Increased matrix metalloproteinase 2 concentration and transcript expression in advanced colorectal carcinomas.

Colorectal cancer is one of the most common malignant tumors and entails a relatively poor prognosis. Clinical outcome depends on the extent of local and metastatic tumor spread. Results of in vivo and in vitro studies suggest that the balance between matrix metalloproteinases (MMPs) and their inhibitors (tissue inhibitors of metalloproteinases TIMPs) is altered in neoplasia, contributing to the invasive and metastatic properties of malignant tumors. We quantified tissue concentrations of MMP-2 and TIMP-2 in 65 malignant colorectal lesions and corresponding normal mucosa by enzyme-linked immunosorbent assay, western blotting, and in situ hybridization. In situ hybridization and western blot analyses demonstrated a clear increase in both stromal expression of MMP-2 transcripts and protein in primary carcinomas. The protein concentration of MMP-2 was higher in all tumor stages, except stage I tumors, than in normal mucosa and adenomas. MMP-2 concentrations were not related to tumor differentiation or to colonic versus rectal location. Surprisingly, the MMP-2 concentration was not increased in metastases. Interestingly, tissue concentrations and epithelial mRNA expression of TIMP-2 decreased significantly in primary colorectal cancer (UICC stages III and IV) but increased in metastases. Therefore an increased ratio of MMP-2 to TIMP-2 is strongly associated with advanced tumor stages, but a decreased ratio was observed in metastases. These findings suggest that the MMP-2:TIMP-2 ratio may prove useful as a marker of local invasion but not of metastasis in colorectal cancer.

Differential effects of ET-1, ET-2, and ET-3 on pancreatic microcirculation, tissue integrity, and inflammation.

The differential effects of endothelin-1, -2, and -3 (ET-1, ET-2, and ET-3) on pancreatic microcirculation, pancreatic tissue integrity, and an initial inflammatory response, which are three distinct characteristics of acute necrotizing pancreatitis, were investigated in a dose-dependent manner in rats using in vivo microscopy. Red blood cell (RBC) velocity and functional capillary density (FCD) were estimated after topical superfusion of the pancreas with ET-1, ET-2, and ET-3 (100, 10, 1 pmol), revealing that ET-1 (100, 10, 1 pmol) or high ET-2 (100 pmol) and ET-3 (100 pmol) cause a dose-related deterioration of exocrine nutritive pancreatic blood flow. Analysis of pancreatic exocrine tissue damage employing the Spormann score displayed that the ET-mediated microcirculatory impairment was paralleled by dose-dependent tissue damage, which was significant compared to the control group (topical superfusion with 1 ml, saline solution 0.9%). Estimation of pancreatic postcapillary leukocyte accumulation by histomorphologically counting choracetate esterase (CAE) stained leukocytes in 50 high-power fields per animal demonstrated a significant increase in postcapillary accumulation of white blood cells, after topical administration of ET-1, ET-2, and ET-3 compared to controls. In contrast to ET-caused effects on microcirculation and tissue impairment, quantitative analysis of postcapillary leukocyte accumulation revealed the most pronounced effect after ET-2 administration but not after ET-1 administration. This demonstrates that ET-1, ET-2, and ET-3 are all able to mediate microcirculatory impairment, tissue damage, and inflammation. However, ET-3-induced damaging effects are less pronounced, while ET-1 most severely alters microcirculation and ET-2 preferentially induces leukocyte-dependent inflammation.

Temporary pancreatic duct occlusion by ethibloc: cause of microcirculatory shutdown, acute inflammation, and pancreas necrosis.

