lipoteichoic-acid and Sepsis

Advances in modern medicine have given very low birth weight (VLBW) infants a better chance of survival; however, these infants remain at high risk for developing nosocomial infections associated with increased morbidity and mortality. The ability of antistaphylococcal immunoglobulins, Altastaph and INH A-2, to augment the neonatal immune system to prevent infections has been studied and evaluated in a 2009 Cochrane review.. Our objective is to evaluate the safety and efficacy of a third antistaphylococcal immunoglobulin, pagibaximab, in the prevention of staphylococcal infection in preterm infants. Three studies of pagibaximab, Phases I, II and III, were examined in terms of study design, pharmacokinetics, development of sepsis and adverse effects.. These studies demonstrated safety and tolerability of pagibaximab with no observed reduction in sepsis. Reported adverse events in both treatment and placebo groups were similar and consistent with events commonly observed in VLBW infants. Antistaphylococcal immunoglobulins alone have been unsuccessful in preventing nosocomial infections. Further investigations need to evaluate any potential immunomodulating products in preterm animal models prior to human studies. Future studies are required to determine how to best augment the immature immune system, likely through the use of multiple immunomodulating agents to successfully prevent infections in preterm infants.

Topics: Animals; Antibodies, Monoclonal; Bacteremia; Clinical Trials as Topic; Cross Infection; Humans; Infant, Newborn; Infant, Premature; Infant, Very Low Birth Weight; Lipopolysaccharides; Sepsis; Staphylococcal Infections; Teichoic Acids

In sepsis and multiple organ dysfunction syndrome (MODS) caused by gram-negative bacteria, lipopolysaccharide (LPS) initiates the early signaling events leading to the deleterious inflammatory response. However, it has become clear that LPS can not reproduce all of the clinical features of sepsis, which emphasize the roles of other contributing factors. Gram-positive bacteria, which lack LPS, are today responsible for a substantial part of the incidents of sepsis with MODS. The major wall components of gram-positive bacteria, peptidoglycan and lipoteichoic acid, are thought to contribute to the development of sepsis and MODS. In this review, the literature underlying our current understanding of how peptidoglycan and lipoteichoic acid activate inflammatory responses will be presented, with a focus on recent advances in this field.

Topics: Animals; Cell Wall; Cytokines; Drug Synergism; Endotoxins; Humans; Lipopolysaccharides; Models, Biological; Peptidoglycan; Receptors, Cell Surface; Sepsis; Signal Transduction; Teichoic Acids

The virulence of pathogenic bacteria is critically dependent on their ability to produce toxins that attack eukaryotic target cells. Microbial toxins are either structural components of the bacterial cell wall (endotoxins) or actively secreted proteins (exotoxins). Sepsis and septic shock, which represent major causes of mortality in modern intensive care medicine, are caused by an inadequate inflammatory and immunological host response to bacterial infection. Emerging evidence suggests that the systemic spread of microbial toxins, rather than bacteremia itself, is the crucial event in the pathogenesis of this dramatic dysregulation. The endothelium, with its diversity of physiological functions is a main target of bacterial toxins. The resulting endothelial dysfunction is believed to contribute to the underlying pathomechanisms and the collapse of homeostasis of organ function. In vitro, bacterial toxins induce subtle alterations of endothelial cell function rather than massive cell damage. Furthermore, bacterial toxins targeting endothelial cells severely alter the behavior of extravascular cells and circulating leukocytes via excessive formation of vasoactive mediators and overexpression of adhesion molecules. Research on the effects of microbial toxins on vascular endothelium has broadened our general understanding of microbial strategies to induce organ damage, even in the absence of viable bacteria. Combining antitoxin strategies with antibiotic therapy may prove to be of benefit to patients suffering from bacterial sepsis in the future.

Topics: Animals; Bacterial Toxins; Endothelium, Vascular; Endotoxins; Exotoxins; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Lipopolysaccharides; Models, Biological; Multiple Organ Failure; Peptidoglycan; Sepsis; Teichoic Acids

Topics: Animals; Antibodies, Bacterial; Bacteria; Bacterial Infections; Cell Membrane; Cell Wall; Erythrocytes; Humans; Kinetics; Lipopolysaccharides; Lymphocytes; Phosphatidic Acids; Receptors, Drug; Sepsis; Teichoic Acids

Cathelicidin LL-37 plays an essential role in innate immunity by killing invading microorganisms and regulating the inflammatory response. These activities depend on the cationic character of the peptide, which is conferred by arginine and lysine residues. At inflammatory foci in vivo, LL-37 is exposed to peptidyl arginine deiminase (PAD), an enzyme released by inflammatory cells. Therefore, we hypothesized that PAD-mediated citrullination of the arginine residues within LL-37 will abrogate its immunomodulatory functions. We found that, when citrullinated, LL-37 was at least 40 times less efficient at neutralizing the proinflammatory activity of LPS due to a marked decrease in its affinity for endotoxin. Also, the ability of citrullinated LL-37 to quench macrophage responses to lipoteichoic acid and poly(I:C) signaling via TLR2 and TLR3, respectively, was significantly reduced. Furthermore, in stark contrast to native LL-37, the modified peptide completely lost the ability to prevent morbidity and mortality in a mouse model of d-galactosamine-sensitized endotoxin shock. In fact, administration of citrullinated LL-37 plus endotoxin actually exacerbated sepsis due to the inability of LL-37 to neutralize LPS and the subsequent enhancement of systemic inflammation due to increased serum levels of IL-6. Importantly, serum from septic mice showed increased PAD activity, which strongly correlated with the level of citrullination, indicating that PAD-driven protein modification occurs in vivo. Because LL-37 is a potential treatment for sepsis, its administration should be preceded by a careful analysis to ensure that the citrullinated peptide is not generated in treated patients.

Topics: Animals; Antimicrobial Cationic Peptides; Cathelicidins; Cell Line; Citrulline; Female; Humans; Hydrolases; Immunity, Innate; Interferon Inducers; Interleukin-6; Lipopolysaccharides; Macrophages; Male; Mice; Poly I-C; Sepsis; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4

In gram-positive sepsis, lipoteichoic acid (LTA) can induce alterations of haemostasis, potentially leading to disseminated intravascular coagulation.. Here, we demonstrate the effects of LTA on haemostasis in an in vitro model of gram-positive sepsis based on rotation thromboelastrography (ROTEM).. In this model, LTA leads to time- and dose-dependent shortening of the clotting time (CT), whereas other ROTEM parameters are unaffected. Following heat shock simulation, the LTA effect was blunted with equal CTs in the presence and in the absence of LTA. In addition, the shortening of CT by LTA was inhibited by addition of the protein synthesis inhibitor.. Our work demonstrates that the ROTEM system is capable of detecting the LTA effect on haemostasis and provides a sensitive in vitro tool for research into the links between gram-positive sepsis and coagulation.

