OBJECTIVEA study was conducted to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of regulated upon activation normal T-cell expressed and secreted (RANTES) and fractalkine in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of Pg-LPS (200, 500, and 1000 ng x mL(-1)) for 1, 6, 12, and 24 h, respectively. Then real time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent method (ELISA) were adopted to detect the protein levels and mRNA levels of RANTES and fractalkine.

RESULTSThe RANTES protein levels and mRNA levels, as well as fractalkine mRNA levels, were significantly higher in all experimental groups of 1, 6, and 12 h than in the control group (P<0.05), except the expression of RANTES mRNA in 200 ng x mL(-1) group of 12 h and RANTES protein in 200 ng x mL(-1) group of 1 h. The expression levels of RANTES mRNA and fractalkine mRNA were highest in 1000 ng x mL(-1) group of 6 h and were 4.88- and 6.20-fold higher, respectively, than those in the control group. The expression levels of RANTES protein, mRNA, and fractalkine mRNA decreased 6 h after stimulation, and were significantly higher than those in the control group (P<0.05) in the RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis 500 ng x mL(-1) group of 24 h. There was a significant difference between the expression of fractalkine mRNA in 1000 ng x mL(-1) group of 6 and 12 h than in the control group (P<0.05).

CONCLUSIONPg-LPS infection might up-regulate the expression of RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Chemokine CX3CL1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; immunology ; isolation & purification ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

In the present study, we analyzed the role of Ginkgo biloba extract in lipopolysaccharide(LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS. G. biloba extract (12 and 24 mg·kg(-1)) and dexamethasone (2 mg·kg(-1)), as a positive control, were given by i.p. injection. The cells in the bronchoalveolar lavage fluid (BALF) were counted. The degree of animal lung edema was evaluated by measuring the wet/dry weight ratio. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activities were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, tumor necrosis factor-a, interleukin-1b, and interleukin-6, were assayed by enzyme-linked immunosorbent assay. Pathological changes of lung tissues were observed by H&E staining. The levels of NF-κB p65 and COX-2 expression were detected by Western blotting. Compared to the LPS group, the treatment with the G. biloba extract at 12 and 24 mg·kg(-1) markedly attenuated the inflammatory cell numbers in the BALF, decreased NF-κB p65 and COX-2 expression, and improved SOD activity, and inhibited MPO activity. The histological changes of the lungs were also significantly improved. The results indicated that G. biloba extract has a protective effect on LPS-induced acute lung injury in mice. The protective mechanism of G. biloba extract may be partly attributed to the inhibition of NF-κB p65 and COX-2 activation.
Acute Lung Injury ; chemically induced ; drug therapy ; metabolism ; Animals ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Count ; Cyclooxygenase 2 ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Gene Expression ; drug effects ; Ginkgo biloba ; chemistry ; Interleukin-1beta ; analysis ; Interleukin-6 ; analysis ; Lipopolysaccharides ; Lung ; immunology ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Peroxidase ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; Pulmonary Edema ; Superoxide Dismutase ; metabolism ; Transcription Factor RelA ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; analysis

This study assessed the roles of chronic stress (CS) in the stimulation of the sympathetic nervous system and explored the underlying mechanisms of periodontitis. Using an animal model of periodontitis and CS, the expression of tyrosine hydroxylase (TH) and the protein levels of the alpha1-adrenergic receptor (alpha1-AR) and beta2-adrenergic receptor (beta2-AR) were assessed. Furthermore, human periodontal ligament fibroblasts (HPDLFs) were stimulated with lipopolysaccharide (LPS) to mimic the process of inflammation. The proliferation of the HPDLFs and the expression of alpha1-AR and beta2-AR were assessed. The inflammatory-related cytokines interleukin (IL)-1beta, IL-6 and IL-8 were detected after pretreatment with the alpha1/beta2-AR blockers phentolamine/propranolol, both in vitro and in vivo. Results show that periodontitis under CS conditions enhanced the expression of TH, alpha1-AR and beta2-AR. Phentolamine significantly reduced the inflammatory cytokine levels. Furthermore, we observed a marked decrease in HPDLF proliferation and the increased expression of alpha1-ARfollowing LPS pretreatment. Pretreatment with phentolamine dramatically ameliorated LPS-inhibited cell proliferation. In addition, the blocking of alpha1-ARsignaling also hindered the upregulation of the inflammatory-related cytokines IL-1beta, IL-6 and IL-8. These results suggest that CS can significantly enhance the pathological progression of periodontitis by an alpha1-adrenergic signaling-mediated inflammatory response. We have identified a potential therapeutic target for the treatment of periodontal disease, particularly in those patients suffering from concurrent CS.
Adrenergic alpha-1 Receptor Antagonists/*therapeutic use ; Animals ; Cells, Cultured ; Cytokines/immunology ; Fibroblasts/immunology/pathology ; Humans ; Lipopolysaccharides/administration & dosage/immunology ; Male ; Periodontal Ligament/cytology/immunology/pathology ; Periodontitis/*drug therapy/*etiology/immunology/pathology ; Phentolamine/*therapeutic use ; Rats ; Rats, Wistar ; Receptors, Adrenergic, alpha-1/analysis/*immunology ; Signal Transduction/drug effects ; *Stress, Physiological/drug effects ; Tyrosine 3-Monooxygenase/analysis/immunology

