Rosanne M. Taylor

Global gene and cell replacement strategies via stem cells

The inherent biology of neural stem cells (NSCs) endows them with capabilities that not only circumvent many of the limitations of other gene transfer vehicles, but that enable a variety of novel therapeutic strategies heretofore regarded as beyond the purview of neural transplantation. Most neurodegenerative diseases are characterized not by discrete, focal abnormalities but rather by extensive, multifocal, or even global neuropathology. Such widely disseminated lesions have not conventionally been regarded as amenable to neural transplantation. However, the ability of NSCs to engraft diffusely and become integral members of structures throughout the host CNS, while also expressing therapeutic molecules, may permit these cells to address that challenge. Intriguingly, while NSCs can be readily engineered to express specified foreign genes, other intrinsic factors appear to emanate spontaneously from NSCs and, in the context of reciprocal donor–host signaling, seem to be capable of neuroprotective and/or neuroregenerative functions. Stem cells additionally have the appealing ability to ‘home in’ on pathology, even over great distances. Such observations help to advance the idea that NSCs – as a prototype for stem cells from other solid organs – might aid in reconstructing the molecular and cellular milieu of maldeveloped or damaged organs.

Interferon-γ regulates the proliferation and differentiation of mesenchymal stem cells via activation of indoleamine 2,3 dioxygenase (IDO).

The kynurenine pathway (KP) of tryptophan metabolism is linked to antimicrobial activity and modulation of immune responses but its role in stem cell biology is unknown. We show that human and mouse mesenchymal and neural stem cells (MSCs and NSCs) express the complete KP, including indoleamine 2,3 dioxygenase 1 (IDO) and IDO2, that it is highly regulated by type I (IFN-β) and II interferons (IFN-γ), and that its transcriptional modulation depends on the type of interferon, cell type and species. IFN-γ inhibited proliferation and altered human and mouse MSC neural, adipocytic and osteocytic differentiation via the activation of IDO. A functional KP present in MSCs, NSCs and perhaps other stem cell types offers novel therapeutic opportunities for optimisation of stem cell proliferation and differentiation.

Learning through inquiry: student difficulties with online course‐based Material

This study investigates the case-based learning experience of 133 undergraduate veterinarian science students. Using qualitative methodologies from relational Student Learning Research, variation in the quality of the learning experience was identified, ranging from coherent, deep, quality experiences of the cases, to experiences that separated significant aspects, such as the online case histories, laboratory test results, and annotated images emphasizing symptoms, from the meaning of the experience. A key outcome of this study was that a significant percentage of the students surveyed adopted a poor approach to learning with online resources in a blended experience even when their overall learning experience was related to cohesive conceptions of veterinary science, and that the difference was even more marked for less successful students. The outcomes from the study suggest that many students are unsure of how to approach the use of online resources in ways that are likely to maximise benefits for learning in blended experiences, and that the benefits from case-based learning such as authenticity and active learning can be threatened if issues closely associated with qualitative variation arising from incoherence in the experience are not addressed.

Neural transplantation of human MSC and NT2 cells in the twitcher mouse model

BACKGROUND Accumulating evidence has demonstrated that the NT2 embryonal carcinoma cell line and multipotential stem cells found in BM, mesenchymal stromal cells (MSC), have the ability to differentiate into a wide variety of cell types. This study was designed to explore the efficacy of these two human stem cell types as a graft source for the treatment of demyelinating disorders such as Krabbes disease and multiple sclerosis (MS). METHODS We examined the engraftment and in vivo differentiation of adult MSC and NT2 cells after transplantation into two demyelinating environments, the neonatal and postnatal twitcher mouse brain. RESULTS Both types of xenografts led to anatomical integration, without tumor formation, and remained viable in the normal and twitcher mouse brain, showing differentiation into neurons, astrocytes and oligodendrocytes. DISCUSSION This study represents a platform for further stem cell transplantation studies in the twitcher model and potentially has important therapeutic implications.

