Search

Staphylococcus aureus is a significant bacterial pathogen that may penetrate through the barrier into the epidermis and dermis of the skin. We hypothesized that the S. aureus cell wall product lipoteichoic acid (LTA) may contribute to the development of inflammation and skin barrier defects; however, the effects of LTA in vivo are not well understood. In this study, we examined the effects induced by intradermal S. aureus LTA. We found that keratinocytes in LTA-treated skin were highly proliferative, expressing 10-fold increased levels of Ki67. Furthermore, we observed that LTA caused damage to the skin barrier with substantial loss of filaggrin and loricrin expression. In addition, levels of the IL-1 family of inflammatory cytokines, as well as the neutrophil-attracting chemokines Cxcl1 and Cxcl2, were increased. Concomitantly, we observed significant numbers of neutrophils infiltrating into the epidermis. Finally, we determined that LTA-induced signals were mediated in part through IL-1, because an IL-1 receptor type 1 antagonist ameliorated the effects of LTA, blocking neutrophil recruitment and increasing the expression of skin barrier proteins. In summary, we show that S. aureus LTA alone is sufficient to promote keratinocyte proliferation, inhibit expression of epidermal barrier proteins, induce IL-1 signaling, and recruit cells involved in skin inflammation., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is recognized by Toll-like receptor 2, expressed on certain mammalian cell surfaces, initiating signaling cascades that include nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase. There are many structural and functional varieties of LTA, which vary according to the different species of gram-positive bacteria that produce them. In this study, we examined whether LTA isolated from Staphylococcus aureus (aLTA) affects the expression of junction proteins in keratinocytes. In HaCaT cells, tight junction-related gene expression was not affected by aLTA, whereas adherens junction-related gene expression was modified. High doses of aLTA induced the phosphorylation of extracellular signal-regulated protein kinases 1 and 2, which in turn induced the epithelial-mesenchymal transition (EMT) of HaCaT cells. When cells were given a low dose of aLTA, however, NF-κB was activated and the total cell population increased. Taken together, our study suggests that LTA from S. aureus infections in the skin may contribute both to the outbreak of EMT-mediated carcinogenesis and to the genesis of wound healing in a dose-dependent manner.

Background and Aim: Few drugs have been found satisfactory in the treatment of nonsteroidal anti-inflammatory drugs (NSAIDs)-induced enteropathy. Toll-like receptor (TLR) 4 and aberrant leukocyte migration to the intestinal mucosa are reported to be involved in the pathology of intestinal enteropathy and TLR2 agonists have been found to evoke hyposensitivity to TLR4 stimulation in vitro. In this study, we investigated whether and how lipoarabinomannan (LAM) or lipoteichoic acid (LTA), TLR2 agonists, attenuated indomethacin (IND)-induced intestinal damage., Methods: LAM (0.5 mg/kg) or LTA (15 mg/kg) was administered intraperitoneally to mice before IND (10 mg/kg) administration. Disease activity was evaluated macroscopically and histologically. In the migration analysis, fluorescence-labeled leukocyte movement in the intestinal microvessels was observed by intravital microscopy. Expression of P-selectin, MAdCAM-1, TLR2, TLR4, and F4/80 was observed immunohistochemically. In the in vitro analysis, RAW264.7 macrophage cells were preincubated with LAM and stimulated with lipopolysaccharide (LPS), and the mRNA expression levels of TLR4, tumor necrosis factor-α, and interleukin-12p40 were measured., Results: Pretreatment with LAM or LTA significantly decreased IND-induced injury as well as decreased leukocyte infiltration. Pretreatment with LAM decreased IND-induced TLR4 expression on F4/80(+) macrophages, the level of P-selectin expression, and leukocyte migration in the small intestinal vessels. In the in vitro study, a single administration of LAM decreased TLR4 mRNA expression and inhibited the increase in mRNA expression of inflammatory cytokines by LPS in a dose-dependent manner., Conclusion: TLR2 agonists attenuated IND-induced small intestinal lesions and leukocyte infiltration probably by suppressing the TLR4 signaling pathway in tissue macrophages., (© 2015 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Objective: Interleukin-6 (IL-6), which is increased in patients who are suffering from septic shock, is an important mediator of the inflammatory response. Here, we examined the priming effect of lipoteichoic acid (LTA) and lipopolysaccharide (LPS) on IL-6 production in a monocyte-like cell line., Methods: THP-1 cells were primed by treatingwith a low or high dose of LTA isolated from Staphylococcus aureus (aLTA) and then re-treated with LPS. IL-6 production, receptor expression, and the variation of signaling molecules were examined by ELISA, reverse transcriptase polymerase chain reaction, and western blotting, respectively., Results: LPS-mediated IL-6 production was dramatically increased in THP-1 cells pretreated with a low dose aLTA, while it was significantly decreased when a high dose of aLTA was given along with LPS. LPS-induced IL-6 production in low dose aLTA priming cells mediated by NF-κB and MAPKs pathways, and Akt functioned as a negative regulator of IL-6 production. Together, the results of this study suggest that different doses of bacterial cell surface components can mediate a diverse range of responses with respect to inflammatory cytokine production.

