Phenotypic and functional analysis of the mesenchymal stromal cell compartment in the thymus

T cells are key cellular components of the adaptive immune system that play a critical role in our defense against pathogens and cancer but also contribute to pathology in the setting of chronic inflammation and autoimmunity. The thymus constitutes the body's major T cell production site, where bone marrow-derived progenitors migrate in a co-ordinated fashion through anatomically distinct cortical and medullary regions that are composed of epithelial and mesenchymal stromal elements. The interaction of cortical (cTEC) and medullary (mTEC) thymic epithelial cells (TEC) with migrating thymocytes is of fundamental importance in driving thymocyte differentiation and their selection towards generation of an immunocompetent but self-tolerant T cell repertoire. While the ontogeny, phenotype and functionality of TEC subsets has been extensively studied, far less is known regarding the thymic mesenchymal cell (TMC) compartment. The vitamin A metabolite, retinoic acid (RA) is a potent regulator of cell proliferation, differentiation and apoptosis that impacts on gene transcription by acting as a trans-activating ligand for a family of transcription factors called RA receptors (RARs). RA-dependent RAR-mediated events (also called RA signaling) have been widely implicated in organ development and epithelial tissue homeostasis but the putative role of endogenous RA signals in regulating TEC and/or thymocyte homeostasis has remained unknown. In this thesis, using comprehensive phenotypic, genetic lineage tracing and microarray analyses we demonstrate that postnatal TMCs comprise two phenotypically distinct and functionally specialized neural crest-derived, gp38+ and gp38- TMC subsets. We characterize gp38- TMC as mural cells with classical pericyte and vascular smooth muscle cell functions that intimately associate with and support thymic vascular endothelium. In contrast, gp38+ TMC were central producers of a range of factors implicated in regulating TEC homeostasis. Our data also provide an initial description of the neural crest cell maturation pathway towards mature gp38+ and gp38- TMC during thymic ontogeny. Using gene expression, biochemical and functional analyses, we further identify gp38+ TMCs together with a subset of mTECs as the major cell populations capable of RA production in the post-natal thymus. In functional experiments involving FTOC, we show that mesenchymal cell-derived RA plays an important role in regulating cTEC numbers and provide initial evidence for a direct role of RA responses in TEC in regulating cTEC homeostasis in vivo. In addition to its direct role in regulating TEC compartments, we demonstrate the presence of endogenous RA sensing by developing thymocytes, and further provide initial data suggesting a key and direct role for RA in thymocyte development in vivo. Collectively, our results significantly broaden the current understanding of TMC heterogeneity, ontogeny and function, and provide valuable insights to the current understanding of vitamin A metabolism and the functionality of RA in the thymus

Diseases of the Brain, Head and Neck, Spine 2020–2023

This open access book offers an essential overview of brain, head and neck, and spine imaging. Over the last few years, there have been considerable advances in this area, driven by both clinical and technological developments. Written by leading international experts and teachers, the chapters are disease-oriented and cover all relevant imaging modalities, with a focus on magnetic resonance imaging and computed tomography. The book also includes a synopsis of pediatric imaging. IDKD books are rewritten (not merely updated) every four years, which means they offer a comprehensive review of the state-of-the-art in imaging. The book is clearly structured and features learning objectives, abstracts, subheadings, tables and take-home points, supported by design elements to help readers navigate the text. It will particularly appeal to general radiologists, radiology residents, and interventional radiologists who want to update their diagnostic expertise, as well as clinicians from other specialties who are interested in imaging for their patient care

Effect of growth factors on the osteoinductive potential of Hydroxyapatite β-Tricalcium Phosphate (HA-TCP).

