Toshiko Yamamoto

DNA from Bacteria, but Not from Vertebrates, Induces Interferons, Activates Natural Killer Cells and Inhibits Tumor Growth

The nucleic acid fraction from cells of 6 species of bacterium and 2 kinds of vertebrate, calf and salmon, was extracted and purified by the same procedures as described previously. When the spleen cells from BALB/c mice were incubated with the nucleic acid fraction from either of the bacteria, natural killer (NK) activity of the cells was remarkably elevated and the cells produced factors to activate macrophages and to inhibit viral growth. It was shown that the factor to activate macrophages was interferon (IFN)‐gamma and that to inhibit viral growth was IFN‐alpha/beta. On the other hand, the nucleic acid fraction from either of the vertebrate cells did not show such activities. Pretreatment of the bacterial nucleic acid fraction with DNase, but not with RNase, abrogated completely the biological activities. The activities of the bacterial nucleic acid were not influenced by the presence of polymyxin B, an inhibitor of lipopolysaccharide (LPS), and the spleen cells from not only BALB/c mice but also LPS‐insensitive C3H/HeJ mice were activated, indicating that the activities of the fraction were not ascribed to LPS contaminated possibly into the fraction, but to DNA itself. Intralesional injection with the bacterial DNA fraction caused regression of mouse IMC tumors, but the injection with the vertebrate DNA fraction did not. These findings prompted us to examine the biological activities of DNA samples from a variety of animals and plants, which were provided from other laboratories or purchased from manufacturers. All of the DNA samples from cells of 5 kinds of bacterium, 2 of virus and 4 of invertebrate augmented NK activity and induced IFN, more or less, in mouse spleen calls, while the DNA from 10 kinds of vertebrate, including 3 of fish and 5 of mammal, showed no such activities. The DNA from 2 species of plants, were also inactive. Possible mechanisms to explain the different biological activities of DNA from different cell sources were discussed based on our previous finding that the particular palindromic sequences with a G‐C motif (s) are required for induction of IFNs and activation of NK cells with synthetic 30‐mer oligonucleotides.

Synthetic Oligonucleotides with Particular Base Sequences from the cDNA Encoding Proteins of Mycobacterium bovis BCG Induce Interferons and Activate Natural Killer Cells

Thirteen kinds of 45‐mer single‐stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30‐mer and 5 kinds of 15‐mer oligonucleotide fragments of an active 45‐mer nucleotide were tested for their activity. One of the 30‐mer oligonucleotides, designated BCG‐A4a, was active, but the other 30‐mer was inactive. All of the 15‐mer oligonucleotide fragments were inactive. The BCG‐A4a also stimulated the spleen cells to produce interferon (IFN)‐α and‐γ An experiment using anti‐IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN‐α produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45‐mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30‐mer oligonucleotides, like BCG‐A4a, to induce IFNs.

Lipofection of Synthetic Oligodeoxyribonucleotide Having a Palindromic Sequence of AACGTT to Murine Splenocytes Enhances Interferon Production and Natural Killer Activity

A synthetic 22‐mer oligodeoxyribonucleotide having an AACGTT palindrome, AAC‐22, induced interferon (IFN) production and augmented the natural killer (NK) activity in murine splenocytes, whereas its analogue, ACC‐22, having an ACCGGT palindrome, did not. The binding of AAC‐22 to splenocytes was not different from that of ACC‐22. Lipofection of AAC‐22 to splenocytes remarkably enhanced IFN production and NK cell activity, whereas that of ACC‐22 caused little enhancement. These results strongly suggest that the prerequisite for IFN production is not the binding of AAC‐22 to the cell surface receptors, but its penetration into the spleen cells.

Protective Immunity Against Bovine Leukaemia Virus (BLV) Induced In Carrier Sheep By Inoculation With A Vaccinia Virus-BLV ENV Recombinant: Association With Cell-Mediated Immunity

The effects of vaccination of sheep with a recombinant vaccinia virus (rVV) expressing the bovine leukaemia virus (BLV) envelope glycoprotein (gp60) were studied by determining BLV titres in peripheral blood leukocytes after vaccination and challenge. The proliferation of BLV was suppressed markedly, not only when rVV was inoculated prior to challenge with BLV, but also when it was inoculated after challenge. These results indicate that vaccination with rVV induces protective immunity that can suppress the growth of BLV in carrier animals. Since rVV induced a strong anti-BLV delayed-type hypersensitivity response without producing detectable levels of binding or neutralizing antibodies, and there was no apparent correlation between the humoral immune response and BLV proliferation, a cell-mediated immune response was assumed to play a major role in protective immunity.

