Making progress and gaining momentum in global 3Rs efforts: how the European pharmaceutical industry is contributing.

Animal research together with other investigational methods (computer modeling, in vitro tests, etc) remains an indispensable part of the pharmaceutical research and development process. The European pharmaceutical industry recognizes the responsibilities inherent in animal research and is committed to applying and enhancing 3Rs principles. New nonsentient, ex vivo, and in vitro methods are developed every day and contribute to reducing and, in some instances, replacing in vivo studies. Their utility is however limited by the extent of our current knowledge and understanding of complex biological systems. Until validated alternative ways to model these complex interactions become available, animals remain indispensable in research and safety testing. In the interim, scientists continue to look for ways to reduce the number of animals needed to obtain valid results, refine experimental techniques to enhance animal welfare, and replace animals with other research methods whenever feasible. As research goals foster increasing cross-sector and international collaboration, momentum is growing to enhance and coordinate scientific innovation globally-beyond a single company, stakeholder group, sector, region, or country. The implementation of 3Rs strategies can be viewed as an integral part of this continuously evolving science, demonstrating the link between science and welfare, benefiting both the development of new medicines and animal welfare. This goal is one of the key objectives of the Research and Animal Welfare working group of the European Federation of Pharmaceutical Industries and Associations.

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.

Correlation between plasma Flt3-ligand concentration and hematopoiesis during G-CSF-induced CD34+ cell mobilization.

This study aimed to correlate blood Flt3-ligand (FL) concentration with CD34(+) cell number in blood and bone marrow (BM) during granulocyte colony-stimulating factor (G-CSF) mobilization. Nonhuman primates were injected with 10 microg/kg of G-CSF (Lenograstim) daily over a period of 5 days. Daily blood sampling and repeated BM sampling showed that FL concentration before mobilization was negatively correlated to the absolute number of BM CD34(+) cells, but also to the number of G-CSF-mobilized CD34(+) cells on days 3-5 of treatment. This showed that FL concentration in the blood reflected BM status before mobilization, and suggested that this parameter could be used as a predictive indicator of G-CSF-induced CD34(+) cell mobilization.

Human mesenchymal stem cells favour healing of the cutaneous radiation syndrome in a xenogenic transplant model.

It has been suggested that human mesenchymal stem cells (hMSC) could be used to repair numerous injured tissues. We have studied the potential use of hMSC to limit radiation-induced skin lesions. Immunodeficient NOD/SCID mice were locally irradiated to the leg (30 Gy, dose rate 2.7 Gy/min) using a (60)Co source to induce a severe skin lesion. Cultured bone marrow hMSC were delivered intravenously to the mice. The irradiated skin samples were studied for the presence of the human cells, the severity of the lesions and the healing process. Macroscopic analysis and histology results showed that the lesions were evolving to a less severe degree of radiation dermatitis after hMSC transplant when compared to irradiated non-transplanted controls. Clinical scores for the studied skin parameters of treated mice were significantly improved. A faster healing was observed when compared to untreated mouse. Immunohistology and polymerase chain reaction analysis provided evidence that the human cells were found in the irradiated area. These results suggest a possible use of hMSC for the treatment of the early phase of the cutaneous radiation syndrome. A successful transplant of stem cells and subsequent reduction in radiation-induced complication may open the road to completely new strategies in cutaneous radiation syndrome therapy.

Use of flt3 ligand to evaluate residual hematopoiesis after heterogeneous irradiation in mice.

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.

Comparison of autologous cell therapy and granulocyte-colony stimulating factor (G-CSF) injection vs. G-CSF injection alone for the treatment of acute radiation syndrome in a non-human primate model.

PURPOSE: To compare the efficacy of autologous cell therapy after irradiation combined with granulocyte-colony stimulating factor (G-CSF) injections with G-CSF treatment alone in a heterogeneous model of irradiation representative of an accidental situation. MATERIAL AND METHODS: Non-human primates were irradiated at 8.7 Gy whole-body dose with the right arm shielded to receive 4.8 Gy. The first group of animals received G-CSF (lenograstim) injections starting 6 h after irradiation, and a second group received a combination of G-CSF (lenograstim) injections and autologous expanded hematopoietic cells. Animals were followed up for blood cell counts, circulating progenitors, and bone marrow cellularity. RESULTS: No significant differences were seen between the two treatment groups, whatever the parameter observed: time to leukocyte or platelet recovery and duration and severity of aplasia. CONCLUSION: Our results indicated that identical recovery kinetic was observed when irradiated animals are treated with G-CSF independently of the reinjection of ex vivo expanded autologous hematopoietic cells. Thus G-CSF injections might be chosen as a first-line therapeutic strategy in the treatment of accidental acute radiation victims.

Application of autologous hematopoietic cell therapy to a nonhuman primate model of heterogeneous high-dose irradiation.

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.

Comparative study on Tp53 gene mutations in lung tumors from rats exposed to 239Pu, 237Np and 222Rn.

The tumor suppressor gene Tp53 was analyzed by polymerase chain reaction-amplification of genomic DNA extracted from paraffin-embedded tissue sections of rat lung tumors to compare mutations that occurred after inhalation exposures to plutonium dioxide, neptunium dioxide, or radon and radon progenies. Exons 5 to 8 of the gene were amplified in 16 plutonium-, 23 neptunium- and 15 radon-induced lung tumors, and their polymerase chain reaction products were examined for mutations by single strand conformational polymorphism analysis and direct sequencing method. Two point mutations were detected in the plutonium-induced tumors, i.e., a guanine to adenine transition at codon 219 of exon 6 and a cytosine to thymine transition at codon 266 of exon 8. Although only one point mutation was found at codon 175 of exon 5 (cytosine to thymine transition) from neptunium-induced tumors, no mutations were detectable from radon-induced tumors. These results indicate that the abnormalities of the Tp53 gene might not be so critical for the pulmonary carcinogenesis after the inhalation of different alpha emitters, even though the presence and frequencies of the Tp53 gene mutations were different.

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

BACKGROUND: 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. METHODS: Hematopoietic cells and MSCs were expanded from bone marrow aspirates. MSCs were transduced with the gene encoding for the green fluorescent protein (e-GFP), in order to track them following infusion. Twelve animals were studied. Nine animals received total-body irradiation at 8 Gy from a neutron/gamma source thus resulting in heterogeneous exposure; three animals were sham-irradiated. The animals were treated with expanded hematopoietic stem cells and MSCs, expanded hematopoietic stem cells alone, or MSCs alone. Unmanipulated bone marrow cell transplants were used as controls. RESULTS: Depending on the neutron/gamma ratio, an acute radiation sickness of varying severity but of similar nature resulted. GFP-labeled cells were found in the injured muscle, skin, bone marrow and gut of the treated animals via PCR up to 82 days post-infusion. CONCLUSIONS: This is the first evidence of expanded MSCs homing in numerous tissues following a severe multi-organ injury in primates. Localization of the transduced MSCs correlated to the severity and geometry of irradiation. A repair process was observed in various tissues. The plasticity potential of the MSCs and their contribution to the repair process in vivo remains to be studied.

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.