Immune Response of Mosquitoes to Filarial Worms

描述(申请人提供)：蚊媒疾病的死灰复燃正在对全球健康造成破坏性影响，迫切需要制定新的控制战略。正在积极研究的一项新战略是利用转基因技术控制媒介传播的疾病，即通过基因转化改变媒介能力，以降低蚊子在种群中传播特定病原体的能力。然而，如果我们要评估蚊子转化作为一种可行的疾病控制方法的使用，我们必须更好地了解蚊子的先天免疫反应，这种免疫反应支配着媒介能力。黑色素包裹是一种细胞先天免疫反应，导致蚊子对丝虫和疟疾寄生虫的抗药性表型。通过我们的父母资助(蚊子对丝虫的免疫反应)，我们正在研究黑色素包裹的生物化学和遗传调节，但在这些研究过程中，我们确定，如果我们要阐明免疫识别和防御反应的启动机制，有必要对蚊子循环免疫反应细胞(血细胞)中的基因表达进行详细研究。一只蚊子只有很少的血细胞(&lt；1,200)，而且它们不适合体外培养，甚至不能在体外短期维持；因此，我们构建了代表从两种蚊子免疫激活的血细胞中获得的mRNA的cDNA文库。从这些文库中产生的有限数量的EST表明核糖体克隆的比例很低，并揭示了一些与已知天然免疫功能的蛋白质相匹配的情况。这些数据使我们相信，更多EST的系统开发将有助于构建信息丰富的微阵列，这些微阵列可用于测试关于血细胞在影响载体能力中所起作用的特定假设。父母拨款的预算并不是为了支付产生所需EST数量所需的成本，更不用说与寡核苷酸微阵列构建相关的成本了；而且，重要的是，我们不能预期在我们的模型中使用全面的转录组方法来评估基因调控的可行性。这些额外工具提供的多维数据将大大加强父赠款的具体目标。因此，使用R21的技术应用机制来加强我们由NIAID资助的研究是合适的，方法是(1)从免疫激活的血细胞cDNA文库中生成大型EST数据集，(2)构建代表这些EST的寡核苷酸微阵列，以及(3)使用这些微阵列对血细胞表达谱进行数据挖掘，以确定蚊子对丝虫免疫反应的不同转录模式。

DESCRIPTION (provided by applicant): The resurgence of mosquito-borne diseases is having a devastating impact on global health and an urgent need exists to develop new control strategies. A new strategy being actively investigated is the use of transgenics for the control of vector-borne diseases, i.e., to change vector competence by genetic transformation to reduce the ability of mosquitoes within a population to transmit a particular pathogen. However, if we are to evaluate the use of mosquito transformation as a viable disease control methodology it is imperative that we gain a better understanding of the mosquito's innate immune response that governs vector competence. Melanotic encapsulation is a cellular innate immune response that is responsible for the resistant phenotype of mosquitoes to filarial worms and malaria parasites. Through support from our parent grant (Immune Response of Mosquitoes to Filarial Worms) we are studying the biochemistry and genetic regulation of melanotic encapsulation, but during the course of these studies we have determined that detailed studies of gene expression in the circulating immune reactive cells (hemocytes) of mosquitoes is necessary if we are to clarify mechanisms of immune recognition and the initiation of defense responses. Very few hemocytes (<1,200) are present in an individual mosquito and they are not amenable to in vitro culture or even short-term in vitro maintenance; consequently, we have constructed cDNA libraries representing mRNA obtained from immune-activated hemocytes from two mosquito species. Generation of a limited number of ESTs from these libraries indicated a low percentage of ribosomal clones and revealed a number of matches to proteins with known innate immunity functions. These data convinced us that the systematic development of additional ESTs would facilitate the construction of informative microarrays that could be used to test specific hypotheses about the role hemocytes play in influencing vector competence. The budget of the parent grant was not designed to cover the costs required to generate the number of ESTs required, let alone the costs associated with oligonucleotide microarray construction; and, importantly, we could not have anticipated the feasibility of a comprehensive transcriptome approach to assess gene regulation in our model. The specific aims of the parent grant would be significantly enhanced by the multi-dimensional data from these additional tools. It is therefore appropriate to use the R21 mechanism for technology application to enhance our NIAID-funded research by (1) generating a large EST data set from immune-activated hemocyte cDNA libraries, (2) constructing oligonucleotide microarrays representing these ESTs, and (3) using these microarrays in data mining of hemocyte expression profiles to identify distinct patterns of transcription underlying the immune response of mosquitoes against filarial worms.