In Vivo Activity of Repurposed Amodiaquine as a Host-Targeting Therapy for the Treatment of Anthrax.

Anthrax is caused by Bacillus anthracis and can result in nearly 100% mortality due in part to anthrax toxin. Antimalarial amodiaquine (AQ) acts as a host-oriented inhibitor of anthrax toxin endocytosis. Here, we determined the pharmacokinetics and safety of AQ in mice, rabbits, and humans as well as the efficacy in the fly, mouse, and rabbit models of anthrax infection. In the therapeutic-intervention studies, AQ nearly doubled the survival of mice infected subcutaneously with a B. anthracis dose lethal to 60% of the animals (LD60). In rabbits challenged with 200 LD50 of aerosolized B. anthracis, AQ as a monotherapy delayed death, doubled the survival rate of infected animals that received a suboptimal amount of antibacterial levofloxacin, and reduced bacteremia and toxemia in tissues. Surprisingly, the anthrax efficacy of AQ relies on an additional host macrophage-directed antibacterial mechanism, which was validated in the toxin-independent Drosophila model of Bacillus infection. Lastly, a systematic literature review of the safety and pharmacokinetics of AQ in humans from over 2 000 published articles revealed that AQ is likely safe when taken as prescribed, and its pharmacokinetics predicts anthrax efficacy in humans. Our results support the future examination of AQ as adjunctive therapy for the prophylactic anthrax treatment.

Teixobactin Provides Protection against Inhalation Anthrax in the Rabbit Model.

The use of antibiotics is a vital means of treating infections caused by the bacteria Bacillus (B.) anthracis. Importantly, with the potential future use of multidrug-resistant strains of B. anthracis as bioweapons, new antibiotics are needed as alternative therapeutics. In this blinded study, we assessed the protective efficacy of teixobactin, a recently discovered antibiotic, against inhalation anthrax infection in the adult rabbit model. New Zealand White rabbits were infected with a lethal dose of B. anthracis Ames spores via the inhalation route, and blood samples were collected at various times to assess antigenemia, bacteremia, tissue bacterial load, and antibody production. Treatments were administered upon detection of B. anthracis protective antigen in the animals' sera. For comparison, a fully protective dose of levofloxacin was used as a positive control. Rabbits treated with teixobactin showed 100% survival following infection, and the bacteremia was completely resolved by 24-48 h post-treatment. In addition, the bacterial/spore loads in tissues of the animals treated with teixobactin were either zero or dramatically less relative to that of the negative control animals. Moreover, microscopic evaluation of the tissues revealed decreased pathology following treatment with teixobactin. Overall, these results show that teixobactin was protective against inhalation anthrax infection in the rabbit model, and they indicate the potential of teixobactin as a therapeutic for the disease.

Experimental Infection of Syrian Hamsters With Aerosolized Nipah Virus.

Background: Nipah virus (NiV) is a paramyxovirus (genus Henipavirus) that can cause severe respiratory illness and encephalitis in humans. Transmission occurs through consumption of NiV-contaminated foods, and contact with NiV-infected animals or human body fluids. However, it is unclear whether aerosols derived from aforesaid sources or others also contribute to transmission, and current knowledge on NiV-induced pathogenicity after small-particle aerosol exposure is still limited. Methods: Infectivity, pathogenicity, and real-time dissemination of aerosolized NiV in Syrian hamsters was evaluated using NiV-Malaysia (NiV-M) and/or its recombinant expressing firefly luciferase (rNiV-FlucNP). Results: Both viruses had an equivalent pathogenicity in hamsters, which developed respiratory and neurological symptoms of disease, similar to using intranasal route, with no direct correlations to the dose. We showed that virus replication was predominantly initiated in the lower respiratory tract and, although delayed, also intensely in the oronasal cavity and possibly the brain, with gradual increase of signal in these regions until at least day 5-6 postinfection. Conclusion: Hamsters infected with small-particle aerosolized NiV undergo similar clinical manifestations of the disease as previously described using liquid inoculum, and exhibit histopathological lesions consistent with NiV patient reports. NiV droplets could therefore play a role in transmission by close contact.

