Added benefit of raxibacumab to antibiotic treatment of inhalational anthrax.

Although antibiotics treat bacteremia in inhalational anthrax, pathogenesis is mainly driven by bacterial exotoxins. Raxibacumab, an IgG1 monoclonal antibody, binds the protective antigen (PA) of Bacillus anthracis, thus blocking toxin effects and leading to improved survival in the rabbit and monkey models of inhalational anthrax. To assess raxibacumab's added benefit over levofloxacin (LVX) alone, rabbits surviving to 84 h after a challenge with 200 times the median (50%) lethal dose of B. anthracis spores were randomized to receive 3 daily intragastric LVX doses of 50 mg/kg of body weight, with the first LVX dose administered just prior to administration of a single intravenous dose of placebo or 40 mg/kg raxibacumab. The percentages of animals alive at 28 days following the last LVX dose were compared between the 2 treatment groups using a two-sided likelihood-ratio chi-square test. The 82% survival rate for the LVX-raxibacumab combination was higher than the 65% survival rate for LVX alone (P=0.0874). There were nearly 2-fold fewer deaths for the combination (7 deaths; n=39) than for LVX alone (13 deaths; n=37), and the survival time was prolonged for the combination (P=0.1016). Toxin-neutralizing-activity titers were similar for both treatment groups, suggesting that survivors in both groups were able to mount a toxin-neutralizing immune response. Microscopic findings considered consistent with anthrax were present in animals that died or became moribund on study in both treatment groups, and there were no anthrax-related findings in animals that survived. Overall, raxibacumab provided a meaningful benefit over antibiotic alone when administered late in the disease course.

Mutation spectra of Kras and Tp53 in urethral and lung neoplasms in B6C3F1 mice treated with 3,3',4,4'-tetrachloroazobenzene.

3,3',4,4'-tetrachloroazobenzene (TCAB) is a contaminant formed during manufacture of various herbicide compounds. A recent National Toxicology Program study showed B6C3F1 mice exposed to TCAB developed a treatment-related increase in lung carcinomas in the high-dose group, and urethral carcinomas, an extremely rare lesion in rodents, in all dose groups. As the potential for environmental exposure to TCAB is widespread, and the mechanisms of urethral carcinogenesis are unknown, TCAB-induced urethral and pulmonary tumors were evaluated for alterations in critical human cancer genes, Kras and Tp53. Uroplakin III, CK20, and CK7 immunohistochemistry was performed to confirm the urothelial origin of urethral tumors. TCAB-induced urethral carcinomas harbored transforming point mutations in K-ras (38%) and Tp53 (63%), and 71% displayed nuclear TP53 expression, consistent with formation of mutant protein. Transition mutations accounted for 88% of Tp53 mutations in urethral carcinomas, suggesting that TCAB or its metabolites target guanine or cytosine bases and that these mutations are involved in urethral carcinogenesis. Pulmonary carcinomas in TCAB-exposed animals harbored similar rates of Tp53 (55%) and Kras (36%) mutations as urethral carcinomas, suggesting that TCAB may induce mutations at multiple sites by a common mechanism. In conclusion, TCAB is carcinogenic at multiple sites in male and female B6C3F1 mice through mechanisms involving Tp53 and Kras mutation.

Accumulation of CD11b⁺Gr-1⁺ cells in the lung, blood and bone marrow of mice infected with highly pathogenic H5N1 and H1N1 influenza viruses.

Infection with pathogenic influenza viruses is associated with intense inflammatory disease. Here, we investigated the innate immune response in mice infected with H5N1 A/Vietnam/1203/04 and with reassortant human H1N1 A/Texas/36/91 viruses containing the virulence genes hemagglutinin (HA), neuraminidase (NA) and NS1 of the 1918 pandemic virus. Inclusion of the 1918 HA and NA glycoproteins rendered a seasonal H1N1 virus capable of inducing an exacerbated host innate immune response similar to that observed for highly pathogenic A/Vietnam/1203/04 virus. Infection with 1918 HA/NA:Tx/91 and A/Vietnam/1203/04 were associated with severe lung pathology, increased cytokine and chemokine production, and significant immune cell changes, including the presence of CD11b(+)Gr-1(+) cells in the blood, lung and bone marrow. Significant differential gene expression in the lung included pathways for cell death, apoptosis, production and response to reactive oxygen radicals, as well as arginine and proline metabolism and chemokines associated with monocyte and neutrophil/granulocyte accumulation and/or activation. Arginase was produced in the lung of animals infected with A/Vietnam/1204. These results demonstrate that the innate immune cell response results in the accumulation of CD11b(+)Gr-1(+) cells and products that have previously been shown to contribute to T cell suppression.

