Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Malar J ; 18(1): 14, 2019 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-30665411

RESUMO

BACKGROUND: Glucose-6-phosphate dehydrogenase deficiency (G6PDd), haemoglobin C (HbC) and S (HbS) are inherited blood disorders (IBD) common in populations in malaria endemic areas. All are associated to some degree with protection against clinical malaria whilst additionally G6PDd is associated with haemolysis following treatment with 8-aminoquinolines. Measuring the prevalence of these inherited blood disorders in affected populations can improve understanding of disease epidemiology. Current methodologies in epidemiological studies commonly rely on individual target amplification and visualization; here a method is presented to simultaneously detect the polymorphisms and that can be expanded to include other single nucleotide polymorphisms (SNPs) of interest. METHODS: Human DNA from whole blood samples was amplified in a novel, multiplex PCR reaction and extended with SNP-specific probes in an allele specific primer extension (ASPE) to simultaneously detect four epidemiologically important human markers including G6PD SNPs (G202A and A376G) and common haemoglobin mutations (HbS and HbC). The products were hybridized to magnetic beads and the median fluorescence intensity (MFI) was read on MAGPIX® (Luminex corp.). Genotyping data was compared to phenotypical data generated by flow cytometry and to established genotyping methods. RESULTS: Seventy-five samples from Burkina Faso (n = 75/78, 96.2%) and 58 samples from The Gambia (n = 58/61, 95.1%) had a G6PD and a HBB genotype successfully assigned by the bead-based assay. Flow cytometry data available for n = 61 samples further supported the concordance between % G6PD normal/deficient cells and genotype. CONCLUSIONS: The bead based assay compares well to alternative measures of genotyping and phenotyping for G6PD. The screening is high throughput, adaptable to inclusion of multiple targets of interest and easily standardized.


Assuntos
Anemia Falciforme/diagnóstico , Técnicas de Genotipagem/métodos , Deficiência de Glucosefosfato Desidrogenase/diagnóstico , Doença da Hemoglobina C/diagnóstico , Polimorfismo de Nucleotídeo Único , Adolescente , Adulto , Burkina Faso , Criança , Glucosefosfato Desidrogenase/genética , Hemoglobina C/genética , Hemoglobina Falciforme/genética , Humanos , Malária/complicações , Masculino , Pessoa de Meia-Idade , Adulto Jovem
2.
PLoS One ; 13(1): e0190272, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29324864

RESUMO

BACKGROUND: Primaquine (PQ) actively clears mature Plasmodium falciparum gametocytes but in glucose-6-phosphate dehydrogenase deficient (G6PDd) individuals can cause hemolysis. We assessed the safety of low-dose PQ in combination with artemether-lumefantrine (AL) or dihydroartemisinin-piperaquine (DP) in G6PDd African males with asymptomatic P. falciparum malaria. METHODS AND FINDINGS: In Burkina Faso, G6PDd adult males were randomized to treatment with AL alone (n = 10) or with PQ at 0.25 (n = 20) or 0.40 mg/kg (n = 20) dosage; G6PD-normal males received AL plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. In The Gambia, G6PDd adult males and boys received DP alone (n = 10) or with 0.25 mg/kg PQ (n = 20); G6PD-normal males received DP plus 0.25 (n = 10) or 0.40 mg/kg (n = 10) PQ. The primary study endpoint was change in hemoglobin concentration during the 28-day follow-up. Cytochrome P-450 isoenzyme 2D6 (CYP2D6) metabolizer status, gametocyte carriage, haptoglobin, lactate dehydrogenase levels and reticulocyte counts were also determined. In Burkina Faso, the mean maximum absolute change in hemoglobin was -2.13 g/dL (95% confidence interval [CI], -2.78, -1.49) in G6PDd individuals randomized to 0.25 PQ mg/kg and -2.29 g/dL (95% CI, -2.79, -1.79) in those receiving 0.40 PQ mg/kg. In The Gambia, the mean maximum absolute change in hemoglobin concentration was -1.83 g/dL (95% CI, -2.19, -1.47) in G6PDd individuals receiving 0.25 PQ mg/kg. After adjustment for baseline concentrations, hemoglobin reductions in G6PDd individuals in Burkina Faso were more pronounced compared to those in G6PD-normal individuals receiving the same PQ doses (P = 0.062 and P = 0.022, respectively). Hemoglobin levels normalized during follow-up. Abnormal haptoglobin and lactate dehydrogenase levels provided additional evidence of mild transient hemolysis post-PQ. CONCLUSIONS: Single low-dose PQ in combination with AL and DP was associated with mild and transient reductions in hemoglobin. None of the study participants developed moderate or severe anemia; there were no severe adverse events. This indicates that single low-dose PQ is safe in G6PDd African males when used with artemisinin-based combination therapy. TRIAL REGISTRATION: Clinicaltrials.gov NCT02174900 Clinicaltrials.gov NCT02654730.


