Gαq modulates the energy metabolism of osteoclasts.

Introduction: The bacterial protein toxin Pasteurella multocida toxin (PMT) mediates RANKL-independent osteoclast differentiation. Although these osteoclasts are smaller, their resorptive activity is high which helps in efficient destruction of nasal turbinate bones of pigs. Methods: The proteome of bone marrow-derived macrophages differentiated into osteoclasts with either RANKL or PMT was analysed. The results were verified by characterizing the metabolic activity using Seahorse analysis, a protein translation assay, immunoblots, real-time PCR as well as flow cytometry-based monitoring of mitochondrial activity and ROS production. A Gαq overexpression system using ER-Hoxb8 cells was used to identify Gαq-mediated metabolic effects on osteoclast differentiation and function. Results: PMT induces the upregulation of metabolic pathways, which included strong glycolytic activity, increased expression of GLUT1 and upregulation of the mTOR pathway. As OxPhos components were expressed more efficiently, cells also displayed increased mitochondrial respiration. The heterotrimeric G protein Gαq plays a central role in this hypermetabolic cell activation as it triggers mitochondrial relocalisation of pSerSTAT3 and an increase in OPA1 expression. This seems to be caused by a direct interaction between STAT3 and OPA1 resulting in enhanced mitochondrial respiration. Overexpression of Gαq mimicked the hypermetabolic phenotype observed for PMT-induced osteoclasts and resulted in higher glycolytic and mitochondrial activity as well as increased bone resorptive activity. In addition, rheumatoid arthritis (RA) patients showed an increase in GNAQ expression, especially in the synovial fluid. Discussion: Our study suggests that Gαq plays a key role in PMT-induced osteoclastogenesis. Enhanced expression of GNAQ at the site of inflammation in RA patients indicates its pathophysiological relevance in the context of inflammatory bone disorders.

Canibacter oris - a fairly unknown pathogenic agent of bite wound infections.

Here, we report on the second case of bite wound infection by Canibacter oris. This bacterium belongs to the family of Microbacteriaceae in the order of Microbacterales in the class of Actinobacteria, which are prevalent in the oral flora. Possibly this bacterium has been overlooked until now, because it cannot be recognized by conventional differentiation methods. MALDI-TOF as well as PCR are able to identify this pathogen.

Hsa-miR-99b/let-7e/miR-125a Cluster Regulates Pathogen Recognition Receptor-Stimulated Suppressive Antigen-Presenting Cells.

Antigen-presenting cells (APCs) regulate the balance of our immune response toward microbes. Whereas immunogenic APCs boost inflammation and activate lymphocytes, the highly plastic cells can switch into a tolerogenic/suppressive phenotype that dampens and resolves the response. Thereby the initially mediated inflammation seems to prime the switch of APCs while the strength of activation determines the grade of the suppressive phenotype. Recently, we showed that pathogen recognition receptor-mediated pro-inflammatory cytokines reprogram differentiating human blood monocytes in vitro toward an immunosuppressive phenotype through prolonged activation of signal transducer and activator of transcription (STAT) 3. The TLR7/8 ligand R848 (Resiquimod) triggers the high release of cytokines from GM-CSF/IL-4-treated monocytes. These cytokines subsequently upregulate T cell suppressive factors, such as programmed death-ligand 1 (PD-L1) and indolamin-2,3-dioxygenase (IDO) through cytokine receptor-mediated STAT3 activation. Here, we reveal an essential role for the microRNA (miR, miRNA) hsa-miR-99b/let-7e/miR-125a cluster in stabilizing the suppressive phenotype of R848-stimulated APCs on different levels. On the one hand, the miR cluster boosts R848-stimulated cytokine production through regulation of MAPkinase inhibitor Tribbles pseudokinase 2, thereby enhancing cytokine-stimulated activation of STAT3. One the other hand, the STAT3 inhibitor suppressor of cytokine signaling-1 is targeted by the miR cluster, stabilizing the STAT3-induced expression of immunosuppressive factors PD-L1 and IDO. Finally, hsa-miR-99b/let-7e/miR-125a cluster regulates generation of the suppressive tryptophan (Trp) metabolite kynurenine by targeting the tryptophanyl-tRNA synthetase WARS, the direct competitor of IDO in terms of availability of Trp. In summary, our results reveal the hsa-miR-99b/let-7e/miR-125a cluster as an important player in the concerted combination of mechanisms that stabilizes STAT3 activity and thus regulate R848-stimulated suppressive APCs.

