Proteasome inhibition overcomes TRAIL resistance in human hepatoblastoma cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is responsible for cell death in many cancer cells while being non-toxic for most normal cells. In this study, we investigated the role of TRAIL in human hepatoblastoma (HB) cells and analyzed different approaches to reverse TRAIL resistance in these tumors. Death receptors DR4 and DR5 expression was found on all analyzed primary HB samples and on the cell lines HuH6 and HepT1 by immunofluorescence staining. Recombinant TRAIL alone did not induce in vitro cytotoxicity. Decoy receptor blocking by antibodies led to moderate effects in HepT1 but not in HUH6 cells, whereas FLIP knock-down using siRNA rendered HUH6 cells but not HepT1 cells sensible to TRAIL. Bcl-2 inhibition with ABT-737 enhanced TRAIL-mediated apoptosis in all HB cells. Strongest cytotoxic TRAIL effects were seen in HB cell lines with synchronous proteasome inhibition using bortezomib. FLIP and Bcl-2 contributed to the TRAIL resistance in HB. Overcoming TRAIL resistance in HB by proteasome inhibitors has been identified a possible additive to improve treatment results in HB patients with drug resistant tumors.

Successful establishment of an orthotopic hepatoblastoma in vivo model in NOD/LtSz-scid IL2Rγnull mice.

Investigation of hepatoblastoma in experimental conditions contributes relevantly to a detailed understanding of tumor biology and the investigation of new treatment approaches. Most systematical analyses currently use subcutaneous xenografts. We established a reproducible intrahepatic model with the hepatoblastoma-cell lines HuH6 and HepT1. The cells were stably transfected with a plasmid vector encoding for Gaussia luciferase. HuH6 and HepT1 were injected intrasplenically in NOD/LtSz-scid IL2Rγnull mice. Mice were splenectomized in order to avoid intrasplenical tumor growth. Multifocal intrahepatic tumor growth was observed in 85% (11/13) of HuH6 tumors and 55% (5/9) of HepT1 tumors. Serum Alpha-fetoprotein and Gaussia luciferase increased 5 weeks after tumor-cell inoculation. Tumors were detected by MRI at this time point. Immunhistochemical analysis such as vascularity (CD31), proliferation index (Ki-67), cytokeratin 7 and distribution of ß-catenin in intrahepatic tumors were different to subcutaneous tumors. We established a reproducible xenograft model for intrahepatic hepatoblastoma growth with a high tumor incidence. Monitoring of tumor cell viability was optimized by measuring GLuc. This model enables further experimental investigations of HB in a more physiological milieu as emphasized by the ß-catenin distribution.

CD155 is involved in NK-cell mediated lysis of human hepatoblastoma in vitro.

NK cells are involved in the lysis of different solid tumors and leukemias. NK-activity is thereby regulated by activating and inhibitory receptors. Until now, nothing is known about the NK-activity against hepatoblastoma and the involved receptors. We tested NK cells for cytotoxicity against HB in vitro. Expression levels of activating NK ligands were analysed on 13 primary HB samples as well as on 3 HB cell lines. All HB cell lines showed low HLA-class-I-expression. CD155 expression was strong on primary HB samples and cell lines. NKG2D-ligands (MICA/B, ULBP1-3) were heterogeneous expressed in primary samples and cell cultures. There were no differences between the various histological subtypes. NK cells showed strong cytotoxicity in vitro which was significantly increased through interleukin-2 and -15 stimulation (p less than 0.001). Blockade of CD155 resulted in decreased lysis rates. Our findings show that NK cells exert high activity against hepatoblastoma in vitro and that CD155 is involved in the NK mediated killing of HB. The inclusion of a NK-based immunotherapy into novel treatment strategies might be a promising alternative especially for advanced tumors.

Differential expression of glutamine synthetase and cytochrome P450 isoforms in human hepatoblastoma.

