Recombinant AAV-mediated Gene Therapy Approaches to Treat Progressive Familial Intrahepatic Cholestasis Type 3

Among contemporary gene transfer vehicles, non-pathogenic recombinant adeno-associated viral vectors (rAAV) show exceptional promise for liver-targeted therapeutic approaches. The broad focus of studies described in this thesis was the development of rAAV-mediated gene therapy to treat Progressive Familial Intrahepatic Cholestasis type 3. This autosomal recessive condition, caused by mutations of ABCB4, results in deficient hepatocanalicular phosphatidylcholine translocation and leads to progressive cholestatic liver disease with approximately 50% of patients requiring liver transplantation before reaching adulthood. Using an Abcb4-knockout mouse model, in vivo liver transduction with rAAV2/8 vectors encoding hABCB4 led to increased biliary phosphatidylcholine in disease-free heterozygous, but not in homozygous adults with established liver disease, despite varying vector genome size and routes of administration. Maximal transduction was achieved prior to onset of liver disease, optimally in neonates. However, loss of transgene expression occurs following neonatal vector delivery, due to rAAV episomal degradation during rapid liver growth. A novel, hybrid rAAV-piggyBac transposon vector strategy was devised to sustain hABCB4 expression in neonatally-treated homozygotes. Successful correction of liver disease was demonstrated in Abcb4-/- mice up to 9 months post-inoculation, with preliminary results indicating reduction in disease-related hepatocarcinogenic risk. These results demonstrate that rAAV-mediated gene therapy has the potential to offer patients with this heritable cholestatic liver disease an effective alternative treatment to liver transplantation, but also illustrate the importance of addressing challenges, such as the impact of liver pathology on vector performance, which is vital before this potential can be realised for this and related conditions

Histone H3.3 Variant Dynamics in the Germline of Caenorhabditis elegans

Germline chromatin undergoes dramatic remodeling events involving histone variants during the life cycle of an organism. A universal histone variant, H3.3, is incorporated at sites of active transcription throughout the cell cycle. The presence of H3.3 in chromatin indicates histone turnover, which is the energy-dependent removal of preexisting histones and replacement with new histones. H3.3 is also incorporated during decondensation of the Drosophila sperm pronucleus, indicating a direct role in chromatin remodeling upon fertilization. Here we present a system to monitor histone turnover and chromatin remodeling during Caenorhabditis elegans development by following the developmental dynamics of H3.3. We generated worm strains expressing green fluorescent protein– or yellow fluorescent protein–fused histone H3.3 proteins, HIS-71 and HIS-72. We found that H3.3 is retained in mature sperm chromatin, raising the possibility that it transmits epigenetic information via the male germline. Upon fertilization, maternal H3.3 enters both male and female pronuclei and is incorporated into paternal chromatin, apparently before the onset of embryonic transcription, suggesting that H3.3 can be incorporated independent of transcription. In early embryos, H3.3 becomes specifically depleted from primordial germ cells. Strikingly, the X chromosome becomes deficient in H3.3 during gametogenesis, indicating a low level of histone turnover. These results raise the possibility that the asymmetry in histone turnover between the X chromosome and autosomes is established during gametogenesis. H3.3 patterns are similar to patterns of H3K4 methylation in the primordial germ cells and on the X chromosome during gametogenesis, suggesting that histone turnover and modification are coupled processes. Our demonstration of dynamic H3.3 incorporation in nondividing cells provides a mechanistic basis for chromatin changes during germ cell development

Supporting rural midwifery practice using a mobile health (mHealth) intervention: a qualitative descriptive study.

