The TLR7 agonist imiquimod selectively inhibits IL-4-induced IgE production by suppressing IgG1/IgE class switching and germline ε transcription through the induction of BCL6 expression in B cells.

Imiquimod (IMQ) is a selective toll-like receptor 7 (TLR7) agonist. TLR7 activation leads to the production of IFN-γ and pro-inflammatory cytokines by innate immune cells. However, the role of TLR7 in B cells is not fully understood. In this study, we investigated the direct effect of in vitro stimulation with IMQ on Ab production and isotype switching in B cells. IMQ selectively diminished IL-4-induced IgE and IgG1 production in anti-CD40-activated mouse B cells. IMQ also inhibited germline ε transcripts (GLTε)/GLTγ1 and post-switch ε transcripts (PSTε)/PSTγ1 expression, while enhancing GLTγ2c and PSTγ2c expression in anti-CD40/IL-4-stimulated B cells. Interestingly, IMQ abrogated IL-4-induced circle transcripts ε-γ1 (CTε-γ1) expression, indicative of sequential switching from IgG1 to IgE. Furthermore, IMQ repressed IL-4-induced surface IgE/IgG1 expression while increasing surface IgG2c expression. The selective inhibition of IgE synthesis was not due to IMQ-induced production of IFN-γ or IL-12 in the same culture. IMQ also enhanced BCL6 expression, a transcriptional repressor for the GLTε promoter, in anti-CD40/IL-4-stimulated B cells. In addition, BCL6 siRNA restored IMQ-mediated suppression of GLTε transcription. Therefore, these results indicate that TLR7 engagement by IMQ inhibits IL-4-induced GLTε transcription by enhancing BCL6 expression and inhibits IL-4-induced sequential switching from IgM to IgE via IgG1, thus resulting in the downregulation of IgE production by B cells.

[Eukaryotic expression, purification of human IgECepsilon2-4 protein and its target fishing].

Objective To construct eukaryotic expression vectors of human IgE heavy chain 2-4 region (IgECepsilon2-4) and purify the recombinant protein, and then capture its interacted proteins by surface plasmon resonance (SPR). Methods Three recombinant eukaryotic expression vectors of IgECepsilon2-4 containing different signal peptides were constructed and transiently transfected into HEK293FT suspension cells separately. The recombinant plasmid with the highest-level expression was selected to express the recombinant protein in a huge amount, and then the recombinant protein was purified by Ni-NTA affinity chromatography. The interaction between high-affinity IgE receptor (FcepsilonR I) of KU812 cell surface and IgECepsilon2-4 was identified by immunofluorescence cytochemistry. The unknown proteins that specifically interacted with IgECepsilon2-4 were captured from human serum by SPR technique. Results The recombinant plasmid containing the signal peptide III showed the highest expression (6.2 mg/L). Highly purified recombinant protein IgECepsilon2-4 was obtained by affinity purification. Immunofluorescence cytochemistry showed that the recombinant protein IgECepsilon2-4 could be combined with the surface receptor of KU812 cells. Thirty-nine kinds of proteins which were likely to interact with IgECepsilon2-4 were captured from human serum by SPR. Conclusion We obtained the purified recombinant protein IgECepsilon2-4 that could be combined with KU812 cell surface receptor. Target fishing experiment revealed that the recombinant protein IgECepsilon2-4 was likely to interact with 39 kinds of proteins in human serum.

Dissociation between skin test reactivity and anti-aeroallergen IgE: Determinants among urban Brazilian children.

