Growth retardation and depigmentation of hair after high-dose busulfan and congenic hematopoietic cell transplantation in mice.

Busulfan, a myeloablative but non-immunosuppressive alkylating agent, is used extensively in clinical bone marrow transplantation (BMT), but the effects of high-dose administration have not been previously evaluated in preclinical BMT settings with young murine recipients. We compared the survival and growth of C57BL/6 mice given graded single doses of busulfan (10-100 mg/kg) or total body irradiation (TBI; 900 cGy) at age 9 days and hematopoietic cell transplantation (HCT; transplantation of congenic bone marrow and spleen cells) 24 h later. The 30-day survival was 87-100% in mice transplanted after 10-40 mg/kg busulfan and 79% after TBI, but fell to 54% and 33%, respectively, after 80 mg/kg and 100 mg/kg busulfan, suggesting that this latter dosage range represents the LD50 for single-dose busulfan in young C57BL/6 mice given stem cell rescue. The weights of 10-week-old mice given HCT after lower doses of busulfan ranged from 87% of control at 10 mg/kg to 64-69% of control in mice conditioned with 35-65 mg/kg busulfan or TBI. Impairment of weight gain was most striking (approximately 50% of control) in mice transplanted after 80-100 mg/kg busulfan. Despite retardation of somatic growth, the brain weights of busulfan-conditioned mice remained at least 90% of control, and there were no obvious neuropathological alterations in the brains of these animals. All mice treated with at least 20 mg/kg busulfan or TBI lost hair by 3-4 weeks after transplant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low-dose parenteral busulfan provides an extended window for the infusion of hematopoietic stem cells in murine hosts

OBJECTIVE: Myeloablative total body irradiation (TBI) in the setting of autologous transplantation of genetically modified hematopoietic stem cells (HSC) is associated with substantial toxicity. Nonmyeloablative doses of TBI are less toxic, but result in low-level engraftment of genetically modified HSCs. As an alternative to TBI, escalating doses of parenteral busulfan were tested for their hematologic toxicity, their ability to promote donor leukocyte engraftment, and the time window for such engraftment. MATERIALS AND METHODS: Hematologic toxicity of busulfan was assessed in C57BL6 mice after single nonmyeloablative doses of intraperitoneal busulfan ranging from 1 to 40 mg/kg by serial complete blood counts monitored up to 40 days. The level of donor engraftment attainable after nonmyeloablative busulfan was determined by infusion of 20 million congenic murine bone marrow nucleated cells (BMNC) following 5 to 40 mg/kg of busulfan. To determine the effects of delayed HSC infusions, BMNCs were infused 1, 10, 15, and 20 days after a single dose of 10 mg/kg of busulfan. RESULTS: Busulfan doses from 1 to 40 mg/kg produced hematologic toxicity that was most pronounced in the 2nd to 3rd week. In transplantation experiments, dose-dependent donor leukocyte engraftment was attained with levels >70% after only 20 mg/kg of busulfan. Similar levels of engraftment were achieved even when infusion of BMNCs was delayed up to 20 days after busulfan injection. CONCLUSION: Nonmyeloablative parenteral busulfan produced transient myelosuppressive effects, clinically relevant levels of engraftment, and an extended time window for HSC infusion in murine hosts.     

Myelosuppressive conditioning is required to achieve engraftment of pluripotent stem cells contained in moderate doses of syngeneic bone marrow

We have investigated the requirement for whole body irradiation (WBI) to achieve engraftment of syngeneic pluripotent hematopoietic stem cells (HSCs). Recipient B6 (H-2b; Ly-5.2) mice received various doses of WBI (0 to 3.0 Gy) and were reconstituted with 1.5 x 10(7) T-cell- depleted (TCD) bone marrow cells (BMCs) from congenic Ly-5.1 donors. Using anti-Ly-5.1 and anti-Ly-5.2 monoclonal antibodies and flow cytometry, the origins of lymphoid and myeloid cells reconstituting the animals were observed over time. Chimerism was at least initially detectable in all groups. However, between 1.5 and 3 Gy WBI was the minimum irradiation dose required to permit induction of long-term (at least 30 weeks), multilineage mixed chimerism in 100% of recipient mice. In these mice, stable reconstitution with approximately 70% to 90% donor-type lymphocytes, granulocytes, and monocytes was observed, suggesting that pluripotent HSC engraftment was achieved. About 50% of animals conditioned with 1.5 Gy WBI showed evidence for donor pluripotent HSC engraftment. Although low levels of chimerism were detected in untreated and 0.5-Gy-irradiated recipients in the early post-BM transplantation (BMT) period, donor cells disappeared completely by 12 to 20 weeks post-BMT. BM colony assays and adoptive transfers into secondary lethally irradiated recipients confirmed the absence of donor progenitors and HSCs, respectively, in the marrow of animals originally conditioned with only 0.5 Gy WBI. These results suggest that syngeneic pluripotent HSCs cannot readily engraft unless host HSCs sustain a significant level of injury, as is induced by 1.5 to 3.0 Gy WBI. We also attempted to determine the duration of the permissive period for syngeneic marrow engraftment in animals conditioned with 3 Gy WBI. Stable multilineage chimerism was uniformly established in 3-Gy-irradiated Ly-5.2 mice only when Ly-5.1 BMC were injected within 7 days of irradiation, suggesting that repair of damaged host stem cells or loss of factors stimulating engraftment may prevent syngeneic marrow engraftment after day 7.     