The study evaluated the effects of repeated oral exposure to LPS and lipoteichoic acid (LTA) on immune responses of dairy cows. Thirty pregnant Holstein cows were randomly assigned to two treatment groups. Cows received orally either 2 ml of 0.85% sterile saline solution (control group), or 2 ml of sterile saline solution containing three doses of LPS from Escherichia coli 0111 : B4 along with a flat dose of LTA from Bacillus subtilis. Blood and saliva samples were collected and analyzed for serum amyloid A (SAA); LPS-binding protein (LBP); anti-LPS plasma IgA, IgG and IgM; TNF-α; and IL-1. Results showed greater concentrations of IgA in the saliva of treated cows compared with the controls (P < 0.01). Treated cows had lower plasma concentrations of anti-LPS IgA, IgG and IgM Abs, and TNF-α than the controls (P < 0.05). There was a tendency for the concentrations of plasma LBP (P = 0.06) and haptoglobin (P = 0.10) to be lesser in the treatment group, although no differences were found in the concentration of plasma SAA and IL-1 (P > 0.10). Overall, the results of this study indicate that repeated oral administration with LPS and LTA stimulates innate and humoral immune responses in periparturient dairy cows.

Background: Previously, we had identified a specific whole blood-derived microRNAs (miRNAs) signature in mice following in vivo injection of lipopolysaccharide (LPS) originated from Gram-negative bacteria. This study was designed to profile the circulating miRNAs expression in mice exposed to lipoteichoic acid (LTA) which is a major component of the wall of Gram-positive bacteria., Results: C57BL/6 mice received intraperitoneal injections of 100 μg of LTA originated from Bacillus subtilis, Streptococcus faecalis, and Staphylococcus aureus were killed 6 h and the whole blood samples were obtained for miRNA expression analysis using a miRNA array (Phalanx miRNA OneArray® 1.0). Up-regulated expression of miRNA targets in the whole blood, serum and white blood cells (WBCs) of C57BL/6 and Tlr2-/- mice upon LTA treatment in 10, 100, or 1000 ug concentrations was quantified at indicated time (2, 6, 24, and 72 h) using real-time RT-PCR and compared with that in the serum of C57BL/6 mice injected with 100 ug of LPS. A significant increase of 4 miRNAs (miR-451, miR-668, miR-1902, and miR-1904) was observed in the whole blood and the serum in a dose- and time-dependent fashion following LTA injection. Induction of miRNA occurred in the serum after 2 h and persisted for at least 6 h. No increased expression of these 4 miRNAs was found in the WBCs. Higher but not significant expression level of these 4 miRNAs were observed following LTA treatment in the serum of Tlr2-/-against that of C57BL6 mice. In contrast, LPS exposure induced moderate expression of miR-451 but not of the other 3 miRNA targets., Conclusions: We identified a specific circulating miRNA signature in mice exposed to LTA. That expression profile is different from those of mice exposed to LPS. Those circulating miRNAs induced by LTA or LPS treatment may serve as promising biomarkers for the differentiation between exposures to Gram-positive or Gram-negative bacteria.

