Glycemic response to low sugar apple juice treated with invertase, glucose oxidase and catalase.

BACKGROUND/OBJECTIVES: Investigating the effect on post-prandial glycemic and venous serum insulin response of an apple drink following the conversion of its glucose to gluconate. SUBJECTS/METHODS: In a double-blind randomized placebo-controlled clinical trial with cross-over design, 30 male adults with impaired fasting glucose (IFG) received a drink of 500 ml: 1. Verum: Apple juice treated with invertase, glucose oxidase/catalase (glucose 0.05 g; gluconate 18.2 g); 2. CONTROL: Untreated apple juice (free glucose 8.5 g; bound glucose 6.7 g; gluconate below detection limit). Postprandial fingerprick capillary blood glucose and venous serum insulin were measured twice at baseline and at times 0 (start of drink), 15, 30, 45, 60, 90 and 120 min. Gastrointestinal symptoms, stool consistency and satiety were also assessed. RESULTS: The incremental area under the curve (iAUC120) of glucose levels (primary parameter) was significantly lower after verum (mean ± SD: 63.6 ± 46.7 min × mmol/l) compared to control (mean ± SD: 198 ± 80.9 min × mmol/l) (ANOVA F = 137.4, p < 0.001; α = 0.05). Also, iAUC120 of venous serum insulin levels (secondary parameter) was significantly lower after verum (mean ± SD: 2045 ± 991 min × mmol/l) compared to control (3864.3 ± 1941 min × mmol/l), (ANOVA F = 52.94, p < 0.001; α = 0.025). Further parameters of glucose metabolism and ISI = 2/[AUC venous serum insulin × AUC glucose +1] were also improved after verum compared to control. Verum increased stool frequency and decreased stool consistency, as assessed by Bristol stool form scale. CONCLUSIONS: By enzymatic treatment of apple juice its sugar content could be reduced by 21% and postprandial glycemic and venous serum insulin response by 68 and 47%, respectively resulting in a reduction of glycemic load by 74.6% without any adverse gastrointestinal side-effects.

Dipeptidase 1 (DPEP1) is a marker for the transition from low-grade to high-grade intraepithelial neoplasia and an adverse prognostic factor in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Improvements in the understanding of its molecular mechanism and the characterisation of CRC-specific biomarkers facilitating early detection are considered to increase overall survival. METHODS: A meta-analysis of microarray and Serial Analysis of Gene Expression (SAGE) has been performed to identify differentially regulated genes in CRC. Dipeptidase 1 (DPEP1/MDP/RDP) and Syntenin-2 (SDCBP2/SITAC18) were found to be differentially expressed in tumour tissue compared with normal mucosa. Expression of DPEP1 was assessed in a validation set of 87 normal mucosa samples, 20 hyperplastic polyps, 46 CR adenomas with low- and high-grade intraepithelial neoplasia (IEN) and 217 well-documented CRCs by immunohistochemistry and partially by immunoblotting and real-time PCR. RESULTS: Expression of DPEP1 was specifically increased in human CRC tissue samples compared with normal mucosa (P<0.0001, Mann-Whitney U-test), showing a striking upregulation in high-grade compared with low-grade IEN. Furthermore, high DPEP1 expression was found to strongly correlate with histological stage (P<0.0001, chi-square test) as well as localisation (P<0.0001, chi-square test) and has been recognised as an independent adverse prognostic factor, showing significant prognostic values with an ROC (receiver operating characteristic)-AUC of 0.9230. CONCLUSION: Dipeptidase 1 has been identified as an excellent marker of high-grade IEN and CRC, and may thus be applied for screening of early neoplastic lesions and for prognostic stratification.

Controlled ribozyme targeting demonstrates an antiapoptotic effect of carcinoembryonic antigen in HT29 colon cancer cells.

