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Porphyromonas gingivalis is a Gram-negative anaerobic periodontal microorganism strongly associated with tissue-destructive processes in human periodontitis. Following oral infection with P. gingivalis, the periodontal bone loss in mice is reported to require the engagement of Toll-like receptor 2 (TLR2). Serine-glycine lipodipeptide or glycine aminolipid classes of P. gingivalis engage human and mouse TLR2, but a novel lipid class reported here is considerably more potent in engaging TLR2 and the heterodimer receptor TLR2/TLR6. The novel lipid class, termed Lipid 1256, consists of a diacylated phosphoglycerol moiety linked to a serine-glycine lipodipeptide previously termed Lipid 654. Lipid 1256 is approximately 50-fold more potent in engaging TLR2 than the previously reported serine-glycine lipid classes. Lipid 1256 also stimulates cytokine secretory responses from peripheral blood monocytes and is recovered in selected oral and intestinal Bacteroidetes organisms. Therefore, these findings suggest that Lipid 1256 may be a microbial TLR2 ligand relevant to chronic periodontitis in humans.

Toll-like receptor 2 (TLR2) activation has been implicated in the pathogenesis of periodontal disease but the identity of the TLR2 agonists has been an evolving story. The serine/glycine lipids produced by Porphyromonas gingivalis are reported to engage human TLR2 and will promote the production of potent pro-inflammatory cytokines. This investigation compared the recovery of serine/glycine lipids in periodontal organisms, teeth, subgingival calculus, subgingival plaque, and gingival tissues, either from healthy sites or periodontally diseased sites. Lipids were extracted using the phospholipid extraction procedure of Bligh and Dyer and were analyzed using liquid chromatography/mass spectrometry for all serine/glycine lipid classes identified to date in P. gingivalis. Two serine/glycine lipid classes, Lipid 567 and Lipid 1256, were the dominant serine/glycine lipids recovered from oral Bacteroidetes bacteria and from subgingival calculus samples or diseased teeth. Lipid 1256 was the most abundant serine/glycine lipid class in lipid extracts from P. gingivalis, Tannerella forsythia, and Prevotella intermedia whereas Lipid 567 was the most abundant serine/glycine lipid class recovered in Capnocytophaga species and Porphyromonas endodontalis. Serine/glycine lipids were not detected in lipid extracts from Treponema denticola, Aggregatibacter actinomycetemcomitans, or Fusobacterium nucleatum. Lipid 1256 was detected more frequently and at a significantly higher mean level in periodontitis tissue samples compared with healthy/gingivitis tissue samples. By contrast, Lipid 567 levels were essentially identical. This report shows that members of the Bacteroidetes phylum common to periodontal disease sites produce Lipid 567 and Lipid 1256, and these lipids are prevalent in lipid extracts from subgingival calculus and from periodontally diseased teeth and diseased gingival tissues.

Multiple reaction monitoring-MS analysis of lipid extracts from human carotid endarterectomy and carotid artery samples from young individuals consistently demonstrated the presence of bacterial serine dipeptide lipid classes, including Lipid 654, an agonist for human and mouse Toll-like receptor (TLR)2, and Lipid 430, the deacylated product of Lipid 654. The relative levels of Lipid 654 and Lipid 430 were also determined in common oral and intestinal bacteria from the phylum Bacteroidetes and human serum and brain samples from healthy adults. The median Lipid 430/Lipid 654 ratio observed in carotid endarterectomy samples was significantly higher than the median ratio in lipid extracts of common oral and intestinal Bacteroidetes bacteria, and serum and brain samples from healthy subjects. More importantly, the median Lipid 430/Lipid 654 ratio was significantly elevated in carotid endarterectomies when compared with control artery samples. Our results indicate that deacylation of Lipid 654 to Lipid 430 likely occurs in diseased artery walls due to phospholipase A2 enzyme activity. These results suggest that commensal Bacteriodetes bacteria of the gut and the oral cavity may contribute to the pathogenesis of TLR2-dependent atherosclerosis through serine dipeptide lipid deposition and metabolism in artery walls.

