Immature Liver Research Articles

Crystallographic Analysis of Human Serum Albumin Complexed with 4Z,15E-Bilirubin-IXα

Bilirubin, an insoluble yellow-orange pigment derived from heme catabolism, accumulates to toxic levels in individuals with impaired or immature liver function. The resulting jaundice may be managed with phototherapy to isomerize the biosynthetic 4Z,15Z-bilirubin-IXα to more soluble and excretable isomers, such as 4Z,15E-bilirubin. Bilirubin and its configurational isomers are transported to the liver by human serum albumin (HSA) but their precise binding location(s) on the protein have yet to be determined. To investigate the molecular details of their interaction, we co-crystallised bilirubin with HSA. Strikingly, the crystal structure—determined to 2.42 Å resolution—revealed the 4Z,15E-bilirubin-IXα isomer bound to an L-shaped pocket in sub-domain IB. We also determined the co-crystal structure of HSA complexed with fusidic acid, an antibiotic that competitively displaces bilirubin from the protein, and showed that it binds to the same pocket. These results provide the first crystal structure of a natural bilirubin pigment bound to serum albumin, challenge some of the present conceptions about HSA–bilirubin interactions, and provide a sound structural framework for finally resolving the long-standing question of where 4Z,15Z-bilirubin-IXα binds to the protein.

Gene Delivery to the Juvenile Mouse Liver Using AAV2/8 Vectors

Recombinant adeno-associated viral (rAAV) vectors have shown promise for use in liver-targeted gene delivery, but their effects have not been extensively investigated in the immature liver. Understanding the impact of liver growth on the efficacy of transduction is essential, because many monogenic liver diseases that are amenable to gene therapy will require treatment early in life. Here we show that rAAV2/8 transduces the neonatal mouse liver with high efficiency. With just one doubling in liver weight, however, there is a rapid reduction in vector genome numbers, irrespective of form, and the loss of episomal vector is almost complete by 2 weeks. Stable transgene expression is observed in a small percentage of hepatocytes, often in two- to eight-cell clusters, suggestive of genomic integration. Delivery at serially older ages was associated with progressively improved episome persistence and transgene expression. Vector re-administration was possible following initial neonatal administration, albeit at reduced efficacy because of an anticapsid humoral immune response. We also found that intraperitoneal (IP) delivery of rAAV2/8 was highly effective at all ages, and that promoter selection is the critical determinant of the intensity and pattern of transgene expression across the hepatic lobule. We conclude that successful use of rAAV to treat liver disease in early childhood will require optimally efficient vector constructs and probable re-administration.

Accumulation of Hedgehog-Responsive Progenitors Parallels Alcoholic Liver Disease Severity in Mice and Humans

Improving outcomes in alcoholic liver disease (ALD) necessitates better understanding of how habitual ethanol (EtOH) consumption alters normal regenerative mechanisms within the liver. Hedgehog (Hh) pathway activation promotes expansion of progenitor populations in other tissues. We evaluated the hypothesis that chronic EtOH exposure activates Hh signaling in liver. Hh signaling, liver progenitors, transforming growth factor (TGF)-beta induction, and liver damage were compared in mice fed chow, high-fat diets (HF), or HF + EtOH for 4 weeks. Susceptibility to TGF-beta-mediated apoptosis was compared in Hh-responsive liver cells (eg, immature cholangiocytes and oval cells) and mature hepatocytes (which are unresponsive to Hh). Hepatic accumulation of Hh-responsive cells were compared in controls and ALD patients and correlated with a discriminant function (DF) that predicts subacute mortality. Hh signaling and numbers of Hh-responsive cells were increased in HF mice and greatest in HF+EtOH mice. In both, progenitor and stromal cell populations harbored Hh-responsive cells. More ductular-type progenitors and fibrosis markers were noted in HF+EtOH mice than in HF mice. The former also expressed more TGF-beta-1. TGF-beta-1 treatment selectively promoted the viability of Hh-responsive immature liver cells and caused mature hepatocytes that survived to produce Hh ligands. Hh-responsive cells were increased in ALD patients. Lobular accumulation of Hh-responsive immature ductular cells was greater in those with a DF >32 than those with a DF <32. Hh signaling is increased in ALD and may influence ALD outcomes by promoting hepatic accumulation of immature ductular cells.

