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Understanding immune responses to native antigens in response to natural infections can lead to improved approaches to vaccination. This study sought to characterize the humoral immune response to anthrax toxin components, capsule and spore antigens in individuals (n = 46) from the Kayseri and Malatya regions of Turkey who had recovered from mild or severe forms of cutaneous anthrax infection, compared to regional healthy controls (n = 20). IgG antibodies to each toxin component, the poly-γ-D-glutamic acid capsule, the Bacillus collagen-like protein of anthracis (BclA) spore antigen, and the spore carbohydrate anthrose, were detected in the cases, with anthrax toxin neutralization and responses to Protective Antigen (PA) and Lethal Factor (LF) being higher following severe forms of the disease. Significant correlative relationships among responses to PA, LF, Edema Factor (EF) and capsule were observed among the cases. Though some regional control sera exhibited binding to a subset of the tested antigens, these samples did not neutralize anthrax toxins and lacked correlative relationships among antigen binding specificities observed in the cases. Comparison of serum binding to overlapping decapeptides covering the entire length of PA, LF and EF proteins in 26 cases compared to 8 regional controls revealed that anthrax toxin-neutralizing antibody responses elicited following natural cutaneous anthrax infection are directed to conformational epitopes. These studies support the concept of vaccination approaches that preserve conformational epitopes., Competing Interests: The authors have declared that no competing interests exist.

Edema toxin (ET), composed of edema factor (EF) and protective antigen (PA), is a virulence factor of Bacillus anthracis that alters host immune cell function and contributes to anthrax disease. Anthrax vaccine precipitated (AVP) contains low but detectable levels of EF and can elicit EF-specific antibodies in human recipients of AVP. Active and passive vaccination of mice with EF can contribute to protection from challenge with Bacillus anthracis spores or ET. This study compared humoral responses to ET in recipients of AVP ( n = 33) versus anthrax vaccine adsorbed (AVA; n = 66), matched for number of vaccinations and time postvaccination, and further determined whether EF antibodies elicited by AVP contribute to ET neutralization. AVP induced higher incidence (77.8%) and titer (229.8 ± 58.6) of EF antibodies than AVA (4.2% and 7.8 ± 8.3, respectively), reflecting the reported low but detectable presence of EF in AVP. In contrast, PA IgG levels and ET neutralization measured using a luciferase-based cyclic AMP reporter assay were robust and did not differ between the two vaccine groups. Multiple regression analysis failed to detect an independent contribution of EF antibodies to ET neutralization in AVP recipients; however, EF antibodies purified from AVP sera neutralized ET. Serum samples from at least half of EF IgG-positive AVP recipients bound to nine decapeptides located in EF domains II and III. Although PA antibodies are primarily responsible for ET neutralization in recipients of AVP, increased amounts of an EF component should be investigated for the capacity to enhance next-generation, PA-based vaccines., (Copyright © 2017 American Society for Microbiology.)

Purpose: The aim of this study is to determine whether exposure to hazardous chemicals alters chemokine or cytokine production in macrophages and link these events to changes in intracellular signaling pathways and activation of specific gene promoters., Methods: RAW 264.7 mouse macrophages were treated with selected toxic industrial chemicals (TICs) and examined for changes in immune function. Luminex multiplex technology was used to assess changes in cytokine/chemokine expression and activation of kinase signaling pathways. In addition, a panel of macrophage cell lines with promoter-specific luciferase reporter genes were generated and treated with the TICs, and transcriptional responses to these chemicals were detected by changes in luminescence., Results: Changes in expression of cytokines and chemokines were linked to changes in the activation state of intracellular signaling pathways. Overall, the findings reveal that sublytic levels of TICs can alter the profile of cytokines and chemokines expressed by macrophages, with a pattern that suggests immunosuppression. The data demonstrate that critical changes in immune function correlate with activation of kinase signaling pathways in macrophages., Conclusions: These data provide insight into the effects of sublytic doses of selected TICs on macrophage function, with a particular emphasis on identifying changes in expression of cytokines and chemokines. These altered patterns in immune function were linked to changes in the activation state of intracellular signaling pathways. The data strongly suggest that small amounts of TICs can have subtle, yet very critical, effects on macrophages.

In an effort to better understand the mechanisms by which Bacillus anthracis establishes disease, experiments were undertaken to identify the genes essential for intracellular germination. Eighteen diverse genetic loci were identified via an enrichment protocol using a transposon-mutated library of B. anthracis spores, which was screened for mutants delayed in intracellular germination. Fourteen transposon mutants were identified in genes not previously associated with B. anthracis germination and included disruption of factors involved in membrane transport, transcriptional regulation, and intracellular signaling. Four mutants contained transposon insertions in gerHA, gerHB, gerHC, and pagA, respectively, each of which has been previously associated with germination or survival of B. anthracis within macrophages. Strain MIGD101 (named for macrophage intracellular germination defective 101) was of particular interest, since this mutant contained a transposon insertion in an intergenic region between BAs2807 and BAs2808, and was the most highly represented mutant in the enrichment. Analysis of B. anthracis MIGD101 by confocal microscopy and differential heat sensitivity following macrophage infection revealed ungerminated spores within the cell. Moreover, B. anthracis MIGD101 was attenuated in cell killing relative to the parent strain. Further experimental analysis found that B. anthracis MIGD101 was defective in five known B. anthracis germination pathways, supporting a mechanism wherein the intergenic region between BAs2807 and BAs2808 has a global affect on germination of this pathogen. Collectively, these findings provide insight into the mechanisms supporting B. anthracis germination within host cells.

