Involvement of stakeholders during the preparedness phase of post-accident situation management.

The Steering Committee for Post-accident Management Preparedness (CODIRPA) was commissioned by the French Government in 2005 with the aim of establishing the main principles to be set up for population protection and recovery in the long term. From the beginning, one of the main principles was the pluralistic nature of the working groups (WGs), including scientific and technical experts, representatives from state departments, nuclear operators, and representatives of civil society (i.e. stakeholders). Stakeholders were mainly associated with the various WGs of CODIRPA. In order to foster the involvement of stakeholders from civil society in the works of CODIRPA, a new organisation was implemented with two WGs: one mainly composed of technical experts for tackling technical issues, and one for evaluating the proposals made by the experts from the stakeholders' point of view. This article presents the results of this new strategy.

Application of rodes software to experimental biokinetic data for dose assessment in mice and rats.

H Miloudi, M Locatelli, G Autret, D Balvay, A Desbrée, E Blanchardon, J M Bertho: application of RODES software to experimental biokinetic data for dose assessment in mice and rats. In support of experimental studies of chronic, long-term contamination in rodents, voxel-based computer models were built representing adult mice and juvenile, adult and elderly rats of both sexes. RODES software was created to calculate absorbed radiation doses to organs with these specific anatomical models. Absorbed doses were then calculated starting from previously published biokinetic data. Whole body doses showed less than 5% differences between calculation with RODES and calculation with the ICRP Publication 108 model for long term exposure to 90Sr of mice. Similar results were obtained for long term exposure to 137Cs. Dose distribution for 90Sr internal contamination also showed that the dose to the skeleton is six fold more as compared to the whole body dose while radiation dose to other organs is less than the mean whole body dose. These results underline the importance of using specific anatomical models according to the age and the sex of experimental animals.

DNA damage induced by Strontium-90 exposure at low concentrations in mesenchymal stromal cells: the functional consequences.

90Sr is one of the radionuclides released after nuclear accidents that can significantly impact human health in the long term. 90Sr accumulates mostly in the bones of exposed populations. Previous research has shown that exposure induces changes in bone physiology both in humans and in mice. We hypothesize that, due to its close location with bone marrow stromal cells (BMSCs), 90Sr could induce functional damage to stromal cells that may explain these biological effects due to chronic exposure to 90Sr. The aim of this work was to verify this hypothesis through the use of an in vitro model of MS5 stromal cell lines exposed to 1 and 10 kBq.mL-1 of 90Sr. Results indicated that a 30-minute exposure to 90Sr induced double strand breaks in DNA, followed by DNA repair, senescence and differentiation. After 7 days of exposure, MS5 cells showed a decreased ability to proliferate, changes in cytokine expression, and changes in their ability to support hematopoietic progenitor proliferation and differentiation. These results demonstrate that chronic exposure to a low concentration of 90Sr can induce functional changes in BMSCs that in turn may explain the health effects observed in following chronic 90Sr exposure.

A rapid multiparametric method for victim triage in cases of accidental protracted irradiation or delayed analysis.

The aim of this study was to develop a rapid and simple method for the triage of radiation accident victims, according to the severity of radiation-induced damage, especially when the radiological nature of the accident is recognised late or gives rise to a protracted irradiation. The work was based on the recent radiation accident in Dakar (Senegal). A score was developed based on the complete blood count and the plasma Flt3 ligand concentration of each of the 63 potential victims of this accident. The three victims who received the highest radiation dose, as defined by cytogenetic analysis, were easily detected by this score analysis. A correlation was obtained between the score and the radiation dose. This score might allow a triage of radiation accident victims based on simple parameters that are easy and fast to obtain. This score is simple to interpret and might allow the identification of victims at risk of developing an acute radiation syndrome. Interestingly, as the parameters used in this score evolve according to the development of the haematopoietic syndrome, this score may be applicable for several weeks after the radiation accident, even in a case of protracted exposure, as happened in the Dakar accident.

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.

Follow-up of stable chromosomal aberrations in gamma-ray irradiated non-human primates.

