Fc-γ receptor-mediated crosslinking co-defines the immunostimulatory activity of anti-human CD96 antibodies.

New strategies that augment T-cell responses are required to broaden the therapeutic arsenal against cancer. CD96, TIGIT and CD226 are receptors that bind to a communal ligand, CD155, and transduce either inhibitory or activating signals. Whereas the function of TIGIT and CD226 is established, the role of CD96 remains ambiguous. Using a panel of engineered antibodies, we discovered that the T-cell stimulatory activity of anti-CD96 antibodies requires antibody crosslinking and is potentiated by Fc-gamma receptors. Thus, soluble 'Fc silent' anti-CD96 antibodies failed to stimulate human T cells, whereas the same antibodies were stimulatory after coating onto plastic surfaces. Remarkably, the activity of soluble anti-CD96 antibodies was reinstated by engineering the Fc domain to a human IgG1 isotype and was dependent on antibody trans-crosslinking by Fc-γRI. In contrast, neither human IgG2 nor variants with increased Fc-γ receptor IIB binding possessed stimulatory activity. Anti-CD96 antibodies acted directly on T cells and augmented gene expression networks associated with T-cell activation, leading to proliferation, cytokine secretion and resistance to regulatory T-cell suppression. Furthermore, CD96 expression correlated with survival in HPV+ head and neck squamous cell carcinoma and its crosslinking activated tumor-infiltrating T cells, thus highlighting the potential of anti-CD96 antibodies in cancer immunotherapy. 

General anaesthetic and airway management practice for obstetric surgery in England: a prospective, multi-centre observational study

There are no current descriptions of general anaesthesia characteristics for obstetric surgery, despite recent changes to patient baseline characteristics and airway management guidelines. This analysis of data from the direct reporting of awareness in maternity patients' (DREAMY) study of accidental awareness during obstetric anaesthesia aimed to describe practice for obstetric general anaesthesia in England and compare with earlier surveys and best-practice recommendations. Consenting patients who received general anaesthesia for obstetric surgery in 72 hospitals from May 2017 to August 2018 were included. Baseline characteristics, airway management, anaesthetic techniques and major complications were collected. Descriptive analysis, binary logistic regression modelling and comparisons with earlier data were conducted. Data were collected from 3117 procedures, including 2554 (81.9%) caesarean deliveries. Thiopental was the induction drug in 1649 (52.9%) patients, compared with propofol in 1419 (45.5%). Suxamethonium was the neuromuscular blocking drug for tracheal intubation in 2631 (86.1%), compared with rocuronium in 367 (11.8%). Difficult tracheal intubation was reported in 1 in 19 (95%CI 1 in 16-22) and failed intubation in 1 in 312 (95%CI 1 in 169-667). Obese patients were over-represented compared with national baselines and associated with difficult, but not failed intubation. There was more evidence of change in practice for induction drugs (increased use of propofol) than neuromuscular blocking drugs (suxamethonium remains the most popular). There was evidence of improvement in practice, with increased monitoring and reversal of neuromuscular blockade (although this remains suboptimal). Despite a high risk of difficult intubation in this population, videolaryngoscopy was rarely used (1.9%)

Human group IIA secreted phospholipase A2 : a protein with many functions

Fluorescently labelled cysteine mutants were used to show increased binding of hgIIA to PMA activated human THP-1 macrophage cells.  Inhibition and competition studies suggest that the hgIIA enzyme is binding via electrostatic interactions to heparan sulphate proteoglycans that show increased cell surface expression on THP-1 activation.  On binding, hgIIA is rapidly internalised via an energy-dependent endocytic mechanism.  Inhibitory studies and confocal microscopy implicate both the clathrin- and caveolae-mediated endocytic pathways as well as macropinocytosis.  Entry via macropinocytosis results in the increase in cell volume, and highlights a possible novel signalling pathway utilised by the hgIIA enzyme.  On internalisation, hgIIA accumulates in the nucleus where we have shown using Western blotting that it can act to increase the LPS induced expression of COX-2 in a synergistic manner.  All results presented were also observed using the catalytically inactive hgIIA mutant H48N,S74C, demonstrating that in addition to hgIIA’s catalytic-dependent role in the antibacterial response, it also possesses other functions independent of activity that allows it to interact with and have subsequent affect on host cell function.  The hgIIA protein may thus play a pro-resolving role in inflammation as increased COX-2 levels leads to increased production of the pro-resolving inflammatory mediator prostaglandin D2.  This response would be of beneficial effect in preventing tumour progression as it would reduce macrophage production of pro-inflammatory cytokines and reactive oxygen and nitrogen species that may potentiate tumour development and metastasis. The accumulated evidence suggest that the hgIIA protein has a variety of physiological roles in addition to its antibacterial properties, including acting as a bridging molecule that facilitates the uptake of anionic cell debris, while nuclear targeting affects the expression of genes involved in the inflammatory response.</p

