Normothermic extracorporeal liver perfusion for donation after cardiac death (DCD) livers.

BACKGROUND: The susceptibility of extended criteria livers to ischemia reperfusion injury is a major obstacle in organ cold preservation. Normothermic extracorporeal liver perfusion (NELP) has been investigated to reduce ischemic damage, restore physiologic function, and assess viability of the liver prior to transplant. The goal of this study is to compare physiological parameters of livers maintained continuously on NELP to those preserved in cold solution. METHODS: Livers from 9 female landrace pigs were subjected to either 20 minutes (W20-NELP), 40 minutes (W40-NELP), or 60 minutes (W60-NELP) of warm ischemia followed by 6 hours of NELP followed by a 2-hour NELP evaluation phase. This was compared with 3 livers subjected to 40 minutes of warm ischemia time followed by 6 hours of cold storage (W40-Cold) and a 2-hour NELP evaluation phase. Groups were compared with the 2-way analysis of variance test. RESULTS: NELP stabilized transaminases accompanied by significant improvement in bile production and decline in lactate and INR values in all W-NELP groups. Histologic analysis demonstrated significant improvement from 0 hour (mild-to-moderate sinusoidal dilation and zone 3 necrosis) to the end of the NELP run (minimal necrosis and mild IRI). Comparison of W40-NELP and W40-Cold revealed greater bile production and oxygen extraction ratio in W40-NELP. In contrast, markers of cellular and functional damage were increased in the W40-Cold group. CONCLUSION: NELP improves metabolic and functional parameters of livers with either short or extended warm ischemia times compared with livers subjected to comparable cold ischemia times.

Ischemia-reperfusion injury in rat steatotic liver is dependent on NFκB P65 activation.

BACKGROUND: Steatotic liver grafts tolerate ischemia-reperfusion (I/R) injury poorly, contributing to increased primary graft nonfunction following transplantation. Activation of nuclear factor kappa-B (NFκB) following I/R injury plays a crucial role in activation of pro-inflammatory responses leading to injury. METHODS: We evaluated the role of NFκB in steatotic liver injury by using an orthotopic liver transplant (OLT) model in Zucker rats (lean to lean or obese to lean) to define the mechanisms of steatotic liver injury. Obese donors were treated with bortezomib to assess the role of NF-κB in steatotic liver I/R injury. Hepatic levels of NF-κB and pro-inflammatory cytokines were analyzed by ELISA. Serum transaminase levels and histopathological analysis were performed to assess associated graft injury. RESULTS: I/R injury in steatotic liver results in significant increases in activation of NF-κB (40%, p<0.003), specifically the p65 subunit following transplantation. Steatotic donor pretreatment with proteasome inhibitor bortezomib (0.1mg/kg) resulted in significant reduction in levels of activated NF-κB (0.58±0.18 vs. 1.37±0.06O.D./min/10 µg protein, p<0.003). Bortezomib treatment also reduced expression of pro-inflammatory cytokines MIP-2 compared with control treated steatotic and lean liver transplants respectively (106±17.5 vs. 443.3±49.9 vs. 176±10.6 pg/mL, p=0.02), TNF-α (223.8±29.9 vs. 518.5±66.5 vs. 264.5±30.1 pg/2 µg protein, p=0.003) and IL-1ß (6.0±0.91 vs. 19.8±5.2 vs. 5±1.7 pg/10 µg protein, p=0.02) along with a significant reduction in ALT levels (715±71 vs. 3712.5±437.5 vs. 606±286 U/L, p=0.01). CONCLUSION: These results suggest that I/R injury in steatotic liver transplantation are associated with exaggerated activation of NFκB subunit p65, leading to an inflammatory mechanism of reperfusion injury and necrosis. Proteasome inhibition in steatotic liver donor reduces NFκB p65 activation and inflammatory I/R injury, improving transplant outcomes of steatotic grafts in a rat model.

Glutathione, lactobionate, and histidine: cryptic inhibitors of matrix metalloproteinases contained in University of Wisconsin and histidine/tryptophan/ketoglutarate liver preservation solutions.

