Clinical and molecular significance of flow cytometric analysis for reactive oxygen species production and residual p67phox expression in p67phox-deficient chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency disease caused by molecular defects in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. p67phox-CGD is an autosomal recessive CGD, which is caused by a defect in the cytosolic components of NADPH oxidase, p67phox, encoded by NCF2. We previously established a flow cytometric analysis for p67phox expression, which allows accurate assessment of residual protein expression in p67phox-CGD. We evaluated the correlation between oxidase function and p67phox expression, and assessed the relevancy to genotypes and clinical phenotypes in 11 patients with p67phox-CGD. Reactive oxygen species (ROS) production by granulocytes was evaluated using dihydrorhodamine-1,2,3 (DHR) assays. p67phox expression was evaluated in the monocyte population. DHR activity and p67phox expression were significantly correlated (r = 0.718, p < 0.0162). Additionally, DHR activity and p67phox expression were significantly higher in patients carrying one missense variant in combination with one nonsense or frameshift variant in the NCF2 gene than in patients with only null variants. The available clinical parameters of our patients (i.e., age at disease onset, number of infectious episodes, and each infection complication) were not linked with DHR activity or p67phox expression levels. In summary, our flow cytometric analysis revealed a significant correlation between residual ROS production and p67phox expression. More deleterious NCF2 genotypes were associated with lower levels of DHR activity and p67phox expression. DHR assays and protein expression analysis by using flow cytometry may be relevant strategies for predicting the genotypes of p67phox-CGD.

Acute Pulmonary Edema Due to Arteriovenous Shunt Placement After Lung Transplantation.

Lung transplant recipients often have complications of immunosuppressant-induced nephropathy, which may require renal replacement therapy. We report a case of unilateral lung edema and pulmonary hypertension due to arteriovenous fistula placement in a patient with unilateral chronic lung allograft dysfunction after bilateral living-donor lobar lung transplantation. Lung transplant recipients with limited residual vascular beds, such as lobar graft or severe deviation in lung perfusion, are vulnerable to the acute increase in blood flow due to arteriovenous fistula placement and pulmonary edema can easily develop regardless of the left ventricular function. Hence, careful volume control is required.

Repeated Necrotizing Lymphadenitis with MEFV Gene Mutations.

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.

Role of E148Q in familial Mediterranean fever with an exon 10 mutation in MEFV.

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.

Clinical Significance of Serum Soluble TNF Receptor I/II Ratio for the Differential Diagnosis of Tumor Necrosis Factor Receptor-Associated Periodic Syndrome From Other Autoinflammatory Diseases.

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.

Atypical Familial Mediterranean Fever Presenting with Recurrent Migratory Polyarthritis.

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.

B-cell-specific accumulation of inclusion bodies loaded with HLA class II molecules in patients with mucolipidosis II (I-cell disease).

BACKGROUND: I-cell disease is characterized by the presence of vacuole-like inclusions in lymphocytes. However, the nature and clinical significance of these inclusions have seldom been characterized. In this study, the authors tried to elucidate the distribution in different lymphocyte subpopulations, and the histological nature of the inclusions. METHODS: Blood samples from three unrelated patients were analyzed. Lymphocyte subpopulations were separated using monoclonal antibodies conjugated to immunomagnetic beads. Cytochemical studies were performed using FITC-conjugated lectins. The expressions of surface and cytoplasmic class II molecules were analyzed by flow cytometry. RESULTS: Virtually all B cells from the patients contained the inclusions. In contrast, CD4+ T cells, CD8+ T cells, natural killer cells, monocytes, or neutrophils did not contain the inclusions. Both fibroblasts and B cells from I-cell patients were stained intensely by multiple FITC-conjugated lectins with distinct binding profiles. The inclusions of B cells were stained intensely by fluorescence-conjugated antibodies against class II antigens. CONCLUSIONS: Inclusions in I-cell disease reflect the accumulation of HLA class II molecules within B cells. These results suggest a potential role for N-acetylglucosamine-1-phosphotransferase in immune functions. Furthermore, the fact that only B cells contain the inclusions provides a novel diagnostic aid for the diagnosis of I-cell disease.

Characterization of skin blister fluids from children with Epstein-Barr virus-associated lymphoproliferative disease.

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.

Longitudinal analysis of serum interleukin-18 in patients with familial Mediterranean fever carrying MEFV mutations in exon 10.

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±6084pg/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 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.

Microarray analysis of circulating microRNAs in familial Mediterranean fever.

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.

Prolonged neutropenia due to antihuman neutrophil antigen 2 (CD177) antibody after bone marrow transplantation.

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.

Increased CD69 Expression on Peripheral Eosinophils from Patients with Food Protein-Induced Enterocolitis Syndrome.

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.

Tumor necrosis factor-α modifies the effects of Shiga toxin on glial cells.

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.

The critical role of lipopolysaccharide in the upregulation of aquaporin 4 in glial cells treated with Shiga toxin.

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.

Late-Onset Combined Immunodeficiency with a Novel IL2RG Mutation and Probable Revertant Somatic Mosaicism.

Primary immunodeficiency disease (PID) is caused by mutations of more than two hundred immunity-related genes. In addition to the heterogeneity of the diseases, the atypical presentation of each disease caused by hypomorphic mutations or somatic mosaicism makes genetic diagnosis challenging. Next-generation sequencing tests all genes simultaneously and has proven its innovative efficacy in genomics. We describe a male PID patient without any family history of immunodeficiency. This patient suffered from recurrent infections from 1 year of age. Laboratory analysis showed hypogammaglobulinemia. T, B, and NK cells were present, but the T cell proliferative response decreased. Whole-exome sequencing analysis identified an IL2RG p.P58T missense mutation. CD8(+) and CD56(+) cells showed revertant somatic mosaicism to the wild-type allele. A late-onset and atypical presentation of the X-linked severe combined immunodeficiency (X-SCID) phenotype might be associated with revertant somatic mosaicism in T and NK cells. This patient is the seventh reported case of X-SCID with revertant somatic mosaicism. His classical clinical management did not result in a molecular diagnosis because of the atypical presentation. The coverage that is provided by whole-exome sequencing of most PID genes effectively excluded differential diagnoses other than X-SCID. As next-generation sequencing becomes available in clinical practice, it will enhance our knowledge of PID and rescue currently undiagnosed patients.

Basophil activation by mosquito extracts in patients with hypersensitivity to mosquito bites.

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.

A Novel In-Frame Deletion in the Leucine Zipper Domain of C/EBPε Leads to Neutrophil-Specific Granule Deficiency.

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.

Identification of Disease-Promoting HLA Class I and Protective Class II Modifiers in Japanese Patients with Familial Mediterranean Fever.

OBJECTIVES: The 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. METHODS: Genotypes 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. RESULTS: The 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, p<0.00179). The protective effect of DRB1*15:02 was completely disappeared in the co-existence of B*40:01. The HLA effects were generally augmented in the patients without a canonical MEFV variant allele M694I, in accordance with the notion that the lower penetrance of the mutations is owing to the larger contribution of modifier genes in the pathogenesis, with a few exceptions. Further, 42.9% of 14 colchicine-resistant patients and 13.5% of 156 colchicine-responders possessed B*35:01 allele, giving OR of 4.82 (p = 0.0041). CONCLUSIONS: The differential effects of HLA class I and class II polymorphisms were identified for Japanese FMF even in those with high-penetrance MEFV mutations.

Munc13-4 deficiency with CD5 downregulation on activated CD8+ T cells.

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.