Ascaris Suum Antigen Research Articles

The long-lasting Ascaris suum antigens in the lungs shapes the tissue adaptation modifying the pulmonary architecture and immune response after infection in mice

Ascariasis is the most prevalent helminth affecting approximately 819 million people worldwide. The acute phase of Ascariasis is characterized by larval migration of Ascaris spp., through the intestinal wall, carried to the liver and lungs of the host by the circulatory system. Most of the larvae subsequently transverse the lung parenchyma leading to tissue injury, reaching the airways and pharynx, where they can be expectorated and swallowed back to the gastrointestinal tract, where they develop into adult worms. However, some larvae are trapped in the lung parenchyma inciting an inflammatory response that causes persistent pulmonary tissue damage long after the resolution of infection, which returns to tissue homeostasis. However, the mechanism by which chronic lung disease develops and resolves remains unknown. Here, using immunohistochemistry, we demonstrate that small fragments and larval antigens of Ascaris suum are deposited and retained chronically in the lung parenchyma of mice following a single Ascaris infection. Our results reveal that the prolonged presence of Ascaris larval antigens in the lung parenchyma contributes to the persistent immune stimulation inducing histopathological changes observed chronically following infection, and clearly demonstrate that larval antigens are related to all phases of tissue adaptation after infection: lung injury, chronic inflammation, resolution, and tissue remodeling, in parallel to increased specific humoral immunity and the recovery of lung function in mice. Additional insight is needed into the mechanisms of Ascaris antigen to induce chronic immune responses and resolution in the host lungs following larval migration.

An innovative approach in the detection of Toxocara canis excretory/secretory antigens using specific nanobodies

Human toxocariasis is a zoonosis resulting from the migration of larval stages of the dog parasite Toxocara canis into the human paratenic host. Despite its well-known limitations, serology remains the most important tool to diagnose the disease. Our objective was to employ camelid single domain antibody fragments also known as nanobodies (Nbs) for a specific and sensitive detection of Toxocara canis excretory/secretory (TES) antigens. From an alpaca immune Nb library, we retrieved different Nbs with specificity for TES antigens. Based on ELISA experiments, these Nbs did not show any cross-reactivity with Ascaris lumbricoides, Ascaris suum, Pseudoterranova decipiens, Anisakis simplex and Angiostrongylus cantonensis larval antigens. Western blot and immunocapturing revealed that Nbs 1TCE39, 1TCE52 and 2TCE49 recognise shared epitopes on different components of TES antigen. The presence of disulphide bonds in the target antigen seems to be essential for recognition of the epitopes by these three Nbs. Three separate sandwich ELISA formats, using monovalent and bivalent Nbs, were assessed to maximise the detection of TES antigens in solution. The combination of biotinylated, bivalent Nb 2TCE49 on a streptavidin pre-coated plate to capture TES antigens, and Nb 1TCE39 chemically coupled to horseradish peroxidase for detection of the captured TES antigens, yielded the most sensitive ELISA with a limit of detection of 0.650 ng/ml of TES antigen, spiked in serum. Moreover, the assay was able to detect TES antigens in sera from mice, taken 3 days after the animals were experimentally infected with T. canis. The specific characteristics of Nbs make this ELISA not only a promising tool for the detection of TES antigens in clinical samples, but also for a detailed structural and functional study of TES antigens.

IgG Induced by Vaccination With Ascaris suum Extracts Is Protective Against Infection.

