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Biomedical subjects

M J Pearse

Publications and source records attributed to M J Pearse.

At least 37 records · Page 2Linked to original sources
PubMed1996

Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhesion molecule 2 promoter.

Genetic engineering of donor animals in xenotransplantation research has been directed largely toward obtaining expression of various immunoregulatory molecules on vascular endothelium, the initial target of recipient antibody and complement. However, specific high-level expression of transgenes throughout the vascular tree in adult animals has proved difficult to achieve, perhaps because of the inherent heterogeneity of endothelium. Using the promoter of the gene for intercellular adhesion molecule 2 (ICAM-2), which is constitutively expressed on all vascular endothelium, we have developed a system for endothelial cell gene targeting in vivo. A 334-basepair fragment from the 5' flanking region of the human ICAM-2 gene was used to drive the expression of human CD59 in transgenic mice. Strong and uniform expression of CD59 was observed on the endothelial cells of all blood vessels in the heart, kidney, lung, liver, and pancreas in the three lines of mice examined. Little or no expression was seen in other cell types, with the exception of neutrophils and monocytes. These results suggest that this small promoter region contains most of the signals necessary to endow it with endothelial cell specificity, making it a potentially valuable tool in areas ranging from xenotransplantation to gene therapy.

Animals↗
PubMed1996

Expression of functional decay-accelerating factor (CD55) in transgenic mice protects against human complement-mediated attack.

Transgenic mice expressing human CD55 were generated by microinjection of a CD55-minigene under the control of the mouse H2K(b) (MHC class I) promoter. Offspring were tested for transgene integration by PCR analysis, and for CD55 expression on peripheral blood leukocytes (PBLs) by flow cytometry. Expression levels of 15 founders ranged from 30 to 80% of that on human neutrophils. Immunohistochemical analysis of kidney, heart, liver, and lung tissue demonstrated staining for CD55 on endothelial surfaces as well as general diffuse staining throughout the tissues. The capacity of the transgenically expressed CD55 to prevent human C3 deposition on the surface of mouse splenocytes was assessed by flow cytometry. Cells from hemizygous mice incubated with 10% fresh human serum as a source of natural antibody and complement bound approximately 65% less C3 than control littermates. No further protection was seen using cells from homozygous littermates, and the protective effect was abrogated by prior incubation with an OFFi-CD55 monoclonal antibody. Similarly, transgenic mice were afforded significant protection from human serum-mediated lysis, determined using an LDH release assay. Hearts perfused with human plasma showed no increase in survival time in a modified Langendorff perfusion system, however deposition of human C3c was greatly reduced in transgenic hearts.

Animals↗
PubMed1996

The alpha-1,3-galactosyltransferase knockout mouse. Implications for xenotransplantation.

Organ xenografts in discordant combinations such as pig-to-man undergo hyperacute rejection due to the presence of naturally occurring human anti-pig xenoantibodies. The galactose alpha(1,3)-galactose epitope on glycolipids and glycoproteins is the major porcine xenoantigen recognized by these xenoantibodies. This epitope is formed by alpha(1,3)-galactosyltransferase, which is present in all mammals except man, apes, and Old World monkeys. We have generated mice lacking this major xenoantigen by inactivating the alpha(1,3)-galactosyltransferase gene. These mice are viable and have normal organs but develop cataracts. Substantially less xenoantibody from human serum binds to cells and tissues of these mice compared with normal mice. Similarly, there is less activation of human complement on cells from mice lacking the galactose alpha(1,3)-galactose epitope. These mice confirm the importance of the galactose alpha(1,3)-galactose epitope in human xenoreactivity and the logic of continuing efforts to generate pigs that lack this epitope as a source of donor organs.

Animals↗
PubMed1996

Inhibition of NF-kappaB activation by a dominant-negative mutant of IkappaBalpha.

The activity of the transcription factor NF-kappaB is tightly regulated by the inhibitory molecule IkappaBalpha. Upon stimulation, IkappaBalpha is rapidly degraded and NF-kappaB translocates to the nucleus to induce gene expression. The IkappaBalpha degradation is preceded by phosphorylation, suggesting that this event plays a role in the activation of NF-kappaB. In this study, we have mutated three potential phosphorylation sites in porcine IkappaBalpha and found that expression of the Ser32 mutant of IkappaBalpha (IS32A), but not Tyr42 or Ser262 mutants or wild-type IkappaBalpha, blocked the activation of NF-kappaB by TNF-alpha. These results suggest that the Ser32 residue, a potential casein kinase II phosphorylation site, is critical for NF-kappaB activation.

Animals↗
PubMed1994

The diagnostic use of the polymerase chain reaction for the detection of Mycobacterium tuberculosis.

