Aerosol Challenge Research Articles

Bronchial lesions of mouse model of asthma are preceded by immune complex vasculitis and induced bronchial associated lymphoid tissue (iBALT)

We systematically examined by immune histology the lungs of some widely used mouse models of asthma. These models include sensitization by multiple intraperitoneal injections of soluble ovalbumin (OVA) or of OVA with alum, followed by three intranasal or aerosol challenges 3 days apart. Within 24 h after a single challenge there is fibrinoid necrosis of arterial walls with deposition of immunoglobulin (Ig) and OVA and infiltration of eosinophilic polymorphonuclear cells that lasts for about 3 days followed by peribronchial B-cell infiltration and slight reversible goblet cell hypertrophy (GCHT). After two challenges, severe eosinophilic vasculitis is present at 6 h, increases by 72 h, and then declines; B-cell proliferation and significant GCHT and hyperplasia (GCHTH) and bronchial smooth muscle hypertrophy recur more prominently. After three challenges, there is significantly increased induced bronchus-associated lymphoid tissue (iBALT) formation, GCHTH, and smooth muscle hypertrophy. Elevated levels of Th2 cytokines, IL-4, IL-5, and IL-13, are present in bronchial lavage fluids. Sensitized mice have precipitating antibody and positive Arthus skin reactions but also develop significant levels of IgE antibody to OVA but only 1 week after challenge. We conclude that the asthma like lung lesions induced in these models is preceded by immune complex-mediated eosinophilic vasculitis and iBALT formation. There are elevations of Th2 cytokines that most likely produce bronchial lesions that resemble human asthma. However, it is unlikely that mast cell-activated atopic mechanisms are responsible as we found only a few presumed mast cells by toluidine blue and metachromatic staining limited to the most proximal part of the main stem bronchus, and none in the remaining main stem bronchus or in the lung periphery.

Characterization of a Cynomolgus Macaque Model of Pneumonic Plague for Evaluation of Vaccine Efficacy.

The efficacy of a recombinant plague vaccine (rF1V) was evaluated in cynomolgus macaques (CMs) to establish the relationship among vaccine doses, antibody titers, and survival following an aerosol challenge with a lethal dose of Yersinia pestis strain Colorado 92. CMs were vaccinated with a range of rF1V doses on a three-dose schedule (days 0, 56, and 121) to provide a range of survival outcomes. The humoral immune response following vaccination was evaluated with anti-rF1, anti-rV, and anti-rF1V bridge enzyme-linked immunosorbent assays (ELISAs). Animals were challenged via aerosol exposure on day 149. Vaccine doses and antibody responses were each significantly associated with the probability of CM survival (P < 0.0001). Vaccination also decreased signs of pneumonic plague in a dose-dependent manner. There were statistically significant correlations between the vaccine dose and the time to onset of fever (P < 0.0001), the time from onset of fever to death (P < 0.0001), the time to onset of elevated respiratory rate (P = 0.0003), and the time to onset of decreased activity (P = 0.0251) postinfection in animals exhibiting these clinical signs. Delays in the onset of these clinical signs of disease were associated with larger doses of rF1V. Immunization with ≥ 12 μg of rF1V resulted in 100% CM survival. Since both the vaccine dose and anti-rF1V antibody titers correlate with survival, rF1V bridge ELISA titers can be used as a correlate of protection.

Intranasal administration of CpG oligodeoxynucleotides reduces lower airway inflammation in a murine model of combined allergic rhinitis and asthma syndrome