Complete substitution of cyclophosphamide by fludarabine and ATG in a busulfan-based preparative regimen for children and adolescents with beta-thalassemia

Children and adolescents with homozygous beta-thalassemia can be cured by transplantation of normal stem cells after eradication of the thalassemic hematopoietic system. In an attempt to achieve durable engraftment and to minimize regimen-related toxicity (RRT), we have initiated a fludarabine-based pilot protocol not containing cyclophosphamide. Between 1999 and 2004, five children with beta-thalassemia major were enrolled. Median age at transplantation was 11.5 years (range 4-14 years). Three patients received conditioning with fludarabine (30 mg/m2/day x 6), oral busulfan (3.5 mg/kg/day x 4), and ATG rabbit Fresenius (10 mg/kg/day x 4). Two children received intravenous busulfan instead of oral busulfan at a dose of 2 x 1.4 mg/kg/day x 4 days. All children were transplanted with a fresh bone marrow graft from an HLA-identical sibling. Mean cell doses given were 3.7 x 10(8) nucleated cells/kg BW (range 2.4-6.2 x 10(8)/kg). Overall, 5/5 patients achieved donor engraftment and are alive and well. No GVHD exceeding grade I was observed, and 2/5 children maintained donor chimerism at 100%. One patient maintains mixed hematopoietic donor chimerism being between 94 and 97% nearly 5 years after transplant.     

Common lymphoid progenitors rapidly engraft and protect against lethal murine cytomegalovirus infection after hematopoietic stem cell transplantation

Lymphoid deficiency after allogeneic hematopoietic cell transplantation (HCT) results in increased susceptibility to infection; however, transplantation of mature lymphocytes frequently results in a serious complication known as graft-versus-host disease (GVHD). Here we demonstrate in mice that both congenic as well as allogeneic transplantation of low numbers of highly purified common lymphoid progenitors (CLPs)-a rare population of lymphoid-lineage-committed bone marrow cells-accelerates immune reconstitution after lethal irradiation and rescue with hematopoietic stem cells (HSCs). After congenic transplantation, 3 x 10(3) CLPs protected against murine cytomegalovirus (MCMV) infection at a level roughly equivalent to 107 unfractionated lymph node cells. In the allogeneic model of matched unrelated donor HSC transplantation, cotransplantation of 3 x 10(3) CLPs protected thymus-bearing as well as thymectomized hosts from MCMV infection and attenuated disease severity. Immunohistochemistry in combination with antibody depletion of T and natural killer (NK) cells confirmed that CLP-derived as well as residual host lymphocytes contribute to antiviral protection. Importantly, transplantation of allogeneic CLPs provided a durable antiviral immunity without inducing GVHD. These data support the potential for composing grafts with committed progenitors to reduce susceptibility to viral infection following HCT.     

Marrow transplantation for thalassemia

Ten patients with homozygous beta thalassemia, aged from 1 year 7 months to 13 years, underwent bone marrow transplantation from siblings or parents. The first case received 12 mg/kg busulfan, 120 mg/kg cyclophosphamide, and 300 cGy total body irradiation before transplantation; he survives, with a graft, more than 680 days after transplantation. The other nine patients received 16 mg/kg busulfan and 200 mg/kg cyclophosphamide. Two died of transplantation-related complications on days 30 and 55. Seven survive 170 to 580 days after transplantation. Three of the seven surviving patients have durable engraftment (greater than 230 to greater than 550 days) while four patients have autologous hematopoietic recovery. Four of five patients who had less than 50 prior transfusions achieved engraftment. Only one of five patients who had more than 50 prior transfusions achieved engraftment (P less than 0.05). The six-month actuarial survival was 80%; six-month actuarial disease-free survival was 40%. These data demonstrate that bone marrow transplantation may cure thalassemia, but engraftment may be jeopardized among patients who have been heavily transfused or have received marrow from a donor who is not HLA-identical.     

