Objectives
- To characterise the innate immune responses in infected individual in relation to patient age, exposure (thus acute versus chronic), infection, and evidence of pathology.
- Identify innate immune receptors involved in the recognition of schistosome infections, associated downstream signalling events, and their involvement in observed levels of immune reactivity, or hypo-responsiveness.
Description of work
Immune responses will be measured in groups of patients with different exposure histories and morbidity as defined in WP1. Blood will be drawn (4mls) from all participants for whole blood assays; in a subset 15mls of blood will be drawn to allow detailed studies on PBMC.
Whole blood/PBMCs will be stimulated in vitro individually, or in combination with:
- extracts of parasites containing ligands of innate immune cells known to stimulate or regulate innate immune responses. I.e. material released from cercariae (0-3hRP), released by eggs (ESP), and previously defined schistosome molecules (i.e. LysoPS, and LFNP-III).
- soluble parasite extracts (i.e. SWAP), conventionally used as stimulants of lymphocyte responsiveness.
- known microbial ligands for TLRs (LPS, PAM3Cys, MALP2, CpG, R848, and flagellin).
Cell culture supernatants at 24 and 72 hours will be collected and frozen. Cytokine levels (e.g. IFN-γ, IL-5, IL-10, IL-12, IL-13, TNF-α will be determined by ELISA, or Luminex assay.
The expression of gene transcripts in whole blood samples will be studied using real time PCR. This micro-assay using 200 μl of blood was developed for human immunoepidemiological studies by partner 2. Samples will be preserved in buffers and stored for further processing by developing country scientists in partner 1 and 2 laboratories. The expression of 20 genes involved innate immune responses and immunoregulation will be studies (e.g. TLRs, costimulatory molecules and signalling molecules).
Blood and PBMC samples from limited numbers of patients also will be analysed for surface expression of different innate immune receptors, and markers of activation and regulation by flow cytometry of labelled cells. We will again be directed by data obtained in parallel experiments in the infection models (WP3) but also by the availability of commercially available antibodies. We will detect different TLRs, and surface expressed markers of activation and regulation on cells labelled with 2, 3, or more fluorescently-conjugated antibodies to ascribe expression of different receptors and so on, to individual cell types. Analysis will take place in the laboratories of partners 1, 2 and 5.