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Introduction Traumatic brain injuries are of concern to the sports and military communities because of the age of the participants and costly burden to society. To markedly reduce the impact of traumatic brain injury and its sequela (TBI-S), it is necessary to determine the initial vulnerability of individuals as well as identify new technologies that indicate early signs of TBI-S. Materials and Methods Currently, diverse methods have been used by the authors and others in laboratory settings to reveal early signs of persistent TBI-S including simulation modeling of the effect of rapid deceleration on the deviatoric strain (shear force) imposed on specific brain regions, auditory evoked potential (AEP) measurements to determine injury to the auditory cortex optokinetic nystagmus (OKN) measures sensitive to vestibular trauma, and optical coherence tomography (OCT) measures that reveal changes in central visual function obtained noninvasively by examination of the retina. Results Simulation studies provided technical information on maximal deviatoric strain at the base of the sulci and interface of gray and white matter consistent with results from neuropathology and from magnetic resonance imaging. The AEP and OKN reveal measurable injury to similar regions below the Sylvian fissure including auditory cortex and midbrain, and the OCT reveals changes to the retina consistent with forceful deceleration effects. Conclusions The studies and results are consistent with prior work demonstrating that noninvasive tests may be sensitive to the presence of TBI-S, potentially in the training field as advances in the portability of test instruments are underway. When combined with baseline data gathered from individuals in quantitative form, key variances can emerge. Therefore, it is hypothesized that AEP, OKN, and OCT, taken together, may yield faster objective and quantitative neurophysiological measures serving as a “signature” of neural injury and more indicative of potentially persistent TBI-S—recommending larger scale longitudinal studies.

During the COVID-19 pandemic associated with high incidence, transmissibility, and mortality, this chapter focuses on three phases of the disease: initial exposure, initiation of the immune response to the agent, and finally, an inflammatory/autoimmune-like presentation with pulmonary, neurological, and renal failure and disseminated intravascular coagulation which occurs in a small proportion of the patients. The elegant demonstration of the site of interaction between the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which is the causative agent of COVID-19, and the ACE (angiotensin-converting enzyme) 2 receptor of cells distributed throughout the body has enabled research efforts to develop pharmacological and immune countermeasures to the viral phase of the disease. This chapter rapidly reviews the molecular and structural organization of SARS-CoV-2 and its interaction with ACE2. It is followed by a discussion over the role of the major histocompatibility complex (MHC) in recognition of the virus. The importance of rapid compartmentation of the viral genome into the target cells as opposed to the binding constant of the virus for the ACE receptor is discussed. Host factors affecting the immune response to the virus are examined, and the subsequent inflammatory dysregulation enabling the cytokine storm leading to system organ failure is described. Finally, the similarities of the clinical effects of the murine hepatitis virus-JHM (a coronavirus) on multi-organ systems (liver, brain, clotting cascade) as described by Perlman and colleagues permit insights regarding the role of the interaction between the host and the virus in developing the clinical presentation of the inflammatory/autoimmune disorders that occur in multiple sclerosis, neuromyelitis optica, and more interestingly, during the third phase of COVID-19.

Objectives:To assemble an algorithm that will describe a “Signature” predictive of an individual’s vulnerability to persistent traumatic brain injury (TBI)Subjects and Methods:Studies of athletes and warriors who are subjected to repeated head impacts with rapid acceleration/deceleration forces are used to assist in the diagnosis and management of TBI-affected individuals. Data from multiple areas, including clinical, anatomical, magnetic resonance imaging, cognitive function, and biochemical analyses, are integrated to provide a Signature of persistent TBI.Results:Studies to date indicate that susceptibility to TBI results from an interaction between host genetic and structural vulnerability factors and force and torque of impact on the head and torso. The host factors include molecular markers affecting immune and inflammatory responses to stress/insult as well as anatomical features such as the degree of transcortical fiber projections and vascular malformations. The host response to forceful impact includes the release of intracellular neural proteins and nucleic acids into the cerebrospinal fluid and vascular compartment as well as mobilization of cytokines and macrophages into the central nervous system with subsequent activation of microglia and inflammatory responses including autoimmune processes. Maximum impact to the base of the sulci via a “water hammer effect” is consistent with the localization of microvascular and inflammatory responses in the affected brain region.Conclusions:An assessment of an individuals’ predisposition to persistent TBI with delayed cognitive deficits and behavioral changes requires an understanding of host vulnerability (genetic factors and brain structure) and external stressors (force and torque of impact as well as repetitive head injury and time interval between impacts). An algorithm that has utility in predicting vulnerability to TBI will include qualitative and quantitative measures of the host factors weighted against post impact markers of neural injury. Implementation of the resulting “Signature” of vulnerability at early stages of injury will help inform athletes and warriors, along with commanders and management, of the risk/benefit approaches that will markedly diminish health care costs to the nation and suffering to this population. This report attempts to define a strategy to create such an algorithm.

Full-spectrum warfare places a heavy cognitive burden on the soldier that may exceed normal adaptive stress response mechanisms. It is crucial to understand the effects of battlefield stressors on ...

The current study was designed to determine whether exposure of mice to aerosolized jet fuel (JP8 + 100) resulted in changes in the cellular distribution or immunoreactivity of the enzyme glutathione S-transferase (GST), a biomarker of toxicant exposure. Male mice were exposed to JP8 + 100 at 1000 mg/m3 or 2500 mg/m3 in aerosol for 1 h per day for 7 days and then sacrificed. The retinas were studied by immunohistochemical methods. The JP8 + 100 exposure caused a marked increase in the immunoreactivity of anti-GSTM antibodies with the radial glial cells of the retina, the Müller cells. These results are consistent with the hypothesis that JP8 + 100 acts as a toxicant to mouse retina by permitting the flux of materials across the blood-retina barrier. The findings are relevant to humans because recent studies indicate that Air Force personnel assigned to clean and maintain fuel pods may be exposed to concentrations of JP8 + 100 exceeding 1000 mg/m3.

