RAGE/NF-κB Pathway Mediates Lipopolysaccharide-Induced Inflammation in Alveolar Type I Epithelial Cells Isolated from Neonate Rats

Alveolar type I epithelial cells (AECIs) play an important role in the pathogenesis of acute lung injury. The receptor for advanced glycation end-products (RAGEs) is expressed at a high basal level in AECIs, and its soluble isoform is suggested as a marker of AECI injury. However, the molecular mechanism by which RAGE mediates inflammatory injury in AECIs remains elusive. In this study, we established lipopolysaccharide (LPS)-induced inflammation in AECIs isolated from neonate rats as the experimental model and investigated the role of RAGE/NF-κB signaling in mediating inflammatory response in AECIs. We found that LPS increased RAGE expression and the secretion of tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in AECIs in a dose-dependent manner. Knockdown of RAGE significantly decreased TNF-α and IL-1β levels in conditioned medium of AECIs. Electrophoretic mobility shift assay (EMSA) showed that NF-κB activation was increased in AECIs treated by LPS. However, knockdown of RAGE inhibited both basic and LPS-induced NF-κB activity in AECIs. Finally, NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced LPS-induced upregulation of RAGE expression at both protein and messenger RNA (mRNA) levels in AECIs. Our results suggest that RAGE mediates inflammatory response in AECIs via activating NF-κB, and RAGE/NF-κB pathway presents potential target for the prevention and therapy of acute lung injury.     

Anti-septic Effects of Fisetin In Vitro and In Vivo

Sepsis is a state of disrupted inflammatory homeostasis that is initiated by infection. High mobility group box 1 (HMGB1) protein acting as a late mediator of severe vascular inflammatory conditions, such as sepsis and endothelial cell protein C receptor (EPCR), is involved in vascular inflammation. Fisetin, an active compound from the family Fabaceae, was reported to have antiviral, neuroprotective, and anti-inflammatory activities. Here, we determined the anti-septic effects of fisetin on HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo, for the first time. First, we monitored the effects of post-treatment fisetin on lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-mediated release of HMGB1 and HMGB1-mediated regulation of pro-inflammatory responses in human umbilical vein endothelial cells (HUVECs) and septic mice. Post-treatment fisetin was found to suppress LPS-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangements. Fisetin also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice. Fisetin induced potent inhibition of phorbol-12-myristate 13-acetate (PMA) and CLP-induced EPCR. Fisetin also inhibited the expression and activity of tumor necrosis factor-α converting enzyme, induced by PMA in endothelial cells. In addition, fisetin inhibited the production of tumor necrosis factor-α and the activation of AKT, nuclear factor-κB, and extracellular regulated kinases 1/2 by HMGB1 in HUVECs. Fisetin also down-regulated CLP-induced release of HMGB1, production of interleukin 1β, and reduced septic mortality. Collectively, these results suggest that fisetin may be a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.     

Complex interactions between the components of the PI3K/AKT/mTOR pathway,and with components of MAPK,JAK/STAT and Notch-1 pathways,indicate their involvement in meningioma development

We investigated the significance of PI3K/AKT/mTOR pathway and its interactions with MAPK, JAK/STAT and Notch pathways in meningioma progression. Paraffin-embedded tissue from 108 meningioma patients was analysed for the presence of mutations in PIK3CA and AKT1. These were correlated with the expression status of components of the PI3K/AKT/mTOR pathway, including p85α and p110γ subunits of PI3K, phosphorylated (p)-AKT, p-mTOR, p-p70S6K and p-4E-BP1, as well as of p-ERK1/2, p-STAT3 and Notch-1, clinicopathological data and patient survival. A mutation in PIK3CA or AKT1 was found in around 9 % of the cases. Higher grade meningiomas displayed higher nuclear expression of p-p70S6K; higher nuclear and cytoplasmic expression of p-4E-BP1 and of Notch-1; lower cytoplasmic expression of p85αPI3K, p-p70S6K and p-ERK1/2; and lower PTEN Histo-scores (H-scores). PTEN H-score was inversely correlated with recurrence probability. In univariate survival analysis, nuclear expression of p-4E-BP1 and absence of p-ERK1/2 expression portended adverse prognosis, whereas in multivariate survival analysis, p-ERK1/2 expression emerged as an independent favourable prognostic factor. Treatment of the human meningioma cell line HBL-52 with the PI3K inhibitor LY294002 resulted in reduction of p-AKT, p-p70S6K and p-ERK1/2 protein levels. The complex interactions established between components of the PI3K/AKT/mTOR pathway, or with components of the MAPK, JAK/STAT and Notch-1 pathways, appear to be essential for facilitating and fuelling meningioma progression.     

