Masayoshi Masuko

The coxsackievirus-adenovirus receptor protein as a cell adhesion molecule in the developing mouse brain

In an attempt to elucidate the molecular mechanisms underlying neuro-network formation in the developing brain, we analyzed 130 proteolytic cleavage peptides of membrane proteins purified from newborn mouse brains. We describe here the characterization of a membrane protein with an apparent molecular mass of 46 kDa, a member of the immunoglobulin superfamily of which the cDNA sequence was recently reported, encoding the mouse homologue of the human coxsackievirus and adenovirus receptor (mCAR). Western and Northern blot analyses demonstrated the abundant expression of mCAR in the mouse brain, the highest level being observed in the newborn mouse brain, and its expression was detected in embryos as early as at 10. 5 days post-coitus (dpc), but decreased rapidly after birth. On in situ hybridization, mCAR mRNA expression was observed throughout the newborn mouse brain. In primary neurons from the hippocampi of mouse embryos the expression of mCAR was observed throughout the cells including those in growth cones on immunohistochemistry. In order to determine whether or not mCAR is involved in cell adhesion, aggregation assays were carried out. C6 cells transfected with mCAR cDNA aggregated homophilically, which was inhibited by specific antibodies against the extracellular domain of mCAR. In addition to its action as a virus receptor, mCAR may function naturally as an adhesion molecule involved in neuro-network formation in the developing nervous system.

Genetic variants in C5 and poor response to eculizumab.

BACKGROUND Eculizumab is a humanized monoclonal antibody that targets complement protein C5 and inhibits terminal complement-mediated hemolysis associated with paroxysmal nocturnal hemoglobinuria (PNH). The molecular basis for the poor response to eculizumab in a small population of Japanese patients is unclear. METHODS We assessed the sequences of the gene encoding C5 in patients with PNH who had either a good or poor response to eculizumab. We also evaluated the functional properties of C5 as it was encoded in these patients. RESULTS Of 345 Japanese patients with PNH who received eculizumab, 11 patients had a poor response. All 11 had a single missense C5 heterozygous mutation, c.2654G → A, which predicts the polymorphism p.Arg885His. The prevalence of this mutation among the patients with PNH (3.2%) was similar to that among healthy Japanese persons (3.5%). This polymorphism was also identified in a Han Chinese population. A patient in Argentina of Asian ancestry who had a poor response had a very similar mutation, c.2653C → T, which predicts p.Arg885Cys. Nonmutant and mutant C5 both caused hemolysis in vitro, but only nonmutant C5 bound to and was blocked by eculizumab. In vitro hemolysis due to nonmutant and mutant C5 was completely blocked with the use of N19-8, a monoclonal antibody that binds to a different site on C5 than does eculizumab. CONCLUSIONS The functional capacity of C5 variants with mutations at Arg885, together with their failure to undergo blockade by eculizumab, account for the poor response to this agent in patients who carry these mutations. (Funded by Alexion Pharmaceuticals and the Ministry of Health, Labor, and Welfare of Japan.).

Expression of Coxsackievirus and Adenovirus Receptor in Hearts of Rats With Experimental Autoimmune Myocarditis

The expression of coxsackievirus and adenovirus receptor (CAR) was dominant in the brains and hearts of mice until the newborn phase. There is no detailed information concerning the relation between the expression of CAR and development of hearts. It is also uncertain whether CAR is able to be induced in adult hearts after cardiac injury. We demonstrated that CAR was abundant in the hearts of newborn rats but was barely detectable in the hearts of adult rats. The expression of CAR in rat hearts with experimental autoimmune myocarditis, which was induced by immunization of purified cardiac myosin, was serially investigated. Active myocarditis was observed from day 15 after immunization. By immunohistochemistry, cardiomyocytes were strongly stained for CAR antibody from days 24 to 42. CAR mRNA was also detected from days 18 to 30 by using reverse transcription-polymerase chain reaction. In the next experiment, the induction of CAR on isolated cardiomyocytes was investigated. CAR was barely detectable in cultured cardiomyocytes by Western blot analysis after isolation. This molecule gradually appeared along with the creation of clusters and beating of cardiomyocytes. Furthermore, the induction of CAR in cultured cardiomyocytes increased after supplement with conditioned medium of rat splenocytes activated by concanavalin A. In conclusion, rat CAR is expressed strongly in the hearts of newborn rats and is suppressed in those of adult rats. The expression of CAR is enhanced during the active phase of experimental autoimmune myocarditis and is induced by inflammatory mediators. CAR may play a role in cell-to-cell contact and adhesion of cardiomyocytes.

