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1.
Cell Rep ; 42(8): 112977, 2023 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-37573505

RESUMO

Arthropod-borne viruses (arboviruses) transmitted by Aedes aegypti mosquitoes are an increasing threat to global health. The small interfering RNA (siRNA) pathway is considered the main antiviral immune pathway of insects, but its effective impact on arbovirus transmission is surprisingly poorly understood. Here, we use CRISPR-Cas9-mediated gene editing in vivo to mutate Dicer2, a gene encoding the RNA sensor and key component of the siRNA pathway. The loss of Dicer2 enhances early viral replication and systemic viral dissemination of four medically significant arboviruses (chikungunya, Mayaro, dengue, and Zika viruses) representing two viral families. However, Dicer2 mutants and wild-type mosquitoes display overall similar levels of vector competence. In addition, Dicer2 mutants undergo significant virus-induced mortality during infection with chikungunya virus. Together, our results define a multifaceted role for Dicer2 in the transmission of arboviruses by Ae. aegypti mosquitoes and pave the way for further mechanistic investigations.


Assuntos
Aedes , Arbovírus , Infecção por Zika virus , Zika virus , Animais , Humanos , Arbovírus/genética , Arbovírus/metabolismo , Mosquitos Vetores , Zika virus/genética , RNA Interferente Pequeno/metabolismo
2.
Nat Ecol Evol ; 6(5): 565-578, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35273366

RESUMO

Host-pathogen interactions impose recurrent selective pressures that lead to constant adaptation and counter-adaptation in both competing species. Here, we sought to study this evolutionary arms-race and assessed the impact of the innate immune system on viral population diversity and evolution, using Drosophila melanogaster as model host and its natural pathogen Drosophila C virus (DCV). We isogenized eight fly genotypes generating animals defective for RNAi, Imd and Toll innate immune pathways as well as pathogen-sensing and gut renewal pathways. Wild-type or mutant flies were then orally infected with DCV and the virus was serially passaged ten times via reinfection in naive flies. Viral population diversity was studied after each viral passage by high-throughput sequencing and infection phenotypes were assessed at the beginning and at the end of the evolution experiment. We found that the absence of any of the various immune pathways studied increased viral genetic diversity while attenuating virulence. Strikingly, these effects were observed in a range of host factors described as having mainly antiviral or antibacterial functions. Together, our results indicate that the innate immune system as a whole and not specific antiviral defence pathways in isolation, generally constrains viral diversity and evolution.


Assuntos
Proteínas de Drosophila , Vírus de RNA , Animais , Antivirais/metabolismo , Dicistroviridae , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Imunidade Inata , Vírus de RNA/metabolismo
3.
Nat Commun ; 12(1): 2290, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33863888

RESUMO

Arthropod-borne viruses pose a major threat to global public health. Thus, innovative strategies for their control and prevention are urgently needed. Here, we exploit the natural capacity of viruses to generate defective viral genomes (DVGs) to their detriment. While DVGs have been described for most viruses, identifying which, if any, can be used as therapeutic agents remains a challenge. We present a combined experimental evolution and computational approach to triage DVG sequence space and pinpoint the fittest deletions, using Zika virus as an arbovirus model. This approach identifies fit DVGs that optimally interfere with wild-type virus infection. We show that the most fit DVGs conserve the open reading frame to maintain the translation of the remaining non-structural proteins, a characteristic that is fundamental across the flavivirus genus. Finally, we demonstrate that the high fitness DVG is antiviral in vivo both in the mammalian host and the mosquito vector, reducing transmission in the latter by up to 90%. Our approach establishes the method to interrogate the DVG fitness landscape, and enables the systematic identification of DVGs that show promise as human therapeutics and vector control strategies to mitigate arbovirus transmission and disease.


Assuntos
Antivirais/administração & dosagem , Vírus Defeituosos/genética , Mosquitos Vetores/efeitos dos fármacos , Infecção por Zika virus/tratamento farmacológico , Zika virus/genética , Aedes/efeitos dos fármacos , Aedes/virologia , Animais , Chlorocebus aethiops , Biologia Computacional , Evolução Molecular Direcionada , Modelos Animais de Doenças , Feminino , Aptidão Genética , Genoma Viral/genética , Células HEK293 , Humanos , Camundongos , Controle de Mosquitos/métodos , Mosquitos Vetores/virologia , Fases de Leitura Aberta/genética , RNA Viral/genética , Células Vero , Infecção por Zika virus/transmissão , Infecção por Zika virus/virologia
4.
Cell Rep ; 33(11): 108506, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33326778

