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S C Robson

Publications and source records attributed to S C Robson.

At least 91 records · Page 5Linked to original sources
PubMed1999

Molecular characterization of the 5' non-coding region of South African GBV-C/HGV isolates: major deletion and evidence for a fourth genotype.

GB virus C/hepatitis G virus (GBV-C/HGV) has been characterised as a novel flavivirus, and to date three known genotypes have been cloned. Greater genetic variation of GBV-C/HGV has been demonstrated in West African isolates, but no major deletions have been shown in the 5' non-coding region (NCR). The 5'NCR regulates protein translation via an internal ribosomal entry site (IRES). We cloned, sequenced, and analysed a 344-bp polymerase chain reaction (PCR) product, representing >60% of the 5'NCR, from 32 GBV-C/HGV PCR-positive volunteers. Wild-type virus amplicons were detected in all samples. However, 5/32 (15.6%) also amplified another fragment of between 205 and 231 bp. Sequence analysis showed all cloned PCR fragments to be GBV-C/HGV-specific. A typical deletion of 113-131 bp with minor variation was detected in isolates generating the smaller bands. RNA secondary structure analysis showed the deletions to be over domains II and III. This finding suggests that nucleotides 303-444 may be non-essential for 5'NCR functioning. Phylogenetic analysis demonstrated a novel fourth South African genotype, distinct from genotypes 1-3 with DNA distances of >0.1000. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) values for the wild-type and mutant samples were normal. This study documents the first major deletion in the 5'NCR of GBV-C/HGV, and suggests that bases 303-444 may not be essential for viral replication and ribosomal entry. A fourth GBV-C/HGV genotype appears to predominate in South Africa.

5' Untranslated Regions↗
PubMed1999

Lysophosphatidic acid activates nuclear factor kappa B and induces proinflammatory gene expression in endothelial cells.

The cellular phospholipid, lysophosphatidic acid (LPA), released by activated platelets and fibroblasts or, at high levels, from ovarian and cervical carcinomas is a powerful serum mitogen that may modulate several signaling pathways in endothelial cells (EC). Hence, LPA could function in a paracrine manner during EC-platelet interactions at sites of vascular injury. Here, we demonstrate activation of the transcription factor nuclear factor kappa B (NF-kappaB) in EC following exposure to LPA. EC activation was further characterized by increased levels of mRNA transcripts encoding E-selectin, Intercellular Adhesion Molecule-1, Interleukin-8 and Monocyte Chemoattractant Protein-1. These effects were inhibited by preincubating EC either in the presence of mepacrine (to block phospholipase A2) or of pertussis toxin (to increase ADP-ribosylation of Gi proteins). No inhibition was observed in the presence of putative LPA receptor antagonists suramin or thrombospondin. LPA induces a proinflammatory activation of endothelial cells that (i) involves Gi proteins; (ii) depends on phospholipase A2 activity; (iii) is associated with the activation of NF-kappaB and (iv) results in increased expression of proinflammatory genes. We propose that LPA release by activated platelets may directly modulate vascular inflammatory responses.

Animals↗
PubMed1999

Analysis of CD39/ATP diphosphohydrolase (ATPDase) expression in endothelial cells, platelets and leukocytes.

Purinergic signaling may influence hemostasis, inflammatory responses and apoptosis. Therefore, hydrolysis of extracellular ATP and ADP by the ATP diphosphohydrolase (ATPDase) could regulate these processes. We have previously demonstrated the identity between the vascular ATPDase and CD39. Here we show that levels of CD39 expression correlate with ATPDase activity in human endothelial cells (EC), platelets and selected monocyte, NK, and megakaryocyte cell lines. Western blotting revealed one to three isoforms of CD39/ATPDase: mobility variations of major protein resulted from post-translational modifications. Northern blotting and primer extension indicated two major mRNA transcripts and one transcription start point, respectively. In addition, mRNAs specific for purinergic P2 receptors were detected in all of the investigated cells, suggesting that the coexpressed CD39/ATPDase may regulate purinergic signaling. Thrombotic and inflammatory responses may be modulated by the expression of CD39/ATPDase.

