Immunohistochemical detection of polyductin and co-localization with liver progenitor cell markers during normal and abnormal development of the intrahepatic biliary system and in adult hepatobiliary carcinomas

The longest open reading frame of PKHD1 (polycystic kidney and hepatic disease 1), the autosomal recessive polycystic kidney disease (ARPKD) gene, encodes a single-pass, integral membrane protein named polyductin or fibrocystin. A fusion protein comprising its intracellular C-terminus, FP2, was previously used to raise a polyclonal antiserum shown to detect polyductin in several human tissues, including liver. In the current study, we aimed to investigate by immunohistochemistry the detailed polyductin localization pattern in normal (ductal plate [DP], remodelling ductal plate [RDP], remodelled bile ducts) and abnormal development of the primitive intrahepatic biliary system, known as ductal plate malformation (DPM). This work also included the characterization of polyductin expression profile in various histological forms of neonatal and infantile cholestasis, and in cholangiocellular carcinoma (CCC) and hepatocellular carcinoma (HCC). We detected polyductin expression in the intrahepatic biliary system during the DP and the RDP stages as well as in DPM. No specific staining was found at the stage of remodelled bile ducts. Polyductin was also detected in liver biopsies with neonatal cholestasis, including mainly biliary atresia and neonatal hepatitis with ductular reaction as well as congenital hepatic fibrosis. In addition, polyductin was present in CCC, whereas it was absent in HCC. Polyductin was also co-localized in some DP cells together with oval stem cell markers. These results represent the first systematic study of polyductin expression in human pathologies associated with abnormal development of intrahepatic biliary tree, and support the following conclusions: (i) polyductin expression mirrors developmental properties of the primitive intrahepatic biliary system; (ii) polyductin is re-expressed in pathological conditions associated with DPM and (iii) polyductin might be a potential marker to distinguish CCC from HCC.

Peptides containing T cell epitopes, derived from Sm14, but not from paramyosin, induce a Th1 type of immune response, reduction in liver pathology and partial protection against Schistosoma mansoni infection in mice

Sm14 and paramyosin are two major Schistosoma mansoni vaccine candidate antigens. Recently, we have identified Sm14 and paramyosin epitopes that are recognized by T cells of resistant individuals living in endemic areas for schistosomiasis. Herein, mice were immunized with these peptides separately or in association in order to evaluate their vaccine potential. Immunization of mice with Sm14 peptides alone or mixed with paramyosin peptides was able to induce 26%-36.7% or 28%-29.2% of worm burden reduction, 67% or 46% of intestinal eggs reduction and also 54%-61% or 43%-52% of liver pathology reduction, respectively. Protection was associated with a Th1 type of immune response induced by Sm14 peptide immunization. In contrast, paramyosin peptide vaccination did not engender protective immunity or liver pathology reduction and immunization was associated with a Th2 type of immune response. (C) 2008 Elsevier B.V. All rights reserved.

Biliary and pancreatic dysgenesis in mice harboring a mutation in Pkhd1

Autosomal recessive polycystic kidney disease is a hereditary fibrocystic disease that involves the kidneys and the biliary tract. Mutations in the PKHD1 gene are responsible for typical forms of autosomal recessive polycystic kidney disease. We have generated a mouse model with targeted mutation of Pkbd1 by disrupting exon 4, resulting in a mutant transcript with deletion of 66 codons and expression at similar to 30% of wild-type levels. Pkhd1(del4/d3l4) mice develop intrahepatic bile duct proliferation with progressive cyst formation and associated periportal fibrosis. In addition, these mice exhibit extrahepatic manifestations, including pancreatic cysts, splenomegaly, and common bile duct dilation. The kidneys are unaffected both histologically and functionally. Fibrocystin is expressed in the apical membranes and cilia of bile ducts and distal nephron segments but is absent from the proximal tubule. This pattern is unchanged in orthologous models of autosomal dominant polycystic kidney disease due to mutation in Pkd1 or Pkd2. Mutant fibrocystin in Pkhd1(del4/d3l4) mice also retains this expression pattern. The hypomorphic Pkhd1(del4/d3l4) mouse model provides evidence that reduced functional levels of fibrocystin are sufficient for cystogenesis and fibrosis in the liver and pancreas, but not the kidney, and supports the hypothesis of species-dependent differences in susceptibility of tissues to Pkbdl mutations.

