PubMed Health⌕ Search

Biomedical subjects

K G Xanthopoulos

Publications and source records attributed to K G Xanthopoulos.

At least 19 recordsLinked to original sources
PubMed2001

Induction of therapeutic antitumor antiangiogenesis by intratumoral injection of genetically engineered endostatin-producing Semliki Forest virus.

Antiangiogenic therapy using Semliki Forest virus (SFV) carrying Endostatin gene for malignant brain tumor was investigated to improve the therapeutic efficacy. The efficiency of SFV-mediated gene delivery was first evaluated for B 16 cells and compared with the efficiency in cells of endothelial origin (HMVECs). HMVECs are more susceptible to SFV infection than B 16 cells. For the in vivo treatment model, phosphate-buffered saline, SFV-LacZ, retrovirus vector GCsap-Endostatin, and SFV-Endostatin were injected to mice bearing B 16 brain tumors. A very significant inhibition of tumor growth was observed in the group that had been treated with SFV-Endostatin. A marked reduction of intratumoral vascularization was seen in the tumor sections from the SFV-Endostatin group compared with tumor sections from the SFV-LacZ or GCsap-Endostatin groups. Moreover, at day 7 after intravenous administration of SFV-Endostatin, the serum level of endostatin was augmented more than 3-fold compared to that after intravenous administration of GCsap-Endostatin. The results indicated that treatment with SFV-Endostatin inhibited the angiogenesis with established tumors. Gene therapy with Endostatin delivered via SFV may be a candidate for the development of new therapy for brain tumors.

Animals↗
PubMed2001

Enhancement of antitumor immune response in glioma models in mice by genetically modified dendritic cells pulsed with Semliki forest virus-mediated complementary DNA.

OBJECT: The aim of this study was to further investigate dendritic cell (DC)-based immunotherapy for malignant glioma to improve its therapeutic efficacy. METHODS: Dendritic cells were isolated from the bone marrow and pulsed with phosphate-buffered saline, tumor RNA, tumor lysate, Semliki Forest virus (SFV)-LacZ, SFV-mediated B16 complementary (c)DNA, or SFV-mediated 203 glioma cDNA, respectively, to treat mice bearing tumors of the 203 glioma cell line. The results indicated that pre-immunization with DCs pulsed with the same type of cDNA as in the tumor by a self-replicating RNA vector (that is, SFV) protected mice from tumor challenge, and that therapeutic immunization prolonged the survival of mice with established tumors. The SFV induced apoptosis in DCs and their death facilitated the uptake of apoptotic cells by other DCs, thus providing a potential mechanism for enhanced immunogenicity. CONCLUSIONS: Therapy with DCs that have been pulsed with SFV-mediated tumor cDNA may be an excellent procedure for the development of new cancer vaccines.

Animals↗
PubMed2000

Induction of a therapeutic antitumor immunological response by intratumoral injection of genetically engineered Semliki Forest virus to produce interleukin-12.

OBJECT: The authors investigated immunogene therapy for malignant glioma to determine whether its therapeutic efficacy could be improved. METHODS: Four groups of 203-glioma-bearing mice were treated with injections of phosphate-buffered saline, Semliki Forest virus (SFV)-LacZ, retrovirus vector DFG-interleukin (IL)-12, and SFV-IL12, respectively. The results indicated that therapeutic immunization with SFV-IL12 prolonged the survival of mice with established tumors. Semliki Forest virus induces apoptotic death to glioma cells, which facilitates the uptake of apoptotic cells by dendritic cells, providing a potential mechanism for enhanced immunogenicity. CONCLUSIONS: Immunogene therapy with IL-12 via SFV may be an excellent candidate for the development of new cancer vaccines.

Animals↗
PubMed1999

Synergistic activation of the human Btk promoter by transcription factors Sp1/3 and PU.1.

