The Medication Level Variability Index (MLVI) Predicts Poor Liver Transplant Outcomes: A Prospective Multi-Site Study.

Nonadherence to immunosuppressant medications is a leading cause of poor long-term outcomes in transplant recipients. The Medication Level Variability Index (MLVI) provides a vehicle for transplant outcome risk-stratification through continuous assessment of adherence. The MALT (Medication Adherence in children who had a Liver Transplant) prospective multi-site study evaluated whether MLVI predicts late acute rejection (LAR). Four hundred pediatric (1-17-year-old) liver transplant recipients were enrolled and followed for 2 years. The a-priori hypothesis was that a higher MLVI predicts LAR. Predefined secondary analyses evaluated other outcomes such as liver enzyme levels, and sensitivity analyses compared adolescents to pre-adolescents. In the primary analysis sample of 379 participants, a higher prerejection MLVI predicted LAR (mean prerejection MLVI with LAR: 2.4 [3.6 standard deviation] versus without LAR, 1.6 [1.1]; p = 0.026). Fifty-three percent of the adolescents with MLVI>2 in year 1 had LAR by the end of year 2, as compared with 6% of those with year 1 MLVI≤2. A higher MLVI was significantly associated with all secondary outcomes. MLVI, a marker of medication adherence that uses clinically derived information, predicts LAR in pediatric liver transplant recipients.

Differing effects of rapamycin or calcineurin inhibitor on T-regulatory cells in pediatric liver and kidney transplant recipients.

In a cross-sectional study, we assessed effects of calcineurin inhibitor (CNI) or rapamycin on T-regulatory (Treg) cells from children with stable liver (n = 53) or kidney (n = 9) allografts several years posttransplant. We analyzed Treg number, phenotype, suppressive function, and methylation at the Treg-specific demethylation region (TSDR) using Tregs and peripheral blood mononuclear cells. Forty-eight patients received CNI (39 as monotherapy) and 12 patients received rapamycin (9 as monotherapy). Treg numbers diminished over time on either regimen, but reached significance only with CNI (r =-0.424, p = 0.017). CNI levels inversely correlated with Treg number (r =-0.371, p = 0.026), and positively correlated with CD127+ expression by Tregs (r = 0.437, p = 0.023). Patients with CNI levels >3.6 ng/mL had weaker Treg function than those with levels <3.6 ng/mL, whereas rapamycin therapy positively correlated with Treg numbers (r = 0.628, p = 0.029) and their expression of CTLA4 (r = 0.726, p = 0.041). Overall, CTLA4 expression, TSDR demethylation and an absence of CD127 were important for Treg suppressive function. We conclude that rapamycin has beneficial effects on Treg biology, whereas long-term and high dose CNI use may impair Treg number, function and phenotype, potentially acting as a barrier to attaining host hyporesponsiveness to an allograft.

Cognitive and academic outcomes after pediatric liver transplantation: Functional Outcomes Group (FOG) results.

This multicenter study examined prevalence of cognitive and academic delays in children following liver transplant (LT). One hundred and forty-four patients ages 5-7 and 2 years post-LT were recruited through the SPLIT consortium and administered the Wechsler Preschool and Primary Scale of Intelligence, 3rd Edition (WPPSI-III), the Bracken Basic Concept Scale, Revised (BBCS-R), and the Wide Range Achievement Test, 4th edition (WRAT-4). Parents and teachers completed the Behavior Rating Inventory of Executive Function (BRIEF). Participants performed significantly below test norms on intelligence quotient (IQ) and achievement measures (Mean WPPSI-III Full Scale IQ = 94.7 ± 13.5; WRAT-4 Reading = 92.7 ± 17.2; WRAT-4 Math = 93.1 ± 15.4; p < 0001). Twenty-six percent of patients (14% expected) had 'mild to moderate' IQ delays (Full Scale IQ = 71-85) and 4% (2% expected) had 'serious' delays (Full Scale IQ ≤ 70; p < 0.0001). Reading and/or math scores were weaker than IQ in 25%, suggesting learning disability, compared to 7% expected by CDC statistics (p < 0.0001). Executive deficits were noted on the BRIEF, especially by teacher report (Global Executive Composite = 58; p < 0.001). Results suggest a higher prevalence of cognitive and academic delays and learning problems in pediatric LT recipients compared to the normal population.

