Impact of immunosuppressive therapy on hepatitis C infection after renal transplantation.

BACKGROUND: Among patients after renal transplantation (NTx), hepatitis C virus (HCV) infection is a risk factor for graft loss and patient death caused by hepatic decompensation. Also, HCV has been implicated in the pathogenesis of glomerular diseases in native and transplanted kidneys. Therefore, the aim of this retrospective cohort study was to determine the effects of the widely used calcineurin inhibitors (CNI) cyclosporine A (CsA) and tacrolimus (Tac) on hepatitis C virus replication, inflammatory activity, development of liver fibrosis, and long-term renal graft function. SUBJECTS AND METHODS: A cohort of 71 patients with HCV infection after kidney transplantation under immunosuppression with either CsA or Tac were analyzed for viral kinetics and serum transaminases. In addition, presence of liver fibrosis was detected by non-invasive measurements using the FibroScan. Graft function was determined biochemically. Patients with interferon therapy prior to transplantation were excluded from the study in order to avoid any impact of the antiviral therapy on outcomes. RESULTS: In the early period after transplantation, hepatitis C viral load was lower in patients treated with Tac as compared to CsA. This effect became negligible 3 months after transplantation. However, hepatic inflammatory activity was reduced in the CsA-treated group. Extent of liver fibrosis was similar in both groups of HCV-infected patients as well as in a control group of non-HCV-infected patients after renal transplantation (NTx), respectively. Renal function and glomerular filtration rate, as calculated by the modification of diet in renal disease (MDRD) formula, were significantly better in patients treated with Tac. CONCLUSIONS: During long-term immunosuppression, the CNIs cyclosporine A versus tacrolimus showed no significant differences in HCV-infected patients after renal transplantation with respect to viral replication and development of liver fibrosis. However, function of the renal graft is significantly better preserved in patients receiving tacrolimus.

Locoregional therapy for hepatocellular carcinoma: radioembolization with yttrium-90 microspheres.

Compared with other malignant tumours, hepatocellular carcinoma (HCC) exhibits particular characteristics regarding its supplying vessels and tumour biology. If a potentially curative surgical approach, such as resection or liver transplantation, is due to technical or prognostical reasons no option, these characteristics are a fundamental prerequisite for the possibility to effectively treat this tumour by local ablation methods. Microsphere and particle technology with selective transport of tumoricidal substances or radiation represents a new generation of therapeutics in interventional oncology. With the intrahepatic application of radioactive microspheres via the hepatic artery (radioembolization) local ablation can be performed even of diffuse and multifocal liver tumours, which hitherto, could only be approached with systemic therapy. The present standard for radioembolization, is the use of yttrium-90 glass or resin microspheres. The indications, technique and current results of radioembolization with yttrium-90 microspheres for the treatment of HCC are discussed in this review.

Analytical performance characteristics and clinical utility of a novel assay for total hepatitis C virus core antigen quantification.

The detection and quantification of hepatitis C virus (HCV) core antigen in serum or plasma by the use of different assay formats have previously been shown to represent useful markers of viral replication. In the present study, the intrinsic performance characteristics and the potential clinical utility of a novel assay for the quantification of total HCV core antigen were comprehensively assessed by using clinical serum samples and specimens contained in various evaluation panels. The Architect HCV Ag assay showed a specificity of 100%. The intra- and interassay coefficients of variation ranged from 3.6 to 8.0% and from 4.7 to 9.5%, respectively. Except for HCV genotype 2 isolates, the analytical sensitivity was always less than 10 fmol core antigen/liter, corresponding to approximately 500 to 3,000 IU of HCV RNA/ml. Linearity was guaranteed throughout the dynamic range (10 to 20,000 fmol/liter). When seroconversion panels were tested, the assay was not inferior to HCV RNA detection and reduced the preseroconversion period by 4 to 16 days. The results obtained by core antigen and HCV RNA quantification for 385 clinical specimens were correlated by regression analysis (r = 0.857), but the calculated conversion equation differed significantly from the line of identity. Monitoring of viral kinetics by use of either core antigen or RNA concentrations in 38 HCV-infected patients undergoing antiviral combination therapy resulted in very similarly shaped curves in all cases. Finally, the Architect HCV Ag assay was also shown to enable high-throughput screening of in vitro HCV RNA replication. With these results taken together, the Architect HCV Ag assay proved to be a specific, reproducible, highly sensitive, and clinically applicable test format which will find its future place in the context of virological HCV diagnostics.

