Quantitation and identification of human monocytic colony-stimulating factor in human serum by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) system for the quantitation of human monocytic colony-stimulating factor (hM-CSF) was established, which was based on the "dual antibody immunometric sandwich" principle using horse and rabbit polyvalent antibodies against human urinary colony-stimulating factor (CSF-HU). The minimal detectable level of hM-CSF was 10 U/mL, and the assays showed good reproducibility. As measured by this method, the average serum hM-CSF level of 20 normal adults was 540 +/- 110 U/mL (range, 300 to 800 U/mL). The peak of hM-CSF measured by ELISA was identical to that measured by bioassay when semipurified CSF-HU was fractionated by reversed-phase high performance liquid chromatography (HPLC). This method detected two types of hM-CSF, which had approximate molecular weights of 85 Kd (CSF-HU) and 45 Kd in human serum and urine; the ratio of 85:45 Kd was very high in serum and the amounts of the two types were nearly equal in urine. After anticancer chemotherapy, the serum hM- CSF level of one half of the patients with hematological malignancy was elevated according to the reduction in neutrophil number, while it was almost in the normal range in the other half of the patients, indicating the possibility that anticancer chemotherapy damaged the hM- CSF-producing cells. This ELISA method may be useful for monitoring the serum hM-CSF level after anticancer chemotherapy.     

Possible dominant-negative mutation of the SHIP gene in acute myeloid leukemia.

The SH2 domain-containing inositol 5'-phosphatase (SHIP) is crucial in hematopoietic development. To evaluate the possible tumor suppressor role of the SHIP gene in myeloid leukemogenesis, we examined primary leukemia cells from 30 acute myeloid leukemia (AML) patients, together with eight myeloid leukemia cell lines. A somatic mutation at codon 684, replacing Val with Glu, was detected in one patient, lying within the signature motif 2, which is the phosphatase active site. The results of an in vitro inositol 5'-phosphatase assay revealed that the mutation reduced catalytic activity of SHIP. Leukemia cells with the mutation showed enhanced Akt phosphorylation following IL-3 stimulation. K562 cells transfected with the mutated SHIP-V684E cDNA showed a growth advantage even at lower serum concentrations and resistance to apoptosis induced by serum deprivation and exposure to etoposide. These results suggest a possible role of the mutated SHIP gene in the development of acute leukemia and chemotherapy resistance through the deregulation of the phosphatidylinositol-3,4,5-triphosphate (PI(3,4,5)P3)/Akt signaling pathway. This is the first report of a mutation in the SHIP gene in any given human cancer, and indicates the need for more attention to be paid to this gene with respect to cancer pathogenesis.     

Development of 18 novel polymorphic microsatellite markers for the mysid crustacean Mesopodopsis orientalis

Conservation Genetics Resources - We have developed microsatellite DNA markers for Mesopodopsis orientalis (Tattersall 1908), a widely distributed mysid crustacean in shallow waters of the coastal...     

Gene delivery system of pDNA using the blood glycoprotein fetuin

Fetuin is a biocompatible plasma protein and strongly enhances phagocytosis of bacteria, DNA and apoptotic cells by peripheral blood cells such as monocytes, macrophages and dendritic cells. We developed a novel gene delivery system: ternary complexes constructed with pDNA, polyethylenimine (PEI) and fetuin. Without covalent binding, fetuin was able to coat pDNA–PEI complexes, and stable anionic nanoparticles formed at a weight ratio greater than 30. Optimised pDNA–PEI–fetuin complexes significantly decreased the cytotoxicity of pDNA–PEI complexes in the melanoma cell line B16F10. Furthermore, the pDNA–PEI–fetuin complexes had higher transgene efficiency compared to that of commercial lipofectin previously reported in B16F10 cells despite an anionic surface. The pDNA–PEI–fetuin complexes did not agglutinate with erythrocytes. The pDNA–PEI–fetuin complexes had high gene expression in the spleen after intravenous administration in mice. Thus, the pDNA–PEI–fetuin complexes were a useful in vivo gene delivery system with tropism for the spleen.     