Protection against inhalation anthrax by immunization with Salmonella enterica serovar Typhi Ty21a stably producing protective antigen of Bacillus anthracis.

The national blueprint for biodefense concluded that the United States is underprepared for biological threats. The licensed anthrax vaccine absorbed vaccine, BioThrax, requires administration of at least 3-5 intramuscular doses. The anthrax vaccine absorbed vaccine consists of complex cell-free culture filtrates of a toxigenic Bacillus anthracis strain and causes tenderness at the injection site and significant adverse events. We integrated a codon-optimized, protective antigen gene of B. anthracis (plus extracellular secretion machinery), into the chromosome of the licensed, oral, live-attenuated typhoid fever vaccineTy21a to form Ty21a-PA-01 and demonstrated excellent expression of the gene encoding protective antigen. We produced the vaccine in a 10-L fermenter; foam-dried and vialed it, and characterized the dried product. The vaccine retained ~50% viability for 20 months at ambient temperature. Sera from animals immunized by the intraperitoneal route had high levels of anti-protective antigen antibodies by enzyme-linked immunosorbent assay and anthrax lethal toxin-neutralizing activity. Immunized mice were fully protected against intranasal challenge with ~5 LD50 of B. anthracis Sterne spores, and 70% (7/10) of vaccinated rabbits were protected against aerosol challenge with 200 LD50 of B. anthracis Ames spores. There was a significant correlation between protection and antibody levels determined by enzyme-linked immunosorbent assay and toxin-neutralizing activity. These data provide the foundation for achievement of our ultimate goal, which is to develop an oral anthrax vaccine that is stable at ambient temperatures and induces the rapid onset of durable, high-level protection after a 1-week immunization regimen.

Phospholipase A2-activating protein is associated with a novel form of leukoencephalopathy.

Leukoencephalopathies are a group of white matter disorders related to abnormal formation, maintenance, and turnover of myelin in the central nervous system. These disorders of the brain are categorized according to neuroradiological and pathophysiological criteria. Herein, we have identified a unique form of leukoencephalopathy in seven patients presenting at ages 2 to 4 months with progressive microcephaly, spastic quadriparesis, and global developmental delay. Clinical, metabolic, and imaging characterization of seven patients followed by homozygosity mapping and linkage analysis were performed. Next generation sequencing, bioinformatics, and segregation analyses followed, to determine a loss of function sequence variation in the phospholipase A2-activating protein encoding gene (PLAA). Expression and functional studies of the encoded protein were performed and included measurement of prostaglandin E2 and cytosolic phospholipase A2 activity in membrane fractions of fibroblasts derived from patients and healthy controls. Plaa-null mice were generated and prostaglandin E2 levels were measured in different tissues. The novel phenotype of our patients segregated with a homozygous loss-of-function sequence variant, causing the substitution of leucine at position 752 to phenylalanine, in PLAA, which causes disruption of the protein's ability to induce prostaglandin E2 and cytosolic phospholipase A2 synthesis in patients' fibroblasts. Plaa-null mice were perinatal lethal with reduced brain levels of prostaglandin E2 The non-functional phospholipase A2-activating protein and the associated neurological phenotype, reported herein for the first time, join other complex phospholipid defects that cause leukoencephalopathies in humans, emphasizing the importance of this axis in white matter development and maintenance.

A Replication-Defective Human Type 5 Adenovirus-Based Trivalent Vaccine Confers Complete Protection against Plague in Mice and Nonhuman Primates.

Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models.

A mutated cholera toxin without the ADP-ribosyltransferase activity induces cytokine production and inhibits apoptosis of splenocytes in mice possibly via toll-like receptor-4 signaling.

