Fc gamma receptor mediated modulation of osteoclastogenesis

Osteoporosis is a condition that results from substantially weakened bone, increasing an individual’s risk of fracture. Post-menopausal osteoporosis is the most common form of the condition, affecting 30% of post-menopausal women over the age of 50. Following the menopause, female oestrogen levels decline and this perturbs bone homeostasis by promoting an environment that is biased towards bone erosion. Osteoclasts are the cells responsible for eroding bone and are normally inhibited by oestrogen. However, the decline in oestrogen production results in increased osteoclast differentiation and activity. This rapidly decreases the bone mineral density and results in fracture-prone bone. Osteoclasts are derived from mononuclear myeloid progenitors found in the blood and bone marrow, which fuse to form large multinucleated cells that reside in the bone cavity. These progenitor cells are also responsible for replenishing monocytes, macrophages and dendritic cells. One class of receptors present on the surface of these cells, which are capable of dictating a cells function, are Fcγ receptors and modulation of Fcγ receptors has been shown to inhibit the differentiation of human monocytes to osteoclasts. This thesis investigates Fcγ receptor modulation on murine osteoclastogenesis and in order to stimulate Fcγ receptors, both IgG and IgG complexes were used. IgG complexes were generated using Staphylococcus aureus Protein A (SpA) in combination with IgG to form SpA-IgG complexes (SIC). We show that IgG and SIC are capable of engaging with Fcγ receptors resulting in the inhibition of osteoclast differentiation. Furthermore, both IgG and SIC inhibit the transcription of mRNA essential for the fusion of progenitors and enzymes for the erosion of bone matrix. Therefore, IgG and SIC are capable of inhibiting murine osteoclastogenesis. The murine model of osteoporosis was used to further investigate the ability of SIC to inhibit murine osteoclast differentiation. Previous studies have shown that when SpA is administered in vivo it is capable of binding circulating IgG to form SIC. We used this property to test the ability of SpA to bind to the surface of monocytes. SpA was found to bind with highest affinity to blood Ly6Chigh monocytes, which are known to differentiate in vitro to OCs. IgG and SIC were also able to inhibit the in vitro osteoclastogenesis of Ly6Chigh monocytes. It was hypothesised that SpA would co-opt IgG and inhibit the in vivo differentiation of progenitors to osteoclasts in the ovariectomy model of osteoporosis. To generate this animal model the ovaries were removed from the mice in order to simulate the menopause and induce bone loss. To assess the percentage of bone present after ovariectomy, we used micro-computer tomography and discovered that SpA was unable to prevent bone loss associated with ovariectomy. Therefore, SpA can bind to the surface of osteoclast progenitors but is unable to inhibit bone loss in the model of osteoporosis. In addition to studying the role of Fcγ receptor modulation of osteoclastogenesis, the role of Bcl-3 (a negative regulator of NF-κB) in osteoclast differentiation and bone remodelling was also investigated. NF-κB is an essential signalling molecule and transcription factor involved in osteoclast differentiation. Previous research has shown that in the absence of Bcl-3 (Bcl-3-/-) aberrant cytokine responses to LPS and TNF- occur. Therefore, RANKL stimulation of WT and Bcl-3-/- osteoclast precursors was done to determine whether Bcl 3 /- animals responded aberrantly to RANKL. WT and Bcl-3-/- animals were able to generate in vitro osteoclasts, which were phenotypically and transcriptionally similar. However, comparison of in vivo osteoclast progenitors revealed that Bcl-3-/- animals had reduced CD115+ osteoclast progenitors compared to WT animals. Examination of the trabecular bone present in the proximal tibia revealed that Bcl-3-/- animals had a higher percentage of bone present that WT controls. Therefore, Bcl-3 does not effect in vitro osteoclast differentiation but further work needs to be done to understand the role of Bcl 3 in bone remodelling. This thesis aimed to investigate whether SpA-IgG complexes or Bcl-3 could represent a novel avenue of therapeutic intervention in osteoporotic disease. In summation, SpA is able to form IgG complexes that can inhibit the differentiation of OCs in vitro; however, treatment of osteoporotic animals with SpA was unable to halt bone loss. This suggests that SpA-IgG complexes are able to modulate Fcγ receptors in vitro and skew progenitors from differentiation into osteoclasts but cannot overcome the prevailing pro-osteoclastogenic environment that results from ovariectomy. The presence of osteoclast progenitors was also shown to be partially dependent on Bcl-3 and as such Bcl-3 may be a novel target for therapeutic agents to target osteoclast progenitors in diseases like osteoporosis. However, the role of Bcl-3 in bone remodelling requires further investigation

