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1.
Front Vet Sci ; 10: 1130182, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-36876006

RESUMEN

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. The U.S Food and Drug Administration (FDA) has approved multiple human CAR T therapies, but translation of these therapies into veterinary medicine faces many challenges. In this review, we discuss considerations for veterinary use including CAR design and cell carrier choice, and discuss the future promise of translating CAR therapy into veterinary oncology.

2.
PLoS One ; 16(12): e0260756, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34914760

RESUMEN

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. Here we asked whether linear stiffness also regulates lipid uptake by macrophages. We cultured murine bone marrow-derived macrophages (BMMs) on polyacrylamide gels modeling stiffness of healthy (1kPa) and diseased (10-150kPa) blood vessels. In unprimed BMMs, increased linear stiffness increased uptake of oxidized (oxLDL) and acetylated (acLDL) low density lipoproteins and generation of reactive oxygen species, but did not alter phagocytosis of bacteria or silica particles. Macrophages adapted to stiff growth surfaces had increased mRNA and protein expression of two key lipoprotein receptors: CD36 and scavenger receptor b1. Regulation of the lipoprotein receptor, lectin-like receptor for ox-LDL, was more complex: mRNA expression decreased but surface protein expression increased with increased stiffness. Focal adhesion kinase was required for maximal uptake of oxLDL, but not of acLDL. Uptake of oxLDL and acLDL was independent of rho-associated coiled coil kinase. Through pharmacologic inhibition and genetic deletion, we found that transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive ion channel, plays an inhibitory role in the uptake of acLDL, but not oxLDL. Together, these results implicate mechanical signaling in the uptake of acLDL and oxLDL, opening up the possibility of new pharmacologic targets to modulate lipid uptake by macrophages in vivo.


Asunto(s)
Lipoproteínas LDL/metabolismo , Macrófagos/patología , Especies Reactivas de Oxígeno/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Transporte Biológico , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Propiedades de Superficie
4.
Sci Adv ; 5(3): eaav9788, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30944865

RESUMEN

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 TLR5 -/- mice diminish the germinal center immune response induced by PLGA nanovaccines. The PLGA nanovaccines, unexpectedly, further changed gut microbiota. By chronically treating mice with antibiotics, we show that disrupting gut microbiome leads to poor vaccine response in an obesity-independent manner. We next demonstrate that the low immune response can be rescued by an immunomodulatory Pyr-pHEMA nanogel vaccine, which functions through TLR2 stimulation, enhanced trafficking, and induced stronger germinal center response than alum-supplemented PLGA nanovaccines. The study highlights the potential for immunomodulation under gut-mediated metabolic syndrome conditions using advanced nanomaterials.


Asunto(s)
Modelos Animales de Enfermedad , Microbioma Gastrointestinal/efectos de los fármacos , Inmunidad/efectos de los fármacos , Síndrome Metabólico/prevención & control , Nanogeles , Polietilenglicoles , Polietileneimina , Vacunas/administración & dosificación , Análisis de Varianza , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Materiales Biocompatibles/administración & dosificación , Microbioma Gastrointestinal/inmunología , Inmunidad/inmunología , Factores Inmunológicos/administración & dosificación , Factores Inmunológicos/inmunología , Síndrome Metabólico/inmunología , Ratones Noqueados , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 5/genética , Receptor Toll-Like 5/inmunología , Receptor Toll-Like 5/metabolismo , Vacunas/inmunología
5.
Br J Cancer ; 120(2): 207-217, 2019 01.
Artículo en Inglés | MEDLINE | ID: mdl-30518816

RESUMEN

BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.


