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W Uckert

Publications and source records attributed to W Uckert.

At least 19 recordsLinked to original sources
PubMed2006

The translocation motif of hepatitis B virus improves protein vaccination.

Cell-penetrating peptides (CPPs) have been shown to improve antigen loading of dendritic cell vaccines. Here we asked whether fusion of a CPP to a protein improves its immunogenicity when this fusion protein is directly applied as vaccine. We used the cell-penetrating translocation motif (TLM) derived from the hepatitis B virus, because no size limitation of cargos has been observed. Increased immunogenicity was observed when TLM was fused to ovalbumin (TLM-ova). TLM-ova was found to be superior to ova in inducing proliferation and cytotoxicity of ova-specific CD8+ T cells in vitro and in vivo. Using ovalbumin-expressing thymoma cells (EG7-ova), an improved anti-tumor immune response was observed for TLM-ova vaccination versus vaccination with ova. Moreover, TLM-ova vaccination induced a higher titer of anti-ovalbumin IgG2a antibodies compared to ova. These data demonstrate that CPP-protein vaccines can improve cellular as well as humoral immune responses.

Amino Acid Motifs↗
PubMed2003

[Overexpression of NPM-ALK induces different types of malignant lymphomas in IL-9 transgenic mice].

Anaplastic large cell lymphoma (ALCL) comprises approximately 25 % of all non-Hodgkin lymphomas in children and young adults. 40% of these tumours have a translocation t(2;5)(p23;q35), which fuses the nucleophosmin gene (NPM) to the anaplastic lymphoma kinase gene (ALK) resulting in a hybrid protein which contributes to the pathogenensis of ALCL. To further analyse the transforming activity in an animal model, a cDNA encoding the protein product, NPM-ALK, was incorporated into a retrovirus construct and introduced into mouse bone marrow progenitors by infection. In a bone marrow gene transfer and transplantation protocol the hematopoietic compartments of lethally irradiated IL-9 transgenic mice were reconstituted with npm-alk infected progenitor cells. IL-9 transgenic mice were chosen, because IL-9, a pleiotropic T helper 2 cytokine, is expressed in most cases of human ALCL and was shown to have an oncogenic potential at least on T cells. Reconstituted mice developed NPM-ALK positive lymphomas including lymphoblastic lymphomas of T-cell type (T-LB), mature and immature plasmacytoma (PZ) and plasmoblastic/anaplastic diffuse large B-cell lymphoma after 10-30 weeks. The combined overexpression of NPM-ALK and IL-9 exerts cooperative oncogenic activity in the transformation of murine lymphoid cells leading to accelerated and enhanced development of T-LB. Many animals developed plasmacytic/plasmoblastic neoplasms, of which the most aggressive tumours share many features with human anaplastic/plasmoblastic diffuse large B-cell lymphoma.

Animals↗
PubMed2001

Suicide gene therapy for pediatric tumors.

Tumor gene therapy is potentially very specific and efficacious. Suicide genes are promising tools in the arsenal of tumor gene therapy. However, problems of tumor targeting, low in vivo efficacy of nucleic acid transfer, and recent reports of adverse effects hinder the translation of this approach into clinical practice. Therefore vector design, tumor targeting, mechanisms of cell kill and killing of untransfected tumor cells must be improved. Once these problems are solved in vitro and in animal models, gene therapy holds great promise for pediatric oncology given the abundance of specific targets in pediatric tumors. This review describes the current state of preclinical research in tumor suicide gene therapy, provides an outline of pediatric suicide gene therapy protocols, and identifies potential targets in pediatric malignancies.

Adolescent↗
PubMed2000

Modulation of Moloney leukemia virus long terminal repeat transcriptional activity by the murine CD4 silencer in retroviral vectors.

We investigated whether CD4 gene regulatory sequences might be useful for developing transcriptionally targeted Moloney murine leukemia virus (Mo-MLV)-based retroviral vectors for gene expression specifically in CD4(+) cells. We could modulate Mo-MLV long terminal repeat (LTR) activity by inserting a 438-bp-long fragment containing the murine CD4 silencer in the LTR of the vector; both beta-galactosidase and green fluorescent protein reporter gene activities were strongly down-regulated in both murine and human CD8(+) cells, but not in CD4(+) lymphoid cell lines and freshly isolated lymphocytes transduced with this vector, compared with the findings using a control vector carrying wild-type LTRs. Titration experiments on NIH-3T3 cells revealed that inclusion of the CD4 silencer in the LTRs did not reduce the titer of the vectors. These findings indicate that a cellular silencer can be successfully included in retroviral vectors, where it maintains its transcription-regulatory function, thus suggesting a novel approach to transcriptional targeting.