Assuntos
Antimaláricos/administração & dosagem , Glucosefosfato Desidrogenase/genética , Malária Falciparum/tratamento farmacológico , Primaquina/administração & dosagem , Adulto , Antimaláricos/efeitos adversos , Burkina Faso , Humanos , Masculino , Primaquina/efeitos adversos , Adulto Jovem
3.
Mol Biochem Parasitol ; 199(1-2): 29-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25827756

RESUMO

The transmission of malaria parasites depends on the presence of sexual stages (gametocytes) in the blood, making the ratio and densities of female and male gametocytes important determinants of parasite fitness. This manuscript describes the development of reverse transcriptase quantitative PCR (RT-qPCR) assays to separately quantify mature female and male gametocytes of the human malaria parasite Plasmodium falciparum, and reveals that Pfs25 mRNA is expressed only in female gametocytes. The female (Pfs25) and male (Pfs230p) gametocyte specific RT-qPCR assays have lower detection limits of 0.3 female and 1.8 male gametocytes per microlitre of blood, respectively, making them more sensitive than microscopy. Accurate quantification of the ratio and densities of female and male gametocytes will increase understanding of P. falciparum transmission and improve the evaluation of transmission blocking interventions.


Assuntos
Antígenos de Protozoários/análise , Perfilação da Expressão Gênica , Parasitologia/métodos , Plasmodium falciparum/classificação , Plasmodium falciparum/genética , Proteínas de Protozoários/análise , Antígenos de Protozoários/genética , Proteínas de Protozoários/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
4.
Cell Rep ; 10(4): 600-15, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25640182

RESUMO

Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, we identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.


Assuntos
Enterovirus/efeitos dos fármacos , Enterovirus/metabolismo , Itraconazol/farmacologia , Receptores de Esteroides/metabolismo , Replicação Viral/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular Tumoral , Humanos
5.
Clin Infect Dis ; 60(3): 357-65, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25414262

RESUMO

BACKGROUND: Artemisinin combination therapy effectively clears asexual malaria parasites and immature gametocytes but does not prevent posttreatment malaria transmission. Ivermectin (IVM) may reduce malaria transmission by killing mosquitoes that take blood meals from IVM-treated humans. METHODS: In this double-blind, placebo-controlled trial, 120 asymptomatic Plasmodium falciparum parasite carriers were randomized to receive artemether-lumefantrine (AL) plus placebo or AL plus a single or repeated dose (200 µg/kg) of ivermectin (AL-IVM1 and AL-IVM2, respectively). Mosquito membrane feeding was performed 1, 3, and 7 days after initiation of treatment to determine Anopheles gambiae and Anopheles funestus survival and infection rates. RESULTS: The AL-IVM combination was well tolerated. IVM resulted in a 4- to 7-fold increased mortality in mosquitoes feeding 1 day after IVM (P < .001). Day 7 IVM plasma levels were positively associated with body mass index (r = 0.57, P < .001) and were higher in female participants (P = .003), for whom An. gambiae mosquito mortality was increased until 7 days after a single dose of IVM (hazard rate ratio, 1.34 [95% confidence interval, 1.07-1.69]; P = .012). Although we found no evidence that IVM reduced Plasmodium infection rates among surviving mosquitoes, the mosquitocidal effect of AL-IVM1 and AL-IVM2 resulted in 27% and 35% reductions, respectively, in estimated malaria transmission potential during the first week after initiation of treatment. CONCLUSIONS: We conclude that IVM can be safely given in combination with AL and can reduce the likelihood of malaria transmission by reducing the life span of feeding mosquitoes. CLINICAL TRIALS REGISTRATION: NCT0160325.


