Purpose Familial hemophagocytic lymphohistiocytosis type 3 (FHL3) is a genetic disorder that results in immune dysregulation. It requires prompt and accurate diagnosis. A natural killer (NK) cell degranulation assay is often used to screen for FHL3 patients. However, we recently encountered two cases of late-onset FHL3 carrying novel UNC13D missense mutations: in these cases, the degranulation assays using freshly isolated and interleukin (IL)-2-activated NK cells yielded contradictory results. Since the defective degranulation of CD57 + cytotoxic T lymphocytes (CTLs) in these cases was helpful for making the diagnosis, we assessed whether the CD57 + CTL degranulation assay more effectively identified FHL3 patients than the NK cell assays. Methods Forty additional patients with hemophagocytic lymphohistiocytosis were prospectively screened for FHL3 by measuring the perforin expression in NK cells and the the expression of Munc13-4, syntaxin-11, and Munc18-2 in platelets, and by performing NK cell and CTL degranulation assays. The results were confirmed by genetic analysis. Results The freshly isolated NK cell degranulation assay detected FHL3 patients with high sensitivity (100%) but low specificity (71%). The IL-2-stimulated NK cell assay had improved specificity, but 3 out of the 31 non-FHL3 patients still showed degranulation below the threshold level. The CD57 + CTL degranulation assay identifying FHL3 patients with high sensitivity and specificity (both 100%). Conclusions The CD57 + CTL degranulation assay more effectively identified FHL3 patients than the NK cell-based assays.

Department of Pediatrics, Faculty of Medicine, Kyoto University, Kyoto, Japan Department of Pediatrics, Tenri Hospital, Tenri, Japan Department of Diagnostic Pathology, Faculty of Medicine, Kyoto University, Kyoto, Japan Department of Dermatology, Faculty of Medicine, Kyoto University, Kyoto, Japan Department of Life Sciences, Hokkaido Pharmaceutical University School of Pharmacy, Sapporo, Japan Department of Pediatrics, Faculty of Medicine, Tohoku University, Sendai, Japan Kazusa DNA Research Institute, Kisarazu, Japan

Objective: The objective of this study was to estimate gonadotropin concentrations in small for gestational age (SGA) male infants with the reactivation of the hypothalamic–pituitary–gonadal axis during the first few months of life that is important for genital development. Study Design: We prospectively examined 15 SGA and 15 appropriate for gestational age (AGA) preterm male infants between 2013 and 2014 at Kyoto University Hospital. Gonadotropin concentrations (luteinizing hormone (LH) and follicle-stimulating hormone (FSH)) were measured in serial urine samples from the postnatal days 7 to 168 and compared between SGA and AGA infants using the Mann–Whitney test. Results: A longitudinal analysis showed that SGA infants had higher LH and lower FSH concentrations ( P =0.004 and P =0.006, respectively) than AGA infants. Conclusion: Male infants who are SGA at birth because of fetal growth restriction have gonadotropin secretion abnormalities in the first few months of life.

OBJECTIVES:To assess the long-term safety and efficacy of canakinumab in Japanese patients with cryopyrin-associated periodic syndrome (CAPS).METHODS:In this open-label phase 3 study, Japanese patients aged ≥2 years with CAPS received canakinumab 2-8 mg/kg subcutaneously every 8 weeks. The duration of the core treatment phase was 24 weeks followed by 22 months extension phase. The primary objective was the proportion of patients free of clinical and serologic relapse at week 24.RESULTS:The study enrolled 19 Japanese patients (median age, 14 years; range, 2-48 years) with CAPS [MWS, 7 (36.8%); NOMID, 12 (63.2%)] for a median of 109 weeks. Fifteen patients (79%) achieved a complete response by day 15, 18 (94.7%) by week 24 and all by week 48. At the end of the study, 18 (95%) were free from relapse and 11 (57.9%) were assessed as having no disease activity by the PGA. Thirteen (68%) patients (MWS, 4; NOMID, 9) had their canakinumab dose increased during the trial. All patients experienced at least one adverse event (AE), the most common being infections (100%) and 5 (26.3%) reported serious AEs. No deaths were reported and the only patient who discontinued the study early withdrew consent.CONCLUSIONS:Regular canakinumab treatment every 8 weeks at dose levels from 2-8 mg/kg, based on the clinical need, represents a successful strategy to induce rapid and complete response while maintain long-term disease control in Japanese patients with CAPS. The safety profile of canakinumab was consistent with that observed from previous studies.

