Downregulation of ST3GAL5 is associated with muscle invasion, high grade and a poor prognosis in patients with bladder cancer.

In patients with bladder cancer (BC), the association between ST3 ß-galactoside α-2,3-sialyltransferase 5 (ST3GAL5) expression and clinical outcomes, particularly regarding muscle-invasive disease, high tumor grade and prognosis, remain unknown. In the present study, the expression of ST3GAL5 and its association with clinical outcomes in patients with BC was analyzed using various public bioinformatics databases. The difference in ST3GAL5 expression between BC and healthy bladder tissues was also evaluated using data from the Oncomine database, The Cancer Genome Atlas and Gene Expression Omnibus database. The differences in ST3GAL5 expression between muscle invasive BC (MIBC) and non-muscle invasive BC (NMIBC), and high- and low-grade BC were also analyzed. Furthermore, genes that were positively co-expressed with ST3GAL5 in patients with BC were identified from the intersection between the Oncomine, Gene Expression Profiling Interactive Analysis 2 and UALCAN databases. Enrichment analysis by Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, Reactome pathway enrichment analyses and a gene-concept network was performed using R package. Gene set enrichment analysis was also performed to assess the signaling pathways influenced by the high and low expression of ST3GAL5 in BC. The results indicated that ST3GAL5 expression was significantly lower in BC tissues compared with normal bladder tissues (P<0.05). Furthermore, ST3GAL5 expression in MIBC and high-grade BC was significantly lower compared with NMIBC and low-grade BC (P<0.05), respectively. The results from Kaplan-Meier survival analysis result demonstrated that ST3GAL5 downregulation was associated with poor survival in patients with BC (P<0.05). Taken together, these findings suggested that ST3GAL5 may be considered as an anti-oncogene in BC, could represent a potential predictive and prognostic biomarker for BC and may be a molecular target for tumor therapy.

Synthetic Human TLR9-LRR11 Peptide Attenuates TLR9 Signaling by Binding to and thus Decreasing Internalization of CpG Oligodeoxynucleotides.

Toll-like receptor (TLR) 9 is an endosomal receptor recognizing bacterial DNA/CpG-containing oligodeoxynucleotides (CpG ODN). Blocking CpG ODN/TLR9 activity represents a strategy for therapeutic prevention of immune system overactivation. Herein, we report that a synthetic peptide (SP) representing the leucine-rich repeat 11 subdomain of the human TLR9 extracellular domain could attenuate CpG ODN/TLR9 activity in RAW264.7 cells by binding to CpG ODN and decreasing its internalization. Our results demonstrate that preincubation with SP specifically inhibited CpG ODN- but not lipopolysaccharide (LPS)- and lipopeptide (PAM3CSK4)-stimulated TNF-α and IL-6 release. Preincubation of SP with CpG ODN dose-dependently decreased TLR9-driven phosphorylation of IκBα and ERK and activation of NF-κB/p65. Moreover, SP dose-dependently decreased FAM-labeled CpG ODN internalization, whereas non-labeled CpG ODN reversed the inhibition. The KD value of SP-CpG ODN binding was within the micromolar range. Our results demonstrated that SP was a specific inhibitor of CpG ODN/TLR9 activity via binding to CpG ODN, leading to reduced ODN internalization and decreased activation of subsequent pathways within cells. Thus, SP could be used as a potential CpG ODN antagonist to block TLR9 signaling.

KDM6B induces epithelial-mesenchymal transition and enhances clear cell renal cell carcinoma metastasis through the activation of SLUG.