Assuntos
Culicidae , Inseticidas/uso terapêutico , Ivermectina/uso terapêutico , Malária Falciparum/prevenção & controle , Malária Falciparum/transmissão , Animais , Antimaláricos/uso terapêutico , Combinação Arteméter e Lumefantrina , Artemisininas/uso terapêutico , Método Duplo-Cego , Combinação de Medicamentos , Etanolaminas/uso terapêutico , Feminino , Fluorenos/uso terapêutico , Humanos , Malária Falciparum/tratamento farmacológico , Masculino
6.
J Virol ; 89(3): 1913-8, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25410869

RESUMO

PI4KIIIß recruitment to Golgi membranes relies on GBF1/Arf and ACBD3. Enteroviruses such as poliovirus and coxsackievirus recruit PI4KIIIß to their replication sites via their 3A proteins. Here, we show that human rhinovirus (HRV) 3A also recruited PI4KIIIß to replication sites. Unlike other enterovirus 3A proteins, HRV 3A failed to bind GBF1. Although HRV 3A was previously shown to interact with ACBD3, our data suggest that PI4KIIIß recruitment occurred independently of both GBF1 and ACBD3.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Interações Hospedeiro-Patógeno , Proteínas de Membrana/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Rhinovirus/fisiologia , Proteínas do Core Viral/metabolismo , Humanos , Ligação Proteica
7.
J Infect Dis ; 210(9): 1456-63, 2014 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24829466

RESUMO

BACKGROUND: The development of drugs and vaccines to reduce malaria transmission is an important part of eradication plans. The transmission-reducing activity (TRA) of these agents is currently determined in the standard membrane-feeding assay (SMFA), based on subjective microscopy-based readouts and with limitations in upscaling and throughput. METHODS: Using a Plasmodium falciparum strain expressing the firefly luciferase protein, we present a luminescence-based approach to SMFA evaluation that eliminates the requirement for mosquito dissections in favor of a simple approach in which whole mosquitoes are homogenized and examined directly for luciferase activity. RESULTS: Analysis of 6860 Anopheles stephensi mosquitoes across 68 experimental feeds shows that the luminescence assay was as sensitive as microscopy for infection detection. The mean luminescence intensity of individual and pooled mosquitoes accurately quantifies mean oocyst intensity and generates comparable TRA estimates. The luminescence assay presented here could increase SMFA throughput so that 10-30 experimental feeds could be evaluated in a single 96-well plate. CONCLUSIONS: This new method of assessing Plasmodium infection and transmission intensity could expedite the screening of novel drug compounds, vaccine candidates, and sera from malaria-exposed individuals for TRA. Luminescence-based estimates of oocyst intensity in individual mosquitoes should be interpreted with caution.


Assuntos
Anopheles/parasitologia , Proteínas de Fluorescência Verde , Luciferases , Malária Falciparum/transmissão , Plasmodium falciparum/fisiologia , Animais , Feminino , Humanos , Medições Luminescentes , Microscopia , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/fisiologia , Plasmodium falciparum/genética
8.
Antiviral Res ; 103: 17-24, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24393668

RESUMO

Proteolytical cleavage of the picornaviral polyprotein is essential for viral replication. Therefore, viral proteases are attractive targets for anti-viral therapy. Most assays available for testing proteolytical activity of proteases are performed in vitro, using heterologously expressed proteases and peptide substrates. To deal with the disadvantages associated with in vitro assays, we modified a cell-based protease assay for picornavirus proteases. The assay is based on the induction of expression of a firefly luciferase reporter by a chimeric transcription factor in which the viral protease and cleavage sites are inserted between the GAL4 binding domain and the VP16 activation domain. Firefly luciferase expression is dependent on cleavage of the transcription factor by the viral protease. This biosafe assay enables testing the effect of compounds on protease activity in cells while circumventing the need for infection. We designed the assay for 3C proteases (3C(pro)) of various enteroviruses as well as of viruses of several other picornavirus genera, and show that the assay is amenable for use in a high-throughput setting. Furthermore, we show that the spectrum of activity of 3C(pro) inhibitor AG7088 (rupintrivir) not only encompasses enterovirus 3C(pro) but also 3C(pro) of foot-and-mouth disease virus (FMDV), an aphthovirus. In contrary, AG7404 (compound 1), an analogue of AG7088, had no effect on FMDV 3C(pro) activity, for which we provide a structural explanation.


