RESUMEN
Glycogen synthase kinase-3 (GSK-3) is a multitasking protein kinase that regulates numerous critical cellular functions. Not surprisingly, elevated GSK-3 activity has been implicated in a host of diseases including pathological inflammation, diabetes, cancer, arthritis, asthma, bipolar disorder, and Alzheimer's. Therefore, reagents that inhibit GSK-3 activity provide a means to investigate the role of GSK-3 in cellular physiology and pathophysiology and could become valuable therapeutics. Finding a potent inhibitor of GSK-3 that can selectively target this kinase, among over 500 protein kinases in the human genome, is a significant challenge. Thus there remains a critical need for the identification of selective inhibitors of GSK-3. In this work, we introduce a novel small organic compound, namely COB-187, which exhibits potent and highly selective inhibition of GSK-3. Specifically, this study 1) utilized a molecular screen of 414 kinase assays, representing 404 unique kinases, to reveal that COB-187 is a highly potent and selective inhibitor of GSK-3; 2) utilized a cellular assay to reveal that COB-187 decreases the phosphorylation of canonical GSK-3 substrates indicating that COB-187 inhibits cellular GSK-3 activity; and 3) reveals that a close isomer of COB-187 is also a selective and potent inhibitor of GSK-3. Taken together, these results demonstrate that we have discovered a region of chemical design space that contains novel GSK-3 inhibitors. These inhibitors will help to elucidate the intricate function of GSK-3 and can serve as a starting point for the development of potential therapeutics for diseases that involve aberrant GSK-3 activity.
Asunto(s)
Compuestos de Bifenilo/farmacología , Glucógeno Sintasa Quinasa 3 beta/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/metabolismo , Animales , Compuestos de Bifenilo/síntesis química , Diseño de Fármacos , Pruebas de Enzimas , Expresión Génica , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Células HEK293 , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Fosforilación , Inhibidores de Proteínas Quinasas/síntesis química , Proteínas Quinasas/genética , Células RAW 264.7 , Relación Estructura-Actividad , Especificidad por Sustrato , Células THP-1 , Acetato de Tetradecanoilforbol/farmacología , Tiadiazoles/química , Tiadiazoles/farmacologíaRESUMEN
Clostridioides difficile infection (CDI) with high morbidity and high mortality is an urgent threat to public health, and C. difficile pathogenesis studies are eagerly required for CDI therapy. The major surface layer protein, SlpA, was supposed to play a key role in C. difficile pathogenesis; however, a lack of isogenic slpA mutants has greatly hampered analysis of SlpA functions. In this study, the whole slpA gene was successfully deleted for the first time via CRISPR-Cas9 system. Deletion of slpA in C. difficile resulted in smaller, smother-edged colonies, shorter bacterial cell size, and aggregation in suspension. For life cycle, the mutant demonstrated lower growth (changes of optical density at 600 nm, OD600) but higher cell density (colony-forming unit, CFU), decreased toxins production, and inhibited sporulation. Moreover, the mutant was more impaired in motility, more sensitive to vancomycin and Triton X-100-induced autolysis, releasing more lactate dehydrogenase. In addition, SlpA deficiency led to robust biofilm formation but weak adhesion to human host cells.IMPORTANCEClostridioides difficile infection (CDI) has been the most common hospital-acquired infection, with a high rate of antibiotic resistance and recurrence incidences, become a debilitating public health threat. It is urgently needed to study C. difficile pathogenesis for developing efficient strategies as CDI therapy. SlpA was indicated to play a key role in C. difficile pathogenesis. However, analysis of SlpA functions was hampered due to lack of isogenic slpA mutants. Surprisingly, the first slpA deletion C. difficile strain was generated in this study via CRISPR-Cas9, further negating the previous thought about slpA being essential. Results in this study will provide direct proof for roles of SlpA in C. difficile pathogenesis, which will facilitate future investigations for new targets as vaccines, new therapeutic agents, and intervention strategies in combating CDI.
