Antibacterial resistance, macrophage influx, and activation induced by bacterial rRNA with dimethyldioctadecylammonium bromide.

Intraperitoneally injected rRNA from Pseudomonas aeruginosa combined with dimethyldioctadecylammonium bromide (DDA) increased nonspecifically the resistance of mice against an intraperitoneal challenge with extracellular (P. aeruginosa, Escherichia coli) and intracellular (Listeria monocytogenes) bacteria. This study concerns the mechanism underlying the nonspecific resistance. RNA with DDA (RNA-DDA) induced a cell influx and activated peritoneal macrophages (M phi) as judged by the decreased 5'-nucleotidase and alkaline phosphodiesterase activities in M phi lysates, the enhanced O2- release, and the increased antitumor activity in comparison with unstimulated M phi. RNA without DDA did not enhance the resistance and did not influence the peritoneal cell numbers or M phi properties. DDA without RNA enhanced the resistance of mice only slightly; it induced a cell influx, yielding elicited M phi as judged by the decreased 5'-nucleotidase activity and increased alkaline phosphodiesterase activity, the slightly enhanced O2- release, and the absence of increased antitumor activity. Both RNA-DDA and DDA M phi showed an enhanced capacity to ingest and kill L. monocytogenes in vitro, DDA M phi being slightly less effective than RNA-DDA M phi with respect to killing. We conclude that the enhanced killing capacity of M phi for L. monocytogenes is characteristic of both elicited DDA M phi and activated RNA-DDA M phi. The relationship between nonspecific resistance, peritoneal cell numbers, and antibacterial M phi activity is discussed. In addition, it is shown that RNA and DDA retain their activity when they are injected apart, suggesting that they activate M phi by sequential action.

RNase-sensitive and RNase-insensitive protective components isolated from Listeria monocytogenes.

Crude ribosomes were isolated from Listeria monocytogenes serotype 4b and separated into two fractions by molecular sieve chromatography. Chemical analysis indicated that fraction I contained cell envelope components while fraction II contained the ribosomes. Both fractions protected mice against Listeria, but only in combination with the adjuvant dimethyldioctadecylammonium bromide (DDA). RNase-treatment, but not proteinase K-treatment destroyed the protective properties of fraction II, and RNA purified from fraction II also induced protection. Protection induced by fraction I was not affected by either RNase- or proteinase K-treatment. Both subcutaneous and intraperitoneal, but not intravenous administration of fraction I, fraction II, or purified RNA induced significant protection against intraperitoneal infection, the intraperitoneal route of administration being the most effective. All preparations induced high levels of protection 3 to 7 days after administration, but protection was already decreased after 14 days. Protection induced with RNA appeared to be biphasic, because it also protected mice 1 day, but not 2 days after administration. Protection induced with both fraction I and RNA was at least in part non-specific, because both preparations also protected mice against L. monocytogenes serotype 3, Streptococcus pneumoniae and Pseudomonas aeruginosa. Results are discussed in relation to previous work with analogous preparations from P. aeruginosa.

Ribonuclease-sensitive ribosomal vaccines.

This paper presents an analysis of the protective properties of the components in ribonuclease (RNase)-sensitive ribosomal vaccines, in particular the ribonucleic acid (RNA). The protective activities in mice of purified ribosomes derived from Pseudomonas aeruginosa and from Listeria monocytogenes were compared. Both ribosomal vaccines had to be combined with the adjuvant dimethyldioctadecylammonium bromide (DDA) in order to be protective, and both lost their activity after RNase treatment. The ribosomal vaccines as well as RNA purified from the ribosomes induced non-specific protection. Intraperitoneal injection of RNA with DDA induced an influx of peritoneal cells. Furthermore, RNA with DDA activated macrophages as shown by, a.o., enhanced phagocytic activity and killing capacity for L. monocytogenes. The results suggest that the observed macrophage activation is probably T-cell-independent. With regard to the ribosomal vaccine of P. aeruginosa it is concluded that RNA also contributed to the protective activity by increasing the humoral response against suboptimal concentrations of contaminating cell surface antigens. In conclusion, it is proposed that ribosomal vaccines may be considered as a combination of a non-specific immunomodulator (RNA) with pathogen-specific cell surface antigens. This concept of ribosomal vaccines is discussed in relation to the literature concerning RNase-sensitive ribosomal vaccines.

Protective activities of ribosomal ribonucleic acid and lipopolysaccharide of Pseudomonas aeruginosa: a comparative study.