Faced with freshwater shortages, water authorities are increasingly utilizing wastewater reclamation to augment supplies. However, concerns over emerging trace contaminants that persist through wastewater treatment need to be addressed to evaluate potential risks. In the present study, perfluorinated surfactant residues were characterized in recycled water from four California wastewater treatment plants that employ tertiary treatment and one that treats primary sewage in a wetland constructed for both treatment and wildlife habitat. Effluent concentrations were compared with surface and groundwater from a creek where recycled water was evaluated as a potential means to augment flow (Upper Silver and Coyote Creeks, San Jose, CA). In the recycled water, 90-470 ng/l perfluorochemicals were detected, predominantly perfluorooctanoate (PFOA; 10-190 ng/l) and perfluorooctanesulfonate (PFOS; 20-190 ng/l). No significant removal of perfluorochemicals was observed in the wetland (total concentration ranged 100-170ng/l across various treatment stages); in this case, 2-(N-ethylperfluorooctanesulfonamido) acetic acid (N-EtFOSAA), perfluorodecanesulfonate (PFDS), and PFOS were dominant. Though there is currently no wastewater discharge into the creeks, perfluorochemicals were found in the surface water and underlying groundwater at a total of 20-150 ng/l with PFOS and PFOA again making the largest contribution. With respect to ecotoxicological effects, perfluorochemical release via recycled water into sensitive ecosystems requires evaluation.

The multifunctional serine protease thrombin has been shown to be neurotoxic in vitro and in vivo and is demonstrable in the Alzheimer disease (AD) brain. We have documented that in AD the cerebral microvasculature is a source of inflammatory and neurotoxic proteins. The objective of this study was to determine if injured brain endothelial cells could be a source of neurotoxic thrombin. Brain endothelial cells were incubated with either sodium nitroprusside (SNP, 10 microM), inflammatory proteins (IL-1beta, IL-6, TNFalpha, LPS, IFNgamma) or the PKC inhibitor bisindolymaleimide (1 microM) for 24 h and conditioned media collected. Endothelial cell conditioned medium was incubated with purified apolipoprotein E4 (apoE4) for 24 h, and then analyzed for neurotoxic activity against primary cortical cultures and for apoE4 fragments by western blot. Endothelial cell conditioned medium collected after treatment with either SNP, inflammatory proteins, or the PKC inhibitor bisindolymaleimide, demonstrated a significant (p < 0.005) level of thrombin activity, the presence of apoE4 fragments, and was capable of evoking neuronal cell death. These data demonstrate that endothelial cell injury results in thrombin release and suggest that the brain microcirculation could be a source of neurotoxic factors in AD.

Morphological facilitation was examined in immediate (Experiment 1) and long-term (Experiment 2) lexical decision with English materials. For the target (payment), related primes consisted of base-alone (pay), affix-plus-base (prepay), or base-plus-affix (payable) combinations, thereby defining position of overlap. In addition, modality of presentation varied for primes and targets (Experiment 1). At short lags, the advantage for prepay-payment over payable-payment type pairs was significant when primes were visual (V) and targets were auditory (A), marginal under AV conditions, and nonexistent under VV conditions. At long lags, the magnitude of VV did not vary with position of overlap. Morphological facilitation was stable across changes in modality following prefixed and simple forms, reflecting lexical architecture. By contrast, the absence of facilitation following suffixed primes presented cross-modally implicates modality-specific processing.

Hearing loss is the most common occupational disease in the United States, with noise serving as the presumed causative agent in most instances. This investigation characterizes the exposure conditions that facilitate the potentiation of noise-induced hearing loss (NIHL) by carbon monoxide (CO). Auditory function was compared in rats exposed 4 weeks earlier to noise alone, CO alone, combined exposure, and air in the exposure chamber. This interval between exposure and auditory threshold assessment was selected to permit recovery of temporary threshold shifts. The compound action potential (CAP) threshold evoked by pure tone stimuli was used as a measure of auditory sensitivity. The no adverse effect level (NOAEL) with respect to potentiation of NIHL was found to be 300 ppm CO. Potentiation of NIHL by CO increases linearly as CO concentration increases between 500 -1500 ppm. Benchmark dose software (version 1. 1B) published by the U.S. EPA National Center for Environmental Assessment was employed to determine a benchmark concentration of CO that produced either a 5-dB potentiation of NIHL or an increase in auditory threshold equivalent to 10% of the effect of noise alone. The lower bound for these benchmark concentrations were 320 and 194 ppm CO, respectively. Unlike CO dose, the relationship between noise severity and potentiation of NIHL by CO shows a nonlinear relationship. The greatest potentiation was observed at moderate noise exposures (100 dB, 2-h, octave band-limited noise, or OBN) that produce limited permanent threshold shifts. Repeated exposures to 95-dB noise for 2-h periods in combination with 1200 ppm CO also yielded potentiation of NIHL, though such effects were not observed following a single combined exposure. These results underscore the potential risk of hearing loss from combined exposure to noise and CO, and the risks associated with repeated exposure.