Role of cytokine gene polymorphisms in acute rejection and renal impairment after liver transplantation.

Although immunosuppressive regimens are effective, rejection occurs in up to 50% of patients after orthotopic liver transplantation (OLT), and there is concern about side effects from long-term therapy. Knowledge of clinical and immunogenetic variables may allow tailoring of immunosuppressive therapy to patients according to their potential risks. We studied the association between transforming growth factor-beta, interleukin-10, and tumor necrosis factor alpha (TNF-alpha) gene polymorphisms and graft rejection and renal impairment in 121 white liver transplant recipients. Clinical variables were collected retrospectively, and creatinine clearance was estimated using the formula of Cockcroft and Gault. Biallelic polymorphisms were detected using polymerase chain reaction-based methods. Thirty-seven of 121 patients (30.6%) developed at least 1 episode of rejection. Multivariate analysis showed that Child-Pugh score (P =.001), immune-mediated liver disease (P =.018), normal pre-OLT creatinine clearance (P =.037), and fewer HLA class 1 mismatches (P =.038) were independently associated with rejection. Renal impairment occurred in 80% of patients and was moderate or severe in 39%. Clinical variables independently associated with renal impairment were female sex (P =.001), pre-OLT renal dysfunction (P =.0001), and a diagnosis of viral hepatitis (P =.0008). There was a significant difference in the frequency of TNF-alpha-308 alleles among the primary liver diseases. After adjustment for potential confounders and a Bonferroni correction, the association between the TNF-alpha-308 polymorphism and graft rejection approached significance (P =.06). Recipient cytokine genotypes do not have a major independent role in graft rejection or renal impairment after OLT. Additional studies of immunogenetic factors require analysis of large numbers of patients with appropriate phenotypic information to avoid population stratification, which may lead to inappropriate conclusions.

Surgical complications and long-term outcome in pediatric liver transplantation.

BACKGROUND/AIMS: Liver transplantation has been widely accepted for the treatment of children with end-stage liver disease over the last 10 years particularly with the advent of reduced-size liver transplant technique. This study reviewed the perioperative and long-term results in the pediatric program of the Queensland Liver Transplant Service, Brisbane, Australia. METHODOLOGY: Retrospective analysis was performed in 153 children who received 176 liver grafts between 1985 and 1995, including 109 (62%) reduced-size and 67 (38%) whole liver grafts. Median follow-up period was 5.3 years. RESULTS: One-, 5-, and 10-year patient and graft survival rates were 82% and 74%, 75% and 63%, and 70% and 60%, respectively. Normal physical and intellectual development was observed in 98% of survivors. There were no significant differences in patient or graft survival rates between transplants using reduced-size and whole liver grafts. Portal vein thrombosis was the most common vascular complication, occurring in 8%. Hepatic artery thrombosis occurred in 7%, including 11% of children less than 1 year old and 8% of those under 10 kg. Biliary complication was found in 16% and posttransplant gastrointestinal perforation in 19%. CONCLUSIONS: Liver transplantation has the potential to cure and allow development in children with end-stage liver disease.

Detection of male DNA in the liver of female patients with primary biliary cirrhosis.

BACKGROUND/AIMS: Primary biliary cirrhosis is a chronic cholestatic liver disease characterized by progressive inflammatory destruction of bile ducts, with eventual hepatic fibrosis and cirrhosis. Since primary biliary cirrhosis affects predominantly middle-aged women and has pathological similarities to hepatic graft-versus-host-disease, we investigated whether fetal cell microchimerism might be involved in the development of this disease. METHODS: The presence of Y-chromosome-specific sequences was analyzed by polymerase chain reaction using peripheral blood mononuclear cells from women with primary biliary cirrhosis (n=18) and healthy (control) women (n=18), and by in situ hybridization of liver biopsy sections from women with primary biliary cirrhosis (n=19) and women with chronic hepatitis C or alcoholic liver disease (n=20). RESULTS: Male cells were detected in liver biopsy specimens of 8 of 19 patients (42%) with primary biliary cirrhosis. Y-chromosome-containing cells were not seen in any of the liver biopsy specimens from women with chronic hepatitis C or alcoholic liver disease. Male cells were detected in peripheral blood mononuclear cells from one healthy control at a level of 1 male cell per 10(6) female cells, but were not detected in peripheral blood mononuclear cells of women with primary biliary cirrhosis. CONCLUSIONS: The presence of male cells in the liver of women with primary biliary cirrhosis raises the possibility that fetal cell microchimerism may be involved in the pathogenesis of this chronic liver disease.