The repopulation potential of fetal liver hematopoietic stem cells in mice exceeds that of their liver adult bone marrow counterparts

Varying, limiting numbers of unseparated or purified cells (Ly-5.1), either from 14.5-day-old fetal liver (FL) or from adult bone marrow (BM) were coinjected with 10(5) unseparated BM cells (Ly-5.2) into lethally irradiated adult C57B1/6 recipients (Ly-5.2). The kinetics of donor cell repopulation of the lymphoid and myeloid compartments by Ly- 5.1+ donor hematopoietic stem cells (ie, competitive repopulation units [CRU]) were monitored at various time points after the transplantation by Ly-5 analysis of the peripheral white blood cells (WBC). Recipients that had received on average less than 2 adult BM or FL CRU did not show a significant difference in the level of donor-reconstitution when analyzed 4 weeks after the transplantation, However, at 8 and 16 weeks, the FL recipients showed a significantly higher percentage of donor- derived nucleated peripheral blood cells than did the recipients of adult BM cells. Analysis of individual mice showed that approximately 80% of the recipients of FL CRU showed an increase in mature WBC output between 4 and 8 weeks after transplantation, whereas this occurred in less than 40% in the recipients of adult BM cells. In addition to this effect on mature cell output, the cellularity of the reconstituted BM was significantly higher in recipients of FL CRU than in recipients of adult BM CRU, even at 7 to 9 months after transplantation, which is consistent with an increased clonal expansion of FL CRU. When marrow cells from primary recipients of FL CRU were injected into secondary recipients, a significantly higher percentage of these mice showed donor-reconstitution of their lymphoid and myeloid compartments (P < .01) and to a greater extent (P < .008) as compared with mice that had received marrow cells from primary recipients of similar numbers of adult BM CRU. Taken together, these results show that individual FL CRU exhibit a greater proliferative activity in vivo than similar cells from adult BM that is accompanied by a greater production of daughter CRU.     

In vivo stem cell function of interleukin-3-induced blast cells

The treatment of mice with high doses of 5-fluorouracil (5-FU) results in an enrichment of primitive hematopoietic progenitors. Using this procedure, we obtained a new class of murine hematopoietic colonies that had very high secondary plating efficiencies in vitro and could differentiate into not only myeloid cells but also into lymphoid lineage cells. The phenotypes of interleukin-3 (IL-3) induced blast colony cells were Thy-1-positive and lineage-marker-negative. We examined whether these blast colony cells contained primitive hematopoietic stem cells in vivo and could reconstitute hematopoietic tissues in lethally irradiated mice. Blast colony cells could generate macroscopic visible spleen colonies on days 8 and 12, and 5 x 10(3) blast cells were sufficient to protect them from lethally irradiation. It was shown that 6 or 8 weeks after transplantation of 5 x 10(3) blast cells, donor male cells were detected in the spleen and thymus of the female recipients but not in the bone marrow by Southern blot analysis using Y-encoded DNA probe. After 10 weeks, bone marrow cells were partially repopulated from donor cells. In a congenic mouse system, donor-derived cells (Ly5.2) were detected in the thymus and spleen 6 weeks after transplantation. Fluorescence-activated cell sorter analyses showed that B cells and macrophages developed from donor cells in the spleen. In the thymus, donor-derived cells were found in CD4, CD8 double-positive, single-positive, and double-negative populations. Reconstitution of bone marrow was delayed and myeloid and lymphoid cells were detected 10 weeks after transplantation. These results indicate that IL-3-induced blast cells contain the primitive hematopoietic stem cells capable of reconstituting hematopoietic organs in lethally irradiated mice.     

