Comparative genomic analysis of toxin-negative strains of Clostridium difficile from humans and animals with symptoms of gastrointestinal disease

Background: Clostridium difficile infections (CDI) are a significant health problem to humans and food animals. Clostridial toxins ToxA and ToxB encoded by genes tcdA and tcdB are located on a pathogenicity locus known as the PaLoc and are the major virulence factors of C. difficile. While toxin-negative strains of C. difficile are often isolated from faeces of animals and patients suffering from CDI, they are not considered to play a role in disease. Toxin-negative strains of C. difficile have been used successfully to treat recurring CDI but their propensity to acquire the PaLoc via lateral gene transfer and express clinically relevant levels of toxins has reinforced the need to characterise them genetically. In addition, further studies that examine the pathogenic potential of toxin-negative strains of C. difficile and the frequency by which toxin-negative strains may acquire the PaLoc are needed. Results: We undertook a comparative genomic analysis of five Australian toxin-negative isolates of C. difficile that lack tcdA, tcdB and both binary toxin genes cdtA and cdtB that were recovered from humans and farm animals with symptoms of gastrointestinal disease. Our analyses show that the five C. difficile isolates cluster closely with virulent toxigenic strains of C. difficile belonging to the same sequence type (ST) and have virulence gene profiles akin to those in toxigenic strains. Furthermore, phage acquisition appears to have played a key role in the evolution of C. difficile. Conclusions: Our results are consistent with the C. difficile global population structure comprising six clades each containing both toxin-positive and toxin-negative strains. Our data also suggests that toxin-negative strains of C. difficile encode a repertoire of putative virulence factors that are similar to those found in toxigenic strains of C. difficile, raising the possibility that acquisition of PaLoc by toxin-negative strains poses a threat to human health. Studies in appropriate animal models are needed to examine the pathogenic potential of toxin-negative strains of C. difficile and to determine the frequency by which toxin-negative strains may acquire the PaLoc

Joint Research Day, UClan, Burnley 2018

The Joint Research Day between Uclan and ELHT took place on the 27th of November, at Victoria Mills, Burnley. The event brought together researchers and clinicians to showcase recent research, share new ideas about clinical problems that need tackling and seek collaborative interest between ELHT and UCLan staff. Uclan researchers from various academic/ research disciplines such as engineering, computer science, psychology, and health participated. The event was an opportunity to: • Hear about current local research projects, • Get involved in planned research, • Develop research ideas, • Develop collaborative partnerships. The program included • Keynote lectures by Professor St John Crean, Pro Vice Chancellor, Uclan (the second keynote speaker to be announced), • Presentations and posters of local studies. • Workshops

Developing liver specific vasculature to support the growth of liver progenitor cells for liver tissue engineering

