RESUMEN
We developed a lipid nanoparticle formulation (LNPK15) to deliver siRNA to a tumor for target gene knock down. LNPK15 is highly PEGylated with 3.3% 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-(polyethylene glycol-2000) (PEG-DSPE) and shows a long duration: the half-lives of siRNA in LNPK15 were 15.2 and 27.0h in mice and monkeys, respectively. Although LNPK15 encapsulating KRAS-targeting siRNA (LNPK15/KRAS) had very weak KRAS gene knock down activity in MIA PaCa-2 cells in vitro, LNPK15/KRAS showed a strong anti-tumor efficacy in MIA PaCa-2 tumor xenograft mice after intravenous administration at 5mg/kg twice weekly. KRAS mRNA and protein knock down was observed in tumor tissue, suggesting on-target anti-tumor efficacy. In order to elucidate the in vitro-in vivo discrepancy, we performed ex vivo knock down assay using serum samples obtained after intravenous administration of LNPK15/KRAS to mice and monkeys. The collected samples were added to MIA PaCa-2 cells, and KRAS gene knock down was evaluated after a 24-h incubation period. The knock down efficacy was weak (≈20%) with serum samples at initial sampling point (2h), and it became much stronger (â¼90%) with serum samples at later time points. Lipid composition of LNPK15 in the serum samples was also investigated. Among the five lipids incorporated in LNPK15, PEG-DSPE was degraded more rapidly than siRNA and the other lipids in both mice and monkeys. In vitro lipase treatment of LNPK15/KRAS also hydrolyzed PEG-DSPE and enhanced knock down activity. From these results, it was concluded that LNPK15 acquires increased knock down activity after undergoing PEG-DSPE hydrolysis in vivo, and that is the key mechanism to achieve both long circulation and potent knock down efficiency. We also proposed an in vitro assay system using lipase for quality control of LNP to ensure biological activity.
Asunto(s)
Nanopartículas/administración & dosificación , Neoplasias/terapia , Fosfatidiletanolaminas/administración & dosificación , Polietilenglicoles/administración & dosificación , Proteínas Proto-Oncogénicas p21(ras)/genética , ARN Interferente Pequeño/administración & dosificación , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Humanos , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Ratones SCID , Neoplasias/genética , Neoplasias/metabolismo , Fosfatidiletanolaminas/farmacocinética , Polietilenglicoles/farmacocinética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , ARN Interferente Pequeño/farmacocinéticaRESUMEN
Patients treated with immune checkpoint blockade (ICB) sometimes experience immune-related adverse events (irAEs), requiring immuno-suppressive drugs such as corticosteroids despite the possibility that immunosuppression may impair the antitumor effects of ICB. Here, we address the dilemma of using corticosteroids for the treatment of irAEs induced by ICB. ICB augments neoantigen-specific CD8+ T cell responses, resulting in tumor regression. In our model, simultaneous, but not late, administration of corticosteroids impaired antitumor responses with reduction of CD8+ T cell proliferation. Secondary challenge using tumors with/without the neoantigen showed selective progression in tumors lacking the neoantigen when corticosteroids were administered. Corticosteroids decreased low- but not high-affinity memory T cells by suppressing fatty acid metabolism essential for memory T cells. In a small cohort of human melanoma patients, overall survival was shorter after treatment with CTLA-4 blockade in patients who received early corticosteroids or had low tumor mutation burden. Together, low-affinity memory T cells are dominantly suppressed by corticosteroids, necessitating careful and thoughtful corticosteroid use.
Asunto(s)
Corticoesteroides/farmacología , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Memoria Inmunológica , Inmunosupresores/farmacología , Animales , Biomarcadores , Linfocitos T CD8-positivos/metabolismo , Antígeno CTLA-4/antagonistas & inhibidores , Antígeno CTLA-4/metabolismo , Diferenciación Celular/inmunología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Melanoma/diagnóstico , Melanoma/tratamiento farmacológico , Melanoma/etiología , Melanoma/metabolismo , Ratones , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Receptor de Muerte Celular Programada 1/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismoRESUMEN
One of the extraordinary aspects of nonlinear wave evolution which has been observed as the spontaneous occurrence of astonishing and statistically extraordinary amplitude wave is called rogue wave. We show that the eigenvalues of the associated equation of nonlinear Schrödinger equation are almost constant in the vicinity of rogue wave and we validate that optical rogue waves are formed by the collision between quasi-solitons in anomalous dispersion fiber exhibiting weak third order dispersion.
