RESUMEN
Benzo[a]pyrene (B[a]P) is a typical complete carcinogen in tobacco, but its mechanism of inducing the development of chronic pneumonia and consequent lung cancer is unclear. Here we elucidated the role of myeloid-derived suppressor cells (MDSCs) in developing B[a]P-induced chronic lung inflammation and efficacy of immunotherapy in preventing subsequent malignant transformation. Our study showed that as B[a]P could induce the accumulation of MDSCs in lung tissues and enhance the immunosuppressive effect regulated by cytokines and metabolites, thereby promoting the formation of immunosuppressive microenvironment, where effector T cells were exhausted, NK cells were dysfunctional, regulatory T (Treg) cells were expanded, polarized alveolar macrophages were transformed from M1 to M2. Subsequently, we performed the immunotherapy to block TNFÉ only or both TNFÉ and PD-1 at the early- or middle-stage of B[a]P-induced chronic lung inflammation to ameliorate the immunosuppressive microenvironment. We found that TNFÉ antagonist alone or with PD-1 blocker was shown to exert therapeutic effects on malignant transformation at the early stage of B[a]P-induced chronic lung inflammation. Taken together, our findings demonstrated that B[a]P-induced chronic lung inflammation resulted in the accumulation of MDSCs in lung tissues and exercise their immunosuppressive functions, thereby developing an immunosuppressive microenvironment, thus TNFÉ antagonist alone or with PD-1 blocker could prevent or retard the malignant transformation of B[a]P-induced chronic lung inflammation.
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Neoplasias Pulmonares , Células Supresoras de Origen Mieloide , Neumonía , Humanos , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/prevención & control , Neumonía/inducido químicamente , Neumonía/tratamiento farmacológico , Neumonía/prevención & control , Receptor de Muerte Celular Programada 1/metabolismo , Microambiente Tumoral , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
The use of chimeric antigen receptor-modified T cells (CAR T cells) is an effective therapy for advanced cancer, especially hematological malignancies, and this method has attracted widespread attention in the last several years. The type, number and vitality of the effector cells clearly play important roles in this approach. In this study, to expand the possibility of curing cancer through adoptive cell therapy (ACT), we developed a novel method for effectively obtaining abundant T cells in vitro. The fusion proteins of three cytokines, SA-hIL-2, SA-hIL-7 and SA-hIL-21, were anchored onto biotin magnetic beads to increase the number of cytokines on the surface of the magnetic beads, which increased the local concentration of cytokines and thus promoted the binding of cytokines to T cells. Next, we examined the effects of these modified magnetic beads on the proliferation rate of T cells and CD19 CAR T cells. In this study, we report the expression and purification of the active bifunctional fusion proteins SA-hIL-2, SA-hIL-7 and SA-hIL-21, which were bound to biotin magnetic beads to develop a platform that was employed to increase the local concentration of cytokines. When the cells had been cultured for 14 days, the proliferation rate of the CD3+ T cells in the group that received cytokine-coupled biotin magnetic beads (Beads-SA-CK) was higher than that of the cells in the groups that received soluble cytokines (Soluble-SA-CK) and that of the cells in the standard group (Standard-CK). We speculate that this difference may be the result of the increased expression of Bcl-2 and the increased phosphorylation of Stat5. Moreover, our results preliminarily indicate that compared with the other two treatments, Soluble-SA-CK and Standard-CK, adding cytokine-coupled biotin magnetic beads more effectively increases the proliferation rate of CD19 CAR-T cells. As expected, the CD19 CAR-T cells stimulated by Beads-SA-CK had a stronger anticancer effect than the cells stimulated by the other two treatments. An effective method of preparing abundant T cells in vitro was developed, and it may provide a novel strategy for ACT.