Given the relationship between allergic rhinitis (AR) and asthma, it can be hypothesized that reducing upper airway inflammation by targeting oligodeoxynucleotides with CpG motifs (CpG-ODN) specifically to the upper airway via intranasal administration in a small volume (10μL) might improve lower airway (asthma) outcomes. The goal of this study was to investigate the therapeutic efficacy of 10μL of intranasal versus intradermal administration of CpG-ODN in suppressing lower airway inflammation and methacholine-induced airway hyperreactivity (AHR) in mice subjected to ovalbumin (OVA)-induced combined allergic rhinitis and asthma syndrome (CARAS). OVA-sensitized BALB/c mice were subjected to upper-airway intranasal OVA exposure three times per week for 3weeks. Then, CpG-ODN was administered to a subset of these mice 1h after intranasal OVA exposure, followed by five days of OVA aerosol challenges, thereby targeting OVA to the lower airways. Immunologic variables and nasal symptoms were evaluated. The results showed that the CARAS mice exhibited significant increases in bronchoalveolar lavage fluid (BALF) and splenocytes Th2-associated cytokine production, OVA-specific serum IgE, and AHR, as well as nose and lung pathologies. Intranasal administration of CpG-ODN significantly reduced Th2-associated cytokine production, the percentage of eosinophils in the BALF, the IL-4 and IL-5 concentrations in the supernatants of cultured OVA-challenged splenic lymphocytes, the serum OVA-specific IgE levels, the peribronchial inflammation score in the lungs, and the severity of nose pathology and nasal symptoms. However, intradermal administration of CpG-ODN did not significantly reduce the aforementioned parameters. In conclusion, intranasal treatment with CpG-ODN attenuated AR and significantly alleviated lower airway inflammation and AHR in the CARAS model. CpG-ODN therapy was more effective when administered intranasally than when administered intradermally. The current study supports the development of CpG-ODN nasal spray as a novel therapeutic agent for CARAS.

Pathogenesis of Bolivian Hemorrhagic Fever in Guinea Pigs.

Machupo virus, the cause of Bolivian hemorrhagic fever, is a highly lethal viral hemorrhagic fever with no Food and Drug Administration-approved vaccines or therapeutics. This study evaluated the guinea pig as a model using the Machupo virus-Chicava strain administered via aerosol challenge. Guinea pigs (Cavia porcellus) were serially sampled to evaluate the temporal progression of infection, gross and histologic lesions, and sequential changes in serum chemistry and hematology. The incubation period was 5 to 12 days, and complete blood counts revealed leukopenia with lymphopenia and thrombocytopenia. Gross pathologic findings included congestion and hemorrhage of the gastrointestinal mucosa and serosa, noncollapsing lungs with fluid exudation, enlarged lymph nodes, and progressive pallor and friability of the liver. Histologic lesions consisted of foci of degeneration and cell death in the haired skin, liver, pancreas, adrenal glands, lymph nodes, tongue, esophagus, salivary glands, renal pelvis, small intestine, and large intestine. Lymphohistiocytic interstitial pneumonia was also present. Inflammation within the central nervous system, interpreted as nonsuppurative encephalitis, was histologically apparent approximately 16 days postexposure and was generally progressive. Macrophages in the tracheobronchial lymph node, on day 5 postexposure, were the first cells to demonstrate visible viral antigen. Viral antigen was detected throughout the lymphoid system by day 9 postexposure, followed by prominent spread within epithelial tissues and then brain. This study provides insight into the course of Machupo virus infection and supports the utility of guinea pigs as an additional animal model for vaccine and therapeutic development.

Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice.

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.

Immune complex disease in guinea pig lungs: elicitation with pigeon serum.

Immune complex- and T cell-mediated reactions to organic antigens appear to contribute to the pathogenesis of hypersensitivity pneumonitis in humans. Because pigeon serum is one of the reagents used by clinicians to diagnose this disease, we assessed its potential to elicit immune complex-mediated pulmonary inflammation in guinea pigs. Animals were immunized with different concentrations of pigeon serum protein emulsified in complete Freund's adjuvant, and serums were collected at 4-day intervals after the booster injection. The largest amounts of tissue-fixing (IgG1) and complement-fixing (IgG2) antibodies to pigeon serum were detected in guinea pigs immunized with 1.0 mg of pigeon serum protein 20 to 24 days after the secondary immunization. Therefore, the responses of these animals and of recipients of serum from these animals to aerosol challenge with either homologous (pigeon serum) or heterologous (bovine gamma globulin) immunogen was investigated. Actively and passively immunized guinea pigs developed pulmonary inflammation only after exposure to aerosolized pigeon serum. However, lesions were not observed in the lungs of complement-deficient recipients of immune serum that had inhaled homologous immunogen. These observations suggest that such pigeon serum-elicited pulmonary inflammation in guinea pigs is a manifestation of a complement-dependent, humoral-immune mechanism of pathogenesis and thus is consistent with an immune complex disease.

