Paul Bradfield

We recently developed a molecule (GT-73) that blocked leukocyte transendothelial migration from blood to the peripheral tissues, supposedly by affecting the platelet endothelial cell adhesion molecule (PECAM-1) function. GT-73 was tested in an LPS-induced acute respiratory distress syndrome (ARDS) mouse model. The rationale for this is based on the finding that the mortality of COVID-19 patients is partly caused by ARDS induced by a massive migration of leukocytes to the lungs. In addition, the role of tert-butyl and methyl ester moieties in the biological effect of GT-73 was investigated. A human leukocyte, transendothelial migration assay was applied to validate the blocking effect of GT-73 derivatives. Finally, a mouse model of LPS-induced ARDS was used to evaluate the histological and biochemical effects of GT-73. The obtained results showed that GT-73 has a unique structure that is responsible for its biological activity; two of its chemical moieties (tert-butyl and a methyl es...

Recruitment of circulating monocytes is critical for tumour angiogenesis. However, how human monocyte subpopulations extravasate to tumours is unclear. Here we show mechanisms of extravasation of human CD14CD16patrolling and CD14CD16intermediate proangiogenic monocytes (HPMo), using human tumour xenograft models and live imaging of transmigration. IFNγ promotes an increase of the chemokine CX3CL1 on vessel lumen, imposing continuous crawling to HPMo and making these monocytes insensitive to chemokines required for their extravasation. Expression of the angiogenic factor VEGF and the inflammatory cytokine TNF by tumour cells enables HPMo extravasation by inducing GATA3-mediated repression of CX3CL1 expression. Recruited HPMo boosts angiogenesis by secreting MMP9 leading to release of matrix-bound VEGF-A, which amplifies the entry of more HPMo into tumours. Uncovering the extravasation cascade of HPMo sets the stage for future tumour therapies.

Atherosclerosis, caused in part by monocytes in plaques, continues to be a disease that afflicts the modern world. Whilst significant steps have been made in treating this chronic inflammatory disease, questions remain on how to prevent monocyte and macrophage accumulation in atherosclerotic plaques. Junctional Adhesion Molecule C (JAM-C) expressed by vascular endothelium directs monocyte transendothelial migration in a unidirectional manner leading to increased inflammation. Here we show that interfering with JAM-C allows reverse-transendothelial migration of monocyte-derived cells, opening the way back out of the inflamed environment. To study the role of JAM-C in plaque regression we used a mouse model of atherosclerosis, and tested the impact of vascular JAM-C expression levels on monocyte reverse transendothelial migration using human cells. Studies in-vitro under inflammatory conditions revealed that overexpression or gene silencing of JAM-C in human endothelium exposed to flo...

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycocon-jugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD 1) and selenomethi-onyl (Se-SnDl) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnDl and Se-SnDl have been crystallized in the absence of ligand, and SnDl has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnDl and Se-SnDl were isomorphous, of space group P3t2\ or P3221, with unit cell dimensions a = b = 38.9 A, c = 152.6 k,a = (3 = 90°, y = 120°, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P21212, with unit cell dimensions a = 40.9 A, b = 97.6 A, c = 101.6 A, a = /3 = y = 90°.

Rapid mobilization of leucocytes through endothelial and epithelial barriers is key in immune system reactivity. The underlying mechanisms that regulate these processes have been the basis for many recent studies. Traditionally, leucocyte extravasation had been believed to occur through a paracellular route, which involves localized disruption of endothelial cell junctions. However, more recently, a transcellular route has been described involving the passage through the endothelial cell body. Leucocytes are also able to migrate through epithelium to monitor mucosal tissues and microenvironments. A number of adhesion molecules are known to regulate transmigration of leucocytes through epithelial and endothelial layers. Paracellular and transcellular leucocyte transmigration are regulated by adhesion molecules such as PECAM-1 (platelet–endothelial cell adhesion molecule 1), CD99, VE-cadherin (vascular endothelial cadherin) and JAM (junctional adhesion molecule) proteins. The purpose ...

Exploring the role of junctional adhesion molecules (JAMs) has proven to be varied and controversial. The purpose of this review is to discuss the new and exciting roles of these IgSF molecules and how they have evolved to contribute to diverse functions from development to inflammation. In particular, recent research has focused on JAM subfamily members JAM-A, -B, and -C with newly described roles in leukocyte trafficking during inflammation and angiogenesis. However, research on all JAM family members has demonstrated recurring themes with striking similarities in the many diverse processes they are now known to regulate.

Leukocyte transendothelial migration is one of the most important step in launching an inflammatory immune response and chronic inflammation can lead to devastating diseases. Leukocyte migration inhibitors are considered as promising and potentially effective therapeutic agents to treat inflammatory and auto-immune disorders. In this study, based on previous trioxotetrahydropyrimidin based integrin inhibitors that suboptimally blocked leukocyte adhesion, twelve molecules with a modified scaffold were designed, synthesized, and tested in vitro for their capacity to block the transendothelial migration of immune cells. One of the molecules, namely, methyl 4-((2-(tert-butyl)-6-((2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene) methyl) phenoxy) methyl) benzoate, (compound 12), completely blocked leukocyte transendothelial migration, without any toxic effects on immune or endothelial cells (IC50 = 2.4 µM). In vivo, compound 12 exhibited significant therapeutic effects in inflammatory bowel disease (IBD)/Crohn's disease, multiple sclerosis, fatty liver disease, and rheumatoid arthritis models. A detailed acute and chronic toxicity profile of the lead compound in vivo did not reveal any toxic effects. Such a type of molecule might therefore provide a unique starting point for designing a novel class of leukocyte transmigration blocking agents with broad therapeutic applications in inflammatory and auto-immune pathologies.

Sialoadhesin is a macrophage-restricted cell surface receptor, consisting of 17 immunoglobulin domains, which mediates cell adhesion via the recognition of specific sialylated glycocon-jugates. A functional fragment of sialoadhesin, comprising the N-terminal immunoglobulin domain, has been expressed in Chinese hamster ovary cells as both native (SnD 1) and selenomethi-onyl (Se-SnDl) stop protein. The successful production of 86% selenomethionine-incorporated protein represents a rare example of production of selenium-labeled protein in mammalian cells. SnDl and Se-SnDl have been crystallized in the absence of ligand, and SnDl has also been crystallized in the presence of its ligand 2,3 sialyllactose. The ligand-free crystals of SnDl and Se-SnDl were isomorphous, of space group P3t2\ or P3221, with unit cell dimensions a = b = 38.9 A, c = 152.6 k,a = (3 = 90°, y = 120°, and diffracted to a maximum resolution of 2.6 A. Cocrystals containing 2,3 sialyllactose diffracted to 1.85 A at a synchrotron source and belong to space group P21212, with unit cell dimensions a = 40.9 A, b = 97.6 A, c = 101.6 A, a = /3 = y = 90°.