Decreased Basophil Activation against House Dust Mite after Japanese Cedar Pollen Subcutaneous Immunotherapy: A Retrospective Study.

BACKGROUND: Allergen-specific immunotherapy (AIT), an established treatment for allergic diseases, prevents the development of other allergic manifestations. Although the mechanisms remain unclear, AIT has been shown to reduce basophil activation (BA) against nontarget allergens. OBJECTIVES: The aim of this study was to assess immunological changes in Dermatophagoides farinae (Der f) after Japanese cedar pollen (JCP)-based subcutaneous immunotherapy (SCIT) monotherapy. METHOD: The data of 16 patients (age: 6-37 years) with JCP-induced allergic rhinitis who were sensitive to Der f (serum Der f-specific immunoglobulin E [IgE] level >0.34 kUA/L) and received JCP-based SCIT for 5 years were reviewed retrospectively. BA by Der f and JCP extracts and serum-specific IgE and immunoglobulin G4 (IgG4) levels against these allergens were evaluated before and after completing 5 years of JCP-based SCIT monotherapy. RESULTS: The areas under the dose-response curves of BA by Der f and JCP extracts were significantly reduced (p = 0.02 and p = 0.002, respectively). JCP-specific IgE levels decreased and JCP-specific IgG4 levels increased significantly (p < 0.001 for both), whereas Der f-specific IgE and IgG4 levels did not change significantly. CONCLUSIONS: JCP-based SCIT monotherapy reduced Der f-specific BA. These findings suggest that JCP-based SCIT has the potential to modulate immune response toward nontarget allergens.

Breakpoints in complex chromosomal rearrangements correspond to transposase-accessible regions of DNA from mature sperm.

Constitutional complex chromosomal rearrangements (CCRs) are rare cytogenetic aberrations arising in the germline via an unknown mechanism. Here we analyzed the breakpoint junctions of microscopically three-way or more complex translocations using comprehensive genomic and epigenomic analyses. All of these translocation junctions showed submicroscopic genomic complexity reminiscent of chromothripsis. The breakpoints were clustered within small genomic domains with junctions showing microhomology or microinsertions. Notably, all of the de novo cases were of paternal origin. The breakpoint distributions corresponded specifically to the ATAC-seq (assay for transposase-accessible chromatin with sequencing) read data peak of mature sperm and not to other chromatin markers or tissues. We propose that DNA breaks in CCRs may develop in an accessible region of densely packaged chromatin during post-meiotic spermiogenesis.

TREC/KREC Newborn Screening followed by Next-Generation Sequencing for Severe Combined Immunodeficiency in Japan.

PURPOSE: The aim of this study is to evaluate the usefulness of T cell receptor excision circle (TREC) and/or kappa-deleting recombination excision circle (KREC) measurements integrated with diagnostic next-generation sequencing (NGS) analysis using a severe combined immunodeficiency (SCID) newborn screening (NBS) program. METHODS: TREC and/or KREC values were measured in 137,484 newborns between April 2017 and December 2021 using EnLite TREC (n = 80,791) or TREC/KREC kits (n = 56,693). For newborns with positive screening results, diagnostic NGS analysis was performed with a 349-gene panel to detect genetic mutations associated with primary immunodeficiencies (PIDs). RESULTS: A total of 145 newborns (0.11%) had abnormal TREC and/or KREC values, and a genetic diagnosis was established in 2 patients with SCID (1 in 68,742 newborns) (IL2RG-SCID and reticular dysgenesis) and 10 with non-SCID PIDs with T and/or B cell deficiencies (1 in 13,748 newborns) using NGS analysis. Furthermore, TREC values of 2849 newborns were measured and confirmed the significant correlation between the results of both TREC and TREC/KREC kits (P < 0.001) and naïve T cell counts. CONCLUSIONS: We performed the first large-scale TREC and TREC/KREC NBS programs in Japan. Our NBS programs followed by the diagnostic NGS analysis for newborns with abnormal TREC and/or KREC values are useful for the early identification and rapid molecular evaluation of not only SCID but also different non-SCID PIDs.

Evaluation of oral immunotherapy efficacy and safety by maintenance dose dependency: A multicenter randomized study.