Aerosol Use in the Pulmonary Function Lab.

Aerosolized medications are frequently used in the pulmonary function laboratory. The 2 most common implementations are bronchodilators and bronchial challenge agents. Bronchodilator administration is not well standardized, largely because of the various methods of delivery available for clinical practice. Metered-dose inhalers used with spacer devices are the most common route for bronchodilator administration, but many laboratories use small-volume nebulizers. Interpretation of pre- and post-bronchodilator studies is confounded by the definitions of airway obstruction and bronchodilator responsiveness. Protocols for administering bronchial challenge aerosols (methacholine, mannitol, hypertonic saline) are well defined but are susceptible to some of the same problems that limit comparison of bronchodilator techniques. Bronchial challenges with inhaled aerosols are influenced not only by the delivery device but by the patient's breathing pattern, particularly in protocols that include deep inspiratory efforts.

Effects of Swimming on the Inflammatory and Redox Response in a Model of Allergic Asthma.

In this study we hypothesized that swimming during sensitization phase could result in a preventive effect in mice with allergic asthma. Swiss mice were divided into 4 groups: Control and Swimming (non-sensitized), OVA and OVA+Swimming (sensitized). The allergic inflammation was induced by 2 intraperitoneal injections and 4 aerosol challenges using ovalbumin. Swimming sessions were performed at high intensity over 3 weeks. 48 h after the last challenge mice were euthanized. Swimming decreased OVA-increased total IgE, IL-1, IL-4, IL-5 and IL-6 levels, as well as the number of total cells, lymphocytes and eosinophils in bronchoalveolar lavage fluid, (p<0.05). Simultaneously, swimming also increased IL-10 and glutathione levels in the Swimming and OVA+Swimming groups (p<0.05). The levels of glutathione peroxidase and catalase were increased only in the Swimming group when compared to all groups (p<0.05). 21 days of swimming resulted in an attenuation of pulmonary allergic inflammation followed by an increase of glutathione levels in the OVA group. Swimming only increased the levels of glutathione peroxidase and catalase in non-sensitized mice (p<0.05). These data suggest that the pulmonary anti-inflammatory effects produced by 3 weeks of high-intensity swimming in this model of OVA-induced asthma may be, at least partly, modulated by reduced oxidative stress and increased IL-10 production.

T cell-mediated protection and epitope mapping with a trivalent alphavirus-like replicon particle vaccine (VIR2P.1167)

Abstract Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses cause debilitating and sometimes fatal illness. These pathogens are highly infectious when aerosolized, and thus favorable candidates for weaponization, yet no licensed vaccines exist. In a subcutaneous model of infection protection correlates with neutralizing antibody response, but studies suggest that protection from aerosol challenge is partially mediated by cellular immunity. We have previously shown that a trivalent (VEEV/EEEV/WEEV) virus-like replicon particle (triVRP) vaccine protected mice against lethal aerosol challenge. To investigate the cell-mediated response, splenocytes from triVRP- and mock-vaccinated mice were cultured for 6 days ex vivo in the presence of VEEV, EEEV or WEEV peptides. Fifteen-mer peptide pool libraries against E1 and E2 glycoproteins were used for T cell epitope mapping. Splenocytes were analyzed via flow cytometry for T cell-specific markers and cytokine production. Restimulation with specific regions of the VEEV E2 protein induced a memory CD8+CD44+ T cell response, as well as an increase in proliferating, non-cytokine producing CD8+CD44+ T cells. Thus, triVRP-vaccinated mice developed a cell-mediated, peptide-specific memory response. Studies are ongoing to precisely map T cell epitopes within VEEV, EEEV and WEEV glycoproteins. Future studies will evaluate the importance of these responses to protection from lethal aerosol alphavirus challenge.

