Dynamical Generation of Elementary Fermion Mass: First Lattice Evidence.

Using lattice simulations we demonstrate from first principles the existence of a nonperturbative mechanism for elementary particle mass generation in models with gauge fields, fermions, and scalars, if an exact invariance forbids power divergent fermion masses and fermionic chiral symmetries broken at UV scale are maximally restored. We show that in the Nambu-Goldstone phase a fermion mass term, unrelated to the Yukawa operator, is dynamically generated. In models with electroweak interactions weak boson masses are also generated, opening new scenarios for beyond the standard model physics.

Microparticle-loaded neonatal porcine Sertoli cells for cell-based therapeutic and drug delivery system.

Neonatal porcine Sertoli cells (NPSC) are immune privileged cells showing innate phagocytic and antibacterial activities. NPSC have been shown capable of immunoaltering the body's response and possess lung homing capacity. These properties encourage investigation of NPSC as functional components of cell-based therapeutic protocols to treat lung infections and related complications. In this work, for the first time, NPSC were tailored to carry an antibiotic drug loaded into poly(d,l lactic) acid microparticles (MP). A loading protocol was developed, which afforded 30% drug uptake and high stability over time, with little or no effects on NPSC viability, morphology, reactive oxygen species production and DNA integrity. FSH receptor integrity, and TGFß (transforming growth factor ß) and AMH (anti-Müllerian hormone) expressions were unchanged after 1month of cryopreservation. Protein tyrosine kinase activation due to phagocytosis may have had resulted in changes in inhibin B expression. The activity of MP-loaded or NPSC alone against Pseudomonas aeruginosa was maintained throughout 1month of storage. NPSC couple an innate antibacterial activity with the capacity to embody drug loaded MP. We showed for the first time that engineered NPSC can be cryopreserved with no loss of their basic properties, thereby possibly representing a novel approach for cell-based therapeutic and drug delivery system.

Targeting the PI3K/Akt/mTOR signaling pathway in B-precursor acute lymphoblastic leukemia and its therapeutic potential.

B-precursor acute lymphoblastic leukemia (B-pre ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. The prognosis of B-pre ALL has improved in pediatric patients, but the outcome is much less successful in adults. Constitutive activation of the phosphatidylinositol 3-kinase (PI3K), Akt and the mammalian target of rapamycin (mTOR) (PI3K/Akt/mTOR) network is a feature of B-pre ALL, where it strongly influences cell growth and survival. RAD001, a selective mTORC1 inhibitor, has been shown to be cytotoxic against many types of cancer including hematological malignancies. To investigate whether mTORC1 could represent a target in the therapy of B-pre ALL, we treated cell lines and adult patient primary cells with RAD001. We documented that RAD001 decreased cell viability, induced cell cycle arrest in G0/G1 phase and caused apoptosis in B-pre ALL cell lines. Autophagy was also induced, which was important for the RAD001 cytotoxic effect, as downregulation of Beclin-1 reduced drug cytotoxicity. RAD001 strongly synergized with the novel allosteric Akt inhibitor MK-2206 in both cell lines and patient samples. Similar results were obtained with the combination CCI-779 plus GSK 690693. These findings point out that mTORC1 inhibitors, either as a single agent or in combination with Akt inhibitors, could represent a potential therapeutic innovative strategy in B-pre ALL.

Cytotoxic activity of the novel Akt inhibitor, MK-2206, in T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive neoplastic disorder arising from T-cell progenitors. T-ALL accounts for 15% of newly diagnosed ALL cases in children and 25% in adults. Although the prognosis of T-ALL has improved, due to the use of polychemotherapy schemes, the outcome of relapsed/chemoresistant T-ALL cases is still poor. A signaling pathway that is frequently upregulated in T-ALL, is the phosphatidylinositol 3-kinase/Akt/mTOR network. To explore whether Akt could represent a target for therapeutic intervention in T-ALL, we evaluated the effects of the novel allosteric Akt inhibitor, MK-2206, on a panel of human T-ALL cell lines and primary cells from T-ALL patients. MK-2206 decreased T-ALL cell line viability by blocking leukemic cells in the G(0)/G(1) phase of the cell cycle and inducing apoptosis. MK-2206 also induced autophagy, as demonstrated by an increase in the 14-kDa form of LC3A/B. Western blotting analysis documented a concentration-dependent dephosphorylation of Akt and its downstream targets, GSK-3α/ß and FOXO3A, in response to MK-2206. MK-2206 was cytotoxic to primary T-ALL cells and induced apoptosis in a T-ALL patient cell subset (CD34(+)/CD4(-)/CD7(-)), which is enriched in leukemia-initiating cells. Taken together, our findings indicate that Akt inhibition may represent a potential therapeutic strategy in T-ALL.

