mayol@crssa.net Counteracting the hematopoietic syndrome following accidental or intentional irradiation remains an important therapeutic challenge. However, the pathophysiology of accidental irradiation has been recently revisited. Indeed irradiation has to be considered as a global illness that is more complex than the juxtaposition of single syndromes. This was especially illustrated in the Tokäı-Mura case where the two highly irradiated victims died from a complex multiorgan distress then failure syndrome (MODS/MOFS) in spite of transient hematopoietic chimerism following hematopoietic stem cell transplantation. Radiation burns were especially involved in this MOF development. In fact, the pathophysiology of MODS strongly overlaps the systemic inflammatory response syndrome that is mainly the consequence of extensive endothelial cell damage. Clearly, there is a need to develop new therapeutic strategies to counter such radiation-induced extrahematological toxicity, in addition to correcting hematopoietic disorders. Regarding hematopoiesis, the rationale of cytokine/hematopoietic growth factor use is the heterogeneity of bone marrow damage in most documented accidents. Today, early administration of granulopoietic factors is recommended to stimulate residual hematopoiesis in victims irradiated at intermediate dose levels. In complement our group proposes the emergency antiapoptotic cytokine (EACK) therapy in case of high dose irradiation, which consists in preservating and stimulating residual hematopoietic stem and progenitor cells following irradiation. We selected the stem cell factor + Flt-3 ligand + thrombopoietin + interleukin-3 + PegG-CSF antiapoptotic combination that is capable of abrogating thrombocytopenia and reducing neutropenia when given as an early single administration in highly irradiated monkeys (7 Gy gamma) 2 hrs after total body irradiation. Regarding extrahematological toxicity, injection of Keratinocyte growth factor or erythropoietin could be a flexible cytokine complementary/alternative approach to cell therapy (i.e. mesenchymal stem cells grafting). We recently re-evaluated the benefit of using erythropoietin (Epo) as a pleiotropic cytokine to counteract hematologic and extra-hematologic toxicity following lethal irradiation. B6D2F1 mice were globally exposed to 9 Gy gamma (LD90%/30days) and then injected with SFT3 at 2 hours + 24 hours with or without Epo (1000-3000 UI/kg) at 2 hours + 8 days. Epo synergized with SFT3 to rescue lethally irradiated mice from radiation-induced death (60%, 95% and 5% respectively for SFT3, SFT3+Epo and controls at 30 days) whereas Epo alone exhibited no protective effect. Interestingly, hematopoietic parameters did not significantly differ between SFT3 and SFT3+Epo groups. This suggests predominant extra-hematological targets for Epo. Ongoing studies aim at improving EACK strategy in terms of tolerance and efficacy.

Counteracting the hematopoietic syndrome following accidental or intentional irradiation remains an important therapeutic challenge. However, the pathophysiology of accidental irradiation has been recently revisited. Indeed irradiation has to be considered as a global illness that is more complex than the juxtaposition of single syndromes. This was especially illustrated in the Tokaï-Mura case where the two highly irradiated victims died from a complex multiorgan distress then failure syndrome (MODS/MOFS) in spite of transient hematopoietic chimerism following hematopoietic stem cell transplantation. Radiation burns were especially involved in this MOF development. In fact, the pathophysiology of MODS strongly overlaps the systemic inflammatory response syndrome that is mainly the consequence of extensive endothelial cell damage. Clearly, there is a need to develop new therapeutic strategies to counter such radiation-induced extrahematological toxicity, in addition to correcting hematopoietic disorders. Regarding hematopoiesis, the rationale of cytokine/hematopoietic growth factor use is the heterogeneity of bone marrow damage in most documented accidents. Today, early administration of granulopoietic factors is recommended to stimulate residual hematopoiesis in victims irradiated at intermediate dose levels. In complement our group proposes the emergency antiapoptotic cytokine (EACK) therapy in case of high dose irradiation, which consists in preservating and stimulating residual hematopoietic stem and progenitor cells following irradiation. We selected the stem cell factor + Flt-3 ligand + thrombopoietin + interleukin-3 + PegG-CSF antiapoptotic combination that is capable of abrogating thrombocytopenia and reducing neutropenia when given as an early single administration in highly irradiated monkeys (7 Gy gamma) 2 hrs after total body irradiation. Regarding extrahematological toxicity, injection of Keratinocyte growth factor or erythropoietin could be a flexible cytokine complementary/alternative approach to cell therapy (i.e. mesenchymal stem cells grafting). We recently re-evaluated the benefit of using erythropoietin (Epo) as a pleiotropic cytokine to counteract hematologic and extra-hematologic toxicity following lethal irradiation. B6D2F1 mice were globally exposed to 9 Gy gamma (LD90%/30days) and then injected with SFT3 at 2 hours + 24 hours with or without Epo (1000-3000 UI/kg) at 2 hours + 8 days. Epo synergized with SFT3 to rescue lethally irradiated mice from radiation-induced death (60%, 95% and 5% respectively for SFT3, SFT3+Epo and controls at 30 days) whereas Epo alone exhibited no protective effect. Interestingly, hematopoietic parameters did not significantly differ between SFT3 and SFT3+Epo groups. This suggests predominant extra-hematological targets for Epo. Ongoing studies aim at improving EACK strategy in terms of tolerance and efficacy.