Mature Human Hepatocytes fromEx VivoDifferentiation of Alginate-Encapsulated Hepatoblasts

Alginate gel was used to provide encapsulation to support the growth and eventually the differentiation of hepatic progenitors cells derived from human fetal livers. The encapsulated cells aggregated into spheroids within a few days in culture and continued to grow for at least 4 weeks in a serum-free medium. The hepatic progenitor cells in the spheroids undergo differentiation, as indicated by the appearance of functions of mature hepatocytes such as the detoxification of ammonia, albumin secretion, expression of the adult cytochrome P450 isozyme CYP3A4 and enzymatic activity typical of CYP2C9. Along with the expression of mature hepatic markers, these progenitor cells lost features typical of immature liver cells such as epithelial cell adhesion molecule. In addition to the acquisition of mature biochemical functions, the spheroids also developed a bile ducts, suggesting that they had differentiated into tissues resembling those in an intact liver.

Canine neonatal mortality in four large breeds

The high mortality in canine neonates are related to many factors, including prolonged labour, maternal neglect or carelessness, lack of milk, plus congenital abnormalities and acquired disorders in the neonate [1,3-5]. The immature status of the newborn puppies makes them vulnerable and totally depending on intensive care from the dam. The puppies are highly susceptible to hypothermia, due to poorly developed thermoregulatory mechanisms. They cannot induce peripheral vasoconstriction or react to low temperature by shivering. The energy requirement is high, but the energy reserves are low and the immature liver is inefficient in generating energy. This makes the neonate predisposed to hypoglycaemia. Due to immature kidney function there is an increased risk of dehydration. The neonates have a high percentage of body water (82%) compared to adults and have a greater loss of water through their lungs and skin due to a large surface to volume ratio, which further increase the risk of dehydration [7-9].

Neonatal Pig Liver–Derived Progenitors for Insulin-Producing Cells: AnIn VitroStudy

Beta (beta)-cell replacement represents an attractive approach for the possible cure of type 1 insulin-dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cells for IDDM substitution therapy, we have focused on the neonatal pig liver, which is putatively enriched in multipotent stem cells. We then isolated cells measuring 10 to 15 microm in diameter, identified as small cells, characterized by a high proliferation rate and positive staining for immature liver and pancreatic endocrine cell markers (i.e., insulin and pancreatic duodenal homeobox). The ability of these cells to transdifferentiate into pancreatic beta-like cells under culture conditions with exendin-4 (Ex-4) or high glucose concentration was examined. We observed that insulin secretion was not physiological in basal conditions, although it became responsive to glucose after 5 days of exposure to Ex-4. This beta-cell-like phenotype remained physiologically stable, even after stimulus withdrawal. Based on these observations, we contend that the proposed cell and tissue model might offer several advantages as a candidate for substitution cell therapy in IDDM, because the neonatal pig liver seems enriched in cells, with a mixed pancreas-liver phenotype, that are easier to purify and grow in culture and are more functional than other beta-like cells upon in vitro single short-term stimulation challenge.

Vitamin K Prophylaxis for Preterm Infants: A Randomized, Controlled Trial of 3 Regimens

Preterm infants may be at particular risk from either inadequate or excessive vitamin K prophylaxis. Our goal was to assess vitamin K status and metabolism in preterm infants after 3 regimens of prophylaxis. Infants <32 weeks' gestation were randomized to receive 0.5 mg (control) or 0.2 mg of vitamin K1 intramuscularly or 0.2 mg intravenously after delivery. Primary outcome measures were serum vitamin K1, its epoxide metabolite (vitamin K1 2,3-epoxide), and undercarboxylated prothrombin assessed at birth, 5 days, and after 2 weeks of full enteral feeds. Secondary outcome measures included prothrombin time and factor II concentrations. On day 5, serum vitamin K1 concentrations in the 3 groups ranged widely (2.9-388.0 ng/mL) but were consistently higher than the adult range (0.15-1.55 ng/mL). Presence of vitamin K1 2,3-epoxide on day 5 was strongly associated with higher vitamin K1 bolus doses. Vitamin K1 2,3-epoxide was detected in 7 of 29 and 4 of 29 infants from the groups that received 0.5 mg intramuscularly and 0.2 mg intravenously, respectively, but in none of 32 infants from group that received 0.2 mg intramuscularly. After 2 weeks of full enteral feeding, serum vitamin K1 was lower in the infants who received 0.2 mg intravenously compared with the infants in the control group. Three infants from the 0.2-mg groups had undetectable serum vitamin K1 as early as the third postnatal week but without any evidence of even mild functional deficiency, as shown by their normal undercarboxylated prothrombin concentrations. Vitamin K1 prophylaxis with 0.2 mg administered intramuscularly maintained adequate vitamin K status of preterm infants until a median age of 25 postnatal days and did not cause early vitamin K1 2,3-epoxide accumulation. In contrast, 0.2 mg administered intravenously and 0.5 mg administered intramuscularly led to vitamin K1 2,3-epoxide accumulation, possibly indicating overload of the immature liver. To protect against late vitamin K1 deficiency bleeding, breastfed preterm infants given a 0.2-mg dose of prophylaxis should receive additional supplementation when feeding has been established.