We analyzed protein expression in the cytosolic fraction prepared from whole lung tissue in male Swiss-Webster mice exposed 1 h/day for seven days to aerosolized JP-8 jet fuel at concentrations of 1000 and 2500 mg/m3, simulating military occupational exposure. Lung cytosol samples were solubilized and separated via large scale, high resolution two-dimensional electrophoresis (2-DE) and gel patterns scanned, digitized and processed for statistical analysis. Significant quantitative and qualitative changes in tissue cytosol proteins resulted from jet fuel exposure. Several of the altered proteins were identified by peptide mass fingerprinting, confirmed by sequence tag analysis, and related to impaired protein synthetic machinery, toxic/metabolic stress and detoxification systems, ultrastructural damage, and functional responses to CO2 handling, acid-base homeostasis and fluid secretion. These results demonstrate a significant but comparatively moderate JP-8 effect on protein expression and corroborate previous morphological and biochemical evidence. Further molecular marker development and mechanistic inferences from these observations await proteomic analysis of whole tissue homogenates and other cell compartment, i.e., mitochondria, microsomes, and nuclei of lung and other targets.

Lead is a potent neuro- and nephrotoxin in humans and a renal carcinogen in rats. Previous studies have detected lead-induced increases in the activities of specific detoxification enzymes in distinct kidney cell types preceding irreversible renal damage. While preferential susceptibility of the highly vascularized cortex to the effects of lead is clear, lead effects on the medullary region have remained unexplored. The present study was undertaken to investigate the extent to which regional renal protein expression differs and to determine which, if any, regionally distinct protein markers indicative of lead's renotoxic mechanism might be detected in kidney cortical and medullary cytosols. We examined protein expression in these two functionally and anatomically distinct regions, and identified several proteins that are differentially expressed in those regions and were significantly altered by lead. Kidney cytosols from rats injected with lead acetate (114 mg/kg, three consecutive daily injections) were separated by two-dimensional electrophoresis. Lead exposure significantly (P

Glutathione S-transferases (GST) are a family of detoxification isoenzymes that catalyze the conjugation of xenobiotics and their metabolites with reduced glutathione. Lead exposure in rats is known to induce GST isoenzymes in the liver and kidney. These changes in expression have potential use as biomarkers of lead exposure. Because two-dimensional electrophoresis (2-DE) enables one to analyze both protein abundance changes and chemical changes in protein structure, 2-DE was used to determine the effect of in vivo lead exposure on GST isoform expression in rat kidney cytosols. Male Sprague-Dawley rats were exposed to inorganic lead, and proteins were separated by conventional ISO-DALT and NEPHGE-DALT techniques and blotted for immunological identification. Lead exposure caused detectable inductions in both GSTP1 and GSTM1 and quantifiable charge modification in GSTP1. These preliminary data confirm the utility of 2-D electrophoretic GST analysis as indicative of lead exposure and toxicity and support its use for further elaboration of lead's effects on renal protein expression.

This article describes the utility of Spizizen medium in effecting the release of 2,4 dinitrotoluene (DNT) from plasticized propellant in aged clay soil and of added Pseudomonas organisms in enhancing the rate of degradation of DNT in clay and sandy soils. DNT is an environmental problem because of its toxicity to mammals. It is proposed that the citrate in Spizizen medium chelates metals that form the aggregates of humin in aged clay soils, thereby releasing propellant components. Lack of awareness of DNT mobilization by citrate or other polyanions may lead to a potential underestimation of the 2,4 DNT present in aged contaminated soil and a potential increased release of DNT following exposure to solvents containing citrate or other polyanions. DNT in contaminated sandy soils was rapidly degraded when Pseudomonas and Spizizen medium were added (85% degraded in 20 days). Pseudomonas isolated/Torn soils at the Badger Plant have particular utility for the in situ degradation of 2,4 DNT in clay and sandy soils because they metabolize Spizizen medium, thrive in diverse climates, and have been selected for their ability to grow in soils contaminated with DNT. Mixture of either the contaminated clay soil or the contaminated sandy soil with uncontaminated prairie silt loam (20:80 ratio) facilitated management of the treatment process. This observation is important because of the widespread distribution, and hence low cost, of this soil type in the central United States.

Glutathione S transferases (GST) are a family of enzymes that detoxify electrophilic xenobiotics. This enzyme family was examined in kidneys of mice exposed to mercuric chloride, a known nephrotoxin, because GST have been shown to protect cells against toxicant-induced damage and may serve as biomarkers for toxicant exposure. The purpose of this study was to determine the effect of mercuric chloride on GST activity, isoenzyme levels, and cellular localization in the kidney of Swiss Webster mice. The cellular localizations of alpha, mu, and pi class GST in the kidneys of control and mercuric chloride treated mice were studied immunohistochemically. The GST isoenzyme levels were measured by high-performance liquid chromatography. The mice treated with mercuric chloride had (1) increased amounts of GSTA1/A2 protein in kidney homogenates as compared with controls when analyzed by chromatography and electrophoresis; (2) two new isoforms of the alpha isoenzyme in kidney as demonstrated by Western blot, polyacrylamide gel electrophoresis, and high-performance liquid chromatography, and (3) increased reactivity between antibodies, against GSTA1/A2 or GSTM1 isoenzymes, and cells in the proximal and distal renal tubules as shown by immunohistochemical techniques. The authors conclude that the GSTA1/A2 may protect those cells in the proximal and distal tubules of the renal cortex from toxicant effects of mercuric chloride. This would be one general mechanism for cell protection against a wide variety of toxicants including heavy metals and halogenated aromatics.