Clear cell renal cell carcinoma (ccRCC) is one of the most common kidney cancers; epithelial-mesenchymal transition (EMT) is associated with carcinoma invasion and metastasis. There have been several studies about the molecular regulation of EMT, but the relationship between histone demethylase and EMT is little known. Here, we reported KDM6B has high expression level in ccRCC and is positively correlated with poor ccRCC prognosis. KDM6B, also known as JMJD3, is a histone demethylase, can remove repressive histone H3K27me3 marks from chromatin, thereby activating gene expression. We found that the knockdown of KDM6B could inhibit ccRCC tumorigenesis in vitro; furthermore, KDM6B could induce EMT in ccRCC cells by activating the expression of master transcription factor SLUG. ChIP assays revealed that KDM6B stimulated SLUG expression by demethylate histone H3K27me3. The knockdown of KDM6B strongly inhibited ccRCC cell invasion in vitro, while the overexpression of KDM6B shown the opposite trend. Meanwhile, our analysis of the ccRCC tissue found that KDM6B expression was significantly corresponded with lymph node metastasis. Together, our data provide a novel epigenetic mechanism regulating tumor cell invasion and EMT, and provide a biomolecule for ccRCC diagnosis and prognosis.

Correlation between ApoE gene polymorphisms and the occurrence of urolithiasis.

The aim of the present study was to investigate the correlation between apolipoprotein E (ApoE) gene polymorphisms and the occurrence of urolithiasis and dyslipidemia. A total of 180 Uyghur individuals, including 90 urolithiasis patients and 90 healthy controls, were enrolled in this study. The blood lipid profiles of the patients and controls were investigated and compared, and the composition of the urinary calculi was determined. The polymorphisms of the ApoE alleles were analyzed using polymerase chain reaction-restriction fragment length polymorphism analysis. Three common genotypes of the ApoE gene, E3/3, E3/4 and E4/4, were detected in the urolithiasis patients and control group. In the patient group, 28 patients with the E3/3 genotype (30.1%), 58 patients with the E3/4 genotype (64.4%) and four patients with the E4/4 genotype (4.5%) were identified. By contrast, in the control group, 52 patients with the E3/3 genotype (57.8%), 35 patients with the E3/4 genotype (38.9%) and three patients with the E4/4 genotype (3.3%) were identified. The frequency of the E3/4 genotype was found to be significantly higher in the patient group when compared with the control group (χ2=12.96; P<0.001). In addition, the frequency of the E4 allele was significantly higher in the patient group when compared with the control group (χ2= 6.61; P=0.025). In conclusion, the occurrence of urolithiasis was found to be associated with ApoE gene polymorphisms, and the E4 allele may be a potential susceptibility factor for urolithiasis.

Low dose of interferon-α improves the clinical outcomes of docetaxel in patients with castration-resistant prostate cancer: A pilot study.

The aim of this study was to test whether a low dose of interferon-α-2b (IFN-α2b) enhances the clinical outcome of docetaxel (DXT) in patients with castration-resistant prostate cancer (CRPC). A prospective controlled trial of 40 CRPC patients receiving 5 mg of prednisone twice daily was conducted, where patients were randomly assigned to be administered 75 mg/m2 DXT plus 3 mIU/m2 IFN-α2b (group A, n=20) or 75 mg/m2 DXT alone (group B, n=20). The prostate-specific antigen (PSA) response, tumor response, progression-free survival (PFS) and overall survival (OS) were evaluated. There was no statistically significant difference in PSA response rate between groups A and B (65 vs. 47.4%, P=0.341). The tumor response rate in group A was significantly greater compared with that in group B (55 vs. 21.1%, P=0.048). The median PFS was longer in group A compared with that in group B (10 vs. 8 months, P=0.043). There was no statistically significant difference in median OS between the two groups (19 vs. 17 months, P=0.348), but one patient displayed a complete tumor response in group A. In groups A and B, transient grade 3 to 4 neutropenia was observed in nine and six patients, grade 3 to 4 anemia was observed in three and five patients, and grade 3 to 4 general fatigue was observed in four and one patient(s), respectively. The proportion of patients with grade 3 to 4 toxicity was not statistically different between the two groups. A low dosage of IFN-α2b may improve the antitumor activity of DXT with an acceptable toxicity profile in patients with CRPC.

Interstitial cells of Cajal mediate excitatory sympathetic neurotransmission in guinea pig prostate.