Assuntos
Antivirais/isolamento & purificação , Antivirais/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Picornaviridae/efeitos dos fármacos , Picornaviridae/enzimologia , Proteínas Virais/antagonistas & inibidores , Animais , Linhagem Celular , Cisteína Endopeptidases , Genes Reporter , Humanos , Luciferases de Vaga-Lume/análise , Luciferases de Vaga-Lume/genética , Inibidores de Proteases/isolamento & purificação , Inibidores de Proteases/farmacologia
9.
Lancet Infect Dis ; 14(2): 130-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24239324

RESUMO

BACKGROUND: Primaquine is the only available drug that clears mature Plasmodium falciparum gametocytes in infected human hosts, thereby preventing transmission of malaria to mosquitoes. However, concerns about dose-dependent haemolysis in people with glucose-6-phosphate dehydrogenase (G6PD) deficiencies have limited its use. We assessed the dose-response association of single-dose primaquine for gametocyte clearance and for safety in P falciparum malaria. METHODS: We undertook this randomised, double-blind, placebo-controlled trial with four parallel groups in Jinja district, eastern Uganda. We randomly allocated Ugandan children aged 1-10 years with uncomplicated falciparum malaria and normal G6PD enzyme function to receive artemether-lumefantrine, combined with either placebo or with 0.1 mg/kg, 0.4 mg/kg, or 0.75 mg/kg (WHO reference dose) primaquine base. Randomisation was done with computer-generated four-digit treatment assignment codes allocated to random dose groups in block sizes of 16. Study staff who provided care or assessed outcomes and the participants remained masked to the intervention group after assignment. The primary efficacy endpoint was the non-inferiority of the mean duration of gametocyte carriage in the test doses compared with the reference group of 0.75 mg primaquine per kg, with a non-inferiority margin of 2.5 days. The primary safety endpoint was the superiority of the arithmetic mean maximum decrease in haemoglobin concentration from enrolment to day 28 of follow-up in the primaquine treatment groups compared with placebo, with use of significance testing of pairwise comparisons with a cutoff of p=0.05. The trial is registered with ClinicalTrials.gov, number NCT01365598. FINDINGS: We randomly allocated 468 participants to receive artemether-lumefantrine combined with placebo (119 children) or with 0.1 mg/kg (116), 0.4 mg/kg (116), or 0.75 mg/kg (117) primaquine base. The mean duration of gametocyte carriage was 6.6 days (95% CI 5.3-7.8) in the 0.75 mg/kg reference group, 6.3 days (5.1-7.5) in the 0.4 mg/kg primaquine group (p=0.74), 8.0 days (6.6-9.4) in the 0.1 mg/kg primaquine group (p=0.14), and 12.4 days (9.9-15.0) in the placebo group (p<0.0001). No children showed evidence of treatment-related haemolysis, and the mean maximum decrease in haemoglobin concentration was not associated with the dose of primaquine received-it did not differ significantly compared with placebo (10.7 g/L, SD 11.1) in the 0.1 mg/kg (11.4 g/L, 9.4; p=0.61), 0.4 mg/kg (11.3 g/L, 10.0; p=0.67), or 0.75 mg/kg (12.7 g/L, 8.2; p=0.11) primaquine groups. INTERPRETATION: We conclude that 0.4 mg/kg primaquine has similar gametocytocidal efficacy to the reference 0.75 mg/kg primaquine dose, but a dose of 0.1 mg/kg was inconclusive for non-inferiority. Our findings call for the prioritisation of further trials into the efficacy and safety of doses of primaquine between 0.1 mg/kg and 0.4 mg/kg (including the dose of 0.25 mg/kg recently recommended by WHO), in view of the potential for widespread use of the drug to block malaria transmission. FUNDING: Wellcome Trust and the Bill & Melinda Gates Foundation.


Assuntos
Antimaláricos/uso terapêutico , Malária Falciparum/tratamento farmacológico , Primaquina/uso terapêutico , Combinação Arteméter e Lumefantrina , Artemisininas/uso terapêutico , Criança , Pré-Escolar , Relação Dose-Resposta a Droga , Método Duplo-Cego , Esquema de Medicação , Combinação de Medicamentos , Etanolaminas/uso terapêutico , Feminino , Fluorenos/uso terapêutico , Seguimentos , Humanos , Lactente , Masculino , Carga Parasitária , Curva ROC , Resultado do Tratamento , Uganda
10.
J Virol ; 88(5): 3048-51, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24371067

RESUMO

Coxsackieviruses require phosphatidylinositol-4-kinase IIIß (PI4KIIIß) for replication but can bypass this need by an H57Y mutation in protein 3A (3A-H57Y). We show that mutant coxsackievirus is not outcompeted by wild-type virus during 10 passages in vitro. In mice, the mutant virus proved as virulent as wild-type virus, even when mice were treated with a PI4KIIIß inhibitor. Our data suggest that upon emergence, the 3A-H57Y mutant has the fitness to establish a resistant population with a virulence similar to that of wild-type virus.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Enterovirus/fisiologia , Aptidão Genética , Mutação , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Infecções por Coxsackievirus/metabolismo , Infecções por Coxsackievirus/virologia , Enterovirus/patogenicidade , Interações Hospedeiro-Patógeno , Camundongos , Proteínas Virais/genética , Proteínas Virais/metabolismo , Virulência/genética , Replicação Viral
11.
Sci Rep ; 3: 3418, 2013 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-24301557

