Faculty Bibliography

Laminin, as a key component of the basement membrane extracellular matrix (ECM), regulates tissue morphogenesis. Here, we show that multiple laminin isoforms promiscuously bind to growth factors (GFs) with high affinity, through their heparin-binding domains (HBDs) located in the α chain laminin-type G (LG) domains. These domains also bind to syndecan cell-surface receptors, promoting attachment of fibroblasts and endothelial cells. We explore the application of these multifunctional laminin HBDs in wound healing in the type-2 diabetic mouse. We demonstrate that covalent incorporation of laminin HBDs into fibrin matrices improves retention of GFs and significantly enhances the efficacy of vascular endothelial cell growth factor (VEGF-A165) and platelet-derived growth factor (PDGF-BB) in promoting wound healing in vivo, under conditions where the GFs alone in fibrin are inefficacious. This laminin HBD peptide may be clinically useful by improving biomaterial matrices as both GF reservoirs and cell scaffolds, leading to effective tissue regeneration.

A machine learning assisted method is presented for molecular simulation of systems with rugged free energy landscapes. The method is general and can be combined with other advanced sampling techniques. In the particular implementation proposed here, it is illustrated in the context of an adaptive biasing force approach where, rather than relying on discrete force estimates, one can resort to a self-regularizing artificial neural network to generate continuous, estimated generalized forces. By doing so, the proposed approach addresses several shortcomings common to adaptive biasing force and other algorithms. Specifically, the neural network enables (1) smooth estimates of generalized forces in sparsely sampled regions, (2) force estimates in previously unexplored regions, and (3) continuous force estimates with which to bias the simulation, as opposed to biases generated at specific points of a discrete grid. The usefulness of the method is illustrated with three different examples, chosen to highlight the wide range of applicability of the underlying concepts. In all three cases, the new method is found to enhance considerably the underlying traditional adaptive biasing force approach. The method is also found to provide improvements over previous implementations of neural network assisted algorithms.

Islet encapsulation may allow transplantation without immunosuppression, but thus far islets in large microcapsules transplanted in the peritoneal cavity have failed to reverse diabetes in humans. We showed that islet transplantation in confined well-vascularized sites like the epididymal fat pad (EFP) improved graft outcomes, but only conformal coated (CC) islets can be implanted in these sites in curative doses. Here, we showed that CC using polyethylene glycol (PEG) and alginate (ALG) was not immunoisolating because of its high permselectivity and strong allogeneic T cell responses. We refined the CC composition and explored PEG and islet-like extracellular matrix (Matrigel; MG) islet encapsulation (PEG MG) to improve capsule immunoisolation by decreasing its permselectivity and immunogenicity while allowing physiological islet function. Although the efficiency of diabetes reversal of allogeneic but not syngeneic CC islets was lower than that of naked islets, we showed that CC (PEG MG) islets from fully MHC-mismatched Balb/c mice supported long-term (>100 days) survival after transplantation into diabetic C57BL/6 recipients in the EFP site (750-1000 islet equivalents/mouse) in the absence of immunosuppression. Lack of immune cell penetration and T cell allogeneic priming was observed. These studies support the use of CC (PEG MG) for islet encapsulation and transplantation in clinically relevant sites without chronic immunosuppression.

UNLABELLED:-MMP-IGF-1 loaded into the micro-beads was retained within the fibrin micro-beads. In vitro, the growth factor modified fibrin micro-beads enhanced cell attachment and the migration of human urinary tract smooth muscle cells, however, no change of the cellular metabolic activity was seen. These bioactive micro-beads were mixed with genipin-crosslinked homogenized collagen, acting as a carrier. The collagen concentration, the degree of crosslinking, and the mechanical behavior of this bioactive collagen-fibrin injectable were comparable to reference samples. This novel injectable showed no burst release of the growth factor, had a positive effect on cell behavior and may therefore induce smooth muscle regeneration in vivo, necessary for the functional treatment of stress and other urinary incontinences. STATEMENT OF SIGNIFICANCE:-MMP-IGF-1). These bioactive fibrin micro-beads induced human smooth muscle cell migration in vitro. Thus, this injectable bulking agent is apt to be a good candidate for regeneration of urethral sphincter muscle, ensuring a long-lasting treatment for urinary incontinence.

Lassa virus (LASV) causes severe hemorrhagic fever with high mortality, yet no vaccine currently exists. Antibodies targeting viral attachment proteins are crucial for protection against many viral infections. However, the envelope glycoprotein (GP)-1 of LASV elicits weak antibody responses due to extensive glycan shielding. Here, we explored a novel vaccine strategy to enhance humoral immunity against LASV GP1. Using structural information, we designed a recombinant GP1 immunogen, and then encapsulated it into oxidation-sensitive polymersomes (PS) as nanocarriers that promote intracellular MHCII loading. Mice immunized with adjuvanted PS (LASV GP1) showed superior humoral responses than free LASV GP1, including antibodies with higher binding affinity to virion GP1, increased levels of polyfunctional anti-viral CD4 T cells, and IgG-secreting B cells. PS (LASV GP1) elicited a more diverse epitope repertoire of anti-viral IgG. Together, these data demonstrate the potential of our nanocarrier vaccine platform for generating virus-specific antibodies against weakly immunogenic viral antigens.

