STUDY OF MINIMAL RESIDUAL DISEASE BY MULTICOLOR FLOW CYTOMETRY IN MULTIPLE MYELOMA AFTER AUTOLOGOUS HEMATOPOIETIC STEM CELL TRANSPLANTATION

The frequency of achieving complete remission, as well as overall and disease-free survival, in multiple myeloma (MM) had increased due to introduction in MM treatment regimens of high-dose chemotherapy with following autologous hematopoietic stem cell transplantation (ASCT). However the number of relapses remains high, caused by persistence of residual tumor cells, i.e., the presence of minimal residual disease (MRD). One of the methods for MRD study is multicolor flow cytometry (MFC) where abnormal expression of surface antigens on myeloma plasma cells (PC) is determined. The aim of our study was to investigate the MRD by MFC before and after ASCT, the frequency of MRD-negative status achievement in complete remission (CR) patients at +100 days after ASCT and the frequency of abnormal expressed antigens on myeloma plasma cells. The study included40 MMpatients in CR at +100 days after ASCT and showed that the most common aberrations of PC were: abnormal absence of CD19 and/or CD27, decreased expression of CD38 and abnormal presence of CD56. The proportion of myeloma PCs from all bone marrow cells decreased significantly after ASCT: 20 % of patients acquired MRD-negative status, 10 % had a decrease in the number of abnormal PCs by one fold. Analysis of probability of immunochemical relapse showed that the worst prognosis was in patients with MRD-positive status before and after ASCT. During the MRD monitoring within 3-18 months, MRD-relapses were detected with the subsequent development of immunochemical relapse. The detection MRD in the dynamics is more informative than the study at only one step of therapy. It may help to select more adequate treatment for patient with multiple myeloma in each specific case

Sequence-Specific Binding of Recombinant Zbed4 to DNA: Insights into Zbed4 Participation in Gene Transcription and Its Association with Other Proteins

Zbed4, a member of the BED subclass of Zinc-finger proteins, is expressed in cone photoreceptors and glial Müller cells of human retina whereas it is only present in Müller cells of mouse retina. To characterize structural and functional properties of Zbed4, enough amounts of purified protein were needed. Thus, recombinant Zbed4 was expressed in E. coli and its refolding conditions optimized for the production of homogenous and functionally active protein. Zbed4’s secondary structure, determined by circular dichroism spectroscopy, showed that this protein contains 32% α-helices, 18% β-sheets, 20% turns and 30% unordered structures. CASTing was used to identify the target sites of Zbed4 in DNA. The majority of the DNA fragments obtained contained poly-Gs and some of them had, in addition, the core signature of GC boxes; a few clones had only GC-boxes. With electrophoretic mobility shift assays we demonstrated that Zbed4 binds both not only to DNA and but also to RNA oligonucleotides with very high affinity, interacting with poly-G tracts that have a minimum of 5 Gs; its binding to and GC-box consensus sequences. However, the latter binding depends on the GC-box flanking nucleotides. We also found that Zbed4 interacts in Y79 retinoblastoma cells with nuclear and cytoplasmic proteins Scaffold Attachment Factor B1 (SAFB1), estrogen receptor alpha (ERα), and cellular myosin 9 (MYH9), as shown with immunoprecipitation and mass spectrometry studies as well as gel overlay assays. In addition, immunostaining corroborated the co-localization of Zbed4 with these proteins. Most importantly, in vitro experiments using constructs containing promoters of genes directing expression of the luciferase gene, showed that Zbed4 transactivates the transcription of those promoters with poly-G tracts

Subpopulations of mobilized hematopoietic stem cells in patients with hematological malignances and donors: expression of CD38, HLA-DR and CD143

The study objective is to investigate the features of subpopulational composition of mobilized hematopoietic stem cells in peripheral blood (PB) and leukocyte concentrates (LC) in adult patients with oncohematological pathology and donors.Materials and methods. In 80 patients with hemoblastoses, expression of CD38, HLA-DR and CD143 (angiotensin-converting enzyme) was measured in PB and LC CD34+CD45low cells. The control group included 10 PB and 14 LC samples from healthy donors. Analysis of PB was performed prior to mobilization of hematopoietic stem cells (HSC) and on the day of leukapheresis prior to HSC collection. LC samples were examined at day 1 after HSC collection.Results. CD143 is expressed on CD34+CD45low cells both prior to mobilization and after it in all patients and donors, but CD34+CD45lowCD143+ cell counts varied depending on diagnosis and mobilization regimen. CD143+ expression on CD34+CD45low cells was significantly higher in patients who received combination of chemotherapy and granulocyte colony-stimulating factor compared to donors and patients with multiple myeloma who received only granulocyte colony-stimulating factor. Along with elevated CD34+CD45low cell count after hematopoiesis stimulation, CD34+CD45lowCD143+ cell counts also increased. It was shown that mobilized HSC almost completely lacks a fraction of early CD34+CD45low progenitor cells not expressing CD38, HLA-DR. Prior to hematopoiesis stimulation among CD34+CD45low cells, CD38+HLADR–cell fractions are prevalent, but after mobilization CD38–HLA-DR+ cell counts increased. No differences between CD34+CD45lowCD143+cell counts in patients with multiple myeloma depending on disease status, sex, age or number of chemotherapy courses prior to HSC mobilizationwere observed.Conclusion. Expression of angiotensin-converting enzyme on CD34+ cells in PB before and after HSC mobilization and in LC was observed. The cell counts varied depending on diagnosis and mobilization regimen