Confirmation of the role of the Mayo Risk Score as a predictor of resource utilization after orthotopic liver transplantation for primary biliary cirrhosis.

Resource utilization is an important consideration when patients are selected for orthotopic liver transplantation (OLT). The Mayo Risk Score has been proposed to help predict optimum time for OLT. We assessed the relation between Mayo risk score, Child-Pugh score, and resource utilization and outcome after OLT for primary biliary cirrhosis. The mean Mayo risk score was greater in patients who died than in the survivors (8.6 +/- 1.4 v 7.1 +/- 1.8; P <.05). There was a positive correlation between Mayo risk score and the 4 resource variables studied (intraoperative blood requirements, time ventilated, and duration of intensive care unit and hospital stays). Patients with a Mayo risk score greater than 7.8 used almost twice the resources of patients with a risk score less than 7.8. A positive correlation also existed between Child-Pugh score and duration of hospital stay. The mean Child-Pugh score in patients who died was greater than that in survivors (10.7 +/- 2.0 v 8.5 +/- 2.8, P =.03). This study confirms that Mayo Risk score is an important predictor of resource utilization and outcome after OLT.

Liver transplantation for familial amyloidotic polyneuropathy in Australia.

Familial amyloidotic polyneuropathy type 1 (FAP-1) is a type of systemic amyloidosis caused by mutant transthyretin (mTTR) that is mainly produced in the liver. Most patients have progressive peripheral and autonomic neuropathy. Ten patients with FAP underwent orthotopic liver transplantation (OLT) at the Queensland Liver Transplant Service (Princess Alexandra Hospital, Brisbane, Australia). Nine patients are still alive, and one patient died of cardiac failure 10 days after OLT. Some symptoms of FAP were alleviated in some of the patients. OLT seems to be a worthwhile treatment for FAP, because it halts the progression of symptoms and achieves improvement in some patients.

Liver transplantation in Japanese and Australian/New Zealand children with biliary atresia: a 10-year comparative study.

OBJECTIVE: To compare Japanese with Australian/New Zealand (ANZ) children with biliary atresia who were treated by liver transplantation, and evaluate the indications for and timing of transplantation. DESIGN: Retrospective study. SETTING: Queensland Liver Transplant Service (QLTS), Australia. SUBJECTS: 43 Japanese and 30 ANZ children with biliary atresia who required transplantation between 1985 and 1992. INTERVENTIONS: The 43 Japanese children had 52 transplants, and the 30 ANZ children had 33. MAIN OUTCOME MEASURES: Morbidity, mortality, and long term survival. RESULTS: The Japanese children had significantly lower serum albumin concentrations than the ANZ children preoperatively (mean (SD) 32 (7) g/L compared with 37 (5), p<0.05). The actuarial survival at 7 years of the ANZ children was significantly higher than that of the Japanese children (79% compared with 49%, p<0.05). There were 24 deaths (17 Japanese, 40%, and 7 ANZ, 23%); 2 of the ANZ and 7 of the Japanese children died more than a year after transplantation. All 26 children who were well-nourished at the time of transplantation defined as a Z-score (weight or height minus mean weight or height for age, sex, and race, divided by the SD) of -1 or more were alive at 1 month compared with 11 of the 47 poorly-nourished children (Z-score <-1). Survival among the Japanese declined after 1 year, and there was no association with Z-scores. Overall, Z-scores for weight improved significantly after transplantation, whereas those for height improved a little, but not significantly so. Japanese children were significantly shorter than ANZ children, and their Z-scores for height did not improve after transplantation. CONCLUSION: liver transplantation should be done as soon as possible for children with biliary atresia to maximise survival and growth.

Role of donor leukocyte chimerism in establishing the etiology of neutropenia after liver transplantation.

