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• W1999824352 abstract "To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([3H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitrostudies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 ± 60versus 195 ± 48 ohm·cm2 in controls,p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration. To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([3H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitrostudies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 ± 60versus 195 ± 48 ohm·cm2 in controls,p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration. trefoil factor family rat TFF intestinal fatty acid-binding protein rat FABPi polymerase chain reaction base pair(s) bromodeoxyuridine analysis of variance Krebs-Henseleit epidermal growth factor Gastrointestinal mucosal integrity depends on the dynamic equilibrium between aggressive factors such as luminal acid, enzymes, and bacteria and host defense mechanisms such as mucus secretion, rapid cell turnover, and efficient blood supply. When an injury does occur, it is usually rapidly repaired by an initial process involving cell migration (restitution), increased proliferation, and subsequent remodelling. There is increasing evidence that a group of molecules, termed the trefoil factor family (TFF),1are involved in gastrointestinal defense and repair, possibly acting by more than one mechanism. Three TFF members have been identified in mammals: TFF1 (previously termed pS2), TFF2 (previously spasmolytic polypeptide), and TFF3 (previously intestinal trefoil factor). All three TFF members contain one or two highly conserved trefoil domains, are located clustered within a 50-kilobase sequence on chromosome 21q22.3 (1Gott B. Beck S. Machado J.C. Carneiro F. Schmitt H. Blin N. Eur. J. Hum. Genet. 1996; 4: 308-315Crossref PubMed Scopus (67) Google Scholar), and are remarkably resistant to proteolytic digestion (2Playford R.J. Marchbank T. Chinery R. Evison R. Pignatelli M. Boulton R.A. Thim L. Hanby A.H. Gastroenterology. 1995; 108: 108-116Abstract Full Text PDF PubMed Scopus (254) Google Scholar), probably in part because of their extensive intrachain disulfide cysteine bridging. Under nondamaged circumstances, expression of the three TFF mammalian homologues are geographically distinct. TFF1 and TFF2 are both primarily located in the stomach, whereas TFF3 is predominantly present in the mucous cells of the small and large intestine (3Tomasetto C. Rio M. Gautier C. Wolf C. Hareuveni M. Chambon P. Lathe R. EMBO J. 1990; 9: 407-414Crossref PubMed Scopus (203) Google Scholar, 4Hanby A.M. Poulsom R. Singh S. Elia G. Jeffery R.E. Wright N.A. Gastroenterology. 1993; 105: 1110-1116Abstract Full Text PDF PubMed Scopus (165) Google Scholar, 5Podolsky D.K. Lynch-Devaney K. Stow J.L. Oates P. Murgue B. DeBeaumont M. Sands B.E. Mahida Y.R. J. Biol. Chem. 1993; 268: 6694-6702Abstract Full Text PDF PubMed Google Scholar). Up-regulation of expression of all three TFF members occurs at sites of damage in conditions such as peptic ulcer and Crohn's disease (6Wright N.A. Poulsom R. Stamp G.W.H. Van Norden S. Sarraf C. Elia G. Ahnen D. Jeffrey R.E. Longcroft J.M. Pike C. Rio M.-C. Chambon P. Gastroenterology. 1993; 104: 12-20Abstract Full Text PDF PubMed Google Scholar, 7Alison M.R. Chinery R. Poulsom R. Ashwood P. Longcroft J.M. Wright N.A. J. Pathol. 1995; 175: 405-414Crossref PubMed Scopus (176) Google Scholar). The temporal relationship to acute injury varies with each peptide, however, suggesting they may have different pathophysiological roles (7Alison M.R. Chinery R. Poulsom R. Ashwood P. Longcroft J.M. Wright N.A. J. Pathol. 1995; 175: 405-414Crossref PubMed Scopus (176) Google Scholar, 8Wong W.M. Poulsom R. Wright N.A. Gut. 1999; 44: 890-895Crossref PubMed Scopus (186) Google Scholar). This idea is also supported by the finding that mice that have had the TFF1 gene functionally deleted (“knock-out”) have a markedly different phenotype (gastric adenomas and carcinomas; Ref. 9Lefebvre O. Chenard M.P. Masson R. Linares J. Dierich A. LeMeur M. Wendling C. Tomasetto C. Chambon P. Rio M.C. Science. 1996; 274: 259-262Crossref PubMed Scopus (456) Google Scholar) compared with mice who have had TFF2 and TFF3 deleted (giving an essentially normal phenotype under nonstressed situations; Refs. 10Mashimo H. Wu D.C. Podolsky D. Science. 1996; 274: 262-265Crossref PubMed Scopus (607) Google Scholar and 11Taupin D. Farrell J.J. Koh T. Podolsky D.K. Wang T.C. MacCallum P. Gastroenterology. 