Topics: Blood Coagulation; Disseminated Intravascular Coagulation; Female; Humans; Lipopolysaccharides; Male; Models, Biological; Sepsis; Teichoic Acids; Thrombelastography

Macrophage stimulation by pathogen-associated molecular patterns (PAMPs) like lipopolysaccharide (LPS) or lipoteichoic acid (LTA) drives a proinflammatory phenotype and induces a metabolic reprogramming to sustain the cell's function. Nevertheless, the relationship between metabolic shifts and gene expression remains poorly explored. In this context, the metabolic enzyme ATP citrate lyase (ACLY), the producer of citrate-derived acetyl-coenzyme A (CoA), plays a critical role in supporting a proinflammatory response. Through immunocytochemistry and cytosol-nucleus fractionation, we found a short-term ACLY nuclear translocation. Protein immunoprecipitation unveiled the role of nuclear ACLY in NF-κB acetylation and in turn its full activation in human PBMC-derived macrophages. Notably, sepsis in the early hyperinflammatory phase triggers ACLY-mediated NF-κB acetylation. The ACLY/NF-κB axis increases the expression levels of proinflammatory genes, including

Topics: Acetylation; Aged; ATP Citrate (pro-S)-Lyase; Cell Nucleus; Cytosol; Female; Gene Expression Regulation; Humans; Inflammation; Lipopolysaccharides; Macrophages; Male; Middle Aged; Mitochondrial Proteins; NF-kappa B; Organic Anion Transporters; Sepsis; Teichoic Acids; Up-Regulation; Young Adult

The goal of our work was to titer the IgG, IgM and IgA in Pentaglobin® (a preparation enriched in IgM), targeting specific surface antigens of Gram-positive and Gram-negative bacteria as well as a C. albicans strain. Lipopolysaccharides from Gram-negative bacteria, peptidoglycan and lipoteichoic acid from the other microorganisms were extracted and used in several ELISA assays in order to determine the titration of immunoglobulins in Pentaglobin® directed towards the aforementioned surface antigens. Our results showed an overall immunoglobulin titer of at least 10

Topics: Anti-Bacterial Agents; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Immunoglobulin A; Immunoglobulin M; Immunoglobulins, Intravenous; Lipopolysaccharides; Peptidoglycan; Sepsis; Teichoic Acids

Previously, we generated and screened a panel of monoclonal antibodies (mAbs) against methicillin-resistant Staphylococcus aureus (MRSA) to identify protective mAbs in mouse infection models. One of these mAbs, ZBIA3H, bound to lipoteichoic acid (LTA) and exerted protective effects in a mouse sepsis model. To reinforce the ability of the mAb to protect against infection, combination therapies with the mAb and antibiotics need to be examined. Therefore, herein, we studied the efficacy of ZBIA3H (in combination or alone) in a mouse sepsis model. ZBIA3H improved the survival rate in the mouse models of sepsis induced by highly virulent or refractory S. aureus (community-acquired MRSA strain MW2, vancomycin-intermediate S. aureus strain Mu3, or vancomycin-resistant S. aureus strain VRS1). Furthermore, ZBIA3H remarkably improved the survival rate in combination with antimicrobial agents (vancomycin, daptomycin, or linezolid) in mouse sepsis models. From these results we conclude that anti-LTA mAb ZBIA3H or its humanized form is a promising mAb individually, or in combination with antibiotics, against clinical refractory infection of S. aureus.

Topics: Animals; Anti-Bacterial Agents; Antibodies, Bacterial; Antibodies, Monoclonal; Daptomycin; Disease Models, Animal; Drug Therapy, Combination; Humans; Linezolid; Lipopolysaccharides; Methicillin-Resistant Staphylococcus aureus; Mice; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Vancomycin

Topics: Animals; Animals, Outbred Strains; Bacterial Outer Membrane Proteins; Candida albicans; Candidiasis; CD5 Antigens; Cytokines; Disease Models, Animal; Disease Susceptibility; Interferon-gamma; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mycoses; Sepsis; Species Specificity; Spleen; Teichoic Acids; Zymosan

Topics: Adult; Aged; Antibodies; CD11b Antigen; Cells, Cultured; Complex Mixtures; Female; Healthy Volunteers; Humans; Immunoglobulin A; Immunoglobulin G; Immunoglobulin M; Immunomodulation; Inflammation; Lipopolysaccharides; Male; Middle Aged; Monocytes; Receptors, IgG; Sepsis; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

Topics: Animals; Antigens, CD; Antigens, Differentiation, T-Lymphocyte; Conserved Sequence; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Immunity, Innate; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Models, Animal; Pathogen-Associated Molecular Pattern Molecules; Peptides; Protein Binding; Receptors, Scavenger; Sepsis; Teichoic Acids

Bacterial infections affect more than 2 million people annually. Of these, systemic infections caused by bacteria in critically ill patients may lead to life-threatening conditions such as sepsis. We have developed a point-of-care (POC) device called Septiflo that can detect and stratify the Gram status of bloodstream bacterial infections in less than 10 min from a drop of human plasma. It works on the principle of identifying pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharides (LPS) and lipoteichoic acid (LTA) that are released into the bloodstream at the onset of Gram-negative and Gram-positive bacterial infections, respectively. The biomarkers are captured on a membrane without a receptor, and the Gram status specificity is conferred by the ligands attached to gold nanoparticles (AuNPs) used as signal amplification probes. The ultrasensitive colorimetric results are read by eye down to a 100-fg/ml detection limit without an instrument. No cross-interference between the PAMPs is seen during Gram stratification. Septiflo results also display better performance than commercial enzyme-linked immunosorbent assays (ELISAs). Tests performed on 60 clinical samples from patients showed a correlation accuracy of 70% against procalcitonin (PCT), an accepted surrogate biomarker for sepsis. A direct comparison with eubacterial PCR yielded up to 94% accuracy in 31 patients at a chosen cutoff level for LPS and LTA and area under the curve (AUC) values of 0.927 and 0.885, respectively, though blood culture was negative for most samples. The high sensitivity, low cost, and simple bedside utility of the assay may aid in better sepsis management apparently at the presymptomatic stage, lowering empirical therapy, medical costs, antimicrobial resistance, and mortality.

