Embedded fragments from U.S. military personnel--chemical analysis and potential health implications.

BACKGROUND: The majority of modern war wounds are characterized by high-energy blast injuries containing a wide range of retained foreign materials of a metallic or composite nature. Health effects of retained fragments range from local or systemic toxicities to foreign body reactions or malignancies, and dependent on the chemical composition and corrosiveness of the fragments in vivo. Information obtained by chemical analysis of excised fragments can be used to guide clinical decisions regarding the need for fragment removal, to develop therapeutic interventions, and to better anticipate future medical problems from retained fragment related injuries. In response to this need, a new U.S Department of Defense (DoD) directive has been issued requiring characterization of all removed fragments to provide a database of fragment types occurring in combat injuries. OBJECTIVES: The objective of this study is to determine the chemical composition of retained embedded fragments removed from injured military personnel, and to relate results to histological findings in tissue adjacent to fragment material. METHODS: We describe an approach for the chemical analysis and characterization of retained fragments and adjacent tissues, and include case examples describing fragments containing depleted uranium (DU), tungsten (W), lead (Pb), and non-metal foreign bodies composed of natural and composite materials. Fragments obtained from four patients with penetrating blast wounds to the limbs were studied employing a wide range of chemical and microscopy techniques. Available adjacent tissues from three of the cases were histologically, microscopically, and chemically examined. The physical and compositional properties of the removed foreign material surfaces were examined with energy dispersive x-ray fluorescence spectrometry (EDXRF), scanning electron microscopy (SEM), laser ablation inductively-coupled plasma mass-spectrometry (LA-ICP-MS), and confocal laser Raman microspectroscopy (CLRM). Quantitative chemical analysis of both fragments and available tissues was conducted employing ICP-MS. RESULTS: Over 800 fragments have been characterized and included as part of the Joint Pathology Center Embedded Fragment Registry. Most fragments were obtained from penetrating wounds sustained to the extremities, particularly soft tissue injuries. The majority of the fragments were primarily composed of a single metal such as iron, copper, or aluminum with traces of antimony, titanium, uranium, and lead. One case demonstrated tungsten in both the fragment and the connected tissue, together with lead. Capsular tissue and fragments from a case from the 1991 Kuwait conflict showed evidence of uranium that was further characterized by uranium isotopic ratios analysis to contain depleted uranium. CONCLUSIONS: The present study provides a systematic approach for obtaining a full chemical characterization of retained embedded fragments. Given the vast number of combat casualties with retained fragments, it is expected that fragment analysis will have significant implications for the optimal short and long-term care of wounded service members.

Evolution of biomedical research during combat operations.

BACKGROUND: The implementation of a human research protection program in Afghanistan and the mobilization of the combat casualty research team have made it possible to design and efficiently conduct multifaceted, multisite, and prospective research studies in a combat environment. Still, to conduct research in such an environment, several unique challenges must be overcome. METHODS: This article describes the development and conduct of three ongoing trauma-related biomedical research studies in Afghanistan, highlighting the challenges and lessons learned within the context of these studies. RESULTS: Key challenges include the process of developing and getting approval for in-theater research protocols, the informed consent process, and logistics of conducting a biomedical research study in an austere environment. Despite these challenges, important lessons learned that can contribute to the success of a protocol include the need for clear operating procedures, judicious selection for which data points must be collected in-theater, and the importance anticipating the "fog and friction" of war. CONCLUSION: As we continue the journey toward more sophisticated research capabilities in combat, this article will help inform the design and conduct of future research performed in a theater of war. Conducting biomedical research in a combat zone is an important but difficult element of military medicine.

Biological responses in rats exposed to cigarette smoke and Middle East sand (dust).

