PubMed Health⌕ Search

Biomedical subjects

R P Darveau

Publications and source records attributed to R P Darveau.

At least 19 recordsLinked to original sources
PubMed2006

Regulation of cementoblast function by P. gingivalis lipopolysaccharide via TLR2.

Although cementoblasts express Toll-like receptors (TLR)-2 and -4, little is known regarding the possible participation of cementoblasts in the inflammatory response. We investigated the effects of Porphyromonas gingivalis lipopolysaccharide (LPS), tetra- and penta-acylated lipid A species (designated PgLPS(1435/1449) and PgLPS(1690), respectively), on gene expression of osteoclastogenesis-associated molecules in murine cementoblasts. Real-time quantitative RT-PCR analysis revealed that receptor activator of NF-kappaB ligand (RANKL), interleukin-6, Regulated on activation, normal T-cell expressed, and secreted (RANTES), macrophage inflammatory protein-1alpha, and monocyte chemoattractant protein-1 were rapidly and dramatically induced upon stimulation with PgLPS(1690), but only slightly induced with PgLPS(1435/1449). Osteoprotegerin, which was expressed constitutively, was not altered significantly. ELISA demonstrated synthesis of corresponding proteins. PgLPS(1690) significantly induced transcripts for NF-kappaB, and this activation was inhibited by pre-treatment with anti-TLR-2 but not with TLR-4 antibodies. These results suggest that cementoblasts participate in the recruitment of osteoclastic precursor cells by up-regulation of chemokines/cytokines.

Analysis of Variance↗
PubMed2005

Lipopolysaccharide heterogeneity: innate host responses to bacterial modification of lipid a structure.

The innate host response system is composed of various mechanisms designed to detect and facilitate host responses to microbial components, such as lipopolysaccharides (LPS). To enable this to occur, innate systems contain multiple pattern recognition receptors (i.e., LBP, CD14, and TLRs), which identify certain features within bacterial LPS that are foreign to the host, as well as essential and uniquely specific for bacteria. Innate host identification of unique bacterial components or patterns, therefore, relies on the inability of bacteria to alter these essential or critical components dramatically. Historically, LPS have been viewed as essential outer-membrane molecules containing both a highly variable outer region (O-segment) as well as a relatively conserved inner region (lipid A). However, over the last decade, new evidence has emerged, revealing that increased natural diversity or heterogeneity within specific components of LPS, such as lipid A-resulting in minor to moderate changes in lipid A structure-can produce dramatic host responses. Therefore, examples of natural lipid A heterogeneity, and the mechanisms that control it, represent a novel approach in which bacteria modulate host responses and may thereby confer specific advantages to certain bacterial species under changing environmental host conditions.

Animals↗
PubMed2004

Cementoblast gene expression is regulated by Porphyromonas gingivalis lipopolysaccharide partially via toll-like receptor-4/MD-2.

Lipopolysaccharides are potent inflammatory mediators considered to contribute to destruction of periodontal tissues. Here, we hypothesized that Porphyromonas gingivalis lipopolysaccharide (P-LPS) treatment would regulate gene expression in murine cementoblasts through Toll-like receptor 4. Real-time (RT)-PCR and Northern blot analysis indicated that P-LPS decreased expression of transcripts for osteocalcin (OCN) and receptor activator of nuclear factor kappaB ligand (RANKL). In contrast, a dose-dependent up-regulation in mRNA levels for osteopontin (OPN) and osteoprotegerin (OPG) was observed. Similarly, ELISA demonstrated decreased RANKL and increased OPG levels. A monoclonal antibody specific for mouse TLR-4/MD-2 partially neutralized the P-LPS effect on cementoblasts. These results indicate that exposure of cementoblasts to P-LPS can alter cell function by regulating markers of osteoclastic activity (e.g., RANKL/OPG), thereby potentially affecting the inflammation-associated resorption of mineralized tissues.

Animals↗
PubMed2001

Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system.

