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We herein report a 36-year-old man with repeated necrotizing lymphadenitis due to MEFV gene mutations. The patient's chief complaints were a fever and painful cervical lymphadenopathy. We diagnosed him with necrotizing lymphadenitis based on the pathological findings of the lymph nodes and the exclusion of other differential diseases. The same episode recurred four times. We speculated the involvement of autoinflammatory backgrounds and detected MEFV gene mutations of E148Q (homo), P369S, and R408Q. Considering the elevation of interleukin-18, these mutations probably played roles in the repeated necrotizing lymphadenitis.

Background. Immunoglobulin A vasculitis (IgAV) is the most common vasculitis of childhood. However, its etiology remains unknown. In the Mediterranean region, 10% of patients with IgAV harbor homozygous and compound heterozygous mutations in the Mediterranean fever (MEFV) gene. Thus, such mutations may be involved in the development of IgAV. Objective. We present a Japanese patient with IgAV harboring MEFV gene mutation. Further, a comprehensive literature analysis was performed to validate the clinical characteristics of Japanese patients with IgAV harboring MEFV mutation. Methods. A 5-year-old girl presented with IgAV. She experienced prolonged abdominal pain, which was steroid-resistant. When treatment with colchicine was started, her abdominal pain disappeared immediately. The serum interleukin-18 levels of the patient and other patients with IgAV and FMF were evaluated using enzyme-linked immunosorbent assay. Moreover, all exons of the MEFV gene were analyzed using the direct sequence method. A comprehensive search of Japanese patients with IgAV harboring MEFV gene mutations in PubMed, Ichushi-Web, and Medical Online was conducted. Results. The serum interleukin-18 level of the patient was higher than that of other patients with IgAV and was similar to that of patients with FMF harboring M694I mutation. In addition, the patient presented with E148Q/M694I mutation. In previous studies, only four patients presented with IgAV harboring MEFV gene mutation in Japan. Conclusion. MEFV gene mutations can be masked if the symptoms of IgAV are prolonged or if patients are refractory to treatment. Moreover, the prevalence of IgAV associated with MEFV gene mutation is low in Japan.

We herein report a 36-year-old man with repeated necrotizing lymphadenitis due to MEFV gene mutations. The patient's chief complaints were a fever and painful cervical lymphadenopathy. We diagnosed him with necrotizing lymphadenitis based on the pathological findings of the lymph nodes and the exclusion of other differential diseases. The same episode recurred four times. We speculated the involvement of autoinflammatory backgrounds and detected MEFV gene mutations of E148Q (homo), P369S, and R408Q. Considering the elevation of interleukin-18, these mutations probably played roles in the repeated necrotizing lymphadenitis.

A 38-year-old Japanese man without any significant past medical history was referred to our clinic to undergo further examination for a "refractory infection in his joints". He suffered recurrent migratory polyarthritis starting from bilateral knees to his right elbow. Certain antibiotic therapies appeared to improve his symptoms, but the symptoms recurred due to the migratory nature of arthritis. A diagnosis of familial Mediterranean fever (FMF) was considered and diagnostic tests were performed. Not many differential diagnoses exist for migratory polyarthritis, particularly when it has a recurrent nature. The administration of antibiotics without sufficient diagnostic consideration can cause a delay in making an accurate diagnosis and thereby also cause a delay in administering appropriate treatment.

BACKGROUND Familial Mediterranean fever (FMF) is an autosomal recessive disease caused by mutations in the MEFV gene. Mutations in exon 10 are associated with typical FMF. Most Japanese patients with typical FMF are compound heterozygotes of M694I in exon 10 and E148Q in exon 2. However, the pathogenic role of E148Q remains controversial. METHODS We assessed symptoms and serum cytokines among patients with FMF and their family members. They were divided into three subgroups, based on MEFV mutations: individuals carrying M694I and E148Q (group A, n = 14), individuals carrying M694I, but not E148Q (group B, n = 10), and individuals carrying E148Q, but not M694I (group C, n = 11). RESULTS All but one individual in group A had typical FMF phenotypes, whereas no individual in groups B and C exhibited any episodes of fever or serositis. The serum levels of interleukin-18 during the afebrile phase were significantly elevated in group A (2,806 ± 2,107 pg/mL), compared to those in groups B (499 ± 369 pg/mL) and C (427 ± 410 pg/mL). No difference in interleukin-6 levels was observed among the three groups. CONCLUSIONS These findings indicated that E148Q may contribute to disease development of FMF in Japanese patients carrying the heterozygous M694I mutation in MEFV and that genetic testing of both parents would lead to better counseling for their children.

Objectives: Genetic analysis of TNFRSF1A can confirm the diagnosis of tumor necrosis factor receptor-associated periodic syndrome (TRAPS), but interpretation of the pathogenesis of variants of unknown significance is sometimes required. The aim of this study was to evaluate the clinical significance of serum soluble tumor necrosis factor receptor type I (sTNFR-I)/II ratio to differentiate TRAPS from other autoinflammatory diseases. Methods: Serum sTNFR-I and sTNFR-II levels were measured using an enzyme-linked immunosorbent assay in patients with TRAPS (n = 5), familial Mediterranean fever (FMF) (n = 14), systemic juvenile idiopathic arthritis (s-JIA) (n = 90), and Kawasaki disease (KD) (n = 37) in the active and inactive phase, along with healthy controls (HCs) (n = 18). Results: In the active phase, the serum sTNFR-I/II ratio in patients with s-JIA, KD, and FMF was significantly elevated compared with that in HCs, whereas it was not elevated in patients with TRAPS. In the inactive phase, the serum sTNFR-I/II ratio in patients with s-JIA and FMF was significantly higher compared with that in HCs, and the ratio was lower in TRAPS patients than in patients with s-JIA and FMF. Conclusions: Low serum sTNFR-I/II ratio in the active and inactive phase might be useful for the differential diagnosis of TRAPS and other autoinflammatory diseases.