Novel technique for the direct flow cytofluorometric analysis of human basophils in unseparated blood and bone marrow, and the characterization of phenotype and peroxidase of human basophils

BACKGROUND No technique has been reported to analyze directly the antigen expression on basophil leukocytes when using a flow cytometer; therefore, the exact phenotype of human basophils and the character of the peroxidase in basophils are not well understood. METHODS Human blood basophils were purified by using an antibody against high-affinity Fc epsilon receptor (hFcepsilonR) and a MACS magnetic cell sorting system and then cytochemically stained. The phenotype and peroxidase of the human basophils were flow cytofluorometrically analyzed directly in unseparated blood and bone marrow samples as hFcepsilonR+/MBP+ (major basic protein)/Hist+ (histamine) light-density cells distributed in the high sidescatter area of lymphocytes on light scattergrams. RESULTS The peroxidase granules of human basophils were stained by an anti-eosinophil peroxidase (EPO) antibody. The human blood basophils had common granulocyte markers plus CD25, i.e., they were CD11a/ CD11b/CD11c/CD25/CD38/CD13/CD33/hFcepsi lonR/MBP/Hist/ EPO positive, CD71 dim positive, CD14/CD15 partially positive, and CD2/CD3/CD7/CD122/CD16/CD56/CD57/ CD10/CD19/CD20/CD22/HLA-DR/MPO (myeloperoxidase)/CD23 negative. Further examination was done to analyze the expression of colony-stimulating factor receptors on three lineages of granulocytes, i.e., basophils, eosinophils, and neutrophils. The neutrophils were CD114 (G-CSFR)/CD116 (GM-CSFR)/CD124 [interleukin (IL)-4R]/CD126 (IL-6R) positive and CD123 (IL-3R)/CD125 (IL-5R) negative. In contrast, the eosinophils and basophils were CD116/CD123/CD125/CD126 positive and CD114/CD124 negative. CONCLUSIONS This novel technique for directly characterizing human basophil leukocytes with flow cytometry may be a convenient way to screen the expression of surface antigens and the cytoplasmic expression of CD antigens and other proteins in human blood basophils and to analyze alterations of the character of basophils by cytokines and other biological substances in vivo and in vitro.

Primary familial polycythaemia associated with a novel point mutation in the erythropoietin receptor.

Primary familial and congenital polycythaemia (PFCP) is a rare disease characterized by congenital erythrocytosis inherited in an autosomal dominant fashion. Recently, mutations in the erythropoietin receptor (EpoR) have been identified in PFCP families. We describe a Japanese family with an autosomal dominant inheritance of PFCP. An in vitro colony assay demonstrated hypersensitivity of erythroid progenitors to erythropoietin (Epo) in affected family members. Sequence analysis of RT‐PCR products amplified from the C‐terminal region of EpoR transcripts in affected family members revealed that they were all heterozygous for C and T bases at position 5986, which suggested a genetic mutation (C to T) on one allele of EpoR. This mutation gave rise to a translation termination codon TAG at amino acid 435. Thus, the resulting EpoR is a truncated protein product lacking all 74 amino acids downstream of the mutation. To date, all genetic mutations affecting a family with PFCP, including this one, have been located in the cytoplasmic negative regulatory region of the EpoR. All mutations gave rise to truncated Epo receptors between Tyrosine 427 and Tyrosine 455. The phosphotyrosines in this region of EpoR have been demonstrated to be binding sites for SHP‐1 phosphatase. Therefore PFCP is presumably brought about as a result of genetic mutations which cause the loss of the SHP‐1 binding site in the cytoplasmic region of EpoR.

Kinetics of pDCs, mDCs, γδT cells and regulatory T cells in association with graft versus host disease after hematopoietic stem cell transplantation

Introduction:  Although the roles of each low‐frequency immunocompetent cells such as dendritic cells (DCs), γδT cells, and Treg cells in induction of acute or chronic graft versus host disease (GVHD) have been discussed in several reports, there are few papers dealing with an evaluation of these immunocompetent cells together and simultaneously in patients with hematopoietic stem cell transplantation (HSCT) and explored the kinetics of these cells in association with GVHD.

