Controlling embryonic stem cell proliferation and pluripotency: the role of PI3K-and GSK-3- dependent signalling

Abstract ESCs (embryonic stem cells) are derived from the inner cell mass of pre-implantation embryos and are pluripotent, meaning they can differentiate into all of the cells that make up the adult organism. This property of pluripotency makes ESCs attractive as a model system for studying early development and for the generation of specific cell types for use in regenerative medicine and drug screening. In order to harness their potential, the molecular mechanisms regulating ESC pluripotency, proliferation and differentiation (i.e. cell fate) need to be understood so that pluripotency can be maintained during expansion, while differentiation to specific lineages can be induced accurately when required. The present review focuses on the potential roles that PI3K (phosphoinositide 3-kinase) and GSK-3 (glycogen synthase kinase 3)-dependent signalling play in the co-ordination and integration of mouse ESC pluripotency and proliferation and contrast this with our understanding of their functions in human ESCs. Control of ESC (embryonic stem cell) fate: an overview ESCs are derived from early pre-implantation embryos and, when cultured appropriately, can be maintained in a proliferative, self-renewing and pluripotent state almost indefinitely. Pluripotency is the ability to differentiate into all of the cells found in an adult organism, while self-renewal describes the generation of a daughter stem cell from its mother. In the case of ESCs, self-renewal occurs symmetrically, such that when an undifferentiated ESC divides and pluripotency is maintained, both its progeny will be undifferentiated Over the last 5-10 years, our understanding of the molecular components involved in maintaining pluripotency of mESCs (mouse ESCs) has increased dramatically, from a simple 'prelude' where STAT3 (signal transducer and activator of transcription 3) activation by LIF (leukaemia Key words: cell cycle, embryonic stem cell, glycogen synthase kinase 3 (GSK-3), phosphoinositide 3-kinase (PI3K), pluripotency, proliferation, self-renewal. Abbreviations used: CDK, cyclin-dependent kinase; ESC, embryonic stem cell; Esrrb, oestrogenrelated receptor β; GSK-3, glycogen synthase kinase-3; hESC, human ESC; LIF, leukaemia inhibitory factor; MEK, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase; mESC, mouse ESC; miRNA, microRNA; mTOR, mammalian target of rapamycin; PI3K, phosphoinositide 3-kinase; siRNA, short interfering RNA. 1 To whom correspondence should be addressed (email [email protected]). inhibitory factor) was all that seemed necessary, to a complex 'symphony' where extrinsic factors, intracellular signals, transcription factors, epigenetic regulators and miRNAs (microRNAs) have all been implicated The ESC cell cycle mESCs proliferate rapidly in culture and display unique cell-cycle kinetics, distinct from those of somatic cells, dividing approximately every 11-16 h and exhibiting a shortened G 1 -phase INK4a [13] and neither do mESCs arrest following DNA damag

Safety, immunogenicity, and reactogenicity of BNT162b2 and mRNA-1273 COVID-19 vaccines given as fourth-dose boosters following two doses of ChAdOx1 nCoV-19 or BNT162b2 and a third dose of BNT162b2 (COV-BOOST): a multicentre, blinded, phase 2, randomised trial

