COORDINATION OF MANAGEMENT OF THE ACUTE RADIATION SYNDROME.

The acute radiation syndrome (ARS) constitutes the most challenging, immediate medical consequence of exposure to high doses of ionizing radiation in an emergency situation. This report highlights some of the currently available medical guidelines and recommendations on the clinical management of ARS, comments recent trends regarding the approval of targeted pharmaceuticals for ARS, and suggests further initiatives for international collaboration aiming at continuously updating the medical knowledge base of this syndrome.

Case Report: Industrial X-Ray Injury Treated With Non-Cultured Autologous Adipose-Derived Stromal Vascular Fraction (SVF).

Local cutaneous injuries induced by ionizing radiation (IR) are difficult to treat. Many have reported local injection of adipose-derived stromal vascular fraction (SVF), often with additional therapies, as an effective treatment of IR-induced injury even after other local therapies have failed. The authors report a case of a locally recurrent, IR-induced wound that was treated with autologous, non-cultured SVF without other concurrent therapy. A nondestructive testing technician was exposed to 130 kVp x rays to his non-dominant right thumb on 5 October 2011. The wound healed 4 mo after initial conservative therapy with oral/topical α-tocopherol, oral pentoxifylline, naproxen sodium, low-dose oral steroids, topical steroids, hyperbaric oxygen therapy (HBOT), oral antihistamines, and topical aloe vera. Remission lasted approximately 17 mo with one minor relapse in July 2012 after minimal trauma and subsequent healing. Aggressive wound breakdown during June 2013 required additional therapy with HBOT. An erythematous, annular papule developed over the following 12 mo (during which time the patient was not undergoing prescribed treatment). Electron paramagnetic resonance (EPR) done more than 2 mo after exposure to IR revealed dose estimates of 14 ± 3 Gy and 19 ± 6 Gy from two centers using different EPR techniques. The patient underwent debridement of the 0.5 cm papular area, followed by SVF injection into and around the wound bed and throughout the thumb without complication. Eleven months post SVF injection, the patient has been essentially asymptomatic with an intact integument. These results raise the possibility of prolonged benefit from SVF therapy without the use of cytokines. Since there is currently no consensus on the use of isolated SVF therapy in chronic, local IR-induced injury, assessment of this approach in an appropriately powered, controlled trial in experimental animals with local radiation injury appears to be indicated.

Regulation of TNFRSF6 (Fas) expression in ataxia telangiectasia cells by ionizing radiation.

Several studies have shown that ionizing radiation induces transcription of the TNFRSF6 (Fas) gene, leading to augmented TNFRSF6 protein levels at the surface of irradiated cells. We have examined TNFRSF6 expression in an apparently normal lymphocyte line and in a lymphocyte cell line derived from a patient with ataxia telangiectasia (AT) before and after exposure to radiation (0-10 Gy). Plasma membranes were isolated from normal lymphocytes and AT cells and subjected to Western blot analysis, using a TNFRSF6-specific monoclonal antibody to probe resolved proteins transferred onto nitrocellulose membranes. In both cell types, the presence of a 48-kDa band corresponding to the molecular mass of TNFRSF6 was revealed. Analysis of FITC-conjugated anti-TNFRSF6 antibody-stained normal lymphocytes and AT cells confirmed TNFRSF6 expression in both cell types. In MTT assays, AT cells treated with agonistic anti-TNFRSF6 Ab (CH.11) displayed a 25.9% decrease in cell viability, relative to cells treated with isotype-matched IgM Ab, suggesting the presence of a biologically active TNFRSF6 receptor at the AT cell surface. Exposure to cycloheximide (0-5 microg/ml), a metabolic inhibitor, enhanced sensitivity of AT cells to CH.11. Normal lymphocytes exhibited increased levels of apoptosis (approximately 34% cell death relative to cells treated with isotype-matched IgM Ab) when exposed to CH.11; however, the degree of cell death was not altered significantly with increasing concentrations of cycloheximide. When AT cells were exposed to 0.1, 0.5, 2 and 10 Gy, the activities of caspases 3 and 8 increased in a dose-dependent manner at 24 h postirradiation and reached a plateau by 72 h. A similar trend for activation of caspase 3 and 8 was observed in normal lymphocytes after irradiation. To assess the roles of TNFRSF6 and/or caspase 8 in radiation-induced cell death of AT and normal lymphocytes, and to determine whether hyper-radiosensitivity in AT cells is correlated with increased activity of these two components of the TNFRSF6 pathway, AT and normal lymphocytes were irradiated in the presence of ZB4, an anti-TNFRSF6 blocking antibody, and a caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was determined by Annexin V staining using flow cytometry. Incubation with ZB4 anti-TNFRSF6 antibody did not alter the fraction of apoptotic cells in either AT cells or normal lymphocytes treated with doses of radiation ranging from 0-10 Gy. In contrast, apoptosis was significantly reduced in both cell lines in the presence of Z-IETD-FMK when samples were exposed to low-dose (< or = 2 Gy) radiation. Relative to control samples (those not incubated with Z-IETD-FMK), no difference in the level of apoptosis was observed in AT or normal lymphocytes treated with 10 Gy. These data indicate that: (a) despite radiation-induced up-regulation of TNFRSF6 at the cell surface, the death-promoting receptor does not play a role in radiation-mediated cytotoxicity; (b) apoptosis in lymphocytes irradiated with low (< or = 2 Gy) but not high doses (>2 Gy) proceeds at least in part through activation of caspase 8; and (3) since blocking anti-TNFRSF6 antibody (ZB4) did not reduce levels of apoptosis in irradiated AT cells to those of normal lymphocytes, TNFRSF6 is unlikely to play a significant role in the hyper-radiosensitivity exhibited by cells having the AT phenotype.

