To investigate the effects of adenosine triphosphate (ATP) on expression of inflammatory factors induced by lipopolysaccharide (LPS) in endothelial progenitor cells (EPCs), and to elucidate the possible mechanisms.
 Methods: Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation, RT-PCR was performed to detect the expression of inflammatory factors induced by LPS (1 mg/mL) in EPCs, the effect of low concentration (5 μmol/L) of ATP on expression of IL-1β, MCP-1 and ICAM-1, and the effect of different concentrations (5, 50 μmol/L) of ATP on the expression of Toll-like receptor (TLR) 4, myeloid differentiation primary response protein 88 (MyD88) and CD14. Western blot was performed to detect expression of TLR4 regulated proteins MyD88 and CD14 or to detect the low concentration (1, 5 μmol/L) of ATP on the expression of TLR4, MyD88 and CD14 and the NF-κB signaling pathway.
 Results: EPCs highly expressed TLR4, and its ligand LPS (1 mg/mL) significantly upregulated mRNA expression of IL-1β, MCP-1 and ICAM-1 and protein expression of MyD88 and CD14 in a time-dependent manner (P<0.01), accompanied by activation of ERK and NF-κB signal pathway. ATP at low concentration (5 μmol/L) significantly inhibited LPS-induced mRNA expression of IL-1β, MCP-1 and ICAM-1(P<0.05), downregulated the LPS-induced protein expression of TLR4, MyD88 and CD14 in EPCs (P<0.05), and suppressed LPS-induced activation of NF-κB signaling pathway (P<0.05).
 Conclusion: ATP at low concentration may suppress LPS-induced expression of inflammatory factors in EPCs through negative regulation of the TLR4 signaling pathway.
Adenosine Triphosphate ; pharmacology ; Endothelial Progenitor Cells ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Leukocytes, Mononuclear ; cytology ; Lipopolysaccharide Receptors ; genetics ; Lipopolysaccharides ; pharmacology ; Myeloid Differentiation Factor 88 ; genetics ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; genetics

OBJECTIVETo evaluate the effect of Chinese medicine Haoqin Qingdan Decoction (, HQD) for febrile disease dampness-heat syndrome (FDDHS).

METHODSForty mice were divided into four groups, including normal control, FDDHS (induced by Radix et Rhizoma Rhei recipe and influenza virus A1 FM1 model), HQD, and the ribavirin groups (10 in each). The normal control and FDDHS groups were administered normal saline. HQD and the ribavirin groups were administered HQD and ribavirin intragastrically once daily at a dose of 64 g/(kg d) and 0.07 g/(kg d), respectively for 7 days. Lethargy, rough hair, diarrhea, tongue color and sole color were evaluated for pathological changes in morphology. The tongue and lung tissues were collected for histology. The CD14 and toll-like receptor 4 (TLR4) expression levels were measured using real-time quantitative polymerase chain reaction.

RESULTSMore than 80% of the FDDHS mice showed hypokinesia and lethargy, and pathological changes associated with rough hair, diarrhea, tongue color and sole color. With advanced treatment for 7 days, the thick greasy tongue fur of the HQD and ribavirin groups were thinner than that of the FDDHS group (P<0.05), and it was the thinnest in the ribavirin group as compared with that in other groups (P<0.05). The CD14 and TLR4 expression levels in the lung tissues of HQD and ribavirin groups significantly delined compared with the model group (P<0.05 or P<0.01). CD14 was down-regulated more remarkably in the HQD group compared with the ribavirin group (P<0.05), whereas the converse was true with TLR4 (P<0.05).

CONCLUSIONSWe established a FDDHS mouse model showing systemic clinical symptoms. Both HQD and ribavirin can inhibit the expression of CD14 and TLR4 in FDDHS mice, while the effect of ribavirin might be much more violent. The expression changes of CD14 and TLR4 consistently refers to lipopolysaccharide, the commonly and hotly inducing factor in FDDHS.

Animals ; Behavior, Animal ; Disease Models, Animal ; Down-Regulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fever ; drug therapy ; pathology ; Gene Expression Profiling ; Lipopolysaccharide Receptors ; genetics ; metabolism ; Lung ; drug effects ; pathology ; Mice, Inbred BALB C ; Ribavirin ; pharmacology ; therapeutic use ; Syndrome ; Toll-Like Receptor 4 ; genetics ; metabolism

BACKGROUNDAdenoid hypertrophy (AH) is associated with pediatric chronic rhinosinusitis (pCRS), but its role in the inflammatory process of pCRS is unclear. It is thought that innate immunity gene expression is disrupted in the epithelium of patients with chronic rhinosinusitis (CRS), including antimicrobial peptides and pattern recognition receptors (PRRs). The aim of this preliminary study was to detect the expression of innate immunity genes in epithelial cells of hypertrophic adenoids with and without pCRS to better understand their role in pCRS.