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Técnicas de Cultivo de Célula/métodos , Proliferación Celular , Linfocitos T/fisiología , Animales , Antígenos CD19/genética , Antígenos CD19/metabolismo , Citocinas/metabolismo , Citotoxicidad Inmunológica , Humanos , Inmunoterapia Adoptiva/métodos , Células K562 , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/citologíaRESUMEN
Mucosal-associated invariant T (MAIT) cells participate in both protective immunity and pathogenesis of diseases. Most murine MAIT cells express an invariant TCRVα19-Jα33 (iVα19) TCR, which triggers signals crucial for their development. However, signal pathways downstream of the iVα19TCR and their regulation in MAIT cells are unknown. Diacylglycerol (DAG) is a critical second messenger that relays the TCR signal to multiple downstream signaling cascades. DAG is terminated by DAG kinase (DGK)-mediated phosphorylation and conversion to phosphatidic acid. We have demonstrated here that downregulation of DAG caused by enhanced DGK activity impairs late-stage MAIT cell maturation in both thymus and spleen. Moreover, deficiency of DGKζ but not DGKα by itself causes modest decreases in MAIT cells, and deficiency of both DGKα and ζ results in severe reductions of MAIT cells in an autonomous manner. Our studies have revealed that DAG signaling is not only critical but also must be tightly regulated by DGKs for MAIT cell development and that both DGKα and, more prominently, DGKζ contribute to the overall DGK activity for MAIT cell development.
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Diacilglicerol Quinasa/inmunología , Diacilglicerol Quinasa/metabolismo , Diglicéridos/inmunología , Diglicéridos/metabolismo , Células T Invariantes Asociadas a Mucosa/inmunología , Células T Invariantes Asociadas a Mucosa/metabolismo , Animales , Ratones , Transducción de Señal/inmunología , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: In view of the current difficulty of clinically diagnosing osteoarticular tuberculosis, our aim was to use mass spectrometry to establish diagnostic models and to screen and identify serum proteins which could serve as potential diagnostic biomarkers for early detection of osteoarticular tuberculosis. METHODS: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to select an osteoarticular tuberculosis-specific serum peptide profile and establish diagnostic models. Further, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to identify potential serum biomarkers that could be used for auxiliary diagnosis of osteoarticular tuberculosis, and then clinical serum samples were used to verify these biomarkers by enzyme-linked immunosorbent assay (ELISA). RESULTS: We established four diagnostic models that can distinguish osteoarticular tuberculosis from rheumatoid arthritis, ankylosing spondylitis, osteoarticular infections, and healthy adults. The models were osteoarticular tuberculosis-rheumatoid arthritis, osteoarticular tuberculosis-ankylosing spondylitis, osteoarticular tuberculosis-osteoarticular infections, and osteoarticular tuberculosis-healthy adult, and their accuracy was 76.78%, 79.02%, 83.77%, and 88.16%, respectively. Next, we selected and identified 18 proteins, including complement factor H-related protein 1 (CFHR1) and complement factor H-related protein 2 (CFHR2), which were upregulated in the tuberculosis group only. CONCLUSIONS: We successfully established four diagnostic models involving osteoarticular tuberculosis, rheumatoid arthritis, ankylosing spondylitis, osteoarticular infections, and healthy adults. Furthermore, we found that CFHR1 and CFHR2 may be two valuable auxiliary diagnostic indicators for osteoarticular tuberculosis. These results provide reference values for rapid and accurate diagnosis of osteoarticular tuberculosis.
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Biomarcadores/sangre , Proteínas Sanguíneas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Tuberculosis Osteoarticular/sangre , Adulto , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Proteínas Sanguíneas/metabolismo , Cromatografía Liquida , Proteínas Inactivadoras del Complemento C3b/análisis , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Espondilitis Anquilosante/sangre , Espondilitis Anquilosante/diagnóstico , Espectrometría de Masas en Tándem/métodos , Tuberculosis Osteoarticular/diagnósticoRESUMEN
Immunotherapy may be an effective way to prevent postoperative recurrence of renal cell carcinoma. Streptavidin-interleukin-2 (SA-IL-2) surface-modified tumor cell vaccine developed through our protein-anchor technology could induce specific antitumor T-cell responses, but this immunotherapy cannot completely eradicate the tumor. These effector T cells highly expressed programmed death receptor-1 (PD-1), and the expression of programmed death ligand-1 (PD-L1) in the tumor environment also was upregulated after SA-IL-2-modified vaccine therapy. PD-1/PD-L1 interaction promotes tumor immune evasion. Adding PD-1 blockade to SA-IL-2-modified vaccine therapy increased the number of CD4+ , CD8+ and CD8+ interferon-γ+ but not CD4+ Foxp3+ T cells. PD-1 blockade could rescue the activity of tumor-specific T lymphocytes induced by the SA-IL-2-modified vaccine. Combination therapy delayed tumor growth and protected mice against a second Renca cells but not melanoma cells challenge. Taken together, PD-1 blockade could reverse immune evasion in the treatment with SA-IL-2-modified vaccine, and eventually induce a stronger specific antitumor immune response against renal cell carcinoma.