Adenosine Diphosphate↗
PubMed1999

Modulation of nucleoside [correction of nucleotide] triphosphate diphosphohydrolase-1 (NTPDase-1)cd39 in xenograft rejection.

BACKGROUND: There is increasing evidence showing that extracellular nucleosides [corrected] may be important mediators of vascular inflammation. Nucleoside [corrected] triphosphate diphosphohydrolase-1 (NTPDase-1, identical to CD39), the major vascular endothelial ectonucleotidase, is responsible for the hydrolysis of both extracellular ATP and ADP in the blood plasma to AMP. Studies were therefore conducted to evaluate the role of vascular NTPDase-1/cd39 in modulating platelet activation and vascular injury in cardiac xenografts. MATERIALS AND METHODS: Cardiac xenografts from both wild-type and cd39 knockout mice (C57BL/6 x 129 Svj) were transplanted into Lewis rats. Alterations in cd39 mRNA transcripts and NTPDase activity expression were evaluated in wild-type grafts in untreated rats and then following complement depletion and immunosuppression. Rejection responses were studied with both mutant and wild-type grafts in the following models: presensitization with or without complement depletion, complement depletion alone, and with chronic immunosuppression to induce long-term graft survival. RESULTS: NTPDase biochemical activity in wild-type xenografts rapidly decreased after transplantation but soon rebounded with graft survival. Elevated levels of cd39 mRNA with associated increases in NTPDase activity were observed in all long-term surviving wild-type grafts. Hyperacute xenograft rejection times were comparable in wild-type and mutant grafts but cd39-deficient grafts were subject to more rapid rejection and exhibited pronounced vascular injury in complement-depleted, presensitized rats. The cd39-deficient grafts in immunosuppressed recipients were subject to increased intravascular platelet sequestration and fibrin deposition; this resulted in focal myocardial infarction in long-term surviving mutant xenografts. CONCLUSIONS: Augmentation of NTPDase-1 activity may be an important adaptive response for graft survival. Our results suggest that NTPDase-1/cd39 influences pathways of vascular injury in cardiac xenografts.

Adenosine Triphosphatases↗
PubMed1998

Disseminated intravascular coagulation in association with the delayed rejection of pig-to-baboon renal xenografts.

BACKGROUND: Intravascular fibrin deposition and platelet sequestration occur with porcine xenograft rejection by baboons. Disseminated intravascular coagulopathy may arise either as a direct consequence of the failure to fully deplete xenoreactive natural antibodies and block complement, or because of putative cross-species molecular incompatibilities in this discordant species combination. METHODS: Three baboons were conditioned with retrovirally transduced autologous bone marrow to induce tolerance to swine antigens. Xenoreactive natural antibodies and complement were depleted by plasmapheresis and the use of Gal alpha1-3Gal column adsorptions; baboons were then splenectomized and underwent renal xenografting from inbred, miniature pigs. Soluble complement receptor type-1 with protocol immunosuppression (mycophenolate mofetil, 15-deoxyspergualin, steroids, and cyclosporine) was administered. RESULTS: A bleeding diathesis was clinically evident from days 5 to 12 after transplantation in two baboons. Low levels of circulating C3a, C3d, and iC3b were measured despite the absence of functional circulating complement components. Profound thrombocytopenia with abnormalities in keeping with disseminated intravascular coagulopathy were observed. Prolongation of prothrombin and partial thromboplastin times was accompanied by evidence for tissue factor-mediated coagulation pathways, high levels of thrombin generation (prothrombin fragment F(1+2) production and thrombin-antithrombin complex formation), fibrinogen depletion, and production of high levels of the fibrin degradation product D-dimer. Importantly, these disturbances resolved rapidly after the excision of the rejected xenografts in two surviving animals. Histopathological examination of the rejected xenografts confirmed vascular injury, fibrin deposition, platelet deposition, and localized complement activation. CONCLUSIONS: Systemic coagulation disturbances are associated with delayed xenograft rejection.

Animals↗
PubMed1998

Isolation, characterisation and embryonic expression of WNT11, a gene which maps to 11q13.5 and has possible roles in the development of skeleton, kidney and lung.