Analysis of the human Bruton's agammaglobulinemia tyrosine kinase (Btk) gene promoter revealed that 280 bp upstream of the transcriptional start site is sufficient for a cell restricted expression pattern. Here, the interplay of the transcription factors Sp1, Sp3, and PU.1 binding to this promoter area was analysed. All three proteins are able to independently activate the promoter in Drosophila Schneider (SL2) cells lacking endogenous Sp- or PU.1-like activities. Furthermore, PU.1 is able to act synergistically with Sp1 as well as Sp3 to transactivate the promoter. This transactivation is mediated through adjacent binding sites rather than through the more distant Sp binding site, suggesting a possible direct interaction between PU.1 and Sp1/3. Expression of Btk was found in ES cells and levels of expression were the same as in ES cells with a targeted deletion of the Sp1 gene, suggesting that Sp3 acts as a positive regulator of Btk in vivo, in the absence of Sp1.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1999

CCAAT/enhancer binding protein epsilon is critical for effective neutrophil-mediated response to inflammatory challenge.

Targeted mutation of CCAAT/enhancer binding protein (C/EBP) epsilon in mice results in early death, primarily due to spontaneous infection with Pseudomonas aeruginosa. Functional analysis of C/EBPepsilon-deficient neutrophils, in an in vivo model of peritoneal inflammation, shows multiple defects. Reduction of phagocytotic killing by C/EBPepsilon-deficient neutrophils is a result of decreased uptake of opsonized bacteria as well as little to no expression of secondary granule proteins. Abnormalities in neutrophil migration detected in a chemical peritonitis model are likely secondary to abnormal CD11b integrin and L-selectin expression on C/EBPepsilon-deficient neutrophils. Alterations in neutrophil cytokine expression in response to inflammation show decreased levels of interleukin-1 receptor antagonist (IL-1Ra) and increased levels of tumor necrosis factor-alpha (TNF-alpha) expression by C/EBPepsilon-deficient neutrophils. Additionally, TNF-alpha expression is increased in nonactivated, circulating C/EBPepsilon-deficient neutrophils. Overall, C/EBPepsilon-deficient neutrophils are severely functionally impaired, evoking an abnormal microenvironment, which may contribute to the loss of normal responses to inflammatory stimuli. Similarities between the C/EBPepsilon-deficient mouse model and the human disease, specific granule deficiency, will be discussed.

Animals↗
PubMed1999

Hunting with traps: genome-wide strategies for gene discovery and functional analysis.

With sequence analysis of the human genome well underway, there is an increasingly urgent challenge to understand the fundamental function and interplay of genes that build and maintain an organism. Several approaches will be critical for interpreting gene function, including random cDNA sequencing, expression profiling in different tissues, genetic analysis of human or model organism phenotypes, and creation of transgenic or "knockout" animals. Traditional gene-trapping approaches, in which genes are randomly disrupted with DNA elements inserted throughout the genome, have been used to generate large numbers of mutant organisms for genetic analysis. Recent modifications of gene-trapping methods and their increased use in mammalian systems are likely to result in a wealth of new information on gene function. Various trapping strategies allow genes to be segregated based on criteria like the specific subcellular location of an encoded protein, the tissue expression profile, or responsiveness to specific stimuli. Genome-wide gene-trapping strategies, which integrate gene discovery and expression profiling, can be applied in a massively parallel format to produce living assays for drug discovery.

Genes↗
PubMed1998

Biological role of the CCAAT/enhancer-binding protein family of transcription factors.

CCAAT/enhancer-binding proteins (C/EBPs) comprise a family of transcription factors that are critical for normal cellular differentiation and function in a variety of tissues. The prototypic C/EBP is a modular protein, consisting of an activation domain, a dimerization bZIP region, and a DNA-binding domain. All family members share the highly conserved dimerization domain, required for DNA binding, by which they form homo- and heterodimers with other family members. C/EBPs are least conserved in their activation domains and vary from strong activators to dominant negative repressors. The pleiotropic effects of C/EBPs are in part because of tissue- and stage-specific expression. Dimerization of different C/EBP proteins precisely modulates transcriptional activity of target genes. Recent work with mice deficient in specific C/EBPs underscores the effects of these factors in tissue development, function, and response to injury.