Asunto(s)
Alarminas/genética , Carcinoma Epitelial de Ovario/genética , ADN Mitocondrial/genética , Trampas Extracelulares/genética , Anciano , Ascitis/genética , Ascitis/patología , Plaquetas/metabolismo , Carcinoma Epitelial de Ovario/patología , Trampas Extracelulares/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Humanos , Elastasa de Leucocito/genética , Persona de Mediana Edad , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neutrófilos/metabolismo , Neutrófilos/patología , Supervivencia sin Progresión , Microambiente Tumoral/genética
6.
Bone Marrow Transplant ; 53(12): 1508-1517, 2018 12.
Artículo en Inglés | MEDLINE | ID: mdl-29795424

RESUMEN

Successful and sustained CD4+ T-cell reconstitution is associated with increased survival after hematopoietic cell transplantation (HCT), but opportunistic infections may adversely affect the time and extent of immune reconstitution. Human herpesvirus 6B (HHV-6B) efficiently infects CD4+ T cells and utilizes as a receptor CD134 (OX40), a member of the TNF superfamily that antagonizes regulatory T-cell (Treg) activity. Reactivation of HHV-6B has been associated with aberrant immune reconstitution and acute graft-versus-host disease (aGVHD) after HCT. Given that Treg counts are negatively correlated with aGVHD severity, we postulate that one mechanism for the poor CD4+ T-cell reconstitution observed shortly after transplant may be HHV-6B infection and depletion of peripheral (extra-thymic) CD4+ T cells, including a subpopulation of Treg cells. In turn, this may trigger a series of adverse events resulting in poor clinical outcomes such as severe aGVHD. In addition, recent evidence has linked HHV-6B reactivation with aberrant CD4+ T-cell reconstitution late after transplantation, which may be mediated by a different mechanism, possibly related to central (thymic) suppression of T-cell reconstitution. These observations suggest that aggressive management of HHV-6B reactivation in transplant patients may facilitate CD4+ T-cell reconstitution and improve the quality of life and survival of HCT patients.


Asunto(s)
Enfermedad Injerto contra Huésped/etiología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 6/patogenicidad , Linfocitos T Reguladores/metabolismo , Acondicionamiento Pretrasplante/efectos adversos , Enfermedad Injerto contra Huésped/patología , Trasplante de Células Madre Hematopoyéticas/métodos , Humanos , Calidad de Vida , Acondicionamiento Pretrasplante/métodos
7.
Int Immunol ; 30(6): 267-278, 2018 05 24.
Artículo en Inglés | MEDLINE | ID: mdl-29800294

RESUMEN

Macrophages participate in immunity, tissue repair and tissue homeostasis. Activation of Toll-like receptors (TLRs) by conserved exogenous or endogenous structures initiates signaling cascades that result in the release of cytokines such as tumor necrosis factor α (TNFα). Extracellular substrate stiffness is known to regulate functions of non-immune cells through a process called mechanotransduction, yet less is known about how physical cues affect macrophage function or TLR signaling. To investigate this question, we cultured murine primary bone marrow-derived macrophages (BMMs) and RAW264.7 cells on fibronectin-coated polyacrylamide (PA) gels of defined stiffnesses (1, 20 and 150 kPa) that approximate the physical properties of physiologic tissues. BMMs on all gels were smaller and more circular than those on rigid glass. Macrophages on intermediate stiffness 20 kPa PA gels were slightly larger and less circular than those on either 1 or 150 kPa. Secretion of the pro-inflammatory cytokine, TNFα, in response to stimulation of TLR4 and TLR9 was increased in macrophages grown on soft gels versus more rigid gels, particularly for BMMs. Inhibition of the rho-associated coiled-coil kinase 1/2 (ROCK1/2), key mediators in cell contractility and mechanotransduction, enhanced release of TNFα in response to stimulation of TLR4. ROCK1/2 inhibition enhanced phosphorylation of the TLR downstream signaling molecules, p38, ERK1/2 and NFκB. Our data indicate that physical cues from the extracellular environment regulate macrophage morphology and TLR signaling. These findings have important implications in the regulation of macrophage function in diseased tissues and offer a novel pharmacological target for the manipulation of macrophage function in vivo.