Treatment of canine fucosidosis by intracisternal enzyme infusion

The blood brain barrier is the major obstacle to treating lysosomal storage disorders of the central nervous system such as canine fucosidosis. This barrier was overcome by three, monthly injections of recombinant canine α-l-fucosidase enzyme were given intracisternally. In dogs treated from 8 weeks of age enzyme reached all areas of central nervous system as well as the cervical lymph node, bone marrow and liver. Brainstem and spinal cord samples from regions adjacent to the injection site had highest enzyme levels (39-73% of normal). Substantial enzyme activity (8.5-20% of normal controls) was found in the superficial brain compared to deeper regions (2.6-5.5% of normal). Treatment significantly reduced the fucosyl-linked oligosaccharide accumulation in most areas of CNS, liver and lymph node. In the surface and deep areas of lumbar spinal cord, oligosaccharide accumulation was corrected (79-80% reduction) to near normal levels (p<0.05). In the spinal meninges (thoracic and lumbar) enzyme activity (35-39% of normal control) and substrate reduction (58-63% affected vehicle treated samples) reached levels similar to those seen in phenotypically normal carriers (p<0.05).The procedure was safe and well-tolerated, treated (average 16%) dogs gained more weight (p<0.05) and there was no antibody formation or inflammatory reaction in plasma and CSF following treatments. The capacity of early ERT to modify progression of biochemical storage in fucosidosis is promising as this disease is currently only amenable to treatment by bone marrow transplantation which entails unacceptably high risks for many patients.

Murine epidermal Langerhans cells and keratinocytes express functional P2X7 receptors

Please cite this paper as: Murine epidermal Langerhans cells and keratinocytes express functional P2X7 receptors. Experimental Dermatology 2010; 19: e151–e157.

The P2X7 receptor mediates the uptake of organic cations in canine erythrocytes and mononuclear leukocytes: comparison to equivalent human cell types

We previously demonstrated that canine erythrocytes express the P2X7 receptor, and that the function and expression of this receptor is greatly increased compared with human erythrocytes. Using 86Rb+ (K+) and organic cation flux measurements, we further compared P2X7 in erythrocytes and mononuclear leukocytes from these species. Concentration response curves of BzATP- and ATP-induced 86Rb+ efflux demonstrated that canine P2X7 was less sensitive to inhibition by extracellular Na+ ions compared to human P2X7. In contrast, canine and human P2X7 showed a similar sensitivity to the P2X7 antagonists KN-62 and Mg2+. KN-62 and Mg2+ also inhibited ATP-induced choline+ uptake into canine and human erythrocytes. BzATP and ATP but not ADP or NAD induced ethidium+ uptake into canine monocytes, T- and B-cells. ATP-induced ethidium+ uptake was twofold greater in canine T-cells compared to canine B-cells and monocytes. KN-62 inhibited the ATP-induced ethidium+ uptake in each cell type. P2X7-mediated uptake of organic cations was 40- and fivefold greater in canine erythrocytes and lymphocytes (T- and B-cells), respectively, compared to equivalent human cell types. In contrast, P2X7 function was threefold lower in canine monocytes compared to human monocytes. Thus, P2X7 activation can induce the uptake of organic cations into canine erythrocytes and mononuclear leukocytes, but the relative levels of P2X7 function differ to that of equivalent human cell types.

Transcriptome analysis identifies pathways associated with enhanced maternal performance in QSi5 mice

