RESUMEN
Despite intense interest in antiviral T cell priming, the routes by which virions move in lymph nodes (LNs) are imperfectly understood. Current models fail to explain how virus-infected cells rapidly appear within the LN interior after viral infection. To better understand virion trafficking in the LN, we determined the locations of virions and infected cells after administration to mice of vaccinia virus or Zika virus. Notably, many rapidly infected cells in the LN interior were adjacent to LN conduits. Through the use of confocal and electron microscopy, we clearly visualized virions within conduits. Functionally, CD8+ T cells rapidly and preferentially associated with vaccinia virus-infected cells in the LN paracortex, which led to T cell activation in the LN interior. These results reveal that it is possible for even large virions to flow through LN conduits and infect dendritic cells within the T cell zone to prime CD8+ T cells.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ganglios Linfáticos/inmunología , Activación de Linfocitos/inmunología , Virión/inmunología , Animales , Linfocitos T CD8-positivos/virología , Femenino , Ganglios Linfáticos/ultraestructura , Ganglios Linfáticos/virología , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica de Transmisión , Virus Vaccinia/inmunología , Virus Vaccinia/fisiología , Virión/fisiología , Virión/ultraestructura , Virosis/inmunología , Virosis/virología , Virus Zika/inmunología , Virus Zika/fisiologíaRESUMEN
Complement protein C3dg, a key linkage between innate and adaptive immunity, is capable of stimulating both humoral and cell-mediated immune responses, leading to considerable interest in its use as a molecular adjuvant. However, the potential of C3dg as an adjuvant is limited without ways of controllably assembling multiple copies of it into vaccine platforms. Here, we report a strategy to assemble C3dg into supramolecular nanofibers with excellent compositional control, using ß-tail fusion tags. These assemblies were investigated as therapeutic active immunotherapies, which may offer advantages over existing biologics, particularly toward chronic inflammatory diseases. Supramolecular assemblies based on the Q11 peptide system containing ß-tail-tagged C3dg, B cell epitopes from TNF, and the universal T cell epitope PADRE raised strong antibody responses against both TNF and C3dg, and prophylactic immunization with these materials significantly improved protection in a lethal TNF-mediated inflammation model. Additionally, in a murine model of psoriasis induced by imiquimod, the C3dg-adjuvanted nanofiber vaccine performed as well as anti-TNF monoclonal antibodies. Nanofibers containing only ß-tail-C3dg and lacking the TNF B cell epitope also showed improvements in both models, suggesting that supramolecular C3dg, by itself, played an important therapeutic role. We observed that immunization with ß-tail-C3dg caused the expansion of an autoreactive C3dg-specific T cell population, which may act to dampen the immune response, preventing excessive inflammation. These findings indicate that molecular assemblies displaying C3dg warrant further development as active immunotherapies.
Asunto(s)
Complemento C3d/inmunología , Nanofibras/química , Psoriasis/prevención & control , Vacunas/inmunología , Adyuvantes Inmunológicos/química , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Linfocitos B/inmunología , Células Cultivadas , Epítopos/química , Epítopos/inmunología , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Vacunas/químicaRESUMEN
Collagen-rich tissues in the cornea exhibit unique and highly organized extracellular matrix ultrastructures, which contribute to its high load-bearing capacity and light transmittance. Corneal collagen fibrils are controlled during development by small leucine-rich proteoglycans (SLRPs) that regulate the fibril diameter and spacing in order to achieve the unique optical transparency. Cyclodextrins (CDs) of varying size and chemical functionality for their ability to regulate collagen assembly during vitrification process are screened in order to create biosynthetic materials that mimic the native cornea structure. Addition of ßCD to collagen vitrigels produces materials with aligned fibers and lamellae similar to native cornea, resulting in mechanically robust and transparent materials. Biochemistry analysis revealed that CD interacts with hydrophobic amino acids in collagen to influence assembly and fibril organization. To translate the self-assembled collagen materials for cornea reconstruction, custom molds for gelation and vitrification are engineered to create ßCD/Col implants with curvature matching that of the cornea. Acellular ßCD/Col materials are implanted in a rabbit partial keratoplasty model with interrupted sutures. The implants demonstrate tissue integration and support re-epithelialization. Therefore, the addition of CD molecules regulates collagen self-assembly and provides a simple process to engineer corneal mimetic substitutes with advanced structural and functional properties.