© 2015 Dr. Aaron Matthew DingleTissue engineering is the combination of organ/tissue specific cells in matrices or scaffolds to grow new tissues. Tissue engineering and the related area of cell therapy hold much promise for the repair, replacement and regeneration of organs and tissues damaged by disease and trauma. To date, liver tissue replacement research has focused on cell therapies – particularly hepatocyte transplantation into the diseased liver which specifically aims to reduce the need for liver transplantation, and aims to treat the myriad of end stage liver diseases and metabolic disorders. Despite the technological advances, a major limitation to the clinical success of hepatocyte cell therapy and other liver tissue engineering strategies is the inability to generate a vascular supply capable of supporting the engineering of large, three-dimensional (3D) tissues and organs. The liver sinusoidal endothelial cell (LSEC) makes up the liver specific microvascular network (sinusoids) and plays an integral role in liver development, liver homeostasis and liver regeneration. Vascularisation itself is rarely addressed in liver tissue engineering; let alone the incorporation of LSECs in liver constructs. In this study murine LSECs were used to construct liver specific blood vessels and were identified by their surface markers (LYVE1+/ CD31-), as opposed to capillary endothelial cells (LYVE1-/ CD31+), and lymphatics (LYVE1+/ CD31+). LSECs were tested in vitro and in vivo with and without the addition of murine liver progenitor cells (LPCs). Liver progenitor cells are a native liver progenitor cell, capable of differentiating into both hepatocytes and cholangiocytes. Whilst hepatocytes are the gold standard for liver tissue engineering, LPCs offer an alternative, rarely investigated source of hepatocytes for tissue engineering. LPCs are responsible for liver regeneration during end stage liver disease, when hepatocyte proliferation has been impaired. As LSECs and LPCs play important roles in liver regeneration, both cell types were investigated for their possible applications in liver tissue engineering. LSECs and LPCs were cultured as 3-D multicellular spheroids of one cell type-termed homospheres, or co-cultured together as heterospheres in vitro, for subsequent in vivo implantation in a vascularized tissue engineering chamber. This thesis demonstrates that LSECs and LPCs are capable of forming homogeneous homospheres, and heterogeneous spheroids of co-cultured heterospheres. Furthermore, LSECs form vascular structures in vitro when cultured as homospheres heterospheres. The ability to generate vascular structures through co-culturing with endothelial cells in vitro is termed pre-vascularization, and is currently at the forefront of vascular tissue engineering. The thesis also demonstrates that LSECs were capable of integrating into native vasculature when implanted in vivo to form a liver specific vasculature at an ectopic site in healthy SCID mice. The liver specific vasculature was identifiable as LYVE1+/ CD31- and through the detection of DiI labeled LSECs. Implantation of LSECs also increased the generation of native neo-vasculature (LYVE1-/CD31+)- this was significant when the matrix Matrigel was used in the tissue engineering chamber. However, the generation of liver specific vasculature and increased native vasculature did not support significant survival of LPCs. Whilst the results indicate that heterospheres of LSECs and LPCs improve the survival and spontaneous hepatocyte differentiation of LPCs, the overall survival was inconsistent throughout the in vivo experimental groups in SCID mice. Finally, LPC spheroids were implanted in a mouse model of the metabolic disorder methylmalonic aciduria (MMA). LSECs were not used in the final study; as LPC survival was inconsistent and low regardless of the presence/absence of LSECs. Instead, the total number of LPCs implanted was increased 4 fold. Again, survival of LPCs was low and appeared to elicit an immune reaction in MMA mice. Ultimately, the final study did not demonstrate any functional disease reversal, due to poor LPC survival. This is the first liver tissue engineering study that has used LSECs with LPCs. Isolated LSECs demonstrated significant promise in their ability to generate a liver-specific vasculature in vivo for liver tissue engineering, however the use of LPCs presented a number of issues, which require further investigation – the most significant of which was their inconsistent and generally poor survival in vivo

Management of symptomatic neuromas: a narrative review of the most common surgical treatment modalities in amputees

Symptomatic neuromas are an all-too-common complication following limb amputation or extremity trauma, leading to chronic and debilitating pain for patients. Surgical resection of symptomatic neuromas has proven to be the superior method of intervention, but traditional methods of neuroma resection do not address the underlying pathophysiology leading to the formation of a future symptomatic neuroma and lead to high reoperation rates. Novel approaches employ the physiology of peripheral nerve injury to harness the regeneration of nerves to their advantage. This review explores the underlying pathophysiology of neuroma formation and centralization of pain signaling. It compares the traditional surgical approach for symptomatic neuroma resection and describes three novel surgical strategies that harness this pathophysiology of neuroma formation to their advantage. The traditional resection of symptomatic neuromas is currently the standard of care for amputation patients, but new techniques including the regenerative peripheral nerve interface, targeted muscle reinnervation, and intraosseous transposition have shown promise in improving patient pain outcomes for postamputation pain and residual limb pain. Symptomatic neuromas are a chronic and debilitating complication following amputation procedures and trauma, and the current standard of care does not address the underlying pathophysiology leading to the formation of the neuroma. New techniques are under development that may provide improved patient pain outcomes and a higher level of care for symptomatic neuroma resection