Topics: Bacteremia; Bacteria; Biological Assay; Biomarkers; Colorimetry; Critical Illness; Gold; Humans; Ligands; Lipopolysaccharides; Metal Nanoparticles; Pilot Projects; Point-of-Care Testing; Procalcitonin; Sensitivity and Specificity; Sepsis; Teichoic Acids

Lipoteichoic acid (LTA) and lipopolysaccharide (LPS) are bacterial lipids that stimulate pro-inflammatory cytokine production, thereby exacerbating sepsis pathophysiology. Proprotein convertase subtilisin/kexin type 9 (PCSK9) negatively regulates uptake of cholesterol by downregulating hepatic lipoprotein receptors, including low-density lipoprotein (LDL) receptor (LDLR) and possibly LDLR-related protein-1 (LRP1). PCSK9 also negatively regulates Gram-negative LPS uptake by hepatocytes, however this mechanism is not completely characterized and mechanisms of Gram-positive LTA uptake are unknown. Therefore, our objective was to elucidate the mechanisms through which PCSK9 regulates uptake of LTA and LPS by investigating the roles of lipoproteins and lipoprotein receptors. Here we show that plasma PCSK9 concentrations increase transiently over time in septic and non-septic critically ill patients, with highly similar profiles over 14 days. Using flow cytometry, we demonstrate that PCSK9 negatively regulates LDLR-mediated uptake of LTA and LPS by HepG2 hepatocytes through an LDL-dependent mechanism, whereas LRP1 and high-density lipoprotein do not contribute to this uptake pathway. Bacterial lipid uptake by hepatocytes was not associated with cytokine production or hepatocellular injury. In conclusion, our study characterizes an LDL-dependent and LDLR-mediated bacterial lipid uptake pathway regulated by PCSK9, and provides evidence in support of PCSK9 inhibition as a potential therapeutic strategy for sepsis.

Topics: Enterococcus hirae; Escherichia coli; Flow Cytometry; Hep G2 Cells; Hepatocytes; Humans; Lipopolysaccharides; Lipoproteins, LDL; Low Density Lipoprotein Receptor-Related Protein-1; Proprotein Convertase 9; Receptors, LDL; Sepsis; Teichoic Acids

Teichoic acid (TA), a crucial cell wall constituent of the pathobiont Streptococcus pneumoniae, is bound to peptidoglycan (wall teichoic acid, WTA) or to membrane glycolipids (lipoteichoic acid, LTA). Both TA polymers share a common precursor synthesis pathway, but differ in the final transfer of the TA chain to either peptidoglycan or a glycolipid. Here, we show that LTA exhibits a different linkage conformation compared to WTA, and identify TacL (previously known as RafX) as a putative lipoteichoic acid ligase required for LTA assembly. Pneumococcal mutants deficient in TacL lack LTA and show attenuated virulence in mouse models of acute pneumonia and systemic infections, although they grow normally in culture. Hence, LTA is important for S. pneumoniae to establish systemic infections, and TacL represents a potential target for antimicrobial drug development.

Topics: Animals; Cell Line; Cell Wall; Disease Models, Animal; Humans; Ligases; Lipopolysaccharides; Male; Mice; Microscopy, Electron; Mutagenesis; Peptidoglycan; Pneumonia, Pneumococcal; Sepsis; Streptococcus pneumoniae; Teichoic Acids; Virulence

The Acute Respiratory Distress Syndrome (ARDS), remains a significant source of morbidity and mortality in critically ill patients. Pneumonia and sepsis are leading causes of ARDS, the pathophysiology of which includes increased pulmonary microvascular permeability and hemodynamic instability resulting in organ dysfunction. We hypothesized that N-ethylmaleimide sensitive factor (NSF) regulates exocytosis of inflammatory mediators, such as Angiopoietin-2 (Ang-2), and cytoskeletal stability by modulating myosin light chain (MLC) phosphorylation. Therefore, we challenged pulmonary cells, in vivo and in vitro, with Gram Positive bacterial cell wall components, lipoteichoic acid (LTA), and peptidoglycan (PGN) and examined the effects of NSF inhibition.. Mice were pre-treated with an inhibitor of NSF, TAT-NSF700 (to prevent Ang-2 release). After 30min, LTA and PGN (or saline alone) were instilled intratracheally. Pulse oximetry was assessed in awake mice prior to, and 6 hour post instillation. Post mortem, tissues were collected for studies of inflammation and Ang-2. In vitro, pulmonary endothelial cells were assessed for their responses to LTA and PGN.. Pulmonary challenge induced signs of airspace and systemic inflammation such as changes in neutrophil counts and protein concentration in bronchoalveolar lavage fluid and tissue Ang-2 concentration, and decreased physiological parameters including oxygen saturation and pulse distention. TAT-NSF700 pre-treatment reduced LTA-PGN induced changes in lung tissue Ang-2, oxygen saturation and pulse distention. In vitro, LTA-PGN induced a rapid (<2 min) release of Ang-2, which was significantly attenuated by TAT-NSF700 or anti TLR2 antibody. Furthermore, TAT-NSF700 reduced LTA-PGN-induced MLC phosphorylation at low concentrations of 1-10 nM.. TAT-NSF700 decreased Ang-2 release, improved oxygen saturation and pulse distention following pulmonary challenge by inhibiting MLC phosphorylation, an important component of endothelial cell retraction. The data suggest that inhibition of NSF in pneumonia and sepsis may be beneficial to prevent the pulmonary microvascular and hemodynamic instability associated with ARDS.

Topics: Angiopoietin-1; Animals; Bacterial Infections; Blood Vessels; Cell Line; Cell Wall; Cytoskeleton; Disease Models, Animal; Exocytosis; Gram-Positive Bacteria; Humans; Inflammation; Lipopolysaccharides; Lung; Male; Mice; Mice, Inbred BALB C; Microcirculation; N-Ethylmaleimide-Sensitive Proteins; Oxygen; Peptidoglycan; Phosphorylation; Pneumonia; Respiratory Distress Syndrome; Sepsis; Teichoic Acids; Vascular Diseases

In spite of advances in neonatal care and the new generation of antibiotics, neonatal sepsis is still a major cause of morbidity and mortality. Early diagnosis of neonatal sepsis is difficult because clinical signs are non-specific. Thus, new biomarkers are still needed for diagnosis. Gelsolin is an actin-binding plasma protein. Furthermore, extracellular gelsolin binds lipopolysaccharide and lipoteichoic acid, which are major virulence factors of Gram-negative and Gram-positive bacteria. The result of this binding is the inhibition of gelsolin's F-actin depolymerizing activity. Thus, gelsolin inhibits the release of IL-8 from human neutrophils subjected to lipoteichoic acid, lipopolysaccharide and heat-inactivated bacteria treatment. Our hypothesis is that pGSN levels decrease in neonatal infants with sepsis and this decrease might be used as a reliable biological marker. Forty patients who were diagnosed with severe sepsis at a neonatal intensive care unit were enrolled in the sepsis group. Twenty patients who were followed for prematurity were enrolled in the control group. The pGSN level at the time of diagnosis in the sepsis group was 33.98±11.44μg/ml, which was significantly lower than that of control group (60.05±11.3μg/ml, P<0.001) and after treatment (53.38±31.26μg/ml, P=0.003). Area under ROC curve was 0.96 (p: 0.0001, 95% CI; 0.90-0.99). Sensitivity was 90.32 (95% CI; 74.2-97.8), specificity was 95 (95% CI; 75.1-99.2). Plasma gelsolin significantly decreased in septic patient and recovery of decreased gelsolin levels correlated with clinical improvement. Thus, plasma gelsolin may be a usable marker for severe sepsis.

