PubMed Health⌕ Search

Biomedical subjects

E F Wagner

Publications and source records attributed to E F Wagner.

At least 109 records · Page 6Linked to original sources
PubMed1993

Human foamy virus proteins accumulate in neurons and induce multinucleated giant cells in the brain of transgenic mice.

Human foamy virus (HFV) is a retrovirus encoding structural genes and, like human immunodeficiency virus and human T cell leukemia virus I, several ancillary reading frames collectively termed the be1 genes. We have previously shown that HFV transgenic mice develop an encephalopathy with neuronal loss in hippocampus and cerebral cortex. We have now raised and characterized rabbit antisera to various recombinant portions of gag, pol, env, and bel-1, the viral trans-activator. Immunoreactivity for gag and bel-1 was observed in nuclei and processes of hippocampal and cortical neurons before the onset of morphological lesions and correlated with the appearance of HFV mRNA. Astrocyte-derived multinucleated giant cells containing HFV proteins were present in the brain of transgenic mice coexpressing full-length HFV genes but not in mice expressing truncated gag and env, suggesting that these genes contain a fusogenic domain. Expression of full-length structural genes decreased the life expectancy of transgenic mice, implying an adjuvant role for these proteins in HFV-induced brain damage.

Animals↗
PubMed1992

Development of a lethal mast cell disease in mice reconstituted with bone marrow cells expressing the v-erbB oncogene.

An animal model for malignant mastocytosis is described in mice reconstituted with bone marrow cells expressing the v-erbB oncogene. The lethal mast cell disease is characterized by massive infiltration of bone marrow, spleen, and several other visceral organs by connective tissue mast cells, which normally reside in the skin and the peritoneal cavity. As is frequently found in malignant mastocytosis, the v-erbB-induced mast cell disease was accompanied in some primary recipients by an acute myelogenous leukemia (AML) that killed all secondary recipients regardless of whether the AML was already evident in the primary host. The infiltrating mast cells stained strongly positive with berberine sulfate, suggesting that they were terminally differentiated and in vitro they showed only a weak proliferative capacity. The leukemias were clonal but apparently of different origin than the malignant mast cells, implying the transformation of two independent cell populations. Leukemic cells expressed various myeloid-specific markers as well as the B220 antigen, normally associated with the B-cell lineage. However, the Ig heavy chain genes were still in germ line configuration. In culture, these cells proliferated in the absence of exogenous growth factors and had the capacity to differentiate into mature myeloid cells. Preliminary experiments suggest that v-erbB may use parts of a signal transduction pathway normally coupled to the c-kit receptor. The v-erbB-induced malignant mast cell disease should provide a useful animal model for elucidating the cause for malignant mastocytosis in humans and to explore possible therapeutic strategies.

Alpharetrovirus↗
PubMed1992

Factor-dependent erythroid cell lines derived from mice transplanted with hematopoietic cells expressing the v-src oncogene.

Transplantation of spleen cells from primary reconstituted mice expressing the v-src oncogene to secondary and tertiary irradiated recipients resulted in the emergence of erythroid precursors with a transformed phenotype. When cultured in methyl cellulose, these precursors generated colonies of undifferentiated cells that could be expanded into continuously growing factor-dependent cell lines in liquid culture. All lines tested had a similar phenotype and expressed the v-src oncogene. In addition they responded to factors that regulate normal erythroid development, namely erythropoietin (Epo), interleukin-3 (IL-3), and mast cell growth factor (MGF), the ligand to the c-kit encoded receptor. When cells from one of the lines were maintained in the absence of factor, a "factor independent" subpopulation emerged that appeared to grow in an autocrine fashion. Conditioned medium from these cells stimulated their own growth as well as the growth of broad spectrum of normal precursors. Studies with neutralizing antibodies indicated that the predominant colony-stimulating factor produced by these cells is IL-3.

Animals↗
PubMed1992

Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants.

Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.

3T3 Cells↗
PubMed1992

Embryonic stem (ES) cells lacking functional c-jun: consequences for growth and differentiation, AP-1 activity and tumorigenicity.

The proto-oncogene c-jun encodes the major component of the transcription factor AP-1 and is thought to have important functions in cell proliferation and differentiation as well as in the cellular response to a variety of external stimuli. To investigate directly the role of c-jun in growth, differentiation and tumorigenicity we generated mouse embryonic stem (ES) cell lines in which both copies of the c-jun gene have been inactivated by homologous recombination. The disruption of both copies of the c-jun gene had no apparent effect on ES cell viability, growth rate and in vitro differentiation potential. Transcriptional activation of the c-jun, junB and c-fos genes following TPA/serum induction was unaffected and efficient transactivation of AP-1 reporter constructs was demonstrated in these cells. Remarkably, subcutaneous injection of ES cells lacking c-Jun into syngeneic mice led to a drastic reduction in the formation of teratocarcinomas. We propose that whereas most of the functions of c-Jun in ES cells appear to be complemented by other Jun proteins in vitro, functional c-Jun protein is essential for efficient tumor growth in vivo.