PURPOSE: Clinical studies suggest that carcinoembryonic antigen (CEA) is associated with metastatic progression of colon cancer. However, the biological function of CEA is not well understood. We have established an approach that allows studying of CEA function within the intact pathophysiological context of human colon cancer cells. EXPERIMENTAL DESIGN: We expressed CEA-targeted ribozymes under control of a tet-off promoter system in human HT29 colon cancer cells. This approach allows regulation of CEA levels on the mRNA and protein level by 50% and enables screening analysis of CEA-mediated changes of gene expression by cDNA microarray analysis. RESULTS: Comprehensive analysis of 273 genes revealed that CEA affects expression of various groups of cancer-related genes, in particular cell cycle and apoptotic genes. Although cell cycle gene expression showed a balanced bidirectional dysregulation, apoptotic genes were unidirectionally down-regulated by CEA. In parallel phenotypic studies, CEA did not affect cell cycle or proliferation rate. However, CEA significantly protected HT29 cells from undergoing apoptosis under various conditions, including confluent growth, UV light, IFN-gamma treatment, and treatment with 5-fluorouracil. CONCLUSIONS: Our study suggests that CEA has an important regulatory role in apoptosis, and we propose that CEA is a survival factor for colon cancer cells.

The carcinoembryonic antigen and its prognostic impact on immunocytologically detected intraperitoneal colorectal cancer cells.

BACKGROUND: Carcinoembryonic antigen (CEA) has been suggested to promote colon cancer progression. In this study we analyzed the prognostic impact of CEA expression on intraperitoneally detected single colon cancer cells. METHODS: Peritoneal lavage samples of 135 colorectal cancer patients were immunocytologically analyzed, including a staining of cellular CEA; serum CEA levels were measured; and 5-year survival rates were calculated according to immunocytological findings and CEA expression. RESULTS: The worst survival rate of 20% was found in patients suffering from CEA-expressing intraperitoneal tumor cells (P = 0.0006). The prognostic impact of an intraperitoneal tumor cell finding significantly increased when serum CEA levels were elevated: only 23% survived 5 years in contrast to a 85% 5-year survival rate of patients who neither had signs of dissemination nor showed elevated serum CEA values (P = 0.0010). CONCLUSIONS: This study shows that the determination of CEA expression improves the prognostic impact of an intraperitoneal tumor cell finding.

Disseminated tumor cells in pancreatic cancer patients detected by immunocytology: a new prognostic factor.

Using an immunocytological approach, we previously showed that disseminated cancer cells are frequently found in peritoneal cavity and bone marrow samples of gastrointestinal and pancreatic cancer patients. Recently, we demonstrated that the detection of isolated tumor cells could serve as a new prognostic factor in gastric and colorectal cancer. Thus far, no conclusive data concerning the clinical implication of minimal residual disease in pancreatic cancer exist. In this study, we investigated peritoneal lavage and bone marrow samples of 80 pancreatic cancer patients to determine the predictive value of immunocytologically detected disseminated tumor cells. Therefore, immunocytological findings were correlated with the clinical follow-up data (median observation time, 10.7 months; range, 2-61 months), and the findings in peritoneal cavity and bone marrow samples were compared. Fifty-two % of the patients showed minimal residual disease at least in one compartment (39% positive lavage and 38% positive bone marrow samples). The detection rate of isolated tumor cells increased in parallel to the tumor stage. The presence of tumor cells in the peritoneal cavity significantly correlated with the survival time of the patients (P = 0.0035). In bone marrow samples, a strong trend was seen (P = 0.06). The evaluation of both compartments increased the number of positive patients and resulted in a highly significant correlation: all patients who were positive in at least one compartment died within 18 months, whereas negative patients showed a 5-year survival rate of 30% (P<0.0001). We recommend immunocytological investigation of peritoneal cavity and bone marrow samples as a new prognostic marker in pancreatic cancer patients.

A human carcinoma model in athymic rats reflecting solid and disseminated colorectal metastases.

