RESUMO
The global incidence of dengue fever has gradually increased in recent years, posing a serious threat to human health. In the absence of specific anti-dengue drugs, understanding the interaction of Dengue virus (DENV) with the host is essential for the development of effective therapeutic measures. Autophagy is often activated during DENV infection to promote viral replication, but the mechanism of how DENV's own proteins induce autophagy has not been clarified. In this study, we first preliminarily identified DENV-2 NS1 as the most likely viral protein for DENV-2-induced autophagy with the help of molecular docking techniques. Further experimental results confirmed that DENV-2 NS1 regulates DENV-2 infection of HUVEC-induced autophagy through the AMPK/ERK/mTOR signaling pathway. Mechanistically, DENV-2 NS1 mainly interacted with AMPK by means of its Wing structural domain, and NS1 bound to all three structural domains on the AMPKα subunit. Finally, the experimental results showed that DENV-2 NS1 promoted the interaction between LKB1 and AMPKα1 and thus activated AMPK by both increasing the expression of LKB1 and binding LKB1. In conclusion, the results of this study revealed that DENV-2 NS1 protein served as a platform for the interaction between AMPK and LKB1 after DENV-2 infection with HUVEC, and pulled AMPK and LKB1 together to form a complex. LKB1 to form a complex, promoting LKB1 action on the kinase structural domain of AMPKα1, which in turn promotes phosphorylation of the Thr172 site on the AMPK kinase structural domain and activates AMPK, thereby positively regulating the AMPK/ERK/mTOR signaling pathway and inducing autophagy. The present discovery improves our understanding of DENV-2-induced host autophagy and contributes to the development of anti-dengue drugs.
Assuntos
Vírus da Dengue , Dengue , Humanos , Proteínas Quinases Ativadas por AMP/metabolismo , Autofagia , Vírus da Dengue/fisiologia , Simulação de Acoplamento Molecular , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas não Estruturais Virais/metabolismoRESUMO
BACKGROUND: Dengue virus type 2 (DENV-2) was used to infect primary human umbilical vein endothelial cells (HUVECs) to examine autophagy induced by activation of the adenosine monophosphate-activated protein kinase (AMPK)/extracellular signal-regulated kinase (ERK)/mammalian target of rapamycin (mTOR) signaling pathway following tripartite motif-containing 22 (TRIM22)-mediated DENV-2 infection to further reveal the underlying pathogenic mechanism of DENV-2 infection. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was used to screen putative interference targets of TRIM22 and determine the knockdown efficiency. The effect of TRIM22 knockdown on HUVEC proliferation was determined using the CCK8 assay. Following TRIM22 knockdown, transmission electron microscopy (TEM) was used to determine the ultrastructure of HUVEC autophagosomes and expression of HUVEC autophagy and AMPK pathway-related genes were measured by qRT-PCR. Moreover, HUVEC autophagy and AMPK pathway-related protein expression levels were determined by western blot analysis. Cell cycle and apoptosis were assessed by flow cytometry (FCM) and the autophagosome structure of the HUVECs was observed by TEM. RESULTS: Western blot results indicated that TRIM22 protein expression levels increased significantly 36 h after DENV-2 infection, which was consistent with the proteomics prediction. The CCK8 assay revealed that HUVEC proliferation was reduced following TRIM22 knockdown (P < 0.001). The TEM results indicated that HUVEC autolysosomes increased and autophagy was inhibited after TRIM22 knockdown. The qRT-PCR results revealed that after TRIM22 knockdown, the expression levels of antithymocyte globulin 7 (ATG7), antithymocyte globulin 5 (ATG5), Beclin1, ERK, and mTOR genes decreased (P < 0.01); however, the expression of AMPK genes (P < 0.05) and P62 genes (P < 0.001) increased. FCM revealed that following TRIM22 knockdown, the percentage of HUVECs in the G2 phase increased (P < 0.001) along with cell apoptosis. The effect of TRIM22 overexpression on HUVEC autophagy induced by DENV-2 infection and AMPK pathways decreased after adding an autophagy inhibitor. CONCLUSIONS: In HUVECs, TRIM22 protein positively regulates autophagy and may affect autophagy through the AMPK/ERK/mTOR signaling pathway. Autophagy is induced by activation of the AMPK/ERK/mTOR signaling pathway following TRIM22-mediated DENV-2 infection of HUVECs.
