Cryopreservation of marrow, purging and autologous bone marrow transplantation in childhood

Since 1984 bone marrow from 42 children with acute lymphoblastic leukaemia, non Hodgkin's lymphoma and neuroblastoma was cryopreserved. In 5 cases (c-ALL, NHL and B type) the marrow was purged by using a cocktail of three monoclonal antibodies (VIL A1, VIB C5, VIB-E3). Up to now 13 children (ALL/10, neuroblastoma/3) were autografted (one of them after purging) after supralethal chemoradiotherapy. Except one child with early death all patients had engraftment: a level of 1.0.10(9)/l leukocytes was reached at days 10-33 (median, 19); platelet level over 60.10(9)/l at days 32-60 (median, 41). 2 children died on treatment related complications, one on infection after full haematological restitution, 2 patients alive with relapse, 8/13 alive in CCR and well.     

No narcosis for bone marrow harvest in autologous bone marrow transplantation

A prospective study with mild general analgesia and sedation together with local anesthesia during bone marrow harvest was performed. Thirty-one patients underwent 33 bone marrow collections. Pretreatment consisted of 100 mg meperidine i.m. and 20 mg diazepam i.m. 1 h before start of procedure. Eight patients got additional meperidine and diazepam during the procedure, all patients got lidocaine 1% locally. A mean volume of 1.321 was obtained with 42.5 punctures. Twenty-two patients had no complications, 4 vomited, 4 had easily correctable hypotension of short duration, one got oxygen for cyanosis of short duration. Acceptance was good in 23 patients, in 6 reasonably well, in two bad. Only one patient experienced pain problems, due to suction. Anxiety was no major problem due to good information before the procedure and mild sedation. This form of anesthesia for bone marrow collection is a safe procedure, it is generally well accepted by the patient and it can be performed on an out-patient basis.     

Cryopreservation of bone marrow for the autologous transplantation in children

In preparing the autologous transplantation of children a method for cryoconservation of bone-marrow was developed by means of investigating the donor's bone-marrow. This method is adapted to our conditions, can easily be practised and is cell-preserving. Quantity and quality of the stored bone-marrow cells were evaluated concerning their proliferation capability by means of CFU-c assays. The highest recovery in CFU-c (78%) and cells (98%) was observed if isolated mononuclear cells with cryoprotective addition of 5% DMSO, 20% of human albumin, and 20% of serum were slowly frozen at a controllable rate, stored in liquid oxygen and thawed very quickly. According to the elaborated method the remission marrow was taken from 15 children affected with malignant diseases for autologous reinfusion. The data gained here confirm the experimental experiences.     

Fetal cord blood's potential for bone marrow transplantation

Approximately 18 years ago, the authors were able to produce an apparently successful bone marrow transplant by using umbilical cord blood. In view of the Chernobyl disaster and the subsequent problems of treatment with marrow transplantation, this study undertook to explore further the potential use of umbilical cord blood as a source of marrow cells. Specimens of umbilical cord blood were collected from 13 routine obstetrical deliveries. All specimens grew erythroid and granulocytic-monocytic colonies. The formation of these various hematopoietic colonies from umbilical cord blood was at least equivalent to bone marrow, and in some instances over 5 times more effective. There appeared to be a statistically significant correlation between the numbers of colony-forming units (CFU-E) and the male infants. The weight of the infants also showed a statistically significant correlation with the burst forming units, erythroid (BFU-E) and the granulocytic-monocytic colony (CFU-GM). The BFU-E also appeared to be greater in number when the time between collection and plating was shorter.     

Increased survival of normal cells during laser photodynamic therapy: implications for ex vivo autologous bone marrow purging

Laser light-induced, dye-mediated photolysis of leukemic cells was tested in an in vitro model for its efficacy in eliminating occult tumor cells for ex vivo autologous bone marrow purging. Merocyanine 540 (MC540) was mixed with acute promyelocytic leukemia (HL-60) cells in the presence of human albumin. This cell-dye mixture was irradiated with 514 nm argon laser light. Results show that in the presence of 0.1%, 0.25% and 0.5% albumin, laser light doses of 62.4 J/cm2, 93.6 J/cm2 and 109.2 J/cm2, respectively, were required for a 5 log reduction in the survival of leukemic cells. Under identical conditions, 80% to 84% of the normal bone marrow cells and 41% of the granulocyte-macrophage colony forming cells survived. The number of surviving stromal cells was reduced (1+) compared to the untreated control (4+). Mixing of irradiated bone marrow cells with equal number of HL-60 cells did not interfere with the killing of HL-60 cells treated with MC540 and laser light. The non-specific cytotoxicity of laser light alone was less than 6% for normal bone marrow cells. These results suggest that the concentration of human albumin plays an important role in laser light-induced phototoxicity. This laser light-induced selective photolysis of leukemic cells can be used in ex vivo purging of tumor cell-contaminated bone marrow grafts to achieve very high survival rates of normal bone marrow cells and granulocyte-macrophage colony forming cells.     

