From NSAIDs to Glucocorticoids and Beyond.

Our interest in inflammation and its treatment stems from ancient times. Hippocrates used willow bark to treat inflammation, and many centuries later, salicylic acid and its derivative aspirin's ability to inhibit cyclooxygenase enzymes was discovered. Glucocorticoids (GC) ushered in a new era of treatment for both chronic and acute inflammatory disease, but their potentially dangerous side effects led the pharmaceutical industry to seek other, safer, synthetic GC drugs. The discovery of the GC-inducible endogenous anti-inflammatory protein annexin A1 (AnxA1) and other endogenous proresolving mediators has opened a new era of anti-inflammatory therapy. This review aims to recapitulate the last four decades of research on NSAIDs, GCs, and AnxA1 and their anti-inflammatory effects.

Magnetically responsive layer-by-layer microcapsules can be retained in cells and under flow conditions to promote local drug release without triggering ROS production.

Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm-2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery.

Endogenous Annexin-A1 Negatively Regulates Mast Cell-Mediated Allergic Reactions.

Mast cell stabilizers like cromoglycate and nedocromil are mainstream treatments for ocular allergy. Biochemical studies in vitro suggest that these drugs prevent mast cell degranulation through the release of Annexin-A1 (Anx-A1) protein. However, the direct effect of Anx-A1 gene deletion on mast cell function in vitro and in vivo is yet to be fully investigated. Hence, we aim to elucidate the role of Anx-A1 in mast cell function, both in vivo and in vitro, using a transgenic mouse model where the Anx-A1 gene has been deleted. Bone marrow-derived mast cells (BMDMCs) were cultured from wild-type animals and compared throughout their development to BMDMCs obtained from mice lacking the Anx-A1 gene. The mast cell differentiation, maturity, mediator, and cytokine release were explored using multiple biochemical techniques, such as Western blots, ELISA, and flow cytometry analysis. Electron microscopy was used to identify metachromatic granules content of cells. For in vivo studies, Balb/C wild-type and Anx-A1-deficient mice were divided into the following groups: group 1, a control receiving only saline, and group 2, which had been sensitized by prior exposure to short ragweed (SRW) pollen by topical contact with the conjunctival mucosae. Allergic conjunctivitis was evaluated blind after 24 h by trained observers scoring clinical signs. Electron micrographs of BMDMCs from Anx-A1-null mice revealed more vacuoles overall and more fused vacuoles than wild-type cells, suggesting enhanced secretory activity. Congruent with these observations, BMDMCs lacking the Anx-A1 gene released significantly increased amounts of histamine both spontaneously as well as in response to Ig-E-FcεRI cross-linking compared to those from wild-type mice. Interestingly, the spontaneous release of IL-5, IL-6, IL-9, and monocyte chemoattractant protein-1 (MCP-1) were also markedly increased with a greater production observed upon IgE cross-linking. This latter finding is congruent with augmented calcium mobilization in BMDMCs lacking the Anx-A1 gene. In vivo, when compared to wild-type animals, Anx-A1-deficient mice exposed to SRW pollen displayed exacerbated signs and symptoms of allergic conjunctivitis. Taken together, these results suggest Anx-A1 is an important non-redundant regulator of mast cell reactivity and particularly in allergen mediated allergic reactions.

Of platelets and aggregometers: personal reminiscences of Gus Born (1921-2018).

This paper recounts the author's personal reminiscences of the late Gustav Born and details some of his major influences on the field of platelet biology and mechanisms of hemostasis. In particular, it focuses on his development of the 'Born aggregometer' and the differences that are seen in the aggregation response to certain stimuli when aggregation is recorded using other techniques such as the impedance method.

Benzolamide improves oxygenation and reduces acute mountain sickness during a high-altitude trek and has fewer side effects than acetazolamide at sea level.

Acetazolamide is the standard carbonic anhydrase (CA) inhibitor used for acute mountain sickness (AMS), however some of its undesirable effects are related to intracellular penetrance into many tissues, including across the blood-brain barrier. Benzolamide is a much more hydrophilic inhibitor, which nonetheless retains a strong renal action to engender a metabolic acidosis and ventilatory stimulus that improves oxygenation at high altitude and reduces AMS. We tested the effectiveness of benzolamide versus placebo in a first field study of the drug as prophylaxis for AMS during an ascent to the Everest Base Camp (5340 m). In two other studies performed at sea level to test side effect differences between acetazolamide and benzolamide, we assessed physiological actions and psychomotor side effects of two doses of acetazolamide (250 and 1000 mg) in one group of healthy subjects and in another group compared acetazolamide (500 mg), benzolamide (200 mg) and lorazepam (2 mg) as an active comparator for central nervous system (CNS) effects. At high altitude, benzolamide-treated subjects maintained better arterial oxygenation at all altitudes (3-6% higher at all altitudes above 4200 m) than placebo-treated subjects and reduced AMS severity by roughly 50%. We found benzolamide had fewer side effects, some of which are symptoms of AMS, than any of the acetazolamide doses in Studies 1 and 2, but equal physiological effects on renal function. The psychomotor side effects of acetazolamide were dose dependent. We conclude that benzolamide is very effective for AMS prophylaxis. With its lesser CNS effects, benzolamide may be superior to acetazolamide, in part, because some of the side effects of acetazolamide may contribute to and be mistaken for AMS.

