Generation of primary feline chimeric antigen receptor T cells.

Objective: The purpose of this study was to develop procedures to engineer feline chimeric antigen receptor (CAR) T cells.

Methods: 6 healthy cats were used in this study. Blood was collected, and CD3+ primary T cells were enriched by magnetic activated cell sorting, expanded, and used to generate CAR T cells.

Racing CARs to veterinary immuno-oncology.

Chimeric antigen receptors (CARs) have demonstrated remarkable promise in human oncology over the past two decades, yet similar strategies in veterinary medicine are still in development. CARs are synthetically engineered proteins comprised of a specific antigen-binding single chain variable fragment (ScFv) fused to the signaling domain of a T cell receptor and co-receptors. Patient T cells engineered to express a CAR are directed to recognize and kill target cells, most commonly hematological malignancies.

Macrophage uptake of oxidized and acetylated low-density lipoproteins and generation of reactive oxygen species are regulated by linear stiffness of the growth surface.

Macrophages are key players in the development of atherosclerosis: they scavenge lipid, transform into foam cells, and produce proinflammatory mediators. At the same time, the arterial wall undergoes profound changes in its mechanical properties. We recently showed that macrophage morphology and proinflammatory potential are regulated by the linear stiffness of the growth surface.

Molecular regulation of TLR signaling in health and disease: mechano-regulation of macrophages and TLR signaling.

Immune cells encounter tissues with vastly different biochemical and physical characteristics. Much of the research emphasis has focused on the role of cytokines and chemokines in regulating immune cell function, but the role of the physical microenvironment has received considerably less attention. The tissue mechanics, or stiffness, of healthy tissues varies dramatically from soft adipose tissue and brain to stiff cartilage and bone.

Immunomodulatory nanogels overcome restricted immunity in a murine model of gut microbiome-mediated metabolic syndrome.

Biomaterials-based nanovaccines, such as those made of poly(lactic-co-glycolic acid) (PLGA), can induce stronger immunity than soluble antigens in healthy wild-type mouse models. However, whether metabolic syndrome can influence the immunological responses of nanovaccines remains poorly understood. Here, we first show that alteration in the sensing of the gut microbiome through Toll-like receptor 5 (TLR5) and the resulting metabolic syndrome in mice diminish the germinal center immune response induced by PLGA nanovaccines.

The effect of cell debris within biologic scaffolds upon the macrophage response.

All biomaterials, including biologic scaffolds composed of extracellular matrix (ECM), elicit a host immune response when implanted. The type and intensity of this response depends in part upon the thoroughness of decellularization and removal of cell debris from the source tissue. Proinflammatory responses have been associated with negative downstream remodeling events including scar tissue formation, encapsulation, and seroma formation.

Safe Recombinant Outer Membrane Vesicles that Display M2e Elicit Heterologous Influenza Protection.

Recombinant, Escherichia coli-derived outer membrane vesicles (rOMVs), which display heterologous protein subunits, have potential as a vaccine adjuvant platform. One drawback to rOMVs is their lipopolysaccharide (LPS) content, limiting their translatability to the clinic due to potential adverse effects. Here, we explore a unique rOMV construct with structurally remodeled lipids containing only the lipid IVa portion of LPS, which does not stimulate human TLR4.

Dendritic cells in host response to biologic scaffolds.

Tissue regeneration and repair require a highly complex and orchestrated series of events that require inflammation, but can be compromised when inflammation is excessive or becomes chronic. Macrophages are one of the first cells to contact and respond to implanted materials, and mediate the inflammatory response. The series of events following macrophage association with biomaterials has been well-studied.

Molecular mechanisms of regulation of Toll-like receptor signaling.

TLRs play a critical role in the detection of microbes and endogenous "alarmins" to initiate host defense, yet they can also contribute to the development and progression of inflammatory and autoimmune diseases. To avoid pathogenic inflammation, TLR signaling is subject to multilayer regulatory control mechanisms, including cooperation with coreceptors, post-translational modifications, cleavage, cellular trafficking, and interactions with negative regulators. Nucleic acid-sensing TLRs are particularly interesting in this regard, as they can both recognize host-derived structures and require internalization of their ligand as a result of intracellular sequestration of the nucleic acid-sensing TLRs.

