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1.  Semi-Quantitative Parameter Analysis of DCE-MRI Revisited: Monte-Carlo Simulation, Clinical Comparisons, and Clinical Validation of Measurement Errors in Patients with Type 2 Neurofibromatosis 
PLoS ONE  2014;9(3):e90300.
Purpose
To compare semi-quantitative (SQ) and pharmacokinetic (PK) parameters for analysis of dynamic contrast enhanced MR data (DCE-MRI) and investigate error-propagation in SQ parameters.
Methods
Clinical data was collected from five patients with type 2-neurofibromatosis (NF2) receiving anti-angiogenic therapy for rapidly growing vestibular schwannoma (VS). There were 7 VS and 5 meningiomas. Patients were scanned prior to therapy and at days 3 and 90 of treatment. Data was collected using a dual injection technique to permit direct comparison of SQ and PK parameters. Monte Carlo modeling was performed to assess potential measurement errors in SQ parameters in persistent, washout, and weakly enhancing tissues. The simulation predictions for five semi-quantitative parameters were tested using the clinical DCE-MRI data.
Results
In VS, SQ parameters and Ktrans showed close correlation and demonstrated similar therapy induced reductions. In meningioma, only the denoised Signal Enhancement Ratio (Rse1/se2(DN)) showed a significant therapy induced reduction (p<0.05). Simulation demonstrated: 1) Precision of SQ metrics normalized to the pre-contrast-baseline values (MSErel and ∑MSErel) is improved by use of an averaged value from multiple baseline scans; 2) signal enhancement ratio Rmse1/mse2 shows considerable susceptibility to noise; 3) removal of outlier values to produce a new parameter, Rmse1/mse2(DN), improves precision and sensitivity to therapy induced changes. Direct comparison of in-vivo analysis with Monte Carlo simulation supported the simulation predicted error distributions of semi-quantitative metrics.
Conclusion
PK and SQ parameters showed similar sensitivity to anti-angiogenic therapy induced changes in VS. Modeling studies confirmed the benefits of averaging baseline signal from multiple images for normalized SQ metrics and demonstrated poor noise tolerance in the widely used signal enhancement ratio, which is corrected by removal of outlier values.
doi:10.1371/journal.pone.0090300
PMCID: PMC3942428  PMID: 24594707
2.  Early reduction in tumour [18F]fluorothymidine (FLT) uptake in patients with non-small cell lung cancer (NSCLC) treated with radiotherapy alone 
Purpose
Changes in tumour 3′-deoxy-3′-[18F]fluorothymidine (FLT) uptake during concurrent chemo-radiotherapy in patients with non-small cell lung cancer (NSCLC) have been reported, at variable time points, in two pilot positron emission tomography (PET) studies. The aim of this study was to assess whether FLT changes occur early in response to radiotherapy (RT) without concurrent chemotherapy and whether such changes exceed test-retest variability.
Methods
Sixteen patients with NSCLC, scheduled to have radical RT, underwent FLT PET once/twice at baseline to assess reproducibility and/or after 5–11 RT fractions to evaluate response. Primary and nodal malignant lesions were manually delineated on CT and volume, mean and maximum standardized uptake values (SUVmean and SUVmax) estimated. Analysis included descriptive statistics and parameter fitting to a mixed-effects model accounting for patients having different numbers of evaluable lesions.
Results
In all, 35 FLT PET scans from 7 patients with a total of 18 lesions and 12 patients with a total of 30 lesions were evaluated for reproducibility and response, respectively. SUVmean reproducibility in primary tumours (SD 8.9 %) was better than SUVmax reproducibility (SD 12.6 %). In nodes, SUVmean and SUVmax reproducibilities (SD 18.0 and 17.2 %) were comparable but worse than for primary tumours. After 5–11 RT fractions, primary tumour SUVmean decreased significantly by 25 % (p = 0.0001) in the absence of significant volumetric change, whereas metastatic nodes decreased in volume by 31 % (p = 0.020) with a larger SUVmean decrease of 40 % (p < 0.0001). Similar changes were found for SUVmax.
Conclusion
Across this group of NSCLC patients, RT induced an early, significant decrease in lesion FLT uptake exceeding test-retest variability. This effect is variable between patients, appears distinct between primary and metastatic nodal lesions, and in primary tumours is lower than previously reported for concurrent chemo-RT at a similar time point. These results confirm the potential for FLT PET to report early on radiation response and to enhance the clinical development of novel drug-radiation combinations by providing an interpretable, early pharmacodynamic end point.