Lipopolysaccharide (LPS) is a key inflammatory mediator. Due to its ability to potently activate host inflammatory and innate defense responses, it has been proposed to function as an important molecule that alerts the host of potential bacterial infection. However, although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, causes a highly unusual host innate host response. It is an agonist for human monocytes and an antagonist for human endothelial cells. Correspondingly, although it activates p38 MAP kinase in human monocytes, P. gingivalis LPS does not activate p38 nor ERK MAP kinase in endothelial cells. In fact, P. gingivalis LPS is an effective inhibitor of Escherichia coli LPS induced p38 phosphorylation. These data show that P. gingivalis LPS modulates host defenses in endothelial cells by interfering with MAP kinase activation. In addition, P. gingivalis LPS is unusual in that it engages TLR-2 but not TLR-4 when examined in stably transfected CHO cell lines. We propose that, since LPS is a key ligand for the human innate host defense system, these unusual properties of P. gingivalis LPS are associated with the bacterium's role in the pathogenesis of periodontitis.

Animals↗
PubMed2001

Soluble CD14 levels in gingival crevicular fluid of subjects with untreated adult periodontitis.

BACKGROUND: This study determined soluble CD14 (sCD14) levels in gingival crevicular fluid (GCF) and their potential relationship to periodontal conditions in adult periodontitis. METHODS: GCF was collected from 15 patients with untreated adult periodontitis. sCD14 levels were determined by ELISA and presented as total amount (ng/site) and concentration (microg/ml). The periodontal examination consisted of plaque index (PI), bleeding index (BI), probing depth (PD), and clinical attachment level (CAL). PD and CAL were measured with an electronic probe. RESULTS: sCD14 was detected in all 15 subjects and was found in 59% (62/105) of the sampled sites. The percentage of sites with sCD14 varied greatly, ranging from 14% to 100%. The mean total amount of sCD14 was 1.71+/-0.40, range 0.03 to 5.41 ng/site; the concentration of sCD14 was 14.04+/-4.15, range 0.16 to 51.74 microg/ml. No significant difference in clinical data was found between the sites with and without detectable levels of sCD14. However, on the basis of the individual profile of sCD14 levels, i.e., those individuals with >50% of the sites containing sCD14 and mean levels of sCD14 >5.0 microg/ml, the 15 subjects were divided into a high sCD14 group (9 subjects) and a low sCD14 group (6 subjects). Compared to the high group, the low group showed greater mean PD and a higher percentage of sites with PD > or = 5.0 mm (P <0.05). Consistent with this, sCD14 concentrations showed a negative correlation with PD (r(s) = -0.636, P = 0.0174). CONCLUSIONS: The present study shows that sCD14 levels in GCF varied greatly among subjects with untreated adult periodontitis. Individuals with higher levels of sCD14 in GCF and more sites containing sCD14 had fewer deep pockets. The negative correlation between GCF sCD14 levels and probing depth implies a crucial role of sCD14 in bacterially induced periodontal destruction. The relationship between GCF sCD14 levels and probing depth warrants further investigations.

Adult↗
PubMed2000

CD14 employs hydrophilic regions to "capture" lipopolysaccharides.

CD14 participates in the host innate inflammatory response to bacterial LPS obtained from Escherichia coli and other Gram-negative bacteria. Evidence from several laboratories suggests that different regions of the amino-terminal portion of the molecule may be involved in LPS binding. In this report a series of single-residue serine replacement and charge reversal mutations were generated to further elucidate the mechanism by which this protein may bind a multitude of different LPS ligands. Single-residue CD14 mutation proteins were examined for their ability to bind LPS obtained from E. coli, Porphyromonas gingivalis, and Helicobacter pylori and facilitate the activation of E-selectin from human endothelial cells. In addition, the single-residue CD14 mutation proteins were employed to perform monoclonal epitope-mapping studies with three LPS-blocking Abs that bound tertiary epitopes. Evidence that several different hydrophilic regions of the amino-terminal region of CD14 are involved in LPS binding was obtained. Epitope-mapping studies revealed that these hydrophilic regions are located on one side of the protein surface. These studies suggest that CD14 employs a charged surface in a manor similar to the macrophage scavenger receptor to "capture" LPS ligands and "present" them to other components of the innate host defense system.

Amino Acid Sequence↗
PubMed2000

Inducible expression of human beta-defensin 2 by Fusobacterium nucleatum in oral epithelial cells: multiple signaling pathways and role of commensal bacteria in innate immunity and the epithelial barrier.

