Tekla M. Lee-Fowler

The Tyrosine Kinase Inhibitor Masitinib Blunts Airway Inflammation and Improves Associated Lung Mechanics in a Feline Model of Chronic Allergic Asthma

Background: Blockade of tyrosine kinase signaling by masitinib, a c-kit/PDGF receptor tyrosine kinase inhibitor, can modulate allergic airway inflammation, but effects on lung mechanics have not been well characterized. We hypothesized masitinib would decrease airway eosinophilia and consequently improve pulmonary mechanics in a feline allergic asthma model. Methods: Asthma was induced in 12 cats using Bermuda grass allergen (BGA). Cats received 50 mg/day oral masitinib or placebo. Bronchoalveolar lavage fluid (BALF) was analyzed for eosinophils, total protein (TP) and BGA-specific IgE. Ventilator-acquired mechanics after methacholine (MCh) challenge determined MCh concentration needed to increase baseline airway resistance by 200% (EC200Raw), positive end expiratory occlusion pressure (PEEP) and end inspiratory breath hold pressure (Pplat). An inverse correlate of respiratory system compliance Pplat-PEEP was also calculated. Data were analyzed using the Wilcoxon test, with one-tailed significance set at p < 0.1. Results: After 4 weeks, percent eosinophils in BALF was lower in masitinib-treated cats (7 ± 9%) versus controls (30 ± 27%, p = 0.023). BALF TP significantly differed (p = 0.047) between groups, decreasing with masitinib and increasing with placebo. BALF BGA-specific IgE was unaffected by masitinib. Both groups showed an improvement in EC200Raw (masitinib, p = 0.015; control, p = 0.078) but no significant change in PEEP after 4 weeks. Masitinib-treated cats demonstrated decreased Pplat (p = 0.033) and Pplat-PEEP (p = 0.075) at week 4, suggesting an improvement in respiratory compliance. Conclusions: Masitinib reduced BALF eosinophilia and TP, indicating improved airway inflammation and edema, and improved Pplat and Pplat-PEEP, suggesting benefit to respiratory compliance influenced by airway inflammation/edema. Masitinib deserves further study in humans with chronic allergic asthma.

Comparison of intradermal skin testing (IDST) and serum allergen-specific IgE determination in an experimental model of feline asthma.

Intradermal skin testing (IDST) and allergen-specific IgE determination are used to determine allergen sensitization. In cats, studies have found poor correlation between the two tests. However, these studies were mainly conducted in pet cats sensitized to unknown allergens with unknown dose and duration of exposure. We hypothesized that in an experimental model of allergic sensitization where these variables are controlled, IDST would demonstrate greater sensitivity and specificity than would serum allergen-specific IgE determination. A model of feline asthma employing Bermuda grass allergen (BGA) or house dust mite allergen (HDMA) was used to test the hypothesis. Thirteen cats were assigned to undergo sensitization to BGA, HDMA or saline (placebo). Bronchoalveolar lavage fluid confirmed development of an asthmatic phenotype. Serum collection and IDST were performed on D0, D28 and D50. A portion of serum was pooled, and an aliquot heat inactivated (HI) to destroy IgE. Individual, pooled, and pooled HI samples were used for allergen-specific IgE determination using an Fc epsilon R1 alpha-based ELISA; pooled samples were also analyzed using an enzymoimmunometric assay. Sensitivity (SE), specificity (SP), and positive and negative predictive values (PPV and NPV) were calculated for IDST and for BGA- and HDMA-specific IgE. Combined results for IDST found SE=90.9%, SP=86.7%, PPV=83.3%, and NPV=92.9%. For ELISA-based serum IgE testing, the SE=22.7%, SP=100%, PPV=100% and NPV=63.8%. The enzymoimmunometric assay did not detect sensitizing IgE, but did detect IgE reactivity to a variety of irrelevant allergens (even in HI samples). Sensitivity of IDST was greater than sensitivity of serum IgE measurement supporting use as a screening test for aeroallergens. Both IDST and allergen-specific IgE determination via ELISA were specific; either test can be used to guide selection of allergens for immunotherapy. The enzymoimmunometric assay was unreliable and cannot be recommended.

Lung histopathology, radiography, high-resolution computed tomography, and bronchio-alveolar lavage cytology are altered by Toxocara cati infection in cats and is independent of development of adult intestinal parasites.