Carcinogenesis is often linked to aberrant activation of Wnt/ß-catenin signalling, in many cases caused by activating CTNNB1 mutations (encoding ß-catenin). Recently, ß-catenin was established as a decisive regulator of hepatic glutamine synthetase (GS) and cytochrome P450 (CYP) expression in mouse hepatocarcinogenesis. This study was aimed to analyse the connection of ß-catenin signalling and GS/CYP expression in human paediatric tumours. Samples from 23 paediatric tumours were analysed for activating mutations in CTNNB1. Protein expression of the model ß-catenin target GS and of various CYP isoforms was analysed and correlated with CTNNB1 mutational status and histological findings. Activating CTNNB1 mutations were frequent in hepatoblastoma (80%) and nephroblastoma (31%). In CTNNB1-mutated hepatoblastoma, expression of GS was only detected in tumour areas with epithelial, not with mesenchymal differentiation. Particularly high expression of glutamine synthetase was found in hepatoblastoma cells directly neighbouring a mesenchymal-type tumour area or stroma cells, associated with above-average cell proliferation. GS expression was not observed in CTNNB1-mutated nephroblastoma. Hepatoblastoma with activated ß-catenin expressed different CYPs relevant for the metabolism of cytostatic drugs, but with high interindividual variance and heterogeneity within a single tumour. GS and different CYPs are co-expressed in hepatoblastoma with activated ß-catenin. Moreover, other factors like histological subtype of tumour cells and cell-cell-interactions at the borders between different areas of the tumours affect expression of these ß-catenin target genes. Analysis of CYP expression in resected tumour tissue might be useful for the selection of appropriate cytostatics for post-operative chemotherapy.

Inhibition of glutathione-S-transferase as a treatment strategy for multidrug resistance in childhood rhabdomyosarcoma.

Multidrug resistance (MDR) is a common problem in the treatment of childhood rhabdomyosarcoma (RMS). A complete reversal of MDR is currently not possible. The aim of this study was to investigate the role of glutathione-S-transferase (GST) as mechanism of MDR in childhood RMS and to analyze possible reversal strategies. Female athymic mice underwent xenotransplantation with embryonal or alveolar RMS cells and were treated with vincristine. Gene expression analysis using Affymetrix HU-Gene 1.0 arrays revealed 2314 differentially expressed genes between the groups in alveolar RMS and 1387 in embryonal RMS. Ingenuity pathway analysis revealed a cluster of 5 overexpressed genes of the GST family in animals treated with vincristine, putative mediating the development of MDR. In order to analyze possible GST activity after chemotherapy with other commonly used drugs (doxorubicin, topotecan), cell culture experiments with alveolar and embryonal RMS cells were carried out. Specific GST activity was quantified using the clorodinitrobenzol conjugation with glutathione. Increased GST activity was found after incubation with cytotoxic agents in all cell lines. Highest induction of GST activity was found in embryonal RMS (up to 12-fold). After incubation with the GST inhibitors, tumor cell viability was decreased depending on the type of tumor cell and inhibitor used. We detected a novel mechanism for MDR in childhood RMS mediated via genes and proteins of the GST family. Reversal of these effects may be achieved by GST inhibitors in part. The GST family represents a promising target for further treatment strategies in childhood RMS.

Adenovirus-mediated cytosine deaminase/5-fluorocytosine suicide gene therapy of human hepatoblastoma in vitro.

BACKGROUND: Multidrug resistance is a key factor for the sobering outcome of relapsed and metastatic human hepatoblastoma (HB). Gene directed treatment approaches were recently identified as possible treatment options against advanced HB, in which standard chemotherapy regimens are partially insufficient. The aim of this study was to systematically analyze the effects of suicide gene therapy in three HB cell lines using a yeast-derived cytosine deaminase (YCD)-combined yeast uracil phosphoribosyltransferase (YUPRT)-based adenovirus-mediated gene transfer. PROCEDURE: YCD and YUPRT were fused to form the bifunctional suicide gene SuperCD. Adeonoviral vectors were used for transduction. Tumor cells transduced at MOI 50 were incubated with 5-fluorocytosine (5-FC) in ascending concentrations. RESULTS: Transduction rates were 87.8% (+6.7) in the mixed HB cell line HUH6, 98.6% (+1.4) in the epithelial HB cell line HepT1 and 93.6% (+0.6) in the multifocal HB embryonal cell line HepT3, respectively. In HepT3 and HepT1 cells suicide gene therapy with SuperCD/5-FC was highly effective leading to HB cell damage far above those of application of the prodrug 5-FC only. In HUH6 cells the approach had no effect due to a lack in activity of the CMV promoter being employed for transcription of the SuperCD transgene. CONCLUSION: Assuming employment of fully active promoters, the SuperCD/5-FC approach may serve as a potentially useful anti-tumor strategy against advanced HB.