Native cholera toxin (CT) and its mutated form (CT-2*) without ADP-ribosyltransferase activity differ in their immunomodulatory effects on host cells, and the mechanisms of these differences are poorly understood. In this study, we demonstrated that CT-2* induced higher levels of cytokine production and down-regulated ex-vivo apoptosis of splenocytes from C57BL/6 mice. After exposure of the splenocytes ex-vivo to CT or CT-2* (2µg/ml) for 48h, CT-2* stimulated expression of the toll-like receptor (TLR-4) gene was much higher and the cells produced increased levels of interleukin (IL)-12, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, compared to splenocytes of mice exposed to native CT. We confirmed these findings by observing that CT-2*, induced much lower levels of IL-12, IFN-γ, and TNF-α in a TLR-4 knockout macrophage cell line derived from C57BL/6 mice. In addition, while CT is known to stimulate apoptosis in splenocytes, we observed that CT-2* significantly down-regulated apoptosis (4.2%), compared to splenocytes exposed to CT (18.7%) or PBS (negative control, 8.5%). On the contrary, we noted both native CT and CT-2* to exhibit similar levels of apoptosis in TLR-4(-/-) cell line. Overall, the evidence supports the conclusion that CT-2* modulated cytokine production and apoptosis in splenocytes of mice possibly through the TLR-4 signaling pathway.

Comparative Burkholderia pseudomallei natural history virulence studies using an aerosol murine model of infection.

Melioidosis is an endemic disease caused by the bacterium Burkholderia pseudomallei. Concerns exist regarding B. pseudomallei use as a potential bio-threat agent causing persistent infections and typically manifesting as severe pneumonia capable of causing fatal bacteremia. Development of suitable therapeutics against melioidosis is complicated due to high degree of genetic and phenotypic variability among B. pseudomallei isolates and lack of data establishing commonly accepted strains for comparative studies. Further, the impact of strain variation on virulence, disease presentation, and mortality is not well understood. Therefore, this study evaluate and compare the virulence and disease progression of B. pseudomallei strains K96243 and HBPUB10303a, following aerosol challenge in a standardized BALB/c mouse model of infection. The natural history analysis of disease progression monitored conditions such as weight, body temperature, appearance, activity, bacteremia, organ and tissue colonization (pathological and histological analysis) and immunological responses. This study provides a detailed, direct comparison of infection with different B. pseudomallei strains and set up the basis for a standardized model useful to test different medical countermeasures against Burkholderia species. Further, this protocol serves as a guideline to standardize other bacterial aerosol models of infection or to define biomarkers of infectious processes caused by other intracellular pathogens.

Pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor.

Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

Antibacterial role for natural killer cells in host defense to Bacillus anthracis.

Natural killer (NK) cells have innate antibacterial activity that could be targeted for clinical interventions for infectious disease caused by naturally occurring or weaponized bacterial pathogens. To determine a potential role for NK cells in immunity to Bacillus anthracis, we utilized primary human and murine NK cells, in vitro assays, and in vivo NK cell depletion in a murine model of inhalational anthrax. Our results demonstrate potent antibacterial activity by human NK cells against B. anthracis bacilli within infected autologous monocytes. Surprisingly, NK cells also mediate moderate antibacterial effects on extracellular vegetative bacilli but do not have activity against extracellular or intracellular spores. The immunosuppressive anthrax lethal toxin impairs NK gamma interferon (IFN-γ) expression, but neither lethal nor edema toxin significantly alters the viability or cytotoxic effector function of NK cells. Compared to human NK cells, murine NK cells have a similar, though less potent, activity against intracellular and extracellular B. anthracis. The in vivo depletion of murine NK cells does not alter animal survival following intranasal infection with B. anthracis spores in our studies but significantly increases the bacterial load in the blood of infected animals. Our studies demonstrate that NK cells participate in the innate immune response against B. anthracis and suggest that immune modulation to augment NK cell function in early stages of anthrax should be further explored in animal models as a clinical intervention strategy.

Structure-based redesign of an edema toxin inhibitor.

