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Immune reconstitution was studied prospectively in 86 children who underwent allogeneic haematopoietic stem cell transplantation (HSCT). We analysed the risk of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) reactivation in correlation with the kinetics of immune recovery and in relation to other potential risk factors that may influence the reactivation of these viruses including: diagnosis, type of HSCT, source of stem cells, type of conditioning, or the occurrence of graft-versus-host disease (GvHD). The absolute number of lymphocyte subpopulations in peripheral blood was evaluated in seven timepoints following HSCT. Significantly lower values of both CD3+ and CD3+CD8+ lymphocytes on day +14 and significantly higher values of both these subsets on day +168 post-transplant in patients with CMV reactivation were observed. Significantly lower values of CD3+CD4+ subpopulation were noted in patients with CMV reactivation on day +28 post allo-HSCT. Significantly lower lymphocyte values in the group with EBV reactivation comparing with the group without EBV reactivation were confirmed only in the case of pan-B lymphocytes (CD19+) subpopulation on day +21, +28, and +84 post allo-HSCT. We identified the impact of CMV reactivation on occurrence of the intestinal acute GvHD, which occurred more frequently in the group with CMV reactivation compared with patients without reactivation. Higher incidence of chronic GvHD was also observed in patients with CMV reactivation compared to the group without reactivation. EBV reactivation occurred more frequently in patients receiving transplants from matched unrelated donors, in particular after peripheral blood stem cell transplantation and while implementing antithymocyte globulin as GvHD prophylaxis.

Hematopoietic chimerism was monitored in 18 patients with various diseases after gender-mismatched allogeneic stem cell transplantation (alloSCT). To detect host and donor cells, FISH analysis of sex chromosomes was applied. X and Y chromosomes were detected simultaneously in interphase nuclei by two-color probes. Chimerism was examined sequentially in post-transplant peripheral blood and bone marrow as well as in purified T cells. Patients with complete donor or decreasing host chimerism have not rejected or relapsed but experienced a high incidence of acute graft versus host disease (aGvHD). The clinical value of stable mixed chimerism detection remains uncertain. However, it appears to be associated with a lower risk of aGvHD. Three patients with an increase in host cells rejected their grafts. The immunotherapy was introduced to four other patients with increasing host chimerism. All of them responded, however, one relapsed in CNS despite the conversion to complete donor chimerism in both bone marrow and peripheral blood. We concluded that two-color FISH analysis of sex chromosomes was a valuable tool for chimerism monitoring and provided significant clinical data.

The BCL-2 protein plays an important role in controlling apoptosis. Disorders of this process can lead to the emergence and development of acute lymphoblastic leukemia (ALL) and can determine the resistance of leukemic cells to chemotherapy. The levels of BCL-2 mRNA were determined in 20 children with pre-B ALL using RT-polymerase chain reaction and the percentage of BCL-2+ cells in 51 patients using flow cytofluorometry. Similar levels of BCL-2 mRNA (P=0.18) with a higher percentage of cells BCL-2+ (P=0.04) were shown in the bone marrow of patients with pre-B ALL compared to normal peripheral blood mononuclear cells. We could not find any connection between the level of BCL-2 mRNA or the percentage of BCL-2+ cells and selected clinical features. A high percentage of BCL-2+ cells and high levels of BCL-2 mRNA did not affect the 5-year overall survival probability nor the 5-year relapse-free survival probability. These results may indicate a high activity of mechanisms promoting the development of the final form of the BCL-2 protein from mRNA in leukemic cells. A high BCL-2 level does not affect the clinical course or worsen the prognosis in children with ALL.

Apoptosis is a programmed active and selective mechanisms of cell death allowing for the removal of cells in the living organisms. It is initiated by a number of stimuli, which are subject to complicated regulating system. It is proved that apoptosis plays a significant role in immunological disorders in uremic patients. The aim of the study was to estimate activity of apoptosis in peripheral blood mononuclear cells of patients with chronic renal failure treated in our Department.70 patients (13 on HD, 14 on PD and 24 pre-dialysis uremic children) were enrolled in the study. The control group comprised 19 children. We evaluated activity of mononuclear cells apoptosis exposed to Annexin V-FITC, Fas Ligand and Bcl-2 proteins activity were investigated also.The results show that cell apoptosis was increased in pre-dialysis uremic patients (both phases early and late). The activity of Fas Ligand protein was similar in every group and activity of Bcl-2 protein was significant higher in HD and PD patients.The highest intensity of mononuclear cell apoptosis in pre-dialysis children was observed. The significantly enhanced activity of Bcl-2 protein in dialysis patients probably protected from apoptosis.

Dendritic cells (DC) are important antigen-presenting cells (APC) of immune system that induce and modulate primary and secondary immune responses; they also induce of central and peripheral tolerance T lymphocytes to self-antigens. They interact with T, B and NK cells and promote activation, differentiation and effectory functions of these cells. The total number of 91 patients: 53 males and 38 females, aged from 1 month to 18 years (median 9 years) treated in the Department of Children Bone Marrow Transplantation, Oncology and Hematology, Medical University of Wroclaw, Poland (ALL--39, AML--15, NHL--8, HD--10, LCAL--4, solid tumors--15 children) were examined. Peripheral blood was collected at time of diagnosis, during chemotherapy, at time of relapse or progression disease and analyzed with used flow cyto-metric analysis. The subpopulations of CD were examined with use of specific markers for distinct blood dendritic cell populations: BDCA1 BDCA2, BDCA3 (Miltenyi Biotec). Both of activated DC CD83+ HLA-DR+ and lymphocytes T CD3+CD26+ were stained of monoclonal antibodies CD83 (Pharmingen), CD11c, HLA-DR, CD3, CD26, CD19 (BD Bisciencies). The results of our study showed increased level of blood DC in ALL and AML patients at time of diagnosis, relapse or during progressive disease in comparison with control group of healthy children, as well as in other patients. Those yields explained positive correlation with value of activated lymphocytes T (p0.05).

Adoptive immunotherapy with interleukin-2 (IL-2) may control minimal residual disease (MRD) and prevent relapse after autologous hematopoietic cell transplantation (AHCT). The objective of this study was to determine the immunologic effects of intermediate doses of intravenous (i.v.) IL-2 after AHCT in children with poor-prognosis solid tumors. Eleven patients received a median five cycles consisting of escalating doses of IL-2 i.v. for 5 days after a median time interval of 94 days post-AHCT. The phenotype of lymphocyte subsets was investigated before and after each cycle, and parallel determination of natural killer (NK) cell activity was performed. Immunotherapy induced a significant increase in total lymphocyte count (TLC), T, NK, and, to some extent, B cells. Among NK cells, CD56+ bright cells expanded more than CD56+ dim cells. High expansion of CD56+ cells with CD94 inhibitory receptor was observed, whereas no difference was recorded in the number of CD3+ CD56+ and CD8+ CD57+ cells. NK activity stabilized after the first cycle of IL-2 and remained elevated during the study period. Cycles of IL-2 immunotherapy induced repeated significant expansion of T cells and NK cells, mostly of the immature CD56+ bright phenotype. Despite enhanced NK activity, relapses occurred frequently, which might have been due to increased CD94 activation and a poor response from the cytotoxic NK T cells and CD8+ CD57+ T cells.