Masahiro Migita

Characterization of the human IL-5 receptors on eosinophils

Interleukin 5 (IL-5) receptors on the cell surface of human eosinophils and other hematopoietic cells were characterized using radiolabeled recombinant IL-5. The binding of 35S-labeled murine IL-5 to eosinophils from normal human peripheral blood was rapid and saturable within a 30-min incubation at both 4 and 37 degrees C. The binding of 35S-labeled murine IL-5 to eosinophils was inhibited by an excess of unlabeled murine and human IL-5 or by an anti-murine IL-5 monoclonal antibody (NC17) but not by other human cytokines. Scatchard plot analysis revealed that human eosinophils have a single class of high affinity receptor (Kd 170-330 pM; number of binding sites: 260-380/cell). IL-5 receptors on eosinophils from four patients with eosinophilia displayed similar characteristics. Affinity cross-linking experiments resulted in the identification of human IL-5 receptor on eosinophils with a molecular mass of 55-60 kDa. Among the various cells besides eosinophils and cell lines that we could test, a subline of HL-60 (YY-1 cells) was found to display a significant number of IL-5 receptor. These results suggest that IL-5 may act on limited types of cells in the human system.

Conversion of Normal Ly-1-Positive B-Lineage Cells into Ly-1-Positive Macrophages in Long-Term Bone Marrow Cultures

We obtained eight different cell lines in the long-term bone marrow culture system that showed a germ-line configuration of the joining (J) region segments of the Ig heavy-chain (IgH) genes. Their surface markers were CD45R+, Ly-1+, Lyb-2+, cIgM-, sIgM-, Ia-, Thy-1-, Mac-1-, and IL-2R (Tac)+. Use of very young mice and the presence of IL-5 were important for preferential promotion of the survival of B-lineage lymphocytes bearing the Ly-1 markers. When we treated two of them (J8 and J10) with 5-azacytidine for 24 h followed by co-culture with stromal cells and IL-.5, they became Ly-1+, sIgM+ B cells, and Ly-1+, Mac-1+ macrophagelike cells, respectively. After other early lymphoid lines (J1, J8, and J13) were maintained by co-culture with ST2 and IL-5 for more than a year, they showed a heterogeneous DNA rearrangement profile of the J region segment of the IgH gene, although only J13 rearranged the κ-light chain gene. Northern blot analysis revealed that these cell lines expressed Cμ-mRNA, and λ5-mRNA, consistent with normal pre-B cells. Intriguingly, J1, J8, and J13 expressed c-fms mRNA constitutively. When J13 cells were co-cultured with ST2 and GM-CSF in place of ST2 and IL-5, they acquired Mac-1 expression and retained Ly-1 expression. They were morphologically macrophages, nonspecific-esterase-positive, and showed phagocytosis of latex beads. These results support evidence for a close relationship between the myeloid and Ly-1+ B-cell pathways of differentiation, and indicate that our IL- 5-dependent clones are multipotential intermediates in differentiation from pro-B cells to B cells and macrophages.

Biochemical and functional characterization of soluble form of IL-5 receptor α (sIL-5Rα) Development of ELISA system for detection of sIL-5Rα

Abstract Interleukin-5 (IL-5) mediates pleiotropic functions in various types of cells through its specific receptor (IL-5R) which is composed of two distinct subunits, α and β. In mice, the α subunit (IL-5Rα) specifically binds IL-5 with low affinity. The β subunit (IL-5Rβ) does not bind IL-5 by itself, but constructs the high affinity receptor with IL-5Rα. We have isolated cDNA encoding the soluble form of IL-5Rα (sIL-5Rα). To elucidate the biochemical and functional properties of sIL-5Rα, we developed an expression system for sIL-5Rα cDNA in insect cell line Sf21 using baculovirus expression vector and obtained conditioned medium containing large quantities of mouse sIL-5Rα. Mouse sIL-5Rα was purified from the conditioned medium by using anti-IL-5Rα mAb-coupled beads. Immunoaffinity-purified sIL-5Rα with an approximate molecular mass of 42 kDa inhibited the binding of 125 I-labeled IL-5 to IL-5R. By using purified sIL-5Rα, we prepared rabbit anti-sIL-5Rα antibody and developed a sandwich ELISA for detection of sIL-5Rα. Significant amounts of sIL-5Rα were detected in sera and ascitic fluids of mice bearing tumors (BCL 1 and MOPC104E) that responded to IL-5 for DNA synthesis, but not in sera of normal mice. Interestingly, elevated levels of serum sIL-5Rα were observed in NZB and NZW mice. The sIL-5Rα may, therefore, have an immunoregulatory role in vivo.

