Characteristics of laryngeal symptoms induced in patients with allergic rhinitis in an environmental challenge chamber

Ann Allergy Asthma Immunol xxx (2016) 1e6

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Characteristics of laryngeal symptoms induced in patients with allergic rhinitis in an environmental challenge chamber Takeshi Suzuki, MD, PhD *; Yoshitaka Okamoto, MD, PhD *; Syuji Yonekura, MD, PhD *; Yusuke Okuma, MD, PhD *; Toshioki Sakurai, MD, PhD y; Daiju Sakurai, MD, PhD * * Department y

of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan Division of Head and Neck Surgery, Chiba Cancer Center Hospital, Chiba, Japan

A R T I C L E

I N F O

Article history: Received for publication December 19, 2015. Received in revised form March 8, 2016. Accepted for publication March 9, 2016.

A B S T R A C T

Background: People with allergic rhinitis often have laryngeal symptoms (LSs) in addition to nasal symptoms during the pollen-scattering season. Objective: To clarify the characteristics of the LSs induced by pollen exposure using an environmental challenge chamber. Methods: Cypress pollen exposure using an environmental challenge chamber for 25 participants with cypress polleneinduced allergic rhinitis was performed for 3 hours for 2 consecutive days in 3 study courses: namely, pollen exposure under normal nasal breathing and pollen or sham pollen exposure with nasal blockage, which eliminated any allergic reactions in the nasal mucosa. The nasal and LSs scores and the levels of serum inflammatory mediators, including eosinophil cationic protein (ECP), were monitored. Laryngeal examinations and physiologic lung tests were also conducted. Results: Various LSs were reported, and these LSs were significantly elevated during pollen exposure and even under sham exposure with artificial nasal blockage. The pollen exposure with artificial nasal blockage exaggerated the LSs in 32% of the participants and also increased the serum ECP levels. The serum ECP levels did not change after sham exposure. The findings of both laryngeal examinations and lung tests failed to reveal any significant changes. Conclusions: Nasal obstruction could induce significant LSs even without pollen exposure. LSs were enhanced by pollen exposure and allergic reactions in the larynx could thus be involved in this enhancement. Trial Registration: clinicaltrials.gov Identifier: UMIN000015667. Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Introduction In addition to nasal symptoms, laryngeal symptoms (LSs), including a dry cough and subjective feelings, consisting of itching, irritation, or tingling of the larynx and the sensation of sticking sputum, are often reported (33%e82%) in individuals with allergic rhinitis (AR) during the pollen season.1e5 Some clinical reports in Japan have indicated that people with symptoms of AR exhibit more severe LSs but milder nasal symptoms in the cypress pollen season than they do in the cedar pollen season.6,7 This finding may be because the diameter of the cypress pollen is smaller and may therefore reach the larynx more easily or the allergic potential of Reprints: Yoshitaka Okamoto, MD, PhD, Department of Otolaryngology, Head and Neck Surgery, Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuoku, Chiba, 260-8670 Japan; E-mail: [email protected]. Disclosures: Authors have nothing to disclose. Funding Sources: This work was supported by the grant from the Japanese Ministry of Health, Labour, and Welfare.

cypress pollen might be stronger than cedar pollen. It is also well known that histamine1 (H1)-receptor antagonists are often effective for improving the nasal and laryngeal symptoms of these patients.3 The presence of these typical LSs is therefore proposed as a part of the diagnostic criterion for allergic laryngitis (AL).8 However, the existence of AL is still underresearched and poorly understood because there are few common findings in the larynx.9,10 It is also difficult to assess individuals with AL because of the instability of their LSs attributable to spatial and temporal changes in pollen levels that are caused by variable weather conditions. Because LSs usually accompany nasal symptoms, they may not be caused by AL. Instead, they may be a secondary effect of oral breathing due to a nasal obstruction, postnasal discharge,1,11,12 or chemical mediators released during an AR reaction in the nose.2,13 Allergen challenge testing using an environmental challenge chamber (ECC) in which a constant level of pollen is used to induce symptoms in participants with AR under well-controlled, stable conditions to examine the efficacy of various treatments.14e17 This

http://dx.doi.org/10.1016/j.anai.2016.03.011 1081-1206/Ó 2016 American College of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