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Vacunas contra el Cáncer/administración & dosificación , Carcinoma de Células Renales/terapia , Inmunoterapia/métodos , Interleucina-2/inmunología , Neoplasias Renales/terapia , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Animales , Antígeno B7-H1/inmunología , Antígeno B7-H1/metabolismo , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Vacunas contra el Cáncer/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Renales/metabolismo , Línea Celular Tumoral , Terapia Combinada , Interferón gamma/inmunología , Interferón gamma/metabolismo , Neoplasias Renales/inmunología , Neoplasias Renales/metabolismo , Ratones Endogámicos BALB C , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismoRESUMEN
Thymus is crucial for generation of a diverse repertoire of T cells essential for adaptive immunity. Although thymic epithelial cells (TECs) are crucial for thymopoiesis and T cell generation, how TEC development and function are controlled is poorly understood. We report here that mTOR complex 1 (mTORC1) in TECs plays critical roles in thymopoiesis and thymus function. Acute deletion of mTORC1 in adult mice caused severe thymic involution. TEC-specific deficiency of mTORC1 (mTORC1KO) impaired TEC maturation and function such as decreased expression of thymotropic chemokines, decreased medullary TEC to cortical TEC ratios, and altered thymic architecture, leading to severe thymic atrophy, reduced recruitment of early thymic progenitors, and impaired development of virtually all T-cell lineages. Strikingly, temporal control of IL-17-producing γδT (γδT17) cell differentiation and TCRVγ/δ recombination in fetal thymus is lost in mTORC1KO thymus, leading to elevated γδT17 differentiation and rearranging of fetal specific TCRVγ/δ in adulthood. Thus, mTORC1 is central for TEC development/function and establishment of thymic environment for proper T cell development, and modulating mTORC1 activity can be a strategy for preventing thymic involution/atrophy.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejos Multiproteicos/metabolismo , Linfocitos T/fisiología , Serina-Treonina Quinasas TOR/metabolismo , Timo/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Quimiocinas/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones Endogámicos C57BL , Proteína Reguladora Asociada a mTOR , Timo/inmunologíaRESUMEN
Alveolar macrophages (AMÏ) have the capacity of local self-renewal through adult life; however, mechanisms that regulate AMÏ self-renewal remain poorly understood. We found that myeloid-specific deletion of Raptor, an essential component of the mammalian/mechanistic target of rapamycin complex (mTORC)1, resulted in a marked decrease of this population of cells accompanying altered phenotypic features and impaired phagocytosis activity. We demonstrated further that Raptor/mTORC1 deficiency did not affect AMÏ development, but compromised its proliferative activity at cell cycle entry in the steady-state as well as in the context of repopulation in irradiation chimeras. Mechanically, mTORC1 confers AMÏ optimal responsiveness to GM-CSF-induced proliferation. Thus, our results demonstrate an essential role of mTORC1 for AMÏ homeostasis by regulating proliferative renewal.
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Homeostasis/inmunología , Macrófagos Alveolares/citología , Complejos Multiproteicos/inmunología , Serina-Treonina Quinasas TOR/inmunología , Animales , Proliferación Celular/fisiología , Citometría de Flujo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
BACKGROUND: Serological tests are indispensable in the diagnosis of early infection. At present, only procalcitonin (PCT) and C-reactive protein (CRP) are commonly used in clinical practice. Recently, serum amyloid A1 (SAA1) and heparin binding protein (HBP) have been shown to be new biomarkers, because SAA1 is highly sensitive and specific for viral infections, and HBP is predictive for septic shock. In this study, PCT, CRP, HBP, and SAA1 were detected in different combinations to improve the diagnostic accuracy of early infection using the biotin-avidin amplifying system-based time-resolved fluorescent immunoassay (BA-TRFIA). METHODS: A time-resolved fluorescent immunoassay for PCT, CRP, HBP, and SAA1 was developed and then tested in a clinical setting. All experiments were carried out using the DR6608 time-resolved fluorescent immunoassay analyzer. RESULTS: The cutoff values of PCT, CRP, HBP, and SAA1 were 0.05 µg/L, 5.59 mg/L, 3.83 µg/L, and 1.56 mg/L, respectively. The area under the ROC curve (AUC) showed that PCT Ë SAA1 Ë CRP Ë HBP > 0.8. A methodological comparison of the results showed that a combination of the four biomarkers had the highest accuracy for the diagnosis of infectious diseases. CONCLUSION: The time-resolved fluorescent immunoassay-based combined detection of PCT, CRP, HBP, and SAA1 was shown to significantly improve the diagnostic accuracy of early infection. Thus, our results indicate that combined detection based on BA-TRFIA may represent a promising strategy in the clinical diagnosis of infection.