Biotin-responsive basal ganglia disease: a case diagnosed by whole exome sequencing.

Using whole exome sequencing, we confirmed a diagnosis of biotin-responsive basal ganglia disease (BBGD) accompanied by possible Kawasaki Disease. BBGD is an autosomal-recessive disease arising from a mutation of the SLC19A3 gene encoding the human thiamine transporter 2 protein, and usually manifests as subacute to acute encephalopathy. In this case, compound heterozygous mutations of SLC19A3, including a de novo mutation in one allele, was the cause of disease. Although a large number of genetic neural diseases have no efficient therapy, there are several treatable genetic diseases, including BBGD. However, to achieve better outcome and accurate diagnosis, therapeutic analysis and examination for disease confirmation should be done simultaneously. We encountered a case of possible Kawasaki disease, which had progressed to BBGD caused by an extremely rare genetic condition. Although the prevalence of BBGD is low, early recognition of this disease is important because effective improvement can be achieved by early biotin and thiamine supplementation.

Acute myeloblastic leukemia (ANLL-M2) with t(8;21)(q22;q22) variant expressing lymphoid but not myeloid surface antigens with a high number of G-CSF receptors

Leukemic cells from an 8-year-old girl with ANLL-M2 expressed precursor B-cell antigen CD19, but none of the myeloid antigens CD11b, CD13, CD14 and CD33. After culture, the cells expressed CD11b and CD13. The cells carried a high number of granulocyte colony-stimulating factor (G-CSF) receptors. In chromosome analysis, metaphase cells were obtained only in the case of culture with G-CSF. The karyotype was a variant of t(8;21)(q22;q22). Southern blot analysis revealed rearrangement of the AMLI gene located on chromosome 21. These observations may suggest that even without myeloid surface antigens and with precursor B-cell antigen, ANLL-M2 with t(8;21)(q22;q22) has apparent myeloid characteristics.

Enzyme-linked immunosorbent assay for apolipoprotein B on dried blood spot derived from newborn infant: its application to neonatal mass screening for hypercholesterolemia

Hypercholesterolemia is a risk factor for atherosclerotic cardiovascular disease, and early diagnosis and treatment should be given attention. We developed a simple and sensitive enzyme-linked immunosorbent assay (ELISA) for apolipoprotein B (apoB) on a dried blood spot (DBS). The specificity of this assay was investigated by constructing curves for other proteins. The cross-reactivity was negligible. The mean intra- and intercoefficients of variation were 5.2 and 7.8%, respectively. We used phosphate buffer with Triton X-100 for extracting apoB on DBS. The elution of apoB was stable up to 30 days after bleeding. The correlation between plasma and DBS apoB was r = 0.94, p less than 0.005, n = 55. Using this method, we screened 2,500 babies between 5 and 7 days of age. The mean +/- SD of DBS apoB was 75 +/- 15 U. Seven hypercholesterolemic neonates were detected. Family studies of these neonates disclosed one definite and two suspected heterozygotes for familial hypercholesterolemia. Four other neonates showed no related familial background. This ELISA method for assaying apoB should prove to be a useful tool for large mass screening for hypercholesterolemia, particularly in the newborn.

Mass screening to identify babies with dyslipoproteinemia by measuring the levels of apoA-I, apoB and the ratio of apoB to apoA-I on dried blood spots

Abstract We screened 18-month-old babies with dyslipoproteinemia by measuring the levels of apolipoprotein (apo) A-I, apoB and the ratio of apoB to apoA-I on a dried blood spot (DBS). We used simple and efficient radial immunodiffusion assays for apoA-I and apoB on DBS. ApoA-I on DBS closely correlated with plasma apoA-I ( r =0.96, n =107). ApoB levels on DBS also correlated well with plasma apoB ( r =0.90, n =107). The elutions of apoA-I and apoB from DBS were stable for up to 7 days and 14 days, at room temperature, respectively. DBS were obtained by ear-prick from 18-month-old babies at public health-care centers. The values were arbitrarily considered abnormal when the apoB and apoB/apoA-I levels were more than + 2.5 SD above the mean, and the apoA-I level was less than - 2.5 SD under the mean. Values were abnormal in I85 babies; of those we reinvestigated 159 babies (86%). Among them, II babies were diagnosed as cases of familial hypercholesterolemia, through the family studies. Others were non-familial type Ha (54 infants), type IIb (3) and type IV (12) hyperlipidemic and hypoalphalipoproteinemic (58) subjects. The remaining 2I babies (13%) had normal plasma lipid and apolipoprotein levels. This screening is useful for detecting dyslipoproteinemic babies.