The serine-glycine dipeptide lipid classes, including lipid 430 and lipid 654, are produced by the periodontal pathogen Porphyromonas gingivalis and can be detected in lipid extracts of diseased periodontal tissues and teeth of humans. Both serine-glycine lipid classes were previously shown to engage human and mouse Toll-like receptor 2 (TLR2) and to inhibit mouse osteoblast differentiation and function through engagement of TLR2. It is not clear if other lipids related to serine-glycine lipids are also produced by P. gingivalis. The goal of this investigation was to determine whether P. gingivalis produces additional lipid classes similar to the serine-glycine lipids that possess biological properties. P. gingivalis (ATCC 33277) was grown in broth culture, and lipids were extracted and fractionated by high-performance liquid chromatography (HPLC). Lipids were separated using semipreparative HPLC, and specific lipid classes were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) and liquid chromatography-multiple reaction monitoring (LC-MRM) mass spectrometric approaches. Two glycine lipid classes were identified, termed lipid 567 and lipid 342, and these lipid classes are structurally related to the serine-glycine dipeptide lipids. Both glycine lipid classes were shown to promote TLR2-dependent tumor necrosis factor alpha (TNF-α) release from bone marrow macrophages, and both were shown to activate human embryonic kidney (HEK) cells through TLR2 and TLR6 but not TLR1. These results demonstrate that P. gingivalis synthesizes glycine lipids and that these lipids engage TLR2 similarly to the previously reported serine-glycine dipeptide lipids.

This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there is continued interest in ways to kill spores without compromising food quality. High hydrostatic pressure (HP) under appropriate conditions can inactivate bacterial spores. With growing use of HP for food pasteurization, advancement of HP for commercial production of sterile low-acid foods requires understanding of mechanisms of spores' interactions with HP. While much is known about HP germination and inactivation of spores of Bacillus species, how HP germinates and inactivates clostridial spores is less well understood. In this work we have tried to remedy this information deficit by examining germination of spores of Clostridium difficile and Clostridium perfringens by several HP and temperature levels. The results may give insight that could facilitate more efficient methods for spore eradication in food sterilization or pasteurization, biodecontamination, and health care.

The keystone periodontal pathogen Porphyromonas gingivalis produces phosphorylated dihydroceramide lipids (sphingolipids) such as phosphoethanolamine dihydroceramide (PE DHC) and phosphoglycerol dihydroceramide (PG DHC) lipids. Phosphorylated DHCs (PDHCs) from P. gingivalis can affect a number of mammalian cellular functions, such as potentiation of prostaglandin secretion from gingival fibroblasts, promotion of RANKL-induced osteoclastogenesis, promotion of apoptosis, and enhancement of autoimmunity. In P. gingivalis , these lipids affect anchoring of surface polysaccharides, resistance to oxidative stress, and presentation of surface polysaccharides (anionic polysaccharides and K-antigen capsule). In addition to phosphorylated dihydroceramide lipids, serine dipeptide lipids of P. gingivalis are implicated in alveolar bone loss in chronic periodontitis through interference with osteoblast differentiation and function and promotion of osteoclast activity. As a prerequisite for designation as bacterial virulence factors, bacterial sphingolipids and serine dipeptide lipids are recovered in gingival/periodontal tissues, tooth calculus, human blood, vascular tissues, and brain. In addition to P. gingivalis , other bacteria of the genera Bacteroides , Parabacteroides , Porphyromonas , Tannerella , and Prevotella produce sphingolipids and serine dipeptide lipids. The contribution of PDHCs and serine dipeptide lipids to the pathogenesis of periodontal and extraoral diseases may be an underappreciated area in microbe-host interaction and should be more intensively investigated.