BACKGROUND: The dissociation between specific IgE and skin prick test reactivity to aeroallergens, a common finding in populations living in low and middle-income countries, has important implications for the diagnosis and treatment of allergic diseases. Few studies have investigated the determinants of this dissociation. In the present study, we explored potential factors explaining this dissociation in children living in an urban area of Northeast Brazil, focusing in particular on factors associated with poor hygiene. METHODS: Of 1445 children from low income communities, investigated for risk factors of allergies, we studied 481 with specific IgE antibodies to any of Blomia tropicalis, Dermatophagoides pteronyssinus, Periplaneta americana and Blatella germanica allergens. Data on demographic, environmental and social exposures were collected by questionnaire; serum IgG and stool examinations were done to detect current or past infections with viral, bacterial, protozoan and intestinal helminth pathogens. We measured atopy by skin prick testing (SPT) and specific IgE (sIgE) to aerollergens in serum (by ImmunoCAP). SIgE reactivity to B. tropicalis extract depleted of carbohydrates was measured by an in-house ELISA. Total IgE was measured by in house capture ELISA. SNPs were typed using Illumina Omni 2.5. RESULTS: Negative skin prick tests in the presence of specific IgE antibodies were frequent. Factors independently associated with a reduced frequency of positive skin prick tests were large number of siblings, the presence of IgG to herpes simplex virus, Ascaris lumbricoides and Trichuris trichiura infections, living in neighborhoods with infrequent garbage collection, presence of rodents and cats in the household and sIgE reactivity to glycosylated B. tropicalis allergens. Also, SNP on IGHE (rs61737468) was negatively associated with SPT reactivity. CONCLUSIONS: A variety of factors were found to be associated with decreased frequency of SPT such as unhygienic living conditions, infections, total IgE, IgE response to glycosylated allergens and genetic polymorphisms, indicating that multiple mechanisms may be involved. Our data, showing that exposures to an unhygienic environment and childhood infections modulate immediate allergen skin test reactivity, provide support for the "hygiene hypothesis".

Histone H3k9 and H3k27 Acetylation Regulates IL-4/STAT6-Mediated Igε Transcription in B Lymphocytes.

IL-4 activates STAT6 and causes the subsequent up-regulation of Ig heavy chain germline Igε via chromatin remodeling involved in B lymphocytes development. STAT6 acts as a molecular switch to regulate the higher-order chromatin remodeling via dynamically orchestrating co-activators (CBP/Tudor-SN) and co-repressors (HDAC1/PSF). Here, we demonstrated that STAT6/Tudor-SN/PSF form a complex, balancing the acetylation and deacetylation states to co-regulate IL-4/STAT6 gene transcription. In addition, we confirmed that IL-4 treatment increased the HATs activity in Ramos cells. As "active" markers, the expression of H3K9ac and H3K27ac increased after treatment with IL-4. However, transcriptional repressors such as H3K9me3 and H3K27me3 decreased in response to IL-4 stimulation. Moreover, IL-4 treatment enhanced H3 acetylation at the Igε promoter regions. Our results revealed that the Igε gene transcription is regulated by histone modifications in the IL-4/STAT6 pathway. The study will provide novel insights into the pathogenesis of allergic diseases.

Generating allergen-specific human IgEs for immunoassays by employing human ε gene knockin mice.

BACKGROUND: Antigen-specific human IgEs are important reagents in immunoassays to quantify antigen-specific IgEs in allergic patients, but they are not easy to prepare. METHODS: We constructed a knockin homozygous mouse strain, referred to as HεκKI strain, whose gene segment encoding γ1 constant region has been replaced by that encoding human ε constant region and gene segment encoding κ constant region replaced by that encoding human κ constant region. The mice were tested for their ability to produce antigen-specific chimeric human IgE (with mouse variable regions) upon the immunization with ovalbumin and papain. Subsequently, the spleen cells from the immunized mice were used as the source of B cells for the preparation of hybridomas, which secreted monoclonal human IgE antibodies specific for the antigens. RESULTS: The HεκKI mice expressed human IgE (ε, κ) in serum at levels 10- to 30-fold higher than those of mouse IgE. Upon immunization with an antigen, the mice yielded splenic B cells for preparing hybridomas that secrete chimeric human IgE specific for the antigen. Purified IgEs from those hybridomas could activate a basophilic cell line to undergo degranulation upon the stimulation with their respective antigens. CONCLUSIONS: We have developed a human ε gene and κ gene knockin mouse strain, which is useful for producing various antigen-specific chimeric human IgEs for potential use as standards in immunoassays.

TRIF signaling is essential for TLR4-driven IgE class switching.