BACKGROUND: Generally, oral immunotherapy (OIT) aims for daily administration. Recently, the efficacy of treatment with OIT at a low dose has been reported. However, the optimal dose and the evaluation of dose-dependent OIT outcome have not been described. METHODS: A multicenter, parallel, open-labeled, prospective, non-placebo controlled, randomized study enrolled 101 Japanese patients for treatment with OIT. We hypothesized that target dose OIT would induce short-term unresponsiveness (StU) earlier than reduced dose OIT. StU was defined as no response to 6200 mg whole egg, 3400 mg milk, and 2600 mg wheat protein, as evaluated by oral food challenge after 2-week ingestion cessation. To compare the two doses of OIT efficacy, the maximum ingestion doses during the maintenance phase of OIT were divided into 100%-dose or 25%-dose groups against their target StU dose, respectively. A total of 51 patients were assigned to the 100%-dose group [hen's egg (HE) = 26, cow's milk (CM) = 13, wheat = 12] and 50 to the 25%-dose group (HE = 25, CM = 13, wheat = 12). Primary outcome was established by comparing StU at 1 year. Secondary outcome was StU at 2 years and established by comparing allergic symptoms and immunological changes. RESULTS: The year 1 StU rates (%) for the 100%- and 25%-dose groups were 26.9 vs. 20.0 (HE), 7.7 vs. 15.4 (CM), and 50.0 vs. 16.7 (wheat), respectively. The year 2 StU rates were 30.8 vs. 36.0 (HE), 7.7 vs. 23.1 (CM), and 58.3 vs. 58.3 (wheat), respectively. There were no statistically significant differences in StU between years 1 and 2. The total allergic symptom rate in the 25%-dose group was lower than that in the 100%-dose group for egg, milk, and wheat. Antigen-specific IgE levels for egg-white, milk, and wheat decreased at 12 months. CONCLUSIONS: Reduced maintenance dose of egg OIT showed similar therapeutic efficacy to the target dose. However, we were not able to clearly demonstrate the efficacy, particularly for milk and wheat. Reducing the maintenance dose for eggs, milk, and wheat may effectively lower the symptoms associated with their consumption compared to the target OIT dose. Furthermore, aggressive reduction of the maintenance dose might be important for milk and wheat, compared to the 25%-dose OIT. TRIAL REGISTRATION: UMIN000009373, Multicenter Oral Immunotherapy for Hen's Egg, Cow's Milk, and Wheat-Allergic Children at Outpatient Clinic.

Changes in passively-sensitized basophil activation to αS1-casein after oral immunotherapy.

INTRODUCTION: Immune response to cow's milk allergen (CMA) has been analyzed mostly using crude milk antigen or a mixture of various caseins. This study aimed to assess the changes in the immunological response against αS1-casein during oral immunotherapy (OIT) and to investigate the mechanism of tolerance. METHODS: We have performed rush OIT to 39 patients with CMA and obtained the serum samples up to 3 years after OIT. Immunoglobulin E (IgE) and IgG4 antibodies specific to highly purified αS1-casein as well as passively-sensitized basophil activation were evaluated using the serial samples. Furthermore, we examined whether basophil activation led by the pre-OIT serum was suppressed by the post-OIT serum, or by the tolerant serum obtained from naturally outgrown patients. RESULTS: Specific IgE to αS1-casein was significantly reduced after OIT. Specific IgG4 (sIgG4) to αS1-casein was also detected in most of the pre-OIT sera, which was not significantly increased after OIT. Activation of passively-sensitized basophils to αS1-casein was significantly reduced after 2 years (14% ± 19%) and 3 years (19% ± 18%) post-OIT compared with pre-OIT (%CD63high basophils; 51% ± 27%). Furthermore, the addition of post-OIT or tolerant serum to pre-OIT serum significantly suppressed the basophil activation. This suppression was abrogated by washing the supernatant after passive sensitization, but not by depleting IgG antibodies from post-OIT or tolerant sera, nor by blocking FcγRIIb using an anti-FcγR antibody. CONCLUSIONS: αS1-casein-sIgG4 plays a minor role in tolerance mechanisms in cases of CMA; humoral factors other than antigen-sIgG4 may be involved.

Changes in the characteristics of patients with latex allergy from 1999 to 2014.

OBJECTIVES: We conducted a multicenter study using the same questionnaire in 1999 and 2014 to investigate changes in the characteristics of patients with latex allergy. METHODS: We mailed questionnaires on latex allergy to hospitals in Japan that were members of the Japanese Latex Allergy Society. RESULTS: We compared the 25 responses received in 2014 and the 81 responses received in 1999. With regard to the age distribution, the number of patients with latex allergy in their 20s declined significantly from 1999 to 2014 (P=0.004). The largest proportion of latex allergy cases was observed among those aged <10 years. The incidence of cases caused by medical rubber gloves decreased significantly (P=0.004). Moreover, latex-fruit syndrome increased from 15% to 40% (P=0.006). CONCLUSIONS: Our findings indicate that the frequency of occurrence of latex allergy in people in their 20s decreased from 1999 to 2014. The largest proportion of latex allergy cases was observed among those aged <10 years. Future measures to protect children are required.