Epidemiological studies of &lt;i&gt;Fasciola gigantica&lt;/i&gt; infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe

During the period between January 1999 and December 2000, the distribution and seasonal patterns of Fasciola gigantica infections in cattle in the highveld and lowveld communal grazing areas of Zimbabwe were determined through monthly coprological examination. Cattle faecal samples were collected from 12 and nine dipping sites in the highveld and lowveld communal grazing areas respectively. Patterns of distribution and seasonal fluctuations of the intermediate host-snail populations and the climatic factors influencing the distribution were also determined by sampling at monthly intervals for a period of 24 months (November 1998 to October 2000) in six dams and six streams in the highveld and in nine dams in the lowveld communal grazing areas. Each site was sampled for relative snail density and the vegetation cover and type, physical and chemical properties of water, and mean monthly rainfall and temperature were recorded. Aquatic vegetation and grass samples 0-1 m from the edges of the snail habitats were collected monthly to determine the presence or absence of F. gigantica metacercariae. Snails collected at the same time were individually checked for the emergence of larval stages of F. gigantica. A total of 16264 (calves 5418; weaners 5461 and adults 5385) faecal samples were collected during the entire period of the study and 2500 (15.4%) of the samples were positive for F. gigantica eggs. Significantly higher prevalences were found in the highveld compared to the lowveld (P < 0.001), for adult cattle than calves (P < 0.01) and in the wet season over the dry season (P < 0.01). Faecal egg output peaked from August/September to March/April for both years of the study. Lymnaea natalensis, the snail intermediate host of F. gigantica was recorded from the study sites with the highveld having a significantly higher abundance of the snail species than the lowveld (P < 0.01). The snail population was low between December and March and started to increase in April reaching a peak in September/October. The number of juvenile snails peaked between April and August. The mean number of snails collected was negatively correlated with rainfall and positively correlated with temperature. Mean number of snails collected was also positively correlated with Potamogeton plant species and negatively correlated with Cyperus plant species. However, none of the L. natalensis collected from the habitats were found shedding Fasciola cercariae. Metacercariae were found on herbage from the fringes of the snail habitats between February and August for both years, with most of the metacercariae concentrated on herbage 0-1 m from the banks of the habitats. Based on the findings of this study, anthelmintic treatment should be administered in December/January to control chronic and mature fasciolosis. A second treatment should be given in April/May to reduce pasture contamination and subsequently snail infection, as this is the time the snail population starts to build up. To control acute fasciolosis due to the immature liver flukes a third treatment should be given in August. The first application of molluscicides to control the snail intermediate hosts can be done in June the time when the snail is harbouring the parasite and a second application in September in order to kill new generations of infected snails

Pharmacology Review: Sodium Phenylacetate and Sodium Benzoate in the Treatment of Neonatal Hyperammonemia