Lignocellulose-Degrading Microbial Communities in Landfill Sites Represent a Repository of Unexplored Biomass- Degrading Diversity Emma

The microbial conversion of lignocellulosic biomass for biofuel production represents a renewable alternative to fossil fuels. However, the discovery of new microbial enzymes with high activity is critical for improving biomass conversion processes. While attempts to identify superior lignocellulose-degrading enzymes have focused predominantly on the animal gut, biomass-degrading communities in landfill sites represent an unexplored resource of hydrolytic enzymes for biomass conversion. Here, to address the paucity of information on biomass-degrading microbial diversity beyond the gastrointestinal tract, cellulose (cotton) “baits” were incubated in landfill leachate microcosms to enrich the landfill cellulolytic microbial community for taxonomic and functional characterization. Metagenome and 16S rRNA gene amplicon sequencing demonstrated the dominance of Firmicutes, Bacteroidetes, Spirochaetes, and Fibrobacteres in the landfill cellulolytic community. Functional metagenome analysis revealed 8,371 carbohydrate active enzymes (CAZymes) belonging to 244 CAZyme families. In addition to observing biomass-degrading enzymes of anaerobic bacterial “cellulosome” systems of members of the Firmicutes, we report the first detection of the Fibrobacter cellulase system and the Bacteroidetes polysaccharide utilization locus (PUL) in landfill sites. These data provide evidence for the presence of multiple mechanisms of biomass degradation in the landfill microbiome and highlight the extraordinary functional diversity of landfill microorganisms as a rich source of biomass-degrading enzymes of potential biotechnological significance

μCT trait analysis reveals morphometric differences between domesticated temperate small grain cereals and their wild relatives

Wheat and barley are two of the founder crops domesticated in the Fertile Crescent, and currently represent crops of major economic importance in temperate regions. Due to impacts on yield, quality and end-use, grain morphometric traits remain an important goal for modern breeding programmes and are believed to have been selected for by human populations. To directly and accurately assess the three-dimensional (3D) characteristics of grains, we combine X-ray microcomputed tomography (μCT) imaging techniques with bespoke image analysis tools and mathematical modelling to investigate how grain size and shape vary across wild and domesticated wheat and barley. We find that grain depth and, to a lesser extent, width are major drivers of shape change and that these traits are still relatively plastic in modern bread wheat varieties. Significant changes in grain depth are also observed to be associated with differences in ploidy. Finally, we present a model that can accurately predict the wild or domesticated status of a grain from a given taxa based on the relationship between three morphometric parameters (length, width and depth) and suggest its general applicability to both archaeological identification studies and breeding programmes.Agências financiadoras: Biotechnology and Biological Sciences Research Council (BBSRC) grant, 'National Capability in Crop Phenotyping' (BB/J004464/1); (BB/CAP1730/1) BBSRC grant, 'MAGIC CARPET' (BB/M011666/1) NERC (NE/M010805/1) ERC (339941)info:eu-repo/semantics/publishedVersio

Brenneria goodwinii growth in vitro is improved by competitive interactions with other bacterial species associated with Acute Oak Decline

Brenneria goodwinii, Rahnella victoriana and Gibbsiella quercinecans are three bacterial species frequently isolated together from oak displaying symptoms of Acute Oak Decline (AOD), which include weeping patches on trunks. All three bacterial species play a role in lesion formation in the current episode of AOD in Britain, although B. goodwinii is the most dominant. The ongoing research into stem lesion formation characteristic of this polybacterial syndrome has been focussed primarily on the pathogenicity, identification and taxonomy of these bacteria. As all three species were newly classified within the past ten years, there are many unanswered questions regarding their ecology and interactions with each other. To determine the effect of bacterial interactions on fitness in vitro, we examined pairwise (diculture) and multispecies (triculture) interactions between B. goodwinii, R. victoriana and G. quercinecans in oak leaf media microcosms. Additionally, the effect of co-culturing on the evolution of these species was determined and the evolved B. goodwinii strains were examined further by whole genome sequencing. Our results indicate that B. goodwinii thrived in monoculture with significantly higher viable cell counts than the other two species. Additionally, B. goodwinii performed well in pairwise culture with mutually competitive interactions observed between B. goodwinii and R. victoriana, and between B. goodwinii and G. quercinecans. In the multispecies triculture, B. goodwinii and R. victoriana appeared to exhibit co-ordinated behaviour to outcompete G. quercinecans. After four weeks B. goodwinii grown in co-culture with the other two species developed greater evolved fitness than the strain grown in monoculture as reflected by the increased viable cell counts. The competitive interactions taking place between the threes species indicated evolving improved fitness of B. goodwinii in vitro, that gave it a growth advantage over both R. victoriana and G. quercinecans which showed no significant changes in fitness. Overall, B. goodwinii gains greater benefit in terms of fitness from in vitro competitive interaction with the other two species