Temporary obliteration of the pancreatic duct has been suggested to be beneficial in chronic pancreatitis, segmental pancreatic transplantation, and following Roux-Y pancreaticojejunostomy. Little is known, however, as to whether obliteration of the duct alters exocrine pancreatic physiology. Therefore we studied in male inbred Lewis rats the immediate effects of Ethibloc-induced duct obliteration (Ethibloc: Ethicon, Norderstedt, Germany) on pancreatic microcirculation, inflammation, and tissue injury (n = 8), and compared these effects with those caused by experimental pancreatitis (4% sodium taurocholate; n = 8). Animals receiving an intraductal infusion of saline served as controls (n = 8). Duct occlusion with Ethibloc resulted in a marked decrease (p < 0.05) in capillary red blood cell (RBC) velocity and functional capillary density (FCD) to 88 +/- 39 microm/s (baseline 716 +/- 40 microm/s) and 72 +/- 33 cm(-1) (baseline 493 +/- 21 cm(-1)), respectively, which was even more pronounced when compared with that observed in experimental pancreatitis (333 +/- 62 microm/s and 195 +/- 44 cm(-1), respectively). In parallel, the manifestation of tissue damage was found to be more severe after Ethibloc; and chloracetate esterase staining showed a larger number of infiltrating leukocytes [555 +/- 86/high power field (HPF) versus pancreatitis: 160 +/- 12/HPF; p < 0.05). We conclude that intraductal application of Ethibloc induces significant microcirculatory failure and a marked inflammatory response, which are even more pronounced when compared with the changes observed with experimental pancreatitis. Based on these results and the fact that there is no direct proof for a benefit of temporary duct occlusion by Ethibloc, it is proposed that the procedure be reevaluated for its use in pancreatic surgery.

Reoperations at the ileostomy in Crohn's disease reflect inflammatory activity rather than surgical stoma complications alone.

After ileostomy construction for Crohn's disease reoperations due to ileal recurrences are thought to be unusually rare, whereas reconstructions of the ileostomy due to stoma complications are considered to be unusually frequent. It remains unclear why the natural course of a disease as well as outstanding results of a standardized surgical procedure should be perverted. Therefore reconstructions of the ileostomy in 92 patients colectomized during a 12.5-year period and followed up for 5.4 years were analyzed concerning preoperative indication and postoperative histology. In 28 patients (30.4%) a total of 42 reoperations were necessary. The clinical indication was prestomal recurrence in 5 reoperations (11.9%) and stoma complications in 37 (88.1%). In contrast, ileal recurrence was demonstrated histologically in 28 specimens (66.7%) and healthy ileum in the rest. There was a statistically significant association between fibrotic recurrence and stoma stenosis/retraction and a trend for association between penetrating recurrence and peristomal ulceration. The cumulative risk for a first reoperation due to clinical recurrence was calculated at 3.3% and 14.0% at 5 and 10 years postoperatively, whereas the corresponding figures for stoma complications were 25.7% and 40.0%. In contrast, the cumulative risk that a recurrence was found histologically on the occasion of the reoperation was 23.0% and 35.0%, while the probability that the ileum was healthy in the case of a stoma complication remained low. In conclusion, most reoperations after ileostomy-construction in Crohn's disease are associated histologically with recurrent inflammation. The accentuation of the inflammatory recrudescence at the stoma itself or the prestomal ileum is decisive for the clinical presentation as stoma complication or intestinal complication. These findings reinforce both well known characteristics of the inflammatory disease and of established surgery.

Stage-specific alternative cplicing of CD44 and alpha 6 beta 1 integrin in colorectal tumorigenesis.

Recent reports suggest that cancerogenesis induces changes in alternative processing of human genes. However, little is known about the regulation of alternative splicing during malignant transformation. Therefore, we examined changes in alternative splicing of two different adhesion molecules, alpha 6 beta 1 integrin and CD44, in multiple stages of colon tumorigenesis. Using semiquantitative RT-PCR it is shown that the alternatively spliced isoforms of both adhesion molecules, alpha 6A and -B and CD44v6, are significantly upregulated in colorectal adenoma (n = 20) compared to normal colon mucosa (n = 32) (P < 0.01). Although beta1 isoforms were expressed in almost all tissues, there was a significant increase in the intensity of gene expression of beta 1A compared to beta 1B (P <0.05) in adenoma tissue. Interestingly, CD44v6 and alpha 6 variant isoforms were downregulated in carcinoma tissue (n = 28) compared to adenoma. These results establish a link between neoplastic transformation and alternative splicing of cell adhesion molecules. Furthermore, these data suggest that colon epithelial cells carrying splice variants of adhesion molecules might acquire a selective growth advantage during early tumorigenesis.

Comparable expression of matrix metalloproteinases 1 and 2 in pouchitis and ulcerative colitis.

