Ablative radiotherapy improves survival but does not cure autochthonous mouse models of prostate and colorectal cancer.

BACKGROUND: Genetically engineered mouse models (GEMMs) of cancer are powerful tools to study mechanisms of disease progression and therapy response, yet little is known about how these models respond to multimodality therapy used in patients. Radiation therapy (RT) is frequently used to treat localized cancers with curative intent, delay progression of oligometastases, and palliate symptoms of metastatic disease. METHODS: Here we report the development, testing, and validation of a platform to immobilize and target tumors in mice with stereotactic ablative RT (SART). Xenograft and autochthonous tumor models were treated with hypofractionated ablative doses of radiotherapy. RESULTS: We demonstrate that hypofractionated regimens used in clinical practice can be effectively delivered in mouse models. SART alters tumor stroma and the immune environment, improves survival in GEMMs of primary prostate and colorectal cancer, and synergizes with androgen deprivation in prostate cancer. Complete pathologic responses were achieved in xenograft models, but not in GEMMs. CONCLUSIONS: While SART is capable of fully ablating xenografts, it is unable to completely eradicate disease in GEMMs, arguing that resistance to potentially curative therapy can be modeled in GEMMs.

Mice can be used to model the types of cancer seen in people to investigate the effects of cancer therapies, such as radiation. Here, we apply radiation therapy treatments that are able to cure cancer in humans to mice that have cancer of the prostate or colorectum. We show that the mice do not experience many side effects and that the tumours reduce in size, but in some cases show progression after treatment. Our study demonstrates that mice can be used to better understand how human cancers respond to radiation treatment, which can lead to the development of improved treatments and treatment schedules.

A C57L/J Mouse Model of the Delayed Effects of Acute Radiation Exposure in the Context of Evolving Multi-Organ Dysfunction and Failure after Total-Body Irradiation with 2.5% Bone Marrow Sparing.

The objective of the current study was to establish a mouse model of acute radiation syndrome (ARS) after total-body irradiation with 2.5% bone marrow sparing (TBI/BM2.5) that progressed to the delayed effects of acute radiation exposure, specifically pneumonitis and/or pulmonary fibrosis (DEARE-lung), in animals surviving longer than 60 days. Two hundred age and sex matched C57L/J mice were assigned to one of six arms to receive a dose of 9.5 to 13.25 Gy of 320 kV X-ray TBI/BM2.5. A sham-irradiated cohort was included as an age- and sex-matched control. Blood was sampled from the facial vein prior to irradiation and on days 5, 10, 15, 20, 25, and 30 postirradiation for hematology. Respiratory function was monitored at regular intervals throughout the in-life phase. Animals with respiratory dysfunction were administered a single 12-day tapered regimen of dexamethasone, allometrically scaled from a similar regimen in the non-human primate. All animals were monitored daily for up to 224 days postirradiation for signs of organ dysfunction and morbidity/mortality. At euthanasia due to criteria or at the study endpoint, wet lung weights were recorded, and blood sampled for hematology and serum chemistry. The left lung, heart, spleen, small and large intestine, and kidneys were processed for histopathology. A dose-response curve with the estimated lethal dose for 10-99% of animals with 95% confidence intervals was established. The median survival time was significantly prolonged in males as compared to females across the 10.25 to 12.5 Gy dose range. Animal sex played a significant role in overall survival, with males 50% less likely to expire prior to the study endpoint compared to females. All animals developed pancytopenia within the first one- to two-weeks after TBI/BM2.5 followed by a progressive recovery through day 30. Fourteen percent of animals expired during the first 30-days postirradiation due to ARS (e.g., myelosuppression, gastrointestinal tissue abnormalities), with most deaths occurring prior to day 15. Microscopic findings show the presence of radiation pneumonitis as early as day 57. At time points later than day 70, pneumonitis was consistently present in the lungs of mice and the severity was comparable across radiation dose arms. Pulmonary fibrosis was first noted at day 64 but was not consistently present and stable in severity until after day 70. Fibrosis was comparable across radiation dose arms. In conclusion, this study established a multiple organ injury mouse model that progresses through the ARS phase to DEARE-lung, characterized by respiratory dysfunction, and microscopic abnormalities consistent with radiation pneumonitis/fibrosis. The model provides a platform for future development of medical countermeasures for approval and licensure by the U.S. Food and Drug Administration under the animal rule regulatory pathway.

