Biomedical subjects

A M Hanby

Publications and source records attributed to A M Hanby.

At least 55 records · Page 3Linked to original sources

PubMed1996

Transgenic mice that overexpress the human trefoil peptide pS2 have an increased resistance to intestinal damage.

pS2 is a member of the trefoil peptide family, all of which are overexpressed at sites of gastrointestinal injury. We hypothesized that they are important in stimulating mucosal repair. To test this idea, we have produced a transgenic mice strain that expresses human pS2 (hpS2) specifically within the jejunum and examined the effect of this overexpression on proliferation and susceptibility to indomethacin-induced damage. A transgenic mouse was produced by microinjecting fertilized oocytes with a 1.7-kb construct consisting of rat intestinal fatty acid binding protein promoter (positions -1178 to +28) linked to full-length (490 bp) hpS2 cDNA. Screening for positive animals was by Southern blot analysis. Distribution of hpS2 expression was determined by using Northern and Western blot analyses and immunohistochemical staining. Proliferation of the intestinal mucosa was determined by assessing the crypt cell production rate. Differences in susceptibility to intestinal damage were analyzed in animals that had received indomethacin (85 mg/kg s.c.) 0-30 h previously. Expression of hpS2 was limited to the enterocytes of the villi within the jejunum. In the nondamaged intestine, villus height and crypt cell production rate were similar in transgenic and negative (control) litter mates. However, there was a marked difference in the amount of damage caused by indomethacin in control and transgenic animals in the jejunum (30% reduction in villus height in controls vs. 12% reduction in transgenic animals, P < 0.01) but the damage sustained in the non-hpS2-expressing ileal region was similar in control and transgenic animals. These studies support the hypothesis that trefoil peptides are important in stimulating gastrointestinal repair.

Animals↗

PubMed1996

An inbred colony of oncogene transgenic mice: diversity of tumours and potential as a therapeutic model.

Transgenic mice carrying the activated rat c-neu oncogene under transcriptional control of the MMTV promoter were backcrossed to BALB/c mice, with the aim of developing a model for cancer therapy. A total of 86 of 268 transgene-positive mice in the first five generations developed 93 histologically diverse tumours (median age of onset 18 months). The cumulative incidence of breast tumours at 24 months was 18%, and overall tumour incidence 31%. As well as expected c-neu expressing breast cancers, lymphomas and Harderian gland carcinomas developed. Virgin mice had fewer mammary tumours than those with two litters. Breast carcinomas metastasised to the lungs, and lymphomas were widely disseminated. The tumours showed a range of architectural patterns, which resembled human breast cancers or lymphomas. This diversity was reflected in S-phase fraction and aneuploidy. Breast tumours transplanted to nude mice showed variable responses to interferon (IFN)-alpha and gamma. A tumour transplanted to BALB/c mice responded to interleukin (IL)-12. There was significant decline in transgene positivity with successive generations. The diversity, histological and biological resemblance to human cancer suggests that the model has potential for evaluating novel therapies. However, further genetic and environmental manipulations are required to increase tumour incidence and decrease age of onset.

Animals↗

PubMed1996

The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract.

BACKGROUND: While it is clear that luminal epidermal growth factor (EGF) stimulates repair of the damaged bowel, its significance in maintaining normal gut growth remains uncertain. If EGF is important in maintaining normal gut growth, the EGF receptor (EGF-R) should be present on the apical (luminal) surface in addition to the basolateral surface. AIMS/SUBJECTS/METHODS: This study examined the distribution of the EGF-R in the epithelium throughout the human gastro-intestinal tract using immunohistochemistry, electron microscopy, and western blotting of brush border preparations. RESULTS: Immunostaining of the oesophagus showed circumferential EGF-R positivity in the cells of the basal portions of the stratified squamous epithelium but surface cells were EGF-R negative. In the normal stomach, small intestine, and colon, immunostaining localised the receptor to the basolateral surface with the apical membranes being consistently negative. EGF-R positivity within the small intestine appeared to be almost entirely restricted to the proliferative (crypt) region. Western blotting demonstrated a 170 kDa protein in whole tissue homogenates but not in the brush border vesicle preparations. CONCLUSIONS: As the EGF-R is located only on the basolateral surfaces in the normal adult gastrointestinal tract, the major role of luminal EGF is probably to stimulate repair rather than to maintain normal gut growth.

Blotting, Western↗

PubMed1996

H-RYK, an unusual receptor kinase: isolation and analysis of expression in ovarian cancer.

BACKGROUND: Protein tyrosine kinases play an important role in cellular metabolism as key components of signal transduction pathways. They are involved in cellular growth, differentiation, and development. Receptor tyrosine kinases (EGF receptor and c-erbB2) have been shown to be important in the pathogenesis of cancer. In ovarian cancer, overexpression of c-erbB2, a type I receptor, has been correlated with an adverse effect on survival of patients. MATERIAL AND METHODS: An unusual receptor tyrosine kinase, H-RYK, has been isolated from a complimentary DNA library of SKOV-3, an epithelial ovarian cancer cell line, using a polymerase chain reaction-mediated approach. RESULTS: The primary structure of the predicted amino acid sequence of the protein shows a novel NH2-terminal region. The catalytic region shows homology to other tyrosine kinases, the closest homology being with v-sea (39%). A significant alteration in the catalytic domain is that the highly conserved "DFG" triplet in subdomain VII is altered to "DNA." The gene was mapped to chromosome 3q22. A single transcript of 3.0 kb is expressed in heart, brain, lung, placenta, liver, muscle, kidney, and pancreas by Northern analysis with maximal expression in skeletal muscle. In situ hybridization analysis on human tissues demonstrated localization of message in the epithelial and stromal compartment of tissues such as brain, lung, colon, kidney, and breast. There was minimal to absent expression of H-RYK on surface epithelium of ovaries. In benign (3) and borderline tumors of the ovary (5), there was expression in the stromal compartment. However, in malignant tumors (24) there was increased expression predominantly confined to the epithelium. Polyclonal antisera raised against synthetic peptides recognize a 100-kD protein in ovarian cancer cells and other cell lines. In contrast to other receptor tyrosine kinases, the receptor did not phosphorylate in an in vitro kinase assay. CONCLUSIONS: The expression of this unusual receptor tyrosine kinase in epithelial ovarian cancer suggests that it may be involved in tumor progression, which needs further investigation.

Adenocarcinoma↗

PubMed1996

Distribution and expression of pancreatic secretory trypsin inhibitor and its possible role in epithelial restitution.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent serine protease inhibitor that prevents excessive digestion of the gastrointestinal mucus but may also directly affect epithelial function. We therefore examined the distribution of PSTI in the human adult and fetus using immunohistochemistry and in situ hybridization and examined its effects on cell proliferation and migration in vitro. PSTI peptide and mRNA were found in the exocrine pancreas, mucus-producing cells of the normal gastrointestinal tract, acinar component of the normal breast, and surface epithelial cells at the edge of benign gastric ulcers. Peptide, but not message, was identified in the renal proximal tubule, probably reflecting reabsorption of filtered peptide. Purified human PSTI did not affect proliferation of the human colonic cell line HT-29 but caused a threefold increase in the rate of migration in an in vitro wounding model of restitution. This effect was inhibited by co-administering a PSTI-neutralizing antibody, a transforming growth factor-beta-neutralizing antibody, or an epidermal growth factor receptor-blocking antibody. As PSTI is widely distributed in several human organ systems and stimulates cell migration in vitro, we conclude that PSTI is likely to have additional roles to that of preserving the gastrointestinal mucous layer from excessive digestion.

Adult↗

PubMed1996

Downregulation of E-cadherin in the reparative epithelium of the human gastrointestinal tract.

E-cadherin, an epithelial adhesion molecule, is critical for the maintenance of cell polarity and differentiation. We studied the distribution of E-cadherin in normal gut and in enteric ulceration to test the hypothesis that the motility of regenerative epithelium over ulcers is associated with a decrease in E-cadherin expression. Sections of normal stomach, small intestine, and colon were examined for E-cadherin distribution using the antibody HECD-1 and compared with the pattern seen in peptic ulceration and Crohn's disease. A subset was examined by in situ hybridization using 35S radiolabeled E-cadherin riboprobes. A wounding system employing the HT-29 cell line was used as an in vitro model of early healing. In the normal gut uniform strong basolateral staining was seen. Areas of ulceration showed a patchy reduction in membrane localized E-cadherin in regenerating epithelium, even though E-cadherin mRNA was demonstrable in this population. In wounded confluent HT29 layers, migrating cells also showed reduced E-cadherin immunostaining. These data support the notion that the motility of restitutive epithelial cells may relate to altered patterns of E-cadherin and that this may play an important role in the reconstitution of epithelial integrity after mucosal injury.

Cadherins↗

PubMed1996

Bcl-2 expression in colorectal tumors: evidence of different pathways in sporadic and ulcerative-colitis-associated carcinomas.

Colorectal cancers (CRCs) differ in their age at presentation, distribution, histological features, and prognosis. If tumor biology reflects genetic events, these tumors might be expected to show differences in their genetic pathways. In this study, we investigated the role of Bcl-2 in the development of three different tumor groups. Using markers at eight different microsatellite locl, we characterized one group of 34 left-sided sporadic CRCs as replication error negative (RER-) and another group of 18 left-sided sporadic CRCs as replication error positive (RER+). These tumors, together with a third group of 22 left-sided ulcerative-colitis-associated CRCs (UCACRCs), were then examined by immunohistochemistry for Bcl-2 overexpression. Of 34 of the RER- tumors, 21 (62%) and 10 of 18 (56%) of the RER+ tumors were positive for Bcl-2 overexpression. In contrast, only 5 of 22 (23%) of the UCACRCs showed similar overexpression. Our results show a significantly lower frequency of Bcl-2 overexpression in UCACRCs as compared with sporadic CRCs (P < 0.005) but no difference between sporadic left-sided RER+ and RER- CRCs. These data provide additional evidence that UCACRCs may develop along a pathway that is different from that of sporadic CRCs.

Carcinoma↗

PubMed1995

Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule.

FAT, a new member of the human cadherin super-family, has been isolated from the T-leukemia cell line J6. The predicted protein closely resembles the Drosophila tumor suppressor fat, which is essential for controlling cell proliferation during Drosophila development. The gene has the potential to encode a large transmembrane protein of nearly 4600 residues with 34 tandem cadherin repeats, five EGF-like repeats, and a laminin A-G domain. The cytoplasmic sequence contains two domains with distant homology to the cadherin catenin-binding region. Northern blotting analysis of J6 mRNA demonstrated full-length, approximately 15-kb, FAT message in addition to several 5'-truncated transcripts. In addition to its presence in J6 cells, in situ hybridization revealed FAT mRNA expression in epithelia and in some mesenchymal compartments. Furthermore, higher levels of expression were observed in fetal, as opposed to adult, tissue, suggesting that its expression may be developmentally regulated in these tissues. FAT shows homologies with a number of proteins important in developmental decisions and cell:cell communication and is the first fat-like protein reported in vertebrates. The gene encoding FAT was located by in situ hybridization on chromosome 4q34-q35. We propose that this family of molecules is likely to be important in mammalian developmental processes and cell communication.

Amino Acid Sequence↗

PubMed1995

Nuclear and cytoplasmic bcl-2 expression in endometrial hyperplasia and adenocarcinoma.

The bcl-2 proto-oncogene, which inhibits programmed cell death (apoptosis), has recently been found to be cyclically expressed in human endometrium. In order to investigate its role in endometrial hyperplasia and neoplasia, bcl-2 expression was studied in 25 cases of endometrial carcinoma and 20 cases of endometrial hyperplasia (eight simple, two complex, and ten atypical hyperplasias). Uniform intense cytoplasmic bcl-2 expression was found in all cases of non-atypical hyperplasia, and less strong positivity in eight out of ten cases of atypical hyperplasia. In well-differentiated carcinomas, nine out of ten showed weak to moderate bcl-2 expression, whereas six out of seven poorly differentiated carcinomas were bcl-2-negative. Moderately differentiated tumours were an intermediate group, with six out of eight being positive. Widespread localization of bcl-2 protein to the chromosomes of dividing cells was also demonstrated, regardless of cytoplasmic bcl-2 expression, with rare staining of interphase nuclei. Our findings suggest a role for bcl-2 in the natural history of endometrial neoplasia and studies are needed to determine its usefulness as a prognostic marker. The finding of bcl-2 localization to chromosomes has important implications for its mode and site of action.

