Stem cell therapy: from bench to bedside.

Several countries have increased efforts to develop medical countermeasures to protect against radiation toxicity due to acts of bioterrorism as well as cancer treatment. Both acute radiation injuries and delayed effects such as cutaneous effects and impaired wound repair depend, to some extent, on angiogenesis deficiency. Vascular damage influences levels of nutrients, oxygen available to skin tissue and epithelial cell viability. Consequently, the evolution of radiation lesions often becomes uncontrolled and surgery is the final option--amputation leading to a disability. Therefore, the development of strategies designed to promote healing of radiation injuries is a major therapeutic challenge. Adult mesenchymal stem cell therapy has been combined with surgery in some cases and not in others and successfully applied in patients with accidental radiation injuries. Although research in the field of radiation skin injury management has made substantial progress in the past 10 y, several strategies are still needed in order to enhance the beneficial effect of stem cell therapy and to counteract the deleterious effect of an irradiated tissue environment. This review summarises the current and evolving advances concerning basic and translational research based on stem cell therapy for the management of radiological burns.

Numerical dosimetric reconstruction of a radiological accident in South America in April 2009.

A severe irradiation accident involving a victim occurred in April 2009 in South America. The victim has found a (192)Ir source fallen from a gammagraphy device and has put it in the left pocket of his pants. Very quickly, an erythema and a blister appeared on the left leg of the victim involving hospitalisation. Following the request of the IAEA assistance, the Ionizing Radiation Dosimetry Laboratory of IRSN was asked to perform a numerical dosimetric reconstruction. A personalised voxel phantom of the victim has been constructed thanks to the Simulation of External Source Accident with Medical images tool developed by the laboratory, and a calculation of the dose with the MCNPX computer code allowed to determine the boundary of the necrotic dose at 25 Gy. On the basis of these calculations, the physicians have performed exeresis of the necrotic region on the left leg on 4 May 2009. Associated with mesenchymal stem cell injection, the leg of the victim was healthy on December 2009.

[Radiation burn "innovating therapeutic approach"]. / Brûlure par irradiation « approche thérapeutique innovante ¼.

Radiation burn is a determinist effect of localized irradiation. The lesion is in good correlation with absorbed dose. Radiation burn is different from thermal burn. The evolution is spatiotemporal unpredictable with successive inflammatory waves and recurrence of necrosis. The conventional surgical treatment is rarely efficient because each surgical operative act seems to stimulate the inflammatory waves and fibro-necrosis process. The lesion can escape to this conventional surgical treatment. The new therapeutic approach combines surgery and cellular therapy with local administration of autologous mesenchymal stem cells. From 5 years, cell therapy have been an adjuvant treatment of surgery. This association is a therapeutic innovation, it's now the recommendation for conservative surgery of this very serious radiation burn.

Mesenchymal stem cells improve small intestinal integrity through regulation of endogenous epithelial cell homeostasis.

Patients who undergo pelvic or abdominal radiotherapy may develop acute and/or chronic side effects resulting from gastrointestinal tract (GIT) alterations. In this study, we address the question of the regenerative capability of mesenchymal stem cells (MSC) after radiation-induced GIT injury. We also propose cellular targets of MSC therapy. We report that the infusion of human bone marrow-derived MSC (hMSC) provides a therapeutic benefit to NOD/SCID mice undergoing radiation-induced GIT failure. We observed that hMSC treatment brings about fast recovery of the small intestine (structure and function) in mice with reversible alterations and extends the life of mice with irreversible GIT disorders. The effects of hMSC are a consequence of their ability to improve the renewal capability of small intestinal epithelium. hMSC treatment favors the re-establishment of cellular homeostasis by both increasing endogenous proliferation processes (Ki67 immunostaining) and inhibiting apoptosis (TUNEL staining) of radiation-induced small intestinal epithelial cells. Our results suggest that MSC infusion may be used as a therapeutic treatment to limit radiation-induced GIT damage.

Molecular modifications of cholesterol metabolism in the liver and the brain after chronic contamination with cesium 137.

