Glycyrrhizin acid and licorice flavonoids are the component of Glycyrrhiza uralensis Fisch root that has been used for various medicinal purposes in traditional oriental medicine for thousands of years. Macrophages as a principal component of immune system play an important role in the initiation, modulation and final activation of immune response against pathogens. In the present study, glycyrrhizin acid and licorice flavonoids was investigated the anti-inflammatory effect on lipopolysaccharide (LPS)-induced macrophage cell line of RAW264.7. Well-grown RAW264.7 cells were collected and randomly divided into the blank control group, the LPS(1 mg x L(-1)) group, the dexamethasone (5 mg x L(-1)) with LPS group, the glycyrrhizin acid (400, 80, 16 mg x L(-1)) with LPS group and the licorice flavonoids (200, 40, 8 mg x L(-1)) with LPS group. RAW264.7 cells were cultured in 24-well plates, pre-incubated for 4 h with different concentrations of dexamethasone, glycyrrhizin acid, or licorice flavonoids. Then cells were stimulated for 20 h with LPS. The supernatant of culture medium was collected from each well and determinated the concentrations of cytokines by means of BioPlex mouse cytokines assay. Compared with the control group, the LPS group could significantly induced relatively high levels of granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor( GM-CSF), macrophage inflammatory protein-1 alpha (MIP-1α), macrophage inflammatory protein-1 beta (MIP-1β), regulated upon activation normal T cell expressed and secreted factor (RANTES), tumor necrosis factor alpha ( TNF-α), monocyte chemotactic protein 1 (MCP-1), chemokine (C-X-C motif) ligand 1 (KC), eotaxin, interleukin(IL)-1α, IL-1β, IL-3, IL-4, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, and IL-17 secretion (P < 0.05). The glycyrrhizin acid significantly inhibited IL-1β, IL-3, IL-5, IL-6, IL-10, IL-12 (p40), IL-12 (p70), IL-13, Eotaxin and TNF-α secreted by LPS-stimulated RAW264.7 cells (P < 0.05). The expression levels of IL-6 and Eotaxin were observably decreased in the licorice flavonoids with LPS group (P < 0.05). The data presented here suggested that the glycyrrhizin acid and licorice flavonoids modulate various cytokines secreted by macrophages and were important anti-inflammatory constituent of Licorice.
Animals ; Cell Line ; Cytokines ; genetics ; immunology ; Flavonoids ; pharmacology ; Glycyrrhiza ; chemistry ; Glycyrrhizic Acid ; pharmacology ; Lipopolysaccharides ; immunology ; Macrophages ; drug effects ; immunology ; Mice

OBJECTIVETo explore the important role of the terminal residues containing sialic acid (SA) in Campylobacter jejuni (CJ) lipopolysaccharide (LPS) as the critical antigen to induce nerve damage, and also to identify immunopathological evidence for the hypothesis of molecular mimicry and cross-immunity between CJ LPS and gangliosides.

METHODSA mutant of Pen O:19 CJ with neuB1 gene inactivated and LPS outer core terminal residues losing SA was to be constructed. PCR and RT-PCR were used to confirm the mutant. Capability of CJ LPS binding to cholera toxin B subunit (CTB) was tested. Guinea pigs were systematically immunized with LPS of the wild and the mutant strains, respectively. Titers of anti-LPS and anti-ganglioside GM(1) IgG antibodies in sera of immunized guinea pigs were detected by ELISA. Pathological study for sciatic nerves of both Guinea pigs either immunized systematically or perineural injection with their immunized serum was finished.

RESULTS(1) The mutant of CJ O:19 strain with inactivated neuB1 gene was successfully constructed and lost transcriptional activity of neuB1 gene in the mutant strain was confirmed by PCR and RT-PCR. SA was well demonstrated by both acidic ninhydrin reaction and periodate-resorcinol reaction in the LPS of wild strain but not in the mutant LPS; (2) Compared with the titers before immunization, the titers of anti-GM(1) IgG antibody increased in sera of guinea pigs immunized with LPS of the wild strain. However there were no detectable anti-GM(1) IgG antibody in sera of the animals immunized with mutant LPS and PBS. (3) The incidence of pathological fibers of sciatic nerves in wild CJ LPS group (17.3%) was significantly higher than the mutant CJ LPS group (chi(2) = 125, P < 0.01); the difference between the mutant CJ LPS group and control group was not statistically significant (chi(2) = 1.633, P > 0.05). (4) After perineural injection with immunized serum, the incidence of pathological fibers of sciatic nerves in wild strain group (67.8%) was also significantly higher than the incidence of mutant group (P < 0.01).

