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Experimental mouse studies are important to gain a comprehensive, quantitative and mechanistic understanding of the biological factors that modify individual risk of radiation-induced health effects, including age at exposure, dose, dose rate, organ/tissue specificity and genetic factors. In this study, neonatal Ptch1+/– mice bred on CD1 and C57Bl/6 background received whole-body irradiation at postnatal day 2. This time point represents a critical phase in the development of the eye lens, cerebellum and dentate gyrus (DG), when they are also particularly susceptible to radiation effects. Irradiation was performed with γ rays (60Co) at doses of 0.5, 1 and 2 Gy, delivered at 0.3 Gy/min or 0.063 Gy/min. Wild-type and mutant mice were monitored for survival, lens opacity, medulloblastoma (MB) and neurogenesis defects. We identified an inverse genetic background-driven relationship between the radiosensitivity to induction of lens opacity and MB and that to neurogenesis deficit in Ptch1+/– mutants. In fact, high incidence of radiation-induced cataract and MB were observed in Ptch1+/–/CD1 mutants that instead showed no consequence of radiation exposure on neurogenesis. On the contrary, no induction of radiogenic cataract and MB was reported in Ptch1+/–/C57Bl/6 mice that were instead susceptible to induction of neurogenesis defects. Compared to Ptch1+/–/CD1, the cerebellum of Ptch1+/–/C57Bl/6 mice showed increased radiosensitivity to apoptosis, suggesting that differences in processing radiation-induced DNA damage may underlie the opposite strain-related radiosensitivity to cancer and non-cancer pathologies. Altogether, our results showed lack of dose-rate-related effects and marked influence of genetic background on the radiosensitivity of Ptch1+/–mice, supporting a major contribution of individual sensitivity to radiation risk in the population. [ABSTRACT FROM AUTHOR]

One harmful long-term effect of ionizing radiation is cataract development. Recent studies have been focused on elucidating the mechanistic pathways involved in this pathogenesis. Since accumulating evidence has established a role of microRNAs in ocular diseases, including cataract, the goal of this work was to determine the microRNA signature of the mouse lens, at short time periods postirradiation, to understand the mechanisms related to radio-induced cataractogenesis. To evaluate the differences in the microRNA profiles, 10-week-old Patched1 heterozygous (Ptch1+/–) mice, bred onto two different genetic backgrounds (CD1 and C57Bl/6J), received whole-body 2 Gy γ-ray irradiation, and 24 h later lenses were collected. Next-generation sequencing and bioinformatics analysis revealed that genetic background markedly influenced the list of the deregulated microRNAs and the mainly predicted perturbed biological functions of 2 Gy irradiated Ptch1+/– mouse lenses. We identified a subset of microRNAs with a contra-regulated expression between strains, with a key role in regulating Toll-like receptor (TLR)-signaling pathways. Furthermore, a detailed analysis of miRNome data showed a completely different DNA damage response in mouse lenses 24 h postirradiation, mainly mediated by a marked upregulation of p53 signaling in Ptch1+/–/C57Bl/6J lenses that was not detected on a CD1 background. We propose a strict interplay between p53 and TLR signaling in Ptch1+/–/C57Bl/6J lenses shortly after irradiation that could explain both the resistance of this strain to developing lens opacities and the susceptibility of CD1 background to radiation-induced cataractogenesis through activation of epithelial-mesenchymal transition. [ABSTRACT FROM AUTHOR]