Asunto(s)
Proteínas Bacterianas , Biopelículas , Clostridioides difficile , Infecciones por Clostridium , Eliminación de Gen , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Infecciones por Clostridium/microbiología , Biopelículas/crecimiento & desarrollo , Antibacterianos/farmacología , Virulencia/genética , Sistemas CRISPR-Cas , Adhesión Bacteriana/genética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismoRESUMEN
Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA induces the production of inflammatory cytokines and chemokines that trigger beta cell apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was designed to evaluate and describe potential protective effects of phenylmethimazole (C10), a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM. We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and cytokine/chemokine production in murine pancreatic beta cells in culture. We found that C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10 (1) possesses novel beta cell protective activity which may have potential clinical relevance in T1DM and (2) may be a useful tool in achieving a better understanding of the role that dsRNA-mediated responses play in the pathogenesis of T1DM.
Asunto(s)
Diabetes Mellitus Tipo 1/prevención & control , Enterovirus/patogenicidad , Células Secretoras de Insulina/efectos de los fármacos , Metimazol/análogos & derivados , ARN Bicatenario/efectos adversos , Tionas/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Citocinas/sangre , Diabetes Mellitus Tipo 1/virología , Enterovirus/metabolismo , Femenino , Inflamación/tratamiento farmacológico , Inflamación/patología , Metimazol/farmacología , Ratones , Ratones Endogámicos NOD , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal , Receptor Toll-Like 3/genética , Receptor Toll-Like 3/metabolismo , Regulación hacia ArribaRESUMEN
Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.
RESUMEN
Previous studies revealed that phenylmethimazole (C10) inhibits IRF3 signaling, preventing dsRNA-induction of type 1 interferon gene expression, production, and downstream signaling. In the present study, we investigated the molecular basis for C10 inhibition of dsRNA-stimulated IRF3 signaling. IRF-3 Trans-AM assays were used to measure C10 effects on dsRNA induction of IRF3 DNA binding. Green fluorescent protein-labeled IRF3 was used to measure C10 effects on dsRNA-induced IRF3 nuclear translocation. Native PAGE, SDS PAGE, and western blotting were used to identify effects of C10 on IRF3 homodimer formation and phosphorylation, respectively. There was a significant impairment of dsRNA-induced IRF3 DNA binding activity in human embryonic kidney and pancreatic cancer cells with C10 treatment. C10 also blocked dsRNA-induced IRF3 nuclear translocation and homodimer formation without blocking serine 396 phosphorylation of IRF3. Together, these results indicate that C10 interferes with IRF3 signaling by blocking dsRNA-induced IRF3 homodimer formation, a prerequisite for nuclear translocation and DNA binding activities.
Asunto(s)
Núcleo Celular/metabolismo , Factor 3 Regulador del Interferón/metabolismo , Metimazol/análogos & derivados , Multimerización de Proteína/efectos de los fármacos , ARN Bicatenario/farmacología , Tionas/farmacología , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , ADN/metabolismo , Células HEK293 , Humanos , Metimazol/farmacología , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacosRESUMEN
BACKGROUND: Dendritic cells (DCs) are antigen presenting cells capable of inducing specific immune responses against microbial infections, transplant antigens, or tumors. Interestingly, microenvironment conditions such as those present in tumor settings might induce a DC phenotype that is poorly immunogenic and with the capability of promoting angiogenesis. We hypothesize that this plasticity may be caused not only by the action of specific cytokines or growth factors but also by the properties of the surfaces with which they interact, such as extracellular matrix (ECM) components. RESULTS: Herewith we studied the effect of different surfaces and soluble factors on the biology of DCs. To accomplish this, we cultured murine myeloid(m) DCs on surfaces coated with fibronectin, collagen I, gelatin, and Matrigel using poly-D-lysine and polystyrene as non-biological surfaces. Further, we cultured these cells in the presence of regular DC medium (RPMI 10% FBS) or commercially available endothelial medium (EGM-2). We determined that mDCs could be kept in culture up to 3 weeks in these conditions, but only in the presence of GM-CSF. We were able to determine that long-term DC cultures produce an array of angiogenic factors, and that some of these cultures still retain the capability to induce T cell responses. CONCLUSIONS: Altogether these data indicate that in order to design DC-based vaccines or treatments focused on changing the phenotype of DCs associated with diseases such as cancer or atherosclerosis, it becomes necessary to fully investigate the microenvironment in which these cells are present or will be delivered.
