Antimicrobial Peptides & Innate Immunity in Otitis Media

说明(申请人提供)：抗菌多肽(AP)是粘膜主要防御机制的关键成分，在呼吸道、胃肠道和泌尿生殖道的巨大上皮表面提供一线灭活细菌、真菌和病毒的作用。这些阳离子多肽的强大抗菌活性，往往是协同作用的，也可以是相加的，为天然免疫系统提供了高效的作用机制。最近，人们开始认识到，生活在这些粘膜表面的共生微生物刺激上皮细胞产生这些天然免疫的效应器，从而有助于宿主维持防御性粘膜屏障和动态平衡。后一点与中耳炎(OM)特别相关，因为OM不是由高毒力微生物引起的；相反，OM是由共生细菌的子集引起的，这些细菌通常构成儿童鼻咽(NP)的正常菌群。然而，当宿主的呼吸道防御受到损害时，这些细菌可能会表现为机会性病原体，并进入现在防御不力的中耳。众所周知，NP的定植是OM病程的第一步。在中耳受到侵袭之前，鼻咽部的细菌负荷显著增加。此外，与不容易患中耳炎的儿童相比，易患中耳炎的儿童更多地被在急性和慢性OM中占主导地位的细菌种类所定植，从而强化了这样的概念，即在NP中保持相对较低的细菌负荷对中耳的健康很重要，特别是在病毒感染的时候。极有可能的是，将儿童NP中的细菌负荷维持在非传染性的“定植”水平主要是由天然免疫系统调节的，包括呼吸道分泌物中存在的AP和天然抗体的作用，而不是获得性免疫的效应者。在特定个体中，先天免疫系统的相对有效性也会影响中耳炎倾向。相反，先天防御机制的失调(即由于并发的上呼吸道(URT)病毒感染或遗传易感性，导致先天免疫系统不充分或反应迟钝)可能会使细菌负荷增加到更容易感染的水平。因此，定义和研究包括鼻咽、咽鼓管和中耳在内的最上呼吸道的天然免疫系统是很重要的。为了实现这一目标，在本申请中，我们建议对最上呼吸道产生的AP进行表征和研究。具体地说，我们希望研究它们在鼻咽细菌定植中的作用，并确定它们的表达是上调还是下调，以响应OM的主要病毒和细菌病原体。此外，我们建议使用成熟的栗鼠模型来确定它们在实验性OM诱导和分解过程中的相对表达水平。最后，我们将分离的重组或合成龙猫AP(包括最近发现的龙猫β防御素-1)输送到龙猫宿主的最上呼吸道，试图抑制该模型中发生的OM的发展。

DESCRIPTION (provided by applicant): Antimicrobial polypeptides (APs) are key components of the primary defense mechanisms of mucus membranes, providing first-line inactivation of bacteria, fungi and viruses on the vast epithelial surfaces that line the respiratory, gastrointestinal and urogenital tracts. The potent antimicrobial activity of these cationic polypeptides, which is often synergistic and can be additive, provides the highly effective mechanism of action of the innate immune system. Recently, it has begun to be appreciated that the commensal microorganisms living on these mucosal surfaces stimulate epithelial cells to produce these effectors of innate immunity and thus contribute to host maintenance of defensive mucosal barriers and homeostasis. This latter point is particularly relevant to otitis media (OM) because OM is not caused by highly virulent microorganisms; OM is instead caused by a subset of the commensal bacteria that typically comprise the normal flora of the pediatric nasopharynx (NP). However, when host airway defenses are compromised, these bacteria can behave as opportunistic pathogens and gain access to the now poorly defended middle ear. It is well known that colonization of the NP is thus the very first step in the disease course of OM. Immediately preceding invasion of the middle ear, the bacterial load in the nasopharynx increases significantly. Moreover, otitis prone children are more heavily colonized by the bacterial species that are predominant in acute and chronic OM compared to their non-otitis prone counterparts, thus reinforcing the notion that maintaining a relatively low bacterial load in the NP is important to the health of the middle ear, particularly during times of viral compromise. It is highly likely that maintenance of the bacterial load in a child's NP at a noninfectious "colonizing" level is regulated on a day-to-day basis primarily by the innate immune system, including action of the APs and natural antibodies present in airway secretions, and not by effectors of acquired immunity. The relative effectiveness of the innate immune system in a given individual would then influence otitis proneness as well. Conversely, dysregulation of innate defense mechanisms (i.e. due to concurrent upper respiratory tract (URT) viral infection or genetic predisposition to an inadequate or hypo-responsive innate immune system) may allow an increase in bacterial load to a more 'infectious-prone' level. It is thereby important to define and study the innate immune system of the uppermost airway, which includes the nasopharynx, Eustachian tube and middle ear. Toward this goal, in the present application we propose to characterize and study the APs produced in the uppermost airway. Specifically, we wish to investigate their role in bacterial colonization of the nasopharynx and determine if their expression is up- or down-regulated in response to the predominant viral and bacterial pathogens of OM. Moreover, we propose to determine their relative level of expression during induction and resolution of experimental OM using well-established chinchilla models. Finally, we will deliver isolated recombinant or synthetic chinchilla APs (including the recently identified chinchilla beta defensin-1) to the uppermost airway of the chinchilla host in an attempt to inhibit the development of OM that occurs in this model.