Nicolas Dudoignon

Mesenchymal stem cells home to injured tissues when co-infused with hematopoietic cells to treat a radiation-induced multi-organ failure syndrome

Recent studies have suggested that ex vivo expansion of autologous hematopoietic cells could be a therapy of choice for the treatment of bone marrow failure. We investigated the potential of a combined infusion of autologous ex vivo expanded hematopoietic cells with mesenchymal (MSCs) for the treatment of multi‐organ failure syndrome following irradiation in a non‐human primate model.

Renal Anemia Induced by Chronic Ingestion of Depleted Uranium in Rats

Kidney disease is a frequent consequence of heavy metal exposure and renal anemia occurs secondarily to the progression of kidney deterioration into chronic disease. In contrast, little is known about effects on kidney of chronic exposure to low levels of depleted uranium (DU). Study was performed with rats exposed to DU at 40 mg/l by chronic ingestion during 9 months. In the present work, a approximately 20% reduction in red blood cell (RBC) count was observed after DU exposure. Hence, three hypotheses were tested to determinate origin of RBC loss: (1) reduced erythropoiesis, (2) increased RBC degradation, and/or (3) kidney dysfunction. Erythropoiesis was not reduced after exposure to DU as revealed by erythroid progenitors, blood Flt3 ligand and erythropoietin (EPO) blood and kidney levels. Concerning messenger RNA (mRNA) and protein levels of spleen iron recycling markers from RBC degradation (DMT1 [divalent metal transporter 1], iron regulated protein 1, HO1, HO2 [heme oxygenase 1 and 2], cluster of differentiation 36), increase in HO2 and DMT1 mRNA level was induced after chronic exposure to DU. Kidneys of DU-contaminated rats had more frequently high grade tubulo-interstitial and glomerular lesions, accumulated iron more frequently and presented more apoptotic cells. In addition, chronic exposure to DU induced increased gene expression of ceruloplasmin (x12), of DMT1 (x2.5), and decreased mRNA levels of erythropoietin receptor (x0.2). Increased mRNA level of DMT1 was associated to decreased protein level (x0.25). To conclude, a chronic ingestion of DU leads mainly to kidney deterioration that is probably responsible for RBC count decrease in rats. Spleen erythropoiesis and molecules involved in erythrocyte degradation were also modified by chronic DU exposure.

Use of Flt3 Ligand to Evaluate Residual Hematopoiesis after Heterogeneous Irradiation in Mice

Abstract Prat, M., Demarquay, C., Frick, J., Dudoignon, N., Thierry, D. and Bertho, J. M. Use of Flt3 Ligand to Evaluate Residual Hematopoiesis after Heterogeneous Irradiation in Mice. Radiat. Res. 166, 504–511 (2006). We evaluated the possibility of using plasma Flt3 ligand (FL) concentration as a biological indicator of bone marrow function after heterogeneous irradiation. Mice were irradiated with 4, 7.5 or 11 Gy with 25, 50, 75 or 100% of the bone marrow in the field of irradiation. This model of irradiation resulted in graded and controlled damage to the bone marrow. Mice exhibited a pancytopenia correlated with both the radiation dose and the percentage of bone marrow irradiated. The FL concentration in the blood increased with the severity of bone marrow aplasia. Nonlinear regression analysis showed that the FL concentration was strongly correlated with the total number of residual colony-forming cells 3 days after irradiation, allowing a precise estimate of residual hematopoiesis. Moreover, the FL concentration on day 3 postirradiation was correlated with the duration and severity of subsequent pancytopenia, suggesting that variations in FL concentrations might be used as a predictive indicator of bone marrow aplasia, especially by the use of linear regression equations describing these correlations. Our results provide a rationale for the use of FL concentration as a biological indicator of residual hematopoiesis after heterogeneous irradiation.

Application of Autologous Hematopoietic Cell Therapy to a Nonhuman Primate Model of Heterogeneous High-Dose Irradiation

Abstract Bertho, J-M., Prat, M., Frick, J., Demarquay, C., Gaugler, M-H., Dudoignon, N., Clairand, I., Chapel, A., Gorin, N-C., Thierry, D. and Gourmelon, P. Application of Autologous Hematopoietic Cell Therapy to a Nonhuman Primate Model of Heterogeneous High-Dose Irradiation. Radiat. Res. 163, 557– 570 (2005). We developed a model of heterogeneous irradiation in a nonhuman primate to test the feasibility of autologous hematopoietic cell therapy for the treatment of radiation accident victims. Animals were irradiated either with 8 Gy to the body with the right arm shielded to obtain 3.4 Gy irradiation or with 10 Gy total body and 4.4 Gy to the arm. Bone marrow mononuclear cells were harvested either before irradiation or after irradiation from an underexposed area of the arm and were expanded in previously defined culture conditions. We showed that hematopoietic cells harvested after irradiation were able to expand and to engraft when reinjected 7 days after irradiation. Recovery was observed in all 8-Gy-irradiated animals, and evidence for a partial recovery was observed in 10-Gy-irradiated animals. However, in 10-Gy-irradiated animals, digestive disease was observed from day 16 and resulted in the death of two animals. Immunohistological examinations showed damage to the intestine, lungs, liver and kidneys and suggested radiation damage to endothelial cells. Overall, our results provide evidence that such an in vivo model of heterogeneous irradiation may be representative of accidental radiation exposures and may help to define the efficacy of therapeutic interventions such as autologous cell therapy in radiation accident victims.

