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We have evaluated the feasibility of large-scale isolation of CD133+ progenitors from healthy mobilized adult donors for potential clinical use in autologous and allogeneic transplantation. A total of 11 healthy volunteer adult donors were mobilized with G-CSF. CD133 + stem cells were isolated from a single leukapheresis using the Clinimacs method. The median percentage of CD133 before positive selection was 0.75% (range 0.39-2.03%). After selection, the median purity and recovery was 94% (range 85.2-98.0%) and 69% (range 44-100%), respectively. The median log10 T-cell depletion obtained by CD133+ positive selection was 4.2 (range 3.8-4.7). The CD133+ progenitors were highly enriched in colony-forming units (CFU) and transplantation into NOD/SCID mice resulted in a high engraftment rate. Transplantation of sorted CD133 +/CD34 + cells into NOD/SCID mice showed a higher engraftment compared to CD133-/CD34+ cells. Mobilized peripheral CD133+ stem cells can be purified in large scale for potential clinical use. The biological function of the cells is not impaired. The majority of the NOD/SCID repopulating cells are within the CD133 +/CD34 + subpopulation. Therefore, clinical studies using purified CD133 + stem cells can be envisoued to further clarify the role of CD133+ stem cells in hematopoietic reconstitution after transplantation. [ABSTRACT FROM AUTHOR]

Presents a study that investigated the feasibility and efficacy of large-scale T cell depletion from granulocyte colony-stimulating factor mobilized peripheral blood stem cells (PBSC). Importance of T cell depletion to graft engineering strategies; Sources of hematopoietic stem cells; Comparison between bone marrow and PBSC transplantation; Ways of preventing graft-versus-host disease.

Bone Marrow Transplantation (2002) 30, S11–S15. doi:10.1038/sj.bmt.1703744 [ABSTRACT FROM AUTHOR]

The purpose of this paper was to define the histologic distribution, clinical features, and treatment response of childhood non-Hodgkin lymphoma (NHL) in northeastern Brazil. We reviewed medical records and histopathologic studies of 98 children treated for NHL from 1980 to 1987 at a major pediatric cancer center in Recife, Brazil. Treatment outcome was evaluated in relation to tumor burden (stage and serum LDH) and type of therapy (LSA2L2 vs other multiagent chemotherapy). There was a striking predominance of the small noncleaved cell (Burkitt) subtype, which occurred in 92 of the 98 children and adolescents diagnosed with NHL. Subsequent analyses focused on these patients. The majority (n = 84) had advanced (stage III/IV) disease at diagnosis. The abdomen was the most common site of disease (84 cases); jaw involvement was rare (three cases). Five-year event-free survival (excluding treatment refusals) was significantly better for patients with limited vs advanced stage disease (75 +/- 14% vs 42 +/- 6%; P < 0.04). Elevated serum LDH (>500 U/l) was associated with a poorer outcome (P = 0.008). The type of chemotherapy did not affect EFS (P = 0.95). Only 39% of patients are long-term survivors, reflecting the high rate of septic deaths (25% of patients) and parental refusal/abandonment of therapy (10%). Epstein-Barr virus (EBV) was detected in tumor cells from eight of the 11 cases studied. In clinical presentation, these cases resemble sporadic Burkitt lymphoma, yet in their apparent responsiveness to LSA2L2 therapy and association with EBV, they do not. Childhood NHL in northeastern Brazil is predominantly of the Burkitt subtype, and is associated with clinical features that appear to distinguish it from the endemic and sporadic forms of this tumor. These cases may represent a third or intermediate subtype of Burkitt lymphoma. [ABSTRACT FROM AUTHOR]

Objectives: To generate non-haematopoietic tissues from mobilized haematopoietic CD133+ stem cells. Materials and methods: Mobilized peripheral blood CD133+ cells from adult healthy donors were used. In vitro ability of highly enriched CD133+ cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. Results: We found that a recently identified population of CD45+ adherent cells generated in vitro after culture of highly purified CD133+ cells for 3–5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. Conclusion: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies. [ABSTRACT FROM AUTHOR]

Background We sought to develop a method for the clinical large-scale depletion of αβ T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and γδ T lymphocytes. Methods Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-αβ T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS® device. Results The median T-cell depletion was 3.9 log (range 3.5-4.1 log). The recovery of the γδ and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%. Discussion This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of αβ-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r null) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of αβ T cells from mobilized peripheral blood in clinical trials. [ABSTRACT FROM AUTHOR]

Background The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. Methods T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. Results The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19×10 10 (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log 10 depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log 10 depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34 + stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2×10 8 (range 2-10×10 8 ). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. Discussion Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft. [ABSTRACT FROM AUTHOR]

Background Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. Methods Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3 + cells were first depleted from the apheresis products. CD56 + cells were then enriched from the CD3 + cell-depleted products. Results The median percentage of CD3 - CD56 + NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3 + CD56 - T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. Discussion We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses. [ABSTRACT FROM AUTHOR]