BACKGROUND: The quantitation of donor leukocyte chimerism may aid in establishing the etiology of neutropenia after liver transplantation. METHODS: The incidence and clinical and laboratory characteristics of severe neutropenia were studied in adults who have undergone liver transplantation at our institution over the last 4 years. RESULTS: Severe neutropenia developed in 5 of 156 patients (3%). The clinical and pathological features were nonspecific. In two patients with a delayed diagnosis of graft-versus-host disease (GVHD), donor leukocytes comprised > or = 50% of the circulating peripheral blood mononuclear cells. In a third patient, an earlier diagnosis of GVHD was suspected on the basis of a donor leukocyte count of 3-10% in the peripheral blood. In contrast, donor leukocyte chimerism was < or = 0.01% in two patients with probable drug-induced neutropenia CONCLUSIONS: The determination of donor leukocyte chimerism has an important role in the investigation of neutropenia after liver transplantation, allowing early diagnosis and treatment of GVHD.

Clinicopathologic findings of recurrent primary sclerosing cholangitis after orthotopic liver transplantation.

Whether primary sclerosing cholangitis (PSC) occurs after orthotopic liver transplantation is controversial, largely because the pre-transplant diagnosis of PSC is based on nonspecific radiological and histological findings. We reviewed clinical, radiological, and histological records of 53 patients who underwent liver transplantation for PSC between 1985 and 1998. Three patients with patent hepatic arteries and no evidence of chronic rejection had radiological and histological findings that may have been due to recurrent PSC. Bile duct stricturing in these patients proved permanent and progressive and affected both the quality of life and graft survival. The first patient, who is 110 months after transplantation, has had repeated episodes of cholangitis for the last year. The second patient underwent excision of a strictured hepatic duct 45 months after transplantation and was ultimately retransplanted 95 months after initial transplantation. The third patient underwent left hemihepatectomy of an atrophied lobe 50 months after transplantation. Although the patient population assessed in this study is limited, putative recurrent PSC in the allografts has led either to graft loss or to clinically significant hepatobiliary complications of the graft.

3000 head injuries: a prospective study of patients admitted to Brisbane neurosurgical units.

A prospective epidemiological study of 3095 patients with head injury admitted to Brisbane neurosurgical units is presented. Falls were the commonest cause of injury overall (42%) but traffic accidents were the leading cause of severe head injury Glasgow Coma Scale ([GCS] 8 or less) and had a higher mortality (5.6%). Outcome was closely related to GCS, presence or absence and type of skull fracture, computed tomography (CT) scan findings and age. Overall mortality was 4.4%. Mortality for mild head injury (GCS 13-15) was 0.4%, moderately severe head injury (GCS 9-12) 10.5% and severe head injury (GCS 3-8) 34.5%. The poor outcome in old patients who fall and sustain a mild head injury is highlighted. Low risk criteria are identified and recommendations regarding admission and management policies are made.

Human liver transplant perfusate: an abundant source of donor liver-associated leukocytes.

In vitro studies designed to examine the mechanisms of immune tolerance after liver transplantation in humans have been hampered by the difficulty in obtaining sufficient numbers of donor liver-associated leukocytes (LALs). We have investigated whether the ex vivo perfusion of donor livers releases a population of LALs that can be readily retrieved from the waste fluid. The mean number of cells recovered after Ficoll-Hypaque density-gradient separation was 2.6 +/- 0.5 x 10(8) cells, with a viability of 94% +/- 2%. The perfusate lymphocytes comprised mainly T cells (39% +/- 2%) with a very low CD4/CD8 ratio and natural killer (NK) cells (56% +/- 6%) with an increase in the proportion of the CD3-CD56+CD16- subset. The activation marker CD69 was present on the majority of the perfusate lymphocytes. These are the phenotypic characteristics that have been previously reported for lymphocytes isolated from hepatic sinusoids. In mixed lymphocyte reactions, the perfusate cells showed a marked increase in the ability to stimulate allogeneic responder cells, resulting in 353% +/- 78% (P = .003) greater incorporation of [3H]thymidine in responder cells when compared with stimulation by donor peripheral blood mononuclear cells. The results show that large numbers of viable donor lymphocytes can be readily isolated from the liver perfusate solution. These cells have the characteristics of liver-associated lymphocytes with a predominance of activated NK and CD8+ T cells. This population can now be used in in vitro assays to elucidate the influence of donor leukocytes on the development of graft acceptance.

Peripheral blood chimerism following human liver transplantation.