The Wnt gene family encodes a set of signalling molecules, thought to play an important role in key processes of embryonic development. In vertebrates as a whole 20 different Wnt genes have been identified to date, however, a complement of only 16 have been identified in man and for some of these the complete coding sequences are unavailable. We have recently isolated the full-length cDNA sequence of a new human WNT gene, WNT11, investigated its genomic organisation and performed detailed expression studies in early human embryos. These have shown that the expression of human WNT11 is restricted to the perichondrium of the developing skeleton, lung mesenchyme, the tips of the ureteric buds and other areas of the urogenital system and the cortex of the adrenal gland. This, for the first time, provides information for the embryonic expression of human WNT11. We have mapped WNT11 to 11q13.5 and this together with its expression in the perichondrium of the developing skeleton, makes it a plausible candidate gene for HBM, which has been previously linked to markers from this region.

Amino Acid Sequence↗
PubMed1998

Extracellular ATP and ADP activate transcription factor NF-kappa B and induce endothelial cell apoptosis.

Inflammation within the vasculature is associated with endothelial cell (EC) perturbation, loss of vascular ATP-diphosphohydrolase activity, and platelet microthrombus formation with release of ATP and ADP into the micro-environment. The nature and effects of purinergic stimulation of EC under these circumstances remain largely undetermined. ATP and ADP activated EC transcribed mRNA from certain transcription factor NF-kappa B target genes and expressed E-selectin protein on cell membranes. Band shift analysis and reporter assays confirmed the activation of NF-kappa B in response to both ATP and ADP. Apoptosis was shown to occur in response to purinergic signaling, potentially through the activation of P2z/P2x7 receptors. Induction of EC activation responses and apoptosis in response to stimulation with ATP and ADP is associated with activation of NF-kappa B.

Adenosine Diphosphate↗
PubMed1998

Expression of human thrombomodulin cofactor activity in porcine endothelial cells.

BACKGROUND: Xenograft rejection may predispose to vascular thrombosis because of putative cross-species' functional incompatibilities between natural anticoagulants present on the donor endothelium and host activated coagulation factors. For example, porcine thrombomodulin expressed on porcine aortic endothelial cells (PAEC) does not provide the expected thrombomodulin (TM)-cofactor activity for human protein C in the presence of human thrombin. In addition, TM may be down-regulated after cellular activation. Our aim was to express human TM cofactor activity in PAEC and to study the proinflammatory effect of tumor necrosis factor-alpha (TNF-alpha) on stable expressed human thrombomodulin in vitro. METHODS AND RESULTS: Retroviral transduction of PAEC with the gene encoding for human thrombomodulin (hTM) resulted in expression of high levels of specific TM cofactor activity on PAEC (0.62 microg/ml activated protein C/10(5) cells). High-level expression of hTM resulted in a 620-fold higher activation of human protein C in the presence of human thrombin when compared with mock-transduced PAEC (0.0001 microg/ml/10(5) cells; P<0.001). Transduced PAEC expressing hTM also bound more human thrombin than control PAEC, as determined by inhibition of thrombin-induced platelet activation (P<0.05). We noted that exposure to TNF-alpha significantly reduced exogenous hTM cofactor activity on transduced PAEC in a time- and dose-dependent fashion; this occurred despite the relatively stable expression of hTM mRNA and hTM antigen in these cells. Treatment of transduced PAEC with selected antioxidants could protect against the loss of hTM cofactor activity directly associated with the oxidative stress induced by TNF-alpha activation responses. CONCLUSIONS: Our data show that the functional deficiency of the anticoagulant protein C pathway in PAEC may be corrected by viral transduction of these cells. As analysis of the hTM function showed modulation under conditions of cellular activation, we suggest that expression of hTM mutants resistant to oxidation may have greater therapeutic utility in the genetic modification of porcine xenografts.

Animals↗
PubMed1998

Human post-implantation embryo collection: medical and surgical techniques.