Semen quality of male idiopathic infertile smokers and nonsmokers: an ultrastructural study.

This retrospective study was aimed at evaluating the effects of cigarette consumption on semen parameters in a group of men with idiopathic infertility. The semen quality of 2 groups of men with idiopathic infertility, smokers (n = 118) and nonsmokers (n = 153), were compared. Conventional semen analysis was performed and sperm morphology was assessed by transmission electron microscopy (TEM). TEM data were elaborated by means of a mathematical formula based on a Bayesian technique able to furnish a fertility index (FI), and the percentages of sperm apoptosis, necrosis, and immaturity. Values of normality recommended by World Health Organization guidelines were used as a control for conventional semen analysis, and values from sperm of 25 men of proven fertility were used for TEM indices. Infertile smoker and nonsmoker patients showed similar sperm parameters, although sperm motility and TEM analysis values in both groups were significantly impaired compared with controls. Smoker patients were then classified as mild (>or=1 and 10 and <20 cigarettes/day), or heavy smokers (>or=20 cigarettes/d). Sperm concentration and FI were significantly (P < .05) different among the 3 considered smoker classes. Comparing the pairs of smoker classes, sperm concentration and FI in heavy smokers were significantly lower (P < .05) than that observed in mild smoker and nonsmoker groups. Although semen quality in males with idiopathic infertility seems not to be dramatically affected by cigarette consumption, heavy smokers show significantly lower sperm concentration and FI: another strong reason to stop smoking.

Even routine painful procedures can be harmful for the newborn.

Stressful events can damage neonatal brain through a complexity of events including free radical (FR) generation. We examined whether pain provoked by a routine heel prick can generate an increase in potentially harmful FR in neonatal blood. To this aim, advanced oxidation protein products (AOPP) and total hydroperoxide (TH) concentrations were measured at the beginning (sample A) and at the end (sample B) of each sampling in 64 babies (corrected age: 37.2+/-2.7 weeks) who underwent heel prick for routine blood tests. We scored pain of every procedure in all newborns. No differences were detected between AOPP and TH blood concentrations at the beginning and at the end of heel prick sampling, considering the whole cohort of babies. Conversely, a significant increase was observed between AOPP and TH blood concentrations considering only those babies who showed the highest pain intensity. When babies' pain was high (ABC score >or=4), mean AOPP and TH blood levels increased significantly; in this case, mean AOPP values increased from 53.5microm/l (SD=41.6) to 63.2microm/l (SD=44.3) and TH values from 218.3UCarr (SD=89.2) to 228.7UCarr (SD=93.3), with a significant p value of 0.02 and 0.036, respectively. A significant correlation was also found between AOPP blood levels ratio (sample B/sample A) in each baby, and the correspondent level of pain. These data show that even common routine procedures can be potentially harmful for the newborn if they provoke a high level of pain.

Retrospective assessment of potential negative synergistic effects of varicocele and tobacco use on ultrastructural sperm morphology.

