Fatal polycythemia induced in mice by dysregulated erythropoietin production by hematopoietic cells.

C57BL/6J murine bone marrow cells, infected with a retroviral vector (MP Zen) carrying a monkey erythropoietin cDNA, were transplanted into lethally irradiated syngeneic recipients to study the effect of erythropoietin production by hemopoietic cells. High levels of erythropoietin were recorded in the plasma (median value: 1.2 u/ml) and in media conditioned by peritoneal, spleen, and bone marrow cells from recipient mice. In transplanted mice, the hematocrit was elevated (90 +/- 5%) and the mice died at a mean of 71 days after transplantation. In the blood, platelet counts were usually low and nucleated blood cells slightly elevated. Spleen weight increased 5-fold and bone marrow cellularity decreased slightly. There was a 9.9-fold increase in erythroblast numbers, a 2-fold reduction of lymphocytes, and no variation of the myeloid cells when the total cellularity of bone marrow, spleen, peripheral blood, and peritoneal cells were considered. Calculation of the total numbers of progenitor cells in these organs revealed a 18-fold increase in erythroid colony-forming units (CFU-E) but no significant variation of the erythroid burst-forming units (BFU-E), and myeloid progenitor cell numbers. A variable proportion of CFU-E, (12% or 24% in bone marrow or spleen, respectively) was able to proliferate in unstimulated cultures. Erythropoietic amplification occurred in the spleen and there was a redistribution of the BFU-E and myeloid cells from the bone marrow to the spleen. No significant extramedullary erythropoiesis was seen. This study emphasizes the erythroid specificity of erythropoietin and shows that elevated dysregulated erythropoietin production by hemopoietic cells leads to a fatal polycythemia without erythroid neoplastic transformation.     

A myelosclerotic syndrome in mice engrafted with cells producing high levels of leukemia inhibitory factor (LIF)

DBA/2 mice engrafted with FDC-P1 cells producing high levels of the leukemia-inhibitory factor (LIF) developed high circulating levels of LIF and a fatal syndrome including the accumulation of excess osteoblasts in the marrow and new bone formation. The mice developed a neutrophil leucocytosis, an enlarged spleen, and excess numbers of hemopoietic cells in the spleen and liver. Marrow cellularity was reduced with selective survival of granulocytic cells, but the frequency of hemopoietic progenitor cells in both the marrow and spleen was higher than in control mice. Megakaryocyte numbers were reduced in marrows with pronounced sclerosis. The disease state may represent a useful model of myelosclerosis, but it remains to be established whether the hemopoietic abnormalities in these mice are direct effects of LIF or secondary changes following occlusion of the marrow by osteosclerotic tissue.     

Demand-control of proliferative activity in the hemopoietic system of lethally irradiated mice during transfusion-induced regeneration

The extent of cell proliferation in the hemopoietic system after bone marrow transfusion of fatally irradiated mice depends on the regeneration of proliferative capacity. This may be modified by the demand for differentiated cells in the peripheral blood. This demand was suppressed by induction of transfusion plethora prior to 800 rad whole body irradiation and bone marrow transfusion. Controls were non-plethoric recipients. For 6 days the following parameters were measured: hemopoietic proliferation by the 125-iodo-deoxyuridine (125-IUdR) incorporation technique, CFU-S content and spleen colony histology. There are three general observations from spleen and marrow with respect to 125-IUdR uptake in plethoric mice: (1) initial higher 125-IUdR uptake, (2) reduced rate of increase of 125-IUdR incorporation, (3) this rate of increasing 125-IUdR uptake in spleen was more depressed than in marrow. On day 6 cellularity and CFU-S in spleen was below, and in marrow above that of the control. These data suggest that initially after fatal irradiation of control mice differentiation of transfused CFU-S predominates over proliferation. Later as the mice become anemic and erythropoietin is produced the stimulation to proliferate is greater in the control than in the plethoric mice in which erythrocytic proliferation is suppressed. These data suggest that there are multiple feedback loops that regulate regeneration in the spleen and the bone marrow. These differences may be connected with the microenvironment that preferentially initiates erythropoiesis in the spleen before the marrow and granulopoiesis in the marrow before the spleen.     

