RESUMEN

Astrocytes in the lamina region of the optic nerve head play vital roles in supporting retinal ganglion cell axon health. In glaucoma, these astrocytes are implicated as early responders to stressors, undergoing characteristic changes in cell function as well as cell morphology. Much of what is currently known about individual lamina astrocyte morphology has been learned from rodent models which lack a defining feature of the human optic nerve head, the collagenous lamina cribrosa (LC). Current methods available for evaluation of collagenous LC astrocyte morphology have significant shortcomings. We aimed to evaluate Multicolor DiOlistic labeling (MuDi) as an approach to reveal individual astrocyte morphologies across the collagenous LC. Gold microcarriers were coated with all combinations of three fluorescent cell membrane dyes, DiI, DiD, and DiO, for a total of seven dye combinations. Microcarriers were delivered to 150 µm-thick coronal vibratome slices through the LC of pig, sheep, goat, and monkey eyes via MuDi. Labeled tissues were imaged with confocal and second harmonic generation microscopy to visualize dyed cells and LC collagenous beams, respectively. GFAP labeling of DiOlistically-labeled cells with astrocyte morphologies was used to investigate cell identity. 3D models of astrocytes were created from confocal image stacks for quantification of morphological features. DiOlistic labeling revealed fine details of LC astrocyte morphologies including somas, primary branches, higher-order branches, and end-feet. Labeled cells with astrocyte morphologies were GFAP+. Astrocytes were visible across seven distinct color channels, allowing high labeling density while still distinguishing individual cells from their neighbors. MuDi was capable of revealing tens to hundreds of collagenous LC astrocytes, in situ, with a single application. 3D astrocyte models allowed automated quantification of morphological features including branch number, length, thickness, hierarchy, and straightness as well as Sholl analysis. MuDi labeling provides an opportunity to investigate morphologies of collagenous LC astrocytes, providing both qualitative and quantitative detail, in healthy tissues. This approach may open doors for research of glaucoma, where astrocyte morphological alterations are thought to coincide with key functional changes related to disease progression.

Asunto(s)

Glaucoma , Disco Óptico , Humanos , Porcinos , Animales , Ovinos , Astrocitos/metabolismo , Glaucoma/metabolismo , Células Ganglionares de la Retina/metabolismo

RESUMEN

Allergic contact dermatitis (ACD) is a common T-cell mediated inflammatory skin condition, characterized by an intensely pruritic rash at the site of contact with allergens like poison ivy or nickel. Current clinical treatments use topical corticosteroids, which broadly and transiently suppress inflammation and symptoms of ACD, but fail to address the underlying immune dysfunction. Here, we present an alternative therapeutic approach that teaches the immune system to tolerate contact allergens by expanding populations of naturally suppressive allergen-specific regulatory T cells (Tregs). Specifically, biodegradable poly(ethylene glycol)-poly(lactic-co-glycolic acid) (PEG-PLGA) microparticles were engineered to release TGF-ß1, Rapamycin, and IL-2, to locally sustain a microenvironment that promotes Treg differentiation. By expanding allergen-specific Tregs and reducing pro-inflammatory effector T cells, these microparticles inhibited destructive hypersensitivity responses to subsequent allergen exposure in an allergen-specific manner, effectively preventing or reversing ACD in previously sensitized mice. Ultimately, this approach to in vivo Treg induction could also enable novel therapies for transplant rejection and autoimmune diseases.

Asunto(s)

Alérgenos/inmunología , Dermatitis Alérgica por Contacto/terapia , Tolerancia Inmunológica , Linfocitos T Reguladores/inmunología , Animales , Diferenciación Celular , Dermatitis Alérgica por Contacto/inmunología , Femenino , Interleucina-2/administración & dosificación , Interleucina-2/inmunología , Ratones , Ratones Congénicos , Ratones Endogámicos C57BL , Ratones Transgénicos , Poliésteres/química , Polietilenglicoles/química , Sirolimus/administración & dosificación , Sirolimus/inmunología , Factor de Crecimiento Transformador beta1/administración & dosificación , Factor de Crecimiento Transformador beta1/inmunología