[Infections after bite wounds : For example rat bite fever due to Streptobacillus moniliformis]. / Infektionen nach Bissverletzungen : Zum Beispiel Rattenbissfieber durch Streptobacillus moniliformis.

Rat bite fever due to Streptobacillus moniliformis induces typical but not pathognomonic clinical signs, such as local purulent wound infection followed by maculopapular exanthema, myalgia as well as purulent joint infections. Severe complications, such as osteomyelitis and endocarditis are possible. it seems that this infection is rarely diagnosed but this infection could be much more common because the final diagnostic proof is difficult to achieve. Firstly, the culture of these bacteria is critical because the bacteria are fastidious and secondly the exact differentiation of the isolates is hardly possible by standard laboratory methods. Modern techniques such as mass spectroscopy (MALDI-TOF) and molecular biology allow a precise clarification. Surgical cleansing of infection sites in combination with a rational antibiotic therapy, for example with beta-lactam antibiotics, are generally able to cure the infection if treatment is started early enough. In addition, vaccinations, for example against tetanus and rabies have to be considered in this situation as for all other bite wound infections.

Extracellular Vesicle Subtypes Released From Activated or Apoptotic T-Lymphocytes Carry a Specific and Stimulus-Dependent Protein Cargo.

Extracellular vesicles (EVs) are released from nearly all mammalian cells and different EV populations have been described. Microvesicles represent large EVs (LEVs) released from the cellular surface, while exosomes are small EVs (SEVs) released from an intracellular compartment. As it is likely that different stimuli promote the release of distinct EV populations, we analyzed EVs from human lymphocytes considering the respective release stimuli (activation Vs. apoptosis induction). We could clearly separate two EV populations, namely SEVs (average diameter <200 nm) and LEVs (diameter range between 200 and 1000 nm). Morphology and size were analyzed by electron microscopy and nanoparticle tracking analysis. Apoptosis induction caused a massive release of LEVs, while activated T-cells released SEVs and LEVs in considerably lower amounts. The release of SEVs from apoptotic T-cells was comparable with LEV release from activated ones. LEVs contained signaling proteins and proteins of the actin-myosin cytoskeleton. SEVs carried cytoplasmic/endosomal proteins like the 70-kDa heat shock protein 70 (HSP70) or tumor susceptibility 101 (TSG101), microtubule-associated proteins, and ubiquitinated proteins. The protein expression profile of SEVs and LEVs changed substantially after the induction of apoptosis. After apoptosis induction, HSP70 and TSG101 (often used as exosome markers) were highly expressed within LEVs. Interestingly, in contrast to HSP70 and TSG101, gelsolin and eps15 homology domain-containing protein 3 (EHD3) turned out to be specific for SEVs irrespective of the stimulus causing the EV release. Finally, we detected several subunits of the proteasome (PSMB9, PSMB10) as well as the danger signal HMGB1 exclusively within apoptotic cell-released LEVs. Thus, we were able to identify new marker proteins that can be useful to discriminate between distinct LEV subpopulations. The mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD009074.

IL-1ß As Mediator of Resolution That Reprograms Human Peripheral Monocytes toward a Suppressive Phenotype.

