Epigenomic alterations define lethal CIMP-positive ependymomas of infancy.

Ependymomas are common childhood brain tumours that occur throughout the nervous system, but are most common in the paediatric hindbrain. Current standard therapy comprises surgery and radiation, but not cytotoxic chemotherapy as it does not further increase survival. Whole-genome and whole-exome sequencing of 47 hindbrain ependymomas reveals an extremely low mutation rate, and zero significant recurrent somatic single nucleotide variants. Although devoid of recurrent single nucleotide variants and focal copy number aberrations, poor-prognosis hindbrain ependymomas exhibit a CpG island methylator phenotype. Transcriptional silencing driven by CpG methylation converges exclusively on targets of the Polycomb repressive complex 2 which represses expression of differentiation genes through trimethylation of H3K27. CpG island methylator phenotype-positive hindbrain ependymomas are responsive to clinical drugs that target either DNA or H3K27 methylation both in vitro and in vivo. We conclude that epigenetic modifiers are the first rational therapeutic candidates for this deadly malignancy, which is epigenetically deregulated but genetically bland.

Nerve ablation after bronchial thermoplasty and sustained improvement in severe asthma.

BACKGROUND: Bronchial thermoplasty (BT) is a non-pharmacological intervention for severe asthma whose mechanism of action is not completely explained by a reduction of airway smooth muscle (ASM). In this study we analyzed the effect of BT on nerve fibers and inflammatory components in the bronchial mucosa at 1 year. METHODS: Endobronchial biopsies were obtained from 12 subjects (mean age 47 ± 11.3 years, 50% male) with severe asthma. Biopsies were performed at baseline (T0) and after 1 (T1), 2 (T2) and 12 (T12) months post-BT, and studied with immunocytochemistry and microscopy methods. Clinical data including Asthma Quality of Life Questionnaire (AQLQ) and Asthma Control Questionnaire (ACQ) scores, exacerbations, hospitalizations, oral corticosteroids use were also collected at the same time points. RESULTS: A statistically significant reduction at T1, T2 and T12 of nerve fibers was observed in the submucosa and in ASM compared to T0. Among inflammatory cells, only CD68 showed significant changes at all time points. Improvement of all clinical outcomes was documented and persisted at the end of follow up. CONCLUSIONS: A reduction of nerve fibers in epithelium and in ASM occurs earlier and persists at one year after BT. We propose that nerve ablation may contribute to mediate the beneficial effects of BT in severe asthma. TRIAL REGISTRATION: Registered on April 2, 2013 at ClinicalTrials.gov Identifier: NCT01839591 .

Training and practice in bronchoscopy a national survey in Italy.

BACKGROUND AND AIM: Bronchoscopy is performed in a variety of different settings in Italy. The surveys conducted so far have highlighted the heterogeneity of the procedures and the frequent inability to adhere to the guidelines. The aim of this survey was to analyse procedures, training, and opinions of Italian respiratory physicians performing interventional bronchology in the clinical practice. METHODS: The study was conducted retrospectively on 300 pulmonologists. From January to June 2008, these were invited to participate in an email survey to be sent out monthly to each participant for four consecutive months. RESULTS: Two hundred and one respiratory physicians took part in the study, most of whom (83.5%) work in either Pulmonology or Interventional Pulmonology Units. The year before the survey, 21.2% of the participants had performed fewer than 100 examinations, 42.3% 100 to 300, and 36.6% more than 300 bronchoscopies; 53.9% were familiar with the international guidelines on the topic. Among the responders, 34.1% had received less than 6 months training, 55.3% considered further training in rigid bronchoscopy, laser procedures and thoracoscopy, invaluable for their professional activity. Adequate training for transbronchial needle aspirates, was reported by 49.6% of respondents. CONCLUSIONS: Our data show that interventional bronchoscopy procedures are regularly performed according to current recommendations by over half of the Italian Pulmonologists participating in our survey. The need for more comprehensive basic education and training was put forward by the majority of physicians.

Acute respiratory distress syndrome: an undercover antisynthetase syndrome: a case report and a review of the literature.

We present the case of a 48-year-old male with an acute respiratory distress syndrome which later proved to be an unexpected and initial manifestation of antisynthetase syndrome. Recognising this as a rare combination of an acute respiratory failure and a connective tissue disease in a previously asymptomatic subject is possible only by means of diagnostic exclusion. Based on similar case reports, the only way to reverse the disease and minimise the sequelae is to begin long-term immunosuppressive therapy as soon as possible once the diagnosis has been made. A review of similar cases with antibody anti-Jo-1 is presented with the aim of providing clinicians with useful indications for promptly recognising this poorly-defined and life-threatening emergency.

