High inter-observer reliability in standardized ultrasound measurements of subcutaneous adipose tissue in children aged three to six years.

BACKGROUND: A procedure to measure subcutaneous adipose (SAT) using brightness-mode ultrasound has recently been standardized and applied to various groups of adults including underweight, overweight and obese adults. High reliability of this procedure was found in each of the examined groups. The purpose of this study was to determine inter-observer reliability of the standardized brightness-mode ultrasound measurement of uncompressed SAT in three to six-year-old children. METHODS: Three experienced observers independently captured the ultrasound images at the eight standardized measurement sites in each of the 20 children and evaluated their images using an interactive software that detects the SAT contour and automatically measures multiple thicknesses in each image; the mean of these represents SAT thickness at a given site. The children were aged 4.9 ± 1.0 years; their body mass index ranged from 13.6-17.7 kgm- 2. Sound speed was set to 1450 ms- 1 for SAT. RESULTS: SAT thickness sums with fibrous structures included (DI) ranged from 25.7-86.4 mm, mean DI was 48.1 ± 15.5 mm. For DI, resulting from 160 measurements by each observer, the intra-class correlation coefficient was 0.998 (95% confidence interval 0.980-0.999), standard error of the estimate was 1.1 mm, and 95% limits of agreement were within ±2.1 mm. The median difference in DI was 0.8 mm, i.e. about 1.9% of mean DI. CONCLUSIONS: Inter-observer results in children are comparable to previously described high reliability in adults. This method, which provides a technical thickness measurement accuracy of about 0.1 to 0.2 mm, enables monitoring of subcutaneous adipose tissue in children with a similarly high reliability as was obtained in adults previously. TRIAL REGISTRATION: German Institute of Medical Documentation and Information, German Clinical Trials Register (DRKS) ID: DRKS00010089; Date 24/02/2016.

Epilepsy and Pregnancy: For healthy pregnancies and happy outcomes. Suggestions for service improvements from the Multispecialty UK Epilepsy Mortality Group.

Between 2009 and 2012 there were 26 epilepsy-related deaths in the UK of women who were pregnant or in the first post-partum year. The number of pregnancy-related deaths in women with epilepsy (WWE) has been increasing. Expert assessment suggests that most epilepsy-related deaths in pregnancy were preventable and attributable to poor seizure control. While prevention of seizures during pregnancy is important, a balance must be struck between seizure control and the teratogenic potential of antiepileptic drugs (AEDs). A range of professional guidance on the management of epilepsy in pregnancy has previously been issued, but little attention has been paid to how optimal care can be delivered to WWE by a range of healthcare professionals. We summarise the findings of a multidisciplinary meeting with representation from a wide group of professional bodies. This focussed on the implementation of optimal pregnancy epilepsy care aiming to reduce mortality of epilepsy in mothers and reduce morbidity in babies exposed to AEDs in utero. We identify in particular -What stage to intervene - Golden Moments of opportunities for improving outcomes -Which Key Groups have a role in making change -When - 2020 vision of what these improvements aim to achieve. -How to monitor the success in this field We believe that the service improvement ideas developed for the UK may provide a template for similar initiatives in other countries.

Development of a core outcome set for epilepsy in pregnancy (E-CORE): a national multi-stakeholder modified Delphi consensus study.

OBJECTIVE: To develop a set of core outcomes for studies on pregnant women with epilepsy. DESIGN: Delphi consensus study. POPULATION: Healthcare professionals, and patient representatives with lived experience of epilepsy in the UK. METHODS: We used a modified Delphi method and a consultation meeting to achieve consensus. Potential outcomes were identified by systematic review, and were scored using a Likert scale anchored between 1 (least important) and 5 (most important). We included outcomes that scored ≥4 by >70% of participants, and outcomes that scored ≤2 by <15% of participants. MAIN OUTCOME MEASURES: Outcomes in studies on epilepsy in pregnancy. RESULTS: Seventy-five healthcare professionals completed the first round, 48 (64%) completed the second round, and 37 (49%) completed the third round of the survey. Twenty-four patient representatives participated. The final core outcome set included 31 outcomes in three domains: neurological, offspring, and obstetric. Outcomes in the neurological domain were seizure control in pregnancy and postpartum, status epilepticus, maternal mortality, drowning, sudden unexpected death in epilepsy, postnatal depression, and quality of life. Offspring domain included congenital abnormalities (major and minor), fetal anticonvulsant syndrome, neurodevelopment, autism disorder, neonatal clinical complications, admission to a neonatal intensive care unit, and anthropometric measurements. The obstetric domain included live birth, stillbirth, miscarriage, ectopic, termination of pregnancy, admission to a high dependency or intensive care unit, breastfeeding, mode of delivery, preterm birth, pre-eclampsia, and eclampsia. Outcomes specific for studies on anti-epileptic drugs (AEDs) included maternal AED toxicity, AED compliance, neonatal withdrawal symptoms, and neonatal haemorrhagic disease. CONCLUSION: Embedding this core set in future clinical trials will promote the standardisation of reporting to inform clinical practice. TWEETABLE ABSTRACT: A Delphi method identifying core outcomes for epilepsy in pregnancy. Final core set includes 31 outcomes.

