Characterization of the Testicular Toxicity of 3-Nitro-1,2,4-Triazol-5-One and 2,4-Dinitroanisole in Rats ( Rattus norvegicus).

Nitrotriazolone (3-nitro-1,2,4-triazol-5-one; NTO) and dinitroanisole (2,4-dinitroanisole; DNAN), insensitive energetic materials used in explosive formulations, have induced testicular toxicity and oligospermia in repeated-dose oral toxicity tests. To identify the target site of testicular toxicity of NTO and DNAN, Sprague Dawley rats were orally dosed with NTO (500 mg/kg/d) or DNAN (50 or 100 mg/kg/d) in corn oil for 1, 3, 7, or 14 days. Degeneration of germinal epithelium occurred in multiple tubule stages on days 7 and 14 in treated rats. Degeneration increased in severity with time and was characterized by degeneration/apoptosis of pachytene spermatocytes and round and elongating spermatids, depletion of step 19 spermatids, luminal spermatogenic cell sloughing, multinucleate cells, and pronounced Sertoli cell vacuolation. Serum luteinizing hormone and follicle-stimulating hormone did not differ between NTO- and DNAN-treated and control rats on any sampling day. Serum testosterone levels reduced only in rats given 50 mg/kg/d DNAN for 7 days. These results suggest that the initial site of testicular injury for both NTO and DNAN is the Sertoli cell.

Evaluating mice lacking serum carboxylesterase as a behavioral model for nerve agent intoxication.

Mice and other rodents are typically utilized for chemical warfare nerve agent research. Rodents have large amounts of carboxylesterase in their blood, while humans do not. Carboxylesterase nonspecifically binds to and detoxifies nerve agent. The presence of this natural bioscavenger makes mice and other rodents poor models for studies identifying therapeutics to treat humans exposed to nerve agents. To obviate this problem, a serum carboxylesterase knockout (Es1 KO) mouse was created. In this study, Es1 KO and wild type (WT) mice were assessed for differences in gene expression, nerve agent (soman; GD) median lethal dose (MLD) values, and behavior prior to and following nerve agent exposure. No expression differences were detected between Es1 KO and WT mice in more than 34 000 mouse genes tested. There was a significant difference between Es1 KO and WT mice in MLD values, as the MLD for GD-exposed WT mice was significantly higher than the MLD for GD-exposed Es1 KO mice. Behavioral assessments of Es1 KO and WT mice included an open field test, a zero maze, a Barnes maze, and a sucrose preference test (SPT). While sex differences were observed in various measures of these tests, overall, Es1 KO mice behaved similarly to WT mice. The two genotypes also showed virtually identical neuropathological changes following GD exposure. Es1 KO mice appear to have an enhanced susceptibility to GD toxicity while retaining all other behavioral and physiological responses to this nerve agent, making the Es1 KO mouse a more human-like model for nerve agent research.

Testicular effects of 3-nitro-1,2,4-triazol-5-one (NTO) in mice when exposed orally.

3-Nitro-1,2,4-triazol-5-one (NTO) is currently being investigated in the development of insensitive munitions. Rats orally exposed to NTO have demonstrated testicular toxicity in both subacute and subchronic studies; however, toxicity has not been verified in mice. Also, previous studies have not demonstrated the nature of NTO-induced testicular toxicity due to the prolonged dosing regimen utilized and effects of maturation depletion. In this study, a time-course design was used and the earliest pathological changes in testes of adult BALB/c mice orally dosed with NTO in corn oil suspensions at 0, 500 or 1000 mg/kg-day NTO for 1, 3, 7 or 14 d were evaluated. The earliest NTO-induced testicular changes occurred in the 1000 mg/kg-day group at day 7 and the 500 mg/kg-day group at day 14 as evident by the presence of bi- and multinucleated giant cells (MNGCs) of almost all spermatids in an isolated stage II-III tubule/step 2-3 and a stage IX tubule/step 9 in the 1000 and 500 mg/kg-day groups, respectively. Testicular toxicity was characterized by degeneration and the presence of bi- and MNGCs of spermatids (stages II-III and IX), which progressed to additional germ cell degeneration as dosing duration increased. Occasional step 16 spermatid retention was also noted in stage XII and I tubules in the day 14, 1000 mg/kg-day group. These data indicate that NTO is a testicular toxicant in mice and that spermatids are the most sensitive cell. The presence of retained spermatids warrants further investigation regarding NTO's role as a direct Sertoli cell toxicant.

Fatal outbreak of an emerging clone of extensively drug-resistant Acinetobacter baumannii with enhanced virulence.

