interleukin-8 and Staphylococcal-Infections

The staphylococcal bi-component leukotoxins constitute a family included in the super-family of the beta-sheet-structured pore-forming toxins. They may be produced by Staphylococcus aureus and by Staphylococcus intermedius and their target cells vary according to the molecules. The mode of action proceeds by the sequential binding of the class S proteins, then by that of the class F proteins at the surface of the membranes. Then, the activation of cellular calcium-channels precedes the pore formation which seems to be sensitive to several monovalent cations. The cell response is inflammatory and includes the neosynthesis as well as the secretion of leukotriene B4, interleukin -8, histamine. The injection of leukotoxins to rabbits generates cell chemotaxis , vasodilatation, and tissue necrosis. The association of the production of leukotoxins with clinical syndromes concerns several aspects of the pathology of S. aureus, and confers to these leukotoxins an important role of virulence factors.

Topics: Animals; Bacterial Proteins; Bacterial Toxins; Calcium Channels; Cations, Divalent; Cattle; Cell Membrane Permeability; Chemotaxis, Leukocyte; Cross Infection; Erythrocytes; Exotoxins; Female; Hemolysin Proteins; Histamine Release; Humans; Interleukin-8; Ion Transport; Leukocidins; Leukotriene B4; Male; Mastitis, Bovine; Models, Biological; Necrosis; Neutrophils; Rabbits; Staphylococcal Infections; Staphylococcus aureus; T-Lymphocytes; Vasodilation; Virulence; Vitreous Body

Long-term administration of azithromycin (AZM) in children with cystic fibrosis (CF) has improved outcomes. However, the doses and schedule of administration are not very well studied in children with CF.. A randomized controlled trial was conducted to compare the effect of two doses of azithromycin (5mg/kg/day and 15mg/kg/day) on FEV(1) and pulmonary exacerbations in children with cystic fibrosis. Enrolled children were randomly allocated to receive daily azithromycin (5mg/kg/day or 15mg/kg/day) for 6months. Clinical assessment and FEV(1) measurement were performed monthly.. 56 children (28 in high dose group and 28 in low dose group) were enrolled. 47 (24 and 23 children in low and high dose groups) completed 12months of follow up. There was no difference in clinical scores, FEV(1), pulmonary exacerbation rates between two groups at baseline, 6months and at 12months. Per protocol analysis revealed that pulmonary exacerbation increased after discontinuing AZM and there was significantly more increase after 12months of enrolment in children getting high dose azithromycin. There was no improvement in FEV(1) in either group at the end of treatment period. Children tolerated daily low as well as high dose AZM well for 6months. There was no significant side effect of azithromycin.. In this randomized controlled trial, we did not find differences in the effect of 2 doses (5mg/kg/day or 15mg/kg/day) of AZM on change in percentage predicted FEV(1), clinical scores, Pseudomonas colonization rates, pulmonary exacerbations and need for antibiotics. There was increase in exacerbations after stopping azithromycin in both the groups. Our results also suggest that the decrease in the incidence of LRTI persists only till 6months after discontinuing azithromycin.

Topics: Anti-Bacterial Agents; Azithromycin; Body Weight; Candidiasis; Child; Child, Preschool; Cystic Fibrosis; Female; Forced Expiratory Volume; Humans; Interleukin-8; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Spirometry; Sputum; Staphylococcal Infections; Staphylococcus aureus; Streptococcal Infections; Streptococcus pneumoniae; Treatment Outcome

Staphylococcal menstrual toxic shock syndrome depends on vaginal production of exotoxins. Glycerol monolaurate (GML) inhibits Staphylococcus aureus exotoxin production in vitro. The purpose of this study was to determine whether GML, as a tampon fiber finish, inhibits production of exotoxins and the cytokine interleukin 8 (IL-8) during normal tampon use.. On day 2 of menstruation, when vaginal S. aureus counts are high in colonized women, study participants exchanged their own preferred tampons, after wearing them for 2-6 h, for study tampons with or without GML (assigned randomly and blindly), which they then wore for 4-6 h. The women's own tampons and the study tampons with or without GML were assayed for S. aureus, the exotoxins toxic shock syndrome toxin 1 and alpha-toxin, and IL-8.. A total of 225 women completed the study. S. aureus was present in the tampons of 41 women (18%). Lower numbers of S. aureus and the exotoxins were detected in study tampons with or without GML than in women's own tampons; lower amounts of the exotoxins were present in study tampons with GML than study tampons without GML. The IL-8 level was lower in tampons from women without vaginal S. aureus compared with women with S. aureus and was lower in study tampons with GML than in study tampons without GML.. Tampons that contain GML reduce S. aureus exotoxin production. S. aureus increases vaginal IL-8 levels, and GML reduces production of this proinflammatory cytokine. These results suggest that GML added to tampons provides additional safety relative to menstrual toxic shock syndrome as well as benefits for vaginal health generally, thus supporting the addition of GML to tampons.

Topics: Adolescent; Adult; Bacterial Toxins; Cariostatic Agents; Enterotoxins; Female; Humans; Interleukin-8; Laurates; Menstrual Hygiene Products; Monoglycerides; Staphylococcal Infections; Staphylococcus aureus; Superantigens; Vagina; Young Adult

Interleukin-8 (IL-8) has been reported to be released by activated peritoneal macrophages (PMs) and a variety of other cell types, and it exhibits potent chemotactic activity for polymorphonuclear cells (PMNs). We have previously shown that IL-8 is detectable in the drain dialysate of uremic patients on continuous ambulatory peritoneal dialysis (CAPD) during peritonitis. The levels of IL-8 in infected drain dialysate caused by different microorganisms were variable. In this study, we evaluated the gene expression and release of IL-8 by PMs and PMNs during peritonitis caused by Staphylococcus aureus in uremic patients on CAPD. IL-8 levels were variable in the drain dialysate at the different episodes of peritonitis, even in the same patient. The IL-8 levels were highly correlated with PMN count in drain dialysate (r = 0.9919, p < 0.001). PMs and PMNs obtained from drain dialysate at the onset of peritonitis increased mRNA expression for IL-8 and the amount of IL-8 mRNA from drainage cells was also highly correlated with PMN count. In contrast, cells isolated from drain dialysate without peritonitis failed to express mRNA for IL-8. These data suggest that increased expression of IL-8 may be a feature of peritonitis. The levels of IL-8 during peritonitis were not only related to the etiological microorganism but also to other unknown factor(s).

Topics: Adult; Female; Gene Expression; Humans; Interleukin-8; Kidney Failure, Chronic; Macrophages, Peritoneal; Male; Middle Aged; Neutrophils; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; RNA, Messenger; Staphylococcal Infections; Uremia

Staphylococcus aureus is the principal causative agent of osteomyelitis, a serious bacterial infection of bone that is associated with progressive inflammatory damage. Bone-forming osteoblasts have increasingly been recognized to play an important role in the initiation and progression of detrimental inflammation at sites of infection and have been demonstrated to release an array of inflammatory mediators and factors that promote osteoclastogenesis and leukocyte recruitment following bacterial challenge. In the present study, we describe elevated bone tissue levels of the potent neutrophil-attracting chemokines CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 in a murine model of posttraumatic staphylococcal osteomyelitis. RNA sequencing (RNA-Seq) gene ontology analysis of isolated primary murine osteoblasts showed enrichment in differentially expressed genes involved in cell migration and chemokine receptor binding and chemokine activity following S. aureus infection, and a rapid increase in the expression of mRNA encoding CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7, in these cells. Importantly, we have confirmed that such upregulated gene expression results in protein production with the demonstration that S. aureus challenge elicits the rapid and robust release of these chemokines by osteoblasts and does so in a bacterial dose-dependent manner. Furthermore, we have confirmed the ability of soluble osteoblast-derived chemokines to elicit the migration of a neutrophil-like cell line. As such, these studies demonstrate the robust production of CXCL1, CXCL2, CXCL3, CXCL5, CCL3, and CCL7 by osteoblasts in response to S. aureus infection, and the release of such neutrophil-attracting chemokines provides an additional mechanism by which osteoblasts could drive the inflammatory bone loss associated with staphylococcal osteomyelitis.

Topics: Animals; Chemokine CCL3; Chemokine CCL7; Chemokine CXCL1; Chemokine CXCL2; Chemokines; Interleukin-8; Mice; Neutrophils; Osteoblasts; Osteomyelitis; Staphylococcal Infections; Staphylococcus aureus

The mechanisms underlying innate immune memory (trained immunity) comprise epigenetic reprogramming of transcriptional pathways associated with alterations of intracellular metabolism. While the mechanisms of innate immune memory carried out by immune cells are well characterized, such processes in non-immune cells, are poorly understood. The opportunistic pathogen,. In the current work, we demonstrated the development of innate immune memory in non-immune cells during S. aureus infection employing a combination of techniques including Enzyme-linked immunosorbent assay (ELISA), microscopic analysis, and cytometry.. We observed that training of human osteoblast-like MG-63 cells and lung epithelial A549 cells with β-glucan increased IL-6 and IL-8 production upon a stimulation with. This work improves our understanding of innate immune memory in non-immune cells in the context of

Topics: Animals; Cattle; Female; Humans; Immunity, Innate; Interleukin-6; Interleukin-8; Reactive Oxygen Species; Staphylococcal Infections; Staphylococcus aureus; Trained Immunity

Topics: Humans; Interleukin-6; Interleukin-8; Keratinocytes; Nucleic Acids; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis

Many bacterial and fungal pathogens cause disease across mucosal surfaces, and to a lesser extent through skin surfaces. Pathogens that potentially cause disease vaginally across epithelial cells include Staphylococcus aureus, group A and B streptococci, Escherichia coli, Neisseria gonorrhoeae, and Candida albicans. We have previously shown that staphylococcal and streptococcal superantigens induce inflammatory chemokines from vaginal epithelial cells through the immune costimulatory molecule CD40 through use of a CRISPR cas9 knockout mutant and complemented epithelial cell line. In this study, we show that the potential vaginal pathogens S. aureus, group A and B streptococci, E. coli, an Enterococcus faecalis strain, and C. albicans in part use CD40 to stimulate interleukin-8 (IL-8) production from human vaginal epithelial cells. In contrast, N. gonorrhoeae does not appear to use CD40 to signal IL-8 production. Normal flora Lactobacillus crispatus and an Enterococcus faecalis strain that produces reutericyclin do not induce IL-8. These data indicate that many potential pathogens, but no normal commensals, induce IL-8 to help disrupt the human vaginal epithelial barrier through CD40, thus providing a potential therapeutic target for drug development.

Topics: Candida albicans; Chemokines; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Female; Humans; Interleukin-8; Staphylococcal Infections; Staphylococcus aureus; Vagina

Periprosthetic joint infection (PJI), a devastating complication of total joint replacement, is of incompletely understood pathogenesis and may sometimes be challenging to clinically distinguish from other causes of arthroplasty failure. We characterized human gene expression in 93 specimens derived from surfaces of resected arthroplasties, comparing transcriptomes of subjects with infection- versus non-infection-associated arthroplasty failure. Differential gene expression analysis confirmed 28 previously reported potential biomarkers of PJI, including bactericidal/permeability increasing protein (BPI), cathelicidin antimicrobial peptide (CAMP), C-C-motif chemokine ligand 3 (CCL3), 4(CCL4) and C-X-C-motif chemokine ligand 2 (CXCL2), colony stimulating factor 2 receptor beta (CSF2RB), colony stimulating factor 3 (CSF3), alpha-defensin (DEFA4), Fc fragment of IgG receptor 1B (CD64B), intercellular adhesion molecule 1 (ICAM1), interferon gamma (IFNG), interleukin 13 receptor subunit alpha 2 (IL13RA2), interleukin 17D (IL17D), interleukin 1 (IL1A, IL1B, IL1RN), interleukin 2 receptors (IL2RA, IL2RG), interleukin 5 receptor (IL5RA), interleukin 6 (IL6), interleukin 8 (IL8), lipopolysaccharide binding protein (LBP), lipocalin (LCN2), lactate dehydrogenase C (LDHC), lactotransferrin (LTF), matrix metallopeptidase 3 (MMP3), peptidase inhibitor 3 (PI3), and vascular endothelial growth factor A (VEGFA), and identified three novel molecules of potential diagnostic use for detection of PJI, namely C-C-motif chemokine ligand CCL20, coagulation factor VII (F7), and B cell receptor FCRL4. Comparative analysis of infections caused by staphylococci versus bacteria other than staphylococci and Staphylococcus aureus versus Staphylococcus epidermidis showed elevated expression of interleukin 13 (IL13), IL17D, and MMP3 in staphylococcal infections, and of IL1B, IL8, and platelet factor PF4V1 in S. aureus compared to S. epidermidis infections. Pathway analysis of over-represented genes suggested activation of host immune response and cellular maintenance and repair functions in response to invasion of infectious agents. The data presented provides new potential targets for diagnosis of PJI and for differentiation of PJI caused by different infectious agents.

Topics: Arthritis, Infectious; Biomarkers; Colony-Stimulating Factors; Humans; Interleukin-8; Ligands; Matrix Metalloproteinase 3; Prosthesis-Related Infections; Staphylococcal Infections; Staphylococcus aureus; Synovial Fluid; Transcriptome; Vascular Endothelial Growth Factor A

Staphylococcus aureus (S. aureus) is a major mastitis-causing pathogen in dairy cows. Dairy cows with mastitis suffer from a decrease in milk yield and protein content. Chlorogenic acid (CGA) is a natural product with anti-inflammatory effects. In this study, we examined the function and mechanism of CGA with regard to its anti-inflammatory effects and evaluated its protective function in milk protein synthesis in bovine mammary epithelial cells (BMECs). BMECs were cultured with and without infection by S. aureus and CGA, and extracellular inflammatory cytokines and amino acids in the medium and milk proteins were determined by ELISA. The function of IL-10RA in anti-inflammatory processes and of SF-1 in milk protein synthesis was assessed by gene silencing. The activity of mTORC1, NF-κB, and STAT5 was examined by western blot. S. aureus caused intracellular infection and upregulated TNF-α, IL-1β, IL-6, and IL-8, whereas uptake of amino acids and milk protein synthesis were suppressed. CGA mitigated the S. aureus-induced inflammatory response and milk protein synthesis in vitro and in vivo. CGA alleviated S. aureus-induced inhibition of mTORC1 and STAT5 and upregulated IL-10 and IL-10RA. In addition, SF-1 was predicted to be a transcription factor of the milk protein-encoding genes α-LA, β-LG, and CSN2. S. aureus downregulated SF-1 and CGA reversed the decline in milk protein synthesis due to SF-1 knockdown. Thus, CGA mitigates the inflammatory response that is induced by S. aureus and protects the uptake of amino acids and milk protein synthesis in BMECs.

Topics: Animals; Anti-Inflammatory Agents; Cattle; Chlorogenic Acid; Cytokines; Epithelial Cells; Female; Interleukin-10; Interleukin-6; Interleukin-8; Mammary Glands, Animal; Mastitis, Bovine; Mechanistic Target of Rapamycin Complex 1; Milk Proteins; Staphylococcal Infections; Staphylococcus aureus; STAT5 Transcription Factor; Tumor Necrosis Factor-alpha

Topics: Anti-Bacterial Agents; Biofilms; Daptomycin; Humans; Inflammasomes; Interleukin-10; Interleukin-6; Interleukin-8; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; NLR Family, Pyrin Domain-Containing 3 Protein; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

The study aimed to understand the differential immune response of methicillin susceptible. Retinal pigment epithelium (RPE) and microglia cells (CHME-3) were exposed to MRSA and MSSA strains and analyzed for expression of inflammatory mediators by real-time quantitative PCR and validated by ELISA or immunofluorescence assay. Heatmap and STRING analysis was used to assess the differential immune expression.. Both microglia and RPE expressed TLR-2, TLR-1, TLR-6, and TLR -9 after challenge with MRSA and MSSA strains though the expression varied. MRSA-infected cells induced higher expression of IL-1β, IL-8, 1 L-10, IL-6, and GM-CSF, while TNF-α and IFN-ϒ were downregulated in comparison to MSSA-infected cells. We also demonstrate that MRSA infection leads to increased activation of MMP-9 and MMP-2 in RPE cells, while microglia expressed only MMP-9 in MRSA-infected cells.. MRSA strain can induce an exacerbated immune response in retinal cells. Giving clues for potential targets in immunomodulatory therapies.