The TLR4 ligand LPS causes mouse B cells to undergo IgE and IgG1 isotype switching in the presence of IL-4. TLR4 activates two signaling pathways mediated by the adaptor molecules MyD88 and Toll/IL-IR domain-containing adapter-inducing IFN-ß (TRIF)-related adaptor molecule (TRAM), which recruits TRIF. Following stimulation with LPS plus IL-4, Tram(-/-) and Trif(-/-) B cells completely failed to express Cε germline transcripts (GLT) and secrete IgE. In contrast, Myd88(-/-) B cells had normal expression of Cε GLT but reduced IgE secretion in response to LPS plus IL-4. Following LPS plus IL-4 stimulation, Cγ1 GLT expression was modestly reduced in Tram(-/-) and Trif(-/-) B cells, whereas Aicda expression and IgG1 secretion were reduced in Tram(-/-), Trif(-/-), and Myd88(-/-) B cells. B cells from all strains secreted normal amounts of IgE and IgG1 in response to anti-CD40 plus IL-4. Following stimulation with LPS plus IL-4, Trif(-/-) B cells failed to sustain NF-κB p65 nuclear translocation beyond 3 h and had reduced binding of p65 to the Iε promoter. Addition of the NF-κB inhibitor, JSH-23, to wild-type B cells 15 h after LPS plus IL-4 stimulation selectively blocked Cε GLT expression and IgE secretion but had little effect on Cγ1 GLT expression and IgG secretion. These results indicate that sustained activation of NF-κB driven by TRIF is essential for LPS plus IL-4-driven activation of the Cε locus and class switching to IgE.

Polyclonal hyper-IgE mouse model reveals mechanistic insights into antibody class switch recombination.

Preceding antibody constant regions are switch (S) regions varying in length and repeat density that are targets of activation-induced cytidine deaminase. We asked how participating S regions influence each other to orchestrate rearrangements at the IgH locus by engineering mice in which the weakest S region, Sε, is replaced with prominent recombination hotspot Sµ. These mice produce copious polyclonal IgE upon challenge, providing a platform to study IgE biology and therapeutic interventions. The insertion enhances ε germ-line transcript levels, shows a preference for direct vs. sequential switching, and reduces intraswitch recombination events at native Sµ. These results suggest that the sufficiency of Sµ to mediate IgH rearrangements may be influenced by context-dependent cues.

[Screening and characterization of aptamers of Cepsilon3-Cepsilon4 protein].

In order to obtain nucleotides aptamers bind to IgE, 80 bp nucleotides single-stranded DNA library containing 40 random nucleotides was designed and synthesized. Oligonucleotides that bind to human Cepsilon3-Cepsilon4 protein were isolated from ssDNA pools by the systematic evolution of ligands by exponential enrichment (SELEX) method using nitrocellulose filters as screening medium. Through the optimization of critical PCR and asymmetric PCR parameters including annealing temperature, cycles, and molar ratios of target protein and ssDNA etc, a suitable screening system was established. The aptamers of Cepsilon3-Cepsilon4 protein with high affinity and high specificity were identified by ELISA with biotin-streptavidin-horseradish peroxidase system, and its primary sequence and second structure were analyzed by DNAMAN package and DNA folding sever after being cloned and sequenced. Moreover, target protein was bound to one aptamer and another aptamer modified with biotion together forming a sandwich-like complex, which was captured in microwell to detect IgE concentration using the optimal combination in the sandwich method named enzyme-linked aptamers sorption assay (ELASA). The method could be used for the quantitative detection of human IgE, and whose sensitivity reached to 120 ng x mL(-1).

Immature B cells preferentially switch to IgE with increased direct Sµ to Sε recombination.

Immunoglobulin heavy chain (IgH) class-switch recombination (CSR) replaces initially expressed Cµ (IgM) constant regions (C(H)) exons with downstream C(H) exons. Stimulation of B cells with anti-CD40 plus interleukin-4 induces CSR from Cµ to Cγ1 (IgG1) and Cε (IgE), the latter of which contributes to the pathogenesis of atopic diseases. Although Cε CSR can occur directly from Cµ, most mature peripheral B cells undergo CSR to Cε indirectly, namely from Cµ to Cγ1, and subsequently to Cε. Physiological mechanisms that influence CSR to Cγ1 versus Cε are incompletely understood. In this study, we report a role for B cell developmental maturity in IgE CSR. Based in part on a novel flow cytometric IgE CSR assay, we show that immature B cells preferentially switch to IgE versus IgG1 through a mechanism involving increased direct CSR from Cµ to Cε. Our findings suggest that IgE dysregulation in certain immunodeficiencies may be related to impaired B cell maturation.

PTB-associated splicing factor (PSF) functions as a repressor of STAT6-mediated Ig epsilon gene transcription by recruitment of HDAC1.