In vitro photodynamic therapy in pediatric epithelial liver tumors promoted by hypericin.

Limited treatment results in advanced pediatric liver tumors have emphasised the need for alternative treatment approaches in these malignancies. Photodynamic therapy (PDT) has been proposed as promising treatment approach in various malignancies. Hypericin, a naturally occurring substance found in the St. John's Wort, has regularly and successfully been used for visualisation and as photosensitizer in various tumor models. However, there exist no data on the effects of hypericin as photodynamic agent in pediatric malignant epithelial liver tumors. In this study, we investigated the potential role of hypericin for visualization and treatment in hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) cells. Two HB cell lines (HUH6, HepT1) and one HCC cell line (HepG2) were incubated with ascending concentrations of hypericin. Uptake and fluorescending capability were assessed using fluorescence microscopy and FACS. PDT with white light was performed for varying time intervals. Cell viability, cell proliferation and apoptotic rates were assessed using MTT assay, Ki-67 immunocytochemisty and TUNEL test, respectively. The changes within tumor cells under therapy were monitored using standard cytology. Relevant hypericin uptake was observed in all cell lines according to the applied concentrations. Histological analysis revealed no alterations of cell structure in HB and HCC cells after solely hypericin uptake, but severe alterations were found after PDT. Enhancement of the hypericin concentration (up to 12.5 microM) and illumination time of up to 40 min resulted in a decrease of tumor cell viability (HUH6 99.8+/-2.4%, HepT1 99+/-2%, HepG2 98.4+/-1.6%, p<0.05), proliferative activity and complete apoptosis of all cells in all investigated cell lines. These data show that hypericin might be a useful tool for visualisation and as alternative treatment option in HB and HCC.

Bcl-2 gene silencing in pediatric epithelial liver tumors.

BACKGROUND: Proteins of the Bcl-2 family prevent cells of various tumor types from undergoing apoptosis and thus contribute to their chemotherapy resistance. The phenotype of multidrug resistance is a major factor for poor treatment results of advanced epithelial liver tumors in children. The role of Bcl-2 proteins in these tumors is yet unclear. The purpose of this study was to analyze the influence of Bcl-2 on the chemotherapy resistance of hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Bcl-2 expression was analyzed in the HB cell lines HUH6 and HepT1 as well as in the HCC cell line HepG2 before and after treatment with cisplatin, doxorubicin, taxol, and etoposid. Silencing of the Bcl-2 gene was performed via RNA interference using specific siRNA. Treatment efficiencies of cytotoxic agents were assessed against original and Bcl-2 siRNA transfected tumor cells. RESULTS: The mixed HB cell line HUH6 showed a relevant amount of Bcl-2 expression, which increased after chemotherapy. In these cells Bcl-2 appeared within the nuclei and the cytosol. Treatment with all cytotoxic agents was significantly improved through Bcl-2 siRNA (P < 0.001-0.0054) in this cell line. There was no effect of Bcl-2 siRNA in HepT1 and HepG2 cells. CONCLUSIONS: Bcl-2 seems to play a role in antiapoptotic mechanisms of some HB subtypes. Thus, this gene might serve as target for a gene-directed adjuvant therapy. Further studies seem necessary to clear the susceptibility of pediatric epithelial liver tumors toward the described approach.

In vitro photodynamic therapy of childhood rhabdomyosarcoma.

Treatment of childhood rhabdomyosarcoma is limited by recurrent disease and the development of multidrug resistance. Therefore, novel treatment options are desirable. Photodynamic therapy (PDT) using the photodynamic agent hypericin is proposed as an alternative approach for intraoperative visualization and treatment of this disease. The aim of this study was to investigate in vitro effects of hypericin on childhood rhabdomyosarcoma and to evaluate photodynamic therapy as a possible basis for treatment. Rhabdomyosarcoma cells and fibroblasts (control) were incubated with increasing concentrations of hypericin. in vitro uptake and visualization of hypericin was evaluated by fluorescence microscopy and FACS. For photodynamic therapy, cells were exposed to white light for different time periods. Cytopathologic effects were assessed using standard histology. Cancer cells were investigated for cell viability (MTT assay), proliferative activity (Ki-67 assay), and apoptosis (TUNEL test). A 100% uptake of hypericin was found within the population of rhabdomyosarcoma cells. Uptake of hypericin in the fibroblasts was much less than in rhabdomyosarcoma cells. Hypericin without exposure to white light had no effect on tumor cell viability. After irradiation, PDT resulted in a nearly complete inhibition of cell proliferation of rhabdomyosarcoma cells with a corresponding increase in the frequency of apoptosis. In fibroblasts, PDT was less effective compared to tumor cells. Our data suggest hypericin as a novel tool for visualization and photodynamic therapy of childhood rhabdomyosarcoma.