The total cellular lipids of Porphyromas gingivalis , a known periodontal pathogen, were previously shown to promote dendritic cell activation and inhibition of osteoblasts through engagement of Toll-like receptor 2 (TLR2). The purpose of the present investigation was to fractionate all lipids of P. gingivalis and define which lipid classes account for the TLR2 engagement, based on both in vitro human cell assays and in vivo studies in mice. Specific serine-containing lipids of P. gingivalis , called lipid 654 and lipid 430, were identified in specific high-performance liquid chromatography fractions as the TLR2-activating lipids. The structures of these lipids were defined using tandem mass spectrometry and nuclear magnetic resonance methods. In vitro , both lipid 654 and lipid 430 activated TLR2-expressing HEK cells, and this activation was inhibited by anti-TLR2 antibody. In contrast, TLR4-expressing HEK cells failed to be activated by either lipid 654 or lipid 430. Wild-type (WT) or TLR2-deficient (TLR2 −/− ) mice were injected with either lipid 654 or lipid 430, and the effects on serum levels of the chemokine CCL2 were measured 4 h later. Administration of either lipid 654 or lipid 430 to WT mice resulted in a significant increase in serum CCL2 levels; in contrast, the administration of lipid 654 or lipid 430 to TLR2 −/− mice resulted in no increase in serum CCL2. These results thus identify a new class of TLR2 ligands that are produced by P. gingivalis that likely play a significant role in mediating inflammatory responses both at periodontal sites and, potentially, in other tissues where these lipids might accumulate.

Bacteria alter the biophysical properties of their membrane lipids in response to environmental cues, such as shifts in pH or temperature. In essence, lipid composition determines membrane structure, which in turn influences many basic functions, such as transport, secretion, and signaling. Like other members of the phylum Bacteroidetes, the oral anaerobe Porphyromonas gingivalis possesses the ability to synthesize a variety of novel membrane lipids, including species of dihydroceramides that are distinct, yet similar in structure to sphingolipids produced by the human host. The role of dihydroceramides in the physiology and pathogenic potential of the human microbiota is only beginning to be explored; yet there is increasing data indicating that these lipids play a role in human diseases, such as periodontitis and multiple sclerosis. Here, we report on the identification of a gene (PG1780) in the chromosome of P. gingivalis strain W83 encoding a putative serine palmitoyltransferase, the enzyme that catalyzes the first step in sphingolipid biosynthesis. While we were able to detect dihydroceramides in whole lipid extracts of P. gingivalis cells as well as crude preparations of outer membrane vesicles, sphingolipids were absent in the PG1780 mutant strain. Moreover, we show that the synthesis of sphingolipids plays an essential role in the long-term survival of the organism as well as its resistance to oxidative stress. Further, a PG1780 mutant displayed much lower activity of cell-associated arginine and lysine gingipains, yet slightly higher activity in the corresponding culture supernates, which we hypothesize is due to altered membrane properties and anchoring of these proteases to the cell surface. In addition, we determined that sphingolipid production is critical to the presentation of surface polysaccharides, with the mutant strain displaying less K-antigen capsule and more anionic polysaccharide (APS). Overall, we have discovered that, in addition to their role in pathogenicity, the synthesis of sphingolipids is critical to the cellular homeostasis and persistence of this important dental pathogen.

Background/aim: Porphyromonas gingivalis synthesizes several classes of dihydroceramides and at least one of these lipid classes promotes proinflammatory secretory reactions in gingival fibroblasts as well as alters fibroblast morphology in culture. The purpose of this investigation was to determine whether the dihydroceramide lipids of P. gingivalis are recovered in lipid extracts of subgingival plaque, diseased teeth, and diseased gingival tissue samples. Methods: Lipids were extracted from P. gingivalis, subgingival plaque, subgingival calculus, teeth laden with gross accumulations of subgingival calculus, and gingival tissue samples obtained from chronic severe periodontitis sites. Lipid samples were analyzed by gas chromatography-mass spectrometry as trimethylsilyl derivatives or by electrospray-mass spectrometry as underivatized products. High-performance liquid chromatography fractions of P. gingivalis lipids and gingival tissue lipids were also analyzed by electrospray-mass spectrometry analysis. Results: P. gingivalis phosphorylated dihydroceramides were recovered in lipid extracts of subgingival plaque, subgingival calculus, calculus contaminated teeth, and diseased gingival tissue samples. However, the distribution of phosphorylated dihydroceramides varied between these samples. Conclusion: Subgingival plaque, subgingival calculus, diseased teeth, and gingival tissue are contaminated with phosphorylated dihydroceramides produced by P. gingivalis. The previously reported biological activity of these substances together with the recovery of these lipids at periodontal disease sites argues strongly for their classification as virulence factors in promoting chronic inflammatory periodontal disease.