Electronic supplementary material
The online version of this article (doi:10.1007/s00259-013-2632-3) contains supplementary material, which is available to authorized users.
doi:10.1007/s00259-013-2632-3
PMCID: PMC3955141  PMID: 24504503
[18F]Fluorothymidine PET; Non-small cell lung cancer; Radiotherapy; Early response monitoring
3.  Impact of Gas Delivery Systems on Imaging Studies of Human Cerebral Blood Flow 
Purpose. To compare a semiopen breathing circuit with a non-rebreathing (Hudson mask) for MRI experiments involving gas delivery. Methods and Materials. Cerebral blood flow (CBF) was measured by quantitative phase contrast angiography of the internal carotid and basilar arteries in 18 volunteers (20–31 years). In 8 subjects, gases were delivered via a standard non-rebreathing (Hudson mask). In 10 subjects, gases were delivered using a modified “Mapleson A” semiopen anesthetic gas circuit and mouthpiece. All subjects were given 100% O2, medical air, and carbogen gas (95% O2 and 5% CO2) delivered at 15 L/min in a random order. Results. The Hudson mask group showed significant increases in CBF in response to increased FiCO2 compared to air (+9.8%). A small nonsignificant reduction in CBF (−2.4%) was seen in response to increased inspired concentrations of oxygen (FiO2). The Mapleson A group showed significantly larger changes in CBF in response to both increased inspired concentrations of carbon dioxide (FiCO2) (+32.2%, P < 0.05) and FiO2 (−14.6%, P < 0.01). Conclusions. The use of an anaesthetic gas delivery circuit avoids entrainment of room air and rebreathing effects that may otherwise adversely affect the experimental results.
doi:10.1155/2013/694803
PMCID: PMC3874307  PMID: 24392225
4.  Does living in a food insecure household impact on the diets and body composition of young children? Findings from the Southampton Women’s Survey 
Background
Little is known about food insecurity in the UK. The aims of this study were to assess the prevalence and factors associated with food insecurity in a UK cohort, and to examine whether the diets, reported health and anthropometry of young food insecure children differed from those of other children.
Methods
The Southampton Women’s Survey is a prospective cohort study in which detailed information about the diet, lifestyle and body composition of 3000 women was collected before and during pregnancy. Between 2002-2006, 1618 families were followed up when the child was 3 years old. Food insecurity was determined using the Household Food Security scale. The child’s height and weight were measured; diet was assessed by food frequency questionnaire.
Results
4.6% of the households were food insecure. Food insecurity was more common in families where the mothers were younger, smokers, of lower social class, in receipt of financial benefits, and who had a higher deprivation score (all p<0.05). In comparison with other 3-year-old children, those living in food insecure households were likely to have worse parent-reported health and to have a diet of poorer quality, characterised by greater consumption of white bread, processed meat and chips, and by a lower consumption of vegetables (all p<0.05). They did not differ in height or body mass index.
Conclusions
Our data suggest that there are significant numbers of food insecure families in the UK. The poorer reported health and diets of young food insecure children have important implications for their development and lifelong health.
doi:10.1136/jech.2010.125476
PMCID: PMC3679518  PMID: 21652519
food insecurity; body composition; dietary quality; children
5.  [11C]-(R)PK11195 tracer kinetics in the brain of glioma patients and a comparison of two referencing approaches 
Purpose
Translocator protein (TSPO) is a biomarker of neuroinflammation that can be imaged by PET using [11C]-(R)PK11195. We sought to characterize the [11C]-(R)PK11195 kinetics in gliomas of different histotypes and grades, and to compare two reference tissue input functions (supervised cluster analysis versus cerebellar grey matter) for the estimation of [11C]-(R)PK11195 binding in gliomas and surrounding brain structures.
Methods
Twenty-three glioma patients and ten age-matched controls underwent structural MRI and dynamic [11C]-(R)PK11195 PET scans. Tissue time–activity curves (TACs) were extracted from tumour regions as well as grey matter (GM) and white matter (WM) of the brains. Parametric maps of binding potential (BPND) were generated with the simplified reference tissue model using the two input functions, and were compared with each other. TSPO expression was assessed in tumour tissue sections by immunohistochemistry.
Results
Three types of regional kinetics were observed in individual tumour TACs: GM-like kinetics (n = 6, clearance of the tracer similar to that in cerebellar GM), WM-like kinetics (n = 8, clearance of the tracer similar to that in cerebral WM) and a form of mixed kinetics (n = 9, intermediate rate of clearance). Such kinetic patterns differed between low-grade astrocytomas (WM-like kinetics) and oligodendrogliomas (GM-like and mixed kinetics), but were independent of tumour grade. There was good agreement between parametric maps of BPND derived from the two input functions in all controls and 10 of 23 glioma patients. In 13 of the 23 patients, BPND values derived from the supervised cluster input were systematically smaller than those using the cerebellar input. Immunohistochemistry confirmed that TSPO expression increased with tumour grade.