Morphological and functional studies have confirmed that interstitial cells of Cajal (ICCs) are involved in many enteric motor neurotransmission pathways. Recent investigations have demonstrated that human and guinea pig prostate glands possess a distinct cell type with morphological and immunological similarities to ICCs. These prostate ICCs have a close relationship with nerve bundles and smooth muscle cells. Prostate smooth muscle tone is largely induced by stimulation from the sympathetic nervous system, which releases excitatory norepinephrine (NE) to act on the α1-adrenoceptor. We have performed morphological and functional experiments to determine the role of ICCs in sympathetic neurotransmission in the guinea pig prostate based on the hypothesis that prostate ICCs act as mediators of sympathetic neurotransmission. Immunohistochemistry revealed many close points of contact between ICCs and sympathetic nerve bundles and smooth muscle cells. Double-labeled sections revealed that α1-adrenoceptor and the gap junction protein connexin 43 were expressed in prostate ICCs. Surprisingly, prostate ICCs co-expressed tyrosine hydroxylase and dopamine ß-hydroxylase, two markers of sympathetic neurons. Functionally, the application of NE evoked a large single inward current in isolated prostate ICCs in a dose-dependent manner. The inward current evoked by NE was mediated via the activation of α1-adrenoceptors, because it was abolished by the non-specific α-adrenoceptor antagonist, phentolamine and the specific α1-adrenoceptor antagonist, prazosin. Thus, ICCs in the guinea pig prostate are target cells for prostate sympathetic nerves and possess the morphological and functional characteristics required to mediate sympathetic signals.

[Inhibitory effect of dihydroartemisinin on the growth of human prostate cancer PC-3M cells and its mechanism].

OBJECTIVE: To study the effects of dihydroartemisinin on the apoptosis of and the vascular endothelial growth factor (VEGF) expression in prostate cancer cell line PC-3M in androgen-independent prostate cancer. METHODS: PC-3M cells were treated with different doses (0, 25, 50 and 100 micromol/L) of dihydroartemisinin for 48 hours, their growth activity analyzed by MTT colorimetric assay and flow cytometry, and changes in the activities of caspase-3 and -8 detected by colorimetric assay. The expression of VEGF mRNA was determined by semi-quantitative RT-PCR, and that of the VEGF protein by Western blotting. RESULTS: Compared with the 0 micromol/L control group, the 25, 50 and 100 micromol/L dihydroartemisinin groups showed significantly increased apoptosis of PC-3M cells ([2.92 +/- 0.45]% vs [8.85 +/- 0.74]%, [12.83 +/- 0.84]% and [18.65 +/- 1.24]%, P < 0.01), and dose-dependent increase in the activities of caspase-8 ([0.47 +/- 0.05 ] U/microg vs [1.22 +/- 0.15], [1.76 +/- 0.07] and [2.91 +/- 0.24] U/microg, P < 0.01) and caspase-3 ([0.44 +/- 0.07] U/microg vs [0.95 +/- 0.08], [1.48 +/- 0.14] and [2.92 +/- 0.45] U/microg, P < 0.01). The expressions of VEGF mRNA and protein were decreased in a concentration-dependent manner. CONCLUSION: Dihydroartemisinin can significantly suppress the growth of PC-3M cells, promote their apoptosis and reduce the expressions of VEGF mRNA and protein, which may serve to explain its inhibitory effect on tumor and angiogenesis.

Leucine-rich repeat 11 of Toll-like receptor 9 can tightly bind to CpG-containing oligodeoxynucleotides, and the positively charged residues are critical for the high affinity.