金沢大学附属病院小児科, Background: Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in the MEFV gene. Mutations in exon 10 are associated with typical FMF phenotypes, and patients with exon 10 mutations have higher serum levels of interleukin (IL)-18 both during attacks and afebrile phases, compared to those without exon 10 mutations. However, longitudinal changes of serum IL-18 in FMF have not been fully characterized. Methods: We serially evaluated serum levels of pro-inflammatory cytokines, including IL-18, in 12 patients with FMF carrying exon 10 mutations, all of whom showed typical FMF attacks. Results: Markedly high concentrations of IL-18 were observed in all patients at diagnosis (5099. ±. 6084. pg/mL). Serum IL-18 levels declined progressively after colchicine treatment in 7 patients (group A), whereas 5 patients showed continued elevation of circulating IL-18, despite declines in IL-6 and neopterin (group B). The mean follow-up times in the two groups were 4.7. ±. 3.2 and 4.8. ±. 1.5 years, respectively. The mean serum IL-18 level at the last hospital visit in group B was 4190. ±. 2610[U+202F]pg/mL. There were no differences in onset age, initial IL-18 levels, and colchicine doses between the groups. FMF attacks almost disappeared in both groups, but there were trends towards more frequent subtle symptoms such as abdominal discomfort in group B. Conclusions: Sustained elevation of serum IL-18 may suggest the presence of persistent subclinical inflammation. Therefore, longitudinal examination of serum IL-18 may contribute to better follow-up of FMF patients with exon 10 mutations. © 2017 Elsevier Ltd., Embargo Period 12 moths

Objectives: Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by mutations in MEFV. Mutations in exon 10 are associated with typical FMF phenotypes, whereas the pathogenic role of variants in exons 2 and 3 remains uncertain. Recent evidence suggests that circulating microRNAs (miRNAs) are potentially useful biomarkers in several diseases. Therefore, their expression was assessed in FMF. Methods: The subjects were 24 patients with FMF who were between attacks: eight with exon 10 mutations (group A), eight with exon 3 mutations (group B), and eight without exon 3 or 10 mutations (group C). We also investigated eight cases of PFAPA as disease controls. Exosome-rich fractionated RNA was subjected to miRNA profiling by microarray. Results: Using the expression patterns of 26 miRNAs, we classified FMF (groups A, B, and C) and PFAPA with 78.1% accuracy. In FMF patients, groups A and B, A and C, and B and C were distinguished with 93.8, 87.5, and 100% accuracy using 24, 30, and 25 miRNA expression patterns, respectively. Conclusions: These findings suggest that expression patterns of circulating miRNAs differ among FMF subgroups based on MEFV mutations between FMF episodes. These patterns may serve as a useful biomarker for detecting subgroups of FMF. © 2017 Japan College of Rheumatology, Embargo Period 12 months

Background FMF is recessive systemic auto inflammatory disorder characterized by recurrent fever, peritonitis, pleuritis, pericarditis and arthritis accompanied with skin rash. Mutation of MEFV gene encoding pyrin resulted in inflammasome activation and the uncontrolled production of IL-1β. Overview of pathogenesis, clinical features and management in Japanese patients with FMF had been reported1. However, the differences of clinical features between mutated and non-mutated of MEFV still remain unclear. Objectives We have analyzed 20 Japanese patients with FMF to clarify the association between various clinical features and mutation of MEFV. Methods Genomic DNA were purified from white blood cells in 20 FMF patients, and mutated MEFV has been explored. We have analyzed MEFV, TNFRSF1A, MVK and NLRP3 genes in 20 patients with FMF. Therefore, we excluded another autoinflammatory diseases such as TNF receptor-associated syndrome (TRAPS), mevalonate kinase deficiency and cryopyrin-associated periodic syndrome. Clinical symptoms and laboratory data were analyzed around onset time. Each patient had been treated with colchicine (0.5-2 mg). Results Characteristics of Patients with FMF (13 female/7 male) were as follows; Onset time were 0-53 years old (17.5 ±12.2), and teen aged patients were most. Frequencies of clinical symptoms such as periodic fever, headache, arthralgia, abdominal pain, chest pain, myalgia, and cervical lymph nodes swelling were 20/20, 7/20, 6/20, 5/20,4/20,2/20 and 1/20, respectively (double symptoms were observed). Patients with FMF were divided to 3 groups as follows; Patients with typical compound heterozygous mutations of MEFV (E148Q/M694I) which indicated exon 10 mutation, were 3 cases (group 1). Patients with atypical mutations, except for mutations in exon 10, such as exon 1 (E84K, L110P), 2 (E148Q), 3(P369S, R408Q), 5(S503C) and 9(I591M) were 8 cases (group 2). Patients with no mutations in MEFV gene were 9 cases (group 3). There were no significant differences of age at first visiting hospital (FV), onset age of fever attack (O), duration of fever attack (D) and frequency of fever attack (FF) between group 1, group2 and group 3 ( FV: 22.3 ± 4.5, 26.8 ± 14.6 years old (yo) and 29.5 ± 8.7 yo, O: 11.6 ± 2.4 yo, 18.5 ± 13.8 yo and 18.5 ± 12.0 D: 3.0 ± 1.4 hrs, 6.3 ± 3.2 hrs, and 8.1 ± 8.5 hrs, FF: 1.2 ± 0.2/month (M), 1.2 ± 1.1/M, and 1.1 ± 0.2/M), respectively. Laboratory examinations such as WBC, CRP and serum amyloid A (SAA) were not significantly different between 3 groups. All of those patients were effective for colchicine treatment except for 2 patients in group 1 because of severe diarrhea and alopecia. Finally, 2 patients in group 1 received canakinumab treatment. Mutations of E148Q were found in 9 patients (45%). Conclusion We have examined association between clinical features and mutations of MEFV in 20 Japanese patients, suggesting no positive findings in Japanese patients with FMF. Mutations of E148Q in exon 2 were observed in 16-23 % of normal Japanese patients1, indicating that E148Q is the polymorphism or accelerating factor. References [1] K. Migita et al, Familial Mediterranean fever : overview of pathogenesis, clinical features and management. Immunological Medicine 41:2, 55-61, 2018 Disclosure of Interests None declared

