Development of an in vitro periodontal biofilm model for assessing antimicrobial and host modulatory effects of bioactive molecules

Background: Inflammation within the oral cavity occurs due to dysregulation between microbial biofilms and the host response. Understanding how different oral hygiene products influence inflammatory properties is important for the development of new products. Therefore, creation of a robust host-pathogen biofilm platform capable of evaluating novel oral healthcare compounds is an attractive option. We therefore devised a multi-species biofilm co-culture model to evaluate the naturally derived polyphenol resveratrol (RSV) and gold standard chlorhexidine (CHX) with respect to anti-biofilm and anti-inflammatory properties.<p></p> Methods: An in vitro multi-species biofilm containing <i>S. mitis, F. nucleatum, P. Gingivalis</i> and <i>A. Actinomycetemcomitans</i> was created to represent a disease-associated biofilm and the oral epithelial cell in OKF6-TERT2. Cytotoxicity studies were performed using RSV and CHX. Multi-species biofilms were either treated with either molecule, or alternatively epithelial cells were treated with these prior to biofilm co-culture. Biofilm composition was evaluated and inflammatory responses quantified at a transcriptional and protein level.<p></p> Results: CHX was toxic to epithelial cells and multi-species biofilms at concentrations ranging from 0.01-0.2%. RSV did not effect multi-species biofilm composition, but was toxic to epithelial cells at concentrations greater than 0.01%. In co-culture, CHX-treated biofilms resulted in down regulation of the inflammatory chemokine IL-8 at both mRNA and protein level. RSV-treated epithelial cells in co-culture were down-regulated in the release of IL-8 protein, but not mRNA.<p></p> Conclusions: CHX possesses potent bactericidal properties, which may impact downstream inflammatory mediators. RSV does not appear to have bactericidal properties against multi-species biofilms, however it did appear to supress epithelial cells from releasing inflammatory mediators. This study demonstrates the potential to understand the mechanisms by which different oral hygiene products may influence gingival inflammation, thereby validating the use of a biofilm co-culture model.<p></p&gt

Significant variation in transformation frequency in Streptococcus pneumoniae

The naturally transformable bacterium Streptococcus pneumoniae is able to take up extracellular DNA and incorporate it into its genome. Maintaining natural transformation within a species requires that the benefits of transformation outweigh its costs. Although much is known about the distribution of natural transformation among bacterial species, little is known about the degree to which transformation frequencies vary within species. Here we find that there is significant variation in transformation frequency between strains of Streptococcus pneumoniae isolated from asymptomatic carriage, and that this variation is not concordant with isolate genetic relatedness. Polymorphism in the signalling system regulating competence is also not causally related to differences in transformation frequency, although this polymorphism does influence the degree of genetic admixture experienced by bacterial strains. These data suggest that bacteria can evolve new transformation frequencies over short evolutionary timescales. This facility may permit cells to balance the potential costs and benefits of transformation by regulating transformation frequency in response to environmental conditions

Chemical Analysis of Cellular and Extracellular Carbohydrates of a Biofilm-Forming Strain Pseudomonas aeruginosa PA14

Background: Pseudomonas aeruginosa is a Gram-negative bacterium and an opportunistic pathogen, which causes persisting life-threatening infections in cystic fibrosis (CF) patients. Biofilm mode of growth facilitates its survival in a variety of environments. Most P. aeruginosa isolates, including the non-mucoid laboratory strain PA14, are able to form a thick pellicle, which results in a surface-associated biofilm at the air-liquid (A\ufffdL) interface in standing liquid cultures. Exopolysaccharides (EPS) are considered as key components in the formation of this biofilm pellicle. In the non-mucoid P. aeruginosa strain PA14, the \ufffd\ufffdscaffolding\ufffd\ufffd polysaccharides of the biofilm matrix, and the molecules responsible for the structural integrity of rigid A\ufffdL biofilm have not been identified. Moreover, the role of LPS in this process is unclear, and the chemical structure of the LPS O-antigen of PA14 has not yet been elucidated. Principal Findings: In the present work we carried out a systematic analysis of cellular and extracellular (EC) carbohydrates of P. aeruginosa PA14. We also elucidated the chemical structure of the LPS O-antigen by chemical methods and 2-D NMR spectroscopy. Our results showed that it is composed of linear trisaccharide repeating units, identical to those described for P. aeruginosa Lanyi type O:2a,c (Lanyi-Bergman O-serogroup 10a, 10c; IATS serotype 19) and having the following structure: -4)-a-L-GalNAcA-(1\ufffd3)-a-D-QuiNAc-(1\ufffd3)- a-L-Rha-(1-. Furthermore, an EC O-antigen polysaccharide (EC O-PS) and the glycerol-phosphorylated cyclic b-(1,3)-glucans were identified in the culture supernatant of PA14, grown statically in minimal medium. Finally, the extracellular matrix of the thick biofilm formed at the A-L interface contained, in addition to eDNA, important quantities (at least ,20% of dry weight) of LPS-like material. Conclusions: We characterized the chemical structure of the LPS O-antigen and showed that the O-antigen polysaccharide is an abundant extracellular carbohydrate of PA14. We present evidence that LPS-like material is found as a component of a biofilm matrix of P. aeruginosa.Peer reviewed: YesNRC publication: Ye

