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Busulfan (1,4-butanediol dimethanesulfonate, BU) is relatively unique among other standard chemotherapy compounds in its ability to deplete noncycling primitive stem cells in the host and consequently to allow for high levels of long-term, donor-type engraftment after bone marrow transplantation (BMT). Such a property explains why this drug can be used as an alternative to total body irradiation in preparative regimes for BMT. However, as with radiation, BU conditioning is still troubled by severe toxicities that limit its applications to suboptimal drug doses. These problems stress the need for other BMT-conditioning drugs that are better tolerated and more selectively targeted toward normal and malignant hematopoietic stem cells. We have therefore compared the effects of various novel dimethanesulfonate compounds (related to BU) in terms of their toxicity to different stem cell subsets in vivo and in vitro and their ability to provide for long-term donor bone marrow engraftment using the congenic glucose-6-phosphate isomerase type 1 marker. Introduction of a benzene or cyclohexane ring in some of these drugs affords rigidity to the molecule and restricts the spatial positioning of the alkylating groups. Among 25 different compounds thus far tested at single doses, PL63 [cis-1,2-(2-hydroxyethyl) cyclohexane dimethanesulfonate] proved to be the most effective in providing for hematopoietic engraftment. The transisomer of the same compound gave significantly less engraftment and was comparable with the effects of dimethylbusulfan and Hepsulfam. The engraftment data correlated well with the depletion of different bone marrow stem cell subsets in the host as measured using the cobblestone area forming cell assay. The extent of stem cell depletion could not be explained on the basis of the distance and orientation of the two alkylating groups. Pharmacokinetic data, however, indicate that there is a correlation between biological activity and plasma levels reached. The diverse cytotoxic effects shown by these novel analogues of BU have provided a basis for relating biological activity with pharmacokinetic properties rather than with structural properties such as distance and orientation of the two alkylating groups. The identification of highly active compounds such as PL63 offers an opportunity for further developing other closely related drugs for potential application in clinical BMT conditioning therapy. more...

Thiotepa (TT) has long been considered for inclusion in clinical bone marrow transplant (BMT) conditioning regimens in an attempt to prevent allograft rejection and leukemia relapse. These studies have been encouraged by initial murine experiments showing a clear improvement in allogeneic bone marrow engraftment with addition of TT to total body irradiation (TBI) where it was assumed that TT enhances donor-type chimerism via ablation of competing stem cells in the recipient. The aim of the present study was to re-evaluate the hematological toxicity of TT among different stem cell subsets that included primitive cells capable of long-term repopulation and to assess how the combination of TT with TBI influences the development of donor engraftment in both syngeneic (B6-Gpi-1a --> B6-Gpi-1b) and H-2 compatible allogeneic (BALB.B10 --> B6) BMT models. At 24 h after TT (20 mg/kg) the femoral content of different stem cell subsets was determined from the frequency of transient repopulating, and the more primitive cobblestone area-forming, cells (CAFCs) growing in stroma-supported cultures. This assay showed a large TT-induced depletion (2% survival) of early clones developing at day 7 in culture but survival recovered towards normal for later appearing clones developing from more primitive CAFC subsets. The sparing of these primitive stem cells was reflected as undetectable levels of donor marrow repopulation in recipients given TT followed by syngeneic BMT. Addition of TT to TBI did not significantly improve long-term engraftment of syngeneic marrow while this combination had a dramatic effect in allogeneic BMT by preventing allograft rejection. In this respect TT shares similar properties with cyclophosphamide and suggests that the large improvement of allogeneic stem cell engraftment is attributable to the immune suppressive properties of TT rather than to its toxicity against host primitive stem cells. more...

The role of presensitizing murine recipients with donor spleen cells prior to T cell-depleted or -repleted H-2 compatible allogeneic bone marrow transplantation (BMT) was investigated at two different doses of total body irradiation (TBI). Recipients that were presensitized with 2 x 10(7) irradiated donor spleen cells at 1 week before a sublethal dose of 6 Gy TBI and BMT showed no evidence of donor blood chimerism while unsensitized recipients showed about 80% donor engraftment as determined by blood Gpi phenotyping. After raising the TBI dose to 9.5 Gy an increase in mortality from marrow failure was observed in presensitized animals. No significant engraftment-promoting effect of up to 2 x 10(6) T cells (20% of total marrow dose) was seen either in presensitized or unsensitized mice. It can be concluded that presensitized recipients are more susceptible to acute marrow rejection and that T cells added to the bone marrow did not influence the level of donor engraftment in these recipients. more...