Stimulatory Effect of Intermittent Feeding on Hemopoietic Recovery in Sublethally Gamma-Irradiated Mice

The effect of three-week adaptation to intermittent feeding on the recovery of the hemopoietic functions of mice after sublethal gamma irradiation was investigated. Measurement of oxygen consumption, carbon dioxide output and the respiratory quotient demonstrated an increased metabolic rate in the intermittently fed animals and an accentuation of lipogenic processes. This metabolic state persisted even after irradiation. An improvement in the recovery of hemopoietic functions after irradiation was demonstrated in adapted animals, which was reflected by the increased proliferative activity of the hemopoietic cell populations (more intensive incorporation of ¹²⁵J-UdR into the DNA of cells of the spleen, thymus and femoral bone marrow), by more rapid renewal of spleen weight, more rapid recovery of the femoral bone marrow cellularity and increased levels of granulocytes in peripheral blood.     

Suppression of marrow stromal cells and microenvironmental damage following sequential radiation and cyclophosphamide

Persistent defects in marrow stroma may contribute to hemopoietic insufficiency in patients treated with combined modality therapy for malignancy. To assess the bone marrow failure following combined therapy, mice received intraperitoneal administration of four weekly doses of cyclophosphamide, 160 mg/kg (CY) one week after 1500 rad leg irradiation (LI). This treatment inhibited repopulation of endogenous nucleated cells to less than 60% of normal at one, two, four and six months post-irradiation. Leg irradiation alone suppressed the repopulation to about 75% of normal and cyclophosphamide alone suppressed to 80% of normal. Normal body weight and recovery of peripheral leukocytes and hematocrit to normal levels demonstrated the localized nature of the lesion. Differential marrow counts revealed that 1500 rad LI + CY suppressed erythroblasts, myeloid, and lymphoid cells more than either modality alone. To directly assess the damage of sequential 1500 rad LI + CY on the microenvironment, marrow stromal cells were flushed from the femoral marrow and cultured as adherent cell colonies. They were suppressed to less than 30% of normal for three months following combined modality treatment. Other progenitors, granulocyte-macrophage and spleen colony forming units (CFU-C and CFU-S respectively), were suppressed but exhibited different patterns of partial recovery. We conclude that multiple courses of cyclophosphamide starting one week after 1500 rad LI produced persistent damage to the microenvironment reflected by decreased marrow stromal cells and failure of hemopoietic cells and their progenitors to completely repopulate femoral marrow.     

Effects of low dose rate irradiation on plateau phase bone marrow stromal cells in vitro: Demonstration of a new form of non-lethal,physiologic damage to support of hematopoietic stem cells

The clinical use of low dose rate (LDR) (5–25 rad/min) total body irradiation in bone marrow transplantation patients is well established. We have developed an in vitro system for study of the effects of LDR irradiation on bone marrow stromal cells. Purified mouse bone marrow stromal cell cultures in plateau phase with no detectable hematopoiesis were prepared and were then “engrafted” in vitro by addition of purified nonadherent hematopoietic cells from continuous bone marrow cultures. Hematopoietic cells were added in liquid medium or suspended in an overlay of semisolid 0.4% agar-containing medium. Other agar overlays contained Interleukin-3-dependent cloned multipotential hematopoietic stem cell fine B6SUtA. In parallel experiments, a cloned permanent bone marrow stromal cell fine D2XRII was used in place of purified stromal cell cultures. Stromal cultures were irradiated at 5 rad/min, 20 rad/min, or 200 rad/min, 24 hours or 3 weeks prior to “engraftment.” Two classes of irradiation damage were demonstrated following 1000 rad irradiation at 200 rad/min: 1) Decreased clonagenic survival of trypsinized replated marrow stromal cells (lethal effect), and 2) decreased production by marrow stromal cells or D2XRII cells of colony stimulating factors (CSF)s for granulocyte-macrophage progenitor cells and B6SUtA cells (physiologic effect). Holding the cultures in plateau phase for 3 weeks after irradiation was associated with significantly more repair of the lethal effect compared to the physiologic effect. Cultures irradiated at 5 rad/min or 20 rad/min to doses producing significantly less lethal effect showed a complex alteration of production of growth factors. Cumulative cell production by hemopoietic stem cells added in liquid culture was comparably decreased for all three dose rates. These data demonstrate a distinct physiologic expression of irradiation damage to bone marrow stromal cells that affects cell to cell interaction, responds differently to changes in dose rate, and is repaired with kinetics different from those of the lethal effect of irradiation. The present system should prove valuable for investigation of cellular interactions in hematopoietic stem cell engraftment that are altered by total body irradiation.     