BACKGROUND AND AIMS: Matrix metalloproteinases (MMPs) are implicated in the tissue destruction associated with inflammatory diseases. Proctocolectomy with ileo-anal pouch (IAP) anastomosis is associated with pouchitis, particularly in patients with ulcerative colitis (UC). The aim of this study was to quantify MMP-1 and MMP-2 in inflamed and uninflamed pouches of patients with UC compared with those with active UC. IAP patients with familial adenomatous polyposis (FAP) served as controls. METHODS: Biopsies were taken from 33 patients with IAP (UC, n=25; FAP, n=8) and from 10 UC patients. MMP-1 and MMP-2 were quantified using sandwich enzyme linked immunosorbent assays. In addition, northern and western blotting and in situ hybridisation experiments were performed. RESULTS: In pouchitis (n=11), MMP-1 and MMP-2 concentrations were increased compared with uninflamed pouches of patients with UC (n=14) or FAP (n=8) (MMP-1 17.7 ng/mg protein v 7.8 (UC) v 7.6 (FAP), p

Establishment of an immunocompetent nipple valve anastomosis to prevent faecal reflux after ileocolic resection in dogs.

OBJECTIVE: To construct an immunocompetent nipple valve anastomosis (NVA) to exclude faecal reflux and compare it with an end-to-end anastomosis to see if it would prevent recurrent inflammation caused by intraluminal bacterial antigens in Crohn's disease. DESIGN: Laboratory study. SETTING: Teaching hospital, Germany. ANIMALS: Two groups of six beagle dogs each of which had NVA or end-to-end anastomosis. INTERVENTIONS: Construction of a NVA by stapling the telescoped nipple, and replacing the ileal mucosa on the valve by colonic mucosa; end-to-end anastomosis. MAIN OUTCOME MEASURES: Radiological, bacteriological, angiographic, and morphometric results. RESULTS: Absolute retrograde pressure-competence and free orthograde permeability of the NVA resulted in significantly lower intestinal bacterial counts compared with the end-to-end anastomosis (p < 0.05). Transposition of colonic mucosa was successful as demonstrated by revascularisation from the ileal nipple and looked normal on morphometry. CONCLUSION: If NVA were constructed in patients with Crohn's disease, recurrences should be prevented, which would verify the immunopathogenetic hypothesis of new inflammation.

Complete shutdown of microvascular perfusion upon hepatic cryothermia is critically dependent on local tissue temperature.

Since microvascular dysfunction with complete circulatory arrest and, thus, prolongation of tissue ischaemia is considered a potential mechanism for cell necrosis following hepatic cryosurgery, we determined the temperature necessary for induction of complete nutritive perfusion failure in cryothermia-treated rat livers. After localization of the cryoprobe with seven thermocouples and application of a single or double freeze-thaw cycle, in vivo fluorescence microscopy of the cryoinjured left lobe was performed over a 2-h period using a computer-controlled stepping motor, which guaranteed analysis of the identical liver tissue segments with exact allocation of the thermocouples and thus determination of tissue temperature. Cryothermia resulted in a central non-perfused part of injury, surrounded by a heterogeneously perfused peripheral zone. The non-perfused area after single and double freezing continuously increased over the first 90-min period due to a successive shutdown of perfusion within the peripheral border zone. Analysis of the thermocouples' temperature at the end of freezing revealed the 0 degrees C-front at 11.7 mm (single freeze-thaw cycle) and 12.1 mm (double freeze-thaw cycle) distant from the centre of the cryoprobe, which exactly corresponds with the initial (30 min) expansion of the area with nutritive perfusion failure. The increased non-perfused tissue area at 2 h conformed a critical border temperature between 8.29 +/- 1.63 degrees C and 9.07 +/- 0.24 degrees C. From these findings, we conclude that freezing of liver tissue to temperatures of at least < 0 degrees C causes complete/irreversible perfusion failure, which consequently will result in cell death and tissue necrosis, and may thus be supposed as a prerequisite for the safe and successful application of cryosurgery in hepatic tumour ablation.

Granulocyte elastase and systemic cytokine response after laparoscopic-assisted and open resections in Crohn's disease.

