Akira Kunii

Sensitive colorimetric assay of serum diamine oxidase

A simple and sensitive colorimetric assay for serum diamine oxidase (DAO) activity was based on a coupled reaction with peroxidase and a new chromogen, 10-(carboxymethyl-aminocarbonyl)-3,7-bis(dimethylamino) phenothiazine sodium salt (DA-67). In the presence of peroxidase and DA-67, peroxidase catalyzes the formation of methylene blue having an absorption maximum at 668 nm. The proposed method eliminates the interferences occurring in serum with use of ascorbate oxidase and stops the reaction with sodium diethyldithiocarbamate, leaving the methylene blue in the reaction mixture stable for about 2 h. Low normal basal values of serum DAO can be determined in the range 2.8-9.0 units/l. Since all reagents are commercially available the method is suitable for the clinical laboratory.

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ

Abstract This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.

T-Cell Phenotype Is Associated with Decreased Survival in Non-Hodgkin's Lymphoma

This study was undertaken to determine which if any presentment factors are statistically significant determinants of the clinical outcome in patients with non‐Hodgkins lymphoma. The pretreatment factors in 20 patients with T‐cell lymphoma, including two patients with adult T‐cell leukemia/ lymphoma (ATLL), and 28 patients with B‐cell lymphoma were evaluated. In a stepwise logistic regression analysis, a T‐cell phenotype in addition to high grade histology and pleural involvement demonstrated a statistically significant correlation with decreased response rate, when the analysis did not include patients with ATLL. Analysis by means of the Cox proportional hazards model disclosed that the T‐cell phenotype retained a statistically significant correlation with survival after adjustments for other prognostic factors, whether the study included the patients with ATLL or not. The decreased response rate and survival of Japanese patients with non‐Hodgkins lymphoma in comparison with those reported in Western countries seem to be due to increased intrusion of T‐cell lymphomas. To permit a reliable comparison of reports on new chemotherapeutic regimens from different institutions, the tumor phenotype must be determined in the population studied

Mechanism of endotoxin-type interferon production in mice

Abstract Splenectomized mice, when stimulated by endotoxin 3 days after operation, produced little or no circulating interferon, but the serum interferon-producing ability of these mice was restored by an intraperitoneal injection, prior to an endotoxin stimulus, of not only mouse (syngeneic), but also rat (xenogeneic) spleen cells. The serum interferon produced in the rat-to-mouse chimera exhibited the species specificity of mouse interferon. Interferon production-restoring capacity of rat spleen cells was attributed to glass-adherent cells (macrophages) and not to nonadherent cells (lymphocytes). Although spleen cells of mice pretreated with Bordetella pertussis vaccine produced far less interferon in vitro in response to endotoxin stimulus, they possessed the capacity to restore the interferon-producing ability of splenectomized mice. Culture fluid of spleen cells incubated with endotoxin was able to stimulate interferon production in splenectomized mice. From these observations, it is postulated that in vivo production of the endotoxin-type interferon is brought about through a two-step mechanism.

Immune Interferon Produced In Vitro as a Quantitative Indicator of Cell-Mediated Immunity

The present study was constructed to provide some information on the possibility of utilizing immune interferon as a quantitative indicator of cell‐mediated immunity and to clarify some of the nature of immune interferon‐producing cells (IIPC).

Production of interferon-like substance by mouse spleen cells through contact with BHK cells persistently infected with HVJ.

Abstract A virus inhibitor, an interferon-like substance, was found in the culture fluid of mouse spleen cells cocultivated with BHK-HVJ cells, the BHK cells persistently infected with HVJ, but not in the medium of cocultivation of mouse spleen cells and normal BHK cells. Neither the culture fluid of spleen cells alone nor that of BHK-HVJ cells alone was shown to contain the virus inhibitor. No virus inhibitory activity could be detected in the culture fluid of mouse spleen cells incubated with either the culture fluid of BHK-HVJ cells contained noninfectious HVJ particles or sonicated BHK-HVJ cell suspension. L cells or mouse liver cells, when cocultivated with BHK-HVJ cells, did not release a virus inhibitor. These findings suggest that mouse lymphoid cells have a capacity to produce a virus inhibitor when cocultivated with BHK-HVJ cells, and that nonlymphoid somatic cells may lack this capacity. Interposition of a Millipore filter between BHK-HVJ monolayer and mouse spleen cells or pretreatment of BHK-HVJ cells with anti-HVJ antiserum resulted in a blockade of virus inhibitor production. These findings suggest that the following sequence is necessary for the mouse spleen cells cocultivated with BHK-HVJ cells to produce the virus inhibitor: first, attachment of the spleen cells to BHK-HVJ cells and, second, recognition by the former of virus antigen(s) present on the surface of the latter. The BHK-HVJ cell membrane, isolated by sucrose density gradient centrifuge, was found to be an active inducer of the virus inhibitor. Moreover, some artificial membranous structures, such as HVJ-erythrocyte complex as well as HVJ spike-erythrocyte complex, exhibited a similar activity. This virus inhibitor induced in the present system appears to have all biologic attributes of interferon and its production might be initiated by membrane-membrane interaction between lymphoid cells and HVJ infected cells.

Effects of splenectomy on production of endotoxin-type interferon in mice

Abstract The present study was undertaken to examine the effects of omentumectomy on the production of interferon in response to an endotoxin stimulus and to compare them with those of splenectomy. Omentumectomized mice, administered endotoxin intraperitoneally, failed to produce any detectable amount of circulating interferon; if given endotoxin intravenously, they produced as much interferon as sham-operated controls. The role of the omentum in circulating interferon production in response to an endotoxin stimulus is discussed in comparison with that of the spleen.

Interferon production in L cells persistently infected with hemagglutinating virus of Japan (HVJ)

Abstract The present study shows that when L cells persistently infected with hemagglutinating virus of Japan (HVJ) (L-HVJ cells) were incubated at 32°, the interferon-producing capacity of the cells was suppressed and was restored by the temperature shift-up to 38°. Synthesis of envelope protein antigen was not detected in the L-HVJ cells incubated at 38° which could produce interferon. Moreover, glutamine-starved L-HVJ cells did produce interferon even at 32°. The relationship between the suppressed state of interferon production and synthesis of viral envelope protein is discussed.

The effects of cytochalasin and colchicine on interferon production.

The present study was undertaken to investigate the mechanism of interferon production by mouse spleen cells co-cultivated with BHK-HVJ cells, i.e. baby hamster kidney cells persistently infected with the HVJ (or Sendai) strain of para-influenza I virus. Cytochalasin B appears to inhibit an early stage in the process and colchicine a relatively late stage. It is suggested that microfilaments and microtubules may play in important role at an initial stage of interferon production in this system.

Interferon Susceptibility of Various Cell Lines Persistently Infected with Haemagglutinating Virus of Japan (HVJ)

Various cell lines persistently infected with para-influenza 1 virus, HVJ strain, were less susceptible to the antiviral action of interferon than the same cell lines when not infected with HVJ. When Vero cells persistently infected with a temperature-sensitive strain of HVJ were incubated at 38 degrees C, a non-permissive temperature, they became fully susceptible to interferon, whereas neither the haemadsorbing nor the cell-associated haemagglutinating activity of the virus was expressed. These findings suggest that the lowered interferon susceptibility of virus-carrier cells may be related to the maturation of virus in them. It was found that the low susceptibility of virus-carrier cells to interferon is not due to blocked adsorption of interferon or to inability of the cells to respond tointerferon. Studies with actinomycin D suggest that some step (or steps) before the synthesis of the messenger RNA for the antiviral protein is blocked.