Gen Kano

Mechanisms of allergy and clinical immunologyMechanism of Siglec-8–mediated cell death in IL-5–activated eosinophils: Role for reactive oxygen species–enhanced MEK/ERK activation

BACKGROUND Sialic acid-binding immunoglobulin-like lectin (Siglec)-8 is expressed on human eosinophils, where its ligation induces cell death. Paradoxically, Siglec-8-mediated cell death is markedly enhanced by the presence of the activation and survival factor IL-5 and becomes independent of caspase activity. OBJECTIVE In this report we investigate the mechanism of Siglec-8-mediated cell death in activated eosinophils. METHODS Human peripheral blood eosinophils were treated with agonistic anti-Siglec-8 antibody and IL-5, and cell death was determined by using flow cytometry and morphology. Phosphorylation of mitogen-activated protein kinase (MAPK) was determined by using phosphoLuminex, flow cytometry, and Western blotting. Reactive oxygen species (ROS) accumulation was determined by using dihydrorhodamine fluorescence. RESULTS Costimulation with anti-Siglec-8 and IL-5 significantly increased the rate and proportion of cell death by means of necrosis accompanied by granule release compared with that seen after stimulation with anti-Siglec-8 alone, in which apoptosis predominated. Together with the caspase-independent mode of cell death in costimulated cells, these findings suggest the activation of a specific and distinct biochemical pathway of cell death during anti-Siglec-8/IL-5 costimulation. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and MAPK-ERK kinase (MEK) 1 was significantly enhanced and sustained in costimulated cells compared with that seen in cells stimulated with IL-5 alone; anti-Siglec-8 alone did not cause ERK1/2 phosphorylation. MEK1 inhibitors blocked anti-Siglec-8/IL-5-induced cell death. ROS accumulation was induced by Siglec-8 ligation in a MEK-independent manner. In contrast, an ROS inhibitor prevented the anti-Siglec-8/IL-5-induced enhancement of ERK phosphorylation and cell death. Exogenous ROS mimicked stimulation by anti-Siglec-8 and was sufficient to induce enhanced cell death in IL-5-treated cells. Collectively, these data suggest that the enhancement of ERK phosphorylation is downstream of ROS generation. CONCLUSIONS In activated eosinophils ligation of Siglec-8 leads to ROS-dependent enhancement of IL-5-induced ERK phosphorylation, which results in a novel mode of biochemically regulated eosinophil cell death.

Potential use of procalcitonin concentrations as a diagnostic marker of the PFAPA syndrome

PFAPA syndrome is a clinical entity of unknown etiology characterized by periodic episodes of high fever accompanied by aphthous stomatitis, pharyngitis/tonsillitis, and cervical adenitis [3, 5]. Since specific laboratory abnormalities for the PFAPA syndrome are inexistent, it is usually diagnosed clinically after excluding other probable causes of the fever, such as infection [1]. In PFAPA patients, discriminating between a fever attack due to bacterial infection and a fever attack due to noninfectious inflammation constitutes a major difficulty. Because procalcitonin, a propeptide of calcitonin, is reported to be a sensitive marker of systemic bacterial infection [2, 4], we followed peripheral leukocyte counts, CRP values and procalcitonin concentrations during the fever attacks associated with PFAPA syndrome in the hope of defining reliable criteria for its diagnosis. We determined serum procalcitonin concentrations in six PFAPA syndrome patients (two males and four females) with a median age of 7.5 (range 3–10) years and in 32 controls (bacterial, n=10 and non-bacterial, n=22). Sampling was performed on the third to fifth day of fever. In the PFAPA syndrome patients, febrile episodes started at the median age of 2.5 (range 1–7) years with each episode lasting 5–7 days and recurring every 3–4 weeks. The ethical committees of our institutes approved the study protocol and the guardians of all the patients gave their informed consent. Serum procalcitonin concentrations were measured by using the fully automated enzyme immunoluminescent assay (Wako Pure Chemical Industries, Ltd.), which employs katacalcin monoclonal antibody and calcitonin polyclonal antibody labeled with peroxidase for SphereLight 180 (Olympus Corporation). The detection limit was 0.1 ng/ml and the normal reference was set at <0.5 ng/ml. In PFAPA patients, the correlations between procalcitonin, CRP values and leukocyte counts were examined over 13 febrile episodes. Serum procalcitonin values ranged from 0.20 to 11.36 (median value 1.05) ng/ml in positive control subjects (Table 1), while all the negative controls had undetectable levels. During febrile episodes in PFAPA patients, which were confirmed not to be due to adenoviral or group A streptococcal infections, leukocyte counts and serum concentrations of CRP were invariably and significantly Eur J Pediatr (2007) 166:621–622 DOI 10.1007/s00431-006-0281-2