OBJECTIVES: To investigate, in a retrospective study, whether smoking cigarettes increases the effect of varicocele on sperm morphology. METHODS: The semen quality of 2 groups of patients with varicocele were compared, those who smoked (n = 121) and those who did not (n = 158). The semen parameters were evaluated, and sperm morphology was assessed using transmission electron microscopy and quantitatively elaborated (fertility index, immaturity, necrosis, and apoptosis percentages). RESULTS: In the smoker and nonsmoker varicocele-associated cases, sperm motility and the results from transmission electron microscopy analysis were significantly impaired compared with controls. However, a nonsignificant difference was detected when the semen parameters were compared. Subsequently, we divided the patients into 4 groups: mild (> or = 1 but < or = 10 cigarettes/d), moderate (>10 but <20 cigarettes/d), and heavy (> or = 20 cigarettes/d) smokers and a group of randomly chosen nonsmoker patients with varicocele. The sperm motility, sperm concentration, and fertility index decreased and the percentage of sperm pathologic features increased as the number of cigarettes smoked daily increased. CONCLUSIONS: A detrimental effect of cigarette smoking (>10 cigarettes/d) associated with varicocele on sperm motility and morphology was observed. Because much of reduced fecundity associated with smoking may be reversed within 1 year of cessation, as reported in published studies, effective interventions targeted at helping patients quit smoking should be addressed for the benefit of general health and fertility.

Role of full-length osteoprotegerin in tumor cell biology.

Osteoprotegerin (OPG) is a soluble tumor necrosis factor receptor family member, which potently inhibits RANKL-mediated osteoclastogenesis. Numerous constructs have been created for therapeutic purposes in which the heparin-binding and death homology domains of OPG were removed and the remaining peptide (amino acids 22-194) was fused to the Fc domain of human IgG1 (OPG-Fc). The administration of OPG-Fc efficiently counteracted bone loss in a variety of preclinical models of cancers. However, several in vitro studies have shown that native or recombinant full-length OPG not only neuralizes RANKL, but also the death-inducing ligand TRAIL, suggesting that OPG might potentially counteract the anti-tumor activity of TRAIL. Additional evidence suggests that full-length OPG possesses RANKL- and TRAIL-independent biological properties, mainly related to the promotion of endothelial cell survival and angiogenesis. Finally, breast tumor cells overexpressing OPG have shown increased bone metastatic potential in vivo. The relevance of these apparently conflicting findings in tumor cell biology is highlighted.

Phytothermotherapy: a possible complementary therapy for fibromyalgia patients.

OBJECTIVE: It is a traditional practice in the Alpine region of Trentino and Alto Adige (Italy) to use phytothermotherapeutic treatment with fermenting grass ("hay baths") for rheumatic diseases. However, despite its long history and popularity, a clinical validation of the efficacy and tolerability of the treatment has yet to be found in current literature. Fibromyalgia syndrome (FMS) is characterised by generalised musculoskeletal pain, high tender point counts, sleep disturbance, fatigue, headaches, irritable bowel syndrome, frequent psychological distress and depressed mood. There is no standard therapy regime for FMS and the variety of medical treatments used have given limited benefits. The aim of this study was to assess the efficacy and tolerability of a cycle of phytothermotherapy through a single-blind, controlled, randomised trial, in patients with primary FMS. METHODS: Fifty-six patients with primary FMS according to the ACR criteria were randomly allocated to two groups: 30 were submitted to phytothermotherapy at the thermal resort of Garniga Terme (Trento, Italy) and the other 26 were considered as controls. All patients were evaluated by FIQ, Tender Points Count, HAQ and AIMS1 at baseline, after 10 days, then after 12 and 24 weeks. RESULTS: Patients submitted to phytothermotherapy showed visible and significant improvement of all evaluation parameters at the end of the treatment, which persisted during the follow-up period. No significant difference was found in the control group. Regarding the tolerability, none of the patients presented side effects. CONCLUSIONS: Our results suggest the efficacy and the tolerability of phytothermotherapy in patients with primary FMS.

Familial non-medullary thyroid carcinoma displays the features of clinical anticipation suggestive of a distinct biological entity.

