ABCB5+ mesenchymal stromal cells therapy protects from hypoxia by restoring Ca2+ homeostasis in vitro and in vivo.

BACKGROUND: Hypoxia in ischemic disease impairs Ca2+ homeostasis and may promote angiogenesis. The therapeutic efficacy of mesenchymal stromal cells (MSCs) in peripheral arterial occlusive disease is well established, yet its influence on cellular Ca2+ homeostasis remains to be elucidated. We addressed the influence of ATP-binding cassette subfamily B member 5 positive mesenchymal stromal cells (ABCB5+ MSCs) on Ca2+ homeostasis in hypoxic human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. METHODS: Hypoxia was induced in HUVECs by Cobalt (II) chloride (CoCl2) or Deferoxamine (DFO). Dynamic changes in the cytosolic- and endoplasmic reticulum (ER) Ca2+ and changes in reactive oxygen species were assessed by appropriate fluorescence-based sensors. Metabolic activity, cell migration, and tube formation were assessed by standard assays. Acute-on-chronic ischemia in Apolipoprotein E knock-out (ApoE-/-) mice was performed by double ligation of the right femoral artery (DFLA). ABCB5+ MSC cells were injected into the ischemic limb. Functional recovery after DFLA and histology of gastrocnemius and aorta were assessed. RESULTS: Hypoxia-induced impairment of cytosolic and ER Ca2+ were restored by ABCB5+ MSCs or their conditioned medium. Similar was found for changes in intracellular ROS production, metabolic activity, migratory ability and tube formation. The restoration was paralleled by an increased expression of the Ca2+ transporter Sarco-/endoplasmic reticulum ATPase 2a (SERCA2a) and the phosphorylation of Phospholamban (PLN). In acute-on-chronic ischemia, ABCB5+ MSCs treated mice showed a higher microvascular density, increased SERCA2a expression and PLN phosphorylation relative to untreated controls. CONCLUSIONS: ABCB5+ MSCs therapy can restore cellular Ca2+ homeostasis, which may beneficially affect the angiogenic function of endothelial cells under hypoxia in vitro and in vivo.

KRAS/GNAS-testing by highly sensitive deep targeted next generation sequencing improves the endoscopic ultrasound-guided workup of suspected mucinous neoplasms of the pancreas.

Pancreatic cysts or dilated pancreatic ducts are often found by cross-sectional imaging, but only mucinous lesions can become malignant. Therefore, distinction between mucinous and non-mucinous lesions is crucial for adequate patient management. We performed a prospective study including targeted next generation sequencing (NGS) of cell-free DNA in the diagnostic endoscopic ultrasound (EUS)-guided workup. Pancreatic cyst(s) or main duct fluid obtained by EUS-guided FNA was analysed by carcinoembryonic antigen (CEA), cytology and deep targeted NGS of 14 known gastrointestinal cancer genes (AKT1, BRAF, CTNNB1, EGFR, ERBB2, FBXW7, GNAS, KRAS, MAP2K1, NRAS, PIK3CA, SMAD4, TP53, APC) with a limit of detection down to variant allele frequency of 0.01%. Results were correlated to histopathology and clinical follow-up. One hundred and thirteen patients with pancreatic cyst(s) and/or a dilated pancreatic main duct (≥5 mm) were screened. Sixty-six patients had to be excluded, mainly due to inoperability or small cyst size (≤10 mm). Forty-seven patients were enrolled for further analysis. A final diagnosis was available in 27 cases including 8 negative controls. In 43/47 (91.5%) of patients a KRAS- and/or GNAS-mutation was diagnosed by NGS. 27.0% of the KRAS-mutated and 10.0% of the GNAS-mutated lesions harbored multiple mutations. KRAS/GNAS-testing by NGS, cytology, and CEA had a sensitivity and specificity of 94.7/100%, 38.1/100%, and 42.1/75.0%, respectively. KRAS/GNAS-testing was significantly superior to CEA (P = .0209) and cytology (P = .0016). In conclusion, KRAS/GNAS-testing by deep targeted NGS is a suitable method to distinguish mucinous from non-mucinous pancreatic lesions, suggesting its usage as a single diagnostic test. Results must be confirmed in a larger cohort.

[Minimally invasive and robot-assisted surgery for chronic inflammatory bowel disease : Current status and evidence situation]. / Minimal-invasive und roboterassistierte Chirurgie bei chronisch entzündlicher Darmerkrankung : Aktueller Stand und Evidenzlage.

