Flightless I exacerbation of inflammatory responses contributes to increased colonic damage in a mouse model of dextran sulphate sodium-induced ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by cytokine driven inflammation that disrupts the mucosa and impedes intestinal structure and functions. Flightless I (Flii) is an immuno-modulatory protein is a member of the gelsolin family of actin-remodelling proteins that regulates cellular and inflammatory processes critical in tissue repair. Here we investigated its involvement in UC and show that Flii is significantly elevated in colonic tissues of patients with inflammatory bowel disease. Using an acute murine model of colitis, we characterised the contribution of Flii to UC using mice with low (Flii+/-), normal (Flii+/+) and high Flii (FliiTg/Tg). High levels of Flii resulted in significantly elevated disease severity index scores, increased rectal bleeding and degree of colon shortening whereas, low Flii expression decreased disease severity, reduced tissue inflammation and improved clinical indicators of UC. Mice with high levels of Flii had significantly increased histological disease severity and elevated mucosal damage with significantly increased inflammatory cell infiltrate and significantly higher levels of TNF-α, IFN-γ, IL-5 and IL-13 pro-inflammatory cytokines. Additionally, Flii overexpression resulted in decreased ß-catenin levels, inhibited Wnt/ß-catenin signalling and impaired regeneration of colonic crypts. These studies suggest that high levels of Flii, as is observed in patients with UC, may adversely affect mucosal healing via mechanisms involving Th1 and Th2 mediated tissue inflammation and Wnt/ß-catenin signalling pathway.

Circulating tumour cells: the evolving concept and the inadequacy of their enrichment by EpCAM-based methodology for basic and clinical cancer research.

Increasing evidence suggests that circulating tumour cells (CTCs) are responsible for metastatic relapse and this has fuelled interest in their detection and quantification. Although numerous methods have been developed for the enrichment and detection of CTCs, none has yet reached the 'gold' standard. Since epithelial cell adhesion molecule (EpCAM)-based enrichment of CTCs offers several advantages, it is one of the most commonly used and has been adapted for high-throughput technology. However, emerging evidence suggests that CTCs are highly heterogeneous: they consist of epithelial tumour cells, epithelial-to-mesenchymal transition (EMT) cells, hybrid (epithelial/EMT(+)) tumour cells, irreversible EMT(+) tumour cells, and circulating tumour stem cells (CTSCs). The EpCAM-based approach does not detect CTCs expressing low levels of EpCAM and non-epithelial phenotypes such as CTSCs and those that have undergone EMT and no longer express EpCAM. Thus, the approach may lead to underestimation of the significance of CTCs, in general, and CTSCs and EMT(+) tumour cells, in particular, in cancer dissemination. Here, we provide a critical review of research literature on the evolving concept of CTCs and the inadequacy of their enrichment by EpCAM-based technology for basic and clinical cancer research. The review also outlines future perspectives in the field.

Induction of apoptosis by the medium-chain length fatty acid lauric acid in colon cancer cells due to induction of oxidative stress.

BACKGROUND: Fatty acids are classified as short-chain (SCFA), medium-chain (MCFA) or long-chain and hold promise as adjunctive chemotherapeutic agents for the treatment of colorectal cancer. The antineoplastic potential of MCFA remains underexplored; accordingly, we compared the MCFA lauric acid (C12:0) to the SCFA butyrate (C4:0) in terms of their capacity to induce apoptosis, modify glutathione (GSH) levels, generate reactive oxygen species (ROS), and modify phases of the cell cycle in Caco-2 and IEC-6 intestinal cell lines. METHODS: Caco-2 and IEC-6 cells were treated with lauric acid, butyrate, or vehicle controls. Apoptosis, ROS, and cell cycle analysis were determined by flow cytometry. GSH availability was assessed by enzymology. RESULTS: Lauric acid induced apoptosis in Caco-2 (p < 0.05) and IEC-6 cells (p < 0.05) compared to butyrate. In Caco-2 cells, lauric acid reduced GSH availability and generated ROS compared to butyrate (p < 0.05). Lauric acid reduced Caco-2 and IEC-6 cells in G0/G1and arrested cells in the S and G2/M phases. Lauric acid induced apoptosis in IEC-6 cells compared to butyrate (p < 0.05). Butyrate protected IEC-6 cells from ROS-induced damage, whereas lauric acid induced high levels of ROS compared to butyrate. CONCLUSION: Compared to butyrate, lauric acid displayed preferential antineoplastic properties, including induction of apoptosis in a CRC cell line.