METHODSNine pCRS patients and nine simple AH patients undergoing adenoidectomy were recruited for the study. Adenoidal epithelium was isolated, and real-time quantitative polymerase chain reaction (RT-qPCR) was employed to measure relative expression levels of the following messenger RNAs in hypertrophic adenoid epithelial cells of pediatric patients with and without CRS: Human β-defensin (HBD) 2 and 3, surfactant protein (SP)-A and D, toll-like receptors 1-10, nucleotide-binding oligomerization domain (NOD)-like receptors NOD 1, NOD 2, and NACHT, LRR and PYD domains-containing protein 3, retinoic acid-induced gene 1, melanoma differentiation-associated gene 5, and nuclear factor-κB (NF-κB). RT-qPCR data from two groups were analyzed by independent sample t-tests and Mann-Whitney U-tests.

RESULTSThe relative expression of SP-D in adenoidal epithelium of pCRS group was significantly lower than that in AH group (pCRS 0.73 ± 0.10 vs. AH 1.21 ± 0.15; P = 0.0173, t = 2.654). The relative expression levels of all tested PRRs and NF-κB, as well as HBD-2, HBD-3, and SP-A, showed no statistically significant differences in isolated adenoidal epithelium between pCRS group and AH group.

CONCLUSIONSDown-regulated SP-D levels in adenoidal epithelium may contribute to the development of pCRS. PRRs, however, are unlikely to play a significant role in the inflammatory process of pCRS.

Adenoids ; cytology ; Antimicrobial Cationic Peptides ; metabolism ; Child ; Epithelial Cells ; metabolism ; Female ; Humans ; Immunity, Innate ; genetics ; physiology ; Male ; Receptors, Pattern Recognition ; metabolism ; Sinusitis ; metabolism ; Toll-Like Receptors ; metabolism

When mouse bone marrow-derived macrophages were stimulated with serum amyloid A (SAA), which is a major acute-phase protein, there was strong inhibition of osteoclast formation induced by the receptor activator of nuclear factor kappaB ligand. SAA not only markedly blocked the expression of several osteoclast-associated genes (TNF receptor-associated factor 6 and osteoclast-associated receptor) but also strongly induced the expression of negative regulators (MafB and interferon regulatory factor 8). Moreover, SAA decreased c-fms expression on the cell surface via shedding of the c-fms extracellular domain. SAA also restrained the fusion of osteoclast precursors by blocking intracellular ATP release. This inhibitory response of SAA is not mediated by the well-known SAA receptors (formyl peptide receptor 2, Toll-like receptor 2 (TLR2) or TLR4). These findings provide insight into a novel inhibitory role of SAA in osteoclastogenesis and suggest that SAA is an important endogenous modulator that regulates bone homeostasis.
Adenosine Triphosphate/metabolism ; Animals ; Cell Differentiation ; Cell Line ; Gene Expression Regulation, Developmental ; Humans ; Macrophages/*cytology/metabolism ; Mice ; Osteoclasts/*cytology/metabolism ; RANK Ligand/*metabolism ; Receptor, Macrophage Colony-Stimulating Factor/genetics ; Receptors, Formyl Peptide/metabolism ; Serum Amyloid A Protein/*metabolism ; Toll-Like Receptor 2/metabolism ; Toll-Like Receptor 4/metabolism

In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (T(reg)) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and T(reg) cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-beta expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and T(reg) cell mediated immune responses, although additional data are needed to convincingly prove this observation.
Animals ; Gene Expression ; Humans ; Interleukin-10/genetics ; Mice ; Mice, Knockout ; Th2 Cells/metabolism ; Toll-Like Receptors/*genetics/metabolism ; Trichinella spiralis/genetics/*physiology ; Trichinellosis/genetics/metabolism/*parasitology