Complex lipids of Porphyromonas gingivalis have been identified in lipid extracts from calculus-contaminated root surfaces and in diseased gingival tissues. However, little is known about the biological effects of these complex lipids on host cells. The purpose of this study was to evaluate the effects of P. gingivalis or calculus lipids on prostaglandin secretion from gingival fibroblasts. Lipids were extracted from paired subgingival plaque and teeth samples, and calculus-contaminated root surfaces before and after scaling and root planing, in order to determine the relevant levels of lipid extracts for the treatment of gingival fibroblasts in culture. Primary cultures of gingival fibroblasts were exposed to lipid extracts from either P. gingivalis or calculus/teeth for a period of 7 days. Control and lipid-treated cultures were exposed to human recombinant interleukin-1beta for 48 h and prostaglandin secretion from interleukin-1beta-treated fibroblasts was compared with control and lipid-treated fibroblasts without interleukin-1beta treatment. These experiments demonstrated that P. gingivalis lipids or calculus-tooth lipids potentiate interleukin-1beta-mediated prostaglandin secretory responses from gingival fibroblasts. Additionally, P. gingivalis or calculus-tooth lipid extracts were readily taken up by gingival fibroblasts as measured by bacterial fatty acid recovery in lipid extracts of cultured fibroblasts. These results indicate that bacterial lipid penetration into gingival tissues in combination with a chronic inflammatory response may substantially potentiate prostaglandin secretion from gingival fibroblasts, thereby promoting tissue destructive processes associated with adult periodontitis.

Bacterial hydroxy fatty acids and alpha-hydroxy fatty acids have been demonstrated in complex lipid extracts of subgingival plaque and gingival tissue. However, little is known about the relationship between these hydroxy fatty acids in plaque and gingival tissues or the significance of these complex lipids in promoting inflammatory periodontal disease. The present study determined the percentages of ester-linked and amide-linked hydroxy fatty acids in complex lipids recovered from plaque and gingival tissue samples and the relationship between bacterial hydroxy fatty acids and alpha-hydroxy fatty acids in the lipid extracts. To evaluate a potential role for these hydroxy fatty acids in inflammatory periodontal disease, gingival tissue samples were examined for a relationship between prostaglandin E 2 (PGE 2 ) and hydroxy fatty acids recovered in gingival lipid. This investigation demonstrated that alpha-hydroxy fatty acids are only ester linked in plaque lipids but are largely amide linked in gingival tissue lipids. Furthermore, the level of alpha-hydroxy fatty acid in gingival lipid is directly related to the level of the bacterial hydroxy fatty acid 3-OH iso -branched C 17:0 (3-OH iC 17:0 ) in the same lipid extract. However, the relationship between hydroxy fatty acids in gingival lipids does not parallel the fatty acid relationship observed in plaque lipids. Finally, alpha-hydroxy fatty acid levels in gingival tissue lipids correlate directly with the recovery of PGE 2 in the same tissue samples. These results demonstrate that alpha-hydroxy fatty acid levels in gingival lipids are directly related to both 3-OH iC 17:0 bacterial lipid levels and PGE 2 levels. These results indicate that in periodontal tissues there are unusual host-parasite interactions involving penetration of bacterial lipid in association with an altered gingival lipid metabolism and prostaglandin synthesis.

