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Protracted inhibition of osteoblast (OB) differentiation characterizes multiple myeloma (MM) bone disease and persists even when patients are in long-term remission. However, the underlying pathophysiology for this prolonged OB suppression is unknown. Therefore, we developed a mouse MM model in which the bone marrow stromal cells (BMSCs) remained unresponsive to OB differentiation signals after removal of MM cells. We found that BMSCs from both MM-bearing mice and MM patients had increased levels of the transcriptional repressor Gfi1 compared with controls and that Gfi1 was a novel transcriptional repressor of the critical OB transcription factor Runx2. Trichostatin-A blocked the effects of Gfi1, suggesting that it induces epigenetic changes in the Runx2 promoter. MM-BMSC cell-cell contact was not required for MM cells to increase Gfi1 and repress Runx2 levels in MC-4 before OBs or naive primary BMSCs, and Gfi1 induction was blocked by anti-TNF-α and anti-IL-7 antibodies. Importantly, BMSCs isolated from Gfi1(-/-) mice were significantly resistant to MM-induced OB suppression. Strikingly, siRNA knockdown of Gfi1 in BMSCs from MM patients significantly restored expression of Runx2 and OB differentiation markers. Thus, Gfi1 may have an important role in prolonged MM-induced OB suppression and provide a new therapeutic target for MM bone disease.

Increased osteoclastogenesis and angiogenesis occur in physiologic and pathologic conditions. However, it is unclear if or how these processes are linked. To test the hypothesis that osteoclasts stimulate angiogenesis, we modulated osteoclast formation in fetal mouse metatarsal explants or in adult mice and determined the effect on angiogenesis. Suppression of osteoclast formation with osteoprotegerin dose-dependently inhibited angiogenesis and osteoclastogenesis in metatarsal explants. Conversely, treatment with parathyroid hormone related protein (PTHrP) increased explant angiogenesis, which was completely blocked by osteoprotegerin. Further, treatment of mice with receptor activator of nuclear factor-kappaB ligand (RANKL) or PTHrP in vivo increased calvarial vessel density and osteoclast number. We next determined whether matrix metalloproteinase-9 (MMP-9), an angiogenic factor predominantly produced by osteoclasts in bone, was important for osteoclast-stimulated angiogenesis. The pro-angiogenic effects of PTHrP or RANKL were absent in metatarsal explants or calvaria in vivo, respectively, from Mmp9(-/-) mice, demonstrating the importance of MMP-9 for osteoclast-stimulated angiogenesis. Lack of MMP-9 decreased osteoclast numbers and abrogated angiogenesis in response to PTHrP or RANKL in explants and in vivo but did not decrease osteoclast differentiation in vitro. Thus, MMP-9 modulates osteoclast-stimulated angiogenesis primarily by affecting osteoclasts, most probably by previously reported migratory effects on osteoclasts. These results clearly demonstrate that osteoclasts stimulate angiogenesis in vivo through MMP-9.

Autosomal recessive osteopetrosis (ARO) is a group of genetic disorders that involve defects that preclude the normal function of osteoclasts, which differentiate from hematopoietic precursors. In half of human cases, ARO is the result of mutations in the TCIRG1 gene, which codes for a subunit of the vacuolar proton pump that plays a fundamental role in the acidification of the cell-bone interface. Functional mutations of this pump severely impair the resorption of bone mineral. Although postnatal hematopoietic stem cell transplantation can partially rescue the hematological phenotype of ARO, other stigmata of the disease, such as secondary neurological and growth defects, are not reversed. For this reason, ARO is a paradigm for genetic diseases that would benefit from effective prenatal treatment. Using the oc/oc mutant mouse, a murine model whose osteopetrotic phenotype closely recapitulates human TCIRG1-dependent ARO, we report that in utero transplantation of adult bone marrow hematopoietic stem cells can correct the ARO phenotype in a limited number of mice. Here we report that in utero injection of allogeneic fetal liver cells, which include hematopoietic stem cells, into oc/oc mouse fetuses at 13.5 days post coitum produces a high level of engraftment, and the oc/oc phenotype is completely rescued in a high percentage of these mice. Therefore, oc/oc pathology appears to be particularly sensitive to this form of early treatment of the ARO genetic disorder.

