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Rezafungin is a next-generation, once-a-week echinocandin in development for the treatment of candidaemia and invasive candidiasis and for the prevention of invasive fungal disease caused by Candida, Aspergillus, and Pneumocystisspp after blood and marrow transplantation. We aimed to compare the efficacy and safety of intravenous rezafungin versus intravenous caspofungin in patients with candidaemia and invasive candidiasis.

Introduction: Intra-abdominal infections represent the second most frequently acquired infection in the intensive care unit (ICU), with mortality rates ranging from 20% to 50%. Candidaspp. may be responsible for up to 10–30% of cases. This study assesses risk factors for development of intra-abdominal candidiasis (IAC) among patients admitted to ICU. Methods: We performed a case–control study in 26 European ICUs during the period January 2015–December 2016. Patients at least 18 years old who developed an episode of microbiologically documented IAC during their stay in the ICU (at least 48 h after admission) served as the case cohort. The control group consisted of adult patients who did not develop episodes of IAC during ICU admission. Matching was performed at a ratio of 1:1 according to time at risk (i.e. controls had to have at least the same length of ICU stay as their matched cases prior to IAC onset), ICU ward and period of study. Results: During the study period, 101 case patients with a diagnosis of IAC were included in the study. On univariate analysis, severe hepatic failure, prior receipt of antibiotics, prior receipt of parenteral nutrition, abdominal drain, prior bacterial infection, anastomotic leakage, recurrent gastrointestinal perforation, prior receipt of antifungal drugs and higher median number of abdominal surgical interventions were associated with IAC development. On multivariate analysis, recurrent gastrointestinal perforation (OR 13.90; 95% CI 2.65–72.82, p= 0.002), anastomotic leakage (OR 6.61; 95% CI 1.98–21.99, p= 0.002), abdominal drain (OR 6.58; 95% CI 1.73–25.06, p= 0.006), prior receipt of antifungal drugs (OR 4.26; 95% CI 1.04–17.46, p= 0.04) or antibiotics (OR 3.78; 95% CI 1.32–10.52, p= 0.01) were independently associated with IAC. Conclusions: Gastrointestinal perforation, anastomotic leakage, abdominal drain and prior receipt of antifungals or antibiotics may help to identify critically ill patients with higher probability of developing IAC. Prospective studies are needed to identify which patients will benefit from early antifungal treatment.

Impaction bone grafts (IBG) in two-stage revision for prosthetic hip infection (PHI) might be more susceptible for infection, therefore antibiotic mixing through these grafts has been suggested. However, outcomes have not been compared with IBG without antibiotics and no long-term results are available. Therefore, we evaluated long-term infection-free outcome after the use of IBG without antibiotic supplement in two-stage revision for PHI. Patients were divided into positive (group 1, n = 8) and negative (group 2, n = 28) cultures at re-implantation and followed up to 18 years after re-implantation. Five of 36 patients died from non-orthopaedic causes (median 37, range 24-149 months). Five patients had a re-operation not related to infection (median 39, range 7-140 months). These were censored in the Kaplan-Meier estimator at the last outpatient evaluation. We found an overall re-infection rate of 2.8% within two years, which matches comparative studies in which antibiotic impregnated bone grafts had been used. In group 1, there was one re-infection after 44 months. In group 2, all three infections occurred within 56 months with an estimated infection-free percentage at 10 years of 87% (95% CI 66-96). Follow-up should be extended beyond two years and randomised clinical trials are needed for further comparison with IBG impregnated with antibiotics.

