Günther Winde

Complete reversion and prevention of rectal adenomas in colectomized patients with familial adenomatous polyposis by rectal low-dose sulindac maintenance treatment. Advantages of a low-dose nonsteroidal anti-inflammatory drug regimen in reversing adenomas exceeding 33 months.

PURPOSE: This nonrandomized, controlled Phase II pilot study aims at the lowest effective dose of rectally applied sulindac to achieve and maintain adenoma reversion in colectomized patients with familial adenomatous polyposis (FAP). METHODS: The study group (n = 15) underwent proctoscopic and laboratory follow-up for polyp reversion every 6 to 12 weeks. Polyp reversion was followed by dose reduction in predefined steps. Proliferating cell nuclear antigen/cyclin (PCNA) and KI-67 proliferation indices (PI) were performed by point counting. Prostaglandin (PG)E2 and PGF2α were quantified by time-resolved competitive fluorescence immunoassay. RESULTS: All patients responded to therapy within 6 to 24 weeks. Sixty and 87 percent of patients achieved complete adenoma reversion after 48 weeks at 53 and 67 mg of sulindac per day per patient on average, respectively. Reversion was evident compared with the control group. Dose reduction by one-sixth to one-eighth of the usual oral dose was significant (Manns trend test,P < 0.05). PCNA and KI-67 Pls of adenomatous and flat mucosa were significantly reduced (Wilcoxons test,P<0.05). Correlation of PCNA and KI-67 Pls indicate similar reaction of different tissue structures (Spearmans rank correlation test,P<0.01). Nonsteroidal anti-inflammatory drug-induced redifferentiation from high-grade to low-grade dysplasia occurred in all but two patients. Tissue-PGE2 levels were greatly reduced. Unwanted, curable side effects were rare (gastritis,n=2), and laboratory controls are within detection limits. CONCLUSIONS: Low-dose rectal sulindac maintenance therapy is highly effective in achieving complete adenoma reversion without relapse in 87 percent of patients after 33 months. Rectal FAP phenotype should be crucial for the surgical decision. Colectomy with ileorectal anastomosis and regular chemoprevention might proceed to be a promising alternative to pouch procedures. Chemoprevention with lower incidence of FAP-related tumorsviadysplasia reversion may be possible in the future.

GNAS1 T393C Polymorphism and Survival in Patients with Sporadic Colorectal Cancer

Purpose: Signaling via the G protein Gαs pathway is linked to proapoptotic processes in cancer cell lines. We have recently shown an association between the GNAS1 T393C polymorphism and disease progression in patients with bladder cancer with homozygous TT genotypes displaying increased transcription of Gαs and a more favorable clinical course compared with C-allele carriers. Experimental Design: In the present study, 151 patients with sporadic colorectal cancer were retrospectively genotyped to examine a potential association between T393C genotypes and survival. Moreover, two other single-nucleotide polymorphisms in common haplotype blocks within the gene GNAS1 and their interaction with the T393C polymorphism were investigated. Results: The allele frequency in the patients group was not significantly different from that of healthy blood donors. Kaplan-Meier curves for overall survival (mean follow-up, 43 months) showed that in International Union Against Cancer (UICC) stages I to II, the 5-year survival rate was significantly higher in TT genotypes (87.8%) compared with TC (71.0%) and CC genotypes (50.0%; P = 0.009), whereas no genotype effect could be observed for UICC stages III to IV. In multivariate Cox proportional analysis the T393C polymorphism was an independent prognostic factor for survival. Homozygous CC patients were at highest risk for death (hazard ratio, 12.1; P = 0.006) compared with TT genotypes. Heterozygous patients had an intermediate risk compatible with a gene-dose effect. The two haplotype blocks investigated were not associated with clinical outcome. Conclusions: The results support the role of the T393C polymorphism as a marker for survival in patients with colorectal cancer stages I to II and in the identification of patients who may benefit from adjuvant chemotherapy.