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Péptidos Catiónicos Antimicrobianos/sangre , Infecciones Bacterianas/diagnóstico , Proteína C-Reactiva/análisis , Proteínas Portadoras/sangre , Técnica del Anticuerpo Fluorescente/métodos , Polipéptido alfa Relacionado con Calcitonina/sangre , Proteína Amiloide A Sérica/análisis , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Infecciones Bacterianas/sangre , Biomarcadores/sangre , Proteínas Sanguíneas , Niño , Preescolar , Diagnóstico Precoz , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Virosis/sangre , Virosis/diagnóstico , Adulto JovenRESUMEN
Gold nanobipyramids were synthesized by a seed-mediated growth method and then supported by multi-walled carbon nanotubes (denoted as AuNBP/MWCNTs). The electrocatalytic activity of the AuNBP/MWCNTs on a glassy carbon electrode (GCE) towards direct glucose oxidation and hydrogen peroxide reduction was superior to that of AuNBPs and MWCNTs. The modified GCE, operated at a typical working voltage of +0.15 V (vs. SCE) and in 0.1 M NaOH solution, exhibits a linear response in the 10 µM to 36.7 mM glucose concentration range with a 3.0 µM detection limit (at S/N = 3) and a sensitivity of 101.2 µA mM-1 cm-2. It also demonstrates good sensitivity towards hydrogen peroxide in at pH 7 solution at a working potential of -0.50 V (vs. SCE), with a linear response range from 5.0 µM to 47.3 mM, a sensitivity of 170.6 µA mM-1 cm-2 and a detection limit of 1.5 µM. Graphical abstract A electrochemical sensing platform based on the use of gold nanobipyramids and multi-walled carbon nanotubes nanocomposites (AuNBP/MWCNTs) is described for the determination of glucose and hydrogen peroxide.
RESUMEN
Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin-granulocyte-macrophage-colony stimulating factor (SA-GM-CSF) surface-modified bladder CSCs vaccine previously developed using our protein-anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor-1 (PD-1)/program death ligand 1 (PD-L1) signaling in tumor microenvironment results in tumor-adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8+ T cells, a part of these CD8+ T cells expressed PD-1. Moreover, the CSCs vaccine upregulated the PD-L1 expression of tumor cells, resulting in immune resistance. Adding PD-1 blockade to the CSCs vaccine therapy increased the population of CD4+ , CD8+ and CD8+ IFN-γ+ but not CD4+ Foxp3+ T cells and induced the highest production of IFN-γ. PD-1 blockade could effectively enhance the functions of tumor-specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM-1 cell challenge. These results indicate that PD-1 blockade combined with the GM-CSF-modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.
Asunto(s)
Vacunas contra el Cáncer/farmacología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Células Madre Neoplásicas/inmunología , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/inmunología , Neoplasias de la Vejiga Urinaria/terapia , Animales , Antígeno B7-H1/biosíntesis , Antígeno B7-H1/inmunología , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Carcinoma de Células Transicionales/inmunología , Carcinoma de Células Transicionales/terapia , Línea Celular Tumoral , Citocinas/sangre , Citocinas/inmunología , Células Dendríticas/inmunología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Receptor de Muerte Celular Programada 1/biosíntesis , Receptor de Muerte Celular Programada 1/inmunología , Distribución Aleatoria , Subgrupos de Linfocitos T/inmunologíaRESUMEN
Norcantharidin (NCTD) is a promising antitumor drug with low toxicity. It was reported to be able to regulate immunity, but the mechanism is not yet clear. Here we explored whether NCTD could enhance the antitumor immunity induced by prostate cancer cell vaccine. The results of the in vitro study showed that NCTD induced apoptosis and inhibited proliferation of regulatory T cells (Tregs). Mechanistic research showed that NCTD inhibited Akt activation and activated FOXO1 transcription, resulting in a pro-apoptotic effect. The results of the in vivo study showed that more tumor-infiltrating Tregs existed within peripheral blood and tumor tissue after treatment with the vaccine. Adding NCTD to vaccine treatment could decrease the number of tumor-infiltrating Tregs and increase the number of CD4+ and CD8+ T cells. Combination therapy with NCTD and vaccine was more effective in inhibiting tumor growth than the vaccine alone. In general, this is the first report that NCTD could induce apoptosis of Tregs and enhance the vaccine-induced immunity.