TLR9 is a receptor for sensing bacterial DNA/CpG-containing oligonucleotides (CpG ODN). The extracellular domain (ECD) of human TLR9 (hTLR9) is composed of 25 leucine-rich repeats (LRR) contributing to the binding of CpG ODN. Herein, we showed that among LRR2, -5, -8, and -11, LRR11 of hTLR9 had the highest affinity for CpG ODN followed by LRR2 and -5, whereas LRR8 had almost no affinity. In vitro, preincubation with LRR11 more significantly decreased CpG ODN internalization, subsequent NF-κB activation, and cytokine release than with LRR2 and -5 in mouse peritoneal macrophages treated with CpG ODN. The LRR11 deletion mutant of hTLR9 conferred decreased cellular responses to CpG ODN. Single- or multiple-site mutants at five positively charged residues of LRR11 (LRR11m1-9), especially Arg-337 and Lys-367, were shown to contribute to hTLR9 binding of CpG ODN. LRR11m1-9 showed reduced inhibition of CpG ODN internalization and CpG ODN/TLR9 signaling, supporting the above findings. Prediction of whole hTLR9 ECD-CpG ODN interactions revealed that Arg-337 and Lys-338 directly contact CpG ODN through hydrogen bonding, whereas Lys-347, Arg-348, and His-353 contribute to stabilizing the shape of the ligand binding region. These findings suggested that although all five positively charged residues within LRR11 contributed to its high affinity, only Arg-337 and Lys-338 directly interacted with CpG ODN. In conclusion, the results suggested that LRR11 could strongly bind to CpG ODN, whereas mutations at the five positively charge residues reduced this high affinity. LRR11 may be further investigated as an antagonist of hTLR9.

CpG ODN107 potentiates radiosensitivity of human glioma cells via TLR9-mediated NF-κB activation and NO production.

Radiotherapy is a standard treatment for glioma patient with or without surgery; radiosensitizer can increase tumor sensitivity for radiotherapy. Herein, a synthetic oligodeoxynucleotide containing unmethylated CpG dinucleotides (CpG ODN107) as a radiosensitizer was investigated in vitro and in vivo, and the possible mechanisms were studied in vitro. In the present experiments, the human glioma U87 cell line used herein was resistant to 5 Gy of ß-ray irradiation. The results showed that 10 µg/ml of CpG ODN107 in combination with irradiation significantly inhibited cell proliferation both in MTT assay and colony formation experiments. Tumor growth was inhibited by CpG ODN107 in combination with local irradiation but not by local irradiation or CpG ODN107 alone in human glioma xenograft model in nude mice. The inhibition ratio of tumor growth produced by CpG ODN107 (1.7, 5, and 15 mg/kg) in combination with irradiation was 27.3, 67.0, and 65.5 %, respectively. Further molecular mechanisms were studied in vitro. The results showed that the expressions of iNOS, NO, TLR9 mRNA, and NF-κB p50/p65 increased in the cells treated with CpG ODN107 in combination with irradiation. CpG ODN107 in combination with irradiation did not induce apoptosis but induced cell cycle arrest at G(1) phase. The said results demonstrated that CpG ODN107 possessed a radiosensitizing effect via TLR9-mediated NF-κB activation and NO production in the tumor cells, leading to cell cycle arrest. Therefore, CpG ODN107 is a potential candidate as radiosensitizer for human glioma.

[Primary non-Hodgkin's lymphoma of the testis and penis: clinical analysis of 5 cases].

OBJECTIVE: To improve the clinical diagnosis and treatment of primary non-Hodgkin's lymphoma of male genitalia. METHODS: We retrospectively reviewed the clinical data of 5 cases of primary non-Hodgkin's lymphoma of male genitalia, 4 in the testis and 1 in the penis, we also analyzed the relevant literature and clinical significance of the disease. RESULTS: All the 5 cases were treated by surgery and pathologically confirmed to be non-Hodgkin's lymphoma. Three of them received chemotherapy, and the other 2 (1 in the testis and 1 in the penis) underwent both chemotherapy and radiotherapy after the operation. Follow-up averaged 25 months, during which 1 of the patients died and the other 4 survived. CONCLUSION: Primary non-Hodgkin's lymphoma of male genitalia is an uncommon disease with atypical clinical presentations and poor prognosis, which occurs mostly in elderly males. Definite diagnosis of the disease mainly depends on histopathology and immunohistochemistry. Surgery with multiagent chemotherapy and radiotherapy is advisable for its treatment.