Topics: Anti-Bacterial Agents; Endophthalmitis; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunity; Interleukin-6; Interleukin-8; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Methicillin; Methicillin Resistance; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 1; Toll-Like Receptor 2; Toll-Like Receptor 6; Tumor Necrosis Factor-alpha

Formyl-peptide receptors (FPRs) are important pattern recognition receptors that sense specific bacterial peptides. Formyl-peptide receptors are highly expressed on neutrophils and monocytes, and their activation promotes the migration of phagocytes to sites of infection. It is currently unknown whether FPRs may also influence subsequent processes such as bacterial phagocytosis and killing. Staphylococcus aureus, especially highly pathogenic community-acquired methicillin-resistant S aureus strains, release high amounts of FPR2 ligands, the phenol-soluble modulins.. We demonstrate that FPR activation leads to upregulation of complement receptors 1 and 3 as well as FCγ receptor I on neutrophils and, consequently, increased opsonic phagocytosis of S aureus and other pathogens.. Increased phagocytosis promotes killing of S aureus and interleukin-8 release by neutrophils.. We show here for the first time that FPRs govern opsonic phagocytosis. Manipulation of FPR2 activation could open new therapeutic opportunities against bacterial pathogens.

Topics: Blood Donors; Cells, Cultured; Community-Acquired Infections; Humans; Interleukin-8; Macrophage-1 Antigen; Methicillin-Resistant Staphylococcus aureus; Neutrophils; Phagocytosis; Receptors, Complement 3b; Receptors, Formyl Peptide; Receptors, IgG; Receptors, Lipoxin; Receptors, Pattern Recognition; Staphylococcal Infections

The study aimed to analyze morphological and functional changes of

Topics: Adult; Allylbenzene Derivatives; Anisoles; Anti-Bacterial Agents; Antioxidants; Bacteremia; Blood Cell Count; Female; Humans; Immunity, Innate; Interleukin-8; Male; Nitroblue Tetrazolium; Phagocytes; Phagocytosis; Spectroscopy, Fourier Transform Infrared; Staphylococcal Infections; Staphylococcus aureus; Xanthophylls

Staphylococcus aureus is a leading cause of bacteremia, yet there remains a significant knowledge gap in the identification of relevant biomarkers that predict clinical outcomes. Heterogeneity in the host response to invasive S. aureus infection suggests that specific biomarker signatures could be utilized to differentiate patients prone to severe disease, thereby facilitating earlier implementation of more aggressive therapies.. To further elucidate the inflammatory correlates of poor clinical outcomes in patients with S. aureus bacteremia, we evaluated the association between a panel of blood proteins at initial presentation of bacteremia and disease severity outcomes using 2 cohorts of patients with S. aureus bacteremia (n = 32 and n = 124).. We identified 13 candidate proteins that were correlated with mortality and persistent bacteremia. Prognostic modeling identified interleukin (IL)-8 and CCL2 as the strongest individual predictors of mortality, with the combination of these biomarkers classifying fatal outcome with 89% sensitivity and 77% specificity (P < .0001). Baseline IL-17A levels were elevated in patients with persistent bacteremia (P < .0001), endovascular (P = .026) and metastatic tissue infections (P = .012).. These results demonstrate the potential utility of selected biomarkers to distinguish patients with the highest risk for treatment failure and bacteremia-related complications, providing a valuable tool for clinicians in the management of S. aureus bacteremia. Additionally, these biomarkers could identify patients with the greatest potential to benefit from novel therapies in clinical trials.

Topics: Adult; Aged; Aged, 80 and over; Anti-Bacterial Agents; Bacteremia; Biomarkers; Case-Control Studies; Chemokine CCL2; Endocarditis, Bacterial; Female; Humans; Interleukin-17; Interleukin-8; Male; Middle Aged; Prognosis; Sensitivity and Specificity; Severity of Illness Index; Staphylococcal Infections; Staphylococcus aureus; Survival Analysis

LI-F type peptides are a family of cyclic lipodepsipeptide antibiotics isolated from Paenibacillus polymyxa and display potent activities against positive bacteria including methicillin-resistant S. aureus (MRSA). In this study, we investigated the mechanism of action of LI-F type peptide AMP-jsa9 against a MRSA (S. aureus CICC10790), which is resistant to ciprofloxacin, gentamicin, kanamycin, chloramphenicol, methicillin, and tetracycline. It was found that AMP-jsa9 mainly targets the cell membrane of MRSA and is able to inhibit biofilm formation through killing planktonic bacteria cells. Moreover, AMP-jsa9 can bind to DNA in vitro, which represents another pathway for the action on MRSA. Furthermore, in vivo treatment of scalded mice with AMP-jsa9 resulted in inhibiting MRSA infections and healing of the scalded wound. In addition, it was demonstrated that AMP-jsa9 can effectively inhibit MRSA infections in scalded murine epidermis and that inflammatory cytokines including IL-8, IL-6, tumor necrosis factor alpha (TNF-α), and monocyte chemotactic factor-1 (MCP-1) were reduced; moreover, both protein and gene expression levels of vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (e-NOS) were enhanced, which promote neovascularization and proliferation of new granulation tissue.

Topics: Animals; Anti-Bacterial Agents; Chemokine CCL2; Depsipeptides; Epidermis; Humans; Interleukin-6; Interleukin-8; Methicillin-Resistant Staphylococcus aureus; Mice; Microbial Sensitivity Tests; Staphylococcal Infections; Vascular Endothelial Growth Factor A

Antimicrobial peptides (AMPs) are key elements of plant defense mechanisms, resembling conserved protection strategies also present in mammals. Among the AMPs, plant thionins are particularly interesting due that display antibacterial and antifungal activities. In Arabidopsis thaliana have been described four thionins: Thi2.1, Thi2.2, Thi2.3 and Thi2.4. Work from our group shows that Thi2.1 expressed by bovine endothelial cells has direct antibacterial activity against Staphylococcus aureus mastitis isolates, bacteria able to persist inside bovine mammary epithelial cells (bMECs). Thus, the objective of this work was to analyze the immunomodulatory effects of the AMP thionin Thi2.1 from A. thaliana on bMECs during S. aureus infection. According to the results, S. aureus internalization into bMECs was reduced in cells pre-treated with Thi2.1 at 5 and 10 μg/mL during 24 h, effect related to the participation of TLR2. In addition, bMECs pre-treated with Thi2.1 (24 h) significantly increased TNF-α (~2-fold) and IL-6 (~7-fold), whereas decreased IL-10 gene expression (~0.5-fold). Interestingly, Thi2.1 inhibits the up-regulation induced by S. aureus of TNF-α and IL-10 gene expression, as well as NO production. In addition, Thi2.1 (10 μg/mL) up-regulates the expression of the chemokine IL-8 (~3-fold) in infected bMECs. Some of these effects are related to TLR2 activation. In this sense, Thi2.1 also reduces S. aureus-induced TLR2 gene expression and membrane abundance. In conclusion, Thi2.1 from A. thaliana modulates bMEC innate immune response by inducing the production of pro- and anti-inflammatory molecules while inhibits S. aureus internalization. Some of these effects are mediated by TLR2.

Topics: Animals; Anti-Infective Agents; Antimicrobial Cationic Peptides; Arabidopsis; Arabidopsis Proteins; Cattle; Cells, Cultured; Epithelial Cells; Humans; Immunologic Factors; Interleukin-10; Interleukin-8; Mammary Glands, Human; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Tumor Necrosis Factor-alpha

Antimicrobial resistance needs to be tackled from new angles, and antimicrobial peptides could be future candidates for combating bacterial infections. This study aims to investigate in vitro the bactericidal effects of the lantibiotic gallidermin on Staphylococcus epidermidis and Staphylococcus aureus, possible cytotoxic effects and its impact on host-microbe interactions. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of gallidermin were determined, and cytotoxicity and proinflammatory effects of gallidermin on fibroblasts, red blood cells (RBCs) and in whole blood were investigated. Both MIC and MBC for all four tested strains of S. epidermidis was 6.25 μg/ml. Both MIC and MBC for methicillin-sensitive S. aureus was 12.5 μg/ml and for methicillin-resistant S. aureus (MRSA) 1.56 μg/ml. Gallidermin displayed no cytotoxic effects on fibroblasts, only a high dose of gallidermin induced low levels of CXCL8 and interleukin-6. Gallidermin hemolyzed less than 1% of human RBCs, and did not induce reactive oxygen species production or cell aggregation in whole blood. In cell culture, gallidermin inhibited the cytotoxic effects of the bacteria and totally suppressed the bacteria-induced release of CXCL8 and interleukin-6 from fibroblasts. We demonstrate that gallidermin, expressing low cell cytotoxicity, is a promising candidate for treating bacterial infections caused by S. epidermidis and S. aureus, especially MRSA.

Topics: Adult; Anti-Bacterial Agents; Bacteriocins; Cells, Cultured; Dermis; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Male; Microbial Sensitivity Tests; Peptides; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis

Some of the most common infectious diseases are caused by bacteria that naturally colonise humans asymptomatically. Combating these opportunistic pathogens requires an understanding of the traits that differentiate infecting strains from harmless relatives. Staphylococcus epidermidis is carried asymptomatically on the skin and mucous membranes of virtually all humans but is a major cause of nosocomial infection associated with invasive procedures. Here we address the underlying evolutionary mechanisms of opportunistic pathogenicity by combining pangenome-wide association studies and laboratory microbiology to compare S. epidermidis from bloodstream and wound infections and asymptomatic carriage. We identify 61 genes containing infection-associated genetic elements (k-mers) that correlate with in vitro variation in known pathogenicity traits (biofilm formation, cell toxicity, interleukin-8 production, methicillin resistance). Horizontal gene transfer spreads these elements, allowing divergent clones to cause infection. Finally, Random Forest model prediction of disease status (carriage vs. infection) identifies pathogenicity elements in 415 S. epidermidis isolates with 80% accuracy, demonstrating the potential for identifying risk genotypes pre-operatively.

Topics: Genome-Wide Association Study; Genome, Bacterial; Genotype; Humans; Interleukin-8; Skin Diseases; Staphylococcal Infections; Staphylococcus epidermidis

Literature reveals that interaction with live Staphylococcus aureus (S. aureus) or heat killed S. aureus (HKSA) promotes secretion of CXCL-8 or interleukin-8 (IL-8) from leukocytes, however, the expressions of CXCR1 in murine splenic (SPM), peritoneal macrophages (PM) and resident fresh bone marrow cells (FBMC) have not been identified. Currently, very few studies are available on the functional characterization of CXCR1 in mouse macrophage subtypes and its modulation in relation to acute S. aureus infection. SPM, PM and FBMCs were infected with viable S. aureus or stimulated with HKSA in presence and absence of anti-CXCR1 antibody in this study. We reported here that CXCR1 was not constitutively expressed by macrophage subtypes and the receptor was induced only after S. aureus stimulation. The CXCR1 band was found specific as we compared with human polymorphonuclear neutrophils (PMNs) as a positive control (data not shown). Although, we did not show that secreted IL-8 from S. aureus-infected macrophages promotes migration of PMNs. Blocking of cell surface CXCR1 decreases the macrophage's ability to clear staphylococcal infection, attenuates proinflammatory cytokine production and the increased catalase and decreased superoxide dismutase (SOD) enzymes of the bacteria might indicate their role in scavenging macrophage derived hydrogen peroxide (H

Topics: Animals; Bone Marrow Cells; Catalase; Cytokines; Hot Temperature; Humans; Hydrogen Peroxide; Inflammation; Interleukin-8; Macrophages; Macrophages, Peritoneal; Mice; Neutrophils; Nitric Oxide; Receptors, Interleukin-8; Spleen; Staphylococcal Infections; Staphylococcus aureus; Superoxide Dismutase; Superoxides

The breach of the skin barrier is a critical issue associated with the treatment of individuals with transfemoral amputation (TFA) using osseointegrated, percutaneous titanium implants. Thirty TFA patients scheduled for abutment exchange or removal were consecutively enrolled. The aims were to determine the macroscopic skin signs, the presence of bacteria and the gene expression in abutment-adherent cells and to conduct correlative and comparative analyses between the different parameters. Redness and a granulation ring were present in 47% of the patients. Bacteria were detected in 27/30 patients, commonly in the bone canal. Staphylococcus aureus, coagulase-negative staphylococci, streptococci, and Enterococcus faecalis were the most common. A positive correlation was found between TNF-α expression and the detection of S. aureus. Staphylococcus aureus together with other bacterial species revealed a positive relationship with MMP-8 expression. A negative correlation was demonstrated between the length of the residual femur bone and the detection of a granulation ring and E. faecalis. A positive correlation was revealed between fixture loosening and pain and the radiological detection of endosteal bone resorption. Fixture loosening was also correlated with the reduced expression of interleukin-10 and osteocalcin. It is concluded that several relationships exist between clinical, radiological, microbiological, and molecular assessments of the percutaneous area of TFAs. Further long term studies on larger patient cohorts are required to determine the precise cause-effect relationships and unravel the role of host-bacteria interactions in the skin, bone canal and on the abutment for the longevity of percutaneous implants as treatment of TFA. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 578-589, 2017.

Topics: Adult; Aged; Amputees; Bone-Implant Interface; Cross-Sectional Studies; Enterococcus faecalis; Female; Femur; Humans; Interleukin-10; Interleukin-8; Male; Middle Aged; Osteocalcin; Staphylococcal Infections; Staphylococcus aureus

Topics: Aged; Anti-Bacterial Agents; Biomarkers; Community-Acquired Infections; Diabetes Mellitus; Disease Management; Disseminated Intravascular Coagulation; Female; Fluid Therapy; Humans; Intensive Care Units; Interleukin-8; Male; Middle Aged; Prognosis; Prospective Studies; Respiratory Insufficiency; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Survival Analysis; Tertiary Care Centers; Thailand

The effect of intramammary infusion of recombinant bovine granulocyte-macrophage colony-stimulating factor (rbGM-CSF) and interleukin-8 (rbIL-8) on mononuclear cell populations in quarters, somatic cell count (SCC) and the California Mastitis Test (CMT) score were investigated. From the selected cows with naturally occurring Staphylococcus aureus subclinical mastitis, one quarter of each cow were selected for the infusions of rbGM-CSF (400 μg/5 mL/quarter, n = 9), rbIL-8 (1 mg/5 mL/quarter, n = 9), and phosphate-buffered saline (5 mL/quarter, n = 7). The CMT score of both cytokines post infusion temporarily increased between days 0 and 1 and significantly decreased between days 7 and 14 compared to the preinfusion level. The SCC on day 14 after infusions of rbGM-CSF tended to be lower than that of the control group. The percentage of CD14+ cells increased on days 1 and 2 post infusion of rbGM-CSF. The percentage of CD4+ and CD8+ cells also increased on days 2 and 3, suggesting that the infusion of rbGM-CSF enhanced cellular immunity in the mammary gland. In contrast, the percentage of CD14+ cells decreased on days 0.25 and 1 post infusion of rbIL-8. No significant changes in the percentages of CD4+ and CD8+ cells in milk after infusion of rbIL-8 were evident during the experimental period, which suggested that rbIL-8 had little effect on the function of T cells in the mammary gland. These results indicated that rbGM-CSF and rbIL-8 decreased the CMT score by a different mechanism and may have a potential as therapeutic agents for subclinical mastitis.

Topics: Animals; Asymptomatic Infections; Cattle; Cell Count; Female; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-8; Leukocyte Count; Mammary Glands, Animal; Mastitis, Bovine; Recombinant Proteins; Staphylococcal Infections

Houttuynia cordata (HC) has been commonly used as many traditional remedies in local areas of Japan. Although many pharmacological activities of HC have been reported, the mechanism underlying the effect of HC remains unknown. We conducted the interview survey in Japan to verify how HC was actually used. The interview survey revealed that HC poultice (HCP) prepared from smothering fresh leaves of HC was most frequently used for the treatment of purulent skin diseases including furuncle and carbuncle with high effectiveness. Ethanol extract of HCP (eHCP) showed anti-bacterial effects against methicillin-resistant Staphylococcus aureus (MRSA), and showed an anti-biofilm activity against MRSA. eHCP showed dose-dependent inhibition of S. aureus lipoteichoic acid (LTA)-induced interleukin-8 and CCL20 production in human keratinocyte without any cytotoxicity. These results suggest that HCP is effective for skin abscess and its underlying mechanism might be the complicated multiple activities for both bacteria and host cells.

