Richard Magdeburg

Endoclipping of iatrogenic colonic perforation to avoid surgery

BackgroundColonoscopy is an established tool for the diagnosis and management of colonic and rectal pathology. Even though colonic perforation is rare after colonoscopy, it is a serious and typical complication. The definitive management remains controversial. Both operative and nonoperative techniques have been described in the literature, though the standard treatment for these patients is still an operative repair of the perforation site. Recently, endoscopic clip application was recommended, particularly for iatrogenic perforations, but less is known about the effectiveness of endoluminal repair of colonic perforations with clips.MethodsIn this series, 7589 colonoscopies were performed over a 34-month period in a tertiary-level referral center. Three perforations occurred during 5413 diagnostic colonoscopies. Therapeutic colonoscopy was under taken in 2176 patients, resulting in a total of 27 perforations. Out of 30 patients with colonic perforation, five patients underwent operative management and 25 patients were subsequently treated nonoperatively.ResultsIn 27 patients, endoscopic application of inert metallic clips was used for closure of iatrogenic perforation. Twenty-five of these patients were treated non-operatively, while two patients underwent surgery. The mean postoperative length of hospitalization for patients was 12.2 days, compared to 3.5 days for patients treated conservatively.ConclusionsEndoluminal repair of colonic perforations with clips and further conservative treatment seems to provide a tool that avoids the major additional trauma associated with laparotomy or laparoscopy and minimizes the length of hospitalization.

Imaging Epidermal Growth Factor Receptor Phosphorylation in Human Colorectal Cancer Cells and Human Tissues

In tumor cells, high phosphorylation levels of receptor tyrosine kinases may occur in the absence of exogenous ligands due to autocrine signaling or enhanced tyrosine kinase activity. Here we show that the phosphorylation state of the endogenous epidermal growth factor receptor (EGFR) can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies against the receptor and phosphotyrosine, respectively. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation with EGF or pervanadate, dependent on the level of basal EGFR phosphorylation in the respective cell lines. This basal activity correlated inversely with receptor expression. Using the acceptor photobleaching fluorescence resonance energy transfer imaging approach, a significantly higher phosphorylation state of EGFR was also found in resected human colorectal tumor samples as compared with adjacent healthy tissue. Imaging of EGFR phosphorylation may thus serve as a valuable tool to investigate the role of receptor tyrosine kinase activity in malignant cell growth.

Influence of TNFA on the formation of liver metastases in a syngenic mouse model

The level of TNFα expression is increased after partial hepatectomy, and experimental evidence exists that TNFα plays a key role in liver regeneration. Contradictory results are reported about the influence of TNFα on tumor growth: on the one hand, stimulation of tumor growth in various animal models and, on the other hand, intraperitoneally administered TNFα leads to reduced metastasis formation. TNFα may be one responsible factor for increased metastasis formation after surgical trauma. The objective of our study was to clarify the influence of TNFα on the formation of liver metastases in a syngenic mouse model in vivo. We used a novel marker system, EGFP transfected C26 tumor cells for in vivo observation of metastasis formation by intravital microscopy. We analyzed the effect of intraperitoneal TNFα‐injection on tumor cell adhesion, extravasation and tumor development. The expression of ICAM‐1, VCAM‐1 and E‐Selectin was measured by Western blot and immunohistochemical staining. Tumor load was assessed by determining EGFP in Western blots. GdCl3 was employed 24 and 48 hr before tumor cell injection to selectively deplete the liver of functioning Kupffer cells. We observed significantly more extravasated tumor cells in the TNFα‐pre‐treated animals at early time points with increased expression of adhesion molecules. Measurement of the EGFP levels showed fewer liver metastases in the TNFα‐pretreated animals at day 8. After GdCl3 pretreatment even lower levels of EGFP, i.e., fewer metastases and also lower expression levels of ICAM‐1, VCAM‐1 and E‐Selectin could be observed. TNFα, acts in a bidirectional manner: whereas TNFα facilitates tumor cell adhesion and extravasation of C26 tumor cells by inducing the expression of adhesion molecules, at later time points, TNFα seems to hinder the formation of liver metastases.