Detection of Mycobacterium tuberculosis by microscopy is difficult in specimens containing fewer than 10(4) bacteria/mL and growth in culture can take up to 6 wks. In this study the Polymerase Chain Reaction (PCR) was investigated as a rapid diagnostic technique for the detection of M. tuberculosis. The presence of DNA polymerase inhibitors in sputum specimens poses a potentially serious problem as false negative results can occur. In this study polymerase inhibitors were detected by inclusion of an internal plasmid control in each test. DNA from specimens in which the internal control failed to amplify was purified with a DNA binding matrix before retesting by PCR. A total of 169 sputum specimens were examined and 4 were found to have inhibitors. The correlation between detection of M. tuberculosis by PCR with a combination of culture, Ziehl-Neelsen (ZN) staining and patient history was 97.6%. This study confirms that PCR offers a more sensitive and rapid alternative for the detection of M. tuberculosis to ZN staining and culture, with results being available within 24 hrs of a specimen being received in the laboratory.

Base Sequence↗
PubMed1993

Clusterin levels increase during neuronal development.

The expression of clusterin has been shown to be elevated in several models of experimentally induced programmed cell death and in association with a number of neurodegenerative conditions. In order to test whether this protein is expressed in neurons during development, the expression of clusterin was examined in the developing nervous system, using immunohistochemistry and mRNA analysis. Clusterin expression was observed in the earliest neurons of the cortical plate on embryonic day (E) 12. Thereafter, the intensity of clusterin staining continued to increase in an age-dependent manner, with the greatest intensity of staining being found in the postnatal mature brain. Virtually all neurons were clusterin-positive and there was no evidence for the appearance of clusterin-positive cells specifically during epochs of programmed neuronal death in the embryo. This study suggests that clusterin has a role in neuronal maturation and it is unlikely to be associated exclusively with neuronal cell death.

Animals↗
PubMed1993

Essential fatty acid deficiency increases interstitial IA-positive cells in rat kidney and accelerates renal allograft rejection.

Essential fatty acid deficiency has been reported to result in depletion of interstitial macrophages from rat kidneys and to permit transplantation of these kidneys across a fully allogeneic barrier without need for immunosuppression. In view of the potential for application of this phenomenon to xenografts, this study attempted to confirm the observation. Kidneys from rats fed normal or essential fatty-acid-deficient diets were transplanted to DA recipients. The donors' livers and contralateral kidneys were analyzed for their fatty acid profile in liver phospholipids, and the kidneys were examined by immunohistology for interstitial Ia(+) cells. EFAD resulted in an increase in renal interstitial Ia(+) cells detected by MRC-OX6 (anti-RT1Bpublic) from 13.5 +/- 2.9 (control diet fed rats) to 22.8 +/- 3.6 in rats on a stringent EFAD diet. Graft survival of kidneys from these EFAD rats was significantly shorter than that of kidneys from control diet fed rats. In direct contrast to the original report, this study found that EFAD caused a marked increase in renal interstitial Ia(+) cells and a reduction in allograft survival of EFAD donor kidneys.

Animals↗
PubMed1992

Differential expression of clusterin in inducible models of apoptosis.

Apoptosis (programmed cell death) and necrosis can be readily distinguished morphologically and biochemically. The most striking biochemical change observed in apoptotic cells is the cleavage of the genomic DNA into discrete nucleosome sized fragments, producing a laddering pattern when the DNA is examined electrophoretically. It has recently been shown that RNA and protein products of the testosterone-repressed prostate message-2 gene are induced, coordinate with the onset of cell death. This gene has been isolated from a variety of species and tissues, it is highly conserved, and collectively referred to as clusterin. We have examined a number of inducible leucocyte models of apoptosis, including glucocorticoid and calcium ionophore induced thymocyte death, 'aged' neutrophils and cytotoxic T cells, and found that in these situations that cell death is not associated with up-regulation of clusterin gene expression. The finding that clusterin is not expressed in all cells undergoing apoptosis would suggest that this molecule is not critical to the mechanism of cell death. It does, however, provide the first example of a readily detectable marker which is differentially expressed in cells undergoing apoptosis and adds further weight to the argument that apoptosis is not a uniform phenomena, but is dependent on the nature of the cells involved and the means of induction.

Animals↗
PubMed1992

Endoglin: a 180-kD endothelial cell and macrophage restricted differentiation molecule.