Deficiency of invariant natural killer T cells in coeliac disease.

BACKGROUND: Immunoregulatory invariant natural killer (iNK) T cells rapidly produce interleukin (IL)-4 and other cytokines that suppress a Th1 response and are deficient in some autoimmune diseases. AIM: The aim of this study was to investigate any deficiency of iNK T cells in coeliac disease. METHODS: Blood was collected from 86 subjects with coeliac disease and from 152 healthy control subjects for investigation of Valpha24+ T cells by flow cytometry. iNK T cells were assessed by Valpha24 and alpha-galactosylceramide/CD1d tetramer markers in 23 normal controls and 13 subjects with coeliac disease. Intracellular IL-4 was measured after anti-CD3 antibody stimulation. Duodenal biopsies were obtained in a subgroup of subjects with coeliac disease and control subjects for Valpha24 mRNA expression using relative PCR and for Valpha24+ T cells by immunofluorescence. RESULTS: The mean numbers of circulating Valpha24+ T cells and iNK T cells in coeliac disease were 27% (p<0.001) and 16% (p<0.001), respectively, of levels in control subjects. After in vitro anti-CD3 stimulation, numbers of IL-4+ producing iNK T cells from subjects with coeliac disease were unchanged but increased by 21% in control subjects. In subjects with coeliac disease, Valpha24 mRNA intestinal expression was reduced to 17% (p<0.001) by relative PCR and numbers of intestinal Valpha24+ T cells were 16% (p<0.01) of levels in control subjects. CONCLUSIONS: We conclude that Valpha24+ T cells and iNK T cells are deficient in coeliac disease. We speculate that this deficiency could contribute to the failure of immunological oral tolerance that seems to underlie this disease.

Relationship between dose of methotrexate, apoptosis, p53/p21 expression and intestinal crypt proliferation in the rat.

Previous studies have shown that apoptosis is induced by cytotoxic chemotherapy and precedes hypoproliferation of intestinal crypt cells. However, the relationship between the degree of intestinal apoptosis and crypt cell hypoproliferation may not be directly related. The purpose of this study was to investigate the relationship between apoptosis and hypoproliferation with increasing doses of chemotherapy. Eleven groups of breast cancer-bearing DA rats were treated with two doses of methotrexate (MTX) i. m. at varying concentrations (0.5, 1.5, 2.5 and 5.0 mg/kg) or saline (control). Animals were killed at 6 or 24 h following treatment. The small and large intestines were examined for apoptosis, villous area (small intestine), crypt length and mitotic count per crypt. Immunohistochemical expression of p53 and p21(waf1/cip1) (p21) were examined quantitatively. Data were analysed using Peritz' F-test. Low dose MTX (0.5 mg/kg) did not change p53 expression at 6 h but induced a 15-fold increase in apoptosis in the crypts of the small intestine. This was associated with only a minor reduction in crypt cell proliferation. Higher doses of MTX increased p53 expression and caused a lower (7-fold) but more prolonged peak of apoptosis that was accompanied by reduced villous area, shortened crypts and a more profound reduction in crypt cell proliferation. Unlike the small intestine, apoptosis in the colon was 10-fold lower, proportional to the dose of MTX and did not induce overt damage. Expression of p21 did not change with any dose at either timepoint. We conclude that apoptosis is not always associated with crypt cell hypoproliferation and that the small intestine can recover after low dose MTX despite a heightened peak of apoptosis of crypt cells.

Effect of breast milk and weaning on epithelial growth of the small intestine in humans.

Breast feeding and weaning are important physiologically significant luminal events that influence the growth of the small intestine in humans. A variety of factors including genetic preprogramming, systemic and local hormones, and permissive factors contribute and modulate intestinal growth. Here, we offer a view that integrates some of these factors, especially those relating to breast feeding and weaning.

Improvement in intestinal permeability precedes morphometric recovery of the small intestine in coeliac disease.