The role of oral bacterial infections including periodontal disease in the pathogenesis of rheumatoid arthritis (RA) has gained increasing interest. Among the major periodontal pathogens, Porphyromonas gingivalis has been mostly associated with RA pathogenesis. The aim of this study was to analyze the effect of P. gingivalis total lipid (TL) fraction and dihydroceramides, as potent virulence factors, on human primary chondrocytes. Primary chondrocyte cultures were incubated with P. gingivalis phosphoglycerol dihydroceramide (PG DHC) lipids, the TL fraction or phosphoethanolamine dihydroceramide. Cell morphology changes were determined by phase contrast light microscopy. Early and late apoptosis cell analysis was performed by Annexin-V, active caspases, and 7-Aminoactinomycin D staining, and examined by flow cytometry, and cell necrosis was evaluated by lactate dehydrogenase release. Procaspase-3 activation was determined by Western blot analysis. Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with P. gingivalis TLs and PG DHC lipids. Flow cytometry demonstrated an increase of Annexin-V positive and active caspases positive chondrocytes after incubation with TL and PG DHC fractions but not after phosphoethanolamine dihydroceramide (control lipid) treatment or in untreated control cells. Furthermore, Western blot analysis showed an early cleavage of procaspase-3 after 1 hr. Significant lactate dehydrogenase release following incubation with P. gingivalis lipids was demonstrated. The present data demonstrate that P. gingivalis lipids promote apoptosis in primary human chondrocytes, and thereby may contribute to the joint damage seen in the pathogenesis of RA.

Gram-negative organisms incorporate hydroxy fatty acids into the lipid A moiety of lipopolysaccharide (LPS), and in the case of some members of the family Enterobacteriaceae, hydroxy fatty acids are incorporated exclusively into lipid A. However, a limited number of Bacteroides species have been shown to incorporate several classes of 3-hydroxy fatty acids, particularly 3-hydroxy iC17:0, into constitutive lipids as well as LPS. The present study examined the distribution of hydroxy fatty acids in two periodontal pathogens, Prevotella intermedia and Porphyromonas gingivalis, by employing a phospholipid extraction procedure (E. G. Bligh and W. J. Dyer, Can. J. Biochem. Physiol. 37:911-917, 1959) which partitioned constitutive lipids into the organic solvent phase and LPS into the aqueous phase. The distribution of hydroxy fatty acids within organic solvent and aqueous extracts of these bacterial species was then compared with the distribution in subgingival plaque samples isolated from either gingivitis or severe periodontitis sites as well as the distribution in gingival tissue samples. The organic solvent and aqueous extracts were hydrolyzed under strong alkaline conditions, and the free fatty acids were treated to form pentafluorobenzyl-ester, trimethylsilyl-ether derivatives. Hydroxy fatty acid levels were quantified by using gas chromatography-negative-ion chemical ionization-mass spectrometry. By using this approach, the mean values of the 3-hydroxy iC17:0 recovered within organic solvent extracts of P. gingivalis strains ranged from 56 to 63% of total 3-hydroxy iC17:0. Substantially less 3-hydroxy iC17:0 (< 5%) was recovered in organic solvent extracts of P. intermedia. By comparison, 75% of the 3-hydroxy iC17:0 in periodontitis subgingival plaque samples was recovered in organic solvent extracts, while only 43% of the 3-hydroxy iC17:0 in gingivitis plaque samples from the same patients was recovered in organic solvent extracts. However, 3-hydroxy iC17:0 was recovered essentially only in organic solvent extracts of both healthy or mildly inflamed and periodontitis gingival tissue samples. The preferential recovery of 3-hydroxy iC17:0 in tissue lipids indicates that gingival tissues do not harbor significant levels of subgingival plaque organisms which contain 3-hydroxy iC17:0. Furthermore, these results indicate that LPS from these organisms is not prevalent in gingival tissues. Finally, these results indicate either selective penetration of certain bacterial lipids into gingival tissues or that 3-hydroxy iC17:0 is metabolically transferred from bacterial lipids into gingival tissue lipids.

