Characterisation of phenotypic determinants in rat models of growth retardation

In this thesis, I describe work on the physiological basis of dwarfism in both established (dw/dw) and novel (78N) rat models. In the dwarf (dw/dw) rat, an unknown mutation causes a severe, sub-total growth hormone (GH) deficiency associated with somatotroph hypoplasia, and an increase in lactotroph numbers and prolactin (PRL) storage. It has, recently, been shown that the dw/dw pituitary contains a unique, morphologically distinct lactotroph, the 'intermediate' lactotroph, which has morphological features between those of the type I and II lactotroph subtypes. These 'intermediate' lactotrophs also show functional features of somatotrophic lineage, releasing PRL in response to ghrelin. This female-specific induction of PRL secretion is oestrogen-dependent. The present study reveals that in pregnancy the population of 'intermediate' lactotrophs increases in parallel with the lactotrophic lineage. We have taken two approaches to investigate whether elevated GH releasing factor (GRF) gives rise to the 'intermediate' lactotroph in the dw/dw pituitary. The absence of the 'intermediate' lactotroph in the GRF-insensitive little (lit/lit) mouse, and a possible reduction in 'intermediate' lactotrophs in monosodium glutamate (MSG)-treated dw/dw rats, suggest that the presence of this unique lactotroph subtype in dw/dw pituitary is regulated, in part by GRF. Although small numbers of 'intermediate' lactotrophs are present in pregnant normal female rats, they do not appear to play a significant role. Pregnancy is also associated with an increase in type I lactotrophs, but no change in somatotrophs. The accompanying increase in baseline circulating GH does not elevate plasma insulin like growth factor I (IGF-I) levels, or stimulate skeletal growth, and may be derived from a placental GH-like protein. A novel transgenic rat line---78N---was previously generated in our laboratory. It is a presumed insertional-mutant that exhibits a pleiotropic phenotype associated with neonatal male lethality. Females exhibit juvenile-onset growth retardation. Initial investigation of the cause of the growth retardation showed that the skeletal impairment is GH-independent. In the present study, parallel postmortem examination has revealed kidney abnormalities in both male and female mutants. Morphological analysis showed alteration of the normal cortico-medullary architecture. Molecular analysis of the neonatal male kidney transcriptome revealed numerous differentially expressed genes including kidney androgen-regulated protein (KAP) and neural precursor cells expressed developmentally down-regulated 4 (Nedd4) WW domain binding protein (N4WBP4). The 78N line may be a useful model of idiopathic short stature (ISS) associated with idiopathic nephritic syndrome---minimal change nephropathy (MCD) or disease

Fetus-derived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction.

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.M.A.M.C. was supported by a PhD studentship from the Cambridge Centre for Trophoblast Research. Research was supported by grants from the MRC (MR/J001597/1 and MR/L002345/1), the Medical College of Saint Bartholomew's Hospital Trust, a Wellcome Trust Investigator Award, EpigeneSys (FP7 Health-257082), EpiHealth (FP7 Health-278414), a Herchel Smith Fellowship (N.T.) and NIH grant RO1 DK89989. The contents are the authors' sole responsibility and do not necessarily represent official NIH views. We thank G. Burton for invaluable support, and M. Constância and I. Sandovici (University of Cambridge) for the Meox2-cre mice. We are extremely grateful to all of the participants in the Pregnancy Outcome Prediction study. This work was supported by the NIHR Cambridge Comprehensive Biomedical Research Centre (Women's Health theme) and project grants from the MRC (G1100221) and Sands (Stillbirth and Neonatal Death Charity). The study was also supported by GE Healthcare (donation of two Voluson i ultrasound systems for this study) and by the NIHR Cambridge Clinical Research Facility, where all research visits took place.This is the author accepted manuscript. The final version is available from Nature Publishing Group via https://doi.org/10.1038/ng.369