Adenocarcinoma↗

PubMed1995

Human spasmolytic polypeptide is a cytoprotective agent that stimulates cell migration.

BACKGROUND/AIMS: Gastric epithelium is attacked by acid, pepsin, and ingested agents. When a mucosal lesion occurs, the defect is rapidly closed by cell migration. Because spasmolytic polypeptide is rapidly produced at sites of injury, we postulated that human spasmolytic polypeptide (hSP) was important in mucosal repair. Recombinant hSP was used to test this hypothesis. METHODS: The ulcer healing effect of various doses of hSP administered orally and subcutaneously was examined using an indomethacin (20 mg/kg) restraint rat model of gastric damage. Stability of hSP in gastrointestinal juice was determined using size-exclusion chromatography. The effect of hSP on migration of human colonic carcinoma cell lines HT29 and SW480 was determined using collagen gel invasion and wounded monolayer assays. Proliferation was assessed using [3H]thymidine incorporation and toluidine blue staining. RESULTS: Infusions of hSP at 25 and 50 micrograms.kg-1.h-1 subcutaneously decreased gastric damage by about 50% (P < 0.01) without changing acid secretion. Oral hSP was ineffective. hSP was stable in gastrointestinal juice. hSP stimulated migration of HT29 cells but did not affect proliferation and had no effect on SW480 cells. CONCLUSIONS: hSP may play a key role in the early stages of mucosal repair by stimulating the initial re-epithelialization by cell migration.

Animals↗

PubMed1995

Level of expression of E-cadherin mRNA in colorectal cancer correlates with clinical outcome.

A series of colorectal carcinomas (n = 49) resected from patients with known clinical outcomes were analysed for E-cadherin expression using in situ hybridisation to measure mRNA. Patients surviving 5 years or longer (n = 31) exhibited significantly higher levels of E-cadherin mRNA than those surviving less than 5 years (n = 18, P = 0.003). These preliminary results from this small sample suggest that E-cadherin expression may be a useful prognostic marker in colorectal cancer patients.

Cadherins↗

PubMed1995

Cutaneous squamoproliferative lesions in renal transplant recipients. Differentiation from lesions in immunocompetent patients.

Of 291 immunosuppressed renal transplant recipients (RTRs) with surviving allografts attending the Royal London Hospital, 171 patients (59%) were found to have warty keratoses. On histological analysis, the lesions in 50 patients (17%) showed partial-thickness dysplasia, and 34 (12%) had one or more invasive squamous cell carcinoma (SCC) and/or one or more in situ SCC or full-thickness dysplasia. We examined the claim that squamoproliferative lesions in RTRs possess distinctive histopathological features that differ from those of similar lesions occurring sporadically in the nonimmunosuppressed population. We compared 40 squamoproliferative lesions from RTRs with 40 matched squamoproliferative lesions from nonimmunosuppressed patients; lesions were coded and their source was unknown to the assessors. Two dermatopathologists independently assessed the cases and gave scores for 11 histological features that have been reported to be characteristic of such lesions in the immunosuppressed population. These included a warty architecture, koilocytes, and multinucleate giant cells. Using these criteria, it was not possible to distinguish lesions of immunosuppressed patients from those of immunocompetent people.

Carcinoma in Situ↗

PubMed1995

Expression of trefoil peptides pS2 and human spasmolytic polypeptide in gastric metaplasia at the margin of duodenal ulcers.

