A puromycin selectable cell line for the enrichment of mouse embryonic stem cell-derived V3 interneurons

INTRODUCTION: Spinal V3 interneurons (INs) are a commissural, glutamatergic, propriospinal neuron population that holds great potential for understanding locomotion circuitry and local rewiring after spinal cord injury. Embryonic stem cells hold promise as a cell source. However, the inevitable heterogeneity resulting from differentiation protocols makes studying post-mitotic stem cell-derived neuron populations difficult because proliferative glia quickly overtake a culture. Previously, an induction protocol for V3 INs was established. However, because of the heterogeneous population resulting from the induction protocol, functional characterization of the induced cells was not possible. METHODS: A selectable murine transgenic embryonic stem cell (ESC) line (Sim1-Puro) was generated by recombineering. The expression of the puromycin resistance enzyme, puromycin N-acetyl-transferase (PAC), was knocked into the locus of a post-mitotic V3 IN marker (Sim1), allowing Sim1 gene regulatory elements to control PAC expression. The resulting cell line was characterized for Sim1 expression by in situ hybridization, for glutamatergic marker expression by immunocytochemistry and quantitative real time polymerase chain reaction (qRT-PCR), and for functional maturation by electrophysiology. RESULTS: Puromycin selection significantly enriched the population for V3 INs, allowing long-term characterization. The selected population expressed the neuronal marker β-III tubulin and the glutamatergic neuron marker VGluT2. The selected V3 INs also exhibited appropriate functional maturation, as assessed by electrophysiology, and remained glutamatergic for 2 weeks. CONCLUSION: The Sim1-Puro cell line provides a simple, high throughput method for generating large numbers of V3 INs from mouse ESCs for future in vitro and cell transplantation studies. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s13287-015-0213-z) contains supplementary material, which is available to authorized users

Finely tuned temporal and spatial delivery of GDNF promotes enhanced nerve regeneration in a long nerve defect model

The use of growth factors, such as glial cell line-derived neurotrophic factor (GDNF), for the treatment of peripheral nerve injury has been useful in promoting axon survival and regeneration. Unfortunately, finding a method that delivers the appropriate spatial and temporal release profile to promote functional recovery has proven difficult. Some release methods result in burst release profiles too short to remain effective over the regeneration period; however, prolonged exposure to GDNF can result in axonal entrapment at the site of release. Thus, GDNF was delivered in both a spatially and temporally controlled manner using a two-phase system comprised of an affinity-based release system and conditional lentiviral GDNF overexpression from Schwann cells (SCs). Briefly, SCs were transduced with a tetracycline-inducible (Tet-On) GDNF overexpressing lentivirus before transplantation. Three-centimeter acellular nerve allografts (ANAs) were modified by injection of a GDNF-releasing fibrin scaffold under the epineurium and then used to bridge a 3 cm sciatic nerve defect. To encourage growth past the ANA, GDNF-SCs were transplanted into the distal nerve and doxycycline was administered for 4, 6, or 8 weeks to determine the optimal duration of GDNF expression in the distal nerve. Live imaging and histomorphometric analysis determined that 6 weeks of doxycycline treatment resulted in enhanced regeneration compared to 4 or 8 weeks. This enhanced regeneration resulted in increased gastrocnemius and tibialis anterior muscle mass for animals receiving doxycycline for 6 weeks. The results of this study demonstrate that strategies providing spatial and temporal control of delivery can improve axonal regeneration and functional muscle reinnervation

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells

Evaluation of 309 Environmental Chemicals Using a Mouse Embryonic Stem Cell Adherent Cell Differentiation and Cytotoxicity Assay

