Clinical and genetic characterization of Japanese sporadic cases of periodic Fever, aphthous stomatitis, pharyngitis and adenitis syndrome from a single medical center in Japan.

PURPOSE: To investigate clinical presentation, genetic background and cytokine profile of Japanese sporadic cases of periodic fever, aphthous stomatitis, pharyngitis and adenitis (PFAPA) syndrome. METHODS: Nine PFAPA syndrome patients were recruited. DNA sequence analysis of auto inflammatory disorder susceptibility genes, MEFV, MVK, NLRP3, and TNFRSF1A, were performed. Serum cytokine levels and monocyte IL-1ß levels were measured by ELISA. RESULTS: The study population consisted of six males and three females (mean age of onset 26.8 months). Febrile episodes lasted 3-6 days with symptom-free intervals ranging from 2 to 12 weeks. Fever was accompanied by pharyngitis (n = 8), aphthous stomatitis (n = 4), and cervical adenitis (n = 5). White blood cells and C-reactive protein were increased during the attack phase. Mean IgD serum levels were 7.32 ± 9.51 mg/dl during the attack phase, and were mildly elevated in two patients. Heterozygous MEFV, NLRP3 and TNFRSF1A variants were detected in four, one and three cases, respectively. Serum TNF-α and IL-18 levels were elevated during the attack-free and attack periods compared with controls. Other cytokines, IL-1ß, IL-1ra, IL-6, and sTNFR1, were only increased during the attack phase. Oral prednisolone was administered to eight patients and immediately reduced fever. Tonsillectomy performed in five patients induced cessation of fever in four patients. One case with repeated fever attacks after tonsillectomy showed increased monocyte IL-1ß production, similar to the other active case with genetic variants of auto inflammatory disorder-associated genes. CONCLUSIONS: Japanese PFAPA syndrome patients may have cytokine regulation dysfunction as a result of genetic variants of auto inflammatory disorder-associated genes.

Total IgE at 6 months predicts remittance or persistence of atopic dermatitis at 14 months.

Some patients with infantile atopic dermatitis (AD) achieve remission around 1 year old, but in others it persists. The difference between them is unclear. We performed a birth cohort study to find the markers predicting the outcome of infantile AD. We followed up a cohort (n = 314) from birth to 14 months of age, and cord blood was taken from the participants. Some of them (n = 144) had a physical examination and a blood test at 6 and 14 months of age. The subjects who had AD at 6 months (n = 34) were divided into two groups, named the transient group (those who had no AD at 14 months of age; n = 16) and the persistent group (those who still had AD at 14 months of age; n = 18). Then, laboratory data were compared between these two groups. Percentage of CD8 in cord blood lymphocytes and total IgE at 6 months of age in the persistent group was significantly higher than those of the transient group. The area under the curves of a receiver operating characteristic analysis were 0.792 (p = 0.007) and 0.722 (p = 0.027). In the persistent group, total IgE, percentages of T-helper (Th) 2 and phytohemagglutinin-induced IL-4 production from peripheral blood mononuclear cells at 14 months of age were also significantly higher than those of the transient group. Thus Th2 polarization in the persistent group was confirmed. In clinical use, total IgE at 6 months of age is the most useful predictive marker to know the outcome of infantile AD. The clinical trial registration ID is UMIN000002926.

Structural property of soybean protein P34 and specific IgE response to recombinant P34 in patients with soybean allergy.

Soybean allergy is one of the important food allergies because soybean is widely used in processed foods. P34 has been identified as the main allergen in soybeans. The main objective was to analyze the structural property of recombinant P34 and the P34 antigen-specific IgE response in soybean allergy using recombinant P34. Recombinant P34 was expressed by the BL21 (DE3) strain of Escherichia coli. Purified recombinant P34 showed oligomerization and binding to endotoxin. The binding of recombinant P34 to endotoxin was confirmed by LPS pull-down assay. High-density SDS treatment dissociated oligomeric recombinant P34 and removed endotoxin. Both native P34 and purified recombinant P34 showed almost identical structural properties as determined by circular dichroism analysis. We analyzed recombinant-P34-specific IgE antibodies by the ImmunoCAP System. In ImmunoCAP using recombinant P34, all sera from healthy controls were classified as negative. A correlation was found between the specific IgE antibodies to whole soybean and recombinant P34 (r=0.526, P<0.05). The sera from 3 of 9 (33%) patients with outgrown soybean allergy and 6 of 9 (66%) patients with soybean allergy were classified as positive. SDS-treated recombinant P34 retained its structure and biological activity. Recombinant P34 is a useful tool for the analysis of antigen-specific response in soybean allergy. It may be possible to develop a modified form of recombinant P34 for the diagnosis or treatment of soybean allergy using specific immunotherapy techniques.

