Antibody Profiling in Na?ve and Semi-immune Individuals Experimentally Challenged with Plasmodium vivax Sporozoites

BackgroundAcquisition of malaria immunity in low transmission areas usually occurs after relatively few exposures to the parasite. A recent Plasmodium vivax experimental challenge trial in malaria naïve and semi-immune volunteers from Colombia showed that all naïve individuals developed malaria symptoms, whereas semi-immune subjects were asymptomatic or displayed attenuated symptoms. Sera from these individuals were analyzed by protein microarray to identify antibodies associated with clinical protection.ConclusionClinical protection against experimental challenge in volunteers with previous P. vivax exposure was associated with elevated pre-existing antibodies, an attenuated serological response to the challenge and reactivity to new antigens.     

B-lymphocytes as Key Players in Chemical-Induced Asthma

T-lymphocytes and B-lymphocytes are key players in allergic asthma, with B-lymphocytes producing antigen-specific immunoglobulins E (IgE). We used a mouse model of chemical-induced asthma and transferred B-lymphocytes from sensitized animals into naïve wild type mice, B-lymphocyte knock-out (B-KO) mice or severe combined immunodeficiency (SCID) mice. On days 1 and 8, BALB/c mice were dermally sensitized with 0.3% toluene diisocyanate (TDI) (20µl/ear). On day 15, mice were euthanized and the auricular lymph nodes isolated. B-lymphocytes (CD19⁺) were separated from the whole cell suspension and 175,000 cells were injected in the tail vein of naïve wild type, B-KO or SCID mice. Three days later, the mice received a single oropharyngeal challenge with 0.01% TDI (20µl) or vehicle (acetone/olive oil (AOO)) (controls). Airway reactivity to methacholine and total and differential cell counts in the bronchoalveolar lavage (BAL) fluid were measured 24 hours after challenge. B-lymphocytes of AOO or TDI-sensitized mice were characterized for the expression of surface markers and production of cytokines. We found that transfer of B-cells obtained from mice dermally sensitized to toluene diisocyanate (TDI) into naïve wild type mice, B-KO mice or SCID mice led, within three days, to an acute asthma-like phenotype after an airway challenge with TDI. This response was specific and independent of IgE. These B-lymphocytes showed antigen presenting capacities (CD80/CD86 and CD40) and consisted of B effector (Be)2- (IL-4) and Be1-lymphocytes (IFN-γ). The transferred B-lymphocytes were visualized near large airways, 24 hours after TDI challenge. Thus, B-lymphocytes can provoke an asthmatic response without the action of T-lymphocytes and without major involvement of IgE.     

20-HETE Mediates Ozone-Induced,Neutrophil-Independent Airway Hyper-Responsiveness in Mice

Background

Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.

Methodology/Principal Findings

Male BALB/c mice were exposed to ozone (3 or 6 ppm) or filtered air (controls) for 2 h. Precision cut lung slices (PCLS; 250 µm thickness) containing an intrapulmonary airway (∼0.01 mm² lumen area) were prepared immediately after exposure or 16 h later. After 24 h, airways were contracted to carbachol (CCh). Log EC₅₀ and E_max values were then calculated by measuring the airway lumen area with respect to baseline. In parallel studies, dexamethasone (2.5 mg/kg), or 1-aminobenzotriazol (ABT) (50 mg/kg) were given intraperitoneal injection to naïve mice 18 h prior to ozone exposure. Indomethacin (10 mg/kg) was administered 2 h prior. Cell counts, cytokine levels and liquid chromatography-mass spectrometry (LC-MS) for lipid analysis were assessed in bronchoalveolar lavage (BAL) fluid from ozone exposed and control mice. Ozone acutely induced AHR to CCh. Dexamethasone or indomethacin had little effect on the ozone-induced AHR; while, ABT, a cytochrome P450 inhibitor, markedly attenuated airway sensitivity. BAL fluid from ozone exposed animals, which did not contain an increase in neutrophils or interleukin (IL)-6 levels, increased airway sensitivity following in vitro incubation with a naïve PCLS. In parallel, significant increases in oxidized lipids were also identified using LC-MS with increases of 20-HETE that were decreased following ABT treatment.