Asunto(s)
Células Dendríticas/fisiología , Células Mieloides/fisiología , Proteínas Angiogénicas/metabolismo , Animales , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Colágeno , Colágeno Tipo I/farmacología , Células Dendríticas/efectos de los fármacos , Combinación de Medicamentos , Femenino , Fibronectinas/farmacología , Gelatina/farmacología , Laminina , Activación de Linfocitos/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Polilisina , Poliestirenos , Proteoglicanos , Linfocitos T/inmunologíaRESUMEN
Sepsis is a serious condition that can lead to long-term organ damage and death. At the molecular level, the hallmark of sepsis is the elevated expression of a multitude of potent cytokines, i.e. a cytokine storm. For sepsis involving gram-negative bacteria, macrophages recognize lipopolysaccharide (LPS) shed from the bacteria, activating Toll-like-receptor 4 (TLR4), and triggering a cytokine storm. Glycogen synthase kinase-3 (GSK-3) is a highly active kinase that has been implicated in LPS-induced cytokine production. Thus, compounds that inhibit GSK-3 could be potential therapeutics for sepsis. Our group has recently described a novel and highly selective inhibitor of GSK-3 termed COB-187. In the present study, using THP-1 macrophages, we evaluated the ability of COB-187 to attenuate LPS-induced cytokine production. We found that COB-187 significantly reduced, at the protein and mRNA levels, cytokines induced by LPS (e.g. IL-6, TNF-α, IL-1ß, CXCL10, and IFN-ß). Further, the data suggest that the inhibition could be due, at least in part, to COB-187 reducing NF-κB (p65/p50) DNA binding activity as well as reducing IRF-3 phosphorylation at Serine 396. Thus, COB-187 appears to be a potent inhibitor of the cytokine storm induced by LPS.
Asunto(s)
Antiinflamatorios/farmacología , Síndrome de Liberación de Citoquinas/prevención & control , Citocinas/metabolismo , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Macrófagos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Síndrome de Liberación de Citoquinas/inducido químicamente , Síndrome de Liberación de Citoquinas/enzimología , Síndrome de Liberación de Citoquinas/genética , Citocinas/genética , Regulación hacia Abajo , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Factor 3 Regulador del Interferón/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/enzimología , FN-kappa B/metabolismo , Fosforilación , Sepsis/inducido químicamente , Sepsis/enzimología , Sepsis/genética , Sepsis/prevención & control , Transducción de Señal , Células THP-1RESUMEN
Because of the lack of full characterization of tumor associated antigens for solid tumors, whole antigen use is a convenient approach to tumor vaccination. Tumor RNA and apoptotic tumor cells have been used as a source of whole tumor antigen to prepare dendritic cell (DC) based tumor vaccines, but their efficacy has not been directly compared. Here we compare directly RNA electroporation and pulsing of DCs with whole tumor cells killed by ultraviolet (UV) B radiation using a convenient tumor model expressing human papilloma virus (HPV) E6 and E7 oncogenes. Although both approaches led to DCs presenting tumor antigen, electroporation with tumor cell total RNA induced a significantly higher frequency of tumor-reactive IFN-gamma secreting T cells, and E7-specific CD8+ lymphocytes compared to pulsing with UV-irradiated tumor cells. DCs electroporated with tumor cell RNA induced a larger tumor infiltration by T cells and produced a significantly stronger delay in tumor growth compared to DCs pulsed with UV-irradiated tumor cells. We conclude that electroporation with whole tumor cell RNA and pulsing with UV-irradiated tumor cells are both effective in eliciting antitumor immune response, but RNA electroporation results in more potent tumor vaccination under the examined experimental conditions.
