Endogenous α7 nAChR Agonist SLURP1 Facilitates Escherichia coli K1 Crossing the Blood-Brain Barrier.

Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is critical for the pathogenesis of Escherichia coli (E. coli) K1 meningitis, a severe central nervous system infection of the neonates. However, little is known about how E. coli K1 manipulates α7 nAChR signaling. Here, through employing immortalized cell lines, animal models, and human transcriptional analysis, we showed that E. coli K1 infection triggers releasing of secreted Ly6/Plaur domain containing 1 (SLURP1), an endogenous α7 nAChR ligand. Exogenous supplement of SLURP1, combined with SLURP1 knockdown or overexpression cell lines, showed that SLURP1 is required for E. coli K1 invasion and neutrophils migrating across the blood-brain barrier (BBB). Furthermore, we found that SLURP1 is required for E. coli K1-induced α7 nAChR activation. Finally, the promoting effects of SLURP1 on the pathogenesis of E. coli K1 meningitis was significantly abolished in the α7 nAChR knockout mice. These results reveal that E. coli K1 exploits SLURP1 to activate α7 nAChR and facilitate its pathogenesis, and blocking SLURP1-α7 nAChR interaction might represent a novel therapeutic strategy for E. coli K1 meningitis.

[SBi4211 alleviates gp120-induced central nervous system injury via inhibiting S100B/ RAGE].

OBJECTIVE: To explore the protective effect of SBi4211 (heptamidine), an inhibitor of S100B, against central nervous system injury induced by HIV-1 envelope protein gp120. METHODS: In an in vitro model, U251 glioma cells were co-cultured with SH-SY5Y cells to explore the protective effect of SBi4211 against gp120-induced central nervous system injury. In a gp120 transgenic (Tg) mouse model (8 months old) mimicking HIV-associated neurocognitive disorder (HAND), the effect of treatment with gp120 or both gp120 and SBi4211 on neuronal activity and apoptosis were assessed using Cell Counting kit-8 (CCK-8) and flow cytometry. ELISA, Western blotting and immunohistochemistry were used to determine the expression levels of S100B, RAGE, GFAP, NeuN, Syn, MAP-2 and the inflammatory factors IL-6 and TNF-α. RESULTS: In the cell co-culture system, SBi4211 treatment significantly inhibited gp120-induced expression of S100B, RAGE and GFAP in U251 cells (P < 0.001), reduced the levels of inflammatory factors iNOS, IL-6 and TNF-α (P < 0.001) and enhanced the expressions of neuron-related proteins NeuN, Syn and MAP-2 (P < 0.001). In the transgenic mouse model, SBi4211 treatment significantly reduced the expressions of S100B, RAGE and inflammation levels (P < 0.05), inhibited the activation of astrocytes in the brain, and maintained the integrity of the neurons (P < 0.05). CONCLUSIONS: SBi4211 can protect neurons from gp120-induced neurotoxicity possibly by inhibiting the S100B/ RAGE-mediated signaling pathway.

Oral Immunization with Recombinant Lactobacillus acidophilus Expressing espA-Tir-M Confers Protection against Enterohemorrhagic Escherichia coli O157:H7 Challenge in Mice.

Enterohemorrhagic Escherichia coli O157:H7 (EHEC O157:H7) causes hemorrhagic colitis and the formation of characteristic attaching and effacing (A/E) lesions in humans. Given the severe sequelae of EHEC O157:H7 infection, it is critical to develop effective vaccines for human use. However, for achieving this goal many hurdles need to be addressed, such as the type or subset of antigens, adjuvant, and the delivery route. We developed a candidate vaccine by inserting the bivalent antigen espA-Tir-M composed of espA and the Tir central domain into Lactobacillus acidophilus. The recombinant L. acidophilus (LA-ET) was safe in a cell model and excluded EHEC O157:H7 from LoVo cells at rates of nearly 94 and 60% in exclusion and competition assays, respectively. LA-ET inhibited the induction of A/E lesions by EHEC O157:H7 cells in vitro. Oral immunization with LA-ET induced higher levels of specific mucosal and systemic antibody responses in mice. Moreover, LA-ET enhanced interferon-γ and interleukin-4 and -10 production, which was associated with mixed helper T (Th1/Th2) cell responses, and protected against EHEC O157:H7 colonization and infection in mice at a rate of 80%. Histopathological analyses revealed that orally administered LA-ET reduced or inhibited A/E lesions and toxin-induced systemic injury. These findings demonstrate that LA-ET induces both humoral and cellular immune responses in mice and is therefore a promising vaccine against EHEC O157:H7 infection.