Topics: Aged; Anti-Bacterial Agents; Anti-Inflammatory Agents, Non-Steroidal; Biofilms; Carbuncle; Cell Line, Transformed; Chemokine CCL20; Dose-Response Relationship, Drug; Ethanol; Female; Furunculosis; Houttuynia; Humans; Interleukin-8; Japan; Keratinocytes; Lipopolysaccharides; Male; Methicillin-Resistant Staphylococcus aureus; Microbial Sensitivity Tests; Middle Aged; Phytotherapy; Plant Extracts; Plant Leaves; Staphylococcal Infections; Surveys and Questionnaires; Teichoic Acids

Secreted factors of Staphylococcus aureus can activate host signaling from the epidermal growth factor receptor (EGFR). The superantigen toxic shock syndrome toxin-1 (TSST-1) contributes to mucosal cytokine production through a disintegrin and metalloproteinase (ADAM)-mediated shedding of EGFR ligands and subsequent EGFR activation. The secreted hemolysin, α-toxin, can also induce EGFR signaling and directly interacts with ADAM10, a sheddase of EGFR ligands. The current work explores the role of EGFR signaling in menstrual toxic shock syndrome (mTSS), a disease mediated by TSST-1. The data presented show that TSST-1 and α-toxin induce ADAM- and EGFR-dependent cytokine production from human vaginal epithelial cells. TSST-1 and α-toxin also induce cytokine production from an ex vivo porcine vaginal mucosa (PVM) model. EGFR signaling is responsible for the majority of IL-8 production from PVM in response to secreted toxins and live S. aureus. Finally, data are presented demonstrating that inhibition of EGFR signaling with the EGFR-specific tyrosine kinase inhibitor AG1478 significantly increases survival in a rabbit model of mTSS. These data indicate that EGFR signaling is critical for progression of an S. aureus exotoxin-mediated disease and may represent an attractive host target for therapeutics.

Topics: ADAM Proteins; Animals; Epithelial Cells; ErbB Receptors; Female; Humans; Interleukin-8; Rabbits; Shock, Septic; Signal Transduction; Staphylococcal Infections; Vagina

Staphylococci can sense Substance P (SP) in skin, but this molecule is generally released by nerve terminals along with another neuropeptide, Calcitonin Gene Related Peptide (CGRP). In this study, we investigated the effects of αCGRP on Staphylococci. CGRP induced a strong stimulation of Staphylococcus epidermidis virulence with a low threshold (<10

Topics: Antimicrobial Cationic Peptides; Biofilms; Calcitonin Gene-Related Peptide; Cathelicidins; Gadolinium; Humans; Interleukin-8; Keratinocytes; Nerve Endings; Neuropeptides; Skin; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis; Substance P

The Toll-Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus-induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air-interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub-groups exposed to UV-killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL-8 mRNA expressions were analyzed by quantitative real-time RT-PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL-8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial-corneal tissue interactions and their inflammatory consequences.

Topics: Animals; Cell Differentiation; Corneal Keratocytes; Corneal Stroma; Enzyme Activation; Inflammation; Interleukin-8; Keratitis; Myofibroblasts; Organ Culture Techniques; Rabbits; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Stromal Cells; Toll-Like Receptor 2

Chronic otitis media with effusion (COME) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Staphylococcus epidermidis, typically considered a commensal organism, is very frequently recovered in chronic middle ear fluid and in middle ear biofilms. Although it has been shown to drive inflammation in sinonasal epithelium, the impact of S. epidermidis on COME is markedly understudied. The goal of this study was to examine the in vitro effects of S. epidermidis lysates on murine and human middle ear epithelial cells.. Staphylococcus epidermidis lysates were generated and used to stimulate submerged and differentiated human and murine epithelial cells (MEECs) for 24 to 48 hours. Quantitative real time-polymerase chain reaction, Western blot, enzyme-linked immunosorbent assay, and immunocytochemistry techniques were performed to interrogate the mucin gene MUC5AC and MUC5B expression and protein production, chemokine response, as well as NF-κB activation. Luciferase reporter assays were performed to further evaluate nuclear factor κB (NF-κB) activation and query specific promoter responses after S. epidermidis exposure.. Staphylococcus epidermidis induced a time- and dose-dependent MUC5AC and MUC5B overexpression along with a parallel overexpression of Cxcl2 in mouse MEEC and IL-8 in human MEEC. Further investigations in mMEEC showed a 1.3 to 1.5 induction of the MUC5AC and MUC5B promoters. As potential mechanisms for these responses, induction of an oxidative stress marker, along with early nuclear translocation and activation of NF-κB, was found. Finally, chronic exposure induced marked epithelial thickening of cells differentiated at the air liquid interface.. Staphylococcus epidermidis lysates activate a proinflammatory response in MEEC, including mucin gene expression and protein production. Although typically considered a nonpathogenic commensal organism in the ear, these results suggest that they may play a role in the perpetuation of an inflammatory and mucogenic response in COME.

Topics: Animals; Cell Line; Ear, Middle; Epithelial Cells; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; Mice; Mucins; NF-kappa B; Oxidative Stress; Proteomics; Staphylococcal Infections; Staphylococcus epidermidis

Staphylococcus aureus is a common cause of chronic mammary gland infections in dairy cattle. However, the inflammatory response and duration of infection following pathogen exposure is variable between individual animals. To investigate interanimal differences in immune response, dermal fibroblast cultures were established from skin biopsies collected from 50 early lactation Holstein cows. The fibroblasts ability to produce IL-8 in response to a 24-h treatment with a synthetic toll-like receptor 2/6 agonist (Pam2CSK4) was used to assign a response phenotype to the animals. Five high-responding and 5 low-responding animals were then selected for an intramammary challenge with S. aureus to evaluate differences in the inflammatory response, chronicity of infection, and development of antibodies to the pathogen. All animals exhibited clinical symptoms of mastitis at 24h postchallenge. Animals previously classified as high responders experienced a greater inflammatory response characterized by elevated levels of milk somatic cell count, IL-8, and BSA following the challenge compared with low responders. In addition, antibodies toward the challenge strain of S. aureus reached higher levels in whey from the challenged gland of high responders compared with low responders. Despite the antibody response, all 5 high responders were chronically infected for the 6-wk duration of the study, whereas 2 of the low responders cleared the infection, although 1 of these did become reinfected. The observed differences between animals classified as low and high responders based on their fibroblast responsiveness suggests that this cell type can be used to further examine the causes of interanimal variation in response to mammary infection.

Topics: Animals; Cattle; Cattle Diseases; Cell Count; Female; Fibroblasts; Interleukin-8; Mastitis, Bovine; Milk; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 6

Pseudomonas aeruginosa (PA) and Staphylococcus aureus (SA) are major respiratory pathogens and can concurrently colonize the airways of patients with chronic obstructive diseases, such as cystic fibrosis (CF). Airway epithelial cell signalling is critical to the activation of innate immune responses. In the setting of polymicrobial colonization or infection of the respiratory tract, how epithelial cells integrate different bacterial stimuli remains unknown. Our study examined the inflammatory responses to PA and SA co-stimulations. Immortalised airway epithelial cells (Beas-2B) exposed to bacteria-free filtrates from PA (PAF) induced a robust production of the neutrophil chemoattractant IL-8 while bacteria-free filtrates from SA (SAF) had a minimal effect. Surprisingly, co-stimulation with PAF+SAF demonstrated that SAF strongly inhibited the PAF-driven IL-8 production, showing that SAF has potent anti-inflammatory effects. Similarly SAF decreased IL-8 production induced by the TLR1/TLR2 ligand Pam3CysSK4 but not the TLR4 ligand LPS nor TLR5 ligand flagellin in Beas-2B cells. Moreover, SAF greatly dampened TLR1/TLR2-mediated activation of the NF-κB pathway, but not the p38 MAPK pathway. We observed this SAF-dependent anti-inflammatory activity in several SA clinical strains, as well as in the CF epithelial cell line CFBE41o-. These findings show a novel direct anti-inflammatory effect of SA on airway epithelial cells, highlighting its potential to modulate inflammatory responses in the setting of polymicrobial infections.

Topics: Cell Line; Epithelial Cells; Gene Expression; Hemolysin Proteins; Humans; Interleukin-8; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Pseudomonas aeruginosa; Pseudomonas Infections; Reproducibility of Results; Respiratory Mucosa; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 1; Toll-Like Receptor 2

Measures of ventilation distribution are promising for monitoring early lung disease in cystic fibrosis (CF). This study describes the cross-sectional and longitudinal impacts of pulmonary inflammation and infection on ventilation homogeneity in infants with CF.Infants diagnosed with CF underwent multiple breath washout (MBW) testing and bronchoalveolar lavage at three time points during the first 2 years of life.Measures were obtained for 108 infants on 156 occasions. Infants with a significant pulmonary infection at the time of MBW showed increases in lung clearance index (LCI) of 0.400 units (95% CI 0.150-0.648; p=0.002). The impact was long lasting, with previous pulmonary infection leading to increased ventilation inhomogeneity over time compared to those who remained free of infection (p<0.05). Infection with Haemophilus influenzae was particularly detrimental to the longitudinal lung function in young children with CF where LCI was increased by 1.069 units for each year of life (95% CI 0.484-1.612; p<0.001).Pulmonary infection during the first year of life is detrimental to later lung function. Therefore, strategies aimed at prevention, surveillance and eradication of pulmonary pathogens are paramount to preserve lung function in infants with CF.

Topics: Breath Tests; Bronchoalveolar Lavage; Bronchoalveolar Lavage Fluid; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Disease Progression; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Infant, Newborn; Interleukin-8; Longitudinal Studies; Male; Pneumonia, Bacterial; Pseudomonas aeruginosa; Pseudomonas Infections; Pulmonary Aspergillosis; Pulmonary Ventilation; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus is an etiological agent of human and animal diseases, and it is able to internalize into non-professional phagocytic cells (i.e. bovine mammary epithelial cells, bMECs), which is an event that is related to chronic and recurrent infections. bMECs contribute to host innate immune responses (IIR) through TLR pathogen recognition, whereby TLR2 is the most relevant for S. aureus. In a previous report, we showed that sodium butyrate (NaB, 0.5mM), which is a short chain fatty acid (SCFA), reduced S. aureus internalization into bMECs by modulating their IIR. However, the molecular mechanism of this process has not been described, which was the aim of this study. The results showed that the TLR2 membrane abundance (MA) and mRNA expression were induced by 0.5mM NaB ∼1.6-fold and ∼1.7-fold, respectively. Additionally, 0.5mM NaB induced p38 phosphorylation, but not JNK1/2 or ERK1/2 phosphorylation in bMECs, which reached the baseline when the bMECs were S. aureus-challenged. Additionally, bMECs that were treated with 0.5mM NaB (24h) showed activation of 8 transcriptional factors (AP-1, E2F-1, FAST-1, MEF-1, EGR, PPAR, ER and CBF), which were partially reverted when the bMECs were S. aureus-challenged. Additionally, 0.5mM NaB (24h) up-regulated mRNA expression of the antimicrobial peptides, TAP (∼4.8-fold), BNBD5 (∼3.2-fold) and BNBD10 (∼2.6-fold). Notably, NaB-treated and S. aureus-challenged bMECs increased the mRNA expression of all of the antimicrobial peptides that were evaluated, and this was evident for LAP and BNBD5. In the NaB-treated bMECs, we did not detect significant expression changes for IL-1β and IL-6 and only TNF-α, IL-10 and IL-8 were induced. Interestingly, the NaB-treated and S. aureus-challenged bMECs maintained the anti-inflammatory response that was induced by this SCFA. In conclusion, our results suggest that 0.5mM NaB activates bMECs via TLR2/p38, which leads to improved antimicrobial defense before/after pathogen invasion, and NaB may exert anti-inflammatory effects during infection.

Topics: Animals; Anti-Inflammatory Agents; Antimicrobial Cationic Peptides; beta-Defensins; Biological Transport; Butyric Acid; Cattle; CD36 Antigens; Cells, Cultured; Enzyme Activation; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Female; Interleukin-10; Interleukin-1beta; Interleukin-6; Interleukin-8; JNK Mitogen-Activated Protein Kinases; Mammary Glands, Animal; Oligopeptides; p38 Mitogen-Activated Protein Kinases; Phosphorylation; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Transcription Factors; Tumor Necrosis Factor-alpha

The knowledge of systemic inflammation and local cytokine expression in porcine endocarditis models is limited, though it could provide valuable information about the pathogenesis and comparability to human endocarditis. Analyses of bacteriology and hematology were performed on blood samples from pigs with non-bacterial thrombotic endocarditis (NBTE, n = 11), Staphylococcus aureus infective endocarditis (IE, n = 2), animals with S. aureus sepsis without endocarditis (n = 2) and controls (n = 2). Furthermore, immunohistochemistry was used to examine the local expression of IL-1β and IL-8. Bacterial blood cultures were continuously positive in IE pigs from inoculation to euthanasia, and negative in all other pigs at all times. The total white blood cell counts and total neutrophil counts were massively elevated in pigs with IE. Local IL-1β and IL-8 expression in IE pigs were moderate to high, and high, respectively. In addition, slight local expression of IL-1β and IL-8 was present in some NBTE pigs. In the IE model, both the systemic inflammatory response and the high local expression of IL-8 were comparable to the human disease. Furthermore, the results indicate IL-1β and IL-8 as important contributors in the endocarditis pathogenesis.

Topics: Animals; Disease Models, Animal; Endocarditis, Bacterial; Endocarditis, Non-Infective; Female; Humans; Immunohistochemistry; Interleukin-1beta; Interleukin-8; Leukocyte Count; Myocardium; Sepsis; Staphylococcal Infections; Sus scrofa; Systemic Inflammatory Response Syndrome

Binding to fibronectin (FN) is a crucial pathogenic factor of Staphylococcus aureus mediated by fibronectin-binding protein A (FnBP-A) and extracellular adherence protein (Eap). Recently, we have shown that binding of soluble CD163 (sCD163) to FN linked to these molecules exhibits anti-microbial effects by enhancing phagocytosis and killing activity of S. aureus-infected monocytes. However, it remained unclear whether sCD163 also influences the monocytic activation status. Using genetically modified staphylococcal strains we now identified FnBP-A, but not Eap, as activator of the inflammatory response of monocytes to infection. FnBP-A-mediated inflammatory activation was masked by sCD163 binding to S. aureus promoting efficient pathogen elimination. Thus, sCD163 protects monocytes from overwhelming activation upon staphylococcal infection by dampening the secretion of pro-inflammatory cytokines TNFα, IL-1β, IL-6 and IL-8 and DAMP molecule MRP8/14. Moreover, sCD163 limited expression of pro-apoptotic transcription factor NR4A1 induced during S. aureus infection and inhibited induction of chemokine CXCL2promoting survival of staphylococci in vivo. sCD163-mediated effects were not due to general immunosuppression since MAP kinase activation and ROS production were unaltered during infection of monocytes with sCD163-bound bacteria. Thus, sCD163 promotes a specific defence of the immune system against FnBP-A-mediated inflammatory activation enabling successful pathogen elimination, tissue recovery and resolution of inflammation.

Topics: Adhesins, Bacterial; Antigens, CD; Antigens, Differentiation, Myelomonocytic; ATP-Binding Cassette Transporters; Bacterial Proteins; Calgranulin B; Cells, Cultured; Chemokine CXCL2; Humans; Inflammation; Interleukin-1beta; Interleukin-6; Interleukin-8; Macrophages; Mitogen-Activated Protein Kinases; Monocytes; Nuclear Receptor Subfamily 4, Group A, Member 1; Phagocytosis; Reactive Oxygen Species; Receptors, Cell Surface; RNA-Binding Proteins; Staphylococcal Infections; Staphylococcus aureus; Tumor Necrosis Factor-alpha

To differentiate between the contribution of mammary epithelial cells (MEC) and infiltrating immune cells to gene expression profiles of mammary tissue during early stage mastitis, we investigated in goats the in vivo transcriptional response of MEC to an experimental intra mammary infection (IMI) with Staphylococcus aureus, using a non-invasive RNA sampling method from milk fat globules (MFG). Microarrays were used to record gene expression patterns during the first 24 hours post-infection (hpi). This approach was combined with laser capture microdissection of MEC from frozen slides of mammary tissue to analyze some relevant genes at 30 hpi. During the early stages post-inoculation, MEC play an important role in the recruitment and activation of inflammatory cells through the IL-8 signalling pathway and initiate a sharp induction of innate immune genes predominantly associated with the pro-inflammatory response. At 30 hpi, MEC express genes encoding different acute phase proteins, including SAA3, SERPINA1 and PTX3 and factors, such as S100A12, that contribute directly to fighting the infection. No significant change in the expression of genes encoding caseins was observed until 24 hpi, thus validating our experimental model to study early stages of infection before the occurrence of tissue damage, since the milk synthesis function is still operative. This is to our knowledge the first report showing in vivo, in goats, how MEC orchestrate the innate immune response to an IMI challenge with S. aureus. Moreover, the non-invasive sampling method of mammary representative RNA from MFG provides a valuable tool to easily follow the dynamics of gene expression in MEC to search for sensitive biomarkers in milk for early detection of mastitis and therefore, to successfully improve the treatment and thus animal welfare.