RESUMO

Transgenerational immune priming (TGIP) allows memory-like immune responses to be transmitted from parents to offspring in many invertebrates. Despite increasing evidence for TGIP in insects, the mechanisms involved in the transfer of information remain largely unknown. Here, we show that Drosophila melanogaster and Aedes aegypti transmit antiviral immunological memory to their progeny that lasts throughout generations. We observe that TGIP, which is virus and sequence specific but RNAi independent, is initiated by a single exposure to disparate RNA viruses and also by inoculation of a fragment of viral double-stranded RNA. The progeny, which inherit a viral DNA that is only a fragment of the viral RNA used to infect the parents, display enriched expression of genes related to chromatin and DNA binding. These findings represent a demonstration of TGIP for RNA viruses in invertebrates, broadly increasing our understanding of the immune response, host genome plasticity, and antiviral memory of the germline.


Assuntos
Aedes/virologia , Antivirais/imunologia , Drosophila melanogaster/virologia , Memória Imunológica/imunologia , Animais , Insetos
5.
Nat Microbiol ; 3(12): 1394-1403, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30374170

RESUMO

Immune responses in insects are differentially triggered depending on the infection route used by the pathogen. In most studies involving Drosophila melanogaster and viruses, infection is done by injection, while oral infection, which is probably the most common route of viral entry in nature, remains unexplored. Here, we orally infected adults and larvae from wild-type and RNA interference (RNAi) mutant flies with different RNA viruses. We found that, in contrast with what is observed following virus injection, oral infections initiated at larval or adult stages are cleared in adult flies. Virus elimination occurred despite a larger infectious dose than for injected flies and evidence of viral replication. RNAi mutant flies suffered greater mortality relative to wild-type flies following oral infection, but they also eliminated the virus, implying that RNAi is not essential for viral clearance and that other immune mechanisms act during oral infections. We further showed that information of infection by RNA viruses acquired orally leaves a trace under a DNA form, which confers protection against future reinfection by the same virus. Together, this work presents evidence of clearance and immune priming for RNA viruses in insects and challenges the current view of antiviral immunity in insects.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/virologia , Interferência de RNA/imunologia , Infecções por Vírus de RNA/imunologia , Vírus de RNA/imunologia , Vírus de RNA/patogenicidade , Animais , Antivirais/imunologia , Antivirais/farmacologia , Proteínas Argonautas/genética , Proteínas Argonautas/imunologia , DNA Viral/imunologia , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Feminino , Larva/virologia , Masculino , RNA Helicases/genética , RNA Helicases/imunologia , Ribonuclease III/genética , Ribonuclease III/imunologia , Análise de Sobrevida , Replicação Viral
6.
Cell Host Microbe ; 23(3): 353-365.e8, 2018 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-29503180

RESUMO

The RNAi pathway confers antiviral immunity in insects. Virus-specific siRNA responses are amplified via the reverse transcription of viral RNA to viral DNA (vDNA). The nature, biogenesis, and regulation of vDNA are unclear. We find that vDNA produced during RNA virus infection of Drosophila and mosquitoes is present in both linear and circular forms. Circular vDNA (cvDNA) is sufficient to produce siRNAs that confer partially protective immunity when challenged with a cognate virus. cvDNAs bear homology to defective viral genomes (DVGs), and DVGs serve as templates for vDNA and cvDNA synthesis. Accordingly, DVGs promote the amplification of vDNA-mediated antiviral RNAi responses in infected Drosophila. Furthermore, vDNA synthesis is regulated by the DExD/H helicase domain of Dicer-2 in a mechanism distinct from its role in siRNA generation. We suggest that, analogous to mammalian RIG-I-like receptors, Dicer-2 functions like a pattern recognition receptor for DVGs to modulate antiviral immunity in insects.


Assuntos
Antivirais/imunologia , DNA Viral/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/imunologia , RNA Helicases/metabolismo , Vírus de RNA/imunologia , Ribonuclease III/metabolismo , Animais , Arbovírus/imunologia , Arbovírus/patogenicidade , Culicidae/imunologia , RNA Helicases DEAD-box/metabolismo , Proteínas de Drosophila/genética , Genes Virais/genética , Genoma Viral , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Mutação Puntual , RNA Helicases/genética , Interferência de RNA/imunologia , Infecções por Vírus de RNA , Vírus de RNA/genética , Vírus de RNA/patogenicidade , RNA Interferente Pequeno/genética , RNA Viral/metabolismo , Ribonuclease III/genética , Carga Viral , Replicação Viral
7.
PLoS Negl Trop Dis ; 11(12): e0006152, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29261661