Regulation of transcription requires cooperation between sequence-specific transcription factors and numerous coregulatory proteins. In IL-4/IL-13 signaling several coactivators for STAT6 have been identified, but the molecular mechanisms of STAT6-mediated gene transcription are still not fully understood. Here we identified by proteomic approach that the PTB-associated splicing factor (PSF) interacts with STAT6. In intact cells the interaction was observed only after IL-4 stimulation. The IL-4-induced tyrosine phosphorylation of both STAT6 and PSF is a prerequisite for the efficient association of the two proteins. Functional analysis demonstrated that ectopic expression of PSF resulted in inhibition of STAT6-mediated transcriptional activation and mRNA expression of the Igε germline heavy chain gene, whereas knockdown of PSF increased the STAT6-mediated responses. PSF recruited histone deacetylase 1 (HDAC1) to the STAT6 transcription complex, which resulted in reduction of H3 acetylation at the promoter regions of Ig heavy chain germline Igε and inhibition of STAT6-mediated transcription. In addition, the HDACs inhibitor trichostatin A (TSA) enhanced H3 acetylation, and reverted the PSF-mediated transcriptional repression of Igε gene transcription. In summary, these results identify PSF as a repressor of STAT6-mediated transcription that functions through recruitment of HDAC to the STAT6 transcription complex, and delineates a novel regulatory mechanism of IL-4 signaling that may have implications in the pathogenesis of allergic diseases and pharmacological HDAC inhibition in lymphomas.

Hydrolyzed Konjac glucomannan suppresses IgE production in mice B cells.

BACKGROUND: Oral administration of pulverized Konjac glucomannan (KGM) reduces increased plasma IgE and the amount of epsilon-germline transcript (epsilonGT) in the spleen, as well as preventing the development of dermatitis in mice. To elucidate the mechanism of action of pulverized KGM, we solubilized KGM and studied its effect on IgE in vitro and in vivo. METHODS: Solubilized KGM was prepared by acid hydrolysis, and we analyzed the effective molecular size for the suppression of IgE production and epsilonGT in vitro and the level of plasma IgE induced by immunization with ovalbumin in BALB/c mice. RESULTS: The production of IgE and epsilonGT in splenic cells, but not purified B cells, was inhibited by hydrolyzed KGM (KGM hydrolyzed with 0.25 N HCl; H-KGM) at the optimal size of between 10 and 500 kDa. However, no effect was observed when H-KGM was substituted with unhydrolyzed KGM in vitro. IgE production from purified B cells cocultured with purified monocytes, but not with purified T cells, was inhibited by H-KGM. The release of IFNgamma in cultures of monocytes but in purified B cells with or without T cells was enhanced in the presence of H-KGM. Injection of mice with H-KGM also suppressed the production of plasma IgE and IgG1 but not IgG2a in vivo. CONCLUSION: KGM at an optimal size prevents germline class-switching and IgE production both in vitro and in vivo. H-KGM may be useful as a tool to study the mechanism of action of KGM and as a dietary supplement to prevent atopic diseases.

Flavones suppress type I IL-4 receptor signaling by down-regulating the expression of common gamma chain.

Immunoglobulin E (IgE) production is induced by interleukin (IL)-4 signaling mediated by type I IL-4 receptor (IL-4R) in B cells. We found that flavones inhibited IL-4-induced epsilon germline transcription which is essential for IgE class switching, and the phosphorylation of signal transducer and activator of transcription 6, janus kinase 3, and IL-4Ralpha, whereas IL-4 signaling mediated through type II IL-4R was unaffected by flavones. Furthermore, flavones reduced the expression of common gamma chain, a characteristic constituent subunit of type I IL-4R, suggesting that flavones suppress type I IL-4R signaling.

Class switch recombination to IgE in the bronchial mucosa of atopic and nonatopic patients with asthma.