Improving long-term outcomes after liver transplantation in children.

The objective was to review the current state of knowledge and recommend future research directions related to long-term outcomes for pediatric liver transplant recipients. A 1-day Clinical Research Workshop on Improving Long-Term Outcomes for Pediatric Liver Transplant Recipients was held on February 12, 2007, in Washington, DC. The speaker topics were germane to research priorities delineated in the chapters on Pediatric Liver Diseases and on Liver Transplantation in the Trans-NIH Action Plan for Liver Disease Research. Issues that compromise long-term well-being and survival but are amenable to existing and new research efforts were presented and discussed. Areas of research that further enhanced the research priorities in the Action Plan for Liver Disease Research included collection of longitudinal data to define emerging trends of clinical challenges; identification of risk factors associated with long-term immunosuppression complications; development of tolerance-inducing regimens; definition of biomarkers that reflect the level of clinical immunosuppression; development of instruments for the measurement of health wellness; identification of risk factors that impede growth and intellectual development before and after liver transplantation and identification of barriers and facilitators that impact nonadherence and transition of care for adolescents.

Characterization of hepatic epidermal growth factor receptors in the developing rat.

Binding of 125I-labeled epidermal growth factor (EGF) was characterized in basolateral plasma membranes prepared from the livers of 21-day gestation fetuses, 14-day-old sucklings and adult Sprague-Dawley rats using a self-generating Percoll gradient method. The membrane preparations employed have been previously assayed in terms of plasma membrane protein yield, enrichment of various marker enzymes and sodium-dependent bile acid and amino acid transport. 125I-EGF binding was saturable and time and temperature dependent. Equilibrium analyses showed that the suckling period is characterized by a marked decrease in overall hepatic EGF binding capacity (460 +/- 50 fmol/mg protein) compared to either the fetal period (1290 +/- 160 fmol/mg) or to adults of either sex (males = 1540 +/- 230, females 1010 +/- 130 fmol/mg). Treatment of the suckling rat with parenteral EGF resulted in a 78% reduction in the observed binding capacity when assessed 2 h after growth factor administration. Comparison of binding affinities revealed no significant difference between the suckling and adult preparations (Kd = 0.40 +/- 0.03 vs. 0.39 +/- 0.02 nM, respectively); however, both preparations differed significantly from the fetal group which exhibited a decreased affinity of binding with a higher overall dissociation constant (Kd = 0.68 +/- 0.06 nM). Thus, it appears that major ontogenetic changes occur in the rat hepatic ligand/receptor system for epidermal growth factor. These changes are discussed in the context of transitional events in mammalian development such as birth and weaning.

A liver-specific isoform of the betaine/GABA transporter in the rat: cDNA sequence and organ distribution.

We report the cloning of a 2.2 kb cDNA encoding a Na(+)-and Cl- dependent betaine/GABA (gamma-aminobutyric acid) transporter from rat liver poly(A+) RNA. 5'-RACE revealed an additional 355 bases 5' to the 2.2 kb cDNA sequence. Northern analysis demonstrated two (2.2 kb and 2.6 kb) mRNA isoforms in rat liver. Betaine transporter mRNA was also detected in the brain, spleen, lung, and kidney using the 2.2 kb cDNA clone as a probe. Only the 2.6 kb mRNA from the liver hybridized with the 5'-RACE product.

Growth hormone insensitivity associated with elevated circulating growth hormone-binding protein in children with Alagille syndrome and short stature.

The purpose of this study was to assess GH sensitivity in children with Alagille syndrome (syndromic intrahepatic bile duct paucity) by examining their response to short term administration of recombinant human GH (rhGH). Serum levels of insulin-like growth factor-I (IGF-I) were low despite elevated overnight integrated serum GH concentrations. Administration of rhGH (0.05 mg/kg.day for 3 days) to four growth-retarded children with Alagille syndrome did not significantly alter the serum concentrations of IGF-I and insulin, blood urea nitrogen, or urinary calcium excretion. In contrast, circulating IGF-I increased 2-fold in two children with Alagille syndrome and normal stature. In the control group, consisting of seven prepubertal children with GH deficiency, the predicted changes in response to rhGH in serum concentrations of IGF-I and insulin, urea nitrogen, and urinary calcium excretion were observed. Serum GH-binding protein levels, measured by a ligand-mediated immunofunctional assay, were significantly higher in children with Alagille syndrome than in children with cirrhosis or GH deficiency. We conclude that growth-retarded children with Alagille syndrome are insensitive to GH. The growth disturbances and metabolic defects may be due in part to failure to increase IGF-I concentrations in response to GH, implying that growth-retarded children with Alagille syndrome may benefit from IGF-I treatment.