Repeated dose toxicity and relative potency of 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) compared to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for induction of CYP1A1, CYP1A2 and thymic atrophy in female Harlan Sprague-Dawley rats.

In this study we assessed the relative toxicity and potency of the chlorinated naphthalenes 1,2,3,4,6,7-hexachloronaphthalene (PCN 66) and 1,2,3,5,6,7-hexachloronaphthalene (PCN 67) relative to that of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Chemicals were administered in corn oil:acetone (99:1) by gavage to female Harlan Sprague-Dawley rats at dosages of 0 (vehicle), 500, 1500, 5000, 50,000 and 500,000 ng/kg (PCN 66 and PCN 67) and 1, 3, 10, 100, and 300 ng/kg (TCDD) for 2 weeks. Histopathologic changes were observed in the thymus, liver and lung of TCDD treated animals and in the liver and thymus of PCN treated animals. Significant increases in CYP1A1 and CYP1A2 associated enzyme activity were observed in all animals exposed to TCDD, PCN 66 and PCN 67. Dose response modeling of CYP1A1, CYP1A2 and thymic atrophy gave ranges of estimated relative potencies, as compared to TCDD, of 0.0015-0.0072, for PCN 66 and 0.00029-0.00067 for PCN 67. Given that PCN 66 and PCN 67 exposure resulted in biochemical and histopathologic changes similar to that seen with TCDD, this suggests that they should be included in the WHO toxic equivalency factor (TEF) scheme, although the estimated relative potencies indicate that these hexachlorinated naphthalenes should not contribute greatly to the overall human body burden of dioxin-like activity.

Subacute oral and dermal toxicity of tert-butyl hydroperoxide in Fischer F344/N rats and B6C3F1 mice.

Tert-butyl hydroperoxide (TBHP) is a catalyst frequently used in oxidation and sulfonation reactions in the plastics industry. Since the toxicological evaluation of TBHP remains unknown, the National Toxicology Program (NTP) designed studies to characterize and compare TBHP toxicity by the dermal and oral (gavage) routes in male and female Fischer 344 rats and B6C3F1 mice in 14-day exposures. Rats and mice were administered TBHP at 22, 44, 88, 176 or 352 mg/kg in 0.5% aqueous methylcellulose for the gavage studies. In the dermal studies, mice were administered the same doses as above, while rats were administered four doses (22, 44, 88, 176 mg/kg) in 50% aqueous acetone. Results from the gavage studies revealed treatment-related decreases in survival in male rats and body weights in both male and female rats in the 352 mg/kg group. Clinical signs included post-treatment lethargy, thinness, abnormal breathing, ruffled fur, and/or ataxia which occurred sporadically. The male mice showed a statistically significant decrease in body weight in the 44, 88, 176, and 352 mg/kg groups. The major target organs of toxicity were the forestomach in male and female rats and mice, and the esophagus in male and female rats and in male mice. In addition, there was an increase in the absolute and relative liver weight in female mice with hepatocellular hypertrophy in the top-dose group only. Results from spin trapping experiments revealed the presence of electron paramagnetic resonance signals from radical adducts in the blood and organic extracts of the liver and kidneys of rats treated by gavage with 176 mg/kg TBHP, suggesting the involvement of free- radical generation. The no observed adverse effect level (NOAEL) was considered to be 22 mg/kg in rats and male mice, and 44 mg/kg in female mice. In the dermal studies, there was no effect on survival, body weight, or organ weights in either rats or mice. TBHP administration at the site of application resulted in dermal irritation, hyperkeratosis, hyperplasia, and/or inflammation of the epidermis and inflammation of the dermis at 176 mg/kg and above in male and female rats. Dermal irritation at the site of application was noted in all the mice exposed to 352 mg/kg TBHP. Histopathological lesions in the epidermis and dermis were seen in the 88-352 mg/kg males and in the 176-352 mg/kg females. The NOAEL was found to be 88 mg/kg for male rats and female mice, and 44 mg/kg for female rats and male mice. In conclusion, these studies demonstrate that TBHP is metabolized to free radicals and is a contact irritant affecting skin by the dermal route of exposure, and forestomach and esophagus by oral administration. There was no evidence of systemic absorption by the dermal route of exposure based on lack of pathological findings (Supported by National Institute of Environmental Health Sciences Contract No. N01-ES-65406).

