Martin Riegler

Effects of substance P on human colonic mucosa in vitro.

Previous studies indicated that the peptide substance P (SP) causes Cl--dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10-8 to 10-6 M) induced a rapid, monophasic concentration and Cl--dependent, bumetanide-sensitive short-circuit current ( I sc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1(SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced I sc increase without impairing forskolin- and carbachol-mediated I sc increase. We conclude that SP stimulates Cl--dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.Previous studies indicated that the peptide substance P (SP) causes Cl--dependent secretion in animal colonic mucosa. We investigated the effects of SP in human colonic mucosa mounted in Ussing chamber. Drugs for pharmacological characterization of SP-induced responses were applied 30 min before SP. Serosal, but not luminal, administration of SP (10(-8) to 10(-6) M) induced a rapid, monophasic concentration and Cl--dependent, bumetanide-sensitive short-circuit current (Isc) increase, which was inhibited by the SP neurokinin 1 (NK1)-receptor antagonist CP-96345, the neuronal blocker TTX, the mast cell stabilizer lodoxamide, the histamine 1-receptor antagonist pyrilamine, and the PG synthesis inhibitor indomethacin. SP caused TTX- and lodoxamide-sensitive histamine release from colonic mucosa. Two-photon microscopy revealed NK1 (SP)-receptor immunoreactivity on nerve cells. The tyrosine kinase inhibitor genistein concentration dependently blocked SP-induced Isc increase without impairing forskolin- and carbachol-mediated Isc increase. We conclude that SP stimulates Cl--dependent secretion in human colon by a pathway(s) involving mucosal nerves, mast cells, and the mast cell product histamine. Our results also indicate that tyrosine kinases may be involved in this SP-induced response.

Inhibition of P-glycoprotein-mediated vinblastine transport across HCT-8 intestinal carcinoma monolayers by verapamil, cyclosporine A and SDZ PSC 833 in dependence on extracellular pH

The ability of the multidrug resistance modifiers R- and R,S-verapamil (VPL), cyclosporine A (CsA) and its non-immunosuppressive derivative SDZ PSC 833 (PSC 833) to inhibit P-glycoprotein (P-gp)-mediated transepithelial flux of tritiated vinblastine was investigated using tight and highly resistant (R>1,400 Ω cm2) monolayer cultures of intestinal adenocarcinoma-derived HCT-8 cells grown on permeable tissue-culture inserts. Apical addition of these chemosensitisers inhibited drug flux (137 pmol h−1 cm−2; range, 133–142 pmol h−1 cm−2) in the basal to apical secretory direction at clinically relevant concentrations, with PSC 833 showing the highest activity, exhibiting inhibition at concentrations as low as 10 ng/ml (9 nM). Acidification of the modulator-containing apical compartment to an extracellular pH (pHo) of 6.8 had no influence on MDR reversal by CsA at 1 μg/ml (0.9 μM; flux inhibition, 52%) or by PSC 833 at 100 ng/ml (0.09 μM; flux inhibition, 60%), in contrast to R,S- and R-VPL, which showed decreased inhibition and caused less accumulation of vinblastine in HCT-8 cells under this condition (flux inhibition of 35% and 23%, respectively, at pHo 6.8 vs 50% and 43%, respectively, at pHo 7.5). P-gp-mediated rhdamine 123 efflux from dye-loaded single-cell suspensions of HCT-8 cells as measured by flow cytometry was not impeded at pHo 6.8 in comparison with pHo 7.5 in standard medium, but at low pHo the inhibitory activity of r-VPL (29% vs 60% rhodamine 123 efflux inhibition) was diminished significantly, again without a reduction in the effect of PSC 833 (rhodamine 123 flux inhibition, 75%). In conclusion, drug extrusion across polarised monolayers, which offer a relevant model for normal epithelia and tumour border areas, is inhibited by the apical presence of R,S- and R-VPL, CsA and PSC 833 at similar concentrations described for single-cell suspensions, resulting in increased (2.2- to 3.7-fold) intracellular drug accumulation. Functional apical P-gp expression, the absence of paracellular leakage and modulator-sensitive rhodamine 123 efflux in single HCT-8 cells indicate a P-gp-mediated transcellular efflux in HCT-8 monolayers. In addition to its high MDR-reversing capacity, the inhibitory activity of PSC 833 is not affected by acidic extracellular conditions, which reduce the VPL-induced drug retention significantly. As far as MDR contributes to the overall cellular drug resistance of solid tumours with hypoxic and acidic microenvironments, PSC 833 holds the greatest promise for clinical reversal of unresponsiveness to the respective group of chemotherapeutics.