Intranasal immunization with novel EspA-Tir-M fusion protein induces protective immunity against enterohemorrhagic Escherichia coli O157:H7 challenge in mice.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and hemolytic uremic syndrome in humans. Due to the risks associated with antibiotic treatment against EHEC O157:H7 infection, vaccines represent a promising method for prevention of EHEC O157:H7 infection. Therefore, we constructed the novel bivalent antigen EspA-Tir-M as a candidate EHEC O157:H7 subunit vaccine. We then evaluated the immunogenicity of this novel EHEC O157:H7 subunit vaccine. Immune responses to the fusion protein administered by intranasal and subcutaneous routes were compared in mice. Results showed higher levels of specific mucosal and systemic antibody responses induced by intranasal as compared to subcutaneous immunization. Intranasal immunization enhanced the concentration of interleukin-4, interleukin-10, and interferon-γ, while subcutaneous immunization enhanced only the latter two. In addition, intranasal immunization protected against EHEC O157:H7 colonization and infection in mice at a rate of 90%.Histopathological analysis revealed that vaccination reduced colon damage, especially when administered intranasally. In contrast, subcutaneous immunization elicited a weak immune response and exhibited a low protection rate. These findings demonstrate that intranasal immunization with the fusion protein induces both humoral and cellular immune (Th1/Th2) responses in mice. The novel EspA-Tir-M novel fusion protein therefore represents a promising subunit vaccine against EHEC O157:H7 infection.

[Lactobacillus rhamnosus GG inhibits Cronobacter-induced meningitis in neonatal rats].

OBJECTIVE: To investigate the inhibitory effect of Lactobacillus rhamnosus GG ( LGG) against Cronobacter-induced meningitis in neonatal rats. METHODS: The cell adhesion and invasion capacities of Cronobacter were assayed in Caco-2 cells, and the optimal time length and concentration of the bacterium for infection were determined. The suppressive effects of LGG on the adhesion and invasion of Cronobacter in caco-2 cells were tested by competitive and exclusion experiments, and its inhibitory effect against Cronobacter-induced meningitis was evaluated in neonatal rats. RESULTS: Cronobacter showed aggressive adhesion to caco-2 cells with an optimal infection time of 3 h. LGG produced a concentration-dependent inhibition of Cronobacter adhesion and invasion by competing with and excluding the latter for cell adhesion. In neonatal rats, LGG showed an obvious preventive effect and also a moderate therapeutic effect against Cronobacter-induced meningitis. CONCLUSION: LGG can inhibit Cronobacter entry across the intestinal barrier to achieve preventive and therapeutic effects against Cronobacter-induced meningitis.

Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Cronobacter sakazakii.

Cronobacter sakazakii is an emerging pathogen associated with the ingestion of contaminated reconstituted formula, which causes necrotizing enterocolitis, sepsis, and meningitis in low-birth-weight preterm neonatal infants. Sensitive and specific detection methods are needed to better control C. sakazakii infections. This study aims to develop a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting C. sakazakii in powdered infant formula (PIF). A set of four LAMP primers were designed based on the published C. sakazakii ompA gene sequence. Specificity of the assay was evaluated using a panel of 22 C. sakazakii, 27 Enterobacteriaceae family except C. sakazakii, and 25 other strains. Assay sensitivity was determined using serial dilutions of C. sakazakii American Type Culture Collection 51329 culture ranging from 10(6) colony-forming units (CFU)/mL to extinction. The assay was also tested in experimentally inoculated PIF samples. The ompA-based LAMP assay was able to detect specifically all of the 22 C. sakazakii strains without amplification from 52 non-C. sakazakii strains. The detection limit was 10(1) CFU/mL in pure culture, up to 10-fold more sensitive than that of the ompA-polymerase chain reaction (PCR). When applied to PIF, sensitivity was 10(2) CFU/mL, up to 10-fold that of the ompA-PCR. The ompA-based LAMP assay developed in this study was sensitive, specific, and low cost with great potential for future field detection of C. sakazakii in PIF.

[Loop-mediated isothermal amplification for detecting enterohemorrhagic Escherichia coli O157:H7: a comparison with PCR].

