RESUMEN
We previously demonstrated that the combination of synthetic small-molecule Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic murine challenge models. Combining the TLR4 and TLR7 ligands balances Th1 and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. Here, we demonstrate that the protective response induced in mice by this combined adjuvant is dependent upon TLR4 and TLR7 signaling via myeloid differentiation primary response gene 88 (MyD88), indicating that the adjuvants function in vivo via their known receptors, with negligible off-target effects, to induce protective immunity. The combined adjuvant acts via MyD88 in both bone marrow-derived and non-bone marrow-derived radioresistant cells to induce hemagglutinin-specific antibodies and protect mice against influenza virus challenge. The protective efficacy generated by immunization with this adjuvant and recombinant hemagglutinin antigen is transferable with serum from immunized mice to recipient mice in a homologous, but not a heterologous, H1N1 viral challenge model. Depletion of CD4+ cells after an established humoral response in immunized mice does not impair protection from a homologous challenge; however, it does significantly impair recovery from a heterologous challenge virus, highlighting an important role for vaccine-induced CD4+ cells in cross-protective vaccine efficacy. The combination of the two TLR agonists allows for significant dose reductions of each component to achieve a level of protection equivalent to that afforded by either single agent at its full dose.IMPORTANCE Development of novel adjuvants is needed to enhance immunogenicity to provide better protection from seasonal influenza virus infection and improve pandemic preparedness. We show here that several dose combinations of synthetic TLR4 and TLR7 ligands are potent adjuvants for recombinant influenza virus hemagglutinin antigen induction of humoral and cellular immunity against viral challenges. The components of the combined adjuvant work additively to enable both antigen and adjuvant dose sparing while retaining efficacy. Understanding an adjuvant's mechanism of action is a critical component for preclinical safety evaluation, and we demonstrate here that a combined TLR4 and TLR7 adjuvant signals via the appropriate receptors and the MyD88 adaptor protein. This novel adjuvant combination contributes to a more broadly protective vaccine while demonstrating an attractive safety profile.
Asunto(s)
Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Glicoproteínas de Membrana/inmunología , Factor 88 de Diferenciación Mieloide/inmunología , Infecciones por Orthomyxoviridae/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 7/inmunología , Proteínas Adaptadoras del Transporte Vesicular/genética , Adyuvantes Inmunológicos/farmacología , Animales , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Femenino , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Vacunas contra la Influenza/inmunología , Pulmón/inmunología , Pulmón/virología , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/virología , Células TH1/inmunología , Células Th2/inmunología , Receptor Toll-Like 4/genética , Receptor Toll-Like 7/genética , VacunaciónRESUMEN
UNLABELLED: Current vaccines against influenza virus infection rely on the induction of neutralizing antibodies targeting the globular head of the viral hemagglutinin (HA). Protection against seasonal antigenic drift or sporadic pandemic outbreaks requires further vaccine development to induce cross-protective humoral responses, potentially to the more conserved HA stalk region. Here, we present a novel viral vaccine adjuvant comprised of two synthetic ligands for Toll-like receptor 4 (TLR4) and TLR7. 1Z105 is a substituted pyrimido[5,4-b]indole specific for the TLR4-MD2 complex, and 1V270 is a phospholipid-conjugated TLR7 agonist. Separately, 1Z105 induces rapid Th2-associated IgG1 responses, and 1V270 potently generates Th1 cellular immunity. 1Z105 and 1V270 in combination with recombinant HA from the A/Puerto Rico/8/1934 strain (rPR/8 HA) effectively induces rapid and sustained humoral immunity that is protective against lethal challenge with a homologous virus. More importantly, immunization with the combined adjuvant and rPR/8 HA, a commercially available split vaccine, or chimeric rHA antigens significantly improves protection against both heterologous and heterosubtypic challenge viruses. Heterosubtypic protection is associated with broadly reactive antibodies to HA stalk epitopes. Histological examination and cytokine profiling reveal that intramuscular (i.m.) administration of 1Z105 and 1V270 is less reactogenic than a squalene-based adjuvant, AddaVax. In summary, the combination of 1Z105 and 1V270 with a recombinant HA induces rapid, long-lasting, and balanced Th1- and Th2-type immunity; demonstrates efficacy in a variety of murine influenza virus vaccine models assaying homologous, heterologous, and heterosubtypic challenge viruses; and has an excellent safety profile. IMPORTANCE: Novel adjuvants are needed to enhance immunogenicity and increase the protective breadth of influenza virus vaccines to reduce the seasonal disease burden and ensure pandemic preparedness. We show here that the combination of synthetic Toll-like receptor 4 (TLR4) and TLR7 ligands is a potent adjuvant for recombinant influenza virus hemagglutinin, inducing rapid and sustained immunity that is protective against influenza viruses in homologous, heterologous, and heterosubtypic challenge models. Combining TLR4 and TLR7 ligands balances Th1- and Th2-type immune responses for long-lived cellular and neutralizing humoral immunity against the viral hemagglutinin. The combined adjuvant has an attractive safety profile and the potential to augment seasonal-vaccine breadth, contribute to a broadly neutralizing universal vaccine formulation, and improve response time in an emerging pandemic.