Liver regeneration in FGF‐2‐deficient mice: VEGF acts as potential functional substitute for FGF‐2

Background/Aims: The angiogenic properties, its role in mesoderm differentiation and cell culture studies implicate an important role of fibroblast growth factor (FGF‐2) in liver regeneration. The aim of the study was to evaluate this role in a FGF‐2 knockout mouse model.

Altered Apoptotic Response and Different Liver Structure During Liver Regeneration in FGF-2-Deficient Mice

Background / Aims: To investigate postulated differences related to FGF-2 in liver morphology and expression of apoptosis-related factors after partial hepatectomy (PH). Methods: Homogenous FGF-2-deficient mice (C57BL/6J) with their FGF-2-(+/+) littermates (control) were used to examine the structure of regenerating livers after PH with light and electron microscopy. The regenerative response and BrDu incorporation were monitored. The expression of BclX-l, Bax, Fas, TNF-α, and Caspase-3 were measured by reverse transcription PCR (RT-PCR) and Northern blot analysis. Results: In the FGF-2-(-/-) group, hepatocytes and endothelial cells contain mitochondria with atypical cristae and fragmented endoplasmic reticulum structures compared to control. Sinusoids show irregular basal laminae. These changes are in accordance with a differential expression of apoptosis-related factors: FasL was expressed throughout the entire observation span (days 0 to 10 post-PH). Following PH, tumor necrosis factor alpha (TNFα)-mRNA levels were higher in FGF-2-(+/+) animals, while Fas as well as Bax and BclXl were overexpressed in FGF-2-(-/-) mice. Caspase-3-mRNA was similarly expressed in both groups, but Caspase-3 activity was elevated for 4 days in FGF-2-(-/-) mice. Conclusion: Despite morphologic differences, differences in the time schedule of DNA synthesis and differences in apoptotic response, the dynamics of liver regeneration in FGF-2-(-/-) mice were not impaired.

Analysis of the First 217 Appendectomies of the German NOTES Registry.

Objective: To analyze the feasibility and safety of Natural Orifice Transluminal Endoscopic Surgery (NOTES) appendectomy, and to analyze separately the transvaginal appendectomy (TVAE) and the transgastric appendectomy (TGAE) procedures. Background: Laparoscopic appendectomy has rare but relevant complications, namely incisional hernias and neuralgia at the trocar sites, which can potentially be avoided by the NOTES techniques. Methods: The first 217 data sets of the largest NOTES registry worldwide—the German NOTES registry—were analyzed with respect to demographic data, procedural data, and short-term outcomes. Furthermore, TVAEs were compared with TGAEs. Results: Almost all procedures were performed in hybrid technique (median of percutaneous trocars: 1). Median age (TVAE: 30.5 yrs vs TGAE: 25 yrs; P < 0.017), body mass index (TVAE: 22.8 kg/m2 vs TGAE: 24.1 kg/m2; P < 0.016), and American Society of Anesthesiologists (ASA) classification (I/II/III; TVAE: 57.1%/41.8%/1.0% vs TGAE: 27.8%/69.4%/2.8%; P < 0.003) significantly differed between both access techniques. Whereas the median number of percutaneous trocars (TVAE: 1 vs TGAE: 1; P < 0.450), the need of additional trocars (TVAE: 6.6% vs TGAE: 13.9%; P < 0.156), the intra, and also postoperative rate of complications (TVAE: 0%/5.5% vs TGAE: 0%/11.1%; P < 1.000/0.258), and the median postoperative hospital stay (TVAE: 3 d vs TGAE: 3 d; P < 0.152) were comparable; the median procedural time (TVAE: 35 minutes vs TGAE: 96 minutes; P < 0.001) and conversion to laparotomy rate (TVAE: 0% vs TGAE: 5.6%; P < 0.023) were significantly less after TVAE. Conclusions: The evaluation of the largest patient collective so far indicates that hybrid NOTES appendectomy is a safe procedure, with advantages for the transvaginal technique with respect to procedural time and conversion rate.