Respiratory symptoms are frequently reported in personnel deployed to the Middle East. This project characterized the respiratory toxicity of inhaled Iraqi sand (IS). Adult rats underwent a 6-wk inhalation to air or mainstream cigarette smoke (MSCS) (3 h/d, 5 d/wk) that included exposure to IS or crystalline silica (1 mg/m(3), 19 h/d, 7 d/wk) or air during the last 2 weeks. Assessments included motor activity, whole-body plethysmography, cytological and biochemical analysis of bronchoalveolar lavage fluid, lung metal burden, nasal and lung pathology, and changes in lung protein and gene expression. A number of metals including nickel, manganese, vanadium, and chromium were detected in IS. Elevated lung parenchyma aluminum, silica, barium, manganese, and vanadium concentrations were seen in IS-exposed rats, suggesting that several metals present in IS are bioavailable. Rats exposed to IS only developed mild inflammation in the anterior nose and lung. Silica inhalation was associated with some pulmonary responses that were not seen in IS-exposed rats, such as mild laryngeal and tracheal inflammation, mild tracheal epithelial hyperplasia, and elevated lung silica concentrations. MSCS inhalation with or without co-exposure to either IS or silica resulted in changes consistent with pulmonary inflammation and stress response. Rats exposed to MSCS and silica had more widespread airway lesions when compared with rats exposed to MSCS only. Silica-exposed rats had more robust pulmonary gene expression and proteomic responses than that seen in IS-exposed rat. Our studies show that the respiratory toxicity of IS is qualitatively similar to or less than that seen following short-term silica exposure.

α-Synuclein overexpression enhances manganese-induced neurotoxicity through the NF-κB-mediated pathway.

Exposure to manganese (Mn) occurs in both civilian and military operations. Mn exposure results in a movement disorder termed manganism, which resembles Parkinson's disease (PD). However, the pathogenic mechanisms underlying this disorder are not fully understood. α-Synuclein, a presynaptic protein is implicated in some neurodegenerative disorders, including PD and Mn-induced apoptosis, and its overexpression contributes to the loss of dopaminergic neurons. Although the role of α-synuclein in this process is widely documented, its exact function is not clear. The objective of this study was to evaluate the mechanism(s) of dopaminergic degeneration associated with α-synuclein expression in response to Mn exposure and to assess the role of nuclear factor-κB (NF-κB) activation as an intermediary of Mn-induced neurotoxicity. Rat mesencephalic cells (MES 23.5) overexpressing human α-synuclein show enhanced susceptibility to Mn exposure as evidenced by increased apoptosis and NF-κB nuclear translocation. Pretreatment with antioxidants and the p38 mitogen-activated protein kinase (MAPK) inhibitor SB239063 significantly diminished NF-κB activation, supporting a role for oxidative stress and p38 MAPK in Mn-induced NF-κB activation. In addition, increased nitric oxide generation was evident during NF-κB activation, which was blocked by NF-κB (SN50) and MAPK inhibitors. Mn-induced cell death was attenuated by SN-50 and specific nitric oxide synthase (NOS) inhibitor (1400W); corroborating NOS activation is mediated through NF-κB in the mechanism of cell death. These data indicate that the transcription factor NF-κB, p38 MAPK, and apoptotic signaling cascades are activated by Mn in human α-synuclein-overexpressing cells. Thus, α-synuclein may facilitate Mn-induced neurotoxicity, and along with NF-κB, it may play a role in dopaminergic cell death.

Pharmacokinetics of radiolabeled tungsten ((188)W) in male Sprague-Dawley rats following acute sodium tungstate inhalation.

