Ion concentrations in nasal airway surface liquid: a prediction model for the identification of cystic fibrosis carriers.

BACKGROUND: Cystic fibrosis (CF) carriers seem to have a higher risk to develop chronic rhino-sinusitis (CRS), although the full underlying mechanisms are unknown. Ion concentrations in nasal airway surface liquid (ASL) may be influenced by the heterozygosity for CF gene mutation, with possible impacts on the development of CRS. METHODS: A cheap and feasible standardized technique was designed to measure the ion levels in nasal ASL. With this purpose we collected, under basal conditions, samples from the nasal cavity of 165 adults: 14 homozygous for CF, 83 carriers and 68 healthy controls. Sodium (Na) and Chlorine (Cl) concentrations were then evaluated among different groups. RESULTS: Statistical analysis revealed a significant difference of Na and Cl values between controls and carriers and between controls and homozygotes. Receiver operating characteristic (ROC) curves and derived indicators (Youden's index and Area Under the Curve, AUC) were used to further evaluate the diagnostic capability of Na and Cl concentrations to differentiate heterozygotes from controls. ROC curves demonstrated that the optimal diagnostic cut-off value of Na is at 124, and the optimal cut-off value of Cl is at 103,2. CONCLUSION: ASL sampling can be considered a new diagnostic tool for providing quantitative information on nasal ion composition. According to our findings, Na and Cl concentrations of nasal ASL could represent a useful tool to assess heterozygotes and healthy controls.

Phytoestrogen-Rich Natural Preparation for Treatment of Climacteric Syndrome and Atherosclerosis Prevention in Perimenopausal Women.

The present study evaluated the risks and benefits of phytoestrogen treatment in healthy perimenopausal women in relation to the dynamics of climacteric syndrome and progression of atherosclerosis. Study participants were treated with placebo or phytoestrogen-rich natural preparation Karinat based on grape (Vitis vinifera) seeds, green tea (Camellia sinensis) leaves, hop (Hunulus lupulus) cone powder and garlic (Allium sativum) powder. The dynamics of climacteric syndrome was evaluated by Kupperman Index and Utian Quality of Life Scale. Atherosclerosis progression was evaluated by measuring carotid intima-media thickness. Significant changes of climacteric syndrome's severity in both Karinat and placebo groups (p = 0.005 and p = 0.001) were obtained after 24 months of follow-up. Detailed analysis of Kupperman Index suggested that Karinat possessed a significant effect on nervousness (p = 0.010), weakness (p = 0.020) and formication (p = 0.010). A significant improvement of medical (p = 0.070) and emotional (p = 0.060) components of Kupperman Index and Utian Quality of Life Scale was also observed in Karinat group. However, difference in carotid intima-media thickness between the two groups was not statistically significant at follow-up. A slight positive effect of phytoestrogens on climacteric syndrome manifestations was demonstrated in this study. Karinat can be used for alleviation of climacteric syndrome and cardiovascular disease prevention in perimenopausal women. Copyright © 2017 John Wiley & Sons, Ltd.

RAW 264.7 co-cultured with ultra-high molecular weight polyethylene particles spontaneously differentiate into osteoclasts: an in vitro model of periprosthetic osteolysis.

Wear-particle osteolysis affects prosthesis survival leading to implant loosening up to 70% of revisions. Therapeutic strategies are increasing, however alternative testing methods to experimentally evaluate such treatments are lacking. The aim of this study was to reproduce an in vitro osteolysis model recapitulating the events that, starting from the exposure of macrophages to polyethylene, lead to the establishment of osteoclastogenesis and inflammation. Responses to polyethylene, at 3 and 7 days, in a macrophage cell line, RAW 264.7, were determined by DNA quantification, immunofluorescence, pit assay, gene expression, cytokine production and NF-kB activation. Results showed that 3 days exposure to particles could induce a significant production of Tumor Necrosis Factor alpha (p < 0.0005) and Prostaglandin E2 (p < 0.005) compared to controls. Particles also induced macrophages to spontaneously differentiate into mature and active osteoclasts, in terms of identification of multinucleated cells by Phalloidin staining and by the analysis of osteoclast-specific gene markers. In particular, at 3 days polyethylene induced a significant up-regulation of Nuclear Factor of Activated T-cells, cytoplasmic 1, Receptor Activator of Nuclear factor Kappa-B and Receptor Activator of Nuclear Factor Kappa-B Ligand genes (p < 0.0005) compared to controls. At protein level, the particles induced a significant increase of Receptor Activator of Nuclear Factor Kappa-B Ligand at day 7 over controls (p < 0.0005). Osteoclasts were capable to resorb bone even in absence of differentiating factors. The possible mechanism, beside spontaneous osteoclastogenesis mediated by wear debris, was identified in an autocrine up-regulation of Receptor activator of nuclear factor kappa-B ligand gene expression and protein synthesis. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 510-520, 2017.

