Post-approval trials of new medicines: widening use or deepening knowledge? Analysis of 10 years of etanercept.

OBJECTIVE: To investigate the main aims of the post-approval randomized controlled trials (RCTs) on etanercept and the extent to which they were designed to gain more comparative information. METHODS: A search of the literature (Medline, Embase), trial registries (Clinical Trials.gov, Controlled Trials.com), and market authorization reports from the Food and Drug Administration (FDA) and the European Medicines Agency (EMEA) was carried out to identify all RCTs. A comparison of trial data identified unpublished trials and multiple publications relating to the same study. All RCTs completed and/or published after initial market approval was regarded as post-approval. RESULTS: Up until 2008, we found 84 post-approval trials, 11 (13%) trials on approved extensions of indication, another 30 (36%) trials on the approved indications, and 43 (51%) trials on indications not (yet) approved. Nearly half of the studies on indications not yet approved were initiated and funded by independent sponsors. After the initial approval of etanercept, six head-to-head trials were conducted on the approved indications. Overall, the main objectives of post-approval trials with etanercept were found to confirm efficacy and safety in new indications, and to gather additional information for optimal use on the approved indications. CONCLUSION: Post-approval RCTs on etanercept focus more on studies searching for new indications than on deepening knowledge about use. Ten years after the market entry of etanercept, one of the reasonable demands of clinical practice, for more comparative information, still remains unanswered.

Identifying quality improvement intervention evaluations: is consensus achievable?

BACKGROUND: The diversity of quality improvement interventions (QIIs) has impeded the use of evidence review to advance quality improvement activities. An agreed-upon framework for identifying QII articles would facilitate evidence review and consensus around best practices. AIM: To adapt and test evidence review methods for identifying empirical QII evaluations that would be suitable for assessing QII effectiveness, impact or success. DESIGN: Literature search with measurement of multilevel inter-rater agreement and review of disagreement. METHODS: Ten journals (2005-2007) were searched electronically and the output was screened based on title and abstract. Three pairs of reviewers then independently rated 22 articles, randomly selected from the screened list. Kappa statistics and percentage agreement were assessed. 12 stakeholders in quality improvement, including QII experts and journal editors, rated and discussed publications about which reviewers disagreed. RESULTS: The level of agreement among reviewers for identifying empirical evaluations of QII development, implementation or results was 73% (with a paradoxically low kappa of 0.041). Discussion by raters and stakeholders regarding how to improve agreement focused on three controversial article selection issues: no data on patient health, provider behaviour or process of care outcomes; no evidence for adaptation of an intervention to a local context; and a design using only observational methods, as correlational analyses, with no comparison group. CONCLUSION: The level of reviewer agreement was only moderate. Reliable identification of relevant articles is an initial step in assessing published evidence. Advancement in quality improvement will depend on the theory- and consensus-based development and testing of a generalizable framework for identifying QII evaluations.

Cryopreserved precision-cut rat liver slices: morphology and cytochrome P450 isoforms expression after prolonged incubation.

Precision-cut liver slices are an accepted in vitro system for toxicological investigations. However, cryopreservation of slices would make a more efficient utilisation, particularly of human liver tissue possible. In the present study sections of cryopreserved male rat liver slices were examined immunohistochemically for cytochrome P450 (CYP) isoforms expression after prolonged incubation and after exposure to typical inducers. Morphologically, with just thawed slices no major alterations were seen, but remarkable cell damage was observed even after 2 h of incubation mainly in the middle of the slices and in the periportal and intermediate regions of the lobules. After 24 h of incubation, viable cells were only observed at the edges of the slices or around bigger vessels. In the viable cells of the cryopreserved liver slices after 2 h of incubation CYP expression pattern was similar to that in normal liver specimens: a low CYP1A1, but a strong CYP2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. After 24 h, the immunostaining for CYP2B1 and 3A2 in the viable cells was reduced, but that for CYP1A1 was increased. Incubation with beta-naphthoflavone further elevated CYP1A1 and 2B1 expression. Phenobarbital caused an enhanced CYP2B1 and 3A2 and dexamethasone and pregnenolone 16 alpha-carbonitrile an increased CYP3A2 immunostaining. These results show that also in cryopreserved liver slices and after a prolonged incubation, a distinct expression pattern and an in vitro induction of phase I enzymes can be demonstrated immunohistochemically.

