Fusion of RVG or gh625 to Iduronate-2-Sulfatase for the Treatment of Mucopolysaccharidosis Type II

Mucopolysaccharidosis type II (MPSII) is a lysosomal storage disease caused by a mutation in the IDS gene, resulting in deficiency of the enzyme iduronate-2-sulfatase (IDS) causing heparan sulfate (HS) and dermatan sulfate (DS) accumulation in all cells. This leads to skeletal and cardiorespiratory disease with severe neurodegeneration in two thirds of sufferers. Enzyme replacement therapy is ineffective at treating neurological disease, as intravenously-delivered IDS is unable to cross the blood-brain barrier (BBB). Haematopoietic stem cell transplant is also unsuccessful, presumably due to insufficient IDS enzyme production from transplanted cells engrafting in the brain. We used two different peptide sequences (RVG and gh625), both previously published as BBB-crossing peptides, fused to IDS and delivered via haematopoietic stem cell gene therapy (HSCGT). HSCGT with LV.IDS.RVG and LV.IDS.gh625 was compared to LV.IDS.ApoEII and LV.IDS in MPSII mice at 6-months post-transplant. Levels of IDS enzyme activity in the brain and peripheral tissues were lower in LV.IDS.RVG and LV.IDS.gh625 treated mice than in LV.IDS.ApoEII and LV.IDS treated mice, despite comparable vector copy numbers. Microgliosis, astrocytosis and lysosomal swelling were partially normalised in MPSII mice treated with LV.IDS.RVG and LV.IDS.gh625. Skeletal thickening was normalised by both treatments to wild-type levels. Although reductions in skeletal abnormalities and neuropathology are encouraging, given the low levels of enzyme activity compared to control tissue from LV.IDS and LV.IDS.ApoEII transplanted mice, the RVG and gh625 peptides are unlikely to be ideal candidates for HSCGT in MPSII, and are inferior to the ApoEII peptide that we have previously demonstrated to be more effective at correcting MPSII disease than IDS alone

Apposition to endometrial epithelial cells activates mouse blastocysts for implantation.

How do interactions between blastocyst-stage embryos and endometrial epithelial cells regulate the early stages of implantation in an in vitro model?Mouse blastocyst apposition with human endometrial epithelial cells initiates trophectoderm differentiation to trophoblast, which goes on to breach the endometrial epithelium.In vitro models using mouse blastocysts and human endometrial cell lines have proven invaluable in the molecular characterisation of embryo attachment to endometrial epithelium at the onset of implantation. Genes involved in embryonic breaching of the endometrial epithelium have not been investigated in such in vitro models.This study used an established in vitro model of implantation to examine cellular and molecular interactions during blastocyst attachment to endometrial epithelial cells.Mouse blastocysts developed from embryonic day (E) 1.5 in vitro were hatched and co-cultured with confluent human endometrial adenocarcinoma-derived Ishikawa cells in serum-free medium. A scale of attachment stability based on blastocyst oscillation upon agitation was devised. Blastocysts were monitored for 48 h to establish the kinetics of implantation, and optical sectioning using fluorescence microscopy revealed attachment and invasion interfaces. Quantitative PCR was used to determine blastocyst gene expression. Data from a total of 680 mouse blastocysts are reported, with 3-6 experimental replicates. T-test and ANOVA analyses established statistical significance at P < 0.05, P < 0.01 and P < 0.001.Hatched E4.5 mouse blastocysts exhibited weak attachment to confluent Ishikawa cells over the first 24 h of co-culture, with intermediate and stable attachment occurring from 28 h (E5.5 + 4 h) in a hormone-independent manner. Attached embryos fixed after 48 h (E6.5) frequently exhibited outgrowths, characterised morphologically and with antibody markers as trophoblast giant cells (TGCs), which had breached the Ishikawa cell layer. Beginning co-culture at E5.5 also resulted in intermediate and stable attachment from E5.5 + 4 h; however, these embryos did not go on to breach the Ishikawa cell layer, even when co-culture was extended to E7.5 (P < 0.01). Blastocysts cultured from E4.5 in permeable transwell inserts above Ishikawa cells before transfer to direct co-culture at E5.5 went on to attach but failed to breach the Ishikawa cell layer by E6.5 (P < 0.01). Gene expression analysis at E5.5 demonstrated that direct co-culture with Ishikawa cells from E4.5 resulted in downregulation of trophectoderm transcription factors Cdx2 (P < 0.05) and Gata3 (P < 0.05) and upregulation of the TGC transcription factor Hand1 (P < 0.05). Co-culture with non-endometrial human fibroblasts did not alter the expression of these genes.None.The in vitro model used here combines human carcinoma-derived endometrial cells with mouse embryos, in which the cellular interactions observed may not fully recapitulate those in vivo. The data gleaned from such models can be regarded as hypothesis-generating, and research is now needed to develop more sophisticated models of human implantation combining multiple primary endometrial cell types with surrogate and real human embryos.This study implicates blastocyst apposition to endometrial epithelial cells as a critical step in trophoblast differentiation required for implantation. Understanding this maternal regulation of the embryonic developmental programme may lead to novel treatments for infertility.This work was supported by funds from the charities Wellbeing of Women (RG1442) and Diabetes UK (15/0005207), and studentship support for SCB from the Anatomical Society. No conflict of interest is declared

