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Biomedical subjects

R A Chamuleau

Publications and source records attributed to R A Chamuleau.

At least 37 records · Page 2Linked to original sources
PubMed1998

Does the extend of the culture time of primary hepatocytes in a bioreactor affect the treatment efficacy of a bioartificial liver?

The purpose of this study was to investigate whether the efficacy of our novel extracorporeal bioartificial liver (BAL) to support rats with complete liver ischemia (LIS) could be improved by extending the culture time of freshly isolated porcine hepatocytes from 14 hours to 38 hours. The results showed that survival as well as porcine hepatocyte integrity improved, the onset of coma delayed, and the ammonia levels decreased in LIS rats of the 38 hour group compared to the 14 hour group, but no statistically significant differences were observed. In the 38 hour group, but not the 14 hour group, the onset of hepatic encephalopathy was significantly delayed and ammonia metabolism significantly improved compared to the LIS rats in control groups that only received a glucose infusion or were connected to a BAL without cells. In conclusion, prolonged hepatocyte recovery favoured all investigated parameters, although not all observed effects were statistically significant. More research is required to find out how long primary hepatocytes should be cultured in a bioreactor for optimal BAL support.

Ammonia↗
PubMed1997

Effect of a protein-rich meal on urinary and salivary free amino acid concentrations in human subjects.

The aim of the present study was to investigate whether in healthy volunteers acute changes in plasma free amino acid composition after a protein-rich test meal are reflected in the urinary and salivary concentrations of the corresponding amino acids. The ingestion of a protein-rich meal elicited a significant increase of plasma and urine amino acid concentrations. The postprandial salivary amino acid excretion showed only minor changes. For several amino acids (alanine, arginine, asparagine, glycine, threonine and valine) significant relations were observed between the increase in concentration of these amino acids in venous plasma and urine. In whole saliva, only threonine and valine showed a significant relationship with the corresponding plasma concentration. Our data suggest that the urinary amino acid excretion of several amino acids has the potential for estimating short-term changes in plasma concentrations. Determination of salivary amino acid concentrations seems less appropriate for this purpose.

Adult↗
PubMed1997

Correlation of repressed transcription of alpha-tocopherol transfer protein with serum alpha-tocopherol during hepatocarcinogenesis.

Using a subtraction-enhanced display technique, we identified a rodent alpha-tocopherol transfer protein (alpha-TTP) cDNA which exhibited markedly lower messenger RNA (mRNA) amounts in rat hepatocellular carcinoma (HCC) than in healthy controls. Several lines of evidence have substantiated that abnormal alpha-TTP results in isolated vitamin E deficiency. In this study, we investigated the hepatic mRNA amounts of alpha-TTP during rat hepatic carcinogenesis and liver regeneration on Northern blot, localization of alpha-TTP mRNA in HCC of rats and humans by in situ hybridization, and we analyzed the correlation between alpha-TTP mRNA and alpha-tocopherol. alpha-TTP mRNA concentrations of the rats were decreased at the early stage of hepatic carcinogenesis, and remained 3-5-fold reduced as the tumor progressed. In parallel, serum alpha-tocopherol concentrations were significantly decreased to 40% of those in the controls at the early stages of rat hepatic carcinogenesis (p < 0.01). The 2 data sets were strongly correlated (r = 0.834, p < 0.001). In situ hybridization revealed that a decrease of alpha-TTP mRNA was preferentially localized in the tumor nodules of rats and humans with HCC. Our data suggest that repressed transcription of alpha-TTP is associated with a decrease of serum alpha-tocopherol and with hepatic carcinogenesis.

Aged↗
PubMed1997

Overexpression of prothymosin alpha, concomitant with c-myc, during rat hepatic carcinogenesis.

By using a subtraction-enhanced display technique, we identified a cDNA clone representing alpha-prothymosin, from rat hepatocellular carcinoma (HCC). alpha-Prothymosin has been reported to be involved in cell proliferation and regulated by c-myc gene in vitro. In the present study, we investigated the gene expression pattern of alpha-prothymosin and analyzed its correlation with c-myc during rat hepatic carcinogenesis and liver regeneration. Hepatic alpha-prothymosin mRNA levels, concomitant with c-myc, were increased at the early stage of hepatic carcinogenesis (6 weeks), and remained nearly 10 fold higher as the tumor progressed. In comparison, alpha-prothymosin mRNA levels were only slightly and moderately increased at early (3-6 hr) and at later stage (24-30 hr) of liver regeneration after 70% partial hepatectomy. In situ hybridization revealed that overexpressed alpha-prothymosin mRNA was restricted to the tumor nodules and to tumor cells invading blood vessels. These data provide evidence that overexpression of alpha-prothymosin, concomitant with c-myc, is related to rat hepatic carcinogenesis.

Animals↗
PubMed1997

No attenuation of ischemic and reperfusion injury in Kupffer cell-depleted, cold-preserved rat livers.

Activated Kupffer cells (KC) have been implicated in the damage sustained by preserved liver grafts during ischemia and reperfusion. The aim of this study was to compare ischemia/reperfusion injury in preserved, KC-depleted rat livers and preserved control livers, with special regard to sinusoidal endothelial cell (SEC) injury. Wistar rats were injected with liposome-encapsulated dichloromethylene diphosphonate, 48 hr before hepatectomy, to eliminate KC, or were withheld this pretreatment (controls). Livers were flushed with cold University of Wisconsin solution and after 0, 8, 16, or 24 hr of storage at 4 degrees C, were reperfused in a recirculation system with 200 ml of oxygenated Krebs-Henseleit solution at 37 degrees C for 90 min. Damage to SEC was measured by the uptake of hyaluronic acid (HA) from the perfusate and release of purine nucleoside phosphorylase (PNP). Perfusate samples were, furthermore, analyzed for aspartate aminotransferase (AST) and tumor necrosis factor-alpha. Carbon particles were infused in the perfusate to determine the phagocytotic capacity of KC. Biopsies were taken for histological examination and sections were stained with ED2 monoclonal antibodies to confirm the absence of KC. After 90 min of reperfusion, immediately after cold flush (t0), the uptake of HA was 72.2+/-2.3% and 69.3+/-1.3% in KC-depleted livers and in control livers, respectively (n.s.). After 8 hr of storage, HA uptake was 21.6+/-4.5% and 34.6+/-8.0%, respectively (n.s.). After 16 and 24 hr of storage and reperfusion, no uptake of HA was found in either KC-depleted or control livers, indicating abolished SEC function. PNP activities in the perfusate were higher in control livers (after 8 and 24 hr of storage), presumably due to release from damaged KC. No difference was found in AST and no tumor necrosis factor-alpha was measured in the perfusates of normal and KC-depleted livers. Electron microscopic studies showed that after 8 and 24 hr of storage and reperfusion, KC were activated and were able to phagocytose colloidal carbon. Our conclusion was that the elimination of Kupffer cells did not result in reduction of ischemic and reperfusion damage in livers preserved up to 24 hr, as assessed in vitro by SEC uptake of HA, PNP release, and AST release.

Animals↗
PubMed1997

Memantine, a noncompetitive NMDA receptor antagonist improves hyperammonemia-induced encephalopathy and acute hepatic encephalopathy in rats.

The aim of this study was to investigate the possible role of N-methyl-D-aspartate (NMDA)-receptor overactivity in two different experimental rat models of encephalopathy: subacute encephalopathy caused by severe hyperammonemia in portacaval-shunted rats (AI-PCS rats) and acute hepatic encephalopathy caused by complete liver ischemia (LIS rats). The effect of the noncompetitive NMDA-receptor antagonist memantine (intraperitoneal [i.p.] 10-20 mg/kg bw or intravenous [i.v.] 5 mg/kg bw) was studied on the severity of encephalopathy by assessment of clinical grading and electroencephalogram (EEG) spectral analysis, on plasma ammonia concentrations, amino acid concentrations in cerebrospinal fluid (CSF), intracranial pressure (ICP), and brain water content. Both rat models developed encephalopathy within 3 to 6 hours, associated with increased CSF glutamate and aspartate concentrations and increased ICP and brain water content. Memantine administration in AI-PCS and LIS rats resulted in a significant improvement in clinical grading and less slowing of EEG activity (P < .05), and smaller increases in CSF glutamate (P < .05) concentrations. Moreover, ICP and brain water content were significantly lower in memantine-treated AI-PCS rats than in untreated AI-PCS rats (P < .05). Memantine had no significant effect on ICP and brain water content in LIS rats, and on ammonia concentrations in both models. These results indicate that NMDA-receptor activation might be involved in the pathogenesis of hyperammonemia-induced encephalopathy and of acute hepatic encephalopathy caused by LIS.

Ammonia↗
PubMed1997

L-ornithine vs. L-ornithine-L-aspartate as a treatment for hyperammonemia-induced encephalopathy in rats.

BACKGROUND/AIMS: The effect of L-ornithine (ORN) and L-ornithine-L-aspartate (OA) therapy on "extracerebral" nitrogen metabolism, brain metabolism and neurotransmission has been investigated in portacaval shunted rats with hyperammonemia-induced encephalopathy. METHODS: One day before ammonium-acetate infusion, a portacaval shunt was performed in three experimental groups: 1-control rats, 2-ORN-treated rats and 3-OA-treated rats. Ammonium-acetate was given as an intravenous bolus injection (0.4 mmol.kg bw-1) followed by a constant infusion (1.9 mmol.kg bw-1.h-1) so that steady-state blood ammonia concentrations (500-800 microM) were obtained in the course of 5 h. After 1 h, ammonium-acetate infusion, either L-ornithine or L-ornithine-L-aspartate, was infused for the next 4 h (3.0 mmol.kg bw-1.h-1) in the treated groups. The following parameters were measured: clinical grade of encephalopathy, EEG activity (n = 10 - 20/group), amino acids in plasma (n = 10 - 20/group) and brain dialysate (n = 5 - 9/group), and brain metabolites obtained by in vivo cerebral 1H-MRS (n = 4 - 6/group). RESULTS: ORN and OA treatment resulted in significantly lower blood (34% and 39%) and brain (42% and 22%) ammonia concentrations, significantly higher urea production (39% and 86%) and significantly smaller increases in brain glutamine and lactate concentrations than in controls. These changes were associated with a significantly smaller increase in clinical grade of encephalopathy in ORN- and OA-treated rats, and a significant improvement in EEG activity in ORN-treated rats. OA-treated rats showed a significant increase in aspartate and glutamate concentrations in brain dialysate. CONCLUSIONS: The beneficial effects of both treatments on the manifestations of hyperammonemia-induced encephalopathy can be explained by a reduction in blood and brain ammonia concentrations. It is suggested that when OA is administered, the effect of ornithine is partly counteracted by aspartate, inducing high brain extracellular concentrations of the two excitatory amino acids glutamate and aspartate, and perhaps causing overstimulation of NMDA receptors.

Ammonia↗
PubMed1997

The effects of ammonia and portal-systemic shunting on brain metabolism, neurotransmission and intracranial hypertension in hyperammonaemia-induced encephalopathy.

BACKGROUND/AIMS: The pathogenetic factors contributing to encephalopathy in portacaval shunted rats with hyperammonaemia were studied. METHODS: Hyperammonaemia was induced by ammonium-acetate infusions in portacaval shunted rats (2.8 mmol.kg bw-1.h-1; AI-portacaval shunted rats) and in sham-portacaval shunted rats (6.5 mmol.kg bw-1.h-1; AI-NORM rats). Severity of encephalopathy was quantified by clinical grading and EEG spectral analysis. Changes in brain metabolites were assessed by amino acid analysis of brain cortex homogenates, whereas changes in amino acids with neurotransmitter activity were assessed in cerebrospinal fluid; brain water content was measured by subtracting dry from wet brain weights and intracranial pressure was measured by a pressure transducer connected to a cisterna magna cannula. RESULTS: Although similar increased blood and brain ammonia concentrations were obtained in both experimental groups, only AI-portacaval shunted rats developed encephalopathy, associated with a significant increase in intracranial pressure. Other significant differences were: higher concentrations of brain glutamine and aromatic amino acids, higher concentrations of cerebrospinal fluid glutamine, aromatic amino acids, glutamate and aspartate in AI-portacaval shunted rats than in AI-NORM rats. CONCLUSIONS: These results indicate that hyperammonaemia alone dose not induce encephalopathy, whereas portal-systemic shunting adds an essential contribution to the pathogenesis of encephalopathy. It is hypothesised that the larger increase in brain glutamine in AI-portacaval shunted rats than in AI-NORM rats is responsible for increased brain concentrations of aromatic amino acids, for cell swelling and for extracellular release of glutamate and aspartate. This might promote encephalopathy. If cell swelling is not restricted, intracranial hypertension will develop.

Amino Acids↗
PubMed1997

Transforming growth factor-beta-induced collagen synthesis by human liver myofibroblasts is inhibited by alpha2-macroglobulin.

BACKGROUND: Transforming growth factor-beta (TGFbeta) plays a central role in the stimulation of matrix production during liver fibrosis. The action of TGFbeta in different systems has been shown to be influenced by alpha2-macroglobulin (alpha2M), a serum protein with strong protease-scavenging and cytokine-binding properties. AIMS: In the present study, alpha2M derived from normal human plasma has been tested for its ability to modulate the TGFbeta-induced collagen production by human liver fat-storing cells (FSC), which had transformed into alpha-smooth muscle actin-expressing myofibroblasts in culture. METHODS: Alpha2M has been tested after activation with methylamine (alpha2M-Me), an in vitro equivalent of protease activated alpha2M. The binding of 125I-TGFbeta1 to activated forms of alpha2M was demonstrated by rate electrophoresis. Collagen synthesis was examined in human liver myofibroblast cultures obtained from three different human livers by incorporation of 3H-proline into TCA-precipitable, specific collagenase degradable proteins. Uptake of alpha2M was studied by means of immunofluorescence. RESULTS: TGFbeta (1 ng/ml) significantly stimulated collagen synthesis of controls in the absence of TGFbeta. Alpha2M-Me reduced this TGFbeta-induced collagen synthesis dose-dependently, reaching significant inhibition from 10 microg/ml alpha2M-Me onward. Upon addition of 100 microg/ml alpha2M-Me the effect of TGFbeta was reduced by 60% to 128+/-31% (mean+/-SD) of control values in the absence of TGFbeta. Human liver myofibroblasts endocytosed alpha2M-Me added to the cultures as detected by immunofluorescence. Accordingly, reduction of TGFbeta-activity by alpha2M-Me may be explained by receptor-mediated clearance of alpha2M-TGFbeta complexes by the cells. CONCLUSIONS: TGFbeta-induced collagen formation by human liver myofibroblasts obtained from three different livers is reduced in vitro by activated alpha2M. From these results, we hypothesize that alpha2M may have an antifibrogenic effect in vivo by interference with TGFbeta-induced matrix synthesis during liver fibrosis.

Cells, Cultured↗
PubMed1997

In vitro evaluation of a novel bioreactor based on an integral oxygenator and a spirally wound nonwoven polyester matrix for hepatocyte culture as small aggregates.

BACKGROUND/AIMS: The development of custom-made bioreactors for use as a bioartificial liver (BAL) is considered to be one of the last challenges on the road to successful temporary extracorporeal liver support therapy. We devised a novel bioreactor (patent pending) which allows individual perfusion of high density cultured hepatocytes with low diffusional gradients, thereby more closely resembling the conditions in the intact liver lobuli. METHODS: The bioreactor consists of a spirally wound nonwoven polyester matrix, i.e. a sheet-shaped, three-dimensional framework for hepatocyte immobilization and aggregation, and of integrated hydrophobic hollow-fiber membranes for decentralized oxygen supply and CO2 removal. Medium (plasma in vivo) was perfused through the extrafiber space and therefore in direct hepatocyte contact. Various parameters were assessed over a period of 4 days including galactose elimination, urea synthesis, lidocaine elimination, lactate/pyruvate ratios, amino acid metabolism, pH, the last day being reserved exclusively for determination of protein secretion. RESULTS: Microscopic examination of the hepatocytes revealed cytoarchitectural characteristics as found in vivo. The biochemical performance of the bioreactor remained stable over the investigated period. The urea synthesizing capacity of hepatocytes in the bioreactor was twice that of hepatocytes in monolayer cultures. Flow sensitive magnetic resonance imaging (MRI) revealed that the bioreactor construction ensured medium flow through all parts of the device irrespective of its size. CONCLUSIONS: The novel bioreactor showed encouraging efficiency. The device is easy to manufacture with scale-up to the liver mass required for possible short-term support of patients in hepatic failure.

Amino Acids↗
PubMed1997

Overexpression of hepatic prothymosin alpha, a novel marker for human hepatocellular carcinoma.

Identification of gene products exclusively or abundantly expressed in cancer may yield novel tumour markers. We recently isolated a number of cDNA clones, including alpha-prothymosin, from rat hepatocellular carcinoma (HCC) using a subtraction-enhanced display technique. Alpha-Prothymosin is involved in cell proliferation and is regulated by the oncogene c-myc in vitro. In the present study, we analysed alpha-prothymosin gene expression and its correlation with c-myc in patients with HCC, cirrhosis and adenoma and in normal controls. Hepatic alpha-prothymosin messenger RNA (mRNA) levels were two- to 9.2-fold higher in tumoral tissues than in adjacent non-tumoral tissues in 14 of 17 patients with HCC, regardless of coexisting cirrhosis and viral hepatitis. No marked difference in alpha-prothymosin mRNA levels was present in patients with adenoma and hepatic cirrhosis and in healthy controls. The c-myc mRNA amounts were two- to fivefold increased in 11 of 17 patients with HCC and correlated significantly with those of alpha-prothymosin (P < 0.001). In situ hybridization revealed that increased alpha-prothymosin mRNA was localized in the tumour nodules of the patients with HCC. These data suggest that overexpression of alpha-prothymosin in HCC patients, correlated with c-myc, is possibly involved in the tumorigenic process and may be a novel molecular marker for human HCC.

Adenoma↗
PubMed1997

Rat ferritin-H: cDNA cloning, differential expression and localization during hepatocarcinogenesis.

Elevated serum ferritin levels, especially of the H subunit, accompany many clinical malignancies. By using the subtraction-enhanced display technique, we have recently isolated several cDNA clones which are over-expressed in rat hepatocellular carcinoma induced by diethylnitrosamine. One 830-base-pair clone was 88% similar to human ferritin-H cDNA and encoded a 182 amino acid protein which is 97% homologous to human ferritin-H chain. Hepatic mRNA levels of ferritin-H were increased markedly at the early stage of diethylnitrosamine-induced hepatocarcinogenesis in the rat (6 weeks) and appeared more than 10-fold overexpressed as the tumour progressed. In contrast, hepatic ferritin-H mRNA remained constant during liver regeneration after a 70% partial hepatectomy. In situ hybridization showed that over-expression of ferritin-H was exclusively localized to preneoplastic foci, to tumour nodules and to tumour cells invading blood vessels. These findings suggest that ferritin-H is a highly conserved protein, its over-expression during tumour development is phenotypically correlated with tumour initiation and/or progression, and it is useful as an early marker for hepatocellular carcinoma.

Animals↗
PubMed1997

Semipermeable hollow fiber membranes in hepatocyte bioreactors: a prerequisite for a successful bioartificial liver?

Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure. Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor. The gold standard remains the intact liver. Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer. These conditions are not met in bioreactors based on hollow fiber membranes. Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient. We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion. The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates. The efficacy of this original design was tested in rats with liver ischemia. Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments.

Animals↗
PubMed1997

Immunological consequences of the use of xenogeneic hepatocytes in a bioartificial liver for acute liver failure.

The use of cells from xenogeneic origin in a bioartificial liver can have a number of immunological consequences, not only for the cells in the bioartificial liver but also for the patient receiving the bioartificial liver treatment. The impact of these consequences will depend on the immune status of the patient receiving bioartificial liver treatment, the duration and frequency of the treatment and on the extent of interaction between the patients blood (or plasma) and the xenogeneic liver cells. In an experimental model we infused rats with a culture supernatant of pig hepatocytes and demonstrated using Western blots and immunohistological techniques that antibodies are raised against the very small amounts of the pig hepatocyte-derived proteins present in the culture medium. Potential problems of bioartificial liver destruction and the possibility of hypersensitivity reactions due to the secretion of xenogeneic proteins into the circulation of the patient are discussed. Because the liver has an important role in the clearance of immune complexes it is concluded that precautions should be taken when (repeated) application of a xenogeneic bioartificial liver in patients with liver failure is considered.

Animals↗
PubMed1996

Formation of antibodies to factor VIII in patients with hemophilia A who are treated with interferon for chronic hepatitis C.

OBJECTIVE: To evaluate the risk for development of antibodies to factor VIII (factor VIII inhibitors) during and after interferon therapy in patients with hemophilia A and chronic hepatitis C infection. DESIGN: Patients were divided into two treatment groups and an untreated control group. Test results from the two treatment groups were compared with those from the control group. SETTING: 3 clinical centers in the Netherlands. PATIENTS: 35 men with hemophilia A who had acquired hepatitis C through the use of plasma-derived clotting factor concentrates. MEASUREMENTS: Patients were tested for factor VIII inhibitors before the start of interferon therapy and every 6 months thereafter. RESULTS: 21 patients with hemophilia A received interferon therapy for chronic hepatitis C infection for a mean of 19.5 months (range, 0.5 to 36 months). In 2 patients, inhibitors were detected on one occasion (maximum titer, 1.2 Bethesda units/mL) during interferon therapy. In 3 patients who were known to have had inhibitors before interferon therapy, no anamnestic reaction was seen during treatment. In 3 of 14 untreated controls who were followed for a mean of 28 months (range, 18 to 40 months), inhibitors were detected on one occasion (maximum titer, 2.3 Bethesda units/mL). CONCLUSION: Long-term interferon therapy in patients with hemophilia did not increase the risk for development of factor VIII inhibitors.

Antibody Formation↗
PubMed1996

Isolation of up- and down-regulated cDNAs associated with hepatocellular carcinoma by a subtraction-enhanced display technique.

Identification of gene products exclusively or abundantly expressed in hepatocellular carcinoma (HCC) and in normal liver may yield novel tumor markers. We have isolated 36 up- and down-regulated cDNAs from diethylnitrosamine-induced rat hepatocellular carcinoma and normal liver tissue by using the subtraction-enhanced display technique. Nucleotide sequence analysis revealed that the majority of 20 subtraction-enriched cDNA fragments were well-characterized oncogenes and tumor-associated genes, like c-myc, alpha-prothymosin, p21, glutathione-S transferase (G-ST) and alpha 1-acid glycoprotein (AGP). As demonstrated by Northern blot detection, all of them were preferentially expressed either in HCC or in normal liver (2- to 7-fold). As paradigm, G-ST and AGP were shown to be exclusively overexpressed in tumor nodules by in situ hybridization. In addition, 14 of the remaining 16 novel genes were analysed on Northern blot, 10 of which were differentially expressed in HCC.

Animals↗