Animals↗
PubMed1998

Hepatocytes deficient in CCAAT/enhancer binding protein alpha (C/EBP alpha) exhibit both hepatocyte and biliary epithelial cell character.

To further elucidate the role of CCAAT/Enhancer Binding Protein alpha (C/EBP alpha) in hepatocyte differentiation, we investigated fetal and newborn C/EBP alpha-deficient (C/EBP alpha -/-) mice using confocal microscopy and markers specific for hepatocyte (AFP) and biliary epithelial cell (A6) differentiation. Histologically, in fetal liver of C/EBP alpha -/- mice, pseudoglandular structures appeared starting at 16.5 days of gestation. In newborn livers, the diameters of these structures greatly increased. They were randomly distributed between portal and central veins and interfered with the establishment of normal hepatic plates. However, the portal bile ducts developed normally. The pseudoglandular structures were lined with small hepatocytes with round nuclei and were positive for both AFP and A6 antigens. These data show that C/EBP alpha -/- hepatocytes exhibit biliary epithelial cell characters and suggest an involvement of C/EBP alpha in the control of the switch in the differentiation of bi-potential hepatoblasts along the hepatocyte lineage.

Animals↗
PubMed1998

Generation of a conditionally neo(r)-containing retroviral producer cell line: effects of neo(r) on retroviral titer and transgene expression.

We have developed a method for generating high-titer retroviral producer cell lines conditionally containing a neomycin resistance gene (neo(r)) based on the Cre/loxP system. For this, a bicistronic retroviral splicing vector carrying the green fluorescence protein (GFP) and a marker gene cassette consisting of internal ribosome entry site (IRES) and neo(r) flanked by loxP sites, was constructed and conveniently used to generate a G418 resistant vector producer cell line. Following titer determination and verification of the biological activity of the retroviral supernatants, the selectable expression cassette which was no longer required was excised from the provirus by transient Cre expression using an adenoviral vector. This strategy led to precise excision of neo(r) and generation of retroviral supernatants containing functional 'neo-less' retroviral particles without detrimental effects on the high vector titers found in the parental neo(r)-containing producer lines. GFP expression was significantly increased after the excision of neo(r), in both the producer lines and retrovirally transduced target cells. Reintroduction of neo(r) did not alter GFP expression, suggesting that the neo(r) gene and/or its gene product per se are not acting as a transcriptional silencer.

Adenoviridae↗
PubMed1998

CCAAT/enhancer binding proteins are critical components of the transcriptional regulation of hematopoiesis (Review).

The coordinated expression of four different CCAAT/enhancer binding proteins (C/EBPs), C/EBPalpha, C/EBPbeta, C/EBPdelta, and C/EBPepsilon constitutes a critical component of the myeloid differentiation program. C/EBPs are modular proteins, consisting of an activation domain, DNA binding domain and leucine zipper dimerization region. Recent studies including the analysis of mice deficient in several C/EBP proteins emphasize the effects of these molecules in hematopoiesis. C/EBPalpha is a master regulator of myeloid progenitors, C/EBPbeta plays an important role in macrophage and B-cell development, C/EBPgamma is involved in B-cell development, and C/EBPdelta is upregulated during myelopoiesis. Furthermore, C/EBPepsilon is a regulator of terminal differentiation of eosinophils and functional maturation of neutrophils. The formation of alternative combinations of tissue-specific and cell-stage specific C/EBP dimers may allow differential regulation of target genes in hematopoietic cells and commitment to distinctive hematopoietic lineages.

Animals↗
PubMed1997

Impaired granulopoiesis, myelodysplasia, and early lethality in CCAAT/enhancer binding protein epsilon-deficient mice.