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Protección Cruzada , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/inmunología , Gripe Humana/inmunología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 7/agonistas , Adyuvantes Inmunológicos/síntesis química , Animales , Anticuerpos Antivirales/inmunología , Humanos , Vacunas contra la Influenza/administración & dosificación , Gripe Humana/virología , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Células TH1/inmunología , Células Th2/inmunología , Receptor Toll-Like 4/inmunología , Receptor Toll-Like 7/inmunologíaRESUMEN
Toll-like receptors (TLRs) in the innate immune system recognize specific pathogen-associated molecular patterns derived from microbes. Synthetic small molecule TLR7 agonists have been extensively evaluated as topical agents for antiviral and anticancer therapy, and as adjuvants for vaccine. However, safe and reproducible administration of synthetic TLR7 ligands has been difficult to achieve due to undesirable pharmacokinetics and unacceptable side effects. Here, we conjugated a versatile low molecular weight TLR7 ligand to various polysaccharides in order to improve its water solubility, enhance its potency, and maintain low toxicity. The synthetic TLR7 ligand, 2-methoxyethoxy-8-oxo-9-(4-carboxy benzyl)adenine, designated 1V209, was stably conjugated to primary amine functionalized Ficoll or dextran using benzoic acid functional groups. The conjugation ratios using specified equivalents of TLR7 ligand were dose responsive and reproducible. The zeta potential value of the polysaccharides was decreased in inverse proportion to the ratio of conjugated TLR7 ligand. These conjugates were highly water-soluble, stable for at least 6 months at room temperature in aqueous solution, and easy to lyophilize and reconstitute without altering potency. In vitro studies with murine mononuclear leukocytes showed that the TLR7 agonist conjugated to polysaccharides had 10- to 1000-fold higher potencies than the unconjugated TLR7 ligand. In vivo pharmacodynamics studies after injection indicate that the conjugates induced systemic cytokine production. When the conjugates were used as vaccine adjuvants, they enhanced antigen specific humoral and cellular immune responses to a much greater extent than did unconjugated TLR7 ligands. These results indicated that small molecule TLR7 ligands conjugated to polysaccharides have improved immunostimulatory potency and pharmacodynamics. Polysaccharides can be conjugated to a variety of molecules such as antigens, peptides, and TLR ligands. Therefore, such conjugates could represent a versatile platform for the development of vaccines against cancer and infectious diseases.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Adyuvantes Inmunológicos/química , Células Dendríticas/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Polisacáridos/química , Receptor Toll-Like 7/fisiología , Animales , Médula Ósea/efectos de los fármacos , Médula Ósea/inmunología , Células Cultivadas , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/citología , Células Dendríticas/efectos de los fármacos , Femenino , Humanos , Inmunización , Inflamación/tratamiento farmacológico , Inflamación/patología , Ligandos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Toll-like receptor (TLR) stimulation has been implicated as a major contributor to chronic inflammation. Among these receptors, TLR4 has been described as a key regulator of endogenous inflammation and has been proposed as a therapeutic target. Previously, we discovered by high-throughput screening a group of substituted pyrimido[5,4-b]indoles that activated a nuclear factor-κB reporter in THP-1 human monocytic cells. A biologically active hit compound was resynthesized, and derivatives were prepared to assess structure-activity relationships. The derived compounds activated cells in a TLR4/myeloid differentiation protein 2 (MD2)-dependent and CD14-independent manner, using the myeloid differentiation primary response 88 and Toll/IL-1 receptor domain-containing adapter-inducing interferon-ß pathways. Two lead compounds, 1Z105 and 1Z88, were selected for further analysis based on favorable biologic properties and lack of toxicity. In vivo pharmacokinetics indicated that 1Z105 was orally bioavailable, whereas 1Z88 was not. Oral or parenteral doses of 1Z105 and 1Z88 induced undetectable or negligible levels of circulating cytokines and did not induce hepatotoxicity when administered to galactosamine-conditioned mice, indicating good safety profiles. Both compounds were very effective in preventing lethal liver damage in lipopolysaccharide treated galatosamine-conditioned mice. Orally administered 1Z105 and parenteral 1Z88 prevented arthritis in an autoantibody-driven murine model. Hence, these low molecular weight molecules that target TLR4/MD2 were well tolerated and effective in reducing target organ damage in two different mouse models of sterile inflammation.