PURPOSE: The aim of this study was to assess whether systemic proinflammatory cytokines (IL-6), anti-inflammatory cytokines (IL-4, IL-10), acute phase proteins (C-reactive protein), or granulocyte elastase are valuable indicators for determining the degree of surgical trauma after open vs. laparoscopic-assisted resections in Crohn's disease. METHOD: Eleven patients in each group (open and laparoscopic-assisted surgery) were matched for indication, surgical procedure, and Crohn's disease activity index. Serum IL-4, IL-6, and IL-10 were measured using enzyme-linked immunosorbent assay. Serum C-reactive protein was determined by immunoturbidimetric assay. Plasma granulocyte elastase was determined by immunoactivation immunoassay. Blood was sampled preoperatively, six hours after the operation, and at postoperative Days 1 to 5. RESULTS: IL-4 was not detectable in any sample analyzed. Serum IL-6 and IL-10 levels peaked postoperatively in both groups without significant differences between laparoscopic-assisted (185.6 +/-54.1 pg/ml and 112.1 +/- 19.4 pg/ml, respectively; mean +/-standard error of the mean) and open surgery (431.1 +/-240.4 pg/ml and 196.7 +/- 56.5 pg/ml, respectively). Serum C-reactive protein levels also rose postoperatively, with a peak on the second day, but showed similar values after laparoscopic-assisted (107.1 +/- 12.1 mg/l) and open (128.3 +/- 17.5 mg/l) surgery. Plasma granulocyte elastase levels peaked on the first and second postoperative day and were found elevated almost throughout the five-day observation period. Comparison between the groups revealed significantly (P < 0.02) lower values after laparoscopic-assisted (Day 1, 46.5 +/- 8.9 microg/l; Day 2, 41.9 +/- 5.9 microg/l) when compared with open surgery (Day 1, 89.7 +/- 13.8 microg/l; Day 2, 91.4 +/- 14). CONCLUSIONS: Serum IL-6 and IL-10 may not be ideal measures for evaluation of the degree of tissue trauma in laparoscopic-assisted and open resections in Crohn's disease, probably because of interference with disease-specific cytokine interactions. In contrast, granulocyte elastase has to be considered a strong marker discriminating the different severity of surgical trauma induced by laparoscopic-assisted vs. open resection in Crohn's disease.

Pattern of mucosal adaptation in acute and chronic pouchitis.

PURPOSE: Variant pathological changes have been observed in ileoanal pouches, including inflammation, villous atrophy, and crypt hyperplasia. Therefore, we investigated the type and degree of mucosal adaptation in patients with ulcerative colitis and familial adenomatous polyposis. METHODS: Forty-two patients with ulcerative colitis and 14 patients with familial adenomatous polyposis with ileoanal pouches were assessed. Samples were taken from three months to eight years after creation of an ileoanal pouch. Mucosal architecture was examined by morphometry after microdissection. RESULTS: Structural changes of the mucosa can be categorized into three groups. Compared with preoperative values, patients without pouchitis (73 percent) has only minor decrease of villous length (402 microm vs. 540 microm) and increase in crypt depth (274.5 microm vs. 177 microm). In patients with acute pouchitis (20 percent), a slight increase in villous length (477 microm vs. 402 microm) and pronounced crypt hyperplasia (376 microm vs. 274.5 microm) was observed compared with noninflamed ileoanal pouches. In contrast, in patients with chronic pouchitis (7 percent), severe villous atrophy (62.5 microm) and crypt hyperplasia (543 microm) was found. CONCLUSIONS: Minor structural changes of ileoanal pouch mucosa develop early as an adaptive response to a new environment. Only in a small group of patients with chronic pouchitis does severe villous atrophy and crypt hyperplasia of the ileoanal pouch mucosa develop, most likely as a consequence of mucosal inflammation.

ET-1 induces pancreatitis-like microvascular deterioration and acinar cell injury.

Using in vivo microscopy red blood cell (RBC) velocities, functional capillary density (FCD) and capillary diameters were estimated after inducing acute pancreatitis by intraductal infusion of sodium taurocholate (0.8 ml; 4%) or after topical superfusion of the pancreas with ET-1 (100 pmol). Sodium taurocholate mediated a significant decrease in RBC velocities between 50 and 70%, transient decrease in capillary diameters by 10%, and a sustained decrease in FCD between 60 and 70% paralleled by a dramatic heterogeneity in blood flow. Topical superfusion of the exteriorized pancreas with ET-1 caused a significant decrease in RBC velocities between 65 and 75%, a sustained decrease in capillary diameters by 10%, and a decrease in FCD by 45% accompanied by an increase in flow heterogeneity. Following sodium taurocholate infusion pancreas histology revealed a severe edema and sublobular acinar cell necrosis, while topical ET-1 application displayed a severe edema of the pancreas with focal acinar cell necrosis. Thus, ET-1 mediated a deterioration of the pancreatic microcirculation, which is similar to the microcirculatory failure found in sodium taurocholate-induced experimental pancreatitis and was associated with focal acinar cell necrosis. We are thus inclined to hypothesize that endothelin released by injured endothelial cells during acute biliary pancreatitis promotes microcirculatory failure and ischemia in acute pancreatitis, eventually leading to acinar cell necrosis.