The majority of cases of inflammatory bowel disease (IBD) can be treated by minimally invasive surgery. The advantages of standard laparoscopic approaches compared to open surgery with respect to short-term and long-term outcome parameters have been adequately proven with evidence level 1 for ileocecal resection in Crohn's disease. For many other indications there are at least several larger registry or case-control studies that have shown advantages for laparoscopy. For robot-assisted surgery the feasibility has principally been demonstrated for IBD, whereby at least for ulcerative colitis limited data suggest comparable results to the standard laparoscopic approach. Single port approaches have so far not been able to demonstrate any relevant advantages in IBD surgery. Major advantages for transanal minimally invasive surgery (TAMIS) for performing restorative proctocolectomy could not be demonstrated in two larger case-controlled studies but it was at least shown to be a comparable alternative to standard laparoscopy. Overall, it seems unlikely that the recently described new laparoscopic approaches will result in measurable advantages for the patient in comparison to standard laparoscopy as the access trauma is not significantly changed. In general, the indications to perform minimally invasive surgery must always be based on the basic principles of IBD surgery and contraindications have to be considered in individual cases as the advantages of laparoscopic approaches are levelled out in the long run.

Gallbladder Cancer Presenting as Mirizzi Syndrome Complicated by Rapidly Evolving 23 rRNA Gene-Linezolid Resistance with Vancomycin-Resistant Enterococcus Infection Resulting in Fatal Cholangial Sepsis.

We describe the case of a 71-year-old woman who presented with obstructive jaundice and subhilar bile duct stenosis. MRI showed extensive cholecystolithiasis with an impacted bile stone in the cystic duct suggesting Mirizzi syndrome. Delayed enhancement of the thickened gallbladder wall suggested inflammation instead of carcinoma. After drainage of the obstructed bile duct via ERCP, the patient developed liver abscesses with a nosocomial vancomycin-resistant enterococcus infection treated by linezolid. After 4 weeks, the VRE infection was complicated by a new-onset 23 rRNA gene-mediated linezolid resistance in the same bacterial strain, which was proven via core genome multilocus sequencing. Meropenem and tigecycline were administered according to a resistogram. Furthermore, percutaneous transhepatic biliary drainage of both sides of the liver was necessary. After demission, the patient had to be admitted again due to septic shock. An emergency operation revealed extended, inoperable gallbladder cancer. The patient died a few days later in the intensive care unit. An earlier diagnosis of bile duct infiltrating gallbladder cancer by cholangioscopy or laparoscopy and treatment of vancomycin-resistant enterococcus infection with daptomycin may have changed the clinical course of the disease.

Hybrid transgastric appendectomy is feasible but does not offer advantages compared with laparoscopic appendectomy: Results from the transgastric appendectomy study.

BACKGROUND: Very few transgastric procedures, the original objective of natural orifice translumenal surgery, have been reported in the international Natural Orifice Translumenal Surgery registers. In addition, most cases were controlled mainly by laparoscopy. To show the feasibility of hybrid transgastric appendectomy and to compare results with laparoscopic a prospective, nonrandomized study was conducted. METHODS: From October 2010 to May 2013, patients with acute appendicitis were screened. If the patients met the inclusion criteria, transgastric appendectomy was offered. If the patient decided on laparoscopy, the consenting patients took part in the observational part of the study. The transgastric appendectomy procedure was controlled completely by the gastroscope, although a 3 mm grasper was used to tauten the appendix. Demographic and clinical parameters were collected. Quality of life and pain were measured with a Short Form-8 questionnaire and the Visual Analogue Scale. RESULTS: Of the 273 patients who underwent an appendectomy, 65 agreed to take part in this study. Out of these, 30 (46.15%) underwent transgastric appendectomy and 35 (53.85%) underwent laparoscopy. No intraoperative complications were recorded. The operation duration rate was greater in the transgastric appendectomy group (94.5 minutes vs 69 minutes; P < .001). Conversions to open appendectomy and complications were the same. There were no differences in pain preoperatively or postoperatively. In both the transgastric appendectomy and laparoscopic groups, the quality of life of all the subscales increased markedly after successful resection of the pathology. CONCLUSION: These preliminary results demonstrate the feasibility of transgastric appendectomy. The postoperative course and quality of life are comparable with laparoscopic appendectomy, but no improvement due to transgastric appendectomy could be demonstrated.

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/m vs TGAE: 24.1 kg/m; 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.

Natural orifice transluminal endoscopic surgery in humans: feasibility and safety of transgastric closure using the OTSC system.