Intake of trans Fatty Acids Causes Nonalcoholic Steatohepatitis and Reduces Adipose Tissue Fat Content

We investigated the effects of dietary trans fatty acids, PUFA, and SEA on body and liver fat content, liver histology, and mRNA of enzymes involved in fatty acid metabolism. LDL receptor knockout weaning male mice were fed for 16 wk with diets containing 40% energy as either trans fatty acids (TRANS), PUFA, or SEA. Afterwards, subcutaneous and epididymal fat were weighed and histological markers of nonalcoholic fatty liver disease (NAFLD) were assessed according to the Histological Scoring System for NAFLD. PPAR alpha, PPAR gamma, microsomal triglyceride transfer protein (MTP), carnitine palmitoyl transferase 1 (CPT-1), and sterol regulatory element binding protein-1c (SREBP-1c) mRNA were measured by quantitative RT-PCR. Food intake was similar in the 3 groups, although mice fed the TRANS diet gained less weight than those receiving the PUFA diet. Compared with the PUFA- and SEA-fed mice, TRANS-fed mice had greater plasma total cholesterol (TC) and triglyceride (TG) concentrations, less epididymal and subcutaneous fat, larger livers with nonalcoholic steatohepatitis (NASH)-like lesions, and greater liver TC and TG concentrations. Macrosteatosis in TRANS-fed mice was associated with a higher homeostasis model assessment of insulin resistance (HOMA(IR)) index and upregulated mRNA related to hepatic fatty acid synthesis (SREBP-1 c and PPAR gamma) and to downregulated MTP mRNA. Diet consumption did not alter hepatic mRNA related to fatty acid oxidation (PPAR alpha and CPT-1). In conclusion, compared with PUFA- and SFA-fed mice, TRANS-fed mice had less adiposity, impaired glucose tolerance characterized by greater HOMA(IR) index, and NASH-like lesions due to greater hepatic lipogenesis. These results demonstrate the role of trans fatty acid intake on the development of key features of metabolic syndrome. J. Nutr. 140: 1127-1132, 2010.

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 mere 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 mere 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.

Functional growth hormone (GH) receptors and GH are expressed by preimplantation mouse embryos: A role for GN in early embryogenesis?

The results of this study challenge the widely held view that growth hormone (GH) acts only during the postnatal period. RNA phenotyping shows transcripts for the GH receptor and GH-binding protein in mouse preimplantation embryos of all stages from fertilized eggs (day 1) to blastocysts (day 4). An antibody specific to the cytoplasmic region of the GH receptor revealed receptor protein expression, first in two-cell embryos, the stage of activation of the embryonic genome (day 2), and in all subsequent stages, In cleavage-stage embryos this immunoreactivity was localized mainly to the nucleus, but clear evidence of membrane labeling was apparent in blastocysts. GH receptor immunoreactivity was also observed in cumulus cells associated with unfertilized oocytes but not in the unfertilized oocytes. The blastocyst receptor was demonstrated to be functional, exhibiting the classic bell-shaped dose-response curves for GH stimulation of both 3-O-methyl glucose transport and protein synthesis. Maximal stimulation of 40-50% was seen for both responses at less than 1 ng/ml recombinant GH, suggesting a role for maternal GK. However mRNA transcripts for GH were also detected from the morula stage (day 3) by using reverse transcription-PCR, and GH immunoreactivity was seen in blastocysts. These observations raise the possibility of a paracrine/autocrine GH loop regulating embryonic development in its earliest stages.