BACKGROUND: Class switching from IgM/IgG/IgA to IgE is required for B cells to express IgE. This requires class switch recombination in the Ig heavy-chain gene locus. It is generally believed that class switch recombination occurs in lymphoid tissue, but it was recently shown that class switching to IgE occurs in the nasal mucosa in allergic rhinitis. OBJECTIVE: We aimed to determine whether class switching to IgE also occurs in the bronchial mucosa in asthma, and to look for possible differences/similarities between atopic and nonatopic asthma. METHODS: We have used RT-PCR to examine epsilon immunoglobulin heavy-chain germline gene transcripts (GLTs; epsilonGLTs), epsilon circle transcripts (CTs; Ivarepsilon-Cmu CT or Ivarepsilon-Cgamma CT), and mRNA encoding the heavy chain of IgE (epsilon mRNA) and activation-induced cytidine deaminase (AID) in bronchial biopsies from atopic patients with asthma, nonatopic patients with asthma, atopic controls without asthma, and nonatopic controls without asthma (10 subjects in each group). RESULTS: The varepsilonGLT and AID mRNA were detectable in the bronchial mucosa of subjects in all 4 groups. In contrast, Iepsilon-Cmu CT, Ivarepsilon-Cgamma CT, and epsilon mRNA were detectable in the bronchial mucosa of the majority of both atopic and nonatopic patients with asthma, but rarely in the controls without asthma. CONCLUSION: The bronchial mucosa is a site primed in all individuals for class switching to IgE, because of B-cell expression of epsilonGLT and AID mRNA. However, it is only in patients with asthma, regardless of atopic status, that class switching to IgE occurs. CLINICAL IMPLICATIONS: Our findings reveal prospects for local targeting of the Ig class switch mechanism in the management of atopic and nonatopic asthma.

Regulation of epsilon germline transcription and switch region mutations by IgH locus 3' enhancers in transgenic mice.

Germline (GL) transcription is regulated by specific promoters and immunoglobulin heavy chain (IgH) 3' locus enhancers and is necessary for Ig class-switch recombination (CSR). We have generated different transgenic lines containing the GL epsilon promoter, switch (S) epsilon region, and constant (C) epsilon region with or without the DNase I-sensitive regions (HS) 3A-HS1,2 or HS3B-HS4 3' IgH enhancer pairs. The enhancerless construct was expressed in B cells activated by interleukin (IL)-4 and CD40, thus resembling regulation of the endogenous gene. Both enhancer-containing transgenes efficiently increased expression in B cells and were strongly up-regulated by stimuli. In addition, Sepsilon regions of the transgene containing HS3B-HS4 were mutated in activated, sorted B cells. Such mutations are known to precede CSR and are dependent on activation-induced cytidine deaminase (AID). Our findings show that all elements necessary for recruitment of the recombination machinery are present in the transgene containing HS3 and HS4. These enhancers probably provide something more specific than mere increased accessibility of switch regions. We propose that transcription factors binding the enhancers help to target the recombination machinery to the switch regions.

Regulation of IgE homeostasis, and the identification of potential targets for therapeutic intervention.

Atopic allergies have increased during the past 20-30 years in frequency quite dramatically and in many countries have reached almost epidemic proportions. Allergies have thereby become one of the major medical issues of the western world. Immunoglobulin E (IgE) is here a central player. IgE is the Ig class that is present in the lowest concentration in human plasma. IgG is, for example, 10 000 to 1 million times more abundant than IgE. However, despite of its low plasma levels IgE is a very important inducer of inflammation, due to its interaction with high-affinity receptors on mast cell and basophils. IgE has been conserved as a single active gene in all placental mammals studied, and the expression of this gene is under a very stringent control, most likely due to its very potent inflammatory characteristics. IgE expression is being regulated at many levels: by cytokines, switch region length, positive and negatively acting transcription factors and suppressors of cytokine signaling (SOCS). In addition, the plasma half-life differs markedly for IgG and IgE, with 21 and 2.5 days, respectively. This review summarizes the rapid progress in our understanding of the complex network of regulatory mechanisms acting on IgE and also how this new information may help us in our efforts to control IgE-mediated inflammatory conditions.

Characterization of RNA helicase A as component of STAT6-dependent enhanceosome.

Signal transducer and activator of transcription 6 (STAT6) is a regulator of transcription for interleukin-4 (IL-4)-induced genes. The ability of STAT6 to activate transcription depends on functional interaction with other transcription factors and coactivators. We have characterized the mechanism of STAT6-mediated transcriptional activation by identifying STAT6 transcription activation domain (TAD) interacting nuclear proteins. The first of the identified proteins was coactivator protein p100, which regulates IL-4-induced transcription by connecting STAT6 with other transcriptional regulators. Here, we describe RNA helicase A (RHA) as a novel component of STAT6 transcriptosome. In vitro and in vivo experiments indicated that RHA did not directly interact with STAT6, but p100 protein was found to mediate the assembly of the ternary complex of STAT6-p100-RHA. In chromatin immunoprecipitation studies RHA together with p100 enhanced the binding of STAT6 on the human Igepsilon promoter after IL-4 stimulation. RHA enhanced the IL-4-induced transcription, and the participation of RHA in IL-4-regulated transcription was supported by RNAi experiments. Our results suggest that RHA has an important role in the assembly of STAT6 transcriptosome. As RHA is also known to interact with chromatin modifying proteins, the RHA containing protein complexes may facilitate the entry of transcriptional apparatus to the IL-4 responsive promoters.

