Establishment Of Experimental Methodology Of Leukotriene D4Nasal Provocation Test And The Comparison Of Its Impact On Upper And Lower Airway Inflammation And Responsiveness With That Of Histamine In Allergic Rhinitis Patients With Or Without Asthma

Rationale and objectives：The prevalence of allergic rhinitis and asthma have increased these years,epidemiological data show that the incidence of asthma in allergic rhinitis patients was20%to38%, significantly higher than that of general population2%to5%, and theincidence of allergic rhinitis in asthma patients was78%, much higher than that of generalpopulation5%to20%, therefore, there is a close relationship between allergic rhinitis andasthma, and they seriously affected the quality of life of the patients.In recent years, more studies have gained evidences to the relationship between upperand lower airway inflammation, and support the theory of allergic rhinitis and asthma,which is said "one airway, one disease". Upper or lower airway allergens provocation testscan induce inflammation response in the other end of the airway, the mechanism mayinclude:(1) airway inflammatory mediators extended to the lower airways;(2) the nasalmucosal inflammation makes its function inefficiency, then breathing pattern changes,allergen directly breath into lower airways;(3) nasal-bronchial reflex;(4) inflammatorymediators absorbed in local nasal mucosa or its exposure to allergens active inflammatorycells through blood circulation and bone marrow involved in signal transduction inducemore inflammatory cells in the lower airways, but it is still in exploration.Since Blackley reported nasal provocation with pollen for the first time in1873, it isalso the first medical allergen provocation tests. Later variety of specific or nonspecificagents (histamine, acetylcholine, latex, pollen, dust mites, etc.) were used for nasalprovocation, which was also used as one of the methods for clinical diagnosis or researchof allergic rhinitis. We have already established the method of histamine nasal provocationtest measured by nasal airway resistance and have explored its effect on the airwayinflammation and responsiveness.Studies have shown that the expression or distribution of leukotriene receptor was much higher in both upper and lower airways mucosa cells. The levels of leukotrienes andits metabolites increased in secretions of upper and lower airways when allergic rhinitisand asthma patients were challenged by nasal or bronchial provocation tests or during theexacerbation of natural exposure. It was tested that the application of leukotriene receptorantagonists can reduce the symptoms of allergic rhinitis and asthma.What’s the upper and lower airways response after nasal provocation tests withleukotriene D4and the relationship between airway inflammation and airwayhyperresponsiveness? Through LTD4nasal provocation tests, there may be some foundabout the relationship between allergic rhinitis and asthma, the relationship between upperand lower airway inflammation, and provide more evidence for the treatment of thepatients with allergic rhinitis and asthma.Part Ⅰ: Establishment of Experimental Methodology ofLeukotriene D4Nasal Provocation TestSegment-1Establishment of Methodology of Leukotriene D4NasalProvocation Test Based on the Measurement of Nasal Airway ResistanceBackground: Cysteinyl leukotrienes (LT) play a vital role in the pathogenesis of allergicrhinitis (AR) and asthma, and anti-leukotrine therapy was tested to be effective in somepatients with AR, however, little was reported about nasal physiological response to LTs inpatients with AR.Objective: To establish the methodology and to investigate nasal airway responsiveness,diagnostic value and safety of LTD4nasal provocation test.Methods: LTD4nasal provocation tests were performed in26AR and16normal controls.Nasal airway responsiveness was assessed by the concentration of LTD4causing a60%increase in nasal airway resistance (PC60NAR-LTD4) measured by rhinomanometry and acomposite symptom score. Receiver operating characteristic (ROC) curve was applied toevaluate the diagnostic value of LTD4nasal provocation test. Adverse events wererecorded.Results: PC60NAR-LTD4in AR was significantly lower than that of normal controls (8.80(12.00) vs.17.00(0.00) mcg.mL-1, p=0.005) and the positive rate was73.08%vs.12.5%, p=0.000. Composite symptom score was higher in AR as compared with normalcontrols (1.19±0.94vs.0.12±0.50, p=0.000). The symptom scores consist ofsneezing(0.12±0.34vs.0.00±0.00, p=0.149), rhinorrhea (0.79±0.66vs.0.06±0.25,p=0.000) and chemosis or itching of the eyes (0.06±0.25vs.0.21±0.42, p=0.216). AUCwas0.822,95%CI (0.665,0.961). No serious adverse event was revealed.Conclusion: Most allergic rhinitis patients were nasal hyperactive to LTD4. Theestablished procedure of LTD4nasal provocation test is effective and safety in thediagnosis of AR. Segment-2Establishment of Methodology of Leukotriene D4NasalProvocation Test Based on the Measurement of Nasal Airway ResistanceBackground: The peak nasal inspiratory flow meter is cheap and portable to use, canobjectively evaluate the changes of nasal airway flow in AR patients.Objectives: To evaluate LTD4nasal provocation tests with nasal peak inspiratory flowmeter, and the relationship between nasal airway resistance and nasal peak inspiratoryflow.Methods: A total of41subjects (25patients with allergic rhinitis and16normal controlsubjects) were enrolled, all subjects were underwent LTD4nasal provocation test with themeasurement of nasal airway resistence and peak nasal inspiratory flow. The distributionof PC60NAR and PC20PNIF were calculated. ROC curves were used to assess thediagnostic values.Results: The responsiveness of peak nasal inspiratory flow to LTD4nasal provocationtests was significantly higher in AR than that of normal controls with PC20-PNIF12.80(9.59) μg/ml and17.00(0.41) μg/ml in AR and normal controls. After stimulation, nasalairway resistance in patients with allergic rhinitis increased in varying degrees anddecresed in PNIF, which were negative correlation with PC60or PC20with r=-0.75,p=0.00and r=-0.765, p=0.00, respectively. High diagositc values were revealed by ROCwith AUC:0.827(0.677,0.976) and O.860(0.741,0.979) for PNIF and NAR. No serious adverse effects were reported of leukotriene D4provocaton tests measured by PNIF.Conclusions: Nasal peak inspiratory flow meter was useful for LTD4nasal provocationtests with advantages of more portable and cost less time of the nasal provocationprocedures. Part Ⅱ: Comparison of LTD4and Histamine Nasal Provocation Test inDiagnosis of nasal hyperesponsiveness and the Impact on Upper andLower Airway Inflammation in Allergic RhinitisBackground：Both histamine and leukotrienes involves in the pathogenesis of allergicrhinitis (AR), but nasal responsiveness to leukotriene D4and histamine nasal challengeand the impact on airway inflammation have not been evaluated and compared in patientswith allergic rhinitis.Objectives：To investigate nasal responsiveness to leukotriene D4and histamine nasalchallenge and to compare the different roles of histamine and LTD4in the pathogenesis ofallergic rhinitis and the impact on airway inflammation and lung function.Methods：An open, randomized self crossover control, prospective trial was carried out in25allergic rhinitis patients and16normal volunteers. Participants randomly underwentstepwise nasal challenge test with histamine (0.2-3.2mg·mL-1) and leukotriene D4(4-16mcg·mL-1) with a one-week interval. Nasal airway responsiveness was assessed based onthe increase of nasal airway resistance (NAR) and the score of induced typical nasalsymptoms. Outcomes included the provocative concentration of agent causing a60%increase in NAR (PC60NAR) and symptom scores. Nasal symptoms visual analogue scale(VAS), fractional concentration of exhaled nitric oxide (FeNO), nasal lavage, spirometry,and induced sputum were performed before and after the challenges to evaluate the impactof histamine and LTD4nasal challenge on upper and lower airway inflammation andfunction.Results：The effect of LTD4(PC60NAR-LTD49.240（11.623）mcg·mL-1) was142times to histamine (PC60NAR-his1.312（2.761mg/ml）mg·mL-1, p=0.000) in respect ofincreasing nasal airway resistance in patients with allergic rhinitis. On the contrary, nasalsymptoms score was higher induced by histamine than LD4nasal challenge (4.00(1.00) vs.1.00(2.00), p=0.000). LTD4nasal provocation test had a little higher diagnostic value thanhistamine nasal provocation test revealed by ROC curve (AUC:0.801,95%CI:[0.647,0.954] vs. AUC:0.795,95%CI:[0.630,0.959]). Changes of VAS before and after LTD4and histamine nasal provocation tests were1.81±0.27、3.84±0.53，p=0.002；the changesof FENO（log）0.27±0.69、-0.01±0.60，p=0.175；the changes of inflammation cells insputum were Neu15.61±20.12、Eos-5.95±14.19�'�Neu10.19±24.89、Eos4.04±15.00，with