Christian F. Krieglstein

Impact of dextran sulfate sodium load on the severity of inflammation in experimental colitis.

In dextran sulfate sodium (DSS)-induced inflammatory bowel disease in mice the relationship between the amount of ingested DSS and the severity of colitis has not been systematically investigated. We examined whether (1) the severity of colitis is DSS load-dependent, and (2) there is a critical DSS load required to reliably induce colitis. DSS load was calculated as: (drinking volume (ml) × [DSS (g)/100 ml])/body weight (g). A minimum DSS load ≥ 30 mg/g body weight over 7 days resulted in a significantly elevated colonic myeloperoxidase (MPO) activity, compared to mice receiving less DSS and controls (P < 0.05). Histomorphologic data correlated with MPO activity and revealed significantly higher damage scores once the DSS load was ≥ 30 mg/g body weight. Our findings demonstrate the importance of monitoring DSS load in this model of experimental colitis.

Preoperative Steroid Administration: Effect on Morbidity among Patients Undergoing Intestinal Bowel Resection for Crohn’s Disease

Long-term steroid therapy may predispose to increased perioperative morbidity in patients undergoing surgery with bowel anastomoses. The aim of our study was to review our data to determine if the steroid dosage is associated with the incidence of early complications after bowel resection in patients with prolonged steroid therapy for Crohn’s disease (CD). Altogether, 397 patients underwent bowel resection with primary intestinal anastomoses for CD between 1982 and 2000 in our institution. The mortality and morbidity rates, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and length of postoperative hospitalization in patients who were having high-dose (≥ 20 mg of prednisolone per day, n = 73) and low-dose (< 20 mg prednisolone per day, n = 146) steroid therapy for more than 1 month before surgery were compared with those of patients (n = 177) who were not receiving steroids. Statistical analysis was performed using Fisher’s exact test and Student’s t-test, with p < 0.05 considered significant. The three groups were similar in terms of gender, duration since first diagnosis, American Society of Anesthesiologists classification, and obesity. Mortality, morbidity, anastomotic leakage, wound infections, intraabdominal abscesses, reoperation rate, and average postoperative stay were not statistically different in patients with high-dose, low-dose, or no steroid therapy. The only factor associated with increased morbidity was a low preoperative hemoglobin level. Our results demonstrate that, in patients who are undergoing bowel resection for CD, even high-dose prolonged preoperative systemic steroid therapy is not associated with increased postoperative complications.

Cricopharyngeal myotomy in the treatment of Zenker’s diverticulum

BACKGROUND Transection of the cricopharyngeus muscle supposedly is of crucial therapeutic importance, because of its hypothesized pathogenic role in Zenkers diverticulum (ZD). This retrospective, nonrandomized study evaluates the outcomes of surgical therapy, with reference to cricopharyngeal myotomy (CM). STUDY DESIGN Seventy-nine patients underwent diverticulectomy from 1985 to 1999. Group A (n = 47; men:women, 29:18; mean age +/- SD: 69 +/- 12 [range 35 to 87] years) underwent CM because of clearly discernible hypertrophic transverse fibers. In group B (n = 32; men:women, 22:10; mean age +/- SD 68 +/- 13 [range 36 to 95] years), without such transverse fibers, no CM was carried out. Dysphagia, regurgitation, and diverticular radiomorphology were classified according to ordinal scales. Diverticular volumes were calculated from barium swallows. Postoperative symptoms and outcomes were evaluated by questionnaires sent to the patients. RESULTS The two groups did not differ significantly in severity of preoperative dysphagia and regurgitation, radiomorphology, or median diverticular volume. Both groups experienced postoperative alleviation of symptoms (p < 0.001), persisting slightly in 11 of 47 (23%) group A and 4 of 32 (13%) group B patients (p > 0.05). Of these, seven group A (64%) patients and three group B patients (75%) had additional upper gastrointestinal tract (GIT) diseases. Recurrent diverticula occurred in one patient and postoperative complications in five patients per group (p > 0.05). Diverticular volume and upper GIT comorbidities, but not CM, were significant risk factors for persistent symptoms in the multivariate analysis. CONCLUSIONS CM has no significant influence on postoperative results, if carried out depending on the anatomic state and configuration of the cricopharyngeus muscle. This is suggested by the favorable outcomes of patients, with persistent symptoms being caused by factors other than CM.

Is Colonoscopy Alone Sufficient to Screen for Ulcerative Colitis-associated Colorectal Carcinoma?

