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MIL77-3 is one component of antibody cocktail that is produced in our lab and represents an effective regimen for animals suffering from Zaire Ebolavirus (EBOV) infection. MIL77-3 is engineered to increase its affinity for the FcγRIIIa (CD16a) by deleting the fucose in the framework region. The potential effects of this modification on host immune responses, however, remain largely unknown. Herein, we demonstrated that MIL77-3 recognized secreted glycoproptein (sGP), produced by EBOV, and formed the immunocomplex to potently augment antibody-dependent cytotoxicity of human peripheral blood-derived natural killer cells (pNKs), including CD56dim and CD56bright subpopulations, in contrast to the counterparts (Mab114, rEBOV548, fucosylated MIL77-3). Intriguingly, this effect was not observed when NK92-CD16a cell line was utilized and restored by the addition of beads-coupled or membrane-anchored sGP in combination with MIL77-3. Furthermore, sGP bound to unrecognized receptors on T cells contaminated in pNKs rather than NK92-CD16a cells. Administration of beads-coupled sGP/MIL77-3 complex in mice elicited NK activation. Overall, this work reveals an immune-stimulating function of sGP/MIL77-3 complex by triggering cytotoxic activity of NK cells, highlighting the necessity to evaluate the potential impact of MIL77-3 on host immune reaction in clinical trials. IMPORTANCE: Zaire Ebolavirus (EBOV) is highly lethal and causes sporadic outbreaks. The passive administration of monoclonal antibodies (mAbs) represents a promising treatment regimen against EBOV. Mounting evidence has shown that the efficacy of a subset of therapeutic mAbs in vivo is intimately associated with its capacity to trigger NK activity, supporting glycomodification of Fc region of anti-EBOV mAbs as a putative strategy to enhance Fc-mediated immune effector function as well as protection in vivo. Our work here uncovers the potential harmful influence of this modification on host immune responses, especially for mAbs with cross-reactivity to secreted glycoproptein (sGP) (e.g., MIL77-3), and highlights it is necessary to evaluate the NK-stimulating activity of a fucosylated mAb engaged with sGP when a new candidate is developed.
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Anticuerpos Antivirales , Citotoxicidad Celular Dependiente de Anticuerpos , Ebolavirus , Fiebre Hemorrágica Ebola , Células Asesinas Naturales , Receptores de IgG , Células Asesinas Naturales/inmunología , Humanos , Animales , Ebolavirus/inmunología , Receptores de IgG/inmunología , Receptores de IgG/metabolismo , Ratones , Fiebre Hemorrágica Ebola/inmunología , Fiebre Hemorrágica Ebola/virología , Anticuerpos Antivirales/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Fucosa , Línea CelularRESUMEN
The cytokine interleukin-6 (IL-6) plays a crucial role in autoimmune and inflammatory diseases. Understanding the precise mechanism of IL-6 interaction at the amino acid level is essential to develop IL-6-inhibiting compounds. In this study, we employed computer-guided drug design tools to predict the key residues that are involved in the interaction between IL-6 and its receptor IL-6R. Subsequently, we generated IL-6 mutants and evaluated their binding affinity to IL-6R and the IL-6R - gp130 complex, as well as monitoring their biological activities. Our findings revealed that the R167A mutant exhibited increased affinity for IL-6R, leading to enhanced binding to IL-6R - gp130 complex and subsequently elevated intracellular phosphorylation of STAT3 in effector cells. On the other hand, although E171A reduced its affinity for IL-6R, it displayed stronger binding to the IL-6R - gp130 complex, thereby enhancing its biological activity. Furthermore, we identified the importance of R178 and R181 for the precise recognition of IL-6 by IL-6R. Mutants R181A/V failed to bind to IL-6R, while maintaining an affinity for the IL-6 - gp130 complex. Additionally, deletion of the D helix resulted in complete loss of IL-6 binding affinity for IL-6R. Overall, this study provides valuable insights into the binding mechanism of IL-6 and establishes a solid foundation for future design of novel IL-6 inhibitors.
