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1.
Eur J Public Health ; 28(4): 730-734, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29659793

RESUMO

Background: All European Union (EU) and European Economic Area (EEA) Member States have pledged to ensure political commitment towards sustaining the region's poliomyelitis-free status and eliminating measles. However, there remain significant gaps between policy and practice in many countries. This article reports on an assessment conducted for the European Commission that aimed to support improvements in preparedness and response to poliomyelitis and measles in Europe. Methods: A documentary review was complemented by qualitative interviews with professionals working in International and EU agencies, and in at-risk or recently affected EU/EEA Member States (six each for poliomyelitis and measles). Twenty-six interviews were conducted on poliomyelitis and 24 on measles; the data were subjected to thematic analysis. Preliminary findings were then discussed at a Consensus Workshop with 22 of the interviewees and eight other experts. Results: Generic or disease-specific plans exist in the participating countries and cross-border communications during outbreaks were generally reported as satisfactory. However, surveillance systems are of uneven quality, and clinical expertise for the two diseases is limited by a lack of experience. Serious breaches of protocol have recently been reported from companies producing poliomyelitis vaccines, and vaccine coverage rates for both diseases were also sub-optimal. A set of suggested good practices to address these and other challenges is presented. Conclusions: Poliomyelitis and measles should be brought fully onto the policy agendas of all EU/EEA Member States, and adequate resources provided to address them. Each country must abide by the relevant commitments that they have already made.


Assuntos
Surtos de Doenças/legislação & jurisprudência , Surtos de Doenças/prevenção & controle , Política de Saúde , Sarampo/prevenção & controle , Poliomielite/prevenção & controle , Medicina Preventiva/educação , Europa (Continente)/epidemiologia , União Europeia , Humanos , Sarampo/epidemiologia , Poliomielite/epidemiologia , Vigilância da População , Medicina Preventiva/legislação & jurisprudência
2.
J Innate Immun ; 5(1): 50-9, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23037919

RESUMO

Francisella tularensis causes the zoonotic disease tularemia. Arthropod vectors are important transmission routes for the disease, although it is not known how Francisella survives the efficient arthropod immune response. Here, we used Drosophila melanogaster as a model host for Francisella infections and investigated whether the bacteria are resistant to insect humoral immune responses, in particular to the antimicrobial peptides (AMPs) secreted into the insect hemolymph. Moreover, we asked to what extent such resistance might depend on lipopolysaccharide (LPS) structure and surface characteristics of the bacteria. We analyzed Francisella novicida mutant strains in genes, directly or indirectly involved in specific steps of LPS biosynthesis, for virulence in wild-type and Relish(E20) immune-deficient flies, and tested selected mutants for sensitivity to AMPs in vitro. We demonstrate that Francisella is sensitive to specific fly AMPs, i.e. Attacin, Cecropin, Drosocin and Drosomycin. Furthermore, six bacterial genes, kpsF, manB, lpxF, slt, tolA and pal, were found to be required for resistance to Relish-dependent immune responses, illustrating the importance of structural details of Francisella lipid A and Kdo core for interactions with AMPs. Interestingly, a more negative surface charge and lack of O-antigen did not render mutant bacteria more sensitive to cationic AMPs and did not attenuate virulence in flies.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Francisella tularensis/imunologia , Proteínas de Insetos/metabolismo , Lipídeo A/metabolismo , Lipopolissacarídeos/metabolismo , Açúcares Ácidos/metabolismo , Tularemia/imunologia , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Vetores Artrópodes/imunologia , Modelos Animais de Doenças , Proteínas de Drosophila/genética , Drosophila melanogaster/imunologia , Genes Bacterianos/genética , Imunidade/genética , Proteínas de Insetos/genética , Lipídeo A/química , Lipopolissacarídeos/química , Mutação/genética , Organismos Geneticamente Modificados , Açúcares Ácidos/química , Fatores de Transcrição/genética
3.
PLoS One ; 7(3): e32367, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22412866

RESUMO

Tularemia or vaccination with the live vaccine strain (LVS) of Francisella tularensis confers long-lived cell-mediated immunity. We hypothesized that this immunity depends on polyfunctional memory T cells, i.e., CD4(+) and/or CD8(+) T cells with the capability to simultaneously express several functional markers. Multiparametric flow cytometry, measurement of secreted cytokines, and analysis of lymphocyte proliferation were used to characterize in vitro recall responses of peripheral blood mononuclear cells (PBMC) to killed F. tularensis antigens from the LVS or Schu S4 strains. PBMC responses were compared between individuals who had contracted tularemia, had been vaccinated, or had not been exposed to F. tularensis (naïve). Significant differences were detected between either of the immune donor groups and naïve individuals for secreted levels of IL-5, IL-6, IL-10, IL-12, IL-13, IFN-γ, MCP-1, and MIP-1ß. Expression of IFN-γ, MIP-1ß, and CD107a by CD4(+)CD45RO(+) or CD8(+)CD45RO(+) T cells correlated to antigen concentrations. In particular, IFN-γ and MIP-1ß strongly discriminated between immune and naïve individuals. Only one cytokine, IL-6, discriminated between the two groups of immune individuals. Notably, IL-2- or TNF-α-secretion was low. Our results identify functional signatures of T cells that may serve as correlates of immunity and protection against F. tularensis.