Therapeutic angiogenesis for severe ischemic heart diseases by autologous bone marrow cells transplantation

Conventional therapies for severe ischemic heart disease are limited in applicability. While several angiogenesis researches have shown novel efficacy, safety and feasibility for clinical use, recently we have started the clinical trial of a sole cell therapy using autologous bone marrow mononuclear cells transplantation targeted into ischemic hibernating myocardium. Here, we review the background of bone marrow cell research and introduce therapeutic angiogenesis for severe ischemic heart disease by autologous bone marrow cells transplantation.     

Therapeutic angiogenesis for severe ischemic heart diseases by autologous bone marrow cells transplantation

The effects of exogenous histone H1 on estrogen receptor status of human breast cancer MCF 7 cells were investigated in presence and absence of estrogen. Exogenous histone H1 was significantly cytotoxic in a dose- and time-dependent manner. Cell cycle analysis revealed a significant increase in the percentage of cell accumulation in G₀/G₁ phase. In histone H1-treated cells, a significant decrease in the estrogen receptor content and an increase in the dissociation constant (K_D) of ER was observed compared to control.     

Some theoretical and practical considerations for multivariate statistical cell classification useful in autologous stem cell transplantation and tumor cell purging.

BACKGROUND: As flow cytometric data becomes more complex, it becomes increasingly difficult to classify cells using conventional flow cytometry data techniques based on visual classification of the data by user-drawn regions. This paper shows some simple applications of multivariate statistical classification to classify flow cytometric data. METHODS: Discriminant Function Analysis (DFA) and Logistic Regression (LR) analysis techniques were evaluated with respect to their potential utility in the problem of detecting human breast cancer cells within normal bone marrow cells. Data sets having defined properties were employed to evaluate the potential utility of these statistical classification techniques whose performance was measured by ROC analysis. RESULTS: Two extreme but reasonable situations are presented: (1) data where the separation of cells was obvious by visual inspection and (2) data where major overlaps in the values of the individual FCM parameters made intuitive classification improbable. Both DFA and LR analysis were able to classify the cells of each type with acceptable accuracy and yield.CONCLUSIONS: The excellent empirical performance of both DFA and LR techniques, suggests that they offer promising approaches for classifying multiparameter FCM data using objective rules that may represent an improvement over commonly employed ad hoc approaches.     

Timing of transplantation of autologous bone marrow derived mesenchymal stem cells for treating myocardial infarction

It is still unclear whether the timing of intracoronary stem cell therapy affects the therapeutic response in patients with myocardial infarction.The natural course of healing the infarction and the presence of putative homing signals within the damaged myocardium appear to favor cell engraftment during the transendothelial passage in the early days after reperfusion.However,the adverse inflammatory environment,with its high oxidative stress,might be deleterious if cells are administered too early after reperfusion.Here we highlight several aspects of the timing of intracoronary stem cell therapy.Our results showed that transplantation of bone marrow mesenchymal stem cells at 2 4 weeks after myocardial infarction is more favorable for reduction of the scar area,inhibition of left ventricular remodeling,and recovery of heart function.Coronary injection of autologous bone marrow mesenchymal stem cells at 2 4 weeks after acute myocardial infarction is safe and does not increase the incidence of complications.     

Therapeutic evaluation of interleukin-1 for stimulation of hematopoiesis in primates after autologous bone marrow transplantation

