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1.
Microbes Infect ; 15(6-7): 450-60, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-23628412

RESUMO

The clinical course of infections caused by Brucella is linked to its capacity to modulate the initial immune response of macrophages in order to ensure its intracellular replication. Signal transduction pathways implicated in the survival of Brucella in human cells are not completely elucidated. We herein investigated the involvement of the TLR-MAPK-dependent signaling pathways in the survival of Brucella in primary human monocytes using live clinical strains of Brucella melitensis. B. melitensis caused a delayed, TLR2 dependent MAPK activation. Specific MAPK inhibitors for p38 (SB203580), ERK1/2 (PD98059) and JNK (SP600125) or the anti-TLR2 blocking Ab inhibited both inflammatory and anti-inflammatory responses characterized by TNF-α, IL-6 and IL-10 production. Intracellular replication of B. melitensis was mainly dependent on p38 and JNK activation and not affected by IL-10 levels. These are the first evidence to support that survival of B. melitensis inside human monocytes depends on interplay among the different MAPK family members, activated through TLR2, in spite of an initial pro-inflammatory response.


Assuntos
Brucella melitensis/crescimento & desenvolvimento , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/microbiologia , Transdução de Sinais , Células Cultivadas , Citocinas/metabolismo , Humanos
2.
Mol Immunol ; 52(2): 51-60, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22578382

RESUMO

Calcium (Ca2+) plays an essential role in lymphocyte activation and differentiation by affecting signaling pathways leading to cytokine production. Among the enzymes responding to calcium increase, Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been involved in anergy with a still poorly characterized role. IL-10 produced by different T lymphocyte subpopulations is critical mediator of tolerance. We tested the hypothesis that CaMKII may be involved in IL-10 production. We report that CaMKII upregulates IL-10 production by primary human T lymphocytes stimulated through the antigen receptor or bypassing that. Overexpression of constitutively active mutant forms of Calcineurin or CaMKII specifically increase IL-10 protein product and IL-10 mRNA accumulation in T lymphocytes. By cotransfecting constitutively active CaMKII with luciferase reporter plasmids carrying specific fragments or the whole IL-10 promoter, we show that CaMKII specifically activates IL-10 promoter activity, whereas it inhibits IL-2 and IL-4 promoter. This effect is mediated by the first 500 bp fragment, which contains binding sites for Myocyte Enhancer Factor-2 (MEF2). A constitutively active mutant of CaMKII activated a luciferase reporter plasmid under the control of MEF2, when cotransfected in T lymphocytes stimulated by Ionomycin and PMA, whereas its inhibitor KN-62 inhibited MEF2 binding in cell lysates of the same cells. Moreover, overexpression of MEF2 enhanced by 2.5-fold IL-10 promoter activity. Our data for the first time suggest a distinct role of CaMKII in the induction of anergy in T lymphocytes, by differential regulation of IL-10 and IL-2 gene transcription suggest MEF2 as a molecular target which can integrate different calcium signals.


Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Interleucina-10/biossíntese , Linfócitos T/imunologia , Linfócitos T/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Sequência de Bases , Calcineurina/metabolismo , Inibidores de Calcineurina , Sinalização do Cálcio , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/antagonistas & inibidores , Células Cultivadas , Humanos , Interleucina-10/genética , Proteínas de Domínio MADS/genética , Fatores de Transcrição MEF2 , Fatores de Regulação Miogênica/genética , Regiões Promotoras Genéticas , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/efeitos dos fármacos , Regulação para Cima
3.
Eur J Immunol ; 39(3): 730-40, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19197942

RESUMO

The immune response to pathogen is regulated by a combination of specific PRR, which are involved in pathogen recognition. Pseudomonas aeruginosa, a bacterium that causes life-threatening disease in immuno-compromised host, is recognized by distinct members of the TLR family. We have previously shown that viable P. aeruginosa bacteria are recognized by human monocytes mainly through TLR2. Using ligand-specific blocking antibodies, we herein show that the mannose receptor (MR), a phagocytic receptor for unopsonized P. aeruginosa bacteria, contributes equally to TLR2 in proinflammatory cytokine production by human monocytes in response to P. aeruginosa infection. Synergy of both receptors totally controls the immune response. Viable P. aeruginosa bacteria activate NF-kappaB and MAPK pathways and enhance TLR2-mediated signaling in MR-transfected human embryonic kidney 293 cells. Moreover, MR follows the same kinetics and colocalizes with TLR2 in the endosome during in vivo infection of human macrophages with P. aeruginosa. The studies provide the first demonstration of a significant role for MR, synergistic with TLR2, in activating a proinflammatory response to P. aeruginosa infection.


