A competitive immunoassay to detect a hapten using an enzyme-labelled peptide mimotope as tracer.

Mimotope peptides-peptides which mimic the binding of a hapten to its corresponding monoclonal antibody-were conjugated to peroxidase and used in competitive immunoassay. The established immunoassay was used to quantitatively determine the concentration of hapten. As model system in all the experiments described here, we used the binding of the monoclonal antibody B13-DE1 to fluorescein and the corresponding peptide mimotope.

Efficient cryopreservation of dendritic cells transfected with cDNA of a tumour antigen for clinical application.

Dendritic cells (DCs) are the most potent antigen-presenting cells of the immune system and are currently being investigated in clinical applications as cancer vaccines. An efficient cryopreservation method would greatly contribute to their use in clinical trials. We have established a method for freezing of DCs derived from peripheral blood mononuclear cells using the plasma expander Gelifundol. This enabled us to reduce the concentration of the toxic DMSO to 5%. The method could be performed without the addition of fetal calf serum or any other serum. After freezing, the viability of the DCs was 90%. The cells exhibited all the phenotypic characteristics (CD11c+, HLA-DR+, CD80+, CD83+, CD86+) of DCs, as tested by flow cytometry. Cells transfected with cDNA for the tumour antigen mucin expressed this protein on their surfaces in the same manner as before freezing. The stimulating capacity of a mixed lymphocyte culture was also preserved. These findings offer an efficient method for the cryopreservation of DCs for use in clinical trials.

Mucin gene (MUC1) transfer into human dendritic cells by cationic liposomes and recombinant adenovirus.

BACKGROUND: Dendritic cells (DC) as antigen presenting cells play an important role in immunotherapy of cancer. Mucin, encoded by the gene MUC1, is a human tumor antigen expressed in breast, pancreatic and ovarian cancers. Therefore, MUC1-transfected DC would be an attractive tool in constructing cancer vaccines. MATERIALS AND METHODS: Using two different cationic liposome preparations and, for comparison, a recombinant adenovirus expressing mucin, we tested the efficiency of mucin gene transfer into DC by flow cytometry. We investigated if these transfected DC were able to specifically stimulate autologous peripheral blood lymphocytes (PBL) from healthy donors. RESULTS: Flow cytometry revealed that 5-20% of DC transfected with liposomes Lipofectin and 20-40% of DC transduced with adenovirus expressed the relevant mucin epitopes. The expression of mucin on DC was similar to the expression of mucin found on carcinoma cells. After antigen uptake, DC specifically stimulated autologous PBL. CONCLUSION: We have shown that cationic liposomal gene transfer into human DC was feasible. We could obtain antigen specific stimulation of PBL at a similar rate as with adenoviral MUC1-transduced DC.

Biological sequences integrated: a relational database approach.

Over the last decade the modeling and the storage of biological data has been a topic of wide interest for scientists dealing with biological and biomedical research. Currently most data is still stored in text files which leads to data redundancies and file chaos. In this paper we show how to use relational modeling techniques and relational database technology for modeling and storing biological sequence data, i.e. for data maintained in collections like EMBL or SWISS-PROT to better serve the needs for these application domains. For this reason we propose a two step approach. First, we model the structure (and therefore the meaning of the) data using an Entity-Relationship approach. The ER model leads to a clean design of a relational database schema for storing and retrieving the DNA and protein data extracted from various sources. Our approach provides the clean basis for building complex biological applications that are more amenable to changes and software ports than their file-base counterparts.

Generation of an immortalized human CD4+ T cell clone inhibiting tumor growth in mice.

Tumor antigen-specific T cell clones represent a useful tool in tumor immunology; however, their long-term culture is limited. To generate an immortalized cytotoxic T cell clone against the human tumor antigen mucin, we exposed a previously generated T cell culture to Herpesvirus saimiri. We obtained an immortalized human CD4+ T cell clone, termed SITAM. Clonality of these cells was shown by analysis of the alpha/beta-T cell receptor (TCR) repertoire. Cytolytic activity was demonstrated against several mucin-expressing tumor cell lines and could not be detected against non-mucin-expressing cells. SITAM cells maintained their features stably for 2 years. Furthermore, growth of the tumor cell line Capan-2 in NOD/SCID mice was inhibited when SITAM cells were coinjected subcutaneously with tumor cells. SITAM cells provide an unlimited source of clonal T cells for analysis of tumor recognition and may be of help in TCR-targeted immunotherapy.