RESUMO

Mosquito feeding assays are important in evaluations of malaria transmission-reducing interventions. The proportion of mosquitoes with midgut oocysts is commonly used as an outcome measure, but in natural low intensity infections the effect of oocyst non-rupture on mosquito infectivity is unclear. By identifying ruptured as well as intact oocysts, we show that in low intensity P. falciparum infections i) 66.7-96.7% of infected mosquitoes experienced oocyst rupture between 11-21 days post-infection, ii) oocyst rupture led invariably to sporozoite release, iii) oocyst rupture led to salivary gland infections in 97.8% of mosquitoes, and iv) 1250 (IQR 313-2400) salivary gland sporozoites were found per ruptured oocyst. These data show that infectivity can be predicted with reasonable certainty from oocyst prevalence in low intensity infections. High throughput methods for detecting infection in whole mosquitoes showed that 18s PCR but not circumsporozoite ELISA gave a reliable approximation of mosquito infection rates on day 7 post-infection.


Assuntos
Culicidae/fisiologia , Culicidae/parasitologia , Oocistos/fisiologia , Oocistos/parasitologia , Esporozoítos/fisiologia , Animais , Feminino , Insetos Vetores/parasitologia , Insetos Vetores/fisiologia , Malária/parasitologia , Plasmodium falciparum , Prevalência , Glândulas Salivares/parasitologia , Glândulas Salivares/fisiologia , Esporozoítos/parasitologia
12.
Antimicrob Agents Chemother ; 57(10): 4971-81, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23896472

RESUMO

Despite their high clinical and socioeconomic impacts, there is currently no approved antiviral therapy for the prophylaxis or treatment of enterovirus infections. Here we report on a novel inhibitor of enterovirus replication, compound 1, 2-fluoro-4-(2-methyl-8-(3-(methylsulfonyl)benzylamino)imidazo[1,2-a]pyrazin-3-yl)phenol. This compound exhibited a broad spectrum of antiviral activity, as it inhibited all tested species of enteroviruses and rhinoviruses, with 50% effective concentrations ranging between 4 and 71 nM. After a lengthy resistance selection process, coxsackievirus mutants resistant to compound 1 were isolated that carried substitutions in their 3A protein. Remarkably, the same substitutions were recently shown to provide resistance to inhibitors of phosphatidylinositol 4-kinase IIIß (PI4KIIIß), a lipid kinase that is essential for enterovirus replication, suggesting that compound 1 may also target this host factor. Accordingly, compound 1 directly inhibited PI4KIIIß in an in vitro kinase activity assay. Furthermore, the compound strongly reduced the PI 4-phosphate levels of the Golgi complex in cells. Rescue of coxsackievirus replication in the presence of compound 1 by a mutant PI4KIIIß carrying a substitution in its ATP-binding pocket revealed that the compound directly binds the kinase at this site. Finally, we determined that an analogue of compound 1, 3-(3-fluoro-4-methoxyphenyl)-2-methyl-N-(pyridin-4-ylmethyl)imidazo[1,2-a]pyrazin-8-amine, is well tolerated in mice and has a dose-dependent protective activity in a coxsackievirus serotype B4-induced pancreatitis model.


Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Antivirais/farmacologia , Antivirais/uso terapêutico , Enterovirus/efeitos dos fármacos , Enterovirus/metabolismo , Animais , Enterovirus/patogenicidade , Ativação Enzimática/efeitos dos fármacos , Imunofluorescência , Células HeLa , Humanos , Masculino , Camundongos , Estrutura Molecular , Pancreatite/tratamento farmacológico , Pancreatite/metabolismo , Replicação Viral/efeitos dos fármacos
13.
Antimicrob Agents Chemother ; 57(4): 1952-6, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23335743

RESUMO

Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)--a selective serotonin reuptake inhibitor--inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as the target of this compound.


