Syngeneic Bone Marrow Transplantation Model Research Articles

F-18-fluorothymidine-PET evaluation of bone marrow transplant in a rat model

The success of bone marrow transplantation in hematologic and oncologic diseases depends upon the ability to provide adequate hematologic and immunologic support in a timely manner. There is no current imaging tool for the evaluation of the bone marrow compartment in the peri-transplant period. In this study, we tested our hypothesis that whole-body positron emission tomography using fluorine-18 (F-18)-fluorothymidine has the ability to provide information about the functioning normal bone marrow and its physiologic distribution. Wistar Furth rats were irradiated with 950 cGy followed 2 days later by syngeneic bone marrow transplantation. The animals were subjected to positron emission tomography using F-18-fluorothymidine imaging before and after ablation as well as after 4, 7, and 14 days after bone marrow transplantation. In a different set of syngeneic bone marrow transplant model, positron emission tomography using F-18-fluorodeoxyglucose was carried out for comparison. Imaging results were correlated with marrow histology upon necropsy. F-18-fluorothymidine images showed definitive recovery of marrow within 4 days after transplant. In contrast, F-18-fluorodeoxyglucose was unable to present a corresponding picture. Sternum and humerus marrow in the images were analyzed by drawing three-dimensional regions of interest to obtain quantitative uptake value of tracers, which corroborated the image data. We conclude that positron emission tomography using F-18-fluorothymidine offers a clinically useful noninvasive technique to evaluate bone marrow injury and recovery.

Long-term Haematopoietic Reconstitution and Survival without Interleukin-7 in a Murine Syngeneic Bone Marrow Transplantation Model

We created a syngeneic mouse bone marrow transplantation (BMT) model to examine the effect of endogenous interleukin-7 (IL-7) on long-term (>or=140 days) haematopoietic reconstitution and survival after BMT. Wild-type (WT) IL-7(+/+) and knockout (KO) IL-7(-/-) mice were lethally irradiated and transplanted with bone marrow. Survival is best (85.7%) in the group WT grafts transplanted into WT recipients (WT-->WT) with a trend towards poorer survival in the other groups (WT-->KO: 60%, KO-->WT: 50%, KO-->KO: 45.5%, differences statistically not significant). If the recipient is deficient for IL-7-producing cells, T- and B-cell reconstitution remain incomplete. If the graft lacks IL-7-producing cells there is a significant delay in T- and NK-cell reconstitution. Interestingly, in the absence of IL-7, T-cell reconstitution is neither delayed nor incomplete because of an expansion of TCRalphabeta(+)/CD4(-)/CD8(-) double negative T cells. Long-term survival and lymphocyte reconstitution after syngeneic BMT can occur despite the absence of IL-7.

Bone Marrow-Derived lin−c-kit+Sca-1+ Stem Cells Do Not Contribute to Vasculogenesis in Lewis Lung Carcinoma

The development of tumor vasculature is thought to occur through two complementary processes: sprouting angiogenesis from preexisting blood vessels of the host, vasculogenesis, which involves the spontaneous development of vessels through specific recruitment, differentiation, vascular incorporation of circulating endothelial cells (EC), endothelial progenitor cells (EPC), or potentially bone marrow-derived cells. Recent reports, however, have challenged the belief that bone marrow-derived cells contribute to tumor neovascularization, claiming an exclusive role for sprouting angiogenesis in tumor blood vessel development. In the present study, we explored the recruitment behavior of bone marrow-derived lin− c-kit+Sca-1+ stem cells to subcutaneously implanted Lewis lung carcinoma in a syngeneic bone marrow transplantation model. We observed that although lin− c-kit+Sca-1+ and their derived cells demonstrate significant recruitment to carcinomas in vivo, they do not appear to functionally contribute to tumor neovascularization. Furthermore, our results support the hypothesis that new vessel formation in carcinomas occurs primarily through endothelialization from adjacent and preexisting vasculature.