Effect of different liver resection methods on liver damage and regeneration factors VEGF and FGF-2 in mice

BACKGROUND Different approaches to study liver regeneration in murine models have been proposed. We investigated the effect of different liver resection models on liver damage and regeneration parameters in mice. METHODS We compared the technical aspect of the 2 most commonly used techniques of 50% and 70% liver resection. Liver damage, as determined by the change in serum alanine aminotransferase and aspartate aminotransferase, as well as the regeneration parameters VEGF and FGF-2 were analyzed at 6 time points. A postoperative vitality score was introduced. RESULTS Cholestasis was not observed for either technique. Both resection techniques resulted in full weight recovery of the liver after 240 hours, with no significant difference between sham and resection groups. Postoperative animal morbidity and total protein levels did not differ significantly for either method, indicating early and full functional recovery. However, comparing the mitogenic growth factors FGF-2 and VEGF, a significant increase in serum levels and, therefore, increased growth stimulus, was shown in the extended resection group. CONCLUSION Extended resection led to a greater response in growth factor expression. This finding is important since it shows that growth factor response differs acdording to the extent of resection. We have demonstrated the need to standardize murine hepatic resection models to adequately compare the resulting liver damage.

FRET-FLIM-Mikroskopie der Apoptoseresistenz durch EGFR-Aktivität: Funktionelle Aspekte der Fluoreszenzmikroskopie

Background: Up-regulation and overexpression of the epidermal growth factor (EGFR) has been correlated to many processes related to cancer, including uncontrolled cellular proliferation and autocrine stimulation of tumors producing their own growth factors. Many epithelial tumors express high EGFR densities, which are associated with advanced disease and poor clinical prognosis. We here questioned whether EGFR activity conveys resistance to apoptosis. Methods: In contrast to former assays, which were restricted to the quantitation of protein or RNA expression, we established a new approach to detect the functional activity of EGFR using fluorescence resonance energy transfer (FRET). Labelling of the F4-antibody (specific antibody against an intracellular epitope of EGFR) with Cy 3 and of the phosphotyrosin-antibody Py72 with Cy 5 allows measure FRET which is present in the activated EGFR. We determined EGFR activity in colorectal cell lines. Additionally we measured the amount of EGFR-expression. Apoptosis commitment in colorectal cell lines was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1 enabling to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy/FLIM) in transfected cells. Results: We show that the phosphorylation state of the endogenous EGFR can be quantitatively imaged in tumor cells and tissues by detecting fluorescence resonance energy transfer between fluorophores conjugated to antibodies. Five different human colorectal cell lines were analyzed for activity and expression of EGFR. All cell lines exhibited basal EGFR phosphorylation under serum starvation conditions. Phosphorylation levels increased after stimulation dependent on the level of basal EGFR phosphorylation. The basal activity correlated inversely with receptor expression. Apoptosis commitment was microscopically imaged by fluorescence lifetime imaging microscopy. This approach provides parameters reporting quantitatively the ratio between cleaved versus uncleaved sensor thereby facilitating the comparison of apoptosis commitment between different cells. We show that EGFR kinase inhibitors sensitize colorectal SW-480 tumor cells for 5-fluorouracil induced apoptosis indicating that EGFR-mediated survival signaling contributes to apoptosis resistance via its intrinsic kinase activity. Conclusion: Although fluorescent biosensors are now widely used in cell biology, few studies have attempted to use them in the context of drug screening procedures. We have therefore developed FRET-FLIM assays for quantitative analysis of cell biological processes. Using these optical approaches, we show that colorectal tumor cells display basal EGFR phosphorylation in the absence of exogenous growth factors. This basal EGFR signaling contributes to cell survival upon chemotherapy-induced DNA damage.

Leberregeneration in der FGF-2 defizienten Maus: funktionelle und morphologische Aspekte

Aims We have previously shown that liver regeneration is not impaired in FGF-2 mice. Here we investigate a potential functional substitution of FGF-2 by other growth factors and characterize apoptosis and morphological changes in the liver of FGF-2 mice.