Tumor cell targeted cytokine (TNF-alpha) gene therapy for cancer.
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Animals↗
Biomedical subjects
T Blankenstein
Publications and source records attributed to T Blankenstein.
Evolutionary aspects, structure, and expression of the rat interleukin 4 gene.
The rat interleukin 4 (IL 4) gene has been isolated from a genomic lambda phage library by cross-hybridization to the mouse IL 4 cDNA. Like the mouse and human counterparts, it exists as a single copy gene in the genome and consists of four exons. The overall structure of the IL 4 locus seems highly conserved. This is indicated by the low degree of restriction fragment length polymorphism in a number of laboratory and wild mice and by the conservation of the intron size between human, rat, and mouse IL 4 genes. Furthermore, evolutionary conserved elements are the promoter region, the position of cysteine residues and sequence motifs in the 3' untranslated regions that are believed to be involved in destabilization of the mRNA. In contrast, the predicted amino acid sequence of the rat IL 4 gene shows low homology (57%) with the mouse homologue. The divergence between mouse and rat IL 4 genes is even more pronounced in the carboxy-terminal region (47% homology in the last 68 amino acids). The ratio between replacement and silent mutations in the IL 4 genes of different species suggests a complex pattern of selective forces acting on the IL 4 gene, which includes both selection against and for amino acid substitutions in individual positions. The functional identity with IL 4 has been confirmed by expression of the gene and the demonstration of the ability to induce MHC class II antigen expression on spleen cells.
Amino Acid Sequence↗
Lack of correlation between BglII RFLP in the human interleukin 6 gene and rheumatoid arthritis.
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Arthritis, Rheumatoid↗
Analysis of restriction fragment length polymorphism in lymphokine genes of normal and autoimmune mice.
Analysis of RFLP has been employed in lymphokine genes of autoimmune and normal mice. No polymorphism could be detected in the loci containing IL-2, IL-2 receptor, IL-5, and IFN-gamma in NZB, NZW, BxSB, and MRL/lpr mice when compared with normal mice. Allelic forms were identified in the IL-1 alpha gene of BALB/c and in the IL-4 gene of NZW. The frequency of the Bam HI RFLP in the TNF-alpha gene of NZW which has been proposed to be associated with the development of autoimmune disease in (NZB x NZW)F1 mice has been analyzed in a number of different inbred strains and in wild mice. Since the same allele is inherited in most autoimmune, healthy laboratory and wild mice the TNF-alpha gene does not seem to be one of the causal agents that contributes to the development of autoimmunity in (NZB x NZW)F1 mice.
Animals↗
Correlation between the biologic functions and the generation of lymphokines in T cell clones.
We have recently reported that various murine T cell clones produce IL-1. Based on this observation we have analyzed in the present study the correlation between the biological functions and the generation of different lymphokines in (T,G)-A--L specific CD4+ clones. One subset of clones--the "helper clones"--were found to provide help to primed B cells, in vitro. These cells could be shown to produce IL-1, IL-2, and B cell stimulatory factor 1 (IL-4) activities and to express mRNA encoding for these three cytokines. The second subset of clones, termed "proliferative clones", were unable to help B cells in vitro but expressed vigorous Ag-dependent proliferations. These cells did not express IL-1, IL-2, or IL-4 activities. They produced another lymphokine(s) which may be granulocyte-macrophage-CSF, or some other factor recognized by the HT2 cell line. This study further substantiates the link between T cell activities and lymphokine repertoire with a special emphasis on the potential role(s) of T cell-derived IL-1.
Animals↗
Structure and evolution of mouse interleukin 6 gene.
Restriction fragment length polymorphism in the interleukin 6 gene of murine rodents extending phylogenetically from Mus musculus domesticus to the rat has been analyzed. Most species exhibit distinct restriction site patterns. In contrast, limited polymorphism was found in the tumor necrosis factor alpha gene indicating different selective pressure acting on both genes. The gene encoding interleukin 6 was isolated from a genomic library and the exon/intron organization was determined by restriction analysis and limited DNA sequence analysis. It consists of five exons which distribute over about seven kilobases, thus resembling in structure and organization the human counterpart. Furthermore, no restriction fragment length polymorphisms in the interleukin 6 gene of autoimmune strains NZB, NZW, MRL-lpr/lpr and BxSB could be detected for either EcoRI, BamHI or HindIII.
Animals↗
Recombination between VH pseudogenes.
The peculiar pattern of nucleotide differences between germline VH genes V104A/VAR104 and 122B could be the result of an exchange of genetic information through gene conversion. A palindromic sequence in one of the genes seems to have been the target site for the break that initiated recombination.
Animals↗
Organization of the Ig VH locus in mice and humans.
In order to assess the contribution of the germline VH gene repertoire to antibody diversity, many groups have studied the evolution and the organization of the immunoglobulin VH gene locus in mice and humans. Here, Ulrich Krawinkel, Thomas Christoph and Thomas Blankenstein review recent data on this subject and discuss the potential influence of gene organization on the activation of the VH gene repertoire during the development of the mammalian immune system.
Animals↗
A retroviral expression vector containing murine immunoglobulin heavy chain promoter/enhancer.
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Animals↗
Immunoglobulin VH region genes of the mouse are organized in overlapping clusters.