3T3 Cells↗
PubMed2000

Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus 10A1.

Lentiviral vectors pseudotyped with the envelope glycoproteins (Env) of amphotropic murine leukemia virus (MLV) and the G protein of vesicular stomatitis virus (VSV-G) have been successfully used in recent preclinical gene therapy studies. We report here the generation of infectious HIV-1-derived vector particles pseudotyped with the Env of the molecular clone 10A1 of MLV and with chimeric envelope glycoprotein variants derived from gibbon ape leukemia virus (GaLV) and MLV. Formation of infectious HIV-1 (GaLV) pseudotype vectors was only possible with the substitution of the cytoplasmic tail of GaLV Env with that of MLV. The lentiviral vectors exhibited a host cell range identical with that of MLV(GaLV) and MLV(10A1) vectors, which are known to enter cells either via the GaLV-receptor Glvr-1 (Pit-1) or via the amphotropic receptor Ram-1 (Pit-2) in addition to Glvr-1, respectively. Thus, HIV-1(GaLV) and HIV-1(10A1) pseudotype vectors may be useful for efficient gene transfer into a variety of human tissues like primary human hematopoietic cells.

AIDS Vaccines↗
PubMed2000

Mitochondrial amplification of death signals determines thymidine kinase/ganciclovir-triggered activation of apoptosis.

Previous clinical experience shows that the efficacy of suicide gene transfer in tumor therapy is limited, resulting from inefficient gene transfer or alternatively, from intrinsic resistance of the tumor in vivo. Herpes simplex virus thymidine kinase/ganciclovir (TK/GCV), a paradigmatic suicide gene therapy system, has been described to exert its cytotoxic effect, at least in part, by inducing apoptosis in target cells. Here, we report that mitochondria amplify TK/GCV-induced apoptosis by regulating p53 accumulation and the effector phase of apoptosis. Treatment with TK/GCV led to mitochondrial perturbations including loss of the mitochondrial membrane potential and release of cytochrome c from mitochondria into the cytosol, inducing caspase activation and nuclear fragmentation. Inhibition of TK/GCV-induced mitochondrial perturbations by Bcl-2 overexpression or by the mitochondrion-specific inhibitor bongkrekic acid also strongly inhibited TK/GCV-induced activation of caspases and apoptosis. TK/GCV-induced mitochondrial perturbations depended on caspase activity possibly initiated by death receptor signaling. Perturbation of mitochondrial function mediated accumulation of wild-type p53 protein, since Bcl-2 overexpression, bongkrekic acid, or inhibition of mitochondrial protein synthesis with chloramphenicol strongly reduced TK/GCV-induced accumulation of wild-type p53 protein. These findings suggest that TK/GCV therapy may be less efficient in tumors in which the mitochondrial amplification of TK/GCV-induced apoptosis is blocked, e.g., by Bcl-2 overexpression. Given the low efficacy of currently used gene therapy systems, our data on molecular mechanisms that regulate sensitivity or resistance toward TK/GCV-induced cytotoxicity might have important implications to improve the clinical application of suicide gene therapy.

Anti-Bacterial Agents↗
PubMed2000

Efficient gene transfer into primary human CD8+ T lymphocytes by MuLV-10A1 retrovirus pseudotype.

Efficient and stable gene transfer into primary human T lymphocytes would greatly improve their use for adoptive transfer to treat acquired disorders, viral diseases, and cancer. We have constructed retroviral vector pseudotypes of amphotropic murine leukemia viruses (A-MuLV, MuLV-10A1), gibbon ape leukemia virus (GaLV), and feline endogenous virus (RD114) containing the enhanced green fluorescent protein (GFP) as a marker gene. Transduction of primary human CD8+ T lymphocytes by the different GFP-retrovirus pseudotypes revealed the superiority of MuLV-10A1 in comparison with A-MuLV, GaLV, and RD114, respectively. The superior transduction efficacy of CD8+ T cells by MuLV-10A1 correlates with a longer half-life of this pseudotype in comparison with A-MuLV and, as shown by interference analysis with the human T cell line HUT78, by the utilization of both the A-MuLV receptor (Pit2) and the GaLV receptor (Pit1) for cell entry.

CD8-Positive T-Lymphocytes↗
PubMed2000

Combined chemotherapy of murine mammary tumors by local activation of the prodrugs ifosfamide and 5-fluorocytosine.

