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C W Pugh

Publications and source records attributed to C W Pugh.

52 records · Page 3Linked to original sources
PubMed1994

Expression of a homologously recombined erythopoietin-SV40 T antigen fusion gene in mouse liver: evidence for erythropoietin production by Ito cells.

We have obtained transgenic mice in which an erythropoietin-SV40 virus T antigen fusion gene is homologously recombined into the native Epo locus. This gene is expressed in a tissue-specific manner closely resembling that of the native Epo gene. Immunohistochemical detection of SV40 T antigen has been used to characterize the hepatic cell populations expressing the transgene. In mice stimulated by anaemia or hypobaric hypoxia, SV40 T antigen was demonstrated in two liver cell populations: a subset of hepatocytes and a nonparenchymal cell type. Immunohistochemical and ultrastructural characterization of these cells by light and electron microscopy showed the nonparenchymal cell type to be the Ito cells, which lie in a persinusoidal position within the space of Disse. We therefore conclude that Ito cells are the nonhepatocytic source of liver Epo production. These cells show many similarities to the Epo-producing fibroblastoid interstitial cells of the kidney.

Anemia↗
PubMed1994

Oxygen-regulated control elements in the phosphoglycerate kinase 1 and lactate dehydrogenase A genes: similarities with the erythropoietin 3' enhancer.

Production of the glycoprotein hormone erythropoietin (Epo) in response to hypoxic stimuli is almost entirely restricted to particular cells within liver and kidney, yet the transcriptional enhancer lying 3' to the Epo gene shows activity inducible by hypoxia after transfection into a wide variety of cultured cells. The implication of this finding is that many cells which do not produce Epo contain a similar, if not identical, oxygen-regulated control system, suggesting that the same system is involved in the regulation of other genes. We report that the human phosphoglycerate kinase 1 and mouse lactate dehydrogenase A genes are induced by hypoxia with characteristics which resemble induction of the Epo gene. In each case expression is induced by cobalt, but not by cyanide, and hypoxic induction is blocked by the protein-synthesis inhibitor cycloheximide. We show that the relevant cis-acting control sequences are located in the 5' flanking regions of the two genes, and we define an 18-bp element in the 5' flanking sequence of the phosphoglycerate kinase 1 gene which is both necessary and sufficient for the hypoxic response, and which has sequence and protein-binding similarities to the hypoxia-inducible factor 1 binding site within the Epo 3' enhancer.

Animals↗
PubMed1994

Production of erythropoietin by liver cells in vivo and in vitro.

Several lines of evidence indicate that the liver is the predominant production site for EPO during the early stages of development. Moreover, in adults, it may contribute significantly to EPO production in some species. Independent of age hepatocytes are the major cellular sites of EPO gene expression in the liver, but quantitatively less significant expression also occurs in at least one additional type of nonparenchymal cell. Although some indirect evidence suggests that these nonparenchymal cells producing EPO may be Kupffer cells, their identity remains to be clarified. Regarding the mechanisms of the adaptation of hepatic EPO production to changes in oxygen availability in the organism, experiments with isolated perfused livers, mixed liver cell cultures, hepatoma cells, and, more recently, isolated hepatocytes have provided evidence that inherent cellular oxygen-sensing mechanisms exist and that external factors are not essential. The use of hepatoma cells has allowed investigation of these cellular mechanisms of oxygen-dependent gene control, and further studies in nontransformed hepatocytes may complement these studies.

Aging↗
PubMed1994

Characterisation of functional domains within the mouse erythropoietin 3' enhancer conveying oxygen-regulated responses in different cell lines.

We have analysed sequences within the mouse erythropoietin enhancer which are required for oxygen regulated operation in the erythropoietin producing cell line, HepG2, and in two non-erythropoietin producing cell lines; the lung fibroblastoid cell line a23, and mouse erythroleukaemia (MEL) cells. At least three critical sites were demonstrated within a 96 nucleotide sequence. Oxygen regulated operation was dependent on sites within the first 26 nucleotides. Sequences lying 3' to this region modulated enhancer function but did not themselves convey oxygen regulated operation. In HepG2 cells these 3' sequences co-operated to permit operation of the inducible element at a distance from a promoter, but in MEL cells 3' sequences repressed activity of the inducible element. Though operation of this 3' sequence differed according to the cell type, oxygen regulated operation was dependent on the same two critical sites in the 5' region in both erythropoietin producing and non-erythropoietin producing cells. These findings support the existence of a widespread oxygen sensing system in mammalian cells which is similar to that operating in specific cells to regulate erythropoietin production, and they indicate that the system activates factors with similar DNA sequence specificity in different cells.