Pulsed homodyne quantum tomography usually requires a high detection efficiency, limiting its applicability in quantum optics. Here, it is shown that the presence of low detection efficiency () does not prevent the tomographic reconstruction of quantum states of light, specifically, of Gaussian states. This result is obtained by applying the so-called ‘minimax’ adaptive reconstruction of the Wigner function to pulsed homodyne detection. In particular, we prove, by both numerical and real experiments, that an effective discrimination of different Gaussian quantum states can be achieved. Our finding paves the way to a more extensive use of quantum tomographic methods, even in physical situations in which high detection efficiency is unattainable. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Fibrotic idiopathic interstitial pneumonias are chronic and progressive lung diseases with different prognosis, with idiopathic pulmonary fibrosis (IPF) having the worst prognosis. Many patients need a surgical lung biopsy for the definite diagnosis of IPF but age and the clinical context often contraindicate this procedure. The aim of this study is to identify predictors of survival, apart from lung biopsy, in patients with definite and possible IPF. PATIENTs AND METHODS: We studied 42 patients with HRCT pattern of definite or possible IPF, by assessing the mortality in relationship with baseline HRCT and functional findings. HRCT was assessed both as prevalent pattern (definite vs possible UIP) and as score of the different abnormalities (in particular, honeycombing (HC) and total fibrotic score). Pulmonary function was assessed as baseline FVC, TLC and DLCO values, as well as change over 6 months of follow-up. Both univariate and multivariate analyses were performed in order to detect predictors of mortality. RESULTS: During follow-up, 10 out of 42 patients died. Mortality rate was not different according to the qualitative pattern of fibrosis at HRCT. Among the different HRCT scores, a cut-off of 15% in the HC score differentiated patients with higher mortality rate. A lower baseline FVC, and a greater decrease in pulmonary function after 6 months, were both associated with higher mortality. In a logistic analysis taking in consideration clinical, radiological and functional findings, only baseline FVC and FVC change after 6 months resulted significant predictors of mortality. CONCLUSIONS: Functional evaluation at the baseline and during follow-up is more relevant than HC score for the prognosis of patients with definite and possible IPF. [ABSTRACT FROM AUTHOR]

Background:In pancreatic ductal adenocarcinoma (PDAC), fractalkine receptor CX3CR1 contributes to perineural invasion (PNI). We investigated whether CX3CR1 expression occurs early in PDAC and correlates with tumour features other than PNI.Methods:We studied CX3CR1 and CX3CL1 expression by immunohistochemistry in 104 human PDAC and coexisting Pancreatic Intraepithelial Neoplasia (PanIN), and in PdxCre/LSL-KrasG12D mouse model of PDAC. CX3CR1 expression in vitro was studied by a spheroid model, and in vivo by syngenic mouse graft of tumour cells.Results:In total, 56 (53.9%) PDAC expressed CX3CR1, 70 (67.3%) CX3CL1, and 45 (43.3%) both. CX3CR1 expression was independently associated with tumour glandular differentiation (P=0.005) and PNI (P=0.01). Pancreatic Intraepithelial Neoplasias were more frequently CX3CR1+ (80.3%, P<0.001) and CX3CL1+ (86.8%, P=0.002) than matched cancers. The survival of PDAC patients was better in those with CX3CR1+ tumour (P=0.05). Mouse PanINs were also CX3CR1+ and -CL1+. In vitro, cytokines significantly increased CX3CL1 but not CX3CR1 expression. Differently, CX3CR1 was upregulated in tumour spheroids, and in vivo only in well-differentiated tumours.Conclusion:Tumour differentiation, rather than inflammatory signalling, modulates CX3CR1 expression in PanINs and PDAC. CX3CR1 expression pattern suggests its early involvement in PDAC progression, outlining a potential target for interfering with the PanIN transition to invasive cancer. [ABSTRACT FROM AUTHOR]

As the oldest known magnetic material, magnetite (Fe3O4) has fascinated mankind for millennia. As the first oxide in which a relationship between electrical conductivity and fluctuating/localized electronic order was shown, magnetite represents a model system for understanding correlated oxides in general. Nevertheless, the exact mechanism of the insulator-metal, or Verwey, transition has long remained inaccessible. Recently, three-Fe-site lattice distortions called trimerons were identified as the characteristic building blocks of the low-temperature insulating electronically ordered phase. Here we investigate the Verwey transition with pump-probe X-ray diffraction and optical reflectivity techniques, and show how trimerons become mobile across the insulator-metal transition. We find this to be a two-step process. After an initial 300 fs destruction of individual trimerons, phase separation occurs on a 1.5±0.2 ps timescale to yield residual insulating and metallic regions. This work establishes the speed limit for switching in future oxide electronics. [ABSTRACT FROM AUTHOR]

