Double-labelling immunohistochemistry for MGMT and a “cocktail” of non-tumourous elements is a reliable, quick and easy technique for inferring methylation status in glioblastomas and other primary brain tumours

BACKGROUND: Our aim was to develop a new protocol for MGMT immunohistochemistry with good agreement between observers and good correlation with molecular genetic tests of tumour methylation. We examined 40 primary brain tumours (30 glioblastomas and 10 oligodendroglial tumours) with our new technique, namely double-labelling immunohistochemistry for MGMT and a "cocktail" of non-tumour antigens (CD34, CD45 and CD68). We compared the results with single-labelling immunohistochemistry for MGMT and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA, a recognised molecular genetic technique which we applied as the gold-standard for the methylation status). RESULTS: Double-labelling immunohistochemistry for MGMT produced a visual separation of tumourous and non-tumourous elements on the same histological slide, making it quick and easy to determine whether tumour cell nuclei were MGMT-positive or MGMT-negative (and thereby infer the methylation status of the tumour). We found good agreement between observers (kappa 0.76) and within observer (kappa 0.84). Furthermore, double-labelling showed good specificity (80%), sensitivity (73.33%), positive predictive value (PPV, 83.33%) and negative predictive value (NPV, 68.75%) compared to MS-MLPA. Double-labelling was quicker and easier to assess than single-labelling and it outperformed quantitative computerised image analysis of MGMT single-labelling in terms of sensitivity, specificity, PPV and NPV. CONCLUSIONS: Double-labelling immunohistochemistry for MGMT and a cocktail of non-tumourous elements provides a "one look" method for determining whether tumour cell nuclei are MGMT-positive or MGMT-negative. This can be used to infer the methylation status of the tumour. There is good observer agreement and good specificity, sensitivity, PPV and NPV compared to a molecular gold-standard

Endothelial Cell Processing and Alternatively Spliced Transcripts of Factor VIII: Potential Implications for Coagulation Cascades and Pulmonary Hypertension

Background: Coagulation factor VIII (FVIII) deficiency leads to haemophilia A. Conversely, elevated plasma levels are a strong predictor of recurrent venous thromboemboli and pulmonary hypertension phenotypes in which in situ thromboses are implicated. Extrahepatic sources of plasma FVIII are implicated, but have remained elusive. Methodology/Principal Findings: Immunohistochemistry of normal human lung tissue, and confocal microscopy, flow cytometry, and ELISA quantification of conditioned media from normal primary endothelial cells were used to examine endothelial expression of FVIII and coexpression with von Willebrand Factor (vWF), which protects secreted FVIII heavy chain from rapid proteloysis. FVIII transcripts predicted from database mining were identified by rt-PCR and sequencing. FVIII mAb-reactive material was demonstrated in CD31+ endothelial cells in normal human lung tissue, and in primary pulmonary artery, pulmonary microvascular, and dermal microvascular endothelial cells. In pulmonary endothelial cells, this protein occasionally colocalized with vWF, centered on Weibel Palade bodies. Pulmonary artery and pulmonary microvascular endothelial cells secreted low levels of FVIII and vWF to conditioned media, and demonstrated cell surface expression of FVIII and vWF Ab–reacting proteins compared to an isotype control. Four endothelial splice isoforms were identified. Two utilize transcription start sites in alternate 59 exons within the int22h-1 repeat responsible for intron 2

Home and Online Management and Evaluation of Blood Pressure (HOME BP) using a digital intervention in poorly controlled hypertension: randomised controlled trial

Objective: The HOME BP (Home and Online Management and Evaluation of Blood Pressure) trial aimed to test a digital intervention for hypertension management in primary care by combining self-monitoring of blood pressure with guided self-management. Design: Unmasked randomised controlled trial with automated ascertainment of primary endpoint. Setting: 76 general practices in the United Kingdom. Participants: 622 people with treated but poorly controlled hypertension (>140/90 mm Hg) and access to the internet. Interventions: Participants were randomised by using a minimisation algorithm to self-monitoring of blood pressure with a digital intervention (305 participants) or usual care (routine hypertension care, with appointments and drug changes made at the discretion of the general practitioner; 317 participants). The digital intervention provided feedback of blood pressure results to patients and professionals with optional lifestyle advice and motivational support. Target blood pressure for hypertension, diabetes, and people aged 80 or older followed UK national guidelines. Main outcome measures: The primary outcome was the difference in systolic blood pressure (mean of second and third readings) after one year, adjusted for baseline blood pressure, blood pressure target, age, and practice, with multiple imputation for missing values. Results: After one year, data were available from 552 participants (88.6%) with imputation for the remaining 70 participants (11.4%). Mean blood pressure dropped from 151.7/86.4 to 138.4/80.2 mm Hg in the intervention group and from 151.6/85.3 to 141.8/79.8 mm Hg in the usual care group, giving a mean difference in systolic blood pressure of −3.4 mm Hg (95% confidence interval −6.1 to −0.8 mm Hg) and a mean difference in diastolic blood pressure of −0.5 mm Hg (−1.9 to 0.9 mm Hg). Results were comparable in the complete case analysis and adverse effects were similar between groups. Within trial costs showed an incremental cost effectiveness ratio of £11 ($15, €12; 95% confidence interval £6 to £29) per mm Hg reduction. Conclusions: The HOME BP digital intervention for the management of hypertension by using self-monitored blood pressure led to better control of systolic blood pressure after one year than usual care, with low incremental costs. Implementation in primary care will require integration into clinical workflows and consideration of people who are digitally excluded. Trial registration: ISRCTN13790648