Liver sinusoidal endothelial cells promote the differentiation and survival of mouse vascularised hepatobiliary organoids

The structural and physiological complexity of currently available liver organoids is limited, thereby reducing their relevance for drug studies, disease modelling, and regenerative therapy. In this study we combined mouse liver progenitor cells (LPCs) with mouse liver sinusoidal endothelial cells (LSECs) to generate hepatobiliary organoids with liver-specific vasculature. Organoids consisting of 5x103 cells were created from either LPCs, or a 1:1 combination of LPC/LSECs. LPC organoids demonstrated mild hepatobiliary differentiation in vitro with minimal morphological change; in contrast LPC/LSEC organoids developed clusters of polygonal hepatocyte-like cells and biliary ducts over a 7 day period. Hepatic (albumin, CPS1, CYP3A11) and biliary (GGT1) genes were significantly upregulated in LPC/LSEC organoids compared to LPC organoids over 7 days, as was albumin secretion. LPC/LSEC organoids also had significantly higher in vitro viability compared to LPC organoids. LPC and LPC/LSEC organoids were transplanted into vascularised chambers created in Fah−/−/Rag2−/−/Il2rg−/− mice (50 LPC organoids, containing 2.5x105 LPCs, and 100 LPC/LSEC organoids, containing 2.5x105 LPCs). At 2 weeks, minimal LPCs survived in chambers with LPC organoids, but robust hepatobiliary ductular tissue was present in LPC/LSEC organoids. Morphometric analysis demonstrated a 115-fold increase in HNF4α+ cells in LPC/LSEC organoid chambers (17.26 ± 4.34 cells/mm2 vs 0.15 ± 0.15 cells/mm2, p = 0.018), and 42-fold increase in Sox9+ cells in LPC/LSEC organoid chambers (28.29 ± 6.05 cells/mm2 vs 0.67 ± 0.67 cells/mm2, p = 0.011). This study presents a novel method to develop vascularised hepatobiliary organoids, with both in vitro and in vivo results confirming that incorporating LSECs with LPCs into organoids significantly increases the differentiation of hepatobiliary tissue within organoids and their survival post-transplantation

Enhanced liver progenitor cell survival and differentiation in vivo by spheroid implantation in a vascularized tissue engineering chamber

Liver tissue engineering is hampered by poor implanted cell survival due to inadequate vascularization and cell–cell/cell–matrix interactions. Here, we use liver progenitor cell (LPC) spheroids to enhance cell–cell/cell–matrix interactions, with implantation into an angiogenic in vivo mouse chamber. Spheroids were generated in vitro in methylcellulose medium. Day 2 spheroids were optimal for implantation (22,407 +/−645 cells/spheroid), demonstrating maximal proliferation (Ki67 immunolabeling) and minimal apoptosis (caspase-3 immunolabelling). In vivo chambers established bilaterally on epigastric vessels of immunodeficient mice were implanted with equivalent numbers of LPCs as a cell suspension (200,000 cells), or spheroids (9 spheroids). At day 14, a trend of increased LPC survival was observed in spheroid-implanted chambers [pan-cytokeratin (panCK+) cells, p = 0.38, 2.4 fold increase)], with significantly increased differentiation [cytokeratin 18 (CK18+) cells, p < 0.002, 5.1 fold increase)] compared to cell suspension-implanted chambers. At day 45, both measures were significantly increased in spheroid-implanted chambers (panCK, p < 0.006, 16 fold increase) (CK18, p < 0.019, 6 fold increase). Hepatic acini/plates of CK18 + cells expressed hepatocyte nuclear factor 4-α and β-catenin, indicating ongoing hepatic differentiation. Spheroid cell-delivery significantly increased LPC survival and differentiation compared to conventional cell suspensions. This LPC spheroid/vascularized chamber model has clinical potential to generate three-dimensional vascularized liver tissue for liver replacement