OBJECTIVE: To establish a rapid method of loop-mediated isothermal amplification (LAMP) for detecting enterohemorrhagic Escherichia coli (EHEC) O157:H7. METHODS: Six primers that specifically recognized the rfbE gene of EHEC O157:H7 were designed. Under the optimized reaction conditions, LAMP and PCR were evaluated for the sensitivity and specificity in the detection of 39 laboratory samples of EHEC O157:H7 strains, and their detection results of contaminated fresh pork samples were compared. RESULTS: LAMP assay correctly identified all the 7 EHEC O157:H7 strains and showed negative results for all the 32 non-EHEC O157:H7 strains. The detection limit of LAMP was much lower than that of rfbE-PCR (10 vs 100 cfu/ml). In the detection of the contaminated pork samples, both LAMP and PCR yielded results consistent with those by the conventional detection method. CONCLUSION: The rfbE-based LAMP assay can serve as a rapid, sensitive, specific and low-cost means for detecting EHEC O157:H7 strain.

[Construction of SDF-1P2G54, a specific antagonist of CXCR4].

OBJECTIVE: To obtain a specific antagonist of CXCR4, SDF-1P2G54 by mutating SDF-1 second proline (P) into glycin (G) and removing the α-helix of its C-terminal. METHODS: SDF-1p2g54 gene amplified by PCR was inserted into the vector pET-30a (+) and transformed into Escherichia coli (E. coli) strain BL21. After IPTG induction of E. coli, the expressed recombinant protein was purified with nickel-affinity chromatography column under denaturing conditions and refolded with gradient dilution and ultra-filtration. The chemotactic effect of SDF-1P2G54 on Jurkat cells and its antagonistic effect against SDF-1 were determined by transwell assay; flow cytometry was used to assay the ability of SDF-1P2G54 to induce calcium influx and CXCR4 internalization in MOLT4 cells. RESULTS: The recombinant protein SDF-1P2G54 completely lost the functions to activate CXCR4 or to induce transmembrane migration of Jurkat cells and calcium influx in MOLT4 cells, but maintained a high affinity to CXCR4. SDF-1P2G54 effectively inhibited the chemotactic effect of wild-type SDF-1 to Jurkat cells, and induced rapid CXCR4 internalization in MOLT4 cells. CONCLUSION: SDF-1P2G54 is a new antagonist of CXCR4 with a potential value as an effective inhibitor of HIV-1 infection, cancer metastasis or other major diseases.

Protection against Escherichia coli O157:H7 challenge by immunization of mice with purified Tir proteins.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 infections cause serious public health problems worldwide. The translocation intimin receptor (Tir) is responsible for adhesion and attaching and effacing lesions. In the current study, we used a mitomycin-treated mouse model to evaluate the efficacy of subcutaneous vs intranasal administration of the recombinant Tir as vaccine. Following immunization, mice were infected with E. coli O157:H7 and faces were monitored for shedding. Mice immunized intrasally with purified Tir proteins produced higher IgG and IgA titers in serum and feces, resulting in significant reductions in fecal shedding of EHEC O157 and higher a survival rate (92.9%), compared with subcutaneous or control immunizations. These results demonstrate the potential for the use of Tir proteins in mucosal vaccine formulations to prevent colonization and shedding of E. coli O157:H7. Therefore, purified Tir protects mice against EHEC challenge after intranasal immunization and is worth further clinical development as a vaccine candidate.

Screening test for anti-Helicobacter pylori activity of traditional Chinese herbal medicines.

AIM: To evaluate the anti-Helicobacter pylori (H. pylori) activity of 50 traditional Chinese herbal medicines in order to provide the primary evidence for their use in clinical practice. METHODS: A susceptibility test of water extract from 50 selected traditional Chinese herbal medicines for in vitro H. pylori Sydney strain 1 was performed with broth dilution method. Anti-H. pylori activity of the selected Chinese herbal medicines was evaluated according to their minimum inhibitory concentration (MIC). RESULTS: The water extract from Rhizoma Coptidis, Radix Scutellariae and Radix isatidis could significantly inhibit the H. pylori activity with their MIC less than 7.8 mg/mL, suggesting that traditional Chinese herbal medicines have anti-inflammatory and antibacterial effects and can thus be used in treatment of H. pylori infection. CONCLUSION: Rhizoma Coptidis, Radix Scutellariae and Radix isatidis are the potential sources for the synthesis of new drugs against H. pylori.

[Construction of a recombinant Lactobacillus acidophilus expressing high levels of Helicobacter pylori adhesin Hp0410].

