Apolipoprotein A-IV is a candidate target molecule for the treatment of seasonal allergic rhinitis

Article Outline

• Abstract
• Methods
  • Subjects
  • Symptom-medication score and quality of life score
  • Sample collection
  • Two-dimensional polyacrylamide gel electrophoresis and protein identification
  • Western blot analysis of apolipoprotein A-IV
  • Test for histamine release from basophils containing various apolipoproteins
  • Pathway analysis
  • Statistical analysis
• Results
• Discussion
• Acknowledgment
• Methods
  • 2-DE
  • Protein identification
  • Western blot analysis to validate the apoA-IV spot
  • ApoA-IV protein purification
• Fig E1. 
• Fig E2. 
• Fig E3. 
• Table E1. 
• References
• Copyright

Background

Allergic rhinitis is a global health problem that causes major illnesses and disability worldwide. Allergen-specific immunotherapy (SIT) is the only available treatment that can alter the natural course of allergic disease. However, the precise mechanism underlying allergen-SIT is not well understood.

Objective

The aim of the current study was to identify protein expression signatures reflective of allergen-SIT—more specifically, sublingual immunotherapy (SLIT).

Methods

Serum was taken twice from patients with seasonal allergic rhinitis caused by Japanese cedar: once before the pollen season and once during the season. A total of 25 patients was randomly categorized into a placebo-treated group and an active-treatment group. Their serum protein profiles were analyzed by 2-dimensional electrophoresis.

Results

Sixteen proteins were found to be differentially expressed during the pollen season. Among the differentially expressed proteins, the serum levels of complement C4A, apolipoprotein A-IV (apoA-IV), and transthyretin were significantly increased in SLIT-treated patients but not in placebo-treated patients. Among these proteins, the serum levels of apoA-IV correlated with the clinical symptom-medication scores (r = –0.635; P < .05) and with quality of life scores (r = –0.516; P < .05) in the case of SLIT-treated patients. The amount of histamine released from the basophils in vitro was greatly reduced after the addition of recombinant apoA-IV in the medium (P < .01).

Conclusion

Our data will increase the understanding of the mechanism of SLIT and may provide novel insights into the treatment of allergic rhinitis.

Key words: Sublingual immunotherapy, apoA-IV, HNF4A, proteome

Abbreviations used: ApoA-IV, Apolipoprotein A-IV, 2-DE, Two-dimensional polyacrylamide gel electrophoresis, HNF4A, Hepatocyte nuclear factor 4α, HRP, Horseradish peroxidase, JAU, Japanese allergy unit, JC, Cryptomeria japonica, JRQLQ, Juniper Rhinoconjunctivitis Quality-of-Life Questionnaire, MALDI-TOF/TOF, Matrix laser desorption/ionization 2-stage time-of-flight, QOL, Quality of life, SAR, Seasonal allergic rhinitis, SIT, Specific immunotherapy, SLIT, Sublingual immunotherapy, SMS, Symptom-medication score

Allergic rhinitis is a global health problem that causes major illnesses and disability worldwide. A conservative estimate revealed that allergic rhinitis occurs in over 500 million people around the world.¹ Seasonal allergic rhinitis (SAR) caused by Japanese cedar (Cryptomeria japonica; JC) is the most common allergic disease in Japan. According to a national survey, the prevalence of allergic rhinitis in Japan was 0.16 in 1992 and 0.21 in 2002.² The results of our recent study showed that over 35% of Japanese individuals in the age group 20 to 50 years develop allergic symptoms during JC pollination season.³

Allergen-specific immunotherapy (SIT) is the only available treatment that can alter the natural course of allergic disease by preventing new sensitization/onset and providing long-term remission after discontinuation of treatment.⁴ Many clinical trials have proven the efficacy of SIT in controlling allergic diseases.⁵ Conventional SIT, subcutaneous injection, however, requires frequent hospital visits and is painful, resulting in a low patient compliance. Further, it may cause some adverse events such as anaphylaxis. To overcome these disadvantages, sublingual immunotherapy (SLIT), oral administration of the allergen, was introduced as an alternative method,⁶ and to date, SLIT has been widely used to treat patients with asthma and rhinitis.⁴ The clinical efficacy of SLIT has been widely proven by many studies,⁶, ⁷ and several studies have revealed that there is increased production of blocking antibody, IgG₄,⁸ as well as induction of regulatory T cells⁹ as a result of SLIT. However, the precise mechanism underlying SLIT is not well understood.