Edema factor (EF) toxin of Bacillus anthracis (NIAID category A), and several other toxins from NIAID category B Biodefense target bacteria are adenylyl cyclases or adenylyl cyclase agonists that catalyze the conversion of ATP to 3',5'-cyclic adenosine monophosphate (cAMP). We previously identified compound 1 (3-[(9-oxo-9H-fluorene-1-carbonyl)-amino]-benzoic acid), that inhibits EF activity in cultured mammalian cells, and reduces diarrhea caused by enterotoxigenic Escherichia coli (ETEC) at an oral dosage of 15µg/mouse. Here, molecular docking was used to predict improvements in potency and solubility of new derivatives of compound 1 in inhibiting edema toxin (ET)-catalyzed stimulation of cyclic AMP production in murine monocyte-macrophage cells (RAW 264.7). Structure-activity relationship (SAR) analysis of the bioassay results for 22 compounds indicated positions important for activity. Several derivatives demonstrated superior pharmacological properties compared to our initial lead compound, and are promising candidates to treat anthrax infections and diarrheal diseases induced by toxin-producing bacteria.

Hemodynamic effects of anthrax toxins in the rabbit model and the cardiac pathology induced by lethal toxin.

Anthrax lethal toxin (LeTx) and edema toxin (EdTx) have been shown to alter hemodynamics in the rodent model, while LeTx primarily is reported to induce extensive tissue pathology. However, the rodent model has limitations when used for comparison to higher organisms such as humans. The rabbit model, on the other hand, has gained recognition as a useful model for studying anthrax infection and its pathophysiological effects. In this study, we assessed the hemodynamic effects of lethal toxin (LeTx) and edema toxin (EdTx) in the rabbit model using physiologically relevant amounts of the toxins. Moreover, we further examine the pathological effects of LeTx on cardiac tissue. We intravenously injected Dutch-belted rabbits with either low-dose and high-dose recombinant LeTx or a single dose of EdTx. The animals' heart rate and mean arterial pressure were continuously monitored via telemetry until either 48 or 72 h post-challenge. Additional animals challenged with LeTx were used for cardiac troponin I (cTnI) quantitation, cardiac histopathology, and echocardiography. LeTx depressed heart rate at the lower dose and mean arterial pressure (MAP) at the higher dose. EdTx, on the other hand, temporarily intensified heart rate while lowering MAP. Both doses of LeTx caused cardiac pathology with the higher dose having a more profound effect. Lastly, left-ventricular dilation due to LeTx was not apparent at the given time-points. Our study demonstrates the hemodynamic effects of anthrax toxins, as well as the pathological effects of LeTx on the heart in the rabbit model, and it provides further evidence for the toxins' direct impact on the heart.

Telemetric left ventricular monitoring using wireless telemetry in the rabbit model.

BACKGROUND: Heart failure is a critical condition that affects many people and often results from left ventricular dysfunction. Numerous studies investigating this condition have been performed using various model systems. To do so, investigators must be able to accurately measure myocardial performance in order to determine the degree of left ventricular function. In this model development study, we employ a wireless telemetry system purchased from Data Sciences International to continuously assess left ventricular function in the rabbit model. FINDINGS: We surgically implanted pressure-sensitive catheters fitted to wireless radio-transmitters into the left ventricle of Dutch-belted rabbits. Following recovery of the animals, we continuously recorded indices of cardiac contractility and ventricular relaxation at baseline for a given time period. The telemetry system allowed us to continuously record baseline left ventricular parameters for the entire recording period. During this time, the animals were unrestrained and fully conscious. The values we recorded are similar to those obtained using other reported methods. CONCLUSIONS: The wireless telemetry system can continuously measure left ventricular pressure, cardiac contractility, and cardiac relaxation in the rabbit model. These results, which were obtained just as baseline levels, substantiate the need for further validation in this model system of left ventricular assessment.

Cethromycin-mediated protection against the plague pathogen Yersinia pestis in a rat model of infection and comparison with levofloxacin.