Aerosol cloud formation may occur when certain tungsten munitions strike hard targets, placing military personnel at increased risk of exposure. Although the pharmacokinetics of various forms of tungsten have been studied in animals following intravenous and oral administration, tungsten disposition following inhalation remains incompletely characterized. The objective of this study was to evaluate the pharmacokinetics of inhaled tungstate (WO(4)) in rats. Male, 16-wk-old, CD rats (n = 7 rats/time point) underwent a single, 90-min, nose-only exposure to an aerosol (mass median aerodynamic diameter [MMAD] 1.50 mum ) containing 256 mg W/m(3) as radiolabeled sodium tungstate (Na(2)(188)WO(4)). (188)W tissue concentrations were determined at 0, 1, 3, 7, and 21 days postexposure by gamma spectrometry. The thyroid and urine had the highest (188)W levels postexposure, and urinary excretion was the primary route of (188)W elimination. The pharmacokinetics of tungsten in most tissues was best described with a two-compartment pharmacokinetic model with initial phase half-lives of approximately 4 to 6 h and a longer terminal phase with half-lives of approximately 6 to 67 days. The kidney, adrenal, spleen, femur, lymph nodes, and brain continued to accumulate small amounts of tungsten as reflected by tissue:blood activity ratios that increased throughout the 21-day period. At day 21 all tissues except the thyroid, urine, lung, femur, and spleen had only trace levels of (188)W. Data from this study can be used for development and refinement of pharmacokinetic models for tungsten inhalation exposure in environmental and occupational settings.

Loop system for creating jet fuel vapor standards used in the calibration of infrared spectrophotometers and gas chromatographs.

A whole body inhalation study of mixed jet fuel vapor and its aerosol necessitated the development of a method for preparing vapor only standards from the neat fuel. Jet fuel is a complex mixture of components which partitions between aerosol and vapor when aspirated based on relative volatility of the individual compounds. A method was desired which could separate the vapor portion from the aerosol component to prepare standards for the calibration of infrared spectrophotometers and a head space gas chromatography system. A re-circulating loop system was developed which provided vapor only standards whose composition matched those seen in an exposure system. Comparisons of nominal concentrations in the exposure system to those determined by infrared spectrophotometry were in 92-95% agreement. Comparison of jet fuel vapor concentrations determined by infrared spectrophotometry compared to head space gas chromatography yielded a 93% overall agreement in trial runs. These levels of agreement show the loop system to be a viable method for creating jet fuel vapor standards for calibrating instruments.

BNIP3 up-regulation and mitochondrial dysfunction in manganese-induced neurotoxicity.

The central nervous system (CNS) appears to be the critical target of manganese (Mn), and neurotoxicity has been the focus of most of the health effects of manganese. In brain, the mechanism underlying the Mn-induced cell death is not clear. We have previously demonstrated that NFkappabeta induction and the activation of nitric oxide synthase through reactive oxygen species (ROS) represent a proximate mechanism for Mn-induced neurotoxicity. In this study, an immortalized dopaminergic cells were used to characterize the cell death signaling cascade activated by manganese. Exposure to Mn resulted in a time and concentration-related loss of cell viability as observed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and live/dead cell assay. Mn increased BNIP3 expression within 3h and continued to increase up to 24h exposure followed by a concentration-related apoptotic death as determined by TUNEL. Further, Mn treatment resulted in accumulation of reactive oxygen species and mitochondrial dysfunction with loss of mitochondrial membrane potential and release of cytochrome c. Antioxidants significantly reduced Mn-induced BNIP3 expression and attenuated cell death, demonstrating the role of oxidative stress in BNIP3 induction. Blocking BNIP3 up-regulation with a transcription or a translational inhibitor reduced the response to manganese. Cell death by manganese was reduced in the presence of CsA (PT pore inhibitor). In addition, knockdown of BNIP3 by small interfering RNA (siRNA) improved mitochondrial recovery and reduced neuronal cell loss suggesting that constitutive expression of BNIP3 plays a role in Mn-induced neurotoxicity by regulating mitochondrial functions. These findings indicate a potential detrimental role of BNIP3 in manganese-induced neuronal cell death.

Acute sodium tungstate inhalation is associated with minimal olfactory transport of tungsten (188W) to the rat brain.