The aim of this study was to prospectively investigate the peak levels and kinetics of donor leucocyte chimerism in human recipients following liver transplantation. The peak levels of chimerism were observed within the first 48 hours following transplantation and ranged from 0.15% to 20% of total peripheral blood mononuclear cells. In all but one patient, who developed graft versus host disease, there was an early peak level of chimerism that declined over time such that donor leukocytes were only intermittently detectable after 3 to 4 weeks. In 8 patients who had no episodes of graft rejection, the peak level of donor leukocyte chimerism ranged from 1.3% to 20% (mean +/- SEM; 5.5% +/- 2.1%). In 3 patients who were treated for episodes of acute graft rejection during the first four postoperative weeks, the peak level of donor leukocyte chimerism ranged from 0.15% to 0.2% (0.18 +/- 0.02, P = .012). The results demonstrate a marked variation in the total number of donor leukocytes detectable in the peripheral blood early after liver transplantation and also, that lower levels of chimerism may be associated with lower rates of initial graft acceptance and a higher incidence of acute rejection.

Endotoxin levels in donors and recipients during orthotopic liver transplantation.

BACKGROUND: The hypothesis being tested in this paper is that endotoxin levels in donors and in recipients during liver transplantation influences postoperative outcome. METHODS: Endotoxin levels in systemic venous and portal venous blood were measured using in 46 adult donors and 44 adult recipients (47 liver transplants) during the period 1992-95. Endotoxin was measured using a modification of the Limulus amoebocyte lysate (LAL) assay. RESULTS: In the donor, systemic endotoxin levels were above normal levels at 10.0 +/- 1.3 pg/mL from the start and rose to 15.8 +/- 2.9 pg/mL after dissection of the hilar structures, but fell to 10.6 +/- 0.8 pg/mL just prior to the removal of the liver (control = 7.8 pg/mL). The mean portal venous endotoxin levels were 18.2 +/- 3.4 pg/mL after dissection of the hilar structures and 12.6 +/- 0.9 pg/mL after cannulation of the portal vein. In the recipients, the highest level in the portal venous blood occurred at the end of the anhepatic phase (46.5 +/- 6.7 pg/mL). The systemic venous samples in the recipients were elevated to start with, but fell rapidly to 19.3 +/- 1.5 pg/mL 24 h postoperatively, and to 13.2 +/- 1.0 pg/mL by day 7. The endotoxin concentrations were higher in recipients who developed complications. CONCLUSIONS: Endotoxin is elevated throughout the recipient transplantation procedure and up to 7 days postoperatively. High levels of endotoxin at induction, the anhepatic phase and at certain time points correlated with patients who developed postoperative complications.

High-performance liquid chromatography-tandem mass spectrometry as a reference for analysis of tacrolimus to assess two immunoassays in patients with liver and renal transplants.

The accuracy and imprecision of three assays used for therapeutic monitoring of tacrolimus were tested using blood-containing weighed-in amounts of the drug, an enzyme-linked immunosorbent assay (ELISA), a microparticle enzyme immunoassay (MEIA I), and a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) assay. Accuracy was acceptable for the HPLC-MS2 assay at all concentrations tested (< 10% deviation) and for the ELISA at 1.0 and 4.0 microg/l. Accuracy was not acceptable for the ELISA at 15.0 and 50.0 microg/l or for the MEIA I at all concentrations tested. Imprecision was acceptable for the HPLC-MS2 assay at all concentrations tested (coefficient of variation < 10%), for the ELISA at 15.0 and 50.0 microg/l, and for the MEIA I at 15.0 and 50.0 microg/l. Imprecision was not acceptable for the ELISA at 1.0 and 4.0 microg/l or for the MEIA I at 1.0 and 4.0 microg/l. This assessment with weighed-in amounts of tacrolimus verified the HPLC-MS2 assay as a reference method. The performance of the two immunoassays with HPLC-MS2 was then compared in the clinical setting using blood from patients with liver (n = 30) and renal (n = 37) transplants. In the liver transplant group (127 samples), the range of tacrolimus concentrations measured by HPLC-MS2, ELISA, and MEIA I was 1.9 to 31.8, 2.1 to 35.0, and less than 0.1 to 36.5 mg/l, respectively. In the renal transplant group (129 samples), the ranges were 1.7 to 26.1, 1.9 to 24.4, and 0.9 to 28.5 microg/l, respectively. Compared with the HPLC-MS2, the ELISA had minimal bias (0.1 to 0.2 microg/l) but unacceptable variability in values (SD > 13%). The MEIA I had unacceptable bias (1.7-1.8 microg/l) and variability (SD > 23%). These data indicated that neither the ELISA nor MEIA I is interchangeable with HPLC-MS2. Moreover, in view of the current trend to reduce the therapeutic dose of tacrolimus, quantitative results using the MEIA I would not be obtainable during therapeutic drug monitoring in some patients in whom effective therapeutic concentrations can be less than 5.0 microg/l.