Comparison of conditioning regimens for alveolar macrophage reconstitution and innate immune function post bone marrow transplant

The authors compared efficiency of alveolar macrophage (AM) reconstitution from donor bone marrow post transplant following 4 chemotherapy conditioning regimens and 2 total body irradiation (TBI) regimens. TBI regimens are more effective in inducing AM reconstitution from donor marrow. However, mice conditioned with 13 Gy split-dose TBI or a dual-chemotherapy regimen (25 mg/kg busulfan x 4 days plus cyclophosphamide 100 mg/kg x 2 days) both demonstrate significant AM repopulation from donor marrow. Additionally, both protocols resulted in impaired pulmonary host defense associated with overproduction of prostaglandin E(2) and I(2) by AMs and impaired AM phagocytosis post bone marrow transplant.     

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.     

Reconstitution of normal neutrophil function in chronic granulomatous disease by bone marrow transplantation

Allogeneic bone marrow transplantation was carried out on an 11-year-old boy with chronic granulomatous disease and severe chronic pulmonary insufficiency of restrictive type. After preparative regimen with busulfan (13 mg/kg) and cyclophosphamide (200 mg/kg), the patient received marrow cells from his HLA-identical and MLC-nonreactive sister. Durable sustained engraftment of donor hematopoietic and lymphoid populations occurred, as documented by analysis of genetic markers and complete reversal of the neutrophil function defect. No episode of infection occurred in the post-transplant course and, currently, 40 months after transplantation the patient is in excellent health and growing normally and showing an increasing improvement of his respiratory capacity. The successful outcome in this patient demonstrates that marrow transplantation is at present the only curative approach for this congenital disorder of neutrophil function.     

High-dose cytostatic agents in allogeneic bone marrow transplantation: comparison of the engraftment promoting potential

We investigated the potential of various cytostatic agents for preventing graft rejection following allogeneic bone marrow transplantation. LEW rats received a lethal dose (35 mg/kg) of busulfan followed by injection of 1 x 10(8) F1(CAP x LEW) marrow cells, which are unable to induce a graft-versus-host reaction in LEW recipients. Rejection of the marrow graft was assessed by monitoring haematocrit and granulocyte counts. Due to its weak immunosuppressive activity, busulfan by itself is unable to allow engraftment of allogeneic marrow. Therefore, agents administered in addition to busulfan can be tested for their capacity to prevent marrow graft rejection. 120 mg/kg of cyclophosphamide, 20 mg/kg of ACNU and 240 mg/kg of ifosfamide completely prevented rejection of the allogeneic marrow. Maximum doses of BCNU applicable in conjunction with busulfan reduced the rejection rate to 12% (30 mg/kg) and 17% (40 mg/kg), whereas the antitumour agents thiotepa, melphalan, and carboplatin exhibited a very limited engraftment-promoting potential in this experimental setting. Thus, BCNU (carmustine), ACNU (nimustine), and ifosfamide might be suitable candidates for conditioning of allogeneic bone marrow graft recipients.     

Unrelated partially matched peripheral blood stem cell transplantation with highly purified CD34+ cells in a child with Wiskott-Aldrich syndrome

Stem cell transplantation is the only curative approach to the treatment of Wiskott-Aldrich syndrome. However, using grafts from partially matched unrelated donors is associated with increased risk of graft rejection and graft-versus-host disease. In an attempt to prevent these problems, a 6-year-old boy with Wiskott-Aldrich syndrome lacking a suitable family donor, was transplanted with large numbers of unrelated highly purified CD34+ peripheral blood stem cells mismatched at one C locus. Conditioning consisted of busulfan 16 mg/kg body weight, cyclophosphamide 200 mg/kg body weight and antithymocyte globulin 20 mg/kg body weight x 3 days. The boy had a rapid hematopoietic engraftment and showed immunologic reconstitution by day +92. Although he did not receive prophylactic immunosuppression he did not develop any graft-versus-host disease and is well and alive up to now, 25 months after transplantation.     

Reconstitution of normal megakaryocytopoiesis and immunologic functions in Wiskott-Aldrich syndrome by marrow transplantation following myeloablation and immunosuppression with busulfan and cyclophosphamide

Three patients with Wiskott-Aldrich syndrome received transplants of marrow from their HLA-A, B, C, D identical siblings after myeloablation with busulfan, 2 mg/kg/day x 4 days, followed by immunosuppression with cyclophosphamide, 50 mg/kg/day x 4. Sustained engraftment of lymphoid and hematopoietic elements was documented in each case. Platelet counts in excess of 100,000/cu mm were restored 20--50 days posttransplant and remain in the normal range 6--12 mo later. Platelets exhibit normal size and in vitro aggregation. The patients produce isoagglutinins and antibodies to other polysaccharides. The use of busulfan in moderate dosages as a myeloablative agent, coupled with cyclophosphamide, may offer an improved alternative to the use of lethal total body irradiation as a preparative regimen for complete correction of Wiskott- Aldrich syndrome by marrow transplantation.     

