2 The three strains used during the study period were BCG-Russia (BCG-I strain from Moscow, Serum Institute of India, India);
BCG-Bulgaria (BCG-SL 222 Sofia strain, BB-NCIPD Ltd., Bulgaria); and BCG-Denmark (BCG-SSI 1331, Statens Seruminstitut, Denmark). Other vaccines administered JNK animal study were OPV (at 0, 6, 10 and 14 weeks); DPT, Hib and Hep B (at 6, 10 and 14 weeks); and measles (at 9 months). Cytokine responses were assessed by six-day whole blood culture and ELISA assay, as previously described . Cytokine levels in culture supernatants were measured by ELISA (Beckton Dickinson, UK) after stimulation by crude culture filtrate protein, antigen 85 (cCFP, Ag 85; Colorado State University, USA), tetanus toxoid (TT; Statens Seruminstitut, Denmark) and phytohaemagglutinin (PHA; Sigma, UK). CFP and Ag85 were used to assess mycobacteria-specific immune responses and PHA and TT to assess non-specific effects of BCG strains. IFN-γ
and IL-10 were analysed as representative of type 1 and regulatory activity respectively. Although IL-4 levels are central to the type 2 response, IL-5 and IL-13 are more detectable in supernatants and were therefore measured instead. Results were adjusted according to responses in unstimulated wells. To avoid time dependent effects of assay performance, the sequentially collected samples were tested in a randomised order. Statistical analyses were conducted using Stata/IC 11.1. Infants were grouped according to strain of BCG received. Characteristics of the three groups of infants and mothers were compared using Pearson’s buy CHIR-99021 chi-squared test for categorical variables
and the t-test for continuous variables. Cytokine levels below the threshold of detection were set to zero 3; distributions of cytokine results were highly skewed, a recognised phenomenon in immunological studies ,  and . Cytokine results were therefore transformed to log10(concentration + 1) before analysis. Mean cytokine responses were compared between strain groups using random effects linear regression, anti-logging the regression coefficients to obtain geometric mean ratios (GMRs). Random effects were used to account for potential between-lot variability (since several lots of Adenylyl cyclase vaccine were administered within each BCG strain group). As some cytokine results remained skewed after log10 transformation, analyses were boostrapped  with 10,000 repeats to calculate bias-corrected accelerated confidence intervals. Cytokine responses of infants with and without a BCG scar were compared using the same methods but without random effects (being independent of potential between-lot variability). Odds ratios for associations between BCG strain and scar presence were calculated through random effects logistic regression. BCG scar sizes were compared across strain groups through linear regression.