[Laparoscopic-transperitoneal and lumboscopic-retroperitoneal surgery of the spine. Developments from animal experiments for use in clinical practice]. / Die laparoskopisch-transperitoneale und lumboskopisch-retroperitoneale Chirurgie der Wirbelsäule. Entwicklungen im Tierexperiment zur Anwendung in der klinischen Praxis.

With the use of an in vivo porcine training model we established the transperitoneal laparoscopic approach for the instrumentation of anterior lumbar spine fusion with Bagby-and-Kuslich (BAK) interbody implants as well as "Brantigan" cages. The transperitoneal laparoscopic approach caudally from the aortic bifurcation allows the spine fusion procedure of the caudal but not of the cranial part of the lumbar spine. Because ventral stabilization of the upper lumbar spine is frequently necessary, in particular in trauma patients with spine body fractures, an additional retroperitoneal minimal-invasive (lumboscopic) approach was established using again the in vivo porcine training model. We demonstrate that via this approach spine fusion can easily be performed including the Th12 segment after fenestration of the diaphragm. With the experience from the in vivo experiments, both techniques could safely and successfully be transferred to clinical practice with the advantage of markedly reducing the extent of operative trauma compared with the corresponding open approaches.

High rates of appendicectomy in a developing country: an attempt to contribute to a more rational use of surgical resources.

OBJECTIVE: In view of the high appendicectomy rate in a university teaching hospital in a developing country, we investigated the regional epidemiology of appendicitis. DESIGN: Prospective clinical and pathological study. SETTING: Teaching hospital, Madagascar. SUBJECTS: 165 patients who underwent appendicectomy during a two month period. INTERVENTIONS: Clinical investigation and histopathological examination of removed appendixes. MAIN OUTCOME MEASURES: Appendicitis score, local incidence of appendicectomy, appendicitis, perforation, and negative appendicectomy. RESULTS: There was a high incidence of primary appendicectomy (51.1/10,000 population/year) in relation to the incidence of histologically confirmed appendicitis (7.7/10,000), resulting in a negative appendicectomy rate of 85% (43/10,000). There were considerable sex related differences in the incidence of primary appendicectomy (81/10,000 women/year; 22/10,000 men), appendicectomy for appendicitis (12/10,000 women/year; 4/10,000 men) and non-perforating appendicitis (8/10,000 women/year; 2/10,000 men), but much less in that of perforating appendicitis (4/10,000 women/year; 2/10,000 men). CONCLUSION: There is potential for more economic use of medical resources by reducing the rate of negative appendicectomy. The incidence of non-perforating appendicitis depends on the number of appendicectomies done, which may explain the differences observed in the incidence of appendicitis between industrialised and developing countries.

IgG, albumin, and sCD44 in whole-gut lavage fluid are useful clinical markers for assessing the presence and activity of pouchitis.

Pouchitis is the most significant long-term complication in patients with ileoanal pouch anastomosis (IAP) and is especially frequent in patients with ulcerative colitis. There is an urgent need for simple and objective parameters to assess the presence and activity of pouchitis. Whole-gut lavage fluid (WGLF) was collected from 34 patients [8 with pouchitis (PDAI > or = 7 points) and 26 without pouchitis (Pouchitis Disease Activity Index, PDAI, < 7)]. Patients with active ulcerative colitis (n = 8) served as controls. Concentrations of IgG and sCD44 in WGLF were measured by enzyme-linked immunosorbent assays and those of albumin by immunoturbidimetry. Similar to the case in active ulcerative colitis, concentrations of IgG, albumin, and sCD44 in WGLF were significantly increased in acute pouchitis and reached high specificity (IgG 96%, albumin 96%, sCD44 100%) and acceptable sensitivity (75%) for the diagnosis of acute pouchitis. These parameters were also closely correlated with disease activity as determined by PDAI and endoscopic scoring indices. Assay of protein concentrations in WGLF is thus a simple and objective means for grading inflammation of the pouch and may be useful as a quantitative index of disease activity in clinical studies.