Residual injury to the hemopoietic microenvironment following sequential radiation and busulfan

Our earlier studies in mice showed that sequential radiation and cyclophosphamide suppressed marrow stromal cells (MSC) and prevented local hemopoietic repopulation for several months. Because others have shown that busulfan administration caused marrow aplasia, we studied its ability, combined with radiation, to produce a peristent microenvironmental defect in mice. Intraperitoneal administration of four weekly doses of 20 mg/kg busulfan, starting one week after 1500 rad leg irradiation, produced a severe microenvironmental lesion for 6 months reflected by lack of repopulation in femoral marrow to greater than 50 % of normal by MSC, hemopoietic stem cells (CFU-S), and granulocyte-macrophage precursors. Differential marrow cell counts revealed that precursors of hemopoietic cells were more profoundly affected than their progeny. Hemopoietic stem cells and MSC failed to recover in busulfan-treated mice at 6 months to the same extent as those treated with cyclophosphamide. In addition, the busulfan-treated mice had an excessive number of myeloid blast cells and a severe erythroid depletion suggesting that these animals were preleukemic. We conclude that: 1) sequential radiation and busulfan administration caused long-term microenvironmental damage reflected by incomplete repopulation of the femoral marrow and suppression of MSC, and 2) multiple courses of busulfan prevented hemopoieticrepopulation longer than a similar regimen of cyclophosphamide.     

Residual toxicity in hematopoietic cells following a single dose of methylnitrosourea

The residual injury to the proliferation capability of hemopoietic stem cells (CFU-S) which results from their exposure to leukemogenic agents was evaluated in mice given a single leukemogenic dose of methol nitrosourea (MNU 50 mg/kg body weight, i.v.). Bone marrow cellularity, splenic weight, number of CFU-S and the proportion of cycling to noncycling CFU-S were measured in an effort to detect acute and residual injury to the CFU-S from mice given MNU 21 and 3 days earlier. Marrow cells were also transferred into lethally irradiated mice to observe the self-renewal capability of the CFU-S in the recipient spleen and bone marrow. The results of these measurements show that the CFU-S in marrow from mice given 50 mg/kg of MNU 21 days earlier still have a defective ability for self-renewal, although the total cellularity, number of CFU-S and proportion of cycling and noncycling CFU-S in the donor have returned to the normal range. The relationship of this self-renewal defect to the development of leukemia after this leukemogenic dose of MNU is not known.     

Radiobiological basis of total body irradiation with different dose rate and fractionation: Repair capacity of hemopoietic cells

Total body irradiation (TBI) followed by bone marrow transplantation is being used in the treatment of malignant or non-malignant hemopoietic disorders. It has been believed that the ability of hemopoietic cells to repair sublethal radiation damage is negligible. Therefore, several school of investigators suggested that TBI in a single exposure at extremely low dose rate (5 rad/min) over several hours, or in several fractions in 2–3 days, should yield a higher therapeutic gain, as compared with a single exposure at a high dose rate (26 rad/min). We reviewed the existing data in the literature, in particular, the response of hemopoietic cells to fractionated doses of irradiation and found that the repair capacity of both malignant and non-malignant hemopoietic cells might be greather than has been thought. It is concluded that we should not underestimate the ability of hemopoietic cells to repair sublethal radiation damage in using TBI.     