RESUMEN

Alcoholics suffer from immune dysfunction that can impede vaccine efficacy. If ethanol (EtOH)-induced immune impairment is in part a result of direct exposure of immune cells to EtOH, then reduced levels of exposure could result in less immune dysfunction. As alcohol ingestion results in lower alcohol levels in skin than blood, we hypothesized that the skin immune network may be relatively preserved, enabling skin-targeted immunizations to obviate the immune inhibitory effects of alcohol consumption on conventional vaccines. We employed the two most common chronic EtOH mouse feeding models, the liver-damaging Lieber-DeCarli (LD) and liver-sparing Meadows-Cook (MC) diets, to examine the roles of EtOH and/or EtOH-induced liver dysfunction on alcohol related immunosuppression. Pair-fed mice were immunized against the model antigen ovalbumin (OVA) by DNA immunization or against flu by administering the protein-based influenza vaccine either systemically (IV, IM), directly to liver (hydrodynamic), or cutaneously (biolistic, ID). We measured resulting tissue EtOH levels, liver stress, regulatory T cell (Treg), and myeloid-derived suppressor cell (MDSC) populations. We compared immune responsiveness by measuring delayed-type hypersensitivity (DTH), antigen-specific cytotoxic T lymphocyte (CTL), and antibody induction as a function of delivery route and feeding model. We found that, as expected, and independent of the feeding model, EtOH ingestion inhibits DTH, CTL lysis, and antigen-specific total IgG induced by traditional systemic vaccines. On the other hand, skin-targeted vaccines were equally immunogenic in alcohol-exposed and non-exposed subjects, suggesting that cutaneous immunization may result in more efficacious vaccination in alcohol-ingesting subjects.

Asunto(s)

Vacunas contra la Influenza/inmunología , Hepatopatías Alcohólicas/inmunología , Piel/inmunología , Animales , Etanol/sangre , Femenino , Vacunas contra la Influenza/administración & dosificación , Inyecciones Intradérmicas , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Linfocitos T Reguladores/inmunología

RESUMEN

PURPOSE: Design and evaluate a new micro-machining based approach for fabricating dissolvable microneedle arrays (MNAs) with diverse geometries and from different materials for dry delivery to skin microenvironments. The aims are to describe the new fabrication method, to evaluate geometric and material capability as well as reproducibility of the method, and to demonstrate the effectiveness of fabricated MNAs in delivering bioactive molecules. METHODS: Precise master molds were created using micromilling. Micromolding was used to create elastomer production molds from master molds. The dissolvable MNAs were then fabricated using the spin-casting method. Fabricated MNAs with different geometries were evaluated for reproducibility. MNAs from different materials were fabricated to show material capability. MNAs with embedded bioactive components were tested for functionality on human and mice skin. RESULTS: MNAs with different geometries and from carboxymethyl cellulose, polyvinyl pyrrolidone and maltodextrin were created reproducibly using our method. MNAs successfully pierce the skin, precisely deliver their bioactive cargo to skin and induce specific immunity in mice. CONCLUSIONS: We demonstrated that the new fabrication approach enables creating dissolvable MNAs with diverse geometries and from different materials reproducibly. We also demonstrated the application of MNAs for precise and specific delivery of biomolecules to skin microenvironments in vitro and in vivo.

Asunto(s)

Productos Biológicos/administración & dosificación , Sistemas de Liberación de Medicamentos/instrumentación , Diseño de Equipo/instrumentación , Microinyecciones/instrumentación , Administración Cutánea , Animales , Sistemas de Liberación de Medicamentos/métodos , Humanos , Ratones , Microinyecciones/métodos , Agujas , Reproducibilidad de los Resultados , Piel/metabolismo

RESUMEN

In vivo dendritic cells (DC) targeting is an attractive approach with potential advantages in vaccine efficacy, cost, and availability. Identification of molecular adjuvants to in vivo "modulate " DC to coordinately render improved Th1 and CD8 T cell immunity, and attenuated deleterious Treg effects, is a critical challenge. Here, we report that in vivo genetic targeting of the active transcription factor XBP1s to DC (XBP1s/DC) potentiated vaccine-induced prophylactic and therapeutic antitumor immunity in multiple tumor models. This immunization strategy is based on a genetic vaccine encoding both cytomegalovirus (CMV)-driven vaccine Aghsp70 and DC-specific CD11c-driven XBP1s. The novel targeted vaccine induced durable Th1 and CD8 T cell responses to poorly immunogenic self/tumor antigen (Ag) and attenuated tumor-associated Treg suppressive function. Bone marrow (BM)-derived DC genetically modified to simultaneously overexpress XBP1s and express Aghsp70 upregulated CD40, CD70, CD86, interleukin (IL)-15, IL-15Rα, and CCR7 expression, and increased IL-6, IL-12, and tumor necrosis factor (TNF)-α production in vitro. XBP1s/DC elevated functional DEC205(+)CD8α(+)DC in the draining lymph nodes (DLN). The data suggest a novel role for XBP1s in modulating DC to potentiate tumor vaccine efficacy via overcoming two major obstacles to tumor vaccines (i.e., T cell hyporesponsiveness against poorly immunologic self/tumor Ag and tumor-associated Treg-mediated suppression) and improving DEC205(+)CD8α(+)DC.

