Claus-Dieter Heidecke

Metastatic behaviour of primary human tumours in a zebrafish xenotransplantation model.

BackgroundAberrant regulation of cell migration drives progression of many diseases, including cancer cell invasion and metastasis formation. Analysis of tumour invasion and metastasis in living organisms to date is cumbersome and involves difficult and time consuming investigative techniques. For primary human tumours we establish here a simple, fast, sensitive and cost-effective in vivo model to analyse tumour invasion and metastatic behaviour.MethodsWe fluorescently labelled small explants from gastrointestinal human tumours and investigated their metastatic behaviour after transplantation into zebrafish embryos and larvae. The transparency of the zebrafish embryos allows to follow invasion, migration and micrometastasis formation in real-time. High resolution imaging was achieved through laser scanning confocal microscopy of live zebrafish.ResultsIn the transparent zebrafish embryos invasion, circulation of tumour cells in blood vessels, migration and micrometastasis formation can be followed in real-time. Xenografts of primary human tumours showed invasiveness and micrometastasis formation within 24 hours after transplantation, which was absent when non-tumour tissue was implanted. Furthermore, primary human tumour cells, when organotopically implanted in the zebrafish liver, demonstrated invasiveness and metastatic behaviour, whereas primary control cells remained in the liver. Pancreatic tumour cells showed no metastatic behaviour when injected into cloche mutant embryos, which lack a functional vasculature.ConclusionOur results show that the zebrafish is a useful in vivo animal model for rapid analysis of invasion and metastatic behaviour of primary human tumour specimen.

LC–MS/MS-based quantification of clinically relevant intestinal uptake and efflux transporter proteins

Multidrug transporter proteins are crucial determinants in the pharmacokinetics of many drugs. To evaluate their impact on intestinal drug absorption, we developed and validated quantification methods for 10 uptake transporters (OATP1A2, OATP2B1, PEPT1, ASBT, OCT1, OCT3) and efflux transporters (ABCB1, ABCC2, ABCC3, ABCG2) that have been reported to be expressed and to be of clinical relevance in the human intestine. Quantification was performed by targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based quantification of proteospecific peptides after tryptic digestion using stable isotope labeled internal standard peptides. The chromatography of the respective peptides was performed by gradient elution using a reversed phase (C18) column (Kinetex(®), 100 × 3.0 mm, 2.6 μm) and 0.1% formic acid (FA) and acetonitrile with 0.1% FA as mobile phases at a flow rate of 0.5 ml/min. The MS/MS detection was done in the positive multiple reaction monitoring (MRM) mode by monitoring in each case three mass transitions for the transporter-derived peptides and the internal standard peptides. The assays were validated with respect to specificity, linearity (0.1-25 nM), within-day and between-day accuracy and precision as well as stability according to current bioanalytical guidelines. Finally, the developed methods were used to determine the transporter protein content in human intestinal tissue (jejunum and ileum). The methods were shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to measure transporter proteins in the human intestine.

Concentration of bacteria passing through puncture holes in surgical gloves

BACKGROUND The reasons for gloving-up for surgery are to protect the surgical field from microorganisms on the surgeons hands and protect the surgeon from the patients microorganisms. This study measured the concentration of bacteria passing through glove punctures under surgical conditions. METHODS Double-layered surgical gloves were worn during visceral surgeries over a 4-month period. The study included 128 outer gloves and 122 inner gloves from 20 septic laparotomies. To measure bacterial passage though punctures, intraoperative swabs were made, yielding microorganisms that were compared with microorganisms retrieved from the inner glove layer using a modified Gaschen bag method. RESULTS Depending on the duration of glove wear, the microperforation rate of the outer layer averaged 15%. Approximately 82% of the perforations went unnoticed by the surgical team. Some 86% of perforations occurred in the nondominant hand, with the index finger being the most frequently punctured location (36%). Bacterial passage from the surgical site through punctures was detected in 4.7% of the investigated gloves. CONCLUSION Depending on the duration of wear, surgical gloves develop microperforations not immediately recognized by staff. During surgery, such perforations allow passage of bacteria from the surgical site through the punctures. Possible strategies for preventing passage of bacteria include strengthening of glove areas prone to punctures and strict glove changing every 90 minutes.