ABSTRACTCytokine production assays have been primarily used in research settings studying novel immunodeficiencies. We sought to determine the diagnostic value of cytokine production assays in patients with recurrent and/or severe infectious diseases (IDs) without known immunodeficiencies and unclassified noninfectious inflammatory disorders (NIIDs). We retrospectively examined cytokine production in whole-blood and peripheral blood mononuclear cell samples from 157 adult patients. A cytokine production rate of <5% of that of healthy controls was considered defective. While monocyte-derived cytokine (tumor necrosis factor alpha [TNF-a], interleukin-1ß [IL-1ß], and IL-6) production was rarely affected, 30% of all included patients had deficient production of interferon gamma (IFN-?), IL-17A, or IL-22. Twenty-five percent of the NIID patients displayed defective IFN-? production, whereas IL-17A production was generally unaffected. In the group of ID patients, defective IFN-? production was found in 19% and 14% of the patients with viral and bacterial infections, respectively, and in 38%, 24%, and 50% of patients with mycobacterial, mucocutaneous, and invasive fungal infections, respectively. Defective IL-17A and IL-22 production was mainly confined to ID patients with mucocutaneous fungal infections. In conclusion, cytokine production assays frequently detect defective Th1 responses in patients with mycobacterial or fungal infections, in contrast to patients with respiratory tract infections or isolated bacterial infections. Defective IL-17A and IL-22 production was primarily found in patients with fungal infections, while monocyte-derived cytokine production was unaffected. Thus, lymphocyte-derived cytokine production assays are helpful in the diagnostic workup of patients with recurrent infections and suspected immunodeficiencies and have the potential to reveal immune defects that might guide adjunctive immunomodulatory therapy.

ABSTRACTAlthough Candida glabratais an important pathogenic Candidaspecies, relatively little is known about its innate immune recognition. Here, we explore the potential role of Dectin-2 for host defense against C. glabrata. Dectin-2-deficient (Dectin-2-/-) mice were found to be more susceptible to C. glabratainfections, showing a defective fungal clearance in kidneys but not in the liver. The increased susceptibility to infection was accompanied by lower production of T helper 1 (Th1) and Th17-derived cytokines by splenocytes of Dectin-2-/-mice, while macrophage-derived cytokines were less affected. These defects were associated with a moderate yet significant decrease in phagocytosis of the fungus by the Dectin-2-/-macrophages and neutrophils. Neutrophils of Dectin-2-/-mice also displayed lower production of reactive oxygen species (ROS) upon challenge with opsonized C. glabrataor C. albicans. This study suggests that Dectin-2 is important in host defense against C. glabrataand provides new insights into the host defense mechanisms against this important fungal pathogen.

Biologics that block the T-helper-17 pathway are very effective in the treatment of psoriasis and other inflammatory diseases. However, interleukin-17 is also crucial for antifungal host defense, and clinical trial data suggest an increase in the incidence of Candidainfections during IL-17 inhibitor (IL-17i) therapy. We investigated the innate and adaptive immune responses of psoriasis patients with a history of skin and/or mucosal candidiasis during IL-17i or IL-12/23i therapy, comparing those responses to healthy controls. Psoriasis patients with IL-17i showed significantly lower CD4+Th1-like (CCR6-CXCR3+CCR4-) and Th1Th17-like (CD4+CCR6+CXCR3+CCR4-) cell percentages. Patient cells stimulated with Candida albicansproduced significantly lower IL-6 in the IL-12/23i group and IL-1β production in the IL-17i group, while the release of TNF-α and ROS was similar between patients and controls. IFN-γ and IL-10 production in response to several stimuli after 7 days was particularly decreased in patients receiving IL-17i therapy. Finally, after stimulation with the polarizing cytokines IL-1β and IL-23, the Th17 cytokine response was significantly lower in the IL-17i patient group. These innate and adaptive immune response defects can diminish antifungal host immune response and thereby increase susceptibility to candidiasis in patients treated with IL-17i or IL-12/23i.

ABSTRACTThe monitoring and prediction of treatment responses to invasive aspergillosis (IA) are difficult. We determined whether serum galactomannan index (GMI) trends early in the course of disease may be useful in predicting eventual clinical outcomes. For the subjects recruited into the multicenter Global Aspergillosis Study, serial GMIs were measured at baseline and at weeks 1, 2, and 4 following antifungal treatment. Clinical response and survival at 12 weeks were the outcome measures. GMI trends were analyzed by using the generalized estimation equation approach. GMI cutoffs were evaluated by using receiver-operating curve analyses incorporating pre- and posttest probabilities. Of the 202 study patients diagnosed with IA, 71 (35.1%) had a baseline GMI of =0.5. Week 1 GMI was significantly lower for the eventual responders to treatment at week 12 than for the nonresponders (GMIs of 0.62 ± 0.12 and 1.15 ± 0.22, respectively; P= 0.035). A GMI reduction of >35% between baseline and week 1 predicted a probability of a satisfactory clinical response. For IA patients with pretreatment GMIs of <0.5 (n= 131; 64.9%), GMI ought to remain low during treatment, and a rising absolute GMI to >0.5 at week 2 despite antifungal treatment heralded a poor clinical outcome. Here, every 0.1-unit increase in the GMI between baseline and week 2 increased the likelihood of an unsatisfactory clinical response by 21.6% (P= 0.018). In summary, clinical outcomes may be anticipated by charting early GMI trends during the first 2 weeks of antifungal therapy. These findings have significant implications for the management of IA.