OBJECTIVE: To construct a recombinant Lactobacillus acidophilus that expresses high levels of Helicobacter pylori (Hp) adhesin Hp0410. METHODS: The gene fragment encoding Hp0410 was amplified by PCR from the DNA of H. pylori NCTC11639 strain and cloned into the shuttle plasmid pMG36e to construct pMG36e-Hp0410, which was transformed into Lactobacillus acidophilus by electroporation. The target protein was confirmed with SDS-PAGE and silver nitrate staining and analyzed by Western blotting. The stability of the recombinant plasmid was assessed by drawing the growth curve of the recombinant Lactobacillus acidophilus. RESULTS: A 750-bp fragment was inserted into the pMG36e plasmid and transformed into Lactobacillus lactis. The transformed bacterium expressed the target protein with a relative molecular mass of about 34 kD. Western blotting confirmed that the expressed proteins could be recognized by the serum of patients with Hp infection. The recombinant plasmid pMG36e-Hp0410 exhibited good stability in the presence or absence of erythromycin. CONCLUSIONS: The recombinant Lactobacillus acidophilus with high constitutive expression of Hp0410 has been constructed successfully.

[Effects of Newcastle disease virus on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells].

OBJECTIVE: To investigate the effects of Newcastle disease virus (NDV) infection on the expression of survivin and cell cycle in human tongue squamous carcinoma TSCCa cells. METHODS: The proliferation of TSCCa cells infected with NDV in vitro was evaluated by means of MTT assay, and survivin expression in the infected cells was detected using RT-PCR and Western blotting. Flow cytometry was performed to assess the changes in the cell apoptosis, cell cycle and cell proliferation index (PI) of the cells. RESULTS: NDV infection resulted in decreased survivin expression and increased apoptosis of TSCCa cells, with reduced cell percentage in G2/M and S phases and lowered PI of the cells, showing significant differences from those of the negative control cells (P<0.05). CONCLUSION: NDV infection can inhibit survivin expression, affect the cell cycle of TSCCa cells and induce their apoptosis.

[Clone, expression and immunoreaction of napA gene of Helicobacter pylori].

AIM: To construct a prokaryotic expression system of Helicobacter pylori(Hp) (neutrophil-activating protein) napA gene, analyze nucleic acid sequence and study its immunity and inflammation. METHODS: napA fragment was amplified from Hp NCTC11639 chromosomal DNA by PCR. Its T-A was cloned, sequenced and compared with other Hp strains on the GenBank. Then the gene was cloned into pGEX-4T-1 fusion expression vector, expressed in E.coli and purified by GST-affinity chromatography. The purified product was used to screen 29 stains of mouse anti Hp monoclonal antibodies(mAb) and its immunity and inflammation analyzed with sera of Hp-infected patients by Western blot. RESULTS: napA fragment was composed of 435 base pairs (GenBank No.DQ341279) and the nucleotide homology with other Hp strains on the GenBank was 94%-98%. The molecular weight of the recombinant napA-pGEX-4T-1 expressed in E.coli was 44 kDa. 3 of 29 anti-Hp mAbs were against NAP. Western blot analysis proved that the recombinant NAP was specifically recognized by the sera of Hp-infected patients. CONCLUSION: The recombinant NAP has original immunoreaction. It is of great value to clinical sero-diagnosis and vaccine study of Hp.

Selection of SARS-coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice.

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed.

[Variability analysis of S2 gene of SARS-CoV].

OBJECTIVE: To determine the sequence of S2 gene of SARS-associated coronavirus (SARS-CoV) GD322 and analyze the phyletic evolution of S2 gene. METHOD: S2 gene fragment was amplified from SARS-CoV GD322 genome with RT-PCR and ligated to pGEM-T vector for sequence analysis after transformation of the plasmid into E. coli DH5a. The variability of S2 genes and S2 proteins from 12 strains isolated in the early, intermediate and advanced stages of the SARS outbreak were analyzed and the phylogenetic tree was constructed with Lasergene, Clustal X, DNAman and Treeview. T cell antigen epitopes of S2 protein were predicted on the basis of Internet database. RESULT: With the epidemic spread of SARS-CoV, the S2 genes of the virus tended to become stable. Homology of S2 genes of SARS-CoV isolated in advanced stage of the outbreak reached 99.9%. Prediction of T cell antigen epitope showed that mutation at the 57th amino acid effected T cell antigen epitope. CONCLUSION: S2 gene of GD322 SARS-CoV is relatively stable during the epidemic spread of the virus, and mutation at the 57th amino acids of S2 protein may affect the T cell antigen epitope.