In vitro induction of cytochrome P450 2B1- and 3A1-mRNA and enzyme immunostaining in cryopreserved precision-cut rat liver slices.

With the exception of cytochrome P450 (CYP) 1A1 and its mRNA, in vitro induction of other CYP forms has not been demonstrated in cryopreserved liver slices until now. Therefore precision-cut rat liver slices were cultured after cryopreservation and thawing in William's medium E for up to 24 h in the presence of inducers to demonstrate CYP2B1- and CYP3A1-mRNA induction. CYP-mRNA expression was determined by competitive RT-PCR. Exposure to 100 microM phenobarbital caused a more than 20-fold increase in CYP2B1-mRNA expression within 24 h, reaching concentrations comparable with those of PB-exposed fresh rat liver slices. Exposure to 1 microM pregnenolone 16 alpha-carbonitrile enhanced CYP3A1-mRNA expression by more than 30-fold within 24 h. This is in the same range, although with higher variability, as detected with fresh liver slices. In spite of considerable variability among the thawed slices, the induction factors are high enough for a sensitive detection of an induction at mRNA level. Additionally, immunostaining of respective CYP-forms was performed in sections of few samples, indicating CYP increase in viable cells of cryopreserved slices.

Morphology and cytochrome P450 isoforms expression in precision-cut rat liver slices.

Precision-cut liver slices are a widely accepted in vitro system for the examination of drug metabolism, enzyme induction, or hepatotoxic effects of xenobiotics. The maintenance of the distinct lobular expression and induction pattern of phase I biotransformation enzymes, however, has not been examined systematically so far. Thus, in the present study, both longitudinal and transversal sections of male rat liver slices were investigated morphologically, as well as immunohistochemically for the expression of different cytochrome P450 (CYP) isoforms after prolonged incubation or after exposure to typical inducers. Histopathological examinations revealed an increasing vacuolization of the periportal hepatocytes mainly in the middle of the slices from 6 h of incubation on, paralleled by a loss of glycogen in the respective cells. After 24 h, mainly in the center of the slices, necroses of cells occurred. After 48 h of incubation, typically a central band of coagulative necrosis flanked by superficial layers of viable cells was observed. Freshly prepared slices displayed a CYP subtypes expression as normal liver specimen, a very low centrilobular CYP 1A1 immunostaining, but a strong CYP 2B1 and 3A2 expression predominantly in the central and intermediate lobular zones. From 2 h on, the immunostaining for CYP 2B1 and 3A2 was to some extent reduced. After 24 h of incubation with beta-naphthoflavone, the CYP 1A1 and 2B1 expression was induced mainly in the viable cells around central veins, around some portal fields with bigger vessels and in the cell layers close to the slice surface. At the same sites, phenobarbital led to an increased CYP 2B1 and 3A2 expression and dexamethasone to an elevated CYP 3A2 immunostaining. These results show, that an in vitro induction of phase I enzymes in precision-cut liver slices can be demonstrated also immunohistochemically.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of different cytotoxins on cytochrome P450 isoforms expression and on glycogen content.