The replacement of missing teeth by osseointegrated dental implants is a commonly utilised treatment option in dentistry. However, successful treatment outcomes are dependent on sufficient bone quantity in the proposed surgical site for implant placement (Buser et al., 2004). Surgical augmentation of bone defects is commonly performed prior to or during implant placement. Bone augmentation procedures of the maxillary sinus or guided bone regeneration (GBR) procedures of alveolar ridge defects have utilised a variety of bone graft materials in block or particulate form, either alone or in combination with resorbable or non-resorbable barrier membranes. Objective: The aim of this study was to determine whether Hydroxyapatite β-Tricalcium Phosphate (HA-TCP) either alone or combined with Enamel Matrix Derivative (EMD) or recombinant human Platelet Derived Growth Factor-BB (rhPDGF-BB) is osteoinductive when implanted into a nonosseous site. Methods: Twenty CD-1 adult male mice underwent intramuscular implantation into both hindlimbs of an empty gelatine capsule or a gelatine capsule containing one of the following: 10 mg of uncoated particulate HA-TCP, (Straumann Bone Ceramic®, HA-TCP), EMD coated HA-TCP, (Emdogain®, HATCP + EMD) or rhPDGF-BB coated HA-TCP (HA-TCP + PDGF). Ten animals were sacrificed at four and eight weeks with five specimens from each group retrieved at each time point. The area of graft placement was radiographed and after graft retrieval, a semi-quantitative histological examination was performed with the aim of assessing the inflammatory changes, reparative processes and osteoinduction within the graft site. Results: At both 4 and 8 weeks, histological analysis failed to demonstrate any osteoinductive activity in any of the specimens from the three experimental groups. A minimal chronic inflammatory response and foreign body reaction was seen in the experimental groups which reduced over time. The particles were embedded within fibrous connective tissue and were encapsulated by a dense cellular layer consisting of active fibroblasts and occasional macrophages with the thickness of this layer decreasing over time. At 4 weeks, a greater density of the fibrous connective tissue was demonstrated in the HA-TCP + EMD group (P<0.001) while a greater thickness in the capsule thickness was seen in the HA-TCP group (P=0.022) although no differences were seen after 8 weeks. Greater neovascularisation was seen in the HA-TCP + PDGF group after 8 weeks (P=0.043) while greater amounts of adipose tissue surrounding the particles were detected in the HA-TCP + PDGF group at 4 weeks (P=0.002) and in the HA-TCP + EMD group at eight weeks (P=0.002). Conclusions: The results of this study suggest that the use of commercially available HA-TCP alone or in combination with EMD or rhPDGF-BB is biocompatible but not osteoinductive in the murine model.Thesis (D.Clin.Dent.) -- University of Adelaide, School of Dentistry, 201

Macrophage Involvement in the Remodeling of an Extracellular Matrix Scaffold

The remodeling response to extracellular matrix (ECM) scaffold materials such as porcine small intestinal submucosa (SIS) is characterized by intense mononuclear cell infiltration during the first 4 weeks post-implantation. Persistence of macrophages in wounds is typically diagnosed as chronic inflammation with downstream formation of scar tissue and/or foreign body reaction, but ECM scaffolds remodel into organized site-specific tissue. Macrophages can express either proinflammatory (M1) or immunomodulatory and tissue remodeling (M2) phenotypes. Processing methods used during the manufacturing of ECM scaffolds can influence macrophage phenotype and downstream remodeling outcome.In the first study, human monocyte-derived macrophages were cultured on SIS and carbodiimide (CDI) crosslinked SIS in 20% and 6% oxygen concentrations. Macrophage phenotype was evaluated by expression of M1 (CXCL10 and CCR7) and M2 (ARG-1, CCL13, CCL18, and MRC-1) gene markers, and secretion of CXCL10, CCL13, CCL18, and MMP9. Macrophages cultured on SIS expressed an M2 profile, while macrophages cultured on CDI-SIS expressed a mixed M1/M2 profile. No consistent patterns were observed when comparing oxygen concentrations.The second study used radioactive 14C-labeled scaffolds to measure ECM scaffold degradation in a rodent model of musculoskeletal reconstruction with and without the depletion of macrophages. Tissues were characterized by expression of M1 (iNOS and IFN-ã) and M2 (ARG-1 and IL-10) gene markers, and cell surface markers CD68 (pan-macrophage), CCR7 (M1), and CD163 (M2). Results showed that macrophages are required for early and rapid degradation of SIS scaffolds, and that CDI-SIS is resistant to macrophage-mediated degradation. Furthermore, depletion of macrophages resulted in an attenuated inflammatory response and slowed the rate of scaffold degradation.The third study determined the contractile response and histomorphologic appearance of tissue repaired with SIS, CDI-SIS, or autologous tissue at 26 weeks after implantation. Contractile properties and fatigue resistance of remodeled tissue and of contralateral native tissue were assessed using an in-situ methodology. Muscle fiber-type distribution, blood vessel density and distribution, and innervation were determined. The tissue repaired with SIS showed complete replacement by tissue that histologically and functionally resembled native muscle. CDI-SIS was characterized by chronic inflammatory response and produced little to no measurable tetanic force output