Ionizing radiation alters Fas antigen ligand at the cell surface and on exfoliated plasma membrane-derived vesicles: implications for apoptosis and intercellular signaling.

Resident proteins that reside on the plasma membrane are continually exfoliated from the cell surface. Exfoliation is a selective, energy-dependent process that mediates intercellular communication. Ionizing radiation modulates the expression of many plasma membrane-bound growth regulators, including the "death" ligand, TNFSF6 (formerly known as FasL, CD95L). Here we report that ionizing radiation induces dose-dependent up-regulation of TNFSF6 on plasma membranes purified from SW620 cells, a TNFSF6-expressing colon cancer cell line. Serum-free medium conditioned by exposed and control cells was collected and exfoliated vesicles were obtained by ultracentrifugation. Western blot analysis of vesicles from unexposed cells and from cells treated with 10 Gy showed increased amounts of TNFSF6 compared to that on vesicles from unexposed cells. Cells treated with 4 Gy released vesicles having a low level of TNFSF6 on their surface relative to that on vesicles exfoliated from unexposed cells. When assayed for bioactivity, vesicles from unexposed cells induced the greatest level of apoptosis in TNFRSF6 (formerly known as FAS) receptor-bearing Jurkat cells (cell surviving fraction of 43.7 +/- 6.1; P < 0.05), followed by vesicles collected from cells treated with 4 Gy (79.6 +/- 2.6%; P < 0.05). Despite having a high level of TNFSF6 by Western analysis, vesicles collected from cells exposed to 10 Gy display minimal biological activity (77.9 +/- 3.2%; P < 0.05), suggesting that modification of the vesicle-associated ligand has occurred. Our results indicate that ionizing radiation increases the level of TNFSF6 exfoliated on extracellular vesicles. The data may provide a mechanism for abscopal and bystander effects after irradiation.

The Raf-1 protein mediates insulin-like growth factor-induced proliferation of erythroid progenitor cells.

Previous studies from this and other laboratories have shown that insulin-like growth factor-1 (IGF-I) and insulin-like growth factor-2 (IGF-II) support erythroid colony formation in cultures supplemented with serum substitute and recombinant erythropoietin. Subpopulations of IGF-I- and IGF-II-dependent, erythropoietin-independent colony-forming unit-erythroid (CFU-E)-derived colonies and BFU-E-derived colonies were identified under serum-substituted conditions for adult bone-marrow-derived erythroid progenitors which proliferate in the absence and presence of exogenous anti-erythropoietin receptor monoclonal antibody and in serum-substituted medium that was preadsorbed with anti-erythropoietin IgG. To assess whether Raf-1 is required for the formation of IGF-dependent, erythropoietin-independent human erythroid colonies, 5-15 microM sense or antisense oligomer to raf-1 were added to serum-substituted cultures containing either 2 U/ml recombinant human erythropoietin (rHuEpo) alone or 0-1,000 ng/ml IGF-I or IGF-II with/without 2 U/ml rHuEpo. Both erythropoietin-induced and IGF-induced erythroid colony formation were completely blocked by antisense (but not sense) oligomers to raf-1. Purified human CFU-Es were examined for Raf-1 message and protein. Total RNA was extracted, and raf-1 mRNA was detected on Northern blots. Furthermore, a 74 kD protein, corresponding to Raf-1, was also detected in CFU-Es purified from human adult sources. Together, these studies support the hypothesis that the Raf-1 protein mediates both erythropoietin-induced and IGF-induced signal transduction in human erythroid progenitor cells.