Monolayers of the polypeptide poly(L-lysine) (PL) are used to control the specific adsorption of proteins onto gold surfaces. A PL monolayer modified with biotin is electrostatically adsorbed onto a vapor-deposited gold film that has been coated with a self-assembled monolayer of the alkanethiol 11-mercaptoundecanoic acid (MUA). The immobilized biotin moieties act as specific adsorption sites for the protein avidin. Adsorption of the biopolymers onto the gold surface is monitored with a combination of surface plasmon resonance (SPR) and fluorescence measurements. By varying the percent biotinylation of the lysine residues on the PL prior to deposition, the surface coverage of avidin can be controlled to create either full or partial monolayers. The thickness of a full monolayer of avidin is 41 A, as determined by the SPR measurements. At high surface coverages of avidin, an excess of biotin sites is required to overcome steric hindrance. The PL monolayer and any adsorbed avidin can be easily rinsed from the surface with a low or high pH solution. This removal allows for quantitation of the adsorbed molecules by fluorescence measurements in solution rather than on the gold surface. In this manner, fluorescein-labeled PL and avidin are used to determine absolute surface coverages of 4 x 10 14 lysine residues cm -2 for the PL monolayer and 3 x 10 12 avidin molecules cm -2 for the full avidin monolayer. SPR imaging experiments are employed to verify that UV photopatterning of the MUA/PL bilayers can be used to spatially direct the adsorption of avidin onto the gold surface. The polylysine attachment methodology will be beneficial in the fabrication of adsorption biosensors.

The electrostatic adsorption of poly-L-lysine molecules onto a vapor-deposited gold film modified with a carboxylic acid-terminated alkanethiol monolayer is monitored with the spectroscopic techniques of polarization-modulation Fourier transform infrared (PM-FTIR) spectroscopy and surface plasmon resonance (SPR). The PM-ITIR spectrum of a monolayer of poly-L-lysine (PL) adsorbed onto a self-assembled monolayer of 11-mercaptoundecanoic acid (MUA) indicates that the lysine residues and the MUA carboxylic acid moieties form ammonium-carboxylate ion pairs which electrostatically bind the polypeptide to the surface. The PL molecules can be desorbed from the surface by rinsing with a buffer solution at a pH that destroys the ion pairing (pH 12). Measurements of the shift in the SPR angle upon adsorption were used to determine the thicknesses of the adsorbed monolayers; the MUA and PL monolayers were found to be 17.0 and 10.5 A thick, respectively. These thickness results suggest that the poly-L-lysine monolayer adsorbs onto the packed MUA surface in an extended conformation with the PL backbone lying parallel to the surface. Subsequent exposure ofthe PL monolayer to a solution ofiron phthalocyanine tetracarboxylic acid (FePc) resulted in the adsorption of a third layer onto the surface. The ability of the adsorbed PL molecules to interact with FePc indicates the presence of free lysine residues available for interaction with molecules other than the MUA monolayer.

The glutathione S-transferases (GSTs) constitute a family of cytosolic isoenzymes and a structurally unrelated microsomal enzyme that is involved in the detoxication of electrophilic xenobiotics. These enzymes also participate in the intracellular binding and transport of a broad range of lipophilic compounds including bilirubin, and hormones such as the glucocorticoids and thyroid hormones. The present investigation demonstrates that GSTs are present in neurons of the brainstem, forebrain, and cerebellum. An isoenzyme-specific distribution of GSTs was found in cytoplasm, nuclei, and nucleoli. The regional and cellular distribution of cytosolic GSTs in the brain was studied by immunohistochemistry, spectrophotometric enzyme assay, and reverse-phase HPLC. Polyclonal antibody against microsomal GST was strongly reactive with Purkinje cells throughout the cerebellar cortex, and with neurons in the brainstem and hippocampus. Nuclei of Purkinje cells and of neurons in the brainstem, hippocampus, and cerebral cortex were immunopositive for alpha-class GST 1–1 (YaYa), whereas alpha-class GST 2–2 (YcYc) antibody was consistently immunoreactive with the nucleolus, but not with the nucleus or soma. All alpha-class GST antibodies studied were reactive, to various degrees, with astrocytes and choroid plexus; however, ependymal cells of the subventricular zones were immunonegative. alpha-class GST 8–8 (YkYk) immunoreactivity was specifically localized to endothelial cells and/or astrocytic end feet associated with blood vessels. Reverse-phase HPLC indicated that there were also substantial regional differences in the pattern of alpha-, mu-, and pi-class GST subunit expression. For example, the thalamus/hypothalamus had the highest GST activity and greatest concentration of total GST protein and mu-class GST subunit 6 (Yb3), whereas the brainstem had the greatest concentration of pi-class GST subunit (Yp). This regional variation in GST expression may be reflective of regional differences in cell populations. In cerebellar cortex, the concentration of mu-class GST subunit 4 (Yb2) was greatest in the flocculus and lowest in the vermis. This is of clinical interest because the pattern of expression of mu-class GST subunit 4 (Yb2) in the cerebellum coincides with the known regional susceptibility of this structure to degeneration after exposure to toxic or metabolic insults. The vermis is most susceptible to these insults, whereas the lateral lobes and flocculus are most resistant.(ABSTRACT TRUNCATED AT 400 WORDS)

This report describes the morphological changes observed in the brain of an untreated 27-year-old man with phenylketonuria, cortical blindness, and seizures. Golgi-Kopsch silver, cresyl violet, and hematoxylin and eosin stains were used to study cell structure and organization of the cerebellum, the lateral geniculate nuclei, the visual cortex, frontal cortex, and hippocampus. Extensive neuronal losses occurred in the right lateral geniculate nucleus (LGN), the visual cortex, and hippocampus. The left LGN, cerebellum, and frontal cortex retained neuronal components; there was a reduction in the number of dendritic processes on the Purkinje cells of the PKU subject. The loss of neurons in the LGN and occipital cortex is related to the blindness and the neuronal loss in the hippocampus is related to seizure activity.

