Yuzo Ichimori

β-Amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity

beta-Amyloid protein (A beta) is the major component of the senile plaques in Alzheimers disease (AD), and microglial cells have been shown to be closely associated with these plaques. However, the roles of A beta and microglial cells in pathogenesis of AD remain unclear. Incubation of rat microglial cells with A beta(1-40) caused a significant increase in nitrite, a stable metabolite of nitric oxide (NO), in culture media, while there was no detectable increase in nitrite in astrocyte-rich glial cells or cortical neurons after incubation with A beta(1-40). Nitrite production by microglial cells was also induced by A beta(1-42), but not A beta(25-35). An inhibitor of NO synthase, NG-monomethyl-L-arginine (NMMA), as well as dexamethasone and actinomycin D, dose-dependently inhibited this nitrite production. Among the various cytokines investigated such as interleukin-1, interleukin-6, tumor necrosis factor-alpha and interferon-gamma (IFN-gamma), only IFN-gamma markedly enhanced A beta-dependent nitrite production. Cultured cortical neurons were injured by microglial cells stimulated with A beta in a dose-dependent manner in the presence of IFN-gamma. Neurotoxicity caused by the A beta plus IFN-gamma-stimulated microglial cells was significantly attenuated by NMMA. Thus, although further investigations into the effect of A beta on human microglial cells are needed, it is likely that A beta-induced NO production by microglial cells is one mechanism of the neuronal death in AD.

Increased serum levels of basic fibroblast growth factor in patients with renal cell carcinoma

The serum level and urinary output of basic and acidic fibroblast growth factors (FGFs) were measured by sandwich enzyme immunoassay (EIA) in patients with renal cell carcinoma. In over fifty percent (16/31) of renal cell carcinoma patients, basic FGF was elevated (greater than 30 pg/ml) in their sera. There is relatively good correlation between serum levels of basic FGF and tumor stage or grade, while urinary daily output of basic FGF did not correlate with increased malignancy. The present results indicate that serum basic FGF level of patients with renal cell carcinoma is a useful diagnostic and prognostic marker for renal cell carcinoma. On the other hand, acidic FGF was not detectable in all sera and urine.

Production of human monoclonal antibodies by heterohybridomas

SummaryMouse human-human heterohybridomas secreting human monoclonal antibodies (MoAb) against tetanus toxoid and hepatitis B virus surface antigen were effectively cultivated in a medium containing a serum substitute called GFS, a 55% to 70% ammonium sulphate fraction of serum from adult cattle. A perfusion culture system using a jar fermentor equipped with a cell sedimentation column with a double jacket was developed and applied to produce human MoAb. In this fermentor, maximum cell density of a heterohybridoma reached 1.2×107 cells/ml and MoAb was continuously accumulated at a constant rate for at least 40 days; this led to the production of more than one gram of human MoAb using a culture vessel with a 1-1 working volume.

Immunohistochemical expression of inducible nitric oxide synthase (iNOS) in reversible endotoxic shock studied by a novel monoclonal antibody against rat iNOS.

An antirat monoclonal antibody (mAb) against inducible nitric oxide synthase (iNOS), ANOS11, was used for immunohistochemistry to examine the expression of iNOS in various organs and tissues of adult rats in experimental endotoxic shock induced by lipopolysaccharide (LPS) injection. The phenotype of iNOS‐expressed cells was also examined immunohistochemically using various mAbs. In control rats, very few cells were positive for ANOS11 except in the thymus. After intravenous injection of LPS, the number of iNOS‐positive cells increased rapidly in almost all organs, except the thymus and brain, peaked 6 h after the injection, and decreased slowly. Of the numerous inflammatory cells that infiltrated the lungs, liver, and spleen after LPS injection, many were positive for ANOS11. Besides inflammatory cells, hepatocytes and endothelial cells of the aorta were also positive for ANOS11 but only around 6 h after injection. The cellular composition of iNOS‐positive infiltrated cells changed along with the progression of endotoxic shock. At 4 to 6 h after injection, most iNOS‐positive cells were considered polymorphonuclear leukocytes judging by their positive reactivity to 0X42 and their nuclear morphology. The population of iNOS‐positive macrophages positive for EDI or ED2 increased with time. After 24 h, many iNOS‐positive macrophages were found around the focal necrosis in the liver and spleen. These results indicate that the expression of iNOS in neutrophils, endothelial cells, and hepatocytes precedes that of macrophages in experimental endotoxic shock. The expression of iNOS in various cells and organs is closely associated with the progress and pathological changes of endotoxic shock. J. Leukoc. Biol. 57: 36–44; 1995.