Shiga toxin (STX) is one of the main factors inducing hemorrhagic colitis and hemolytic-uremic syndrome (HUS) in infections with STX-producing Escherichia coli (STEC). Approximately 62% of patients with HUS showed symptoms of encephalopathy in the 2011 Japanese outbreak of STEC infections. At that time, we reported elevated serum concentrations of tumor necrosis factor (TNF)-α in patients with acute encephalopathy during the HUS phase. In the current study, we investigated whether TNF-α augments the effects of STX in glial cell lines and primary glial cells. We found that TNF-α alone or STX in combination with TNF-α activates nuclear factor-κB (NF-κB) signaling and inhibits growth of glial cells. The magnitude of the NF-κB activation and the inhibition of cell growth by the STX and TNF-α combination was greater than that obtained with TNF-α alone or STX alone. Thus, this in vitro study reveals the role of TNF-α in glial cells during STEC infections. © 2016 Elsevier B.V. All rights reserved., Embargo Period 12 months

Background: Food protein-induced enterocolitis syndrome (FPIES) is an uncommon, non-IgE-mediated food allergy. We recently described a significant increase in fecal eosinophil-derived neurotoxin (EDN) after ingestion of the causative food. However, little is known about the activation status of circulating eosinophils in patients with an acute FPIES reaction. Methods: Surface CD69 expression was assessed by flow cytometry on peripheral eosinophils from 5 patients with FPIES before and after ingestion of the causative food. Fecal EDN was measured by enzyme-linked immunosorbent assay. Results: No eosinophil activation was observed before ingestion; however, a significant increase in CD69 expression on eosinophils after an acute FIPES reaction was demonstrated in all of the patients. There was no significant change in absolute eosinophil counts in the peripheral blood. The levels of fecal EDN increased on the day after ingestion of the causative food in all patients. Conclusion: These results suggest that circulating eosinophils as well as eosinophils in the intestinal mucosal tissue are activated in acute FPIES reactions and might be associated with systemic immune events in FPIES. © 2016 S. Karger AG, Basel., Embargo Period 12 months

Background:FMF is recessive systemic autoinflammatory disorder characterized by recurrent fever, peritonitis, pleuritis, pericarditis and arthritis accompanied with headache and abdominal pain. Mutation of MEFV gene encoding pyrin resulted in inflammasome activation and the uncontrolled production of IL-1β. Overview of pathogenesis, clinical features and management in Japanese patients with FMF had been reported. However, the differences of clinical features between mutated and non-mutated of MEFV still remain unclear.Objectives:We have analyzed 31 Japanese patients with FMF in Gifu district to clarify the association between various clinical features and mutation ofMEFV.Methods:Genomic DNA were purified from white blood cells in 31 FMF patients, and mutated MEFV has been explored. We have analyzedMEFV, TNFRSF1A, MVK and NLRP3 genesin 31 patients with FMF except for 1 patient. Therefore, we excluded another autoinflammatory diseases such as TNF receptor-associated syndrome (TRAPS), mevalonate kinase deficiency and cryopyrin-associated periodic syndrome. Clinical symptoms and laboratory data were analyzed around onset time. Each patient had been treated with colchicine (0.5-2 mg).Results:Characteristics of Patients with FMF (22 female/9 male) were as follows; Onset time were 0-56 years-old (21.4 ±11.8), and Frequencies of clinical symptoms such as periodic fever, headache, abdominal pain, arthralgia, chest pain, cervical lymph nodes swelling, and myalgia were 31/31, 9/31, 8/31, 6/31, 5/31, 3/31 and 1/31, respectively (double symptoms were observed). Patients with FMF were divided into 3 groups as follows; Patients with typical compound heterozygous mutations of MEFV (E148Q /M694I) which indicated exon 10 mutation, were 5 cases (G1). Patients with atypical mutations, except for exon 10, such as 133G>A in 3UTR, exon 1 (E84K), 2 (L110P, E148Q), 3(R202Q, P257L, G304R, P369S, R408Q), 5(S503C) and 9(I591M) were 13 cases (G2). Patients with no mutations in MEFV gene were 12 cases (G3). There were no significant differences of age at first visiting hospital (FV)and onset age of fever attack (O) (FV: 29.0 ± 15.6, 27.1 ± 12.5 years-old (yo) and 34.7 ± 12.7 yo, O: 21.0 ± 17.6 yo, 17.8 ± 12.1 yo and 25.2 ± 6.5). But significant differences in duration of fever attack (D) and frequency of fever attack (FF) between G1 and G2 or G3 were observed as follows; (D: 2.2 ± 0.4 days vs 5.5 ± 3.1 days, PMEFVmutations have no family histories. Mutations of E148Q were found in 12 patients (40%).Conclusion:We have examined association between clinical features and mutations of MEFV in 31 patients in Gifu district, suggesting that duration of fever attacks and frequency of fever attacks in G1 are significantly shorter than G2 and G3, respectively in Japanese patients with FMF. Mutations of E148Q in exon 2 were observed in 16-23 % of normal Japanese patients, indicating that E148Q is the polymorphism or accelerating factor.Disclosure of Interests:None declared

Hypersensitivity to mosquito bites (HMB) is a cutaneous disorder belonging to the group of Epstein-Barr virus (EBV)-associated T/natural killer (NK)-cell lymphoproliferative diseases, and is primarily mediated by EBV-infected NK cells. It is characterized by intense local skin reactions accompanied by general symptoms after mosquito bites, and infiltration of EBV-infected NK cells into the bite sites. However, the mechanisms underlying these reactions have not been fully examined. We recently described the activation of circulating basophils by mosquito extracts in vitro in a patient with HMB. To further investigate this finding, we studied four additional patients with HMB. All patients showed typical clinical features of HMB after mosquito bites and they had NK lymphocytosis and high peripheral blood EBV DNA loads. We found evidence of EBV infection in NK cells through in situ hybridization that detected EBV-encoded small RNA-1, and flow cytometry showed HLA-DR expression on almost all NK cells. Basophil activation tests with the extracts of epidemic mosquitoes Culex pipiens pallens and Aedes albopictus showed positive responses to one or both extracts in all samples from patients with HMB, suggesting the presence of mosquito antigen-specific IgE and its binding to basophils. In particular, the extract of Aedes albopictus was able to activate basophils in all available patient samples. These results indicate that basophils and/or mast cells activated by mosquito bites may be involved in initiation and development of severe skin reactions to mosquito bites in HMB. © 2015 The Authors.