Patients with ulcerative colitis (UC) are at increased risk for colorectal carcinoma (CAC). Despite the fact that patients at risk are followed closely by colonoscopy to screen for dysplasia, the prevalence of CAC is still unacceptably high. The aim of this study was to evaluate the prevalence of risk factors for CAC, such as dysplasia, and to determine the relevance of colonoscopic surveillance in the group who went on to develop cancer. A series of 24 patients with UC were diagnosed with CAC. The patients’ records were analyzed retrospectively for duration of UC, prevalence of preoperative dysplasia, and other cancer risk factors (CRFs) (e.g., pancolitis, primary sclerosing cholangitis, early onset of UC, and backwash ileitis). The mean age of the patients at the time of cancer diagnosis was 43 years with an average UC duration of 15 years (6 patients had had UC less than 8 years). CAC was identified preoperatively by colonoscopy in 15 of 24 patients, with an additional 7 of 15 showing flat dysplasia. Five of nine patients without preoperatively diagnosed CAC had flat dysplasia. Overall, 19 patients had additional CRFs, most of them with at least two more CRFs. Despite a regular colonoscopic follow-up for most patients with UC, flat dysplasia was missed in 12 patients preoperatively. Therefore we suggest that patient information should also always include surgical options in each case where significant cancer risk factors are found.

Metallothionein: early marker in the carcinogenesis of ulcerative colitis-associated colorectal carcinoma.

Metallothioneins (MTs) are zinc-binding proteins whose over-expression may lead to sequestration of zinc ions and consequently to functional inactivation of the p53 tumor suppressor gene. The aim of the study was to investigate the potential role of MTs in the carcinogenesis of ulcerative colitis (UC) as well as possible effects on p53 function. The monoclonal antibodies E9 (anti-MT), DO-7, and 1801 (anti-p53) and the polyclonal antibody CM-1 (anti-p53) were used to stain formalin-fixed, paraffin-embedded colon specimens obtained from 14 patients with UC-associated colorectal carcinoma (CAC), 13 with high-grade dysplasia (HGD), 10 with low-grade dysplasia (LGD), and 30 with UC without dysplasia or carcinoma. Statistical significance (p<0.05) was assessed using Fisher’s exact test. Positive MT staining (>20% of tumor, dysplastic, or epithelial cells) was found in most UC and LGD but in only a small percentage of HGD and CAC (p<0.01 for CAC vs. UC and LGD vs. HGD). Positive p53 immunoreactivity was observed predominantly in HGD and CAC but not in LGD and UC (p<0.01 for CAC vs. UC and HGD vs. LGD). In histologically normal tissue neighboring CAC, significant MT expression was found in six of seven specimens with simultaneous lack of p53 expression. MT overexpression may represent an important early step in the development of CAC independent of p53 expression and should be investigated in the long term as an independent cancer risk factor in UC.RésuméLes métallothionéines (MT) sont des protéines liées au zinc dont la surexpression peut provoquer une séquestration des ions zinc et par conséquent une inactivation fonctionnelle du gène de suppression tumorale p53. Le but de cette étude a été d’examiner le rôle potentiel des MT dans la carcinogenèse au cours de la recto-colite hémorragique (RCH), de même que les effets sur la fonction p53. Les anticorps monoclonaux E9 (anti-MT), DO-7 et 1801 (anti-p53) ainsi que l’anticorps polyclonal CM-1 (anti-p53) ont été utilisés pour colorer les pièces de côlon, fixées par le formol et en paraffine, obtenues à partir de 14 patients atteints de RCH et de cancer colorectal (CCR), 13 patients porteurs de dysplasie de haut grade (DHG), 10 patients porteurs de dysplasie de bas grade (DBG) et 30 patients ayant une RCH sans dysplasie ni cancer. La signification statistique recherchée par l’utilisation du test exact de Fisher a été fixée à 0.05. Une coloration MT positive (définie comme >20% des cellules tumorales, dysplasiques ou épithéliales) a été retrouvée dans la majorité des cas de RCH et de DBG, mais seulement dans une minorité de patients porteurs de DHG et/ou CCR (p<0.01 pour CRC vs RCH et DBG vs DHG). L’immunoréactivité du P53 était positive essentiellement pour les patients DHG et CCR, mais pas pour les DBG ou RCH (p<0.01 pour CRC vs RCH et pour DHG vs DBG). Dans le tissu histologiquement normal aux alentours du CCR, on a retrouvé une expression MT significative mais sans expression de P53 dans six des sept pièces. La surexpression MT pourrait représenter une étape précoce importante dans le développement du CCR, indépendamment de l’expression p53 et devrait être recherchée comme un facteur de risque indépendant de cancer au cours d’une RCH, dans le suivi au long cours.ResumenLas melatoninas (MT) son proteínas ligadoras de zinc cuyas superexpresión puede Ilevar al secuestro de los iones de zinc y, en consecuencia, a la inactivación del gen p53 de supresión tumoral. El propósito del presente estudio fue investigar el papel de las MT en la carcinogénesis de la colitis ulcerativa (CU), así como a los posibles efectos sobre la función del p53. Se utilizaron los anticuerpos monoclonales E9 (anti-MT), DO-7 y 1801 (anti-p53) y el anticuerpo policional CM-1 (anti-p53) para teñir especímenes colónicos incluidos en parafina obtenidos de 14 pacientes con carcinoma colorectal asociado a CU (CCA), de 13 pacientes con displasia de alto grado (DAG), de 10 pacientes con displasia de bajo grado (DBG) y de 30 pacientes con CU sin displasia ni carcinoma. La significancia estadística (p<0.05) se determinó por la prueba exacta de Fisher. Se registró tinción positiva para MT (>20% de las células tumorales, displásicas o epiteliales) en la mayoría de las CU y DBG, pero sólo en la minoría de la DAG y CCA (p<0.01 entre CCA vs CU y DBG vs DAG). Se encontró expresión significante de MT en el tejido histológicamente normal vecino del CCA en 6 de 7 especímenes con ausencia de expresión de p53. La superexpresión de MT puede representar una fase temprana en el desarrollo del CCA, independiente de la expresión de p53 y debe ser investigada en el seguimiento a largo plazo como un factor de riesgo independiente en la CU.

