Joseph Albanese

Ionizing Radiation Alters Fas Antigen Ligand at the Cell Surface and on Exfoliated Plasma Membrane-Derived Vesicles: Implications for Apoptosis and Intercellular Signaling

Abstract Albanese, J. and Dainiak, N. 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.

First global consensus for evidence-based management of the hematopoietic syndrome resulting from exposure to ionizing radiation.

OBJECTIVE Hematopoietic syndrome (HS) is a clinical diagnosis assigned to people who present with ≥ 1 new-onset cytopenias in the setting of acute radiation exposure. The World Health Organization convened a panel of experts to evaluate the evidence and develop recommendations for medical countermeasures for the management of HS in a hypothetical scenario involving the hospitalization of 100 to 200 individuals exposed to radiation. The objective of this consultancy was to develop recommendations for treatment of the HS based upon the quality of evidence. METHODS English-language articles were identified in MEDLINE and PubMed. Reference lists of retrieved articles were distributed to panel members before the meeting and updated during the meeting. Published case series and case reports of individuals with HS, published randomized controlled trials of relevant interventions used to treat nonirradiated individuals, reports of studies in irradiated animals, and prior recommendations of subject matter experts were selected. Studies were extracted using the Grading of Recommendations Assessment Development and Evaluation (GRADE) system. In cases in which data were limited or incomplete, a narrative review of the observations was made. No randomized controlled trials of medical countermeasures have been completed for individuals with radiation-associated HS. The use of GRADE analysis of countermeasures for injury to hematopoietic tissue was restricted by the lack of comparator groups in humans. Reliance on data generated in nonirradiated humans and experimental animals was necessary. RESULTS Based upon GRADE analysis and narrative review, a strong recommendation was made for the administration of granulocyte colony-stimulating factor or granulocyte macrophage colony-stimulating factor and a weak recommendation was made for the use of erythropoiesis-stimulating agents or hematopoietic stem cell transplantation. CONCLUSIONS Assessment of therapeutic interventions for HS in humans exposed to nontherapeutic radiation is difficult because of the limits of the evidence.

Literature Review and Global Consensus on Management of Acute Radiation Syndrome Affecting Nonhematopoietic Organ Systems

OBJECTIVES The World Health Organization convened a panel of experts to rank the evidence for medical countermeasures for management of acute radiation syndrome (ARS) in a hypothetical scenario involving the hospitalization of 100 to 200 victims. The goal of this panel was to achieve consensus on optimal management of ARS affecting nonhematopoietic organ systems based upon evidence in the published literature. METHODS English-language articles were identified in MEDLINE and PubMed. Reference lists of retrieved articles were distributed to conferees in advance of and updated during the meeting. Published case series and case reports of ARS, publications of randomized controlled trials of relevant interventions used to treat nonirradiated individuals, reports of studies in irradiated animals, and prior recommendations of subject matter experts were selected. Studies were extracted using the Grading of Recommendations Assessment Development and Evaluation system. In cases in which data were limited or incomplete, a narrative review of the observations was made. RESULTS No randomized controlled trials of medical countermeasures have been completed for individuals with ARS. Reports of countermeasures were often incompletely described, making it necessary to rely on data generated in nonirradiated humans and in experimental animals. A strong recommendation is made for the administration of a serotonin-receptor antagonist prophylactically when the suspected exposure is >2 Gy and topical steroids, antibiotics, and antihistamines for radiation burns, ulcers, or blisters; excision and grafting of radiation ulcers or necrosis with intractable pain; provision of supportive care to individuals with neurovascular syndrome; and administration of electrolyte replacement therapy and sedatives to individuals with significant burns, hypovolemia, and/or shock. A strong recommendation is made against the use of systemic steroids in the absence of a specific indication. A weak recommendation is made for the use of fluoroquinolones, bowel decontamination, loperamide, and enteral nutrition, and for selective oropharyngeal/digestive decontamination, blood glucose maintenance, and stress ulcer prophylaxis in critically ill patients. CONCLUSIONS High-quality studies of therapeutic interventions in humans exposed to nontherapeutic radiation are not available, and because of ethical concerns regarding the conduct of controlled studies in humans, such studies are unlikely to emerge in the near future.

