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J Southgate

Publications and source records attributed to J Southgate.

At least 55 records · Page 3Linked to original sources
PubMed1998

Interleukins 4 and 13 upregulate expression of cd44 in human colonic epithelial cell lines.

Interleukin 4 (IL-4) inhibits carcinoma cell growth and promotes expression of differentiation-associated products by normal and malignant epithelial cells. The effects of IL-4 and IL-13 on expression of the CD44 transmembrane adhesion receptor were examined in human epithelial cell lines of colonic (HT-29, CaCo-2, DLD-1, T84), breast (MCF-7, ZR75-1) and liver (Hep-G2, PLC/PRF/5) origins as well as mitogen-activated and resting peripheral blood lymphocytes (PBL) and T cell lines (Jurkat, HUT78). Liver and Jurkat cells were negative for CD44. Colonic, breast and HUT78 cells expressed CD44 constitutively and all except DLD-1 and HUT78 also expressed CD44 splice variant (CD44v) epitopes. All cell lines expressed IL-4 receptors, but IL-4 and IL-13 induced upregulation of CD44 only in the colonic cell lines. CD44v was also upregulated, but there was no de novo induction of CD44v in variant-negative cells and no de novo expression of CD44 in the CD44(-) lines. CD44 upregulation in mitogen-activated PBL was not increased by IL-4 and IL-13 and was not inhibited by neutralizing antibodies. Other cytokines tested [interferon gamma (IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta1 (TGF-beta1) and IL-6] did not affect CD44 core epitope expression in the cell lines tested.

Alternative Splicing↗
PubMed1998

Uroplakin gene expression by normal and neoplastic human urothelium.

cDNA sequences for human uroplakins UPIa, UPIb, UPII, and UPIII were cloned and used to investigate uroplakin transcription by normal and neoplastic urothelial cells. Normal urothelium expressed mRNA for all four uroplakins, although UPIII could be detected only by ribonuclease protection assay. By in situ hybridization, UPIa and UPII were confined to superficial cells and UPIb was also expressed by intermediate cells. Cultured normal human urothelial cells showed a proliferative basal/intermediate cell phenotype and constitutive expression of UPIb only. Uroplakin expression by transitional cell carcinoma cell lines was related to their differentiated phenotype in vitro. RT4 cells expressed all uroplakins, VM-CUB-3 expressed three uroplakins, RT112 and HT1376 cells expressed only UPIb in high abundance, and COLO232, KK47, and EJ cells had no detectable expression. These results correlated with patterns of uroplakin expression in tumors. UPIa and UPII were detected superficially only in well differentiated transitional cell carcinoma papillae. UPIb was positive in seven of nine and overexpressed in five of nine noninvasive transitional cell carcinomas and was also present in four of eight invasive transitional cell carcinomas. Lymph node metastases retained the same pattern of UPIb expression as the primary tumor. Unlike the three differentiation-regulated uroplakins, UPIb may have an alternative role in urothelial cell/tissue processes.

Amino Acid Sequence↗
PubMed1998

Expression and cytokine regulation of immune recognition elements by normal human biliary epithelial and established liver cell lines in vitro.

BACKGROUND/AIMS: Biliary epithelial cells are targets of immune-mediated attack in conditions such as primary biliary cirrhosis and allograft rejection. This has been attributed to the ability of biliary epithelial cells to express ligands for T cell receptors. We aimed to investigate the expression of immune recognition elements and the effects of pro-inflammatory and anti-inflammatory cytokines on cell surface phenotypes of normal human biliary epithelial cells and established human liver-derived (PLC/PRF/5, HepG2, Hep3B and CC-SW) lines. METHODS: Cells were cultured in the presence or absence of cytokines for 72 h, and expression of cell surface molecules was assessed by flow cytometry and immunofluorescence. RESULTS: All cell lines expressed MHC class I, ICAM-1 (CD54), LFA-3 (CD58) and EGF receptor, and all but Hep3B expressed Fas/Apo-1 (CD95). Unlike hepatocyte-derived cell lines, biliary epithelial cells and CC-SW expressed CD40 and CD44. As expected, IFNgamma and TNFalpha upregulated expression of ICAM-1, MHC class I and MHC class II, particularly in biliary epithelial cells. TGFbeta downregulated these molecules and downregulated CD95 on biliary epithelial cells, but upregulated LFA-3. The Th2 cytokines had little effect, although IL-4 upregulated CD95 expression on biliary epithelial cells. IFNgamma upregulated CD40 expression on biliary epithelial cells, CC-SW and HepG2. CONCLUSIONS: These findings imply that biliary epithelial cells may be capable of interacting with activated T lymphocytes via CD40 and LFA-3, which are thought to be important T cell accessory ligands for T cell activation in a B7-independent manner. Sensitivity to pro-inflammatory cytokines and expression of CD95 may explain why biliary epithelial cells are primary targets for autoimmune attack.

Bile Ducts, Intrahepatic↗
PubMed1998

Induction of a differentiated ciliated cell phenotype in primary cultures of Fallopian tube epithelium.

Human Fallopian tubal epithelial cells in culture lose morphological features associated with the epithelium in situ and the extent to which they retain their in-vivo phenotype or function is unknown. In order to address this question, immunocytochemical markers were identified which distinguish secretory (HMFG2+, LhS28-) from ciliated (HMFG2-, LhS28+) epithelial cells in tissue sections of Fallopian tube. These markers were used to analyse the phenotype of tubal cells in vitro. Primary cultures of human tubal epithelial cells were seeded onto glass and grown to confluence before addition of oestradiol-17beta. In the absence of hormone, tubal epithelial cells expressed cytokeratins and nuclear receptors for oestrogen and progesterone and adopted a homogeneous (HMFG2+, LhS28-) secretory cell phenotype. Following the addition of oestradiol-17beta, a proportion of cells became positive for LhS28. The induction of a ciliated epithelial cell phenotype was confirmed by scanning electron microscopy, where on permeable collagen membranes, approximately one-third of tubal epithelial cells became ciliated in the presence of oestradiol-17beta. We suggest that in vitro, tubal epithelial cells adopt an immature secretory-like phenotype and that oestrogen can induce differentiation to a ciliated epithelial cell phenotype.

Antibodies, Monoclonal↗
PubMed1998

Study of the cell biology and biochemistry of cherubism.

AIMS: To establish whether the multinucleate cells in lesions of patients with cherubism are also osteoclasts and if this is the case whether they were responsive to calcitonin; to carry out cytogenetic studies on two members of the same family affected by cherubism in an attempt to identify any major chromosomal defects; and to perform an in-depth modern biochemical study of four children in the same family. SUBJECTS AND METHODS: Four related children with cherubism were studied. Tissue taken from one of the children at elective decompression of an optic nerve was submitted to in vitro bone resorption studies. Cytogenetic studies were done on two of the children and biochemical studies on all four. RESULTS: The multinucleate cells in the cherubic lesions were shown to be osteoclasts since they synthesised tartrate resistant acid phosphatase, expressed the vitronectin receptor, and resorbed bone. Bone resorption by the cultured multinucleate cells was significantly inhibited by calcitonin. High resolution cytogenetic studies failed to detect any chromosomal abnormalities in two children with cherubism. The biochemistry profile of all four children with cherubism showed that serum calcium, parathyroid hormone, parathyroid related hormone, calcitonin, and alkaline phosphatase were within normal levels. Urine analysis of pyridinium and deoxypyridinium cross links, hydroxyproline, and calcium in relation to urine creatinine were measured to assess bone resorption in these children, and the values were at the upper end of the normal range in all four. CONCLUSIONS: Further studies are required to determine whether calcitonin treatment will control this grossly deforming disease until the time when the physiological changes that occur at puberty rectify the pathology. It is not recommended that biochemical markers of bone resorption are used in isolation to monitor the activity of cherubism in individuals because the results are based on a small number of children and because of reports of marked interindividual variation in the levels of these markers, particularly in children.

Bone Resorption↗
PubMed1998

Culture of ascitic ovarian cancer cells as a clinically-relevant ex vivo model for the assessment of biological therapies.

There are few in vitro models of human ovarian cancer suitable for the assessment of biological therapies. We have established short-term cultures of ovarian carcinoma cells from ascites, maintained in suspension in ascitic fluid to help preserve the original tumor cell microenvironment. We assessed the effects of a potential biological therapeutic agent, interferon-alpha (IFN alpha), on ovarian cell phenotype. In total, eight cultures were established from seven patients. Quantitative changes in cell surface phenotype were determined by flow cytometry for HLA-ABC, HLA-DR, TAG72, CA125, HMFG1 and HMFG2 antigens. The amount of CA125 antigen shed into the culture media was also assessed. Variations in cell surface phenotype between specimens probably reflected the specific cytokine milieu of the ascites as well as idiotypic differences between tumors. Nevertheless, there were consistent phenotypic responses to IFN alpha, with up-regulation of MHC Class I but down-regulation of the HMFG1 and HMFG2 antigens from the cell surface. The results suggest that this approach may be useful in patient selection and for optimizing biological therapies, as it enables patients' individual tumor responses to exogenous cytokine to be studied against the background of the endogenous cytokine milieu.

Aged↗
PubMed1997

Reconstitution of human urothelium from monolayer cultures.

PURPOSE: We established a 3-dimensional organ culture model of urinary tract tissue in which to study the effects of seeding cultured urothelial cells onto de-epithelialized urothelial stroma. MATERIALS AND METHODS: Normal human urinary tract tissues were placed in organ culture or used to establish urothelial cell cultures. At passage 2 cell cultures were harvested and used to reconstitute autologous organ cultures by seeding onto de-epithelialized stroma. Organ cultures were harvested at intervals and analyzed by immunohistology with a panel of antibodies against differentiation associated antigens, cytokeratins, cell adhesion molecules, extracellular matrix components and proliferation associated antigens. RESULTS: Human urothelial tissues were maintained in organ culture for at least 18 weeks and they retained a transitional epithelial morphology with expression of normal in situ antigenic characteristics. Within 2 weeks of reconstitution recombined organ cultures formed a stratified, polarized, transitional-like neo-epithelium that expressed many of the phenotypic and differentiated characteristics of normal tissue. Basement membrane formed at sites of direct contact between urothelial cells and stroma. After an initial stabilization period the proliferation rate of the urothelium of intact and reconstituted organ cultures decreased to the low turnover rate characteristic of normal urothelium in situ, indicating that the cells were responsive to normal growth regulatory controls. CONCLUSIONS: Normal human urothelial cells, which express a proliferative nondifferentiated phenotype in monolayer culture, retain the capacity to differentiate and reform a slow turnover, stratified transitional epithelium.

Cell Adhesion↗
PubMed1997

Cytokeratin 14 as a marker of squamous differentiation in transitional cell carcinomas.

The presence of squamous differentiation in transitional cell carcinomas has been variably related to prognosis and response to radiotherapy. This study sought to establish whether cytokeratin (CK) 14, normally expressed in the basal cells of squamous epithelium, could be used as a reliable marker of the emergence of a squamous phenotype in transitional cell carcinomas. In a series of 42 tumours, CK14 was expressed in areas of squamous morphology, whereas CK20 identified continuing urothelial differentiation. Furthermore, focal positivity for CK14 was present in a proportion of tumours with no morphological evidence of squamous differentiation, suggesting that it is a more sensitive marker of phenotypic switch. Investigation of CK subtypes may be more powerful than morphology alone in clinical studies of transitional cell carcinomas as CK expression profiles have been related to treatment response in other tumour types.

Biomarkers, Tumor↗
PubMed1997

Stromal and vascular invasion in an human in vitro bladder cancer model.

In transitional cell carcinoma (TCC) of the urinary bladder, disease spread may occur through local invasion of the bladder wall and/or via hematogenous dissemination. Although the metastatic phenotype is well studied, little is known about the mechanisms that determine and regulate secondary dispersion via stromal and vascular routes. The aim of this study was to develop an in vitro system to study TCC invasion using normal human urinary tract stroma in organ culture. Human de-epithelialized urinary tract stromas were seeded with established human TCC cell lines (RT4, RT112, and EJ) and maintained in organ culture at an air-liquid interface for up to 8 weeks. The composite tissues were analyzed immunohistochemically using antibodies to a proliferation-associated antigen, cell adhesion molecules, extracellular matrix components, and cytokeratin isotypes. The phenotype of TCC cell lines maintained as monolayer cultures were compared with the composite tissues to assess the regulatory effects of the stroma. On a normal urothelial stroma, the TCC cell lines proliferated and underwent a degree of differentiation related to the grade of the original tumor. RT4 cells formed a stratified epithelium with low proliferation, some characteristics of urothelial differentiation, and no evidence of stromal invasion over the 8-week culture period. RT12 cells formed a stratified epithelium with limited differentiation and by 2 weeks showed intravasation of the subepithelial capillary bed. Anaplastic EJ cells did not stratify but diffusely invaded the stromal matrix and the superficial vasculature. In conclusion, the urothelial stroma can support cytodifferentiation in vitro and influence the behavior of malignant TCC cells. The model has application for determining genetic and epigenetic factors that influence whether a tumor progresses by local and/or hematogenous spread. Furthermore, it may offer a useful approach in evaluating prognostic and diagnostic markers and strategies for preventing invasion.

Antibodies, Monoclonal↗
PubMed1996

The effects of dietary fatty acids on the proliferation of normal human urothelial cells in vitro.

Little is known of the mechanisms by which dietary fatty acids (FAs) may affect normal epithelial cell physiology and thereby directly or indirectly influence tumour incidence and progression. In this study, we have used normal human urothelial cell cultures to investigate whether FAs may modify proliferation of normal human epithelial cells in vitro. FAs were presented as albumin complexes in serum-free medium and the effects on proliferation over a concentration range of 1-100 microM were assayed by thymidine incorporation. Saturated FAs had no effect. At lower concentrations (1-10 microM), mono-unsaturated FAs (MUFAs) and n-3 polyunsaturated FAs (PUFAs) were slightly stimulatory. Concentrations of unsaturated FAs above 10 microM were growth inhibitory in a dose-dependent manner. Oleic acid showed least cytostatic effect, whereas gamma-linolenic acid induced irreversible growth arrest. Although marked morphological effects were observed in irreversibly growth-inhibited cells, the cells remained attached to the substratum and showed no evidence of nuclear pyknosis or apoptosis. The inhibitory effects of single PUFAs could be reduced, but not abolished, by the addition of saturated FAs or MUFAs. Mixtures of different PUFAs were inhibitory in an additive manner. These data suggest that PUFAs have a cytostatic effect on rapidly proliferating epithelial cells which appears unrelated to malignant transformation.

Analysis of Variance↗
PubMed1996

Cytokeratin 20 as an objective marker of urothelial dysplasia.

OBJECTIVE: To investigate the dysregulation of cytokeratin 20 (CK20) expression in urothelial dysplasia and its potential as a diagnostic aid. PATIENTS AND METHODS: Twenty-two patients were selected on the basis that they had undergone one or more biopsies showing dysplasia before the development of a transitional cell carcinoma (TCC): 15 of these patients also had a prior history of TCC. The dysplasia was classified as mild in 12, moderate in 14 or severe dysplasia/carcinoma in situ in 10 patients, ensuring that a spectrum of morphological appearances was represented. Control biopsies were obtained from seven children undergoing bladder reconstructions and 23 patients with recurrent urinary tract infections, haematuria or functional bladder symptoms, but no history of TCC. RESULTS: The expression of CK20 was restricted to superficial 'umbrella' cells and occasional intermediate cells in the control biopsies, even in the presence of severe inflammation. In 31 of the 36 cases of dysplasia complete loss of restriction was seen at least focally with positive expression in all layers of the urothelium. CONCLUSION: The abnormal expression of CK20 is a reliable, positive marker of urothelial dysplasia in the urinary bladder. Immunostaining for CK20 is therefore a useful adjunct to morphology in the diagnosis of dysplasia, of particular value in the distinction from reactive states where diagnostic difficulties are greatest.

Aged↗
PubMed1996

Reverse transcriptase-polymerase chain reaction for expression of tyrosinase to identify malignant melanoma cells in peripheral blood.

BACKGROUND: Circulating tumour cells in the peripheral blood may be important for haematogenous spread of disease. The detection of these cells may therefore be a poor prognostic indicator. Reverse-transcriptase polymerase chain reaction (RT-PCR) of target tumour-specific protein expression has been used as a sensitive and specific method for the detection of these tumour cells. Initial reports by our laboratory and other suggested RT-PCR amplification of the enzyme tyrosinase is a useful method for detection of melanoma cells in peripheral blood [1-3]. PATIENTS AND METHODS: In this report, we have evaluated the application of RT-PCR for tyrosinase mRNA as a detection method for melanoma cells in a series of 24 patients with advanced, metastatic malignant melanoma. A single round RT-PCR method is described. RESULTS: The single round RT-PCR was as sensitive as previously described nested PCR methods, and had the advantage of reduced contamination risks. Blood samples from three out of the twenty-four patients were positive. CONCLUSIONS: The frequency of tumour cell detection in peripheral blood from patients with advanced disease was lower than previously reported. It may be only small numbers of circulating tumour cells are present at any one time in the peripheral blood of patients with malignant melanoma. If this is the case increased sampling will improve detection frequency. Alternatively, dissemination of melanoma through peripheral blood may be a rare event. In our experience, RT-PCR for tyrosinase mRNA as a staging test for melanoma patients must be interpreted cautiously.

Adult↗
PubMed1996

Human Fallopian tubal epithelial cells in vitro: establishment of polarity and potential role of intracellular calcium and extracellular ATP in fluid secretion.

A pure population of human Fallopian tubal epithelial cells has been isolated by enzyme digestion, grown in primary culture and used to explore the biochemical basis of oviduct fluid secretion. Confluence was achieved in 3-7 days. Immunocytochemical labelling for cytokeratins indicated that the cells were epithelial in nature and formed extensive desmosomal contacts, producing a polarized layer in culture. By growing the cells on collagen-impregnated filters, a small transepithelial electrical potential difference could be recorded, with the apical side of the cells negative with respect to the basal side. In addition, the consumption of glucose and the appearance of lactate were greater on the basal than on the apical side of the cells. Because intracellular Ca2+ ([Ca2+]i) is well established as a signal transduction agent in epithelial fluid secretion, the effect of a wide range of agonists on [Ca2+]i in isolated tubal epithelial cells was studied using Fura-2. The only agent which induced a change in [Ca2+]i was extracellular ATP. The transients induced were dependent on both intracellular and extracellular calcium. ATP added to the basal side of the cells of the polarized layer induced a transient increase in the potential difference. The data are consistent with a potential role for extracellular ATP in the regulation of human tubal fluid formation.

Adenosine Triphosphate↗