Topics: Acute-Phase Proteins; alpha 1-Antitrypsin; Animals; C-Reactive Protein; Epithelial Cells; Female; Gene Expression Regulation; Glycolipids; Glycoproteins; Goat Diseases; Goats; Immunity, Innate; Interleukin-8; Lipid Droplets; Mammary Glands, Animal; Mastitis; Oligonucleotide Array Sequence Analysis; Real-Time Polymerase Chain Reaction; RNA; Serum Amyloid A Protein; Serum Amyloid P-Component; Staphylococcal Infections; Staphylococcus aureus

Staphylococcus aureus is a major human pathogen that elaborates several exotoxins. Among these are the bicomponent leukotoxins (BCLs), which include γ-hemolysin, Panton-Valentine leukocidin (PVL), and LukDE. The toxin components are classified as either F or S proteins, which are secreted individually and assemble on cell surfaces to form hetero-oligomeric pores resulting in lysis of PMNs and/or erythrocytes. F and S proteins of γ-hemolysin, PVL and LukDE have ∼ 70% sequence homology within the same class and several heterologous combinations of F and S members from these three bicomponent toxin groups are functional. Recently, an additional BCL pair, LukGH (also called LukAB) that has only 30% homology to γ-hemolysin, PVL and LukDE, has been characterized from S. aureus. Our results showed that LukGH was more cytotoxic to human PMNs than PVL. However, LukGH-induced calcium ion influx in PMNs was markedly attenuated and slower than that induced by PVL and other staphylococcal BCLs. In contrast to other heterologous BCL combinations, LukG in combination with heterologous S components, and LukH in combination with heterologous F components did not induce calcium ion entry or cell lysis in human PMNs or rabbit erythrocytes. Like PVL, LukGH induced IL-8 production by PMNs. While individual components LukG and LukH had no cytolytic or calcium influx activity, they each induced high levels of IL-8 transcription and secretion. IL-8 production induced by LukG or LukH was dependent on NF-κB. Therefore, our results indicate LukGH differs functionally from other staphylococcal BCLs.

Topics: Animals; Apoptosis; Bacterial Proteins; Bacterial Toxins; Blotting, Western; Calcium; Cell Proliferation; Cells, Cultured; Erythrocytes; Exotoxins; Hemolysin Proteins; Hemolysis; Humans; Interleukin-8; Leukocidins; Neutrophils; NF-kappa B; Rabbits; Real-Time Polymerase Chain Reaction; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

Allergy is characterized by eosinophilia and an increased susceptibility to microbial infection. Atopic dermatitis (AD) is typically associated with Staphylococcus aureus (SA) colonization. Some of the mechanisms by which SA and its exotoxins interact with eosinophils remain elusive. CD48, a glycosylphosphatidylinositol-anchored receptor belonging to the CD2 family, participates in mast cells-SA stimulating cross-talk, facilitates the formation of the mast cell/eosinophils effector unit and as expressed by eosinophils, mediates experimental asthma.. To investigate the role of CD48 expressed on human peripheral blood and mouse bone marrow-derived eosinophils (BMEos) in their interaction with heat-killed SA and its three exotoxins, Staphylococcal enterotoxin B (SEB), protein A (PtA) and peptidoglycan (PGN).. Eosinophils were obtained from human peripheral blood and BM of WT and CD48-/- mice. SA was heat killed and eosinophils-SA/exotoxins interactions were analyzed by confocal microscopy, adhesion and degranulation, cell viability, cytokine release and cell signalling. In addition, peritonitis was induced by SEB injection into CD48-/- and WT mice. CD48 expression was studied in AD patients' skin and as expressed on their leucocytes in the peripheral blood.. We provide evidence for the recognition and direct physical interaction between eosinophils and SA/exotoxins. Skin of AD patients showed a striking increase of eosinophil-associated CD48 expression while on peripheral blood leucocytes it was down-regulated. SA/exotoxins enhanced CD48 eosinophil expression, bound to CD48 and caused eosinophil activation and signal transduction. These effects were significantly decreased by blocking CD48 on human eosinophils or in BMEos from CD48-/- mice. We have also explored the role of CD48 in a SEB-induced peritonitis model in CD48-/- mice by evaluating inflammatory peritoneal cells, eosinophil numbers and activation.. These data demonstrate the important role of CD48 in SA/exotoxins-eosinophil activating interactions that can take place during allergic responses and indicate CD48 as a novel therapeutic target for allergy and especially of AD.

Topics: Animals; Antigens, CD; Bacterial Adhesion; Bone Marrow Cells; CD48 Antigen; Cell Degranulation; Dermatitis, Atopic; Enterotoxins; Eosinophils; Gene Expression; Humans; Interleukin-10; Interleukin-8; Leukocytes; Mice; Mice, Knockout; Peritonitis; Protein Binding; Signal Transduction; Skin; Staphylococcal Infections; Staphylococcus aureus

Atopic dermatitis is an inflammatory skin disease that is characterized by a reduced skin barrier function, reduced filaggrin (FLG) expression as well as increased colonization by Staphylococcus aureus.. This study focused on the possible involvement of FLG in epidermal colonization by S. aureus and/or whether it affects the epidermal defence mechanisms, including the expression of antimicrobial peptides (AMPs) and enzymes involved in stratum corneum barrier lipid synthesis. Furthermore, IL-31 has been shown to reduce FLG expression, but its effects on bacterial colonization and on the expression of AMPs and enzymes involved in the barrier lipid synthesis are not known.. We established N/TERT-based epidermal models (NEMs), after FLG knockdown (FLG-KD) and/or cultured with IL-31, that were colonized with S. aureus for 24 h.. Both FLG-KD and IL-31 supplementation resulted in significantly increased epidermal S. aureus colonization, as well as in an up-regulation of S. aureus-induced IL-8 expression. IL-31, but not FLG-KD, prevented S. aureus-induced up-regulation of mRNA expression for the AMPs human β-defensin 2 and -3 and RNAse7, whereas psoriasin expression remained unchanged. Furthermore, the S. aureus colonization induced changes in mRNA expression of ELOVL4 was not affected by FLG-KD, but was blocked by IL-31. Expression of SCD-1 and Gcase mRNA was reduced by IL-31, but not by FLG-KD.. This study shows that NEMs, with FLG-KD and/or cultured in the presence of IL-31, mimic the skin of patients with atopic dermatitis in several aspects, including enhanced bacterial colonization, increased inflammatory and reduced protective responses.

Topics: Adenosine Monophosphate; Animals; Cell Line; Dermatitis, Atopic; Disease Models, Animal; Epidermis; Filaggrin Proteins; Gene Expression Regulation; Gene Expression Regulation, Enzymologic; Gene Knockdown Techniques; Humans; Interleukin-8; Interleukins; Intermediate Filament Proteins; Lipids; Staphylococcal Infections; Staphylococcus aureus

The vaginal epithelium provides a barrier to pathogens and recruits immune defenses through the secretion of cytokines and chemokines. Several studies have shown that mucosal sites are innervated by norepinephrine-containing nerve fibers. Here we report that norepinephrine potentiates the proinflammatory response of human vaginal epithelial cells to products produced by Staphylococcus aureus, a pathogen that causes menstrual toxic shock syndrome. The cells exhibit immunoreactivity for catecholamine synthesis enzymes and the norepinephrine transporter. Moreover, the cells secrete norepinephrine and dopamine at low concentrations. These results indicate that norepinephrine may serve as an autocrine modulator of proinflammatory responses in the vaginal epithelium.

Topics: Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Adrenergic beta-Antagonists; Cell Line, Transformed; Dopamine; Epithelial Cells; Female; Humans; Immunomodulation; Interleukin-6; Interleukin-8; Neuroimmunomodulation; Neuropeptide Y; Norepinephrine; Peptide Fragments; Phentolamine; Propranolol; Shock, Septic; Staphylococcal Infections; Superantigens; Vagina; Vasoactive Intestinal Peptide

The aim of this study was to evaluate the role of trans-resveratrol on Staphylococcus aureus-induced keratitis. Rabbit corneas (intact corneas, abraded corneas and abraded corneas exposed to inactivated S. aureus strains) were placed in an ex vivo culture model. The abraded corneas exposed to S. aureus were divided into two 1-h-treatment sub-groups: corneas treated with trans-resveratrol and corneas treated with vehicle. The tissues were examined by immunohistochemical analyses and quantitative real-time RT-PCR to determine whether resveratrol could reduce TLR2-mediated recognition of S. aureus on epithelial cells and, if so, whether this reduction repressed the expression of inflammatory cytokines. The results demonstrated that resveratrol treatment effectively downregulated cell surface TLR2 on cells stimulated by S. aureus and reduced the expression of interleukin-8 gene. In addition, the corneal culture model tested, which is simple and reproducible, could be an alternative to in vivo animal testing for the development of novel specific therapies.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Cornea; Disease Models, Animal; Gene Expression Profiling; Immunohistochemistry; Interleukin-8; Keratitis; Rabbits; Real-Time Polymerase Chain Reaction; Resveratrol; Staphylococcal Infections; Stilbenes; Toll-Like Receptor 2; Treatment Outcome

Staphylococci are a major contribution for neonatal sepsis, which is the main risk factor for bronchopulmonary dysplasia. This study investigated the expression of pro-inflammatory mediators in endothelial and respiratory cells from newborns exposed to staphylococci.. Human vascular endothelial cells and small airway epithelial cells were incubated with neonatal blood isolates of Staphylococcus epidermidis (n = 14) and Staphylococcus aureus (n = 14). The extracellular release of IL-8, IL-10, sICAM-1, ICAM-1 mRNA and the expression of membrane bound ICAM-1 were assessed by ELISA, RT-PCR and immunofluorescence microscopy.. Staphylococcus epidermidis induced higher levels of IL-8 (mean 38.5 ng/mL) and ICAM-1 mRNA (mean ratio 1.037) in the small airway epithelial cells than S. aureus (IL-8 mean 22.2 ng/mL, p < 0.01 and ICAM-1 mRNA mean ratio 0.715, p < 0.01). In the endothelial cells, ICAM-1 remained more integrated in the cell membranes after exposure to S. epidermidis compared with S. aureus, which induced disintegration and release of soluble ICAM-1 into the supernatants.. Staphylococcus epidermidis induced a higher chemoattractive response than S. aureus. A persistent transmigration of granulocytes into the lung tissue in neonatal S. epidermidis sepsis might contribute to the development of bronchopulmonary dysplasia.

Topics: Biomarkers; Cells, Cultured; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Humans; Infant, Newborn; Intercellular Adhesion Molecule-1; Interleukin-10; Interleukin-8; Real-Time Polymerase Chain Reaction; Respiratory Mucosa; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Staphylococcus epidermidis

We previously showed that Staphylococcus aureus and Pseudomonas aeruginosa stimulate IL-8 expression in human conjunctival epithelial cells through different signal transduction pathways. As in some cell types both the bacteria may induce the release of prostaglandin E2 (PGE2) and PGE2 may affect the expression of IL-8, we aimed at investigating whether in human conjunctival cells infected with S. aureus or P. aeruginosa the activation of IL-8 transcription was mediated by PGE2 and which were the underlying molecular mechanisms. We found that S. aureus, but not P. aeruginosa, triggered IL-8 activation by increasing COX-2 expression and PGE2 levels in a time-dependent manner. Overexpression of nucleotide-binding oligomerization domain-2 (NOD2) resulted to be essential in the enhancement of IL-8 induced by S. aureus. It dramatically activated c-jun NH2-terminal kinase (JNK) pathway which in turn led to COX2 upregulation and ultimately to IL-8 transcription. The full understanding of the S. aureus-induced biochemical processes in human conjunctival epithelium will bring new insight to the knowledge of the molecular mechanisms involved in conjunctiva bacterial infections and develop novel treatment aiming at phlogosis modulation.

Topics: Cell Line; Conjunctiva; Conjunctivitis, Bacterial; Cyclooxygenase 2; Dinoprostone; Epithelial Cells; Humans; Interleukin-8; Nod2 Signaling Adaptor Protein; Pseudomonas aeruginosa; Staphylococcal Infections; Staphylococcus aureus; Transcriptional Activation

Staphylococcus epidermidis (SE) is an important cause of late-onset sepsis in neonates. SE frequently produces a polysaccharide intercellular adhesin (PIA) biofilm, important in the pathogenesis of these infections. Little is known about how the neonatal innate immune system reacts to SE biofilm-associated infections. Our hypothesis was that SE biofilms induce a lower complement activation in neonates as compared with adults.. Cord blood from term infants (n = 15) and blood from adults (n = 6) were studied in an ex vivo whole-blood sepsis model. A PIA biofilm-producing strain (SE1457) and its isogenic mutant (M10), producing a non-PIA biofilm, were used.. Both SE biofilms induced stronger complement activation in adult than in cord blood (P ≤ 0.033). We found lower levels of antibodies toward both PIA (P = 0.002) and the whole bacterium (P = 0.001) in cord vs. adult blood. By contrast, the interleukin-8 (IL-8) and IL-6 secretion were higher in cord than in adult blood (P ≤ 0.002). The PIA biofilm induced stronger complement activation than the non-PIA biofilm.. We conclude that the neonatal complement system exhibits a maturational deficiency. This may reduce the ability of neonates to combat biofilm-associated SE infections.

Topics: Adult; Biofilms; Complement Activation; Enzyme-Linked Immunosorbent Assay; Fetal Blood; Humans; Infant, Newborn; Interleukin-6; Interleukin-8; Polysaccharides, Bacterial; Staphylococcal Infections; Staphylococcus epidermidis; Statistics, Nonparametric

Pheromonicin-SA (Ph-SA) is a newly developed, engineered multidomain peptide that has a bactericidal effect against Staphylococcus aureus. The objective of this study was to characterize innate immune responses by Staph. aureus-stimulated bovine mammary epithelial cells (BMEC) following treatment with Ph-SA. Primary BMEC from one lactating Holstein cow were isolated and exposed to Staph. aureus for 2 h, and then treated with rifampicin or Ph-SA. Total RNA was isolated from BMEC at 0, 2, 6, 12, and 24 h postinfection, and the mRNA expression of selected genes, including toll-like receptor (TLR)2 and TLR4, IL-1β, IL-6, IL-8, tumor necrosis factor α (TNF-α), and lactoferrin, was quantified by real-time PCR. In the rifampicin group, increases in the expression of mRNA for TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection and in the expression of mRNA for TLR2 but not for TLR4 at 12 h postinfection. In the Ph-SA group, increases in the mRNA expression of TLR2, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin were observed at 6 h postinfection, and an increase in TLR4 mRNA expression was observed at 24 h postinfection. At 24 h postinfection, the mRNA expression of TLR4, TNF-α, IL-1β, IL-6, IL-8, and lactoferrin was higher in the Ph-SA group than in the rifampicin group. In conclusion, Ph-SA might promote the expression of mRNA for TLR2, TLR4, the pro-inflammatory cytokines IL-1, IL-6, and TNF-α, the chemotactic factor IL-8, and lactoferrin in Staph. aureus-infected BMEC. Moreover, Ph-SA may be of value as an antibiotic in promoting innate immune responses by Staph. aureus-infected bovine mammary epithelial cells.

Topics: Animals; Anti-Bacterial Agents; Cattle; Cytokines; Epithelium; Female; Interleukin-1beta; Interleukin-6; Interleukin-8; Lactoferrin; Mammary Glands, Animal; Mastitis, Bovine; Recombinant Fusion Proteins; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Mortality of sepsis is still high. Crucial for therapeutic response are the early start of treatment as well as the choice of antibiotics or antibiotic combinations. β-lactam antibiotics with bactericidal mode of action are often recommended in guidelines. But this antibiotic class can trigger the immune system to a maximum by releasing cell wall components or exotoxins. This may lead to a worsening of the patient's clinical situation. In contrast, antibiotics with bacteriostatic action often inhibit bacterial protein synthesis with decrease of production of virulence factors and minimize release of cell wall components. The purpose of this review is to summarise the significance of some bacteriostatic antibiotics and to discuss whether a combination of bactericidal and bacteriostatic agents may improve the course of the illness.