RESUMO

Dengue virus (DENV) causes more human infections than any other mosquito-borne virus. The current lack of antiviral strategies has prompted genome-wide screens for host genes that are required for DENV infectivity. Earlier transcriptomic studies that identified DENV host factors in the primary vector Aedes aegypti used inbred laboratory colonies and/or pools of mosquitoes that erase individual variation. Here, we performed transcriptome sequencing on individual midguts in a field-derived Ae. aegypti population to identify new candidate host factors modulating DENV replication. We analyzed the transcriptomic data using an approach that accounts for individual co-variation between viral RNA load and gene expression. This approach generates a prediction about the agonist or antagonist effect of candidate genes on DENV replication based on the sign of the correlation between gene expression and viral RNA load. Using this method, we identified 39 candidate genes that went undetected by conventional pairwise comparison of gene expression levels between DENV-infected midguts and uninfected controls. Only four candidate genes were detected by both methods, emphasizing their complementarity. We demonstrated the value of our approach by functional validation of a candidate agonist gene encoding a sterol regulatory element-binding protein (SREBP), which was identified by correlation analysis but not by pairwise comparison. We confirmed that SREBP promotes DENV infection in the midgut by RNAi-mediated gene knockdown in vivo. We suggest that our approach for transcriptomic analysis can empower genome-wide screens for potential agonist or antagonist factors by leveraging inter-individual variation in gene expression. More generally, this method is applicable to a wide range of phenotypic traits displaying inter-individual variation.


Assuntos
Aedes/virologia , Vírus da Dengue/genética , Dengue/virologia , Insetos Vetores/virologia , Proteínas de Ligação a Elemento Regulador de Esterol/genética , Transcriptoma , Animais , Sistema Digestório/virologia , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Proteínas de Insetos/genética , Interferência de RNA , RNA Viral/análise , Carga Viral , Replicação Viral
8.
Nat Commun ; 7: 12410, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27580708

RESUMO

Mosquitoes develop long-lasting viral infections without substantial deleterious effects, despite high viral loads. This makes mosquitoes efficient vectors for emerging viral diseases with enormous burden on public health. How mosquitoes resist and/or tolerate these viruses is poorly understood. Here we show that two species of Aedes mosquitoes infected with two arboviruses from distinct families (dengue or chikungunya) generate a viral-derived DNA (vDNA) that is essential for mosquito survival and viral tolerance. Inhibition of vDNA formation leads to extreme susceptibility to viral infections, reduction of viral small RNAs due to an impaired immune response, and loss of viral tolerance. Our results highlight an essential role of vDNA in viral tolerance that allows mosquito survival and thus may be important for arbovirus dissemination and transmission. Elucidating the mechanisms of mosquito tolerance to arbovirus infection paves the way to conceptualize new antivectorial strategies to selectively eliminate arbovirus-infected mosquitoes.


Assuntos
Aedes/virologia , Arbovírus/genética , Vírus Chikungunya/genética , DNA Viral/genética , Vírus da Dengue/genética , Mosquitos Vetores/virologia , Animais , Linhagem Celular , Febre de Chikungunya/transmissão , Febre de Chikungunya/virologia , Vírus Chikungunya/crescimento & desenvolvimento , Chlorocebus aethiops , Cricetinae , DNA Viral/biossíntese , Dengue/transmissão , Dengue/virologia , Vírus da Dengue/crescimento & desenvolvimento , Interferência de RNA , RNA Viral/biossíntese , RNA Viral/genética , Células Vero , Carga Viral
9.
Nat Immunol ; 14(4): 396-403, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23435119

RESUMO

How persistent viral infections are established and maintained is widely debated and remains poorly understood. We found here that the persistence of RNA viruses in Drosophila melanogaster was achieved through the combined action of cellular reverse-transcriptase activity and the RNA-mediated interference (RNAi) pathway. Fragments of diverse RNA viruses were reverse-transcribed early during infection, which resulted in DNA forms embedded in retrotransposon sequences. Those virus-retrotransposon DNA chimeras produced transcripts processed by the RNAi machinery, which in turn inhibited viral replication. Conversely, inhibition of reverse transcription hindered the appearance of chimeric DNA and prevented persistence. Our results identify a cooperative function for retrotransposons and antiviral RNAi in the control of lethal acute infection for the establishment of viral persistence.


