Porphyromonas gingivalis and its lipopolysaccharide differently modulate epidermal growth factor-dependent signaling in human gingival epithelial cells.

Periodontitis is an inflammatory disease induced by pathogenic bacteria such as Porphyromonas gingivalis. Little is known about epidermal growth factor (EGF) signals in human gingival epithelial cells (HGEC), which are major targets of P. gingivalis, and how the expression of proteins participating in EGF signaling-that is, EGF-receptor (EGFR), suppressor of cytokine signaling-3 (SOCS-3), interferon regulatory factor-1 (IRF-1), and signal transducers and activators of transcription (STAT-3)-are modified. This study aimed to assess the effects of P. gingivalis and its purified lipopolysaccharide (LPS-Pg) on EGF signaling. HGEC were infected for 2 h in a dose-dependent manner with P. gingivalis and with heat-killed P. gingivalis, and activated for 2 and 24 h by 1 µg/mL of purified LPS-Pg. Quantitative reverse transcription polymerase chain reaction and Western blotting were performed to measure mRNA and protein levels for SOCS-3, IRF-1 EGF, EGFR, and STAT-3. The tyrosine-phosphorylation status of STAT-3 was also examined. The results showed that infection of HGEC cells with P. gingivalis, but not with heat-killed P. gingivalis, led to significant reductions in expression levels of mRNAs and proteins for SOCS-3, IRF-1, and EGFR, while LPS-Pg over time significantly increased the expression of these mRNAs and proteins. Tyrosine-phosphorylation of STAT-3 was significantly increased during infection with P. gingivalis and activation by LPS-Pg but not modified during infection with heat-killed P. gingivalis. This study highlights that P. gingivalis and its purified LPS differentially modulated the expression of proteins (SOCS-3, IRF-1, EGFR, and STAT-3) interfering with EGF signaling.

Porphyromonas gingivalis and its lipopolysaccharide differentially regulate the expression of cathepsin B in endothelial cells.

Porphyromonas gingivalis infection and cathepsins protease upregulation are independently implicated in atherosclerosis worsening. In this study, we evaluated the effects of P. gingivalis infection and P. gingivalis -purified lipopolysaccharide (Pg-LPS) stimulation on the expression of cathepsin B (CATB) in endothelial cells (ECs). Analysis of the enzymatic activity and expression of CATB were investigated at the messenger RNA, protein and protein-phosphorylation levels. Effects of Toll-like receptors 2 and 4 blocking on CATB activity were also analysed. Our results showed that P. gingivalis and Pg-LPS significantly increased the activity of CATB but with different kinetics. The peak of CATB activity was observed 3 h after P. gingivalis infection but it appeared 48 h after Pg-LPS stimulation. The increase of CATB activity was related to its rapid tyrosine-dephosphorylation during P. gingivalis infection, whereas the levels of CATB messenger RNAs and proteins did not vary after P. gingivalis infection or Pg-LPS stimulation. Inhibition of Toll-like-receptors 2 and 4 differentially decreased P. gingivalis and Pg-LPS CATB activations. These results showed for the first time that P. gingivalis infection rapidly affects ECs and modulates CATB activity, whereas Pg-LPS effects appear to be delayed. This study suggests that direct infection of ECs by P. gingivalis may worsen atherosclerotic plaque formation via activation of the CATB pathway.

Variable cell responses to P. gingivalis lipopolysaccharide.

Porphyromonas gingivalis is a major etiological agent of chronic periodontal diseases, the virulence of which has been attributed to different factors, including lipopolysaccharide (LPS). We investigated the differential responses induced by P. gingivalis LPS stimulation of human umbilical vein endothelial cells and human oral epithelial cells. RT-PCR analysis showed that P. gingivalis LPS used Toll-like receptor 2 (TLR2) to activate epithelial cells and Toll-like receptor 4 (TLR4) to activate endothelial cells. Both cell types were stimulated by P. gingivalis LPS to produce pro-inflammatory cytokines. Cytokine Array assay showed that although patterns of cytokine expression were similar in both cell types, some cytokines were specifically secreted by the endothelial cells, and others were specific to epithelial cells. These results support the idea that the same LPS preparation can act as a TLR2 or TLR4 agonist, depending on TLR expression of the host cell, inducing cytokine profiles that differ according to the cell type.