Biologically active Fas antigen and its cognate ligand are expressed on plasma membrane-derived extracellular vesicles.

Exfoliation of plasma membrane components is a directed process that consumes energy and requires active cell metabolism. Proteins involved in regulating the survival and proliferation of eukaryotic cells are released on exfoliated vesicles. We examine here whether the Fas receptor and its cognate ligand (FasL) are present on vesicles shed from high metastatic potential CX-1 cells and low metastatic potential MIP-101 cells and from HuT 78 cells, respectively. Rates of exfoliation at 2 hours and cumulative levels of extracellular vesicles in serum-free medium conditioned by CX-1 cells are increased by 1.8-fold and 1.6-fold, respectively, relative to that in medium conditioned by MIP-101 cells. Although vesicles shed from both cancer cell lines contain Fas antigen, the amount of Fas per vesicle and the percentage of vesicles containing Fas are increased for vesicles isolated from MIP-101 cells, relative to those from CX-1 cells, as determined by immunogold particle labeling and electron microscopy and by immunofluorescence microscopy and flow cytometry. Results of metabolic labeling with 35S-methionine indicate that Fas biosynthesis is reduced by up to 3.3-fold for CX-1 cells, relative to that of MIP-101 cells, consistent with the finding of decreased Fas on vesicles shed from the plasma membrane of CX-1 cells. Although mRNA for soluble Fas receptor is detectable in both cell lines, depletion of shed vesicles from serum-free medium by ultracentrifugation removes all detectable biological activity. FasL is detected on vesicles exfoliated from HuT 78 cells by immunoelectron microscopy and Western blot analysis. FasL-bearing vesicles induce apoptosis of Fas-expressing cancer cells at the same level as observed by treatment with monoclonal anti-Fas antibody. Furthermore, Fas-bearing extracellular vesicles from MIP-101 but not from CX-1 cells protect the CX-1 cell line from FasL-induced and anti-Fas-mediated apoptosis, indicating that Fas present on shed vesicles is biologically active. We conclude that the Fas antigen and its cognate ligand are exfoliated from the cell surface in a bioactive configuration. Exfoliation may provide a mechanism for long-range signal-directed apoptosis while maintaining Fas/FasL on a membrane surface.

Early plasma membrane events occurring in ultraviolet-B-induced apoptosis.

Whereas nonsolar ultraviolet C radiation primarily affects nuclei (i.e., where it is absorbed by nucleic acids) of eukaryotic cells, ultraviolet radiation of long (320-380 nm) wavelengths (ultraviolet A) and intermediate (290-320 nm) wavelengths (ultraviolet B) primarily affects lipid membranes. We have previously demonstrated that ultraviolet B irradiation alters the surface architecture of human B cells and impairs expression of an erythroid growth factor on their surface and on extracellular vesicles. Here, we examined the effects of ultraviolet B irradiation on the capacity of Chinese hamster ovary cells to undergo the process of exfoliation, and on the capacity of Chinese hamster ovary cells transfected with flt3/flk2 cDNA to express the cytokine flt3/flk2. Our results indicate that the rate of release of shed vesicles from untransfected Chinese hamster ovary cells is decreased after one to two h, at a time when there is electron microscopic evidence for retention of vesicles at the cell surface. These changes at the cell surface precede all other apparent morphological changes (including DNA condensation in the nucleus, swelling of the mitochondria and appearance of apoptotic bodies). Furthermore, plasma membranes and shed extracellular vesicles from ultraviolet B irradiated Chinese hamster ovary cells that have been transfected with flt3/flk2 cDNA fail to express the protein.

Practical and theoretical issues in 1993 concerning radiation effects on the growth of normal and neoplastic hematopoietic cells.