POSTERSCD40 ligand (CD40L) and its receptor CD40 on DC is essential for DCactivation and for induction of antigen-specific T-cells responses.In this study, DC pulsed with tumor-lysate and subsequently transduced with an adenovirus (Ad) encoding murine CD40L were injected intratumorally, subcutaneously or intravenously to treat pre-established HCC-tumors in vivo.Methods: Two Ad were generated: Ad-CD40L and Ad-LacZ (encoding LacZ).DC were obtained from bone marrow of C3H-HeNcrl mice and cultured with GM-CSF and 1L-4.DC were pulsed with tumor lysate on day 5 and transduced with adenoviruses (MOI 250) on day 6.Expression of CD40L, CD80 and CD86 was assayed by flow cytometry and TL-12-expression by ELTSA.Subcutaneous HCC-tumors were induced by inoculation of 106 Hepal 29-cells into the right flank ofmice.When tumor volume was 100-200mm3, injection of DC was performed.Results: Transduction of DC with Ad-CD40L induced a strong 1L-12expression of >200 ng/ml/l06 DC in their supernatant.The intratumoral (i.t.)-injection of CD40L-expressing DC induced a significant tumor inhibition of >60% compared to mice treated with LacZ-transduced DC (p i 0.002).Moreover, tumor growth inhibition was significantly higher after it.-DC-applicationthan after S.C. or i.v.treatment: 17 days after tumor induction, the mean tumor volume was 477.5+120 mm3 in i.t.-treated animals vs. 1050.4f167.2 mm3 in i.v.-treated animals, p = 0.02 I . 2 1 days after tumor induction, the mean of tumor volume was 663.1Zt140.7mm' in i.t.treated animals vs. 1374.3&165.4mm3 in s.c.-treated animals (p= 0.0273).The s.c.-application of DC showed also an inhibition oftumor growth compared to the s.c.-treatment with LacZ-expressing DC (p i 0.05) and was significantly more effective than i.v.-administration of CD40L-transduced DC (531.6Zt111.3mm3 vs. 1050.4&167.2mm3on day 21 respectively, p = 0.0323).Conclusions: Our data show that transduction of tumor lysate pulsed DC with Ad-CD40L yields a high CD40L-and IL-12-expression in DC, which could induce a tumor regression of pre-established S.C. hepatocellular tumors.1.t.-application of these engineered DC was more effective than the s.c. or i.v.-injection of these DC.These results favor it.-injection of CD40L-expressing DC for immunotherapy of hepatocellular tumors.

