PubMed Health⌕ Search

Biomedical subjects

L E Diamond

Publications and source records attributed to L E Diamond.

17 recordsLinked to original sources
PubMed1999

High level transcription of the complement regulatory protein CD59 requires an enhancer located in intron 1.

CD59 is a complement regulatory protein and may also act as a signal-transducing molecule. CD59 transgenic mice have been generated using a CD59 minigene (CD59 minigene-1). Although this minigene contained a 4.6-kilobase pair 5'-flanking region from the human CD59 gene as a promoter, the expression levels of the CD59 mRNA were substantially lower than those observed in humans, suggesting that CD59 gene expression might also require other transcriptional regulatory elements such as an enhancer. To investigate the transcriptional regulation of the CD59 gene, we used three cell lines that express CD59 at different levels. We have identified DNase I-hypersensitive sites in intron 1 in HeLa cells, which express CD59 at high levels, but not in Jurkat (intermediate level) or Raji cells (low level). Furthermore, cell line-specific enhancer activity was detected in a fragment containing these DNase I-hypersensitive sites. The CD59 enhancer was mapped to between -1155 and -888 upstream of the 5'-end of exon 2. To investigate the enhancer activity in vivo, a new CD59 minigene was constructed by the addition of the enhancer fragment into CD59 minigene-1. High expressor CD59 transgenic mice were generated using the new minigene.

Animals↗
PubMed1998

Role of CD46 in measles virus infection in CD46 transgenic mice.

The susceptibility of CD46 (human membrane cofactor protein) transgenic mice to measles virus (MV) infection was investigated. Cell cultures (lung and kidney) established from transgenic and control mice showed that although both could be infected only those from the CD46+ mice gave fusion. A complete round of replication with the release of infectious virus was detected exclusively in the transgenic cell cultures whose permissiveness to MV was markedly less than that of Vero cells. The ability of MV to replicate in vivo in mice was studied using both vaccine and laboratory-adapted wild-type strains of virus. After intraperitoneal and intranasal inoculations of transgenic mice, virus replication could not be detected. In contrast intracerebral inoculation induced infection in both transgenic and nontransgenic mice. Our results from in vitro infection studies support the hypothesis that CD46 is a major host cell factor involved in the MV-induced fusion process and MV entry. The studies further indicate that MV tropism is not governed solely by the expression of the CD46 gene and that the high efficiency of the replicative cycles characteristic of fully permissive host cells requires additional factors, which are lacking in both transgenic and nontransgenic mice.

Administration, Intranasal↗
PubMed1998

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Acute Disease↗
PubMed1997

Humoral responses to pig-to-baboon cardiac transplantation: implications for the pathogenesis and treatment of acute vascular rejection and for accommodation.

Organs transplanted between phylogenetically-disparate species, such as from the pig into the primate, are subject to hyperacute and acute vascular rejection. Hyperacute rejection of a porcine organ by a primate is thought to be initiated by the binding of xenoreactive natural antibodies to Galalpha1-3Gal expressed on the endothelial lining of blood vessels in the xenograft. The factor(s) which initiates acute vascular rejection is uncertain; however, there is some evidence implicating xenoreactive antibodies. The nature of the humoral response which might contribute to acute vascular rejection of a porcine organ was investigated in baboons which received a porcine cardiac xenograft plus immunosuppression with methylprednisolone, azathioprine, and cyclosporine. Following rejection and surgical removal of the xenografts, the serum concentration of xenoreactive antibodies increased in untreated animals but in immunosuppressed animals was similar to the concentration in preimmune serum. The antibodies in the sensitized recipients were specific for Galalpha1-3Gal (70-95%) and other determinants (5-30%). However, cross-blocking studies showed that, following xenotransplantation, the immunosuppressed baboons had no detectable IgM or IgG directed against "new" endothelial antigens. These results indicate that antibodies made by immunosuppressed individuals in response to xenotransplantation are much like xenoreactive natural antibodies and suggest that acute vascular rejection might in some cases be addressed by therapeutic strategies aimed at those antibodies.

Animals↗
PubMed1997

The role of natural anti-Gal alpha 1-3Gal antibodies in hyperacute rejection of pig-to-baboon cardiac xenotransplants.