Asunto(s)
Macrófagos/enzimología , Macrófagos/inmunología , Mecanotransducción Celular/inmunología , Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Quinasas Asociadas a rho/metabolismo , Resinas Acrílicas/farmacología , Animales , Supervivencia Celular/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Células RAW 264.7 , Receptores Toll-Like/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Quinasas Asociadas a rho/antagonistas & inhibidores
8.
J Biomed Mater Res A ; 105(8): 2109-2118, 2017 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-28263432

RESUMEN

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. The relative effects of specific cellular components upon the inflammatory response are not known. The objective of the present study was to determine the effect of different cell remnants that may be present in ECM scaffold materials upon the host innate immune response, both in vitro and in vivo. Collagen scaffolds were supplemented with one of three different concentrations of DNA, mitochondria, or cell membranes. Murine macrophages were exposed to the various supplemented scaffolds and the effect upon macrophage phenotype was evaluated. In vivo studies were performed using an abdominal wall defect model in the rat to evaluate the effect of the scaffolds upon the macrophage response. Murine macrophages exposed in vitro to scaffolds supplemented with DNA, mitochondria, and cell membranes showed increased expression of proinflammatory M1 marker iNOS and no expression of the proremodeling M2 marker Fizz1 regardless of supplementation concentration. A dose-dependent response was observed in the rat model for collagen scaffolds supplemented with cell remnants. DNA, mitochondria, and cell membrane remnants in collagen scaffolds promote a proinflammatory M1 macrophage phenotype in vivo and in vitro. These results reinforce the importance of a thorough decellularization process for ECM biologic scaffold materials. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2109-2118, 2017.


Asunto(s)
Matriz Extracelular/química , Matriz Extracelular/inmunología , Inmunidad Innata , Macrófagos/inmunología , Andamios del Tejido/efectos adversos , Andamios del Tejido/química , Animales , Materiales Biocompatibles/efectos adversos , Materiales Biocompatibles/química , Células Cultivadas , Colágeno/efectos adversos , Colágeno/química , Colágeno/inmunología , ADN/efectos adversos , ADN/química , ADN/inmunología , Inflamación/etiología , Inflamación/inmunología , Macrófagos/citología , Ensayo de Materiales , Ratones Endogámicos C57BL , Mitocondrias/química , Mitocondrias/inmunología , Ingeniería de Tejidos
9.
Semin Immunol ; 29: 41-48, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-28214177

RESUMEN

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. Dendritic cells (DCs) also directly interact with biomaterials, are critical for specific immune responses, and can be activated in response to interactions with biomaterials. Yet, much less is known about the responses by DCs. This review discusses what we know about DC response to biomaterials, the underlying mechanisms involved, and how DCs can be influenced by the macrophage response to biomaterials. Lastly, I will discuss how biomaterials can be manipulated to enhance or suppress DC function to promote a specific desirable immune response - a major goal for implantable biologically active therapeutics.


Asunto(s)
Materiales Biocompatibles/metabolismo , Células Dendríticas/inmunología , Macrófagos/inmunología , Ingeniería de Tejidos , Andamios del Tejido , Animales , Humanos , Inmunidad Celular , Inmunomodulación , Medicina Regenerativa
10.
Mol Ther ; 25(4): 989-1002, 2017 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-28215994

RESUMEN

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. The rOMVs are derived from a genetically engineered B strain of E. coli, ClearColi, which produces lipid IVa, and which was further engineered in our laboratory to hypervesiculate and make rOMVs. We report that rOMVs derived from this lipid IVa strain have substantially attenuated pyrogenicity yet retain high levels of immunogenicity, promote dendritic cell maturation, and generate a balanced Th1/Th2 humoral response. Additionally, an influenza A virus matrix 2 protein-based antigen displayed on these rOMVs resulted in 100% survival against a lethal challenge with two influenza A virus strains (H1N1 and H3N2) in mice with different genetic backgrounds (BALB/c, C57BL/6, and DBA/2J). Additionally, a two-log reduction of lung viral titer was achieved in a ferret model of influenza infection with human pandemic H1N1. The rOMVs reported herein represent a potentially safe and simple subunit vaccine delivery platform.