The Gram-negative plague bacterium, Yersinia pestis, has historically been regarded as one of the deadliest pathogens known to mankind, having caused three major pandemics. After being transmitted by the bite of an infected flea arthropod vector, Y. pestis can cause three forms of human plague: bubonic, septicemic, and pneumonic, with the latter two having very high mortality rates. With increased threats of bioterrorism, it is likely that a multidrug-resistant Y. pestis strain would be employed, and, as such, conventional antibiotics typically used to treat Y. pestis (e.g., streptomycin, tetracycline, and gentamicin) would be ineffective. In this study, cethromycin (a ketolide antibiotic which inhibits bacterial protein synthesis and is currently in clinical trials for respiratory tract infections) was evaluated for antiplague activity in a rat model of pneumonic infection and compared with levofloxacin, which operates via inhibition of bacterial topoisomerase and DNA gyrase. Following a respiratory challenge of 24 to 30 times the 50% lethal dose of the highly virulent Y. pestis CO92 strain, 70 mg of cethromycin per kg of body weight (orally administered twice daily 24 h postinfection for a period of 7 days) provided complete protection to animals against mortality without any toxic effects. Further, no detectable plague bacilli were cultured from infected animals' blood and spleens following cethromycin treatment. The antibiotic was most effective when administered to rats 24 h postinfection, as the animals succumbed to infection if treatment was further delayed. All cethromycin-treated survivors tolerated 2 subsequent exposures to even higher lethal Y. pestis doses without further antibiotic treatment, which was related, in part, to the development of specific antibodies to the capsular and low-calcium-response V antigens of Y. pestis. These data demonstrate that cethromycin is a potent antiplague drug that can be used to treat pneumonic plague.

Characterization of an F1 deletion mutant of Yersinia pestis CO92, pathogenic role of F1 antigen in bubonic and pneumonic plague, and evaluation of sensitivity and specificity of F1 antigen capture-based dipsticks.

We evaluated two commercial F1 antigen capture-based immunochromatographic dipsticks, Yersinia Pestis (F1) Smart II and Plague BioThreat Alert test strips, in detecting plague bacilli by using whole-blood samples from mice experimentally infected with Yersinia pestis CO92. To assess the specificities of these dipsticks, an in-frame F1-deficient mutant of CO92 (Δcaf) was generated by homologous recombination and used as a negative control. Based on genetic, antigenic/immunologic, and electron microscopic analyses, the Δcaf mutant was devoid of a capsule. The growth rate of the Δcaf mutant generally was similar to that of the wild-type (WT) bacterium at both 26 and 37 °C, although the mutant's growth dropped slightly during the late phase at 37 °C. The Δcaf mutant was as virulent as WT CO92 in the pneumonic plague mouse model; however, it was attenuated in developing bubonic plague. Both dipsticks had similar sensitivities, requiring a minimum of 0.5 µg/ml of purified F1 antigen or 1 × 10(5) to 5 × 10(5) CFU/ml of WT CO92 for positive results, while the blood samples were negative for up to 1 × 10(8) CFU/ml of the Δcaf mutant. Our studies demonstrated the diagnostic potential of two plague dipsticks in detecting capsular-positive strains of Y. pestis in bubonic and pneumonic plague.

Recombinant anthrax toxin receptor-Fc fusion proteins produced in plants protect rabbits against inhalational anthrax.

Inhalational anthrax, a zoonotic disease caused by the inhalation of Bacillus anthracis spores, has a ∼50% fatality rate even when treated with antibiotics. Pathogenesis is dependent on the activity of two toxic noncovalent complexes: edema toxin (EdTx) and lethal toxin (LeTx). Protective antigen (PA), an essential component of both complexes, binds with high affinity to the major receptor mediating the lethality of anthrax toxin in vivo, capillary morphogenesis protein 2 (CMG2). Certain antibodies against PA have been shown to protect against anthrax in vivo. As an alternative to anti-PA antibodies, we produced a fusion of the extracellular domain of human CMG2 and human IgG Fc, using both transient and stable tobacco plant expression systems. Optimized expression led to the CMG2-Fc fusion protein being produced at high levels: 730 mg/kg fresh leaf weight in Nicotiana benthamiana and 65 mg/kg in N. tabacum. CMG2-Fc, purified from tobacco plants, fully protected rabbits against a lethal challenge with B. anthracis spores at a dose of 2 mg/kg body weight administered at the time of challenge. Treatment with CMG2-Fc did not interfere with the development of the animals' own immunity to anthrax, as treated animals that survived an initial challenge also survived a rechallenge 30 days later. The glycosylation of the Fc (or lack thereof) had no significant effect on the protective potency of CMG2-Fc in rabbits or on its serum half-life, which was about 5 days. Significantly, CMG2-Fc effectively neutralized, in vitro, LeTx-containing mutant forms of PA that were not neutralized by anti-PA monoclonal antibodies.