Molecular pathogenesis of myeloma for the therapeutic targets

Multiple myeloma(MM) is a neoplasia of plasma cells in bone marrow. High dose chemotherapy followed by stem cell transplantation and new drugs such as thalidomide and bortezomib have improved the survival in MM. However, most of patients with myeloma are incurable so there is a need for the new therapeutic approaches that have been developed against molecular targets and pathway. It has been reported that activation of NF-kappaB pathway was required to survival of myeloma cells and this pathway was the potential target for anti -MM therapy. Recently we reported diverse genetic aberrations that activated NF-kappaB signaling in MM. In this section, the molecular pathogenesis of myeloma, especially the new findings related with NF-kappaB activation, will be reviewed in Japanese.     

Formation of the thalamocortical projection regulated differentially by BDNF- and NT-3-mediated signaling

During development thalamocortical (TC) axons establish lamina-specific connections with cortical cells, and in later developmental stages TC projections are modified by activity-dependent processes. Recent studies have demonstrated that brain-derived neurotrophic factor and neurotrophin-3 are expressed in the cortex with distinct developmental time courses, and are involved not only in the formation of the TC projection but also in the subsequent refinement processes. Evidence further suggests that these actions of neurotrophins are achieved in cooperation with membrane-associated molecules expressed in cortical cells.     

Frequent occurrence of CCND1 deregulation in patients with early stages of plasma cell dyscrasia

Chromosomal translocations involving the immunoglobulin heavy chain gene ( IgH ) and nonrandom protooncogene loci are the hallmark of genetic alterations found not only in multiple myeloma (MM), but also in premalignant stages of MM, including monoclonal gammopathy of undetermined significance (MGUS) and smoldering myeloma (SMM). We studied the frequency of IgH (14q32) rearrangements and their partner chromosomes in 16 Japanese patients with MGUS (13 cases), and SMM (3 cases) by means of interphase double-color fluorescence in situ hybridization (DCFISH) applied to purified plasma cells and using CD138-bead selection. IgH rearrangement was recognized in nine of the patients (56.3%). Protooncogene loci juxtaposed to IgH were identified in seven cases including CCND1 (11q13) in six cases and FGFR3 (4p16) in one. Four out of the six t(11;14)-positive cases showed nuclear staining of the cyclin D1 protein, whereas none of the seven t(11;14)-negative cases did. Moreover, neither MUM1 (6p25)- IgH nor MAFB (20q11)- IgH fusion signals were observed. This suggests to us that cyclin D1 deregulation due to the presence of t(11;14) is involved in the early development of plasma cell neoplasms, and that this event alone is not enough for the development of symptomatic myeloma. (Cancer Sci 2003; 94: 350-354)     

Role of selectins on IgE-mediated skin reaction

Selectins play an important role on leukocytes infiltration into inflammatory tissues. To understand the role of selectins, we investigated the effects of selectin-IgG chimeras and anti selectin antibodies on the murine IgE-mediated skin inflammation model. Biphasic skin reactions were induced by intradermal challenge with ovalbumin (OA) to ears of actively sensitized mice. This reaction was characterized by immediate and late phase responses observed as which were induced via a rapid increase in capillary permeability and leukocyte infiltration, respectively. The expression of E-selectin mRNA was significantly increased to reach its highest level at 2 h after OA challenge. E-, P-, and L-selectin-IgG chimeras inhibited the late phase responses, i.e. ear swelling, neutrophil infiltration and eosinophil infiltration at 24 h after OA challenge in a dose-dependent manner at dose range of 0.1 - 10 mg kg(-1), i.v. Antiselectin antibodies did not inhibit the increase of ear swelling. But anti E- and P-selectin antibodies significantly inhibited neutrophil infiltration and eosinophil infiltration. These results indicate that selectins play an important role on the late phase response of the murine IgE-mediated skin inflammation model by mediating inflammatory cell adhesion to endothelium.