Functioning human lung organoids model pulmonary tissue response from carbon nanomaterial exposures

Human lung organoids (HLOs) are increasingly used to model development and infectious diseases, however their ability to recapitulate functional pulmonary tissue response to nanomaterial (NM) exposures has yet to be demonstrated. Here, we established a lung organoid exposure model that utilises microinjection to present NMs into the lumen of organoids. Our model assures efficient, reproducible and controllable exposure of the apical pulmonary epithelium, emulating real-life human exposure scenario. By comparing the impact of two well studied carbon-based NMs, graphene oxide sheets (GO) and multi-walled carbon nanotubes (MWCNT), we validated lung organoids as tools for predicting pulmonary NM-driven responses. In agreement with established in vivo data, we demonstrate that MWCNT, but not GO, elicit adverse effects on lung organoids, leading to a pro-fibrotic phenotype. Our findings reveal the capacity and suitability of HLOs for hazard assessment of NMs, aligned with the much sought-out 3Rs (animal research replacement, reduction, refinement) framework

Improving the glial differentiation of human Schwann-like adipose-derived stem cells with graphene oxide substrates

There is urgent clinical need to improve the clinical outcome of peripheral nerve injury. Many efforts are directed towards the fabrication of bioengineered conduits, which could deliver stem cells to the site of injury to promote and guide peripheral nerve regeneration. The aim of this study is to assess if graphene and related nanomaterials can be useful in the fabrication of such conduits. A comparison is made between GO and reduced GO substrates. Our results show that the graphene substrates are highly biocompatible, and the reduced GO substrates are more effective in ncreasing the gene expression of the biomolecules involved in the regeneration process compared to the other substrates studied.Comment: 15 pages, 3 figure

Functional Characterization of Muscarinic Receptors in Human Schwann Cells

Functional characterization of muscarinic cholinergic receptors in myelinating glial cells has been well described both in central and peripheral nervous system. Rat Schwann cells (SCs) express different muscarinic receptor subtypes with the prevalence of the M2 subtype. The selective stimulation of this receptor subtype inhibits SC proliferation, improving their differentiation towards myelinating phenotype. In this work, we describe for the first time that human SCs are cholinoceptive as they express several muscarinic receptor subtypes and, as for rat SCs, M2 receptor is one of the most abundant. Human SCs, isolated from adult nerves, were cultured in vitro and stimulated with M2 muscarinic agonist arecaidine propargyl ester (APE). Similarly to that observed in rat, M2 receptor activation causes a decreased cell proliferation and promotes SC differentiation as suggested by increased Egr2 expression with an improved spindle-like shape cell morphology. Conversely, the non-selective stimulation of muscarinic receptors appears to promote cell proliferation with a reduction of SC average cell diameter. The data obtained demonstrate that human SCs are cholinoceptive and that human cultured SCs may represent an interesting tool to understand their physiology and increase the knowledge on how the cholinergic stimulation may contribute to address human SC development in normal and pathological conditions

Renal hemofiltration prevents metabolic acidosis and reduces inflammation during normothermic machine perfusion of the vascularized composite allograft—A preclinical study

From Wiley via Jisc Publications RouterHistory: received 2021-05-17, rev-recd 2021-09-16, accepted 2021-09-24, pub-electronic 2021-11-26Article version: VoRPublication status: PublishedAbstract: Introduction: Recent experimental evidence suggests normothermic machine perfusion of the vascularized composite allograft results in improved preservation compared to static cold storage, with less reperfusion injury in the immediate post‐operative period. However, metabolic acidosis is a common feature of vascularized composite allograft perfusion, primarily due to the inability to process metabolic by‐products. We evaluated the impact of combined limb‐kidney perfusion on markers of metabolic acidosis and inflammation in a porcine model. Methods: Ten paired pig forelimbs were used for this study, grouped as either limb‐only (LO, n = 5) perfusion, or limb‐kidney (LK, n = 5) perfusion. Infrared thermal imaging was used to determine homogeneity of perfusion. Lactate, bicarbonate, base, pH, and electrolytes, along with an inflammatory profile generated via the quantification of cytokines and cell‐free DNA in the perfusate were recorded. Results: The addition of a kidney to a limb perfusion circuit resulted in the rapid stabilization of lactate, bicarbonate, base, and pH. Conversely, the LO circuit became progressively acidotic, correlating in a significant increase in pro‐inflammatory cytokines. Global perfusion across the limb was more homogenous with LK compared to LO. Conclusion: The addition of a kidney during limb perfusion results in significant improvements in perfusate biochemistry, with no evidence of metabolic acidosis