Identification of autoantibodies using human proteome microarrays in patients with IPEX syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is one of the inborn errors of immunity, characterized by impaired function of the regulatory T cells. Clinical manifestations of IPEX syndrome are characterized by various autoimmune diseases with autoantibodies. The comprehensive analysis for autoantibodies using human proteome microarrays in the four patients with IPEX syndrome was performed. The numbers of the highly expressed autoantibody showing relative log2 ratios greater than 1 were 1876, 513, 234 and 831 (mean: 864), respectively. Some novel autoantibodies which could explain the phenotypes of patients, adrenal dysfunction, muscular hypotonia, afibrinogenemia, enteropathy and pancytopenia were identified. Various kinds of autoantibodies targeting testis-specific antigens were also identified. Human proteome microarray is a powerful tool to understand the pathophysiology of IPEX syndrome. The larger cohort analysis using this method will provide further understanding of the impaired immune tolerance in humans.

Tolerability of partially and extensively hydrolysed milk formulas in children with cow's milk allergy.

BACKGROUND AND OBJECTIVES: The safety and tolerability of hydrolysed cow's milk protein-based formulas, particularly partially hydrolysed formulas (pHFs), in children with cow's milk allergy (CMA) remain poorly understood. We evaluated the tolerability of hydrolysed cow's milk-based formulas in children with CMA. METHODS AND STUDY DESIGN: A three-period double-blind crossover evaluation compared the allergic tolerance against three dietary cow's milk-based formulas: extensively hydrolysed cow's milk formula (eHF), pHF, and regular cow's milk formula (rCMF). The primary outcome was the rate of tolerance against a maximum of 20.0 mL of formula. RESULTS: Controlled food challenges were performed in 25 children (18 boys; 7 girls) with a median age of 4.25 years (range: 1-9 years) diagnosed with CMA. The median cow's milk-specific immunoglobulin E level was 31.9 UA/mL (range: 1.16-735 UA/mL). The tolerance rate ratios for rCMF were lower than those for pHF (2 vs 16; p<0.01) and eHF (2 vs 22; p<0.01). The allergic symptom scores induced by intake of pHF and eHF were significantly lower than those of rCMF (p=0.01 and p<0.01, respectively), and the pHF and eHF scores were not significantly different. CONCLUSIONS: Compared to rCMF, the partially and extensively hydrolysed whey and casein formulas evaluated in this study were better tolerated and therefore safer for children with CMA. Although further confirmation from additional centres is needed, our findings suggest the use of pHF in patients with mild CMA. Some children with CMA react to hydrolysed formulas; therefore, food challenge tests in these children should be undertaken with caution.

Analysis of the distribution of rice allergens in brown rice grains and of the allergenicity of products containing rice bran.

To understand the allergenicity of rice bran, the distribution of rice allergens in brown rice grains was analysed and the allergenicity of cosmetics and health foods containing rice bran determined. RAG2 and a 19-kDa globulin were localized in polished rice, while a 52-kDa globulin was localized in rice bran. The 52-kDa globulin was also identified as the most likely causative allergen of rice bran allergy. Several products containing intact rice bran were found to contain the 52-kDa globulin. Our study provides the first data regarding cosmetics and health foods containing potential rice bran allergens. Western blot analysis using a rice-bran-allergic patient's plasma showed that 52-kDa globulin was detected as an IgE-binding protein of rice bran and some rice bran-containing cosmetics and health foods. Our results indicate that patients with rice bran allergy need to be careful about using products containing intact rice bran as a constituent.

Hematopoietic stem cell transplantation for progressive combined immunodeficiency and lymphoproliferation in patients with activated phosphatidylinositol-3-OH kinase δ syndrome type 1.