The word proteome describes the entire collection of proteins in a cell, tissue, or body fluid at a given time. With a proteomic approach, all the proteins present in a biological sample can be visualized simultaneously and identified. This approach is not based on any experimental hypothesis but on correlation-associated network analyses of proteomic profiles, leading to hypotheses regarding relations between structurally and biologically related proteins/peptides. Therefore, this approach can be used to identify proteins associated with SLIT, which could improve our understanding of the mechanism underlying this therapy. In this study, we performed proteomic analysis to identify protein expression signatures that reflect the responsiveness to SLIT and to determine novel therapeutic targets for the treatment of SAR.

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Methods 

Subjects 

Patients with SAR caused by JC pollens were enrolled in this study. All the patients exhibited the following characteristics: (1) JC pollen-specific RAST score ≥2, (2) positive allergic reaction after nasal challenge with JC allergens, (3) JC pollen–induced symptoms of allergic rhinoconjunctivitis (from February to April) in the last 2 years and medication for the treatment of the symptoms, (4) no history of asthma, and (5) no allergen-specific immunotherapy in the past. Total and specific IgE (JC, Dermatophagoides, Dactylis glomerata, Ambrosia artemisiifolia, Candida albicans, and Aspergillus) were measured by using the CAP-RAST method (Pharmacia Diagnostics AB, Uppsala, Sweden) in all patients, and positive allergic sensitization was defined if the levels of 1 or more specific IgE molecules were greater than or equal to 0.70 IU/mL (class 2). A total of 25 patients were randomly categorized into a placebo-treated group and an actively treated group; the patients in the latter group received JC pollen extract. One patient in the placebo group withdrew from the study for personal reasons. The characteristics of the patients are presented in Table I and this article's Table E1 in the Online Repository at www.jacionline.org.

Table I. Characteristics of patients with SAR

Before enrollment, written informed consent was obtained from each patient, and the trial was performed in compliance with the Declaration of Helsinki and Good Clinical Practice. The trial was approved by the ethics committees of the University of Fukui and University of Tsukuba. The members of the ethical committee suggested that the number of samples collected for the active group should be 150% of that collected for the placebo group; thus, we collected samples according to their suggestion. JC pollen extracts were prepared by Torii Pharmaceutical Co, Ltd (Tokyo, Japan). The extracts (2000 Japanese allergy units [JAU]/mL) contained 15 μg Cry j 1 and 2 to 5 μg Cry j 2. Administration of the allergen extract was initiated at a dose of 2 JAU/mL with 50% glycerin as a diluent in November 2004; thereafter, the dose was gradually increased to 1 mL 2000 JAU/mL (final maintenance concentration) and maintained at this concentration until April 2005. The placebo-treated group was administered only the diluent, 50% glycerin. The details of the JC pollen sublingual immunotherapy have been described previously.¹⁰

Symptom-medication score and quality of life score 

The number of paroxysmal sneezes and occasions when the patients blew their noses were recorded daily on forms that were used to record nasal symptoms. On the basis of these numbers, the patients were graded on a scale of 0 to 4 (0, none; 1, 1-5 times; 2, 6-10 times; 3, 11-20 times; and 4, over 20 times). Nasal congestion was also graded on a scale of 0 to 3 (0, no symptoms; 1, mild; 2, moderate; and 3, severe symptoms).¹⁰, ¹¹ During SLIT/placebo treatment, the use of other medications, including oral antihistamine and topical steroids, was also recorded daily. The total symptom-medication score (SMS) was daily calculated on the basis of the abovementioned grades; further, we analyzed the correlation between the scores obtained over 2 weeks, during the peak JC pollination season, and the level of serum proteins. Quality of life (QOL) was assessed by using the modified Japanese version of Juniper Rhinoconjunctivitis Quality-of-Life Questionnaire (JRQLQ).¹⁰, ¹¹, ¹² This questionnaire includes 17 questions in 6 domains designed to measure the effects of rhinoconjunctivitis symptoms on disease-specific QOL.