Acute Lung Injury ; chemically induced ; genetics ; immunology ; Animals ; Cytokines ; blood ; Gene Expression Regulation ; Lipopolysaccharides ; toxicity ; Lung ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; MicroRNAs ; analysis

OBJECTIVETo study the regulatory role of bacterial lipopolysaccharide (LPS ) in the development of bronchial asthma by examining the effects of LPS on serum IL-4, serum IL-8 and pulmonary vascular endothelial growth factor (VEGF) expression in mice with asthma.

METHODSTwenty-seven BALB/c mice were randomly assigned into control, asthma and LPS-treated asthma groups (n=9 each). Serum IL-4 and IL-8 concentrations were measured using ELISA. VEGF expression in lung tissues was examined using the immunohistochemical method.

RESULTSSerum IL-4 and IL-8 concentrations in the asthma group were significantly higher than in the control group (P<0.05). LPS treatment significantly decreased serum IL-4 and IL-8 concentrations compared with the asthma group (P<0.05), although levels were significantly higher than in the control group (P<0.05). Airway VEGF expression in the asthma group was significantly higher than in the control group (P<0.05). LPS treatment significantly decreased airway VEGF expression compared with the asthma group (P<0.05), although concentrations remained higher than in the control group (P<0.05).

CONCLUSIONSLPS can decrease serum IL-4, serum IL-8 and pulmonary VEGF expression in mice with asthma, and thus can possibly reduce both airway inflammation and airway vascular remodeling.

Animals ; Asthma ; drug therapy ; immunology ; Female ; Interleukin-4 ; blood ; Interleukin-8 ; blood ; Lipopolysaccharides ; pharmacology ; Mice ; Mice, Inbred BALB C ; Vascular Endothelial Growth Factor A ; analysis ; physiology

Dominant inflammatory cytokines might be different depending on the underlying causes of acute lung injury (ALI). The role of kertinocyte-derived chemokine (KC), a potent chemoattractant for neutrophils, has not been clearly established in hemorrhage-induced ALI. In this study, lung injury and cytokine expressison were evaluated in LPS- or hemorrhage-induced ALI models of BALB/c mice. The myeloperoxidase activities at 4 hr after hemorrhage and LPS-injection were 47.4+/-13.0 and 56.5+/-16.4 U/g, respectively. NF-kappa B activity peaked at 4 hr after hemorrhage, which was suppressed to the control level by anti-high mobility group B1 (HMGB1) antibody. Lung expressions of TNF-alpha, MIP-2, and IL-1beta were increased by LPS injection. However, there was only a minimal increase in IL-1beta and no expressions of TNF-alpha or MIP-2 in hemorrhage-induced ALI. In contrast, lung KC increased significantly at 4 hr after hemorrhage compared to control levels (83.1+/-12.3 vs. 14.2+/-1.6 pg/mL/mg by ELISA) (P<0.05). By immunohistochemical staining, lung neutrophils stained positive for KC. Increased KC was also observed in bronchoalveolar lavage fluid and plasma. KC plays an important role in hemorrhage-induced ALI.
Acute Lung Injury/etiology/*metabolism ; Animals ; Antibodies/immunology/metabolism ; Chemokine CXCL2/analysis ; Chemokines/analysis/blood/*physiology ; Chickens ; HMGB1 Protein/metabolism ; Humans ; Interleukin-1beta/analysis ; Lipopolysaccharides/toxicity ; Mice ; Mice, Inbred BALB C ; NF-kappa B/metabolism ; Neutrophils/immunology/metabolism ; Peroxidase/analysis ; Shock, Hemorrhagic/*complications ; Time Factors ; Tumor Necrosis Factor-alpha/analysis

AIMTo elucidate the genetic basis for the pronounced resistance that the oral pathogen, Porphyromonas gingivalis (P. gingivalis), exhibits towards the cationic antimicrobial peptide, polymyxin B.

METHODOLOGYA genetic screen of P. gingivalis clones generated by a Tn4400'-based random insertion mutagenesis strategy was performed to identify bacteria harboring novel genetic mutations that render P. gingivalis susceptible to killing by the cationic antimicrobial peptide, polymyxin B (PMB, 50 microg x mL(-1)).