Early detection of metastatic infection in patients with Gram-positive bacteremia is important as morbidity and mortality are higher in the presence of these foci, probably due to incomplete eradication of clinically silent foci during initial treatment. We performed a prospective study in 115 patients with Staphylococcus aureusor Streptococcusspecies bacteremia with at least 1 risk factor for the development of metastatic foci, such as community acquisition, treatment delay, persistently positive blood cultures for >48 hours, and persistent fever >72 hours after initiation of treatment. An intensive search for metastatic infectious foci was performed including 18F-fluorodeoxyglucose-positron emission tomography in combination with low-dose computed tomography scanning for optimizing anatomical correlation (FDG-PETCT) and echocardiography in the first 2 weeks of admission.

Candida albicans is both the most common fungal commensal microorganism in healthy individuals and the major fungal pathogen causing high mortality in at-risk populations, especially immunocompromised patients. In this review, we summarize the interplay between the host innate system and C. albicans, ranging from how the host recognizes, responds, and clears C. albicans infection to how C. albicans evades, dampens, and escapes from host innate immunity.

ABSTRACTCandida albicansis both the most common fungal commensal microorganism in healthy individuals and the major fungal pathogen causing high mortality in at-risk populations, especially immunocompromised patients. In this review, we summarize the interplay between the host innate system and C. albicans, ranging from how the host recognizes, responds, and clears C. albicansinfection to how C. albicansevades, dampens, and escapes from host innate immunity.

ABSTRACTInterleukin-23 (IL-23) is known to play a crucial role in the development and maintenance of T helper 17 cells. It has been previously demonstrated that IL-17 is involved in experimental Lyme arthritis, caused by Borrelia burgdorferibacteria. However, the precise role of the IL-23 receptor (IL-23R) for the B. burgdorferi-induced IL-17 responses or human Lyme disease has not yet been elucidated. IL-23R single nucleotide polymorphism (SNP) rs11209026 was genotyped using the TaqMan assay. Functional studies were performed using peripheral blood mononuclear cells, and cytokines were measured using enzyme-linked immunosorbent assay (ELISA). Dose-dependent production of IL-23 and IL-17 by B. burgdorfericould be observed. Interestingly, when IL-23 bioactivity was inhibited by a specific antibody against IL-23p19, IL-17 production was significantly downregulated. In contrast, production of gamma interferon (IFN-γ) was not affected after the blockade of IL-23 activity. Moreover, individuals bearing a single nucleotide polymorphism in the IL-23R gene (Arg381Gln) produced significantly less IL-17 after B. burgdorferistimulation compared with that of the individuals bearing the wild type. Despite lower IL-17 production, the IL-23R gene polymorphism did not influence the development of chronic Lyme disease in a cohort of patients with Lyme disease. This study demonstrates that IL-23R signaling is needed for B. burgdorferi-induced IL-17 production in vitroand that an IL-23R gene SNP leads to impaired IL-17 production. However, the IL-23R gene polymorphism is not crucial for the pathogenesis of chronic Lyme.

Invading C. albicanshyphae are recognized by macrophages, activate the caspase‐1/IL‐1β pathway, and lead to the activation of IL‐17 pathway to control the C. albicansinfection. In the mucosa, the immune pathways discriminating between colonizing and invasive Candida, thus inducing tolerance or inflammation, are poorly understood. Th17 responses induced by Candida albicanshyphae are central for the activation of mucosal antifungal immunity. An essential step for the discrimination between yeasts and hyphae and induction of Th17 responses is the activation of the inflammasome by C. albicanshyphae and the subsequent release of active IL‐1β in macrophages. Inflammasome activation in macrophages results from differences in cell‐wall architecture between yeasts and hyphae and is partly mediated by the dectin‐1/Syk pathway. These results define the dectin‐1/inflammasome pathway as the mechanism that enables the host immune system to mount a protective Th17 response and distinguish between colonization and tissue invasion by C. albicans.