Effect of anti-CD11b (αM-MAC-1) and anti-CD54 (ICAM-1) monoclonal antibodies on indomethacin induced chronic ileitis in rats

Abstract Leukocyte emigration from blood to sites of inflammation involves sequential interaction of specific adhesion molecules expressed by both leukocytes and endothelial cells. The central steps in leukocyte-endothelial adhesive interactions are leukocyte rolling, sticking, and transmigration. This study investigated the effect of monoclonal antibodies against CD54 (ICAM-1) and CD11b (αM-chain of MAC-1) on intestinal inflammation. Anti-CD54 and anti-CD11b were tested in rats with indomethacin-induced chronic ileitis. Macroscopic changes were assessed by a modified version of the Wallace et al. score. Leukocyte rolling and sticking were investigated by intravital microscopy. Results show that indomethacin administration led to a chronic inflammatory response characterized by significant increase (P<0.05) in rolling (from 5.41±2.87 to 32.41±15.03 100 µm–1 s–1) and sticking (from 0.16±0.18 to 9.11±5.3 100 µm–1 s–1) leukocytes. After antibody treatment only the anti-CD11b group showed significant (P<0.05) reduction in rolling (from 32.41±15.03 to 6.6±2.7 100 µm–1 s–1) and sticking (from 9.11±5.3 to 0.07±0.09 100 µm–1 s–1) leukocytes. This was also the case for macroscopic changes. Indomethacin led to a rise in the Wallace score from 0 to 4.29±0.76 points (P<0.05) and anti-CD11b to a reduction from 4.29±0.76 to 1.29±1.11 points (P<0.05). Anti-CD54 and combined anti-CD11b/CD54 administration was not followed by significant changes. Therefore we suggest that leukocyte-based CD11b but not endothelial-based CD54 contributes most to leukocyte adhesion in the setting of indomethacin-induced ileitis in rats.

“Pancreatic lesion” outside the pancreas: Value of endoscopic ultrasound

Tumors of the small intestine are rare as compared to malignant tumors of the pancreas. Here we report on the case of a 61-year-old man suffering from chronic pancreatitis presenting with a lesion projecting into the pancreatic head shown by both computed tomography and transabdominal ultrasound. Pancreatic cancer was suspected, but endoscopic ultrasound revealed this lesion to be situated in the submucosal layer of the duodenal wall. Surgery was performed since biopsy of this lesion was not diagnostic and a malignant leiomyosarcoma could therefore not be excluded. Limited surgery comprised resection of the duodenal lesion, whereas based on computed tomography alone, exploration of the pancreas would have been performed. Thus, in the present case endoscopic ultrasound leads to a more appropriate, less invasive therapeutic measure.