Optimizing vector application for gene transfer into human hepatoblastoma cells.

Gene targeting is currently of distinct interest as an innovative additive treatment option in various malignancies. Its role in pediatric liver tumors has not yet been evaluated thoroughly. For the first time the authors systematically analyzed both lipid-based transfection as well as transduction with adenovirus vectors (Ad) and Sendai virus vectors (SeVV) in order to optimize gene transfer into hepatoblastoma (HB) cells. Two HB cell lines were infected with Ad or SeVV coding for green fluorescent protein (Ad-GFP, SeVV-GFP); transduction efficiencies and apoptosis were assessed using flow cytometry. Furthermore, lipofection of HB cell lines with plasmid-constructs comprising liver-specific promoters was performed using Lipofectamine 2000 and FuGENE 6; lipofection efficiency was monitored by flow cytometry, microscopy, and luciferase activity. The Ad-GFP showed higher transduction rates (61-86%) than the SeVV-GFP (4-24%) depending on the HB cell line used. Infections with first generation SeVV vectors (SeVV-GFP) led to increased target cell apoptosis (7-43%) compared to Ad-GFP (4-16%). The Lipofectamine 2000 revealed a higher transfection efficiency than the FuGENE 6 for both HB cell lines tested. The liver-specific promoters were found to be differently active in the HB cell lines. This study delineates recombinant adenovirus vectors as a promising tool for gene transduction in the HB cells. Furthermore, enhanced activity of the liver-specific promoters in HUH6 cells compared to HepT1 cells supports the observation of varying biological behavior in histologically differing HB tissues.

Altered protease expression by periarterial trophoblast cells in severe early-onset preeclampsia with IUGR.

Adaptation of uteroplacental arteries in patients with early-onset preeclampsia combined with IUGR is compromised due to insufficient invasion of extravillous trophoblast cells (EVT) into the spiral artery wall. The underlying molecular mechanisms are widely unknown. We investigated expression and possible mechanisms of regulation of different matrix-metalloproteases (MMPs) by EVT in placental bed biopsies from patients with early onset preeclampsia combined with IUGR and healthy pregnant women. Expression of MMP-3 and MMP-7 by EVT was markedly reduced in preeclamptic patients, especially close to spiral arteries. In contrast to healthy pregnancies these cells strongly expressed the receptor for leukemia inhibitory factor (LIF). LIF is known to suppress MMP-expression and is produced by uterine natural killer (uNK) cells which we found to be present in higher concentrations in the placental bed of preeclamptic patients, and accumulating aside the spiral arteries. We speculate that in preeclampsia a maternal immune cell network accumulating and interfering in the placental bed leads to an altered cytokine environment, resulting in disturbed trophoblast cell function such as impaired MMP expression and reduced invasiveness.

Visualization of xenotransplanted human rhabdomyosarcoma after transfection with red fluorescent protein.

BACKGROUND/AIMS: Discosoma sp red fluorescent protein (DsRed2) is a newly developed marker for in vivo labeling studies in different biologic systems. After vector transfection, DsRed2 is expressed in mammalian cells and can be detected by fluorescence microscopy. The aims of this study were to establish a DsRed2-transfected human rhabdomyosarcoma (RMS) cell line and to perform a xenotransplantation on nude mice to use imaging as a tool for further basic research studies on this neoplasm. PROCEDURE: The human alveolar RMS cell line Rh30 was transfected with the pDsRed2-N1 vector by lipofection. The DsRed2-positive cells were sorted out by fluorescence-activated cell sorting analysis 96 hours after transfection and selected in culture with G418. Expression of DsRed2 messenger RNA was assessed using single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Transfected and parental cells were characterized cytologically, cytogenetically, immunohistochemically, and in vivo after subcutaneous injection in NMRI (nu/nu) nude mice. RESULTS: After vector transfection, a pure and stable DsRed2-positive cell line was established by monoclonal growth of the cells. Reverse transcriptase polymerase chain reaction revealed constant expression of DsRed2 messenger RNA in fluorescencing cells. There was no difference between transfected and parental cells by means of cell morphology and desmin expression. Clonal cells (1 x 10(6)) were used for xenotransplantation. Tumors were visualized noninvasively through the skin of the mice using specific emission and excitation filters. Tumor vascularization and vessel growth could be discriminated from tumor tissue using this imaging system. CONCLUSION: This is the first report on successful transfection of an RMS cell line with red fluorescent protein followed by xenotransplantation into nude mice. This model can serve as an imaging tool for in vivo studies investigating tumor biology and metastases of human RMS.

