Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Characterization and genomic analysis of the Lyme disease spirochete bacteriophage ϕBB-1.

Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferentially packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Inhibition of PQS signaling by the Pf bacteriophage protein PfsE enhances viral replication in Pseudomonas aeruginosa.

Quorum sensing, a bacterial signaling system that coordinates group behaviors as a function of cell density, plays an important role in regulating viral (phage) defense mechanisms in bacteria. The opportunistic pathogen Pseudomonas aeruginosa is a model system for the study of quorum sensing. P. aeruginosa is also frequently infected by Pf prophages that integrate into the host chromosome. Upon induction, Pf phages suppress host quorum sensing systems; however, the physiological relevance and mechanism of suppression are unknown. Here, we identify the Pf phage protein PfsE as an inhibitor of Pseudomonas Quinolone Signal (PQS) quorum sensing. PfsE binds to the host protein PqsA, which is essential for the biosynthesis of the PQS signaling molecule. Inhibition of PqsA increases the replication efficiency of Pf virions when infecting a new host and when the Pf prophage switches from lysogenic replication to active virion replication. In addition to inhibiting PQS signaling, our prior work demonstrates that PfsE also binds to PilC and inhibits type IV pili extension, protecting P. aeruginosa from infection by type IV pili-dependent phages. Overall, this work suggests that the simultaneous inhibition of PQS signaling and type IV pili by PfsE may be a viral strategy to suppress host defenses to promote Pf replication while at the same time protecting the susceptible host from competing phages.

Longitudinal map of transcriptome changes in the Lyme pathogen Borrelia burgdorferi during tick-borne transmission.

Borrelia burgdorferi (Bb), the causative agent of Lyme disease, adapts to vastly different environments as it cycles between tick vector and vertebrate host. During a tick bloodmeal, Bb alters its gene expression to prepare for vertebrate infection; however, the full range of transcriptional changes that occur over several days inside of the tick are technically challenging to capture. We developed an experimental approach to enrich Bb cells to longitudinally define their global transcriptomic landscape inside nymphal Ixodes scapularis ticks during a transmitting bloodmeal. We identified 192 Bb genes that substantially change expression over the course of the bloodmeal from 1 to 4 days after host attachment. The majority of upregulated genes encode proteins found at the cell envelope or proteins of unknown function, including 45 outer surface lipoproteins embedded in the unusual protein-rich coat of Bb. As these proteins may facilitate Bb interactions with the host, we utilized mass spectrometry to identify candidate tick proteins that physically associate with Bb. The Bb enrichment methodology along with the ex vivo Bb transcriptomes and candidate tick interacting proteins presented here provide a resource to facilitate investigations into key determinants of Bb priming and transmission during the tick stage of its unique transmission cycle.

Tripartite interactions between filamentous Pf4 bacteriophage, Pseudomonas aeruginosa, and bacterivorous nematodes.

The opportunistic pathogen Pseudomonas aeruginosa PAO1 is infected by the filamentous bacteriophage Pf4. Pf4 virions promote biofilm formation, protect bacteria from antibiotics, and modulate animal immune responses in ways that promote infection. Furthermore, strains cured of their Pf4 infection (ΔPf4) are less virulent in animal models of infection. Consistently, we find that strain ΔPf4 is less virulent in a Caenorhabditis elegans nematode infection model. However, our data indicate that PQS quorum sensing is activated and production of the pigment pyocyanin, a potent virulence factor, is enhanced in strain ΔPf4. The reduced virulence of ΔPf4 despite high levels of pyocyanin production may be explained by our finding that C. elegans mutants unable to sense bacterial pigments through the aryl hydrocarbon receptor are more susceptible to ΔPf4 infection compared to wild-type C. elegans. Collectively, our data support a model where suppression of quorum-regulated virulence factors by Pf4 allows P. aeruginosa to evade detection by innate host immune responses.

Peptidoglycan Production by an Insect-Bacterial Mosaic.

Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes.

Multiple mild traumatic brain injury in the rat produces persistent pathological alterations in the brain.

Multiple mild traumatic brain injury (mmTBI), in certain cases, produces persistent symptoms. However, the molecular mechanisms underlying these symptoms remain unclear. Here, we demonstrate extended pathological changes in the rat brain following mmTBI. Using the lateral fluid percussion (LFP) technique we exposed adult male Wistar rats to a mild TBI (mTBI) once a week for four weeks and compared them to surgical shams. At 90days following the last TBI or sham procedure the animals were cognitively tested in the Morris Water Maze (MWM), euthanized, and the brains removed for immunohistochemistry. At 90days following the last mTBI, NRF-2 staining was significantly decreased in the hilus of the hippocampus and cortex on the injured side, but did not significantly differ from shams on the un-injured side. CD68 positive microglia were significantly increased in the ipsilateral corpus callosum, cortex, and internal capsule of injured animals. Reactive astrocytosis, determined by increased GFAP staining, was also evident in the corpus callosum, cortex, internal capsule and thalamus on the injured side. Interestingly, the corpus callosum thickness at the midline was decreased in injured animals and had evident demyelination when compared to sham animals. Despite these findings, there were no significant differences in neurological assessments at 90days following the last injury. In MWM testing there were not significant differences in the training phase, the time spent in the thigmotaxia zone, or the target quadrant during the probe trial. However, there were significant differences between shams and injured animals in platform zone crossings during the probe trial. These results demonstrate that repetitive head trauma may produce persistent, long-term pathological alterations in brain architecture that may be difficult to detect using standard cognitive and neurological assessments.

Administration of low dose methamphetamine 12 h after a severe traumatic brain injury prevents neurological dysfunction and cognitive impairment in rats.

We recently published data that showed low dose of methamphetamine is neuroprotective when delivered 3 h after a severe traumatic brain injury (TBI). In the current study, we further characterized the neuroprotective potential of methamphetamine by determining the lowest effective dose, maximum therapeutic window, pharmacokinetic profile and gene expression changes associated with treatment. Graded doses of methamphetamine were administered to rats beginning 8 h after severe TBI. We assessed neuroprotection based on neurological severity scores, foot fault assessments, cognitive performance in the Morris water maze, and histopathology. We defined 0.250 mg/kg/h as the lowest effective dose and treatment at 12 h as the therapeutic window following severe TBI. We examined gene expression changes following TBI and methamphetamine treatment to further define the potential molecular mechanisms of neuroprotection and determined that methamphetamine significantly reduced the expression of key pro-inflammatory signals. Pharmacokinetic analysis revealed that a 24-hour intravenous infusion of methamphetamine at a dose of 0.500 mg/kg/h produced a plasma Cmax value of 25.9 ng/ml and a total exposure of 544 ng/ml over a 32 hour time frame. This represents almost half the 24-hour total exposure predicted for a daily oral dose of 25mg in a 70 kg adult human. Thus, we have demonstrated that methamphetamine is neuroprotective when delivered up to 12 h after injury at doses that are compatible with current FDA approved levels.

Treatment with low-dose methamphetamine improves behavioral and cognitive function after severe traumatic brain injury.

