BRAF mutation and RASSF1A expression in thyroid carcinoma of southern Italy

Aim of this work is to provide a detailed comparison of clinical‐pathologic features between well‐differentiated and poorly differentiated tumors according to their BRAF and RASSF1A status. We analyzed RASSF1A methylation by MSP and BRAF mutation by LCRT‐PCR with LightMix® kit BRAF V600E in neoplastic thyroid tissues. Immunohistochemical evaluation of RASSF1A expression was also performed by standard automated LSAB‐HRP technique. An overall higher degree of RASSF1A over‐expression than normal thyroid parenchyma surrounding tumors (P < 0.05) has been found in all malignant well‐differentiated lesions. Moreover, statistically significant higher levels of RASSF1A expression were observed in differentiated cancers associated to an inflammatory autoimmune background (P = 0.01). Amplifiable DNA for LC PCR with LightMix® kit BRAF V600E was obtained in nine PTCs, four FVPTCs, five ATCs, and one control. The V600E mutation was found in 13 of 18 (72%) tumors. BRAF was mutated in 6 of 9 (66%) classical PTC, in 2 of 4 (50%) follicular variant PTC and in all ACs (100%). The overall frequency of RASSF1A promoter methylation observed was 20.5% (9 cases out 44). Hypermethylation of RASSF1A in primary tumors was variable according to histotypes ranging from100% (5/5) in ACs to only 12.5% (4/32) in PTCs. We show a correlation between RASSF1A methylation status and RASSF1A protein expression. Finally, we conclude that BRAF V600E mutation and RASSF1A methylation were pathogenetic event restricted to a subgroup of PTC/FVPTCs in early stage and to clinically aggressive ATCs. J. Cell. Biochem. 114: 1174–1182, 2013. © 2012 Wiley Periodicals, Inc.     

Phylogeographical Studies of Ascaris spp. Based on Ribosomal and Mitochondrial DNA Sequences

Background

The taxonomic distinctiveness of Ascaris lumbricoides and A. suum, two of the world''s most significant nematodes, still represents a much-debated scientific issue. Previous studies have described two different scenarios in transmission patterns, explained by two hypotheses: (1) separated host-specific transmission cycles in highly endemic regions, (2) a single pool of infection shared by humans and pigs in non-endemic regions. Recently, A. suum has been suggested as an important cause of human ascariasis in endemic areas such as China, where cross-infections and hybridization have also been reported. The main aims of the present study were to investigate the molecular epidemiology of human and pig Ascaris from non-endemic regions and, with reference to existing data, to infer the phylogenetic and phylogeographic relationships among the samples.

Methodology

151 Ascaris worms from pigs and humans were characterized using PCR-RFLP on nuclear ITS rDNA. Representative geographical sub-samples were also analysed by sequencing a portion of the mitochondrial cox1 gene, to infer the extent of variability at population level. Sequence data were compared to GenBank sequences from endemic and non-endemic regions.

Principal Findings

No fixed differences between human and pig Ascaris were evident, with the exception of the Slovak population, which displays significant genetic differentiation. The RFLP analysis confirmed pig as a source of human infection in non-endemic regions and as a corridor for the promulgation of hybrid genotypes. Epidemiology and host-affiliation seem not to be relevant in shaping molecular variance. Phylogenetic and phylogeographical analyses described a complex scenario, involving multiple hosts, sporadic contact between forms and an ancestral taxon referable to A. suum.

Conclusions/Significance

These results suggest the existence of homogenizing gene flow between the two taxa, which appear to be variants of a single polytypic species. This conclusion has implications on the systematics, transmission and control programs relating to ascariasis.     

In vivo and in vitro studies of the role of the adrenergic system and follicular wall contractility in the pathogenesis and resolution of bovine follicular cysts

