| Objective1. To evaluate the potential diagnostic value of apolipoprotein E (Apo E) measurements in pediatric patients with invasive bacterial infections.2. In the second part, we analyze the biological actions of antimicrobial and immunomodulatory activity of a new ApoE-dervied pepetide (ApoE23), and evaluated the therapeutic approach of ApoE23against the spesis in mice model.Methods1. The patients with confirmable infections, including bacterial meningitis, virus meningitis, and sepsis associated with bacterial infection, were enrolled in this study, and the subjects who were without confirmable infections were included as control.The investigative data collected from a series of laboratroy tests included the C-reactive protein levels in the peripheral blood, the peripheralblood white blood cell counts, the CSF routine tests and the CSF biochemical profiles, the microbiological results collected from bacterial cultivation in blood and CSF samples, the Streptococcus pneumonia antigens measurment and Gram strain in the CSF samples, and enterovirus RNA detection in the CSF samples. ApoE levels in serum and in CSF were measured by immunoturbidimetry. The diagnostic values of ApoE and the cut-off value of prediction for bacterial infections were established by the receiver operating curve (ROC) method.2. The ApoE mimetic peptide, named ApoE23, was designed by ourself. ApoE23and control peptides (ApoEdp, ApoEll) were synthesized using solid phase synthesis and were purified by high performance liquid chromatography (HPLC).The mass spectrum and electrospray ionization mass spectrum (ESI-MS) were employed to check the produces. The secondary structures of the ApoE23and the control peptides were analyzed by circular dichroism spectra technique aided by K3D3software. The distribution patterns of hydrophobic amino acid residues and hydrophilic amino acid residues of the peptides were analyzed by a-helical wheel picture.3. The antimicriobial activity of peptides were detected by bactericidal method in vitro.4. Cell culture in vitro, Enzyme-Linked Immunosorbent Assay (ELISA)and quantified PCR were employed to measure the TNF-a, IL-6, and IL-10expressions in THP-1cells and human peripheral blood mononuclear cell (PBMC)induced by lipopolysaccharide (LPS)with or without ApoE23intervention.5. The sepsis C57BL mice was established by peritoneal cavity injection of Salmonella typhimurium group B. After tail vein injection of ApoE23, the therapeutic effect were evaluated by calculation the mortality of the septic mice. The bacterial load test in the spleen tissue samples and the LPS, TNF-a, IL-6levels measurement in the plasma samples were carried out to investigate the anti-sepsis mechanisms of ApoE23.Results1. A total number of185pediatric patients aged from1month to13years old were enrolled. Among them,26patients with bacterial meningitis,32patients with virus meningitit,36patients with bacterial sepsis, and91patients without infections were enrolled in the control group. ApoE levels in CSF significantly increased in patients with bacterial meningitis, and serum ApoE markedly elevated in patients with sepsis or with bacterial meningitis compared with patients with other infections and uninfected children (control group). The optimal ApoE cutoff value for CSF was>1.7mg/L with85%(95%confidence interval,0.71-1) sensitivity and100%specificity (95%CI,0.89-1) and was>42mg/L in serum with80%(95%CI,0.64-0.89) sensitivity and93%(95%CI,0.88-0.97) specificity. The CSF ApoE level measurements by immunoturbidimetry showed higher diagnostic values than the CSF WBC count, CSF proteintest, and CSF glucose test. The diagnostic value of the serum ApoE measurement as an indicator of sepsis was higher than that of the CRP test and WBC count of whole blood.2. The a-helical proportion in the secondary constrcture of ApoE23were21.6%,which appeared amphipathic properties and better penetrability than the other ApoE mimetic peptides designed by foreign.3. ApoE23has higher bactericid activity against gram negtive bacterial than that against the gram positive germs. The same bactericid activities of ApoE23against resistant bacterial such as Acinetobacter baumannii were detected. The minimal inhibitory concentrations of inhibition by50%(MIC50) of ApoE23was in the range of1-1.25μmol/L, which is lower than the MIC50of the ApoEdp reported by now. However, no antimicrobial activities against enterococcus and Candida albicans were detected.4. ApoE23down-regulated the mRNA and protein expression levels of TNF-a and IL-6induced by LPS in THP-1cells and human PBMC. ApoE23also down regulated the expression of IL-10in mRNA and protein levels in THP-1cells. But, in the human PBMC only IL-10protein was reduced.5. Twenty-four hours after the live Salmonella typhimurium groups B (106CFU/mouse)were inculated by peritoneal cavity injection in the C57BL mice, the mortality of mice accepted physiological saline therapy was60%. No mouse died in the group with ApoE23treatment. Moreover, our results showed that the load of bacteria in the spleen tissue and the concentrations of plasma LPS, TNF-a, IL-6in the mice with ApoE23therapy were lower than that in the mice with physiological saline treatment. The morphological and pathological changes in the spleen, lung, liver and small intestine were slighter in the mice with ApoE23treatment than that in mice with physiological saline treatment.Conclutions1. CSF and serum ApoE detection provided with a good specificity and sensibility marker for prediction the invasive bacterial infections in pediatric patients.2. ApoE23has good antibacterial characteristics including narrow antibacterial spectrum, low MIC50, showing antibacterial activity against intracellular growth of bacteria and the resistant bacteria.3. ApoE23can regulate the response reaction induced by LPS in the immune cells cultured in vitro.4. ApoE23has both of antibacterial effect and immunomodulatory effect, which can significantly reduce the mortality of the sepsis mice. ApoE23is a potential anti-sepsis drug. |
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