Lymphangiogenesis and lymphovascular invasion diminishes prognosis in esophageal cancer.

BACKGROUND Tumor-associated lymphatic networks are the primary routes for tumor cell dissemination and metastasis. Behind the background of possible lymphangiogenesis-associated therapies, the clinical impact of lymphangiogenesis (measured by lymphatic microvessel density [LMVD]) and specific lymphovascular invasion (LVI) in esophageal cancer remains unclear. The aim of this study was to evaluate the clinical role of lymphangiogenesis and LVI in a large cohort of esophageal cancer. METHODS For the specific assessment of LMVD and LVI, 393 tissue samples from a prospective tissue databank of esophageal adenocarcinomas, squamous cell carcinomas, and their precursor lesions were included into this study. LMVD and LVI were assessed by immunostaining for podoplanin, a selective marker of lymphatic endothelium. In addition the peritumoral inflammatory stroma reaction (ISR) was assessed. RESULTS Patients with high LMVD had a significant increased risk to develop LVI (P = .00123; coefficient of regression [CR] 0.27) and lymph node metastasis (P = .00233), independent of the tumors histology. During a follow-up of 52 months, patients with high LMVD had a significantly reduced overall survival (OS; P < .001; 5-year OS 30% vs 54%) and disease-free survival (DFS; P < .001; 5-year DFS 28% vs 48%). OS (P < .001; 5-year OS 14% vs 60%) and DFS (P < .001; 5-year DFS 14% vs 49%) were significantly reduced in patients with present LVI. In invasive cancer, LMVD was significantly increased compared with precursor lesions (P = .008). The amount of ISR correlated significantly with LMVD. CONCLUSION Our data provide evidence for a clinically significant role of specific lymphangiogenesis in esophageal cancer. Patients with high lymphangiogenic tumor activity represent candidates for lymphangiogenesis-associated therapies.

Effects of extracellular pH on intracellular pH-regulation and growth in a human colon carcinoma cell-line

Mechanisms of intracellular pH (pHi) regulation seem to be involved in cellular growth and cell division. Little is known about how extracellular acidosis, known to occur in central regions of solid tumors, or alkaline conditions affect pHi regulation in colonic tumors. pHi changes in the colonic adenocarcinoma cell-line SW-620 were recorded by spectrofluorimetric monitoring of the pH-sensitive, fluorescent dye BCECF, and proliferative activity was assessed by [3H]thymidine uptake. Resting pHi in Hepes-buffered solution was 7.53 +/- 0.01 (n = 36). Both 1 mM amiloride and Na(+)-free solution inhibited pHi recovery from acidification and decreased pHi in resting cells. In HCO3-/CO2-buffered media resting pH1 was 7.42 +/- 0.01 (n = 36). Recovery from acidification was Na(+)-dependent, CI(-)-independent, and only partially blocked by 1 mM amiloride. In the presence of amiloride and 200 microM H2DIDS pHi recovery was completely inhibited. In Na(+)-free solution pHi decreased from 7.44 +/- 0.04 to 7.29 +/- 0.03 (n = 6) and no alkalinization was observed in CI(-)-free medium. Addition of 5 microM tributyltin bromide (an anion/OH-exchange ionophore) caused pHi to decrease from 7.43 +/- 0.05 to 7.17 +/- 0.08 (n = 5). The effects of pH0 on steady-state pHi, pHi recovery from acidification and proliferative activity after 48 h were investigated by changing buffer [CO2] and [HCO3-]. In general, increases in pH0 between 6.7 and 7.4 increased pHi recovery, steady-state pHi and growth rates. In summary, SW-620 cells have a resting pHi > 7.4 at 25 degrees C, which is higher than other intestinal cells. Acid extrusion in physiological bicarbonate media is accomplished by a pHi-sensitive Na+/H+ exchanger and a pHi-insensitive Na(+)-HCO3-cotransporter, both of which are operational in control cells at the resting pHi. No evidence for activity of a CI-/HCO3- exchanger was found in these cells, which could account for the high pHi observed and may explain why the cells continue to grow in acidic tumor environments.