It is often difficult to assess small bowel recovery in adults with coeliac disease on a gluten-free diet (GFD). This prospective study compares changes in intestinal permeability with changes in intestinal biopsy at various intervals after commencing a GFD. Intestinal permeability was measured by lactulose/rhamnose absorption from 1 week to 24 months after commencing a GFD. Intestinal morphometry was measured by villus area, crypt length and mitotic count per crypt at diagnosis and after commencing a GFD. Median intestinal permeability values decreased from 0.47 (n = 35) at diagnosis to 0.25 (n = 17) after 1 week and to 0.16 (n = 18) after 2 months of a GFD. Rhamnose absorption improved significantly at an early stage, from 6.6% (untreated) to 15.4% at 3 months of a GFD, whereas the decrease in lactulose permeation took longer: from 3.4% (untreated) to 0.8% after 12 months of a GFD. Mean villus area (n = 29) was reduced to 16% of control values at diagnosis, and improved to a maximum of 48% after 6 months on a GFD, but did not change thereafter. Mean crypt length and mitotic count per crypt were increased by 222% and 356% respectively at diagnosis, and these parameters remained elevated at 172% and 216% above control values after 6 months of a GFD. We conclude that intestinal permeability improves within 2 months after starting a GFD, but that measurable intestinal biopsy improvement requires ingestion of a GFD for at least 3-6 months, and even then remains incomplete.

The role of small intestinal bacterial overgrowth, intestinal permeability, endotoxaemia, and tumour necrosis factor alpha in the pathogenesis of non-alcoholic steatohepatitis.

BACKGROUND: Small intestinal bacterial overgrowth may contribute to the development of non-alcoholic steatohepatitis, perhaps by increasing intestinal permeability and promoting the absorption of endotoxin or other enteric bacterial products. AIMS: To investigate the prevalence of small intestinal bacterial overgrowth, increased intestinal permeability, elevated endotoxin, and tumour necrosis factor alpha (TNF-alpha) levels in patients with non-alcoholic steatohepatitis and in control subjects. PATIENTS AND METHODS: Twenty two patients with non-alcoholic steatohepatitis and 23 control subjects were studied. Small intestinal bacterial overgrowth was assessed by a combined (14)C-D-xylose and lactulose breath test. Intestinal permeability was assessed by a dual lactulose-rhamnose sugar test. Serum endotoxin levels were determined using the limulus amoebocyte lysate assay and TNF-alpha levels using an ELISA. RESULTS: Small intestinal bacterial overgrowth was present in 50% of patients with non-alcoholic steatosis and 22% of control subjects (p=0.048). Mean TNF-alpha levels in non-alcoholic steatohepatitis patients and control subjects were 14.2 and 7.5 pg/ml, respectively (p=0.001). Intestinal permeability and serum endotoxin levels were similar in the two groups. CONCLUSIONS: Patients with non-alcoholic steatohepatitis have a higher prevalence of small intestinal bacterial overgrowth, as assessed by the (14)C-D-xylose-lactulose breath test, and higher TNF-alpha levels in comparison with control subjects. This is not accompanied by increased intestinal permeability or elevated endotoxin levels.

Chemotherapy for cancer causes apoptosis that precedes hypoplasia in crypts of the small intestine in humans.

BACKGROUND AND AIMS: The mechanism of gastrointestinal damage (mucositis) induced by cancer chemotherapy remains uncertain. The aims of this study were to define the time course and mechanism of small intestinal damage following chemotherapy in humans. METHODS: Patients receiving chemotherapy underwent upper gastrointestinal endoscopy (a maximum of two per patient) with duodenal biopsy prior to chemotherapy and again at 1, 3, 5, and 16 days after chemotherapy. Tissue was taken for morphometry, disaccharidase assays, electron microscopy, and for assessment of apoptosis using the Tdt mediated dUTP-biotin nick end labelling (TUNEL) method. Villus area, crypt length, and mitotic index were measured by a microdissection technique. RESULTS: Apoptosis increased sevenfold in intestinal crypts at one day, and villus area, crypt length, mitotic count per crypt, and enterocyte height decreased at three days after chemotherapy. Disaccharidase activities remained unchanged. Electron microscopy showed increased open tight junctions of enterocytes at day 3, consistent with more immature cells. All indices improved by 16 days. CONCLUSION: Small intestinal mucositis is associated with apoptosis in crypts that precedes hypoplastic villous atrophy and loss of enterocyte height.

Activation of the gut-associated lymphoid tissue with expression of interleukin-2 receptors that peaks during weaning in the rat.

