An Expert Consensus to Standardize Assessment of Bowel Cleansing for Clinical Trials of Bowel Preparations for Crohn's Disease.

BACKGROUND: Despite regular need for colonoscopy in patients with Crohn's disease (CD), the efficacy and tolerability of bowel preparation (BP) agents is rarely assessed in this population. Assessing BP quality with existing scales may be challenging in CD due to presence of inflammation, bowel resection, and strictures. AIMS: To provide recommendations for assessing BP quality in clinical trials for CD using a modified Research and Development/University of California, Los Angeles appropriateness process. METHODS: Based on systematic reviews and a literature search, 110 statements relating to BP quality assessment in CD were developed. A panel of 15 gastroenterologists rated the statements as appropriate, uncertain, or inappropriate using a 9-point Likert scale. RESULTS: Panelists considered it appropriate that central readers, either alone or with local assessment, score BP quality in clinical trials. Central readers should be trained on scoring BP quality and local endoscopists on performing high-quality video recording. Both endoscope insertion and withdrawal phases should be reviewed to score BP quality in each colonic segment and segments should align with endoscopic disease activity indices. The Harefield Cleansing Scale and the Boston Bowel Preparation Scale were considered appropriate. The final score should be calculated as the average of all visualized segments. Both total and worst segment scores should also be assessed. CONCLUSIONS: We developed a framework for assessing BP quality in patients with CD based on expert feedback. This framework could support the development or refinement of BP quality scales and the integration of BP quality assessment in future CD studies.

Naïve orangutans (Pongo abelii and Pongo pygmaeus) individually acquire nut-cracking using hammer tools.

Nut-cracking with hammer tools (henceforth: nut-cracking) has been argued to be one of the most complex tool-use behaviors observed in nonhuman animals. So far, only chimpanzees, capuchins, and macaques have been observed using tools to crack nuts in the wild (Boesch and Boesch, 1990; Gumert et al., 2009; Mannu and Ottoni, 2009). However, the learning mechanisms behind this behavior, and the extent of nut-cracking in other primate species are still unknown. The aim of this study was two-fold. First, we investigated whether another great ape species would develop nut-cracking when provided with all the tools and appropriate conditions to do so. Second, we examined the mechanisms behind the emergence of nut-cracking by testing a naïve sample. Orangutans (Pongo abelii and Pongo pygmaeus) have the second most extensive tool-use repertoire among the great apes (after chimpanzees) and show flexible problem-solving capacities. Orangutans have not been observed cracking nuts in the wild, however, perhaps because their arboreal habits provide limited opportunities for nut-cracking. Therefore, orangutans are a valid candidate species for the investigation of the development of this behavior. Four nut-cracking-naïve orangutans at Leipzig zoo (P. abelii; Mage = 16; age range = 10-19; 4F; at the time of testing) were provided with nuts and hammers but were not demonstrated the nut-cracking behavioral form. Additionally, we report data from a previously unpublished study by one of the authors (Martina Funk) with eight orangutans housed at Zürich zoo (six P. abelii and two P. pygmaeus; Mage = 14; age range = 2-30; 5F; at the time of testing) that followed a similar testing paradigm. Out of the twelve orangutans tested, at least four individuals, one from Leipzig (P. abelii) and three from Zürich (P. abelii and P. pygmaeus), spontaneously expressed nut-cracking using wooden hammers. These results demonstrate that nut-cracking can emerge in orangutans through individual learning and certain types of non-copying social learning.

Recommendations for Standardizing Clinical Trial Design and Endoscopic Assessment in Postoperative Crohn's Disease.

BACKGROUND: The lack of standardized methods for clinical trial design and disease activity assessment has contributed to an absence of approved medical therapies for the prevention of postoperative Crohn's disease (CD). We developed recommendations for regulatory trial design for this indication and for endoscopic assessment of postoperative CD activity. METHODS: An international panel of 19 gastroenterologists was assembled. Modified Research and Development/University of California Los Angeles methodology was used to rate the appropriateness of 196 statements using a 9-point Likert scale in 2 rounds of voting. Results were reviewed and discussed between rounds. RESULTS: Inclusion of patients with a history of completely resected ileocolonic CD in regulatory clinical trials for the prevention of postoperative recurrence was appropriate. Given the absence of approved medical therapies, a placebo-controlled design with a primary end point of endoscopic remission at 52 weeks was appropriate for drug development for this indication; however, there was uncertainty regarding the appropriateness of a coprimary end point of symptomatic and endoscopic remission and the use of currently available patient-reported outcome measures. The modified Rutgeerts Score, endoscopic assessment of the anastomosis, and a minimum of 5cm of neoterminal ileum were also appropriate; although the appropriateness of other indices including the Simple Endoscopic Score for CD for endoscopic assessment of postoperative CD activity was uncertain. CONCLUSIONS: A framework for regulatory trial design for the prevention of postoperative CD recurrence and endoscopic assessment of disease activity has been developed. Research to empirically validate end points for these trials is needed.