Recombinant Human Prolactin Improves Antitumor Effects of Murine Natural Killer Cells in vitro and in vivo

Objective: To verify the effect of prolactin on natural killer (NK) cells in vivo and its implications for NK cell immunotherapy. Methods: Recombinant human prolactin (rhPRL; 30 µg, i.p.) was administered to BALB/c mice and severe combined immunodeficiency (SCID) mice 1 day before harvesting splenocytes for ⁵¹Cr release assay to examine the effects of rhPRL on NK cells of normal mice. rhPRL (10 µg, i.p., every other day for a total of 5 injections) was administered to BALB/c mice after syngeneic bone marrow transplantation (SBMT) to determine its effects on NK cell reconstitution. CT26 tumor cells were injected into BALB/c mice intravenously on day 0, and interleukin (IL)-15-cocultured activated syngeneic NK cells (IL-15-NK) from SCID mice were intravenously injected into tumor-bearing BALB/c mice on day 1. The improvement of antimetastasis effects and survival of tumor-bearing mice by rhPRL were checked. Results: BALB/c and SCID mice receiving one rhPRL injection exhibited a significant increase in cellular cytotoxicity against YAC-1 target tumor cells; the specific lysis was enhanced from 12.5 to 17.3% in BALB/c mice and from 27.8 to 51.2% in SCID mice. BALB/c mice continuously receiving rhPRL injections exhibited significant increases in NK cell (DX5-positive) contents and cellularity in both the bone marrow and spleen in the SBMT model. The bone marrow NK cell contents were improved from 1.53 to 3.13% after rhPRL injection. NK cells from SCID mice were then cultured with recombinant human IL-15 (rhIL-15; 6,000 IU/ml), rhPRL (10 ng/ml) or rhIL-15 plus rhPRL for 25 days. The cytotoxicity and cellularity were enhanced by rhPRL when tested on day 10, when comparing the rhIL-15 plus rhPRL group with the rhIL-15 group or rhPRL group, respectively. In the adoptive cellular immunotherapy study, the IL-15-NK plus IL-15 plus rhPRL group showed significantly lower numbers of lung metastases and longer survival than the IL-15-NK plus IL-15 group; the mean survival interval was prolonged from 31.5 to 53.7 days. Conclusion: These results indicate that rhPRL is possibly an important regulator of NK cells and a potential biologic product for immunotherapy.

Detection and quantitation of genetically marked acute myeloid leukemia by competitive polymerase chain reaction after autologous bone marrow transplantation: A preclinical model for minimal residual disease

Preclinical models and methods aimed at detecting and quantitating minimal residual disease (MRD) after autologous bone marrow transplantation (BMT) for acute myeloid leukemia (AML) could facilitate assessment of innovative therapeutic strategies for their antileukemic potential. Among the various techniques exploited to identify MRD, polymerase chain reaction (PCR) proved to be a valuable tool in instances in which clonogeneic markers are involved during the evolution of disease. In human AML, however, detection of MRD by PCR is limited to a minority of subgroups, as clonospecific markers are absent or presently unknown. Although gene labeling has proved to be efficient in detecting marker-devoid leukemia cells in preclinical models, detection and quantitation by PCR have not yet been considered. We therefore developed an experimental model in which detection and quantitation of genetically marked murine AML cells are based on a highly sensitive two-step nested PCR and competitive PCR protocol, respectively. We further demonstrated its applicability to a murine syngeneic BMT model that was designed to monitor minimal numbers of gene-tagged AML cells at various time intervals after transplantation. Our results showed that detection and quantitation could reproducibly be achieved at levels as low as one in 10 6 and 10 5 cells, respectively.

In vivo radiosensitizing effects of recombinant interleukin 6 on radiation resistant BCL-1 B-lineage leukemia cells in a murine syngeneic bone marrow transplant model system.