2000; 118 (abstr.): 823Abstract Full Text PDF Google Scholar). To gain further insight into the function of the TFF family and TFF3 in particular, we have now established a model that chronically overexpresses rat TFF3 in the proximal small intestine of mice. We use this model to determine the effect of TFF3 overexpression under basal circumstances and its ability to influence sensitivity to damage using both in vitro and in vivo models of injury. Materials were obtained from Sigma Chemicals (Poole, Dorset, UK) unless otherwise stated. Genetic modification of animals and all animal procedures were approved by the appropriate local and national authorities. All of the animals were kept on standard chow diet ad libitum and were killed by cervical dislocation. Nucleotides −1178 to +28 of the rat FABPi promoter inserted into the EcoRI-SmaI sites of pUC13 was kindly donated by Jeff Gordon (Washington University, St. Louis, MO; Ref. 12Sweetser D.A. Birkenmeier E.H. Klisak I.J. Zollman S. Sparkes R.S. Mohandas T. Lusis A.J. Gordon J.I. J. Biol. Chem. 1987; 262: 16060-16071Abstract Full Text PDF PubMed Google Scholar). The details of the production and map are shown in Fig.1. The final 1.6-kilobase cassette was microinjected into over 200 recently fertilized oocytes (strain C57 × CBA). The forward primer comprised bases 997–1017 of the FABPi sequence, and the reverse primer comprised bases 374–394 of the rTFF3, resulting in a 550-bp product. These primers detect the construct but do not cross-hybridize with native normal mouse DNA. Ear snips were collected in 0.25 ml of “ear buffer” (50 mm Tris, pH 8.0, 20 mmNaCl, 0.1% (v/v) SDS) and incubated in 100 μg/ml proteinase K at 55 °C for 16 h. Samples were diluted in water, boiled for 20 min, and stored at −20 °C prior to PCR analysis. Of 150 live-born mice, PCR analysis identified one founder mouse that had incorporated the construct. A line (RPTMrITF/1) was established from this founder. For all studies, heterozygotes were used because this allowed negative littermates to act as controls. To establish the expression of rTFF3 RNA in transgenic positive animals, total RNA was prepared from the intestine of several heterozygote-positive mice and -negative littermates. Total RNA was isolated from ileum and jejunum using the RNAgents/total RNA isolation system (Promega, Southampton, UK). RNA integrity was established by formaldehyde-agarose gel electrophoresis. RNA was transferred to Nylon filters (Hybond-N; Amersham Pharmacia Biotech) by capillary transfer and cross-linked by UV irradiation (UV Stratalinker 2400, Stratagene, Cambridge, UK). Northern blot analyses were performed using a specific 110-bp probe to the 3′-untranslated region of the rTFF3 cDNA, which did not interact with the native mouse TFF3 RNA. The probe was labeled with 32P using a nick translation kit (Life Technologies, Inc.). Filters were hybridized using QuickHyb (Stratagene) and washed as follows twice in 2× SSC + 0.1% SDS for 15 min at room temperature and once in 0.5× SSC + 0.1% SDS for 30 min at 60 °C. Equal loading of RNA into the wells was confirmed by stripping the filters and reprobing with a 558-bp32P-labeled DNA sequence corresponding to the coding region of 18 S RNA from positions 28 to 586 (kindly donated by A. Gandarillas, Imperial Cancer Research Fund). Expression of rTFF3 peptide was confirmed by immunohistochemical staining using methods described previously (14Taupin D.R. Pang K.C. Green S.P. Giraud A.S. Peptides. 1995; 15: 1001-1005Crossref Scopus (42) Google Scholar). The antibody used for these immunostaining studies, rITF1#7, is a rabbit polyclonal antibody raised against the C-terminal decapeptide of rTFF3 and detects the presence of both the rat and mouse forms of TFF3. The samples were fixed in neutral buffered formalin and embedded in paraffin wax. 4-μm sections were cut onto poly-l-ornithine-coated slides and rehydrated. The slides were incubated with normal goat serum (1:20) for 30 min, primary antibody (1:1000) for 1 h, and biotinylated goat anti-rabbit IgG (1:200) for 30 min. The location of rTFF3 antibody binding was visualized using the avidin-biotin method (Vector), and a brown reaction product was obtained with a peroxidase substrate (diaminobenzidine and phosphate-buffered saline in addition to 0.3% hydrogen peroxide). This study was performed to examine the effect of rTFF3 expression on base-line and post-indomethacin morphometry (microdissected villi), proliferation (BrdUrd staining), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), cell migration up the crypt and villus (using double labeling with BrdUrd and [3H]thymidine), and cell adhesion