Ex Vivo Gene Therapy Approaches for the Treatment of Globoid Cell Leukodystrophy

Globoid cell leukodystrophy (GLD) is a rare lysosomal storage disorder (LSD) due to the deficiency of the lysosomal enzyme Galactocerebrosidase (GALC). The enzymatic deficiency results in intracellular storage of undegraded metabolites in the nervous system, leading to progressive dysmyelination. We are testing the feasibility and efficacy of a gene therapy strategy based on hematopoietic stem/progenitor cells (HSPC) and lentiviral vectors (LV) in the murine model of GLD. Differently from what observed with other lysosomal enzymes, GALC gene transfer and expression in HSPC causes apoptosis and functional impairment of the transduced cells due to an inbalance of the intracellular content in bioactive sphingolipids consequent to de novo enzyme expression. Differentiated cells of the myeloid and lymphoid lineages are not affected by GALC expression, suggesting a unique sensitivity of HSPC to enzyme toxicity. To overcome this issue, we explored strategies aimed at de-targeting vector expression from HSPC, while permitting GALC over-expression in differentiated cells. The first approach aims at a transcriptional regulation of GALC expression by the microglia/macrophage specific promoter CD11b. The second strategy exploits endogenous microRNAs for post-transcriptional regulation of GALC expression. The use of LV containing the target sequence of miRNA126, expressed only in HSPC, allows expressing GALC only in the differentiated progeny of transduced HSPC. Although these two approaches proved to be very promising in protecting HSPC from enzyme toxicity both in vitro and in vivo in heterozygous GLD mice, gene-corrected HSPC failed to repopulate lethally irradiated homozygous GLD mice. This poor result might be explained by the existence of a niche defect in homozygous GLD mice, which might hamper the engraftment of HSPC

Expression of flavin-containing monooxygenases in the human brain

Flavin-containing Monooxygenases (FMOs) were found in the human brain basal-ganglia, an area affected by neurodegenerative disorders. Full length radiolabelled cDNA probes encoding each of the five known FMOs were used in northern blot hybridisation. FMO3 and FMO4 mRNAs were detected in thalamus, substantia nigra, subthalamic nucleus and corpus callosum regions. In-situ hybridisation analysis of brain sections from normal and Parkinsons disease individuals was carried out using cRNA FMO3 and FMO4 probes. Both mRNAs were detected in dopaminergic neurons of the substantia nigra and red nucleus, neurons of the subthalamic nucleus, neurons of the thalamus and pyramidal cells of Ammons horn. Neither mRNA was found in the crus cerebre, internal capsule or superior colliculus of mid brain, or the internal capsule, putamen, dentate gyrus of the hippocampus. FMO3 was detected in higher levels than FMO4. Immunocytochemistry confirmed FMO3 is present in the same neuronal types as its mRNA. To confirm expression in human brain (and not other cross-reacting FMO mRNAs), RT-PCR of RNA from the thalamus of human subjects was undertaken. Each of the five known FMO DNA sequences were amplified using specific primers. Neither FMO1 or FMO2 sequences were amplified. The PCR product for FMO3 was of the expected size. Restriction digestion analysis and Southern blotting confirmed FMO3 is expressed in human brain. FMO5 gave a positive result which was an RT-PCR of RNA from human subthalamic nucleus. Due to the species difference in FMO expression it was of interest to see if other primates express FMO3 in brain. Substantia nigra, thalamus, midbrain and central brain regions were dissected from Orang-utan and Gorilla brain and RNA prepared. Each FMO sequence was amplified using RT-PCR reactions. No amplification products were observed for FMO1, FMO2 or FMO5. However, FMO3 amplification products were detected and a faint amplification product for FMO4 was observed

Deep Learning-Based Machinery Fault Diagnostics

This book offers a compilation for experts, scholars, and researchers to present the most recent advancements, from theoretical methods to the applications of sophisticated fault diagnosis techniques. The deep learning methods for analyzing and testing complex mechanical systems are of particular interest. Special attention is given to the representation and analysis of system information, operating condition monitoring, the establishment of technical standards, and scientific support of machinery fault diagnosis