Conclusions/Significance

These data show that ozone acutely induces AHR to CCh independent of inflammation and is insensitive to steroid treatment or cyclooxygenase (COX) inhibition. BAL fluid from ozone exposed mice mimicked the effects of in vivo ozone exposure that were associated with marked increases in oxidized lipids. 20-HETE plays a pivotal role in mediating acute ozone-induced AHR.     

Reference values for exhaled nitric oxide (reveno) study

Background

Human airway surface liquid (ASL) has abundant antimicrobial peptides whose potency increases as the salt concentration decreases. Xylitol is a 5-carbon sugar that has the ability to lower ASL salt concentration, potentially enhancing innate immunity. Xylitol was detected for 8 hours in the ASL after application in airway epithelium in vitro. We tested the airway retention time of aerosolized iso-osmotic xylitol in healthy volunteers.

Methods

After a screening spirometry, volunteers received 10 ml of nebulized 5% xylitol. Bronchoscopy was done at 20 minutes (n = 6), 90 minutes (n = 6), and 3 hours (n = 5) after nebulization and ASL was collected using microsampling probes, followed by bronchoalveolar lavage (BAL). Xylitol concentration was measured by nuclear magnetic resonance spectroscopy and corrected for dilution using urea concentration.

Results

All subjects tolerated nebulization and bronchoscopy well. Mean ASL volume recovered from the probes was 49 ± 23 μl. The mean ASL xylitol concentration at 20, 90, and 180 minutes was 1.6 ± 1.9 μg/μl, 0.6 ± 0.6 μg/μl, and 0.1 ± 0.1 μg/μl, respectively. Corresponding BAL concentration corrected for dilution was consistently lower at all time points. The terminal half-life of aerosolized xylitol obtained by the probes was 45 minutes with a mean residence time of 65 minutes in ASL. Corresponding BAL values were 36 and 50 minutes, respectively.

Conclusion

After a single dose nebulization, xylitol was detected in ASL for 3 hours, which was shorter than our in vitro measurement. The microsampling probe performed superior to BAL when sampling bronchial ASL.     

Intrinsic and antigen-induced airway hyperresponsiveness are the result of diverse physiological mechanisms.

Airway hyperresponsiveness (AHR) is a defining feature of asthma. We have previously shown, in mice sensitized and challenged with antigen, that AHR is attributable to normal airway smooth muscle contraction with exaggerated airway closure. In the present study we sought to determine if the same was true for mice known to have intrinsic AHR, the genetic strain of mice, A/J. We found that A/J mice have AHR characterized by minimal increase in elastance following aerosolized methacholine challenge compared with mice (BALB/c) that have been antigen sensitized and challenged [concentration that evokes 50% change in elastance (PC(50)): 22.9 +/- 5.7 mg/ml for A/J vs. 3.3 +/- 0.4 mg/ml for antigen-challenged and -sensitized mice; P < 0.004]. Similar results were found when intravenous methacholine was used (PC(30) 0.22 +/- 0.08 mg/ml for A/J vs. 0.03 +/- 0.004 mg/ml for antigen-challenged and -sensitized mice). Computational model analysis revealed that the AHR in A/J mice is dominated by exaggerated airway smooth muscle contraction and that when the route of methacholine administration was changed to intravenous, central airway constriction dominates. Absorption atelectasis was used to provide evidence of the lack of airway closure in A/J mice. Bronchoconstriction during ventilation with 100% oxygen resulted in a mean 9.8% loss of visible lung area in A/J mice compared with 28% in antigen-sensitized and -challenged mice (P < 0.02). We conclude that the physiology of AHR depends on the mouse model used and the route of bronchial agonist administration.     

