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Background: Invariant natural killer T (iNKT) cells are known as CD1d-restricted T cells that express the invariant T-cell receptors (TCR) Vα24 and Vβ11 in humans and specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells presenting glycolipid antigens and indirect cytotoxicity by activating other cytotoxic immune cells or regulating CD1d-positive immunosuppressive cells in the tumor microenvironment. Although we previously reported that αGalCer-activated NKT cells exert a potent perforin-dependent cytotoxic activity against a wide variety of human tumor cell lines, the direct recognition of CD1d-negative tumors is controversial and the mechanism is unknown. Here we clarify whether iNKT cells recognize and exhibit cytotoxicity toward leukemia cells in a CD1d-independent manner and identify the molecule that recognizes CD1d-negative leukemia cells. Methods: Purified iNKT cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteer donors. PBMCs were cultured in complete RPMI 1640 medium for 9-14 days in the presence of 100 U/mL of recombinant human IL-2 and 200 ng/mL of αGalCer. The iNKT cells were then isolated with an autoMACS Pro separator using FITC-labeled anti-Vα24 antibody (clone, C15) and anti-FITC microbeads. We evaluated the cytotoxic activity of iNKT cells toward CD1d-negative leukemia cells within four days after isolation using a CD107a assay for degranulation, cytometric bead array for cytokine production, and cytotoxicity assay in vitro and in vivo. For in vivo cytotoxicity assays, NOG mice were inoculated with 1 × 106 K562-luc cells on day 0 and with 4 × 106 human iNKT cells on day 1. Gene knock-out (KO) was performed using a CRISPR/Cas9 system. T-cell or NK receptor-KO iNKT cells were used for experiments three or four days after electroporation of the Cas9 protein and guide RNA CRISPR ribonucleoprotein complex. Patient-derived leukemia cells were obtained from PBMCs or bone marrow mononuclear cells of pre-treatment pediatric patients. All studies were approved by the institutional review board and the Animal Care and Use Committee of Chiba University. Results: We observed that iNKT cells degranulated and released Th1 cytokines when co-cultured with CD1d-negative leukemia cells (K562, HL-60, REH, and CD1d-KO U937) as well as αGalCer-loaded CD1d-positive leukemia cells (Jurkat), and showed in vitro cytotoxicity toward these CD1d-negative leukemia cells. This CD1d-independent degranulation decreased over time after isolation and was not restored with re-stimulation by αGalCer. The cytotoxicity of iNKT cells toward K562 cells was confirmed in vivo by comparsion with survival curves of K562-inoculated NOG mice given iNKT cells or PBS alone (log-rank, p= 0.016). To identify the receptors contributing to the CD1d-independent recognition and cytotoxicity against CD1d-negative leukemia cells, we first focused on costimulatory receptors, which are also known as activating NK receptors and are expressed on iNKT cells such as NKG2D, DNAM-1, 2B4, LFA-1, and CD2, and analyzed cytotoxicity after blocking these receptors with antibodies. We found that all costimulatory receptors that we assessed contributed to cytotoxicity toward CD1d-negative leukemia cells. Next, we analyzed cytotoxicity of TCR-KO iNKT cells toward CD1d-negative leukemia cells to confirm the contribution of TCR to CD1d-independent recognition. Notably, TCR-KO iNKT cells showed decreased degranulation, Th1 cytokine release, and cytotoxicity toward K562 cells more so than iNKT cells with KO of NK receptors such as LFA-1(CD11a) or CD2. To assess the clinical application potential of adoptive iNKT cell immunotherapy for leukemia treatment, we analyzed degranulation of iNKT cells using patient-derived leukemia cells. We found iNKT cells degranulation using cells from four out of five myeloid leukemia cases, but only one out of eight BCP-ALL cases (p = 0.032). Conclusion: Primary iNKT cells activated by αGalCer can recognize and show anti-tumor effects toward leukemia cells in an unrestricted manner via CD1d. The TCR also has an important role in recognizing CD1d-negative leukemia cells and multiple NK receptors assist in cytotoxicity. Adoptive iNKT cell immunotherapy may be effective in treating myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

The consumption of probiotics by pregnant and lactating women may prevent the onset of allergic disorders in their children by increasing the concentrations of immunoactive agents such as cytokines in breast milk. Prebiotics such as fructo-oligosaccharides (FOS) increase the number of beneficial organisms such as bifidobacteria. Thus, prebiotics may have an effect similar to that of probiotics. The objective of the present study was to carry out a comprehensive analysis of mRNA expression in human milk cells to identify changes in the concentrations of cytokines in breast milk after the consumption of FOS (4 g × 2 times/d) by pregnant and lactating women. The microarray analysis of human milk cells demonstrated that the expression levels of five genes in colostrum samples and fourteen genes in 1-month breast milk samples differed more than 3-fold between the FOS and control groups (sucrose group). The mRNA expression level of IL-27, a cytokine associated with immunoregulatory function, was significantly higher in 1-month breast milk samples obtained from the FOS group than in those obtained from the control group. In addition, the protein concentrations of IL-27 in colostrum and 1-month breast milk samples were significantly higher in the FOS group than in the control group. In conclusion, the consumption of FOS by pregnant and lactating women increases the production of IL-27 in breast milk. Future studies will address the association of this phenomenon with the onset of allergic disorders in children.