Absorption of uranium through the entire gastrointestinal tract of the rat.

The aim was to determine the gastrointestinal segments preferentially implicated in the absorption of uranium. The apparent permeability to uranium (233U) was measured ex vivo in Ussing chambers to assess uranium passage in the various parts of the small and large intestines. The transepithelial electrical parameters (potential difference, short-circuit current, transepithelial resistance and tissue conductance) were also recorded for each segment. Determination of in vivo uranium absorption after in-situ deposition of 233U in digestive segments (buccal cavity, ileum and proximal colon) and measurements of uranium in peripheral blood were then made to validate the ex vivo results. In addition, autoradiography was performed to localize the presence of uranium in the digestive segments. The in vivo experiments indicated that uranium absorption from the digestive tract was restricted to the small intestine (with no absorption from the buccal cavity, stomach or large intestine). The apparent permeability to uranium measured with ex vivo techniques was similar in the various parts of small intestine. In addition, the experiments demonstrated the existence of a transcellular pathway for uranium in the small intestine. The study indicates that uranium absorption from the gastrointestinal tract takes place exclusively in the small intestine, probably via a transcellular pathway.

Reinjection of Ex Vivo–Expanded Primate Bone Marrow Mononuclear Cells Strongly Reduces Radiation-Induced Aplasia

To assess the therapeutic efficacy of ex vivo-expanded hematopoietic cells in the treatment of radiation-induced pancytopenia, we have set up a non-human primate model. Two ex vivo expansion protocols for bone marrow mononuclear cells (BMMNC) were studied. The first consisted of a 7-day culture in the presence of stem cell factor (SCF), Flt3-ligand, thrombopoietin (TPO), interleukin-3 (IL-3), and IL-6, which induced preferentially the expansion of immature hematopoietic cells [3.1 +/- 1.4, 10.0 +/- 5.1, 2.2 +/- 1.9, and 1.0 +/- 0.3-fold expansion for mononuclear cells (MNC), colony-forming units-granulocyte-macrophage (CFU-GM), burst-forming units erythroid (BFU-E), and long-term culture initiating cells (LTC-IC) respectively]. The second was with the same cytokine combination supplemented with granulocyte colony-stimulating factor (G-CSF) with an increased duration of culture up to 14 days and induced mainly the production of mature hematopoietic cells (17.2 +/- 11.7-fold expansion for MNC and no detectable BFU-E and LTC-IC), although expansion of CFU-GM (13.7 +/- 18.8-fold) and CD34+ cells (5.2 +/- 1.4-fold) was also observed. Results showed the presence of mesenchymal stem cells and cells from the lymphoid and the megakaryocytic lineages in 7-day expanded BMMNC. To test the ability of ex vivo-expanded cells to sustain hematopoietic recovery after radiation-induced aplasia, non-human primates were irradiated at a supralethal dose of 8 Gy and received the product of either 7-day (24 h after irradiation) or 14-day (8 days after irradiation) expanded BMMNC. Results showed that the 7-day ex vivo-expanded BMMNC shortened the period and the severity of pancytopenia and improved hematopoietic recovery, while the 14 day ex vivo-expanded BMMNC mainly produced a transfusion-like effect during 8 days, followed by hematopoietic recovery. These results suggest that ex vivo expanded BMMNC during 7 days may be highly efficient in the treatment of radiation-induced aplasia.

Short-term effects of depleted uranium on immune status in rat intestine.