P. gingivalis regulates the expression of Cathepsin B and Cystatin C.

Porphyromonas gingivalis is a major etiological agent of periodontitis that could affect the expression of Cathepsins B and C by disrupting the balance between these enzymes and their inhibitor, Cystatin C. We tested this hypothesis by infecting human oral epithelial cells with P. gingivalis or activating solely by its lipopolysaccharide. The mRNA level, the enzymatic activity, and the protein expression of Cathepsin B were increased (three-fold) in a dose-dependent manner, while those of Cystatin C decreased (five-fold). No changes were observed for Cathepsin C. Although activation by lipopolysaccharides led to a delayed imbalance (2 days) between Cathepsin B and Cystatin C, this imbalance took place very rapidly during the infection (< 6 hrs), indicating that the whole bacterium contains components that initiate rapid changes in the transcription rates of Cathepsin B and Cystatin C and selectively modify the molecular pathways that lead to this imbalance.

Prevalence of periodontal pathogens in subgingival lesions, atherosclerotic plaques and healthy blood vessels: a preliminary study.

BACKGROUND AND OBJECTIVE: Previous studies have reported different periodontal bacteria in atherosclerotic lesions, but their involvement in plaque formation remains unclear. The aim of the present study was to investigate the presence of 20 periodontal bacteria in atherosclerotic samples and healthy blood vessels (used as controls) and to clarify their relationship in regard to clinical and bacteriological periodontal status. MATERIAL AND METHODS: The day before vascular surgery the patients had a thorough periodontal examination and bacteriological samples were taken from periodontally diseased sites. Atheromatous plaques, internal mammary arteries and saphenous veins were harvested during surgery. A DNA-DNA hybridization procedure was used to screen periodontal and vascular samples for the 20 selected bacterial species. RESULTS: Periodontal samples from the severe periodontitis group were found to have a higher prevalence and biomass of bacterial species than the moderate periodontitis group. In vessel samples, the prevalence of the same 20 bacterial species analyzed together was similar in the two groups, except for saphenous veins. CONCLUSION: The presence of periodontal pathogens in atherosclerotic plaques and in apparently healthy vessels appeared to reflect a higher level of bacteremia rather than infection of endothelial cells.

Human osteoblast response to pulsed laser deposited calcium phosphate coatings.

Octacalcium phosphate (OCP) and Mn(2+)-doped carbonate hydroxyapatite (Mn-CHA) thin films were deposited on pure, highly polished and chemically etched Ti substrates with pulsed laser deposition. The coatings exhibit different composition, crystallinity and morphology that might affect their osteoconductivity. Human osteoblasts were cultured on the surfaces of OCP and Mn-CHA thin films, and the cell attachment, proliferation and differentiation were evaluated up to 21 days. The cells showed a normal morphology and a very good rate of proliferation and viability in every experimental time. Alkaline phosphatase activity was always higher than the control and Ti groups. From days 7 to 21 collagen type I production was higher in comparison with control and Ti groups. The level of transforming growth factor beta 1 (TGF-beta1) was lower at 3 and 7 days, but reached the highest values during following experimental times (14 and 21 days). Our data demonstrate that both calcium phosphate coatings favour osteoblasts proliferation, activation of their metabolism and differentiation.

Expression of RNAs encoding for alpha and beta integrin subunits in periodontitis and in cyclosporin A gingival overgrowth.

BACKGROUND: Variation of integrin expression in healthy and diseased gingiva revealed a potential biological role for these cell matrix receptors during gingival remodeling. AIM: Here we determined the level of RNA and tissue localization of different integrin subunits in periodontitis and cyclosporin A-induced gingival overgrowth. METHODS: The level of expression was determined by Reverse Transcriptase Polymerase Chain Reaction in 12 periodontitis-affected patients, four patients exhibiting severe cyclosporin A-induced gingival overgrowth and seven healthy patients as controls. RESULTS: The RNA encoding for beta1, alpha2 and alpha5 integrin subunits were reduced in periodontitis gingiva. The reduction observed was stronger in cyclosporin A-treated patients as compared to the healthy controls, while RNA encoding for alpha1 subunit was increased. The RNA encoding for alpha6 integrin was only reduced in cyclosporin A-treated gingiva. Immunohistochemistry showed that i) integrin alpha2 expression is restricted to the gingival epithelium of cyclosporin A-treated patients, ii) the reduction of alpha6 integrin expression in cyclosporin A-treated gingiva is due to loss of expression at focal contacts and iii) beta1 integrin is evenly distributed in the three populations with an intensity decrease in periodontitis and cyclosporin A-treated gingiva. CONCLUSION: Taken together these results showed a role for the integrin receptors in periodontal diseases and cyclosporin A-induced gingival overgrowth.

