Children aged three-12 years were recruited for a study assessing complications of cerebral malaria, which was conducted at Mulago Hospital in Kampala, Uganda . Eighty-five children with cerebral malaria (CM) and 76 children with uncomplicated malaria (UM) were recruited. Children with CM were included in the study if they were admitted to Mulago Hospital, and met the WHO criteria for CM: coma (Blantyre coma scale ≤2, Glasgow coma scale ≤8), P. falciparum-smear positive, and no other signs of encephalopathy. Lumbar puncture was performed to rule out meningitis or encephalitis. Children with UM were enrolled at the acute-care clinic or a malaria outpatient clinic that is sponsored by the University of California San Francisco (UCSF). Children were enrolled as UM patients if they exhibited the signs and symptoms of malaria (fever, chills, vomiting, headache), were P. falciparum-smear positive, had no malaria-related complications (e.g. seizures, coma, severe anaemia, or respiratory distress), no other sign of acute illness, and were not hospitalized for their malarial illness. Details of study enrolment have been reported previously . Genetic testing was requested from study participants upon follow-up testing at two years after enrolment. If consent was obtained from the study participant parent or guardian, testing for TLR polymorphisms was performed on the filter paper samples collected at initial enrolment.
Exclusion criteria for children in all three groups was (1) a history of meningitis or encephalitis, or any other brain disorder (including CM); (2) a history of developmental delay; (3) prior admission to the hospital for malnutrition; (4) a history of chronic illness. Ethical approval for these studies was granted by the institutional review boards for human studies at Makerere University Faculty of Medicine, University Hospitals of Cleveland, Case Western Reserve University, Indiana Wesleyan University, and the University of Minnesota. Upon admission, study participants' blood was collected onto Whatman filter paper (Whatman Corporation, Florham Park, NJ). Genomic DNA was extracted from dried blood spots using the QIAamp 96 spin blood kit (QIAGEN, Valencia, CA). Presence of P. falciparum was determined by light microscopy of thin and thick blood smear with two independent readings. A third independent reading was performed if necessary to resolve any discrepancies between the initial two readings.
Polymerase chain reaction (PCR)
Genomic DNA was extracted from blood using the QIAamp 96 spin blood kit (QIAGEN, Valencia, CA). PCR was performed using a master mix consisting of 1× PCR buffer, 125μM dNTPs, 2.5 mM MgCl2, 125nM primers, and 0.8 units Taq polymerase in a reaction volume of 25μl. PCR primers and amplification conditions have been described previously . TLR2 Δ22 genotypes were assigned based on size discrimination of PCR products on a 4% agarose gel. TLR2 GTn genotypes were assigned based on size discrimination of PCR products run on a 6% polyacrylamide gel as described elsewhere .
Cloning and sequencing
PCR amplification products from local donors were purified using the QIAquick PCR purification kit (QIAGEN, Valencia, CA). Purified PCR products were sent to MWG Biotech, High Point, NC for sequencing. Sequences were analysed using the Sequencher software (Gene Codes Corporation, Ann Arbor, MI).
Serum levels of IFNγ, TNF, IL-1β, IL-10, and IL6 in children with uncomplicated malaria and cerebral malaria were compared according to TLR2 genotype. Serum samples for cytokine measurement were obtained at the time of admission (CM) or outpatient enrollment (UM). Cytokine testing was done using cytometric bead array (CBA) technology using the Bioplex-Luminex system (Austin TX), or ELISA as previously described [6, 20].
Allele and genotype frequencies were calculated for the different polymorphisms, and the Hardy-Weinberg exact test (estimation of p-values by the Markov chain method) was performed for each population using Arlequin version 3.01. Haplotypes were statistically inferred using the Expectation - Maximization method in Arlequin. Chi squared test was used to determine allelic, genotypic, and haplotypic associations using SPSS for Windows (version 13.0). Genotypic tests were performed for additive, dominant, recessive, and heterozygote advantage models. Odds ratios were performed for the different genotype models using an online calculator. All cytokines were log transformed to create a normalized distribution, and comparisons for the different genotype models were made using the T-test using SPSS for windows (version 13.0). Because of errors due to multiple comparisons, Bonferroni correction assuming two individuals tests was applied. At a false positive rate of 5%, the adjusted significance threshold becomes p < .025.
TLR2 expression by flow cytometry
Venous blood was collected from 18 malaria naïve North American volunteers. Peripheral blood mononuclear cells were isolated by Ficoll-Paque separation (GE Healthcare, Piscataway, NJ) and stained with TLR2-FITC (clone TL2.1) and CD14-APC (clone 61D3, eBiosciences, San Diego, CA). Cells were read on an LSRII flow cytometer (BD Biosciences, Franklin Lakes, NJ). Monocytes were distinguished by scatter characteristics and CD14 positivity. Median fluorescence intensity of isotype controls were subtracted from each sample to determine relative TLR2 fluorescence. Ethical approval for these studies was granted by the institutional review board for University Hospitals of Cleveland, Case Western Reserve University.
Peripheral blood mononuclear cells (PBMC) were cultured in RPMI 10% human serum AB, 2 mM L-glut, 20 mM HEPES, and pen/strep. PBMC were plated out at 1 × 106 cells/ml and stimulated with Pam3Cys (200 ng/ml) for 24 h. Human monocytes were isolated by immunomagnetic purification using the Miltenyi monocyte isolation kit II (miltneyi) to obtain unlabelled monocytes which were 80-95% pure. Purified monocytes were cultured for six days in RPMI supplemented with 2 mM L-glut, 20 mM HEPES, pen/strep, and 10% autologous serum to allow differentiation into monocyte derived macrophages. Cells were washed three times with PBS, and adherent cells were removed with cell dissociation buffer (vendor) for flow cytometry.
Plasmodium falciparum strain 3D7 parasites were cultured and the parasite life cycle was synchronized as described . Trophozoites and schizonts were obtained by magnetic purification. Late stage parasite cultures were washed three times with Macs buffer (PBS, pH 7.2, 0.5% FBS, and 2 mM EDTA), and resuspended at a concentration of 2 × 109 cells/ml. Parasite cultures were passed through Miltenyi LD columns (Miltenyi), allowing uninfected RBCs and rings to pass through the column. Miltenyi columns were washed two times 1 ml Macs buffer, and late stage parasites were eluted with 3 ml Macs buffer. Trophs/schizonts were washed three times and counted. Schizonts were lysed by sonication (40 V, 10 mins). Following sonication, schizonts were brought up to the appropriate concentration in RPMI. Schizonts or intact trophozoites were added to macrophage cultures at a ratio of 10:1.
Approximately 1 × 106 monocytes were isolated for qPCR. Total RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). 100 ng of total RNA was reverse transcribed into cDNA using commercial reagents (Invitrogen) and non-specific Oligo-DT primers. Quantitative PCR was carried out using the following gene specific primers; TLR2 - forward 5'-ATTGTGCCCATTGCTCTTTC-3', reverse, 5'-CTTCCTTGGAGAGGCTGATG-3'; GAPDH - forward 5'-AAGATCATCAGCAATGCCTCCTGC-3', reverse 5'-ATGGACTGTGGTCATGAGTCCTTC-3'. Samples were analysed on a 7300 Real Time PCR System (Applied Biosystems), and normalized using the comparative threshold method [25, 26]. HEK cell mRNA was used as a negative control for TLR2 expression.