Efficacy of cornstarch therapy in type III glycogen-storage disease.

Type III glycogen-storage disease (GSD-III), due to decreased activity of the glycogen debranching enzyme amylo-1,6 glucosidase, may cause hepatic dysfunction, growth failure, and myopathy. The prevention of hypoglycemia by nocturnal intragastric formula infusion has been shown to enhance growth and improve the metabolic abnormalities associated with GSD-III. Cornstarch therapy was effective in preventing hypoglycemia in a few patients with GSD-III who were previously treated with nocturnal enteral formula infusion, but oral cornstarch had not been evaluated as an initial treatment. We studied three patients with GSD-III who exhibited growth failure, elevated serum aminotransferase concentrations, and asymptomatic hypoglycemia. Cornstarch therapy was associated with maintenance of normoglycemia, increased growth velocity, and decreased serum aminotransferase concentrations in all patients. Our experience suggests that cornstarch therapy can be effective as an initial treatment for patients with GSD-III.

Expansion of transplanted hepatocytes during liver regeneration.

BACKGROUND: Successful clinical application of hepatocyte transplantation has been limited by poor engraftment of the recipient liver by transplanted hepatocytes. METHODS: To address the hypothesis that liver regeneration induced by an acute hepatotoxic injury promotes expansion of transplanted hepatocytes, we injected beta-galactosidase-labeled hepatocytes intrasplenically into mice 24 hr after treatment with carbon tetrachloride (CCl4) and into untreated controls. RESULTS: Macroscopic examination of whole liver segments identified clusters of transplanted hepatocytes uniformly spread on the capsular surface of the recipient liver and in the liver core following the distribution pattern of portal vein branches. Frozen sections showed that although the degree of initial engraftment of transplanted hepatocytes was similar in CCl4-treated and control livers, there was a fourfold increase of engrafted hepatocytes in CCl4-treated livers 10 days after transplantation which persisted to 28 days. CONCLUSIONS: We conclude that the number of transplanted hepatocytes increases in response to regeneration signal triggered by an acute hepatocyte-specific liver injury.

Long-term survival after liver transplantation.

BACKGROUND: Liver transplantation (LT) remains a high-risk operation, especially during the first months after LT when technical complications and preexisting illness exert their influence on survival. However, there are late deaths. The authors have reviewed their experience to identify factors impacting on long-term survival. METHODS: A total of 150 patients who had undergone liver transplantation over an 11-year period were reviewed. Thirty-three patients died after LT (22%). Of these, 18 of 33 (55%) died in the first 3 postoperative months. One hundred thirty-two patients survived beyond 3 months, and 15 patients (11%) suffered late deaths. This review concentrates on the latter group. RESULTS: The primary cause of death was sepsis in 11 of 15 (73%). In two, sepsis complicated retransplantation in chronically debilitated patients. Two additional patients had late-presenting postoperative complications (bile leak or abscess, intestinal obstruction with perforation). In two cases, pneumocystis carinii pneumonia occurred; noncompliance or unplanned discontinuation of prophylaxis was directly responsible. Multiple organ system failure from presumed immunoincompetence developed in four patients; one had undergone bone marrow transplantation for aplastic anemia (AA) after fulminant hepatic failure (FHF). Lymphoproliferative disease (LPD) was the cause of death in 3 of 15 cases (20%). In only three cases was the cause of death related to the patient's primary disease (chronic hepatitis, Alper's syndrome or seizures, and AA with FHF). Pretransplant diagnosis, and UNOS status at the time of LT did not influence the long-term survival. CONCLUSIONS: Long-term survival in patients who have undergone LT was compromised by immunosuppressive complications and sepsis. Early mortality factors, such as UNOS status, age at LT, primary diagnosis, and technical complications do not predict late deaths. In children who adhere to their medical regimen and have good initial allograft function, late postoperative infection, especially with Ebstein-Barr virus, accounts for most of the late mortality. Improved and decreased immunosuppression may further improve these long-term results.