SummaryBackground The thromboxane A2 receptor (TBXA2R) gene is associated with asthma, but no functional genetic variations are known to associate with the disease or its related phenotypes. Objective To investigate the association of TBXA2R polymorphisms with asthma susceptibility and related phenotypes and to identify functionally relevant polymorphisms. Methods We performed comprehensive sequencing of the TBXA2R gene in 48 Japanese control subjects and found a set of variants (SNP1 G>T rs2238634, SNP2 T>G rs2238633, SNP3 C>T rs2238632 and SNP4 G>A rs2238631) in intron 1 in linkage disequilibrium with c.795 T>C rs1131882, which was previously reported to be associated with asthma and related phenotypes. To investigate the effect of four common haplotypes (H1, H2, H3 and H4) on transcriptional activity, we performed a luciferase assay in primary bronchial smooth muscle cells (BSMCs) and human airway epithelial cells (BEAS-2B). We also studied the haplotype association with lung function, TBXA2R mRNA levels, and eosinophil fraction/count in peripheral blood in childhood-onset asthma patients and/or controls. Results H2 and H4, containing minor alleles of SNP2 and SNP3, had significantly higher transcriptional activities than H1 consisting of major alleles (P < 0.001 in BSMCs and BEAS-2B). Homozygotes for redefined haplotype h2 corresponding to minor alleles of SNP2 and SNP3 were associated with lower lung function in childhood-onset asthma patients compared to other zygotes (baseline Forced expiratory volume in one second (FEV1)/ Forced vital capacity (FVC) and Forced expiratory flow between 25% and 75% of the FVC (%FEF25–75%): P = 0.00201 and 0.0128, respectively, and post-bronchodilator FEV1/FVC and %FEF25–75%: P = 0.00224 and 0.0393 respectively). Haplotype h2 was also associated with higher mRNA levels in control peripheral blood cells and higher blood eosinophil fractions and counts in female controls. Conclusions and Clinical Relevance Genetic variants were identified in the TBXA2R gene that influenced transcriptional activity and were associated with asthma-related phenotypes. Thromboxane pathways may therefore play important roles in airway inflammation and remodelling in asthma patients.

Background: Nebulized drugs for asthma treatment are often mixed together in order to simplify inhalation regimens, although not recommended. We therefore evaluated aerosol characteristics and physicochemical stability of the admixture of an inhaled corticosteroid suspension with a beta2-agonist solution. Methods: An 8-stage cascade impactor was used to measure the particle size distribution of admixture of Pulmicort® Respules® (budesonide, 0.5 mg/2 mL) with Meptin® Inhalation Solution Unit (procaterol hydrochloride, 30 Mg/0.3 mL) from a jet nebulizer, PARI LC Plus®. Concentration of each drug was assayed with high- pressure liquid chromatography. Physicochemical compatibility was also assessed up to 24 hours after mixing. Results: With regard to budesonide, impactor parameters such as mass median aerodynamic diameter (MMAD) and respirable mass (RM) were comparable between admixtures and single-drug preparations (2.92 ± 0.03 vs 2.99 ± 0.14 µm, 146.8 ± 2.9 vs 147.6 ± 8.2 µg, respectively). On the other hand, delivery rates of procaterol increased when admixed with budesonide suspension, resulting in significantly higher RM (15.1 ± 0.8 vs 10.2 ± 0.5 µg, p < 0.01). Variations from initial concentration in the percentages of drug remaining at any time point were less than 10%, and there were no appreciable changes in pH of the admixtures for up to 24 hours. Conclusions: There is a possibility that admixture might influence of aerodynamic characteristics of procaterol, but not budesonide. In vivo data will be needed for the clinical implications of our findings.

Background There have been several reports on neonates with milk allergy in a neonatal ward. This type of allergy is mostly categorized as a non-IgE-mediated food allergy. Although most cases of milk allergy occur in the first few months of life, the differences in clinical characteristics between premature and full-term neonates are still unclear. Objective This study aimed to clarify the differences in clinical characteristics of milk allergy between premature and full-term neonates. Methods We retrospectively evaluated 2,116 neonates admitted to the Department of Neonatology, Chiba Kaihin Municipal Hospital, between 2001 and 2007. Results We identified 24 neonates strongly suspected of having milk allergy because of symptoms, such as bloody stools, repeated vomiting, diminished sucking and abdominal distension, as well as objective laboratory findings of eosinophilia in stool cytology and/or positive results for a rectal milk challenge test. Twelve of these 24 neonates were premature (median gestational age, 31 ± 3 weeks; median birth weight, 1,656 ± 592 g) and the other 12 were full-term (median gestational age, 38 ± 1 weeks; median birth weight, 2,760 ± 560 g). There were no differences in symptoms and time to start of feeding between premature and full-term neonates, but there was a significant difference in the median postnatal age at onset (premature neonates: 23 days; vs. full-term neonates: 3.5 days; p Conclusion All premature neonates developed a milk allergy after 32 weeks of corrected gestational age, suggesting that the development of milk allergy requires a certain degree of immunological maturation.

Moyamoya syndrome is a progressive cerebrovascular disease that is characterized by stenosis of the terminal portion of the internal carotid artery and its main branches, in combination with an accompanying disease. We herein describe an 8-year-old boy exhibiting transient loss of consciousness, who had recurrent seizures in infancy with progressive brain calcification. On admission, he was alert but magnetic resonance angiography showed bilateral stenosis of the whole internal carotid artery and proliferation of vascular collaterals, and brain CT revealed calcification on bilateral putamen. Given that this fulfilled diagnostic criteria, we finally diagnosed him as having moyamoya syndrome, though the etiology was unclear. Interestingly, a whole vessel survey revealed vascular stenosis of abdominal aorta and renal arteries, in which the former has not been reported in moyamoya syndrome. We considered that brain calcification was gradually formed by decreased cerebral vascular flow from infancy, and stenosis of abdominal aorta was possibly extended from renal arteries. This is, moyamoya syndrome with brain calcification and stenosis of abdominal aorta, suggesting that morphological screening of whole vessels containing cerebral and abdominal arteries should be considered in cases of slowly progressive brain calcification.