Dendritic cells provide a therapeutic target for synthetic small molecule analogues of the parasitic worm product, ES-62

ES-62, a glycoprotein secreted by the parasitic filarial nematode Acanthocheilonema viteae, subverts host immune responses towards anti-inflammatory phenotypes by virtue of covalently attached phosphorylcholine (PC). The PC dictates that ES-62 exhibits protection in murine models of inflammatory disease and hence a library of drug-like PC-based small molecule analogues (SMAs) was synthesised. Four sulfone-containing SMAs termed 11a, 11e, 11i and 12b were found to reduce mouse bone marrow-derived dendritic cell (DC) pathogen-associated molecular pattern (PAMP)-induced pro-inflammatory cytokine production, inhibit NF-κB p65 activation, and suppress LPS-induced up-regulation of CD40 and CD86. Active SMAs also resulted in a DC phenotype that exhibited reduced capacity to prime antigen (Ag)-specific IFN-γ production during co-culture with naïve transgenic TCR DO.11.10 T cells in vitro and reduced their ability, following adoptive transfer, to prime the expansion of Ag-specific T lymphocytes, specifically TH17 cells, in vivo. Consistent with this, mice receiving DCs treated with SMAs exhibited significantly reduced severity of collagen-induced arthritis and this was accompanied by a significant reduction in IL-17+ cells in the draining lymph nodes. Collectively, these studies indicate that drug-like compounds that target DCs can be designed from parasitic worm products and demonstrate the potential for ES-62 SMA-based DC therapy in inflammatory disease

Closed incision negative pressure wound therapy versus conventional dressings following soft-tissue sarcoma excision: a prospective, randomized controlled trial

Aims: The primary objective of this study was to compare the postoperative infection rate between negative pressure wound therapy (NPWT) and conventional dressings for closed incisions following soft-tissue sarcoma (STS) surgery. Secondary objectives were to compare rates of adverse wound events and functional scores. Methods: In this prospective, single-centre, randomized controlled trial (RCT), patients were randomized to either NPWT or conventional sterile occlusive dressings. A total of 17 patients, with a mean age of 54 years (21 to 81), were successfully recruited and none were lost to follow-up. Wound reviews were undertaken to identify any surgical site infection (SSI) or adverse wound events within 30 days. The Toronto Extremity Salvage Score (TESS) and Musculoskeletal Tumor Society (MSTS) score were recorded as patient-reported outcome measures (PROMs). Results: There were two out of seven patients in the control group (28.6%), and two out of ten patients in the intervention group (20%) who were diagnosed with a SSI (p > 0.999), while one additional adverse wound event was identified in the control group (p = 0.593). No significant differences in PROMs were identified between the groups at either 30 days (TESS, p = 0.987; MSTS, p = 0.951) or six-month (TESS, p = 0.400) follow-up. However, neoadjuvant radiotherapy was significantly associated with a SSI within 30 days of surgery, across all patients (p = 0.029). The mean preoperative modified Glasgow Prognostic Score (mGPS) was also significantly higher among patients who developed a postoperative adverse wound event (p = 0.028), including a SSI (p = 0.008), across both groups. Conclusion: This is the first RCT comparing NPWT with conventional dressings following musculoskeletal tumour surgery. Postoperative wound complications are common in this group of patients and we observed an overall SSI rate of 23.5%. We propose proceeding to a multicentre trial, which will help more clearly define the role of closed incision NPWT in STS surgery