AEOL10150: a novel therapeutic for rescue treatment after toxic gas lung injury

New therapeutics designed as rescue treatments after toxic gas injury such as from chlorine (Cl(2)) are an emerging area of interest. We tested the effects of the metalloporphyrin catalytic antioxidant AEOL10150, a compound that scavenges peroxynitrite, inhibits lipid peroxidation, and has SOD and catalase-like activities, on Cl(2)-induced airway injury. Balb/C mice received 100ppm Cl(2) gas for 5 min. Four groups were studied: Cl(2) only, Cl(2) followed by AEOL10150 1 and 9 h after exposure, AEOL10150 only, and control. Twenty-four hours after Cl(2) gas exposure airway responsiveness to aerosolized methacholine (6.25-50mg/ml) was measured using a small-animal ventilator. Bronchoalveolar lavage (BAL) was performed to assess airway inflammation and protein. Whole lung tissue was assayed for 4-hydroxynonenal. In separate groups, lungs were collected at 72 h after Cl(2) injury to evaluate epithelial cell proliferation. Mice exposed to Cl(2) showed a significantly higher airway resistance compared to control, Cl(2)/AEOL10150, or AEOL10150-only treated animals in response to methacholine challenge. Eosinophils, neutrophils, and macrophages were elevated in BAL of Cl(2)-exposed mice. AEOL10150 attenuated the increases in neutrophils and macrophages. AEOL10150 prevented Cl(2)-induced increase in BAL fluid protein. Chlorine induced an increase in the number of proliferating airway epithelial cells, an effect AEOL10150 attenuated. 4-Hydroxynonenal levels in the lung were increased after Cl(2) and this effect was prevented with AEOL10150. AEOL10150 is an effective rescue treatment for Cl(2)-induced airway hyperresponsiveness, airway inflammation, injury-induced airway epithelial cell regeneration, and oxidative stress.     

B Cells Play Key Roles in Th2-Type Airway Immune Responses in Mice Exposed to Natural Airborne Allergens

Humans are frequently exposed to various airborne allergens. In addition to producing antibodies, B cells participate in immune responses via various mechanisms. The roles of B cells in allergic airway inflammation and asthma have been controversial. We examined the functional importance of B cells in a mouse model of asthma, in which mice were exposed repeatedly to common airborne allergens. Naïve wild-type BALB/c mice or B cell-deficient JH^−/− mice were exposed intranasally to a cocktail of allergen extracts, including Alternaria, Aspergillus, and house dust mite, every other day for two weeks. Ovalbumin was included in the cocktail to monitor the T cell immune response. Airway inflammation, lung pathology, and airway reactivity were analyzed. The airway exposure of naïve wild type mice to airborne allergens induced robust eosinophilic airway inflammation, increased the levels of Th2 cytokines and chemokines in the lung, and increased the reactivity to inhaled methacholine. These pathological changes and immune responses were attenuated in B cell-deficient JH^−/− mice. The allergen-induced expansion of CD4⁺ T cells was impaired in the lungs and draining lymph nodes of JH^−/− mice. Furthermore, lymphocytes from JH^−/− mice failed to produce Th2 cytokines in response to ovalbumin re-stimulation in vitro. Our results suggest that B cells are required for the optimal development of Th2-type immune responses and airway inflammation when exposed to common airborne allergens. The therapeutic targeting of B cells may be beneficial to treat asthma in certain patients.     

A novel activity on thymocytes cells exerted by the rattlesnake (Crotalus durissus cumanensis) venom

Introduction: The thymus is active mainly during the neonatal and pre-adolescent periods. Objective: To test naïve thymocytes proliferation and monocytes stimulation. Materials and methods: We collected fresh thymus tissue from neonate mice after surgery. Suspension cells were coated onto Ficoll-Hypaque support. The obtained cells (thymocytes) were cultured measuring the proliferation of naïve T cells stimulated by Crotalus durissus cumanensis (Cdc) venom at sub-lethal doses (20 ng). Then, we supplemented the wells with AlamarBlue™ and incubated them for 5 h to test their proliferation. Mononuclear cells from mice peripheral blood were collected and layered onto the support of the Ficoll-Hypaque solution. We added the thymocytes actively dividing (25 x 10⁵ cells) from cultures stimulated with Cdc venom at 20 ng/well to cultured monocytes freshly obtained from the Ficoll-Hypaque separation. Both cell populations were incubated for 36 h until monocytes matured to macrophages.Results: The naïve thymocytes rapidly proliferated after stimulation with the Cdc venom (NTCdc) and these successively induced the maturation and function of monocytes progenitor cells to mature macrophages, which ingested Chinese ink. Conclusions: The naïve thymocytes proliferated by stimulation with the Cdc venom and subsequently the NT/Cdc induced the rapid maturation and function of monocytes progenitor cells becoming mature macrophages with their phenotypic characteristics.     