Polymorphonuclear leukocytes are essential for host defense to infectious diseases. CCAAT/enhancer binding protein epsilon (C/EBP epsilon) is preferentially expressed in granulocytes and lymphoid cells. Mice with a null mutation in C/EBP epsilon develop normally and are fertile but fail to generate functional neutrophils and eosinophils. Opportunistic infections and tissue destruction lead to death by 3-5 months of age. Furthermore, end-stage mice develop myelodysplasia, characterized by proliferation of atypical granulocytes that efface the bone marrow and result in severe tissue destruction. Thus, C/EBP epsilon is essential for terminal differentiation and functional maturation of committed granulocyte progenitor cells.

Animals↗
PubMed1997

Semliki Forest virus-mediated production of retroviral vector RNA in retroviral packaging cells.

Retroviral vectors are efficient tools for gene transfer studies. Their major advantage is that they can permanently integrate the transgene into the target cell's genome. However, because of the compulsory nuclear expression phase of their life cycle, it can be difficult for retroviruses to carry complex expression cassettes. In a attempt to mimic the structural features of most eukaryotic genes and obtain a potentially self-amplifying system for retrovirus production, we tested the feasibility of Semliki Forest virus (SFV) expression to mediate cytoplasmic synthesis of retrovirus vector RNA. An equivalent of a retrovirus virion RNA (retrovirus vector cassette, RVC) was cloned under the SFV 26S promoter, and full-length chimeric SFV-RVC RNA was produced in vitro. This RNA was introduced into retrovirus packaging cells, either via electroporation or transduction in SFV virions, and supernatants were analyzed for the presence of biologically active retroviruses. We demonstrate that this strategy can be used for cytoplasmic retrovirus production. The resulting viral particles are fully functional; they can transduce target cells, undergo reverse transcription, and integrate into genomic DNA. We also demonstrate that the SFV virion-based RVC delivery into packaging cells can yield high transient titers, in this case more than 10(5) G418R cfu/ml. This study shows that a simple, one-plasmid, heterologous viral RNA production system can be used to create functional retroviral RNA outside the cell nucleus.

3T3 Cells↗
PubMed1997

Targeted expression of Cre recombinase to adipose tissue of transgenic mice directs adipose-specific excision of loxP-flanked gene segments.

Functional analysis of mammalian genes relies, in part, on targeted mutations generated by homologous recombination in mice. We have developed a strategy for adipose-specific inactivation of loxP-floxed gene segments. Transgenic mice have been established that express Cre recombinase under the control of the adipose-specific aP2 enhancer/promoter. Crossing of the aP2/ Cre mice with any loxP-floxed gene will facilitate its functional analysis in adipose tissue.

Adipose Tissue↗
PubMed1997

CCAAT/enhancer binding protein epsilon is preferentially up-regulated during granulocytic differentiation and its functional versatility is determined by alternative use of promoters and differential splicing.

CCAAT/enhancer binding protein (C/EBP) epsilon is a recently cloned member of the C/EBP family of transcription factors and is expressed exclusively in cells of hematopoietic origin. The human C/EBPepsilon gene is transcribed by two alternative promoters, Palpha and Pbeta. A combination of differential splicing and alternative use of promoters generates four mRNA isoforms, of 2.6 kb and 1.3-1.5 kb in size. These transcripts can encode three proteins of calculated molecular mass 32.2 kDa, 27.8 kDa, and 14.3 kDa. Accordingly, Western blots with antibodies specific for the DNA-binding domain, that is common to all forms, identify multiple proteins. C/EBPepsilon mRNA was greatly induced during in vitro granulocytic differentiation of human primary CD34(+) cells. Retinoic acid treatment of HL60 promyelocytic leukemia cells for 24 hr induced C/EBPepsilon mRNA levels by 4-fold, while prolonged treatment gradually reduced mRNA expression to pretreatment levels. Transient transfection experiments with expression vectors for two of the isoforms demonstrated that the 32.2-kDa protein is an activator of transcription of granulocyte colony-stimulating factor receptor promoter, while the 14.3-kDa protein is not. Thus, C/EBPepsilon is regulated in a complex fashion and may play a role in the regulation of genes involved in myeloid differentiation.