Polyamine synthesis inhibitors, such as a-difluoromethylornithine (DFMO), inhibit tumor cell growth in vitro and in vivo. However, upon cessation of treatment, tumor growth resumes. We hypothesized that incorporation of radioactive polyamines might kill the growth-arrested cells. This hypothesis was previously tested in rat 9L brain tumor cells in which DFMO increased both the uptake and the retention of [3H] putrescine. In these rat cells, DFMO-induced retention of high-specific-activity [3H] putrescine for 20 days resulted in several logs killing. In the present studies all of the 5 different human glioma cell lines tested with DFMO treatment also showed enhanced uptake of exogenous [3H] putrescine, reduced cell counts and enhanced killing of colony forming cells (CSF). Extending the time of DFMO treatment of cells that had taken up high-specific-activity (80 Ci/mmol) [3H] putrescine further increased the killing. A 10-day extension resulted in a 10,000-fold reduction in cumulative cell growth. A 5-day extension resulted in a 2-3 log decrease in numbers of surviving CFC. These data further support the hypothesis and suggest that DFMO-induced cell cycle arrest enhances cellular retention of [3H] putrescine, increasing the effective internal radiation dose enough to cause proliferative death. In a clinical setting, the short (approximately 1 microm) path-length of the tritium beta particle should limit effects to the tumor cells and spare adjacent normal cells. These results support the concept that treatment with the combination of polyamine inhibitors and radioactive polyamines might be a useful adjunct to current therapies for glioblastoma multiforme.

Nonstimulated fetal liver (FL) from 14.5-day gestation mice had no natural killer (NK) cell activity and <3% expressed NK1.1. Even after short-term (3-4 day) culture of FL with the late-acting cytokines, interleukin (IL)-15 or IL-2, little or no NK activity was detected. However, longer-term (13 day) culture with IL-2 plus stroma derived from bone marrow (BM) of adult mice, resulted in extensive proliferation and differentiation to mature NK cells. Cell numbers began to increase after 4 days, and by day 13, they had increased 40-fold and 69% of the cells were NK1.1+ with high NK activity and 5%-10% were NK1.1- B220+. With stroma, but no IL-2, equivalent proliferation occurred, but differentiated cells were predominantly NK1.1- B220+, not NK cells. Culture for 13 days without stroma, but with either IL-2, IL-15, FLTK3-ligand (L) or stroma-conditioned medium, resulted in less than fivefold expansion, and minimal NK activity. Culture with combinations of FLTK3-L or ckit-L plus IL-15 or IL-2 increased both cell number and NK activity, but the increase in cell number was less than that seen with stroma plus IL-2. By limiting dilution assay on stroma plus IL-2, the precursor frequency was 1/(2660+/-292) whole FL cells and the absolute number, but not the frequency, increased during culture on stroma without IL-2. The NK cell progenitors were found in sorted NK1.1- and Sca-1+ c-kit+ lineage- subpopulations at a frequency of 1/(156+/-52.5). Together, these data suggest that the NK lineage cells in FL are primarily in early stages of development. They are highly proliferative, respond to early acting cytokines and express stem cell markers.

Bone marrow stem cells develop into hematopoietic and mesenchymal lineages but have not been known to participate in production of hepatocytes, biliary cells, or oval cells during liver regeneration. Cross-sex or cross-strain bone marrow and whole liver transplantation were used to trace the origin of the repopulating liver cells. Transplanted rats were treated with 2-acetylaminofluorene, to block hepatocyte proliferation, and then hepatic injury, to induce oval cell proliferation. Markers for Y chromosome, dipeptidyl peptidase IV enzyme, and L21-6 antigen were used to identify liver cells of bone marrow origin. From these cells, a proportion of the regenerated hepatic cells were shown to be donor-derived. Thus, a stem cell associated with the bone marrow has epithelial cell lineage capability.