Assuntos
Francisella tularensis/imunologia , Linfócitos T/imunologia , Tularemia/imunologia , Adulto , Fatores Etários , Idoso , Antígenos de Bactérias/imunologia , Citocinas/metabolismo , Epitopos/imunologia , Feminino , Humanos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Fatores Sexuais , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Tularemia/metabolismo , Tularemia/prevenção & controle , Adulto Jovem
4.
Eur J Immunol ; 41(4): 974-80, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21442618

RESUMO

The efficacy of many vaccines against intracellular bacteria depends on the generation of cell-mediated immunity, but studies to determine the duration of immunity are usually confounded by re-exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine-induced cell-mediated immunity. Here, lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1-3 or 27-34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP-1ß, IFN-γ, IL-10, and IL-5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN-γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN-γ by CD4(+) CCR7(+) , CD4(+) CD62L(+) , CD8(+) CCR7(+) , and CD8(+) CD62L(+) cells significantly increased in samples from vaccinated donors. In conclusion, cell-mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline.


Assuntos
Vacinas Bacterianas/imunologia , Francisella tularensis/imunologia , Adulto , Antígenos de Bactérias/imunologia , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Citocinas/imunologia , Feminino , Humanos , Linfócitos/citologia , Linfócitos/imunologia , Masculino , Vacinas Atenuadas/imunologia
5.
Infect Immun ; 78(7): 3118-28, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20479082

RESUMO

Francisella tularensis is a highly virulent, facultative intracellular human pathogen whose virulence mechanisms are not well understood. Occasional outbreaks of tularemia and the potential use of F. tularensis as a bioterrorist agent warrant better knowledge about the pathogenicity of this bacterium. Thus far, genome-wide in vivo screens for virulence factors have been performed in mice, all however restricted by the necessity to apply competition-based, negative-selection assays. We wanted to individually evaluate putative virulence determinants suggested by such assays and performed directed screening of 249 F. novicida transposon insertion mutants by using survival of infected fruit flies as a measure of bacterial virulence. Some 20% of the genes tested were required for normal virulence in flies; most of these had not previously been investigated in detail in vitro or in vivo. We further characterized their involvement in bacterial proliferation and pathogenicity in flies and in mouse macrophages. Hierarchical cluster analysis of mutant phenotypes indicated a functional linkage between clustered genes. One cluster grouped all but four genes of the Francisella pathogenicity island and other loci required for intracellular survival. We also identified genes involved in adaptation to oxidative stress and genes which might induce host energy wasting. Several genes related to type IV pilus formation demonstrated hypervirulent mutant phenotypes. Collectively, the data demonstrate that the bacteria in part use similar virulence mechanisms in mammals as in Drosophila melanogaster but that a considerable proportion of the virulence factors active in mammals are dispensable for pathogenicity in the insect model.


Assuntos
Drosophila melanogaster/microbiologia , Francisella/patogenicidade , Animais , Feminino , Francisella/genética , Técnicas de Silenciamento de Genes , Genes Bacterianos/genética , Infecções por Bactérias Gram-Negativas/microbiologia , Macrófagos/microbiologia , Masculino , Camundongos , Mutagênese Insercional/genética , Fenótipo , Fatores de Virulência/genética
6.
Proc Natl Acad Sci U S A ; 106(24): 9779-84, 2009 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-19497884

RESUMO

The Drosophila NF-kappaB transcription factor Relish is an essential regulator of antimicrobial peptide gene induction after gram-negative bacterial infection. Relish is a bipartite NF-kappaB precursor protein, with an N-terminal Rel homology domain and a C-terminal IkappaB-like domain, similar to mammalian p100 and p105. Unlike these mammalian homologs, Relish is endoproteolytically cleaved after infection, allowing the N-terminal NF-kappaB module to translocate to the nucleus. Signal-dependent activation of Relish, including cleavage, requires both the Drosophila IkappaB kinase (IKK) and death-related ced-3/Nedd2-like protein (DREDD), the Drosophila caspase-8 like protease. In this report, we show that the IKK complex controls Relish by direct phosphorylation on serines 528 and 529. Surprisingly, these phosphorylation sites are not required for Relish cleavage, nuclear translocation, or DNA binding. Instead they are critical for recruitment of RNA polymerase II and antimicrobial peptide gene induction, whereas IKK functions noncatalytically to support Dredd-mediated cleavage of Relish.