Transitioning from Gamma Rays to X Rays for Comparable Biomedical Research Irradiations: Energy Matters.

Many studies in biomedical research and various allied fields, in which cells or laboratory animals are exposed to radiation, rely on adequate radiation dose standardization for reproducibility and comparability of biological data. Due to increasing concerns regarding international terrorism, the use of radioactive isotopes has recently been met with enhanced security measures. Thus, a growing number of researchers have considered transferring their studies from gamma-ray to kilovoltage X-ray irradiators. Current commercially-available X-ray biological irradiators produce radiation beams with reasonable field geometry and overall dose-homogeneity; however, they operate over a wide range of different energies, both between different models and for a specific unit as well. As a result, the contribution from Compton scattering and the photoelectric effect also varies widely between different irradiators and different beam qualities. The photoelectric effect significantly predominates at the relatively low X-ray energies in which these irradiators operate. Consequently, a higher dose is delivered to bony tissues and the adjacent hematopoietic cells of the bone marrow. The increase in average radiation absorbed dose to the bone marrow compartment of the mouse can be as high as 30%, causing higher hematological sensitivity of animals when exposed to kilovoltage X rays. Adjusting the radiation dose to simply provide biological equivalency is complicated due to steep dose gradients within the marrow tissue and the qualitatively different outcomes depending on the spatial location of critical stem and progenitor populations in relationship to bone. These concerns may be practically addressed by efforts to implement X rays of the highest possible beam energy and penetration and increased awareness that radiation damage to hematopoietic cells will not be identical to data obtained from standard 137Cs gamma rays.

Ex Vivo Selection of Transduced Hematopoietic Stem Cells for Gene Therapy of ß-Hemoglobinopathies.

Although gene transfer to hematopoietic stem cells (HSCs) has shown therapeutic efficacy in recent trials for several individuals with inherited disorders, transduction incompleteness of the HSC population remains a hurdle to yield a cure for all patients with reasonably low integrated vector numbers. In previous attempts at HSC selection, massive loss of transduced HSCs, contamination with non-transduced cells, or lack of applicability to large cell populations has rendered the procedures out of reach for human applications. Here, we fused codon-optimized puromycin N-acetyltransferase to herpes simplex virus thymidine kinase. When expressed from a ubiquitous promoter within a complex lentiviral vector comprising the ßAT87Q-globin gene, viral titers and therapeutic gene expression were maintained at effective levels. Complete selection and preservation of transduced HSCs were achieved after brief exposure to puromycin in the presence of MDR1 blocking agents, suggesting the procedure's suitability for human clinical applications while affording the additional safety of conditional suicide.

A survey of changing trends in modelling radiation lung injury in mice: bringing out the good, the bad, and the uncertain.