Twenty years after Chernobyl accident, the daily ingestion of foodstuff grown on contaminated grounds remains the main source for internal exposure to ionizing radiations, and primarily to cesium 137 ((137)Cs). Though the effects of a long-term internal contamination with radionuclides are poorly documented, several non-cancerous pathologies have been described in this population. However, lipid metabolism was never investigated after chronic internal contamination although disturbances were observed in externally-exposed people. In this regard, we assessed the effects of a chronic ingestion of (137)Cs on hepatic and cerebral cholesterol metabolism. To mimic a chronically-exposed population, rats were given (137)Cs-supplemented water at a post-accidental dose (150 Bq/rat/day) during 9 months. The plasma profile, and brain and liver cholesterol concentrations were unchanged. A decrease of ACAT 2, Apo E, and LXRmRNA levels was recorded in the liver. In the brain, a decrease of CYP27A1 and ACAT 1 gene expression was observed. These results clearly show that cholesterol metabolism is not disrupted by a chronic ingestion of (137)Cs, although several molecular alterations are observed. This work would be interestingly completed by studying the influence of (137)Cs in models likely more sensitive to contaminants, such as the fetus or individuals susceptible to a lipidic disease.

Cell therapy based on adipose tissue-derived stromal cells promotes physiological and pathological wound healing.

OBJECTIVE: We hypothesized that adipose tissue may contain progenitors cells with cutaneous and angiogenic potential. METHODS AND RESULTS: Adipose tissue-derived stroma cells (ADSCs) were administrated to skin punched wounds of both nonirradiated and irradiated mice (20 Gy, locally). At day 14, ADSCs promoted dermal wound healing and enhanced wound closure, viscolesticity, and collagen tissue secretion in both irradiated and nonirradiated mice. Interestingly, GFP-positive ADSCs incorporated in dermal and epidermal tissue in vivo and expressed epidermal markers K5 and K14. Cultured ADSCs in keratinocyte medium have been shown to differentiate into K5- and K14-positive cells and produced high levels of KGF. At Day 7, ADSCs also improved skin blood perfusion assessed by laser Doppler imaging, capillary density, and VEGF plasma levels in both irradiated and nonirradiated animals. GFP-positive ADSCs incorporated into capillary structures in vivo and expressed the endothelial cell marker CD31. Finally, in situ interphase fluorescence hybridization showed that a small number of ADSCs have the potential to fuse with endogenous keratinocytes. CONCLUSIONS: ADSCs participate in dermal wound healing in physiological and pathological conditions by their ability to promote reepithelialization and angiogenesis. Hence, adipose lineage cells represent a new cell source for therapeutic dermal wound healing.

[Uranium: properties and biological effects after internal contamination]. / Uranium: propriétés et effets biologiques après contamination interne.

Uranium is a radionuclide present in the environment since the origin of the Earth. In addition to natural uranium, recent deposits from industrial or military activities are acknowledged. Uranium's toxicity is due to a combination of its chemical (heavy metal) and radiological properties (emission of ionizing radiations). Acute toxicity induces an important weight loss and signs of renal and cerebral impairment. Alterations of bone growth, modifications of the reproductive system and carcinogenic effects are also often seen. On the contrary, the biological effects of a chronic exposure to low doses are unwell known. However, results from different recent studies suggest that a chronic contamination with low levels of uranium induces subtle but significant levels. Indeed, an internal contamination of rats for several weeks leads to detection of uranium in many cerebral structures, in association with an alteration of short-term memory and an increase of anxiety level. Biological effects of uranium on the metabolisms of xenobiotics, steroid hormones and vitamin D were described in the liver, testis and kidneys. These recent scientific data suggest that uranium could participate to increase of health risks linked to environmental pollution.

Lessons from recent accidents in radiation therapy in France.

Many accidents in radiotherapy have been reported in France over the last years. This is due to the recent legal obligation to declare to the national safety authorities any significant incident relative to the use of ionising radiation including medical applications. The causes and consequences of the most serious events in radiotherapy are presented in this paper. Lessons can be learned from possible technical dysfunctions, from human errors or organisational weaknesses as to how such events can be prevented. The technical aspects are addressed here: in particular, dosimetric issues.

