Repetitive DNA polymorphisms in following chimerism after allogeneic bone marrow transplantation.

Information about the chimeric status of patients is of great importance in comparison of different conditioning and prophylactic regimens as well as for the post-bone marrow transplantation (BMT) therapies. In some cases, mixed chimerism (MC) can also be predictive of relapse. Analysis of the short tandem repeats (STR) loci by polymerase chain reaction (PCR) is a choice method for this purpose. In this study, we monitored 15 patients after BMT. Twelve of them underwent classical-conditioning regimen while the remaining three patients were subjected to non-myeloablative conditioning (minitransplantation). Evaluation of chimerism was performed using five STR and one variable number of tandem repeats (VNTR) locus. Four additional loci were PCR-amplified in cases of minitransplantation. Samples were analyzed by electrophoresis in an ALFexpress sequencer. MC was detected in seven cases of which it was predictive of relapse for two patients, who suffered from acute lymphocytic leukemia (ALL). The PCR-STR method proved to be a fast and relatively simple method, while the tested STR loci showed a high level of informativeness.

[MHC tetramers: tracking specific immunity]. / MHC-tetrameri: na tragu specificnoj imunosti.

In an adaptive immune response, antigen is recognized by two distinct sets of highly variable receptor molecules: (1) immunoglobulins, that serve as antigen receptors on B cells and (2) the antigen-specific receptors on T cells. T cells play important role in the control of infection and in the development of protective immunity. These cells can also mediate anti-tumor effects and, in case of autoimmune syndromes, contribute to the development and pathology of disease. The specificity of T cells is determined by T cell receptors (TCR). Understanding of the success of immune responses requires the direct measurement of antigen-specific T lymphocytes. Cell with major histocompatibility complex (MHC) class I molecules are able to present antigens to antigen-specific CD8+ cytotoxic T lymphocytes. MHC class I molecules present small peptides (epitopes) processed from intracellular antigens such as viruses and intracellular bacteria. MHC class I molecules in humans are designated as human leukocyte antigen (HLA) class I and divided into HLA-A, -B and -C. CD8+ T cells recognize MHC class I molecules and after activation produce proteins that destroy infected cells. MHC class II molecules receive their peptides mainly from extracellular and soluble antigens and present them to the CD4+ T helper cells. A recently described technique that can be used in flow cytometry enables us to quantify ex vivo antigen-specific T cells by binding of soluble tetramer MHC-peptide complexes attached to fluorochrome. Quantitative analyses of antigen-specific T cell populations provide important information on the natural course of immune responses. The interaction of T cell receptors on T lymphocytes with tetrameric MHC-peptide complexes mimics the situation on the cell surface, and allows for reliable binding. Tetramers consist of four biotinylated HLA-peptide epitope complexes bound to streptavidin conjugated with fluorescent dye. Tetramer technology has sensitivity of detection as little as 0.02% of total cytotoxic T cell pool or T helper cell pool (i.e. approximately 1 in 50.000 lymphocytes). The combination of this technology with intracellular cytokine staining methods opens up significantly better ways of studying these cells than previously possible, allowing immunologists to look at their life cycle (activation and proliferation), manner of death (aging and apoptosis) and effector function (cytotoxic potential and cytokine production). MHC tetramers class I have yielded useful insights into in vivo dynamic and function of antigen-specific CD8+ T cells in viral infections, parasitic infections, cancer, autoimmune disease and transplantation. This knowledge is of special interest for immunotherapy, diagnostic monitoring of T cell mediated immunity, and the development of new vaccination strategies. There is some possibility for cell therapy with antigen-specific CD8+ T cells for various diseases including cancer and viral infections. Targeted immunotherapy of selective deletion of auto--or alloreactive T cells with MHC tetramers may be important for the treatment of autoimmune disease, or to prevent the rejection of transplanted organs. The utility of this technique for the immunotherapy in vivo needs to be confirmed and modified in further research. Understanding how antigen-specific cells develop and function in different circumstances and pathologies will be the key to unravelling the secrets of cellular immune system.

Determination of polymorphism at eight STR loci in the Croatian population using automated detection.

Population study was carried out on the sample of 167 unrelated donors from the wider area of the Croatia's capital, Zagreb, using the short tandem reapet (STR) loci: TH01, VWFA31, FES/FPS, F13A01, D1S1656, D12S391, D18S535 and D22S683.