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Interchangeable combinations of Fusobacterium nucleatum Fev1 lipopolysaccharide (LPS) with its split products by acetic acid hydrolysis, i.e. lipid A (LA) and degraded polysaccharide (PS), amplified the blastogenic response in murine spleen cell cultures as measured by [3H]thymidine uptake. Athymic murine spleen cells precultured with LPS-Fev1 for 48 h (stage 1), washed twice and cultured together with fresh cells and either LA or PS for 72 h (stage 2) gave a synergistic response over that found in spleen cell cultures of thymic mice. Spleen cells pre-cultured with LA or PS and with fresh cells and LPS-Fev1 added to stage 2 cultures gave less significant amplification compared with precultures of LPS and either LA or PS together with fresh cells added to stage 2. Precultures with LA, PS or LPS-Fev1 and with pokeweed mitogen (PWM) and fresh cells added produced an additional increment of synergy which was most pronounced in spleen cell cultures of normal mice.

Puriified lipopolysaccharides (LPS) from strains of Bacteroides, Fusobacterium and Veillonella incubated with guinea pig serum, were tested for chemotatic activity against rabbit polymorphonuclear leukocytes (PMNs) in modified Boyden chambers. Comparisons were made to a Salmonella LPS (S. enteriditis S-795). Submicrogram amounts of LPS induced positive chemotaxis, and a typical dose-response relationship up to certain dose levels was observed. The difference in chemotactic activity between the Veillonella LPS and LPS-S-795 was not statistically significant. The Fusobacterium LPS showed either a non-significant or a highly significantly lower chemotatic capacity than LPS-S-795. The Bacteroides LPS were also clearly chemotactic, but considerable less when compared to the Salmonella LPS. When serum was not added, the LPS preparation showed no chemotactic activity.

Lipopolysaccharides (LPS) extracted from strains of Bacteroides fragilis, Bacteroides melaninogenicus and Bacteroides oralis with phenol-water, trichloroacetic acid, EDTA or liquid phenol-chloroform-petroleum ether (PCP) and isolated by ultracentrifugation, varied considerably in their quantitative chemical composition. Negligible yields of LPS were obtained by PCP-extraction. All preparations were more or less serologically active. All methods (except PCP) extracted the same O-antigenic determinants from B. fragilis. Endotoxic activity, as measured by primary skin inflammations in rabbits, was low but was present in all preparations. Proteins (and/or lipoproteins) co-precipitated with LPS in the ultracentrifuge.

The clearance of bacterial lipopolysaccharide (LPS) from Bacteroides fragilis was studied, using wound chambers implanted subcutaneously in rabbits. The primary skin inflammatory reaction, the Limulus amoebocyte lysate test and the haemagglutination inhibition test all demonstrated a more rapid elimination of LPS from the wound chambers after the second injection, compared to the elimination rate after the first injection given three days earlier. The clearance rate of LPS was significantly higher (0.003 greater than or equal to p greater than or equal to 0.0006) and the number of accumulated leukocytes in the inflammatory exudate significantly lower (p less than or equal to 0.05) after the second injection. Antibodies to B. fragilis LPS in the exudate before the second endotoxin injection was of the 19S IgM class. This suggests that phagocytes in the granulation tissue lining the chamber walls may be of importance in the elimination of endotoxin.

By chromatographic separation on Sephadex gels a peptide, termed the lipopolysaccharide-induced chemotactic factor (LPS-CF), has been isolated from inflammatory exudate. The exudate was obtained from Teflon chambers implanted subcutaneously in rabbits 3 h after LPS from Bacteroides fragilis ss. fragilis had been injected. Three chemotactic peaks were eluted by fractionation of the exudate on Sephadex G-200 columns; one major peak with molecular weight of approximately 16,000 and two minor peaks with molecular weights of approximately 68,000 and 7,000. Refiltration of the major peak on G-75 showed the same elution profile as that found on G-200 columns. By addition of 8 M urea to the elution fluid only the major and the low molecular weight peaks appeared. The molecular weight of the major chemotactic peak was calculated to 16,000 on Sephadex gels, and also using SDS-polyacrylamide gel electrophoresis and equilibrium centrifugation. The chemotactic factor was quite heat-stable and was also non-dialyzable, and freezing and thawing as well as storage at 4 degrees C for several weeks did not impede its activity. This chemotactic factor is probably identical to the cytotaxic fragment split off from C5 upon interaction with LPS.

The atypical Bacteroides fragilis lipopolysaccharide is chemotactic for polymorphonuclear leukocytes. The chemotactic activity is mediated by complement activated through the alternative pathway. This mediator has been isolated. It is a polypeptide with a molecular weight of 16,000 and is most likely the complement component C5a.

Wound chambers implanted subcutaneously in rabbits proved suitable for measurements of leukocyte chemotaxis. Injection of bacterial lipopolysaccharide (LPS) in the chambers the sixth day after implantation was followed by a marked increase of polymorphonuclear leukocytes in the wound chamber fluid, the number of which was dependent on the time after application of LPS. Up to a certain amount of LPS, the concentration of leukocytes in the chamber fluid was dose-dependent. The histopathological appearance of the granulation tissue lining the chamber wall one day after the injection of LPS from Veillonella revealed aggregation of blood cells plugging the lumina of small vessels and many eosinophilic leukocytes.