Sample collection 

Serum was collected from each patient twice: before the pollen season and the initiation of SLIT (November 2004) and during the pollen season (May to April 2005). Serum samples were centrifuged at 3000g for 10 minutes and stored at –80°C until use.

Two-dimensional polyacrylamide gel electrophoresis and protein identification 

Two-dimensional polyacrylamide gel electrophoresis (2-DE) was performed with the IPGphor IEF System (GE Healthcare, Piscataway, NJ) and Ettan DALT six (GE Healthcare) as described previously.¹³ Labeled proteins were visualized with a Typhoon 9400 Imager (GE Healthcare), and 24 images of paired samples were analyzed with the DeCyder Software Platform version 4.0 (GE Healthcare). The detailed Methods are available in this article's Online Repository at www.jacionline.org.

The data concerning the changes in the amounts of proteins in each spot were combined, and the spots, which indicated significant changes in the amount of proteins before and after the treatment, were analyzed.

Protein spots that satisfied both the following criteria were subjected to protein identification: (1) those in which at least a 1.1-fold increase or decrease in protein expression was observed, and (2) those that indicated significant differences in the protein expression levels before and after SLIT (paired t test, P < .05). Differentially expressed protein spots were subjected to nano-HPLC, and samples were separated on a Paradigm MS4 LC system (Michrom Bioresources, Auburn, Calif).¹⁴ The purified samples were analyzed by using matrix-assisted laser desorption/ionization 2-stage time-of-flight (MALDI-TOF/TOF) mass spectrometry; all the imaging analysis was performed on an Ultraflex II (Bruker Daltonics, Billerica, Mass). Detailed Methods can be accessed in the Online Repository.

Western blot analysis of apolipoprotein A-IV 

The protocol of the Western blot analysis to validate the apolipoprotein A-IV (apoA-IV) spot is described in the Methods in the Online Repository. For the quantification of apoA-IV, the protein concentration in the serum was measured by a protein assay (Bio-Rad, Hercules, Calif). Then the protein concentrations in the serum samples were adjusted to 4 μg/μL, and these prepared samples were separated on 10% SDS-polyacrylamide gels. The separated proteins were transferred onto nylon membranes (PVDF; GE Healthcare) by a semidry electrical transfer (Bio-Rad). Nonspecific binding sites were blocked for 1 hour at room temperature with 1% blocking reagent in PBS-Tween 20 (0.1%; Roche, Indianapolis, Ind). The membranes were incubated with mAb (dilution 1:2000; antihuman apoA-IV mouse IgG antibody; BML, Saitama, Japan) for 1 hour at room temperature. After washing, the membranes were incubated with antimouse IgG (H + L-chain)–horseradish peroxidase (HRP) goat IgG antibody (MBL, Nagoya, Japan) for 1 hour at room temperature. After washing 3 times, the membranes were incubated with Immobilon western chemiluminescent HRP substrate (Millipore, Billerica, Mass) for 1 minute. The chemiluminescent images were then analyzed with a LAS-4000UVmini and Multi Gauge Version 3.0 (Fujifilm Life Science, Tokyo, Japan).