C/EBP homologous protein expression in alveolar macrophages promotes airway inflammation in an ovalbumin-based mouse model of asthma (HYP2P.329)

Abstract Lungs from asthmatic subjects are in a state of chronic Th2-driven inflammation with substantial shifts in myeloid cell function. We have recently shown that C/EBP homologous protein (Chop), a transcription cofactor linked with Integrated Stress Responses, is highly expressed in myeloid cells under chronic inflammatory processes. Therefore, we hypothesized that Chop plays a central role in the polarization of Th2 inflammation in a murine aerosol sensitization and challenge model. Increased expression of Chop was observed in alveolar macrophages (AMΦ) from ovalbumin (OVA)-challenged mice, compared to naïve controls. Chop expression appears to be mediated by Th2 cytokines as treatment of naïve AMΦ by a combination of IL-4 and IL-13 promoted a substantial increase in Chop expression. Interestingly, Chop deletion impaired AMΦ M2 polarization upon exposure to IL-4 and IL-13, suggesting an upstream role for Chop in promoting M2 responses. Using an OVA-based mouse model of asthma, we show that systemic Chop deletion blocked airway hyperresponsiveness, eosinophilia, and ovalbumin-specific IgE levels in mice following sensitization and challenge. Moreover, OVA-challenged Chop deficient mice exhibited decreased influx of F4/80+ AMΦ into the lungs accompanied by reduced accumulation of CD4+ T cells and increased numbers of CD8+ T cells. These results indicate a potential role for Chop in AMΦ in promoting Th2 inflammation in asthma.

A novel DNA vaccine against Mycobacterium tuberculosis infection (VAC4P.1109)

Abstract Background: Mycobacterium tuberculosis (M.tb.) pandemic continues to grow together with serious issues of increasing drug resistance, HIV co-infection and mortality. Since the existing BCG vaccine does not provide significant protection in adults, novel protective strategies should be explored with urgency. Methods: DNA vaccines that express TB antigens Ag85B, ESAT-6, Rv2660c and fusion BER, were investigated in BALB/c mice intramuscularly with electroporation (EP). Immunogenicity and efficacy assessments of these DNA vaccines were then determined before or after aerosol challenge of 100CFU M.tb H37Rv strain. Results: Compared with BCG and other DNA/EP vaccines, pBER/EP induced high frequency of Ag85B-specific CD8+ T cells (up to 41%) in tetramer assay. Moreover, pBER/EP also elicited significantly higher frequencies of antigen-specific CD4+ T cells by ELIspot and ICS assays. Surprisingly, these T cell responses were also significantly higher than those induced by a heterologous DNA/EP prime and vaccinia-85B boost regimen. Critically, pBER/EP conferred significantly better protection against M.tb H37Rv challenge when compared to other DNA/EP vaccines, with lower CFU count in the lungs (p&amp;lt;0.01) and the absence of lung pathology. Conclusions: pBER/EP is a promising DNA vaccine candidate that does not have major issues of safety and pre-existing anti-vector immunity (e.g. vaccinia vector). Our findings warrant further investigation of pBER/EP for TB prevention and immunotherapy.

Inhibition of CD23-mediated IgE transcytosis suppresses the initiation and development of airway allergic inflammation (HYP4P.308)

Abstract The human low affinity IgE receptor, CD23 (FceRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains completely unknown whether CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo. To prove this, we first showed that epithelial CD23 in wild-type (WT) mice transcytosed either IgE or ovalbumin (OVA)-IgE complexes across the airway epithelial barrier. Neither type of transcytosis was observed in CD23-knockout (KO) mice. The OVA sensitization and aerosol challenge of the irradiated WT/WT or CD23KO/WT chimeric mice that CD23 expressed on radioresistant airway structural cells, mainly in epithelial cells, resulted in airway eosinophilia, lung damages including collagen deposition and significantly increased goblet cells, and increased airways hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, of the bone-marrow chimeric mice (WT/CD23KO) significantly reduced OVA-driven allergic airway inflammation. Finally inhalation of a CD23-blocking B3B4 antibody to target exposed CD23 in AECs of the sensitized WT mice before airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Our study further suggests that the functional inhibition of CD23 on AECs is a potential therapeutic means to intervene the asthma development.