Effect of Mycobacterium bovis BCG Vaccination on Mycobacterium-Specific Cellular Proliferation and Tumor Necrosis Factor Alpha Production from Distinct Guinea Pig Leukocyte Populations

ABSTRACT In this study, we focused on three leukocyte-rich guinea pig cell populations, bronchoalveolar lavage (BAL) cells, resident peritoneal cells (PC), and splenocytes (SPC). BAL cells, SPC, and PC were stimulated either with live attenuated Mycobacterium tuberculosis H37Ra or with live or heat-killed virulent M. tuberculosis H37Rv (multiplicity of infection of 1:100). Each cell population was determined to proliferate in response to heat-killed virulent H37Rv, whereas no measurable proliferative response could be detected upon stimulation with live mycobacteria. Additionally, this proliferative capacity (in SPC and PC populations) was significantly enhanced upon prior vaccination with Mycobacterium bovis BCG. Accordingly, in a parallel set of experiments we found a strong positive correlation between production of antigen-specific bioactive tumor necrosis factor alpha (TNF-α) and prior vaccination with BCG. A nonspecific stimulus, lipopolysaccharide, failed to induce this effect on BAL cells, SPC, and PC. These results showed that production of bioactive TNF-α from mycobacterium-stimulated guinea pig cell cultures positively correlates with the vaccination status of the host and with the virulence of the mycobacterial strain.

Chemical properties and immunobiological activities of streptococcal lipoteichoic acids

Lipoteichoic acids (LTAs) were chromatographically purified from crude phenol-water extract of whole cells of some streptococcal species, which included Streptococcus pyogenes Sv, Streptococcus mutans 6715, and Streptococcus sanguis ATCC 10556. Among these, special attention was paid to S. pyogenes LTA for analyses of chemical composition and biological activities. All LTA preparations contained equimolar amounts of glycerol and phosphorus. Chemical analyses showed that S. pyogenes LTA contained glycerophosphate, alanine, glucose, and fatty acids (as palmitic acid) at molar ratio of 1 : 0.1 : 0.1 : 0.25. The crude phenol-water extract and isolated LTA from S. pyogenes Sv were found to be mitogenic for spleen cells of BALB/c and BALB/c (nu/nu) mice, but not for thymus cells of BALB/c mice. The mitogenicity of deacylated LTA (dLTA) was significantly lower than that of LTA. It was also found that various LTA preparations possessed polyclonal B cell activation ability and adjuvant activity both in vivo and in vitro, as demonstrated by using hemolytic plaque assay. LTA, but not dLTA, induced macrophage activation which resulted in tumor cytotoxicity in mice. Limulus lysate activity of S. pyogenes LTA was approximately 1,000 fold lower than that of Escherichia coli lipopolysaccharide. These results indicate that streptococcal LTA possesses various immunobiological activities that modulate lymphoreticular system in vivo and in vitro.

Characterization of a Monoclonal Antibody Specific for Lipoteichoic Acid from Various Gram-Positive Bacteria

A hybrid cell line, 3G6, producing monoclonal antibody (mAb) against the polyglycerophosphate (PGP) backbone of lipoteichoic acids has been derived by the polyethylene glycol‐induced fusion of mouse myeloma cells and spleen cells from mice immunized with partially purified glucosyltransferase from culture supernatant of Streptococcus mutans strain 6715. Immunodiffusion tests and ELISA revealed that the antibody reacted with purified PGP from group A Streptococcus pyogenes strain Sv as well as crude phenol‐water and saline extracts of various gram‐positive bacteria except for a few species such as biotype B S. sanguis, Micrococcus sp., and Actinomyces viscosus. Whole cells of serotype b S. mutans and Staphylococcus epidermidis were agglutinated upon addition of 3G6 mAb, while those of most other species were not significantly affected by this procedure. A hapten inhibition study showed that glycerophosphate was only a potent inhibitor of passive hemagglutination reactions between LTA coated sheep erythrocytes and 3G6 mAb.