Chitin is a skeletal cell wall polysaccharide of the inner cell wall of fungal pathogens. As yet, little about its role during fungus-host immune cell interactions is known. We show here that ultrapurified chitin from Candida albicans cell walls did not stimulate cytokine production directly but blocked the recognition of C. albicans by human peripheral blood mononuclear cells (PBMCs) and murine macrophages, leading to significant reductions in cytokine production. Chitin did not affect the induction of cytokines stimulated by bacterial cells or lipopolysaccharide (LPS), indicating that blocking was not due to steric masking of specific receptors. Toll-like receptor 2 (TLR2), TLR4, and Mincle (the macrophage-inducible C-type lectin) were not required for interactions with chitin. Dectin-1 was required for immune blocking but did not bind chitin directly. Cytokine stimulation was significantly reduced upon stimulation of PBMCs with heat-killed chitin-deficient C. albicans cells but not with live cells. Therefore, chitin is normally not exposed to cells of the innate immune system but is capable of influencing immune recognition by blocking dectin-1-mediated engagement with fungal cell walls.

Disseminated candidiasis remains a life-threatening disease in the ICU. The development of invasive disease with Candida albicansis dependent on multiple factors, such as colonization and efficient host defense at the mucosa. In the present review, we describe the host defense mechanisms against Candidathat are responsible for counteracting mucosal invasion, and eliminating the pathogen once invasion has taken place.

The role of the IL-17 pathway in antifungal host defense is controversial. Several studies suggested that IL-17 is crucial for the protection against Candidainfection, whereas other studies reported that IL-17 may contribute to inflammatory pathology and worsening of fungal disease. To address these discrepancies, we assessed the differential role of IL-17 pathway in two models of fungal sepsis intravenous infection with live Candida albicans, in which fungal growth is the main cause of mortality, and zymosan-induced multiple organ failure, in which the inflammatory pathology drives the mortality. First, IL-17 receptor-deficient (IL-17RA−/−) mice showed increased mortality and higher fungal loads in the kidneys in the model of disseminated candidiasis, partly caused by lower neutrophil recruitment in the IL-17RA−/−mice. Second, IL-17RA−/−mice were not protected against the multiorgan failure induced by zymosan. These data demonstrate that IL-17 does not have a major contribution to the inflammatory pathology leading to organ failure in fungal sepsis and support the concept that the IL-17 pathway is protective in antifungal host defense.

IL‐17A production induced by Mycobacterium tuberculosisthrough TLR4 and dectin‐1 is dependent on the steps necessary for the induction of endogenous IL‐1β. In the present study, we dissected the pathways that trigger the IL‐17A responses by MTB. Dectin‐1 and TLR4 were shown to be involved in MTB‐induced IL‐17A production, and blockade of the NOD2, TLR2, or MR had no effect on IL‐17A. The MAPK Erk, known to mediate transcription of IL‐1β mRNA, was strongly involved in the IL‐17A production induced by MTB. The intracellular enzymes caspase‐1 and serine proteases, which process pro‐IL‐1β into the active IL‐1β, were also crucial for the induction of IL‐17A. Lastly, the MTB‐induced IL‐17A response was strongly dependent on signaling through the IL‐1R but not the IL‐6R pathway. In conclusion, the MTB‐induced IL‐17A response relies strongly on the endogenous IL‐1 pathway and IL‐1R signaling. TLR4 and dectin‐1 are the main receptors responsible for mediating the signals responsible for IL‐17A production by MTB. These findings contribute to a better understanding of the host response to mycobacteria and provide the opportunity to explore potential, novel, therapeutic strategies against TB.