Conclusion
The three types of [11C]-(R)PK11195 kinetics in gliomas are determined in part by tracer delivery, and indicated that kinetic analysis is a valuable tool in the study of gliomas with the potential for in vivo discrimination between low-grade astrocytomas and oligodendrogliomas. Supervised cluster and cerebellar input functions produced consistent BPND estimates in approximately half of the gliomas investigated, but had a systematic difference in the remainder. The cerebellar input is preferred based on theoretical and practical considerations.
doi:10.1007/s00259-013-2447-2
PMCID: PMC3738844  PMID: 23715902
Translocator protein; [11C]-(R)PK11195; Kinetic analysis; Reference tissue; Glioma; PET
6.  Role of Nuclear Factor-κB in Oxidative Stress Associated with Rabies Virus Infection of Adult Rat Dorsal Root Ganglion Neurons 
Journal of Virology  2012;86(15):8139-8146.
Recent studies in an experimental model of rabies showed major structural changes in the brain involving neuronal processes that are associated with severe clinical disease. Cultured adult rat dorsal root ganglion (DRG) neurons infected with the challenge virus standard-11 strain of rabies virus (CVS) showed axonal swellings and immunostaining for 4-hydroxy-2-nonenal (4-HNE), indicating evidence of lipid peroxidation associated with oxidative stress and reduced axonal growth compared to that of mock-infected DRG neurons. We have evaluated whether nuclear factor (NF)-κB might act as a critical bridge linking CVS infection and oxidative stress. On Western immunoblotting, CVS infection induced expression of the NF-κB p50 subunit compared to that of mock infection. Ciliary neurotrophic factor, a potent activator of NF-κB, had no effect on mock-infected rat DRG neurons and reduced the number of 4-HNE-labeled puncta. SN50, a peptide inhibitor of NF-κB, and CVS infection had an additive effect in producing axonal swellings, indicating that NF-κB is neuroprotective. The fluorescent signal for subunit p50 was quantitatively evaluated in the nucleus and cytoplasm of mock- and CVS-infected rat DRG neurons. At 24 h postinfection (p.i.), there was a significant increase in the nucleus/cytoplasm ratio, indicating increased transcriptional activity of NF-κB, perhaps as a response to stress. At both 48 and 72 h p.i., there was significantly reduced nuclear localization of NF-κB. CVS infection may induce oxidative stress by inhibiting nuclear activation of NF-κB. A rabies virus protein may directly inhibit NF-κB activity. Further investigations are needed to gain a better understanding of the basic mechanisms involved in the oxidative damage associated with rabies virus infection.
doi:10.1128/JVI.00550-12
PMCID: PMC3421658  PMID: 22623795
7.  Southampton mealtime assistance study: design and methods 
BMC Geriatrics  2013;13:5.
Background
Malnutrition is common in older people in hospital and is associated with adverse clinical outcomes including increased mortality, morbidity and length of stay. This has raised concerns about the nutrition and diet of hospital in-patients. A number of factors may contribute to low dietary intakes in hospital, including acute illness and cognitive impairment among in-patients. The extent to which other factors influence intake such as a lack of help at mealtimes, for patients who require assistance with eating, is uncertain. This study aims to evaluate the effectiveness of using trained volunteer mealtime assistants to help patients on an acute medical ward for older people at mealtimes.
Methods/design
The study design is quasi-experimental with a before (year one) and after (year two) comparison of patients on the intervention ward and parallel comparison with patients on a control ward in the same department. The intervention in the second year was the provision of trained volunteer mealtime assistance to patients in the intervention ward. There were three components of data collection that were repeated in both years on both wards. The first (primary) outcome was patients’ dietary intake, collected as individual patient records and as ward-level balance data over 24 hour periods. The second was clinical outcome data assessed on admission and discharge from both wards, and 6 and 12 months after discharge. Finally qualitative data on the views and experience of patients, carers, staff and volunteers was collected through interviews and focus groups in both years to allow a mixed-method evaluation of the intervention.
Discussion
The study will describe the effect of provision of trained volunteer mealtime assistants on the dietary intake of older medical in-patients. The association between dietary intake and clinical outcomes including malnutrition risk, body composition, grip strength, length of hospital stay and mortality will also be determined. An important component of the study is the use of qualitative approaches to determine the views of patients, relatives, staff and volunteers on nutrition in hospital and the impact of mealtime assistance.
Trial registration
Trial registered with ClinicalTrials.gov NCTO1647204
doi:10.1186/1471-2318-13-5
PMCID: PMC3547699  PMID: 23294981
Nutrition; Older; Volunteer; Mealtime assistance; Dietary intake; Hospital
8.  Post-mortem imaging as an alternative to autopsy in the diagnosis of adult deaths: a validation study 
Lancet  2012;379(9811):136-142.
Summary
Background
Public objection to autopsy has led to a search for minimally invasive alternatives. Imaging has potential, but its accuracy is unknown. We aimed to identify the accuracy of post-mortem CT and MRI compared with full autopsy in a large series of adult deaths.
