The procoagulant response of cytomegalovirus infected endothelial cells.

The report describes the effect of an in vitro infection of human umbilical vein endothelial cells with human Cytomegalovirus (CMV). The parameters studied are cellular procoagulant activity, secretion of plasminogen activator inhibitor (PAI-1) and urokinase-type plasminogen activator (u-PA), activation and internalization of factor X and Merocyanine 540 staining. The infection does not result in an increase in PAI-1 and u-PA secretion, but it brings about a procoagulant response, which is relatively rapid compared to the tissue factor mediated response induced by inflammatory mediators. The time course and the coagulation factor dependency suggest a facilitated interaction of coagulation factors on the surface of infected cells. Chromogenic activity measurements after the addition of purified factor X and electron microscopic examination of the cells after addition of colloidal gold-factor X conjugates both point to an internalization of factor X and/or Xa after interaction with the endothelial cell surface. Merocyanine 540 staining suggests that CMV infection leads to membrane perturbations.

Influence of a cytomegalovirus infection on functions and arachidonic acid metabolism of rat peritoneal macrophages.

An intraperitoneal (rat) cytomegalovirus (RCMV) infection in the rat caused an influx of mononuclear cells, which have been altered in functions and arachidonic acid (AA) metabolism. Phagocytosis has been increased considerably 3 days postinfection (p.i.), whereas the release of prostacyclin, thromboxane A2, 12-hydroxyheptadecatrienoic acid (HHT), 5-hydroxyeicosatetraenoic acid (5-HETE), and leukotriene B4 (LTB4) was inhibited for more than 80%. The release of superoxide anions and the chemiluminescence response (CL) upon opsonized zymosan stimulation did not differ from those observed in resident peritoneal macrophages. Additionally, the levels of cyclic nucleotides (cAMP and cGMP) were low in both resident and influx macrophages (day 3 p.i.). In contrast, peritoneal macrophages harvested on day 10 p.i. still showed a high level of phagocytosis. However, the intracellular level of cyclic AMP had decreased fivefold, whereas CL response and superoxide anion release were inhibited significantly. Moreover, the production of prostacyclin, LTB4, and 5-HETE was still suppressed in contrast to thromboxane synthesis, which has selectively been restored in these macrophages. A direct regulatory role of AA metabolites in changes in macrophage functions that were due to a RCMV infection could not be demonstrated.

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.

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.

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.