Characterising the Immune Response Against T. cruzi in the GI tract and a Possible Role for Anti-inflammatory Cytokines TGF-b and IL-10

最近的论文Francisco等人(2015)通过体内成像发现GI，特别是结肠和胃，是即使在化疗泊沙唑治疗的情况下，在慢性阶段T.ruzi复制活跃的关键部位。尽管进行了药物治疗，但这种模式的复制反映了查加斯病患者临床上看到的药物失败的类型。这一观察与我们对肠道组织的经典免疫学理解有关，肠组织是在早期发育过程中建立起来的耐受性部位。该项目提案有几个目标：1.确定携带寄生虫的特定组织/细胞类型2。确定转化生长因子-b在克氏锥虫中的作用第一个目标是确定寄生虫的永久细胞库的特征，可以推断它间歇性地释放受感染的免疫细胞，导致短暂的感染，包括心脏在内的其他组织，在那里可以观察到大部分临床疾病的病理。识别这种受感染的细胞类型将允许更好地针对未来的候选药物，目标是全球无菌治疗。对克氏毛滴虫免疫反应的表型进行了调查，得出了相互矛盾的结论。人们普遍认为Th1应答，特别是高表达的干扰素-γ和肿瘤坏死因子-α与脊椎动物宿主细胞内寄生虫的控制有关。不太清楚的是，抗炎细胞因子转化生长因子-b的作用。Ferrao等人(2015)的研究发现，转化生长因子-b是克氏毛滴虫成功进入宿主细胞和随后的无鞭毛体复制的主要决定因素。在你的Fransico等人(2015)的论文中明确指出的结肠是寄生虫生存的场所，众所周知，它具有非常丰富的转化生长因子-b耐受性环境，以防止免疫病理。这项建议试图表明，在模型中的其他小鼠组织清除泊沙唑期间，胃肠道中的器官是否提供了支持克氏毛滴虫复制的优势。目的1-进一步表征克氏毛滴虫在泊沙唑治疗的小鼠模型中的感染定位-福尔马林固定和石蜡包埋的寄生虫负担器官的切片，用曙红和苏木精染色。400x光学显微镜搜索Roffe等人之前拍摄的无鞭毛虫巢。(2012)。-带有GFP基因插入和表达的克氏锥虫。从慢性感染小鼠模型中取出胃肠道组织。组织匀浆后通过FACS分选机。抗细胞类型表面标记的单抗，以识别含有绿色荧光蛋白T.克鲁兹的细胞。2-确定抗炎细胞因子转化生长因子-b的作用-以递增的浓度注射抗转化生长因子-b抗体，直到观察到治疗和未治疗的泊沙唑小鼠模型之间的差异。在一个类似的实验中也观察到了对转化生长因子-β的抑制(Vitsky等人，2009年)。本文报道的最高剂量注射50 mg/kg对小鼠口腔和食道组织中的转化生长因子-β活性有明显的抑制作用。在目前的方案中，我将用非常低的0.1 mg/kg剂量的抗转化生长因子-b单抗进行泊沙唑+免疫抑制实验，并以逐渐增加的剂量重新进行实验，直到对照组和单抗处理组之间的显着统计学差异变得明显。

The recent paper Francisco et.al, (2015) identifies through in vivo imaging the GI, specifically the colon and stomach, as key sites in which T. cruzi replication is active at the chronic stage even in the presence of chemotherapeutic posacoazole treatment. The replication in this model in spite of drug treatment mirrors the types of drug failure seen in the clinic in patients with Chagas disease. This observation links with our classical immunological understanding of the intestinal tissue as a site set up in early development to be tolerogenic. This project proposal has several aims:1. Defining the specific tissue/cell type harbouring the parasite2. Establishing a role for TGF-b in T. cruzi persistenceThe first aim will allow characterisation of the permanent cellular reservoir of the parasite which can be inferred to intermittently release infected immune cells, that lead to the transient infection other tissues including the heart, where much of the clinical disease pathology is observed. Identification of this infected cell type would allow better targeting of future drug candidates with the goal being global sterile cure. The phenotype of the immune response to T. cruzi has been investigated with conflicting conclusions drawn. It is widely accepted that a Th1 response, particularly high expression of IFN-y and TNF-a is associated with control of the intracellular parasite in vertebrate hosts. What is less clear is the role of the anti-inflammatory cytokine TGF-b. Studies by Ferrao et.al, (2015) identifying TGF-b as a major determinant of successful T. cruzi host cell entry and subsequent amastigote replication. The colon, specifically identified in your Fransico et.al, (2015) paper as a site of parasite survival is well known to have a very TGF-b rich tolerogenic environment to prevent immunopathology. This proposal seeks to show whether organs in the GI tract provide an advantage sustaining T. cruzi parasite replication during posacoazole clearance of the other mouse tissues in the model. Aims1 - Further characterising the T. cruzi localisation of infection in the posacoazole treated mouse model-Formalin fixed and paraffin embedded sections of parasite burdened organ stained with eosin and haematoxylin. 400x light microscope search for amastigote nests imaged previously by Roffe et al. (2012).-T. cruzi with GFP gene insertion and expression. GI tract tissue removed from chronically infected mouse model. Tissue homogenised and put through a FACS sorter. mAb against cell type surface markers to identify cells harbouring the GFP T. cruzi. 2 - Identifying the role of anti-inflammatory cytokine TGF-b-Inject anti TGF-b Abs at progressively increasing concentrations until a difference between treated and non-treated posacoazole mouse model is observed. Inhibition of TGF-b is observed in a similar experiment by (Vitsky et.al, 2009). The highest dose reported in this paper 50mg/kg injected clearly yielded a good inhibition of TGF-b activity as demonstrated by the dysregulation in the oral and oesophageal tissues in the mice. In the current proposal I would run the posacoazole + immunosuppression experiment with a very low 0.1mg/kg dose of the anti-TGF-b mAb and rerun the experiment with gradually increasing dosages until a significant statistical difference between the control and mAb treatment groups became apparent.