Fructooligosaccharides (FOS) can selectively stimulate the growth of bifidobacteria. Here, we investigated the effect of maternal FOS ingestion on maternal and neonatal gut bifidobacteria. In a randomized, double-blind, placebo-controlled study, we administered 8 g/day of FOS or sucrose to 84 women from the 26th week of gestation to one month after delivery. The bifidobacteria count was detected using quantitative PCR in maternal (26 and 36 weeks of gestation) and neonatal (one month after delivery) stools. Maternal stool frequency was recorded from 24 to 36 weeks of gestation. The number of fecal Bifidobacterium spp. and Bifidobacterium longum in the FOS group was significantly higher than that in the placebo group at 36 weeks of gestation (2.7 × 1010/g vs. 1.1 × 1010/g and 2.3 × 1010/g vs. 9.7 × 109/g). In their neonates, these numbers did not differ between the groups. Also, stool frequency in the FOS group was slightly higher than that in the placebo group two weeks after the intervention (1.0 vs. 0.8 times/day), suggesting a potential constipation alleviation effect. In conclusion, the maternal FOS ingestion showed a bifidogenic effect in pregnant women but not in their neonates.

To our knowledge, this is the first report of the use of real-time reverse transcription–polymerase chain reaction to assess changes in viral load in a patient with congenital rubella syndrome (CRS). Rubella-specific antibody titers were also determined. The patient was a male neonate born to a primipara with rubella infection at 10 weeks of gestation. He had no symptoms at birth, but rubella virus was detected in his pharynx, blood, and urine. His mental and physical development was normal for 1 year; however, he was diagnosed with deafness at 13 months of age. Thus, the patient was diagnosed with CRS. Rubella infection in the pharynx was almost constant until 5 months of age; however, it increased dramatically at 6 months of age. No infection was detected at 13 months. Rubella-specific immunoglobulin M titer was consistently low until 9 months of age and then decreased gradually until it became negative at 20 months of age. Rubella-specific immunoglobulin G titer was high at birth. However, it decreased at 3 months and increased again at 4 months. This titer peaked at ∼9 months and then decreased again at 13 months. This case shows that the period after the decline in maternal antibody titers, not the neonatal period, may be the most contagious period in patients with CRS.

This preliminary prospective study suggests that background factors may differ among allergic diseases. The beneficial interventions for reducing development of allergic rhinitis (AR) are also effective for the prevention of subsequent onset of bronchial asthma (BA).To determine the risk factors associated with onset of AR in atopic children in a prospective study.All patients with atopic dermatitis (AD) or food allergy with or without BA who visited the Pediatric Unit of Chiba University Hospital from 2005 to 2006 were enrolled in the study and received allergy examinations every 3-6 months.A total of 100 patients were followed up for more than 2 years. Among the 60 patients without BA at entry to the study, 12 developed BA during the follow-up period. Development of AR preceded BA in 10 of the 12 patients (83.3%). In the background factors at the entry, positive sensitization to house dust mite (HDM) was significantly related to development of BA. Among the 48 patients without AR, 20 developed AR. High titers of serum HDM-specific IgE and high eosinophil counts in blood, and detection of eosinophils in nasal smears at the entry were significantly related to development of AR.

Summary In Japan, loratadine, fexofenadine, cetirizine and epinastine are currently approved for paediatric use. However, none of these drugs is officially approved for use in children aged under 2 years, and therefore older second-generation antihistamines such as ketotifen and oxatomide are sometimes prescribed for these patients. Because ketotifen and oxatomide have relatively strong sedative effects, one should be cautious when using these antihistamines in young children. In fact, there are reports describing the development of West syndrome, an intractable epilepsy, in 4-month-old infants taking these drugs. Recent clinical trials in Japanese children with allergic rhinitis have shown no serious adverse effects associated with several new second-generation antihistamines, supporting previous overseas reports. New second-generation antihistamines should be approved in the near future for young children in Japan.

Matrix metalloproteinase 9 (MMP9) gene has been shown to be involved in the pathogenesis of allergic rhinitis (AR) and asthma. Previous studies suggested that single-nucleotide polymorphisms (SNPs) of the MMP9 gene conferred a risk for childhood asthma. However, whether the SNPs confer a risk for AR has not been previously investigated. The objective of this study was to investigate whether SNPs of the MMP9 gene are associated with risk of seasonal AR (pollinosis), perennial AR and allergen sensitization. A total of 670 school children were recruited in Japan and genotyped for functional polymorphism in the promoter (-1590C/T: rs3918242) and three amino-acid substitutions (R297Q: rs17576; P574R: rs2250889; R668Q: rs17577). Serum levels of total and specific IgE were determined. Disease status and other clinical characteristics of the subjects were investigated using a questionnaire. Associations between the MMP9 SNPs and both AR and serum IgE levels were evaluated. -1590C/T showed significant association with cedar pollinosis (corrected P (Pcor)=0.039). R668Q was in strong linkage disequilibrium (LD) with -1590C/T and showed significant association with cedar pollinosis (Pcor=0.023) and serum cedar pollen-specific IgE level (Pcor=0.022). A haplotype associated with -1590T and 668Q showed a significant association with cedar pollinosis, orchard grass pollinosis and cedar pollen-specific IgE (Pcor=0.0012, Pcor=0.0059 and Pcor=0.0041, respectively). R297Q and P574R were in weak LD with the rest of the SNPs and did not show significant association with disease. Compared with wild-type MMP9 protein (279R-574P-668R), a variant enzyme (279R-574P-668Q) that showed association with pollinosis had lower activity. However, lower enzyme activity was not associated with disease risk because another variant (279Q-574R-668R) showed lower enzyme activity but was not associated with pollinosis. The -1590T allele and its corresponding haplotype was associated with higher promoter activity and with pollen-specific IgE levels and pollinosis, suggesting that -1590C/T may have more impact on sensitization and disease development than R668Q. Our results suggest that the MMP9 gene confers susceptibility to cedar pollinosis in Japanese children. The MMP9 gene may be associated with pollinosis through sensitization processes.

