C286, an orally available retinoic acid receptor ß agonist drug, regulates multiple pathways to achieve spinal cord injury repair.

Retinoic acid receptor ß2 (RARß2) is an emerging therapeutic target for spinal cord injuries (SCIs) with a unique multimodal regenerative effect. We have developed a first-in-class RARß agonist drug, C286, that modulates neuron-glial pathways to induce functional recovery in a rodent model of sensory root avulsion. Here, using genome-wide and pathway enrichment analysis of avulsed rats' spinal cords, we show that C286 also influences the extracellular milieu (ECM). Protein expression studies showed that C286 upregulates tenascin-C, integrin-α9, and osteopontin in the injured cord. Similarly, C286 remodulates these ECM molecules, hampers inflammation and prevents tissue loss in a rodent model of spinal cord contusion C286. We further demonstrate C286's efficacy in human iPSC-derived neurons, with treatment resulting in a significant increase in neurite outgrowth. Additionally, we identify a putative efficacy biomarker, S100B, which plasma levels correlated with axonal regeneration in nerve-injured rats. We also found that other clinically available retinoids, that are not RARß specific agonists, did not lead to functional recovery in avulsed rats, demonstrating the requirement for RARß specific pathways in regeneration. In a Phase 1 trial, the single ascending dose (SAD) cohorts showed increases in expression of RARß2 in white blood cells correlative to increased doses and at the highest dose administered, the pharmacokinetics were similar to the rat proof of concept (POC) studies. Collectively, our data suggests that C286 signalling in neurite/axonal outgrowth is conserved between species and across nerve injuries. This warrants further clinical testing of C286 to ascertain POC in a broad spectrum of neurodegenerative conditions.

Metallomic mapping of gut and brain in heavy metal exposed earthworms: A novel paradigm in ecotoxicology.

This study explored the uptake of lead in the epigeic earthworm Dendrobaena veneta exposed to 0, 1000, and 2500 µg Pb/g soil. The soil metal content was extracted using strong acid digestion and water leaching, and analysed by means of Inductively Coupled Plasma Mass Spectrometry (ICP-MS) to estimate absolute and bioavailable concentrations of metals in the soil. The guts and heads of lead-exposed earthworms were processed into formalin-fixed and paraffin embedded sections for high-resolution multi-element metallomic imaging via Laser Ablation ICP-MS (LA-ICP-MS). Metallomic maps of phosphorus, zinc, and lead were produced at 15-µm resolution in the head and gut of D. veneta. Additional 4-µm resolution metallomic maps of the earthworm brains were taken, revealing the detailed localisation of metals in the brain. The Pb bioaccumulated in the chloragogenous tissues of the earthworm in a dose-dependent manner, making it possible to track the extent of soil contamination. The bioaccumulation of P and Zn in earthworm tissues was independent of Pb exposure concentration. This approach demonstrates the utility of LA-ICP-MS as a powerful approach for ecotoxicology and environmental risk assessments.

Anti-cancer pro-inflammatory effects of an IgE antibody targeting the melanoma-associated antigen chondroitin sulfate proteoglycan 4.

Outcomes for half of patients with melanoma remain poor despite standard-of-care checkpoint inhibitor therapies. The prevalence of the melanoma-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) expression is ~70%, therefore effective immunotherapies directed at CSPG4 could benefit many patients. Since IgE exerts potent immune-activating functions in tissues, we engineer a monoclonal IgE antibody with human constant domains recognizing CSPG4 to target melanoma. CSPG4 IgE binds to human melanomas including metastases, mediates tumoricidal antibody-dependent cellular cytotoxicity and stimulates human IgE Fc-receptor-expressing monocytes towards pro-inflammatory phenotypes. IgE demonstrates anti-tumor activity in human melanoma xenograft models engrafted with human effector cells and is associated with enhanced macrophage infiltration, enriched monocyte and macrophage gene signatures and pro-inflammatory signaling pathways in the tumor microenvironment. IgE prolongs the survival of patient-derived xenograft-bearing mice reconstituted with autologous immune cells. No ex vivo activation of basophils in patient blood is measured in the presence of CSPG4 IgE. Our findings support a promising IgE-based immunotherapy for melanoma.

