Biomedical subjects

S Finke

Publications and source records attributed to S Finke.

13 recordsLinked to original sources

PubMed2005

Recombinant rhabdoviruses: vectors for vaccine development and gene therapy.

The establishment of methods to recover rhabdoviruses from cDNA, so-called reverse genetics systems, has made it possible to genetically engineer rhabdoviruses and to study all aspects of the virus life cycle by introducing defined mutations into the viral genomes. It has also opened the way to make use of the viruses in biomedical applications such as vaccination, gene therapy, or oncolytic virotherapy. The typical gene expression mode of rhabdoviruses, a high genetic stability, and the propensity to tolerate changes in the virus envelope have made rhabdoviruses attractive, targetable gene expression vectors. This chapter provides an overview on the possibilities to manipulate biological properties of the rhabdoviruses that may be important for further development of vaccine vectors and examples of recombinant rhabdoviruses expressing foreign genes and antigens.

Animals↗

PubMed2000

Differential transcription attenuation of rabies virus genes by intergenic regions: generation of recombinant viruses overexpressing the polymerase gene.

Gene expression of nonsegmented negative-sense RNA viruses involves sequential synthesis of monocistronic mRNAs and transcriptional attenuation at gene borders resulting in a transcript gradient. To address the role of the heterogeneous rabies virus (RV) intergenic regions (IGRs) in transcription attenuation, we constructed bicistronic model RNAs in which two reporter genes are separated by the RV N/P gene border. Replacement of the 2-nucleotide (nt) N/P IGR with the 5-nt IGRs from the P/M or M/G border resulted in attenuation of downstream gene transcription to 78 or 81%, respectively. A severe attenuation to 11% was observed for the 24-nt G/L border. This indicated that attenuation in RV is correlated with the length of the IGR, and, in particular, severe downregulation of the L (polymerase) gene by the 24 nt IGR. By reverse genetics, we recovered viable RVs in which the strongly attenuating G/L gene border of wild-type (wt) RV (SAD L16) was replaced with N/P-derived gene borders (SAD T and SAD T2). In these viruses, transcription of L mRNA was enhanced by factors of 1.8 and 5.1, respectively, resulting in exaggerated general gene expression, faster growth, higher virus titers, and induction of cytopathic effects in cell culture. The major role of the IGR in attenuation was further confirmed by reintroduction of the wt 24-nt IGR into SAD T, resulting in a ninefold drop of L mRNA. The ability to modulate RV gene expression by altering transcriptional attenuation is an advantage in the study of virus protein functions and in the development of gene delivery vectors.

Base Sequence↗

PubMed1999

Phase I clinical study applying autologous immunological effector cells transfected with the interleukin-2 gene in patients with metastatic renal cancer, colorectal cancer and lymphoma.

Natural killer-like T lymphocytes termed cytokine-induced killer (CIK) cells have been shown to eradicate established tumours in a severe combined immune deficient (SCID) mouse/human lymphoma model. Recently, we demonstrated that CIK cells transfected with cytokine genes possess an improved proliferation rate and a significantly higher cytotoxic activity as compared to non-transfected cells. Here, in a phase I clinical protocol, autologous CIK cells were generated from peripheral blood obtained by leukapheresis in patients with metastatic renal cell carcinoma, colorectal carcinoma and lymphoma. CIK cells were transfected with a plasmid containing the interleukin-2 (IL-2) gene via electroporation. Transfected cells generated IL-2 in the range of 330-1800 pg 10(-6) cells 24 h(-1) with a mean of 836 pg 10(-6) cells 24 h(-1). Ten patients received 1-5 intravenous infusions of IL-2-transfected CIK cells; five infusions with transfected CIK cells were given. In addition, the same patients received five infusions with untransfected CIK cells for control reasons. In three patients, WHO grade 2 fever was observed. Based on polymerase chain reaction of peripheral blood transfected cells could be detected for up to 2 weeks after infusion. There was a significant increase in serum levels of interferon gamma (IFN-gamma), granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta (TGF-beta) during treatment. Interestingly, there was also an increase in CD3+ lymphocytes in the blood of patients during therapy. In accordance, a partial increase in cytotoxic activity in peripheral blood lymphocytes (PBLs) was documented when patient samples before and after therapy were compared. Concerning clinical outcome, six patients remained in progressive disease, three patients showed no change by treatment, and one patient with lymphoma developed a complete response. In conclusion, we were able to demonstrate that CIK cells transfected with the IL-2 gene can be administered without major side-effects and are promising for future therapeutic trials.