Introduction: Hypertensive disorders in pregnancy account for 12% of all maternal deaths globally. The risks of sub-optimal outcomes from these disorders might be greater in rural and remote locations. These potential risks might be related to poor intra and inter-professional communications due to geographic and digital isolation. Studies in low- and middle-income countries (LMICs) suggest that improving communications is essential and that mobile health (mHealth) interventions can improve outcomes. However, for such interventions to be successful they must involve midwives in any design and software development. This study explored how a mHealth intervention might support midwives in the management of women with pre-eclampsia in Scottish rural and remote locations. Methods: A qualitative descriptive approach was adopted. Rural and remote practicing community midwives (n = 18) were recruited to participate in three focus groups. The data were gathered through digital recordings of conversations at these focus groups. Recordings were transcribed and thematically analysed. Themes were agreed by consensus with the research team in an iterative process. Results: Five principal themes were identified: 'working in isolation'; 'encountering women with pre-eclampsia in rural and remote settings'; 'learning on the move'; 'using audio-visual resources' and 'unease with advances in technology'. Conclusion: Geographic and digital isolation poses significant challenges to rural midwifery practice in a high income country such as Scotland. Midwives need to be involved in the development of mHealth interventions for them to be acceptable and tailored to their needs in a rural and remote context. The study highlights how mHealth interventions can support Continuous Professional Development (CPD) whilst on the move with no internet connectivity. However, pride in current practice and unease with advances in mobile technology are barriers to the adoption of a mHealth intervention. mHealth interventions could be of value to other specialised healthcare practitioners in these regions, including GPs, to manage women with complications in their pregnancies

Therapeutic exercises for affecting post-treatment swallowing in people treated for advanced-stage head and neck cancers: a Cochrane systematic review.

Head and neck cancer encompasses tumours of the upper aero-digestive tract (oral cavity, pharynx, larynx, nasal cavity, paranasal sinuses and salivary glands). There are 550,000 new cases, globally, each year, the majority of these are mucosal small cell carcinoma. Smoking, alcohol abuse and viral infection are associated with the disease. Although survival rates are improving, morbidity has correspondingly increased, specifically speech and swallowing. Dysphagia may be temporary/long term or permanent and this has implacations with food and fluid intake, social participation and psychological wellbeing. The objective of this research was to establish evidence for the effects of therapeutic swallowing exercises, undertaken before, during and/or immediately after head and neck (HNC) treatment, on swallowing

AN EXPLORATORY STUDY OF LECTURERS' VIEWS OF OUT-OF-CLASS ACADEMIC COLLABORATION AMONG STUDENTS

This article reports an exploratory study of lecturers' perceptions of out-of-class academic collaboration (OCAC) among students at a large Singapore university. Two types of OCAC were investigated: collaboration initiated by students, e.g., groups decide on their own to meet to prepare for exams, and collaboration required by teachers, e.g., teachers assign students to do projects in groups. Data were collected via one-on-one interviews with 18 faculty members from four faculties at the university. Findings suggest that OCAC, especially of a teacher-required kind, is fairly common at the university. Faculty members' views on factors affecting the success of OCAC are discussed for the light they might shed on practices to enhance the effectiveness of OCAC

Peripheral proinsulin expression controls low-avidity proinsulin-reactive CD8 T Cells in type 1 diabetes

Low-avidity autoreactive CD8 T cells (CTLs) escape from thymic negative selection, and peripheral tolerance mechanisms are essential for their regulation. We report the role of proinsulin (PI) expression on the development and activation of insulin-specific CTLs in the NOD mouse model of type 1 diabetes. We studied insulin B-chain–specific CTL from different T-cell receptor transgenic mice (G9Cα−/−) expressing normal PI1 and PI2 or altered PI expression levels. In the absence of PI2 (Ins2−/−), CTL in pancreatic lymph nodes (PLNs) were more activated, and male G9Cα−/− mice developed T1D. Furthermore, when the insulin-specific CTLs developed in transgenic mice lacking their specific PI epitope, the CTLs demonstrated increased cytotoxicity and proliferation in vitro and in vivo in the PLNs after adoptive transfer into NOD recipients. Dendritic cell–stimulated proliferation of insulin-specific T cells was reduced in the presence of lymph node stromal cells (LNSCs) from NOD mice but not from mice lacking the PI epitope. Our study shows that LNSCs regulate CTL activation and suggests that exposure to PI in the periphery is very important in maintenance of tolerance of autoreactive T cells. This is relevant for human type 1 diabetes and has implications for the use of antigen-specific therapy in tolerance induction