Age, Obesity and Hyperglycaemia: Activation of innate immunity initiates a series of molecular interactions involving anionic surfaces leading to Covid-19 morbidity and mortality.

Obesity and type 2 diabetes are major factors in Covid-19 causing a progression to excessive morbidity and mortality. An important characteristic of these conditions is poor glycaemic control leading to inappropriate chemical reactions and the production of glycated proteins in which positively charged lysine and arginine residues are neutralised. We propose that this protein glycation primes the inflammatory system as the presence of aspartate and glutamate residues in any glycated zwitterionic proteins will thus increase its anionic characteristics. As a result, these macromolecules will be recognised by the innate immune system and identified as originating from infection or cell damage (sterile inflammation). Many proteins in the body exist to non-specifically target these anionic macromolecules and rely heavily on positively charged (cationic) binding-sites to produce a relatively non-specific interaction as the first step in the body’s response. Proteins involved in this innate immunity are collectively referred to as damage associated molecular pattern molecules or pathogen associated molecular pattern molecules. A crucial player in this process is RAGE (Receptor for Advanced Glycation End products). RAGE plays a central role in the inflammatory response and on ligand binding stimulates many aspects of inflammation including the production of the key inflammatory mediator NF-κB, and the subsequent production of inflammatory cytokines. This process has the potential to show a positive feedback loop resulting in a dramatic response within the tissue. We propose that protein glycation primes the inflammatory system by generating negatively charged surfaces so that when a SARS-Cov-2 infection occurs within the lung the further release of negatively-charged macromolecules due to cell damage results in a potentially catastrophic inflammatory response resulting in the cytokine storm associated with covid-19 morbidity and mortality. That part of the population that do not suffer from inflammatory priming (Phase 1), such as the young and the non-obese, should not be subjected to the catastrophic inflammatory response seen in others (Phase 2). This hypothesis further highlights the need for improved dietary intake to minimise the inflammatory priming resulting from poor glycaemic control

Could anionic LDL be a ligand for RAGE and TREM2 in addition to LOX-1 and thus exacerbate lung disease and dementia?

We recently highlighted the potential of protein glycation to generate anionic (electronegative) surfaces. We hypothesised that these anionic proteins are perceived by the innate immune system as arising from infection or damaged cell components, producing an inflammatory response within the lung involving the receptor RAGE. We now review two other pathologies linked to the innate immune response, cardiovascular disease and dementia that involve receptors LOX-1 and TREM2 respectively. Remarkable similarities in properties between RAGE, LOX-1 and TREM2 suggest that electronegative LDL may act as a pathogenic anionic ligand for all three receptors and exacerbate lung inflammation and dementia.</p

A catalytically independent physiological function for human acute phase protein group IIA phospholipase A2: cellular uptake facilitates cell debris removal