Background: Long-term follow-up of diisocyanate-induced occupational asthma has been occasionally reported. Methods: We studied the outcome of toluene diisocyanate (TDI)-induced asthma in 46 patients at diagnosis and after a follow-up of 11 ± 3.6 years. Symptoms, anti-asthma therapy, forced expiratory volume in 1 s (FEV1) and bronchial hyperresponsiveness to methacholine were assessed. Results: A significant improvement in FEV1 (% predicted) and PD20FEV1 methacholine was observed at follow-up in comparison with diagnosis. Anti-asthma treatment was performed by 42% of patients at diagnosis and by 70% at follow-up. At the time of follow-up, 32 subjects had been removed from exposure for 6.0 ± 6.9 years, whereas 14 subjects continued to work with reduced exposure to TDI. There was a significant reduction in the prevalence of attacks of shortness of breath and dyspnoea at follow-up, but only in unexposed patients. PD20FEV1 was significantly improved only in patients with a lower FEV1 at diagnosis and in those who have ceased work. Logistic regression analysis, using different models with some independent variables, showed that there were no significant determinants of improvement in FEV1 at follow-up, while a shorter duration of symptoms before diagnosis was a significant predictor of improvement in PD20FEV1 at follow-up. Conclusions: Asthma-like symptoms, bronchial hyperresponsiveness and airway obstruction improved, but did not normalize, after a long-term follow-up with cessation or reduction in TDI exposure, mainly in subjects with an early diagnosis of occupational asthma and in patients with a lower baseline FEV1 no longer exposed to TDI. Copyright © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background: Few data are reported on the effects of a reduction of exposure to specific sensitizers in occupational asthma (OA). The objective of this study was to evaluate the clinical outcome of subjects with OA, comparing the effect of a reduction with that of the persistence or cessation of occupational exposure to the specific sensitizer. Subjects and Methods: Forty-one subjects with OA due to different sensitizers were diagnosed via a specific inhalation challenge. After a follow-up interval of 3.5 years, subjects were reexamined by clinical assessment, bronchial hyperresponsiveness (BH) and induced sputum. Results: At follow-up, subjects who had reduced occupational exposure (n = 22) showed a significant improvement in BH and a nonsignificant improvement in sputum eosinophilia (from 5.3 to 1.1%, n.s.), while subjects still exposed (n = 10) showed a significant decrease in FEV1. Subjects who ceased work (n = 9) showed a trend of improvement in BH and sputum eosinophilia. Logistic analysis showed that the major determinant of improvement in BH at follow-up was the severity of BH at diagnosis, with a minimal contribution from the duration of exposure and treatment with inhaled corticosteroids during follow-up; reduction of work exposure did not enter into any model. Conclusion: The reduction of occupational exposure could not be considered to be as effective as work cessation, which remained the best treatment for OA. However, it was not associated with a deterioration of FEV1 as observed in subjects with persistent exposure. Copyright © 2011 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background. Oxidative stress plays a role in the pathogenesis of many chronic inflammatory lung diseases. Exhaled breath condensate (EBC) collection is a noninvasive method to investigate pulmonary oxidative stress biomarkers such as malondialdehyde (MDA). Subjects and Methods. We measured MDA levels in EBC in a large number of patients (N = 194) with respiratory diseases: asthma (N = 64), bronchiectasis (BE, N = 19), chronic obstructive pulmonary disease (COPD, N = 73), idiopathic pulmonary fibrosis (IPF, N = 38). Fourteen healthy nonsmoking subjects were included as controls. Results. Excluding IPF subjects, MDA levels were significantly higher in all disease groups than in control group. MDA was significantly higher in COPD than asthmatic and BE subjects. Among asthmatics, corticosteroids-treated subjects had lower MDA levels than untreated subjects. COPD subjects showed an inverse correlation between MDA concentrations and FEV1 % (rho: -0.24, P < .05). Conclusions. EBC-MDA is increased in subjects with chronic airway disorders, particularly in COPD, and it is related to FEV1 reduction. [ABSTRACT FROM AUTHOR]

