R Musilová

Masaryk University, Brünn, South Moravian, Czech Republic

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Publications (7)9.86 Total impact

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    ABSTRACT: Anticancer immunotherapy using dendritic cell-based vaccines is a strategy aimed at the induction and maintenance of immune responses against cancer cells. Clinical applications of dendritic cells (DCs) require stringent adherence to Good Manufacturing Practice (GMP) methods and rigorous standardization of DC-based vaccine preparation. Recently, closed systems for DC culture have been developed with a goal to minimize the risk of contamination. Here, we compare the yield, immunophenotype, and functional properties of DCs generated in Lifecell X-Fold culture bags and in plastic wells, both from adherence-selected monocytes, and review the current literature on closed systems for DC generation. We found that both the overall yield and the yield of CD83+ cells in cell culture bags was lower than in the standard culture method. No statistically significant differences were observed in the expression of DC immunophenotypic markers. The capability of DCs cultured in bags and in wells to induce the proliferation of allogeneic mononuclear cells were equivalent. The performance of DCs in mixed lymphocyte reaction correlated significantly (p = 0.005) with the CD83 expression but not with the CD80, CD86, HLA-DR, CD1a, and CD1c expression. We conclude that the immunophenotype and stimulatory properties of DCs cultured in closed cell culture bags are similar to those generated by conventional method using cell culture wells.
    Hematology 07/2004; 9(3):199-205. DOI:10.1080/10245330410001701486 · 1.25 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are professional antigen-presenting cells and are frequently used in current immunotherapy protocols. The administration of DCs loaded with tumor-associated proteins or peptides results in the induction of immune responses against different types of malignant cells. Methods for large-scale generation of DCs in a sufficient quality and quantity have permitted their use in clinical experiments. DC-based vaccines have already shown promise in follicular non-Hodgkin's lymphoma, and to some extent, in other hematological malignancies. Several strategies have been developed to boost their potency as a new and relatively non-toxic treatment modality. Our review focuses on clinical trials using DCs in the treatment of hematologic malignancies and on recent studies of the immunophenotype, development, and maturation of DCs may have an important impact on designing DC-based antitumor vaccines.
    Hematology 05/2003; 8(2):97-104. DOI:10.1080/1024533031000084204 · 1.25 Impact Factor
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    ABSTRACT: Dendritic cells (DCs) are antigen-presenting cells that play a critical role in the induction of cytotoxic T-lymphocytes. An optimal method for the generation of DC for clinical use remains to be established. The aim of our study was to find an optimal cytokine combination for DC generation from peripheral blood stem cells (PBSC) and peripheral blood mononuclear cells (PBMC) in serum-free conditions. Serial immunophenotyping enabled us to observe changes in DC content during the culture as well as the development of maturation and activation markers. As a source for DC culture, we used PBSC from patients with multiple myeloma after stem cell mobilization using cyclophosphamide and G-CSF, or PBMC from healthy donors without mobilization. The cells were cultured in a serum-free medium with different cytokine combinations including GM-CSF, TNF-alpha, Flt-3, CD40L, IFN-gamma, IL-1alpha, IL-6, PGE1, and IL-4. The cell cultures were evaluated by immunophenotyping. For PBMC, interleukin-12 assay was performed. For PBSC, the yield of DC as determined by CD83+ cell count ranged from 0. 6 x 10(5) to 30.1 x 10(4) (mean: 9.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated nucleated cells from apheresis. This yield corresponded to (0.3-19.1) x 10(5) (mean: 4.3 x 10(5)) per 1 x 10(6) of CD34+ cells in the apheresis products. For PBMC, the yield was (0.4-24.8) x 10(4) (mean: 2.4 x 10(4)) of DC generated per 1 x 10(6) of initially plated mononuclear cells from venous blood. The cultured cells expressed the mature immunophenotype. No significant differences in cell yield or immunophenotype were detected when comparing different cytokine combinations.
    Vaccine 03/2003; 21(9-10):877-82. DOI:10.1016/S0264-410X(02)00535-2 · 3.62 Impact Factor
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    ABSTRACT: 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.
    Neoplasma 02/2003; 50(5):345-9. · 1.87 Impact Factor
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    ABSTRACT: 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.
    Neoplasma 02/2002; 49(5):300-6. · 1.87 Impact Factor
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    ABSTRACT: Idiotype vaccination can be used as immunotherapy for B-cell malignancies, including multiple myeloma. Malignant plasma cells produce monoclonal immunoglobulin that can be detected in the serum and/or urine and is tumour-specific. To isolate the myeloma Id protein we used affinity chromatography on a protein G column. Id was eluted with 0.2M glycine-HCl. The protein was more than 90 % pure as determined by SDS-polyacrylamide gel electrophoresis. Id protein was conjugated with KLH in the presence of 0.1% glutaraldehyde. The conjugation product was administered in the preclinical experiment to test vaccination as a part of the clinical protocol under preparation.
    Transfuze a Hematologie Dnes 01/2002; 8(3):91-95.