Immunohistochemistry results following reperfusion.

<p>(A) Recipient serum levels of aspartate aminotransferase (AST) following transplantation. (B) Neutrophil infiltration, platelet activation and expression of vWF post-reperfusion. (C) Correlation between post-reperfusion cell death and WIT.</p

Multivariate analysis of pre vs. post biopsies and ceramides distribution.

<p>(A) OPLS-DA score plot visualizing the grouping of pre vs. post for all biopsies. (B) S-plot illustrating ceramides correlation to groups for model in A. (C) OPLS-DA score plot showing group separation between pre- and post-transplant for none steatotic grafts. (D) S-plot indicating ceramides correlation to groups for model in C.</p

Immunohistochemistry results prior to transplantation.

<p>(A) Leukocyte infiltration and FasL expression in liver allografts prior to transplantation. Biopsies from DBD and DCD liver biopsies were stained with monoclonal antibodies against neutrophil elastase, CD3 and FasL. (B) Expression of ICAM-1 in liver allografts prior to transplantation.</p

Study design.

<p>Two independent cohorts of immunohistochemistry and targeted lipids analysis found that DBD and DCD liver allografts have different pathways for I/R injury.</p

Heat-map showing distinct ceramides profiles of DBD and DCD tissue in 46 transplant samples.

<p>Values are mean amounts per donor group at pre and post-transplantation stages. A clustering analysis (dendrogram) shows which lipids differ most; red depicts increased amounts and green decreased.</p

CD107a expression in liver isolated CD8+ T-cells.

<p>(A) In vitro CD107a expression on liver (DBD and DCD) CD8+ T-cells was analysed after a co-culture with K562 cell line. Mann-Whitney U or Spearman correlation test was used (B) Gating of liver mononuclear cells to analyse CD107a expression on CD8+ T-cells. ns = not significant, * p<0.05.</p

Intrahepatic T-cells respond to HMGB1 <i>in vitro</i>.

<p>(A, B) Biopsies obtained from liver allografts before (left) and one-hour after (right) reperfusion. (A) Haematoxylin and eosin staining of a representative biopsy from DBD or DCD livers showing hepatocellular necrosis (plain arrow) and neutrophil aggregation (arrowhead) in post-reperfusion samples; (B) Immuno-histochemical staining for HMGB1 of the biopsies shown in (A) reveals an increased proportion of hepatocytes showing nuclear expression of HMGB1 in post-reperfusion samples compared to the pre-reperfusion counterparts but no cytoplasmic expression; (C) Production of IFN-γ from T-cells of a representative donor co-cultured with syngeneic DC at the ratio of 20:1 for 7 days. Percentage of IFN-γ-producing cells gated on CD8+CD3+ T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml; (D) Proliferative response of T-cells in unstimulated cultures (negative control), in presence of anti-CD3/CD28 (positive control) or in presence of HMGB1 at 100ng/ml. 1 = negative, 3 = positive control and 2 = HMGB1; (E) Quantitative analysis of IFN-γ concentration in supernatants of cultures. NT = negative control, S = aCD3/CD28 stimulation, HMGB1 = cultures stimulated in presence of 100 or 10ng/ml of HMGB1. Depiction of IFN-γ concentration in pg/ml (left) and ratio of increase reported to the negative control (right). Kruskal Wallis test was used.</p

Clinical parameters of recipients.

<p>Clinical parameters of recipients.</p

Cytokine production of T-cells in peripheral blood, LD, DBD and DCD livers.

<p>Hepatic mononuclear cells isolated from liver perfusate of living donors (LD) and matched PBMC were stimulated <i>ex vivo</i> and their cytokine production (n = 9 to 17) was assessed by intracellular staining. Frequency of IL-2 (A), IFN-γ (B) and IL-17 producing CD4 (left) and CD8 (right) T-cells (C). ** p<0.01, *** p<0.001. Furthermore, HMC isolated from DBD, DCD or LD livers have been stimulated <i>ex vivo</i> and cytokine production assessed by intracellular staining. Gating strategy determined using unstimulated cells. Numbers indicate percentage of cells detected; (D-F) Cytokine production of HMC isolated from indicated liver donors. Frequency of CD4 or CD8 T-cells expressing IL-2 (D), IFN-γ, E) and IL-17 (F). Statistical analysis: Mann Whitney U or Kruskal Wallis test was applied, ns = non-significant, **p<0.01, ***p<0.001.</p

The donation status of the liver allograft influences the cytokine production in culture.

<p>Cytokines in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-α (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC culture were measured. Left column: levels of IL-2 (A), TNF-ells. Numbers indicate epatocellular injury iα (B), IFN-γ(B), IFN-in the supernatants of day-seven HMC un-stimulated and stimulated HMC. Mann Whitney U or Kruskal Wallis test was performed. ns = non-significant, * p<0.05, **p<0.01, ***p<0.001.</p