Ammonia is present in all body fluids and exists primarily as ammonium ion at physiologic pH. Hyperammonemia is defined as a blood ammonia concentration greater than about 100 mcmol/L in neonates or 50 mcmol/L in children and adults (precise cut-offs vary, depending on individual laboratory normative ranges). The concentration of ammonia is 10 times higher in tissue than in blood. A 5- to 10-fold increase in blood ammonia concentration usually is toxic to the nervous system. Hyperammonemia in the neonatal period, especially when due to inborn errors of metabolism, can progress rapidly and cause severe neurologic damage or early death. Hyperammonemia can be caused by inborn errors of metabolism as well as by a variety of acquired conditions (Tables 1 and 2). Urgent treatment is required because of the potential for irreversible neurologic sequelae that can, in many cases, be prevented by prompt diagnosis and institution of therapy. | Urea cycle defects | || | Amino acid transporter deficiencies | | Organic acidemias | | Fatty acid oxidation defects | | Pyruvate carboxylase deficiency | | Mitochondrial disorders | | Hyperinsulinism/hyperammonemia syndrome (glutamate dehydrogenase mutations) | | Delta1-pyrroline-5-carboxylate synthase deficiency | Table 1. Inborn Errors of Metabolism Associated With Hyperammonemia | Sampling artifact | || | Cardiovascular | | Perinatal asphyxia | | Liver failure | | Bacterial colonization (urease-positive organisms) | | Iatrogenic | Table 2. Causes of Acquired Hyperammonemia The combination of sodium phenylacetate (NAPA) and sodium benzoate (NABZ) in a 10%/10% solution is an intravenously administered United States Food and Drug Administration (FDA)-approved drug used as adjunctive therapy for …

7H-Dibenzo[ c, g]carbazole and 5,9-dimethyldibenzo[ c, g]carbazole exert multiple toxic events contributing to tumor promotion in rat liver epithelial ‘stem-like’ cells

Immature liver progenitor cells have been suggested to be an important target of hepatotoxins and hepatocarcinogens. The goal of the present study was to assess the impact of 7H-dibenzo[ c, g]carbazole (DBC) and its tissue-specific carcinogenic N-methyl ( N-MeDBC) and 5,9-dimethyl (DiMeDBC) derivatives on rat liver epithelial WB-F344 cells, in vitro model of liver progenitor cells. We investigated the cellular events associated with both tumor initiation and promotion, such as activation of aryl hydrocarbon receptor (AhR), changes in expression of enzymes involved in metabolic activation of DBC and its derivatives, effects on cell cycle, cell proliferation/apoptosis and inhibition of gap junctional intercellular communication (GJIC). N-MeDBC, a tissue-specific sarcomagen, was only a weak inhibitor of GJIC or inducer of AhR-mediated activity, and it did not affect either cell proliferation or apoptosis. DBC was efficient GJIC inhibitor, while DiMeDBC manifested the strongest AhR inducing activity. Accordingly, DiMeDBC was also the most potent inducer of cytochrome P450 1A1 (CYP1A1) and CYP1A2 expression among the three compounds tested. Both DBC and DiMeDBC induced expression of CYP1B1 and aldo-keto reductase 1C9 (AKR1C9). N-MeDBC failed to significantly upregulate CYP1A1/2 and it only moderately increased CYP1B1 or AKR1C9. Only the potent liver carcinogens, DBC and DiMeDBC, caused a significant increase of p53 phosphorylation at Ser15, an increased accumulation of cells in S-phase and apoptosis at micromolar concentrations. In addition, DiMeDBC was found to stimulate cell proliferation of contact-inhibited WB-F344 cells at 1 μM concentration, which is a mode of action that might further contribute to its hepatocarcinogenicity. The present data seem to suggest that the AhR activation, induction of enzymes involved in metabolic activation, inhibition of GJIC or stimulation of cell proliferation might all contribute to the hepatocarcinogenic effects of DBC and DiMeDBC.

Migratory responses of murine hepatic myeloid, lymphoid-related, and plasmacytoid dendritic cells to CC chemokines.

Dendritic cell (DC) trafficking is regulated by chemokine receptor expression and responsiveness to chemokines. The authors compared CC chemokine receptor (CCR) expression by mouse liver myeloid, "lymphoid-related," and plasmacytoid DC subsets and their responsiveness to CC chemokines. CCR mRNA expression by liver DC subsets was evaluated by RNase protection assay. In vitro migration of the DC in response to recombinant murine CC chemokines was assayed using Transwell chambers. Immature liver DC did not respond to any CC chemokines tested, despite expression of mRNA encoding appropriate receptors for their ligands. CCR7 expression by each liver DC subset was strongly enhanced in response to maturation. The migratory capacity of liver plasmacytoid DC was similar to that of liver myeloid and lymphoid-related DC. These findings suggest that targeting of CCR7 and its ligands may be a potential approach for manipulation of liver DC trafficking to secondary lymphoid tissue after liver transplantation.