Grooming network cohesion and the role of individuals in a captive chimpanzee group.

Social network analysis offers new tools to study the social structure of primate groups. We used social network analysis to investigate the cohesiveness of a grooming network in a captive chimpanzee group (N = 17) and the role that individuals may play in it. Using data from a year-long observation, we constructed an unweighted social network of preferred grooming interactions by retaining only those dyads that groomed above the group mean. This choice of criterion was validated by the finding that the properties of the unweighted network correlated with the properties of a weighted network (i.e. a network representing the frequency of grooming interactions) constructed from the same data. To investigate group cohesion, we tested the resilience of the unweighted grooming network to the removal of central individuals (i.e. individuals with high betweenness centrality). The network fragmented more after the removal of individuals with high betweenness centrality than after the removal of random individuals. Central individuals played a pivotal role in maintaining the network's cohesiveness, and we suggest that this may be a typical property of affiliative networks like grooming networks. We found that the grooming network correlated with kinship and age, and that individuals with higher social status occupied more central positions in the network. Overall, the grooming network showed a heterogeneous structure, yet did not exhibit scale-free properties similar to many other primate networks. We discuss our results in light of recent findings on animal social networks and chimpanzee grooming.

Anti-tumorigenic and anti-angiogenic effects of natural conifer Abies sibirica terpenoids in vivo and in vitro.

AIM: The natural terpenoid compound was explored in vitro and in vivo to investigate the anti-HCC properties. METHODS: For our study we used Abisilin®- a novel natural pharmacological terpenoid compound extracted and purified from coniferous Pinaceae trees. Anti-tumorigenic properties of different concentrations of Abisilin® were tested on murine hepatoma Hepa 1-6 cell lines. The analysis of proliferation and apoptosis was performed using immunofluorescence microscopy, FACS and qPCR. As an in vivo approach, we tested Abisilin® (400mg/kg/day, 14 days, orally) in xenograft mouse models of liver cancer and investigated tumor growth, proliferation, apoptosis and angiogenesis by means of Western blotting, immunofluorescence microscopy and qPCR. RESULTS: Application of Abisilin® for 24h at a dosage ranging from 0.03 to 0.045mg significantly reduced the number of viable Hepa 1-6 cells and induced apoptotic cell death with microscopic evidence of changes in cell morphology, and positive TUNEL, cleaved caspase 3 and Annexin V/Propidium Iodide (PI) stainings. Furthermore, treatment with Abisilin® strongly inhibited proliferation, impaired mitosis and prompted cell cycle arrest by down-regulation of the Cyclin D1, E1 and A2 expression levels. In Hepa 1-6 xenograft in vivo model, Abisilin® considerably decreased the xenograft tumor size and tumor volume. Consistently with in vitro Abisilin® administration elicited apoptosis and inhibit proliferation in the xenograft tumor. We also found that Abisilin® remarkably decrease microvessel density, diminished tumor angiogenesis and reduced expression of ICAM-1. Moreover, the expression of pAMPK, a cellular energy sensor, was up-regulated after Abisilin® application. CONCLUSIONS: Anti-proliferative, pro-apoptotic activity and anti-angiogenic potential of natural conifer terpenoids might turn these compounds into an attractive drug candidate for combination therapy against liver cancer.

Postprandial responses in the African rhombig egg eater (Dasypeltis scabra).