This study presents clinical findings after oral ingestion of Toxocara cati eggs which resulted in rapid pulmonary lung migration and parenchymal disease, noted on clinically relevant diagnostic methods. Further, the study investigated the efficacy of pre-infection applications of preventative medication on larval migration through the lungs. A third aim of the study was to determine if adult cats infected with T. cati developed lung disease. Cats in infected groups were administered five oral doses of L3 T. cati larvae. Four-month-old specific pathogen free (SPF) kittens were divided into three groups (six per group): an infected untreated group, an uninfected untreated control group, and an infected treated group (topical moxidectin and imidacloprid, Advantage Multi for Cats, Bayer Healthcare LLC). Six 2- to 3-year-old adult multiparous female SPF cats were an infected untreated adult group. The cats were evaluated by serial CBCs, bronchial-alveolar lavage (BAL), fecal examinations, thoracic radiographs, and thoracic computed tomography (CT) scans and were euthanized 65 days after the initial infection. Adult T. cati were recovered in infected untreated kittens (5/6) and infected untreated adults (5/6) in numbers consistent with natural infections. Eggs were identified in the feces of most but not all cats with adult worm infections. No adult worms were identified in the uninfected controls or the infected treated group. All cats in the infected groups, including treated cats and untreated cats without adult worms, had lung pathology based on evaluation of radiography, CT scans, and histopathology. The infected cats demonstrated a transient peripheral eosinophilia and marked eosinophilic BAL cytology, but normal bronchial reactivity based on in vivo CT and in vitro ring studies. Lung lesions initially identified by CT on day 11 were progressive. Thoracic radiographs in infected cats had a diffuse bronchial-interstitial pattern and enlarged pulmonary arteries. Pulmonary arterial, bronchial, and interstitial disease were prominent histological findings. Infected treated cats had a subtle attenuation but not prevention of lung disease compared to infected cats. Significant lung disease in kittens and adult cats is associated with the early arrival of T. cati larvae in the lungs and is independent of the development of adult worms in the intestine. These data suggest that while the medical prevention of the development of adult parasites after oral exposure to T. cati is obviously beneficial, this practice even with good client compliance will not prevent the development of lung disease which can alter clinical diagnostic methods.

Evaluation of biomarkers in bronchoalveolar lavage fluid for discrimination between asthma and chronic bronchitis in cats

OBJECTIVE To compare concentrations of interleukin (IL)-4, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha and total nitric oxide (NO) metabolites in bronchoalveolar lavage fluid (BALF) for discrimination between asthma and chronic bronchitis in cats. ANIMALS 97 cats. PROCEDURES Cats screened with cytologic examination of BALF included 13 client-owned cats with naturally developing asthma, 8 client-owned cats with chronic bronchitis, 23 research cats with experimentally induced asthma, 33 research cats with experimentally induced nonseptic suppurative inflammation of the airways, and 20 healthy control cats. Banked unconcentrated BALF supernatant samples were assayed for concentrations of IL-4, IFN-gamma, TNF-alpha, and total NO metabolites. RESULTS Concentrations of IL-4 and IFN-gamma in BALF were less than the limits of detection for most cats, precluding statistical analysis. No significant differences were detected among groups for TNF-alpha concentrations. Concentrations of total NO metabolites were significantly higher in cats with clinical chronic bronchitis, compared with research cats with nonseptic suppurative inflammation or research cats with asthma. CONCLUSIONS AND CLINICAL RELEVANCE There were no significant differences in tested biomarkers between cats with asthma and healthy control cats. None of the measured cytokines or NO metabolites were useful for discriminating between cats with naturally developing asthma and those with chronic bronchitis.

Evaluation of subcutaneous versus mucosal (intranasal) allergen-specific rush immunotherapy in experimental feline asthma

Rush immunotherapy (RIT) is effective for the treatment of experimental feline allergic asthma. In humans, the safety profile of immunotherapy is improved by delivering allergen by a mucosal route. We hypothesized that mucosal (intranasal) RIT would have similar efficacy to subcutaneous RIT with improved safety. Twelve cats sensitized and challenged with Bermuda grass allergen (BGA) were randomized to receive subcutaneous (SC) or intranasal (IN) RIT. Increasing doses of BGA (20-200 microg) were administered over 24h followed by 200 microg BGA weekly as maintenance. Adverse reactions were recorded. Clinical respiratory scores after BGA aerosol challenge, bronchoalveolar lavage fluid (BALF) % eosinophils, and cytokine concentrations were measured before RIT (day 1) and at months 1, 3 and 6 (M1, M3, M6). More adverse events were recorded with SC RIT (n=12) compared with IN RIT (n=6). Respiratory scores were lower by M6 compared with D1 in both the groups. The % BALF eosinophils declined significantly after RIT for both groups (mean+/-SEM, SC RIT D1 62+/-12, M6 9+/-4; IN RIT D1 54+/-9, M6 14+/-6). The BALF IL-4:IFN-gamma ratio significantly decreased over time in the IN RIT group (mean+/-SEM, D1 2.4+/-0.2, M6 1.0+/-0.2). While both protocols decreased eosinophilic airway inflammation, the SC RIT protocol did not cause life-threatening adverse events and demonstrated more consistent resolution of clinical signs after allergen challenge. Either protocol could be considered for the treatment of feline allergic asthma.