54 Vitamin K Status in Preterm Infants: A Randomised Controlled Trial to Compare Three Regimes of Prophylaxis

Background: Neonatal vitamin K stores are precarious. Without adequate vitamin K prophylaxis preterm infants may be at particular risk of vitamin K deficiency bleeding (VKDB), but the optimal dose and route is unclear. Objective: To compare the vitamin K status of preterm infants during the first week of life and when on full enteral feeds, following three regimes of vitamin K prophylaxis after delivery.Design/Methods: Infants born at < 32 weeks gestation were randomised to receive vitamin K1 0.5 mg intramuscularly (IM) (group 1: control), 0.2 mg IM (group 2) or 0.2 mg intravenously (IV) (group 3) on day 1. Cord blood was obtained; venous blood samples at 5 days postnatal and 2 weeks after establishment of full enteral feeds were analysed for serum vitamin K1, vitamin K1 2,3-epoxide, descarboxyprothrombin (PIVKA-II), and prothrombin time.Results: Of 98 infants enrolled, 90 had a day 5 sample and 80 had a second sample obtained at a median (IQR) of 25 (22–31) days. Baseline characteristics (mean ±SD) for groups 1 (n=31), 2 (n=34) and 3 (n=33) were respectively: gestational age 28.3 ±2.5, 28.6 ± 2.3, and 28.1 ±2.6 weeks; birthweight 1025 ±379, 1138 ±379, and 1060 ±371 g.Serum vitamin K concentrations (ng/mL)Compared with the control group, day 5 vitamin K concentrations were significantly lower in group 2 (p = 0.04), and at the time of established feeds they were lower in group 3 (p = 0.03). Three infants (one in group 2; two in group 3) had undetectable levels of vitamin K at the time of the second sample, however in each case PIVKA-II was undetected. Eleven of ninety (12%) infants (seven in group 1; four in group 3) had detectable concentrations of vitamin K epoxide on day 5 (p = 0.007).Conclusions: Preterm infants given 0.2 mg or 0.5 mg vitamin K1 at birth have very high serum concentrations during the first week of life. The presence of vitamin K epoxide is significantly associated with a higher dose (0.5 mg) of vitamin K given IM and with a reduced dose (0.2 mg) given IV, and may reflect vitamin K overload of the immature liver by these regimes of prophylaxis. With a reduced dose given IV or IM, vitamin K can fall to undetectable levels by as early as the fourth postnatal week. The risk of subsequent late VKDB may be increased in these infants unless further vitamin K supplements are given.

Physiologically Based Pharmacokinetic (PBPK) Modeling of Caffeine and Theophylline in Neonates and Adults: Implications for Assessing Children's Risks from Environmental Agents

Children's risks can differ from those in adults for numerous reasons, one being differences in the pharmacokinetic handling of chemicals. Immature metabolism and a variety of other factors in neonates can affect chemical disposition and clearance. These factors can be incorporated into physiologically based pharmacokinetic (PBPK) models that simulate the fate of environmental toxicants in both children and adults. PBPK models are most informative when supported by empirical data, but typically pediatric pharmacokinetic data for toxicants are not available. In contrast, pharmacokinetic data in children are readily available for therapeutic drugs. The current analysis utilizes data for caffeine and theophylline, closely related xanthines that are both cytochrome P-450 (CYP) 1A2 substrates, in developing PBPK models for neonates and adults. Model development involved scale-up of in vitro metabolic parameters to whole liver and adjusting metabolic function for the ontological pattern of CYP1A2 and other CYPs. Model runs were able to simulate the large differences in half-life and clearance between neonates and adults. Further, the models were able to reproduce the faster metabolic clearance of theophylline relative to caffeine in neonates. This differential between xanthines was found to be due primarily to an extra metabolic pathway available to theophylline, back-methylation to caffeine, that is not available to caffeine itself. This pathway is not observed in adults exemplifying the importance of secondary or novel routes of metabolism in the immature liver. Greater CYP2E1 metabolism of theophylline relative to caffeine in neonates also occurs. Neonatal PBPK models developed for these drugs may be adapted to other CYP1A2 substrates (e.g., arylamine toxicants). A stepwise approach for modeling environmental toxicants in children is proposed.