Neuronal cells from murine trisomy 16 fetuses have increased levels of class I MHC H-2Kk. To determine whether this increased level of H-2Kk protein product resulted from an increased synthesis of mRNA, a 33 base antisense cDNA probe complementary to a region in exon 2 of the H-2Kk sequence (nucleotide 392–424) was synthesized. This probe was used to examine, by in situ hybridization and immunohistochemistry, the neural distribution of H-2Kk mRNA and protein product. A marked elevation of the H-2Kk mRNA and protein were localized in mts16 neuronal populations that were susceptible to dysgenesis. The results implicate the expression of the H-2Kk in the neuropathology of mts16 and its human counterpart, Down syndrome.

The diagnosis of neurological disease (ND) traditionally involves the association of specific symptoms in a patient with a clinical examination, consideration of the patient's prior medical history, and clinical laboratory testing (e.g., electroencephalography, electromyography). The decoding of the human genome has made possible the identification of proteomic and genomic biomarkers associated with specific neurological disorders. The identification of multiple biomarkers, including determination of chemical nature and concentration, provides a disease “signature” that is not achievable through testing for any single marker alone. Biomarker signatures can augment conventional clinical evaluation and testing by improving diagnosis, prognosis, monitoring progression, and management of ND. Biomarker signature profiling for a patient can allow for personalized medicine catered to individual need. The need for biosensors in the detection of ND is illustrated by recent events. Two cases of bovine spongiform encephalopathy (BSE) in Canada have cost their beef industry millions of dollars and resulted in precluding shipment of cattle from Canada to the United States. The ability to determine absence of prion disease in live cattle could greatly increase confidence in cattle imports. In a separate example, Tysabri, an immunosuppressant drug effective in lengthening periods of remission in patients with multiple sclerosis (MS), was withdrawn from the market because of occurrence of progressive multifocal leukoencephalopathy (PML) in treated individuals. PML in the treated MS individuals was caused by proliferation of JC virus. The ability to detect JC virus production during treatment with Tysabri could remedy this problem. Biosensors designed to detect analytes indicative of disease are currently available. Ranging from single target detection to more complex multitarget signature profiling, these biosensors are offered for a wide range of disorders. Many focus on the detection of cancer and ND. The platforms used for cancer detection are directly applicable to ND determination because the sensors detect analytes or genetic mutations present in both disease types. Specific products and technologies that are already being used for or can be readily adapted to ND diagnosis, prognosis, and treatment are discussed. A select list of ND-related biomarkers for current and future study is presented. Keywords: proteomics; genomics; multiplexed sensor; microarray; neurological disease; biomarker; immunosensor; cancer; degenerative disease

Technologies required for strategic actionable net-centric biological defense systems consist of : 1) multiplexed multi-array sensors for threat agents and for signatures of the host response to infection; 2) novel vaccines and restricted access antivirals/bacterials to reduce emergence of drug resistant strains pre- and post-event; 3) telemedicine capabilities to deliver post-event care to 20,000 victims of a biological strike; and 4) communication systems with intelligent software for resource allocation and redundant pathways that survive catastrophic attack. The integrated system must detect all threat agents with minimal false positive/negative events, a seamless integrated broad-band communications capability that enables conversion of data to actionable information, and novel pre- and post-event treatments. The development of multiplexed multi-array sensors, appropriate vaccines and antibiotics, and integrated communication capabilities are critical to sustaining normal health, commerce, and international activities.

Developments in the continental United States (dissemination of Anthrax-contaminated items through the U.S. mail in October 2001) and on the international scene (emergence of Congo-Crimean hemorrhagic fever in the Afghanistan-Pakistan border region) have alerted the defense community to the real threat of biological and chemical agents to civilian and military populations. Current strategies have had a stove-piped appearance, with efforts directed towards the development of individualized communications systems, separate detectors for each threat agent, tradition-based vaccines, and antibiotics for post-event care delivery. What is needed is an integrated sensor alert system that can selectively and simultaneously detect all threat agents with little to no false positive/negative events, a seamlessly integrated communications network capability that enables the conversion of data to actionable information, and novel pre- and post-event treatments. New technologies required for an effective response to a biological or chemical attack include: multi-array sensors for threat agents; sensors for validating signatures of the host response to infection; a telemedicine system to deliver post-event care for up to 20,000 victims of a biological or chemical strike; an intelligent software network that permits meaningful resource allocations and redundant pathways that survive a catastrophic attack; and novel vaccines and restricted access antivirals/antibacterials to reduce the emergence of drug resistant strains pre- and post-event. Multi-array sensor platforms and alert systems must permit rapid screening of many samples for the presence of signature molecules and be small and lightweight with low power requirements. Genomic sequences of threat agents naturally present in different global regions must be determined to permit attribution of an attack to the source of an agent. A communications system that allows for the integration of information on emerging disease, resource allocation inventory, public safety coordination, perimeter management and extended telemedicine care is yet to be realized. Databases that provide information on the normal distribution of naturally occurring threat agents and the normal incidence of diseases in communities must be established. These databases should be integrated into a sensor and communication system that will provide an early alert of a biological or chemical attack, and optimize consequence management actions to neutralize the impact of the attack. This sensor and communication strategy is critical to sustaining normal health, commerce and international activities.