CONCLUSIONA mutant of CJ O:19 strain neuB1 gene inactivated and SA component of terminal structure of LPS lost was successfully constructed. And it no longer expressed SA component which is the normal terminal structure of LPS in wild strain. The capability of the wild strain to induce increased titers of anti-GM(1) antibody and immune-mediated nerve damage was simultaneously lost for the mutant strain. It could be a strong immunopathologic evidence to identify the molecular mimicry hypothesis between CJ LPS and ganglioside epitope in nerve on the pathogenesis of CJ related GBS. The terminal residues containing SA should be as the basic GM1-like structure in CJ LPS.

Animals ; Antibodies, Bacterial ; blood ; immunology ; Antigens, Bacterial ; genetics ; immunology ; Campylobacter jejuni ; genetics ; immunology ; G(M1) Ganglioside ; immunology ; Guinea Pigs ; Lipopolysaccharides ; chemistry ; immunology ; Molecular Mimicry ; Mutagenesis ; N-Acetylneuraminic Acid ; chemistry ; immunology ; Peripheral Nervous System Diseases ; immunology ; microbiology

Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria. Minute amounts of LPS released from infecting pathogens can initiate potent innate immune responses that prime the immune system against further infection. However, when the LPS response is not properly controlled it can lead to fatal septic shock syndrome. The common structural pattern of LPS in diverse bacterial species is recognized by a cascade of LPS receptors and accessory proteins, LPS binding protein (LBP), CD14 and the Toll-like receptor4 (TLR4)-MD-2 complex. The structures of these proteins account for how our immune system differentiates LPS molecules from structurally similar host molecules. They also provide insights useful for discovery of anti-sepsis drugs. In this review, we summarize these structures and describe the structural basis of LPS recognition by LPS receptors and accessory proteins.
Amino Acid Sequence ; Animals ; Binding Sites ; Carbohydrate Sequence ; Humans ; Immunity, Innate ; Lipopolysaccharides/*chemistry/immunology/pharmacology ; Molecular Sequence Data ; Toll-Like Receptor 4/*chemistry/immunology/metabolism

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

Lipooligosacharide (LOS) of Neisseria gonorrhoeae (gonococci, GC) is involved in the interaction of GC with host cells. Deletion of the alpha-oligosaccharide (alpha-OS) moiety of LOS (lgtF mutant) significantly impairs invasion of GC into epithelial cell lines. GC opacity (Opa) proteins, such as OpaI, mediate phagocytosis and stimulate chemiluminescence responses in neutrophils in part through interaction with members of the carcinoembryonic antigen (CEA) family, which includes CEACAM3 (CD66d), a human neutrophil specific receptor for phagocytosis of bacteria. In the present work, we examined the effects of OpaI-expressing lgtF mutant on phagocytosis by HeLa-CEACAM3 cells and chemiluminescence responses in neutrophils. The results showed that lgtF mutant even expressing OpaI completely lost the ability to promote either phagocytosis mediated by CEACAM3 interaction in HeLa cells or chemiluminescence responses in neutrophils. These data indicated that Opa proteins in the lgtF mutant, which might result from the conformational change, cannot be functional.
Antigens, Bacterial ; chemistry ; genetics ; immunology ; metabolism ; Carbohydrate Sequence ; Carcinoembryonic Antigen ; genetics ; immunology ; Gene Expression Regulation ; HeLa Cells ; Host-Pathogen Interactions ; Humans ; Lipopolysaccharides ; chemistry ; immunology ; Luminescent Measurements ; Mutation ; Neisseria gonorrhoeae ; genetics ; metabolism ; pathogenicity ; Neutrophils ; immunology ; microbiology ; Phagocytosis