Hypertonic saline reduces metalloproteinase expression in liver during pancreatitis

P>We recently demonstrated that hypertonic saline reduces inflammation and mortality in acute pancreatitis. The present study investigated the effects of hypertonic saline in metalloproteinase (MMP) regulation and pancreatitis-associated hepatic injury. Wistar rats were divided into four groups: (i) control, not subjected to insult or treatment; (ii) no treatment (NT), induction of pancreatitis (retrograde infusion of 2.5% sodium taurocholate (1.0 mL/kg)), but no further treatment; (iii) normal saline (NS), induction of pancreatitis and treatment with normal saline (0.9% NaCl, 34 mL/kg, i.v. bolus, 1 h after the induction of pancreatitis); and (iv) hypertonic saline (HS), induction of pancreatitis and treatment with hypertonic saline (7.5% NaCl, 4 mL/kg administered over a period of 5 min, 1 h after the induction of pancreatitis). In all four groups, 4, 12 and 24 h after the induction of pancreatitis, liver tissue samples were assayed to determine levels of MMP-2, MMP-9, 47 kDa heat shock protein (HSP47) and collagen (Type I and III). Compared with the control group, MMP-9 expression and activity was increased twofold in the NS and NT groups 4 and 12 h after the induction of pancreatitis, but remained at basal levels in the HS group. In contrast, MMP-2 expression was increased twofold 12 h after the induction of pancreatitis only in the NS group, whereas the expression of HSP47 was increased 4 h after the induction of pancreatitis in the NS and NT groups. Greater extracellular matrix remodelling occurred in the NS and NT groups compared with the HS group, probably as a result of the hepatic wound-healing response to repeated injury. However, the collagen content in hepatic tissue remained at basal levels in the HS group. In conclusion, the results of the present study indicate that hypertonic saline is hepatoprotective and reduces hepatic remodelling, maintaining the integrity of the hepatic extracellular matrix during pancreatitis. Hypertonic saline-mediated regulation of MMP expression may have clinical relevance in pancreatitis-associated liver injury.

Increased tissue resistance in the nude mouse against Candida albicans without altering strain-dependent differences in susceptibility

Strain differences in tissue responses to infection with Candida albicans were examined in nude mice having susceptible (CBA/CaH) and resistant (BALB/c) parentage. Homozygous (nu/nu) mice of both strains were more resistant to systemic infection with C. albicans than heterozygous (nu/+) littermates as indicated by a reduction in both the severity of tissue damage and colony counts in the brain and kidney. However, the tissue lesions in nu/nu CBA/CaH mice were markedly more severe than those in nu/nu mice with the BALB/c background. This pattern was reflected in the greater fungal burden in the CBA/CaH strain. Analysis of cDNA from infected tissues using a competitive polymerase chain reaction excluded interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha), and interleukin 6 (IL-6) as mediators of the enhanced resistance of the nude mice. The results confirm that the different patterns of lesion severity in BALB/c and CBA/CaH mice do not involve T lymphocyte-mediated pathology, and are consistent with the hypothesis that strain-dependent tissue damage is not dependent on the effector function of macrophages or their precursors.