Dendritic cells (DCs) comprise two functionally distinct subsets: plasmacytoid DCs (pDCs) and myeloid DCs (mDCs). pDCs are specialized in rapid and massive secretion of type I interferon (IFN-I) in response to nucleic acids through Toll like receptor (TLR)-7 or TLR-9. In this report, we characterized a CD56(+) DC population that express typical pDC markers including CD123 and BDCA2 but produce much less IFN-I comparing with pDCs. In addition, CD56(+) DCs cluster together with mDCs but not pDCs by genome-wide transcriptional profiling. Accordingly, CD56(+) DCs functionally resemble mDCs by producing IL-12 upon TLR4 stimulation and priming naïve T cells without prior activation. These data suggest that the CD56(+) DCs represent a novel mDC subset mixed with some pDC features. A CD4(+)CD56(+) hematological malignancy was classified as blastic plasmacytoid dendritic cell neoplasm (BPDCN) due to its expression of characteristic molecules of pDCs. However, we demonstrated that BPDCN is closer to CD56(+) DCs than pDCs by global gene-expression profiling. Thus, we propose that the CD4(+)CD56(+) neoplasm may be a tumor counterpart of CD56(+) mDCs but not pDCs.
Biomarkers ; metabolism ; CD56 Antigen ; genetics ; immunology ; Cell Lineage ; genetics ; immunology ; Dendritic Cells ; immunology ; metabolism ; pathology ; Gene Expression ; Hematologic Neoplasms ; genetics ; immunology ; pathology ; Humans ; Immunophenotyping ; Interferon Type I ; biosynthesis ; metabolism ; Interleukin-12 ; biosynthesis ; metabolism ; Interleukin-3 Receptor alpha Subunit ; genetics ; immunology ; Lectins, C-Type ; genetics ; immunology ; Membrane Glycoproteins ; genetics ; immunology ; Myeloid Cells ; immunology ; metabolism ; pathology ; Receptors, Immunologic ; genetics ; immunology ; Terminology as Topic ; Toll-Like Receptor 4 ; genetics ; immunology ; Toll-Like Receptor 7 ; genetics ; immunology ; Toll-Like Receptor 9 ; genetics ; immunology

OBJECTIVETo investigate the composition, function, and regulatory mechanisms of the secreted phosphoprotein 1 (SPP1) gene in metastatic prostate cancer.

METHODSWe obtained the data about the whole genomic expression profiles on prostate cancer metastasis from the GEO database, and performed data-mining and bioinformatic analysis using BRB-Array Tools and such softwares as Protparam, MotifScan, SignalP 4.0, TMHMM, NetPhos2.0, PredictProtein, GO, KEGG, and STRING.

RESULTSTotally, 73 co-expressed differential genes in prostate cancer metastasis were identified, 21 up-regulated and 52 down-regulated (P <0.01). Bioinformatic analysis indicated that the highly expressed SPP1 gene encoded 314 amino acids and contained 2 N-glycosylation sites, 8 casein kinase II phosphorylation sites and 3 protein kinase C phosphorylation sites, playing essential roles in extracellular matrix (ECM) binding, ossification, osteoblast differentiation, cell adhesion, PI3K-Akt signaling pathway, focal adhesion, Toll-like receptor signaling pathway, and ECM-receptor interaction.

CONCLUSIONThe bioinformatic method showed a high efficiency in analyzing microarray data and revealing internal biological information. SPP1 may play an important role in prostate cancer metastasis and become a novel biomarker for the diagnosis of prostate cancer metastasis and a new target for its treatment.

Computational Biology ; Data Mining ; Down-Regulation ; Humans ; Male ; Microarray Analysis ; Osteopontin ; chemistry ; genetics ; secretion ; Phosphatidylinositol 3-Kinases ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; Signal Transduction ; Toll-Like Receptors ; metabolism

OBJECTIVETo explore the pathological mechanisms of Guizhi Decoction () syndrome and the therapeutic molecular mechanisms of the Guizhi Decoction, Mahuang Decoction (), Sangju Decoction ( ) and Yinqiao Powder (), as well as the potentially biological basis that Guizhi Decoction is most effective only for the patients with Guizhi Decoction syndrome in clinical practice.

METHODSWe first got serum samples from the patients suffering from both upper respiratory tract infection and Guizhi Decoction syndrome identified by the doctors of Chinese medicine (CM) in the clinic. Four formulas with therapeutic actions of pungent warmth or pungent coolness for superficial syndromes were chosen and four kinds of rat serum samples each containing one of the above-mentioned herbal formulas were collected, then the effects of Guizhi Decoction syndromes' patient serum as well as the effects of sera containing the formulas after being stimulated by the patient serum samples on both the mRNA expression of certain toll-like receptor (TLR) subtypes and the release of some inflammatory cytokines in RAW264.7 cells were tested and analyzed in vitro.

RESULTSThe expression of TLR-3, TLR-4 and TLR-9 mRNA among the 9 tested TLR subforms were up-regulated in the macrophages stimulated by the sera from untreated upper respiratory infection patients with the Guizhi Decoction syndrome (symptomcomplex). The products such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α and interferon (IFN)-β from stimulated macrophages through TLR signaling pathways were also increased correspondingly. Interestingly, the changes induced by the Guizhi Decoction syndrome patients' sera were masked significantly after the macrophages were incubated with the sera from donors treated with Guizhi Decoction. Similarly, the three other exterior-releasing formulas were all effective in reversing the up-regulated changes of certain TLR subforms to different degrees, but both the number of targeted TLRs and efficacy of them seemed to be inferior to that of Guizhi Decoction.

CONCLUSIONEvidence from these experiments might contribute to the scientific explanation of both the pharmacological mechanisms of Guizhi Decoction and also the CM theory that Guizhi Decoction is specifically prescribed for the treatment of Guizhi Decoction syndrome (The gearing formula to the symptom-complex).