The use of the lidocaine-monoethylglycinexylidide test in the liver transplant recipient.

The lidocaine-monoethylglycinexylidide (MEGX) test is used to monitor liver function in liver transplant recipients. Serial studies have been undertaken after 155 allografts. The initial MEGX concentration is significantly correlated with the donor MEGX concentration. It is also influenced by the recipient's pretransplant bilirubin concentration, being lowest among patients with very high bilirubin levels. Use of segmental grafts is also accompanied by low MEGX concentrations. The flow-dependent clearance of lidocaine makes it a sensitive indicator of disturbed liver blood flow, with decreased MEGX concentrations occurring in hepatic artery thrombosis and rejection and as a result of cardiac failure and pulmonary effusions. Significant hepatic ischemia resulting in delayed initial function or cholestasis also is associated with low MEGX concentrations. The initial median MEGX concentrations were lowest among patients who required retransplantation or who died within 2 months of allografting.

The management of difficult abdominal closure after pediatric liver transplantation.

Between January 1985 and December 1994, 164 liver transplantations were performed on 141 children. There were 100 reduced-size and 64 whole-liver grafts. Primary closure of the abdominal wound was not possible in 21 patients because of liver size, bowel edema, and distension. Temporary SILASTIC patch closure of the abdominal wound was used. For 16 of the 21 patients, removal of the SILASTIC patch and abdominal wall closure were completed by the seventh postoperative day; for the others, these were accomplished by the end of 2 weeks. The method is recommended when primary wound closure is not possible.

Sensitive, specific quantitative analysis of tacrolimus (FK506) in blood by liquid chromatography-electrospray tandem mass spectrometry.

The capacity of liquid chromatography-tandem mass spectrometry (LC-MS2) to detect and define individual components in a complex mixture has been utilized to develop a quantitative assay of the potent immunosuppressant drug, tacrolimus. Trough blood concentrations were measured in 175 samples obtained over several weeks after liver transplantation from seven patients. The assay was linear over the range of 0.2 to 100 micrograms/L. Imprecision was <8%, and accuracy was 99-101%. The turnaround time for a batch of 20 samples was 2.5 h. No interference from any of the other drugs being administered to the patients was evident. An ELISA also performed on the same samples overestimated the concentrations substantially, as indicated by a plot of the difference between the results for the two methods vs their mean. The favorable characteristics of the LC-MS2 assay, especially its sensitivity and specificity, will facilitate detailed pharmacokinetic studies of tacrolimus, particularly under circumstances in which metabolism is perturbed by either hepatic dysfunction or drug interactions.

Renal glomerular lesions in unselected patients with cirrhosis undergoing orthotopic liver transplantation.

Renal biopsies were obtained from 23 patients at the time of orthotopic liver transplantation. Twelve biopsies showed minor glomerular abnormalities, 2 exhibited IgA nephropathy and one showed mesangiocapillary glomerulonephritis type I. The remaining 8 patients had glomerular lesions diagnosed as hepatic glomerulosclerosis (HGS). Immunofluorescence, available in 6 of the 8 biopsies with HGS, revealed granular deposits of immunoglobulins and complement in glomerular capillary walls and/or the mesangium. IgA was seen in 5 biopsies with HGS, but the staining for this protein was no more intense than that for the other immunoglobulins in 4 of these. Electron microscopy in HGS revealed partial mesangial interposition, hypertophy of mesangial and endothelial cells, granular material in a widened subendothelial space, slender projections of endothelial cytoplasm extending into the subendothelial space, and clusters of vesicles in the mesangium and glomerular capillary walls. These ultrastructural abnormalities have not hitherto been reported as a group of associated pathological changes. The renal biopsies were obtained from patients with advanced hepatic disease not selected because of urinary abnormalities or renal dysfunction. The frequency of lesions in this group of patients therefore probably reflects the true incidence of glomerular lesions in cirrhosis and related conditions. Progressive decline in renal function was not observed in any patient during follow up which ranged from 11 days to 55 mths.