BACKGROUND: Methamphetamine increases the release and blocks the reuptake of dopamine. The moderate activation of dopamine receptors may elicit neuroprotective effects. We have recently demonstrated that low doses of methamphetamine reduce neuronal loss after ischemic injury. On the basis of this finding, we hypothesized that methamphetamine could also prevent neuronal loss and improve functional behavior after severe traumatic brain injury (TBI). METHODS: The rat lateral fluid percussion injury model was used to generate severe TBI. Three hours after injury, animals were treated with saline or methamphetamine. Neurological severity scores and foot fault assessments were used to determine whether treatment enhanced recovery after injury. The potential for methamphetamine treatment to improve cognitive function was assessed using the Morris water maze. Forty-eight hours after injury, paraffin-embedded brain sections were TUNEL stained to measure apoptotic cell death. Sections were also stained with antibody to doublecortin to quantify immature neurons within the dentate gyrus. RESULTS: Treatment with low-dose methamphetamine significantly reduced both behavioral and cognitive dysfunction after severe TBI. Methamphetamine-treated animals scored significantly lower on neurological severity scores and had significantly less foot faults after TBI compared with saline-treated control rats. Furthermore, methamphetamine treatment restored learning and memory function to near normal ability after TBI. At 48 hours after injury, apoptotic cell death within the hippocampus was significantly reduced, and the presence of immature neurons was significantly increased in methamphetamine-treated rats compared with saline-treated controls. CONCLUSION: Treatment with low-dose methamphetamine after severe TBI elicits a robust neuroprotective response resulting in significant improvements in behavioral and cognitive functions.

Low dose methamphetamine mediates neuroprotection through a PI3K-AKT pathway.

High doses of methamphetamine induce the excessive release of dopamine resulting in neurotoxicity. However, moderate activation of dopamine receptors can promote neuroprotection. Therefore, we used in vitro and in vivo models of stroke to test the hypothesis that low doses of methamphetamine could induce neuroprotection. We demonstrate that methamphetamine does induce a robust, dose-dependent, neuroprotective response in rat organotypic hippocampal slice cultures exposed to oxygen-glucose deprivation (OGD). A similar dose dependant neuroprotective effect was observed in rats that received an embolic middle cerebral artery occlusion (MCAO). Significant improvements in behavioral outcomes were observed in rats when methamphetamine administration delayed for up to 12 h after MCAO. Methamphetamine-mediated neuroprotection was significantly reduced in slice cultures by the addition of D1 and D2 dopamine receptor antagonist. Treatment of slice cultures with methamphetamine resulted in the dopamine-mediated activation of AKT in a PI3K dependant manner. A similar increase in phosphorylated AKT was observed in the striatum, cortex and hippocampus of methamphetamine treated rats following MCAO. Methamphetamine-mediated neuroprotection was lost in rats when PI3K activity was blocked by wortmannin. Finally, methamphetamine treatment decreased both cleaved caspase 3 levels in slice cultures following OGD and TUNEL staining within the striatum and cortex in rats following transient MCAO. These data indicate that methamphetamine can mediate neuroprotection through activation of a dopamine/PI3K/AKT-signaling pathway.

Air pollution is associated with brainstem auditory nuclei pathology and delayed brainstem auditory evoked potentials.

We assessed brainstem inflammation in children exposed to air pollutants by comparing brainstem auditory evoked potentials (BAEPs) and blood inflammatory markers in children age 96.3±8.5 months from highly polluted (n=34) versus a low polluted city (n=17). The brainstems of nine children with accidental deaths were also examined. Children from the highly polluted environment had significant delays in wave III (t(50)=17.038; p<0.0001) and wave V (t(50)=19.730; p<0.0001) but no delay in wave I (p=0.548). They also had significantly longer latencies than controls for interwave intervals I-III, III-V, and I-V (all t(50)>7.501; p<0.0001), consisting with delayed central conduction time of brainstem neural transmission. Highly exposed children showed significant evidence of inflammatory markers and their auditory and vestibular nuclei accumulated α synuclein and/or ß amyloid(1-42). Medial superior olive neurons, critically involved in BAEPs, displayed significant pathology. Children's exposure to urban air pollution increases their risk for auditory and vestibular impairment.

Chronic low-level Pb exposure during development decreases the expression of the voltage-dependent anion channel in auditory neurons of the brainstem.