Although 14.5-day murine fetal liver (FL) has few, if any, mature natural killer (NK) cells, culture of FL with recombinant human IL-2 (rhIL-2) and stroma from irradiated NK longterm bone marrow cultures (NK-LTBMC) allows proliferation and differentiation of NK cell progenitors. Using this system, NK cell progenitors were found in both CD34+ and CD34- sorted subpopulations of FL. The CD34 antigen was expressed by 14+/-1.3% of whole FL cells, while mature NK cells cultured from NK cell precursors in FL did not express the CD34 antigen. Anti-TER-119 mAb reacted with 84%+/-10.3% of the FL cells, and NK cell progenitors were enriched in the TER-119- subpopulation. After coculture with rhIL-2 and stroma, neither TER-119- nor TER-119+ cells expressed antigens associated with T cells (CD3, CD4, and CD8) or myeloid cells (Gr-1 and Mac-1). Only the TER-119 subpopulation generated NK1.1+ (77%) and B220+ (87%) cells. Within the TER-119 subpopulation, both CD34+ and CD34- cells generated cytolytic and NK1.1+ cells after culture. By a limiting dilution assay (LDA) of the Lin (i.e., negative for NK1.1, CD3, CD4, CD8, B220, Gr-1, and TER-119) CD34 positive or negative subpopulations, the calculated mean frequency of NK cell progenitors was about 1/100 for the CD34+Lin- subpopulation and about 1/(200-300) for the CD34-Lin- subpopulation. In kinetic studies, we found that NK1.1 antigen expression continued to increase with time in culture for both the CD34+Lin- and CD34-Lin- fractions. In contrast, the percentage of CD34+ cells decreased rapidly and produced CD34- cells, and the CD34- population remained CD34-. These data suggest that both CD34+ and CD34- subpopulations of FL can differentiate into NK cells when cocultured for 13 days with irradiated NK-LTBMC stroma and rhIL-2, and that CD34+ progenitors differentiate to CD34- precursors, which in turn differentiate to CD34- mature NK cells.

For over 40 years, the association between hemopoietic chimerism and donor-specific tolerance for allografts has been recognized. However, toxicity associated with lethal conditioning has prevented the clinical application of bone marrow (BM) chimerism to induce tolerance. We previously demonstrated that engraftment could be achieved with less than total recipient myeloablation (700 cGy) and that the incidence of engraftment correlated with the dose of total body irradiation (TBI). Administration of cyclophosphamide (CyP) on Day +2 reduced the minimum TBI dose sufficient to permit engraftment to 500 cGy. In the current study, addition of antilymphocyte globulin (ALG) to the TBI/CyP-based conditioning approach reduced the radiation required for engraftment to < or = 300 cGy. B10 (H-2b) mice conditioned with ALG on day -3, 300 cGy of TBI with transplantation of B10.BR (H-2k) or BALB/c (H-2d) BM on day 0, and CyP on day +2 exhibited evidence of donor chimerism (49.6 +/- 3.7% and 38.2 +/- 2.4%, respectively) in 97% of recipients. ALG eliminated CD4+ and CD8+ cells and decreased NK1.1+ cells in the peripheral circulation at the time of transplantation. Moreover, T and NK cells in the host BM were significantly decreased compared with cells of recipients conditioned with TBI alone. CyP delayed repopulation of host thymocytes, providing time for the establishment of donor chimerism before production of mature T cells. Chimeric animals exhibited stable multilineage chimerism and donor-specific tolerance to skin grafts and in in vitro assays. This model may provide a clinically acceptable approach for the induction of donor-specific transplantation tolerance.

