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1.
Virol Sin ; 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31512106

RESUMO

Enterovirus A71 (EV-A71) is the major pathogen responsible for the severe hand, foot and mouth disease worldwide, for which few effective antiviral drugs are presently available. Interferon-α (IFN-α) has been used in antiviral therapy for decades; it has been reported that EV-A71 antagonizes the antiviral activity of IFN-α based on viral 2Apro-mediated reduction of the interferon-alpha receptor 1 (IFNAR1); however, the mechanism remains unknown. Here, we showed a significant increase in IFNAR1 protein induced by IFN-α in RD cells, whereas EV-A71 infection caused obvious down-regulation of the IFNAR1 protein and blockage of IFN-α signaling. Subsequently, we observed that EV-A71 2Apro inhibited IFNAR1 translation by cleavage of the eukaryotic initiation factor 4GI (eIF4GI), without affecting IFNAR1 mRNA levels induced by IFN-α. The inhibition of IFNAR1 translation also occurred in puromycin-induced apoptotic cells when caspase-3 cleaved eIF4GI. Importantly, we verified that 2Apro could activate cellular caspase-3, which was subsequently involved in eIF4GI cleavage mediated by 2Apro. Furthermore, inhibition of caspase-3 activation resulted in the partial restoration of IFNAR1 in cells transfected with 2A or infected with EV-A71, suggesting the pivotal role of both viral 2Apro and caspase-3 activation in the disturbance of IFN-α signaling. Collectively, we elucidate a novel mechanism by which cellular caspase-3 contributes to viral 2Apro-mediated down-regulation of IFNAR1 at the translation level during EV-A71 infection, indicating that caspase-3 inhibition could be a potential complementary strategy to improve clinical anti-EV-A71 therapy with IFN-α.

2.
Front Immunol ; 10: 1647, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379845

RESUMO

Background: Antibody-dependent cellular cytotoxicity (ADCC), which mainly mediated by natural killer (NK) cells, may play a critical role in human immunodeficiency virus type-1 (HIV-1) disease progression. However, the potential mechanisms that affecting NK-mediated ADCC response are still not well-elucidated. Methods: Antigen-antibody complex model of Ab-opsonized P815 cells was adopted to induce a typical non-specific ADCC response. The capacities of HIV-1 specific NK-ADCC were measured by using the combination model of gp120 protein and plasma of HIV-1 elite controllers. The levels of plasma cytokine were measured by ELISA. Anti-IL-2 blocking antibody was used to analyze the impact of activated CD56+ T cells on NK-ADCC response. Results: IL-2, IL-15, IFN-α, and IFN-ß could effectively enhance the non-specific and HIV-1-specific NK-ADCC responses. Compared with healthy controls, HIV-1-infected patients showed decreased plasma IL-2 levels, while no differences of plasma IFN-α, IL-15, and IFN-ß were presented. IL-2 production was detected from CD56+ T cells activated through antibody-dependent manner. The capability of NK-ADCC could be weakened by blocking IL-2 secretion from activated CD56+ T cells. Although no difference of frequencies of CD56+ T cells was found between HIV-1-infected patients and healthy controls, deficient IL-2 secretion from activated CD56+ T were found in chronic HIV-1 infection. Conclusions: The impaired ability of activated CD56+ T cells to secreting IL-2 might contribute to the attenuated NK cell-mediated ADCC function in HIV-1 infection.