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T. Suzuki et al. / Ann Allergy Asthma Immunol xxx (2016) 1e6

method is expected to offer consistent results.18 However, before the present study, no reports have been published on the use of an ECC to investigate the effects of pollen exposure on the larynx. The present study used an ECC to evaluate the LSs and the objective outcome measures caused by pollen exposure to examine their characteristics. Methods The study was conducted at Chiba University Hospital in compliance with the Ethical Guidelines for Clinical Studies and the Declaration of Helsinki (2013 revision). The ethics committee at Chiba University approved the protocol. Participants received a detailed explanation of the study and of the possible adverse effects and provided their written informed consent before participation in the study. The clinical registration number for this study is UMIN000015667. Participants Participants ranging in age from 20 to 65 years with seasonal AR caused by cypress pollen were enrolled in the study. All participants had a history of rhinitis for at least 2 consecutive cypress pollen seasons and met the following inclusion criteria: a serum cypress pollen specific IgE score of 0.71 kU/L or higher (class 2) by an ImmunoCAP test (Phadia, Uppsala, Sweden) and a positive allergen-specific intradermal skin test result using 0.02 mL of cypress (wheal diameter 10 mm or erythema 20 mm). The results were read after 15 minutes, and wheal reaction was measured in millimeters according to the standard guideline.19 Participants were excluded if they had a history of nasal diseases, including AR induced by allergens other than cedar and cypress pollen, asthma, cough variant asthma, gastroesophageal reflux disease, postnasal discharge, or a cold within 2 weeks of the study. Participants with abnormal results for a respiratory function test (<70% predicted forced expiratory volume in 1 second) or LSs; women who were pregnant, planned to become pregnant, or were breastfeeding; and participants who reported the use of antiallergic drugs, such as antihistamines, leukotriene receptor antagonists, corticosteroids, chemical mediator release inhibitors, decongestants, or allergen immunotherapy, within 4 weeks before each experiment were also excluded from the study. Study Protocol The placebo-controlled study was performed in an ECC at Chiba University from September to October 2013, which is the period outside the normal cypress pollen dispersal season (March to May). As previously described,18 the ECC has an area of 71.8 m2 and can accommodate up to 50 participants. Pollen is supplied from an exposure room above the ceiling through holes where 9 fans agitate the pollen particles. The pollen was collected through the mesh floor

Blood and nasal fluid sampling Inspection / laryngoscopy Physiologic lung test Acoustic analysis Laryngeal symptoms recorded using a handheld device

of the ECC to avoid accumulation. The pollen level is monitored at 56 points within the ECC using laser particle technology. Three experiments were performed with washout periods of more than 2 weeks. Pollen exposure in the ECC was performed for 3 hours, from 9 AM to 12 PM, for 2 consecutive days in each experiment (Fig 1). According to our previous study, the severity of nasal symptoms in the chamber peaked at approximately 2 hours after the start of cedar pollen exposure and then plateaued. The nasal symptoms induced by pollen exposure in the ECC outside pollen season were weak on the first day of exposure and increased after consecutive daily exposure.17,18 These nasal symptoms persisted for 3 days after single exposure.18 The hygrothermal conditions in the ECC were adjusted to 24 C and 60% humidity. Exposure to cypress pollen at concentrations of 8,000 grains/m3 induced comparatively milder nasal symptoms than to cedar pollen at the same concentration.20 In the present study, the effects of cypress pollen exposure were examined at concentrations of 12,000 grains/m3. The participants were instructed to avoid any other drug therapy during the experiment; however, when necessary, a rescue drug (fexofenadine) was given. Subjective assessments of the nasal symptom severity (sneezing, rhinorrhea, nasal congestion, and itchy nose) and LSs (itching, irritation, or tingling of the larynx and sputum sticking sensation) were recorded every 30 minutes using an e-diary (Willcom Co, Tokyo, Japan), which allowed the symptoms to be precisely evaluated during the period of exposure in the ECC on days 1 and 2. The following descriptions were given to the participants: itching, the state of feeling an uncomfortable sensation in the larynx that causes the desire to scratch; irritation, the state of feeling annoyed, impatient, or slightly angry but with no itching or tingling; and tingling, the state of feeling pain, such as a prickling or stinging sensation. The symptom severity was evaluated on a 4-point scale, with responses ranging from 0 to 3, as follows: 0, none; 1, mild; 2, moderate; and 3, severe. The total nasal symptom score (TNSS) and total laryngeal symptom score (TLSS) were used to assess the subjective symptoms (a 12-point scale with 4 categories). The number of coughing and throat-clearing episodes was also recorded as an assessment for LSs 1 hour before the initiation of the experiment on day 1 and at the closing time on the second day of exposure. A laryngeal examination using an endoscopic fiberscope was conducted for participants with severe LSs just after pollen exposure to evaluate the existence of laryngeal change. In addition, a 9-item questionnaire, which evaluated LSs according to a 4-point scale (Fig 2A), was distributed before the experiments on day 1, just after the experiment on day 2, and at 9 PM on days 3 and 4 to identify other specific laryngeal symptoms. Blood samples, nasal fluid specimens, and gargle fluid specimens were collected, and physiologic lung tests (forced expiratory volume in 1 second), including an oral fractional exhaled nitric oxide test and an acoustic analysis (jitter, shimmer, and harmonics to noise ratio) to