Background: Lipopolysaccharide (LPS) is recognized as the most potent microbial mediator presaging the threat of invasion of Gram-negative bacteria that implicated in the pathogenesis of sepsis and septic shock. This study was designed to examine the microRNA (miRNA) expression in whole blood from mice injected with intraperitoneal LPS., Methods: C57BL/6 mice received intraperitoneal injections of varying concentrations (range, 10-1000 μg) of LPS from different bacteria, including Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Salmonella enterica, and Serratia marcescens and were killed 2, 6, 24, and 72 h after LPS injection. Whole blood samples were obtained and tissues, including lung, brain, liver, and spleen, were harvested for miRNA expression analysis using an miRNA array (Phalanx miRNA OneArray® 1.0). Upregulated expression of miRNA targets in the whole blood of C57BL/6 and Tlr4(-/-) mice injected with LPS was quantified using real-time RT-PCR and compared with that in the whole blood of C57BL/6 mice injected with lipoteichoic acid (LTA) from Staphylococcus aureus., Results: Following LPS injection, a significant increase of 15 miRNAs was observed in the whole blood. Among them, only 3 miRNAs showed up-regulated expression in the lung, but no miRNAs showed a high expression level in the other examined tissues. Upregulated expression of the miRNA targets (let-7d, miR-15b, miR-16, miR-25, miR-92a, miR-103, miR-107 and miR-451) following LPS injection on real-time RT-PCR was dose- and time-dependent. miRNA induction occurred after 2 h and persisted for at least 6 h. Exposure to LPS from different bacteria did not induce significantly different expression of these miRNA targets. Additionally, significantly lower expression levels of let-7d, miR-25, miR-92a, miR-103, and miR-107 were observed in whole blood of Tlr4(-/-) mice. In contrast, LTA exposure induced moderate expression of miR-451 but not of the other 7 miRNA targets., Conclusions: We identified a specific whole blood-derived miRNA signature in mice exposed to LPS, but not to LTA, from different gram-negative bacteria. These whole blood-derived miRNAs are promising as biomarkers for LPS exposure.

Mastitic milk is associated with increased bovine protease activity, such as that from plasmin and somatic cell enzymes, which cause proteolysis of the caseins and may reduce cheese yield and quality. The aim of this work was to characterize the peptide profile resulting from proteolysis in a model mastitis system and to identify the proteases responsible. One quarter of each of 2 cows (A and B) was infused with lipoteichoic acid from Staphylococcus aureus. The somatic cell counts of the infused quarters reached a peak 6h after infusion, whereas plasmin activity of those quarters also increased, reaching a peak after 48 and 12h for cow A and B, respectively. Urea-polyacrylamide gel electrophoretograms of milk samples of cow A and B obtained at different time points after infusion and incubated for up to 7 d showed almost full hydrolysis of β- and α(S1)-casein during incubation of milk samples at peak somatic cell counts, with that of β-casein being faster than that of α(S1)-casein. Two-dimensional gel electrophoretograms of milk 6h after infusion with the toxin confirmed hydrolysis of β- and α(S1)-casein and the appearance of lower-molecular-weight products. Peptides were subsequently separated by reversed-phase HPLC and handmade nanoscale C(18) columns, and identified by matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Twenty different peptides were identified and shown to originate from α(s1)- and β-casein. Plasmin, cathepsin B and D, elastase, and amino- and carboxypeptidases were suggested as possible responsible proteases based on the peptide cleavage sites. The presumptive activity of amino- and carboxypeptidases is surprising and may indicate the activity of cathepsin H, which has not been reported in milk previously., (Copyright © 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Activation of prophenoloxidase and synthesis of antimicrobial peptides (AMPs) are two important innate immune mechanisms in insects. In the current study, we investigated immune responses activated by three major bacterial components, lipopolysaccharide (LPS) (including rough mutants of LPS), lipoteichoic acid (LTA), and peptidoglycan (PG), in the larvae of a lepidopteran insect, Manduca sexta. We found that two DAP (diaminopimelic acid)-type PGs from Escherichia coli and Bacillus subtilis were much more potent than LPS and LTA from the respective bacteria as well as a Lysine-type PG in activation of prophenoloxidase in M. sexta larval plasma in vitro. Transcription levels of AMP genes, such as Attacin, Lebocin and Moricin genes, in the hemocytes and fat body of larvae were significantly induced by smooth LPS (TLR4grade) and rough mutants of LPS (TLRgrade), synthetic lipid A, LTA, and PG. LPS from E. coli and LTA from B. subtilis activated AMP expression to significantly higher levels than PGs from the respective bacterial strains, and smooth LPS were more potent than lipid A and rough mutants of LPS in activation of AMP expression. Our results demonstrated for the first time that LTA can activate AMP expression, and different moieties of LPS may synergistically activate AMP expression in M. sexta., (2010 Elsevier Ltd. All rights reserved.)