The effect of stem cell factor (SCF) given at 24, 12 and 2 h before either 5-fluorouracil (5-FU) or total-body irradiation (TBI) was investigated on a range of bone marrow hemopoietic cell subsets that included primitive stem cells capable of long-term repopulation in bone marrow transplant (BMT) recipients. At 24 h after treatment, the femoral content of transient and permanent repopulating stem cell subsets was assessed from the frequency of early- and late-developing cobblestone area-forming cells (CAFCs) growing in stroma-associated cultures. At this time untreated 3 x 10(6) congenically marked donor bone marrow cells (B6-Gpi-Ia-->B6-Gpi-Ib) were transplanted and the level of erythroid engraftment was followed over 1 year. Analysis of the frequencies of CAFCs in host bone marrow after treatment with SCF demonstrated a remarkable increase in the number of early-developing CAFC subsets by about 10-fold. At the same time SCF conferred a sensitization of these subsets after treatment with 5-FU, which indicated an enhanced proliferative activity. The SCF-induced increase in the number of progenitor cells, however, was the more dominant process in the irradiated animals, resulting in less overall depletion of CAFCs. These contrasting results provide an explanation for the sensitization by SCF of 5-FU-induced lethality and its converse protection against radiation-induced lethality as reported by others. Nevertheless, the number of the more primitive CAFC subsets appearing at 28 and 35 days in culture and their sensitivity to 5-FU or radiation remained unaffected by this short SCF treatment. The number of CAFCs that remained in the bone marrow largely predicted the subsequent patterns of donor marrow engraftment in the treated BMT recipients: SCF enhanced short-term engraftment after treatment with 5-FU while it reduced the need for short-term engraftment after irradiation. Only irradiation afforded long-term engraftment through depletion of primitive host stem cells, and this was moderately improved by prior treatment with SCF. more...

Variations in hyperthermic sensitivity among different hematopoietic progenitor and stem cell populations of the bone marrow have been previously described for clonogenic subsets responsible for short-term hematopoiesis. However, less is known of the heat sensitivity of more primitive stem cells capable of long-term repopulation in irradiated recipients. In the present study, control and heat-treated (60 minutes at 43 degrees C) donor bone marrow cells from congenic B6-Gpi-1a mice were transplanted at different cell doses (10(4), 10(5), 10(6), and 10(7) nucleated cells) in pre-irradiated (6 Gy) B6-Gpi-1b mice. The development and levels of donor marrow engraftment were determined from blood Gpi phenotyping, and the bone marrow dose required for equivalent long-term engraftment at 20 weeks provided an estimate of the surviving fraction corresponding to primitive stem cells of long-term repopulating ability (LTRA). Comparison with previous bone marrow cell survival values demonstrates that LTRA cells are less sensitive to hyperthermic treatment than other hematopoietic subsets, confirming a relationship between the heat sensitivity and the hierarchical structure of the hematopoietic stem cell compartment. more...

A number of different alkylating chemotherapeutic agents--busulphan, dimethylbusulphan (DMB), isopropylmethane sulphonate (IMS), melphalan, cyclophosphamide (CY) and bischloroethylnitrosourea (BCNU)--were investigated for their cytotoxic effects on different haemopoietic cell populations in host mice and for their ability to induce short- and long-term engraftment of transplanted bone marrow. At 24 h after drug treatment the femoral content of transient and permanent repopulating stem cell subsets was assessed, respectively, from the frequency of early- (day 5-15) and late- (day 25-35) developing cobblestone area-forming cells (CAFCs), growing in vitro in long-term bone marrow cultures (LTBMCs). At this time a fixed complement of 10(7) congenically marked donor bone marrow cells (B6-Gpi-1a-->B6-Gpi-1b) was infused in the drug-treated mice and erythroid engraftment was followed over 36 weeks. Diverse effects on early- and late-developing CAFC frequencies were found for the different drugs; these were generally related to the pattern of donor bone marrow engraftment in treated recipients. Melphalan was more toxic to early-developing than to late-developing CAFC subsets, and the transplant only offered an early wave of blood chimerism followed by return of host cells. CY and BCNU had minimal to moderate effects on CAFC content and engraftment with no apparent preference for any particular haemopoietic cell subset. IMS also had a relatively low toxic effect on host marrow CAFC frequencies but appeared exceptional in its ability to allow for more donor-type engraftment. The dimethane sulphonate compounds busulphan and DMB were especially potent at depleting late CAFC subsets and ensured high and lasting levels of donor bone marrow engraftment. These studies support the value of CAFC measurements for predicting the fate and growth of transplanted bone marrow cells in recipients pretreated with a variety of cytotoxic agents. more...