The vast landscape of environmental chemicals has motivated the need for alternative methods to traditional whole-animal bioassays in toxicity testing. Embryonic stem (ES) cells provide an in vitro model of embryonic development and an alternative method for assessing developmental toxicity. Here, we evaluated 309 environmental chemicals, mostly food-use pesticides, from the ToxCast™ chemical library using a mouse ES cell platform. ES cells were cultured in the absence of pluripotency factors to promote spontaneous differentiation and in the presence of DMSO-solubilized chemicals at different concentrations to test the effects of exposure on differentiation and cytotoxicity. Cardiomyocyte differentiation (α,β myosin heavy chain; MYH6/MYH7) and cytotoxicity (DRAQ5™/Sapphire700™) were measured by In-Cell Western™ analysis. Half-maximal activity concentration (AC50) values for differentiation and cytotoxicity endpoints were determined, with 18% of the chemical library showing significant activity on either endpoint. Mining these effects against the ToxCast Phase I assays (∼500) revealed significant associations for a subset of chemicals (26) that perturbed transcription-based activities and impaired ES cell differentiation. Increased transcriptional activity of several critical developmental genes including BMPR2, PAX6 and OCT1 were strongly associated with decreased ES cell differentiation. Multiple genes involved in reactive oxygen species signaling pathways (NRF2, ABCG2, GSTA2, HIF1A) were strongly associated with decreased ES cell differentiation as well. A multivariate model built from these data revealed alterations in ABCG2 transporter was a strong predictor of impaired ES cell differentiation. Taken together, these results provide an initial characterization of metabolic and regulatory pathways by which some environmental chemicals may act to disrupt ES cell growth and differentiation

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.

BACKGROUND: A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. METHODS: This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. FINDINGS: Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0-75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4-97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8-80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3-4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. INTERPRETATION: ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials. FUNDING: UK Research and Innovation, National Institutes for Health Research (NIHR), Coalition for Epidemic Preparedness Innovations, Bill & Melinda Gates Foundation, Lemann Foundation, Rede D'Or, Brava and Telles Foundation, NIHR Oxford Biomedical Research Centre, Thames Valley and South Midland's NIHR Clinical Research Network, and AstraZeneca

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Background A safe and efficacious vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), if deployed with high coverage, could contribute to the control of the COVID-19 pandemic. We evaluated the safety and efficacy of the ChAdOx1 nCoV-19 vaccine in a pooled interim analysis of four trials. Methods This analysis includes data from four ongoing blinded, randomised, controlled trials done across the UK, Brazil, and South Africa. Participants aged 18 years and older were randomly assigned (1:1) to ChAdOx1 nCoV-19 vaccine or control (meningococcal group A, C, W, and Y conjugate vaccine or saline). Participants in the ChAdOx1 nCoV-19 group received two doses containing 5 × 1010 viral particles (standard dose; SD/SD cohort); a subset in the UK trial received a half dose as their first dose (low dose) and a standard dose as their second dose (LD/SD cohort). The primary efficacy analysis included symptomatic COVID-19 in seronegative participants with a nucleic acid amplification test-positive swab more than 14 days after a second dose of vaccine. Participants were analysed according to treatment received, with data cutoff on Nov 4, 2020. Vaccine efficacy was calculated as 1 - relative risk derived from a robust Poisson regression model adjusted for age. Studies are registered at ISRCTN89951424 and ClinicalTrials.gov, NCT04324606, NCT04400838, and NCT04444674. Findings Between April 23 and Nov 4, 2020, 23 848 participants were enrolled and 11 636 participants (7548 in the UK, 4088 in Brazil) were included in the interim primary efficacy analysis. In participants who received two standard doses, vaccine efficacy was 62·1% (95% CI 41·0–75·7; 27 [0·6%] of 4440 in the ChAdOx1 nCoV-19 group vs71 [1·6%] of 4455 in the control group) and in participants who received a low dose followed by a standard dose, efficacy was 90·0% (67·4–97·0; three [0·2%] of 1367 vs 30 [2·2%] of 1374; pinteraction=0·010). Overall vaccine efficacy across both groups was 70·4% (95·8% CI 54·8–80·6; 30 [0·5%] of 5807 vs 101 [1·7%] of 5829). From 21 days after the first dose, there were ten cases hospitalised for COVID-19, all in the control arm; two were classified as severe COVID-19, including one death. There were 74 341 person-months of safety follow-up (median 3·4 months, IQR 1·3–4·8): 175 severe adverse events occurred in 168 participants, 84 events in the ChAdOx1 nCoV-19 group and 91 in the control group. Three events were classified as possibly related to a vaccine: one in the ChAdOx1 nCoV-19 group, one in the control group, and one in a participant who remains masked to group allocation. Interpretation ChAdOx1 nCoV-19 has an acceptable safety profile and has been found to be efficacious against symptomatic COVID-19 in this interim analysis of ongoing clinical trials

