Takato Katsuki

Identification of the target cells in human B lymphocytes for transformation by epstein-barr virus

Abstract Lymphocytes from human umbilical cord blood were purified and then separated into rosette-forming cells (T cells) and non-rosette-forming cells (non-T cells). Non-rosette-forming cells were further divided by the technique of rosette formation, after neuraminidase treatment, into surface immunoglobulin (SIg)-carrying cells (B cells) and cells lacking SIg but carrying Fc receptors (null cells). The three cell subsets, T, B, and null cells, were examined for susceptibility to transformation by the B95-8 strain of Epstein-Barr virus (EBV) using the criteria of colony formation by transformed cells and/or transformation efficiency as judged by the days required for the first appearance of transformation. Not only the T cells but also the null cells were unsusceptible to transformation by EBV. In contrast, B cells were highly susceptible. In a study of the quantitative relationship between the target cells for viral transformation and those B cells which possessed SIg after an acid pH treatment, 10 lymphocyte preparations from three cord blood samples and seven adult peripheral blood samples were tested individually. The fraction size of transformable cells was determined by the growth-curve procedure for transformed cells while the procedure of direct membrane immunofluorescence with anti-IgM (μ-specific) serum and polyvalent anti-Ig serum was used to determine the fractions of SIg and SIg(M) cells. The actual fraction of EBV target cells was nearly equal to that of SIg(M) B cells but not to that of the total SIg B cells. Thirty-four lymphocyte preparations from eight cord and 26 adult peripheral blood samples were examined for the percentage of SIg B cells and for susceptibility to EBV transformation as assayed by the colony-formation procedure. EBV susceptibility and the fraction of SIg(M) B cells, but not of total SIg B cells, correlated nicely, suggesting that SIg(M)-bearing cells were probably the major target among the B lymphocytes for transformation by EBV.

High Correlation between Lipid Peroxide Radical and Tumor-promoter Effect: Suppression of Tumor Promotion in the Epstein-Barr Virus/B-Lymphocyte System and Scavenging of Alkyl Peroxide Radicals by Various Vegetable Extracts

We examined the ability of hot‐water extracts of 66 vegetables and plants to suppress tumor promotion, as well as to scavenge lipid peroxide radicals in vitro. To assess the effect against tumor promotion (transformation) in vitro, we used the phorbol myristate acetate/Epstein‐Barr virus/ B‐lymphocyte system. To assess the lipid radical‐scavenging effect, the luminol‐enhanced cliemi‐luminescence method using the tot‐butyl hydroperoxide/heme system was used, which generates more alkyl peroxide radical (ROO·) than alkyl (R·) and alkoxyl <RO) radicals. The results showed a significant correlation between the anti‐tumor‐promoting effect and the lipid radical‐scavenging effect (r=0.82). We found that boiled extracts of green leaves of carrot, crucifers, and beans (black bean, red bean, mung bean, and soybean) had the greatest anti‐tumor‐promoter and radical‐scavenging activities. Cold‐water extracts of vegetables generally exhibited only about 10% or less of the activity of the hot‐water extracts.

UNIQUE PATTERN OF EPSTEIN-BARR VIRUS SPECIFIC ANTIBODIES IN RECURRENT PAROTITIS

Sera from 34 children with recurrent parotitis were measured by indirect immunofluorescence technique for antibody levels to several Epstein-Barr virus (EBV) antigens. IgG antibodies to EBV-capsid antigen (VCA) and EBV-associated nuclear antigen, often at high titres, were found in 29 of these patients, of whom 20 also had IgA antibody to VCA. Antibody level to early antigen complex (R and D) rose in 19 patients, of whom 18 had antibody to the R component alone. The abnormal patterns of EBV antibodies persisted for 3-14 months during and after the illness in 8 patients. These observations suggest that EBV infection may be important in the pathogenesis of recurrent parotitis.

Enhancement of Epstein-Barr virus-induced transformation of human lymphocytes by teleocidin

Abstract Teleocidin, a naturally occurring tumor promoter, enhanced colony formation of human umbilical cord blood lymphocytes in soft agar iduced by the B95-8 strain of Epstein-Barr virus. Colonies were formed most efficiently in the presence of teleocidin at a concentration of 0.1 ng/ml. Possible mechanisms of this enhancement are discussed.