Animals↗
PubMed1993

Inducible operation of the erythropoietin 3' enhancer in multiple cell lines: evidence for a widespread oxygen-sensing mechanism.

Adaptive responses to hypoxia occur in many biological systems. A well-characterized example is the hypoxic induction of the synthesis of erythropoietin, a hormone which regulates erythropoiesis and hence blood oxygen content. The restricted expression of the erythropoietin gene in subsets of cells within kidney and liver has suggested that this specific oxygen-sensing mechanism is restricted to specialized cells in those organs. Using transient transfection of reporter genes coupled to a transcriptional enhancer lying 3' to the erythropoietin gene, we show that an oxygen-sensing system similar, or identical, to that controlling erythropoietin expression is wide-spread in mammalian cells. The extensive distribution of this sensing mechanism contrasts with the restricted expression of erythropoietin, suggesting that it mediates other adaptive responses to hypoxia.

Animals↗
PubMed1993

Oxygen-dependent expression of the erythropoietin gene in rat hepatocytes in vitro.

Since in juvenile rats the liver is the predominant site of erythropoietin (EPO) gene expression, we have used primary cultures of juvenile rat hepatocytes to establish and in vitro system for investigation of oxygen-dependent EPO formation. When isolated hepatocytes were incubated at reduced oxygen tensions for 18-48 h, we found increased secretion of EPO protein and elevated levels of EPO mRNA, as determined by RNas protection. This increase was maximal at 3% O2, where EPO mRNA levels after 18 h were approximately 15-fold higher than at 20% O2. The increase in EPO mRNA at low oxygen tensions was specific insofar as [3H]uridine incorporation, as a measure of total RNA synthesis, was reduced by approximately 50% at 3% O2, and it appeared to involve gene transcription since it was abolished in the presence of actinomycin D (35 microM). Significant increases in EPO mRNA were also observed in cells kept at 20% oxygen in the presence of cobalt chloride (50 microM) and nickel chloride (400 microM), but EPO mRNA levels achieved under these conditions were less than 7% of those in cells incubated at 3% oxygen. No increase in EPO mRNA levels was observed in cultures incubated at 20% O2 in the presence of cyclic dibutyryl-AMP (10 microM-3 mM), cyclic 8-bromoGMP (10 microM-1 mM), cyclohexyladenosine (1 microM), 5'-N-ethylcarboxamidoadenosine (1 microM) and phorbol 12-myristate 13-acetate (3 nM). In the presence of 10% carbon monoxide, used to block haem proteins in their oxy conformation, EPO mRNA levels in hepatocytes incubated at low oxygen tensions were reduced to 63%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Identification of the renal erythropoietin-producing cells using transgenic mice.

Regulation of erythropoietin production by the kidneys is central to the control of erythropoiesis. Uncertainty about the identity of the renal cells involved has been a major obstacle to understanding this mechanism. We have used sequence from the mouse erythropoietin locus to direct expression of a marker gene, SV40 T antigen, to these cells in transgenic mice. The transgenic constructs contained an oligonucleotide marker (Epo-M) or SV40 sequence (Epo-TAg) in the 5' untranslated region of the mouse erythropoietin gene, flanked on each side by 9 and 7.5 kb of DNA from the mouse erythropoietin locus. Anemia-inducible expression of Epo-M and Epo-TAg was observed in the kidney. In one of thirteen lines, homologous integration of Epo-TAg into the mouse erythropoietin locus occurred. In transgenic mice bearing Epo-TAg at homologous and heterologous insertion sites, renal expression was restricted to a population of cells in the interstitium of the cortex and outer medulla. Immunohistochemical characterization by light and electron microscopy shows that these are the fibroblast-like type I interstitial cells.

Animals↗
PubMed1991

Functional analysis of an oxygen-regulated transcriptional enhancer lying 3' to the mouse erythropoietin gene.

Erythropoietin, the major hormone controlling red-cell production, is regulated in part through oxygen-dependent changes in the rate of transcription of its gene. Using transient transfection in HepG2 cells, we have defined a DNA sequence, located 120 base pairs 3' to the poly(A)-addition site of the mouse erythropoietin gene, that confers oxygen-regulated expression on a variety of heterologous promoters. The sequence has the typical features of a eukaryotic enhancer. Approximately 70 base pairs are necessary for full activity, but reiteration restores activity to shorter inactive sequences. This enhancer operates in HepG2 and Hep3B cells, but not in Chinese hamster ovary cells or mouse erythroleukemia cells, and responds to cobalt but not to cyanide or 2-deoxyglucose, thus reflecting the physiological control of erythropoietin production accurately.