Allogeneic hematopoietic cell transplantation for paroxysmal nocturnal hemoglobinuria

BACKGROUND: Although allogeneic hematopoietic cell transplantation (HCT) has a potential to cure patients with paroxysmal nocturnal hemoglobinuria (PNH), appropriate indication and conditioning regimen for HCT have not been established. PATIENTS AND METHODS: Between July 1999 and December 2001, five patients with PNH underwent allogeneic HCT: three for refractory hemolysis and two for aggravating cytopenia. Four patients with hypercellular marrow received Bu-Fludara-ATG (busulfan 4 mg/kg/d for 2 d, fludarabine 30 mg/m2/d for 6 d, and ATG 20 mg/kg/d for 4 d) for conditioning therapy and one patient with hypocellular marrow was conditioned with Cy-ATG (cyclophosphamide 50 mg/kg/d for 4 d and ATG 30 mg/kg/d for 3 d). Three patients received stem cell graft from matched sibling donor and two patients from 1-antigen mismatched unrelated donor. RESULTS: One patient who was conditioned with Bu-Fludara-ATG failed to engraft and died at post-transplant day 62. The other four patients showed three lineage engraftment and normal expression of CD55 and CD59 antigens by flow cytometric analysis. They are alive with stable engraftment and full donor chimerism between post-transplant day 510 and 1116. Acute graft vs. host disease (GVHD) of grade II or more occurred in two patients and extensive chronic GVHD in four. CONCLUSION: HCT using related or unrelated donor could eradicate PNH clones and may cure patients with the disease. Further studies are needed to establish the role of allogeneic HCT, especially with reduced intensity conditioning therapy, in the treatment of PNH.     

Organ-selective homing defines engraftment kinetics of murine hematopoietic stem cells and is compromised by Ex vivo expansion

Hematopoietic reconstitution of ablated recipients requires that intravenously (IV) transplanted stem and progenitor cells "home" to organs that support their proliferation and differentiation. To examine the possible relationship between homing properties and subsequent engraftment potential, murine bone marrow (BM) cells were labeled with fluorescent PKH26 dye and injected into lethally irradiated hosts. PKH26(+) cells homing to marrow or spleen were then isolated by fluorescence-activated cell sorting and assayed for in vitro colony-forming cells (CFCs). Progenitors accumulated rapidly in the spleen, but declined to only 6% of input numbers after 24 hours. Although egress from this organ was accompanied by a simultaneous accumulation of CFCs in the BM (plateauing at 6% to 8% of input after 3 hours), spleen cells remained enriched in donor CFCs compared with marrow during this time. To determine whether this differential homing of clonogenic cells to the marrow and spleen influenced their contribution to short-term or long-term hematopoiesis in vivo, PKH26(+) cells were sorted from each organ 3 hours after transplantation and injected into lethally irradiated Ly-5 congenic mice. Cells that had homed initially to the spleen regenerated circulating leukocytes (20% of normal counts) approximately 2 weeks faster than cells that had homed to the marrow, or PKH26-labeled cells that had not been selected by a prior homing step. Both primary (17 weeks) and secondary (10 weeks) recipients of "spleen-homed" cells also contained approximately 50% higher numbers of CFCs per femur than recipients of "BM-homed" cells. To examine whether progenitor homing was altered upon ex vivo expansion, highly enriched Sca-1(+)c-kit+Lin- cells were cultured for 9 days in serum-free medium containing interleukin (IL)-6, IL-11, granulocyte colony-stimulating factor, stem cell factor, flk-2/flt3 ligand, and thrombopoietin. Expanded cells were then stained with PKH26 and assayed as above. Strikingly, CFCs generated in vitro exhibited a 10-fold reduction in homing capacity compared with fresh progenitors. These studies demonstrate that clonogenic cells with differential homing properties contribute variably to early and late hematopoiesis in vivo. The dramatic decline in the homing capacity of progenitors generated in vitro underscores critical qualitative changes that may compromise their biologic function and potential clinical utility, despite their efficient numerical expansion.     