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Interleucina-6 , Simulación del Acoplamiento Molecular , Unión Proteica , Receptores de Interleucina-6 , Interleucina-6/metabolismo , Interleucina-6/genética , Humanos , Receptores de Interleucina-6/metabolismo , Receptores de Interleucina-6/genética , Receptores de Interleucina-6/química , Receptor gp130 de Citocinas/metabolismo , Receptor gp130 de Citocinas/genética , Receptor gp130 de Citocinas/química , Mutagénesis Sitio-Dirigida , Sitios de Unión , Factor de Transcripción STAT3/metabolismo , Fosforilación , MutaciónRESUMEN
Background: CD2v, a critical outer envelope glycoprotein of the African swine fever virus (ASFV), plays a central role in the hemadsorption phenomenon during ASFV infection and is recognized as an essential immunoprotective protein. Monoclonal antibodies (mAbs) targeting CD2v have demonstrated promise in both diagnosing and combating African swine fever (ASF). The objective of this study was to develop specific monoclonal antibodies against CD2v. Methods: In this investigation, Recombinant CD2v was expressed in eukaryotic cells, and murine mAbs were generated through meticulous screening and hybridoma cloning. Various techniques, including indirect enzyme-linked immunosorbent assay (ELISA), western blotting, immunofluorescence assay (IFA), and bio-layer interferometry (BLI), were employed to characterize the mAbs. Epitope mapping was conducted using truncation mutants and epitope peptide mapping. Results: An optimal antibody pair for a highly sensitive sandwich ELISA was identified, and the antigenic structures recognized by the mAbs were elucidated. Two linear epitopes highly conserved in ASFV genotype II strains, particularly in Chinese endemic strains, were identified, along with a unique glycosylated epitope. Three mAbs, 2B25, 3G25, and 8G1, effectively blocked CD2v-induced NF-κB activation. Conclusions: This study provides valuable insights into the antigenic structure of ASFV CD2v. The mAbs obtained in this study hold great potential for use in the development of ASF diagnostic strategies, and the identified epitopes may contribute to vaccine development against ASFV.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Anticuerpos Monoclonales , Mapeo Epitopo , FN-kappa B , Animales , Virus de la Fiebre Porcina Africana/inmunología , FN-kappa B/metabolismo , FN-kappa B/inmunología , Porcinos , Ratones , Fiebre Porcina Africana/inmunología , Fiebre Porcina Africana/virología , Anticuerpos Monoclonales/inmunología , Proteínas del Envoltorio Viral/inmunología , Epítopos/inmunología , Anticuerpos Antivirales/inmunología , Ratones Endogámicos BALB CRESUMEN
Natural killer (NK) cells represent key player in immune surveillance to eliminate transformed or malignant cells. One of mechanisms of action of NK cells is antibody-dependent cell-mediated cytotoxicity (ADCC) by recognizing tumor antigens on the surface of cancer cells. However, the heterogeneity of tumor antigens and the scarcity of membrane surface targets significantly restrict this strategy. Recently, we constructed a new cargo by tethering a low pH insertion peptide (pHLIP) to the C terminus of the ectodomain of programed death ligand-1 (PD-L1) and demonstrated its ability to modulate immune responses. Herein, the potential application of PD-L1-pHLIP in cancer therapy was determined. pHLIP tethering had no effect on the binding capacity of PD-L1 protein to an anti-PD-L1 antibody (i.e. avelumab). Association of pHLIP rendered PD-L1 segment display on the surface of cellular membrane in the acidic buffer instead of the neutral solution. Importantly, plate-coated or beads-coupled PD-L1-pHLIP enable robust activation and expression of cytotoxic mediators of NK cells via engaging avelumab. Overall, this work provides proof of concept that recombinant PD-L1 protein decorated on the cellular membrane driven by pHLIP in combination with appropriate monoclonal antibody has potentials to elicit NK cytotoxicity, which may represent a novel and promising therapeutic avenue in cancer.
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Influenza A viruses (IAVs) continue to pose a huge threat to public health, and their prevention and treatment remain major international issues. Neuraminidase (NA) is the second most abundant surface glycoprotein on influenza viruses, and antibodies to NA have been shown to be effective against influenza infection. In this study, we generated a monoclonal antibody (mAb), named FNA1, directed toward N1 NAs. FNA1 reacted with H1N1 and H5N1 NA, but failed to react with the NA proteins of H3N2 and H7N9. In vitro, FNA1 displayed potent antiviral activity that mediated both NA inhibition (NI) and blocking of pseudovirus release. Moreover, residues 219, 254, 358, and 388 in the NA protein were critical for FNA1 binding to H1N1 NA. However, further validation is necessary to confirm whether FNA1 mAb is indeed a good inhibitor against NA for application against H1N1 and H5N1 viruses.
