Kazushi Tamura

Prediction of Intravenous Immunoglobulin Unresponsiveness in Patients With Kawasaki Disease

Background— In the present study, we developed models to predict unresponsiveness to intravenous immunoglobulin (IVIG) in Kawasaki disease (KD). Methods and Results— We reviewed clinical records of 546 consecutive KD patients (development dataset) and 204 subsequent KD patients (validation dataset). All received IVIG for treatment of KD. IVIG nonresponders were defined by fever persisting beyond 24 hours or recrudescent fever associated with KD symptoms after an afebrile period. A 7-variable logistic model was constructed, including day of illness at initial treatment, age in months, percentage of white blood cells representing neutrophils, platelet count, and serum aspartate aminotransferase, sodium, and C-reactive protein, which generated an area under the receiver-operating-characteristics curve of 0.84 and 0.90 for the development and validation datasets, respectively. Using both datasets, the 7 variables were used to generate a simple scoring model that gave an area under the receiver-operating-characteristics curve of 0.85. For a cutoff of 0.15 or more in the logistic regression model and 4 points or more in the simple scoring model, sensitivity and specificity were 86% and 67% in the logistic model and 86% and 68% in the simple scoring model. The kappa statistic is 0.67, indicating good agreement between the logistic and simple scoring models. Conclusions— Our predictive models showed high sensitivity and specificity in identifying IVIG nonresponders among KD patients.

Novel dinucleotide repeat polymorphism in the first exon of the STAT-6 gene is associated with allergic diseases.

Background T helper‐type 2 cytokines, such as interleukin‐4 (IL‐4) and IL‐13, may play a central role in allergic diseases. The protein known as ‘signal transducers and activators of transcription 6’ (STAT‐6) is a key transcription factor involved in both IL‐4‐ and IL‐13‐mediated biological responses.

Linkage and Association Studies of STAT6 Gene Polymorphisms and Allergic Diseases

Background: Signal transducer and activator of transcription 6 (STAT6) is a key transcription factor involved in both interleukin-4 (IL-4) and IL-13-mediated biological responses, such as allergies. Recently, we reported that the polymorphism of the STAT6 gene exon 1 was associated with allergic diseases, while another group studied the G2964A variant of the STAT6 gene’s association with atopic asthma. We undertook an association study between these variants of the STAT6 gene and allergic diseases, including atopic dermatitis, bronchial asthma, and food-related anaphylaxis in a Japanese population. Methods: STAT6 gene polymorphisms were genotyped by polymerase chain reaction (PCR) fragment length polymorphism analysis, and PCR-SSCP analysis in 106 allergic and 66 control subjects. Results: The 2964A variant was in significant linkage disequilibrium with the dinucleotide repeat polymorphism, the 13-GT repeat allele of STAT6 exon 1 (p < 0.0000000003). There was no association between the STAT6 2964A variant and allergic subjects in a Japanese population (p = 0.2724). The genotype of 13/15-GT repeat allele heterozygosity was significantly associated with allergic subjects (p = 0.0006), as previously reported. In one major genotype of the STAT6 exon 1 (15 GT repeat homozygosity), wild-type 2964G allele homozygosity was significantly associated with allergic subjects (p = 0.0382). Conclusions: Our findings indicate that in combination the dinucleotide repeat polymorphism of the STAT6 exon 1 gene and the 2964A variant may be useful markers for predicting allergic diseases in a Japanese population.

Increased serum monocyte chemoattractant protein-1, macrophage inflammatory protein-1β, and interleukin-8 concentrations in hemophagocytic lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is characterized by hypercytokinemia caused by macrophage and T cell activation. We analyzed the serum concentrations of monocyte chemoattractant protein (MCP)‐1, macrophage inflammatory protein (MIP)‐1β, and interleukin (IL)‐8 to investigate the roles of these chemokines in the pathophysiology of HLH.

Hemophagocytic lymphohistiocytosis associated with uncontrolled inflammatory cytokinemia and chemokinemia was caused by systemic anaplastic large cell lymphoma: A case report and review of the literature

To the Editor: Hemophagocytic syndrome or hemophagocytic lymphohistiocytosis (HLH) induced by lymphoma has been reported in Asian adult patients and was called lymphoma-associated hemophagocytic syndrome. However, lymphoma-associated hemophagocytic syndrome was rarely reported in adults and children of Western countries. Especially, HLH associated with anaplastic large-cell lymphoma (ALCL) was well known, but uncertain in pathophysiology and rarely reported in children. On the other hand, anaplastic large kinase (ALK)-positive ALCL was predominant in children and young adults. ALK-positive ALCL showed a better prognosis than ALK-negative ALCL. We present here a case of a 3-year-old boy with HLH who suffered from systemic ALCL. No family history of immunodeficiency was observed. He showed prolonged fever, pleural effusion, liver dysfunction, and pancytopenia. The extranodular diseases including brain, lung, skin, liver, and soft tissue were observed by magnetic resonance imaging (Fig. 1A). Bone marrow picture showed the erythrophagocytosis. The biopsy of lymph node showed the aggregation of large cells positively stained by antibodies of CD30 or ALK. The karyotypic analysis showed t(2;5)(p23;q35) by G-banding method and the transcript of NPM-ALK was confirmed by real-time–polymerase chain reaction (RT-PCR) and direct sequencing (data not shown). Bone marrow sample also showed the transcript of NPM-ALK by real-time–PCR from the first diagnosis. The infections of Epstein-Barr virus or cytomegalovirus were denied by PCR analysis. The disease was severe and fatal compared with ALCL without HLH. Systemic chemotherapy for ALCL with immunosuppressive therapy using steroid and cyclosporine were repeatedly FIGURE 1. A, A gadolinium-enhanced MRI imaging of the brain. High-intensity area was estimated to be the invaded lesion by tumor cells. B, A picture of May-Giemsa staining of cytospine of cerebrospinal fluid. Tumor cells were observed. MRI indicates magnetic resonance imaging.