During infection pathogen-associated molecular patterns activate immune cells to initiate a cascade of reactions leading to inflammation and the activation of the adaptive immune response culminating in the elimination of foreign pathogens. However, shortly after activation of the host defense machinery, a return to homeostasis is preferred to prevent inflammation-induced tissue damage. This switch from the initial immunogenic to the subsequent tolerogenic phase after clearance of the infection can be mediated through highly plastic peripheral monocytes. Our studies reveal that an early encounter with toll-like receptor 7/8-ligand R848 mediates a strong pro-inflammatory monocytic phenotype that primes its own reprogramming toward an immunosuppressive one. Previously, we showed that these R848-treated antigen-presenting cells (APCs) fail to activate allogeneic T cells and induce regulatory T cells (Tregs) through signal transducer and activator of transcription 3 (STAT3)-dependent PD-L1. Here, we further demonstrate that R848-treated APCs suppress CD3/CD28-mediated and dendritic cell-mediated T cell activation and that adenosine and indoleamine 2,3-dioxygenase/kynurenin pathways are involved in tolerance induction. Reprogramming of monocytes after R848 stimulation requires the pro-inflammatory cytokine IL-1ß and a boosted IL-6 release. The subsequent autocrine prolonged activation of STAT3 induces direct upregulation of tolerogenic factors which finally downregulate proliferation of activated T cells and mediate Tregs. Thereby our study suggests that inflammatory cytokines, such as IL-1ß and IL-6, should be considered as mediators of resolution of inflammation.

Human monocytes downregulate innate response receptors following exposure to the microbial metabolite n-butyrate.

INTRODUCTION: Hyporesponsiveness of human lamina propria immune cells to microbial and nutritional antigens represents one important feature of intestinal homeostasis. It is at least partially mediated by low expression of the innate response receptors CD11b, CD14, CD16 as well as the cystine-glutamate transporter xCT on these cells. Milieu-specific mechanisms leading to the down-regulation of these receptors on circulating monocytes, the precursor cells of resident macrophages, are mostly unknown. METHODS: Here, we addressed the question whether the short chain fatty acid n-butyrate, a fermentation product of the mammalian gut microbiota exhibiting histone deacetylase inhibitory activity, is able to modulate expression of these receptors in human circulating monocytes. RESULTS: Exposure to n-butyrate resulted in the downregulation of CD11b, CD14, as well as CD16 surface expression on circulating monocytes. XCT transcript levels in circulating monocytes were also reduced following exposure to n-butyrate. Importantly, treatment resulted in the downregulation of protein and gene expression of the transcription factor PU.1, which was shown to be at least partially required for the expression of CD16 in circulating monocytes. PU.1 expression in resident macrophages in situ was observed to be substantially lower in healthy when compared to inflamed colonic mucosa. CONCLUSIONS: In summary, the intestinal microbiota may support symbiosis with the human host organism by n-butyrate mediated downregulation of protein and gene expression of innate response receptors as well as xCT on circulating monocytes following recruitment to the lamina propria. Downregulation of CD16 gene expression may at least partially be caused at the transcriptional level by the n-butyrate mediated decrease in expression of the transcription factor PU.1 in circulating monocytes.

Microbiological Assessment of Bile and Corresponding Antibiotic Treatment: A Strobe-Compliant Observational Study of 1401 Endoscopic Retrograde Cholangiographies.

The aim of this study was to determine the antibiotic susceptibility profiles of bacteria in bile samples and to analyze the clinical relevance of the findings as only limited information about risk factors for elevated frequence of bacterial and fungal strains in routinely collected bile samples has been described so far.A prospective cohort study at a tertiary care center was conducted. Seven hundred forty-four patients underwent 1401 endoscopic retrograde cholangiographies (ERCs) as indicated by liver transplantation (427/1401), primary sclerosing cholangitis (222/1401), choledocholithiasis only (153/1401), obstruction due to malignancy (366/1401), or other conditions (233/1401). Bile samples for microbiological analysis were obtained in all patients.The 71.6% (823/1150) samples had a positive microbiological finding, and 57% (840/1491) of the bacterial isolates were gram-positive. The main species were Enterococcus spp (33%; 494/1491) and Escherichia coli (12%; 179/1491). Of the samples, 53.8% had enteric bacteria and 24.7% had Candida spp; both were associated with clinical and laboratory signs of cholangitis (C-reactive proteins 35.0 ±â€Š50.1 vs 44.8 ±â€Š57.6; 34.5 ±â€Š51.2 vs 52.9 ±â€Š59.7; P < 0.001), age, previous endoscopic intervention, and immunosuppression. Multi-resistant (MR) strains were found in 11.3% of all samples and were associated with clinical and laboratory signs of cholangitis, previous intervention, and immunocompromised status. In subgroup analysis, strain-specific antibiotic therapy based on bile sampling was achieved in 56.3% (89/158) of the patients. In cases with a positive bile culture and available blood culture, blood cultures were positive in 29% of cases (36/124), and 94% (34/36) of blood cultures had microbial species identical to the bile cultures.Bactobilia and fungobilia can usually be detected by routine microbiological sampling, allowing optimized, strain-specific antibiotic treatment. Previous endoscopic intervention, clinical and laboratory signs of cholangitis, and age are independent risk factors. MR bacteria and fungi are an evolving problem in cholangitis, especially in immunocompromised patients.