Accidental busulfan overdose during conditioning for stem cell transplantation

High dose busulfan is widely used in preparative regimens for bone marrow transplantation. We describe three cases of accidental busulfan overdosing. Two adults received a single dose of 8 and 18 mg/kg busulfan, respectively. Doses of 9 x 4 mg/kg were ingested by a 14-year-old girl, who experienced seizures. In all cases, no severe liver toxicity including veno-occlusive disease was observed. Plasma samples were obtained from two patients. Busulfan plasma concentrations were far above published values after high-dose busulfan treatment. Busulfan was eliminated by a first-order process. All patients survived these high doses of busulfan and successful transplantation was possible. Two patients died from refractory GvHD on days 91 and 80 after transplantation. One patient is alive in remission after an observation time of 18 months. These cases show that busulfan overdosing may occur and pharmacokinetic evaluation is warranted to estimate risk of early and late toxicity.     

Congenital sideroblastic anemia successfully treated by allogeneic bone marrow transplantation.

Allogeneic bone marrow transplantation (BMT) was carried out on a 34-month-old boy with congenital sideroblastic anemia. The patient had been red blood cell transfusion dependent since the age of 7 weeks. He did not respond to therapy with pyridoxine and developed secondary progressive hemosiderosis. The preparatory regimen consisted of busulfan (3.5 mg/kg for 4 days) and cyclophosphamide (50 mg/kg for 4 days). Full engraftment of donor bone marrow was achieved and effective hemopoiesis is still maintained 3 years after BMT.     

Neonatal bone marrow transplantation of ADA-deficient SCID mice results in immunologic reconstitution despite low levels of engraftment and an absence of selective donor T lymphoid expansion

Adenosine deaminase (ADA)–deficient severe combined immune deficiency (SCID) may be treated by allogeneic hematopoietic stem cell transplantation without prior cytoreductive conditioning, although the mechanism of immune reconstitution is unclear. We studied this process in a murine gene knockout model of ADA-deficient SCID. Newborn ADA-deficient pups received transplants of intravenous infusion of normal congenic bone marrow, without prior cytoreductive conditioning, which resulted in long-term survival, multisystem correction, and nearly normal lymphocyte numbers and mitogenic proliferative responses. Only 1% to 3% of lymphocytes and myeloid cells were of donor origin without a selective expansion of donor-derived lymphocytes; immune reconstitution was by endogenous, host-derived ADA-deficient lymphocytes. Preconditioning of neonates with 100 to 400 cGy of total body irradiation before normal donor marrow transplant increased the levels of engrafted donor cells in a radiation dose–dependent manner, but the chimerism levels were similar for lymphoid and myeloid cells. The absence of selective reconstitution by donor T lymphocytes in the ADA-deficient mice indicates that restoration of immune function occurred by rescue of endogenous ADA-deficient lymphocytes through cross-correction from the engrafted ADA-replete donor cells. Thus, ADA-deficient SCID is unique in its responses to nonmyeloablative bone marrow transplantation, which has implications for clinical bone marrow transplantation or gene therapy.     

Non-radiotherapy conditioning with stem cell transplantation from alternative donors in children with refractory severe aplastic anemia

Conditioning including total body/lymphoid irradiation is widely used to prevent graft rejection in patients with refractory severe aplastic anemia (SAA) undergoing hemopoietic cell transplantation (HCT) from alternative donors and or after graft manipulation. To reduce regimen-related toxicity we transplanted three children with refractory SAA after conditioning with radiotherapy-free regimens. Conditioning included fludarabine 175-180 mg/m2 in all patients. In addition, patient 1 (failing two previous grafts) received thiotepa 10 mg/kg and Campath-1H 60 mg/m2; patient 2 cyclophosphamide 120 mg/kg, thiotepa 15 mg/kg and OKT-3 0.1 mg/kg/day for 4 weeks; and patient 3 cyclophosphamide 120 and ATG 90 mg/kg. Stem cell source was unmanipulated marrow from the same unrelated donor as for the two previous transplantations in patient 1 and CD34+-purified peripheral blood stem cells from an HLA-matched unrelated donor and from the haploidentical mother in patients 2 and 3. Only patient 1 received graft-versus-host disease (GVHD) prophylaxis with cyclosporine A and mycophenolate mofetil. Follow-up is now 30, 51, and 15 months. None of the patients developed GVHD. All patients have normal counts with complete donor chimerism. Fludarabine-based conditioning is powerfully immunosuppressive and may be used for children with refractory SAA undergoing HCT from alternative donors even after rejection following previous HCT.