Background: Bifidobacteria are a natural part of the bacterial flora in the human body and have a symbiotic bacteria-host relationship with human beings. Aging is associated with reduced number of beneficial colonic bifidobacteria and impaired immunity. Lipoteichoic acid is a major constituent of the cell wall of bifidobacteria which is important for bacterial survival, growth, and function. The possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria is presently unknown., Objective: The aim of the present study was to investigate possible anti-aging effects of lipoteichoic acid isolated from bifidobacteria on senescent mice artificially induced by chronic injection of d-galactose and explore potential anti-aging's mechanisms., Methods: Mice were artificially induced senescence by consecutive injection of d-galactose (100mg/kg) once daily for 7weeks and lipoteichoic acid from bifidobacterium bifidum, was simultaneously administered to them once a week by intraperitoneal infusion. Mice were sacrificed, blood and other samples were collected at the indicated time. Anti-oxidation activity in brain, histology of tissue, gene expression, lymphocyte's DNA damage and cytokine production of lymphocytes in vitro and in vivo were measured., Results: Lipoteichoic acid could significantly improve general appearance of the aging model mice, improve anti-oxidation activity in brain, increase IL-2 level and decrease TNF-alpha level in vitro and in vivo, respectively. Besides, LTA remarkably inhibited DNA damage in the both splenic lymphocytes and circulating lymphocytes. Moreover, LTA could decrease p16 expression while increase c-fos expression in the d-galactose treated mice., Conclusion: Taken together, the results indicated, for the first time, that LTA could suppress the aging process via the following several mechanisms, including enhancement of anti-oxidation activity in brain, improvement of immune function and alteration of gene expression.

Objective: Pneumonia is characterized by an acute inflammatory response in the lung, which is frequently associated with changes in coagulation and fibrinolysis in the bronchoalveolar space. Here, we compared the effects of lipoteichoic acid (LTA), a major cell wall component of Gram-positive bacteria, and lipopolysaccharide (LPS), in the human bronchoalveolar space., Design: Controlled in vivo volunteer study., Setting: Clinical research unit., Subjects: Twenty-three healthy nonsmoking male volunteers., Interventions: Sterile saline was instilled into a lung subsegment followed by bronchoscopic instillation of either LTA (Staphylococcus aureus, at a dose of 4, 20, or 100 ng/kg body weight) or LPS (Escherichia coli, 4 ng/kg body weight) into the contralateral lung. Bronchoalveolar lavage fluid was obtained 6 hours thereafter., Measurements and Main Results: Bronchial instillation of LTA- or LPS-activated bronchoalveolar coagulation, as reflected by increases in the levels of thrombin-antithrombin complexes, d-dimer, and soluble tissue factor. Concurrently, LTA and LPS inhibited anticoagulant mechanisms, as indicated by reductions in antithrombin, Protein C, and Activated Protein C concentrations together with elevated levels of soluble thrombomodulin. Both LTA and LPS administration was associated with an inhibition of pulmonary fibrinolysis, as measured by a reduction in plasminogen activator activity and elevated levels of plasminogen activator inhibitor type I., Conclusions: This study is the first to describe the effects of LTA on hemostasis in humans, demonstrating that LTA induces similar changes in the human bronchoalveolar space as LPS, characterized by activation of coagulation with concurrent inhibition of anticoagulant and fibrinolytic pathways.

Background: Bacterial infection of the pleural space often causes adherence of the pleural membranes by fibrous tissue, probably mediated by inflammation initiated by bacterial cell-wall motifs, including lipoteichoic acid-T (LTA-T). We postulated that therapeutically administered LTA-T might produce a similar effect, achieving control of malignant pleural effusion (pleurodesis)., Methods: Patients with histocytologically proven symptomatic malignant pleural effusions were included in this phase I toxicity and dose-escalation study, An indwelling pleural catheter was placed in the pleural effusion to drain the fluid fully. A control dose of intrapleural saline was administered after complete drainage (day 1) and pleural-fluid production was recorded for 7 days. On day 7 a single dose of intrapleural LTA-T (increasing in each patient) was administered and pleural-fluid production was monitored for a further 7 days. Long-term fluid control was recorded. This study is registered as an International Standard Randomised Controlled Trial, ISRCTN44367564., Findings: Between November, 2004, and November, 2005, 14 patients were enrolled on the trial at the Oxford Centre for Respiratory Medicine (Oxford, UK). 13 of 14 patients received escalated doses of LTA-T. A dose-limiting toxic effect (ie, systemic inflammation) occurred at 3000 microg, and a therapeutic dose of 750-1500 microg was established. Toxic effects were mild and had no consistent pattern at the therapeutic dose. Pleural-fluid production decreased significantly after a dose of at least 750 microg LTA-T, compared with saline control (mean fluid production after saline control 1244 mL [SD 933], mean fluid production after LTA-T 394 mL [SD 375], mean difference -850 mL [SD 699], p=0.028), and six of seven (86%) patients achieved pleural-fluid control at 1 month with no further intervention., Interpretation: The toxic effects of intrapleural LTA-T seem to be mild and favourable when compared with the toxicity profiles of standard pleurodesis agents. There is early evidence of LTA-T-induced pleurodesis efficacy, suggesting that this might be a viable therapeutic strategy for the control of malignant pleural effusion.

