Daniel B. Hawcutt

Human and Mouse Enteric Nervous System Neurosphere Transplants Regulate the Function of Aganglionic Embryonic Distal Colon

BACKGROUND & AIMS Recent advances have raised the possibility of treating enteric nervous system (ENS) disorders with transplanted progenitor cells (ENSPC). Although these cells have been shown to migrate and differentiate after transplantation, no functional effects have been demonstrated. We therefore aimed to investigate whether embryonic mouse and neonatal human ENSPC can regulate the contractility of aganglionic bowel. METHODS Embryonic mouse and neonatal human ENSPC were grown as neurospheres before transplantation into aganglionic embryonic mouse hindgut explants and culture for 8-12 days. Engraftment and neural differentiation were confirmed using immunofluorescence and transmission electron microscopy. The contraction frequency of transplanted bowel was measured and compared with that of embryonic day 11.5 embryonic ganglionic and aganglionic bowel cultured for the same period. Calcium movement was measured at spatially defined points in bowel wall smooth muscle. Neural modulation of bowel contractility was assessed using tetrodotoxin. RESULTS Both mouse and human ENSPC migrated and differentiated after neurosphere transplantation. Transmission electron microscopy demonstrated the existence of synapses. Transplantation restored the high contraction frequency of aganglionic bowel to the lower rate of ganglionic bowel. Calcium imaging demonstrated that neurosphere transplantation coordinates intracellular free calcium levels. Both these effects were reversed by the addition of tetrodotoxin, indicating the functional effect of neurosphere-derived neurons. CONCLUSIONS Neonatal human gut is a source of ENSPC that can be transplanted to restore the contractile properties of aganglionic bowel by a neurally mediated mechanism. This may aid development of a stem cell-based treatment for Hirschsprungs disease.

Properties of secondary and tertiary human enteric nervous system neurospheres

UNLABELLED Advances in enteric nervous system (ENS) stem cell biology have raised the possibility of treating Hirschsprungs disease with ENS stem/progenitor cell (ENSPC) transplantation. This study aimed to expand ENSPC numbers by the growth and redissociation of neurospheres and assess their differential potential. METHODS Human ENS neurospheres were cultured as previously described and redissociated to generate secondary and tertiary neurospheres. Neurospheres were assessed for the presence of neuronal (PGP9.5), glial (S100), and stem cell (p75, nestin markers). The degree of immunofluorescence was quantified using the ImageJ program. Secondary/tertiary neurospheres were transplanted into mouse distal colon grown in tissue culture. RESULTS Secondary/tertiary neurospheres could be generated with exponentially increasing numbers. Tertiary neurospheres showed a significant increase in the proportion of p75 staining but a significant decrease in the proportion of S100 staining. After transplantation, secondary/tertiary neurosphere-derived cells positive for PGP9.5 and S100 could be identified. CONCLUSIONS It is possible to exponentially expand neurosphere and therefore ENSPC numbers by repeated dissociation and culture. There is a loss of S100-positive cells in secondary/tertiary neurospheres, but the ENSPCs remain capable of differentiating into neurons and glia when transplanted into an embryonic gut environment.

Pharmacogenetic Markers of Drug Efficacy and Toxicity

The action of a drug is dictated by its pharmacokinetic and pharmacodynamics properties, both of which can vary in different individuals because of environmental and genetic factors. Pharmacogenetics, the study of genetic factors determining drug response, has the potential to improve clinical outcomes through targeting therapies, individualizing dosing, preventing adverse drug reactions, and potentially rescuing previously failed therapies. Although there have been significant advances in pharmacogenetics over the last decade, only a few have been translated into clinical practice. However, with new rapid genotyping technologies, regulatory modernization, novel clinical trial designs, systems approaches, and integration of pharmacogenetic data into decision support systems, there is hope that pharmacogenetics, as an important component of the overall drive towards personalized medicine, will advance more quickly in the future. There will continue to be a need for collaboration between centers all over the world, and multisector working, capitalizing on the current data revolution.

Paediatric pharmacogenomics: an overview

Pharmacogenomics research is becoming more prevalent in both academia and the pharmaceutical industry. While some discoveries have been integrated into practice and are benefiting patient care, these successes have been limited given the vast amount of research undertaken. However, the advances in high-throughput genomic technologies, better study designs and improved understanding of complexity, means that pharmacogenomic determinants of drug response will continue to be identified. It is important to develop an understanding of the basis of pharmacogenomics in clinical teams to allow accurate interpretation of the findings, and facilitate their implementation into clinical care (if appropriate). This article explains the science behind pharmacogenomics, and describes some of the challenges that have been encountered in the field, with a specific focus on paediatrics.