Asunto(s)
Escherichia coli/inmunología , Vesículas Extracelulares/inmunología , Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/genética , Antígenos Virales/inmunología , Diferenciación Celular , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Modelos Animales de Enfermedad , Escherichia coli/metabolismo , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/ultraestructura , Inmunoglobulina G , Ratones , Ratones Endogámicos BALB C , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/metabolismo , Receptor Toll-Like 2/inmunología , Receptor Toll-Like 2/metabolismo
11.
J Immunol ; 197(4): 1343-52, 2016 08 15.
Artículo en Inglés | MEDLINE | ID: mdl-27421483

RESUMEN

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. Thus, there are at least two independent cleavage events that regulate TLR9. In this study, we investigated whether an N-terminal fragment of TLR9 could be responsible for regulation of the mature or negative-regulatory form. We show that TLR9(471-1032), corresponding to the proteolytically cleaved form, does not function on its own. Furthermore, activity is not rescued by coexpression of the N-terminal fragment (TLR9(1-440)), inclusion of the hinge region (TLR9(441-1032)), or overexpression of UNC93B1, the last of which is critical for trafficking and cleavage of TLR9. TLR9(1-440) coimmunoprecipitates with full-length TLR9 and TLR9(471-1032) but does not rescue the native glycosylation pattern; thus, inappropriate trafficking likely explains why TLR9(471-1032) is nonfunctional. Lastly, we show that TLR9(471-1032) is also a dominant-negative regulator of TLR9 signaling. Together, these data provide a new perspective on the complexity of TLR9 regulation by proteolytic cleavage and offer potential ways to inhibit activity through this receptor, which may dampen autoimmune inflammation.


Asunto(s)
Receptor Toll-Like 9/inmunología , Receptor Toll-Like 9/metabolismo , Animales , Western Blotting , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Inmunoprecipitación , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Reacción en Cadena de la Polimerasa , Proteolisis
12.
J Leukoc Biol ; 100(5): 927-941, 2016 11.
Artículo en Inglés | MEDLINE | ID: mdl-27343013

RESUMEN

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. This review summarizes the regulatory mechanisms of TLRs, including regulation of their access to ligands, receptor folding, intracellular trafficking, and post-translational modifications, as well as how altered control mechanism could contribute to inflammatory and autoimmune disorders.


Asunto(s)
Transducción de Señal/inmunología , Receptores Toll-Like/inmunología , Inmunidad Adaptativa , Alarminas/inmunología , Animales , Dimerización , Endosomas/inmunología , Glicosilación , Humanos , Inmunidad Innata , Infecciones/inmunología , Ligandos , Ácidos Nucleicos/inmunología , Fosforilación , Unión Proteica , Dominios Proteicos , Pliegue de Proteína , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Relación Estructura-Actividad , Receptores Toll-Like/ultraestructura , Ubiquitinación
13.
J Leukoc Biol ; 100(3): 525-33, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26957214

RESUMEN

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. We demonstrated that tyrosine phosphorylation is essential for mouse Toll-like receptor-9 protein stability and signaling. Upon inhibition of tyrosine kinases with piceatannol, Toll-like receptor-9 tyrosine phosphorylation induced by CpG deoxyribonucleic acid was inhibited, which correlated with decreased signaling. Furthermore, inhibition of Src kinases with 1-tert-Butyl-3-(4-chlorophenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine also inhibited response to CpG deoxyribonucleic acid. Toll-like receptor-9 protein stability was also sensitive to autophagy, the cellular stress response pathway, and infection by a deoxyribonucleic acid virus. Whereas autophagy induced by rapamycin or low serum levels caused a preferential loss of the mature p80 proteolytic cleavage product, infection with herpes simplex virus-1 and induction of cell stress with tunicamycin caused preferential loss of full-length Toll-like receptor-9, which is localized to the endoplasmic reticulum. Our data reveal new information about the stability and signaling of Toll-like receptor-9 and suggest that immune evasion mechanisms may involve targeted loss of innate sensing receptors.