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Anticuerpos Monoclonales , Subtipo H1N1 del Virus de la Influenza A , Neuraminidasa , Neuraminidasa/inmunología , Neuraminidasa/metabolismo , Neuraminidasa/antagonistas & inhibidores , Anticuerpos Monoclonales/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Humanos , Animales , Anticuerpos Antivirales/inmunología , Ratones , Subtipo H5N1 del Virus de la Influenza A/inmunología , Ratones Endogámicos BALB C , Antivirales/farmacología , Proteínas Virales/inmunología , Proteínas Virales/metabolismo , Subtipo H3N2 del Virus de la Influenza A/inmunología , Subtipo H7N9 del Virus de la Influenza A/inmunologíaRESUMEN
Marburg virus (MARV) is one of the filovirus species that cause deadly hemorrhagic fever in humans, with mortality rates up to 90%. Neutralizing antibodies represent ideal candidates to prevent or treat virus disease. However, no antibody has been approved for MARV treatment to date. In this study, we identified a novel human antibody named AF-03 that targeted MARV glycoprotein (GP). AF-03 possessed a high binding affinity to MARV GP and showed neutralizing and protective activities against the pseudotyped MARV in vitro and in vivo. Epitope identification, including molecular docking and experiment-based analysis of mutated species, revealed that AF-03 recognized the Niemann-Pick C1 (NPC1) binding domain within GP1. Interestingly, we found the neutralizing activity of AF-03 to pseudotyped Ebola viruses (EBOV, SUDV, and BDBV) harboring cleaved GP instead of full-length GP. Furthermore, NPC2-fused AF-03 exhibited neutralizing activity to several filovirus species and EBOV mutants via binding to CI-MPR. In conclusion, this work demonstrates that AF-03 represents a promising therapeutic cargo for filovirus-caused disease.
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Ebolavirus , Fiebre Hemorrágica Ebola , Marburgvirus , Humanos , Anticuerpos Antivirales , Simulación del Acoplamiento Molecular , Glicoproteínas , Fiebre Hemorrágica Ebola/prevención & control , Ebolavirus/químicaRESUMEN
Marburg virus (MARV), one member of the Filoviridae family, cause sporadic outbreaks of hemorrhagic fever with high mortality rates. No countermeasures are currently available for the prevention or treatment of MARV infection. Monoclonal antibodies (mAbs) are promising candidates to display high neutralizing activity against MARV infection in vitro and in vivo. Recently, growing evidence has shown that immune effector function including antibody-dependent cell-mediated cytotoxicity (ADCC) is also required for in vivo efficacy of a panel of antibodies. Glyco-engineered methods are widely utilized to augment ADCC function of mAbs. In this study, we generated a fucose-knockout MARV GP-specific mAb named AF-04 and showed that afucosylation dramatically increased its binding affinity to polymorphic FcγRIIIa (F176/V176) compared with the parental AF-03. Accordingly, AF-04-mediated NK cell activation and NFAT expression downstream of FcγRIIIa in effector cells were also augmented. In conclusion, this work demonstrates that AF-04 represents a novel avenue for the treatment of MARV-caused disease.
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Marburgvirus , Anticuerpos Monoclonales/uso terapéuticoRESUMEN
Smallpox is an infectious disease caused by the variola virus, and it has a high mortality rate. Historically it has broken out in many countries and it was a great threat to human health. Smallpox was declared eradicated in 1980, and Many countries stopped nation-wide smallpox vaccinations at that time. In recent years the potential threat of bioterrorism using smallpox has led to resumed research on the treatment and prevention of smallpox. Effective ways of preventing and treating smallpox infection have been reported, including vaccination, chemical drugs, neutralizing antibodies, and clinical symptomatic therapies. Antibody treatments include anti-sera, murine monoclonal antibodies, and engineered humanized or human antibodies. Engineered antibodies are homologous, safe, and effective. The development of humanized and genetically engineered antibodies against variola virus via molecular biology and bioinformatics is therefore a potentially fruitful prospect with respect to field application. Natural smallpox virus is inaccessible, therefore most research about prevention and/or treatment of smallpox were done using vaccinia virus, which is much safer and highly homologous to smallpox. Herein we summarize vaccinia virus epitope information reported to date, and discuss neutralizing antibodies with potential value for field application.