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with different cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ], carbon tetrachloride [CCl4], or thioacetamide [TAA]) or the respective solvents 24 or 48 hours before sacrifice. Effects of the cytotoxins on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers were assessed by immunohistochemistry. Additionally, effects on glycogen content within the hepatocytes were examined. In the livers AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ led to a perivenous necrosis of single cells only, whereas CCl4 and TAA caused complete necrosis of the centrilobular parenchyma. Treatment with each of the four cytotoxins led to necrosis and fatty degeneration of single or groups of hepatocytes within the intrasplenic transplants. This effect was most pronounced with CCl4 and TAA. The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 in the periportal areas was even decreased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. TAA and, more pronounced, CCl4 administration caused a strong reduction in the expression of all three P450 isoforms. Spleens of control rats displayed almost no P450 isoforms expression, independent of the treatment with the cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. AAL administration slightly reduced the P450 2B1 and 3A2 expression in the transplants. BBZ and, much more pronounced, CCl4 and TAA treatment diminished the staining for all three P450 isoforms. AAL administration led to a marked decrease in the glycogen content of the hepatocytes of the periportal zones of the liver lobules, whereas after BBZ, CCl4 and TAA treatment a strong perivenous reduction in the glycogen content was seen. Similarly, within the intrasplenic transplants a remarkable decline in the glycogen content of the hepatocytes was caused by the treatment with each of the four cytotoxins. Especially after AAL and BBZ treatment the glycogen depletion within both livers and transplants was much more pronounced than the effects on morphology or P450 isoforms expression. It can be concluded that the effects of cytotoxins like AAL, BBZ, CCl4 or TAA seen in normal orthotopic liver are exerted in a similar way also in intrasplenic liver cell transplants.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of various mitogens and cytotoxins on cytochrome P450 (P450) isoforms expression and on P450 mediated monooxygenase functions.

Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with various mitogens (fluorene [FEN], fluorenone [FON] and 2-acetylaminofluorene [AAF]) or cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ] and carbon tetrachloride [CCl4]) or the respective solvents 24 or 48 hours before sacrifice. The expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers was assessed by immunohistochemistry and P450 mediated monooxygenase functions in spleen and liver 9000 g supernatants by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (EMND). The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a moderate P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. Correspondingly, regular EROD, ECOD and EMND activities were observed. Each of the three mitogens increased the P450 1A1 expression in the hepatocytes of the perivenous zones of the liver lobules. FON administration caused an additional P450 1A1 immunostaining in the periportal areas, and AAF treatment a P450 1A1 expression in bile duct epithelia. Also the staining for P450 2B1 and 3A2 in the hepatocytes of the perivenous and intermediate zones of the liver lobules was intensified after treatment with any of the mitogens. The three model reactions were significantly increased within the livers after FEN and FON administration, whereas after AAF treatment only ECOD was enhanced, EROD remained unaffected and EMND was decreased. The cytotoxin AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ only produced a perivenous necrosis of single cells, whereas CCl4 caused complete necrosis of the centrilobular parenchyma. Immunohistochemically, AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 was even decreased in the periportal areas. Due to AAL treatment EROD and EMND activities were not affected and ECOD activity was increased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. After BBZ treatment, EROD and EMND activities were decreased, ECOD activity was not affected. CCl4 administration caused a strong reduction in the expression of all three P450 isoforms and in the activity of all three model reactions. Spleens of control rats displayed almost no P450 isoforms expression and P450 mediated monooxygenase functions, without as well as after treatment with the mitogens or cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants.

The granulated convoluted tubules of the rat submandibular glands under experimental conditions.

Granulated convoluted tubules are the specific ductal segment of the submandibular glands of mice and rats. These tubules are functionally integrated into hormonal circuits, produce regulatory peptides as well as epidermal and nerve growth factor. The experimental studies on adult male Wistar and Fischer 344 rats demonstrate acute cytotoxic lesions of the granulated tubules by means of a single dose of 2-acetylaminofluorene. After only a few administrations of the compound the intralobular duct tree is lined by an atrophic epithelium with loss of specific structures, the EGF immunoreactivity and the susceptibility to further cytolethal effects. The early selective damage of the granulated convoluted tubules indicates that the growth factor production and certain drug-metabolizing/drug-excreting capacities are situated within the same ductal segment. It is considered that other systemically administered compounds might also influence this growth factor-producing ductal segment, though less dramatically than 2-acetylaminofluorene.