Immunogenicity and protective efficacy of DMT liposome-adjuvanted tuberculosis subunit CTT3H vaccine

Different strategies have been proposed for the development of protein subunit vaccine candidates for tuberculosis (TB), which shows better safety than other types of candidates and the currently used Bacillus Calmette-Guérin (BCG) vaccine. In order to develop more effective protein subunits depending on the mechanism of cell-mediated immunity against TB, a polyprotein CTT3H, based on 5 immunodominant antigens (CFP10, TB10.4, TB8.4, Rv3615c, and HBHA) with CD8+ epitopes of Mycobacterium tuberculosis, was constructed in this study. We vaccinated C57BL/6 mice with a TB subunit CTT3H protein in an adjuvant of dimethyldioctadecylammonium/monophosphoryl lipid A/trehalose 6,6′-dibehenate (DDA/MPL/TDB, DMT) liposome to investigate the immunogenicity and protective efficacy of this novel vaccine. Our results demonstrated that DMT liposome-adjuvanted CTT3H vaccine not only induced an antigen-specific CD4+ Th1 response, but also raised the number of PPD- and CTT3H-specific IFN-γ+ CD8+ T cells and elicited strong CTL responses against TB10.4, which provided more effective protection against a 60 CFU M. tuberculosis aerosol challenge than PBS control and DMT adjuvant alone. Our findings indicate that DMT-liposome is an effective adjuvant to stimulate CD8+ T cell responses and the DMT-adjuvanted subunit CTT3H vaccine is a promising candidate for the next generation of TB vaccine.

Temporal changes in glutaredoxin 1 and protein s-glutathionylation in allergic airway inflammation.

IntroductionAsthma is a chronic inflammatory disorder of the airways, involving oxidative stress. Upon oxidative stress, glutathione covalently binds to protein thiols to protect them against irreversible oxidation. This posttranslational modification, known as protein S-glutathionylation, can be reversed by glutaredoxin 1 (Glrx1) under physiological condition. Glrx1 is known to increase in the lung tissues of a murine model of allergic airway inflammation. However, the temporal relationship between levels of Glrx1, protein S-glutathionylation, and glutathione in the lungs with allergic airway inflammation is not clearly understood.MethodsBALB/c mice received 3 aerosol challenges with ovalbumin (OVA) following sensitization to OVA. They were sacrificed at 6, 24, 48, or 72 h, or 8 days (5 mice per group), and the levels of Glrx1, protein S-glutathionylation, glutathione, and 25 cytokines/chemokines were evaluated in bronchoalveolar lavage fluid (BALF) and/or lung tissue.ResultsLevels of Glrx1 in BALF were significantly elevated in the OVA 6 h (final challenge) group compared to those in the control, with concurrent increases in protein S-glutathionylation levels in the lungs, as well as total glutathione (reduced and oxidized) and oxidized glutathione in BALF. Protein S-glutathionylation levels were attenuated at 24 h, with significant increases in Glrx1 levels in lung tissues at 48 and 72 h. Glrx1 in alveolar macrophages was induced after 6 h. Glrx1 levels concomitantly increased with Th2/NF-κB-related cytokines and chemokines in BALF.ConclusionsThe temporal relationships of Glrx1 with protein S-glutathionylation, glutathione, and cytokines/chemokines were observed as dynamic changes in lungs with allergic airway inflammation, suggesting that Glrx1 and protein–SSG redox status may play important roles in the development of allergic airway inflammation.

Effects of Swimming on the Inflammatory and Redox Response in a Model of Allergic Asthma.