Background: In developed countries, increasing food allergy prevalence and concern regarding food allergies have been reported. Although the use of complementary and alternative medicine (CAM) for the treatment of allergic diseases has increased in some Western countries, the actual proportion and patterns of CAM use for pediatric food allergies in Japan are still unknown. Methods: Fourteen allergy centers in Japan participated in the study using a questionnaire survey regarding the use of CAM by pediatric patients. A diagnosis of food allergy was made at each hospital by pediatric allergists. Results: Surveys were completed by parents/guardians, and data were collected for a total of 962 pediatric food-allergic patients. Overall, 8.4% of the participants used CAM to treat a food allergy. The major CAM therapies used were herbal teas (22.2%), including several Japanese herbal teas, Chinese herbal medicine (18.5%) and lactic acid bacteria (16%). Among the participants using CAM to treat food allergy, 13.6% thought that the CAM being used was very effective, while 11.1% of participants thought that CAM caused some type of side effect. Conclusions: Our study is the first large-scale national survey regarding the use of CAM in pediatric patients with food allergies in Japan. Unlike in the USA, which has a higher rate of CAM use (17%), approximately 8.4% of food-allergic patients used CAM in Japan. Interestingly, the major types of CAM used in Japan differed from those used in the USA. Cultural differences and food customs may affect the use of CAM.

BACKGROUND Several patient-/caregiver-completed questionnaires have been utilized for the assessment of asthma control. However, due to the diversity in medical/social circumstances, they may not be optimal for use in all countries. The Japanese pediatric asthma guideline (JPGL) aims at higher levels of control compared with other international guidelines, based on a strong social demand for best care. We developed a new control test to help meet that demand. METHODS A 34-item working questionnaire was developed based on input from pediatric asthma specialists and the caregivers of preschool children with asthma. The questionnaire was administered to caregivers of 565 patients aged

Matrix metalloproteinases (MMPs) are a class of extra-cellular and membrane-bound proteases involved in a wide array of physiological and pathological processes including tissue remodeling, inflammation, and cytokine secretion and activation. MMP-13 has been shown to be involved in lung diseases such as acute lung injury, viral infections, and chronic obstructive pulmonary disease; however, the molecular pathogenesis of MMP-13 in these conditions is not well understood. In this study, we investigated the mechanisms and roles of MMP-13 secretion in human small airway epithelial cells (SAECs) and functional polymorphisms of the MMP13 gene. Polyinosinic-polycytidylic acid (poly(I:C)) and interferon β (IFN-β) stimulated the secretion of MMP-13 from SAECs by more than several hundred-fold. Stimulation of the secretion by poly(I:C) was abolished by SB304680 (p38 inhibitor), LY294002 (PI3K inhibitor), Janus kinase (JAK) inhibitor I, RNA-activated protein kinase (PKR) inhibitor, and Bay 11-7082 (NF-κB inhibitor), while stimulation by IFN-β was inhibited by all except Bay 11-7082. These data suggested that the secretion of MMP-13 was mediated through IFN receptor pathways independently of nuclear factor kappa B (NF-κB) and that poly(I:C) stimulated IFN secretion in an NF-κB-dependent manner from SAECs, leading to IFN-stimulated MMP-13 secretion. Chemical MMP-13 inhibitors and MMP-13 small interfering RNA (siRNA) inhibited IFN-stimulated secretion of interferon gamma-inducible protein 10 (IP-10) and regulated on activation, normal T-cell expressed and secreted (RANTES), suggesting that MMP-13 is involved in the secretion of these virus-induced proinflammatory chemokines. We identified a novel functional polymorphism in the promoter region of the MMP13 gene. The MMP13 gene may play important roles in defense mechanisms of airway epithelial cells.

The number of patients with juvenile-onset Sjögren's syndrome (SS) has recently increased. However, there is no drug that is safe and effective for the xerostomia that occurs in patients of this age group. We evaluated the efficacy and safety of orally administered pilocarpine hydrochloride for juvenile-onset SS patients. Five female patients, aged from 9 to 16 years, received 5-10 mg/day for 4 weeks. On days 1 and 28, salivary production was measured by the Saxon test, and patients completed subjective self-evaluations of xerostomia symptoms and were asked about changes in water intake and overall improvement of dry mouth on day 28. After 4 weeks of pilocarpine administration, salivary production increased significantly in all patients, and overall status was assessed as "improved" in all patients. One patient had excessive sweating. No serious adverse events or laboratory examination abnormalities correlated with pilocarpine administration were found. In conclusion, the results of this study suggest that orally administered pilocarpine is safe and effective for treating xerostomia in juvenile-onset SS patients. This is the first report of the efficacy of pilocarpine for juvenile SS patients; further evaluations are needed to confirm our result.