Mini-microabscess syndrome in liver transplant recipients

Cytomegalovirus (CMV) is a significant cause of morbidity in immunosuppressed patients. It is characterized in the liver by parenchymal microabscesses, usually containing CMV-infected cells. However, not all hepatic microabscesses are due to CMV infection. In 1992, we described ''mini'' microabscess (MMA) syndrome, a distinct clinical syndrome that occurs in transplanted livers. This report analyzes the clinical and laboratory features of 57 cases of MMA syndrome occurring in 52 patients and compares these with 19 biopsy-proven cases of CMV infection. The diagnosis of MMA syndrome can only be made histologically. The microabscesses are smaller and more numerous than in CMV infection, and there are no viral inclusions present. CMV DNA could not be detected in liver biopsy specimens with MMAs by using ''nested'' polymerase chain reaction (PCR), indicating that MMA syndrome is not caused by CMV infection. The pattern of liver enzyme and bilirubin elevation is predominantly hepatocellular, with transaminase levels elevated, on average, six to eight times the upper limit of normal. The clinical features of MMA syndrome are that it predominantly affects female (40 of 52 patients) orthotopic liver transplant (OLT) recipients of all ages (range, 11 months to 66.9 years). MMA syndrome is unrelated to the indication for initial OLT and tends to occur later after transplantation than CMV infection (median, 91 days post-OLT vs. 32 days for CMV hepatitis). Although the etiology of MMA syndrome is not clear, it does not appear to adversely affect graft or patient survival.

Kinetics of elimination and distribution in blood and liver of biocompatible ferrofluids based on Fe(3)O(4) nanoparticles: An EPR and XRF study

In this study, we evaluated the biodistribution and the elimination kinetics of a biocompatible magnetic fluid, Endorem (TM), based on dextrancoated Fe(3)O(4) nanoparticles endovenously injected into Winstar rats. The iron content in blood and liver samples was recorded using electron paramagnetic resonance (EPR) and X-ray fluorescence (XRF) techniques. The EPR line intensity at g=2.1 was found to be proportional to the concentration of magnetic nanoparticles and the best temperature for spectra acquisition was 298 K. Both EPR and XRF analysis indicated that the maximum concentration of iron in the liver occurred 95 min after the ferrofluid administration. The half-life of the magnetic nanoparticles (MNP) in the blood was (11.6 +/- 0.6) min measured by EPR and (12.6 +/- 0.6) min determined by XRF. These results indicate that both EPR and XRF are very useful and appropriate techniques for the study of kinetics of ferrofluid elimination and biodistribution after its administration into the organism. (c) 2007 Elsevier B.V. All rights reserved.

Real-time and Doppler US after pediatric segmental liver transplantation

Background Accurate diagnosis of portal vein (PV) stenosis by real-time and color Doppler US (CD-US) after segmental liver transplantation in children can decrease morbidity by avoiding unnecessary biopsy, PV hypertension, thrombosis and loss of the graft. Objective To evaluate CD-US parameters for the prediction of PV stenosis after segmental liver transplantation in children. Materials and methods We retrospectively reviewed 61 CD-US examinations measuring the diameter at the PV anastomosis, velocities at the anastomosis (PV1) and in the segment proximal to the anastomosis (PV2), and the PV1/PV2 velocity ratio. The study group comprised patients with stenosis confirmed by angiography and the control group comprised patients with a good clinical outcome. Results PV stenosis was seen in 12 CD-US examinations. The mean PV diameter was smaller in the study group (2.6 mm versus 5.7 mm) and a PV diameter of < 3.5 mm was highly predictive of stenosis (sensitivity 100%, specificity 91.8%). Conclusion A PV diameter of < 3.5 mm is a highly predictive CD-US parameter for the detection of hemodynamically significant stenosis on angiography.

Amino acids change liver growth factors gene expression in malnourished rats

Background: Glutamine and proline are metabolized in the liver and may collaborate on its regeneration. Parenteral nutrition (PN) containing either glutamine or proline was given to partially hepatectomized rats. The total RNA content and growth factor gene expression in hepatic remnants was measured, to determine the effects of these amino acid supplementation on the expression of growth factors during liver regeneration. Methods: Wistar rats nourished (HN) and malnourished (HM) were hepatectomized and divided in two groups: 20 receiving PN enriched with Alanyl-Glutamine (HN-Gln and HM-Gln) and 20 PN enriched with proline+alanine (HN-Pro and HM-Pro). The control groups comprised 7 nourished (CN) and 7 malnourished (CM) rats that didn`t undergo surgery. Growth factor and thymidine kinase mRNA levels were measured by RT-PCR. Results: In nourished rats, total hepatic RNA levels were lower in the HN-Gln and HN-Pro groups (0.75 and 0.63 mu g/mg tissue, respectively) than in control group (1.67 mu g/mg tissue) (P<0.05). In malnourished rats, total hepatic RNA content was higher in the HM-Pro group than FIN-Pro, HM-Gln, and CM (3.18 vs. 0.63, 0.93 and 1.10 mu g/mg, respectively; P<0.05). Hepatocyte growth factor mRNA was more abundant in the HM-Gln group when compared to CM (031 vs. 0.23 arbitrary units) and also in HM-Pro in relation to HM-Gln, HN-Pro, and CM (0.46 vs. 033 and 0.23, respectively, P<0.05). Conclusions: Proline or glutamine supplementation in malnourished rats improves total RNA content in the remnant hepatic tissue. Amino acids administration increased HGF gene expression after partial hepatectomy in malnourished rats, with a greater effect of proline than glutamine.

Long-Term Results of Percutaneous Bilioenteric Anastomotic Stricture Treatment in Liver-Transplanted Children

The purpose of this study was to evaluate the mid- and long-term results of percutaneous transhepatic cholangiography (PTC) and biliary drainage in children with isolated bilioenteric anastomotic stenosis (BAS) after pediatric liver transplantation. Sixty-four children underwent PTC from March 1993 to May 2008. Nineteen cholangiograms were normal; 10 showed intrahepatic biliary stenosis and BAS, and 35 showed isolated BAS. Cadaveric grafts were used in 19 and living donor grafts in 16 patients. Four patients received a whole liver, and 31 patients received a left lobe or left lateral segment. Roux-en-Y hepaticojejunostomy was performed in all patients. Indication for PTC was based on clinical, laboratory, and histopathologic findings. In patients with isolated BAS, dilation and biliary catheter placement, with changes every 2 months, were performed. Patients were separated into 4 groups according to number of treatment sessions required. The drainage catheter was removed if cholangiogram showed no significant residual stenosis and normal biliary emptying time after a minimum of 6 months. The relationship between risk factors (recipient`s weight < 10 kg, previous exposure to Cytomegalovirus, donor-recipient sex and weight relations, autoimmune disease as indication for transplantion, previous Kasai`s surgery, use of reduced liver grafts, chronic or acute rejection occurrence) and treatment was evaluated. Before PTC, fever was observed in 46%, biliary dilation in 23%, increased bilirubin in 57%, and increased gamma-glutamyltransferase (GGT) in 100% of patients. In the group with BAS, 24 of 35 (69%) patients had histopathologic findings of cholestasis as did 9 of 19 (47%) patients in the group with normal PTC. Of the 35 patients, 23 (65.7%) needed 1 (group I), 7 needed 2 (group II), 4 needed 3 (group III), and 1 needed 4 treatment sessions (group IV). The best results were observed after 1 treatment session, and the mean duration of catheter placement and replacement was 10 months. The primary patency rate was 61.2%, and the recurrence rate was 34.3% (group I). Seven patients (7 of 35; 20%) had their stricture treated with a second treatment session (group II). The average drainage time in group II was 24 months. During a period > 20 months, 4 patients (4 of 35; 11.4%) required 1 additional treatment session (group III), and 1 patient (1 of 35; 2.9%) had a catheter placed at the end of the study period (group IV). Drainage time in group I was significantly shorter than those in groups II, III, and IV (p < 0.05). There was no statistically significant relation between therapeutic response and the selected risk factors (p > 0.05). The majority of complications, such as catheter displacement and leakage, were classified as minor; however, 2 patients (5.7%) with hemobilia were noted. Complications increased according to the need for reintervention. In conclusion, balloon dilation and percutaneous drainage placement is safe and effective, and it has long-term patency for children with BAS after liver transplantation. Because of prolonged treatment time, reintervention may be necessary, thereby increasing the complication rate. Balloon dilation and percutaneous drainage placement should be considered as the first treatment option because of its minimally invasive nature.

Human leukemia inhibitory factor upregulates LDL receptors on liver cells and decreases serum cholesterol in the cholesterol-fed rabbit

In a previous study, we found that the cytokine (human) leukemia inhibitory factor (hLIF) significantly reduced plasma cholesterol levels and the accumulation of lipid in aortic tissues of cholesterol-fed rabbits after 4 weeks of treatment. The mechanisms by which this occurs were investigated in the present study. This involved examining the effect of hLIF on (1) the level of plasma cholesterol at different times throughout the 4-week treatment and diet period; (2) smooth muscle cell (SMC) and macrophage-derived foam cell formation in vitro; and (3) LDL receptor expression and uptake in the human hepatoma cell line HepG2. At time zero, an osmotic minipump (2-mL capacity; infusion rate, 2.5 mu L/h; 28 days) containing either hLIF (30 mu g.kg(-1).d(-1)) or saline was inserted into the peritoneal cavity of New Zealand White rabbits (N=24). Rabbits were divided into four groups of six animals each. Group 1 received a normal diet/saline; group 2, a normal diet/hLIF; group 3, a 1% cholesterol diet/saline; and group 4, a 1% cholesterol diet/hLIF. hLIF had no effect on the plasma lipids or artery wall of group 2 rabbits (normal diet). However, in group 4 rabbits, plasma cholesterol levels and the percent surface area of thoracic aorta covered by fatty streaks was decreased by approximate to 30% and 80%, respectively, throughout all stages of the 4-week treatment period. In vitro, hLIF failed to prevent lipoprotein uptake by either SMCs or macrophages (foam cell formation) when the cells were exposed to P-VLDL for 24 hours. In contrast, hLIF (100 ng/mL) added to cultured human hepatoma HepG2 cells induced a twofold or threefold increase in intracellular lipid accumulation in the medium containing 10% lipoprotein-deficient serum or 10% fetal calf serum, respectively. This was accompanied by a significant non-dose-dependent increase in LDL receptor expression in hLIF-treated HepG2 cells incubated with LDL (20 mu g/mL) when compared with controls (P

Midterm and long-term results of percutaneous endovascular treatment of venous outflow obstruction after pediatric liver transplantation

PURPOSE: To evaluate retrospectively the midterm and long-term results of percutaneous endovascular treatment of venous outflow obstruction after pediatric liver transplantation. MATERIALS AND METHODS: During a 9-year period, 18 children with obstruction of a hepatic vein (HV) or inferior vena cava (IVC) anastomosis underwent percutaneous transluminal angioplasty (PTA) with balloon dilation or stent placement in case of PTA failure after liver transplantation. Patients` body weights ranged from 7.7 kg to 42.6 kg (mean, 18.8 kg +/- 9). Potential predictors of patency were compared between balloon dilation and stent placement groups. RESULTS: Forty-two procedures were performed (range, 1-11 per patient; mean, 2). Technical and initial clinical success were achieved in all cases. Major complications included one case of pulmonary artery stent embolization and one case of hemothorax. Three children (25%) with HV obstruction were treated with PTA and nine (75%) were treated with stent placement. Three children with IVC obstruction (75%) were treated with PTA and one (25%) was treated with a stent. There were two children with simultaneous obstruction at the HV and IVC; one was treated with PTA and the other with a stent. Cases of isolated HV stenosis have a higher probability of patency with balloon-expandable stent treatment compared with balloon dilation (P < .05). Follow-up time ranged from 7 days to 9 years (mean, 42 months +/- 31), and the primary assisted patency rate was 100% when stent placement was performed among the first three procedures. CONCLUSIONS: In cases of venous outflow obstruction resulting from HV and/or IVC lesions after pediatric liver transplantation, percutaneous endovascular treatment with balloon dilation or stent placement is a safe and effective alternative treatment that results in long-term patency.