Aging process, adherence to Mediterranean diet and nutritional status in a large cohort of nonagenarians: Effects on endothelial progenitor cells.

BACKGROUND AND AIM: Adherence to the Mediterranean Diet (MD) has been associated with a longer and better life. The aim of this study was to examine the effects of adherence to the MD, and of nutritional habits on endothelial progenitor (EPCs) and circulating progenitor (CPCs) cells in a cohort of nonagenarians enrolled within the Mugello Study, an epidemiological study aimed at investigating both clinically relevant geriatric items and various health issues, including those related to nutritional status. METHODS AND RESULTS: Four hundred twenty-one nonagenarians (306 F, 115 M, mean age: 93.1 ± 3.2 years) were evaluated. Adherence to MD was assessed through the Mediterranean Diet Score. Elderly subjects who were in the fourth quartile of the Mediterranean diet score showed significantly higher EPCs than subjects grouped into the other three quartiles. After adjustment for confounders, elderly subjects who were in the highest quartile of adherence to the MD score reported to have EPCs' levels significantly higher than those who reported lower values of adherence to the MD. Furthermore, by analyzing different food categories, it was reported that daily consumption of olive oil and a higher consumption of fruit and vegetables showed higher CPCs CD34+ and EPCs CD34+/KDR+ than subjects with not daily or lower consumption. CONCLUSION: Our results support the hypothesis that the adherence to MD, as well as a daily consumption of olive oil and fruit and vegetables, characteristics of MD, may protect against the development of endothelial dysfunction through increasing EPCs and CPCs in older age.

BRAF and RAS mutations as prognostic factors in metastatic colorectal cancer patients undergoing liver resection.

BACKGROUND: Despite major advances in the management of metastatic colorectal cancer (mCRC) with liver-only involvement, relapse rates are high and reliable prognostic markers are needed. METHODS: To assess the prognostic impact of BRAF and RAS mutations in a large series of liver-resected patients, medical records of 3024 mCRC patients were reviewed. Eligible cases undergoing potentially curative liver resection were selected. BRAF and RAS mutational status was tested on primary and/or metastases by means of pyrosequencing and mass spectrometry genotyping assay. Primary endpoint was relapse-free survival (RFS). RESULTS: In the final study population (N=309) BRAF mutant, RAS mutant and all wild-type (wt) patients were 12(4%), 160(52%) and 137(44%), respectively. Median RFS was 5.7, 11.0 and 14.4 months respectively and differed significantly (Log-rank, P=0.043). At multivariate analyses, BRAF mutant had a higher risk of relapse in comparison to all wt (multivariate hazard ratio (HR)=2.31; 95% CI, 1.09-4.87; P=0.029) and to RAS mutant (multivariate HR=2.06; 95% CI, 1.02-4.14; P=0.044). Similar results were obtained in terms of overall survival. Compared with all wt patients, RAS mutant showed a higher risk of death (HR=1.47; 95% CI, 1.05-2.07; P=0.025), but such effect was lost at multivariate analyses. CONCLUSIONS: BRAF mutation is associated with an extremely poor median RFS after liver resection and with higher probability of relapse and death. Knowledge of BRAF mutational status may optimise clinical decision making in mCRC patients potentially candidate to hepatic surgery. RAS status as useful marker in this setting might require further studies.

Dynamic Reorganization of Vortex Matter into Partially Disordered Lattices.

We report structural evidence of dynamic reorganization in vortex matter in clean NbSe(2) by joint small-angle neutron scattering and ac susceptibility measurements. The application of oscillatory forces in a transitional region near the order-disorder transition results in robust bulk vortex lattice configurations with an intermediate degree of disorder. These dynamically originated configurations correlate with intermediate pinning responses previously observed, resolving a long-standing debate regarding the origin of such responses.

First Report of Plum pox virus Strain M Isolates in Apricot in Sicily, Italy.