BACKGROUND: NOTES is a technique in which an operation is performed within the body without a skin incision by using a natural body orifice to provide access. The principal challenge of transgastric NOTES procedures is still the feasibility and safety of access closure. Currently, there are very limited data regarding the closure of transgastric NOTES in humans, and the standard method and device for closure has not been defined. Herein, we evaluate the feasibility and safety of gastric closure after NOTES procedures in humans with the over-the-scope clip (OTSC). METHODS: Review of collected data of patients underwent transgastric NOTES in prospective clinical studies between April 2010 and March 2014 focused on the gastric closure with the OTSC. RESULTS: A total of 43 patients underwent transgastric NOTES: 36 patients with an acute appendicitis, six patients with a prophylactic bilateral salpingo-oophorectomy, and one patient with uterus myomatosus. In all 43 cases, the incision of the gastric wall and the endoscopic access to the abdominal cavity succeeded without any difficulty. After performing transgastric procedures, it was possible to close the access by OTSC in all cases. There were all in all three adverse events: one major (Clavien-Dindo Grade III) and two minor (Clavien-Dindo Grades I and II). CONCLUSION: Even if we could show for the first time in more than 40 consecutive patients that there is a safe approach for closing the transgastric access, it is absolutely necessary that further investigation in clinical settings has to be done to establish clear indications and guidelines for the use of transgastric NOTES.

Endoscopic application of polysaccharide powder for hemostasis in anticoagulated pigs (with video).

BACKGROUND: Acute GI bleeding remains a clinical problem of daily importance. Increasing numbers of patients with compromised coagulation challenge the established methods of endoscopic hemostasis. Therefore, new powders for the treatment of GI bleeding have been developed. OBJECTIVE: To clarify the efficacy of a newly available anticoagulant powder in stopping hemorrhage. DESIGN: A randomized prospective comparative study regarding the potential of an endoscopically applicable polysaccharide powder in pigs receiving antithrombotic medication. SETTING: A professional veterinary animal laboratory. PATIENTS: Twenty-two pigs were anticoagulated with heparin (n = 8), aspirin (n = 8), or no antithrombotic medication (n = 6), in a randomized order. INTERVENTIONS: A bleeding ulcer with continuous bleeding (Forrest I b) was established in the stomach. Endoscopic hemostasis was performed using the powder. MAIN OUTCOME MEASUREMENTS: Time to hemostasis and the amount of powder used were recorded. Follow-up occurred for 3 days, final hemoglobin measurement, followed by autopsy with control for post-interventional bleeding. RESULTS: Endoscopic hemostasis was successful in all cases. Post-interventional bleeding was not recorded. LIMITATIONS: Animal study, artificial superficial bleeding source. CONCLUSION: Polysaccharide powder is able to stop active bleeding from Forrest I b lesions in pigs receiving antithrombotic therapy.

New methods of magnet-based instrumentation for NOTES.

INTRODUCTION: Laparoscopic surgery has displaced open surgery as the standard of care for many clinical conditions. NOTES has been described as the next surgical frontier with the objective of incision-free abdominal surgery. The principal challenge of NOTES procedures is the loss of triangulation and instrument rigidity, which is one of the fundamental concepts of laparoscopic surgery. To overcome these problems necessitates the development of new instrumentation. material and methods: We aimed to assess the use of a very simple combination of internal and external magnets that might allow the vigorous multiaxial traction/counter-traction required in NOTES procedures. The magnet retraction system consisted of an external magnetic assembly and either small internal magnets attached by endoscopic clips to the designated tissue (magnet-clip-approach) or an endoscopic grasping forceps in a magnetic deflector roll (magnet-trocar-approach). RESULTS: We compared both methods regarding precision, time and efficacy by performing transgastric partial uterus resections with better results for the magnet-trocar-approach. DISCUSSION: This proof-of-principle animal study showed that the combination of external and internal magnets generates sufficient coupling forces at clinically relevant abdominal wall thicknesses, making them suitable for use and evaluation in NOTES procedures, and provides the vigorous multiaxial traction/counter-traction required by the lack of additional abdominal trocars.

Differences in the endoscopic closure of colonic perforation due to diagnostic or therapeutic colonoscopy.

