Biology subjects

Roy, K.

Publications and source records attributed to Roy, K..

44 records · Page 3Linked to original sources

bioRxiv2020

Nano-Emulsion Adjuvant Enhances Cross-presentation by Lipid Body Formation Independent of Glycolysis

Here, we report that a carbomer-based adjuvant, Adjuplex(R) (ADJ), stimulated robust CD8 T-cell responses to subunit antigens by modulating multiple steps in the cytosolic pathway of cross-presentation, and afforded effective immunity against virus and intracellular bacteria. Cross-presentation induced by TLR agonists requires a critical switch to anabolic metabolism, but ADJ enhanced cross presentation without this metabolic switch in DCs and NLRP3-driven caspase 1 activity. Instead, ADJ induced in DCs, an unique metabolic state, typified by dampened oxidative phosphorylation and basal levels of glycolysis. In the absence of increased glycolytic flux, induction of ROS and lipid bodies (LBs) and alterations in LB composition mediated by ADJ were critical for DC cross-presentation. These findings challenge the prevailing metabolic paradigm by suggesting that DCs can perform effective DC cross-presentation, independent of glycolysis to induce robust T cell-dependent protective immunity to intracellular pathogens. These findings have implications in the rational development of novel adjuvants.

immunology↗

bioRxiv2020

Development of Systemic Immune Dysregulation in a Rat Trauma Model with Biomaterial-Associated Infection

Orthopedic biomaterial-associated infections remain a large clinical challenge, particularly with open fractures and segmental bone loss. Invasion and colonization of bacteria within immune-privileged canalicular networks of the bone can lead to local, indolent infections that can persist for years without symptoms before eventual catastrophic hardware failure. Host immunity is essential for bacterial clearance and an appropriate healing response, and recent evidence has suggested an association between orthopedic trauma and systemic immune dysregulation and immunosuppression. However, the impact of a local infection on this systemic immune response and subsequent effects on the local response is poorly understood and has not been a major focus for addressing orthopedic injuries and infections. Therefore, this study utilized a model of orthopedic biomaterial-associated infection to investigate the effects of infection on the long-term immune response. Here, despite persistence of a local, indolent infection lacking outward symptoms, there was still evidence of long-term immune dysregulation with systemic increases in MDSCs and decreases in T cells compared to non-infected trauma. Further, the trauma only group exhibited a regulated and coordinated systemic cytokine response, which was not present in the infected trauma group. Locally, the infection group had attenuated macrophage infiltration in the local soft tissue compared to the non-infected group. Our results demonstrate widespread impacts of a localized orthopedic infection on the systemic and local immune responses. Characterization of the immune response to orthopedic biomaterial-associated infection may identify key targets for immunotherapies that could optimize both regenerative and antibiotic interventions, ultimately improving outcomes for these patients.

bioengineering↗

bioRxiv2019

A Multi-Niche Microvascularized Human Bone-Marrow-on-a-Chip

The human bone marrow (hBM) is a complex organ critical for hematopoietic and immune homeostasis, and where many cancers metastasize. Yet, understanding the fundamental biology of the hBM in health and diseases remain difficult due to complexity of studying or manipulating the BM in humans. Accurate in vitro models of the hBM microenvironment are critical to further our understanding of the BM niche and advancing new clinical interventions. Although, in vitro culture models that recapitulate some key components of the BM niche have been reported, there are no examples of a fully human, in vitro, organoid platform that incorporates the various niches of the hBM - specifically the endosteal, central marrow, and perivascular niches - thus limiting their physiological relevance. Here we report an hBM-on-a-chip that incorporates these three niches in a single micro-physiological device. Osteogenic differentiation of hMSCs produced robust mineralization on the PDMS surface ("bone layer") and subsequent seeding of endothelial cells and hMSCs in a hydrogel network ("central marrow") created an interconnected vascular network ("perivascular niche") on top. We show that this multi-niche hBM accurately mimics the ECM composition, allows hematopoietic progenitor cell proliferation and migration, and is affected by radiation. A key finding is that the endosteal niche significantly contributes to hBM physiology. Taken together, this multi-niche micro-physiological system opens up new opportunities in hBM research and therapeutics development, and can be used to better understand hBM physiology, normal and impaired hematopoiesis, and hBM pathologies, including cancer metastasis, multiple myelomas, and BM failures.

bioengineering↗

bioRxiv2019

In-vitro and in-vivo Characterization of a Multi-Stage Enzyme-Responsive Nanoparticle-in-Microgel Pulmonary Drug Delivery System

Although the lung is an obvious target for site-specific delivery of many therapeutics for respiratory airway diseases such as asthma, COPD, and cystic fibrosis, novel strategies are needed to avoid key physiologic barriers for efficient delivery and controlled release of therapeutics to the lungs. Specifically, deposition into the deep lung requires particles with a 1-5 {micro}m aerodynamic diameter; however, particles with a geometric diameter less than 6 {micro}m are rapidly cleared by alveolar macrophages. Additionally, epithelial, endothelial, and fibroblast cells prefer smaller (< 300 nm) nanoparticles for efficient endocytosis. Here we address these contradictory design requirements by using a nanoparticle-inside-microgel system (Nano-in-Microgel). Using an improved maleimide-thiol based Michael Addition during (water-in-oil) Emulsion (MADE) method, we fabricated both trypsin-responsive and neutrophil elastase-responsive polymeric Nano-in-Microgel to show the versatility of the system in easily exchanging enzyme-responsive crosslinkers for disease-specific proteases. By varying the initial macromer concentration, from 20-50 % w/v, the size distribution means ranged from 4-8 {micro}m, enzymatic degradation of the microgels is within 30 minutes, and in vitro macrophage phagocytosis is lower for the higher % w/v. We further demonstrated that in vivo lung delivery of the multi-stage carriers through the pulmonary route yields particle retention up to several hours and followed by clearance within in naive mice. Our results provide a further understanding of how enzymatically-degradable multi-stage polymeric carriers can be used for pulmonary drug delivery.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC=\"FIGDIR/small/650911v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (15K):\norg.highwire.dtl.DTLVardef@1dcd1dcorg.highwire.dtl.DTLVardef@55e23eorg.highwire.dtl.DTLVardef@788c02org.highwire.dtl.DTLVardef@30161f_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Modification of primary amines to higher order amines reduces in vivo hematological and immunotoxicity of cationic nanocarriers through TLR4 and complement pathways