Integrating Diverse Datasets Improves Developmental Enhancer Prediction

Gene-regulatory enhancers have been identified using various approaches, including evolutionary conservation, regulatory protein binding, chromatin modifications, and DNA sequence motifs. To integrate these different approaches, we developed EnhancerFinder, a two-step method for distinguishing developmental enhancers from the genomic background and then predicting their tissue specificity. EnhancerFinder uses a multiple kernel learning approach to integrate DNA sequence motifs, evolutionary patterns, and diverse functional genomics datasets from a variety of cell types. In contrast with prediction approaches that define enhancers based on histone marks or p300 sites from a single cell line, we trained EnhancerFinder on hundreds of experimentally verified human developmental enhancers from the VISTA Enhancer Browser. We comprehensively evaluated EnhancerFinder using cross validation and found that our integrative method improves the identification of enhancers over approaches that consider a single type of data, such as sequence motifs, evolutionary conservation, or the binding of enhancer-associated proteins. We find that VISTA enhancers active in embryonic heart are easier to identify than enhancers active in several other embryonic tissues, likely due to their uniquely high GC content. We applied EnhancerFinder to the entire human genome and predicted 84,301 developmental enhancers and their tissue specificity. These predictions provide specific functional annotations for large amounts of human non-coding DNA, and are significantly enriched near genes with annotated roles in their predicted tissues and lead SNPs from genome-wide association studies. We demonstrate the utility of EnhancerFinder predictions through in vivo validation of novel embryonic gene regulatory enhancers from three developmental transcription factor loci. Our genome-wide developmental enhancer predictions are freely available as a UCSC Genome Browser track, which we hope will enable researchers to further investigate questions in developmental biology. © 2014 Erwin et al

Validating the portal population of the United Kingdom Multiple Sclerosis Register

MS Society. Award 64

A multicentre, randomised controlled trial to compare the clinical and cost-effectiveness of Lee Silverman Voice Treatment versus standard NHS Speech and Language Therapy versus control in Parkinson’s disease: a study protocol for a randomised controlled trial

Abstract: Background: Parkinson’s disease (PD) affects approximately 145,519 people in the UK. Speech impairments are common with a reported prevalence of 68%, which increase physical and mental demands during conversation, reliance on family and/or carers, and the likelihood of social withdrawal reducing quality of life. In the UK, two approaches to Speech and Language Therapy (SLT) intervention are commonly available: National Health Service (NHS) SLT or Lee Silverman Voice Treatment (LSVT LOUD®). NHS SLT is tailored to the individuals’ needs per local practice typically consisting of six to eight weekly sessions; LSVT LOUD® comprises 16 sessions of individual treatment with home-based practice over 4 weeks. The evidence-base for their effectiveness is inconclusive. Methods/design: PD COMM is a phase III, multicentre, three-arm, unblinded, randomised controlled trial. Five hundred and forty-six people with idiopathic PD, reporting speech or voice problems will be enrolled. We will exclude those with a diagnosis of dementia, laryngeal pathology or those who have received SLT for speech problems in the previous 2 years. Following informed consent and completion of baseline assessments, participants will be randomised in a 1:1:1 ratio to no-intervention control, NHS SLT or LSVT LOUD® via a central computer-generated programme, using a minimisation procedure with a random element, to ensure allocation concealment. Participants randomised to the intervention groups will start treatment within 4 (NHS SLT) or 7 (LSVT LOUD®) weeks of randomisation. Primary outcome: Voice Handicap Index (VHI) total score at 3 months. Secondary outcomes include: VHI subscales, Parkinson’s Disease Questionnaire-39; Questionnaire on Acquired Speech Disorders; EuroQol-5D-5 L; ICECAP-O; resource utilisation; adverse events and carer quality of life. Mixed-methods process and health economic evaluations will take place alongside the trial. Assessments will be completed before randomisation and at 3, 6 and 12 months after randomisation. The trial started in December 2015 and will run for 77 months. Recruitment will take place in approximately 42 sites around the UK. Discussion: The trial will test the hypothesis that SLT is effective for the treatment of speech or voice problems in people with PD compared to no SLT. It will further test whether NHS SLT or LSVT LOUD® provide greater benefit and determine the cost-effectiveness of both interventions. Trial registration: International Standard Randomised Controlled Trials Number (ISRCTN) Registry, ID: 12421382. Registered on 18 April 2016