This study was aimed at evaluating the in vitro and in vivo haematopoietic potential in macaque skeletal muscle cells. Biopsy samples showed the presence of CD34(+) (7.6%), CD90(+) (8.4%), CD117(+), CD31(+), side population (SP) cells (7-10%) and a low number of CD45(+) cells. In clonogenic and long-term culture-initiating cell assays, no haematopoietic potential could be detected in either total mononuclear cells or SP cells. Regarding in vivo studies, two animals were transplanted with unfractionated fresh muscle cells after lethal irradiation. Both animals died early after transplant without any evidence of haematopoietic reconstitution. In two other monkeys, harvested muscle cells were frozen and secondarily marked using a green fluorescent protein (GFP)-lentiviral vector. After sublethal irradiation, both animals were transplanted with GFP-expressing muscle cells followed by a bone marrow rescue. Both animals had haematopoietic reconstitution at days 22 and 25, but no GFP-expressing haematopoietic cells could be detected by flow cytometry, either in the blood or in clonogenic cells from marrow aspirates. Using PCR assays, GFP(+) cells were detected in a single marrow sample of one animal at 41 days after transplantation. These results strongly suggest that as opposed to murine muscle, the non-human primate skeletal muscle does not harbour cells with a straightforward haematopoietic potential.

Ex vivo expansion of residual autologous hematopoietic stem and progenitor cells collected from victims soon after accidental irradiation (autologous cell therapy) may represent an additional or alternative approach to cytokine therapy or allogeneic transplantation. Peripheral blood CD34(+) cells could be a useful source of cells for this process provided that collection and ex vivo expansion of hematopoietic stem and progenitor cells could be optimized. Here we investigated whether mesenchymal stem cells could sustain culture of irradiated peripheral blood CD34(+) cells. In vitro irradiated (4 Gy Co-60 gamma rays) or nonirradiated mobilized peripheral blood CD34(+) cells from baboons were cultured for 7 days in a serum-free medium supplemented with stem cell factor + thrombopoietin + interleukin 3 + FLT3 ligand (50 ng/ml each) in the presence or absence of mesenchymal stem cells. In contrast to cultures without mesenchymal stem cells, irradiated CD34(+) cells cultured with mesenchymal stem cells displayed cell amplification, i.e. CD34(+) (4.9-fold), CD34(++) (3.8-fold), CD34(++)/Thy-1(+) (8.1-fold), CD41(+) (12.4-fold) and MPO+ (50.6-fold), although at lower levels than in nonirradiated CD34+ cells. Fourteen times more clonogenic cells, especially BFU-E, were preserved when irradiated cells were cultured on mesenchymal stem cells. Moreover, we showed that the effect of mesenchymal stem cells is related mainly to the reduction of apoptosis and involves cell-cell contact rather than production of soluble factor(s). This experimental model suggests that mesenchymal stem cells could provide a crucial tool for antologous cell therapy applied to accidentally irradiated Victims. (c) 2005 by Radiation Research Society.

Radiation-induced (RI) tissue injuries can be caused by radiation therapy, nuclear accidents or radiological terrorism. Notwithstanding the complexity of RI pathophysiology, there are some effective approaches to treatment of both acute and chronic radiation damages. Cytokine therapy is the main strategy capable of preventing or reducing the acute radiation syndrome (ARS), and hematopoietic growth factors (GF) are particularly effective in mitigating bone marrow (BM) aplasia and stimulating hematopoietic recovery. However, first, as a consequence of RI stem and progenitor cell death, use of cytokines should be restricted to a range of intermediate radiation doses (3 to 7 Gy total body irradiation). Second, ARS is a global illness that requires treatment of damages to other tissues (epithelial, endothelial, glial, etc.), which could be achieved using pleiotropic or tissue-specific cytokines. Stem cell therapy (SCT) is a promising approach developed in the laboratory that could expand the ability to treat severe radiation injuries. Allogeneic hematopoietic stem cell transplantation (BM, mobilized peripheral blood and cord blood) transplantation has been used in radiation casualties with variable success due to limiting toxicity related to the degree of graft histocompatibility and combined injuries. Ex vivo expansion should be used to augment cord blood graft size and/or promote very immature stem cells. Autologous SCT might also be applied to radiation casualties from residual hematopoietic stem and progenitor cells (HSPC). Stem cell plasticity of different tissues such as liver or skeletal muscle, may also be used as a source of hematopoietic stem cells. Finally, other types of stem cells such as mesenchymal, endothelial stem cells or other tissue committed stem cells (TCSC), could be used for treating damages to nonhematopoietic organs.