Periaqueductal grey and spinal cord pathology contribute to pain in Parkinson's disease.

Pain is a key non-motor feature of Parkinson's disease (PD) that significantly impacts on life quality. The mechanisms underlying chronic pain in PD are poorly understood, hence the lack of effective treatments. Using the 6-hydroxydopamine (6-OHDA) lesioned rat model of PD, we identified reductions in dopaminergic neurons in the periaqueductal grey (PAG) and Met-enkephalin in the dorsal horn of the spinal cord that were validated in human PD tissue samples. Pharmacological activation of D1-like receptors in the PAG, identified as the DRD5+ phenotype located on glutamatergic neurons, alleviated the mechanical hypersensitivity seen in the Parkinsonian model. Downstream activity in serotonergic neurons in the Raphé magnus (RMg) was also reduced in 6-OHDA lesioned rats, as detected by diminished c-FOS positivity. Furthermore, we identified increased pre-aggregate α-synuclein, coupled with elevated activated microglia in the dorsal horn of the spinal cord in those people that experienced PD-related pain in life. Our findings have outlined pathological pathways involved in the manifestation of pain in PD that may present targets for improved analgesia in people with PD.

The Prrx1eGFP Mouse Labels the Periosteum During Development and a Subpopulation of Osteogenic Periosteal Cells in the Adult.

The identity of the cells that form the periosteum during development is controversial with current dogma suggesting these are derived from a Sox9-positive progenitor. Herein, we characterize a newly created Prrx1eGFP reporter transgenic mouse line during limb formation and postnatally. Interestingly, in the embryo Prrx1eGFP-labeled cells become restricted around the Sox9-positive cartilage anlage without themselves becoming Sox9-positive. In the adult, the Prrx1eGFP transgene live labels a subpopulation of cells within the periosteum that are enriched at specific sites, and this population is diminished in aged mice. The green fluorescent protein (GFP)-labeled subpopulation can be isolated using fluorescence-activated cell sorting (FACS) and represents approximately 8% of all isolated periosteal cells. The GFP-labeled subpopulation is significantly more osteogenic than unlabeled, GFP-negative periosteal cells. In addition, the osteogenic and chondrogenic capacity of periosteal cells in vitro can be extended with the addition of fibroblast growth factor (FGF) to the expansion media. We provide evidence to suggest that osteoblasts contributing to cortical bone formation in the embryo originate from Prrx1eGFP-positive cells within the perichondrium, which possibly piggyback on invading vascular cells and secrete new bone matrix. In summary, the Prrx1eGFP mouse is a powerful tool to visualize and isolate periosteal cells and to quantify their properties in the embryo and adult. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Planar cell polarity proteins determine basal cell height in the later stage embryonic mouse epidermis'.

Background: Complex organ formation requires the coordinated morphogenesis of adjacent tissue layers. Here, a role for the planar cell polarity (PCP) proteins Fz6 and Celsr1 in generating squamous basal cells in the later stage embryonic epidermis of the mouse is reported, which impacts upon the shape of overlying suprabasal cells. Methods: The depth of the epidermis and basal layer as well as cell proliferation index was scored from immunostained wax sections taken from different mouse embryos mutant in planar cell polarity signalling and their wild-type littermates. Orientation of epidermal cell division in Celsr1 Crash/Crash mutants was determined from thick frozen immunostained sections. Immunostained wax sections of wild-type skin explants cultured using the Lumox method enabled any changes in epidermal and basal layer depth to be measured following the release of surface tension upon dissection of skin away from the whole embryo.   Results: Increased numbers of columnar and cuboidal basal epidermal cells were observed in fz6 and Celsr1 mouse mutants including Celsr1 Crash/Crash which correlated with more rounded suprabasal cells and a thicker epidermis. Conclusions: Altogether these data support tissue intrinsic roles for PCP proteins in 'outside-in' (radial) skin architecture.