Effect of colony-stimulating factor-producing tumor on hemopoiesis in splenectomized mice

The colony-stimulating factor-producing BMA-1 tumor was transplanted into splenectomized mice. These mice showed as much granulocytosis as non-splenectomized mice bearing the tumor. The increase in the number of peripheral leukocytes induced per tumor weight was similar in the two groups. The hemopoietic cell population in the central bone marrow (tibia) was not increased by splenectomy. The total numbers of spleen colony-forming cells and granulocyte-macrophage colony-forming cells in the whole body of tumor-bearing splenectomized mice were 29% and 58% of those in the tumor-bearing non-splenectomized mice, respectively. The expansion of hemopoiesis to the peripheral bone marrow in tumor-bearing splenectomized mice was confirmed by histological examination of the tail bone. These results suggest that the lack of increase in the bone marrow cellularity is due to the space limitation, and that this is well compensated for by a great expansion of hemopoietic marrow to the peripheral bones in splenectomized mice.     

In vitro quantitation of lethal and physiologic effects of total body irradiation on stromal and hematopoietic stem cells in continuous bone marrow cultures from Rf mice

The role of stromal-supportive cells in hematopoietic stem cell responses to irradiation is poorly understood. The effects of in vivo total body irradiation (TBI) and interval from TBI to explant of marrow on: stromal cell proliferation in vitro; stromal cell support of hematopoiesis in continuous bone marrow culture; and generation of WEHI-3 growth factor (GF)-dependent lines of hematopoietic progenitor cells were evaluated. Continuous marrow cultures from non-irradiated control RfM/UN, C57BL/6J, C3H/HeJ, and N:NIH (Swiss) mice generated pluripotential hematopoietic stem cells (CFUs) and committed granulocyte-macrophage progenitor cells (GM-CFUc) for over 20 weeks. Explant of marrow at 2, 4, 5, or 6 months after single fraction TBI (300-800 rad) was associated with decreased longevity of hemopoiesis (2-12 weeks), and a decrease in the proliferative capacity of fibroblastic adherent-stromal colony forming cells (CFUf) as measured by colony size at 14 days and number of colonies per 10(6) cells plated. In contrast, explant of marrow 8 to 24 months after TBI produced cultures with longevity that was indistinguishable from age-matched control cultures (19-24 weeks). Marrow from irradiated first and second generation recipients of serially transferred marrow demonstrated a similar 7-month in vivo recovery period; however, the plateau maximum duration of hemopoiesis did not return to control levels. Purified stromal cell cultures were prepared by corticosteroid-deprivation of explanted marrow for 28 days and were then engrafted in vitro with marrow from C57BL/6J or RfM/UN mice that had been irradiated 1 month previously. Hemopoiesis in these cultures was restored, and they produced GM-CFUc and granulocytes for 15-24 weeks. Thus, healthy stroma supported growth of recently irradiated hemopoietic cells in vitro. Nonadherent cells removed from the above continuous marrow cultures generated clonal non-leukemogenic WEHI-3 GF-dependent hemopoietic progenitor cell lines with a frequency concordant with radiation effects on culture longevity, and this was increased by the presence of purified healthy stromal cultures. Indirect effects of x-irradiation on hemopoietic stem cells through damage and repair in the stromal cell compartment can be effectively studied with the present bone marrow culture system.     