Within this millennium there has been resurgence in funding and research dealing with animal models of radiation-induced lung injury to identify and establish predictive biomarkers and effective mitigating agents that are applicable to humans. Most have been performed on mice but there needs to be assurance that the emphasis on such models is not misplaced. We therefore considered it timely to perform a comprehensive appraisal of the literature dealing with radiation lung injury of mice and to critically evaluate the validity and clinical relevance of the research. A total of 357 research papers covering the period of 1970-2015 were extensively reviewed. Whole thorax irradiation (WTI) has become the most common treatment for studying lung injury in mice and distinct trends were seen with regard to the murine strain, radiation dose, intended pathology investigated, length of study, and assays. Recently, the C57BL/6 strain has been increasingly used in the majority of these studies with the notion that they are susceptible to pulmonary fibrosis. Nonetheless, many of these investigations depend on animal survival as the primary end point and neglect the importance of radiation pneumonitis and the anomaly of lethal pleural effusions. A relatively large variation in survival times of C5BL/6 mice is also seen among different institutions pointing to the need for standardization of radiation treatments and environmental conditions. An analysis of mitigating drug treatments is complicated by the fact that the majority of studies are limited to the C57BL/6 strain with a premature termination of the experiments and do not establish whether the treatment actually prevents or simply delays the progression of radiation injury. This survey of the literature has pointed to several improvements that need to be considered in establishing a reliable preclinical murine model of radiation lung injury. The lethality end point should also be used cautiously and with greater emphasis on other assays such as non-invasive lung functional and imaging monitoring in order to quantify specific pulmonary injury that can be better extrapolated to radiation toxicity encountered in our own species.

Microdosimetric and Biological Effects of Photon Irradiation at Different Energies in Bone Marrow.

To ensure reliability and reproducibility of radiobiological data, it is necessary to standardize dosimetry practices across all research institutions. The photoelectric effect predominates over other interactions at low energy and in high atomic number materials such as bone, which can lead to increased dose deposition in soft tissue adjacent to mineral bone due to secondary radiation particles. This may produce radiation effects that deviate from higher energy photon irradiation that best model exposure from clinical radiotherapy or nuclear incidences. Past theoretical considerations have indicated that this process should affect radiation exposure of neighboring bone marrow (BM) and account for reported differences in relative biological effectiveness (RBE) for hematopoietic failure in rodents. The studies described herein definitively estimate spatial dose distribution and biological effectiveness within the BM compartment for (137)Cs gamma rays and 320 kVp X rays at two levels of filtration: 1 and 4 mm Cu half-value layer (HVL). In these studies, we performed: 1. Monte Carlo simulations on a 5 µm resolution model of mouse vertebrae and femur derived from micro-CT images; 2. In vitro biological experiments irradiating BM cells plated directly on the surface of a bone-equivalent material (BEM); and 3. An in vivo study on BM cell survival in irradiated live mice. Simulation results showed that the relative dose increased in proximity to bone at the lower radiation energies and produced averaged values of relative dose over the entire BM volume within imaged trabecular bone of 1.17, 1.08 and 1.01 for beam qualities of 1 mm Cu HVL, 4 mm Cu HVL and (137)Cs, respectively. In accordance with Monte Carlo simulations, in vitro irradiation of BM cells located on BEM and in vivo whole-body irradiation at a prescribed dose to soft tissue of 6 Gy produced relative cell killing of hematopoietic progenitors (CFU-C) that significantly increased for the 1 mm Cu HVL X rays compared to radiation exposures of higher photon energies. Thus, we propose that X rays of the highest possible kVp and filtration be used to investigate radiation effects on the hematopoietic system, as this will allow for better comparisons with high-energy photon exposures applied in radiotherapy or as anticipated in a nuclear event.

Whole-thorax irradiation induces hypoxic respiratory failure, pleural effusions and cardiac remodeling.

To study the mechanisms of death following a single lethal dose of thoracic radiation, WAG/RijCmcr (Wistar) rats were treated with 15 Gy to the whole thorax and followed until they were morbid or sacrificed for invasive assays at 6 weeks. Lung function was assessed by breathing rate and arterial oxygen saturation. Lung structure was evaluated histologically. Cardiac structure and function were examined by echocardiography. The frequency and characteristics of pleural effusions were determined. Morbidity from 15 Gy radiation occurred in all rats 5 to 8 weeks after exposure, coincident with histological pneumonitis. Increases in breathing frequencies peaked at 6 weeks, when profound arterial hypoxia was also recorded. Echocardiography analysis at 6 weeks showed pulmonary hypertension and severe right ventricular enlargement with impaired left ventricular function and cardiac output. Histologic sections of the heart revealed only rare foci of lymphocytic infiltration. Total lung weight more than doubled. Pleural effusions were present in the majority of the irradiated rats and contained elevated protein, but low lactate dehydrogenase, when compared with serum from the same animal. Pleural effusions had a higher percentage of macrophages and large monocytes than neutrophils and contained mast cells that are rarely present in other pathological states. Lethal irradiation to rat lungs leads to hypoxia with infiltration of immune cells, edema and pleural effusion. These changes may contribute to pulmonary vascular and parenchymal injury that result in secondary changes in heart structure and function. We report that conditions resembling congestive heart failure contribute to death during radiation pneumonitis, which indicates new targets for therapy.