Topics: Animals; Anti-Bacterial Agents; Bacteria; Bacterial Infections; beta-Lactams; Cell Wall; Critical Care; Cross Infection; Drug Therapy, Combination; Exotoxins; Guideline Adherence; Humans; Immunization; Interleukin-8; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Virulence Factors

5-Lipoxygenase (5-LOX) plays key roles in infection and allergic responses. Herein, four 5-LOX-derived lipids comprising 5-hydroxyeicosatetraenoic acid (HETE) attached to phospholipids (PLs), either phosphatidylethanolamine (PE) or phosphatidylcholine (18:0p/5-HETE-PE, 18:1p/5-HETE-PE, 16:0p/5-HETE-PE, and 16:0a/5-HETE-PC), were identified in primary human neutrophils. They formed within 2 minutes in response to serum-opsonized Staphylococcus epidermidis or f-methionine-leucine-phenylalanine, with priming by lipopolysaccharide, granulocyte macrophage colony-stimulating factor, or cytochalasin D. Levels generated were similar to free 5-HETE (0.37 ± 0.14 ng vs 0.55 ± 0.18 ng/10(6) cells, esterified vs free 5-HETE, respectively). They remained cell associated, localizing to nuclear and extranuclear membrane, and were formed by fast esterification of newly synthesized free 5-HETE. Generation also required Ca(2+), phospholipase C, cytosolic and secretory phospholipase A(2), 5-LOX activating protein, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1. 5-HETE-PLs were detected in murine S epidermidis peritonitis, paralleling neutrophil influx, and in effluent from Gram-positive human bacterial peritonitis. Formation of neutrophil extracellular traps was significantly enhanced by 5-LOX inhibition but attenuated by HETE-PE, whereas 5-HETE-PE enhanced superoxide and interleukin-8 generation. Thus, new molecular species of oxidized PL formed by human neutrophils during bacterial infection are identified and characterized.

Topics: Aged; Aged, 80 and over; Animals; Arachidonate 5-Lipoxygenase; Bacterial Infections; Eicosanoids; Female; Gram-Positive Bacterial Infections; Humans; Hydroxyeicosatetraenoic Acids; In Vitro Techniques; Interleukin-8; Male; Mice; Mice, Inbred C57BL; Middle Aged; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Peritonitis; Phospholipids; Plasmalogens; Signal Transduction; Staphylococcal Infections; Staphylococcus epidermidis; Superoxides; Tandem Mass Spectrometry; Tetradecanoylphorbol Acetate

The pore-forming toxin Panton-Valentine leukocidin (PVL) is carried by community-acquired methicillin-resistant Staphylococcus aureus and associated with necrotizing pneumonia together with poor prognosis of infected patients. Although the cell-death-inducing properties of PVL have previously been examined, the pulmonary immune response to PVL is largely unknown. Using an unbiased transcriptional profiling approach, we show that PVL induces only 29 genes in mouse alveolar macrophages, which are associated with TLR signaling. Further studies indicate that PVL directly binds to TLR2 and induces immune responses via NF-κB in a TLR2, CD14, MyD88, IL-1R-associated kinase 1, and TNFR-associated factor 6-dependent manner. PVL-mediated inflammation is independent of pore formation but strongly depends on the LukS subunit and is suppressed in CD14/TLR2(-/-) cells. In vivo PVL or LukS induced a robust inflammatory response in lungs, which was diminished in CD14/TLR2(-/-) mice. These results highlight the proinflammatory properties of PVL and identify CD14/TLR2 as an essential receptor complex for PVL-induced lung inflammation.

Topics: Animals; Bacterial Toxins; Cell Line; Exotoxins; Humans; Immunity, Innate; Inflammation Mediators; Interleukin-8; Leukocidins; Lipopolysaccharide Receptors; Methicillin-Resistant Staphylococcus aureus; Mice; Mice, Inbred C57BL; Mice, Knockout; Pneumonia; Signal Transduction; Staphylococcal Infections; Toll-Like Receptor 2; Up-Regulation

Pulsed-field gel electrophoresis (PFGE) clonal type USA200 is the most widely disseminated Staphylococcus aureus colonizer of the nose and is a major cause of toxic shock syndrome (TSS). Exoproteins derived from these organisms have been suggested to contribute to their colonization and causation of human diseases but have not been well-characterized. Two representative S. aureus USA200 isolates, MNPE (α-toxin positive) and CDC587 (α-toxin mutant), isolated from pulmonary post-influenza TSS and menstrual vaginal TSS, respectively, were evaluated. Biochemical, immunobiological, and cell-based assays, including mass spectrometry, were used to identify key exoproteins derived from the strains that are responsible for proinflammatory and cytotoxic activity on human vaginal epithelial cells. Exoproteins associated with virulence were produced by both strains, and cytolysins (α-toxin and γ-toxin), superantigens, and proteases were identified as the major exoproteins, which caused epithelial cell inflammation and cytotoxicity. Exoprotein fractions from MNPE were more proinflammatory and cytotoxic than those from CDC587 due to high concentrations of α-toxin. CDC587 produced a small amount of α-toxin, despite the presence of a stop codon (TAG) at codon 113. Additional exotoxin identification studies of USA200 strain [S. aureus MN8 (α-toxin mutant)] confirmed that MN8 also produced low levels of α-toxin despite the same stop codon. The differences observed in virulence factor profiles of two USA200 strains provide insight into environmental factors that select for specific virulence factors. Cytolysins, superantigens, and proteases were identified as potential targets, where toxin neutralization may prevent or diminish epithelial damage associated with S. aureus.

Topics: Animals; Chromatography, High Pressure Liquid; Cytotoxins; Electrophoresis, Gel, Pulsed-Field; Enterotoxins; Epithelial Cells; Exotoxins; Female; Humans; Immunoblotting; Immunoglobulin G; Interleukin-8; Lung; Rabbits; Shock, Septic; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Staphylococcal Infections; Staphylococcus aureus; Superantigens; Swine; Vagina; Virulence Factors

Staphylococcus aureus is one of the most prevalent pathogens to cause mastitis in dairy cattle. Intramammary infection of dairy cows with S. aureus is often subclinical, due to the pathogen's ability to evade the innate defense mechanisms, but this can lead to chronic infection. A sub-population of S. aureus, known as small colony variant (SCV), displays atypical phenotypic characteristics, causes persistent infections, and is more resistant to antibiotics than parent strains. Therefore, it was hypothesized that the host immune response will be different for SCV than its parental or typical strains of S. aureus. In this study, the local and systemic immune protein responses to intramammary infection with three strains of S. aureus, including a naturally occurring bovine SCV strain (SCV Heba3231), were characterized. Serum and casein-depleted milk cytokine levels (interleukin-8, interferon-γ, and transforming growth factor-β1), as well as serum haptoglobin concentrations were monitored over time after intramammary infection with each of the three S. aureus strains. Furthermore, comparative proteomics was used to evaluate milk proteome profiles during acute and chronic phases of S. aureus intramammary infection.. Serum IL-8, IFN-γ, and TGF-β1 responses differed in dairy cows challenged with different strains of S. aureus. Changes in overall serum haptoglobin concentrations were observed for each S. aureus challenge group, but there were no significant differences observed between groups. In casein-depleted milk, strain-specific differences in the host IFN-γ response were observed, but inducible IL-8 and TGF-β1 concentrations were not different between groups. Proteomic analysis of the milk following intramammary infection revealed unique host protein expression profiles that were dependent on the infecting strain as well as phase of infection. Notably, the protein, component-3 of the proteose peptone (CPP3), was differentially expressed between the S. aureus treatment groups, implicating it as a potential antimicrobial peptide involved in host defense against S. aureus intramammary infection.. Intramammary infection of dairy cattle with S. aureus causes an up-regulation of serum and milk immune-related proteins, and these responses vary depending on the infecting strain.

Topics: Animals; Area Under Curve; Cattle; Female; Haptoglobins; Interferon-gamma; Interleukin-8; Mastitis, Bovine; Milk; Proteomics; Staphylococcal Infections; Staphylococcus aureus; Transforming Growth Factor beta; Up-Regulation

With the rapid rise in the incidence of multidrug resistant infections, there is substantial interest in host defense peptides as templates for production of new antimicrobial therapeutics. Natural peptides are multifunctional mediators of the innate immune response, with some direct antimicrobial activity and diverse immunomodulatory properties. We have previously developed an innate defense regulator (IDR) 1, with protective activity against bacterial infection mediated entirely through its effects on the immunity of the host, as a novel approach to anti-infective therapy. In this study, an immunomodulatory peptide IDR-1002 was selected from a library of bactenecin derivatives based on its substantially more potent ability to induce chemokines in human PBMCs. The enhanced chemokine induction activity of the peptide in vitro correlated with stronger protective activity in vivo in the Staphylococcus aureus-invasive infection model, with a >5-fold reduction in the protective dose in direct comparison with IDR-1. IDR-1002 also afforded protection against the Gram-negative bacterial pathogen Escherichia coli. Chemokine induction by IDR-1002 was found to be mediated through a Gi-coupled receptor and the PI3K, NF-kappaB, and MAPK signaling pathways. The protective activity of the peptide was associated with in vivo augmentation of chemokine production and recruitment of neutrophils and monocytes to the site of infection. These results highlight the importance of the chemokine induction activity of host defense peptides and demonstrate that the optimization of the ex vivo chemokine-induction properties of peptides is a promising method for the rational development of immunomodulatory IDR peptides with enhanced anti-infective activity.

Topics: Amino Acid Sequence; Animals; Antimicrobial Cationic Peptides; Bacterial Infections; Cell Line; Cells, Cultured; Chemokine CCL2; Chemokine CCL7; Chemokine CXCL1; Chemokines; Female; Humans; Interleukin-8; Leukocytes; Leukocytes, Mononuclear; Macrophages; Mice; Mice, Inbred C57BL; Molecular Sequence Data; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Staphylococcal Infections; Staphylococcus aureus

Staphylococcal superantigen-like proteins (SSLs) constitute a family of exoproteins exhibiting structural similarities to superantigens and enterotoxins but no superantigenic activity. In this article, we present evidence that SSL5 specifically binds to matrix metalloproteinase 9 (MMP-9) and inhibits its enzymatic activity. When human neutrophil cell lysate was applied to recombinant His-tagged SSL5 conjugated to Sepharose, the bound fraction gave a major band of approximately 100 kDa in SDS-polyacrylamide gel electrophoresis. This protein was identified as the proform of MMP-9 (proMMP-9) by peptide mass fingerprinting analysis. The recombinant SSL5-Sepharose also bound to proMMP-9 secreted by interleukin 8 (IL-8)-stimulated neutrophils and HT1080 fibrosarcoma cells. Surface plasmon resonance analysis revealed that recombinant SSL5 bound to proMMP-9 with rather high affinity (dissociation constant [K(D)] = 1.9 nM). Recombinant SSL5 was found to effectively inhibit MMP-9-catalyzed hydrolysis of gelatin and a synthetic fluorogenic peptide in a noncompetitive manner (K(i) = 0.097 nM), as assessed by zymography and the fluorescence quenching method. Finally, the transmigration of neutrophils across Matrigel basement membranes in response to N-formyl-methionyl-leucyl-phenylalanine (FMLP) was suppressed by the presence of recombinant SSL5. We discuss possible roles that SSL5 may play in immune evasion of staphylococci by inhibiting MMP and interfering with leukocyte trafficking.

Topics: Electrophoresis, Polyacrylamide Gel; Exotoxins; Humans; Interleukin-8; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Neutrophils; Peptide Mapping; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus; Superantigens

Although transmembrane C-type lectins (CLs) are known to initiate immune signaling, the participation and mechanism of action of soluble CLs have remained enigmatic. In this study, we found that M-ficolin, a conserved soluble CL of monocyte origin, overcomes its lack of membrane-anchor domain by docking constitutively onto a monocyte transmembrane receptor, G protein-coupled receptor 43 (GPCR43), to form a pathogen sensor-cum-signal transducer. On encountering microbial invaders, the M-ficolin-GPCR43 complex activates the NF-κB cascade to upregulate IL-8 production. We showed that mild acidosis at the local site of infection induces conformational changes in the M-ficolin molecule, which provokes a strong interaction between the C-reactive protein (CRP) and the M-ficolin-GPCR43 complex. The collaboration among CRP-M-ficolin-GPCR43 under acidosis curtails IL-8 production thus preventing immune overactivation. Therefore, we propose that a soluble CL may become membrane-associated through interaction with a transmembrane protein, whereupon infection collaborates with other plasma protein to transduce the infection signal and regulate host defense. Our finding implies a possible mechanism whereby the host might expand its repertoire of immune recognition-cum-regulation tactics by promiscuous protein networking. Furthermore, our identification of the pH-sensitive interfaces of M-ficolin-CRP provides a powerful template for future design of potential immunomodulators.

Topics: Acidosis; Animals; C-Reactive Protein; Cell Line; Chlorocebus aethiops; COS Cells; Escherichia coli Infections; Ficolins; Humans; Immunity, Innate; Interleukin-8; Lectins; Macromolecular Substances; Membrane Proteins; Monocytes; Pseudomonas Infections; Receptor Cross-Talk; Receptors, Cell Surface; Salmonella Infections; Signal Transduction; Staphylococcal Infections; U937 Cells; Up-Regulation

To assess the effects of Pseudomonas aeruginosa and Staphylococcus aureus infection on lower airway inflammation and clinical status in young children with cystic fibrosis (CF).. We studied 111 children age < 6 years who had 2 P aeruginosa-positive oropharyngeal cultures within 12 months. We examined bronchoalveolar lavage fluid (BALF) inflammatory markers (ie, cell count, differential, interleukin [IL]-8, IL-6, neutrophil elastase), CF-related bacterial pathogens, exotoxin A serology, and clinical indicators of disease severity.. Young children with CF with both upper and lower airway P aeruginosa infection had higher neutrophil counts, higher IL-8 and free neutrophil elastase levels, increased likelihood of positive exotoxin A titers, and lower Shwachman scores compared with those with positive upper airway cultures only. S aureus was associated with increased lower airway inflammation, and the presence of both P aeruginosa and S aureus had an additive effect on concentrations of lower airway inflammatory markers. BALF markers of inflammation were increased with the number of different bacterial pathogens detected.. Young children with CF who have upper and lower airway P aeruginosa infection have increased endobronchial inflammation and poorer clinical status compared with those with only upper airway P aeruginosa infection. The independent and additive effects of S aureus on inflammation support the significance of polymicrobial infection in early CF lung disease.

Topics: Biomarkers; Bronchoalveolar Lavage Fluid; Bronchoscopy; Child, Preschool; Cross-Sectional Studies; Cystic Fibrosis; Exotoxins; Female; Humans; Inflammation; Interleukin-8; Leukocyte Count; Leukocyte Elastase; Lung; Male; Neutrophils; Oropharynx; Pseudomonas aeruginosa; Pseudomonas Infections; Severity of Illness Index; Staphylococcal Infections; Staphylococcus aureus

Hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by atopic manifestations and susceptibility to infections with extracellular pathogens, typically Staphylococcus aureus, which preferentially affect the skin and lung. Previous studies reported the defective differentiation of T helper 17 (Th17) cells in HIES patients caused by hypomorphic STAT3 mutations. However, the apparent contradiction between the systemic Th17 deficiency and the skin/lung-restricted susceptibility to staphylococcal infections remains puzzling. We present a possible molecular explanation for this enigmatic contradiction. HIES T cells showed impaired production of Th17 cytokines but normal production of classical proinflammatory cytokines including interleukin 1beta. Normal human keratinocytes and bronchial epithelial cells were deeply dependent on the synergistic action of Th17 cytokines and classical proinflammatory cytokines for their production of antistaphylococcal factors, including neutrophil-recruiting chemokines and antimicrobial peptides. In contrast, other cell types were efficiently stimulated with the classical proinflammatory cytokines alone to produce such factors. Accordingly, keratinocytes and bronchial epithelial cells, unlike other cell types, failed to produce antistaphylococcal factors in response to HIES T cell-derived cytokines. These results appear to explain, at least in part, why HIES patients suffer from recurrent staphylococcal infections confined to the skin and lung in contrast to more systemic infections in neutrophil-deficient patients.

Topics: Animals; Antigens, Bacterial; Antigens, Fungal; beta-Defensins; Chemokines; Cytokines; Epithelial Cells; Humans; Interleukin-8; Job Syndrome; Keratinocytes; Lung Diseases; Receptors, Interleukin-1; Respiratory Mucosa; Staphylococcal Infections; Staphylococcal Skin Infections; STAT3 Transcription Factor; T-Lymphocytes, Helper-Inducer

Staphylococcus aureus is a major cause of community-acquired and nosocomial infections including the life-threatening conditions endocarditis, necrotizing pneumonia, necrotizing fasciitis, and septicemia. Toll-like receptor (TLR)-2, a membrane-bound microbial sensor, detects staphylococcal components, but macrophages lacking TLR2 or both TLR2 and TLR4 remain S. aureus responsive, suggesting that an alternative microbial recognition receptor might be involved. The cytoplasmic sensor nucleotide-binding oligomerization domain containing (NOD) 2/caspase recruitment domain (CARD) 15 detects muramyl dipeptide from bacterial peptidoglycans and mediates cytokine responses to S. aureus in vitro, but the physiological significance of these observations is not well defined. Here we show that NOD2-deficient mice exhibit a delayed but ultimately exacerbated ulcerative response and impaired bacterial clearance after s.c. infection with S. aureus. NOD2-dependent recognition of S. aureus and muramyl dipeptide is facilitated by alpha-toxin (alpha-hemolysin), a pore-forming toxin and virulence factor of the pathogen. The action of NOD2 is dependent on IL-1beta-amplified production of IL-6, which promotes rapid bacterial killing by neutrophils. These results significantly broaden the physiological importance of NOD2 in innate immunity from the recognition of bacteria that primarily enter the cytoplasm to the detection of bacteria that typically reside extracellularly and demonstrate that this microbial sensor contributes to the discrimination between commensal bacteria and bacterial pathogens that elaborate pore-forming toxins.