Hepatoblastoma in children of extremely low birth weight: A report from a single perinatal center

Background/Purpose: The incidence of hepatoblastoma (HB) in children of low birth weight is increasing. In the authors' institute, 5 infants of extremely low birth weight (ELBW) were found to have HB. The purpose of this study was to identify the characteristics of these infants to elucidate the pathogenesis of HB arising in ELBW infants. Methods: Birth weight (BW) ranged from 554 to 750 g (mean, 654 g) and gestational age from 23 to 29 weeks (mean, 25.8 weeks). Medical records of the 5 patients were reviewed, and perinatal treatments were compared with those of ELBW infants without HB. Results: One patient with intraabdominal hemorrhage had emergency operation, which was followed by early postoperative death. The parents of one child refused treatment because of associated severe anomalies. He died of the growing tumor 4 months after diagnosis. The remaining 3 patients had radical operation performed after intraarterial chemoembolization and systemic chemotherapy. One died of hepatic failure 7 months after operation. Two are alive 5 and 9 months after operation. The incidence of HB among ELBW infants was estimated to be about 0.5% in our institute. The mean durations of mechanical ventilation, oxygen inhalation, and hospitalization during the neonatal periods in cases of HB were significantly longer than those in BW matched control infants (P <.01). Conclusions: ELBW children have a high risk for HB. In follow-up of ELBW infants, serum alpha-fetoprotein or abdominal ultrasonography may be useful to detect early HB. The children with HB received perinatal treatments for a significantly longer time, which suggests that perinatal intensive and long-term medical treatments may be involved in the tumorigenesis in the highly sensitive immature liver. J Pediatr Surg 38:134-137. Copyright 2003, Elsevier Science (USA). All rights reserved.

Case report

A five-year-old, 2000 lb (909 kg) beef bull was presented to the Oklahoma State University Food Animal Clinic because of weight loss during the previous eight weeks, and lameness and increasing recumbency of two weeks? duration. Physical examination revealed a subsolar abscess, oral lesions, mild diarrhea, modest generalized lymphadenopathy and an enlarged left scrotum. Further diagnostic testing revealed liver fluke infestation, scrotal hydrocele, ascites and a positive gp-51 bovine leukemia virus serology. The ascites, weight loss, diarrhea, lymphadenopathy and hydrocele were attributed to migration of immature liver flukes. The lameness and recumbency were caused by a subsolar abscess and, therefore, unrelated to the primary problem. Bovine papular stomatitis was suspected to have caused the oral lesions, due to their physical appearance and rapid resolution. The bull was treated twice, at eight- week intervals, with ivermectin which contained clorsulon. Body condition markedly improved over the next 24 days, and the scrotal hydrocele resolved.

Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA:colloidal gold by hep G2 cells.

Previously, we have identified a membrane-mediated uptake and translocation of 1,3,5(10)-estratrien-3, 17beta-diol 6-(O-carboxymethyl)oxime:(125)I-labeled BSA (E6(125)I-BSA) in vivo in immature female rat liver from the plasma membrane (P3 fraction) to the mitochondria and/or lysosomes (P2 fraction). To further investigate this unique effect, current experiments have involved the use of 1,3,5(10)-estratrien-3, 17beta-diol 17-hemisuccinate:( 125)I-BSA (E17(125)I-BSA) to demonstrate the presence of binding sites and translocation of the ligand in human hepatoblastoma (Hep G2) cells. In addition, an estrogen-BSA:colloidal gold conjugate, E17 BSA:Au, was used to directly visualize this uptake in Hep G2 cells. Hep G2 cells displayed high-affinity, stereospecific binding of E17(125)I-BSA. This same ligand was also translocated from the P3 fraction to the P2 fraction. In contrast, (125! )I-BSA was minimally removed from the culture medium. Electron micrographs of Hep G2 cells labeled with E17 BSA:Au demonstrated uptake of this ligand by clathrin-coated pits, indicative of receptor-mediated endocytosis. Furthermore, this ligand was also found in larger vesicles and multivesicular bodies, suggesting the involvement of the compartment of uncoupling of receptor and ligand (CURL), but never in the nucleus. As early as 30 min post-exposure, the ligand could be viewed in organelles, many of which had vesiculated interiors, resembling rounded, vesiculated mitochondria. Labeled BSA was detected mainly in the extracellular compartment, with few multivesicular bodies containing the labeled BSA. The translocation of E17 BSA:Au was virtually eliminated by 100 nM unlabeled E17 BSA or free 17beta-estradiol, but not 17alpha-E6 BSA, 17alpha-estradiol or P6 BSA, and also by exposure of the cells to reduced temperature. These experiments are the first t! o visually demonstrate membrane binding and specific uptake of an estrogen-containing ligand while allowing the intracellular structures responsible to be seen. Furthermore, they identify a potentially new pathway of receptor-mediated endocytosis; namely, the shuttling of estrogens to the mitochondria, in addition to the classical lysosomal pathway.