molecule distribution (E-cadherin immunohistochemistry). Six groups (n = 6/group) of control and rTFF3 transgenic animals were injected with [3H]thymidine (0.5 mCi/kg, intraperitoneally; Amersham Pharmacia Biotech) 17 h before killing and BrdUrd (50 mg/kg, intraperitoneally) 1 h before killing, i.e. the “double labeling technique,” to determine the differences in proliferation and the cell migration between groups. All animals also received a single dose of indomethacin (85 mg/kg, subcutaneously) at various time points either before or after the [3H]thymidine and BrdUrd, so that at the time of killing, changes in morphology, morphometry, and cell migration could be assessed 0, 6, 12, 18, 24, and 30 h after injection of indomethacin. Following killing, the various sections of the intestine were dissected free, and the weights and lengths of the small and large intestine of transgenic and negative littermates were recorded. To maintain consistency between animals, the lengths of the small intestine and the colon were expressed as 100%, and two serial 1-cm samples were taken from the following regions of the small intestine: 5% of small intestine length (defined as duodenum), 30% of small intestine length (defined as jejunum), and 90% of small intestine length (defined as ileum). The proximal 1-cm segments of each section were collected in Carnoy's solution, left at room temperature for 4 h, and then stored in 70% alcohol until further assessment of morphology and morphometry. The second 1-cm segments were fixed in neutral buffered formalin for subsequent analysis of migration, proliferation, E-cadherin expression, and apoptosis. Using methods described previously (15Goodlad R.A. Cell Biology: A Laboratory Handbook. Academic Press, San Diego1994: 205-216Google Scholar), small pieces of the Carnoy's-fixed jejunal and ileal tissue samples were hydrated, hydrolyzed, stained with the Feulgen reaction, and transferred to 45% (v/v) acetic acid, and the crypts and villi were teased apart under a stereo dissecting microscope. The tissues were then transferred to a glass microscope slide, flattened gently beneath a coverslip, and examined under a compound microscope. The villus height, crypt depth, and cross-sectional surface area were assessed by tracing the outline of the crypts and villi using a precalibrated drawing tube. The tracings were then scanned and analyzed using a flatbed scanner connected to an Apple Macintosh computer running the National Institutes of Health public domain program IMAGE. Twenty individual crypts and villi were assessed in each animal at each site, and the mean values from these 20 measurements were used in the subsequent ANOVA. This technique utilizes the fact that because the BrdUrd was given shortly before death, whereas the [3H]thymidine was given 17 h prior, the BrdUrd provides a marker for the proliferative zone within the crypt, whereas [3H]thymidine-positive cells provide an index for the distance traveled by cells up the villus over this 16-h period. The neutral buffered formalin-fixed samples were embedded in paraffin wax, and 5-μm-thick sections were cut on gelatin-coated slides. The sections were hydrolyzed in 1 m HCl for 8 min and 60 °C and then stained for BrdUrd with MAS 250 (Sera-Lab, Loughborough, UK) and peroxidase-conjugated anti-rat IgG. A brown reaction product was obtained with a peroxidase substrate (diaminobenzidine and phosphate-buffered saline in addition to 0.3% hydrogen peroxide). Autoradiography was then carried out on these same sections; slides were dipped in K2 (Ilford Ltd., Mobbersley, UK) emulsion dissolved in water warmed to 45 °C, left to dry on a cold plate, and exposed at 4 °C for 4 weeks. The slides were immersed in D-19 developer (Kodak, Paris, France) for 4 min, immersed in 1% acetic acid for 30 s, and finally fixed twice in 30% sodium thiosulfate for 4 min. The slides were then washed in cold tap water for 1 h and counterstained using Giemsa solution. For each animal, at each of the two sites, the positions of [3H]thymidine- and BrdUrd-labeled cells were recorded from 20 individual crypts and villi. The highest labeled position was determined for both the [3H]thymidine- and the BrdUrd-labeled cells and used in the subsequent ANOVA. The rate of cell migration was determined by the difference between the highest [3H]thymidine- and BrdUrd-labeled cells. The difference between these cell positions shows the maximal distance that cells migrated within the 16-h period of the experiment. Tissue sections were cut and stained for BrdUrd as described above and counterstained with hematoxylin. The slides were examined until well orientated crypts were found. The number of labeled mitotic cells/crypt were recorded for each individual animal. Twenty crypts/animal were scored at each site, and the mean number of labeled cells was used in the subsequent ANOVA. All tissues were scored by a person unaware of the experimental conditions. In vitrostudies suggest that addition of TFF peptides may result in a down-regulation of membranous E-cadherin expression (16Liu D. El-Harirry I. Karayiannakis A.J. Wilding J. Chinery R. Kmiot W. McCrea P.D. Gullick W.J. Pignatelli M. Lab. Invest. 