The ability of total body irradiation (TBI) to eradicate clonogenic leukemia cells from B-lineage acute lymphoblastic leukemia patients prior to bone marrow transplantation (BMT) is greatly hampered by their inherent or acquired radiation resistance. The radiorefractory nature of these cells is believed to contribute to the high relapse rate subsequent to TBI and BMT in patients with B-lineage acute lymphoblastic leukemia (ALL). A method by which clonogenic leukemia cells could be radiosensitized in vivo could be clinically beneficial. In the present study, we used a highly radiation resistant subclone of the murine B-lineage leukemia cell line BCL-1 in a syngeneic BMT model to investigate if any of the B-cell stimulatory cytokines interleukin 2, interleukin 4, interleukin 5, or interleukin 6 could have radiosensitizing effects. All untreated BALB/c mice (N = 33) inoculated with 1 x 10(6) BCL-1 cells died of disseminated leukemia within 24 days with a median survival of 13.3 days. TBI (700 cGy = LD100/30 for BALB/c mice) followed by syngeneic BMT (N = 70) extended the median survival to 23.6 days (P < 0.001 by log-rank test). A single intraperitoneal bolus injection of 100 ng, 500 ng, or 2500 ng recombinant murine interleukin 6(rmIL-6) 2-4 hours before TBI extended the median survival to 32.5 days, 31.0 days, and 30.5 days, respectively (P < 0.01 by log-rank test for all dose groups). The improved survival was not due to any direct anti-leukemic activity of rmIL-6 and all control BALB/c mice (N = 15) that received the same doses of rmIL-6 but did not undergo TBI and BMT died of BCL-1 leukemia within 28 days with a median survival of 13.6 days. In contrast to rmIL-6, recombinant murine interleukin 5 (rmIL-5) had minimal radiosensitizing effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of nephrotoxic drugs on the development of radiation nephropathy after bone marrow transplantation

Purpose : Chronic renal failure is a significant cause of late morbidity in bone marrow transplant patients whose conditioning regimen includes total body irradiation (TBI). Radiation is a major cause of this syndrome (bone marrow transplant nephropathy), but it may not be the only cause. These studies use a rat syngeneic bone marrow transplant model to determine whether nephrotoxic agents used in conjunction with bone marrow transplantation (BMT) could be enhancing or accelerating the development of radiation nephropathy. Methods and Materials : Rats received 11–17 Gy TBI in six fractions over 3 days followed by syngeneic bone marrow transplant. In conjunction with the bone marrow transplants, animals received either no drugs, cyclosporine, amphotericin, gentamicin, or busulfan. Drugs were given in schedules analogous to their use in clinical bone marrow transplantation. Drug doses were chosen so that the drug regimen alone caused detectable acute nephrotoxicity. Animals were followed for 6 months with periodic renal function tests. Results : Gentamicin had no apparent interactions with TBI. Amphotericin increased the incidence of engraftment failure, but did not enhance radiation nephropathy. Cyclosporin with TBI caused late morbidity that appeared to be due to neurological problems, but did not enhance radiation nephropathy. Busulfan resulted in a significant enhancement of radiation nephropathy. Conclusion : Of the nephrotoxins used in conjunction with bone marrow transplantation only radiation and busulfan were found to be risk factors for bone marrow transplant nephropathy.

FK506 inhibits the differentiation of developing thymocytes but not negative selection of T cell receptor Vβ5 + and Vβ;11 + T lymphocytes in vivo

