Biomedical subjects

R Kircheis

Publications and source records attributed to R Kircheis.

9 recordsLinked to original sources

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↗

PubMed1998

Cytokine gene-modified tumor cells for prophylactic and therapeutic vaccination: IL-2, IFN-gamma, or combination IL-2 + IFN-gamma.

Murine melanoma cells were engineered to express interleukin-2 (IL-2), interferon-gamma (IFN-gamma) or both cytokines at various dose levels by means of the adenovirus-enhanced transferrinfection (AVET) method. The gene-modified cells were tested for their potency to induce an antitumor immune response in two experimental settings with different tumor load. In a prophylactic vaccination model, both IL-2 and IFN-gamma showed a dose-dependent protection against tumor cell challenge in two melanoma models. In the therapeutic vaccination model, where mice with measurable tumors were treated, immunization with IL-2 or IFN-gamma gene-modified cells led to complete tumor regression in 30% or 20% of the tumor-bearing animals respectively. The combination of IL-2 + IFN-gamma resulted in complete tumor regression in up to 50% of the tumor-bearing mice.

Animals↗

PubMed1998

Sustained cytokine delivery for anticancer vaccination: liposomes as alternative for gene-transfected tumor cells.

Vaccination with tumor cells genetically engineered to produce interleukin (IL)-2 is an attractive strategy to enhance antitumor immune responses. The improved antitumor immunity upon vaccination with IL-2 gene-modified tumor cells may be due to the prolonged presence of the cytokine at the vaccination site. Because liposomes have been used for sustained delivery of a variety of agents, we compared the protective effect of vaccines consisting of IL-2 gene-modified B16 melanoma cells to that of vaccines composed of IL-2 liposomes and irradiated melanoma cells. The results indicate that both approaches equally protect against a lethal challenge with B16 melanoma cells. More than 20% of the protected animals developed vitiligo at the vaccination and/or tumor challenge site.

Animals↗

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

Coupling of cell-binding ligands to polyethylenimine for targeted gene delivery.

Recently the high transfection potential of the cationic polymer polyethylenimine (PEI) was described (Boussif O et al. Proc Natl Acad Sci USA 1995; 92: 7297-7301). To combine the promising DNA delivering activity of PEI with the concept of receptor-mediated gene delivery, cell-binding ligands (transferrin or antiCD3 antibody) were incorporated by covalent linkage to PEI. DNA complexes of PEI or ligand-PEI conjugates were tested for transfection of cultured neuroblastoma Neuro 2A cells, melanoma B16 or H225 cells, erythroid leukemic K562 cells and T cell leukemia Jurkat E6.1 cells. Depending on the cell line, incorporation of the cell-binding ligand resulted in an up to 1000-fold increased transfection efficiency. This activity depends on ligand-receptor interaction and was observed also at low PEI cation:DNA anion ratios where ligand-free PEI lacks efficiency. Depending on the cell-binding ligand, specific targeting (CD3 antibody, Jurkat cells) can be achieved. Gene transfer can be augmented by the addition of an endosome-destabilizing influenza peptide, but is not dependent on the presence of additional endosomolytic agents. Application of transferrin-PEI for the production of murine interleukin-2 in B16 cells resulted in exceptionally high secretion rates of 19 micrograms IL-2 protein per 10(6) cells per 24 h.

Animals↗

PubMed1992

Biological activity of mutants of human tumour necrosis factor-alpha.

Point mutations in different regions of the tumour necrosis factor-alpha (TNF-alpha) molecule influence anti-tumour cytotoxic/cytostatic activities as well as haemorrhagic tumour necrosis, tumour regression and lethal toxicity in mice. Mutations in the C-terminal region in positions 150 and 155 markedly decrease cytotoxicity for murine L929 fibroblasts and human MCF7 mammary carcinoma cells. Competitive binding experiments with 125I-labelled TNF-alpha revealed that the loss of cytotoxicity is caused by a loss of target cell binding. In contrast to the reduced activity against L929 and MCF7 cells, neither binding to nor cytostatic activity against the human myeloid leukaemia cell lines HL60 and U937 are affected. This target cell type-dependent behaviour is probably due to the fact that L929 and MCF7 cells express different types of TNF receptor compared with myeloid leukaemia cells. While a mutation in position 127 decreases the overall activity of TNF-alpha, a deletion of four N-terminal amino acids does not reduce biological activity. In vivo the TNF mutants differed in their anti-tumour effects and lethal toxicity, but a segregation of anti-tumour activity and toxicity was not observed.

