Objectives
Use isolated molecules to:
- deviate the in vitro immune response of cells obtained from local lymphoid tissues of two infection models.
- deviate the in vitro immune response of PBMCs from patients with different exposure regimes and pathology.
- manipulate the in vivo immune response in the two infection models to enhance, or regulate immune responses and pathology, and aid the induction of RA vaccine mediated protection.
Description of work
The isolated molecules will be tested in vitro and in vivo for their ability to modulate the immune response using the two models of experimental infection and PBMCs from infected patients. The two approaches will be conducted simultaneously, although the model studies are likely to be faster to give results. The technologies to test the immunological activities of the isolated molecules are already described in WP2 and WP3.
In vitro experimental studies: Isolated fractions of parasites and synthetic derivatives where available, will be used to stimulate immune cells obtained from the skin, sdLN and blood of mice exposed to multiple doses of S. mansoni, or from the mesenteric LN, spleen, and blood of mice exposed to chronic S. mansoni infection. Cells will be stimulated in vitro with titrated concentrations of the isolated fractions, either individually, or in combination with known activating, or regulatory preparations (parasite antigens, or microbial molecules). Responsiveness will be measured by cell proliferation and cytokine production.
In vitro human studies: New patients will be recruited from areas not previously covered by the study: a max of 50 patients from each area will be sought. The assays will be performed as above; isolated molecules will be used to stimulate PBMCs obtained from patients with different history of exposure, or pathology, either individually or in combination. Responsiveness will be judged by cytokine production measured by ELISA and by gene expression determined by real time PCR and whenever possible by flow cytometry.
In vivo experimental studies: The most reactive isolated molecules will be used to deviate the in vivo immune response in the multiple exposure and chronic infection models, and a model of vaccination (RA vaccine). The molecules will be tested for their ability to enhance established immune regulation, or lead to its reversal following administration to mice at selected time points; In the multi exposure model, measures will be made of pathology at the site of exposure, and the cytokine responses of pinnae and sdLN cells. In the chronic model, granuloma size and deposition of collagen as an index of fibrosis will be measured in addition to the antigen-driven cytokine response of cells from the mesenteric LN and the spleen. In the RA vaccine model, we will measure the induction of protective immunity.