A review of depleted uranium biological effects: in vitro and in vivo studies.

The use of depleted uranium in armor-penetrating munitions remains a source of controversy because of the numerous unanswered questions about its long-term health effects. Although no conclusive epidemiologic data have correlated DU exposure to specific health effects, studies using cultured cells and laboratory rodents continue to suggest the possibility of leukemogenic, genetic, reproductive, and neurological effects from chronic exposure. Until issues of concern are resolved with further research, the use of depleted uranium by the military will continue to be controversial.

Successful Use of Ceftolozane-Tazobactam to Treat a Pulmonary Exacerbation of Cystic Fibrosis Caused by Multidrug-Resistant Pseudomonas aeruginosa.

Ceftolozane-tazobactam, a novel ß-lactam/ß-lactamase inhibitor, was recently approved for the treatment of complicated urinary tract and intraabdominal infections, as monotherapy and in combination with metronidazole, respectively. Ceftolozane-tazobactam exhibits a wide spectrum of activity against both gram-positive bacteria, gram-negative bacteria including multidrug-resistant (MDR) Pseudomonas aeruginosa, and some anaerobic bacteria. Although not currently approved for any pulmonary indication, studies have demonstrated excellent distribution to epithelial lining fluid, indicating that it may be an alternative agent to use in the treatment of respiratory tract infections caused by MDRP. aeruginosa. Unfortunately, data are lacking regarding the use of ceftolozane-tazobactam in the treatment of respiratory tract infections including patients with cystic fibrosis (CF). We describe the first case report, to our knowledge, of a 25-year-old white man successfully treated with ceftolozane-tazobactam for a pulmonary exacerbation of his CF caused by MDRP. aeruginosa. He was admitted for his fourth hospitalization in 7 months for a pulmonary exacerbation of his CF. After blood and sputum were cultured, prednisone, cefepime, inhaled tobramycin, and intravenous ciprofloxacin were started. On day 4, after no signs of clinical improvement, respiratory cultures revealed nonmucoid MDRP. aeruginosa, susceptible only to colistin. ß-Lactam therapy was subsequently changed to ceftolozane-tazobactam 3 g intravenously every 8 hours while continuing ciprofloxacin and inhaled tobramycin. Ceftolozane-tazobactam susceptibility was determined by the Etest method (minimum inhibitory concentration 1.5 µg/ml). By day 3 of therapy, the patient showed signs of clinical improvement and was discharged after completion of a 12-day course of antibiotics. Until additional research is available, we hope this evidence will provide consideration of ceftolozane-tazobactam for this novel off-label indication.

Safety and immunogenicity of a mutagenized, live attenuated Rift Valley fever vaccine, MP-12, in a Phase 1 dose escalation and route comparison study in humans.

Rift Valley fever (RVF) poses a risk as a potential agent in bioterrorism or agroterrorism. A live attenuated RVF vaccine (RVF MP-12) has been shown to be safe and protective in animals and showed promise in two initial clinical trials. In the present study, healthy adult human volunteers (N=56) received a single injection of (a) RVF MP-12, administered subcutaneously (SQ) at a concentration of 10(4.7) plaque-forming units (pfu) (SQ Group); (b) RVF MP-12, administered intramuscularly (IM) at 10(3.4)pfu (IM Group 1); (c) RVF MP-12, administered IM at 10(4.4)pfu (IM Group 2); or (d) saline (Placebo Group). The vaccine was well tolerated by volunteers in all dose and route groups. Infrequent and minor adverse events were seen among recipients of both placebo and RVF MP-12. One subject had viremia detectable by direct plaque assay, and six subjects from IM Group 2 had transient low-titer viremia detectable only by nucleic acid amplification. Of the 43 vaccine recipients, 40 (93%) achieved neutralizing antibodies (measured as an 80% plaque reduction neutralization titer [PRNT80]) as well as RVF-specific IgM and IgG. The highest peak geometric mean PRNT80 titers were observed in IM Group 2. Of 34 RVF MP-12 recipients available for testing 1 year following inoculation, 28 (82%) remained seropositive (PRNT80≥1:20); this included 20 of 23 vaccinees (87%) from IM Group 2. The live attenuated RVF MP-12 vaccine was safe and immunogenic at the doses and routes studied. Given the need for an effective vaccine against RVF virus, further evaluation in humans is warranted.

Embedded weapons-grade tungsten alloy shrapnel rapidly induces metastatic high-grade rhabdomyosarcomas in F344 rats.

