Takahiro Yasumi

High Incidence of NLRP3 Somatic Mosaicism in Patients With Chronic Infantile Neurologic, Cutaneous, Articular Syndrome: Results of an International Multicenter Collaborative Study

OBJECTIVE Chronic infantile neurologic, cutaneous, articular (CINCA) syndrome, also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease. Although heterozygous germline gain-of-function NLRP3 mutations are a known cause of this disease, conventional genetic analyses fail to detect disease-causing mutations in ∼40% of patients. Since somatic NLRP3 mosaicism has been detected in several mutation-negative NOMID/CINCA syndrome patients, we undertook this study to determine the precise contribution of somatic NLRP3 mosaicism to the etiology of NOMID/CINCA syndrome. METHODS An international case-control study was performed to detect somatic NLRP3 mosaicism in NOMID/CINCA syndrome patients who had shown no mutation during conventional sequencing. Subcloning and sequencing of NLRP3 was performed in these mutation-negative NOMID/CINCA syndrome patients and their healthy relatives. Clinical features were analyzed to identify potential genotype-phenotype associations. RESULTS Somatic NLRP3 mosaicism was identified in 18 of the 26 patients (69.2%). Estimates of the level of mosaicism ranged from 4.2% to 35.8% (mean ± SD 12.1 ± 7.9%). Mosaicism was not detected in any of the 19 healthy relatives (18 of 26 patients versus 0 of 19 relatives; P < 0.0001). In vitro functional assays indicated that the detected somatic NLRP3 mutations had disease-causing functional effects. No differences in NLRP3 mosaicism were detected between different cell lineages. Among nondescript clinical features, a lower incidence of mental retardation was noted in patients with somatic mosaicism. Genotype-matched comparison confirmed that patients with somatic NLRP3 mosaicism presented with milder neurologic symptoms. CONCLUSION Somatic NLRP3 mutations were identified in 69.2% of patients with mutation-negative NOMID/CINCA syndrome. This indicates that somatic NLRP3 mosaicism is a major cause of NOMID/CINCA syndrome.

Aicardi-Goutières Syndrome Is Caused by IFIH1 Mutations

Aicardi-Goutières syndrome (AGS) is a rare, genetically determined early-onset progressive encephalopathy. To date, mutations in six genes have been identified as etiologic for AGS. Our Japanese nationwide AGS survey identified six AGS-affected individuals without a molecular diagnosis; we performed whole-exome sequencing on three of these individuals. After removal of the common polymorphisms found in SNP databases, we were able to identify IFIH1 heterozygous missense mutations in all three. In vitro functional analysis revealed that IFIH1 mutations increased type I interferon production, and the transcription of interferon-stimulated genes were elevated. IFIH1 encodes MDA5, and mutant MDA5 lacked ligand-specific responsiveness, similarly to the dominant Ifih1 mutation responsible for the SLE mouse model that results in type I interferon overproduction. This study suggests that the IFIH1 mutations are responsible for the AGS phenotype due to an excessive production of type I interferon.

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells.

We investigated the antitumor effects of FR901228, a HDAC inhibitor, on human osteosarcoma cells, in vitro and in vivo to explore its possible utility in the treatment of pediatric bone cancers. FR901228 caused marked growth inhibition with a 50% inhibitory concentration of 1.2–7.3 nM and induction of apoptosis in all eight osteosarcoma cell lines tested. These effects of FR901228 were also observed in vivo xenograft models on BALB/c nude mice, and treatment with 5.6 mg/kg/day resulting in a >70% reduction in the mean final tumor volume compared with the mean initial tumor volume. TUNEL assays demonstrated extensive apoptosis in tumor sections of mice treated with FR901228. Induction of apoptosis was preceded by increased expression of Fas ligand (FasL) mRNA, resulting in expression of membrane-bound FasL, which was followed by sequential activation of caspase-8 and -3. The level of apoptosis induction was reduced using a neutralizing anti-FasL antibody and overexpression of either the dominant-negative FADD or the viral FLICE inhibitory protein. Furthermore, treatment with a suboptimal dose of FR901228 greatly sensitized osteosarcoma cells to agonistic anti-Fas antibody-mediated apoptosis. These findings suggest that FR901228 is a highly promising antitumor agent against osteosarcoma, inducing apoptosis by the activation of the Fas/FasL system.