Cathepsin C, matrix metalloproteinases, and their tissue inhibitors in gingiva and gingival crevicular fluid from periodontitis-affected patients.

Successive active phases observed in periodontal diseases may be explained either by a sudden activation of the pro-forms of tissue-stored degradative enzymes such as metalloproteinases (MMPs) or by an imbalance between metalloproteinases and their tissue inhibitors (TIMPs). To discriminate between these two hypotheses, we quantified the levels, the percentage of active form, and the activities of four metalloproteinases (MMPs -1, -2, -3, and -9), as well as the levels of two tissue inhibitors of metalloproteinases (TIMP-1 and -2) and the activity of cathepsin C in tissue extract supernatants and their corresponding gingival crevicular fluid samples collected from periodontitis-affected and healthy patients. Our results supported evidence that tissue destruction results from an imbalance of metalloproteinases over their tissue inhibitors rather than from a sudden activation of the pro-forms of these enzymes. A significant reduction in the activity of cathepsin C also contributed to the degradative process.

Expression of matrix metalloproteinases in healthy and diseased human gingiva.

BACKGROUND, AIMS: The aim of our study was to investigate the patterns of several metalloproteinases (MMP-1, MMP-2 and MT1-MMP) mRNAs expression using a semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and to correlate them with clinical parameters and bacteriological diagnosis in healthy versus diseased human gingiva. METHODS: To identify the cell origin of MMP production, in situ hybridization (ISH) was also performed for the MMPs on the same samples. 17 gingival biopsies were collected (13 affected by advanced periodontitis and 4 healthy used as controls) and plaque index, gingival index, pocket depth and bleeding on probing were measured. Subgingival microbial samples were also collected to be analysed by a DNA probe technique. The biopsies were processed both for RT-PCR and ISH. We also investigated a model for bacterial induced MMP expression in human gingival fibroblasts (HGF) infected by Eikenella corrodens. RESULTS: We found an expression of the mRNA encoding MMP-1 only in diseased gingiva but at low levels relative to beta-actin (mean+/-SD: diseased versus healthy: 0.013+/-0.024 versus 0). Although the frequencies and levels of mRNA encoding for MMP-2 or MT1-MMP are not significantly different between each group (mean+/-SD: 0.329+/-0.344 versus 0.137+/-0.219 for MMP-2; 0.485+/-0.374 versus 0.466+/-0.296 for MT1-MMP), using ISH, we observed an expression of both mRNAs in fibroblasts of pathological specimens at sites that histologically showed signs of chronic inflammation and connective tissue remodelling. In vitro infection of HGF by Eikenella corrodens stimulated 3-fold the production of the mRNA encoding MMP-2 while other mRNAs remained unchanged. CONCLUSION: Our results did not reveal significant differences in the expression of mRNAs encoding for the MMPs between healthy and periodontitis-affected patients, reflecting the great heterogeneity in the periodontal status of individuals. However, they indicate that gingival fibroblasts are an active source of MMP-2 production in response to a periopathogen.

Expression of mRNAs encoding for alpha and beta integrin subunits, MMPs, and TIMPs in stretched human periodontal ligament and gingival fibroblasts.

The biological mechanisms of tooth movement result from the cellular responses of connective tissues to exogenous mechanical forces. Among these responses, the degradation of the extracellular matrix takes place, but the identification of the molecular basis as well as the components implicated in this degradation are poorly understood. To contribute to this identification, we subjected human fibroblasts obtained from the periodontal ligament (PDLs) and from the gingiva (HGFs) to a continuous stretch to quantify the mRNAs encoding for various metalloproteinases (MMPs), their tissue inhibitors (TIMPs), and alpha and beta integrin subunits. Both cell lines reacted by inducing the expression of the mRNAs encoding for MMP-1, MMP-2, TIMP-1, and TIMP-2, while other mRNAs did not vary (MT1-MMP, TIMP-3) or were not expressed (MMP-9). PDLs expressed selectively the mRNAs encoding for alpha4 and alphav, with no difference measurable under stretching, while the mRNAs encoding for alpha6 and beta1 were increased and the one encoding for alpha5 was decreased. HGFs increased the mRNAs encoding for alpha2, alpha6, beta1, and beta3 and decreased the one encoding for alpha3. Analysis of our data indicated that stretched HGFs and PDLs induced the same pattern of mRNAs encoding for MMPs and TIMPs but differed for those encoding various integrin subunits, known to act as protein receptors in mechanotransduction.