Artesunate enhances the antibacterial effect of {beta}-lactam antibiotics against Escherichia coli by increasing antibiotic accumulation via inhibition of the multidrug efflux pump system AcrAB-TolC.

OBJECTIVES: Occasionally, we found that artesunate enhanced the antibacterial effects of antibiotics in vitro. Therefore, the enhancement of various ß-lactam antibiotics by artesunate against Escherichia coli and the possible mechanism were investigated in the present study. METHODS: Antibacterial effects were observed using the serial 2-fold dilution method and dynamic bacterial growth. Daunomycin accumulation within E. coli was observed using fluorospectrophotometry and laser confocal scanning microscopy. AcrAB-TolC, AmpC and TEM-1 mRNA expression was observed using a PCR method. Antisense oligonucleotides (as-ODNs) targeting AcrB were designed and used to block AcrB gene expression within E. coli ATCC 35218. RESULTS: Although artesunate itself had no antibacterial ability, artesunate significantly increased the antibacterial effect of ß-lactam antibiotics against E. coli ATCC 35218 and an E. coli clinical strain. Artesunate increased daunomycin accumulation within E. coli ATCC 35218 in a dose-dependent manner and reduced the mRNA expression of AcrAB-TolC, an important multidrug efflux system for Gram-negative bacteria. The bacterial number was significantly reduced by as-ODN targeting AcrB, but did not further decrease after additional artesunate treatment. In contrast, artesunate lost its enhancement of ß-lactam antibiotics against E. coli AG100A, a strain lacking the gene encoding AcrAB, and artesunate did not increase daunomycin accumulation within E. coli AG100A. After the transformation of pET28a-AcrB into E. coli AG100A, artesunate regained enhancement of ß-lactam antibiotics. Furthermore, artesunate did not inhibit the expression of AmpC and TEM-1 mRNA. CONCLUSIONS: Artesunate enhances the antibacterial effect of various ß-lactam antibiotics against E. coli, which might be associated with the suppression of a major multidrug resistance system, AcrAB-TolC.

Artesunate protects sepsis model mice challenged with Staphylococcus aureus by decreasing TNF-alpha release via inhibition TLR2 and Nod2 mRNA expressions and transcription factor NF-kappaB activation.

Gram-positive bacteria have become the most common organisms responsible for the development of sepsis. Staphylococcus aureus (S. aureus) is the major gram-positive pathogen in both community-acquired and nosocomial infections. The Mortality associated with nosocomial infections caused by S. aureus may vary but are generally high. In the present study, we found that artesunate (AS) could protect mice against a lethal challenge with heat-killed S. aureus in a dose-dependent manner, and AS in combination with ampicillin sodium-sulbactam sodium (AMPS) could further increase survival of mice challenged with live S. aureus than AMPS alone. This protection was associated with reductions of serum at TNF-alpha level. In in vitro experiments, AS-pretreatment strongly inhibited TNF-alpha release from murine peritoneal macrophage induced by heat-killed S. aureus or peptidoglycan in a dose-dependent manner. AS reduced the Toll like receptor 2 (TLR2) and nucleotide-binding oligomerization domain containing 2 (Nod2) mRNA expressions up-regulated by heat-killed S. aureus and inhibited NF-kappaB activation induced by heat-killed S. aureus. In conclusion, our results demonstrated that AS-mediated protection on septic mice challenged with S. aureus was associated with its reduction on TNF-alpha release via inhibition of TLR2 and Nod2 mRNA expressions and transcription factor NF-kappaB activation.

Antimalarial artesunate protects sepsis model mice against heat-killed Escherichia coli challenge by decreasing TLR4, TLR9 mRNA expressions and transcription factor NF-kappa B activation.