3.
Gastroenterology ; 156(8): 2230-2241.e11, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30742832

RESUMO

BACKGROUND & AIMS: We performed a nationwide, retrospective study to determine the incidence and causes of drug-induced liver injury (DILI) in mainland China. METHODS: We collected data on a total of 25,927 confirmed DILI cases, hospitalized from 2012 through 2014 at 308 medical centers in mainland China. We collected demographic, medical history, treatment, laboratory, disease severity, and mortality data from all patients. Investigators at each site were asked to complete causality assessments for each case whose diagnosis at discharge was DILI (n = 29,478) according to the Roussel Uclaf Causality Assessment Method. RESULTS: Most cases of DILI presented with hepatocellular injury (51.39%; 95% confidence interval [CI] 50.76-52.03), followed by mixed injury (28.30%; 95% CI 27.73-28.87) and cholestatic injury (20.31%; 95% CI 19.80-20.82). The leading single classes of implicated drugs were traditional Chinese medicines or herbal and dietary supplements (26.81%) and antituberculosis medications (21.99%). Chronic DILI occurred in 13.00% of the cases and, although 44.40% of the hepatocellular DILI cases fulfilled Hy's Law criteria, only 280 cases (1.08%) progressed to hepatic failure, 2 cases underwent liver transplantation (0.01%), and 102 patients died (0.39%). Among deaths, DILI was judged to have a primary role in 72 (70.59%), a contributory role in 21 (20.59%), and no role in 9 (8.82%). Assuming the proportion of DILI in the entire hospitalized population of China was represented by that observed in the 66 centers where DILI capture was complete, we estimated the annual incidence in the general population to be 23.80 per 100,000 persons (95% CI 20.86-26.74). Only hospitalized patients were included in this analysis, so the true incidence is likely to be higher. CONCLUSIONS: In a retrospective study to determine the incidence and causes of DILI in mainland China, the annual incidence in the general population was estimated to be 23.80 per 100,000 persons; higher than that reported from Western countries. Traditional Chinese medicines, herbal and dietary supplements, and antituberculosis drugs were the leading causes of DILI in mainland China.


Assuntos
Causas de Morte , Doença Hepática Induzida por Substâncias e Drogas/epidemiologia , Doença Hepática Terminal/induzido quimicamente , Falência Hepática Aguda/induzido quimicamente , Sistema de Registros , Doença Aguda , Adulto , Distribuição por Idade , Idoso , Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Doença Hepática Induzida por Substâncias e Drogas/etiologia , China/epidemiologia , Doença Crônica , Estudos de Coortes , Intervalos de Confiança , Doença Hepática Terminal/epidemiologia , Doença Hepática Terminal/fisiopatologia , Feminino , Humanos , Incidência , Falência Hepática Aguda/epidemiologia , Falência Hepática Aguda/fisiopatologia , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Medição de Risco , Índice de Gravidade de Doença , Distribuição por Sexo , Taxa de Sobrevida , Adulto Jovem
4.
Antiviral Res ; 160: 10-16, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30315876

RESUMO

Activation of the ERK signaling cascade in host cells has been demonstrated to be essential for enterovirus A71 (EV-A71) replication. Our previous study showed that MEK kinase, which specially activated downstream ERK kinase, is an important and potential target against EV-A71. Furthermore, we reported that a series of substituted 3-benzylcoumarins designed and synthesized as well as verified for inhibiting the MEK-ERK cascade were found to be effective on anti-EV-A71. In this study, we further demonstrated that two substituted 3-benzylcoumarins designated as 13 and 14 were more effective anti-MEK/ERK activity, less cytotoxicity and stronger antiviral effect represented by inhibition of viral-induced CPE, the expression of viral proteins and the replication of the viral genome, as well as the production of progeny virions, compared to those of U0126, an available MEK inhibitor, and sorafenib, a multiple-targeted kinase inhibitor in clinical use. Moreover, we explored that the likely mechanism of action of these two test compounds were to block EV-A71 2A dependent IRES-driven activity essential for successful viral replication. Hence, our results suggest that two substituted 3-benzylcoumarins 13 and 14 could be candidates as potential anti-EV-A71 agents.


Assuntos
Antivirais/farmacologia , Cumarínicos/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Biossíntese de Proteínas/efeitos dos fármacos , Proteínas Virais/antagonistas & inibidores , Replicação Viral/efeitos dos fármacos , Antivirais/química , Cumarínicos/química , Inibidores de Cisteína Proteinase/química , Enterovirus Humano A/crescimento & desenvolvimento , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia
5.
Nat Commun ; 9(1): 3746, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30218073

RESUMO

In response to myeloablative stresses, HSCs are rapidly activated to replenish myeloid progenitors, while maintaining full potential of self-renewal to ensure life-long hematopoiesis. However, the key factors that orchestrate HSC activities during physiological stresses remain largely unknown. Here we report that Med23 controls the myeloid potential of activated HSCs. Ablation of Med23 in hematopoietic system leads to lymphocytopenia. Med23-deficient HSCs undergo myeloid-biased differentiation and lose the self-renewal capacity. Interestingly, Med23-deficient HSCs are much easier to be activated in response to physiological stresses. Mechanistically, Med23 plays essential roles in maintaining stemness genes expression and suppressing myeloid lineage genes expression. Med23 is downregulated in HSCs and Med23 deletion results in better survival under myeloablative stress. Altogether, our findings identify Med23 as a gatekeeper of myeloid potential of HSCs, thus providing unique insights into the relationship among Med23-mediated transcriptional regulations, the myeloid potential of HSCs and HSC activation upon stresses.