In asthma genetics, the association of highly replicated susceptibility genes lacks consistency across populations. To identify genuine associations, we investigated the reproducibility of the 23 most promising asthma and asthma-related candidate genes in a moderately sized sample from the Japanese population. We compared the frequency of 33 polymorphisms in unrelated cases and controls and tested for their association with asthma, atopy and serum total IgE levels using allele frequency, codominant, dominant and recessive genotype models. On the basis of the consistency of our findings with previous meta-analyses and large replication studies, IL13, TNF, ADAM33, IL4RA and TBXA2R might represent common major asthma and asthma-related trait genes. Individual gene assessment was extended to the interactions between two polymorphisms using our original method. Interactions between TBXA2R and ADAM33, and IL4RA and C3 were suggested to increase the risk for childhood and all asthma (adult and childhood asthma combined). The confirmation of previously reported associations between gene polymorphisms and phenotypes was problematic when as few as several hundred samples per group were used. Stratification of the subjects by environmental factors or other confounding factors may be necessary to improve the sensitivity and reliability of association results.

A new version of the Japanese pediatric guideline for the treatment and management of bronchial asthma was published in Japanese at the end of 2011. The guideline sets the pragmatic goal for clinicians treating childhood asthma as maintaining a "well-controlled level" for an extended period in which the child patient can lead a trouble-free daily life, not forgetting the ultimate goal of obtaining remission and/or cure. Important factors in the attainment of the pragmatic goal are: (i) appropriate use of anti-inflammatory drugs; (ii) elimination of environmental risk factors; and (iii) educational and enlightening activities for the patient and caregivers regarding adequate asthma management in daily life. The well-controlled level refers to a symptom-free state in which no transient coughs, wheezing, dyspnea or other symptoms associated with bronchial asthma are present, even for a short period of time. As was the case in the previous versions of the guideline, asthmatic children younger than 2 years of age are defined as infantile asthma patients. Special attention is paid to these patients in the new guideline: they often have rapid exacerbation and easily present chronic asthmatic conditions after the disease is established.

Background House dust extract is used in conventional immunotherapy for house dust-mite (HDM) allergic rhinitis in Japan. However, an alternative administration route is desired. The aims of the present double blind, placebo-controlled trial were to evaluate the therapeutic efficacy and safety of sublingual immunotherapy (SLIT) with house dust extract in pediatric patients with HDM allergic rhinitis. Methods The study population comprised 31 subjects (21 males and 10 females) aged from 7 to 15 years old. Twenty patients (the active group) received house dust extract and 11 received placebo via sublingual administration. Extract or placebo (1 ml) was administered at 10-fold dilution once weekly for 40 weeks. During the study period, the subjects recorded their daily nasal symptoms and use (dose and frequency) of other medications in a nasal allergy diary. Results The symptom scores in the active group began to decrease about 24 weeks after initiation of treatment and significant differences between the active and placebo groups were observed after 30 weeks. The average scores for the last four weeks of the study were significantly lower than those for the first four weeks in the active group but not in the placebo group. The only local adverse effect was a bitter taste reported by one patient. There were no other local or systemic adverse effects associated with SLIT. Conclusions Our results suggest that SLIT with house dust extract for more than 30 weeks is safe and effective treatment for HDM allergic rhinitis in children.

Lower numbers of Vgamma9Vdelta2 T cells in cord blood (CB) than in adult peripheral blood (PB), as well as their impaired ability to produce interferon-gamma (IFN-gamma) in response to stimulation, are associated with functional deficiency in the immune system in newborns. In this study, we stimulated CB Vgamma9 T cells with their T-cell receptor-specific ligand, isopentenyl pyrophosphate (IPP), plus exogenous costimulatory cytokines such as interleukin-2 (IL-2), IL-12 and tumour necrosis factor-alpha (TNF-alpha), which are known to play important roles in the activation of PB gammadelta T cells. Our data show that CB Vgamma9 T cells are able to produce IFN-gamma at levels comparable to PB Vgamma9 T cells by the addition of TNF-alpha in the presence of IPP and IL-2; however, under the same culture conditions, IL-12 does not efficiently activate CB Vgamma9 T cells to produce IFN-gamma. The frequency of TNF-alpha receptor II-positive Vgamma9T cells and the expression levels of TNF-alpha receptor II are similar in CB and PB; in contrast, the frequency of IL-12 receptor betaI (IL-12RbetaI) -positive Vgamma9T cells and expression levels of IL-12RbetaI are significantly lower in CB than PB. TNF-alpha but not IL-12 increases the expression of IL-2Rbeta on CB Vgamma9 T cells. These results provide new insights into the role of TNF-alpha in the activation of CB Vgamma9 T cells.

The regulatory IL-10 and TGF-beta1 cytokine gene polymorphisms have been associated with allergic diseases in different populations, like Caucasian, Chinese and Indians. We investigated the association between the polymorphisms IL-10 A-1082G, C-819T, C-627A and TGF-beta1 T+869C, G+915C, C-509T and food allergy in Japanese children. One hundred and eleven children with food allergy and 115 atopic control children without food allergy were recruited. DNA samples from these subjects were genotyped by using PCR. The odds ratio of IL-10 -1082 AA genotype was 2.5 (95% CI, 1.0-6.4) for food allergy risk when compared with atopic control subjects (p = 0.03). There were no significative differences in the frequency of TGF-beta1 gene polymorphisms between both groups. Our results indicate that IL-10 A-1082G gene polymorphism is associated with food allergy susceptibility in atopic Japanese children.