Colon carcinoma cell lines stimulate monocytes and lamina propria mononuclear cells to produce IL-10

Cytokines released from tumour cells may have function as signals to neighbouring immune and inflammatory cells. Several studies have shown that the immunoregulatory cytokines IL‐10 and transforming growth factor‐beta 1 (TGF‐β1) as well as prostaglandin‐E2 (PGE2) play an important role in tumour‐induced immunosuppression. The aim of the study was to investigate the effect of colon carcinoma cell lines on IL‐10 production in peripheral monocytes (PBMC) and lamina propria mononuclear cells (LPMC). We examined four colon carcinoma cell lines (HT‐29, Caco‐2, Colo‐320 and HCT‐116) and determined their production of TGF‐β1, IL‐10 and PGE2. Peripheral monocytes were isolated by density gradient centrifugation and LPMC were isolated from surgical specimens using a collagenase digestion method. Monocytes and LPMC were cultured with colon carcinoma cell conditioned medium or in co‐culture with colon carcinoma cells. Supernatants were then determined for the production of IL‐10 by ELISA assays. All colon carcinoma cell lines stimulated peripheral monocytes as well as LPMC to produce markedly increased levels of IL‐10. Colon cancer cells secreted negligible levels of IL‐10, but high amounts of TGF‐β1 and PGE2. Neutralization of TGF‐β1 by administration of anti‐TGF‐β as well as neutralization of PGE2 with anti‐PGE2 antisera reduced the IL‐10 production of monocytes markedly, indicating that tumour cell‐derived TGF‐β1 and PGE2 are major factors for IL‐10 stimulation. In vitro stimulation of monocytes with TGF‐β1 and PGE2 could confirm that TGF‐β1 as well as PGF2 at picogram concentrations were able to prime monocytes for enhanced IL‐10 production. Our results demonstrate that colon carcinoma cell lines enhance the ability of monocytes and intestinal macrophages to produce IL‐10. The stimulation of monocyte IL‐10 by colon cancer cell‐derived TGF‐β1 and PGE2 may act as a tumour‐protecting mechanism by impairing the activation of anti‐tumour cytokines.

Early Identification of Peritonitis by Peritoneal Cytokine Measurement

AbstractPURPOSE: The assessment of plasma cytokine levels adds a useful tool to the diagnostic measures in severe inflammatory diseases. Proinflammatory cytokine levels in abdominal fluid after abdominal surgery have been shown to far exceed plasma cytokine levels. Thus, we investigated the local release of interleukin 1β, interleukin 6, and tumor necrosis factor-α in patients after colorectal surgery during the early postoperative period to evaluate whether it may serve as an indicator of evolving peritonitis. METHOD: In a prospective, observational pilot study, the first 12 consecutive patients who did not develop any postoperative complications (Group I), and the first 12 patients with secondary peritonitis caused by an anastomotic leakage (Group II), were included in the study. Interleukin 6, interleukin 1β, and tumor necrosis factor-α levels were determined in the abdominal exudate and compared between the groups within the first four days after colorectal surgery. RESULTS: Abdominal fluid interleukin 6 levels in Group II patients were higher (162,500 ± 105,800 pg/ml) as early as the first postoperative day compared with Group I (27,940 ± 13,860 pg/ml; P < 0.0001); this lasted for the whole observation period. The same applies to tumor necrosis factor-α levels (461.4 ± 167.8 pg/ml vs. 175.8 ± 178.6 pg/ml on day 1; P = 0.0007). The difference in interleukin 1β cytokine levels became statistically significant on the third postoperative day. Moreover, abdominal fluid cytokine levels rose in Group II, whereas they remained virtually unchanged or even tended to decrease over time in Group I. CONCLUSION: We suggest that the estimation of the peritoneal cytokine levels might be an additional diagnostic tool that can support the early recognition of peritonitic complications in colorectal surgery.