UW solution and HTK solution are both used for cold preservation of liver allografts. Although they are about equally effective, their compositions are very different, and they were formulated using different rationales. The authors recently showed an important role for MMPs in liver preservation injury and consequently postulated that these preservation solutions contain cryptic inhibitors of MMP activity. To determine this possibility, the ability of these solutions to inhibit MMP activity was studied. The source of MMP2 and MMP9 was human liver effluents obtained at the time of liver transplantation or commercially available human recombinant MMP2 and MMP9. MMP2 and MMP9 showed gelatinolytic activity at 37 degrees C and also at 4 degrees C, although activity at 4 degrees C was reduced. Activity was inhibited by University of Wisconsin (UW) and Histidine/Tryptophan/ Ketoglutarate (HTK) solutions. Examination of individual ingredients disclosed that reduced glutathione (GSH) and lactobionate in UW solution and histidine in HTK solution were the cryptic inhibitors. HTK solution was a more effective inhibitor than UW solution. GSH inhibited the activity of both enzymes, but was a much more effective inhibitor of MMP9 than MMP2. Oxidized glutathione(GSSG) was a much less effective inhibitor of the enzymes. The inhibitor constants (K(i)) of GSH for MMP2 and MMP9 were 34 micromol/L and 3 micromol/L, respectively. The authors conclude that MMP inhibition is a cryptic property of both commonly used liver preservation solutions and contributes importantly to their action. Furthermore, GSH appears to be an effective inhibitor of gelatinases at concentrations at which it is normally present in extracellular fluid.

Evidence that actin disassembly is a requirement for matrix metalloproteinase secretion by sinusoidal endothelial cells during cold preservation in the rat.

Cold preservation induces the secretion of matrix metalloproteinases (MMPs) by hepatic sinusoidal endothelial cells (SECs). These enzymes are important mediators of cold preservation injury. The purpose of this study was to determine if low temperature caused actin disassembly in SECs and whether actin disassembly was required for secretion of MMPs under these conditions. To establish the basis of interpreting the effect of low temperature, isolated SECs were exposed to cytochalasin B with or without pretreatment with phalloidin. Cytochalasin B produced actin disassembly and resulted in the secretion of MMPs. Both were retarded by phalloidin pretreatment. Low temperature (4 degrees C) also induced actin disassembly and MMP secretion and pretreatment with phalloidin again retarded actin disassembly and MMP secretion. Cycloheximide had no effect on these results. Actin disassembly began with 30 minutes of exposure of isolated SECs to cold and reached a final state at 8 hours, at which time no actin stress fibers were visible, and the normally fusiform SECs were fully rounded. Increased MMP activity in the supernatant was also present at 30 minutes and continued to rise sharply in the first hour; thereafter the rate of rise diminished. The study shows that secretion of MMPs during cold preservation is dependent on the induction of actin disassembly by low temperature. The rapid appearance of increased MMP activity after exposure to cold and the studies using cycloheximide indicate that the MMPs originate from preformed MMPs rather than newly synthesized MMPs.

Cholesterol microcrystals associated with concanavalin A-binding glycoproteins contribute artifactually to nucleating activity assays.

Concanavalin A (Con A) affinity chromatography is the standard procedure to separate cholesterol nucleating biliary proteins from lipids and pigment. We observed that even after extensive washing following application of bile, lipid contaminants remain. We have determined the contribution of lipid contamination to cholesterol nucleation and assessed a modified procedure to remove lipids from the column. Human gallbladder bile was spiked with [3H]cholesterol and [14C]phospholipid and applied to two sets of Con A-Sepharose columns. One column was washed in the usual manner with Tris-HCl buffer and the other with buffer containing 10 mM taurocholate prior to eluting bound glycoproteins with alpha-D-methylmannopyranoside. Eluted proteins were added to heated abnormal bile at a final concentration of 250 micrograms/ml to study the effect on cholesterol nucleation. A separate aliquot (20 microliter) of the protein solutions was counted for radioactivity. Cholesterol nucleating activity was less in samples from columns washed with 10 mM taurocholate than in samples from columns not washed with the bile salt. Lipid radioactivity was associated with Con A-binding proteins prepared without taurocholate, but not in those prepared with taurocholate wash. Light microscopy revealed the presence of cholesterol microcrystals and vesicles in some Con A-binding protein preparations prepared without a taurocholate wash. However, pellets from ultracentrifuged Con A preparations prepared without a bile salt wash revealed cholesterol crystals in all samples (n = 6). Washing with taurocholate did not affect recovery of protein mass and appearance of bands on SDS-PAGE gel showed an identical pattern in the two groups. This modified procedure to prepare potential nucleating proteins from gallbladder bile should eliminate erroneous results that may arise due to lipid contamination.