Effect in vivo of multiple injections of purified murine and recombinant human macrophage colony-stimulating factor to mice

Hematopoietic efficacy in vivo of multiple injections of purified murine L-cell and recombinant human macrophage colony-stimulating factors (M-CSF; specific activity, greater than 2 x 10(7) units/mg) was assessed in mice. Injections i.v. of sterile saline or 20,000 units of M-CSF were administered once (at 0 h), twice (at 0 and 12 h), or three times (at 0, 12, and 24 h) to C57BL/6 x DBA/2 F1 mice. Numbers and cycling rates of marrow and spleen granulocyte-macrophage, erythroid, and multipotential progenitor cells were assessed 32-36 h after the first injection. Marrow, spleen, and peripheral blood cellularity was assessed at intervals of up to 105 h. Progenitor cell cycling rates were significantly increased after one and two injections of M-CSF but were reduced to a slow or noncycling state after three injections. For marrow cells, the third injection resulted in a significant suppression of hematopoietic progenitor cell cycling compared to the control group. No significant changes were noted for number of progenitors per femur or spleen, for marrow, spleen, or peripheral blood cellularity, or for differential cell counts in these organs after any of the M-CSF treatment schedules. Suppression of progenitor cell proliferation noted after three injections of M-CSF may at least partially explain why repeated injections of 20,000 units of M-CSF fails to increase bone marrow, spleen, or blood cellularity even though one injection of M-CSF increases cycling rates of the hematopoietic progenitors.     

Effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse

This paper reports the effect of cyclophosphamide on the bone marrow hematopoiesis in the mouse. Cyclophosphamide 0.12 mg/g body weight was injected into the mice once and the observation lasted for 2 weeks. After the injection, peripheral leukocytes were reduced to the lowest level on day 4 and then increased higher than the control on day 7 to 14. The number of nucleated cell in the bone marrow was the lowest at the 48th hour and gradually became normal within two weeks. The pluripotent hemopoietic stem cells--CFU-S (colony forming unit-spleen) were depleted abruptly in 24 hours, then reproliferated exponentially to a peak on day 3, followed by a second decrease and came back to normal level on day 11 to 14. The changes of granulocytic progenitor cell CFU-D (colony forming unit-diffusion chamber) and CFU-C (colony forming unit-culture) were quite similar to that of CFU-S but their proliferation peak was on day 4. The peripheral leukocyte drop was slower and the return to normal was earlier than the hemopoietic cells. So the recovery of leukocyte count does not mean a real reconstruction of hematopoiesis. The bone marrow stroma observed by CFU-F (colony forming unit-fibroblastoid) assay and marrow microcirculation were also damaged and did not recover to normal during the observation. The bone marrow stroma and microcirculation showed a more serious damage.     

Some effects of chemotherapeutic drugs

Summary cis-Platinum is a relatively new active anticancer drug. In the study described in this paper, its toxicity was tested in the hematopoietic and renal systems of mice after six injections of 3 mg per kg body weight at 10-day intervals.Acute hematopoietic toxicity was studied by determining the survival of pluripotent (CFU-S) and granulo-macrophagic unipotent (GM-CFC) stem cells. The number of nucleated cells in the bone marrow and in the spleen and the number of granulocytes in the blood were determined.Renal toxicity was studied by histological examination of kidneys from treated mice compared with control animals.The number of stem cells in the bone marrow and in the spleen decreased during the treatment. One year after treatment, the autorepopulating ability of CFU-S was still diminished in spite of normal numbers of these cells.No renal damage could be demonstrated by light microscopy when the protocol described was used.Abbreviations used in this paper CFU-S pluripotent hemopoietic stem cells assessed by the spleen colony technique - GM-CFC granulo-macrophagic progenitor cells - BFU-E erythroid progenitor cells - E/G ratio ratio of erythroid and granulocytic colonies in the recipient spleen and assessed by histological examination - Ara-C cytosine arabinoside     

Friend virus-infected long-term bone marrow cultures produce colony stimulating factor dependent and independent granulocyte-macrophage progenitor cells for over four years in vitro