Neutrophil-specific granule deficiency (SGD) is a rare autosomal recessive primary immunodeficiency characterized by neutrophil dysfunction, bilobed neutrophil nuclei and lack of neutrophil-specific granules. Defects in a myeloid-specific transcription factor, CCAAT/enhancer binding protein-ε (C/EBPε), have been identified in two cases in which homozygous frameshift mutations led to loss of the leucine zipper domain. In this study, we report a 55-y-old woman affected with SGD caused by a novel homozygous 2-aa deletion (ΔRS) in the leucine zipper domain of the C/EBPε gene. The patient showed characteristic neutrophil abnormalities and recurrent skin infections; however, there was no history of deep organ infections. Biochemical analysis revealed that, in contrast to the two frameshift mutations, the ΔRS mutant maintained normal cellular localization, DNA-binding activity, and dimerization, and all three mutants exhibited marked reduction in transcriptional activity. The ΔRS mutant was defective in its association with Gata1 and PU.1, as well as aberrant cooperative transcriptional activation of eosinophil major basic protein. Thus, the ΔRS likely impairs protein-protein interaction with other transcription factors, resulting in a loss of transcriptional activation. These results further support the importance of the leucine zipper domain of C/EBPε for its essential function, and indicate that multiple molecular mechanisms lead to SGD.

金沢大学附属病院小児科, Epstein-Barr virus (EBV)-associated T- or natural killer (NK)-cell lymphoproliferative disease (LPD) is a heterogeneous group of disorders characterized by chronic proliferation of EBV-infected lymphocytes. Patients may present with severe skin manifestations, including hypersensitivity to mosquito bites (HMB) and hydroa vacciniforme (HV)-like eruption, which are characterized by blister formation and necrotic ulceration. Skin biopsy specimens show inflammatory reactions comprising EBV-infected lymphocytes. However, blister fluids have not been fully assessed in patients with this disease. Blister fluids were collected from three patients with EBV-associated LPD: two with HMB and one with HV. Immunophenotyping of blister lymphocytes and measurement of tumor necrosis factor (TNF)-α in blister fluids were performed. The patients with HMB and HV exhibited markedly increased percentages of NK and γδ T cells, respectively, in both peripheral blood and blister fluids. These NK and γδ T cells strongly expressed the activation marker human leukocyte antigen-DR and were considered to be cellular targets of EBV infections. TNF-α was highly elevated in all blister fluids. Severe local skin reactions of EBV-associated LPD may be associated with infiltrating EBV-infected lymphocytes and a high TNF-α concentration in blister fluids. © 2018 Japanese Dermatological Association., Embargo Period 12 months

Familial hemophagocytic lymphohistiocytosis (FHL) is characterized by uncontrolled activation of T cells and macrophages and hypercytokinemia. We have recently described a significant increase in a subpopulation of CD8(+) T cells with downregulation of CD5 during the acute phase of FHL type2 (FHL2; perforin deficiency), which declines after successful treatment, with a concomitant reduction in serum cytokine level. This unusual subset of CD8(+) T cells, however, has not been characterized in patients with other subtypes of FHL. Herein, we describe a patient with FHL3 (Munc13-4 deficiency) carrying compound heterozygous mutations in the UNC13D gene. He had high serum levels of pro-inflammatory cytokines and significantly increased activated CD8(+) T cells with downregulation of CD5 during the acute phase, similar to that found in FHL2. This immunophenotypic feature may serve as a useful marker of immune dysregulation in FHL3 in addition to FHL2.

Familial Mediterranean fever (FMF) is an autoinflammatory disease caused by MEditerranean FeVer gene (MEFV) mutations. In Japan, patients with FMF have been previously reported, including a mild or incomplete form. Several factors are presumed to contribute to the variable penetrance and to the phenotypic variability of FMF. We conducted the current study to investigate the correlation of variable clinical presentations and MEFV genotypic distributions in Japanese FMF patients. We analyzed demographic, clinical, and genetic data for 311 FMF patients enrolled in the study. Clinically, we classified FMF into 2 phenotypes: 1) the “typical” form of FMF, and 2) the “atypical” form of FMF according to the Tel Hashomer criteria. Patients with the typical FMF phenotype had a higher frequency of febrile episodes, a shorter duration of febrile attacks, more frequent thoracic pain, abdominal pain, a family history of FMF, and MEFV exon 10 mutations. Conversely, patients with the atypical FMF phenotype had a lower frequency of fever episodes and more frequent arthritis in atypical distribution, myalgia, and MEFV exon 3 mutations. Multivariate analysis showed that the variable associated with typical FMF presentation was the presence of MEFV exon 10 mutations. Typical FMF phenotype frequencies were decreased in patients carrying 2 or a single low-penetrance mutations compared with those carrying 2 or a single high-penetrance mutations (M694I), with an opposite trend for the atypical FMF phenotype. In addition, patients having more than 2 MEFV mutations had a younger disease onset and a higher prevalence of thoracic pain than those carrying a single or no mutations. Thus, MEFV exon 10 mutations are associated with the more typical FMF phenotype. In contrast, more than half of the Japanese FMF patients without MEFV exon 10 mutations presented with an atypical FMF phenotype, indicating that Japanese FMF patients tend to be divided into 2 phenotypes by a variation of MEFV mutations.