Non-medullary thyroid carcinoma (NMTC) is mostly sporadic, but familial clustering is described. We aimed to compare the features of patients with sporadic and familial NMTC (FNMTC) patients and to assess whether FNMTC patients with parent-child relationship exhibit the 'anticipation' phenomenon (earlier age at disease onset and increased severity in successive generations). Among 300 NMTCs followed in the Section of Endocrinology (University of Siena, Italy), 34 (11.3%) patients, all with the papillary histotype, (16 kindred), met the criteria of FNMTC. Twenty-seven of them (79.4%) exhibited a parent-child relationship and seven (20.6%) a sibling relationship. These patients were compared with 235 patients with sporadic papillary thyroid cancer (PTCs). To analyze the features of FNMTC of the first and second generations, we cumulated the series of Siena with 32 additional FNMTC patients (15 kindred) from the Department of Endocrinology-Endocrine Oncology, Thessaloniki, Greece. Significant difference between sporadic PTC and FNMTC patients included more frequent tumor multifocality (P=0.001) and worse final outcome in FNMTC patients (P=0.001). Among 47 FNMTC with parent-child relationship, we found an earlier age at disease presentation (P<0.0001), diagnosis (P<0.0001), and disease onset (P=0.04) in the second generation when compared with the first generation. Patients in the second generation were more frequently males (P=0.02); their tumors were more frequently multifocal (P=0.003) and bilateral (P=0.01), had higher rate of lymph node metastases at surgery (P=0.02) and worse outcome (P=0.04) when compared with the first generation. In conclusion, FNMTC displays the features of clinical 'anticipation' with the second generation acquiring the disease at an earlier age and having more advanced disease at presentation.

Nuclear phosphatidylinositol 3,4,5-trisphosphate, phosphatidylinositol 3-kinase, Akt, and PTen: emerging key regulators of anti-apoptotic signaling and carcinogenesis.

Inositol lipid-derived second messengers have long been known to have an important regulatory role in cell physiology. Phosphatidylinositol 3-kinase (PI3K) synthesizes the second messenger 3,4,5'-phosphatidylinositol trisphosphate (Ptdlns 3,4,5P3) which controls a multitude of cell functions. Down-stream of PI3K/PtdIns 3,4,5P3 is the serine/threonine protein kinase Akt (protein kinase B, PKB). Since the PI3K/ PtdIns 3,4,5P3 /Akt pathway stimulates cell proliferation and suppresses apoptosis, it has been implicated in carcinogenesis. The lipid phosphatase PTEN is a negative regulator of this signaling network. Until recently, it was thought that this signal transduction cascade would promote its anti-apoptotic effects when activated in the cytoplasm. Several lines of evidence gathered over the past 20 years, have highlighted the existence of an autonomous nuclear inositol lipid cycle, strongly suggesting that lipids are important components of signaling pathways operating at the nuclear level. PI3K, PtdIns(3,4,5)P3, Akt, and PTEN have been identified within the nucleus and recent findings suggest that they are involved in cell survival also by operating in this organelle, through a block of caspase-activated DNase and inhibition of chromatin condensation. Here, we shall summarize the most updated and intriguing findings about nuclear PI3K/ PtdIns(3,4,5)P3/Akt/PTEN in relationship with carcinogenesis and suppression of apoptosis.

Sperm aneuploidies and low progressive motility.

BACKGROUND: Patients with poor semen quality show increased sperm disomy and diploidy rates. Oligozoospermia and teratozoospermia are known to influence sperm aneuploidy, but there is still a debate about whether aneuploidies are associated with reduced motility. METHODS: Ejaculates from a large group of patients were examined by light microscopy to evaluate sperm concentration, motility and morphology, and by fluorescence in-situ hybridization (FISH) to analyse the presence of aneuploidies. Statistical analysis was performed to compare differences and to evaluate the relationship between sperm aneuploidy rate and semen quality. RESULTS: Five groups were established following the motility parameter, and total aneuploidy rates were statistically significantly higher in the groups where motility was <30% compared to the controls. A homogeneous group of men with asthenozoospermia showed higher FISH values compared to control data, although the difference was not statistically significant. Motility and sperm morphology were each found to be statistically related to aneuploidy using a multiple linear regression analysis, whereas sperm concentration was only related to aneuploidy by the equation of a hyperbolic curve. CONCLUSIONS: In conclusion, biological and statistical data from the present research support the idea that the presence of aneuploidies could also be associated with reduced sperm motility.

TEM, FISH and molecular studies in infertile men with pericentric inversion of chromosome 9.