Asunto(s)
Estrés del Retículo Endoplásmico , Procesamiento Proteico-Postraduccional , Receptor Toll-Like 9/química , Receptor Toll-Like 9/fisiología , Tirosina/metabolismo , Animales , Ratones , Ratones Noqueados , Fosforilación , Estabilidad Proteica , Proteolisis , Transducción de Señal
14.
Vaccine ; 34(10): 1252-8, 2016 Mar 04.
Artículo en Inglés | MEDLINE | ID: mdl-26827663

RESUMEN

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. Here, we engineer three M2e-OMV vaccines and show that all elicit strong IgG titers, with high IgG2a:IgG1 ratios, in BALB/c mice. Additionally, the administration of one M2e-OMV construct containing tandem heterologous M2e peptides (M2e4xHet-OMV) resulted in 100% survival against lethal doses of the mouse-adapted H1N1 influenza strain PR8. Passive transfer of antibodies from M2e4xHet-OMV vaccinated mice to unvaccinated mice also resulted in 100% survival to challenge, indicating that protection is driven largely via antibody-mediated immunity. The potential mechanism through which M2e-OMVs initiated the immune response was explored and it was found that the constructs triggered TLR1/2, TLR4, and TLR5. Our data indicate that OMVs have potential as a platform for influenza A vaccine development due to their unique adjuvant profile and intrinsic pathogen-mimetic nature.


Asunto(s)
Vesículas Extracelulares/inmunología , Vacunas contra la Influenza/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Proteínas de la Matriz Viral/inmunología , Animales , Anticuerpos Antivirales/sangre , Escherichia coli/metabolismo , Femenino , Inmunidad Innata , Inmunoglobulina G/sangre , Subtipo H1N1 del Virus de la Influenza A , Ratones Endogámicos BALB C , Nanopartículas , Receptores Toll-Like/agonistas , Vacunas Sintéticas/inmunología
15.
J Med Chem ; 58(9): 3922-43, 2015 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-25901531

RESUMEN

Grp94 is involved in the regulation of a restricted number of proteins and represents a potential target in a host of diseases, including cancer, septic shock, autoimmune diseases, chronic inflammatory conditions, diabetes, coronary thrombosis, and stroke. We have recently identified a novel allosteric pocket located in the Grp94 N-terminal binding site that can be used to design ligands with a 2-log selectivity over the other Hsp90 paralogs. Here we perform extensive SAR investigations in this ligand series and rationalize the affinity and paralog selectivity of choice derivatives by molecular modeling. We then use this to design 18c, a derivative with good potency for Grp94 (IC50 = 0.22 µM) and selectivity over other paralogs (>100- and 33-fold for Hsp90α/ß and Trap-1, respectively). The paralog selectivity and target-mediated activity of 18c was confirmed in cells through several functional readouts. Compound 18c was also inert when tested against a large panel of kinases. We show that 18c has biological activity in several cellular models of inflammation and cancer and also present here for the first time the in vivo profile of a Grp94 inhibitor.


Asunto(s)
Adenina/análogos & derivados , Retículo Endoplásmico/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Glicoproteínas de Membrana/antagonistas & inhibidores , Purinas/química , Adenina/química , Adenina/farmacocinética , Adenina/farmacología , Sitio Alostérico , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Diferenciación Celular , Línea Celular , Línea Celular Tumoral , Femenino , Factor II del Crecimiento Similar a la Insulina/metabolismo , Ligandos , Glicoproteínas de Membrana/metabolismo , Ratones Desnudos , Simulación del Acoplamiento Molecular , Mioblastos/citología , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Unión Proteica , Purinas/farmacocinética , Purinas/farmacología , Receptor ErbB-2/metabolismo , Relación Estructura-Actividad , Distribución Tisular , Receptor Toll-Like 9/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesis
16.
Biomacromolecules ; 16(2): 564-77, 2015 Feb 09.
Artículo en Inglés | MEDLINE | ID: mdl-25531946

