Department of Radiation Biology
Uniformed Services University
of the Health Sciences
Bldg. 42, 8901 Wisconsin Ave.
Bethesda, MD 20889-5603
Vijay K. Singh, PhD
Associate Professor and Acting Chair,
Department of Radiation Biology
List of recent publications
The primary research interests of my laboratory fall into the following areas: 1) Development of myeloid progenitors as a radiation countermeasure, 2) Investigation of anti-ceramide antibody as a radiation mitigator, 3) Role of Toll-like receptor ligands in radioprotection/mitigation, 4) Role of granulocyte colony-stimulating factor (G-CSF) in radioprotection and cytokines as efficacy biomarkers for radiation countermeasures, and 5) Tocopherol succinate mobilized progenitors as a bridging therapy for radiation victims.
Our overall goal is to elucidate the mechanism of action for radiation countermeasures at the cellular and molecular levels, and investigate cell signaling pathways such as cytokine expression, NF-?B stimulation, p53 pathway, and toll-like receptor activation. Our ultimate goal is to develop fieldable radiation countermeasures and understand their mechanism of action.
Figure 1. Schematic representation of NF-κB signaling pathways. TLR ligands interact with TLR receptors activating NV-κB. Alternatively, radiation exposure results in reactive oxygen species (ROS), which also activates NF-κB.
Ionizing radiation, in moderate to high doses, can cause significant damage to biological systems, resulting in morbidity and mortality. In humans, acute radiation syndrome (ARS) can occur following radiation exposure greater than 1 Gy at a high dose rate. The consequences of increasing radiation doses include hematopoetic, gastrointestinal (GI) and cerebrovascular syndromes. My lab focuses on the hematopoetic and GI syndromes.
Figure 2A. DNA strand breaks in splenocytes of irradiated mice treated with G-CSF antibody.
In collaboration with Cellerant Therapeutics to develop myeloid progenitors as a radiomitigator, my lab has worked with cryopreservable, culture-derived mouse progenitor cells (mMPC). These cells have the potential to mitigate radiation injury in unmatched recipients across a broad range of lethal radiation doses, even when administration is delayed up to 7 days after irradiation. The flexibility in time of administration relative to irradiation, in addition to the extraordinary efficacy in promoting survival, make mMPC one of the most promising radiation countermeasures for ARS among all candidate therapeutics currently under development.
In collaboration with Prof. Richard Kolesnick of the Memorial Sloan-Kettering Cancer Center in New York, I am investigating acid sphinomyelinase (ASMase)-mediated ceramide, which regulates apoptosis in response to ionizing radiation in select cell types. Our aim is to develop 2A2 for preclinical testing for use as a radiation mitigator against GI syndrome.
We have developed an approach to predict dose ranges of Toll-like receptor ligands using common pro-inflammatory cytokines as efficacy biomarkers. When using these biomarkers while investigating CBLB502, a truncated derivative of the FliC flagellin protein of Salmonella enterica, we determined the appropriate human dose (0.3–0.45 µg/kg).
I also investigated two other TLR ligands as radiation countermeasures, CBLB613 and CBLB612, which are naturally occurring Mycoplasma-derived lipopeptides. CBLB613 was observed for toxicity, radioprotection, radiomitigation, pharmacokinetics and immunogenicity. CBLB613 provided significant protection to mice against lethal doses of radiation, reduced radiation-induced cytopenia and increased bone cellularity. It also stimulated the induction of IL-ß, IL-6, IL-10, IL-12, KC and G-CSF cytokines. CBLB613 is not immunogenic in mice, indicating its capability as a radioprotectant and radiomitigator for humans. CBLB612 induces high levels of G-CSF and mobilizes progenitors in peripheral circulation.
Figure 2B. Analysis of p53 up-regulated modulator of apoptosis (PUMA) in irradiated mice treated with isotope.
My lab has evaluated the role of G-CSF on survival and tissue injury after total-body gamma-irradiation (figure 2A). G-CSF stimulates granulopoisesis, which increase the levels of circulating polymorphonuclear leukocytes. We analyzed mice exposed to irradiation post administration of the neutralizing antibody to G-CSF. The neutralizing antibody exacerbates the deleterious effects of radiation, indicating the important role G-CSF plays in post-radiation recovery. Administration of the G-CSF antibody significantly increased mortality in irradiated mice. Our results show that when a neutralizing antibody was administered to mice prior to irradiation, radiation-induced DNA damage increased.
