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1 Department of Microbiology, Molecular Biology, and Biochemistry, University of Idaho, Moscow, ID 83844-3052, USA
2 Department of Statistics, University of Idaho, Moscow, ID 83844-3052, USA
Correspondence
Scott A. Minnich
sminnich{at}uidaho.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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, interferon-; IL, interleukin; i.n., intranasal(ly); i.p., intraperitoneal(ly); PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; TNF-
, tumour necrosis factor-; TTD, time to death |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Although plague incidence has declined dramatically due to public health measures, its ability to be transmitted by aerosol has resulted in designation of Y. pestis as a potential terrorist weapon. A formaldehyde-killed whole-cell vaccine in previous use has been discontinued, in part due to its failure to protect against pneumonic plague (Anderson et al., 1996:, Russell et al., 1995). An improved vaccine employing V and F1 antigens is in clinical trials (Leary et al., 1997; Russell et al., 1995; Williamson et al., 2000, 2007). Nonetheless, mass vaccinations have economic and public health consequences and an effective incidence-response approach with improved therapeutics could be a viable alternative.
The hallmark of a Yersinia infection is the lack of an inflammatory response due to bacterial repression and evasion of innate immunity. The repressive mechanisms responsible depend on bacterial injection of host cells with type III secretion effectors. For example, the Y. pestis secreted effector YopJ is a potent inhibitor of the NF-
B and MAPK signalling pathways (Sweet et al., 2007), and YopE has been shown to strongly inhibit activation of JNK, ERK and NF-B with a coincident suppression of interleukin (IL)-8 production (Viboud & Bliska, 2005; Viboud et al., 2003). The Yersinia V-antigen has been reported to induce IL-10, which represses innate immunity (Brubaker, 2003; Overheim et al., 2005; Philipovskiy et al., 2005) but this activity has been challenged in more recent reports (Pouliot et al., 2007). Other mechanisms include the modification and/or loss of Toll-like receptor (TLR) ligands, denoted as pathogen-associated molecular pattern (PAMP) molecules (Kaufman et al., 2004). Known modifications include loss of flagellin synthesis (TLR5 ligand, Minnich & Rohde, 2007) and temperature-dependent modification of lipid A, the TLR4 ligand (Mukhopadhyay et al., 2004; Rebeil et al., 2004; Roy & Mocarski, 2007).
Lipid A has a conserved molecular backbone consisting of a phosphorylated glucosamine disaccharide and species variation in the amount and length of secondary fatty acid/acyl side chains (Bruneteau & Minka, 2003; Raetz & Whitfield, 2002; Rebeil et al., 2004). Although LPS interacts with TLR4 (Lathem et al., 2005), relatively low LPS concentrations can be pyrogenic, toxic and lethal. Certain synthetic modifications of the lipid A backbone retain TLR4 stimulation but abrogate toxicity. Lipid A mimetics, such as aminoalkyl glucosaminide 4-phosphates (AGPs), vary in the length of secondary acyl side chains and the functional group on the aglycon component. The mechanism of AGP-mediated protection is well documented and involves TLR4 stimulation of chemokine and cytokine expression, leading to a classic Th1 immune response. In murine studies, various AGPs delivered intranasally provided protection against organisms such as Listeria monocytogenes or influenza virus (Cluff et al., 2005) . One of these compounds, CRX-524, and a newer AGP (CRX-527), elicited the highest cytokine responses. These compounds have 10-carbon acyl side chains; CRX-524 has a hydrogen on the aglycon moiety while CRX-527 has a carboxyl group at this position (Cluff et al., 2005).
Considering that dampened innate immunity is a signature of Y. pestis pathogenesis, the present study was designed to ascertain whether artificial induction of innate immunity using lipid A mimetics alters the course of infection. Specifically, the potential application of CRX-524 and CRX-527 against lethal high doses of Y. pestis CO92 in a mouse pneumonic plague model was assessed.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1x108 c.f.u. ml–1) were mixed with glycerol (20 %, v/v) and stored at –80 °C for use in challenge; doses were quantified by plate counts. All experiments were performed under CDC-certified BSL-3 conditions at the University of Idaho.
Animals.