Characterization of the dose response relationship for lung injury following acute radiation exposure in three well-established murine strains: developing an interspecies bridge to link animal models with human lung.

Approval of radiation countermeasures through the FDA Animal Rule requires pivotal efficacy screening in one or more species that are expected to react with a response similar to humans (21 C.F.R. § 314.610, drugs; § 601.91, biologics). Animal models used in screening studies should reflect the dose response relationship (DRR), clinical presentation, and pathogenesis of lung injury in humans. Over the past 5 y, the authors have characterized systematically the temporal onset, dose-response relationship (DRR), and pathologic outcomes associated with acute, high dose radiation exposure in three diverse mouse strains. In these studies, C57L/J, CBA/J, and C57BL/6J mice received wide field irradiation to the whole thorax with shielding of the head, abdomen, and forelimbs. Doses were delivered at a rate of 69 cGy min using an x-ray source operated at 320 kVp with half-value layer (HVL) of 1 mm Cu. For all strains, radiation dose was associated significantly with 180 d mortality (p < 0.0001). The lethal dose for 50% of animals within the first 180 d (LD50/180) was 11.35 Gy (95% CI 11.1-11.6 Gy) for C57L/J mice, 14.17 Gy (95% CI 13.9-14.5 Gy) for CBA/J mice, and 14.10 Gy (95% CI 12.2-16.4 Gy) for C57BL/6J mice. The LD50/180 in the C57L/J strain was most closely analogous to the DRR for clinical incidence of pneumonitis in non-human primates (10.28 Gy; 95% CI 9.9-10.7 Gy) and humans (10.60 Gy; 95% CI 9.9-12.1 Gy). Furthermore, in the C57L/J strain, there was no gender-specific difference in DRR (p = 0.5578). The reliability of the murine models is demonstrated by the reproducibility of the dose-response and consistency of disease presentation across studies.Health Phys. 106(1):000-000; 2014.

Do variations in mast cell hyperplasia account for differences in radiation-induced lung injury among different mouse strains, rats and nonhuman primates?

The role of mast cell infiltrates in the pathology of radiation damage to the lung has been a subject of continuing investigation over the past four decades. This has been accompanied by a number of proposals as to how mast cells and the secretory products thereof participate in the generation of acute inflammation (pneumonitis) and the chronic process of collagen deposition (fibrosis). An additional pathophysiology examines the possible connection between mast cell hyperplasia and pulmonary hypertension through the release of vasoactive mediators. The timing and magnitude of pneumonitis and fibrosis are known to vary tremendously among different genetic mouse strains and animal species. Therefore, we have systematically compared mast cell numbers in lung sections from nine mouse strains, two rat strains and nonhuman primates (NHP) after whole thorax irradiation (WTI) at doses ranging from 10-15 Gy and at the time of entering respiratory distress. Mice of the BALB/c strain had a dramatic increase in interstitial mast cell numbers, similar to WAG/Rij and August rats, while relatively low levels of mast cell infiltrate were observed in other mouse strains (CBA, C3H, B6, C57L, WHT and TO mice). Enumeration of mast cell number in five NHPs (rhesus macaque), exhibiting severe pneumonitis at 17 weeks after 10 Gy WTI, also indicated a low response shared by the majority of mouse strains. There appeared to be no relationship between the mast cell response and the strain-dependent susceptibility towards pneumonitis or fibrosis. Further investigations are required to explore the possible participation of mast cells in mediating specific vascular responses and whether a genetically diverse mast cell response occurs in humans.