Pancreatic pseudocysts – when and how to treat?

Pancreatic pseudocysts are a well-known complication of acute or chronic pancreatitis, with a higher incidence in the latter. Currently several classification systems are in use that are based on the origin of the pseudocyst, their relation to pancreatic duct anatomy and a possible pseudocyst-duct communication. Diagnosis is accomplished most often by CT scanning, by endoscopic retrograde cholangiopancreaticography (ERCP) or by ultrasound, and rapid progress in the improvement of diagnostic tools has enabled detection with high sensitivity and specificity. There are different therapeutic strategies: endoscopic transpapillary or transmural drainage, percutaneous catheter drainage, or open surgery. The feasibility of endoscopic drainage is highly dependent on the anatomy and topography of the pseudocyst, but provides high success and low complication rates. Percutaneous drainage is used for infected pseudocysts. However, its usefulness in chronic pancreatitis-associated pseudocysts is questionable. Internal drainage and pseudocyst resection are frequently used as surgical approaches with a good overall outcome, but a somewhat higher morbidity and mortality compared with endoscopic intervention. We therefore conclude that pseudocyst treatment in chronic pancreatitis can be effectively achieved by both endoscopic and surgical means.

Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics

Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes.

Metabolic biomarker signature to differentiate pancreatic ductal adenocarcinoma from chronic pancreatitis

Objective Current non-invasive diagnostic tests can distinguish between pancreatic cancer (pancreatic ductal adenocarcinoma (PDAC)) and chronic pancreatitis (CP) in only about two thirds of patients. We have searched for blood-derived metabolite biomarkers for this diagnostic purpose. Design For a case–control study in three tertiary referral centres, 914 subjects were prospectively recruited with PDAC (n=271), CP (n=282), liver cirrhosis (n=100) or healthy as well as non-pancreatic disease controls (n=261) in three consecutive studies. Metabolomic profiles of plasma and serum samples were generated from 477 metabolites identified by gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry. Results A biomarker signature (nine metabolites and additionally CA19-9) was identified for the differential diagnosis between PDAC and CP. The biomarker signature distinguished PDAC from CP in the training set with an area under the curve (AUC) of 0.96 (95% CI 0.93–0.98). The biomarker signature cut-off of 0.384 at 85% fixed specificity showed a sensitivity of 94.9% (95% CI 87.0%–97.0%). In the test set, an AUC of 0.94 (95% CI 0.91–0.97) and, using the same cut-off, a sensitivity of 89.9% (95% CI 81.0%–95.5%) and a specificity of 91.3% (95% CI 82.8%–96.4%) were achieved, successfully validating the biomarker signature. Conclusions In patients with CP with an increased risk for pancreatic cancer (cumulative incidence 1.95%), the performance of this biomarker signature results in a negative predictive value of 99.9% (95% CI 99.7%–99.9%) (training set) and 99.8% (95% CI 99.6%–99.9%) (test set). In one third of our patients, the clinical use of this biomarker signature would have improved diagnosis and treatment stratification in comparison to CA19-9.

Selective depletion of alveolar macrophages in polymicrobial sepsis increases lung injury, bacterial load and mortality but does not affect cytokine release.