BACKGROUND: Activated phosphatidylinositol-3-OH kinase δ syndrome type 1 (APDS1) is a recently described primary immunodeficiency syndrome characterized by recurrent respiratory tract infections, lymphoid hyperplasia, and Herpesviridae infections caused by germline gain-of-function mutations of PIK3CD. Hematopoietic stem cell transplantation (HSCT) can be considered to ameliorate progressive immunodeficiency and associated malignancy, but appropriate indications, methods, and outcomes of HSCT for APDS1 remain undefined. OBJECTIVE: Our objective was to analyze the clinical manifestations, laboratory findings, prognosis, and treatment of APDS1 and explore appropriate indications and methods of HSCT. METHODS: We reviewed retrospectively the medical records of cohorts undergoing HSCT at collaborating facilities. RESULTS: Thirty-year overall survival was 86.1%, but event-free survival was 39.6%. Life-threatening events, such as severe infections or lymphoproliferation, were frequent in childhood and adolescence and were common indications for HSCT. Nine patients underwent HSCT with fludarabine-based reduced-intensity conditioning. Seven patients survived after frequent adverse complications and engraftment failure. Most symptoms improved after HSCT. CONCLUSION: Patients with APDS1 showed variable clinical manifestations. Life-threatening progressive combined immunodeficiency and massive lymphoproliferation were common indications for HSCT. Fludarabine-based reduced-intensity conditioning-HSCT ameliorated clinical symptoms, but transplantation-related complications were frequent, including graft failure.

Oral Immunotherapy Using Partially Hydrolyzed Formula for Cow's Milk Protein Allergy: A Randomized, Controlled Trial.

BACKGROUND: Partially hydrolyzed cow's milk protein-based formula (pHF) possesses low allergenicity. Here, we investigate the safety and efficacy of oral immunotherapy using pHF for children with cow's milk protein allergy (CMPA). OBJECTIVES: A randomized, double-blind, controlled single-center trial was conducted to evaluate the efficacy and safety of pHF oral immunotherapy in children with CMPA. METHODS: Participants were randomized into double-blind pHF-pHF and extensively hydrolyzed cow's milk protein-based formula (eHF)-pHF groups. During this phase, the pHF-pHF group received pHF and the eHF-pHF group received eHF. During the open phase, all participants received pHF. The primary end point was a change in thresholds between baseline and the end of the first phase. Secondary end points were changes in thresholds between baseline and the end of the second phase, and casein-specific immunoglobulin (Ig)E, IgG4, and basophil activation. RESULTS: Twenty-five children, aged 1-9 years, were randomized into pHF-pHF and eHF-pHF groups. The threshold between baseline and the end of the first phase was significantly elevated in the pHF-pHF group (p = 0.048), but not in the eHF-pHF group. The threshold between other phases did not change significantly in either group. There were significant decreases in casein-specific IgE antibody levels between baseline and the second phase in the eHF-pHF group (p = 0.014). No participants suffered systemic allergic reactions requiring adrenaline or systemic corticosteroids after receiving the formulas. CONCLUSIONS: The results of this trial suggest that, in children with CMPA, tolerance to cow's milk might be safely enhanced by intake of pHF, relative to that of eHF.

The transcription factor EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction.

Mutations of DOCK8 in humans cause a combined immunodeficiency characterized by atopic dermatitis with high serum IgE levels. However, the molecular link between DOCK8 deficiency and atopic skin inflammation is unknown. Here we show that CD4+ T cells from DOCK8-deficient mice produce large amounts of IL-31, a major pruritogen associated with atopic dermatitis. IL-31 induction critically depends on the transcription factor EPAS1, and its conditional deletion in CD4+ T cells abrogates skin disease development in DOCK8-deficient mice. Although EPAS1 is known to form a complex with aryl hydrocarbon receptor nuclear translocator (ARNT) and control hypoxic responses, EPAS1-mediated Il31 promoter activation is independent of ARNT, but in collaboration with SP1. On the other hand, we find that DOCK8 is an adaptor and negative regulator of nuclear translocation of EPAS1. Thus, EPAS1 links DOCK8 deficiency to atopic skin inflammation via IL-31 induction in CD4+ T cells.

Effect of Japanese cedar-specific sublingual immunotherapy on allergen-specific TH2 cell counts in blood.

BACKGROUND: The contribution of antigen-specific TH cells in peripheral blood to immunologic mechanisms underlying sublingual immunotherapy (SLIT) remains unclear, partly because of the lack of a standardized method for the analysis of this rare lymphocyte subset. OBJECTIVE: To clarify the role of antigen-specific TH cells during SLIT using a sensitive method analyzing activation marker CD154-positive TH cells with multicolor flow cytometry. METHODS: We assessed antigen-specific TH cells using multicolor flow cytometry based on the expression of the activation marker CD154 and intracellular cytokines in patients with Japanese cedar pollinosis receiving SLIT at baseline and during the first pollen season after the initiation of SLIT. RESULTS: A total of 18 patients between 12 and 44 years of age were enrolled in the present study. Of these, 8 patients received SLIT (SLIT group) and 10 patients received symptomatic treatment only (control group). Although seasonal pollen exposure significantly increased the number of Japanese cedar-specific interleukin 5- and interleukin 4-producing TH cells in the control group (P < .01 for both), SLIT ameliorated this increase in the SLIT group (P = .64 and P = .84, respectively). CONCLUSION: The present study indicates that allergen-specific TH2 cells in peripheral blood are involved in mechanisms underlying SLIT. The analysis of antigen-specific TH cells using multicolor flow cytometry based on the expression of the activation marker CD154 represents a sensitive and relatively simple, standardized method for monitoring peripheral antigen-specific TH cells during allergen-specific immunotherapy.