A multiple dose IL-1 therapy was evaluated for its capability to stimulate hematopoiesis in normal primates and to restore hematopoiesis after autologous bone marrow transplantation. The administration of IL-1 to normal animals over a dose range of 0.5 to 10 ug/kg/d led to a 7-12 fold increase in peripheral blood neutrophil and monocyte counts after 24 hours. This increase in the mature peripheral blood myeloid cells was followed by changes in the myeloid composition of the bone marrow, where the percentage of myeloid elements increased along with a transient increase in myeloid progenitor cell activity. IL-1 treatment also led to an initial decrease in platelet counts of 10-30% during the first 3 days of treatment. However, a striking finding was a significant and long lasting stimulation of increased platelet production with platelet counts increasing to 77% of baseline 3 days after cessation of treatment and remaining elevated for the next 10 days. The therapeutic potential of the IL-1 regimen to restore hematopoiesis was further evaluated in an established autologous bone marrow transplantation model. In monkeys receiving IL-1 doses, 1.0 and 5.0 ug/kg/d, neutrophil counts recovered to >0.5 x 10e9/1 on day 16, one day earlier than control, but the recovery to baseline neutrophil counts occurred 5 days sooner than control. IL-1 therapy had its greatest effect on the restoration of platelet counts after transplantation, reaching >100 x 10e9/l by day 21, two weeks earlier than control. This work demonstrates that IL-1 therapy stimulates myelopoiesis but its most promising clinical application is the stimulation of platelet production.Views presented in this paper are those of the authors: no endorsement by the Department of the Navy or the Defense Nuclear Agency has been given or should be inferred. This work was supported by the Naval Medical Research and Development Command, Research Task No. 63706N MM095.003.1007 and the Defense Nuclear Agency Work Unit No. 132082.     

Development of immunity in human severe primary T cell deficiency following haploidentical bone marrow stem cell transplantation

Recent advances in the prevention of graft-vs-host disease (GVHD) have allowed the use of haploidentical bone marrow cells for correction of lethal genetic defects of the immune system. Sequential analyses of blood lymphocyte phenotypes and functions were done before and after transplantation of haploidentical marrow stem cells into 17 infants with severe primary T cell deficiencies. The marrow was depleted of post-thymic T cells and most other mature marrow cells by soy lectin agglutination and sheep erythrocyte rosetting. The studies were performed to define the time course and extent of appearance of immune function, and to identify factors leading to resistance to engraftment. No pretransplant immunosuppression was used. T cell function was detected between 34 and 287 days after transplantation, but a sharp rise usually occurred between 84 and 115 days, and normal function was reached between 113 and 210 days. Fifteen of the patients are alive from 6 to 41 mo post-transplantation, 12 have improved or have normal T lymphocyte function, and nine have proven T cell chimerism. Increased immunoglobulins of several isotypes have been noted in 11 patients and specific antibodies in seven patients, although B cell chimerism has been detected in only one patient. B cell function required 2 to 2.5 yr for normalization. No GVHD occurred in 14 patients, and the other three had only transient mild skin rashes. Two patients died of viral infections. Failure to engraft was correlated with some pre-transplant lymphocyte responses to mitogens and allogeneic cells (three cases), but not with the presence of pre-transplant natural killer cell function (five cases) nor with the presence of purine salvage pathway enzyme deficiencies (four cases). The latter, however, was associated with poor lymphoid function in two patients. These studies indicate that the thymic microenvironment of most infants with severe combined immunodeficiency disease is capable of differentiating donor stem cells to mature and functioning T lymphocytes which can cooperate with apparently normal host B cells for antibody production.     

Randomized study of recombinant interleukin-2 after autologous bone marrow transplantation for acute leukemia in first complete remission

Immunological control of acute leukemia may be achieved after allogeneic transplant. Despite promising preliminary results, the impact of immunotherapy with interleukin-2 (r-IL-2) on patients with acute leukemia (AL), in first complete remission (CR1) remains unclear. We conducted a prospective multicenter randomized trial to compare outcome in patients with AL in CR1, treated with autologous bone marrow transplantation (BMT) with or without postgraft r-IL-2. One hundred and thirty patients with AL in CR1 (myeloblastic (AML): N = 78; lymphoblastic (ALL): N = 52) were randomized at time of BMT to receive (N = 65) or not (N = 65) r-IL-2. r-IL-2 (RU 49637 from Roussel Uclaf) was started after hematological recovery, as a five cycle regimen (12 M IU/m2/day continuous infusion on day 1-5, 15-17, 29-31,43-45 and 57-59). The two groups were balanced for patient and transplant characteristics. Analysis was based on an intent to treat. Thirty-eight (59%) of the 65 patients randomized into the study group started r-IL-2 at a median of sixty-eight days (23-140) after transplant and received 77% (16-100) of the scheduled dosage. They received a median of 120 x 10(6) IU/m2 (25-156) over 10 (3-13) days during a total median period of 56 (3-78) days. With a median follow-up of 7 years (5.4-8.1 years), 79 patients relapsed (study group: 43 (66%); control group: 36 (55%): p = NS). Survival and leukemia-free survival estimates were 33% (23-45) versus 43% (22-52) and 29% (19-41) versus 36% (24-51) respectively for study and control groups (all p = NS). These results show that leukemic control after autologous BMT is not increased by r-IL-2 therapy. Further studies should investigate more appropriate r-IL-2 schedules and the possibilities offered by better antigen recognition and activated effector cells.     