Asunto(s)
Vacunas contra el Cáncer , Células Dendríticas/citología , Neoplasias/metabolismo , Neoplasias/terapia , ARN/metabolismo , Rayos Ultravioleta , Animales , Antígenos de Neoplasias , Apoptosis , Células de la Médula Ósea/citología , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Papillomaviridae/metabolismo , Linfocitos T/metabolismoRESUMEN
Epithelial ovarian cancer is the most frequent cause of gynecologic malignancy-related mortality in women. To identify genes up-regulated in ovarian cancer, PCR-select cDNA subtraction was done and Drosophila Eyes Absent Homologue 2 (EYA2) was isolated as a promising candidate. The transcriptional coactivator eya controls essential cellular functions during organogenesis of Drosophila. EYA2 mRNA was found to be up-regulated in ovarian cancer by real-time reverse transcription-PCR, whereas its protein product was detected in 93.6% of ovarian cancer specimens by immunohistochemistry (n = 140). EYA2 was amplified in 14.8% of ovarian carcinomas, as detected by array-based comparative genomic hybridization (n = 88). Most importantly, EYA2 overexpression was significantly associated with short overall survival in advanced ovarian cancer (n = 99, P = 0.0361). EYA2 was found to function as transcriptional activator in ovarian cancer cells by Gal4 assay and to promote tumor growth in vivo in xenograft models. Therefore, this study suggests an important role of EYA2 in ovarian cancer and its potential application as a therapeutic target.
Asunto(s)
Neoplasias Ováricas/genética , Transactivadores/genética , Animales , Procesos de Crecimiento Celular/genética , ADN de Neoplasias/genética , Progresión de la Enfermedad , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos BALB C , Proteínas Nucleares , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Pronóstico , Proteínas Tirosina Fosfatasas , Transactivadores/biosíntesis , Activación Transcripcional/genética , Regulación hacia ArribaRESUMEN
We tested whether tumor cells were killed by replication-incompetent recombinant herpes simplex virus (HSV) d120 lacking immediate early gene ICP4 and whether HSVd120-killed tumor cells could be used directly for tumor vaccination. Vaccine efficacy was tested in TC-1, a murine adenocarcinoma transformed with HPV16 E6 and E7, and ID8-Vegf, a murine epithelial ovarian cancer model. HSVd120 killed tumor cells by apoptosis. Tumor cells infected by HSVd120 were engulfed more avidly by immature DCs and induced DC maturation more efficiently than tumor cells killed by ultraviolet B (UVB) radiation. HSVd120 infection induced stronger upregulation of GRP94 than UVB in cells undergoing apoptosis. Immunization of mice with HSVd120-killed cells elicited stronger antitumor T cell response, including tumor reactive interferon-gamma secreting and cytotoxic T cells, and resulted in significantly stronger delay in tumor growth than immunization with UVB-killed tumor cells. Thus, the use of replication-incompetent HSV strains lacking ICP4 offers possible advantages in the preparation of whole tumor cell antigen for direct tumor vaccination.
Asunto(s)
Adenocarcinoma/tratamiento farmacológico , Antígenos de Neoplasias/metabolismo , Vacunas contra el Cáncer/inmunología , Glicoproteínas de Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Simplexvirus/genética , Animales , Antígenos de Neoplasias/inmunología , Vacunas contra el Cáncer/uso terapéutico , Células Dendríticas/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Inmediatas-Precoces/genética , Glicoproteínas de Membrana/inmunología , Ratones , Linfocitos T/inmunología , Regulación hacia Arriba , Vacunación/métodos , Replicación Viral/genéticaRESUMEN
The role of the NKG2D immunoreceptor and its ligands in antitumor immune response is incompletely understood. Here, we report that effector immune cells infiltrating ovarian carcinoma are mostly CD8+ lymphocytes lacking CD28 but expressing the NKG2D costimulatory receptor. Human ovarian carcinoma expresses the novel NKG2D ligand lymphocyte effector cell toxicity-activating ligand (Letal). Letal was found to be an independent prognosticator of improved survival in advanced ovarian cancer. Higher levels of tumor-derived Letal were associated with stronger lymphocyte infiltration. Letal exerted marked costimulatory effects and induced type-1 polarization in CD8+CD28- tumor-infiltrating lymphocytes ex vivo. Letal engagement increased the expression of the glucose transporter Glut-1, enhanced glucose up-take, and protected CD8+ lymphocytes from cisplatin-induced killing. Letal also down-regulated the expression of Fas in CD8+ cells and rendered them resistant to Fas ligand-induced apoptosis. Our results indicate that Letal promotes tumor immune surveillance by promoting the survival and intratumoral expansion of antitumor cytotoxic lymphocytes. We propose that Letal could be used for the ex vivo expansion of apoptosis-resistant tumor-reactive cytotoxic lymphocytes for adoptive transfer.