OBJECTIVE: T-helper 1 (Th1) and Th17 lymphocytes are involved in experimental autoimmune encephalomyelitis, the model of multiple sclerosis (MS). We characterized the Th1/Th17 cell populations in peripheral blood (PB), their interferon (IFN) receptor expression sensitivity to IFN-beta in MS patients. METHODS: In 30 untreated patients with active MS (AMS) and 32 with inactive MS (IMS), and in 22 healthy subjects, we measured intracellular cytokine expression, interleukin-17-producing myelin basic protein-stimulated PB lymphocytes, surface IFN type I receptor chain1 (IFN-alphaR1) expression, IFN-beta-dependent signal transducer and activator of transcription 1 (STAT1) phosphorylation, and apoptosis of anti-CD3 monoclonal antibody-stimulated PB lymphocytes. RESULTS: Th17 cell percentage increased around sevenfold in AMS compared with IMS or healthy subjects, but there was no change in Th1 cells. Th17 cells in AMS were myelin basic protein specific. The longitudinal follow-up of 18 MS patients shifting between AMS and IMS showed that the percentage of Th17 but not Th1 cells always increased in AMS. IFN-alphaR1 expression, IFN-beta-induced STAT1 activation, and apoptosis were significantly greater in Th17 than Th1 cells. IFN-alphaR1 expression and IFN-beta-dependent STAT1 activation progressively increased in vitro with a highly significant positive correlation only in developing Th17 but not in Th0 or Th1 cells. INTERPRETATION: Evidence that an expansion of peripheral Th17 cells, a Th subset that can infiltrate brain parenchyma and damage cells, is associated with disease activity in MS. The greater IFN-alphaR1 level expressed by Th17 compared with Th1 cells might make them a selective target for IFN-beta therapy. Ann Neurol 2009;65:499-509. [ABSTRACT FROM AUTHOR]

Background and aim: Mesenchymal stem cells from bone marrow (MSCs) may have the potential to differentiate in vitro and in vivo into hepatocytes. We investigated whether transplanted human MSCs (hMSCs) may engraft the liver of non-obese diabetic severe combined immuno-deficient (NOD/SCID) mice and differentiate into cells of hepatic lineage. Methods: Ex vivo expanded, highly purified and functionally active hMSCs from bone marrow were transplanted (caudal vein) in sublethally irradiated NOD/SCID mice that were either exposed or not to acute liver injury or submitted to a protocol of chronic injury (single or chronic intraperitoneal injection of CCI4, respectively). Chimeric livers were analysed for expression of human transcripts and antigens. Results: Liver engraftment of cells of human origin was very low in normal and acutely injured NOD/SCID mice with significantly higher numbers found in chronically injured livers. However, hepatocellular differentiation was relatively rare, limited to a low number of cells (ranging from less than 0.1% to 0.23%) as confirmed by very low or not detectable levels of human transcripts for α-fetoprotein, CK1B, CK19 and albumin in either normal or injured livers. Finally, a significant number of cells of human origin exhibited a myofibroblast-like morphology. Conclusions: Transplanted hMSCs have the potential to migrate into normal and injured liver parenchyma, particularly under conditions of chronic injury, but differentiation into hepatocyte-like cells is a rare event and pro-fibrogenic potential of hMSC transplant should be not under-evaluated. [ABSTRACT FROM AUTHOR]