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In this study, we test the therapeutic effects of rapamycin in a murine model of SLE-like experimental lupus nephritis induced by chronic graft-versus-host disease (cGVHD). Our results suggest that rapamycin treatment reduced autoantibody production, inhibited T lymphocyte and subsequent B cell activation, and reduced inflammatory cytokine and chemokine production, thereby protecting renal function and alleviating histological lupus nephritis by reducing the occurrence of albuminuria. To explore the potential mechanism of rapamycin's reduction of kidney damage in mice with lupus nephritis, a series of functional assays were conducted. As expected, rapamycin remarkably inhibited the lymphocytes' proliferation within the morbid mice. Interestingly, significantly increased proportions of peripheral CD4+FOXP3+ and CD4+CD25high T cells were observed in rapamycin-treated group animals, suggesting an up-regulation of regulatory T cells (Tregs) in the periphery by rapamycin treatment. Furthermore, consistent with the results regarding changes in mRNA abundance in kidney by real-time PCR analysis, intracellular cytokine staining demonstrated that rapamycin treatment remarkably diminished the secretion of Th1 and Th2 cytokines, including IFN-γ, IL-4 and IL-10, in splenocytes of the morbid mice. However, the production of IL-2 from splenocytes in rapamycin-treated mice was significantly higher than in the cells from control group animals. These findings suggest that rapamycin treatment might alleviate systemic lupus erythematosus (SLE)-like experimental lupus nephritis through the recovery of IL-2 production, which promotes the expansion of regulatory T cells while inhibiting effector T cell activation. Our studies demonstrated that, unlike other commonly used immunosuppressants, rapamycin does not appear to interfere with tolerance induction but permits the expansion and suppressive function of Tregs in vivo.
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AIM: To investigate the impact of deficiency of LIG4 gene on site-specific integration in CHO cells. RESULTS: CHO cells are considered the most valuable mammalian cells in the manufacture of biological medicines, and genetic engineering of CHO cells can improve product yield and stability. The traditional method of inserting foreign genes by random integration (RI) requires multiple rounds of screening and selection, which may lead to location effects and gene silencing, making it difficult to obtain stable, high-yielding cell lines. Although site-specific integration (SSI) techniques may overcome the challenges with RI, its feasibility is limited by the very low efficiency of the technique. Recently, SSI efficiency has been enhanced in other mammalian cell types by inhibiting DNA ligase IV (Lig4) activity, which is indispensable in DNA double-strand break repair by NHEJ. However, this approach has not been evaluated in CHO cells. In this study, the LIG4 gene was knocked out of CHO cells using CRISPR/Cas9-mediated genome editing. Efficiency of gene targeting in LIG4-/--CHO cell lines was estimated by a green fluorescence protein promoterless reporter system. Notably, the RI efficiency, most likely mediated by NHEJ in CHO, was inhibited by LIG4 knockout, whereas SSI efficiency strongly increased 9.2-fold under the precise control of the promoter in the ROSA26 site in LIG4-/--CHO cells. Moreover, deletion of LIG4 had no obvious side effects on CHO cell proliferation. CONCLUSIONS: Deficiency of LIG4 represents a feasible strategy to improve SSI efficiency and suggests it can be applied to develop and engineer CHO cell lines in the future.