Xenoreactive natural antibodies in humans and higher primates are directed predominantly at Gal alpha 1-3Gal. These antibodies are thought to initiate hyperacute rejection of porcine organ xenografts. The contribution of anti-Gal alpha 1-3Gal antibodies to the xenoractive natural antibody repertoire and to the initiation of hyperacute rejection was tested in a pig-to-baboon cardiac xenograft model. Anti-Gal alpha 1-3Gal antibodies were depleted from baboons by extracorporeal absorption of anti-Gal alpha 1-3Gal antibodies from plasma using columns with a matrix bearing Gal alpha 1-3Galb1-4GlcNAc. Specific removal of anti-Gal alpha 1-3Gal antibodies was achieved prior to transplantation as demonstrated by immunoassay. Porcine hearts were then transplanted into these baboons and the outcome of the transplants was analysed. Immunofluorescence revealed little deposition of baboon antibodies in the grafts. The porcine hearts did not undergo hyperacute rejection even though complement activity was approximately 90% of baseline at the time of transplantation. These findings demonstrate that anti-Gal alpha 1-3Gal antibodies constitute a major fraction of xenoreactive natural antibodies in primate blood and that these antibodies contribute significantly to the pathogenesis of hyperacute xenograft rejection.

Acute Disease↗
PubMed1996

Characterization of transgenic pigs expressing functionally active human CD59 on cardiac endothelium.

The critical shortage of human donor organs has generated interest in the potential for porcine to human xenotransplantation. The initial immunological barrier to xenotransplantation is hyperacute rejection, which is mediated by xenoreactive antibodies and complement, and results in rapid and irreversible tissue destruction. While endogenous complement regulatory proteins (CRPs) protect cells from injury caused by autologous complement, they are relatively species specific and most likely ineffectual in this setting. This has led to the hypothesis that expression of human CRPs in transgenic pigs may affect susceptibility to complement-mediated tissue injury in a porcine-to-human xenograft. Using specific lines of transgenic pigs that express low levels of human CD59, a CRP that acts at the terminal stage of the complement cascade, we present evidence that shows that the human CD59 protein inhibits membrane attack complex assembly and reduces tissue damage when the heart is transplanted to a baboon. Examination by immunohistochemistry of transgenic porcine hearts after transplantation revealed markedly reduced deposition of C5b and MAC, but a similar level of C3 deposition as compared with transplanted control hearts. This finding supports the concept that the species specific function of CRPs contributes to the humoral barrier to xenotransplantation and, given the low level of human CD59 protein expression in the porcine heart, argues that the human protein contributes a unique rather than an additive function in regulation of complement in a xenogeneic setting.

Animals↗
PubMed1995

Transgenic expression of human complement regulatory proteins in mice results in diminished complement deposition during organ xenoperfusion.

Complement activation is an essential step in the hyperacute rejection of a vascularized xenograft. Endothelial cell-associated complement regulatory proteins limit complement activation in most settings, but are not able to limit the extensive complement activation that occurs in xenografts, at least in part due to their species specificity. To overcome this problem we and others have sought to express human complement regulatory proteins in the organs of potential donor animals. As an initial step toward evaluating this concept we tested organs from transgenic mice expressing human CD59 and/or decay-accelerating factor (DAF) in two in vitro perfusion systems for the ability to control activation of heterologous complement. In the first system, mouse hearts were perfused on a Langendorff circuit with 50% human plasma. Immunopathologic analysis of heart biopsies revealed deposition of human IgG, IgM, and C4 in both control and transgenic organs. The hearts from mice transgenic for human CD59 had substantially less and in some cases no membrane attack complex (MAC) and hearts from CD59/DAF transgenic mice had substantially less or no C5b and MAC. In the second system, mouse hearts were perfused with baboon blood through arterial lines inserted into baboons. Immunopathologic analysis of serial biopsies revealed the deposition of IgG, IgM, and C4 in control and transgenic hearts. Compared with controls, less MAC was deposited in many CD59-expressing hearts and less C5b and MAC in DAF-expressing hearts. These results demonstrate that human complement regulatory proteins expressed in a xenogeneic organ are able to contribute to the control of complement activation in that organ and support the concept that expression of these human molecules would help protect a xenogeneic organ transplanted into a human.

Animals↗
PubMed1995

Human CD59 expressed in transgenic mouse hearts inhibits the activation of complement.