A rapid, simple high capacity cholesterol crystal growth assay.

Cholesterol crystal "nucleation time", more recently referred to as cholesterol crystal observation time, is defined as the first appearance of cholesterol crystals from isotropic crystal-free biles on light microscopy. This test is used to assess the potency of nucleating agents. Crystal appearance has conventionally been determined by polarizing light microscopy and crystal growth by counting the number of crystals. In this study we adapted a spectrophotometric method to a microtiter plate reader to generate cholesterol crystal growth curves. Model biles were prepared with a cholesterol saturation of 1.2 to 1.3 and total lipid concentration of 10.7 g/dl (taurocholate, 125 mM; cholesterol, 16.8-18.4 mM; phospholipid, 43 mM). Pronucleating IgM samples were used to establish and validate the assay. Cholesterol crystal growth curves were generated by reading absorption at 630 nm daily on a Dynatech microplate reader. Results were correlated to cholesterol crystal counts as determined by polarizing light microscopy. Standard curves generated from absorbencies of known masses of cholesterol crystals were used to quantify the mass of cholesterol crystals formed over the observation period. The assay was applied to known pronucleating biliary immunoglobulins. Results obtained were similar to our previous report that biliary IgM is more potent than biliary IgG. We conclude that using microplates and a microtiter plate reader provides a rapid high capacity method for detecting cholesterol crystal growth to assess potential nucleating agents in nucleation assays.

Diffusion of substances into human cholesterol gallstones.

BACKGROUND/AIMS: The possibility that substances penetrate gallstones and accumulate after stones have formed has not been examined. The specific aims of this study were to determine whether cholesterol gallstones are permeable and, if so, the effect of molecular weight on permeability. METHODS: Cholesterol gallstones from patients with multiple stones were collected during surgery and incubated in fluorescein solution or in solutions of fluoresceinated albumin or immunoglobulin (Ig) G. To determine egress from the stones, some stones were removed from the fluoresceinated solution after incubation and placed in bicarbonate buffer. The total area of the stone and the area of dye that had diffused into the calculi were calculated. To determine mass of penetrating IgG, stones were powdered after incubation, and IgG was measured by an enzyme-linked immunosorbent assay. RESULTS: All substances penetrated stones. Although all compounds tested diffused back out of the stones when they were replaced in buffer, proteins did so more slowly than fluorescein. CONCLUSIONS: Substances of different molecular weights can diffuse into and out of cholesterol gallstones. These findings must be taken into account when considering the role of substances contained in stones on stone formation and growth.

Effect of human biliary immunoglobulins on the nucleation of cholesterol.

We have previously identified that either biliary immunoglobulin IgA or IgM is a pronucleating protein which can accelerate the precipitation of cholesterol from bile. In this study we purified the biliary immunoglobulins (IgA, IgG, and IgM) to homogeneity by affinity chromatography to investigate the relative cholesterol nucleating potency of each immunoglobulin. Each immunoglobulin was added to slow nucleating heated abnormal biles in a dose-response manner to give a final concentration of protein in the range of 62.5-625 micrograms/ml bile. Cholesterol-nucleating activity was measured by noting the first day of cholesterol crystal formation as well as the number of crystals formed over the observation period. Biliary IgM and IgG appear to be more potent pronucleators than IgA. Isolated serum IgM from patients with Waldenstrom's macroglobulinemia as well as serum IgG from patients with and without cholesterol gallstones were shown to have pronucleating activity and acted in a dose-response manner. Commercial IgG unlike commercial IgM retains nucleating activity. The concentration of biliary immunoglobulins was measured by an enzyme-linked immunoassay (ELISA) in the gallbladder bile of patients with and without cholesterol gallstones. Biliary IgG concentrations in bile were higher in cholesterol gallstones patients than in pigmented gallstone patients and controls. We conclude that immunoglobulins particularly IgG and IgM are important pronucleating proteins and could play a role in the pathogenesis of cholesterol gallstones.

