Tetsuro Kataoka

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.

A SYNTHETIC ADJUVANT EFFECTIVE IN INDUCING ANTITUMOR IMMUNITY

Guinea pigs which were injected repeatedly with a mixture of a 3MKC1 extract of line 10 tumor and 6‐O‐(2‐tetradecyl‐hexadecanoyl)‐muramyl‐dipeptide (B30‐MDP) rejected a tumor graft of line 10. Such an adjuvant effect of B30‐MDP was also demonstrated when X‐ray‐treated line 10 cells were mixed with B30‐MDP dissolved in phosphatebuffered saline and inoculated into the animals.

Restriction by the Major Histocompatibility Complex of Antigen‐Induced T Lymphocyte Proliferation in New Inbred Guinea Pig Strains

Employing new inbred guinea pig strains, JY 1, JY 2 and JY 3, established in this Institute in addition to strains 2 and 13, the authors investigated histocompatibility restriction in macrophage‐T lymphocyte interaction. These five strains are known to possess distinct major histocompatibility complex (MHC) gene profiles (1, 2). This fact was supported by our results concerning the mixed leukocyte reaction (MLR) and cytotoxicity test with alloantisera. Using various combinations of T lymphocytes and peritoneal exudated cells (PECs) from these strains, in vitro proliferative responses of T lymphocytes from BCC‐immune animals to PPD‐pulsed normal PEC were tested. Successful activation of T cell response was observed not only in syngeneic combinations but also in allogeneic combinations among strains JY 1, JY 3 and strain 13 which share common Ia antigens detected by strain 2 anti‐strain 13 alloantiserum. Because JY 1 and JY 3 seem to share a common B antigen differing from strain 13, it was suggested that identification in the I region of MHC is sufficient for effective antigen‐presentation by the macrophage. Although a part of Ia is shared, no T lymphocyte activation was observed in the combination between JY 2 and JY 1 or JY 3, whereas strong MLR occurred in these allogeneic combinations. At the present stage of the study, it can be said that disparity in the part(s) of Ia antigens which is responsible for strong MLR cannot lead to effective T cell‐macrophage interaction. These results support the concept that functional activation of primed, proliferating T lymphocyte requires the participation of gene products of macrophages coded for by the I region in MHC. By employing JY 1, JY 2 and strain 2, which appear to possess distinct B and Ia antigens, it was shown that the T lymphocyte and macrophage interactions essential for mitogen‐induced T lymphocyte proliferation are not restricted by histocompatibility.

Effect of a synthetic adjuvant for inducing anti-tumour immunity

An acylated derivative of muramyl dipeptide (MDP), 6-O-(2-tetradecyl-hexadecanoyl)-muramyl-dipeptide (B30-MDP) is a strong adjuvant effective in inducing cell-mediated immunity. We used B30-MDP as an adjuvant for induction of anti-tumour immunity. Guinea-pigs which were injected repeatedly with a mixture of X-ray-treated leukaemic cells and B30-MDP dissolved in phosphate buffered saline resisted a challenge of leukaemia cells and showed no sign of leukocytosis. The immunity induced was tumour-specific and retained for more than 100 days. These results suggest that B30-MDP is useful as a simple but potent immunotherapeutic tool.

Fluorometric Cell Detection Method Using Complement-Mediated Cytolytic Reaction and Imaging Sensor System

Abstract A rapid and sensitive cell detection method using complement-mediated cytolytic reaction and an image processing system was developed. The present system consists of SIT video camera, fluorescent microscope and personal computer system equipped with image memory board. Target cell cytolysis caused by 3C4 monoclonal antibody and complement was determined using DNA (or RNA) specific fluorescent dye, propidium iodide. Target cells, guinea pig hepatocarcinoma cells, could be detected quantitatively when its content was above 2%. Measurement time was approximately 5 minutes.

Establishment of Macrophage-Like Cell Lines of the Guinea Pig

Several macrophage-like cell lines have been established from mice and applied to studies on macrophages from various standpoints such as functional and biochemical research and cell differentiation (1,4,9, 12-15). However, no report on such a cell line from guinea pig has been published. In this paper, we describe three macrophage-like cell lines established from the guinea pig for the first time. Peritoneal cells and bone marrow cells from strain 13 guinea pigs were suspended in RPMI 1640 medium (Flow Lab. Inc., Md., U.S.A.) supplemented with 10% heat-inactivated fetal bovine serum (Flow Lab. Inc.), penicillin G (100 units/ml) and streptomycin (100,ug/ml), and seeded at a concentration of about 4 X 106 cells per plastic dish (Falcon 3002; Becton, Dickinson Co., Oxnard, Calif., U.S.A.). One hour later, the medium was discarded and unattached cells were removed by vigorous pipetting with the same medium without serum. The adherent cells were then infected with SV40 (small plaque type) (11) or BK virus (BKV; Gardner strain) (6) at a multiplicity of 10 or 100 plaque-forming units per cell. After adsorption for 3 hr, the cultures were fed with growth medium and incubated in a CO2 incubator at 37 C. The medium was changed once a week until the transformed foci emerged. Two transformants, called KI and K3, were obtained from peritoneal cells infected with SV40, and one transformant, called K2, was obtained from bone marrow cells infected with BKV. KI and K2 transformants emerged 3 and 7 months after the infection, respectively, and K3 appeared about one month after the infection. All three cell lines have been maintained in cultures for more than 20 passages. The average doubling time of KI and K2 was roughly 48 hr and that of K3 was about 24 hr. Phase contrast microscopic photographs of the transformed cells and those of ingested latex beads are shown in Fig. 1. These cell lines were tested to determine whether or not they express virusinduced tumor antigen (T antigen) and histocompatibility antigens of the guinea pig, possess Fcand C3-receptors, produce lysozyme, and are phagocytotic. As control cells, normal peritoneal macrophages from strain 13 guinea pig and Line 10 hepatoma cells of strain 2 guinea pig (17) were employed. T antigens of SV40 and BKV were assayed by the immunofluorescent antibody test using antiserum

Induction of calcium influx in immature T lymphocytes by anti-FT-1 monoclonal antibody.

The functional role of FT-1 antigen was investigated by measuring changes in intracellular [Ca2+]i after the addition of anti-FT-1 monoclonal antibody to murine T leukemic cells and embryonic thymocytes. Anti-FT-1 induced a rapid and sustained increase in cytosolic free [Ca2+]i and this effect was completely blocked by EGTA and La3+. These results suggest that FT-1 molecule is functionally associated with a plasma membrane calcium ion channel in embryonic thymocytes.

Tumor cell detection method using complement-mediated cytolytic reaction and imaging sensor system

A novel tumor-detection system consisting of complementmediated cytolytic reaction and an image processing system was developed for the simple and rapid determination of tumor cells.The present system consists of a CCD image sensor, image memory board, personal computer, and microscope.When monoclonal antibody 3C4, which is specific to the guinea pig hepatoma L-10, was added to cell suspension, only L-10 cytolysis occurred. Cytolysis caused a decrease in brightness of the cells observed by phase-contrast microscopy. The phase contrast image of the cells before cytolysis was converted to a digitalized signal and stored in computer memory. After cytolysis, a brightness threshold above that of lysed cells was subtracted from the digitalized signal and compared to the signal stored before reaction.L-10 cells in mixed cell suspension were determined specifically by the system. Measurement time was only 2 sec and overall time, including reaction time, was approximately 30 min. Since this method does not require a cell washing process, automation of the whole system is possible.