The Italian National External quality assessment program in molecular genetic testing: results of the VII round (2010-2011).

Since 2001 the Istituto Superiore di Sanità established a quality assurance programme for molecular genetic testing that covers four pathologies: Cystic Fibrosis (CF), Beta Thalassemia (BT), Fragile X Syndrome (FX), and Familial Adenomatous Polyposis Coli (APC). Since 2009 this activity is an institutional activity and participation is open to both public and private laboratories. Seven rounds have been performed until now and the eighth is in progress. Laboratories receive 4 DNA samples with mock clinical indications. They analyze the samples using their routine procedures. A panel of assessors review the raw data and the reports; all data are managed through a web utility. In 2010 the number of participants was 43, 17, 15, 5 for CF, BT, FX, APC schemes respectively. Genotyping results were correct in 96%, 98.5%, 100%, and 100% of CF, BT, FX, and APC samples, respectively. Interpretation was correct in 74%, 91%, 88%, and 60% of CF, BT, FX, and APC reports, respectively; however in most of them it was not complete but a referral to genetic counseling was given. Reports were satisfactory in more than 60% of samples in all schemes. This work presents the 2010 results in detail comparing our data with those from other European schemes.

Effects of smoking regular or light cigarettes on brachial artery flow-mediated dilation.

BACKGROUND AND PURPOSE: To compare the effects of regular cigarettes (RCs) and light cigarettes (LCs) on brachial artery flow-mediated dilation (FMD) and sublingual glyceryl trinitrate-induced dilation (GTN), markers of endothelial dependant and independent function, respectively. METHODS: 206 subjects (age 51.5 ± 12.8 yr, 122 men) had their smoking habits recorded and FMD and GTN measured by B-mode ultrasound. Cigarettes were categorized as RCs or LCs according to their content of tar, nicotine and CO. The chronic effect was assessed in current smokers of RCs (n = 85) or LCs (n = 53) and in never smokers (NS; n = 68). The acute effect was assessed in current smokers by measuring FMD before and 10-min after smoking a single regular (n = 29) or light (n = 51) cigarette. RESULTS: FMD was significantly lower in consumers of RCs (6.26%, 95% C.I. 5.58, 6.94) or LCs (5.59%, 95% C.I. 4.74, 6.45) compared to NS (8.68%, 95% C.I. 7.92, 9.44) (both P < 0.0001), but did not differ (P > 0.05) when compared to each other. GTN was similar in the three groups. Analyses adjusted for clinical confounders and for markers involved in oxidative stress, arginine/nitric oxide pathway, and inflammation provided identical results. Smoking a single cigarette, either regular or light, reduced FMD (-0.88% and -1.17%, respectively, both P < 0.05), without significant difference between cigarette type. RCs and LCs produced analogous chronic and acute effects when FMD was calculated with respect to the last 60 s of the low-flow phase (FMD60s). CONCLUSIONS: LCs impair endothelial-dependant vasodilation as much as RCs. Thus, smoking LCs cannot be considered an alternative to the only safe choice of a complete and permanent smoking cessation.

The metabolic syndrome predicts carotid intima-media thickness no better than the sum of individual risk factors in a lipid clinic population.

OBJECTIVE: To assess whether the diagnosis 'metabolic syndrome' (MS) predicts the degree of subclinical atherosclerosis better than its component parts or the total number of vascular risk factors (VRFs) in patients attending a lipid clinic. METHODS: Carotid intima-media thickness (C-IMT) was measured by B-mode ultrasound in 1804 patients (56+/-13 years; 52% women). To investigate whether the increased subclinical carotid atherosclerosis often ascribed to MS may be explained by a real interaction between the components or simply by a sum of VRFs, observed C-IMTs were compared with those predicted by the sum of individual components. Values for C-IMT of MS patients were also compared with those of controls matched for number of VRFs or for SCORE predicted risk (SPR). RESULTS: Carotid IMT values were significantly higher in patients with MS (n=362) than in those not so diagnosed (IMT(mean), 1.07+/-0.37 vs. 0.95+/-0.33; IMT(max), 1.98+/-0.93mm vs. 1.67+/-0.82mm, both p<0.0001), but were not higher than those predicted by the sum of individual risk factors. The linear regression lines of the correlations between C-IMT and total number of VRFs overlapped in patients with and without MS. In patients with and without MS matched for age, sex and total number of VRFs, or matched for age, sex and SPR the C-IMT differences disappeared. CONCLUSIONS: In patients attending a lipid clinic, 'metabolic syndrome' appears not to correlate with C-IMT to a greater extent than what is expected from its component parts or from the patient's total number of VRFs.