In this study we hypothesized that swimming during sensitization phase could result in a preventive effect in mice with allergic asthma. Swiss mice were divided into 4 groups: Control and Swimming (non-sensitized), OVA and OVA+Swimming (sensitized). The allergic inflammation was induced by 2 intraperitoneal injections and 4 aerosol challenges using ovalbumin. Swimming sessions were performed at high intensity over 3 weeks. 48 h after the last challenge mice were euthanized. Swimming decreased OVA-increased total IgE, IL-1, IL-4, IL-5 and IL-6 levels, as well as the number of total cells, lymphocytes and eosinophils in bronchoalveolar lavage fluid, (p<0.05). Simultaneously, swimming also increased IL-10 and glutathione levels in the Swimming and OVA+Swimming groups (p<0.05). The levels of glutathione peroxidase and catalase were increased only in the Swimming group when compared to all groups (p<0.05). 21 days of swimming resulted in an attenuation of pulmonary allergic inflammation followed by an increase of glutathione levels in the OVA group. Swimming only increased the levels of glutathione peroxidase and catalase in non-sensitized mice (p<0.05). These data suggest that the pulmonary anti-inflammatory effects produced by 3 weeks of high-intensity swimming in this model of OVA-induced asthma may be, at least partly, modulated by reduced oxidative stress and increased IL-10 production.

Inhibition of CD23-mediated IgE transcytosis suppresses the initiation and development of allergic airway inflammation.

The epithelial lining of the airway tract and allergen-specific IgE are considered essential controllers of inflammatory responses to allergens. The human low affinity IgE receptor, CD23 (FcεRII), is capable of transporting IgE or IgE-allergen complexes across the polarized human airway epithelial cell (AEC) monolayer in vitro. However, it remains unknown whether the CD23-dependent IgE transfer pathway in AECs initiates and facilitates allergic inflammation in vivo, and whether inhibition of this pathway attenuates allergic inflammation. To this end, we show that in wild-type (WT) mice, epithelial CD23 transcytosed both IgE and ovalbumin (OVA)-IgE complexes across the airway epithelial barrier, while neither type of transcytosis was observed in CD23 knockout (KO) mice. In chimeric mice, OVA sensitization and aerosol challenge of WT/WT (bone-marrow transfer from the WT to WT) or CD23KO/WT (CD23KO to WT) chimeric mice, which express CD23 on radioresistant airway structural cells (mainly epithelial cells) resulted in airway eosinophilia, including collagen deposition and a significant increase in goblet cells, and increased airway hyperreactivity. In contrast, the absence of CD23 expression on airway structural or epithelial cells, but not on hematopoietic cells, in WT/CD23KO (the WT to CD23KO) chimeric mice significantly reduced OVA-driven allergic airway inflammation. In addition, inhalation of the CD23-blocking B3B4 antibody in sensitized WT mice before or during airway challenge suppressed the salient features of asthma, including bronchial hyperreactivity. Taken together, these results identify a previously unproven mechanism in which epithelial CD23 plays a central role in the development of allergic inflammation. Further, our study suggests that functional inhibition of CD23 in the airway is a potential therapeutic approach with which to inhibit the development of asthma.

Genetic Deletion of β-Arrestin-2 and the Mitigation of Established Airway Hyperresponsiveness in a Murine Asthma Model

β-Arrestin-2 (βarr2) is a ubiquitously expressed cytosolic protein that terminates G protein-coupled receptor signaling and transduces G protein-independent signaling. We previously showed that mice lacking βarr2 do not develop an asthma phenotype when sensitized to, and challenged with, allergens. The current study evaluates if an established asthma phenotype can be mitigated by deletion of βarr2 using an inducible Cre recombinase. We sensitized and challenged mice to ovalbumin (OVA) and demonstrated that on Day (d) 24 the allergic asthma phenotype was apparent in uninduced βarr2 and wild-type (WT) mice. In a second group of OVA-treated mice, tamoxifen was injected on d24 to d28 to activate Cre recombinase, and OVA aerosol challenge was continued through d44. The asthma phenotype was assessed using lung mechanics measurements, bronchoalveolar lavage cell analysis, and histological assessment of mucin and airway inflammation. Compared with their respective saline-treated controls, OVA-treated WT mice and mice expressing the inducible Cre recombinase displayed a significant asthma phenotype at d45. Whereas tamoxifen treatment had no significant effect on the asthma phenotype in WT mice, it inhibited βarr2 expression and caused a significant reduction in airway hyper-responsiveness (AHR) in Cre-inducible mice. These findings suggest that βarr2 is actively required for perpetuation of the AHR component of the allergic asthma phenotype. Our finding that βarr2 participates in the perpetuation of AHR in an asthma model means that targeting βarr2 may provide immediate and potentially long-term relief from daily asthma symptoms due to AHR irrespective of inflammation.