We previously showed that immunization of mice with murine fibroblasts transfected with the thyrotropin receptor (TSHR) and a murine major histocompatibility complex (MHC) class II molecule induces immune thyroid disease with the humoral and histological features of human Graves' disease in about 20% of mice. In this model, based on the proliferative response of T cells from hyperthyroid mice to a panel of overlapping TSHR peptides, we now demonstrate that TSHR 121–140 peptide contains an immunodominant T cell epitope. Supporting this conclusion, spleen cells from mice immunized with TSHR 121–140 peptide showed a strong proliferative response to fibroblasts transfected with the TSHR and a murine I-A k molecule, but not either alone. Also, intranasal administration of 100 μg of TSHR 121–140 peptide led to suppressed proliferative response of lymph node cells to the peptide. Interestingly, however, administration of this peptide enhanced, rather than suppressed, the frequency and severity of Graves' disease induced by the immunization of the fibroblasts transfected with the TSHR and a murine I-A k molecule. Spleen cells from hyperthyroid mice that were pretreated with intranasal peptide tended to produce lesser amounts of IL-4, IL-10 and IFN-gamma than those from normothyroid control mice. Although precise mechanisms of this enhancement remain to be determined, the results suggest that attempts to treat Graves' disease by intranasal administration of an immunodominant TSHR T cell epitope may aggravate, not prevent, the disease.

Our objective was to investigate the efficacy of intravenous alendronate for the treatment of glucocorticoid-induced osteoporosis (GIOP) in children with autoimmune diseases. Five children with autoimmune disease and GIOP were treated with 5 mg intravenous alendronate once every 3 months. After 1 and 2 years, we evaluated the changes in the Z score of the femoral neck bone mineral density (BMD), serum bone alkaline phosphatase, and urinary deoxypyridinoline. Six patients with GIOP, whose BMD could be observed over a 1-year period without alendronate treatment, were defined as controls. After 1 and 2 years of treatment, intravenous treatment significantly inhibited bone loss. The efficacy of alendronate demonstrated a significant correlation with a high level of bone turnover markers before alendronate treatment. Intravenous alendronate is considered to be a good choice for the treatment of GIOP because of its excellent efficacy. In addition, our study suggests that the efficacy of alendronate depends on the bone turnover of patients before treatment. Intervention with bisphosphonates during periods of high bone turnover may be recommended.

Background. The contribution that genetic polymorphisms of Toll-like receptor (TLR) 4 and of CD14-both of which recognize respiratory syncytial virus (RSV) in the innate immune response-make to RSV bronchiolitis in the Japanese population has not yet been clarified. Methods. This study genotyped 2 TLR4 mutations, Asp299Gly and Thr399Ile, and 2 single-nucleotide polymorphisms (SNPs) of CD14, -550 C/T and -159 C/T, in 54 children with RSV bronchiolitis and in 203 control subjects. CD14 SNPs and the serum level of soluble CD14 (sCD14) also were examined in 67 cord-blood specimens and in serum samples from 73 6-year-old children. Results. No TLR4 mutations were found. The frequencies of both the CC genotype and the C allele of CD14 -550 C/T were significantly higher in children with RSV bronchiolitis than in the control subjects. The serum level of sCD14 was significantly higher in children with the CC genotype of CD14 -550 C/T than in those with the CT and TT genotypes. Conclusions. CD14 -550 C/T, which is related to the serum level of sCD14, is associated with the development of RSV bronchiolitis in the Japanese population. This study's results indicate that, in different ethnic groups, different genetic factors contribute to the development of RSV bronchiolitis.

Ellis-van Creveld (EVC) syndrome is a rare autosomal recessive disorder characterized by hypoplastic nails, polydactyly, and achondroplasia. Patients usually exhibit normal cognitive function and no remarkable developmental delay. We herein present an unusual case of EVC syndrome. A Japanese 2-year-old boy was born at term, but immediately developed severe respiratory failure due to thorax deformity, postaxial polydactyly and nail hypoplasia. We identified a novel pattern of germinal compound heterozygous nonsense EVC2 mutations of c.1814CA (p. S605X) and c.2653CT (p. R885X), leading to the diagnosis of EVC syndrome. Interestingly, he also had severe developmental delay, and suddenly developed excessive abdominal distension at the age of 2. On surgery, extensive necrotic bowel with chronic intestinal pseudo-obstruction was noted. This is, to our knowledge, a most severe phenotype of EVC syndrome, illustrating that the specific pattern of EVC2 compound heterozygous mutations may cause severe developmental delay and intestinal malfunction.

We studied the molecular evolution of the C-terminal 3rd hypervariable region in the attachment glycoprotein gene of human respiratory syncytial virus subgroup B (HRSV-B) genotypes BA9 and BA10. We performed time-scaled phylogenetic analyses using Bayesian Markov chain Monte Carlo methods. We also performed a genetic distance analysis (p-distance analysis), positive and negative selection analyses, and a Bayesian skyline plot (BSP) analysis. We found that genotype BA9 diverged from the common ancestor of genotypes BA7, BA8, and BA10, while genotype BA10 diverged from the ancestor of genotypes BA7 and BA8. Strains of both genotypes were distributed worldwide. BA9 and BA10 diverged between 1999 and 2001. Both BA9 and BA10 evolved rapidly (about 4.8×10(-3)substitutions/site/year) and formed three distinct lineages in a 10-year period. BA10 strains belonging to lineage 3 had large genetic distances (p-distance>0.07). Thus, it may be possible to classify these strains as a new genotype, BA11. No positive selection site was detected in either genotype. Phylodynamic analyses showed that the effective population size of BA10 decreased gradually since 2010 and BA9 slightly decreased since 2009. The results suggested that the recently prevalent HRSV-B genotypes BA9 and BA10 evolved uniquely, leading to epidemics of HRSV-B worldwide over a 15-year period.