Porcine-to-human xenotransplantation offers a potential solution to the critical shortage of human organs. The major immunological barrier to xenotransplantation between these species is a rapid rejection process mediated by preformed natural antibodies and complement. Xenogeneic organ grafts are especially susceptible to complement mediated injury because complement regulatory proteins, which ordinarily protect cells from inadvertent injury during the activation of complement, function poorly in regulating activation of heterologous complement. Removal of xenoreactive antibodies or systemic inhibition of complement activity has been shown to prolong graft survival. As an alternative to the systemic inhibition of complement activity, we have established a model system using transgenic animals to test whether the expression of human membrane bound complement regulatory proteins on mouse endothelial cells can inhibit the activation of human complement. CD59, which acts at the terminal stage of complement activation by inhibiting the formation of the membrane attack complex, was used as a paradigm for this model. A CD59 construct containing the putative CD59 gene promoter linked to the CD59 coding region was used to demonstrate expression of the human CD59 protein in various tissues of transgenic mice, including endothelial cells in the heart. In addition, we show that the transgenic CD59 protein is biologically active as determined by the ability to inhibit the formation of membrane attack complex in transgenic mouse hearts perfused ex vivo with human plasma. These results demonstrate that expression of membrane bound complement regulatory proteins can achieve complement inhibition in a xenogeneic organ and suggest that this approach may be useful for successful xenotransplantation between discordant species.

Animals↗
PubMed1990

The c-myc-regulated gene mrl encodes plasminogen activator inhibitor 1.

The DNA sequence of the c-myc-regulated gene mrl (G. C. Prendergast and M. D. Cole, Mol. Cell. Biol. 9:124-134, 1989) reveals that it encodes plasminogen activator inhibitor 1 (PAI-1), a regulator of extracellular proteolysis. Comparison of the human and mouse PAI-1 promoters and cDNA 3' noncoding regions revealed several highly conserved sequence domains, potential targets for c-myc and other factors influencing PAI-1 expression. We discuss possible roles for PAI-1 in normal and neoplastic cell growth control.

Animals↗
PubMed1989

Radiation and chemical activation of ras oncogenes in different mouse strains.

A survey of a large series of radiation- or chemically induced thymic lymphomas in (AKR X RF)F1, RF/J, 129/J, and C57BL/6J mouse strains for activated ras oncogenes showed that of the tumors containing transforming activity, in more than 75% of the cases this activity segregated with either K-ras or the N-ras gene. H-ras activity was never detected. The genetic background of the host influenced susceptibility to tumor induction and oncogene activation. The K-ras gene was preferentially activated over the N-ras gene (approximately 2:1) whether the inducing agent was radiation or the chemical N-nitrosomethylurea. The activating mutation for the K-ras gene was consistently identified as a GGT to GAT transition in codon 12. In contrast, several different mutations of the N-ras gene were identified and localized to codons 12, 13, or 61. Assessment of the allelic composition of the ras locus shows that some proportion of the tumors lost the normal ras allele.

Animals↗
PubMed1988

T-cell receptor gene rearrangement in primary tumors: effect of genetic background and inducing agent.

The status of T-cell receptor beta and gamma genes has been assessed in a series of primary tumors induced by a chemical carcinogen or by gamma-irradiation using two inbred strains of mice. It appears that these well-characterized regimens of carcinogenesis yield T-cell tumors showing gene rearrangements consistent with a clonal origin of the tumors. Individual rearranged bands seem to represent orthodox, intralocus recombination events. A variety of rearrangement phenotypes are observed, most strikingly for the gamma genes, and differences in the degree of T-cell receptor gene rearrangements observed can be categorized according to the inducing agent and to the genetic background of the mice, with the implication that premalignant thymocytes have been captured in different stages of T-cell development. Additionally, primary tumors were shown to express significant levels of mature beta gene mRNA.

Animals↗
PubMed1988

Concomitant K- and N-ras gene point mutations in clonal murine lymphoma.

We have surveyed a panel of induced murine lymphomas for c-ras gene mutations. The K-ras gene seems to be preferentially activated in our system, and there are at least two examples of concomitant K- and N-ras gene mutations in the same tumor. This indicates that in some cases additional ras mutations may contribute to tumorigenesis and is evidence for a role of ras activation in tumor progression.

Alleles↗
PubMed1987

Differential expression of surface markers on thymic lymphomas induced by two carcinogenic agents in different mouse strains.

Expression of several thymocyte surface antigens was monitored in a murine model system of thymic lymphoma induction in two different strains of mice. RF/J mice are sensitive to tumor induction by N-nitrosomethylurea (NMU) and by gamma-irradiation, while 129/J mice form tumors only upon NMU treatment. Latency periods for tumor formation were characteristically different depending upon the inducing agent and the mouse strain. We observed differences in thymic leukemia antigen and H-2K expression according to the mode of tumor induction and in relation to the mouse strain, implying multiple factors involved in target cell selection and tumor progression.

Animals↗