Background: Resident tissue macrophages exert important functions during severe systemic infection and contribute to changes in local as well as systemic immune responses. Alveolar macrophages (AM) play a crucial role in airway diseases and in the defense against microorganisms invading the body via the bronchopulmonary tract. It has been postulated that AM are involved in the development of acute local disorders as a consequence of extrapulmonary stimuli like pancreatitis, peritonitis, or trauma. Objective: The aim of this study was to analyze the local and systemic role of AM during sepsis using selective AM depletion in the murine colon ascendens stent peritonitis (CASP) model of polymicrobial sepsis. Methods: 48 h prior to CASP surgery, AM of female C57BL/6 mice were selectively depleted by intratracheal application of clodronate liposomes (Lipo-clod). For control purposes, phosphate-buffered saline (PBS) liposomes (Lipo-PBS) were used. Results: CASP led to significantly elevated levels of local and systemic cytokines independent of the presence of AM. In contrast, levels of gut-derived bacteria in bronchoalveolar lavage and lung of septic mice were significantly higher in Lipo-clod-treated animals compared to Lipo-PBS-treated animals. After CASP-induced sepsis, local barrier dysfunction in the lung was detected; AM depletion resulted in severely enhanced development of acute lung injury. Consequently, Lipo-clod-treated animals showed strongly reduced survival rates after CASP. Conclusions: Contrarily to other macrophage populations, AM do not significantly contribute to local and systemic cytokine release during polymicrobial abdominal sepsis. AM have important protective functions for local clearance of gut-derived bacteria and attenuation of lung injury.

The Organic Anion–Transporting Peptide 2B1 Is Localized in the Basolateral Membrane of the Human Jejunum and Caco-2 Monolayers

The organic anion-transporting polypeptide (OATP) 2B1 which is ubiquitously expressed in the human body is assumed to play an important role in the cellular uptake of many drugs. Although the expression and function of this solute carrier transporter is well characterized in the human liver and other tissues, little is known about its localization and functional relevance in the intestine. Thus, it was the aim of this study to investigate its localization and function in the human jejunum and in the frequently used intestinal Caco-2 cell line. The basolateral membrane of jejunal tissue from 6 individuals showed a significant enrichment of OATP2B1 (17-fold) and the known basolateral proteins ABCC3 and Na/K-ATPase compared to the apical membrane as derived from targeted proteomics analysis. On the contrary, apical localization could be confirmed for ABCB1, ABCC2, and PEPT1. Basolateral localization of OATP2B1 could also be verified in Caco-2 cells. Bidirectional transport studies with established OATP2B1 substrates (sulfasalazine and pravastatin) across freshly exercised human jejunum and Caco-2 cell monolayers demonstrated a markedly higher transport from the basal to the apical compartment than in the opposite direction. Our data provide evidence for a basolateral localization of OATP2B1 which may improve our understanding of intestinal drug absorption.

The Ussing Chamber Assay to Study Drug Metabolism and Transport in the Human Intestine

The Ussing chamber is an old but still powerful technique originally designed to study the vectorial transport of ions through frog skin. This technique is also used to investigate the transport of chemical agents through the intestinal barrier as well as drug metabolism in enterocytes, both of which are key determinants for the bioavailability of orally administered drugs. More contemporary model systems, such as Caco‐2 cell monolayers or stably transfected cells, are more limited in their use compared to the Ussing chamber because of differences in expression rates of transporter proteins and/or metabolizing enzymes. While there are limitations to the Ussing chamber assay, the use of human intestinal tissue remains the best laboratory test for characterizing the transport and metabolism of compounds following oral administration. Detailed in this unit is a step‐by‐step protocol for preparing human intestinal tissue, for designing Ussing chamber experiments, and for analyzing and interpreting the findings.