The protraction of total body irradiation (TBI) to a continuous low dose-rate has been investigated for its effect on donor marrow engraftment in murine bone marrow transplant (BMT) models of varying histocompatibility. Three different BMT combinations were used: syngeneic [B6-Gpi-1a-->B6-Gpi-1b], H-2 compatible allogeneic [BALB.B (H-2b)-->B6 (H-2b)] and H-2 mismatched allogeneic [BALB/c (H-2d)-->B6 (H-2b)]. TBI was delivered over a range of doses at either a high (HDR, 40 cGy/min) or low (LDR, 2 cGy/min) dose rate followed by infusion of 10(7) bone marrow cells from syngeneic or allogeneic donors. The level of donor (Gpi-1a) engraftment was determined from blood Gpi-typing at different times after TBI and BMT. Radiation dose-response relationships corresponding to long-term haemopoietic engraftment at 20 weeks showed a dose-sparing effect of LDR that became more prominent with increasing genetic disparity between donor and host. For fully allogeneic (H-2 incompatible) BMT, a dose as high as 16Gy LDR was still not sufficient for achieving chimerism in all recipients. In many cases allogeneic BMT gave transient blood chimerism enabling the recipient to survive the acute effects of high dose TBI with full long-term repopulation from surviving stem cells of the host. Radiation cell survival curves were obtained for the frequency of alloreactive precursors of proliferating T-lymphocytes (pPTL) remaining in the spleen at 1 day after TBI. A radiation dose-sparing effect of LDR was also found for pPTL depletion. These data suggest that radiation damage repair during LDR irradiation in an immunocyte target cell population is mainly responsible for enhanced graft rejection thus rendering protracted TBI less effective for application in clinical allogeneic BMT. more...

Fractionated and low-dose-rate total-body irradiation (TBI) were compared with single-dose high-dose-rate TBI for induction of long-term hemopoietic chimerism in a murine syngeneic bone marrow transplantation model. At 5 months after TBI and bone marrow transplantation, the degree of stable blood chimerism was determined from the proportion of stem cell-derived donor (B6-Gpi-1a) and host (B6-Gpi-1b) blood erythrocytes. This end point was used to construct radiation dose-response curves for long-term donor marrow engraftment corresponding to ablation of primitive bone marrow stem cells of the host. Increasing dose fractionation and decreasing dose rate had the effect of restoring host hemopoiesis and required higher TBI doses for equal donor engraftment. Most of the dose recovery occurred within the first 6 h between fractions, consistent with the kinetics of sublethal damage repair. The late chimerism data were fitted to the linear-quadratic model using indirect and direct analysis for a fixed threshold response. Both analyses gave relatively low alpha/beta ratios (below 2 Gy), within the range normally seen in late-responding tissues. The dose-rate data gave a repair half-time of 2 h as estimated by the incomplete-repair model. These estimates contrast with the much higher alpha/beta values and lower repair half-times derived from acute hemopoietic failure as indicated by LD50/30, with the implication that separate target cell populations with differing radiosensitivities are involved in these two bone marrow end points. more...

To study the effects of donor T lymphocytes on engraftment and graft-versus-host disease in relation to recipient total-body irradiation, we have returned small numbers of T cells to T-cell-depleted bone marrow transplanted across a minor histocompatibility barrier in mice (B10.BR-->CBA). T-cell-depleted B10.BR marrow (10(7) cells) was transplanted into CBA recipients prepared with TBI doses ranging from 4 to 14 Gy. Selected animals also received 10(4) (0.1%) and 10(5) (1.0%) measured B10.BR T lymphocytes. The extent of donor marrow engraftment was determined from hemoglobin and carbonic anhydrase phenotyping of peripheral blood at 3 months posttransplant. Toxicity was assessed from breathing-rate measurements, histopathology, and animal survival. Addition of T cells had a profound effect on survival related to radiation dose. The TBI doses resulting in an LD50 at 12 weeks were 6.9 Gy, 9.3 Gy, and 13.0 Gy for animals receiving 10(5), 10(4), and no T cells, respectively. Mortality was associated with pulmonary dysfunction as measured by an elevation of breathing rates. Autopsy and histological analysis revealed extensive damage to the lung parenchyma. In contrast to the toxicity data, addition of T cells to the donor marrow had no effect on the TBI dose required for equivalent erythroid engraftment. These results demonstrate that in combination with TBI small numbers of T cells in the transplanted marrow do not aid engraftment but do significantly increase the risk of pulmonary toxicity. more...

