Low antigen-dependent activity of T cells after repeated stimulation using dendritic cells and expansion with interleukin-2.

Both CD8+ and CD4+ T cells with specific activity against tumor antigens are needed for an efficient antitumor immune response. Activation and proliferation of T cells require cellular interactions including adhesion, recognition of peptides presented by MHC molecules to the T cells receptor, and costimulation. In a series of experiments we attempted to generate and expand specific T cells by repeated stimulation using antigen-loaded autologous dendritic cells (DCs). DCs were obtained from peripheral blood mononuclear cells (PBMC) in the presence of IL-4 and GM-CSF. TNF-a was added to induce maturation. A conjugate of myeloma idiotypic protein with keyhole limpet hemocyanin was used as antigen. Nonadherent peripheral blood mononuclear cells were cultured in the presence of Il-2 and IL-7. Autologous DCs were added to the lymphocyte cultures on days 3, 10, and 17. The lymphocytes were stimulated by high concentration of IL-2 between days 21 and 27. Lymphocytes harvested on day 27 proliferated in response to antigen-loaded DC but failed to do so if less than 0.3 x 10(6) DCs were added for stimulation during culture. However, no cytotoxic activity against autologous DCs was detected and IFN-g production in the T cell cultures was low at the end of culture. In conclusion, the generation and expansion of T cells using repeated stimulation by autologous DCs is feasible but defective cytotoxic response of these cells occurs, possibly as a consequence of repeated frequent exposure to antigen.

Detection of 13q abnormalities in multiple myeloma using immunomagnetically selected plasma cells.

Accurate prognostic evaluation of patients with multiple myeloma (MM) is required for their stratification for more adequate therapy. Chromosomal G-banding and interphase fluorescence in situ hybridization (FISH) on cell-nonspecific samples and on myeloma cells selected by magnetic-activated cell separation (MACS) were used to study 13 samples from 12 multiple myeloma (MM) patients. Bone marrow (BM) samples were analysed using three approaches. Standard mitotic samples were prepared and analysed after G-banding. Interphase FISH was performed to detect the 13q14 deletion in unselected BM cells. In parallel, myeloma cells were selected from the BM using the CD138-specific antibody. The high-purity myeloma cell suspension was then analysed by interphase FISH for the 13q14 deletion. Magnetic separation yielded enriched myeloma cell suspensions with the mean viability of 98.0% (range: 97.0%-99.0%), and the purity of 97.6% (range: 87.2%-99.2%) as detected morphologically, and 85.2% (range: 44.8%-98.4%) as detected by immunophenotyping for CD138+ cells. Interphase FISH revealed the 13q14.3 deletion in 5 of 13 (38.5%) of cell-nonspecific samples and in 9 of 13 (69.2%) of enriched myeloma cell suspensions. In conclusion, interphase FISH on immunomagnetically selected MM cells increases the detection of the 13q14 deletion in BM samples from the patients with MM.

[Molecular analysis of Wilson disease]. / Molekulární analýza Wilsonovy choroby.

BACKGROUND: Wilson disease is an autosomal recessive disorder, characterized by cooper accumulation and intoxication of the organism. Molecular basis of the disease represent mutations in the gene for the copper-transporting ATPase (ATP7B). METHODS AND RESULTS: The submitted paper deals with results of molecular-genetic examination in 130 unrelated families in which Wilson disease was diagnosed. By denaturing gradient gel electrophoresis (DGGE), the exons with abnormal sequences were detected. Followed by sequencing, 17 causal mutations and 9 silent polymorphism were found. Five novel mutations were detected. After analysis of 260 mutant alleles, 214 (82.3%) were identified. The most frequent mutation, H1069Q, occurred in our population with the frequency of 65.8%. Incidence of other mutations, however, did not exceed 5%. CONCLUSIONS: DNA analysis of the Wilson disease offers prompt and reliable results in affected families. It can help to identify asymptomatic and heterozygote siblings at genetic counselling.