Asunto(s)
Antígenos CD28/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Ováricas/inmunología , Receptores Inmunológicos/metabolismo , Adenocarcinoma Mucinoso/inmunología , Adenocarcinoma Mucinoso/patología , Animales , Apoptosis , Carcinoma Endometrioide/inmunología , Carcinoma Endometrioide/patología , Supervivencia Celular , Cistadenocarcinoma Seroso/inmunología , Cistadenocarcinoma Seroso/patología , Citotoxicidad Inmunológica , Femenino , Humanos , Ligandos , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK , Neoplasias Ováricas/patología , Receptores de Células Asesinas NaturalesRESUMEN
In the present study, we investigated the ability of replication-restricted herpes simplex virus (HSV) 1716 lacking ICP34.5 to infect endothelium and disrupt tumor vasculature. HSV-1716 efficiently infected and killed mouse endothelial cell lines H5V and MS1 cells, as well as human umbilical vein endothelial cells in vitro. Capillary tube formation by endothelial cells was inhibited by HSV-1716 in vitro and in vivo. Following intratumoral administration of oncolytic HSV-1716, HSV-glycoproteins could be detected in CD31-positive tumor vascular endothelium by immunostaining. Viral DNA was recovered from highly purified microdissected tumor vascular endothelium. Furthermore, endothelium of tumors treated with HSV-1716 exhibited expression of tissue factor, a marker of endothelial damage. Importantly, HSV antigen and DNA were also detected in endothelium distant from foci of active tumor infection. After intravascular inoculation of HSV-1716, viral glycoproteins were detected in association to tumor endothelium, but not vascular endothelium of different organs. Purified tumor endothelial cells showed high proliferative capability and were susceptible to HSV-1716 infection and killing ex vivo while endothelium from normal organs was not. We conclude that oncolytic HSV-1716 exerts direct antiangiogenic effects, which may contribute to the overall therapeutic efficacy of the virus.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Carcinoma/terapia , Vectores Genéticos/farmacología , Neoplasias Ováricas/terapia , Simplexvirus/genética , Inhibidores de la Angiogénesis/genética , Animales , Carcinoma/genética , Células Cultivadas , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/patología , Femenino , Humanos , Ratones , Neoplasias Ováricas/genética , Células Tumorales Cultivadas , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3(+/+)) and TLR3 knockout (TLR3(-/-)) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice.
Asunto(s)
Infecciones por Coxsackievirus/complicaciones , Diabetes Mellitus Tipo 1/virología , Receptor Toll-Like 3/metabolismo , Animales , Infecciones por Coxsackievirus/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Enterovirus Humano B/aislamiento & purificación , Femenino , Ratones Endogámicos NOD , Ratones Noqueados , Páncreas/virología , Distribución AleatoriaRESUMEN
NKG2D serves as one of the most potent activating receptors for effector lymphocytes. in peripheral tissues. Here we report the characterization of Letal, the first human trans-membrane NKG2D ligand lacking an immunoglobulin-like alpha-3 ectodomain. Letal is constitutively expressed by a variety of normal tissues, and is upregulated in tumor cells of different origins. Unlike other NKG2D ligands, Letal mRNA expression progressively decreased after treatment of tumor cells with retinoic acid. Simultaneous T-cell receptor activation and engagement of Letal stimulated proliferation of CD8(+) cells and dramatically increased IL-2 and IFNgamma secretion. In addition, Letal induced the killing of cancer cells by CD8(+) and NK cells. These results suggest that Letal delivers activating signals to NK cells and promotes tumor immune surveillance by inducing the expansion of anti-tumor cytotoxic lymphocytes.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Ligandos , Activación de Linfocitos , Neoplasias Ováricas/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/inmunología , Citotoxicidad Inmunológica , Femenino , Humanos , Inmunoglobulina G/inmunología , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Células K562 , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Subfamilia K de Receptores Similares a Lectina de Células NK , Neoplasias Ováricas/tratamiento farmacológico , ARN Mensajero/metabolismo , Receptores Inmunológicos/genética , Receptores de Células Asesinas Naturales , Homología de Secuencia de Aminoácido , Tretinoina/uso terapéutico , Fosfolipasas de Tipo C/farmacologíaRESUMEN