Assuntos
Proteínas de Transporte/metabolismo , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/metabolismo , Enterovirus Humano D/efeitos dos fármacos , Enterovirus Humano D/metabolismo , Fluoxetina/farmacologia , Inibidores de Captação de Serotonina/farmacologia , Proteínas não Estruturais Virais/metabolismo , Proteínas de Transporte/genética , Enterovirus Humano B/genética , Enterovirus Humano D/genética , Proteínas não Estruturais Virais/genética
14.
Cell Res ; 22(11): 1576-92, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22945356

RESUMO

RNA viruses can rapidly mutate and acquire resistance to drugs that directly target viral enzymes, which poses serious problems in a clinical context. Therefore, there is a growing interest in the development of antiviral drugs that target host factors critical for viral replication, since they are unlikely to mutate in response to therapy. We recently demonstrated that phosphatidylinositol-4-kinase IIIß (PI4KIIIß) and its product phosphatidylinositol-4-phosphate (PI4P) are essential for replication of enteroviruses, a group of medically important RNA viruses including poliovirus (PV), coxsackievirus, rhinovirus, and enterovirus 71. Here, we show that enviroxime and GW5074 decreased PI4P levels at the Golgi complex by directly inhibiting PI4KIIIß. Coxsackievirus mutants resistant to these inhibitors harbor single point mutations in the non-structural protein 3A. These 3A mutations did not confer compound-resistance by restoring the activity of PI4KIIIß in the presence of the compounds. Instead, replication of the mutant viruses no longer depended on PI4KIIIß, since their replication was insensitive to siRNA-mediated depletion of PI4KIIIß. The mutant viruses also did not rely on other isoforms of PI4K. Consistently, no high level of PI4P could be detected at the replication sites induced by the mutant viruses in the presence of the compounds. Collectively, these findings indicate that through specific single point mutations in 3A, CVB3 can bypass an essential host factor and lipid for its propagation, which is a new example of RNA viruses acquiring resistance against antiviral compounds, even when they directly target host factors.


Assuntos
1-Fosfatidilinositol 4-Quinase/antagonistas & inibidores , Antivirais/farmacologia , Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Proteínas não Estruturais Virais/genética , 1-Fosfatidilinositol 4-Quinase/metabolismo , Animais , Benzimidazóis/farmacologia , Linhagem Celular Tumoral , Chlorocebus aethiops , Farmacorresistência Viral/genética , Enterovirus Humano B/genética , Células HeLa , Humanos , Indóis/farmacologia , Oximas , Fenóis/farmacologia , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Mutação Puntual , Interferência de RNA , RNA Interferente Pequeno , Sulfonamidas , Replicação Viral/efeitos dos fármacos
15.
Diabetes ; 61(8): 2030-6, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22596052

RESUMO

Enteroviruses of the human enterovirus B species (HEV-Bs) (e.g., coxsackie B viruses [CVBs] and echoviruses) have been implicated as environmental factors that trigger/accelerate type 1 diabetes, but the underlying mechanism remains elusive. The aim of this study was to gain insight into the cytokines and chemokines that are produced by human pancreatic islets upon infection with CVBs. To this end, we studied the response of human islets of Langerhans upon mock or CVB3 infection. Using quantitative PCR, we showed that upon CVB3 infection, transcription of interferon (IFN), IFN-stimulated genes, and inflammatory genes was induced. Analysis of secreted cytokines and chemokines by Luminex technology confirmed production and secretion of proinflammatory cytokines (e.g., interleukin [IL]-6 and tumor necrosis factor-α) as well as various chemotactic proteins, such as IFN-γ-induced protein 10, macrophage inflammatory protein (MIP)-1α, MIP-1ß, and IL-8. Infection with other HEV-Bs induced similar responses, yet their extent depended on replication efficiency. Ultra violet-inactivated CVB3 did not induce any response, suggesting that virus replication is a prerequisite for antiviral responses. Our data represent the first comprehensive overview of inflammatory mediators that are secreted by human islets of Langerhans upon CVB infection and may shed light on the role of enteroviruses in type 1 diabetes pathogenesis.


Assuntos
Quimiocinas/biossíntese , Infecções por Coxsackievirus/metabolismo , Citocinas/biossíntese , Enterovirus Humano B , Ilhotas Pancreáticas/metabolismo , Diabetes Mellitus Tipo 1/virologia , Humanos
16.
J Virol ; 84(15): 7535-42, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20504936