Patientensicherheit in der Viszeralmedizin

Die Einfuhrung neuer diagnostischer und therapeutischer Verfahren hat die Medizin in den letzten Jahrzehnten zunehmend komplexer gemacht. Diese Komplexitat geht mit erhohten Anforderungen an eine interdisziplinare Patientenbetreuung einher und fugt immer mehr Schnittstellen und Ubergabepunkte in den Behandlungsablauf ein. Die Zeiten, in denen ein einzelner Arzt von den ersten Symptomen uber die Diagnostik und Therapie bis hin zur Entlassung und ambulanten Nachbetreuung alle Diagnoseund Behandlungsschritte in einer Hand hielt, sind lange vorbei. Kaum ein medizinischer Bereich ist so von Interdisziplinaritat und Schnittstellenproblematiken gekennzeichnet wie die Viszeralmedizin. Nicht nur die Diagnostik wird von der Bildgebung uber das Labor bis hin zu genetischen Untersuchungen immer komplexer und in mehr Hande gelegt, sondern auch die Grenzen zwischen chirurgischer und gastroenterologischer Behandlung verschwinden zunehmend und die Facher werden komplementar. Dies sind die Grunde, warum die Patientensicherheit, gerade an Schnittstellen der Behandlung, in den letzten Jahren immer mehr in den Vordergrund geruckt ist. Das hat uns bewogen, dieses Thema den Herausgebern sowie dem Karger Verlag ans Herz zu legen und diesem Schwerpunkt eine eigene Ausgabe der Zeitschrift VISZERALMEDIZIN zu widmen. Das Spektrum der Artikel zur Patientensicherheit reicht hierbei von spezifischen Hygienemasnahmen [1], die heute im OP und in der Endoskopie erforderlich sind und zunehmend in ambulanter Praxis und in der Klinik als Qualitatsmerkmale angesehen werden, bis zum Management von Teamressourcen im OP und in der Endoskopie [2]. Die zunehmende Notwendigkeit, nicht nur Komplikationen, sondern auch Critical Incidents, also Vorfalle, die zu Komplikationen fuhren konnten, in einer Weise zu dokumentieren, die Prozessablaufe nachhaltig verbessert, wird im Kapitel zum «Critical Incident Reporting»-System behandelt [3]. Eine Entwicklung, die die Medizin aus der Flugsicherheit ubernommen hat, sind Checklisten, deren nachhaltiger Erfolg inzwischen in vielen Bereichen des OP-Managements und der Notfallmedizin belegt werden konnte. Deshalb widmen sich eigene Kapitel diesem Thema im Zusammenhang mit dem Patientenmanagement in der Endoskopie [4], im Operationssaal [5] und auch bei der notfallmasigen Versorgung von Patienten [6]. Das interdisziplinare Gesprach ist in der Regel ein fester Bestandteil der Zeitschrift VISZERALMEDIZIN. Anhand der verschiedenen Artikel kommt in diesem Themenheft jedoch bereits deutlich zum Ausdruck, dass zwischen den jeweiligen Abteilungen im Bereich Patientensicherheit grose Unterschiede bestehen. Ein interdisziplinares Gesprach im Sinne einer Podiumsdiskussion hatte lediglich die verschiedenen Masnahmen dargestellt, die aufgrund der unterschiedlichen Bedingungen der einzelnen Facher von Chirurgen und Endoskopikern zur Starkung der Patientensicherheit angewendet werden, wurde aber fur den Leser mutmaslich keinen Zugewinn bezuglich einer Problemlosung darstellen. Wir bitten daher um Ihr Verstandnis fur unsere Entscheidung, dass diese Rubrik diesmal im Heft entfallt. Die Bereitschaft von Patienten oder Angehorigen, im medizinischen Schadensfall auch ein gerichtliches Verfahren anzustreben, ist lediglich ein Aspekt der zunehmenden Fokussierung auf die Patientensicherheit. Vielmehr jedoch setzt sich heute eine arztliche Haltung durch, die die Sicherheit der Patienten in den Vordergrund ruckt und Fehler oder «BeinaheFehler» als Chance zur Verbesserung der eigenen Prozessablaufe und nicht als Niederlage allwissender Experten betrachtet. Moge diese Haltung und die Nutzung der entsprechenden Werkzeuge im OP und in der Endoskopie eine weite Verbreitung finden. Wir als Gastherausgeber hoffen, mit der Betreuung dieses Themenhefts der VISZERALMEDIZIN hierzu einen kleinen Beitrag geleistet zu haben.