Inter-laboratory validation of PCR-based HPV detection in pathology specimens

The detection and typing of human papilloma virus (HPV) in pathology specimens is gaining increasingly in importance. In the context of the initiative for quality assurance in pathology (QuIP) of the German Society of Pathology and the Professional Association of German Pathologists, four panel laboratories with experience and expertise in polymerase chain reaction (PCR)-based HPV detection were selected to establish an inter-laboratory trial. In a first step, these laboratories performed an internal testing of their own methodologies, which comprised DNA sequencing, multiplex nested PCR and hybridization techniques. Material from 39 samples including paraffin sections and DNA preparations of tissues and plasmids were evaluated by each panel institute according to their own protocols. Despite the different methodologies, a high degree of inter-laboratory reliability was achieved. In this report, we summarise the results. Pretested specimens are available for the external trail and can be ordered from the steering institute via provitro GmbH Berlin (). Supplementary data are online available at (rubric "Forschung"), which includes a web-based photo gallery of HPV-associated lesions and their potential association with specific virus types. The initiative is intended to foster the quality assurance of molecular HPV analysis in pathology and its correlation with morphological changes.     

Genetic influences on mannan-binding lectin (MBL) and mannan-binding lectin associated serine protease-2 (MASP-2) activity

The lectin pathway of the complement system is activated when Mannan-binding lectin (MBL) in complex with MASP-2 binds microorganisms. Polymorphisms in both genes are responsible for low serum levels, which associate with increased risk of infection and autoimmune disease. The present study includes 1215 MBL measurements and 1214 MASP-2 activity measurements in healthy Danish adult twins. Total MASP-2 activity was estimated by C4 cleaving activity of samples diluted in an excess of MBL. Twin-twin correlations were higher in monozygotic (MZ) than in dizygotic (DZ) twins for both traits. Heritabilites of MBL levels and MASP-2 activity were estimated using structural equation modeling allowing assessment of the contribution of common genes affecting both traits. The estimated heritability was 0.77 [95% CI 0.64;0.91] for MBL levels and 0.75 [95% CI 0.59;0.81] for MASP-2 activity with the presence of additive genetic factors, shared environmental factors, and non-shared environmental factors. The genetic correlation, i.e., common genetic factors affecting MBL and MASP-2 activity was estimated to r(g) = 0.34 [0.25;0.42]. The data indicate a strong genetic influence for the serum levels of MBL and for MASP-2 activity with a significant genetic correlation between the two traits.     

The natural history of vestibular schwannoma growth—prospective 40-year data from an unselected national cohort

BackgroundOptimal management of vestibular schwannoma (VS) is still debated and thus international consensus has not been achieved. Treatment options are observation, radiotherapy, and surgery. Knowledge on the natural history of tumor growth is essential for choice of treatment modality. The aim is to present intra-/extrameatal tumor growth and management data from a prospective, unselected national cohort of patients diagnosed with VS during the period 1976–2015.MethodsSince 1976, all data from patients diagnosed with sporadic VS in Denmark have been referred to our national treatment center, where they have been entered prospectively into the national database. Data on tumor localization, growth, and treatment were retrieved. Growth definition: >2 mm by linear measurement, in accordance with the Tokyo 2001 consensus-meeting recommendations.Results3637 cases of VS were diagnosed, in which 1304 patients had surgery and 21 received radiotherapy post diagnosis. 2312 patients were observed with mean follow-up of 7.33 years. Of these, 434(19%; 102 intra-and 332 extrameatal tumors) changed to active treatment during the observation period due to tumor growth. 5 years after diagnosis, 21% of the intrameatal tumors exhibited growth during observation, whereas 37% of extrameatal tumors had grown, increasing to 25% intrameatal and 42% extrameatal after 10 years. Following growth, the intrameatal tumors were mostly observed further and the extrameatal mostly underwent surgery. Tumor growth occurred mainly within the first 5 years post diagnosis.ConclusionThis natural history study documents the growth occurrence of both intra-and extrameatal VS during the first 12 years after diagnosis and should be used in patient counseling, management, and treatment decision making.     

