OBJECTIVETo observe the production of interleukin (IL)-1beta, IL-6 and tumor necrosis factor alpha (TNFalpha) from stimulated human fibroblast with abstract from cell wall of Actinomyces naeslundii ATCC19246.

METHODSThe abstract from the cell wall from Actinomyces naeslundii were extracted and purified with the method of purifying lipoteichoic acid(LTA) and stimulated the THP-1 with three different concentrations (1, 10, 100 mg/mL). The level of IL-1beta, IL-6 and TNFalpha in the supernatant was quantitatively analyzed by ELISA. Results Abstracts at the concentrations of 10, 100 mg/mL significantly produced IL-1beta, IL-6 and TNFalpha, especially 10 mg/mL.

CONCLUSIONThe abstract from cell wall of Actinomyces naeslundii may significantly increase IL-1beta, IL-6 and TNFalpha level in the supernatant of THP-1, and the increasing level is different with the concentrations.

Actinomyces ; Fibroblasts ; Humans ; Interleukin-1 ; Interleukin-1beta ; Interleukin-6 ; Lipopolysaccharides ; Teichoic Acids ; Tumor Necrosis Factor-alpha

OBJECTIVETo investigate the expression of Toll like receptor 2 (TLR2) and interleukin-1 beta (IL-1 beta) of cultured human periodontal ligament cells (HPDLCs) activated by Enterococcus faecalis (E. faecalis) lipoteichoic acid (LTA).

METHODSHPDLCs that were obtained from the periodontal tissues of healthy humans were maintained in proper condition. Flow cytometry was used to detect the expression of TLR2 on normal HPDLCs and infectious HPDLCs which were incubated with 0.1, 1, 10 microg mL(-1) E. faecalis LTA for 24 h. IL-1 beta was detected by enzyme linked immunosorbent assay (ELISA) after incubating with LTA of the above concentration for 12, 24 and 48 h or pretreated with TLR2 neutralizing antibody for 1 h and then co-cultured with 1 microg mL(-1) LTA for 24 h.

RESULTSE. faecalis LTA promoted the expression of TLR2 in normal HPDLCs. The difference had statistical significance (P<0.05). IL-1 beta secretion could be detected 12h after stimulation with LTA and increasingly escalate within 48h (P<0.05). TLR2 neutralizing antibody had no evident effect on IL-1 beta generation stimulating by E. faecalis LTA.

CONCLUSIONE. faecalis LTA can increase the expression of TLR2 and IL-1 beta in normal HPDLCs.

Cell Line ; Enterococcus faecalis ; Humans ; Interleukin-1beta ; Lipopolysaccharides ; Periodontal Ligament ; Teichoic Acids ; Toll-Like Receptor 2

BACKGROUND: Toll-like receptors (TLRs) are expressed by human epidermal keratinocytes and are involved in immune responses. OBJECTIVE: The goal of this was to investigate the expression of TLR2 in response to bacterial antigens, cytokines, and different calcium concentrations. METHODS: The expression of TLR2 was assessed after stimulation by lipoteichoic acid (LTA) and streptolysin O (SLO). In addition, TLR2 expression was evaluated after treatment with IFN-gamma and TNF-alpha, and different concentrations of calcium. The expression levels of TLR2 mRNA and protein were studied using RT-PCR and Western blot analysis. RESULTS: Cultured human epidermal keratinocytes constitutively expressed TLR2 and the expression was stimulated by LTA and SLO; in addition, IFN-gamma and TNF-alpha upregulated TLR2 expression. However, the changes in TLR2 expression associated with the calcium concentrations were insignificant. CONCLUSION: TLR2 expression increased with the concentration and duration of bacterial pathogens and this increase was amplified by several cytokines, from activated keratinocytes and other cells.
Antigens, Bacterial ; Bacterial Proteins ; Blotting, Western ; Calcium ; Cytokines ; Humans ; Keratinocytes ; Lipopolysaccharides ; RNA, Messenger ; Streptolysins ; Teichoic Acids ; Toll-Like Receptor 2 ; Toll-Like Receptors ; Tumor Necrosis Factor-alpha