Identification of the Pangenome and Its Components in 14 Distinct Aggregatibacter actinomycetemcomitans Strains by Comparative Genomic Analysis

Aggregatibacter actinomycetemcomitans is genetically heterogeneous and comprises distinct clonal lineages that may have different virulence potentials. However, limited information of the strain-to-strain genomic variations is available.The genome sequences of 11 A. actinomycetemcomitans strains (serotypes a-f) were generated de novo, annotated and combined with three previously sequenced genomes (serotypes a-c) for comparative genomic analysis. Two major groups were identified; serotypes a, d, e, and f, and serotypes b and c. A serotype e strain was found to be distinct from both groups. The size of the pangenome was 3,301 genes, which included 2,034 core genes and 1,267 flexible genes. The number of core genes is estimated to stabilize at 2,060, while the size of the pangenome is estimated to increase by 16 genes with every additional strain sequenced in the future. Within each strain 16.7-29.4% of the genome belonged to the flexible gene pool. Between any two strains 0.4-19.5% of the genomes were different. The genomic differences were occasionally greater for strains of the same serotypes than strains of different serotypes. Furthermore, 171 genomic islands were identified. Cumulatively, 777 strain-specific genes were found on these islands and represented 61% of the flexible gene pool.Substantial genomic differences were detected among A. actinomycetemcomitans strains. Genomic islands account for more than half of the flexible genes. The phenotype and virulence of A. actinomycetemcomitans may not be defined by any single strain. Moreover, the genomic variation within each clonal lineage of A. actinomycetemcomitans (as defined by serotype grouping) may be greater than between clonal lineages. The large genomic data set in this study will be useful to further examine the molecular basis of variable virulence among A. actinomycetemcomitans strains

Influence of angiogenetic factors and matrix metalloproteinases upon tumour progression in non-small-cell lung cancer

We attempted to investigate immunohistochemical expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PD-ECGF), c-erbB-2, matrix metalloproteinase-2 (MMP-2), and MMP-9 using surgical specimens of 119 non-small-cell lung carcinoma (NSCLC) cases and to evaluate the relationship between the expression levels of each molecule and clinicopathological factors or prognosis. VEGF expression levels were significantly associated with the local invasion (P = 0.0001), lymph node involvement (pN-factor) (P = 0.0019), pathological stage (p-stage) (P = 0.0027) and lymphatic permeation (P = 0.0389). PD-ECGF expression levels were associated with pN-factor (P = 0.0347). MMP-2 expression levels were associated with pN-factor (P = 0.004) and lymphatic permeation (P = 0.0056). Also, MMP-9 expression levels showed a significant correlation to local invasion (P = 0.0012), pN-factor (P = 0.0093) and p-stage (P = 0.0142). Multivariate analysis showed VEGF to be the most related to local invasion (P = 0.0084), and MMP-2 was the only factor with significant independent impact on lymphatic permeation (P = 0.0228). Furthermore, log-rank analysis showed significant association with poor survival by VEGF, bFGF, MMP-2 and MMP-9. Especially, combined overexpression of VEGF and MMP-2 revealed poor prognosis, our study might provide a basis for the better evaluation of biological characteristics and a new therapeutic strategy based on chemotherapy. © 2001 Cancer Research Campaign http://www.bjcancer.co

Luteinising hormone-releasing hormone analogue reverses the cell adhesion profile of EGFR overexpressing DU-145 human prostate carcinoma subline