Malignant rhabdoid tumor (MRT) is a rare, highly aggressive pediatric malignancy that primarily develops during infancy and early childhood. Despite the existing standard of intensive multimodal therapy, the prognosis of patients with MRT is dismal; therefore, a greater understanding of the biology of this disease is required to establish novel therapies. In this study, we identified a highly tumorigenic sub-population in MRT, based on the expression of CD146 (also known as melanoma cell adhesion molecule), a cell adhesion molecule expressed by neural crest cells and various derivatives. CD146+ cells isolated from four MRT cell lines by cell sorting exhibited enhanced self-renewal and invasive potential in vitro. In a xenograft model using immunodeficient NOD/Shi-scid IL-2Rγ-null mice, purified CD146+ cells obtained from MRT cell lines or a primary tumor exhibited the exclusive ability to form tumors in vivo. Blocking of CD146-related mechanisms, either by short hairpin RNA knockdown or treatment with a polyclonal antibody against CD146, effectively suppressed tumor growth of MRT cells both in vitro and in vivo via induction of apoptosis by inactivating Akt. Furthermore, CD146 positivity in immunohistological analysis of 11 MRT patient samples was associated with poor patient outcomes. These results suggest that CD146 defines a distinct sub-population in MRT with high tumorigenic capacity and that this marker represents a promising therapeutic target.

OBJECTIVES:The aim of this study was to present the genetic and clinical data of the largest cohort of Turkish cryopyrin-associated periodic syndromes (CAPS) patients.METHODS:This is a two-centre descriptive study of Turkish children with clinical diagnosis of CAPS. NLRP3 analyses were performed by Sanger sequencing and by massively parallel sequencing. ASC dependent NF-κB activation and transfection-induced THP-1 cell death assays determined the functional consequences of the detected variants. Disease activity and response to anti interleukin 1 (anti-IL-1) treatment was also assessed.RESULTS:Heterozygous germline NLRP3 mutation was detected in 8 of 14 enrolled patients (57.1%). Two novel somatic mutations Y560H and G307D were found which induced both THP-1 cell death and ASC dependent NF-kB activation. With anti-IL-1 treatment the disease activity was improved in all patients except one. Except two patients with macrophage activation syndrome (MAS) attack, there were no serious adverse events requiring hospitalisation.CONCLUSIONS:CAPS should be considered in all patients with typical symptoms even if Sanger-based genetic analysis is negative, since a considerable number of patients have mosaicism. Treatment should be patient-tailored and MAS should be considered as a rare complication.

NOMID, also known as CINCA syndrome, is a dominantly inherited autoinflammatory disease caused by NLRP3 mutations. The pathophysiology of NOMID is explained by gain of function mutation of NLRP3, which activates NLRP3 inflammasome and produce an excess of IL-1β. This mechanism is supported by clinical observation that anti-IL-1 therapy is effective on its systemic inflammation. However, one of its characteristic features, epiphyseal overgrowth, is considered to be resistant to anti-IL-1 therapy, which raises a question that other mechanism than NLRP3 inflammasome may play a role in the epiphyseal overgrowth. In this study, we investigated the effect of mutated NLRP3 on chondrocytes using induced pluripotent stem cells (iPSCs) derived from NOMID patients, and tried to identify drugs to treat the abnormal chondrocytes overgrowth.