New biological indicators to evaluate and monitor radiation-induced damage: an accident case report.

The aim of this work was to use several new biological indicators to evaluate damage to the main physiological systems in a victim exposed accidentally to ionizing radiation. Blood samples were used for biological dosimetry and for measurement of the plasma concentrations of several molecules: Flt3 ligand to assess the hematopoietic system, citrulline as an indicator of the digestive tract, and several oxysterols as lipid metabolism and vascular markers. The cytogenetic evaluation estimated the dose to the victim to be between 4.2 and 4.8 Gy, depending on the methodology used. Monitoring the Flt3 ligand demonstrated the severity of bone marrow aplasia. In contrast, the citrulline concentration showed the absence of gastrointestinal damage. Variations in oxysterol concentrations suggested radiation-induced damage to the liver and the cardiovascular system. These results were correlated with those from classic biochemical markers, which demonstrated severe damage to the hematopoietic system and suggested the appearance of subclinical damage to the liver and cardiovascular system. These results demonstrate for the first time the importance of a multiparameter biological approach in the evaluation of radiation damage after accidental irradiation.

New approach to radiation burn treatment by dosimetry-guided surgery combined with autologous mesenchymal stem cell therapy.

The therapeutic management of severe radiation burns remains a challenging issue. Conventional surgical treatment (excision and skin autograft or rotation flap) often fails to prevent unpredictable and uncontrolled extension of the radiation necrotic process. We report here an innovative therapeutic strategy applied to the victim of a radiation accident (December 15, 2005) with an iridium gammagraphy radioactive source (192Ir, 3.3 TBq). The approach combined numerical dosimetry-guided surgery with cellular therapy using mesenchymal stem cells. A very severe buttock radiation burn (2000 Gy at the center of the skin surface lesion) of a 27-year-old Chilean victim was widely excised (10 cm in diameter) using a physical and anatomical dose reconstruction in order to better define the limit of the surgical excision in apparently healthy tissues. A secondary extension of the radiation necrosis led to a new excision of fibronecrotic tissues associated with a local cellular therapy using autologous expanded mesenchymal stem cells as a source of trophic factors to promote tissue regeneration. Bone marrow-derived mesenchymal stem cells were expanded according to a clinical-grade technique using closed culture devices and serum-free medium enriched in human platelet lysate. The clinical evolution (radiation pain and healing progression) was favorable and no recurrence of radiation inflammatory waves was observed during the 11 month patient's follow-up. This novel multidisciplinary therapeutic approach combining physical techniques, surgical procedures and cellular therapy with adult stem cells may be of clinical relevance for improving the medical management of severe localized irradiations. It may open new prospects in the field of radiotherapy complications.

Comparison of the effects of enriched uranium and 137-cesium on the behaviour of rats after chronic exposure.

PURPOSE: A radionuclide that accumulates in the central nervous system is likely to exert both a chemical and a radiological effect. The present study aimed at assessing the behavioral effect of two radionuclides previously shown to accumulate in the central nervous system after chronic exposure--uranium and cesium. MATERIALS AND METHODS: Rats were exposed for 9 months to drinking water contaminated with either enriched uranium at a dosage of 40 mg U x l(-1) or 137-cesium at a dosage of 6500 Bq x l(-1), which correspond to the highest concentrations measured in some wells in the south of Finland (uranium) or in the milk in Belarus in the year following the Chernobyl accident (137-cesium). RESULTS: At this level of exposure, 137-cesium had no effect on the locomotor activity measured in an open-field, on immobility time in a forced swimming test, on spontaneous alternation in a Y-maze and on novel object exploration in an object recognition test. Enriched uranium exposure specifically reduced the spontaneous alternation measured in the Y-maze after 3 and 9 months exposure although it did not affect the other parameters. CONCLUSION: Enriched uranium exposure altered the spatial working memory capacities and this effect was correlated with previously described accumulation of uranium in the hippocampus which is one of the cerebral areas involved in this memory system.