Control of ovine hepatic growth hormone receptor and insulin-like growth factor I by thyroid hormones in utero.

By use of RNase protection assays, hepatic growth hormone receptor (GHR) and insulin-like growth factor I (IGF-I) mRNA abundances were measured in sheep fetuses after experimental manipulation of fetal plasma thyroid hormone concentrations by fetal thyroidectomy (TX) and exogenous infusion of triiodothyronine (T(3)) and cortisol. TX abolished the normal prepartum rise in hepatic GHR abundance but had little effect on hepatic GHR gene expression at 127-130 days (term 145 +/- 2 days). By contrast, it upregulated basal IGF-I expression in immature fetal liver by increasing both Class 1 and Class 2 transcript abundance but had no further effects on IGF-I gene mRNA levels at 142-145 days. Raising plasma T(3) to prepartum values by exogenous infusion of either T(3) or cortisol into immature intact fetuses prematurely raised hepatic GHR and IGF-I mRNA abundances to values similar to those seen in intact fetuses at 142-145 days. In TX fetuses, cortisol infusion increased hepatic GHR mRNA but not total IGF-I mRNA abundance at 127-130 days. These findings show that thyroid hormones have an important role in the regulation of hepatic GHR and IGF-I gene expression in fetal sheep during late gestation and suggest that T(3) mediates the maturational effects of cortisol on the hepatic somatotropic axis close to term.

Preferential induction of Th1 responses by functionally mature hepatic (CD8alpha- and CD8alpha+) dendritic cells: association with conversion from liver transplant tolerance to acute rejection.

Liver grafts are accepted across major histocompatibility barriers in mice without immunosuppressive therapy. Potentially tolerogenic immature donor dendritic cells (DC) may play a key role in this phenomenon, but recovery of purified DC from normal livers for functional analysis is inherently difficult. Administration of in vitro propagated immature donor DC to recipients of different types of allograft can prolong transplant survival. By contrast, marked increases in donor liver DC as the result of Flt3 ligand (FL) administration and the resulting augmentation of allostimulatory activity within host lymphoid tissue, is associated with acute graft rejection. Here, we compared the capacity of in vitro generated normal liver immature DC and FL-treated donor liver DC to induce alloimmune CD4+ T helper (Th) 1/Th2 and CD8+ T cytotoxic (Tc) 1/Tc2 responses, in vitro and in vivo. B10 (H2b, IAb) immature liver DC were propagated from normal hepatic nonparenchymal cells in granulocyte macrophage-colony stimulating factor (GM-CSF) for 6-8 days. Freshly isolated DC from livers of FL-treated mice (FL-liver DC) were cultured overnight (o/n) in GM-CSF, and both myeloid (CD11c+ CD8alpha-) and lymphoid DC (CD11c+ CD8alpha+) flow-sorted for functional analysis. Proliferative activity and production of interferon (IFN)-gamma, interleukin (IL)-4, and IL-10 by naive C3H (H2k, IEk) T cells in response to DC stimulation was assessed by [3H]thymidine incorporation, and by multicolor flow cytometric analysis, respectively, after 3-day mixed leukocyte reactions. To investigate their in vivo trafficking, B10 DC were injected subcutaneously into normal C3H mice. Sections of lymphoid tissue were immunostained for donor MHC class II+ (IAb+) cells, and for IFN-gamma, IL-4, and IL-10 production. Donor cells and clusters of specific cytokine-secreting cells were enumerated. Both in vitro propagated normal liver-derived DC, and freshly isolated bulk FL-liver DC showed an immature phenotype (MHC class II(lo), CD40-, CD80-, and CD86-) and were weak stimulators of naive allogeneic T cells. After o/n incubation in GM-CSF, both CD8alpha- and CD8alpha+ FL-liver DC exhibited marked up-regulation of surface MHC class II and costimulatory molecules, and acquired potent stimulatory activity for Th1 (mainly) and Th2 cells. Both in vitro propagated immature DC and o/n-cultured mature FL-liver DC homed in vivo to host lymphoid tissues, but with different kinetics. Whereas the mature allogeneic FL-liver DC induced IFN-gamma+ clusters in splenic T-cell areas within 2 days, the IFN-gamma response to immature DC was much slower and weaker. FL-treated donor livers that are rejected acutely contain markedly enhanced numbers of myeloid (CD8alpha-) and lymphoid (CD8alpha+) DC, many of which are capable of maturing rapidly into strong inducers of Th1 and Tcl responses. Substantial differences in quantity, and both the phenotypic and functional characteristics of the DC constituency of donor livers, may contribute significantly toward the distinct outcomes of liver transplant tolerance and rejection.