Assuntos
Proteínas Quinases Ativadas por AMP , MAP Quinases Reguladas por Sinal Extracelular , Humanos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Células Endoteliais da Veia Umbilical Humana , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Sirolimo/farmacologia , Soro Antilinfocitário/farmacologia , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/farmacologia , Autofagia , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/farmacologia , Proteínas Repressoras/metabolismo , Antígenos de Histocompatibilidade Menor/farmacologiaRESUMO
Several different vaccines have been produced for human use to prevent the highly pathogenic H5N1 influenza. Some studies reported that the clinical effectiveness of influenza vaccines in older adults may be lower than in younger adults. In this study, a meta-analysis of the immunogenicity of H5N1 influenza vaccines in elderly adults was performed. Database search was conducted in EMBASE, PubMed, the Cochrane Library, Chinese VIP, Wanfang and CBM. A total of 3951 elderly adults from 10 articles were included in the meta-analysis. Compared to a single dose, two doses of H5N1 vaccines resulted in the higher seroconversion and seroprotection. For all groups treated with adjuvanted vaccines, there were significant increases (1.55- to 2.16-fold) in the seroconversion rates (SCRs) and seroprotection rates (SPRs) after two immunizations. Oil-in-water emulsion (OE)-adjuvanted 7.5 µg vaccine caused higher antibody responses than 3.75 µg of vaccine (SCR: risk ratio (RR) = 1.26 (1.19, 1.33); SPR: RR = 1.25 (1.14, 1.36)). Elderly adults exhibited slightly lower antibody responses only when given 7.5 µg of OE-adjuvanted vaccine (SCR: RR = 1.06 (1.01, 1.11)) than younger adults. After treatment with the 7.5 µg of OE-adjuvanted vaccines, the most commonly reported adverse events were injection site pain, swelling and erythema, with the incidence of 32%, 3% and 2%, respectively, and no serious adverse events were found. These data demonstrate that two doses of 7.5 µg of OE-adjuvanted H5N1 vaccine are well tolerated and induce a robust antibody response in elderly adults.
Assuntos
Virus da Influenza A Subtipo H5N1 , Vacinas contra Influenza , Influenza Humana , Adjuvantes Imunológicos , Idoso , Anticorpos Antivirais , Testes de Inibição da Hemaglutinação , Humanos , Vacinas contra Influenza/efeitos adversos , Influenza Humana/prevenção & controleRESUMO
Inoculation with vaccine is the major intervention currently used to prevent influenza infections. However, it will be a challenge to produce and implement a new vaccine when a novel highly pathogenic influenza virus emerges in humans as significant infections. H7 subtype influenza viruses have similar epitopes on hemagglutinin, which can induce cross-reactive antibodies. In this study, a meta-analysis of the cross-reactivity of antibodies induced by one H7 subtype influenza vaccine against other H7 subtypes was performed. Database search was conducted in PubMed, Cochrane Library, EMBASE, MEDLINE, Chinese Biological Medicine Database (CBM), and Wanfang. A total of 9 articles comprising 811 human subjects were included in this meta-analysis. All assessed H7 influenza vaccines induced vaccine strain-specific protective antibodies [seroconversion rate (SCR) = 0.74, 95% CI (0.65, 0.82); seroprotection rate (SPR) = 0.81, 95% CI (0.78, 0.83)]. All H7 influenza virus monovalent vaccines exhibited cross-reactivity tested by hemagglutinin inhibition test (HI), microneutralization test (MN) and immunosorbent assay (ELISA) to other H7 subtype viruses. H7N1, H7N3, H7N7, and H7N9 vaccines elicited cross-reactive antibodies against other H7 subtype influenza viruses [SCR = 0.66, 95% CI (0.50, 0.82); SPR = 0.79, 95% CI (0.67, 0.91)]. The pooled SCR (95%CI) of cross-reactivity of H7N1 and H7N3 vaccines were 0.88 (0.85, 0.91) and 0.40 (0.26, 0.54), respectively. The consolidated SPR (95%CI) of H7N1 and H7N7 vaccines were 0.89 (0.86, 0.92) and 0.93 (0.81, 1.06). All H7 vaccines induced cross-reactive antibodies against H7N9 viruses [SCR = 0.69, 95% CI (0.52, 0.86); SPR = 0.85, 95% CI (0.76, 0.94)]. H7 vaccines can be used to limit influenza infection when a new highly pathogenic H7 virus appears.