Ikaros dominant negative isoform (Ik6) induces IL-3-independent survival of murine pro-B lymphocytes by activating JAK-STAT and up-regulating Bcl-xl levels

Ikaros is an essential regulator of lymphocyte differentiation. Mice transgenic for the Ikaros dominant negative (DN) mutation rapidly develop lymphoid malignancies. Various human leukemias have also been reported to express Ikaros DN isoforms, but its role in leukemogenesis is yet to be defined. We demonstrate that overexpressed Ikaros DN (Ik6) prolonged the survival of two different murine pro-B cell lines in cytokine deprived condition, and this was associated with increased expression of Bcl-xl. A survey of the upstream controller(s) of Bcl-xl expression revealed Ik6 overexpression enhanced the phosphorylation of JAK2 and STAT5. Interestingly, the Ik6 expressing cell lines showed reduced expression of B-cell differentiation surface marker CD45R (B220), which is also known as a JAK2 inhibitor. Although further evaluation with human clinical materials are required, these results propose a putative role of Ik6 in the development of B-lineage acute lymphoblastic leukemia, by activating the JAK2-STAT5 pathway and thus stimulating the production of Bcl-xl.

A novel missense mutation (1060G → C) in the phosphoglycerate kinase gene in a Japanese boy with chronic haemolytic anaemia, developmental delay and rhabdomyolysis

Summary. We report the case of a 3‐year‐old Japanese boy with phosphoglycerate kinase 1 (PGK1) deficiency (Online Mendelian Inheritance in Man entry 311800). The patient had anaemia and jaundice at birth, necessitating exchange transfusions for 2 d. After one red blood cell transfusion at age 2 months, his Hb level was 8–9 g/dl, his reticulocyte counts were 300–500 × 109/l, and his total bilirubin level was 25·65–42·75 µmol/l. The patient suffered two episodes of respiratory infection‐associated haemolytic crisis and rhabdomyolysis during early infancy. At age 3·0 years, his developmental milestones (developmental quotients measured using the Tsumori–Inage methods) score was 49% (normal 74–131%), and his height was below average by −2·0 standard deviations. The diagnosis of PGK1 deficiency was made based on his remarkably low (< 10% of normal) erythrocyte PGK enzyme activity level and the identification of a novel missense (1060G→C) PGK1 gene mutation. This mutation results in the Ala‐353Pro amino acid substitution, which has been designated PGK Kyoto. The patient developed the full clinical symptoms of PGK1 deficiency including haemolytic anaemia, myopathy, central nervous system disorder and growth retardation, which is unusual.

Cerebral thrombotic complications in adolescent leukemia/lymphoma patients treated with L-asparaginase-containing chemotherapy

We described the cerebral thrombotic complications developed in 2 adolescent patients treated with L-asparaginase-containing regimens. For determining risk factors, we retrospectively analysed hemostatic markers in 19 pediatric patients with leukemia or lymphoma who were treated with either 1 of the 2 L-asparaginase-containing regimens; 11 were treated with VLP1 and the remaining 8 were treated with the VLAD protocol. The data indicated that low coagulation factors in association with increased plasma D-dimer levels during or post-L-asparaginase administration combined with fresh frozen plasma infusion might have activated coagulation processes in these patients. Careful management is required to prevent such episodes in patients with markedly decreased coagulation factors and increased D-dimer levels following L-asparaginase administration.

Chlamydia pneumoniae infection-related hemophagocytic lymphohistiocytosis and acute encephalitis and poliomyelitis-like flaccid paralysis.

A 3‐year‐old male presented with Chlamydia pneumoniae infection‐related hemophagocytic lymphohistiocytosis (HLH). The patient developed an episode of HLH with severe skin eruption following C. pneumoniae pneumonia. Symptoms responded to steroid/cyclosporine A therapy, but the patient slowly lost consciousness and developed systemic flaccid paralysis. He was diagnosed with encephalitis/myelitis by brain and spinal MRI. Neurological symptoms and signs gradually resolved. We thought that the immune response to C. pneumoniae infection triggered the development of HLH, associated with unusual neurological complications. This report describes a novel case of C. pneumoniae‐associated HLH and with poliomyelitis like flaccid paralysis. Pediatr Blood Cancer 2011;56:853–855.

Complicated pathophysiology behind rituximab-induced persistent hypogammaglobulinemia.