Augmented cell death with Bloom syndrome helicase deficiency.

Bloom syndrome (BS) is a rare autosomal genetic disorder characterized by lupus-like erythematous telangi-ectasias of the face, sun sensitivity, infertility, stunted growth, upper respiratory infection, and gastrointestinal infections commonly associated with decreased immuno-globulin levels. The syndrome is associated with immuno-deficiency of a generalized type, ranging from mild and essentially asympto-matic to severe. Chromosomal abnormalities are hallmarks of the disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BS is caused by mutations in BLM, a member of the RecQ helicase family. We determined whether BLM deficiency has any effects on cell growth and death in BLM-deficient cells and mice. BLM-deficient EB-virus-transformed cell lines from BS patients and embryonic fibroblasts from BLM-/- mice showed slower growth than wild-type cells. BLM-deficient cells showed abnormal p53 protein expression after irradiation. In BLM-/- mice, small body size, reduced number of fetal liver cells and increased cell death were observed. BLM deficiency causes the up-regulation of p53, double-strand break and apoptosis, which are likely observed in irradiated control cells. Slow cell growth and increased cell death may be one of the causes of the small body size associated with BS patients.

Genetic variations in MyD88 adaptor-like are associated with atopic dermatitis.

Toll-like receptors (TLRs) are important pathogen-associated molecular pattern recognition receptors involved in initiating immune responses. The adaptor protein MyD88 adaptor-like (Mal), involved in signaling downstream of TLRs, plays a crucial role in mediating NF-κB activation. The association of Mal polymorphisms with allergic diseases has not previously been defined. The objective of this study was to detect polymorphisms in the Mal gene and to investigate their association with allergic diseases. Mal gene polymorphisms were genotyped in 310 subjects. The functional effects of Mal variants were analyzed in vitro. One Mal polymorphism, c.303 G>A (Q101Q), was found at a significantly lower frequency in atopic dermatitis patients (p=0.016). Q101Q is in linkage disequilibrium with -103 A>G (rs1893352) and c.539 C>T (S180L) (rs8177374) in the HapMap database. The A allele of -103 A>G showed significantly reduced transcription of Mal compared with the G allele. In addition, three rare variants were identified in this study, c.394 G>A (E132K), c.428 G>A (R143Q) and c.570 G>C (E190D), and were shown to lead to loss-of-function of Mal. It is possible that gene polymorphisms in Mal could affect atopic dermatitis by influencing the innate immune system. We show that Q101Q, which is in linkage disequilibrium with -103 A>G and S180L, may play a protective role against atopic dermatitis. Furthermore, we propose that loss-of-function variants of Mal could predispose individuals to atopic dermatitis or other immunological disorders.

A family having type 2B von Willebrand disease with an R1306W mutation: Severe thrombocytopenia leads to the normalization of high molecular weight multimers.

In type 2B von Willebrand disease (2B VWD), abnormal von Willebrand factor (VWF) spontaneously binds to platelets. This leads to the clearance of the high molecular weight multimers (HMWM) of VWF and results in thrombocytopenia. Herein we report a family of 2B VWD with an R1306W mutation which caused thrombocytopenia with giant platelets. The most important finding in this study is dynamic changes in VWF values in association with platelet counts. When the proband (2 years of age) had severe thrombocytopenia, his HMWM were normal, however, hematological examination showed a low level of VWF and a lack of HMWM after platelet count recovered. His affected sister also exhibited similar phenomenona. These results suggest that the severe thrombocytopenia leads to decreased clearance of VWF HMWM and restoration of VWF HMWM in plasma. We must consider 2B VWD in the case of recurrent thrombocytopenia following infection or other stress condition.