Prevalence of six periodontal pathogens detected by DNA probe method in HIV vs non-HIV periodontitis.

OBJECTIVE: The aim of the study was to examine the prevalence of selected periodontal pathogens associated with HIV and non-HIV related periodontal lesions. METHODS: Subgingival plaque samples were obtained from both HIV-seropositive and HIV-seronegative patients affected with periodontal disease. DNA probes were used to detect Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Bacteroides forsythus, Eikenella corrodens and Campylobacter rectus. RESULTS: A actinomycetemcomitans, P. intermedia and B. forsythus (P < 0.05) were more prevalent in HIV-seronegative patients with rapidly progressive periodontitis. Only C. rectus was slightly more prevalent in HIV-seropositive subjects with periodontal diseases, but this was not significant.

Hybricomb. A novel diagnostic tool for DNA probing.

A technique is described in which unlabeled DNA probes are immobilized on the plastic surface of a 12-tooth comb and used to capture homologs by a hybridization procedure. In the examples described, cellular DNA from cervical biopsies is chemically labeled using the Chemi-probe system. The sample containing labeled DNA is then hybridized with the immobilized unlabeled probe (reverse hybridization). By means of this technique, human papillomavirus sequences can be detected with a sensitivity comparable to that of radioactive probe procedures. DNA hybrids are visualized as colored spots on each tooth by moving the comb through the reagent solutions of the prefilled developing plate. The whole procedure requires less than 50 minutes hands-on time, and the results are obtained in a few hours. Application of the technique to the detection and typing of mycoplasma DNA is also reported.

Active gamma-carboxylated human factor IX expressed using recombinant DNA techniques.

Factor IX (Christmas factor), a vitamin K-dependent plasma protein made in the liver, functions in the middle phase of the intrinsic pathway of blood coagulation. A functional deficiency of factor IX underlies haemophilia B, a chromosome X-linked recessive disease for which the major therapeutic approach is replacement treatment using factor IX concentrates. The cloning and characterization of the gene for human factor IX would mean that human factor IX could be produced in greater yield and purity through using recombinant DNA techniques. We have now used a human factor IX cDNA clone, inserted into a vaccinia virus-derived vector, to infect human hepatoma cells which normally produce no factor IX, and mouse fibroblasts. Fully active factor IX was produced by the hepatoma cells, whereas the fibroblasts produced a protein less active than natural factor IX, even in the presence of high levels of vitamin K. Human factor IX is extensively post-translationally modified, and thus represents probably the most complex protein produced in active form by recombinant DNA techniques to date. Our study also illustrates the potential of vaccinia virus-based vectors for expressing significant amounts of complex, clinically useful proteins in eukaryotic cells, in addition to its already demonstrated usefulness for producing live recombinant vaccines.

Transcriptional analysis of the adenovirus-5 EIII promoter: absence of sequence specificity for stimulation by EIa gene products.

To identify the adenovirus-5 EIII promoter sequences that are involved in basal level of transcription, a series of promoter deletion mutants were analyzed in vivo by transfection into HeLa cells and in vitro using a HeLa whole cell extract system. Three regions within the EIII promoter were shown to be important for efficient transcription: the TATA sequence, an upstream element centered at -55/-57, and an additional element located between -111 and -233. In vivo transcriptional analysis of EIII promoter deletions in the presence of adenovirus EIa gene products have demonstrated that the same three regions are required for EIa-stimulated transcription. We conclude that there is no sequence element in the EIII promoter between -15 and -233 that is uniquely required for the stimulation of EIII transcription by EIa gene products.

Individual products of the adenovirus 12S and 13S EIa mRNAs stimulate viral EIIa and EIII expression at the transcriptional level.

Recombinant plasmids containing mutant or wild-type adenovirus serotype 2 EIa genes that produce the 12S mRNA alone, the 13S mRNA alone, or both mRNAs were cotransfected into HeLa cells with plasmids containing the viral EIIa or EIII transcription units. The amount of RNA produced from the EIIa and EIII promoters was increased by the products of both the 13S and the 12S RNAs. By measuring the level of specific transcription in nuclei isolated from transfected cells we directly demonstrate that the increased amount of EIIa RNA is due to stimulation of the rate of transcription.

