Interleukin-6 and microRNA profiles induced by oral bacteria in human atheroma derived and healthy smooth muscle cells.

BACKGROUND: Atherosclerosis is an inflammatory disease with possible contributions from bacterial antigens. We aimed to investigate the role of oral bacteria as inducers of inflammatory cascades in smooth muscle cells from carotid endarterectomy patients (AthSMCs) and healthy controls (HSMCs). FINDINGS: Inactivated Streptococcus mitis, S. sanguinis, S. gorgonii, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis were used to stimulate inflammation in HSMCs and AthSMCs. Tumor necrosis factor-α (TNFα) was used as a positive control in all stimulations. Interleukin-6 (IL-6) levels were determined from cell culture supernatants and microRNA expression profiles from cells after 24 h of bacterial stimulation. Genome wide expression (GWE) analyses were performed after 5 h stimulation. All studied bacteria induced pro inflammatory IL-6 production in both SMCs. The most powerful inducer of IL-6 was A. actinomycetemcomitans (p < 0.001). Of the 84 studied miRNAs, expression of 9 miRNAs differed significantly (p ≤ 0.001) between HSMCs and AthSMCs stimulated with inactivated bacteria or TNFα. The data was divided into two groups: high IL-6 producers (A. actinomytectemcomititans and TNFα) and low IL-6 producers (streptococcal strains and P. gingivalis). The expression of 4 miRNAs (miR-181-5p, -186-5p, -28-5p and -155-5p) differed statistically significantly (p < 0.001) between healthy HSMCs and AthSMCs in the low IL-6 producer group. According to multidimensional scaling, two gene expression clusters were seen: one in HSMCs and one AthSMCs. CONCLUSIONS: Our results suggest that inactivated oral bacteria induce inflammation that is differently regulated in healthy and atherosclerotic SMCs.

Bacterial signatures in thrombus aspirates of patients with myocardial infarction.

BACKGROUND: Infectious agents, especially bacteria and their components originating from the oral cavity or respiratory tract, have been suggested to contribute to inflammation in the coronary plaque, leading to rupture and the subsequent development of coronary thrombus. We aimed to measure bacterial DNA in thrombus aspirates of patients with ST-segment-elevation myocardial infarction and to check for a possible association between bacteria findings and oral pathology in the same cohort. METHODS AND RESULTS: Thrombus aspirates and arterial blood from patients with ST-segment-elevation myocardial infarction undergoing primary percutaneous coronary intervention (n=101; 76% male; mean age, 63.3 years) were analyzed with real-time quantitative polymerase chain reaction with specific primers and probes to detect bacterial DNA from several oral species and Chlamydia pneumoniae. The median value for the total amount of bacterial DNA in thrombi was 16 times higher than that found in their blood samples. Bacterial DNA typical for endodontic infection, mainly oral viridans streptococci, was measured in 78.2% of thrombi, and periodontal pathogens were measured in 34.7%. Bacteria-like structures were detected by transmission electron microscopy in all 9 thrombus samples analyzed; whole bacteria were detected in 3 of 9 cases. Monocyte/macrophage markers for bacteria recognition (CD14) and inflammation (CD68) were detected in thrombi (8 of 8) by immunohistochemistry. Among the subgroup of 30 patients with myocardial infarction examined by panoramic tomography, a significant association between the presence of periapical abscesses and oral viridans streptococci DNA-positive thrombi was found (odds ratio, 13.2; 95% confidence interval, 2.11-82.5; P=0.004). CONCLUSIONS: Dental infection and oral bacteria, especially viridans streptococci, may be associated with the development of acute coronary thrombosis.

Behavior of titanium dioxide nanoparticles in Lemna minor growth test conditions.

Titanium dioxide nanoparticles (TiO(2) NPs) have raised concern of environmental risks due to their widespread applications, but little is known about the potential toxicity of TiO(2) NPs to aquatic plants. The aim of this work was to study the effects of TiO(2) NPs on Lemna minor and to study the behavior of TiO(2) NPs under modified ISO 20079 test conditions. TiO(2) NPs had a tendency to aggregate in ISO (Steinberg) growth medium, but modification of the standard growth medium enabled the exposure of L. minor to TiO(2) NPs. By dilution of the growth medium (1:10), and exposure under semi-static conditions with medium renewal every second or third day, the size of TiO(2) particles remained rather stable throughout the test period. TiO(2) NPs showed no adverse effect on the growth rate or chlorophyll a content of L. minor, even at a high exposure concentration of 5 mg L(-1) and extended exposure time of 14 days. TiO(2) NPs attached onto L. minor cell walls, but no cellular uptake was observed. Although TiO(2) NPs were not toxic to L. minor, the potential transfer of TiO(2) NPs in aquatic food chains, e.g. attached to the plant leaves and other biological surfaces may be of importance, causing exposure of other organisms and contributing to the environmental fate of nanoparticles.