Bovine follicular cysts (FCs) are a common cause of economic loss in modern dairy herds. Their aetiopathogenesis is not completely understood, even though an inadequate hypothalamic release of GnRH at the time of ovulation is considered to be their main cause. Much evidence, however, suggests a role for adrenergic innervation in ovarian functions, such as follicular development, steroid hormone secretion, and follicular contractility, the latter being an event important for ovulation. Moreover, in humans, polycystic ovary syndrome, a disease very similar to bovine follicular cysts, is characterised by increased density of adrenergic nerves. Given these premises, the aim of our study was to evaluate the effectiveness and mode of action of a novel strategy for the treatment of bovine follicular cysts. In the in vivo experiment, 170 Friesian cows diagnosed with follicular cysts were assigned to four groups (groups A, B, C, and D) to assess the effects of epidural administration of a β-adrenergic antagonist (carazolol) alone or in combination with a GnRH analogue (lecirelin). The four groups underwent the following treatments: Group A was administered lecirelin in combination with carazolol; Group B was administered carazolol; Group C was administered lecirelin; and Group D was administered only normal saline solution. In the in vitro experiment, strips of the walls of cystic follicles recovered post-mortem were suspended in an organ bath, connected to an isometric force transducer and exposed to increasing doses of epinephrine or to the same treatment after exposure to carazolol for 15 min (n = 10). The amplitude and frequency of the contractile activity were recorded. None of the control cows was observed in oestrus or was submitted to AI. The combination of lecirelin and carazolol induced a significant increase in the number of cows in oestrus (88%) compared to lecirelin alone or to carazolol alone (P < 0.05 and P < 0.01, respectively). The combination of lecirelin and carazolol and lecirelin alone were significantly more efficacious than carazolol alone (P < 0.01 and P < 0.05, respectively). In the in vitro experiment, epinephrine increased the amplitude of the contractions of the strips in a dose-dependent manner. This response was significantly enhanced in strips pre-treated with carazolol. The treatments had no effect on the frequency of contractions. In conclusion, our work demonstrates that the epidural administration of a GnRH analogue and a β-adrenergic blocker is an effective means of treating cows with cystic ovarian disease. Moreover, it confirms, from a clinical point of view, that alterations of the adrenergic system and of the contractility of the follicular wall can be considered aetiopathogenic factors involved in the development of FCs. The results of this study lay the basis for a new therapeutic approach to FCs.     

Oxidative status of plasma and muscle in rabbits supplemented with dietary vitamin E

Thirty New Zealand white rabbits, mean weight 2 kg, were divided into three equal groups balanced for body weight and randomly assigned to a diet containing 60 (C), 150 (T1) or 375 (T2) mg/kg of all-rac-alpha-tocopheryl acetate. After 29 days, the animals were slaughtered. alpha-Tocopherol was assayed in muscle (longissimus dorsi) and plasma; triglycerides and cholesterol (total, high density lipoprotein, low density lipoprotein) were analysed in plasma; reactive oxygen metabolites (ROMs) were analysed in serum; and thiobarbituric acid-reactive substances (TBARS) were analysed in muscle. There were no body weight and food intake differences between the groups. The plasma vitamin E and vitamin E:lipid ratio were significantly higher in groups T1 and T2 than in C, but increases were not linearly related to dietary levels. Muscle alpha-tocopherol concentrations in the treated groups were significantly higher than in C, and linearly related (R =.67) to the vitamin E:lipid ratio. ROM and vitamin E levels in blood were inversely related (R =.74), with ROMs significantly lower in the treated groups than in C. The 60-mg/kg dose of C recommended by the National Research Council was unable to control ROM production. Lipid oxidation in muscle was significantly lower in T2 than in the other groups, and TBARS correlated significantly with muscle vitamin E (R =.61) and serum ROM (R =.73). These data suggest that vitamin E supplemented at 375 mg/kg diet can effectively control ROM production and improve muscle lipostability. ROM assay provides a useful indirect estimate of the oxidative status of muscle in vivo.     

Handgrip-induced airway dilation in asthmatic patients with bronchoconstriction induced by MCh inhalation.

We investigated the effects of static and rhythmic handgrip on the time course of recovery of airway resistance measured with the interrupter technique (Rint) following bronchoconstriction induced by methacholine (MCh) inhalation in 17 asthmatic patients. On three separate occasions, a 100 +/- 5% increase in baseline Rint was induced by MCh inhalation. Subsequently, patients either rested [control trials (CTs)] or performed 3-min bouts of static or rhythmic handgrip. Respiratory and cardiovascular variables were continuously monitored. Rint changes were assessed at 1-min intervals for 30 min after rest and both types of handgrip. Plasma catecholamine concentrations were also determined at scheduled intervals. Bronchoconstriction increased ventilation (P < 0.01) but did not affect cardiovascular variables and plasma catecholamine concentrations. Handgrip provoked an increase in cardiovascular variables (P < 0.01) and plasma norepinephrine concentrations (P < 0.05) but caused no additional changes in ventilation. Rint only partially recovered within 30 min after CTs, whereas it consistently decreased 1 min after both handgrip paradigms and remained lower than after CTs (P always <0.01) for the whole 30-min observation period. Sympathetic activation and withdrawal of cholinergic input to the airway smooth muscle reflexly induced by activation of skeletal muscle and carotid sinus receptors may be the primary events accounting for the bronchodilator response induced by handgrip. Mediators co-released in response to sympathetic activation may also have contributed.     