Bacterial DNA (bDNA) and lipopolysaccharide (LPS) are potent activators of immune cells such as monocytes and macrophages, which contribute to systemic inflammatory response syndrome (SIRS) and sepsis. Unfortunately, many experimental inflammatory antagonist-based therapies have failed in sepsis trials, and currently there is only one adjuvant therapy in clinical use, e.g. activated protein C. Artesunate (AS), a water-soluble derivative of dihydroartemisinin, has recently been demonstrated to protect against LPS-induced human umbilical vein endothelial cell (HUVEC) activation and injury by inhibiting tumor necrosis factor-alpha (TNF-alpha) mRNA expression. In the present study, heat-killed Escherichia coli was used to induce sepsis in the animal models. We observed that AS could protect mice against a lethal challenge with heat-killed E. coli in a dose-dependent manner. This protection was associated with reductions in serum TNF-alpha and measurable endotoxin levels. In addition, the treatment of murine peritoneal macrophage cells with AS strongly inhibited the release of TNF-alpha and IL-6 induced by CpG oligodeoxynucleotide (CpG ODN), LPS, or heat-killed E. coli in a dose-dependent manner. Experiments using affinity sensor technology revealed that AS could not directly bind to CpG ODN or LPS. Moreover, AS could not neutralize LPS in vitro. Further, flow cytometry revealed that AS could not alter the binding of CpG ODN to cell surfaces but could promote CpG ODN accumulation within RAW264.7 cells. Furthermore, AS reduced the expressions of TLR4 and TLR9 mRNA that were stimulated by LPS, CpG ODN, or heat-killed E. coli and inhibited heat killed E. coli-induced NF-kappaB activation. In conclusion, our results demonstrated that AS-mediated protection against a lethal heat-killed E. coli challenge was associated with a reduction in proinflammatory cytokine release and endotoxin levels via a mechanism involving a decrease in TLR4, TLR9 mRNA expression and NF-kappaB activation.

[Clone, expression and identification of penicillin binding protein 2a of methicillin-resistant Staphylococcus aureus isolated from patients].

OBJECTIVE: To clone, express and identify the mecA fragment which encoded penicillin binding protein 2a (PBP2a) from methicillin-resistant staphylococcus aureus (MRSA) isolated from patients by gene recombination method. METHODS: According to the sequence of mecA gene recorded in GenBank, the primer of mecA fragment which encoded amino acids 25 - 668 of PBP2a was designed. Then the mecA fragment was amplified by PCR and cloned into pQE30 plasmid. After being identified by enzyme digestion and sequencing, the recombinant plasmid was transferred into E. coli M15 [pREP4], and then its expression was induced by 1 mmol/L Isopropy-beta-D-Thiogalactoside (IPTG). The expression product was analyzed by SDS-PAGE, protein sequencing and mass spectroscopy. RESULTS: The recombinant pQE30- mecA had been successfully constructed. The result of sequencing showed that the mecA fragment had 1932 bases, including 9 bases undergoing mutation. After being induced for 6 hours by IPTG, the soluble protein in M15 (pQE30- mecA), with a relative molecular weight of 74 x 10(3), was found by SDS-PAGE. The soluble protein had been confirmed to be PBP2a after identification. CONCLUSION: The soluble PBP2a of MRSA isolated from patients is expressed successfully by gene recombinant technology.

The newly identified CpG-N ODN208 protects mice from challenge with CpG-S ODN by decreasing TNF-alpha release.

Administration of an excess of oligodeoxynucleotides containing immunostimulatory CpG motifs (CpG-S ODNs) may induce systemic inflammatory response syndrome (SIRS) and sepsis. Therefore, it is important to develop neutralizing CpG ODNs (CpG-N ODNs), which can be used to reduce the release of cytokines induced by the presence of CpG-S ODNs. In the present study, CpG-N ODN208 (5'-TGCCGCGGCAGA-3'), a neutralizing twelve-oligodeoxynucleotide molecule recently identified in our laboratory, inhibited TNF-alpha release from human peripheral blood mononuclear cells (hPBMCs) and murine RAW264.7 cells induced by CpG-S ODN exposure in a dose- and time-dependent manner. Flow cytometry revealed that CpG-N ODN208 decreased cell-surface binding and internalization of 6-FAM-CpG-S ODN. However, the decreased cell-surface binding and internalization of CpG-S ODN could not completely account for the decreased TNF-alpha release. RT-PCR experiments revealed that CpG-N ODN treatment could down-regulate the CpG-S ODN-induced upregulation of Toll-like receptor 9 (TLR9) mRNA expression. This finding suggested that the decreased cytokine release following CpG-N ODN treatment might be related to decreased TLR9 mRNA expression. In in vivo experiments, no protection was found when the ratio of CpG-N ODN to CpG-S ODN delivered to mice was 3:1. However, at a 5:1 ratio, CpG-N ODN208 could protect mice from an ordinarily lethal dose of CpG-S ODN. Furthermore, we found that CpG-N ODN208 treatment decreased serum TNF-alpha levels in mice injected with sublethal doses of CpG-S ODN whether the CpG-N ODN208 was added prior to or concurrent with the CpG-S ODN. Our results demonstrated that CpG-N ODN-mediated protection against a lethal challenge by CpG-S ODN was associated with the reduction of TNF-alpha release.

