Elena Muresu

Functionalized carbon nanotubes as immunomodulator systems

In view of the broad potential biomedical applications of carbon nanotubes (CNTs) different studies were performed to assess their effect on the immune system. However, the work performed to date was able to give a restricted view looking only at some activation markers and cytokine expression. The immune system is rarely limited to few molecule interactions being instead always a balance of switching several genes on and off. Whole genome expression (microarray) is a technology able to give the full picture on genome expression. Here we describe a microarray genome-wide study on Jurkat cells, a T lymphocyte cell line, and THP1, a monocytic cell line, representative of both types of immune response, the adaptive and innate, respectively. Since any structure or molecule modification may lead to very different immune reactions, we treated the two cell lines with four types of functionalized multi-walled CNTs that differ in terms of functionalization and diameter. After having assessed the internalization and the lack of toxicity of CNTs in both cell types, we used the Affymetrix technology to analyze the expression of about 32,000 transcripts. Three of the tested nanotubes (i.e., ox-MWCNT-1, ox-MWCNT-NH3(+)-1, and ox-MWCNT-NH3(+)-2) activated immune-related pathways in monocytes but not in T cells. In view of these charateristics they were named as monocyte activating CNTs (MA-CNTs). Molecular pathways upregulated by MA-CNTs included IL6, CD40, dendritic cell maturation, tumor necrosis factor-(TNF)-α/TNFR1-2, NFKB signaling and T helper 1 chemokine pathways (CXCR3 and CCR5 ligand pathways). These pathways are commonly activated during acute inflammatory processes as those associated with immune-mediated tumor rejection and pathogen clearance. One of them (i.e., ox-MWCNT-2) downregulated genes associated with ribosomal proteins in both monocytes and T cells. We validated our findings at gene expression level by performing real-time PCR assessing the most highly modulated genes in monocytes. To confirm the results at protein level, the secretion of IL1β, TNFα, IL6 and IL10 by THP1 and primary monocytes was assessed by ELISA, corroborating gene-expression data. Our results provide new insights into the whole gene expression modulation by different CNTs on immune cells. Considering the well known drug carrier ability of CNTs, our findings demonstrate that MA-CNTs here behave as cell specific immunostimulatory systems, giving very interesting future perspectives for their application also as immunotherapeutic agents and/or vaccine adjuvants.

Prevalence study of Legionella spp. contamination in ferries and cruise ships

BackgroundIn the last years, international traffic volume has significantly increased, raising the risk for acquisition of infectious diseases. Among travel-associated infections, increased incidence of legionellosis has been reported among travellers.Aim of our study was: to describe the frequency and severity of Legionella spp. contamination in ferries and cruise ships; to compare the levels of contamination with those indicated by the Italian ministerial guidelines for control and prevention of legionellosis, in order to assess health risks and to adopt control measures.MethodA prevalence study was carried out on 9 ships docked at the seaports of northern Sardinia in 2004. Water samples were collected from critical sites: passenger cabins, crew cabins, kitchens, coffee bars, rooms of the central air conditioning system. It was performed a qualitative and quantitative identification of Legionella spp. and a chemical, physical and bacteriological analysis of water samples.ResultsForty-two percent (38/90) water samples were contaminated by Legionella spp.. Positive samples were mainly drawn from showers (24/44), washbasins (10/22). L. pneumophila was isolated in 42/44 samples (95.5%), followed by L. micdadei (4.5%).Strains were identified as L. pneumophila serogroup 6 (45.2%; 19 samples), 2–14 (42.9%), 5 (7.1%) and 3 (4.8%). Legionella spp. load was high; 77.8% of the water samples contained > 104 CFU/L.Low residual free chlorine concentration (0–0,2 mg/L) was associated to a contamination of the 50% of the water samples.ConclusionLegionella is an ubiquitous bacterium that could create problems for public health.We identified Legionella spp. in 6/7 ferries. Microbial load was predominantly high (> 104 CFU/L or ranging from 103 to 104 CFU/L). It is matter of concern when passengers are subjects at risk because of Legionella spp. is an opportunist that can survive in freshwater systems; high bacterial load might be an important variable related to diseases occurrence.High level of contamination required disinfecting measures, but does not lead to a definitive solution to the problem. Therefore, it is important to identify a person responsible for health safety in order to control the risk from exposure and to apply preventive measures, according to European and Italian guidelines.