Test for histamine release from basophils containing various apolipoproteins 

Recombinant apolipoprotein A-I and apolipoprotein E were purchased from BioVision (Mountain View, Calif), and recombinant apolipoprotein C-III was purchased from Abnova Co (Taipei, Taiwan). Recombinant apoA-IV was expressed in COS-7 cells and purified by using the QIAexpress Ni-NTA Fast Start Kit (Qiagen, Valencia, Calif). Detailed Methods for the production of recombinant apoA-IV can be accessed in the Online Repository. Anticoagulated blood obtained from 6 patients with JC pollinosis was subjected to gradient centrifugation to obtain peripheral mononuclear cells by using Lymphoprep (Axis-Shield, Oslo, Norway). The basophils were enriched by using the Basophil Isolation Kit II (Miltenyi Biotec, Gladbach, Germany) and autoMACS (Miltenyi Biotec) according to the manufacturer's instructions. The enriched basophils (>98%) were seeded on a 96-well plate at a density of 5 × 10⁴ cells/well and incubated in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% FCS (Gibco, Grand Island, NY), 100 U/mL penicillin-G potassium salt (Sigma, St Louis, Mo), and 100 μg/mL streptomycin sulfate salt (Sigma) at 37°C under 5% CO₂ for 30 minutes. The basophils were further incubated with or without the apolipoproteins (apolipoprotein A-I, apoA-IV, apolipoprotein C-III, and apolipoprotein E; final concentration of apolipoproteins, 1 μg/mL) for 30 minutes; subsequently, Cry j 1 (Hayashibara Biochemical Laboratories, Okayama, Japan) was added to each well at a final concentration of 0.1 μg/mL. After 30 minutes of incubation, histamine concentrations in the cells and supernatants were determined by using a Histamine ELISA kit (Oxford Biomedical Research, Oxford, Mich) according to the manufacturer's instructions. Histamine release (%) was calculated as follows:

The percent inhibition of histamine release by apolipoproteins was calculated as follows:

Pathway analysis 

To investigate whether the differentially expressed proteins belong to specific pathways or networks, we used the IPA version 7.1 software (Ingenuity Systems, Mountain View, Calif). This web-based software enables the identification of biologic networks relevant to each researcher's experiment. A data set containing protein identifiers and the corresponding expression values was uploaded onto the Ingenuity pathways knowledge base. These uploaded proteins (referred to as focus genes) were then used as starting points for generating biological networks, and a network was constructed such that it was enriched with the proteins of interest.

Statistical analysis 

The differences in the protein levels before and after SLIT were analyzed for statistical significance by the paired t test. The statistical significance of the difference in apoA-IV levels observed by Western blot analysis was calculated by the Wilcoxon signed-rank sum test. Pairwise correlations between SMS and serum levels of apoA-IV were calculated by using the Pearson correlation. Significance was defined as P < .05.

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Results 

A representative 2-DE image of the serum samples from a SLIT-treated patient is shown in Fig 1. Sixteen spots were differentially expressed in the samples obtained before and after the pollen seasons (Fig 1; Table II). Before 2-DE analysis of the samples of patients with SAR, we evaluated the reproducibility and deviation of the spot intensity by repeated experiments with same samples labeled with 2 different dyes (Cy5 and Cy3). The variation in the 2-DE analysis was less than 1.1-fold. Thus, a protein spot showing a greater than 1.1-fold change in expression with a statistically significant difference of P <. 05 in the samples obtained before and after the pollen season was considered to be differentially expressed spot. Fig 2 shows the output of the DeCyder differential analysis software in the case of spot 10 in actively treated patients. The graphical data showed that the amount of apoA-IV protein increased after SLIT. These altered spots were excised and analyzed by MALDI-TOF/TOF analysis. Fifteen spots were successfully identified by MALDI-TOF/TOF analysis. These identified spots corresponded to 7 proteins (Table II). We classified these 7 proteins on the basis of their function into 4 categories: lipid transporters, complement factors, protease inhibitors, and transporters. An example of MALDI-TOF/TOF analysis is shown in this article's Fig E1 in the Online Repository at www.jacionline.org (spot 10, ApoA-IV). Among these identified proteins, the serum levels of complement C4A, apoA-IV, and transthyretin were significantly increased in the patients belonging to the actively treated group (P < .05), but this trend was not observed in the patients belonging to the placebo-treated group. The results of pathway analysis with the 3 SLIT-related proteins are shown in Fig 3. The IPA software generates a large global molecular network on the basis of hundreds of thousands of curated direct and indirect physical and functional interactions between orthologous mammalian genes/proteins from published, peer-reviewed content in the Ingenuity knowledge base. As shown in Fig 3, hepatocyte nuclear factor 4α (HNF4A) was identified as the hub protein in the network, indicating that these 3 proteins were regulated by the common transcription factor HNF4A.