We studied the efficacy of drugs indicated for mitochondrial dysfunction in the treatment of 21 patients with acute encephalopathy with onset of febrile convulsive status epilepticus at our hospital from January 2006 to December 2014. Among them, 11 patients had been treated with a mitochondrial drug cocktail consisting of vitamin B1, vitamin C, biotin, vitamin E, coenzyme Q10, and l-carnitine (prescription group) and 10 patients were not treated with the cocktail (non-prescription group). We retrospectively reviewed age, trigger, clinical form, treatment start time, and sequelae. Clinical form was classified into a biphasic group presenting acute encephalopathy with biphasic seizures and late reduced diffusion (AESD) and a monophasic group. Sequelae were classified as (A) no sequelae group or (B) sequelae group, and differences in the interval between diagnosis and treatment were also evaluated. The sequelae were not different between the mitochondrial drug cocktail prescription and non-prescription groups, but significantly better in the group administered the mitochondrial drug cocktail within 24h (P=0.035). We expect that early treatment with a mitochondrial drug cocktail could prevent sequelae in acute encephalopathy with onset of febrile convulsive status epilepticus.

SUMMARYHeptavalent pneumococcal conjugate vaccine (PCV7) was introduced to Japan in 2010. We investigated the impact of PCV7 on childhood community-acquired pneumonia (CAP) and pneumococcal pneumonia (PP). Children aged Streptococcus pneumoniaein cultured blood and/or sputum samples of CAP patients. The incidence of CAP andS. pneumoniaeisolated from PP patients was compared before (April 2008–March 2009) and after (April 2012–March 2013) the introduction of PCV7 immunization. The annual incidence of CAP was reduced [incidence rate ratio 0·81, 95% confidence interval (CI) 0·73–0·90]. When comparing post-vaccine with pre-vaccine periods, the odds ratio for PP incidence was 0·60 (95% CI 0·39–0·93,P= 0·024). PCV7-covered serotypes markedly decreased (66·6% in pre-vaccinevs. 15·6% in post-vaccine,P< 0·01), and serotypes 6C, 15A, 15C and 19A increased. Multidrug-resistant international clones in the pre-vaccine period (Spain6B-2/ST90, Taiwan19F-14/ST236) decreased, while Sweden15A-25/ST63 was the dominant clone in the post-vaccine period. A significant reduction in the incidence of both CAP hospitalizations and culture-confirmed PP of vaccine serotypes was observed at 2 years after PCV7 vaccination.

Constitutional 11q interstitial deletion syndrome presents with congenital anomalies including microcephaly with craniostenosis, minor dysmorphic features, vitreoretinopathy, and renal anomalies. This syndrome is occasionally associated with neuroblastoma (NB) as a life-threatening complication, which is important for clinical care. Although the corresponding locus to NB has been predicted to exist in 11q22-23 by previous deletion studies related to NB, the causative haploinsufficient genes have not yet been identified. We herein reported for the first time the simultaneous coexistence of adrenal NB and abdominal prevertebral ganglioneuroma in a 6-year-old girl with a constitutional hemizygous 11q14.1-23.3 deletion. Of the 11 haploinsufficient genes predicted with an in silico database, we focused on NCAM1 and CADM1 as the genes accountable for NB and ganglioneuroma. The deletion range, especially the 11q22.3 involvement, needs to be determined in 11q deletion cases in order to predict susceptibility to peripheral nerve tumors involving NB and ganglioneuroma.

Prednisolone combined with intravenous immunoglobulin (IVIG) is the standard option for the initial treatment of Kawasaki disease (KD) in Japan, as described in the 2012 Japanese guidelines for KD. However, there is still debate regarding treatment in patients with refractory KD initially treated with IVIG plus prednisolone, especially in those with comorbid coronary artery aneurysm (CAA). Here, we present the case of a 3 year-old-girl initially treated with IVIG plus prednisolone, whose clinical and laboratory parameters improved after plasma exchange therapy (PE). She was admitted to a local hospital on day 4 of illness and diagnosed as having severe KD (Kobayashi score, 7). Therefore, prednisolone plus IVIG was administered as initial treatment. The initial course was ineffective. She was treated with methylprednisolone pulse therapy, and a second and third round of additional IVIG, but low-grade fever persisted. After these treatments the patient’s general condition had improved and major KD symptoms improved by day 13 of illness, despite a high serum C-reactive protein level (19.9 mg/dl) and hypoalbuminemia (1.8 g/dl). Expanding medium-sized aneurysm was detected on the same day, so she was immediately referred to our hospital for further treatment. The CAA continued to expand, so PE was performed for 3 consecutive days and prednisolone was gradually reduced from day 15 of illness. After that, she became afebrile and CAA progression stopped. Laboratory findings such as serum C-reactive protein and albumin levels were dramatically improved after PE. This case suggests that PE might be effective for refractory KD initially treated with IVIG plus glucocorticoids and may stop the CAA development. Doctors must remain aware that patients treated with glucocorticoids may have masked clinical conditions, and take care not to underestimate the signs of refractory KD.

Background: There are still no definite treatments for refractory Kawasaki disease (KD). In this pilot study, we evaluated the safety and efficacy of a new protocol consisting of sivelestat sodium hydrate (SSH) combined with additional intravenous immune globulin (IVIG) for KD patients who were resistant to initial IVIG therapy. Methods: We prospectively collected clinical data of KD patients who were resistant to initial IVIG (2g/kg for one day) and received SSH (0.2mg/kg/hour for consecutive 5 days) combined with additional IVIG (2g/kg for one day) as a second-line therapy at Chiba University hospital between December 2006 and March 2014. We defined patients who remained febrile (37.5°C or more of an axillary temperature) after 36 to 48 hours after start of initial IVIG therapy or who had recrudescent fever associated with other symptoms of Kawasaki disease as being resistant to initial IVIG. Results: Thirty five KD patients were enrolled in this study. No serious adverse effect was noted. The median total duration of fever was 8 days (range 6 to 17 days) and the incidence of coronary artery lesion (CAL) was 5.7% (2 of 35 patients). Among a total of 35 patients, 24 (69%) of them responded promptly to be afebrile 36 to 48 hours after the start of the additional IVIG with SSH. One of these 24 patients developed CAL. The other 11 (31%) failed to become afebrile 36 to 48 hours after the start of the additional IVIG with SSH therapy. Of these 11 patients, one developed CAL. Before initial IVIG, there was no difference in demographic and laboratory data except the age, body weight and % Neutrophils. However, after initial IVIG therapy, there appeared significant difference in % Neutrophils and C-reactive protein levels and both of which were higher in additional IVIG with SSH therapy non-responders than in responders. Conclusions: Additional IVIG combined with SSH for the additional treatment of KD patients who were refractory to initial IVIG therapy was safe and the incidence of CAL is acceptable considering the severity of patients in this study.

