Association of pre- and early post-transplant serum amino acids and metabolites of amino acids and liver transplant outcome.

The aim of the present study was to investigate association of serum amino (AA) acids and metabolites of AAs with post-transplant outcome in liver transplant recipients. Eighty-nine patients with end-stage liver diseases and available pre- and early post-transplant serum were characterised as patients with (GI) and without one-year mortality (GII) and patients with and without early graft dysfunction (EAD). A panel of pre- and early post-transplant serum levels of AAs and early and metabolites of tryptophan were measured using tandem mass spectrometry. Patient groups had significantly higher pre-transplant serum levels of phenylalanine, tryptophan, and tryptophan metabolites than healthy controls (for all p<0.001). Pre-transplant serum levels of all these parameters were significantly higher in GI than in GII (for all p<0.001). GI had a higher MELD score and re-transplantation number than GII (p≤0.005 for both investigations). Serum bilirubin on day 5 and serum phenylalanine on day 10 post-transplant were associated parameters of mortality, whereas day 1post-transplant phenylalanine and kynurenine and female gender were associated parameters of EAD. Our results indicate that pre- and early post-transplant levels of phenylalanine, tryptophan and metabolites of tryptophan are increased in patients and are associated with EAD and one-year mortality in liver transplant recipients.

LPS from bovine serum albumin drives TNF-α release during ex-vivo placenta perfusion experiments, contaminates the perfusion system but can be effectively removed by oxidative cleaning.

INTRODUCTION: The dual ex-vivo perfusion of human placental tissue is useful to study inflammatory pathways. We found significant TNF-α release in negative controls similar in concentration to lipopolysaccharide (LPS) stimulated placentas. The aim of the current study was to (i) identify sources driving TNF-α release and (ii) develop an approach to control for it. METHOD: (i) To determine sources leading to TNF-α release, solutions frequently circulated through the perfusion system and perfusion media with different bovine serum albumin (BSA) quality were exposed to mouse macrophage cell lines (RAW264.7) and subsequently measured for TNF-α expression. (ii) To assess memory effects and validate cleaning procedures, sham perfusion experiments were conducted either in the presence or absence of exogenous LPS, in new tubing that was contaminated, cleaned and analyzed for the effectiveness of LPS removal. Oxidative and acid-base cleaning were tested for their effectiveness to reduce LPS contamination. RESULTS: TNF-α release, observed in negative control experiments, was attributed to the use of LPS-contaminated BSA as well as inadequate cleaning of the perfusion system. Once introduced in the perfusion system, LPS accumulated and created a memory effect. Oxidative but not acid-base depyrogenation effectively reduced LPS levels to concentrations that were in accordance with FDA guidelines (<0.5 EU/mL) for medical equipment redeemed appropriate for re-use. DISCUSSION: LPS contamination of the placenta perfusion model could have confounding effects on experimental outcomes leading to misinterpretation of data. To circumvent LPS contamination LPS-free BSA and oxidative depyrogenation cleaning techniques should be implemented in future placental perfusion studies.

Immunological mechanisms to establish embryo tolerance in early bovine pregnancy.

A well-balanced immunological interaction between mother and the semi-allogenic embryo is of particular importance. The objective of the present study was to analyse mechanisms of immune tolerance in bovine pregnancy during peri-implantation. Simmental heifers inseminated with either cryopreserved spermatozoa or seminal plasma were killed 12, 15 or 18 days after oestrus. Uteri were flushed for the recovery of conceptuses and the ipsilateral intercaruncular endometrium was sampled for gene expression analysis. Indoleamine 2,3-dioxygenase (IDO) mRNA, coding for the initial enzyme of the kynurenine pathway, was 18-fold (P < 0.001) more abundant in the endometrium of Day 18 pregnant v. non-pregnant animals. Tandem mass spectrometry revealed a decrease of endometrial l-tryptophan (P = 0.0008), but an increase of l-kynurenine concentration (P = 0.005) from Day 12 to Day 18, suggesting increasing IDO activity (P < 0.03). An in vitro coculture model of endometrial cells showed an induction of IDO expression following interferon-τ exposure primarily in stroma cells, which was confirmed by in situ hybridisation localising IDO mRNA mainly in deep stroma cells. Immunohistochemical analysis revealed fewer CD45-positive leucocytes in the zona basalis of pregnant animals. Elevated IDO activity may reduce the presence of leucocytes in the pregnant endometrium, providing a possible mechanism for protecting the semi-allogenic conceptus from maternal rejection.

Excessive tryptophan catabolism along the kynurenine pathway precedes ongoing sepsis in critically ill patients.

It has recently been shown that an increased plasma level of the tryptophan catabolite kynurenine is an early indicator for the development of sepsis in major trauma patients. We examined the predictive value of kynurenine pathway activity for ongoing sepsis in patients being admitted to a surgical intensive care unit for different reasons. In addition, we asked whether an accumulation of kynurenines in patients' plasma depends on reduced renal clearance. We conducted a prospective observational study including 100 consecutive patients and monitored laboratory variables, physiological and adverse events, sepsis and outcome. Using tandem mass spectrometry, we quantified the five indoleamines tryptophan, serotonin (5-HT), kynurenine, quinolinic acid and kynurenic acid at baseline and twice a week during the intensive care unit stay. Among the patients enrolled, 50 did not develop sepsis in the intensive care unit (non-septic), 18 patients did not have sepsis at baseline but developed sepsis later on (pre-septic) and 32 patients already fulfilled the criteria of severe sepsis and septic shock at baseline (septic). In general, non-septic critically ill patients showed activation of the kynurenine pathway, but septic shock coincided with an exacerbation of kynurenine pathway activity even in the absence of renal failure. Importantly, plasma concentrations of quinolinic acid (area under the curve 0.832 [95% confidence interval 0.710 to 0.954]) and the Quin/Trp ratio (area under the curve 0.835 [95% confidence interval; 0.719 to 0.952]) showed the best discrimination between non-septic and pre-septic patients at baseline. These findings open new avenues for further investigations on the pathophysiology of sepsis.