Knowledge of the genetic control of early human development is limited with most information being extrapolated from studies in animal models. There is compelling evidence that undertaking gene expression studies in human embryos can be expected to dramatically enhance our understanding of embryonic formation and malformation. Such studies require the systematic and coordinated collection, storage and study of human embryos. We have successfully collected intact embryos from cases undergoing termination of pregnancy (TOP). Embryonic material was collected from 62% of attempts using a technique of surgical aspiration carried out under ultrasound guidance. Collection rates were lower after medical termination of pregnancy (41%) although the proportion of undisrupted embryos was identical with the two methods (26%). Surgical aspiration provided intact embryos between Carnegie stages (CS) 16-22 while earlier developmental stages were collected from medical terminations. Our collection of over 60 intact specimens, spanning Carnegie stages 10 to 22 (about 21 to 53 days of development) covers a huge window of critical developmental events and hence represents an exciting and valuable research resource.

Abortion, Induced↗
PubMed1998

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type II activation.

BACKGROUND: Xenoreactive natural antibodies (XNAs) and complement mediate hyperacute rejection of discordant xenografts. Inhibition of complement alone results in some prolongation of graft survival, but delayed xenograft rejection still precludes long-term graft survival. In vitro data provide evidence for the direct proinflammatory activation of endothelial cells (ECs) by XNAs. These antibodies are primarily directed against galactose alpha(1-3)-galactose (alpha-gal), the major xenoantigen in the pig to primate xenotransplant model. Previous studies have shown EC activation by XNAs but failed to address the question of whether alpha-gal-specific ligands can induce EC activation. The aim of this study was to investigate whether agonist binding to the alpha-gal epitope by alpha-gal-specific lectins as compared with XNAs or elicited xenoreactive antibodies can directly elicit type II porcine aortic EC (PAEC) activation (i.e., activation that requires protein synthesis). METHODS AND RESULTS: The tetravalent, alpha-gal-binding Bandeiraea simplicifolia lectin I (BS-I), the wholly alpha-gal-specific BS-I isolectin B4, and elicited primate anti-pig xenoreactive antibodies (decomplemented cynomolgus monkey anti-porcine serum) induced E-selectin protein expression in PAECs. This induction was alpha-gal-specific, as preincubation with synthetic alpha-gal carbohydrate or adsorption of lectin or serum to rabbit, but not human, red blood cells removed the activating component. E-selectin expression, induced by BS-I, was inhibited in the presence of genistein, a tyrosine kinase inhibitor, and by mepacrine, an inhibitor of phospholipase A2. Human and primate XNAs lacked this activity when tested at relevant concentrations; however, stimulation of PAECs with affinity-purified human XNA (IgM and IgG) resulted in slightly increased interleukin-8 and P-selectin mRNA levels but had no apparent effects on E-selectin transcription. BS-I strongly induced E-selectin, P-selectin, intercellular adhesion molecule-1, and interleukin-8 mRNA in an NF-kappaB-dependent manner. CONCLUSIONS: Several agonists that specifically bind to alpha-gal can evoke type II EC activation. Hence, anti-Gal antibodies may contribute directly to xenograft rejection in the absence of complement activation.

Animals↗
PubMed1998

Alpha-galactosyl epitope-mediated activation of porcine aortic endothelial cells: type I activation.

BACKGROUND: The galactose alpha(1-3)galactose (alpha-gal) epitope associated with membrane glycoproteins and glycolipids represents a major determinant recognized on porcine cells by human xenoreactive natural antibodies (XNA). Together, bound XNA and complement rapidly induce porcine aortic endothelial cell (PAEC) activation; this process is associated with cellular shape changes, transient development of intercellular gaps, and loss of ATDPase and thrombomodulin, with release of heparan sulfate. The aim of this study was to evaluate patterns of type I endothelial cell activation (i.e., activation that does not require protein synthesis) following ligation of alpha-gal epitopes with anti-Gal antibodies and alpha-gal-specific lectins. METHODS AND RESULTS: PAEC incubated in the presence of the alpha-gal binding, Bandeiraea simplicifolia lectin (BS-I) underwent cellular shape changes associated with the formation of intercellular gaps. PAEC exposure to BS-I was also associated with the tyrosine phosphorylation of a protein (apparent molecular mass of approximately 130 kDa), not observed following lipopolysaccharide, tumor necrosis factor, or XNA stimulation. This lectin-induced tyrosine phosphorylation was not affected by cytochalasin D (inhibitor of actin filament polymerization), by genistein (inhibitor of tyrosine kinases), or by staurosporine (inhibitor of tyrosine phosphorylation and protein kinase C). In addition, incubation of PAEC with BS-I and monoclonal anti-Gal IgM induced p42/44 map kinase and activated the transcription factor NF-kappaB. CONCLUSIONS: Agonist binding of alpha-gal can evoke endothelial cell activation independently of complement activation. These observations have implications for the survival of xenografts.