Lipoteichoic acid (LTA) is a major outer cell wall component of Gram-positive bacteria that has been implicated as an important factor in the inflammatory response following bacterial infection. In vitro data indicate roles for TLR2, platelet-activating factor receptor (PAFR), CD14, and LPS-binding protein (LBP) in cellular responsiveness to LTA, whereas the mechanisms contributing to LTA effects in vivo have never been investigated. Using mice deficient for LBP, CD14, TLR2, TLR4, or PAFR, we now examined the role of these molecules in pulmonary inflammation induced by highly purified LTA in vivo. Although pulmonary LBP increased dose-dependently following administration of LTA, the inflammatory response was unaltered in LBP-/- mice. TLR2 proved to be indispensable for the initiation of an inflammatory response, as polymorphonuclear cell influx, TNF-alpha, keratinocyte-derived chemokine, and MIP-2 release were abolished in TLR2-/- mice. Minor effects such as moderately decreased TNF-alpha and MIP-2 levels were observed in the absence of CD14, indicating a role for CD14 as a coreceptor. Quite surprisingly, the absence of TLR4 greatly diminished pulmonary inflammation and the same phenotype was observed in PAFR-/- animals. In contrast to all other mice studied, only TLR4-/- and PAFR-/- mice displayed significantly elevated IL-10 pulmonary concentrations. These data suggest that TLR2 is the single most important receptor signaling the presence of LTA within the lungs in vivo, whereas TLR4 and PAFR may influence lung inflammation induced by LTA either by sensing LTA directly or through recognition and signaling of endogenous mediators induced by the interaction between LTA and TLR2.

Lipoteichoic acid (LTA), an immunostimulatory component of the cell walls of gram positive bacteria, has pro-inflammatory effects in vitro and in vivo. However, one in vivo study concluded that LTA had no noticeable effects on leukocyte recruitment. In this study we investigated the effects of highly purified LTA, prepared by butanol extraction (Bu-LTA) at room temperature, on in vivo leukocyte adhesion. Using intravital microscopy we measured adhesion of leukocytes in mesenteric post-capillary venules of rats and mice. Topical superfusion of Bu-LTA (1 microg/ml) in rats significantly (p<0.05) increased adhesion within 30 min. By contrast, hot phenol-extracted LTA did not increase adhesion. Alkaline hydrolysis of Bu-LTA removed alanine residues and prevented adhesion. Also, pre-administration of anti-rat beta2-integrin antibody abolished Bu-LTA-induced adhesion. Finally, intraperitoneal injection of Bu-LTA (100 microg/ml) into mice also significantly (p<0.01) increased leukocyte adhesion measured at 60 min. In conclusion, Bu-LTA with intact alanine residues promotes beta2-integrin-dependent leukocyte adhesion in vivo.

Background: FcepsilonRI on the surface of mast cells (MCs) plays a central role in allergic responses. Recent evidence shows that exposure to microbial components corresponds with a significant reduction in the risk for allergic diseases. Although many reports suggest that this is due to changes in T-cell functions, how MC functions are altered by bacterial infection remains unknown., Objective: We sought to elucidate the effect of bacterial infection on MC function and expression of Fc receptors, such as FcepsilonRI., Methods: Isolated human pulmonary MCs and a human MC line (LAD2) were stimulated with bacterial components, and the function and surface expression of Fc receptors were measured., Results: Lipoteichoic acid (LTA) and peptidoglycan, but not LPS, flagellin, or 3CpG-oligodeoxynucleotide, reduced the expression of FcepsilonRI on LAD2 cells. An antibody to Toll-like receptor (TLR) 2 partially blocked the effect of LTA but not peptidoglycan. Both LTA and peptidoglycan reduced MC degranulation caused by an antigen-specific IgE. Furthermore, exposure of pulmonary MCs to LTA reduced both FcepsilonRI expression and IgE-induced degranulation. None of the bacterial components affected the expression of other Fc receptors, such as Fcgamma receptors or Fcalpha receptor I., Conclusions: Our results indicate that LTA reduces the surface expression of FcepsilonRI through TLR2 and suggests that TLR2 ligands could be used as a novel therapy for controlling allergic disorders., Clinical Implications: By knowing how bacterial components modulate MC function, we can expand our possibilities for therapeutic interventions of allergic diseases.