Short-term toxicity study of ST-20 (NSC-741804) by oral gavage in Sprague-Dawley rats.

ST-20 (sodium 2,2-dimethylbutyrate) is a potential therapeutic agent for treatment of ß-thalassemia and sickle cell disease. A subchronic oral toxicity study was conducted in Sprague-Dawley rats (10/sex/dose) at gavage dosages of 0 (vehicle control), 200, 600, or 1,000 mg/kg, once daily for up to 15 days followed by a 14-day recovery. Ataxia (females), rough coat/thin appearance (males), and decreased body weights were observed at 1,000 mg/kg. Functional observational battery (FOB) deficits were observed more frequently in females and included decreased body tone, rectal temperature, emotional reactivity, neuromotor-neuromuscular activity (as exhibited by a deficit in visual/tactile placing accuracy, ataxia, hind limb dragging, and decreased grip strength), and rearing. ST-20 caused a decrease in WBC/RBC counts and RBC parameters; increase in reticulocytes and red cell inclusion bodies; decrease in total protein, globulin, and glucose; and increase in AG ratio. Micronucleated polychromatic erythrocytes of the bone marrow increased significantly in males at 1,000 mg/kg. Mean liver and kidney weights increased, and hepatocellular hypertrophy was observed in males at 1,000 mg/kg. Toxicologic findings were fully recovered during the 14-day recovery period. In conclusion, the no-observed adverse effect level for FOB and general toxicity was 200 mg/kg following gavage administration of ST-20 for up to 15 consecutive days.

Cynomolgus macaque model for pneumonic plague.

A recombinant vaccine (rF1V) is currently being developed for protection against pneumonic plague. An essential component in evaluating efficacy of the rF1V vaccine is the development of a well-understood animal model that shows similarity to human disease. The objective of this study was to determine the inhaled median lethal dose (LD50), evaluate the pathophysiology of disease and identify appropriate study endpoints in a cynomolgus macaque (CM) model of pneumonic plague. Eighteen CMs were challenged by head-only aerosol exposure with seven dosages of Yersinia pestis CO92. An LD50 of 24 colony forming units was estimated using Probit analysis. Disease pathology was evaluated by blood culture, clinical pathology, histopathology and telemetry. CMs that died became febrile following challenge and died 34-92 h after onset of fever. Bacteremia, increased respiration and heart rate, decreased blood pressure and loss of diurnal rhythm were also observed in conjunction with onset of fever. Histopathological examinations revealed significant findings in the lungs (intra-alveolar neutrophils and fibrinous pleuritis) consistent with pneumonic plague. These data indicate that the disease pathology observed in CMs following aerosol exposure to Y. pestis CO92 is similar to that of pneumonic plague in humans. Thus, the CM is an appropriate model to evaluate efficacy of a recombinant F1V vaccine candidate.

A genetic approach to the prediction of drug side effects: bleomycin induces concordant phenotypes in mice of the collaborative cross.

The antineoplastic drug bleomycin leads to the side effect of pulmonary fibrosis in both humans and mice. We challenged genetically diverse inbred lines of mice from the Collaborative Cross with bleomycin to determine the heritability of this phenotype. Sibling pairs of mice from 40 lines were treated with bleomycin. Lung disease was assessed by scoring lung pathology and by measuring soluble collagen levels in lavage fluid. Serum micro ribonucleic acids (miRNAs) were also measured. Inbred sibling pairs of animals demonstrated high coinheritance of the phenotypes of disease susceptibility or disease resistance. The plasma levels of one miRNA were clearly correlated in sibling mice. The results showed that, as in humans, the lines that comprise the Collaborative Cross exhibited wide genetic variation in response to this drug. This finding suggests that the genetically diverse Collaborative Cross animals may reveal drug effects that might be missed if a study were based on a conventional mouse strain.

A critical role for CD8 T cells in a nonhuman primate model of tuberculosis.