An NMR-based metabonomic investigation on effects of milk and meat protein diets given to 8-year-old boys

The objective of the study was to investigate the ability of an NMR-based metabonomic approach, applied to biofluids, to explore and identify overall exogenous and endogenous biochemical effects of a short-time high intake of milk protein or meat protein given to prepubertal children, the aim being to compare relative differences and not an absolute quantification. A total of twenty-four 8-year-old boys were asked to take 53 g protein as milk (n 12) or meat daily (n 12). At baseline and after 7 d, urine and serum samples were collected and high-resolution 1H NMR spectra were acquired on these using a 800 MHz spectrometer. The milk diet reduced the urinary excretion of hippurate, while the meat diet increased the urinary excretion of creatine, histidine and urea. The NMR measurements on serum revealed minor changes in the lipid profile, which most probably should be ascribed to an increase in the content of SCFA in the blood after consumption of the milk diet. The meat diet had no effect on the metabolic profile of serum. The study for the first time demonstrates the capability of proton NMR-based metabonomics to identify the overall biochemical effects of consumption of different animal proteins. The urine metabolite profile is more susceptible to perturbations as a result of short diet interventions than the serum metabolite profile. The milk diet-induced reduction in urinary excretion of hippurate suggests alterations in gut microflora, which may be useful information for further studies elucidating the effects of bioactive components in milk.     

Investigation of the detoxification mechanism of formaldehyde-treated tetanus toxin

The tetanus vaccine is based on the extremely potent tetanus neurotoxin (TeNT), which is converted by treatment with formaldehyde and lysine into the non-toxic, but still immunogenic tetanus toxoid (TTd). This formaldehyde-induced detoxification, which to a large extend determines the quality and properties of the vaccine component, occurs through partly unknown chemical modifications of the toxin. The aim of this study was to gain knowledge of the detoxification mechanism in the generation of the tetanus vaccine. Two approaches were chosen: (i) the effect of changes in the concentrations of lysine and formaldehyde in the detoxification process and (ii) characterisation of the chemically detoxified TTd. (i) We examined a number of TTd components that was produced by varying the concentrations of formaldehyde and lysine during the inactivation. Toxicity tests showed that the detoxification failed when the lysine or formaldehyde concentration was < or =1/5 or < or =1/10, respectively, of the standard level. Gel-electrophoretic analyses showed that inter-chain cross-linking was formaldehyde-dependent and, furthermore, revealed that inter-chain cross-linking was not the only requirement for the inactivation. In addition, the measurable amount of tyrosine correlated inversely with the degree of inter-chain cross-linking. (ii) To study the formaldehyde-induced chemical modifications, the TTd was investigated using protein chemical techniques in combination with mass spectrometry (MS). Using off-line liquid chromatography (LC)-MS, the most pronounced chemical modifications were characterised as unstable Schiff-bases (+12 Da) located on lysine residues and the N-termini of peptides throughout the molecule. Several arginine residues were also found with +12 Da modifications due to Schiff-base formation or as a consequence of degenerative fragmentation of lysine/formaldehyde adducts or cross-links during MS. A few tyrosine residues were similarly observed with a mass increase of 12 Da. Even though it cannot be ruled out that this is a residual mass of higher molecule adducts or cross-links to tyrosine, amino acid analysis and MS data indicated that the modification forms a ring structure from a carbon in the aromatic ring to the backbone N(alpha). In addition, several mono-varepsilon-methyllysines (+14 Da) were observed as a likely consequence of reductive methylation of the Schiff-bases. A substantial part (87%) of the known TeNT sequence, including the active site, was covered using the off-line LC-MS approach to investigate the tryptic digested TTd. In contrast to the results obtained from the gel-electrophoretic experiments, neither intra/inter-chain cross-links nor cross-links to external lysines were observed in the MS analysis. Instability of the cross-links during separation and/or MS is likely to explain their absence in the analyses. The biological relevance of the observed modifications is discussed in relation to 3D mapping analyses. Proposals for the TeNT detoxification are discussed, although no direct evidence for the exact mechanism could be obtained.     

The Implementation of Cognitive Therapy in STAR*D

The Sequenced Treatment Alternatives to Relieve Depression (STAR*D) project will provide symptomatic and functional outcome data to evaluate the theoretical principles and clinical beliefs that currently guide the treatment of nonpsychotic major depression. Cognitive Therapy (CT) for depression has been chosen as a switch or augmentation treatment for patients who have failed an adequate trial of the antidepressant citalopram. We describe the rationale, organization, and role of CT in STAR*D. We discuss the issues involved in developing and implementing CT in a large, multisite, effectiveness study: therapist selection, training, certification, quality assurance, and post-training supervision. We conclude with a discussion of the implications of our implementation procedures on interpreting the results of the STAR*D study.