Phenotypic and functional heterogeneity of human peripheral blood T lymphocytes producing colony stimulating factor. A clonal and precursor frequency analysis

In this report we describe the precursor frequency and the subset distribution of peripheral blood human T cells producing lymphokine(s) acting on the proliferation and differentiation of bone marrow precursors of the granulocyte/macrophage series, as assessed in a liquid microculture assay. Because the sensitivity of this system was similar to that of the classic colony formation assay in semi-solid (methylcellulose) medium, it is likely that the lymphokine activity measured in this assay corresponds to the colony-stimulating factor (CSF) activity. Single human T lymphocytes, isolated from peripheral blood by E rosetting and Ficoll-Hypaque gradients, were seeded into microculture wells by limiting dilution or micromanipulation techniques and were incubated under culture conditions that allow clonal expansion of essentially all T cells. After 15 to 20 days, microcultures were stimulated with PHA and CSF activity was assayed in culture supernatants 24 hr thereafter. About 45% (1/2.3) of peripheral blood T cells were found to give rise to CSF-producing progenies. Moreover, when fluorescence-activated cell sorter-purified T4+ and T4- (or T8- and T8+) were analyzed, the frequency of the precursors of CSF-producing cells was 1/1.5 in the T4+ subset, whereas approximately one-third of the T8+ cells had this functional potential. To additionally characterize T cells responsible for CSF production, unfractionated T cells as well as T4+ and T4- cells were cloned by single cell micromanipulation. The resulting clones were analyzed simultaneously for the production of IL 2, production of CSF and for cytolytic activity in a lectin-dependent assay. It was found that 25/48 clones obtained from unfractionated T cells produced CSF, whereas 23/48 and 19/48 produced IL 2 or had cytolytic activity respectively. Six of the 25 CSF-producing clones had only this functional capability, whereas the remaining clones in addition displayed cytolytic activity (4/25), IL 2 production (10/25), or both (5/25). A similar functional heterogeneity was observed among T4+ and T4- clones, thus indicating that T cells producing CSF are functionally heterogeneous within both the T4+ and T8+ subsets.     

CHy-banding patterns and chromatin organization inAegilops andTriticum species (Poaceae)

The distribution of CHy-banded heterochromatin was studied in the chromosomes ofAegilops longissima, Ae. speltoides, Triticum monococcum, andT. turgidum. Interphase nuclei were measured after Feulgen staining at different thresholds of optical density; the curves so obtained indicated the relationship among the species with respect to the different fractions of the genomic DNA. The karyological and cytophotometric analyses indicate differences betweenAe. speltoides andAe. longissima, the latter species being enriched in heterochromatin. Similar results were demonstrated for the genusTriticum, in whichT. turgidum showed more heterochromatin when compared withT. monococcum. The results suggest that the B genome of the cultivated wheats possesses a type of heterochromatin that resembles the type present inAe. longissima.     

Robust vaccine-elicited cellular immune responses in breast milk following systemic simian immunodeficiency virus DNA prime and live virus vector boost vaccination of lactating rhesus monkeys

Breast milk transmission of HIV remains an important mode of infant HIV acquisition. Enhancement of mucosal HIV-specific immune responses in milk of HIV-infected mothers through vaccination may reduce milk virus load or protect against virus transmission in the infant gastrointestinal tract. However, the ability of HIV/SIV strategies to induce virus-specific immune responses in milk has not been studied. In this study, five uninfected, hormone-induced lactating, Mamu A*01(+) female rhesus monkey were systemically primed and boosted with rDNA and the attenuated poxvirus vector, NYVAC, containing the SIVmac239 gag-pol and envelope genes. The monkeys were boosted a second time with a recombinant Adenovirus serotype 5 vector containing matching immunogens. The vaccine-elicited immunodominant epitope-specific CD8(+) T lymphocyte response in milk was of similar or greater magnitude than that in blood and the vaginal tract but higher than that in the colon. Furthermore, the vaccine-elicited SIV Gag-specific CD4(+) and CD8(+) T lymphocyte polyfunctional cytokine responses were more robust in milk than in blood after each virus vector boost. Finally, SIV envelope-specific IgG responses were detected in milk of all monkeys after vaccination, whereas an SIV envelope-specific IgA response was only detected in one vaccinated monkey. Importantly, only limited and transient increases in the proportion of activated or CCR5-expressing CD4(+) T lymphocytes in milk occurred after vaccination. Therefore, systemic DNA prime and virus vector boost of lactating rhesus monkeys elicits potent virus-specific cellular and humoral immune responses in milk and may warrant further investigation as a strategy to impede breast milk transmission of HIV.