Lead (Pb) exposure is a risk factor for neurological dysfunction. How Pb produces these behavioral deficits is unknown, but Pb exposure during development is associated with auditory temporal processing deficits in both humans and animals. Pb disrupts cellular energy metabolism and efficient energy production is crucial for auditory neurons to maintain high rates of synaptic activity. The voltage-dependent anion channel (VDAC) is involved in the regulation of mitochondrial physiology and is a critical component in controlling mitochondrial energy production. We have previously demonstrated that VDAC is an in vitro target for Pb, therefore, VDAC may represent a potential target for Pb in the auditory system. In order to determine whether Pb alters VDAC expression in central auditory neurons, CBA/CaJ mice (n=3-5/group) were exposed to 0.01mM, or 0.1mM Pb acetate during development via drinking water. At P21, immunohistochemistry reveals a significant decrease for VDAC in neurons of the Medial Nucleus of the Trapezoid Body. Western blot analysis confirms that Pb results in a significant decrease for VDAC. Decreases in VDAC expression could lead to an upregulation of other cellular energy producing systems as a compensatory mechanism, and a Pb-induced increase in brain type creatine kinase is observed in auditory regions of the brainstem. In addition, comparative proteomic analysis shows that several proteins of the glycolytic pathway, the phosphocreatine circuit, and oxidative phosphorylation are also upregulated in response to developmental Pb exposure. Thus, Pb-induced decreases in VDAC could have a significant effect on the function of auditory neurons.

Long-term air pollution exposure is associated with neuroinflammation, an altered innate immune response, disruption of the blood-brain barrier, ultrafine particulate deposition, and accumulation of amyloid beta-42 and alpha-synuclein in children and young adults.

Air pollution is a serious environmental problem. We investigated whether residency in cities with high air pollution is associated with neuroinflammation/neurodegeneration in healthy children and young adults who died suddenly. We measured mRNA cyclooxygenase-2, interleukin-1beta, and CD14 in target brain regions from low (n = 12) or highly exposed residents (n = 35) aged 25.1 +/- 1.5 years. Upregulation of cyclooxygenase-2, interleukin-1beta, and CD14 in olfactory bulb, frontal cortex, substantia nigrae and vagus nerves; disruption of the blood-brain barrier; endothelial activation, oxidative stress, and inflammatory cell trafficking were seen in highly exposed subjects. Amyloid beta42 (Abeta42) immunoreactivity was observed in 58.8% of apolipoprotein E (APOE) 3/3 < 25 y, and 100% of the APOE 4 subjects, whereas alpha-synuclein was seen in 23.5% of < 25 y subjects. Particulate material (PM) was seen in olfactory bulb neurons, and PM < 100 nm were observed in intraluminal erythrocytes from lung, frontal, and trigeminal ganglia capillaries. Exposure to air pollution causes neuroinflammation, an altered brain innate immune response, and accumulation of Abeta42 and alpha-synuclein starting in childhood. Exposure to air pollution should be considered a risk factor for Alzheimer's and Parkinson's diseases, and carriers of the APOE 4 allele could have a higher risk of developing Alzheimer's disease if they reside in a polluted environment.

Arsenic exposure in pregnant mice disrupts placental vasculogenesis and causes spontaneous abortion.

Arsenic is an abundant toxicant in ground water and soil around areas with extractive industries. Human epidemiological studies have shown that arsenic exposure is linked to developmental defects and miscarriage. The placenta is known to utilize vasculogenesis to develop its circulation. The hypothesis tested here states the following: arsenic exposure causes placental dysmorphogenesis and defective placental vasculogenesis resulting in placental insufficiency and subsequent spontaneous abortion. To test this hypothesis, pregnant mice were exposed to sodium arsenite (AsIII) through drinking water from conception through weanling stages. Neonatal assessment of birth rates, pup weights, and litter sizes in arsenic exposed and control mothers revealed that AsIII-exposed mothers had only 40% the fecundity of controls. Preterm analysis at E12.5 revealed a loss of fecundity at E12.5 from either 20 ppm or greater exposures to AsIII. There was no loss of fecundity at E7.5 suggesting that spontaneous abortion occurs during placentation. Histomorphometry on E12.5 placentae from arsenic-exposed mice revealed placental dysplasia especially in the vasculature. These results suggest that arsenic toxicity is causative for mammalian spontaneous abortion by virtue of aberrant placental vasculogenesis and placental insufficiency.