Immune checkpoint blockade exhibits considerable antitumor activity, but previous studies have reported instances of severe treatment-related adverse events. We sought to explore local immune checkpoint blockade, with an antibody (Ab) form that would be retained intra- or peritumorally, limiting systemic exposure. To accomplish this, we conjugated the checkpoint blockade Abs to an extracellular matrix (ECM)-super-affinity peptide derived from placenta growth factor-2 (PlGF-2_123-144). We show enhanced tissue retention and lower Ab concentrations in blood plasma after PlGF-2_123-144 conjugation, reducing systemic side effects such as the risk of autoimmune diabetes. Peritumoral injections of PlGF-2_123-144-anti-CTLA4 (cytotoxic T lymphocyte antigen 4) and PlGF-2_123-144-anti-PD-L1 (programmed death ligand 1) Abs delayed tumor growth and prolonged survival compared to the unmodified Abs in genetically engineered murine tumor models of melanoma and breast cancer. The PlGF-2_123-144-Abs increased tumor-infiltrating activated CD8⁺ and CD4⁺ T cells, resulting in a delay of distant tumor growth as well. This simple and translatable approach of engineered ECM-binding Abs may present a viable and safer approach in checkpoint blockade.

We evaluated whether the genetic background of embryonic stem cells (ESCs) affects the properties suitable for three-dimensional (3D) synthetic scaffolds for cell self-renewal. Inbred R1 and hybrid B6D2F1 mouse ESC lines were cultured for 7 days in hydrogel scaffolds with different properties derived from conjugating 7.5, 10, 12.5, or 15% (wt/vol) vinylsulfone-functionalized three-, four-, or eight-arm polyethylene glycol (PEG) with dicysteine-containing crosslinkers with an intervening matrix metalloproteinase-specific cleavage sites. Cell proliferation and expression of self-renewal-related genes and proteins by ESCs cultured in feeder-free or containing 2D culture plate or 3D hydrogel were monitored. As a preliminary experiment, the E14 ESC-customized synthetic 3D microenvironment did not maintain self-renewal of either the R1 or B6D2F1 ESCs. The best R1 cell proliferation (10.04 vs. 0.16-4.39; p < 0.0001) was observed in the four-arm 7.5% PEG-based hydrogels than those with other properties, whereas the F1 ESCs showed better proliferation when they were embedded in the three-arm 10% hydrogels. Self-renewal-related gene and protein expression by ESCs after feeder-free 3D culture was generally maintained compared with the feeder-containing 2D culture, but expression patterns and quantities differed. However, the feeder-free 3D culture yielded better expression than the feeder-free 2D culture. In conclusion, genetic background determined the suitability of hydrogel scaffolds for self-renewal of ESCs, which requires customization for the mechanical properties of each cell line. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2261-2268, 2017.

BACKGROUND:Newborns display distinct immune responses, leaving them vulnerable to infections and impairing immunization. Targeting newborn dendritic cells (DCs), which integrate vaccine signals into adaptive immune responses, might enable development of age-specific vaccine formulations to overcome suboptimal immunization. OBJECTIVE:Small-molecule imidazoquinoline Toll-like receptor (TLR) 8 agonists robustly activate newborn DCs but can result in reactogenicity when delivered in soluble form. We used rational engineering and age- and species-specific modeling to construct and characterize polymer nanocarriers encapsulating a TLR8 agonist, allowing direct intracellular release after selective uptake by DCs. METHODS:Chemically similar but morphologically distinct nanocarriers comprised of amphiphilic block copolymers were engineered for targeted uptake by murine DCs in vivo, and a range of TLR8 agonist-encapsulating polymersome formulations were then synthesized. Novel 96-well in vitro assays using neonatal human monocyte-derived DCs and humanized TLR8 mouse bone marrow-derived DCs enabled benchmarking of the TLR8 agonist-encapsulating polymersome formulations against conventional adjuvants and licensed vaccines, including live attenuated BCG vaccine. Immunogenicity of the TLR8 agonist adjuvanted antigen 85B (Ag85B)/peptide 25-loaded BCG-mimicking nanoparticle formulation was evaluated in vivo by using humanized TLR8 neonatal mice. RESULTS:T-cell numbers. CONCLUSION/CONCLUSIONS:TLR8 agonist-encapsulating polymersomes hold substantial potential for early-life immunization against intracellular pathogens. Overall, our study represents a novel approach for rational design of early-life vaccines.

Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants.

Nanoparticle delivery systems are known to enhance the immune response to soluble antigens (Ags) and are thus a promising tool for the development of new vaccines. Our laboratory has engineered two different nanoparticulate systems in which Ag is either encapsulated within the core of polymersomes (PSs) or decorated onto the surface of nanoparticles (NPs). Previous studies showed that PSs are better at enhancing CD4 T cells and antibody titers, while NPs preferentially augment cytotoxic CD8 T cells. Herein, we demonstrate that the differential activation of T cell immunity reflects differences in the modes of intracellular trafficking and distinct biodistribution of the Ag in lymphoid organs, which are both driven by the properties of each nanocarrier. Furthermore, we found that Ags within PSs promoted better CD4 T cell activation and induced a higher frequency of CD4 T follicular helper (Tfh) cells. These differences correlated with changes in the frequency of germinal center B cells and plasma cell formation, which reflects the previously observed antibody titers. Our results show that PSs are a promising vector for the delivery of Ags for B cell vaccine development. This study demonstrates that nanocarrier design has a large impact on the quality of the induced adaptive immune response.