Gene expression was measured by real time PCR (RT-PCR) using the Corbet Research Rotor gene 6000 with the QuantiTech SYBR Green kit (QIAGEN). The FOXP3 sequences used were: forward primer 5′-ACCTGGAAGAACGCCAT and reverse primer Onalespib order 5′-TGTTCGTCCATCCTCCTTTC both at a final concentration of 0.4 μM. FOXP3 copy numbers were expressed in relation to human acidic ribosomal protein (HuPO), the house keeping gene. The standards were prepared as above using blood donated by an adult and the RT-PCR product
pooled and purified using the QIAquick PCR Purification kit (QIAGEN). The DNA was then quantified using the nanodrop and FOXP3 copy numbers calculated using the Avogadro constant formula. Statistical analyses: For paired comparisons between two time points random effects models were used to allow for the clustering effect of subject. For the antibody responses
Abiraterone solubility dmso where there were 7 time points a generalised estimating equation was used with an exchangeable correlation structure. Responses were appropriately transformed and in the absence of a suitable transformation the data was ranked. All regressions were adjusted for possible confounding affects of sex, but due to well balanced groups there was very little evidence of confounding. Where appropriate, time and dose group interactions were tested. Significance was measured at the 5% level and all analyses were performed in Stata 11 (Statacorp) and figures drawn using Matlab 7.9 (The MathWorks Inc.). The number of participants and their loss to the study at different time points are shown in Fig. 1. The overall refusal rate was 11.5%, loss to follow up due to the participant travelling was 17.4% and 3.8% of the children received an unscheduled measles vaccine. The two dose PD184352 (CI-1040) regimen was safe since side effects were mild and infrequent. They did not differ in frequency or timing between group 1 and group 2 either at 4 months of age or at 9 months of age. The most frequent complaints were diarrhoea and fever with a mean prevalence of 7.9 ± 2.4% and 6.6 ± 2.7% respectively. Before vaccination at 4 months of age median HAI titres were log2 2 (IQR 0–3) and log2 3 (IQR 1–4) in
groups 1 and 2 respectively (Fig. 2 and Supplementary Table). At 9 months before the second measles vaccination the median HAI titre in group 2 was log2 3 (IQR 1–6) which is significantly higher than that of group 1 which was zero; 77% of group 2 children had detectable antibody and 66% had protective levels whereas antibody was detected in only 6% of group 1 children. Two weeks after the second dose of E-Z vaccine antibody titres had risen sharply in group 2 with all but one child reaching protective levels whereas only 25/65 (36.4%) of group1 children attained these levels after their first measles vaccination. At 18 months of age antibody titres in group 2 (median 4, IQR 3–5) fell significantly lower than those in group 1 (median 6, IQR 5–7) but then stabilised between 18 and 36 months.