Topics: Actins; Area Under Curve; Biomarkers; Female; Gelsolin; Humans; Infant, Newborn; Interleukin-8; Lipopolysaccharides; Male; Morbidity; Neonatal Sepsis; Neutrophils; Prospective Studies; ROC Curve; Sensitivity and Specificity; Sepsis; Teichoic Acids

Sepsis is characterized by dysregulated systemic inflammation and cytokine storm. Angiopoietin-2 (Ang-2) is known to closely correlate with severity of sepsis-related acute lung injury and mortality. The aim of this study was to clarify the mechanisms involved in Ang-2 secretion to better understand the pathophysiology of sepsis.. The concentration of Ang-2 was assessed in culture medium of pulmonary microvascular endothelial cells in the presence or absence of Gram-positive bacteria cell wall components [lipoteichoic acid (LTA) and peptidoglycan (PGN)] stimulation at different time points ranging from 15 minutes to 24 hours. Constitutive and LTA-PGN-stimulated Ang-2 level changes were also assessed after cells were pretreated with different pathway inhibitors for 1 hour.. Two distinctive mechanisms of Ang-2 secretion, constitutive and stimulated secretion, were identified. Constitutive secretion resulted in slow but continuous increase in Ang-2 in culture medium over time. It was regulated by 3'5'-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-Ca. We demonstrated characteristics and involved pathways for two distinctive secretory mechanisms, constitutive and stimulated, of Ang-2 in pulmonary microvascular endothelial cells. Considering the close correlation of Ang-2 with sepsis outcomes, our findings provide a better understanding of an important mechanism associated with sepsis pathophysiology and identify possible therapeutic targets to improve outcomes in the potentially lethal disease.

Topics: Angiopoietin-2; Cell Wall; Cells, Cultured; Endothelial Cells; Gram-Positive Bacteria; Humans; Lipopolysaccharides; Lung; Peptidoglycan; Sepsis; Teichoic Acids

Nerve injury-induced protein (Ninjurin [Ninj]) 1 is an adhesion molecule originally identified in Schwann cells after nerve injury, whereas it is also expressed in leukocytes, epithelium, endothelium, and various organs, and is induced under inflammatory conditions. Its contribution to inflammation was so far restricted to the nervous system and exclusively attributed to its role during leukocyte migration. We hypothesized a proinflammatory role for Ninj1 also outside the nervous system. To elucidate its impact during inflammation, we analyzed expression levels and its contribution to inflammation in septic mice and studied its effect on inflammatory signaling in vitro. The effect on inflammation was analyzed by genetic (only in vitro) and pharmacologic repression in septic mice (cecal ligation and puncture) and cell culture, respectively. Repression of Ninj1 by an inhibitory peptide or small interfering RNA attenuated LPS-triggered inflammation in macrophages and endothelial cells by modulating p38 phosphorylation and activator protein-1 activation. Inhibition of Ninj1 in septic mice reduced systemic and pulmonary inflammation as well as organ damage, and ameliorated survival after 24 hours. Ninj1 is elevated under inflammatory conditions and contributes to inflammation not only by mediating leukocyte migration, but also by modulating Toll-like receptor 4-dependent expression of inflammatory mediators. We assume that, owing to both mechanisms, inhibition reduces systemic inflammation and organ damage in septic mice. Our data contribute to a better understanding of the complex inflammatory mechanisms and add a novel therapeutic target for inflammatory conditions such as sepsis.

Topics: Animals; Cell Adhesion Molecules, Neuronal; Cell Movement; Disease Models, Animal; Endothelial Cells; Gene Expression Regulation; Humans; Leukocytes, Mononuclear; Lipopolysaccharides; Male; Mice; Mice, Inbred C57BL; Nerve Growth Factors; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly I-C; Primary Cell Culture; Sepsis; Signal Transduction; Systemic Inflammatory Response Syndrome; Teichoic Acids; Toll-Like Receptor 4; Transcription Factor AP-1; Tumor Necrosis Factor-alpha

Sepsis, a life-threatening syndrome with increasing incidence worldwide, is triggered by an overwhelming inflammation induced by microbial toxins released into the bloodstream during infection. A well-known sepsis-inducing factor is the membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS), signalling via Toll-like receptor-4. Although sepsis is caused in more than 50% cases by Gram-positive and mycoplasma cells, the causative compounds are still poorly described. In contradicting investigations lipoproteins/-peptides (LP), lipoteichoic acids (LTA), and peptidoglycans (PGN), were made responsible for eliciting this pathology. Here, we used human mononuclear cells from healthy donors to determine the cytokine-inducing activity of various LPs from different bacterial origin, synthetic and natural, and compared their activity with that of natural LTA and PGN. We demonstrate that LP are the most potent non-LPS pro-inflammatory toxins of the bacterial cell walls, signalling via Toll-like receptor-2, not only in vitro, but also when inoculated into mice: A synthetic LP caused sepsis-related pathological symptoms in a dose-response manner. Additionally, these mice produced pro-inflammatory cytokines characteristic of a septic reaction. Importantly, the recently designed polypeptide Aspidasept(®) which has been proven to efficiently neutralize LPS in vivo, inhibited cytokines induced by the various non-LPS compounds protecting animals from the pro-inflammatory activity of synthetic LP.

Topics: Animals; Anti-Bacterial Agents; Cytokines; Disease Models, Animal; Endotoxemia; Endotoxins; Female; Gram-Negative Bacteria; HEK293 Cells; Humans; Leukocytes, Mononuclear; Lipopolysaccharides; Lipoproteins; Macrophages; Mice; Peptides; Peptidoglycan; Sepsis; Staphylococcus aureus; Teichoic Acids

Lipoteichoic acid (LTA) is the key pathogenic factor of gram-positive bacteria and contributes significantly to organ dysfunction in sepsis, a frequent complication in critical care patients. We hypothesized that LTA directly affects cardiomyocyte function, thus contributing to cardiac failure in sepsis. This study was designed to evaluate the effects of LTA on contractile properties and calcium-transients of isolated adult rat cardiomyocytes. When myocytes were exposed to LTA for 1h prior to analysis, the amplitudes of calcium-transients as well as sarcomere shortening increased to 130% and 142% at 1 Hz stimulation frequency. Relengthening of sarcomeres as well as decay of calcium-transients was accelerated after LTA incubation. Exposure to LTA for 24 h resulted in significant depression of calcium-transients as well as of sarcomere shortening compared to controls. One of the major findings of our experiments is that LTA most likely affects calcium-handling of the cardiomyocytes. The effect is exacerbated by reduced extracellular calcium, which resembles the clinical situation in septic patients. Functionally, an early stimulating effect of LTA with increased contractility of the cardiomyocytes may be an in vitro reflection of early hyperdynamic phases in clinical sepsis. Septic disorders have been shown to induce late hypodynamic states of the contractile myocardium, which is also supported at the single-cell level in vitro by results of our 24h-exposure to LTA.