Assuntos
Diferenciação Celular/genética , Autorrenovação Celular/genética , Células-Tronco Hematopoéticas/citologia , Complexo Mediador/genética , Células Mieloides/citologia , Estresse Fisiológico/genética , Animais , Transplante de Medula Óssea , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Células Mieloides/metabolismo , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo
6.
J Virol Methods ; 248: 87-91, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28629711

RESUMO

A versatile single-step method is described for constructing a picornavirus replicon RNA with precise ends to facilitate improved understanding of viral genome function and mimic native virus replication in host cells as far as possible. The key innovation in this new approach is the use of a bridge primer to both introduce a ribozyme sequence for cis-cleavage of RNA to generate precise 5' ends of EV71 RNA and also mediate overlapping assembly of two fragments. Using an EV71 replicon as a test case, precise ends for the viral replicon were shown to be important for efficient virus replication. Thus, our work provides a novel efficient way to generating higher efficient viral replicon with precise ends and this novel method can be applied to other picornaviruses' research.


Assuntos
Biologia Molecular/métodos , Picornaviridae/genética , RNA Viral/genética , Replicon , Linhagem Celular , Primers do DNA , Genoma Viral , RNA Catalítico , Replicação Viral
7.
Antiviral Res ; 143: 13-21, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28351508

RESUMO

In a previous study the ERK1/2 pathway was found to be crucially involved in positive regulation of the enterovirus A 71(EV-A71) IRES (vIRES), thereby contributing to the efficient replication of an important human enterovirus causing death in young children (<5yrs) worldwide. This study focuses on unraveling more about the detailed mechanism of ERK's involvement in this regulation of vIRES. Through the use of siRNAs and specifically pharmacological inhibitor U0126, the ERK cascade was shown to positively regulate EV-A71-mediated cleavage of eIF4GI that established the cellular conditions which favour vIRES-dependent translation. Site-directed mutagenesis of the viral 2A protease (2Apro) was undertaken to show that the positive regulation of virus replication by the ERK cascade was mediated through effects on both the cis-cleavage of the viral polyprotein by 2Apro and its trans-cleavage of cellular eIF4GI. This ERK-2Apro linked network coordinating vIRES efficiency was also found in other important human enteroviruses. This identification of the ERK cascade as having a key role in maintaining the 2Apro proteolytic activity required to maximize enterovirus IRES activity, expands current understanding of the diverse functions of the ERK signaling cascade in the regulation of viral translation, therefore providing a potentially comprehensive drug target for anti-enterovirus infection.


Assuntos
Antivirais/farmacologia , Enterovirus Humano A/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Sítios Internos de Entrada Ribossomal/fisiologia , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismo , Butadienos/antagonistas & inibidores , Linhagem Celular Tumoral , Enterovirus Humano A/enzimologia , Infecções por Enterovirus/tratamento farmacológico , Infecções por Enterovirus/virologia , Células HEK293 , Humanos , Sítios Internos de Entrada Ribossomal/genética , Mutagênese Sítio-Dirigida , Nitrilos/antagonistas & inibidores , Poliproteínas , RNA Interferente Pequeno , Rabdomiossarcoma , Transdução de Sinais/efeitos dos fármacos , Proteínas Virais/genética , Replicação Viral/efeitos dos fármacos
8.
Infect Genet Evol ; 45: 83-89, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27566335

RESUMO

Currently, it is still controversial that if the pathogenicity of EV-A71 causing severe or mild hand, foot, and mouth disease (HFMD) is associated with viral nucleotide or amino acid sequence(s). In this study, 19 clinical strains were detected in samples from diagnosed patients of EV-A71-caused HFMD with mild or severe symptoms. Then, VP1-2A fragment sequences of 19 EV-A71 isolates were determined, the phylogenetic analysis, based on VP1 sequences of 19 EV-A71 stains in this study and which of 62 EV-A71 strains with different clinical phenotypes reported before, were carried out. Our results showed that no difference in the genotype and evolution distribution was observed among the EV-A71 strains mentioned above. Furthermore, two EV-A71 isolates, which with much close evolutionary relationship but different clinical manifestations, were purified by plaque assay, the complete genome sequencing was done, and deduced amino acid sequence analysis of 11 proteins coded by EV-A71 was carried out. Eight variable amino acid sites were found and further verified with those of 62 strains reported before. Our study provides further evidence that the potential pathogenicity of EV-A71 causing severe or mild HFMD seems not to be associated with viral genotype and even the amino acid substitution.