OBJECTIVE:Whether hormone supplementation is necessary for infants with transient hypothyroxinemia of prematurity (THOP) remains controversial, and further analysis of the hypothalamus-pituitary-thyroid axis of infants with THOP is necessary.STUDY DESIGN:Thyrotropin-releasing hormone (TRH) stimulation tests were performed at 2 weeks of age in 50 infants with a gestational age of 30 weeks or less, and the data were analyzed retrospectively.RESULT:Subjects were divided into three groups; group A consisted of euthyroid infants, group B consisted of infants with THOP and group C consisted of hypothyroid infants. The basal and peak thyroid-stimulating hormone level of group C in response to TRH stimulation tests was significantly higher than the others, but no differences were observed between groups A and B.CONCLUSION:The response of infants with THOP to the TRH stimulation test was not different from that of euthyroid infants, which suggested that their hypothalamic-pituitary-thyroid axis was appropriately regulated in infants with THOP.

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 Crohn's 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.

Adverse prognostic impact of KIT mutations in childhood CBF-AML: the results of the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 trial

Multicentric carpotarsal osteolysis syndrome (MCTO) is a rare autosomal dominant disorder, characterized by aggressive osteolysis of the carpal and tarsal bone, and progressive nephropathy leading to end-stage renal disease. Recently, heterozygous mutations in MAFB gene within a short region of the amino-terminal transcriptional activation domain had been reported to cause MCTO. Although affected patients suffer from early childhood with a clinical appearance mimicking juvenile idiopathic arthritis, most of anti-rheumatic agents are ineffective to control their pain.

AimPhysical growth in neurologically healthy preterm infants affects motor development. This study investigated the separate relationships between muscle and fat in infancy and later motor development and physical growth.MethodsMuscle thickness and subcutaneous fat thickness of the anterior thigh were measured using ultrasound images obtained from neurologically healthy preterm infants at birth, 3, 6, 12 and 18months' corrected age. We also obtained the Pediatric Evaluation of Disability Inventory and Alberta Infant Motor Scale scores at 18months' corrected age to assess motor ability and motor delay.ResultsThirty preterm infants completed the study protocol. There was a significant positive correlation between motor ability and increments in subcutaneous fat thickness during the first 3 and 6months' corrected age (r=0.48 and 0.40, p<0.05, respectively), but not between motor ability and muscle thickness growth in any of the periods. A secondary, logistic regression analysis showed that increments in subcutaneous fat thickness during the first 3months were a protective factor for motor delay.ConclusionSubcutaneous fat accumulation in early infancy is more strongly associated with motor development and delay than muscle growth.

Using serum-containing culture, we examined whether AGM-S3 stromal cells, alone or in combination with hematopoietic growth factor(s), stimulated the proliferation of CD34 + cells from patients with juvenile myelomonocytic leukemia (JMML). AGM-S3 cells in concert with stem cell factor plus thrombopoietin increased the numbers of peripheral blood CD34 + cells to approximately 20-fold of the input value after 2 weeks in nine JMML patients with either PTPN11 mutations or RAS mutations, who received allogeneic hematopoietic transplantation. Granulocyte-macrophage colony-stimulating factor (GM-CSF) also augmented the proliferation of JMML CD34 + cells on AGM-S3 cells. The expansion potential of CD34 + cells was markedly low in four patients who achieved spontaneous hematological improvement. A large proportion of day-14-cultured CD34 + cells were negative for CD38 and cryopreservable. Cultured JMML CD34 + CD38 − cells expressed CD117, CD116, c-mpl, CD123, CD90, but not CXCR4, and formed GM and erythroid colonies. Day-7-cultured CD34 + cells from two of three JMML patients injected intrafemorally into immunodeficient mice stimulated with human GM-CSF after transplantation displayed significant hematopoietic reconstitution. The abilities of OP9 cells and MS-5 cells were one-third and one-tenth, respectively, of the value obtained with AGM-S3 cells. Our culture system may provide a useful tool for elucidating leukemogenesis and for therapeutic approaches in JMML.