BACKGROUND: Severe Acinetobacter baumannii infections in immunocompetent patients are uncommon, and the virulence mechanisms of this organism are not fully understood. METHODS: Following an outbreak of fatal A. baumannii infections in a cohort of relatively immunocompetent patients (low comorbidity and illness severity scores), isolates were investigated with comparative genomics and in animal models. RESULTS: Two unrelated A. baumannii clades were associated with the outbreak. The clone associated with the majority of patient deaths, clade B, is evolutionarily distinct from the 3 international clonal complexes, belongs to multilocus sequence type (MLST) 10, and is most closely related to strains isolated from the Czech Republic, California, and Germany in 1994, 1997, and 2003, respectively. In 2 different murine models, clade B isolates were more virulent than comparator strains, including the highly virulent reference strain AB5075. The most virulent clade B derivative, MRSN 16897, was isolated from the patient with the lowest combined comorbidity/illness severity score. Clade B isolates possess a unique combination of putative virulence genes involved in iron metabolism, protein secretion, and glycosylation, which was leveraged to develop a rapid and specific clinical assay to detect this clade that cannot be distinguished by MLST. CONCLUSIONS: Clade B warrants continued surveillance and investigation.

Evaluation of Gallium Citrate Formulations against a Multidrug-Resistant Strain of Klebsiella pneumoniae in a Murine Wound Model of Infection.

Skin and soft tissue infections (SSTIs) are a common occurrence in health care facilities with a heightened risk for immunocompromised patients. Klebsiella pneumoniae has been increasingly implicated as the bacterial agent responsible for SSTIs, and treatment can be challenging as more strains become multidrug resistant (MDR). Therefore, new treatments are needed to counter this bacterial pathogen. Gallium complexes exhibit antimicrobial activity and are currently being evaluated as potential treatment for bacterial infections. In this study, we tested a topical formulation containing gallium citrate (GaCi) for the treatment of wounds infected with K. pneumoniae. First, the MIC against K. pneumoniae ranged from 0.125 to 2.0 µg/ml GaCi. After this in vitro efficacy was established, two topical formulations with GaCi (0.1% [wt/vol] and 0.3% [wt/vol]) were tested in a murine wound model of MDR K. pneumoniae infection. Gross pathology and histopathology revealed K. pneumoniae-infected wounds appeared to close faster with GaCi treatment and were accompanied by reduced inflammation compared to those of untreated controls. Similarly, quantitative indications of infection remediation, such as reduced weight loss and wound area, suggested that treatment improved outcomes compared to those of untreated controls. Bacterial burdens were measured 1 and 3 days following inoculation, and a 0.5 to 1.5 log reduction of CFU was observed. Lastly, upon scanning electron microscopy analysis, GaCi treatment appeared to prevent biofilm formation on dressings compared to those of untreated controls. These results suggest that with more preclinical testing, a topical application of GaCi may be a promising alternative treatment strategy for K. pneumoniae SSTI.

Validation of a novel murine wound model of Acinetobacter baumannii infection.

Patients recovering from traumatic injuries or surgery often require weeks to months of hospitalization, increasing the risk for wound and surgical site infections caused by ESKAPE pathogens, which include A. baumannii (the ESKAPE pathogens are Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species). As new therapies are being developed to counter A. baumannii infections, animal models are also needed to evaluate potential treatments. Here, we present an excisional, murine wound model in which a diminutive inoculum of a clinically relevant, multidrug-resistant A. baumannii isolate can proliferate, form biofilms, and be effectively treated with antibiotics. The model requires a temporary, cyclophosphamide-induced neutropenia to establish an infection that can persist. A 6-mm-diameter, full-thickness wound was created in the skin overlying the thoracic spine, and after the wound bed was inoculated, it was covered with a dressing for 7 days. Uninoculated control wounds healed within 13 days, whereas infected, placebo-treated wounds remained unclosed beyond 21 days. Treated and untreated wounds were assessed with multiple quantitative and qualitative techniques that included gross pathology, weight loss and recovery, wound closure, bacterial burden, 16S rRNA community profiling, histopathology, peptide nucleic acid-fluorescence in situ hybridization, and scanning electron microscopy assessment of biofilms. The range of differences that we are able to identify with these measures in antibiotic- versus placebo-treated animals provides a clear window within which novel antimicrobial therapies can be assessed. The model can be used to evaluate antimicrobials for their ability to reduce specific pathogen loads in wounded tissues and clear biofilms. Ultimately, the mouse model approach allows for highly powered studies and serves as an initial multifaceted in vivo assessment prior to testing in larger animals.

Protocol Improvement for RNA Extraction From Compromised Frozen Specimens Generated in Austere Conditions: A Path Forward to Transcriptomics-Pathology Systems Integration.

