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AimsThis work aimed to characterize spore inner membrane (IM) properties and the mechanism of spore killing by wet heat and H2O2 with spores overexpressing the 2Duf protein, which is naturally encoded from a transposon found only in some Bacillus strains with much higher spore resistance than wild-type spores.Methods and ResultsKilling of Bacillus subtilis spores by wet heat or hydrogen peroxide (H2O2) was slower when 2Duf was present, and Ca-dipicolinic acid release was slower than killing. Viabilities on rich plates of wet heat- or H2O2 -treated spores +/- 2Duf were lower when NaCl was added, but higher with glucose. Addition of glucose but not Casamino acids addition increased treated spores’ viability on minimal medium plates. Spores with 2Duf required higher heat activation for germination, and their germination was more wet-heat resistant than that of wild-type spores, processes that involve IM proteins. IM permeability and lipid mobility were lower in spores with 2Duf, although IM phospholipid composition was similar in spores +/- 2Duf.ConclusionsThese results and previous work suggests that wet heat and H2O2 kill spores by damaging an IM enzyme or enzymes involved in oxidative phosphorylation.

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.

Oral and gut Bacteroidetes produce unique classes of serine-glycine lipodipeptides and glycine aminolipids that signal through host Toll-like receptor 2. These glycine lipids have also been detected in human arteries, but their effects on atherosclerosis are unknown. Here, we sought to investigate the bioactivity of bacterial glycine lipids in mouse models of atherosclerosis. Lipid 654 (L654), a serine-glycine lipodipeptide species, was first tested in a high-fat diet (HFD)-fed Ldlr

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.

Phospholipases A2 (PLA2) are a diverse group of enzymes that cleave the fatty acids of membrane phospholipids. They play critical roles in pathogenesis of neurodegenerative diseases such as multiple sclerosis by enhancing oxidative stress and initiating inflammation. The levels of PLA2 activity in MS patients compared to controls and role of inhibiting PLA2 activity on severity scores in different experimental models are not comprehensively assessed in the light of varying evidence from published studies. The objective of this systematic review is to determine the association between PLA2 activity and multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). We performed a systematic review of six studies that assessed PLA2 activity in MS patients compared to controls and nine studies that assessed PLA2 activity in EAE. sPLA2 nor Lp-PLA2 activity were not increased in MS compared to controls in five of those six studies. A difference in sPLA2 activity was only found in a study that measured the enzyme activity in urine. However, inhibiting cPLA2 or sPLA2 led to lower clinical severity or no signs of EAE in mice, and a lower incidence of EAE lesions compared to animals without cPLA2 inhibition. These findings indicate that PLA2 appears to play a role in the pathogenesis of EAE.

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.

Among several virulence factors produced by the periodontal pathogen Porphyromonas gingivalis (Pg), a recently identified novel class of dihydroceramide lipids that contains a long acyl-chain has the potential to play a pathogenic role in periodontitis because of its higher level of tissue penetration compared to other lipid classes produced by Pg. However, the possible impact of Pg ceramides on osteoclastogenesis is largely unknown. In the present study, we report that the phosphoglycerol dihydroceramide (PGDHC) isolated from Pg enhanced osteoclastogenesis in vitro and in vivo. Using RAW264.7 cells, in vitro assays indicated that PGDHC can promote RANKL-induced osteoclastogenesis by generating remarkably larger TRAP+ multinuclear osteoclasts compared to Pg LPS in a TLR2/4-independent manner. According to fluorescent confocal microscopy, co-localization of non-muscle myosin II-A (Myh9) and PGDHC was observed in the cytoplasm of osteoclasts, indicating the membrane-permeability of PGDHC. Loss- and gain-of-function assays using RNAi-based Myh9 gene silencing, as well as overexpression of the Myh9 gene, in RAW264.7 cells showed that interaction of PGDHC with Myh9 enhances RANKL-induced osteoclastogenesis. It was also demonstrated that PGDHC can upregulate the expression of dendritic cell-specific transmembrane protein (DC-STAMP), an important osteoclast fusogen, through signaling that involves Rac1, suggesting that interaction of PGDHC with Myh9 can elicit the cell signal that promotes osteoclast cell fusion. Taken together, our data indicated that PGDHC is a Pg-derived, cell-permeable ceramide that possesses a unique property of promoting osteoclastogenesis via interaction with Myh9 which, in turn, activates a Rac1/DC-STAMP pathway for upregulation of osteoclast cell fusion.