both p>0.05. The changes of spirometry parameters were without significantstatistical diffenrences. No serious adverse event was found in both methods ofprovocation tests.Conclusion：Patients with allergic rhinitis were nasal hyperreactivity to both histamineand leukotriene D4. LTD4play a vital role in the early-phase of nasal allergy reaction byincreasing nasal airway resistance, while histamine also caused sneezing and rhinorrhea.Totally, LTD4nasal provocation test has a little higher diagnostic value than histaminenasal provocation test in the diagnostic of nasal airway hyperreactivity in allergic rhinitispatients. Part Ⅲ: Predictive Value of Fractional Exhaled Nitric Oxide andEosinophils in Induced Sputum for Bronchial Hyperresponsiveness inAllergic RhinitisObjective：To investigate the predictive and diagnostic value of fractional exhaled nitricoxide (FeNO) in patients with allergic rhinitis accompanied with asymptomatic bronchialhyperresponsiveness (BHR) or chronic persistent asthma.Methods：Fractional exhaled nitric oxide (FeNO), nasal lavage, eosinophils (EOS) in induced sputum and methacholine bronchial provocation test (BPT) were performed in20patients with allergic rhinitis (AR)(male/female9/11, aged18-50years),15patients withAR and asymptomatic BHR (AR+BHR)（male/female7/8, aged18-50years）and20patients with AR and chronic persistent asthma (AR+asthma)(male/female8/12, aged18-50years). Correlation between FeNO and EOS was analyzed. Receiver operatingcharacteristic (ROC) curve was applied to evaluate the predictive and diagnostic value ofFeNO.Results：The median and interquartile range of FeNO values in AR, AR+BHR andAR+asthma were24.0(20.0) ppb,46.5(38.0) ppb, and61.5(36.0) ppb, respectively, withsignificant statistically difference among the groups (p<0.01). Receiver OperatingCharacteristic (ROC) curve was obtained by using FeNO values to assess the diagnosisvalues. In AR+BHR group, the area under curve (AUC) was0.842, p=0.002, thesensitivity was0.750and specificity was0.737, while the cut-off value was set as34.0ppb. For the diagnoses of AR+asthma, AUC was0.887(p=0.000), the sensitivity was0.750, and the specificity was0.831, when taking41.0ppb as the cut-off value. Thepercentages of EOS were2.43%±3.56%,7.36%±4.98%,18.58%±11.26%in AR group,AR+BHR group and AR+asthma group, respectively, with significant statisticallydifferences among the groups (p<0.01).Conclusions：Lower airway inflammation might exist in some extents in patients withallergic rhinitis. The measurement of FeNO is simple and stable, with reliable predictiveand diagnostic value for patients with allergic rhinitis accompany with BHR and chronicpersistence asthma. Part Ⅳ: Effect of LTD4Nasal Provocation Test on BronchialHyperresponsiveness in Patients with Aallergic Rhinitis and AsthmaBackground: Allergic rhinitis and asthma are often co-existence and have impact on eachother. Leukotrienes play a vital role in both allergic rhinitis and asthma, but the effect ofLTD4nasal provocation on lower airway hypereactivity has not been evaluated. Objective: To evaluate the effect of LTD4nasal provocation test on lower airwayhypereactivity in patients with allergic rhinitis and asthma.Methods: A total of15patients with allergic rhinitis and asthma underwent LTD4nasalprovocation test and methacholine bronchial provocation test before and after nasalprovocation. The distribution of PD20FEV1was calculated to evaluate the effect of LTD4nasal provocation on lower airway hypereactivity in patients with allergic rhinitis andasthma, nasal lavage and induced sputum were performed.Results: House dust mite was the major allergen accounted for10/15of the patients.PC60NAR-LTD4was9.80（9.00）mcg/ml. PD20FEV1-MCH were0.203(0.905),0.191(0.295) mg, p=0.910before and after nasal provocation. Although there was a decline ofPD20FEV1after nasal chanllege, but it was without significant statistical. Compared with thebaseline, eosinophil ratio decreased while neutrophil ratio increased24h after nasalprovocation. The baseline of Eos in nasal lavage was38.36±23.14, Neu48.70±24.86,after chanllege they were50.30±24.46and60.64±23.14. The baseline of Eos inducedsputum was17.51±11.05, Neu25.29±16.52, after chanllege they were55.19±22.2853.83±23.27respectively.Conclusion: LTD4envolved in the pathogenetic of allergic rhinitis and asthma, but couldnot induced the increase of bronchial hypereactivity after LTD4nasal provocation.