Human gingival epithelial cells (HGE) express two antimicrobial peptides of the beta-defensin family, human beta-defensin 1 (hBD-1) and hBD-2, as well as cytokines and chemokines that contribute to innate immunity. In the present study, the expression and transcriptional regulation of hBD-2 was examined. HBD-2 mRNA was induced by cell wall extract of Fusobacterium nucleatum, an oral commensal microorganism, but not by that of Porphyromonas gingivalis, a periodontal pathogen. HBD-2 mRNA was also induced by the proinflammatory cytokine tumor necrosis factor alpha (TNF-alpha) and phorbol myristate acetate (PMA), an epithelial cell activator. HBD-2 mRNA was also expressed in 14 of 15 noninflamed gingival tissue samples. HBD-2 peptide was detected by immunofluorescence in HGE stimulated with F. nucleatum cell wall, consistent with induction of the mRNA by this stimulant. Kinetic analysis indicates involvement of multiple distinct signaling pathways in the regulation of hBD-2 mRNA; TNF-alpha and F. nucleatum cell wall induced hBD-2 mRNA rapidly (2 to 4 h), while PMA stimulation was slower ( approximately 10 h). In contrast, each stimulant induced interleukin 8 (IL-8) within 1 h. The role of TNF-alpha as an intermediary in F. nucleatum signaling was ruled out by addition of anti-TNF-alpha that did not inhibit hBD-2 induction. However, inhibitor studies show that F. nucleatum stimulation of hBD-2 mRNA requires both new gene transcription and new protein synthesis. Bacterial lipopolysaccharides isolated from Escherichia coli and F. nucleatum were poor stimulants of hBD-2, although they up-regulated IL-8 mRNA. Collectively, our findings show inducible expression of hBD-2 mRNA via multiple pathways in HGE in a pattern that is distinct from that of IL-8 expression. We suggest that different aspects of innate immune responses are differentially regulated and that commensal organisms have a role in stimulating mucosal epithelial cells in maintaining the barrier that contributes to homeostasis and host defense.

Adolescent↗
PubMed1999

P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses.

Cells in the mucosal barrier are equipped to sense and respond to microbes in the lumen and translate this molecular information into signals that can reach local or distant sites. The interaction of P-fimbriated Escherichia coli with human uroepithelial cells is a model to study the molecular mechanism of epithelial cell activation by mucosal pathogens. Here, we examine the role of lipopolysaccharide (LPS) as a co-stimulatory molecule in epithelial cell activation by P-fimbriated E. coli. P-fimbriated clinical isolates or recombinant strains were shown to trigger a fimbriae-dependent epithelial cell cytokine response. Mutational inactivation of the msbB sequences that control lipid A myristoylation drastically impaired monocyte stimulation but not epithelial responses to P-fimbriated bacteria. Polymyxin B or bactericidal/permeability increasing factor (BPI) neutralized the effects of lipid A in the monocyte assay, but did not reduce epithelial responses. Finally, isolated LPS of the smooth, rough and deep rough chemotypes were poor epithelial cell activators. The cells were shown to lack surface CD14 or CD14 mRNA as well as the CD14 co-receptor function and were also very poor LPS responders in the presence of human serum. These results demonstrate that epithelial cell responses to P-fimbriated E. coli are CD14 and LPS independent, and suggest that attaching pathogens can overcome the LPS unresponsiveness of epithelial cells by fimbriae-dependent activation mechanisms.

Acyltransferases↗
PubMed1999

Escherichia coli and Porphyromonas gingivalis lipopolysaccharide interactions with CD14: implications for myeloid and nonmyeloid cell activation.

Porphyromonas gingivalis, a gram-negative bacterium, is an etiologic agent for adult periodontitis. Lipopolysaccharide (LPS) released from this bacterium can react with numerous host cell types. P. gingivalis LPS stimulates tumor necrosis factor alpha and interleukin-1beta secretion from monocytes (myeloid) but does not elicit E-selectin expression from human endothelial cells (nonmyeloid). In contrast, Escherichia coli LPS facilitates expression of these inflammatory mediators through CD14-dependent pathways on both myeloid and nonmyeloid cells. LPS binding studies have revealed that although P. gingivalis and E. coli LPSs bind to CD14 differently, this fact does not adequately explain the lack of endothelial cell activation by P. gingivalis LPS. Rather, LPS binding site and blocking monoclonal antibody epitope mapping studies have suggested that CD14 presents a charged surface that captures different microbial ligands by electrostatic interactions. We propose that human endothelial cells do not respond to P. gingivalis LPS because of their inability to "recognize" CD14-P. gingivalis LPS complexes.