Here, we studied the effect of aminoguanidine (AG) treatment, a nitric oxide synthase (NOS)-2 inhibitor, during the immune response against intranasal administration of ovalbumin (OVA) mixed with cholera toxin (CT) in BALB/c mice. NOS-2 mRNA was detected by reverse transcription-PCR (RT-PCR) in samples of lungs and turbinates early post-inoculation of the antigen. Animals intranasally treated with AG, showed an increase in the levels of seric specific IgG and IgM. A higher IgG1/IgG2a ratio against OVA was also observed in sera of same animals. Moreover, high levels of specific IgA were detected in samples of pulmonar washings obtained from treated animals. On the contrary, treated animals showed a lower DTH response while splenocytes obtained from the same animals showed a reduced proliferative capability against OVA compared to controls. Finally, RT-PCR analysis showed increased expression of TGF-beta in turbinates, lungs and cells from pulmonar washings obtained from AG treated mice. Taken together, these data suggest a role of nitric oxide (NO) in modulating the primary immune response against intranasal antigens.
Asunto(s)
Toxina del Cólera/inmunología , Guanidinas/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Ovalbúmina/inmunología , Administración Intranasal , Animales , Toxina del Cólera/administración & dosificación , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/inmunología , Inmunidad Mucosa/efectos de los fármacos , Inmunidad Mucosa/inmunología , Pulmón/inmunología , Ratones , Ovalbúmina/administración & dosificación , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Cornetes Nasales/inmunologíaAsunto(s)
Ojo/virología , Interleucina-15/genética , Interleucina-18/genética , Queratitis Herpética/genética , Óxido Nítrico Sintasa/genética , Animales , ADN Viral/análisis , Ojo/química , Ojo/patología , Expresión Génica , Herpesvirus Humano 1 , Interleucina-15/metabolismo , Interleucina-18/metabolismo , Queratitis Herpética/enzimología , Queratitis Herpética/inmunología , Ratones , Ratones Endogámicos BALB C , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
We have previously shown that intratumor administration of HSV-1716 (an ICP34.5 null mutant) resulted in significant reduction of tumor growth and a significant survival advantage in a murine model of ovarian cancer. Herewith we report that oncolytic HSV-1716 generates vaccination effects in the same model. Upon HSV-1716 infection, mouse ovarian tumor cells showed high levels of expression viral glycoproteins B and D and were highly phagocyted by dendritic cells (DCs). Interestingly, increased phagocytosis of tumor-infected cells by DCs was impaired by heparin, and anti-HSV glycoproteins B and D, indicating that viral infection enhances adhesive interactions between DCs and tumor apoptotic bodies. Moreover, HSV-1716 infected cells expressed high levels of heat shock proteins 70 and GRP94, molecules that have been reported to induce maturation of DCs, increase cross-presentation of antigens and promote antitumor immune response. After phagocytosis of tumor-infected cells, DCs acquired a mature status in vitro and in vivo, upregulated the expression of costimulatory molecule and increased migration towards MIP-3beta. Furthermore, HSV-1716 oncolytic treatment markedly reduced vascular endothelial growth factor (VEGF) levels in tumor-bearing animals thus abrogating tumor immunosuppressive milieu. These mechanisms may account for the highly enhanced antitumoral immune responses observed in HSV-1716 treated animals. Oncolytic treatment induced a significantly higher frequency of tumor-reactive IFNgamma producing cells, and induced a robust tumor infiltration by T cells. These results indicate that oncolytic therapy with HSV-1716 facilitates antitumor immune responses.