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Sistemas CRISPR-Cas , Edición Génica , Animales , Células CHO , Sistemas CRISPR-Cas/genética , Cricetinae , Cricetulus , Reparación del ADN por Unión de Extremidades/genética , ADN Ligasa (ATP)/genéticaRESUMEN
Ebola virus, a member of the Filoviridae family, utilizes the attachment factors on host cells to support its entry and cause severe tissue damage. TIM-1 has been identified as a predominant attachment factor via interaction with phosphatidylserine (PS) localized on the viral envelope and glycoprotein (GP). In this study, we give the first demonstration that TIM-1 enhances the cellular entry of three species of Ebola virus, as well as those harboring GP mutations (A82V, T544I, and A82V T544I). Furthermore, two TIM-1 variants (i.e., TIM-1-359aa and TIM-1-364aa) had comparable effects on promoting Zaire Ebola virus (EBOV) attachment, internalization, and infection. Importantly, recombinant TIM-1 ectodomain (ECD) protein could decrease the infectivity of Ebola virus and display synergistic inhibitory effects with ADI-15946, a monoclonal antibody with broad neutralizing activity to Ebola virus. Of note, EBOV strains harboring GP mutations (K510E and D552N), which were refractory to antibody treatment, were still sensitive to TIM-1 protein-mediated impairment of infectivity, indicating that TIM-1 protein may represent an alternative therapeutic regimen when antibody evasion occurs. IMPORTANCE The viral genome has acquired numerous mutations with the potential to increase transmission during the 2013-to-2016 outbreak of Ebola virus. EBOV strains harboring GP mutations (A82V, T544I, and A82V T544I), which have been identified to increase viral infectivity in humans, have attracted our attention. Herein, we give the first report that polymorphic TIM-1 enhances the infectivity of three species of Ebola virus, as well as those harboring GP mutations (A82V, T544I, and A82V T544I). We show that recombinant TIM-1 ECD protein could decrease the infectivity of Ebola virus with or without a point mutation and displays synergistic inhibitory effects with ADI-15946. Furthermore, TIM-1 protein potently blocked cell entry of antibody-evading Ebola virus species. These findings highlight the role of TIM-1 in Ebola virus infection and indicate that TIM-1 protein represents a potential therapeutic avenue for Ebola virus and its mutated species.
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Ebolavirus , Fiebre Hemorrágica Ebola , Ebolavirus/genética , Genoma Viral , Fiebre Hemorrágica Ebola/epidemiología , Humanos , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismo , Internalización del VirusRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Renal cell carcinoma (RCC) is the most common cancer of the urinary system, the current treatments for RCC are unsatisfactory. Paeonol is the main pharmacologically active ingredient of the traditional Chinese medicine (TCM) moutan cortex (Paeonia suffruticosa Andrews) and Paeonia albiflora Pall, and has been used in TCM to treat various diseases including cancer. However, the underlying therapeutic mechanisms of paeonol in RCC have not been investigated yet. AIM OF THE STUDY: This study aimed to explore the potential antitumor effects and mechanisms of paeonol on RCC based on network pharmacology and experimental validation. MATERIALS AND METHODS: Network pharmacological analysis was performed to predict the potential targets and mechanism of paeonol against RCC. The antitumor effects and the priority targets of paeonol against RCC were further assessed by in vitro experiments. RESULTS: 104 intersection targets shared by paeonol and RCC were collected, 15 hub genes were obtained, among these genes, VEGFA expression was higher in RCC, and the higher expression of IL-6 or lower expression of AKT1, JUN, MAPK1, and MAPK8 were correlated to the shorter overall survival (OS) in RCC patients. GO and KEGG analyses suggested that the genes were mainly enriched in the positive regulation of cell death and apoptosis pathway. In vitro experiments showed that paeonol inhibited 786-O cell proliferation, migration, invasion, and promoted apoptosis. When 786-O cells were treated with paeonol, the expression of Bax increased while Bcl-2 and VEGFA decreased. CONCLUSION: The present study demonstrated that paeonol might play an essential role in RCC by regulating cell proliferation, apoptosis, metastasis, and invasion through the Bcl-2/Bax signaling pathway and VEGFA, providing a theoretical and experimental scientific basis for future investigations of the antitumor effects of paeonol against RCC.
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Acetofenonas/farmacología , Antineoplásicos Fitogénicos/farmacología , Carcinoma de Células Renales/tratamiento farmacológico , Fitoterapia , Acetofenonas/administración & dosificación , Acetofenonas/química , Antineoplásicos Fitogénicos/química , Apoptosis , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Estructura Molecular , Farmacología en Red , Mapas de Interacción de Proteínas , Reproducibilidad de los ResultadosRESUMEN
Phagocytic resistance plays a key role in tumor-mediated immune escape, so phagocytosis immune checkpoints are a potential target for cancer immunotherapy. CD47 is one of the important phagocytosis immune checkpoints; thus, blocking the interaction between CD47 and signal regulatory protein α (SIRPα) may provide new options for cancer treatment. Using computer-aided targeted epitope mammalian cell-displayed antibody library, we screened and obtained an engineered SIRPα variant fragment crystallizable fusion protein, FD164, with higher CD47-binding activity than wild-type SIRPα Compared with wild-type SIRPα, FD164 has approximately 3-fold higher affinity for binding to CD47, which further enhanced its phagocytic effect in vitro and tumor suppressor activity in vivo. FD164 maintains the similar antitumor activity of the clinical research drug Hu5F9 in the mouse xenograft model. Furthermore, FD164 combined with rituximab can significantly improve the effect of single-agent therapy. On the other hand, compared with Hu5F9, FD164 does not cause hemagglutination, and its ability to bind to red blood cells or white blood cells is weaker at the same concentration. Finally, it was confirmed by computer structure prediction and alanine scanning experiments that the N45, E47, 52TEVYVK58, K60, 115EVTELTRE122, and E124 residues of CD47 are important for SIRPα or FD164 recognition. Briefly, we obtained a high-affinity SIRPα variant FD164 with balanced safety and effectiveness. SIGNIFICANCE STATEMENT: Up to now, few clinically marketed drugs targeting CD47 have been determined to be effective and safe. FD164, a potential signal regulatory protein α variant fragment crystallizable protein with balanced safety and effectiveness, could provide a reference for the development of antitumor drugs.