PURPOSE: The purpose of this study was to examine a new approach to retrospective biological dosimetry, by using a long-term animal model to determine the stability of translocation frequency after in vivo irradiation. While the frequency of dicentrics is known to decrease over time, the persistence of more stable chromosomal aberrations such as translocations could be useful if their stability were definitively proved. MATERIALS AND METHODS: Four monkeys (Macaca fascicularis) were exposed to two different doses of ionizing radiation: 2 Gy whole body irradiation for two and 4 Gy for two others. Blood samples were obtained at various times after irradiation. Both total and two-way translocations were detected by fluorescence in situ hybridization. Translocations were scored in stable cells, that is, those without dicentrics, rings or fragments. The course of translocation frequency was analysed at four time-points: one hour (H1), 2 months (M2), 10 months (M10) and 31 months (M31) after irradiation. RESULTS: We observed two separate trends in translocation frequency: Total translocation frequency decreased slightly in animals irradiated with a dose of 2 Gy, while two-way translocation frequency was relatively stable in all irradiated animals. CONCLUSIONS: We confirmed the long-term stability of translocations and found that it seems to depend on the type of the translocation recorded. Overall translocations were stable for up to 31 months regardless of dose, but two-way translocations were more stable than those that were non-reciprocal, especially in stable cells.

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.

Level of Flt3-ligand in plasma: a possible new bio-indicator for radiation-induced aplasia.

PURPOSE: To follow plasma Flt3-ligand (FL) concentrations in irradiated animals in order to evaluate it as an indicator of bone marrow damage for the management of accidental radiation-induced aplasia. MATERIALS AND METHODS: Non-human primates were irradiated at doses ranging from 2 to 8 Gy, using whole- or partial-body irradiation. Plasma FL concentrations and blood cell counts were determined daily. RESULTS: FL concentrations increased as early as day 2 after irradiation, whatever the irradiation dose. Increase in plasma FL concentration on day 5 post-irradiation was correlated with radiation dose and with the severity of radiation-induced aplasia. During the course of aplasia, FL concentrations in plasma were inversely correlated with neutrophil counts. A peak in FL concentration appeared before the neutrophil nadir, and the subsequent decrease in FL concentration was correlated with the recovery of blood-cell populations. CONCLUSIONS: Monitoring plasma FL concentration can be used as an indicator of radiation-induced marrow aplasia, and this may be of use in accidental irradiation situations.

Prospective X-ray densitometry and ultrasonography study of the hand bones of patients with rheumatoid arthritis of recent onset.

OBJECTIVE: Bone demineralization observed in early rheumatoid arthritis is not easily measured. To measure bone loss and to discriminate between rheumatoid arthritis and other rheumatic diseases, we used two methods: dual-energy X-ray absorptiometry and ultrasonography. METHODS: From a population-based recruitment, 32 patients with early peripheral polyarthritis (median disease duration: 4 months) were studied. Clinical, laboratory, functional, hand-bone assessments were made at the entry an at months 6 and 12. Bone X-ray densitometry measurements were made on 16 areas of the hand. Speed of sound was measured across the proximal phalanges of the four fingers. X-rays of both hands were scored according to the modified Sharp's score. At 12 months, patients were classified as rheumatoid arthritis (N = 15; 9 F) or as other rheumatic diseases. RESULTS: We found: 1) significantly decreased bone mineral density (BMD) of the whole hand, in the rheumatoid arthritis group versus the other rheumatic diseases group, at 6 and 12 months (P < 0.05); 2) no significant decrease of bone mineral density (BMD) in other areas in the rheumatoid arthritis group; 3) no significant change of ultrasounds in either group; and 4) no significant correlation between the decrease of BMD in the rheumatoid arthritis group and clinical, biological or radiologic parameters, except for IFNgamma, whose production in whole blood cell culture was lower at entry in the rheumatoid arthritis group. CONCLUSION: DEXA bone assessment in rheumatoid arthritis was able t detect bone loss in the whole hand at 6 months.

