Tropoelastin, the precursor of elastin, undergoes a rapid monomer to multimer association in an inverse temperature transition. This association culminates in the rapid formation of stable, optically distinct droplets of tropoelastin. Light scattering and microscope measurements reveal that these droplets are 2-6 microm in diameter. Scanning electron microscopy confirms that the droplets are spherical. Three-dimensional confocal image stacks based on the autofluorescence of tropoelastin reveal that droplets are loaded with hydrated tropoelastin. Droplets are viable intermediates in synthetic elastin macroassembly. Dense clusters of aggregated droplets and partially formed fibers develop when droplets are incubated in the presence of a lysyl oxidase. Lysine-reacting chemical and enzyme-assisted cross-linking conditions generate cross-linked beads due to interactions between multiple, surface-exposed lysine epsilon-amino groups. Droplets represent an efficient mechanism for the bolus delivery during elastogenesis of quantized packages of preaccreted tropoelastin.

In forming elastic fibers, microfibrils act as the scaffold sites for depositing the elastin precursor tropoelastin. We examined key binding interactions that promote massive tropoelastin association through coacervation. Using a segment of the microfibril protein fibrillin-1, PF2, known to bind full-length tropoelastin, we mapped its interaction site to the N-terminal region of tropoelastin bounded by domains 2 and 18. Precise contact residues between domain 4 of tropoelastin and domain 16 of fibrillin-1 were discovered through a novel combination of transglutaminase cross-linking and mass spectroscopy, with contact sites at residues K38 of tropoelastin and Q669 of fibrillin-1. This is the first report of a role for this region of tropoelastin in microfibril interactions. The addition of PF2 thermodynamically facilitated the coacervation of tropoelastin, resulting in smaller changes in entropy and enthalpy values for the coacervating system. A novel multicomponent in vitro tropoelastin assembly reaction system demonstrated that amassed tropoelastin was spatially and preferentially directed to surfaces coated with PF2 as expected for organized three-dimensional distribution during tissue elastogenesis. This study underscores the role of this part of fibrillin-1 as an anchor point for tropoelastin at the microfibril-elastin junction during the initial stages of elastic fiber assembly.

TL1A is an attractive therapeutic target for the treatment of mucosal inflammation associated with inflammatory bowel disease (IBD) and asthma. Blockade of the TL1A pathway has been shown to reduce inflammatory responses while leaving baseline immunity intact, and to be beneficial in animal models of colitis and asthma. Given the therapeutic potential of blocking this pathway in IBD and asthma, we developed C03V, a human antibody that binds with high affinity to soluble and membrane-bound TL1A. In an assay measuring apoptosis induced by exogenous TL1A, C03V was 43-fold more potent than the next most potent anti-TL1A antibody analyzed. C03V also potently inhibited endogenous TL1A activity in a primary cell-based assay. This potency was linked to the C03V-binding epitope on TL1A, encompassing the residue R32. This residue is critical for the binding of TL1A to its signaling receptor DR3 but not to its decoy receptor DcR3, and explains why C03V inhibited TL1A-DR3 binding to a much greater extent than TL1A-DcR3 binding. This characteristic may be advantageous to preserve some of the homeostatic functions of DcR3, such as TL1A antagonism. In colitis models, C03V significantly ameliorated microscopic, macroscopic and clinical aspects of disease pathology, and in an asthma model it significantly reduced airways inflammation. Notable in both types of disease model was the reduction in fibrosis observed after C03V treatment. C03V has the potential to address unmet medical needs in asthma and IBD.