Sulforaphane Inhibits IL-1β-Induced Proliferation of Rheumatoid Arthritis Synovial Fibroblasts and the Production of MMPs,COX-2, and PGE2

This study was performed to define the effects of sulforaphane on interleukin-1β (IL-1β)-induced proliferation of rheumatoid arthritis synovial fibroblasts (RASFs), the expression of matrix metalloproteinases (MMPs) and cyclooxygenase (COX), and the production of prostaglandin E2 (PGE2) by RASFs. The proliferation of RASFs was evaluated with CCK-8 reagent in the presence of IL-1β with/without sulforaphane. The expression of MMPs, tissue inhibitor of metalloproteinase-1, COXs, intracellular mitogen-activated protein kinase signalings, including p-ERK, p-p38, p-JNK, and nuclear factor-kappaB (NF-kB), and the production of PGE2 were examined by Western blotting or semi-quantitative RT-PCR and ELISA. Sulforaphane inhibits unstimulated and IL-1β-induced proliferation of RASFs; the expression of MMP-1, MMP-3, and COX-2 mRNA and protein; and the PGE2 production induced by IL-1β. Sulforaphane also inhibits the phosphorylation of ERK-1/2, p-38, and JNK and activation of NF-kB by IL-1β. These results indicate that sulforaphane inhibits the proliferation of synovial fibroblasts, the expression of MMPs and COX-2, and the production of PGE2, which are involved in synovitis and destruction of RA, and suggest that sulforaphane might be a new therapeutic agent for RA.     

Geniposide Plays an Anti-inflammatory Role via Regulating TLR4 and Downstream Signaling Pathways in Lipopolysaccharide-Induced Mastitis in Mice

Geniposide is a medicine isolated from Gardenia jasminoides Ellis, which is a traditional Chinese herb that is widely used in Asia for the treatment of inflammation, brain diseases, and hepatic disorders. Mastitis is a highly prevalent and important infectious disease. In this study, we used a lipopolysaccharide (LPS)-induced mouse mastitis model and LPS-stimulated primary mouse mammary epithelial cells (mMECs) to explore the anti-inflammatory effect and the mechanism of action of geniposide. Using intraductal injection of LPS as a mouse model of mastitis, we found that geniposide significantly reduced the infiltration of inflammatory cells and downregulated the production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6). To further investigate the anti-inflammatory mechanism, we used LPS-stimulated mMECs as an in vitro mastitis model. The results of enzyme-linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR) showed that geniposide inhibited the expression of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. Western blot analysis demonstrated that geniposide could suppress the phosphorylation of inhibitory kappa B (IκBα), nuclear factor-κB (NF-κB), p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK). Geniposide also inhibited the expression of toll-like receptor 4 (TLR4) in the LPS-stimulated mMECs. In conclusion, geniposide exerted its anti-inflammatory effect by regulating TLR4 expression, which affected the downstream NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways. Thus, geniposide may be a potential drug for mastitis therapy.     

Stevioside Plays an Anti-inflammatory Role by Regulating the NF-κB and MAPK Pathways in S. aureus-infected Mouse Mammary Glands

Mastitis is an inflammatory disease caused by microbial infection. Staphylococcus aureus is one of the primary bacteria responsible for mastitis. Stevioside is isolated from Stevia rebaudiana and is known to have therapeutic functions. This study was designed to evaluate the effects of stevioside in a mouse model of S. aureus-induced mastitis. In this study, the mouse mammary gland was infected with S. aureus to induce the mastitis model. The stevioside was administered intraperitoneally after the S. aureus infection was established. Hematoxylin–eosin (HE) staining, ELISA, Western blot, and q-PCR methods were used. The results show that stevioside significantly reduced the inflammatory cell infiltration and the levels of TNF-α, IL-1β, and IL-6 and the respective expression of their messenger RNAs (mRNAs). Further studies revealed that stevioside downregulated the TLR2, NF-κB, and (mitogen-activated protein kinase) MAPK signaling pathways in the S. aureus-infected mouse mammary gland. Our results demonstrate that stevioside reduced the expression of TNF-α, IL-1β, and IL-6 by inhibiting the phosphorylation of proteins in the NF-κB and MAPK signaling pathways dose-dependently, but that their mRNA expression was not obviously changed.     

