderation analysis to estimate the proportion of PM10 effects on BP that was mediated by miRNA expression. PM10 exposure 24 and 48 h before the recruitment day was associated with increased systolic BP (β=1.22 mmHg, P=0.019; β=1.24 mmHg, P=0.019, respectively) and diastolic BP (β=0.67 mmHg, P=0.044; β=0.91 mmHg, P=0.007, respectively). We identified nine miRNAs associated with PM10 levels 48 h after exposure. A conditional indirect effect (CIE=−0.1431) of PM10 on diastolic BP, which was mediated by microRNA-101, was found in individuals with lower values of mean body mass index. Conclusions Our data provide evidence that miRNAs are a molecular mechanism underlying the BP-related effects of air pollution exposure, and indicate miR-101 as epigenetic mechanism to be further investigated.

Acute particulate matter affects cardiovascular autonomic modulation and IFN-γ methylation in healthy volunteers

Aims: Air particulate matter (PM) is associated with increased cardiovascular morbidity and mortality. Altered autonomic functions play a key role in PM‐induced cardiovascular disease. However, previous studies have not address the impact of PM on sympathetic and parasympathetic control of heart function, independently, and using controlled conditions, i.e., increasing titration of PM of known composition, in absence of other potential confounding factors. To fill this gap, here we used symbolic analysis that is capable of detecting non‐mutual changes of the two autonomic branches, thus considering them as independent, and concentrations of PM as they could be measured at peak levels in Milan during a polluted winter day. Methods and results: In this randomized, cross‐over study, we enrolled 12 healthy subjects who underwent two random sessions: inhalation of filtered air mixture or inhalation of filtered air containing particulate mixture (PM 10, PM 2.5, PM 1.0 and PM 0.5 &mgr;m). ECG and respiration for autonomic analysis and blood sample for DNA Methylation were collected at baseline (T1), after air exposure (T2) and after 2 h (T3). Spectral and symbolic analysis of heart rate variability (HRV) were performed for autonomic control of cardiac function, while alterations in DNA methylation of candidate genes were used to index pro‐inflammatory modifications. In the PM expose group, autonomic analysis revealed a significant decrease of 2UV%, index of parasympathetic modulation (14% vs 9%, p = 0.0309), while DNA analysis showed a significant increase of interferon &ggr; (IFN‐ &ggr;) methylation, from T1 to T3. In a mixed model using T1, T2 and T3, fine and ultrafine PM fractions showed significant associations with IFN‐ &ggr; methylation and parasympathetic modulation. Conclusions: Our study shows, for the first time, that in healthy subjects, acute exposure to PM affects parasympathetic control of heart function and it increases methylation of a pro‐inflammatory gene (i.e. methylation of interferon &ggr;). Thus, our study suggests that, even in absence of other co‐factors and in otherwise healthy individuals, PM per se is sufficient to trigger parasympathetic dysautonomia, independently from changes in sympathetic control, and inflammation, in a dose‐dependent manner. HIGHLIGHTSIn healthy subjects, acute exposure to PM affects vagal autonomic control of the heart.PM exposure increases methylation of a pro‐inflammatory gene, i.e. methylation of interferon &ggr; (IFN‐ &ggr;).PM per se is able to trigger autonomic deregulation and inflammation in a dose‐dependent manner.

Hydroquinone induces DNA hypomethylation-independent overexpression of retroelements in human leukemia and hematopoietic stem cells

Hydroquinone (HQ) is an important benzene-derived metabolite associated with acute myelogenous leukemia risk. Although altered DNA methylation has been reported in both benzene-exposed human subjects and HQ-exposed cultured cells, the inventory of benzene metabolite effects on the epigenome is only starting to be established. In this study, we used a monocytic leukemia cell line (THP-1) and hematopoietic stem cells (HSCs) from cord blood to investigate the effects of HQ treatment on the expression of the three most important families of retrotransposons in the human genome: LINE-1, Alu and Endogenous retroviruses (HERVs), that are normally subjected to tight epigenetic silencing. We found a clear tendency towards increased retrotransposon expression in response to HQ exposure, more pronounced in the case of LINE-1 and HERV. Such a partial loss of silencing, however, was generally not associated with HQ-induced DNA hypomethylation. On the other hand, retroelement derepression was also observed in the same cells in response to the hypomethylating agent decitabine. These observations suggest the existence of different types of epigenetic switches operating at human retroelements, and point to retroelement activation in response to benzene-derived metabolites as a novel factor deserving attention in benzene carcinogenesis studies.

Short-term particulate matter exposure influences nasal microbiota in a population of healthy subjects

Background Exposure to air pollutants, such as particulate matter (PM), represents a growing health problem. The aim of our study was to investigate whether PM could induce a dysbiosis in the nasal microbiota in terms of &agr;‐diversity and taxonomic composition. Methods We investigated structure and characteristics of the microbiota of 40 healthy subjects through metabarcoding analysis of the V3–V4 regions of the 16s rRNA gene. Exposure to PM10 and PM2.5 was assessed with a personal sampler worn for 24 h before sample collection (Day −1) and with measurements from monitoring stations (from Day −2 to Day −7). Results We found an inverse association between PM10 and PM2.5 levels of the 3rd day preceding sampling (Day −3) and &agr;‐diversity indices (Chao1, Shannon and PD_whole_tree). Day −3 PM was inversely associated also with the majority of analyzed taxa, except for Moraxella, which showed a positive association. In addition, subjects showed different structural profiles identifying two groups: one characterized by an even community and another widely dominated by the Moraxella genus. Conclusions Our findings support the role of PM exposure in influencing microbiota and altering the normal homeostasis within the bacterial community. Whether these alterations could have a role in disease development and/or exacerbation needs further research. HighlightsWe evaluated whether PM could induce a dysbiosis in the nasal microbiota.PM exposure reduces the diversity within the microbiota community.PM alters microbiota homeostasis potentially influencing disease development.

Fibrin clot structure is affected by levels of particulate air pollution exposure in patients with venous thrombosis

BACKGROUND Particulate air pollution is a risk factor for cardiovascular diseases and thrombosis. Long-term exposure to particulate matter with a diameter<10μm (PM10) has been associated with an increased risk of venous thrombosis. OBJECTIVES The aim of this study was to investigate whether or not particulate air pollution alters fibrin clot structure and thus modulates thrombosis risk. METHODS We investigated fibrin polymerization by turbidity (maximum absorbance mOD), clot structure by confocal microscopy (fibre number per μm) and fibrin pore size by permeability (Ks×10(-10)cm(2)) in 103 patients with deep vein thrombosis and 121 healthy controls, for whom levels of air pollution exposure had been recorded. Exposure groups were defined by mean PM10 concentrations over the 730days before the event. RESULTS We found a higher average number of fibres per clot area in patients than controls, but no difference in Ks or fibre thickness. When the two groups were divided into high or low exposure to PM10, a significantly denser fibrin clot network structure with thicker fibres (higher maximum absorbance, p<0.05), decreased permeability (lower Ks value, p<0.05) and higher average fibre numbers per clot area (p<0.05) was observed in patients in the high exposure group compared to those with low exposure. There were no significant differences in fibrin clot structure between the two exposure levels in healthy subjects. CONCLUSIONS PM10 levels are associated with altered fibrin clot structure in patients with deep vein thrombosis but not in controls, suggesting that air pollution may trigger differences in fibrin clot structure only in patients predisposed to thrombotic disease.