RESUMEN

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 0.772-22.08 µm in diameter, whereas the uncrosslinked microparticles formed a core with an outer petal-like structure of 2.49-15 µm in diameter. It was discovered that the morphological structure of the self-assembled microparticles had a profound influence on their biological properties. At a 1:1 feed ratio of UArg-PEA to HA, the uncrosslinked microparticles showed no cytotoxicity toward NIH 3T3 fibroblasts at concentrations up to 20 µg/mL, and the crosslinked particles exhibited no cytotoxicity at concentrations up to 10 µg/mL. The UArg-PEA/HA hybrid microparticles exhibited a significantly lower macrophage-induced proinflammatory response (via TNF-α) than that from a pure hyaluronic acid control while retaining the beneficial anti-inflammatory IL-10 production by HA. The UArg-PEA/HA microparticles also stimulated size-dependent induction of arginase activity. Therefore, self-assembling these two types of biomaterials in a favorable nontoxic aqueous environment, having complementary biological properties like those of the currently reported UArg-PEA/HA hybrid microparticles, may provide a new class of biomaterials to improve the overall tissue microenvironment for promoting wound healing.


Asunto(s)
Materiales Biocompatibles/química , Ácido Hialurónico/química , Polisacáridos/química , Electricidad Estática , Animales , Materiales Biocompatibles/metabolismo , Ácido Hialurónico/metabolismo , Macrófagos/metabolismo , Ratones , Células 3T3 NIH , Polisacáridos/metabolismo , Propiedades de Superficie , Viscosidad
17.
PLoS One ; 9(11): e112802, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25426709

RESUMEN

Recombinant subunit vaccine engineering increasingly focuses on the development of more effective delivery platforms. However, current recombinant vaccines fail to sufficiently stimulate protective adaptive immunity against a wide range of pathogens while remaining a cost effective solution to global health challenges. Taking an unorthodox approach to this fundamental immunological challenge, we isolated the TLR-targeting capability of the probiotic E. coli Nissle 1917 bacteria (EcN) by engineering bionanoparticlate antigen carriers derived from EcN outer membrane vesicles (OMVs). Exogenous model antigens expressed by these modified bacteria as protein fusions with the bacterial enterotoxin ClyA resulted in their display on the surface of the carrier OMVs. Vaccination with the engineered EcN OMVs in a BALB/c mouse model, and subsequent mechanism of action analysis, established the EcN OMV's ability to induce self-adjuvanted robust and protective humoral and T(H)1-biased cellular immunity to model antigens. This finding appears to be strain-dependent, as OMV antigen carriers similarly engineered from a standard K12 E. coli strain derivative failed to generate a comparably robust antigen-specific TH1 bias. The results demonstrate that unlike traditional subunit vaccines, these biomolecularly engineered "pathogen-like particles" derived from traditionally overlooked, naturally potent immunomodulators have the potential to effectively couple recombinant antigens with meaningful immunity in a broadly applicable fashion.


Asunto(s)
Anticuerpos Antibacterianos/biosíntesis , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Escherichia coli/inmunología , Células TH1/inmunología , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/genética , Vacunas Bacterianas/administración & dosificación , Vacunas Bacterianas/genética , Membrana Celular/química , Membrana Celular/inmunología , Escherichia coli/química , Proteínas de Escherichia coli/administración & dosificación , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/inmunología , Femenino , Expresión Génica , Proteínas Hemolisinas/administración & dosificación , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/inmunología , Inmunidad Celular/efectos de los fármacos , Inmunidad Humoral/efectos de los fármacos , Inmunización , Ratones , Ratones Endogámicos BALB C , Probióticos/química , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Especificidad de la Especie , Células TH1/citología , Vacunas de Subunidad , Vacunas Sintéticas
18.
Immunol Lett ; 162(2 Pt A): 3-9, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25284610

RESUMEN

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. A variety of human TLR5 gene alleles exist that also associated with inflammatory conditions and may do so via effects on the gut microbiome and altered host-microbial crosstalk.