Actins↗
PubMed1998

Isolation from human fetal liver of cells co-expressing CD34 haematopoietic stem cell and CAM 5.2 pancytokeratin markers.

BACKGROUND/AIMS: Ductal plate and bile duct cells in developing human liver express haematopoietic stem cell markers, such as c-kit and CD34, in association with cytokeratin markers CAM 5.2 and CK 18. The identification of such ductal plate cells as likely progenitors for both bile duct epithelial cells and hepatocytes and their possible reappearance as oval cells in the regenerating liver have generated much interest in their pluripotential capacities. This study aimed to isolate cells from human fetal liver that co-express haematopoietic stem cell and epithelial cell markers. METHODS: Human fetal liver was harvested following legal termination of pregnancy at week 14-22. CD34+ mononuclear cells were isolated from liver cell suspensions with immunomagnetic beads. Immunofluorescent staining, using anticytokeratin CAM 5.2 against CK 8 and 18, was performed on permeabilised CD34+ cells for flow cytometry and fluorescent microscopy. CD34+ cells were also stained for other stem cell markers (HLA-DR, c-kit) and committed haematopoietic cell markers (CD33, CD38). RESULTS: Approximately 0.9% (range 0.07-4.0%) of the mononuclear cells isolated were CD34+ cells. The number of mononuclear cells isolated correlated with fetal liver weight (r=0.508). About 3-8% of these CD34+ cells stained positively for CAM 5.2. In addition, CD34+ cells were positive for HLA-DR, but only a small percentage was positive for c-kit. Staining for the committed haematological markers, CD33 and CD38, was consistently negative. CONCLUSIONS: This study describes an immunoaffinity method for the enrichment from human fetal liver of cells that co-express haematopoietic stem cell and epithelial cell markers. Such cellular subsets may correspond to pluripotential ductal plate and bile duct cells.

Antigens, CD34↗
PubMed1998

A novel mammalian wnt gene, WNT8B, shows brain-restricted expression in early development, with sharply delimited expression boundaries in the developing forebrain.

Our current knowledge of mammalian forebrain development is meagre. The comparatively few relevant anatomical landmarks are, however, being supplemented by gene expression studies which are able to identify subsets of anatomical structures. We previously described cloning, subchromosomal localization and preliminary structural characterization of the human WNT8B gene, the first mammalian Wnt8b gene to be reported. Wnt genes encode intercellular signalling molecules which play a variety of critical roles in early development, including, in several cases, a presumed role in brain development. In the current report we present the full-length cDNA sequence and genomic organization of the human Wnt8b gene and report studies of expression of the Wnt8b gene in human and mouse embryos. The human and mouse expression patterns appeared identical and were restricted to the developing brain, with the great majority of expression being found in the developing forebrain. In the latter case expression was confined to the germinative neuroepithelium of three sharply delimited regions: the dorsomedial wall of the telencephalic ventricles (which includes the developing hippocampus), a discrete region of the dorsal thalamus and the mammillary and retromammillary regions of the posterior hypothalamus. Expression in the developing hippocampus may suggest a role for WNT8B in patterning of this region and subchromosomal localization of the human gene to 10q24 may suggest it as a candidate gene for partial epilepsy in families where the disease has been linked to markers in this region.

Amino Acid Sequence↗
PubMed1998

Bilateral renal agenesis, cardiac hypertrophy and pancytopenia, a new syndrome?