Radiation has multiple effects on eukaryotic cells, ranging from altered gene expression and cell-cell signaling to induction of programmed cell death (i.e., apoptosis). These changes may lead to neoplastic transformation of the cell and diverse effects on differentiation and growth. The Belarusians were exposed to greater levels of radiation from the Chernobyl nuclear reactor melt-down than any other population. Medical consequences of this exposure are reviewed, focusing on the appearance of thyroid cancer and the possible increased risk of future hematologic malignancies. Since circulating hematopoietic cells must be relaced continually at high rates, the effects of ionizing radiation are reviewed at the cellular level, utilizing the hematopoietic system as a model tissue. An overview of normal hematopoiesis (as understood in 1993) is provided, and the effects of ionizing radiation on hematopoietic stem cell compartments are reviewed. Hematopoietic growth factors (i.e., cytokines) that are associated with the plasma membrane (i.e., membrane-bound cytokines), or that are released as "soluble" molecules into the microenvironment and circulation, may "protect" organisms from radiation injury and may accelerate hematopoietic recovery following radiation exposure. Cloned hematopoietic cytokines, individually or in combination, may be useful in the treatment of radiation accident victims in the future.

Morbidity in a large cohort study of children born to mothers exposed to radiation from Chernobyl.

Reproductive health was reviewed in four oblasts of the Republic of Belarus that were either heavily exposed (Mogilev and Gomel) or lightly exposed (Brest and Vitebsk) to ionizing radiation after the meltdown of a nuclear reactor in Chernobyl. A retrospective analysis was conducted on pregnancies occurring between January 1, 1982, and December 31, 1990, and a comparison of results was made between pregnancy outcomes prior to and after the meltdown for individuals residing in heavily exposed and lightly exposed oblasts. Pregnant women who resided in heavily exposed oblasts appeared to be at risk for development of toxemia, renal insufficiency and anemia. Neonates born in heavily contaminated areas were apparently at risk for development of anemia and congenital malformations and perinatal death. In addition, a cohort of 757 neonates, 0-18 months old, with a normal physical examination, was identified for laboratory analysis of hematological, immunological, endocrinological and nutritional status. Decreased levels of copper and zinc were documented in erythrocytes from neonates from heavily contaminated oblasts, findings that may be related more to inadequate nutrition than to radiation exposure. Increased absolute "null" lymphocyte number and diminished absolute T lymphocyte number with a reduction in the "helper" (i.e., T4) subclass of T cells were evident in neonates born-in heavily exposed oblasts. Geographic differences in reproductive health and immune status are apparent in Belarus that may be related to radiation exposure. Additional studies are required to exclude confounding variables and possible selection bias.

Early identification of radiation accident victims for therapy of bone marrow failure.

Ionizing radiation damages the lymphohematopoietic system via direct effects on viability and/or function of hematopoietic stem/progenitor cells and via abnormal production of cytokines (i.e., growth factors). Other tissues that have a rapid turnover (including the gastrointestinal tract and skin) are also profoundly affected by acute radiation exposure. A major issue in selection of appropriate therapy for bone marrow failure (i.e., the bone marrow syndrome) is early assessment of radiation dose. Although several biological markers are available for assessing dose received, the absolute polymorphonuclear neutrophil (PMN) and/or lymphocyte counts, together with clinical presentation (i.e., time to onset of nausea and vomiting, etc.) still provide the most practical and timely assessment of radiation dose. Limited information is available regarding CD34-positive cell frequency as a measure of radiation-induced damage to the bone marrow. Since a subpopulation of radioresistant hematopoietic stem cells may persist after exposure to high-dose radiation, the primary goal of therapy is to provide an adequate number of lymphohematopoietic stem cells for a finite (rather than indefinite) period, after which endogenous stem cells may reinstate lymphohematopoiesis. A model is presented which describes the hypothesis that stem cell clonal repopulation over time is distinct in transplant recipients who have received moderate compared to high-dose radiation exposures. Since some individuals receiving high levels of radiation and presenting with rapidly declining PMN counts spontaneously recover lymphohematopoiesis, better tools (including CD34-positive cell analysis) must be developed to select the appropriate therapy for exposed individuals.

Induction of Erythropoiesis by MHC-Mediated Cognate Interactions between B- and T-Cells.