Human group IIA phospholipase A2 (IIA PLA2) is an acute phase protein first identified at high concentrations in synovial fluid from patients with rheumatoid arthritis. Its physiological role has since been debated; the enzyme has a very high affinity for anionic phospholipid interfaces but expresses almost zero activity with zwitterionic phospholipid substrates, because of a lack of interfacial binding. We have prepared the cysteine-containing mutant (S74C) to allow the covalent attachment of fluorescent reporter groups. We show that fluorescently labeled IIA was taken up by phorbol 12-myristate 13-acetate-activated THP-1 cells in an energy-dependent process involving cell surface heparan sulfate proteoglycans. Uptake concurrently involved significant cell swelling, characteristic of macropinocytosis and the fluorescent enzyme localized to the nucleus. The endocytic process did not necessitate enzyme catalysis, ruling out membrane phospholipid hydrolysis as an essential requirement. The enzyme produced supramolecular aggregates with anionic phospholipid vesicles as a result of bridging between particles, a property that is unique to this globally cationic IIA PLA2. Uptake of such aggregates labeled with fluorescent anionic phospholipid was dramatically enhanced by the IIA protein, and uptake involved binding to heparan sulfate proteoglycans on activated THP-1 cells. A physiological role for this protein is proposed that involves the removal of anionic extracellular cell debris, including anionic microparticles generated as a result of trauma, infection, and the inflammatory response, and under such conditions serum levels of IIA PLA2 can increase approximately 1000-fold. A similar pathway may be significant in the uptake into cells of anionic vector DNA involving cationic lipid transfection protocols.<br/

Catalytic and non-catalytic functions of human IIA phospholipase A2

Group IIA phospholipase A2 (PLA2) is a low-molecular-mass secreted PLA2 enzyme that has been identified as an acute phase protein with a role in the inflammatory response to infection and trauma. The protein is possibly unique in being highly cationic and having a global distribution of surface arginine and lysine residues. This structure supports two functions of the protein. (1) An anti-bacterial role where the enzyme is targeted to the anionic cell membrane of Gram-positive bacteria and phospholipid hydrolysis assists in bacterial killing. (2) A proposed non-catalytic role in which the protein forms supramolecular aggregates with anionic phospholipid vesicles or debris. These aggregates are then internalized via interactions with cell surface heparin sulphate proteoglycans and macropinocytosis for disposal by macrophages

The role of three-dimensional in vitro models in modelling the inflammatory microenvironment associated with obesity in breast cancer

Abstract Obesity is an established risk factor for breast cancer in postmenopausal women. However, the underlying biological mechanisms of how obesity contributes to breast cancer remains unclear. The inflammatory adipose microenvironment is central to breast cancer progression and has been shown to favour breast cancer cell growth and to reduce efficacy of anti-cancer treatments. Thus, it is imperative to further our understanding of the inflammatory microenvironment seen in breast cancer patients with obesity. Three-dimensional (3D) in vitro models offer a key tool in increasing our understanding of such complex interactions within the adipose microenvironment. This review discusses some of the approaches utilised to recapitulate the breast tumour microenvironment, including various co-culture and 3D in vitro models. We consider how these model systems contribute to the understanding of breast cancer research, with particular focus on the inflammatory tumour microenvironment. This review aims to provide insight and prospective future directions on the utility of such model systems for breast cancer research

A cyclic peptide inhibitor of C-terminal binding protein dimerization links metabolism with mitotic fidelity in breast cancer cells

Identification of direct modulators of transcription factor protein–protein interactions is a key challenge for ligand discovery that promises to significantly advance current approaches to cancer therapy. Here, we report an inhibitor of NADH-dependent dimerization of the C-terminal binding protein (CtBP) transcriptional repressor, identified by screening genetically encoded cyclic peptide libraries of up to 64 million members. CtBP dimers form the core of transcription complexes associated with epigenetic regulation of multiple genes that control many characteristics of cancer cells, including proliferation, survival and migration. CtBP monomers also have distinct and critical cellular function, thus current experimental tools that deplete all forms of a targeted protein (e.g. siRNA) do not allow the cellular consequences of this metabolically regulated transcription factor to be deciphered. The most potent inhibitor from our screen (cyclo-SGWTVVRMY) is demonstrated to disrupt CtBP dimerization in vitro and in cells. This compound is used as a chemical tool to establish that the NADH-dependent dimerization of CtBPs regulates the maintenance of mitotic fidelity in cancer cells. Treatment of highly glycolytic breast cancer cell lines with the identified inhibitor significantly reduced their mitotic fidelity, proliferation and colony forming potential, whereas the compound does not affect mitotic fidelity of cells with lower glycolytic flux. This work not only links the altered metabolic state of transformed cells to a key determinant of the tumor cell phenotype, but the uncovered compound also serves as the starting point for the development of potential therapeutic agents that target tumors by disrupting the CtBP chromatin-modifying complex