Somatic NLRP3 mosaicism in Muckle-Wells syndrome. A genetic mechanism shared by different phenotypes of cryopyrin-associated periodic syndromes

UNLABELLED : Familial cold autoinflammatory syndrome, Muckle-Wells syndrome (MWS), and chronic, infantile, neurological, cutaneous and articular (CINCA) syndrome are dominantly inherited autoinflammatory diseases associated to gain-of-function NLRP3 mutations and included in the cryopyrin-associated periodic syndromes (CAPS). A variable degree of somatic NLRP3 mosaicism has been detected in ≈35% of patients with CINCA. However, no data are currently available regarding the relevance of this mechanism in other CAPS phenotypes. OBJECTIVE To evaluate somatic NLRP3 mosaicism as the disease-causing mechanism in patients with clinical CAPS phenotypes other than CINCA and NLRP3 mutation-negative. METHODS NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. Apoptosis-associated Speck-like protein containing a CARD (ASC)-dependent nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB) activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. RESULTS A variable degree (5.5-34.9%) of somatic NLRP3 mosaicism was detected in 12.5% of enrolled patients, all of them with a MWS phenotype. Six different missense variants, three novel (p.D303A, p.K355T and p.L411F), were identified. Bioinformatics and functional analyses confirmed that they were disease-causing, gain-of-function NLRP3 mutations. All patients treated with anti-interleukin1 drugs showed long-lasting positive responses. CONCLUSIONS We herein show somatic NLRP3 mosaicism underlying MWS, probably representing a shared genetic mechanism in CAPS not restricted to CINCA syndrome. The data here described allowed definitive diagnoses of these patients, which had serious implications for gaining access to anti-interleukin 1 treatments under legal indication and for genetic counselling. The detection of somatic mosaicism is difficult when using conventional methods. Potential candidates should benefit from the use of modern genetic tools.

Breastfeeding and the prevalence of allergic diseases in schoolchildren: Does reverse causation matter?

Kusunoki T, Morimoto T, Nishikomori R, Yasumi T, Heike T, Mukaida K, Fujii T, Nakahata T. Breastfeeding and the prevalence of allergic diseases in schoolchildren: Does reverse causation matter?
Pediatr Allergy Immunol 2010: 21: 60–66.
© 2010 John Wiley & Sons A/S

Reduced numbers and proapoptotic features of mucosal-associated invariant T cells as a characteristic finding in patients with inflammatory bowel disease

Background:Mucosal-associated invariant T (MAIT) cells are innate-like T cells involved in the homeostasis of mucosal immunity; however, their role in inflammatory bowel disease (IBD) is unclear. Methods:Flow cytometry was used to enumerate peripheral blood MAIT cells in 88 patients with ulcerative colitis (UC), 68 with Crohns disease (CD), and in 57 healthy controls. Immunohistochemistry identified MAIT cells in intestinal tissue samples from patients with UC (n = 5) and CD (n = 10), and in control colon (n = 5) and small intestine (n = 9) samples. In addition, expression of activated caspases by MAIT cells in the peripheral blood of 14 patients with UC and 15 patients with CD, and 16 healthy controls was examined. Results:Peripheral blood analysis revealed that patients with IBD had significantly fewer MAIT cells than healthy controls (P < 0.0001). The number of MAIT cells in the inflamed intestinal mucosae of patients with UC and CD was also lower than that in control mucosae (P = 0.0079 and 0.041, respectively). The number of activated caspase-expressing MAIT cells in the peripheral blood of patients with UC and CD was higher than that in healthy controls (P = 0.0061 and 0.0075, respectively), suggesting that the reduced MAIT cell numbers in IBD are associated with an increased level of apoptosis among these cells. Conclusions:The number of MAIT cells in the peripheral blood and inflamed mucosae of patients with UC and CD was lower than that in non-IBD controls. Also, MAIT cells from patients with IBD exhibited proapoptotic features. These data suggest the pathological involvement and the potential for therapeutic manipulation of these cells in patients with IBD.

Robust and Highly-Efficient Differentiation of Functional Monocytic Cells from Human Pluripotent Stem Cells under Serum- and Feeder Cell-Free Conditions.

Monocytic lineage cells (monocytes, macrophages and dendritic cells) play important roles in immune responses and are involved in various pathological conditions. The development of monocytic cells from human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) is of particular interest because it provides an unlimited cell source for clinical application and basic research on disease pathology. Although the methods for monocytic cell differentiation from ESCs/iPSCs using embryonic body or feeder co-culture systems have already been established, these methods depend on the use of xenogeneic materials and, therefore, have a relatively poor-reproducibility. Here, we established a robust and highly-efficient method to differentiate functional monocytic cells from ESCs/iPSCs under serum- and feeder cell-free conditions. This method produced 1.3×106±0.3×106 floating monocytes from approximately 30 clusters of ESCs/iPSCs 5–6 times per course of differentiation. Such monocytes could be differentiated into functional macrophages and dendritic cells. This method should be useful for regenerative medicine, disease-specific iPSC studies and drug discovery.