Assuntos
Enterovirus/genética , Doença de Mão, Pé e Boca/virologia , Aminoácidos , Proteínas do Capsídeo/genética , Estudos de Coortes , Enterovirus/classificação , Genoma Viral/genética , Doença de Mão, Pé e Boca/diagnóstico , Doença de Mão, Pé e Boca/fisiopatologia , Humanos , Fenótipo , Filogenia , RNA Viral/análise , RNA Viral/genética , Análise de Sequência de RNA
9.
Bioorg Med Chem Lett ; 19(24): 6918-21, 2009 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-19889538

RESUMO

A series of novel oxazaphosphorine prodrugs of 9-(2-phosphonomethoxyethyl)adenine (PMEA, adefovir) were synthesized and their anti-hepatitis B virus (HBV) activity was evaluated in HepG2 2.2.15 cells, with adefovir dipivoxil as a reference drug. In the cell assays, compounds 7b and 7d exhibited anti-HBV activity comparable to that of adefovir dipivoxil, while compound 7c, with an IC(50) value of 0.12 microM, was found to be three times more potent than the reference compound. In vitro stability studies showed that (S(P),S)-7c, the diastereomer of compound 7c, was stable in human blood plasma but underwent rapid metabolism to release the parent drug PMEA in liver microsomes. The possible metabolic pathway of (S(P),S)-7c in human liver microsomes was described. These findings suggest that compound (S(P),S)-7c is a promising anti-HBV drug candidate for further development.


Assuntos
Adenina/análogos & derivados , Antivirais/química , Vírus da Hepatite B/efeitos dos fármacos , Organofosfonatos/química , Compostos Organofosforados/química , Pró-Fármacos/química , Adenina/síntese química , Adenina/química , Adenina/farmacologia , Antivirais/síntese química , Antivirais/farmacologia , Linhagem Celular Tumoral , Desenho de Drogas , Humanos , Organofosfonatos/síntese química , Organofosfonatos/farmacologia , Compostos Organofosforados/síntese química , Compostos Organofosforados/farmacologia , Pró-Fármacos/síntese química , Pró-Fármacos/farmacologia
10.
Chem Res Toxicol ; 22(5): 824-34, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19358519

RESUMO

Dauricine is one type of the bisbenzyltetrahydroisoquinoline alkaloid derivative with antiarrhythmic effects. Severe liver toxicity was observed in experimental animals treated with analogues of dauricine, which may be caused by covalent binding of reactive metabolite(s) to critical macromolecules in tissues. The study described herein aimed at characterizing pathways of dauricine bioactivation and the CYP enzyme involved. In incubations of dauricine with NADPH- and GSH-supplemented human liver microsomes, four GSH conjugates with [M + H]+ ions at m/z 930, 916, 916, and 902, respectively, were detected by liquid chromatography-ion trap mass spectrometry. The structures of the four metabolites were determined to be GSH conjugates of dauricine, 2-N-demethyl dauricine, 2'-N-demethyl dauricine, and N-demethyl-O-demethyl dauricine. GSH conjugation took place with a strong preference at C-17, suggesting that the phenol moiety of dauricine and its metabolites underwent oxidation to quinone methide intermediates. The formation of the GSH conjugates was found to require the presence of NADPH. To identify the CYP isoforms that are responsible for bioactivation, dauricine was also incubated with recombinant human CYP450 enzymes. The formation of GSH was only observed with the incubation of CYP3A4. In addition, the level of these GSH conjugates in human microsomes was reduced upon the addition of a CYP3A4 inhibitor ketoconazole. The same GSH conjugates were also observed in rat bile following a single oral dose of 40 mg/kg dauricine. These studies suggest that the CYP3A4 mediated quinone methide formation was associated with dauricine bioactivation.