Single nucleotide polymorphisms are randomly dispersed and mostly synonymous in partial rpoB and cpn60 genes of Campylobacter showae human isolates.

The partial 16S rRNA, rpoB, and cpn60 genes congruently allow this study to identify all the eight isolates as the species Campylobacter showae. To our knowledge, this is the first report to reveal the interspecies and intraspecies sequence variations present in the three genes of the C. showae isolates.

Chlamydia pneumoniae infection in polarized epithelial cell lines.

We set up a polarized cell culture model to study the pathogenicity of a common respiratory tract pathogen, Chlamydia pneumoniae. Immunofluorescence staining of ZO-1 (a tight junction protein) and Na(+)K(+) ATPase (a protein pump localized at the basolateral membrane in the polarized epithelial cells), as well as TER measurements, suggested that the filter-grown Calu-3 cells, but not the A549 cells, were polarized when grown on collagen-coated membranes. Both the flat and the filter-grown cultures were infected with C. pneumoniae. Infection in the polarized Calu-3 cultures produced more C. pneumoniae genome equivalents than infection in the flat cultures. However, this progeny was not as infective as that in the flat cultures. The maximum amount of C. pneumoniae was detected at 6 days postinfection in the filter-grown A549 cells, indicating a slower developmental cycle than that observed in the flat A549 cultures. The effect of cycloheximide on the growth of C. pneumoniae in the polarized cells was negligible. Furthermore, the infection in the polarized Calu-3 cells was resistant to doxycycline, and several cytokines were released mainly on the apical side of the polarized cells in response to C. pneumoniae infection. These findings indicate that the growth of chlamydiae was altered in the filter-grown epithelial culture system. The diminished production of infective progeny of C. pneumoniae, together with the resistance to doxycycline and polarized secretion of cytokines from the infected Calu-3 cells, suggests that this model is useful for examining epithelial cell responses to C. pneumoniae infection, and it might better resemble in vivo infection in respiratory epithelial cells.

Effects of Norway spruce (Picea abies) resin on cell wall and cell membrane of Staphylococcus aureus.

Resin salve prepared from Norway spruce (Picea abies) has been used for centuries in traditional medicine to treat skin diseases. The authors studied with transmission and scanning electron microscopy, and with electron physiology, changes in cell wall and cell membrane of Staphylococcus aureus after exposure of the bacterial cultures to resin. After exposure, cell wall thickening, cell aggregation, changed branching of fatty acids, and dissipation of membrane potential of the bacterial cells were observed. The authors conclude that spruce resin affects the cell viability via changes in the cell wall and membrane, and impairs, thereby, the synthesis of energy in the bacteria.

Comparison of polymerase chain reaction methods, in situ hybridization, and enzyme immunoassay for detection of Chlamydia pneumoniae in atherosclerotic carotid plaques.

Chlamydia pneumoniae has been associated with cardiovascular diseases and has been shown by different methods to be present in atherosclerotic lesions. However, not all studies have found C. pneumoniae in atherosclerotic tissues. We compared polymerase chain reaction (PCR) methods, in situ hybridization (ISH), and measurement of chlamydial lipopolysaccharide (cLPS) by enzyme immunoassay (EIA) from homogenized atherosclerotic tissue in the detection of C. pneumoniae. In a study population of 110 patients with carotid artery disease, cLPS was found in 22.2%, and DNA by PCR was found in 34.3% and by ISH in 39.4% of the samples. Poor repeatability was shown to complicate PCR, and the technical problems inherent in ISH were not insignificant. In contrast, the cLPS EIA test was fast and easy to perform. If the sensitivity could be increased, for example, by testing multiple tissue pieces, cLPS EIA might provide a standardized commercial method for the detection of chlamydia in tissue samples, and it, thus, merits further characterization and validation in different patient populations.