At the heart of the phenome-to-genome approach is high throughput assays, which are liable to produce false results. This risk can be mitigated by minimizing the sample bias, specifically, recycling the same tissue specimen for both phenotypic and genotypic investigations. Therefore, our aim is to suggest a methodology of obtaining robust results from frozen specimens of compromised quality, particularly if the sample is produced in conditions with limited resources. For example, generating samples at the International Space Station (ISS) is challenging because the time and laboratory footprint allotted to a project can get expensive. In an effort to be economical with available resources, snap-frozen euthanized mice are the straightforward solution; however, this method increases the risk of temperature abuse during the thawing process at the beginning of the tissue collection. We found that prolonged immersion of snap frozen mouse carcass in 10% neutral buffered formalin at 4°C yielded minimal microscopic signs of ice crystallization and delivered tissues with histomorphology that is optimal for hematoxylin and eosin (H&E) staining and fixation on glass slides. We further optimized a method to sequester the tissue specimen from the H&E slides using an incubator shaker. Using this method, we were able to recover an optimal amount of RNA that could be used for downstream transcriptomics assays. Overall, we demonstrated a protocol that enables us to maximize scientific values from tissues collected in austere condition. Furthermore, our protocol can suggest an improvement in the spatial resolution of transcriptomic assays.

Characterization and treatment of spontaneous recurrent seizures following nerve agent-induced status epilepticus in mice.

PURPOSE: To develop and characterize a mouse model of spontaneous recurrent seizures following nerve agent-induced status epilepticus (SE) and test the efficacy of existing antiepileptic drugs. METHODS: SE was induced in telemeterized male C57Bl6/J mice by soman exposure, and electroencephalographic activity was recorded for 4-6 weeks. Mice were treated with antiepileptic drugs (levetiracetam, valproic acid, phenobarbital) or corresponding vehicles for 14 d after exposure, followed by 14 d of drug washout. Survival, body weight, seizure characteristics, and histopathology were used to characterize the acute and chronic effects of nerve agent exposure and to evaluate the efficacy of treatments in mitigating or preventing neurological effects. RESULTS: Spontaneous recurrent seizures manifested in all survivors, but the number and frequency of seizures varied considerably among mice. In untreated mice, seizures became longer over time. Moderate to severe histopathology was observed in the amygdala, piriform cortex, and CA1. Levetiracetam provided modest improvements in neurological parameters such as reduced spike rate and improved histopathology scores, whereas valproic acid and phenobarbital were largely ineffective. CONCLUSIONS: This model of post-SE spontaneous recurrent seizures differs from other experimental models in the brief latency to seizure development, the occurrence of seizures in 100 % of exposed animals, and the lack of damage to CA4/dentate gyrus. It may serve as a useful tool for rapidly and efficiently screening novel therapies that would be effective against severe epilepsy cases.

A Porcine Wound Model of Acinetobacter baumannii Infection.

Objective: To better understand Acinetobacter baumannii pathogenesis and to advance drug discovery against this pathogen, we developed a porcine, full-thickness, excisional, monospecies infection wound model. Approach: The research was facilitated with AB5075, a previously characterized, extensively drug-resistant A. baumannii isolate. The model requires cyclophosphamide-induced neutropenia to establish a skin and soft tissue infection (SSTI) that persists beyond 7 days. Multiple, 12-mm-diameter full-thickness wounds were created in the skin overlying the cervical and thoracic dorsum. Wound beds were inoculated with 5.0 × 104 colony-forming units (CFU) and covered with dressing. Results: A. baumannii was observed in the wound bed and on the dressing in what appeared to be biofilm. When bacterial burdens were measured, proliferation to at least 106 CFU/g (log106) wound tissue was observed. Infection was further characterized by scanning electron microscopy (SEM) and peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) staining. To validate as a treatment model, polymyxin B was applied topically to a subset of infected wounds every 2 days. Then, the treated and untreated wounds were compared using multiple quantitative and qualitative techniques to include gross pathology, CFU burden, histopathology, PNA-FISH, and SEM. Innovation: This is the first study to use A. baumannii in a porcine model as the sole infectious agent. Conclusion: The porcine model allows for an additional preclinical assessment of antibacterial candidates that show promise against A. baumannii in rodent models, further evaluating safety and efficacy, and serve as a large animal in preclinical assessment for the treatment of SSTI.

The Galleria mellonella larvae as an in vivo model for evaluation of Shigella virulence.