Developments in the continental U.S. (Anthrax letters disseminated October 2001 CONUS) and the international scene (Congo Crimean Fever emergence in Afghanistan-Pakistan border region OCONUS) have alerted the defense community to the threat of biological and chemical agents on civilian and military populations. The objective of the program will lead to the protection of U.S. and allied forces from biological/chemical threats. The following focus areas are being developed and integrated in our Program: ( I ) scientific validation; (2) medical countermeasures; and (3) communication and integrated conversion of data to knowledge. We are developing binder elements (high affinity antibodies, polynucleotide probes, and aptamers) for the sensors and platforms (MEMS, electronic tongue, hand held redox systems) for these sensor systems. To rapidly determine whether individuals have been exposed to threat agents, archival data sets have been established through a partnership with the Texas Department of Health. Large scale, real-time symptomatic diagnoses of patients from emergency medical facilities are being electronically collected and sent to an archival data facility for identification of emergent disease. The ability to diagnose emergent disease can be reduced to twenty-four hours from the week to several weeks currently required.

We have completed all analyses. The results indicate the great importance of including sham controls in all JP8 studies. The most significant effect was the increase of hippocampal DOPAC during the exposure period, indicative of increased dopamine release and turnover. We conclude that with this significant exception, JP8 exposure at this level caused no global alterations in neurotransmitter levels.

GlutathioneS-transferases (GST, EC 2.5.1.18) are a family of phase II detoxification enzymes involved in the conjugation of glutathione to a highly diverse group of compounds. The purpose of this study was to evaluate the dose–response effects of lead acetate administration on the expression of rat kidney GST. Sprague–Dawley rats were injected with doses of lead acetate ranging from 0.11 to 114 mg/kg (0.3 to 300 μmol/kg) for three consecutive days and sacrificed 24 h later. Kidney GST activity, GST isoform HPLC profiles, blood lead analysis, and electron microscopy were performed. A dose of 1.1 mg/kg lead acetate resulted in a blood lead level of 26 μg/dl and produced a significant increase in GST activity which continued to increase with dose up to 38 mg/kg. Morphological changes were detected at 3.8 mg/kg and increasing severity of cellular damage paralleled dose, blood lead levels, and changes in body weight. Individual GST isoforms exhibited different thresholds and maxima; rGSTP1 and rGSTM1 had thresholds of 1.1 and 3.8 mg/kg, respectively, very similar rates of increase with dose, and a maximum yield that was 450% above control at a dose of 38 mg/kg for both enzymes. rGSTA1 and rGSTA3 showed similar thresholds (1.1 mg/kg) and maximal fold increase (275%) but varied in the relative response to each dose. These results indicate that renal GST increases occur at lead levels which are environmentally significant, that these changes precede cellular damage, and suggest that GST may serve as a tissue biomarker of lead exposure.

The rodent kidney is a target of many xenobiotics and is typified by regionally specific structure and function. This renders distinct regions of the kidney differentially susceptible to toxic exposure and effect. To characterize these differences at the proteome level, protein patterns from male rat kidney cortex and medulla cytosols were examined by two-dimensional electrophoresis (2-DE) and image analysis and prominent proteins identified immunologically or by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and electrospray/ionization-tandem mass spectrometry (ESI-MS/MS) sequence tag identification. An average of 727 protein spots were resolved and matched to the cortex cytosol reference pattern, and 716 in the medulla. Of this total, 127 proteins were found to differ in abundance (86 higher in cortex; 41 higher in medulla) (P < 0.001). Of those proteins that were detectable in both cortex and medulla, the abundance of 97 differed significantly while 30 proteins were found to be unique to one region or the other (26 in cortex, 4 in medulla). Twenty protein spots were identified and their regional differences are discussed. These results both confirm and expand our understanding of the molecular heterogeneity characterizing structurally and functionally distinct regions of the kidney and serve as a useful foundation for future nephrotoxicologic studies.

The effect of acute exposure to lead acetate on the expression of glutathione S-transferase (GST) subunits and the levels of reduced and oxidized glutathione (GSH) and malondialdehyde (MDA) in rat kidney and liver was determined. The purpose of this study was to determine if GSH depletion and/or oxidative stress were responsible for changes in the expression of some or all GSTs that followed lead exposure. In kidney, all GST subunits increased following injection of lead. The level of kidney GSH was not changed at either 0.5 or 1 h after lead exposure, but increased 3, 6, 12 and 24 h after a single injection of lead. MDA levels (a marker of lipid peroxidation) did not change in kidney following lead injection. Immunohistochemical markers of oxidative stress and nitric oxide production were also unchanged by lead administration. Therefore. we conclude that the increases in GST levels in kidney following lead exposure were not dependent on oxidative stress. In liver, lead injection caused GSH depletion (61% of control 12 h after lead treatment) and increased MDA production (2.5-fold increase 6 h after lead exposure), while GSTA1, GSTA2, GSTM1 and GSTM2 did not increase. Analysis of the effects of lead on GST mRNA and GST cellular localization were performed by Northern blot and immunohistochemical techniques. Immunoperoxidase light microscopy and immunogold electron microscopy revealed that the increase in kidney GSTM1 and GSTP1 occurred in nuclei, cytoplasm and microvilli of proximal tubules. Northern blot analysis of GSTA2 and GSTP1 mRNAs showed that their increase following lead exposure was inhibited by actinomycin D, suggesting transcriptional induction. This study demonstrates that acute lead exposure causes dramatic changes in the subcellular distribution and expression of rat kidney GSTs, and that these changes are not a result of oxidative stress.