Assuntos
Lectinas Tipo C/imunologia , Lectinas de Ligação a Manose/imunologia , Monócitos/imunologia , Infecções por Pseudomonas/imunologia , Receptores de Superfície Celular/imunologia , Receptor 2 Toll-Like/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Interleucina-1beta/biossíntese , Interleucina-1beta/imunologia , Interleucina-6/biossíntese , Interleucina-6/imunologia , Lectinas Tipo C/metabolismo , Receptor de Manose , Lectinas de Ligação a Manose/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/imunologia , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , Monócitos/microbiologia , Fagocitose/imunologia , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Serina-Treonina Quinases/metabolismo , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/imunologia , Receptores de Superfície Celular/metabolismo , Receptor 2 Toll-Like/metabolismo , Transfecção , Fator de Necrose Tumoral alfa/biossíntese , Fator de Necrose Tumoral alfa/imunologia , Quinase Induzida por NF-kappaB
4.
Mol Immunol ; 46(3): 345-54, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19058854

RESUMO

Signal transduction by the cAMP/cAMP-dependent protein kinase A (PKA) pathway is triggered through multiple receptors and is important for many processes in a variety of cells. In T cells, the engagement of the TCR-CD3 complex induces cAMP, a second messenger that controls immune response. IL-10, produced by a variety of lymphocyte subpopulations, is an important regulator of this response exerting a wide range of immunomodulatory actions. Elevation of cAMP has been shown to increase IL-10 production by monocytes. However, the mechanism of cAMP mediated regulation of IL-10 production by T lymphocytes remains unclear. In this study using normal peripheral T lymphocytes stimulated either through the TCR-CD3 complex or the TCR-CD3 and the CD28 molecule, we show that IL-10 is produced mainly by memory T lymphocytes after either way of stimulation and is drastically inhibited (70-90%) by cAMP elevating agents. cAMP mediated inhibition was reversed by the use of the specific PKA inhibitor Rp-8-Br-cAMP but not by the addition of exogenous rhIL-2, indicating that the inhibitory effect depends on PKA activation and is not secondary to IL-2 inhibition. Inhibition is taking place at both transcriptional and posttranscriptional level. Transfection of a luciferase reporter plasmid carrying the IL-10 promoter in T cells, revealed that TCR/CD28-induced activation was inhibited by 60% by cAMP elevation. The most sensitive part to cAMP mediated inhibition was a fragment of 135 bp upstream of TATA box, which contains multiple binding sites for MEF-2. Overexpression of MEF-2 in the same cells increased IL-10 promoter activity by 2.5-fold. Stimulation through TCR/CD28 increased MEF-2 binding in its corresponding binding sites which was inhibited by 80% in the presence of cAMP elevating agents. These results suggest that the inhibitory effect of cAMP on IL-10 production by normal peripheral T lymphocytes is cell type and stimulus specific, exerted on multiple levels and involves MEF2 transcription factor.


Assuntos
AMP Cíclico/farmacologia , Interleucina-10/biossíntese , Proteínas de Domínio MADS/metabolismo , Fatores de Regulação Miogênica/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Sequência de Bases , Antígenos CD28/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-10/genética , Interleucina-2/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Fatores de Transcrição MEF2 , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/enzimologia , TATA Box/genética
6.
Scand J Infect Dis ; 40(5): 368-72, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18418797

RESUMO

Clonal analysis and PCR screening for the presence of Panton-Valentine leukocidin (PVL) genes was performed among 694 methicillin-resistant Staphylococcus aureus (MRSA) cases collected during a 2-y period in Greece. The detection rate of PVL-positive MRSA is high, both in the community and in hospital. Clonal analysis revealed the predominance among the PVL-positive strains of the clonal complex CC80 (ST80-IV) and the emergence of ST377 clone carrying agr1 allele and SCCmec type V.


Assuntos
Toxinas Bacterianas/genética , Exotoxinas/genética , Leucocidinas/genética , Resistência a Meticilina/genética , Infecções Estafilocócicas/epidemiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Análise por Conglomerados , Infecções Comunitárias Adquiridas/epidemiologia , Infecções Comunitárias Adquiridas/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Impressões Digitais de DNA , DNA Bacteriano/genética , Eletroforese em Gel de Campo Pulsado , Grécia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Reação em Cadeia da Polimerase/métodos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Fatores de Virulência/genética
7.
Chemotherapy ; 53(6): 392-6, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17934258

RESUMO

BACKGROUND: A study was conducted at the University Hospital of Patras between January 2002 and December 2003 to investigate antibiotic resistance patterns and clonality of Salmonella enterica in southwestern Greece. METHODS: Ninety-five isolates recovered from different outpatients were characterized by specific antisera and were tested for their susceptibility to various antimicrobial agents. Clones were identified by pulsed-field gel electrophoresis (PFGE) of XbaI chromosomal DNA digests. RESULTS: Five serotypes were characterized among 95 isolates, recovered mainly from children, with the predominance of Salmonella enteritidis followed by Salmonella typhimurium. The strains were further distinguished by PFGE to 16 clones. The majority of S.enteritidis (61 strains) belonged to clone A, already characterized in Greece, while S.typhimurium (18 isolates) belonged to 3 clones, exhibiting multiresistant phenotypes. CONCLUSIONS: One clone S.enteritidis, type A, circulates in southwestern Greece, mainly during summer, while clonality was also observed among S.typhimurium.


