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Biomedical subjects

A M Hanby

Publications and source records attributed to A M Hanby.

At least 73 records · Page 4Linked to original sources
PubMed1994

The kinetic organization of the ulcer-associated cell lineage (UACL): delineation of a novel putative stem-cell region.

We have examined the proliferative organization of the ulcer-associated cell lineage (UACL), in a series of human ileal tissues involved by Crohn's disease, using antibodies to trefoil peptides and Ki-67. The UACL arises in conjunction with endodermal damage and is likely to play a role in subsequent tissue repair. Our study supports the view that the UACL initially forms through extrusion from the base of a parent intestinal crypt without indigenous mitotic activity. Eventually a duct forms extending to the gut surface and within this duct a proliferative zone develops. We also confirm that the trefoil peptides pS2 and hSP, which play a major role in the repair process, are spatially segregated within the UACL with a zone of overlap in the duct. Using triple antibody labelling techniques we have demonstrated that the zone of overlap of these peptides coincides with the proliferative region and provides evidence for a novel stem cell zone.

Cell Division↗
PubMed1994

Isolation and characterization of an epithelial-specific receptor tyrosine kinase from an ovarian cancer cell line.

A protein receptor tyrosine kinase (RTK 6) has been isolated from a complementary DNA library of SKOV-3, an epithelial ovarian cancer cell line, using a polymerase chain reaction (PCR)-mediated approach. The primary structure of the predicted amino acid sequence of the protein shows a novel NH2-terminal region which has homology to a factor VIII-like domain. The juxtamembrane region is proline and glycine rich and is the longest for any known receptor kinase. The COOH-terminal catalytic domain has all of the canonical sequence motifs of a receptor tyrosine kinase with homology to the TRK-2H protein (49%). A single transcript of 4.5 kilobases is expressed at low levels in heart, placenta, lung, liver, muscle, kidney, and pancreas, with high levels of expression in the brain. Ribonuclease protection assay showed a varying level of expression of message in a panel of eight ovarian cancer cell lines compared to placenta. In situ hybridization analysis demonstrated localization of mRNA in the epithelial cells of the ovary, kidney, small bowel, lung, thymus, and brain. There was a lower level of message in normal, benign, and borderline tumors of the ovary compared to malignant tumors of the ovary. Polyclonal antisera raised against a COOH-terminal synthetic peptide recognize a M(r) 140,000 protein in ovarian cancer cells, which autophosphorylates in an in vitro kinase assay.

Amino Acid Sequence↗
PubMed1993

hP1.B, a human P-domain peptide homologous with rat intestinal trefoil factor, is expressed also in the ulcer-associated cell lineage and the uterus.

The six-cysteine P-domain motif forms the basic repeat unit of a growing family of mucin-associated peptides. A precursor for a human secretory polypeptide has been discovered by molecular cloning and deduced to have a single P-domain, termed hP1.B. The pre-pro-peptide has 67% amino acid identity with rat intestinal trefoil factor. We find, using the techniques of RNA analysis and in situ hybridization, that this P-domain peptide is expressed in the human gastrointestinal tract, where a number of pathological conditions affect its expression, and surprisingly find it is expressed in the uterus also. In the intestine, hP1.B is expressed by goblet cells, but in Crohn disease this peptide is synthesized and secreted additionally by the ulcer-associated cell lineage that is known to secrete two other trefoil peptides, pS2 and spasmolytic polypeptide (hSP). In the stomach, hP1.B mRNA is relatively scarce but is more abundant in foci of intestinal metaplasia and near to ulceration. Mucin-rich epithelial cells in hyperplastic polyps of the colon also express this peptide. The discovery of this P-domain peptide and its expression in association with mucins support the hypothesis that P-domains with mucins may subserve related functions in the maintenance and repair of mucosal function.

Amino Acid Sequence↗
PubMed1993

Epstein-Barr virus-related oral T-cell lymphoma associated with human immunodeficiency virus immunosuppression.

Epstein-Barr virus (EBV) is generally held to infect B cells and epithelial cells, although there are now reports of EBV infection in normal T cells and neoplastic T-cell diseases. In patients with human immunodeficiency virus (HIV) infection, EBV is associated with the benign epithelial lesion, hairy leukoplakia, and has been reported in up to 80% of acquired immunodeficiency syndrome (AIDS)-related B-cell lymphoma. This study shows the presence of EBV in malignant oral T-cell lymphoma in three AIDS patients, two of whom had concurrent manifestation of hairy leukoplakia. The T-cell lineage of the tumor cells was determined by positive immunophenotyping for T-cell markers and lack of B-cell or nonhematopoietic (cytokeratin) determinants. All tumors contained monoclonal T-cell populations shown by polymerase chain reaction, which showed amplification of T-cell receptor gamma chain DNA without evidence of Ig heavy chain gene rearrangement. Furthermore, these lesions showed the presence of EBV DNA and expression of EBV latent gene products in the tumor cells. EBV involvement in AIDS-related T-cell lymphoma has not been widely reported and may represent a further manifestation of opportunistic EBV infection arising in the HIV-immunocompromised host.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Expression and purification of a trefoil peptide motif in a beta-galactosidase fusion protein and its use to search for trefoil-binding sites.

The cysteine-rich trefoil motif of rat intestinal trefoil factor (rITF) was cloned and expressed in Escherichia coli. A 270-bp cDNA fragment including the signal sequence and the trefoil motif was cloned into the expression vector pAX5+ to direct the expression of a beta-galactosidase collagen-hinged fusion protein in E. coli. Cultures harbouring the recombinant plasmid produced a soluble novel protein with a molecular mass of 134.5 kDa, as predicted for the trefoil-motif-containing fusion protein. Purification of the rITF moiety was achieved by p-aminophenyl-thio-beta-D-galactoside(APTG)-affinity chromatography, collagenase digestion of the hybrid molecule, and removal of the beta-galactosidase-hinge molecule by a further APTG-affinity step. It was demonstrated that intrachain disulphide-bond formation in rITF occurred during the procedure, so no refolding steps were required. Analysis by immunoblotting revealed that the fusion protein and the cleaved trefoil-motif-containing protein were recognised by an antibody raised against the chemically synthesised peptide. The trefoil motif present in the fusion protein was used to localise putative trefoil-binding sites in sections of frozen rat tissue. Binding was demonstrated using the beta-galactosidase portion of the fusion protein as a reporter moiety, either directly with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside, or indirectly using a monoclonal antibody to beta-galactosidase and indirect immunohistochemistry. Binding sites were localised to the foveolar and surface epithelium of rat stomach, the collecting ducts of the kidney and within colonic crypts. The presence of a trefoil motif was necessary for binding. The use of beta-galactosidase fusion proteins for histochemical localisation of peptide-binding sites should prove more generally useful.

Animals↗
PubMed1993

The expression of the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) in 'gastric metaplasia' of the proximal duodenum: implications for the nature of 'gastric metaplasia'.

Expression of pS2 protein (an oestrogen-induced gene discovered in the MCF-7 breast carcinoma cell line) and its homologue human spasmolytic polypeptide (hSP) was analysed, using immunohistochemistry and in situ hybridization to their mRNAs, in the proximal duodenum of 17 partial gastrectomy specimens removed from individuals with chronic peptic ulceration. Eight were found to have gastric-type metaplasia. In gastric metaplasia, mRNAs for pS2 and hSP, and pS2 peptide antibody were co-localized in the cells covering the duodenal villi. pS2 immunostaining was diffusely cytoplasmic in nature. A similar pattern was seen in Brunner's gland ducts. The trefoil peptide localization in gastric metaplasia closely resembles that seen in superficial gastric epithelium and the distal Brunner's gland duct, which in turn shares morphological similarities with gastric epithelium. We therefore conclude that gastric metaplasia may be the result of an expansion of the surface component of the Brunner's gland duct. The function of these trefoil peptides is at present unknown, but their distribution elsewhere suggests an involvement in reparative mechanisms. The similarities between gastric foveolar and Brunner's gland duct epithelium may derive from common restitution-enhancing features pertinent to a locally harsh environment.

Brunner Glands↗
PubMed1993

Bcl-2 expression in adult and embryonic non-haematopoietic tissues.

The B-cell leukaemia/lymphoma-2 (bcl-2) proto-oncogene is unusual as its product appears to provide survival advantage to B cells by blocking apoptosis. In this study, the expression of bcl-2 has been examined in normal non-haematopoietic tissues, embryos, and psoriatic skin by immunohistochemical staining. Bcl-2 protein expression is mainly observed in cell populations with a long life and/or proliferating ability such as duct cells in exocrine glands, basal keratinocytes, cells at the bottom of colon crypts, and neurons. In the skin of both adult and embryo and also embryonic kidney and cartilage, bcl-2 expression was observed in cells which were undergoing morphological transition from undifferentiated stem cells to committed precursor cells. The finding of bcl-2 expression in the terminal differentiated syncytial trophoblast, but not cytotrophoblast, and in some cells responsive to hormone stimulation such as in the endometrium and myometrium suggests that the gene expression may be related to hormone responsiveness. As no bcl-2 localization was seen in the benign hyperproliferative skin condition psoriasis, this does not suggest a straight-forward link to proliferation. These observations support the view that the bcl-2 gene may have an important role in cell development, maturation, and the path to terminal differentiation.

Adult↗
PubMed1993

Beta 1 and beta 4 integrin expression in methacarn and formalin-fixed material from in situ ductal carcinoma of the breast.

The integrins are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Previous distribution studies on integrin expression have been limited by the requirement of cryostat sectioned tissues, and consequent poor resolution. We have examined 40 examples of ductal carcinoma in situ (DCIS) for the expression of both beta 1 and beta 4 integrin chains. These showed strong polarized membrane staining of residual myoepithelial cells (correlating with expression of smooth muscle specific actin) and of the basement membrane region with beta 1 and beta 4 antibodies respectively. In 12 out of 40 cases, the DCIS was negative for the beta 1 chain and a variable pattern of reactivity was seen in the remaining cases. The beta 4 chain was detected focally and weakly in the tumour cells of 7/40 DCIS and strongly in one; all of these cases were also positive for the beta 1 chain. Of the 22 cases where co-existent invasion was present, the infiltrating component showed either a similar degree or a diminution of the extent of immunostaining when compared with the in situ component; only one showed enhanced staining (beta 1 only). This study demonstrates that two of the main beta chains, beta 1 and beta 4, can be effectively demonstrated on methacarn and cold (4 degrees C) formalin-fixed tissues by avidin-biotin indirect immunoperoxidase staining and that the results are similar to those achieved using frozen tissue.

Acetates↗
PubMed1993

Spasmolytic polypeptide is a major antral peptide: distribution of the trefoil peptides human spasmolytic polypeptide and pS2 in the stomach.

BACKGROUND: The regional differences in the distribution of the trefoil peptides pS2 and human spasmolytic polypeptide (hSP) within the stomach are not known. The aim of this study was to gain insight into these functionally obscure molecules by characterizing their distribution. METHODS: Tissue from gastrectomy specimens removed for peptic ulceration was examined to chart the distribution of hSP and pS2, using immunohistochemistry and in situ hybridization to chart their messenger RNAs (mRNAs). RESULTS: Colocalization of pS2 and hSP was noted in the gastric foveolar and surface epithelium throughout the stomach. In the gastric antrum and pylorus, an extremely strong hSP mRNA signal was present within pyloric-type glands; strong hSP immunostaining was also seen at this site and in mucous-neck cells. Neither pS2 mRNA nor pS2 peptide were shown within the deep portions of the pyloric glands. Within areas of intestinal metaplasia, a few goblet cells immunostained for pS2 and putative ulcer associated cell lineage was seen with a pattern of trefoil peptide localization similar to the ileum. CONCLUSION: The detailed function of these trefoil peptides is unknown, but their distribution suggests involvement in repair-enhancing mechanisms. hSP may be an important antral peptide and both of these peptides may play a specific reparative role.

Epithelium↗
PubMed1993

Expression of gelatinase A and TIMP-2 mRNAs in desmoplastic fibroblasts in both mammary carcinomas and basal cell carcinomas of the skin.

AIMS: To compare the localisation of mRNAs for the basement membrane degrading enzyme gelatinase A (72 kilodalton type IV collagenase) and its inhibitor TIMP-2 in carcinomas of the breast and basal cell carcinomas of the skin which have little or no ability to metastasize. METHODS: In situ hybridisation was performed on formalin fixed, paraffin wax embedded blocks using 35S-labelled riboprobes on 16 mammary carcinomas, three fibroadenomas, and a benign phyllodes tumour, and on 15 basal cell carcinomas of the skin (BCC). RESULTS: Labelling for both mRNAs was detectable in 14 of 16 mammary carcinomas and in 13 of 15 BCC, most often over organising desmoplastic fibroblasts in the stroma around invasive epithelial aggregates. Some sparse labelling was seen over malignant epithelial cells in six of the mammary carcinomas but not in the BCC. Some expression of gelatinase A mRNA was also seen in fibroblasts of breast lobules adjacent to the mammary carcinomas and around engulfed adnexal elements in the BCC, but not in unaffected breast tissues, fibroadenomas, the phyllodes tumour or unaffected skin. CONCLUSIONS: Maximal expression of gelatinase A and TIMP-2 mRNAs occurs in malignant neoplasms as part of the host response to the presence of established neoplastic cells rather than as an initial response to invasion. The degree to which this is present suggests this may be a highly relevant mechanism modulating tumour differentiation, growth and progression, possibly entailing uptake via specific receptors on the tumour cell surface.