Human ascariasis has a global and cosmopolitan distribution, and has been characterized as the most prevalent neglected tropical disease worldwide. The development of a preventive vaccine is highly desirable to complement current measures required for this parasitic infection control and to reduce chronic childhood morbidities. In the present study, we describe the mechanism of protection elicited by a preventive vaccine against ascariasis. Vaccine efficacy was evaluated after immunization with three different Ascaris suum antigen extracts formulated with monophosphoryl lipid A (MPLA) as an adjuvant: crude extract of adult worm (ExAD); crude extract of adult worm cuticle (CUT); and crude extract of infective larvae (L3) (ExL3). Immunogenicity elicited by immunization was assessed by measuring antibody responses, cytokine production, and influx of tissue inflammatory cells. Vaccine efficacy was evaluated by measuring the reductions in the numbers of larvae in the lungs of immunized BALB/c mice that were challenged with A. suum eggs. Moreover, lung physiology and functionality were tested by spirometry to determine clinical efficacy. Finally, the role of host antibody mediated protection was determined by passive transfer of serum from immunized mice. Significant reductions in the total number of migrating larvae were observed in mice immunized with ExL3 61% (p < 0.001), CUT 59% (p < 0.001), and ExAD 51% (p < 0.01) antigens in comparison with non-immunized mice. For the Ascaris antigen-specific IgG antibody levels, a significant and progressive increase was observed with each round of immunization, in association with a marked increase of IgG1 and IgG3 subclasses. Moreover, a significant increase in concentration of IL-5 and IL-10 (pre-challenge) in the blood and IL-10 in the lung tissue (post-challenge) was induced by CUT immunization. Finally, ExL3 and CUT-immunized mice showed a marked improvement in lung pathology and tissue fibrosis as well as reduced pulmonary dysfunction induced by Ascaris challenge, when compared to non-immunized mice. Moreover, the passive transfer of specific IgG antibodies from ExL3, CUT, and ExAD elicited a protective response in naïve mice, with significant reductions in parasite burdens in lungs of 65, 64, and 64%, respectively. Taken together, these studies indicated that IgG antibodies contribute to protective immunity.

Treatment of canine asthma by high selective vagotomy

This study evaluated the effect of high selective bilateral vagotomy of hilus pulmonis with video-assisted thoracoscopy on asthma. Eight dogs with skin sensitive to Ascaris suum antigens were randomly divided into groups A and B. Asthma was induced by aerosol inhalation of A suum antigens. Respiratory rate and peak airway pressure were significantly increased (P<.05) in both groups. Dynamic compliance was dramatically increased (P<.05) in both groups. Two days later, bilateral vagotomy of hilus pulmonis under thoracoscopic guidance was performed on dogs in group A; dogs in group B underwent bilateral sham vagotomy plus thoracoscopy. Five days after treatment, all dogs had rechallenge with a second aerosol inhalation. Dogs in group A did not show typical asthmatic symptoms, and no significant changes were found in respiratory rate, peak airway pressure, and dynamic compliance (P > .05). Dogs in group B still had typical symptoms, and respiratory rate and peak airway pressure were increased and dynamic compliance decreased significantly (P<.05 for all). Significant differences in respiratory rate, peak airway pressure, and dynamic compliance were observed between groups. Moreover, inflammatory cells in the lungs and bronchoalveolar lavage fluid of group A were dramatically reduced relative to group B (P<.05). There were no significant changes in heart rate and mean arterial pressure after vagotomy, indicating that vagotomy did not affect the cardiac plexus of vagus. High selective bilateral vagotomy of hilus pulmonis with thoracoscope can effectively control asthma in dogs.

Development of a model of Ascaris Suum antigen-induced pulmonary inflammation in nonhuman primates

Airway inflammation is one of the hallmarks of asthma and can contribute to airway hyperresponsiveness. Using cynomolgus monkeys (Macaca fascicularis) that are naturally sensitized to Ascaris suum antigen in the wild, we developed a reproducible model of acute airway inflammation following segmental A.suum antigen challenge. A pediatric bronchoscope was used to perform bronchoalveolar lavage (BAL) for the collection of BAL cells and fluid (BALF). The bronchoscope was also used for the administration of a segmental lung A.suum challenge. Baseline BAL was performed on one lung prior to segmental antigen challenge on the opposite lung. BAL on the challenged lung was performed 24 hours post-challenge to assess pulmonary inflammation. As compared to baseline lavage, a single airway challenge with A.suum antigen, at a dose of 0.50 or 0.75 mg/challenge, resulted in a reproducible pulmonary inflammation. The inflammation was characterized by an increase in total BAL cell counts and increased eosinophils. From concentrated BALF samples, taken 24 hours post A.suum antigen challenge, there was also a trend towards increased IL-5 and eotaxin as measured by ELISA. In some studies, animals were treated with two doses of the steroid, dexamethasone (DEX), 1 mg/kg by IM injection, prior to antigen challenge. In these studies, administration of DEX prior to antigen challenge prevented airway inflammation. These data show that segmental challenge with A. suum antigen produced an acute pulmonary eosinophilic inflammation that was prevented by pretreatment with the steroid, dexamethasone. This model should be useful for testing the efficacy of selected drug candidates, as compared to proven anti-inflammatory therapy, in blocking the pulmonary inflammatory response to A. suum in a large animal model.

