Biomedical subjects

Peter Ruck

Publications and source records attributed to Peter Ruck.

14 recordsLinked to original sources

PubMed2006

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.

Adenoviridae↗

PubMed2006

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.

Animals↗

PubMed2006

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.

Apoptosis↗

PubMed2005

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.

Flow Cytometry↗

PubMed2005

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.

Animals↗

PubMed2004

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.

Fluorescent Antibody Technique↗

PubMed2004

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.

Animals↗

PubMed2003

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.

Cell Nucleus↗

PubMed2002

Stem-like cells in hepatoblastoma.

BACKGROUND: The principles of tumor biology suggest that hepatoblastoma is derived from a pluripotent stem cell. Our studies were undertaken to investigate this tumor for the presence of cells with morphologic and immunophenotypic features of the oval cells of rodents that are thought to be closely related to hepatic stem cells. PROCEDURE: Hepatoblastomas of various subtypes were investigated by electron microscopy and immunoelectron microscopy with antibodies against cytokeratin 7, in the liver a marker of biliary differentiation, and albumin, a marker of hepatocytic differentiation. Immunohistochemical investigations were performed with the antibodies OV-1 and OV-6, which recognize antigens associated with oval cells. RESULTS: OV-1 stained scattered cells in seven of 12 tumors investigated and OV-6 in nine. Small epithelial cells (SEC) with the ultrastructural features of the oval cells were found by electron microscopy. They were characterized by small size (7-18 microm), often an oval shape, tonofilament bundles, and tight junctions or desmosome-like junctions. SEC were found in small numbers in areas of fetal differentiation and in moderate numbers in areas of embryonal differentiation. In small cell hepatoblastoma, nearly all the tumor cells exhibited SEC-like features. Immunoelectron microscopy revealed coexpression of cytokeratin 7 and albumin by SEC. CONCLUSIONS: SEC with ultrastructural and immunophenotypic features exhibited by oval cells, i.e., hepatic stem-like cells, are found in hepatoblastoma. Their numbers vary with the differing degrees of differentiation seen in the various subtypes. The findings further support the hypothesis that hepatoblastoma is derived from a pluripotent stem cell.

Antigens, Surface↗

PubMed2002

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.

Amniotic Fluid↗

PubMed2002

MR imaging-detected breast lesions: histopathologic correlation of lesion characteristics and signal intensity data.

OBJECTIVE: The aim of our study was to differentiate benign from malignant breast lesions that had been detected exclusively on MR imaging by analyzing qualitative and quantitative lesion characteristics. MATERIALS AND METHODS: We performed 51 MR imaging-guided breast interventions (41 preoperative lesion localizations and 10 large-core needle biopsies) in 45 patients with exclusively MR imaging-detected lesions. All patients had previously undergone diagnostic dynamic contrast-enhanced MR imaging of the breast with a double breast coil at 1.0 T (n = 36) or 1.5 T (n = 15). The diagnostic MR images were evaluated on a workstation. Lesion morphology (size, shape, margin type, enhancement pattern), signal intensity parameters (time to peak enhancement, maximum slope of enhancement curve, washout, relative water content), and scores analogous to the Breast Imaging Reporting and Data System (BI-RADS) categories were correlated with histology. RESULTS: Histology revealed malignancy in 37.3% (19/51) of the lesions. The positive predictive value for malignancy of exclusively MR imaging-detectable lesions increased as the analogous BI-RADS category increased. Late inhomogeneous contrast enhancement was the only morphologic criterion that was statistically significantly correlated with malignancy. Malignant and benign lesions did not differ significantly in any of the quantitatively evaluated signal intensity parameters. Carcinomas showed a tendency toward faster and stronger enhancement and stronger washout. CONCLUSION: The classification of exclusively MR imaging-detectable breast lesions according to a combination of morphologic and perfusion parameters including the late enhancement pattern helps identify the lesions for which interventional MR imaging is required. Quantitative signal intensity data alone do not suffice.

Adult↗

PubMed2002

Ultrasonographic findings correspond to clinical, endoscopic, and histologic findings in inflammatory bowel disease and other enterocolitides.

OBJECTIVE: To compare results obtained by abdominal ultrasonography with clinical findings, including endoscopic and histologic findings, to evaluate the location and activity of inflammatory bowel disease, including disease controls in children. METHODS: Ninety-two ultrasonographic scans and 41 colonoscopic examinations with biopsies were performed in 78 patients (1 month to 17.8 years of age) with Crohn's disease (n = 26), ulcerative colitis (n = 21), inflammatory bowel disease of indeterminate type (n = 2), and disease controls (other intestinal disorders, including infectious and ischemic lesions; n = 29). Laboratory parameters for inflammatory bowel disease were determined, and disease activity was assessed by a combination of clinical and laboratory data. Bowel wall thickness and echo texture were recorded in a standardized way by ultrasonography and compared with endoscopic and histologic findings in a segment-by-segment comparison. RESULTS: Sensitivity and specificity of ultrasonography in detecting patients with severe macroscopic lesions depicted on endoscopy were 77% and 83%, respectively. Sensitivity and specificity of ultrasonography in detecting patients with severe histologic inflammation were 75% and 82%. There was a statistically significant correlation between maximal bowel wall thickness and disease activity score (P < .01). CONCLUSIONS: Abdominal ultrasonography may be helpful in evaluating the location, severity, and inflammatory activity of inflammatory bowel disease in children and young adults.

Adolescent↗

PubMedDate not supplied

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.

Aged↗

PubMedDate not supplied

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.

ATP Binding Cassette Transporter, Subfamily B, Mem↗