Epidermal growth factor attenuates Clostridium difficile toxin A- and B-induced damage of human colonic mucosa

Epidermal growth factor (EGF) exhibits a cytoprotective effect on gastrointestinal epithelia via a receptor-mediated mechanism. We investigated the effect of EGF on Clostridium difficile toxin A (TxA)- and toxin B (TxB)-induced damage of human colon. Ussing-chambered colonic mucosa was exposed serosally to EGF before and during luminal exposure to TxA and TxB. Resistance was calculated from potential difference and short-circuit current. Epithelial damage was assessed by light microscopy and alteration of F-actin by fluoresceinated phalloidin. Luminal exposure of colonic strips to TxA and TxB caused a time- and dose-dependent decrease in electrical resistance, necrosis and dehiscence of colonocytes, and disruption and condensation of enterocyte F-actin. These effects were inhibited by prior, but not simultaneous, serosal application of EGF (20 nM). Administration of the tyrosine kinase inhibitor genistein (10-6 M) inhibited the protective effects of EGF. We conclude that EGF protects against TxA and TxB probably by stabilizing the cytoskeleton, the main target of these toxins.

HER-2 status in primary oesophageal cancer, lymph nodes and distant metastases

Some 10–15 per cent of patients with oesophageal cancer overexpress human epidermal growth factor receptor (HER) 2 at the primary tumour site, leading to the hope that specific targeted systemic therapy might favourably influence clinical and subclinical disease at locoregional and distant sites. This approach is based on primary tumour characteristics, without knowledge of expression patterns at metastatic sites. In oesophageal cancer, concordance between HER‐2 status at the primary tumour and other sites is unknown.

Intracellular pH regulation of human colonic crypt cells

In order to investigate the regulation of intracellular pH (pHi) in freshly isolated human colonocytes, we have used a newly developed technique for the rapid isolation and covalent attachment of these cells to glass surfaces and microspectrofluorimetric measurement of the pH-sensitive fluorescence of 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded specimens in a perfusion chamber (37°C). In N-2-hydroxyethylpiperazine — N′ — 2 — ethanesulphonic — acid — (HEPES)-buffered Ringer solution (HBS) a baseline pHi of 7.35±0.03 (mean ± SD; n=42) was found for human colonocytes and in HBS, NH4Cl-prepulse-induced intracellular acidification in colonocytes is reversed rapidly by the ubiquitous amiloride-sensitive (1 mmol/l) Na+/H+ exchanger. Switching from HBS to HCO3−buffered solution (BBS) led to a transient intracellular acidification (7.29±0.09), followed by a recovery to a final resting pHi of 7.43±0.03. One-third of the acid extrusion in BBS is amiloridesensitive; the remaining two-thirds are caused by the dihydroderivative of 4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid (H2DIDS)-sensitive HCO3−-dependent mechanisms. The functional activity of an acid-extruding Na+/HCO3−cotransporter in human colonocytes was observed in response to the reintroduction of Na+ into amiloride-containing Na+/Cl−-free BBS. In addition, the mechanism leading to alkalinization (7.56±0.05) in Cl−-free BBS was identified as Na+-dependent Cl−/HCO3−exchange, by its H2DIDS sensitivity and the specific requirement for Cl− and Na+. The intrinsic buffering capacity (βi) of the human colonocytes was calculated from pH changes induced by sequential NH4Cl-loading steps during blockage of acid/base transporters. With βi=80 mmol · l−1 · pH unit−1 for the pH interval ranging from 6.9 to 7.1 (n=8) the colonocytes exhibited a relatively high intrinsic buffering in comparison with other cell types. In conclusion, the freshly isolated human colonocytes express a Na+/H+ exchanger, a Na+/HCO3−cotransporter and a Na+-dependent Cl−/HCO3−exchanger, all of them likely to be involved in the regulation of pH homeostasis in vivo in the presence of widely varying extracellular conditions. Maintenance of a stable pHi of human colonocytes seems to be facilitated by a comparatively high βi at physiological pH values.

The value of protecting the longitudinal staple line with invaginating sutures during esophageal reconstruction by gastric tube pull-up.