Topics: Animals; Bacterial Toxins; Hemolysin Proteins; Immunity, Innate; Interleukin-1beta; Interleukin-6; Interleukin-8; Mice; Mice, Inbred C57BL; Neutrophil Activation; Nod2 Signaling Adaptor Protein; Skin; Staphylococcal Infections; Staphylococcus aureus

Interleukin (IL)-22 production triggered by innate immune mechanisms has been identified as key to efficient intestinal anti-bacterial host defence and preservation of homeostasis. We hypothesized that glucocorticoid therapy may impair IL-22 expression, which should promote intestinal epithelial damage with the potential of subsequent bacterial translocation. High-dose corticosteroid therapy in Crohn's disease has been associated with an increased rate of abscess formation and ultimately with a higher risk of developing postoperative infectious complications, including abdominal sepsis. Thus, we sought to investigate effects of the prototypic glucocorticoid dexamethasone on IL-22 production in the context of bacterial infection. Enhanced IL-22 plasma levels were detectable in rat sepsis. Moreover, heat-inactivated Staphylococcus epidermidis, used as a prototypic activator of innate immunity, induced robust production of IL-22 by human peripheral blood mononuclear cells (PBMC). Here, we report for the first time that dexamethasone mediates remarkable suppression of IL-22 as detected in S. epidermidis-activated PBMC and rat sepsis, respectively. The data presented herein suggest that insufficient IL-22 function may contribute to impaired intestinal host defence in the context of corticosteroid therapy.

Topics: Animals; Case-Control Studies; Cells, Cultured; Depression, Chemical; Dexamethasone; Gene Expression; Glucocorticoids; Humans; Interleukin-10; Interleukin-22; Interleukin-8; Interleukins; Male; Models, Animal; Peritonitis; Random Allocation; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staphylococcal Infections; Staphylococcus epidermidis

The impact of Staphylococcus aureus in the pathogenesis of chronic rhinosinusitis is not well understood. Therefore, we investigated primary human nasal epithelial cell cultures for their ability to produce IL-8, growth-related oncogene-alpha, and IL-6 via stimulation with trypsin and culture supernatants of different S. aureus strains and phenotypes. Inhibition of cytokine synthesis was performed using a glucocorticoid, a serine protease inhibitor, and a cysteine protease inhibitor. Finally, signal transduction pathways were analyzed by quantifying phosphorylated forms of MAPKs (PI3K, ERK, and p38) and DNA-binding assays that quantified NF-kappaB and its inhibition using BAY11-7085. In vitro studies showed that the induction of IL-8, growth-related oncogene-alpha, and IL-6 by S. aureus culture supernatants was significantly inhibited by the serine protease inhibitor. In contrast, steroids and the cysteine protease inhibitor had little effect. Activation of NF-kappaB was observed after cell treatment with trypsin and bacterial supernatants, and was inhibited by BAY11-7085 and the serine protease inhibitor. S. aureus serine proteases were identified to modulate chemokine synthesis and activate NF-kappaB in nasal epithelial cells, and may therefore be relevant for the pathophysiology of chronic rhinosinusitis.

Topics: Cells, Cultured; Chemokine CXCL1; Electrophoretic Mobility Shift Assay; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Immunomodulation; Interleukin-6; Interleukin-8; Metalloendopeptidases; Middle Aged; Nasal Mucosa; NF-kappa B; Rhinitis; Serine Proteases; Signal Transduction; Sinusitis; Staphylococcal Infections; Staphylococcus aureus

In this study we tested how a combination of early and late paraclinic markers could predict early onset neonatal sepsis (EONS).. The first 24 hours after the suspicion of EONS, we measured interleukine (IL)-6, IL-8, IL-10, IL-18, tumor necrosis factor-alpha (TNF-alpha), interferon gamma (INF-gamma), procalcitonin (PCT) and C-reactive protein (CRP) at 8-hour intervals on 123 neonates clinically suspected for EONS. The neonates were divided into two groups. The sepsis group: 1A with blood culture verified bacteraemia and 1B strongly suspected sepsis (29 patients). The no sepsis group: 2A treated with antibiotics (37 patients) and 2B not treated with antibiotics (57 patients).. Combined evaluation of each of the early markers with PCT > 25 ng/ml for prediction of EONS at time 0, gave the following sensitivities and specificities: IL-6 > 250 pg/ml: 71% and 88%; IL-8 > 900 pg/ml: 50% and 88%; IL-10 > 40 pg/ml: 43% and 87%; and immature/total (I/T) ratio > 0.35: 59% and 88%. The results of IL-18, TNF-alpha and IFN-gamma did not predict EONS.. IL-6 combined with PCT values is a fair way to evaluate EONS at the time of suspicion of infection. The "old" early marker, I/T ratio, is almost as efficient as IL-6. By combining an early and a late marker it may be possible to reduce the diagnostic "non-conclusive" period of paraclinic values.

Topics: Anti-Bacterial Agents; Bacteremia; Biomarkers; C-Reactive Protein; Calcitonin; Calcitonin Gene-Related Peptide; Cytokines; Escherichia coli Infections; Female; Humans; Infant, Newborn; Inflammation Mediators; Interferon-gamma; Interleukin-10; Interleukin-18; Interleukin-6; Interleukin-8; Leukocyte Count; Male; Neutrophils; Protein Precursors; Retrospective Studies; Sensitivity and Specificity; Sepsis; Staphylococcal Infections; Streptococcal Infections; Streptococcus agalactiae; Tumor Necrosis Factor-alpha

Staphylococcus aureus, but not E. coli pathogens frequently cause subclinical, chronic infections of the mammary gland. We examined here, if inadequate activation of the bovine TLR2 and TLR4 pathogen receptors by ligands derived from S. aureus pathogens might contribute to molecular mechanisms underpinning the escape strategies from mammary immune defence of this pathogen. We show that infections with live E. coli, but not S. aureus pathogens induce strongly IL-8 and TNFalpha gene expression in the udders. Yet, preparations of heat-killed bacteria from both pathogens activate equally well bovine TLR2 and TLR4 receptors to induce NF-kappaB activation, as shown in the HEK293 reconstitution system of TLR-signal transduction. LTA prepared from the S. aureus strain used to infect the cows activates the bovine TLR2 as strongly as the entire, heat-killed pathogen. Both pathogens induce in primary bovine mammary epithelial cells (pbMEC) IL-8 and TNFalpha gene expression, but S. aureus to less than 5% of the degree caused by E. coli. This impaired proinflammatory activation is paralleled by a complete lack of NF-kappaB activation in pbMEC by S. aureus or LTA. In contrast, E. coli and LPS activate strongly NF-kappaB in these cells. A large proportion of this activation is attributable to TLR-mediated signalling, since a dual transdominant negative DN-MyD88-DN-TRIF factor blocks >80% of the pathogen-related NF-kappaB activation in pbMEC. Our results prove that impaired binding of TLR-ligands from the pathogenic S. aureus strain are not the cause for the inadequate mammary immune response elicited by this pathogen. Rather, the pathogen causing subclinical mastitis impairs NF-kappaB activation in MEC thereby severely weakening the immune response in the udder.

Topics: Animals; Animals, Domestic; Cattle; Epithelial Cells; Escherichia coli; Escherichia coli Infections; Immunity; Interleukin-8; Mammary Glands, Animal; NF-kappa B; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Recent reports have shown that Staphylococcus aureus infection increases the expression of cytokines and cell adhesion molecules in endothelial cells and enhances leucocyte migration, thereby resulting in bacterial elimination. In this study, we analysed the production of the chemokine interleukin (IL)-8 in human umbilical vein endothelial cells (HUVEC) infected with several S. aureus strains by using reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. We found that the avirulent strains (00-51 and 00-62) increased IL-8 production but the virulent strains (A17 and A151) decreased it at both the mRNA and protein levels. We considered that the inhibition of IL-8 production depended on certain inhibitory factor(s) secreted by bacteria. This was because S. aureus also abolished IL-8 expression in HUVEC treated with cytochalasin D, and the addition of culture supernatants of strains A17 and A151 decreased IL-8 production in HUVEC. This factor(s) in the bacterial culture supernatant inhibited both basal and tumour necrosis factor (TNF)-alpha-induced IL-8 production. In contrast, no inhibitory effect was observed on monocyte chemotactic protein-1 (MCP-1) production. These results indicate that S. aureus can down-regulate IL-8 release in endothelial cells through the secretion of inhibitory factor(s), and this may result in decreased neutrophil recruitment, thus interfering with the host immune response to bacterial infection.

Topics: Bacteriological Techniques; Cells, Cultured; Chemokine CCL2; Culture Media; Depression, Chemical; Endothelial Cells; Endothelium, Vascular; Humans; Interleukin-8; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Tumor Necrosis Factor-alpha; Virulence

In sepsis, Gram-positive and Gram-negative bacteria provoke similar inflammatory processes. Whereas lipopolysaccharides (LPSs) are acknowledged as the principal immunostimulatory components of Gram-negative bacteria, the effect of the Gram-positive cell wall component lipoteichoic acid (LTA) is less well characterized. In the present study, we investigated the effect of highly purified LTA from Staphylococcus aureus on cytokine generation by isolated human neutrophils.. Isolated human neutrophils from healthy volunteers.. Incubation of neutrophils with purified LTA from S. aureus in the absence or presence of interleukin (IL)-10, anti-CD14, or anti-Toll-like-receptor antibodies.. Measurement of tumor necrosis factor (TNF)-alpha, IL-1beta, and IL-8 by enzyme-linked immunosorbent assay. Analysis of IL-8 mRNA by reverse transcriptase polymerase chain reaction.. The LTA challenge provoked a dramatic release of cytokines, with an early appearance of TNF-alpha and IL-1beta and a delayed liberation of IL-8. The first phase of IL-8 production was induced directly by LTA, whereas the second phase was endogenously mediated by TNF-alpha, as it was largely abrogated by neutralizing anti-TNF-alpha antibodies. In contrast, IL1-beta was not involved in LTA-induced IL-8 generation. Interestingly, the late phase of IL-8 generation could also be attenuated by exogenous IL-10, probably as a consequence of its downregulatory effects on TNF-alpha generation. When investigating the mechanism of LTA-induced cellular activation, activity-neutralizing antibodies demonstrated that CD14 was involved in LTA-mediated neutrophil cytokine generation. Using antibodies that neutralize the activity of Toll-like receptor 2 (TLR2) or 4 (TLR4), we also show that CD14-dependent, LTA-induced neutrophil activation did not proceed via TLR2- or TLR4-mediated pathways. In conclusion, LTA is a potent activator of human neutrophil cytokine generation, with the synthesis of the chemokine IL-8 being largely dependent on TNF-alpha generation in an autocrine fashion. This LTA-induced effect was inhibited by IL-10, dependent on CD14, and independent of TLR 2 or 4.

Topics: Analysis of Variance; Cells, Cultured; Cytokines; Humans; Interleukin-10; Interleukin-8; Lipopolysaccharide Receptors; Lipopolysaccharides; Neutrophils; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Summary The effect of intramammary injection of recombinant bovine interleukin-8 (rbIL-8, 1 mg/10 ml of saline) on quarter milk levels of somatic cell count (SCC), chemiluminescence (CL) activity and counts of total bacteria and Staphylococcus aureus (S. aureus) was investigated, using 10 Holstein cows with an early stage or a late stage of subclinical mastitis naturally infected with S. aureus. In the late-stage group, milk SCC and CL activity had significant rises with maximum levels at 6 h, following maintained high levels thereafter post-cytokine injection. The counts in milk total bacteria and S. aureus were insignificantly decreased, being increased back on day 7 post-cytokine injection. Thus, the cytokine was inefficient for the late-stage subclinical mastitis. However, in the early-stage group milk SCC and CL activity declined to under pre-injection levels on day 7 after marked and significant rises at 6 h and day 1 post-cytokine injection. The milk total bacterial count decreased significantly on days 0.25 and 2. Furthermore, the milk S. aureus count was decreased significantly on days 1, 2, 3 and 7 by the cytokine injection. These results suggest that the rbIL-8 has a potential as a therapeutic agent of the subclinical mastitis of dairy cows, if the cytokine is applied at an initial stage of infection.

Topics: Animals; Cattle; Cell Count; Colony Count, Microbial; Female; Injections; Interleukin-8; Luminescent Measurements; Mammary Glands, Animal; Mastitis, Bovine; Milk; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus; Treatment Outcome

Staphylococcus aureus colonization is common in atopic keratoconjunctivitis, potentially activating epithelial cells via toll-like receptor 2 (TLR-2) and the receptor for platelet-activating factor (PAFR).. To examine human conjunctival epithelial cells for the expression of TLR-2 in vitro and in vivo and to evaluate the role of TLR-2 in S aureus-mediated activation of these cells.. Conjunctival epithelial cells isolated from cadaveric tissues were stimulated with interferon gamma (IFN-gamma) or a commercial S aureus cell wall extract (Staphylococcus aureus-CWE) (with or without anti-TLR-2 blocking antibody or PAFR antagonist) and were analyzed for tumor necrosis factor alpha (TNF-alpha) and interleukin 8 (IL-8) release; surface expression of TLR-2, intercellular adhesion molecule-1, HLA, and CD14; and TLR-2 messenger RNA expression. Ocular surface cells collected via impression cytology were examined for TLR-2 expression via flow cytometry.. Expression of TLR-2 was up-regulated on conjunctival epithelial cells by IFN-gamma and Staphylococcus aureus-CWE. Expression of TLR-2 messenger RNA was increased by IFN-gamma. Staphylococcus aureus-CWE up-regulated intercellular adhesion molecule 1, HLA, and CD14 expression and increased TNF-alpha and IL-8 release in a dose-dependent manner. Anti-TLR-2 significantly inhibited TNF-alpha release, whereas PAFR antagonist significantly inhibited IL-8 release. Toll-like receptor 2 was expressed on conjunctival epithelial cells from 4 of 5 patients with atopic keratoconjunctivitis, 3 of 5 with seasonal allergies, and 0 of 3 without allergies.. Conjunctival epithelial cells express TLR-2 and may play an active role in the chronic ocular inflammatory response to S aureus through pathways that involve TLR-2 and PAFR.

Topics: Adolescent; Adult; Blotting, Northern; Conjunctiva; Conjunctivitis; Epithelial Cells; Female; HLA-DR Antigens; Humans; Intercellular Adhesion Molecule-1; Interferon-gamma; Interleukin-8; Lipopolysaccharide Receptors; Male; Membrane Glycoproteins; Middle Aged; Receptors, Cell Surface; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 2; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Infection of the bovine mammary gland, in addition to causing animal distress, is a major economic burden of the dairy industry. Greater understanding of the initial host response to infection may lead to more accurate selection of resistant animals or to novel prophylactic or therapeutic intervention strategies. The epithelial cell plays a role in the host response by alerting the immune system to the infection and providing a signal as to where the infection is located. To understand this process better, a cDNA microarray approach was used to search for potential signals produced by mammary epithelial cells in response to exposure to Escherichia coli lipopolysaccharide (LPS). Total RNA from separate cultures of epithelial cells from 4 Holstein cows was harvested 6 h after LPS challenge or control conditions. For each cow, RNA from control or LPS-exposed cells was transcribed to cDNA and labeled with Cy3 or Cy5, then pooled and applied to a bovine total leukocyte (BOTL) microarray slide containing 1278 unique transcripts. Dye reversal was used so that RNA from two of the control cultures was labeled with Cy3 while RNA from the other two control cultures was labeled with Cy5. From the resulting microarray data we selected 4 of the 9 genes significantly (P < 0.02) induced (>1.25-fold) in response to LPS exposure for more detailed analysis. The array signal intensity for 3 of these genes, RANTES/CCL5, IL-6 and T-PA, was relatively low, but quantitative real-time RT-PCR (Q-RT-PCR) analysis revealed that they were induced 208-fold, 10-fold and 3-fold, respectively. The gene that showed the greatest fold induction by microarray analysis (2.5-fold) was CXCL5. This gene had a relatively strong signal intensity on the array and was easily detected by northern blot analysis, which indicated a 10-fold induction. This cell culture model system provides evidence for an important role of the mammary epithelial cell in initiating the innate response to infection.