Lipopeptides promote innate immune response and are related to disease pathology. To investigate the newly emerging roles of lipopeptides, accurate measurements of stereoisomers with multiple chiral centers are essential yet challenging. This work uses (3R)- and (3S)-(15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, abbreviated as l-serine-(R+S)-Lipid 654, to develop a method that combines chiral liquid chromatography, a diastereomeric mixture of isotopically labeled internal standards, and multiple reaction monitoring mass spectrometry. The new method allows for simultaneously determining the absolute configuration and quantity of stereoisomers of bacteria-derived lipopeptides. Total lipid extracts of nine evaluated bacteria strains had different amounts, but only the (R)-isoform of l-serine-Lipid 654. The developed method also allowed for the first quantitative analysis of hydrolysis of a nonphospholipid as a novel substrate of honey bee venom phospholipase A2.

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.

Background Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. Methods Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2−/−) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. Results Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2−/− mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. Discussion Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS. Electronic supplementary material The online version of this article (10.1186/s12974-019-1540-2) contains supplementary material, which is available to authorized users.

Gingival augmentation surgical procedures are commonly used to restore gingival tissues at sites where gingival recession is problematic for the patient or where gingival augmentation/manipulation is required to achieve optimal dental therapeutic results. The goals of this chapter are first to review the types of gingival augmentation procedures commonly used today and next, to summarize the principles and techniques for performing traditional gingival augmentation surgical procedures. Also summarized in this chapter are modifications of gingival augmentation procedures to achieve secondary benefits such as increased vestibule depth, removal of a high frenum attachments, or augmentation of keratinized gingival tissue adjacent to implants. These examples provide a rationale to enhance therapeutic outcomes by adapting the surgical principles of gingival augmentation surgery to specific dental problems.

A periodontal pathogen, Porphyromonas gingivalis, produces two serine dipeptide lipid classes that we labeled lipid 654 and lipid 430, and both contain l -serine as the terminal amino acid. The lipid 654 and lipid 430 classes are each comprised of three species with differing fatty acid substitutions, but the most abundant species demonstrate unit masses of either 654 or 430, respectively. Recently we observed that the lipid 654 can be hydrolyzed by specific lipases to lipid 430. However, a substantial percentage of the naturally occurring lipid 654 cannot be enzymatically hydrolyzed to lipid 430. The observed partial hydrolysis could be due to the presence of a mixture of stereoisomers. Testing this theory requires structural verification of our so-called 654 and 430 by total synthesis. We present herein details of the convergent synthesis of lipids 430 and 654, which confirm the proposed structure of P. gingivalis lipid 654 to be (3R and 3S)- l -serine-2. The bis(fatty acid) (3R)- l -serine-2 was prepared as well as the synthetic precursor, serine dipeptide mono-fatty acid (3R)- l -serine-1, which is the structure of lipid 430. We also synthesized the (3S)- l -serine-2 diastereomer as well as (3S)- l -serine-1. Using these synthetic standards, we confirmed that PLA2-mediated hydrolysis of lipid 654 is enantioselective in that only the (3R)- l -serine-2, but not (3S)- l -serine 2 is enzymatically hydrolyzed.