Staphylococcus aureus infections are known triggers for skin inflammation and can modulate immune responses. The present studies used model systems consisting of platelet-activating factor receptor-positive and -negative (PAF-R-positive and -negative) cells and PAF-R-deficient mice to demonstrate that staphylococcal lipoteichoic acid (LTA), a constituent of Gram-positive bacteria cell walls, acts as a PAF-R agonist. We show that LTA stimulates an immediate intracellular Ca2+ flux only in PAF-R-positive cells. Intradermal injections of LTA and the PAF-R agonist 1-hexadecyl-2-N-methylcarbamoyl glycerophosphocholine (CPAF) induced cutaneous inflammation in wild-type but not PAF-R-deficient mice. Systemic exposure to LTA or CPAF inhibited delayed-type hypersensitivity (DTH) reactions to the chemical dinitrofluorobenzene only in PAF-R-expressing mice. The inhibition of DTH reactions was abrogated by the addition of neutralizing antibodies to IL-10. Finally, we measured levels of LTA that were adequate to stimulate PAF-R in vitro on the skin of subjects with infected atopic dermatitis. Based on these studies, we propose that LTA exerts immunomodulatory effects via the PAF-R through production of the Th2 cytokine IL-10. These findings show a novel mechanism by which staphylococcal infections can inhibit Th1 reactions and thus worsen Th2 skin diseases, such as atopic dermatitis.

Objectives: Several case reports have described complete hematological remissions for patients with otherwise untreated acute myelogenous leukemia (AML) who receive hematopoietic growth factor therapy during complicating bacterial infections. This may be caused by indirect cytokine effects, but direct effects of infecting agents on the malignant cells are also possible because bacterial molecules can bind to specific receptors expressed by normal and malignant leukocytes. Lipoteichoic acid (LTA) is a cell wall component of gram-positive bacteria, and it can activate normal immunocompetent cells through binding to specific cell membrane receptors., Methods: We investigated effects of LTA derived from Enterococcus faecalis on in vitro cultured (i) normal peripheral blood mononuclear cells (PBMC); (ii) remaining T cells derived from patients with hematologic malignancies and chemotherapy-induced leukopenia; and (iii) native human AML cells., Results: Increased interleukin 1beta (IL1beta) and IL8 release by in vitro cultured normal PBMC was observed after stimulation with LTA at concentrations > or =5 microg/mL; these levels were lower than for lipopolysaccharide (LPS)-stimulated cells and LTA antagonized LPS-induced cytokine release by normal PBMC. In most cases LTA did not alter T-cell proliferation for patients with chemotherapy-induced leukopenia. The LTA effects on AML blasts were investigated for 62 consecutive patients. LTA altered either cytokine (granulocyte-macrophage colony-stimulating factor + stem cell factor + IL3)-dependent proliferation or the release of IL1beta/IL8 for 23 patients; the effects were divergent but increased proliferation/cytokine levels were most commonly observed., Conclusion: The LTA derived from E. faecalis can modulate the functional characteristics of normal leukocytes and native human AML blasts.

The innate immune system recognizes pathogen-associated molecular patterns (PAMP) to cope with evolving infections. Toll-like receptors (TLRs) play a pivotal role in recognition of PAMPs. In the course of infection not a single but rather a full panel of different microbial components interacts with distinct TLRs simultaneously. Only limited information is available on effects of combinations of TLR agonists. Here, we have analyzed the effects of lipoteichoic acid (LTA), CpG-DNA and combinations thereof on innate immune cells in vitro. Although proinflammatory cytokines like TNF-alpha were induced by these agonists in quite similar amounts, CpG DNA was superior in its potency to induce IL-12p40 reflecting important differences in the biological valence of LTA and CpG-DNA. When given in combination, LTA and CpG-DNA were additive in induction of TNF-alpha, IL-6 and nitric oxide in RAW 264 macrophages, peritoneal macrophages and dendritic cells. Additive effects were also observed in regard to TNF-alpha mRNA. In contrast, LTA suppressed IL12p40 secretion induced by CpG-DNA in RAW cells and peritoneal macrophages but not in dendritic cells. Intracellular signal cascades (NFkappaB and p38 MAP kinase) showed additive effects after simultaneous triggering. mRNA expression ofTLRs showed only minor regulation after CpG or LTA application and thus does not account for the additive/suppressive effects observed. These results indicate that the consequences of interaction of innate immune cells with microbial pattern depend on the responding cell type and might be differential for certain effector mechanisms. Thus, the pathogen-characteristic panel of TLR ligands will induce pathogen-specific innate responses decisive for the inflammatory reactions.

Objective: Streptococcus (S.) pyogenes is common cause of acute tonsillitis. Lipoteichoic acid (LTA), which is a common constitute of the cell surface of most gram positive bacteria, is known to act as a substance of bacterial site for adherence to epithelium and antiserum to LTA is reported to inhibit bacterial attachment to epithelial cells in vitro. Cholera toxin subunit B (CT-B) is known to be a mucosal adjuvant. The purpose of this study is to investigate whether intranasal immunization with LTA and CT-B may be a possible candidate for vaccine formulation., Methods: Six-week-old male BALB/c mice were assigned to three experimental groups, mice immunized with LTA and CT-B, with LTA alone and with phosphate buffered saline (PBS) as a control. Immunizations were performed intranasally every 2 days for 2 weeks in every group. At the 21 days after immunization, sera, pharyngeal washings and pharyngeal epithelial cells were taken. The levels of serum IgG and pharyngeal IgA antibodies to LTA were measured by enzyme-linked immunosorbent assay (ELISA). The adherence rates of S. pyogenes pretreated by pharyngeal washings to pharyngeal epithelial cells from the mice were determined by in vitro adherence assay., Results: The serum anti-LTA IgG antibody levels of either mice immunized with LTA and CT-B or mice immunized with LTA alone were significantly higher than those of mice administered with PBS alone. The pharyngeal anti-LTA IgA antibody levels of the mice immunized with LTA and CT-B were significantly higher than those of either mice with LTA alone or mice with PBS alone. The streptococcal adherence rates to pharyngeal epithelial cells were significantly decreased by pretreatment with pharyngeal washings from the mice immunized with LTA and CT-B as compared with pretreatment with those from either mice with PBS or mice with LTA alone., Conclusions: These data shows that intranasal immunization with LTA and CT-B evokes a good pharyngeal IgA response as well as systemic IgG response to LTA and inhibits streptococcal adherence to pharyngeal epithelial cells, suggesting that intranasal immunization with LTA and CT-B may be an effective approach to prevent streptococcal tonsillitis.

Exposure of macrophages to LPS induces a state of hyporesponsiveness to subsequent stimulation with LPS termed LPS desensitization or tolerance. To date, it is not known whether similar mechanisms of macrophage refractoriness are induced on contact with components of Gram-positive bacteria. In the present study, we demonstrate that pretreatment with highly purified lipoteichoic acid (LTA) results in suppression of cytokine release on restimulation with LTA in vitro and in vivo in both C3H/HeN and C3H/HeJ mice, but not in macrophages from Toll-like receptor (TLR)-2-deficient mice. Furthermore, desensitization in response to LPS or LTA exposure also inhibits responses to the other stimulus ("cross-tolerance"), suggesting that signaling pathways shared by TLR2 and TLR4 are impaired during tolerance. Finally, we show that LPS- or LTA-induced cross-tolerance is not transferred to hyporesponsive cells cocultured with LPS/LTA-responsive macrophages, showing that soluble mediators do not suffice for tolerance induction in neighboring cells.

We have examined the ability of peptidoglycan (PepG) and lipoteichoic acid (LTA) isolated from Staphylococcus aureus to induce the release of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and IL-10 in whole human blood and identified the cellular origins of these cytokines. Both PepG and LTA induced transient increases in TNF-alpha and IL-10 in plasma, with peak values at 6 and 12 h, respectively. IL-6 values increased throughout the experimental period (24 h). The TNF-alpha, IL-6, and IL-10 release induced by PepG and LTA was dose dependent. Only PepG was a potent inducer of TNF-alpha secretion. After stimulation of whole blood with PepG or LTA, very pure populations of monocytes (CD14 positive), T cells (CD2 positive), B cells (CD19 positive), and granulocytes (CD15 positive) were isolated by immunomagnetic separation and analyzed by reverse transcription-PCR for mRNA transcripts encoding TNF-alpha, IL-6, and IL-10. The TNF-alpha mRNA results were inconclusive. In contrast, PepG induced IL-6 and IL-10 mRNA accumulation in both T cells and monocytes. LTA, as well as lipopolysaccharide, induced IL-6 and IL-10 mRNA production in monocytes and possibly in T cells. Whether granulocytes and B cells produce cytokines in response to bacterial stimuli remains obscure. Blockade of the CD14 receptors with monoclonal antibodies (18D11) had no influence on the PepG-induced release of TNF-alpha but attenuated the LTA-induced release of the same cytokine. In conclusion, our data indicate that circulating T cells and monocytes contribute to cytokine production in sepsis caused by gram-positive bacteria.