Trichinella spiralis: Monoclonal antibody against the muscular larvae for the detection of circulating and fecal antigens in experimentally infected rats

In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were infected with 15 ML per gram of body weight and blood samples were collected weekly for 10 weeks. Antibodies were studied using an indirect ELISA. For detection of circulating antigens and coproantigens, a sandwich ELISA was developed with the use of polyclonal rabbit antibodies obtained against the total extract of ML and an IgM monoclonal antibody (Mab) against ES antigens of ML. No reactivity was observed between Mab and the total worm antigens of Angiostrongylus cantonensis, Ascaris suum, Echinococcus granulosus, Fasciola hepatica, Strongyloides stercoralis, Taenia solium, Toxocara canis and Trichuris trichiura. The IgM Mab recognized antigens of 45, 49, and 55 kDa in ES antigens and was unable to bind ES antigens deglycosylated with trifluoromethanesulphonic acid (TFMS) indicating that a glycan structure is present in the epitope recognized by this Mab. The sensitivity of sandwich ELISA was 1 ng/mL. Circulating antigens were detected in all infected rats between 3 and 8 weeks post infection and coproantigens were found during the first two days post infection. Antibodies were detected since the third week post infection through the end of experiment. These results suggested that antigen detection by our sandwich ELISA could be a useful complementary laboratory test for antibody detection.

Possible Regulatory Role of Galectin-9 on Ascaris suum-Induced Eosinophilic Lung Inflammation in Mice

Background: Galectin-9 (Gal-9) is a member of the galectin family of lectins that exhibit binding affinity for β-galactosides. We found a T cell line-derived Gal-9 with novel eosinophil chemoattractant activity, but its role in eosinophilic inflammation of the lung is unknown. We evaluated the role of Gal-9 in Ascaris suum-induced eosinophilic lung inflammation in mice. Methods: To evaluate the role of Gal-9 in Ascaris suum-induced eosinophilic lung inflammation, we developed a mouse model of eosinophilic pneumonia induced by the Ascaris suum antigen, and analyzed eosinophilic inflammation in Gal-9-deficient mice. The therapeutic effects of recombinant Gal-9 on lung inflammation were also examined in this mouse model. To evaluate lung inflammation, numbers of inflammatory cells and cytokine levels in the bronchoalveolar lavage fluid (BALF) were estimated by flow cytometry and enzyme-linked immunosorbent assay, respectively. Results: The BALF of this mouse model of eosinophilic pneumonia induced by the Ascaris suum antigen contained increased numbers of inflammatory cells and elevated Gal-9 levels. Compared with wild-type mice, the BALF of Gal-9-deficient mice contained higher numbers of both eosinophils and T helper type 2 (Th2) cells. Th2 cytokines and eotaxin levels were also higher, and levels of CD4+CD25+Foxp3+ regulatory T cells were lower in Gal-9-deficient mice than in wild-type mice. Intranasal administration of recombinant Gal-9 prevented eosinophilic inflammation of the lung and upregulated the release of endogenous Gal-9. Conclusions: Our findings suggest that Gal-9 negatively regulates Th2-mediated eosinophilic inflammation of the lung and that Foxp3+ regulatory T cells might be involved in suppressing allergic inflammation.

Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

BackgroundPrevious studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of <2500 daltons.ObjectiveTo investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep.MethodsAllergic sheep without (acute responder) and with late airway responses (LAR; dual responder) were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR) to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment.ResultsThe inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4), and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4)GlcNS6S (1→4) IdoU2S (1→4) AMan-6S]) which lacked anti-coagulant activity.ConclusionsThese results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti-allergic and anti-coagulant activity are distinctly different.

Antigen challenge influences various transcription factors of rat bronchus: Protein/DNA array study

Although the transcription factors are required for expression of the proinflammatory cytokines and immune proteins which are involved in airway hyperresponsiveness and inflammation of bronchial asthma, the antigen-induced alterations of the transcription factors in bronchi have not yet been revealed. Therefore, in order to profile the alteration pattern of the transcription factors after antigen challenge in bronchi, we used protein/DNA arrays. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen. Half, 1, 2 and 4 h after the last antigen challenge, protein/DNA array was performed with nuclear extract of bronchial tissue. Twenty-one the transcription factors exhibited an activation after the last antigen challenge in rat bronchial tissue. Among them, upstream transcription factor-1 (USF-1) and CAAT box general (CBF) were markedly activated after the last antigen challenge. Conversely, 4 transcription factors were inactivated after the last challenge. In development of bronchial asthma, some of the transcription factors may have an ability to modulate the transcription of inflammatory proteins such as cytokines, inflammatory enzymes, etc. Furthermore, the transcription factors, such as USF-1 and CBF, which have not been taken notice so far are also presumed to play an important role in the development of bronchial asthma.

Production of Monoclonal Antibody AgainstToxocara catiSecond-Stage Larvae and Its Application for the Detection of Circulating Antigens

Toxocariasis is an important zoonotic disease caused by the second-stage larvae of Toxocara species. Human toxocariasis is usually diagnosed by serological methods. We prepared monoclonal antibody specific for Toxocara cati (T. cati) and performed the following experiments using this antibody to increase the sensitivity of our capture ELISA for antigen detection. The isotype of monoclonal antibody was IgG3. Using this monoclonal antibody, a capture ELISA was developed. The produced monoclonal antibody reacted well with excretory-secretory (ES) antigen of T. cati with protein nature but not with other T. cati related antigens. A monoclonal antibody was raised against ES antigen of T. cati larval stage. No cross-reaction was found with antigens of Ascaris suum, Dirofilaria immitis, Trichostrongylus orientalis, encysted larvae of Trichinella spiralis, and metacercariae of Centrocestus armatus. The system had enough sensitivity to detect 5 ng/mL of circulating antigens in the serum. The results indicate that the new antigen detection system provides a useful method for the diagnosis of visceral and ocular larva migrans caused by T. cati and fulfils the requirements of a serological method for diagnosis of toxocariasis.

Serological, clinical and epidemiological evaluation of toxocariasis in urban areas of south Brazil

Toxocariasis is a worldwide public-health problem that poses major risks to children who may accidentally ingest embryonated eggs of Toxocara. The objectives of this study were to investigate the occurrence of anti-Toxocara spp. antibodies in children and adolescents and the variables that may be involved, as well as environmental contamination by Toxocara spp. eggs, in urban recreation areas of north central mesoregion, Paraná State, Brazil. From June 2005 to March 2007. a total of 376 blood samples were collected by the Public Health Service from children and adolescents one to 12 years old, of both genders. Samples were analyzed by the indirect ELISA method for detection of anti-Toxocara antibodies. Serum samples were previously absorbed with Ascaris suum antigens, and considered positive with a reagent reactivity index >1. Soil samples from all of the public squares and schools located in the four evaluated municipalities that had sand surfaces (n = 19) or lawns (n = 15) were analyzed. Of the 376 serum samples, 194 (51.6%) were positive. The seroprevalence rate was substantially higher among children aging one to five years (p = 0.001) and six to eight years (p = 0.022). The clinical signs and symptoms investigated did not show a statistical difference between seropositive and seronegative individuals (p > 0.05). In 76.5% of the investigated recreation places, eggs of Toxocara were detected in at least one of the five collected samples. Recreation areas from public schools were 2.8 times more contaminated than from public squares. It is important to institute educational programs to inform families and educators, as well as to improve sanitary control of animals and cleaning of the areas intended for recreation in order to prevent toxocariasis.