The antimalarial artemisinin synergizes with antibiotics to protect against lethal live Escherichia coli challenge by decreasing proinflammatory cytokine release.

In the present study artemisinin (ART) was found to have potent anti-inflammatory effects in animal models of sepsis induced by CpG-containing oligodeoxy-nucleotides (CpG ODN), lipopolysaccharide (LPS), heat-killed Escherichia coli 35218 or live E. coli. Furthermore, we found that ART protected mice from a lethal challenge by CpG ODN, LPS, or heat-killed E. coli in a dose-dependent manner and that the protection was related to a reduction in serum tumor necrosis factor alpha (TNF-alpha). More significantly, the administration of ART together with ampicillin or unasyn (a complex of ampicillin and sulbactam) decreased mortality from 100 to 66.7% or 33.3%, respectively, in mice subjected to a lethal live E. coli challenge. Together with the observation that ART alone does not inhibit bacterial growth, this result suggests that ART protection is achieved as a result of its anti-inflammatory activity rather than an antimicrobial effect. In RAW264.7 cells, pretreatment with ART potently inhibited TNF-alpha and interleukin-6 release induced by CpG ODN, LPS, or heat-killed E. coli in a dose- and time-dependent manner. Experiments utilizing affinity sensor technology revealed no direct binding of ART with CpG ODN or LPS. Flow cytometry further showed that ART did not alter binding of CpG ODN to cell surfaces or the internalization of CpG ODN. In addition, upregulated levels of TLR9 and TLR4 mRNA were not attenuated by ART treatment. ART treatment did, however, block the NF-kappaB activation induced by CpG ODN, LPS, or heat-killed E. coli. These findings provide compelling evidence that ART may be an important potential drug for sepsis treatment.

Lipopolysaccharide could be internalized into human peripheral blood mononuclear cells and elicit TNF-alpha release, but not via the pathway of toll-like receptor 4 on the cell surface.

Lipopolysaccharide (LPS), the principal component of the outer membrane of Gram-negative bacteria, stimulates various cell types to release numerous proinflammatory mediators such as TNF-alpha, IL-6 and IL-12, which may damage cells and lead to organ injury, even sepsis and septic shock. Toll-like receptor 4 (TLR4) has been identified as the receptor involved in the recognition of LPS, but the role of LPS uptake in activating signal transduction remains controversial. In the present study, TNF-alpha was used as a marker of macrophages/ monocytes activated by LPS, and CQ was used as an inhibitor of endosome mature in order to definitude what stage of the signal transduction elicited by LPS was interrupted. We found that there indeed existed internalization of LPS and internalization partially participated in LPS signaling since CQ inhibited cytokine release, and decreased accumulation of FITC-LPS in hPBMCs. In contrast, anti-hTLR4 antibody could decrease cytokine release, but had no inhibition on accumulation of FITC-LPS. This result revealed that inhibition of cytokine release was related to reduction of FITC-LPS accumulation in the cells. But TLR4 on the cell surface couldn't participate in internalization of LPS. Thus, LPS signaling and internalization couldn't be viewed as mutually independent processes.