A Glutamate-Dependent Redox System in Blood Cells Is Integral for Phagocytosis in Drosophila melanogaster

SummaryGlutamate transport is highly regulated as glutamate directly acts as a neurotransmitter [1–3] and indirectly regulates the synthesis of antioxidants [4, 5]. Although glutamate deregulation has been repeatedly linked to serious human diseases such as HIV infection and Alzheimer’s [6–8], glutamate’s role in the immune system is still poorly understood. We find that a putative glutamate transporter in Drosophila melanogaster, polyphemus (polyph), plays an integral part in the fly’s immune response. Flies with a disrupted polyph gene exhibit decreased phagocytosis of microbial-derived bioparticles. When infected with S. aureus, polyph flies show an increase in both susceptibility and bacterial growth. Additionally, the expression of two known glutamate transporters, genderblind and excitatory amino acid transporter 1, in blood cells affects the flies’ ability to phagocytose and survive after an infection. Consistent with previous data showing a regulatory role for glutamate transport in the synthesis of the major antioxidant glutathione, polyph flies produce more reactive oxygen species (ROS) as compared to wild-type flies when exposed to S. aureus. In conclusion, we demonstrate that a polyph-dependent redox system in blood cells is necessary to maintain the cells’ immune-related functions. Furthermore, our model provides insight into how deregulation of glutamate transport may play a role in disease

Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action

A workshop sponsored by the Human Toxicology Project Consortium (HTPC), “Building Shared Experience to Advance Practical Application of Pathway-Based Toxicology: Liver Toxicity Mode-of-Action” brought together experts from a wide range of perspectives to inform the process of pathway development and to advance two prototype pathways initially developed by the European Commission Joint Research Center (JRC): liver-specific fibrosis and steatosis. The first half of the workshop focused on the theory and practice of pathway development; the second on liver disease and the two prototype pathways. Participants agreed pathway development is extremely useful for organizing information and found that focusing the theoretical discussion on a specific AOP is helpful. It is important to include several perspectives during pathway development, including information specialists, pathologists, human health and environmental risk assessors, and chemical and product manufacturers, to ensure the biology is well captured and end use is considered

Ten-year mortality, disease progression, and treatment-related side effects in men with localised prostate cancer from the ProtecT randomised controlled trial according to treatment received

Background The ProtecT trial reported intention-to-treat analysis of men with localised prostate cancer randomly allocated to active monitoring (AM), radical prostatectomy, and external beam radiotherapy. Objective To report outcomes according to treatment received in men in randomised and treatment choice cohorts. Design, setting, and participants This study focuses on secondary care. Men with clinically localised prostate cancer at one of nine UK centres were invited to participate in the treatment trial comparing AM, radical prostatectomy, and radiotherapy. Intervention Two cohorts included 1643 men who agreed to be randomised and 997 who declined randomisation and chose treatment. Outcome measurements and statistical analysis Analysis was carried out to assess mortality, metastasis and progression and health-related quality of life impacts on urinary, bowel, and sexual function using patient-reported outcome measures. Analysis was based on comparisons between groups defined by treatment received for both randomised and treatment choice cohorts in turn, with pooled estimates of intervention effect obtained using meta-analysis. Differences were estimated with adjustment for known prognostic factors using propensity scores. Results and limitations According to treatment received, more men receiving AM died of PCa (AM 1.85%, surgery 0.67%, radiotherapy 0.73%), whilst this difference remained consistent with chance in the randomised cohort (p = 0.08); stronger evidence was found in the exploratory analyses (randomised plus choice cohort) when AM was compared with the combined radical treatment group (p = 0.003). There was also strong evidence that metastasis (AM 5.6%, surgery 2.4%, radiotherapy 2.7%) and disease progression (AM 20.35%, surgery 5.87%, radiotherapy 6.62%) were more common in the AM group. Compared with AM, there were higher risks of sexual dysfunction (95% at 6 mo) and urinary incontinence (55% at 6 mo) after surgery, and of sexual dysfunction (88% at 6 mo) and bowel dysfunction (5% at 6 mo) after radiotherapy. The key limitations are the potential for bias when comparing groups defined by treatment received and changes in the protocol for AM during the lengthy follow-up required in trials of screen-detected PCa. Conclusions Analyses according to treatment received showed increased rates of disease-related events and lower rates of patient-reported harms in men managed by AM compared with men managed by radical treatment, and stronger evidence of greater PCa mortality in the AM group. Patient summary More than 95 out of every 100 men with low or intermediate risk localised prostate cancer do not die of prostate cancer within 10 yr, irrespective of whether treatment is by means of monitoring, surgery, or radiotherapy. Side effects on sexual and bladder function are better after active monitoring, but the risks of spreading of prostate cancer are more common