Evaluation of antibiotic resistance to orally administrable antibiotics in staphylococcal bone and joint infections in one of the largest university hospitals in Germany: is there a role for fusidic acid?

Bone and joint infections (BJIs) are often difficult to treat. Staphylococcus spp. is the major pathogen causing these infections, which is often associated with biofilm formation on prosthetic materials. Therapeutic measures are complex, ranging from surgical intervention to initial intravenous and supportive long-term oral antibiotic therapy. The options for oral antimicrobial therapy are limited, mainly due to the resistance profile of the causative pathogen and the unfavourable pharmacodynamic and pharmacokinetic properties of most antibiotics in biofilm. Data analysis over a 5-year period was performed on staphylococci isolated from BJI patients in the Orthopaedic Department of the University Hospital Heidelberg (Heidelberg, Germany) to assess the plausibility of fusidic acid (FA)-based alternative oral treatment regimens. Six percent of BJIs were caused by meticillin-resistant Staphylococcus aureus (MRSA), and multiresistance was common. Over 75% of MRSA in BJIs were resistant to the commonly used rifampicin (RIF)-based combinations. Resistance to FA-based combinations was high. However, over 80% were susceptible to the combination RIF+FA. In coagulase-negative staphylococci, resistance to RIF-based combinations was similar to FA-based combinations. Almost two-thirds of the isolates tested were susceptible to RIF+FA. These data suggest FA as a possible option as a substitution for RIF or as a combination companion in case of resistance or unavailability.

Inhibition of Histone Deacetylases Permits Lipopolysaccharide-Mediated Secretion of Bioactive IL-1ß via a Caspase-1-Independent Mechanism.

Histone deacetylase (HDAC) inhibitors (HDACi) are clinically approved anticancer drugs that have important immune-modulatory properties. We report the surprising finding that HDACi promote LPS-induced IL-1ß processing and secretion in human and murine dendritic cells and murine macrophages. HDACi/LPS-induced IL-1ß maturation and secretion kinetics differed completely from those observed upon inflammasome activation. Moreover, this pathway of IL-1ß secretion was dependent on caspase-8 but was independent of the inflammasome components NACHT, LRR, and PYD domains-containing protein 3, apoptosis-associated speck-like protein containing a carboxyl-terminal caspase-recruitment domain, and caspase-1. Genetic studies excluded HDAC6 and HDAC10 as relevant HDAC targets in this pathway, whereas pharmacological inhibitor studies implicated the involvement of HDAC11. Treatment of mice with HDACi in a dextran sodium sulfate-induced colitis model resulted in a strong increase in intestinal IL-1ß, confirming that this pathway is also operative in vivo. Thus, in addition to the conventional inflammasome-dependent IL-1ß cleavage pathway, dendritic cells and macrophages are capable of generating, secreting, and processing bioactive IL-1ß by a novel, caspase-8-dependent mechanism. Given the widespread interest in the therapeutic targeting of IL-1ß, as well as the use of HDACi for anti-inflammatory applications, these findings have substantial clinical implications.

Granzyme A produces bioactive IL-1ß through a nonapoptotic inflammasome-independent pathway.