The immunologic reactivity of a lipopolysaccharide (LPS)-protein complex isolated from a potassium thiocyanate extract of a Pasteurella multocida (capsular type A and somatic type 3) strain was evaluated in mice. The LPS-protein complex provided 100% protection in mice against a challenge with the homologous strain. However, when the complex was fractionated into LPS and protein moieties by phenol-water treatment, both components lacked immunogenicity. The complex and extracted components were mitogenic for mouse B lymphocytes with the protein moiety the most active. Although immune serum against the LPS-protein complex protected mice against challenge thereby indicating a role for humoral immunity, the LPS-protein complex of P. multocida was also found to induce cell-mediated immunity. This cell-mediated immunity was demonstrated in mice immunized with the complex by: (1). mitogenic responses of T lymphocytes, (2). induction of delayed type hypersensitivity reaction in the hind footpads, and (3). enhanced resistance to challenge infection with Salmonella enteritidis.
Animals ; Antibodies, Bacterial/blood/immunology ; Bacterial Proteins/chemistry/*immunology ; Chemical Fractionation ; Hypersensitivity, Delayed ; Immune Sera/immunology ; Immunity, Cellular ; Immunization, Passive ; Lipopolysaccharides/chemistry/*immunology ; Lymphocyte Activation ; Mice ; Pasteurella Infections/immunology/*prevention & control ; Pasteurella multocida/*chemistry/immunology ; Salmonella Infections, Animal/immunology/prevention & control ; Salmonella enteritidis/growth & development/immunology ; Spleen/cytology/immunology/microbiology

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

Withaminimas A-F (1-6), six new withaphysalin-type withanolides were isolated from the aerial parts of Physalis minima L.. The structures of these compounds were elucidated through a variety of spectroscopic techniques including HR-MS, NMR, and ECD. Compound 1 belongs to rare 18-norwithanolides, and 2-3 were 13/14-secowithanolides. According to the traditional usage of P. minima, inhibitory effects on nitric oxide (NO) production in lipopolysaccaride-activated RAW264.7 macrophages were evaluated, and compounds 1-4 exhibited significant inhibitory effects with IC values among 3.91-18.46 μmol·L.
Animals ; Anti-Inflammatory Agents ; chemistry ; pharmacology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; Mice ; Molecular Structure ; Physalis ; chemistry ; RAW 264.7 Cells ; Structure-Activity Relationship ; Withanolides ; chemistry ; pharmacology

Scutellariae Radix (SR), the root of Scutellaria baicalensis Georgi, is used as an antipyretic drug and has been demonstrated to have anti-inflammatory activity. SR is divided into two specifications, "Ku Qin" (KQ) and "Zi Qin" (ZQ), for use against different symptoms (upper energizer heat or lower portion of the triple energizer), according to the theory of traditional Chinese medicine (TCM). However, differences in the efficacies of these two specifications have not been determined. In the present study, we aimed to characterize the differences in the anti-inflammatory activities between KQ and ZQ and to explore how their differences are manifested in lipopolysaccharide (LPS)-induced macrophages. Our results showed that, in RAW264.7 cells (a mouse macrophage cell line derived from ascites), KQ and ZQ displayed anti-inflammatory effects by inhibiting the release of nitric oxide (NO), inducible NOS (iNOS), and nuclear factor-κB (NF-κB) in a dose-dependent manner without distinction. In NR8383 cells (a rat alveolar macrophage cell line), KQ and ZQ displayed similar effects on NO, iNOS, and NF-κB as seen in RAW264.7 cells, but KQ showed a higher inhibition rate for NO and iNOS than that shown by ZQ at the same concentration. These results indicated that there were differences in efficacy between KQ and ZQ in treating lung inflammation. Our findings provided an experimental evidence supporting the different uses of KQ and ZQ in clinic, as noted in ancient herbal records.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cell Line ; Lipopolysaccharides ; pharmacology ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; RAW 264.7 Cells ; Rats ; Scutellaria baicalensis ; chemistry

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology