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Biomedical subjects

W F Doe

Publications and source records attributed to W F Doe.

At least 55 records · Page 3Linked to original sources
PubMed1992

Enhanced mucosal cytokine production in inflammatory bowel disease.

Proliferation, maturation, chemotaxis, and activation of neutrophils and monocytes are mediated largely by cytokines, including colony-stimulating factors and lymphokines. Cytokines produced in the intestinal mucosa contribute to the increased migration of neutrophils and monocytes into the lesion of inflammatory bowel disease and to the activation of these inflammatory cells. Lamina propria mononuclear cells isolated from colon tissue from 14 patients with inflammatory bowel disease (IBD) and from histologically normal controls were studied. Cells from IBD-affected tissue produced significantly more colony-stimulating factor activity (1402 +/- 252 U) per 2 x 10(6) cells than those from normal mucosa (362 +/- 85 U), mainly because of the increased production of granulocyte colony-stimulating factor and interleukin 1. This was accompanied by increases in the amount of specific messenger RNA for these two cytokines in lamina propria mononuclear cells from mucosa of patients with Crohn's disease (CD) compared with normal controls. By contrast, there was a substantial reduction in interleukin 3 production in CD and in ulcerative colitis lamina propria mononuclear cells, and this was reflected in significantly reduced expression of interleukin 3 messenger RNA in CD cells. Of the agents used in the therapy of IBD, hydrocortisone and 5-aminosalicylic acid, but not cyclosporin A, markedly suppressed in vitro production of cytokines by lamina propria mononuclear cells, suggesting that their therapeutic efficacy in vivo may be due in part to down-regulation of cytokine production in the inflamed mucosa.

Aminosalicylic Acids↗
PubMed1992

IL-2 production by intestinal lamina propria cells in normal inflamed and cancer-bearing colons.

Biologically significant levels of IL-2 activity were produced by isolated lamina propria mononuclear cells (LPMC) from normal intestine (n = 12), cancer-bearing colons (n = 35) and inflammatory bowel disease (IBD) affected tissue (n = 12). The levels of IL-2 produced were similar for all three sources of LPMC (normal 252 +/- 48 U/ml, IBD-affected mucosa 197 +/- 42 U/ml and colon cancer 285 +/- 43 U/ml). These levels were significantly greater than those produced by peripheral blood mononuclear cells (20 +/- 5 U/ml, P less than 0.01) on a per cell basis. In mucosa from cancer-bearing colons the amount of IL-2 produced by LPMC was unaffected by the invasiveness of the colon cancer. LPMC IL-2 production was markedly suppressed by drugs used in IBD therapy. 5-Aminosalicylic acid (5-ASA) reduced activity in a dose-dependent fashion. At a dose equivalent to the faecal therapeutic level of 0.5 mg/ml activity, IL-2 production by LPMC was suppressed to 3.4% of controls. Similarly, exposure of LPMC to cyclosporin A (CyA) and hydrocortisone (HC) at therapeutic levels reduced IL-2 activity to less than 1% of controls. The major producers of IL-2 activity were shown to be CD3+ T lymphocytes and those bearing the activation markers IL-2R and TFR. Suppression of mucosal IL-2 production represents an important therapeutic mechanism of drugs used in the management of IBD including HC, 5-ASA and CyA. These results suggest that mucosal T cells produce appreciable levels of IL-2 activity that may be important in maintaining immune homeostasis in the normal intestine, provide anti-neoplastic cytotoxic activity and contribute to the inflammatory events that characterize the mucosal lesions of IBD.

Aminosalicylic Acids↗
PubMed1991

Cell associated urokinase activity and colonic epithelial cells in health and disease.

It is not known if urokinase-type plasminogen activator (uPA) is associated with normal colonic epithelial cells. The aims of this study were to determine if normal colonic epithelial cells have uPA activity and whether this is concentrated at the cell membrane. In addition, the contribution of colonic epithelial cell associated uPA activity to disease related pertubations of mucosal uPA activity were examined. A highly enriched population of colonic epithelial cells was isolated from resected colon or biopsy specimens by an enzymatic technique. uPA activity was measured in cell homogenates by a specific and sensitive colorimetric method and expressed relative to cellular DNA. In two experiments subcellular fractionation of colonic epithelial cells was performed by nitrogen cavitation followed by ultracentrifugation over a linear sucrose gradient. The fractions collected were analysed for uPA and organelle-specific enzyme activities. Normal colonic epithelial cells have cell associated uPA activity (mean (SEM) 5.6 (1.1) IU/mg, n = 18). This colocalised with fractions enriched for leucine-beta-naphthylamidase and 5'-nucleotidase, markers of plasma membrane. uPA activities in epithelial cells from cancerous colons (9.8 (3.1) n = 7) or from mucosa affected by inflammatory bowel disease (3.8 (0.7) n = 15) were not significantly different from normal (paired t test), while that in epithelial cells from greatly inflamed mucosa was similar to that from autologous normal or mildly inflamed areas (4.4 (1.2) v 5.9 (3.6), n = 9). Thus normal colonic epithelial cells have cell associated uPA activity which is concentrated on the plasma membranes, suggesting the presence of uPA receptors. Increased mucosal levels of uPA previously reported in patients with inflammatory bowel disease are not due to increased colonic epithelial cell associated uPA.