Occurrence of del(GIB6-D13S1830) mutation in Italian non-syndromic hearing loss patients carrying a single GJB2 mutated allele.

Molecular screening for GJB2 (connexin 26) mutations represents the standard diagnostic approach for the genotype definition of non-syndromic deafness. Nevertheless, a single GJB2 pathogenic mutation is detectable in a relevant number of cases, therefore failing to explain the phenotype. We aimed at assessing the occurrence of the recently described del(GIB6-D13S1830) mutation, occurring in the connexin 30 gene, in a group of Italian hearing-impaired patients carrying a single GJB2 mutated allele. A total of 59 non-syndromic hearing loss (NSHL) patients were screened for GJB2 mutations. Among these, nine NSHL patients were found to be heterozygous for a single GJB2 mutation. These patients, heterozygotes for different GJB2 mutated alleles (35delG, L90P, M34T, V153I), together with 11 additional 35delG/neg cases previously described, were studied for the presence of the del(GIB6-D13S1830) mutation. Two double heterozygotes del(GIB6-D13S1830)/35delG were identified. In both cases the degree of hearing loss was profound. Furthermore, GJB2 molecular screening led to the identification of a novel change (T55G) occurring in compound heterozygosity with the V37I mutation. In conclusion, our data suggest a significant frequency of del(GIB6-D13S1830) mutation in Italian hearing-impaired subjects (10% of unexplained GJB2 heterozygotes) similar to that reported in other European countries.

Atypical familial motor neuropathy in patients with mutant TTR Ile68Leu.

Two sisters from an Italian family shared progressive motor symptoms, preceding the onset of sensory and autonomic disturbances. The familial occurrence of axonal and slowly progressive polyneuropathy led us to consider these patients as candidates for TTR molecular analysis. We found a missense mutation causing Ile68Leu TTR substitution in both. The aims of this work are to report the possibility of a motor onset of amyloid polyneuropathy and to suggest the search for TTR mutations in familial cases of axonal polyneuropathy. Second, to stress the possible occurrence of amyloid within the spinal canal as the potential pathogenesis and responsible for motor presentation.

Exploring the clinical and epidemiological complexity of GJB2-linked deafness.

GJB2 mutation analysis was performed in 179 unrelated subjects with sporadic or familial hearing loss (HL). Among 57 families, 18 showed a vertical transmission of HL, the disease being present in two or three generations. Besides 155 nonsyndromic cases, 24 patients presenting with extra-auditory clinical signs were included in the molecular study. GJB2 mutation analysis was also performed in 19 subjects with an anamnestic history of perinatal risks factors for acquired HL. The 35delG mutation accounted for 22.1% of analyzed chromosomes in sporadic cases and 39.4% in familial cases; 35delG prevalence reached 41% in autosomal recessive and 44.4% in pseudodominant pedigrees. Two novel GJB2 mutations were identified in compound heterozygosity with 35delG allele (D159V, 284ins/dup[CACGT]). Two 35delG homozygous subjects were identified among HL cases classified as environmental in origin. Four patients 35delG heterozygous (35delG/V95M, 35delG/L90P, 35delG/167delT, and 35delG/?) and two homozygous presented with extra-auditory clinical signs involving different organs (skin, vascular system, hemopoietic lineages, and thyroid). In a high proportion of 35delG heterozygous HL patients (52%), no second GJB2 mutation was detected. The reported data highlight the complexity of the genetic epidemiology of GJB2-linked deafness, further enlarging the spectrum of situations in which GJB2 mutation analysis should be performed. The presence of extra-auditory signs in a significant portion of GJB2-mutated patients suggests the possibility that GJB2 loss of function could contribute to clinical phenotypes presenting in association with deafness. This hypothesis deserves further investigation. The failure to identify a presumed partnering GJB2 mutation in a high proportion of deaf patients remains a challenging problem to be clarified.