Day 1

Day 2

Pollen or sham exposure 180 min

Pollen or sham exposure 180 min

Detailed laryngeal symptoms are recoded in the questionnaire Figure 1. Study schedule.

Day 3

Day 4

T. Suzuki et al. / Ann Allergy Asthma Immunol xxx (2016) 1e6

B 1

2. Irritation of the larynx

2

3. Sputum sticking sensation

3

4. Tingling of the larynx

4

5. Coughing

5

6. Hoarseness

6

7. Clearing of the throat

7

8. Foreign-body sensation in the larynx

8

9. Sputum production

9

*

A 1. Itching of the larynx

3

Pollen Sham

0

0.2

0.4

Questionnaire item

0.6

0.8

1

Score

Figure 2. A 9-item questionnaire on laryngeal symptoms (A) and the mean subjective assessments (B). *P < .05.

evaluate the effects of the lower respiratory tract function and dysphonia, were performed using a handheld electrochemical analyzer (NObreath; Bedfont Scientific Ltd, Kent, United Kingdom), SpiroShift SP-370COPD (Fukuda Denshi Co Ltd, Tokyo, Japan), and Computerized Speech Lab model 4500 (KayPENTAX, Lincoln Park, New Jersey), respectively, before the experiment on day 1 and within a half hour after pollen exposure on day 2. Influence of Nasal Allergic Reactions and Nasal Obstruction in LSs LSs that occurred in response to pollen and sham pollen exposure through oral breathing, with an artificial nasal obstruction, were compared. The participants were prevented from performing normal nasal breathing by an artificial nasal obstruction in the form of a nasal plug, which was made of cotton and surrounded by a plastic wrap. The plug was suitable for placement in the nostrils of each participant. The plug made it impossible for the participants to breathe through their noses. The participants were then exposed to cypress pollen (pollen exposure: experiment 1) or nothing (sham pollen exposure: experiment 2). The participants were not informed about whether they were in the pollen exposure or the sham exposure group. Analysis of Blood, Nasal Fluid, and Gargle Fluid Samples The participants’ serum eosinophil cationic protein (ECP) levels were measured by enzyme-linked immunosorbent assay. Hansel staining was used to visualize eosinophils in the nasal fluid. The cytokine (interleukin [IL] 2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17, interferon g [IFN-g], and tumor necrosis factor a) and chemokine (IFN-geinducible protein 10 and eotaxin) levels were assayed in the gargle fluid via a cytometric bead array (BDCBA Human Flex sets; BD Biosciences, Oxford, United Kingdom) according to the manufacturer’s instructions. The plates were read using a BD FACSCalibur instrument with the CELLQuestpro software program (BD Biosciences, San Jose, California). The results were analyzed using the BD FCAP Array version 1.0.1 software program (BD Biosciences). Adverse Events The participants recorded their experience of any adverse events in a questionnaire at days 1, 2, 3, and 4. Statistical Analysis Statistical significance was determined by Wilcoxon signed-rank test using the GraphPad Prism4 software program (GraphPad, La