Background: Radical surgery with lymphadenectomy offers the best chance of curing esophageal cancer, but it carries considerable risks. Generally, the resected esophagus is replaced with a gastric tube. Rupture of the gastric tube staple line is a rare but serious surgical complication. One unresolved issue is whether oversewing of the longitudinal gastric staple line is necessary to avoid staple line rupture or insufficiency. Patients and Methods: Between 2000 and February 2008, 199 patients underwent esophageal resection for cancer or perforation at the Vienna General Hospital, Medical University of Vienna. Data were collected prospectively. Of these patients, 151 (75.9%) underwent reconstruction by pulling up a gastric tube. These comprised the study population. In 83 patients (55.0%) the longitudinal gastric staple line was not oversewn (group A). In 68 patients (45.0%) the staple line was reinforced by invaginating sutures (group B). Results: The mean age of the patients was 62.0 ± 10.6 years (median: 63.1 years). Males comprised 75.5% of the population. Adenocarcinoma was diagnosed in 77 patients (51.0%), 63 patients (41.7%) suffered from a squamous cell carcinoma, 10 patients (6.6%) had esophageal perforation, and in 1 patient (0.7%) a gastrointestinal stromal tumor was diagnosed. In group A, a leak within the staple line was observed in 4 of 83 patients (4.9%). No leak was found in group B (p = 0.09). Major surgical complications included anastomotic leakage (21 patients; 13.9%), gastric tip necrosis (3 patients; 2%), postoperative ileus (3 patients; 2.0%) and chylothorax (7 patients; 4.6%). Two major intraoperative complications (1.3%) were splenic injury and aortic bleeding. Conclusion: A remarkable but not statistically significant difference was found regarding staple line rupture between study groups. However, all leaks were seen in patients without a staple line suture.

Peptic esophageal stricture: is surgery still necessary?

B a l b a n demonstriert 1. neuerdings den yon 0 p p e n h e l m in der vorigen Sitzung vorgeste]lten Fall yon U l z e r a t i o n e n d e s h a r t e n u n d w e i e h e n G a u m e n s . Es wurde damals die Vermutung ausgesprochen, dab es sich um eine Kombination yon Lues mit Tuberkulose handeln kSnnte. Der therapeutische Effekt hat jedoch gezeigt, d a d es sieh um relne Gummen handelte, denn nach der dritten halben t tgSalizyl-Injektion war die Affektion fast vS1]ig geheilt. 0 p p e n h e i m : Vor 14 Tagen wurde der Fall yon mir als u l z e r 5 s e S y p h i 1 is des harten und weichen Gaumens vorgestellt, wobei ich mit Riicksicht auf kleine miMare Gesehwiirchen i n der Umgebung der Ulzerationen und auf kleine graue KnStchen den Verdacht ausslorach, es kSnnte sieh um eine n a c h t r ~ g l i c h e M i s e h i n f e k t i o n m i t T u b e r k u l o s e h a n d e l n . Naeh dem E r f o l g der Quecksilberbehandlung mug die Diagnose Tuberkulose fallen und darauf hingewiesen werden, wie ~ihnJ lich sieh Syphilis und Tuberkulose der Mundschleimhaut dokumentieren kSnnen.