Bedeutung der Interaktion der Thrombozyten und Leukozyten in der Pathogenese der experimentellen Kolitis

Introduction: There is an important role for the interactions between circulating blood cells and the endothelium within the microcirculation of the intestine in initiating and maintaining inflammatory bowel disease. Recent studies suggest a key role in inflammation not only for leukocytes but also for other cell types, including platelets. The aim of the study was to define the mechanisms responsible for platelet-leukocyte and platelet-endothelial interactions that occur in experimental colitis. Methods: Colitis was induced in wild-type (WT) C57Bl/6J and ICAM-1 knockout (−/−) mice with 3% dextran sulfate sodium (DSS) in drinking water for 7 days (d0–6). Control animals received water. At day 6 platelets were isolated from genotype-matched donors, fluorescently-labeled ex vivo with carboxyflurescein succinimidylester (CFSE) and injected into the recipient. Leukocytes were visualized with rhodamine 6G in vivo and both cell types in colonic venules were monitored at the same time by intravital microscopy. Results: Compared with controls, DSS-treated WT mice showed significantly increased adhesion of platelets, with 2,5% of platelets adhering to endothelial cells directly and 97,5% binding to adherent leukocytes. ICAM-1 deficiency significantly decreased platelet-leukocyte adhesion. WT mice rendered neutropenic with anti-neutrophil serum (ANS) exhibited reduced platelet-leukocyte binding, but significantly increased platelet-endothelial adhesion. The increase in platelet-endothelial adhesion in neutropenic animals was prevented by both, a single dose (2 mg/kg) of either P-selectin or PSGL-1 blocking monoclonal antibody. Conclusions: In DSS-induced colitis the accumulation of platelets in venules is dependent on both, leukocyte adhesion as well as on mechanisms involving the adhesion molecules P-selectin and PSGL-1. (DFG VO 998/1-1 and NIH DK43785)

Störung des HLA-vermittelten Antigentransports in Enterozyten von Patienten mit chronisch-entzündlichen Darmerkrankungen

Introduction: The pathogenesis of inflammatory bowel disease (IBD; e.g. ulcerative colitis (UC) and Crohn‘s disease, CD) is still unknown. Recent reports suggest a possible disorder of oral tolerance caused by a dysfunctional MHC regulated antigen presentation of enterocytes to lymphocytes of the mucosa-associated lymphatic system (MALT). Therefore the objective of our study was to investigate the intracellular MHC distribution of enterocytes of patients with IBD and compare them with enterocytes of a healthy control group (CG). In IBD-patients both regular enterocytes (NE) and enterocytes with cytosolic antigen intake (RACE, rapid antigen uptake into the cytosol enterocytes) were examined in this case. Method: Fresh mucosal specimens of MC (ileum [il]), CU (colon [co]) patients and controls (CG-ileum, CG-colon, each n = 5) were incubated with egg albumin antigen (OVA). Following MHC-DP, -DQ, -DR (MHC-II) and OVA as well as β 2-microglobulin, a structure protein of MHC-I and OVA were localized using an immunogold double-labeling method with mono- and polyclonal antibodies by electron microscopy. Distinguished between antigen-loaded and antigen-free MHC-positive vesicles the number of this vesicle per cut cell face were determined. As a measure of the concentration of MHC-I being in the rough endoplasmic reticulum (RER) the number of gold points were counted per membrane length. Statistical analysis was performed using χ2- or Fischer‘s exact test with p < 0.05 considered significant. Results: Compared to control group normal enterocytes of patients with MC and CU showed a significantly stronger enrichment of antigen-loaded (MC: 22.7 ± 8.8 vs. 2.1 ± 1.9; CU: 70.8 ± 15.3 vs. 0) and antigen-free (MC: 32.1 ± 10.9 vs. 14.6 ± 6.5; CU: 54.1 ± 17.5 vs. 6.2 ± 5.0) vesicles. In contrast to that the MHC-II-positive vesicles were significantly reduced at RACE of MC (2.8 ±2.4) and CU (25.5 ±8.1) compared with NE as well as compared to CG-enterocytes. In the RER of RACE a significant stronger expression of MHC-I (MC: 30.3 ±6.1; CU: 24.3 ± 4.7) were found compared to control enterocytes (MC: 6.9 ± 2.1; CU: 11.2 ± 3.5) and NE (MC: 2.1 ± 0.6; CU: 1.5 ± 0.5) of MC and CU. Conclusion: The decrease of antigen presentation provided by MHC-II proteins in RACE cells at simultaneous activation of MHC-I proteins in the RER possibly represents a morphological correlative for a changed oral tolerance in patients with IBD.