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Antígenos de Diferenciación/genética , Antígenos de Diferenciación/inmunología , Antígeno CD47/inmunología , Receptores Inmunológicos/genética , Receptores Inmunológicos/inmunología , Animales , Anticuerpos Monoclonales Humanizados/efectos adversos , Anticuerpos Monoclonales Humanizados/uso terapéutico , Antígenos de Diferenciación/efectos adversos , Antígenos de Diferenciación/química , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Linfoma de Burkitt/tratamiento farmacológico , Linfoma de Burkitt/inmunología , Linfoma de Burkitt/patología , Antígeno CD47/química , Células CHO , Línea Celular , Cricetulus , Diseño de Fármacos , Epítopos/química , Epítopos/genética , Epítopos/inmunología , Hemaglutinación/efectos de los fármacos , Inmunoterapia , Ratones SCID , Modelos Moleculares , Fagocitosis/efectos de los fármacos , Fagocitosis/inmunología , Receptores Inmunológicos/química , Proteínas Recombinantes de Fusión/efectos adversos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Rituximab/uso terapéutico , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Programmed cell death-ligand 1 (PD-L1)/PD-1 axis is critical for maintenance of immune homeostasis by limiting overactivation of effector T-cell responses. The impairment of PD-L1/PD-1 signals play an important role in the pathogenesis of inflammatory diseases, making this pathway an ideal target for novel therapeutics to induce immune tolerance. Given weakly acidic environment as a putative hallmark of inflammation, in this study we designed a new cargo by linking the ectodomain of murine PD-L1 to the N terminus of pHLIPs, a low pH-responding and membrane-insertion peptide, and demonstrated its potent immune-suppressive activity. Specifically, PD-L1-pHLIP spanned the cellular membrane and perfectly recognized its ligand PD-1 in acidic buffer. Immobile PD-L1-pHLIP actively inhibited T-cell proliferation and IFN-γ production. Importantly, soluble PD-L1-pHLIP retained its function to dampen T-cell responses under acidic condition instead of neutral aqueous solution. Overall, these data suggest that PD-L1-pHLIP has potentials to be a novel therapeutic avenue for T-cell-mediated inflammatory diseases.
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Antígeno B7-H1/metabolismo , Receptor de Muerte Celular Programada 1/metabolismo , Linfocitos T/inmunología , Animales , Antígeno B7-H1/genética , Células Cultivadas , Ingeniería Genética , Células HEK293 , Humanos , Concentración de Iones de Hidrógeno , Terapia de Inmunosupresión , Activación de Linfocitos , Proteínas de la Membrana/genética , Ratones , Dominios Proteicos/genética , Transducción de SeñalRESUMEN
Highly invasive and rapidly fatal, small-cell lung cancer (SCLC) has been an insurmountable gulf since discovery. Innate immunity plays a vital role in anti-tumor response, among which macrophages contribute to an indispensable character. Here, we found that macrophage infiltration in SCLC reduced significantly in a stage-dependent manner, attributed to the decreased expression of CCL2, a potent chemoattractant for monocytes. Validated by ChIP-qPCR and MassArray methylation analysis, CCL2 expression was inhibited by EZH2-mediated H3K27me3 in the enhancer regions and DNMT1-mediated DNA methylation in the promoter regions, the process of which could be reversed by small-molecular compounds, EPZ011989 and Decitabine. Direct cell-cell contact between SCLC cells and macrophages skewed the phenotype of macrophages to be more M1-like. Furthermore, in an ectopic engraft model of SCLC, disruption of EZH2/DNMT1 function using the combination treatment of EPZ011989 and Decitabine potently abrogated the inhibition of macrophage infiltration and thus suppressed tumor growth, the effect of which was impaired by CCL2 neutralization or macrophage depletion. Overall, this work provides new insights into the role of macrophages in SCLC and establishes a rationale for constructing novel therapeutic avenues for SCLC patients.