RESUMEN
Rapid lymphocyte cell division places enormous demands on the protein synthesis machinery. Flow cytometric measurement of puromycylated ribosome-associated nascent chains after treating cells or mice with translation initiation inhibitors reveals that ribosomes in resting lymphocytes in vitro and in vivo elongate at typical rates for mammalian cells. Intriguingly, elongation rates can be increased up to 30% by activation in vivo or fever temperature in vitro. Resting and activated lymphocytes possess abundant monosome populations, most of which actively translate in vivo, while in vitro, nearly all can be stalled prior to activation. Quantitating lymphocyte protein mass and ribosome count reveals a paradoxically high ratio of cellular protein to ribosomes insufficient to support their rapid in vivo division, suggesting that the activated lymphocyte proteome in vivo may be generated in an unusual manner. Our findings demonstrate the importance of a global understanding of protein synthesis in lymphocytes and other rapidly dividing immune cells.
Asunto(s)
Biosíntesis de Proteínas , Ribosomas , Ratones , Animales , Ribosomas/metabolismo , Linfocitos , Citometría de Flujo , MamíferosRESUMEN
Butyrate-a metabolite produced by commensal bacteria-has been extensively studied for its immunomodulatory effects on immune cells, including regulatory T cells, macrophages and dendritic cells. However, the development of butyrate as a drug has been hindered by butyrate's poor oral bioavailability, owing to its rapid metabolism in the gut, its low potency (hence, necessitating high dosing), and its foul smell and taste. Here we report that the oral bioavailability of butyrate can be increased by esterifying it to serine, an amino acid transporter that aids the escape of the resulting odourless and tasteless prodrug (O-butyryl-L-serine, which we named SerBut) from the gut, enhancing its systemic uptake. In mice with collagen-antibody-induced arthritis (a model of rheumatoid arthritis) and with experimental autoimmune encephalomyelitis (a model of multiple sclerosis), we show that SerBut substantially ameliorated disease severity, modulated key immune cell populations systemically and in disease-associated tissues, and reduced inflammatory responses without compromising the global immune response to vaccination. SerBut may become a promising therapeutic for autoimmune and inflammatory diseases.
Asunto(s)
Artritis Experimental , Disponibilidad Biológica , Butiratos , Profármacos , Serina , Animales , Profármacos/farmacología , Profármacos/uso terapéutico , Profármacos/farmacocinética , Profármacos/química , Ratones , Serina/metabolismo , Butiratos/farmacología , Butiratos/uso terapéutico , Butiratos/química , Butiratos/administración & dosificación , Administración Oral , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/inmunología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Ratones Endogámicos C57BL , Enfermedades Neuroinflamatorias/tratamiento farmacológico , FemeninoRESUMEN
Urinary tract infections (UTIs) are a major public health problem affecting millions of individuals each year. Recurrent UTIs are managed by long-term antibiotic use, making the alarming rise of antibiotic resistance a substantial threat to future UTI treatment. Extended antibiotic regimens may also have adverse effects on the microbiome. Here, we report the use of a supramolecular vaccine to provide long-term protection against uropathogenic Escherichia coli, which cause 80% of uncomplicated UTIs. We designed mucus-penetrating peptide-polymer nanofibers to enable sublingual (under the tongue) vaccine delivery and elicit antibody responses systemically and in the urogenital tract. In a mouse model of UTI, we demonstrate equivalent efficacy to high-dose oral antibiotics but with significantly less perturbation of the gut microbiome. We also formulate our vaccine as a rapid-dissolving sublingual tablet that raises response in mice and rabbits. Our approach represents a promising alternative to antibiotics for the treatment and prevention of UTIs.