Amino Acid Sequence↗

PubMedDate not supplied

Differences in the biological activity of TNF alpha and TNF beta correlate with their different abilities for binding to the target cells.

TNF alpha and TNF beta were compared regarding their binding to different types of target cells, cytotoxic/cytostatic activity against murine and human tumor cell lines as well as human capillary endothelial cells, their ability to induce differentiation in myeloid leukemia cell lines, and induction of hemorrhagic tumor necrosis and tumor regression as well as lethal toxicity in tumor-bearing mice. The results show considerable quantitative differences in the biological activity between TNF alpha and TNF beta depending on the type of target cell which has been used. TNF beta was 3 fold more cytotoxic than TNF alpha against murine L929 fibroblasts and 3-5 times more active concerning the induction of hemorrhagic tumor necrosis, complete tumor regression and more toxic in tumor-bearing mice. In contrast to this, TNF beta was markedly less cytotoxic against human capillary endothelial cells and the human mammary carcinoma cell line MCF7 and much less cytostatic against the human myeloid leukemia cell lines HL60 and U937. The lesser antiproliferative effect of TNF beta correlated with a lower ability for induction of differentiation in these cell lines. Competitive radioligand binding assays showed that TNF beta was about 4 fold more effective than TNF alpha in competing with 125I-labeled TNF alpha for the binding to murine L929 fibroblasts. But it was 15-20 times less effective in binding to the human MCF7 cells and the human myeloid leukemia cell lines HL60 and U937. This revealed that, at least for these targets, the differences in the biological activity between TNF alpha and TNF beta are due to different abilities for binding to the target cells. Possible mechanisms for these different binding abilities are discussed.

Animals↗

PubMedDate not supplied

Early embryonic cells activate the alternative complement system.

Murine embryonic stem cells, embryonic carcinoma cells and pre-implantation embryos were found to be extremely sensitive to cytolysis by normal human serum as compared to matured cells. The cytolytic activity to embryonic cells was not removed by pre-absorption of serum with spleen lymphocytes. Conditions which block both complement activation pathways or, selectively, the alternative pathway completely abrogated the activity of human serum against embryonic cells whereas the activity was retained under conditions which block the classical complement pathway, indicating that embryonic cells activate the alternative complement system (ACS). The cytotoxic effect to murine embryonic cells was reproduced using syngeneic murine serum. Concerning the mechanism of ACS-activation, the expression of regulators of complement activation and of membrane bound sialic acid was analysed. Embryonic cells express mRNA for Crry similarly to other cells but additionally express Cr2-transcripts not found in most adult cells. Embryonic cells have strikingly low levels of membrane-bound sialic acid compared to adult cells.

Animals↗

PubMedDate not supplied

Selective lysis of early embryonic cells by the alternative pathway of complement--a possible mechanism for programmed cell death in embryogenesis.

Early embryonic cells and early mouse embryos were shown to activate the alternative pathway of complement, and to be highly sensitive to complement-mediated cytolysis (Kircheis et al, In Vivo 9: 85-98, 1995). Under further development embryonic cells become resistant. The induction of resistance to the alternative pathway of complement correlates with: a) altered splicing of Cr2-transcript and b) changes in the acidic glycolipids under differentiation. Early embryonic cells have low amounts of sialic acid-containing glycolipids or express mainly GM3. The induction of differentiation changes the glycolipid pattern leading to an increase in membrane-bound sialic acid. The importance of membrane-bound sialic acid in the restriction of complement activation is demonstrated by increased sensitivity to complement after pre-treatment of cells with neuraminidase. The results indicate that there is target-specific lysis of early embryonic cells by the alternative pathway of complement. Early embryonic cells activate the alternative pathway of complement by expressing activators and low levels of membrane-bound sialic acid. Induction of differentiation changes the glycolipid pattern, leading to an increase in membrane-bound sialic acid sufficient to restrict complement-activation on the cell surface.

Animals↗