To examine the influence of FK506 on lymphocyte development, we employed a syngeneic bone marrow transplantation model using MHC-disparate B10 (H-2 b, I-A b) and B10.BR (H-2 k, I-A k, I-E k) mice. B10 mice, which do not express class II I-E, do not delete any known T cell receptor (TCR)-Vβ;, while B10.BR mice (MHC class II I-E k, I-A k) delete Vβ;5 + and Vβ;11 + TCR. Continuous daily treatment of syngeneically reconstituted B10 mice with FK506 delayed the development of thymocytes from the CD4 +CD8 + to CD4 +CD8 − stage, while no effect was observed at the earlier CD4 −CD8 − to CD4 +CD8 + stage. At the same time, there was a significant reduction in TCR high thymocytes compared with untreated, syngeneically reconstituted controls. These results suggest that FK506 treatment interfered with thymic positive selection. We also examined whether FK506 treatment would influence negative selection. Levels of expression of Vβ5 + and Vβ11 + T cells in FK506-treated B10.BR → B10.BR recipients were similar to those observed in unmanipulated, syngeneically reconstituted B10.BR → B10.BR controls. This was not due to the inhibition of clonal proliferation by FK506, since 35 days after drug withdrawal complete recovery of the peripheral Thy1.2 + population was observed, while the percentages of Vβ5 + and Vβ11 +Thy1.2 + T cells were maintained at values similar to controls. Surprisingly, clonal proliferation stimulated by monoclonal antibody against Vβ5 and Vβ11 TCRs was observed in CsA-treated, syngeneically reconstituted B10.BR mice but not in FK506-treated mice, suggesting that CsA may be more likely to induce autoreactivity. Differences in thymic architecture between FK506- and CsA-treated animals further suggested that the drugs may differ in their effects on T cell development in vivo.

Radiation Dose-Fractionation and Dose-Rate Relationships for Long-Term Repopulating Hemopoietic Stem Cells in a Murine Bone Marrow Transplant Model

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.

Synergism of interleukin-2 and cyclosporine A in induction of a graft- versus-tumor effect without graft-versus-host disease after syngeneic bone marrow transplantation

Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD.

Synergism of Interleukin-2 and Cyclosporine A in Induction of a Graft-Versus-Host Effect Without Graft-Versus-Host Disease After Syngeneic Bone Marrow Transplantation

Abstract Interleukin-2 (IL-2) therapy generates killer cells with major histocompatibility complex (MHC)-unrestricted cytotoxicity against most tumors but not normal tissues. Cyclosporine A (CsA) has been reported to break tolerance to self and to induce killer cells with specificity against class II MHC (Ia) antigens both on the host and the tumor cells, resulting in a mild graft-versus-host disease (GVHD) in an autologous bone marrow transplantation (BMT) setting in the rat. We used these two agents in a syngeneic BMT model in a strain of mice that does not develop GVHD with CsA. Therapy with either agent alone was ineffective, whereas a combination of CsA plus IL-2 after BMT induced a potent graft-versus-tumor (GVT) effect against a melanoma and an acute myeloid leukemia. The antitumor effect could be adoptively transferred by infusing spleen cells harvested from mice treated with CsA plus IL-2 into secondary recipients that received chemoradiotherapy. The cytotoxicity of these cells was not influenced by treatment of tumor cells with gamma-interferon or Ia antibody. The cytotoxic effect was mediated by Thy 1+ and asialo GM 1+ cells. There was no GVHD either in the primary recipients of CsA and IL-2 or in those receiving the adoptively transferred spleen cells. Our findings show that combination therapy with CsA and IL-2 after syngeneic BMT induces a potent GVT effect in a non-MHC-restricted manner, and point to the existence of differences between the mechanisms of GVT and GVHD.

Effects of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of murine late spleen colony-forming cells (day-12 CFU-S) and hematopoietic repopulating cells to mafosfamide (ASTA Z 7557)