Cell Membrane↗
PubMed1990

Inhibition of cancer cell urokinase plasminogen activator by its specific inhibitor PAI-2 and subsequent effects on extracellular matrix degradation.

Isotopically labeled [( 3H]serine, [3H]proline, and [35S]sulfate) subendothelial cell basement membranes were used to determine the role of urokinase plasminogen activator (uPA) and its specific inhibitor plasminogen activator inhibitor 2 (PAI-2) in colon cancer cell extracellular matrix degradation. Recombinant PAI-2 irreversibly inhibited low and high molecular weight purified human uPA in addition to both colon cancer cell-associated and secreted uPA, particularly if pro-uPA had been preactivated. Two selected lines (COLO394 and LIM1215) preferentially degraded differently labeled matrices in a time- and plasminogen-dependent manner. This process was inhibitable by PAI-2 in the medium at levels which suggested that some degree of "shielding" of cell surface uPA from inhibitor occurred. The ability of PAI-2 to regulate the invasive phenotype of cells which express cell surface or receptor-bound uPA is discussed.

Cell Line↗
PubMed1990

Plasminogen activator inhibitor 2 (PAI-2) is not inactivated by exposure to oxidants which can be released from activated neutrophils.

Purified recombinant human monocyte plasminogen activator inhibitor 2 (PAI-2) retained inhibitory activity after exposure to a number of oxidants, including hypochlorite anion (OCl-), chloramine-T (CT) and hydrogen peroxide (H2O2). Analysis of PAI-2 exposed to oxidants by gel filtration chromatography and SDS-PAGE indicated that although the protein could no longer be detected by silver staining, this was not due to fragmentation of the PAI-2 molecule. The sensitivity of a number of serine protease inhibitors (serpins), (eg. alpha 1 proteinase inhibitor (alpha 1PI) and plasminogen activator inhibitor 1 (PAI-1] to oxidative inactivation has been attributed to oxidation of reactive site methionine residues and/or tertiary structural modifications. The relevance of these phenomena and the potential for PAI-2 to be used as a therapeutic inhibitor of urokinase (uPA)-dependent proteolysis during inflammation and tumour metastasis is discussed.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of antigen-presenting dendritic cells from the mouse intestinal lamina propria.

A method was developed for the isolation of antigen-presenting dendritic cells, and macrophages, from mouse intestinal lamina propria and Peyer's patches. Peyer's patches, and the lamina propria of both the small and large intestine, contained cells with potent stimulatory activity in the allogeneic mixed leucocyte reaction. These cells were separated from macrophages by fibronectin adherence and further enriched by density centrifugation. The isolated stimulatory cells expressed high levels of class II major histocompatibility complex (MHC) antigens, and resembled splenic dendritic cells in both morphology and function. Macrophages were recovered from the lamina propria but not Peyer's patches. These cells also expressed class II MHC antigens, but failed to stimulate the mixed leucocyte reaction and, instead, induced indomethacin-sensitive suppression.

Animals↗
PubMed1989

Isolation of colonic crypts that maintain structural and metabolic viability in vitro.

The aim of this study was to develop a method by which colonic epithelial cells can be isolated from resected mucosa or colonoscopic biopsy specimens and viability maintained in the short term. The principles of the technique are to digest the lamina propria from the epithelium with Dispase and collagenase, to disrupt the epithelium by trituration, and to purify the epithelial cells by seiving and differential sedimentation. Whole and partial crypts were isolated with consistently high purity of 93.5% +/- 1.2% (excluding red cells). Structural integrity was confirmed by light and electron microscopy, exclusion of trypan blue, minimal leakage of lactic dehydrogenase over 5 h (4.1% +/- 1.7%), and 51Cr leakage of less than 2% per hour over 16 h. Functional integrity was supported by continued deoxyribonucleic acid synthesis [( 3H]thymidine uptake) over 16 h and the formation of epithelial monolayer cultures on plastic. Thus, this simple method yields a highly enriched cell population that maintains high viability in vitro for at least 16 h. Such cells may be useful for the study of the biology of colonic epithelial cells.