[AN EVALUATION OF SPONTANEOUS HISTAMINE RELEASE AND THE LOW RESPONDERS IN A BASOPHIL HISTAMINE RELEASE TEST].

BACKGROUND: We evaluated the clinical significance of the spontaneous histamine release ratio (SHR/T) and low responders in the automated basophil histamine release test (AllerportⓇ HRT). METHODS: This study analyzed the outcomes of 101 oral food challenges (OFC) with egg, milk or wheat (challenge-positive: n=79) in relation to the SHR/T. The traditional HRT low responders (n=27) were separated into two groups:"LOW"responders (n=10), who showed a ≥10% concentration-dependent maximum histamine release in response to the anti-human IgE stimulation, and"NON"responders who did not fulfill the criteria (n=17). RESULTS: Among the 34 patients with ≥20% SHR/T, 32 patients (94%) had a positive OFC with a low threshold dose which provoked severe symptoms. Among the"LOW"responders, four cases showed ≥10% allergen-specific maximum histamine release. On the other hand, concentration-dependent histamine release was not seen in the"NON"responders, suggesting the basophil function was not detected in this subgroup. CONCLUSION: The present study suggested that SHR/T could be an indicator of basophil activation and hypersensitivity in vivo. We also suggested that significant basophil functions might be detected among the "LOW"responders, but not among the"NON"responders.

Three infants with rotavirus gastroenteritis complicated by severe gastrointestinal bleeding.

Rotavirus gastroenteritis causes substantial morbidity and mortality worldwide in children. We report three infants with rotavirus gastroenteritis complicated by various severity of gastrointestinal bleeding. Two patients (cases 1 and 2) recovered completely without any specific treatments. One patient (case 3) died despite extensive treatments including a red blood cell transfusion and endoscopic hemostatic therapy. Rotavirus genotypes G1P[8] and G9P[8] were detected in cases 2 and 3, respectively. Rotavirus antigenemia levels were not high at the onset of melena, suggesting that systemic rotaviral infection does not play an important role in causing melena.

Multicolor flow-cytometric analysis of milk allergen-specific T-helper type 2 cells revealed coexpression of interleukin-4 with Foxp3.

BACKGROUND: Allergen-specific T-helper type 2 (TH2) cells play an important role in the development of allergic inflammation; however, investigations of the properties of allergen-specific T cells have been challenging in humans. Despite clear evidence that forkhead box p3 (Foxp3) is expressed in conventional effector T cells, its function has remained unknown. OBJECTIVE: To characterize allergen-specific TH2 cells in milk allergy, with particular focus on the expression of Foxp3. METHODS: Twenty-one children with milk allergy and 11 children without milk allergy were studied. Peripheral blood mononuclear cells from subjects were stimulated with milk allergen for 6 hours and analyzed using multicolor flow cytometry to identify CD154(+) allergen-specific T-helper cells. Simultaneously, the expression of intracellular cytokines and Foxp3 was analyzed. RESULTS: The milk allergy group had significantly larger numbers of milk allergen-specific interleukin (IL)-4- and IL-5-producing CD4(+) T cells than the control group. Subjects in the milk allergy group had significantly more CD154(+)CD4(+) IL-10-producing cells and CD154(+)Foxp3(+)CD4(+) cells than those in the control group. In addition, the number of milk allergen-specific CD154(+)Foxp3(+)CD4(+) cells strongly correlated with that of CD154(+)IL4(+)CD4(+) cells. Bcl-2 expression in CD154(+)IL-4(+)Foxp3(+) T-helper cells was significantly lower compared with that in total CD4 cells. CONCLUSION: Increased numbers of IL-4-producing allergen-specific T-helper cells were found in patients with milk allergy. In addition, Foxp3 was coexpressed with IL-4 in allergen-specific TH2 cells from patients. This coexpression was associated with lower Bcl-2 levels and could contribute to the phenotype and function of TH2 cells.