First outbreak of community-acquired MRSA USA300 in France: failure to suppress prolonged MRSA carriage despite decontamination procedures

The first French outbreak of community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 clone was investigated. After outbreak investigation, hygiene measures were implemented in all family households and childminders’ homes. Several decontamination procedures were performed, which used a combination of topical mupirocin, total body application of chlorhexidine, chlorhexidine gargle (if >6 years old) and a course of antibiotic therapy in cases of infection or decontamination failure. Patients were followed up for MRSA skin and soft tissue infections (SSTIs) and carriage. Strains were characterised by antimicrobial drug resistance profile, pulsed-field gel electrophoresis (PFGE) and DNA microarrays. Between June 2011 and June 2012, six children and six adults among the ten corresponding relatives developed 28 SSTIs. None of the family members, including the index case, had any contact with foreigners or individuals known to have SSTIs. After infection control measures and prolonged decontamination have been implemented with a high adherence, six patients remained sustained CA-MRSA USA300 carriers, including one who developed mupirocin resistance and six who experienced minor CA-MRSA-related SSTIs. A baby was identified as an MRSA carrier 2 months after delivery. CA-MRSA decontamination using mupirocin and chlorhexidine in the community setting may also be a questionable strategy, associated with failure and resistance to both agents. Close monitoring of CA-MRSA SSTIs is required in France and in other European countries where MRSA USA300 has recently emerged. We showed that a closed management based on hygiene measures reinforcement, decolonisation and extended screening may fail to suppress CA-MRSA carriage and subsequent infections.     

Curcumin Inhibits TLR2/4-NF-κB Signaling Pathway and Attenuates Brain Damage in Permanent Focal Cerebral Ischemia in Rats

Toll-like receptors 2 and 4 (TLR2/4) and the downstream nuclear factor-kappa B (NF-κB) signaling pathway, which mediate the inflammatory reaction in cerebral ischemia, were demonstrated to be involved in the extension of cerebral infarction and the aggravation of ischemic brain damage. Reports showed that curcumin provides neuroprotection against ischemic brain damage. In this study, we investigated whether curcumin inhibits the activation of TLR2/4-NF-κB signaling pathway in rats of permanent focal cerebral ischemia. Adult male Sprague-Dawley rats underwent permanent middle cerebral artery occlusion (pMCAO). Curcumin was administered by intraperitoneal injection twice at 2 and 12 h after the onset of ischemia. Neurological deficit scores, cerebral infarct size, morphological characteristic, and cerebral water content were measured after 24 h of pMCAO. The enzymatic activity of myeloperoxidase (MPO) was assessed after 24 h of pMCAO. Expression of TLR2 and TLR4 in ischemic brain was determined by western blot. Expression of NF-κB p65 in ischemic brain was detected by immunohistochemistry and western blot. The release of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in blood was examined by ELISA. Curcumin significantly reduced neurological deficit scores, cerebral infarct size, neuronal damage, cerebral water content, and MPO activity. It also inhibited the expression of TLR2/4 and decreased the expression and activity of NF-κB p65 in rat brain. In addition, curcumin attenuated the release of TNF-α and IL-1β in blood. Our results suggest that curcumin reduces inflammatory reaction and brain damage in a rat model of permanent focal cerebral ischemia. The neuroprotective effect and anti-inflammatory property of curcumin in cerebral ischemia might be associated with the inhibition of TLR2/4-NF-κB signaling pathway.     

The Effect of Taurine on the Relationship Between NO,ADMA and Homocysteine in Endotoxin-Mediated Inflammation in HUVEC Cultures

The aim of our study was to investigate the effect of taurine on the relationship between nitric oxide (NO), asymmetric dimethylarginine (ADMA) and homocysteine (Hcy) in endotoxin-induced human umblical vein endothelial cell (HUVEC) cultures. For this reason, four groups were formed (n?=?12). Control group consists of HUVEC cultures without any treatment. Lipopolysaccharide (LPS) and LPS?+?taurine groups were treated with 10 μg/mL endotoxin, 5 μg/mL taurine and endotoxin?+?taurine (same doses), respectively. Nitrite/nitrate (NOx), ADMA and Hcy levels were measured. There was a significant increase of NOx, ADMA and Hcy in endotoxemia (p?p?ADMA levels to the control level both in taurine and taurine?+?LPS group compared to LPS. Hcy levels increased significantly compared to taurine group (p?ADMA–NO relationship whereas no beneficial effect was observed in Hcy levels (p?相似文献   

Use of the enzyme-linked immunosorbent assay for the early diagnosis ofMycoplasma pneumoniae infection