Amino Acid Sequence↗
PubMed1999

Escherichia coli msbB gene as a virulence factor and a therapeutic target.

A mutation in the msbB gene of Escherichia coli results in the synthesis of E. coli lipopolysaccharide (LPS) that lacks the myristic acid moiety of lipid A. Although such mutant E. coli cells and their purified LPS have a greatly reduced ability to stimulate human immune cells, a minor reduction in the mouse inflammatory response is observed. When the msbB mutation is transferred into a clinical isolate of E. coli, there is a significant loss in virulence, as assessed by lethality in BALB/c mice. When a cloned msbB gene is provided to functionally complement the msbB mutant, virulence returns, providing direct evidence that the msbB gene product is an important virulence factor in a murine model of E. coli pathogenicity. In the genetic background of the clinical E. coli isolate, the msbB mutation also results in filamentation of the cells at 37 degrees C but not at 30 degrees C, a reduction in the level of the K1 capsule, an increase in the level of complement C3 deposition, and an increase in both opsonic and nonopsonic phagocytosis of the msbB mutant, phenotypes that can help to explain the loss in virulence. The demonstration that the inhibition of msbB gene function reduces the virulence of E. coli in a mouse infection model warrants further investigation of the msbB gene product as a novel target for antibiotic therapy.

Acyltransferases↗
PubMed1998

Treponema pallidum and Borrelia burgdorferi lipoproteins and synthetic lipopeptides activate monocytic cells via a CD14-dependent pathway distinct from that used by lipopolysaccharide.

Lipoproteins of Treponema pallidum and Borrelia burgdorferi possess potent proinflammatory properties and, thus, have been implicated as major proinflammatory agonists in syphilis and Lyme disease. Here we used purified B. burgdorferi outer surface protein A (OspA) and synthetic lipopeptides corresponding to the N-termini of OspA and the 47-kDa major lipoprotein immunogen of T. pallidum to clarify the contribution of CD14 to monocytic cell activation by spirochetal lipoproteins and lipopeptides. As with LPS, mouse anti-human CD14 Abs blocked the activation of 1,25-dihydroxyvitamin D3-matured human myelomonocytic THP-1 cells by OspA and the two lipopeptides. The existence of a CD14-dependent pathway was corroborated by using undifferentiated THP-1 cells transfected with CD14 and peritoneal macrophages from CD14-deficient BALB/c mice. Unlike LPS, cell activation by lipoproteins and lipopeptides was serum independent and was not augmented by exogenous LPS-binding protein. Two observations constituted evidence that LPS and lipoprotein/lipopeptide signaling proceed via distinct transducing elements downstream of CD14: 1) CHO cells transfected with CD14 were exquisitely sensitive to LPS but were lipoprotein/lipopeptide nonresponsive; and 2) substoichiometric amounts of deacylated LPS that block LPS signaling at a site distal to CD14 failed to antagonize activation by lipoproteins and lipopeptides. The combined results demonstrate that spirochetal lipoproteins and lipopeptides use a CD14-dependent pathway that differs in at least two fundamental respects from the well-characterized LPS recognition pathway.

Acute-Phase Proteins↗
PubMed1998

Lipid A diversity and the innate host response to bacterial infection.

Lipopolysaccharide, a component of the outer membrane of Gram-negative bacteria, is a potent immunostimulatory molecule which activates the innate host defense system. Over the past few years progress has been made in identifying the molecular mechanisms of host recognition of lipid A (a component of lipopolysaccharide), the identification of the genes required for Escherichia coli lipid A biosynthesis, and the role of lipid A acylation when viable bacteria are presented to host cells. Recent data indicate that bacteria can regulate this molecule in response to different host microenvironments. Host factors that induce lipid A modifications and the resultant changes in host response remain to be determined.