CD135 (Flk2/Flt3) expression by human thymocytes delineates a possible role of FLT3-ligand in T-cell precursor proliferation and differentiation.

The expression of the Flk2/Flt3 molecule (CD135), the receptor for Flt3 ligand (FL), was investigated in the human thymus. Results showed that there is a high level of expression of CD135 by thymocyte populations, especially by intrathymic T-cell precursor populations. As these results suggested a role for FL in the regulation of thymic T-cell precursor differentiation and/or proliferation, we used an in vitro model of thymic stromal cell cultures in order to delineate the activity of FL on human CD7(high)CD3-CD4-CD8- triple negative intrathymic T-cell precursors. Results showed that FL, either alone or in combination with stem cell factor (SCF) induced the proliferation of CD7(high) precursors, but to a lower extent than interleukin-7 (IL-7) or IL-7 + SCF, used as positive controls. In the presence of FL + SCF, CD7(high) cells developed mainly towards a CD11b+ phenotype whereas IL-7 + SCF preferentially induce a CD3+TcRgammadelta+CD8+ phenotype. Taken together, these data suggest that FL may play a role in inducing the proliferation of CD7(high) human intrathymic T-cell precursors, but also in the induction of a myeloid differentiation pathway within the human thymus.

Presence of primitive lymphoid progenitors with NK or B potential in ex vivo expanded bone marrow cell cultures.

OBJECTIVE: In previous work, we showed that CD34+ bone marrow cells can be successfully expanded along the myeloid pathway in stroma- and serum-free conditions in the presence of SCF+IL-3+IL-6+Flt3-l+G-CSF+MGDF. Due to the lack of phenotypically detectable lymphoid cells, it was necessary to address the question of the lymphoid potential of the expanded populations under these conditions. MATERIALS AND METHODS: The present report describes a long-term culture system that supports human B- and NK-cell differentiation from the day 14 fraction without further selection of the more primitive cells. In NK proliferation assays, the cells were maintained over stroma cells in the presence of IL-2 for 4-5 weeks. NK initiating cells (NK-IC) were determined by a limiting dilution assay. In B-cell cultures, the expanded cells were maintained over MS5 in the presence of Flt3-l for 4-8 weeks. RESULTS: NK cells rose from 0.2%+/-0.04% at culture initiation to 71%+/-6% at week 5. These cells displayed cytolytic activity. NK-IC evaluation showed a mean 18-fold expansion in the day 14 expanded fraction as compared to the initial day 0 fraction. Similarly, CD19+ cells rose from 0.1% at culture initiation to 30%+/-1% at week 6. Cells produced under these B-LTC conditions were CD34-CD19+CD10+. We also demonstrated that the CD34+/Lin- sorted cells from the day 14 fraction gave rise to NK and B cells. CONCLUSION: This culture system permits the revelation of a population that, although poorly represented in terms of phenotypically detectable cells, nevertheless retains high levels of lymphoid NK and B potential after 14 days expansion. Such data suggest the persistence, or expansion, of lymphoid progenitors and, hence, the multipotentiality of the expanded progenitor/stem cells.

Human thymic stromal cell irradiation reduces intra-thymic T cell precursor proliferation: evidence for a soluble mediator.

PURPOSE: To investigate the effects of ionizing radiations on human thymic stromal cells and the consequences of stromal cell irradiation on T cell precursor proliferation. MATERIAL AND METHODS: An in vitro model of thymic stromal cell culture was used. These cultures are able to support early CD7high T cell precursor proliferation and differentiation, mainly towards a CD3+TcRgamma+CD8+ pathway, in the presence of interleukin-7 and stem cell factor. Thymic stromal cell cultures were exposed to 10 Gy 60Co gamma-rays, 24 h before the seeding of CD7high T cell precursors. RESULTS: Irradiation of thymic stromal cells induced a reduction in CD7high T cell precursors, without modifying their differentiation. This effect was reproduced by the addition of supernatants from irradiated stromal cell cultures in sham-irradiated cultures. CONCLUSIONS: These results suggest that gamma-irradiation induces modifications in the production of soluble factors by thymic stromal cells, which in turn modify their ability to support T cell precursor proliferation. To the authors' knowledge this is the first direct demonstration that gamma-irradiation modifies the supportive function of thymic stromal cells.