Adolescent↗

PubMed1999

Generation of bovine respiratory syncytial virus (BRSV) from cDNA: BRSV NS2 is not essential for virus replication in tissue culture, and the human RSV leader region acts as a functional BRSV genome promoter.

In order to generate recombinant bovine respiratory syncytial virus (BRSV), the genome of BRSV strain A51908, variant ATue51908, was cloned as cDNA. We provide here the sequence of the BRSV genome ends and of the entire L gene. This completes the sequence of the BRSV genome, which comprises a total of 15,140 nucleotides. To establish a vaccinia virus-free recovery system, a BHK-derived cell line stably expressing T7 RNA polymerase was generated (BSR T7/5). Recombinant BRSV was reproducibly recovered from cDNA constructs after T7 RNA polymerase-driven expression of antigenome sense RNA and of BRSV N, P, M2, and L proteins from transfected plasmids. Chimeric viruses in which the BRSV leader region was replaced by the human respiratory syncytial virus (HRSV) leader region replicated in cell culture as efficiently as their nonchimeric counterparts, demonstrating that all cis-acting sequences of the HRSV promoter are faithfully recognized by the BRSV polymerase complex. In addition, we report the successful recovery of a BRSV mutant lacking the complete NS2 gene, which encodes a nonstructural protein of unknown function. The NS2-deficient BRSV replicated autonomously and could be passaged, demonstrating that NS2 is not essential for virus replication in cell culture. However, growth of the mutant was considerably slower than and final infectious titers were reduced by a factor of at least 10 compared to wild-type BRSV, indicating that NS2 provides a supporting factor required for full replication capacity.

5' Untranslated Regions↗

PubMed1999

Virus promoters determine interference by defective RNAs: selective amplification of mini-RNA vectors and rescue from cDNA by a 3' copy-back ambisense rabies virus.

Typical defective interfering (DI) RNAs are more successful in the competition for viral polymerase than the parental (helper) virus, which is mostly due to an altered DI promoter composition. Rabies virus (RV) internal deletion RNAs which possess the authentic RV terminal promoters, and which therefore are transcriptionally active and can be used as vectors for foreign gene expression, are poorly propagated in RV-infected cells and do not interfere with RV replication. To allow DI-like amplification and high-level gene expression from such mini-RNA vectors, we have used an engineered 3' copy-back (ambisense) helper RV in which the strong replication promoter of the antigenome was replaced with the 50-fold-weaker genome promoter. In cells coinfected with ambisense helper virus and mini-RNAs encoding chloramphenicol acetyltransferase (CAT) and luciferase, mini-RNAs were amplified to high levels. This was correlated with interference with helper virus replication, finally resulting in a clear predominance of mini-RNAs over helper virus. However, efficient successive passaging of mini-RNAs and high-level reporter gene activity could be achieved without adding exogenous helper virus, revealing a rather moderate degree of interference not precluding substantial HV propagation. Compared to infections with recombinant RV vectors expressing CAT, the availability of abundant mini-RNA templates led to increased levels of CAT mRNA such that CAT activities were augmented up to 250-fold, while virus gene transcription was kept to a minimum. We have also exploited the finding that internal deletion model RNAs behave like DI RNAs and are selectively amplified in the presence of ambisense helper virus to demonstrate for the first time RV-supported rescue of cDNA after transfection of mini-RNA cDNAs in ambisense RV-infected cells expressing T7 RNA polymerase.