The anti-CD20 monoclonal antibody rituximab has made a avorable impact on the prognosis of B-cell non-Hodgkin lymhoma (B-NHL), as a first-line therapy for adult cases and a econd-line therapy for pediatric cases. Additionally, rituximab has mproved the outcome of autoimmune diseases such as systemic upus erythematosus (SLE) and idiopathic thrombocytopenic purura. Complications of retuximab therapy are usually well tolerated nd manageable. Although hypogammaglobulinemia occurring fter retuximab therapy is an axiom, this hypogammaglobulinemia s usually transient and thus often unrecognized in both maligancy and autoimmune settings, with the increase of infection risk ppearing to be marginal [1]. However, it is also known that a inority of patients develop severe hypogammaglobulinemia that ersists for several years even after the completion of rituximab herapy (rituximab-induced persistent hypogammaglobulinemia RPHg]). RPHg is relatively common in malignancy patients who eceived rituximab as part of their regimen for hematopoietic stem ell transplantation (SCT) or as maintenance after SCT [2] but is ess common in patients who received rituximab for treatment of utoimmune diseases [3]. Curiously, hypogammaglobulinemia can rise even after the recovery of peripheral CD19+ B-cell numbers. imilarities in the B-cell phenotype of RPHg to that of common ariable immune deficiency (CVID), including the lack of expresion of IgG2, IgG3, and IgG4 and a profound decrease in CD27+IgD+ witched memory B cells, have been reported [4], raising the posibility that RPHg and CVID pathophysiology may partially overlap. nlike in patients with CVID, however, severe and/or recurrent nfections seem to be uncommon in patients with RPHg [3]; hether patients with RPHg have other clinical manifestations of VID, such as susceptibility to autoimmune diseases or neoplasms, s unknown. Here we report on an adolescent patient who developed RPHg ollowing treatment for B-NHL and suffered unusual complications ith sustained massive lymphadenopathy and recurrent episode f a severe form of histiocytic necrotizing lymphadenitis (Kikuchiujimoto disease [KFD]). Comprehensive analysis of the patient’s -cell phenotype was performed to evaluate possible linkages to VID pathophysiology.

Hb bristol-alesha presenting thalassemia-type hyperunstable hemoglobinopathy

Hemoglobin (Hb) Bristol-Alesha is caused by a GTG→ ATG mutation at codon 67 in the Hb ß chain, resulting in abnormal ß globin chains with mutated molecules from normal ß67 valine (Val) to ß67 methionine (Met) or ß67 aspartate (Asp). We describe a Japanese child with this rare hemoglobinopathy and a very unstable Hb molecule phenotype. The diagnosis of hemolytic anemia was made when the patient was 6 months of age. Development of marked splenomegaly necessitated red blood cell transfusions twice a month. After splenectomy when the patient was 4 years of age, laboratory findings of hemolytic anemia became more prominent. Specific abnormal Hb molecules initially were not detected, and the α/ß globin synthesis ratio was abnormal at 2.22. After splenectomy, we identified the presence of abnormal ß-globin chains with a ß67Val:ß67Met:ß67 Asp molecule ratio of 74:11:15. We speculate that the high fraction of the ß67Met molecule in this patient, compared with that in previously reported cases, caused extreme Hb instability, which resulted in thalassemic hyperunstable hemoglobinopathy and very severe clinical findings.

Systemic juvenile idiopathic arthritis mimics multicentric Castleman’s disease

An 11-year-old girl presented with fever and a large cervical lymphadenopathy. Indicators of inflammation were remarkable: she had extremely high levels of serum interleukin-6 (IL-6) (398 pg/ml) in addition to hypergammaglobulinemia and hypoalbuminemia. Computed tomography (CT) revealed swollen systemic lymph nodes. Two weeks after the onset of symptoms she developed polyarthralgia. Biopsy of the cervical lymph node revealed massive infiltration of plasma cells without hyaline vascular changes. She was diagnosed with systemic juvenile idiopathic arthritis (JIA). The patient’s symptoms and hypercytokinemia disappeared soon after corticosteroid treatment was started. This case demonstrates that overproduction of IL-6 is common to systemic JIA and multicentric Castleman’s disease.

Pulmonary veno-occlusive disease in an 11-year-old girl: diagnostic pitfalls.

Pulmonary veno‐occlusive disease (PVOD) is a rare chronic lung disease that is difficult to diagnose due to non‐specific clinical findings. Little is known about the pathogenesis of PVOD. Reported herein is the case of an 11‐year‐old girl who initially presented with ‘bat‐wing’ shadows on chest radiography. This finding, coupled with prominent hemosiderosis in bronchoalveolar lavage fluid, initially led to a misdiagnosis of idiopathic pulmonary hemosiderosis. Oral prednisolone dramatically improved signs and symptoms initially, but her condition then gradually deteriorated during maintenance therapy with corticosteroids and other immunosuppressants. PVOD was suspected but not confirmed owing to a lack of hallmark radiographic findings and contraindications for lung biopsy. Three years later, while arranging for lung transplantation, the patient experienced sudden onset of fatal massive pulmonary edema. PVOD was confirmed at autopsy. This case provides insights regarding an unfamiliar presentation of PVOD and may help physicians to avoid diagnostic pitfalls.