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• Fig 1. 

  Upregulated and downregulated proteins by 2-DE analysis. Upregulated or downregulated protein spots that were altered in the actively treated and placebo-treated groups are marked with circles. The spot numbers correspond to the numbers in Table II.

Table II. Differently expressed proteins identified by 2-DE analysis

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• Fig 2. 

  DeCyder differential analysis software output. Quantitative assessments of protein spots were obtained with the DeCyder-based quantization software. The y-axis represents the standardized log abundance of protein expression. The graphical data show the abundance of the protein corresponding to spot 10 in the actively treated group. The spot number corresponds to the number in Table II.

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• Fig 3. 

  Results of the network analysis by Ingenuity pathways analysis. The complement factor C4A, apoA-IV, and transthyretin are shown in red.

Subsequently, we analyzed the correlation between the fold change in the levels of these proteins and the nasal SMS. The average SMS during the peak JC pollination season was lower in the patients belonging to the SLIT-treated group than in those belonging to the placebo-treated group (122.0 ± 62.0 vs 166.4 ± 89.0); however, this difference was not significant (P = .36). The average medication score during the peak JC pollination season was 39.4 ± 12.5 in the SLIT-treated group and 56.0 ± 16.1 in the placebo-treated group; however, the 2 groups did not differ significantly in this regard (P = .42). On the basis of the JRQLQ scores, we found that the QOL of the patients belonging to the SLIT-treated group was superior to that of the patients belonging to the placebo-treated group (9.5 ± 8.3 vs 15.9 ± 19.6; P = .048).

To confirm the results of 2-DE, we performed Western blotting with apoA-IV–specific antibody. The spot identified by Western blotting was identical to the one detected by 2-DE and MALDI-TOF/TOF analysis (see this article's Fig E2 in the Online Repository at www.jacionline.org). The serum concentration of apoA-IV in each sample was assessed by Western blotting (see this article's Fig E3 in the Online Repository at www.jacionline.org). The average fold change before and after treatment was 3.27 ± 1.3 in the SLIT-treated group and 1.57 ± 1.0 in the placebo-treated group, and the apoA-IV levels were significantly higher in actively treated group compared with the placebo-treated group (P < .05).

The levels of apoA-IV showed significant reverse correlation with SMS (Fig 4; r = –0.635; P = .011) and JRQLQ (r = –0.516; P = .049) in the case of patients belonging to the actively treated group; thus, when the levels of apoA-IV were high, a low SMS and QOL score were obtained. However, the serum levels of complement C4A and transthyretin did not show significant correlation with the SMS (P > .05).

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• Fig 4. 

  Correlation of the fold change in apoA-IV with the SMS. A statistically significant correlation was observed between the levels of apoA-IV and the SMS (r = –0.635; P = .011 in the actively treated group.

Next, to evaluate the effect of apoA-IV on histamine release from the basophils, we examined the rate of histamine release from the basophils obtained from the patients with SAR in the presence and absence of apoA-IV (Fig 5). The histamine release from basophils was greatly reduced after the addition of recombinant apoA-IV in the medium; however, this was not observed when extracts from an empty vector (41.7 ± 16.0 vs 70.0 ± 13.7; P < .01) were added to the medium. The addition of recombinant apolipoprotein A-I, apolipoprotein C-III, and apolipoprotein E did not reduce histamine release from the basophils (apolipoprotein A-I, 62.5 ± 25.3; apolipoprotein C-III, 78.3 ± 29.2; and apolipoprotein E, 85.0 ± 37.0 vs control medium, 74.3 ± 26.9; P > .05).

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• Fig 5. 

  Histamine release from basophils with or without apoA-IV. The graph shows the histamine release rate from the basophils of SAR patients with or without apoA-IV.