Methods
This study was undertaken at two UK centres in Manchester and Oxford between April, 2006, and November, 2008. We used whole-body CT and MRI followed by full autopsy to investigate a series of adult deaths that were reported to the coroner. CT and MRI scans were reported independently, each by two radiologists who were masked to the autopsy findings. All four radiologists then produced a consensus report based on both techniques, recorded their confidence in cause of death, and identified whether autopsy was needed.
Findings
We assessed 182 unselected cases. The major discrepancy rate between cause of death identified by radiology and autopsy was 32% (95% CI 26–40) for CT, 43% (36–50) for MRI, and 30% (24–37) for the consensus radiology report; 10% (3–17) lower for CT than for MRI. Radiologists indicated that autopsy was not needed in 62 (34%; 95% CI 28–41) of 182 cases for CT reports, 76 (42%; 35–49) of 182 cases for MRI reports, and 88 (48%; 41–56) of 182 cases for consensus reports. Of these cases, the major discrepancy rate compared with autopsy was 16% (95% CI 9–27), 21% (13–32), and 16% (10–25), respectively, which is significantly lower (p<0·0001) than for cases with no definite cause of death. The most common imaging errors in identification of cause of death were ischaemic heart disease (n=27), pulmonary embolism (11), pneumonia (13), and intra-abdominal lesions (16).
Interpretation
We found that, compared with traditional autopsy, CT was a more accurate imaging technique than MRI for providing a cause of death. The error rate when radiologists provided a confident cause of death was similar to that for clinical death certificates, and could therefore be acceptable for medicolegal purposes. However, common causes of sudden death are frequently missed on CT and MRI, and, unless these weaknesses are addressed, systematic errors in mortality statistics would result if imaging were to replace conventional autopsy.
Funding
Policy Research Programme, Department of Health, UK.
doi:10.1016/S0140-6736(11)61483-9
PMCID: PMC3262166  PMID: 22112684
11.  Role of Oxidative Stress in Rabies Virus Infection of Adult Mouse Dorsal Root Ganglion Neurons▿  
Journal of Virology  2010;84(9):4697-4705.
Rabies virus infection of dorsal root ganglia (DRG) was studied in vitro with cultured adult mouse DRG neurons. Recent in vivo studies of transgenic mice that express the yellow fluorescent protein indicate that neuronal process degeneration, involving both dendrites and axons, occurs in mice infected with the challenge virus standard (CVS) strain of rabies virus by footpad inoculation. Because of the similarities of the morphological changes in experimental rabies and in diabetic neuropathy and other diseases, we hypothesize that neuronal process degeneration occurs as a result of oxidative stress. DRG neurons were cultured from adult ICR mice. Two days after plating, they were infected with CVS. Immunostaining was evaluated with CVS- and mock-infected cultures for neuron specific β-tubulin, rabies virus antigen, and amino acid adducts of 4-hydroxy-2-nonenal (4-HNE) (marker of lipid peroxidation and hence oxidative stress). Neuronal viability (by trypan blue exclusion), terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining, and axonal growth were also assessed with the cultures. CVS infected 33 to 54% of cultured DRG neurons. Levels of neuronal viability and TUNEL staining were similar in CVS- and mock-infected DRG neurons. There were significantly more 4-HNE-labeled puncta at 2 and 3 days postinfection in CVS-infected cultures than in mock-infected cultures, and axonal outgrowth was reduced at these time points in CVS infection. Axonal swellings with 4-HNE-labeled puncta were also associated with aggregations of actively respiring mitochondria. We have found evidence that rabies virus infection in vitro causes axonal injury of DRG neurons through oxidative stress. Oxidative stress may be important in vivo in rabies and may explain previous observations of the degeneration of neuronal processes.
doi:10.1128/JVI.02654-09
PMCID: PMC2863745  PMID: 20181692
12.  Effect of physiological doses of oral vitamin B12 on plasma homocysteine – A randomized, placebo-controlled, double-blind trial in India 
Background:
Vitamin B12 (B12) deficiency is common in Indians and a major contributor to hyperhomocysteinemia, which may influence fetal growth, risk of type 2 diabetes and cardiovascular disease.
Objective:
To study the effect of physiological doses of B12 and folic acid on plasma total homocysteine (tHcy).
Design:
A cluster randomized, placebo-controlled, double-blind, 2x3 factorial trial, using the family as the randomization unit. Vitamin B12 was given as 2 or 10 μg capsules, with or without 200 μg folic acid, forming six groups (B0F0, B2F0, B10F0, B0F200, B2F200, B10F200). Plasma tHcy was measured before and after 4 and 12 mo of supplementation.
