Low-dose radiation (LDR) induces hematopoietic hormesis: LDR-induced mobilization of hematopoietic progenitor cells into peripheral blood circulation

OBJECTIVE: The aim of this study was to investigate the stimulating effect of low-dose radiation (LDR) on bone marrow hematopoietic progenitor cell (HPC) proliferation and peripheral blood mobilization. METHODS: Mice were exposed to 25- to 100-mGy x-rays. Bone marrow and peripheral blood HPCs (BFU-E, CFU-GM, and c-kit+ cells) were measured, and GM-CSF, G-CSF, and IL-3 protein and mRNA expression were detected using ELISA, slot blot hybridization, and Northern blot methods. To functionally evaluate LDR-stimulated and -mobilized HPCs, repopulation of peripheral blood cells in lethally irradiated recipients after transplantation of LDR-treated donor HPCs was examined by WBC counts, animal survival, and colony-forming units in the recipient spleens (CFUs-S). RESULTS: 75-mGy x-rays induced a maximal stimulation for bone marrow HPC proliferation (CFU-GM and BFU-E formation) 48 hours postirradiation, along with a significant increase in HPC mobilization into peripheral blood 48 to 72 hours postradiation, as shown by increases in CFU-GM formation and proportion of c-kit+ cells in the peripheral mononuclear cells. 75-mGy x-rays also maximally induced increases in G-CSF and GM-CSF mRNA expression in splenocytes and levels of serum GM-CSF. To define the critical role of these hematopoietic-stimulating factors in HPC peripheral mobilization, direct administration of G-CSF at a dose of 300 microg/kg/day or 150 microg/kg/day was applied and found to significantly stimulate GM-CFU formation and increase c-kit+ cells in the peripheral mononuclear cells. More importantly, 75-mGy x-rays plus 150 microg/kg/day G-CSF (LDR/150-G-CSF) produced a similar effect to that of 300 microg/kg/day G-CSF alone. Furthermore, the capability of LDR-mobilized donor HPCs to repopulate blood cells was confirmed in lethally irradiated recipient mice by counting peripheral WBC and CFUs-S. CONCLUSION: These results suggest that LDR induces hematopoietic hormesis, as demonstrated by HPC proliferation and peripheral mobilization, providing a potential approach to clinical application for HPC peripheral mobilization.     

Cytogenetic follow-up studies of recipients of T-cell depleted allogeneic bone marrow

Serial cytogenetic studies of bone marrow and blood cells were made in leukaemic patients who had received an allogeneic bone marrow graft from a donor of unlike sex. The donor marrow was treated with the monoclonal antibody Campath-1 before infusion. The persistence of a significant proportion of dividing recipient cells in marrow and blood was observed after grafting. These recipient cells showed evidence of radiation damage of both the stable and unstable types. More than one transient clone of chromosomally abnormal recipient cells was observed in three cases. The differences between the cytogenetic findings in patients receiving donor marrow from which T cells have been removed and those cases previously studied in our Leukaemia Unit who had received untreated donor marrow are discussed.     

Substitution of cyclophosphamide and busulfan by fludarabine, treosulfan and melphalan in a preparative regimen for children and adolescents with Shwachman-Diamond syndrome

Allogeneic hematopoietic stem cell transplantation (HSCT) is the only definitive treatment for severe bone marrow dysfunction and clonal disorders in patients diagnosed with Shwachman-Diamond syndrome (SDS). In an attempt to minimize regimen-related toxicity (RRT), we have initiated a fludarabine/treosulfan/melphalan-based pilot protocol avoiding the combination of busulfan and cyclophosphamide. Median age at transplantation was 9.6 years (range 1.5-17 years). All three patients received conditioning with fludarabine (30 mg/m2/day x 6), treosulfan (12 g/m2/day x 3) and melphalan (140 mg/m2/day x 1). CAMPATH-1H (0.1 mg/kg x 2) was added in two cases, while rabbit ATG (Genzyme; 3 x 2.5 mg/kg) was given to the cord blood recipient. One patient was transplanted with a non-manipulated marrow graft from an HLA-identical sibling, one with a marrow graft from a 10/10 matched unrelated donor, and one with a 9/10 matched unrelated umbilical cord blood (UCB) unit. Mean cell doses given were 3.6 x 10(8) nucleated cells/kg BW for the bone marrow recipients and 4.2 x 10(7) nucleated cells/kg BW for UCB recipient. Overall, two of three patients are alive and display 100% donor chimerism. Acute graft-versus-host disease grade II was seen in one patient, while no GVHD exceeding grade I occurred in the remaining two.     