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Discussion 

This is the first proteomic study to investigate proteins involved in inhalant allergen immunotherapy. The serum protein levels of complement C3, α-1-antitrypsin, apolipoprotein E, and apolipoprotein A-I differed in both the actively treated group and placebo-treated group, suggesting that these 4 proteins may be altered by JC pollen exposure. On the contrary, the levels of apoA-IV, complement C4A, and transthyretin were increased in the actively treated group, whereas such a trend was not observed in the case of the placebo-treated patients. These data indicate that apoA-IV, complement C4A, and transthyretin may be altered with the SLIT treatment.

Several patients had allergy to multiple allergens, and it is possible that reaction to a particular allergen can be influenced by exposure to other allergens. We measured specific IgE levels against 6 major aeroallergens in all patients. Of these patients, 63% of the patients had allergy only to pollen from JC, and the rest had allergy to mites and other aeroallergens. Among the allergens tested, the pollens of D glomerata and A artemisiifolia are not dispersed by air currents between February and April in Japan. One patient had allergy to C albicans, and none of the patients had allergy to Aspergillus. Five patients had allergy to mites, but they did not exhibit any symptoms related to perennial allergic rhinitis or asthma. The ratio of patients with allergy to aeroallergens other than JC in the placebo-treated group (0.33) did not differ from that in the actively treated group (0.40; P = 1, Fisher exact test); therefore, the allergic reaction obtained against the other allergens did not have a significant impact on the results of our proteomic study.

Among 3 proteins altered during SLIT, the apoA-IV serum levels correlated only with the clinical symptom scores, and identified apoA-IV was detected on the same molecular weight and isoelectric point as native apoA-IV, which was theoretically calculated from the database. Therefore, we think apoA-IV we identified by 2-DE was similar to native apoA-IV. ApoA-IV is a 46-kd glycoprotein that is produced mainly in the small intestine and liver.¹⁵ Although the precise function of apoA-IV has not been completely elucidated, several functions have been proposed, such as lipid transport and metabolism,¹⁶, ¹⁷ satiety,¹⁸ and antiatherogenic effects.¹⁹, ²⁰ Several lines of evidence also suggested that apoA-IV has anti-inflammatory effects. Vowinkel et al²¹ showed in their experimental colitis model that apoA-IV knockout mice exhibited a significantly greater inflammatory response than their wild-type littermates, and this inflammation was reversed by exogenous administration of apoA-IV to knockout mice. It has also been shown that expression of human apoA-IV in apolipoprotein E knockout mice significantly reduced the development of atherosclerosis and release of cytokines such as IL-4, IFN-γ, and TNF-α induced by repeated injections of LPS.²² In the current study, increased levels of apoA-IV were observed in the actively treated group, and apoA-IV inhibited the release of histamine from basophils. These results combined with the results of previous studies indicate that the anti-inflammatory effects of apoA-IV and inhibition of histamine release by apoA-IV may contribute to the effect of SLIT for the treatment of allergic rhinitis.

The complement component system provides innate defense against microbial pathogens and acts in conjunction with antibody-mediated immunity. It has been reported that complement-activation products such as C3a and C5a, known as anaphylatoxins, contribute to inflammation in allergic rhinitis. Andersson et al²³ showed that an allergen challenge test administered to subjects with allergy induced nasal symptoms and concomitantly increased their C3a and C5a levels. We observed that the acidic complement component C4A was increased in the actively treated group compared with the control placebo group, and the levels of 2 C3 isoforms were upregulated and 1 was downregulated in the actively treated group. The human C4 complement components are encoded by 2 genes, acidic C4A and basic C4B, located on chromosome 6p21.3. C4 deficiencies are reported to be associated with Mycobacterium leprae infection and autoimmune diseases²⁴; further, increased levels of C4a, the C4 fragment formed by the cleavage reaction, were observed in the case of patients with aspirin-induced asthma compared with those with aspirin-tolerant asthma.²⁵ To date, the mechanisms underlying the control of C4A gene transcription are poorly understood; however, C4A, apoA-IV, and transthyretin were reported to be regulated by a common transcription factor, HNF4A (Fig 3).²⁶, ²⁷, ²⁸ Furthermore, apoA-IV and transthyretin possess HNF4A-binding sites in their regulatory regions.²⁸, ²⁹, ³⁰ HNF4A is a liver-enriched transcription factor, and many acute phase proteins have HNF4A-binding sites in their regulatory elements, and changes in the expression of these acute phase proteins alter the serum protein composition, which facilitates recovery from insult or stress. A recent study showed that HNF4A is responsible for the transcriptional regulatory changes in a cell injury model in which IL-1β, IL-6, and TNF-α are induced.³⁰ Therefore, we speculated that HNF4A may be upregulated by SLIT, and as a result, the SAR symptoms may be relieved, but the precise mechanism underlying HNF4A upregulation is unknown.

