Study design and participants
231 blood samples were obtained from children at the final time point in a double-blind placebo-controlled randomized trial on children aged 39–73 months in four semi-urban villages, Akinlalu, Ipetumodu, Moro and Edunabon, near Ile-Ife, Osun State, Nigeria. Details of the study area, design and participants were published previously . This current sub-study was to examine immune responses associated with P. falciparum infection and examine the impact of A. lumbricoides. Data were available on children’s age and infection status for P. falciparum and A. lumbricoides infection. Children who suffered from severe malaria were treated and excluded from the study and therefore only individuals with uncomplicated malaria were included (malaria parasitaemia and fever >37.5°C) . The study protocol was approved by the Ethics and Research Committee, Obafemi Awolowo University Teaching Hospitals’ Complex, Ile-Ife, Nigeria.
Isolation of peripheral blood mononuclear cells
Ten ml of blood (in tubes containing heparin) was obtained from 231 children, ranging in age from 39–73 months. Peripheral blood mononuclear cells (PBMCs) and plasma were obtained following histopaque (Sigma-Aldrich, St Louis, MO, USA) density gradient centrifugation. PBMCs were collected and stored in liquid nitrogen and plasma samples were stored at −80°C.
Human IFN-γ, IL-10, TGF-β, TNF, IL-4 and IL-12p70 Opti-EIA kits (BD Biosciences) and human IL-17, RANTES, MMP-8 and TIMP-1 DuoSet ELISA Developmental Kits (R&D, Minneapolis, MN, USA) were used to quantify cytokine, chemokine and metalloproteinase levels in plasma samples as described per manufacturers instructions. NO levels were also measured in plasma using the Greiss Reagent System (Promega, Madison, WI, USA).
Flow cytometry and in vitro culture
The following mAbs were used for cells surface staining and intracellular cytokine staining: FITC-conjugated anti- CD4, CD14, CD36, CD56, IFN-γ; PE-conjugated anti- γδTCR, CD54, IL-10; and APC-conjugated anti- CD3, CD11c, HLA-DR, IL-2 and TNF (eBiosciences, San Diego, CA, USA). Isotype controls included FITC-conjugated mouse IgG1, IgM, IgG2a, IgG2b; PE-conjugated mouse IgG1, IgG2b and APC-conjugated mouse IgG1, IgG2b and rat IgG2a (eBiosciences). PBMCs were thawed and cultured in complete RPMI containing 10% FCS (foetal calf serum), 1% L-glutamine and 1% penicillin/streptomycin solution (Bio-sciences Ltd, Co. Dublin, Ireland). For intracellular cytokine staining, PBMCs were stimulated with 50 ng/ml PMA (Phorbol 12-myristate 13-acetate) and 1 mg/ml ionomycin for 4 h and to block cytokine secretion, 10 mg/ml Brefeldin A (Sigma) was added to the culture media. Cells were then washed and stained with cell surface mAbs, fixed with 4% PFA and permeabilized with 0.2% saponin (Sigma), before incubation with antibodies for IL-12, IFN-γ, IL-10 and TNF cytokines. Appropriately labelled isotype-matched antibodies were used as controls. Acquisition was performed using a FACSCalibur flow cytometer (BD Biosciences), and analysis of results performed using FlowJo software (Tree Star). A sample of gating strategy for T Cells in shown in Additional file 1.
PBMCs (1 × 106 cells/ml) from a cohort of children were also cultured on a 24-well plate with mycoplasma free P. falciparum parasites (at a ratio of 1:5), which were extracted from cell culture by saponin lysis (0.15%), were kindly provided by Dr Alison Creasey, University of Edinburgh, Scotland. After three days, cell culture supernatants were harvested and frozen for subsequent measurement of IFN-γ, IL-10, and IL-5 by commercial ELISA.
All data were analysed using SPSS 15 for windows. Percentage data were normalized prior to analysis by Arcsin transformation. Skewed data were normalized prior to analysis by log transformation. For differences between multiple groups, one-way ANOVA with Post-hoc testing by Tukey’s HSD test was used. For data with more than one factor, factorial ANOVA was used. Association between variables was assessed using regression analysis. For differences between two treatments 2-tailed Student t-test was used. In all tests, p <0.05 was deemed significant.