Assuntos
Drosophila melanogaster/genética , Drosophila melanogaster/virologia , Interferência de RNA , Infecções por Vírus de RNA/virologia , Vírus de RNA/genética , Transcrição Reversa , Animais , Sequência de Bases , Linhagem Celular , Vírus de DNA/química , Vírus de DNA/genética , Vírus de DNA/metabolismo , Modelos Animais de Doenças , Feminino , Ordem dos Genes , Modelos Biológicos , Dados de Sequência Molecular , Vírus de RNA/química , Vírus de RNA/metabolismo , RNA Interferente Pequeno/genética , Retroelementos , Carga Viral , Replicação Viral/genética
10.
Methods Mol Biol ; 721: 107-22, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21431681

RESUMO

At the current rate of technological progress, high-throughput sequencing of nucleic acids has become a commodity. These techniques are perfectly suitable for viral small RNAs sequencing and contribute to the understanding of many aspects of virus biology in the context of host-pathogen interaction. However, the generation of high quality data is still an issue and the preparation of small RNAs libraries that accurately reflect the viral siRNAs in the sample remains a challenge. In this chapter we describe how to clone and sequence libraries of viral small RNAs from infected insect samples (mosquito, drosophilidae, insect-derived cell lines).


Assuntos
Clonagem Molecular/métodos , RNA Interferente Pequeno/genética , RNA Viral/genética , Análise de Sequência de RNA/métodos , Vírus/genética , Sequência de Bases , Biologia Computacional , Primers do DNA/genética , Primers do DNA/metabolismo , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Marcação por Isótopo , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/isolamento & purificação , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , RNA Interferente Pequeno/química , RNA Interferente Pequeno/isolamento & purificação , RNA Interferente Pequeno/metabolismo , RNA Viral/química , RNA Viral/isolamento & purificação , RNA Viral/metabolismo , Transcrição Reversa
11.
Proc Natl Acad Sci U S A ; 107(45): 19390-5, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-20978209

RESUMO

Activation of innate antiviral responses in multicellular organisms relies on the recognition of structural differences between viral and cellular RNAs. Double-stranded (ds)RNA, produced during viral replication, is a well-known activator of antiviral defenses and triggers interferon production in vertebrates and RNAi in invertebrates and plants. Previous work in mammalian cells indicates that negative-strand RNA viruses do not appear to generate dsRNA, and that activation of innate immunity is triggered by the recognition of the uncapped 5' ends of viral RNA. This finding raises the question whether antiviral RNAi, which is triggered by the presence of dsRNA in insects, represents an effective host-defense mechanism against negative-strand RNA viruses. Here, we show that the negative-strand RNA virus vesicular stomatitis virus (VSV) does not produce easily detectable amounts of dsRNA in Drosophila cells. Nevertheless, RNAi represents a potent response to VSV infection, as illustrated by the high susceptibility of RNAi-defective mutant flies to this virus. VSV-derived small RNAs produced in infected cells or flies uniformly cover the viral genome, and equally map the genome and antigenome RNAs, indicating that they derive from dsRNA. Our findings reveal that RNAi is not restricted to the defense against positive-strand or dsRNA viruses but can also be highly efficient against a negative-strand RNA virus. This result is of particular interest in view of the frequent transmission of medically relevant negative-strand RNA viruses to humans by insect vectors.


Assuntos
Imunidade Inata/genética , Interferência de RNA/imunologia , Vesiculovirus/imunologia , Animais , Linhagem Celular , Drosophila/virologia , Genoma Viral , Insetos Vetores , Vírus de RNA/imunologia , RNA de Cadeia Dupla/análise , RNA Viral
12.
Nature ; 458(7236): 346-50, 2009 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-19204732

RESUMO

Multicellular organisms evolved sophisticated defence systems to confer protection against pathogens. An important characteristic of these immune systems is their ability to act both locally at the site of infection and at distal uninfected locations. In insects, such as Drosophila melanogaster, RNA interference (RNAi) mediates antiviral immunity. However, the antiviral RNAi defence in flies seems to be a local, cell-autonomous process, as flies are thought to be unable to generate a systemic RNAi response. Here we show that a recently defined double-stranded RNA (dsRNA) uptake pathway is essential for effective antiviral RNAi immunity in adult flies. Mutant flies defective in this dsRNA uptake pathway were hypersensitive to infection with Drosophila C virus and Sindbis virus. Mortality in dsRNA-uptake-defective flies was accompanied by 100-to 10(5)-fold increases in viral titres and higher levels of viral RNA. Furthermore, inoculating naked dsRNA into flies elicited a sequence-specific antiviral immune response that required an intact dsRNA uptake pathway. These findings suggest that spread of dsRNA to uninfected sites is essential for effective antiviral immunity. Notably, infection with green fluorescent protein (GFP)-tagged Sindbis virus suppressed expression of host-encoded GFP at a distal site. Thus, similar to protein-based immunity in vertebrates, the antiviral RNAi response in flies also relies on the systemic spread of a virus-specific immunity signal.