Asunto(s)
Infecciones por Escherichia coli , Nanofibras , Infecciones Urinarias , Vacunas , Ratones , Conejos , Animales , Infecciones por Escherichia coli/prevención & control , Infecciones Urinarias/prevención & control , Antibacterianos/farmacología , Antibacterianos/uso terapéuticoRESUMEN
Current treatments for chronic immune-mediated diseases such as psoriasis, rheumatoid arthritis, or Crohn's disease commonly rely on cytokine neutralization using monoclonal antibodies; however, such approaches have drawbacks. Frequent repeated dosing can lead to the formation of anti-drug antibodies and patient compliance issues, and it is difficult to identify a single antibody that is broadly efficacious across diverse patient populations. As an alternative to monoclonal antibody therapy, anti-cytokine immunization is a potential means for long-term therapeutic control of chronic inflammatory diseases. Here we report a supramolecular peptide-based approach for raising antibodies against IL-17 and demonstrate its efficacy in a murine model of psoriasis. B-cell epitopes from IL-17 were co-assembled with the universal T-cell epitope PADRE using the Q11 self-assembling peptide nanofiber system. These materials, with or without adjuvants, raised antibody responses against IL-17. Exploiting the modularity of the system, multifactorial experimental designs were used to select formulations maximizing titer and avidity. In a mouse model of psoriasis induced by imiquimod, unadjuvanted nanofibers had therapeutic efficacy, which could be enhanced with alum adjuvant but reversed with CpG adjuvant. Measurements of antibody subclass induced by adjuvanted and unadjuvanted formulations revealed strong correlations between therapeutic efficacy and titers of IgG1 (improved efficacy) or IgG2b (worsened efficacy). These findings have important implications for the development of anti-cytokine active immunotherapies and suggest that immune phenotype is an important metric for eliciting therapeutic anti-cytokine antibody responses.
Asunto(s)
Diseño de Fármacos , Interleucina-17/antagonistas & inhibidores , Psoriasis/inmunología , Vacunas de Subunidad/inmunología , Vacunas de Subunidad/farmacología , Animales , Modelos Animales de Enfermedad , Femenino , Inmunoterapia Activa/métodos , Ratones , Ratones Endogámicos C57BLRESUMEN
The current paradigm that subunit vaccines require adjuvants to optimally activate innate immunity implies that increased vaccine reactogenicity will invariably be linked to improved immunogenicity. Countering this paradigm, nanoparticulate vaccines have been reported to act as delivery systems for vaccine antigens and induce immunity without the need for exogenous adjuvants or local inflammation; however, the mechanisms underlying the immunogenicity of nanoparticle vaccines are incompletely identified. Here, we show that antigens displayed on self-assembling nanofiber scaffolds and delivered intranasally are presented by CD103+ and CD11b+ lung dendritic cells that up-regulate CD80 and migrate into the draining lymph node (LN). This was accompanied by a nearly exclusive priming and accumulation of antigen-specific TH17 cells occurring independently in both LN and lung. Thus, self-assembling peptide nanofiber vaccines may represent a novel, needle- and adjuvant-free means of eliciting protective immunity against fungal and bacterial infections at skin and mucosal barrier surfaces.
Asunto(s)
Nanofibras , Adyuvantes Inmunológicos , Células Dendríticas , Pulmón , Vacunas de SubunidadRESUMEN
Biomaterials employed to raise therapeutic immune responses have become a complex and active field. Historically, vaccines have been developed primarily to fight infectious diseases, but recent years have seen the development of immunologically active biomaterials towards an expanding list of non-infectious diseases and conditions including inflammation, autoimmunity, wounds, cancer, and others. This review structures its discussion of these approaches around a progression from single-target strategies to those that engage increasingly complex and multifactorial immune responses. First, the targeting of specific individual cytokines is discussed, both in terms of delivering the cytokines or blocking agents, and in terms of active immunotherapies that raise neutralizing immune responses against such single cytokine targets. Next, non-biological complex drugs such as randomized polyamino acid copolymers are discussed in terms of their ability to raise multiple different therapeutic immune responses, particularly in the context of autoimmunity. Last, biologically derived matrices and materials are discussed in terms of their ability to raise complex immune responses in the context of tissue repair. Collectively, these examples reflect the tremendous diversity of existing approaches and the breadth of opportunities that remain for generating therapeutic immune responses using biomaterials.
Asunto(s)
Autoinmunidad/efectos de los fármacos , Materiales Biocompatibles/uso terapéutico , Citocinas/antagonistas & inhibidores , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Vacunas/inmunología , Animales , Autoinmunidad/inmunología , Citocinas/inmunología , Humanos , Cicatrización de Heridas/inmunologíaRESUMEN
Tissue-engineering strategies offer promising tools for repairing cartilage damage; however, these strategies suffer from limitations under pathological conditions. As a model disease for these types of nonideal systems, the inflammatory environment in an osteoarthritic (OA) joint limits the efficacy of engineered therapeutics by disrupting joint homeostasis and reducing its capacity for regeneration. In this work, we investigated a sugar-based drug candidate, a tributanoylated N-acetyl-d-glucosamine analogue, called 3,4,6-O-Bu3GlcNAc, that is known to reduce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in osteoarthritis. 3,4,6-O-Bu3GlcNAc not only inhibited NFκB signaling but also exerted chondrogenic and anti-inflammatory effects on chondrocytes isolated from patients with osteoarthritis. 3,4,6-O-Bu3GlcNAc also increased the expression of extracellular matrix proteins and induced cartilage tissue production in three-dimensional in vitro hydrogel culture systems. To translate these chondrogenic and anti-inflammatory properties to tissue regeneration in osteoarthritis, we implanted 3,4,6-O-Bu3GlcNAc-loaded poly(lactic-co-glycolic acid) microfiber scaffolds into rats. The drug-laden scaffolds were biocompatible, and when seeded with human OA chondrocytes, similarly promoted cartilage tissue formation. 3,4,6-O-Bu3GlcNAc combined with the appropriate structural environment could be a promising therapeutic approach for osteoarthritis.