Complex Negative Regulation of TLR9 by Multiple Proteolytic Cleavage Events.

TLR9 is an innate immune receptor important for recognizing DNA of host and foreign origin. A mechanism proposed to prevent excessive response to host DNA is the requirement for proteolytic cleavage of TLR9 in endosomes to generate a mature form of the receptor (TLR9(471-1032)). We previously described another cleavage event in the juxtamembrane region of the ectodomain that generated a dominant-negative form of TLR9.

TLR9 stability and signaling are regulated by phosphorylation and cell stress.

Innate sensing of pathogens elicits protective immune responses through pattern recognition receptors, including Toll-like receptors. Although signaling by Toll-like receptors is regulated at multiple steps, including localization, trafficking, proteolytic cleavage, and phosphorylation, the significance of post-translational modifications and cellular stress response on Toll-like receptor stability and signaling is still largely unknown. In the present study, we investigated the role of cytoplasmic tyrosine motifs in Toll-like receptor-9 stability, proteolytic cleavage, and signaling.

Recombinant M2e outer membrane vesicle vaccines protect against lethal influenza A challenge in BALB/c mice.

Currently approved influenza vaccines predominantly protect through antibodies directed against the highly variable glycoprotein hemagglutinin (HA), necessitating annual redesign and formulation based on epidemiological prediction of predominant circulating strains. More conserved influenza protein sequences, such as the ectodomain of the influenza M2 protein, or M2e, show promise as a component of a universal influenza A vaccine, but require a Th1-biased immune response for activity. Recently, recombinant, bacterially derived outer membrane vesicles (OMVs) demonstrated potential as a platform to promote a Th1-biased immune response to subunit antigens.

Electrostatically self-assembled biodegradable microparticles from pseudoproteins and polysaccharide: fabrication, characterization, and biological properties.

Electrostatically self-assembling hybrid microparticles derived from novel cationic unsaturated arginine-based poly(ester amide) polymers (UArg-PEA) and anionic hyaluronic acid (HA) were fabricated into sub-micron-sized particles in aqueous medium with subsequent UV crosslinking treatment to stabilize the structure. These hybrid microparticles were characterized for size, charge, viscosity, chemical structure, morphology, and biological properties. Depending on the feed ratio of cationic UArg-PEA to anionic HA, the crosslinked microparticles formed spherical structures of 0.

Linking genetic variation in human Toll-like receptor 5 genes to the gut microbiome's potential to cause inflammation.

Immunodeficiencies can lead to alterations of the gut microbiome that render it pathogenic and capable of transmitting disease to naïve hosts. Here, we review the role of Toll-like receptor (TLR) 5, the innate receptor for bacterial flagellin, in immune responses to the normal gut microbiota with a focus its role on adaptive immunity. Loss of TLR5 has profound effects on the microbiota that include greater temporal instability of major lineages and upregulation of flagellar motility genes that may be linked to the reduced levels of anti-flagellin antibodies in the TLR5(-/-) host.

Discordant results obtained with Francisella tularensis during in vitro and in vivo immunological studies are attributable to compromised bacterial structural integrity.

Francisella tularensis (Ft) is a highly infectious intracellular pathogen and the causative agent of tularemia. Because Ft can be dispersed via small droplet-aerosols and has a very low infectious dose it is characterized as a category A Select Agent of biological warfare. Respiratory infection with the attenuated Live Vaccine Strain (LVS) and the highly virulent SchuS4 strain of Ft engenders intense peribronchiolar and perivascular inflammation, but fails to elicit select pro-inflammatory mediators (e.

Participation of MyD88 and interleukin-33 as innate drivers of Th2 immunity to Trichinella spiralis.

Trichinella spiralis is a highly destructive parasitic nematode that invades and destroys intestinal epithelial cells, injures many different tissues during its migratory phase, and occupies and transforms myotubes during the final phase of its life cycle. We set out to investigate the role in immunity of innate receptors for potential pathogen- or danger-associated molecular patterns (PAMPs or DAMPs). Focusing on the MyD88-dependent receptors, which include Toll-like receptors (TLRs) and interleukin-1 (IL-1) family members, we found that MyD88-deficient mice expelled worms normally, while TLR2/4-deficient mice showed accelerated worm expulsion, suggesting that MyD88 was active in signaling pathways for more than one receptor during intestinal immunity.