Assuntos
Drosophila melanogaster/imunologia , Drosophila melanogaster/virologia , Interferência de RNA/imunologia , Vírus de RNA/imunologia , Animais , Linhagem Celular , Drosophila melanogaster/genética , Drosophila melanogaster/microbiologia , Micrococcus luteus/imunologia , Pectobacterium carotovorum/imunologia , Vírus de RNA/fisiologia , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/imunologia , RNA de Cadeia Dupla/metabolismo , Sindbis virus/genética , Sindbis virus/crescimento & desenvolvimento , Sindbis virus/imunologia , Especificidade por Substrato
13.
Blood Purif ; 26(3): 300-10, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18451643

RESUMO

BACKGROUND/AIMS: This study aimed at evaluating oxidative stress (OS) markers (i) in a cross-sectional study of hemodialysis (HD) patients to investigate potential regional effects of these markers and (ii) in a prospective crossover study to evaluate vitamin E-coated membrane (VE) effects. METHODS: At baseline, OS parameters including low-density lipoprotein (LDL) oxidizability were measured in HD patients from five dialysis facilities. Patients were then randomly assigned to two treatment groups: group I patients (n = 33) switching to VE, and group II patients (n = 29) still using reference polysulfone (PS) membrane. After 3 months, patients were switched from VE to PS and vice versa for 6 months. The same OS parameters were measured after each period. RESULTS: At baseline, the cross-sectional analysis of LDL oxidizability showed a regional effect. By contrast, the crossover study did not show beneficial effects of VE on this parameter. CONCLUSION: Regional variations of LDL oxidizability in HD patients exist and may explain discrepancies in interventional therapy on OS.


Assuntos
Antioxidantes/farmacologia , Materiais Revestidos Biocompatíveis , Lipoproteínas LDL/química , Membranas Artificiais , Diálise Renal , Vitamina E/farmacologia , Idoso , Aminoácidos/química , Antioxidantes/administração & dosagem , Feminino , Glutationa/sangue , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/terapia , Peroxidação de Lipídeos , Lipídeos/sangue , Lipoproteínas LDL/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo , Polímeros , Estudos Prospectivos , Espécies Reativas de Oxigênio/sangue , Diálise Renal/instrumentação , Diálise Renal/métodos , Sulfonas , Fatores de Tempo , Vitamina E/administração & dosagem
14.
Mediators Inflamm ; 2006(2): 36735, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16883063

RESUMO

The severity of cystic fibrosis (CF) pulmonary disease is not directly related to CFTR genotype but depends upon several parameters, including neutrophil-dominated inflammation. Identification of agents modulating inflammation constitutes a relevant goal. Myeloperoxidase (MPO) is involved in both microbicidal and proinflammatory neutrophil activities. The aim of this study was to evaluate whether the -463GA MPO promoter polymorphism is linked to clinical severity of CF-associated pulmonary inflammation. This polymorphism significantly affects the level of MPO gene expression in leukocytes and the G allele is more expressing than the A allele. We show that MPO genotype significantly influences the severity of pulmonary disease in early stages, prior to the development of chronic lung infections, with GG genotype being associated with more severe CF disease. Our findings indicate that the level of MPO gene expression influences the CF pathogenesis, presumably reflecting cellular damage by MPO-generated oxidants or other activity of MPO in airway inflammation.


Assuntos
Fibrose Cística/sangue , Fibrose Cística/enzimologia , Regulação Enzimológica da Expressão Gênica , Peroxidase/genética , Polimorfismo Genético , Regiões Promotoras Genéticas , Adolescente , Adulto , Alelos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Genótipo , Humanos , Pulmão/metabolismo , Masculino , Fatores Sexuais
15.
Nephrol Dial Transplant ; 21(6): 1555-63, 2006 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-16476719

