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

最近的论文弗朗西斯科et.al（2015）通过体内成像确定了胃肠道，特别是结肠和胃，作为T. Cruzi复制在慢性阶段是活跃的，即使在化疗剂泊沙唑治疗的存在下。尽管进行了药物治疗，但该模型中的复制反映了临床上在查加斯病患者中观察到的药物失败类型。这一观察结果与我们经典的免疫学理解有关，即肠组织是在早期发育中形成的致耐受性位点。本项目提案有几个目标：1。确定寄生虫的特定组织/细胞类型2。建立TGF-β在T.克鲁兹持续性第一个目标将允许对寄生虫的永久细胞储存库进行表征，可以推断寄生虫间歇性释放受感染的免疫细胞，从而导致包括心脏在内的其他组织的短暂感染，其中观察到大部分临床疾病病理学。这种感染细胞类型的鉴定将允许更好地靶向未来的候选药物，目标是全球无菌治愈。T.对cruzi的调查得出了相互矛盾的结论。Th 1应答，特别是IFN-γ和TNF-α的高表达与脊椎动物宿主中细胞内寄生虫的控制有关，这一点已被广泛接受。不太清楚的是抗炎细胞因子TGF-b的作用。Ferrao的研究et.al，（2015）将TGF-b确定为成功T. Cruzi宿主细胞进入和随后的无鞭毛体复制。众所周知，在您的Fransico et.al（2015）论文中特别确定的结肠是寄生虫存活的部位，具有非常丰富的TGF-b耐受性环境，以防止免疫病理学。这项提议旨在表明胃肠道中的器官是否提供了维持T。模型中泊沙唑清除其它小鼠组织期间克氏寄生虫复制。目的1-进一步研究T.泊沙唑处理的小鼠模型中感染的Cruzi定位-用伊红和苏木精染色的负载寄生虫的器官的福尔马林固定和石蜡包埋切片。400倍光学显微镜搜索先前由Roffe et al.（2012）成像的无鞭毛体巢。T. cruzi与GFP基因的插入和表达。从慢性感染的小鼠模型中取出胃肠道组织。组织均质化并通过FACS分选仪。针对细胞类型表面标志物的mAb，以鉴定携带GFP T的细胞。克鲁兹2 -鉴定抗炎细胞因子TGF-β的作用-以逐渐增加的浓度注射抗TGF-β Ab，直到观察到治疗的和未治疗的泊沙唑小鼠模型之间的差异。在类似的实验中观察到TGF-β的抑制（Vitsky et.al，2009）。本文报道的最高剂量50 mg/kg注射清楚地产生了良好的TGF-β活性抑制，如小鼠口腔和食管组织中的失调所证明的。在当前的建议中，我将使用非常低的0.1 mg/kg剂量的抗TGF-B mAb进行泊沙唑+免疫抑制实验，并使用逐渐增加的剂量进行实验，直到对照组和mAb治疗组之间的显著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.