RESUMO

The genus Enterovirus, belonging to the family Picornaviridae, includes well-known pathogens, such as poliovirus, coxsackievirus, and rhinovirus. Brefeldin A (BFA) impedes replication of several enteroviruses through inhibition of Golgi-specific BFA resistance factor 1 (GBF1), a regulator of secretory pathway integrity and transport. GBF1 mediates the GTP exchange of Arf1, which in activated form recruits coatomer protein complex I (COP-I) to Golgi vesicles, a process important in transport between the endoplasmic reticulum and Golgi vesicles. Recently, the drugs AG1478 and Golgicide A (GCA) were put forward as new inhibitors of GBF1. In this study, we investigated the effects of these putative GBF1 inhibitors on secretory pathway function and enterovirus replication. We show that both drugs induced fragmentation of the Golgi vesicles and caused dissociation of Arf1 and COP-I from Golgi membranes, yet they differed in their effect on GBF1 localization. The effects of AG1478, but not those of GCA, could be countered by overexpression of Arf1, indicating a difference in their molecular mechanism of action. Consistent with this idea, we observed that GCA drastically reduced replication of coxsackievirus B3 (CVB3) and other human enterovirus species, whereas AG1478 had no effect at all on enterovirus replication. Time-of-addition studies and analysis of RNA replication using a subgenomic replicon both showed that GCA suppresses RNA replication of CVB3, which could be countered by overexpression of GBF1. These results indicate that, in contrast to AG1478, GCA inhibits CVB3 RNA replication by targeting GBF1. AG1478 and GCA may be valuable tools to further dissect enterovirus replication.


Assuntos
Enterovirus Humano B/efeitos dos fármacos , Enterovirus Humano B/fisiologia , Inibidores Enzimáticos/farmacologia , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Piridinas/farmacologia , Quinolinas/farmacologia , Tirfostinas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Cricetinae , Complexo de Golgi/efeitos dos fármacos , Humanos , Quinazolinas
17.
Viral Immunol ; 23(1): 99-104, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20121407

RESUMO

Previous studies have shown that enteroviral RNA can be detected in blood at the onset of type 1 diabetes (T1D). The infection may play a role in triggering T1D and genetic host factors may contribute to this process. We investigated (1) whether enterovirus is present at the onset of T1D in peripheral blood mononuclear cells (PBMC), plasma, throat, or stool, and (2) whether enteroviral presence is linked with HLA-DR type and/or polymorphisms in melanoma differentiation-associated gene 5 (MDA5) and 2'-5' oligoadenylate synthetase 1 (OAS1), factors of antiviral immunity. To this end, PBMC, plasma, throat, and stool samples from 10 T1D patients and 20 unrelated controls were tested for the presence of enteroviruses (RT-PCR), for HLA-DR type, and polymorphisms in MDA5 and OAS1. Enterovirus RNA was detected in PBMC of 4/10 T1D patients, but none of 20 controls. Plasma was positive in 2/10 T1D patients and none of 20 controls, suggesting that enteroviruses found at the onset of T1D are mainly present in PBMC. All throat samples from positive T1D patients were virus-negative and only 1 fecal sample was positive. The negative results for all throat and most stool samples argues against acute infection. Enterovirus presence was linked with HLA-DR4, but not with polymorphisms in MDA5 or OAS1.


Assuntos
Sangue/virologia , Diabetes Mellitus Tipo 1/virologia , Infecções por Enterovirus/complicações , Infecções por Enterovirus/virologia , Enterovirus/isolamento & purificação , Leucócitos Mononucleares/virologia , RNA Viral/isolamento & purificação , 2',5'-Oligoadenilato Sintetase/genética , Adolescente , Criança , Pré-Escolar , RNA Helicases DEAD-box/genética , Enterovirus/genética , Fezes/virologia , Feminino , Antígenos HLA-DR/genética , Humanos , Helicase IFIH1 Induzida por Interferon , Masculino , Faringe/virologia , Plasma/virologia , Polimorfismo Genético , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
18.
Diabetes ; 59(5): 1182-91, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20071599

RESUMO

OBJECTIVE: Type 1 diabetes is a chronic endocrine disorder in which enteroviruses, such as coxsackie B viruses and echoviruses, are possible environmental factors that can trigger or accelerate disease. The development or acceleration of type 1 diabetes depends on the balance between autoreactive effector T-cells and regulatory T-cells. This balance is particularly influenced by dendritic cells (DCs). The goal of this study was to investigate the interaction between enterovirus-infected human pancreatic islets and human DCs. RESEARCH DESIGN AND METHODS: In vitro phagocytosis of human or porcine primary islets or Min6 mouse insuloma cells by DCs was investigated by flow cytometry and confocal analysis. Subsequent innate DC responses were monitored by quantitative PCR and Western blotting of interferon-stimulated genes (ISGs). RESULTS: In this study, we show that both mock- and coxsackievirus B3 (CVB3)-infected human and porcine pancreatic islets were efficiently phagocytosed by human monocyte-derived DCs. Phagocytosis of CVB3-infected, but not mock-infected, human and porcine islets resulted in induction of ISGs in DCs, including the retinoic acid-inducible gene (RIG)-I-like helicases (RLHs), RIG-I, and melanoma differentiation-associated gene 5 (Mda5). Studies with murine Min6 insuloma cells, which were also efficiently phagocytosed, revealed that increased ISG expression in DCs upon encountering CVB-infected cells resulted in an antiviral state that protected DCs from subsequent enterovirus infection. The observed innate antiviral responses depended on RNA within the phagocytosed cells, required endosomal acidification, and were type I interferon dependent. CONCLUSIONS: Human DCs can phagocytose enterovirus-infected pancreatic cells and subsequently induce innate antiviral responses, such as induction of RLHs. These responses may have important consequences for immune homeostasis in vivo and may play a role in the etiology of type 1 diabetes.