Actins↗
PubMed1993

Hyperplastic polyps: a cell lineage which both synthesizes and secretes trefoil-peptides and has phenotypic similarity with the ulcer-associated cell lineage.

Hyperplastic polyps are common benign lesions of uncertain histogenesis, which occur in the colon in populations at risk for colorectal carcinoma. They contain neutral/MUC1 gene-related mucin which in turn is closely associated with the trefoil-peptide pS2, a major component of the ulcer-associated cell lineage, previously termed pseudopyloric metaplasia. We have examined 17 hyperplastic polyps for expression of the trefoil-peptides pS2 and human spasmolytic polypeptide by in situ hybridization and immunohistochemistry, as well as by using antisera to epidermal growth factor/urogastrone and its receptor and to epitopes of the product of the MUC1 gene to characterize any further similarity between these lesions and the ulcer-associated cell lineage and thus help elucidate the nature of the lesions. Our investigations show both human spasmolytic polypeptide and pS2 messenger RNA within the polyps, whereas only pS2 peptide could be demonstrated immunohistochemically. Epidermal growth factor/urogastrone, its receptor, and antisera to the MUC1 gene also showed widespread staining of these polyps. We suggest that hyperplastic polyps are formed of a lineage that both synthesizes and secretes trefoil-peptides and the MUC1 mucin and that hyperplastic polyps may be related to the phenotypically similar ulceration-associated cell lineage.

Cell Line↗
PubMed1992

Abnormal expression of wild type p53 protein in normal cells of a cancer family patient.

Mutations in the p53 gene are the commonest specific genetic change in human cancer. In normal tissues, p53 protein is present in such low quantities that it is not readily detectable by immunochemical techniques. However, in many tumour cells large amounts of p53 protein accumulate and can be seen by simple immunohistochemical staining; this is generally attributed to the accumulation of stabilised, mutant protein. We have found a mother and daughter, who both have a history of breast cancer, who show strong immunohistochemical staining of p53 in most of their normal epithelial and mesenchymal cells. Their family has a history of multiple cancers developing at an early age. Detailed protein analysis and gene sequencing of material obtained from cultured cells, grown from a skin biopsy taken from the daughter, suggest that her cells contained large quantities of normal (unmutated) p53. We suggest that this phenotype defines a new inherited cancer susceptibility syndrome that is distinct from the germ-line mutations in p53 found in some Li-Fraumeni families. This new syndrome affects p53 tumour suppressor function through an indirect mechanism that stabilises normal p53. It remains to be established whether this mechanism also contributes to the accumulation of p53 in sporadic cancers.

Adult↗
PubMed1992

Localization of intestinal trefoil-factor mRNA in rat stomach and intestine by hybridization in situ.

A cDNA encoding rat intestinal trefoil factor (rITF) was prepared by reverse transcription and PCR amplification. The sequence obtained was well conserved with that of other trefoil peptides. An antisense riboprobe produced from the clone was used to localize the sites of ITF expression in the rat gastrointestinal tract using hybridization in situ. We found rITF mRNA in goblet cells in the small intestine and colon; a gradient of signal strength greatest near the crypt base was sometimes present. We found no evidence for rITF expression in Brunner's glands, the pancreas, or most regions of the gastric mucosa. Surprisingly, strong signals for rITF mRNA were detected in a region of stomach at the junction of the squamous fore-stomach with the glandular gastric mucosa. This region, which may correspond to the cardiac region, formed part of a larger area of cells staining positive for acid mucins. We hypothesize that concerted expression occurs of particular trefoil peptides with specific mucins, and that this organization reflects a functional relationship between mucins and trefoil peptides.