Diet and nutrients are contributing factors that influence blood cadmium levels.

Studies suggested the intake of Cd from diet can be approximately equivalent to that from smoking. Moreover, a mutual metabolic influence between Cd and nutrients has been reported. The purpose of this study was to evaluate the relationship between blood cadmium concentration (BCdC) and food consumption, nutrients intake (Ca, Fe, Zn, vitamin C, and vitamin D), tobacco smoking, and some other variables (age, body mass index, and residence) in 243 adults living in the Italian island of Sardinia (Sassari Province). Specifically, we hypothesized that offal consumption contributes to Cd intakes and blood levels. The BCdC was quantified by graphite furnace atomic absorption spectrometry, and information on personal data was collected through questionnaires. Smoke significantly contributed to the BCdC (P < .001). Nonsmoker subjects who eat offal showed significantly higher BCdC (P = .04). Moreover, slightly higher BCdCs were also observed in nonsmoker subjects who eat rice, fish, and bread. The BCdC positively correlated with age of subjects (r = 0.144; P = .025) and offal daily intake in nonsmokers (r = 0.393; P < .001). The intake of Ca was negatively correlated (r = -0.281; P = .001) with the BCdC in females. The multiple linear regression analysis showed smoking > consumption of offal > body mass index ≈ age as the most important risk factors for the BCdC in the selected population.

IS200 fingerprint of Salmonella enterica serotype Typhimurium human strains isolated in Sardinia

A collection of Salmonella enterica serotype Typhimurium human strains isolated in Northern Sardinia (Italy) was examined for the insertion sequence IS200, phage type, antibiotic profile, ribotyping polymorphisms and plasmid profile. All clinical isolates studied contained from 4 to 10 copies of the IS200 element. IS200 permitted to discriminate Typhimurium strains and to identify five IS200 types, some of them circulating in Sardinia at least since 1900. Strains belonging to phage DT104 predominated and correlated with a specific IS200 pattern.

High HPV-51 Prevalence in Invasive Cervical Cancers: Results of a Pre-Immunization Survey in North Sardinia, Italy

Background Human Papilloma virus (HPV) is recognized as the etiological agent of benign and malignant ano-genital lesions. The most prevalent genotypes associated with cervical carcinoma are HPV-16 and -18 worldwide. However, recent studies have emphasized the role of other genotypes, such as HPV-51, in the pathogenesis of cervical dysplasia. The aim of the study was to estimate the burden of HPV-51 infection in invasive cervical malignant lesions in Northern Sardinia, Italy. Methods/Principal Findings An observational, retrospective, prevalence, mono-center study was carried out to evaluate the presence of HPV genotypes in tissues biopsies of cervical lesions (CIN-1, CIN-2, CIN-3 and invasive carcinoma) gathered from 1996 to 2009. Biological samples were collected from women admitted consecutively to a tertiary university hospital situated in Sassari, Italy. Molecular methods were used to identify 28 oncogenic HPV types. A total of 155 formalin-fixed and paraffin-embedded cervical tissue samples were analyzed. Approximately half of the cervical lesions were classified as invasive carcinoma. HPV-DNA was detected in 71% of the samples, with a higher frequency (100%) in those categorized as invasive neoplasia. Mono- or co-infections were demonstrated in 45.8% and 25.8% of the cervical samples, respectively. Overall, the most prevalent HPV types were -16 (49%) and -51 (19.4%), with an increased frequency of detection associated with the severity of the cervical lesions. Conclusions/Significance This survey highlights for the first time the relevant role of HPV-51 infection in the pathogenesis of invasive cervical cancer prior to the introduction of a vaccination program. Although a selection bias could have influenced the results, other recent studies have described the impact of HPV-51. This remarkable epidemiological element should be carefully evaluated, particularly in the view of opting for preventive vaccines, whose cross-protection patterns determine their efficacy in protecting against infection from HPV types that are not included in the vaccine itself.