Red cell salvage and reinfusion in pediatric bone marrow donors.

We evaluated the use of a semi-automated processing technique to salvage red blood cells from pediatric bone marrow donors to minimize the risk of severe anemia following bone marrow harvest and ABO incompatibility in the recipient. Sixty healthy, HLA-matched, pediatric donors of bone marrow hematopoietic cells with a median age 8.0 years (2-19) were studied. Thirteen of the donor-recipient pairs were ABO incompatible. There were 60 recipients with a median age of 8.6 years (2 months to 20.8 years). Bone marrow was harvested under general anesthesia, filtered in the operating room and then transferred to the stem cell laboratory for processing. Samples were obtained for cell count, CD34+ quantification, colony assay, viability, and bacteriologic cultures before and after processing. The cells were processed in a semi-automated closed system (Stericel, Terumo) by density gradient separation with Ficoll-Hypaque and then washed. Two aliquots were obtained: one containing the mononuclear cell layer to be infused to the recipient and the other the washed red cells to be infused to the donor. The median volume harvested was 608 +/- 40.42 ml (278-1409), while the final volume infused was 174 +/- 10.75 ml (30.2-380) P < 0.0001, representing a decrease of 72% of the volume infused. The nucleated cell count harvested was 1.6 x 10(10) +/- 0.1 (0.56-3.2), while the count infused was 6.9 x 10(9) +/- 0.1 (0.12-5.4) P < 0.0001. The median mononuclear cell count (MNC) per kg harvested was 0.67 x 10(8) +/- 0.05 (0.18-2.0) vs an infused cell number of 1.3 x 10(8) MNC/kg +/- 0.1 (0.6-33.6) P < 0. 0001. The CD34+ cells harvested were 2.8 x 10(6)/kg +/- 0.1 (0.25-10.2) vs an infused number of 6.0 x 10(6)/kg +/- 0.5 (0.84-31.0) P < 0.0001. The viability before and after processing was 99%. Red cell salvage performed in a semi-automated closed system is safe and reduces the risk of post-bone marrow harvest anemia in pediatric donors, decreases the volume infused into the donor and enriches the mononuclear and CD34+ cell population, without affecting hematopoietic reconstitution.     

Transplant of rhesus-positive bone marrow in a rhesus-negative woman having anti-rhesus D alloantibodies

A woman affected by acute myeloblastic leukemia was grafted with HLA A, B and D compatible rhesus-positive bone marrow from her brother. Before grafting, she had anti-D alloantibodies (1/512 IAT, 2.9 micrograms/ml). To prevent the destruction of donor red blood cells, four plasma exchanges and a conditioning regimen (total-body irradiation 800 rad, cyclophosphamide, methotrexate) were carried out to decrease anti-D from 2.9 to less than 0.02 micrograms/ml on day 0. The anti-D level was 0.8 micrograms/ml on day 12 and was decreased to 0.2 micrograms/ml by eight plasma exchanges until day 35. Anti-D antibodies were undetectable with Lalezari's technique on day 45. Engraftment was obtained on day 25 (3,000 leukocytes/mm3 and 50% erythroblasts in bone marrow). The patient died from aspergillosis and graft-versus-host disease on day 54. This observation shows that an engraftment of rhesus-positive bone marrow in a recipient with anti-D antibody is possible.     