IL-10 Synergizes with IL-4 and IL-13 in Inhibiting Lysosomal Enzyme Secretion by Human Monocytes and Lamina Propria Mononuclear Cells from Patients with Inflammatory Bowel Disease

Tissue injury and inflammation in inflammatorybowel disease (IBD) are associated with enhancedmonocytic lysosomal enzyme release. In this study,peripheral monocytes and lamina propria mononuclearcells (LPMNC) were isolated from IBD patients andnormal controls. Cells were stimulated withlipopolysaccharide after treatment with IL-13, IL-4, andIL-10, and enzyme secretion was assessed by using thecorresponding p-nitrophenyl glycosides as substrates.Molecular forms of cathepsin D were examined to describethe mode of enzyme release. IL-10 and IL-4 stronglydown-regulate enzyme secretion in IBD monocytes. IBD monocytes showed a diminished responsiveness tothe inhibitory effect of IL-13. Impaired monocyteresponse was not found with combinations of IL-13 andIL-10 or IL-4 and IL-10. LPMNC from involved IBD mucosa showed significantly higher enzyme secretioncompared with LPMNC from noninvolved IBD mucosa butresponded inefficiently to either IL-4, IL-13, or IL-10alone. However, combined treatment with IL-10 and IL-4 or IL-10 and IL-13 strongly suppressedenzyme release by these cells. Both the precursor andmature forms of cathepsin D were elevated in IBDpatients. While IL-13 reduced mainly the precursor form, the effect of IL-4 and IL-10 concerns both theprecursor and mature form of cathepsin D. Our resultsfavor the potent clinical utility of combined treatment,thus improving chances of developing effective treatments for human IBD.

Immunohistochemical demonstration of the calcium-binding proteins MRP8 and MRP14 and their heterodimer (27E10 antigen) in Crohn's disease

Monospecific antibodies against the calcium-binding proteins MRP8 and MRP14 and their heterodimer MRP8/14 (epitope 27E10) were used to investigate immunohistochemically the distribution of these proteins in routinely processed small and large bowel tissues from patients with Crohns disease. MRP8, MRP14, and complex MRP8/14 were demonstrated in most granulocytes and macrophages in active Crohns disease. Additionally, a strong complex MRP8/14 immunoreactivity was present in epithelial cells of the terminal ileum adjacent to ulcerative and fissuring lesions, whereas epithelial cells in large bowel tissues were consistently negative. Our results morphologically confirm the clinical finding of increased MRP8/14 serum levels in patients with active Crohns disease; there is evidence that the serum MRP8/14 increase is caused by active secretion from granulocytes, monocytes, and epithelial cells.

Combined analysis of hypoxia-inducible factor 1 alpha and metallothionein indicates an aggressive subtype of colorectal carcinoma

PurposeHypoxia-inducible factor 1 (HIF-1) is a hypoxia-induced transcription factor that regulates gene expression in critical pathways involved in tumour growth and metastasis. Metallothionein (MT) is a group of small molecular weight cysteine-rich proteins with a broad variety of functions. The present study aimed to analyse the prognostic impact of HIF-1α and MT expression in colorectal cancer and to evaluate a possible link of combined HIF-1α and MT expression with colorectal cancer progression.Materials and methodsWe investigated the relationship of HIF-1α and MT with each other and clinicopathological parameters including proliferative activity (Ki67) and apoptosis (terminal desoxyribonucleotide transferase-mediated dUTP nick-end labelling) using immunohistochemistry.ResultsHIF-1α expression was identified as an independent prognostic parameter in multivariate survival analysis and characterised an aggressive cancer phenotype. In addition, HIF-1α was significantly linked to an increased expression of MT.ConclusionsHIF-1α expression qualified as an independent prognostic and characterised an aggressive cancer phenotype associated with an increased expression of MT. Our study suggests that MT can be added to the complex biological pathways induced by hypoxia in human cancer tissue.

sTNF-RII: is it useful for the early diagnosis of rejection and for prognosis after renal transplantation?