Cooperation of isoforms of laminin-332 and tenascin-CL during early adhesion and spreading of immortalized human corneal epithelial cells.

The repair of corneal wounds requires both epithelial cell adhesion and migration. We have studied the early adhesion process of immortalized human corneal epithelial (HCE) cells and show by field emission scanning electron microscopy (FESEM) that the cells first adhere via foot-like process to the growth substratum and later present lamellar spreading. During early adhesion indirect immunofluorescence showed that the cells codeposited laminin (Lm) -332 and the large subunit of tenascin-C (Tn-CL) as a demarcated plaque beneath the cells. Instead, unprocessed Lm-332 (alpha 3'32) was found in a wider area in cells showing lamellar spreading and was also prominently expressed in the cytoplasm of the migrating marginal cells in the in vitro wounded HCE cultures. Confocal laser scanning microscopy (CLSM) showed that the Golgi apparatus was located to the vicinity of the Lm-332/Tn-CL-containing adhesion plaque and accordingly treatment of the cells with demecolcine, dispersing the Golgi apparatus, prevented the formation of plaques. This suggests that formation of the adhesion plaque depends on a direct vectorial secretion of Lm-332 and Tn-CL to the culture substratum. Instead, cytochalasin B treatment disrupted microfilaments and arborized the cells but did not affect the deposition of Tn-CL/Lm-332 as a plaque beneath the cells. The suggestion was supported by immunoprecipitation experiments which showed that Tn-CL and Lm alpha 3' chain were found in cell-free matrices on the culture substratum of spreading cells but not at all (Tn-CL) or much less (Lm-332) in the culture medium. Quantitative cell adhesion experiments showed that HCE cells did not adhere to plain Tn-C coat and that integrin (Int) alpha(3)beta(1) mediated the adhesion of HCE cells to purified Lm-332 and to Lm-332/Tn-C while Int beta4 did not mediate adhesion to these proteins. Taken together, our data suggest that Lm-332 and Tn-CL cooperate in early adhesion process of HCE cells. Furthermore, the results show that Lm-3'32 isoform functions in the spreading of the cells beyond the early adhesion stage and appears to emerge into HCE cells starting to migrate in experimental wounds.

Destruction of Deinococcus geothermalis biofilm by photocatalytic ALD and sol-gel TiO2 surfaces.

The aim of the present work was to explore possibilities of photocatalytic TiO2 coating for reducing biofilms on non-living surfaces. The model organism, Deinococcus geothermalis, known to initiate growth of durable, colored biofilms on machine surfaces in the paper industry, was allowed to form biofilms on stainless steel, glass and TiO2 film coated glass or titanium. Field emission electron microscopy revealed that the cells in the biofilm formed at 45 degrees C under vigorous shaking were connected to the surface by means of numerous adhesion threads of 0.1-0.3 microm in length. Adjacent cells were connected to one another by threads of 0.5-1 microm in length. An ultrastructural analysis gave no indication for the involvement of amorphous extracellular materials (e.g., slime) in the biofilm. When biofilms on photocatalytic TiO2 surfaces, submerged in water, were exposed to 20 W h m(-2) of 360 nm light, both kinds of adhesion threads were completely destroyed and the D. geothermalis cells were extensively removed (from >10(7) down to below 10(6) cells cm(-2)). TiO2 films prepared by the sol-gel technique were slightly more effective than those prepared by the ALD technique. Doping of the TiO2 with sulfur did not enhance its biofilm-destroying capacity. The results show that photocatalytic TiO2 surfaces have potential as a self-cleaning technology for warm water using industries.

Laminin-10 and Lutheran blood group glycoproteins in adhesion of human endothelial cells.