IgM and IgG antibodies toMycoplasma pneumoniae were measured in 147 sera from four groups of patients by means of an indirect enzyme-linked immunosorbent assay (ELISA) and the results compared with those obtained by other methods. A good correlation was demonstrated between the complement fixation test and ELISA-IgM and to a lesser extent ELISA-IgG; for the metabolic inhibition test the reverse was the case. The indirect haemagglutination test appeared to detect mainly IgM antibodies. Low levels of IgM antibodies were dectected by ELISA in 60 sera of children not suffering from pneumonia. However, if only high titres (> 800) were regarded as indicative ofMycoplasma pneumoniae infection, a presumptive diagnosis could have been made in 42 of 73 single acute phase sera from patients. Comparable results were obtained with IHA. The diagnostic level of IgM antibodies was reached during the second week of the disease. It is concluded that examination of a single serum sample by either ELISA-IgM or IHA may assist in early diagnosis ofMycoplasma pneumoniae infection.     

Improve immunogenicity of DNA vaccine against foot-and-mouth disease virus: Role of intron and probably viral 3C protease as biological adjuvants

Foot-and-mouth disease (FMD) is one of the most important diseases with heavy economic losses. The causative agent of the disease is a virus, named as FMD virus, belonging to the picornavirus family. There is no treatment for the disease and vaccination is the main control strategy. Several vaccination methods have been introduced against FMD including DNA vaccines. In this study, two genetic constructs, which were defined by absence and presence of an intron, were tested for their ability to induce the anti-FMD virus responses in mouse. Both constructs encoded a fusion protein consisting of viral (P12A and 3C) and EGFP proteins under the control of CMV promoter. The protein expression was studied in the COS-7 cells transfected with the plasmids by detecting EGFP protein. Cell death was induced in the cells expressing the P12A3C-EGFP, but not the EGFP, protein. This might be explained by the protease activity of the 3C protein which cleaved critical proteins of the host cells. Mice injected with the intron-containing plasmid induced 16-fold higher antibody level than the intronless plasmid. In addition, serum neutralization antibodies were only induced in the mice injected with intron-containing plasmid. In conclusion, the use of intron might be a useful strategy for enhancing antibody responses by DNA vaccines. Moreover, cell death inducing activity of the 3C protein might suggest applying it along with DNA vaccines to improve immunogenicity.     

Antibody-dependent cell-mediated cytotoxicity againstEscherichia coli O antigens

Antibodies againstEscherichia coli O antigen from rabbits immunized with formalin-killed bacteria were tested for cytotoxic capacity in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay with human lymphocytes as effector cells and autologous papainized erythrocytes coated with O antigen as target cells. The cytotoxic titres were compared with the titres obtained with three methods of antibody quantitation. It was found that ADCC recorded antibodies with similar sensitivity as the enzyme-linked immunosorbent assay (ELISA) for IgG, but was much more sensitive than the ammonium sulphate precipitation (ASP) and indirect haemagglutination (IHA) usingβ-mercaptoethanol reduced sera. The ADCC titres were found to correlate very well with the titres obtained with ASP, ELISA and IHA for IgG but not for IgM, which is in accordance with a previous notion that ADCC is primarily mediated via IgG antibodies. ADCC should be considered as a possible immunopathologic mechanism in renal parenchymal damage in connection with urinary tract infections.     

The utility of IgG,IgM, and CD138 immunohistochemistry in the evaluation of autoimmune liver diseases

Primary biliary cirrhosis (PBC) and autoimmune hepatitis (AIH) present with distinct clinical features. The term “PBC–AIH overlap syndrome (OS)” has been adopted to describe the condition characterized by occurrence of both PBC and AIH, although this clinical entity is difficult to define. This study aimed to assess the utility of IgG, IgM, and CD138 immunohistochemistry in the evaluation of AIH, PBC, and OS. Immunohistochemistry was performed with anti-human IgG, IgM, and CD138 to detect specific plasma cells in the liver. Predominant IgG staining was observed in AIH (85.7 %), while equivocal (46.1 %) or predominant (38.5 %) IgM staining was observed in PBC. In OS, equivocal (20 %) or predominant (80 %) IgG staining was observed. The IgM/IgG ratio was significantly higher in PBC than in AIH or OS (P < 0.005). Histological findings revealed significantly higher IgM expression in PBC at cholangitis activity grades 2–3 compared to those at cholangitis activity grades 0–1. In contrast, a significantly higher IgG expression was observed in PBC at hepatitis activity and fibrosis grades 2–3 compared to those at hepatitis activity and fibrosis grades 0–1. Taken together, periportal plasmacytic infiltrates with variable immunohistochemistry patterns of IgG and IgM expression characterized different autoimmune liver diseases.