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.

The warrior in the machine: neuroscience goes to war.

Ever since Stone Age men discovered that knapping flint produced sharp stone edges that could be used in combat as well as for cooking and hunting, technological advances of all kinds have been adapted and adopted by the military.The opportunities provided by modern neuroscience are proving no exception, but their application in a military context is accompanied by complex practical and ethical considerations.

Anti-allergic cromones inhibit histamine and eicosanoid release from activated human and murine mast cells by releasing Annexin A1.

BACKGROUND AND PURPOSE: Although the 'cromones' (di-sodium cromoglycate and sodium nedocromil) are used to treat allergy and asthma, their 'mast cell stabilising' mechanism of pharmacological action has never been convincingly explained. Here, we investigate the hypothesis that these drugs act by stimulating the release of the anti-inflammatory protein Annexin-A1 (Anx-A1) from mast cells. EXPERIMENTAL APPROACH: We used biochemical and immuno-neutralisation techniques to investigate the mechanism by which cromones suppress histamine and eicosanoid release from cord-derived human mast cells (CDMCs) or murine bone marrow-derived mast cells (BMDMCs) from wild type and Anx-A1 null mice. KEY RESULTS: CDMCs activated by IgE-FcRε1 crosslinking, released histamine and prostaglandin (PG) D2, which were inhibited (30-65%) by 5 min pre-treatment with cromoglycate (10 nM) or nedocromil (10 nM), as well as dexamethasone (2 nM) and human recombinant Anx-A1 (1-10 nM). In CDMCs cromones potentiated (2-5 fold) protein kinase C (PKC) phosphorylation and Anx-A1 phosphorylation and secretion (3-5 fold). Incubation of CDMCs with a neutralising anti-Anx-A1 monoclonal antibody reversed the cromone inhibitory effect. Nedocromil (10 nM) also inhibited (40-60%) the release of mediators from murine bone marrow derived-mast cells from wild type mice activated by compound 48/80 and IgE-FcRε1 cross-linking, but were inactive in such cells when these were prepared from Anx-A1 null mice or when the neutralising anti-Anx-A1 antibody was present. CONCLUSIONS AND IMPLICATIONS: We conclude that stimulation of phosphorylation and secretion of Anx-A1 is an important component of inhibitory cromone actions on mast cells, which could explain their acute pharmacological actions in allergy. These findings also highlight a new pathway for reducing mediator release from these cells.

Anti-inflammatory drugs, eicosanoids and the annexin A1/FPR2 anti-inflammatory system.

The action of anti-inflammatory and anti-allergic drugs on the eicosanoid system is briefly reviewed. In addition to the aspirin-like drugs, which directly inhibit the cyclo-oxygenase enzymes, other drugs such as the glucocorticoids and the cromones also inhibit the formation of eicosanoids. In the latter cases this is bought about through the release of a protein factor that acts through formyl peptide receptors on the target cell surface. Of growing interest, is the observation that this receptor is also a target for other eicosanoids, such as lipoxins and resolvins that modulate host defence systems.

Modulation of experimental autoimmune encephalomyelitis by endogenous annexin A1.