Eight- to ten-week-old pathogen-free female BALB/c mice were obtained from Simonsen Laboratories. C.C3-Tlr4Lps-d/J (background BALB/c) mice were the result of F1–F6 generations of harem-mating male to female C.C3-Tlr4Lps-d/J breeding stock (Jackson Laboratories). Each experimental group was housed in a mouse containment facility (BioZone MiniRack), provided food and water ad libitum, and handled in accordance with the University of Idaho's Animal Care and Use Committee guidelines. Mice were evaluated daily for signs of illness (ruffled fur, apparent weight loss, decreased mobility), and subjects succumbing to infection were promptly removed.
AGPs.
AGPs (CRX-524 and CRX-527) were provided by GlaxoSmithKline and stored at 4 °C in 2 % glycerol. On the day of use, each was diluted to 1 µg µl–1, stored on ice, and used within 5 h. Both compounds were tested for toxicity by intranasal (i.n.) administration of 5–25 µg.
Mouse protection studies.
Timing of experimental treatments was referenced relative to the day of Y. pestis CO92 challenge (see below), which was designated day 0. AGPs were evaluated for their effect when applied on day –1, day +1, or on both days. Mice were anaesthetized with 3 % isoflurane (EZ-Anaesthesia 2000, Euthanex) and AGPs were administered to prone-positioned animals. Animals received an i.n. delivery of either 10 µg or 20 µg CRX-527 and/or CRX-524 in 20 µl sterile 2 % glycerol (10 µl per nostril); controls received PBS. Treated mice were returned to their cages and observed until subsequent experimental procedures. In challenge experiments, an aliquot of Y. pestis CO92 stock culture was thawed and adjusted to the desired concentrations. Anaesthetized mice were challenged with a designated bacterial i.n. dose (5 µl per nostril) or the same volume of PBS. Challenged animals were returned to their cages and observed for distress and death. In a subset of Y. pestis CO92 challenge experiments, gentamicin (Sigma) was administered. The antibiotic (10 mg ml–1 in sterile distilled water) was given twice as 200 µg intraperitoneal (i.p) doses [12 mg (kg body weight)–1], 12 h apart on day +1 or day +2 (Byrne et al., 1998; Frean et al., 2003). Control animals received an equivalent volume of PBS.
Quantification of cytokines and bacteria in lung tissue.
Mice were given an i.n. treatment of 10 µg CRX-524 and 10 µg CRX-527 and at designated times were euthanized with an overdose of sodium pentobarbital (150 mg kg–1). Animal lungs were removed and pooled by group, weighed, and homogenized in 2 ml PBS containing 1 µl Protease Inhibitors Mix ml–1 (Amersham). Homogenates were clarified by centrifugation (16 000 g, 2 min), filter-sterilized, and stored at –80 °C. Filtrates were thawed on ice and analysed for tumour necrosis factor- (TNF-), IL-12p70, and IFN-, using the Quantikine ELISA (R&D Systems). Samples were tested in duplicate, with group mean±SEM reported. To determine the effect of AGP treatment on Y. pestis CO92 levels in lung tissue, mice were given i.n. AGPs or PBS as above, and challenged with Y. pestis CO92 (10 or 100 LD50) 24 h later. Mice were euthanized at various times, lungs were removed, pooled by group, homogenized, and the c.f.u. of Y. pestis per g of tissue was determined by plate counts on BHI agar. Aliquots of lung tissue homogenates from infected animals were also analysed for cytokines as described above.
Statistics.
Chi-squared analyses of treatment group survival data were conducted using the web-based R Projects for Statistical Computing (http://www.r-project.org/index.html), with P values of 
0.05 considered to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Initial experiments showed that the LD50 for Y. pestis CO92 via the i.n. route was 2x104 c.f.u. (results not shown), consistent with the results of other investigators (Andrews et al., 1996; Lathem et al., 2005, 2007). Control animals receiving 
10 LD50 succumbed after 3–4 days (Fig. 1). Symptoms associated with severe infection and impending death included ruffled fur, rapid respiration, anorexia, apparent weight loss and crusty eyes.