Cetrorelix, a luteinising hormone-releasing hormone (LHRH) analogue, has been shown to limit growth of the human androgen-independent prostate cell line DU-145, although other inhibitory actions may also be affected. Both growth and invasion of DU-145 cells are linked to autocrine epidermal growth factor receptor (EGFR) signalling. Invasiveness requires not only cells to migrate to conduits, but also reduced adhesiveness between tumour cells to enable separation from the tumour mass. Thus, we investigated whether Cetrorelix alters the DU-145 cell–cell adhesion and if this occurs via altered EGFR signalling. Pharmacologic levels of Cetrorelix limited the invasiveness of a highly invasive DU-145 subline overexpressing full-length EGFR (DU-145 WT). Extended exposure of the cells to Cetrorelix resulted in increased levels of the cell–cell adhesion complex molecules E-cadherin, α- and β-catenin, and p120. Puromycin blocked the increases in E-cadherin and β-catenin levels, suggesting that de novo protein synthesis is required. The Cetrorelix effect appears to occur via transmodulation of EGFR by a protein kinase C (PKC)-dependent mechanism, as there were no changes in DU-145 cells expressing EGFR engineered to negate the PKC transattenuation site (DU-145 A654); downregulation of EGFR signalling produced a similar upregulation in adhesion complex proteins, further suggesting a role for autocrine signalling. Cetrorelix increased the cell–cell adhesiveness of DU-145 WT cells to an extent similar to that seen when autocrine EGFR signalling is blocked; as expected, DU-145 A654 cell–cell adhesion also was unaffected by Cetrorelix. The increased adhesiveness is expected as the adhesion complex molecules moved to the cells' periphery. These data offer direct insight into the possible crosstalk pathways between the LHRH and EGFR receptor signalling. The ability of Cetrorelix to downregulate EGFR signalling and subsequently reverse the antiadhesiveness found in metastatic prostate cancer highlights a novel potential target for therapeutic strategies

Patterning of the Dorsal-Ventral Axis in Echinoderms: Insights into the Evolution of the BMP-Chordin Signaling Network

Formation of the dorsal-ventral axis of the sea urchin embryo relies on cell interactions initiated by the TGF beta Nodal. Intriguingly, although nodal expression is restricted to the ventral side of the embryo, Nodal function is required for specification of both the ventral and the dorsal territories and is able to restore both ventral and dorsal regions in nodal morpholino injected embryos. The molecular basis for the long-range organizing activity of Nodal is not understood. In this paper, we provide evidence that the long-range organizing activity of Nodal is assured by a relay molecule synthesized in the ventral ectoderm, then translocated to the opposite side of the embryo. We identified this relay molecule as BMP2/4 based on the following arguments. First, blocking BMP2/4 function eliminated the long-range organizing activity of an activated Nodal receptor in an axis rescue assay. Second, we demonstrate that BMP2/4 and the corresponding type I receptor Alk3/6 functions are both essential for specification of the dorsal region of the embryo. Third, using anti-phospho-Smad1/5/8 immunostaining, we show that, despite its ventral transcription, the BMP2/4 ligand triggers receptor mediated signaling exclusively on the dorsal side of the embryo, one of the most extreme cases of BMP translocation described so far. We further report that the pattern of pSmad1/5/8 is graded along the dorsal-ventral axis and that two BMP2/4 target genes are expressed in nested patterns centered on the region with highest levels of pSmad1/5/8, strongly suggesting that BMP2/4 is acting as a morphogen. We also describe the very unusual ventral co-expression of chordin and bmp2/4 downstream of Nodal and demonstrate that Chordin is largely responsible for the spatial restriction of BMP2/4 signaling to the dorsal side. Thus, unlike in most organisms, in the sea urchin, a single ventral signaling centre is responsible for induction of ventral and dorsal cell fates. Finally, we show that Chordin may not be required for long-range diffusion of BMP2/4, describe a striking dorsal-ventral asymmetry in the expression of Glypican 5, a heparin sulphated proteoglycan that regulates BMP mobility, and show that this asymmetry depends on BMP2/4 signaling. Our study provides new insights into the mechanisms by which positional information is established along the dorsal-ventral axis of the sea urchin embryo, and more generally on how a BMP morphogen gradient is established in a multicellular embryo. From an evolutionary point of view, it highlights that although the genes used for dorsal-ventral patterning are highly conserved in bilateria, there are considerable variations, even among deuterostomes, in the manner these genes are used to shape a BMP morphogen gradient