BACKGROUND: Autoimmune diseases, like multiple sclerosis, are triggered by uncontrolled activation of cells of the immune system against self-antigen present, for instance, in the central nervous system. We have reported novel biological functions for Annexin A1, an effector of endogenous anti-inflammation, to produce positive actions on the adaptive immune system by reducing the threshold of T cell activation. In this study, we investigated the potential modulatory role of Annexin A1 in the development of experimental autoimmune encephalomyelitis, a model of multiple sclerosis. METHODS: Male control C57/BL6 and AnxA1 null mice were immunized subcutaneously with an emulsion consisting of 300 microg of MOG35-55 in PBS combined with an equal volume of CFA. Lymph node cells obtained from mice immunized with MOG33-55 for 14 days were re-stimulated in vitro with MOG33-55 (100 microg/ml) for 4 days and the Th1/Th17 cytokine profile measured by ELISA. Spinal cords were processed either to isolate the infiltrated T cells or fixed and stained with haematoxylin and eosin. Statistical analyses were performed using two-tailed, unpaired Student's t tests or ANOVA. RESULTS: Our results show a direct correlation between Annexin A1 expression and severity of EAE. Analysis of MOG35-55-induced EAE development in Annexin A1 null mice showed decreased signs of the disease compared to wild type mice. This defect was significant at the peak of the disease and accompanied by reduced infiltration of T cells in the spinal cord. Finally, analysis of the T cell recall response in vitro following stimulation with MOG35-55 showed a decrease proliferation of Annexin A1 null T cells, with a significantly reduced Th1/Th17 phenotype, compared to wild type cells. CONCLUSION: Together these findings suggest that Annexin A1 null mice have an impaired capacity to develop EAE. Furthermore strategies aiming at reducing Annexin A1 functions or expression in T cells might represent a novel therapeutic approach for multiple sclerosis.

Lifestyle drugs: pharmacology and the social agenda.

The precise definition of 'lifestyle' drugs is a subject of debate. However, they are generally defined as drugs taken to satisfy a non-medical or non-health-related goal. Although the term has been used only recently, the media debate that surrounds these agents, fuelled by the release of medicines such as Viagra (sildenafil), is intense. The increasing availability of drugs that can be used to alter our appearance, our physical and mental capabilities or even our characters is changing the social fabric of our culture and poses a difficult challenge to our healthcare systems. It is also revolutionizing the traditional doctor-patient relationship and raises important issues about the rights to, and limits of, self-diagnosis and self-medication.

Annexin 1: a paracrine/juxtacrine mediator of glucorticoid action in the neuroendocrine system.

Glucocorticoids (GCs) play an essential role in the maintenance of homeostasis. In normal circumstances their secretion is tightly regulated by a complex servo mechanism through which the steroids suppress the synthesis and release of ACTH and its hypothalamic releasing factors (CRH and AVP) and thereby reduce the positive drive to the adrenal cortex. The feedback actions of GCs on hormone release develop rapidly (within minutes), well before any changes in hormone synthesis are apparent. By using immunoneutralization, gene targeting and pharmacological strategies in in vivo and in vitro models, we have identified annexin 1, a Ca(2+)- and phospholipid-binding protein, as a key mediator of the early inhibitory actions of GCs on peptide release. This brief review outlines this work and describes molecular and cellular studies which have provided insight into the mechanism of annexin 1-dependent GC signalling in the neuroendocrine system.

The development of COX2 inhibitors.

Aspirin, arguably the world's favourite drug, has been around since the late nineteenth century, but it wasn't until the late 1970s that its ability to inhibit prostaglandin production by the cyclooxygenase enzyme was identified as the basis of its therapeutic action. Early hints of a second form of the cyclooxygenase that was differentially sensitive to other aspirin-like drugs ultimately ushered in an exciting era of drug discovery, culminating in the introduction of an entirely new generation of anti-inflammatories. This article reviews the story of this discovery and looks at the future of cyclooxygenase pharmacology.

Attenuation of glucocorticoid functions in an Anx-A1-/- cell line.

The Ca(2+)- and phospholipid-binding protein Anx-A1 (annexin 1; lipocortin 1) has been described both as an inhibitor of phospholipase A(2) (PLA(2)) activity and as a mediator of glucocorticoid-regulated cell growth and eicosanoid generation. Here we show that, when compared with Anx-A1(+/+) cells, lung fibroblast cell lines derived from the Anx-A1(-/-) mouse exhibit an altered morphology characterized by a spindle-shaped appearance and an accumulation of intracellular organelles. Unlike their wild-type counterparts, Anx-A1(-/-) cells also overexpress cyclo-oxygenase 2 (COX 2), cytosolic PLA(2) and secretory PLA(2) and in response to fetal calf serum, exhibit an exaggerated release of eicosanoids, which is insensitive to dexamethasone (10(-8)- 10(-6) M) inhibition. Proliferation and serum-induced progression of Anx-A1(+/+) cells from G(0)/G(1) into S phase, and the associated expression of extracellular signal-regulated kinase 2 (ERK2), cyclin-dependent kinase 4 (cdk4) and COX 2, is strongly inhibited by dexamethasone, whereas Anx-A1(-/-) cells are refractory to the drug. Loss of the response to dexamethasone in Anx-A1(-/-) cells occurs against a background of no apparent change in glucocorticoid receptor expression or sensitivity to non-steroidal anti-inflammatory drugs. Taken together, these observations suggest strongly that Anx-A1 functions as an inhibitor of signal-transduction pathways that lead to cell proliferation and may help to explain how glucocorticoids regulate these processes.