Hemophagocytic lymphohistiocytosis (HLH) is characterized by uncontrolled activation of T cells and macrophages with overproduction of cytokines. Familial HLH type 2 (FHL2) is the most common form of primary HLH and is caused by mutations in PRF1. We have recently described a significant increase in the subpopulation of CD8+ T cells with clonal expansion and CD5 down-regulation in Epstein-Barr virus associated-HLH, which represented a valuable tool for its diagnosis. However, this unusual phenotype of CD8+ T cells has not been investigated fully in patients with FHL2. We performed immunophenotypic analysis of peripheral blood and measured serum pro-inflammatory cytokines in five patients with FHL2. All patients showed significantly increased subpopulations of activated CD8+ T cells with down-regulation of CD5, which were negligible among normal controls. Analysis of T-cell receptor Vβ repertoire suggested the reactive and oligoclonal expansion of these cells. The proportion of the subset declined after successful treatment concomitant with reduction in the serum levels of cytokines in all patients except one who continued to have a high proportion of the subset and died. These findings suggest that down-regulation of CD5 on activated CD8+ T cells may serve as a useful marker of dysregulated T cell activation and proliferation in FHL2. © 2013 American Society for Histocompatibility and Immunogenetics.

Peeling skin disease (PSD) is an autosomal recessive skin disorder caused by mutations in CDSN and is characterized by superficial peeling of the upper epidermis. Corneodesmosin (CDSN) is a major component of corneodesmosomes that plays an important role in maintaining epidermis integrity. Herein, we report a patient with PSD caused by a novel homozygous large deletion in the 6p21.3 region encompassing the CDSN gene, which abrogates CDSN expression. Several genes including C6orf15, PSORS1C1, PSORS1C2, CCHCR1, and TCF19 were also deleted, however, the patient showed only clinical features typical of PSD. The deletion size was 59.1 kb. Analysis of the sequence surrounding the breakpoint showed that both telomeric and centromeric breakpoints existed within Alu-S sequences that were oriented in opposite directions. These results suggest an Alu-mediated recombination event as the mechanism underlying the deletion in our patient.

Background DiGeorge syndrome is a congenital malformation characterized by variable defects of the thymus, heart and parathyroid glands. Athymic patients are classified as exhibiting complete DiGeorge syndrome. Some of these patients may also exhibit oligoclonal T-cell expansion, generalized rash and lymphadenopathy at some point after birth. This rare condition is known as atypical complete DiGeorge syndrome, resembles Omenn syndrome, and has not been fully characterized. Methods The clinical and immunophenotypic features of atypical complete DiGeorge syndrome were assessed in two affected Japanese infants. T-cell receptor (TCR) Vβ repertoire was analyzed on flow cytometry and complementarity-determining region 3 spectratyping. Results Both patients had no detectable thymus tissue and profound T-cell lymphopenia soon after birth. Progressive increase of activated T cells, however, as well as eosinophilia, high serum IgE level, generalized rash, and lymphadenopathy were observed during early infancy. A highly restricted TCR Vβ repertoire was demonstrated both in CD4+ and CD8+ T cells. Conclusions The Omenn syndrome-like manifestations might be associated with the oligoclonal proliferation of activated T cells. Analysis of the immunophenotype and TCR Vβ repertoire is helpful to establish the early diagnosis of atypical complete DiGeorge syndrome.

We describe a patient who presented with prolonged neutropenia due to anti-human neutrophil antigen (HNA)-2 (CD177) antibody after allogeneic bone marrow transplantation. A granulocyte immunofluorescence test showed bimodal expression of antineutrophil antibody that resulted from specific binding of anti-HNA-2 to CD177+ neutrophils from healthy donors. The patient did not respond to granulocyte colony stimulating factor, which is able to upregulate CD177 expression on neutrophils. The low percentage of CD177+ cells in the few remaining neutrophils supports the possibility of elimination of CD177-upregulated neutrophils. These findings might explain an inferior response to neutrophil growth factors in neutropenia secondary to anti-HNA-2 antibody. © 2016 Wiley Periodicals, Inc., Embargo Period 12 months

Chronic active Epstein-Barr virus (EBV) disease (CAEBV) is a systemic EBV-positive lymphoproliferative disorder characterized by fever, lymphadenopathy, and splenomegaly. Patients with CAEBV may present with cutaneous symptoms, including hypersensitivity to mosquito bites and hydroa vacciniforme (HV)-like eruptions. HV is a rare photodermatosis characterized by vesicles and crust formation after exposure to sunlight, with onset in childhood, and is associated with latent EBV infection. While γδ T cells have recently been demonstrated to be the major EBV-infected cell population in HV, the immunophenotypic features of EBV-infected γδ T cells in CAEBV with HV-like eruptions or HV remain largely undetermined. We describe three patients with CAEBV whose γδ T cells were found to be the major cellular target of EBV. HV-like eruptions were observed in two of these patients. A clonally expanded subpopulation of γδ T cells that were highly activated and T cell receptor Vγ9- and Vδ2-positive cells was demonstrated in all patients. We also show that the clonally expanded γδ T cells infiltrated into the HV-like eruptions in one patient from whom skin biopsy specimens were available. These results suggest the pathogenic roles of clonally expanded γδ T cells infected by EBV in patients with CAEBV and HV-like eruptions. © 2012 The Japanese Society of Hematology.

A 38-year-old woman presented with 2 days history of left-flank pain. She had similar episodes of abdominal pain as well as chest pain several times, but symptoms disappeared spontaneously. Each time she developed pain, there was no fever. After ruling out common causes of recurrent abdominal pain, familial Mediterranean fever (FMF) was considered as a potential diagnosis. Genetic tests revealed multiple heterozygote mutations, which may be associated with FMF. Patients with Mediterranean fever mutations may present with atypical presentations without fever, like in this case. Astute clinical suspicion is required to make an accurate diagnosis.