The success of chemotherapeutic intervention is limited because the necessary high local drug doses cannot be achieved without systemic toxicity. Application of suicide genes (SGs) and direct conversion of prodrugs (PDs) to toxic metabolites in situ by SGs may enhance the efficacy of chemotherapy. To evaluate this strategy in two murine breast cancer models, TS/A and GR, we injected cellulose sulfate capsules harboring cat kidney cells expressing the SGs cytosine deaminase and cytochrome P450 2B1 (CYP2B1) intratumorally. The PDs 5-fluorocytosine and ifosfamide were administered in 3-day intervals. The effect of in situ chemotherapy with each PD alone and the combination was analyzed over a period of 100 days. The results reveal that for TS/A tumors, the antitumoral effect mediated by CYP2B1 is more efficient than that of cytosine deaminase, whereas for GR tumors, both systems worked equally well. Furthermore, we find additive toxicity using both SG/PD systems for both TS/A and GR tumors.

Animals↗
PubMed1999

Herpes simplex virus thymidine kinase/ganciclovir-induced apoptosis involves ligand-independent death receptor aggregation and activation of caspases.

Suicide gene therapy systems such as the herpes simplex thymidine kinase/ganciclovir system (TK/GCV) may kill cancer cells by apoptosis through as yet undefined mechanisms. Here we show that TK/GCV treatment induces p53 accumulation and increases cell surface expression of CD95 and tumor necrosis factor receptor, which is likely to involve p53-mediated translocation of CD95 to the cell surface. TK/GCV-induced apoptosis involves CD95-L-independent CD95 aggregation leading to the formation of a Fas-associated death domain protein (FADD) and caspase-8-containing, death-inducing signaling complex. Dominant negative FADD, the caspase-8 inhibitor zIETD-fmk [Z-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone], and zVAD-fmk (Z-Val-Ala-Asp-fluoromethylketone) partially abrogate TK/GCV-induced apoptosis. In addition to apoptosis induction, TK/GCV treatment strongly sensitizes for CD95-L-, TNF-, and TNF-related, apoptosis-inducing, ligand (TRAIL)-induced cell death in constitutively resistant cells. These findings may be used to increase the efficacy of TK/GCV and other suicide gene therapy systems for the treatment of cancer.

Adaptor Proteins, Signal Transducing↗
PubMed1998

RNA levels of human retrovirus receptors Pit1 and Pit2 do not correlate with infectibility by three retroviral vector pseudotypes.

The gibbon ape leukemia virus (GaLV) and the amphotropic murine leukemia virus (A-MuLV) infect human cells via specific receptors, Pit1 and Pit2, respectively. mRNA levels of these receptors were determined by Northern analysis and for Pit2 in addition by quantitative RT-PCR. Pit1 and Pit2 were expressed in different amounts in human tissues and cell lines; Pit1-specific mRNA was generally more abundant than Pit2 mRNA. No correlation was found between Pit1 and Pit2 RNA levels and infectibility by GaLV and A-MuLV pseudotyped vectors, respectively. GaLV and A-MuLV revealed a partial reciprocal interference. MuLV-10A1 can utilize both Pit1 and Pit2 for entry into cells but could not infect any of the 14 human cell lines more efficiently than A-MuLV or GaLV. Interference assays suggested that MuLV-10A1 has a higher affinity for and infected most cells predominantly by Pit2. However, at least in one cell line it used Pit1 more efficiently for entry. We conclude that (1) Pit1 and Pit2 mRNA levels in human cells are not indicative of the infectibility by GaLV and A-MuLV pseudotypes, respectively; (2) A-MuLV can infect target cells as efficiently as can GaLV, although Pit2 RNA is less abundant than Pit1 RNA; (3) factor(s) in addition to the presence of Pit1 and Pit2 are involved in retroviral infection; and (4) MuLV-10A1 pseudotype does not infect human cells more efficiently than do A-MuLV and GaLV pseudotypes.

Base Sequence↗
PubMed1998

Double suicide gene (cytosine deaminase and herpes simplex virus thymidine kinase) but not single gene transfer allows reliable elimination of tumor cells in vivo.

Suicide genes such as cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) encode products that convert nontoxic substances (prodrugs) into toxic metabolites. Suicide gene transfer is currently being used in cancer therapy or can be used as a safety modality. To analyze the reliability of suicide genes as a safety modality for a vaccination study with viable cytokine/B7 gene-modified tumor cells, the individual and combined efficacy of the two suicide genes was compared for in vitro and in vivo cell killing of a murine mammary adenocarcinoma cell line (TS/A). To adapt the system to an in vivo gene delivery situation, bulk cultures cotransfected with the CD and TK gene were used instead of selected clones. In vitro, both CD and TK conferred sensitivity to the respective prodrug but the combined cytotoxic effects of both gene products were always superior. For in vivo analysis BALB/c mice were injected subcutaneously with CD- and TK-modified TS/A cells, treated with prodrugs, and tumor size was evaluated for a period of 100 days. In the in vivo situation the combination of both enzyme/prodrug systems was again most effective. The highest single concentration of 5-FC (500 mg/kg) or GCV (100 mg/kg) was not able to fully protect the animals from developing tumors, whereas a combination of 5-FC (250 mg/kg) and GCV (50 mg/kg) resulted in complete tumor eradication. In nude mice treated in the same way, most CD/TK tumors could not be eliminated. Furthermore, BALB/c mice cured of TS/A-CD/TK tumors developed a systemic tumor immunity against challenge with parental TS/A cells. These findings indicate that reliable tumor elimination by the suicide genes depends on T cells. The cooperative effect of both suicide genes was confirmed in vitro with the human renal cell carcinoma line RCC26. We conclude that TK and CD together, but neither gene alone, act as a safety mechanism for the elimination of tumor cells in a reliable fashion and suggest that a rapid and quantitative antigen release by effective TK- and CD-mediated tumor destruction is necessary for T cell immunity to develop.