Biosensor technology for real-time detection of the cystic fibrosis W1282X mutation in CFTR.

In the present paper, biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect the Trp1282Ter mutation (W1282X) of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene. We first immobilized on a SA5 sensor chip a single-stranded biotinylated oligonucleotide containing the sequence involved in this mutation, and the efficiency of hybridization of oligonucleotide probes differing in length was determined. Second, we immobilized on different SA5 sensor chips biotinylated polymerase-chain reaction (PCR) products from a normal subject as well as from heterozygous and homozygous W1282X samples. The results obtained show that both allele-specific 10- and 12-mer oligonucleotides are suitable probes to detect W1282X mutations of the cystic fibrosis gene under standard BIA experimental conditions. During the association phase performed at 25 degrees C, discrimination between mismatched and full matched hybrids was readily and reproducibly observed by using the 10-mer W1282X probes. By contrast, when the 12-mer DNA probes were employed, discrimination between mismatched and full matched hybrids was observed during the dissociation phase. Taken together, the results presented suggest that BIA is an easy, speedy, and automatable approach to detect point mutations leading to cystic fibrosis. By this procedure, it is possible to perform real-time monitoring of hybridization between target single stranded PCR products obtained by using as substrates DNA isolated from normal or heterozygous subjects, and homozygous W1282X CF samples and oligonucleotide probes, therefore enabling a one-step, non-radioactive protocol to perform diagnosis.

Comparison of structures from frequencies of genes and surnames in the population of Ferrara.

The genetic structure based on isonymy and on gene frequencies of 7 enzyme systems was studied in a sample of 1361 individuals residing in the Ferrara Province in the Po delta (northern Italy). The sample was divided into two subsamples. The first, or indigenous, sample (n = 885) was composed of persons born and residing in the same commune of the province; the second, or migrant, sample (n = 476) was composed of persons who immigrated from a different commune. The study of the seven polymorphic genetic systems shows that there is no significant difference in gene and genotype frequencies between the two subsamples. On the other hand, the migration indicator derived from isonymy of family names is significantly larger in the migrant group than in the indigenous group. Isonymy techniques permit the detection of recent migration even under equality of gene frequencies.

Genetic structures in the Po Delta: principal components, systemic functions and the relative age of the beta-thalassemia polymorphism.

The principal component representations of the genetic structure of the human population of the Po Delta, obtained from 7 polymorphic loci, are compared with the representations obtained from the systemic function of gene frequencies devised by Womble 1951. It is noted that, when tridimensional representations are used, some consistency is visible in the results of the two methods for the description of the genetic population structure in the area under study. Both methods indicate that the present structure of the balanced polymorphism for beta-thalassemia in the area appears to be more recent than the structure of the neutral polymorphisms studied.

Kinship structures and migration in the Po Delta.

The kinship analysis of seven genetic systems in the province of Ferrara permits some considerations on the possible chronology of emergence of their polymorphisms in the area. It is proposed that, assuming neutrality of these systems, and under several restrictions, the emergence by migration of the polymorphisms in the seven systems ACP, ESD, GLO, GPT, PGD, PGM1, PGP might have had the following sequence: PGP and GLO and possibly PGD; PGM1 and GPT; ACP and ESD. All polymorphisms must be older than the beta-thalassemia polymorphism in the area.

Genetic structure of the human population in the Po delta.

The genetic structure of the population of Ferrara Province in the Po delta in Italy was investigated using chi 2 analysis, kinship analysis, analysis of correspondences, and geographical mapping of principal components of gene frequencies. chi 2 Analysis tests for Hardy-Weinberg equilibrium and for heterogeneity of gene and phenotype frequencies; kinship analysis tests for association between indicators of genetic and geographic proximity; analysis of correspondences relates localities and genetic systems in an eigenvectorial space; and geographic mapping displays the principal components of gene frequencies in the real space. In 1,364 adults in 26 residential units, seven presumably neutral isoenzyme systems were typed; ACP1 ESD, GLO I, GPT, PGD, PGM1 and PGP. It was found that average kinship for these neutral systems is correlated with geographic distance in this small area, but not as strongly as kinship for beta-thalassemia. A north-south gradient was observed for ESD. Analysis of correspondences indicated GPT, PGM1, and GLO I as the systems contributing most to differentiation within the province. The maps obtained from principal components of gene frequencies were consistent with the migrational history of the area.