3' Untranslated Regions↗

PubMed1998

Generation of cytokine-induced killer cells using exogenous interleukin-2, -7 or -12.

Immunologic effector cells termed cytokine-induced killer (CIK) cells are generated in vitro from peripheral blood lymphocytes by addition of interferon-gamma, interleukin (IL)-2, IL-1 and an antibody against CD3. CIK cells have been shown to eradicate established tumors in a SCID mouse/human lymphoma model. CIK cells are dependent on exogenous cytokines such as IL-2, IL-7, or IL-12. We studied the effect of these cytokines in detail. Cellular proliferation was analyzed using an MTT proliferation assay, surface antigen expression via flow cytometry, cytotoxic activity using an LDH release assay, and apoptosis via flow cytometric analysis. IL-2, IL-7 and IL-12 led to significant growth of lymphocytes. Cells grown in IL-2 and IL-7 showed higher proliferation rates than cells grown in IL-12 according to the MTT assay. Concerning surface antigen expression, exogenous IL-7 led to a decrease in IL-7 receptor expression (4.8% from 60.4%) and exogenous IL-2 to a decrease in IL-2 receptor expression (61.2% from 73.2%). CD28 expression was higher in cells grown in IL-7 (77.3%) than in cells grown in IL-2 (62.5%). IL-12 led to a decrease in ICAM-1 adhesion molecule expression (57.7% from 76.7%) and an increase in CD56 expression compared with exogenous IL-7. IL-7 led to higher number of CD4-positive cells than IL-2 (53.0% vs 49.5%). No significant difference was found between IL-2, IL-7 and IL-12 in cytotoxic activity measured in an LDH release assay. Small amounts of apoptotic cells were found with all cytokines. However, the percentage of necrotic cells was higher with exogenous IL-12 than with IL-2 or IL-7. In summary, CIK cells can be generated using exogenous IL-2, IL-7 or IL-12. No difference in cytotoxic activity was found. However, significant differences were found in cell proliferation rates, antigen expression and percentage of necrotic cells.

Animals↗

PubMed1998

Increase of proliferation rate and enhancement of antitumor cytotoxicity of expanded human CD3+ CD56+ immunologic effector cells by receptor-mediated transfection with the interleukin-7 gene.

Cytokine-induced killer (CIK) cells have been shown to eradicate established tumors in a SCID mouse-human lymphoma model. CIK cells depend on exogenous addition of cytokines such as interleukin-2 (IL-2), interleukin-7 (IL-7) or interleukin-12 (IL-12) for proliferation. In this study, we used the adenovirus-enhanced CD3 receptor-mediated gene transfer for transfection with the IL-7 gene. An episomally replicating plasmid was used containing cDNA of the human IL-7 gene under the control of a CMV promoter for transfection of CIK cells. Biosynthesis of IL-7 was demonstrated by RT-PCR, an enzyme-linked immunosorbent assay (ELISA) and using a bioassay. Transfected cells produced IL-7 in the range between 200 and 1100 pg/10(6) cells in 24 h. IL-7 was shown to be biologically active, since transfected CIK cells showed an improved proliferation rate as compared with nontransfected cells. Expression of IL-7 altered the secretion of other cytokines by CIK cells, in particular the production of TNF alpha increased after transfection. In contrast, nontransfected CIK cells fed with IL-7 showed no increase in TNF alpha secretion. No significant differences were found in expression of surface antigens linked to the cytotoxic activity of CIK cells. Cytotoxic activity against various tumor cell lines (eg renal cell carcinoma, malignant melanoma and colon carcinoma) was tested. Transfected cells possessed a significantly higher cytotoxic activity as compared with nontransfected cells. Receptor-mediated gene transfer effectively delivers expression plasmids for therapeutic genes into CIK cells and CIK cells transfected with an IL-7 gene expression construct may be valuable for adoptive immunotherapy.