Asunto(s)
Inflamación/tratamiento farmacológico , Antígeno 96 de los Linfocitos/fisiología , Receptor Toll-Like 4/fisiología , Animales , Artritis Experimental/prevención & control , Galactosamina/toxicidad , Células Hep G2 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ligandos , Receptores de Lipopolisacáridos/fisiología , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/fisiología , Transducción de Señal , Relación Estructura-ActividadRESUMEN
The Toll-like receptors (TLRs) are critical components of the innate immune system that regulate immune recognition in part through NF-κB activation. A human cell-based high throughput screen (HTS) revealed substituted 4-aminoquinazolines to be small molecular weight activators of NF-κB. The most potent hit compound predominantly stimulated through the human TLR4/MD2 complex, and had less activity with the mouse TLR4/MD2. There was no activity with other TLRs and the TLR4 activation was MD-2 dependent and CD14 independent. Synthetic modifications of the quinazoline scaffold at the 2 and 4 positions revealed trends in structure-activity relationships with respect to TLR dependent production of the NF-κB associated cytokine IL-8 in human peripheral blood mononuclear cells, as well as IL-6 in mouse antigen presenting cells. Furthermore, the hit compound in this series also activated the interferon signaling pathway resulting in type I interferon production. Substitution at the O-phenyl moiety with groups such as bromine, chlorine and methyl resulted in enhanced immunological activity. Computational studies indicated that the 4-aminoquinazoline compounds bind primarily to human MD-2 in the TLR4/MD-2 complex. These small molecules, which preferentially stimulate human rather than mouse innate immune cells, may be useful as adjuvants or immunotherapeutic agents.
Asunto(s)
Interleucina-6/metabolismo , Interleucina-8/metabolismo , Leucocitos Mononucleares/metabolismo , FN-kappa B/metabolismo , Quinazolinas/química , Receptor Toll-Like 4/metabolismo , Receptores Toll-Like/metabolismo , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunidad Innata , Leucocitos Mononucleares/citología , Ligandos , Ratones , Modelos Moleculares , Estructura Molecular , Quinazolinas/metabolismo , Transducción de Señal , Relación Estructura-ActividadRESUMEN
Nitrogen-containing bisphosphonates (NBPs) are taken by millions for bone disorders but may cause serious inflammatory reactions. In this study, we used a murine peritonitis model to characterize the inflammatory mechanisms of these agents. At dosages comparable to those used in humans, injection of NBPs into the peritoneum caused recruitment of neutrophils, followed by an influx of monocytes. These cellular changes corresponded to an initial increase in IL-1α, which preceded a rise in multiple other proinflammatory cytokines. IL-1R, IL-1α, and IL-1ß were required for neutrophil recruitment, whereas other MyD88-dependent signaling pathways were needed for the monocyte influx. Mice deficient in mast cells, but not mice lacking lymphocytes, were resistant to NBP-induced inflammation, and reconstitution of these mice with mast cells restored sensitivity to NBPs. These results document the critical role of mast cells and IL-1 in NBP-mediated inflammatory reactions.
Asunto(s)
Alendronato/toxicidad , Difosfonatos/toxicidad , Imidazoles/toxicidad , Interleucina-1alfa/fisiología , Interleucina-1beta/fisiología , Mastocitos/fisiología , Peritonitis/inducido químicamente , Animales , Quimiotaxis/fisiología , Ácido Clodrónico/toxicidad , Interleucina-1alfa/deficiencia , Interleucina-1alfa/genética , Interleucina-1beta/deficiencia , Interleucina-1beta/genética , Leucocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/fisiología , Neutrófilos/inmunología , Pamidronato , Peritonitis/inmunología , Peritonitis/patología , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/fisiología , Ácido ZoledrónicoRESUMEN
Nitrogen bisphosphonates (NBPs) are commonly prescribed for osteoporosis but have also been found to induce inflammatory reactions and to delay the progression of breast cancer. The inflammatory and anticancer effects of the NBPs might be associated with an ability to modulate innate immune signaling. In mice, intraperitoneal NBP administration causes a rapid influx of neutrophils and monocytes that is dependent on the myeloid differentiation primary response gene 88 (MyD88) mediator of Toll-like receptor (TLR) and IL-1 signaling. Bone marrow chimeras demonstrate that this inflammatory response is partially dependent on TLR4 expression by hematopoietic cells and the IL-1 receptor on radioresistant cells. In vitro, NBPs directly stimulate neither murine bone marrow-derived mononuclear cells nor human peripheral blood mononuclear cells, but rather prime them to produce increased amounts of cytokines when exposed to IL-1 or TLR ligands. This potentiation is mediated by a reduction in IL-1 receptor-associated kinase-M, a negative regulator of MyD88-dependent signaling. In vivo, this property renders the NBPs as effective adjuvants that enhance both cellular and antibody responses to antigens.
Asunto(s)
Difosfonatos/farmacología , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/fisiología , Inflamación/etiología , Inflamación/metabolismo , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Inmunidad Adaptativa/efectos de los fármacos , Adyuvantes Inmunológicos/farmacología , Animales , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Difosfonatos/toxicidad , Humanos , Inflamación/inmunología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/metabolismo , Peritonitis/etiología , Peritonitis/inmunología , Peritonitis/metabolismo , Receptores de Interleucina-1/deficiencia , Receptores de Interleucina-1/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 4/deficiencia , Receptor Toll-Like 4/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic inflammatory disease marked by bone and cartilage destruction. Current biologic therapies are beneficial in only a portion of patients; hence small molecules targeting key pathogenic signaling cascades represent alternative therapeutic strategies. Here we show that c-Jun N-terminal kinase (JNK) 1, but not JNK2, is critical for joint swelling and destruction in a serum transfer model of arthritis. The proinflammatory function of JNK1 requires bone marrow-derived cells, particularly mast cells. Without JNK1, mast cells fail to degranulate efficiently and release less IL-1ß after stimulation via Fcγ receptors (FcγRs). Pharmacologic JNK inhibition effectively prevents arthritis onset and abrogates joint swelling in established disease. Hence, JNK1 controls mast cell degranulation and FcγR-triggered IL-1ß production, in addition to regulating cytokine and matrix metalloproteinase biosynthesis, and is an attractive therapeutic target in inflammatory arthritis.