Recent reports indicate that Porphyromonas gingivalis mediates alveolar bone loss or osteoclast modulation through engagement of Toll-like receptor 2 (TLR2), though the factors responsible for TLR2 engagement have yet to be determined. Lipopolysaccharide (LPS) and lipid A, lipoprotein, fimbriae, and phosphorylated dihydroceramides of P. gingivalis have been reported to activate host cell responses through engagement of TLR2. LPS and lipid A are the most controversial in this regard because conflicting evidence has been reported concerning the capacity of P. gingivalis LPS or lipid A to engage TLR2 versus TLR4. In the present study, we first prepared P. gingivalis LPS by the Tri-Reagent method and evaluated this isolate for contamination with phosphorylated dihydroceramide lipids. Next, the lipid A prepared from this LPS was evaluated for the presence of phosphorylated dihydroceramide lipids. Finally, we characterized the lipid A by the matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) and electrospray-MS methods in order to quantify recovery of lipid A in lipid extracts from diseased teeth or subgingival plaque samples. Our results demonstrate that both the LPS and lipid A derived from P. gingivalis are contaminated with phosphorylated dihydroceramide lipids. Furthermore, the lipid extracts derived from diseased teeth or subgingival plaque do not contain free lipid A constituents of P. gingivalis but contain substantial amounts of phosphorylated dihydroceramide lipids. Therefore, the free lipid A of P. gingivalis is not present in measurable levels at periodontal disease sites. Our results also suggest that the TLR2 activation of host tissues attributed to LPS and lipid A of P. gingivalis could actually be mediated by phosphorylated dihydroceramides.

Porphyromonas gingivalis produces unusual sphingolipids that are known to promote inflammatory reactions in gingival fibroblasts and Toll-like receptor 2 (TLR2)-dependent secretion of interleukin-6 from dendritic cells. The aim of the present study was to examine whether P. gingivalis lipids inhibit osteoblastic function. Total lipids from P. gingivalis and two fractions, phosphoglycerol dihydroceramides and phosphoethanolamine dihydroceramides, were prepared free of lipid A. Primary calvarial osteoblast cultures derived from 5- to 7-day-old CD-1 mice were used to examine the effects of P. gingivalis lipids on mineralized nodule formation, cell viability, apoptosis, cell proliferation, and gene expression. P. gingivalis lipids inhibited osteoblast differentiation and fluorescence expression of pOBCol2.3GFP in a concentration-dependent manner. However, P. gingivalis lipids did not significantly alter osteoblast proliferation, viability, or apoptosis. When administered during specific intervals of osteoblast growth, P. gingivalis total lipids demonstrated inhibitory effects on osteoblast differentiation only after the proliferation stage of culture. Reverse transcription-PCR confirmed the downregulation of osteoblast marker genes, including Runx2, ALP, OC, BSP, OPG, and DMP-1, with concurrent upregulation of RANKL, tumor necrosis factor alpha, and MMP-3 genes. P. gingivalis total lipids and lipid fractions inhibited calvarial osteoblast gene expression and function in vivo , as determined by the loss of expression of another osteoblast differentiation reporter, pOBCol3.6GFPcyan, and reduced uptake of Alizarin complexone stain. Finally, lipid inhibition of mineral nodule formation in vitro was dependent on TLR2 expression. Our results indicate that inhibition of osteoblast function and gene expression by P. gingivalis lipids represents a novel mechanism for altering alveolar bone homeostasis at periodontal disease sites.

Gram-negative bacteria recovered from necrotic pulps of teeth with periapical lesions have been shown to promote bone resorption through the effects of their lipopolysaccharide (LPS). Recently, it was shown that microflora of root-filled teeth with persisting periapical lesions consisted mainly of single species of Gram-positive bacteria. But Gram-positive bacteria do not contain LPS and their role in the development of periapical lesions is not clearly understood. The purpose of this study was to evaluate the effects of muramyl dipeptide (MDP), a cell wall component of both Gram-negative and Gram-positive bacteria, on cytokine release from monocytes. Human monocyte cultures were treated with MDP or LPS and interleukin-1 beta, and tumor necrosis factor-alpha levels in the supernatants were estimated. MDP and LPS stimulated cytokine release, but the effect of MDP was significantly less than that of LPS.