The potential of matrix-assisted laser desorption/ionization mass spectrometry in the quality control of water buffalo mozzarella cheese.

Adulteration by addition of bovine milk to water buffalo milk employed for mozzarella cheese production is often observed. Water buffalo milk and mozzarella cheese were analysed by matrix-assisted laser desorption/ionization mass spectrometry in order to achieve their rapid and accurate characterization and to evaluate possible fraudulence in mozzarella cheese production.

Stem cell characteristics in glioblastoma are maintained by the ecto-nucleotidase E-NPP1.

Glioblastomas are highly aggressive brain tumours and are characterised by substantial cellular heterogeneity within a single tumour. A sub-population of glioblastoma stem-like cells (GSCs) that shares properties with neural precursor cells has been described, exhibiting resistance to therapy and therefore being considered responsible for the high recurrence rate in glioblastoma. To elucidate the underlying cellular processes we investigated the role of phosphatases in the GSC phenotype, using an in vitro phosphatome-wide RNA interference screen. We identified a set of genes, the knockdown of which induces a significant decrease in the glioma stem cell marker CD133, indicating a role in the glioblastoma stem-like phenotype. Among these genes, the ecto-nucleotidase ENPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) was found to be highly expressed in GSCs compared with normal brain and neural stem cells. Knockdown of ENPP1 in cultured GSCs resulted in an overall downregulation of stem cell-associated genes, induction of differentiation into astrocytic cell lineage, impairment of sphere formation, in addition to increased cell death, accumulation of cells in G1/G0 cell cycle phase and sensitisation to chemotherapeutic treatment. Genome-wide gene expression analysis and nucleoside and nucleotide profiling revealed that knockdown of ENPP1 affects purine and pyrimidine metabolism, suggesting a link between ENPP1 expression and a balanced nucleoside-nucleotide pool in GSCs. The phenotypic changes in E-NPP1-deficient GSCs are assumed to be a consequence of decreased transcriptional function of E2F1. Together, these results reveal that E-NPP1, by acting upstream of E2F1, is indispensable for the maintenance of GSCs in vitro and hence required to keep GSCs in an undifferentiated, proliferative state.

A simple and reliable liquid chromatography-tandem mass spectrometry method for the determination of aflatoxin M1 in milk.

A new chromatographic method is proposed for the analysis of aflatoxin M(1) in milk. The method is based on liquid-liquid extraction followed by LC-MS/MS analysis. Liquid-liquid extraction (LLE) is performed on the defatted milk plus sodium chloride by using ethyl acetate as an extraction solvent. Accuracy and precision were evaluated at the LOQ (15 ng kg(-1)) spiked sample as well as with three other different naturally contaminated reference materials. The mean overall recovery (n = 24) was 95% with a confidence interval of 1.9% and a CV% of 4.5%. The performance of the proposed method was compared with that of the Official ISO Method based on the use of immunoaffinity chromatography columns (IAC): LLE protocol could be considered a valid alternative to the LC-IAC. In general it showed better accuracy with lower data dispersion. Moreover, the sample preparation is very simple and straightforward, potentially being applicable as a high-throughput method which, on account of its simplicity and low cost, may be applied to the analysis of a large number of samples in the occasion of outbreaks of large-scale contamination.

Simultaneous determination of lincomycin and five macrolide antibiotic residues in honey by liquid chromatography coupled to electrospray ionization mass spectrometry (LC-MS/MS).

A sensitive and specific method based on high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), for the simultaneous determination of lincomycin and five macrolide antibiotics in honey, was developed and validated. The analytes were extracted with Tris buffer 0.1 M, pH 10.5, and cleaned-up by a single solid-phase extraction step on OASIS HLB column. The chromatographic separation of analytes was performed on a Synergi Hydro-RP reversed-phase column using a gradient programme of aqueous 0.01 M ammonium acetate, pH 3.5, and acetonitrile as the mobile phase, at a flow rate 0.25 ml min-1. The detection of analytes was achieved by positive ionization electrospray in multiple reaction-monitoring mode. Two characteristic transitions were monitored for each substance. The following analytical parameters were validated according to the guidelines laid down by European Commission Decision 2002/657/EC (European Commission 2002): linearity, specificity, decision limit (CCalpha), detection capability (CCbeta), repeatability, within-laboratory reproducibility, recovery and ruggedness.

Detection of veterinary drugs in foodstuffs using gel permeation.