1997; 77: 557-563PubMed Google Scholar). We therefore examined changes in the distribution of E-cadherin in the study groups; 4-μm sections of the paraffin-embedded tissue samples were cut onto poly-l-ornithine-coated slides and rehydrated. The endogenous peroxidase activity was blocked with 3% hydrogen peroxide, and sections were microwaved for 10 min at 1200 Watts in citric acid buffer, pH 6. The slides were incubated with normal swine serum (1:20) for 30 min followed by a primary monoclonal antibody directed against E-cadherin. All of the sections were tested with two different monoclonal antibodies: HECD-1, directed against the extracellular domains 1 and 2 of human E-cadherin, (10 μg/ml, 17), and a rat-anti-mouse E-cadherin monoclonal antibody (Sigma catalog number U3254). Antibodies were incubated with slides for 16 h at 4 °C. Labeling was performed using a commercial streptavidin-biotin-peroxidase technique (DAKO, Ely, UK). The sections were examined blind and scored for intensity (strong, weak, or negative) and distribution (basolateral only, apical, or circumferential). Three nonadjacent sections 4 μm thick were cut from each paraffin-embedded tissue sample onto poly-lysine-coated glass slides. Cells containing DNA strand breaks, a marker of apoptosis, were detected by staining using the terminal deoxyuridine nucleotidyl nick end labeling method as described previously (18Moss S.F. Attia L. Scholes J.V. Walters J.R. Holt P.R. Gut. 1996; 39: 811-817Crossref PubMed Scopus (146) Google Scholar). For each block at least 10 well orientated-crypt villus units were evaluated by light microscopy (×400 magnification) by an observer unaware of the experimental conditions. The total number of cells/crypt and villus were counted, as were the number of positively staining cells/crypt and villus. The number of positively stained cells/100 cells was expressed as the apoptotic index (%). This in vitro method measures the short circuit current (Isc) across mucosal preparations under voltage-clamp (0 mV) conditions. The changes in resistance following challenge with luminal thus provides an indirect method of assessing tissue sensitivity to injurious agents (19Rutten M.J. Ito S. Mechanisms of Mucosal Protection of the Upper GI Tract. Raven Press, New York1984: 987-998Google Scholar). Transgenic mice and negative littermates (n = 6/group/site) were killed, and four adjacent segments of either jejunum and ileum intestine were placed immediately into Krebs-Henseleit (KH) solution (118 mm NaCl, 4.7 mm KCl, 2.5 mm CaCl2, 1.2 mm MgSO4,1.2 mm KH2PO4, 25 mmNaHCO3, and 11.1 mm glucose). Full thickness tissue sections were then placed in between the two halves of a modified Ussing chamber (0.2 cm2 of exposed area) and bathed on each side with 5 ml of oxygenated (95% O2, 5% CO2) KH solution at 37 °C, pH 7.4. Tissues were voltage-clamped at 0 mV, and the resultant basalIsc was recorded continuously, on top of which 1-mV voltage pulses were applied at 100-s intervals throughout the period of experimentation. The resultant deflections in the current were recorded, and the resistance (in ohms·cm2) was determined using Ohms Law. The mean resistance calculated from six consecutive voltage pulses, 10 min prior to washes with either KH (both sides for 10 min) or an acid wash (50 mmHCl apical and KH basolateral for 10 min) was denoted as 100% for each preparation. Following 10 min of wash (as described above), tissues were bathed once again with fresh KH. The individual resistance measurements were calculated as a percentage of the mean pre-wash values for a further 60 min. For statistical analyses, the data from all experiments are expressed as the means ± S.E. For the study on base-line parameters and sensitivity to indomethacin, the data were analyzed by two-way ANOVA using the presence of transgene and the time since administration of indomethacin as factors. Where a significant effect (p < 0.05) was found, individuals t tests were performed, based on the group means and residual obtained from the ANOVA, a method equivalent to repeated measures analyses. For the voltage-clamp study, basal Isc and resistance were compared between the transgenic and control groups using unpaired two-tailed t testing. The changes in resistance following HCl exposure were analyzed by two-way ANOVA using the presence of transgene and the time since acid exposure as factors (zero time point not included as zero variance). Subsequent analyses were performed as for the indomethacin study. Positive heterozygote offspring were detected using PCR (Fig.2 A). As expected when using this promoter (20Playford R.J. Marchbank T. Goodlad R.A. Chinery R. Poulsom R. Hanby A.H. Wright N.