The African rhombic egg eater (Dasypeltis scabra) is a colubrid snake feeding exclusively on bird eggs. Frequency of feeding is governed by the seasonal availability of bird eggs; i.e., long fasting intervals change with relatively short periods when plenty of food is available. Intermittent feeding snakes show a remarkable postprandial increase of metabolic rate and digestive organ size. The postprandial increase in metabolic rate (specific dynamic action, SDA) in snakes is affected by meal size, temperature, and meal composition. A major portion of SDA in snakes is allocated to gastric function and the breakdown of the meal. We hypothesize that SDA in egg eaters is lower than in other snake species, because egg eaters feed on "liquid" food that does not require enzymatic breakdown in the stomach. We also hypothesized that other components of the postprandial response of egg eaters (e.g., size changes of the intestine and the liver) do not differ from other snakes. The standard metabolic rate and metabolic response to feeding were measured using closed-chamber respirometry. Size changes of small intestine and liver were measured using high-resolution transcutaneous ultrasonography. Standard metabolic rates of fasting egg eaters were in the same range of mass specific values as known from other snakes. Within 24h after feeding, oxygen consumption doubled and peaked at 2 days after feeding. At the same time, the size of the small intestine and the cross-sectional diameter of the liver increased. Within 2 days after feeding, the size of the mucosal epithelium doubled its thickness. Liver size increased significantly within 24h reaching maximum size 2-4 days after feeding. The size of both organs returned to fasting values within 7-10 days after feeding. The postprandial response of African rhombic egg eaters shows the same pattern and dynamics as known from other snake species. However, the factorial increase of metabolic rate during SDA is the lowest reported for any snake. A comparison with literature data supports the idea that SDA is mainly determined by gastric function and that it is low in egg eaters because they do not have to break down solid meals in the stomach as other snake species do.

XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer.

PURPOSE: Capecitabine has demonstrated high efficacy as first-line treatment for metastatic colorectal cancer (MCRC). Oxaliplatin shows synergy with fluorouracil (FU), with little toxicity overlap. The XELOX regimen (capecitabine plus oxaliplatin), established in a previous dose-finding study, should improve on infused oxaliplatin with FU and leucovorin (FOLFOX) regimens. The present studies further characterize efficacy and safety of the XELOX regimen. PATIENTS AND METHODS: The antitumor activity of XELOX was investigated in a colon cancer xenograft model. Patients with MCRC received first-line XELOX in 3-week treatment cycles: intravenous oxaliplatin 130 mg/m(2) (day 1) followed by oral capecitabine 1,000 mg/m(2) twice daily (day 1, evening, to day 15, morning). RESULTS: A preclinical study confirmed that capecitabine has supra-additive activity with oxaliplatin. In the clinical study, 53 of 96 patients (55%) achieved an objective response, and 30 (31%) experienced disease stabilization for >/= 3 months following treatment. After 24 months' minimum follow-up, median time to disease progression (TTP) and median overall survival were 7.7 and 19.5 months, respectively. XELOX safety was predictable and similar to the FOLFOX4 regimen, except that myelosuppression was uncommon with XELOX (grade 3 or 4 neutropenia, 7%). Most adverse events were mild to moderate, the most common being acute sensory neuropathy (85%). Sixty-day, all-cause mortality was 2%. CONCLUSION: XELOX is a highly effective first-line treatment for MCRC. Response rates, TTP, and overall survival are similar to those observed with FU/leucovorin/oxaliplatin combinations. XELOX provides a more convenient regimen, likely to be preferred by both patients and healthcare providers. Capecitabine has the potential to replace FU/LV in combination with oxaliplatin for MCRC.

Capecitabine/oxaliplatin, a safe and active first-line regimen for older patients with metastatic colorectal cancer: post hoc analysis of a large phase II study.

PURPOSE: The tumor-activated fluoropyrimidine capecitabine achieves response rates superior to those of bolus 5-fluorouracil/leucovorin (5-FU/LV) as first-line treatment for metastatic colorectal cancer (CRC), with favorable safety and fewer hospitalizations. Capecitabine is also at least as effective as bolus 5-FU/LV in the adjuvant setting, again with a favorable safety profile. Improved outcomes with capecitabine versus bolus 5-FU/LV in the adjuvant setting have been shown in overall trial populations and in patients aged >or= 70 years. Capecitabine/oxaliplatin (XelOx) is a safe and active combination for the first-line treatment of metastatic CRC. PATIENTS AND METHODS: This post hoc analysis of a large phase II trial compared data from older and younger patients treated with first-line XelOx: oxaliplatin 130 mg/m(2) intravenously on day 1 followed by oral capecitabine 1,000 mg/m(2) twice daily for 14 days every 3 weeks. RESULTS: The median age of the overall population (N = 96) was 64 years (range, 34-79 years), including 52 younger patients (< 65 years of age) and 44 older patients (>or= 65 years of age). Both age groups received a median of 8 cycles (range, 1-26 cycles) of XelOx. The XelOx regimen had similar high activity in both groups, with response rates of 58% (95% CI, 43%-71%) and 52% (95% CI, 37%-68%) in younger and older patients, respectively. In addition, time to disease progression and overall survival were similar in both groups (P > 0.5 for both outcomes). The XelOx regimen also had a favorable safety profile, with no clinically relevant differences between older and younger patients. The overall incidence of adverse events (including grade 3/4), dose reductions, and withdrawals because of adverse events were similar in both groups. CONCLUSION: In the context of an aging population, XelOx provides a highly effective and tolerable first-line treatment for patients with metastatic CRC.

Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC).

Intestinal epithelial cells (IEC) form the largest surface of the human body and are of pivotal importance to digest and absorb nutrients. Furthermore these cells play a critical role shielding the organism against microorganisms and toxins present in the intestinal lumen. It is therefore not surprising that a large group of researchers take great interest in the study of these cells. However, to date it is a challenge to purify viable primary human intestinal epithelial cells and it has been even more fastidious to maintain IEC in culture ex-vivo as IEC undergo apoptosis within hours due to loss of cell anchorage ('anoikis') following the isolation process. Over recent years the authors aimed to continuously improve the isolation technique for primary IEC, allowing a simple, effective and rapid isolation of highly purified non-apoptotic human IEC. In this study the newly improved method is presented and applied to establish ex-vivo cultures of highly purified, fully viable primary IEC displaying important functional properties, making these cells amenable for ex-vivo research on primary human intestinal epithelial cells.

TNF-alpha induces apoptosis of parietal cells.

Helicobacter pylori infection can be associated with chronic gastric inflammation and hypochlorhydria with increased levels of the proinflammatory cytokines. The current study investigated the effects of TNF-alpha on programmed death of gastric parietal cells. TNF-alpha induced apoptosis of parietal cells in isolated perfused rat stomachs at 10ng/mL. In isolated and highly enriched rat parietal cells, 10ng/mL TNF-alpha induced a 2.6-fold increase in the apoptotic rate. The 55kDa protein of TNFR-1 but not the 75kDa of TNFR-2 was detected by Western blot analysis. TNF-alpha-induced apoptosis of isolated parietal cells was inhibited by pretreatment with different NF-kappaB-inhibitors, nitric oxide synthase inhibitors and with antisense-oligodeoxynucleotides against the p65 subunit of NF-kappaB. Investigation of downstream signaling pathways of apoptosis revealed that TNF-alpha induced the expression of iNOS, but failed to stimulate the activity of caspase 3. The TNF-alpha effect on gastric parietal cells may contribute to the atrophy and hypochlorhydria of the gastric mucosa observed during chronic H. pylori infection.

First-line therapy with daily versus thrice-weekly interferon alfa-2b plus ribavirin for chronic hepatitis C.

OBJECTIVES: Combination therapy with interferon and ribavirin is the most effective treatment for chronic hepatitis C today. Before pegylated interferons became available, higher and more frequent doses of interferon were expected to be more effective than the standard regimen of three million units thrice weekly. In fact, daily dosing is still proposed for non-pegylated interferon. The aim of this study was to compare the efficacy and safety of daily versus thrice-weekly interferon alfa-2b in combination with ribavirin as first-line treatment of chronic hepatitis C. METHODS: A total of 116 treatment-naive patients were randomised to receive either interferon alfa-2b three million units daily or thrice-weekly in combination with ribavirin for 24 weeks. Patients with hepatitis C virus (HCV) genotype 1 who were HCV-RNA negative at 24 weeks continued treatment with thrice-weekly interferon plus ribavirin for another 24 weeks. Sustained virological response was defined as an undetectable HCV-RNA level 24 weeks after treatment was completed (end of follow-up). RESULTS: In an intention-to-treat analysis, HCV-RNA was undetectable at the end of treatment in 71% and 74% of patients treated with daily and thrice-weekly interferon, respectively. At the end of follow-up, HCV-RNA was undetectable in 47% and 57% of patients treated with daily and thrice-weekly interferon, respectively. Sustained virological response rates were almost twice as high in patients with genotypes 2 and 3 as in patients with genotype 1 but were not different between treatment groups. CONCLUSIONS: This study could not show any difference between daily and thrice-weekly standard interferon plus ribavirin in achieving end-of-treatment and sustained virological responses in chronic hepatitis C.