Background. The renal minimal lesion disease induced in rats by adriamycin (ADR) is generally thought to be consequent to a direct cytotoxic effect of this drug on glomerular epithelial cells. Only recently an altered synthesis of mediators, including reactive oxygen species and monocyte-macrophage cytokines, has been hypothesized. Methods. A mouse strain (nude) bearing a congenital thymic aplasia is a suitable experimental animal to evaluate the role of immune reactions in the development of ADR nephropathy, provided mouse susceptibility to its toxic effect. Therefore, experimental mice were divided into three groups (G) each receiving adriamycin 7.5 mg/kg b.w.: GA (15 heterozygous nu/O mice with normal immune system); GB (15 homozygous nu/nu athymic mice); GC (15 homozygous nu/nu mice which were also splenectomized, irradiated, and treated with anti-asialo Gml antibody to abolish NK and decrease macrophage activity). All animals were maintained under pathogen-free conditions. Urinary proteins, albumin and TNF-α excretion were measured. Results. After 14 days the proteinuria was 43.8± 1.7 μg/min in GA, 30.2±2.9 μg/min in GB (P<0.05) and 12.2±2.8 μg/min in GC (GA vs GC, P<0.0001; GB vs GC, P<0.05). Albuminuria gave a similar profile. TNF-α urinary excretion was significantly higher in GA (17.3±3.2 mU/min) than in GB (5±0.6 mU/min, P<0.001) and GC (3.2±0.9 mU/min, P<0.001). A significant correlation was found in GA between urinary TNF-α and protein losses (r2=0.63 P<0.0001). Kidney tissue homogenates failed to show in each experimental group any evidence of mRNA encoding for TNF-α, which was detectable in peripheral mononuclear cells from GA and GB, but undetectable in GC mice. Segmental effacements of glomerular epithelial cell foot process were observed by electron-microscopy in GA only, while they were minimal in GB and absent in GC. Iron colloidal staining for anionic sites on frozen sections always showed a normal pattern. Conclusions. Nude mice bearing cellular immunity deficiency are protected from proteinuria following ADR toxicity. An impaired synthesis and release of lymphomonocyte mediators including TNF-α could be envisaged. [ABSTRACT FROM PUBLISHER]

Abstract Background Tuberculosis (TB) surveillance systems have some pitfalls outside of a National Tuberculosis Program and lack of efficient surveillance hampers accurate epidemiological quantification of TB burden. In the present study we assessed the quality of surveillance at the University Hospital in Pisa (UHP), Italy, and TB incidence rates over a ten year period (1999–2008). Methods Assessment of underreporting was done by record-linkage from two sources: databases of TB diagnoses performed in the UHP and the Italian Infectious Disease Surveillance (IIDS) system. Two different databases were examined: a) TB diagnoses reported in the Hospital Discharge Records (HDR) from three Units of UHP (Respiratory Pathophysiology, Pulmonology and Infectious Diseases Units) (TB database A); b) TB diagnoses reported in HDR of all Units of UHP plus TB positive cases obtained by the Laboratory Register (LR) of UHP (TB database B). For the TB database A, the accuracy of TB diagnosis in HDR was assessed by direct examination of the Clinical Record Forms of the cases. For the TB database B, clinical and population data were described, as well as the trend of incidence and underreporting over 10 yrs. Results In the first study 293 patients were found: 80 patients (27%) with a confirmed TB diagnosis were underreported, 39 of them were microbiologically confirmed. Underreporting was related to age (Reported vs Non Reported, mean age: 49.27 ± 20 vs 55 ± 19, p vs negative cases 18.7 vs 81.2%, p = 0.001), microbiological confirmation (49% vs 51%, p vs non-cavitary cases: 12.5 vs 87.5%, p = 0.001) but not to nationality. In the second study, 666 patients were found. Mean underreporting rate was 69.4% and decreased over time (68% in 1999, 48% in 2008). Newly diagnosed TB cases were also found to decrease in number whereas immigration rate increased. Underreporting was related to nationality (Immigrants vs Italians: 18% vs 68%, p vs 75%, p vs Day Hospital: 70% vs 16%, p vs in the following position: 39,5% vs 45% p Conclusions TB is underreported in Pisa, particularly in older patients and those without microbiological confirmation. The TB code in first position of HDR seems fairly accurate in confirming TB diagnosis.