Influenza gain-of-function experiments: their role in vaccine virus recommendation and pandemic preparedness.

In recent years, controversy has arisen regarding the risks and benefits of certain types of gain-of-function (GOF) studies involving avian influenza viruses. In this article, we provide specific examples of how different types of data, including information garnered from GOF studies, have helped to shape the influenza vaccine production process-from selection of candidate vaccine viruses (CVVs) to the manufacture and stockpiling of safe, high-yield prepandemic vaccines for the global community. The article is not written to support a specific pro- or anti-GOF stance but rather to inform the scientific community about factors involved in vaccine virus selection and the preparation of prepandemic influenza vaccines and the impact that some GOF information has had on this process.

Characteristics of a widespread community cluster of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza in Australia.

BACKGROUND: Oseltamivir resistance in A(H1N1)pdm09 influenza is rare, particularly in untreated community cases. Sustained community transmission has not previously been reported. METHODS: Influenza specimens from the Asia-Pacific region were collected through sentinel surveillance, hospital, and general practitioner networks. Clinical and epidemiological information was collected on patients infected with oseltamivir-resistant viruses. RESULTS: Twenty-nine (15%) of 191 A(H1N1)pdm09 viruses collected between May and September 2011 from Hunter New England (HNE), Australia, contained the H275Y neuraminidase substitution responsible for oseltamivir resistance. Only 1 patient had received oseltamivir before specimen collection. The resistant strains were genetically very closely related, suggesting the spread of a single variant. Ninety percent of cases lived within 50 kilometers. Three genetically similar oseltamivir-resistant variants were detected outside of HNE, including 1 strain from Perth, approximately 4000 kilometers away. Computational analysis predicted that neuraminidase substitutions V241I, N369K, and N386S in these viruses may offset the destabilizing effect of the H275Y substitution. CONCLUSIONS: This cluster represents the first widespread community transmission of H275Y oseltamivir-resistant A(H1N1)pdm09 influenza. These cases and data on potential permissive mutations suggest that currently circulating A(H1N1)pdm09 viruses retain viral fitness in the presence of the H275Y mutation and that widespread emergence of oseltamivir-resistant strains may now be more likely.

Increased detection in Australia and Singapore of a novel influenza A(H1N1)2009 variant with reduced oseltamivir and zanamivir sensitivity due to a S247N neuraminidase mutation.

A novel influenza A(H1N1)2009 variant with mildly reduced oseltamivir and zanamivir sensitivity has been detected in more than 10% of community specimens in Singapore and more than 30% of samples from northern Australia during the early months of 2011. The variant, which has also been detected in other regions of the Asia-Pacific, contains a S247N neuraminidase mutation. When combined with the H275Y mutation, as detected in an oseltamivir-treated patient, the dual S247N+H275Y mutant had extremely high oseltamivir resistance.

Oseltamivir-resistant influenza viruses circulating during the first year of the influenza A(H1N1) 2009 pandemic in the Asia-Pacific region, March 2009 to March 2010.

During the first year of the influenza A(H1N1) 2009 pandemic, unprecedented amounts of the neuraminidase inhibitors, predominantly oseltamivir, were used in economically developed countries for the treatment and prophylaxis of patients prior to the availability of a pandemic vaccine. Due to concerns about the development of resistance, over 1,400 influenza A(H1N1) 2009 viruses isolated from the Asia-Pacific region during the first year of the pandemic (March 2009 to March 2010) were analysed by phenotypic and genotypic assays to determine their susceptibility to the neuraminidase inhibitors. Amongst viruses submitted to the World Health Organization Collaborating Centre for Reference and Research in Melbourne, Australia,oseltamivir resistance was detected in 1.3% of influenza A(H1N1) 2009 strains from Australia and 3.1% of strains from Singapore, but none was detected in specimens received from other countries in Oceania or south-east Asia, or in east Asia. The overall frequency of oseltamivir resistance in the Asia-Pacific region was 16 of 1,488 (1.1%). No zanamivir-resistant viruses were detected. Of the 16 oseltamivir-resistant isolates detected, nine were from immunocompromised individuals undergoing oseltamivir treatment and three were from immunocompetent individuals undergoing oseltamivir treatment. Importantly, four oseltamivir-resistant strains were from immunocompetent individuals who had not been treated with oseltamivir, demonstrating limited low-level community transmission of oseltamivir-resistant strains. Even with increased use of oseltamivir during the pandemic, the frequency of resistance has been low, with little evidence of community-wide spread of the resistant strains. Nevertheless, prudent use of the neuraminidase inhibitors remains necessary, as does continued monitoring for drug-resistant influenza viruses.