Study on established lymphoid cells in maple syrup urine disease. Correlation with clinical heterogeneity

SummaryBranched-chain keto acid dehydrogenase complex (BCKAD) was measured in lymphoid cells established from five patients with maple syrup urine disease (MSUD) and six control subjects. Two other MSUD lymphoid cell lines obtained from The Human Genetic Mutant Cell Repository were used as references. One day after subculture, the cells grew logarithmically up to 4–5 days. With this cell growth, BCKAD activity increased greatly in controls, but not in MSUD cells. The maximum BCKAD activity of MSUD cells was less than 7% and 13%–16% of the control in classic and variatant types, respectively. Leucine added to culture medium at the concentration of 10–20 mM significantly inhibited cell growth in MSUD cells alone, and with increasing concentration and impaired enzyme activity in a cell line, the effect became more prominent. The effects of isoleucine and valine were mild and did not differ between control and MSUD cells.

Preoperative in vitro chemosensitivity test of esophageal cancer with endoscopic specimens

Background. From January 1990 to June 1991, the authors tested in vitro chemosensitivity before surgery with endoscopic biopsy specimens from 23 patients with intrathoracic esophageal cancer.

Ornithine transcarbamylase (OTC) in white blood cells

Summary: A radiochemical assay method of ornithine transcarbamylase (OTC) was developed using labeled carbamyl phosphate as a substrate. The enzyme activities determined by this method in peripheral white blood cells from ten normal subjects were 1.32 ± 0.95 nmoles/mg/hr and the apparent Kms, when assayed at pH 8.5, were 6.4 mM for ornithine and 0.6 mM for carbamyl phosphate. On the contrary, the apparent Kms of human liver OTC were 0.6 mM for ornithine and 0.12 mM for carbamyl phosphate. The average OTC activity of granulocytes was 1.0 nmoles/mg/hr, whereas that of mononuclear cells was 0.4 nmoles/mg/hr.Lymphoid cell lines were established from three normal subjects and an OTC-deficient infant. All these cell lines demonstrated no OTC activity. When arginine was removed from the medium and replaced by ornithine, the lymphoid cells were unable to grow in culture. On autoradiography, the lymphoid cells showed labeling at incubation in the presence of 14C-citrulline, but not with 14C-ornithine.Speculation: The wide range of OTC activities observed in white blood cells from normal subjects might be due to a difference of the enzyme activities between granulocytes and mononuclear cells and in turn due to an individual difference of the two cell population. OTC is either absent or inactivated in lymphoid cell lines, when grown in a culture medium with arginine. The enzyme of the peripheral white blood cells might be of a different genetic origin from that of the liver.

Ornithine Transcarbamylase (OTC) in White Blood Cells and Jejunal Mucosa

The presence of OTC in white blood cells was demonstrated by Wolfe and Gatfield(1) and Snodgrass et al.(2), but was denied by Rabier et al (3). OTC deficiency was diagnosed using white blood cells by Wolfe and Gatfield (1) and Krieger et al (4). On the other hand, Snodgrass et al (2) reported that OTC deficiency in the liver can not be inferred from the measurements of the enzyme’s activity in peripheral white blood cells, because the latter parameter was normal in their patients. The presence of OTC in jejunal mucosa and reliability of this material for detecting OTC deficiency were demonstrated by Levin et al (5) and Cathelineau et al (6). However, the nature of OTC in intestinal mucosa was not clearly defined. We would like to describe some kinetic studies on OTC in white blood cells and jejunal mucosa.

Three Phases in Transformation of Human Lymphocytes by Epstein‐Barr Virus, as Revealed by Colony Formation in Soft Agar

Human cord lymphocytes were examined for colony formation in soft agar medium at various times after infection with Epstein‐Barr virus (EBV). The transformation process could be divided into the following three sequential phases: (1) A pre‐early phase, lasting about 10 hr post infection (pi), during which the number of transformed colonies was virus dose dependent. (2) An early phase, between about 10 and 60 hr pi, during which cloning efficiency was markedly lower than that of the pre‐early phase. (3) A later phase, starting at about 60 hr pi, during which cloning efficiency was restored and the number of colony formers increased in an exponential fashion. EBV‐associated nuclear antigen‐positive cells first appeared at the beginning (12 hr pi) of the early phase and increased continuously thereafter. Increased DNA synthesis began at about 48 hr pi. These results suggest that the cloning efficiency of the infected cells changes during the transformation process, possibly reflecting altered cell sensitivity to agar.