We report a fetus with an unusual combination of features including bilateral renal agenesis, hydrops, cardiac hypertrophy and pancytopenia. There was haematological evidence of apoptosis with nuclear degeneration of megakaryocytes. The combination of structural anomalies associated with pancytopenia has many features reminiscent of Fanconi's anaemia although Mitomycin C studies were normal. There was no evidence of infection as a cause for the pancytopenia. We suggest that fetal blood sampling should be considered in similar cases with renal agenesis. Some may have a milder form of pancytopenia and may not present with hydrops thereby not prompting further haematological investigation.

Abnormalities, Multiple↗
PubMed1998

Tyrosine phosphorylation following lectin mediated endothelial cell stimulation.

Terminal alpha (1,3) galactosyl galactoside epitopes (alpha-gal) on membrane glycoproteins expressed by vascular endothelial cells represent the major xenoreactive antigens in pig to primate xenotransplantation. In other discordant xenotransplantation combinations, such as from guinea pig to rat, carbohydrate epitopes other than alpha-gal may be targeted by xenoreactive antibodies (XNA). We have shown that agonist binding to alpha-gal epitopes induces proinflammatory activation of porcine aortic endothelial cells (PAEC). Binding of alpha-gal epitopes by Bandeiraea simplicifolia isolectin B4 results in both type I and type II PAEC activation. This includes the phosphorylation of tyrosine residue(s) of a protein with an apparent molecular weight of 130 kDa (p130). In order to investigate whether binding of other carbohydrate epitopes could induce a similar phosphorylation event, several lectins with different carbohydrate specificities were used to stimulate PAEC and human umbilical endothelial cells (HUVEC). In addition to BS-IB4 binding to alpha-gal, lectins binding to sialic acid isolated from Sambucus nigra (SNA), Maackia amurensis (MAA), Wheat germ agglutinin (WGA), and lectin from jack bean (Concanavalin A, ConA), that binds to mannose residues within the core structure of N-glycosylated proteins all induced the phosphorylation of the p130 protein(s). Lectins with affinity to alpha bound N-acetylgalactosamine, Dolichos biflorus (DOB), and Sophora japonoca (SOJ) did not induce this phosphorylation event. A similar negative result was obtained with Ulex europaeus lectin I, which binds to fucose residues. Conclusively, endothelial cell activation can be observed upon binding of various lectins to the glycosylated moiety of surface glycoproteins. These carbohydrate epitopes against which XNA may exist in certain models might represent minor xenoantigens from porcine to primates or may comprise the major xenoepitopes in other discordant xenograft models. Binding of XNA and subsequently the elicited xenoreactive antibodies to carbohydrate epitopes may therefore contribute to xenograft rejection even in the absence of complement inactivation.

Animals↗
PubMed1998

Regulation of monocyte tissue factor activity by allogeneic and xenogeneic endothelial cells.

The regulation of tissue factor (TF) activity by the cell associated tissue factor pathway inhibitor (TFPI) during monocyte (Mo) and endothelial cell (EC) interactions is not fully understood. This report describes co-ordinate induction of TF antigen (TF-Ag) and membrane-associated TFPI-Ag on human Mo following coculture with human aortic (HAEC) or porcine aortic EC (PAEC) or after stimulation with TNFalpha. We show that both allo- and xenogeneic EC induce human Mo-TF antigen in short-term culture. However, the TF activity of TNFalpha-primed Mo is suppressed when these cells are cocultured with HAEC [by 40.3 +/- 6.3% (p<0.02)] or PAEC [by 50.5 +/- 10.6% (p<0.001)]. Antibody (Ab) blocking studies confirm that TFPI is the principal anticoagulant associated with this suppression of TF-activity. Our data indicate that anti-TF activity originates, at least in part, from the activated human Mo in the coculture; additionally, specific generation of TFPI by Mo is observed under the xenogeneic culture conditions. As Mo associated TF, induced by allo- or xenogeneic EC interactions, is regulated by cell-associated TFPI, we propose that infiltrating Mo may modulate the thrombotic process at sites of vascular injury in association with both allo- and xenograft rejection.

Animals↗