Alpha v beta 5 and related receptors in human B lymphocyte development

CD23 is a multi-functional protein which exists in membrane-bound and soluble forms. Its functions include acting as the low affinity receptor for IgE and generating pro-inflammatory cytokine release in monocytes. CD23 has been found to interact with αvβ5 and this interaction greatly enhances growth of the B cell precursor cell line SMS-SB. This interaction may have a role in the development of normal human B cells and in cancer as the integrin is expressed on both precursor and ALL cells but not on normal mature B cells. One of the aims of this investigation was to expand on the finding that CD23 peptides containing an RKC motif had the same positive growth effect on SMS-SB cells as CD23. Other B cell lines – representative of both precursor and mature stages – were studied to ascertain whether this proliferative effect was dependent upon cell differentiation stage and/or presence of the αvβ5 integrin. It was found that peptides containing the basic RKC motif were mitogenic only for precursor B cells which were expressing αvβ5. Details of these peptides and their varying effects on the different cell lines are in Chapter 4. Stimulation of SMS-SB cells, presumably via the αvβ5, results in signalling through PI3K and subsequent phosphorylation of Akt. The growth of SMS-SB cells observed following stimulation with peptides containing the RKC motif was abrogated by the PI3K inhibitor LY294002 and western blotting revealed that phosphorylation of Akt was enhanced by stimulation with RKS containing peptides. Among CD23’s receptors is the integrin αvβ3. This integrin can form a signalling complex with CD47. Ligation of CD47 by anti-CD47 antibodies induces apoptosis in some cell lines. To determine whether a pattern exists between response to this stimulation and expression of αvβ3 integrin, cell lines with and without the integrin were tested. It was found that the myeloma cell lines KMS11 and H929 were responsive to this stimulus. Since these cell lines differ in their expression of αvβ3 (H929 cells express αvβ3 whereas KMS11 do not) it does not appear that any connection between the presence of the integrin and response via CD47 exists and therefore this signalling mechanism would appear to occur independently of the complex formed by CD47 and αvβ3

Multifunctional cytokine production reveals functional superiority of memory CD4 T cells

T cell protective immunity is associated with multifunctional memory cells that produce several different cytokines. Currently, our understanding of when and how these cells are generated is limited. We have used an influenza virus mouse infection model to investigate whether the cytokine profile of memory T cells is reflective of primary responding cells or skewed towards a distinct profile. We found that, in comparison to primary cells, memory T cells tended to make multiple cytokines simultaneously. Analysis of the timings of release of cytokine by influenza virus‐specific T cells, demonstrated that primary responding CD4 T cells from lymphoid organs were unable to produce a sustained cytokine response. In contrast CD8 T cells, memory CD4 T cells, and primary responding CD4 T cells from the lung produced a sustained cytokine response throughout the restimulation period. Moreover, memory CD4 T cells were more resistant than primary responding CD4 T cells to inhibitors that suppress T cell receptor signalling. Together, these data suggest that memory CD4 T cells display superior cytokine responses compared to primary responding cells. These data are key to our ability to identify the cues that drive the generation of protective memory CD4 T cells following infection

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

Susceptibility of hamsters to clostridium difficile isolates of differing toxinotype

Clostridium difficile is the most commonly associated cause of antibiotic associated disease (AAD), which caused ~21,000 cases of AAD in 2011 in the U.K. alone. The golden Syrian hamster model of CDI is an acute model displaying many of the clinical features of C. difficile disease. Using this model we characterised three clinical strains of C. difficile, all differing in toxinotype; CD1342 (PaLoc negative), M68 (toxinotype VIII) and BI-7 (toxinotype III). The naturally occurring non-toxic strain colonised all hamsters within 1-day post challenge (d.p.c.) with high-levels of spores being shed in the faeces of animals that appeared well throughout the entire experiment. However, some changes including increased neutrophil influx and unclotted red blood cells were observed at early time points despite the fact that the known C. difficile toxins (TcdA, TcdB and CDT) are absent from the genome. In contrast, hamsters challenged with strain M68 resulted in a 45% mortality rate, with those that survived challenge remaining highly colonised. It is currently unclear why some hamsters survive infection, as bacterial and toxin levels and histology scores were similar to those culled at a similar time-point. Hamsters challenged with strain BI-7 resulted in a rapid fatal infection in 100% of the hamsters approximately 26 hr post challenge. Severe caecal pathology, including transmural neutrophil infiltrates and extensive submucosal damage correlated with high levels of toxin measured in gut filtrates ex vivo. These data describes the infection kinetics and disease outcomes of 3 clinical C. difficile isolates differing in toxin carriage and provides additional insights to the role of each toxin in disease progression