For decades, cationic polymer nanoparticles have been investigated for nucleic acid delivery. Despite promising in vitro transfection results, most formulations have failed to translate into the clinic due to significant in vivo toxicity - especially when delivered intravenously. To address this significant problem, we investigated the detailed mechanisms that govern the complex in vivo systemic toxicity response to common polymeric nanoparticles. We determined that the toxicity response is material dependent. For branched polyethylenimine (bPEI) nanoparticles - toxicity is a function of multiple pathophysiological responses - triggering of innate immune sensors, induction of hepatic toxicity, and significant alteration of hematological properties. In contrast, for chitosan-based nanoparticles - systemic toxicity is primarily driven through innate immune activation. We further identified that modification of primary amines to secondary and tertiary amines using the small molecule imidazole-acetic-acid (IAA) ameliorates in vivo toxicity from both nanocarriers by different, material-specific mechanisms related to Toll-like receptor 4 activation (for bPEI) and complement activation driven neutrophil infiltration (for chitosan), respectively. Our results provide a detailed roadmap for evaluating in vivo toxicity of nanocarriers and identifies potential opportunities to reduce toxicity for eventual clinical translation.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC=\"FIGDIR/small/647305v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (27K):\norg.highwire.dtl.DTLVardef@63c7a7org.highwire.dtl.DTLVardef@c8ac52org.highwire.dtl.DTLVardef@a9589dorg.highwire.dtl.DTLVardef@1f8ec62_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2019

Functionalized microcarriers improve T cell manufacturing by facilitating migratory memory T cell production and increasing CD4/CD8 ratio

Adoptive cell therapies (ACT) using chimeric antigen receptor (CAR) T cells have shown promise in treating cancer, but manufacturing large numbers of high quality cells remains challenging. Critically, current T cell expansion technologies only partially recapitulate the in vivo microenvironment found in the human lymph nodes. In these organs, T cells expand at high cell density with autocrine/paracrine signaling, as well as signals from the extracellular matrix (ECM). Here we describe a T cell expansion system using degradable gelatin microcarriers functionalized with anti-CD3 and anti-CD28 monoclonal antibodies (mAbs), which address several of these shortcomings. We show that using this system, we can achieve approximately 2-fold greater expansion compared to functionalized magnetic beads, the current industry standard. Furthermore, carriers generated higher numbers of CCR7+CD62L+ migratory, central memory T cells and CD4+ T cells across multiple donors. Both these phenotypes have emerged as important for establishing durable and effective responses in patients receiving T cell immunotherapies. We further demonstrate that carriers can achieve greater memory cell yield compared to beads across a range of IL2 concentrations from 20 U/mL to 100 U/mL. These differences were greater at lower IL2 concentrations, indicating that the carriers are more efficient. We optimized this system using a design of experiments (DOE) approach and found that the carrier concentration affects the memory cell yield in a quadratic manner, where high or low concentrations are detrimental to memory formation. Finally, we show that carriers do not hinder CAR transduction and can maintain the CD4 and memory phenotype advantages in CAR-transduced T cells.

bioengineering↗

bioRxiv2018

Effects of BMP-2 dose and delivery of microvascular fragments on healing of bone defects with concomitant volumetric muscle loss

Traumatic composite bone-muscle injuries, such as open fractures, often require multiple surgical interventions and still typically result in long-term disability. Clinically, a critical indicator of composite injury severity is vascular integrity; vascular damage alone is sufficient to assign an open fracture to the most severe category. Challenging bone injuries are often treated with bone morphogenetic protein 2 (BMP-2), an osteoinductive growth factor, delivered on collagen sponge. Previous studies in a composite defect model found that a minimally bridging dose in the segmental defect model was unable to overcome concomitant muscle damage, but the effect of BMP dose on composite injuries has not yet been studied. Here, we test the hypotheses that BMP-2-mediated functional regeneration of composite extremity injuries is dose dependent and can be further enhanced via co-delivery of adipose-derived microvascular fragments (MVF), which have been previously shown to increase tissue vascular volume. Although MVF treatment did not improve healing outcomes, we observed a significant BMP-2 dose-dependent increase in regenerated bone volume and biomechanical properties. While high dose BMP-2 delivery can induce heterotopic ossification (HO) and increased inflammation, the maximum 10 g dose used in this study did not result in HO and was associated with a lower circulating inflammatory cytokine profile than the low dose (2.5 g) group. These data support the potential benefits of an increased, though still moderate, BMP-2 dose for treatment of bone defects with concomitant muscle damage. Future work to improve vascularization may further improve healing.

bioengineering↗

bioRxiv2017

A synthetic stroma-free germinal center niche using material-surface driven polyvalent signaling efficiently induces antigen-specific humoral immunity ex vivo

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial \"follicular T helper cells (TFH)\" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40; and by mimicking artificial \"follicular dendritic cells (FDC)\" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

bioengineering↗