A multicentre, randomised controlled trial to compare the clinical and cost-effectiveness of Lee Silverman Voice Treatment versus standard NHS Speech and Language Therapy versus control in Parkinson’s disease: a study protocol for a randomised controlled trial

Abstract: Background: Parkinson’s disease (PD) affects approximately 145,519 people in the UK. Speech impairments are common with a reported prevalence of 68%, which increase physical and mental demands during conversation, reliance on family and/or carers, and the likelihood of social withdrawal reducing quality of life. In the UK, two approaches to Speech and Language Therapy (SLT) intervention are commonly available: National Health Service (NHS) SLT or Lee Silverman Voice Treatment (LSVT LOUD®). NHS SLT is tailored to the individuals’ needs per local practice typically consisting of six to eight weekly sessions; LSVT LOUD® comprises 16 sessions of individual treatment with home-based practice over 4 weeks. The evidence-base for their effectiveness is inconclusive. Methods/design: PD COMM is a phase III, multicentre, three-arm, unblinded, randomised controlled trial. Five hundred and forty-six people with idiopathic PD, reporting speech or voice problems will be enrolled. We will exclude those with a diagnosis of dementia, laryngeal pathology or those who have received SLT for speech problems in the previous 2 years. Following informed consent and completion of baseline assessments, participants will be randomised in a 1:1:1 ratio to no-intervention control, NHS SLT or LSVT LOUD® via a central computer-generated programme, using a minimisation procedure with a random element, to ensure allocation concealment. Participants randomised to the intervention groups will start treatment within 4 (NHS SLT) or 7 (LSVT LOUD®) weeks of randomisation. Primary outcome: Voice Handicap Index (VHI) total score at 3 months. Secondary outcomes include: VHI subscales, Parkinson’s Disease Questionnaire-39; Questionnaire on Acquired Speech Disorders; EuroQol-5D-5 L; ICECAP-O; resource utilisation; adverse events and carer quality of life. Mixed-methods process and health economic evaluations will take place alongside the trial. Assessments will be completed before randomisation and at 3, 6 and 12 months after randomisation. The trial started in December 2015 and will run for 77 months. Recruitment will take place in approximately 42 sites around the UK. Discussion: The trial will test the hypothesis that SLT is effective for the treatment of speech or voice problems in people with PD compared to no SLT. It will further test whether NHS SLT or LSVT LOUD® provide greater benefit and determine the cost-effectiveness of both interventions. Trial registration: International Standard Randomised Controlled Trials Number (ISRCTN) Registry, ID: 12421382. Registered on 18 April 2016