Beneficial cross-protection of allergen-specific immunotherapy on airway eosinophilia using unrelated or a partial repertoire of allergen(s) implicated in experimental feline asthma

The study hypothesis was that in experimentally asthmatic cats rush immunotherapy (RIT) using allergens not completely matched with sensitizing allergen(s) would at least partially attenuate the asthmatic phenotype and modulate the aberrant immune response. In phase I, cats sensitized to Bermuda grass allergen (BGA), house dust mite allergen (HDMA) or placebo received BGA RIT. In phase II, cats dually sensitized to BGA and HDMA received RIT using BGA, HDMA or placebo. Efficacy of RIT was assessed using percentage bronchoalveolar lavage fluid (BALF) eosinophils. Additionally, a variety of immunologic assays were performed. Eosinophilic airway inflammation significantly decreased over time in asthmatic cats given RIT using sensitizing allergen or unrelated allergen (P<0.001). In dually sensitized cats, single allergen RIT but not placebo reduced airway eosinophilia (P=0.038). Differences in allergen-specific lymphocyte proliferation, in the number of IL-10 producing cells and in the percentage T regulatory cells were detected between asthmatic cats getting RIT and controls. Cross-protection manifested by reduced airway eosinophilia was noted in cats treated with RIT allergens which did not completely match allergen used in asthma induction. However, the mechanism of immunologic tolerance may differ when improperly matched allergens to the sensitizing allergens are used in RIT.

Endotracheal nebulization of N-acetylcysteine increases airway resistance in cats with experimental asthma

N-acetylcysteine (NAC), a mucolytic and antioxidant, is speculated to cause bronchoconstriction in cats when delivered via aerosol. We hypothesized that in cats with experimental asthma, aerosol delivery of NAC (400 mg cumulative dose) via an endotracheal tube would increase airflow limitation as measured by ventilator-acquired mechanics. After endotracheal drug delivery, airway resistance and inspiratory plateau pressure (Pplat) measurements were obtained in six mechanically ventilated asthmatic cats. Results demonstrated significantly increased airway resistance (P=0.0007) compared with aerosolized saline control; Pplats were not significantly different (P=0.059). All cats exhibited at least one adverse effect: excessive airway secretions (n=3), spontaneous cough (n=2), unilateral strabismus (n=1) and post-anesthetic death (n=1). No adverse reactions were noted with saline aerosol; cough was noted in one cat with methacholine challenge. In conclusion, airway resistance and adverse reactions were documented in all cats after NAC aerosol delivery. Further studies must be performed to evaluate if it is an effective mucolytic and/or antioxidant in cats and to determine if bronchodilator pre-treatment will negate NAC-induced bronchoconstriction.

Effect of pre-cardiac and adult stages of Dirofilaria immitis in pulmonary disease of cats: CBC, bronchial lavage cytology, serology, radiographs, CT images, bronchial reactivity, and histopathology.