Traditional biochemical assays for studying toll-like receptor 9.

Understanding the mechanistic basis of receptor activation and regulation can offer therapeutic targets for disease treatment. Evidence is emerging for a role of the normally foreign responsive Toll-like receptors (TLRs) in the development of autoimmunity through response to self-patterns. Regulatory mechanisms governing this class of receptors are poorly understood, and failures within this system likely contribute to development of autoimmunity.

TLR9 is important for protection against intestinal damage and for intestinal repair.

Toll-like receptors (TLRs) are innate receptors critical for host defense, and play a role in normal biological processes. For example, host DNA, a TLR9 ligand, stimulates epithelial repair following skin wounding. TLR signaling also plays a crucial role in regulating intestinal homeostasis.

Multifunctional role of dextran sulfate sodium for in vivo modeling of intestinal diseases.

Background: Inflammatory bowel diseases (IBDs) are chronic, relapsing disorders that affect the gastrointestinal tract of millions of people and continue to increase in incidence each year. While several factors have been associated with development of IBDs, the exact etiology is unknown. Research using animal models of IBDs is beginning to provide insights into how the different factors contribute to disease development.

Heterozygous carriage of a dysfunctional Toll-like receptor 9 allele affects CpG oligonucleotide responses in B cells.

Toll-like receptors (TLR) are employed by the innate immune system to detect microbial pathogens based on conserved microbial pathogen molecules. For example, TLR9 is a receptor for CpG-containing microbial DNA, and its activation results in the production of cytokines and type I interferons from human B cells and plasmacytoid dendritic cells, respectively. Both are required for mounting an efficient antibacterial or antiviral immune response.

Heat shock protein gp96 regulates Toll-like receptor 9 proteolytic processing and conformational stability.

Nucleic acid-sensing Toll-like receptors (TLRs) initiate innate immune responses to foreign RNA and DNA, yet can detect and respond to host DNA. To avoid autoimmune pathologies, nucleic acid sensing TLRs are tightly regulated. TLR9 primarily resides in the endoplasmic reticulum, traffics to endosomes, is proteolytically processed and responds to DNA.

Cutting edge: a TLR9 cytoplasmic tyrosine motif is selectively required for proinflammatory cytokine production.

Compartmentalization of nucleic acid sensing TLR9 has been implicated as a mechanism to prevent recognition of self nucleic acid structures. Furthermore, recognition of CpG DNA in different endosomal compartments leads to the production of the proinflammatory cytokine TNF-α, or type I IFN. We previously characterized a tyrosine-based motif at aa 888-891 in the cytoplasmic tail of TLR9 important for appropriate intracellular localization.

Antimicrobial peptides inhibit polyinosinic-polycytidylic acid-induced immune responses.

Viral proteins and nucleic acids stimulate TLRs to elicit production of cytokines, chemokines, and IFNs. Because of their immunostimulatory activity, several TLR agonists are being developed as vaccine adjuvants and cancer immunotherapeutics. However, TLR signaling is modified by disease state, which could enhance or impair therapeutic efficacy.

Negative regulation of signaling by a soluble form of toll-like receptor 9.

Nucleic acid structures are highly conserved through evolution and when self nucleic acids are aberrantly detected by toll-like receptors (TLRs) they contribute to autoimmune disease. For this reason, multiple regulatory mechanisms exist to prevent immune responses to self nucleic acids. TLR9 is a nucleic acid-sensing TLR that is regulated at multiple levels including association with accessory proteins, intracellular localization and proteolytic processing.

Early life environment and developmental immunotoxicity in inflammatory dysfunction and disease.

Components of the innate immune system such as macrophages and dendritic cells are instrumental in determining the fate of immune responses and are, also, among the most sensitive targets of early life environmental alterations including developmental immunotoxicity (DIT). DIT can impede innate immune cell maturation, disrupt tissue microenvironment, alter immune responses to infectious challenges, and disrupt regulatory responses. Dysregulation of inflammation, such as that observed with DIT, has been linked with an increased risk of chronic inflammatory diseases in both children and adults.