Sharka or plum pox disease is one of the most economically important virus diseases of stone fruits. Plum pox virus (PPV), the causal agent, is a member of the genus Potyvirus of the family Potyviridae transmitted by aphids in a non-persistent manner and by grafting. To date, nine PPV strains have been described on the basis of their biological, serological, and molecular properties: M and D are the most widespread and economically important strains, PPV-Rec and PPV-C have been reported mainly in Europe, PPV-EA confined to Egypt, PPV-T to Turkey, PPV-W from Canada, Ukraine, Latvia, and Russia, PPV-CR detected in Russia, and finally a putative PPV strain infecting plum in Albania described as the ancestor of the M. PPV-M is responsible for major epidemics in many Italian regions and despite phytosanitary measures, the infection rate increases each year. The D and Rec isolates are sporadically reported while PPV-C, once signaled in Apulia, has been successfully eradicated. Except for a report from the 1980s, which is no longer traceable, Sicily was considered free from the virus (2). In 2012, two new foci of sharka in a coastal area of Catania in Sicily were first reported by the national plant protection service to the European Commission (DG-SANCO). In spring 2013, plants of different varieties of apricot (Prunus armeniaca) and peach (P. persica) showing typical symptoms of flower color break, yellowing and leaf deformation, chlorotic spots or rings, and malformation on fruits were tested positive to PPV by DAS-ELISA using polyclonal antibodies. In order to characterize two isolates from apricot varieties (Carmen Top and Ninfa), total RNAs, extracted using the RNeasy Plant Mini Kit (Qiagen) from ELISA-positive samples, were analyzed by RT-PCR with primers P1/P2, targeting the 3'-terminal region of the coat protein (CP) gene (5) followed by RFLP analysis after digestion with Rsa1. Subsequently total RNAs were analyzed with the type-specific primers P1/PM and P1/PD (3), P3M/P4b and P3D/P4b amplifying the N-terminal region of the CP gene (1) and, finally, with primers mD5/mD3, mM5/mM3, and mD5/mM3, amplifying the region 3'NIb-5'CP, including the recombination site of Rec isolates (4). Only primer pairs P1/P2, P1/PM, P3M/P4b, and mM5/mM3 produced amplicons of the expected size (243, 198, 466, and 459 bp, respectively). The RFLP assay confirmed both isolates belonging to the M strain. Moreover, no reaction was obtained with primer pair mD5/mM3, excluding isolates belonging to Rec-type. Isolate characterization was completed by direct sequencing in both directions of the of P1/P2 and P3M/P4b amplicons obtained from apricot samples L9-1 (Carmen Top isolate) and 9-335 (Ninfa isolate). The P1/P2 sequences (KJ994235, KJ994237) showed 98% similarity with PPV-M or PPV-Rec isolates. The P3M/P4b sequences (KJ994236, KJ994238) confirmed that Sicilian isolates belong to the PPV-M strain showing 99% similarity with those already present in GenBank, thus ruling out the possibility of an infection with a PPV-Rec isolate. This outbreak of the Marcus strain of PPV in Sicily represents a high risk for the expanding production of stone fruit in southern Italy. An eradication plan was quickly activated by the regional phytosanitary service. References: (1) T. Candresse et al. Phytopathology 101:611, 2011. (2) EPPO. PQR-EPPO database on quarantine pests (available online). http://www.eppo.int , 2014. (3) A. Olmos et al. J. Virol. Methods 68:127, 1997. (4) Z. Subr et al. Acta Virol. 48:173, 2004. (5) T. Wetzel et al. J. Virol. Methods 33:355, 1991.

Higher uric acid levels are associated with better functional recovery in elderly patients receiving cardiac rehabilitation.