Human rhinovirus species C (HRV-C) was recently discovered and this virus has been associated with various acute respiratory illnesses (ARI). However, the molecular evolution of the major antigens of this virus, including VP1, VP2 and VP3, is unknown. Thus, we performed complete VP1, VP2, and VP3 gene analyses of 139 clinical HRV-C strains using RT-PCR with newly designed primer sets and next-generation sequencing. We assessed the time-scale evolution and evolutionary rate of these genes using the Bayesian Markov chain Monte Carlo method. In addition, we calculated the pairwise distance and confirmed the positive/negative selection sites in these genes. The phylogenetic trees showed that the HRV-C strains analyzed using these genes could be dated back approximately 400 to 900 years and these strains exhibited high evolutionary rates (1.35 to 3.74 × 10−3 substitutions/site/year). Many genotypes (>40) were confirmed in the phylogenetic trees. Furthermore, no positively selected site was found in the VP1, VP2 and VP3 protein. Molecular modeling analysis combined with variation analysis suggested that the exterior surfaces of the VP1, VP2 and VP3 proteins are rich in loops and are highly variable. These results suggested that HRV-C may have an old history and unique antigenicity as an agent of various ARI.

Pseudohypoparathyroidism type Ib (PHP1B) is caused by proximal tubular resistance to parathyroid hormone that occurs in most cases in the absence of Albright's Hereditary Osteodystrophy (AHO). Familial forms of PHP1B are caused by maternally inherited microdeletions within STX16, the gene encoding syntaxin 16, or within GNAS, a complex genetic locus on chromosome 20q13.3 encoding Gsα and several splice variants thereof. These deletions lead either to a loss-of-methylation affecting GNAS exon A/B alone or to epigenetic changes involving multiple differentially methylated regions (DMRs) within GNAS. Broad GNAS methylation abnormalities are also observed in most sporadic PHP1B (sporPHP1B) cases. However, with the exception of paternal uniparental disomy involving chromosome 20q (patUPD20q), the molecular mechanism leading to this disease variant remains unknown. We now investigated 23 Japanese sporPHP1B cases, who presented with hypocalcemia, hyperphosphatemia, elevated PTH levels, and occasionally with TSH elevations and mild AHO features. Age at diagnosis was 10.6 ± 1.45 years. Calcium, phosphate, and PTH were 6.3 ± 0.23 mg/dL, 7.7 ± 0.33 mg/dL, and 305 ± 34.5 pg/mL, respectively, i.e. laboratory findings that are indistinguishable from those previously observed for Caucasian sporPHP1B cases. All investigated patients showed broad GNAS methylation changes. Eleven individuals were homozygous for SNPs within exon NESP and a pentanucleotide repeat in exon A/B. Two of these patients furthermore revealed homozygosity for numerous microsatellite markers on chromosome 20q raising the possibility of patUPD20q, which was confirmed through the analysis of parental DNA. Based on this and our previous reports, paternal duplication of the chromosomal region comprising the GNAS locus appears to be a fairly common cause of sporPHP1B that is likely to occur with equal frequency in Caucasians and Asians.

Hedgehog signaling is a pivotal developmental pathway that comprises hedgehog, PTCH1, SMO, and GLI proteins. Mutations in PTCH1 are responsible for Gorlin syndrome, which is characterized by developmental defects and tumorigenicity. Although the hedgehog pathway has been investigated extensively in Drosophila and mice, its functional roles have not yet been determined in human cells. In order to elucidate the mechanism by which transduction of the hedgehog signal is regulated in human tissues, we employed human fibroblasts derived from three Gorlin syndrome patients and normal controls. We investigated GLI1 transcription, downstream of hedgehog signaling, to assess native signal transduction, and then treated fibroblasts with a recombinant human hedgehog protein with or without serum deprivation. We also examined the transcriptional levels of hedgehog-related genes under these conditions. The expression of GLI1 mRNA was significantly higher in Gorlin syndrome-derived fibroblasts than in control cells. Hedgehog stimulation and nutritional deprivation synergistically enhanced GLI1 transcription levels, and this was blocked more efficiently by vismodegib, a SMO inhibitor, than by the natural compound, cyclopamine. Messenger RNA profiling revealed the increased expression of Wnt signaling and morphogenetic molecules in these fibroblasts. These results indicated that the hedgehog stimulation and nutritional deprivation synergistically activated the hedgehog signaling pathway in Gorlin syndrome fibroblasts, and this was associated with increments in the transcription levels of hedgehog-related genes such as those involved in Wnt signaling. These fibroblasts may become a significant tool for predicting the efficacies of hedgehog molecular-targeted therapies such as vismodegib.

We summarized the Therapeutic Guidelines for Atopic Dermatitis 2002, and outlined important points with reference to medical care and management of atopic dermatitis.