Tolerance induced by chronic inhaled antigen in a murine asthma model is not mediated by endotoxin

Ovalbumin (OVA)-sensitized wildtype (WT) and endotoxin-resistant (ER) mice developed similar degrees of airways eosinophilia and serum OVA-specific IgE levels after acute aerosolized OVA challenge. WT mice demonstrated methacholine hyperreactivity, whereas ER mice showed no change in responsiveness. With chronic aerosolized OVA challenge, both WT and ER mice developed local tolerance, with resolution of airway eosinophilia but persistence of anti-OVA IgE in serum. Thus, the development of local tolerance with chronic aerosol exposure to OVA is independent of any potential effects of endotoxin in the OVA aerosol solution.     

Abacavir-Reactive Memory T Cells Are Present in Drug Na?ve Individuals

BackgroundFifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population.MethodsTo determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling.ResultsAbacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells.ConclusionsWe propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection.     

Evaluation of the Murine Immune Response to Xenopsylla cheopis Flea Saliva and Its Effect on Transmission of Yersinia pestis

Background/Aims

Arthropod-borne pathogens are transmitted into a unique intradermal microenvironment that includes the saliva of their vectors. Immunomodulatory factors in the saliva can enhance infectivity; however, in some cases the immune response that develops to saliva from prior uninfected bites can inhibit infectivity. Most rodent reservoirs of Yersinia pestis experience fleabites regularly, but the effect this has on the dynamics of flea-borne transmission of plague has never been investigated. We examined the innate and acquired immune response of mice to bites of Xenopsylla cheopis and its effects on Y. pestis transmission and disease progression in both naïve mice and mice chronically exposed to flea bites.

Methods/Principal Findings

The immune response of C57BL/6 mice to uninfected flea bites was characterized by flow cytometry, histology, and antibody detection methods. In naïve mice, flea bites induced mild inflammation with limited recruitment of neutrophils and macrophages to the bite site. Infectivity and host response in naïve mice exposed to flea bites followed immediately by intradermal injection of Y. pestis did not differ from that of mice infected with Y. pestis without prior flea feeding. With prolonged exposure, an IgG1 antibody response primarily directed to the predominant component of flea saliva, a family of 36–45 kDa phosphatase-like proteins, occurred in both laboratory mice and wild rats naturally exposed to X. cheopis, but a hypersensitivity response never developed. The incidence and progression of terminal plague following challenge by infective blocked fleas were equivalent in naïve mice and mice sensitized to flea saliva by repeated exposure to flea bites over a 10-week period.

Conclusions

Unlike what is observed with many other blood-feeding arthropods, the murine immune response to X. cheopis saliva is mild and continued exposure to flea bites leads more to tolerance than to hypersensitivity. The immune response to flea saliva had no detectable effect on Y. pestis transmission or plague pathogenesis in mice.     

Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice

Airway hyperresponsiveness, airway inflammation, and reversible airway obstruction are physiological hallmarks of asthma. These responses are increasingly being studied in murine models of antigen exposure and challenge, using whole body plethysmography to noninvasively assess airway hyperresponsiveness. This approach infrequently has been correlated with indexes of airway hyperresponsiveness measured by invasive means. Furthermore, correlation with quantitative histological data for tissue infiltration by inflammatory and immune cells, particularly in the wall of airways, during daily airway challenge is lacking. To address these uncertainties, we used C57BL/6 mice that were immunized with ovalbumin or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle 8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14-22. We assessed airway hyperresponsiveness to methacholine noninvasively on days 14, 15, 18, or 22; we studied the same mice 24 h later while they were anesthetized for invasive analyses of airway hyperresponsiveness. Plasma total IgE concentration was significantly higher in the ovalbumin-treated mice compared with the vehicle-treated mice, but this did not correlate with eosinophil number. Peak airway hyperresponsiveness measured by either approach correlated early during daily antigen challenge (days 14 and 15), but this correlation was lost later during subsequent daily antigen challenges (days 18 and 22). On days 14 and 15, peak airway hyperresponsiveness correlated with transmigration of neutrophils and macrophages, but not lymphocytes, in the peribronchovascular connective tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between noninvasive and invasive measures of peak airway hyperresponsiveness was inconsistent.     