Bacterial components are recognized by the immune system through activation of the inflammasome, eventually causing processing of the proinflammatory cytokine interleukin-1? (IL-1?), a pleiotropic cytokine and one of the most important mediators of inflammation, through the protease caspase-1. Synthesis of the precursor protein and processing into its bioactive form are tightly regulated, given that disturbed control of IL-1? release can cause severe autoinflammatory diseases or contribute to cancer development. We show that the bacterial Pasteurella multocida toxin (PMT) triggers Il1b gene transcription in macrophages independently of Toll-like receptor signaling through RhoA/Rho-kinase-mediated NF-?? activation. Furthermore, PMT mediates signal transducer and activator of transcription (STAT) protein-controlled granzyme A (a serine protease) expression in macrophages. The exocytosed granzyme A enters target cells and mediates IL-1? maturation independently of caspase-1 and without inducing cytotoxicity. These findings show that macrophages can induce an IL-1?-initiated immune response independently of inflammasome activity.

Risk factors and outcome in patients with primary sclerosing cholangitis with persistent biliary candidiasis.

BACKGROUND: Candidiasis is commonly observed in patients with primary sclerosing cholangitis (PSC), but the clinical risk factors associated with its presence have not been fully investigated. In this study, we aimed to analyse the incidence, risk factors, and transplantation-free survival in primary sclerosing cholangitis (PSC) patients with persistent biliary candidiasis. METHODS: We retrospectively analysed patients diagnosed with PSC who were admitted to our department during 2002 to 2012. One-hundred fifty patients whose bile cultures were tested for fungal species were selected, and their clinical and laboratory parameters were investigated. The results of endoscopic retrograde cholangiography (ERC) and bile cultures were analysed using chart reviews. The cases of biliary candidiasis were sub-classified as transient or persistent. RESULTS: Thirty out of 150 (20.0%) patients had biliary candidiasis. Although all patients demonstrated comparable baseline characteristics, those with biliary candidiasis showed significantly reduced transplantation-free survival (p < 0.0001) along with a markedly elevated frequency of cholangiocarcinoma (CCA) (p = 0.04). The patients were further sub-classified according to the transient (15/30) or persistent (15/30) nature of their biliary candidiasis. A subgroup analysis showed reduced survival with a greater necessity for orthotopic liver transplantation (OLT) only in patients with persistence of Candida (p = 0.007). The survival in the patients with transient biliary candidiasis was comparable to that in candidiasis-free patients. In a multivariate regression analysis that included Mayo risk score (MRS), sex, age, dominant stenosis, inflammatory bowel disease, autoimmune hepatitis overlap syndrome, and number of times ERC was performed, biliary candidiasis was an independent risk factor for reduced survival (p = 0.008). Risk factors associated with acquisition of biliary candidiasis were age at PSC diagnosis and number of ERCs. CONCLUSIONS: The persistence of biliary candidiasis is associated with markedly reduced transplantation-free survival in PSC patients. By contrast, actuarial survival in patients with transient biliary candidiasis approaches that for patients without any evidence of biliary candidiasis. Further studies on the treatment of persistent biliary candidiasis in patients with PSC are warranted.

Bacteriobilia and fungibilia are associated with outcome in patients with endoscopic treatment of biliary complications after liver transplantation.

BACKGROUND AND STUDY AIMS: To determine the importance of bacteriobilia and fungibilia in patients with endoscopic treatment of biliary complications after orthotopic liver transplantation (OLT). PATIENTS AND METHODS: In a prospective study at a tertiary center, 213 patients underwent 857 endoscopic retrograde cholangiographies (ERCs) after OLT. Findings at first ERC were: anastomotic stricture in 24.4%, nonanastomotic stricture in 18.3%, leakage in 11.3%, and gallstones in 4.7%. RESULTS: Bile samples from first ERC showed Gram-positive bacterial isolates in 102/180 (57%) and Gram-negative in 44/180 (24%). Main species were Enterococcus spp. (40%; 72/180) and Escherichia coli (10%; 18 /180). Enteric bacteria (present in 47%) and Candida spp. (present in 18%) were both associated with clinical signs of cholangitis, but not with laboratory signs of inflammation. Multiresistant strains (present in 12% of samples) showed no association with clinical or laboratory parameters. Detection of microbiological isolates was independent of endoscopic findings and treatment. In patients with successful endoscopic intervention, the actuarial survival free of retransplantation was significantly lower in those with detection of enteric bacteria, being 51.8 months (95% confidence interval [CI] 42.9-60.6) vs. 62.9 months (95% CI 59.1-66.7); P = 0.025). Fungibilia was associated with significantly lower actuarial retransplantation-free survival, independently of successful endoscopic treatment (mean 35.1 months [95% CI 23.5-46.7] vs. 53.1 months [(95% CI 48.0-58.2]; P = 0.019). CONCLUSIONS: Bacteriobilia and fungibilia can frequently be detected by routine microbiological sampling in patients after OLT. Regular bile sampling is recommended since the presence of difficult-to-treat multiresistant strains is unpredictable. Survival is affected by this altered biliary microbiological environment after OLT.