Seroprevalence of 2009 pandemic influenza A(H1N1) virus in Australian blood donors, October - December 2009.

Assessment of the severity of disease due to the 2009 pandemic influenza A(H1N1) in Australian states and territories has been hampered by the absence of denominator data on population exposure. We compared antibody reactivity to the pandemic virus using haemagglutination inhibition assays performed on plasma specimens taken from healthy adult blood donors (older than 16 years) before and after the influenza pandemic that occurred during the southern hemisphere winter. Pre-influenza season samples (April ­ May 2009, n=496) were taken from donation collection centres in North Queensland (in Cairns and Townsville); post-outbreak specimens (October ­ November 2009, n=779) were from donors at seven centres in five states. Using a threshold antibody titre of 40 as a marker of recent infection, we observed an increase in the influenza-seropositive proportion of donors from 12% to 22%, not dissimilar to recent reports of influenza A(H1N1)-specific immunity in adults from the United Kingdom. No significant differences in seroprevalence were observed between Australian states, although the ability to detect minor variations was limited by the sample size. On the basis of these figures and national reporting data, we estimate that approximately 0.23% of all individuals in Australia exposed to the pandemic virus required hospitalisation and 0.01% died. The low seroprevalence reported here suggests that some degree of prior immunity to the virus, perhaps mediated by broadly reactive T-cell responses to conserved influenza viral antigens, limited transmission among adults and thus constrained the pandemic in Australia.

The impact of the pandemic influenza A(H1N1) 2009 virus on seasonal influenza A viruses in the southern hemisphere, 2009.

Data collected over winter 2009 by five World Health Organisation National Influenza Centres in the southern hemisphere were used to examine the circulation of pandemic and seasonal influenza A strains during the first pandemic wave in the southern hemisphere.There is compelling evidence that the pandemic influenza A(H1N1) 2009 virus significantly displaced seasonal influenza A(H1N1) and, to a lesser extent, A(H3N2) viruses circulating in the southern hemisphere. Complete replacement of seasonal influenza A strains, however, was not observed during the first pandemic wave.

Influenza in the Pacific

Influenza A and B viruses cause significant human disease worldwide through regular outbreaks and epidemics of seasonal influenza, and occasional pandemics when a novel influenza A virus emerges. Whereas Australia and New Zealand have well-established systems for community and laboratory-based surveillance of influenza, most other countries of the Pacific are only beginning to develop such systems with the support of various global and regional agencies and networks. Here we describe the role of the World Health Organization Global Influenza Surveillance Network and other organizations in laboratory-based influenza surveillance in the region and review some of the available data on seasonal and pandemic influenza in the developed and developing countries of the Pacific. The particular features of the Pacific Island countries and territories as small dispersed island communities, together with the greater susceptibility of indigenous people to the severe effects of influenza, highlight the importance of developing local laboratory-based surveillance systems. Such systems will improve the understanding, detection and control of seasonal influenza while also providing early warning of the emergence of potential pandemic viruses.

Immunity to avian influenza A viruses.

While the basic principles of immunity to the influenza A viruses are probably similar for all vertebrates, detailed understanding is based largely on experiments in laboratory mice. Elements of the innate response limit early virus replication, although high pathogenicity strains can trigger effusive cytokine/chemokine production and lethal shock. Virus clearance is normally mediated via CD8+ effector T cells but, in their absence, the class-switched antibody response can ultimately achieve the same goal. Protection against reinfection is optimally provided by antibody (IgG and IgA) specific for the homologous viral haemagglutinin, and priming against the neuraminidase and the low abundance, conserved M2 protein can also have an effect. Influenza virus-specific plasma cells and CD8+ T cells persist in the long term and the recall of the CD8+ T cell response can lead to earlier virus clearance. The characteristics of the aging immune system and possible, novel vaccine strategies are also considered.