Protection Afforded by Fluoroquinolones in Animal Models of Respiratory Infections with Bacillus anthracis, Yersinia pestis, and Francisella tularensis.

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 µg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

Testing the efficacy and toxicity of adenylyl cyclase inhibitors against enteric pathogens using in vitro and in vivo models of infection.

Enterotoxigenic Escherichia coli (ETEC) produces the ADP-ribosyltransferase toxin known as heat-labile enterotoxin (LT). In addition to the toxic effect of LT resulting in increases of cyclic AMP (cAMP) and disturbance of cellular metabolic processes, this toxin promotes bacterial adherence to intestinal epithelial cells (A. M. Johnson, R. S. Kaushik, D. H. Francis, J. M. Fleckenstein, and P. R. Hardwidge, J. Bacteriol. 191:178-186, 2009). Therefore, we hypothesized that the identification of a compound that inhibits the activity of the toxin would have a suppressive effect on the ETEC colonization capabilities. Using in vivo and in vitro approaches, we present evidence demonstrating that a fluorenone-based compound, DC5, which inhibits the accumulation of cAMP in intoxicated cultured cells, significantly decreases the colonization abilities of adenylyl cyclase toxin-producing bacteria, such as ETEC. These findings established that DC5 is a potent inhibitor both of toxin-induced cAMP accumulation and of ETEC adherence to epithelial cells. Thus, DC5 may be a promising compound for treatment of diarrhea caused by ETEC and other adenylyl cyclase toxin-producing bacteria.

Emergence of anthrax edema toxin as a master manipulator of macrophage and B cell functions.

Anthrax edema toxin (ET), a powerful adenylyl cyclase, is an important virulence factor of Bacillus anthracis. Until recently, only a modest amount of research was performed to understand the role this toxin plays in the organism's immune evasion strategy. A new wave of studies have begun to elucidate the effects this toxin has on a variety of host cells. While efforts have been made to illuminate the effect ET has on cells of the adaptive immune system, such as T cells, the greatest focus has been on cells of the innate immune system, particularly the macrophage. Here we discuss the immunoevasive activities that ET exerts on macrophages, as well as new research on the effects of this toxin on B cells.

Stimulated innate resistance of lung epithelium protects mice broadly against bacteria and fungi.

Pneumonia is a serious problem worldwide. We recently demonstrated that innate defense mechanisms of the lung are highly inducible against pneumococcal pneumonia. To determine the breadth of protection conferred by stimulation of lung mucosal innate immunity, and to identify cells and signaling pathways activated by this treatment, mice were treated with an aerosolized bacterial lysate, then challenged with lethal doses of bacterial and fungal pathogens. Mice were highly protected against a broad array of Gram-positive, Gram-negative, and class A bioterror bacterial pathogens, and the fungal pathogen, Aspergillus fumigatus. Protection was associated with rapid pathogen killing within the lungs, and this effect was recapitulated in vitro using a respiratory epithelial cell line. Gene expression analysis of lung tissue showed marked activation of NF-kappaB, type I and II IFN, and antifungal Card9-Bcl10-Malt1 pathways. Cytokines were the most strongly induced genes, but the inflammatory cytokines TNF and IL-6 were not required for protection. Lung-expressed antimicrobial peptides were also highly up-regulated. Taken together, stimulated innate resistance appears to occur through the activation of multiple host defense signaling pathways in lung epithelial cells, inducing rapid pathogen killing, and conferring broad protection against virulent bacterial and fungal pathogens. Augmentation of innate antimicrobial defenses of the lungs might have therapeutic value for protection of patients with neutropenia or impaired adaptive immunity against opportunistic pneumonia, and for defense of immunocompetent subjects against a bioterror threat or epidemic respiratory infection.