Age-related changes of transforming growth factor beta1 in Japanese children.

BACKGROUND: Transforming growth factor beta1 (TGF beta 1) is an important factor in immunomodulation. The expression of TGF beta 1 has been shown to be influenced by the C-509T polymorphism in the TGF beta 1 gene. We investigated age-related changes of plasma TGF beta 1 levels in a birth-cohort study. In addition, the genotypes of the C-509T polymorphism were investigated in allergic and non-allergic subjects. METHODS: Sixty-four neonates who met the following criteria were enrolled in this cohort study: 1) full-term vaginally delivery; 2) underwent DNA polymorphism analysis; and 3) questionnaire forms were filled out by parents at 0, 6 and 14 months of age. The umbilical cord blood at 0 months and peripheral blood at 6, and 14 months were collected. Plasma TGF beta1 levels were measured at 0, 6 and 14 months of age. Genomic DNA was extracted from their umbilical cord blood. The genotype of the subjects was examined for the presence of C-509T. RESULTS: The plasma TGF beta 1 level at 6 months was the highest of the 3 measurements (at 0, 6, and 14 months of age). The TGF beta 1 levels at 14 months in allergic subjects were significantly higher than those in non-allergic subjects (p = 0.03). All subjects with bronchial asthma (n = 3) had the TT genotype of the C-509T polymorphism. CONCLUSIONS: The plasma TGF beta 1 levels change with age. In addition, TGF beta 1 may play a role in the pathogenesis of bronchial asthma.

Various expression patterns of alpha1 and alpha2 genes in IgA deficiency.

BACKGROUND: IgA deficiency (IgAD) is the most common immunodeficiency, however the pathogenesis in most cases of IgAD is unknown. There are 2 subclasses of IgA, IgA1 and IgA2, and its heavy chains are encoded by 2 different genes, the alpha1 and alpha2 genes. To investigate the molecular pathogenesis of IgA deficiency, it is important to evaluate each of the expressions of IgA1 and IgA2 separately. METHODS: In this study, we report on the reverse transcriptase (RT)-PCR method in which alpha1 and alpha2 mRNAs can be separately evaluated. This method is based on electrophoretic separation using the difference of 39 bases between alpha1 and alpha2 mRNAs. Three selective, 5 partial and 2 secondary IgAD patients were examined. RESULTS: In the 3 selective IgAD patients, no alpha1 or alpha2 mRNA expression was detected. In the 5 partial IgAD patients, various alpha1 and alpha2 mRNA expression patterns were found. One of the partial IgAD patients showed only alpha2 gene expression, but not alpha1 gene expression, and was found to show an alpha1 gene deletion together with gamma 2 and epsilon gene deletions. His plasma IgA2 level was within the normal range. CONCLUSIONS: Patients with an alpha1 gene deletion can be considered as having partial IgAD. Using this method, we identified the second case of alpha1 gene deletion in Japan, and classified IgAD patients on the basis of alpha1 and alpha2 expression.

Hypothermia augments NF-kappaB activity and the production of IL-12 and IFN-gamma.

BACKGROUND: The differentiation of Th1 and Th2 is strictly regulated by humoral and cellular factors. The imbalance between Th1 and Th2 is considered to be the pathogenesis of allergic and autoimmune disorders. It is important to elucidate the effect of environmental factors, such as temperature, on the expression of cytokines of Th1 and Th2. METHODS: We investigated the expression of IFN-gamma, IL-4, IL-5, IL-10 and IL-12 from LPS- or PHA-stimulated PBMCs at 30 degrees C or 37 degrees C using ELISA and Real-time PCR. We measured the change of NF-kappaB activity at 30 degrees C or 37 degrees C with LPS stimulation using the reporter gene assay. RESULTS: IFN-gamma production from LPS-stimulated PBMCs at 30 degrees C was up-regulated compared with 37 degrees C. IL-5 and IL-10 production from PHA-stimulated PBMCs at 30 degrees C were down-regulated compared with 37 degrees C. This augmented IFN-gamma production was caused by the up-regulation of IL-12 production from CD14+ blood monocytes. Both IL-12 mRNA and IL12 protein at 30 degrees C were up-regulated compared with 37 degrees C. NF-kappaB, the key molecule for the expression of IL-12, was also augmented at 30 degrees C compared with 37 degrees C. CONCLUSIONS: Hypothermia up-regulated the expression of IL-12 and IFN-gamma due to the augmented NF-kappaB activity. It is suggested that hypothermia modifies the pattern of cytokine gene expression.