Metals in plasma of nonagenarians and centenarians living in a key area of longevity.

The concentration of calcium (Ca), cobalt (Co), copper (Cu), iron (Fe), magnesium (Mg), manganese (Mn) and selenium (Se) in plasma of 76 nonagenarians (mean age, 89.0±6.3 years), 64 centenarians (mean age, 101±1 years) and 24 middle-aged subjects as controls (mean age 61.2±1.1 years), was determined by sector field inductively coupled plasma mass spectrometry. All the subjects lived in Sardinia, an Italian island, that has the higher prevalence of centenarians than in other European countries. A comparison among the three classes of age showed a significant depletion of Ca, Co, Fe, Mn and Se (all p<0.001) in nonagenarians and centenarians with respect to controls. In particular, the geometric mean (GM) values of Ca, Co, Fe, Mn and Se were: 94.1 μg/ml, 0.46 ng/ml, 1314 ng/ml, 2.47 ng/ml and 111 ng/ml in controls; 87.6 μg/ml, 0.22 ng/ml, 815 ng/ml, 1.07 ng/ml and 88.9 ng/ml in nonagenarians; 87.0 μg/ml, 0.29 ng/ml, 713 ng/ml, 1.27 ng/ml and 81.9 ng/ml in centenarians. The highest inverse relationship with age was observed for Fe (p<0.001; ρ=-0.352) and Se (p<0.001; ρ=-0.417). This trend was also observed when data were sorted by gender. On the other hand, Cu and Mg levels in plasma remained substantially unchanged during aging. As regards Cu, it was significantly higher in females than in males in controls (GM, 1294 ng/ml vs. 1077 ng/ml; p=0.012), in nonagenarians (GM, 1216 ng/ml vs. 1081 ng/ml; p=0.011) as well as in centenarians (GM, 1226 ng/ml vs. 1152 ng/ml; p=0.045) and in hypertensive subjects with respect to healthy people (GM, 1215 ng/ml vs. 1129 ng/ml; p=0.021). These data can be used to enhance knowledge and support the research on: i) metals involved in aging in areas with high rates of human longevity; ii) variables (gender, lifestyle habits and health status) as critical determinants in aging; and iii) mineral intake and supplementation at older age affecting the healthy aging.

Molecular methods for the detection of human papillomavirus infection: new insights into their role in diagnostics and epidemiological surveillance

Human papillomaviruses (HPVs) comprise more than 180 genotypes. HPV infection is mainly diagnosed by molecular methods. The aim of our study was to review the main molecular methods used to diagnose HPV infection, underscoring their characteristics. Several methods have been developed for molecular diagnosis of Papilloma infection, such as those based on PCR technique. Another commercial non-PCR based diagnostic method is Hybrid Capture test; it is the only commercially available HPV DNA detection test approved by the FDA. Several Authors have suggested that viral load and E6/E7 transcripts could be used as surrogate markers of persistent HPV infection, being more specific predictors of progressive disease than the simple presence of HPV DNA. Validating clinical sensitivity and specificity of each technique and improving the interpretation of the results are essential; consequently, there is a clear need for well characterized international quality control panels to compare the various diagnostic methods. HPV DNA testing could be useful both as a primary screening test, alone or in combination with a Pap smear, for the early detection of cervical cancer precursors, and as triage test to select women with minor cytological abnormalities who will need further follow-up and to predict possible treatment failure in women with diagnosed high-grade intraepithelial lesions who have undergone excisional therapy. In the next future surveillance for HPV infections, based on these molecular methods, could represent an important step for the development of primary and secondary prophylactic interventions, such as new vaccines targeted to genotypes who might replace those previously prevalent.