Protein delivery to primary cells by protein transduction domain (PTD) serves as a novel measure for manipulation of the cells for biological study and for the treatment of various human conditions. Although the method has been employed to modulate cellular function in vitro, only limited reports are available on its application in the replacement of deficient signaling molecules into primary cells. We examined the potential of recombinant proteins to compensate for defective cytosolic components of the NADPH oxidase complex in chronic granulomatous disease (CGD) neutrophils in both p47(phox) and p67(phox) deficiency. The p47(phox) or p67(phox) protein linked to Hph-1 PTD was effectively expressed in soluble form and transduced into human neutrophils efficiently without eliciting unwanted signal transduction or apoptosis. The delivered protein was stable for more than 24h, expressed in the cytoplasm, translocated to the membrane fraction upon activation, and, most importantly able to restored reactive oxygen species (ROS) production. Although research on human primary neutrophils using the protein delivery system is still limited, our data show that the protein transduction approach for neutrophils may be applicable to the control of local infections in CGD patients by direct delivery of the protein product.

Background: Balloon angioplasty has recently been adopted as an acceptable form of treatment for stenotic vessel lesions of congenital heart diseases. However, precise mechanisms of restenosis and thrombosis, which are the most common complications after these procedures, are unknown. Methods: We examined the effects of antithrombin III (ATIII) on inflammation, thrombus formation, and remodeling of vascular wall after guidewire-induced injury in the femoral artery of mice. ATIII or saline was administered as a bolus intravenous infusion before injury. Results: Seventy-two hours after injury, approximately half of the saline-treated vessels showed macroscopic thrombus formation. In contrast, no thrombi were seen in the arteries pretreated with ATIII. Significantly higher levels of inflammation were induced in the injured vessels than in the sham-operated controls, as determined by CD11b-positive cell density in the adventitial area. ATIII treatment resulted in marked reduction of inflammatory cell infiltration. Twenty-eight days after injury, similar levels of neointimal proliferation were found in the injured arteries in both groups. Conclusions: Our results suggested that a high dose of ATIII may influence the sequelae of arterial injury by reducing mural thrombus formation and limiting the inflammatory reaction of the vessel wall without altering the process of vascular remodeling.

Most reported cases of familial Mediterranean fever (FMF) involve missense mutations of MEFV concentrated within exon 10. We experienced two independent pedigrees of a unique variant in the MEFV gene that might cause excessive exon 2 skipping due to enhanced alternative splicing. In this study, we tried to elucidate the molecular mechanism of the MEFV variant as a cause of the FMF phenotype. Peripheral blood was obtained from volunteers and two patients with homozygous c.910G>A variant of the MEFV gene. MEFV messenger RNA (mRNA) expression patterns in mononuclear cells and granulocytes were compared using forward and reverse primers from exons 1 and 3, respectively. Expression profiles of pyrin were examined by transfecting wild-type and variant MEFV genes into HEK293T cells. Expression of normal-sized mRNA was extremely reduced in these patients, whereas that of aberrant short mRNA, deleting exon 2 (Δex2), was significantly increased. Immunohistochemical and immunoblotting analyses revealed a truncated immunoreactive pyrin protein in cells transfected with Δex2 cDNA. The MEFV gene c.910G>A variant results in accelerated aberrant splicing with abnormal protein size, presumably leading to anomalous pyrin function. This is the first report to show that an MEFV variant other than missense mutation is responsible for the FMF phenotype.

Background/Aims: In oxidative stress, heme oxygenase-1 (HO-1) plays a pivotal role in maintaining renal function and protecting renal structure, especially in renal tubular epithelial cells. We examined urinary HO-1 (uHO-1) levels to assess whether uHO-1 acts as a sensitive biomarker for detecting tubulointerstitial inflammatory damage in renal diseases. Methods: Immunohistochemical analyses and enzyme-linked immunosorbent assays for uHO-1 were performed using 61 urine samples (supernatants and sediment lysates) from healthy children and renal disease patients. Results: Proximal and distal epithelial cells showed higher uHO-1 levels than squamous and urothelial cells. Inflammatory renal disease patients had higher uHO-1 levels than noninflammatory renal disease patients and controls. In IgA nephropathy, patients with interstitial cellular infiltration showed higher uHO-1 levels than those without it. Among patients with increased urinary β2-microglobulin or N-acetyl-β-D-glucosaminidase levels, uHO-1 levels increased only in those with renal disease and tubulointerstitial inflammatory damage. uHO-1 levels positively correlated with urinary interleukin-6 in inflammatory renal disease patients. Conclusions: These results indicate that uHO-1 is a potentially useful, novel, and noninvasive biomarker for evaluating the degree of tubulointerstitial inflammatory damage in renal disease.

We investigated whether pravastatin ameliorates renal damage induced by cisplatin (CP). Forty-three male Wistar rats were divided into four groups: rats treated with a control diet for 19 days and saline injection on day 14 (group1), group 1 with pravastatin treatment with 19 days (group 2), group 1 with CP injection on day 14 (group 3), and group 2 with CP injection (group 4). Renal function and serum lipids, renal malondialdehyde (MDA) and glutathione (GSH) levels, glutathione peroxidase (GPx) mRNA expression and activity, and kidney triglyceride (TG) concentrations were measured. Histology was evaluated by light microscopy with immunohistochemistry for p53, p53-upregulated modulation of apoptosis (PUMA), and terminal deoxynucleotide transferase dUTP nick end-labeling (TUNEL) staining. CP induced renal tubular damage with a higher MDA level, increased PUMA expression, p53- and TUNEL-positive cells counts, elevation of serum lipids, and decreased GSH level, GPx mRNA expression, and activity. Pravastatin partially ameliorated CP-induced renal injury, based on suppression of the renal MDA and TG levels, decreased p53 expression, and apoptosis in CP-treated rats. These findings suggest that pravastatin has a partial protective effect against CP nephrotoxicity via antioxidant activity as well as attenuation of the p53 response, and lipid-lowering effects.