Results:
Three hundred individuals from 119 families in the Pune Maternal Nutrition Study were randomised. There was no interaction between B12 and folic acid (P=0.14) in relation to tHcy change and their effects were analyzed separately: B0 vs. B2 vs. B10; and F0 vs. F200. At 12 mo, tHcy fell by a mean 5.9 (95% CI: −7.8, −4.1) μmol/L in B2, and by 7.1 (95% CI: −8.9, −5.4) μmol/L in B10, compared to non-significant rise of 1.2 (95% CI: −0.5, 2.9) μmol/L in B0. B2 and B10 did not differ significantly. In F200, tHcy fell by 4.8 (95% CI: −6.3, −3.3) μmol/L compared to 2.8 (95% CI: −4.3, −1.2) μmol/L in F0.
Conclusion:
Daily oral supplementation with physiological doses of B12 is an effective community intervention to reduce tHcy. Folic acid (200 μg/d) showed no additional benefit, neither had any unfavourable effects.
doi:10.1038/ejcn.2010.15
PMCID: PMC2865445  PMID: 20216560
Cyanocobalamin; folic acid; homocysteine; randomised controlled trial; South Asian Indians; vitamin B12
13.  Nutrition in early life, and risk of cancer and metabolic disease: alternative endings in an epigenetic tale? 
The British journal of nutrition  2008;101(5):619-630.
There is substantial evidence which shows that constraints in the early life environment is an important determinant of risk of metabolic and cardiovascular disease. There is emerging evidence that higher birth weight, which reflects a more abundant prenatal environment, is associated with increased risk of cancer, in particular breast cancer and childhood leukaemia. Using specific examples from epidemiology and experimental studies, this review discusses the hypothesis that increased susceptibility to cardiovascular, metabolic disease and cancer have a common origin in developmental changes induced in the developing fetus by aspects of the intra uterine environment including nutrition which involve stable changes to the epigenetic regulation of specific genes. However, the induction of specific disease risk is dependent upon the nature of the environmental challenge and interactions between the susceptibility set by the altered epigenome and the environment throughout the life course.
doi:10.1017/S0007114508145883
PMCID: PMC2649281  PMID: 19079817
14.  Feeding pregnant rats a protein-restricted diet persistently alters the methylation of specific cytosines in the hepatic PPARα promoter of the offspring 
The British journal of nutrition  2008;100(2):278-282.
Induction of an altered phenotype by prenatal under-nutrition involves changes in the epigenetic regulation of specific genes. We investigated the effect of feeding pregnant rats a protein-restricted (PR) diet with different amounts of folic acid on the methylation of individual CpG dinucleotides in the hepatic PPARα promoter in juvenile offspring, and the effect of the maternal PR diet on CpG methylation in adult offspring. Pregnant rats (n 5 / group) were fed 180g / kg casein (Control) or 90g / kg casein (PR) with 1mg / kg folic acid, or 90g / kg casein and 5 mg / kg folic acid (PRF). Offspring were killed on postnatal d34 (n 5 males and females / group) and d80 (n 5 males / group). Methylation of 16 CpG dinucleotides in the PPARα promoter was measured by pyrosequencing. Mean PPARα promoter methylation in the PR offspring (4.5%) was 26% lower than Controls (6.1%) due to specific reduction at CpG dinucleotides 2 (40%), 3 (43%), 4 (33%) and 16 (48 %) (P < 0.05). There was no significant difference in methylation at these CpGs between Control and PRF offspring. Methylation of CpGs 5 and 8 was higher (47% and 63%, respectively, P < 0.05) in the PRF offspring than Control or PR offspring. The methylation pattern in d80 PR offspring was comparable to d34 PR offspring. These data show for the first time that prenatal nutrition induces differential changes to the methylation of individual CpG dinucleotides in juvenile rats which persist in adults.
doi:10.1017/S0007114507894438
PMCID: PMC2564112  PMID: 18186951
Fetal programming; epigenetic; rat; PPARα
15.  The nature of the growth pattern and of the metabolic response to fasting in the rat are dependent upon the dietary protein and folic acid intakes of their pregnant dams and post-weaning fat consumption 
The British journal of nutrition  2007;99(3):540-549.
The nutritional cues which induce different phenotypes from a single genotype in developing offspring are poorly understood. How well prenatal nutrient availability before birth predicts that after birth may also determine the offspring's response to later metabolic challenge. We investigated the effect of feeding pregnant rats diets containing 180 g/ kg (Control) or 90 g/ kg (PR) protein and either 1 or 5 mg/kg folic acid on growth and metabolic response to fasting in their offspring, and also the effect of diets with different fat contents (40 g/kg (Fat4) or 100g/kg (Fat10) after weaning. Offspring of dams fed the PR diet with 5 mg/kg folic acid were significantly lighter than other offspring. The PR offspring fed the Fat4 diet had lower plasma triacylglycerol (TAG) than the Control offspring, but this relationship was reversed when offspring were fed Fat10. Increasing the folic acid content of the Control or PR maternal diets induced opposing effects on plasma TAG, non-esterified fatty acids, β-hydroxybutyrate and glucose concentrations in offspring fed Fat4. The effect was accentuated in offspring fed the Fat10 diet such that these metabolites were increased in the Control offspring, but reduced in the PR offspring. These data show for the first time that maternal dietary folic acid intake alters offspring phenotype depending upon dietary protein intake, and that this effect is modified by fat intake after weaning. Prevention by increased folic acid intake of an altered metabolic phenotype by maternal protein-restriction may be at the expense of somatic growth.