Animals↗
PubMed1991

Imaging of haemodialysis-associated amyloidosis with 123I-serum amyloid P component.

Long-term haemodialysis is frequently complicated by amyloid deposition in which the fibrils consist of beta 2-microglobulin. Dialysis-related amyloid disease causes extensive morbidity and has been associated with deaths in some cases. All amyloid deposits contain amyloid P component that is derived from the normal circulating protein, serum amyloid P component (SAP). We have used scintigraphic imaging after injection of 123I-labelled SAP to assess the distribution of amyloidosis in 38 patients receiving long-term haemodialysis for end-stage renal failure. There was focal localisation of tracer at all sites where histological examination confirmed amyloid deposition. Splenic uptake was seen in 12 patients, indicating splenic amyloidosis, but there was no evidence of other visceral involvement. 6 control subjects who had been dialysed for under 1.5 years showed no localisation of tracer, nor was there any uptake of 123I-labelled human serum albumin in 3 long-term dialysis patients with histologically confirmed amyloidosis and positive 123I-SAP images. Negative scans were also obtained in 5 patients who had been transplanted 0.8-2.4 years previously, despite past evidence of dialysis arthropathy (5) and histologically proven amyloidosis (4). 123I-SAP scintigraphy may be helpful as a non-invasive method for both the diagnosis and monitoring of dialysis-associated amyloidosis.

Amyloidosis↗
PubMed1984

Heterogeneity amongst lymph-borne "dendritic" cells.

Thoracic duct lymph from mesenteric lymphadenectomized rats contains a population of non-lymphocytic, irregularly shaped, Ia-positive cells. These cells are able to stimulate an allogeneic or syngeneic MLR, can act as accessory cells in Concanavalin A-induced lymphocyte mitogenesis and can stimulate the rejection of enhanced allografts. They are thus similar to dendritic cells isolated from lymphoid and peripheral tissues. These cells are not endocytic or glass adherent and do not bear C3 receptors or mannose-fucose receptors. They may possess weak Fc receptor activity. In terms of morphology, cytochemistry and surface markers these cells show marked heterogeneity. The nature of this heterogeneity is described and the relationship of these cells to other potential accessory cells is discussed in terms of lineage and function.

Animals↗
PubMed1983

Characterization of nonlymphoid cells derived from rat peripheral lymph.

Mesenteric lymphadenectomy in rats is followed by union of peripheral and central lymphatics, allowing the collection of intestine-derived peripheral lymph cells via the thoracic duct for several days. These cells include a proportion of nonlymphoid cells (NLC) that show irregular and heterogeneous surface morphology including long pseudopodia and veils. They stain variably for nonspecific esterase and acid phosphatase and are ATPase-positive. Their nuclei are irregular and some contain cytoplasmic inclusions, some of which show peroxidase activity and/or contain DNA. NLC have a range of densitites generally lower than that of lymphocytes. Freshly collected NLC express the leukocyte-common antigen (defined by monoclonal antibody MRC Ox 1) and Ia antigens (I-A and I-E subregion products defined by monoclonal antibodies) but they show a relative lack of other surface markers normally found on rat B or T lymphocytes (W3/13, W3/25, MRC Ox 12 (sIg), MRC Ox 19) or rat macrophages (FcR, C'R, mannose R, W3/25). In general NLC are only weakly adherent to glass or plastic. Although a subpopulation of NLC appear to have had a phagocytic past, freshly collected NLC fail to phagocytose a variety of test particles in vitro. NLC also appear incapable of pinocytosis in vitro. This heterogeneity may represent distinct subpopulations of NLC or different stages in the development of a single cell lineage. Direct cannulation of mesenteric lacteals shows that the majority of NLC are derived from the small intestine and their precursors appear to be present both in lamina propria and Peyer's patches. Kinetic studies, following irradiation or intravenous tritiated thymidine, show that the majority of NLC turn over rapidly in the intestine with a modal time of 3-5 d. Studies with bone marrow chimeras show that they are derived from a rapidly dividing precursor present in normal bone marrow. NLC occur at very low frequencies in normal thoracic duct lymph at all times following cannulation. The evidence presented suggests that NLC closely resemble mouse lymphoid dendritic cells. This conclusion is supported by evidence already obtained showing that NLC are potent stimulators of the semi-allogeneic rat primary mixed leukocyte reaction. In addition to the ceils resembling dendritic cells rare monocytoid cells are found in thoracic duct lymph of lymphadenectomized specific pathogen-free rats. The proportion of these cells increases greatly when the animals are conventionally housed. It seems probable that the physiological function of NLC is to act as accessory cells in the lymph nodes to which they normally drain. Methods for enriching NLC and thus facilitating analysis of their functions are discussed.