Our investigation of tocopherol succinate as a radiation countermeasure demonstrated protection against radiation-induced hematopoetic and gastrointestinal syndromes. TS also modulates thrombocytopenia, neutropenia, and monocytopenia, as well as antioxidant enzymes and oncogene expression. Infusion of whole blood or peripheral blood of mononuclear cells from TS-injected mice improves the chances of extended survival after exposure to escalating doses of radiation.
Figure 2C. Terminal deoxynucleotidl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in irradiated mice treated with isotype antibody.
Our recent results demonstrate the ability of TS-mobilized progenitors to significantly inhibit apoptosis, enhance cell proliferation in vital gastrointestinal and lymphohematopoetic tissues, and suppress bacterial translocation in irradiated mice when administered after radiation exposure.
Recently, we have initiated a study to determine whole-genome expression signatures associated with G-CSF production in responsive cell types to identify gene regulatory networks involved in receptor-mediated activation by various tocols. Our objective is to profile mobilized human CD34+ cells to determine the transcriptomic and proteomic signatures associated with mitigation efficacy.
- 2015 - Dalgard CL, Jacobowitz DM, Singh VK, Saleem KS, Ursano RJ, Starr JM, Pollard HB: A novel analytical brain block tool to enable functional annotation of discriminatory transcript biomarkers among discrete regions of the fronto-limbic circuit in primate brain. Brain Research (in press doi: 10.1016/j.brainres.2014.12.031).
- 2015 - Singh VK, Romaine LP, Seed TM: Medical countermeasures for radiation exposures and related injuries: Characteristics of medicinals, FDA-approval status and inclusion into the strategic national pharmaceutical stockpile. Health Physics (in press).
- 2015 - Singh VK, Newman VL, Seed TM: Colony-stimulating factors for the treatment of acute radiation syndrome: A review. Cytokine 71:22-37.
- 2014 - Rosen, EM, Day R, Singh VK: New approaches to radiation protection. Front. Oncol. 4:381.
- 2014 - Singh VK, Romaine PLP, Newman VL: Biologics as countermeasures for acute radiation syndrome: Where are we now? Expert Opin Biol Ther (in press: doi:10.1517/14712598.2015.986453).
- 2014 - Singh VK, Newman VL, Romaine PLP, Wise SY, Seed TM. Radiation countermeasure agents: an update (2011 - 2014). Exper Opin Ther Pat. 24:1229-1255.
- 2014—Singh VK, Romaine PLP, Newman VL, Seed TM. Tocols induce G-CSF and mobilize progenitors that mitigate radiation injury. Radiation Protection Dosimetry (in press).
- 2014—Seed TM, Inal CE, Singh VK. Radioprotection of hematopoietic progenitors by low dose amifostine prophylaxis. Int J Rad Biol. 90:594-604.
- 2014—Singh VK, Wise SY, Scott JR, Romaine, LP, Newman VL, Fatanmi OO. Radioprotective efficacy of delta-tocotrienol, a vitamin E isoform, is mediated through granulocyte colony-stimulating factor. Life Sci. 98:113–122.
- 2014—Singh VK, Wise SY, Fatanmi OO, Beattie, L, Seed TM. Preclinical development of a bridging therapy for radiation casualities. Health Phys. 689–698.
- 2014—Singh VK, Wise SY, Fatanmi OO, Beattie L, Ducey EJ, Seed TM. Alpha-tocopherol succinate- and AMD3100-mobilized progenitors mitigate radiation combined injury in mice. J. Radiat. Res. 55:41–53.
- 2013—Singh VK, Beattie L, Seed TM. Vitamin E: Tocopherols and tocotrienols as potential radiation countermeasures. J Radiat Res. 54:973–988.
- 2013—Kulkarni S, Singh PK, Ghosh S, Posarac A, Singh VK. Granulocyte colony-stimulating factor antibody abrogates radioprotective efficacy of gamma-tocotrienol, a promising radiation countermeasure. Cytokine 62:278–285.
- 2013—Singh VK, Singh PK, Wise SY, Posarac A, Fatanmi OO.
Radioprotective properties of tocopherol succinate against ionizing radiation in mice. J Radiat Res. 54:210–20. doi: 10.1093/jrr/rrs088.