Engraftment of donor bone marrow in relation to total body irradiation (TBI) dose was studied in syngeneic (B6----B6), MHC-compatible (BALB.B----B6) and MHC-incompatible allogeneic (BALB/c----B6) murine bone marrow transplantation (BMT) models. For each BMT combination radiation dose-response curves were obtained from stable long-term bone-marrow chimerism using Gpi-1 phenotyping and this was compared with the growth of exogenous CFU-S. Syngeneic engraftment required the lowest TBI doses limited to ablation of host haemopoietic stem cells. Resistance against H-2-compatible allogeneic engraftment was evident at low radiation doses (less than 5.5 Gy) but at 6 Gy and above the level of chimerism was comparable to syngeneic transplants, which indicated effective immunosuppression. Higher TBI doses were needed for engraftment as the immunological barrier was increased using fully H-2-incompatible allogeneic transplants. The high TBI dose (9.5 Gy) needed for suppression of spleen endocolonies in the CFU-S assay meant that rejection of exogenous bone marrow was evident only across the larger immunological barriers. When the fully allogeneic combination was reversed (B6----BALB/c) both CFU-S and chimerism data showed less rejection. The steep dose-response relationships show how engraftment is critically dependent on TBI dose, as well as the genetic disparity between donor and host. more...

Murine bone marrow chimera models were used to assess the efficacy of host total body irradiation (TBI) given at different doses, dose rates, and fractionation schemes in providing for engraftment of syngeneic and allogeneic bone marrow. B6-Hbbd congenic and LP mice, respectively, were used as donors (10(7) bone marrow cells) for syngeneic and allogenic (H-2 compatible) transplantation in standard B6 recipients. Stable marrow chimerism was determined from host and donor stem cell-derived hemoglobin phenotypes (Hbbs and Hbbd) on gel electrophoresis at 3 months posttransplant. Partial engraftment of syngeneic marrow was seen at single doses as low as 2 Gy, with the donor component increasing steadily with increasing TBI dose to a level of 100% at 7 Gy. Immunologic resistance of the host appeared to prevent allogeneic engraftment until 5.5 Gy. A very steep radiation dose response was then observed so that the level of chimerism with 6 Gy and above became comparable with syngeneic engraftment. Low dose rate (5 cGy minute-1) and fractionated TBI required higher total doses for equivalent engraftment (radiation dose-sparing) in both syngeneic and allogenic bone marrow transplantation. This displacement in the dose-response curve on fractionation was seen with interfraction intervals of 3 and 6 hours. A further dose-sparing effect was observed on extending the interval to 18 and 24 hours, but only for allogeneic transplantation, and may therefore be related to recovery of immune-mediated graft resistance. The involvement of multiple target cell populations in determining allogenic engraftment rendered the application of the linear-quadratic model for radiation cell survival problematic in this case. The recovery in dose when low dose rate and 6-hour interfraction intervals were applied in either syngeneic or allogeneic BMT is consistent with appreciable sub-lethal damage repair in the primitive self-renewing stem cell population of the host marrow. These results contrast with the poor repair capacity of the 11-day spleen colony-forming units (CFUs) population after fractionated irradiation and support the notion that ablation of early stem cells in the pre-CFUs compartment is essential for long-term marrow engraftment. more...

The LFA-1 molecule is a member of the leukocyte adhesion complex (CD11/CD18) that is critical to adhesion and effector function of T cells and NK cells. Both T cells and NK cells play important roles in bone marrow graft rejection, and anti-LFA-1 MAbs inhibit in vitro T cell and NK cell cytotoxicity. We here describe the in vivo use of a monoclonal antibody to the alpha chain of LFA-1 (CD11a) to prevent rejection of T cell-depleted allogeneic bone marrow. Animals receiving anti-LFA-1 and 1100 cGy total-body irradiation showed improved hemopoietic and immunologic reconstitution one month after BMT when compared with animals receiving TBI alone. Animals in the anti-LFA-1 group showed significantly improved long-term survival, and had no deficits in long-term lymphohemopoietic reconstitution. more...