Alleles↗
PubMed1992

Expression of human foamy virus is differentially regulated during development in transgenic mice.

The human foamy virus (HFV) is a recently characterized member of the spumavirus family. Although no diseases have been unequivocally associated with HFV infection, expression of HFV regulatory genes in transgenic mice induces a characteristic acute neurodegenerative disease and a myopathy. To better characterize the sequence of events leading to disease, and to gain a better understanding of the underlying pathogenetic mechanisms, we have analyzed in detail the transgene expression pattern during development. Transcription of a construct containing all regulatory elements and ancillary genes of HFV was analyzed by in situ hybridization and was shown to occur in two distinct phases. At midgestation, low but widespread expression was first detected in cells of extraembryonic tissues. Later, various tissues originating from embryonic mesoderm, neuroectoderm, and neural crest transcribed the transgene at moderate levels. However, expression decreased dramatically during late gestation and was suppressed shortly after birth. After a latency period of up to 5 weeks, transcription of the transgene resumed in single cells distributed irregularly in the central nervous system and in the skeletal muscle. By the age of 8 weeks, an increasing number of cells displayed much higher expression levels than in embryonic life and eventually underwent severe degenerative changes. These findings demonstrate that HFV transgene expression is differentially regulated in development and that HFV cytotoxicity may be dose-dependent. Such biphasic pattern of expression differs from that of murine retroviruses and may be explained by the specificity of HFV regulatory elements in combination with cellular factors. Future studies of this model system should, therefore, provide novel insights in the mechanisms controlling retroviral latency.

Animals↗
PubMed1992

Human foamy virus: an underestimated neuropathogen?

Human foamy virus (HFV) is a recently characterized retrovirus which was originally isolated from patients with various neoplastic and degenerative diseases. However, until today it has not been possible to identify HFV as the causative agent of any disease and little is known about its prevalence in human populations. Like HTLV and HIV, HFV encodes the three structural retroviral genes, gag, pol and env, and an additional region containing three open reading frames, bel-1 to bel-3. Bel-1 activates transcription of the long terminal repeat of HFV and HIV. In order to study the consequences of expressing HFV regulatory genes and to investigate a possible pathogenic potential of HFV, we have introduced parts of the HFV genome into the germ line of mice. Our studies with transgenic mice demonstrate that HFV transgenes encompassing the bel region are transiently transcribed between midgestation and birth at moderate levels in various tissues. Expression is then suppressed, but resumes after a latency of several weeks in a restricted range of tissues and leads to extensive accumulation of HFV transcripts in single cells. This is associated with a progressive degenerative disease of the central nervous system and of striated muscle. These findings provide the first evidence of a disease induced by HFV and suggest that HFV might also act as a human pathogen in neurological diseases. Moreover, the transgenic mouse model will be useful for studying the molecular basis of HFV-induced neurotoxicity, the role of individual disease-associated HFV genes and the regulation of retroviral latency.

Animals↗
PubMed1991

Fractionation of mouse bone marrow by adherence separates primitive hematopoietic stem cells from in vitro colony-forming cells and spleen colony-forming cells.

Fractionation of mouse bone marrow by adherence to tissue culture plastic was used to characterize the adhesive properties of hematopoietic stem (HS) cells capable of long-term reconstitution. The adherent fraction that represents approximately 13% of the total marrow population was virtually devoid of in vitro colony-forming cells and spleen colony-forming cells but did contain approximately 30% of the total HS cells recovered from the procedure. These cells could be detected by both the competitive repopulation assay and by repopulation of W/Wv recipients. In approximately 60% of the recipients from the competitive repopulation experiments, the contribution of the adherent marrow cells was relatively low early (8 to 10 weeks) after transplantation. With time, however, the hematopoietic contribution from these cells increased, reaching a stable level 20 to 30 weeks posttransplantation. In the remaining recipients (40%), the contribution from adherent cells was already significant within 8 to 10 weeks of transplantation and did not change dramatically throughout the course of the experiment. Adherent bone marrow containing significant numbers of HS cells was unable to protect mice from radiation death, indicating that these early cells in the absence of later-stage progenitors are unable to provide this function.