Successful All-trans Retinoic Acid Treatment of Acute Promyelocytic Leukemia in a Patient with NPM/RAR Fusion

Acute promyelocytic leukemia (APL) is characterized by a reciprocal chromosomal translocation involving the gene for retinoic acid receptor α (RAR). Most APL patients have a t(15;17) translocation that generates the PML-RAR fusion gene, and such patients respond well to treatment with all-trans retinoic acid (ATRA). Some APL cases also involve rearrangements that fuse RAR to partner genes other than PML, including nucleophosmin (NPM), promyelocytic leukemia zinc finger (PLZF), nuclear mitotic apparatus (NUMA), and Stat5b, but the clinical characteristics of APL without PML-RAR have not been fully clarified. We describe a 64-year-old man with NPM-RAR—positive APL who was receiving hemodialysis therapy for chronic uremia. Complete remission was achieved with ATRA monotherapy and was maintained for 18 months with consolidation chemotherapy. These findings suggest that ATRA can be used to treat APL patients with NPM/RAR as well as APL with PML/RAR.

Risk factors for acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation: retrospective analysis of 73 patients who received cyclosporin A.

Cyclosporin A (CsA) has been used most widely as an immunosuppressive agent for preventing graft-versus-host disease (GVHD). To explore the risk factors including CsA blood levels for grades II–IV acute GVHD, we retrospectively analyzed the data of patients who underwent allogeneic hematopoietic stem cell transplantation in our hospital between March 1989 and July 2001. Seventy-three patients (47 males and 26 females) received CsA and short-term methotrexate for GVHD prophylaxis. CsA 1.5 mg/kg was administered as a 3-h infusion twice daily from day 1 until the patient recovered from the toxic gastrointestinal complication. Methotrexate was given at a dose of 15 mg/m2 on day 1 and 10 mg/m2 on days 3, 6 and 11. Grades II–IV acute GVHD occurred in 18 patients (24.7%). Multivariate Cox regression analysis revealed that higher C5 (the whole-blood CsA concentration at 5 h after the start of infusion) before the onset of acute GVHD reduced the onset of grades II–IV acute GVHD with a hazard ratio of 0.994 (95% confidence interval 0.989–0.999) for every increase of 1 ng/ml. Our data indicate that inadequate exposures of CsA can be a vital risk for developing acute GVHD. From our results, we consider that precise monitoring of CsA concentrations and adjustment of CsA dose using the concentration may be effective to prevent the onset of severe acute GVHD. To confirm this finding, further prospective study will be needed.

Noninvasive Tracking of Donor Cell Homing by Near-Infrared Fluorescence Imaging Shortly after Bone Marrow Transplantation

Background Many diseases associated with bone marrow transplantation (BMT) are caused by transplanted hematopoietic cells, and the onset of these diseases occurs after homing of donor cells in the initial phase after BMT. Noninvasive observation of donor cell homing shortly after transplantation is potentially valuable for improving therapeutic outcomes of BMT by diagnosing the early stages of these diseases. Methodology/Principal Findings Freshly harvested near-infrared fluorescence-labeled cells were noninvasively observed for 24 h after BMT using a photon counting device to track their homing process. In a congenic BMT model, the homing of Alexa Fluor 750-labeled donor cells in the tibia was detected less than 1 h after BMT. In addition, subsequent cell distribution in an intraBM BMT model was successfully monitored for the first time using this method. In the allogeneic BMT model, T-cell depletion decreased the near-infrared fluorescence (NIRF) signals of the reticuloendothelial system. Conclusions/Significance This approach in several murine BMT models revealed that the transplanted cells homed within 24 h after transplantation. NIRF labeling is useful for tracking transplanted cells in the initial phase after BMT, and this approach can contribute to in vivo studies aimed at improving the therapeutic outcomes of BMT.

Late Onset Post-Transfusion Hepatitis E Developing during Chemotherapy for Acute Promyelocytic Leukemia

We herein report the case of a leukemia patient who developed hepatitis E seven months after undergoing a transfusion with contaminated blood products. The latency period in this case was significantly longer than that of typical hepatitis E. Recently, chronic infection with hepatitis E virus (HEV) genotype 3 has been reported in immunocompromised patients. There is a possibility that our patient was unable to eliminate the virus due to immunosuppression following chemotherapy and the administration of steroids. The prevalence of HEV in healthy Japanese individuals is relatively high and constitutes a critical source of infection via transfusion. Hepatitis E is an important post-transfusion infection, and immunocompromised patients may exhibit a long latency period before developing the disease.