Functional Analysis of Antimicrobial Peptides in Protection from Bacterial Infection

最初，我们研究了诱导型hBD在体外肺炎支原体感染急性期的作用。我们观察到化学合成的hBD-2和hBD-3，而不是hBD-1，对肺炎分枝杆菌表现出有效的抗菌活性。为了诱导hBD的产生，将人肺鳞状细胞系EBC-1与强效刺激物IL-1β孵育。检测EBC-1的培养上清和总RNA是否表达hBD。western blot检测上清液中hBD-2的含量。此外，IL-1bβ显著上调hBD-2 mRNA，但hBD-1和hBD-3均未明显上调。采用il -1β处理的EBC-1细胞感染肺炎支原体，以确定hBD是否诱导对感染的保护作用。菌落测定表明，il -1β处理的EBC-1显著抑制肺炎支原体的生长。因此，我们的研究结果表明，il -1β处理的EBC-1细胞产生的hBD-2在早期肺炎支原体感染中起保护作用。随后，抗菌肽，特别是CRAMP，在体内感染模型中的作用进行了研究。似乎没有关于抽筋和支原体感染的报道。用化学方法合成了抗菌肽家族中的CRAMP。CRAMP对肺炎支原体具有良好的抑菌活性。为了在体内诱导CRAMP， BALB/c小鼠鼻内感染肺炎支原体，并在感染后6、12、24和48小时取感染小鼠的肺检测CRAMP的表达。Western blot分析显示，感染后24小时肺匀浆中含有CRAMP。此外，采用RT-PCR检测感染肺的CRAMP mRNA。免疫染色显示支气管周围有许多cramp阳性的中性粒细胞，但没有明确的cramp阳性上皮细胞。目前正在研究camp阳性中性粒细胞与保护作用之间的关系

Initially, we examined the role of inducible hBD in the acute phase of in vitro M.pneumoniae infection. We observed that chemically synthesized hBD-2 and hBD-3, but not hBD-1, show effective antimicrobial activity against M.pneumoniae. To induce hBD production, a human pulmonary squamous cell line EBC-1 was incubated with a potent stimulant, IL-1β. Culture supernatant and total RNA from the EBC-1 were examined for hBD expression. hBD-2 in the supernatant was detected by western blot analysis. In addition, hBD-2 mRNA was strongly up-regulated by IL-1bβ, but neither hBD-1 nor hBD-3 was apparently up-regulated. To determine whether hBD induces the protection from infection, IL-1β-treated EBC-1 cell were infected with M.pneumoniae. IL-1β-treated EBC-1 significantly inhibited the growth of M.pneumoniae, as judged by colony assay. Thus, our results suggest that hBD-2 produced by IL-1β-treated EBC-1 cells plays a role in the protection of early stage of M.pneumoniae infection.Subsequently, roles of antimicrobial peptides, in particular CRAMP, in in vivo infection model were examined. There seems to be no reports regarding the CRAMP and Mycoplasma infection. CRAMP, a family of cathelicidin, was chemically synthesized. The CRAMP showed an effective antimicrobial activity against M.pneumoniae. To induce CRAMP in vivo, BALB/c mice were intranasally infected with M.pneumoniae, and infected lungs taken from the mice at 6,12,24 and 48 hours postinfection were examined for CRAMP expression. Western blot analysis showed that the homogenate from the lungs at 24 hours after infection contains CRAMP. In addition, CRAMP mRNA from the infected lungs was detected by using RT-PCR. Immunostaining showed that there are many CRAMP-positive neutrophils in the peribronchial area, but no definitive CRAMP-positive epithelial cells. At present underway is investigation on the association between CRAMP-positive neutrophils and protective effect