Clonal Diversity and Stability of Subgingival Eikenella corrodens

Eikenella corrodens is a commensal subgingival bacterium commonly found in both periodontally nondiseased and diseased subjects. The present study examined the clonal diversity and stability of subgingival E. corrodens over time. Ninety-five subjects were enrolled at the baseline examination, including 44 periodontally nondiseased subjects and 51 subjects with aggressive periodontitis. Twenty-two nondiseased subjects and 11 subjects with aggressive periodontitis were subsequently reexamined after an average interval of 14 months. Two subgingival plaque samples were obtained from each subject to determine the total cultivable bacteria. In addition, multiple E. corrodens isolates from each sample were recovered for clonal analysis by arbitrarily primed PCR. The mean numbers of distinct E. corrodens clones harbored by nondiseased subjects and subjects with aggressive periodontitis were 1.3 and 3.0, respectively. Thirty-nine percent of the nondiseased subjects and 63% of the subjects with aggressive periodontitis harbored multiple clones of E. corrodens. The numbers of distinct E. corrodens clones increased significantly (Mann-Whitney ranking test, P < 0.05) in sites from patients with aggressive periodontitis, in sites with pocket depths of 4 mm or greater, in sites with a clinical attachment loss of 2 mm or greater, and in sites coinfected with Porphyromonas gingivalis. Comparison of E. corrodens clones recovered at the baseline and those recovered at the follow-up examination showed that E. corrodens colonization was not stable. Thirty-eight of the 66 follow-up samples (58%) showed a complete change (including de novo colonization of the sites or complete elimination of the organism from the sites) of the subgingival E. corrodens clonal types between the baseline and the follow-up examinations. Our results suggest a complexity of subgingival microbiota not seen previously

Quantitative Real-time PCR based on single copy gene sequence for detection of periodontal pathogens

7 páginas, 3 figuras, 5 tablas -- PAGS nros. 1054-1060Objective: To develop a method for quantification of Actinobacillus actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg) and Tannerella forsythensis (Tf) from subgingival plaque samples based on TaqMan real-time polymerase chain reaction (PCR) technology. Material and Methods: Bacterial cells from these species were obtained after culturing reference strains and were counted microscopically. Cellular suspensions in Tris-EDTA buffer were used for DNA extraction after boiling for 20 min. Primers for PCR were selected from sequences of the LktC (Aa), Arg-gingipain (Pg) and BspA antigen (Tf) genes in order to yield amplicons below 100 bp. TaqMan-based real-time PCR was adjusted to quantify each species separately. Cycle threshold (CT) values were calculated for each species according to the initial number of copies. A reliability analysis was carried out using intra-class correlation coefficients (ICCs) with a two-way random effects model. Results: A high sensitivity and specificity was obtained for the detection of the three bacterial species. The TaqMan real-time PCR technology yielded a good repeatability in the obtained cycle threshold (CT) values for each initial number of copies, demonstrating coefficients of variation below 5% for each bacteria. The reproducibility of the technique was also demonstrated by the high ICCs (>0.98; p<0.00001) obtained for each bacteria with and without the addition of subgingival plaque. Conclusion: A novel diagnostic method based on TaqMan real-time PCR was developed for the quantification of Aa, Pg and Tf. It has demonstrated good sensitivity and repeatability on pure cultures. Its diagnostic utility should be demonstrated in subgingival plaque samplesPeer reviewe

Comparative Outcomes of Transapical Versus Transfemoral Access for Transcatheter Aortic Valve Replacement in Diabetics

INTRODUCTION: The outcomes of transfemoral (TF) compared with transapical (TA) access for transcatheter aortic valve replacement (TAVR) in diabetics are unknown. METHODS: We queried the NIS database (2011-2014) to identify diabetics who underwent TAVR. We performed a propensity matching analysis comparing TF-TAVR versus TA-TAVR. RESULTS: The analysis included 14.555 diabetics who underwent TAVR. After matching, in-hospital mortality was not different between TF-TAVR and TA-TAVR. (3.5 vs. 4.4%, p = 0.11). TF-TAVR was associated with lower rates of cardiogenic shock (2.7 vs. 4.7%, p = 0.02), use of mechanical circulatory support (2.0 vs. 2.9%, p = 0.03), acute renal failure (17.8 vs. 26.5%, p \u3c 0.001), major bleeding (35.8 vs. 40.7%, p \u3c 0.001) and respiratory complications (1.1 vs. 4.4%, p \u3c 0.001) compared with TA-TAVR. However, TF-TAVR was associated with a higher rate of vascular complications (2.9 vs. 0.9%, p \u3c 0.001), cardiac tamponade (0.5 vs. 0.0%, p \u3c 0.001), complete heart block (10.8 vs. 7.7%, p \u3c 0.001) and pacemaker insertion (11.8 vs. 8.3%, p \u3c 0.001). There was no difference between both groups in acute stroke (1.8 vs. 2.2%, p = 0.39), hemodialysis (2.0 vs. 2.2%, p = 0.71), and ventricular arrhythmias (4.9 vs. 4.2%, p = 0.19). Notably, TF-TAVR was associated with higher mortality, acute stroke, AKI, hemodialysis, PCI, and respiratory complications in complicated diabetics compared with non-complicated diabetics. CONCLUSIONS: This observational analysis showed no difference in-hospital mortality between TF-TAVR and TA-TAVR among diabetic patients. Studies exploring the optimal access for TAVR among diabetics are recommended