A critical role for Dnmt1 and DNA methylation in T cell development, function, and survival.

The role of DNA methylation and of the maintenance DNA methyltransferase Dnmt1 in the epigenetic regulation of developmental stage- and cell lineage-specific gene expression in vivo is uncertain. This is addressed here through the generation of mice in which Dnmt1 was inactivated by Cre/loxP-mediated deletion at sequential stages of T cell development. Deletion of Dnmt1 in early double-negative thymocytes led to impaired survival of TCRalphabeta(+) cells and the generation of atypical CD8(+)TCRgammadelta(+) cells. Deletion of Dnmt1 in double-positive thymocytes impaired activation-induced proliferation but differentially enhanced cytokine mRNA expression by naive peripheral T cells. We conclude that Dnmt1 and DNA methylation are required for the proper expression of certain genes that define fate and determine function in T cells.

CD4 ligation promotes the IL-4-independent development of IL-4-producing clones from naive CD4(+) T cells.

The signals that trigger IL-4-independent IL-4 synthesis by conventional CD4(+) T cells are not yet defined. In this study, we show that coactivation with anti-CD4 mAb can stimulate single naive CD4(+) T cells to form IL-4-producing clones in the absence of APC and exogenous IL-4, independently of effects on proliferation. When single CD4(+) lymph node cells from C57BL/6 mice were cultured with immobilized anti-CD3epsilon mAb and IL-2, 65-85% formed clones over 12-14 days. Coimmobilization of mAb to CD4, CD11a, and/or CD28 increased the size of these clones but each exerted different effects on their cytokine profiles. Most clones produced IFN-gamma and/or IL-3 regardless of the coactivating mAb. However, whereas 0-6% of clones obtained with mAb to CD11a or CD28 produced IL-4, 10-40% of those coactivated with anti-CD4 mAb were IL-4 producers. A similar response was observed among CD4(+) cells from BALB/c mice. Most IL-4-producing clones were derived from CD4(+) cells of naive (CD44(low) or CD62L(high)) phenotype and the great majority coproduced IFN-gamma and IL-3. The effect of anti-CD4 mAb on IL-4 synthesis could be dissociated from effects on clone size since anti-CD4 and anti-CD11a mAb stimulated formation of clones of similar size which differed markedly in IL-4 production. Engagement of CD3 and CD4 in the presence of IL-2 is therefore sufficient to induce a substantial proportion of naive CD4(+) T cells to form IL-4-producing clones in the absence of other exogenous signals, including IL-4 itself.

Regulation of T cell cytokine production by dendritic cells.

Previous work has established that the dendritic cells (DC) of mouse spleen regulate the IL-2 production, and hence the extent of proliferation, of the CD8 T cells they activate. It is now reported here that interaction of primary CD8 T cells with splenic CD8alpha- DC induced much higher production of IL-3, IFN-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF), as well as IL-2, than did interaction with CD8alpha+ splenic DC. Furthermore, the CD8alpha- DC also induced higher levels of IL-2, IL-3 and IL-10 production in primary CD4 T cells, compared with that induced by CD8alpha+ DC. These quantitative differences did not involve qualitative shifts in the type of cytokine produced. Interleukin-4 production remained low in all the primary T cell cultures and restimulation experiments in secondary cultures did not reveal any bias in the cytokine production profile. When exogenous IL-2 was added to the primary cultures to ensure equal proliferation in response to CD8alpha- or CD8alpha+ DC, the higher level of production of IL-3, IFN-gamma and GM-CSF induced by CD8alpha- DC was maintained. Thus, this general control of T cell cytokine production by splenic DC involves factors additional to those that govern activation of T cells into cell cycle.

Differential effects of CD4 and CD8 engagement on the development of cytokine profiles of murine CD4+ and CD8+ T lymphocytes.

A simple culture system devoid of antigen-presenting cells was used to examine the ability of immobilized antibodies to lymphocyte function-associated antigen-1 (LFA-1) (CD11a), CD28 and CD4 or CD8 to modulate the responses of normal murine CD4+ and CD8+ lymph node T cells to immobilized anti-CD3 antibody and interleukin-2 (IL-2). All the antibodies enhanced proliferative responses to limiting anti-CD3 antibody. Both CD4+ and CD8+ cells produced substantial titres of IL-3 and interferon-gamma (IFN-gamma) in primary and secondary cultures regardless of the coactivating antibodies used for priming. By contrast, the combination of anti-CD4 with anti-CD3 antibody stimulated significantly higher titres of IL-4 than any other antibody combination in cultures of CD4+ cells. This CD4-dependent IL-4 response was induced in CD4+ T cells of naive (CD44low) phenotype and was similar in magnitude to the response induced by exogenous IL-4 but, unlike the latter, was not associated with elevated IL-3 synthesis. A comparable effect of anti-CD8 antibodies on CD8+ cells was not observed: although IL-4 production by CD8+ cells was induced by exogenous IL-4, it was not detected following coactivation with anti-CD8 or any other antibodies. We conclude that anti-CD4 antibody is a potent inducer of IL-4-secreting CD4+ T cells whose effects can be distinguished from those of anti-CD8 antibody on CD8+ T cells and from those of IL-4 on either subset.