Dental prevention among Turkish and Moroccan parents in The Hague.

OBJECTIVE: The aim of this study was to determine the extent to which dental prevention among Turkish parents differs from that among Moroccan parents in one of the large cities in the Netherlands. DESIGN: Turkish and Moroccan mothers with a child of approximately six months old, who visited the well baby clinics in the Schilderswijk area of The Hague, were asked to participate in a dental care project. As part of the initial data collection in this project, 170 Turkish and 153 Moroccan mothers were interviewed at home by a woman who spoke their native languages. The interview contained questions about dental prevention, such as fluoride use, feeding bottle use, visits to the dentist, brushing, information about oral care, and about relevant background characteristics. RESULTS: More Moroccan than Turkish mother reported that they did not use a feeding bottle for the baby, went to the dentist twice a year, brushed their own teeth at least twice a day and started to brush their children's teeth at an early age. The Moroccan mothers were more committed to dental prevention than Turkish mothers, even after correction for background characteristics such as education, language skills and the number of years spent in the Netherlands. CONCLUSIONS: Ethnic groups should not be seen as a single group for dental prevention. It is recommended that Turkish parents in particular be encouraged to go to the dentist twice a year, to start brushing their children's teeth at an early age and to wean their children off use of a feeding bottle in bed.

Ciprofibrate--racemate and enantiomers: effects of a four-week treatment on male inbred Fischer rats. A biochemical and morphological study.

Ciprofibrates (racemate and both enantiomers, Raccip, R- and Scip) were administered orally in doses of 1 and 10 mg/kg once daily over 28 days to male inbred Fischer 344 rats, age 90-110 days at the beginning of the experiment. Body mass gain was observed in all groups. The 1 mg groups showed almost no difference to the control group. The 10 mg groups exhibited less body mass gain, most pronounced in the Scip group. Liver masses were increased in a dose dependent manner up to more than 200%, only the 10 mg Scip group was not significantly different from the 1 mg group which exhibited an increase in liver weight to about 175%. Also the kidney weights increased to 130%, whereas thymus and spleen weights were decreased in the high dose groups. Liver microsomal cytochromes P450 (P450) concentrations were not altered in the 1 mg groups and distinctly lowered in the 10 mg groups. Ethoxyresorufin and ethoxycoumarin O-deethylations were lowered in all experimental groups in a dose dependent manner, after administration of the high doses down to 30% of the control levels or less. Pentoxyresorufin O-depentylation, however, was increased in all 1 mg groups. In the high dose groups it was not altered. Ethylmorphine N-demethylation was decreased after administration of the high doses by about 50%, but only Scip decreased this reaction also after administration of the low dose. NADPH/Fe2+-stimulated microsomal luminol and lucigenin amplified chemiluminescence was increased, whereas hydrogen peroxide formation was depressed even by the low doses to 50% of the normal values, to about 25% by the high doses. Microsomal lipid peroxidation, however, was only slightly or not influenced. Glutathion concentrations (in the reduced and the oxidized form) were increased in a dose dependent manner by about 20 to 30%, the concentration of lipid peroxides was not significantly influenced. Thus, the effects of the enantiomers were not different and were similar to those of the racemate. In serum, cholesterol and triglycerides were only moderately lowered. Albumin concentrations were significantly enhanced in all groups, total proteins after 1 mg/kg Raccip only. Serum bilirubins were not altered, and among the indicator enzymes for liver damage only ALAT, alkaline phosphatase and the dehydrogenases were increased, in no case higher than twofold. Histologically distinct effects were seen after administration of both doses, more pronounced after 10 mg/kg, but with no differences between the enantiomers and Raccip: marked hypertrophy of the hepatocytes, reduced staining of the nuclei, strongly acidophilic granulated cytoplama, no basophilia of the cell bodies, loss of glycogen. These changes were most pronounced around the central veins. Hepatocyte apoptoses also were observed. By immunohistochemistry an increased staining was seen for all P450 isoforms tested (1A1, 2B1, 2E1, 3A2 and 4A1), predominantly perivenously and most pronounced after administration of the high doses without differences between Rcip, Scip or Raccip (preliminary results). By electron microscopy a moderate proliferation of peroxisomes after treatment with 1 mg/kg Cips with a ratio between mitochondria and peroxisomes of about 1:1 (controls: 10:1) was observed, and the peroxisomes were a more heterogeneous population. The relative portions of glycogen and both forms of the ER decreased. Treatment with 10 mg/kg Rcip, Scip or Raccip led to a strong increase in the number of peroxisomes, in some hepatocytes the ratio between mitochondria and peroxisomes was 1:3 with an increased heterogeneity among the peroxisomes evidenced by a broad range of electron densities. Most peroxisomes lacked a nucleoid. Thus, the biochemical effects differed only slightly and the morphological effects of the enantiomers were not different and were similar to those of the racemate.