RESULTSP. gingivalis (ATCC 33277) is unusually resistant to the cationic antimicrobial peptide, PMB at relatively high concentrations (200 microg x mL(-1)). Approximately 2,700 independent Tn4400'-derived mutants of P. gingivalis were examined for increased sensitivity to PMB killing at a relatively low dose (50 microg x mL(-1)). A single PMB-sensitive mutant was obtained in this phenotypic screen. We determined that the Tn4400' transposon was integrated into the gene encoding the lipid A 4'-phosphatase, PGN_0524, demonstrating that this insertion event was responsible for its increased susceptibility of this clone to PMB-dependent killing. The resulting mutant strain, designated 0524-Tn4400', was highly sensitive to PMB killing relative to wild-type P. gingivalis, and exhibited the same sensitivity as the previously characterized strain, 0524KO, which bears a genetically engineered deletion in the PGN_0524 locus. Positive ion mass spectrometric structural (MALDI-TOF MS) analyses revealed that lipid A isolates from 0524-Tn4400' and 0524KO strains displayed strikingly similar MALDI-TOF MS spectra that were substantially different from the wildtype P. gingivalis lipid A spectrum. Finally, intact 0524-Tn4400' and 0524KO mutant bacteria, as well as their corresponding LPS isolates, were significantly more potent in stimulating Toll-like receptor 4 (TLR4)-dependent E-selectin expression in human endothelial cells relative to intact wild-type P. gingivalis or its corresponding LPS isolate.

CONCLUSIONThe combined molecular evidence provided in this report suggests that PGN_0524, a lipid A 4'-phosphatase, is the sole genetic element conferring the ability of the periodontopathogen, P. gingivalis, to evade the killing activity of cationic antimicrobial peptides, such as PMB. These data strongly implicate PGN_0524 as a critical virulence factor for the ability of P. gingivalis to evade front-line host innate defenses that are dependent upon cationic antimicrobial peptide activity and TLR 4 sensing.

Anti-Bacterial Agents ; pharmacology ; Chromosome Mapping ; DNA Transposable Elements ; genetics ; Drug Resistance, Bacterial ; genetics ; E-Selectin ; analysis ; immunology ; Endothelial Cells ; immunology ; microbiology ; Gene Deletion ; Humans ; Lipid A ; analysis ; immunology ; Lipopolysaccharides ; analysis ; immunology ; Mutagenesis, Insertional ; genetics ; Open Reading Frames ; genetics ; Phosphoric Monoester Hydrolases ; genetics ; physiology ; Polymyxin B ; pharmacology ; Porphyromonas gingivalis ; enzymology ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Toll-Like Receptor 4 ; analysis ; immunology ; Virulence Factors ; physiology

BACKGROUNDThe coordinated change of haematopoietic supporting microenvironment in bone marrow (BM) is crucial for innate immunity and inflammation. As the precursors of marrow stroma, BM derived mesenchymal stem cells (MSCs) promote haematopoietic function, but their roles in innate immunity or inflammation have not been investigated. Here we investigated the expression of Toll like receptor 4 (TLR-4) and the effect of lipopolysaccharide (LPS) on its expression in BM MSCs in vitro.

METHODSMSCs were harvested from adult rat's BM cells by density gradient centrifugation and adhesive culture. The purity of MSCs were identified with the cell morphological feature and osteogenic capacity, the phenotypes were tested by flow cytometry. Cultured MSCs were treated by LPS (1 microg/ml, 10 microg/ml or 100 microg/ml) for 24 hours. The relative expression levels of TLR-4 mRNA were detected by semiquantitative reverse transcription polymerase chain reaction and costimulatory molecules (CD80, CD86 and MHC-II) expressed on MSCs were analyzed by flow cytometry. The levels of tumor necrosis factor-alpha (TNF-alpha) in supernatants were determined by enzyme linked immunosorbent assay.

RESULTSAfter incubation with LPS, MSCs expressed the higher levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha than the untreated group: LPS 10 microg/ml was the most effective (P < 0.01); the levels of TLR-4 mRNA, costimulatory molecules and TNF-alpha decreased when MSCs were exposed to 100 microg/ml LPS. Except for MHC-II and TNF-alpha (P > 0.05), the levels of CD80, CD86 and TLR-4 mRNA were significantly lower than that in the treated group of 10 microg/ml (P < 0.01).

CONCLUSIONMSCs expressed TLR-4 mRNA. LPS activated the functional expression levels of TLR-4 in MSCs although the activity may depend on the concentration of LPS.