BACKGROUND: We evaluated the effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on the production of tumor necrosis factor-alpha (TNF-alpha) and expression of nuclear factor-kappaB (NF-kappaB) in stimulated THP-1 cells, a human monocyte cell line. MATERIALS AND METHODS: We evaluated the cytotoxic effect of 15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), one of natural PPAR-gamma ligands, using commercial cell proliferation assay. Cells were pretreated with 15d-PGJ(2) and then stimulated with lipopolysaccharide (LPS) or lipoteichoic acid (LTA). The amount of TNF-alpha was measured by using commercial ELISA method. NF-kappaB activation was evaluated by Western blot analysis. RESULTS: 15d-PGJ(2) showed dose-dependent cytotoxic effect on the tested cells after 4 hr of treatment. Stimulation of cells by LPS or LTA induced TNF-alpha production. TNF-alpha production was markedly decreased in the cells pretreated with 15d-PGJ(2) compared to cells treated only with LPS or LTA in a dose-dependent manner. Pretreatment of 15d-PGJ(2) reduced LPS or LTA induced NF-kappaB expression in the nuclear extracts of THP-1 cells. CONCLUSION: 15d-PGJ(2) pretreatment decreased TNF-alpha production from the THP-1 cells stimulated by LPS or LTA, and this assumed to be associated with inhibition of NF-kappaB activation.
Blotting, Western ; Cell Line ; Cell Proliferation ; Enzyme-Linked Immunosorbent Assay ; Humans ; Ligands ; Lipopolysaccharides ; Monocytes ; NF-kappa B ; Peroxisomes ; Teichoic Acids ; Tumor Necrosis Factor-alpha

OBJECTIVETo study the effect and mechanism of soluble dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (sDC-SIGN) on the phagocytosis of Staphylococcus aureus (S. aureus) by immature dendritic cells (imDCs).

METHODSFlow cytometry was employed to examine the effect of sDC-SIGN on the phagocytosis of S. aureus by imDCs. Enzyme-linked immunosorbent assay (ELISA) was used to analyze the binging of sDC-SIGN to S. aureus, lipoteichoic acid (LTA) and lipopolysaccharides (LPS) and investigate the effect of the ligands mannan and LTA and anti-DC-SIGN antibodies 1C6 and 4H3 on the binging of sDC-SIGN to S. aureus.

RESULTSsDC-SIGN inhibited the phagocytosis of S. aureus by imDCs. sDC-SIGN bound to S. aureus in a Ca(2+)-dependent manner. sDC-SIGN concentration-dependently bound to LTA, but not to LTA, and the binging of sDC-SIGN to S. aureus was blocked by mannan, LTA, 1C6 and 4H3.

CONCLUSIONsDC-SIGN preferentially binds to the carbohydrate constituents on S. aureus to affect the binding between membrane-bound DC-SIGN and S. aureus, thus suppressing the phagocytosis of S. aureus by imDCs.

Cell Adhesion Molecules ; metabolism ; Dendritic Cells ; cytology ; metabolism ; Humans ; Lectins, C-Type ; metabolism ; Lipopolysaccharides ; Phagocytosis ; Receptors, Cell Surface ; metabolism ; Staphylococcus aureus ; Teichoic Acids

We recently reported a Philyra pisum lectin (PPL) that exerts mitogenic effects on human lymphocytes, and its molecular characterization. The present study provides a more detailed characterization of PPL based on the results from a monosaccharide analysis indicating that PPL is a glycoprotein, and circular dichroism spectra revealing its estimated alpha-helix, beta-sheet, beta-turn, and random coil contents to be 14.0%, 39.6%, 15.8%, and 30.6%, respectively. These contents are quite similar to those of deglycosylated PPL, indicating that glycans do not affect its intact structure. The binding properties to different pathogen-associated molecular patterns were investigated with hemagglutination inhibition assays using lipoteichoic acid from Gram-positive bacteria, lipopolysaccharide from Gram-negative bacteria, and both mannan and beta-1,3-glucan from fungi. PPL binds to lipoteichoic acids and mannan, but not to lipopolysaccharides or beta-1,3-glucan. PPL exerted no significant antiproliferative effects against human breast or bladder cancer cells. These results indicate that PPL is a glycoprotein with a lipoteichoic acid or mannan-binding specificity and which contains low and high proportions of alpha-helix and beta-structures, respectively. These properties are inherent to the innate immune system of P. pisum and indicate that PPL could be involved in signal transmission into Gram-positive bacteria or fungi.
beta-Glucans ; Breast ; Circular Dichroism ; Fungi ; Glycoproteins ; Gram-Negative Bacteria ; Gram-Positive Bacteria ; Hemagglutination ; Humans ; Immune System ; Lipopolysaccharides ; Lymphocytes ; Mannans ; Polysaccharides ; Sensitivity and Specificity* ; Teichoic Acids ; Urinary Bladder Neoplasms