Background Some high-income countries have deployed fourth doses of COVID-19 vaccines, but the clinical need, effectiveness, timing, and dose of a fourth dose remain uncertain. We aimed to investigate the safety, reactogenicity, and immunogenicity of fourth-dose boosters against COVID-19.Methods The COV-BOOST trial is a multicentre, blinded, phase 2, randomised controlled trial of seven COVID-19 vaccines given as third-dose boosters at 18 sites in the UK. This sub-study enrolled participants who had received BNT162b2 (Pfizer-BioNTech) as their third dose in COV-BOOST and randomly assigned them (1:1) to receive a fourth dose of either BNT162b2 (30 µg in 0·30 mL; full dose) or mRNA-1273 (Moderna; 50 µg in 0·25 mL; half dose) via intramuscular injection into the upper arm. The computer-generated randomisation list was created by the study statisticians with random block sizes of two or four. Participants and all study staff not delivering the vaccines were masked to treatment allocation. The coprimary outcomes were safety and reactogenicity, and immunogenicity (antispike protein IgG titres by ELISA and cellular immune response by ELISpot). We compared immunogenicity at 28 days after the third dose versus 14 days after the fourth dose and at day 0 versus day 14 relative to the fourth dose. Safety and reactogenicity were assessed in the per-protocol population, which comprised all participants who received a fourth-dose booster regardless of their SARS-CoV-2 serostatus. Immunogenicity was primarily analysed in a modified intention-to-treat population comprising seronegative participants who had received a fourth-dose booster and had available endpoint data. This trial is registered with ISRCTN, 73765130, and is ongoing.Findings Between Jan 11 and Jan 25, 2022, 166 participants were screened, randomly assigned, and received either full-dose BNT162b2 (n=83) or half-dose mRNA-1273 (n=83) as a fourth dose. The median age of these participants was 70·1 years (IQR 51·6–77·5) and 86 (52%) of 166 participants were female and 80 (48%) were male. The median interval between the third and fourth doses was 208·5 days (IQR 203·3–214·8). Pain was the most common local solicited adverse event and fatigue was the most common systemic solicited adverse event after BNT162b2 or mRNA-1273 booster doses. None of three serious adverse events reported after a fourth dose with BNT162b2 were related to the study vaccine. In the BNT162b2 group, geometric mean anti-spike protein IgG concentration at day 28 after the third dose was 23 325 ELISA laboratory units (ELU)/mL (95% CI 20 030–27 162), which increased to 37 460 ELU/mL (31 996–43 857) at day 14 after the fourth dose, representing a significant fold change (geometric mean 1·59, 95% CI 1·41–1·78). There was a significant increase in geometric mean anti-spike protein IgG concentration from 28 days after the third dose (25 317 ELU/mL, 95% CI 20 996–30 528) to 14 days after a fourth dose of mRNA-1273 (54 936 ELU/mL, 46 826–64 452), with a geometric mean fold change of 2·19 (1·90–2·52). The fold changes in anti-spike protein IgG titres from before (day 0) to after (day 14) the fourth dose were 12·19 (95% CI 10·37–14·32) and 15·90 (12·92–19·58) in the BNT162b2 and mRNA-1273 groups, respectively. T-cell responses were also boosted after the fourth dose (eg, the fold changes for the wild-type variant from before to after the fourth dose were 7·32 [95% CI 3·24–16·54] in the BNT162b2 group and 6·22 [3·90–9·92] in the mRNA-1273 group).Interpretation Fourth-dose COVID-19 mRNA booster vaccines are well tolerated and boost cellular and humoral immunity. Peak responses after the fourth dose were similar to, and possibly better than, peak responses after the third dose

Investigation of a discus-milling process using a powder mixture of Al and TiO₂

In this study, a powder mixture of Al and TiO₂ was employed to investigate the milling process in a discus mill. In this first report on this novel mechanical mill, several variables, including the milling time and powder charge and their effects on the microstructural evolution of powder particles, are monitored and studied. The study reveals that the dominant parameters of the milling process are the milling time and the starting powder charge, similar to the other high-energy ball-milling processes. The longer the milling time and the smaller the starting powder charge, the more homogeneous the mixing and the finer the microstructure of the powder particles. The reaction between Al and TiO₂ was not observed with a milling period as long as 6 hours, for the present materials. However, the reaction between Al and TiO₂, during the subsequent heat treatment, is influenced by the milling condition. The powders with the longer milling times and finer mixing microstructures also form a finer microstructure, after the reaction between Al and TiO₂ during heat treatment. The methods for achieving an optimal milling efficiency for the Al- TiO₂ system are discussed

Bioradiomic SPECT-CT quantification of active lung inflammation is influenced by heterogeneity of lung tissue