Endotoxic lipopolysaccharides (LPS) isolated from Fusobacterium are similar to those of Salmonella with respect to chemical composition, O-antigenic specificity and endotoxic activity. Bacteroides LPS are highly atypical with respect to the chemical composition, and their endotoxic activity is low. O-Antigenic specificity is present. B. fragilis LPS is chemotactic for polymorphonuclear leukocytes in vitro and in vivo. The chemotactic activity is brought about by activation of the alternative complement pathway.

Preparations of the polymorphonuclear leukocyte (PMN) chemotactic factor isolated from lipopolysaccharide (LPS)-induced inflammatory exudate in rabbits were immunogenic in guinea pigs. Complete fusion of the precipitation lines produced against anti-CF by LPS-CF (molecular weigth 16,000) and material eluted on Sephadex G-200 columns with molecular weights (MW) of 68,000, 16,000 and 7,000 was found. Also, the chemotactically active material with MW of 68,000 and 7,000 eluted on G-75 columns after fractionation of the fraction of MW 16,000 from the G-200 eluate was antigenically identical to LPS-CF in double diffusion in agar. Normal rabbit serum (NRS) incubated with LPS, LPS-induced wound chamber exudate and NRS alone gave lines of precipitation against the anti-LPS-CF sera identical to that of LPS-CF. The capacity of LPS-CF to attract PMNs was significantly higher than that of LPS, and a peak in the number of PMNs in the exudate of wound chambers implanted in rabbits was found 4 h after the local injection of LPS-CF. When injected intraperitoneally in C5 deficient mice, LPS-CF stimulated a PMN migration which was only slightly below that in C5 normal mice. Antisera to LPS-CF inhibited the chemotactic activity of LPS-CF as well as that of LPS-NRS when the supernatants were tested using the Boyden's technique. Also, preincubation of PMNs with LPS-CF suppressed the migration towards a chemotactic gradient of LPS-CF molecules of these PMNs.

Chambers implanted subcutaneously in C5 normal (C5 N) and C5 deficient (C5 D) mice were used to examine the migration of polymorphonuclear leukocytes (PMNs) into the wound chamber fluid in response to injected Bacteroides fragilis lipopolysaccharide (LPS). The difference in PMN migration was highly significant between the two mouse strains, the C5 D mice showing no initial, but a low, delayed migration. The results from the study indicated that chemotaxis plays a major role in the accumulation of PMNs in the acute inflammatory response. Intraperitoneal endotoxin stimulation also showed a significantly lower total number of leukocytes in the exudate from C5 D mice as well as a delayed migration of cells compared to C5 N mice. No leukotactic mediators were elaborated in C5 D serum or exudate upon incubation with LPS when tested in a modified Boyden chamber. However, endotoxin-induced wound chamber fluid in C5 D mice showed an increasing leukotactic activity at the same time as the acute inflammatory response subsided in C5 N mice. Incubation of B. fragilis LPS in C4 deficient (C4 D) guinea pig serum indicated that the LPS was able to activate complement components to generated split products chemotacic for rabbit PMNs via the alternative complement pathway.

Subcutaneously implanted chambers in rabbits were used for testing the migration of polymorphonuclear leukocytes in response to injected LPS isolated from strains of Bacteroides, Fusobacterium and Veillonella. A salmonella LPS was used as reference endotoxin. No differnece in chemotactic activity between the Veillonella LPS and LPS from Salmoneila was found. Fusobacterium LPS whoed insignificantly lower chemotactic capacity than the Salmonella LPS. The Bacteroides LPS were all significantly less chemotactic than the reference endotoxin. An insignificant correlation between the amount of exudate aspirated from the chambers 5 h after injection of the different LPS preparations and the number of leukocytes per microliter of exudate was found.

Lipopolysaccharides (LPS) isolated from oral strains of Veillonella, Fusobacterium and Bacteroides stimulated the release of 45Ca from prelabeled fetal rat bones in culture. There was a typical dose-response relationship between the quantities of released 45Ca and LPS used for stimulation. Bacteroides-LPS proved to be the less active inducer of 45Ca release. LPS had no stimulating effect on the release of 45Ca from devitalized bone. The stimulated 45Ca release was paralleled by an increase in the culture medium of hydroxyproline and lactate. This, together with the findings of numerous osteoclasts in stained histological specimens of the experimental bones, indicates that LPS stimulated the osteoclasts to bone resorption. Heparin, which did not directly induce 45Ca release, potentiated the bone resorption stimulating capability of LPS. The lipid A and the polysaccharide portion of Fusobacterium LPS also stimulated bone resorption and, remarkably, the polysaccharide portion showed the greatest activity. This may explain the mode of action of LPS lacking a typical lipid A. It is suggested that stimulation of osteoclasts by LPS may result from activation of complement components by lipid A or its polysaccharide portion.

Phenol-water extracted lipopolysaccharides (LPS) from Veillonella, Fusobacterium nucleatum, Bacteroides fragilis and Bacteroides melaninogenicus were lethal for mice and 11-days-old chick embryos, pyrogenic in rabbits, and gelated Limulus amoebocyte lysate. Mouse lethality was considerably enhanced by actinomycin-D. In all test systems the endotoxin activity of Veillonella and Fusocbacterium LPS was comparable to that of LPS from Salmonella enteritidis, which was included as a reference endotoxin. The endotoxicity of the Bacteroides LPS was very low. While nanograms of the Veillonella and Fusobacterium LPS killed the chick embryos and gelated the Limulus lysates, microgram amounts of the Bacteroides LPS were needed to give positive reaction in the same test systems. As much as 74 microgram of the most active B. fragilis LPS were required to give a typical biphasic fever response in rabbits. A significant correlation was found between all test results (r = 0.90-0.98, p less than 0.001).