In conclusion, we identified proteins associated with SLIT by 2-DE analysis. Our data will increase the understanding of the mechanism of SLIT and may provide novel insights into the treatment of allergic rhinitis.

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We thank Ms Y. Ishikawa of the University of Fukui for the excellent technical assistance. We also thank all of the participants in this study.

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Methods 

2-DE 

The protein concentrations in the serum were measured by a protein assay (Bio-Rad, Hercules, Calif). Serum was diluted in lysis buffer, and samples containing 30 μg solubilized proteins were labeled with 240 pmol fluorescent dyes (Cy2, Cy3 or Cy5; GE Healthcare), which have the same molecular weight and isoelectric point but different excitation and emission wavelengths. The internal standard, which was a mixture of equal volumes of all the samples, was labeled with Cy2. Serum samples obtained before the initiation of SLIT were labeled with Cy3, and those obtained during SLIT from the same patients were labeled with Cy5. These labeled samples were mixed and solubilized in 450 μL of rehydration buffer and loaded onto 24-cm immobilized pH gradient gel strips (GE Healthcare). Isoelectric focusing was conducted at 8000 V for a total of 65 kV/h at 20°C, and 2-DE was run at 2.5 W for 30 minutes and then at 30 W for 3 hours.

Gel image pairs were processed by the Differential In-gel Analysis (DeCyderTM-DIA) software module to codetect and quantify protein spots in the images, considering the internal standard sample as a reference to normalize the data so the rest of the normalized spot maps could be compared. DeCyder biological variation analysis (DeCyderTM-BVA) was used for gel-to-gel matching of the internal standard spot maps from each gel. In the BVA, we initially analyzed 24 images of the paired samples obtained from the patients belonging to the 2 groups—the placebo-treated and actively treated groups.

Protein identification 

Protein spots that satisfied both of the following criteria were subjected to protein identification: (1) protein spots showing at least a 1.1-fold change in expression, and (2) proteins spots showing statistically significant differences in expression before and after SLIT (paired t test; P values < .05). The 2-DE was performed with internal control samples; gels were stained with Dodeca Silver Stain Kits (Bio-Rad Laboratories). Differentially expressed protein spots were excised from the gels with Ettan Spot Picker version 1.10 (GE Healthcare). These excised gels were destained with destaining solution containing 15 mmol/L potassium hexacyanoferrate (III) (Wako, Osaka, Japan) and 50 mmol/L sodium thiosulfate (Sigma, St Louis, Mo) and digested with sequencing-grade modified trypsin (Promega, Madison, Wis), and using these peptide extracts, we performed nano-HPLC sample separation with a Paradigm MS4 LC system (Michrom BioResources, Auburn, Calif)¹⁴ and MALDI-TOF/TOF mass spectrometry with ultraflex II (BRUKER Daltonics, Billerica, Mass).¹³ Molecular mass information obtained by MALDI-TOF/TOF mass spectrometry was searched against the Swiss-Prot protein database (version 56.5, http://www.expasy.org/sprot/) with the MASCOT search program (version 2.2; MatrixScience, Boston, Mass) automatically using the Warp-LC software (BURUKER Daltnics), which attached to the ultraflex II. The following search criteria were used: (1) the taxonomy was Homo sapiens (human being); (2) the specified enzyme was trypsin, with up to 1 missed cleavage permitted; (3) the fixed modifications were carbamidomethylation of cysteine residues and variable modifications were oxidation of methionine residues; and (4) the peptide tolerance and MS/MS tolerance were set at 100 ppm and ± 0.5 d, respectively.