Asunto(s)
Acetilglucosamina/farmacología , Antiinflamatorios/farmacología , Butanos/química , Cartílago/efectos de los fármacos , Regeneración/efectos de los fármacos , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Acetilglucosamina/química , Animales , Antiinflamatorios/química , Condrocitos/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato/farmacología , Interleucina-1beta/farmacología , Ácido Láctico/química , Masculino , Polietilenglicoles/farmacología , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas Sprague-DawleyRESUMEN
Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degradation. Because OA has a multifactorial nature and complex interrelationship of the individual elements of a whole joint, there is a need for comprehensive therapeutic approaches for cartilage tissue engineering, which simultaneously address multiple aspects of disease etiology. In this work, we investigated a multifunctional carbohydrate-based drug candidate, tri-butanoylated N-acetyl-D-galactosamine analog (3,4,6-O-Bu3GalNAc) that induced cartilage tissue production by human mesenchymal stem cells (hMSCs) and human OA chondrocytes by modulating Wnt/ß-catenin signaling activity. The dual effects promoted chondrogenesis of human MSC and reduced inflammation of human OA chondrocytes in in vitro cultures. Translating these findings in vivo, we evaluated therapeutic effect of 3,4,6-O-Bu3GalNAc on the rat model of posttraumatic OA when delivered via local intra-articular sustained-release delivery using microparticles and found this method to be efficacious in preventing OA progression. These results show that 3,4,6-O-Bu3GalNAc, a disease modifying OA drug candidate, has promising therapeutic potential for articular cartilage repair.
Asunto(s)
Cartílago Articular/patología , Sistemas de Liberación de Medicamentos , Galactosamina/uso terapéutico , Inflamación/tratamiento farmacológico , Osteoartritis/tratamiento farmacológico , Regeneración , Animales , Antiinflamatorios/farmacología , Antiinflamatorios/uso terapéutico , Biomarcadores/metabolismo , Cartílago Articular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Condrocitos/patología , Condrogénesis/efectos de los fármacos , Modelos Animales de Enfermedad , Matriz Extracelular/efectos de los fármacos , Matriz Extracelular/metabolismo , Galactosamina/química , Galactosamina/farmacología , Humanos , Inflamación/complicaciones , Inflamación/patología , Masculino , Osteoartritis/complicaciones , Osteoartritis/patología , Ratas Sprague-Dawley , Regeneración/efectos de los fármacosRESUMEN
Replacement of autogenous or allograft bones by artificial graft materials represents a growing area of interest in current bone repair strategies. Bioactive ceramics in particulate form, such as Bioglass (BG) 45S5, stimulate bone mineralization comparable to autologous bone grafts, but have potential issues of particle migration and inflammation. The aim of this study was to employ a chondroitin sulfate- (CS-) based bioadhesive to improve integration of the bioglass (NovaBone Putty) to prevent particle migration and promote bone regeneration. This BG-CS composite can encapsulate bone marrow (BM) to form a mechanically stable construct, BG-CS-BM. Rheological characterization confirmed the formation of CS-BM hydrogel by reacting the CS-based bioadhesive with the BM. Compared to the bioglass, the BG-CS-BM composite demonstrated a superior capacity to maintain construct integrity under both aqueous and turbulent environments in vitro. After implantation for 4 weeks in a critical-size distal femoral bone defect in a rabbit model, there was significantly greater bone growth in BG-CS-BM as compared to bioglass-only and the empty control. Unlike BG-CS-BM, BG-CS recruited BM in situ from the bone defect. BG-CS demonstrated a similar effect in bone formation but at a comparatively slower rate than BG-CS-BM over 6-weeks' implantation.