The zinc finger/RING domain protein Unkempt regulates cognitive flexibility.

Correct orchestration of nervous system development is a profound challenge that involves coordination of complex molecular and cellular processes. Mechanistic target of rapamycin (mTOR) signaling is a key regulator of nervous system development and synaptic function. The mTOR kinase is a hub for sensing inputs including growth factor signaling, nutrients and energy levels. Activation of mTOR signaling causes diseases with severe neurological manifestations, such as tuberous sclerosis complex and focal cortical dysplasia. However, the molecular mechanisms by which mTOR signaling regulates nervous system development and function are poorly understood. Unkempt is a conserved zinc finger/RING domain protein that regulates neurogenesis downstream of mTOR signaling in Drosophila. Unkempt also directly interacts with the mTOR complex I component Raptor. Here we describe the generation and characterisation of mice with a conditional knockout of Unkempt (UnkcKO) in the nervous system. Loss of Unkempt reduces Raptor protein levels in the embryonic nervous system but does not affect downstream mTORC1 targets. We also show that nervous system development occurs normally in UnkcKO mice. However, we find that Unkempt is expressed in the adult cerebellum and hippocampus and behavioural analyses show that UnkcKO mice have improved memory formation and cognitive flexibility to re-learn. Further understanding of the role of Unkempt in the nervous system will provide novel mechanistic insight into the role of mTOR signaling in learning and memory.

Development of an in vitro model of the dentogingival junction using 3D organotypic constructs.

OBJECTIVES: The overall aim was to propose a plausible model of the dentogingival junction (DGJ) to deepen our understanding of the extrinsic influences responsible for the development of the junctional epithelial phenotype. The specific objective was to test the hypothesis that epithelial migration and proliferation would be inhibited by periodontal ligament (PDL) fibroblasts in an in vitro model of the DGJ consisting of 3D organotypic cultures. BACKGROUND: Previously, we showed that 3D organotypic cultures containing human gingival fibroblasts (HGF) supported the development of a multi-layered epithelium, while constructs containing human periodontal ligament fibroblasts (HPDLF) resulted in epithelial atrophy (Lu EMC, Hobbs C, Dyer CJ, Ghuman M, Hughes FJ. J Perio Res., 2020). However, changes in epithelial phenotype have not been studied within an in vitro model of the DGJ. METHODS: The in vitro model of the DGJ comprised of a donor HGF construct (H400 epithelium overlying HGF-collagen matrix) supported by a dimensionally larger recipient collagen bed enriched with HPDLF. Samples were harvested, fixed and processed for immunohistochemistry. The changes in epithelial migration and proliferation following contact with HPDLF were assessed by measuring the horizontal extension of the epithelial outgrowth on the recipient collagen matrix. RESULTS: Within our in vitro model of the DGJ, epithelial migration and proliferation were inhibited following contact with the recipient HPDLF. By contrast, the control set-up showed a relative increase in epithelial growth, where the epithelium came into contact with the recipient HGF. Overall, there were limited changes in the molecular expression of keratin markers. CONCLUSION: This study has proposed a plausible in vitro model of the DGJ to illustrate the role of different fibroblasts in the regulation of dentogingival epithelia. Furthermore, it suggests that the anatomical positional stability of the JE and its apparent resistance to apical migration could be associated with its interaction with the PDL.

A developmental basis for the anatomical diversity of dermis in homeostasis and wound repair.

The dermis has disparate embryonic origins; abdominal dermis develops from lateral plate mesoderm, dorsal dermis from paraxial mesoderm and facial dermis from neural crest. However, the cell and molecular differences and their functional implications have not been described. We hypothesise that the embryonic origin of the dermis underpins regional characteristics of skin, including its response to wounding. We have compared abdomen, back and cheek, three anatomical sites representing the distinct embryonic tissues from which the dermis can arise, during homeostasis and wound repair using RNA sequencing, histology and fibroblast cultures. Our transcriptional analyses demonstrate differences between body sites that reflect their diverse origins. Moreover, we report histological and transcriptional variations during a wound response, including site differences in ECM composition, cell migration and proliferation, and re-enactment of distinct developmental programmes. These findings reveal profound regional variation in the mechanisms of tissue repair. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

The spatiotemporal spread of cervical spinal cord contusion injury pathology revealed by 3D in-line phase contrast synchrotron X-ray microtomography.

Extensive structural changes occur within the spinal cord following traumatic injury. Acute tissue debris and necrotic tissue are broken down, proliferating local glia and infiltrating leukocytes remodel tissue biochemical and biophysical properties, and a chronic cavity surrounded by a scar forms at the injury epicentre. Serial-section 2D histology has traditionally assessed these features in experimental models of spinal cord injury (SCI) to measure the extent of tissue pathology and evaluate efficacy of novel therapies. However, this 2D snapshot approach overlooks slice intervening features, with accurate representation of tissue compromised by mechanical processing artefacts. 3D imaging avoids these caveats and allows full exploration of the injured tissue volume to characterise whole tissue pathology. Amongst 3D imaging modalities, Synchrotron Radiation X-ray microtomography (SRµCT) is advantageous for its speed, ability to cover large tissue volumes at high resolution, and need for minimal sample processing. Here we demonstrate how extended lengths of formalin-fixed, paraffin-embedded (FFPE) rat spinal cord can be completely imaged by SRµCT with micron resolution. Label-free contrast derived from X-ray phase interactions with low-density soft tissues, reveals spinal cord white matter, gray matter, tissue damage and vasculature, with tissue still viable for targeted 2D-histology after 3D imaging. We used SRµCT to quantify tissue pathology after a midline, cervical level (C6), 225 kDyne contusion injury over acute-to-chronic (24 h to 5 weeks) post injury time points. Quantification revealed acute tissue swelling prior to chronic atrophy across the whole imaged region (spanning 2 spinal segments above and below injury), along with rostro-caudal asymmetries in white and gray matter volume loss. 3D volumes revealed satellite damage in tissue far removed from the epicentre, and extensive rostro-caudal spread of damage through the base of the dorsal columns at 24 h post injury. This damage overlapped regions of vasogenic oedema, confirmed with subsequent histology. Tissue damage at later time points in border regions was most prominent in the dorsal columns, where it overlapped sites of damaged venous vasculature. Elaborating rostro-caudal and spatiotemporal asymmetries in reduced traumatic injury models centred on these regions may inform future treatments that seek to limit the spread of tissue pathology to these 'at-risk' regions.

Differential regulation of epithelial growth by gingival and periodontal fibroblasts in vitro.

OBJECTIVES: To investigate the underlying molecular mechanisms by which gingival and periodontal ligament (PDL) fibroblasts regulate epithelial phenotype. BACKGROUND: Fibroblast populations regulate the epithelial phenotype through epithelial-mesenchymal interactions (EMI). Previous studies have proposed that maintenance of the junctional epithelium (JE) is dependent on the differential effects from gingival and PDL tissues. However, these cell populations are undefined and the signalling mechanisms which may regulate JE are unknown. METHODS: Immunohistochemical analyses were performed on formalin-fixed paraffin-embedded sections of dentogingival tissues to identify phenotypic differences in fibroblast populations. The effect of distinct fibroblasts on epithelial phenotype was studied via 3D organotypic cultures, consisting of an H400 epithelium supported by human gingival fibroblasts (HGF) or human periodontal ligament fibroblasts (HPDLF), embedded in collagen gel. To investigate the involvement of Wnt signalling in EMI, the Wnt antagonist rhDKK1 was added to HGF constructs. The gene expression of Wnt antagonists and agonists was tested via RNA extraction and qPCR. Specific gene silencing using RNA interference was performed on HPDLF/HGF constructs. RESULTS: Gingival fibroblasts were characterized by Sca1 expression, and PDL fibroblasts, characterized by Periostin and Asporin expression. Through the construction of 3D organotypic cultures, we showed that HGF supported epithelial multilayering, whilst HPDLF failed to support epithelial cell growth. Furthermore, HGF constructs treated with rhDKK1 resulted in a profound reduction in epithelial thickness. We identified SFRP4 to be highly specifically expressed in HPDLF, at both the mRNA and protein levels. A knockdown of SFRP4 in HPDLF constructs led to an increase in epithelial growth. CONCLUSION: The study demonstrates the presence of phenotypically distinct fibroblast populations within dentogingival tissues and that these specific populations have different influences on the epithelium. Our data suggest that a downregulation of Wnt signalling within PDL may be important in maintaining the integrity and anatomical position of the JE.

Stem Cell Research Lab Resource: Stem Cell LineInduced pluripotent stem cell (iPSC) line MLi-003A derived from an individual with the maximum number of filaggrin (FLG) tandem repeats.

We have generated MLi003-A, a new induced pluripotent stem cell (iPSC) line derived from hair follicle keratinocytes of a healthy male characterized with a maximum number of filaggrin tandem repeats, making this iPSC line the best control for studies on skin barrier function. The characterization of the MLi003-A cell line consisted of molecular karyotyping, high-throughput array-based sequencing composed of Fluidigm microfluidics technology and next-generation sequencing of the filaggrin alleles, and pluripotency and differentiation potentials testing by immunofluorescence of associated markers both in vitro and in vivo. The MLi-003A line has been also tested for ability to differentiate into keratinocytes.

In vivo safety profile of a CSPG4-directed IgE antibody in an immunocompetent rat model.

IgE monoclonal antibodies hold great potential for cancer therapy. Preclinical in vivo systems, particularly those in which the antibody recognizes the host species target antigen and binds to cognate Fc receptors, are often the closest approximation to human exposure and represent a key challenge for evaluating the safety of antibody-based therapies. We sought to develop an immunocompetent rat system to assess the safety of a rodent anti-tumor IgE, as a surrogate for the human therapeutic candidate. We generated a rat IgE against the human tumor-associated antigen chondroitin sulfate proteoglycan 4 (CSPG4) and cross-reactive for the rat antigen. We analyzed CSPG4 distribution in normal rat and human tissues and investigated the in vivo safety of the antibody by monitoring clinical signs and molecular biomarkers after systemic administration to immunocompetent rats. Human and rat CSPG4 expression in normal tissues were comparable. Animals receiving antibody exhibited transient mild to moderate adverse events accompanied by mild elevation of serum tryptase, but not of angiotensin II or cytokines implicated in allergic reactions or cytokine storm. In the long term, repeated antibody administration was well tolerated, with no changes in animal body weight, liver and kidney functions or blood cell counts. This model provides preclinical support for the safety profiling of IgE therapeutic antibodies. Due to the comparable antigen tissue distribution in human and rat, this model may also comprise an appropriate tool for proof-of-concept safety evaluations of different treatment approaches targeting CSPG4.

Biomimetic gradient scaffold of collagen-hydroxyapatite for osteochondral regeneration.

Osteochondral defects remain a major clinical challenge mainly due to the combined damage to the articular cartilage and the underlying bone, and the interface between the two tissues having very different properties. Current treatment modalities have several limitations and drawbacks, with limited capacity of restoration; however, tissue engineering shows promise in improving the clinical outcomes of osteochondral defects. In this study, a novel gradient scaffold has been fabricated, implementing a gradient structure in the design to mimic the anatomical, biological and physicochemical properties of bone and cartilage as closely as possible. Compared with the commonly studied multi-layer scaffolds, the gradient scaffold has the potential to induce a smooth transition between cartilage and bone and avoid any instability at the interface, mimicking the natural structure of the osteochondral tissue. The scaffold comprises a collagen matrix with a gradient distribution of low-crystalline hydroxyapatite particles. Physicochemical analyses confirmed phase and chemical compositions of the gradient scaffold and the distribution of the mineral phase along the gradient scaffold. Mechanical tests confirmed the gradient of stiffness throughout the scaffold, according to its mineral content. The gradient scaffold exhibited good biological performances both in vitro and in vivo. Biological evaluation of the scaffold, in combination with human bone-marrow-derived mesenchymal stem cells, demonstrated that the gradient of composition and stiffness preferentially increased cell proliferation in different sub-regions of the scaffold, according to their high chondrogenic or osteogenic characteristics. The in vivo biocompatibility of the gradient scaffold was confirmed by its subcutaneous implantation in rats. The gradient scaffold was significantly colonised by host cells and minimal foreign body reaction was observed. The scaffold's favourable chemical, physical and biological properties demonstrated that it has good potential as an engineered osteochondral analogue for the regeneration of damaged tissue.

RARß Agonist Drug (C286) Demonstrates Efficacy in a Pre-clinical Neuropathic Pain Model Restoring Multiple Pathways via DNA Repair Mechanisms.

Neuropathic pain (NP) is associated with profound gene expression alterations within the nociceptive system. DNA mechanisms, such as epigenetic remodeling and repair pathways have been implicated in NP. Here we have used a rat model of peripheral nerve injury to study the effect of a recently developed RARß agonist, C286, currently under clinical research, in NP. A 4-week treatment initiated 2 days after the injury normalized pain sensation. Genome-wide and pathway enrichment analysis showed that multiple mechanisms persistently altered in the spinal cord were restored to preinjury levels by the agonist. Concomitant upregulation of DNA repair proteins, ATM and BRCA1, the latter being required for C286-mediated pain modulation, suggests that early DNA repair may be important to prevent phenotypic epigenetic imprints in NP. Thus, C286 is a promising drug candidate for neuropathic pain and DNA repair mechanisms may be useful therapeutic targets to explore.

Induced pluripotent stem cell line heterozygous for p.R501X mutation in filaggrin: KCLi003-A.

We have generated an induced pluripotent stem cell (iPSC) line KCLi003-A (iOP101) from epidermal keratinocytes of a female donor, heterozygous for the loss-of-function mutation p.R501X in the filaggrin gene (FLG), using non-integrating Sendai virus vectors. Derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization and validation of KCLi003-A line included molecular karyotyping, mutation screening using restriction enzyme digestion, next generation sequencing (NGS), while pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and by in vivo teratoma assay.

Induced pluripotent stem cell line heterozygous for p.R2447X mutation in filaggrin: KCLi002-A.

We have generated an induced pluripotent stem cell (iPSC) line KCLi002-A (iOP107) from a female donor, heterozygous for the loss-of-function mutation p.R2447X in the filaggrin gene (FLG). Epidermal keratinocytes were reprogrammed using non-integrating Sendai virus vectors. The entire process of derivation and expansion of iPSCs were performed under xeno-free culture conditions. Characterization of KCLi002-A line included molecular karyotyping, mutation screening using restriction enzyme digestion Sanger sequencing and next generation sequencing (NGS), whereas pluripotency and differentiation potential were confirmed by expression of associated markers in vitro and in vivo teratoma assay.

Induced pluripotent stem cell (iPSC) line from an epidermolysis bullosa simplex patient heterozygous for keratin 5 E475G mutation and with the Dowling Meara phenotype.

We have generated MLi002-A, a new induced pluripotent stem cell (iPSC) line derived from keratinocytes of a skin punch biopsy of a female patient with the severe epidermolysis bullosa simplex Dowling-Meara phenotype and the keratin K5 E475G mutation. Keratinocytes were reprogrammed using non-integrating Sendai virus vectors, and xeno-free culture conditions were used throughout. The characterization of MLi002-A cell line consisted of molecular karyotyping, mutation screening using restriction enzyme digestion and Sanger sequencing, and testing of the pluripotency and differentiation potentials by immunofluorescence of associated markers both in vitro and in vivo. This is the first iPSC model of EB Simplex.