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Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Quimiocina CCL2/genética , Neoplasias Pulmonares/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Macrófagos Asociados a Tumores/inmunología , Adolescente , Adulto , Anciano , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinogénesis/inmunología , Quimiocina CCL2/metabolismo , Islas de CpG/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/efectos de los fármacos , Metilación de ADN/inmunología , Decitabina/farmacología , Decitabina/uso terapéutico , Elementos de Facilitación Genéticos/genética , Proteína Potenciadora del Homólogo Zeste 2/antagonistas & inhibidores , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/inmunología , Femenino , Histonas/genética , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Pulmón/inmunología , Pulmón/patología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Masculino , Ratones , Persona de Mediana Edad , Fagocitosis/efectos de los fármacos , Piridonas/farmacología , Piridonas/uso terapéutico , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Análisis de Matrices Tisulares , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , Adulto JovenRESUMEN
BACKGROUND & AIMS: Methylation of lysines on histones, controlled by various methyltransferases and demethylases, is an important component of epigenetic modifications, and abnormal regulation of such enzymes serves as common events in hepatocellular carcinoma. We determined to identify important methyltransferases and demethylases that might regulate the development of hepatocellular carcinoma by bioinformatics. METHODS: The Oncomine and UALCAN databases were used to retrieve mRNA expression levels of histone lysine methyltransferases and demethylases in hepatocellular carcinoma. Data analyses of genetic alterations, mainly mutations and copy number alterations, were performed on the cBioportal platform. Protein-protein interactions were established in the STRING database. RESULTS: mRNA expression of 8 genes correlated with clinical staging and grading, whereas 4 genes indicated a role in the prognosis, all co-expressed with SEDB1 and WHSC1. Genetically, 12 genes showing an alteration rate higher than 5% were identified, and only 3 were indicative of prognosis. Copy number gains in ASH1L, SETDB1, and KDM5B might partially contribute to the upregulation of their mRNA expression. The close relationship of mutations in MLL2/MLL3 with driver gene mutations in hepatocellular carcinoma provided a rationale for further investigation. CONCLUSIONS: We identified 11 methyltransferases and demethylases for major histone lysines that might be promising research targets in the pathogenesis, development, and prediction of prognosis in hepatocellular carcinoma using bioinformatics.
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Carcinoma Hepatocelular/metabolismo , Biología Computacional , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Neoplasias Hepáticas/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Biología Computacional/métodos , Bases de Datos Genéticas , Epigénesis Genética , Regulación de la Expresión Génica , Variación Genética , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Metilación , Pronóstico , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas , Procesamiento Proteico-PostraduccionalRESUMEN
Objective To elucidate the mechanisms by which elongation factor Tu GTP binding domain containing 2 (Eftud2) enhances the immune function of murine macrophages by bioinformatics analysis. Methods The bone marrow-derived macrophages (BMDMs) of Eftud2 myeloid cell-specific knockout (MKO) mice (n=10) and wild-type (WT) littermates (n=10) were collected and stimulated by lipopolysaccharide (LPS) (100 ng/mL) for 2 hours. Bioinformatics analysis was conducted to examine the differences in gene expression and mRNA transcription levels. The the differences in gene expression and alternative splicing of mRNA transcription in BMDMs were analyzed by DEGseq and rMATS, respectively. The signaling pathways affected were clarified by Kyoto Encyclopedia of Genes and Genomes (KEGG) classification and enrichment methods. Results Compared with WT counterparts, the expression levels of IL-6, IL-1ß, TNF-α, and the genes related to immune response in MKO BMDMs were down-regulated following LPS stimulation. KEGG pathway analysis showed that the differently expressed genes in BMDMs and alternative splicing mainly affected the signal transduction and immune system-related metabolic pathways, and had a strong correlation with PI3K-AKT signaling pathway. The difference in alternative splicing also existed in ubiquitination and endocytosis. Compared with WT counterparts, there were 232 differences in alternative splicing in MKO BMDMs, among which 125 were skipping exons, accounting for the largest proportion. In addition, the analysis of alternative splicing differences also confirmed the previous experimental results, that is, Eftud2 could participate in the activation of inflammatory signaling pathways by enhancing the alternative splicing of key molecules such as MyD88 in TLR4-NF-κB signaling pathway, thereby augmenting the function of macrophages. Conclusion Eftud2 can promote the release of inflammatory cytokines in BMDMs by regulating gene expression and alternative splicing, and consequently enhance the immune function of macrophages.