Jolla, California). P < .05 was considered statistically significant. The values are given as mean (SEM). Results Twenty-five participants were enrolled in the study and participated in experiments 1 and 2 (Table 1). Three participants withdrew because of personal reasons, and 22 of the participants took part in experiment 3. There were no adverse events, and no rescue drugs were used during any of the experiments. Differences in LSs During Pollen and Sham Exposure With a Nasal Plug (Experiments 1 and 2) During pollen exposure with a nasal plug, with the exception of nasal obstruction, nasal symptoms such as sneezing and rhinorrhea were not reported. Eosinophils were not detected in the nasal smear examinations collected after the pollen exposure. Figure 3A shows the changes in the mean subjective assessments of the LS scores during pollen and sham exposure on days 1 and 2 in the ECC. There were significant time-dependent increases in the participants’ TLSSs in response to pollen and sham exposure. The participants’ TLSSs increased significantly during sham exposure, from 0.88 (0.32) at the beginning of sham exposure on day 1 to 2.28 (0.61) at 180 minutes after sham exposure on day 1 (P ¼ .006) (Fig 3A). The TLSSs during pollen exposure were higher than those during sham exposure, and significant differences were observed at 150 (P ¼ .03) and 180 (P ¼ .02) minutes after the start of the exposure on day 1 and at 120 (P ¼ .03) and 150 (P ¼ .02) minutes after exposure on day 2 (Fig 3A). In the nasal plug experiments (experiments 1 and 2), the TLSSs in 8 (32%) of 25 participants increased by more than 1 point after pollen exposure compared with sham exposure. The number of coughing and throat-clearing episodes were examined in the 1 hour before the initiation of the experiments on day 1 and at the closing time on the second day of exposure. They were found to have increased because of pollen exposure (P ¼ .01 and P ¼ .03, respectively). Although the number of throat-clearing episodes was higher during pollen exposure than during sham exposure (P ¼ .04), the number of coughing episodes did not differ to a statistically significant extent between during pollen exposure and during sham exposure (P ¼ .09) (Fig 3B). No abnormal laryngeal findings were observed around the laryngeal inlet, vocal folds, or the surrounding soft-tissue boundaries during the endoscopic

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T. Suzuki et al. / Ann Allergy Asthma Immunol xxx (2016) 1e6

Table 1 Participant Data by Experiment Experiment No.

Pollen Dispersala

Nasal Pluga

Total No. of Participants

No. of Female Participants

Age, Mean (SEM), y

ImmunoCAP score Mean (SEM)

1 2 3

þ þ 

2

3

4

5

6

25

15

42 (2.2)

2.3 (0.1)

17

8

0

0

0

22

13

42 (2.4)

2.3 (0.1)

15

7

0

0

0

Plus sign indicates positive; minus sign, negative.

examination of these participants just after pollen exposure even in the participants with a high TLSS. The serum ECP levels significantly increased by pollen exposure (5.25 [0.88] before and 10.3 [2.14] mL/L after the experiment, respectively; P < .001). No changes were observed after sham exposure. No significant differences were found in the cytokine levels in the gargle fluid, the respiratory function test, or the acoustic analysis (jitter, shimmer, and harmonics to noise ratio) performed before and after experiment 1. When comparing the responses to the 9 questions on LSs after the pollen and sham challenges, there was a significant difference in the scores regarding itching of the larynx (P ¼ .01) (Fig 2B).

Differences in LSs With and Without a Nasal Plug During Pollen Exposure (Experiments 1 and 3) Under normal breathing without a nasal plug, 64% of the participants reported some LSs after pollen exposure. Figure 4 shows the changes in the mean subjective assessments of the nasal and laryngeal symptom scores during pollen exposure on days 1 and 2 in the ECC without a nasal plug. The TLSSs increased from 0.71 (0.36) at the beginning of pollen exposure on day 1 and peaked at 2.18 (0.74) (P ¼ .009). The number of coughing and throat-clearing episodes induced by pollen between experiment 1 and experiment 3 were almost the same at 0.35 (0.17) and 0.41 (0.17), respectively. No abnormal laryngeal findings were observed. The TLSSs tended to be higher in the participants under nasal obstruction with a nasal plug than in those without a nasal plug on day 1, and significant differences were observed at 150 and 180 minutes after pollen exposure (P ¼ .02 and P ¼ .04, respectively). However, no difference in the TLSSs was found on day 2 (Fig 4B).