Protective effect of tumor necrosis factor alpha antibody on experimental necrotizing enterocolitis in the rat.

BACKGROUND: Necrotizing enterocolitis (NEC) is a common and devastating disorder of premature infants. Elevated proinflammatory cytokines, especially tumor necrosis factor alpha (TNF-alpha), have been implicated in the pathogenesis of NEC. The aim of this study was to evaluate the effects of TNF-alpha on the inflammatory response in NEC by immunoneutralizing TNF-alpha with a selective antibody. METHODS: Neonatal Sprague-Dawley rats were divided in 3 groups: group 1 (n = 20), a NEC-like enterocolitis was induced by formula feeding, asphyxia, and cold exposure; group 2 (n = 9), animals were treated like in group 1 and additionally received TNF-alpha antibody intraperitoneally; and group 3 (n = 17), animals were dam-fed (controls). Animals were killed in case of imminent death or after 96 hours. Specimens from small bowel were processed for blinded histologic (H&E) and immunhistologic (myeloperoxidase [MPO]) analysis. RESULTS: In group 1, animals developed severe NEC (mean NEC score, 3.28 +/- 0.32; mean MPO, 65.85 +/- 9.46). In group 2, animals developed mild NEC (mean NEC score, 1.72 +/- 0.41; mean MPO, 34.33 +/- 9.69; P < .05). In group 3, no NEC was induced (mean NEC score, 0.0 +/- 0; mean MPO, 6 +/- 1.32; P < .05). CONCLUSION: Tumor necrosis factor alpha antibody may have an attenuating effect on experimental NEC in rats.

Effects of P-glycoprotein modulation on the chemotherapy of xenotransplanted human hepatoblastoma.

Multidrug resistance (MDR) contributes to limited treatment results in human hepatoblastoma (HB). The MDR1 gene and its product P-glycoprotein (P-gP) has been identified as important factor in this development. In other tumors, P-gP modulation leads to a restored chemosensitivity of the cells. The aim of this study was to analyze the P-gP-modulating effects of PSC 833, a cyclosporine derivate, and verapamil on the chemotherapy of HB in vivo. HB from 2 patients were transplanted subcutaneously into nude mice NMRI (nu/nu). Animals were divided into 7 groups: Group 1 (Control); Group 2 (CDDP); Group 3 (DOXO); Group 4 (DOXO + verapamil); Group 5 (DOXO + PSC 833); Group 6 (CDDP + verapamil); and Group 7 (CDDP + PSC 833). If DOXO was administered (regardless of the combination), the dose was two times 60 mg/m2. If CDDP was administered, the dose was two times 27 mg/m2. When the chemosensitizers were administered, the doses for PSC 833 and for verapamil were four times 5 mg/kg body-weight. In the combined treatment groups the chemosensitizers were given ten minutes prior to CDDP and DOXO. Tumor volume developments and a-fetoprotein (AFP) alterations were assessed. Relative expression levels of the MDR1 gene after treatment were determined using a semiquantitative rT-PCR approach. In a mixed HB, both chemosensitizers combined with DOXO or CDDP produced a significant reduction of tumor growth (p = .0001-.00063) and AFP levels (p = .0006-.0128) compared to tumors treated with DOXO or CDDP only. Treatment results were identical to those in a less differentiated pure embryonal HB, but only in one case (DOXO + PSC 833, p = .031) significant. The chemosensitizers had no influence on the MDR1 gene expression. MDR1 modulators improve the efficiency of DOXO and CDDP treatment in xenotransplanted HB. They do not induce a further increase of drug resistance in the tumors. The data provide evidence that chemosensitizers might improve treatment results in patients with advanced or relapsed HB.

New trends in tumor biology: transfection of a human hepatoblastoma cell line with green fluorescent protein.