Detection of Base Substitution-Type Somatic Mosaicism of the NLRP3 Gene with >99.9% Statistical Confidence by Massively Parallel Sequencing

Chronic infantile neurological cutaneous and articular syndrome (CINCA), also known as neonatal-onset multisystem inflammatory disease (NOMID), is a dominantly inherited systemic autoinflammatory disease and is caused by a heterozygous germline gain-of-function mutation in the NLRP3 gene. We recently found a high incidence of NLRP3 somatic mosaicism in apparently mutation-negative CINCA/NOMID patients using subcloning and subsequent capillary DNA sequencing. It is important to rapidly diagnose somatic NLRP3 mosaicism to ensure proper treatment. However, this approach requires large investments of time, cost, and labour that prevent routine genetic diagnosis of low-level somatic NLRP3 mosaicism. We developed a routine pipeline to detect even a low-level allele of NLRP3 with statistical significance using massively parallel DNA sequencing. To address the critical concern of discriminating a low-level allele from sequencing errors, we first constructed error rate maps of 14 polymerase chain reaction products covering the entire coding NLRP3 exons on a Roche 454 GS-FLX sequencer from 50 control samples without mosaicism. Based on these results, we formulated a statistical confidence value for each sequence variation in each strand to discriminate sequencing errors from real genetic variation even in a low-level allele, and thereby detected base substitutions at an allele frequency as low as 1% with 99.9% or higher confidence.

Differential Requirement for the CD40-CD154 Costimulatory Pathway during Th Cell Priming by CD8α+ and CD8α− Murine Dendritic Cell Subsets

Dendritic cells (DCs) regulate the development of distinct Th populations and thereby provoke appropriate immune responses to various kinds of Ags. In the present work, we investigated the role CD40-CD154 interactions play during the process of Th cell priming by CD8α+ and CD8α− murine DC subsets, which have been reported to differently regulate the Th response. Adoptive transfer of Ag-pulsed CD8α+ DCs induced a Th1 response and the production of IgG2a Abs, whereas transfer of CD8α− DCs induced Th2 cells and IgE Abs in vivo. Induction of distinct Th populations by each DC subset was also confirmed in vitro. Although interruption of CD80/CD86-CD28 interactions inhibited Th cell priming by both DC subsets, disruption of CD40-CD154 interactions only inhibited the induction of the Th1 response by CD8α+ DCs in vivo. CD40-CD154 interactions were not required for the proliferation of Ag-specific naive Th cells stimulated by either DC subset, but were indispensable in the production of IL-12 from CD8α+ DCs and their induction of Th1 cells in vitro. Taken together, in our immunization model of Ag-pulsed DC transfer, CD40-CD154 interactions play an important role in the development of CD8α+ DC-driven Th1 responses but not CD8α− DC-driven Th2 responses to protein Ags.

Changing prevalence and severity of childhood allergic diseases in kyoto, Japan, from 1996 to 2006.

BACKGROUND Published data regarding changes in the prevalence of childhood allergic diseases in Japan have been limited. METHODS To observe changes in the recent trends of the childhood allergy epidemic in Japan, a population-based questionnaire survey of allergic diseases was conducted among 13,215 schoolchildren, aged 7 to 15 years, in Kyoto, Japan in 2006. The results were compared with those obtained in the 1996 survey using the same scale and methods in the same region. RESULTS The prevalences of bronchial asthma (BA), atopic dermatitis (AD), allergic rhinitis (AR), and allergic conjunctivitis (AC) in 1996 and 2006 were 5.1% and 5.0% (p = 0.58), 4.2% and 5.6% (p < 0.0001), 20.3% and 27.4% (p < 0.0001), and 13.3% and 25.2% (p < 0.0001), respectively. Although the distribution of BA severity improved, the severity distribution of AD, AR, and AC all deteriorated. The lifetime prevalence (present prevalence and past history combined) of BA increased from 6.5% to 7.6% (p < 0.0001). The sex ratio analysis showed that the female predominance in the prevalence of AD observed in 1996 disappeared in 2006, indicating a particular rise in AD prevalence among boys. CONCLUSIONS Overall, the results indicate that the rising trend of allergic diseases, especially in AD, AR, and AC, continues among schoolchildren living in Kyoto, Japan. Special attention should be paid to skin and nasoocular symptoms.