Repeated antigen inhalation results in a prolonged airway eosinophilia and airway hyperresponsiveness in primates

The effects of repeated antigen inhalation on airway cellular composition and airway responsiveness were examined in primates. Airway cellular composition was assessed by bronchoalveolar lavage (BAL), and airway responsiveness was measured as the bronchoconstrictor response to cumulative methacholine dose-response determinations over the course of a 10-wk study. Control animals, exposed to repeated vehicle inhalation challenges, were tested in parallel with the antigen-challenged group. Repeated antigen inhalation resulted in a prolonged inflammatory reaction characterized by a large increase in airway eosinophils (3 +/- 1 to 59 +/- 15%, P less than 0.01). Airway eosinophilia was associated with an increase in airway responsiveness as indicated by a leftward shift in the methacholine dose-response curves, an increase in the slope of the dose-response curves, and a decrease in PC100 values (the dose of methacholine required to cause a 100% increase in lung resistance). The number of BAL eosinophils and the level of eosinophil major basic protein in BAL correlated significantly with methacholine PC100 values (r = 0.61, P less than 0.01 and r = 0.64, P less than 0.01, respectively). Histological examination of lung biopsy samples taken at week 10 of the study demonstrated a striking infiltration of eosinophils in the antigen-challenged animals. These results support earlier observations that demonstrated an association between increases in airway eosinophils and increases in airway responsiveness and suggest that eosinophils are involved in the pathogenesis of hyperresponsive airways.     

Altered allergen-induced eosinophil trafficking and physiological dysfunction in airways with preexisting virus-induced injury

Although both asthmatics and allergic rhinitics develop an acute inflammatory response to lower airway allergen challenge, only asthmatics experience airway obstruction resulting from chronic environmental allergen exposure. Hypothesizing that asthmatic airways have an altered response to chronic allergic inflammation, we compared the effects of repeated low-level exposures to inhaled Alternaria extract in sensitized rats with preexisting chronic postbronchiolitis airway dysfunction versus sensitized controls with normal airways. Measurements of air space (bronchoalveolar lavage) inflammatory cells, airway goblet cells, airway wall collagen, airway wall eosinophils, airway alveolar attachments, and pulmonary physiology were conducted after six weekly exposures to aerosolized saline or Alternaria extract. Postbronchiolitis rats, but not those starting with normal airways, had persistent increases in airway wall eosinophils, goblet cell hyperplasia in small airways, and loss of lung elastic recoil after repeated exposure to aerosolized Alternaria extract. Despite having elevated airway wall eosinophils, the postbronchiolitis rats had no eosinophils in bronchoalveolar lavage at 5 days after the last allergen exposure, suggesting altered egression of tissue eosinophils into the air space. In conclusion, rats with preexisting airway pathology had altered eosinophil trafficking and allergen-induced changes in airway epithelium and lung mechanics that were absent in sensitized control rats that had normal airways before the allergen exposures.     