Total Laryngeal Symptom Score (Points)

A

Discussion This is the first study, to our knowledge, to evaluate the LSs of participants with pollen-induced AR by pollen exposure in an ECC and to examine the influence of oral breathing and allergic reaction in the nasal mucosa on the larynx. We found that nasal obstruction itself could induce LSs and that these symptoms were aggravated by pollen exposure. Furthermore, we found that the aggravation of these LSs could be induced by an allergic reaction in the larynx. Because the larynx is sensitive to protect the lower airway, LSs may be induced by a variety of situations. The present study found that only nasal blockade led to the relevant LSs in the participants with pollen-induced AR of the present study. The nasal airway plays important physiologic roles, including filtering inspired air, regulating temperature, and humidifying inspired air.21-23 Although the temperature and humidity in the ECC were controlled, the disruption of natural breathing causes some dryness and a reduction in the temperature of the inhaled air; thus, it is possible that these factors increased the sensitivity of the larynx. Furthermore, some neurologic changes may also be induced in the larynx by a nasal obstruction.24 Kubo et al25 reported that coughing experienced by individuals with pollen-induced AR during the pollen season had the highest correlation with a nasal obstruction according to a multivariate analysis. Our findings also implied an association between LSs and nasal congestion (Fig 4). Nasal congestion is the predominant symptom of the late-phase reaction in AR, and it can have far-reaching effects that extend through the airway and beyond the nose.23 The LSs observed by pollen exposure might have simply been induced by direct irritation caused by the increased number of orally inhaled pollen particles and not by an allergic reaction. Formaldehyde, welding fumes, and acrylate compounds and chemicals used in hairdressing26-28 are known to be causes of nonallergic laryngitis.

B

4

Pollen Sham

Pollen Sham Increased No. (Events per Hour)

a

þ  þ

Class

3 *

* *

2

1

0 30 60 90120150180 Time, min ECC (Day 1)

0 30 60 90120150180 Time, min ECC (Day 2)

9

PM

9

PM

0.9 0.8 0.7

*

0.6 0.5 0.4 0.3 0.2 0.1 Coughing

Throat clearing

Day 3 Day 4

Figure 3. The total laryngeal symptom score (TLSS) every 30 minutes during pollen (experiment 1) and sham (experiment 2) exposure (A) with a nasal plug and the increase in the number of coughing and throat clearing episodes (B). The data are expressed as mean (SE). *P < .05.

T. Suzuki et al. / Ann Allergy Asthma Immunol xxx (2016) 1e6

Total Nasal Symptom Score (Points)

A 6

4 3 2 1 0 30 60 90 120 150 180

B Total Laryngeal Symptom Score (Points)

Normal breathing

5

4

0 30 60 90 120 150 180

Nasal obstruction Normal breathing

3

2

1

0 30 60 90 120 150 180 Time, min ECC (Day 1)

0 30 60 90 120 150 180 Time, min ECC (Day 2)

Figure 4. The total nasal symptom score (A) and total laryngeal symptom score (B) every 30 minutes during pollen exposure with a nasal obstruction (experiment 1) and under normal breathing (experiment 3). Data are expressed as mean (SE). *P < .05.