Evidence in experimental models suggests that many autoimmune diseases can be prevented by transplantation of bone marrow from disease-resistant donors. For potential clinical application, it would be important to avoid the morbidity and mortality associated with lethal conditioning and achieve mixed chimerism using less than complete recipient ablation. We report here for the first time that stable chimerism achieved in NOD mice using a sublethal radiation-based conditioning approach is sufficient to prevent beta-cell destruction and abrogate insulitis in prediabetic NOD mice. The percentage of NOD mouse recipients (8 wk of age) that engrafted with donor bone marrow correlated with the dose of irradiation and number of bone marrow cells transplanted. Engraftment of B10.BR bone marrow occurred in > or = 94% of animals receiving > or = 750 cGy of total body irradiation before bone marrow transplantation and > or = 30 x 10(6) bone marrow cells, while reproducible engraftment did not occur at radiation doses of less than 700 cGy and cellular doses of less than 30 x 10(6) bone marrow cells. All chimeric animals remained free of diabetes (n = 38) for 10 mo following bone marrow transplantation. Moreover, in all animals examined, no insulitis was present from 12 to 36 wk following reconstitution. In striking contrast, 61% (22 of 36) of NOD recipients that were conditioned but did not receive bone marrow developed acute diabetes by 12 mo. Insulitis was present in all remaining animals. These results suggest that allogeneic chimerism achieved using a sublethal conditioning approach can prevent the onset of diabetes and even reverse preexisting insulitis in NOD mice.

This study was designed to test the feasibility and safety of long-term expression of high levels of secreted human interleukin-1 receptor antagonist (hIL-1ra) protein in mice by retroviral transduction of hematopoietic stem cells. The retroviral vector, CRIP-MFG-hIL-1ra (MFG-IRAP), carrying the hIL-1ra gene was used to infect mouse bone marrow (BM) which was subsequently injected into lethally irradiated mice. All of the mice survived and greater than 98% of the white blood cells (WBC) of these mice were of donor type from 2-13 months after transplantation. All of the mice had hIL-1ra protein in their sera (40-1200 ng of hIL-1ra/ml) at all assay periods for at least 15 months after transplantation. Bone marrow from seven of seven primary recipients produced at least one secondary recipient with sustained, high serum levels of hIL-1ra, indicating that hematopoietic stem cells had been successfully transduced. Although the hIL-1ra was biologically active when assayed in vitro, the mice appeared to be well and their WBC counts and hematocrit (HCT) were not significantly different from those of lethally-irradiated mice given BM cells infected with the same vector carrying the lacZ gene. There was also no evidence of alterations of white cell subpopulations. These results demonstrate that systemic production of biologically active hIL-1ra can be obtained by retrovirus-mediated gene transfer to hematopoietic stem cells and that this level of expression and secretion into the serum is compatible with normal BM engraftment, hematopoietic recovery and survival of the lethally irradiated recipient mice. These hIL-1ra-expressing mice represent a model to examine the functions of IL-1 and hIL-1ra and to determine the ability of hIL-1ra to reduce susceptibility to chronic diseases such as rheumatoid arthritis as well as effects of aging such as bone degeneration. The data further suggest that transduction and transplantation of hematopoietic stem cells is a potential method for delivery of hIL-1ra and other secreted therapeutic gene products for systemic diseases.

