OBJECTIVETo study the expression of human beta-defensin-3 (hBD-3) induced by lipopolysaccharide (LPS) in human bronchial epithelial (HBE) cells, and explore the role of hBD-3 in respiratory infection.

METHODSHBE cells were stimulated with different concentrations of LPS (0.01, 0.1, 1 and 10 microg/mL). hBD-3 mRNA expression was detected by RT-PCR 2 hrs later. hBD-3 protein expression was detected by Western blot 4 hrs later.

RESULTShBD-3 mRNA and protein was weakly expressed in normal HBE cells. LPS stimulation resulted in a significant increase of hBD-3 mRNA and protein expression (p<0.01). hBD-3 mRNA and protein expression increased with increasing LPS concentrations. There were significant differences in the hBD-3 mRNA and protein expression in cells stimulated by different concentrations of LPS (p<0.05).

CONCLUSIONSLPS can induce hBD-3 expression in a dose-dependent manner. hBD-3 might play a role in initial defensive reaction against bacterial invasion.

Bronchi ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Lipopolysaccharides ; toxicity ; RNA, Messenger ; analysis ; beta-Defensins ; analysis ; genetics

Lipopolysaccharide (LPS) or glycolipid antigens of Leptospira interrogans have been candidates as serogroup or serotype specific antigen. In this study, therefore, we prepared the LPS and lipid antigens from L. interrogans serovars lai, icterohaemorrhagiae, copenhageni, canicola, pomona, grippotyphosa, and a Korean isolate 30R. The LPS antigens were analyzed by a polyacrylamide gel electrophoresis and lipid antigens by thin-layer chromatography, respectively. The seroreactivity of the antigens were also examined with homologous or heterologous antisera using an enzyme-linked immunosorbent assay. The LPS antigens from serovar lai and the strain 30R were closely related but different from serovar icterohaemorrhagiae. Particularly, the LPS antigens from serovars icterohaemorrhagiae and grippotyphosa were reactive only with the homologous antisera, thus indicating serovar specificity. However, the LPS antigens of the other serovars were reactive to the heterologous antisera. The lipid antigen of serovar icterohaemorrhagiae reacted only with the homologous antisera. In contrast, lipids of other serovars reacted broadly with heterologous antisera, particularly among serovars lai, copenhageni, canicola, pomona, and the strain 30R. The results thus indicated that the LPS and lipid antigens of L. interrogans may contain serovar-specific as well as cross-reactive epitopes.
Antigens, Bacterial/*analysis ; Chromatography, Thin Layer ; Comparative Study ; Electrophoresis, Polyacrylamide Gel ; Leptospira interrogans/*chemistry/immunology ; Lipids/*analysis/immunology ; Lipopolysaccharides/*analysis/immunology ; Support, Non-U.S. Gov't

OBJECTIVETo investigate the effects of bifidobacteria on malondialdehyde (MDA) content and intercellular adhesion molecule-1 (ICAM-1) expression in serum and ileum tissues of young rats with endotoxin-induced intestinal damage, and possible protective mechanisms of bifidobacteria on intestines.

METHODSEigteen-day-old Wistar rats were randomly administered with normal saline (NS), lipopolysaccharide (LPS, 5 mg/kg) or LPS + bifidobacteria. Bifidobacteria (0.5 mL, twice a day) was intragastrically administrated 7 days prior to LPS injection until the end of the experiment. MDA contents in serum and ileum were detected by the TBA method. Expression of ICAM-1 protein and mRNA were evaluated by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR) after 2, 6, 24 and 72 hrs of LPS injection.

RESULTSThe serum and ileum MDA contents in the untreated LPS group increased significantly and reached a peak at 6 hrs of LPS injection when compared with the NS control group (ileum: 99.88 +/- 12.62 nmol/mg prot vs 84.25+/-12.96 nmol/mg prot, P < 0.05; serum: 1.67 +/- 0.30 nmol/mL vs 1.13 +/- 0.20 nmol/mL, P < 0.05). The MDA contents in ileum (92.75 +/- 9.28 nmol/mg prot) and serum (1.17 +/- 0.23 nmol/mL) in the bifidobacteria-treated group at 6 hrs of LPS injection were significantly lower than in the LPS group (P < 0.05). The expression of ICAM-1 protein in the untreated LPS group remarkably increased at 6, 12, 24 and 72 hrs of LPS injection when compared with the NS control group (P < 0.01). The bifidobacteria-treated group displayed lower ICAM-1 protein levels than the untreated LPS group at 72 hrs of LPS injection (P < 0.01). The ICAM-1 mRNA expression in the untreated LPS group significantly increased at 2 hrs of LPS injection when compared with the NS control group (P < 0.01). The ICAM-1 mRNA expression in the bifidobacteria-treated group began to decrease at 2 hrs of LPS injection and was reduced again at 24 hrs after experiencing increase at 6 and 12 hrs of LPS injection when compared with the untreated LPS group (P < 0.01).