Elevated expression of proto-oncogenes during interleukin-5-induced growth and differentiation of murine B lineage cells

Intcrleukin 5 (IL‐5), a lymphokine produced by helper T cells, is involved in the regulation of growth and differentiation of B cells and other hematopoietic cells. To elucidate IL‐5‐mediated intracellular mechanisms, we have established IL‐5‐dependent and ‐independent murine early B cell lines, J6 and MJ88‐1, respectively, and examined the effect of IL‐5 on the expression of proto‐oncogenes during proliferation. Two‐ to 3.5‐fold increases in the levels of c‐myb, c‐myc, c‐fos, and c‐fms mRNA were observed in J6 cells, compared with those in MJ88‐1 cells. Further, a role of IL‐5 in the proto‐oncogene expression during differentiation was examined by using thymidine‐treated murine B‐cell chronic leukemia BCL1‐B20 cells with growth arrest. After 4‐day culture, the amount of IgM secreted from BCL1‐B20 cells was augmented 4‐6 fold in the presence of IL‐5. Although expression of c‐myb, c‐fos, and c‐fms mRNA did not change, only c‐myc mRNA expression was elevated within 30 min of stimulation with IL‐5 and reached a maximal level by 1 hr. Addition of phorbol 12‐myristate 13‐acetate (PMA) or IL‐4 to the culture of BCL1‐B20 cells inhibited both the IL‐5‐mediated augmentation of IgM secretion and the elevated expression of c‐myc mRNA. These findings suggest that the IL‐5 signal may be associated with the up‐regulation of c‐myc expression.

Web-based time schedule system for multiple LMSs on the SSO/portal environment

We developed a web-based time schedule system as an important feature of the university portal along with our universitys long-term ICT (Information and Communication Technologies) plan. By using the system, students and professors can get their own course timetable in collaboration with the Student Information System (SIS). Each course name on the timetable is linked to the corresponding course page on the Learning Management System (LMS) through the Single Sign-On (SSO). The system is adapted to multiple LMSs which can be selected by the course professor. In order to widely cooperate with other systems, the system is designed by using global standards (IMS Enterprise, JSR-168 Portlet, etc.) and open source software (uPortal, CAS, JSF, Hibernate, etc.) as possible as we can. This paper shows the major functions, the measured use of the portal and the time schedule system over eight months, and the implementation especially for supporting multiple LMSs, syllabus and grade books.

Fatal cytomegalovirus myocarditis in a seronegative ALL patient

Fatal cytomegalovirus (CMV) myocarditis occurred in a 2 year old boy with acute lymphoblastic leukemia (ALL) in remission. The patient showed mild hepatic dysfunction and a rapid progress of pancytopenia after complete remission had been achieved. At the fifth week of complete remission, he presented signs of heart failure such as tachycardia, S4 gallop on auscultation and decreased ejection fraction on echocardiography. However, no significant electrocardiographic changes were recognized. In addition to the cardiac dysfunction, the patient presented a marked tachypnea and dyspnea associated with hypoxemia. These were dramatically improved by methylprednisolone pulse therapy (30 mg/kg per day, for 3 days) and CMV high titer immunoglobulin (400 mg/kg per day, for 3 days). On the sixth day after signs of respiratory failure were improved, the patient suddenly presented a paroxysmal atrial tachycardia followed by a fatal ventricular fibrillation. Although we could detect neither a specific IgM antibody, a significant increase of IgG antibody, nor CMV genome by DNA hybridization techniques during the course of the illness, microscopic examination of necropsy specimens of the heart showed a marked disruption and disintegration of muscle bands associated with cytomegalic inclusion bodies. Polymerase chain reaction (PCR) yielded a 305 bp amplification product in the heart and lung tissues, supporting the view that myocarditis was caused by CMV.