The effects of inhibitors of aldehyde dehydrogenase activity on the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorine anticancer agents, e.g. mafosfamide, were examined using two different assay procedures. In the first part of the investigation, the ex vivo sensitivity of murine day-12 spleen colony-forming cells (CFU-S) to mafosfamide was determined in the absence and presence of known inhibitors of aldehyde dehydrogenase activity, viz. diethyldithiocarbamate and cyanamide. These results were compared to those generated for day-8 CFU-S. Day-12 CFU-S were less sensitive to mafosfamide, and to phosphoramide mustard, although the difference in sensitivity to the latter was less marked. Diethyldithiocarbamate and cyanamide each potentiated the cytotoxic action of mafosfamide toward both day-12 and day-8 CFU-S; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Since cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major transport form of mafosfamide, to the relatively nontoxic acid, carboxyphosphamide, the results suggest that intracellular aldehyde dehydrogenase activity is a determinant of the sensitivity of day-12 CFU-S, as well as of day-8 CFU-S, to mafosfamide and other oxazaphosphorines, e.g. cyclophosphamide. In the second part of this investigation, a murine syngeneic bone marrow transplantation model was used to determine the ex vivo sensitivity of murine hematopoietic repopulating cells to mafosfamide in the absence and presence of diethyldithiocarbamate. Specifically, the ability of treated marrow grafts to repopulate the hematopoietic system, and thereby save recipients from the otherwise lethal effect of total body irradiation, was determined. Diethyldithiocarbamate potentiated the cytotoxic action of mafosfamide, but not that of phosphoramide mustard, toward hematopoietic repopulating cells. These observations support our previous contention that aldehyde dehydrogenase activity is an operative determinant with regard to the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines.

Enhanced recovery of gut-associated lymphoid tissue by infusion of buffy coat cells and gut leukocytes in the murine syngeneic bone marrow transplantation model

Ways of accelerating recovery of the mucous membrane immune system in lethally irradiated mice following syngeneic bone marrow transplantation were studied over a 35-day period by quantification of jejunal intraepithelial lymphocytes (IELs) and lamina propria plasma cells. Recovery after a low bone marrow dose allowing 100% animal survival (LBM) was compared with a high (five times minimal) dose (HBM), or a minimal dose augmented with equal numbers of buffy coat cells (LBM + BC) or small gut mucosal lymphocytes (LBM + GL). The maximal decline and subsequent peak repopulation of IELs were: LBM, days 7 through 14, peaking suboptimally by day 28; HBM, day 14, peaking suboptimally but higher than LBM by day 35; LBM + BC, days 11 through 14, peaking at control levels by day 35; and LBM + GL, day 7, peaking at control levels by day 28. The IEL decline was most severe with LBM and HBM treatment and least with LBM + GL. All transplant groups experienced maximal plasma cell decline by day 7. LBM had the most severe depletion, and LBM + GL had the least. Recovery to control levels for the LBM, HBM, LBM + BC and LBM + GL groups occurred by days 28, 21, 21, and 14, respectively. In all instances, greater than 95% of the plasma cells were IgA positive.

Enhanced Recovery of Gut-Associated Lymphoid Tissue by Infusion of Buffy Coat Cells and Gut Leukocytes in the Murine Syngeneic Bone Marrow Transplantation Model

Ways of accelerating recovery of the mucous membrane immune system in lethally irradiated mice following syngeneic bone marrow transplantation were studied over a 35-day period by quantification of jejunal intraepithelial lymphocytes (IELs) and lamina propria plasma cells. Recovery after a low bone marrow dose allowing 100% animal survival (LBM) was compared with a high (five times minimal) dose (HBM), or a minimal dose augmented with equal numbers of buffy coat cells (LBM + BC) or small gut mucosal lymphocytes (LBM + GL). The maximal decline and subsequent peak repopulation of IELs were: LBM, days 7 through 14, peaking suboptimally by day 28; HBM, day 14, peaking suboptimally but higher than LBM by day 35; LBM + BC, days 11 through 14, peaking at control levels by day 35; and LBM + GL, day 7, peaking at control levels by day 28. The IEL decline was most severe with LBM and HBM treatment and least with LBM + GL. All transplant groups experienced maximal plasma cell decline by day 7. LBM had the most severe depletion, and LBM + GL had the least. Recovery to control levels for the LBM, HBM, LBM + BC and LBM + GL groups occurred by days 28, 21, 21, and 14, respectively. In all instances, greater than 95% of the plasma cells were IgA positive.