A controlled, blind study was conducted to define the initial inflammatory response and lung damage associated with the death of precardiac stages of Dirofilaria immitis in cats as compared to adult heartworm infections and normal cats. Three groups of six cats each were used: UU: uninfected untreated controls; PreS I: infected with 100 D. immitis L3 by subcutaneous injection and treated topically with selamectin 32 and 2 days pre-infection and once monthly for 8 months); IU: infected with 100 D. immitis L3 and left untreated. Peripheral blood, serum, bronchial lavage, and thoracic radiographic images were collected from all cats on Days 0, 70, 110, 168, and 240. CT images were acquired on Days 0, 110, and 240. Cats were euthanized, and necropsies were conducted on Day 240 to determine the presence of heartworms. Bronchial rings were collected for in vitro reactivity. Lung, heart, brain, kidney, and liver tissues were collected for histopathology. Results were compared for changes within each group. Pearson and Spearman correlations were performed for association between histologic, radiographic, serologic, hematologic and bronchoalveolar lavage (BAL) results. Infected cats treated with selamectin did not develop radiographically evident changes throughout the study, were heartworm antibody negative, and were free of adult heartworms and worm fragments at necropsy. Histologic lung scores and CT analysis were not significantly different between PreS I cats and UU controls. Subtle alveolar myofibrosis was noted in isolated areas of several PreS I cats and an eosinophilic BAL cytology was noted on Days 75 and 120. Bronchial ring reactivity was blunted in IU cats but was normal in PreS I and UU cats. The IU cats became antibody positive, and five cats developed adult heartworms. All cats with heartworms were antigen positive at one time point; but one cat was antibody positive, antigen negative, with viable adult females at necropsy. The CT revealed early involvement of all pulmonary arteries and a random pattern of parenchymal disease with severe lesions immediately adjacent to normal areas. Analysis of CT 3D reconstruction and Hounsfield units demonstrated lung disease consistent with restrictive pulmonary fibrosis with an interstitial infiltrate, absence of air trapping, and decrease in total lung volume in Group IU as compared to Groups UU and PreS I. The clinical implications of this study are that cats pretreated with selamectin 1 month before D. immitis L3 infection did not become serologically positive and did not develop pulmonary arterial hypertrophy and myofibrosis.

Nebulized lidocaine blunts airway hyper-responsiveness in experimental feline asthma

Nebulized lidocaine may be a corticosteroid-sparing drug in human asthmatics, reducing airway resistance and peripheral blood eosinophilia. We hypothesized that inhaled lidocaine would be safe in healthy and experimentally asthmatic cats, diminishing airflow limitation and eosinophilic airway inflammation in the latter population. Healthy (n = 5) and experimentally asthmatic (n = 9) research cats were administered 2 weeks of nebulized lidocaine (2 mg/kg q8h) or placebo (saline) followed by a 2-week washout and crossover to the alternate treatment. Cats were anesthetized to measure the response to inhaled methacholine (MCh) after each treatment. Placebo and doubling doses of methacholine (0.0625–32.0000 mg/ml) were delivered and results were expressed as the concentration of MCh increasing baseline airway resistance by 200% (EC200Raw). Bronchoalveolar lavage was performed after each treatment and eosinophil numbers quantified. Bronchoalveolar lavage fluid (BALF) % eosinophils and EC200Raw within groups after each treatment were compared using a paired t-test (P <0.05 significant). No adverse effects were noted. In healthy cats, lidocaine did not significantly alter BALF eosinophilia or the EC200Raw. There was no difference in %BALF eosinophils in asthmatic cats treated with lidocaine (36±10%) or placebo (33 ± 6%). However, lidocaine increased the EC200Raw compared with placebo 10 ± 2 versus 5 ± 1 mg/ml; P = 0.043). Chronic nebulized lidocaine was well-tolerated in all cats, and lidocaine did not induce airway inflammation or airway hyper-responsiveness in healthy cats. Lidocaine decreased airway response to MCh in asthmatic cats without reducing airway eosinophilia, making it unsuitable for monotherapy. However, lidocaine may serve as a novel adjunctive therapy in feline asthmatics with beneficial effects on airflow obstruction.

The impact of oral versus inhaled glucocorticoids on allergen specific IgE testing in experimentally asthmatic cats

Glucocorticoids (GCs) are palliative for allergic asthma, but allergen-specific immunotherapy (ASIT), which relies on identification of allergens, represents a potentially curative treatment. The purpose of this study was to determine if oral or inhaled GCs would interfere with identification of sensitizing allergens. The hypothesis was that oral but not inhaled GCs would interfere with accurate allergen-specific IgE identification determined by skin and serum testing in experimentally asthmatic cats. Asthma was induced in 18 cats using Bermuda grass allergen (BGA). Cats (n=6/group) were randomized to receive oral GCs (10mg prednisolone q 24 h), inhaled GCs (600 μg budesonide q 24 h) or placebo (q 24 h PO) for one month. Intradermal skin testing (IDST) and serum BGA-specific IgE were measured prior to, during and after treatment. A paired t test was used to compare groups pre- and post-treatment (P<0.05 significant). IDST reactivity was eliminated in 4/6, 3/6, and 1/6 cats receiving oral GCs, inhaled GCs, and placebo respectively. Two weeks after stopping treatment, IDST was again positive in all cats. Serum IgE reactivity to BGA was not significantly diminished by any treatment. In conclusion, a two-week withdrawal from GCs is adequate for IDST, but may not be necessary for serum IgE testing.