OBJECTIVES: Toll-like receptors (TLRs) detect microbial infections and they can directly induce innate host defense responses. TLR 2 has been shown to be primarily involved in the recognition of peptidoglycans and lipoteichoic acid of gram positive bacteria. TLR 4 recognizes lipopolysaccharides and lipoteichoic acids from both gram-negative and gram-positive bacteria. Both mutations lead a reduced capacity to elicit inflammation and they increase the risk for gram-positive and negative infections. This study was performed to investigate the expressions of TLR 2 and 4 and their mutations in patients suffering with otitis media and middle ear effusion. METHODS: Middle ear fluid samples were collected from 40 otitis media effusion (OME) patients who had ventilating tubesinserted. Bacteria in the effusion fluid were detected by standard bacterial culture. The secreted IgG, IgA and IgM were measured by Enzyme-linked immunosorbent assay. TLR 2 and 4 were assessed by performing RT-PCR. The genomic DNA from each patient was isolated from the middle ear fluid samples that were collected from 60 OME patients, and the presence of mutations was determined by performing restriction digestion and DNA sequencing analysis. RESULTS: Among the 40 middle ear fluid samples, bacteria were detected in 13 middle ear fluid samples. The amounts of IgM, IgA, and IgG were 151.20+/-60.94 ng/mL, 21.59+/-7.96 ng/mL and 11.55+/-16.98 ng/mL, respectively. TLR 2 and 4 were expressed in the middle ear fluid and the expression of TLR 2 was higher than that of TLR 4. However, there was no correlation between the expressions of TLR 2 and 4, and the concentration of immunoglobulin or the presence of bacteria (P>0.05). There ware no mutations of TLR 2 (Arg753Gln, Arg677Trp) and TLR 4 (Asp299Gly, Thr399Ile). CONCLUSION: TLR 2 and 4 were expressed in all the middle ear fluid samples of OME, but the mutations of TLR 2 and 4 were not detected. TLR 2 and 4 may play a vital role in the immunological responses of patients with OME.
Bacteria ; Digestion ; DNA ; Ear, Middle ; Enzyme-Linked Immunosorbent Assay ; Gram-Positive Bacteria ; Humans ; Immunoglobulin A ; Immunoglobulin G ; Immunoglobulin M ; Immunoglobulins ; Inflammation ; Lipopolysaccharides ; Otitis ; Otitis Media ; Otitis Media with Effusion ; Peptidoglycan ; Sequence Analysis, DNA ; Stress, Psychological ; Teichoic Acids ; Toll-Like Receptors

To explore the potential of lipoteichoic acid (LTA) induced cardioprotection against ischemia-reperfusion (I/R) injury in isolated rat hearts and whether endogenous nitric oxide (NO) participates in the protection, the rats were pretreated with LTA (1 mg/kg, i.p.) 24 h before the experiment, and the isolated hearts were subjected to 30 min no-flow normothermic global ischemia and 60 min reperfusion after a 20-min stabilization period by the langendorff method. Cardiac functions were evaluated at the end of stabilization, and at 30 min, 60 min of reperfusion. The amounts of MB isoenzyme of creatine kinase (CK-MB), lactate dehydrogenase(LDH) and total NO oxidation products in the coronary effluent were measured spectrophotometrically at the end of reperfusion. It was revealed that pretreatment with LTA could significantly improve the recovery of cardiac function, reduce the release of CK-MB and LDH, and increase the concentrations of NO in coronary effluent. The protective effects were abrogated by pretreatment of the rats with L-NAME. It was concluded that LTA could induce the delayed cardioprotection against I/R injury, and endogenous NO may be involved in the mechanisms.
Animals ; Cardiotonic Agents ; pharmacology ; Creatine Kinase ; metabolism ; Creatine Kinase, MB Form ; In Vitro Techniques ; Ischemic Preconditioning, Myocardial ; Isoenzymes ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; pharmacology ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Nitric Oxide ; metabolism ; Rats ; Rats, Wistar ; Teichoic Acids ; pharmacology