A New Assay System for Guinea Pig Interferon Biological Activity

We have developed an assay system for guinea pig interferon (IFN) based on reduction of viral cytopathic effect (CPE) in various cell lines. CPE inhibition was detected optimally in the guinea pig fibroblast cell line 104C1 infected with encephalomyocarditis virus (EMCV). The amount of biologically active guinea pig IFN was quantified by estimating viable cell numbers colorimetrically by means of a tetrazolium compound, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-1) and 1-methoxy-5-methylphenazinium methylsulfate (PMS). WST-1 color developed until stopped by the addition of sulfuric acid. This had no effect on the colorimetric assay, and the color was stable for at least 24 h. The acid also inactivated the EMCV and, thus, eliminated the viral hazard. Inhibition of CPE activity was highly correlated with the concentration of culture supernatants from BCG-vaccinated guinea pig splenocytes stimulated in vitro with tuberculin or an immunostimulatory oligoDNA. This assay detected guinea pig IFN and human IFN-alpha, but not IFN-gamma from human, mouse, rat, pig, or dog. This assay system has proved useful for the titration of guinea pig IFN, being easy to perform, free from viral hazard, relatively species specific, highly reproducible, and inexpensive.

Immunological studies of a 21 kDa cellular component efficiently incorporated into rabies virion grown in a BHK-21 cell culture

To investigate cellular components incorporated into the rabies virion, monoclonal antibodies (MAbs) were screened based on their reactivity with additional virion components. Two of the MAbs we prepared recognized a virion‐associated 21 kDa polypeptide (referred to as VAP21) from a BHK‐21 cell. Since the MAbs precipitated the rabies virion and trypsin digestion eliminated the VAP21 antigen from the virion but alkaline treatment (pH 11) did not, VAP21 seems to be anchored into the viral envelope and exposed on the virion surface. Although quantitative immunoblot analyses indicated an apparently increased concentration of VAP21 in the virion, the ratio of the content of VAP21 to that of viral glycoprotein (G) was several times decreased as compared to the ratio of those in the cell. These data suggest that sorting of VAP21 occurs during the viral budding process on the cell but that it might be inefficient, probably due to a more intimate association of VAP21 with the viral envelope proteins. This assumption seems to be consistent with the results of immunofluorescence studies; that is, VAP21 displayed colocalized distribution with viral envelope antigens in the cell. From these results, it is suggested that VAP21 closely associates with the viral envelope proteins in the cell, and this association might cause passive but relatively efficient incorporation of VAP21 into the virion.

Chemical and immunological characterization of a novel amphipathic antigen from biotype B Streptococcus sanguis.

A new type of amphipathic antigen was extracted from whole cells of Streptococcus sanguis ATCC 10557 (biotype B, serotype II) by the phenol/water method. The extract was treated with nuclease P1, and was applied to a column of Sepharose 6B. Each fraction was checked by passive haemagglutination (PHA) and immunodiffusion tests against anti-10557 serum which was obtained by immunizing rabbits with whole cells of strain ATCC 10557. Strong PHA activity was demonstrated in the first hexose-containing peak (peak 1) eluted near the void volume, while the second hexose-containing peak (peak 2) produced a heavy band against anti-10557 serum in an immunodiffusion test. The third peak (peak 3) which partially overlapped with peak 2 reacted with concanavalin A, but not with the antiserum, in agar gel. Peaks 2 and 3 had no PHA activity. Peak 1 contained only 1% phosphorus, indicating that cells of strain ATCC 10557 possess an amphipathic antigen which differs from the lipoteichoic acids that are common in many Gram-positive bacteria. Peak 1 was a fatty acid-substituted heteropolysaccharide composed of glucose, galactose, mannose, glycerol and fatty acids in a molar ratio of approximately 1.0:1.3:2.7:0.3:1.0. PHA activity was inhibited in the presence of polymerized mannose. Peak 2 was composed of glucose, galactose, rhamnose and N-acetylgalactosamine in a molar ratio of approximately 1.0:1.4:0.8:0.8, which was essentially identical to the serotype II carbohydrate antigen reported previously.