Multivariable analysis of pre-operative risk factors for patient death and graft loss in those undergoing liver transplantation for acute liver failure.

<p>Cox Proportional Hazards model using variables found to be significant in univariable analysis (P<0.05) and those thought to be clinically significant. Serum creatinine was centred at a value of 90 μmol/L for the purposes of the analysis. Patient survival data was only considered for patients receiving their first liver transplantation. Values in parentheses are 95% confidence intervals.</p

Univariable analysis of risk factors for patient death and graft loss after liver transplantation for acute liver failure.

<p>Hazard ratios were determined using Cox Proportional Hazards model. Values in parentheses are 95% confidence intervals.</p

Renal replacement therapy and survival in a matched dataset.

<p>Probability of patient (A) and graft survival (B) at one year following liver transplant by pre-operative serum creatinine level and RRT requirement in a matched dataset. In patients not receiving RRT, a pre-operative serum creatinine of greater than 175 μmol/L had a significantly greater risk of graft failure compared to those receiving RRT (C; black line indicates hazard ratio = 1, shaded areas represent 95% CI). Cox proportional hazards model with groups fully matched for baseline covariates used for each analysis.</p

The effect of renal replacement therapy and serum creatinine on survival.

<p>Kaplan-Meier plots demonstrating the effect of both renal replacement therapy (A&B) and pre-operative serum creatinine (C&D) on patient and graft survival respectively in liver transplantation for acute liver failure. P value calculated using log-rank test.</p

The interaction between renal replacement therapy and elevated creatinine on patient and graft survival.

<p>Kaplan-Meier plots demonstrating the interaction between pre-operative creatinine concentration and requirement for RRT on patient (A) and graft survival (B); P value calculated using log-rank test. Probability of death at one year following liver transplant by pre-operative serum creatinine level, RRT and recipient age is shown for patient (C) and graft survival (D). Models use Cox proportional hazards, with co-variable patient characteristics adjusted to haemoglobin concentration of 10 g/dL, requirement of mechanical ventilation and absence of sepsis.</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 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

<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

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

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