Alleles↗
PubMed1991

Efficacy of interpersonal psychotherapy as a maintenance treatment of recurrent depression. Contributing factors.

In earlier reports, we demonstrated that in patients with recurrent unipolar depression, survival time without a new episode of major depression following discontinuation of medication was significantly and positively related to continued interpersonal psychotherapy (IPT). To determine whether the prophylactic benefit of monthly sessions of IPT was a function of specific features of the intervention, we examined the contribution of the quality of IPT sessions to the length of the well interval in this 3-year maintenance trial. Therapy sessions were rated on specificity and purity of interpersonal interventions. Analysis of these ratings indicated that psychotherapy that was more specifically interpersonal was associated with significantly increased survival time. Patients whose therapy sessions were rated above the median on specificity of IPT had a median survival time of almost 2 years, while those below the median had a median survival time of less than 5 months. We concluded that when patient and therapist are able to maintain a high level of interpersonal focus, monthly sessions of IPT have substantial prophylactic benefit.

Adult↗
PubMed1991

Expression of functional c-kit receptors rescues the genetic defect of W mutant mast cells.

Loss-of-function mutations in the gene for the c-kit tyrosine kinase receptor are strongly implicated in the developmental abnormalities of W mutant mice. To dissect further the relationship between kit and the W phenotype, retroviruses carrying the normal murine c-kit gene were constructed. In infected cells, the level of c-kit expression from these vectors varied markedly with different promoter elements, the 5' viral LTR proving to be the most effective. When introduced into cells which normally do not express c-kit, ectopic kit receptors transduced a ligand (Steel factor)-dependent proliferative signal in IL-3-dependent DA-1 myeloid cells and induced transformation in fibroblasts. Primary mutant mast cells were used to examine the effects of reconstituting functional kit expression in cells affected by W mutations. Exogenous c-kit expression rescued the defective proliferative response to Steel factor of cells from both W/Wv and W/W mutant mice. Moreover, functional kit expression also restored the capacity of W/Wv mast cells to survive and differentiate in vivo. These results imply that defective c-kit receptor function is sufficient to generate the W mutant phenotype.

3T3 Cells↗
PubMed1991

A novel target cell for c-fos-induced oncogenesis: development of chondrogenic tumours in embryonic stem cell chimeras.

Embryonic stem (ES) cells were used to investigate the target cell specificity and consequences of c-fos when expressed ectopically during embryonic development. Chimeric mice generated with different ES cell clones selected for high exogenous c-fos expression were not affected during embryonic development; however, a high frequency of cartilage tumours developed as early as 3-4 weeks of age apparently independent of the extent of chimerism. The tumours originated from cartilagenous tissues and contained many chondrocytes. Expression of exogenous c-fos RNA and Fos protein was observed during development but was highest in tumour tissues, predominantly in differentiating chondrocytes. A number of primary and clonal tumour-derived cell lines were established which expressed high levels of c-fos, c-jun as well as the cartilage-specific gene type II collagen and which gave rise to cartilage tumours in vivo, some of which also contained bone. Interestingly, the levels of c-Fos and c-Jun appeared to be coordinately regulated in the cell lines as well as in chimeric tissues. Thus, we demonstrate that chondrogenic cells and earlier progenitors are specially transformed by Fos/Jun and therefore represent a novel mesenchymal target cell for c-fos overexpression.

Animals↗
PubMed1991

Expression of v-src induces aberrant development and twinning in chimaeric mice.

The role of the proto-oncogene c-src in mouse development has been investigated by studying the consequences of expressing its viral homologue, v-src. Embryonic stem (ES) cell lines with differing levels of v-src tyrosine kinase activity have been used to generate chimaeric mice. Whereas a low level of v-src expression is compatible with embryogenesis, chimaeras derived from ES clones with high levels of v-src activity develop abnormally and die on the 8th-9th day of gestation. These abnormalities are characterized by the formation of twin or multiple embryos within the same Reichert's membrane, and by the arrest of embryonic development at the late egg cylinder stage, accompanied by relative expansion of the visceral yolk sac (VYS) and hyperplasia of the VYS endoderm. These results demonstrate for the first time that deregulated expression of the src protooncogene product can induce developmental abnormalities during early embryogenesis.

Animals↗
PubMed1991

Early fetal hematopoietic development from in vitro differentiated embryonic stem cells.