Autologous stem cell therapy (ACT) has been proposed to prevent irradiated victims from bone marrow (BM) aplasia by grafting hematopoietic stem and progenitor cells (HSPCs) collected early after damage, provided that a functional graft of sufficient size could be produced ex vivo. To address this issue, we set up a baboon model of cell therapy in which autologous peripheral blood HSPCs collected before lethal total body irradiation were irradiated in vitro (2.5 Gy, D0 1 Gy) to mimic the cell damage, cultured in small numbers for a week in a serum-free medium in the presence of antiapoptotic cytokines and mesenchymal stem cells (MSCs) and then cografted. Our study shows that baboons cografted with expanded cells issued from 0.75 and 1 x 10(6)/kg irradiated CD34+ cells and MSCs (n=2) exhibited a stable long-term multilineage engraftment. Hematopoietic recovery became uncertain when reducing the CD34+ cell input (0.4 x 10(6)/kg CD34+ cells; n=3). However, platelet recovery was accelerated in all surviving cografted animals, when compared with baboons transplanted with unirradiated, unmanipulated CD34+ cells (0.5-1 x 10(6)/kg, n=4). Baboons grafted with MSCs alone (n=3) did not recover. In all cases, the nonhematopoietic toxicity remained huge. This baboon study suggests that ACT feasibility is limited.

Preservation of hematopoietic stem and progenitor cell survival is required for recovery from radiation-induced myelosuppression. We recently showed that short-term injection of antiapoptotic cytokine combinations into mice soon after lethal gamma irradiation promoted survival. The present study investigated the hematopoietic response of cynomolgus monkeys to a single dose of stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination (4F, each factor given intravenously at 50 microg/kg) administered 2 hours after 5-Gy gamma irradiation. Treated monkeys (n = 4) experienced no thrombocytopenia. Only 1 in 4 displayed a transient period of neutropenia (neutrophil [ANC] count < 0.5 x 10(9)/L), whereas all irradiated controls (n = 4) experienced neutropenia (5-12 days) and thrombocytopenia (platelet [PLT] count < 20 x 10(9)/L, 5-31 days). Treated animals exhibited an impressive 2-wave PLT response that peaked at days 8 and 22 after total body irradiation (TBI). Areas under the curve (AUC) of PLTs, ANCs, white blood cells (WBCs), and red blood cells (RBCs) between days 0 and 90 were significantly higher in treated animals than in controls. Humeral bone marrow-derived clonogenic activity was significantly spared at 24 hours and 4 days after TBI in treated monkeys. No apparent impairment of the hematopoietic status and stem cell pool, in terms of long-term culture-initiating cells (LTC-ICs) and side population (SP) cells, was observed after 15 months. These results strongly suggest that the 4F cytokine combination, as a single dose regimen, could act as an emergency treatment for nuclear accident or terrorism victims.

Preservation of hematopoietic stem and progenitor cell survival is required for recovery from radiation-induced myelosuppression. We recently showed the capacity of a single injection of antiapoptotic cytokines, namely stem cell factor, FLT-3 ligand, megakaryocyte growth and development factor, and interleukin-3 in combination (4F, each factor given intravenously at 50 μg/kg) administered shortly after a 5 Gy gamma total body irradiation to prevent monkeys from myelosuppression. As cytokine efficacy depends on the residual stem cell pool, the aim of the present study was to assess the efficacy of 4F in case of high dose irradiation. Adult monkeys (n=7) were globally and frontally irradiated at 7 Gy gamma using a Co60 source (dose rate 20 cGy/mn). Three animals received 4F 2 hours after irradiation and four control animals were injected with the diluant at the same time. Moreover, one of the treated monkeys was grafted with allogeneic mesenchymal stem cells (MSC) to enhance hematopoietic recovery. Human MSCs were infused directly in humerus simultaneously with 4F injection, allowing cell tracking. Treated monkeys experienced a very short period of thrombocytopenia as compared with untreated animals (platelet [PLT] count < 20 x 109/L: 1 ± 1.7 day versus 27 ± 18 days). Areas under the curve (AUC) of PLTs and red blood cells (RBCs) between days 0 and days 90 were higher in treated animals than in controls. The animal treated with 4F + MSC did not differ from the two 4F monkeys. In contrast with 5 Gy irradiated animals previously studied, both treated and untreated monkeys experienced a prolonged period of neutropenia (neutrophil [ANC] count < 0.5 x 109/L: 12 ± 3 days versus 10.3 ± 7.3) and AUCs of ANC did not significantly differ. This study shows that the 4F treatment, as a single dose regimen, prevents thrombocytopenia in high dose irradiation setting and could act as an emergency treatment for nuclear accident or terrorism victims.