Host-pathogen interactions at epithelial barriers play an important role in health and disease. This also applies to the clinical setting of stem cell transplantation (SCT) in which deregulated sensing of microbes and their cell wall components by pattern recognition receptors (PRRs) can contribute to inflammatory and infectious complications. The role of Candida species herein has recently been rediscovered since a 'loss-of-function' Y238X polymorphism in dectin-1, a C-type lectin receptor recognizing the β-1,3-glucan motif of Candida,resulted in diminished membrane expression and lower cytokine responses upon β-1,3-glucan recognition, and was associated with increased Candida colonization of SCT recipients, rendering them at risk for candidaemia. In addition, Candida colonization was associated with an increased incidence of acute graft-versus-host disease (GvHD), but only in those individuals with the wild-type dectin-1 allele. The Th17 mediated immune responses might provide a common link in these processes, as they have recently been implicated in anti-Candidaimmunity as well as the pathogenesis of GvHD. These new insights suggest that immunogenetics could contribute to a more individualized risk-based strategy for managing SCT recipients, for example concerning antifungal prophylaxis. In addition, modulating host-pathogen interactions by selectively modulating PRR activity could be exploited in SCT to achieve better outcomes.

ABSTRACTThe innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicanscells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicansreleases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

The innate immune system recognizes pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRR) and transduces downstream signaling to activate the host defense. Here we report that in addition to direct PAMP-PRR interactions, live Candida albicans cells can release soluble factors to actively potentiate interleukin-6 (IL-6) and IL-8 production induced in human mononuclear cells by the fungi. Although protease-activated receptor 1 (PAR1) and PAR2 ligation can moderately upregulate Toll-like receptor 4 (TLR4)-mediated IL-8 production, no effect on the C. albicans-induced cytokine was apparent. Similarly, the blockade of PAR signaling did not reverse the potentiation of cytokine production induced by soluble factors released by C. albicans. In conclusion, C. albicans releases soluble factors that potentiate cytokine release in a PAR1/2-independent manner. Thus, human PAR1 and PAR2 have a redundant role in the activation of human cells by C. albicans.

ABSTRACTVeillonella parvulais an anaerobic gram-negative coccus that is part of the normal flora of the animal and human mouth and gastrointestinal and genitourinary tracts. Oral V. parvulais involved in the development of early periodontal disease as well as different types of serious infections. Present data on molecular mechanisms responsible for innate immune response against Veillonellaare very scanty. The aim of this study was to investigate the Toll-like receptor (TLR) pathways responsible for V. parvulalipopolysaccharide (LPS) and to identify the intracellular pathways induced by this recognition. V. parvulaLPS stimulated tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6) release in human peripheral blood mononuclear cells (PBMC) in a dose-dependent manner. Pretreatment of cells with a TLR4 antagonist significantly reduced TNF-a and IL-6 production in PBMC stimulated with either Veillonellaor Escherichia coliLPS. However, V. parvulaLPS was 10- to 100-fold less active than E. coliLPS for cytokine induction. TNF-a, IL-1ß, IL-6, and IL-10 were released in wild-type and TLR2-/-, but not TLR4-/-, mouse macrophage cultures. V. parvulaLPS was able to activate the human PBMC p38 mitogen-activated protein kinase (MAPK). A specific p38 MAPK inhibitor strongly inhibited V. parvulaLPS-induced TNF-a, IL-1ß, IL-6, and IL-10. In conclusion, V. parvulaLPS is able to induce cytokine production in both human and murine in vitro models, although it is less effective than EnterobacteriaceaeLPS. V. parvulaLPS-stimulated cytokine induction, as well as p38 MAPK activation, are TLR4-dependent features.

Toll-like receptor (TLR)-based signaling pathways in the host may be modulated by pathogens during the course of infection. We describe a novel immunomodulatory mechanism in which Aspergillus fumigatus conidia induce attenuation of TLR2- and TLR4-mediated interleukin (IL)-6 and IL-1β proinflammatory responses in human mononuclear cells with suppression of IL-1β mRNA transcription. Background TLR2 and TLR4 mRNA transcription was not influenced. A. fumigatus conidia induced TLR2 internalization and uptake into the phagosome with a resultant decrease in surface receptor expression. A. fumigatus hyphae, on the other hand, selectively downregulated the TLR4-mediated response. These novel immunosuppressive effects may facilitate the invasiveness of A. fumigatus.

The processing of pro-interleukin-1β depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1β (IL-1β). Here we demonstrate that human blood monocytes release processed IL-1β after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1β solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1β production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Background: Interleukin-1β is one of the most important pro-inflammatory cytokines. In contrast to other cytokines, activation of IL-1β requires processing from an inactive precursor by the cysteine protease caspase-1. Caspase-1 forms a protein platform called the inflammasome, together with proteins of the nucleotide-binding oligomerization domain-like receptor (NLR) family. Objective/methods: A review of literature investigating the stimulation of IL-1β production by microbial pathogens and their components. Results/conclusions: To produce IL-1β, macrophages need a double stimulation with Toll like receptor (TLR) ligands that induce gene transcription, and NLR agonists (such as ATP or muramyl dipeptide (MDP)) that activate the inflammasome. Monocytes can release active IL-1β upon stimulation with TLR ligands alone. This probably represents an adaptation of each cell type to its environment.