We studied the metastatic properties of human tumor cells and tumor cell dissemination in a xenograft tumor model for human colorectal carcinoma in athymic rats which shows a reproducible pattern of metastases similar to the clinical situation. Such a model is also attractive for evaluating several therapeutic approaches. The tumor cell lines HT-29 and WiDr which are derived from the same colorectal tumor and exhibit a similar tumorigenic potential after subcutaneous injection were injected into the portal venous system of 4-week-old male nude rats. After injection of WiDr cells no liver metastases were observed; however, 50% of the rats developed liver metastases 4-12 weeks after injection of HT-29. Immunostaining of the liver cryosections at different times after injection revealed a total disappearance of WiDr cells within the first 12 h. A subpopulation of HT-29 (HT29-b) with increased metastatic activity was isolated by double selection and recultivation of cells from induced liver metastases. After a 6- to 12-week period rats injected with HT-29b showed a pattern of metastases with additional lung metastases and in some cases peritoneal carcinosis. In addition to immunohistochemistry cytokeratin 20 reverse transcriptase-polymerase chain reaction was confirmed to be a sensitive and specific tool for the detection of disseminated tumor cells in different compartments.

Comparative analysis of bone marrow and venous blood isolates from gastrointestinal cancer patients for the detection of disseminated tumor cells using reverse transcription PCR.

We investigated 141 bone marrow and 104 venous blood isolates from gastrointestinal cancer patients with a cytokeratin (CK) 20-specific nested reverse transcription PCR for the detection of disseminated tumor cells at time of primary tumor resection. In colorectal cancer patients, 20 of 65 (31%) bone marrow and 9 of 52 (17%) venous blood isolates yielded a CK 20 mRNA-positive result in a stage-dependent manner. The detection rates for gastric cancer patients were 11 of 49 (22%) and 5 of 30 (17%) for bone marrow and venous blood, respectively. In pancreatic cancer patients, positive signals were found in advanced tumor stage. A duplex PCR system improved the feasibility of the test. After analyzing 70 sets of bone marrow and venous blood isolates from colorectal, gastric, and pancreatic cancer patients, we observed a higher detection rate in bone marrow isolates. Survival of patients with CK 20 mRNA-positive findings was significantly shorter than that of negatively tested patients.

Differential expression and function of CD80 (B7-1) and CD86 (B7-2) on human peripheral blood monocytes.

The interaction of CD28 with its ligands is important for T-cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7-1) and CD86 (B7-2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80-negative. CD80 expression is weakly induced after 6-8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4-6 hr in stimulated cells. Reverse transcription-polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80-mRNA. The mRNA of CD80 is induced after 4-6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti-CD80 and CTLA4Ig could partially inhibit antigen-specific (tuberculin) and polyclonal (anti-CD3) lymphoproliferation and interferon-gamma (IFN-gamma) secretion of T cells cocultured with autologous monocytes. IFN-gamma secretion was more sensitive to blocking costimulation than proliferation. The antibody BB-1 did not inhibit proliferation and cytokine secretion, nor did the anti-CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti-CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.

The detection of disseminated tumor cells in bone marrow from colorectal-cancer patients by a cytokeratin-20-specific nested reverse-transcriptase-polymerase-chain reaction is related to the stage of disease.

The dissemination of cancer cells is a prerequisite in the development of micrometastases and solid metastases. Our previous examinations of these cells were based on immunocytological staining of tumor-associated antigens and cytokeratins. We have now developed a highly sensitive and specific detection method based on a nested reverse-transcriptase-polymerase-chain reaction (RT-PCR) of cytokeratin-20 (CK-20) mRNA. Using this method, we examined the bone marrow of 57 patients with colorectal cancer and detected increasing numbers of CK-20-positive samples, depending on the UICC stage. Some 35% of all bone-marrow samples tested positive for CK-20: none were found in colorectal cancer stage 1, 24% were in stage II, 31% in stage III and 71% in stage IV. Investigation of bone-marrow specimens of patients with pancreatic cancer showed that 4 out of 11 patients were positive for CK-20 mRNA. To confirm that sample positivity for CK-20 expression was due to disseminated tumor cells, we examined bone marrow from a control group (n = 16) without apparent carcinoma. In this group, 15 out of 16 donors were CK-20-negative, while one donor with familial adenomatous polyposis showed a CK-20-specific signal.