The role of TLR signaling in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE) is unclear. This role is especially controversial in models of adoptive transfer EAE in which no adjuvant and no TLR ligands are administered. We recently reported that a microbiome-derived TLR2 ligand, Lipid 654 (L654), is present in healthy human serum but significantly decreased in the serum of MS patients. This suggested that microbiome products that gain access to the systemic circulation, rather than being proinflammatory, may normally play an immune-regulatory role by maintaining a state of relative TLR tolerance. Therefore, a loss of microbiome-mediated TLR tolerance, as suggested by lower serum levels of L654, may play a role in the pathogenesis of MS. As proof of concept we asked whether administering low-level TLR2 ligands in adoptive transfer EAE induces TLR2 tolerance and attenuates disease. We administered low-level Pam2CSK4 or L654 to mice receiving encephalitogenic cells and in doing so induced both TLR2 tolerance and attenuation of EAE. Disease attenuation was accompanied in the CNS by a decrease in macrophage activation, a decrease in a specific proinflammatory macrophage population, and a decrease in Th17 cells. In addition, disease attenuation was associated with an increase in splenic type 1 regulatory T cells. Kinetic tolerance induction studies revealed a critical period for TLR2 involvement in adoptive transfer EAE. Overall, these results suggest that inducing TLR tolerance may offer a new approach to treating CNS autoimmune diseases such as MS.

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.

Porphyromonas gingivalis is a periodontal pathogen strongly associated with loss of attachment and supporting bone for teeth. We have previously shown that the total lipid extract of P. gingivalis inhibits osteoblast differentiation through engagement of Toll-like receptor 2 (TLR2) and that serine dipeptide lipids of P. gingivalis engage both mouse and human TLR2. The purpose of the present investigation was to determine whether these serine lipids inhibit osteoblast differentiation in vitro and in vivo and whether TLR2 engagement is involved. Osteoblasts were obtained from calvaria of wild type or TLR2 knockout mouse pups that also express the Col2.3GFP transgene. Two classes of serine dipeptide lipids, termed Lipid 654 and Lipid 430, were tested. Osteoblast differentiation was monitored by cell GFP fluorescence and osteoblast gene expression and osteoblast function was monitored as von Kossa stained mineral deposits. Osteoblast differentiation and function were evaluated in calvarial cell cultures maintained for 21 days. Lipid 654 significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation and this inhibition was dependent on TLR2 engagement. Lipid 430 also significantly inhibited GFP expression, osteoblast gene expression and mineral nodule formation but these effects were only partially attributed to engagement of TLR2. More importantly, Lipid 430 stimulated TNF-α and RANKL gene expression in wild type cells but not in TLR2 knockout cells. Finally, osteoblast cultures were observed to hydrolyze Lipid 654 to Lipid 430 and this likely occurs through elevated PLA2 activity in the cultured cells. In conclusion, our results show that serine dipeptide lipids of P. gingivalis inhibit osteoblast differentiation and function at least in part through engagement of TLR2. The Lipid 430 serine class also increased the expression of genes that could increase osteoclast activity. We conclude that Lipid 654 and Lipid 430 have the potential to promote TLR2-dependent bone loss as is reported in experimental periodontitis following oral infection with P. gingivalis. These results also support the conclusion that serine dipeptide lipids are involved in alveolar bone loss in chronic periodontitis.

The pathophysiology of autoimmune diseases such as Multiple Sclerosis (MS) involves a complex interaction between genetic and environmental factors. Studies of monozygotic twins suggest a significant role for environmental factors in susceptibility to MS. Numerous studies, driven by the "Hygiene Hypothesis," have focused on the role of environmental factors in allergic and autoimmune diseases. The hygiene hypothesis postulates that individuals living in environments that are too "clean" lack the requisite exposure to "immune-tolerizing" microbial products, resulting in poorly regulated immune systems and increased immune-mediated diseases. Interestingly, few studies have linked MS with the hygiene hypothesis. Similarly, although numerous studies have examined the role of the microbiome in autoimmune diseases, there has been no consistent documentation of disease-specific alterations in the MS microbiome. In this review, we present evidence that integrating the hygiene hypothesis and the microbiome allows for the identification of novel pathophysiologic mechanisms in MS. Our central hypothesis is that the microbiome in MS represents a "defective environment" that fails to provide normal levels of "TLR2-tolerizing" bacterial products to the systemic immune system. Consistent with the hygiene hypothesis, we posit that this defective microbiome function results in abnormally regulated systemic innate immune TLR2 responses that play a critical role in both the inflammatory and defective remyelinative aspects of MS. We have completed proof of concept studies that support the inflammatory, remyelinating, and human immune response components of this paradigm. Our studies suggest that induction of TLR2 tolerance may represent a novel approach to treating MS, inhibiting autoimmune inflammation while simultaneously facilitating remyelination.