Restriction fragment length polymorphism has been compared between the Igh loci of C57BL/6 and MOLF/EI (Mus musculus molossinus) mice utilizing probes which detect the C gamma 2b gene and genes from nine VH-gene families. Distinct restriction site patterns were found for the CH genes and for VH families PC7183, Q52 and X24. VH families V31 and J558 showed identical patterns. Mixed patterns of identical and distinct bands were detected in VH families S107, J606, V3660 and VGAM3.8. This indicates that a recombination took place involving the Igh loci of a M. m. molossinus and a progenitor of the C57BL/6 strain. The breakpoint of recombination maps to the chromosomal region carrying VH families S107, J606, VGAM3.8 and V3660. VH families PC7183, Q52 and X24 map 3' to the recombination breakpoint and proximal to the DH-JH-CH region, whereas VH families V31 and J558 accordingly map 5' to the recombination breakpoint and distal to DH-JH-CH. This order of VH families was confirmed by deletion mapping utilizing hybridomas which are haploid either for the Ighb or for the Igha locus. The mapping data indicate that the VH families of the mouse are organized in overlapping clusters. This notion is confirmed by demonstration of the physical linkage of VH genes from families V31 and J558 in the Igha locus.
Animals↗
Evolution of pseudogenes in the immunoglobulin VH-gene family of the mouse.
A quantitative analysis of the complexity of the J558 VH-gene family in the mouse immunoglobulin heavy chain (Igh) gene locus has been performed. Considerable variations in the degree of complexity are observed in various Igh haplotypes derived from laboratory mice and wild mice. The BALB/c strain shows the highest degree of complexity of the J558 VH-gene family when all mice are compared. Multiple gene duplications seem to have occurred in the BALB/c-derived J558 VH-gene family less than 1-2 million years ago. This dating is supported by the divergence in coding and flanking regions of three strongly homologous VH-region genes. Two of these genes were generated by the duplication of a pseudogene about 1.5 X 10(5) years ago. A recent expansion of the J558 VH-gene family and therefore little time for evolutionary drift may explain why most of the pseudogenes in this family exhibit a largely intact structure. We also describe two VH-region genes which represent older pseudogenes in states of progressive disintegration.
Amino Acid Sequence↗
Analysis of immunoglobulin heavy chain V-region genes belonging to the V NP-gene family.
A method was devised to clone immunoglobulin VH-region genes located on selected restriction fragments from genomic DNA directly into M13 vectors for subsequent nucleotide sequence analysis. Ten recombinant M13 clones representing four so far unknown VH-region genes of the VNP-gene family have been analysed. Sequence comparison shows that these genes are closely related to other VH-genes of the VNP gene family. One of the VH-genes exhibits a so far unobserved unusual length of 100 2/3 codons and appears to be functional. Analysis of the variation of the isolated VH-genes suggests that framework and complementarity determining regions are exposed to separate types of selective pressures.
Animals↗
MLV-10A1 retrovirus pseudotype efficiently transduces primary human CD4+ T lymphocytes.
BACKGROUND: Previously, we showed that retroviral vectors pseudotyped with the envelope of the amphotropic murine leukemia virus 10A1 (MLV-10A1) more efficiently transduce primary human CD8+ T lymphocytes when compared with other A-MLV, gibbon ape leukemia virus (GaLV) and feline endogenous retrovirus (RD114) vector pseudotypes. For the success of several gene therapeutic approaches (ADA, HIV) it is important to effectively transduce primary human CD4+ T lymphocytes. METHODS: We have used retroviral vectors encoding the enhanced green fluorescent protein (EGFP) as a marker gene and carrying envelopes of MLV-10A1, A-MLV and GaLV and have analyzed the transduction efficiency of both human CD4+ T cell lines (CEM, H9, HUT78, J16) and primary human CD4+ T lymphocytes using a RetroNectin-assisted transduction protocol and virus-containing supernatant. RESULTS: In CD4+ T cell lines the MLV-10A1 vector pseudotype was most effective and infected up to 85% of cells which then stably expressed GFP over time. MLV-10A1 was also superior and infected approximately 32% of primary human CD4+ T lymphocytes in comparison to GaLV (18%) and A-MLV (12%). The superior efficiency of MLV-10A1 for the transduction of CD4+ T cells correlates with the longer half-life of this pseudotype in comparison to A-MLV and, as previously shown by interference analysis, with the usage of both the A-MLV (Pit2) and the GaLV receptor (Pitl) for cell entry. CONCLUSIONS: MLV-10A1 is a suitable vector for transferring genes with high efficacy into primary human CD4+ T lymphocytes. The use of MLV-10A1 pseudotyped vectors should make it easier to obtain a sufficient number of gene-modified T lymphocytes for an adoptive transfer.
Animals↗
Tumor-cell-targeted cytokine gene transfer in experimental models for cancer therapy.
The genetic manipulation of tumor cells to express immunostimulatory molecules provides a current approach for the analysis of immune reactions against tumor cells in vivo. Experiments with multiple cytokines have demonstrated that an array of different host effector cells can be recruited by different cytokines in vivo, but more studies are necessary to distinguish cytokine-specific effects from as yet uncharacterized influences of different tumor models. A technically feasible clinical application of this approach is to be seen in the generation of vaccines by introducing such immunostimulatory genes into cancer cells and boosting systemic immune reactions against the unmodified cells. The experimental basis of these vaccination studies is critically discussed.
Animals↗