Late and persistent up-regulation of intercellular adhesion molecule-1 (ICAM-1) expression by ionizing radiation in human endothelial cells in vitro.

Adhesion molecules play a key role in cellular traffic through vascular endothelium, in particular during the inflammatory response when leukocytes migrate from blood into tissues. Since inflammation is one of the major consequences of radiation injury, we investigated the effect of ionizing radiation on cell-surface expression of the intercellular adhesion molecule-1 (ICAM-1), the vascular cell adhesion molecule-1 (VCAM-1) and E-selectin in cultured human umbilical vein endothelial cells (HUVEC). Flow cytometry performed on irradiated HUVEC revealed both a time- (from 2 to 10 days) and dose- (from 2 to 10 Gy) dependent up-regulation of basal expression of ICAM-1, and no induction of VCAM-1 or E-selectin. The radiation-induced increase in ICAM-1 expression on HUVEC was correlated with augmented adhesion of neutrophils on irradiated endothelial cells. Interleukin-6 (Il-6) or other soluble factors released by irradiation were not involved in the enhanced ICAM-1 expression by irradiation. Northern blot analysis showed an overexpression of ICAM-1 mRNA from 1 to 6 days after a 10 Gy exposure. Our data suggest that ICAM-1 participates in the radiation-induced inflammatory reaction of the endothelium.

Phenotypic and immunohistological analyses of the human adult thymus: evidence for an active thymus during adult life.

We analyzed cellular content of thymic samples from 26 human healthy donors, ranging from 1 week postnatal to 49 years old. Our results showed that there was an overall decrease in cellular density, beginning early during life, but with two peaks of cellular density, at 9 months and 10 years of age. Histological and immunohistological analyses showed that variations in cellular density were correlated with the morphological changes observed during thymic involution, namely the enlargement of interlobular trabeculae and the development of adipocytic tissue. However, the adult thymus still contained thymocytes, up to 49 years. Phenotypic analysis showed no significant variations according to the age of donors in the distribution of the main thymocyte subsets, both precursors and more mature cells. These results suggest that the human thymus remains active during adult life.

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells.

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.

How do French rheumatologists treat early rheumatoid arthritis?

Although factors that appear to predict long-term outcomes of rheumatoid arthritis have been identified, there is no consensus about the treatment early in the disease. To determine how French office- and hospital-based rheumatologists treat early rheumatoid arthritis, we created three clinical vignettes corresponding to different levels of severity of early rheumatoid arthritis (less than six months' disease duration). Cases 1 and 2 were relatively young patients (35 and 50 years), and Case 1 had numerous poor prognosis factors. Case 3 was 80 years of age. Rheumatologists were asked to indicate which medications they would use at presentation and after one year of a favorable or unfavorable course. The study was conducted by questionnaire (response rate, 58%). Of the 185 rheumatologists who completed the questionnaire, 81% were male and 19% female; mean age was 42 +/- 8 years. In Cases 1 and 2, nonsteroidal antiinflammatory drugs were given by 99% of respondents; second-line drugs were prescribed at presentation by 93% of respondents in Case 1 and 86% in Case 2, and methotrexate was more likely to be used in the presence of poor prognosis factors (23% in case 1 and 7% in Case 2). In the event of an unfavorable course after one year, a larger proportion of rheumatologists prescribed glucocorticoid therapy (65% in Case 1 and 20% in Case 2), and there was a shift from "conventional" to "modern" second-line drugs, with more widespread use of methotrexate (65% in case 1 and 18% in case 2). In the 80-year-old patient, glucocorticoid therapy was used more often than nonsteroidal antiinflammatory drugs and second-line drugs (gold salts, hydroxychloroquine, sulfasalazine) were prescribed by 40% of rheumatologists at presentation and by 67% after one year of an unfavorable course; in the latter situation, methotrexate was selected in 24% of cases. In contrast to conventional recommendations, many French office- or hospital-based rheumatologists use second-line drugs very early and base their choice of medications on the estimated risk of severe disease and on the age of the patient.