Detection of epsilon class switching and IgE synthesis in human B cells.

We observed that mast cells, as other cells expressing the CD40 ligand CD154, can trigger IgE synthesis in B cells in the presence of interleukin (IL)-4. Numerous complementary techniques can be used to follow the succession of molecular events leading to IgE synthesis. This chapter will illustrate how human B cells (naïve or memory) can be purified, stored, and cultivated in medium that is permissive for IgE synthesis and stimulated with IL-4 or IL-13 and CD40 activation, the latter being induced by soluble CD154, anti-CD40 antibodies, or CD154-expressing cells. All these molecules are expressed by mast cells. The quantification of the epsilon-sterile transcript synthesis by polymerase chain reaction or Northern blot, the epsilon excision circles produced during immunoglobulin heavy chain locus rearrangement by polymerase chain reaction, and the IgE production by enzyme-linked immunosorbent assay will be described.

Modification of Cepsilon mRNA expression by EBV-encoded latent membrane protein 1.

To evaluate the effect of expression of latent membrane protein (LMP) 1 encoded by Epstein-Barr virus (EBV) on Cepsilon mRNA expression, mRNA levels were examined by RT-PCR or Northern blot analysis upon transient transfection of LMP1 in the splenocytes derived from Brown-Norway rats with or without immunization with 2,4-dinitrophenyl-conjugated Ascaris suum antigen. Splenocytes were transfected with LMP1 expression vector, pSG5-LMP1, using lipofection method. Cepsilon mRNA levels were considerably increased by transfection with pSG5-LMP1 in the splenocytes derived from the nonimmunized rats; however, Cepsilon mRNA levels were decreased in the splenocytes derived from the immunized rats. Cepsilon mRNA expression in IgE-producing cells are modulated by LMP1, which might depend on the differentiation status of B cells upon exposure to allergen.

Transcription of Ig germline genes in single human B cells and the role of cytokines in isotype determination.

We have developed a critical test of the chromatin accessibility model of Ig isotype determination in which local unfolding of chromatin higher order structure (chromatin accessibility) in the region of specific germline genes in the H chain locus determines the Ab class to be expressed in the B cell. We show that multiple germline genes are constitutively transcribed in the majority of naive human B cells in a population. Thus, because chromatin in its higher order structure cannot be transcribed, the entire Ig H chain locus must be unfolded in naive B cells. We have also established that IL-4 and anti-CD40 act by enhancing transcription in the majority of cells, rather than by activating transcription in more of the cells. Transcriptional activity in the human H chain locus rules out the perturbation of chromatin higher order structure as a factor in isotype determination. We have also found that the levels of germline gene transcription cannot fully account for the levels of secretion of the different Ig isotypes, and that secretion of IgE, in particular, is suppressed relative to that of IgG.

Characterization of VHepsilon gene expressed in PBL from children with atopic diseases: detection of homologous VH1-69 derived transcripts from three unrelated patients.

To elucidate the molecular background of IgE production in early infancy, we analyzed the nucleotide sequences of 36 VH-Cepsilon transcripts expressed in PBL from three infants with allergic diseases. We detected transcripts derived from VH1, VH3, VH4 gene family members, and no bias was observed in the usage of particular VH gene family. However, some VH members, VH1; 1-46, 1-69, VH3; 3-11, 3-21, VH4; 4-39, 4-59 were frequently seen and thus notable. VH4 gene was dominant in one patient with severe atopic dermatitis and food allergy, suggesting the involvement of this gene in pathogenesis of the disease. Even a limited number of clones were analyzed, we also found highly homologous VH1-69 derived sequences from all the three patients, which share the same somatic mutations or polymorphic variations in complementarity-determining region (CDR) 1, and 2 with the same CDR3 (D-JH) sequences including the junctions. These findings might suggest that a rather limited VH gene might be rearranged for specific IgE in early life.