In this report we describe the efficient hematopoietic differentiation of embryonic stem (ES) cells in vitro. When cultured in semisolid medium two of five ES cell lines efficiently generated embryoid bodies (EBs) containing blood islands in which hematopoietic cells from all six myeloid lineages could be detected. Among a variety of growth factors tested, only erythropoietin significantly increased blood island formation. We directly demonstrate the presence of hematopoietic progenitors in the EBs by employing an in vitro precursor assay. Colony-forming cells (CFC) of all myeloid lineages as well as bi- and multipotent (CFC-MIX) progenitors were readily identified, and a detailed time-course analysis of their appearance was performed. Despite a high frequency of CFC-MIX in vitro, we did not observe any spleen colony-forming cells (CFU-S) in vivo. We conclude that hematopoietic differentiation of ES cells under these conditions reflects formation of the complete range of blood cells found in the yolk sac of the early fetus. Therefore this system provides a unique model in which to study the earliest events of hematopoietic development in vitro.

Animals↗
PubMed1991

Expression of human CSF-1 receptor induces CSF-1-dependent proliferation in murine myeloid but not in T-lymphoid cells.

The receptor for human macrophage colony stimulating factor (CSF-1R) was introduced into hematopoietic cell lines of myeloid and T-lymphoid origin, both of which normally do not express the CSF-1R. Infection of an interleukin-3 (IL-3)-dependent mouse myeloid cell line (FDC-P1) with a high titer retroviral vector expressing the human c-fms c-DNA, enabled CSF-1-dependent proliferation in short-term liquid culture assays as well as in clonal culture systems. CSF-1-dependent cell lines could be established after sorting for CSF-1R positive cells. In contrast to FDC-P1 cells, expression of the CSF-1R in CTLL cells, an IL-2-dependent mouse cytotoxic T-cell line, and in T-cell growth factor III/P40-dependent helper T-cells, ST2/K9.4a2, did not lead to CSF-1-dependent proliferation. These observations lead to the conclusion that ectopically expressed CSF-1R may function on certain myeloid cells where it is normally not expressed, suggesting the presence of signal transduction pathways which can be utilized by that foreign receptor. In contrast, it appears that T-lymphoid cells lack such a signalling mechanism, indicating that quite different modes of transducing mitogenic signals from the cell membrane to the nucleus must have developed during myeloid and T-lymphoid differentiation.

Animals↗
PubMed1991

Localization of regulatory elements controlling human MYCN expression.

Expression of the cellular oncogene MYCN is restricted to few cell lineages and is highest during development both in mouse and in humans. In pursuit of elucidating the mechanisms underlying MYCN regulation we introduced a human MYCN clone (pNb-9) with approximately 2.5 kbp 5'- and 6 kbp 3'-flanking genomic sequences into different murine and human cell lines as well as into mice. In all cases we found a correlation between the expression of the exogenous and the endogenous MYCN. Among cell lines, only those expressing the endogenous gene also expressed the transfected gene. In the transgenic mice transcripts of the transgene were present in proportion to the transcripts of the endogenous MYCN gene with the highest level in the brain. Therefore, the genetic information necessary for regulated expression of MYCN appears to be contained in pNb-9. To localize the DNA-regions responsible for regulated expression, we generated MYCN/CAT hybrid genes with different portions of the putative MYCN promoter region linked to the reporter gene. Transient transfections into various murine and human cell lines identified three DNA regions apparently involved in the regulation of expression. One region about 200 bp upstream of the transcriptional start site is responsible for a basal level of MYCN expression. A second region located about 800 bp further upstream appears to be involved in cell type-specific gene activation. The third regulatory element is located at the 3' end of the first exon and/or in the first intron and may mediate tissue-specific down regulation of gene expression.

Animals↗
PubMed1990

Increased proteolytic activity is responsible for the aberrant morphogenetic behavior of endothelial cells expressing the middle T oncogene.

Expression of the polyoma virus middle T (mT) oncogene in vivo is associated with a profound subversion of normal vascular development, which results in the formation of endothelial tumors (hemangiomas). In an attempt to understand the molecular mechanisms responsible for this phenomenon, we have investigated, in an in vitro system, the morphogenetic properties of endothelial cells expressing this oncogene. mT-expressing endothelioma (End) cells grown within fibrin gels formed large hemangioma-like cystic structures. All End cell lines examined expressed high levels of fibrinolytic activity resulting from increased production of urokinase-type plasminogen activator and decreased production of plasminogen activator inhibitors. Neutralization of excess proteolytic activity by exogenously added serine protease inhibitors corrected the aberrant in vitro behavior of End cells and allowed the formation of capillary-like tubules. These results suggest that tightly controlled proteolytic activity is essential for vascular morphogenesis and that physiological protease inhibitors play an important regulatory role in angiogenesis.

Animals↗