ABSTRACTInnate antifungal defense in Drosophila melanogasterrelies on the activation of the Toll molecule and the release of drosomycin, a defensin-like molecule with antifungal properties. Ten human homologues of Toll have been described, with central roles in activation of the innate host defense. In the present study, we report a putative human homologue of the Drosophila-derived drosomycin, designated drosomycin-like defensin (DLD). Synthetic DLD displays a broad spectrum of activity against Aspergillusspp. and other clinically relevant filamentous fungi. These effects are specific for filamentous fungi; no activity has been found against yeasts or gram-positive or gram-negative bacteria. Synthetic DLD also displays immunomodulatory effects on Aspergillus-stimulated cytokine production. In addition, we show the expression of DLD mRNA in several human tissues, particularly in the skin, consistent with its putative role as a defensin against invading microorganisms. This is the first indication of an endogenous human peptide with specific antifungal activity, which is probably central in the defense against infections with molds.

The cell surface of Candida albicans is enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc3Man9GlcNAc2N-glycan is processed by -glucosidases I and II and 1,2-mannosidase to generate Man8GlcNAc2. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1 encode for -glucosidase I, -glucosidase II catalytic subunit, and 1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1 homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41, Carot2, and Camns1 null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated β-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

ABSTRACTThe cell surface of Candida albicansis enriched in highly glycosylated mannoproteins that are involved in the interaction with the host tissues. N glycosylation is a posttranslational modification that is initiated in the endoplasmic reticulum (ER), where the Glc3Man9GlcNAc2N-glycan is processed by a-glucosidases I and II and a1,2-mannosidase to generate Man8GlcNAc2. This N-oligosaccharide is then elaborated in the Golgi to form N-glycans with highly branched outer chains rich in mannose. In Saccharomyces cerevisiae, CWH41, ROT2, and MNS1encode for a-glucosidase I, a-glucosidase II catalytic subunit, and a1,2-mannosidase, respectively. We disrupted the C. albicans CWH41, ROT2, and MNS1homologs to determine the importance of N-oligosaccharide processing on the N-glycan outer-chain elongation and the host-fungus interaction. Yeast cells of Cacwh41?, Carot2?, and Camns1? null mutants tended to aggregate, displayed reduced growth rates, had a lower content of cell wall phosphomannan and other changes in cell wall composition, underglycosylated ß-N-acetylhexosaminidase, and had a constitutively activated PKC-Mkc1 cell wall integrity pathway. They were also attenuated in virulence in a murine model of systemic infection and stimulated an altered pro- and anti-inflammatory cytokine profile from human monocytes. Therefore, N-oligosaccharide processing by ER glycosidases is required for cell wall integrity and for host-fungus interactions.

Bartonella quintana is a gram-negative microorganism that causes trench fever and chronic bacteremia. B. quintana lipopolysaccharide (LPS) was unable to induce the production of proinflammatory cytokines in human monocytes. Interestingly, B. quintana LPS is a potent antagonist of Toll-like receptor 4 (TLR4), as it inhibited both mRNA transcription and the release of tumor necrosis factor alpha, interleukin 1{szligbeta} (IL-1{szligbeta}), and IL-6 by Escherichia coli LPS in human monocytes, at ratios ranging from 1,000:1 to 10:1 (B. quintana LPS to E. coli LPS). Likewise, B. quintana LPS blocked the interaction of E. coli LPS with TLR4 in transfected cell lines. The extent of the inhibitory effect of B. quintana LPS was demonstrated in microarray studies, which showed downregulation of practically all genes induced by LPS in monocytes. Because of the role of TLR4 in inflammation, B. quintana LPS may prove useful as a potent anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.