The role of CD8 T cells in anti-tuberculosis immunity in humans remains unknown, and studies of CD8 T cell-mediated protection against tuberculosis in mice have yielded controversial results. Unlike mice, humans and nonhuman primates share a number of important features of the immune system that relate directly to the specificity and functions of CD8 T cells, such as the expression of group 1 CD1 proteins that are capable of presenting Mycobacterium tuberculosis lipids antigens and the cytotoxic/bactericidal protein granulysin. Employing a more relevant nonhuman primate model of human tuberculosis, we examined the contribution of BCG- or M. tuberculosis-elicited CD8 T cells to vaccine-induced immunity against tuberculosis. CD8 depletion compromised BCG vaccine-induced immune control of M. tuberculosis replication in the vaccinated rhesus macaques. Depletion of CD8 T cells in BCG-vaccinated rhesus macaques led to a significant decrease in the vaccine-induced immunity against tuberculosis. Consistently, depletion of CD8 T cells in rhesus macaques that had been previously infected with M. tuberculosis and cured by antibiotic therapy also resulted in a loss of anti-tuberculosis immunity upon M. tuberculosis re-infection. The current study demonstrates a major role for CD8 T cells in anti-tuberculosis immunity, and supports the view that CD8 T cells should be included in strategies for development of new tuberculosis vaccines and immunotherapeutics.

Neurovirulence evaluation of Venezuelan equine encephalitis (VEE) vaccine candidate V3526 in nonhuman primates.

Assessment of neurovirulence is a standard test for vaccines derived from virulent neurotropic viruses. This study evaluated the potential neurovirulence of V3526, a live attenuated vaccine derived from a full-length infectious clone of Venezuelan equine encephalitis virus (VEEV) Trinidad donkey strain (TrD), a comparator VEEV vaccine (TC-83), TrD, and process control material (PCM) in juvenile rhesus macaques. Following intrathalamic/intraspinal (i.t./i.s. ) or subcutaneous (s.c.) inoculations, animals were observed for periods of 18, 91 or 181 days for paresis, paralysis, neurological disorders and other signs of clinical illness. Blood was collected for measurement of viremia, VEEV neutralizing antibodies, hematologic parameters, and liver enzymes. Gross necropsies and histopathological examinations were conducted with emphasis on detecting lesions in the brain and spinal cord. Elevated temperatures (1-2 degrees C) were noted in several of the TrD and vaccine inoculated animals on Day 6 following inoculation and mean temperatures for the V3526 i.t./i.s. and TC-83 groups were higher than PCM group throughout the study Day 18. No significant differences were seen for weight or clinical chemistry results between vaccine and PCM inoculated groups. Clinically significant signs (Grades 3 or 4) were noted in three of 21 V3526 i.t./i.s. and three of 12 TC-83 inoculated animals, however, these signs resolved within 3 weeks for all V3526 i.t./i.s. and for two of three TC-83 inoculated animals. At Day 18 extensive lesions indicative of a viral infection were seen in brain sections of all four TrD inoculated animals and one of seven V3526 i.t./i.s. inoculated animals. Only scattered lesions, characterized by foci of gliosis and vessels with perivascular inflammation, were found in the sections from four TC-83 and six V3526 i.t./i.s. inoculated animals. The minimal histological changes observed at Day 18 resolved to baseline levels by Day 181 comparable to the PCM group. V3526 was immunogenic and essentially nonneurovirulent when administered via the clinically relevant subcutaneous route.

Immune distribution and localization of phosphoantigen-specific Vgamma2Vdelta2 T cells in lymphoid and nonlymphoid tissues in Mycobacterium tuberculosis infection.

Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model of Mycobacterium tuberculosis infection to assess phosphoantigen-specific Vgamma2Vdelta2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonary M. tuberculosis infection generated diverse distribution patterns of Vgamma2Vdelta2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vgamma2Vdelta2 T cells in tissues were associated with M. tuberculosis infection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vgamma2Vdelta2 T cells were present within TB granulomas. In extrathoracic organs, Vgamma2Vdelta2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vgamma2Vdelta2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the gammadelta T cells present within granulomas. Thus, clonally expanded Vgamma2Vdelta2 T cells appeared to undergo trans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses to M. tuberculosis infection.

A single codon in the nucleocapsid protein C terminus contributes to in vitro and in vivo fitness of Edmonston measles virus.

