Sing Hiem Yap

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.

The effect of beclobric acid and clofibric acid on peroxisomal β-oxidation and peroxisome proliferation in primary cultures of rat, monkey and human hepatocytes

The peroxisome-proliferating effects of clofibric acid and beclobric acid were studied in primary cultures of hepatocytes derived from rat, monkey (Macaca fascicularis) and human liver. Determination of peroxisomal fatty acid beta-oxidation and morphometrical analysis of the peroxisomal compartment were performed after incubation of 1-day-old hepatocyte cultures for 3 days with either compound. In rat liver cell cultures both compounds gave a 10-fold increase in peroxisomal beta-oxidation, a 3-fold increase in the relative number of peroxisomes and a 1.5-fold increase in the mean size of peroxisomes. Beclobric acid gave its maximal effect at a concentration of 10 microM, which is at least one order of magnitude lower than the maximum-effect concentration of clofibric acid. At concentrations greater than 300 microM beclobric acid was cytotoxic. No stimulation of peroxisomal fatty acid beta-oxidation was found in either monkey or human hepatocyte cultures. Morphometrical analysis also showed no increase in the peroxisomal compartment in cultures derived from these species, as indicated by the lack of increase in both relative number and size of peroxisomes. In all three species tested beclobric acid was equally cytotoxic for hepatocytes in vitro. These results are of relevance for the interpretation of the peroxisome-proliferating effects of clofibrate and similar compounds in rats. Since peroxisome proliferation may be correlated to increased hepatic tumour incidences in the rat, the absence of peroxisome proliferation in primates suggests the absence of tumourogenic activity by hypolipidemic compounds in these species.

Etiologic factors of jaundice in severely ill patients: A retrospective study in patients admitted to an intensive care unit with severe trauma or with septic intra-abdominal complications following surgery and without evidence of bile duct obstruction

In order to understand the pathophysiology of jaundice in severely ill patients, we have examined several possible promoting factors in a retrospective study of 86 patients with multiple organ failure admitted to an intensive care unit (ICU). Patients with bile duct obstruction were excluded from this study. Cholestatic jaundice had developed in 19 of 54 patients after trauma and in 20 of 32 patients after septic intra-abdominal complications. No differences were found between the icteric and non-icteric groups of patients with regard to median age, sex distribution, duration of stay in the ICU, number of operations, utilization of gaseous and/or intravenously administered anaesthetics and lipid, and administration of potential hepatotoxic drugs. Twenty-six of 39 icteric patients had a normal renal function. However, a significantly higher number of blood transfusions was found in the icteric as compared to the non-icteric patients. The higher number of blood transfusions and the incidence of initial shock in the icteric trauma patients were probably related to the higher injury severity score. Furthermore, sepsis was found significantly more frequently in the icteric trauma patients, while the number of organ failures when the presence of jaundice was not accounted for was the same in both groups. Nevertheless, the severity of jaundice correlated well with the increasing number of failing organs and the increasing mortality. From these findings we can therefore conclude that jaundice occurring in patients with multiple organ failure is usually not due to the administration of potential hepatotoxic drugs. However, the number of blood transfusions may be an important associated factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct evidence of transcriptional control of fibrinogen and albumin synthesis in rat liver during the acute phase response

Summary Using immunoprecipitation technique we have purified fibrinogen polypeptide mRNAs to homogeneity as demonstrated by translation in a wheat germ cell free system and by hybridization kinetics. By measuring the sequence contents of fibrinogen polypeptide mRNAs and albumin mRNA, using tritiated DNAs complementary to fibrinogen polypeptide mRNAs and to albumin mRNA respectively, a dramatical increase in mRNA content of fibrinogen polypeptides (7 fold) and a decrease in albumin mRNA content (2 fold) have been found in the liver of rats 24 hours after i.m. injection of 1.0 ml turpentine. These results were consistent with the findings in cell free translation under the direction of poly A + RNA prepared from livers of experimental animals and suggest that the fibrinogen and albumin synthesis during the acute phase response is reciprocally regulated at the transcriptional level.

Distribution of mRNAs of fibrinogen polypeptides and albumin in free and membrane-bound polyribosomes and induction of α-fetoprotein mRNA synthesis during liver regeneration after partial hepatectomy

To study the effect of regenerative response of the liver following partial hepatectomy on the synthesis of major plasma proteins (secretory proteins), we have determined the sequence contents and the distribution of albumin and fibrinogen polypeptide mRNAs in rat liver at intervals after partial hepatectomy and sham operation. Using a quantitative technique for the isolation of polyribosomes, we demonstrated that the distribution of RNA between free and membrane-bound polyribosomal fraction was unchanged in these experiments. There was no shift in the polyribosomal population to favor free polyribosomes after partial hepatectomy. However, there was a dramatic increase (5-6-fold) of the fibrinogen polypeptide mRNA concentration during the first 24 h after resection. In contrast, the albumin mRNA concentration decreased (2-3-fold). There were no alpha-fetoprotein mRNA sequences detectable in any liver RNA fraction in these experimental animals. In sham-operated rats with intact livers, similar changes of fibrinogen polypeptide and albumin mRNA concentrations as described in regenerating liver after partial hepatectomy, were observed. These results suggest that albumin and fibrinogen synthesis after partial hepatectomy is reciprocally regulated at the mRNA level and represents a nonspecific acute phase response to surgical trauma.

In vitro infection of primary cultures of cryopreserved adult human hepatocytes with hepatitis B virus

Infection with HBeAg and HBV DNA positive serum in primary cultures of cryopreserved human hepatocytes in the presence of human whole blood in the medium was performed in 8 consecutive experiments. HBsAg and HBV DNA release into the medium was increased in the second week after infection. Via immunostaining, HBcAg was first observed in the nucleus of hepatocytes approximately 3 days after infection. A maximal percentage of HBcAg positive cells in 0.1% of cultured hepatocytes was detected on the 7th day. HBsAg was also first demonstrated on the 3rd day, and predominantly localized in the cytoplasm. About 5% of hepatocytes were HBsAg positive on the 12th day after infection. The percentage of positive cells did not appear to increase after this time. Using in situ cytohybridization and agarose gel electrophoresis and Southern blot analysis, HBV DNA was first detected on the 4th day. In addition, electron microscopic studies revealed the presence of 42 nm virus-like particles in the cytoplasm of infected cells in the second week after infection. This in vitro system provides a model for studying the mechanism of HBV infection, viral replication and maturation. However, further improvement of culture systems is needed, to increase the number of infected cells and for active HBV replication.

Activation of mutagens by hepatocytes and liver 9000 × g supernatant from human origin in the Salmonella typhimurium mutagenicity assay: Comparison with rat liver preparations

The mutagenicity of 10 known genotoxic compounds, of several chemical classes, was measured in Salmonella typhimurium mutagenicity assays comprising isolated human hepatocytes or human liver 9000 X g supernatant (S9) from 4 different individuals, as activating system. The mutagenic activity of several compounds as determined with the Salmonella/hepatocyte suspension assay showed obvious differences when compared with the values obtained in the Salmonella/S9 plate assay. For instance, the mutagenic activity of BZ, DMN and DEN appeared to be much higher in the hepatocyte assay than in the S9 assay. However, 2-AF and 2-AAF were activated more effectively into mutagens in the S9 assay than in the hepatocyte assay. 2-AF was slightly more mutagenic than 2-AAF in the hepatocyte assay, whereas it was far more mutagenic than 2-AAF in the S9 assay. DMN was found more mutagenic than DEN in the hepatocyte assay, whereas in the S9 assay DEN appeared to be slightly more mutagenic. Furthermore, great interindividual differences in the metabolic activation of certain compounds, e.g. BZ and DMN, were observed in the hepatocyte suspension assay, whereas these variations were less evident in the S9 plate assay. Comparison of the mutagenicity data obtained with the human liver preparations, with those obtained with rat liver preparations, showed great interspecies differences in the capacity to activate certain chemicals into mutagens. The use of human liver preparations, in particular isolated human hepatocytes, may be of great value in studies on inter- and intraspecies variations in metabolic activation of genotoxic agents.

Restoration Effects of Glucose Refeeding on Reduced Synthesis of Albumin and Total Protein and on Disaggregated Polyribosomes in Liver of Starved Rats: Evidence of a Post-Transcriptional Control Mechanism

Previous studies demonstrated that fasting is accompanied with a reduction of liver protein and albumin synthesis. A protein-deficient diet also leads to a marked change in liver RNA and protein metabolism. Although the reduction of protein synthesis and the disaggregated polyribosomes during fasting can be corrected by a single feeding of protein or a complete amino acid mixture, no or little changes of amino acid concentrations were found in portal blood and liver cytosol of fasted animals as compared to those of the fed group. To determine the effect of glucose on the reduced rate of protein and albumin synthesis of fasted rats, free and membrane-bound polyribosomes were isolated quantitatively from liver of starved rats (42-66 h) at different intervals after a single feeding of glucose and after giving glucose ad libitum for 24 h. (1) The yield of polyribosomal RNA decreased dramatically after a 42- to 66-hour starvation. A glucose refeeding did not change the RNA content. However, the restoration of polyribosome size could be observed rapidly. (2) At various levels of RNA, there was a decreased protein synthesis in fasted animals. However, the synthesis was enhanced after glucose refeeding. The albumin synthesis was also proportionately increased (10-12% of total protein synthesis of membrane-bound polyribosomes). (3) Glucose refeeding had no influence on the content of albumin mRNA sequence and liver RNA. These findings suggest that the effect of glucose on the restoration of protein and albumin synthesis is a sole post-transcriptional event.

In vitro binding properties of the hepatitis delta antigens to the hepatitis B virus envelope proteins: potential significance for the formation of delta particles.

To investigate the possible existence of (a) reactive binding site(s) on the hepatitis B surface antigen (HBsAg) for the hepatitis delta antigen (delta Ag) in the hepatitis delta virus (HDV), we performed binding studies using recombinant (rec)Small, recMiddle, recLarge HBsAg and recombinant small (S) and large (L) hepatitis delta antigen (recS delta Ag, recL delta Ag). Rec delta Ag was immobilized onto microtiter plates and incubated with recSmall, recMiddle and recLarge HBsAg. Of the three HBsAg proteins only the recMiddle HBsAg was found to bind to recS delta Ag. This binding was inhibited by the addition of synthetic PreS2 peptide but not by small HBsAg, indicating that the S delta Ag exhibits a PreS2 binding site. RecL delta Ag bound to all three forms of HBsAg. The binding of the HBsAg to recL delta Ag was saturable and could be blocked with an excess of HBsAg, but not with BSA. The region of the additional 19 amino acids of the L delta Ag is therefore responsible for the creation of the small HBsAg binding site on the L delta Ag. We therefore suggest that all HBsAg proteins but particularly the small HBsAg in the HDV coat seem to be involved in the interaction with the HDV core particle and that the PreS2 region of the middle HBsAg plays a crucial role in binding to small delta Ag during HDV particle formation, probably to increase the stability of the HDV particle.

The influence of glucocorticoid on albumin synthesis and its messenger RNA in rat in vivo and in hepatocyte suspension culture

Corticosteroids are known to stimulate the synthesis of a number of liver-specific proteins. The reports regarding the effect of glucocorticoid on albumin synthesis in vivo and in vitro are controversial. In an attempt to determine the mechanism by which glucocorticoid exerts its influence on hepatic albumin synthesis and to find an explanation for the conflicting data, we have studied the effect of dexamethasone disodium phosphate on albumin synthesis and albumin messenger RNA as determined by the molecular hybridization technique in hepatocytes in rat in vivo and in suspension culture. In hepatocyte suspension culture, addition of 0.48 microM dexamethasone in medium at zero time led to a significant increase (20%) in incorporation of labeled precursor into albumin as compared to control experiments; this was accompanied by a maintainance of the initial level of full-length albumin mRNA for a 9 h period. In hepatocytes cultured without dexamethasone in the medium there was a progressive loss of albumin mRNA content. Despite this finding, dexamethasone was not able to increase the albumin mRNA content in hepatocyte to a level higher than the initial value. Moreover, administration of this hormone either intraperitoneally or intravenously into rats did not lead to enhanced cell-free albumin synthesis or to an increased level of albumin mRNA. These findings suggest that glucocorticoid does not play an essential role in the regulation of albumin synthesis in vivo. In vitro, however, glucocorticoid leads to a preservation of the initial level of albumin mRNA and thus plays a role in the control of spontaneous dedifferentiation of liver cells in culture.