Eighth International Conference on Paleoceanography

Every three years since 1983, the paleoceanographic community has come together at a different venue to share new data and discoveries at the International Conference on Paleoceanography (ICP). For the recent ICP-8, France was the host country for a conference focused on the theme of “An Ocean View of Global Change.” The Environnements et Paleoenvironnement Oceanique (EPOC) paleoceanography group of the University Bordeaux I acted as the local organizing committee. Scientific presentations at ICP-8 addressed the latest discoveries in paleoceanography and highlighted both emerging and as-yet-unsolved questions on global climate change. Thirty-five speakers, invited by the ICP-8 Science Committee, gave overview talks during morning sessions organized around five major scientific themes. These themes were Cenozoic-Mesozoic Oceans; Carbonate and Silica Systems of the Pleistocene Ocean; Biogeochemical Cycles of the Past; High-Frequency Climate Variability; and Interhemispheric Ocean-Continent-Climate Linkages

Immunofluorescence images of FVIII expression by EC.

<p><b>a</b>: Sequential confocal fluorescence microscopy images in primary human EC. <b>a</b> and <b>b</b>: Representative HPMEC and HPAEC control images using To-Pro-3 nuclear counterstain (first panel, monochrome), murine control IgG₁ (second panel monochrome) and merged images (third panel: To-Pro-3 nuclear counterstain white, control IgG₁ red) using maximum gain used for imaging. <b>c</b>) Comparison of proportion of <b>i</b>) <b>HPMEC</b> and <b>ii</b>) <b>HPAEC</b> expressing vWF and FVIII protein (expression levels defined in methods). <b>d, e, f, g</b>) Sequential confocal fluorescence microscopy images comparing vWF (monochrome in first panel); FVIII (monochrome in second panel, specific mAb as denoted), and merged images (third panel; vWF green, anti-FVIII reacting protein red) in <b>d</b>): HPMEC, and <b>e, f, g</b>): HPAEC. FVIII mAb images displayed here are representative of all FVIII mAbs examined, and all cell lots. Note yellow merged images suggesting degree of FVIII/vWF colocalisation in <b>e, f, g</b>, with white colouring denoting the nuclei (TO-PRO3 nuclear counterstain). Scale bars indicate 5 µm.</p

FVIII splice isoforms.

<p><b>a: Variants identified by ExonMine</b>: Variants 1 and 2 correspond to major and minor RefSeq isoforms; variants 3–5 to expressed sequence tag (EST) sequences deposited in Genbank. Note none of the alternate variants encode the FVIII mAb epitopes. <b>b: Variants identified in EC</b>. Simultaneous expression of variants 1–4 in HPAEC (PA), HPMEC (PM), and HUVEC (H). Gels: φx, HaeIII-digested φx marker, C^A negative water control for HPAEC/HUVEC, C^M negative water control for HPMEC. The apparent difference in size of variant 4 is an artefact due to gel running (note differential site of 194 marker band in first and last lanes). Cartoons: Thin and thick arrows indicate sites of PCR and sequencing oligonucleotide primers respectively. Sequence chromatograms were obtained using nested reverse internal primers in exon 23 (variants 1–3) or exon 3 (variant 4; low concentration first round product sequenced). Note V5 sequences (exons U1-1-2-3) were not amplified from EC in any reaction.</p

Lung expression of FVIII.

<p>Serial sections of frozen normal human lung tissue from two donor blocks (a and b) stained with <b>i)</b> control IgG₁, <b>ii</b>) anti-CD31, or <b>iii</b>) anti-FVIII (C5). The 200x images are representative of data from all five donors.</p