Amino Acid Sequence↗
PubMed1992

An inter-observer and intra-observer variability study on the diagnosis of lymph node biopsy specimens.

One hundred lymph node biopsy specimens were examined on two separate occasions by seven pathologists differing in experience in lymphoreticular pathology. Neither history nor immunohistochemistry was provided and the study, therefore, focused on morphological interpretation alone. The participants evaluated each case using a constructed response form in which the confidence with which they entered each response was also entered. Agreement on various points, between pathologists, between the two rounds, and with the referring centre was assessed. Whilst there was a high level of agreement over a diagnosis of benign vs. malignant and non-Hodgkin lymphoma vs. Hodgkin's disease, there was considerably less agreement over both T vs. B cell phenotype and high vs. low grade. The lack of agreement over grade, an evaluation which is usually made independent of immunohistochemistry, is particularly important, because of the relevance to selection of treatment. Proliferation markers may be more appropriate determinants of treatment choice.

Biopsy↗
PubMed1992

Mammary hamartoma--a review of 35 cases.

Mammary hamartomas are macroscopically well-delineated tumours composed of a variable mixture of epithelial elements, fat and fibrous tissue. Such lesions are an under-recognized entity and, as they can be visualized by mammography, may be seen more frequently with the advent of the UK National Breast Screening Programme. The clinical and pathological features of 35 cases of mammary hamartoma seen at the Imperial Cancer Research Fund Clinical Oncology Unit at Guy's Hospital between 1979 and 1990 have been reviewed. Hormone receptor analysis on nine cases gave high progesterone with low oestrogen levels, probably reflecting their premenopausal status. Immunohistochemistry showed that the positive receptor staining was confined to the epithelial elements. In 25 cases pseudo-angiomatous hyperplasia was evident in the stroma of the lesion. The importance of distinguishing the inter-anastomosing stromal spaces seen in the latter condition from low-grade angiosarcoma is emphasized; the relationship between pseudo-angiomatous hyperplasia and mammary hamartoma is discussed; and the possibility that the former represents a permanently dilated form of the lymphatic labyrinth suggested.

Adolescent↗
PubMed1991

Low expression of beta 1, alpha 2 and alpha 3 subunits of VLA integrins in malignant mammary tumours.

The Very Late Antigens (VLAs) are alpha beta heterodimeric transmembrane proteins mediating cell-substratum as well as cell-cell interactions. Changes in their expression and/or function seem to occur in a number of invasive carcinomas and may at least in part explain their abnormal patterns of growth and differentiation. Using monoclonal antibodies to the beta 1 (DH12, A1A-5), alpha 2 (B1.515) and alpha 3 (E1.56) chains, VLA-2 (alpha 2 beta 1) and VLA-3 (alpha 3 beta 1) were studied on cryostat sections of three fibroadenomas and 43 invasive breast carcinomas (29 ductal, 14 lobular) by the avidin-biotin complex immunoperoxidase technique. In non-neoplastic breast tissue and in fibroadenomas VLA-2 and VLA-3 were expressed by myoepithelial cells and on the basolateral surface of the luminal cells. There was weak or absent expression of alpha 2, alpha 3 and the common beta 1 chain in the majority of invasive carcinomas compared to the adjacent normal breast epithelium and preinvasive (in-situ) carcinomas. In addition, the expression of the alpha 2 chain of VLA-2 was reduced significantly (P less than 0.005) in the poorly differentiated ductal breast carcinomas (Grade III) compared to the well (Grade I) and moderately (Grade II) differentiated ductal tumours. These data give further evidence that loss or down-regulation of VLA-2 and VLA-3 occur relatively frequently in invasive cancers, and, at least in the invasive ductal breast carcinomas. Loss of an extracellular matrix receptor controlling growth and differentiation seems to be one of the abnormalities underlying the progression towards an undifferentiated morphology.

Adenofibroma↗
PubMed1991

Angiosarcoma of the skin overlying an irradiated breast.

A case of angiosarcoma of the breast which developed six and a half years after treatment for carcinoma of the same breast is reported. As a result of radiotherapy the breast manifested signs of chronic lymphedema prior to development of angiosarcoma. Although the aetiology in this case is uncertain, there was a past history of childhood naevus regressing spontaneously. Angiosarcoma is a well known complication following radiotherapy and lymphedema, and is likely to be seen more frequently as conservation treatment is used more commonly for patients with early breast cancer.

Breast Neoplasms↗