Confirmation of bone marrow engraftment by detection of M/N blood group antigens on erythroblasts in erythroid bursts

In order to confirm erythroid engraftment, we examined the changes in blood group antigens expressed on erythroblasts in the haematopoietic colonies of a bone marrow transplant recipient. A 44-year-old female with acute myelogenous leukaemia underwent an allogeneic bone marrow transplantation from her sister donor. Prior to the transplantation, the blood groups of both recipient and donor were analysed, and their M/N groups found to differ, the former being MN and the latter N. Bone marrow mononuclear cells were obtained from the patient on day 21 after bone marrow transplantation and cultured in semi-solid medium. Erythroblasts were collected from the erythroid bursts that had formed, and were subjected to flow cytometry using monoclonal anti-M and anti-N. Anti-N reactive cells accounted for 99.2% of those in the erythroid bursts, while only 0.3% of these cells were anti-M reactive. MN type erythrocytes in the recipient's peripheral blood had been replaced by N type and only 2.0% of erythrocytes were anti-M reactive on the 70th day after BMT. These erythroblasts and erythrocytes were phenotypically N, and originated from the donor haemopoietic stem cells with the success of bone marrow engraftment. From our findings, it appears that the M/N blood group antigens were produced by the erythroid cells themselves.     

Bone marrow cell trafficking following intravenous administration

To address trafficking of transplanted marrow cells immediately after intravenous infusion, we examined the early fate of infused non-adherent, low-density donor bone marrow cells in a syngeneic mouse model. The presence of infused donor cells, marked with indium-111 oxine (111In), with the fluorescent dye PKH26, or by a detectable transgene marker, was evaluated at 3-48 h in a variety of tissues, including peripheral blood. All three cell-marking methods indicated a rapid (< 4 h) influx of cells into the bone marrow, liver, spleen, muscle and other tissues. Moreover, these tissues remained positive for the 48 h observation period. Interestingly, analysis of PKH26-positive cells in non-myeloablated animals demonstrated that approximately 17% of infused donor marrow cells localized to the marrow space within 15 h, whereas a smaller proportion of donor cells (approximately 1-2%) localized to the marrow in recipients preconditioned by irradiation. In an effort to enrich for cells that specifically home to the bone marrow, PKH26-labelled donor marrow cells were recovered from the first host and infused into a secondary recipient. Although this was a phenotypically undefined population of cells, no increase was observed in the relative fraction of PKH26-labelled cells returning or 'homing' to the marrow of the second recipient. Taken together, these data suggest both that marrow engraftment may be mediated by non-specific 'seeding' rather than a specific homing signal, and that efficient targeting of transplanted cells to the marrow is a complex multifaceted process.     

Cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells

Multipotent progenitor cells have the ability to differentiate into most somatic cell types, including cardiac myocytes. We sought to investigate cardiac chimerism after peripheral-blood and bone marrow stem cell transplantation. Between 10 and 17 highly polymorphic short tandem repeat (STR) markers were assayed in DNA obtained from donors' peripheral blood, recipients' peripheral blood before transplantation, and the recipient's heart in every patient. Gender and non-gender STR donor alleles were identified in the recipient heart in three patients. Using a highly sensitive PCR assay to determine donor and recipient genotypes, we confirmed the existence of cardiac chimerism in recipients of peripheral-blood and bone marrow stem cells.     

Successful donor cell engraftment in a recipient of bone marrow from a cadaveric donor

A 12-year-old male with acute lymphocytic leukemia received donor bone marrow from his histocompatible father whose marrow was harvested 40 minutes postmortem after he suffered a myocardial infarction. The marrow was stored in liquid nitrogen for 17 days prior to infusion into the recipient. Trypan blue viability was greater than 99% for the fresh marrow. Progenitor cell assays revealed that 20% of the CFU-MIX, 16% of the BFU-E, 10% of the CFU-E, and 17% of the CFU-GM were spared during the cryopreservation period. Posttransplantation, the recipient had a leukocyte count greater than 10(3)/microL by day 26. Southern blotting analysis documented the donor origin of the peripheral blood mononuclear cells and granulocytes isolated 46 days posttransplantation. Unfortunately, the patient died of complications relating to graft-v-host disease 67 days following transplantation. This case demonstrates the feasibility of cadaveric marrow as a source of donor cells and is the first reported case of documented leukocyte engraftment in a recipient of cadaveric marrow.     