Assuntos
Vírus da Influenza A Subtipo H7N1 , Vírus da Influenza A Subtipo H7N7 , Subtipo H7N9 do Vírus da Influenza A , Vacinas contra Influenza , Influenza Humana , Anticorpos Neutralizantes , Anticorpos Antivirais , Glicoproteínas de Hemaglutininação de Vírus da Influenza , Humanos , Vírus da Influenza A Subtipo H7N3 , Influenza Humana/prevenção & controleRESUMO
Interleukin 31 (IL-31) is a four-helix cytokine made predominantly by Th2 CD4+ T cells. It was initially identified as being associated with the promotion of atopic dermatitis, where increased levels of IL-31 levels have been found and IL-31 induced the expression of proinflammatory cytokines and chemokines in a human bronchial epithelial cell line. However, subsequent study has shown that IL-31RA knockout mice developed exacerbated type 2 inflammation in the lung following infection with Schistosoma mansoni eggs. In this study, we investigated the dynamic expression of IL-31 and IL-31RA during eight consecutive ovalbumin (OVA) challenges and measured the chemokines from lung alveolar epithelial cells induced by IL-31. In addition, we examined the effect deletion of IL-31RA has on lung inflammation and the differentiation of CD4+ T cells. Our results demonstrate that the expression of IL-31 and IL-31RA was elevated after each weekly OVA challenge, although slightly less of both observed after the first week of OVA challenge. IL-31 also promoted the expression of inflammatory chemokines CCL5, CCL6, CCL11, CCL16, CCL22, CCL28, CX3CL1, CXCL3, CXCL14 and CXCL16 in alveolar epithelial cells. Migration of macrophages and T cells was enhanced by culture supernatants of IL-31-stimulated alveolar epithelial cells. Lastly, and in contrast to the IL-31 results, mice deficient in IL-31RA developed exacerbated lung inflammation, increased IL-4-positive cell infiltrates and elevated Th2 cytokine responses in draining lymph nodes. The proliferation of IL-31RA-/- CD4+ T cells was enhanced in vitro after anti-CD3/anti-CD28 antibody stimulation. These data indicate that IL-31/IL-31RA may play dual roles, first as an early inflammatory mediator promoting the secretion of chemokines to recruit inflammatory cells, and subsequently as a late inflammatory suppressor, limiting Th2 cytokine responses in allergic asthma.
RESUMO
This study introduces the use of a waste by-product from wet limestone flue gas desulfurization as a potential material for fluoride removal. Systematic laboratory-scale experiments were tested to identify the fluoride removal performance and determine the underlying mechanism. Flue gas desulfurization (FGD) gypsum removes 93.31% of fluoride from 109â¯mg/L to 7.3â¯mg/L. Fluoride can be efficiently removed at the optimum pH range of 5-11. Kinetics analysis indicates that the theoretical fluoride capacity at 1â¯g/L FGD gypsum is 96.9â¯mg/g. Equilibrium speciation analysis indicates that the decrease of system pH to lower than 5 is unsuitable for the formation of calcium fluoride, and the increase of system pH to higher than 11 opposes calcium release from FGD gypsum. Thermodynamic analysis confirms the feasibility of converting calcium sulphate into calcium fluoride at pHâ¯>â¯5. FGD gypsum and precipitates were characterized to describe their surface morphology, elemental composition and crystalline phase. Results indicate that FGD gypsum removes fluoride through the combination of calcium with fluoride to generate calcium fluoride.