Experimental Neuroinflammation: A Study of Hypoxia and Protein Translation

Multiple sclerosis is a chronic inflammatory disease of the CNS associated with widespread primary demyelination and axonal degeneration. The mechanisms underlying the expression of neurological deficits are incompletely understood. Recent histological findings are consistent with a view that active MS lesions may be hypoxic (i.e. suffer a low oxygen concentration). For example, inflammatory ‘Pattern III’ MS lesions express hypoxia inducible factor-1a (HIF-1a), a master regulator of genes whose function is to bias a cell for survival under hypoxic conditions. Hypoxia also results in a number of other consequences designed to limit energy expenditure, including the inhibition of protein translation by the phosphorylation of the eukaryotic initiation factor (eIF-2a), and the formation of stress granules (SGs). Both hypoxia and the inhibition of protein synthesis could cause neurological deficits and thus contribute to the neurological deficits of MS. The aim of the present study is to explore experimental models of MS for evidence of a) hypoxia and b) inhibition of protein translation. The experimental models of inflammatory demyelinating lesions were induced either by the intraspinal injection of lipopolysaccharide (LPS) into the rat dorsal column (LPS-DC, focal lesion), or by immunization of rats or mice to induce experimental autoimmune encephalomyelitis (EAE, disseminated lesions). Animals were examined daily for the expression of any neurological deficit, and tissue hypoxia was detected during life by the systemic administration of pimonidazole, a marker for hypoxia, several hours before termination. Tissue was taken at different stages of lesion development (1-28 days post injection) and examined immunohistochemically for the presence of hypoxia, determined by the expression of binding for pimonidazole, and for the inhibition of protein translation by examining the expression of eIF-2a and SGs. Other markers of disease activity were also examined, including a marker of microglial/macrophage activation (ED-1), HIF-1a, and glucose transporter-1 (GLUT-1). In LPS lesions, labelling for pimonidazole was most intense at the site of injection, 24 hours later. In EAE, labelling for pimonidazole was present as early as 2 days post immunization, but it was expressed more strongly when animals were exhibiting a neurological deficit, subsiding thereafter. In animals injected with LPS, eIF-2a and SGs were expressed most intensely 24 hours post LPS injection, localised to the spinal motor neurons. In EAE, eIF-2a and SGs were expressed in spinal motor neurons, and in cerebellar neurons, at the onset of neurological deficits. These findings reveal for the first time that inflammatory demyelinating lesions are associated with the presence of tissue hypoxia and markers of the inhibition of protein synthesis. It appears that these phenomena may contribute to the expression of neurological deficits, opening new opportunities for therapy

The intermediate lactotroph: A morphologically distinct, ghrelin-responsive pituitary cell in the dwarf (dw/dw) Rat

Profound somatotroph hypoplasia in the dwarf (dw/dw) rat is accompanied by an estrogen-dependent induction of prolactin secretion by the GH secretagogue, GHRP-6. Using electron microscopy, we demonstrated that the reduction in the somatotroph population in the dw/dw pituitary is accompanied by the presence of a morphologically distinct lactotroph subpopulation. In these cells, which did not coexpress GH, the size, shape, and number of the secretory granules were between those of the type I and type II lactotrophs. We therefore called these cells intermediate lactotrophs. The intermediate lactotrophs accounted for up to 30% of the total prolactin-positive cell population in dw/dw males and up to 12% in females. Using tannic acid to quantify the fusion of secretory granules, we have shown that the intermediate lactotrophs are unresponsive to either GH-releasing factor (GRF) or TRH but exhibit a sexually dimorphic secretory response to acute ghrelin treatment, granular fusions being 4-fold higher in females. No cell matching the morphology of the novel lactotroph subpopulation was observed in the pituitary of the GRF-insensitive lit/lit mouse. However, ablation of GRF neurons with neonatal monosodium glutamate treatment had no effect on the population of intermediate lactotrophs in the dw/dw rat. Thus, the presence of the intermediate lactotrophs in the dw/dw pituitary appears to be independent of the function of the GRF neurons.Icnelia Huerta-Ocampo, Helen C. Christian, Nichola M. Thompson, Muna M. El-Kasti, and Timothy Well

Intensive nutrition support may benefit patients with a rare mitochondrial disorder

Mitochondrial neurogastrointestinal encephalopathy (MNGIE) is a rare, inherited, multisystemic autosomal recessive disorder caused by mutations in the nuclear TYMP gene. This syndrome is characterized by ptosis, external ophthalmoplegia, gastrointestinal dysmotility, cachexia, peripheral neuropathy, and leukoencephalopathy. Our case illustrates a patient diagnosed with MNGIE and cachexia who has benefited from the initiation and maintenance of parenteral nutrition. We highlight the benefits of receiving long-term supplementary home parenteral nutrition under close monitoring for patients with this neurogastrointestinal disease in order to gain weight and maintain good health. © 2021 American Society for Parenteral and Enteral Nutritio