Normalization method of individual IFN-γ specific response.

<p>A. Schematic representation of the <i>in-vitro</i> stimulation assay. Blood samples of 76 pregnant women with primary CMV were stimulated with either CMV peptides or PHA, for 24 hours at 37°C. The amounts of IFN-γ, TNF-α, IL-10 and IL-6 in the supernatant were determined by CBA analysis. B. Comparing normalizations of IFN-γ induced after CMV stimulation to Nil or Mitogen tubes. We compared the ratios between the levels of IFN-γ induced by CMV peptides divided by that of 'nil' (CMV/N) or 'Mitogen' (CMV/M). Blood samples of five patients (P-I to P-V) were taken in two different time points, T1 and T2 (intervals of 5–8 weeks). Delta determined as the ratio at T1 subtracted from the ratio at T2. C. Comparing IFN-γ TB/M to IFN-g CMV/M ratio. Blood samples of 19 women were collected into Nil, CMV and mitogen tubes, and an additional tube containing Mycobacterium tuberculosis (TB) antigens. We compared the IFN-γ induced by CMV peptides to the IFN-γ induced by TB peptides both normalized to the amount of IFN-γ in the Mitogen tube.</p

Clinical and laboratory characteristics of CMV-infected pregnant women- transmitters and non-transmitters.

<p>Clinical and laboratory characteristics of CMV-infected pregnant women- transmitters and non-transmitters.</p

Low IFN-γ RR in non transmitters does not stem primarily from high IFN-γ level caused by mitogen activation.

<p>Comparison of IFN-γ levels after mitogen activation in transmitters and non transmitters. Women with IFN-γ RR <1.8% are marked with filled triangles. T = transmitters, NT = non transmitters.</p

Normalization method of individual IFN-γ specific response.

<p>A. Schematic representation of the <i>in-vitro</i> stimulation assay. Blood samples of 76 pregnant women with primary CMV were stimulated with either CMV peptides or PHA, for 24 hours at 37°C. The amounts of IFN-γ, TNF-α, IL-10 and IL-6 in the supernatant were determined by CBA analysis. B. Comparing normalizations of IFN-γ induced after CMV stimulation to Nil or Mitogen tubes. We compared the ratios between the levels of IFN-γ induced by CMV peptides divided by that of 'nil' (CMV/N) or 'Mitogen' (CMV/M). Blood samples of five patients (P-I to P-V) were taken in two different time points, T1 and T2 (intervals of 5–8 weeks). Delta determined as the ratio at T1 subtracted from the ratio at T2. C. Comparing IFN-γ TB/M to IFN-g CMV/M ratio. Blood samples of 19 women were collected into Nil, CMV and mitogen tubes, and an additional tube containing Mycobacterium tuberculosis (TB) antigens. We compared the IFN-γ induced by CMV peptides to the IFN-γ induced by TB peptides both normalized to the amount of IFN-γ in the Mitogen tube.</p

PD-1 expression in PBMC’s of women with high and low IFN-γ relative response is similar.

<p>The expression of PD-1 in PBMC was performed by TaqMan Real time PCR of PD-1 and GUSB (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147883#sec007" target="_blank">methods</a> section). ∆Ct was calculated (average ∆Ct of PD-1minus the average ∆Ct of GUSB). Relative expression (2^(-∆Ct)±SE) values of women with high and low IFN-γ RR were compared by <i>t</i>-test.</p

Post-test probability of women with low IFN-γ relative response to CMV.

<p>The post-test probability of women with IFN-g RR<1.8% reduced from 40% to 7%-8% in all tested women (n = 75), for women with low IgG avidity (n = 62) and for viremic women (n = 37).</p