Continuing concern regarding the potential health and environmental effects of depleted uranium and lead has resulted in many countries adding tungsten alloy (WA)-based munitions to their battlefield arsenals as replacements for these metals. Because the alloys used in many munitions are relatively recent additions to the list of militarily relevant metals, very little is known about the health effects of these metals after internalization as embedded shrapnel. Previous work in this laboratory developed a rodent model system that mimicked shrapnel loads seen in wounded personnel from the 1991 Persian Gulf War. In the present study, we used that system and male F344 rats, implanted intramuscularly with pellets (1 mm times 2 mm cylinders) of weapons-grade WA, to simulate shrapnel wounds. Rats were implanted with 4 (low dose) or 20 pellets (high dose) of WA. Tantalum (20 pellets) and nickel (20 pellets) served as negative and positive controls, respectively. The high-dose WA-implanted rats (n = 46) developed extremely aggressive tumors surrounding the pellets within 4-5 months after implantation. The low-dose WA-implanted rats (n = 46) and nickel-implanted rats (n = 36) also developed tumors surrounding the pellets but at a slower rate. Rats implanted with tantalum (n = 46), an inert control metal, did not develop tumors. Tumor yield was 100% in both the low- and high-dose WA groups. The tumors, characterized as high-grade pleomorphic rhabdomyosarcomas by histopathology and immunohistochemical examination, rapidly metastasized to the lung and necessitated euthanasia of the animal. Significant hematologic changes, indicative of polycythemia, were also observed in the high-dose WA-implanted rats. These changes were apparent as early as 1 month postimplantation in the high-dose WA rats, well before any overt signs of tumor development. These results point out the need for further studies investigating the health effects of tungsten and tungsten-based alloys.

Depleted uranium-uranyl chloride induces apoptosis in mouse J774 macrophages.

Depleted uranium entering the body as a result of inhalation or embedded fragments becomes associated to a great extent with macrophages. As part of our continuing studies on the health effects of internalized depleted uranium, we investigated the effect of soluble depleted uranium-uranyl chloride on the mouse macrophage cell line, J774. Using a cytochemical staining protocol specific for uranium, we found that uranium uptake by the macrophages increased in a time-dependent manner. Treatment with 1, 10, or 100 microM depleted uranium-uranyl chloride resulted in decreased viability of the J774 cells within 24 h. Flow cytometric analysis of the treated cells with annexin V showed the translocation of phosphatidylserine from the inner face of the plasma membrane to the outer surface indicating the loss of phospholipid symmetry and the beginning of the apoptotic process. Significant differences in annexin V labeling between control cells and cells treated with 100 microM depleted uranium-uranyl chloride were apparent within 2 h. Other events associated with apoptosis, including morphological changes and DNA fragmentation, were also apparent after depleted uranium-uranyl chloride treatment. These results suggest that the uptake and concentration of soluble depleted uranium by macrophages initiates events that results in the apoptotic death of these cells.

Determination of depleted uranium in urine via isotope ratio measurements using large-bore direct injection high efficiency nebulizer-inductively coupled plasma mass spectrometry.

Inductively coupled plasma mass spectrometry (ICP-MS), coupled with a large-bore direct injection high efficiency nebulizer (LB-DIHEN), was utilized to determine the concentration and isotopic ratio of uranium in 11 samples of synthetic urine spiked with varying concentrations and ratios of uranium isotopes. Total U concentrations and (235)U/(238)U isotopic ratios ranged from 0.1 to 10 microg/L and 0.0011 and 0.00725, respectively. The results are compared with data from other laboratories that used either alpha-spectrometry or quadrupole-based ICP-MS with a conventional nebulizer-spray chamber arrangement. Severe matrix effects due to the high total dissolved solid content of the samples resulted in a 60 to 80% loss of signal intensity, but were compensated for by using (233)U as an internal standard. Accurate results were obtained with LB-DIHEN-ICP-MS, allowing for the positive identification of depleted uranium based on the (235)U/(238)U ratio. Precision for the (235)U/(238)U ratio is typically better than 5% and 15% for ICP-MS and alpha-spectrometry, respectively, determined over the concentrations and ratios investigated in this study, with the LB-DIHEN-ICP-MS system providing the most accurate results. Short-term precision (6 min) for the individual (235)U and (238)U isotopes in synthetic urine is better than 2% (N = 7), compared to approximately 5% for conventional nebulizer-spray chamber arrangements and >10% for alpha-spectrometry. The significance of these measurements is discussed for uranium exposure assessment of Persian Gulf War veterans affected by depleted uranium ammunitions.

Depleted uranium: a radiochemical toxicant?

The first large-scale combat use of depleted uranium (DU) weapons occurred during the Gulf War, and some U.S. personnel were wounded by DU fragments. Established fragment removal policies dictated that embedded metal fragments be left in place unless doing so posed unacceptable additional risks. However, questions were raised as to whether these policies are appropriate for a metal that--unlike lead, steel, or others--is chemically toxic and emits low-level radiation. Data from research currently under way indicate that long-term exposure to embedded DU fragments may present a level of risk that requires modification of established policies. Our understanding of DU health effects and of the possible mechanisms by which DU might affect tissues is evolving. Understanding more about the long-term response of tissues exposed to DU could facilitate future development of treatments for DU injuries.