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.

Summary The roles of the microbiome and innate immunity in the pathogenesis of multiple sclerosis (MS) remain unclear. We have previously documented abnormally low levels of a microbiome-derived Toll-like receptor (TLR)2-stimulating bacterial lipid in the blood of MS patients and postulated that this is indicative of a deficiency in the innate immune regulating function of the microbiome in MS. We postulated further that the resulting enhanced TLR2 responsiveness plays a critical role in the pathogenesis of MS. As proof-of-concept, we reported that decreasing systemic TLR2 responsiveness by administering very low-dose TLR2 ligands attenuated significantly the mouse model of MS, experimental autoimmune encephalomyelitis. Studies of Toll-like receptor responses in patients with MS have been conflicting. Importantly, most of these investigations have focused on the response to TLR4 ligation and few have characterized TLR2 responses in MS. In the present study, our goal was to characterize TLR2 responses of MS patients using multiple approaches. Studying a total of 26 MS patients and 32 healthy controls, we now document for the first time that a large fraction of MS patients (50%) demonstrate enhanced responsiveness to TLR2 stimulation. Interestingly, the enhanced TLR2 responders include a significant fraction of those with progressive forms of MS, a subset of patients considered unresponsive to adaptive immune system-targeting therapies. Our results suggest the presence of a pathologically relevant TLR2 related innate immune abnormality in patients with both relapsing–remitting and progressive MS. These findings may have significant implications for understanding the role of innate immunity in the pathogenesis of MS.

Introduction Periapical infections secondary to pulpal necrosis are associated with bacterial contamination of the pulp. Porphyromonas endodontalis , a gram-negative organism, is considered to be a pulpal pathogen. P. gingivalis is phylogenetically related to P. endodontalis and synthesizes several classes of novel complex lipids that possess biological activity, including the capacity to promote osteoclastogenesis and osteoclast activation. The purpose of this study was to extract and characterize constituent lipids of P. endodontalis and evaluate their capacity to promote proinflammatory secretory responses in the macrophage cell line, RAW 264.7, as well as their capacity to promote osteoclastogenesis and inhibit osteoblast activity. Methods Constituent lipids of both organisms were fractionated by high-performance liquid chromatography and were structurally characterized using electrospray mass spectrometry or electrospray-mass spectrometry/mass spectrometry. The virulence potential of P. endodontalis lipids was then compared with known biologically active lipids isolated from P. gingivalis . Results P. endodontalis total lipids were shown to promote tumor necrosis factor alpha secretion from RAW 264.7 cells, and the serine lipid fraction appeared to account for the majority of this effect. P. endodontalis lipid preparations also increased osteoclast formation from RAW 264.7 cells, but osteoblast differentiation in culture was inhibited and appeared to be dependent on Toll-like receptor 2 expression. Conclusions These effects underscore the importance of P. endodontalis lipids in promoting inflammatory and bone cell activation processes that could lead to periapical pathology.

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.

The nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) was shown to play an immunoregulatory role in many immune-related cell types, and activation of PPARγ was reported to be an effective therapeutic approach in murine and human autoimmune disease. However, despite an association between lymphopenia and autoimmunity, there has been no study on the role of T cell PPARγ in lymphopenia-associated autoimmunity. In the present studies, we examined the role of PPARγ in CD4+ T cells in two murine models of lymphopenia-associated autoimmunity. Surprisingly, we found that PPARγ expression in CD4+ CD25− T cells (T effector cells [Teffs]) is actually required for development of autoimmunity under lymphopenic conditions. Mechanistically, the inability of PPARγ-deficient (T-PPAR) Teffs to mediate lymphopenic autoimmunity is associated with a significant decrease in accumulation of Teffs in the spleen, lymph nodes, and tissues after adoptive transfer. This abnormal accumulation of T-PPAR Teffs was associated with defects in both in vivo proliferation and survival. Additionally, T-PPAR Teffs demonstrated decreased cytokine production in inflammatory sites and decreased expression of the homing receptor α4β7. Finally, these abnormalities in T-PPAR Teff function were not elicited by lymphopenia alone but also required the additional activation involved in the mediation of autoimmunity. Thus, in contrast to its documented immunosuppressive role, we identified an unexpected function for PPARγ in Teffs: a role in Teff proliferation and survival in lymphopenia-associated autoimmunity. These findings highlight both the multifunctional role of PPARγ in T cells and the complexity of PPARγ as a potential therapeutic target in autoimmunity.

TNF-α has a multifunctional role in autoimmune diseases as reflected in the variable responses of different human diseases to anti–TNF-α therapy. Recent studies have suggested that TNF-α modulates autoimmunity partially via effects on regulatory T cells (Tregs) and that these effects are mediated through the type II TNFR (TNFR2). We have investigated the requirement for TNFR2-expression on murine natural Tregs (nTregs) and induced Tregs (iTregs) in mediating suppression of colitis. Surprisingly, we find that TNFR2-expression is required for both spleen- and thymus-derived nTreg-mediated suppression, but is not required for iTreg-mediated suppression. Abnormal TNFR2−/− nTreg function was not associated with an in vivo decrease in accumulation, stability, or expression of markers known to be relevant in Treg function. Because iTregs are generated in the presence of TGF-β, we investigated whether activation in the presence of TGF-β could overcome the functional defect in TNFR2−/− nTregs. Although preactivation alone did not restore suppressive function of nTregs, preactivation in the presence of TGF-β did. These results identify potentially critical differences in activation requirements for nTregs versus iTregs. Furthermore, our findings are consistent with reports suggesting that nTregs are activated in sites of inflammation while iTregs are activated in lymph nodes. Finally, by demonstrating that nTregs require TNF-α for optimal function whereas iTregs do not, our results suggest that the enigma of variable responses of different human diseases to anti–TNF-α therapy may relate to whether nTregs or iTregs have the predominant regulatory role in a given disease.

Although nonsteroidal anti-inflammatory drugs (NSAID), including sulindac, have been used extensively as chemopreventive agents for colorectal cancer, results are not consistent. NSAIDs, most reportedly sulindac, often do not cause a complete regression of adenomas and some patients develop resistance to NSAID treatment. In this study, we evaluated the effect of sulindac on colon tumorigenesis in the ApcMin/+ mouse model. Sulindac (180 ppm) given in drinking water for 9 weeks to ApcMin/+ mice significantly reduced the size of colon tumors, but actually caused an increase in colon tumor multiplicity relative to untreated controls (average of 5.5 versus 1.6 tumors per mouse, respectively; P < 0.0001). This indicated that the drug could inhibit colon tumor progression but not initiation. As expected, in the small intestine, sulindac significantly reduced tumor size and multiplicity relative to untreated controls (average of 2.3 versus 42.0 tumors per mouse, respectively; P < 0.0001). Generation of a panel of prostanoids was comparably suppressed in the small intestine and colon by sulindac treatment. Sulindac is also known to exert its growth inhibitory effects through regulation of many noncyclooxygenase targets, including p21, β-catenin, E-cadherin, mitochondrial apoptotic proteins, and peroxisome proliferator–activated receptor-γ. We found that sulindac treatment protected against E-cadherin loss in colon tumors, with associated inhibition of nuclear β-catenin accumulation. Importantly, p21WAF1/cip1 and peroxisome proliferator–activated receptor-γ expression were absent in colon tumors from sulindac-treated mice, suggesting that loss of these proteins is necessary for drug resistance. Together, these observations may be translatable to designing novel clinical therapies using combinations of agents that target multiple molecular pathways to overcome sulindac resistance. Cancer Prev Res; 3(9); 1187–97. ©2010 AACR.