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Antineoplásicos/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Vacunas contra el Cáncer/farmacología , Neoplasias de la Próstata/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Humanos , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Masculino , RatonesRESUMEN
Interferon (IFN) regulates immune responses and antitumor activity. Arginine-glycine-aspartic acid (RGD) peptides can specifically bind to integrin αvß3, a transmembrane receptor that is highly expressed on the surface of various cancer cells. In this study, we expressed recombinant RGD-IFN-α2a-core fusion proteins and assessed their antitumor activity in vitro. Two RGD-IFN-α2a-core fusion proteins and a negative control protein were expressed in vitro. These two RGD-IFN-α2a-core fusion proteins could bind the tumor cell surface specifically and did not bind to normal cells. RGD-IFN-α2a-core fusion protein treatment of tumor cells significantly reduced cell viability and induced apoptosis in a dose-dependent manner. At the 'mRNA' level, both proteins could upregulate CASP3 expression. These data indicate that both laboratory-engineered RGD-IFN-α2a-core fusion proteins could bind the surface of tumor cells and induce apoptosis in vitro. Further studies will investigate the in-vivo antitumor activities of the RGD-IFN-α2a-core fusion proteins.
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Antineoplásicos/farmacología , Interferón-alfa/farmacología , Oligopéptidos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Células A549 , Apoptosis/efectos de los fármacos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Línea Celular Tumoral , Clonación Molecular , Neoplasias del Colon/tratamiento farmacológico , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Neoplasias Pulmonares/tratamiento farmacológico , Oligopéptidos/biosíntesis , Oligopéptidos/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The mechanisms that control invariant natural killer T (iNKT)-cell development and function are still poorly understood. The mechanistic or mammalian target of rapamycin (mTOR) integrates various environmental signals/cues to regulate cell growth, proliferation, metabolism, and survival. We report here that ablation of mTOR complex 1 (mTORC1) signaling by conditionally deleting Raptor causes severe defects in iNKT-cell development at early stages, leading to drastic reductions in iNKT-cell numbers in the thymus and periphery. In addition, loss of Raptor impairs iNKT-cell proliferation and production of cytokines upon α-galactosylceramide stimulation in vitro and in vivo, and inhibits liver inflammation in an iNKT cell-mediated hepatitis model. Furthermore, Raptor deficiency and rapamycin treatment lead to aberrant intracellular localization and functional impairment of promyelocytic leukemia zinc-finger, a transcription factor critical for iNKT-cell development and effector programs. Our findings define an essential role of mTORC1 to direct iNKT-cell lineage development and effector function.
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Diferenciación Celular/inmunología , Factores de Transcripción de Tipo Kruppel/inmunología , Complejos Multiproteicos/inmunología , Células T Asesinas Naturales/inmunología , Serina-Treonina Quinasas TOR/inmunología , Timocitos/inmunología , Análisis de Varianza , Animales , Western Blotting , Trasplante de Médula Ósea , Bromodesoxiuridina , Muerte Celular/inmunología , Proliferación Celular , Inmunoprecipitación de Cromatina , Cartilla de ADN/genética , Citometría de Flujo , Genes Codificadores de los Receptores de Linfocitos T/genética , Espacio Intranuclear/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteína de la Leucemia Promielocítica con Dedos de Zinc , Reacción en Cadena en Tiempo Real de la Polimerasa , Estadísticas no Paramétricas , Timocitos/citologíaRESUMEN
Nucleolin is a ubiquitously expressed protein and participates in many important biological processes, such as cell cycle regulation and ribosomal biogenesis. The activity of nucleolin is regulated by intracellular localization and post-translational modifications, including phosphorylation, methylation, and ADP-ribosylation. Small ubiquitin-like modifier (SUMO) is a category of recently verified forms of post-translational modifications and exerts various effects on the target proteins. In the studies reported here, we discovered SUMOylational modification of human nucleolin protein at Lys-294, which facilitated the mRNA binding property of nucleolin by maintaining its nuclear localization. In response to arsenic exposure, nucleolin-SUMO was induced and promoted its binding with gadd45α mRNA, which increased gadd45α mRNA stability and protein expression, subsequently causing GADD45α-mediated cell death. On the other hand, ectopic expression of Mn-SOD attenuated the arsenite-generated superoxide radical level, abrogated nucleolin-SUMO, and in turn inhibited arsenite-induced apoptosis by reducing GADD45α expression. Collectively, our results for the first time demonstrate that nucleolin-SUMO at K294R plays a critical role in its nucleus sequestration and gadd45α mRNA binding activity. This novel biological function of nucleolin is distinct from its conventional role as a proto-oncogene. Therefore, our findings here not only reveal a new modification of nucleolin protein and its novel functional paradigm in mRNA metabolism but also expand our understanding of the dichotomous roles of nucleolin in terms of cancer development, which are dependent on multiple intracellular conditions and consequently the appropriate regulations of its modifications, including SUMOylation.