Late mortality and pathology were assessed in various mouse strains following total-body irradiation (TBI) and bone marrow transplantation. A, C57BL/6, B6AF1, LP and C3H mice received TBI in two fractions 3 h apart at total doses of between 11 and 15 Gy. They were then transplanted with syngeneic bone marrow cells providing sufficient reconstitution to avoid hemopoietic failure. Long-term survival data revealed both radiation dose- and strain-dependent onset of mortality between 1 and 2 years post-treatment. Renal damage appeared to have contributed to the late mortality in most treatment groups as shown by glomerular lesions, elevated blood urea nitrogen and an accompanying fall in hematocrit. Hemolysis was deduced to be the major cause of anemia, as concluded from results of 51Cr-labeled erythrocyte survival. No decrease in erythropoiesis was evident as seen from spleen and bone marrow 59Fe uptake. These findings are together consistent with the manifestation of a hemolytic uremic syndrome (HUS) with kidney glomeruli representing the principal sites of injury responsible for both renal dysfunction and microangiopathic hemolysis. more...

The efficacy of total body irradiation and busulfan were studied in recipient preparation for bone marrow transplantation. Male C57BL/6 (B6) mice were prepared for BMT with fractionated TBI or busulfan given in 4 equal doses over 3 days. Both TBI and busulfan are potent stem-cell killers. Both agents resulted in an exponential decrease in CFUs survival with increasing dose down to a 1 x 10(-4) CFUs survival. There appeared to be no break in the curve for either agent. Extrapolated fractional CFUs survival, as related to equivalent donor marrow engraftment or to equivalent 30-day survival without marrow transplantation, appeared lower for TBI as compared to busulfan. This may be due to the effect of busulfan and TBI on different stem-cell populations. Erythroid engraftment was tested after transplanting H-2 compatible LP marrow cells into treated B6 recipients. Greater than 80% of animals demonstrated complete engraftment with 3.4 mg busulfan or 1640 cGy TBI. At these 2 doses, the rate of recovery of donor marrow cellularity and CFUs content in treated recipients were identical for both preparative agents such that a selective effect on the host hematopoietic microenvironment harmful to engraftment was not seen. Complete engraftment in 100% of busulfan-prepared animals could not be achieved as such doses resulted in severe and fatal pulmonary vascular injury at 7-12 weeks posttransplant. more...

This study was designed to compare several fractionation and dose rate schedules to optimize the therapeutic ratio for total body irradiation (TBI). C3H/HeJ mice were given TBI and the bone marrow survival fraction was calculated using the CFUS assay. Irradiation was given at two dose rates: low dose rate (LDR) at 5 cGy/min or high dose rate (HDR) at 80 cGy/min in single fraction and fractionated regimens. The fractionated regimens were given as either 120 cGy three times daily, 200 cGy twice daily, or 200 cGy daily. The Do was 80 cGy for the single fraction, HDR group and 85 for the LDR group. For the fractionated regimens, the apparent Do's ranged from 55-65 indicating no sparing effect of fractionation for the normal bone marrow stem cells. Indeed, the Do's were smaller suggesting an increased sensitivity to irradiation with fractionation. Low dose rate (LDR) and fractionation were also studied for their influence on normal tissue toxicity following upper half body irradiation (UHBI). All the fractionated regimens had higher LD50/30 and LD50/30-180 values than those achieved by single fraction LDR alone. There was no significant dose rate effect for LD50/30 when 120 or 200 cGy fractions were used. However, dose rate was important for LD50/30-180 with 200 cGy but not with 120 cGy fractions. These results demonstrate protection of non-hematopoietic tissues with fractionation and low dose rate without protecting hematopoietic stem cells and may have implications for human bone marrow transplantation. more...

Total body irradiation (TBI) and busulfan were compared for late effects in a murine model of bone marrow transplantation (BMT). Male C57BL/6 mice were given fractionated TBI or busulfan given in 4 equal daily doses followed by infusion of 10(7) syngeneic bone marrow cells. Total doses of 16.4 Gy TBI and 3.4 mg busulfan were chosen for their equivalence in inducing near complete engraftment of allogeneic marrow from donor mice of the LP strain. The two treatment groups had a late wave of mortality starting at about 80 weeks after transplantation. Specific tissue damage was manifested in bone marrow stem cells, splenic T-cell precursors, hair greying and cataract formation for both TBI and busulfan but to varying degrees. Severe nephrotoxicity and anemia were observed only after TBI. Although both busulfan and TBI kill early marrow stem cells and are effective preparative agents in bone marrow transplantation, their effects on other stem cell and organ systems are not similar. In addition, many of the injuries seen are late to occur. The delayed expression of injury deserves careful long-term evaluation of BMT recipients before the therapeutic potential of effective preparative regimens can be fully appreciated. more...