Topics: Animals; Calcium; Cells, Cultured; Humans; Intracellular Space; Lipopolysaccharides; Male; Myocytes, Cardiac; Rats; Rats, Sprague-Dawley; Sarcomeres; Sepsis; Single-Cell Analysis; Staphylococcus aureus; Teichoic Acids; Time Factors; Tumor Necrosis Factor-alpha

The current epidemic of infections caused by antibiotic-resistant gram-positive bacteria requires the discovery of new drug targets and the development of new therapeutics. Lipoteichoic acid (LTA), a cell wall polymer of gram-positive bacteria, consists of 1,3-polyglycerol-phosphate linked to glycolipid. LTA synthase (LtaS) polymerizes polyglycerol-phosphate from phosphatidylglycerol, a reaction that is essential for the growth of gram-positive bacteria. We screened small molecule libraries for compounds inhibiting growth of Staphylococcus aureus but not of gram-negative bacteria. Compound 1771 [2-oxo-2-(5-phenyl-1,3,4-oxadiazol-2-ylamino)ethyl 2-naphtho[2,1-b]furan-1-ylacetate] blocked phosphatidylglycerol binding to LtaS and inhibited LTA synthesis in S. aureus and in Escherichia coli expressing ltaS. Compound 1771 inhibited the growth of antibiotic-resistant gram-positive bacteria and prolonged the survival of mice with lethal S. aureus challenge, validating LtaS as a target for the development of antibiotics.

Topics: Acyltransferases; Animals; Anti-Bacterial Agents; Catalytic Domain; Disease Models, Animal; Drug Resistance, Microbial; Enzyme Inhibitors; Lipopolysaccharides; Mice; Microbial Sensitivity Tests; Mutation; Phosphatidylglycerols; Sepsis; Small Molecule Libraries; Staphylococcal Infections; Staphylococcus aureus; Structure-Activity Relationship; Survival Analysis; Teichoic Acids

Lipoteichoic acid (LTA) is a major virulence factor of Gram-positive bacteria including Staphylococcus aureus. Despite its pivotal role in causing sepsis, the systemic immune responses to LTA in human cells are poorly understood. Here, we produced highly-pure and structurally-intact LTA from S. aureus and examined the gene expression profile of LTA-stimulated human peripheral blood mononuclear cells (PBMCs). The LTA preparation did not contain any detectable biologically-active impurities and stimulated Toll-like receptor 2. Protein expression profiling using a cytokine array kit and ELISA revealed expression of MCP-1/CCL2, IL-6, and IL-1β. We performed transcriptional profiling of PBMCs in response to S. aureus LTA using an Affymetrix genechip microarray. A total of 208 genes were significantly (fold change>1.5 and P<0.05) altered, with 157 up-regulated and 51 down-regulated genes in response to S. aureus LTA treatment. The up-regulated genes were involved in recognition (30 genes), cellular adhesion (6 genes), signal transduction (42 genes), co-stimulation (4 genes), chemokines, cytokines and their receptors (51 genes), apoptosis (9 genes), and negative regulation (15 genes). The down-regulated genes were involved in recognition (12 genes), antigen processing and presentation (9 genes), signal transduction (27 genes), and chemotaxis (3 genes). The microarray results were validated using real-time RT-PCR with 21 up-regulated genes and 9 down-regulated genes. Our results provide a more comprehensive overview of the transcriptional changes in PBMCs in response to S. aureus LTA, and contribute to the understanding of the pathophysiological role of S. aureus LTA during the systemic inflammatory response.

Topics: Cells, Cultured; Cytokines; Gene Expression Profiling; Gene Expression Regulation; Humans; Immune Evasion; Inflammation Mediators; Leukocytes, Mononuclear; Lipopolysaccharides; Oligonucleotide Array Sequence Analysis; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids; Virulence Factors

Lipoteichoic acid (LTA) from Staphylococcus aureus (aLTA) and from Lactobacillus plantarum LTA (pLTA) are both recognized by Toll-like receptor 2 (TLR2), but cause different stimulatory effects on the innate immune and inflammatory responses, and their underlying cellular mechanisms are unknown. In this study, comparative proteome analysis was performed using two-dimensional gel electrophoresis and mass spectrometry on protein extracts from human monocyte THP-1 cells stimulated with either aLTA or pLTA. Differentially expressed proteins might be involved in innate immunity and inflammation. Cells treated with aLTA and with pLTA showed different protein expression profiles. Of 60 identified proteins, 10 were present only in treated cells (8 in aLTA-treated only, and 2 in pLTA-treated only), 1 protein (IMPDH2) was suppressed by pLTA, and 49 were up- or down-regulated more than three-fold by aLTA- or pLTA- stimulation. Several proteins involved in immunity or inflammation, antioxidation, or RNA processing were significantly changed in expression by aLTA- or pLTA-stimulation, including cyclophilin A, HLA-B27, D-dopachrome tautomerase, Mn- SOD, hnRNP-C, PSF and KSRP. These data demonstrated that aLTA and pLTA had different effects on the protein profile of THP-1 cells. Comparison of the proteome alterations will provide candidate biomarkers for further investigation of the immunomodulatory effects of aLTA and pLTA, and the involvement of aLTA in the pathogenesis of Staphylococcus aureus sepsis.

Topics: Biomarkers; Cell Line, Tumor; Cyclophilin A; Electrophoresis, Gel, Two-Dimensional; HLA-B27 Antigen; Humans; Immunomodulation; IMP Dehydrogenase; Lactobacillus plantarum; Lipopolysaccharides; Mass Spectrometry; Monocytes; Proteome; Sepsis; Staphylococcus aureus; Teichoic Acids

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naïve WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

Topics: Animals; Cell Movement; Chemotaxis; Down-Regulation; G-Protein-Coupled Receptor Kinase 2; Gene Expression Regulation; Immunity, Innate; Lipopolysaccharides; Mice; Neutrophils; Peritonitis; Receptors, Interleukin-8B; Sepsis; Signal Transduction; Survival Analysis; Teichoic Acids; Toll-Like Receptor 2