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Arsenitos/farmacología , Proteínas de Ciclo Celular/biosíntesis , Proteínas Nucleares/biosíntesis , Fosfoproteínas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Sumoilación/efectos de los fármacos , Teratógenos/farmacología , Células 3T3 , Animales , Proteínas de Ciclo Celular/genética , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Regulación de la Expresión Génica , Células HEK293 , Humanos , Lisina/genética , Lisina/metabolismo , Ratones , Proteínas Nucleares/genética , Fosfoproteínas/genética , Procesamiento Proteico-Postraduccional/genética , Proto-Oncogenes Mas , Estabilidad del ARN/genética , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Sumoilación/genética , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , NucleolinaRESUMEN
UNLABELLED: Both HIV-1 virions and infected cells use their surface regulators of complement activation (RCA) to resist antibody-dependent complement-mediated lysis (ADCML). Blockage of the biological function of RCA members, particularly CD59 (a key RCA member that controls formation of the membrane attack complex at the terminal stage of the complement activation cascades via all three activation pathways), has rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by anti-Env antibodies (Abs) or sera/plasma from patients at different stages of viral infection. In the current study, we used the well-characterized anti-HIV-1 neutralizing Abs (nAbs), including 2G12, 2F5, and 4E10, and non-nAbs, including 2.2C, A32, N5-i5, and N12-i15, to investigate whether the enhancement of ADCML by blockage of CD59 function is mediated by nAbs, non-nAbs, or both. We found that all nAbs and two non-nAbs (N5-i5 and A32) strongly reacted to three HIV-1 laboratory strains (R5, X4, and R5/X4), six primary isolates, and provirus-activated ACH-2 cells examined. In contrast, two non-nAbs, 2.2C and N12-i15, reacted weakly and did not react to these targets, respectively. After blockage of CD59 function, the reactive Abs, regardless of their neutralizing activities, significantly enhanced specific ADCML of HIV-1 virions (both laboratory strains and primary isolates) and provirus-activated latently infected cells. The ADMCL efficacy positively correlated with the enzyme-linked immunosorbent assay-reactive intensity of those Abs with their targets. Thus, blockage of RCA function represents a novel approach to restore activities of both nAbs and non-nAbs in triggering ADCML of HIV-1 virions and provirus-activated latently infected cells. IMPORTANCE: There is a renewed interest in the potential role of non-nAbs in the control of HIV-1 infection. Our data, for the first time, demonstrated that blockage of the biological function of RCA members rendered both HIV-1 virions and infected cells sensitive to ADCML mediated by not only nAbs but also non-nAbs. Our results are significant in developing novel immune-based approaches to restore the functions of nAbs and non-nAbs in the circulation of HIV-1-infected individuals to specifically target and clear HIV-1 virions and infected cells. Our data also provide new insights into the mechanisms by which HIV-1 virions and infected cells escape Ab-mediated immunity and could aid in the design and/or development of therapeutic HIV-1 vaccines. In addition, a combination of antiretroviral therapy with RCA blockage, provirus activators, and therapeutic vaccines may represent a novel approach to eliminate HIV-1 reservoirs, i.e., the infected cells harboring replication-competent proviruses and residual viremia.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD59/inmunología , Proteínas del Sistema Complemento/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/fisiología , Provirus/inmunología , Virión/inmunología , Latencia del Virus/inmunología , Femenino , Humanos , MasculinoRESUMEN
Development of effective immune therapies for cancer patients requires better understanding of hurdles that prevent the generation of effective antitumor immune responses. Administration of α-galactosylceramide (α-GalCer) in animals enhances antitumor immunity via activation of the invariant NKT (iNKT) cells. However, repeated injections of α-GalCer result in long-term unresponsiveness or anergy of iNKT cells, severely limiting its efficacy in tumor eradication. The mechanisms leading to iNKT cell anergy remain poorly understood. We report in this study that the tuberous sclerosis 1 (TSC1), a negative regulator of mTOR signaling, plays a crucial role in iNKT cell anergy. Deficiency of TSC1 in iNKT cells results in resistance to α-GalCer-induced anergy, manifested by increased expansion of and cytokine production by iNKT cells in response to secondary Ag stimulation. It is correlated with impaired upregulation of programmed death-1, Egr2, and Grail. Moreover, TSC1-deficient iNKT cells display enhanced antitumor immunity in a melanoma lung metastasis model. Our data suggest targeting TSC1/2 as a strategy for boosting antitumor immune therapy.