Performance of novel VUV-sensitive Silicon Photo-Multipliers for nEXO

Liquid xenon time projection chambers are promising detectors to search for neutrinoless double beta decay (0$\nu \beta \beta$), due to their response uniformity, monolithic sensitive volume, scalability to large target masses, and suitability for extremely low background operations. The nEXO collaboration has designed a tonne-scale time projection chamber that aims to search for 0$\nu \beta \beta$ of \ce{^{136}Xe} with projected half-life sensitivity of $1.35\times 10^{28}$~yr. To reach this sensitivity, the design goal for nEXO is $\leq$1\% energy resolution at the decay $Q$-value ($2458.07\pm 0.31$~keV). Reaching this resolution requires the efficient collection of both the ionization and scintillation produced in the detector. The nEXO design employs Silicon Photo-Multipliers (SiPMs) to detect the vacuum ultra-violet, 175 nm scintillation light of liquid xenon. This paper reports on the characterization of the newest vacuum ultra-violet sensitive Fondazione Bruno Kessler VUVHD3 SiPMs specifically designed for nEXO, as well as new measurements on new test samples of previously characterised Hamamatsu VUV4 Multi Pixel Photon Counters (MPPCs). Various SiPM and MPPC parameters, such as dark noise, gain, direct crosstalk, correlated avalanches and photon detection efficiency were measured as a function of the applied over voltage and wavelength at liquid xenon temperature (163~K). The results from this study are used to provide updated estimates of the achievable energy resolution at the decay $Q$-value for the nEXO design

An integrated online radioassay data storage and analytics tool for nEXO

Large-scale low-background detectors are increasingly used in rare-event searches as experimental collaborations push for enhanced sensitivity. However, building such detectors, in practice, creates an abundance of radioassay data especially during the conceptual phase of an experiment when hundreds of materials are screened for radiopurity. A tool is needed to manage and make use of the radioassay screening data to quantitatively assess detector design options. We have developed a Materials Database Application for the nEXO experiment to serve this purpose. This paper describes this database, explains how it functions, and discusses how it streamlines the design of the experiment

Severe experimental autoimmune encephalomyelitis (EAE) is ameliorated by human olfactory-derived mesenchymal stromal cell transplantation revealing a role for IL-16

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are being tested in the clinic for the treatment of the demyelinating disease, multiple sclerosis (MS) [1, 2]. Their immunomodulatory action, which targets the inflammatory component of the disease, is thought to make them ideal candidates. We have identified mesenchymal stromal cells from the human olfactory mucosa termed, OM-MSCs[3]. These cells have additional properties suitable for repair since they also enhance CNS myelination and skew microglia to an anti-inflammatory phenotype in vitro [4]. Therefore, OM-MSCs may have therapeutic benefits over BM-MSCsin the treatment of MS. In this investigation, we have compared the reparative properties of OM-MSCs to BM-MSCs in vivousing the experimental autoimmune encephalomyelitis (EAE) model. Although, both MSC types were capable of ameliorating disease, if delivered at early disease onset, when administered during established severe disease, only OM-MSCs significantly improved disease outcome. OM-MSCs mediate this action, in part, by a faster closing of the BBB and a reduction in the recruitment of inflammatory cells, which leads to improved remyelination and axonal survival within demyelinated lesions. OM-MSCs also modulated IL-16 secretion of recruited inflammatory cells. Further in vitroinvestigation of IL-16 revealed a novel inhibitory role on OPC differentiation and myelination. Our data suggests that OM-MSCs may have therapeutic benefits over BM-MSCs in the treatment of MS, especially if administered during progressive disease

Colonisation kinetics of <i>C. difficile</i> CD1342 in hamsters.

<p>To monitor colonisation throughout the infection process hamsters were culled at 1- (A), 3- (B) and 11 (C) days post challenge. <i>C. difficile</i> was recovered from the caecum (CAE) and the colon (COL) either associated with the lumen (LA) or the tissue (TA). Filled bars represent vegetative bacteria whilst empty bars indicate bacteria in spore form. Bacterial recoveries represent the geometric mean plus the standard error of the mean (SEM) of 2 biological replicates, where a total of at least 5 animals were used per time interval.</p