Purpose: It has been shown previously that the sera, from patients with visual paraneoplastic syndrome associated with lung cancer, contain immunoglobulins that are reactive with the tumor and with photoreceptor and large retinal ganglion cells. The purpose of this study is to determine the retinal cell population that reacts with immunoglobulins in the sera of patients with melanoma-associated retinopathy. Methods: Clinical and electrophysiologic studies were used to determine the locus responsible for the visual defect in each patient. Sera from two patients with melanomaassociated retinopathy, from a patient with herpes zoster, and from a patient who had a colon tumor were obtained. The sera were incubated with sections of retina obtained from a healthy 3-year-old child who had died of asphyxiation. The tissue sections subsequently were incubated with biotin-labeled anti-human immunoglobulin G, and then with streptavidin-labeled peroxidase. Finally, the tissue sections were developed to show peroxidase activity in the targeted retinal cells. Results: Clinical and electrophysiologic studies were consistent with a defect in intraretinal transmission distal to the photoreceptors. The immunoglobulins from the patients with the melanoma-associated retinopathy reacted selectively with the bipolar cells of the retina; approximately 30% of the bipolar cells were immunoreactive. The sera from the other two patients were not reactive with any of the retinal cells examined. Conclusions: The sera of patients with the paraneoplastic syndrome, melanomaassociated retinopathy, contain high titer immunoglobulins that are reactive only with a subset of the bipolar retinal cells. The clinical, electrophysiologic, and immunologic studies are all consistent with an intra-retinal transmission defect at the level of the bipolar cells.

The near-microscopic resolution of the mouse brain, by magnetic resonance imaging (MRD at 9.4 T, permits in situ examination of the entire brain and longitudinal studies of neural development. MRI can be utilized to reveal brain structure at a resolution of 100 μm in the X, Y , and Z planes of brain, to differentiate the gray from white (myelin-rich) matter, and to reveal the ventricular compartments. The present report describes the structure of normal BALB/c mouse brain as revealed by imaging at 9.4 T and by histological stains; the structure of normal brain is compared with that from a phenylalanine hydroxylase-deficient mouse mutant line (Pah enu2 ) and those from normal littermates. The brains of patients with phenylketonuria (PKU) were reported to have demyelination and other structural abnormalities revealed by magnetic resonance imaging (MRI). Therefore, high-resolution MRI was used to examine the brain of this mutant, an animal model for the study of human phenylketonuria. Our study revealed no evidence of demyelination or other abnormalities in the brains of Pah enu2 mice. Histologically, the mutant and normal mouse brains appear similar. This is consistent with a recent study from our laboratory which demonstrated that the histology of the brain of an untreated male patient, who died with PKU at the age of 29, was similar to control brain with the exception of changes directly related to visual blindness and seizures experienced by the patient.

The glutathione S-transferases (GSTs) are a family of isoenzymes involved in the detoxication of a variety of electrophilic xenobiotics. The present investigation demonstrates that GST activity and the concentration of cytosolic GSTs in cerebellar cortex of Gunn rats were increased in hyperbilirubinaemic animals compared with non-jaundiced controls. Age-dependent and region-specific increases in GST isoenzymes were seen in three regions of the cerebellar cortex of jaundiced Gunn rats, whereas GST concentrations were not altered in the brainstem, thalamus/hypothalamus, cortex or liver. Cytosolic GST activity was increased 1.3-fold in the flocculus and lateral hemispheres of 20-day-old and 1.7-fold in the flocculus, lateral hemispheres and vermis of 60-day-old jaundiced (jj; homozygous) Gunn rats compared with non-jaundiced (Jj; heterozygous) Gunn rats. H.p.l.c. was used to determine the GST subunit protein concentrations in cytosolic fractions isolated from liver and brain regions of jaundiced and non-jaundiced animals. In all regions of the cerebellum from 20-day-old animals, the levels of Alpha-class GST subunits 2 (Yc1; 3.0-fold) and 8 (Yk; 2.0-fold) were increased in jaundiced rats. In 60-day-old animals, the concentrations of Alpha-class GST subunits 2 (Yc1; 5.0-fold) and 8 (Yk; 3.0-fold), Mu-class subunit 11 (Yo; 2.5-fold) and Pi-class subunit 7 (Yp; 2.0-fold) were increased in all regions of cerebellar cortex of jaundiced animals. In cerebellum of 10-, 20- and 60-day-old non-jaundiced and jaundiced Gunn rats, the flocculus had the highest concentration of Mu-class GST subunit 4 (Yb2) and vermis the lowest; hyperbilirubinaemia increased the concentration of subunit 4 (Yb2; 3- to 5-fold) in the flocculus and lateral hemispheres, but not the vermis, of 20- and 60-day-old rats. Intraperitoneal injection of sulphadimethoxine, a long-acting sulphonamide which displaces bilirubin from its albumin-binding sites and increases the bilirubin levels in tissues, further increased the already elevated concentrations of GST subunits in the lateral regions of cerebellar cortex of hyperbilirubinaemic rats. For example, the concentration of subunit 4 (Yb2) was increased 2.2-fold (compared with non-jaundiced controls) in Gunn rats injected with saline and 7.4-fold in rats injected with 100 mg of sulphadimethoxine/kg body weight. In contrast, GSTs in the vermis of jaundiced animals were not affected by sulphadimethoxine injection. Sulphadimethoxine had no effect on GST concentrations in lateral regions and vermis of heterozygous (Jj) Gunn rats.(ABSTRACT TRUNCATED AT 400 WORDS)

Murine trisomy 16 (mts16) placentas and fetuses, 17-day gestation age, were examined histologically and by magnetic resonance imaging at 9.4 T and compared with control littermate tissues. Placentas were studied by immunohistochemical methods, at 15-days gestational age, for expression of the major histocompatibility complex (MHC) class I H-2Kk cell surface marker. Immunohistochemical studies revealed a markedly increased expression of the MHC marker H-2Kk on cells in the labyrinth of the placenta of mts16. There were differences between the magnetic resonance (MR) images of the trisomic and normal placentas, which may be correlated with the increased expression of H-2Kk in the mts16 placental labyrinth. The decidual and labyrinthine components of the normal placentas showed similar high signal intensities (SI) while in trisomic placentas a marked high SI was characteristic only of the decidual region on proton spin density images. The MRI also revealed a smaller cerebellum in the ts16 fetuses. The potential effects of the compromised structure of the placental labyrinth and the overexpression of the H-2Kk marker on the mts16 neural and placental dysgenesis are discussed.