A preclinical rodent model of radiation-induced lung injury for medical countermeasure screening in accordance with the FDA animal rule.

The purpose of preclinical murine model development is to establish that the pathophysiological outcome of the rodent model of radiation-induced lung injury is sufficiently representative of the anticipated pulmonary response in the human population. This objective is based on concerns that the C57BL/6J strain may not be the most appropriate preclinical model of lethal radiation lung injury in humans. In this study, the authors assessed this issue by evaluating the relationship between morbidity (pulmonary function, histopathologic damage) and mortality among three strains of mice: C57BL/6J, CBA/J, and C57L/J. These different strains display variations in latency and phenotypic expression of radiation-induced lung damage. By comparing the response of each strain to the human pulmonary response, an appropriate animal model(s) of human radiation-induced pulmonary injury was established. Observations in the C57L/J and CBA/J murine models can be extrapolated to the human lung for evaluation of the mechanisms of action of radiation as well as future efficacy testing and approving agents that fall under the "Animal Rule" of the U.S. Food and Drug Administration (FDA) (21 CFR Parts 314 and 601).

Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat ß-thalassemia.

A lentiviral vector encoding ß-globin flanked by insulator elements has been used to treat ß-thalassemia (ß-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting ß-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, ß-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.

A further comparison of pathologies after thoracic irradiation among different mouse strains: finding the best preclinical model for evaluating therapies directed against radiation-induced lung damage.

The human lung is among the most sensitive and critical tissues of concern in localized and systemic radiation exposures, and it is a subject of active preclinical research for evaluating mitigating therapies within the radiation countermeasures program. Our previous study comparing C57BL/6, CBA and C57L mice after whole-thorax irradiation pointed to the problems of late pleural effusions that prevented the full development of lung injury in C57BL/6 mice and suggested that the CBA and C57L strains are more favorable for modeling lung injury in humans (Jackson et al., Radiat. Res. 173, 10-20, 2010). We extended these comparisons to include three other mouse strains (BALB/c, C57BR/J and A/J mice) irradiated with 10, 12.5 or 15 Gy. Most of these mice were unable to survive the first 6 months and presented with a mixture of lung injury and pleural effusions as determined from gross pathology, histology and micro-CT. The independent and varying development of compressive pleural effusions of ill-defined etiology represents a concern for these strains in that they may not satisfy the preclinical requirements for approval of medical countermeasures (e.g. radiation mitigators) for human use. Thus, among the various different mouse strains studied so far for these pathologies, only three (CBA, C3H and C57L) appear to be desirable in exhibiting an early wave of pulmonary dysfunction attributed exclusively to radiation pneumonitis and for further assessment of radioprotective and mitigating therapies. C57L mice are particularly relevant in that they show significant lung damage at lower radiation doses that are closer to what is predicted for humans.

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

Identifying the high radiosensitivity of the lungs of C57L mice in a model of total-body irradiation and bone marrow transplantation.

Pulmonary tissue is sensitive and often treatment-limiting in patients exposed to total-body irradiation (TBI) in preparation for hematopoietic stem cell transplantation. Many rodent strains, however, exhibit a relatively high resistance to radiation lung damage that often requires extra radiation doses to be delivered locally to the thorax to generate significant levels of pulmonary injury. The present study compared the effects of TBI and bone marrow transplantation (BMT) on two mouse strains that are known to differ in lung radiosensitivity after whole-thorax irradiation, namely the relatively resistant CBA mice and the sensitive C57L mice. Evaluation by survival, microcomputerized tomography (micro-CT), lung tissue weights and histopathology showed that the C57L mice responded with severe lethal radiation pneumonitis at 4 months after 12.5 Gy while CBA mice showed only minimal sublethal damage at this dose. C57L mice receiving 10 Gy TBI also had focal fibrotic lesions in the lungs out to 8 months. The manifestation of both pneumonitis and focal fibrosis in the lungs of C57L mice at relatively low radiation doses points to the merits of using this strain in further studies aimed at exploring and ameliorating the high susceptibility of the lung as encountered in clinical TBI.