Reconstitution of lethally irradiated mice with a mixture of syngeneic and allogeneic (A+B-->A) bone marrow results in multilineage mixed allogeneic chimerism, donor-specific transplantation tolerance, superior immunocompetence and resistance to graft-vs-host disease. However, the morbidity and mortality associated with lethal irradiation would be a major limitation to the clinical application of chimerism to induce tolerance for solid organ grafts or treat other nonmalignant hematologic diseases. We report here that durable multilineage mixed allogeneic chimerism and donor-specific transplantation tolerance for skin and primarily vascularized allografts can be achieved across multiple histocompatibility barriers using a nonmyeloablative radiation-based approach. The percentage of B10 mouse recipients that engrafted directly correlated with the degree of disparity between donor and recipient and the dose of total body irradiation administered. Although the occurrence of engraftment following conditioning with doses of total body irradiation of > or = 600 cGy was similar for animals receiving bone marrow disparate at MHC or MHC, minor and hematopoietic (Hh-1) loci (67% vs 78%), the level of donor chimerism was significantly less when multiple histocompatibility barriers were present (94.6 +/- 3.8% vs 37.5 +/- 12.5%). Treatment of the recipient with cyclophosphamide 2 days following allogeneic bone marrow transplantation reduced the dose of radiation sufficient for reliable engraftment to only 500 cGy of total body irradiation, regardless of MHC and Hh-1 disparity. Donor chimerism was stable and present in all lineages, with production of lymphoid (T and B cell), NK, and myeloid (erythrocyte, platelet, granulocyte, and macrophage) cells. Mixed chimeras exhibited donor-specific tolerance in vitro, as assessed by mixed lymphocyte culture (MLR) and cytotoxicity (CML) assays, and in vivo to skin and primarily vascularized cardiac allografts. The observation that engraftment and tolerance can be achieved across multiple histocompatibility barriers using nonmyeloablative recipient conditioning may allow allogeneic bone marrow transplantation to be applied to nonmalignant disease states in which lethal conditioning cannot be justified, including the induction of donor-specific tolerance for solid organ transplantation and the treatment of hemoglobinopathies and enzyme deficiency states.

Transplantation of untreated rat bone marrow into lethally irradiated (950 cGy) mouse recipients results in durable xenogeneic (rat-->mouse) chimerism and confers donor-specific transplantation tolerance for subsequent xenografts. The purpose of the present study was to characterize the minimal dose of total body irradiation (TBI) which would allow engraftment of rat bone marrow in mouse recipients. We report here that durable and stable lymphohaematopoietic cross-species chimerism can be achieved using a less than totally ablative radiation-based conditioning approach. The percentage of B10 mouse recipients which engrafted with rat bone marrow cells correlated with the dose of TBI. Engraftment of rat bone marrow stem cells occurred in all animals receiving 750 cGy prior to bone marrow transplantation, while no engraftment was detected at doses less than 650 cGy. Although most of the recipients were repopulated with mixed mouse and rat multilineage chimerism, some exhibited a predominance of rat cells. Although mixed xenogeneic rat/mouse chimeras prepared by lethal TBI produced only mouse derived RBC (red blood cells), chimeras prepared by sublethal conditioning produced both rat and mouse RBC. Only animals with detectable chimerism exhibited specific functional transplantation tolerance to donor xenoantigens, as assessed in vitro by mixed lymphocyte reaction assay. This model may offer an in vivo approach to study the role of species-specific growth factors in stem cell biology as well as the mechanisms for the induction of tolerance across species barriers.

Studies were undertaken to determine whether the doses of FK506 that are effective for acute GVHD prophylaxis following lethal irradiation and bone marrow transplantation (BMT) would also suppress myeloid cell reconstitution. FK506 (3 mg/kg/day) abrogated acute lethal graft versus host disease (GVHD) in lethally irradiated C57BL/10SnJ (H-2b) recipient mice given histoincompatible BM plus spleen cells from B10.BR (H-2k) donors and this dose was used in all of the studies. Endogenous and exogenous myeloid repopulation was studied in mice given daily injections of either FK506, an equivalent amount of carrier solvent or no treatment throughout the interval between total body irradiation (TBI) and the day of assay. Repopulation was studied after 400 or 500 cGy TBI (endogenous) and after 950 cGy TBI plus injection with syngeneic BM (exogenous). No consistent adverse effects of FK506 were seen during either exogenous or endogenous recovery. Parameters studied included hematocrit (Hct), WBC count, cells per humerus, spleen weight, splenic colony-forming units, % spleen or BM 59Fe uptake and colony forming cells per humerus. Similarly, when lethally irradiated secondary recipients were reconstituted with BM from FK506 treated primary recipients (lethal irradiation plus exogenous BM), no consistent effects were observed. These data suggest that FK506 given to prevent GVHD would not compromise the myeloid recovery that is critical for survival in the interval of time following shortly after BMT.