Olfactory transport of represents an important mechanism for direct delivery of certain metals to the central nervous system (CNS). The objective of this study was to determine whether inhaled tungsten (W) undergoes olfactory uptake and transport to the rat brain. Male, 16-week-old, Sprague-Dawley rats underwent a single, 90-min, nose-only exposure to a Na(2)(188)WO(4) aerosol (256 mg W/m(3)). Rats had the right nostril plugged to prevent nasal deposition of (188)W on the occluded side. The left and right sides of the nose and brain, including the olfactory pathway and striatum, were sampled at 0, 1, 3, 7, and 21 days post-exposure. Gamma spectrometry (n=7 rats/time point) was used to compare the levels of (188)W found on the left and right sides of the nose and brain and blood to determine the contribution of olfactory uptake to brain (188)W levels. Respiratory and olfactory epithelial samples from the side with the occluded nostril had significantly lower end-of-exposure (188)W levels confirming the occlusion procedure. Olfactory bulb, olfactory tract/tubercle, striatum, cerebellum, rest of brain (188)W levels paralleled blood (188)W concentrations at approximately 2-3% of measured blood levels. Brain (188)W concentrations were highest immediately following exposure, and returned to near background concentrations within 3 days. A statistically significant difference in olfactory bulb (188)W concentration was seen at 3 days post-exposure. At this time, (188)W concentrations in the olfactory bulb from the side ipsilateral to the unoccluded nostril were approximately 4-fold higher than those seen in the contralateral olfactory bulb. Our data suggest that the concentration of (188)W in the olfactory bulb remained low throughout the experiment, i.e., approximately 1-3% of the amount of tungsten seen in the olfactory epithelium suggesting that olfactory transport plays a minimal role in delivering tungsten to the rat brain.

Neurobehavioral effects of sodium tungstate exposure on rats and their progeny.

The use of tungsten as a replacement for lead and depleted uranium in munitions began in the mid 1990's. Recent reports demonstrate tungsten solubilizes in soil and can migrate into drinking water supplies and therefore is a potential health risk to humans. This study evaluated the reproductive and neurobehavioral effects of sodium tungstate in Sprague-Dawley rats following 70 days of daily pre- and postnatal exposure. Adult male and female rats were orally dosed with diH(2)O vehicle, 5 or 125 mg/kg/day of sodium tungstate through mating, gestation, and weaning (PND 0-20). Daily administration of sodium tungstate produced no overt evidence of toxicity and had no apparent effect on mating success or offspring physical development. Distress vocalizations were elevated in the highest dose group. There was no treatment related effect on righting reflex latencies, however, the males had significantly shorter latencies than the females. Locomotor activity was affected in both the low and high dose groups of F0 females. Those in the low dose group showed increased distance traveled, more time in ambulatory movements, and less time in stereotypic behavior than controls or high dose animals. The high dose group had more time in stereotypical movements than controls, and less time resting than controls and the lowest exposure group. Maternal retrieval was not affected by sodium tungstate exposure and there were no apparent effects of treatment on F1 acoustic startle response or water maze navigation. Overall, the results of this study suggest pre- and postnatal oral exposure to sodium tungstate may produce subtle neurobehavioral effects in offspring related to motor activity and emotionality. These findings warrant further investigation to characterize the neurotoxicity of sodium tungstate on dams and their developing pups.

Short report: HIV infection among commercial sex workers and injecting drug users in the Czech Republic.

Since the first HIV case was diagnosed in the Czech Republic in 1985, there is a lack of information regarding the epidemiology of HIV infection in most high-risk groups. To determine the prevalence of, and risk factors for, HIV among female and male commercial sex workers (FCSW and MCSW, respectively) and injecting drug users (IDUs), cross-sectional studies were conducted in the cities of Cheb, Usti nad Labem, Ostrava, and Prague of the Czech Republic. A total of 1,277 subjects were enrolled, which included 585 FCSWs, 230 MCSWs, and 462 IDUs. The HIV prevalences were 0.7% (95% CI: 0.2-1.7%), 0.9% (95% CI: 0.1-3.1%), and 0.2% (95% CI: 0.005-1.2%) among FCSWs, MCSWs, and IDUs, respectively. Although low HIV prevalences were found, ongoing sentinel surveillance studies, which address modifiable behavioral and biologic risk factors among high-risk groups, are necessary to guide strategies to stem the tide of the epidemic in this country.

Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax.

Bacillus anthracis is the etiologic agent of anthrax, an acute fatal disease among mammals. It was thought to differ from Bacillus cereus, an opportunistic pathogen and cause of food poisoning, by the presence of plasmids pXO1 and pXO2, which encode the lethal toxin complex and the poly-gamma-d-glutamic acid capsule, respectively. This work describes a non-B. anthracis isolate that possesses the anthrax toxin genes and is capable of causing a severe inhalation anthrax-like illness. Although initial phenotypic and 16S rRNA analysis identified this isolate as B. cereus, the rapid generation and analysis of a high-coverage draft genome sequence revealed the presence of a circular plasmid, named pBCXO1, with 99.6% similarity with the B. anthracis toxin-encoding plasmid, pXO1. Although homologues of the pXO2 encoded capsule genes were not found, a polysaccharide capsule cluster is encoded on a second, previously unidentified plasmid, pBC218. A/J mice challenged with B. cereus G9241 confirmed the virulence of this strain. These findings represent an example of how genomics could rapidly assist public health experts responding not only to clearly identified select agents but also to novel agents with similar pathogenic potentials. In this study, we combined a public health approach with genome analysis to provide insight into the correlation of phenotypic characteristics and their genetic basis.

Etiology, antimicrobial susceptibility profiles, and mortality associated with bacterial meningitis among children in Egypt.

PURPOSE: Surveillance for patients with meningitis is a high priority in order to determine the etiology of disease and design prevention strategies. This study presents data on the causes of bacterial meningitis among children <6 years of age treated in a network of hospitals throughout Egypt. METHODS: Training was provided to standardize the collection of clinical information and optimize recovery of bacterial pathogens. Bacterial isolates were tested for antimicrobial resistance patterns using Kirby Bauer disk diffusion, E-test and/or Beta-lactamase (BL) testing methods. RESULTS: Patients with culture-confirmed bacterial meningitis (228 children<6 years) were identified including 89 (39%) patients with H. influenzae (HI), 68 (30%) with Streptococcus pneumoniae (SP), 30 (13%) with N. meningitidis (NM), 18 (8%) with Mycobacterium tuberculosis (MTB) and 23 (10%) with other bacteria. The overall case fatality ratio was high (24%) and increased among children with TB meningitis (56%). The susceptibility for HI to ampicillin (AMP), chloramphenicol (C) and ceftriaxone (CRO) was 21%, 13%, and 100% respectively. The susceptibility for SP to C and CRO was 79% and 100%, respectively. CONCLUSIONS: HI and SP are the leading causes of bacterial meningitis among children in Egypt. The majority of HI strains tested were resistant to AMP or C suggesting the need for routine use of CRO as first line therapy. Among older children TB emerges as a significant cause of bacterial meningitis in Egypt.

HIV type 1 strains from East and West Africa are intermixed in Sudan.

The genetic subtypes of HIV-1 in the Sudan epidemic have not been characterized. Here we report the partial sequencing and analysis of 30 strains collected from HIV-1-positive patients and blood donors in Khartoum in 1998 and 1999. From analysis of partial pol and env sequences, it was determined that 50% were subtype D and 30% were subtype C. Of interest, some subtype D clustered with those from East Africa whereas others joined subtype D from West Africa. Subtype A, subtype B, and three unique recombinants were also found, some partially unclassifiable. One unclassified strain matched another reported previously from the Democratic Republic of Congo. Sudan borders nine other African countries, and has suffered more than 20 years of civil strife with large population displacements. The intermixing of HIV-1 subtypes previously separated in Africa may be occurring there, with the potential to generate novel new strains by recombination.