Dysfunction of the hypothalamic-pituitary-glandular axes and relation to Child-Pugh classification in male patients with alcoholic and virus-related cirrhosis.

OBJECTIVE: To investigate anterior pituitary function (adrenal, somatotropic, thyroid and gonadal axes, and prolactin) in relation to the Child-Pugh score in male patients with alcoholic and virus-related liver cirrhosis. METHOD: Anterior pituitary function was evaluated in 52 male cirrhotics (26 Child-Pugh class A (CPA), 16 Child-Pugh class B (CPB) and 10 Child-Pugh class C (CPC)) by a combined pituitary stimulation test, and was compared with 50 age-matched controls. RESULTS: A normal cortisol response to corticotropin-releasing hormone (CRH) stimulation was demonstrated in 57.6% of CPA patients, 31.1% of CPB patients and 20% of CPC patients, while basal levels of adrenocorticotropic hormone (ACTH) and cortisol in cirrhotics were comparable to those in controls. Levels of basal growth hormone (P < 0.001) and stimulated growth hormone (P < 0.01) were significantly higher in cirrhotics compared with controls, while levels of insulin-like growth factor 1 (IGF-1) were significantly lower (P < 0.001). Basal prolactin levels were elevated significantly in CPC patients (P < 0.01), while stimulated prolactin as well as basal and stimulated thyroid-stimulating hormone (TSH) levels were comparable. Basal luteinizing hormone levels were significantly higher in CPA (P < 0.001) and CPB (P < 0.001) patients, and stimulated luteinizing hormone levels were significantly lower in CPC patients than in controls (P < 0.005). Basal and stimulated follicle-stimulating hormone (FSH) levels were comparable in all groups. Child-Pugh score was correlated positively to prolactin and was correlated negatively to IGF-1, stimulated luteinizing hormone and free testosterone. CONCLUSIONS: In cirrhotics, the hypothalamic-pituitary-adrenal and -gonadal axes and prolactin secretion are impaired. Growth hormone response to growth hormone-releasing hormone (GHRH) is accelerated in cirrhotics. Thus, elevated basal and stimulated levels of growth hormone probably reflect compensation for low levels of IGF-1, which are associated with deteriorating liver function. The aetiology of cirrhosis was found to have no influence on the degree of alteration of the hypothalamic-pituitary-glandular axes.

Poster exhibitions at national conferences: education or farce?

INTRODUCTION: The so-called poster exhibition is an established element of medical meetings which often receives little attention. The aim of this study was to analyze the organization, acceptance and value of poster exhibitions. METHODS: Interview based study conducted during the annual meeting of a German specialist medical conference. A total of 247 attendees, poster authors and "poster chairpersons" were interviewed. Attendance at poster exhibitions was documented, the poster review and award process analyzed, and abstracts assessed for redundancy of presentation. RESULTS: Participation in poster exhibitions was very low. Despite this, their scientific value was esteemed high by young authors and the poster chairpersons. Almost a third (29.4%) of posters had been displayed at other meetings. Several attendees (55.4%) and poster presenters (49.1%) say they would welcome the opportunity for personal one-on-one discussion at the poster in addition to poster viewing. DISCUSSION: The option of additional personal discussion with the poster presenter may lead to an increase of the rather modest participation of attendees at poster exhibitions. Poster exhibitions are of value in particular for young scientists and poster chairpersons.

Phase III trial in metastatic gastroesophageal adenocarcinoma with fluorouracil, leucovorin plus either oxaliplatin or cisplatin: a study of the Arbeitsgemeinschaft Internistische Onkologie.