Animals ; B7-2 Antigen ; analysis ; Bone Marrow Cells ; immunology ; Cell Differentiation ; Cells, Cultured ; Immunophenotyping ; Lipopolysaccharides ; pharmacology ; Male ; Mesenchymal Stromal Cells ; drug effects ; immunology ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; physiology ; Tumor Necrosis Factor-alpha ; biosynthesis ; Up-Regulation

OBJECTIVEGastrointestinal dysfunction is closely correlated with the destruction of intestinal barrier function induced by serious infection. Platelet activating factor (PAF) may induce intestinal injuries. This study aimed to investigate the effect of PAF on the injury of intestinal mucosal immuno-barrier function in young rats.

METHODSEighteen-day-old Wistar rats were randomized to lipopolysaccharide (LPS) (5 mg/kg), LPS plus PAF receptor antagonist and normal saline injection (Control). PAF receptor antagonist BN52021 5 mg/kg was administered before or 30 minutes after LPS injection (pretreatment or treatment). The ileum specimens (n=8) were harvested at 1.5, 3, 6, 24, 48 and 72 hrs after LPS injection. Double antibody-PEG radioimmunoassay was used to determine the secretory IgA (sIgA) content in intestinal mucosa. Hematoxylin and erosin staining was used for histological evaluation. The ratio of wet and dry weight (W/D) of ileum tissues was calculated.

RESULTSIntestinal villi edema, capillary congestion, extension of the subepithelial lympho channel, and polymorphonuclear infiltration in enteric cavity were noted in the LPS group at 1.5, 3, 6 and 24 hrs after LPS injection. In the PAF receptor antagonist group only villi edema was found. The W/D ratio in the LPS group was significantly higher than that in the Control group at all time points, but it was slightly reduced by the PAF receptor antagonist pretreatment or treatment. The sIgA content was obviously decreased after 1.5, 3, 6, 24 and 48 hrs of LPS challenge compared with that in the Control group (P < 0.01). It reached to a nadir at 6 hrs (0.15 +/- 0.04 microg/mL). The level of sIgA in the PAF receptor antagonist group was higher than that in the LPS group at each time point. There was no statistical difference in the sIgA level between the PAF receptor pretreatment and treatment groups.

CONCLUSIONSPAF plays roles in the injury of intestinal immuno-barrier function. Preventive and remedial use of PAF receptor antagonist BN52021 may relieve intestinal injury.

Animals ; Diterpenes ; pharmacology ; Ginkgolides ; Immunoglobulin A, Secretory ; analysis ; Intestines ; immunology ; pathology ; Lactones ; pharmacology ; Lipopolysaccharides ; toxicity ; Platelet Activating Factor ; physiology ; Rats ; Rats, Wistar

OBJECTIVETo explore the influences of anti-CD47 monoclonal antibody (mAb) B6H12 on the differentiation and function of cultured dendritic cells (DCs).

METHODSHuman peripheral monocyte derived DCs were propagated with granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin-4 (IL-4) in the presence or absence of soluble B6H12. Flow cytometry was used to analyze the immunophenotypes of cells, semi-quantitative RT-PCR and ELISA methods to analyse the mRNA and protein expression levels of interleukin-12 (IL-12). The antigen-presenting function of DCs was determined in one-way mixed leukocyte reaction by Brdu-ELISA technique.

RESULTSThere was a high expression rate (94% approximately 98%) of CD47 molecules in DCs. The cell immunophenotypes in B6H12 mAb treated and untreated DC groups were as follows: CD80(+) (68.14 +/- 7.41)% vs (89.17 +/- 8.59)%; CD86(+) (67.33 +/- 4.71)% vs (87.27 +/- 3.56)%; CD83 (40.08 +/- 14.80)% vs (72.77 +/- 8.68)%; CD1a(+) (66.45 +/- 4.06)% vs (95.93 +/- 3.03)%; and HLA-DR (40.67 +/- 13.48) vs (98.97 +/- 1.01)%, respectively. The expression levels of mRNA and protein of IL-12 were strongly inhibited in B6H12 mAb treated DC (P < 0.01). The quantity of Brdu was also lower in B6H12 mAb treated DC than that in untreated DCs (P < 0.01).

CONCLUSIONThe anti-CD47 McAb exerts a negative effect on the maturation and functions of cultured DCs.

Adult ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; analysis ; Antigens, CD1 ; analysis ; B7-2 Antigen ; analysis ; CD47 Antigen ; analysis ; immunology ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Immunoglobulins ; analysis ; Interleukin-12 ; genetics ; metabolism ; Interleukin-4 ; pharmacology ; Lipopolysaccharides ; pharmacology ; Membrane Glycoproteins ; analysis ; Monocytes ; cytology ; drug effects ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction