Continuous Erythropoietin Receptor Activator (C.E.R.A.) administered at extended administration intervals corrects anaemia in patients with chronic kidney disease on dialysis: a randomised, multicentre, multiple-dose, phase II study.

This dose-finding, open-label study examined the potential of subcutaneous Continuous Erythropoietin Receptor Activator (C.E.R.A.) to correct anaemia at extended administration intervals in 61 erythropoiesis-stimulating agent-naïve patients with chronic kidney disease (CKD) on dialysis. After a 4-week run-in, patients were randomised to C.E.R.A. 0.15, 0.30 and 0.45 microg/kg/week. Within these dose groups, patients were further randomised to once weekly, once every 2 weeks or once every 3 weeks treatment. Mean changes in haemoglobin (Hb) increased with increasing C.E.R.A. dose during a period of 6 weeks where no dose adjustments were permitted. The effect was independent of administration schedule. Erythropoietic responses were sustained until the end of the study (12 weeks) in all groups. In total, 90% of patients in the 0.30 microg/kg/week group and 79% in the 0.45 microg/kg/week group responded to treatment (Hb increase > or =1.0 g/dl), compared with 72% in the 0.15 microg/kg/week group. Faster median response time was associated with increasing dose (51, 38 and 31 days, respectively) and response was unrelated to administration frequency. C.E.R.A. was generally well tolerated. Our results suggest that 0.60 microg/kg twice monthly would be a suitable starting dose of C.E.R.A. for the initiation of anaemia correction in patients with CKD on dialysis. Phase III studies will confirm the feasibility of using C.E.R.A. at extended administration intervals in patients with CKD and anaemia.

Disruption of integrin-dependent adhesion and survival of endothelial cells by recombinant adenovirus expressing isolated beta integrin cytoplasmic domains.

We explored the possibility of using a genetic approach to inhibit integrin-mediated endothelial cell adhesion and survival. We constructed recombinant adenoviruses (Ads) expressing chimeric proteins consisting of the cytoplasmic and transmembrane domains of integrin beta1 (CH1), beta3 (CH3) or the beta1 transmembrane domain alone (CH2) connected to the extracellular domain of L3T4 placed under the control of the CMV promoter (AdCMV) or the endothelial cell specific Tie-1 promoter (AdTie). All constructs were expressed in a dose- and time-dependent manner with over 90% of cells expressing the constructs within 24 h (AdCMVs) or 72 h (AdTies) after infection. Confluent monolayers of HUVEC infected with AdCMVCH1 or AdCMVCH3 detached from the substrate in a time- and dose-dependent manner with over 95% of the cells being detached 2 days (AdCMVs) or 3 to 4 days (AdTies) after infection. Cell detachment was preceded by the disruption of focal adhesions and reorganization of the actin cytoskeleton and was associated with a reduced ligand-binding activity of beta1, while cell surface density of beta1 integrins remained unchanged. Detached cells failed to re-adhere to different matrix proteins, without, however, any specificity toward beta1 or beta3 integrin-mediated adhesion. Upon detachment, HUVEC rapidly died by apoptosis. These results demonstrate that dominant negative inhibition of integrin function is an effective approach to disrupt endothelial cell adhesion and survival in vitro.

Analysis of the tumorigenicity of the X gene of hepatitis B virus in a nontransformed hepatocyte cell line and the effects of cotransfection with a murine p53 mutant equivalent to human codon 249.

Chronic infection with hepatitis B virus (HBV) is associated with the development of human hepatocellular carcinoma (HCC). One of the HBV genes, HBx, may have transforming potential, but this issue is still the subject of controversy. One of the major difficulties in addressing this question is the lack of a suitable in vitro model. We used a nontransformed, differentiated murine hepatocyte cell line (AML12) to transfect the HBx gene and examine its transforming capabilities. Because mutations of the p53 gene, in particular at codon 249, have been implicated in HCC development in geographical areas with high incidence of the tumor, we also studied the putative cooperative role in transformation between HBx and mutated p53 by cotransfecting HBx with a murine p53 mutant equivalent to human ser249 (ser246p53). Transfection with HBx plasmids containing the HBx gene under the control of two different promoters resulted in fewer colonies than in control plasmids. The toxic effect of HBx on colony formation was abolished by cotransfection with 246p53, suggesting that the inhibitory effect requires functionally intact p53. Clonal cell lines that stably expressed HBx messenger RNA (mRNA) (HBX lines) were tested for their growth characteristics and their ability to grow in soft agar and form tumors in nude mice. At passages 19-27 after transfection, one of four HBx-expressing lines showed the capacity for anchorage-independent growth in soft agar and produced poorly differentiated hepatocellular carcinomas in 8 of 13 sites of injection in nude mice. HBX lines as well as clonal cell lines of cells transfected with 246p53 (246 cell lines), cotransfected with HBx and 246p53 (246x lines) or transfected with control plasmids, were analyzed by flow cytometry to determine the fraction of cells in S phase (SPF). 246 and 246X lines had similar SPFs that were approximately twofold greater than control or HBX lines. 246x lines showed morphological changes in culture such as marked cellular heterogeneity, cell crowding, and the presence of multinucleated giant cells, but their tumorigenicity was not increased compared with the HBX lines. These data show that HBx has a weak tumorigenicity in murine hepatocytes and that the addition of mutation of p53 at codon 249 to HBx expression does not increase tumorigenicity in AML12 cells.

Introduction of a murine p53 mutation corresponding to human codon 249 into a murine hepatocyte cell line results in growth advantage, but not in transformation.

The p53 gene is frequently mutated in human tumors; in hepatocellular carcinomas, there is a high frequency of a specific mutation at codon 249 in regions with significant aflatoxin exposure. To assess the role of this p53 mutation in the development of hepatocellular carcinoma, a mutant murine p53 gene, p53ser246, which corresponds to human codon 249, was transfected into a differentiated, nontransformed hepatocyte cell line AML12. Expression of p53ser246 in this line resulted in a growth advantage when compared with either a control vector (which contains a large p53 deletion) or with a different p53 mutant, val135, not found in hepatocellular carcinoma. Overall, there was a threefold increase in colony formation after transfection with p53ser246 as compared with the control or p53val135 vectors, and the p53ser246 plates developed consistently larger colonies. Whereas clones expressing the control or p53val135 constructs showed no significant morphological changes, clones expressing p53ser246 showed increased heterogeneity (large multinucleated cells and areas of small crowded cells) without focus formation. In addition, the ser246 mutation imparted a growth advantage in serum-free media, suggesting less dependence on specific factors present in serum. None of the mutant p53 or control lines were capable of growth in soft agar or tumor formation in nude mice. Thus in this model, in which endogenous wild-type p53 expression is retained, a high level of mutant p53 expression is not sufficient to transform hepatocytes. Our findings indicate that p53ser246 has effects on hepatocytes that may result in a clonal growth advantage and suggest that additional factors are required for the development of hepatocellular carcinoma.

The overexpression of proliferating cell nuclear antigen in biliary cirrhosis in the rat and its relationship with epidermal growth factor receptor.

Chronic bile duct obstruction in the rat leads to biliary cirrhosis but maintained hepatocellular mass. We have previously demonstrated translocation of epidermal growth factor receptor to nuclei. It remained unclear, however, whether this was due to hepatocyte proliferation and/or altered handling of epidermal growth factor receptor. Therefore, in the present investigation we stereologically estimated expression of proliferating cell nuclear antigen, a marker of the S phase of teh cell cycle at 1, 2, 3, 7, 14, 21 and 28 days after bile duct ligation. Proliferating cell nuclear antigen positive hepatocytes averaged 2.1 +/- 3.6% in sham-operated control animals. This increased to 20.7 +/- 6.4, 26.8 +/- 18.7, 31.3 +/- 23.9, 42.3 +/- 16.6 and 24.7 +/- 28.0% 3, 7, 14, 21 and 28 days after bile duct ligation, respectively (p<0.005 by ANOVA). This was correlated with the number of epidermal growth factor receptor positive nuclei (rs = 0.737) and inversely with the maximal binding capacity of epidermal growth factor to a crude plasma membrane fraction (rs = 0.697) reported previously. We conclude that bile duct ligation in the rat induces a significant hepatocellular proliferation as assessed by proliferating cell nuclear antigen expression and that this process could, at least in part, be related to increased nuclear expression of the epidermal growth factor receptor.

Differential effect of micronodular and biliary cirrhosis on epidermal growth factor receptor expression in the rat.

Cirrhosis is characterized by fibrogenesis, hepatocyte necrosis and the formation of regenerative nodules. Modulation of the epidermal growth factor receptor is an early event during regeneration. We have recently demonstrated alterations in the epidermal growth factor receptor during the development of biliary cirrhosis. The aim of the present study was to compare epidermal growth factor receptor distribution, expression and binding in biliary cirrhosis to that occurring in micronodular cirrhosis induced by phenobarbital/CCl4 exposition. Biliary cirrhosis and micronodular cirrhosis had similar functional impairment as assessed by the aminopyrine breath test. Epidermal growth factor receptor binding capacity was reduced in both models (control vs micronodular cirrhosis vs biliary cirrhosis: (mean +/- 1 SD) 60 +/- 22 vs 16 +/- 12 vs 27 +/- 9 fmol/mg protein, p < 0.05), while the binding constant was increased in biliary cirrhosis only. The receptor mass in plasma membrane, determined by Western blotting, was not changed. Distribution of epidermal growth factor receptor was assessed immunohistochemically on tissue sections. In both models, cytoplasmic staining was decreased and basolateral plasma membrane labeling was maintained. Nuclear localization was found in biliary cirrhosis only. In conclusion, in both models, cirrhosis induces an alteration in the binding properties, but not in the number of epidermal growth factor receptors in the plasma membrane. The loss of cytoplasmic epidermal growth factor receptor could reflect alterations in expression and/or in intracellular trafficking. This is supported by the reduced mRNA steady state levels for epidermal growth factor receptor which were found in both models, presumably representing down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal growth factor receptor in chronic bile duct obstructed rats: implications for maintenance of hepatocellular mass.

Changes of the number and properties of the epidermal growth factor (EGF) receptor occur during liver regeneration and may be of importance in the maintenance of hepatocellular mass in liver cirrhosis. We therefore studied the changes in the number and distribution of EGF receptor in the development of liver cirrhosis induced by bile duct ligation. Receptor binding assays demonstrated a marked decrease in the binding capacity of crude plasma membrane fractions from 45 +/- SD 16 to 19 +/- 10 fmol/mg protein (p < 0.001) in control and bile duct ligated livers, respectively while the Kd increased after 3 days of bile duct ligation from 0.5 +/- 0.2 to 1.4 +/- 0.6 nmol/l. Total receptor concentration in the same membrane fractions, as assessed by Western blot analysis, was not changed. The expression of EGF receptor mRNA was reduced to about one third of control levels after 28 days of bile obstruction. Immunohistochemistry, performed using monoclonal antibodies against EGF receptor, showed a strong labeling of cytoplasm (87 +/- 3% positive) and plasma membranes (84 +/- 24%) but no labeling of nuclei in control livers. In bile duct ligated rats, in contrast, cytoplasmic staining was decreased (15 +/- 12%) already after 3 days of bile obstruction; labeling of canalicular membranes and nuclei appeared after 14 days. The shift of EGF receptor from plasma membranes to nuclei supports the notion that EGF receptor is involved in the maintenance of hepatocellular mass in this model of liver cirrhosis. This concept is supported by the finding of decreased mRNA for EGF receptor presumably representing down-regulation as seen in regenerating rat liver.

Effect of food on the bioavailability of low-dose methotrexate in patients with rheumatoid arthritis.

OBJECTIVE: To evaluate the influence of food on the bioavailability of an oral dose of methotrexate in patients with rheumatoid arthritis. METHODS: Methotrexate (15 mg) was given intravenously and orally, with or without a meal, to 10 patients. Blood samples were drawn at specific intervals to evaluate drug levels. RESULTS: Food reduced the peak concentration, from a mean of 0.71 mumoles/liter to 0.49 mumoles/liter (P less than 0.02), and slightly increased the time to peak concentration, from a mean of 1.3 hours to 2.0 hours. Bioavailability was highly variable (range 28-94%) but was not affected by food intake. CONCLUSION: Bioavailability of oral methotrexate shows marked interindividual variation but is not affected by the presence of food.