Excessive microglial cell activation is related to the progression of chronic neuro-inflammatory disorders. Heme oxygenase-1 (HO-1) expression mediated by the NFE2-related factor (Nrf-2) pathway is a key regulator of neuro-inflammation. Nardostachys chinensis is used as an anti-malarial, anti-nociceptive, and neurotrophic treatment in traditional Asian medicines. In the present study, we examined the effects of an ethyl acetate extract of N. chinensis (EN) on the anti-neuro-inflammatory effects mediated by HO-1 up-regulation in Salmonella lipopolysaccharide (LPS)- or Staphylococcus aureus lipoteichoic acid (LTA)-stimulated BV2 microglial cells. Our results indicated that EN suppressed pro-inflammatory cytokine production and induced HO-1 transcription and translation through Nrf-2/antioxidant response element (ARE) signaling. EN markedly inhibited LPS- and LTA-induced activation of nuclear factor-kappa B (NF-κB) as well as phosphorylation of mitogen-activated protein kinases (MAPKs) and signal transducer and activator of transcription (STAT). Furthermore, EN protected hippocampal HT22 cells from indirect neuronal toxicity mediated by LPS- and LTA-treated microglial cells. These results suggested that EN impairs LPS- and LTA-induced neuro-inflammatory responses in microglial cells and confers protection against indirect neuronal damage to HT22 cells. In conclusion, our findings indicate that EN could be used as a natural anti-neuro-inflammatory and neuroprotective agent.
Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Heme Oxygenase-1 ; genetics ; immunology ; Humans ; Lipopolysaccharides ; adverse effects ; Microglia ; cytology ; drug effects ; immunology ; Mitogen-Activated Protein Kinases ; genetics ; immunology ; NF-kappa B ; genetics ; immunology ; Nardostachys ; chemistry ; Neuroprotective Agents ; pharmacology ; Plant Extracts ; pharmacology ; Teichoic Acids ; adverse effects

AIMTo explore the effects of lipoteichoic acid (LTA) induced delayed preconditioning (PC) on hypoxia-reoxygenation (H/R) injury of cultured human coronary artery endothelial cells (HCAECs), and to investigate the potential role of endogenous nitric oxide (NO) participated in the protective mechanism.

METHODSHCAECs were incubated for 2 h in a hypoxic atmosphere and reoxygenated for 4 h in a normoxic atmosphere. The delayed PC was induced by pretreatment with LTA (30 or 300 microg x L(-1)) for 4 h before 24 h recovery. The extent of cellular injury after reoxygenation was assessed by the percentage of cellular injury with Trypan blue exclusion and by the amount of lactate dehydrogenase (LDH) in culture media. The NO level of the culture media was measured spectrophotometrically. Furthermore, HCAECs were exposed to 300 microg x L(-1) of LTA for 4 h, and to detect the expression of eNOS mRNA by RT-PCR method after cells were recovered from different points.

RESULTSLTA pretreatment significantly decreased the percentage of the killed cell and the concentration of LDH in media. Also, LTA pretreatment obviously raised the concentrations of NO in culture media. The protective effects of LTA were abrogated by pretreatment with N-monomethyl-L-arginine (L-NMMA). Moreover, the expression of eNOS mRNA was significantly upregulated after HCAECs exposure to LTA for 4 h following 2 h or 4 h recovery.

CONCLUSIONLTA could induce the delayed protection against H/R induced endothelial injury and dysfunction of cultured HCAECs. NO produced by eNOS acts initially as a trigger and subsequently as a mediator of delayed PC.

Adult ; Cell Death ; Cell Hypoxia ; Cells, Cultured ; Coronary Vessels ; cytology ; metabolism ; Endothelial Cells ; cytology ; metabolism ; Female ; Humans ; Ischemic Preconditioning, Myocardial ; L-Lactate Dehydrogenase ; metabolism ; Lipopolysaccharides ; isolation & purification ; pharmacology ; Myocardial Reperfusion Injury ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase Type III ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Staphylococcus aureus ; chemistry ; Teichoic Acids ; isolation & purification ; pharmacology