Immunoglobulins as nucleating proteins in the gallbladder bile of patients with cholesterol gallstones.

The gallbladder bile of patients with cholesterol gallstones contains pronucleating proteins which accelerate precipitation of cholesterol crystals from bile. In this study we have improved the purification procedure developed earlier for these nucleating proteins and have now identified the nature of these proteins. Gallbladder bile from patients with cholesterol gallstones was applied to concanavalin A affinity columns. The ConA-binding glycoprotein fractions containing the nucleating proteins were then separated by FPLC (fast protein liquid chromatography) using a Superose 12 gel filtration column. Nucleating activity was detected in the high molecular weight (FPLC-1) as well as in the low molecular weight fractions (FPLC-3). Investigation of the high molecular weight fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by electroelution and amino acid sequencing suggested that these proteins were immunoglobulins. Immunostaining of Western blots with specific monoclonal antibodies identified the presence of immunoglobulin (Ig) M and IgA in the FPLC-1 fraction. These immunoglobulins were further purified by affinity chromatography employing an antibody exchanger (ABx) column which specifically binds immunoglobulins. There was no reduction in the cholesterol nucleating activity in the Abx-bound fraction compared to FPLC-1. Additional studies showed that the FPLC-1 fraction was significantly more potent than the ConA glycoproteins from either rapid and slow nucleating biles. Also the number of crystals formed was significantly greater in the FPLC-1 fraction isolated from cholesterol gallstone biles than from the FPLC-1 fraction from control patient biles. Commercially obtained IgM and IgA had no effect on nucleation, but IgM isolated from the serum of patients with Waldenstrom's macroglobulinemia did accelerate the nucleation of cholesterol. We conclude that the IgM and possibly IgA are pronucleating proteins and may be important in the pathogenesis of cholesterol gallstones in man.

Fluorometric assay of protein in native human bile.

Proteins in human native bile samples were determined by a fluorometric assay. Results were compared to biliary proteins quantified by the Lowry and Bradford techniques. The mean protein concentrations in bile as determined by the Lowry, Bradford and fluorometric assays were respectively 7.26 +/- 4.52 (SD), 2.9 +/- 1.42, and 2.12 +/- 1.28 mg/ml (n = 27). Bilirubin was shown to significantly interfere with the Lowry and Bradford assays but not the fluorometric assay. Bile salts remaining in the trichloroacetic acid (TCA) precipitate did not interfere with the fluorometric assay. No cholesterol or phospholipid could be detected in the TCA preparation prior to protein analysis. Proteolytic digestion of proteins in native bile was shown to occur at 37 degrees C and to a lesser extent at 22 degrees C. The fluorometric protein assay is an easy and accurate method to quantitate proteins in native human bile.

Total protein output during rapid reduction of bile salt secretion rates in man.

An investigation was undertaken to study the effect of bile salt secretion on total biliary protein secretion in man. Bile was collected in eight patients from a tube in the bile duct. Collection was started after a meal and continued for six hours, in order to obtain bile salt secretion rates over the entire physiological range. Total protein secretion rates did not vary with change in bile salt secretion or bile flow. The protein pattern assessed by SDS-PAGE did not vary with bile salt secretion. The results indicate that bile salt secretion has little influence on biliary protein secretion under these conditions in man. Changes in bile salt secretion were associated with linear change in bile flow, but there was no relationship between bile flow and protein secretion rates. This argues against convective sieving of plasma proteins into bile.