Human olfactory mesenchymal stromal cell transplantation ameliorates experimental autoimmune encephalomyelitis revealing an inhibitory role for IL16 on myelination

One of the therapeutic approaches for the treatment of the autoimmune demyelinating disease, multiple sclerosis (MS) is bone marrow mesenchymal stromal cell (hBM-MSCs) transplantation. However, given their capacity to enhance myelination in vitro, we hypothesised that human olfactory mucosa-derived MSCs (hOM-MSCs) may possess additional properties suitable for CNS repair. Herein, we have examined the efficacy of hOM-MSCs versus hBM-MSCs using the experimental autoimmune encephalomyelitis (EAE) model. Both MSC types ameliorated disease, if delivered during the initial onset of symptomatic disease. Yet, only hOM-MSCs improved disease outcome if administered during established disease when animals had severe neurological deficits. Histological analysis of spinal cord lesions revealed hOM-MSC transplantation reduced blood–brain barrier disruption and inflammatory cell recruitment and enhanced axonal survival. At early time points post-hOM-MSC treatment, animals had reduced levels of circulating IL-16, which was reflected in both the ability of immune cells to secrete IL-16 and the level of IL-16 in spinal cord inflammatory lesions. Further in vitro investigation revealed an inhibitory role for IL-16 on oligodendrocyte differentiation and myelination. Moreover, the availability of bioactive IL-16 after demyelination was reduced in the presence of hOM-MSCs. Combined, our data suggests that human hOM-MSCs may have therapeutic benefit in the treatment of MS via an IL-16-mediated pathway, especially if administered during active demyelination and inflammation

A reproducible method for the expansion of mouse CD8+ T lymphocytes

Murine adoptive CD8 + T-cell immunotherapy studies require the generation of large numbers of high viability CD8 + cells. Here we report a tissue culture protocol for the reliable expansion of CD8 + T-cells derived from murine spleen to give a 20-fold expansion after 4 days in culture. The cells were transfected with an mRNA GFP construct and transferred into NOD mice. GFP positive cells could be detected 7 days after transfer thus confirming that the cells survive and are functional for up to 1 week

Adoptive transfer of mRNA-Transfected T cells redirected against diabetogenic CD8 T cells can prevent diabetes

Chimeric major histocompatibility complex (MHC) molecules supplemented with T cell receptor (TCR) signaling motifs function as activation receptors and can redirect gene-modified T cells against pathogenic CD8 T cells. We have shown that β2 microglobulin (β2m) operates as a universal signaling component of MHC-I molecules when fused with the CD3-ζ chain. Linking the H-2Kd-binding insulin B chain peptide insulin B chain, amino acids 15–23 (InsB15–23) to the N terminus of β2m/CD3-ζ, redirected polyclonal CD8 T cells against pathogenic CD8 T cells in a peptide-specific manner in the non-obese diabetic (NOD) mouse. Here, we describe mRNA electroporation for delivering peptide/β2m/CD3-ζ genes to a reporter T cell line and purified primary mouse CD8 T cells. The peptide/β2m/CD3-ζ products paired with endogenous MHC-I chains and transmitted strong activation signals upon MHC-I cross-linking. The reporter T cell line transfected with InsB15–23/β2m/CD3-ζ mRNA was activated by an InsB15–23-H-2Kd-specific CD8 T cell hybrid only when the transfected T cells expressed H-2Kd. Primary NOD CD8 T cells expressing either InsB15–23/β2m/CD3-ζ or islet-specific glucose-6-phosphatase catalytic subunit-related protein, amino acids 206–214 (IGRP206–214)/β2m/CD3-ζ killed their respective autoreactive CD8 T cell targets in vitro. Furthermore, transfer of primary CD8 T cells transfected with InsB15–23/β2m/CD3-ζ mRNA significantly reduced insulitis and protected NOD mice from diabetes. Our results demonstrate that mRNA encoding chimeric MHC-I receptors can redirect effector CD8 against diabetogenic CD8 T cells, offering a new approach for the treatment of type 1 diabetes