Liver transplantation for hepatocellular carcinoma after yttrium therapy: a case report.

Yttrium-90 microspheres constitute one of the most recent treatment options for hepatocellular carcinoma (HCC) in the setting of cirrhosis. As such, their spectrum of indication is not yet fully established. Herein, we have reported the case of a patient with HCC beyond the listing criteria for liver transplantation (OLT) who was treated preoperatively with selective transarterial chemoembolization and yttrium-90 microspheres. He was subsequently transplanted with a liver from an 81-year-old donor allocated through Eurotransplant as a "rescue offer." The posttransplant course was uneventful. Pathologic examination revealed a multifocal, well-differentiated pT2 tumor with no vascular invasion. The patient is currently alive and in good condition at 14 months posttransplant, with no evidence of tumor recurrence by a current computed tomography scan. This report provided encouraging information on the potential of yttrium-90 microspheres as a bridging option before OLT for multifocal HCC.

Ribavirin with either standard or pegylated interferon to treat recurrent hepatitis C after liver transplantation.

AIM: To investigate the efficacy of two anti-viral protocols in hepatitis C virus-reinfected liver transplant recipients. METHODS: In this prospective study, 26 liver transplant patients were treated with standard interferon-alpha2b for 12 months or standard interferon-alpha2b for 3 months followed by pegylated interferon-alpha2b for 9 months. Interferon was combined with ribavirin in all patients. The histological course of the study population was compared with an untreated historic control group (n = 38) with similar baseline characteristics. RESULTS: The sustained virological response rates in the standard interferon group and in the pegylated interferon group were 27.3% and 26.7%, respectively. Only 29% of patients with sustained virological response had end of treatment histological response, whereas 47% of viral non-responders showed end of treatment histological response. The percentage of patients with histological improvement was significantly higher in the study population when compared to the controls. Univariate analysis indicated that hepatitis C virus genotype non-1, high baseline alanine aminotransferase, the time interval between liver transplant and interferon therapy and the body mass index predicted sustained virological response. In the multivariate model, baseline alanine aminotransferase and the body mass index remained a significant predictor of sustained virological response. CONCLUSIONS: Both treatment regimens offer similar efficacy profiles. Failure to eradicate hepatitis C virus should not lead to treatment discontinuation if serial liver biopsies demonstrate histological response.

Reversible tumorigenesis in mice by conditional expression of the HER2/c-erbB2 receptor tyrosine kinase.

In the present study we describe the reversible transformation of NIH3T3 fibroblasts by overexpression of the HER2/c-erbB2 receptor tyrosine kinase. Cell lines expressing HER2 under control of a tetracycline-responsive promoter were isolated. Induction of HER2 expression resulted in cellular transformation in vitro as depicted by growth in soft agar and focus formation in tissue culture. Subsequent treatment of these cells with the effector anhydrotetracyline switched-off HER2 expression and induced morphological and functional changes characteristic for non-transformed cells. Subcutaneous transplantation of cells in nude mice resulted in the formation of solid tumors. Interestingly tumor formation was completely suppressed by treatment of the animals with anhydrotetracyline. Our findings indicate that overexpression of HER2 induces the transformed phenotype of NIH3T3 cells and is required for tumor formation and progression in nude mice. By linking the expression of the marker gene secreted placental alkaline phosphatase to the expression of HER2, a sensitive monitoring of tumor development in nude mice was feasible.

Biotransformation of mafosfamide in P388 mice leukemia cells: intracellular 31P-NMR studies.

The intracellular transformation of cis-mafosfamide has been studied in P388 mice leukemia cells using 31P-NMR spectroscopy. For this purpose the cells were entrapped in low-gelling-temperature agarose threads. Internal pH of the cells, determined from the position of the intracellular inorganic phosphate, was 7.2. The cell membrane was permeable to 4-hydroxycyclophosphamide and aldophosphamide and less permeable to phosphoramide mustard. 4-Ketocyclophosphamide and carboxyphosphamide signals were not detectable in cells either sensitive or resistant to oxazaphosphorine treatment.