BACKGROUND/PURPOSE: Enhanced green fluorescent protein (eGFP) is widely used as a marker in different biologic systems. After vector transfection, eGFP is expressed by eukaryotic cells and can be visualized using fluorescent microscopy. The aim of this study was to establish an eGFP-transfected human hepatoblastoma (HB) cell line as tool for further basic research studies. METHODS: The HB cell line HUH6 was transfected with the pEGFP-N1 vector by liposomal transfection. Enhanced green fluorescent protein-positive cells were sorted out by fluorescence-activated cell sort and selected using G418 resistance. Expression of eGFP-messenger RNA was assessed by single-cell reverse transcriptase polymerase chain reaction after laser microdissection. Original and transfected cells were compared biologically and cytomorphologically. RESULTS: Vector transfection produced up to 15% eGFP-positive cells. After fluorescence-activated cell sort and G418 selection, a pure cell line was established with 100% eGFP-positive cells. Reverse transcriptase polymerase chain reaction revealed constant expression of eGFP-messenger RNA in fluorescending cells. Analysis of cell characteristics revealed no differences between transfected and original cells. CONCLUSIONS: For the first time, the authors established an eGFP-transfected HB cell line. This cell line can serve as a promising tool for further studies investigating HB in vitro and in vivo. Our model might also be a basis for similar work on other pediatric solid tumors.

Engagement of the CD137 (4-1BB) costimulatory molecule inhibits and reverses the autoimmune process in collagen-induced arthritis and establishes lasting disease resistance.

Agonistic antibodies against CD137 act as costimulators in the activation of CD8 T cells. They enhance the immune response against syngeneic tumour grafts and suppress T cell-dependent humoral immune responses in vivo. The present study was undertaken to determine whether suppression of antibody production by anti-CD137 mAb affects the development of collagen-induced arthritis (CIA). Male DBA/1J mice were immunized with bovine collagen II (CII) and treated with an agonistic anti-CD137 mAb or an isotype-matched control mAb. Mice were assessed regularly for macro- and microscopic signs of arthritis and for the appearance of collagen-specific antibody production. Interferon (IFN)-gamma determination, FACS analysis of splenocytes and histopathological joint examinations were performed after the animals were killed. Administration of anti-CD137 mAb at the time of collagen immunization blocked the development of disease and inhibited the humoral immune response against CII. Agonistic anti-CD137 mAb exhibited therapeutic efficacy even after the immune response to CII had succeeded and the disease became apparent. Furthermore, it induced a protective memory in the animals, enabling resistance to subsequent challenges with the pathogenic antigen. Our results suggest a key role for CD137 in the pathogenesis of CIA. This model provides insights into immunoregulatory conditions that control the pathogenesis of autoimmune diseases.

Hepatic progenitor cells in human liver cirrhosis: immunohistochemical, electron microscopic and immunofluorencence confocal microscopic findings.

AIM: To investigate whether hepatic progenitor cells (HPC), that reveal the features of oval cells in rodents and small epithelial cells (SEC) in certain human liver disease, were also found in human liver cirrhosis (HLC). METHODS: Surgical liver specimens from 20 cases of hepatitis B virus-positive HLC (15 cases containing hepatocellular carcinoma) were investigated by light microscopic immunohistochemistry (LM-IHC). Among them specimens from 15 cases were investigated by electron microscopy (EM) and those from 5 cases by immunofluorencence confocal laser scanning microscopy (ICLSM). Antibodies against cytokeratin 7 and albumin were used and single and/or double labelling were performed respectively. RESULTS: LM-IHC showed that at the margins of regenerating nodules and in the fibrous septae, a small number of cells in the proliferating bile ductules were positive for CK7 and albumin. At the EM level these HPC were morphologically similar to the SEC described previously, and also similar to the oval cells seen in experimental hepatocarcinogenesis. They were characterized by their small size, oval shape, a high nucleus/cytoplasm ratio, a low organelle content in cytoplasm, and existence of tonofilaments and intercellular junctions. ICLSM revealed that HPC expressed both cytokeratin 7 and albumin. CONCLUSION: HPC with ultrastructural and immunophenotypical features of oval cells, i.e., hepatic stem cell-like cells as noted in other liver diseases, were found in HLC. These findings further support the hypothesis that bipotent hepatic stem cells, that may give rise to biliary epithelial cells and hepatocytes, exist in human livers.