doi:10.1017/S0007114507815819
PMCID: PMC2493056  PMID: 17761015
Low protein diet; fetal programming; folic acid; Growth; Metabolism
16.  Structural Abnormalities in Neurons Are Sufficient To Explain the Clinical Disease and Fatal Outcome of Experimental Rabies in Yellow Fluorescent Protein-Expressing Transgenic Mice▿  
Journal of Virology  2007;82(1):513-521.
Under natural conditions and in some experimental models, rabies virus infection of the central nervous system causes relatively mild histopathological changes, without prominent evidence of neuronal death despite its lethality. In this study, the effects of rabies virus infection on the structure of neurons were investigated with experimentally infected transgenic mice expressing yellow fluorescent protein (YFP) in neuronal subpopulations. Six-week-old mice were inoculated in the hind-limb footpad with the CVS strain of fixed virus or were mock infected with vehicle (phosphate-buffered saline). Brain regions were subsequently examined by light, epifluorescent, and electron microscopy. In moribund CVS-infected mice, histopathological changes were minimal in paraffin-embedded tissue sections, although mild inflammatory changes were present. Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling and caspase-3 immunostaining showed only a few apoptotic cells in the cerebral cortex and hippocampus. Silver staining demonstrated the preservation of cytoskeletal integrity in the cerebral cortex. However, fluorescence microscopy revealed marked beading and fragmentation of the dendrites and axons of layer V pyramidal neurons in the cerebral cortex, cerebellar mossy fibers, and axons in brainstem tracts. At an earlier time point, when mice displayed hind-limb paralysis, beading was observed in a few axons in the cerebellar commissure. Toluidine blue-stained resin-embedded sections from moribund YFP-expressing animals revealed vacuoles within the perikarya and proximal dendrites of pyramidal neurons in the cerebral cortex and hippocampus. These vacuoles corresponded with swollen mitochondria under electron microscopy. Vacuolation was also observed ultrastructurally in axons and in presynaptic nerve endings. We conclude that the observed structural changes are sufficient to explain the severe clinical disease with a fatal outcome in this experimental model of rabies.
doi:10.1128/JVI.01677-07
PMCID: PMC2224401  PMID: 17942540
18.  Induction of altered epigenetic regulation of the hepatic glucocorticoid receptor in the offspring of rats fed a protein-restricted diet during pregnancy suggests that reduced DNA methyltransferase-1 expression is involved in impaired DNA methylation and changes in histone modifications 
The British journal of nutrition  2007;97(6):1064-1073.
Prenatal nutritional constraint induces an altered metabolic phenotype in the offspring which in humans confers an increased risk of non-communicable disease. Feeding a protein-restricted (PR) diet to pregnant rats causes hypomethylation of specific gene promoters in the offspring and alters phenotype. We investigated how altered epigenetic regulation of the hepatic glucocorticoid receptor (GR) 110 promoter is induced in the offspring. Rats were fed a control (180g casein/kg) or a PR (90g casein/kg) diet throughout pregnancy, and chow during lactation. Offspring were killed at postnatal day 34 (n 5 per maternal dietary group). Methylation-sensitive PCR showed GR110 promoter methylation was 33% lower (P<0.001) and GR expression 84% higher (P<0.05) in the PR offspring. RT PCR showed DNA methyltransferase-1 (Dnmt1) expression was 17% lower (P<0.05) in PR offspring, while Dnmt3a/b and methyl domain binding protein-2 expression was not altered. Thus hypomethylation of the GR110 promoter may result from lower capacity to methylate hemimethylated DNA during mitosis. Histone modifications which facilitate transcription were increased at the GR110 promoter (147 to 921%, P<0.001), while those that suppress methylation were decreased (54%, P<0.01) or similar to controls. In human umbilical cord (n 15), there was a 2-fold difference between the highest and lowest level of GR1-CTotal promoter methylation. Dnmt1, but not Dnmt3a, expression predicted 49% (P = 0.003) of the variation in GR1-CTotal promoter methylation. These findings suggest that induction in the offspring of altered epigenetic regulation of the hepatic GR110 promoter, and hence metabolic phenotype, may be due to reduced Dnmt1 expression.
doi:10.1017/S000711450769196X
PMCID: PMC2211425  PMID: 17433129
Fetal programming; epigenetic; rat; glucocorticoid receptor; histones; human
19.  Therapy with Minocycline Aggravates Experimental Rabies in Mice▿  
Journal of Virology  2007;81(12):6248-6253.