Asunto(s)
Presentación de Antígeno/inmunología , Antígenos de Neoplasias/inmunología , Neoplasias/inmunología , Viroterapia Oncolítica/métodos , Simplexvirus/inmunología , Animales , Presentación de Antígeno/genética , Antígenos de Neoplasias/genética , Femenino , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Neoplasias/terapia , Neoplasias/virología , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Neoplasias Ováricas/virología , Simplexvirus/genética , Células Tumorales CultivadasRESUMEN
Cooperation between oncolytic herpes simplex virus (HSV) and host effector immune mechanisms has been previously described. In the present study, we investigated the mechanism underlying such cooperation in a murine syngeneic model of ovarian carcinoma. Therapeutic administration of HSV-1716, a replication-restricted mutant, resulted in significant reduction of tumor growth and a significant survival advantage. Intratumoral injection of HSV-1716 induced expression of IFN-gamma, MIG, and IP-10 in the tumor. This was accompanied by a significant increase in the number of tumor-associated NK and CD8+ T cells expressing CXCR3 and CD25. Ascites from HSV-1716-treated animals efficiently induced in vitro migration of NK and CD8+ T cells, which was dependent on the presence of MIG and IP-10. Murine monocytes and dendritic cells (DCs) were responsible for the production of MIG and IP-10 upon HSV-1716 infection. In monocytes, this was partially abrogated by neutralizing antibodies against IFN-alpha and -beta, thus indicating a role for type-1 IFNs in the reported effect. Human ovarian carcinomas showed high numbers of monocytes and DCs. Upon HSV-1716 infection, human monocyte-derived DCs produced large amounts of IFN-gamma and upregulated MIG and IP-10 expression. These results indicate that HSV-1716 induces an inflammatory response that may facilitate antitumor immune response upon oncolytic therapy.
Asunto(s)
Linfocitos T CD8-positivos/fisiología , Quimiocinas/metabolismo , Terapia Genética , Neoplasias Ováricas/inmunología , Simplexvirus/genética , Animales , Quimiotaxis de Leucocito , Femenino , Humanos , Interferones/metabolismo , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Neoplasias Ováricas/terapia , Receptores de Quimiocina , Regulación hacia ArribaRESUMEN
There is no proof that hematopoietic cells contribute significantly to vasculogenesis in postnatal life. Here we report a novel leukocyte subset within ovarian carcinoma that coexpresses endothelial and dendritic cell markers. Fluorescence-activated cell sorter (FACS) analysis identified a high frequency of VE-cadherin+ CD45+ leukocytes (39% of host cells) in 10 of 10 solid tumors evaluated. This population represented less than 1% of nontumor cells in ascites and peripheral blood. At the protein level, more than 86% of these cells expressed the endothelial markers P1H12, CD34, and CD31 and leukocyte markers CD11c and major histocompatibility complex (MHC) class II. At the mRNA level, we detected TEM1, TEM7, and Thy-1, specific markers of angiogenic endothelium. Finally, this population has the capacity to generate functional blood vessels in vivo. Because of its mixed phenotype, we named this population vascular leukocytes (VLCs). Our data provide an important link between hematopoietic endothelial precursors and vascular development in postnatal life and a possible novel therapeutic target.
Asunto(s)
Leucocitos/fisiología , Neovascularización Patológica/inmunología , Neoplasias Ováricas/irrigación sanguínea , Neoplasias Ováricas/inmunología , Animales , Antígenos CD , Cadherinas/metabolismo , Femenino , Humanos , Antígenos Comunes de Leucocito/metabolismo , Leucocitos/citología , Ratones , Ratones Endogámicos BALB C , Ratones SCID , Células Madre/citología , Células Madre/fisiologíaRESUMEN
Here, we studied the role of nitric oxide (NO) production during the first steps of the respiratory infection of BALB/c mice with herpes simplex virus type 1 (HSV-1), strain F. Nitric oxide synthase II (NOS-II) mRNA and protein were detected by reverse transcription (RT)-PCR and dot blot, respectively in samples of lungs and turbinates early post-infection (p.i.). Immunohistochemical analysis revealed pulmonar macrophages and PMN expressing NOS-II in the lungs of infected animals. Animals intranasally treated with aminoguanidine (AG), a NOS inhibitor, during the first steps of infection, showed a dose-dependent increase in pneumonitis compared to controls. Viral titres in turbinates, lungs, and brains were higher in AG treated mice. Finally, histopathology studies revealed a stronger inflammation in eyes, and lungs of these animals. Taken together, these results suggest a role of NO in controlling primary HSV intranasal infection.