Review on novel concepts of columnar lined esophagus

SummaryBackgroundColumnar lined esophagus (CLE) is a marker for gastroesophageal reflux and associates with an increased cancer risk among those with Barrett’s esophagus. Recent studies fostered the development of integrated CLE concepts.MethodsUsing PubMed, we conducted a review of studies on novel histopathological concepts of nondysplastic CLE.ResultsTwo histopathological concepts—the squamo-oxyntic gap (SOG) and the dilated distal esophagus (DDE), currently model our novel understanding of CLE. As a consequence of reflux, SOG interposes between the squamous lined esophagus and the oxyntic mucosa of the proximal stomach. Thus the SOG describes the histopathology of CLE within the tubular esophagus and the DDE, which is known to develop at the cost of a shortened lower esophageal sphincter and foster increased acid gastric reflux. Histopathological studies of the lower end of the esophagus indicate, that the DDE is reflux damaged, dilated, gastric type folds forming esophagus and cannot be differentiated from proximal stomach by endoscopy. While the endoscopically visible squamocolumnar junction (SCJ) defines the proximal limit of the SOG, the assessment of the distal limit requires the histopathology of measured multilevel biopsies. Within the SOG, CLE types distribute along a distinct zonation with intestinal metaplasia (IM; Barrett’s esophagus) and/or cardiac mucosa (CM) at the SCJ and oxyntocardiac mucosa (OCM) within the distal portion of the SOG. The zonation follows the pH-gradient across the distal esophagus. Diagnosis of SOG and DDE includes endoscopy, histopathology of measured multi-level biopsies from the distal esophagus, function, and radiologic tests. CM and OCM do not require treatment and are surveilled in 5 year intervals, unless they associate with life quality impairing symptoms, which demand medical or surgical therapy. In the presence of an increased cancer risk profile, it is justified to consider radiofrequency ablation (RFA) of IM within clinical studies in order to prevent the progression to dysplasia and cancer. Dysplasia justifies RFA ± endoscopic resection.ConclusionsSOG and DDE represent novel concepts fusing the morphological and functional aspects of CLE. Future studies should examine the impact of SOG and DDE for monitoring and management of gastroesophageal reflux disease (GERD).ZusammenfassungHintergrundZylinderepithel-Ösophagus (engl. columnar lined esophagus; CLE) zeigt gastroösophagealen Reflux und bedingt bei jenen mit einem Barrett Ösophagus ein erhöhtes Krebsrisiko. Rezente Studien beschreiben ein integriertes morphofunktionales CLE Konzept.MethodikDiese PubMed basierte Analyse gibt eine Übersicht zu neuen histopathologischen Konzepten zu CLE ohne Dysplasie.ErgebnisseUnsere neue Vorstellung zu CLE wird anhand von zwei neuen histopathologischen Konzepten dargestellt: dem Mukosasegment zwischen Plattenepithel und oxntischer Magenschleimhaut (engl. squamo-oxntic gap; SOG) und dem dilatierten distalen Ösophagus (engl. dilated distal esophagus; DDE). Als Folge des Reflux entsteht das SOG zwischen dem von Plattenepithel ausgekleideten Ösophagus und des von oxyntischer Mukosa ausgekleideten proximalen Magens. SOG beschreibt die Histologie des CLE im tubulären Ösophagus und DDE, welcher auf Kosten des durch den Reflux verkürzten unteren Ösophagussphinkters entsteht und damit vermehrten Rückfluss des sauren Mageninhalts begünstigt. Morphologische Untersuchungen des Ausgangs der Speiseröhre zeigten, dass der DDE Reflux-geschädigter, dilatierter, magenähnliche Falten bildender Ösophagus ist und in der Endoskopie nicht vom proximalen Magen unterschieden werden kann. Während die proximale Grenze des SOG der endoskopisch definierbaren Platten-Zylinderepithelgrenze entspricht, kann die untere Grenze des SOG nur mittels Fusion von Biopsie-Lokalisation und der Histologie von aus diesem Bereich entnommenen Gewebeproben bestimmt werden. Im SOG ordnen sich die CLE Typen entsprechend einer typischen proximalen-distalen Verteilung mit intestinaler Metaplasie (IM, Barrett Ösophagus) ± Kardia Schleimhaut (CM) an der Platten-Zylinderepithelgrenze und Oxyntokardia (OCM) Mukosa im distalen Abschnitt des SOG. Die Ausrichtung folgt dem Reflux-bedingte pH Gradienten entlang des unteren Ösophagus. Die Diagnose von SOG und DDE erfolgt mittels Endoskopie, Histologie von Multi-Level Biopsien aus dem Ausgang der Speiseröhre sowie Funktionstests und Röntgenuntersuchungen. CM und OCM an sich bedürfen keiner Therapie und sollen in 5 Jahren nachuntersucht werden, nur assoziierte Reflux Beschwerden, welche die Lebensqualität beeinträchtigen, sollen medikamentös oder chirurgisch behandelt werden. Bei entsprechendem Krebsrisiko ist es gerechtfertigt, bei IM ohne Dysplasie eine Radiofrequenzablation (RFA) im Rahmen klinischer Studien zu erwägen, um damit die Entstehung von Dysplasie und Karzinom zu verhindern. Dysplasie rechtfertigt eine RFA ± endoskopischer Resektion.SchlussfolgerungenSOG und DDE sind neue Konzepte, welche Morphologie und Funktion des Zylinderepithel-Ösophagus integrieren. Die Zukunft wird zeigen, welche Bedeutung diese neuen Konzepte für Diagnose und Therapie der gastroösophagealen Refluxkrankheit haben.