Autoradiography↗
PubMed1988

Urokinase-type plasminogen activator in colorectal carcinomas and adenomatous polyps: quantitative expression of active and proenzyme.

Total urokinase-type plasminogen activator (u-PA) content (proenzyme plus active enzyme) was significantly higher in 20 colorectal carcinomas and in 27 adenomatous polyps than in metaplastic polyps and autologous normal mucosa. u-PA content was also markedly increased in adenomatous polyps and autologous colonic mucosa removed from familial polyposis coli patients. Using a new monoclonal antibody technique to distinguish the proenzyme of u-PA from the active enzyme, we found that 70% of the u-PA in polyp and cancer tissue was present in the proenzyme form compared to 47% in normal colonic mucosa. For colon cancers, there was a significant correlation between their stage of invasiveness and the levels of proenzyme. No correlation was observed between the u-PA content of adenomatous polyps and their size or degree of dysplasia. Study of the u-PA content of the colonic mucosa may offer a useful biochemical correlate of epithelial cell transformation in the colon.

Adenoma↗
PubMed1988

Assessment of inflammatory bowel disease activity by technetium 99m phagocyte scanning.

Autologous technetium 99m-labeled phagocyte scanning has been used to assess disease activity in inflammatory bowel disease in 51 consecutive patients. Strong correlations were found between the 24-h fecal excretion of isotope and the histologic score of mucosal biopsy specimens (rS = 0.84, p less than 0.001, where rS is Spearman's rank correlation coefficient), and between the 24-h fecal excretion of isotope and a clinical inflammatory bowel disease activity index based on the Crohn's disease activity index (rS = 0.87, p less than 0.001). To develop a clinically useful and objective measure of inflammatory bowel disease activity that did not require a 24-h stool collection, the intensity of bowel uptake on scanning was graded visually from 0 to 4, a ratio of count rates for the region of interest to the iliac crest reference region was calculated, and the rapidity of labeled phagocyte uptake into inflamed bowel was measured as the peak uptake time. Visual grading of disease activity on the scans was validated by comparing it with the ratio of count rates from inflamed bowel regions of interest and those from the iliac crest reference region. The ratio of count rates showed a significant correlation with the clinical disease activity index (r = 0.75, p less than 0.001). The visual scan grade also correlated well with the clinical activity index (r = 0.87, p less than 0.001). Count rates from hourly scans were also used to calculate the time of peak uptake of counts for a given region of interest. There was a strong negative correlation between this peak uptake time and the fecal excretion of isotope (rS = -0.81, p less than 0.001), a clinical activity index (r = -0.60, p less than 0.001), and the histologic score of the mucosal biopsy specimens (r = -0.84, p less than 0.001). These results indicate that the technetium 99m-labeled phagocyte scan provides an objective assessment of disease activity in inflammatory bowel disease using the visual scan grade, ratio of count rates for the region of inflamed bowel, or by the peak uptake time, thereby avoiding the problems associated with fecal collections. This scanning test may prove to be of significant value in clinical management and in the assessment of treatment response.

Adolescent↗
PubMed1988

Ulcerative colitis--a disease characterised by the abnormal colonic epithelial cell?

The leakiness of the cell membranes of colonic epithelial cells isolated by the collagenase/Dispase technique from normal or diseased colons was assessed in a 4 h 51Cr release assay. Cells from normal, adenoma bearing or cancer bearing colons showed 51Cr release of 8% or less in almost all of 46 cell populations tested. In contrast, cells from mucosa affected by ulcerative colitis [11.9 (4.3%) n = 23] or Crohn's disease [8.4 (2.7%) n = 18] released significantly more 51Cr than the non-inflamed groups. Values are expressed as mean (SD). Overall, release values were greater in ulcerative colitis than Crohn's disease (p less than 0.01). In Crohn's disease, cells obtained from histologically inflamed mucosa released significantly more 51Cr [9.7 (2.5%) n = 11] than those from non-inflamed mucosa [6.4 (1.5%) n = 7, p less than 0.02] whereas, in ulcerative colitis, abnormal release values were found in 8 of 13 cell populations isolated from mucosa showing no histological evidence of active disease. In five patients with distal ulcerative colitis, cells from mucosa not apparently involved demonstrated normal 51Cr release in four of five studies despite abnormal release from cells from involved mucosa suggesting that a diffuse abnormality of the colonic epithelial cell is not usually present. These data indicate that chronic mucosal inflammation per se is associated with abnormalities of the colonic epithelial cell but that, in ulcerative colitis, the abnormality remains in many patients with quiescent disease. Identification of the local factors responsible for such an abnormality may contribute to an understanding of the pathogenesis of ulcerative colitis.

