Adoptive transfer of pre-activated nTreg does not protect animals from IRI.

<p>nTreg were obtained from FoxP3 GFP mouse spleens by flow sorting followed by expansion <i>in vitro</i> for 21 days and further flow-sorting for purity [a–c]. Flow cytometry of spleen and liver confirmed successful transfer [d,e]. There was no difference in injury severity between nTreg supplemented and control animals at 3 hours of reperfusion (n = 4 per group) [f].</p

Treg depletion in the FoxP3.LuciDTR mouse does not increase susceptibility to IRI.

<p>FoxP3.LuciDTR mice or wildtype control (n = 5–10 per group) received 25 ng/g DT 24 hours prior to ischemic insult. ALT release [a] and histological injury score [b] did not differ between groups. DT treatment of DTR animals effected almost total depletion of Treg from the circulation [c,d], spleen [e] and liver [f].</p

Treg antibody depletion does not increase susceptibility to IRI.

<p>Wildtype mice received the CD25 depleting antibody pc61 7 and 2 days or PBS (n = 5–8 per group) prior to ischemia reperfusion injury. Injury severity was no different between Treg intact and Treg depleted animals in terms of ALT [a] or histological injury score [b]. PC61 treatment resulted in significant depletion of CD4+CD25+[c] and CD4+FoxP3+[d] cells. FlowCytomix was used to profile circulating chemo/cytokines. No differences were detected in post-operative rises in CXCL-10/IP-10 [e], KC/CXCL-1 [f], IL-6 [g] and GM-CSF [h] between Treg intact and depleted animals. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and IL-10) were not detected.</p

Adoptive transfer of pre-activated iTreg does not protect animals from IRI.

<p>iTreg were generated <i>in vitro</i> from sorted CD62L high, FoxP3- naïve T cells cultured in the presence of TGFβ and IL-2 for 5 days before flow sorting to maximize purity [a-d]. Successful transfer was confirmed by detection of FoxP3 GFP+cells in spleen [e] and liver [f]. No difference was detected in injury severity between iTreg supplemented and PBS treated control animals at 3 or 24 hours of reperfusion (n = 4–5 per group) [g].</p

Treg mobilization during reperfusion is not enhanced by IPC.

<p>Mice (n = 5–8 per group) were subjected to ischemia reperfusion injury with or without ischemic preconditioning and killed at 3 or 24 hours. Ischemic preconditioning protected the liver from injury both in terms of ALT release [a] and histological injury score [b] measured at 24 hours. CD4+FoxP3+cells were mobilized into the circulation during reperfusion [c]. Hepatic CD4+FoxP3+cells increased over the same time period [d]. Total CD3+lymphocytes were stable throughout reperfusion [e]. FlowCytomix was used to profile circulating chemo/cytokines. Rises were detected in CXCL-10/IP-10 [f], CXCL-1/KC [g], IL-6 [h] and GMCSF [i]. Other analytes (IL-1α, IL-1β, IL-2, IFNγ, IL-17 and IL-17F, and the Treg cytokine IL-10) were not detected.</p

<i>In vivo</i> expansion of Treg does not protect animals from IRI.

<p>Mice received IL-2/JES6-A12 complexes or PBS for three consecutive days prior to hepatic ischemia reperfusion insults (n = 8 per group). Animals were culled at 24 hours of reperfusion, and tissues analysed for Treg frequency. There was significant expansion of splenic [a], circulating [b] and hepatic [c] Treg. Injury severity was similar between Treg expanded and control animals, in terms of ALT and histological injury [d,e]. FlowCytomix was used to profile circulating chemo/cytokines. Levels of IL-2 [f], CXCL-10/IP-10 [g] and IL-6 [h] were elevated. Rises in KC/CXCL-1 [i], and GM-CSF [j] did not differ between groups. Other analytes (IL-1α, IL-1β, IL-10, IL-17, IL-17F, IFNγ, MIP-1α) were not detected.</p

Spearman’s correlation coefficient analysis to determine the relationship between predicted hormone concentrations at 26 weeks’ gestation and ultimate gestation at delivery.

<p>Spearman’s correlation coefficient analysis to determine the relationship between predicted hormone concentrations at 26 weeks’ gestation and ultimate gestation at delivery.</p

Maternal and fetal characteristics and pregnancy outcomes classified as early preterm, preterm and term deliveries.

<p>Maternal and fetal characteristics and pregnancy outcomes classified as early preterm, preterm and term deliveries.</p

Spearman’s correlation coefficient analysis comparing salivary and serum concentrations.

<p>Spearman’s correlation coefficient analysis comparing salivary and serum concentrations.</p

Correlation of salivary and serum hormone concentrations in five individuals.

<p><i>Red lines</i>, serum concentrations; <i>blue lines</i>, saliva concentrations.<i>R</i>, Spearman’s correlation coefficient (rho) value. <i>Sig</i>., significance value. <i>*</i>, <i>p</i> < 0.05; <i>**</i>, <i>p</i> < 0.01.</p