The role of the adhesion molecule CD44 in the development of NK cells was analyzed in a mouse long-term bone marrow culture system. After 4 wk of culture (day 0), recombinant human IL-2 was added and 13 days later the cells generated were shown to have substantial cytotoxic activity against YAC-1 and to be enriched for NK cells, as assessed for NK-1.1 phenotype by flow cytometric analysis. Physical separation between stroma and precursors partially inhibited proliferation and, consequently, a lower number of cytotoxic cells were produced. Similar results were obtained when an anti-CD44 mAb was added together with IL-2 at day 0. The disruption of hyaluronic acid (HA), one of the ligands of CD44, by hyaluronidase or the competition for the binding of CD44 by soluble HA added with IL-2 on day 0 inhibited both proliferation and development of cytotoxicity to a greater degree than did anti-CD44. These results indicate that interaction of CD44 with HA plays an important role in the development of pre-NK cells into cytotoxic effector cells.

The features of a mouse long-term bone marrow culture (LTBMC) system that produces natural killer (NK) cell activity are described. Over a 4-week period in the NK-LTBMC, cellularity dropped from approximately 2.5 x 10(7) to 8 x 10(5) cells/25-cm2 flask. About 3 x 10(5) of these cells were loosely adherent. The cultures at this time contained about one-third the spleen colony forming units, one-tenth the granulocyte macrophage colony forming units and about one-third the transplantable NK progenitor activity of fresh bone marrow (BM), and no detectable NK cell activity. In the 4-week NK-LTBMC, IL-2-responsive precursor cells appeared to be selectively maintained and gave an 8-fold higher activity after culture with human recombinant IL-2 (rIL-2) than did fresh BM. The addition of 50-5,000 IU/ml of rIL-2 resulted, after a minimal 3-day lag, in progressively increased cellularity for as long as 13 days. The percentage and staining intensity of NK-1.1+ cells increased with time after addition of rIL-2. CD3 epsilon + cells were occasionally seen and B220+ cells were present in low numbers at day 7 and slowly increased through day 13. The stroma was necessary for IL-2-dependent development of NK activity.

The macrocytic anemia of W/Wv mice can be cured by injection of +/+ bone marrow cells (BMC) from WBB6F1 mice. However, it has been observed that some W/Wv recipients appear to "lose" their cure with time, an effect that does not appear to be related to the age of the BMC donor. The present study was undertaken to determine the effect of recipient age on W/Wv responses to BMC injection. The effect of aging on erythroid parameters was similar in untreated W/Wv mice and +/+ controls. In both genotypes, hematocrit (HCT) and red blood cell count (RBC) decreased, and the modal red blood cell size (peak) increased between 13 and 150 weeks of age. As anticipated, mean HCT and RBC values were lower and peak values higher in W/Wv mice compared to +/+ controls at every age. However, the rate of decrease in HCT and RBC with age was the same for both genotypes, suggesting that the age effect and W gene effect were independent. Peak values increased slightly more with age for W/Wv than for +/+ controls. When female W/Wv mice in three age groups (23.5, 70, and 91.5 weeks old) were injected with 5 x 10(5) BMC from 20-week-old +/+ female donors and HCT, RBC, and peak were determined monthly, improvement was seen in most W/Wv recipients. However, in the older mice this improvement was slower and often was not sustained; 100% of the youngest recipients, 80% of the middle-aged, and only 30% of the older groups were cured after 3 months. Taken together, these data suggest a latent deficiency of the aging hematopoietic microenvironment that is revealed in W/Wv mice by the stress of continuing erythroid demand on the limited number of normal donor BMC.