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2137-2142Crossref PubMed Scopus (165) Google Scholar), Northern blot analyses (Fig. 2 B) and immunohistochemical staining showed that expression of rTFF3 was restricted to the jejunum of transgenic animals. Expression of rTFF3 was limited to enterocytes on the surface of the villi, with the crypts being negative (Fig. 3).Figure 3Distribution of rat TFF3 in FABPi−1178 to +28-rTFF3 fusion gene transgenic mice as demonstrated by immunostaining. Immunohistochemical staining was performed using a polyclonal antibody directed against TFF3. A brown reaction product was then obtained with a peroxidase substrate. A, control animals showed presence of (mouse) TFF3 in the goblet cells of the jejunal villi (initial magnification, ×100). B, in the transgenic animals, additional positivity (because of the presence of rat TFF3) was seen in the enterocytes of the jejunal villi but, as expected using the FABP promoter, did not result in expression in the crypt region (initial magnification, ×37). C, immunostaining of villi of a transgenic animal that have been cut in cross-section showing the enterocytes stained positive for TFF3, whereas the cells and mesenchyme of the villi core were negative (initial magnification, ×100).View Large Image Figure ViewerDownload Hi-res image Download (PPT) The morphology of microdissected villi in the jejunum of control and transgenic animals under base-line conditions was identical, with long, round ended villi (Fig.4, top panel). The villi obtained from control animals that had received indomethacin were progressively shorter with distal bulbous expansion (Fig. 4,middle panel). This effect of indomethacin was much less marked in the jejunum of transgenic animals (i.e. the rTFF3-expressing region; Fig. 4, bottom panel). Morphometric analyses showed a progresssive fall in the villus height of both the jejunum and ileum, reaching a minimum size after 12 h in the jejunum (Fig. 5, upper panel) and 24 h in the ileum (Fig. 5, lower panel). ANOVA for jejunal villus height gave a significant effect for the presence of transgenic DNA (p < 0.001) and administration of indomethacin (p < 0.001), with a significant interaction between transgenic status, administration of indomethacin, and timing (p < 0.001). This showed that the effect of the presence of transgenic DNA varied depending on the presence of indomethacin and its time since administration.Figure 5Effect of indomethacin on the jejunal morphometry of transgenic and control mice. Experimental protocol is as described in the legend for Fig. 2. Villus heights were assessed in microdissected villi obtained from control (○) and transgenic (●) animals under base-line conditions (0 h) and at various times after receiving the injection of indomethacin (n = 6 animals/group). The results are expressed as the means + S.E. * and **,p < 0.05 and <0.01 versus equivalent site and time of control animals, respectively. Note that y axis of the lower panel has a smaller range, reflecting the fact that ileal villi are shorter. Upper panel, in the jejunum of control animals, indomethacin caused a significant reduction (p < 0.001) in villus height during the period 6–24 h after indomethacin administration. The degree of shortening was much less severe, however, in the jejunum (rTFF3-expressing region) of transgenic littermates. Lower panel, indomethacin also caused significant shortening of the villi of the ileal region of control animals. A similar degree of shortening was also seen in the ileum (non-rTFF3-expressing region) of transgenic animals.