Asunto(s)
Anergia Clonal/inmunología , Melanoma Experimental/inmunología , Células T Asesinas Naturales/inmunología , Esclerosis Tuberosa/inmunología , Animales , Línea Celular Tumoral , Citocinas/inmunología , Citocinas/metabolismo , Proteína 2 de la Respuesta de Crecimiento Precoz/genética , Proteína 2 de la Respuesta de Crecimiento Precoz/inmunología , Proteína 2 de la Respuesta de Crecimiento Precoz/metabolismo , Citometría de Flujo , Expresión Génica/inmunología , Homeostasis/genética , Homeostasis/inmunología , Immunoblotting , Inmunoterapia Adoptiva , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/secundario , Melanoma Experimental/patología , Melanoma Experimental/terapia , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Células T Asesinas Naturales/metabolismo , Células T Asesinas Naturales/trasplante , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Receptor de Muerte Celular Programada 1/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerosis Tuberosa/genética , Esclerosis Tuberosa/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/inmunología , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Latently HIV-1-infected cells are recognized as the last barrier toward viral eradication and cure. To purge these cells, we combined a provirus stimulant with a blocker of human CD59, a key member of the regulators of complement activation, to trigger Ab-dependent complement-mediated lysis. Provirus stimulants including prostratin and histone deacetylase inhibitors such as romidepsin and suberoylanilide hydroxamic acid activated proviruses in the latently HIV-1-infected T cell line ACH-2 as virion production and viral protein expression on the cell surface were induced. Romidepsin was the most attractive provirus stimulant as it effectively activated proviruses at nanomolar concentrations that can be achieved clinically. Antiretroviral drugs including two protease inhibitors (atazanavir and darunavir) and an RT inhibitor (emtricitabine) did not affect the activity of provirus stimulants in the activation of proviruses. However, saquinavir (a protease inhibitor) markedly suppressed virus production, although it did not affect the percentage of cells expressing viral Env on the cell surface. Provirus-activated ACH-2 cells expressed HIV-1 Env that colocalized with CD59 in lipid rafts on the cell surface, facilitating direct interaction between them. Blockage of CD59 rendered provirus-activated ACH-2 cells and primary human CD4(+) T cells that were latently infected with HIV-1 sensitive to Ab-dependent complement-mediated lysis by anti-HIV-1 polyclonal Abs or plasma from HIV-1-infected patients. Therefore, a combination of provirus stimulants with regulators of complement activation blockers represents a novel approach to eliminate HIV-1.
Asunto(s)
Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD59/inmunología , Activación de Complemento , Proteínas del Sistema Complemento/inmunología , Anticuerpos Anti-VIH/inmunología , Infecciones por VIH/inmunología , VIH-1/inmunología , Provirus/inmunología , Adolescente , Adulto , Fármacos Anti-VIH/administración & dosificación , Línea Celular , Femenino , Proteínas gp160 de Envoltorio del VIH/inmunología , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Myosin light chain phosphatase with its regulatory subunit, myosin phosphatase target subunit 1 (MYPT1) modulates Ca(2+)-dependent phosphorylation of myosin light chain by myosin light chain kinase, which is essential for smooth muscle contraction. The role of MYPT1 in vascular smooth muscle was investigated in adult MYPT1 smooth muscle specific knock-out mice. MYPT1 deletion enhanced phosphorylation of myosin regulatory light chain and contractile force in isolated mesenteric arteries treated with KCl and various vascular agonists. The contractile responses of arteries from knock-out mice to norepinephrine were inhibited by Rho-associated kinase (ROCK) and protein kinase C inhibitors and were associated with inhibition of phosphorylation of the myosin light chain phosphatase inhibitor CPI-17. Additionally, stimulation of the NO/cGMP/protein kinase G (PKG) signaling pathway still resulted in relaxation of MYPT1-deficient mesenteric arteries, indicating phosphorylation of MYPT1 by PKG is not a major contributor to the relaxation response. Thus, MYPT1 enhances myosin light chain phosphatase activity sufficient for blood pressure maintenance. Rho-associated kinase phosphorylation of CPI-17 plays a significant role in enhancing vascular contractile responses, whereas phosphorylation of MYPT1 in the NO/cGMP/PKG signaling module is not necessary for relaxation.