The applicability of gel permeation chromatography (GPC) to the purification in foodstuffs in the detection of three classes of drugs, sulfonamides, nitrofurans, and growth promoters, is described. This technique has already found a wide application in the routine analysis of pesticides in milk, eggs and grease but to our knowledge there is no published work on the use of GPC for purifying animal feedstuffs for the detection of the above-mentioned classes of drugs. Three main advantages characterize this method: the simultaneous determination of a wide spectrum of drugs; good performance of the purification system; and high recoveries. The method was tested for a determination limit of 1 ppm in each compound. Average recoveries of spiked samples were: 99% for sulfonamides; 98% for nitrofurans; and 100% for carbadox [methyl-3-(2-quinoxalinylmethylene) carbazate-1,4-dioxide] and olaquindox [N-(2-hydroxyethyl)-3-methyl-2-quinoxalinecarboxamide-1,4-dioxide] . The method was also tested for linearity over the range 1-100 ppm and gave r values between 0.9998 and 0.9999 for each drug. Analyses were performed by HPLC, either using a diode array or windowplot-system detector. The described method is quick and simple, which is very useful in routine analysis.

HCV, HIV, HBV and HDV infections in intravenous drug addicts.

Hepatitis viruses and the acquired immunodeficiency viruses often infect intravenous drug addicts (IVDAs). Our study includes 255 IVDAs (26 females and 229 males, aged 20-35 years) from Cagliari. Of 255 subjects examined, 207 (81.1%) were positive for anti-HCV and 84 (32.9%) for anti-HIV. Nineteen (7.4%) subjects were HBsAg carriers, and 12 of these (63%) had an HDV superinfection. Markers of previous HBV infections were tested in 223 cases and 137 (61.4%) were found positive; of these 14 (10.2%) also had HDV infection. Of the 223 drug addicts examined for all infection markers, 18 (8%) were negative to all markers, 46 (20.6%) were positive to only one, 89 (39.9%) were positive to two, 64 (28.7%) to three and 6 (2.6%) were positive to all. Subjects with a single infection were significantly fewer than those with multiple infections. The correlations studied among the various markers did not point out any statistically significant associations. Even so, a previous HBV infection was more common while active HBV/HDV infections were less common among subjects with anti-HCV; HDV infection was more common among HIV-positive subjects. In HBsAg carriers neither HBV-DNA nor HCV-RNA was detected; HCV-RNA was found more frequently in anti-HIV positive subjects than in subjects with the anti-HCV isolate.

Sexual behaviour and multiple infections in drug abusers.

We have studied the prevalence and the serological profile of HBV, HCV, HDV and HIV infections in 137 Italian subjects addicted to the intravenous use of heroine and correlated the virological findings with sexual behaviour. HBV and HCV viremia were also measured in 114 patients. Anti-HCV was detected in 81% of the addicts, and one or more markers of HBV infection were detected in 62.8% (4.4% were carriers of HBsAg, 58.4% had evidence of past HBV infection and 13.1% of the latter also had HDV markers). Anti-HIV was positive in 23.4%; 26% of those positive for anti-HCV and 4.6% of those positive for HBV markers had no other viral marker: none had only anti-HIV. HBV-DNA was negative in the carriers of HBsAg, and HCV-RNA was not detected in any of the HBsAg carriers who also had circulating anti-HCV. Overall, 34% of the anti-HCV positive addicts had HCV-RNA in their blood. The prevalence of the virus infection correlated with the duration of drug addiction but not with sexual behaviour, and sexual behaviour did not influence the acquisition of any virus. HCV infection was most frequent and probably the first infection to occur, but exposure to HBV was also common despite a low rate of HBsAg carriage. The prevalence of HDV infection was high (50%) in the HBsAg carriers, while the overall prevalence of HIV was lower (23%) than expected. Lack of HBV-DNA and HCV-RNA in carriers of HBV with anti-HCV in serum may indicate that HBV and HCV mutually inhibit their own replication.

Human immunodeficiency virus infection in multi-transfused patients with thalassaemia major.

We investigated the incidence, clinical and immunological characteristics of human immuno-deficiency virus (HIV) infection in a group of multi-transfused patients with thalassaemia major who were exposed to transfusion-associated HIV infection. Seropositivity to HIV by Western blot and immunofluorescence analysis was detected in 26 out of 590 patients. At a follow up 21-40 months later, none of these seropositive patients had developed acquired immuno-deficiency syndrome (AIDS), and six manifested the AIDS related complex (ARC). ARC was unusually mild and consisted of moderate laterocervical and submandibular lymph node enlargement associated with hypergammaglobulinaemia and a reduced CD4/CD8 ratio resulting from the decreased number of CD4 lymphocytes. These findings suggest that multi-transfused patients with thalassaemia major are relatively resistant to the development of severe manifestations of HIV infection, presumably because their immune status is relatively better preserved than that of other infected populations. Longer follow up is, however, necessary to determine whether the incidence of AIDS will be lower in this population or whether overt AIDS merely takes longer to develop.