Group B streptococcus (GBS) is the most important cause of neonatal sepsis, which is mediated in part by TLR2. However, GBS components that potently induce cytokines via TLR2 are largely unknown. We found that GBS strains of the same serotype differ in released factors that activate TLR2. Several lines of genetic and biochemical evidence indicated that lipoteichoic acid (LTA), the most widely studied TLR2 agonist in Gram-positive bacteria, was not essential for TLR2 activation. We thus examined the role of GBS lipoproteins in this process by inactivating two genes essential for bacterial lipoprotein (BLP) maturation: the prolipoprotein diacylglyceryl transferase gene (lgt) and the lipoprotein signal peptidase gene (lsp). We found that Lgt modification of the N-terminal sequence called lipobox was not critical for Lsp cleavage of BLPs. In the absence of lgt and lsp, lipoprotein signal peptides were processed by the type I signal peptidase. Importantly, both the Deltalgt and the Deltalsp mutant were impaired in TLR2 activation. In contrast to released factors, fixed Deltalgt and Deltalsp GBS cells exhibited normal inflammatory activity indicating that extracellular toxins and cell wall components activate phagocytes through independent pathways. In addition, the Deltalgt mutant exhibited increased lethality in a model of neonatal GBS sepsis. Notably, LTA comprised little, if any, inflammatory potency when extracted from Deltalgt GBS. In conclusion, mature BLPs, and not LTA, are the major TLR2 activating factors from GBS and significantly contribute to GBS sepsis.

Topics: Animals; Animals, Newborn; Aspartic Acid Endopeptidases; Bacterial Proteins; Gene Deletion; Humans; Lipopolysaccharides; Lipoproteins; Mice; Mice, Inbred BALB C; Mice, Knockout; Protein Processing, Post-Translational; Protein Structure, Tertiary; Sepsis; Streptococcus agalactiae; Teichoic Acids; Toll-Like Receptor 2; Transferases

Lipoteichoic acid (LTA), as a primary immunostimulus, triggers the systematic inflammatory responses. Our hypothesis is that ApoA-I can neutralize LTA toxicity, like its effect on LPS. BALB/c mice were challenged with LTA, followed by human ApoA-I administration. We found that ApoA-I could attenuate LTA-induced acute lung injury and inflammation and significantly inhibit LTA-induced IL-1beta and TNF-alpha accumulation in the serum (P<0.01 and P<0.05, respectively), as well as in bronchoalveolar lavage (BAL) fluid (P<0.01 and P<0.05, respectively). Moreover, ApoA-I could significantly reduce the L-929 cell mortality caused by LTA-activated macrophages in a dose-dependent fashion. Furthermore, ApoA-I treatment could diminish LTA-mediated NFkappaB nuclear translocation in macrophages. An in vitro binding assay indicated that ApoA-I can bind LTA. These results clearly indicated that ApoA-I can effectively protect against LTA-induced sepsis and acute lung damage. The mechanism might be related to the binding and neutralization of LTA.

Topics: Animals; Apolipoprotein A-I; Cell Line; Humans; Lipopolysaccharides; Male; Mice; Mice, Inbred BALB C; Respiratory Distress Syndrome; Sepsis; Teichoic Acids

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

Septicemia is one of the major health concerns worldwide, and rapid activation of adrenal steroid release is a key event in the organism's first line of defense during this form of severe illness. The family of Toll-like receptors (TLRs) is critical in the early immune response upon bacterial infection, and TLR polymorphisms are frequent in humans. Here, we demonstrate that TLR-2 deficiency in mice is associated with reduced plasma corticosterone levels and marked cellular alterations in adrenocortical tissue. TLR-2-deficient mice have an impaired adrenal corticosterone release after inflammatory stress induced by bacterial cell wall compounds. This defect appears to be mediated by a decrease in systemic and intraadrenal cytokine expression, including IL-1, tumor necrosis factor alpha, and IL-6. Our data demonstrate a link between the innate immune system and the endocrine stress response. The critical role of TLR-2 in adrenal glucocorticoid regulation needs to be considered in patients with inflammatory disease.

Topics: Adrenal Cortex; Adrenocorticotropic Hormone; Animals; Corticosterone; Cytokines; Endotoxemia; Humans; Immunity, Innate; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Mice, Knockout; Models, Biological; NF-kappa B; Receptors, Cell Surface; Sepsis; Teichoic Acids; Toll-Like Receptor 2

Gene- and signal-specific adaptation/tolerance of blood leukocytes to lipopolysaccharide endotoxin (LPS) occurs during human and animal septicemia. These phenotypes can be modeled in vitro. LPS-TLR4-adapted human THP-1 promonocytic cells cross-adapt to lipoteichoic acid (LTA)-TLR2-induced IL-1beta/TNF-alpha production, suggesting disruption of a common intracellular signaling event(s). A plausible explanation for homologous adaptation of TLR4 with heterologous adaptation of TLR2 is a persistent inactivation and degradation of IRAK1 following TLR4 activation. LTA stimulation of TLR2 also produces homologous adaptation of TLR2 with inactivation of IRAK1, but there is no detectable degradation of IRAK1. Strikingly, such LTA-adapted cells still respond to LPS stimulation of TLR4 with rapid activation and degradation of IRAK1, and robust IL-1beta/TNF-alpha production. Moreover, cells adapted to either LTA- or LPS-production of IL-1beta/TNF-alpha normally produce soluble interleukin 1 receptor antagonist (sIL-1Ra) anti-inflammatory protein when stimulated by either agonist. We conclude that: (i) disruption of a unique TLR2 signaling component upstream of IRAK1, but downstream of TLR2 sensing, induces homologous adaptation to LTA; (ii) disruption of IRAK1 may induce homologous adaptation of TLR4 to LPS and cross-adaptation of TLR2 to LTA; and (iii) TLR2/TLR4 signaling events that control sIL-1Ra translation do not adapt to LPS or LTA, indicating that TLR4 and TLR2 can still function. We present a hypothetical model of adaptation based on a signalsome, with IRAK1 evolving after IRAK4 to regulate TLR4 adaptation tightly.

Topics: Adaptation, Biological; Gene Expression Regulation; Humans; Immune Tolerance; Interleukin-1; Interleukin-1 Receptor-Associated Kinases; Leukocytes, Mononuclear; Lipopolysaccharides; Membrane Glycoproteins; Models, Biological; Protein Kinases; Receptors, Cell Surface; RNA, Messenger; Sepsis; Signal Transduction; Teichoic Acids; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptors; Tumor Necrosis Factor-alpha

Our study aimed to characterize the mechanisms underlying the attenuated cardiovascular responsiveness toward the renin-angiotensin system during sepsis. For this purpose, we determined the effects of experimental Gram-negative and Gram-positive sepsis in rats. We found that sepsis led to a ubiquitous upregulation of NO synthase isoform II expression and to pronounced hypotension. Despite increased plasma renin activity and plasma angiotensin (Ang) II levels, plasma aldosterone concentrations were normal, and the blood pressure response to exogenous Ang II was markedly diminished in septic rats. Mimicking the fall of blood pressure during sepsis by short-term infusion of the NO donor sodium nitroprusside in normal rats did not alter their blood pressure response to exogenous Ang II. Therefore, we considered the possibility of an altered expression of Ang II receptors during sepsis. It turned out that Ang II type 1 receptor expression was markedly downregulated in all organs of septic rats. Further in vitro studies with rat renal mesangial cells showed that NO and a combination of proinflammatory cytokines (interleukin-1beta, tumor necrosis factor-alpha, and interferon-gamma) downregulated Ang II type 1 receptor expression in a synergistic fashion. In summary, our data suggest that sepsis causes a systemic downregulation of Ang II type 1 receptors that is likely mediated by proinflammatory cytokines and NO. We suggest that this downregulation of Ang II type 1 receptors is the main reason for the attenuated responsiveness of blood pressure and of aldosterone formation to Ang II and, therefore, contributes to the characteristic septic shock.