Asunto(s)
Músculo Liso Vascular/fisiología , Quinasa de Cadena Ligera de Miosina/fisiología , Animales , Presión Sanguínea/fisiología , Femenino , Hipertensión/etiología , Hipertensión/fisiopatología , Péptidos y Proteínas de Señalización Intracelular , Masculino , Arterias Mesentéricas/fisiología , Ratones , Ratones Noqueados , Proteínas Musculares/metabolismo , Cadenas Ligeras de Miosina/metabolismo , Quinasa de Cadena Ligera de Miosina/deficiencia , Quinasa de Cadena Ligera de Miosina/genética , Fosfatasa de Miosina de Cadena Ligera , Óxido Nítrico/metabolismo , Fosfoproteínas/metabolismo , Fosforilación , Transducción de Señal , Vasoconstricción/fisiología , Vasodilatación/fisiologíaRESUMEN
Klf4 is a transcription factor of the family of Kruppel-like factors and plays important roles in stem cell biology; however, its function during embryogenesis is unknown. Here, we report the characterization of a Klf4 homologue in Xenopus laevis during embryogenesis. Klf4 is transcribed both maternally and zygotically and the transcript is ubiquitous in embryos during germ-layer formation. Klf4 promotes endoderm differentiation in both Nodal/Activin-dependent and -independent manners. Moreover, Klf4 regulates anteroposterior body axis patterning via activation of a subset of genes in the Spemann organizer, such as Noggin, Dkk1 and Cerberus, which encode Nodal, Wnt and BMP antagonists. Loss of Klf4 function leads to the failure of germ-layer differentiation, the loss of responsiveness of early embryonic cells to inducing signals, e.g. Nodal/Activin, and the loss of transcription of genes involved in axis patterning. We conclude that Klf4 is required for germ-layer differentiation and body axis patterning by means of rendering early embryonic cells competent to differentiation signals.
Asunto(s)
Tipificación del Cuerpo/fisiología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario/fisiología , Factores de Transcripción de Tipo Kruppel/metabolismo , Proteínas de Xenopus/metabolismo , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Embrión no Mamífero/citología , Desarrollo Embrionario/genética , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMEN
We developed a novel trimeric sTNFRII fusion protein, named sTNFRII-gAD, which exhibited a higher in vitro antagonistic efficacy for TNFα in comparison with sTNFRII-Fc. This study aimed to investigate the arthritic protection of sTNFRII-gAD in a rat collagen-induced arthritis (CIA). The rats were injected intradermally with collagen type II at days 0 and 7. Three days after the second injection (day 10), the rats were intraperitoneally given sTNFRII-gAD or sTNFRII-Fc, or PBS. Effects of treatments were examined with respect of CIA incidence, severity and pathological changes. Serum TNFα, IL-17A and regulatory T cell (Treg) in periphery were determined at days 10 and 16, respectively. Our results showed that sTNFRIIgAD significantly reduced CIA incidence and severity (p < 0.05); meanwhile it led to a dramatic improvement in cartilage and bone damage. Moreover, the increase in serum anti-CII and IL-17A, and the reduction in Treg population were inhibited (p < 0.05) by sTNFRII-gAD or sTNFRII-Fc. Serum TNFα was found to be accumulated in the groups treated with sTNFRII-gAD or sTNFRII-Fc compared with the group treated with PBS (p < 0.05). It is noteworthy that sTNFRII-gAD displayed a better efficacy than sTNFRII-Fc in CIA incidence, pathological changes in cartilage and the elevation of anti-CII antibody, indicating that sTNFRII-gAD is potentially a more efficacious anti-TNFα agent for rheumatoid arthritis.