Assuntos
Anti-Infecciosos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica , Quinase I-kappa B/fisiologia , Peptídeos/genética , Fatores de Transcrição/metabolismo , Animais , Drosophila , Proteínas de Drosophila/química , Epistasia Genética , Quinase I-kappa B/química , Fosforilação , Regiões Promotoras Genéticas , Serina/metabolismo
7.
Dev Comp Immunol ; 33(5): 690-6, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19135474

RESUMO

The Rel/NF-kappaB transcription factor Relish is a major regulator of the antimicrobial response in Drosophila. Upon immune challenge, Relish is cleaved to generate two fragments, the DNA-binding transcription factor REL-68 and the IkappaB-like REL-49. Using transgenic fly strains we show here that overexpression of REL-68 separately from REL-49 is sufficient to activate strong constitutive transcription of the Diptericin gene, but little constitutive or inducible transcription of Attacin and Cecropin, two other Relish target genes. Their transcription may therefore require additional modifications of Relish. However, phosphorylation of the conserved serine residue S431 is not involved in such modifications. This is unlike p65 and Dorsal, which are modulated by phosphorylation at their homologous site. In contrast to other IkappaB proteins, overexpression of REL-49 had no inhibitory effect on Relish-dependent transcription. Instead, we propose that the C-terminal IkappaB-like domain executes a scaffolding and recruiting function for full activation of Relish.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Sequência de Aminoácidos , Animais , Cecropinas/imunologia , Cecropinas/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/imunologia , Drosophila melanogaster/genética , Drosophila melanogaster/imunologia , Proteínas de Insetos/imunologia , Proteínas de Insetos/metabolismo , Dados de Sequência Molecular , Fosforilação/imunologia , Fosforilação/fisiologia , Alinhamento de Sequência , Fatores de Transcrição/genética , Fatores de Transcrição/imunologia
8.
Cell Microbiol ; 10(6): 1327-38, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18248629

RESUMO

Drosophila melanogaster is a widely used model organism for research on innate immunity and serves as an experimental model for infectious diseases. The aetiological agent of the zoonotic disease tularaemia, Francisella tularensis, can be transmitted by ticks and mosquitoes and Drosophila might be a useful, genetically amenable model host to elucidate the interactions between the bacterium and its arthropod vectors. We found that the live vaccine strain of F. tularensis was phagocytosed by Drosophila and multiplied in fly haemocytes in vitro and in vivo. Bacteria injected into flies resided both inside haemocytes and extracellularly in the open circulatory system. A continuous activation of the humoral immune response, i.e. production of antimicrobial peptides under control of the imd/Relish signalling pathway, was observed and it may have contributed to the relative resistance to F. tularensis as flies defective in the imd/Relish pathway died rapidly. Importantly, bacterial strains deficient for genes of the F. tularensis intracellular growth locus or the macrophage growth locus were attenuated in D. melanogaster. Our results demonstrate that D. melanogaster is a suitable model for the analysis of interactions between F. tularensis and its arthropod hosts and that it can also be used to identify F. tularensis virulence factors relevant for mammalian hosts.


Assuntos
Drosophila melanogaster/microbiologia , Francisella tularensis , Tularemia/metabolismo , Tularemia/microbiologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/farmacologia , Drosophila melanogaster/imunologia , Francisella tularensis/crescimento & desenvolvimento , Francisella tularensis/patogenicidade , Genes de Insetos/genética , Hemócitos/microbiologia , Imunidade Inata , Peptídeos/genética , Peptídeos/metabolismo , Peptídeos/farmacologia , Transdução de Sinais , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Virulência
9.
EMBO J ; 25(13): 3068-77, 2006 Jul 12.
Artigo em Inglês | MEDLINE | ID: mdl-16763552

RESUMO

Jun N-terminal kinase (JNK) signaling is a highly conserved pathway that controls both cytoskeletal remodeling and transcriptional regulation in response to a wide variety of signals. Despite the importance of JNK in the mammalian immune response, and various suggestions of its importance in Drosophila immunity, the actual contribution of JNK signaling in the Drosophila immune response has been unclear. Drosophila TAK1 has been implicated in the NF-kappaB/Relish-mediated activation of antimicrobial peptide genes. However, we demonstrate that Relish activation is intact in dTAK1 mutant animals, and that the immune response in these mutant animals was rescued by overexpression of a downstream JNKK. The expression of a JNK inhibitor and induction of JNK loss-of-function clones in immune responsive tissue revealed a general requirement for JNK signaling in the expression of antimicrobial peptides. Our data indicate that dTAK1 is not required for Relish activation, but instead is required in JNK signaling for antimicrobial peptide gene expression.