Animals↗
PubMed1998

Antigenic components of Actinobacillus actinomycetemcomitans lipopolysaccharide recognized by sera from patients with localized juvenile periodontitis.

The dominant antigen of Actinobacillus actinomycetemcomitans recognized by high-titer sera from patients with localized juvenile periodontitis is the serotype antigen located in the O-side chains of lipopolysaccharide. Whether such sera contain antibodies reactive with other epitopes in lipopolysaccharide, as is the case for patients with rapidly progressive periodontitis, remains unknown. We prepared and characterized by gas liquid chromatography lipopolysaccharide, lipid A, core carbohydrate with no or few O-side chains (core) and high-molecular-mass carbohydrate-rich in O-side chains (oligosaccharide) from A. actinomyce-temcomitans ATCC 43718 (serotype b, Y4). Using enzyme-linked immunosorbent assay (ELISA), sera from 36 patients with localized juvenile periodontitis were surveyed using whole-cell sonicate as plate antigen. The seven highest titer sera were selected for further study. Specific IgG antibody binding was observed to intact lipopolysaccharide and to all the lipopolysaccharide fractions. The mean titers were highest for intact lipopolysaccharide (138.8 ELISA units), and lipid A (122 ELISA units), followed by the core fraction (81 ELISA units) and the oligosaccharide fraction (69.5 ELISA units). ELISA inhibition revealed that the core fraction at a concentration of 10 micrograms/test well inhibited antibody binding to A. actinomycetemcomitans lipopolysaccharide by a mean value of 56.7%. To further characterize antibody binding to the core fraction, ELISA inhibition was performed using as inhibitor the core carbohydrate fraction of the Re mutant of Salmonella minnesota, which is known to contain only alpha-keto-3-deoxyoctonate residues and phosphate. This fraction at 10 micrograms/test well inhibited binding of antibodies from 6 of 7 test sera with a mean value of 49.2%. Thus, sera from patients with localized juvenile periodontitis contain antibodies that bind to the O-side chains of lipopolysaccharide, as has been previously reported, but they also contain antibodies that bind to lipid A and to lipopolysaccharide core polysaccharide epitopes, specifically to alpha-keto-3-deoxyoctonate moieties. The humoral immune response to A. actinomycetemcomitans in patients with localized juvenile periodontitis is more complex than previously reported and is very similar to that of patients with rapidly progressive periodontitis.

Adolescent↗
PubMed1998

Local chemokine paralysis, a novel pathogenic mechanism for Porphyromonas gingivalis.

Periodontitis, which is widespread in the adult population, is a persistent bacterial infection associated with Porphyromonas gingivalis. Gingival epithelial cells are among the first cells encountered by both P. gingivalis and commensal oral bacteria. The chemokine interleukin 8 (IL-8), a potent chemoattractant and activator of polymorphonuclear leukocytes, was secreted by gingival epithelial cells in response to components of the normal oral flora. In contrast, P. gingivalis was found to strongly inhibit IL-8 accumulation from gingival epithelial cells. Inhibition was associated with a decrease in mRNA for IL-8. Antagonism of IL-8 accumulation did not occur in KB cells, an epithelial cell line that does not support high levels of intracellular invasion by P. gingivalis. Furthermore, a noninvasive mutant of P. gingivalis was unable to antagonize IL-8 accumulation. Invasion-dependent destruction of the gingival IL-8 chemokine gradient at sites of P. gingivalis colonization (local chemokine paralysis) will severely impair mucosal defense and represents a novel mechanism for bacterial colonization of host tissue.

Dental Plaque↗
PubMed1997

Regulation of lipid A modifications by Salmonella typhimurium virulence genes phoP-phoQ.

Bacterial pathogenesis requires proteins that sense host microenvironments and respond by regulating virulence gene transcription. For Salmonellae, one such regulatory system is PhoP-PhoQ, which regulates genes required for intracellular survival and resistance to cationic peptides. Analysis by mass spectrometry revealed that Salmonella typhimurium PhoP-PhoQ regulated structural modifications of lipid A, the host signaling portion of lipopolysaccharide (LPS), by the addition of aminoarabinose and 2-hydroxymyristate. Structurally modified lipid A altered LPS-mediated expression of the adhesion molecule E-selectin by endothelial cells and tumor necrosis factor-alpha expression by adherent monocytes. Thus, altered responses to environmentally induced lipid A structural modifications may represent a mechanism for bacteria to gain advantage within host tissues.