RESUMO

BACKGROUND: In haemodialysis (HD) patients, advanced oxidation protein products (AOPP) were previously ascribed to oxidized plasma proteins, resulting mainly from increased myeloperoxidase (MPO) activity. The aim of the present study was to assess the mechanisms leading to the generation of AOPP during the course of chronic kidney disease including end-stage renal disease, with particular focus on AOPP and MPO characterization in the plasma at decreasing levels of kidney function. METHODS: Phagocyte activation was evaluated by whole blood NADPH oxidase and MPO activities. In plasma, MPO protein concentration was quantified by ELISA and catalytic activity assayed by the spectrophotometric detection of phenol and 4-aminoantipyrine (AAP) co-oxidation in the presence of hydrogen peroxide (H(2)O(2)). RESULTS: In HD patients, plasma AOPP concentration was linked to neutrophil oxidative activity. Such an association was not found in control subjects or predialysis patients, suggesting that in the latter, AOPP generation did not mainly result from MPO released by activated neutrophils. Similarly, plasma AOPP correlated with plasma MPO protein concentration in HD patients, but not in control subjects or predialysis patients, suggesting that in the latter AOPP did not predominantly result from MPO activity. This interpretation was supported by the observation of a greater degree of co-oxidation of phenol and AAP in the absence of H(2)O(2) in predialysis patients than in HD patients or control subjects. The contribution of MPO dramatically differed between predialysis and HD patients (2+/-5 vs 46+/-6%; P<0.001). CONCLUSION: Our observations suggest that AOPP generation in predialysis patients mainly results from MPO-independent oxidation mechanisms.


Assuntos
Falência Renal Crônica/urina , Peroxidase/fisiologia , Toxinas Biológicas/urina , Uremia/etiologia , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Falência Renal Crônica/enzimologia , Masculino , Pessoa de Meia-Idade , Oxirredução , Peroxidase/metabolismo , Fagócitos/metabolismo , Fenol/metabolismo , Proteínas/metabolismo , Diálise Renal , Uremia/enzimologia
16.
J Immunol ; 174(10): 6381-90, 2005 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-15879139

RESUMO

In the present study, we provide evidence that procaspase-3 is a novel target of proteinase 3 (PR3) but not of human neutrophil elastase (HNE). Human mast cell clone 1 (HMC1) and rat basophilic leukemia (RBL) mast cell lines were transfected with PR3 or the inactive mutated PR3 (PR3S203A) or HNE cDNA. In both RBL/PR3 and HMC1/PR3, a constitutive activity of caspase-3 was measured with DEVD substrate, due to the direct processing of procaspase-3 by PR3. No caspase-3 activation was observed in cells transfected with the inactive PR3 mutant or HNE. Despite the high caspase-3 activity in RBL/PR3, no apoptosis was detected as demonstrated by an absence of 1) phosphatidylserine externalization, 2) mitochondria cytochrome c release, 3) upstream caspase-8 or caspase-9 activation, or 4) DNA fragmentation. In vitro, purified PR3 cleaved procaspase-3 into an active 22-kDa fragment. In neutrophils, the 22-kDa caspase-3 activation fragment was present only in resting neutrophils but was absent after apoptosis. The 22 kDa fragment was specific of myeloid cells because it was absent from resting lymphocytes. This 22-kDa fragment was not present when neutrophils were treated with pefabloc, an inhibitor of serine proteinase. Like in HMC1/PR3, the 22-kDa caspase-3 fragment was restricted to the plasma membrane compartment. Double immunofluorescence labeling after streptolysin-O permeabilization further showed that PR3 and procaspase-3 could colocalize in an extragranular compartment. In conclusion, our results strongly suggest that compartmentalized PR3-induced caspase-3 activation might play specific functions in neutrophil survival.


Assuntos
Apoptose , Caspases/metabolismo , Proteínas de Membrana/metabolismo , Neutrófilos/enzimologia , Serina Endopeptidases/fisiologia , Animais , Apoptose/genética , Apoptose/imunologia , Caspase 3 , Caspases/genética , Compartimento Celular/genética , Linhagem Celular Tumoral , Membrana Celular/enzimologia , Membrana Celular/genética , Proliferação de Células , Células Clonais , Ativação Enzimática/genética , Precursores Enzimáticos/genética , Humanos , Hidrólise , Peso Molecular , Mieloblastina , Neutrófilos/citologia , Neutrófilos/metabolismo , Fragmentos de Peptídeos/metabolismo , Ratos , Fase de Repouso do Ciclo Celular/genética , Serina Endopeptidases/genética , Frações Subcelulares/metabolismo , Transfecção , Regulação para Cima/genética
17.
Am J Kidney Dis ; 45(1): 39-47, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15696442

