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 IFNalpha/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.We have demonstrated that a DNA-rich fraction, extracted fromMycobacterium bovis BCG and designated MY-1 (22), exhibited strong antitumor activities against 98 3

Specific palindromic sequences in synthetic oligonucleotides are required to induce IFN and augment IFN-mediated natural killer activity. To study the mechanism of IFN induction by oligonucleotides containing palindromic sequences, we investigated the possible target molecules of the oligonucleotides. Oligo-1, a 30mer single-stranded oligonucleotide with oligoG sequences next to the active palindromic sequence (AACGTT), had more activity than oligonucleotides with oligoA, oligoC, or oligoT sequences. The activity of oligo-1 was inhibited by a guanine homo-oligomer (G30), dextran sulfate, and polyvinyl sulfate. Oligo-1 bound to plastic-adherent mouse splenocytes, and the binding was inhibited by G30, dextran sulfate, and polyvinyl sulfate. Oligo-1 inhibited acetyl-LDL binding to the scavenger receptor on mouse splenocytes. These findings suggest that the binding of an extrapalindromic sequence to the scavenger receptor is required for the immunostimulatory activity of oligo-1.

A nucleic acid‐rich fraction extracted and purified from BCG (MY‐1) augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factor(s) which showed anti‐viral activity and rendered normal macrophages cytotoxic towards tumor cells. These cellular responses were induced by the MY‐1 digested preliminarily with RNase, but not by the MY‐1 digested with DNase, indicating that DNA contained in MY‐1 was essential for the responses. The function of the factor to activate macrophages was destroyed by treatment with a small amount of anti‐interferon (IFN)‐γ antiserum or under acidic conditions (pH 2), but not by treatment with anti‐IFN‐α/β antiserum, while the anti‐viral activity was destroyed almost completely by treatment with anti‐IFN‐α/β antiserum. It appears that DNA from BCG stimulated mouse spleen cells in vitro, resulting in augmentation of NK activity and production of IFN‐α/β and ‐γ.

Thirteen kinds of 45

Based on the previous finding that certain 30‐mer single‐stranded oligodeoxyribonucleotides (oligonucleotides) having particular 6‐mer palindromic sequences could induce interferon‐alpha and ‐gamma, and enhance natural killer activity, the present study was carried out to clarify the entire relationship between the activity and the sequence of 30‐mer oligonucleotides. The results indicated that the activity depended critically on the presence of particular palindromic sequences including the 5 ‐CG‐3 motif(s). The size and the number of palindromes as well as the extra‐palindromic sequences also influenced the activity. An oligonucleotide with a 10‐mer palindrome and extra‐palindromic oligoguanylate sequences showed the strongest activity among the oligonucleotides tested.