Tandem Repeats of Lactoferrin‐Derived Anti‐Hepatitis C Virus Peptide Enhance Antiviral Activity in Cultured Human Hepatocytes

Previously, we found that bovine and human lactoferrin (LF) specifically inhibited hepatitis C virus (HCV) infection in cultured non‐neoplastic human hepatocyte‐derived PH5CH8 cells, and we identified 33 amino acid residues (termed C‐s3–33; amino acid 600–632) from human LF that were primarily responsible for the binding activity to the HCV E2 envelope protein and for the inhibiting activity against HCV infection. Since the anti‐HCV activity of C‐s3–33 was weaker than that of human LF, we speculated that an increase of E2 protein‐binding activity might contribute to the enhancement of anti‐HCV activity. To test this possibility, we made two repeats [(C‐s3–33)2] and three repeats [(C‐s3–33)3] of C‐s3–33 and characterized them. Far‐Western blot analysis revealed that the E2 protein‐binding activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33, and that the binding activity of (C‐s3–33)3 was stronger than that of (C‐s3–33)2. Using an HCV infection system in PH5CH8 cells, we demonstrated that the anti‐HCV activities of (C‐s3–33)2 and (C‐s3–33)3 became stronger than that of the C‐s3–33. Furthermore, using a recently developed infection system with a VSV pseudotype harboring the green fluorescent protein gene and the native E1 and E2 genes, we demonstrated that the antiviral activities of (C‐s3–33)2 and (C‐s3–33)3 were stronger than that of C‐s3–33. These results suggest that tandem repeats of LF‐derived anti‐HCV peptide are useful as anti‐HCV reagents.

Formation of Vesicular Stomatitis Virus Pseudotypes Bearing Surface Proteins of Hepatitis B Virus

ABSTRACT It has been difficult to propagate and titrate hepatitis B virus (HBV) in tissue culture. We examined whether vesicular stomatitis virus (VSV) pseudotypes bearing HBV surface (HBs) proteins infectious for human cell lines could be prepared. For this, expression plasmids for three surface proteins, L, M, and S, of HBV were made. 293T cells were then transfected with these plasmids either individually or in different combinations. 293T cells expressing HBs proteins were infected with VSVΔG*-G, a recombinant VSV expressing green fluorescent protein (GFP), to make VSV pseudotypes. Culture supernatants together with cells were harvested and sonicated for a short time. The infectivities of freshly harvested supernatants were determined by quantifying the number of cells expressing GFP after neutralization with anti-VSV serum and mouse monoclonal antibodies (MAbs) against HBs protein. Among 14 cell lines tested for susceptibility to HBV pseudotype samples, HepG2, JHH-7, and 293T cells were judged to be the most susceptible. Namely, the infectious units (IU) of the culture supernatant samples neutralized with anti-VSV in the absence and presence of anti-HBs S MAbs and titrated on HepG2 cells ranged from 1,000 to 4,000 IU/ml and 200 to 400 IU/ml, respectively, suggesting the presence of VSVΔG*(HBV) pseudotypes. This infectivity was inhibited by treatment with lactoferrin or dextran sulfate. Pretreatment of the cells with trypsin or tunicamycin inhibited plating of the pseudotype samples. The HBV pseudotypes can be used to analyze early steps of HBV infection, including the entry mechanism of HBV.

Successful induction of immune tolerance by continuous infusion of recombinant factor VIII in a haemophilia A patient with high-inhibitor titres

Summary.  The successful and persistent abolition of the inhibitor is of significant clinical benefit, as it allows for the restoration of the usual treatment with clotting factor concentrate. We describe a successful induction of immune tolerance by continuous infusion of recombinant factor VIII (rFVIII) in a 5‐year‐old boy with severe haemophilia A and high‐responding inhibitor. He had previously been subjected to immune tolerance induction (ITI) with rFVIII at 100 units (U) kg−1 three times weekly. One year after the beginning of therapy tolerance was not achieved and a high titer of inhibitor was detected (15 Bethesda Units). The patient had a sudden onset of severe neck pain. The diagnosis of spinal epidural haematoma was revealed by magnetic resonance imaging, and emergency laminectomy with evacuation of the haematoma was required. The patient received sequential therapy for surgery first as bolus rFVIII injection of 500 U kg−1 in order to overwhelm the inhibitor and then as continuous infusion at 6 to 12 U kg−1 hour−1 to avoid bleeding episodes in the postoperative period. After the 3 weeks of continuous infusion, the inhibitor became undetectable. Thereafter, prophylactic treatment with rFVIII was started three times weekly, and the inhibitor has remained undetectable for 6 months. The, present case suggests that continuous infusion of rFVIII may be an effective therapy to induce immune tolerance.

NOTCH1 mutation in a female with myeloid/NK cell precursor acute leukemia

A 6‐year‐old Japanese female was diagnosed as having myeloid/NK cell precursor acute leukemia (MNKL) using immunocytochemical analysis. The patient was treated by cord blood transplantation from an HLA 1‐locus mismatched unrelated donor after chemotherapy comprising cytosine arabinoside, idarubicin, etoposide, and L‐asparaginase. We detected a nonsense mutation, C7412A, resulting in S2471X, where X is a terminal codon, in the PEST domain of NOTCH1 in this patient. The presence of the NOTCH1 activating mutation in MNKL might suggest a possible role in the leukemogenesis of MNKL. Pediatr Blood Cancer. 2010;55:1406–1409.