C3H/HeJ mouse long-term bone marrow cultures infected at initiation with a cloned polycythemic strain of Friend spleen focus forming virus in a cloned N-tropic murine leukemia virus helper virus coat, persistently produced: colony-forming unit spleen (CFUs) for 55 weeks that formed macroscopic spleen colonies in syngeneic or allogeneic C57B10.Br/J mice; and L-cell or WEHI-3 cell conditioned medium-dependent granulocyte-macrophage colony forming unit culture (GM-CFUc); and morphologically normal granulocytes for over 245 weeks. Colony stimulating factor (CSF)-independent colony forming progenitor cells were first detectably produced in vitro at 75 weeks, and when subcultured generated karyotypically distinct permanent factor-independent tumorigenic cell lines. Nonadherent cells removed from long-term marrow cultures at 19 but not at 77 weeks reconstituted donor origin hematopoiesis in C57B10.Br/J mice as measured by B-cell lineage surface immunoglobin allotype. Nonadherent cells removed at 77 weeks produced lethal splenomegaly and marrow infiltration with culture origin cells in C57B10.Br/J mice. Despite generation of clonal malignant cell lines, L-cell DSF (CSF-1, M-CSF) responsive GM-CFUc that were simultaneously produced over 4 years in the same long-term marrow cultures, grew to 7 day colonies in semisolid medium and terminally differentiated. Thus, adherent stromal cells in Friend virus-infected long-term bone marrow cultures simultaneously support CSF-responsive and malignant CSF-independent hematopoietic progenitor cells.     

Postirradiation treatment with granulocyte colony-stimulating factor and preirradiation WR-2721 administration synergize to enhance hemopoietic reconstitution and increase survival.

These studies tested whether WR-2721 could be used to protect hemopoietic stem cells, which after irradiation could be stimulated by granulocyte colony-stimulating factor (G-CSF) to proliferate and reconstitute the hemopoietic system. Female C3H/HeN mice were administered WR-2721 (4 mg/mouse, i.p.) 30 min before 60Co irradiation and G-CSF (2.5 micrograms/mouse/day, s.c.) from days 1-16 after irradiation. In survival studies, saline, G-CSF, WR-2721, and WR-2721 + G-CSF treatments resulted in LD50/30 values of 7.85 Gy, 8.30 Gy, 11.30 Gy, and 12.85 Gy, respectively. At these LD50/30 values, the dose reduction factor (DRF) of 1.64 obtained in combination-treated mice was more than additive between the DRF's of G-CSF-treated mice (1.06) and WR-2721-treated mice (1.44). Bone marrow and splenic multipotent hemopoietic stem cell (CFU-s) and granulocyte-macrophage progenitor cell (GM-CFC) recoveries were also accelerated most in mice treated with WR-2721 + G-CSF. In addition, mice treated with WR-2721 + G-CSF exhibited the most accelerated peripheral blood white cell, platelet, and red cell recoveries. These studies (a) demonstrate that therapeutically administered G-CSF accelerates hemopoietic reconstitution from WR-2721-protected stem and progenitor cells, increasing the survival-enhancing effects of WR-2721 and (b) suggest that classic radioprotectants and recombinant hemopoietic growth factors can be used in combination to reduce risks associated with myelosuppression induced by radiation or radiomimetic drugs.     

Human myeloma marrow cells in immunologically deficient mice

Intact human bone marrow cells from 7 patients with myelomatosis were inoculated intravenously into adolescent CBA mice rendered immunologically deficient by thymectomy followed by total body irradiation (600 rad). Each inoculum of human myeloma marrow cells and subsequent passages of intact mouse marrow and spleen cells resulted in the presence of morphological changes in the marrow, spleen and peripheral blood of a proportion of these mice which were closely similar to those seen in the human donor. A substantial amount of human immunoglobulin (IgG and IgA) was detected in the sera of some of the mice showing morphological changes. Mice prepared identically but remaining uninoculated or receiving intact human bone marrow cells from 3 patients with no evidence of haematological malignancy showed none of these changes when examined after similar intervals.     