Abstract Changes in soluble tumour necrosis factor receptor II (sTNF‐RII) correlate with transplant rejection, and it increases in the course of sepsis. These changes might help to identify rejection early, and thus lead to more effective treatment. Serum and urine sTNF‐RII levels were measured in 70 patients during the first 3 weeks after kidney transplantation and correlated with clinical and laboratory findings. Retrospectively, three groups were identified: I. stable transplant function (n= 23), II. at least one rejection episode (n=38) and III. other complications (infection or reperfusion injury) (n= 9). The pre‐operative maximum for serum sTNF‐RII was 22.4±10.7 ng/ml. In group I it decreased to 9.5±6.7 ng/ml on day 6 after transplantation (P<0.01), while in group II sTNF‐RII serum levels were significantly higher on day 6 (24.9±15.0 ng/ml, P<0.01). High levels of sTNF‐RII in serum (>40 ng/ml for at least 2 days) predicted a higher risk of an unfavourable outcome. High serum levels of sTNF‐RII are not specific but seem to be a prognostic indicator of a complicated course; sTNF‐RII in urine has no diagnostic value.

Density gradient centrifugation of colonic fluid after segmental lavage: a method of purification of exfoliative epithelial colonic cells for cytological interpretation and image cytometry in patients with long-standing ulcerative colitis

Objectives:Patients with extensive, long-standing ulcerative colitis (UC) have an increased risk for developing colorectal cancer. In this study, we wanted to establish a method for retrieving cytological material after segmental colonic lavage for further cytopathological investigations and for performing DNA image cytometry.Methods:Ten patients with long-standing and extensive ulcerative colitis and 10 patients without macroscopic abnormalities were investigated. After segmental colonic lavage during routine colonoscopy a three-layer (1.146, 1.075, and 1.046 g/ml, respectively) density gradient centrifugation of the retrieved colonic fluid was performed for isolation and purification of the epithelial cells. For identification of the epithelial cells flow cytometry with monoclonal antibody against cytokeratin and counterstaining with propidium iodine was performed. The smears obtained were stained for routine cytopathological interpretation and for DNA image cytometry.Results:In eight of 10 UC patients and in nine of 10 control group patients adequate cytological material could be obtained. The band on top of the density gradient at 1.046 g/ml could be identified as the epithelial cells. Atypical cells were found in smears of three UC patients. In these patients and in one additional patient aneuploid stemlines could be detected. In smears of control group patients neither atypical cells nor aneuploidy were present.Conclusions:Isolation and purification of epithelial cells after segmental colonic lavage by using density gradient centrifugation was performed. This cytological material is adequate for cytopathological interpretation and for DNA image cytometry. Information about atypical cells and DNA aneuploidy as an additional marker of malignant transformation in UC patients was obtained. The combination of cytological examination and DNA image cytometry might improve the detection of UC patients with high risk for colorectal cancer.

Outcome of surgical intervention for rectoneovaginal fistulas in Mayer-Rokitansky-Kuester-Hauser syndrome.

Creation of a neovagina to treat vaginal atresia or aplasia in Mayer-Rokitansky-Kuester-Hauser syndrome must always be followed by long-term application of dilators to avoid shrinkage. However, rectoneovaginal fistulas are caused by chronic alteration and consecutive necrosis of the posterior neovaginal wall. We evaluated retrospectively the postoperative outcome of rectal wall and neovaginal reconstruction using a standardized surgical technique in an exclusive collection of women. Eight women with a mean age of 28 years (range 22–31 years) were treated for rectoneovaginal fistulas in our clinic. Preoperatively, proctoscopy, sphincter manometry, endoluminal rectal ultrasonography, and colonoscopy were performed; and regular postoperative follow-up by digital examination and rectoscopy were obligate. The standard surgical procedure via a perineal approach included fistulectomy and closure of the mucosa and rectal wall followed by a levatorplasty. All but one woman had a temporary colostomy. After 2 weeks the patients were allowed to wear vaginal dilators of a smaller size. Within the mean follow-up period of 20 months, reintervention was necessary twice because of late fistula relapse detected by proctoscopy, barium enema, and subjective symptoms. Morbidity was 25% (n= 2) due to secondary superficial wound healing or urinary tract infection. The average time of the hospital stay was 13 days (10–14 days). One patient complained of vaginal shrinkage and underwent local estrogen therapy with a good functional result 3 months later. Proper fistulectomy and surgical reconstruction with interpositioning of well perfused muscle layers achieved good functional outcome with an acceptable number of minor morbidities. Local estrogen treatment is helpful for avoiding scarification and decreasing the neovaginal size.