Assuntos
Farmacorresistência Bacteriana , Infecções por Salmonella/microbiologia , Salmonella enterica/isolamento & purificação , Animais , Técnicas de Tipagem Bacteriana , Eletroforese em Gel de Campo Pulsado , Grécia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Infecções por Salmonella/epidemiologia , Salmonella enterica/genética
8.
J Clin Microbiol ; 44(5): 1881-3, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16672430

RESUMO

Analysis of 177 methicillin-resistant Staphylococcus aureus (MRSA) strains for the presence of egc and tst revealed that 60 strains carried at least one of the tested genes. MRSA strains were classified by pulsed-field gel electrophoresis into four clones belonging to agr groups I and III. Toxin genotypes were related by clonal type and agr group.


Assuntos
Enterotoxinas/genética , Genes Bacterianos , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Genótipo , Humanos , Resistência a Meticilina , Família Multigênica , Staphylococcus aureus/classificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Superantígenos/genética , Transativadores/genética
9.
Infect Immun ; 71(8): 4614-22, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12874341

RESUMO

Pseudomonas aeruginosa, an opportunistic pathogen, causes infections associated with a high incidence of morbidity and mortality in immunocompromised hosts. Production of tumor necrosis factor alpha (TNF-alpha), primarily by cells of monocytic lineage, is a crucial event in the course of these infections. During in vivo infections with P. aeruginosa, both lipopolysaccharide (LPS) and extracellular slime glycolipoprotein (GLP) produced by mucoid and nonmucoid strains are released. In the present study, we sought to explore the relative contributions of these two bacterial products to TNF-alpha production by human monocytes. To this end, fresh human monocytes and THP-1 human monocytic cells were stimulated with P. aeruginosa LPS or GLP. GLP was found to be a more potent stimulus for TNF-alpha production (threefold higher) by human monocytes than LPS. Moreover, its effect was comparable to that of viable bacteria. Quantitative mRNA analysis revealed predominantly transcriptional regulation. Electrophoretic mobility shift assays and transfection assays demonstrated activation of NF-kappa B and activator protein 1 (AP-1). NF-kappa B activation by GLP was rapid and followed the same time course as that by viable bacteria, suggesting that bacteria could directly activate NF-kappa B through GLP. Moreover P. aeruginosa GLP induced the formation of AP-1 complex with delayed kinetics compared with NF-kappa B but much more efficiently than the homologous LPS. These results identify GLP as the most important stimulant for TNF-alpha production by human monocytes. Activation of NF-kappa B and AP-1 by P. aeruginosa GLP may be involved not only in TNF-alpha induction but also in many of the inflammatory responses triggered in the course of infection with P. aeruginosa.


Assuntos
Proteínas de Bactérias/toxicidade , NF-kappa B/metabolismo , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/patogenicidade , Fator de Transcrição AP-1/metabolismo , Fator de Necrose Tumoral alfa/genética , Proteínas de Bactérias/química , Linhagem Celular , Expressão Gênica/efeitos dos fármacos , Humanos , Cinética , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Monócitos/microbiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/biossíntese , Virulência
10.
J Microbiol Methods ; 53(3): 417-22, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12689720

RESUMO

Characterization of clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and representatives of three MRSA clones from other hospitals was performed by arbitrarily primed polymerase chain reaction (AP-PCR) and antibiotic susceptibility patterns. All isolates were mecA-positive and two main antibiotypes have been characterized. Two major clones were identified with AP-PCR related to the aforementioned antibiotypes. The combination of antibiotypes with AP-PCR patterns successfully identified the two major clones in our hospital, which are identical with the MRSA clones previously characterized in Athens and in Central and North Greece.


Assuntos
Impressões Digitais de DNA/métodos , Resistência a Meticilina , Reação em Cadeia da Polimerase/métodos , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/classificação , Antibacterianos/uso terapêutico , Técnicas de Tipagem Bacteriana , Contagem de Colônia Microbiana , Farmacorresistência Bacteriana Múltipla , Grécia/epidemiologia , Humanos , Testes de Sensibilidade Microbiana/métodos , Sensibilidade e Especificidade , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/epidemiologia , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação
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