Abstract Background Several studies demonstrated that epidermal growth factor receptor (EGFR) gene copy number (GCN) correlates to the response to tyrosine kinase inhibitors in non small cell lung cancer (NSCLC) and to anti-EGFR monoclonal antibodies (MoAbs) in metastatic colorectal cancer (CRC). In the presence of lung nodules, cytology is often the only possible diagnostic approach. Chromogenic in situ hybridization (CISH) is an alternative technique to fluorescence in situ hybridization (FISH), but its feasibility in detecting EGFR GCN in cell blocks from fine-needle aspiration cytology (FNAC) of lung nodules has not yet been established. Methods We evaluated the feasibility of CISH on 33 FNAC from 20 primary NSCLC (5 squamous carcinomas, 8 large cell carcinomas and 7 adenocarcinomas) and 13 lung metastases from CRC. Results Of the 33 FNAC analyzed by CISH, 27 (82%) presented a balanced increase in EGFR gene and chromosome 7 number: 10 cases (30%) showed a low polysomy, 15 (45%) a high polysomy and 2 (6%) NSCLC were amplified. No significant differences between NSCLC and CRC lung metastases were found in relation to disomic or polysomic status. In addition, no correlation between EGFR GCN and EGFR immunohistochemical overexpression was found. Furthermore, we compared CISH results with those obtained by FISH on the same samples and we found 97% overall agreement between the two assays (k = 0.78, p < 0.0001). Two cases were amplified with both assays, whereas 1 case of NSCLC was amplified by FISH only. CISH sensitivity was 67%, the specificity and positive predictive value (PPV) was 100%, and the negative predictive value (NPV) was 97%. Conclusions Our study shows that CISH is a valid method to detect EGFR GCN in cell blocks from FNAC of primary NSCLC or metastatic CRC to the lung.

Abstract Background Aberrant signaling by ErbB-2 (HER 2, Neu), a member of the human Epidermal Growth Factor (EGF) receptor family, is associated with an aggressive clinical behaviour of carcinomas, particularly breast tumors. Antibodies targeting the ErbB-2 pathway are a preferred therapeutic option for patients with advanced breast cancer, but a worldwide deficit in the manufacturing capacities of mammalian cell bioreactors is foreseen. Methods Herein, we describe a multi-platform approach for the production of recombinant Single chain Fragments of antibody variable regions (ScFvs) to ErbB-2 that involves their functional expression in (a) bacteria, (b) transient as well as stable transgenic tobacco plants, and (c) a newly developed cell-free transcription-translation system. Results An ScFv (ScFv800E6) was selected by cloning immunoglobulin sequences from murine hybridomas, and was expressed and fully functional in all the expression platforms, thereby representing the first ScFv to ErbB-2 produced in hosts other than bacteria and yeast. ScFv800E6 was optimized with respect to redox synthesis conditions. Different tags were introduced flanking the ScFv800E6 backbone, with and without spacer arms, including a novel Strep II tag that outperforms conventional streptavidin-based detection systems. ScFv800E6 was resistant to standard chemical radiolabeling procedures (i.e. Chloramine T), displayed a binding ability extremely similar to that of the parental monovalent Fab' fragment, as well as a flow cytometry performance and an equilibrium binding affinity (Ka approximately 2 × 108 M-1) only slightly lower than those of the parental bivalent antibody, suggesting that its binding site is conserved as compared to that of the parental antibody molecule. ScFv800E6 was found to be compatible with routine reagents for immunohistochemical staining. Conclusion ScFv800E6 is a useful reagent for in vitro biochemical and immunodiagnostic applications in oncology, and a candidate for future in vivo studies.

We present equilibrium and out-of-equilibrium studies of the Verwey transition in magnetite. In the equilibrium optical conductivity, we find a steplike change at the phase transition for photon energies below about 2 eV. The possibility of triggering a nonequilibrium transient metallic state in insulating magnetite by photo excitation was recently demonstrated by an x-ray study. Here we report a full characterization of the optical properties in the visible frequency range across the nonequilibrium phase transition. Our analysis of the spectral features is based on a detailed description of the equilibrium properties. The out-of-equilibrium optical data bear the initial electronic response associated to localized photoexcitation, the occurrence of phase separation, and the transition to a transient metallic phase for excitation density larger than a critical value. This allows us to identify the electronic nature of the transient state, to unveil the phase transition dynamics, and to study the consequences of phase separation on the reflectivity, suggesting a spectroscopic feature that may be generally linked to out-of-equilibrium phase separation. [ABSTRACT FROM AUTHOR]