Clinical, magnetic resonance imaging (MRI), and serologic studies were performed on 11 patients with diffuse central nervous system (CNS) systemic lupus erythematosus and 8 patients with focal CNS lupus. MRI of patients with diffuse clinical disease showed symmetrically distributed areas of increased signal intensity in the subcortical white matter; these resolved after treatment with high-dose methylprednisolone. These patients' sera contained elevated levels of antineurofilament antibodies. Patients with focal CNS lupus had areas of increased signal intensity and atrophic changes in regions corresponding to the major cerebral vessels. These MRI abnormalities did not improve after treatment with high-dose steroids. The sera of patients with focal CNS lupus had elevated levels of cardiolipin and lupus anticoagulant but normal levels of antineurofilament antibody. Our findings suggest that results of a combined clinical, MRI, and serologic evaluation of patients with CNS lupus may predict the response of patients to high-dose steroid therapy.

Biotechnology is advancing at a rapid pace with numerous applications in medicine, industry, agriculture and environmental remediation. Recognizing this, government, industrial and academic research and development investment in biotechnology has expanded rapidly. The past decade has seen the emergence of applications of this technology with a dual-use potential. Military applications focus on four major areas: biomedical technology, such as vaccine development and medical diagnostics; detection of toxins, chemicals and pathogens; material biotechnology; and biological decontamination, including biodegradation and bioremediation. This conference emphasizes the non-medical applications of biotechnology. The first two sessions focus on the synthesis and properties of molecules that may be used in detectors. The traditional approach to detection of chemical and biological agents relied on the development of specific assays or analyses for known agents. Advances in molecular biology have made possible the production of large quantities of toxins which were previously available in minute quantities, and the molecular engineering of toxins and pathogens with specific pharmacologic and physical-chemical properties.

We used Western blot analysis to examine the occurrence and titer of antibody to cytoskeletal neurofilament protein antigens in patients with neuropsychiatric manifestations of systemic lupus erythematosus (SLE) and in controls. Twenty-two patients with neuropsychiatric SLE (NPSLE) had an increased incidence of antineurofilament antibody (ANFA) compared with 34 patients with SLE without neuropsychiatric symptoms, 78 patients with other disease processes, and 22 healthy controls. ANFA were found to be directed against the 205,000- and 160,000-dalton proteins of the neurofilament triplet. Patients with a diffuse NPSLE clinical presentation had the greatest frequency of serum ANFA (7 of 12, 58%) compared with all other groups examined. Magnetic resonance imaging and serum anticardiolipin antibody testing were also performed in selected patients with NPSLE. Patients with a focal clinical presentation of NPSLE, positive magnetic resonance imaging findings, and negative serum ANFA had significantly elevated levels of anticardiolipin antibody.

The purpose of this study was to determine the selectivity with which polyspecific antibodies directed against large retinal ganglion cells destroy axons in the cat's optic nerve. Immune serum prepared against large ganglion cells isolated from ox retinas was injected into 1 eye of each of 2 cats. After more than 1 month, the cats were perfused with mixed aldehydes and the optic nerves were prepared for transmission electron microscopy. On the basis of a large sample of micrographs of transverse thin sections, we estimated that each of the nerves that issued from treated eyes contained approximately 62,500 necrotic fibers, amounting to 42-46% of the total fiber population in 1 case and 39-42% in the other. The diameters of 2900-5000 intact axons from each of 4 nerves (2 from immune-treated eyes and 2 from untreated eyes) were measured. Comparisons of histograms of axon diameter for nerves from treated and untreated eyes revealed that 90-100% of large axons with diameters above 3.5-4.0 micron were eliminated by the antibodies. Between 65 and 70% of medium-sized fibers were also eliminated. The number of small axons--those with diameters less than 1.2-1.6 micron--did not differ appreciably from normal. These results suggest that the immune serum destroyed virtually all alpha cell axons and a substantial fraction of beta cell axons but did not reduce the number of small fibers that largely stem from the gamma class of retinal ganglion cells.

The effect of poly- l -lysine and polyglycols on the electronic spectral properties of cobalt-tetracarboxy-phthalocyanine, compound (I), was studied in order to determine the effect of the polymers on the molecular stacking properties of compound (I). In the present study we have coupled, both covalently and electrostatically, the poly- l -lysine to compound (I). The electrostatic interaction with the polylysine resulted in (i) a bathochromic shift of absorbance by compound (I) from 680 to 695 nm, and (ii) the generation of shoulders in the 590 and 580 nm region. The bathochromic shift indicates that the polylysine either misaligns the units of compound (I) in the molecular stack or alters the angular relationship of the planar compound (I) molecules. The covalent linkage with the polylysine resulted in stabilization of the monomeric form of compound (I) with no hypochromism in the 680 nm region. The reaction of the polyglycols, dextran and DEAE dextran with compound (I) prior to the addition of the polylysine, stabilized the polymeric stacked form of compound (I) in the presence of the polylysine. Ammonium ion and ethanolamine resulted in the appearance of peaks in the 730–760 nm region; a trihydroxy containing primary amine and monomeric lysine generated no such peak at wavelengths above 700 nm. The polyglycol binding capacity of compound (I) facilitated the separation of the unbound compound (I) from the polymeric complexes.