The major inducible 70-kDa heat shock protein (hsp72) increases measles virus (MV) transcription and genome replication. This stimulatory effect is attributed to hsp72 interaction with two highly conserved hydrophobic domains in the nucleocapsid protein (N) C terminus of Edmonston MV. These domains are known as Box-2 and Box-3. A single amino acid substitution in Box-3 of Edmonston MV (i.e., N522D) disrupts hsp72 binding. The prevalence of the N522D substitution in contemporary wild-type MV isolates suggests that this sequence has been positively selected. The present work determined if the N522D substitution enhances viral fitness and the degree to which any fitness advantage is influenced by hsp72 levels. Both parent Edmonston MV (Ed N) and an N522D substitution mutant (Ed N-522D) exhibited similar growth on Vero and murine neuroblastoma cells and in cotton rat lung, although Ed N-522D virus exhibited an attenuated in vitro response to hsp72 overexpression. In contrast, mixed infections showed a significantly reduced in vitro and in vivo fitness of Ed N-522D virus. Results support the involvement of additional selectional pressures that maintain the circulation of virus containing N-522D despite the cost to viral fitness.

Hyperthermic pre-conditioning promotes measles virus clearance from brain in a mouse model of persistent infection.

Nervous tissue subjected to hyperthermic pre-conditioning is resistance to numerous insults although in vitro, the same treatment can increase gene expression and cytopathic effect of neurotropic paramyxoviruses, including measles virus (MV). The present work determined whether the in vivo relationship between hyperthermic pre-conditioning and MV infection would be to increase neuropathogenicity or, conversely, to promote clearance. Balb/c mice 36 h of age were exposed to a 41 degrees C hyperthermic treatment for 30 min. Intracranial inoculation of mice with Edmonston MV was performed at 6 h following the heat treatment, a time point exhibiting elevated levels of the major inducible 70-kDa heat shock protein in brain, a hallmark of pre-conditioning. Forty-seven percent of the non-heated animals supported a persistent cytopathic infection at 21-day post infection (PI) based upon the quantitative detection of viral RNA in brain using real time RT-PCR. Cytopathic effect in the infected brains was proportionate to viral RNA burden. In contrast, infected stress conditioned mice lacked significant cytopathic effect and clearance was demonstrated in 95% of the animals. Analysis of shorter post-infection intervals showed that levels of viral RNA in brain were equivalent between stress conditioned and non-conditioned mice at 2 and 7 days PI, with clearance being first evident in both groups at 14 days. The temporal onset and progression of clearance was correlated to splenocyte blastogenic responsiveness to purified MV antigen but not the production of MV-specific antibody. Collectively, these results support the hypothesis that stress conditioning enhances the efficacy of cell-mediated immune responses known to mediate viral clearance from brain.

Pathology of inhalation anthrax in cynomolgus monkeys (Macaca fascicularis).

Anthrax is considered a serious biowarfare and bioterrorism threat because of its high lethality, especially by the inhalation route. Rhesus macaques (Macaca mulatta) are the most commonly used nonhuman primate model of human inhalation anthrax exposure. The nonavailability of rhesus macaques necessitated development of an alternate model for vaccine testing and immunologic studies. This report describes the median lethal dose (LD(50)) and pathology of inhalation anthrax in cynomolgus macaques (Macaca fascicularis). Gross and microscopic tissue changes were reviewed in 14 cynomolgus monkeys that died or were killed after aerosol exposure of spores of Bacillus anthracis (Ames strain). The LD(50) and 95% confidence intervals were 61800 (34000 to 110000) colony-forming units. The most common gross lesions were mild splenomegaly, lymph node enlargement, and hemorrhages in various organs, particularly involving the meninges and the lungs. Mediastinitis, manifested as hemorrhage or edema, affected 29% of the monkeys. Microscopically, lymphocytolysis occurred in the intrathoracic lymph nodes and spleens of all animals, and was particularly severe in the spleen and in germinal centers of lymph nodes. Hemorrhages were common in lungs, bronchial lymph nodes, meninges, gastrointestinal tract, and mediastinum. These results demonstrate that the Ames strain of B. anthracis is lethal by the inhalation route in the cynomolgus macaque. The LD(50) of the Ames strain of B. anthracis was within the expected experimental range of previously reported values in the rhesus monkey in an aerosol challenge. The gross and microscopic pathology of inhalation anthrax in the cynomolgus monkey is remarkably similar to that reported in rhesus monkeys and humans. The results of this study are important for the establishment of an alternative nonhuman primate model for evaluation of medical countermeasures against inhalational anthrax.