Assuntos
Benzilisoquinolinas/metabolismo , Bile/química , Indolquinonas/metabolismo , Microssomos Hepáticos/química , Tetra-Hidroisoquinolinas/metabolismo , Administração Oral , Animais , Benzilisoquinolinas/administração & dosagem , Benzilisoquinolinas/química , Cromatografia Líquida de Alta Pressão , Citocromo P-450 CYP3A/metabolismo , Glutationa/química , Glutationa/metabolismo , Humanos , Indolquinonas/química , Cetoconazol/farmacologia , Ratos , Espectrometria de Massas por Ionização por Electrospray , Tetra-Hidroisoquinolinas/administração & dosagem , Tetra-Hidroisoquinolinas/química
11.
Rapid Commun Mass Spectrom ; 22(12): 1843-52, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18470870

RESUMO

The metabolism of lafutidine in human liver microsomes was studied using liquid chromatography/ion trap mass spectrometry with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) sources. A total of 14 metabolites were identified including hydroxylated lafutidine and sulfonyl lafutidine as the major metabolites. The chemical properties and the MS(n) behaviors of lafutidine and all of its identified metabolites were studied in detail. Lafutidine had a fragmentation pattern as a result of homolytic bond cleavage in the MS/MS spectrum. This cleavage can form an odd-electron ion with the loss of furan-2-ylmethyl radical (-81 Da with a proton shift), which then sequentially loses neutral groups in the MS(3) spectrum. This fragmentation sequence was also observed from the metabolites with the unchanged sulfinyl moiety. When the sulfinyl moiety was oxidized to the sulfonyl moiety, this fragmentation sequence did not exist, which could be used to identify S-oxidation metabolites of lafutidine. In general, N-oxides could produce distinct [M+H-O](+) ions under LC/APCI-MS due to the thermal activation in the desolvation region of the API source, which could be used to identify N-oxidation metabolites of lafutidine. In order to avoid the possibility of false positives, the MS/MS spectrum of the [M+H-O](+) ion was compared with that of the non-N-oxidation metabolites or parent drug in the APCI source. If they were consistent, the structure could be finally confirmed. The exact masses for lafutidine and lafutidine N-oxide fragment ions were determined using an LTQ/Orbitrap mass spectrometer.


Assuntos
Acetamidas/metabolismo , Cromatografia Líquida/métodos , Antagonistas dos Receptores Histamínicos H2/metabolismo , Espectrometria de Massas/métodos , Microssomos Hepáticos/metabolismo , Piperidinas/metabolismo , Piridinas/metabolismo , Acetamidas/química , Antagonistas dos Receptores Histamínicos H2/química , Humanos , Estrutura Molecular , Peso Molecular , Piperidinas/química , Piridinas/química
12.
J Mass Spectrom ; 43(8): 1099-109, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18324609

RESUMO

The metabolism of arbidol in humans was studied using liquid chromatography-electrospray ionization (ESI) ion trap mass spectrometry (ITMS) after an oral dose of 300-mg arbidol. A total of 17 metabolites were identified including the glucuronide arbidol and the glucuronide sulfinylarbidol as the major metabolites. Arbidol and its metabolites have some common fragmentation patterns as a result of a homolytic bond cleavage. This cleavage will form odd-electron ions with the loss of a radical. The arbidol fragmentation sequence is first to lose dimethylamine (45 Da), followed by the loss of acetaldehyde (44 Da), and then the phenylthio radical (109 Da). This fragmentation sequence is also observed from N-demethylarbidol, sulfonylarbidol, and N-demethylsulfonylarbidol. However, for sulfinylarbidol and N-demethylsulfinylarbidol, the fragmentation sequence is reversed so that the phenylsulfiny radical (125 Da) was lost first, followed by the loss of dimethylamine (45 Da), and then acetaldehyde (44 Da). The exact masses for arbidol and sulfinylarbidol fragment ions were determined by a quadrupole/time-of-flight mass spectrometer (Q-TOF MS). The phase II metabolites, such as sulfate and glucuronide conjugates of arbidol, N-demethylarbidol, sulfonylarbidol, and N-demethylsulfonylarbidol were identified by observing the neutral loss of 80 Da (SO(3)) or 176 Da (glucuronic acid) from the MS(2) spectra. The sulfate and glucuronide conjugates such as sulfinylarbidol and N-demethylsulfinylarbidol had an unusual fragmentation pattern, in which the phenylsulfinyl radical (125 Da) was lost before the loss of SO(3) group (80 Da) or glucuronic acid (176 Da) occurred.


Assuntos
Antivirais/urina , Indóis/urina , Espectrometria de Massas por Ionização por Electrospray/métodos , Administração Oral , Antivirais/farmacocinética , Cromatografia Líquida de Alta Pressão , Humanos , Indóis/farmacocinética , Masculino
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