Functional and quality of life outcomes of localised prostate cancer treatments (prostate testing for cancer and treatment [ProtecT] study)

Objective To investigate the functional and quality of life (QoL) outcomes of treatments for localised prostate cancer and inform treatment decision-making. Patients and Methods Men aged 50–69 years diagnosed with localised prostate cancer by prostate-specific antigen testing and biopsies at nine UK centres in the Prostate Testing for Cancer and Treatment (ProtecT) trial were randomised to, or chose one of, three treatments. Of 2565 participants, 1135 men received active monitoring (AM), 750 a radical prostatectomy (RP), 603 external-beam radiotherapy (EBRT) with concurrent androgen-deprivation therapy (ADT) and 77 low-dose-rate brachytherapy (BT, not a randomised treatment). Patient-reported outcome measures (PROMs) completed annually for 6 years were analysed by initial treatment and censored for subsequent treatments. Mixed effects models were adjusted for baseline characteristics using propensity scores. Results Treatment-received analyses revealed different impacts of treatments over 6 years. Men remaining on AM experienced gradual declines in sexual and urinary function with age (e.g., increases in erectile dysfunction from 35% of men at baseline to 53% at 6 years and nocturia similarly from 20% to 38%). Radical treatment impacts were immediate and continued over 6 years. After RP, 95% of men reported erectile dysfunction persisting for 85% at 6 years, and after EBRT this was reported by 69% and 74%, respectively (P < 0.001 compared with AM). After RP, 36% of men reported urinary leakage requiring at least 1 pad/day, persisting for 20% at 6 years, compared with no change in men receiving EBRT or AM (P < 0.001). Worse bowel function and bother (e.g., bloody stools 6% at 6 years and faecal incontinence 10%) was experienced by men after EBRT than after RP or AM (P < 0.001) with lesser effects after BT. No treatment affected mental or physical QoL. Conclusion Treatment decision-making for localised prostate cancer can be informed by these 6-year functional and QoL outcomes

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

BMP-2 Induces Versican and Hyaluronan That Contribute to Post-EMT AV Cushion Cell Migration

<div><p>Distal outgrowth and maturation of mesenchymalized endocardial cushions are critical morphogenetic events during post-EMT atrioventricular (AV) valvuloseptal morphogenesis. We explored the role of BMP-2 in the regulation of valvulogenic extracellular matrix (ECM) components, versican and hyaluronan (HA), and cell migration during post-EMT AV cushion distal outgrowth/expansion. We observed intense staining of versican and HA in AV cushion mesenchyme from the early cushion expansion stage, Hamburger and Hamilton (HH) stage-17 to the cushion maturation stage, HH stage-29 in the chick. Based on this expression pattern we examined the role of BMP-2 in regulating versican and HA using 3D AV cushion mesenchymal cell (CMC) aggregate cultures on hydrated collagen gels. BMP-2 induced versican expression and HA deposition as well as mRNA expression of <i>versican</i> and <i>Has2</i> by CMCs in a dose dependent manner. Noggin, an antagonist of BMP, abolished BMP-2-induced versican and HA as well as mRNA expression of <i>versican</i> and <i>Has2</i>. We further examined whether BMP-2-promoted cell migration was associated with expression of versican and HA. BMP-2- promoted cell migration was significantly impaired by treatments with versican siRNA and HA oligomer. In conclusion, we provide evidence that BMP-2 induces expression of versican and HA by AV CMCs and that these ECM components contribute to BMP-2-induced CMC migration, indicating critical roles for BMP-2 in distal outgrowth/expansion of mesenchymalized AV cushions. </p> </div

BMP-2 treatment increases versican expression by AV endocardial cushion mesenchymal cells (CMCs).