ABSTRACTBartonella quintanais a gram-negative microorganism that causes trench fever and chronic bacteremia. B. quintanalipopolysaccharide (LPS) was unable to induce the production of proinflammatory cytokines in human monocytes. Interestingly, B. quintanaLPS is a potent antagonist of Toll-like receptor 4 (TLR4), as it inhibited both mRNA transcription and the release of tumor necrosis factor alpha, interleukin 1β (IL-1β), and IL-6 by Escherichia coliLPS in human monocytes, at ratios ranging from 1,000:1 to 10:1 (B. quintanaLPS to E. coliLPS). Likewise, B. quintanaLPS blocked the interaction of E. coliLPS with TLR4 in transfected cell lines. The extent of the inhibitory effect of B. quintanaLPS was demonstrated in microarray studies, which showed downregulation of practically all genes induced by LPS in monocytes. Because of the role of TLR4 in inflammation, B. quintanaLPS may prove useful as a potent anti-TLR4 agent with therapeutic potential in both infections and autoimmune inflammation.

Mycobacterium paratuberculosishas been suggested to be involved in the pathogenesis of Crohn's disease (CD). The importance of microorganisms in CD is supported by the association of CD with mutations in the intracellular pathogen recognition receptor (PRR) nucleotide‐binding oligomerization domain 2 (NOD2). The aim of this study is to investigate the PRR involved in the recognition of M. paratuberculosis. Methods used include in vitro stimulation of transfected cell lines, murine macrophages, and human PBMC. M. paratuberculosisstimulated human TLR2 (hTLR2)‐Chinese hamster ovary (CHO) cells predominantly and hTLR4‐CHO cells modestly. Macrophages from TLR2 and TLR4 knockout mice produced less cytokines compared with controls after stimulation with M. paratuberculosis. TLR4 inhibition in human PBMC reduced cytokine production only after stimulation with live M. paratuberculosis. TLR‐induced TNF‐α, IL‐1β, and IL‐10 production is mediated through MyD88, whereas Toll‐IL‐1R domain‐containing adaptor inducing IFN‐β (TRIF) promoted the release of IL‐1β. hNOD2‐human embryo kidney (HEK) cells, but not hNOD1‐HEK cells, responded to stimulation with M. paratuberculosis. PBMC of individuals homozygous for the 3020insC NOD2 mutation showed a 70% defective cytokine response after stimulation with M. paratuberculosis. These results demonstrate that TLR2, TLR4, and NOD2 are involved in the recognition of M. paratuberculosisby the innate immune system.

Objective.To compare the rate of surgical site infection (SSI) before and after an intervention period in which an optimized policy for antibiotic prophylaxis was implemented. To demonstrate that a more prudent, restrictive policy would not have a detrimental effect on patient outcomes.Design.Before-after trial with prospective SSI surveillance in the Dutch nosocomial surveillance network (Preventie Ziekenhuisinfecties door Surveillance [PREZIES]), using the criteria of the Centers for Disease Control, including postdischarge surveillance for up to 1 year.Methods.During a preintervention period and a postintervention period (both 6-13 months), 12 Dutch hospitals collected data on antimicrobial prophylaxis and SSI rates. The study was limited to commonly performed surgical procedures in 4 specialties: vascular, intestinal, gynecological and orthopedic surgery. Selected risk factors for analysis were sex, age, American Society of Anesthesiologists classification, wound contamination class, duration of surgery, length of hospital stay before surgery, and urgency of surgery (elective or acute).Results.A total of 3,621 procedures were included in the study, of which 1,668 were performed before the intervention and 1,953 after. The overall SSI rate decreased from 5.4% to 4.5% (P= .22). Among the procedures included in the study, the largest proportion (55%) were total hip arthroplasty, and the smallest proportion (2%) were replacement of the head of the femur. SSI rates varied from 0% for vaginal hysterectomy to 21.1% for femoropopliteal or femorotibial bypass surgery. Crude and adjusted odds ratios showed that there were no significant changes in procedure-specific SSI rates after the intervention (P>.1).Conclusions.An optimized and restrictive antibiotic prophylaxis policy had no detrimental effect on the outcome of clean and clean contaminated surgery, as measured by SSI rate.