RESUMO

BACKGROUND: Inflammation and oxidative stress are established risk factors for atherosclerosis, but whether they contribute to the accelerated atherogenesis associated with chronic kidney disease (CKD) remains to be assessed at the predialysis stage. METHODS: We prospectively examined the relationship between plasma levels of C-reactive protein (CRP), fibrinogen, and advanced oxidation protein products (AOPPs), as selected markers of inflammation and oxidative stress, and incident first occlusive atherosclerotic cardiovascular (CV) events (ASCVEs) in a single-center cohort of 80 uremic predialysis patients without diabetes with a creatinine clearance ranging from 20 to 40 mL/min/1.73 m2 . RESULTS: During follow-up (median, 7 years), 21 patients developed coronary, cerebral, or peripheral artery occlusive accidents, an incidence of 44/1,000 patient-years. Except for older age, their conventional risk factors did not differ compared with the 59 patients who remained free of such accidents. Conversely, plasma levels of CRP (4.3 +/- 2.7 versus 2.3 +/- 2 mg/L; P = 0.005), fibrinogen (5.6 +/- 1.4 versus 4.4 +/- 1.2 mg/L; P = 0.0009), and AOPPs (58 +/- 20 versus 42 +/- 14 micromol/L; P = 0.0002) were significantly greater at baseline, although serum creatinine levels did not differ between the 2 groups. By multivariate Cox regression analysis, age and CRP, fibrinogen, and AOPP levels were significant independent predictors of ASCVEs. Risk factor-adjusted hazard ratios were as follows: age, 1.13 (95% confidence interval, 1.04 to 1.22; P = 0.002); CRP level, 1.37 (95% confidence interval, 1.05 to 1.79; P = 0.02); fibrinogen level, 2.23 (95% confidence interval, 1.20 to 4.13; P = 0.011); and AOPP level, 1.68 (95% confidence interval, 1.12 to 2.51; P = 0.011). CONCLUSION: CRP, fibrinogen, and AOPP levels independently predict ASCVEs in patients with CKD in the predialysis phase and might directly contribute to the uremia-associated accelerated atherogenesis.


Assuntos
Arteriosclerose/sangue , Proteínas Sanguíneas/metabolismo , Doenças Cardiovasculares/sangue , Diabetes Mellitus/sangue , Diálise/métodos , Biomarcadores/sangue , Doença Crônica , Estudos de Coortes , Feminino , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Estresse Oxidativo/fisiologia , Estudos Prospectivos , Insuficiência Renal/sangue , Fatores de Risco
18.
Kidney Int ; 66(4): 1606-12, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15458457

RESUMO

BACKGROUND: Inflammation and oxidative stress have been incriminated in the pathogenesis of IgA nephropathy (IgAN). The aim of the present study was to assess whether markers reflecting these pathophysiologic processes, namely C-reactive protein (CRP) and advanced oxidation protein products (AOPP), would allow-in conjunction with clinical and histopathologic parameters-to predict disease progression. METHODS: Between 1994 and 1997, 120 adult patients with biopsy-proven IgAN were included in a prospective cohort study, and followed until the end of 2002 or start of dialysis. In every patient, we determined plasma levels of CRP and AOPP. These parameters were included, together with clinical data, in a multivariate Cox proportional hazard regression analysis, with halving of baseline creatinine clearance as the primary renal end point. RESULTS: A total of 51 patients reached the renal end point, including 30 who had to start dialysis. With multivariate analysis, the most potent independent risk factors of poor renal outcome were proteinuria > or =1 g/day [proportional hazard risk (HR) = 23.7, P= 0.0001], hypertension (HR = 8.13, P= 0.008), and AOPP plasma level (HR = 1.09 per 10 micromol/L, P= 0.042), whereas angiotensin II inhibitors were protective (HR = 0.19, P= 0.001). CONCLUSION: Our data support the role of oxidative stress in the pathogenesis of IgAN and suggest that patients with proteinuria > or =1 g/day should be eligible for early implemented antioxidant and/or anti-inflammatory therapeutic strategies, with AOPP plasma level as a surrogate marker to evaluate their effects.


Assuntos
Glomerulonefrite por IGA/diagnóstico , Glomerulonefrite por IGA/mortalidade , Estresse Oxidativo , Proteinúria/diagnóstico , Proteinúria/mortalidade , Adulto , Proteínas Sanguíneas/metabolismo , Proteína C-Reativa/metabolismo , Progressão da Doença , Feminino , Humanos , Hipertensão Renal/diagnóstico , Hipertensão Renal/mortalidade , Masculino , Pessoa de Meia-Idade , Oxirredução , Valor Preditivo dos Testes , Prognóstico , Modelos de Riscos Proporcionais , Estudos Prospectivos
19.
Biochim Biophys Acta ; 1689(2): 91-102, 2004 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-15196590