<p>(A-F) Versican immunostaining in HH stage-24 AV CMC aggregate cultures on collagen gels. The CMC aggregates were cultured in Medium 199 alone (control) (A), or with BMP-2, 20 ng/ml (B), BMP-2, 50 ng/ml (C), BMP-2, 200 ng/ml (D), BMP-2 200 ng/ml plus noggin, 500 ng/ml (E) or noggin, 500 ng/ml (F), dissolved in Medium 199. As low as 20 ng/ml of BMP-2 promoted versican immunostaining in cultured CMC aggregates. (G) Quantitative evaluation of versican immunointensity in AV CMC aggregate cultures. Immunointensity of the CMC aggregates treated with concentrations as low as 20 ng/ml of BMP-2 was significantly higher than the control (**<i>p</i><0.01). Conversely, immunointensity in CMC aggregates cultured with noggin was significantly lower than the control (*<i>p</i><0.05). (H) Quantitative evaluation of <i>versican</i> mRNA expression in CMC aggregate cultures. QRT-PCR data revealed an approximately 7-fold increase of <i>versican</i> expression in AV CMC aggregate cultures incubated with BMP-2 over the control cultures (**<i>p</i><0.01). The potential of BMP-2 to stimulate <i>versican</i> expression was abolished when noggin was added to the cultures (*<i>p</i><0.05). Vertical bars indicate ± SD of the mean. *<i>p</i><0.05 ; **<i>p</i><0.01 as compared to the control (M199).</p

Collagen gel images of HH stage-24 AV CMC aggregate cultures treated with HA oligomers.

<p>Upper panels show the surface of the gel and the lower panels show a depth of 240µm within the gel. Higher magnification views shown in the insets in Figures e, f, h and k indicate CMCs in the focal plane at 240µm below the surface of the gels (arrows in e, f, h and k). Note that there are no mesenchymal cells in the focal plane at 240µm below the surface of the gels in Figures g and l. CMCs treated with BMP-2 (200 ng/ml) migrated deeper into the collagen gels (b and arrows in f) than the control cultures (M199, a and arrows in e). BMP-2-stimulated CMC migration was significantly reduced when HA oligomers (100 µg/ml) were added to the medium (c and g). HA oligomer treatments alone reduced mesenchymal cell migration relative to the control cultures (j and l), whereas chitin oligomers (control oligomers) (100 µg/ml) did not alter CMC migration (i, arrows in k). Chitin oligomers did not reduce BMP-2-stimulated CMC migration (d, arrows in h). </p

BMP-2 treatment increases HA deposition and <i>Has2</i> expression by AV endocardial cushion mesenchymal cells (CMCs).

<p>(A-F) HA staining by using hyaluronan-binding protein (HABP) in HH stage-24 AV CMC aggregate cultures on collagen gels. The CMC aggregates were cultured in Medium 199 alone (control) (A), or with BMP-2, 20ng/ml (B), BMP-2, 50ng/ml (C), BMP-2, 200 ng/ml (D), BMP-2 200 ng/ml plus noggin, 500 ng/ml (E) or noggin, 500 ng/ml (F), dissolved in Medium 199. As low as 20 ng/ml of BMP-2-stimulated HA staining in cultured CMC aggregates. (G) Quantitative evaluation of HA staining in AV CMC aggregate cultures. Staining intensity of the CMC aggregates treated with as low as 20 ng/ml of BMP-2 was significantly higher than the control (**<i>p</i><0.01). Conversely, staining intensity in CMC aggregates cultured with noggin was significantly lower than the control (*<i>p</i><0.05). (H) Quantitative evaluation of mRNA expression of <i>Has2</i> in CMC aggregate cultures. QRT-PCR data revealed an approximately 5-fold increase of <i>Has2</i> in CMC aggregate cultures treated with BMP-2 over control cultures (**<i>p</i><0.01). BMP-2-stimulated <i>Has2</i> expression was abolished when noggin was added to the cultures (*<i>p</i><0.05). Vertical bars indicate ± SD of the mean. *<i>p</i><0.05; **<i>p</i><0.01 as compared to the control (M199). </p