Enhancement of in vitro erythropoiesis by peripheral blood mononuclear cells from allogeneic marrow recipients in the early post-transplant period

The effect of peripheral blood mononuclear cells (PBMCs) from bone marrow recipients on in vitro growth of erythroid burst-forming units (BFU-E) was studied. PBMCs were obtained from 5 allogeneic, 1 syngeneic and 1 autologous bone marrow recipient(s) at different intervals after transplantation. The number of BFU-E was significantly increased when donor bone marrow cells were co-cultured with PBMCs obtained from allogeneic marrow recipients in the early post-transplant period. No effect was observed using PBMCs obtained in the later post-transplant period, PBMCs from a syngeneic marrow recipient, or PBMCs from an autologous marrow recipient. The BFU-E enhancing activity was present among T cells and was abolished by treating them with OKT3 or OKT4 antibody and complement. These observations suggest that chimeric T lymphocytes, probably of the helper/inducer subset, from allogeneic marrow recipients in the early post-transplant period have a potent enhancing effect on in vitro erythropoiesis.     

Cytogenetic studies on recipients of allogeneic bone marrow using the sex chromosomes as markers of cellular origin

S ummary . In 45 patients whose donor was of unlike sex, the sex chromosomes were used as markers of the cellular origin of myeloid and lymphoid tissues after allogeneic bone marrow transplantation (BMT). Successful engraftment was characterized by the appearance of dividing donor cells in marrow within 2 weeks of grafting and in mitogen stimulated blood cultures by 3 weeks. Leukaemic relapse was identified in eight cases and was associated with different patterns of cellular origin of the myeloid and lymphoid tissues. At the time of relapse the marrow contained either a mixed population of normal donor and leukaemic recipient cells, or only recipient cells. Thus, in this series, leukaemic relapse was not found occurring in donor cells. The importance of defining the origin of cells in interphase as well as in metaphase was demonstrated. In all but one case, the dividing lymphoid population remained of donor origin during relapse.     

Hematopoietic engraftment following transplantation of bone marrow cells carrying a Philadelphia (Ph′)-like chromosome

A phenotypically normal donor for bone marrow transplantation was found to have a previously unreported karyotype, 46, XY, t(18q+; 22q-), resulting in a Ph′-like chromosome. Identification of the Ph′-like chromosome in cultures of skin fibroblasts, phytohemaglutinin-stimulated peripheral blood cells, and bone marrow cells from the marrow donor, but not in cell cultures from siblings or parents, indicated that it represented an acquired somatic mutation. Demonstration of the Ph′-like chromosome in the marrow graft recipient's blood and bone marrow cells after transplantation provided a unique and definitive marker of engraftment. Hematopoiesis appeared normal in both the donor and recipient after transplantation. This study indicates that a mutation creating a Ph′-like chromosome in hematopoietic cells need not produce hematologic abnormality. Presence of this translocation did not appear to interfere with normal hematopoietic or lymphoid differentiation and replication in the transplant setting.     

Preparation of red-blood-cell-depleted marrow for ABO-incompatible marrow transplantation by density-gradient separation using the IBM 2991 blood cell processor

Thirty patients who had major ABO blood group incompatibility with their HLA-matched donors underwent allogeneic marrow transplantation after removal of red blood cells (RBC) from donor marrow by Ficoll-Diatrazoate (F-D) separation using the IBM 2991 blood cell processor. We selected the IBM 2991 because we were interested in obtaining information about RBC-depleted mononuclear cells for monoclonal antibody and complement incubation of marrow. The median residual marrow RBC volume was 2.6 ml (1.2% of the original volume) and marrow infusion was well tolerated in every instance. The median doses of nucleated and mononuclear cells were 8.7 X 10(7)/kg and 2.2 X 10(7)/kg recipient weight, respectively, representing median marrow total nucleated and mononuclear cell losses of 63.4% and 52.9%, respectively. The median CFU-GM loss was 52.4%. Four patients died 13-21 days after marrow infusion and were unevaluable for engraftment. One patient failed to achieve engraftment and received a second transplant on day 36 from a second donor. One patient with myelofibrosis had poor engraftment and died on day 177 with low peripheral blood counts. For evaluable patients, no significant differences were observed in the rate of recovery of peripheral blood granulocyte or platelet counts between those receiving RBC-depleted marrow or ABO-matched unprocessed marrow. However, posttransplant red cell transfusion requirements were increased and transfusion independence delayed in patients receiving RBC-depleted marrow as compared to patients receiving unprocessed marrow. We concluded that red cell depletion using the IBM 2991 was effective in removing red cell, but resulted in significant and variable hematopoietic cell losses which may have contributed to graft failure in at least one patient. Such cell losses appear to be inherent in both manual and semiautomated methods for F-D cell separation.     

Use of minisatellite DNA probes for recognition and characterization of relapse after allogeneic bone marrow transplantation

Restriction fragment length polymorphisms can be used to distinguish blood and marrow cells from close relatives. We used two probes that recognize a series of dispersed and highly polymorphic tandem-repetitive minisatellite regions in the human genome that can be detected via a shared 10-15 base pair core sequence similar to the generalized recombination sequence (chi) of E. coli. We have studied the resulting individual-specific DNA fingerprints in 15 patients before and after allogeneic bone marrow transplantation performed for chronic myeloid leukaemia and in two patients transplanted for acute leukaemia. Early engraftment could be demonstrated at 3 weeks post-transplant based on the recognition of cells of donor origin. One patient who failed to engraft had only recipient type marrow cells 3 months post-transplant. Nine patients who relapsed after transplantation had only cells of recipient origin. In one patient who relapsed after transplantation with T-cell depleted donor marrow, fractionation studies showed that his T-cells at relapse were of recipient origin. We conclude that these minisatellite probes are valuable for characterizing the origin of different cell populations after marrow transplantation and could be useful for characterizing relapse when donor and recipient are of the same sex.     

Origin of bone marrow stromal mechanocytes in radiochimeras and heterotopic transplants.

Strain histocompatibility antigens (detected by indirect immunofluorescence reaction) and sex chromosomes were used as markers of donor and recipient cells in monolayer cultures of bone marrow from radiochimeras and from heterotopic transplants. In contrast to macrophages and hemopoietic cells all bone marrow fibroblasts precursors in radiochimeras were of recipient origin; in heterotopic transplants they were of donor origin. These data point to the decisive role of stromal mechanocytes, and not macrophages or hemopoietic cells, in creating the bone marrow hematopoietic microenvironment.     

Allogeneic bone marrow transplantation-mediated transfer of specific immunity against Toxocara canis associated with excessive IgE

A girl with myelodysplastic syndrome (RAEB-T) received HLA-identical bone marrow from her younger brother after myeloablative treatment with busulfan and cyclophosphamide. After bone marrow transplantation, fever, exanthema, pruritus, and a pulmonary infiltrate were treated symptomatically. Bacterial cultures remained negative. Leukocyte engraftment began on day 10, and all blood cell populations proved to be of donor origin on FISH analysis. Increasing IgE levels (21 000 U/ml) on day 14 after BMT, positive RAST, specific IgG-antibodies, and missing Toxocara (T.) canis antigens in the recipient indicated donor-derived seroconversion. Before BMT, the recipient had been negative for T. canis in routine parasitological screening, and the donor proved to be positive for T. canis antibody by ELISA. This report suggests that the transfer of IgE immunity in the absence of detectable antigens may be responsible for IgE-mediated symptoms consistent with toxocara infection and confirms the need for parasite screening in donor medical examinations.     