Acylation↗
PubMed1997

Recognition of antigenic epitopes in lipopolysaccharide and protein from Actinobacillus actinomycetemcomitans by serum antibodies in untreated rapidly progressive periodontitis patients.

Actinobacillus actinomycetemcomitans has been associated with early-onset periodontitis, including the localized juvenile and rapidly progressive forms. The immunodominant antigens of A. actinomycetemcomitans recognized by rapidly progressive periodontitis patients remain unidentified. Sera from 22 patients with rapidly progressive periodontitis and 20 periodontally normal subjects were tested by enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G antibodies to whole-cell sonicate, protein, purified lipopolysaccharide and lipopolysaccharide fractions of A. actinomycetemcomitans. The median titers of rapidly progressive periodontitis patients and control subjects to whole-cell sonicate were 25.0 and 14.5 ELISA units, respectively (not significantly different). Binding of antibody from patient sera occurred to both the lipopolysaccharide and the protein fractions, with greater binding to lipopolysaccharide than to protein. We show for the first time that patient sera contain antibodies that bind specifically to antigenic epitopes in lipid A and in the core carbohydrate of lipopolysaccharide that were previously considered to be inaccessible and unavailable, as well as to epitopes in the O side chains. Sera manifesting antibody titers 2-fold or greater than the median titer for control sera were judged to be seropositive. More patients were seropositive for lipid A than for any of the other antigen preparations studied, and the median titer for patient sera to lipid A but to none of the other purified lipopolysaccharide fractions was significantly elevated relative to control values. Of 22 patients, 10 were seropositive to whole-cell sonicate, 7 to protein, 8 to lipopolysaccharide, 7 to the high-molecular-weight lipopolysaccharide-polysaccharide fraction rich in O side chains, and 16 to lipid A. The core carbohydrate did not adhere to the test plate surface, and this precluded ELISA measurements. However, when the core carbohydrate was used in the ELISA inhibition assay, it reduced antibody binding to lipopolysaccharide-coated plates by up to 45%, thereby demonstrating antibody binding to core carbohydrate. The core carbohydrate fraction from the Re mutant of Salmonella minnesota known to contain no O-side chains also inhibited binding of specific antibody to plates coated with A actinomycetemcomitans lipopolysaccharide. Overall, there was extreme variation in responses among patients to the various antigen preparations, with no single pattern dominating. Lipopolysaccharide and its components appear to be the immunodominant epitopes, since most rapidly progressive periodontitis patients are seropositive for lipopolysaccharide and/or its components and they have titers relative to those for proteins.

Adult↗
PubMed1997

Identification of CD14 residues involved in specific lipopolysaccharide recognition.

CD14 is a key molecule responsible for the innate host inflammatory response to microbial infection. It is able to bind a wide variety of microbial ligands and facilitate the activation of both myeloid and nonmyeloid cells. However, its specific contribution to the innate recognition of bacteria is not known. Presently there is no information on the contribution of individual CD14 residues to Escherichia coli lipopolysaccharide (LPS) binding or on the molecular basis of the interaction between CD14 and LPS from other bacteria. LPS obtained from Porphyromonas gingivalis, a bacterium associated with chronic inflammatory disease, binds CD14 and activates myeloid cells but does not facilitate the activation of nonmyeloid cells. The transfer and binding of these two LPS species to soluble CD14 recombinant globulin proteins with single point mutations was examined. Functional activity of the mutant proteins was monitored by E-selectin expression on human umbilical cord endothelial cells. The analysis identified a charge reversal mutation in a single residue, E47, that demonstrated selective binding to E. coli LPS but not to P. gingivalis LPS. E-selectin activation assays indicated that proteins with mutations at position E47 maintained their structural integrity. Other mutations, including a charge reversal mutation of residue E58, did not significantly reduce the binding of either LPS ligand or the ability of the molecule to facilitate E-selectin activation. These data demonstrate that CD14 can selectively recognize different LPS ligands.

Amino Acid Sequence↗