Bacterial lipopolysaccharide (LPS) plays a major role in the development of periapical bone resorption. Although the chemical properties of LPS are altered by treatment with an alkali such as calcium hydroxide, the effects of calcium hydroxide on the biological properties of LPS are not known. The purpose of this study was to investigate whether treatment of LPS with calcium hydroxide alters its biological action as measured by human monocyte secretion of prostaglandin E2. Monocyte cell cultures were stimulated with LPS or calcium hydroxide-treated LPS and culture supernatants were analyzed for prostaglandin E2 content using gas chromatography-mass spectrometry. Prostaglandin E2 was identified in supernatants of LPS-stimulated monocytes but not in those stimulated with calcium hydroxide-treated LPS. It was concluded that the treatment with calcium hydroxide may alter biological properties of bacterial LPS.

Novel phosphorylated dihydroceramide (PDHC) lipids produced by the periodontal pathogen Porphyromonas gingivalis include phosphoethanolamine (PE DHC) and phosphoglycerol dihydroceramides (PG DHC) lipids. These PDHC lipids mediate cellular effects through Toll-like receptor 2 (TLR2) including promotion of IL-6 secretion from dendritic cells and inhibition of osteoblast differentiation and function in vitro and in vivo. The PE DHC lipids also enhance (TLR2)-dependent murine experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. The unique non-mammalian structures of these lipids allows for their specific quantification in bacteria and human tissues using multiple reaction monitoring (MRM)-mass spectrometry (MS). Synthesis of these lipids by other common human bacteria and the presence of these lipids in human tissues have not yet been determined. We now report that synthesis of these lipids can be attributed to a small number of intestinal and oral organisms within the Bacteroides, Parabacteroides, Prevotella, Tannerella and Porphyromonas genera. Additionally, the PDHCs are not only present in gingival tissues, but are also present in human blood, vasculature tissues and brain. Finally, the distribution of these TLR2-activating lipids in human tissues varies with both the tissue site and disease status of the tissue suggesting a role for PDHCs in human disease.

Apical periodontitis and its concomitant periapical osteolysis is caused by pulpal infection, and bacterial lipopolysaccharide (LPS) is known to play a major role in the bone resorption process. Little is known concerning the effect of root canal intervisit dressings on residual LPS in root canals after bacterial cell lysis. The purpose of this study was to evaluate the effects of calcium hydroxide on bacterial LPS. Free hydroxy fatty acids were quantified in samples of LPS treated with calcium hydroxide. Calcium hydroxide treatment of LPS was shown to release elevated quantities of hydroxy fatty acids. It was concluded that calcium hydroxide hydrolyzed the lipid moiety of bacterial LPS, resulting in the release of free hydroxy fatty acids. This result suggests that calcium hydroxide-mediated degradation of LPS may be an important reason for the beneficial effects obtained with calcium hydroxide use in clinical endodontics.

The purpose of this investigation was to examine gamma interferon potentiation of lipopolysaccharide (LPS) responses in human monocytes by using phenol-water-extracted (unfractionated) and highly purified LPS preparations isolated from Bacteroides intermedius and Salmonella typhimurium. Phenol-water-extracted LPS preparations from these bacteria were further purified by chromatography over Sepharose-CL-4B. LPS enrichment in pooled column fractions was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantitation of hydroxy-fatty acid and 2-keto-3-deoxyoctulosonic acid content, protein contamination, and anthrone-reactive material. Monocyte stimulation by LPS, measured as prostaglandin E (PGE) release, was assessed with and without gamma interferon treatment. Cells were either treated simultaneously with gamma interferon and LPS or pretreated with gamma interferon prior to LPS stimulation. PGE release from counterflow-isolated monocytes was quantitated during the 0- to 24-h and 24- to 48-h culture intervals. Contrary to previous results from this laboratory, phenol-water-extracted LPS preparations from B. intermedius and S. typhimurium were similar in their capacities to stimulate PGE release from monocytes. Molecular sieve chromatography was found to remove substantial amounts of high-molecular-weight polysaccharide contaminants only from the B. intermedius LPS but did not significantly alter the potency of either B. intermedius or S. typhimurium LPS. Gamma interferon cotreatment did not potentiate the release of PGE with any of the LPS preparations tested. However, 24-h pretreatment of monocytes with gamma interferon followed by a 24-h exposure to LPS resulted in significant potentiation of PGE release over LPS alone. In addition, B. intermedium preparations were approximately threefold more potent than similarly prepared LPS isolates from S. typhimurium following gamma interferon pretreatment. These results indicate that gamma interferon can selectively potentiate the effects of B. intermedius LPS in human monocyte isolates.