The increase in seroprevalence to Toxocara canis in asthmatic children is related to cross-reaction with Ascaris suum antigens

It has been suggested that the presence of Toxocara canis larvae in lungs is an underlying factor in the onset of asthma. Although the association of asthma and seropositivity to Toxocara has been observed, there are no studies that indicate whether these antibodies are specific against T. canis antigens. Seroprevalence to T. canis excretion-secretion antigens (TcES Ag) were compared between asthmatic children (n=285) and non-asthmatic children (n=152), using IgG-ELISA and IgE-ELISA. The recognition patterns of TcES Ag were determined using Western blot (WB). IgG-ELISA seroprevalence was 30.8% for asthmatic children and 19.7% for non-asthmatic children (p<0.05). IgE-ELISA seroprevalence was 7.7% for asthmatic children and 6.5% for non-asthmatic children, respectively (p>0.05). Sera of both groups positive to IgG-ELISA recognised 11 TcES Ag bands for IgG. No differences between the groups were observed regarding recognition patterns; the asthmatic group, however, presented significantly higher cross-reaction to Ascaris suum somatic antigens (AsS Ag) than the non-asthmatic group. Sixty-three sera from asthmatic children positive to IgG-ELISA were evaluated by WB for IgE and 58.7% revealed a recognition pattern for IgE. In the group of non-asthmatic children positive to IgG-ELISA, 80% presented IgE band recognition. No differences were observed between the groups regarding recognition patterns. The results observed suggest that differences in seroprevalence determined by IgG-ELISA between groups of asthmatic and non-asthmatic children reported by other authors occur because of a higher frequency of cross-reaction in asthmatic children.

Preclinical Safety and Pharmacology of an Anti-Human Interleukin-13 Monoclonal Antibody in Normal Macaques and in Macaques with Allergic Asthma

Interleukin-13 (IL-13) plays a central role in chronic airway diseases, including asthma. These studies were conducted to evaluate the safety of administration of a human anti-IL-13 monoclonal antibody (mAb) to normal macaques and in macaques with allergic asthma. In addition, serum and bronchioalveolar lavage fluid were collected from allergic cynomolgus macaques in order to identify potential surrogate markers of IL-13 pharmacology that could be useful for subsequent clinical trials. In vitro studies demonstrated that the anti-IL-13 mAb inhibited the pharmacological actions of both human and cynomolgus macaque IL-13. Allergic macaques were treated systemically with 10 mg/kg anti-IL-13 mAb 1 day prior to inhaled Ascaris suum antigen challenge. Normal macaques were dosed intravenously with anti-IL-13 once per week for 3 weeks at doses of 10 or 50 mg/kg. Treatment of macaques with the anti-IL-13 mAb was not associated with any toxicologically significant findings. A slight treatment-related but nonadverse decrease in platelet counts was observed in both the normal and allergic macaques. In allergic macaques, the anti-IL-13 mAb treatment did not affect lung function, lung eosinophilia, or serum or BAL immunoglobulin E (IgE) concentrations but did produce a reduction in BAL and serum eotaxin concentrations (p < .05) at 6 h post antigen challenge. This study shows that administration of an anti-IL-13 mAb was well tolerated in both normal and allergic asthmatic macaques and that serum eotaxin concentrations may be a useful early in vivo marker for evaluating IL-13 inhibition in patients with asthma.

Interleukin-13 Neutralization by Two Distinct Receptor Blocking Mechanisms Reduces Immunoglobulin E Responses and Lung Inflammation in Cynomolgus Monkeys

Interleukin (IL)-13 is a key cytokine driving allergic and asthmatic responses and contributes to airway inflammation in cynomolgus monkeys after segmental challenge with Ascaris suum antigen. IL-13 bioactivity is mediated by a heterodimeric receptor (IL-13Ralpha1/IL-4Ralpha) and can be inhibited in vitro by targeting IL-13 interaction with either chain. However, in cytokine systems, in vitro neutralization activity may not always predict inhibitory function in vivo. To address the efficacy of two different IL-13 neutralization mechanisms in a primate model of atopic disease, two humanized monoclonal antibodies to IL-13 were generated, with highly homologous properties, differing in epitope recognition. Ab01 blocks IL-13 interaction with IL-4Ralpha, and Ab02 blocks IL-13 interaction with IL-13Ralpha1. In a cynomolgus monkey model of IgE responses to A. suum antigen, both Ab01 and Ab02 effectively reduced serum titers of Ascaris-specific IgE and diminished ex vivo Ascaris-triggered basophil histamine release, assayed 8 weeks after a single administration of antibody. The two antibodies also produced comparable reductions in pulmonary inflammation after lung segmental challenge with Ascaris antigen. Increased serum levels of IL-13, lacking demonstrable biological activity, were seen postchallenge in animals given either anti-IL-13 antibody but not in control animals given human IgG of irrelevant specificity. These findings demonstrate a potent effect of IL-13 neutralization on IgE-mediated atopic responses in a primate system and show that IL-13 can be efficiently neutralized by targeting either the IL-4Ralpha-binding epitope or the IL-13Ralpha1-binding epitope.

PROXIMAL AND DISTAL LUNG MECHANICS IN ANTIGEN CHALLENGED ALLERGIC CYNOMOLGUS MONKEYS

We used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and the effects of antigen challenge on the proximal and distal lung. In addition, we treated animals with two oral doses of placebo or betamethasone (1 mg/kg) 18 and 2 hours prior to antigen challenge. FOT provides indices of proximal (Newtonian airway resistance RN) and distal airway status (tissue damping (G) and elastance (H)). Ascaris suum antigen challenge caused a significant increase in RN, G and H. Pretreatment with betamethasone significantly reduced the peak increase in RN and the area under the curve of the FOT response over 60 minutes. G and H responses were unaffected by steroid treatment. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre‐treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to explain the apparent disconnect between the empirical and subjective measures of efficacy for some asthma treatments.

CONTINUOUS MEASURES OF BREATHING AND PHASE ANGLE IN CONSCIOUS CYNOMOLGUS MONKEYS

We evaluated an inductance plethysmography (IP) system in 3 allergic cynomolgus monkeys for measurement of breathing and phase angle (Φ) as a surrogate for airway resistance. Initial studies in anesthetized, intubated animals compared a standard pneumotachograph measure of ventilation with IP to estimate the accuracy of the measurement. Data showed excellent agreement between IP and the standard approach. 24 hours of baseline unanesthetized breathing was then recorded. After 24 hours, monkeys were re‐anesthetized, exposed to an aerosolized ascaris suum antigen and immediately returned to their cages. A subsequent 24 hours of recording provided data from each animal during and following a bronchoconstrictor response. Data indicate that ventilatory pattern is distinctly altered for a period of at least 60 minutes post challenge at which point breathing recovers to normal values. These data agree with our AUC measures in anesthetized animals after antigen exposure. The Φ for each breath was also calculated. During bronchoconstriction, a distinct different angle was measurable. Upon recovery, Φ returned to normal and remained stable, regardless breathing pattern. We conclude that this system is a useful tool to measure breathing in unencumbered, conscious animals for a long period and may be useful in evaluating bronchoconstrictor events.

Lovastatin inhibits bronchial hyperresponsiveness by reducing RhoA signaling in rat allergic asthma