Animals↗
PubMed1983

The distribution, ontogeny and origin in the rat of Ia-positive cells with dendritic morphology and of Ia antigen in epithelia, with special reference to the intestine.

Ia antigens were localized in cryostat sections of rat intestine and other tissues by an indirect immunoperoxidase technique using monoclonal antibodies that recognize the rat antigens homologous to the gene products of the I-A and I-E subregions of the mouse major histocompatibility complex (MHC). Two categories of Ia+ cells were characterized, namely epithelial cells and bone marrow-derived cells with dendritic morphology. In the small intestine Ia antigen was present in the distal 2/3 of the absorptive epithelium but absent from the bases of the villi, the crypts and the epithelium covering the Peyer's patches. The distribution in nude rats was similar, indicating that T lymphocytes are not obligatory for its expression. In ontogeny Ia antigen was absent in the epithelium of neonatal gut, appearing at about 4 weeks of age and reaching adult levels at about 6 weeks. Different rat strains showed large differences in the amount of Ia antigen expressed by villus epithelium and the traits for the level of expression were shown to map outside the MHC. The levels of expression of Ia antigen in the proximal tubules of the kidney followed that of the gut epithelium in the different strains and in both tissues was mostly intracellular. Studies with chimeras showed that the Ia antigen in epithelial cells was not acquired from bone marrow-derived cells. The second category of cell studied had a characteristic dendritic morphology and was present in large numbers in the lamina propria of the villi and in the crypts. In the Peyer's patches these cells were present both in the subepithelial dome region and within the epithelium itself. These Ia+ dendritic cells were present in nude rat jejunum and appeared in normal fetal gut by 18 days gestation and were also shown to migrate into antigen-free grafts of fetal gut. This suggests that they do not require stimulation from antigens, bacterial products or T lymphocytes in order to localize in the gut or to express Ia antigen. Studies with other cell surface markers suggest that the Ia+ cells with dendritic morphology represent a range of cell types, some with similarities to macrophages and others to nonphagocytic dendritic cells.

Age Factors↗
PubMed1981

The rat mixed lymphocyte reaction: roles of a dendritic cell in intestinal lymph and T-cell subsets defined by monoclonal antibodies.

Cells present in the intestinal lymph of rats were obtained in large numbers by removing the mesenteric, portal and caecal lymph nodes and cannulating the thoracic duct 6 weeks later. About 1% of the cells present in the thoracic duct lymph of these mesenteric lymphadenectomized rats had striking dendritic morphology, were strongly Ia+ but labelled weakly with monoclonal antibodies that recognize rat B or T cells. It was found that intestinal lymph was highly enriched for cells that stimulated allogeneic T cells in the mixed lymphocyte reaction (MLR) and cells with stimulator activity co-purified with dendritic cells. Thus, these dendritic cells appear phenotypically and functionally similar to the dendritic cells that have been described in the mouse spleen and rat lymph node. The ability of the intestinal lymph cells to stimulate rat T cells was used to determine which of the two subsets of these cells were the prime responders in the rat MLR. These subsets, defined by monoclonal antibodies, have been shown by previous work to display close functional analogies to the Lyt 2+ and Lyt 2- subsets in the mouse and to the two human T-cell subsets that have been defined by monoclonal antibodies. It was found that the T-cell subset that contains the helper cells for antibody responses proliferated when irradiated, fully allogeneic or semi-allogeneic thoracic duct cells were used as stimulators, but the subset containing suppressor T cells did so only in the fully allogeneic system. Detailed studies showed that in the absence of helper cells in the responder population T cells in the stimulator population of helper phenotype were responsible for proliferation of the suppressor T-cell subset observed in fully allogeneic MLRs. Proliferation of the suppressor T-cell subset could be obtained using semi-allogeneic stimulators, provided that the F1 cells were derived from a source containing dendritic cells but it was shown that, as in the case with fully allogeneic stimulators, the helper T cells in the stimulator population were playing an active role. These results demonstrate that proliferation of the suppressor T-cell subset in the rat MLR is dependent on blastogenic activity provided by the helper T-cell subset and suggest that in some situations this blastogenic activity may arise through the recognition, by the helper cells, of environmental antigens presented on dendritic cells. It has been reported that in the human MLR both T-cell subsets proliferate but that only the helper subset does so when antigen-primed cells are stimulated with specific antigen. The present experiments, by emphasizing the activity of helper T cells in the stimulator population in the MLR, cast doubt on the implication that recognition of alloantigens in vitro differs in an essential way from that of soluble antigens.

Animals↗