Motheaten (me/me) mice deficient in SHP-1 are less susceptible to focal cerebral ischemia.

We have demonstrated previously that the protein tyrosine phosphatase SHP-1 seems to play a role in glial development and is upregulated in non-dividing astrocytes after injury. The present study examines the effect of loss of SHP-1 on the CNS response to permanent focal ischemia. SHP-1 deficient (me/me) mice and wild-type littermates received a permanent middle cerebral artery occlusion (MCAO). At 1, 3, and 7 days after MCAO, infarct volume, neuronal survival and cell death, gliosis, and inflammatory cytokine levels were quantified. SHP-1 deficient me/me mice display smaller infarct volumes at 7 days post-MCAO, increased neuronal survival within the ischemic penumbra, and decreased numbers of cleaved caspase 3+ cells within the ischemic core compared with wild-type mice. In addition, me/me mice exhibit increases in GFAP+ reactive astrocytes, F4-80+ microglia, and a concomitant increase in the level of interleukin 12 (IL-12) over baseline compared with wild-type. Taken together, these results demonstrate that loss of SHP-1 results in greater healing of the infarct due to less apoptosis and more neuronal survival in the ischemic core and suggests that pharmacologic inactivation of SHP-1 may have potential therapeutic value in limiting CNS degeneration after ischemic stroke.

Lipopolysaccharide-activated SHP-1-deficient motheaten microglia release increased nitric oxide, TNF-alpha, and IL-1beta.

Accumulating evidence suggests a deleterious role for activated microglia in facilitating neuronal death by producing neurocytotoxic substances during injury, infection, or neurodegenerative diseases. After cochlear ablation, abnormal microglial activation accompanied by increased neuronal loss within the auditory brainstem occurs in motheaten (me/me) mice deficient in the protein tyrosine phosphatase SHP-1. To determine whether abnormally activated microglia contribute to neuronal death in me/me mice, primary microglial cultures from me/me and wild-type mouse cortices were stimulated by the bacterial endotoxin lipopolysaccharide (LPS) to evaluate the secretion of the neurotoxic mediators nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-1beta (IL-1beta). Me/me microglia release significantly greater amounts of all three mediators compared with wild-type microglia. However, the increased release of these compounds in microglia lacking SHP-1 does not appear to occur through activation of extracellular signal-regulated kinase (ERK), p38 kinase subgroups of mitogen-activated protein (MAP) kinases, or increases in NF-kappaB-inducing kinase (NIK). These results suggest that abnormal microglial activation and release of neurotoxic compounds may potentiate neuronal death in deafferented cells and can thus potentiate neurodegeneration in the me/me brainstem. Our data also indicate that SHP-1 is engaged in signaling pathways in LPS-activated microglia, but not through regulation of the ERK and p38 MAP kinases.

The effect of lead on the avian auditory brainstem.

Lead (Pb) continues to be a significant environmental toxin and remains an integral part of many industrial processes, hobbies, and tobacco smoke. Pb has been shown to be a potent toxin to the CNS and low levels of Pb (below the CDC established toxic blood level of 10 microg/dl) have been correlated with decreases in the IQ of children. Pb exposure is a risk factor for dyslexia, and significantly, dyslexics have deficits in auditory temporal processing, including backward masking and amplitude modulation detection. Importantly, Pb-exposed children have been found to be deficient in various aspects of auditory temporal processing, including backward masking. Auditory temporal information is vital for appropriate speech detection and it is not known where within the auditory axis temporal processing takes place, nor is it understood how Pb exposure modifies the cells of the auditory system. To address these questions, we have developed an animal model of auditory temporal processing using chickens and have established that Pb exposure during development results in deficits in backward masking in avians. The current study was undertaken to identify the cellular changes induced by Pb exposure in the auditory brainstem of chickens that are likely anatomical correlates of the observed deficits in backward masking. We found Pb exposure had no effect on neuron number or glial cells within the auditory brainstem. However, Pb exposure does result in significant decreases in the amount of the medium weight neurofilament protein (NFM) as well as decreased NFM phosphorylation within the axons connecting auditory nuclei in the avian brainstem. Because the amount of neurofilament can affect the conduction velocities of axons, these results may provide an anatomical link between Pb exposure, auditory temporal processing deficits, and dyslexia.