Hepatic taurine transport: a Na+-dependent carrier on the basolateral plasma membrane.

Highly purified rat basolateral liver plasma membrane vesicles were used to examine the mechanism and the driving forces for hepatic uptake of the beta-amino acid, taurine. An inwardly directed 100 mM NaCl gradient stimulated the initial rate of taurine uptake and energized a transient twofold accumulation of taurine above equilibrium ("overshoot"). In contrast, uptake was slower and no overshoot was detected in the presence of a KCl gradient. A negative intravesicular electrical potential generated by the presence of permeant anions or an outwardly directed K+ gradient with valinomycin increased Na+-stimulated taurine uptake. External Cl- stimulated Na+-dependent taurine uptake independent of effects on the transmembrane electrical potential difference. Na+-dependent taurine uptake showed a sigmoidal dependence on extravesicular Na+ concentration, suggesting multiple Na+ ions are involved in the translocation of each taurine molecule. Na+-dependent taurine uptake demonstrated Michaelis-Menten kinetics with a maximum velocity of 0.537 nmol.mg protein-1.min-1 and an apparent Km of 174 microM. [3H]taurine uptake was inhibited by the presence of excess unlabeled taurine, beta-alanine, or hypotaurine but not by L-glutamine or L-alanine. In summary, using basolateral liver plasma membrane vesicles, we have shown that hepatic uptake of taurine occurs by a carrier-mediated, secondary active transport process specific for beta-amino acids. Uptake is electrogenic, stimulated by external Cl-, and requires multiple Na+ ions for the translocation of each taurine molecule.

Enhanced uptake of taurine by basolateral plasma membrane vesicles isolated from developing rat liver.

Inasmuch as taurine biosynthesis is decreased during early postnatal life, an efficient mechanism for taurine uptake by the liver must be present to maintain intracellular stores of this beta-amino acid. Therefore, basolateral liver plasma vesicles prepared from 14-day and adult rats were used to examine taurine transport during development. For both age groups, the presence of an inwardly directed Na+ gradient stimulated the initial rate of taurine uptake and caused a transient accumulation of taurine above equilibrium. For all time points before equilibrium, taurine uptake was significantly greater with membrane vesicles from 14-day compared to adult rat livers. In contrast, no age-related differences in Na+-independent uptake as measured with a K+ gradient were detected. With equal intravesicular and extravesicular Na+ concentrations, taurine uptake remained significantly greater in the younger age group. For both age groups, Na+-dependent taurine uptake was saturable but the apparent Km and Vmax for Na+-dependent taurine uptake were significantly greater in membrane vesicles from 14-day compared to adult rats. These findings suggest that an increased number of functional carriers for taurine are present in developing compared to adult basolateral plasma membrane perhaps reflecting the needs of the immature liver for this essential nutrient.

Determinants of bile formation during development: ontogeny of hepatic bile acid metabolism and transport.

The studies cited in this brief review stress that the development of hepatic transport processes is extraordinarily complex. Important changes in hepatic morphology and synthetic capacity are required before maturation of membrane carriers for bile acids. Transport systems at both poles of the hepatocyte develop independently. An increase in bile acid synthesis at several stages during the development appears to be an ontogenic event that is programmed to occur in concert with functional maturation of the enterohepatic circulation. Expression of specific membrane transporters for bile acids can be observed in fetal liver and postnatal ileum during periods of expansion of the bile acid pool. It is likely that specific defects, such as congenitally absent or defective bile acid transport proteins, will eventually be discovered in rare patients with undefined cholestatic syndromes. The absence of active ileal bile acid transport has recently been demonstrated in several children with congenital bile acid malabsorption. Whether bile acids can actually induce or regulate production of their own carriers during development has not been determined, but an increase in bile acid pool through feeding of exogenous bile acid has been shown to stimulate an increase in plasma membrane carriers for bile acids in adult rat liver. Thus, a number of factors, including available driving forces for transport, bile acid pool size and composition, effectiveness of intracellular compartmentation and transfer, and the function of membrane carriers, can all contribute to low rates of bile flow and bile acid secretion, depending on the stage of development.