A novel polymorphism, E254K, in the 5-lipoxygenase gene associated with bronchial asthma.

Cysteinyl-leukotrienes are important pro-inflammatory mediators in bronchial asthma (BA) and are derived from arachidonic acid by the action of 5-lipoxygenase. We identified a novel polymorphism, c.760 G>A (E254K), in exon 6 of the 5-lipoxygenase gene (5-LO). This substitution was detected in 11 out of 180 patients with BA, but not in any of the 150 non-allergic subjects. The frequency of c.760 G>A showed a significant difference between BA and non-allergic subjects (P=0.0007). The c.760 G>A polymorphism existed at the surface edge of the C-terminal catalytic domain, and the E-to-K substitution changed the charge of the side chain from negative to positive. Thus, our results suggest that E254K in the 5-LO might be associated with BA.

Induction of α1 and α2 gene expression in selective immunoglobulin A deficiency.

Immunoglobulin A deficiency (IgAD) is the most common immunodeficiency, but the pathogenesis of most cases of IgAD is poorly understood. The gene and protein expression levels of members of the IgA subclasses in IgAD patients were analyzed by a reverse transcriptase (RT)-PCR method that could differentiate between α1 and α2 gene expression. Three selective, 5 partial and 2 secondary IgAD patients were examined. Peripheral blood mononuclear cells which were unstimulated or stimulated with TGF-ß1 and PMA for 24 h were cultured. The IgA1/IgA2 expression ratios were measured by zone densitometry. Three bands appeared (the α1 and α2 genes and a hetero-duplex formation), owing to the difference of 39 bases between α1 and α2 mRNAs. In the controls, there were no significant differences in the IgA1/IgA2 ratios between unstimulated and stimulated cells. In selective IgAD patients, both α1 and α2 gene expression was induced following stimulation, and α1 gene expression was induced more dominantly than in the other IgAD patients following stimulation. Based on our results, suppression of α1 gene expression may be related to the pathogenesis of IgAD.

IL-10 plays an important role as an immune-modulator in the pathogenesis of atopic diseases.

Interleukin (IL)-10 has anti-inflammatory activities in various immune reactions and plays an important role in the regulation of immune diseases. In the present study, we examined the role of IL-10 in atopic diseases. Peripheral blood mononuclear cells (PBMCs) from healthy control subjects, patients with atopic dermatitis and patients with bronchial asthma were cultured with lipopolysaccharide (LPS). The production of IL-10, IL-12 or IFN-γ by PBMCs stimulated with LPS was measured. Next, we investigated whether the haplotype in the IL-10 gene promoter region had an effect on the production of IL-10 by PBMCs. PBMCs from patients were cultured with phytohemagglutinin, to which recombinant human IL-10 had been added. IL-12, IFN-γ and IL-4 production by PBMCs was measured. ß-lactoglobulin (BLG)-specific T cell clones were cultured with BLG peptide (P-17), antigen-presenting cells and recombinant human IL-10. The antigen-induced proliferation of the T cell clones and cytokine production were assayed. Results demonstrated that IL-10 production by LPS-stimulated PBMCs was lower in atopic patients than in healthy control subjects. Three different haplotypes in the IL-10 gene promoter region were detected. These haplotypes did not correlate with IL-10 production by PBMCs. IL-10 inhibited Th1 cytokine production by PBMCs, and also inhibited the antigen-induced proliferation of T cell clones and Th2 cytokine production. In conclusion, IL-10 inhibits both the production of Th1 and Th2 cytokines and the antigen-induced proliferation of T cell clones. Thus, IL-10 modulates other cytokines and plays an important role as an immune-modulator in the pathogenesis of atopic diseases.

Suppression of IFN-gamma production in atopic group at the acute phase of RSV infection.