A MoAb RMAC8 was generated by immunizing Balb/c mice with cultured human umbilical vein endothelial cells (HUVE). The molecule recognized had a mol. wt of 180 kD non-reduced, 95 kD after reduction and 66 kD in its reduced and N-deglycosylated form. Sequential immunoprecipitation studies with the MoAb 44G4, which recognizes the O- and N-glycosylated homodimer endoglin, showed that both MoAbs recognize the same molecule on HUVE and phorbol myristate acetate (PMA)-stimulated U937 cells. The distribution of the RMAC8-recognized molecule was the same as that described for endoglin, i.e. arterial and venous endothelium, myelomonocytic and pre-B leukaemia cells and cell lines; however, unlike 44G4, RMAC8 also reacted weakly with monocytes and strongly with in vitro differentiated macrophages as well as peritoneal and alveolar macrophages. This distribution of endoglin was confirmed by Northern blot analysis using a full length endoglin cDNA probe. These studies suggest that endoglin is a differentiation marker on macrophages.

Antibodies, Monoclonal↗
PubMed1991

Basic fibroblast growth factor upregulates steady-state levels of laminin B1 and B2 chain mRNA in cultured neuroepithelial cells.

The growth of purified populations of murine neuroepithelial cells isolated from 10 day embryonic (E10) telencephalon and mesencephalon can be specifically enhanced by supplementing growth culture media with basic fibroblast growth factor (bFGF). One effect of bFGF on cultured neuroepithelial cells was to enhance the amount of laminin expressed at the protein level as detected by immunofluorescence. This was correlated with significant upregulation of steady-state levels of laminin B1 and B2 chain expression as analyzed at the mRNA level. When E12 neuroepithelial cells were split into precursor neuronal or glial subpopulations on the basis of differential expression of major histocompatibility class-1 antigens, only the glial progenitor fraction was found to be capable of detectable laminin synthesis. It is thus possible that a primary action of FGF is to increase the synthesis and release of extracellular matrix molecules from neural cells which act back in a paracrine manner to stimulate differentiation.

Animals↗
PubMed1989

CD45R as a primary signal transducer stimulating IL-2 and IL-2R mRNA synthesis by CD3-4-8- thymocytes.

CD45R is a high molecular weight (p205/220) form of a series of transmembrane glycoproteins, collectively known as CD45 and present in some form on all lymphoid cells. We have proposed that CD45R+ thymocytes, a minority (15 to 30%) of total thymocytes, represent the generative thymic lineage whereas CD45 p180+ thymocytes are destined for intrathymic death. To test this hypothesis, we prepared human thymus fractions enriched for the expression of CD45R by exhaustive depletion of CD45 p180+ cells, as well as progenitor CD3-4-8- "multinegative" thymocytes which are predominantly CD45R+. Northern analysis of RNA extracted from CD45 p180- and multinegative thymus fractions demonstrated that these populations are enriched for cells able to synthesize mRNA encoding IL-2 and IL-2R after mitogenic stimulation, as compared to unfractionated thymus, consistent with the properties expected for generative thymocytes. Postulating that the CD45R glycoprotein might represent an important signal delivery molecule, we analyzed the ability of mAb specific for CD45 epitopes to synergize with suboptimal amounts of PHA and PMA in the stimulation of IL-2 mRNA production by multinegative thymocytes. We found that CD45R-specific mAb synergizes strongly with PHA/PMA to stimulate IL-2 and IL-2R mRNA expression. In contrast, mAb to CD45 common determinants were unable to synergize. Multinegative thymocytes depleted of all CD45 p180+ cells were compared to total multinegative cells and found to synthesize fourfold greater levels of IL-2 mRNA after stimulation with anti-CD45R mAb. This CD45 p180- multinegative subset is enriched for cells expressing a high density of CD45R, and for CD45- thymus cells, suggesting a possible enrichment for nonlymphoid cells which may play a role in the stimulation process. Our results suggest that the extended amino acid insert of CD45R plays a fundamental role in transmembrane signalling, and that CD45R may be a primary signal transducer for developing thymic progenitor cells.

Adjuvants, Immunologic↗
PubMed1989

Efficient radioiodination and immunopurification of surface proteins from small numbers of cells.

This report describes a method for the cell surface radioiodination and immunoprecipitation of the T cell receptor from as few as 7.5 x 10(4) lymphoid cells. The membranes of cells were labelled using the lactoperoxidase/H2O2 method. In order to minimize the loss of labelled cells after iodination, T cell receptor-negative 'filler' cells were added to the washes and the washing volume was kept small. Before immunoprecipitation, the solid-phase Staphylococcus aureus was precoated with the specific monoclonal antibody to ensure optimal recovery of antibody-antigen complexes. These simple modifications greatly increased the efficiency of the iodination and immunoprecipitation procedures, thus enabling direct analysis of membrane proteins from small numbers of cells.

Antibodies, Monoclonal↗