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). Therefore, we initially assessed the protective effect of i.n. CRX-524 and/or CRX-527 against three challenge doses (10, 100 or 200 LD50) of Y. pestis CO92 administered 24 h later (Fig. 1
). BALB/c mice treated with CRX-527 (10 µg), or CRX-524 and CRX-527 (10 µg of each) and then challenged with 10 LD50 of Y. pestis CO92 had delayed time to death (TTD) or completely recovered (Fig. 1a). Only 23 % of the control mice receiving PBS survived to day 3; none were alive on day 4. In contrast, 93 % of animals treated with the combined CRX-524 and CRX-527 survived to day 3, 50 % survived to day 4, and 34 % recovered. Seventy per cent of the mice treated with CRX-527 alone survived to day 3 and 28 % survived to day 4. The superiority of combining both AGPs (10 µg each) appears to be a dose effect (see below).
Fig. 1(b) shows that, when challenge doses of 100 LD50 were used, AGP treatment extended TTD and either 20 µg of individual AGPs or CRX-527 and CRX-524 combined (10 µg each) enhanced survival compared to PBS-treated control mice (P<0.01). Although 20 µg CRX-527 generated a slightly higher level of protection, overall there was no statistical difference compared to treatment with 10 µg CRX-527 plus 10 µg CRX-524 combined (P=0.54). At a higher challenge dose (200 LD50), CRX-524 plus CRX-527 (10 µg of each) induced significant delayed TTD compared to control animals (P<0.01) (Fig. 1c). Fifty per cent of treated animals were alive on day 4 compared to 100 % mortality in the control group. Despite the delayed TTD, AGP-treated animals succumbed by day 7.
Treatment with AGPs following bacterial challenge was not protective (Fig. 1d). Animals receiving a single AGP treatment 24 h after challenge (day +1) were not protected and responded similarly to controls receiving only PBS. Animals that received two AGP treatments, one 24 h prior to challenge (day –1), plus one 24 h after challenge (day +1), had a similar response to animals receiving a single day –1 CRX-524 plus CRX-527 treatment. Thus, AGP treatment 24 h prior to bacterial challenge provided pathogen dose-dependent protection against Y. pestis.
AGP-induced protection against pneumonic plague is TLR4-dependent
We assessed whether AGP-induced protection against pneumonic plague is TLR4-dependent by comparing the effects of i.n. AGP treatment in BALB/c TLR4–/– and BALB/c TLR4+/+ mice (Fig. 2). Consistent with data for parental mice in Fig. 1, when administered 24 h prior to a moderate Y. pestis challenge (15 LD50), CRX-524 plus CRX-527 (10 µg each) resulted in a delayed TTD and 10 % survival. In comparison, all TLR4–/– mice succumbed to infection by day 4. Interestingly, 9 of 10 AGP-treated TLR4–/– mice were dead by 2.5 days, representing an accelerated TTD compared to PBS-treated TLR4+/+ animals. TLR4–/– control animals treated with PBS showed the same TTD as AGP-treated TLR4–/– mice (data not shown).
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, 2007). Because AGP pre-treatment enhanced survival and delayed TTD, we examined the effects of 20 µg of combined AGPs (CRX-524 and CRX-527) on Y. pestis bacterial burdens in the mouse lung following identical challenges with 10 LD50 (Fig. 3). Y. pestis numbers in lungs from control animals receiving i.n. PBS remained relatively stable at 1x105 c.f.u. g–1 for the first 24 h and then increased to more than 1x108 c.f.u. g–1 at 48 h. Pooled lung tissue from AGP-treated animals had Y. pestis numbers similar to controls immediately following challenge, but levels dropped to 1x103 c.f.u. g–1 by 24 h. After 48 h, Y. pestis numbers in lungs from AGP-treated mice rose approximately tenfold, but remained four orders of magnitude less than in PBS-treated control animals.
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, IL-12p70 and IFN-. Cytokine expression levels were compared during the course of infection in mice that received a 10 LD50 challenge of Y. pestis CO92 versus mice pre-treated with AGPs 24 h before the 10 LD50 Y. pestis challenge. Additionally, we examined the time-course of cytokine induction in non-infected mice that received AGP treatment (Fig. 4).