However, elevated serum ECP levels were observed in the present study. ECP is one of the basic proteins in eosinophil granules.29 Not only ECP in the nasal secretion but also serum ECP has been reported to be a sensitive measure for allergic reactions.30,31 Under the unified airway theory, cytokines and other inflammatory mediators produced in the nose by allergic reactions may extend to other parts of the airway, including the larynx, either directly or through blood circulation.23,32 In the present study, however, the closure of the nose with a plug ruled out the influence of an allergic reaction in the nose. The stable results of the physiologic lung tests and the lack of lower respiratory tract symptoms in the nonasthmatic participants also excluded an allergic reaction in the bronchi. Most pollen grains measured 20 to 40 mm in diameter and were thus unable to reach the lower airways.33,34 These results suggest that the allergic reaction in the larynx of participants with pollen-induced AR was therefore not related to nasal or bronchial allergic reactions. Itching sensation around the larynx, which was observed among the participants in the present study, also supported that the allergic reaction occurred around the larynx. The delayed allergic reaction by pollen exposure on day 1 could influence the LSs on day 2. In addition, the exaggerated nasal congestion on day 2 compared with that on day 1 due to a carryover effect influenced the induction of LSs on day 2. Although we could not clearly prove that the allergic reaction took place in the larynx because of the absence of any abnormal objective signs, repeated pollen exposure during the natural pollen season may have resulted in clear changes occurring in the larynx.

5

The contribution of the oral tonsil and other pharyngeal mucosal lymphoid tissues to the development of LSs thus cannot be ruled out. However, no abnormal signs were found and no symptoms were reported at the pharynx or oral cavity after pollen exposure. LSs could be improved by treatment of the nasal obstruction, even if no abnormal signs were observed in the larynx. Several animal studies have suggested the existence of an allergic reaction of the larynx. In histologic experiments using anesthetized guinea pig models for AR, Iwae et al35 found a type I allergic reaction in the larynx. The guinea pigs underwent tracheostomy, were perfused by antigen, were washed with saline after 3 minutes, and then underwent laryngectomy. The number of mast cells in the subglottic epithelial layer of the AR model was significantly higher than that of the control model. A high degree of infiltration by eosinophils, edematous change, and degranulation of mast cells was observed in the laryngeal mucosa after the antigen challenge. In addition, the histamine concentration in the lavage fluid significantly increased after the antigen challenge. Naito et al5 also found a significantly increased number of eosinophilia in the larynx after Japanese cedar pollen exposure using rat models of AR. These results suggest the existence of an allergic reaction in the larynx, especially in the subglottic epithelial layer. However, the anatomical and physiologic differences in the upper airway among species cannot be denied. To date, only a few antigen challenge tests for the human larynx have been reported. Reidy et al36 were unable to find a direct association between exposure to dust mite antigen and laryngeal changes with regard to the appearance of the larynx using videostroboscopic examinations, LSs as evaluated using the Voice Handicap Index, or the laryngeal function using acoustic analyses and speech aerodynamic testing. Roth et al37 found a greater phonation threshold pressure, which reflects the minimum subglottic pressure required to initiate and sustain phonation, in participants with prior vocal symptoms associated with increased allergy symptoms after antigen exposure compared with sham exposure. The authors concluded that a primary allergic response in the larynx existed. However, these studies used allergen extracts for provocation using an oral nebulizer, which differed from natural allergen exposure, and additionally the influence of AR could not be ruled out. In the present study, we found the possible contribution of an allergic reaction in the larynx to LSs and that LSs were enhanced by nasal obstruction. Our findings should thus facilitate further research on this phenomenon, including examinations of allergic and nonallergic participants, antihistamine examinations, and physiologic and histologic examinations to elucidate the role of allergic reaction in the larynx. In addition, a further validation analysis of LSs is needed to make an accurate diagnosis of AL; however, the relevant LSs have been previously reported in the pertinent Japanese literature.7,38 Acknowledgement We thank Karen Sak for her editorial assistance and Chie Imai, Dr Heizaburo Yamamoto, Dr Tomohisa Iinuma, Dr Yuji Ohki, Dr Sawako Hamasaki, and Dr Tomoyuki Arai for assistance with the data collection. References [1] Krouse JH, Altman KW. Rhinogenic laryngitis, cough, and the unified airway. Otolaryngol Clin North Am. 2010;43:111e121. [2] Hellings PW, Fokkens WJ. Allergic rhinitis and its impact on otorhinolaryngology. Allergy. 2006;61:656e664. [3] Duncan RB, Duncan DD. Otolaryngeal allergy in Wellington. 1971-1975. N Z Med J. 1977;85:45e52. [4] Naito K. Foreign body sensation of the larynx in laryngeal allergy patients. J Jpn Bronchoesophagol Soc. 2001;52:120e124.

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