Asunto(s)
Artritis/metabolismo , Degranulación de la Célula , Interleucina-1beta/biosíntesis , Mastocitos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Transducción de Señal , Animales , Artritis/genética , Artritis/inmunología , Artritis/patología , Células de la Médula Ósea/inmunología , Células de la Médula Ósea/metabolismo , Células de la Médula Ósea/patología , Colagenasas/biosíntesis , Colagenasas/genética , Colagenasas/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mastocitos/inmunología , Mastocitos/patología , Ratones , Ratones Noqueados , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/inmunología , Proteína Quinasa 9 Activada por Mitógenos/genética , Proteína Quinasa 9 Activada por Mitógenos/inmunología , Proteína Quinasa 9 Activada por Mitógenos/metabolismo , Receptores Fc/genética , Receptores Fc/inmunología , Receptores Fc/metabolismoRESUMEN
Although the mechanisms for sustained chemokine gradients and recurring cell infiltration in sterile peritonitis have not been elucidated, toll-like receptors (TLRs) have been implicated. To abate the deleterious recruitment of neutrophils in sterile inflammation, we repeatedly administered a TLR7 ligand that hyposensitized to TLR7 and receptors that converged on the MyD88-signaling intermediary and reduced cellular infiltration in murine autoimmune models of multiple sclerosis and arthritis. To reduce potential adverse effects, a polyethylene glycol polymer was covalently attached to the parent compound (Tolerimod1). The proinflammatory potency of Tolerimod1 was 10-fold less than the unconjugated TLR7 ligand, and Tolerimod1 reduced neutrophil recruitment in chemically induced peritonitis and colitis. The effects of Tolerimod1 were mediated by the radioresistant cells in radiation chimeric mice and by mast cells in reconstituted mast cell-deficient mice (Kit(W-sh)). Although the Tolerimod1 had weak proinflammatory agonist activity, it effectively reduced neutrophil recruitment in sterile peritoneal inflammation.
Asunto(s)
Mastocitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/inmunología , Polietilenglicoles/metabolismo , Polietilenglicoles/farmacología , Purinas/farmacología , Receptor Toll-Like 7/metabolismo , Animales , Autoinmunidad , Línea Celular , Inflamación/tratamiento farmacológico , Ligandos , Mastocitos/citología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/metabolismo , Neutrófilos/metabolismo , Peritonitis/inmunología , Permeabilidad , Peroxidasa/metabolismo , Polímeros/química , Factor de Células Madre/genéticaRESUMEN
Inflammatory arthritis is associated with the release of a network of key cytokines. In T cell receptor transgenic K/BxN mice interleukin (IL)-1 plays a key role in joint swelling and destruction, as suggested by the ability of anti-IL-1receptor (IL-1R) antibody treatment to delay the onset and slow the progression of this disease. This mechanism is dependent on the signaling pathway intermediary myeloid differentiation factor 88 (MyD88), such that neither IL-1R nor MyD88-deficient mice developed visually detectable synovitis after transfer of arthritogenic sera. The Toll-like receptors (TLRs) share the same signaling pathway through MyD88 as the IL-1R. The administration of a TLR-4 ligand, lipopolysaccharide, concomitant with arthritogenic serum in IL-1 receptor-deficient mice resulted in acute paw swelling, but not in MyD88-deficient mice. Also, serum transferred arthritis was not sustained in TLR-4 mutant mice compared with controls. These results suggest that innate immune functions via TLR-4 might perpetuate inflammatory mechanisms and bypass the need for IL-1 in chronic joint inflammation.