Revisiting strain-related differences in radiation sensitivity of the mouse lung: recognizing and avoiding the confounding effects of pleural effusions.

The mouse has been used extensively to model radiation injury to the lung, a major dose-limiting organ for radiotherapy. Substantial differences in the timing and sensitivity of this tissue between mouse strains have been reported, with some strains, including C57BL/6, being designated as "fibrosis-prone". Pleural effusions have also been reported to be a prominent problem in many mouse strains, but it remains unclear how this affects the lung function and survival of the standard C57BL/6 mouse. The purpose of this investigation was to re-evaluate this strain in comparison with C57L and CBA mice after whole-thorax irradiation at doses ranging from 10 to 15 Gy. Breathing rate measurements, micro-computerized tomography, lung tissue weight, pleural fluid weight and histopathology showed that the most prominent features were an early phase of pneumonitis (C57L and CBA) followed by a late incidence of massive pleural effusions (CBA and C57BL/6). A remarkable difference was seen between the C57 strains: The C57L mice were exquisitely sensitive to early pneumonitis at 3 to 4 months while C57BL/6 mice showed a delayed response, with most mice presenting with large accumulations of pleural fluid at 6 to 9 months. These results therefore caution against the routine use of C57BL/6 mice in radiation lung experiments because pleural effusions are rarely observed in patients as a consequence of radiotherapy. Future experiments designed to investigate genetic determinants of radiation lung damage should focus on the high sensitivity of the C57L strain (in comparison with CBA or C3H mice) and the possibility that they are more susceptible to pulmonary fibrosis as well as pneumonitis.

Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia.

The interaction of stem cells with their bone marrow microenvironment is a critical process in maintaining normal hematopoiesis. We applied an approach to resolve the spatial organization that underlies these interactions by evaluating the distribution of hematopoietic cell subsets along an in vivo Hoechst 33342 (Ho) dye perfusion gradient. Cells isolated from different bone marrow regions according to Ho fluorescence intensity contained the highest concentration of hematopoietic stem cell (HSC) activity in the lowest end of the Ho gradient (i.e., in the regions reflecting diminished perfusion). Consistent with the ability of Ho perfusion to simulate the level of oxygenation, bone marrow fractions separately enriched for HSCs were found to be the most positive for the binding of the hypoxic marker pimonidazole. Moreover, the in vivo administration of the hypoxic cytotoxic agent tirapazamine exhibited selective toxicity to the primitive stem cell subset. These data collectively indicate that HSCs and the supporting cells of the stem cell niche are predominantly located at the lowest end of an oxygen gradient in the bone marrow with the implication that regionally defined hypoxia plays a fundamental role in regulating stem cell function.

Primitive hematopoietic cell populations reside in the spleen: Studies in the pig, baboon, and human.