Comparative genomic hybridization analysis of hepatoblastoma reveals high frequency of X-chromosome gains and similarities between epithelial and stromal components.

Hepatoblastoma (HB) is the most common liver tumor in childhood and differs in its environmental risk factors and genetic background from hepatocellular carcinoma. HB is associated with inherited conditions such as familial adenomatous polyposis and Beckwith-Wiedemann syndrome, suggesting the importance of genetic abnormalities in the pathogenesis and progression of this disease. It has a very polymorphous morphology. A diverse range of cytogenetic alterations has been reported to date, the most frequent being trisomy 2 and trisomy 20. Thirty-five HB specimens from 31 patients (22 purely epithelial, 4 purely mesenchymal, 9 mixed) were examined by comparative genomic hybridization (CGH), a technique that enables us to screen the entire tumor genome for genetic losses and gains. Our aims were as follows: (1) to characterize chromosome abnormalities that appear in this tumor and (2) to identify possible differences between different histologic subtypes of HB. We found significant gains of genetic material, with very little difference in the number and type of alterations between the different histologic components of HB. The most frequent alterations were gains of Xp (15 cases, 43%) and Xq (21 cases, 60%). This finding was also confirmed by fluorescent in situ hybridization performed on nuclei extracted from 6 specimens. Other common alterations were 1p-, 2q+, 2q-, 4q-, and 4q+. We found no difference between different histologic subtypes, a finding that may be in agreement with the hypothesis of a common clonal origin for the different components. An hitherto-unreported high frequency of X chromosome gains may support the assumption that X-linked genes are involved in the development of this neoplasm.

Treatment of periprosthetic soft tissue infection of the groin following vascular surgical procedures by means of a polyvinyl alcohol-vacuum sponge system.

Deep groin infections after prosthetic vascular surgical procedures represent a serious complication of surgical practice. Septicemia and/or erosive hemorrhage can both be consequences. In this situation, removal of the graft appears to be the only option. However, if the infection is detected early (type Szilagyi III), local treatment to eradicate the infection could serve as an alternative. Twenty-four patients with confirmed infection of the soft tissue adjacent to the prosthetic material in the groin were treated locally by implantation of a vacuum sponge system. Duration of this treatment was 2 weeks. All patients showed excellent tissue granulation of the wound area and the microbial stains were negative at the end of therapy. In 21 patients the wound could be primarily closed after explantation of the sponge. Three patients underwent open treatment because of a skin defect. After 12 months, the wounds had healed well in all patients. Histologic evaluation revealed a physiological healing process. Deep soft tissue infections of the groin adjacent to prosthetic vascular material (type Szilagyi III) can be treated effectively and safely with the vacuum sponge system. The treatment is inexpensive, easy to perform, and the initial vascular reconstruction can be preserved.

Cytokine concentrations in the amniotic fluid during parturition at term: correlation to lower uterine segment values and to labor.

BACKGROUND: In previous studies a significant increase in interleukin (IL)-6 and IL-8 concentrations in the lower uterine segment parallel to cervical dilatation at term could be found, however only a weak correlation to duration of labor was detected. This study compares amniotic fluid concentrations of interleukin (IL)-6 and IL-8 with those in the lower uterine segment, and the duration of labor. METHODS: Amniotic fluid and lower uterine segment specimens were obtained from 29 patients during cesarean section at term. The patients were divided into groups according to cervical dilatation (< 2 cm, 2-3.9 cm, 4-6 cm, > 6 cm) and to labor (0 h, > 0-12 h, > 12 h). Interleukin-6 and IL-8 concentrations were determined by enzyme immunoassay. RESULTS: Amniotic fluid IL-6 already increased at 2-3.9 cm (p = 0.02), while the steepest increase in IL-8 was observed at > 6 cm (p = 0.003). No correlation with lower uterine segment values was observed for either of the cytokines. However, the amniotic fluid IL-6 concentration correlated with labor (p = 0.0008). CONCLUSION: The increase in the concentration of IL-6 in the amniotic fluid earlier than its increase in the lower uterine segment supports the hypothesis that IL-6 plays a crucial role for the promotion of labor in the first place, and afterwards for the biochemical degradation processes in the lower uterine segment. The fact that the greatest increase in IL-8 concentration occurs only at > 6 cm indicates that this chemotactic cytokine has only minor significance in the initiation of parturition by its concentration in the amniotic fluid.