Recent reports suggest that commensal bacteria may play a down-regulatory role in autoimmune disease. In the present studies, we demonstrate that phosphorylated dihydroceramides, uniquely structured lipids derived from the common human oral bacterium Porphyromonas gingivalis and from bacteria commonly found in the gastrointestinal tract and other organs, are capable of enhancing autoimmunity. We have previously reported that these lipids have proinflammatory effects on human fibroblasts in vitro and, in preliminary studies, have recovered these lipids from surgically removed human carotid atheroma, suggesting that they may play a role in human inflammatory disease. To investigate whether these lipids have functional effects on autoimmunity, we administered phosphorylated dihydroceramides to mice with the murine model of multiple sclerosis, experimental allergic encephalomyelitis (EAE). We find that these lipids, and particularly the phosphoethanolamine dihydroceramide (PE DHC) fraction, significantly enhanced EAE. Mechanistically, PE DHC enhances EAE in mice lacking natural killer T cells, fails to enhance EAE in Toll-like receptor 2 (TLR2)-deficient mice and, in vitro, induces dendritic cell interleukin-6 secretion in a TLR2-dependent manner. Finally, PE DHC-treated mice with EAE demonstrate a decreased percentage of spinal cord Foxp3+ T cells, suggesting that these lipids may affect regulatory aspects of adaptive immune responses. Overall, our results suggest that phosphorylated dihydroceramides derived from common human bacteria function as TLR2 ligands and may play a previously unrecognized role in human autoimmune diseases.

Porphyromonas gingivalis dihydroceramides are found in extracts of calculus-contaminated root surfaces, diseased gingival tissue, and atherosclerotic plaques. These ceramides have been shown to promote inflammatory secretory responses in gingival fibroblasts. Little is known about their effects on the vascular system. We tested the hypothesis that P. gingivalis lipids induce apoptosis of human endothelial cells, and investigated the effects of extracted and purified P. gingivalis lipids on human umbilical vein endothelial cells. P. gingivalis phosphoglycerol dihydroceramides induced apoptosis, but not necrosis, in endothelial cells. Early apoptotic cells showed exposure of phosphatidylserine on the cell surface, followed by the cleavage of procaspases 3, 6, and 9. The release of apoptosis-inducing factor was increased, suggesting mitochondrial involvement. Different caspase inhibitors and cAMP elevation blocked DNA fragmentation. Moreover, N-acetylcysteine significantly reduced apoptosis, suggesting a role for reactive oxygen species in this process. Analysis of these data indicates that dihydroceramides may be important virulence factors of P. gingivalis.

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.

Purpose: The purpose of this study was to compare jet injection to a syringe and needle in terms of the difference in discomfort and pharmacokinetics after the subcutaneous administration of midazolam. Patients and Methods: Using a prospective, randomized, double-blinded study design, 14 subjects were administered midazolam on two separate occasions (at least 2 weeks apart). The subjects were randomly distributed into two groups: syringe and needle (saline)/jet injector (midazolam) or syringe and needle (midazolam)/jet injector (saline). The subjects were randomly assigned to receive either EMLA (eutectic mixture of local anesthesics) or a placebo at the injection site for the first administration and the other topical agent on the second visit. Each subject received one subcutaneous injection in the deltoid region per arm per day. Each injection contained the same volume of solution. Subjects completed visual analog scale (VAS) questionnaires assessing the discomfort of the injection. Blood samples were taken at specified intervals over 2 hours for determination of midazolam levels. Results: The discomfort associated with the injection was less with the Biojector 2000 (Bioject Inc, Portland, OR) although this was not statistically significant. However, persistent discomfort was significantly greater at the needle site. The mean peak plasma level of midazolam was achieved more rapidly with the Biojector 2000 than with the syringe and needle (P < .05). However, the peak plasma level after jet injection or injection with a syringe and needle was not statistically different. Conclusion: The results of the study show that the Biojector 2000 is a needle-free injection system that can be used for the administration of a premedicant before induction of anesthesia. It has several advantages, including the potential reduction of anxiety associated with the “fear of needles” and occupational injuries.