Establishment of hybridomas secreting human monoclonal antibodies against tetanus toxin and hepatitis B virus surface antigen

Mouse-human heterohybrids (M X H) were constructed and compared with other cell lines (human or mouse) as parental cells to obtain hybrids secreting human monoclonal antibody (MoAb). One of the M X H lines, HM-5, was far superior to the others and useful for establishing hybrids secreting human MoAb. Using HM-5 as a parental cell line, we have obtained 2 hybrids secreting human anti-tetanus toxoid MoAb with neutralizing activity and a hybrid secreting human anti-hepatitis B virus surface antigen (HBsAg) MoAb which recognizes the a-determinant of HBsAg.

Translation of interleukin 2 mRNA from human peripheral blood leukocytes in Xenopus oocytes

Abstract Interleukin 2 was induced in cultures of human peripheral blood leukocytes by combined treatment with 12-O-tetradecanoylphorbol-13-acetate and a T-cell mitogen, Concanavalin A. Poly(A)-containing mRNA was isolated from these cultures and fractionated by sucrose density gradient centrifugation. When injected into Xenopus laevis oocytes, the mRNA preparation gave rise to interleukin 2 activity in the culture supernatant of the oocytes. The sucrose density gradient centrifugation analysis showed that the interleukin 2 mRNA sedimented at 10–12S, which suggests that it contains about 900–1,100 nucleotides.

Monoclonal antibodies to human interferon-γ. I. Antibodies to a synthetic carboxyl-terminal peptide

Two types of hybridomas secreting monoclonal antibodies (MAB) against human interferon-gamma (HuIFN-gamma) were obtained by somatic cell hybridization between mouse myeloma P3U1 cells and spleen cells from BALB/c mice immunized with a conjugate of a synthetic carboxyl-terminal peptide (residues 131-146) of HuIFN-gamma and bovine thyroglobulin. One of the antibodies bound to recombinant HuIFN-gamma produced in E. coli as well as to natural HuIFN-gamma, while the others bound only to recombinant HuIFN-gamma. These 2 types of MAB did not neutralize the anti-viral activity of HuIFN-gamma. They were useful for effectively purifying recombinant HuIFN-gamma and quantitatively determining it by an enzyme-linked immunosorbent assay.

Establishment of hybridoma secreting human monoclonal antibody against hepatitis B virus surface antigen.

The HAT (hypoxanthine, aminopterin, thymidine) sensitive and ouabain resistant human B lymphoblastoid cell line TAW-925 was obtained from 6-thioguanine resistant B lymphoblastoid cell line WI-L2. Hybridomas were obtained at a high frequency (10(-4)-10(-5) when TAW-925 was hybridized with cells transformed with Epstein-Barr virus. Using TAW-925 as a parental cell line, we have obtained a hybridoma which stably secretes human monoclonal antibody against hepatitis B virus surface antigen.

PHOTOACTIVE METHYLENE BLUE DYE DERIVATIVES SUITABLE FOR COUPLING TO PROTEIN

Methylene blue is a very strong photoactive dye that has an absorption peak (668 nm) that corresponds well to a popular low‐cost diode laser. However, it has not been used in photodynamic tumor therapy and immunodiagnostics because it cannot be covalently coupled to protein. Therefore, methylene blue derivatives having a succinimido or maleimido functional group were synthesized and coupled to antibody, serum albumin and transfemn proteins. Incorporation of dye into antibody protein at high ratios (more than three per molecule) caused precipitation and loss of antibody activity. Inclusion of one or more carboxylic acid residues in the methylene blue derivative before coupling to protein alleviated the precipitation problem, and up to 36 methylene blue dye molecules could be attached to an antibody fragment using bovine serum protein as a carrier. Methylene blue derivatives and protein complexes formed from them oxidized luminol when stimulated with red light. The new dye conjugates were used in an optically pumped chemiluminescence immunoassay for α‐fetoprotein. These compounds and techniques should also be useful for photodynamic tumor therapy where it is desired to attach a red‐absorbing photoactive dye to antibody protein.

A hyman hybrid hybridoma producing a bispecific monoclonal antibody that can target tumor cells for attack by Pseudomonas aeruginosa exotoxin A

By fusing a human hybridoma producing an IgG2ķ antibody against human A431 epidermoid carcinoma cells with an Epstein-Barr virus-transformed human B lymphocyte producing an IgG2ķ antibody against Pseudomonas aeruginosa exotoxin A, we established a hybrid hybridoma producing a bispecific monoclonal antibody reacting with both A431 cells and the exotoxin. Human IgG was purified from the culture supernatant of the hybrid hybridoma, and the bispecific monoclonal antibody in the IgG preparation was further separated from the two parental antibodies by hydroxyapatite high-performance liquid chromatography. The human bispecific monoclonal antibody thus obtained efficiently targeted the antibody-reative cells, A431, for attack by the exotoxin in vitro.