Ultrastructural localization of adenosine triphosphatase activity in lymphocytes activated in vitro by phytohaemagglutinin

The ultrastructural localization of Ca 2+, Mg2+ -activated ATPase was studied in phytohaemagglutinin activated lymphocytes and in normal unstimulated lymphocytes. Cells, fixed in paraformaldehyde-glutaraldehyde, were incubated in a medium containing 3 mM A TP, 5 mM CaCl2 and 2.4 mM Pb(N03h in 0.1 M tris buffer at pH 8.5, the optimum pH for histochemical demonstration of this enzyme. Reaction product was localized in the endoplasmic reticulum, nuclear membrane, Golgi apparatus and mitochondria and on the membrane surrounding large electron-dense bodies. Cytoplasmic vesicles and the plasma membrane were negative. Activity in unstimulated lymphocytes showed a similar localization but the amount of endoplasmic reticulum was much less than in activated lymphocytes. The pH of the medium was critical for the localization of the enzyme. At pH 7.5, the cytoplasmic reaction was almost completely inhibited but a dense precipitate was present on the outer surface of the plasma membrane. The reaction was stimulated by either Ca2+ or Mg2+ and was greatly decreased in the absence of these cations or in the presence of p-chloromercuribenzoate or N-ethylmaleimide. Oligomycin inhibited selectively the reaction in mitochondria but not the reaction at other sites. While the reaction in mitochondria showed complete substrate specificity, a mild reaction was obtained at the other sites with uridine diphosphate or sodium p-glycophosphate as substrate. A TP was, however, the preferential substate.peer-reviewe

Reduction of low- and high-grade cervical abnormalities associated with high uptake of the HPV bivalent vaccine in Scotland

In Scotland, a national HPV immunisation programme began in 2008 for 12-13 year olds, with a catch-up campaign from 2008-2011 for those under the age of 18. To monitor the impact of HPV immunisation on cervical disease at the population level, a programme of national surveillance was established.  We analysed colposcopy data from a cohort of women born between 1988-1992 who entered the Scottish Cervical Screening Programme (SCSP) and were aged 20-21 in 2008-2012.  By linking datasets from the SCSP and colposcopy services, we observed a significant reduction in diagnoses of cervical intraepithelial neoplasia 1 (CIN 1) (RR 0.71, 95% CI 0.58 to 0.87, p=0.0008), CIN 2 (RR 0.5, 95% CI 0.4, 0.63, p<0.0001) and CIN 3 (RR 0.45, 95% CI 0.35 to 0.58, p< 0.0001) for women who received 3 doses of vaccine compared with unvaccinated women.  To our knowledge, this is one of the first studies to show a reduction of low and high grade cervical intraepithelial neoplasia associated with high uptake of the HPV bivalent vaccine at the population level. These data are very encouraging for countries that have achieved high HPV vaccine uptake

The Potential of Biobanked Liquid Based Cytology Samples for Cervical Cancer Screening Using Raman Spectroscopy.

Patient samples are unique and often irreplaceable. This allows biobanks to be a valuable source of material. The aim of this study was to assess the ability of Raman spectroscopy to screen for histologically confirmed cases of Cervical Intraepithelial neoplasia (CIN) using biobanked liquid based cytology (LBC) samples. Two temperatures for long term storage were assessed; 80°C and -25°C. The utility of Raman spectroscopy for the detection of CIN was compared for fresh LBC samples and biobanked LBC samples. Two groups of samples were used for the study with one group associated with disease (CIN 3) and the other associated with no disease (cytology negative). The data indicates that samples stored at -80°C are not suitable for assessment by Raman spectroscopy due to a lack of cellular material and the presence of cellular debris. However, the technology can be applied to fresh LBC samples and those stored at -25°C and is, moreover, effective in the discrimination of negative samples from those where CIN 3 has been confirmed. Pooled fresh and biobanked samples are also amenable to the technology and achieve a similar sensitivity and specificity for CIN 3. This study demonstrates that cervical cytology samples stored within biobanks at temperatures that preclude cell lysis can act as a useful resource for Raman spectroscopy and will facilitate research and translational studies in this area

Droplet digital PCR quantification suggests that higher viral load correlates with improved survival in HPV-positive oropharyngeal tumours

Background: Although HPV-positive oropharyngeal cancer (OPC) patients have improved prognosis compared to HPV negative patients; there remains an HPV-positive group who have poor outcomes. Biomarkers to stratify discrete patient outcomes are thus desirable. Our objective was to analyse viral load (VL) by droplet digital PCR (ddPCR), in HPV-positive patients with OPC on whom clinical outcome data were available. Methods: In a cohort of patients that had previously tested HPV positive via conventional PCR, VL was determined using ddPCR assays for HPV16 L1 and E6 genes. VL was classed as “medium/high” if more than 5.57 copies or 8.68 copies of the HPV 16 L1 or E6 gene were detected respectively. Effect of VL on overall survival and hazard of death &amp; disease progression was performed with adjustments made for sex, age, deprivation, smoking, alcohol consumption and stage. Results: L1 VL ranged from 0.0014–304 gene copies per cell with a mean of 30.9; comparatively E6 VL ranged from 0.0012–356 copies per cell with a mean of 37.9. Univariate analysis showed those with a medium/high VL had a lower hazard of death; this was significant for L1 (p = 0.02) but not for E6 (p = 0.67). The ratio of E6 to L1 deviated from n = 1 in most samples but had no influence on clinical outcomes. Conclusions: HPV viral load may be informative for the further stratification of clinical outcomes in HPV positive OPC patient

Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields

We investigated the interaction between magnetic carbon nanotubes (CNTs) and mesenchymal stem cells (MSCs), and their ability to guide these intravenously injected cells in living rats by using an external magnetic field. MATERIALS & METHODS: Multiwalled CNTs were used to treat MSCs derived from rat bone marrow. Cytotoxicity induced by nanotubes was studied using the WST-1 proliferation and Hoechest 33258 apoptosis assays. The effects of nanotubes on MSCs were evaluated by monitoring the effects on cellular growth rates, immunophenotyping and differentiation, and on the arrangement of cytoskeletal actin. MSCs loaded with nanotubes were injected in vivo in the portal vein of rats driving their localization in the liver by magnetic field. An histological analysis was performed on the liver, lungs and kidneys of all animals. RESULTS: CNTs did not affect cell viability and their ability to differentiate in osteocytes and adipocytes. Both the CNTs and the magnetic field did not alter the cell growth rate, phenotype and cytoskeletal conformation. CNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the biodistribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). CONCLUSION: Multiwalled CNTs hold the potential for use as nanodevices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for the manipulation/guidance of MSCs in regenerative medicine and cell transplantation

A prospective cohort study of human papillomavirus-driven oropharyngeal cancers: implications for prognosis and immunisation

Aims: Oropharyngeal cancer (OPC) is increasing on a global scale, including the component driven by high-risk human papillomavirus (HR-HPV); contemporary data that provides insight into the prognosis of this disease in addition to the fraction attributable to HR-HPV are essential to inform primary and secondary disease management strategies. Materials and methods: A population-based cohort of 235 patients diagnosed with OPC between 2013 and 2015 in Scotland was assessed for HPV status using molecular genotyping. Associations between HR-HPV status and key clinical and demographic variables were estimated using the Pearson chi-squared test. Rates of overall survival and progression-free survival were estimated and visualised using Kaplan–Meier curves. Results: HPV DNA (largely HPV 16) was identified in 60% of cases. After adjustment for age, gender, deprivation, smoking, alcohol consumption and tumour stage, patients with HR-HPV-positive OPC had an 89% reduction in the risk of death (hazard ratio = 0.11, 95% confidence interval 0.05–0.25) and an 85% reduction in the risk of disease progression (hazard ratio = 0.15, 95% confidence interval 0.07–0.30). HPV positivity was not associated with age, deprivation or smoking status, whereas those who reported excess alcohol consumption were less likely to be positive for HR-HPV. Conclusions: The prevalence of HR-HPV-associated OPC is high in Scotland and strongly associated with dramatically improved clinical outcomes, including survival. Demographic/behavioural variables did not reliably predict HPV positivity in this cohort, which underlines the importance of laboratory confirmation. Finally, the dominance of HPV 16 in OPC indicates the significant impact of prophylactic immunisation on this disease

Use of barbed suture without fashioning the “classical” Wirsung-jejunostomy in a modified end-to-side robotic pancreatojejunostomy

Background: The treatment of the pancreatic stump is a critical step of pancreatoduodenectomy (PD). Robot-assisted surgery (RAS) can facilitate minimally invasive challenging abdominal procedures, including pancreatojejunostomy. However, one of the major limitations of RAS stems from its lack of tactile feedback that can lead to pancreatic parenchyma laceration during knot tying or during traction on the suture. Moreover, a Wirsung-jejunostomy is not always easy to execute, especially in cases with small diameter duct. Herein, we describe and video-report the technical details of a robotic modified end-to-side invaginated robotic pancreatojejunostomy (RmPJ) with the use of barbed suture instead of the “classical” Wirsung-jejunostomy. Methods: The RmPJ technique consists of a double layer of absorbable monofilament running barbed suture (3–0&nbsp;V-Loc), the outer layer is used to invaginate the pancreatic stump. Thereafter, a small enterotomy is made in the jejunum exactly opposite to the location of the pancreatic duct for stent insertion (usually 5 Fr) inside the duct. The internal layer provides a second barbed running suture placed between the pancreatic capsule/parenchyma and the jejunal seromuscular layer. Results: A total of 14 patients underwent robotic PD with RmPJ at our Institution. The mean console time was (281.36 ± 31.50&nbsp;min), while the mean operative time for fashioning the RmPJ was 37.31 ± 7.80&nbsp;min. Ten out of 14 patients were discharged within postoperative day 8. No clinically relevant pancreatic fistulas were encountered, while two patients developed biochemical leaks. Conclusions: RmPJ is feasible and reproducible irrespective of pancreatic duct size and parenchyma, and can enhance the surgical workflow of this operation. Specifically, the use of barbed sutures allows the exploitation of the potential advantages of the RAS, while minimizing the negative effect caused by the main disadvantage of the robotic approach, its absence of tactile feedback, by ensuring uniform tension on the continuous suture lines used, especially during the reconstructive phase of the operation