Research Project
10150769
Neurocysticercosis (NCC), the infection of the central nervous system (CNS) caused by the metacestode larva of Taenia solium, the pork tapeworm, is endemic in most developing countries and identified as the most common cause of acquired epilepsy worldwide. The parasites cause a chronic neuroinflammation and pathological studies reveal reactive astrogliosis, fibrosis, angiogenesis, alteration of the brain blood barrier permeability and overexpression of both inflammatory and anti-inflammatory cytokines. Yet to this end we poorly understand the mechanisms underlying the pathology in NCC patients, and have minimal clinical means to prevent neurological complications in these patients. Effective treatment for NCC remains a challenge, as the severity of disease symptoms is thought to be a result of pathologic inflammatory response induced by the degenerating larvae. We have pioneered an in vitro model of T. solium larval development, from the infectious stage (the oncosphere), through large 60-day post-oncospheres. During these stages and until the parasite reaches a mature larva or cysticerci, the parasite itself changes its protein expression profile, however we have little to no information on the molecules secreted by each stage. TGF-? plays a pivotal role in a large spectrum of infections with protozoa and helminths. Besides the importance of host TGF-? signaling in the regulation of host-parasite interactions, much evidence has shown that helminth parasites might directly influence the TGF-? dependent pathway via the expression of TGF-? receptor and ligand homologues. Based on these studies, we will take advantage of our in vitro model and examine the excretory/secretory (E/S) products of the different larval stages of development of T. solium to test for immunomodulatory functions, starting with TGF-?. E/S from the five different stages of T. solium larval development (oncosphere, postoncospheres at 15, 30 and 60 days of growth and mature cysts), to proteomic analysis by mass spectrometry, characterize each stage?s secretome and compare the spectrum of secreted molecules between the stages. We will interrogate this new resource to identify homologues of members of the TGF-? superfamily. Additionally, E/S proteins from the different development stages of the larvae will be fractionated using both gel filtration and anion exchange Fast Protein Liquid Chromatography to provide a resource for use in subsequent in vitro assays in order to functionally test for and identify new immunomodulators. We will then test T. solium E/S for TGF-? like activity utilizing in the first instance a sensitive TGF-? reporter cell line. Positive E/S will then have its fraction profile tested to narrow down the identification of candidate molecules to screen in vitro, and the active fraction(s) will be subject to mass spectrometry. Subsequently, E/S will be tested in in vitro cultures of naïve CD4+ Tcells with IL-2 and anti-CD3 in order to assess the induction of the transcription factor Foxp3, that indicates that these cells have been induced to Tregs by a TGF-? homologue or mimic. Molecules with positive activity will be identified and recombinantly expressed, their activity will be characterized using the reporter MFB-F11 bioassay and in in vitro regulatory T cell induction assays. To identify TGF-? mimic proteins in the T. solium larvae has the potential both to change our understanding of parasite adaptation to the host and to develop possible therapies for immune mediated disease. In addition, understanding developmental signals required for parasite maturation may open new avenues for pharmaceutical treatment of infection.