Asunto(s)

Vacunas contra el Cáncer/genética , Vacunas contra el Cáncer/inmunología , Proteínas de Unión al ADN/genética , Células Dendríticas/inmunología , Marcación de Gen , Neoplasias Experimentales/inmunología , Factores de Transcripción/genética , Animales , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/genética , Proteínas de Unión al ADN/metabolismo , Células Dendríticas/metabolismo , Femenino , Expresión Génica , Orden Génico , Interferón gamma/biosíntesis , Ganglios Linfáticos/inmunología , Ganglios Linfáticos/metabolismo , Ratones , Ratones Endogámicos BALB C , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/mortalidad , Factores de Transcripción del Factor Regulador X , Análisis de Supervivencia , Linfocitos T Reguladores , Canales Catiónicos TRPC/inmunología , Células TH1/inmunología , Factores de Transcripción/metabolismo , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteína 1 de Unión a la X-Box

RESUMEN

The skin contains readily accessible dendritic cells (DCs) with potent antigen presentation function and functional plasticity enabling the integration of antigen specificity with environmentally responsive immune control. Recent studies challenge the established paradigm of cutaneous immune function by suggesting that lymph node-resident DCs, rather than skin-derived DCs (sDCs), are responsible for eliciting T cell immunity against cutaneous pathogens including viral vectors. We show that cutaneous delivery of lentivirus results in direct transfection of sDCs and potent and prolonged antigen presentation. Further, sDCs are the predominant antigen-presenting cells for the induction of potent and durable CD8(+) T cell immunity. These results support the classical paradigm of cutaneous immune function and suggest that antigen presentation by sDCs contributes to the high potency of lentivector-mediated genetic immunization.

Asunto(s)

Linfocitos T CD8-positivos/inmunología , Células Dendríticas/inmunología , Inmunización/métodos , Lentivirus/inmunología , Piel/inmunología , Animales , Presentación de Antígeno/inmunología , Biolística , Movimiento Celular/inmunología , Proliferación Celular , Células Dendríticas/citología , Citometría de Flujo , Vectores Genéticos , Lentivirus/genética , Infecciones por Lentivirus/inmunología , Activación de Linfocitos/inmunología , Ratones , Ratones Transgénicos , Proteínas Recombinantes/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/citología , Piel/virología , Transfección

RESUMEN

Dendritic cells (DCs) play a key role in the induction and control of immunity. Genetic engineering of DCs is a promising approach for the development of a broad range of immunomodulatory strategies, for purposes ranging from genetic immunization to tolerance induction. The development of DC-based immunotherapies is limited by the inability to efficiently transfect DCs using naked DNA. Here we demonstrate that after plasmid DNA delivery, the transgene expression level controlled by the human immediate-early cytomegalovirus promoter (hIE-CMVp) is higher in mature DCs than in immature DCs and is further increased after terminal differentiation of DCs by agonist anti-CD40 monoclonal antibody (mAb) or after DC interaction with CD4(+) T cells. CD40 signaling of DCs resulted in nuclear translocation of the transcription factors nuclear factor-kappaB (NF-kappaB), activator of protein-1 (AP-1), and cyclic adenosine monophosphate (cAMP)-responsive element, necessary for the activation of hIE-CMVp. Transgene expression by DCs diminished after the inhibition of these transcription factors or the blockade of adhesion molecules involved in the DC-T-cell synapse. Importantly, CD40 signaling of DCs results in the highly efficient expression and presentation of transgenic antigens and the induction of "in vivo" cytotoxic T-cell (CTL) responses specific for transgenic antigen peptides, demonstrating the functional potential of genetically engineered DCs.

Asunto(s)

ADN Recombinante/genética , Células Dendríticas/citología , Células Dendríticas/metabolismo , Proteínas Recombinantes/genética , Animales , Presentación de Antígeno , Antígenos , Antígenos CD40/metabolismo , Diferenciación Celular , Citomegalovirus/genética , Células Dendríticas/inmunología , Expresión Génica , Humanos , Inmunoterapia/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Ovalbúmina/inmunología , Regiones Promotoras Genéticas , Transducción de Señal , Linfocitos T Citotóxicos/inmunología