Evaluation of radon levels in indoor gymnasia of Palermo (Sicily) and Sassari (Sardinia)

Background : In the last decades, there has been increased worldwide interest in the management of health risks from indoor radon. Methods : From 2006 to 2008, a survey on air radon levels was carried out in a total of 57 indoor gymnasia respectively located in the urban area of Palermo (Sicily) and Sassari (Sardinia). Results : The indoor radon levels were generally low with different geometric means in the two geographic areas (14.3 Bq/m3 in Palermo and 36 Bq/m3 in Sassari, respectively). Overall, in both groups increasing values of radon were found during the night and the early morning, with radon concentrations significantly lower during working time than at other times. The analysis of structural parameters showed that direct contact with the soil significantly correlated with increased levels of radon in gymnasia located in Palermo’s area (p<0.05). Furthermore, higher radon levels in Sicilian structures were also associated, although not significantly, with lack of sumps (19.3 vs 12.9 Bq/m3), location below the ground level (18.8 vs 8.7 Bq/m3), lack of windows (25 vs 13.2 Bq/m3), presence of forced ventilation (15.5 vs 7.3 Bq/m3) and with cracks or moisture tracks (15.7 vs 13.2 Bq/m3). Conclusions : Radon tends to accumulate in indoor sites and it’s monitoring, especially in buildings with potentially overcrowded conditions, could be of public health interest. Mitigation measures and minimization of the number of structural and functional risk parameters should be strongly encouraged in order to limit radon accumulation especially in countries with higher radium concentration in the soil.

Cloriti e clorati nelle acque potabili della Sardegna centro-settentrionale

Introduzione: l’approvvigionamento idropotabile della Sardegna e basato quasi esclusivamente su acqua superficiale particolarmente esposta a contaminazione ambientale per il cui trattamento sono richiesti complessi sistemi di potabilizzazione. In questo contesto, l’utilizzo del biossido di cloro in sostituzione dell’ipoclorito di sodio, pur determinando la significativa riduzione dei THM, potrebbe generare ioni clorito e clorato potenzialmente dannosi per l’uomo. Obiettivi: e stata condotta un‘indagine al fine di valutare la presenza di cloriti e clorati e rilevare indicazioni utili al loro contenimento nelle acque in uscita dagli impianti di potabilizzazione della Sardegna centro-settentrionale. Materiali e metodi: in due distinte serie di campionamenti, sono stati rilevate sia concentrazioni di disinfettante in pre ed in post clorazione sia, mediante cromatografia liquida per scambio ionico secondo la procedura EPA Metodo 300.0, cloriti e clorati nei vari step della potabilizzazione di 12 impianti. Risultati: il dosaggio medio totale di ClO2 e stato di 4,63 mg/l (2,87 mg/l in preclorazione e 1,93 in postclorazione). La percentuale media di abbattimento dei cloriti da parte dei filtri a carbone attivo ha evidenziato un calo di efficacia, essendo passata da un 62,3% rilevato nel primo campionamento ad un 23,6% rilevato nel secondo campionamento. La concentrazione media di cloriti e clorati in uscita dagli impianti e stata rispettivamente di 1,66 mg/l (min. 0,72 mg/l, max. 2,98 mg/l, dev. st. 0,69) e di 1,05 mg/l (min. 0,47 mg/l, max. 1,58 mg/l, dev. st. 0,36). La concentrazione media di cloro residuo attivo libero (C.R.A.L.) e stata di 0,59 mg/l (min. 0,19 mg/l, max. 1,26 mg/l). Conclusioni: Le concentrazioni di cloriti rilevate appaiono piuttosto elevate rispetto al valore di parametro del D.L.vo 31/2001 e, unitamente alle concentrazioni dei clorati, sono indicative della necessita di adottare, quanto prima, articolati e specifici interventi per il loro contenimento.