Pulmonary vasoconstriction produced by protamine and protamine-heparin complex in the isolated cat lung perfused with blood or dextran.

Injections of protamine sulphate, protamine-heparin complex, and the supernatant from this mixture produced a rise in pulmonary artery pressure during constant flow perfusion in the isolated cat lung. The rise in pressure occurred with both blood and dextran perfusates. This suggests that the pulmonary vasoconstriction produced by protamine may be partly due to a direct action on the pulmonary vasculature or to the release of vasoactive substances from the lungs.

alpha-/beta-Triglycidyl-urazol (TGU, NSC 332488, I.N.N.: ANAXIRONE): a new chemotherapeutic agent.

Triglycidyl-urazol (TGU) was a rational drug development from the original triepoxide triglycidyl-triazinetrione (TGT). It was selected for further studies because of its superior physico-chemical properties as well as its improved therapeutic range in animals. Like the parent compound, TGU exerts antitumour activity in a wide spectrum of experimental tumours, including those resistant to cyclophosphamide. Its biochemical reactivity is very high, a fact which may contribute to its rapid plasma clearance after parenteral administration. Bone marrow suppression constitutes the dose-limiting toxicity in animals and man with a vague suggestion of cumulative effects. Other toxicities are generally mild and rapidly reversible. The new chemical structure, its reproducible experimental antitumour activity combined with an acceptable and manageable toxicology warranted the introduction of the compound into the clinic. Phase I studies in patients have largely confirmed the predicted toxicities and probable antitumour activity in man (7, 12, 13). Consequently, clinical phase II studies in various tumour types are now under way.

Anticancer drugs: estrophilic cisplatin derivatives.

Our approach to develop platinum complexes with a selective effect on the hormone-dependent MC by exchanging the two NH3 groups of cisplatin by an 1,2-bis(4-hydroxy-phenyl)ethylenediamine derivative which possesses estrogenic activity, was successful. The most interesting compound of this new platinum complex series, meso-(1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine)dichloroplati num(II) (meso-1-PtCl2) and its water soluble sulfatoplatinum(II) derivative (meso-1-PtSO4) were significantly more active on the DMBA-MC and the receptor positive MXT-MC than cisplatin and the related ligand 1,2-bis(2,6-dichloro-4-hydroxyphenyl)ethylenediamine (meso-1). According to our proposed mechanism of action, meso-1-PtX (X = Cl2 or SO4) should be enriched in the nuclei of mammary tumor cells, which possess an intact ER system (i.e. an intact cytoplasma nucleus translocation process), thereby causing very strong tumor growth inhibition. Preliminary studies of meso-1-PtSO4 on the DMBA-MC confirm this assumption. In the tumor tissue we found higher Pt-levels than in uterine tissue, which also contains ERs. The Pt-levels of the tumor tissue are much higher than those of skeletal muscle and of blood. In therapeutic dosages meso-1-PtSO4 does not cause kidney damages in rats.

Hexadecylphosphocholine in the topical treatment of skin metastases in breast cancer patients.

Widespread local recurrence of breast cancer, untreatable by surgery or radiation therapy, can present a serious therapeutic problem predominantly in patients refractory to systemic therapy. In a phase I trial hexadecylphosphocholine, a new agent with high membrane affinity and antineoplastic activity was applied topically to affected skin areas of breast cancer patients. The results provide evidence that hexadecylphosphocholine may be an active agent in the topical treatment of skin metastases.

Molecular and cellular effects of hexadecylphosphocholine (Miltefosine) in human myeloid leukaemic cell lines.