BACKGROUND: Weaning exposes the intestinal mucosa to food and bacterial antigens at an age when the immune system is believed to be immature and functionally defective. The purpose of this study was to investigate changes in activation and phenotype of immune cells of the gut-associated lymphoid tissue during weaning. METHODS: Litters of infant rats were studied from pre- to postweaned life. The activation status, assessed by interleukin-2 receptor (IL-2R) expression, and phenotype of cells in the gut-associated lymphoid tissue were examined by immunostaining. RESULTS: Interleukin-2 receptor expression peaked two to four-fold at midweaning (day 21) in mesenteric lymph nodes, jejunal lamina propria, Peyer's patches, and intraepithelial lymphocytes, compared with adult animals (day 70). CD45+ cells expanded in the lamina propria, epithelium, and lymphocyte-filled villi. With CD45 as the denominator, 10% to 50% of lymphocytes in the lamina propria and epithelium were alphabetaT-cell receptor (TCR)+, but the remaining cells had a null phenotype, because there were low numbers of gammadeltaTCR+ T cells, B cells, and macrophages. Natural killer cells peaked at midweaning in the lamina propria (9%) and epithelium (20%) but were less than 5% of CD45+ cells after weaning. CONCLUSIONS: Rather than being immature or functionally inactive, the gut-associated lymphoid tissue reacts appropriately during weaning with expression of IL-2R and expansion of alphabetaTCR+ T-cells.

Transforming growth factor-beta levels in maternal milk and expression in postnatal rat duodenum and ileum.

After birth, the gastrointestinal tract of the neonate is exposed to food and bacterial and environmental antigens. Maternal milk components may play a role in regulation of mucosal immune activity to luminal antigens. In this study we determine the ontogeny of transforming growth factor (TGF)-beta1-producing cells in the rat pup small intestine and assess maternal milk concentrations of TGF-beta. Intestinal tissue samples of duodenum and ileum were collected, processed, and stained for TGF-beta1, and in situ hybridization for TGF-beta1 mRNA was also performed on the duodenum. TGF-beta levels in milk were assayed by ELISA. TGF-beta2 levels in milk were high at d 6, and declined thereafter at d 10 and 19. TGF-beta1 was not detected. In contrast, the cell number and intensity of staining of TGF-beta1 peptide in the small intestine was low in 3- and 10-d-old rats and increased markedly by 19 d of life. In the duodenum mRNA levels mirrored this trend. TGF-beta1 expression in the lamina propria was absent before d 19, and increased progressively over time. Maternal milk TGF-beta2 levels are high in early milk and decrease during the weaning period. In contrast, endogenous TGF-beta production in the small intestine increases during the weaning period.

Comparison of culture, histopathology and urease testing for the diagnosis of Helicobacter pylori gastritis and susceptibility to amoxycillin, clarithromycin, metronidazole and tetracycline.

Gastric biopsy specimens were taken from 737 patients undergoing upper gastrointestinal endoscopy and assessed for Helicobacter pylori infection. The diagnostic utilities of H. pylori culture (733 patients), detection of urease production (724 patients) and histopathological examination (469 patients) were compared. Since each of these techniques may fail to diagnose patients infected with H. pylori, an attempt was made to estimate the true rate of infection using a mathematical approach that combined the results of culture, histopathology and urease testing; 34% of the 733 patients were thought to be infected. Using this figure as a benchmark, the sensitivity, specificity, positive predictive value and negative predictive value of H. pylori culture were 73.2%, 100%, 100% and 86.3%, respectively, compared with 58.7%, 100%, 100% and 89.6%, respectively for urease production and 77.0%, 100%, 100% and 82.4%, respectively for histopathology. Thus, histopathological examination was the single most reliable test. A combination of histopathological examination and H. pylori culture diagnosed 99.5% of patients that were estimated to be truly infected. The minimum inhibitory concentrations of a number of antibiotics were measured for 135 isolates of H. pylori. All isolates were susceptible to amoxycillin and tetracycline whereas 5.2% were resistant to clarithromycin and 60% were resistant to metronidazole.

Epithelial growth of the small intestine in human infants.