Pericentric inversions involving the secondary constriction (qh) region of chromosome 9 are considered to be normal variants of human karyotype. A number of investigators have suggested that chromosomal anomalies can contribute to human infertility causing spermatogenetic derangement. The present study was aimed at verifying the influence of chromosome 9 inversion on human spermatogenesis. Semen samples of 18 male carriers of chromosome 9 inversion, analysed by light microscopy, revealed that five patients were azoospermic. PCR analysis demonstrated that two of them also had Y microdeletions. The other 13 showed generally normal sperm concentrations and reduced motility. The morphological characteristics of sperm were studied by TEM and the data were elaborated by a mathematical formula. Sperm pathologies resulted more frequently in the studied group compared to controls, particularly apoptosis. Partial sequences of the A-kinase anchoring protein (Akap) 4 and 3 genes were performed in all patients, as a previous study by our group highlighted Dysplasia of Fibrous Sheath (DFS) defect in two men with inv 9 investigations. The possible effect of chromosome 9 inversion on meiotic chromosome segregation was investigated by FISH, which showed an increased incidence of diploidy. We hypothesized that this inversion could have variable effects on spermatogenesis, from azoospermia to severely altered sperm morphology, motility and meiotic segregation.

Effects of simulated microgravity on the morphology and function of neonatal porcine cell clusters cultured with and without Sertoli cells.

Human islet allografts are well known to induce full and sustained remission of hyperglycemia, with complete normalization of key metabolic parameters. Nevertheless, acquiring human islets, even from cadaveric human donor pancreases, remains a significant impediment to successful transplantation therapy for diabetes. To overcome this difficulty, neonatal porcine cell clusters (NPCCs) have been considered for human islet substitutes because they are easily obtained by collagenase digestion of the neonatal piglet pancreas. Currently, the major hurdle in using NPCCs for xenograft is the delay (time lag) in achieving the posttransplant normalization of blood glucose levels in animal diabetic recipients. The present work is the first attempt to evaluate whether incubation of NPCCs in simulated microgravity, in the presence or absence of Sertoli cells (SC), may reduce the maturation time lag of beta-cells by differentiation acceleration in vitro, thereby expediting production, viability, and acquisition of functional competence of pretransplantation beta-cell-enriched islets. Following a 3-day incubation period, NPCCs maintained in conventional culture, NPCCs incubated in simulated microgravity in the HARV biochamber, and NPCCs plus co-incubated SC in simulated microgravity were examined for viability, morphology, and insulin secretion. Results show that NPCCs grown alone in the HARV biochamber are superior in quality, both in terms of viability and functional competence, when compared to other culture pretreatment protocols. This finding strongly suggests that NPCC pretreatment in simulated microgravity may enhance the transplantation success of NPCCs in the diabetic recipient.

Ultrastructural and DNA fragmentation analyses in swim-up selected human sperm.

Seventeen sperm samples were evaluated by transmission electron microscopy (TEM) before and after swim-up separation. DNA-fragmentation was tested by terminal d-UTP nick end labeling (TUNEL) in unselected and selected semen samples, and the results were analyzed in relation to sperm ultrastructural characteristics detected by TEM. A significant improvement in mean numbers and percentages of structurally normal sperm was observed after swim-up selection, corresponding to a significant decrease in the percentage of necrotic and apoptotic sperm, while the percentage of sperm with immature nuclei did not change significantly. TUNEL indicated a significant decrease in chromatin-fragmented sperm after swim-up. Swim up selection based on sperm motility excludes many sperm with ultrastructural evidence of necrosis (absent or reacted acrosome, disrupted chromatin, broken plasma membrane) and apoptosis (misshapen nuclei with marginated chromatin), as confirmed by TUNEL analysis. Nevertheless, immature sperm with elliptical or roundish nuclei, misshapen acrosomes and uncondensed chromatin remain part of fertilizing pool.

Ultrastructural sperm study in infertile males with microdeletions of Y chromosome.

A retrospective study to detect specific Y chromosome microdeletions and to evaluate sperm ultrastructural characteristics in infertile men was set up. We selected 219 infertile men referred to Regional Referral Center for Male Infertility, Siena, Italy for semen analysis from January 1999 to April 2004. Family history, lymphocyte karyotype determination, Y microdeletion screening, physical examination, hormonal assays, semen analysis were carried out. Sperm concentration and progressive motility, ultrastructural analysis of sperm organelles, PCR amplification of sequence tagged sites for Y microdeletion screening were performed. Different Y-chromosome deletions were found, mainly in the AZFb and AZFc regions. Severe alterations of sperm ultrastructure, affecting whole sperm population, were detected in carriers of Y-deletions. Our data confirms the highest frequency of Y deletions in azoospermic patients. In all other patients with Y microdeletions, sperm ultrastructural defects affected the whole sperm population and were mainly related to apoptosis or immaturity.