Topics: Adrenal Glands; Angiotensin II; Animals; Blood Pressure; Cells, Cultured; Cytokines; Down-Regulation; Drug Antagonism; Glomerular Mesangium; Lipopolysaccharides; Liver; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Rats; Receptor, Angiotensin, Type 1; Receptor, Angiotensin, Type 2; Receptors, Angiotensin; Renin-Angiotensin System; RNA, Messenger; Sepsis; Teichoic Acids; Tissue Distribution

Despite the fact that gram-positive infections constitute around 50% of all cases leading to septic shock, little is yet known about the mechanisms involved. This study was carried out to find out more about the effects of cell wall components peptidoglycan (PepG) and lipoteichoic acid (LTA) of the gram-positive bacterium Streptococcus pyogenes in the pig. Specific pathogen-free pigs (20 kg bodyweight) were pretreated with metyrapone (a cortisol-synthesis inhibitor) and then were given 2-h infusions of 160 microg/kg of PepG (n = 5), 160 microg/kg LTA (n=5), or a combination of both (LTA + PepG, 160 microg/kg each, n = 5). Four hours after start of the infusions, the PepG, LTA, and LTA + PepG groups showed decreases in mean arterial pressure (change of -11%, -25%, and -47% from baseline, respectively), dynamic lung compliance (-18%, -24%, and -38%), arterial oxygen tension (-10%, -16%, and -37%), changes in blood leukocyte numbers (+11%, -27%, and -67%), and increases in pulmonary vascular resistance index (+7%, +106%, and +307% from baseline) and metabolic acidosis (base excess values decreased with 1.8, 2.3 and 8.1 units). The differences between the PepG and LTA + PepG groups were statistically significant (P < 0.05, Kruskal-Wallis tests), but not between LTA and LTA + PepG groups. However, no changes in systemic nitric oxide (NO) production could be detected, which is much in contrast to studies on lower order animals. Moreover, comparison of the results obtained using this model with those obtained in a model of endotoxin-induced septic shock showed distinct difference in the mechanisms by which gram-positive and gram-negative bacterial components exert their actions. For example, a marked fall in systemic blood pressure and dynamic lung compliance is seen in both models, but in the present gram-positive sepsis model, much less interleukin-8 and tumor necrosis factor-alpha are produced. In conclusion, this study showed that PepG and LTA act synergistically to cause respiratory failure and septic shock in the pig. The infusion of the combination of PepG and LTA in the pig could serve as a new, well-controlled model for studies of gram-positive sepsis.

Topics: Animals; Drug Synergism; Endothelin-1; Endotoxins; Female; Hydrocortisone; Interleukin-8; Leukocyte Count; Lipopolysaccharides; Lung Compliance; Male; Metyrapone; Nitric Oxide; Oxygen; Peptidoglycan; Pulmonary Circulation; Sepsis; Shock, Septic; Specific Pathogen-Free Organisms; Streptococcus pyogenes; Swine; Teichoic Acids; Tumor Necrosis Factor-alpha; Vascular Resistance

In this study, gram-positive Staphylococcus aureus lipoteichoic acid (LTA) dose-dependently (0.1-1.0 microg/ml) and time-dependently (10-60 min) inhibited platelet aggregation in human platelets stimulated by agonists. LTA also dose-dependently inhibited phosphoinositide breakdown and intracellular Ca+2 mobilization in human platelets stimulated by collagen. LTA (0.5 and 1.0 microg/ml) also significantly inhibited thromboxane A2 formation stimulated by collagen in human platelets. Moreover, LTA (0.1-1.0 microg/ml) dose-dependently decreased the fluorescence of platelet membranes tagged with diphenylhexatrience. Rapid phosphorylation of a platelet protein of Mr. 47,000 (P47), a marker of protein kinase C activation, was triggered by PDBu (30 nM). This phosphorylation was markedly inhibited by LTA (0.5 and 1.0 microg/ml) within a 10-min incubation period. These results indicate that the antiplatelet activity of LTA may be involved in the following pathways: LTA's effects may initially be due to induction of conformational changes in the platelet membrane, leading to a change in the activity of phospholipase C, and subsequent inhibition of phosphoinositide breakdown and thromboxane A2 formation, thereby leading to inhibition of both intracellular Ca+2 mobilization and phosphorylation of P47 protein. Therefore, LTA-mediated alteration of platelet function may contribute to bleeding diathesis in gram-positive septicemic and endotoxemic patients.

Topics: Calcium Signaling; Cell Membrane; Collagen; Cytosol; Dose-Response Relationship, Drug; Endotoxemia; Enzyme Activation; Gram-Negative Bacterial Infections; Gram-Positive Bacterial Infections; Hemorrhagic Disorders; Humans; L-Lactate Dehydrogenase; Lipopolysaccharides; Membrane Fluidity; Membrane Lipids; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Peptides; Phorbol 12,13-Dibutyrate; Phosphatidylinositols; Phosphorylation; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Glycoprotein GPIIb-IIIa Complex; Protein Kinase C; Protein Processing, Post-Translational; Sepsis; Shock, Septic; Staphylococcus aureus; Teichoic Acids; Thromboxane A2; Thromboxane B2

To determine the sensitivity and specificity of a novel antibody test for the diagnosis of intravascular catheter-related infections due to coagulase-negative staphylococci.. Sixty-seven patients diagnosed as having central venous catheter (CVC)-associated sepsis based on strict clinical criteria, including positive blood cultures, were compared to 67 patients with a CVC in situ who exhibited no evidence of sepsis. An ELISA serological test based on a novel short-chain lipoteichoic acid antigen isolated from coagulase-negative staphylococci (CNS) was used to determine the patient's serological response (IgG and IgM) to CVC sepsis caused by CNS. The specificity and sensitivity of the test was determined.. There was a significant increase in the antibody levels (IgG and IgM) to the short-chain lipoteichoic acid in patients with CVC-associated staphylococcal sepsis as compared to the control patients.. This new serological method may offer a useful diagnostic test for intravascular catheter infections caused by staphylococci.