Asunto(s)
Artritis Reumatoide/etiología , Proteínas de Drosophila , Glicoproteínas de Membrana/fisiología , Receptores de Superficie Celular/fisiología , Receptores de Interleucina-1/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Antígenos de Diferenciación/fisiología , Artritis Reumatoide/sangre , Modelos Animales de Enfermedad , Interleucina-1/fisiología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/fisiología , Receptores Inmunológicos/fisiología , Receptor Toll-Like 4 , Receptores Toll-LikeRESUMEN
OBJECTIVE: To study the immune response caused by the intravesical administration of the immunomodulator R-837 in various formulations and to estimate its therapeutic potential for bladder cancer. METHODS: Female C57BL/6 mice were intravesically treated with different formulations of R-837, a Toll-like receptor 7 agonist used for treating genital warts and skin malignancy. The tested formulation mixtures contained different ratios of lactic acid, a thermosensitive poloxamer polymer (Lutrol F127) and 2-(hydroxypropyl)-beta-cyclodextrin (HPbetaCD). Induction of tumor necrosis factor alpha (TNFalpha) and keratinocyte-derived chemokine (KC) was analyzed by Luminex microbeads assay. The therapeutic potential of intravesical administration of R-837 was assessed in an orthotopic, syngeneic mouse model of bladder cancer using MB49 cells. RESULTS: Intravesical administration of R-837 in lactic acid alone induced systemic and bladder TNFalpha and KC in a dose-dependent manner. Formulations including poloxamer decreased systemic absorption of R-837 and significantly reduced systemic and local induction of KC. Addition of HPbetaCD in the poloxamer formulation particularly reversed levels of systemic and local levels of TNFalpha and KC. Histological examination showed that poloxamer-HPbetaCD formulation allowed infiltration of mononuclear cells into urothelium and lamina propria. In studies using orthotopic mouse bladder cancer, the tumor loads in R-837-treated mice were significantly lower than those in vehicle-treated or non-treated mice. CONCLUSION: The optimized poloxamer-HPbetaCD formulation of R-837 shows therapeutic potential for bladder cancer while avoiding adverse side-effects.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Aminoquinolinas/farmacología , Química Farmacéutica/métodos , Glicoproteínas de Membrana/agonistas , Receptor Toll-Like 7/agonistas , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , 2-Hidroxipropil-beta-Ciclodextrina , Administración Intravesical , Animales , Cistitis/prevención & control , Citocinas/metabolismo , Modelos Animales de Enfermedad , Excipientes/farmacología , Femenino , Imiquimod , Ácido Láctico/farmacología , Ratones , Ratones Endogámicos C57BL , Polietilenos/farmacología , Polipropilenos/farmacología , beta-Ciclodextrinas/farmacologíaRESUMEN
Autoimmune diabetes mellitus (DM) results from the destruction of pancreatic islet cells by activated T lymphocytes, which have been primed by activated dendritic cells (DC). Individualized therapy with ex vivo DC manipulation and reinfusion has been proposed as a treatment for DM, but this treatment is limited by cost, and requires specialized facilities. A means of in situ modulation of the DC phenotype in the host would be more accessible. Here we report a novel innate immune modulator, 1Z1, generated by conjugating a TLR7 ligand to six units of polyethylene glycol (PEG), which skews DC phenotype in vivo. 1Z1 was less potent in inducing cytokine production by DC than the parent ligand in vitro and in vivo. In addition, this drug only modestly increased DC surface expression of activation markers such as MHC class II, CD80, and CD86; however, the expression of negative regulatory molecules, such as programmed death ligand 1 (PD-L1), and interleukin-1 receptor-associated kinase M (IRAK-M) were markedly increased. In vivo transfer of 1Z1 treated DC into prediabetic NOD mice delayed pancreatic insulitis. Daily administration of 1Z1 effectively prevented the clinical onset of hyperglycemia and reduced histologic islet inflammation. Daily treatment with 1Z1 increased PD-L1 expression in the CD11c(+) population in peri-pancreatic lymph nodes; however, it did not induce an increase in regulatory T cells. Pharmaceutical modulation of DC maturation and function in situ, thus represents an opportunity to treat autoimmune disease.
Asunto(s)
Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Diabetes Mellitus Tipo 1/inmunología , Diabetes Mellitus Tipo 1/metabolismo , Tolerancia Inmunológica , Receptor Toll-Like 7/metabolismo , Traslado Adoptivo , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/terapia , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Inmunidad Innata , Inmunomodulación , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Fenotipo , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptor Toll-Like 7/genéticaRESUMEN
Observational data show that nonsteroidal anti-inflammatory drug (NSAID) use is associated with a lower rate of breast cancer. We evaluated the effect of etodolac, an FDA-approved NSAID reported to inhibit cyclooxygenase (COX) enzymes and the retinoid X receptor alpha (RXR), on rationally identified potential biomarkers in breast cancer. Patients with resectable breast cancer planned for initial management with surgical resection were enrolled and took 400 mg of etodolac twice daily prior to surgery. Protein and gene expression levels for genes related to COX-2 and RXRα were evaluated in tumor samples from before and after etodolac exposure. Thirty subjects received etodolac and 17 subjects were assayed as contemporaneous or opportunistic controls. After etodolac exposure mean cyclin D1 protein levels, assayed by immunohistochemistry, decreased (P = 0.03). Notably, pre- versus post cyclin D1 gene expression change went from positive to negative with greater duration of etodolac exposure (r = -0.64, P = 0.01). Additionally, etodolac exposure was associated with a significant increase in COX-2 gene expression levels (fold change: 3.25 [95% CI: 1.9, 5.55]) and a trend toward increased ß-catenin expression (fold change: 2.03 [95% CI: 0.93, 4.47]). In resectable breast cancer relatively brief exposure to the NSAID etodolac was associated with reduced cyclin D1 protein levels. Effect was also observed on cyclin D1 gene expression with decreasing levels with longer durations of drug exposure. Increased COX-2 gene expression was seen, possibly due to compensatory feedback. These data highlight the utility of even small clinical trials with access to biospecimens for pharmacodynamic studies.