Topics: Animals; Cattle; Cell Culture Techniques; Chemokine CXCL5; Chemokines, CXC; Epithelial Cells; Escherichia coli Infections; Female; Gene Expression Profiling; Gene Expression Regulation; Interleukin-8; Leukocytes; Lipopolysaccharides; Mammary Glands, Animal; Mastitis, Bovine; Oligonucleotide Array Sequence Analysis; Reverse Transcriptase Polymerase Chain Reaction; Staphylococcal Infections

Leukotriene B(4) (LTB(4)) and interleukin-8 (IL-8) are inflammatory mediators involved in the neutrophil response to pulmonary bacterial colonization in cystic fibrosis (CF). The aim of this study was to investigate whether the LTB(4) and IL-8 levels in exhaled breath condensate (EBC) could be related to the type of bacterial colonization in CF patients. The pH level in EBC was analyzed as an estimate of airway acidification. Forty children were evaluated: 10 CF patients with P. aeruginosa, 10 CF patients with S. aureus, 10 not colonized CF patients, and 10 healthy children. LTB(4) and IL-8 in EBC were analyzed by specific enzyme immunoassay kits (EIA). The pH of EBC was measured with a pH-meter after deareation by bubbling with argon. Exhaled LTB(4) was higher in CF children with P. aeruginosa compared to those with S. aureus (P < 0.01), not colonized (P < 0.001), and healthy children (P < 0.01). Exhaled IL-8 was elevated in CF patients colonized by P. aeruginosa compared with other subgroups (vs. not colonized, P < 0.05; vs. healthy children, P < 0.001). IL-8 levels were higher in CF children with S. aureus than in healthy children (P < 0.05). There was an increase in IL-8 levels in not colonized CF patients compared with healthy children (P < 0.05). EBC pH was higher in healthy children compared to CF patients not colonized (P < 0.05). Our data suggest that EBC is suitable for evaluating neutrophil inflammatory mediators (LTB(4), IL-8, and pH) involved in the response to pulmonary bacterial colonization in CF children.

Topics: Biomarkers; Breath Tests; Child; Cystic Fibrosis; Humans; Hydrogen-Ion Concentration; Immunoenzyme Techniques; Interleukin-8; Leukotriene B4; Neutrophils; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Function Tests; Sputum; Staphylococcal Infections; Staphylococcus aureus

Mammary gland epithelial cells are likely to be important effectors in defending against mastitis, yet little is known about their response mechanisms. Here, we describe a cryopreserved bovine mammary epithelial cell model to study the infection response. Primary cell cultures from four Holstein cows were prepared, and frozen after two passages. The cell cultures from each cow were then thawed and maintained separately, yet simultaneously, and exposed to treatments that included infection with Staphylococcus aureus or exposure to LPS from Escherichia coli. A clear inflammatory response was shown by a significant (P < 0.05), dose dependent, increase of lactoferrin and IL-8 secretion within 24h in response to S. aureus or LPS. Marked increases (P < 0.05) in lactoferrin, TNF-alpha and serum amyloid A (SAA) mRNA expression were also observed. The results indicate the usefulness of our model to study infection responses of mammary epithelial cells, where all cells are simultaneously exposed to the same infection pressure. These responses can be studied over time, and most importantly, biological replication is provided by the four different genotypes being investigated individually. Finally, the results indicate that mammary epithelial cells play an important role in inflammatory response, through the production of pro-inflammatory cytokines, an acute phase protein, and lactoferrin.

Topics: Animals; Apolipoproteins; Blotting, Northern; Cattle; Cell Culture Techniques; Cryopreservation; Epithelial Cells; Female; Interleukin-8; Lactoferrin; Lipopolysaccharides; Mastitis, Bovine; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Serum Amyloid A Protein; Staphylococcal Infections; Staphylococcus aureus

Mediastinitis after open heart operation is an infrequent, but life-threatening complication with a reported incidence rate between 1% and 4%. Hospital mortality is estimated at 10% to 35%. The aim of the present work was to study the systemic inflammatory reaction as judged by complement activation and cytokine and chemokines release in patients with mediastinitis after open heart operation.. Seven patients with clinical signs of mediastinitis were included. Three patients had undergone coronary artery bypass grafting, whereas 4 patients had combined coronary artery bypass grafting, valve replacement, or valvuloplasty. Blood samples were drawn before induction of anesthesia and at the time of reoperation, and thereafter daily during the hospital stay. Controls comprised similar patients with an uneventful postoperative course.. The terminal SC5b-9 complement complex concentration in the mediastinitis patients was substantially higher compared with the controls (p < 0.001), and the terminal SC5b-9 complement complex values showed no overlap between the two groups. Interleukin-8, stromal cell-derived factor-1alpha and IL-6 concentrations were also significantly higher in the mediastinitis group than in the control group (p < 0.001), but with considerable overlap between the groups. Interleukin-1beta, interleukin-10, and monocyte chemoattractant protein-1 concentrations were slightly higher in the mediastinitis group, and no differences were seen for the tumor necrosis factor-alpha.. During mediastinitis, the complement is activated and the cytokines and chemokines, interleukin-6, interleukin-8, and stromal cell-derived factor-1alpha are released. These proteins may be involved in the pathogenesis of this complication. Terminal SC5b-9 complement complex may be an indicator to discriminate mediastinitis patients from those with uneventful course.

Topics: Aged; Anti-Bacterial Agents; Chemokine CXCL12; Chemokines; Chemokines, CXC; Complement Activation; Complement Membrane Attack Complex; Complement System Proteins; Coronary Artery Bypass; Cytokines; Drug Therapy, Combination; Female; Glycoproteins; Heart Valve Prosthesis Implantation; Heart Valves; Humans; Interleukin-6; Interleukin-8; Male; Mediastinitis; Middle Aged; Postoperative Complications; Prognosis; Reoperation; Staphylococcal Infections

Combination therapy that includes fusidic acid, an antimicrobial agent highly active against staphylococci, has been recommended in the treatment of patients with Staphylococcus aureus meningitis. The aim of this study was to evaluate the pharmacokinetic, CSF bactericidal and anti-inflammatory properties of fusidic acid.. The pharmacokinetics, treatment efficacy and parameters of the meningeal inflammatory response were studied in rabbits, using an experimental meningitis model against S. aureus (MICs of fusidic acid and methicillin were 0.125 and 1 mg/L, respectively).. Fusidic acid entered the CSF, with peak values within 0.5-1 h of the intravenous bolus injection/infusion and with a percentage penetration (AUCCSF/AUCserum) into uninfected and purulent CSF of 1.9% +/- 0.7 and 4.5% +/- 0.7, respectively. Rabbits treated with antibiotics [fusidic acid 80 mg/kg/6 h (n = 6), methicillin 80 mg/kg/3 h (n = 7) and the two combined (n = 6)] had significantly higher bacterial kill rates than untreated controls (n = 6, P < 0.05). Combination therapy was less effective, with significantly less killing after 6 h of treatment than methicillin alone (P < 0.05). CSF white blood cells and CSF levels of interleukin-8 (IL-8), glucose, lactate and protein were altered during staphylococcal meningitis, but with no significant difference between antibiotic-treated and untreated rabbits.. Antagonism between methicillin and fusidic acid was observed in staphylococcal meningitis.

Topics: Animals; Anti-Bacterial Agents; Drug Therapy, Combination; Fusidic Acid; Glucose; Interleukin-8; Leukocyte Count; Meningitis, Bacterial; Methicillin; Microbial Sensitivity Tests; Penicillins; Rabbits; Staphylococcal Infections; Staphylococcus aureus

The responses of five lactating East Friesian milk ewes to experimental mammary infection with Staphylococcus epidermidis and of five control ewes were examined over a period of 10 weeks. Infection caused an influx of neutrophils into milk, the numbers of which started to rise 4 h post infection and peaked 24 h after infection. The initial response was accompanied by mild fever and mild leucopaenia in blood (8 h after infection). No other signs of systemic infection were observed. Milk appeared normal at all times, although the milk yield of infected ewes tended to decline. Staphylococci were absent in milk from four ewes at 2 d and at 3 d after infection, but re-emerged intermittently in four of five ewes at subsequent samplings. Cytokines in milk were measured by ELISA. IL-8 was elevated in infected glands at 2 h and peaked at 8 h. In the four ewes intermittently shedding bacteria, IL-8 remained elevated until the final sampling at 10 weeks. IL-1beta was transiently elevated at 1 d and 2 d and showed a pronounced peak in one sheep. Milk samples from this ewe were bacteriologically negative, somatic cell count (SCC) was within the normal range and the concentrations of IL-1beta, as well as IL-8, were similiar to the control group (n=5) from 1 week after infection until the final sampling. Histological examination revealed leucocytic infiltrates in the four glands remaining infected at the end of the experiment, and a high level of CD5+ lymphocytes in three ewes. The results suggest that the relationship between the initial neutrophil influx and the proinflammatory cytokines may be responsible for determining the course of infection. Subclinical mastitis due to coagulase-negative staphylococci leads to minor changes in milk yield and milk constituents.

Topics: Animals; CD5 Antigens; Cell Count; Female; Immunohistochemistry; Interleukin-1; Interleukin-8; Lactation; Lymphocyte Count; Lymphocytes; Mastitis; Milk; Sheep; Sheep Diseases; Staphylococcal Infections; Staphylococcus epidermidis

Cultured primary human keratinocytes were screened for their expression of various members of the toll-like receptor (TLR) family. Keratinocytes were found to constitutively express TLR1, TLR2, TLR3, TLR5, and TLR9 but not TLR4, TLR6, TLR7, TLR8, or TLR10 as shown by polymerase chain reaction analysis. The expression of the crucial receptor for signaling of staphylococcal compounds TLR2 was also confirmed by immunohistochemistry, in contrast to TLR4, which showed a negative staining pattern. Next, we analyzed the activation of the proinflammatory nuclear transcription factor kappaB by Staphylococcus aureus strain 8325-4. Using nuclear extract gel shifts, RelA staining, and luciferase reporter transfection plasmids we found a clear induction of nuclear factor kappaB translocation by the bacteria. This translocation induced the transcription of nuclear factor kappaB controlled genes such as inducible nitric oxide synthetase, COX2, and interleukin-8. Transcription of these genes was followed by production of increased amounts of interleukin-8 protein and NO. Inhibition experiments using monoclonal antibodies and the specific platelet activating factor receptor inhibitor CV3988 showed that nuclear factor kappaB activation by S. aureus was TLR2 but not TLR4 or platelet activating factor receptor dependent. In line, the purified staphylococcal cell wall components lipoteichoic acid and peptidoglycan, known to signal through TLR2, also showed nuclear factor kappaB translocation in human keratinocytes, indicating a crucial role of the staphylococcal cell wall in the innate immune stimulation of human keratinocytes. These results help to explain the complex activation of human keratinocytes by S. aureus and its cell wall components in various inflammatory disorders of the skin.

Topics: Cells, Cultured; Gene Expression; Humans; Immunohistochemistry; Interleukin-8; Keratinocytes; Membrane Glycoproteins; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Phospholipid Ethers; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Protein Biosynthesis; Receptors, Cell Surface; Receptors, G-Protein-Coupled; RNA, Messenger; Signal Transduction; Staphylococcal Infections; Staphylococcus aureus; Toll-Like Receptor 1; Toll-Like Receptor 10; Toll-Like Receptor 2; Toll-Like Receptor 3; Toll-Like Receptor 4; Toll-Like Receptor 5; Toll-Like Receptor 7; Toll-Like Receptor 8; Toll-Like Receptor 9; Toll-Like Receptors; Transcriptional Activation; Virulence

Bone infection or osteomyelitis is characterized by uncontrolled inflammation and destructive bone loss although little is known about immunopathogenesis of infection. We investigated control of chemokine secretion from osteoblasts infected with either Mycobacterium tuberculosis, which normally elicits a granulomatous host response, or Staphylococcus aureus, which drives a host response dominated by neutrophil influx. We show that M. tuberculosis infection of cultured and primary osteoblasts induces extensive secretion of the chemokines interleukin (IL)-8, inducible protein (IP) 10, RANTES, and monocyte chemoattractant protein (MCP) 1 within 72 h (1630 +/- 280 pg/ml per 4 x 10(5) cells, 74,130 +/- 8480 pg/ml per 4 x 10(5) cells, 18,330 +/- 3040 pg/ml per 4 x 10(5) cells, and 138,670 +/- 13,340 pg/ml per 4 x 10(5) cells, respectively, for MG-63 osteoblasts). S. aureus infection also results in secretion of these chemokines but secretion is delayed and of lesser magnitude (210 +/- 10 pg/ml per 4 x 10(5) cells, 11,570 +/- 1240 pg/ml per 4 x 10(5) cells, 930 +/- 34 pg/ml per 4 x 10(5) cells, and 13,770 +/- 720 pg/ml per 4 x 10(5) cells for IL-8, IP-10, RANTES, and MCP-1, respectively). The minimal up-regulation of secretion of the neutrophil attractant IL-8 in staphylococcal infection is both striking and unexpected. In both infections, chemokine secretion was dependent on the presence of live organisms. Differences in kinetics and magnitude of chemokine secretion are associated with distinct patterns of mRNA expression, as assessed by ribonuclease protection assay (RPA) and reverse-transcription polymerase chain reaction (RT-PCR). In addition, nuclear localization of the transcription factor activator protein (AP) 1 in M. tuberculosis-infected osteoblasts also is distinct as compared with S. aureus-infected cells. In summary, this study shows that osteoblasts have an important pathogen-specific role in control of chemokine gene expression and secretion during the human immune response to osteomyelitis.

Topics: Base Sequence; Cell Line; Chemokine CCL2; Chemokine CCL5; Chemokine CXCL10; Chemokines; Chemokines, CXC; Gene Expression Regulation; Humans; Interleukin-8; Osteoblasts; Osteomyelitis; RNA, Messenger; Staphylococcal Infections; Transcription Factor AP-1; Tuberculosis, Osteoarticular

Staphylococcus aureus strains lacking agr- and sarA-dependent gene products or specific MSCRAMM (microbial surface components recognizing adhesive matrix molecules) adhesins were compared for the ability to activate inflammatory responses in the lung. The mutants were evaluated for virulence in a mouse model of pneumonia and by quantifying their ability to stimulate interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression in respiratory epithelial cells. In a neonatal mouse, only strains with intact agr and sarA loci were consistently associated with invasive, fatal pulmonary infection (P < 0.001) and sarA was specifically required to cause bacteremia (P < 0.001). The agr and/or sarA mutants were, nonetheless, fully capable of producing pneumonia and were as proficient as the wild-type strain in stimulating epithelial IL-8 expression, a polymorphonuclear leukocyte chemokine, in airway cells. In contrast, agr and especially sarA mutants induced less epithelial GM-CSF expression, and MSCRAMM mutants lacking fibronectin binding proteins or clumping factor A, a ligand for fibrinogen, were unable to stimulate epithelial GM-CSF production. The ability to induce IL-8 expression was independent of the adherence properties of intact bacteria, indicating that shed and/or secreted bacterial components activate epithelial responses. While conserved staphylococcal components such as peptidoglycan are sufficient to evoke inflammation and cause pneumonia, the agr and sarA loci of S. aureus are critical for the coordination of invasive infection of the lungs.

Topics: Animals; Bacterial Adhesion; Bacterial Proteins; Cell Line, Transformed; Coagulase; Gene Expression; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Mutation; Pneumonia, Staphylococcal; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Trans-Activators; Transcription Factors; Virulence

Nasal polyps frequently appear in patients with cystic fibrosis (CF). The aims of this study were to focus on what problems (symptoms, endoscopic findings, and laboratory correlates) nasal polyps cause the CF patient, and how these correlate to the total health situation of this patient group.. The clinical histories, endoscopic investigations of the nasal cavity, and analyses of nasal lavage fluid of 44 patients with CF complicated with nasal polyposis have been compared with those of 67 CF control subjects. The patients were examined at annual control examinations (with pulmonary tests, working capacity, liver tests, and bacterial and blood tests) from 1995 to 1996 at Stockholm Cystic Fibrosis Center, Huddinge University Hospital. All patients were > 2 years of age. The endoscopic findings were related to the actual pulmonary function, inflammatory blood parameters, colonizing pathogens, antibodies (Staphylococcus aureus and Pseudomonas aeruginosa), and genotype.. The patients with nasal polyps differed with respect to chronic colonization of P aeruginosa in sputum samples and had a higher occurrence of serum antibodies against the same species. The two groups did not differ in pulmonary functions, inflammatory parameters, or genotype. The polyps found were mainly small (within the meatus media) and gave no significant increase in ongoing clinical symptoms such as rhinorrhea, nasal obstruction, or hyposmia. Neither was any significantly marked finding concerning the nose (mucosal swellings, secretion, etc.) made in the polyp patients. The patients with CF scored slightly lower in a smell identification test in comparison with the healthy control group. The nasal lavage fluid was analyzed (in 93 of the 111 patients) for the occurrence of P aeruginosa (by polymerase-chain reaction [PCR]), interleukin [IL]-5, IL-8, and lysozyme. The lysozyme and IL-8 content was equal in the two CF groups but increased in comparison with the healthy control group. P aeruginosa was not detected with PCR in any nasal lavage fluid. No measurable levels of IL-5 in the nasal lavage were found.. There was a higher frequency of chronic colonization of P aeruginosa in the lower respiratory tract in patients with nasal polyps. Otherwise, nonsevere nasal polyposis was not an indicator of lower respiratory tract morbidity in CF patients.