Arsenic exposure exacerbates atherosclerotic plaque formation and increases nitrotyrosine and leukotriene biosynthesis.

A correlation between arsenic and cardiovascular disease (CVD) has been established through epidemiological studies, although the mechanisms are unknown. Using a mouse model that develops atherosclerotic lesions on a normal chow diet, we have confirmed a connection between long-term arsenic intake and CVD. Our results reveal a significant increase in the degree of atherosclerotic plaque stenosis within the innominate artery of ApoE-/-/LDLr-/- mice treated with 10 ppm sodium arsenite (133 microM) in drinking water for 18 weeks compared to controls. Immunohistochemistry shows nitrotyrosine formation, a marker of reactive nitrogen species generation, is significantly higher within the atherosclerotic plaque of arsenic-treated mice. In addition, there is a significant increase in the 5-lipoxygenase (5-LO) product, leukotriene E4 (LTE4), in the serum of arsenic-treated mice. This is supported by induction of the 5-LO protein and subsequent increases in LTE4 synthesis in bovine aortic endothelial cells. This increase in LTE4 is partially inhibited by inhibitors of nitric oxide synthase, suggesting a link between reactive nitrogen species and arsenic-induced inflammation. Furthermore, there is a significant increase in prostacyclin (PGI2) in the serum of arsenic-treated mice. We conclude that changes in specific inflammatory mediators such as LTE4 and PGI2 are related to arsenic-induced atherosclerosis. In addition, amplified synthesis of reactive species such as peroxynitrite results in increased protein nitration in response to arsenic exposure. This finding is consistent with the pathology seen in human atherosclerotic plaques.

Focal cerebral ischemia upregulates SHP-1 in reactive astrocytes in juvenile mice.

The role of the tyrosine phosphatase SHP-1 in the hematopoietic system has been well studied; however, its role in the central nervous system (CNS) response to injury is not well understood. Previous studies in our laboratory have demonstrated increased immunoreactivity for SHP-1 in a subset of reactive astrocytes that do not appear to enter the cell cycle following deafferentation of the chicken auditory brainstem. In order to determine whether mammalian astrocytes also upregulate SHP-1 immunoreactivity following CNS injury, a mouse model of focal cerebral ischemia was utilized to study SHP-1 expression. The brains of 3-week-old mice were analyzed at four time points following permanent middle cerebral artery occlusion (MCAO): 1, 3, 7, and 14 days. Our results demonstrate consistent infarct volumes within surgical groups, and infarct volumes decrease as a function of time from 1 day (maximum infarct volume) to 14 days (minimum infarct volume) post-MCAO. In addition, SHP-1 protein levels are upregulated following cerebral ischemia and this increase peaks at 7 days post-MCAO. Analysis of confocal images further reveals that immunoreactivity for SHP-1 occurs predominantly in GFAP+ reactive astrocytes, although a small percentage of F4-80+ microglia are also double labeled for SHP-1 at early times post-MCAO. These SHP-1+ reactive astrocytes do not appear to enter the cell cycle (as defined by PCNA immunoreactivity), confirming our previous studies in the avian auditory brainstem. These results suggest that SHP-1 plays an important role in the regulation of glial activation and proliferation in the ischemic CNS.