PURPOSE: This study was designed to compare fluorouracil, leucovorin, and oxaliplatin with fluorouracil, leucovorin, and cisplatin in patients with advanced gastric cancer. PATIENTS AND METHODS: Patients with previously untreated advanced adenocarcinoma of the stomach or esophagogastric junction were randomly assigned to receive either fluorouracil 2,600 mg/m(2) via 24-hour infusion, leucovorin 200 mg/m(2), and oxaliplatin 85 mg/m(2) (FLO) every 2 weeks or fluorouracil 2,000 mg/m(2) via 24-hour infusion, leucovorin 200 mg/m(2) weekly, and cisplatin 50 mg/m(2) every 2 weeks (FLP). The primary end point was progression-free survival (PFS). RESULTS: Two hundred twenty patients (median age, 64 years; metastatic, 94%) were randomly assigned. FLO was associated with significantly less (any grade) anemia (54% v 72%), nausea (53% v 70%), vomiting (31% v 52%), alopecia (22% v 39%), fatigue (19% v 34%), renal toxicity (11% v 34%), thromboembolic events (0.9% v 7.8%), and serious adverse events related to the treatment (9% v 19%). FLP was associated with significantly less peripheral neuropathy (22% v 63%). There was a trend toward improved median PFS with FLO versus FLP (5.8 v 3.9 months, respectively; P = .077) and no significant difference in median overall survival (10.7 v 8.8 months, respectively). However, in patients older than 65 years (n = 94), treatment with FLO resulted in significantly superior response rates (41.3% v 16.7%; P = .012), time to treatment failure (5.4 v 2.3 months; P < .001), and PFS (6.0 v 3.1 month; P = .029) and an improved OS (13.9 v 7.2 months) as compared with FLP, respectively. CONCLUSION: FLO reduced toxicity as compared with FLP. In older adult patients, FLO also seemed to be associated with improved efficacy.

Cell-cell contacts prevent anoikis in primary human colonic epithelial cells.

BACKGROUND & AIMS: Colonic epithelial cells (CECs) receive important survival signals from the extracellular matrix and undergo detachment-induced apoptosis (anoikis) as soon as they lose their cell-matrix anchorage. In contrast to the established role of cell-matrix contact, the role of cell-cell contacts as a physiologic survival factor for CECs is less clear. METHODS: Intact CEC crypts gently centrifuged to form a cell aggregate in which cell-cell contacts were maintained. Induction of apoptosis was assessed by Western Blot analysis, colorimetric assays, DNA electrophoresis, 4',6-diamidino-2-phenylindole staining, and flow cytometry. Activation of survival pathways was analyzed by Western blot. The role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK)/extracellular signal-regulated kinase (Erk)1/2, epidermal growth factor receptor, phosphatidylinositol 3-kinase (PI3-K), and Src signaling was investigated using specific inhibitors. RESULTS: Despite a complete loss of cell-matrix adhesion after CEC isolation, activation of caspases was blocked and anoikis was prevented when cell-cell contacts were preserved. CECs with preserved cell-cell contacts exhibited a rapid dephosphorylation of focal adhesion kinase. Aggregated CECs had stable levels of active beta-catenin and phosphorylated Akt, Erk1/2, and epidermal growth factor receptor, but CECs undergoing anoikis rapidly degraded beta-catenin and dephosphorylated Akt. Inhibition of Src- and PI3-K-dependent signaling reversed the antiapoptotic effect of cell-cell contact preservation, while inhibition of the MEK pathway had no effect. CONCLUSIONS: Integrity of cell-cell contacts compensates for the loss of cell-matrix contact-mediated survival signals in CECs and prevents apoptosis. Cell-cell contact-triggered CEC survival involves antiapoptotic signaling through beta-catenin-, Src-, and PI3-K/Akt- but not through MEK- and focal adhesion kinase-dependent pathways.

The loss of sarco/endoplasmic reticulum calcium transport ATPase 3 expression is an early event during the multistep process of colon carcinogenesis.

Calcium accumulation in the endoplasmic reticulum is accomplished by sarco/endoplasmic reticulum calcium transport ATPases (SERCA enzymes). To better characterize the role of SERCA3 in colon carcinogenesis, its expression has been investigated in colonic epithelium, benign lesions, adenomas, and adenocarcinomas. In addition, the regulation of SERCA3 expression was analyzed in the context of the adenomatous polyposis coli/beta-catenin/T-cell factor 4 (TCF4) pathway and of specificity protein 1 (Sp1)-like factor-dependent transcription. We report that SERCA3 expression increased along the crypts as cells differentiated in normal colonic mucosa and in hyperplastic polyps, was moderately and heterogeneously expressed in colonic adenomas with expression levels inversely correlated with the degree of dysplasia, was barely detectable in well and moderately differentiated adenocarcinomas, and was absent in poorly differentiated tumors. Inhibition of Sp1-like factor-dependent transcription blocked SERCA3 expression during cell differentiation, and SERCA3 expression was induced by the expression of dominant-negative TCF4 in colon cancer cells. These data link SERCA3 expression to the state of differentiation of colonic epithelial cells, and relate SERCA3 expression, already decreased in adenomas, to enhanced adenomatous polyposis coli/beta-catenin/TCF4-dependent signaling and deficient Sp1-like factor-dependent transcription. In conclusion, intracellular calcium homeostasis becomes progressively anomalous during colon carcinogenesis as reflected by deficient SERCA3 expression.