Participation of different cell types in the restitutive response of the rat liver to periportal injury induced by allyl alcohol

Background/Aim: Restitution of periportal liver necrosis induced by allyl alcohol involves proliferation and differentiation of putative liver stem cells. The participation of different non-epithelial cell types required to restore the liver cord structure in this process has not been well documented. The aim of the study was to determine the anatomic relationships among cells of liver lineage, extracellular matrix, and non-parenchymal cells during repair of periportal liver injury. Methods: Periportal liver injury in rats was induced by intraperitoneal injection of allyl alcohol. Cells of the liver lineage, as well as Kupffer cells, hepatic stellate cells, macrophages, and the extracellular matrix components fibronectin and laminin were localized using immunohistologic methods for 7 days after injury. Results: During the first day there was loss of periportal hepatocytes, as well as sinusoidal nonparenchymal cells, including macrophages, Kupffer cells and hepatic stellate cells. After day 1 macrophages appeared within the necrotic zone, increased until days 3–4, and then decreased to a few cells within reappearing sinusoids. At days 2–5 there was first proliferation of small “null” intraportal cells, which later acquired markers of ductular (OV-6, CKPan ) and liver cell differentiation (alphafetoprotein, carbamoylphosphate synthetase-I), eventually assuming mature hepatocyte morphology. There was also moderate bile duct hyperplasia with extension of small newly-formed ducts from the intraportal zone into the immediate periportal zone. Kupffer cells and hepatic stellate cells became enlarged at the borders of the necrotic and non-necrotic central zone and then appeared to migrate into the oval cell population expanding across the periportal zone. During therestitution phase, hepatic stellate cells were closely associated with the proliferating oval cells, surrounding small aggregates of oval cells which appeared to be forming liver cords. Kupffer cells also stained for fibronectin, and fibronectin was seen at the intersection of the injured portal and uninjured central zones and around the expanding oval cells. In some intraportal zones, the laminin surrounding the bile ducts was lost. It was speculated that this may permit proliferating ductular cells to migrate out of the bile ducts into the periportal zone. By days 6 and 7 most of the injured liver was restored to normal, with a few foci of chronic inflammation remaining. Conclusions: There is a close anatomic relationship between immature liver lineage cells (oval/duct cells) and non-parenchymal cells during the restitutive repair of periportal injury. The nature of this relationship to the possible production of growth factors and expression of growth factor receptors by the cells involved during the restitution process is discussed.

Mesenchymal hamartoma of the liver—New insight into histogenesis

Background/Purpose: Mesenchymal hamartoma (MH) of the liver is thought to develop from the ductal plates of the prenatal liver. This immunohistochemical study was performed to gain insight into the pathophysiology of its development. Methods: Specimens from four MHs with adjacent liver, in one case from a biopsy and from the resected lesion after 6 years follow-up, were investigated with immunostaining on cryostat sections with antibodies against cytokeratins, vimentin, desmin and alpha-actin, as well as von Willebrand factor (factor VIII), fibroblast growth factor (FGF) receptors, FGF-1 (acidic FGF), FGF-2 (basic FGF), and the proliferation-associated Ki67 antigen. Results: Fibrous tissue of MH stained positive not only for vimentin, but also for desmin and alpha-actin, whereas cytokeratins and factor VIII showed specific staining in biliary cysts and endothelial cells, respectively. All mesenchymal cells expressed proteins of the FGF receptor family. Although FGF-1 was only scarcely detectable, there was an accumulation of FGF-2 in borderline areas of liver to MH. Multiple Ki67-positive mesenchymal cells could be identified in these regions in all three MHs. However, we could not detect any proliferative activity in the MHs after follow-up. Conclusions: The proliferative process in MH is still active during early childhood. FGF-2 may have a role in promoting this process. The positivity for desmin and alpha-actin of the lesions suggests that fat-storing (Ito) cells of the immature liver may be involved in the development of MH.