Binding of tumor antigen mucin (MUC1) derived peptides to the heat shock protein DnaK.

The human epithelial mucin encoded by the gene MUC1 is a tumor-associated antigen expressed on breast, pancreatic, colon and ovarian cancer cells recognized by cytotoxic T-cells and antibodies. Underglycosylated as well as glycosylated mucin-peptide epitopes are promising targets for vaccination against cancer. Heat shock proteins of 70 kDa (HSP70), also highly expressed in tumor cells, can function as chaperones for peptides and proteins and are involved in antigen processing. The involvement of HSP70 molecules in mucin antigen binding, processing and presentation has not yet been examined. Here we present first results concerning the relative binding affinities of various mucin-derived peptides to the bacterial 70 kDa heat shock protein DnaK. Interestingly, longer mucin peptides reveal a higher affinity to DnaK than short peptides. The non-glycosylated mucin-derived peptides of 5-8 amino acids length were able to compete with a high affinity (unrelated) reference peptide at millimolar concentrations. Glycosylation of the investigated short peptides lowers their binding affinity to DnaK, depending on the position of the carbohydrate. The binding affinity is not influenced by free charges at unprotected ends. The peptide (MUC1)5 consisting of five repeating units has an affinity enhanced by a factor of three as compared to the peptide with only one repeating unit. Mucin-peptide-HSP-complexes could be the basis of developing new kinds of tumor vaccines.

Reverse transformation of human mammary carcinoma cells.

Exposure of cells derived from human mammary carcinoma cell line, MaTu, to daunorubicin started a selection process which reproducibly gave rise to sublines with different phenotypes. One subline exhibited a fibroblast-like morphology (MaTu/c7), while others retained the epitheloid phenotype of the parental cells (MaTu/p). Among the latter was clone 8 (MaTu/c8) which displayed piling-up structures not seen in MaTu/p cells. Striking differences were detected on immunocytochemistry using the anti-cytokeratin 19 antibody A53-B/A2 which positively reacted with cells from MaTu/c7, but not with those of MaTu/c8 and MaTu/p. In contrast, the anti-blood group H 2 antibody A46-B/B10 positively stained cells from MaTu/c8 and MaTu/p, but not those of MaTu/c7. Assays for tumorigenicity in nude mice demonstrated that MaTu/c7 is far less tumorigenic than MaTu/p, while MaTu/c8 showed a pattern distinguishing it from MaTu/p cells. Cross-resistance assays showed decreasing drug resistance in the order MaTu/c8 > MaTu/c7 > MaTu/p. These data suggest drug-induced differentiation with reversion of the neoplastic phenotype in MaTu/c7 and some form of malignant progression in MaTu/c8. This model system may be helpful for understanding cancer development, especially its relation to differentiation.

Production and characterization of monoclonal antibodies against urea derivatives.

A panel of monoclonal antibodies was generated against the urea-based hapten N-(2-N-chloroacetylaminobenzyl)-N'-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Cross reactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.

Fast isolation of RNA to detect expression of tumor markers.

The expression status of several tumor-related proteins is of great interest in clinical examination and research. As a completion to conventional antibody staining, RT-PCR is often used today. Reliable isolation of RNA from a low number of cells is very often a critical stage of such an examination. We demonstrate here a simple and fast method to isolate RNA from only 10,000 cells and applied it to the detection of CEA, c-ERB-B2, and mdr-1 as often studied models for tumor markers.

The accessibility of thiophosphorylated groups in DNA fragments to the enzymatic activity of ligases and restriction endonuclease Bbs I.

The aim of this paper was to test the possibility to ligate and hydrolyse DNA sequences containing thiomodified ends and bonds. T4 DNA ligase was shown to ligate DNA fragments regardless of whether it contains phosphorylated or thiophosphorylated 5'-end. But the cleavage of an internally thiomodified phosphodiester bond was found to be totally inhibited when using the non-palindromic restrictase Bbs I. The special properties of this restriction endonuclease should allow the development of an oriented cloning strategy when combined with T4 ligase and a thiophosphorylation of DNA fragments.