Allergen-induced increase in airway responsiveness and inflammation in mild asthma

The relationship between airway responsiveness to methacholine and inflammatory cells in bronchoalveolar lavage (BAL) was determined in patients with history of rhinitis and/or mild bronchial asthma either at baseline (10 patients) or 3-4 h after allergen inhalation challenge (11 patients). At baseline, airway responsiveness did not correlate with any BAL cell population. When data obtained after allergen challenge were included in the analysis, 44% of the variability of airway responsiveness was explained by a multiple regression model with BAL eosinophils as a directly correlated (P = 0.002) independent variable and with BAL macrophages as an inversely correlated (P = 0.045) independent variable. Changes in airway responsiveness after allergen challenge were predicted (82% of variance explained) by a model with BAL eosinophils and BAL lymphocytes as directly correlated (P = 0.0002 and P = 0.03, respectively) independent variables. We conclude that, in stable asymptomatic asthma, baseline airway responsiveness does not correlate with the presence in the airways of inflammatory and immunoeffector cells that can be recovered by BAL. Nevertheless the allergen-induced increase in airway responsiveness is associated with an influx of eosinophils and lymphocytes in the bronchial lumen.     

Prior Infection of Chickens with H1N1 or H1N2 Avian Influenza Elicits Partial Heterologous Protection against Highly Pathogenic H5N1

There is a critical need to have vaccines that can protect against emerging pandemic influenza viruses. Commonly used influenza vaccines are killed whole virus that protect against homologous and not heterologous virus. Using chickens we have explored the possibility of using live low pathogenic avian influenza (LPAI) A/goose/AB/223/2005 H1N1 or A/WBS/MB/325/2006 H1N2 to induce immunity against heterologous highly pathogenic avian influenza (HPAI) A/chicken/Vietnam/14/2005 H5N1. H1N1 and H1N2 replicated in chickens but did not cause clinical disease. Following infection, chickens developed nucleoprotein and H1 specific antibodies, and reduced H5N1 plaque size in vitro in the absence of H5 neutralizing antibodies at 21 days post infection (DPI). In addition, heterologous cell mediated immunity (CMI) was demonstrated by antigen-specific proliferation and IFN-γ secretion in PBMCs re-stimulated with H5N1 antigen. Following H5N1 challenge of both pre-infected and naïve controls chickens housed together, all naïve chickens developed acute disease and died while H1N1 or H1N2 pre-infected chickens had reduced clinical disease and 70–80% survived. H1N1 or H1N2 pre-infected chickens were also challenged with H5N1 and naïve chickens placed in the same room one day later. All pre-infected birds were protected from H5N1 challenge but shed infectious virus to naïve contact chickens. However, disease onset, severity and mortality was reduced and delayed in the naïve contacts compared to directly inoculated naïve controls. These results indicate that prior infection with LPAI virus can generate heterologous protection against HPAI H5N1 in the absence of specific H5 antibody.     

Infectious Prions in Pre-Clinical Deer and Transmission of Chronic Wasting Disease Solely by Environmental Exposure

Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.     

The effects of inhaled house dust mite on airway barrier function and sensitivity to inhaled methacholine in mice

Asthma is functionally characterized by increased airway sensitivity and reactivity. Multiple mechanisms are believed to underlie these functional disorders, including impairment of airway wall barrier function. One proposed mechanism of impaired barrier function is through the direct consequence of proteolytic properties of inhaled allergens, including house dust mite (HDM). Here, we have observed the direct effects of HDM on airway barrier function and response to nebulized or intravenous methacholine. HDM na?ve BALB/c mice were anesthetized, exposed to intranasal or intratracheal HDM (15 or 100 μg), and allowed to recover for 30 min or 2 h before methacholine challenge. A separate group of mice was exposed to intratracheal poly-L-lysine (PLL; 100 μg) for a duration of 30 min. This group served as a positive control for the presence of impaired barrier function and airway hypersensitivity. Negative control mice received saline challenges. Outcomes included assessment of lung mechanics in response to nebulized or intravenous methacholine as well as clearance of intratracheally instilled technetium-labeled ((99m)Tc) DTPA to evaluate airway epithelial barrier function. We found that PLL produced a leftward shift in the dose-response curve following nebulized but not intravenous methacholine challenge. This was associated with a significantly faster clearance of (99m)Tc-DTPA, indicating impairment in airway barrier function. However, HDM exposure did not produce changes in these outcomes when compared with saline-exposed mice. These findings suggest that direct impact on airway barrier function does not appear to be a mechanism by which HDM produces altered airway sensitivity in airway disease.