Physical growth in neurologically healthy preterm infants affects motor development. This study investigated the separate relationships between muscle and fat in infancy and later motor development and physical growth. Muscle thickness and subcutaneous fat thickness of the anterior thigh were measured using ultrasound images obtained from neurologically healthy preterm infants at birth, 3, 6, 12 and 18 months' corrected age. We also obtained the Pediatric Evaluation of Disability Inventory and Alberta Infant Motor Scale scores at 18 months' corrected age to assess motor ability and motor delay. Thirty preterm infants completed the study protocol. There was a significant positive correlation between motor ability and increments in subcutaneous fat thickness during the first 3 and 6 months' corrected age (r = 0.48 and 0.40, p < 0.05, respectively), but not between motor ability and muscle thickness growth in any of the periods. A secondary, logistic regression analysis showed that increments in subcutaneous fat thickness during the first 3 months were a protective factor for motor delay. Subcutaneous fat accumulation in early infancy is more strongly associated with motor development and delay than muscle growth.

In recent years, responsible genes for autoinflammatory diseases have been increasingly known and clinical phenotype-genotype correlations of these diseases have been explored through international clinical databases such as EUROFEVER project. However, clinical features of genetic disorders could be affected by countries and races of the patients. Actually, patients with Familial Mediterranean fever in Japan show the different distribution of both clinical features and MEFV mutations. Up to now, however, no nationwide research of autoinflammatory diseases with standardized contents which can integrate with data of other international studies has been performed in Japan.

CINCA syndrome, also known as NOMID, is a rare autoinflammatory disease caused by the NLRP3 mutations. It has been known that conventional genetic analysis failed to detect disease-causing mutations in approximately 40% of patients. We have recently identified NLRP3 somatic mosaicism on 70% of these ”mutation-negative” patients in the international collaborative study (Tanaka N. and Izawa K. et al., Arthritis Rheum, 2011), and found no significant differences on systemic inflammation between heterozygous germline mutations and somatic mosaicism. This raises a question how a small number of NLRP3-mutated cells cause systemic inflammation as severely as 100% of germline mutations.

OBJECTIVESCryopyrin-associated periodic syndrome (CAPS), a rare hereditary auto-inflammatory disease, is associated with mutations in the NLRP3 gene resulting in elevated interleukin-1β (IL-1 β) release. CAPS generally occurs in early childhood with most patients presenting with periodic fever, skin rash, osteoarthropathy, aseptic meningitis, sensorineural hearing loss and optic neuritis. Canakinumab, a fully human anti-IL-1β monoclonal antibody which binds selectively to IL-1β, has demonstrated good efficacy with CAPS. This is the first study to evaluate the safety and efficacy of canakinumab in Japanese patients with CAPS.METHODSIn this open-label study, 19 Japanese CAPS patients aged ≥2 years received canakinumab either 150 mg s.c. or 2 mg/kg for patients with a body weight ≤ 40 kg every 8 weeks for 24 weeks. The primary objective was to assess the proportion of patients who were free of relapse at week 24.RESULTSA complete response was achieved in 18 (94.7%) patients with some requiring a dose and/or a frequency adjustment to attain full clinical response. The majority of patients (14/18; 77.8%) were in remission, i.e. free of relapse at week 24. Auto-inflammatory disease activity as assessed by physician's global assessment declined from baseline to end of the study (score of absent in 10.5% at baseline versus 31.6% at end of the study). Two patients had serious adverse events (SAEs), which resolved with standard treatment. One patient reported a mild injection-site reaction. No malignancies or deaths were reported during the study.CONCLUSIONSCanakinumab 150 mg s.c. every 8 weeks was well-tolerated, highly efficacious and offered a convenient dosing regimen for treating Japanese patients with CAPS.