Background Numerous gaseous microemboli (GME) are delivered into the arterial circulation during cardiopulmonary bypass (CPB). These emboli damage end organs through multiple mechanisms that are thought to contribute to neurocognitive deficits after cardiac surgery. Here, we use hypobaric oxygenation to reduce dissolved gases in blood and greatly reduce GME delivery during CPB. Methods Variable subatmospheric pressures were applied to 100% oxygen sweep gas in standard hollow fiber microporous membrane oxygenators to oxygenate and denitrogenate blood. GME were quantified using ultrasound while air embolism from the surgical field was simulated experimentally. We assessed end-organ tissues in swine postoperatively using light microscopy. Results Variable sweep gas pressures allowed reliable oxygenation independent of carbon dioxide removal while denitrogenating arterial blood. Hypobaric oxygenation produced dose-dependent reductions of Doppler signals produced by bolus and continuous GME loads in vitro. Swine were maintained using hypobaric oxygenation for 4 hours on CPB with no apparent adverse events. Compared with current practice standards of oxygen/air sweep gas, hypobaric oxygenation reduced GME volumes exiting the oxygenator (by 80%), exiting the arterial filter (95%), and arriving at the aortic cannula (∼100%), indicating progressive reabsorption of emboli throughout the CPB circuit in vivo. Analysis of brain tissue suggested decreased microvascular injury under hypobaric conditions. Conclusions Hypobaric oxygenation is an effective, low-cost, common sense approach that capitalizes on the simple physical makeup of GME to achieve their near-total elimination during CPB. This technique holds great potential for limiting end-organ damage and improving outcomes in a variety of patients undergoing extracorporeal circulation.

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.

Although patients with refractory periodontitis have been widely reported, no clear biologic profile of these patients has been noted. The purpose of the present study was to evaluate host responsiveness of a well-defined group of refractory periodontitis patients by determining the effect of a lipopolysaccharide (LPS) challenge on monocyte surface receptor density and on the release of inflammatory mediators. Venous blood was obtained from 7 refractory periodontitis, 8 stable periodontal maintenance, and 8 gingivitis patients with no evidence of periodontitis. Mononuclear cells were cultured in either control media or media treated with Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), or Salmonella typhimurium (S. typh) LPS. At 0 and 24 hours supernatants were assayed for prostaglandin-E2 (PGE2) and interleukin-1 beta (Il-1 beta) release by ELISA. Using flow cytometry the density of specific monocyte surface receptors were assayed with Mo3e and LeuM3 monoclonal antibodies (mAb); T-cell CD4/CD8 ratios were assayed with OKT-3, OKT-4, and OKT-8 mAb. After 24 hours incubation with Pg or S. typh LPS, the upregulation of the Mo3e receptor was significantly decreased for refractory periodontitis patients (P < 0.05) when compared to gingivitis and to stable maintenance patients. In refractory periodontitis patients the T-cell CD4/CD8 ratio was decreased. Upon stimulation with Pg or S. typh LPS, monocytes from stable maintenance and refractory periodontitis patients released more Il-1 beta (P < 0.05) and PGE2 (P = 0.13 and 0.15) than monocytes from gingivitis subjects.(ABSTRACT TRUNCATED AT 250 WORDS)