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

Evaluating the contribution of a wildlife health capacity building program on orangutan conservation

One Health is increasingly being used as a tool in ecosystem protection. The Orangutan Veterinary Advisory Group (OVAG) is working to address One Health concerns in Pongo spp. (orangutan) welfare and conservation. Orangutans are vital contributors to the ecosystem health of their range areas. Strengthening national capacity is crucial to make a lasting difference in the currently bleak outlook for orangutan species survival. OVAG is a capacity strengthening and expertise network that brings together all those working with orangutans, in- and ex-situ, to share knowledge, skills, and to collectively learn. Using the One Health paradigm embedded to enhance professional development, the OVAG network is successfully supporting conservation outcomes and impact. As part of our adaptive management approach, and to assess individual and organizational change attributable to the capacity strengthening work of OVAG, we evaluated technical skill test data, program satisfaction data, and asked participants to complete a self-reflective survey. This pilot study of our work demonstrates statistically significant improvements in conservation medicine (t = 5.481, p < 0.0001) and wildlife clinical skills knowledge (t = 3.923, p < 0.001) for those in the OVAG program. Most consider OVAG participation to be either critical or very useful in their conservation medicine decision-making process, with a perceived positive impact on their skills at handling multiple situations. Additionally, participant feedback shows a sense of being able to drive positive change locally and nationally (within their own countries) as a consequence of OVAG participation. The authors hope the OVAG model including its associated capacity support mechanisms and pedagogical approaches can be used as a template for other One Health efforts

A genetic and phenotypic approach to understanding reading ability and associated health and lifestyle outcomes in the national child development study

Reading ability is a polygenic trait, which is also influenced by environment. Despite the global social and economic cost of poor reading skills, molecular genetic research regarding reading ability as a quantitative trait is still in its infancy. Modern molecular methods have the potential to reveal much about such complex traits, including their genetic basis and how they relate to other traits. Chapter 1 explores the literature around reading ability, including how reading skills are defined, and the predictors and outcomes associated with them. Chapter 2 reviews the current state of knowledge on the genetics of reading ability, including studies from family, twin, and molecular genetic designs. This thesis has utilised the National Child Development Study (NCDS) to investigate longitudinal reading, the genetics of reading, and associations with other traits in detail. Chapter 3 reviews the cohort in more detail, including the data available and a review of existing reading research in the dataset. Chapter 4 outlines the genetics methods that are used to carry out the empirical research in the remainder of the thesis, including genotype imputation, genome-wide association studies, and linkage disequilibrium score based genetic correlations. The NCDS is a UK based birth cohort study, which has been running since 1958. A subsample of NCDS participants have provided genetic data, however this genomic dataset has been poorly documented. In Chapter 5, I present an updated version of the NCDS genomic dataset, which has been imputed to the Haplotype Reference Consortium v1.1 reference panel, for use in future research (n = 6431). This contribution will allow other researchers to carry out analysis using modern molecular methods on a dataset imputed to a reference panel which has been shown to be more effective than the previous reference panel the NCDS was imputed to, the 1000 Genomes reference panel. The NCDS contains a series of different reading measures, including objective tests, teacher ratings, and self-report measures, from childhood though to midlife. In Chapter 5, these measures have been analysed to develop a series of reliable and valid age-specific indices which measure reading ability quantitatively, with potential to be used in future reading research. Chapter 6 uses reading composites as the phenotype outcome for a series of genetic analyses, which replicate several previous findings, showing that the reading composites generated in the NCDS, while not gold standard reading assessments, are still appropriate measures of reading ability. Genome-wide association analysis was conducted on reading ability composites derived at different ages through childhood to young adulthood. Downstream analysis showed the reading measures at different ages to be strongly genetically correlated with each other, with reading and language variables from other samples, and moderately genetically correlated with a series of additional traits related to social, educational, occupational and health outcomes, replicating several previous findings. Positive genetic correlations were found between reading and cognitive performance, educational attainment, and employment status, whereas negative genetic correlations were found between reading and occupational conditions, wellbeing measures, and health outcomes. This study replicated an earlier intriguing finding of a genetic correlation between reading and pain, which was then explored further by interrogating genetic correlations between reading and additional pain related variables, such as painkiller use. Given the richness of the NCDS longitudinal data, in Chapter 7 I further explored the association between reading ability and pain in an effort to disentangle potential causal relationships between the two. Logistic and linear regression analyses were used to analyse the association between childhood reading ability and experiencing pain in childhood and adulthood. Several pain types were tested, including abdominal pain and headache in childhood, and back pain, eye pain, headache, migraine, and rheumatism in adulthood. Numerous significant associations were discovered at multiple age points, with all significant associations indicating that better readers are less likely to experience pain. Further analysis indicated that socioeconomic status (SES) may act as a mediator for the relationship between reading and some pain types. Evidence of full mediation was found for the relationship between reading and back pain, while evidence regarding a mediation effect between reading and rheumatism was unclear. SES, measured by occupation, did not mediate the relationship between reading and eye pain, and the results indicated that reading ability and SES acted together to influence headache. I conducted post-hoc analysis in an attempt to further untangle the relationship between reading and back pain, including physical demands of the participant’s job as a covariate. The mediation effect remained, indicating that mediation was due to other elements of SES. Chapter 8 explores the further implications of this research. This thesis shows that non-validated reading measures can be combined in order to generate a reliable and valid quantitative reading composite that is suitable for further research in reading, including genetic research. This is significant, as the NCDS is an exceptionally rich longitudinal dataset, which has the potential to be used to study gene by environment interactions in future reading research using molecular methods, an area with potential which is still in its infancy. This work has added to a small amount of existing molecular evidence which suggests that reading is genetically stable over time, with high genetic correlations found between reading at ages seven, eleven, sixteen, and an overall reading composite including measures from childhood through to age 33. Additionally, this thesis replicated previously identified genetic correlations between reading ability and health, including pain. Phenotypic analysis has indicated that, in some cases, the relationship is not due to SES, and therefore it is possible that pain and reading ability share a biological mechanism; future research using multivariate genetic methods (expanding to other neurodevelopmental traits) are needed to identify these common biological causes. Finally, this thesis has shown that the richness of the NCDS dataset, combined with genetic information, proves it valuable for behaviour genetic reading research. With much still to discover about the behaviour genetics of reading ability and its transactions with the environment, this thesis will hopefully act as a starting point for further work in this area, which should include a thorough investigation of gene by environment correlation and interaction in reading ability

