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Biomedical subjects

S Afford

Publications and source records attributed to S Afford.

12 recordsLinked to original sources
PubMed2000

Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation.

We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC.

Animals↗
PubMed2000

Hepatocyte growth factor activator (HGF-A) and its zymogen in human placenta.

HGF-activator (HGF-A) is a circulating serine protease known to be responsible for activation of hepatocyte growth factor (HGF). Active HGF is thought to be an important regulator of trophoblast growth. In vitro, HGF-A is produced via proteolytic cleavage of its zymogen by thrombin. Immunocytochemistry and Western immunoblotting were performed using human placental tissue from all three trimesters with an antibody that recognizes both HGF-A and its zymogen. Western immunoblotting revealed a 97 kDa band equivalent to the zymogen in placenta from all three trimesters. A smaller 34 kDa band equivalent to HGF-A was only seen in first and second trimester placenta. The anti-HGF-A/zymogen antibody demonstrated immunostaining in placental villi and membranes throughout gestation. Within first trimester villi immunostaining was strongest within the syncytio- and cytotrophoblast layers, but was also seen within stromal and endothelial cells. Likewise, in third trimester placenta the syncytio-cytotrophoblast layer showed the strongest immunoreactivity. In vitro, HGF can induce trophoblast DNA synthesis and the localization of HGF-A to the peri-villous trophoblast layer (which expresses c-met, the HGF receptor) suggests that it may be responsible for activation of pro-HGF at this site. This adds further weight to the hypothesis that HGF in vivo is an important regulator of trophoblast growth.

Blotting, Western↗
PubMed2000

Apoptosis.

Apoptosis is the genetically regulated form of cell death that permits the safe disposal of cells at the point in time when they have fulfilled their intended biological function. Examples of apoptosis can be cited throughout the whole of the animal and plant kingdoms. It is a vitally important process during normal development and the adult life of many living organisms. In humans, dysregulation of apoptosis can result in inflammatory, malignant, autoimmune, and neurodegenerative diseases. In addition, infectious agents, including viruses, exploit cellular apoptosis in the host to evade the immune system. This review gives a brief historical perspective of some of the landmark discoveries in apoptosis research. The morphological and biochemical stages of apoptosis are then covered, followed by an overview of how it can be studied in the laboratory. Finally, the implications for therapeutic intervention in disease treatment are discussed.

Animals↗
PubMed1998

Ontogeny of hepatocyte growth factor (HGF) and its receptor (c-met) in human placenta: reduced HGF expression in intrauterine growth restriction.

Severe intrauterine growth restriction (IUGR) is characterized by abnormal placentation. Mouse gene knockout studies show that an absence of either hepatocyte growth factor (HGF) or its receptor, c-met, leads to intrauterine death secondary to severe IUGR with deficient placentation. In this study, immunocytochemistry localized HGF protein throughout placental villi across gestation, whereas c-met protein was localized only to the perivillous trophoblast and vascular endothelium. Within the IUGR placentae, a reduction in HGF immunostaining within the villous stroma was observed. HGF mRNA was strongly expressed in the perivascular tissue around the stem villous arteries throughout gestation, with weaker expression within the villous stroma and the terminal villi. c-met mRNA expression was limited to the perivillous trophoblast, particularly in the first trimester, with only a faint hybridization signal from the villous stroma. Placental mRNA expression was examined quantitatively using a ribonuclease protection assay: HGF and c-met mRNA expression increased from the first to the second trimester, reaching a zenith before decreasing again through the third trimester to term. HGF mRNA levels were significantly reduced in the IUGR placentae (P = 0.036), whereas c-met mRNA expression was within the normal range for gestation. These findings suggest that HGF derived from the perivascular tissue of stem villous arteries may play an important role in controlling normal villous development. Whereas reduced expression of HGF within IUGR placentae does not prove a causative link with abnormal villous development, the association lends support to this possibility.

Adult↗
PubMed1996

Hepatic expression of macrophage inflammatory protein-1 alpha and macrophage inflammatory protein-1 beta after liver transplantation.

Two local events that are crucial for T cell emigration into tissue are (1) activation of T cell integrins to permit binding to endothelial counter-receptors and (2) directed migration through the endothelium and into tissue in response to chemotactic factors. Because the chemokines macrophage inflammatory protein-1 alpha (MIP-1 alpha) and MIP-1 beta can activate adhesion and induce migration of T cells in vitro, we investigated their expression in human liver allografts to determine whether they might be involved in regulating the recruitment of T cells to allografts in vivo. Both chemokines were expressed strongly by infiltrating leukocytes during rejection and could be detected immunohistochemically on biliary epithelium, an important target for T cell mediated graft damage. Both chemokines, but particularly MIP-1 beta, were detected on the vascular and sinusoidal endothelium of rejecting liver allografts, where they were coexpressed with the T cell beta 1-integrin receptor vascular cell adhesion molecule-1. In situ hybridization with complementary ribonucleic acid probes showed no MIP-1 alpha or MIP-1 beta mRNA in normal liver but dramatic expression of both chemokines in infiltrating leukocytes and graft endothelium during rejection. Expression was reduced after successful corticosteroid treatment of rejection but persisted in patients progressing to chronic rejection. Increased MIP-1 alpha and MIP-1 beta mRNA expression was already found in biopsies taken at the end of the transplant operation, suggesting that early induction of chemokines, possibly in response to graft reperfusion, might promote the subsequent development of graft rejection. These data demonstrate for the first time that MIP-1 alpha and MIP-1 beta are (1) expressed in human liver allografts, (2) produced by endothelial cells in vivo, and (3) induced early after transplantation. They suggest that MIP-1 alpha and MIP-1 beta produced by graft infiltrating leukocytes and graft endothelium might play a crucial role in regulating T cell recruitment to liver allografts in vivo.