Purpose: To investigate the role of lipoteichoic acid (LTA), one of the cell wall components in gram-positive bacteria in uveitis., Methods: Intraocular inflammation in rabbit eyes was induced by intravitreal or intravenous injections of LTA from Staphylococcus aureus or Streptococcus sanguis. The inflammation was monitored progressively with the laser flare-cell photometer, and examined by periodic clinical observations. Histological examinations were performed 24 hours after administration, and aqueous protein concentrations and cell counts were also determined., Results: Intraocular inflammation appeared within 6-9 hours of LTA intravitreal injection. became maximal at about 24-48 hours postinjection, and lasted for nearly 6 days. Intraocular inflammation was also induced by intravenous injection of LTA at a higher dose. Inflammation reached a peak 4-5 hours after injection, and rapidly disappeared in 24 hours. No cellular response was observed in intravenous LTA-treated eyes., Conclusions: This study demonstrates that LTAs from gram-positive bacteria have the biological activity to induce intraocular inflammation in rabbits by intravitreal or intravenous injection. Therefore, we suggest that LTA may play a role in the pathogenesis of uveitis as one of the etiological factors.

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.

This study describes activation of serum complement by lipoteichoic acid (LTA) from Streptococcus mutans OMZ 176, while in solution. Serum from 16 healthy students was taken. Test samples were incubated with increasing doses (1-5,000 micrograms/ml) of LTA or lipopolysaccharide (LPS) from Escherichia coli 0111:B4 for 1 h at 37 degrees C; then assayed for degradation of C3, C4 or factor B by crossed immunoelectrophoresis. Each preparation caused a significant (p < 0.05) dose-dependent conversion of C3. The response curves obtained were not statistically different. LPS was a stronger activator of the alternative pathway than LTA, as judged from analysis of C3 degradation in the presence of Mg2+/EGTA, and from their effects on factor B cleavage. LTA caused, however, pronounced alterations in the shape of C4 precipitation in the gels. Functional (hemolytic) assays showed that, when tested at 200 micrograms/ml, LTA and LPS triggered significant (p < 0.05) consumptions of both classical and alternative pathway proteins. LPS was a significantly (p < 0.05) stronger activator than LTA. Apparently, the C3 degradation found for this LTA involved the alternative pathway to a small extent; thus some other mechanism of fluid-phase C3 cleavage seemed also to be operative.

Lipoteichoic acid (LTA) was prepared from type III group B streptococci and administered by topical oral application or intravenous or intratracheal injection in weanling and adult white New Zealand rabbits. Tritiated [3H]LTA in tissues and body fluids was measured by scintillation spectrometry. Five minutes to 120 h after intravenous injection of 10 mg (17 x 10(6) dpm) of [3H]LTA, none was present in blood. Combined urine and fecal excretion peaked at 24 h and decreased over 5 days. There was no effect on collagen-induced platelet aggregation. [3H]LTA concentrations were greatest in colon, bone, stomach, and skin 1 day after intravenous injection. After a 5-mg oral dose (8.5 x 10(6) dpm) in an adult animal, fecal excretion peaked at 24 h and decreased after 4 days. No systemic absorption was noted. No [3H]LTA was found in any of seven tissues examined at autopsy 3 days after 1 to 5 mg/kg oral doses in weanling animals with normal or traumatized buccal mucosa. No effect was noted on platelet aggregation or serum complement, there was no increase in the incidence of nephrocalcinosis and the buccal mucosa remained histologically normal. Intratracheal injection of 0.5 to 2.5 mg/kg of LTA resulted in no tachypnea or alteration in blood gases. All animals remained healthy after LTA administration. The absence of toxicity and absorption in animals suggests that studies could be performed in humans to evaluate the safety and efficacy of oral LTA.

Fibronectin binds to Streptococcus pyogenes, and this binding is inhibited by lipoteichoic acid (LTA). Previous studies have shown that LTA can be released from S. pyogenes by treatment with penicillin. Penicillin released LTA from both S. pyogenes and Staphylococcus aureus; however, the binding of fibronectin correlated with the amount of LTA released only in the case of S. pyogenes. Contrarily, clindamycin decreased the ability of S. aureus to bind fibronectin without affecting the binding of fibronectin to S. pyogenes. Further studies indicated that LTA does not inhibit the binding of fibronectin to S. aureus. Fibronectin bound to S. pyogenes could be released from the cell surface by penicillin. Immunological analysis of the released fibronectin indicated that LTA was the only surface component which could be detected as a soluble complex with the released fibronectin. These studies suggest that LTA plays a central role in the binding of fibronectin to S. pyogenes and is not involved in the binding of fibronectin to S. aureus.