Asunto(s)
Biomarcadores de Tumor/metabolismo , Neoplasias de la Mama/metabolismo , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Etodolaco/administración & dosificación , Administración Oral , Anciano , Biomarcadores de Tumor/genética , Neoplasias de la Mama/cirugía , Ciclina D1/genética , Ciclina D1/metabolismo , Ciclooxigenasa 2/genética , Inhibidores de la Ciclooxigenasa 2/farmacología , Etodolaco/farmacología , Femenino , Expresión Génica/efectos de los fármacos , Humanos , Persona de Mediana Edad , Periodo Preoperatorio , Receptor alfa X Retinoide/antagonistas & inhibidores , beta Catenina/genéticaRESUMEN
BACKGROUND: Synchronous tumors can be independent primary tumors or a primary-metastatic (clonal) pair, which may have clinical implications. Mutational profiling of tumor DNA is increasingly common in the clinic. We investigated whether mutational profiling can distinguish independent from clonal tumors in breast and other cancers, using a carefully defined test based on the Clonal Likelihood Score (CLS = 100 x # shared high confidence (HC) mutations/ # total HC mutations). METHODS: Statistical properties of a formal test using the CLS were investigated. A high CLS is evidence in favor of clonality; the test is implemented as a one-sided binomial test of proportions. Test parameters were empirically determined using 16,422 independent breast tumor pairs and 15 primary-metastatic tumor pairs from 10 cancer types using The Cancer Genome Atlas. RESULTS: We validated performance of the test with its established parameters, using five published data sets comprising 15,758 known independent tumor pairs (maximum CLS = 4.1%, minimum p-value = 0.48) and 283 known tumor clonal pairs (minimum CLS 13%, maximum p-value <0.01), across renal cell, testicular, and colorectal cancer. The CLS test correctly classified all validation samples but one, which it appears may have been incorrectly classified in the published data. As proof-of-concept we then applied the CLS test to two new cases of invasive synchronous bilateral breast cancer at our institution, each with one hormone receptor positive (ER+/PR+/HER2-) lobular and one triple negative ductal carcinoma. High confidence mutations were identified by exome sequencing and results were validated using deep targeted sequencing. The first tumor pair had CLS of 81% (p-value < 10-15), supporting clonality. In the second pair, no common mutations of 184 variants were validated (p-value >0.99), supporting independence. A plausible molecular mechanism for the shift from hormone receptor positive to triple negative was identified in the clonal pair. CONCLUSION: We have developed the statistical properties of a carefully defined Clonal Likelihood Score test from mutational profiling of tumor DNA. Under identified conditions, the test appears to reliably distinguish between synchronous tumors of clonal and of independent origin in several cancer types. This approach may have scientific and clinical utility.
Asunto(s)
Neoplasias de la Mama/diagnóstico , Mama/patología , Carcinoma Ductal de Mama/diagnóstico , Mutación , Metástasis de la Neoplasia/diagnóstico , Neoplasias Primarias Múltiples/diagnóstico , Anciano , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patología , Células Clonales , Análisis Mutacional de ADN , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Metástasis de la Neoplasia/genética , Metástasis de la Neoplasia/patología , Neoplasias Primarias Múltiples/genética , Neoplasias Primarias Múltiples/patologíaRESUMEN
Pulmonary administration of Toll-like receptor (TLR) ligands protects hosts from inhaled pathogens. However, systemic side effects induced by TLR stimulation limit clinical development. Here, a small-molecule TLR7 ligand conjugated with phospholipid, 1V270 (also designated TMX201), was tested for innate immune activation and its ability to prevent pulmonary infection in mice. We hypothesized that phospholipid conjugation would increase internalization by immune cells and localize the compound in the lungs, thus avoiding side effects due to systemic cytokine release. Pulmonary 1V270 administration increased innate cytokines and chemokines in bronchial alveolar lavage fluids, but neither caused systemic induction of cytokines nor B cell proliferation in distant lymphoid organs. 1V270 activated pulmonary CD11c+ dendritic cells, which migrated to local lymph nodes. However, there was minimal cell infiltration into the pulmonary parenchyma. Prophylactic administration of 1V270 significantly protected mice from lethal infection with Bacillus anthracis, Venezuelan equine encephalitis virus and H1N1 influenza virus. The maximum tolerated dose of 1V270 by pulmonary administration was 75 times the effective therapeutic dose. Therefore, pulmonary 1V270 treatment can protect the host from different infectious agents by stimulating local innate immune responses while exhibiting an excellent safety profile.