Necrosis in human spermatozoa. I. Ultrastructural features and FISH study in semen from patients with uro-genital infections.

Ultrastructural characteristics and meiotic segregation in spermatozoa from twelve patients affected by uro-genital bacterial infections were investigated. The sperm quality was examined by light and transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) analysis was performed in eight out of twelve individuals in order to investigate the meiotic behaviour of chromosomes namely gonosomes and chromosome 18. TEM analysis highlighted a severely altered sperm morphology, typical of apoptosis and in particular, necrosis. We define the ultrastructural characteristics of necrosis as involving the acrosome, chromatin, mitochondrial helix, axonemal structure and plasma membrane. Based on our observations, it is possible to hypothesize that infection acts at the testicular level causing sperm death, due to necrosis itself or by necrosis proposed as the final step of apoptosis. Moreover, FISH analysis revealed the presence of altered meiotic segregation in these patients. The high rate of diploidy and gonosomes disomy in our group of patients suggests the possibility of a negative effect of infection and/or inflammation on sperm morphogenesis.

Necrosis in human spermatozoa. II. Ultrastructural features and FISH study in semen from patients with recovered uro-genital infections.

Inflammation of the male genital tract is a potential cause of male sterility. The quality of spermatozoa from ten patients with recovered uro-genital infections was examined by transmission electron microscopy (TEM); fluorescence in situ hybridization (FISH) was performed on sperm nuclei in six our of ten patients to investigate the frequency of aneuploidies. TEM analysis demonstrated the presence of a high percentage of necrosis in all patients, whereas apoptosis was present in only five of them. Meiotic segregation was altered in all analysed semen samples. Recovery from infections does not seem to coincide with improved sperm quality, probably because a persistent inflammatory state demonstrated by a high percentage of sperm necrosis sometimes associated with the presence of white blood cells (WBC) in the seminal plasma, is present. The effects of infections of the male genital tract could proceed in the absence of microbial agents due to immunological mechanisms involving the pattern of chemical products typical of inflammation. Our results suggest that the presence of necrosis, sometimes associated with apoptosis, could be considered to be an indicator of male genital tract inflammation. However, further studies are necessary to test the correlation between biochemical parameters and ultrastructural and molecular markers of inflammation.

Significance of nuclear phospholipase C signaling through type 1 IGF receptor.

The existence of a nuclear polyphosphoinositol metabolism independent from that at the plasma membrane is now widely recognized. Specific changes in the nuclear phosphatidylinositol (Ptdlns) metabolism have been implicated in cell growth, differentiation, and neoplastic transformation. Here we shall review the main features of nuclear inositol lipid signaling through type I IGF receptor, focusing the attention on the role of inositide-specific phospholipase C (PI-PLC) beta1 in cell proliferation and differentiation, given its peculiar localization in the nuclear compartment.

TRAIL promotes the survival, migration and proliferation of vascular smooth muscle cells.

Human and rat primary sub-cultured vascular smooth muscle cells (VSMCs) showed clear expression of the death receptors TRAIL-R1 and TRAIL-R2; however, recombinant soluble TRAIL did not induce cell death when added to these cells. TRAIL tended to protect rat VSMCs from apoptosis induced either by inflammatory cytokines tumor necrosis factor-alpha + interleukin-1beta + interferon-gamma or by prolonged serum withdrawal, and promoted a significant increase in VSMC proliferation and migration. Of note, all the biological effects induced by TRAIL were significantly inhibited by pharmacological inhibitors of the ERK pathway. Western blot analysis consistently showed that TRAIL induced a significant activation of ERK1/2, and a much weaker phosphorylation of Akt, while it did not affect the p38/MAPK pathway. Taken together, these data strengthen the notion that the TRAIL/TRAIL-R system likely plays a role in the biology of the vascular system by affecting the survival, migration and proliferation of VSMCs.