- 2013—Singh VK, Wise SY, Singh PK, Posarac A, OO Fatanmi, Ducey EJ, Bolduc DL, Elliott TB, Seed TM. Alpha-tocopherol succinate-mobilized progenitors improve intestinal integrity after whole body irradiation. Int J Rad Biol. 89:334–345. doi: 10.3109/09553002.2013.762137.
- 2012—Krivokrysenko V, Shakhov A, Singh V, Bone F, Kononov Y, Shyshynova i, Cheney A, Maitra R, Purmal A, Whitnall M, Gudkov AV, Feiinstein E. Identification of G-CSF and IL-6 as candidate biomarkers of CBLB502 efficacy as a medical radiation countermeasure. J Pharmacol Exp Ther. 343:497–508. doi: 10.1124/jpet.112.196071.
- 2012—Grace MB, Singh VK, Rhee JG, Jackson WE III, Kao T-C, Whitnall MH. 5-AED enhances survival of irradiated mice in a G-CSF-dependent manner, stimulates innate immune cell function, reduces radiation-induced DNA damage and induces genes that modulate cell cycle progression and apoptosis. J Rad Res. 53:840–853. doi: 10.1093/jrr/rrs060.
- 2012—Singh VK, Christensen J, Fatanmi OO, Gille D, Ducey EJ, Wise SY, Karsunky H, Sedello AK. Myeloid progenitors: A radiation countermeasure that is effective when initiated days after irradiation.Radiat Res. 177:781–791.
- 2012—ShakhovAN, SinghVK, BoneF, CheneyA, KononovY, et al. Prevention and mitigation of acute radiation syndrome in mice by synthetic lipopeptide agonists of Toll-like receptor 2 (TLR2). PLoS ONE 7: e33044, doi:10.1371/ journal.pone.0033044.
- 2012—Singh VK, Ducey EJ, Brown DS, Whitnall MH. A review of radiation countermeasure work ongoing at the Armed Forces Radiobiology Research Institute. Int J Radiat Biol. 88:296–310.
- 2012—Singh VK, Ducey EJ, Fatanmi OO, Singh PK, Brown DS,Purmal A, Shakhova VV, Gudkov AV, Feinstein E, Shakhov A. CBLB613: A TLR 2/6 agonist, natural lipopeptide of Mycoplasma arginini, as a novel radiation countermeasure. Radiat Res. 177:628–642.
- 2012—Singh VK, Fatanmi OO, Singh PK, Whitnall MH. Role of radiation-induced granulocyte colony-stimulating factor in recovery from whole body gamma-irradiation.Cytokine. 58:406–414.
- 2012—Singh PK, Wise SY, Ducey EJ, Fatanmi OO, Elliott TB, Singh VK.a-Tocopherol succinate protects mice against radiation-induced gastrointestinal injury. Radiat Res. 177:133–45.
- 2012—Singh VK, Wise SY, Singh PK, Ducey EJ, Fatanmi OO, Seed TM. a-Tocopherol succinate and AMD3100-mobilized progenitors mitigate radiation-induced gastrointestinal injury in mice. Exp Hematol. 40:407–417.
- 2011—Singh VK, Brown, DS, Singh, PK, Seed, TM. Progenitor cells as a bridging therapy for radiation casualties. Defence Science Journal 61: 118–124, 2011.
- 2011—Singh PK, Wise SY, Ducey EJ, Brown DS, Singh VK. Radioprotective efficacy of tocopherol succinate is mediated through granulocyte-colony stimulating factor. Cytokine. 56:411–421, 2011.
- 2011—Singh VK, Singh PK, Wise SY, Seed TM. Mobilized progenitor cells as a bridging therapy for radiation casualties: A brief review of tocopherol succinate-based approaches, Int Immunopharmacol. 11:842–47, 2011.
- 2011—Singh VK, Parekh VI, Brown DS, Kao TC, Mog SR. Tocopherol succinate: modulation of antioxidant enzymes and oncogene expression, and hematopoietic recovery, Int J Radiat Oncol Biol Phys. 79:571–8, 2011.
- 2010—Singh VK, Brown DS, Kao T-C. Alpha-tocopherol succinate protects mice from gamma-radiation by induction of granulocyte-colony stimulating factor, Int J Radiat Biol. 86:12–21, 2010.
- 2010—Singh VK, Brown DS, Kao T-C, Seed TM. Preclinical development of a bridging therapy for radiation casualties, Exp Hematol. 38:61–70, 2010.