Linked to the development of 3-Amino-1-propanesulfonic acid site allergic diseases [19,20]. Actually, E. coli consists 
of a diverse group of bacteria, most of which is harmless and an important part of a healthy human intestinal tract by producing vitamin K2 and by preventing the establishment of pathogenic bacteria [21]. E. coli ATCC 25922 is a nonpathogenic strain of E. coli, which is BSL-1 certified to make it useful for various laboratory experiments [22]. It is not only well-characterized as a control Gram-negative bacterium, but most widely studied as a prokaryotic model organism in the fields of biotechnology and microbiology served as the host organism [23,24]. As yet, to the best of our knowledge, no study has been conducted to elucidate the contribution of E. coli in vivo to allergic rhinitis and/or asthma. Therefore, herein we adopted E. coli ATCC25922 by three approaches of intestinal infection prior to ovalbumin (OVA)-induced allergic airway inflammation in mice, examined its immunomodulatory efficacy, as well as elucidated the underlying mechanisms. We aimed to provide experimental evidence for the beneficial effect of E. coli against AAD, and to consider how our knowledge of these inverse interactions to be harnessed to improve people’s health.Materials and Methods Animals and ReagentsSpecific pathogen free (SPF) female Balb/c mice when 4,5 days and 4,5 weeks of age were obtained from Shandong University School of Medicine (Jinan, China) and maintained under SPF conditions with free access to sterile water and food in individual ventilated cages. All experiments were approved by the Animal Care Committee of Shandong University (NO. ECAESDUSM 20123011). E. coli (ATCC, 25922, USA) was used in this study for in vivo experimentation. OVA (Sigma-Aldrich, A5503, USA) as the allergen, and aluminum hydroxide (Thermo Scientific Imject Alum, 77161, USA) as the immunologic adjuvant were obtained for sensitization challenge. Staining reagents of Wright-Giemsa and alcian blue-periodic acid Stiff come from YiLi Bioscience and Technology (Beijing, P.R. China). Serum OVA-specific IgE (Chondrex Assay Kit, 3010, USA) was assayed to aid the diagnosis of the allergic state. Mouse IL-4, IL-10, IFN-gamma (IFN-c) and IL-2 enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, 88-7711, USA) and regulatory T cell staining kit (eBioscience, 88-8115, USA) were all purchased from eBioscience.(106infN+OVA) group, neonatal mice were infected with a lower dose of oral 106 CFU E. coli and then sensitized challenged with OVA. (E) (108infA+OVA) group, adult mice when 4,5 weeks of age were infected with oral 108 CFU E. coli and then sensitized challenged with OVA. In our preliminary experiments, we have performed the control group of the adult mice, but we did not find any statistical differences between the controls of the neonatal mice and that of the adult mice in pathologic analysis or immune responses. Considering that there were 
no positive results between the controls, thus we dropped the assessment of the controls of adult mice in our study. Each experimental group consisted of 10 animals. The detail experimental protocol was as following: get 86168-78-7 without on need of anesthesia, mice in a sober state of nature were intragastric (i.g.) administrated with 100 ml volume of live E. coli ATCC 25922 on days 0, 5, 10, 15, 20, 25 and 30, using an 8-gavage needle tightly connected to a 1 ml syringe interface. Mice were infected with approximately 108 or 106 CFU live E. coli as neonates (1.Linked to the development of allergic diseases [19,20]. Actually, E. coli consists of a diverse group of bacteria, most of which is harmless and an important part of a healthy human intestinal tract by producing vitamin K2 and by preventing the establishment of pathogenic bacteria [21]. E. coli ATCC 25922 is a nonpathogenic strain of E. coli, which is BSL-1 certified to make it useful for various laboratory experiments [22]. It is not only well-characterized as a control Gram-negative bacterium, but most widely studied as a prokaryotic model organism in the fields of biotechnology and microbiology served as the host organism [23,24]. As yet, to the best of our knowledge, no study has been conducted to elucidate the contribution of E. coli in vivo to allergic rhinitis and/or asthma. Therefore, herein we adopted E. coli ATCC25922 by three approaches of intestinal infection prior to ovalbumin (OVA)-induced allergic airway inflammation in mice, examined its immunomodulatory efficacy, as well as elucidated the underlying mechanisms. We aimed to provide experimental evidence for the beneficial effect of E. coli against AAD, and to consider how our knowledge of these inverse interactions to be harnessed to improve people’s health.Materials and Methods Animals and ReagentsSpecific pathogen free (SPF) female Balb/c mice when 4,5 days and 4,5 weeks of age were obtained from Shandong University School of Medicine (Jinan, China) and maintained under SPF conditions with free access to sterile water and food in individual ventilated cages. All experiments were approved by the Animal Care Committee of Shandong University (NO. ECAESDUSM 20123011). E. coli (ATCC, 25922, USA) was used in this study for in vivo experimentation. OVA (Sigma-Aldrich, A5503, USA) as the allergen, and aluminum hydroxide (Thermo Scientific Imject Alum, 77161, USA) as the immunologic adjuvant were obtained for sensitization challenge. Staining reagents of Wright-Giemsa and alcian blue-periodic acid Stiff come from YiLi Bioscience and Technology (Beijing, P.R. China). Serum OVA-specific IgE (Chondrex Assay Kit, 3010, USA) was assayed to aid the diagnosis of the allergic state. Mouse IL-4, IL-10, IFN-gamma (IFN-c) and IL-2 enzyme-linked immunosorbent assay (ELISA) kits (eBioscience, 88-7711, USA) and regulatory T cell staining kit (eBioscience, 88-8115, USA) were all purchased from eBioscience.(106infN+OVA) group, neonatal mice were infected with a lower dose of oral 106 CFU E. coli and then sensitized challenged with OVA. (E) (108infA+OVA) group, adult mice when 4,5 weeks of age were infected with oral 108 CFU E. coli and then sensitized challenged with OVA. In our preliminary experiments, we have performed the control group of the adult mice, but we did not find any statistical differences between the controls of the neonatal mice and that of the adult mice in pathologic analysis or immune responses. Considering that there were no positive results between the controls, thus we dropped the assessment of the controls of adult mice in our study. Each experimental group consisted of 10 animals. The detail experimental protocol was as following: without on need of anesthesia, mice in a sober state of nature were intragastric (i.g.) administrated with 100 ml volume of live E. coli ATCC 25922 on days 0, 5, 10, 15, 20, 25 and 30, using an 8-gavage needle tightly connected to a 1 ml syringe interface. Mice were infected with approximately 108 or 106 CFU live E. coli as neonates (1.