BACKGROUND: Findings in studies in rodents have suggested that epithelial growth of the small intestine is dependent on activation of the immune system. The purpose of this study was to compare changes of postnatal epithelial growth with immunologic activity in humans. METHODS: Duodenal biopsies were obtained by endoscopy from 74 infants. Villus area, crypt length, and mitotic count were measured, using a microdissection technique. Enterocyte height, intraepithelial lymphocytes and mucosal mast cells were recorded in histologic sections, and soluble interleukin-2 receptor levels were measured in sera. These data were compared with those from 77 adult control subjects. RESULTS: Mean +/- SD villus area was similar in infants compared with that in adults (0.364 +/- 0.108 mm2 vs. 0.339 +/- 0.1 mm2); but mean crypt length was 31% longer (270 +/- 56 microm vs. 206 +/- 29 microm; p < 0.0001), and mitotic count was 68% higher (4.2 +/- 2.8 vs. 2.5 +/- 1 per crypt; p < 0.0001) in infants. Enterocyte height was lower during infancy (27.0 +/- 3.4 microm vs. 30.9 +/- 4.6 microm; p < 0.0001). There was no evidence of a trophic effect on the small intestine of breast feeding compared with the effect of bottle feeding. Counts of intraepithelial lymphocytes but not mucosal mast cells were significantly less in infants. Mean soluble interleukin-2 receptor levels peaked during early infancy, compared with levels in adults (1,820 +/- 596 U/ml vs. 695 +/- 359 U/ml). CONCLUSION: These results indicate that epithelial proliferation is increased during infancy at an age when immunologic activity is high.

Effect of high-dose chemotherapy on intestinal permeability in humans.

1. Mucositis is a common side-effect of chemotherapy which is difficult to assess except by invasive means such as upper gastrointestinal endoscopy. Differential absorption of mono- and di-saccharides, such as rhamnose and lactulose, is a non-invasive measure of intestinal damage. 2. The purpose of the study was to assess the duration and severity of intestinal damage in patients undergoing high-dose chemotherapy and autologous blood stem-cell transplantation for malignant disease. 3. Thirty-five patients were studied before treatment and at 7, 28, 60 and 90 days after treatment. 4. The median lactulose/rhamnose ratios before treatment and at 7 and 90 days post-treatment were 0.09, 0.62 and 0.06 respectively. Altered permeability was due to both increased lactulose permeation and decreased rhamnose absorption. These abnormalities suggest a defect in tight-junction integrity as well as a decrease in surface area of small bowel. 5. We conclude that chemotherapy given for malignant disease is associated with a transient abnormality in intestinal sugar permeability, which peaks at 7 days after treatment and is composed of both mono- and di-saccharide absorption abnormalities.

Postnatal changes in mucosal immune response: a physiological perspective of breast feeding and weaning.

There are profound changes of immune activity during infancy from suppression during breast feeding, activation with weaning, and later intrinsic down-regulation after weaning. Breast feeding, as well as protecting against infections, seems to have a fundamental role in modifying the immune system against certain disease states. Transforming growth factor (TGF)beta in breast milk may mediate this immunosuppressive effect. Although the infant immune system is not in an adult state, the notion that the infant immune system is immature is difficult to reconcile with evidence that most infants respond appropriately to immunization and to infections. The systemic immune system of neonates may be subject to Th2 immune deviation, while the mucosal immune system, particularly of the gastrointestinal tract and probably the respiratory tract, is up-regulated with physiological inflammation during infancy. Weaning is associated with a peak of intestinal immune activation which includes mucosal mast cells and T cells. The physiological effects of this activation are promotion of epithelial growth of the small intestine and initial activation of mechanisms leading to subsequent down-regulation of the physiological heightened immune activity. This coincides with the development of mucosal (oral) tolerance to food and bacterial antigens.

Dependence of epithelial growth of the small intestine on T-cell activation during weaning in the rat.

BACKGROUND & AIMS: Intestinal crypt hyperplasia is associated with local T-cell activation in both clinical and experimental examples of immunologically mediated enteropathy. This suggests that T cell-derived factors may be trophic for epithelial proliferation in the intestine postnatally. The purpose of this study was to investigate T-cell activity during weaning in the rat and to investigate immune dependence of intestinal growth on T-cell activation. METHODS: The expression of interleukin-2 receptor (IL-2R) by mesenteric lymph node T cells was investigated from days 14 to 160 of life. Rats were treated with monoclonal antibodies against the IL-2R that were nonblocking (control) or blocking (experimental) from day 7, and intestinal growth was assessed at days 19, 25, and 29 of life. RESULTS: The mean +/- SEM of T cells expressing the IL-2R during weaning (days 15-28) was 6.1% +/- 0.3% compared with 3.3% +/- 0.3% at other ages (P < 0.001). The small intestine in rats treated with blocking antibody had reduced crypt length and mitotic count compared with control animals. CONCLUSIONS: Weaning is associated with activation of T cells and blockade of the IL-2R reduces intestinal growth.