Assuntos
Células Dendríticas/imunologia , Enterovirus/fisiologia , Imunidade Inata/imunologia , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/virologia , Fagocitose/imunologia , Animais , Células Cultivadas , Enterovirus/efeitos dos fármacos , Humanos , Células Secretoras de Insulina/imunologia , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/imunologia , Ilhotas Pancreáticas/virologia , Camundongos , Suínos , Células Tumorais Cultivadas
19.
J Virol ; 83(22): 11940-9, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19740986

RESUMO

The replication of enteroviruses is sensitive to brefeldin A (BFA), an inhibitor of endoplasmic reticulum-to-Golgi network transport that blocks activation of guanine exchange factors (GEFs) of the Arf GTPases. Mammalian cells contain three BFA-sensitive Arf GEFs: GBF1, BIG1, and BIG2. Here, we show that coxsackievirus B3 (CVB3) RNA replication is insensitive to BFA in MDCK cells, which contain a BFA-resistant GBF1 due to mutation M832L. Further evidence for a critical role of GBF1 stems from the observations that viral RNA replication is inhibited upon knockdown of GBF1 by RNA interference and that replication in the presence of BFA is rescued upon overexpression of active, but not inactive, GBF1. Overexpression of Arf proteins or Rab1B, a GTPase that induces GBF1 recruitment to membranes, failed to rescue RNA replication in the presence of BFA. Additionally, the importance of the interaction between enterovirus protein 3A and GBF1 for viral RNA replication was investigated. For this, the rescue from BFA inhibition of wild-type (wt) replicons and that of mutant replicons of both CVB3 and poliovirus (PV) carrying a 3A protein that is impaired in binding GBF1 were compared. The BFA-resistant GBF1-M832L protein efficiently rescued RNA replication of both wt and mutant CVB3 and PV replicons in the presence of BFA. However, another BFA-resistant GBF1 protein, GBF1-A795E, also efficiently rescued RNA replication of the wt replicons, but not that of mutant replicons, in the presence of BFA. In conclusion, this study identifies a critical role for GBF1 in CVB3 RNA replication, but the importance of the 3A-GBF1 interaction requires further study.


Assuntos
Enterovirus Humano B/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , RNA Viral/biossíntese , Replicação Viral/fisiologia , Fator 1 de Ribosilação do ADP/fisiologia , Animais , Brefeldina A/farmacologia , Linhagem Celular , Enterovirus Humano B/efeitos dos fármacos , GTP Fosfo-Hidrolases/fisiologia , Células HeLa , Humanos , Replicação Viral/efeitos dos fármacos
20.
Traffic ; 10(3): 316-23, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19170981

RESUMO

ADP-ribosylation factor (Arf) proteins are small guanosine triphosphatases (GTPases) that act as major regulators of intracellular vesicular trafficking and secretory organelle pathway integrity. Like all small monomeric GTPases, Arf proteins cycle between a GDP-bound and a GTP-bound state, and this cycling is catalysed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins. While the class I Arfs, especially Arf1, have been studied extensively, little is known as yet about the function and regulation of class II Arfs, Arf4 and Arf5. In this study, we show that Arf proteins show class-specific dynamic behaviour. Moreover, unlike class I Arfs, membrane association of class II Arfs is resistant to inhibition of large Arf GEFs by Brefeldin A. Through the construction of Arf chimeric proteins, evidence is provided that the N-terminal amphipathic helix and a class-specific residue in the conserved interswitch domain determine the membrane-binding properties of class I and class II Arf proteins. Our results show that fundamental differences exist in behaviour and regulation of these small GTPases.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Membrana Celular/metabolismo , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Sequência de Aminoácidos , Animais , Brefeldina A/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Haplorrinos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...