Annette Zipfel

Cultivation of porcine hepatocytes in polyurethane nonwovens as part of a biohybrid liver support system.

Many patients suffering from end-stage liver disease cannot be transplanted within reasonable time due to the shortage of donor organs. Bioartificial liver support systems may contribute to the liver regeneration or bridging the time until a liver graft for transplantation becomes available. Nonwovens with integrated oxygenation capacity have been developed and manufactured by melt blow technology using thermoplastic polyurethane. Capillary membranes for oxygenation were integrated into the nonwoven during the processing. The polyurethane nonwoven structures with adapted pore size and high pore volume allow high cell densities in the hepatocyte culture. The three-dimensional cell culture was housed by a flow bioreactor system and was integrated in a closed loop circulation with monitoring possibilities for pressure, pH, temperature, ammonia, and oxygen. Hepatocytes were isolated from rats or pigs by collagenase perfusion and infused into the medium-perfused circulation. Cells showed high viability and hepatocyte specific cytochrome P450-dependent metabolic function in culture (MEGX test).

TNF-α and its receptors mediate graft rejection and loss after liver transplantation

Abstract Tumour necrosis factor-α (TNF-α) plays a pivotal role in the immune response and mediates inflammation by its receptors (TNF-RI and TNF-RII), as observed during rejection episodes and impaired graft function after liver transplantation. TNF-α and its receptors were analysed by an ELISA technique in serum samples from 77 consecutive liver transplantations in 63 patients. Samples were collected preoperatively from donors and recipients and then daily in the first two postoperative weeks. Peak levels of TNF-α and its soluble receptors (sTNF-RI and sTNF-RII) in the first and second postoperative week correlated with the extent of reperfusion injury. Impaired graft functions correlated with high sTNF-RI levels preoperatively (> 5 ng/ml, p = 0.01) and in the postoperative period (> 16 ng/ml, p = 0.02). Significantly increased TNF-α (> 25 pg/ml, p = 0.009) and sTNF-RI levels (> 5 ng/ml, p = 0.05) were found in donors of grafts with a high rejection risk. Elevated levels of TNF-α in the postoperative period correlated with an increased rejection risk (> 90 pg/ml, p = 0.02). The activity of the immune system with high concentrations of TNF-α and its receptors both in the recipient and the transplant donor seems to play an essential role in allograft development.

RANTES in the postoperative course after liver transplantation

Abstract RANTES (regulated upon activation, normal T‐cell expressed and secreted), an inflammatory cytokine, promotes accumulation and activation of leukocytes. In 67 liver transplantations, systemic concentrations of RANTES were correlated to graft survival and incidence of rejection. RANTES levels either increased to highly elevated levels at day 14 (84 ± 64 ng/ml; group 1; n = 43) or remained within the limit of healthy controls (19 ± 11 ng/ml at day 14; group 2; n = 24). The 100‐day graft function rate was 0.91 in group 1 and 0.63 in group 2 (P = 0.002). The risk ratio for rejection during the first 100 days was increased 2.2‐fold in group 2 compared to group 1 (P = 0.02). High postoperative release of RANTES after liver transplantation, a beneficial factor, may reflect a general systemic immunological activation. It can be concluded that high early systemic RANTES levels may play a role in immunological recognition leading to a tolerance of the liver graft.

Endotoxemia in organ donors: graft function following liver transplantation.

Abstract Translocation of endotoxin (LPS) to the portal‐venous system is produced by multiple factors. In the case of normal liver function, LPS is rapidly cleared from the portal blood by Kupffer cells; in impaired liver function, LPS can reach the systemic circulation. The objective of this study was to investigate whether elevated donor endotoxin levels affect graft function in the recipient. LPS levels in donor plasma were measured in 14 consecutive liver transplantations. Grafts with donor LPS levels ≤ 12 pg/ml had a function probability of 100% after 600 days (n = 10). LPS concentrations of > 12 pg/ml in donor plasma led to loss of function in 75% of the liver grafts (n = 4; P = 0.003; Wilcoxon). Elevated LPS values in donor plasma seem to impair the prognosis of the grafts and could predict poor graft function as early as at the time of brain death.

Release of TNF-α from lipopolysaccharide (LPS)-stimulated Kupffer cells in serum- and nutrient-free medium

In monocytes/macrophages LPS stimulation occurs by the binding of LPS and the serum component LPS-binding protein (LBP) to CD14. This study was conducted to investigate whether this mechanism also occurs in Kupffer cells. Rat Kupffer cells were stimulated for up to 8 h by LPS (0, 100 ng/ml, 10 μg/ml) in RPMI medium or in nutrient-free Krebs–Henseleit (KH) buffer. Some incubations were performed without serum, while in others serum was provided. TNF-α concentrations of the supernatants were measured by ELISA. LPS stimulation of Kupffer cells yielded the following results. In KH without any additives a considerable amount of TNF-α was released. Incubation in RPMI without serum caused twice as much TNF-α to be released as when KH was used. The addition of autologous serum to RPMI did not increase TNF-α response. These results provide evidence that a substantial part of TNF-α release by LPS-stimulated Kupffer cells occurs in a serum- and thus LBP-independent way.

The quality of a liver graft

Abstract Because transplantation success is influenced by the quality of the graft, the objective of this study was to find parameters to evaluate transplant livers in the recipient centre. In 64 liver grafts, the venous effluates of a portal back‐table flush were investigated for various parameters. Amongst them, glutathione S‐transferase (GST), glutamate dehydrogenase (GLDH) and the leucocyte count were found superior in predicting graft survival. Using the combination of these parameters, 100‐day graft survival of between 95% (all parameters positive) and 0% (all parameters negative) was predicted. We concluded that good liver grafts are characterized by a low width of injury (cytosolic component: GST), a low depth of injury (mitochondrial component: GLDH), as well as by a potential to induce tolerance (passenger leucocytes). Perfusate analysis seems to be a valuable tool to recognize problematic grafts in advance and to quantify the “graft factor” in considerations concerning quality control.

The Postoperative Course of γ-Glutamyl Transpeptidase – a Marker of Cytomegalovirus (CMV) Replication Risk?

Abstract Cytomegalovirus (CMV) infection is a common complication in the postoperative course of liver transplantation. In order to start early prophylactic therapy, but to avoid unnecessary treatment, or expensive screening, a desirable goal in post-transplant monitoring is to find appropriate markers in standard laboratory diagnostics. In the present study, the results of a 6-week CMV replication monitoring schedule by the pp65 antigenemia assay in 100 liver graft recipients were included. The activities of transaminases, glutamate dehydrogenase and γ-glutamyl transpeptidase (γ-GT) were measured by routine laboratory methods. In contrast to the transaminases, the serum activity of γ-GT increased during the first postoperative week. The maximum levels were 246 ± 211 U/l in patients without (n= 46) and 140 ± 89 U/l in patients with early CMV replication (n = 54; p = 0.02). Patients with γ-GT levels below 200 U/l on the 5th postoperative day (n = 72) had a CMV replication risk of 65%, whereas those patients with γ-GT levels above this threshold had a risk of 30% (n = 28; p = 0.0007; relative risk = 2.9). These findings provide a routinely usable marker for the identification of patients at an increased risk of CMV replication. It can be considered that these phenomena may be caused by an additional immunosuppressive effect of the CMV virus.