Porphyromonas gingivalis, a recognized periodontal pathogen, is a source of sphinganine bases, fatty acids, free ceramides as well as complex lipids that potentiate interleukin-1b-mediated secretory responses in gingival fibroblasts. The purpose of this study is the structural verification of the sphinganine bases and fatty acids that had been proposed as major components of the complex lipids found in P. gingivalis. The putative C17, C18, and C19 sphinganine bases were prepared from Garner's aldehyde (1) or from a protected serine Weinreb's amide (2). We confirmed that isobranched sphinganine bases are the major structural feature of the ceramides observed from P. gingivalis. We also prepared a C17 unsaturated fatty acid, along with an isobranched C17 3-hydroxy fatty acid, and determined that the major component of the active lipids was the latter.

Lipopolysaccharides (LPS) from Bacteroides species are less potent than enterobacterial LPS preparations when compared by pyrogenicity and bone resorption assays and by the localized Schwartzman reaction. In this investigation, LPS preparations from Bacteroides intermedius and Bacteroides gingivalis were examined for their capacity to stimulate PGE2 release from human monocytes relative to identical doses of Salmonella typhimurium LPS. In addition, the capacity of recombinant gamma interferon (IFN-γ) to potentiate PGE2 release was examined with each LPS preparation. Monocytes, separated by counter flow centrifugation, were exposed to LPS (1 ng/ml, 100 ng/ml and 10 ng/ml) alone or in combination with IFN-γ (10 Units/ml). PGE2 levels (measured by RIA) were determined in monocyte culture supernatants exposed during the following incubation periods: 0-8, 8-24, 24-48, and 48-72 h. Monocytes treated with Salmonella LPS released 2 to 3-fold greater amounts of PGE2 than cells treated with Bacteroides species LPS preparations. Although IFN-y alone did not stimulate PGE2 release, Bacteroides-elicited PGE2 release was siginificantly increased when monocytes were cultured concomitantly with IFN-γ. PGE2 release elicited by Salmonella LPS was not significantly affected by interferon. These findings indicate that IFN-γ can markedly potentiate the release of PGE2 from monocytes stimulated with Bacteroides LPS. Furthermore, the observed weakness of these LPS preparations relative to Salmonella LPS can be largely overcome by co-treating monocytes with IFN-γ.

Lipopolysaccharides (LPS) prepared from the suspected periodontal pathogens Actinobacillus actinomycetemcomitans (A. a.), Bacteroides gingivalis, B. intermedius and Wolinella recta were compared to Salmonella typhimurium LPS for their capacity to stimulate prostaglandin E2 (PGE2) release from human monocytes. Counterflow isolated monocytes were cultured with control medium or media containing 10 micrograms/ml LPS. Media were then exchanged every 24 hours for a total of 72 hours. Salmonella and Wolinella LPS preparations demonstrated seven-fold greater PGE2 release than B. gingivalis and two-fold greater than A. a. and B. intermedius. PGE2 release was found to decrease over time with all LPS preparations except Wolinella. The potency of the LPS preparations is tentatively ranked as follows: Wolinella greater than or equal to Salmonella greater than A. a. greater than B. intermedius greater than or equal to B. gingivalis. These findings demonstrate that LPS preparations from suspected periodontal pathogens are capable of stimulating PGE2 release from human monocytes. The high potency and prolonged stimulation of PGE2 release with Wolinella LPS suggests unusual toxic properties that may exert a greater influence in the pathogenesis of destructive periodontal diseases.