Asunto(s)
Adenina/análogos & derivados , Carbunco/tratamiento farmacológico , Bacillus anthracis/inmunología , Enfermedades Transmisibles/tratamiento farmacológico , Células Dendríticas/efectos de los fármacos , Virus de la Encefalitis Equina Venezolana/inmunología , Encefalomielitis Equina Venezolana/tratamiento farmacológico , Subtipo H1N1 del Virus de la Influenza A/inmunología , Gripe Humana/tratamiento farmacológico , Pulmón/efectos de los fármacos , Infecciones por Orthomyxoviridae/tratamiento farmacológico , Ácidos Fosfatidicos/efectos adversos , Fosfolípidos/administración & dosificación , Purinas/administración & dosificación , Receptor Toll-Like 7/agonistas , Adenina/administración & dosificación , Adenina/efectos adversos , Adenina/síntesis química , Administración Intranasal , Animales , Carbunco/inmunología , Líquido del Lavado Bronquioalveolar/inmunología , Enfermedades Transmisibles/inmunología , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Encefalomielitis Equina Venezolana/inmunología , Femenino , Humanos , Inmunidad Innata , Gripe Humana/inmunología , Inyecciones Espinales , Ligandos , Pulmón/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Infecciones por Orthomyxoviridae/inmunología , Ácidos Fosfatidicos/administración & dosificación , Ácidos Fosfatidicos/síntesis química , Fosfolípidos/efectos adversos , Fosfolípidos/síntesis química , Purinas/efectos adversos , Purinas/síntesis químicaRESUMEN
BACKGROUND: The Toll-like receptor 7 (TLR7) activator imiquimod (IMQ) is safe and effective in treating actinic keratosis; however, an intermittent treatment regimen is necessary because of excessive local reactions. OBJECTIVES: To evaluate in vitro potency, pharmacodynamics/pharmacokinetics, toxicity and efficacy in vivo of the newly developed TLR7 ligand-phospholipid conjugate, TMX-202, in a gel formulation. MATERIAL AND METHODS: The effects of TMX-202 were assessed both in vitro on a murine macrophage cell line and in primary bone marrow-derived dendritic cells and in vivo on mice (C57BL/6-wild type, Myd88(-/-) and Tlr7(-/-)). RESULTS: TMX-202 was more potent than IMQ in vitro using murine and human cells. In contrast, in vivo it showed less systemic pro-inflammatory activity and better safety than IMQ. Moreover, the TMX-202 gel formulation exhibited at least comparable efficacy to Aldara in a mouse model for skin proliferative diseases. CONCLUSION: TMX-202 is safe and efficacious without causing excessive adverse effects, suggesting that it may be an alternative to Aldara for the treatment of proliferative skin conditions.
Asunto(s)
Adenina/análogos & derivados , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Proliferación Celular/efectos de los fármacos , Glicerofosfolípidos/farmacología , Glicerofosfolípidos/uso terapéutico , Queratosis Actínica/tratamiento farmacológico , Glicoproteínas de Membrana/genética , Receptor Toll-Like 7/genética , Adenina/sangre , Adenina/farmacología , Adenina/uso terapéutico , Aminoquinolinas/sangre , Aminoquinolinas/farmacología , Animales , Antineoplásicos/sangre , Línea Celular , Factores Quimiotácticos/sangre , Células Dendríticas/fisiología , Geles/farmacología , Geles/uso terapéutico , Glicerofosfolípidos/sangre , Humanos , Imiquimod , Interferón gamma/metabolismo , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Queratinocitos/fisiología , Queratosis Actínica/genética , Leucocitos Mononucleares/efectos de los fármacos , Macrófagos/fisiología , Dosis Máxima Tolerada , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Factor 88 de Diferenciación Mieloide/genética , Proteínas Proto-Oncogénicas c-myc/genética , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
BACKGROUND: The only therapeutic options that exist for squamous cell lung carcinoma (SCC) are standard radiation and cytotoxic chemotherapy. Cancer stem cells (CSCs) are hypothesized to account for therapeutic resistance, suggesting that CSCs must be specifically targeted. Here, we analyze the transcriptome of CSC and non-CSC subpopulations by RNA-seq to identify new potential therapeutic strategies for SCC. METHODS: We sorted a SCC into CD133- and CD133+ subpopulations and then examined both by copy number analysis (CNA) and whole genome and transcriptome sequencing. We analyzed The Cancer Genome Atlas (TCGA) transcriptome data of 221 SCCs to determine the generality of our observations. RESULTS: Both subpopulations highly expressed numerous mRNA isoforms whose protein products are active drug targets for other cancers; 31 (25%) correspond to 18 genes under active investigation as mAb targets and an additional 4 (3%) are of therapeutic interest. Moreover, we found evidence that both subpopulations were proliferatively driven by very high levels of c-Myc and the TRAIL long isoform (TRAILL) and that normal apoptotic responses to high expression of these genes was prevented through high levels of Mcl-1L and Bcl-xL and c-FlipL-isoforms for which drugs are now in clinical development. SCC RNA-seq data (nâ=â221) from TCGA supported our findings. Our analysis is inconsistent with the CSC concept that most cells in a cancer have lost their proliferative potential. Furthermore, our study suggests how to target both the CSC and non-CSC subpopulations with one treatment strategy. CONCLUSIONS: Our study is relevant to SCC in particular for it presents numerous potential options to standard therapy that target the entire tumor. In so doing, it demonstrates how transcriptome sequencing provides insights into the molecular underpinnings of cancer propagating cells that, importantly, can be leveraged to identify new potential therapeutic options for cancers beyond what is possible with DNA sequencing.