Early inhibition of IL-1ß expression by IFN-γ is mediated by impaired binding of NF-κB to the IL-1ß promoter but is independent of nitric oxide.

The significance of bacterial RNA recognition for initiating innate immune responses against invading pathogens has only recently started to be elucidated. Bacterial RNA is an important trigger of inflammasome activation, resulting in caspase-1-dependent cleavage of pro-IL-1ß into the active form. It was reported previously that prolonged treatment with IFN-γ can inhibit IL-1ß production at the level of both transcription and Nlrp3 inflammasome activation in an NO-dependent manner. As a result of the delayed kinetics of NO generation after IFN-γ stimulation, these effects were only observed at later time points. We report that IFN-γ suppressed bacterial RNA and LPS induced IL-1ß transcription in primary murine macrophages and dendritic cells by an additional, very rapid mechanism that was independent of NO. Costimulation with IFN-γ selectively attenuated binding of NF-κB p65 to the IL-1ß promoter, thus representing a novel mechanism of IL-1ß inhibition by IFN-γ. Transcriptional silencing was specific for IL-1ß because expression of other proinflammatory cytokines, such as TNF, IL-6, and IL-12p40, was not affected. Furthermore, by suppressing IL-1ß production, IFN-γ impaired differentiation of Th17 cells and production of neutrophil chemotactic factor CXCL1 in vitro. The findings provide evidence for a rapid immune-modulating effect of IFN-γ independent of NO.

IRAK4 turns IL-10+ phospho-FOXO+ monocytes into pro-inflammatory cells by suppression of protein kinase B.

IRAK4, a serine/threonine kinase is a central adaptor protein in TLR signaling. To better understand the clinical significance of IRAK4 deficiency we examined the impact of IRAK4 on bacterial recognition in human monocytes. We show that IRAK4 knockdown modulates monocyte-derived cytokine secretion in response to Staphylococcus aureus and Streptococcus pneumoniae, resulting in decreased IL-12 and elevated IL-10 production, a finding also reproducible with ligands for TLR2 and TLR4. In contrast, silencing of MyD88 leads to a complete loss of cytokine secretion, indicating that IRAK4 acts as a differential regulator of bacteria/TLR-induced cytokine secretion downstream of MyD88. Further analysis revealed that this modulatory function results from IRAK4-mediated suppression of protein kinase B (PKB/Akt). Release of suppression upon IRAK4 silencing (but not MyD88 knockdown) increases phosphorylation of PKB/Akt, counteracts NF-κB activation and finally results in a monocyte phenotype with tolerogenic features, thus unleashing Akt- and mTOR-dependent release of IL-10, along with concomitant phosphorylation of FOXO transcription factors. In line with these observations IRAK4-deficient monocytes failed to induce allogeneic CD8(+) and CD4(+) T-cell responses, an effect reverted by neutralization of IL-10. Taken together, our data highlight an unexpected role of IRAK4, Akt, and mTOR in the regulation of tolerance in human monocytes.

Functional variation reflects intra-strain diversity of Staphylococcus aureus small colony variants in the host-pathogen interaction.