BACKGROUND AND AIMS: Whether uric acid (UA) serves as risk factor for cardiovascular diseases or as antioxidant defense has not yet been completely clarified. In this study we investigated the effects of UA on functional recovery in patients receiving cardiac rehabilitation. METHODS AND RESULTS: 306 patients, 209 men and 97 women, age range 25-87 years (mean 68 ± 11), performed the 6-min walk test (6mWT) before and after the rehabilitation, and the increase in walking distance was considered as the outcome measure of the study. Baseline UA serum levels ranged from 1.0 to 10.9 mg/dL (mean 5.2 ± 1.7). As there was a significant (p = 0.005) age*UA levels interaction, patients were divided into two subgroups, less then 65 years (n. 103, 68 men and 35 women, mean age 56 ± 9) and 65 years or more (n. 203, 141 men and 62 women, mean age 74 ± 5). After adjusting for relevant confounders, higher UA levels remained independent positive predictors of the increase in walking distance in older (p < 0.001) but not in younger patients (p = 0.807). CONCLUSIONS: Our findings show an independent association of higher UA levels with better functional recovery after cardiac rehabilitation selectively in elderly patients, suggesting that higher UA levels might reflect the decline in antioxidant defenses that occurs with advancing age. Future studies aimed at understanding the several contradictions concerning UA should, probably, address the issue within this perspective.

Characterization of Bois noir isolates by restriction fragment length polymorphism of a Stolbur-specific putative membrane protein gene.

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

Mediterranean Diet, Food Consumption and Risk of Late-Life Depression: The Mugello Study.

OBJECTIVES: To investigate eating habits and adherence to Mediterranean Diet (MD) in relation to the risk of depression in a cohort of nonagenarians enrolled within the Mugello Study, an epidemiological study aimed at investigating both clinically relevant geriatric items and various health issues, including those related to nutritional status. DESIGN: Cross-sectional study. SETTING: Homes and nursing homes in the Mugello area, Florence, Italy. PARTICIPANTS: Subjects aged 90-99 years [N=388 (271F; 117M) mean age: 92.7±3.1]. MEASUREMENTS: All subjects were evaluated through questionnaires and instrumental examinations. Adherence to MD was assessed through the Mediterranean Diet Score. A shorter version of the Geriatric Depression Scale (GDS) was used to detect the possible presence of depressive symptoms. In addition, cognitive and functional status was assessed using the Mini-Mental State Examination, the Clock Drawing Test, as well as the Basic and Instrumental Activities of Daily Living test. RESULTS: Depressed subjects (DS) (GDS score≥5, 43.8%) were older, females and widows, than non-depressed subjects (NDS). DS reported a slightly but not statistically significant lower MD score than NDS (33.9±3.9 vs. 34.6±3.3, p=0.149). Subjects who reported to consume a greater amount of olive oil and fruit were associated with a lower risk of depression (OR=0.35, 95%CI=0.20-0.59, p<0.001 and OR=0.46, 95%CI=0.26-0.84, p=0.011, respectively) after adjustment for many possible confounders. Similar results were obtained for women, while no statistically significant differences emerged for men. CONCLUSION: Our results support the hypothesis that a diet rich in olive oil and fruit, characteristics of MD, may protect against the development of depressive symptoms in older age.

Higher uric acid serum levels are associated with better muscle function in the oldest old: Results from the Mugello Study.

BACKGROUND: Sarcopenia is the progressive loss of muscle mass and strength that occurs with advancing age and plays a pivotal role in the causal pathway leading to frailty, disability and, eventually, to death among older persons. As oxidative damage of muscle proteins has been shown to be a relevant contributory factor, in this study we hypothesized that uric acid (UA), a powerful endogenous antioxidant, might exert a protective effect on muscle function in the oldest old and we tested our hypothesis in a group of nonagenarians who participated in the Mugello Study. METHODS: 239 subjects, 73 men and 166 women, mean age 92.8years±SD 3.1, underwent the assessment of UA serum level and isometric handgrip strength, a widely used clinical measure of sarcopenia. RESULTS: Mean UA serum level was 5.69mg/dL±SD 1.70 and mean handgrip strength was 15.0kg±SD 6.9. After adjusting for relevant confounders, higher UA serum levels remained independent positive predictors of isometric handgrip strength (ß 1.24±SE(ß) 0.43, p=0.005). CONCLUSION: Our results show that higher UA serum levels are associated with better muscle function in the oldest old and, accordingly, might slow down the progression of sarcopenia.