CONCLUSIONSThe serum and ileum MDA contents and the expression of ICAM-1 protein and mRNA increased in young rats with endotoxin-induced intestinal damage. Bifidobacteria supplementation can decrease MDA contents and inhibit ICAM-1 expressions, thus providing protections for intestines.

Animals ; Bifidobacterium ; Ileum ; chemistry ; metabolism ; Intercellular Adhesion Molecule-1 ; analysis ; genetics ; Lipopolysaccharides ; toxicity ; Malondialdehyde ; analysis ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Time Factors

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopolysaccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs (miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 microg/ml of E. coli (Ec) LPS or 5 microg/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875-3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.
Bacteria ; Biofilms ; Cell Wall ; Epithelial Cells ; Fusobacterium nucleatum ; Humans ; Inflammation ; Lipopolysaccharides ; Microarray Analysis ; MicroRNAs ; Periodontitis ; Porphyromonas gingivalis ; Prevotella intermedia ; RNA

OBJECTIVETo investigate the effect of Angelicae Pubescentis Radix (APR) on the activity of endocannabinoid hydrolase and N-acylethanolamine-hydrolyzing acid amidase (NAAA), and to demonstrate the mechanism of anti-inflammatory effect of APR by in vitro lipopolysaccharide (LPS)-induced inflammation model.

METHODAPR essential oil was extracted by steam distillation, and the chemical components were identified by GC-MS. Enzymatic activity was performed by using recombinant NAAA-overexpressing protein and detected by LC-MS. Lipids were extracted by methonal/chloroform mixure and analyzed by LC-MS. mRNA and protein expression levels of proinflammatory genes were examined by Real time-PCR and ELISA assay kit, respectively. The content of nitro oxide (NO) was detected by Griess reaction.

RESULTTwenty active components were identified from APR essential oil which inhibited NAAA activity in a dose-dependent manner. On the LPS-induced RAW264.7 cells, APR essential oil reversed LPS-suppressed N-palmitoylethanolamide (PEA) contents in a dose-dependent manner and reduced LPS-induced proinflammatory genes, TNF-alpha and IL-6. Moreover, APR essential oil reduced the mRNA expression of iNOS, subsequently reduced the release of NO, a classic inflammatory marker.

CONCLUSIONThe research demonstrated that the effect of APR on inflammation is mediated by the inhibition of NAAA activity, which increase the cellular endobioactor PEA levels and decrease proinflammatory factor. The results suggest that APR can serve as a nature NAAA inhibitor.

Amidohydrolases ; antagonists & inhibitors ; Angelica ; chemistry ; Animals ; Anti-Inflammatory Agents ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Lipopolysaccharides ; pharmacology ; Mice ; Oils, Volatile ; analysis ; pharmacology

OBJECTIVETo investigate the mechanism of polymyxin B (PMB) antagonizing the biological activity of lipopolysaccharide (LPS).

METHODSThe affinity of PMB for LPS and lipid A was assayed by biosensor, and the neutralization of PMB for LPS (2 ng/ml) was detected by kinetic turbidimetric limulus test. The releases of TNF-alpha and IL-6 in murine peritoneal macrophages a (PMphi) after exposure to LPS (100 ng/ml) were detected, and the expression levels of TLR4, TNF-alpha and IL-6 mRNA in PMphi induced by LPS (100 ng/ml) were measured by RT-PCR.

RESULTSPMB had high-affinity to LPS and lipid A with dissociation equilibrium constants of 18.9 nmol/L and 11.1 nmol/L, respectively, and neutralized LPS in a dose-dependent manner. Furthermore, PMB could markedly inhibit the expressions of TLR4, TNF-alpha and IL-6 mRNA and the release of cycokines in LPS-stimulated murine peritoneal macrophages.

CONCLUSIONSPMB neutralizes LPS and inhibites the expression and release of cycokines in macrophages, in which the affinity of PMB for lipid A plays an important role.

Animals ; Cytokines ; analysis ; Limulus Test ; Lipid A ; antagonists & inhibitors ; Lipopolysaccharides ; antagonists & inhibitors ; Macrophages ; chemistry ; Mice ; Polymyxin B ; pharmacology

BACKGROUNDToll-like receptors play an important role in the human immune system. This study was conducted to investigate the expression profiles and function of Toll-like receptor (TLR) 1 - 9 in human corneal epithelium.

METHODSThe expression of TLR1 - 9 mRNA in 20 human donor corneal epithelia samples abraded during photorefractive keratotomy (PRK) and cultivated telomerase-immortalized human corneal epithelial cells (THCEs) was examined by semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis. Human peripheral blood mononuclear cells (PBMCs) were used as positive controls. The expression of the TLR2 and TLR4 proteins was detected by Western analysis. ELISA was used to detect IL-8 secretion from THCEs challenged with ligands for TLR3 and TLR4 with and without antibody blockade.