Bile Ducts↗
PubMed1996

Localisation of hepatocyte growth factor and its receptor (c-met) protein and mRNA in human term placenta.

Successful pregnancy depends upon placental growth and development, which follows a specific spatial and temporal sequence. Hepatocyte Growth Factor (HGF) is a potent mitogen, morphogen and motogen to both endothelial and epithelial cell types and is linked to a tyrosine kinase, proto-oncogene, c-met receptor. In 'normal' third trimester placentae (n = 5) full thickness biopsies (obtained at Caesarean section), immunolocalisation and in situ hybridisation studies were performed for HGF and c-met., respectively. HGF immunoreactive protein was present in mesenchymal core, the vaculosyncytial membrane (syncytotrophoblast) and the vascular endothelial cells of villous trophoblast. The HGF mRNA was present particularly strongly in the perivascular stromal cells surrounding the villous vasculature and the amnion/chorionic membranes. Immunoreactive c-met protein was strongly localised to the endothelial cells lining the villous vasculature and the vasculosyncytial membrane. A relatively weak and diffuse hybridisation signal for c-met mRNA was present throughout the villous trophoblast, most pronounced in the vasculosyncytial membrane. These results indicate that HGF may serve as a paracrine mediator to control placental development and growth.

DNA Probes↗
PubMed1994

Differential cellular synthesis of insulin-like growth factor binding protein-1 (IGFBP-1) and IGFBP-3 within human liver.

The two major insulin-like growth factor-binding protein (IGFBP) species in the human circulation, IGFBP-1 and IGFBP-3, are synthesized in large amounts by liver. To determine which hepatic cell populations in human liver were responsible for the synthesis and release of these IGFBPs, we 1) performed in situ hybridization with specific complementary RNA (RNA) probes for human IGFBP-1 or-3 or performed immunohistochemical analysis to reveal the sites of messenger RNA (mRNA) presence and peptide translation, respectively, in sections of normal liver derived from organ donors; and 2) examined the release of IGFBP species by Western ligand and immunoblots of medium conditioned by isolated cultures of hepatocytes, lipocytes, and Kupffer cells. In situ hybridization showed that IGFBP-1 mRNA was distributed widely among the parenchymal cell population, which also showed immunohistochemical staining for IGFBP-1 peptide. Conversely, IGFBP-3 mRNA and immunoreactive peptide were mainly localized to Kupffer cells, which were positively identified by immunoreactivity with antiserum against the glycoprotein marker, CD68. Isolated hepatocytes released two species of IGFBP of 28 and 30-32 kilodaltons, which were recognized immunologically as IGFBP-1. Isolated Kupffer cells released only a 43- to 46-kilodalton IGFBP immunologically recognized as IGFBP-3. Lipocyte cultures released no detectable IGFBP species. The results suggest that IGFBP-1 and IGFBP-3 are derived from separate cell populations in human liver.

Adolescent↗
PubMed1991

The effect of inducing agents on the numbers of interphase fibrillar centers in the U937 promonocytic cell line.

The mean numbers of interphase fibrillar centers have been determined in triplicate experiments, using the argyrophil (AgNOR) method. Promonocytic U937 cells were incubated with each of three inducing agents, namely, interleukin-6, granulocyte-macrophage colony-stimulating factor, and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. The cells were examined at 24, 48, and 72 h during the induction periods and their doubling time and mean AgNOR score were calculated. After 72 h, the cells were maintained in culture for a further 24 h in the absence of an inducing agent and these parameters were determined again. It was found that whereas the unstimulated U937 cells had a mean value in the region of 50 AgNORs per nucleus, this diminished to about 20 after a 72 h incubation, but rose to 30 or more when the inducing agents had been withdrawn for 24 h. These observation confirm the results of previous studies using melanoma and HL60 cell lines: however, it has now been demonstrated that a variety of agents can modulate the numbers of fibrillar centers in a very similar way in a single cell line. Furthermore, we have shown that the "undifferentiated" U937 cell AgNOR score recovers when the agents no longer act upon the cells; this implies that fibrillar center numbers are intimately related to differentiation state in cell lines, as in the case in, for example, tumor specimens.

Cell Count↗
PubMedDate not supplied

Hepatocyte growth factor concentration in maternal and umbilical cord blood samples and expression in fetal liver.

OBJECTIVE: To determine whether human placenta secretes hepatocyte growth factor (HGF) and could influence fetal liver development. METHODS: Expression of HGF and c-met mRNA in paired samples of first- and second-trimester fetal liver and placenta was compared using a quantitative ribonuclease protection assay. Serum HGF concentration in 30 samples of paired umbilical and maternal blood from term pregnancies was evaluated using an enzyme-linked immunosorbent assay. RESULTS: HGF and c-met mRNA were expressed at similar levels in liver and placenta, with expression increasing from 9 to 16 weeks' gestation. Median serum HGF values were 1.4 ng/mL (maternal venous), 1.2 ng/mL (cord venous), and 1.3 ng/mL (cord arterial). The maternal venous HGF levels were significantly higher than fetal venous levels (P =.02). CONCLUSIONS: This study does not support the hypothesis that the placenta secretes HGF, because maternal serum levels were higher than fetal and there was no significant difference between umbilical arterial and venous samples. Fetal liver expresses abundant HGF mRNA during the first and second trimester and expression increases in line with receptor (c-met) expression, suggesting that hepatic growth and development are independent of placental HGF.

Electrophoresis, Polyacrylamide Gel↗