It is well acknowledged that genetic variation accounts for the intra-species variability in Staphylococcus aureus isolates. Similarly, deficiency in DNA repair and the resulting increase in genomic mutations determine intra-strain variability in S. aureus small colony variants (SCV). The aim of this study was to investigate whether intra-strain diversity would be associated with an alteration of the host-pathogen interaction. To this end, biofilm formation and immune stimulatory capacity were compared in consecutive SCV isolates originating from a single patient. Despite the relatedness of the isolates, the results revealed significant differences in biofilm formation and immune stimulation determined by Toll-like receptor-2 (TLR2) activity. Variation in the extent of biofilm production could be attributed to differences in the expression of protein A (SpA) and agrA. TLR2 activity only partially correlated with these parameters. Although transiently increased functional activity correlated with clinical remission and was abrogated in MRSA superinfection, we can only speculate that changes in the SCV phenotype reflect alterations in the microbial environment and/or treatment. Taken together, our study provides in vivo evidence for the functional consequences of intra-strain variation in S. aureus.

Integration of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in blood culture diagnostics: a fast and effective approach.

Sepsis is a major cause of mortality in hospitalized patients worldwide, with lethality rates ranging from 30 to 70 %. Sepsis is caused by a variety of different pathogens, and rapid diagnosis is of outstanding importance, as early and adequate antimicrobial therapy correlates with positive clinical outcome. In recent years, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has become a powerful tool in microbiological diagnostics. The direct identification of micro-organisms in a positive blood culture by MALDI-TOF MS can shorten the diagnostic procedure significantly. Therefore, the aim of the present study was to evaluate whether identification rates could be improved by using the new Sepsityper kit from Bruker Daltonics for direct isolation and identification of bacteria from positive blood cultures by MALDI-TOF MS compared with the use of conventional separator gel columns, and to integrate the MALDI-TOF MS-based identification method into the routine course of blood culture diagnostics in the setting of a microbiological laboratory at a university hospital in Germany. The identification of Gram-negative bacteria by MALDI-TOF MS was significantly better using the Sepsityper kit compared with a separator gel tube-based method (99 and 68 % correct identification, respectively). For Gram-positive bacteria, only 73 % were correctly identified by MALDI-TOF with the Sepsityper kit and 59 % with the separator gel tube assay. A major problem of both methods was the poor identification of Gram-positive grape-like clustered cocci. As differentiation of Staphylococcus aureus from coagulase-negative staphylococci is of clinical importance, a PCR was additionally established that was capable of identifying S. aureus directly from positive blood cultures, thus closing this diagnostic gap. Another benefit of the PCR approach is the possibility of directly detecting the genes responsible for meticillin resistance in staphylococci and for vancomycin resistance in enterococci, which is of high importance for early adequate treatment. Both of the described methods were finally integrated into a protocol for fast and effective identification of bacteria from positive blood cultures.

Distinct functions of the mitogen-activated protein kinase-activated protein (MAPKAP) kinases MK2 and MK3: MK2 mediates lipopolysaccharide-induced signal transducers and activators of transcription 3 (STAT3) activation by preventing negative regulatory effects of MK3.

In LPS-treated macrophages, activation of STAT3 is considered to be crucial for terminating the production of inflammatory cytokines. By analyzing the role of MAPK-activated protein kinase (MK) 2 and MK3 for LPS-induced STAT3 activation in macrophages, the present study provides evidence that MK2 is crucial for STAT3 activation in response to LPS because it prevents MK3 from impeding IFNß gene expression. Accordingly, LPS-induced IFNß gene expression is down-regulated in MK2-deficient macrophages and can be reconstituted by additional ablation of the MK3 gene in MK2/3(-/-) macrophages. This is in contrast to LPS-induced IL-10 expression, which essentially requires the presence of MK2. Further analysis of downstream signaling events involved in the transcriptional regulation of IFNß gene expression suggests that, in the absence of MK2, MK3 impairs interferon regulatory factor 3 protein expression and activation and inhibits nuclear translocation of p65. This inhibition of p65 nuclear translocation coincides with enhanced expression and delayed degradation of IκBß, whereas expression of IκBα mRNA and protein is impaired in the absence of MK2. The observation that siRNA directed against IκBß is able to reconstitute IκBα expression in MK2(-/-) macrophages suggests that enhanced expression and delayed degradation of IκBß and impaired NFκB-dependent IκBα expression are functionally linked. In summary, evidence is provided that MK2 regulates LPS-induced IFNß expression and downstream STAT3 activation as it restrains MK3 from mediating negative regulatory effects on NFκB- and interferon regulatory factor 3-dependent LPS signaling.