The molecular and cellular effects of the anti-neoplastic alkylphospholipid hexadecylphosphocholine (Miltefosine, MIL) on parameters associated with growth and differentiation of human myeloid leukaemic cell lines U937, KG1 and KG1a were investigated. On a cellular level, MIL has dose-dependent differentiation-inducing growth-promoting and cytotoxic activities exemplified by induction of respiratory burst activity, stimulation of interleukin-3 (IL-3)/granulocyte-macrophage colony stimulating factor (GM-CSF)-dependent growth of the KG1 cell line in soft agar culture, inhibition of cellular net growth and finally cell death. By northern blot analysis, transcription of functional receptors for IL-3, GM-CSF, G-CSF and FcRI were studied. It was shown that MIL has stimulatory activity on IL-3 and GM-CSF receptor gene transcription. In addition, the transcription of proliferation- and differentiation-associated proteins, namely histone subtypes, c-myc and NF-kappa B p50, were studied. MIL suppressed c-myc and enhanced NF-kappa B p50 transcription in the U937 cell line, comparable to the well-characterised differentiation-inducing phorbolester 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that the interaction of MIL with its molecular target(s) in myeloid cells induces molecular and cellular effects associated with induction of differentiation, distinct from its cytotoxic activity.

Opposite effect of miltefosine on the antineoplastic activity and haematological toxicity of cyclophosphamide.

The effect of pretreatment with miltefosine (MIL) on the antineoplastic activity of cyclophosphamide (CPA) was evaluated in subcutaneous benzo(a)pyrene-induced sarcomas (BPS) of the rat. MIL alone had no antineoplastic effect on this autochthonous tumour, but enhanced the chemotherapeutic effect of CPA. Conversely, MIL counteracted the myelotoxicity of CPA in normal adult rats. Although the nadir of the leucocyte count remained unchanged, the recovery phase was considerably shortened, an effect which resembled the pharmacological action of GM-CSF.

D-21266, a new heterocyclic alkylphospholipid with antitumour activity.

The aim of this study was to determine the antitumour effects of D-21266 in a rodent tumour model. Hexadecylphosphocholine (INN: Miltefosine) represents the first anticancer agent which was specifically formulated for topical use in cancer patients. The development as an oral drug was hampered by the gastrointestinal toxicity. Hexadecylphosphocholine derivatives were sought with a better therapeutic index. Octadecyl-(1,1-dimethyl-4-piperidylio) phosphate (D-21266) was identified as a suitable candidate. This compound is highly active in vitro inhibiting the growth of a number of human cancer cell lines. Mammary carcinomas were induced in Sprague-Dawley rats using DMBA, and oral doses of D-21266, in various schedules, were given to the animals. A high antineoplastic potency was observed without inducing loss of body weight at highly effective doses. The antitumour effect could be enhanced by introducing a dose schedule consisting of a high loading dose followed by a low maintenance dose, both of which are only marginally active when given alone. Therefore, D-21266 with its favourable pharmacological and toxicological profile, warrants evaluation in the clinic. However, the concept of clinical trials requires new approaches to dose finding and response evaluation, because the dose-response relationship of this compound is distinctly different from that of classical cytostatic agents.

Pharmacological studies with cetrorelix (SB-75), a potent antagonist of luteinising hormone-releasing hormone.

The antitumour and hormone-suppressive effects of the luteinising hormone-releasing hormone LH-RH antagonist Cetrorelix (D-20761) and its pamoate salt (D-20762) were investigated in the model of the DMBA-induced mammary carcinoma of female rats and by testosterone determinations in normal male rats. Treatment with single high doses of D-20761 induced a rapid decrease of tumour weights with a dose-dependent duration of action. Strong antitumour effects were also observed by applying different multiple dose schedules, including a initial high dose (3.16 mg/kg, s.c.) followed by a low maintenance dose (31.6 micrograms/kg, s.c.). The stability of the molecule against degrading enzymes led to the idea of using the poorly soluble pamoate salt for facilitating a sustained release of active compound. This salt indeed induced a prolonged suppression of tumour growth and of testosterone levels. In conclusion, we found that Cetrorelix is a highly effective LH-RH antagonist which should be further developed for the treatment of hormone-dependent diseases.

Evidence for abrogation of oncogene-induced radioresistance of mammary cancer cells by hexadecylphosphocholine in vitro.

Hexadecylphosphocholine (HePC), an experimental and clinical antitumour agent of the alkyllysophospholipid group, was tested for its radiosensitising effect on a panel of nine human mammary cancer cell lines in vitro. Growth inhibition by ionising radiation and recovery from it were not influenced by pretreatment with HePC in most cases, except for two cell lines expressing an activated ras oncogene. In the latter we found an enhanced radioresistance that was abolished by pretreatment with HePC. Our results suggest that HePC may act as a radiosensitiser for cells carrying an activated ras oncogene.