Objectives: To compare the pro-inflammatory cytokine profiles and the cytokine kinetics in patients with secondary macrophage activation syndrome (MAS) due to systemic-onset juvenile idiopathic arthritis (s-JIA) and in both active and inactive disease states of s-JIA (but no MAS), with those demonstrated in EBV-induced haemophagocytic lymphohistiocytosis (HLH) and Kawasaki disease (KD), and to investigate the significance of IL-18 in the pathogenesis of s-JIA. Methods: Five patients with MAS complicating s-JIA (MAS/s-JIA), 10 with HLH due to EBV infection (EBV-HLH), 22 with KD and 28 healthy controls were analysed. Cytokine concentrations (IL-18, IL-6, neopterin and TNF-α receptor Types I and II) were quantified in serum by ELISA. Results: were compared with clinical features of MAS/s-JIA, including ferritin concentrations. Results. Serum IL-18 concentrations in MAS/s-JIA patients were significantly higher than those in EBV-HLH or KD patients (P

X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain (γc) and usually characterized by the absence of T and natural killer (NK) cells. Here, we report an atypical case of XSCID presenting with autologous T and NK cells and Omenn syndrome-like manifestations. The patient carried a splice-site mutation (IVS1+5G>A) that caused most of the mRNA to be incorrectly spliced but produced normally spliced transcript in lesser amount, leading to residual γc expression and development of T and NK cells. The skin biopsy specimen showed massive infiltration of revertant T cells. Those T cells were found to have a second-site mutation and result in complete restoration of correct splicing. These findings suggest that the clinical spectrum of XSCID is quite broad and includes atypical cases mimicking Omenn syndrome, and highlight the importance of revertant mosaicism as a possible cause for variable phenotypic expression.

Hemophagocytic lymphohistiocytosis (HLH) is a severe and often fatal condition characterized by uncontrolled activation of T cells and macrophages. In Epstein-Barr virus (EBV)-associated HLH (EBV-HLH), the pathogenic roles of ectopic EBV infection in the T-cell population and of clonal proliferation of EBV-infected T cells has been described. However, the immunophenotype of EBV-infected T cells has not been fully characterized. Here we describe a case of EBV-HLH presenting with a massive clonal proliferation of CD8(+) T cells with TCR VB14. Analysis of in situ hybridization for EBV-encoded small RNA1 showed that only CD8(+) T cells harbored EBV in this patient. The EBV-infected TCR VB14(+) CD8(+) T cells exhibited unique immunophenotypic features including lacked CD5 expression and a markedly bright expression of HLA-DR. After initiation of treatment with prednisolone, etoposide, and cyclosporin A, the percentage of infected cells declined progressively in parallel with other serum markers such as ferritin. These findings suggest that lacking expression of CD5 on CD8(+) T cells with specific TCR VB may serve as a useful marker of dysregulated T-cell activation and proliferation in EBV-HLH.

We observed a patient with X-linked severe combined immunodeficiency (X-SCID) with Omenn syndrome-like manifestations. X-linked inheritance, absence of CD132 expression and impaired response to interleukin-2 (IL-2) indicated that the case is typical of X-SCID due to gamma(c) defect. However, this case was unusual in that circulating natural killer (NK) cells were increased and nearly half of these NK cells exhibited the CD56(bright) CD16(-) phenotype. A missense mutation was found within exon 5 of the IL2RG gene. The identical mutation was detected within NK, CD4(+) T and B cells. Engraftment of maternally derived NK cells or gene reversion was ruled out. The erythroderma-like skin lesion was characterized by infiltration of the dermis by CD56(bright) NK cells admixed with CD1a(+) dendritic cells (DC). Expression of mRNA for inflammatory cytokines was significantly enhanced within the skin. This may be the first human case to demonstrate that close cell-to-cell contact between DC and NK cells provides an effective alternative pathway for NK cell differentiation/activation in vivo.

Summary Haematopoietic stem cell transplantation (HSCT) is performed for treatment of a broad spectrum of illnesses. Reconstitution of an intact immune system is crucial after transplantation to avoid infectious complications, and above all, the establishment of T cell receptor (TCR) diversity is the most important goal in the procedure. Until recently, little has been known of the mechanism of T cell reconstitution in the very early period after HSCT. In this study, we analysed TCR repertoires sequentially in four patients with severe combined immunodeficiency (SCID) before and after HSCT. In all patients, the TCR repertoires were extremely abnormal before HSCT, whereas after transplantation there was progressive improvement in TCR diversity, based on analysis of the TCR Vβ repertoire and CDR3 size distributions. Somewhat unexpectedly, there was a significant but transient expansion of TCR diversity 1 month after transplantation in all cases. Clonotypic analysis of TCRs performed in one case showed that many T cell clones shared identical CDR3 sequences at 1 month and that the shared fraction decreased progressively. These results indicate that early expansion of TCR diversity may reflect transient expansion of pre-existing mature T cells from the donor blood, independent of de novo T cell maturation through the thymus.

Background In 2011, there was an outbreak of Shiga toxin-producing Escherichia coli (STEC) infections in Japan. Approximately 62 % of patients with hemolytic-uremic syndrome also showed symptoms of encephalopathy. To determine the mechanisms of onset for encephalopathy during STEC infections, we conducted an in vitro study with glial cell lines and primary glial cells. Results Shiga toxin 2 (Stx-2) in combination with lipopolysaccharide (LPS), or LPS alone activates nuclear factor-κB (NF-κB) signaling in glial cells. Similarly, Stx-2 in combination with LPS, or LPS alone increases expression levels of aquaporin 4 (AQP4) in glial cells. It is possible that overexpression of AQP4 results in a rapid and increased influx of osmotic water across the plasma membrane into cells, thereby inducing cell swelling and cerebral edema. Conclusions We have showed that a combination of Stx-2 and LPS induced apoptosis of glial cells recently. Glial cells are indispensable for cerebral homeostasis; therefore, their dysfunction and death impairs cerebral homeostasis and results in encephalopathy. We postulate that the onset of encephalopathy in STEC infections occurs when Stx-2 attacks vascular endothelial cells of the blood–brain barrier, inducing their death. Stx-2 and LPS then attack the exposed glial cells that are no longer in contact with the endothelial cells. AQP4 is overexpressed in glial cells, resulting in their swelling and adversely affecting cerebral homeostasis. Once cerebral homeostasis is affected in such a way, encephalopathy is the likely result in STEC patients. Electronic supplementary material The online version of this article (doi:10.1186/s12929-015-0184-5) contains supplementary material, which is available to authorized users.