Prioritising neonatal medicines research: UK Medicines for Children Research Network scoping survey

BackgroundThe dosing regimen and indications for many medicines in current use in neonatology are not well defined. There is a need to prioritise research in this area, but currently there is little information about which drugs are used in UK neonatal units and the research needs in this area as perceived by UK neonatologists.MethodsThe Neonatal Clinical Studies Group (CSG) of the Medicines for Children Research Network (MCRN) undertook a 2 week prospective scoping survey study to establish which medicines are used in UK neonatal units; how many babies are receiving them; and what clinicians (and other health professionals) believe are important issues for future research.Results49 out of 116 units responded to at least one element of the survey (42%). 37 units reported the number of neonates who received medicines over a 2 week period. A total of 3924 medicine-patient pairs were reported with 119 different medicines. 70% of medicine-patient pairs involved medicines that were missing either a license or dose for either term or preterm neonates. 4.3% of medicine-patient pairs involved medicines that were missing both license and dose for any neonate. The most common therapeutic gap in need of additional research identified by UK neonatologists was chronic lung disease (21 responding units), followed by patent ductus arteriosus and vitamin supplements (11 responding units for both)ConclusionThe research agenda for neonatal medicines can be informed by knowledge of current medicine use and the collective views of the neonatal community.

Early morning salivary cortisol and cortisone, and adrenal responses to a simplified low-dose short Synacthen test in children with asthma.

To examine serum cortisol responses to a simplified low‐dose short Synacthen test (LDSST) in children treated with inhaled corticosteroids (ICS) for asthma and to compare these to early morning salivary cortisol (EMSC) and cortisone (EMSCn) levels.

ST13 polymorphisms and their effect on exacerbations in steroid-treated asthmatic children and young adults

The clinical response to inhaled corticosteroids (ICS) is associated with single nucleotide polymorphisms (SNPs) in various genes. This study aimed to relate variations in genes in the steroid pathway and asthma susceptibility genes to exacerbations in children and young adults treated with ICS.

Adrenal responses to a low-dose short synacthen test in children with asthma.

Corticosteroids are known to cause adrenal suppression. The aim of this study was to assess clinical factors affecting responses to a low dose short synacthen test (LDSST) in asthmatic children using corticosteroids.

Pharmacogenetics of warfarin in a paediatric population: time in therapeutic range, initial and stable dosing and adverse effects.

Warfarin is used in paediatric populations, but dosing algorithms incorporating pharmacogenetic data have not been developed for children. Previous studies have produced estimates of the effect of polymorphisms in Cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex subunit 1 (VKORC1) on stable warfarin dosing, but data on time in therapeutic range, initial dosing and adverse effects are limited. Participants (n=97) were recruited, and routine clinical data and salivary DNA samples were collected from all participants and analysed for CYP2C9*2, *3 and VKORC1-1639 polymorphisms.VKORC1 -1639 was associated with a greater proportion of the first 6 months’ treatment time spent within the target International Normalised Ratio (INR) range, accounting for an additional 9.5% of the variance in the proportion of time. CYP2C9*2 was associated with a greater likelihood of INR values exceeding the target range during the initiation of treatment (odds ratio (OR; per additional copy) 4.18, 95% confidence interval (CI) 1.42, 12.34). CYP2C9*2 and VKORC1-1639 were associated with a lower dose requirement, and accounted for almost 12% of the variance in stable dose. VKORC1-1639 was associated with an increased likelihood of mild bleeding complications (OR (heterozygotes vs homozygotes) 4.53, 95% CI 1.59, 12.93). These data show novel associations between VKORC1-1639 and CYP2C9*2 and INR values in children taking warfarin, as well as replicating previous findings with regard to stable dose requirements. The development of pharmacogenomic dosing algorithms for children using warfarin has the potential to improve clinical care in this population.

Rationale and design of the multiethnic Pharmacogenomics in Childhood Asthma consortium

AIM International collaboration is needed to enable large-scale pharmacogenomics studies in childhood asthma. Here, we describe the design of the Pharmacogenomics in Childhood Asthma (PiCA) consortium. MATERIALS & METHODS Investigators of each study participating in PiCA provided data on the study characteristics by answering an online questionnaire. RESULTS A total of 21 studies, including 14,227 children/young persons (58% male), from 12 different countries are currently enrolled in the PiCA consortium. Fifty six percent of the patients are Caucasians. In total, 7619 were inhaled corticosteroid users. Among patients from 13 studies with available data on asthma exacerbations, a third reported exacerbations despite inhaled corticosteroid use. In the future pharmacogenomics studies within the consortium, the pharmacogenomics analyses will be performed separately in each center and the results will be meta-analyzed. CONCLUSION PiCA is a valuable platform to perform pharmacogenetics studies within a multiethnic pediatric asthma population.