OBJECTIVE: We previously observed high levels (>40%) of multilineage hematopoietic cell chimerism following spleen transplantation across full MHC barriers in immunosuppressed miniature swine. We therefore investigated the spleen as a source of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Specific cell-surface markers were used to identify HPCs in the spleen and bone marrow (BM) of young adult (n = 15) and fetal (n = 9) miniature swine by flow cytometry. Hoechst dye-effluxing side population (SP) cells were analyzed in adult spleen, BM, and blood for their expression of c-kit. Functional HPC activity of varying repopulation potential in vitro was investigated by the ability of spleens and BM to give rise to colony-forming units (CFUs) and cobblestone area-forming cells (CAFCs) in long-term stromal cultures. Studies were also carried out on baboon and human spleens and BM. RESULTS: Spleen c-kit+ cells co-expressed more lymphoid markers, but equal myeloid markers, when compared with BM c-kit+ cells. BM and spleen both contained significant percentages of c-kit+ SP cells. Although the frequency of early-forming CFUs in the spleen was only 0.1 to 1.3% of that in the BM, the frequency of CAFCs developing after 8 weeks in culture was comparable to that of BM. Secondary CFUs in long-term culture-initiating cell assays confirmed the presence of long-term repopulating cells at comparable frequencies in spleen and BM. Similar findings were found with regard to baboon and human spleen cells. CONCLUSION: The adult spleen is a relatively rich source of very primitive HPCs, possibly hematopoietic stem cells (HSCs), and may be of therapeutic value.

Addition of treosulfan to a nonmyeloablative conditioning regimen results in enhanced chimerism and immunologic tolerance in an experimental allogeneic bone marrow transplant model.

Treosulfan (L-threitol-1,4-bismethanesulfonate) is an alkylating agent with routine clinical application in the treatment of ovarian cancer. In this murine study we show that this drug also has the ability to deplete primitive hematopoietic stem cells in a dose-dependent manner as determined by the cobblestone area-forming cell assay and is similar to its parent compound busulfan. Because busulfan is frequently used as part of the conditioning regimen before stem cell transplantation, we investigated an alternative nonmyeloablative protocol in an allogeneic bone marrow transplantation model in which low-dose treosulfan was added to an immune-suppressive regimen consisting of T cell-depleting antibodies, fludarabine, and thymic irradiation. Although this treatment protocol produced minimal myelosuppression, the addition of treosulfan proved to be important for allowing stable multilineage and mixed chimerism in C57BL/6 recipients of major histocompatibility complex-mismatched B10.A bone marrow without evidence of graft-versus-host disease. Donor lymphocyte infusion performed at 10 weeks after bone marrow transplantation had the effect of transforming the state of mixed chimerism to full donor-type cells, again without evidence of graft-versus-host disease. Donor-specific immunologic tolerance in the mixed chimeric animals was indicated by the acceptance of donor-type and rejection of third-party skin grafts. Thus, low-dose treosulfan may be considered as a useful component of a truly nonmyeloablative conditioning protocol in providing for mixed hematopoietic chimerism and, consequently, in establishing a platform for adoptive immunotherapy.

Graft-versus-host-reactive donor CD4 cells can induce T cell-mediated rejection of the donor marrow in mixed allogeneic chimeras prepared with nonmyeloablative conditioning.

Murine mixed hematopoietic chimerism can be achieved following nonmyeloablative conditioning with cyclophosphamide, T cell-depleting monoclonal antibodies, and thymic irradiation. Donor lymphocyte infusions (DLIs) 35 days after bone marrow transplantation (BMT) convert mixed to full donor chimerism and mediate graft-versus-lymphoma effects without graft-versus-host disease. We evaluated the role of T-cell subsets in DLIs in converting mixed to full donor chimerism in a fully major histocompatibility complex-mismatched strain combination. Whereas DLIs administered on day 35 converted 100% of mixed chimeras to full donor chimerism, conversion was less frequent when either CD4 or CD8 cells were depleted, indicating that both subsets contribute to the conversion. Surprisingly, administration of CD8-depleted DLIs led to complete loss of donor chimerism in a high proportion (54%) of recipients compared with CD4-plus CD8-depleted DLIs (15%) or CD4-depleted DLIs (0%) (P <.05). DLIs administered at early time points after BMT (eg, day 21) also precipitated rejection of donor marrow by recipient alphabeta T cells, in association with donor CD4 cell expansion and high production of interleukin 2 (IL-2), IL-4, and interferon-gamma. Thus, DLIs can paradoxically induce marrow rejection by residual host alphabeta T cells. These results have implications for the timing of and use of subset depletion of DLIs in recipients of nonmyeloablative transplants.