Lethal graft-versus-host disease: modification with allogeneic cultured donor cells

The use of the bone marrow culture technique was studied as a means to prepare donor marrow for bone marrow transplantation to avoid lethal graft-versus-host disease (GVHD). Preliminary experiments demonstrated the rapid loss of theta-positive cells in such cultures, so that theta- positive cells were not detected after 6 days. Initial experiments in C3H/HeJ (H-2k, Hbbd) recipients prepared with 900 rad demonstrated improved survival when 3-day cultured C57BL/6 (H-2b, Hbbs) donor cells were used in place of hind limb marrow for transplantation. However, hemoglobin typing of recipient animals revealed only short-term donor engraftment, with competitive repopulation of recipient marrow occurring. Subsequent experiments were done in 1,200-rad prepared recipients, with long-term donor engraftment demonstrated. The majority of 1,200-rad prepared animals receiving cultured allogeneic cells died of GVHD, but animals receiving 28-day cultured cells had an improved 90- day survival and a delay in GVHD development over animals receiving hind limb marrow or marrow from shorter times in culture. In addition, animals receiving anti-theta-treated, 3-day nonadherent cells had an improved survival (44%) over animals receiving anti-theta-treated hind limb marrow (20%). These experiments demonstrate modest benefit for the use of cultured cells in bone marrow transplantation across major H-2 histocompatibility complex differences.     

Donor-recipient incompatibility at CD31-codon 563 is a major risk factor for acute graft-versus-host disease after allogeneic bone marrow transplantation from a human leucocyte antigen-matched donor

Disparities at minor histocompatibility antigens (mHA) are thought to be responsible for acute graft-versus-host disease (aGVHD) in patients receiving bone marrow transplantation (BMT) from a human leucocyte antigen (HLA)-matched donor. Although some mHA have been identified in humans, their role in aGVHD has not. Patients (n = 150) receiving a BMT from an HLA-matched donor were investigated for a correlation between aGVHD and donor/recipient incompatibility for seven polymorphisms previously proposed for mHA (HA-1, H-Y, CD31-codon 125, CD31-codon 563, HPA-1, HPA-3 and HPA-5). Only mismatch at CD31-codon 563 predicted grade II-IV aGVHD. The risk derived from CD31-codon 563 mismatch was the same as that derived from the use of bone marrow from an unrelated donor. We suggest that donor/recipient compatibility at CD31-codon 563 should be added to HLA-typing for donor selection and/or adjustment of aGVHD prophylaxis.     

异体骨髓移植供者骨髓采集方法和安全性探讨

目的：评价异体骨髓移植供者骨髓采集方法的安全性。方法：在连续续硬外或全身麻醉下对408例供者进行骨髓采集术，术中回输预先采集的供者自体血。结果：术中抽取骨髓血的中位数为1108ml，输血输液量的中位数为4000ml，骨髓采集术中术后不良反应主要有低血压，骨痛和发热，发生率分别为2．5％，100％及26．5％，全部供者均未见严重并发症，结论：连续硬膜外麻醉下进行骨髓采集术安全可靠，并发症发生率较少，术中采用预贮式自体输血有很大优势。     

Donor cell leukemia after allogeneic peripheral blood stem cell transplantation: a case report and literature review

A 49-year-old male developed recurrent acute myeloid leukemia 27 months after allogeneic peripheral blood stem cell transplantation (PBSCT) from an HLA-identical brother. The immunophenotype of the blastic cell population was incompatible with that of the pre-transplant blast cells; a mutation in C/EBPA gene was found in the pre-transplant blast cells that was not present in the post-transplant blast cells, and short tandem repeat analysis of marrow cells, which included 71% blasts, showed complete donor chimera. Thus, this recipient developed donor cell leukemia (DCL). The donor was healthy when DCL developed in the recipient as well as before donation of the peripheral blood stem cells. Only five cases of DCL after PBSCT have been reported in the literature. As a mechanism for the development of DCL, a vigorous proliferative demand on the donor cells, which often correlates with a higher likelihood of replication error or mutation, has been proposed. Peripheral blood stem cells might have an advantage in that they are associated with a low incidence of DCL development because PBSCT recipients receive a higher total cell dose than recipients of bone marrow or cord blood cells.