Mitogenic short-term effects on hepatocytes and adrenocortical cells: phenobarbital and reserpine compared to carcinogenic and non-carcinogenic fluorene derivatives.

The adrenal cortex has a low physiologic cell renewal and shows only a moderate cell replication even after contralateral adrenalectomy. Although rather unsusceptible to the malignancy-inducing action of carcinogens, a single oral dose of various tumorigenic xenobiotics induced an additive mitotic response of adrenocortical cells studied after 48 h. Presently we report on three different response patterns in rats. First, a selective mitostimulation of the zona glomerulosa occured after reserpine associated with a loss of body weight, thymus and liver weight. These are unspecific stress effects and occur also after exogenous ACTH. Second, hepatomitogenic and liver-enlarging congeners, e.g. fluorene (FEN), fluorenone (FON) and 4-benzoyl-FON, but also the genotoxic 2-acetylaminofluorene (2-AAF) and 2,4,7-trinitro-FON induced a selective mitotic response of the zona fasciculata (ZF). After the lowest effective dose of FEN or FON the afore-mentioned effects occured simultaneously, but were absent in the high dose group (only studied with fluorene). The 2-benzyl and 2-benzoyl-substituted derivatives were ineffective at all. Third, a bizonal response was found only after phenobarbital (PB) or the lowest effective FEN dose. The preventive action of a low PB dose on the 2-AAF-induced ZF response indicates a modified metabolism. We conclude that the rapid mitotic ZF response is an endogenously mediated net effect of interactions between metabolic and various adaptive mechanisms. The latter are reported to be activated in a stressor-dependent manner and converge in the adrenals. In this way the early mitotic ZF response could reflect indirectly 'specific' proliferation-prone properties of xenobiotics.

Influence of 2-acetylaminofluorene (2-AAF) on biotransformation and lipid peroxidation in salivary glands and liver from male rats.

Salivary glands proved to be active in biotransformation. In microsomes of rat salivary glands 7-ethoxyresorufin O-deethylation (EROD) and 7-pentoxyresorufin O-depentylation (PEROD) were detectable, but with much lower activities than in the liver. Beside the well-known induction of EROD or PEROD in the liver by beta-naphthoflavone (BNF) or phenobarbital (PB), respectively, a marked rise in EROD rate of salivary glands was observed after BNF treatment. Administration of 2-AAF caused an increase in EROD rates in liver microsomes, but a decrease in microsomes of salivary glands. This decrease in EROD rate was accompanied by selective cytotoxic damages in the convoluted granulated tubules of the submandibular glands. No cytotoxic damage occurred in the submandibular glands after a combined administration of the inducer BNF and 2-AAF. This indicates relations between these toxic effects of 2-AAF and changes of 2-AAF-metabolism in BNF-induced rats, maybe in the liver and/or in the submandibular glands themselves.

Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of beta-naphthoflavone, phenobarbital and dexamethasone on cytochrome P450 isoforms expression and on glycogen storage.

In the present study, the effect of beta-naphthoflavone (BNF), phenobarbital (PB) and dexamethasone (DEX) on the expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and on glycogen storage was investigated in intrasplenic liver cell explants in comparison to adult liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Four months after surgery, transplant recipients and age matched controls were orally treated with BNF (1 x 50 mg/kg body weight (b.wt.)), PB (1 x 50 mg/kg b.wt.), DEX (for 3 days 4 mg/kg b.wt. per day), or the respective solvents (dimethylsulfoxide or 0.9% NaCl). The animals were sacrificed 24 (BNF, DEX) or 48 (PB) hours after the last treatment. The livers of both solvent treated transplant recipients and control rats displayed only in few liver lobules a slight P450 1A1, but in all lobules a strong P450 2B1 and 3A2 expression, which was all mainly located in the hepatocytes around the central veins (zone III, according to Rappaport). After BNF administration a P450 1A1 expression was induced in the hepatocytes of the peripheral regions of the liver lobules (zone I, according to Rappaport), whereas the staining of the hepatocytes around the central veins disappeared. Also the staining for P450 2B1 in the hepatocytes of zone III became slightly more pronounced. Following PB treatment the P450 1A1 expression in the hepatocytes of the central regions (zone III), as seen in few lobules after solvent treatment only, was reduced, whereas the staining for P450 2B1 and 3A2 was more pronounced in the hepatocytes of the intermedial and central regions of the liver lobules (zone II and III). DEX treatment diminished P450 1A1 and 2B1 expression within the livers of both transplant recipients and control rats. In contrast, the staining for P450 3A2 was enhanced in all regions of the liver lobules. Transplantation of fetal liver tissue suspensions into the spleens did not influence the inducibility of P450 isoforms expression within the respective livers of the animals. Spleens of control rats displayed no P450 isoforms expression without as well as with induction. In the explant containing spleens, however, similar to normal liver, the transplanted hepatocytes displayed nearly no P450 1A1, but a strong P450 2B1 and 3A2 expression. After BNF treatment a staining for P450 1A1 was induced and also the P450 2B1 expression was slightly more pronounced. PB treatment caused an increase in the staining for P450 2B1 and 3A2 and DEX administration for P450 3A2 within the transplanted hepatocytes. Additionally, after DEX treatment some bile ducts of the explants displayed a slight staining for P450 1A1, 2B1 and 3A2. All hepatocytes within the livers of both solvent treated transplant recipients and control rats displayed a slightly PAS-positive cytoplasma and, in most cases, homogeneously distributed, fine-grained, strongly PAS-stained granules indicating glycogen storage. No regional variance in the glycogen content of the hepatocytes was seen within the liver lobules, but there was a marked difference between the individual hepatocytes of the same lobular region in the extent of glycogen accumulation. The hepatocytes within the explants displayed the same type of glycogen storage as did the adult liver cells. BNF treatment did not display any effect on the glycogen accumulation in livers and intrasplenic liver cell explants. After PB administration, only in livers, but not in the transplants, the glycogen content in the hepatocytes around the central veins was slightly reduced. DEX treatment lead to an excessive storage of fat within the hepatocytes of both livers and spleens. Thus, the glycogen was displaced, leading to a "spoke-wheel" like pattern of glycogen storage. Additionally, within the hepatocytes of both livers and liver cell explants a higher amount of glycogen seemed to be stored and the granules appeared to be more coarse-grained. (ABSTRACT

[Pharmacotherapeutic compass 1998]. / Farmacotherapeutisch kompas 1998.