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In contrast to the finding in the jejunum, there was no difference in the degree of shortening of the ileal villi between control and trangenic animals caused by indomethacin (Fig. 5,lower panel). ANOVA gave a significant effect caused by indomethacin administration (p < 0.001) but none caused by the presence of transgenic DNA (p = 0.747), with no interaction between transgene and indomethacin (p = 0.768; Fig. 5, lower panel). For both the jejunal and ileal regions, assessment using villus surface area as the parameter of damage gave similar results to using villus height (data not shown). There was no significant effect on the crypt depths caused by the presence of transgenic DNA or indomethacin administration (data not shown). There was no significant difference in proliferation, as assessed by the number of BrdUrd-labeled mitotic cells/crypt, between transgenic mice and their negative littermates at any time point in either the jejunum or the ileum (effect of presence of transgenic DNA on ANOVA, p= 0.514 for jejunum and p = 0.388 for ileum). Administration of indomethacin caused a significant increase in the number of mitoses compared with base line at 6 and 12 h post-indomethacin administration and was followed by a decrease at 30 h post-indomethacin (Fig. 6,upper panel). Administration of indomethacin also caused a significant increase in the rate of migration compared with base line at 6, 12, and 18 h in transgenic and control animals (Fig. 6, lower panel). However, there was no significant difference in the rate of migration, as assessed by the change in the cell position over 16 h, between transgenic mice and their negative littermates at any time point in either jejunum or ileum (effect of presence of transgenic DNA on ANOVA,p = 0.122 for jejunum and p = 0.231 for ileum). Base-line apoptotic index of combined crypt and villus values was 1.54% in the jejunum of controls and 2.72% in transgenic animals (group SEM 1.03). ANOVA showed no significant effect of the presence of transgenic DNA (p = 0.133) and no effect of indomethacin (p = 0.299), with no interaction (p = 0.314). Subanalyses of the crypt and villi apoptotic indices also showed no significant differences between groups (data not shown). Both the anti-human and anti-mouse monoclonal antibodies used gave identical results; the enterocytes on the villi and within the crypts showed the presence of strong E-cadherin staining on basolateral membranes. The expression of E-cadherin was not influenced by the presence of transgene or indomethacin (Fig. 7). There was no significant difference between theIsc values in the jejunum of transgenic and control animals (6.38 ± 2.8 versus 3.75 ± 1.2 μA/cm2, p = 0.418). The ileal samples also showed no significant differences (3.05 ± 0.98 μA/cm2 in transgenic versus 3.96 ± 1.4 μA/cm2 in control animals, p = 0.61). Basal resistance measurements in the jejunal tissue of transgenic animals (rTFF3-expressing intestinal region) were higher than the equivalent region of controls (326 ± 60 versus195 ± 48 ohm·cm2, p < 0.05). In contrast, there was no significant difference in the basal resistances between transgenic and control animals in the ileal (non-rTFF3-expressing) region. The fall in resistance of jejunal tissue following exposure to HCl administration was significantly less in the rTFF3-expressing jejunal region of transgenic animals (effect of transgene p < 0.001; Fig.8). There was no significant difference between transgenic animals and controls, however, in the sensitivity to HCl exposure in the non-rTFF3 ileal region (ANOVA effect of trangene,p = 0.11; Fig. 8). Ectopic expression of the rat homologue of TFF3 in the jejunum of mice resulted in a normal phenotype under nondamaged circumstances but reduced the amount of initial injury sustained using in vivoand in vitro models. This protective effect of rTFF3 in the transgenic animals was limited to regions that overexpressed the peptide and, based on results from the in vivo experiment, did not appear to be mediated by alteration in proliferation, migration, or apoptosis. The trefoil peptides form a family of molecules that share a motif comprising six-cysteine residues linked by three intrachain disulfide bonds. This configuration, termed a trefoil or P domain, is distinct from those found in other peptide families such as epidermal growth factor. Three members of this family, TFF1, TFF2, and TFF3, are found in mammals, and the amino acid sequence is h" @default.
• W1999824352 created "2016-06-24" @default.
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• W1999824352 date "2001-06-01" @default.
• W1999824352 modified "2023-10-14" @default.
• W1999824352 title "Effect of Ectopic Expression of Rat Trefoil Factor Family 3 (Intestinal Trefoil Factor) in the Jejunum of Transgenic Mice" @default.
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