Replication of Brucella melitensis inside primary human monocytes depends on mitogen activated protein kinase signaling.

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.

Calcium/calmodulin-dependent protein kinase II regulates IL-10 production by human T lymphocytes: a distinct target in the calcium dependent pathway.

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.

Synergistic regulation of Pseudomonas aeruginosa-induced cytokine production in human monocytes by mannose receptor and TLR2.

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.

cAMP regulates IL-10 production by normal human T lymphocytes at multiple levels: a potential role for MEF2.

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.

Emergence of a new clone carrying Panton-Valentine leukocidin genes and staphylococcal cassette chromosome mec type V among methicillin-resistant Staphylococcus aureus in Greece.

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.

Molecular epidemiology and antibiotic resistance patterns of Salmonella enterica from southwestern Greece.

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.

Occurrence of the enterotoxin gene cluster and the toxic shock syndrome toxin 1 gene among clinical isolates of methicillin-resistant Staphylococcus aureus is related to clonal type and agr group.

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.

Pseudomonas aeruginosa slime glycolipoprotein is a potent stimulant of tumor necrosis factor alpha gene expression and activation of transcription activators nuclear factor kappa B and activator protein 1 in human monocytes.

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.

Polymerase chain reaction fingerprints of methicillin-resistant Staphylococcus aureus clinical isolates in Greece are related to certain antibiotypes.

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.