05). PA-specific gamma interferon (IFN-␥) and interleukin-4 (IL-4) CD4؉ cell frequencies and T cell stimulation indices were sustained through 50.5 months (the last time point measured). PA-specific memory B cell frequencies were highly variable but, in general, were detectable in peripheral blood mononuclear cells (PBMC) by 2 months, were significantly above control levels by 7 months, and remained detectable in the HuAVA and 1:5 and 1:20 AVA groups through 42 months (the last time point measured). HuAVA and diluted AVA elicited a combined Th1/Th2 response and robust immunological priming, with sustained production of high-avidity PA-specific functional antibody, long-term immune cell competence, and immunological memory (30 months for 1:20 AVA and 52 months for 1:10 AVA). Vaccinated animals surviving inhalation anthrax developed high-magnitude anamnestic anti-PA IgG and TNA responses.

The purpose of this study was to investigate whether coexposure to lipopolysacchride (LPS) will heighten the inflammatory response and other pulmonary lesions in mice exposed to cigarette smoke, and thus to evaluate the potential use of this LPS-compromised mouse model as a model for chronic obstructive pulmonary disease (COPD) investigation. AKR/J male mice were exposed to HEPA-filtered air (sham control group), cigarette smoke (smoke group), LPS (LPS group), or smoke plus LPS (smoke-LPS group) by nose-only inhalation. Lungs were collected at the end of the 3-wk exposure and processed for microarray analysis. Clustering and network analysis showed decreased heat-shock response and chaperone activity, increased immune and inflammatory response, and increased mitosis in all three exposed groups. Two networks/function modules were exclusively found in the smoke-LPS group, that is, the downregulated muscle development/muscle contraction process and the upregulated reactive oxygen species production process. Notably, the number of genes and function modules/networks associated with inflammation was reduced in the smoke-LPS group compared to the LPS group. The most upregulated gene in the smoke group, MMP12, is a matrix metalloproteinase that preferentially degrades elastin and has been implicated in COPD development. NOXO1, which was upregulated in all three treatment groups, positively regulates the expression of a subunit of NADPH oxidase (NOX1), a major source of reactive oxygen species, and may play an important role in the pathogenesis of COPD. Serum amyloid A1, which is an acute-phase systemic inflammation marker and can be induced by LPS exposure, was significantly upregulated in the LPS and smoke-LPS groups. MARCO, a scavenger receptor expressed in macrophages that may play a significant role in LPS-induced inflammatory response, was upregulated in the LPS group and the smoke-LPS group, but not in the smoke group. In conclusion, gene expression profiling identified genes and function modules that may be related to COPD pathogenesis and may be useful as biomarkers to monitor COPD progression. In addition, an LPS-compromised mouse model showed potential as a useful tool for studying cigarette smoke-associated COPD.

The anthrax lethal toxin neutralization assay (TNA) will likely be used to correlate the protection offered by new anthrax vaccines in animal models to the immunogenicity that will be provided in humans. TNA data are being generated in several different laboratories to measure the immune responses in rabbits, nonhuman primates, and humans. In order to compare data among species and laboratories, a collaborative study was conducted in which 108 samples from the three species were analyzed in seven independent laboratories. Six of the seven laboratories had participated in an interlaboratory technology transfer of the TNA. Analysis of the titration curves generated by samples from each species indicated that the behaviors of the samples from all species were similar; the upper and lower asymptotes and the slopes of the curves were less than 30% divergent from those for human reference material. Dilutional linearity was consistent among samples from each species, with spike to effective dilution at 50% inhibition (ED 50 ) slopes of less than 1.2 for all species. Agreement among the laboratories with consensus values was within 10% of the ED 50 s for all samples and within 7.5% of the quotients of the test sample ED 50 and the reference standard ED 50 (NF 50 s) for all samples. The relative standard deviations obtained when data from all laboratories and for all species were combined were 45% for the ED 50 s and 35% for the NF 50 s. These precision data suggest that the NF 50 readout may normalize the values generated by different laboratories. This study demonstrates that the TNA is a panspecies assay that can be performed in several different laboratories with a high degree of quantitative agreement and precision.

A new tool beginning to have wider application in toxicology studies is transcript profiling using microarrays. Microarrays provide an opportunity to directly compare transcript populations in the tissues of chemical-exposed and unexposed animals. While several studies have addressed variation between microarray platforms and between different laboratories, much less effort has been directed toward individual animal differences especially among control animals where RNA samples are usually pooled. Estimation of the variation in gene expression in tissues from untreated animals is essential for the recognition and interpretation of subtle changes associated with chemical exposure. In this study hepatic gene expression as well as standard toxicological parameters were evaluated in 24 rats receiving vehicle only in 2 independent experiments. Unsupervised clustering demonstrated some individual variation but supervised clustering suggested that differentially expressed genes were generally random. The level of hepatic gene expression under carefully controlled study conditions is less than 1.5-fold for most genes. The impact of individual animal variability on microarray data can be minimized through experimental design.