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and IFN- had the highest increases (max. 3500 pg and 4000 pg per g lung tissue, respectively), while IL-12p70 levels had a modest rise in infected animals, reaching 250 pg g–1 by 72 h post-infection, the time when most animals succumbed to the infection.
Animals that were primed with 10 µg of each AGP 24 h prior to a Y. pestis 10 LD50 challenge had significantly higher levels of IL-12p70 and IFN- (700 pg and 4500 pg per g lung tissue, respectively) at the onset of infection compared to the untreated animals discussed above. In contrast, the TNF- level rose dramatically in the first 8 h post-AGP treatment, reaching 20 000 pg g–1 but dropped almost tenfold (2000 pg g–1) by the time of i.n. Y. pestis challenge (24 h after AGP application, time 0).
Finally, all three cytokines were assayed in AGP-treated non-infected animals. In general, the same timing was observed for each cytokine induction compared to AGP-treated animals that were infected with 10 LD50 Y. pestis. The TNF- level peaked during the first 8 h after AGP treatment, and then dropped, significantly. Both IL-12p70 and INF- had a sharp rise at 18 h post AGP treatment and showed similar patterns of fluctuation over the next 3 days. However, we also observed a significant difference in the level of cytokines from these animals vs AGP-treated/infected animals, especially during the first hour post-Y. pestis challenge (see Discussion).
AGPs enhance the effectiveness of antimicrobial therapy
The optimal AGP treatment identified in this study (i.n., 10 µg each CRX-524 and CRX-527) provided a delayed TTD or increased survival of animals in a pathogen dose-dependent manner. Although 100 % protection was not provided, even at the lower dose of 10 LD50 (Fig. 1a), the partial protection suggested that interventions with additional therapies, such as antibiotics, during this time might improve survival rates. Byrne et al. (1998) provide evidence that alternative antibiotics, i.e. those currently not specifically FDA-recommended for plague therapy, such as gentamicin and ciprofloxacin, give some protection in Y. pestis-infected mice. For example, gentamicin therapy (12 mg kg–1 every 6 h for 5 days) initiated 42 h after challenge with 100 LD50, resulted in 30 % survival. Because a single AGP treatment provided 15 % protection with this challenge (100 LD50), we examined combined AGP and gentamicin treatments.
Percentage survival was determined after a 200 LD50 Y. pestis challenge in the following groups of animals (n=8): (i) PBS-treated controls, (ii) animals that received two i.p. injections of gentamicin (12 mg kg–1) on day +1 or +2, (iii) animals that received AGPs on day –1, and (iv) animals that received AGPs on day –1 and two i.p. injections of gentamicin (12 mg kg–1) on either day +1 or day +2 post-infection. As shown in Fig. 5, neither AGPs alone nor gentamicin alone provided protection, although some animals had an extended TTD relative to PBS-treated control animals. However, a combination of AGPs provided on day –1 and gentamicin given on either day +1 or day +2 showed 14 % survival at this high challenge dose.
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demonstrated that a second dose of CRX-524 plus CRX-527 on day +1 (24 h post-challenge) provided no additional protection from a challenge of 100 LD50, we repeated this experiment but provided a supplementary i.p. gentamicin treatment (two i.p. injections 12 h apart at 12 mg kg–1 dose) on day +1. This therapeutic regimen produced dramatic results: 100 % of the mice given this treatment were alive on day 7 and 83 % recovered completely (Fig. 6). Providing a single AGP treatment and two doses of gentamicin (as above) on day +1 produced a survival rate of 46 %. These experiments showed that, combined, AGP and gentamicin had a positive synergistic effect.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Prophylactic AGP treatment, specifically a 20 µg combination of CRX-524 and CRX-527, extended TTD or provided protection in a murine pneumonic plague model, depending upon the challenge dose. The observed AGP effects were confirmed to be a consequence of TLR4 signalling. This confirmation was achieved by showing that AGP-pretreated TLR4–/– mice were highly susceptible to 15 LD50 of Y. pestis CO92 when compared to their BALB/c TLR4+/+ counterparts. The TLR4–/– mice did not survive beyond day 3, compared to 50 % survival of the wild-type mice on day 4 and an overall 15 % survival rate.