Ribosomal ribonucleic acid (RNA) and lipopolysaccharide (LPS) from P. aeruginosa were compared with respect to their protective activities in mice against an infection with P. aeruginosa. This study is concentrated on the protective activity of RNA. RNA isolated from purified ribosomes did not contain LPS as determined with the Limulus test. Injection of RNA with the adjuvant dimethyldioctadecylammonium bromide (DDA) protected mice against P. aeruginosa without inducing LPS-specific antibodies. C3H/HeJ mice which are relatively insensitive to the protective activity of LPS could be protected with RNA. The protective activities of RNA and LPS from a mutant strain of P. aeruginosa, PAC 605, containing defective lipopolysaccharide, were compared with the protective activities of RNA and LPS from the parent strain, PAC IR. The protective activity of LPS from PAC 605 was 1000 fold lower than the protective activity of LPS from PAC IR. RNA preparations of both strains induced similar percentages of survival. The protective activity of ribosomal RNA from P. aeruginosa was nonspecific since mice were also protected against a heterologous serotype of P. aeruginosa and against Escherichia coli. RNA from ribosomes of P. aeruginosa, E. coli and the non-lipopolysaccharide containing Saccharomyces cerevisiae had similar protective activities. No protection was obtained with the ribonucleic acid from the E. coli phage MS2. It is concluded that ribosomal RNA has protective activities distinct from those of LPS.

Serotype-nonspecific protection induced by ribonucleic acid isolated from the ribosomal vaccine of Pseudomonas aeruginosa.

A ribosomal vaccine of Pseudomonas aeruginosa and a vaccine containing purified lipopolysaccharide (LPS) were compared with respect to their capacity to protect mice against a lethal challenge with P. aeruginosa. The route of vaccination appeared to be important for the protective activity of the ribosomal vaccine. Optimal protection was measured if both the immunizing and the challenge injection were given intraperitoneally. The ribosomal vaccine protected mice as early as 1 day after vaccination, and the protection lasted at least 6 days. LPS-specific antibodies were detectable 6 but not 2 days after vaccination. The ribosomal vaccine protected mice also against a heterologous serotype of Pseudomonas. Injection of purified LPS did not protect mice earlier than at day 3, and the protection induced by LPS was serotype specific. Ribonucleic acid (RNA) isolated from the ribosomal vaccine had the same protective properties as the ribosomes. RNA induced serotype-nonspecific protection as quickly as 1 day after injection, and the protection lasted at least 6 days. However, the capacity to induce antibodies to LPS was lost or reduced. It is concluded that the serotype-nonspecific protection induced by RNA and the serotype-specific protection induced by LPS are due to different mechanisms. Experiments with combined vaccines containing RNA and LPS demonstrated that the addition of RNA to LPS resulted in a slight increase in LPS-specific antibodies. The data presented indicate that both the serotype-specific protection induced by LPS and the serotype-nonspecific protection induced by RNA contribute to the protective activity of the ribosomal vaccine.

Evidence for the presence of lipopolysaccharide in a ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa.

To obtain information about the nature of the immunogens in the ribosomal vaccine (fraction II) of Pseudomonas aeruginosa, we studied the specificity of rabbit antibodies to fraction II. Crossed immunoelectrophoresis demonstrated the presence of antibodies which precipitated with ribosomal antigens, but not with lipopolysaccharide (LPS). By means of an enzyme-linked immunosorbent assay, antibodies to LPS were detected in antibodies to fraction II. Antibodies to fraction II could protect mice against a lethal challenge with P. aeruginosa. Absorption experiments demonstrated that the protective ability of antibodies to fraction II was due to antibodies to cell envelope antigens, whereas antibodies to ribosomal antigens did not contribute to the protection. Antibodies to LPS could be detected in mice 1 week after a single vaccination with fraction II. It was concluded that the protective activity of fraction II was due, at least in part, to the presence of LPS in the ribosomal vaccine. Treatment of fraction II with ribonuclease decreased the protective activity of the ribosomal vaccine. Addition of synthetic polyadenylic acid-polyuridylic acid restored the protective activity of ribonuclease-treated fraction II, indicating that RNA in the ribosomal vaccine might act as an adjuvant or a carrier in the presentation of the of the contaminating cell envelope antigens. The protective activity and the toxicity of fraction II were compared with the protective activity and the toxicity of fraction I, which contained cell envelope components, including LPS, and of purified LPS. The results indicated that protection by the ribosomal vaccine was associated with a slightly higher toxicity than was protection by fraction I, whereas purified LPS was the most toxic vaccine.

Ribonuclease-sensitive ribosomal vaccine of Pseudomonas aeruginosa.

In mice, active protection against Pseudomonas aeruginosa could be induced with two fractions derived from a crude preparation of ribosomes from P. aeruginosa. The two fractions were obtained by gel filtration chromatography of the crude ribosomal preparation on Sepharose CL-2B. In fraction I, less than 1% of the ribonucleic acid (RNA) applied to the column was recovered. Fraction II contained RNA and protein in a ratio of 1.94. The presence of ribosomes in this fraction was confirmed by analysis on a sucrose density gradient. The protection by fraction I was not affected by treatment with ribonuclease; in contrast, incubation of fraction II with ribonuclease completely abolished active protection. Fraction I contained lipopolysaccharide (LPS) as was indicated by the presence of 2-keto-3-deoxyoctonic acid. No LPS was found in fraction II. The adjuvant dimethyl dioctadecyl ammonium bromide enhanced the protection by fraction II; however, immunity by a low dose of fraction I was abolished by dimethyl dioctadecyl ammonium bromide. Protection by fractions I and II appeared to be restricted to the homologous serotype of P. aeruginosa. These results indicate that RNA is required for protection by fraction II. Active protection by fraction I is likely due to LPS.