Chronic contamination with 137cesium in rat: effect on liver cholesterol metabolism.

After the Chernobyl nuclear accident, epidemiological studies on human populations living in 137Cs-contaminated areas revealed the increase frequencies of thyroid cancer and evoked the apparition of cardiovascular diseases, hormonal effect, liver alteration, and lipid disorder. Actually, it raises a problem of public safety for the populations living on these territories that are exposed to low levels of 137Cs during a long period through food. Then it is necessary to study potential effect of this chronic contamination. To mimic this situation, the authors investigate the potential biological effects of chronic exposure to 137Cs at a postaccidental dose (150 Bq/rat/day) on hepatic metabolism of cholesterol in rat. Plasma lipid level, gene expression and activity were analyzed. It was observed that in 137Cs-exposed rats, gene expression of low-density lipoprotein receptor (LDLr), apolipoprotein B (apoB), and liver X receptor alpha (LXRalpha) are increased (95%, p < .05; 34%, p < .05; 20%, p < 0.05, respectively), whereas transporter adenosine triphosphate-binding cassette transporter G5 (ABCG5) is decreased (42%, p < .05). In addition, cytochrome P450 27A1 (CYP27A1) activity is increased (34%, p < .05) in contaminated rat liver. In conclusion, the results suggest that 137Cs contamination at low-level induces molecular modifications of the liver cholesterol metabolism without leading to a dysregulation of its homeostasis. These results suggest that chronic long term exposure at low-level of 137Cs may evolve to lipid disorder.

Study of the tools available in biological dosimetry to estimate the dose in cases of accidental complex overexposure to ionizing radiation: the Lilo accident.

PURPOSE: To compare the efficiency of different cytogenetic tools in estimating the doses received by four people involved in the Lilo accident and to monitor the dose estimate over 4.5 years. MATERIALS AND METHODS: Several young Georgian frontier guards handled at least one of the 12 Caesium sources found in a former Russian military camp. Overexposure lasted from July 1996 to May 1997. The Institute for Radiological Protection and Nuclear Safety (IRSN) obtained blood samples taken at several intervals post-exposure from the four most highly-exposed people. Dose estimation was performed using dicentric and translocation scoring. RESULTS: The first dose estimations performed by dicentric scoring gave whole-body doses ranging from 0.4 to 1.3 Gy. Overexposure was complex and several mathematical models were used to take this complexity into account. This could provide information concerning the circumstances of overexposure. Concerning follow-up, the yield of dicentrics decreased by about 50% in the first 4 months following the end of overexposure whereas translocations were stable over the period of analysis. CONCLUSION: It has been useful to compare cytogenetic results with clinical results. The results presented here reveal good stability of translocations. However the first dose estimation was not attempted until 6 months after the last exposure.

PCC-FISH in skin fibroblasts for local dose assessment: biodosimetric analysis of a victim of the Georgian radiological accident.

We propose a new method of biodosimetry that could be applied in cases of localized irradiation. The approach is based on excess chromosome segments determination by the PCC-FISH technique in fibroblasts isolated from skin biopsy. Typically, 0 to 10 Gy ex vivo gamma-irradiated human skin biopsies were dissociated and fibroblasts were isolated and grown for several days. Cells next underwent PCC-FISH painting of whole chromosome 4, and the number of excess chromosome segments per metaphase was determined. An ex vivo reference curve correlating the number of excess chromosome segments per metaphase to the radiation dose was established and used to assess the dose delivered to the skin of one of the victims of the radiological accident that occurred at Lia in Georgia in December 2001. Specifically, the victim suffering from moist desquamation underwent skin excision in Hospital Percy (France). Measurement of excess chromosome segments per metaphase was done in fibroblasts isolated and grown from removed wounded skin and subsequent conversion to radiation doses was performed. The radiation dose map obtained was shown to be in accordance with clinical data and physical dosimetry as well as with conventional biodosimetry. These results demonstrated that PCC-FISH painting applied to skin fibroblasts may be a suitable technique for dose estimation. To assess its worth, this approach needs to be extended to future accidents involving localized radiation exposure.