Several studies have suggested that respiratory syncytial virus (RSV) bronchiolitis induced the change of cytokine production profile in childhood. We sought to determine whether the RSV-induced cytokine production was affected by the patient's atopic background. We quantified interferon-gamma (IFN-gamma) and interleukin (IL)-4 in the supernatant of peripheral blood mononuclear cells (PBMCs) cultured for 24 h and in the presence of phytohemaglutinin (PHA), IL-12, or IL-18, from 14 infants who were divided into two groups, those who are non-atopic and an atopic group. In RSV-infected infants with atopic diseases, IFN-gamma production from IL-12- or especially IL-18-stimulated PBMCs was subtotally suppressed in the acute phase, whereas in RSV-infected infants without atopic diseases IFN-gamma production was not suppressed on acute phase. The IFN-gamma suppression observed in the atopic group is not caused by the immaturity of an infant's immune system since reduced IFN-gamma production to RSV is not observed in the infants of non-atopic group. IFN-gamma suppression in regard to RSV infection might be caused by some genetic factor involved in the development of atopic disease such as IL-18 signal cascade.

Age-related changes in intracellular cytokine profiles and Th2 dominance in allergic children.

The unbalanced T helper response has been pointed out in allergic diseases. Especially in childhood, it is important to consider the development of acquired immunity. We investigated the relationship between age and Th1, Th2, Tc1 or Tc2 cells. In addition, Th1, Th2, Tc1 or Tc2 cells in allergic diseases were compared with control subjects. Thirty-four healthy controls (0-40 years old), 200 samples of cord blood, nine patients with atopic dermatitis (AD) (1-3 years old) and five patients with bronchial asthma (BA) (2-6 years old) were studied. Surface staining with CD4, CD8 and intracellular staining with anti-interferon-gamma (IFN-gamma) and anti-interleukin (IL)-4 were carried out, and analyzed by using flow cytometry. In the healthy controls, the percentages of Th1, Tc1 or Th2 showed positive correlation with age. The absolute numbers of Th1 or Tc1 also correlated with age. Cord blood with a family history of allergic disease showed no significant difference compared to that without a family history. The percentage of Th2 in AD and BA patients was significantly higher than in the age-matched healthy controls. The increase in Th1, Th2 and Tc1 with age might reflect on the development of acquired immunity. Age matching is important when evaluating the cytokine profiles of T cells. In allergic diseases, although cord blood showed a Th1-dominant pattern, it changed to Th2 dominance in childhood, and this may reflect on some genetic background.

A novel single-nucleotide substitution, Glu 4 Lys, in the leukotriene C4 synthase gene associated with allergic diseases.

Cysteinyl leukotrienes (cysLTs) play important roles in bronchial asthma, and can mediate bronchial smooth muscle constriction and increase mucous secretion, vascular permeability and cellular infiltration. We identified a novel heterozygous single-nucleotide substitution 10G>A (Glu 4 Lys) in the first exon of the leukotriene C4 synthase gene (LTC4S). This substitution was detected in 5 of 141 allergic patients, but not in 110 nonallergic subjects. There was a difference in the Glu 4 Lys frequency between the allergic patients and nonallergic subjects (Fisher's exact test, p=0.0460). The five patients with Glu 4 Lys had allergic diseases such as bronchial asthma and/or allergic dermatitis. Furthermore, a familial analysis of Glu 4 Lys revealed a link with allergic diseases. Thus, our results suggest that Glu 4 Lys in the LTC4S might be associated with allergic diseases.

Relatively common mutations of the Bloom syndrome gene in the Japanese population.

Bloom syndrome (BS) is a rare autosomal recessive genetic disorder characterized by lupus-like erythematous facial telangiectasia, sun sensitivity, infertility, stunted growth and a high predisposition to various types of cancer. Chromosomal abnormalities are hallmarks of this disorder, and high frequencies of sister chromatid exchanges and quadriradial configurations in lymphocytes and fibroblasts are diagnostic features. BLM is the causative gene for BS. We investigated the mutation in the BLM gene in 4 Japanese BS kindreds. Taken together with previously documented mutations, 2 kindreds were homozygous for 631delCAA and 2 were compound heterozygous for 631delCAA. The silent mutation of A1055C (Thr to Thr) was detected in control Japanese individuals. The 6-bp deletion/7-bp insertion at position 2,281, which most Askenazi Jewish BS patients carry, was not detected in 200 Japanese alleles. These results suggest that 631delCAA is a relatively common mutation among the Japanese BS patients.