The Central Medical Pharmaceutical Committee of the Health Insurance Council informs the medical profession annually about the effects of drugs through the Pharmacotherapeutical Compass. The 1998 edition now contains a chapter on pharmacokinetics as well. Compared with previous editions the main alterations of the contents concern an introduction and advice on the antidepressants, two protocols with respect to the medical treatment of patients suffering from epilepsy, advice with respect to oral drugs for the treatment of inflammatory bowel disease, an introduction and advice regarding the treatment of allergic rhinitis, the treatment of patients suffering from AIDS with antiretroviral drugs, the treatment of genital herpes, the taking of insulin lispro by patients with diabetes and the taking of bisphosphonates to prevent or to treat osteoporosis. Two corrections to the 1998 edition are given.

Influence of different transplantation methods on liver cell survival in spleens of rats.

Isolated hepatocytes, liver tissue suspensions, or liver tissue cylinders from biopsies were transplanted into the spleens of adult male rats. Donors were syngenic fetuses, syngenic or allogenic adult rats, or autologous material was obtained from the rat's own liver. The outcome of the different transplantation procedures was evaluated at 1 and 6 months after surgery. Additionally the influence of a 30% hepatectomy (HX) on the result of the transplantation was investigated. When fetal material was transplanted, the best results in sequence with respect to the number of viable hepatocytes within the spleens were obtained (1) after transplantation of syngenic fetal liver tissue suspensions, (2) syngenic fetal liver tissue cylinders and (3) syngenic fetal isolated hepatocytes. HX only improved the results with transplantation of syngenic fetal isolated hepatocytes. After transplantation of syngenic fetal liver tissue suspensions and isolated hepatocytes, but not after transplantation of syngenic fetal liver tissue cylinders, the number of hepatocytes was higher at 6 months than at 1 month after surgery. Concerning syngenic adult liver material, only transplantation of isolated hepatocytes lead to a remarkable and increasing number of surviving hepatocytes at both 1 and 6 months after surgery. These results were further improved by HX. With syngenic adult liver, the other transplantation methods yielded no or nearly no viable hepatocytes in the spleens. In comparison to the results after transplantation of syngenic fetal liver tissue suspensions, transplantation of syngenic adult isolated hepatocytes was less efficient, but still yielded more viable hepatocytes than the transplantation of syngenic fetal isolated hepatocytes. After transplantation of autologous liver tissue suspensions, autologous liver tissue cylinders or allogenic adult liver material only few surviving hepatocytes were observed. At 1 month after transplantation of syngenic fetal liver material, syngenic adult isolated hepatocytes or autologous liver tissue cylinders into the spleens 40-80% of the explants consisted of bile ducts independent from the transplantation method. At 6 months after surgery the bile ducts were much less and in some cases no longer visible. After transplantation of autologous liver tissue suspensions or allogenic adult liver material only very few bile ducts were seen, but anyhow in those cases only poor results were obtained. Thus, with respect to transplantation outcome and long-term liver cell survival, intrasplenic transplantation of both syngenic fetal liver tissue suspensions and syngenic adult isolated hepatocytes seem to be the most suitable methods and should be chosen for further investigations on explant morphology and function.

Developmental expression of cytochrome P450 isoforms after transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats.