Adenoviridae↗

PubMed1997

A CXCR4/CD4 pseudotype rhabdovirus that selectively infects HIV-1 envelope protein-expressing cells.

We show that a cellular virus receptor functions in the envelope of a virus, allowing selective infection of cells displaying the receptor ligand. A G-deficient rabies virus (RV) pseudotyped with CD4- and CXCR4-derived proteins selectively infected cells expressing HIV-1 envelope protein. Envelope protein or CD4 antibodies blocked virus entry. Pseudotype virus formation was most efficient with chimeric receptor proteins possessing the cytoplasmic tail of the RV G spike protein (CXCR4-RV and CD4-RV). While CXCR4-RV was incorporated when expressed alone, CD4-RV incorporation required CXCR4-RV as a carrier protein, indicating a mechanism by which oligomeric surface proteins are sorted into the RV envelope. Viral vectors bearing virus receptors in their envelope may be useful reagents for targeting virus-infected cells in vivo.

Amino Acid Sequence↗

PubMed1997

Lymphocyte apoptosis: induction by gene transfer techniques.

Efficient gene transfer of lymphocytes has been shown to be extremely difficult. The molecular background for this gene transfer resistance is not completely understood. We reasoned that apoptosis may play a role in this gene transfer resistance of lymphocytes. We show that transfection of lymphocytes via nonviral vectors leads to induction of apoptosis in a significant proportion of cells. Since apoptosis may be mediated via the TNF alpha and TNF alpha receptor pathway, we studied the amount of TNF secreted by transfected lymphocytes. The percentage of apoptotic lymphocytes correlated well with TNF alpha secretion. TNF secretion was dependent on the gene transfection method used. High amounts of TNF secretion were detected using receptor-mediated gene transfer and lipofection. In contrast, only low amounts of TNF were detected after electroporation and retroviral gene transfer. In receptor-mediated gene transfer, TNF secretion was due to the use of anti-CD3 antibody. Induction of apoptosis and increase in necrosis was blocked using an anti-TNF antibody. This blockage led to a significant increase in the proliferation rate of lymphocytes transfected with the interleukin-2 or interleukin-7 gene. In conclusion, gene transfer techniques led to TNF secretion, apoptosis and necrosis of lymphocytes. This could be blocked using an anti-TNF antibody. Blockage of apoptosis after gene transfer should have an impact on the use of lymphocytes transfected with cytokine genes as immunologic effector cells in cancer gene therapy protocols.

Antibodies, Monoclonal↗

PubMed1997

Ambisense gene expression from recombinant rabies virus: random packaging of positive- and negative-strand ribonucleoprotein complexes into rabies virions.

We have recovered from cDNA a rabies virus (RV) containing identical, transcriptionally active promoters at its genome (negative-strand) and antigenome RNA and directing efficient expression of a chloramphenicol acetyltransferase (CAT) reporter gene from the antigenome. Transcription of the antigenome CAT gene was terminated by a modified RV gene junction able to mediate transcription stop and polyadenylation but not reinitiation of downstream transcripts. While in standard RV-infected cells genome and antigenome RNAs were present in a 49:1 ratio, the ambisense virus directed synthesis of equal amounts of genome and antigenome RNA (1:1). Total replicative synthesis was reduced by a factor of less than 2, revealing an unexpectedly high level of replication activity of the transcriptionally active promoter in the absence of the parental antigenome promoter. Successful packaging of ambisense ribonucleoprotein complexes (RNPs) into virions demonstrated that the parental 5' end of the RV genome RNA does not contain putative signals required for incorporation into virions. As determined both for standard RV and ambisense RV, virus particles contained genome and antigenome RNPs in the same ratios as those present in infected cells (49:1 and 1:1, respectively), indicating indiscriminate incorporation of RNPs independent of signals in the RNA. Ambisense expression of multiple foreign genes from RV vectors may circumvent problems with transcriptional attenuation of rhabdovirus housekeeping genes.