Asunto(s)
Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/terapia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Células Madre Neoplásicas/metabolismo , Antígeno AC133 , Animales , Antígenos CD/metabolismo , Apoptosis/genética , Carcinoma de Células Escamosas/patología , Variaciones en el Número de Copia de ADN , ADN de Neoplasias/genética , Glicoproteínas/metabolismo , Humanos , Neoplasias Pulmonares/patología , Proteínas de la Membrana/genética , Ratones , Mutación , Células Madre Neoplásicas/clasificación , Células Madre Neoplásicas/patología , Péptidos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Transcriptoma , Trasplante HeterólogoRESUMEN
A cell-based high-throughput screen to identify small molecular weight stimulators of the innate immune system revealed substituted pyrimido[5,4-b]indoles as potent NFκB activators. The most potent hit compound selectively stimulated Toll-like receptor 4 (TLR4) in human and mouse cells. Synthetic modifications of the pyrimido[5,4-b]indole scaffold at the carboxamide, N-3, and N-5 positions revealed differential TLR4 dependent production of NFκB and type I interferon associated cytokines, IL-6 and interferon γ-induced protein 10 (IP-10) respectively. Specifically, a subset of compounds bearing phenyl and substituted phenyl carboxamides induced lower IL-6 release while maintaining higher IP-10 production, skewing toward the type I interferon pathway. Substitution at N-5 with short alkyl substituents reduced the cytotoxicity of the leading hit compound. Computational studies supported that active compounds appeared to bind primarily to MD-2 in the TLR4/MD-2 complex. These small molecules, which stimulate innate immune cells with minimal toxicity, could potentially be used as adjuvants or immune modulators.
Asunto(s)
Indoles/síntesis química , Pirimidinas/síntesis química , Receptor Toll-Like 4/agonistas , Animales , Células Cultivadas , Quimiocina CXCL10/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Inmunidad Innata/efectos de los fármacos , Indoles/química , Indoles/farmacología , Interleucina-6/metabolismo , Interleucina-8/metabolismo , Ligandos , Lipopolisacáridos/farmacología , Antígeno 96 de los Linfocitos/agonistas , Antígeno 96 de los Linfocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , FN-kappa B/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Relación Estructura-Actividad , Receptor Toll-Like 4/metabolismoRESUMEN
The Toll-like receptors (TLR) have been advocated as attractive therapeutic targets because TLR signaling plays dual roles in initiating adaptive immune responses and perpetuating inflammation. Paradoxically, repeated stimulation of bone marrow mononuclear cells with a synthetic TLR7 ligand 9-benzyl-8-hydroxy-2-(2-methoxyethoxy) adenine (called 1V136) leads to subsequent TLR hyporesponsiveness. Further studies on the mechanism of action of this pharmacologic agent demonstrated that the TLR7 ligand treatment depressed dendritic cell activation, but did not directly affect T cell function. To verify this mechanism, we utilized experimental allergic encephalitis (EAE) as an in vivo T cell dependent autoimmune model. Drug treated SJL/J mice immunized with proteolipid protein (PLP)(139-151) peptide had attenuated disease severity, reduced accumulation of mononuclear cells in the central nervous system (CNS), and limited demyelination, without any apparent systemic toxicity. Splenic T cells from treated mice produced less cytokines upon antigenic rechallenge. In the spinal cords of 1V136-treated EAE mice, the expression of chemoattractants was also reduced, suggesting innate immune cell hyposensitization in the CNS. Indeed, systemic 1V136 did penetrate the CNS. These experiments indicated that repeated doses of a TLR7 ligand may desensitize dendritic cells in lymphoid organs, leading to diminished T cell responses. This treatment strategy might be a new modality to treat T cell mediated autoimmune diseases.
Asunto(s)
Adenina/análogos & derivados , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Innata/efectos de los fármacos , Inmunosupresores/farmacología , Glicoproteínas de Membrana/agonistas , Receptor Toll-Like 7/agonistas , Adenina/farmacología , Adenina/uso terapéutico , Animales , Células Presentadoras de Antígenos/efectos de los fármacos , Células Presentadoras de Antígenos/inmunología , Células Cultivadas , Quimiocinas/metabolismo , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Femenino , Inmunosupresores/uso terapéutico , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/inmunología , Microglía/fisiología , Infiltración Neutrófila/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Médula Espinal/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Receptor Toll-Like 7/metabolismoRESUMEN
Ultra-deep targeted sequencing (UDT-Seq) can identify subclonal somatic mutations in tumor samples. Early assays' limited breadth and depth restrict their clinical utility. Here, we target 71 kb of mutational hotspots in 42 cancer genes. We present novel methods enhancing both laboratory workflow and mutation detection. We evaluate UDT-Seq true sensitivity and specificity (> 94% and > 99%, respectively) for low prevalence mutations in a mixing experiment and demonstrate its utility using six tumor samples. With an improved performance when run on the Illumina Miseq, the UDT-Seq assay is well suited for clinical applications to guide therapy and study clonal selection in heterogeneous samples.