Induction of apoptosis before shedding of human intestinal epithelial cells.

OBJECTIVES: Human intestinal epithelial cells (IECs) derive from stem cells at the crypt base and migrate along the so-called crypt-villus axis toward the intestinal lumen. As they reach the luminal surface in the colon or the villus tip in the small intestine, IECs are shed and their life cycle is terminated. The role of apoptosis during IEC migration along the crypt-villus axis has been subject to studies with conflicting results. In this study we use a novel approach to identify the initiation of apoptosis within normal human IECs. METHODS: Normal mucosa from the large and small human intestine was analyzed employing a novel antibody directed against activated caspase-3--an early marker of apoptosis. RESULTS: IECs initiate the apoptotic cascade as they approach the area of shedding before displaying evident morphological features of apoptosis. IECs of the small bowel also show caspase-3 activation in the small intestinal crypt base, whereas IECs of the colononic crypt base rarely show evidence of ongoing apoptosis. CONCLUSIONS: These findings indicate that apoptosis is initiated in human IECs as they reach the luminal surface/villus tip and before shedding. Furthermore, they show that different sections of the intestinal tract vary significantly in the rate of IEC apoptosis, possibly reflecting their difference in susceptibility to epithelial cell neoplasia.

Autocrine fibronectin-induced migration of human colonic fibroblasts.

OBJECTIVES: A central event during wound repair is the migration of activated fibroblasts to the wound area. Thus far, the mechanisms inducing migration of colonic lamina propria fibroblasts (CLPF) have not been studied in detail. Previously, we have shown that CLPF secrete factors that are essential to their ability to migrate in response to different growth factors. METHODS: Primary human CLPF were obtained from endoscopic biopsies or surgical specimens taken from normal mucosa areas of patients undergoing surveillance colonoscopy or surgery for colorectal carcinoma. Migration assays of CLPF were performed in the modified 48-well Boyden chamber. RESULTS: Conditioned medium of CLPF collected after 24-h stimulated migration of CLPF (22 +/- 2 cells/ hpf). Filtration of conditioned medium through a 300-kDa filter reduced the migration-inducing potential in subsequent migration assays to 2 +/- 1 cells/hpf, filtration through a 100-kDa filter abolished migration of CLPF completely, indicating that large molecules such as extracellular matrix components could be responsible for the induction of CLPF migration. Enzyme-linked immunosorbent assays revealed the presence of fibronectin in conditioned medium (17.3 microg/ml). Immunoprecipitation of fibronectin in conditioned medium of CLPF reduced the migration-inducing potential by 63%. Addition of fibronectin to fibronectin-depleted conditioned medium reconstituted the migration. Dose-response assays with fibronectin (1-100 microg/ml) diluted in nonconditioned medium induced migration of CLPF in a dose-dependent manner. Maximum migration was induced with 25 microg/ml fibronectin (37 +/- 5 cells/hpf). CONCLUSION: Fibronectin is an autocrine and paracrine factor essential for intestinal fibroblast migration. Fibronectin induces migration of intestinal fibroblasts and is essential for their ability to migrate in response to different growth factors. A detailed understanding of the regulation of the migration of intestinal fibroblasts is necessary to gain further insights in the pathophysiology of stricture and fistula formation.

Immunomodulatory consequences of oral administration of Lactobacillus rhamnosus strain GG in healthy volunteers.

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either 'self' or 'non-self' preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-gamma, TNF-alpha) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-alpha, IL-6 and in part IFN-gamma cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole 'non-self' intestinal flora was higher than by 'self' intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.

Expression of endomembrane calcium pumps in colon and gastric cancer cells. Induction of SERCA3 expression during differentiation.

Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.