Duodenal ulcers are associated with gastric metaplasia in the duodenum, both at the ulcer margin and at more distant sites in the duodenal bulb. pS2 and human spasmolytic polypeptide (hSP) are secretory peptides expressed in gastric epithelial cells and in gastric metaplasia. As these peptides may be important in ulcer healing, this study investigated the possibility that the expression of pS2 and hSP is increased in gastric metaplasia at the margin of duodenal ulcers. Duodenal bulb biopsy specimens from 12 duodenal ulcer patients were assessed. Sections were immunostained with monoclonal antibodies for pS2 and hSP. Cytoplasmic stain intensities were measured by an image analysis system and expressed as integrated optical density (IOD) units, In situ hybridisation for pS2 and hSP mRNA was carried out on parallel sections. Duodenal sections were also stained with diatase periodic acid Schiff/alcian blue to localise areas of gastric metaplasia. pS2 antigen staining in the duodenum was restricted to surface epithelial cells, and hSP to acinar and ductular components of Brunner's gland. mRNA localisation corresponded to immunostaining cells. In gastric metaplasia, pS2 expression was greater at the ulcer margin than away from the ulcer, as judged by the intensity of antibody staining (mean IOD units (SEM), 20.6 (3.3) v 9.5 (3.0); p < 0.001). There was a trend towards greater hSP staining at the ulcer margin but this did not achieve statistical significance. These findings support the putative role of pS2 and possible hSP in mucosal healing and providy further evidence for an autocrine 'ulcer-gastric metaplasia-repair' loop involving these trefoil peptides.

Brunner Glands↗

PubMed1995

Influence of inflammatory bowel disease on the distribution and concentration of pancreatic secretory trypsin inhibitor within the colon.

Gastrointestinal epithelia contain a powerful protease inhibitor called pancreatic secretory trypsin inhibitor (PSTI). Patients with inflammatory bowel disease have changes in mucus structure suggestive of increased proteolysis. We therefore examined the distribution and concentration of PSTI in the colon of normal subjects and patients with inflammatory bowel disease. In normal subjects (N = 12), mucosal levels of PSTI were approximately 200 ng/mg protein in all regions of the colon and was localized to goblet and endocrine cells. Mucosal PSTI levels in the (affected) left side of the colon of patients with active (N = 12) or quiescent (N = 10) ulcerative colitis were reduced (approximately 80% of control in descending colon, 55% of control in sigmoid colon, and 50% of control in rectum, all P < 0.01), whereas levels in the (unaffected) right side of the colon were normal. PSTI levels were also reduced to approximately 65% of control in colonic tissue affected by Crohn's disease (N = 6, P = 0.01) and immunostaining showed PSTI positivity within the ulcer-associated cell lineage. As the mucous layer is important in preserving mucosal integrity, our finding of prolonged reduction in mucosal PSTI levels after an episode of ulcerative colitis probably represents a long-term reduction in a mucosal defense mechanisms that could lead to increased susceptibility to episodes of inflammation.

Colitis, Ulcerative↗

PubMed1994

Expression of the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) in Barrett's metaplasia and the native oesophageal epithelium: delineation of epithelial phenotype.

The distribution of the two trefoil peptides pS2 and human spasmolytic polypeptide (hSP) and their mRNAs was investigated in 90 selected oesophageal biopsies, 23 of which contained epithelium with wholly gastric cardiac-type morphology, 52 specialized (intestinal)-type metaplasia, and 15 non-metaplastic oesophageal epithelium. No fundic-type epithelium was represented. The cardiac-type epithelium resembled true gastric antral epithelium, with hSP and pS2 mRNA localization to the superficial/foveolar compartment and hSP mRNA alone in deeper glands. The pattern of peptide distribution was broadly in line with the mRNA, but hSP peptide was generally not demonstrable in the surface epithelium. pS2 immunostaining was diffusely cytoplasmic, whereas in specialized-type mucosa it was aggregated and cytoplasmic. hSP mRNA was demonstrable in surface epithelium of incomplete- but not complete-type intestinal (specialized) metaplasia. Deep glands with morphological features of pyloric glands/ulcer-associated cell lineage (UACL) typically contained only hSP peptide and its mRNA. In three biopsies containing specialized epithelium, small foci morphologically identical to true small intestinal surface epithelium were seen in which only pS2 mRNA and peptide were found and then only in rare goblet cells. Neither squamous epithelium nor oesophageal glands contained demonstrable pS2/hSP mRNA or peptide. The function of these proteins in the metaplastic mucosa of Barrett's oesophagus is unknown but they may reflect an epithelium responding to repeated insult. Their localization facilitates definition of the disparate epithelium types seen in this portion of the gut, with both native gastric and intestinal epithelia, and may help to pinpoint high-risk epithelium in Barrett's oesophagus, a potentially preneoplastic condition.