OBJECTIVE: To describe the experience of a single center regarding the feasibility of endoscopic closure of iatrogenic colonic perforations and to elucidate differences between the efficacy of endoscopic clip closure due to diagnostic or therapeutic colonoscopy. MATERIAL AND METHODS: A retrospective institutional computer-based search of records of colonoscopic perforation occurring between January 2004 and December 2011 was undertaken. Data on patients undergoing colonoscopy were entered into a clinical database. To further improve the detection of all cases of colon perforations, the authors also searched their separate surgical database for every patient with a colon perforation treated or operated on in the surgery department. Statistical significance was tested using either Fortran-Subroutine Fytest, chi-square testing or t-test. RESULTS: Over 8 years, 22,924 patients underwent colonoscopy, of which 105 consecutive patients suffered iatrogenic perforation. Clip application was possible in 62 patients (81.58%) after perforation due to a therapeutic colonoscopy, whereas clip application was only possible in 9 patients (31.03%) after perforation due to a diagnostic colonoscopy. 4 out of 9 patients (44.44%) in the diagnostic group compared with 7 out of 62 patients (11.29%) after clipping a perforation during a therapeutic colonoscopy were sent to surgery. CONCLUSIONS: The authors' data indicate significant differences in the potential for and success of endoscopic closure of iatrogenic perforations occurring during diagnostic or therapeutic colonoscopy. The frequency of surgery was significantly greater after clipping a perforation during a diagnostic colonoscopy.

The EURO-NOTES clinical registry for natural orifice transluminal endoscopic surgery: a 2-year activity report.

BACKGROUND: The EURO-NOTES Clinical Registry (ECR) was established as a European database to allow the monitoring and safe introduction of Natural Orifice Transluminal Endoscopic Surgery (NOTES). The aim of this study was to analyze different techniques applied and relative results during the first 2 years of the ECR. METHODS: The ECR was designed as a voluntary database with online access. All members of the European Society for Gastrointestinal Endoscopy and the European Association for Endoscopic Surgery were requested to participate in the registry. Demographic and therapy data as well as data on the postoperative course are recorded in the ECR in an anonymous way. RESULTS: A total of 533 patients who underwent NOTES procedures were included in the study. Four different hybrid techniques for 435 cholecystectomies were described, registering postoperative complications in 2.8% of patients, addition of a single trocar in 5.3%, and conversions to laparoscopy in 0.5%. Both flexible endoscopic and rigid laparoscopic cholecystectomy techniques proved to be safe and effective with minor differences. There was a shorter operative time in the rigid laparoscopic group. Thirty-three appendectomies were reported by transgastric and transvaginal techniques, with transvaginal techniques scoring shorter operative time and hospital stay, but with a frequent need to add more trocars. Overall complications occurred in 14.7% of patients but they did not differ significantly among the different techniques. One transvaginal and 31 transanal sigmoidectomies were included for prolapse and diverticulitis, with four postoperative complications (12.5%), but none needing further treatment. Twenty peroral esophageal myotomies were included with three postoperative complications (15.0%), but none needing further treatment. CONCLUSIONS: Five years since the introduction of NOTES into clinical practice, hybrid techniques have gained considerable clinical application. Several NOTES hybrid cholecystectomy and appendectomy techniques are practicable and safe alternatives to laparoscopic procedures. Also, sigmoidectomies and peroral esophageal myotomies were described, proving feasibility and safety. Nevertheless, the real benefit of NOTES for patients still needs to be assessed.

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.

First experiences with A circular stapled gastro-jejunostomy by a new transorally introducible stapler system in laparoscopic Roux-en-Y gastric bypass.

BACKGROUND: The only effective treatment for patients with morbid obesity is surgery. Laparoscopic bariatric surgery has become quite popular in attempts to decrease the morbidity associated with laparotomy. The aim of this study was to assess the safety and efficiency by using SurgASSIST(R) for performing the gastrojejunostomy in laparoscopic Roux-en-Y gastric bypass (RYGBP). The variables were compared with the results using the standard laparoscopic circular end-to-end anastomotic stapler (CEEA(R)). METHODS: After randomization, the gastro-jejuostomy of RYGBP was performed in ten patients by transabdominal introduced circular stapler (group A) and in ten patients by transorally introduced circular stapler (SurgASSIST(R); group B) via five-port laparoscopy. A prospective 12-month postoperative follow-up including documentation of minor and major complication as well as weight loss and body composition is done every 8 weeks. RESULTS: The average body mass index (BMI, 52 kg/m(2)) and the other baseline characteristics were equally distributed in both groups. There was no difference in reduction of BMI, excess weight loss, and fat mass in both groups. The rate of port site wound infection in group A was significantly higher (p = 0.03) when compared to group B. There was no anastomotic leak or stricture postoperatively in both groups. CONCLUSIONS: Performing of a gastrojejunostomy in RYGBP by SurgASSIST is a safe and feasible method in comparison to conventional circular stapler systems. The advantage of SurgASSIST is the avoidance to introduce the stapler through the abdominal wall and, by this, a possible port site wound infection. Further prospective studies have to be performed to verify the advantages of the SurgASSIST in comparison to conventional circular stapler systems.

Association between EGF, TGF-beta1, VEGF gene polymorphism and colorectal cancer.