Shigella spp. causing bacterial diarrhea and dysentery are human enteroinvasive bacterial pathogens that are orally transmitted through contaminated food and water and cause bacillary dysentery. Although natural Shigella infections are restricted to humans and primates, several smaller animal models are used to analyze individual steps in pathogenesis. No animal model fully duplicates the human response and sustaining the models requires expensive animals, costly maintenance of animal facilities, veterinary services and approved animal protocols. This study proposes the development of the caterpillar larvae of Galleria mellonella as a simple, inexpensive, informative, and rapid in-vivo model for evaluating virulence and the interaction of Shigella with cells of the insect innate immunity. Virulent Shigella injected through the forelegs causes larvae death. The mortality rates were dependent on the Shigella strain, the infectious dose, and the presence of the virulence plasmid. Wild-type S. flexneri 2a, persisted and replicated within the larvae, resulting in haemocyte cell death, whereas plasmid-cured mutants were rapidly cleared. Histology of the infected larvae in conjunction with fluorescence, immunofluorescence, and transmission electron microscopy indicate that S. flexneri reside within a vacuole of the insect haemocytes that ultrastructurally resembles vacuoles described in studies with mouse and human macrophage cell lines. Some of these bacteria-laden vacuoles had double-membranes characteristic of autophagosomes. These results suggest that G. mellonella larvae can be used as an easy-to-use animal model to understand Shigella pathogenesis that requires none of the time and labor-consuming procedures typical of other systems.

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model.

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.

AB5075, a Highly Virulent Isolate of Acinetobacter baumannii, as a Model Strain for the Evaluation of Pathogenesis and Antimicrobial Treatments.

UNLABELLED: Acinetobacter baumannii is recognized as an emerging bacterial pathogen because of traits such as prolonged survival in a desiccated state, effective nosocomial transmission, and an inherent ability to acquire antibiotic resistance genes. A pressing need in the field of A. baumannii research is a suitable model strain that is representative of current clinical isolates, is highly virulent in established animal models, and can be genetically manipulated. To identify a suitable strain, a genetically diverse set of recent U.S. military clinical isolates was assessed. Pulsed-field gel electrophoresis and multiplex PCR determined the genetic diversity of 33 A. baumannii isolates. Subsequently, five representative isolates were tested in murine pulmonary and Galleria mellonella models of infection. Infections with one strain, AB5075, were considerably more severe in both animal models than those with other isolates, as there was a significant decrease in survival rates. AB5075 also caused osteomyelitis in a rat open fracture model, while another isolate did not. Additionally, a Tn5 transposon library was successfully generated in AB5075, and the insertion of exogenous genes into the AB5075 chromosome via Tn7 was completed, suggesting that this isolate may be genetically amenable for research purposes. Finally, proof-of-concept experiments with the antibiotic rifampin showed that this strain can be used in animal models to assess therapies under numerous parameters, including survival rates and lung bacterial burden. We propose that AB5075 can serve as a model strain for A. baumannii pathogenesis due to its relatively recent isolation, multidrug resistance, reproducible virulence in animal models, and genetic tractability. IMPORTANCE: The incidence of A. baumannii infections has increased over the last decade, and unfortunately, so has antibiotic resistance in this bacterial species. A. baumannii is now responsible for more than 10% of all hospital-acquired infections in the United States and has a >50% mortality rate in patients with sepsis and pneumonia. Most research on the pathogenicity of A. baumannii focused on isolates that are not truly representative of current multidrug-resistant strains isolated from patients. After screening of a panel of isolates in different in vitro and in vivo assays, the strain AB5075 was selected as more suitable for research because of its antibiotic resistance profile and increased virulence in animal models. Moreover, AB5075 is susceptible to tetracycline and hygromycin, which makes it amenable to genetic manipulation. Taken together, these traits make AB5075 a good candidate for use in studying virulence and pathogenicity of this species and testing novel antimicrobials.

Rapid release of tissue enzymes into blood after blast exposure: potential use as biological dosimeters.

Explosive blast results in multiple organ injury and polytrauma, the intensity of which varies with the nature of the exposure, orientation, environment and individual resilience. Blast overpressure alone may not precisely indicate the level of body or brain injury after blast exposure. Assessment of the extent of body injury after blast exposure is important, since polytrauma and systemic factors significantly contribute to blast-induced traumatic brain injury. We evaluated the activity of plasma enzymes including aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and creatine kinase (CK) at different time points after blast exposure using a mouse model of single and repeated blast exposures to assess the severity of injury. Our data show that activities of all the enzymes in the plasma were significantly increased as early as 1 h after blast exposure. The elevated enzyme activity remained up to 6 h in an overpressure dose-dependent manner and returned close to normal levels at 24 h. Head-only blast exposure with body protection showed no increase in the enzyme activities suggesting that brain injury alone does not contribute to the systemic increase. In contrast to plasma increase, AST, ALT and LDH activity in the liver and CK in the skeletal muscle showed drastic decrease at 6 h after blast exposures. Histopathology showed mild necrosis at 6 h and severe necrosis at 24 h after blast exposures in liver and no changes in the skeletal muscle suggesting that the enzyme release from the tissue to plasma is probably triggered by transient cell membrane disruption from shockwave and not due to necrosis. Overpressure dependent transient release of tissue enzymes and elevation in the plasma after blast exposure suggest that elevated enzyme activities in the blood can be potentially used as a biological dosimeter to assess the severity of blast injury.