Treatment with AGPs also showed a dramatic effect on survival of Y. pestis in lung tissue. Lung homogenates of AGP-treated animals challenged 24 h later with 10 LD50 of Y. pestis showed four orders of magnitude fewer organisms per gram of lung tissue compared to AGP-untreated animals at 48 h post-infection. Guthrie et al. (1984) and Sheppard et al. (2005) showed that stimulation of neutrophils with a TLR ligand can result in neutrophil ‘priming’. Priming results in mobilization of secretory vesicles and cytokine secretion, but fails to induce degranulation and reactive oxygen species (ROS) production. Priming promotes recruitment of cytosolic oxidase factors with plasma- and vesicle-membrane bound NADPH-oxidase, resulting in immediate production of ROS upon secondary stimulus. Thus, clearing of Y. pestis following AGP treatment is consistent with AGP-induced activation of neutrophils in lung tissue and perhaps enhanced phagocytosis. More recently, it has been shown that priming of human neutrophils with hexa-acylated LPS derived from Y. pestis grown at 21 °C can overcome the type III secretion system inhibition of ROS production (S. Kobayashi, personal communication). In contrast, neutrophils exposed to tetra-acylated LPS from Y. pestis, the primary form of LPS from cultures grown at 37 °C, showed a marked reduction in ROS production upon secondary stimulation. Montminy et al. (2006) show that artificial expression of hexa-acylated lipid A attenuates Y. pestis virulence. AGP-induced clearing of Y. pestis in lung tissue is consistent with this emerging model of innate immune system priming via TLR activation.
Analysis of lung homogenates from mice pre-treated with AGPs demonstrated an upregulation of the pro-inflammatory cytokines TNF-, IL-12p70 and IFN-. Treatment of human monocytes with either CRX-527 or monophosphoryl lipid A (MPL, the parent molecule of AGPs), has been shown to result in high-level TNF- induction (11-fold increase) through a mechanism that requires TLR4 (Cluff et al., 2005; Martin et al., 2003). The in vivo use of AGPs as TLR4 ligands in this present study is similar to that observed by Honko & Mizel (2004) using the TLR5 ligand flagellin. These authors showed that i.n. flagellin treatment results in a peak of TNF- induction after 4–6 h, followed by a reduction to baseline levels by 24 h. Similarly, we showed that IL-12p70 and IFN- levels in homogenates from AGP-treated lungs increased beginning 18 h after AGP treatment and did not decrease as dramatically as TNF levels. Instead, both IL-12p70 and IFN- were maintained at much higher levels throughout the 72 h time interval measured. Parent et al. (2006) demonstrated that IFN-, TNF- and nitric oxide synthase 2 are necessary to protect against a Y. pestis pneumonic infection in mice while Lathem et al. (2005) showed that naive mice infected with Y. pestis only have an increase in lung inflammatory cytokines IFN-, TNF- and IL-12p70 late in the infection, similar to what we observed in this study among control animals receiving 10 LD50 i.n. We believe that induction of these pro-inflammatory cytokines is consistent with protection against a challenge 24 h following treatment.
IFN- and IL-12p70 cytokine expression in lung homogenates showed a fourfold decrease in the AGP-treated animals immediately after challenge with 10 LD50 Y. pestis CO92. Possible explanations for this decrease include the immunosuppressive activity of the organism, an artificial effect on cytokine levels as a consequence of isoflurane anaesthesia (Flondor et al., 2007), or a combination of both. Preliminary experiments addressing this point indicate that isoflurane exposure 24 h after AGP stimulation does significantly reduce cytokine levels. Should this be the case, AGP protection might be artificially reduced in our model of infection. We are currently examining this effect in more detail.