2- Deoxy–D-Glucose (2-DG) is being developed as a potential anticonvulsant and disease-modifying agent for epilepsy patients; however, during preclinical development, cardiac toxicity has been encountered in rats. This study was performed to determine whether cardiac troponin (cTnI and cTnT), Atrial Natriuretic Peptide (ANP), Brain Natriuretic Peptide (BNP), N-terminal pro-brain natriuretic peptide (NT-proBNP) and/or creatine kinase (CK) could be useful as indicators of 2-deoxy-D-glucose (2-DG) cardiac toxicity. In addition, this study also investigated the association of cardiac histopathological changes with these biomarkers. F344 rats (four/sex/group/sacrifice point) were gavaged with either vehicle or 2-DG (50, 125, or 375 mg/kg BID; total daily dose of 100, 250 or 750 mg/kg/day) for 7, 14, 21, or 45 days followed by a 15 day recovery. Dose dependent increases in NT pro-BNP and BNP plasma concentrations were observed. Following recovery period, the NT pro-BNP and BNP concentrations returned to baseline levels. There were no remarkable increases in CK, ANP, cTnI, or cTnT concentrations. There were no gross cardiac lesions observed at the necropsy. Microscopic findings of vacuolar degeneration and hypertrophy of the endothelial cells of the endocardium were present in the heart at doses of 250 and 750 mg/kg/day. Microscopic findings, in general, were associated with increases in NT pro-BNP levels. Cardiac toxicity appeared to be reversible. In conclusion, NT-proBNP and BNP are potential early biomarkers for 2-DG induced cardiac toxicity that can be useful to monitor 2-DG therapy in clinical trials.

A nthrax vaccine adsorbed (AVA; BioThrax; Emergent Bio Solutions Inc., Lansing, MI) is the only Food and Drug Administration (FDA)-approved vaccine in the United States for prevention of anthrax in humans. The primary immunogen in AVA is anthrax toxin protective antigen (PA). Serum anti-PA antibody levels are accurate immune correlates of protection in nonhuman primate (NHP) models of inhalation anthrax and for predicted probability of survival in humans (1-3). There is a significant lack of data in humans regarding the onset, duration, quantitative analysis, and functional activity of humoral antibody and cell-mediated immunity (CMI) responses following priming and boosting with AVA.In 2012, the preexposure schedule for AVA was approved as a priming series of three 0.5-ml intramuscular (IM) injections at 0, 1, and 6 months (3-IM) with subsequent boosters at 12 and 18 months and annually thereafter for those at continued risk of infection (http://www.fda.gov/BiologicsBloodVaccines/Vaccines /ApprovedProducts/ucm304758.htm; http://www.fda.gov /downloads/BiologicsBloodVaccines/BloodBloodProducts /ApprovedProducts/LicensedProductsBLAs/UCM074923 .pdf). In 2013, AVA received market approval in the European Union (EU) using a 3-IM priming series and 3-yearly booster schedule (http://emergentbiosolutions.com/sites/default/files /BioThrax_Germany.pdf).These recent changes in the FDA-approved priming schedule and route of administration for AVA and EU approval of an alternate schedule warranted detailed characterization of their immunological impact. Serological noninferiority analyses for peak anti-PA IgG and lethal toxin neutralization activity (TNA) in response to the 3-IM priming schedule and alternative booster schedules were reported previously, and the safety profile of AVA administered IM in humans was confirmed to be similar to that for other alum-containing vaccines (4-6). Less frequent AVA injection doses resulted in a reduction in some injection site adverse events (AEs), and IM administration resulted in reduced frequency, duration, and severity of AEs (5-11). The potential for increasing the intervals between booster doses requires an assessment of sustained antibody functional activity, CMI, and the ability to develop rapid protective anamnestic responses (5,6,12).In a rhesus macaque model of inhalation anthrax, the AVA

Anthrax toxin (ATx) is composed of the binary exotoxins lethal toxin (LTx) and edema toxin (ETx).Results showed that macaque anti-AVA sera neutralized LTx in vitro, even when PA was prebound to cells. Neutralization titers in surviving versus nonsurviving animals and between prechallenge and postchallenge activities were highly correlated. These data demonstrate that AVA stimulates a myriad of antibodies that recognize multiple neutralizing epitopes and confirm that change, loss, or occlusion of epitopes after PA is processed from PA83 to PA63 at the cell surface does not significantly affect in vitro neutralizing efficacy. Furthermore, these data support the idea that the full-length PA83 monomer is an appropriate immunogen for inclusion in next-generation anthrax vaccines.

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