Asunto(s)
Flagelina/metabolismo , Inflamación/inmunología , Mucosa Intestinal/inmunología , Microbiota , Receptor Toll-Like 5/metabolismo , Inmunidad Adaptativa , Animales , Formación de Anticuerpos/genética , Flagelina/genética , Flagelina/inmunología , Regulación Bacteriana de la Expresión Génica/genética , Predisposición Genética a la Enfermedad , Variación Genética , Humanos , Inmunidad Innata , Inflamación/genética , Inflamación/microbiología , Mucosa Intestinal/microbiología , Ratones , Ratones Noqueados , Receptor Toll-Like 5/genética
19.
PLoS One ; 8(3): e58513, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23554897

RESUMEN

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.g., TNF, IL-1ß, IL-6, IL-12, and IFN-γ) within the first ~72 h. This in vivo finding is discordant with the principally TH1-oriented response to Ft frequently observed in cell-based studies wherein the aforementioned cytokines are produced. An often overlooked confounding factor in the interpretation of experimental results is the influence of environmental cues on the bacterium's capacity to elicit certain host responses. Herein, we reveal that adaptation of Ft to its mammalian host imparts an inability to elicit select pro-inflammatory mediators throughout the course of infection. Furthermore, in vitro findings that non-host adapted Ft elicits such a response from host cells reflect aberrant recognition of the DNA of structurally-compromised bacteria by AIM2-dependent and -independent host cell cytosolic DNA sensors. Growth of Ft in Muller-Hinton Broth or on Muller-Hinton-based chocolate agar plates or genetic mutation of Ft was found to compromise the structural integrity of the bacterium thus rendering it capable of aberrantly eliciting pro-inflammatory mediators (e.g., TNF, IL-1ß, IL-6, IL-12, and IFN-γ). Our studies highlight the profound impact of different growth conditions on host cell response to infection and demonstrate that not all in vitro-derived findings may be relevant to tularemia pathogenesis in the mammalian host. Rational development of a vaccine and immunotherapeutics can only proceed from a foundation of knowledge based upon in vitro findings that recapitulate those observed during natural infection.


Asunto(s)
Citocinas/inmunología , Francisella tularensis/inmunología , Infecciones del Sistema Respiratorio/inmunología , Tularemia/inmunología , Animales , Vacunas Bacterianas/inmunología , Vacunas Bacterianas/farmacología , Francisella tularensis/patogenicidad , Humanos , Inflamación/inmunología , Inflamación/microbiología , Inflamación/patología , Ratones , Ratones Noqueados , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/patología , Tularemia/microbiología , Tularemia/patología , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/farmacología
20.
Infect Immun ; 81(4): 1354-63, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23403558

RESUMEN

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. A direct role for PAMPs in TLR activation was not supported in a transactivation assay involving a panel of murine and human TLRs. Mice deficient in the IL-1 family receptor for the DAMP, IL-33 (called ST2), displayed reduced intestinal Th2 responses and impaired mast cell activation. IL-33 was constitutively expressed in intestinal epithelial cells, where it became concentrated in nuclei within 2 days of infection. Nuclear localization was an innate response to infection that occurred in intestinal regions where worms were actively migrating. Th2 responses were also compromised in the lymph nodes draining the skeletal muscles of ST2-deficient mice, and this correlated with increased larval burdens in muscle. Our results support a mechanism in which the immune system recognizes and responds to tissue injury in a way that promotes Th2 responses.


Asunto(s)
Interleucinas/metabolismo , Factor 88 de Diferenciación Mieloide/metabolismo , Células Th2/inmunología , Trichinella spiralis/inmunología , Animales , Interleucina-33 , Interleucinas/inmunología , Mucosa Intestinal/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/inmunología
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