Cytokines and their receptors: an overview.

Cytokines participate in the induction and effector phases of all immune and inflammatory responses. They are therefore obvious candidates for exploitation as drugs or drug targets to promote, limit, or alter these responses in infection, allergy, autoimmunity, and other disease states. Although some cytokines and related molecules are already in clinical use, the full therapeutic potential of this class of hormones has yet to be realized, and this will depend in part on understanding their normal functions and regulation in health and disease. An overview is given of the cytokines and their receptors, their genetic and structural relationships, and the regulation of their activities by naturally occurring antagonists and other physiologic mechanisms. Some of the ways in which new therapeutic strategies are being developed from knowledge of the structure, function, and regulation of these various components of the cytokine network are outlined.

Nature versus nurture in T cell cytokine production.

Both extrinsic and intrinsic factors influence the development of cytokine expression patterns in T lymphocytes. The models proposed to accommodate these factors are often separated into two types: deterministic (or instructional) and probabilistic (or stochastic). In this review we compare these two types of models and examine how they account for different stages of T cell cytokine responses to antigen stimulation. We conclude by showing how a reconciliation of the two types of models may be possible, perhaps through the regulatory potential of heritable epigenetic mechanisms. This type of reconciliation may open up new avenues for manipulating T cell cytokine expression and redirecting immune responses.

The activated type 1-polarized CD8(+) T cell population isolated from an effector site contains cells with flexible cytokine profiles.

The capacity of activated T cells to alter their cytokine expression profiles after migration into an effector site has not previously been defined. We addressed this issue by paired daughter analysis of a type 1-polarized CD8(+) effector T cell population freshly isolated from lung parenchyma of influenza virus-infected mice. Single T cells were activated to divide in vitro; individual daughter cells were then micromanipulated into secondary cultures with and without added IL-4 to assess their potential to express type 2 cytokine genes. The resultant subclones were analyzed for type 1 and 2 cytokine mRNAs at day 6-7. When the most activated (CD44(high)CD11a(high)) CD8(+) subpopulation from infected lung was compared with naive or resting (CD44(low)CD11a(low)) CD8(+) cells from infected lung and from normal lymph nodes (LNs), both clonogenicity and plasticity of the cytokine response were highest in the LN population and lowest in the activated lung population, correlating inversely with effector function. Multipotential cells were nevertheless detected among clonogenic CD44(high)CD11a(high) lung cells at 30-50% of the frequency in normal LNs. The data indicate that activated CD8(+) T cells can retain the ability to proliferate and express new cytokine genes in response to local stimuli after recruitment to an effector site.

Methodological issues in volumetric magnetic resonance imaging of the brain in the Edinburgh High Risk Project.

The Edinburgh High Risk Project is a longitudinal study of brain structure (and function) in subjects at high risk of developing schizophrenia in the next 5-10 years for genetic reasons. In this article we describe the methods of volumetric analysis of structural magnetic resonance images used in the study. We also consider potential sources of error in these methods: the validity of our image analysis techniques; inter- and intra-rater reliability; possible positional variation; and thresholding criteria used in separating brain from cerebro-spinal fluid (CSF). Investigation with a phantom test object (of similar imaging characteristics to the brain) provided evidence for the validity of our image acquisition and analysis techniques. Both inter- and intra-rater reliability were found to be good in whole brain measures but less so for smaller regions. There were no statistically significant differences in positioning across the three study groups (patients with schizophrenia, high risk subjects and normal volunteers). A new technique for thresholding MRI scans longitudinally is described (the 'rescale' method) and compared with our established method (thresholding by eye). Few differences between the two techniques were seen at 3- and 6-month follow-up. These findings demonstrate the validity and reliability of the structural MRI analysis techniques used in the Edinburgh High Risk Project, and highlight methodological issues of general concern in cross-sectional and longitudinal studies of brain structure in healthy control subjects and neuropsychiatric populations.