ObjectivesThe genotype-phenotype correlation of MEFV remains unclear for the familial Mediterranean fever (FMF) patients, especially without canonical MEFV mutations in exon 10. The risk of FMF appeared to be under the influence of other factors in this case. The contribution of HLA polymorphisms to the risk of FMF was examined as strong candidates of modifier genes.MethodsGenotypes of HLA-B and -DRB1 loci were determined for 258 mutually unrelated Japanese FMF patients, who satisfied modified Tel-Hashomer criteria, and 299 healthy controls. The effects of carrier status were evaluated for the risk of FMF by odds ratio (OR). The HLA effects were also assessed for clinical forms of FMF, subsets of FMF with certain MEFV genotypes and responsiveness to colchicine treatment.ResultsThe carriers of B*39:01 were increased in the patients (OR = 3.25, p = 0.0012), whereas those of DRB1*15:02 were decreased (OR = 0.45, p = 0.00050), satisfying Bonferroni’s correction for multiple statistical tests (n = 28, pConclusionsThe differential effects of HLA class I and class II polymorphisms were identified for Japanese FMF even in those with high-penetrance MEFV mutations., Article, PLOS ONE.10(5):e0125938(2015)

X-linked thrombocytopenia (XLT) is caused by mutations in the WAS gene and characterized by thrombocytopenia with minimal or no immunodeficiency. Patients with XLT usually exhibit persistent thrombocytopenia, and intermittent thrombocytopenia has been described only in two families. Here, we report a patient with intermittent XLT carrying a novel missense mutation (Ala56Thr). He showed residual expression of Wiskott–Aldrich syndrome protein in the lymphocytes and platelets. There appeared to be an association between normal platelet numbers and a post infectious state. Our findings further support the importance of analysis of Wiskott–Aldrich syndrome protein in male patients who exhibit fluctuating courses of thrombocytopenia. Pediatr Blood Cancer 2014;61:746–748. © 2013 Wiley Periodicals, Inc.

Monocytes play key roles both in innate and adaptive antigen-specific immunity and they constitute critical components of the immune responses. Although most of the monocyte-derived cytokines exhibit proinflammatory functions in vivo, heme oxygenase-1 (HO-1), an inducible heme-degrading enzyme, exerts potent anti-inflammatory effect through production of carbon monoxide and bilirubin. We compared HO-1 production by monocytes in vivo in various acute inflammatory illnesses and in normal controls. Freshly isolated monocytes produced little HO-1 as detected by immunohistochemistry, but it was rapidly induced in vitro upon stimulation. HO-1 production by monocytes was selective because it was not induced in other leukocyte populations, including granulocytes and lymphocytes. Monocytes from acute inflammatory illnesses, such as Kawasaki disease and acute infectious diseases, viral or bacterial, produced significant levels of HO-1, as detected by flow cytometry, immunohistochemistry, and reverse transcription polymerase chain reaction. Quantitative analysis of HO-1 mRNA expression by real-time polymerase chain reaction revealed that monocytes from controls exhibited low, but significant levels of HO-1 mRNA, indicating that circulating monocytes produce HO-1 constantly, in response to basal level of oxidative stress encountered daily. Significantly elevated HO-1 mRNA levels seen in acute inflammatory illnesses suggest that monocyte HO-1 production serve as potent anti-inflammatory agent to control excessive cell or tissue injury in the presence of oxidative stress and cytokinemia.

Whereas most peripheral CD8+ αβ T cells highly express CD8αβ heterodimer in healthy individuals, there is an increase of CD8α+βlow or CD8αα αβ T cells in HIV infection or Wiskott-Aldrich syndrome and after bone marrow transplantation. The significance of these uncommon cell populations is not well understood. There has been some question as to whether these subsets and CD8α+βhigh cells belong to different ontogenic lineages or whether a fraction of CD8α+βhigh cells have down-regulated CD8β chain. Here we assessed clonality of CD8αα and CD8α+βlow αβ T cells as well as their phenotypic and functional characteristics. Deduced from surface antigens, cytotoxic granule constituents, and cytokine production, CD8α+βlow cells are exclusively composed of effector memory cells. CD8αα cells comprise effector memory cells and terminally differentiated CD45RO−CCR7−memory cells. T-cell receptor (TCR) Vβ complementarity-determining region 3 (CDR3) spectratyping analysis and subsequent sequencing of CDR3 cDNA clones revealed polyclonality of CD8α+βhigh cells and oligoclonality of CD8α+βlow and CD8αα cells. Importantly, some expanded clones within CD8αα cells were also identified within CD8α+βhigh and CD8α+βlow subpopulations. Furthermore, signal-joint TCR rearrangement excision circles concentration was reduced with the loss of CD8β expression. These results indicated that some specific CD8α+βhigh αβ T cells expand clonally, differentiate, and simultaneously down-regulate CD8β chain possibly by an antigen-driven mechanism. Provided that antigenic stimulation directly influences the emergence of CD8αα αβ T cells, these cells, which have been previously regarded as of extrathymic origin, may present new insights into the mechanisms of autoimmune diseases and immunodeficiencies, and also serve as a useful biomarker to evaluate the disease activities.

The blood–brain barrier (BBB) selectively controls the homeostasis of the central nervous system (CNS) environment using specific structural and biochemical features of the endothelial cells, pericytes, and glial limitans. Glial cells, which represent the cellular components of the mature BBB, are the most numerous cells in the brain and are indispensable for neuronal functioning. We investigated the effects of Shiga toxin on glial cells in vitro. Shiga toxin failed to inhibit cell proliferation but attenuated expression of heat shock protein 70, which is one of the chaperone proteins, in cultured and primary glial cells. Furthermore, the combination of Shiga toxin and a heat shock procedure induced cell apoptosis and decreased cell proliferation in both cells. Thus, we speculate that glial cell death in response to the combination of Shiga toxin and heat shock might weaken the BBB and induce central nervous system complications.