Local secretion of parathyroid hormone-related protein by an osteoblastic osteosarcoma (UMR 106-01) cell line results in growth inhibition.

Parathyroid hormone-related protein (PTHrP) has been implicated as being important in the growth of tumor cells responsive to the peptide. We utilized a rat osteoblastic osteosarcoma cell line, UMR 106-01, which has PTHrP receptors and a PTHrP-responsive adenylate cyclase/cAMP messenger system, to produce a modified cell line that overexpresses PTHrP. The human PTHrP cDNA sequence was transfected by electroporation into UMR 106-01 cells and the stable cell lines UMR-36 and UMR-34 were established. The modified cell line, UMR-36, had increased levels of PTHrP mRNA compared with control cell lines and secreted PTHrP into the culture medium at levels of 0.01-0.1 pmol/10(7) cells in 12 h. The secreted peptide was biologically active as indicated by its ability to activate adenylate cyclase. The number of UMR-36 cells following 9 days in culture was reduced by up to 80% compared with control lines, which was associated with decreased (3)H-thymidine incorporation into genomic DNA. Addition of 1000-fold excess of the PTHrP antagonist, PTHrP(7-34), to UMR-36 cells resulted in the escape of growth inhibition and increased rate of growth. In vivo, tumors derived from UMR-36 cells were smaller in size compared with tumors derived from control cells. In conclusion, increased autocrine secretion of, and responsiveness to, PTHrP results in inhibited growth kinetics of an osteoblast-like bone tumor cell line in vitro and in vivo.

Modulation of glucose transport by parathyroid hormone and insulin in UMR 106-01, a clonal rat osteogenic sarcoma cell line.

This study characterizes the actions of insulin and parathyroid hormone (PTH) on the glucose transport system in the rat osteogenic sarcoma cell line UMR 106-01, which expresses a number of features of the osteoblast phenotype. Using [1,2-3H]2-deoxyglucose (2-DOG) as a label, UMR 106-01 cells were shown to possess a glucose transport system which was enhanced by insulin. In contrast, PTH influenced glucose transport in a biphasic manner with a stimulatory effect at 1 h and a more potent inhibitory effect at 16 h on basal and insulin-stimulated 2-DOG transport. To explore the mechanism of PTH action, a direct agonist of cAMP-dependent protein kinase (PKA) was tested. 8-Bromo-cAMP had no acute stimulatory effect but inhibited basal and insulin-stimulated 2-DOG transport at 16 h. This result suggested that the prolonged, but not the acute, effect of PTH was mediated by the generation of cAMP. Further studies with the cell line UMR 4-7, a UMR 106-01 clone stably transfected with an inducible mutant inactive regulatory subunit of PKA, confirmed that the inhibitory but not the stimulatory effect of PTH was mediated by the PKA pathway. Northern blot data indicated that the prolonged inhibitory effects of PTH and 8-bromo-cAMP on glucose transport were likely to be mediated in part by reduction in the levels of GLUT1 (HepG2/brain glucose transporter) mRNA.

Production of parathyroid hormone-related protein by a rat parathyroid cell line.

Parathyroid hormone-related protein (PTHrP), the peptide associated with humoral hypercalcemia of malignancy, has been identified in fetal and adult parathyroid glands. We here report a sub-clone of a rat parathyroid cell line which secretes a single peptide species corresponding in size to PTHrP(1-84). Biological activity of the secretion product was blocked by a specific antiserum against PTHrP, but not by parathyroid hormone (PTH) antiserum. Secretion of PTHrP by these cells was regulated by extracellular calcium in the physiological range. A single messenger RNA species for PTHrP was identified, though PTH mRNA could not be shown in these cells. Hybrid CAT genes containing 700-1000 bp of 5'-flanking DNA from the human PTH or PTHrP genes were transfected into these cells, and the PTHrP gene was expressed at 10-fold higher levels than the PTH gene. These cells thus provide a valuable model system for investigation expression of PTHrP in a non-transformed cell line.