We have previously shown that the expression of membrane burst-promoting activity (mBPA), an erythropoietic cytokine, by B-lymphocytes is augmented by the addition of allogeneic effector cells to the B-cells. Here, we have examined immune mechanisms involved in the induction/promotion of erythropoiesis as assessed by the capacity of autologous and allogeneic peripheral blood lymphocytes to augment burst-forming unit-erythroid (BFU-E) in normal human bone marrow cells in vitro. Preincubation of mBPA-expressing human B-cells with monoclonal antibodies to major histocompatibility complex (MHC) antigens, abrogated erythropoietic activity of both autologous and allogeneic lymphocytes, suggesting that MHC antigens play a role in regulating the expression of the erythroid growth factor. Inhibition of BFU-E proliferation was also evident when antibodies to MHC class-I or class-II antigens were added directly to marrow culture. Furthermore, addition of anti-CD4 antibody to the cultures of PBL and autologous target BM cells markedly reduced erythroid proliferation induced by PBL. By contrast, anti-CD8 and control (UPC-10) monoclonal antibodies had no effect. These results provide evidence that MHC-mediated cognate interactions between T- and B-lymphocytes may participate in the control of erythropoiesis, either directly or by modulating mBPA function.

Utility of biological membranes as indicators for radiation exposure: alterations in membrane structure and function over time.

In addition to interacting with genomic DNA, ionizing radiation may directly and indirectly alter the structure and function of components of the plasma membrane of eukaryotic cells. Water radiolysis generates reactive species, including superoxide, hypochlorous acid and chloride radicals that may in turn react with biological membranes, as well as with cellular DNA. Reaction of plasma membrane lipids with molecular oxygen results in lipid peroxidation of both reconstituted membranes and biological membranes, an effect that increases with decreasing dose rate. Both ionizing radiation and ultraviolet light alter functions of membrane-anchored molecules, including adhesion molecules, histocompatibility complex antigens and membrane-bound growth factors. The latter growth factors represent a repertoire of growth and differentiation signals that are expressed in a nondiffusible fashion at the cell surface, and in soluble forms appearing after cleavage of their extracellular domain. The importance of cell-cell signaling via the membrane-anchored form of growth factors is becoming increasingly recognized. Expression of membrane-bound hematopoietic cytokines by eukaryotic cells is impaired after exposure to ultraviolet light, a defect in cell-cell signaling that may lead to impaired hematopoiesis. While studies suggest that permanent changes in membrane structure and function may result from radiation-induced injury to the plasma membrane and reconstituted "pure" membranes, reversibility of these defects over time requires additional study.

Human macrophage colony-stimulating factor is expressed at and shed from the cell surface.

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF-alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane-derived vesicles.

Pancytopenia in allogeneic marrow transplant recipients: role of cytomegalovirus.

We describe the clinical course of three cytomegalovirus-antibody-positive allogeneic marrow graft recipients who developed progressive pancytopenia during the third month post-transplant. Bone marrow biopsy cores were hypocellular without evidence of disease recurrence. Haemopoietic progenitor assays demonstrated a decrease of all assayable progenitors. Cytomegalovirus was identified from the blood and urine of all three patients. However, none of the patients developed other manifestations of cytomegalovirus infection such as pneumonitis, hepatitis and enteritis. The therapeutic use of ganciclovir and intravenous immunoglobulins resulted in prompt resolution of both viraemia and viruria in all three patients, and of pancytopenia in two patients.

Modulation of hematopoiesis by lymphocyte membrane-derived components.

Membrane bound erythroid burst-promoting activity (mBPA) is an integral membrane protein that is present on normal B-cells and some activated T-cells, that induces burst-forming units-erythroid (BFU-E) when cultured with human erythropoietin (rHuEpo). Plasma membranes and vesicles shed from the leukemic A-1 cell line express mBPA. This activity derived from both A-1 cells and normal B-cells can be immunoadsorbed by the D3A4 antibody raised against mBPA. In this study, we demonstrate that interferon-gamma (IFN-gamma) suppresses BFU-E proliferation when added directly to culture of normal human bone marrow cells and in the absence and presence of A-1 cells. However, FACS analysis reveals that IFN-gamma enhances the surface expression of mBPA on A-1 cells. The role of IFN-gamma in modulating erythropoiesis in vitro is discussed with respect to the role of shedding membrane-derived vesicles from the B-cell surface.