Hyper-IgM syndrome with putative dominant negative mutation in activation-induced cytidine deaminase.

BACKGROUND: Hyper-IgM immunodeficiency is an immunologic disorder characterized by normal or increased serum IgM levels and reduced serum IgG and IgA levels caused by the disruption of Ig class switching in B cells. The gene encoding activation-induced cytidine deaminase (AID) is responsible for the autosomal recessive form of hyper-IgM syndrome. OBJECTIVE: To investigate the relationship between the AID gene mutation and the clinical phenotype, we analyzed the AID gene in a female Japanese patient with the autosomal recessive form of hyper-IgM syndrome. METHODS: Genomic DNA and cDNA were extracted from neutrophils and analyzed by means of PCR. The AID gene was expressed as a glutathione S-transferase fusion protein. RT-PCR was performed after stimulation of the patient's PBMCs with phorbol myristate acetate and TGF-beta. RESULTS: Despite significantly low serum IgG levels, our patient had not shown a predisposition to any severe infections, even without Ig replacement therapy. We identified a point mutation resulting in the stop codon in exon 5 of the AID gene (R190X) in the patient. No other mutations of the AID gene were detected in the patient. The same mutation was not detected in other members of her family. The mutant allele fused with the glutathione S-transferase protein was expressed stably at the same level as the normal allele. The AID gene expression in the patient was induced by phorbol myristate acetate and TGF-beta. CONCLUSION: The mutation of the AID gene is assumed to be of the dominant negative form. This is the first report of a dominant negative form of the mutation in vivo.

A novel single-nucleotide substitution, Leu 467 Pro, in the interferon-gamma receptor 1 gene associated with allergic diseases.

We identified a novel heterozygous single-nucleotide substitution 1400 T right curved arrow C (Leu 467 Pro) in the seventh exon of the interferon-gamma receptor 1 (IFNGR1) gene. This substitution was detected in 6 of the 89 allergic patients but not in the 72 non-allergic subjects. There was a difference in the L467P frequency between the allergic patients and the non-allergic subjects (Fisher's exact test: p=0.033). The 6 patients with L467P have allergic diseases such as bronchial asthma and/or allergic rhinitis. Furthermore, a familial analysis for L467P revealed a linkage between allergic diseases and L467P. Serum IgE levels of the patients with L467P were higher than those of the non-allergic subjects (p=0.001). Our previous studies have been shown that interferon-gamma (IFN-gamma) production by PBMCs in the allergic patients was lower than that in the non-allergic subjects. In this study, although IFN-gamma production in the allergic patients with L467P was equivalent to that in the non-allergic subjects, their serum IgE levels were high and they had allergic diseases. Our results suggest that some allergic patients have IFNGR dysfunction, and that L467P in the IFNGR1 gene is one of candidate susceptibility genes for allergic diseases.

Detection of the genes induced in activated lymphocytes by modified differential display.

Activated lymphocytes induced by mitogens or antigens express various sets of genes, including those involved in the expression of cytokines, surface molecules, and nuclear proteins. To detect inducible genes in activated lymphocytes, we used the RNA arbitrarily primed polymerase chain reaction (RAP-PCR) method, which is modified by original differential display. By this method we identified eight clones; four contained sequences almost identical to that of the genes for heat shock 90Kd protein1 alpha, STAT2, Ig kappa constant region and interferon receptor 1, two had 70% homology to mitochondrial ATP synthase and bromodomain-containing 2 genes, and two had less than 40% homology to known DNA sequences. By RT-PCR, the heat shock 90 Kd protein1 alpha, STAT2 and interferon receptor 1 genes showed increased expression in phytohemagglutinin (PHA)-stimulated or antigen stimulated T cell line. Differential display is a useful method for detection of inducible genes from a small amount of material and at various time points, although the homology of PCR primer influences the displayed genes.