Highly sensitive northern hybridization of rare mRNA using a positively charged nylon membrane.

The effect of different RNA fixation methods using Hybond-N and Hybond-N+ nylon membranes in Northern blot experiments was analyzed with human glyceraldehyde-3-phosphate dehydrogenase and human interleukin-3 receptor alpha-subunit as radioactive labeled DNA probes. Highest sensitivity could be achieved by mild alkaline fixation of RNA to positively charged Hybond-N+ nylon membrane. Fixation by UV light resulted in reduced sensitivity in comparison with alkaline treatment. Fixation of RNA to Hybond-N or -N+ membranes by baking for 2 h at 80 degrees C resulted in a strong decrease in sensitivity and should be avoided when using modified nylon membranes in Northern hybridizations.

A new platelet aggregating material (PAM) in an experimentally induced rat fibrosarcoma.

The present work concerns our studies to search for factor(s) which may influence the hemostatic process in or around metastasis of tumours. We studied the platelet aggregating property of a methyl cholanthrene induced experimental tumour. Platelet aggregating material was found to be different from the known aggregating agents like thrombin, ADP, collagen, thromboxane A2 and trypsin. It depends on a critical level of calcium for its action. PAM is a high molecular weight substance which contains sialic acid. It is trypsin and plasmin insensitive. The activity of this substance is not being destroyed by phospholipase-C. Metabolic study indicates that PAM acts by mitochondrial energy metabolic pathway of the platelets.

Three abnormal fibrinogen variants with the same amino acid substitution (gamma 275 Arg----His): fibrinogens Bergamo II, Essen and Perugia.

We report on three unrelated individuals with the same uncommon type of dysfibrinogenemia, originating from Bergamo, Essen and Perugia. None of them showed bleeding symptoms while the Bergamo patient and members of her family presented with a thrombotic tendency. The presence of a defective fibrinogen was suggested by prolonged thrombin and reptilase times. Furthermore, fibrinogen concentrations of less than 0.28 g/L were determined by the functional assay whereas values of 1.5-2.4 g/L were measured by heat precipitation or electroimmunoassay. Fibrinogen was isolated by affinity chromatography on insoluble fibrin monomer. The rate of fibrinopeptide release by thrombin was normal while the fibrin polymerization reaction was strongly delayed. An abnormal peptide (gamma 265-310) was isolated by high-performance liquid chromatography after cyanogen bromide cleavage of the purified gamma-chain of fibrinogen Bergamo II and Essen. The same peptide was also isolated following cyanogen bromide treatment of the intact fibrinogen Perugia. Sequence analyses of these peptides demonstrated the same amino acid exchange in all three fibrinogens: gamma 275 arginine----histidine. The described fibrinogen variants appear to possess a molecular defect which has thus far only been observed in fibrinogen Haifa.

Induction of the c-myc but not the cH-ras promoter by platinum compounds.

The recombinant plasmids p324, p330 and p323 carrying the aminoglycoside phosphotransferase (aph) gene and 5' flanking c-myc sequences linked to the reporter gene chloramphenicol acetyl-transferase (cat) were introduced into the mouse erythroleukaemic cell line F412B2TK- and stable transfectants resistant to geneticin G418 were obtained. The effects of cis-platin and two novel platinum compounds, D19466 (lobaplatin) and D17872, on c-myc promoter regions were studied using a large range of drug concentrations and correlated with cytotoxicity data. It was found that cis-platin and D19466 show a similar pattern of cytotoxicity and activation of c-myc promoter-driven cat gene expression with maximum effect (increased expression of 7.4- and 8.1-fold, respectively) at a concentration of 5 x 10(-5) M, while the less cytotoxic D17872 only slightly activates cat expression at the same concentration. However, when the F412B2TK- cell line was transfected with a plasmid carrying 5' flanking sequences of the c-Hras1 gene with promoter/enhancer function, linked to the cat reporter, no similar inductive effect was observed with any of the platinum drugs used. These data suggest that platinum compounds and possibly other DNA-damaging agents may specifically influence the expression of certain genes.