Minocycline is a tetracycline derivative with antiapoptotic and anti-inflammatory properties, and the drug has been shown to have beneficial effects in a variety of models of neurological disorders. The potentially neuroprotective role of minocycline was assessed in experimental in vitro and in vivo models of rabies virus infection. In this study, 5 nM minocycline did not improve the viability of embryonic mouse cortical and hippocampal neurons infected in vitro with the attenuated SAD-D29 strain of rabies virus, based on assessments using trypan blue exclusion. Two-day-old ICR mice were inoculated in the right hind limb thigh muscle with SAD-D29, and they received daily subcutaneous injections of either 50 mg/kg minocycline or vehicle (phosphate-buffered saline). Infected minocycline-treated mice experienced an earlier onset of neurologic signs and greater mortality (83% versus 50%) than those receiving vehicle (log rank test, P = 0.002 and P = 0.003, respectively). Immunohistochemical analysis of rabies virus antigen distribution was performed at early time points and in moribund mice. There were greater numbers of infected neurons in the regional brain areas of minocycline-treated mice than in vehicle-treated mice, which was significant in the CA1 region of the hippocampus. There was less apoptosis (P = 0.01) and caspase 3 immunostaining (P = 0.0008) in the midbrains of mice treated with minocycline than in mice treated with vehicle, consistent with a neuroprotective role of neuronal apoptosis that may have had a mild effect of inhibiting viral spread. Reduced infiltration of CD3+ T cells was observed in the pons/medulla of moribund mice that received minocycline therapy (P = 0.008), suggesting that the anti-inflammatory actions of minocycline may intensify the neurologic disease. These findings indicate that minocycline has important detrimental effects in the therapy of experimental rabies. Empirical therapy with minocycline should therefore be approached with caution in cases of human rabies and possibly other viral encephalitides until more experimental data become available.
doi:10.1128/JVI.00323-07
PMCID: PMC1900124  PMID: 17409147
20.  Body mass index cut offs to define thinness in children and adolescents: international survey 
BMJ : British Medical Journal  2007;335(7612):194.
Objective To determine cut offs to define thinness in children and adolescents, based on body mass index at age 18 years.
Design International survey of six large nationally representative cross sectional studies on growth.
Setting Brazil, Great Britain, Hong Kong, the Netherlands, Singapore, and the United States.
Subjects 97 876 males and 94 851 females from birth to 25 years.
Main outcome measure Body mass index (BMI, weight/height2).
Results The World Health Organization defines grade 2 thinness in adults as BMI <17. This same cut off, applied to the six datasets at age 18 years, gave mean BMI close to a z score of −2 and 80% of the median. Thus it matches existing criteria for wasting in children based on weight for height. For each dataset, centile curves were drawn to pass through the cut off of BMI 17 at 18 years. The resulting curves were averaged to provide age and sex specific cut-off points from 2-18 years. Similar cut offs were derived based on BMI 16 and 18.5 at 18 years, together providing definitions of thinness grades 1, 2, and 3 in children and adolescents consistent with the WHO adult definitions.
Conclusions The proposed cut-off points should help to provide internationally comparable prevalence rates of thinness in children and adolescents.
doi:10.1136/bmj.39238.399444.55
PMCID: PMC1934447  PMID: 17591624
21.  Rabies Virus Infection of Primary Neuronal Cultures and Adult Mice: Failure To Demonstrate Evidence of Excitotoxicity†  
Journal of Virology  2006;80(20):10270-10273.
Cultures derived from the cerebral cortices and hippocampi of 17-day-old mouse fetuses infected with the CVS strain of rabies virus showed loss of trypan blue exclusion, morphological apoptotic features, and activated caspase 3 expression, indicating apoptosis. The NMDA (N-methyl-d-aspartate acid) antagonists ketamine (125 μM) and MK-801 (60 μM) were found to have no significant neuroprotective effect on CVS-infected neurons, while the caspase inhibitor Ac-Asp-Glu-Val aspartic acid aldehyde (25 μM) exerted a marked neuroprotective effect. Glutamate-stimulated increases in levels of intracellular calcium were reduced in CVS-infected hippocampal neurons. Ketamine (120 mg/kg of body weight/day intraperitoneally) given to CVS-infected adult mice produced no beneficial effects. We have found no supportive evidence that excitotoxicity plays an important role in rabies virus infection.
doi:10.1128/JVI.01272-06
PMCID: PMC1617316  PMID: 17005706
22.  Hormone replacement therapy and false positive recall in the Million Women Study: patterns of use, hormonal constituents and consistency of effect 
Breast Cancer Research  2005;8(1):R8.