Deletion of chromosome 2 is an early event in the development of radiation-induced myeloid leukemia in SJL/J mice

In this study we have analyzed the chromosomal changes in the preleukemic phase in SJL/J mice treated with radiation and acute myeloid leukemias (AMLs) induced by radiation alone or with additional corticosteroid treatment. SJL/J mice exposed to 300 rad whole body irradiation developed a low incidence of AML (20-25%) that could be markedly increased (to 50-70%) by additional coleukemogenic treatment with corticosteroids. Partial deletion in one chromosome 2 was found in 100% of bone marrow and spleen cells of leukemic animals in both treatment modalities, whereas the age-matched controls exhibited a normal karyotype. Five types of deletion were observed according to site and size, but region D through G was the common missing part in all five types of chromosome 2 deletion. The occurrence of chromosome 2 deletion was also tested among bone marrow cells removed from 17 mice, 4 months after exposure to 300 rad whole body irradiation, long before the time when AML development is expected. About 80% of the mice tested had different levels of deleted chromosome 2 among their bone marrow population. Cytological and histological examination of bone marrow and spleen of most tested animals showed a normal hematologic picture. These results suggest that the marker chromosome is related to the process of radiation-induced initiation of AML in SJL/J mice.     

A phase I trial of monoclonal antibody M195 in acute myelogenous leukemia: specific bone marrow targeting and internalization of radionuclide

Ten patients with myeloid leukemias were treated in a phase I trial with escalating doses of mouse monoclonal antibody (mAb) M195, reactive with CD33, a glycoprotein found on myeloid leukemia blasts and early hematopoietic progenitor cells but not on normal stem cells. M195 was trace-labeled with iodine-131 (131I) to allow detailed pharmacokinetic and dosimetric studies by serial sampling of blood and bone marrow and whole-body gamma-camera imaging. Total doses up to 76 mg were administered safely without immediate adverse effects. Absorption of M195 onto targets in vivo was demonstrated by biopsy, pharmacology, flow cytometry, and imaging; saturation of available sites occurred at doses greater than or equal to 5 mg/m2. The entire bone marrow was specifically and clearly imaged beginning within hours after injection; optimal imaging occurred at the lowest dose. Bone marrow biopsies demonstrated significant dose-related uptake of M195 as early as 1 hour after infusion in all patients, with the majority of the dose found in the marrow. Tumor regressions were not observed. An estimated 0.33 to 1.0 rad/mCi 131I was delivered to the whole body, 1.1 to 6.1 rad/mCi was delivered to the plasma, and up to 34 rad/mCi was delivered to the red marrow compartment. 131I-M195 was rapidly modulated, with a majority of the bound immunoglobulin G (IgG) being internalized into target cells in vivo. These data indicate that whole bone marrow ablative doses of 131I-M195 can be expected. The rapid, specific, and quantitative delivery to the bone marrow and the efficient internalization of M195 into target cells in vivo also suggest that the delivery of other isotopes such as auger or alpha emitters, toxins, or other biologically important molecules into either leukemia cells or normal hematopoietic progenitor cells may be feasible.     

Comparative in vitro effects of cyclophosphamide derivatives on murine bone marrow-derived stromal and hemopoietic progenitor cell classes

We investigated the in vitro effects of ASTA-Z-7595, ASTA-Z-7557, ASTA-Z-7654, and 4-hydroperoxycyclophosphamide (4HC) on murine stromal fibroblastoid colony-forming units, committed hemopoietic progenitors (erythroid burst-forming units and granulocyte/macrophage colony-forming units), and pluripotent hemopoietic stem cells assayed by the spleen colony-forming unit (CFU-s) assay. In general, the drugs showed a time-and dose-dependent effect on colony-forming unit survival, and the relative toxicities were in the order in which the drugs are listed above. We found a relative sparing of day 12 CFU-s compared with day 7 CFU-s and committed hemopoietic and stromal progenitors, although colony size of day 12 CFU-s was reduced. Our results support two possible mechanisms for delayed or inadequate hemopoietic reconstitution in clinical studies using bone marrow purged with 4-hydroperoxycyclophosphamide or ASTA-Z-7557, i.e., damage to (a) transplantable stromal cells or (b) the hemopoietic stem cells.