Topics: Adolescent; Adult; Antibodies, Bacterial; Child; Child, Preschool; Cystic Fibrosis; Endoscopy; Female; Humans; Interleukin-5; Interleukin-8; Male; Muramidase; Nasal Lavage Fluid; Nasal Polyps; Pseudomonas aeruginosa; Pseudomonas Infections; Respiratory Tract Infections; Risk Factors; Staphylococcal Infections

The prompt recruitment of neutrophils to the site of infection is essential for the defense of the bovine mammary gland against invading pathogens and is determinant for the outcome of the infection. Escherichia coli is known to induce clinical mastitis, characterized by an intense neutrophil recruitment leading to the eradication of the bacteria, whereas Staphylococcus aureus induces subclinical mastitis accompanied by a moderate neutrophil recruitment and the establishment of chronic mastitis. To elicit the neutrophil recruitment into the udder, inflammatory mediators must be produced after recognition of the invading pathogen. To our knowledge, those mediators have never been studied during S. aureus mastitis, although understanding of the neutrophil recruitment mechanisms could allow a better understanding of the differences in the pathogeneses elicited by E. coli and S. aureus. Therefore, we studied, at several time points, the accumulation of neutrophils and the presence of the chemoattractant complement fragment C5a and of the cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha, and IL-8 in milk after inoculation of E. coli or S. aureus in lactating bovine udders. The low levels of C5a and the absence of cytokines in milk from S. aureus-infected cows, compared to the high levels found in milk from E. coli-infected animals, mirror the differences in the severities of the two inflammatory reactions. The cytokine deficit in milk after S. aureus inoculation in the lactating bovine mammary gland could contribute to the establishment of chronic mastitis. This result could help in the design of preventive or curative strategies against chronic mastitis.

Topics: Animals; Cattle; Complement C5a; Escherichia coli Infections; Female; Haptoglobins; Interleukin-1; Interleukin-8; Mastitis; Serum Albumin, Bovine; Staphylococcal Infections; Staphylococcus aureus; Tumor Necrosis Factor-alpha

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.

Topics: Animals; Biopsy; DNA; Enzyme-Linked Immunosorbent Assay; Escherichia coli; Escherichia coli Infections; Female; Flow Cytometry; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-1; Interleukin-8; Leukocyte Count; Mammary Glands, Animal; Mastitis; Neutrophils; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sheep; Sheep Diseases; Staphylococcal Infections; Staphylococcus aureus

The aim of this prospective study was to evaluate if patients with endocarditis display a more extensive endothelial activation than those with bacteraemia but without endocarditis. Sixty-five patients with blood culture-verified Staphylococcus aureus bacteraemia were included and serum samples collected on admission were analysed by enzyme immunoassays. Elevated serum concentrations of adhesion molecules were found in most of the patients with S. aureus bacteraemia. Patients with endocarditis (n = 15) showed significantly higher serum E-selectin (median 156 ng/ml) and VCAM-1 (median 1745 ng/ml) concentrations compared with those with S. aureus bacteraemia but without endocarditis (80 ng/ml and 1172 ng/ml, respectively; P = 0.01 and P = 0.003). No significant difference was found between the groups concerning ICAM-1 (median 451 ng/ml versus 522 ng/ml). In addition, serum tumour necrosis factor-alpha (TNF-alpha) concentrations were significantly correlated (P < 0.002) to serum levels of E-selectin, ICAM-1 and VCAM-1.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Bacteremia; Child; E-Selectin; Endocarditis; Female; Granulocyte Colony-Stimulating Factor; Humans; Intercellular Adhesion Molecule-1; Interleukin-1; Interleukin-6; Interleukin-8; Male; Middle Aged; Prospective Studies; Staphylococcal Infections; Staphylococcus aureus; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Radiolabelled interleukin-8 (IL-8) is a promising agent for the imaging of infection and inflammation. Several experiments were performed to explore further the imaging potential of radiolabelled IL-8. IL-8 was radioiodinated via the Bolton-Hunter method. Rabbits with focal infection (Escherichia coli, Staphylococcus aureus) or sterile inflammation (zymosan) were injected intravenously with 18.5 MBq (0.5 mCi) of 123I-IL-8. In separate studies, rabbits were injected intravenously with 111In-granulocytes with or without 125I-IL-8. Gamma camera images were obtained at 5 min, 1, 4 and 8 h post-injection (p.i.). Biodistribution was determined at 8 h p.i. In all models, the biodistribution of 123I-IL-8 was characterized by rapid blood clearance and high uptake in infection and sterile inflammation. All foci could be clearly visualized within 4 h p.i. Ex vivo abscess-to-contralateral muscle ratios increased to 114.7+/-23.0 (E. coli), 52.3+/-24.5 (S. aureus) and 49.8+/-8.3 (zymosan) at 8 h p.i. In the circulation, most 123I-IL-8 was bound to erythrocytes. The abscess uptake of 125I-IL-8 reached high levels despite reduced migration of granulocytes towards the site of infection due to the anti-inflammatory activity of intravenously injected IL-8. IL-8 could be injected without induction of neutropenia at a dosage of 2 ng kg(-1). In conclusion, the characteristics of radiolabelled IL-8 for imaging of infection and sterile inflammation are highly encouraging and warrant further optimization for clinical application.

Topics: Animals; Antigens, CD; Escherichia coli Infections; Female; Humans; Inflammation; Interleukin-8; Iodine Radioisotopes; Neutrophils; Rabbits; Radionuclide Imaging; Radiopharmaceuticals; Receptors, Interleukin; Receptors, Interleukin-8A; Recombinant Proteins; Staphylococcal Infections; Staphylococcus aureus; Tissue Distribution; Zymosan

Human immunodeficiency virus (HIV)-infected patients are at increased risk of contracting bacterial infections, mainly pneumonia. Despite this, little is known about immunopathogenic mechanisms in HIV-related bacterial pneumonia. This paper investigates the presence of the neutrophil chemotactic mediators, interleukin-8 (IL_8) and leukotriene B4 (LTB4), in bronchoalveolar lavage (BAL) fluid from 27 HIV-infected patients with bacterial pneumonia. Significantly elevated levels of IL-8 were found in BAL fluid of patients with bacterial pneumonia [529 pg ml-1 (296-1161 pg ml-1)] compared to matched patients with Pneumocystis carinii pneumonia (PCP) [59 pg ml-1 (42-254 pg ml-1)] and healthy controls [58 pg ml-1 (37-82 pg ml-1)]. Levels of LTB4 were not elevated during bacterial pneumonia when compared to PCP patients and healthy controls. Furthermore, a positive correlation was found between IL-8 levels in BAL fluid and relative BAL neutrophilia (r = 0.60, P = 0.001) in bacterial pneumonia. In conclusion, elevated IL-8 levels in BAL fluid were found in patients suffering from bacterial pneumonia, which may account for the influx of neutrophils to the lung, whereas LTB4 appears not to be an important chemotactic factor in this setting.

Topics: Bronchoalveolar Lavage Fluid; CD4 Lymphocyte Count; Chemotaxis, Leukocyte; Haemophilus Infections; HIV Infections; Humans; Interleukin-8; Leukotriene B4; Neutrophils; Pneumococcal Infections; Pneumonia, Bacterial; Pneumonia, Pneumocystis; Staphylococcal Infections

This study was designed to assess monoclonal antibodies (MAbs) directed against tumor necrosis factor-alpha (TNF-alpha) (anti-TNF) or interleukin-8 (anti-IL-8) as radioactive agents for the detection of Staphylococcus aureus-or Klebsiella pneumoniae-infected thighs in mice. At 5 min (acute infection) or 20 h (established) post-infection, 20 micrograms of the 99mTc-labeled MAbs were injected. At various time intervals, the accumulation of the radiotracer in the infected thighs was assessed and expressed as a target-to-nontarget (T/NT) ratio. The binding of 99mTc-labeled MAbs to circulating mononuclear cells and granulocytes was quantitated 20 h after injection. The pharmacokinetics of the MAbs, in relation to the control agents 99mTc-labeled polyclonal human immunoglobulin (IgG) and a 99mTc-labeled nonspecific IgG1 MAb, were also studied. In acute infections, 99mTc-anti-TNF accumulated to a higher extent (p < 0.05) in S. aureus-infected thighs in mice until 4 h after the injection than 99mTc-IgG and was higher at 0.25 h in K. pneumoniae-infected mice (p < 0.03) compared with 99mTc-IgG. In established S. aureus and K. pneumoniae infections, 99mTc-anti-IL-8 detected the infection more intensely than 99mTc-IgG until 1 h after injection. In both S. aureus and K. pneumoniae infections, localization of sites of infection correlates (p < 0.05) with increased binding of the 99mTc-labeled MAbs to granulocytes and mononuclear cells in both acute and established infections. It was concluded that 99mTc-labeled MAbs, directed against TNF-alpha and IL-8, accumulate in bacterial infections in mice to a higher extent than does 99mTc-IgG after infection and is related to the binding of the antibodies to blood leukocytes. With these 99mTc-labeled MAbs, information might be gained about the development of an infection.

Topics: Animals; Antibodies, Monoclonal; Humans; Immunoglobulin G; Interleukin-8; Isotope Labeling; Klebsiella Infections; Klebsiella pneumoniae; Leukocytes; Male; Mice; Muscle, Skeletal; Radionuclide Imaging; Staphylococcal Infections; Technetium; Tissue Distribution; Tumor Necrosis Factor-alpha

As collections of lower respiratory tract specimens from young children with cystic fibrosis (CF) are difficult, we determined whether oropharyngeal cultures predicted lower airway pathogens. During 1992-1994, 75 of 90 (83%) infants with CF diagnosed by neonatal screening had 150 simultaneous bronchoalveolar lavage (BAL) and oropharyngeal specimens collected for quantitative bacterial culture at a mean age of 17 months (range, 1-52). Ten children undergoing bronchoscopy for stridor served as controls. Total and differential cell counts and interleukin-8 concentrations were measured in BAL fluid. A subset of bacterial pathogens were typed by pulsed field gel electrophoresis. A non-linear relationship with inflammatory markers supported a diagnosis of lower airway infection when > or = 10(5) colony-forming units/ml were detected. This criterion was met in 47 (31%) BAL cultures from 37 (49%) children. Staphylococcus aureus (19%), Pseudomonas aeruginosa (11%), and Hemophilus influenzae (8%) were the major lower airway pathogens. In oropharyngeal cultures, S. aureus (47%), Escherichia coli (23%), H. influenzae (15%), and P. aeruginosa (13%) predominated. The sensitivity, specificity, and positive and negative predictive values of oropharyngeal cultures for pathogens causing lower respiratory infections were 82%, 83%, 41%, and 97%, respectively. When there was agreement between paired oropharyngeal and BAL cultures, genetic fingerprinting showed some strains of the same organism were unrelated. We conclude that oropharyngeal cultures do not reliably predict the presence of bacterial pathogens in the lower airways of young CF children.

Topics: Bacteriological Techniques; Bronchoalveolar Lavage Fluid; Child, Preschool; Cystic Fibrosis; Escherichia coli Infections; Female; Haemophilus Infections; Haemophilus influenzae; Humans; Infant; Interleukin-8; Male; Oropharynx; Pneumonia, Bacterial; Predictive Value of Tests; Pseudomonas Infections; Respiratory Tract Infections; Staphylococcal Infections

Our objective was to determine the incidence of peritonitis episodes with an impaired initial cell reaction (IICR:neutrophil number < 100 x 10(6)/L) over a period of ten years, and to find possible explanations for this unusual presentation of peritonitis. A retrospective review of the files of continuous ambulatory peritoneal dialysis (CAPD) patients included in the CAPD program 1984 and 1993 was done. Analysis of cytokine and prostanoid patterns during four peritonitis episodes with an IICR was compared to 12 episodes with a normal initial cell reaction (NICR). Dialysate cell numbers and immunoeffector characteristics of peritoneal cells were compared in 7 IICR patients in a stable situation and a control group of 70 stable CAPD patients. The setting was a CAPD unit in the Academic Medical Center in Amsterdam. Thirty-five CAPD patients who had one or more peritonitis episodes with an IICR and a control group of 249 CAPD patients were included in the study. The incidence of peritonitis with an IICR was 6%. These episodes occurred more than once in 51% of the patients who presented with IICR. In 72% the cell reaction was only delayed: a cell number exceeding 100 x 10(6)/L was reached later. Staphylococcus aureus was significantly more frequently the causative microorganism compared to all peritonitis episodes (PE) that occurred during the study period. Patients with IICR had lower dialysate cell counts in a stable situation, compared to a control group (p < 0.01). This was caused by a lower number of macrophages and CD4 positive lymphocytes. The phagocytosis capacity of the macrophages appeared to be normal. In a comparison of four PE with an IICR and 12 episodes with an NICR, the tumor necrosis factor-alpha (TNF-alpha) response was similar and occurred on day 1, also pointing to normally functioning macrophages. However, the maximal appearance rates of interleukin-6 (IL-6) and IL-8 occurred later in the episodes with IICR compared to NICR (day 2 vs day 1, p < 0.05). No differences were found in vasodilating prostaglandins, mesothelial cell markers (cancer antigen 125, phospholipids, hyaluronan), and mesothelial cell numbers in the stable situation nor during peritonitis. Peritonitis can present as abdominal pain in the absence of a cloudy dialysate. In some of the patients this presentation occurred more than once. This impaired, most often delayed, cell reaction was associated with a delayed secondary cytokine response. As IL-6 and IL-8 can be synthesiz

Topics: Adolescent; Adult; Aged; Bacterial Infections; Female; Humans; Interleukin-6; Interleukin-8; Kidney Failure, Chronic; Leukocyte Count; Macrophage Activation; Male; Middle Aged; Neutrophils; Peritoneal Dialysis, Continuous Ambulatory; Peritonitis; Prostaglandins; Retrospective Studies; Staphylococcal Infections; Tumor Necrosis Factor-alpha

Invasion by gram-positive and gram-negative bacterial organisms is characterized immunopathologically by the activation of mononuclear phagocytic cells, leading to the elaboration of macrophage-derived regulatory and chemotactic factors, and the resultant influx of inflammatory leukocytes. Little is known regarding the mechanisms by which gram-positive organisms initiate macrophage activation and subsequent inflammation. In this investigation, we postulated that lipoteichoic acid (LTA) purified from two different gram-positive bacterial species was an important signal for the expression of chemotactic cytokines from human peripheral blood monocytes (PBM). In initial experiments, we demonstrated that cell-associated interleukin-8 (IL-8) was expressed by mononuclear phagocytes present in inflamed areas of endocardium in cases of acute Staphylococcus aureus endocarditis. We next demonstrated that LTA purified from either Staphylococcus aureus or Streptococcus pyogenes induced the time- and dose-dependent expression of IL-8 mRNA and protein from human PBM. The expression of IL-8 mRNA from LTA- but not lipopolysaccharide (LPS)-treated PBM was superinduced by concomitant treatment with cycloheximide, indicating that the expression of IL-8 mRNA from LTA-treated PBM was negatively controlled by repressor proteins. Furthermore, mRNA stability studies indicated that IL-8 mRNA was less stable in the presence of LTA than in the presence of LPS. Our findings indicate that LTA can induce the secretion of the polymorphonuclear leukocyte chemotactic factor IL-8 and that LTA may be an important cellular mediator of inflammatory cell recruitment that characterizes immune responses to gram-positive bacterial infections.

Topics: Cycloheximide; Endocarditis, Bacterial; Gene Expression; Humans; In Vitro Techniques; Interleukin-8; Lipopolysaccharides; Monocytes; RNA, Messenger; Staphylococcal Infections; Staphylococcus aureus; Streptococcus pyogenes; Teichoic Acids

Topics: Bacterial Infections; Blood Bactericidal Activity; Chemotactic Factors; Exotoxins; Humans; Interleukin-8; Leukotriene B4; Neutrophils; Pseudomonas Infections; Staphylococcal Infections