Toll-like receptors (TLRs) have been identified as a major class of pattern-recognition receptors. Recognition of pathogen-associated molecular patterns (PAMPs) by TLRs, either alone or in heterodimerization with other TLR or non-TLR receptors, induces signals responsible for the activation of innate immune response. Recent studies have demonstrated a crucial involvement of TLRs in the recognition of fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. By studying fungal infection in knock-out mice deficient in either TLRs or TLRassociated adaptor molecules, it appeared that specific TLRs such as TLR2 and TLR4 play differential roles in the activation of the various arms of the innate immune response. Recent data also suggest that TLRs offer escape mechanisms to certain pathogenic microorganisms, especially through TLR2-driven induction of antiinflamatory cytokines. These recent developments provide crucial information for understanding the mechanisms of fungal recognition by cells of the immune system, and provide hope for designing new therapeutical approaches to fungal infections.

The different manifestations of Candida albicansinfection are dictated by an underlying defect in the immune response of the host. Protective immunity to disseminated candidiasis, the manifestation of C. albicansinfection discussed in this review, has traditionally been ascribed to innate immunity with emphasis on the role of granulocytes. Lately, however, immunological studies have learned that host defence against disseminated candidiasis is based on a complex interplay between innate and cell-mediated immunity. Despite the availability of new antifungal agents, mortality associated with disseminated C. albicansinfection remains high. Immunotherapy that augments host defence is an important strategic option in the battle against disseminated candidiasis. Here, the authors review the chronological events in the pathogenesis of disseminated candidiasis that aid in predicting the impact of existing immunotherapy and the development of future immunomodulating strategies.

The nucleotide oligomerization domain 2 (NOD2) 3020insC (NOD2fs) mutation increases susceptibility to Crohn’s disease (CD), but the mechanism remains controversial. Loss‐of‐function and gain‐of‐function phenotypes have been described as a result of NOD2fs. Here, we show that dendritic cells (DC) derived from CD patients homozygous for this mutation respond normally to purified Toll‐like receptor (TLR) ligands but fail to up‐regulate the costimulatory molecules CD80 and CD86 in response to the NOD2 ligand muramyl dipeptide (MDP). Moreover, they lack MDP‐induced enhancement of TLR‐mediated tumor necrosis factor α, interleukin (IL)‐12, and IL‐10 production, which is observed in control DC with intact NOD2. These data indicate that the NOD2fs mutation results in a loss‐of‐function phenotype in human myeloid DC and imply decreased immune regulation by IL‐10 as a possible mechanism for this mutation in CD.

Toll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicans by TLRs. In contrast to Candida blastoconidia, which stimulated large amounts of gamma interferon (IFN-γ), the tissue-invasive Candida hyphae did not stimulate any IFN-γ by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-γ was TLR4 dependent, as shown by the significantly decreased IFN-γ production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor α (TNF-α) than macrophages of control mice did when stimulated with Candida blastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-α in response to both Candida blastoconidia and hyphae. Candida hyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candida stimulation, whereas Candida recognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candida blastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.

ABSTRACTToll-like receptors (TLR) are crucial for an efficient antifungal defense. We investigated the differential recognition of blastoconidia and hyphae of Candida albicansby TLRs. In contrast to Candidablastoconidia, which stimulated large amounts of gamma interferon (IFN-γ), the tissue-invasive Candidahyphae did not stimulate any IFN-γ by human peripheral blood mononuclear cells (PBMC) or murine splenic lymphocytes. After stimulation with blastoconidia, the production of IFN-γ was TLR4 dependent, as shown by the significantly decreased IFN-γ production in anti-TLR4-treated PBMC and in splenic lymphocytes from TLR4-defective ScCr mice. In addition, peritoneal macrophages from ScCr mice produced less tumor necrosis factor α (TNF-α) than macrophages of control mice did when stimulated with Candidablastoconidia, but not with hyphae, indicating that TLR4-mediated signals are lost during hyphal germination. In contrast, macrophages from TLR2 knockout mice had a decreased production of TNF-α in response to both Candidablastoconidia and hyphae. Candidahyphae stimulated production of interleukin-10 through TLR2-dependent mechanisms. In conclusion, TLR4 mediates proinflammatory cytokine induction after Candidastimulation, whereas Candidarecognition by TLR2 leads mainly to anti-inflammatory cytokine release. TLR4-mediated proinflammatory signals are lost during germination of Candidablastoconidia into hyphae. Phenotypic switching during germination may be an important escape mechanism of C. albicans, resulting in counteracting host defense.