Topics: Adolescent; Adult; Aged; Catheterization, Central Venous; Enzyme-Linked Immunosorbent Assay; Female; Humans; Immunoglobulin G; Immunoglobulin M; Lipopolysaccharides; Male; Middle Aged; Sensitivity and Specificity; Sepsis; Staphylococcal Infections; Teichoic Acids

Cell adhesion molecule expression (CAM) and IL-8 release in lung microvascular endothelium facilitate neutrophil accumulation in the lung. This study investigated the effects of lipoteichoic acid (LTA), a cell wall component of Gram-positive bacteria, alone and with LPS or TNF-alpha, on CAM expression and IL-8 release in human lung microvascular endothelial cells (HLMVEC). The concentration-dependent effects of Staphylococcus aureus (S. aureus) LTA (0.3-30 microg/ml) on ICAM-1 and E-selectin expression and IL-8 release were bell shaped. Streptococcus pyogenes (S. pyogenes) LTA had no effect on CAM expression, but caused a concentration-dependent increase in IL-8 release. S. aureus and S. pyogenes LTA (30 microg/ml) abolished LPS-induced CAM expression, and S. aureus LTA reduced LPS-induced IL-8 release. In contrast, the effects of S. aureus LTA with TNF-alpha on CAM expression and IL-8 release were additive. Inhibitory effects of LTA were not due to decreased HLMVEC viability, as assessed by ethidium homodimer-1 uptake. Changes in neutrophil adhesion to HLMVEC paralleled changes in CAM expression. Using RT-PCR to assess mRNA levels, S. aureus LTA (3 microg/ml) caused a protein synthesis-dependent reduction (75%) in LPS-induced IL-8 mRNA and decreased the IL-8 mRNA half-life from >6 h with LPS to approximately 2 h. These results suggest that mechanisms exist to prevent excessive endothelial cell activation in the presence of high concentrations of bacterial products. However, inhibition of HLMVEC CAM expression and IL-8 release ultimately may contribute to decreased neutrophil accumulation, persistence of bacteria in the lung, and increased severity of infection.

Topics: Cell Adhesion Molecules; Drug Interactions; E-Selectin; Endothelium, Vascular; Half-Life; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Lipopolysaccharides; Lung; Lung Diseases; Microcirculation; Neutrophil Infiltration; RNA Stability; RNA, Messenger; Sepsis; Streptococcus; Streptococcus pyogenes; Teichoic Acids; Tumor Necrosis Factor-alpha

This study was done to investigate the influence of Gram-negative and Gram-positive sepsis on the expression of the three isoforms of nitric oxide synthase (NOS) gene in rat liver and kidney. Male Sprague-Dawley rats were treated with lipopolysaccharide (LPS, 10 mg/kg i.v.) as an in vivo model for Gram-negative sepsis or lipoteichoic acid (LTA, 10 mg/kg i.v.) as an in vivo model for Gram-positive sepsis. Animals were killed 12 h and 24 h after i.v. treatment. NOS mRNA of the three isoforms was determined by RNase protection assay. NOS II gene expression was strongly induced after LPS or LTA treatment in rat liver and kidney, indicating the efficacy of this treatment to induce sepsis. We found no change of NOS I gene expression after LPS or LTA injection in rat liver and kidney. NOS III gene expression was increased about 8-fold 12 h and about 5-fold 24 h after induction of sepsis in the rat liver whereas in the kidney there was no significant increase in NOS III gene expression. After correction for length NOS III mRNA was about 4- and 40-fold more abundant 12 h and 24 h after LPS treatment than NOS II mRNA in the liver, respectively. Twelve and 24 h after LTA treatment NOS III mRNA was about 18- and 140-fold more abundant than NOS II in the liver. These findings suggest that NOS III is an even more potent source of NO than NOS II in the liver after stimulation with LPS or LTA.

Topics: Animals; Gene Expression Regulation; Injections, Intravenous; Isoenzymes; Kidney; Lipopolysaccharides; Liver; Male; Nitric Oxide Synthase; Rats; Rats, Sprague-Dawley; Sepsis; Teichoic Acids

A sandwich immunoassay has been developed for the detection of lipoteichoic acid (LTA), a major cell wall constituent of gram-positive bacteria, from whole blood and ISOLATOR supernate. Monoclonal antibodies were produced to purified LTA from Streptococcus mutans, BHT and were further characterized for crossreactivity with gram-positive and negative bacteria and for reactivity to substituted and unsubstituted LTA. Eight monoclonal antibodies were identified that reacted exclusively with gram-positive bacteria. Those antibodies able to capture 3H-LTA were chosen to develop a sandwich immunoassay. The assay has a sensitivity of 0.2 ng LTA/mL in PBS, 0.5 ng/mL in whole blood and 2.0 ng/mL in whole blood that has been processed through the ISOLATOR. Further development of this assay may lead to the rapid detection of LTA from body fluids.

Topics: Antigens, Bacterial; Enzyme-Linked Immunosorbent Assay; Gram-Negative Bacteria; Gram-Positive Bacteria; Humans; Immunoassay; Lipopolysaccharides; Sepsis; Teichoic Acids

Sixty-three strains of Group D streptococci and viridans streptococci isolated from blood cultures during a two year period were typed to the species level with conventional biochemical tests and API Strep. Streptococcus faecalis was the most common species isolated followed by S. sanguis, S. mitis and S. constellatus (S. milleri). One of the two isolates of S. faecium was a contamination. The reported increasing frequency of this organism and other Group D and viridans streptococci as well as the association of S. bovis with malignant bowel disease indicate the need for full identification of streptococcal isolates from blood cultures. Pronounced surface hydrophobicity as measured with the salt aggregation test (SAT) was expressed by 59/63 (94%) of the blood culture isolates whereas strains isolated from commercial fermentation products and strains passaged several times were hydrophilic. In the presence of human serum albumin which binds to lipoteichoic acid only one strain decreased in surface hydrophobicity. The surface hydrophobicity of two strains even slightly increased indicating that lipoteichoic acid but marginally contributes to surface hydrophobicity of streptococcal cells from these species.

Topics: Adolescent; Adult; Aged; Blood; Child; Child, Preschool; Enterococcus faecalis; Humans; Infant; Lipopolysaccharides; Middle Aged; Phosphatidic Acids; Sepsis; Streptococcal Infections; Streptococcus; Streptococcus sanguis; Surface Properties; Teichoic Acids

Topics: Antibodies, Bacterial; Endocarditis, Bacterial; Humans; Lipopolysaccharides; Osteomyelitis; Peptidoglycan; Phosphatidic Acids; Sepsis; Staphylococcal Infections; Staphylococcus aureus; Teichoic Acids