Introduction
Current and recent users of hormone replacement therapy (HRT) have an increased risk of being recalled to assessment at mammography without breast cancer being diagnosed ('false positive recall'), but there is limited information on the effects of different patterns of HRT use on this. The aim of this study is to investigate in detail the relationship between patterns of use of HRT and false positive recall.
Methods
A total of 87,967 postmenopausal women aged 50 to 64 years attending routine breast cancer screening at 10 UK National Health Service Breast Screening Units from 1996 to 1998 joined the Million Women Study by completing a questionnaire before screening and were followed for their screening outcome.
Results
Overall, 399 (0.5%) participants were diagnosed with breast cancer and 2,629 (3.0%) had false positive recall. Compared to never users of HRT, the adjusted relative risk (95% CI) of false positive recall was: 1.62 (1.43–1.83), 1.80 (1.62–2.01) and 0.76 (0.52–1.10) in current users of oestrogen-only HRT, oestrogen-progestagen HRT and tibolone, respectively (p (heterogeneity) < 0.0001); 1.65 (1.43–1.91), 1.49 (1.22–1.81) and 2.11 (1.45–3.07) for current HRT used orally, transdermally or via an implant, respectively (p (heterogeneity) = 0.2); and 1.84 (1.67–2.04) and 1.75 (1.49–2.06) for sequential and continuous oestrogen-progestagen HRT, respectively (p (heterogeneity) = 0.6). The relative risk of false positive recall among current users appeared to increase with increasing time since menopause, but did not vary significantly according to any other factors examined, including duration of use, hormonal constituents, dose, whether single- or two-view screening was used, or the woman's personal characteristics.
Conclusion
Current use of oestrogen-only and oestrogen-progestagen HRT, but not tibolone, increases the risk of false positive recall at screening.
doi:10.1186/bcr1364
PMCID: PMC1413983  PMID: 16417651
23.  Recombinant rabies virus vaccine strain SAD-L16 inoculated intracerebrally in young mice produces a severe encephalitis with extensive neuronal apoptosis 
Abstract
Seven-day-old ICR mice were infected by intracerebral inoculation with recombinant rabies virus vaccine strain SAD-L16. Infected mice developed severe and fatal encephalitis with rabies virus-infected neurons in widespread regions of the brain. There was extensive neuronal death with predominant features of apoptosis, as assessed by light and electron microscopy, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining, and immunohistochemical staining for activated caspase-3. Although SAD-L16 is a neuroattenuated rabies virus, it is fully capable of spreading efficiently and inducing widespread neuronal apoptosis in the immature mouse brain.
PMCID: PMC1142176  PMID: 15971673
24.  Influence of personal characteristics of individual women on sensitivity and specificity of mammography in the Million Women Study: cohort study 
BMJ : British Medical Journal  2004;329(7464):477.
Objectives To examine how lifestyle, hormonal, and other factors influence the sensitivity and specificity of mammography.
Methods Women recruited into the Million Women Study completed a questionnaire about various personal factors before routine mammographic screening. A sample of 122 355 women aged 50-64 years were followed for outcome of screening and incident breast cancer in the next 12 months. Sensitivity and specificity were calculated by using standard definitions, with adjustment for potential confounding factors.
Results Breast cancer was diagnosed in 726 (0.6%) women, 629 in screen positive and 97 in screen negative women; 3885 (3.2%) were screen positive but had no subsequent diagnosis of breast cancer. Overall sensitivity was 86.6% and specificity was 96.8%. Three factors had an adverse effect on both measures: use of hormone replacement therapy (sensitivity: 83.0% (95% confidence interval 77.4% to 87.6%), 84.7% (73.9% to 91.6%), and 92.1% (87.6% to 95.0%); specificity: 96.8% (96.6% to 97.0%), 97.8% (97.5% to 98.0%), and 98.1% (98.0% to 98.2%), respectively, for current, past, and never use); previous breast surgery v no previous breast surgery (sensitivity: 83.5% (75.7% to 89.1%) v 89.4% (86.5% to 91.8%); specificity: 96.2% (95.8% to 96.5%) v 97.4% (97.3% to 97.5%), respectively); and body mass index < 25 v ≥ 25 (sensitivity: 85.7% (81.2% to 89.3%) v 91.0% (87.5% to 93.6%); specificity: 97.2% (97.0% to 97.3%) v 97.4% (97.3% to 97.6%), respectively). Neither sensitivity nor specificity varied significantly according to age, family history of breast cancer, parity, past oral contraceptive use, tubal ligation, physical activity, smoking, or alcohol consumption.
Conclusions The efficiency, and possibly the effectiveness, of mammographic screening is lower in users of hormone replacement therapy, in women with previous breast surgery, and in thin women compared with other women.
PMCID: PMC515195  PMID: 15331472

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