A 2-year follow-up of an anti-HIV immune reaction in HIV-1 gp160-immunized healthy seronegative humans: evidence for persistent cell-mediated immunity.

The first trial of an anti-HIV immunization, using a recombinant vaccinia virus expressing gp160 (rV) for priming and paraformaldehyde-fixed rV-infected PBLs and soluble gp 160 for boosting, clearly showed an in vitro HIV-protective immune reaction. This result led us to carry out an additional 2 year Phase I clinical trial in 25 HIV-seronegative volunteers, using HIV gp 160 antigens for immunization in four different protocols. The 2 year trial showed (a) the safety of the preparations, (b) a transient humoral immunity following each boost, and (c) a long-lasting memory T-cell response. Memory cytotoxic T-lymphocytes (CTLs) induced by gp 160 antigen with or without vaccinia vector lysed HLA class I restricted target cells expressing HIV-1 env antigens. These results are consistent with CTLs being an effective component of an AIDS vaccine to control cell-to-cell viral replication, dissemination in the organism, and subsequent evolution toward AIDS.

One-year follow-up of vaccine therapy in HIV-infected immune-deficient individuals: a new strategy.

Immunization of AIDS/ARC patients with autologous cells expressing HIV antigens, although providing clinical and biological benefits, fails to restore cellular immunity. The latter result is due partly to the antiproliferative effect of HIV-1 on activated T-cells (immune suppression), which leads to blockade of specific immune reactions. To overcome immune suppression, a new vaccine strategy was designed consisting of an immunization against HIV-1 combined with components of the T-cell-suppressive (antiproliferative) network. This new vaccine treatment proved to be innocuous in mice, monkeys, and two non-HIV-infected humans. A Phase I clinical trial was performed in six patients previously under cellular immunotherapy and still presenting a cellular immune defect. Preliminary results confirmed, after a 1-year follow-up of the patients, the safety of the new vaccine, which also partially restored the cellular immune response, including anti-HIV HLA-restricted cell-mediated cytotoxicity, delayed hypersensitivity to recall antigens, and proliferation of T-cells specifically activated by recall antigens.

Synergistic effect of interleukin 1 and soluble CD23 on the growth of human CD4+ bone marrow-derived T cells.

Low-affinity Fc epsilon receptor (Fc epsilon RII/CD23) is expressed by various human cells and known to be cleaved into soluble fragments (sCD23). Several biological activities were ascribed to these molecules. In this study, we have assessed the effect of recombinant 25-kDa sCD23 (rsCD23) on human bone marrow-derived T cells. Our results show that rsCD23 in synergy with recombinant interleukin 1 enhances mitogenic responsiveness of CD4+ T cells but does not affect CD8+ cell growth. Furthermore, rsCD23 synergizes autologous marrow cells in enhancement of CD4+ cell growth while CD23 monoclonal antibodies decrease accessory cell effect. Together, these data confirm cytokine-like activity of sCD23 on human T cell lineage.

Human thymic epithelial cells produce interleukin-3.

Interleukin-3 (IL-3) is a hematopoietic growth factor suggested to be produced by activated T lymphocytes. Meanwhile, supernatants from human thymic stroma could promote the proliferation of myeloid stem cells. Thus, we investigated whether IL-3 accounts for this activity. Therefore, human thymic epithelial cells (TEC), fibroblasts, and adherent cells were isolated, and their culture supernatants assayed for myeloid colony promotion. Only supernatants from thymic epithelial cells supported colony-forming unit growth in semisolid media. This effect decreased following anti-IL-3 monoclonal antibody addition to these cultures. Furthermore, in situ hybridization showed the presence of IL-3 mRNA in epithelial cells. Effect of TEC culture conditions on IL-3 production by these cells was also studied. Together, these data show that IL-3 production is not the exclusive property of human activated T lymphocytes.