Papillary stenosis and sclerosing cholangitis in an immunodeficient child.

Sclerosing cholangitis, an inflammatory disease of the biliary tree that occurs infrequently in childhood, has been recognized in combination with papillary stenosis in adults with the acquired immunodeficiency syndrome. A 10-yr-old child with a familial immunodeficiency syndrome characterized by defective T-cell function and deficiencies of immunoglobulins A and G developed papillary stenosis and sclerosing cholangitis associated with cryptosporidium enteritis. The patient presented with fever, jaundice, right upper quadrant pain, and elevated serum concentrations of transaminases and alkaline phosphatase. The pain and jaundice resolved after endoscopic sphincterotomy, but the biochemical abnormalities persisted. This case demonstrates that the combination of papillary stenosis and sclerosing cholangitis can occur in children as well as adults and may be associated with immunodeficiency syndromes other than the acquired immunodeficiency syndrome. Endoscopic sphincterotomy can provide symptomatic treatment for papillary stenosis in children with this condition, although the effect of sphincterotomy on the natural history of the sclerosing cholangitis is uncertain.

Resistance to growth hormone in children with chronic liver disease.

Malnutrition adversely affects mortality and morbidity before and after liver transplantation. Outcome might be improved if liver transplant recipients were in a better nutritional state at the time of transplantation. In this review, we will examine the potential use of GH and IGF-I to improve nutritional status in patients with cirrhosis. Patients with cirrhosis have low circulating IGF-I levels in the face of elevated serum GH concentrations. IGFBP-3 levels are low while IGFBP-1 levels are high. In patients with cirrhosis, IGF-I levels do not increase in response to treatment with GH. Patients with cirrhosis are insensitive to GH, and rhGH treatment is not likely to reverse malnutrition. The pathobiology of GH insentivity may reflect decreased nutritional intake, low GH receptor density, decreased IGF-I half-life and hepatic insensitivity to insulin.

Fasting enhances taurine transport by rat liver plasma membrane vesicles.

Hepatic taurine stores are maintained by biosynthesis from the sulfur-containing amino acids, methionine and cysteine, and by uptake via a Na(+)- and Cl(-)-dependent transport system, which is specific for beta-amino acids. We hypothesized that liver stores of taurine are maintained by enhanced hepatic transport during fasting when dietary sources for taurine and its precursors are diminished. Liver plasma membrane vesicles, enriched for the basolateral domain, were prepared from adult male rats fasted for 72 h and from control rats. The maximum velocity for Na(+)-dependent taurine uptake was twofold greater for the fasted group compared with the control group (0.87 +/- 0.09 vs. 0.31 +/- 0.03 nmol.mg protein-1.min-1). The apparent Michaelis constant for taurine was also greater for fasted compared with control (154.0 +/- 0.5 vs. 80.0 +/- 2.0 microM). gamma-Aminobutyric acid, but not alanine or glutamine, abolished the effect of fasting on hepatic taurine transport. To determine the effect of fasting independent of changes in the lipid microenvironment, taurine uptake was measured in proteoliposomes reconstituted by inserting detergent-solubilized membrane proteins into asolectin vesicles. Taurine uptake by proteoliposomes reconstituted from membranes prepared from the fasted group was significantly greater than from the control group. We conclude that Na(+)-dependent taurine transport is enhanced in liver plasma membranes prepared from fasted rats. Our findings imply that enhanced taurine uptake with fasting is due to either an increased number of functional carriers or activation of existing transporters.

An ontogenically regulated 48-kDa protein is a component of the Na(+)-bile acid cotransporter of rat liver.