Alteration of the enterohepatic recirculation of bile acids in rats after exposure to ionizing radiation.

The aim of this work was to study acute alterations of the enterohepatic recirculation (EHR) of bile acids 3 days after an 8-Gy radiation exposure in vivo in the rat by a washout technique. Using this technique in association with HPLC analysis, the EHR of the major individual bile acids was determined in control and irradiated animals. Ex vivo ileal taurocholate absorption was also studied in Ussing chambers. Major hepatic enzyme activities involved in bile acid synthesis were also measured. Measurements of bile acid intestinal content and intestinal absorption efficiency calculation from washout showed reduced intestinal absorption with significant differences from one bile acid to another: absorption of taurocholate and tauromuricholate was decreased, whereas absorption of the more hydrophobic taurochenodeoxycholate was increased, suggesting that intestinal passive diffusion was enhanced, whereas ileal active transport might be reduced. Basal hepatic secretion was increased only for taurocholate, in accordance with the marked increase of CYP8B1 activity in the liver. The results are clearly demonstrate that concomitantly with radiation-induced intestinal bile acid malabsorption, hepatic bile acid synthesis and secretion are also changed. A current working model for pathophysiological changes in enterohepatic recycling after irradiation is thus proposed.

Feasibility and limits of bone marrow mononuclear cell expansion following irradiation.

PURPOSE: To define the ability of bone marrow mononuclear cells (BMMNC) to expand after irradiation and to determine the amount of apoptosis in irradiated expanded cells. MATERIALS AND METHODS: Non-human primate BMMNC were irradiated in vitro at doses ranging from 0 to 4 Gy and were cultured during 1 week in the presence of interleukin 3, interleukin 6, stem cell factor, thrombopoietin and fms-like tyrosine kinase-3 ligand. The expansion yield of BMMNC, colony-forming cells and CD34(+) cells were compared with non-irradiated control cultures. Apoptosis in expanded cells was also defined by annexin V/propidium iodine staining. RESULTS: Irradiation of BMMNC up to 1 Gy did not modify the ability of haematopoietic cells to expand. At higher doses, expansion of haematopoietic cells is reduced as compared with non-irradiated cultures but it remains significant. This reduction in expansion of BMMNC was related to radiation-induced apoptosis. CONCLUSION: The results suggest that it is possible to expand haematopoietic cells after irradiation doses at least up to 2 Gy. This suggests a possible use of cell therapy for the treatment of radiation accident victims.

IL-1beta, TNFalpha and IL-6 induction in the rat brain after partial-body irradiation: role of vagal afferents.

PURPOSE: To evaluate the central nervous system neuroimmune and inflammatory responses during the prodromal phase of the acute irradiation syndrome in rat brains after partial-body exposure (head-protected) and to investigate the potential neural signalling pathways from the irradiated periphery to the non-irradiated brain. MATERIAL AND METHODS: The study included four groups of rats: one irradiated group and one sham irradiated group, each containing non-vagotomized and vagotomized rats. In vagotomized rat groups, the subdiaphragmatic vagal section surgery was carried out 45 days before the irradiation exposure. The rats were partial-body irradiated with the head shielded with (60)Co gamma-rays to a dose of 15 Gy. They were sacrificed 6 h after the end of exposure. The hypothalamus, hippocampus, thalamus and cortex were then collected, and the concentrations of IL-1beta, TNFalpha and IL-6 in each were measured by ELISA assays. RESULTS: Six hours after irradiation, IL-1beta levels had increased in the hypothalamus, thalamus and hippocampus, and TNFalpha and IL-6 levels had increased significantly in the hypothalamus. Vagotomy before irradiation prevented these responses. CONCLUSIONS: It was concluded that the hypothalamus, hippocampus, thalamus and cortex react rapidly to peripheral irradiation by releasing pro-inflammatory mediators. The results also show that the vagus nerve is one of the major ascending pathways for rapid signalling to the brain with respect to partial body irradiation.