HPV DNA in intraepithelial neoplasia and carcinoma of the vulva and penis.

Surgical specimens of 15 patients with early and 12 patients with advanced squamous cell carcinoma of the vulva and the penis were examined for the presence of human papillomavirus (HPV) type 6, 11, 16, and 18 DNA by Southern blotting (SB) and polymerase chain reaction (PCR) analysis. By SB, HPV type 16 DNA was detected in all early carcinomas and 2 of 12 cases of advanced squamous cell carcinoma (ISCC) of the vulva and penis. PCR revealed HPV DNA in four additional cases of vulvar and penile ISCC negative by SB. Three cases contained HPV16 and one HPV18. Two cases of vulvar and penile Buschke-Löwenstein (BL) tumor with malignancy and one case of vulvar verrucous carcinoma were also examined by both techniques. While BL tumors were associated with DNA of HPV6 or 11, no HPV association was found for verrucous carcinoma. Our results confirm that the detection rate of HPV DNA in early vulvar and penile carcinomas is much higher than in invasive carcinomas. In addition, we have shown that in the lower genital tract, 50% of cases of ISCC are HPV16 correlated. The absence of HPV DNA (types 6, 11, 16, and 18) in the remaining 50% of cases of ISCC thus suggests that vulvar and penile ISCC may have more than one pathogenetic pathway.

TP53 mutations and mdm2 protein overexpression in cholangiocarcinomas.

Tumor suppressor protein p53 is a positive regulator of MDM2 gene expression and the mdm2 protein can bind to p53, preventing the transactivation of p53 responsive genes, thus mimicking TP53 mutation. The authors looked for alterations that could affect, directly and indirectly, p53 function in 13 patients with extrahepatic cholangiocarcinoma. Molecular analysis by single strand conformation polymorphism and DNA sequencing revealed that TP53 gene mutations occurred in only 2 of 13 cholangiocarcinomas. High levels of mdm2 protein were found, by immunohistochemical staining, in 61% of the cholangiocarcinomas and in almost all specimens (70%) displaying stabilized p53 protein in the absence and in the presence of TP53 mutations. The finding of co-overexpressed mdm2 and p53 proteins in cholangiocarcinomas indicates that they can upregulate the expression of mdm2 protein to a level sufficient for binding and accumulating p53 in a presumably inactive complexed form. The presence of TP53 mutations or upregulation of MDM2 gene expression in 9 of the 13 cholangiocarcinomas strongly supports that the impairment of the p53 pathway is an important and specific step in cholangiocarcinoma pathogenesis. At variance with other authors, no alteration of p16ink4/CDKN2 gene was observed in all 13 cholangiocarcinomas.

Epstein-Barr virus genomes in undifferentiated and squamous cell nasopharyngeal carcinomas in Italian patients.

Although undifferentiated carcinoma (UC) and squamous cell carcinoma (SCC) of the nasopharynx are regarded as two distinct histopathologic and clinical entities, it is unclear whether, like UC, SCC carries Epstein-Barr virus (EBV) genomes. We used the polymerase chain reaction (PCR) on paraffin-embedded biopsy specimens to test for the presence of EBV DNA in 20 cases of UC and 9 cases of SCC. Multiple copies of the viral genome were regularly detected in all UCs; however, of the nine cases of SCC, seven had no detectable EBV DNA and two contained viral genomes in a low copy number. In parallel, a marked difference in the serum levels of anti-EBV antibodies between patients with UC and SCC was found. Our findings provide evidence for the specific association of EBV with UC in Italian patients and prove by means of a highly sensitive molecular technique that SCC is occasionally related to EBV DNA. Because of the absence of EBV DNA in most cases of SCC and the minimal viral DNA copy number in the few EBV-associated cases of SCC, a different pathway of oncogenic transformation and growth of the nasopharyngeal epithelium is suggested for SCC and UC.

[Blood viscosity and triglyceridemia. Findings using a co-axial cylindrical viscosimeter at low "shear rates"]. / Viscosità ematica e trigliceridemia. Indagini con viscosimetro a cilindri coassiali a basse "shear rates".