Although 100 % protection was not achieved by AGP treatment alone, the treatment provided an extended TTD even with high-dose pathogen challenges, thereby providing a window of opportunity for intervention during the early stages of disease. Using controlled conditions, we showed that a subtherapeutic antimicrobial regimen in conjunction with AGPs provided nearly 100 % protection against a challenge with 200 LD50 Y. pestis CO92. Currently, only streptomycin, tetracycline or doxycycline are approved by the United States Food and Drug Administration for treatment of plague, although it has been shown that gentamicin and levofloxacin are equally effective when given early to infected mice (Heine et al., 2007; Inglesby et al., 2000). Previous results demonstrate that aggressive gentamicin treatment at 12 mg (kg body weight)–1 (the dose we employed) every 6 h for 5 days (Byrne et al., 1998) rescues only 30 % of mice suffering from pneumonic plague when given late in the infection. Even when a higher dose of gentamicin (20 mg kg–1) is administered in the same regimen, only 85 % survival is seen (Byrne et al., 1998). In contrast to this intensive injection schedule, we observed dramatic effects when gentamicin was combined with AGP treatment. Using two i.p. injections of gentamicin (12 mg kg–1 dose) 12 h apart combined with AGP treatment, we observed a protection level of 83 % or higher, equivalent to the sole use of a high dose of gentamicin [20 mg (kg body weight)–1] injected every 6 h for 5 days. Other antibiotics such as streptomycin, ciprofloxacin and ofloxacin are more effective (Byrne et al., 1998), suggesting that AGP-mediated TLR4 induction will enhance their effect as well, perhaps by reducing the number of antibiotic doses required. Because Y. pestis is highly amenable to genetic manipulation, which could include introduction of antibiotic resistance, TLR induction may be prudent as an initial therapy if there is a question regarding antibiotic resistance or if antibiotic susceptibility profiles are unknown. In the event of a 50 kg aerosolized release of Y. pestis over a large urban area, 150 000 cases of pneumonic plague are predicted (Frean et al., 2003; Inglesby et al., 2000). Such a scenario would tax supplies of first-line antibiotics, requiring utilization of alternative drugs including gentamicin. Combining antibiotic and AGP therapies would enhance therapeutic options.
When used as the sole treatment, optimal AGP protective activity is time-dependent and effective only when given relatively early in relation to Y. pestis exposure. However, even many animals not receiving prophylactic AGPs were rescued following exposure to 100 LD50. Post-treatment of these animals with AGPs and two injections of subtherapeutic levels of gentamicin rescued 46 % of this population, reducing Y. pestis lethality without protracted and intensive treatment. Individuals receiving lower doses of Y. pestis would be expected to fare much better with a combined AGP and antibiotic post-bacterial exposure therapy.
Our findings are consistent with similar in vivo and in vitro studies showing that protection against bacterial pathogens can be enhanced by IL-12 administration (Pammit et al., 2004) or other TLR inducers (Honko & Mizel, 2004; Montminy et al., 2006). The advantage of inducing the non-specific innate immune response is in its potential for protection against a broad spectrum of infectious agents. Thus, early administration of TLR inducers, such as AGPs, prior to the identification of the specific agent in a suspected release may be advantageous not only against Y. pestis but against other agents as well.
In summary, this study has demonstrated that AGPs could be considered for addition to the repertoire of potential agents for use as prophylactic or post-exposure treatment for pneumonic plague. It has also delineated some of the conditions that may be most appropriate for efficacious use of these compounds. We showed that the effects are dose- (compound and agent) and time-dependent, require TLR4 activation, and that the effects can be enhanced with suboptimal antimicrobial therapy. Ongoing work in our laboratories is aimed at optimizing the conditions to maximize their effectiveness and to evaluate their activity against other potential routes of infection or agents. Because lipid A mimetics are effective vaccine adjuvants, we have also determined that their formulation with Y. pestis protective antigens induces rapid and sustained adaptive immunity.
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ACKNOWLEDGEMENTS |
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Edited by: P. van der Ley
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 13 February 2008;
revised 3 April 2008;
accepted 8 April 2008.
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, n=35), 10 µg CRX-524 plus 10 µg CRX-527 (*, n=29), or PBS (
, n=21) on day –1, and challenged with 10 LD50 Y. pestis CO92 24 h later (day 0). (b) Mice (n=15 per group) were treated i.n. with 20 µg CRX-524 (
), 20 µg CRX-527 (
) or PBS (
) on day –1, and challenged with 100 LD50 Y. pestis CO92 on day 0. On day +1, mice were given a second i.n. dose: 10 µg CRX-524 plus 10 µg CRX-527 (
: n=7) were treated intranasally with 10 µg CRX-524 plus 10 µg CRX-527 (*, 