Efficacy of experimental inactivated and live Rhodococcus equivaccines for thoroughbred Arabian mares in mice

The aim of this study was to determine the efficacy of inactive Rhodococcus equi vaccine candidates included that bacterin+aluminum hydroxide (Al(OH) 3 ), bacterin+VapA+(Al(OH) 3 ), bacterin+Montanide IMS 3012 (IMS), bacterin+VapA+IMS, and live vaccine using mice as a model. The efficacy of vaccine was evaluated according to clinical findings, humoral and cellular immunity (levels of INF-g and IL-4), and results of microbiological culture from internal organs in dead or sacrificed mice. Inactive R. equi vaccines were subcutaneously administered to mice three times at 15-day intervals and live vaccine was intraperitoneally injected once. Fifteen days after the last vaccination, aerosol challenges were carried out with the pathogenic R. equi VapA + K2002 strain in all groups. Two mice were sacrificed from each challenge groups on days 1, 3, 5, and 7. The antibody titers of vaccinated mice were found to be significantly higher than those of the controls. The largest number of INF-g positive samples were detected in the bacterin+VapA+IMS and bacterin+IMS groups. IL-4 positivity was determined only in live vaccine groups. The lowest reisolation rate of R. equi from internal organs was observed in the bacterin+VapA+IMS group. It was concluded that R. equi vaccines, and especially bacterin+VapA+IMS, are useful to protect mice against R. equi infection.

First genomic analysis of dendritic cells from lung and draining lymph nodes in murine asthma.

Asthma is the consequence of allergic inflammation in the lung compartments and lung-draining lymph nodes. Dendritic cells initiate and promote T cell response and drive it to immunity or allergy. However, their modes of action during asthma are poorly understood. In this study, an allergic inflammation with ovalbumin was induced in 38 mice versus 42 control animals. After ovalbumin aerosol challenge, conventional dendritic cells (CD11c/MHCII/CD8) were isolated from the lungs and the draining lymph nodes by means of magnetic cell sorting followed by fluorescence-activated cell sorting. A comparative transcriptional analysis was performed using gene arrays. In general, many transcripts are up- and downregulated in the CD8− dendritic cells of the allergic inflamed lung tissue, whereas few genes are regulated in CD8+ dendritic cells. The dendritic cells of the lymph nodes also showed minor transcriptional changes. The data support the relevance of the CD8− conventional dendritic cells but do not exclude distinct functions of the small population of CD8+ dendritic cells, such as cross presentation of external antigen. So far, this is the first approach performing gene arrays in dendritic cells obtained from lung tissue and lung-draining lymph nodes of asthmatic-like mice.

Protection of non-human primates against glanders with a gold nanoparticle glycoconjugate vaccine

The Gram-negative Burkholderia mallei is a zoonotic pathogen and the causative agent of glanders disease. Because the bacteria maintain the potential to be used as a biothreat agent, vaccine strategies are required for human glanders prophylaxis. A rhesus macaque (Macaca mulatta) model of pneumonic (inhalational) glanders was established and the protective properties of a nanoparticle glycoconjugate vaccine composed of Burkholderia thailandensis LPS conjugated to FliC was evaluated. An aerosol challenge dose of ∼1×104CFU B. mallei produced mortality in 50% of naïve animals (n=2/4), 2–3 days post-exposure. Although survival benefit was not observed by vaccination with a glycoconjugate glanders vaccine (p=0.42), serum LPS-specific IgG titers were significantly higher on day 80 in 3 vaccinated animals who survived compared with 3 vaccinated animals who died. Furthermore, B. mallei was isolated from multiple organs of both non-vaccinated survivors, but not from any organs of 3 vaccinated survivors at 30 days post-challenge. Taken together, this is the first time a candidate vaccine has been evaluated in a non-human primate aerosol model of glanders and represents the initial step for consideration in pre-clinical studies.