Adenocarcinoma↗
PubMed1997

Successful retroviral mediated transduction of a reporter gene in human dendritic cells: feasibility of therapy with gene-modified antigen presenting cells.

In this study we have analyzed the feasibility of gene transfer in human dendritic cells (DCs). DCs were generated from T and B cell-depleted peripheral blood mononuclear cells cultured for 7 days in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). The cells showed morphologic and immunophenotypical features typical of DCs, including expression of major histocompatibility complex (MHC) class I and II molecules, CD1a, CD80, CD86, CD13, CD33, CD40, and CD54. The cells showed high stimulatory activity in both allogeneic and autologous mixed lymphocyte reaction (MLR). The bacterial reporter gene lacZ coding for beta-galactosidase (beta-gal) was introduced in DCs by three sequential cycles of infection using a MFG retroviral vector system. After 7 days of culture 35-67% of the cells showed high expression of beta-gal activity, proving successful gene transfer. Stable integration of the lacZ gene was demonstrated by genomic DNA-polymerase chain reaction (PCR) up to 20 days after gene transfer. The percentage of transduction was similar when DCs were further purified by immunomagnetic separation according to CD1a-expression. We conclude that human DCs can be efficiently gene modified, further broadening the spectrum of possible DC-based clinical applications.

Antigen Presentation↗
PubMed1994

Conditional expression of human TNF-alpha: a system for inducible cytotoxicity.

Tumour necrosis factor-alpha (TNF-alpha) is currently being used in clinical trials for cancer treatment, but toxic side effects, due to systemic administration and high doses, are observed. Inducible expression of TNF may permit selective killing of tumour cells in gene therapy protocols without need for prolonged and/or high-level TNF expression. A conditional TNF expression vector has been constructed in which the coding sequences of human TNF have been placed under the transcriptional control of the glucocorticoid-regulated murine mammary tumour virus long terminal repeat (MMTV-LTR). Negligible levels of TNF expression, associated with no phenotypic alterations, are observed in cells transfected with MMTV-TNF vectors in the absence of glucocorticoid. Expression levels could be stimulated by the addition of the synthetic glucocorticoid dexamethasone. Increasing expression levels of TNF were associated with enhanced cytotoxicity. Our results suggest the potential use of inducible TNF systems for the treatment of tumours in gene therapy protocols.

Animals↗
PubMed1994

Retrovirus-mediated gene transfer in cancer therapy.

Retroviral vectors are one of the most promising systems for the transfer and the expression of therapeutic genes in human gene therapy protocols. This review will focus both on the advantages and intricacies of retroviral vectors themselves as well as on the application of these vector systems in experimental and clinical cancer therapy protocols. Therefore, the retrovirus life cycle and the general features of retroviral vectors, including possible targeting strategies with retroviral vectors, are overviewed. These topics are followed by the presentation of genes with emphasis on their potential as tools in somatic cell cancer therapy (cytokines, lymphokines, colony-stimulating growth factors, suppressor genes, antisense oncogenes, suicide genes). Finally, a prospect on the application of retroviral vectors will be described.

Cytokines↗
PubMed1990

Use of molecular cloned viral genomes for studies into interrelationship between type D retroviruses from monkey and human cells.

Type D retroviruses constitute a group of RNA tumor viruses so far isolated exclusively from primates. 4 groups of isolates are known to date (Mason-Pfizer virus, agents of simian acquired immunodeficiency syndrome--SAIDS, endogenous viruses of Old World and New World monkeys, and some isolates from cell lines--PMF virus). The members of the latter group are well characterized at protein-chemical and immunological levels. Briefly described in this paper are the results of our studies into the molecular structure of the viral genome of PMFV in relation to other members of the group (SAIDS-retrovirus/NE, MPMV, and SRV-1).

Animals↗