Animals ; Cell Survival ; drug effects ; genetics ; Cytokines ; secretion ; Drugs, Chinese Herbal ; pharmacology ; Female ; Gene Expression Regulation ; drug effects ; Healthy Volunteers ; Humans ; Inflammation Mediators ; metabolism ; Inhibitory Concentration 50 ; Macrophages ; drug effects ; metabolism ; Male ; Mice ; Middle Aged ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Syndrome ; Toll-Like Receptors ; genetics ; metabolism

OBJECTIVEThe aim of this study was to explore the roles and clinical significance of innate immune receptors and cytokine in children with measles.

METHODThe children with measles hospitalized in the department of infectious diseases, Children's Hospital of Fudan University during 2009-2011 were enrolled into measles group, while the healthy children examined in well baby clinic were enrolled into control group. The mRNA expression of TLR2/3/4/7, melanoma differentiation-associated gene-5 (MDA-5), retinoic acid-inducible gene I (RIG-I), IFN-α/β and IL-10 in peripheral blood mononuclear cells were detected by real-time PCR. The protein levels of IFN-α, IFN-β and IL-10 in plasma were measured using ELISA. SPSS 13.0 software was applied to analyze the difference between two groups.

RESULTData from a total of 98 patients in measles group and 59 children in control group were collected. The mRNA expressions of TLR2, MDA-5 and RIG-I had no statistical significance between two groups (P > 0.05, respectively). The relative mRNA expressions of TLR3, TLR4, TLR7 in measles group (2.25 ± 0.74, 2.05 ± 0.72, 2.12 ± 0.29) were significantly lower than those in control group (2.09 ± 0.78, 1.90 ± 0.75, 1.87 ± 0.68) (P < 0.01; respectively). Both IFN-α and IFN-β had significantly decreased mRNA expressions in measles patients (2.41 ± 1.31, 2.47 ± 1.26) compared with those in controls (2.22 ± 0.48, 2.35 ± 0.64)(P < 0.01 respectively); however, IL-10 mRNA levels significantly increased (2.49 ± 0.58 vs. 2.62 ± 0.95) (P < 0.001). The IL-10 levels in plasma in measles group were significantly higher during the whole period of fever [<5 d group: 29.89 (25.82-38.15) ng/L and ≥ 5 d group:34.55 (28.26-38.70) ng/L] than that in control group [25.15 (24.20-27.38) ng/L] (P < 0.05 respectively).

CONCLUSIONTLR3/4/7 mRNA expression was low in peripheral blood mononuclear cells of measles patients. Levels of IL-10 were significantly raised in the early stage after infection and lasted for a long time, and reduced IFN-α levels in plasma were associated with the fever durations of measles patients. These results indicated that multiple TLRs and cytokines may participate in the immune response after measles virus infection.

Case-Control Studies ; Child ; Child, Preschool ; Cytokines ; blood ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunity, Innate ; Infant ; Infant, Newborn ; Leukocytes, Mononuclear ; immunology ; metabolism ; Male ; Measles ; immunology ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Real-Time Polymerase Chain Reaction ; Toll-Like Receptors ; genetics ; metabolism

TLR2 activity plays an important role in the pathogenesis of autoimmune diseases, tumor carcinogenesis and cardio-cerebrovascular diseases. To establish a TLR2 receptor-based cell screening model, NF-kappaB promoter-driven luciferase reporter plasmids were transfected into human embryonic kidney cells (HEK293) stably expressing human TLR2 and co-receptors CD14, TLR1 and TLR6. Single clones were then isolated and characterized. Using this screening system, a human TLR2-binding peptide C8 was obtained from the Ph.D.-7 Phage Display Peptide Library through biopanning and rapid analysis of selective interactive ligands (BRASIL). The binding characteristic of C8 with human TLR2 was evaluated by ELISA, flow cytometry and immunofluorescence. The NF-kappaB luciferase activity assay showed that C8 could activate the TLR2/TLR1 signaling pathway and induce the production of cytokines TNF-alpha and IL-6. In conclusion, the TLR2 receptor-based cell screening system is successfully established and a new TLR2-binding peptide is identified by using this system.
Bacteriophages ; Drug Evaluation, Preclinical ; Genes, Reporter ; HEK293 Cells ; Humans ; Interleukin-6 ; metabolism ; Lipopolysaccharide Receptors ; metabolism ; Luciferases ; genetics ; metabolism ; Peptide Library ; Peptides ; metabolism ; pharmacology ; Promoter Regions, Genetic ; Protein Binding ; Signal Transduction ; drug effects ; Toll-Like Receptor 1 ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 6 ; metabolism ; Transfection ; Tumor Necrosis Factor-alpha ; metabolism