We evaluated the effects of 16,16-dimethyl prostaglandin E2 (dm-PGE2), with and without syngeneic bone marrow transplantation (BMT) on the survival and hematopoietic recovery of mice given 14-20 Gy total body irradiation (TBI). Survival of mice given combined dm-PGE2 and BMT was improved significantly over that of mice given either treatment alone. The 30-day survival after 14, 15, 16 or 18 Gy TBI for combined treatment was 97, 90, 20 or 10 percent, respectively. The corresponding 30-day survival rates for mice given BMT alone were 69, 60, 7 or 0 percent, respectively. For dm-PGE2 alone, 30-day survival was 63, 20, 10 or 0 percent, respectively. Deaths in both dm-PGE2 treated groups generally occurred after day 10 whereas deaths in the BMT group occurred before day 10. All irradiated controls were dead on or before day 10; after larger doses, deaths clustered around day 5. After 20 Gy TBI, all mice in all groups were dead by day 7. Studies of white blood cell recovery 1-9 days after 14 Gy TBI showed improvement with BMT, whereas dm-PGE2 did not enhance recovery. Nucleated cells per humerus, spleen weight, and spleen iron uptake (erythropoiesis) were also improved by BMT but not dm-PGE2.

By utilizing a unique AKR(Rb6.15)Ald (AKRb) mouse line we have been able to determine the incidence and frequency of cells with and without abnormalities of chromosome number (specifically trisomy 6.15) in spontaneous and transplanted lymphoma. The transplanted lymphoma cells were easily distinguished from the normal cells of AKR/J recipients by the presence in diploid cells of 2 metacentric and 36 acrocentric chromosomes. The majority of the cells from 17 normal control AKRb mice were diploid and trisomy 6.15 (3 metacentric and 36 acrocentric chromosomes) was not seen. In 73% of the 59 AKRb mice with spontaneous lymphoma, the majority of cells were diploid. In 54% of the mice none of the cells had trisomy 6.15, and in 22% of these mice a majority (greater than 50%) of cells had trisomy 6.15. In order to determine which of the cells from AKRb mice with spontaneous lymphoma were malignant, they were transplanted into normal young AKR/J recipient mice, and when lymphoma developed in recipients, their cells were analyzed. The majority of first passage lymphoma cells from 30 recipient mice were donor-type and the majority of these donor cells were diploid. Ten (17%) of these recipients had a majority of cells with trisomy 6.15. In order to further determine whether or not trisomic lymphoma cells had a different proliferative rate than diploid lymphoma cells, eight first passage recipient mice were used to start sequential passage lines. In two diploid lines and four trisomic lines, the same relative frequency of the trisomy was maintained in the sequential passages. However, in two trisomic lines, the frequency of lymphoma cells with three metacentric chromosomes diminished after passage. These studies suggest that aneuploidy is not necessary for proliferation of lymphoma and that diploid cells are a major part of the malignant cell population.

Whether the hematocrit normally declines in the aged or whether such a decline represents inapparent disease in addition to aging is a matter of dispute. Female B6D2f1 mice were studied at ages 3, 13, or 27-28 months, and there was no difference in hematocrit between the younger groups. The hematocrit of 45 aged mice was slightly lower than that of 66 younger mice; mean 43% vs 49% (p less than .001). However, rather unexpectedly, the total red cell mass was not decreased in the aged; rather, the plasma volume was expanded. Survival of mature red blood cells did not differ significantly between young and aged mice. Mice were bled 0.4 ml from the orbital sinus for 4 days, reducing the hematocrit of all groups to a nadir of 20-25%. Recovery of hematocrit began more slowly in aged than in young mice. That this reflected a difference in erythropoiesis rather than a difference in plasma volume equilibration was suggested by studies with 59Fe. 59Fe was given following the second bleed, and 1 day later RBC 59Fe was more than twice as high in young mice than in groups of aged mice. Aged mice that did not appear healthy had been excluded. Aged mice were divided into a group with significant amounts of gray hair and/or patches of hair loss and two groups with normal-appearing hair; the latter was subdivided into those weighing less (25-26 g) or more (30-34 g) than most aged mice. Neither hair condition nor weight influenced hematocrit or response to bleeding. These results suggest, but do not prove, that a mild "dilutional" anemia and a blunted erythropoietic response to hemorrhage may be an expected part of the murine aging process.