自体骨髓单个核细胞移植治疗缺血性下肢血管病变患者254例

目的探讨自体骨髓单个核细胞移植在下肢缺血性疾病治疗中的应用并评价其疗效。方法回顾性总结分析解放军第四六三医院细胞治疗中心2003年11月至2008年8月住院的具有完整随访资料的下肢缺血性疾病患者254例,经动员后采集及分离自体骨髓,行下肢自体骨髓单个核细胞移植术。随访时间为1年。术后随访指标：3、6、12个月后复查皮温、深感觉、经皮氧分压和踝肱比,随访疼痛、冷感和跛行距离,观察溃疡和坏疽情况。采用F检验分析差异的统计学意义。结果完成随访254例患者。（1）患者的疼痛缓解率为61．8％（157例／254例）,冷感缓解率缓解率74．0％（188例／254例）,跛行好转率40．2％（102例／254例）,溃疡好转率59．0％（36例／61例）,坏疽截肢6例,脱落愈合5例,无变化8例,扩大7例;（2）患者下肢皮温由32．89℃±2．19℃上升至35．52℃±2．26℃（t=13．32,P=0．000）,深感觉缓解由（26．20±15．78）mV下降至（20．34±10．86）mV（t=4．901,P=0．000）,经皮氧分压由（26．46±18．49）mmHg上升至（34．14±14．99）mmHg（t=5．157,P=0．000）,踝肱比由0．62±0．36上升至0．84±0．24（t=8．104,P=0．000）。结论骨髓单个核细胞移植治疗下肢缺血性血管病有效,是一种简单的、有效的治疗下肢动脉缺血性疾病的方法。     

Preparing the patient for bone marrow transplantation: nursing care issues

The phases of bone marrow transplantation can be identified as the pre-transplant period, the immediate post-transplant period, and the late post-transplant period. The pre-transplant period is characterized by identification of the appropriate type of transplant to be done and, if necessary, finding an appropriate donor; entry of the patient into the transplant unit; administration of the preparative chemotherapy/irradiation regime; management of early toxicities; and pre-transplant supportive care. Nurses play an integral role during the entire transplant process. During the pre-transplant phase, nursing expertise is exemplified in the administration of chemotherapy, management of side effects, teaching of transplant procedures to patient and family, and supportive care. This paper reviews the patient care issues during the pre-transplant phase of bone marrow transplantation and identifies nursing management strategies.     

HLA compatibility for patients with thalassemia: implications for bone marrow transplantation

In order to evaluate the possibility of a patient with thalassemia finding an HLA-identical sibling donor, we performed an analysis of HLA antigens in families of thalassemic patients. The pattern distribution was not significantly different from the expected ratio 25:50:25. When the siblings were subdivided according to the age of the patients (under or over 5 years), the above pattern remained unmodified for both the age groups. The average size of the 129 thalassemic families was 2.4. Thus, taking into account that thalassemic patients have an average of 1.4 siblings and that the HLA genotype distribution is expected as such, HLA-matched siblings are available for 33% of the patients. Because an additional 8.5% of the patients were found HLA-phenotypically identical to one parent, the chance for a patient with thalassemia to find a suitable donor for bone marrow transplantation would be increased to 41.5%. Our preliminary data cannot be extrapolated to the overall Italian thalassemic population; however, it can be inferred that for a patient with thalassemia, the chance of finding a suitable donor for bone marrow transplantation is not reduced.     

Expression of vascular cell adhesion molecule-1 indicates the differentiation potential of human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are a mixture of cells differing in differentiation potential including mesenchymal stem cells, and so far no CD antigens were found to be predictable for the differentiation property of each BMSC. Here we attempted to isolate differentiation-associated CD antigens using 100 immortalized human BMSC (ihBMSC) clones. Among 13 CD antigens analyzed, only CD106/Vascular cell adhesion molecule-1 (VCAM-1) showed a clear correlation with the differentiation potential of each clone; CD106-positive ihBMSC clones were less osteogenic and more adipogenic than CD106-negative clones. This association was confirmed in primary BMSCs sorted by CD106, showing that the CD106-positive fraction contained less osteogenic and more adipogenic cells than the CD106-positive fraction. The evaluation of CD106 fraction of BMSC strains in early passages predicted clearly the osteogenic and adipogenic potential after in vitro induction of differentiation, indicating the usefulness of CD106 as a differentiation-predicting marker of BMSC.