Animals↗

PubMed1996

Highly stable expression of a foreign gene from rabies virus vectors.

A reverse genetics approach was applied to generate a chimeric nonsegmented negative strand RNA virus, rabies virus (RV) of the Rhabdoviridae family, that expresses a foreign protein. DNA constructs containing the entire open reading frame of the bacterial chloramphenicol acetyltransferase (CAT) gene and an upstream RV cistron border sequence were inserted either into the nontranslated pseudogene region of a full-length cDNA copy of the RV genome or exchanged with the pseudogene region. After intracellular T7 RNA polymerase-driven expression of full-length antigenome RNA transcripts and RV nucleoprotein, phosphoprotein and polymerase from transfected plasmids, RVs transcribing novel monocistronic mRNAs and expressing CAT at high levels, were recovered. The chimeric viruses possessed the growth characteristics of standard RV and were genetically stable upon serial cell culture passages. CAT activity was still observed in cell cultures infected with viruses passaged for more than 25 times. Based on the unprecedented stability of the chimeric RNA genomes, which is most likely due to the structure of the rhabdoviral ribonucleoprotein complex, we predict the successful future use of recombinant rhabdovirus vectors for displaying foreign antigens or delivering therapeutic genes.

Animals↗

PubMedDate not supplied

[Results of prevention of retinal detachment by using photocoagulation].

Prophylactic retinal photocoagulation (in order to prevent the retinal detachment) was performed in the Ophthalmological Department of Medical School in Poznań in the period 1980-1984 and 1987-1988 in 114 eyes, in the Ophthalmological Department in Halle in the period 1982-1983 in 160 eyes. Qualified for coagulation were following changes: holes in the upper quadrants, tears, lattice-like degeneration and vitreoretinal adhesions in the fellow-eye, in high myopia, in aphakia, in cases with subjective symptoms (flashes) or in cases with suspected family history (familial incidence of retinal detachment). In our material the percentage of retinal detachment in the fellow-eye amounted 3.5 p.c. In the Poznań Department in spite of performed prophylactic intervention retinal detachment occurred in 3 eyes, in Halle in 7.

Argon↗

PubMedDate not supplied

Increase of cytotoxic sensitivity of primary human melanoma cells transfected with the interleukin-7 gene to autologous and allogeneic immunologic effector cells.

Patients with metastatic melanoma have a very poor prognosis. In many cases, the tumor recurs after surgical excision. Therefore, it might be beneficial for cancer patients to induce an immune attack against the tumor by inserting a cytokine gene into the tumor cells. Here, 14 primary cell cultures could be established from 45 patients with malignant melanoma. Primary cell cultures were transfected via electroporation with the gene encoding for human interleukin-7 (IL-7). Transfection resulted in the production of biologically active IL-7 with an average of 850 pg/mL per 10(6) cells per 24 hours. Irradiation with 10,000 cGy, which inhibited tumor cell growth in vitro, increased the amount of released IL-7 to an average amount of 1050 pg/mL per 10(6) cells per 24 hours. No significant differences in the phenotype were observed in the IL-7-transfected cells compared with nontransfected cells. The expression of HLA class I and II, ICAM-1, and of a melanoma-associated antigen remained unaltered. Transfection with IL-7 had no significant effect on the proliferation of melanoma cells as measured in a MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. There was no significant change in the cytokine profile after transfection or irradiation of the cells, but one cell culture expressed a high amount of IL-6 (about 2 ng/mL). IL-6 was expressed in nontransfected cells and was not altered by transfection. Interestingly, transfected cells from primary melanoma cultures possessed a higher sensitivity to immunologic effector cells compared with nontransfected cells. This was true for allogeneic as well as autologous melanoma cells. Our results show the feasibility of a gene transfer into primary human melanoma cells, different from retroviral transduction. IL-7-transfected cells might be of value in vaccination protocols for melanoma patients.

Cytotoxicity, Immunologic↗