Recovery from radiation-induced (RI) myelosuppression depends on hematopoietic stem and progenitor cell survival and the active proliferation/differentiation process, which requires early cytokine support. Single cytokine or late-acting growth factor therapy has proved to be inefficient in ensuring reconstitution after severe RI damage. This work was aimed at evaluating the in vivo survival effect of combinations of early-acting cytokines whose antiapoptotic activity has been demonstrated in vitro: stem cell factor (SCF [S]), FMS-like tyrosine kinase 3 ligand (FLT-3 ligand [F]), thrombopoietin (TPO [T]), interleukin-3 (IL-3 [3]), and stromal derived factor-1 (SDF-1). B6D2F1 mice underwent total body irradiation at 8 Gy cesium Cs 137 gamma radiation (ie, lethal dose 90% at 30 days) and were treated soon after irradiation, at 2 hours and at 24 hours, with recombinant murine cytokines, each given intraperitoneally at 50 microg/kg per injection. All treatments induced 30-day survival rates significantly higher than control (survival rate, 8.3%). 4F (SFT3) and 5F (4F + SDF-1) were the most efficient combinations (81.2% and 87.5%, respectively), which was better than 3F (SFT, 50%), TPO alone (58.3%), and SDF-1 alone (29.2%) and also better than 4F given at 10 microg/kg per injection (4F10, 45.8%) or as a 50 microg/kg single injection at 2 hours (4Fs, 62.5%). Despite delayed death occurring mainly from day 150 on and possible long-term hematopoiesis impairment, half the 30-day protective effects of 4F and 5F were preserved at 300 days. Our results show that short- and long-term survival after irradiation depends on appropriate multiple cytokine combinations and at optimal concentrations. The proposal is made that an emergency cytokine regimen could be applied to nuclear accident victims as part of longer cytokine treatment, cell therapy, or both.

Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 2–10 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34+cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng·mL–1(15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-alpha(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34+cells after 6 days in a serum free culture medium (SFM) (kCD34+= 4.3 and 6.3 respectively for 4F and successive 4F + TNF-alpha/ 4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (kCD34+= 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident.Key words: apoptosis, cytokine, hematopoiesis, irradiation, bone marrow aplasia.[Journal translation]

Abstract: Bone marrow aplasia observed following ionizing radiation exposure (Total Body Irradiation; gamma dose range: 2-10 Gy) is a result, in particular, of the radiation-induced (RI) apoptosis in hematopoietic stem and progenitor cells (HSPC). We have previously shown in a baboon model of mobilized peripheral blood CD34+ cell irradiation in vitro that RI apoptosis in HSPC was an early event, mostly occurring within the first 24 hours, which involves the CD95 Fas pathway. Apoptosis may be significantly reduced with a combination of 4 cytokines (4F): Stem Cell Factor (SCF), FLT-3 Ligand (FL), thrombopoietin (TPO), and interleukin-3 (IL-3), each at 50 ng x mL(-1) (15% survival versus <3% untreated cells, 24 h post-irradiation at 2.5 Gy). In this study we show that addition of TNF-alpha(800 IU/ml) induces an increase in 4F efficacy in terms of cell survival 24 h after incubation (26% survival after 24 h irradiation exposure at 2.5 Gy) and amplification (k) of CD34+ cells after 6 days in a serum free culture medium (SFM) (kCD34+ = 4.3 and 6.3 respectively for 4F and successive 4F + TNF-a/ 4F treatments). In addition, the 4F combination allows culture on pre-established allogenic irradiated stromal cells in vitro at 4 Gy (kCD34+ = 4.5). Overall this study suggests (i) the potential therapeutic interest for an early administration of anti-apoptotic cytokines with or without hematopoiesis inhibitors (emergency cytokine therapy) and (ii) the feasibility in the accidentally irradiated individual, of autologous cell therapy based on ex vivo expansion in order to perform autograft of residual HSPC collected after the accident.