Recent evidence suggests that the Na(+)-coupled carrier mechanism for bile acids on the hepatocyte basolateral plasma membrane is a polypeptide in the molecular weight range of 48,000-50,000. In this study we used a strategy for the identification and isolation of this transport protein based on the observation that Na(+)-dependent transport activity is abruptly expressed in fetal rat liver just before birth [Suchy et al. Am. J. Physiol. 251 (Gastrointest. Liver Physiol. 14): G665-G673, 1986]. Analysis of basolateral plasma membranes by SDS-PAGE revealed that a protein of apparent molecular weight 48,000 was absent from fetal rat liver on day 19 of gestation, barely detectable on day 20, and thereafter increased progressively with postnatal development. Monospecific, polyclonal antibodies raised against the 48-kDa protein but not preimmune antibodies significantly inhibited the initial rate of Na(+)-dependent taurocholate uptake by isolated rat hepatocytes. In contrast, Na(+)-independent taurocholate transport and uptake of another anion, 35SO4(2-), were not affected by antibody treatment. When an extract containing the total complement of basolateral proteins was incorporated into asolectin liposomes, Na+ gradient-dependent uptake of taurocholate was observed, including a 2- to 2.5-fold accumulation of substrate above its equilibrium concentration (overshoot). However, if the membrane extract was first selectively depleted of the 48-kDa protein by immunoprecipitation with the anti-48-kDa antibody before reconstitution, Na(+)-dependent stimulation of taurocholate transport was completely abolished. These studies indicate that an ontogenically regulated 48-kDa protein is a component of the basolateral Na(+)-dependent transport system for bile acids.

Pustular pyoderma gangrenosum associated with ulcerative colitis in childhood. Report of two cases and review of the literature.

Pyoderma gangrenosum is a cutaneous disorder associated with systemic diseases such as ulcerative colitis, Crohn's disease, rheumatoid arthritis, and blood dyscrasias. We are reporting two cases of pustular pyoderma gangrenosum associated with ulcerative colitis. One patient had inactive bowel disease when she developed her third episode of pustules, erosions, and nodules on the left leg. The other patient exhibited a widespread painful vesiculopustular eruption that coincided with the onset of her colitis. Both patients presented with pustules as the primary manifestation of their pyoderma gangrenosum. Histologic examination of skin from both patients revealed an acute perifollicular inflammation. Pyoderma gangrenosum should be considered in the differential diagnosis of pustular disorders in children with underlying conditions such as ulcerative colitis.

Growth hormone insensitivity in children with biliary atresia.

Malnutrition is a critical predictor of mortality and morbidity in children with biliary atresia who undergo orthotopic liver transplantation. Growth hormone (GH) enhances nitrogen retention in patients with chronic obstructive lung disease, sepsis, and in fasted adult volunteers. The goal of this study was to assess the acute response to recombinant human GH (rhGH) treatment in children with biliary atresia to determine whether GH therapy was likely to improve pretransplant nutritional status. Five children, aged 10-32 months, with biliary atresia and persistent cholestasis despite surgical attempts to reestablish bile flow, were studied. All five children had portal hypertension, conjugated hyperbilirubinemia, and decreased serum albumin concentrations. Length, weight, and growth velocity were decreased in all five children. Despite adequate energy and protein intake, fat stores were depleted in all five subjects, and somatic protein stores were diminished in all except one child. Baseline serum concentrations of insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 (IGFBP-3) were low (8.4 +/- 2 ng/ml and 0.2 +/- 0.1 mg/l respectively). In the four children who completed the study, serum IGF-I and IGFBP-3 levels did not change after treatment with rhGH (0.1 mg/kg/day) for 4 days. Our findings indicate that children with biliary atresia awaiting liver transplantation are insensitive to GH and that treatment with GH is unlikely to promote anabolism. A rationale exists for examining the effect of treatment with IGF-I, which mediates the anabolic effects of GH.

Biliary atresia--surgical management and treatment options as they relate to outcome.

Results show that the use of sequential surgical treatment, employing Kasai portoenterostomy in infancy, followed by selective liver transplantation for children with progressive hepatic deterioration yields improved overall survival. All children with successful Kasai portoenterostomy procedures who do not require OLT are survivors. Using newer transplant techniques, the 5-year survival rate for children who receive transplants with a primary diagnosis of biliary atresia was 82%. This yields an overall survival rate of 86% in this entire study population. Limited donor availability and increased complications after liver transplantation in infants less than 1 year of age mitigate against the use of primary liver transplantation without prior portoenterostomy for infants with biliary atresia. At present, these two operative procedures should be used as sequential and complementary modes of treatment rather than as competitive procedures. When biliary atresia is not recognized in infancy and established cirrhosis has resulted, primary transplantation should be offered as the initial surgical treatment.