Recent studies revealed an importance of a monomeric GTP-binding protein, RhoA, in contraction of bronchial smooth muscle (BSM). RhoA and its downstream have been proposed as a new target for the treatment of airway hyperresponsiveness in asthma. Statins are known to inhibit the functional activation of RhoA via the depletion of geranylgeranylpyrophosphate. To determine the beneficial effects of statins on the airway hyperresponsiveness in allergic bronchial asthma, we investigated the effects of systemic treatment with lovastatin on the augmented BSM contraction and activation of RhoA in rats with allergic bronchial asthma. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen. Animals were also treated with lovastatin (4 mg kg(-1) day(-1) ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a marked BSM hyperresponsiveness to ACh with the increased expression and translocation of RhoA. Lovastatin treatments significantly attenuated both the augmented contraction and RhoA translocation to the plasma membrane. Lovastatin also reduced the increased cell number in bronchoalveolar lavage fluids and histological changes induced by antigen exposure, whereas the levels of immunoglobulin E in sera and interleukins-4, -6, and -13 in bronchoalveolar lavage fluids were not significantly changed. These findings suggest that lovastatin ameliorates antigen-induced BSM hyperresponsiveness, an important factor of airway hyperresponsiveness in allergic asthmatics, probably by reducing the RhoA-mediated signaling.

Modeling of asthma, COPD and cystic fibrosis in sheep

Sheep naturally allergic to Ascaris suum antigen have been used to study the pathophysiology of asthma and more recently allergic rhinitis, chronic obstructive pulmonary disease (COPD) and cystic fibrosis. The utility of the model as it relates to the study of these diseases is discussed.

Glucocorticoids Ameliorate Antigen-Induced Bronchial Smooth Muscle Hyperresponsiveness by Inhibiting Upregulation of RhoA in Rats

To determine the mechanism(s) of the inhibitory effect of glucocorticoids on airway hyperresponsiveness in allergic bronchial asthma, the effects of systemic treatment with glucocorticoids on bronchial smooth muscle hyperresponsiveness and RhoA upregulation were investigated in rats with allergic bronchial asthma. Rats were sensitized and repeatedly challenged with 2,4-dinitrophenylated Ascaris suum antigen. Animals were also treated with prednisolone or beclomethasone (each 10 mg/kg, i.p.) once a day during the antigen inhalation period. Repeated antigen inhalation caused a marked bronchial smooth muscle hyperresponsiveness to acetylcholine with an upregulation of RhoA. Augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain were observed in bronchial smooth muscles of the antigen-exposed animals. Systemic treatment with either glucocorticoid used inhibited the bronchial smooth muscle hypercontraction until the level of the sensitized control rats that received saline inhalation instead of antigen challenge. Interestingly, both glucocorticoids also inhibited the upregulation of RhoA and augmented acetylcholine-induced activation of RhoA and phosphorylation of myosin light chain. In conclusion, glucocorticoids ameliorated the augmented bronchial smooth muscle contraction by inhibiting upregulation of RhoA. These effects of glucocorticoids may account for, in part, their beneficial effects in the treatment of asthma.

Differential Effects of Dexamethasone on the Proximal and Distal Lung Response to Antigen Challenge in Allergic Cynomolgus Monkeys

The proximal and distal portions of the lungs may respond differently to antigen challenge and bronchodilator treatment. This difference may contribute to differences in actual and perceived efficacy of therapies. In this study we used the forced oscillation technique (FOT) to measure impedance in the pulmonary system and discern the effects of antigen challenge on proximal (large airway) and distal (small airway and lung parenchyma) portions of the lung. In addition we treated the animals with two i.m. injections of either a saline control or dexamethasone (0.5 mg/kg) 18 and 1 hour(s) before the antigen challenge. The FOT technique was used to measure indices of proximal airway status, Newtonian airway resistance (RN), and distal airway status, including tissue damping (G) and tissue elastance (H). Challenging the animals with Ascaris Suum antigen caused a significant increase in both the proximal and distal lung measures. Pretreatment with dexamethasone significantly reduced the peak increase in RN but not G or H. In addition, the area under the curve (AUC) of the FOT response over 60 minutes was significantly reduced for the RN but again, G and H were not significantly reduced. These data indicate that, using the FOT, we can dissociate the response of proximal and distal airways to an antigen challenge. Moreover, steroid pre-treatment can reduce the bronchoconstrictor response to inhaled antigen but this effect is primarily via effects on the proximal airways with little effect on the distal airways and parenchymal component of pulmonary impedance. These data may help to provide a mechanism for evaluation of novel therapies for small airway dysfunction.