In the companion study ( Dev. Brain Res. , 34 (1987) 223–233), we showed that a monocular injection of antibodies against ox large retinal ganglion cells produces a 53% (low concentration) to 82% (high concentration) loss of Y-cells in lateral geniculate nucleus (LGN) layers A and A 1 that receive inputs from the antibody-injected eye. At the same time, the percentage of LGN X-cells is unaffected. In the present study, we investigated the effect of this monocular antibody-induced reduction of LGN Y-cells on the development of ocular dominance in striate cortex. Four-week-old kittens were given an intraocular injection of either a low (110 μg/33 μl volume) or a high (333 μg/33 μl volume) concentration of antibodies and single-cell recordings were carried out in striate cortex 33–65 weeks later. Following an injection of either antibody concentration, we found only slight abnormalities in striate cortex ocular dominance compared to normal adult cats. There was a small, but significant, decrease in the percentage of binocularly driven cells and a concomitant increase in the percentage of cells driven exclusively by the normal or control-injected eye. No ocular dominance abnormalities were found in kittens injected monocularly with control γ-globulins, indicating that the changes are due to effects of the antibodies. The changes in cortical ocular dominance produced by early antibody treatment are very different from those produced by rearing with monocular deprivation (MD) despite a similar loss of LGN Y-cells in the two conditions. This suggests that an initial (primary) monocular loss of LGN Y-cells in young kittens is not sufficient to alter cortical ocular dominance in a manner similar to that found after MD. The results thus argue against the hypothesis that the MD-induced loss of response to the deprived eye in striate cortex is secondary to a Y-cell loss in the LGN.

The cat's superior colliculus (SC) receives direct inputs from retinal W-cells (a W-D input) and Y-cells (Y-D input) and an indirect Y-cell input via the lateral geniculate nucleus and visual cortex (Y-I input). In previous studies we have shown that intraocular injection of antibodies raised against large retinal ganglion cells produces a dose-dependent reduction in the Y retinogeniculate pathway. Furthermore, when a sufficiently high antibody concentration is used, there is a substantial loss of the Y pathway and no apparent loss of the W pathway. In the present study, we used the antibodies to investigate the contributions of the Y and W pathways to functional organization within the SC. Binocular injections of low (330 micrograms/100 microliters) or high (1,000 micrograms/100 microliters) antibody concentrations were made. The antibody-mediated effects on SC cells' response properties were compared directly with effects of early binocular deprivation, which have been attributed to a loss of Y-I input. Extracellular single-cell recordings were made from the SC, and cells were classified as receiving Y-D, Y-I, or W-D inputs on the basis of their response latencies to electrical stimulation of the optic chiasm and optic tract. Injections of the low antibody concentration produced no significant effects on inputs to the SC. However, injections of the high antibody concentration resulted in a 70% reduction in SC cells with a Y-D input and an 82% reduction in SC cells with a Y-I input. There was no effect on the percentage of cells with a W-D input. Binocular deprivation produced a 76% reduction in the percentage of cells with Y-I input. Visual response properties of SC cells also were assessed. Injections of the high antibody concentration produced a 55% reduction in cells that respond with a directional preference and a 51% reduction in cells that respond to high-velocity stimuli. Binocular deprivation produced a 78% reduction in the proportion of directional cells and a 25% reduction in cells that respond to the ipsilateral eye. Taken together, the results of this and previous studies using cortical lesions, visual deprivation, and immunoablation suggest that Y-D input is the primary basis for responses to high stimulus velocity, Y-I input is an important basis for directional responses and response through the ipsilateral eye, and W-D input is important for responses to low stimulus velocity.(ABSTRACT TRUNCATED AT 400 WORDS)

– The buoyant density, ultrastructure and protein composition of dendrodendritic (DD), axodendritic (AD) and axosomatic (AS) synaptosomes were compared. DD synaptosomes prepared from either bovine superior colliculi or olfactory bulbs sedimented in 0.32 m-sucrose at 1000 g whereas AD and AS synaptosomes from frontal and temporal lobe cortices sedimented at 10.000 g. In gradients of Renografin the DD contacts banded at a density of 1.12 whereas AD and AS banded at 1.07. The DD contacts appeared as large clusters of serial contacts, and each individual terminal had a mean surface area of 1.48 μm2; the mean area of AD and AS terminals was less than 0.35 μm2. The protein compositions of DD, AD and AS synaptosomes were quite similar, with minor exceptions. These findings suggest that different synaptosmal types evolved from common membrane constituents.

Subcellular fractions were prepared from brains obtained at post-mortem from patients with Tay-Sachs disease and age-related human control subjects. The isopycnic density of synapses from Tay-Sachs tissue was increased and that of lysosomes was decreased. The ganglioside content of each fraction from Tay-Sachs brain was elevated and the phospholipid and cholesterol contents were reduced, compared with similar density control fractions. Membranous cytoplasmic bodies (MCB's), in addition to high concentrations of ganglioside, contain several proteins, one of which is present in unusually high amounts and has a molecular weight of approximately 63,000. Hexosaminidase A activity was markedly reduced in each fraction from Tay-Sachs brain and the elevated hexosaminidase B was found primarily in the MCB fraction. To determine the effect of excess gangliosides on neural membranes, mixed gangliosides (0.05%) were added to homogenates of pig brain. The gangliosides were increased whereas the phospholipid and cholesterol content was reduced in synaptic membranes prepared from these homogenates. A hypothesis is presented which relates the altered buoyant density and lipid composition of membranes from Tay-Sachs brain to the detergent effect of accumulated gangliosides.