RESUMO

We previously described the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of hemodialyzed patients (HD). The present study was carried out to further investigate how myeloperoxidase (MPO)-catalyzed reactions could contribute to AOPP generation in the plasma. First, patterns of plasma protein oxidation obtained after in vitro incubation of control plasma with hypochlorous acid (HOCl) were compared to those from HD patients and control plasma. The use of various analytical techniques enabled localising and identifying the main oxidized proteins with albumin (HSA) after protein separation by size-exclusion chromatography and SDS-PAGE electrophoresis. The characterization of the oxidation level of the individual plasma proteins in terms of carbonyl groups and 3-nitrotyrosine formations was performed by immunoblotting. Secondly, to highlight the significance of AOPP index monitored by spectrophotometry, spectra were established for plasma fractions from HD patients and compared to data for control plasma and HOCl-treated plasma. The corresponding absorbance difference spectra were matched with external standards such as dityrosine, nitrotyrosine and pentosidine and elaborated chromophoric probe models. Indeed, HSA was chlorinated by HOCl reagent or HOCl generated via the MPO/H(2)O(2)/Cl(-) system and was nitrated by tetranitromethane. Increased absorbances at the range of 340 nm were observed both with chlorinated and nitrated HSA. Finally, our results indicate that HOCl, and not NO(2)(*), generated via MPO activity, could represent one of the pathways for AOPP production in plasma proteins exposed to activated phagocytes.


Assuntos
Biomarcadores/sangue , Proteínas Sanguíneas/metabolismo , Peroxidase/metabolismo , Diálise Renal/métodos , Insuficiência Renal/metabolismo , Espectrofotometria/métodos , Proteínas Sanguíneas/química , Humanos , Ácido Hipocloroso/química , Nitritos/metabolismo , Oxirredução , Estresse Oxidativo , Peroxidase/sangue , Insuficiência Renal/sangue , Albumina Sérica/química , Albumina Sérica/metabolismo
20.
Kidney Int ; 64(1): 82-91, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12787398

RESUMO

UNLABELLED: AOPP-induced activation of human neutrophil and monocyte oxidative metabolism: A potential target forN-acetylcysteine treatment in dialysis patients. BACKGROUND: Oxidative stress largely contributes to hemodialysis-associated lethal complications, thus explaining the urgent need of antioxidant-based therapeutic strategies in hemodialysis patients. We previously identified advanced oxidation protein products (AOPP) in the uremic plasma as exquisite markers of oxidative stress and potent mediators of monocyte activation. The present study was aimed at searching whether (1) AOPP can also trigger activation of polymorphonuclear neutrophils (PMN), and (2) whether AOPP-induced activation could be inhibited by N-acetylcysteine (NAC), a widely used compound which has been shown to prevent oxidative injury to kidney. METHODS: Both human serum albumin (HAS) AOPP (i.e., HOCl-modified HSA in vitro preparations and AOPP extracted from plasma of hemodialysis patients) were tested for their capacity to trigger phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and myeloperoxidase (MPO)-dependent activities as measured by lucigenin- and luminol-amplified chemiluminescence (CL), respectively, as compared to receptor-dependent [opsonized zymosan or receptor-independent phorbol myristate acetate (PMA)]. The effect of PMN priming by platelet-activating factor (PAF), and the effect of NAC on normal monocyte and on normal or hemodialysis patient's (N = 16) PMN oxidative responses were compared. RESULTS: HSA-AOPP triggered in a HOCl dose-dependent manner both NADPH-oxidase- and MPO-dependent CL of PMN. This latter was further enhanced by PAF priming. Plasma-derived AOPP obtained from hemodialysis patients also triggered PMN respiratory burst. NAC significantly reduced HSA-AOPP-mediated responses of normal monocyte and of normal and uremic PMN but had no significant effect on opsonized zymosan- or PMA-induced CL responses. CONCLUSION: This dual potential of NAC to inhibit phagocyte oxidative responses induced by HSA-AOPP without affecting those mediated by compounds mimicking pathogens supports the proposal of a therapeutic trial with NAC aimed at reducing oxidative stress-related inflammation in hemodialysis patients.


Assuntos
Proteínas Sanguíneas/metabolismo , Monócitos/metabolismo , Neutrófilos/metabolismo , Acetilcisteína/farmacologia , Acetilcisteína/uso terapêutico , Proteínas Sanguíneas/administração & dosagem , Proteínas Sanguíneas/farmacologia , Relação Dose-Resposta a Droga , Humanos , NADP/metabolismo , Neutrófilos/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Oxigênio/metabolismo , Peroxidase/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Albumina Sérica/farmacologia , Uremia/metabolismo , Uremia/patologia
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