In the event of ingestion, the digestive tract is the first biological system exposed to depleted uranium (DU) intake via the intestinal lumen. However, little research has addressed the biological consequences of a contamination with depleted uranium on intestinal properties such as the barrier function and/or the immune status of this tissue. The aim of this study was to determine if the ingestion of depleted uranium led to changes in the gut immune system of the intestine. The experiments were performed at 1 and 3 d following a per os administration of DU to rats at sublethal dose (204 mg/kg). Several parameters refering to the immune status, such as gene and protein expressions of cytokines and chemokines, and localization and density of immune cell populations, were assessed in the intestine. In addition, the overall toxicity of DU on the small intestine was estimated in this study, with histological appearance, proliferation rate, differentiation pattern, and apoptosis process. Firstly, the results of this study indicated that DU was not toxic for the intestine, as measured by the proliferation, differentiation, and apoptosis processes. Concerning the immune properties of the intestine, the ingestion of depleted uranium induced some changes in the production of chemokines and in the expression of cytokines. A diminished production of monocyte chemoattractant protein-1 (MCP-1) was noted at 1 day post exposure. At 3 d, the increased gene expression of interferon γ (IFNγ) was associated with an enhanced mRNA level of Fas ligand, suggesting an activation of the apoptosis pathway. However, no increased apoptotic cells were observed at 3 d in the contaminated animals. There were no changes in the localization and density of neutrophils, helper T lymphocytes, and cytotoxic T lymphocytes after DU administration. In conclusion, these results suggest that depleted uranium is not toxic for the intestine after acute exposure. Nevertheless, DU seems to modulate the expression and/or production of cytokines (IFNγ) and chemokines (MCP-1) in the intestine. Further experiments need to be performed to determine if a chronic contamination at low dose leads in the long term to modifications of cytokines/chemokines patterns, and to subsequent changes in immune response of the intestine.

Survival, lung clearance, dosimetry and gross pathology of rats exposed to either NpO2 or PuO2 aerosols.

Purpose : To compare survival, lung dosimetry and gross pathology after inhalation exposure of rats to either NpO 2 or industrial PuO 2 aerosols with similar granulometric parameters. Because the specific alpha activity ratio Pu/Np is about 600, a much more homogeneous lung irradiation was expected for NpO 2. Materials and methods : Male Sprague Dawley rats were exposed once and their lung burdens were measured by X-ray spectrometry at different times post-exposure up to death. The time-course of doses delivered to the lungs were estimated, taking into account individual lung clearance parameters and body and lung weights. Gross lung pathologies were scored at autopsy. Results : In the range of initial lung deposits (ILD) studied (0.1-4 kBq), lung clearance impairment and reduced lifespan were only observed after exposure to NpO 2. For similar ILD or doses, the highest incidences of lung lesions assumed to be tumours were observed for NpO 2 with a saturation of lung tumour induction for doses larger than 8 Gy (ILD: 1.5kBq). Up to 22Gy (ILD: 3.5kBq), such saturation was not observed for PuO 2. Conclusions : NpO 2 appears much more toxic than PuO 2. Before saturation, lung tumour incidence increased nearly linearly with dose, the slope of the curve for NpO 2 being about twice as steep as that for PuO 2

Evaluation of risk factors for lung tumour induction in rats exposed to either NpO(2) or PuO(2) aerosols.

Purpose : To compare the incidence of each lung tumour type after inhalation exposure of rats to either NpO 2 or industrial PuO 2 aerosols, which have a similar size. Materials and methods : Male Sprague-Dawley rats were exposed once and followed during their whole life span. At the end of their life, the whole lungs were fixed, embedded and cut into thin sections for histological analysis. The presence of tumours was evaluated on three distinct levels of the lobes for phenotype determination to establish dose-effect relationships. Results : In the range of lung doses studied (0.05 to more than 50 Gy), the general trend was an increased frequency of all types of tumours after inhalation exposure to neptunium compared with plutonium. The linearity of the lower part of the dose-effect relationships for all malignant lung tumours leads to the conclusion that NpO 2 is 3.3-fold more carcinogenic than PuO 2. Conclusions : According to a linear extrapolation of the data on malignant lung tumour incidence collected among all studies reported on actinide oxide carcinogenesis, the risk of lung tumour appears to vary over a factor of about 10 depending on the nature and/or size of the aerosol. This variation has to be taken into account for a realistic assessment of tumour risk.

Lung carcinogenesis in rats after inhalation exposure to (237)NpO2.

The results of several studies of experimental carcinogenesis suggest that, after inhalation of alpha-particle emitters, lung tumor incidence varies depending on the exposure rate and dose distribution in the tissue. In the case of transuranics, the main influencing factor would be the specific alpha-particle activity of the inhaled actinide. To confirm these results, long-term studies were performed using male Sprague-Dawley rats exposed to (237)NpO(2) by inhalation. The initial lung burdens of the animals ranged from 0. 1 to about 7 kBq. The rats were followed during their life span and weighed regularly, and their lung burdens were determined in vivo and at death to estimate the lung dose. At death, the incidence of lung tumors and their malignancy and histological types were analyzed. The analysis revealed a typically linear-quadratic dose response for incidence of malignant lung neoplasm and a differential dose response for various types of tumors. Although these results confirm the influence of the activity of the inhaled actinide oxide, further experiments are needed to be able to compare a more homogeneous population of animals.