In the present study, the developmental expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, and the ability to store glycogen was investigated in intrasplenic liver cell explants in comparison to adult and fetal liver. Fetal liver tissue suspensions were transplanted into the spleens of adult male syngenic Fisher inbred rats. Animals were sacrificed at 3 days, 1, 2, 4 weeks, 2, 4, 6 months and 1 year after transplantation. Spleens and livers of transplant recipients were compared to those of sham operated and control rats. Three days after transplantation little bulks of hepatocytes and only few bile ducts were seen in the red pulp of the transplant containing spleens. A massive hypertrophy and proliferation of bile ducts and also an augmentation in the number of hepatocytes were observed 4 weeks after transplantation. One month later, however, the bile ducts had become more and more atrophic, while instead the number of hepatocytes continuously increased. One year after surgery large masses of hepatocytes with apparent cord structure and only few but well preserved bile ducts were seen. Within the livers of adult rats, P450 1A1 was only slightly expressed by some hepatocytes around the central veins. P450 2B1 and 3A2 isoforms expression was much stronger, but also predominantly located in the hepatocytes of the central zone of the liver lobule. Hepatocytes of fetal livers displayed a moderate P450 1A1 expression. In some cells also a very mild staining for P450 2B1 and 3A2 was observed. Within the hepatocytes of the intrasplenic liver cell explants P450 1A1 was still expressed 3 days after transplantation, disappeared at 1 week after surgery, but reappeared at 4 weeks after transplantation. After 2, 4 and 6 months no staining for P450 1A1 was detectable any more. One year after transplantation again a slight P450 1A1 expression appeared. With P450 2B1 and 3A2 a mild to moderate expression was seen already at 3 days after transplantation. Four weeks after surgery nearly all of the hepatocytes were stained for P450 2B1 and 3A2, but there were marked differences between the individual cells in the extent of the expression of these two P450 subtypes, like it was also the case with normal adult liver. Within hepatocytes of the fetal livers strongly stained glycogen granules were seen, which, in comparison to adult livers, were rather coarse-grained. Three days after transplantation the glycogen granules in the transplanted hepatocytes were still coarse-grained, but from 1 week after transplantation on, they became more and more fine-grained. As it was also the case with normal adult liver cells, there were marked differences between the individual transplanted hepatocytes in their glycogen content. These results demonstrate that transplanted liver cells originating from syngenic fetal liver tissue suspensions can survive in host organs like the spleen for at least 1 year. They proliferate, differentiate, are able to store glycogen, and express different P450 isoforms, like normal adult liver cells.

[Intramural hemangioma of the portal vein]. / Intramurales Hämangiom der Vena portae.

Capillary hemangiomas occur rather ubiquitously, but extremely rare within the wall of blood vessels. For this reason the authors report on a small capillary hemangioma of sinusoidal type. It has been observed fortuitously within the wall of the portal vein at autopsy of a 79-year-old woman. Based on site and structural characteristics the benign neoplasia is supposed to have developed from a local malformation, probably an abortive liver anlage.

2-Acetylaminofluorene-produced selective cytotoxic damage of a ductal compartment and its repair in the submandibular gland of rats.

Salivary glands of rodents are rarely affected with spontaneous and induced malignancies. This may be linked with low physiologic cell renewal and the infrequency of cytolethal actions by xenobiotics. The genotoxic 2-acetylaminofluorene, carcinogenic for other organs, causes acute damage in salivary ducts. In the submandibular glands the damage is limited to the granulated convoluted tubules. They produce and release regulatory peptides including epidermal growth factor and nerve growth factor. The partial chemical sialoadenectomy is repaired by sequential cell proliferation in the basal cell layer of interlobular ducts and in dilated intralobular ducts (day 4 and 6), in intermediate duct-like structures (day 6 and 8), and lastly in acini (day 8 and 12). This is associated with a transient loss of structural characteristics of striated ducts and acini (up to day 6) and of the immunoreactivity for S-100 protein (up to day 4). Actin immunoreactivity at the acinar base is increased from day 6 to 20. Analogous to the late postnatal differentiation of the granulated convoluted tubules, their structural characteristics and immunoreactivity for epidermal growth factor do not recover within 20 days. The acute lesion of the endocrine ductal segment is suggested to be causally involved with other systemic effects following treatment with 2-acetylaminofluorene. First, hypophagia with loss of body, liver and thymus weight may result from disturbed saliva production. Second, previous studies have shown a mitotic burst of the biliary epithelium and bloodborne lymphocyte-stimulating activities. Either effect could be brought about by regulatory peptides (see above), probably after elevated circulatory release from necrotic granulated convoluted tubules.