Assuntos
Proteínas de Drosophila/fisiologia , Drosophila melanogaster/imunologia , MAP Quinase Quinase 4/fisiologia , NF-kappa B/fisiologia , Animais , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/microbiologia , Drosophila melanogaster/fisiologia , Ativação Enzimática , Imunidade Inata , Larva/imunologia , Larva/microbiologia , Mutação , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Fatores de Transcrição/fisiologia
10.
EMBO J ; 24(19): 3423-34, 2005 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-16163390

RESUMO

The Imd signaling cascade, similar to the mammalian TNF-receptor pathway, controls antimicrobial peptide expression in Drosophila. We performed a large-scale RNAi screen to identify novel components of the Imd pathway in Drosophila S2 cells. In all, 6713 dsRNAs from an S2 cell-derived cDNA library were analyzed for their effect on Attacin promoter activity in response to Escherichia coli. We identified seven gene products required for the Attacin response in vitro, including two novel Imd pathway components: inhibitor of apoptosis 2 (Iap2) and transforming growth factor-activated kinase 1 (TAK1)-binding protein (TAB). Iap2 is required for antimicrobial peptide response also by the fat body in vivo. Both these factors function downstream of Imd. Neither TAB nor Iap2 is required for Relish cleavage, but may be involved in Relish nuclear localization in vitro, suggesting a novel mode of regulation of the Imd pathway. Our results show that an RNAi-based approach is suitable to identify genes in conserved signaling cascades.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Proteínas Inibidoras de Apoptose/genética , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , DNA/genética , Primers do DNA , Proteínas de Drosophila/imunologia , Drosophila melanogaster/imunologia , Drosophila melanogaster/microbiologia , Eletroforese em Gel de Poliacrilamida , Escherichia coli/imunologia , Biblioteca Gênica , Imuno-Histoquímica , Proteínas Inibidoras de Apoptose/metabolismo , Proteínas de Insetos/metabolismo , Luciferases , Modelos Biológicos , Dados de Sequência Molecular , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Transdução de Sinais/imunologia
11.
Proc Natl Acad Sci U S A ; 100(10): 5991-6, 2003 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-12732719

RESUMO

The NF-kappaB-like transcription factor Relish plays a central role in the innate immune response of Drosophila. Unlike other NF-kappaB proteins, Relish is activated by endoproteolytic cleavage to generate a DNA-binding Rel homology domain and a stable IkappaB-like fragment. This signal-induced endoproteolysis requires the activity of several gene products, including the IkappaB kinase complex and the caspase Dredd. Here we used mutational analysis and protein microsequencing to demonstrate that a caspase target site, located in the linker region between the Rel and the IkappaB-like domain, is the site of signal-dependent cleavage. We also show physical interaction between Relish and Dredd, suggesting that Dredd indeed is the Relish endoprotease. In addition to the caspase target site, the C-terminal 107 aa of Relish are required for endoproteolysis and signal-dependent phosphorylation by the Drosophila IkappaB kinase beta. Finally, an N-terminal serine-rich region in Relish and the PEST domain were found to negatively regulate Relish activation.


Assuntos
Caspases/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/imunologia , Fatores de Transcrição/genética , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Primers do DNA , Proteínas de Drosophila/metabolismo , Deleção de Genes , Regulação da Expressão Gênica , Genes Reporter , Cinética , Dados de Sequência Molecular , Fosforilação , Reação em Cadeia da Polimerase , Deleção de Sequência , Fatores de Transcrição/metabolismo , beta-Galactosidase/genética
12.
Science ; 296(5566): 359-62, 2002 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-11872802

RESUMO

Components of microbial cell walls are potent activators of innate immune responses in animals. For example, the mammalian TLR4 signaling pathway is activated by bacterial lipopolysaccharide and is required for resistance to infection by Gram-negative bacteria. Other components of microbial surfaces, such as peptidoglycan, are also potent activators of innate immune responses, but less is known about how those components activate host defense. Here we show that a peptidoglycan recognition protein, PGRP-LC, is absolutely required for the induction of antibacterial peptide genes in response to infection in Drosophila and acts by controlling activation of the NF-kappaB family transcription factor Relish.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/imunologia , Imunidade Inata , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Processamento Alternativo , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas de Transporte/genética , Núcleo Celular/metabolismo , Drosophila/genética , Drosophila/metabolismo , Drosophila/microbiologia , Escherichia coli/imunologia , Éxons , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Genes de Insetos , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Lipopolissacarídeos/imunologia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Mutação , Peptidoglicano/imunologia , Peptidoglicano/metabolismo , Peptidoglicano/farmacologia , Fenótipo , Isoformas de Proteínas , RNA de Cadeia Dupla
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