BackgroundHighly fecund mouse strains provide an ideal model to understand the factors affecting maternal performance. The QSi5 inbred strain of mice was selected for high fecundity and low inter-litter interval, and is very successful at weaning large numbers of offspring when compared to other inbred strains.ResultsPost-natal pup weight gain was used to estimate mammary gland output and to compare the performance of QSi5 mice to CBA mice. Cumulative litter weights and individual pup weight gain was significantly higher throughout the first eight days of lactation in QSi5 mice compared to CBA mice. Morphometric analysis of mammary glands during pregnancy in QSi5 mice revealed a 150 percent greater ductal side branching compared to CBA mice (P < 0.001). Ontology and pathway classification of transcript profiles from the two strains identified an enrichment of genes involved in a number of pathways, including the MAPK, tight junction, insulin signalling and Wnt signalling. Eleven of these genes, including six genes from the MAPK signalling pathway, were identified as associated with postnatal growth. Further, positive mediators of Wnt signalling, including Wnt4, Csnk2a1 and Smad4, were over-represented in the QSi5 strain profile, while negative regulators, including Dkkl1, Ppp2r1a and Nlk, were under-represented. These findings are consistent with the role of Wnt and MAPK signalling pathway in ductal morphogenesis and lobuloalveolar development suggesting enhanced activity in QSi5 mice. A similar pattern of phenotype concordance was seen amongst 12 genes from the tight junction pathway, but a pattern did not emerge from the insulin signalling genes. Amongst a group of differentially expressed imprinted genes, two maternal imprinted genes that suppress growth induced via the IGF signalling pathway, Grb10 and Igf2r, were under-represented in QSi5 mice. Whereas Peg3 and Plagl1, both paternally imprinted genes that enhance neonatal growth, were over-represented in QSi5 mice.ConclusionWe propose that the combined action of at least three major signalling pathways involved in mammary gland development and milk secretion, namely Wnt, MAPK and tight junction pathways, contribute to the superior maternal performance phenotype in QSi5 mice. Additionally, favourable expression patterns of the imprinted genes Peg3, Plagl1, Grb10 and Igf2r may also contribute.

TGF-β1 prevents up-regulation of the P2X7 receptor by IFN-γ and LPS in leukemic THP-1 monocytes.

The P2X7 receptor is an extracellular ATP-gated cation channel critical in inflammation and immunity, and can be up-regulated by IFN-γ and LPS. This study aimed to examine the effect of TGF-β1 on the up-regulation of P2X7 function and expression in leukemic THP-1 monocytes differentiated with IFN-γ and LPS. Cell-surface molecules including P2X7 were examined by immunofluorescence staining. Total P2X7 protein and mRNA was assessed by immunoblotting and RT-PCR respectively. P2X7 function was evaluated by ATP-induced cation dye uptake measurements. Cell-surface P2X7 was present on THP-1 cells differentiated for 3days with IFN-γ and LPS but not on undifferentiated THP-1 cells. ATP induced ethidium(+) uptake into differentiated but not undifferentiated THP-1 cells, and the P2X7 antagonist, KN-62, impaired ATP-induced ethidium(+) uptake. Co-incubation of cells with TGF-β1 plus IFN-γ and LPS prevented the up-regulation of P2X7 expression and ATP-induced ethidium(+) uptake in a concentration-dependent fashion with a maximum effect at 5ng/ml and with an IC(50) of ~0.4ng/ml. Moreover, ATP-induced YO-PRO-1(2+) uptake and IL-1β release were abrogated in cells co-incubated with TGF-β1. TGF-β1 also abrogated the amount of total P2X7 protein and mRNA induced by IFN-γ and LPS. Finally, TGF-β1 prevented the up-regulation of cell-surface CD86, but not CD14 and MHC class II, by IFN-γ and LPS. These results indicate that TGF-β1 prevents the up-regulation of P2X7 function and expression by IFN-γ and LPS in THP-1 monocytes. This suggests that TGF-β1 may limit P2X7-mediated processes in inflammation and immunity.

Canine Fucosidosis: A Neuroprogressive Disorder

The lysosomal storage disease, canine fucosidosis, is caused by the absence of the lysosomal enzyme canine α-L-fucosidase with storage of undegraded fucose-rich material in different organs. Canine fucosidosis is a severe, progressive, fatal neurological disease which results in death or euthanasia and is the only available animal model for this human disease. We analysed the progressive neuropathology from birth to severe clinical disease and related this to the clinical signs. At birth no vacuolation was observed in fucosidosis brain; however, a complex storage presence with vacuolation was well established by 4 months of age, before the clinical signs of motor dysfunction which occurred at 10–12 months of age. Purkinje cell loss, neuronal loss, gliosis, perivascular storage and demyelination accompanied disease progression. Increased vacuolation (15.3-fold increase compared to controls) coincided with advanced motor and mental deterioration in late-stage disease. Significant loss of myelin commenced early, with greatest impact in the cerebellum, and was severe in late disease (1.6- to 1.9-fold decrease) compared to controls (p < 0.05) contributing to clinical signs of motor and mental dysfunction. This detailed description and quantification of the CNS pathology in canine fucosidosis will inform monitoring of the onset, progression and response of this disease to therapy.