Asunto(s)
Biología Computacional , Expresión Génica , Macrófagos , Animales , Citocinas/genética , Citocinas/inmunología , Expresión Génica/genética , Expresión Génica/inmunología , Lipopolisacáridos/farmacología , Macrófagos/inmunología , Ratones , FN-kappa B/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismoRESUMEN
The generation of large numbers of plasma cells (PCs) is a main factor in systemic lupus erythematosus (SLE). We hypothesize that Hspa13, a member of the heat shock protein family, plays a critical role in the control of PC differentiation. To test the hypothesis, we used lipopolysaccharide (LPS)-activated B cells and a newly established mouse line with a CD19cre-mediated, B cell-specific deletion of Hspa13: Hspa13 cKO mice. We found that Hspa13 mRNA was increased in PCs from atacicept-treated lupus-prone mice and in LPS-stimulated plasmablasts (PBs) and PCs. A critical finding was that PBs and PCs [but not naïve B cells and germinal center (GC) B cells] expressed high levels of Hspa13. In contrast, the Hspa13 cKO mice had a reduction in BPs, PCs, and antibodies induced in vitro by LPS and in vivo by sheep red blood cells (SRCs)- or 4-hydroxy-3-nitrophenylacetyl (NP)-immunization. Accordingly, the Hspa13 cKO mice had reduced class-switched and somatically hypermutated antibodies with defective affinity maturation. Our work also showed that Hspa13 interacts with proteins (e.g., Bcap31) in the endoplasmic reticulum (ER) to positively regulate protein transport from the ER to the cytosol. Importantly, Hspa13 mRNA was increased in B220+ cells from patients with multiple myeloma (MM) or SLE, whereas Hspa13 cKO led to reduced autoantibodies and proteinuria in both pristane-induced lupus and lupus-prone MRL/lpr mouse models. Collectively, our data suggest that Hspa13 is critical for PC development and may be a new target for eliminating pathologic PCs.
Asunto(s)
Anticuerpos/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Lupus Eritematoso Sistémico/metabolismo , Mieloma Múltiple/metabolismo , Células Plasmáticas/metabolismo , Animales , Anticuerpos/inmunología , Afinidad de Anticuerpos , Estudios de Casos y Controles , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Proteínas HSP70 de Choque Térmico/sangre , Proteínas HSP70 de Choque Térmico/genética , Humanos , Lipopolisacáridos/farmacología , Lupus Eritematoso Sistémico/sangre , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos MRL lpr , Ratones Noqueados , Mieloma Múltiple/sangre , Mieloma Múltiple/inmunología , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/inmunología , Transporte de Proteínas , Proteínas Recombinantes de Fusión/farmacología , Vías SecretorasRESUMEN
Alternative splicing (AS) of mRNA is known to be involved in regulation of immune cell differentiation and activation. Elongation factor Tu GTP binding domain containing 2 (Eftud2) is an AS factor to potentially modulate innate immune response in macrophages. In this study, we investigate its involvement in the pathogenesis of colitis-associated cancer (CAC). Using an established mouse model of CAC, we show that Eftud2 is constantly overexpressed in the colonic tissues as well as infiltrating macrophages. Myeloid-specific knockout of Eftud2 remarkably suppresses chronic intestinal inflammation and tumorigenesis, which is associated with decreased production of inflammatory cytokines and tumorigenic factors. Repression of colonic inflammation and colorectal tumor development in Eftud2-deficient mice is due to the impaired activation of NF-κB signaling in LPS-challenged macrophages. Furthermore, the alteration of Eftud2-mediated AS involving the components of TLR4-NF-κB cascades underlies the impairment of NF-κB activation. Overall, these findings provide new insights into the tight link between inflammation and cancer and modulation of AS in innate immune signals may be a potentially therapeutic avenue for CAC treatment.