Adolescent↗
PubMed1988

Lysis of colonic epithelial cells by allogeneic mononuclear and lymphokine activated killer cells derived from peripheral blood and intestinal mucosa: evidence against a pathogenic role in inflammatory bowel disease.

A sensitive 4 h 51Cr-release cytotoxicity assay has been developed using as targets colonic epithelial cells obtained by Dispase-collagenase digestion of resected mucosa or colonoscopic biopsies. Peripheral blood mononuclear cells (MNC) from most healthy donors showed low, but significant levels of cytotoxicity for normal epithelial cell target cells of 8.7 (4.4) % (mean (SD] and similar levels were found in 14 ulcerative colitis (6.5 (4.4) %) and 16 Crohn's disease (6.2 (5.2) %) patients. Neither drug therapy nor disease activity influenced the results. The sensitivity of colonic epithelial cells isolated from inflamed and histologically normal mucosa to lysis by peripheral blood MNC from a single donor was not affected by the underlying disease. Anti-epithelial cell activity did not correlate with anti-K562 activity and the cytotoxic cell was plastic non-adherent and Leu-11b-. None of 15 MNC populations isolated from mucosa of normal, tumour bearing, or chronically inflamed intestine exhibited significant lysis of colonic epithelial cells despite killing of K562 target cells in 10. Lymphokine activated killer (LAK) cells, generated by interleukin-2 stimulation in vitro of nine intestinal and seven peripheral blood MNC populations, exhibited high levels of lysis of K562 cells but, on every occasion, failed to lyse colonic epithelial cells. These data indicate that spontaneously cytotoxic or LAK cells are unlikely to play a role in the generation of colonic epithelial cell injury by direct cytotoxicity in inflammatory bowel disease.

Adolescent↗
PubMed1987

Immunohistochemical characterisation of macrophages in human liver and gastrointestinal tract: expression of CD4, HLA-DR, OKM1, and the mature macrophage marker 25F9 in normal and diseased tissue.

This report describes the immunocytochemical characterisation of macrophages in sections of human liver, gastrointestinal tract, and associated lymphoid tissue and the inflammatory lesions of Crohn's disease. 25F9 is an antigen reported to be induced during the maturation of blood monocytes in vitro. The antigen was concentrated in cytoplasmic vesicular structures of isolated gastrointestinal macrophages. Similar labelled cells were observed in the apical regions of lamina propria in both small and large intestine in vivo. Their numbers and size were greatly increased in specimens of colon from patients with melanosis coli. Mucosal inflammatory lesions in specimens from patients with Crohn's disease did not contain 25F9-positive cells. The antigen was absent from giant cells and epithelioid cells in granulomata but was expressed on histiocytes in submucosal microgranulomata. In lymphoid organs, 25F9-positive cells were found in germinal centres, in the dome region of Peyer's patch, and in the medulla, but were largely excluded from T cell areas. In reactive nodes from Crohn's disease patients, the number of labelled cells in germinal centres and T cell areas was greatly increased. 25F9 was absent from the majority of typical liver Kupffer cells, but was expressed on cytoplasmic granules in a minor subpopulation of larger, more rounded cells in the liver. The results suggest that 25F9 is a marker for endocytosis rather than maturation. In parallel sections, resident macrophages of both liver and gastrointestinal tract labelled with Leu 3a/OKT4 (CD4) and with OKIa (HLA-DR antigen) but did not express OKM1 (type III complement receptor). By contrast, OKM1 was present on inflammatory cells, epithelioid cells, and giant cells in mucosal lesions of Crohn's disease.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1987

Proenzyme to urokinase-type plasminogen activator in human colon cancer: in vitro inhibition by monocyte minactivin after proteolytic activation.