RESULTSThe expression of TLR1 - 9 at the mRNA level was detected in the epithelia of 20 patients and in THCE. Significant differences among individuals were observed. One patient was found to lack of the expression of TLR3, 4, 6 and 8, whereas another did not express TLR5. The expression of TLR2 and TLR4 protein was detected in human corneal epithelial cells. As THCE cells express TLR1 - 9, cells were challenged with lipopolysaccharides (LPS) and poly I:C to determine whether TLR4 and TLR3 were functional. The results showed that secretion of IL-8 by cells stimulated with LPS and Poly I:C was 7 to 10 fold greater than secretion by unchallenged cells. Blocking TLR4 with an anti-TLR4 antibody significantly inhibited the LPS-induced IL-8 production by THCE (P < 0.05).

CONCLUSIONHuman corneal epithelial cells express multiple TLRs and are able to recognize LPS and poly I:C. Different expression profiles among individuals suggest that differences in the susceptibilities and sensitivities to bacterial and viral infection in human populations relate to different patterns of TLR expression.

Blotting, Western ; Epithelium, Corneal ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Poly I-C ; pharmacology ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Toll-Like Receptors ; analysis ; genetics ; physiology

Animals ; Bronchoalveolar Lavage Fluid ; chemistry ; Chemokine CCL2 ; analysis ; Immunohistochemistry ; Lipopolysaccharide Receptors ; analysis ; genetics ; Lipopolysaccharides ; toxicity ; Lung ; pathology ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha ; analysis ; Ventilators, Mechanical ; adverse effects

Lipopolysaccharides (LPS) contamination in herbal crude polysaccharides is inevitable. The present study was performed to explore the effect of polymyxin B on abolishing the influence of LPS contamination in mononuclear cells. LPS was pretreated with polymyxin B sulfate (PB) at different concentrations for 1, 5 or 24 h, and then used to stimulate RAW264.7 and mouse peritoneal macrophages (MPMs). The nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in cell culture supernatant, as the indications of cell response, were assayed. Bupleurum chinensis polysaccharides (BCPs) with trace amount contamination of LPS was treated with PB. 30 μg·mL of PB, treating LPS (10 and 1 000 ng·mL in stimulating RAW264.7 and MPMs respectively) at 37 °C for 24 h, successfully abolished the stimulating effect of LPS on the cells. When the cells were stimulated with LPS, BCPs further promoted NO production. However, pretreated with PB, BCPs showed a suppression of NO production in MPMs and no change in RAW264.7. In the in vitro experiments, LPS contamination in polysaccharide might bring a great interference in assessing the activity of drug. Pretreatment with PB (30 μg·mL) at 37 °C for 24 h was sufficient to abolish the effects of LPS contamination (10 and 1 000 ng·mL).
Animals ; Bupleurum ; chemistry ; Drug Contamination ; Drugs, Chinese Herbal ; analysis ; pharmacology ; Lipopolysaccharides ; analysis ; antagonists & inhibitors ; Macrophages ; drug effects ; metabolism ; Mice ; Nitric Oxide ; metabolism ; Polymyxin B ; analysis ; pharmacology ; Polysaccharides ; analysis ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

Tuberculosis has traditionally been confirmed by AFB staining or culturing Mycobacterium tuberculosis from clinical specimens. However, because of the low sensitivity of the sputum smear examination and of the delayed report in culturing, the conventional methods for detection of M. tuberculosis have not been always satisfactory. In an attempt to develop an alternate tool, this study was initiated to produce monoclonal antibodies (MAb) to lipoarabinomannan B (LAM-B) antigen and to use the antibodies in detecting mycobacteria. In this study, five monoclonal antibodies specific to LAM-B were produced; LAM701 (IgG3), LAM138 (IgM), LAM204 (IgM), LAM302 (IgM), and LAM604 (IgM). A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting LAM-B and other mycobacterial antigens using the monoclonal antibodies. With the MAb LAM701, the minimal detectable concentration was 1.0 ng/ml for LAM-B, and 1.0 microgram/ml for M. tuberculosis whole cells, respectively. When 14 clinical specimens proven to contain AFB by smear staining or culture were examined, ten (71.4%) were positive by the sandwich ELISA; in contrast, sputum smear examination gave positive results in only six (42.9%) specimens. Meanwhile, none of 25 specimens with no evidence of AFB were positive by the sandwich ELISA using the MAb LAM701. Although further evaluations are required, this study suggests that the monoclonal antibodies to LAM-B may be useful in detecting mycobacteria from clinical specimens.
Antibodies, Monoclonal/*biosynthesis ; Antigens, Bacterial/*analysis ; Enzyme-Linked Immunosorbent Assay ; Human ; Lipopolysaccharides/chemistry/*immunology ; Mycobacterium tuberculosis/*immunology ; Sputum/*microbiology ; Support, Non-U.S. Gov't ; Tuberculosis/*diagnosis/microbiology