Alternative Splicing↗
PubMed1996

Cell specific expression of human Bruton's agammaglobulinemia tyrosine kinase gene (Btk) is regulated by Sp1- and Spi-1/PU.1-family members.

Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase involved in the human disease X-linked agammaglobulinemia (XLA). The gene is expressed in all hematopoietic cells with the exception of T-cells and plasma cells. For this expression pattern the first 280 bp upstream of the major transcriptional start site seems to be sufficient. In vitro footprinting analysis within this part of the promoter revealed two Sp1 binding sites as well as a PU-box. The transcription factor Spi-1/PU.1 as well as the closely related factor Spi-B bound to the PU-box in B-cells. In the erythroleukemia cell line K562, due to the absence of Spi-B, only PU.1 bound to the Btk promoter. Mutation of either site reduced the expression in transient transfection experiments. However, mutation of the PU box had no effect in the T-cell line Jurkat, where none of the Spi-1 family members is expressed. In addition Spi-B as well as PU.1 were able to transactivate Btk expression. In fetal liver of PU.1-/- mice, which lack lymphoid and myeloid cells, expression of Btk was reduced two- to threefold but not abolished. Collectively this study shows that expression of the Btk gene is regulated by the combined action of Sp1- and PU.1-family members.

Agammaglobulinaemia Tyrosine Kinase↗
PubMed1996

Increased hepatic cell proliferation and lung abnormalities in mice deficient in CCAAT/enhancer binding protein alpha.

CCAAT/enhancer binding protein alpha (C/EBPalpha) is a transcription factor that has been implicated in the regulation of cell-specific gene expression mainly in hepatocytes and adipocytes but also in several other terminally differentiated cells. It has been previously demonstrated that the C/EBPalpha protein is functionally indispensable, as inactivation of the C/EBPalpha gene by homologous recombination in mice results in the death of animals homozygous for the mutation shortly after birth (Wang, N., Finegold, M. J., Bradley, A., Ou, C. N., Abdelsayed, S. V., Wilde, M. D., Taylor, L. R., Wilson, D. R., and Darlington, G. J. (1995) Science 269, 1108-1112). Here we show that C/EBPalpha -1-mice have defects in the control of hepatic growth and lung development. The liver architecture is disturbed, with acinar formation, in a pattern suggestive of either regenerating liver or pseudoglandular hepatocellular carcinoma. Pulmonary histology shows hyperproliferation of type II pneumocytes and disturbed alveolar architecture. At the molecular level, accumulation of glycogen and lipids in the liver and adipose tissues is impaired, and the mutant animals are severely hypoglycemic. Levels of c-myc and c-jun RNA are specifically induced by several fold in the livers of the C/EBPalpha -/- animals, indicating an active proliferative stage. Furthermore, immunohistologic detection with an antibody to proliferating cell nuclear antigen/cyclin shows a 5-10 times higher frequency of positively stained hepatocytes in C/EBPalpha -/- liver. These results suggest a critical role for C/EBPalpha in vivo for the acquisition of terminally differentiated functions in liver including the maintenance of physiologic energy homeostasis.

3T3 Cells↗
PubMed1996

Suicide gene therapy for plasma cell tumors.

Suicide gene therapy for plasma cell tumors was attempted in severe combined immunodeficient (SCID) mice injected with human myeloma cell lines. Initially, a ganciclovir-induced bystander effect was observed in vitro using myeloma cells transduced with a herpes simplex thymidine kinase (HSVtk) gene. Transduced cells injected subcutaneously (SC) into SCID mice could be eradicated by the administration of ganciclovir (GCV). Furthermore, an in vivo bystander effect was noticed when mice received mixtures of HSVtk-positive and nontransduced cells. Unexpectedly, a "distant bystander" effect was observed as tumors in regions inoculated with only nontransduced cells were significantly smaller and had increased frequency of apoptotic figures and decreased mitotic frequency in GCV-treated mice transplanted with HSVtk-positive cells at a different region compared with control mice.

Animals↗