Marked increases of plasminogen activator activity were observed in human colon cancer tissue, compared to corresponding normal tissues. This increase was attributable to urokinase-type activator (HPA52), with no increase evident in the level of the tissue-type plasminogen activator (HPA66). Human monocyte minactivin specifically inhibited HPA52 activity in cancer tissue homogenates and in colon cancer cell supernatants, an effect that was greatly enhanced by preincubation with plasminogen, indicating that the predominant form of HPA52 in tissue and the form that is secreted in vitro is the proenzyme. Inactivation of HPA52 by minactivin was shown to be dependent on proteolytic activation of HPA52 proenzyme. Utilization of HPA52 activity by tumors in vivo could therefore be dependent upon a protease, such as plasmin, to generate the extracellular proteolytic activity necessary to digest the intercellular matrix and permit invasion of normal tissue structures by colon cancer cells.

Adult↗
PubMed1986

Monoclonal antibodies inhibitory to human plasmin. Definitive demonstration of a role for plasmin in activating the proenzyme of urokinase-type plasminogen activator.

Four monoclonal antibodies raised against purified human plasminogen were characterized for their effects on the activation of plasminogen and on three enzymic properties of plasmin: (a) thioesterolysis, (b) fibrinolysis, (c) conversion of high-Mr urokinase to its low-Mr form. None of the monoclonal antibodies inhibited plasminogen (plg) activation by urokinase. The monoclonal antibodies characterized in this study fell into three groups. Anti-plg 1 inhibited (a), (b) and (c), while anti-plg 2 inhibited activities (a), (b) and (c) to varying degrees but also formed complexes with plasmin that were stable to sodium dodecyl sulphate. Anti-plg 3 and anti-plg 4 inhibited only activity (c). Selective use of these monoclonal antibodies demonstrated unequivocally that plasmin mediates the activation of the proenzyme form of urokinase-type plasminogen activator. Besides their use in affinity chromatography, therefore, these antibodies are valuable for defining the role of plasmin in the mechanisms of extracellular matrix degradation.

Antibodies, Monoclonal↗
PubMed1986

Technetium-99m autologous phagocyte scanning: a new imaging technique for inflammatory bowel disease.

A method to determine the extent of active inflammatory bowel disease using selective labelling of autologous neutrophils and monocytes by phagocytosis of a technetium-99m (99mTc) stannous oxide colloid is described. Unlike leucocyte scanning techniques using Indium-III (IIIIn), the 99mTc colloid scan uses a cheap, readily available isotope, which specifically labels phagocytes. Scan results in 20 patients with inflammatory bowel disease were compared with barium examinations and colonoscopic appearances. There was close agreement in 15 of 20 patients as to the extent of mucosal disease. In four cases the scan showed more extensive disease than was suggested by barium examination. The scan showed terminal ileal Crohn's disease in three patients in whom the barium studies of the ileum had been reported as normal. In four patients with inactive disease and normal barium examinations no activity was seen on the scans. The 99mTc phagocyte scan is a sensitive, reliable means of determining the extent of active inflammatory bowel disease and can be used to quantify disease activity.

Adolescent↗
PubMed1985

Isolation of plasma membrane from human blood monocytes. Subcellular fractionation and marker distribution.

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface-labelled with a radiolabelled monoclonal antibody, 125I-WVH-1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5'-nucleotidase and leucine 2-naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), beta-hexosaminidase and beta-glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I-WVH-1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD-splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non-specific esterase in human mononuclear phagocytes is controversial. No evidence was found for alpha-naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane-bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.

Antibodies, Monoclonal↗
PubMed1985

Preparation and characterization of human bone marrow-derived macrophages.

Bone marrow-derived macrophages were prepared from human bone marrow mononuclear cells following cultivation in GCT-conditioned medium (GCT-CM) and purification by adherence to fibronectin-coated flasks. The growth of bone marrow mononuclear cells in GCT-CM was dependent on the shape of the culture vessels, being increased in round-bottomed versus flat-bottomed wells. Proliferation was confined to nonadherent cells; like blood monocytes, bone marrow-derived macrophages did not incorporate [3H]thymidine in response to GCT-CM or human serum. Purified macrophages from this source expressed nonspecific esterase and OKM1, OKla, FMC 17, 32, and 34 and 25F9 antigens but lacked Mo2. They expressed high levels of an inactivator of plasminogen activator, minactivin, and gave a substantial metabolic burst in response to phorbol myristate acetate or opsonized (but not unopsonized) zymosan. Bone marrow-derived macrophages acted as accessory cells in the response of T lymphocytes to phytohemagglutinin. The results suggest that liquid bone marrow cultures are useful in the study of the differentiation of human mononuclear phagocytes.

Antibodies, Monoclonal↗