Distribution of 59Fe into various bone groups of the complete murine skeleton was studied using two methods of dividing up the bones: 1) our previously reported technique of simply cutting up a skinned, eviscerated carcass and 2) separating bones from skeletons cleaned of overlying tissue by beetles, Dermestes species. The total percentage of injected 59Fe recovered in the sum of all skeletal parts, the percentage of total skeletal 59Fe found in each bone group, and the overall accuracy of determining these values were quite similar for the two techniques. The only statistically significant difference shown was a modest decrease in the percentage of total skeletal iron found in ribs plus sternum plus cervical and thoracic spine in beetle-cleaned as compared to cut up groups and we would not consider this to be of biological significance. Cutting up carcasses is the simpler of the two techniques but there are circumstances in which beetle digestion would be advantageous. In addition, we collected data on the reproducibility and precision of determining the percentage of 59Fe injected which is found in a "pulled off" foreleg plus scapula and of the distribution of 59Fe within three cut up pieces from the leg and within the scapula. These data can be used as a measure of overall changes in marrow mass and/or distribution, or at least they can be used as a screening procedure to detect such. This simple procedure adds potentially useful values for fully interpreting hematopoietic changes in the mouse.

Electronic sizing with a Coulter Counter was used to measure the frequency and number of cells with a large nuclear volume in tissues from AKR/J mice with spontaneous, long passaged, or first passage leukemia. The mean percentage of cells with a large nuclear volume was 1% in the blood, 4% in the spleen, 10% in the bone marrow, and 5% in the thymus of nonleukemic AKR/J mice. The percentage of cells with a large nucleus increased to 14-83% in all four tissues from AKR/J mice with spontaneous leukemia. In mice with a long passaged leukemia, a progressive increase in the percentage of cells with a large nucleus in the spleen and blood was associated with an increase in the number of cells/tissue with a large nucleus. However, in the bone marrow, an increase in percentage of cells with a large nuclear volume appeared to be the result of a decrease in the number of cells/humerus with a small nuclear volume. In the thymus, there was only a slight increase in the percentage of cells with a large nucleus. In mice with a first passage leukemia, there was a progressive increase, until death of the animals (21 +/- 1.9 days), in thymus weight and in the percentage of cells with a large nucleus. Also, the number of cells with a large nucleus increased in the spleen and bone marrow until day 12. Thereafter, there was a decrease in the total number of cells and number of cells with a large nucleus.

Mice were given sub-lethal (200-600 cGy) or near-lethal (800 cGy) whole body irradiation and the effect of injecting syngeneic marrow on subsequent hematopoietic recovery was studied. Marrow cell injection enhanced erythropoietic recovery after sub-lethal irradiation as reflected in hematocrit values and rate of appearance of 59Fe-labeled red cells in blood. However, this enhanced erythropoiesis was only seen in the spleen, and 59Fe uptake in marrow was reduced. When the irradiation dose was kept constant and the marrow dose increased from 10(5) to 10(6) to 10(7) cells, there was a somewhat erratic increase in spleen 59Fe and a decrease in marrow 59Fe uptake. When marrow cell number was kept constant and the dose of irradiation was increased from 200 to 400 to 600 to 800 cGy, there was an exponential increase in spleen 59Fe uptake but the marrow 59Fe uptake changed from depressed after lower doses to increased after 800 cGy. Cell injection after sub-lethal irradiation did not increase or decrease granulocytopoiesis. Injection of irradiated marrow cells also reduced marrow erythropoiesis and this was evident after both sub- and near-lethal irradiation. However, injection of irradiated cells did not increase splenic erythropoiesis. Following splenectomy, the depressed marrow erythropoiesis attending injection of viable cells was virtually eliminated but no increase was seen. These data suggest that the injection of autologous or syngeneic marrow may not be effective as a means of accelerating hematopoietic recovery after irradiation unless near-lethal or lethal dose have been received.