Developing High Purity Embryonic Stem Cell-Derived V2a Interneurons for In Vitro Investigation and Transplantation Following Spinal Cord Injury

Functional recovery following spinal cord injury has been attributed to plasticity in local interneuron populations, which are able to create novel circuits that bypass the inhibitory lesion to activate pre-existing motor pathways. Cell replacement strategies and growth factor delivery platforms often attempt to enhance these natural mechanisms of regeneration. The work of this thesis sought to overcome the major obstacle to manipulating specific interneurons for therapy: an inability to obtain large, enriched interneuron subpopulations for drug screening and transplantation. We focused on the generation of V2a interneurons, which normally coordinate a wide range of locomotor tasks in both the spinal cord and respiratory centers of the hindbrain. They are an ipsilaterally projecting excitatory glutamatergic inteneuron population that is defined by expression of the homeodomain protein Chx10 during development. Using CRISPR/Cas9-mediated homologous recombineering, we generated a transgenic mouse embryonic stem cell (ESC) line that enabled us to positively select for Chx10+ ESC-derived V2a interneurons (ESC-V2as) by treating differentiated cells with puromycin. ESC-V2a cultures remained free of proliferative cells, matured into normal glutamatergic neurons, and were capable of forming synapses onto motor neurons in adherent monolayer culture. For further in vitro investigation and transplantation studies, we cultured ESC-V2as and progenitor populations as spherical aggregates to generate a cellular microenvironment more closely related to that in vivo. We used this platform to test whether V2a IN axon growth could be stimulated in a dose-dependent manner by glial-derived neurotrophic factor (GDNF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and platelet-derived neurotrophic factor (PDGF) and/or by co-culture with ESC-derived progenitor motor neurons (ESC-pMNs). Although ESC-V2as did not variably respond to the growth factors applied when cultured in isolation, co-culture with ESC-pMNs improved ESC-V2a neurite extension, survival, maturation, and electrophysiological activity. Application of optimized concentrations of GDNF and PDGF, which act specifically on ESC-pMNs but not ESC-V2a INs, enhanced these positive effects. Finally, we sought to determine the feasibility and efficacy of transplanting neural progenitor cells (NPCs) or ESC-pMNs enriched with ESC-V2as in a respiratory model of spinal cord injury. Despite the historic difficulty in transplanting post-mitotic neuronal populations, ESC-V2as were able to survive and migrate from injury site when co-transplanted with progenitors, and in the case of V2a/NPC groups, resulted in improved recovery of diaphragm activity compared to control groups. Taken together, this thesis establishes in vitro and in vivo methodologies to investigate possible mechanisms of V2a interneuron rewiring following spinal cord injury and a platform to optimize combinatorial treatment strategies moving forward

Assessment of racial and ethnic differences of atopic dermatitis severity and treatment patterns in a diverse outpatient cohort in the United States: a retrospective observational study

Previous population-based studies in the United States found racial/ethnic differences of atopic dermatitis (AD) severity and treatment patterns. It is unclear whether these differences are from differences of disease characteristics or disparities. To examine racial/ethnic differences in severity and treatment patterns in a diverse outpatient patient cohort of AD patients (n = 833). There were no significant associations of highest-reported body surface area (BSA; Fisher\u27s exact test, P = 0.19 and P = 0.44) or physician\u27s global assessment (PGA; P = 0.63 and P = 0.57) with race or ethnicity; nor interactions of race/ethnicity with gender or age as predictors of BSA or PGA. Asian and multiracial/other patients were more likely than White or Black patients to use topical calcineurin inhibitors (Chi-square, P = 0.01). Dupilumab use differed by race (Multiracial/other = 35.0%; White = 20.1%; Asian = 15.7%; Black = 13.6%; Chi-square, P = 0.03), but not ethnicity (P = 0.88). Use of oral corticosteroids (Chi-square, P = 0.74), immunosuppressants (P = 0.98) or GABAergics (P = 0.16) or NBUVB (P = 0.42) did not differ by race. There were no interactions of race/ethnicity with gender or age as predictors of treatment use. Similar treatment patterns were observed across racial/ethnic groups. Though, topical calcineurin inhibitors were more commonly used in Asian and multiracial/other patients; dupilumab use was more common in multiracial/other patients