Regulation of TNFRSF6 (Fas) Expression in Ataxia Telangiectasia Cells by Ionizing Radiation1

Abstract Albanese, J. and Dainiak, N. 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 μg/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 (≤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 (≤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.

Local, regional and national responses for medical management of a radiological/nuclear incident.

Radiological and nuclear devices may be used by terrorists or may be the source of accidental exposure. A tiered approach has been recommended for response to a terrorist event wherein local, regional, state and federal assets become involved sequentially, as the magnitude in severity of the incident increases. State-wide hospital plans have been developed and published for Connecticut, New York and California. These plans address delineation of responsibilities of various categories of health professionals, protection of healthcare providers, identification and classification of individuals who might have been exposed to and/or contaminated by radiation and, in the case of Connecticut response plan, early management of victims. Regional response programs such as the New England Regional Health Compact (consisting of 6 member states) have been developed to manage consequences of radiation injury. The Department of Homeland Security is ultimately responsible for managing both health consequences and the crisis. Multiple US national response assets may be called upon for use in radiological incidents. These include agencies and programs that have been developed by the Department of Energy, the Environmental Protection Agency and the Department of Defense. Coordination of national, regional and state assets with local response efforts is necessary to provide a timely and efficient response.

Impact of Mass Casualties Resulting from Radiation Exposure on Healthcare Systems

Detonation of a nuclear device in a major metropolitan area constitutes the greatest vulnerability for health care facilities. Such a catastrophe would strain the capabilities and capacities of a health care system, and impair its ability to provide an acceptable standard of care. Accommodation for a large and sudden influx of victims constitutes a formidable challenge for health care facilities that are already operating at near-full capacity. Health care facilities must reestablish priorities concerning communication with the public, and must modify their role in delivering community services. The need for radiological surveys, decontamination of victims, and medical management of potentially contaminated patients adds to this adverse impact. Caregivers must understand basic guidelines for prevention of contamination of themselves and others. Mitigation of these events requires a successful integration of the health care system response with the overall community response, and with state, regional, and federal response efforts. Attention must be directed toward establishment of a command and control system, conduction of radiological, or nuclear compatible triage, management of radiation-associated injuries, and arrangement for appropriate follow-up of psychosocial issues.

Ionizing radiation (Ir) Alters functional activity of cell surface fas and fas ligand (Fasl) In human bone marrow stromal cells

Abstract Hematopoietic homeostasis requires a network of cytokines that balances proliferation and differentiation with apoptosis of stem/progenitor cells. We have reported that Fas and FasL, principal mediators of apoptosis, are modulated at the cell surface by IR (Albanese and Dainiak, Radiat Res 153:49, 2000). Here, we examined expression and functional activity of Fas and FasL in a cytokine-producing, human bone marrow stromal cell line (HS-5), before and after exposure to IR. Plasma membranes were purified from HS-5 cells, solubilized in Triton-X 100 and subjected to Western blot analysis. Proteins of 40 and 48 kDal, corresponding to FasL and Fas, respectively, were observed, a finding confirmed by flow cytometry and RT-PCR. Despite the presence of Fas receptor, anti-Fas antibodies DX-2 and CH.11 (100–500 ng/ml), failed to induced cell death in HS-5 cells, as determined by the MTT assay. Hs-5 cells exposed to IR (0–10 Gy), displayed a dose-dependent increase in levels of surface Fas and FasL, as assessed by flow cytometry. To examine the biological activity of FasL expressed by HS-5 cells, irradiated and non-irradiated HS-5 cells (effectors) were co-cultured with Jurkat cells (targets), a cell line sensitive to FasL. Viability of target cells was determined using the MTT assay following a 24 hr co-incubation period. We observed that Jurkat cell viability increased in a dose-dependent manner in samples co-cultivated with HS-5 cells exposed to 0–10 Gy of IR. A maximum increase (relative to non-irradiated HS-5 cells) in target cell survival was observed in Jurkat cell samples co-cultered with HS-5 cells that were previously irradiated (10 Gy). These findings suggest that HS-5 cells express FasL at their surface in a biologically active form and that functional activity of FasL is diminished following exposure to IR. Whether the reduction in apoptosis-inducing activity of FasL results from direct damage by IR to the protein or to the proteins lipid microenvironment is unclear. The finding that Fas is expressed by HS-5 cells in an inactive form may explain why these cells fail to undergo apoptosis when grown in monolayer.