Examination of blood viscosity at low shear rates using a co-axial cylinder viscometer showed a significant difference between the means of values observed in hypertrigliceridemic patients compared with that of control subjects. This result differs from what has been reported by most workers although generally greater shear rates have been used. Calculation of the "r" coefficient and plotting of the regression line for each shear rate showed that there is no linear correlation between blood viscosity and triglyceridaemia, whose variations occur quite independently. It is suggested that the absence of a correlation between the two parameters examined may depend on various factors, of which the most important are those pertaining to the rheological properties of red blood cells and to the structure and chemical and physical characteristics of the triglyceride molecule and of the lipoproteins and chylomicrons which transport them.

Comparison of different methods of RNA isolation for plum pox virus detection by reverse transcription-polymerase chain reaction.

The diagnosis of plum pox virus (PPV) is still considered one of the most important aspects of the "sharka" problem. In fact, different studies demonstrated an uneven distribution of the virus in infected trees due to a high variability in virus concentration. These aspects complicate the PPV diagnosis. To date, biological, serological and molecular assays have been successively developed in order to obtain sensitive and efficient PPV detection techniques. In particular, the polymerase chain reaction (PCR) technique seems to be promising and can be considered the most sensitive and reliable one. Preparation of viral RNA is still a fundamental step in reverse transcription-PCR (RT-PCR) technique, especially when applied to large scale testing, i.e., for certification purposes. In order to find the most rapid and efficient procedure, we have compared three different procedures of extraction of viral RNA to be processed RT-PCR. Their common characteristics is their capacity to extract the RNA from a small amount of plant tissue without organic solvents in the extraction fluid. The procedures were as follows: an immuno-capture (IC) method using a specific antiserum, a silica-capture (SC) method using a non-specific matrix, and a simple and rapid RNA extraction (RE) method. They all were followed by one-tube RT-PCR. The obtained results show that all the three techniques allowed a successful amplification and detection of PPV in tested samples except the SC-PCR method which proved less effective. In fact, the IC-PCR and RE-PCR methods amplified and detected PPV in all isolates tested, while the SC-PCR method was able to reveal the presence of the virus in apricot and infected control samples only.

Detection of plum pox virus in apricot seeds.

Twelve different apricot selection trees from a germplasm collection naturally infected with plum pox virus (PPV) were chosen to investigate the role of seeds in the epidemiology of this dangerous pathogen. All the considered plants showed typical symptoms on leaves and fruits and were positive in enzyme-linked immunosorbent assay (ELISA). The virus was characterized by immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) followed by restriction fragment length polymorphism (RFLP) analysis with RsaI enzyme as a PPV-D isolate. The presence of PPV was checked on fully ripe seeds and seedlings. One half of the seed stock was analysed immediately by ELISA and IC-RT-PCR tests: the cotyledons containing also the embryo were separated from the teguments. The other half of the seed stock was germinated and maintained in an insect-proof screenhouse over a 2-year period. PPV was detected by ELISA only in the seed coat while by IC-RT-PCR also in cotyledons. Seedlings from infected seeds did not show any typical symptoms and were PPV-negative in serological and molecular assays. So far, the presence of PPV in seeds seems to play no role in its epidemiology.

[Varicocele in young recruits]. / Il varicocele nei giovani di leva.

The authors, after an examination of the literature on the subject, present the results of a retrospective study in which the incidence of varicocele among selected young soldiers population called up at SARVAM (Viterbo) from May 1993 until October 1995 is evaluated.

[Surgical treatment of hydatid cysts of the hepatic dome ruptured into the thoracic cavity]. / Trattamento chirurgico delle cisti idatidee della cupola epatica rotte in cavo toracico.

Two cases of hydatid cyst of the hepatic dome, complicated by rupture in the thoracic cavity, are reported. The Authors stress the frequency of human hydatidosis, still high in Italy, and the severity of the above mentioned complication. Surgical treatment is also discussed.