INTRODUCTION: Up to the present, EGF 61 A/G, TGF-beta1 -509 T/C, and VEGF 936 T/C gene polymorphisms have been analyzed in other cancer entities than colorectal cancer. We have now investigated the frequency of these gene polymorphisms among colorectal cancer patients. MATERIAL AND METHODS: A total of 157 colorectal cancer patients and 117 cancer-free healthy people were recruited at the Surgical Department of the Universitätsklinikum Mannheim. All patients and healthy people are Caucasians. Genomic DNA was isolated from peripheral blood, and gene polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). RESULTS: The distribution of EGF 61 G/G homozygotes among colorectal cancer patients was more frequent than that in the control group (33.1% versus 11.1%; Odds Ratio [OR]=3.962; 95% Confidence Interval [CI]=2.036-7.708). The frequency of the "G" allele in the colorectal cancer patient group was also higher than that in the control group (51.3% versus 33.3%; OR=2.105; 95% CI=1.482-2.988). No difference could be found for the TGF-beta1 and VEGF genotypes among colorectal cancer patients and healthy controls. CONCLUSIONS: The EGF 61 G/G genotype and the G allele are significantly related to colorectal cancer. The TGF-beta1 -509 T/C and VEGF 936 T/C gene polymorphisms are not related to colorectal cancer.

Endoclipping of iatrogenic colonic perforation to avoid surgery.

BACKGROUND: Colonoscopy 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. METHODS: In 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. RESULTS: In 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. CONCLUSIONS: Endoluminal 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.

Quantitative imaging of apoptosis commitment in colorectal tumor cells.

We have studied caspase-3 activation by combined DNA damage induction and EGFR kinase inhibition in order to identify potential EGFR-mediated survival signals conferring resistance to apoptosis in human colorectal tumor cells. The onset of apoptosis was microscopically imaged with a newly developed caspase-3 substrate sensor based on EGFP and tHcred1, enabling us to monitor caspase-3 activation in cells by fluorescence lifetime imaging microscopy or fluorescence correlation spectroscopy. Both optical approaches provide parameters quantitatively reporting the ratio between cleaved and uncleaved sensor, thereby facilitating the comparison of caspase-3 activation between different cells. Using these methods, 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.

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.

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-alpha, 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 (TNFalpha)-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.

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. METHODS: Liver regeneration after left hemihepatectomy (partial hepatectomy, PH) was evaluated in homozygous FGF-2 deficient (-/-) mice (male C57BL/6J) and their FGF-2 competent (+/+) littermates (controls) (day 0-10). RESULTS: FGF-2-(-/-) mice displayed normal dynamics in liver regeneration. FGF-2 protein was overexpressed 4 days post PH in controls. BrdU incorporation showed a biphasic pattern in FGF-2-(-/-) mice, whereas it decreased continuously after one peak (day 2) in controls. In FGF-2-(-/-) livers hepatic growth factor mRNA post PH was 1 day longer decreased and markedly less elevated thereafter compared with control. Vascular endothelial growth factor (VEGF) mRNA levels were clearly increased in FGF-2-(-/-) mice pre- and postoperatively in contrast to controls. VEGF protein levels in livers of FGF-2-(-/-) mice were elevated preoperatively, but similar in both groups after PH. With SU5416, a VEGF-receptor inhibitor, liver regeneration in FGF-2-(-/-) mice was reduced significantly, whereas it remained unchanged in controls. CONCLUSIONS: Liver regeneration dynamics in FGF-2-(-/-) mice were comparable with controls, potentially due to a functional substitution of FGF-2 by VEGF.

Gas gangrene pyaemia with myocardial abscess formation--fatal outcome from a rare infection nowadays.

We report a case of sudden death after gas gangrene. A 67-year-old male patient with diabetes mellitus and chronic renal failure (on haemodialysis three times a week) presented in the surgical emergency department with a severe swelling and crepitation in the right groin. No signs of trauma were present-except for a well-healed, 1-year-old scar after femoro-popliteal bypass surgery. Two days earlier, he had presented to the internal medicine department with epigastric pain and had left against medical advice. On readmission the patient was initially conscious and in a stable cardiopulmonary condition but developed sudden cardiocirculatory failure and underwent resuscitation. Despite all resuscitation measures, including the administration of high doses of catecholamines and the treatment of hyperkalemia, the patient died. Autopsy revealed septicaemia with rod-shaped gram-positive bacteria, typical of Clostridium perfringens, evidenced by multiple areas of myonecrosis. Abscess formation was found in the myocardium. Clostridial gas gangrene is a rare clinical condition. Unless immediate diagnosis and adequate therapy measures are taken, the outcome and chances for survival are poor as demonstrated by this case.