Western blot analysis to validate the apoA-IV spot 

The serum protein concentration was measured by a protein assay (Bio-Rad). 2-DE was performed with the IPGphor IEF System (GE Healthcare) and Hoefer SE 600 Ruby standard vertical electrophoresis (GE Healthcare). The protein concentration was adjusted to 300 μg in 250 μL rehydration buffer and loaded onto 13-cm immobilized pH gradient gel strips (GE Healthcare). Isoelectric focusing was performed at 8000 V for a total of 65 kV/h at 20°C, and 2-DE was run at 2.5 W for 30 minutes and then at 30 W for 3 hours. Separated proteins were transferred onto nylon membranes (PVDF; GE Healthcare) by a semidry electrical transfer (Bio-Rad). Then the total protein separated by 2-DE was also stained with the Deep Purple Total Protein Stain (GE Healthcare) and visualized with a Typhoon 9400 Imager (GE Healthcare). Next, nonspecific binding sites on the membranes were blocked for 1 hour at room temperature in 5% skim milk (Morinaga Nyugyou, Tokyo, Japan) in PBS-Tween 20 (0.05%). Membranes were incubated with mAb (dilution 1:2000; antihuman apoA-IV mouse IgG antibody; BML, Saitama, Japan) overnight at room temperature. After washing, the membranes were incubated with antimouse IgG HRP-linked sheep antibody (GE Healthcare) overnight at room temperature. After washing 3 times, the membranes were incubated with the Western blotting luminol reagent (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) for 1 minute; then the chemiluminescent images were analyzed with the Typhoon 9400 Imager (GE Healthcare).

ApoA-IV protein purification 

The vector expressing apoA-IV in mammalian cells was developed on the basis of pBudCE4.1 (Invitrogen, San Diego, Calif). PCR was performed by using the primer pair 5′-CAG TCG ACG ATG TTC CTG AAG GCC GTG GTC and 5′-GGG ATC CCA GCT CTC CAA AGG GGC CA with human liver cDNA as a PCR template. The PCR product was digested with SalI and BamHI (TOYOBO, Tokyo, Japan), then subcloned into pBudCE4.1 (Invitrogen). The accuracy of the sequence was confirmed by the direct sequencing.

COS-7 cells were cultured in RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% FCS (Gibco, Grand Island, NY), 100 U/mL penicillin G potassium salt (Sigma), and 100 μg/mL streptomycin sulfate salt (Sigma), and these cells were transfected with apoA-IV expression constructs with the Effectene Transfection Reagent (Qiagen, Chatsworth, Calif) according to the manufacturer's protocol. After a 48-hour transfection, purification of the His-tagged protein from the cultured cells was performed by using the QIAexpress Ni-NTA Fast Start Kit (Qiagen) according to the manufacturer's instructions.

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Fig E1. 

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• An example of nano-HPLC sample separation and MALDI-TOF/TOF analysis (spot 10). A, An example of a chromatograph of nano-HPLC. The y-axis represents intensity, which shows the relative abundance of the separated peptide at each separation time. B, An example of MALDI-TOF/TOF analysis. The MS/MS data show underlined amino acid sequences, which form part of apoA-IV.

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Fig E2. 

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• Western blotting with apoA-IV–specific antibody. A, 2-DE image of the total protein stained with Deep Purple total protein stain. B, 2-DE image of the total protein merged with the image detected with the apoA-IV–specific antibody. The arrow indicates the spot corresponding to apoA-IV.

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Fig E3. 

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• Representative Western blot image of apoA-IV in actively treated and placebo-treated groups.

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Table E1. 

Characteristics of patients

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