Genomic instability in human osteoblast cells after exposure to depleted uranium: delayed lethality and micronuclei formation.

It is known that radiation can induce a transmissible persistent destabilization of the genome. We have established an in vitro cellular model using HOS cells to investigate whether genomic instability plays a role in depleted uranium (DU)-induced effects. Transmissible genomic instability, manifested in the progeny of cells exposed to ionizing radiation, has been characterized by de novo chromosomal aberrations, gene mutations, and an enhanced death rate. Cell lethality and micronuclei formation were measured at various times after exposure to DU, Ni, or gamma radiation. Following a prompt, concentration-dependent acute response for both endpoints, there was de novo genomic instability in progeny cells. Delayed reproductive death was observed for many generations (36 days, 30 population doublings) following exposure to DU, Ni, or gamma radiation. While DU stimulated delayed production of micronuclei up to 36 days after exposure, levels in cells exposed to gamma-radiation or Ni returned to normal after 12 days. There was also a persistent increase in micronuclei in all clones isolated from cells that had been exposed to nontoxic concentrations of DU. While clones isolated from gamma-irradiated cells (at doses equitoxic to metal exposure) generally demonstrated an increase in micronuclei, most clonal progeny of Ni-exposed cells did not. These studies demonstrate that DU exposure in vitro results in genomic instability manifested as delayed reproductive death and micronuclei formation.

Health effects of embedded depleted uranium.

The health effects of embedded fragments of depleted uranium (DU) are being investigated to determine whether current surgical fragment-removal policies are appropriate for this metal. The authors studied rodents implanted with DU pellets as well as cultured human cells exposed to DU compounds. Results indicate that uranium from implanted DU fragments distributes to tissues distant from implantation sites, including bone, kidney, muscle, and liver. Despite levels of uranium in kidney that would be nephrotoxic after acute exposure, no histological or functional kidney toxicity was observed with embedded DU, indicating that the kidney adapts when exposed chronically. Nonetheless, further studies of the long-term health impact are needed. DU is mutagenic and transforms human osteoblastic cells into a tumorigenic phenotype. It alters neurophysiological parameters in rat hippocampus, crosses the placental barrier, and enters fetal tissue. Preliminary data also indicate decreased rodent litter size when animals are bred 6 months or longer after DU implantation.

Primary induction of human CD8+ cytotoxic T lymphocytes and interferon-gamma-producing T cells after smallpox vaccination.

This study measured the ability of a standard smallpox vaccine, given by scarification (by bifurcated needle), to induce primary human vaccinia virus-specific cytotoxic and interferon (IFN)-gamma-producing T lymphocyte responses. Because protection against smallpox may be mediated in part by T cell memory responses induced by vaccination, an analysis of the induction of primary human cytotoxic T lymphocytes (CTL) and IFN-gamma-producing T cell responses was performed. Although smallpox is no longer an epidemic threat under natural conditions, vaccination is still recommended for persons working with vaccinia viruses in the laboratory and for those who may be at risk from the potential use of smallpox virus as a bioterrorism agent. The results demonstrate that smallpox vaccine given by bifurcated needle induces strong vaccinia virus-specific CD8(+) CTL and IFN-gamma-producing T cell responses and provide baseline information useful for planning the immunologic assessment of future smallpox vaccines.

Constitutive NO synthase regulates the Na+/Ca2+ exchanger in human T cells: role of [Ca2+]i and tyrosine phosphorylation.

For many types of cells, heat stress leads to an increase in intracellular free calcium concentration ([Ca2+](i)) that has been shown to trigger a wide variety of cellular responses. In T lymphocytes, for example, heat stress stimulates pathways that make them more susceptible to Fas/CD95-mediated apoptosis. Because of our interest in understanding more about the response of lymphocytes to various stressors, we used human peripheral and Jurkat T lymphocytes to investigate the effect of heat stress on calcium homeostasis. We found that peripheral and Jurkat T cells both exhibit cNOs activity but not iNOs activity. Heat stress increased NO production, which was inhibited by LNNA (a cNOs inhibitor) but not L-NIL (an iNOs inhibitor). Heat stress increased [Ca2+](i) in Jurkat T cells by decreasing the K(m) of the cell surface membrane Na+/Ca2+ exchanger for extracellular Ca2+. Heating also increased cNOs phosphorylation at tyrosine residues. In cells incubated with LNNA, heat stress promoted an increase in [Ca2+](i) and a decrease in [Na+](i) greater than in cells heated without LNNA, a larger decrease in K(m) of the Na+/Ca2+ exchanger for extracellular Ca2+, and decreased phosphorylation of cNOs. Our results suggest that cNOs plays an important regulatory role after heat stress. Heating appears to increase the phosphorylation of cNOs that is complexed with the Na+/Ca2+ exchanger to decrease its activity. This process is related to increased expression of Fas/CD95 on the cell surface, which might explain the apoptotic diathesis of lymphocytes after heat stress.