RESUMEN
We recently reported that repeated 0.5-Gy gamma irradiation attenuates the pathology of collagen-induced arthritis. In this study, to investigate the mechanism further, we focused on changes in Treg/Th17 cells and changes in the production of antibody against an external antigen in response to gamma irradiation as well as on the radiosensitivity of Treg cells. DBA/1J mice were immunized with type II collagen to induce arthritis and exposed to low-dose gamma rays (0.5 Gy/week for 5 weeks). Production of IL6 and IL17 as well as autoantibody was suppressed by irradiation in the early phase of collagen-induced arthritis. The percentage of Treg cells was significantly increased by irradiation at 4, 6 and 8 weeks after the immunization. We also investigated the effect of repeated gamma radiation on the production of antibodies against an external antigen in ovalbumin-immunized BALB/c mice. We found that repeated 0.5-Gy gamma irradiation enhanced antibody production, accompanied by an increase of the antibody-producing plasma cell population and increased Th2-type cytokine secretion. We also found that the radiosensitivity of Treg cells did not differ from that of other T cells. These results suggest that a major mechanism of attenuation of the pathology of collagen-induced arthritis by repeated 0.5-Gy gamma irradiation is up-regulation of Treg cells concomitantly with suppression of IL6 and IL17 production.
Asunto(s)
Artritis Experimental/inmunología , Colágeno/toxicidad , Rayos gamma , Linfocitos T Reguladores/efectos de la radiación , Regulación hacia Arriba/efectos de la radiación , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBARESUMEN
Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Stimulation of TCR induces ATP release from T cells through gap junction hemichannels and maxianion channels, at least in part. However, the mechanisms of ATP release from activated T cells are not fully understood. Here, we studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that not only anion channel and gap junction hemichannel inhibitors, but also exocytosis inhibitors suppressed the ATP release. These results suggest that ATP release from murine T cells is regulated by various mechanisms, including exocytosis. An inhibitor of exocytosis, bafilomycin A, significantly blocked TCR signaling, such as Ca(2+) elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y(6) receptor antagonist significantly inhibited production of pro-inflammatory cytokines from external antigen-restimulated splenocytes, indicating that vesicular exocytosis-mediated purinergic signaling has a significant role in TCR-dependent cytokine production. We also detected vesicular ATP in murine T cells and human T lymphoma Jurkat cells, both of which also expressed mRNA of SLC17A9, a vesicular nucleotide transporter. Knockdown of SLC17A9 in Jurkat cells markedly reduced ATP release and cytosolic Ca(2+) elevation after TCR stimulation, suggesting involvement of SLC17A9-dependent vesicular exocytosis in ATP release and T cell activation. In conclusion, vesicular exocytosis of ATP appears to play a role in T cell activation and immune responses.
Asunto(s)
Adenosina Trifosfato/química , Proteínas de Transporte de Nucleótidos/química , Linfocitos T/metabolismo , Animales , Calcio/química , Citosol/metabolismo , Exocitosis , Humanos , Interleucina-2/metabolismo , Células Jurkat , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Receptores de Antígenos de Linfocitos TRESUMEN
Extracellular nucleotides and their metabolites activate ionotropic P2X and metabotropic P2Y receptors on the surface of various types of cells. Here, we investigated the involvement of P2X and P2Y receptor-mediated signaling in TCR-dependent T cell activation. Murine T cells were activated by stimulation of TCR, and both CD25 expression and interleukin (IL)-2 production were observed in activated T cells. Ecto-nucleotidase apyrase and P2Y6 antagonist MRS2578 significantly blocked the increases of both CD25 expression and IL-2 production, and P2X7 antagonists A438079 and oxidized ATP inhibited IL-2 production rather than CD25 expression, suggesting the involvement of P2Y6 and P2X7 receptors in different processes of T cell activation. MRS2578 also blocked TCR-dependent elevation of cytosolic Ca2+ in T cells. The P2X7 and P2Y6 receptors were expressed in murine CD4 T cells. In conclusion, our results indicate that activation of P2Y6 and P2X7 receptors contributes to T cell activation via TCR.