Introduction: COPD is a heterogenous disease. Molecular imaging of inflammation could define endotypes, but heterogeneity of lung structure complicates this process. We aimed to develop methods to quantify inflammatory cytokines in heterogenous lung tissue as potential targets for therapy.Methods: using SPECT-CT imaging, we developed techniques to quantify cytokine activity. Five patients with COPD and 5 healthy volunteers were recruited under ethically approved informed consent. They underwent SPECT-CT of the lungs at 6 (+/- 1) and 24 (+/- 4) hours after infusion of 99mTc-anti-TNF-α to quantify TNF-α activity in the lungs. Quantification was normalised to aortic arch signal to account for biological clearance.Results: median normalised SPECT counts (CN) were higher in the healthy group at both time points. Strong correlations were seen between CN and both blood vessel density and emphysema quantification (figure 1). A regression model to correct for emphysema revealed higher CN at both time points in the COPD group, but differences were not statistically significant.Conclusions: molecular imaging of inflammatory cytokines is affected by key confounding factors, and analysis techniques should account for structural heterogeneity

Towards precision medicine in COPD: bioradiomic SPECT-CT quantification of active lung inflammation

Introduction: multiple inflammatory endotypes exist as targets for therapy in COPD but novel precision medicine approaches are needed to define these. We aim to develop non-invasive imaging methods to quantify specific inflammatory cytokines in the lung as potential targets for therapy.Methods: using SPECT-CT, we developed techniques to quantify cytokine activity. Five patients with COPD and 5 healthy volunteers were recruited and gave informed consent. They underwent SPECT-CT of the lungs at 6 (+/- 1) and 24 (+/- 4) hours after infusion of 99mTc-anti-TNF-α to quantify TNF-α activity in the lungs. Quantification (figure 1) was normalised to aortic arch signal to account for biological clearance.Results: isotope signals were quantified at 6 and 24 hours. Median normalised lung SPECT counts (CN) were higher in the healthy group at both time points. However, the increase in CN at 24 hours calculated as a percentage of the 6-hour scan (a measure of tissue bound signal) was higher in the COPD group at 64.88% +/- (SD) 31.04 compared with 35.38% +/- 34.33 in the healthy group - a significant change in the COPD group (p=0.029, paired t-test) but not the healthy.Conclusions: this proof-of-concept study provides early evidence that molecular imaging of inflammatory cytokines is possible in the lungs of patients with COPD

A review of patients following a diagnosis of unclassified interstitial lung disease (ILD) at a tertiary centre

Introduction: a proportion of patients with ILD remain unclassified after multidisciplinary discussion (MDD). Without a definitive diagnosis there is currently a lack of licensed treatment options for this group. We aimed to review outcomes in this cohort of patients.Method: UK single centre retrospective review of all patients with unclassified ILD diagnosis following ILD MDD between 12/11/2012 and 19/6/2017 with a minimum 2 year follow up. Data extracted from electronic records. Ethical approval obtained.Results: 145 patients included with a mean (SD) follow-up period of 3.9 years (1.2). Baseline demographics were as follows: males 62.1% with mean (SD); age 70.7 years (9.2), FVC% 81.4 (21.4) and DLCO% 55.4 (17.1).At censor, 40.7% of patients had been reclassified to an alternative diagnosis. The median time taken to re-classify patients was 90 days with a wide range (16-2052 days). Common review diagnosis were (n); idiopathic pulmonary fibrosis (29), hypersensitivity pneumonitis (10) and connective tissue disease related ILD (3). Repeat CT and MDD re-discussion led to re-classification in 23% of patients. 15% of patients had a surgical lung biopsy and this led to subsequent re-classification of ILD in 82% of people biopsied.In patients with serial FVC% (n=101), 25% had a >10% decline over a mean interval of 2.5 years. 40% of patients with >10% FVC decline remained unclassified. No baseline characteristics were found to predict progression.Conclusion: these results highlight the importance of MDD follow up of patients with unclassified ILD over time to both review diagnosis and monitor for disease progression to enable appropriate disease targeted therapy