Alveolar macrophage lipid burden correlates with clinical improvement in patients with pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and non-PAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients

Alveolar macrophage lipid burden correlates with clinical improvement in patients with pulmonary alveolar proteinosis

Pulmonary alveolar proteinosis (PAP) is a life-threatening, rare lung syndrome for which there is no cure and no approved therapies. PAP is a disease of lipid accumulation characterized by alveolar macrophage foam cell formation. While much is known about the clinical presentation, there is a paucity of information regarding temporal changes in lipids throughout the course of disease. Our objectives were to define the detailed lipid composition of alveolar macrophages in PAP patients at the time of diagnosis and during treatment. We performed comprehensive mass spectrometry to profile the lipid signature of alveolar macrophages obtained from three independent mouse models of PAP and from PAP and non-PAP patients. Additionally, we quantified changes in macrophage-associated lipids during clinical treatment of PAP patients. We found remarkable variations in lipid composition in PAP patients, which were consistent with data from three independent mouse models. Detailed lipidomic analysis revealed that the overall alveolar macrophage lipid burden inversely correlated with clinical improvement and response to therapy in PAP patients. Specifically, as PAP patients experienced clinical improvement, there was a notable decrease in the total lipid content of alveolar macrophages. This crucial observation suggests that the levels of these macrophage-associated lipids can be utilized to assess the efficacy of treatment. These findings provide valuable insights into the dysregulated lipid metabolism associated with PAP, offering the potential for lipid profiling to serve as a means of monitoring therapeutic interventions in PAP patients

Statin as a novel pharmacotherapy of pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is a syndrome of reduced GM-CSF-dependent, macrophage-mediated surfactant clearance, dysfunctional foamy alveolar macrophages, alveolar surfactant accumulation, and hypoxemic respiratory failure for which the pathogenetic mechanism is unknown. Here, we examine the lipids accumulating in alveolar macrophages and surfactant to define the pathogenesis of PAP and evaluate a novel pharmacotherapeutic approach. In PAP patients, alveolar macrophages have a marked increase in cholesterol but only a minor increase in phospholipids, and&nbsp;pulmonary surfactant has an increase in the ratio of cholesterol to phospholipids. Oral statin therapy is associated with clinical, physiological, and radiological improvement in autoimmune PAP patients, and ex vivo statin treatment reduces cholesterol levels in explanted alveolar macrophages. In Csf2rb-/- mice, statin therapy reduces cholesterol accumulation in alveolar macrophages and ameliorates PAP, and ex vivo statin treatment increases cholesterol efflux from macrophages. These results support the feasibility of statin as a novel pathogenesis-based pharmacotherapy of PAP

Effectiveness and cost-effectiveness of a 12-month automated text message intervention for weight management in postpartum women with overweight or obesity: protocol for the Supporting MumS (SMS) multisite, parallel-group, randomised controlled trial

Introduction The reproductive years can increase women’s weight-related risk. Evidence for effective postpartum weight management interventions is lacking and engaging women during this life stage is challenging. Following a promising pilot evaluation of the Supporting MumS intervention, we assess if theory-based and bidirectional text messages to support diet and physical activity behaviour change for weight loss and weight loss maintenance, are effective and cost-effective for weight change in postpartum women with overweight or obesity, compared with an active control arm receiving text messages on child health and development.Methods and analysis Two-arm, parallel-group, assessor-blind randomised controlled trial with cost-effectiveness and process evaluations. Women (n=888) with body mass index (BMI) ≥25 kg/m2 and within 24 months of giving birth were recruited via community and National Health Service pathways through five UK sites targeting areas of ethnic and socioeconomic diversity. Women were 1:1 randomised to the intervention or active control groups, each receiving automated text messages for 12 months. Data are collected at 0, 6, 12 and 24 months. The primary outcome is weight change at 12 months from baseline, compared between groups. Secondary outcomes include weight change (24 months) and waist circumference (cm), proportional weight gain (&gt;5 kg), BMI (kg/m2), dietary intake, physical activity, infant feeding and mental health (6, 12 and 24 months, respectively). Economic evaluation examines health service usage and personal expenditure, health-related quality of life and capability well-being to assess cost-effectiveness over the trial and modelled lifetime. Cost–utility analysis examines cost per quality-adjusted life-years gained over 24 months. Mixed-method process evaluation explores participants’ experiences and contextual factors impacting outcomes and implementation. Stakeholder interviews examine scale-up and implementation.Ethics and dissemination Ethical approval was obtained before data collection (West of Scotland Research Ethics Service Research Ethics Committee (REC) 4 22/WS/0003). Results will be published via a range of outputs and audiences.<br/