Adenosine diphosphate ribosyltransferase and protein acceptors associated with cytoplasmic free messenger ribonucleoprotein particles.

ADP-ribosyltransferase activity has been characterized in free messenger ribonucleoprotein particles (mRNP) from mouse plasmacytoma cells. This enzymatic activity appears to be associated with the free mRNP and not due to nuclear contamination. The enzyme activity is not stimulated by added DNA or histone H1 and represents 34 per cent of the total cellular ADP-ribosyltransferase activity while the DNA contamination in free mRNP is less than 4 per cent of the total cellular DNA. Moreover, the ADP-ribosyltransferase specific activity per mg of DNA is about 75-fold higher in free mRNP than in the nuclei. During CsCl gradient centrifugation of the cytoplasmic fraction, the ADP-ribosylated material separates out at a buoyant density similar to that of free mRNP. This ADP-ribosyltransferase activity is inhibited by thymidine, nicotinamide and 3-aminobenzamide, while it is highly stimulated by exogenous pancreatic RNase. The in vitro synthesized acid insoluble material is rendered partly soluble by treatment by a proteolytic enzyme or by snake venom phosphodiesterase resulting in phosphoribosyl-AMP formation: the pancreatic RNase does not solubilize this material. Several ADP-ribosylated proteins are detected by lithium dodecylsulfate gel electrophoresis. Such an ADP-ribosyltransferase activity has also been detected in free mRNP from rat liver. It is suggested that this ADP-ribosylation of specific free mRNP proteins may be associated with free mRNP structure and/or with some chemical covalent type of modification rendering mRNA available for translation.

The adenovirus-2 EIIa early gene promoter: sequences required for efficient in vitro and in vivo transcription.

A series of deletion mutants extending from -250 toward the capsite has been constructed in the early promoter region of the adenovirus 2 EIIa gene and tested both in vitro, and in vivo after transfection of HeLa cells, for the ability to act as a template for transcription. A region between positions -94 and -63 upstream from the major EIIa early cap site is essential both in vivo and in vitro for efficient promoter function. By cotransfection of the EIIa deletion mutants with the EIa transcription unit it has been possible to demonstrate that deletion to position -94 does not affect induction of transcription of the EIIa early gene by the EIa transcription unit, but deletion to position -63 results in loss of detectable levels of EIIa early specific RNA. Thus, sequences upstream from position -94 of the EIIa early gene are not involved in the induction of the EIIa early gene by the EIa transcription unit.

Specific in vitro initiation of transcription on the adenovirus type 2 early and late EII transcription units.

Three transcription units are present in the adenovirus type 2 region EII. Transcription units EIIaE and EIIaL encode the mRNA for the 72,000-dalton DNA binding protein, early and late in the lytic cycle, respectively, and transcription unit EIIb encodes the mRNA for the protein that binds to the 5' termini of adenovirus DNA. By using a cell-free transcription system in the presence of purified RNA polymerase B (or II), we have obtained specific initiation of transcription from both the EIIaE promoter, which does not contain a T-A-T-A box, and the EIIaL promoter, which does. In addition, we have identified a new EII T-A-T-A box promoter that is located close to the early non-T-A-T-A box promoter and is used both in vivo and in vitro.

Protein kinases and their protein substrates in free messenger ribonucleoprotein particles and polysomes from mouse plasmacytoma cells.

In free messenger ribonucleoprotein particles (mRNP) and polysomes from plasmacytoma cells, a phosphorylated protein/protein kinase system has been characterized by a combination of oligo(dT)-cellulose chromatography and CsCl isopycnic gradient centrifugation. The presence phosphorylated in vivo has been detected in both types of particles. Endogenous protein phosphorylation occurs in vitro by particle-associated cAMP-independent protein kinase(s) using [gamma-32P]ATP and [gamma-32P]GTP. These kinases are sensitive to hemin action. Analysis of mRNP proteins by gel electrophoresis and autoradiography showed strong analogies between the phosphorylation patterns obtained in vivo and in vitro, suggesting a substrate specificity for the associated enzymes. The phosphorylated proteins have been compared to initiation factors and ribosomal proteins. We have partially purified the cAMP-independent protein kinase activities responsible for the endogenous phosphorylation in free mRNP and polysomes; two activities were identified in free mRNP whereas three activities were found to be associated with polysomes.