Barrett Esophagus↗

PubMed1994

The production and characterization of a new monoclonal antibody to the trefoil peptide human spasmolytic polypeptide.

Human spasmolytic polypeptide (hSP) is a member of the growing family of trefoil peptides which are expressed in discrete regions of the body, most notably the gastrointestinal tract. Much of the research into the localization of the spasmolytic polypeptide has relied on hybridization in situ to detect its mRNA, due to the absence of a suitable antibody. The aim of the present study was to develop and characterize a monoclonal antibody against the human spasmolytic polypeptide, using a combination of immunohistochemistry and hybridization in situ. After immunoblotting, the antibody detected a 14 kDa protein in gastrointestinal tissue extracts from the stomach and small intestine only. Using immunohistochemistry, human spasmolytic polypeptide showed a distinctive staining pattern in the duodenum which co-localized with its mRNA. The co-localization of the immunoreactive peptide with its mRNA provides good evidence that the antibody truly recognized human spasmolytic polypeptide.

Animals↗

PubMed1994

Influence of inflammation and atrophy on pancreatic secretory trypsin inhibitor levels within the gastric mucosa.

BACKGROUND/AIMS: Gastrointestinal epithelia contain a powerful protease inhibitor called pancreatic secretory trypsin inhibitor (PSTI). Gastric mucus obtained from patients with atrophic gastritis or gastric ulceration shows changes suggestive of excessive proteolytic digestion. Therefore, the aim of this study was to examine whether mucosal PSTI levels are affected by gastritis and/or ulceration. METHODS: Mucosal PSTI levels were measured in 12 patients with normal gastric histology and 26 patients with gastritis and/or gastric ulceration. RESULTS: In control subjects, mean gastric PSTI concentrations were 990 ng/mg protein (95% confidence interval, 819-1195) in the antrum and 445 ng/mg protein (95% confidence interval, 395-502) in the body. Immunostaining for PSTI was strongly positive in the pyloric glands of the antrum and in the foveolar/surface and mucus neck cells of the gastric body. Tissue levels of PSTI were reduced by 40% in biopsy specimens showing superficial gastritis (P = 0.035) and by 75% in biopsy specimens showing atrophic gastritis and/or gastric ulceration (P < 0.001). Immunostaining was also reduced in specimens showing atrophic gastritis. CONCLUSIONS: The decrease in tissue PSTI levels associated with gastric atrophy probably represents a permanent reduction in one of the mucosal defense mechanisms. This may lead to the maintenance and even expansion of the gastritic process with time.

Aged↗

PubMed1994

Bcl-2 protein localizes to the chromosomes of mitotic nuclei and is correlated with the cell cycle in cultured epithelial cell lines.

bcl-2 gene expression confers a survival advantage by preventing cells from entering apoptosis. In contrast to the previously described cytoplasmic localization of Bcl-2 in epithelial cells in vivo, in this study we have demonstrated, in a series of human epithelial cell lines, that Bcl-2 also localizes to mitotic nuclei. Both immunocytochemical and immunoelectron microscopical examinations localize this protein to nuclei and in particular to chromosomes. Nuclear Bcl-2 expression in these cell lines is correlated with the cell cycle. There is relatively strong expression during mitosis, most intense during prophase and metaphase, declining in telophase and then the protein becomes undetectable soon after separation of the two daughter cells. The expression and distribution of Bcl-2 is influenced by treatment with excessive thymidine. These results indicate that Bcl-2 may protect the cells from apoptosis occurring during mitosis and suggest a possible role for the protein in cell immortalization.

Cell Cycle↗