Laminin alpha5-chain, a constituent of laminins-10 and -11, is expressed in endothelial basement membranes. In this study we evaluated the roles of alpha5 laminins and Lutheran blood group glycoproteins (Lu), recently identified receptors of the laminin alpha5-chain, in the adhesion of human dermal microvascular and pulmonary artery endothelial cells. Field emission scanning electron microscopy and immunohistochemistry showed that the endothelial cells spread on laminin-10 and formed fibronectin-positive fibrillar adhesion structures. Immunoprecipitation results suggested that the cells produced fibronectin, which they could use as adhesion substratum, during the adhesion process. When the protein synthesis during the adhesion was inhibited with cycloheximide, the formation of fibrillar adhesions on laminin-10 was abolished, suggesting that laminin-10 does not stimulate the formation of any adhesion structures. Northern and Western blot analyses showed that the cells expressed M(r) 78,000 and 85,000 isoforms of Lu. Quantitative cell adhesion assays showed that in the endothelial cell adhesion to laminin-10, Lu acted in concert with integrins beta(1) and alpha(v)beta(3), whereas in the adhesion to laminin-10/11, Lu and integrin beta(1) were involved. In the cells adhering to the alpha5 laminins, Lu and the integrins showed uniform cell surface distribution. These findings indicate that alpha5 laminins stimulate endothelial cell adhesion but not the formation of fibrillar or focal adhesions. Lu mediates the adhesion of human endothelial cells to alpha5 laminins in collaboration with integrins beta(1) and alpha(v)beta(3).

Attachment of oral gram-negative anaerobic rods to a smooth titanium surface: an electron microscopy study.

PURPOSE: Attachment of bacteria to titanium may differ not only between bacterial species but also between strains within a species. The aim of the present in vitro study was to examine differences in bacterial attachment using 4 gram-negative anaerobic species of bacteria that are considered potential periodontal pathogens. MATERIALS AND METHODS: The attachment of clinical and laboratory strains (n = 23) representing 2 Fusobacterium nucleatum subspecies, Porphyromonas gingivalis, and Prevotella intermedia to smooth, commercially pure titanium was examined using scanning electron microscopy. RESULTS: All bacterial strains were attached to the smooth titanium surface by their outer membrane. F nucleatum cells were poorly attached to the titanium, unlike P gingivalis or P intermedia cells, but only slight differences were observed in the quantity of attached cells between the strains within each bacterial group. DISCUSSION: In favorable conditions, some anaerobes can attach directly to an inert titanium surface. Microbial adhesion and subsequent colonization on the dental implant surface can lead to infection of the peri-implant tissue. CONCLUSION: The results indicated that the avidity of bacterial attachment to a smooth titanium surface varies between species of oral gram-negative anaerobes but not between strains.

Podocytes are firmly attached to glomerular basement membrane in kidneys with heavy proteinuria.

Glomerular epithelial cells (podocytes) play an important role in the pathogenesis of proteinuria. Podocyte foot process effacement is characteristic for proteinuric kidneys, and genetic defects in podocyte slit diaphragm proteins may cause nephrotic syndrome. In this work, a systematic electron microscopic analysis was performed of the structural changes of podocytes in two important nephrotic kidney diseases, congenital nephrotic syndrome of the Finnish type and minimal-change nephrotic syndrome (MCNS). The results showed that (1) podocyte foot process effacement was present not only in proteinuric glomeruli but also in nonproteinuric MCNS kidneys; (2) podocytes in proteinuric glomeruli did not show detachment from the basement membrane or cell membrane ruptures; (3) the number of pinocytic membrane invaginations in the basal and apical parts of the podocytes was comparable in proteinuric and control kidneys; (4) in proteinuric kidneys, the podocyte slit pore density was decreased by 69 to 80% and up to half of the slits were so "tight" that no visible space between foot processes was seen; thus, the filtration surface area between podocytes was dramatically reduced; and (5) in the narrow MCNS slit pores, nephrin was located in the apical part of the podocyte foot process, indicating vertical transfer of the slit diaphragm complex in proteinuria. In conclusion, these results suggest that protein leakage in the two nephrotic syndromes studied occurs through defective podocyte slits, and the other structural alterations commonly seen in electron microscopy are secondary to, not a prerequisite for, the development of proteinuria.

Paenibacillus stellifer sp. nov., a cyclodextrin-producing species isolated from paperboard.

The microflora isolated from food-packaging board is dominated by paenibacilli; a number of these micro-organisms have been characterized using a polyphasic approach. The highest 16S rRNA gene similarity was found between these isolates and Paenibacillus azotofixans ATCC 35681(T) (97.7 %). The main fatty acid of the paperboard isolates was C(16 : 0) (34-45 %); straight-chain fatty acids made up 41-60 % of the total cellular fatty acids, thus distinguishing these strains from other Paenibacillus species. The paperboard isolates produced cyclodextrins from starch. The spore surface had a characteristic ribbed ornamentation. Spores and vegetative cells frequently had pilus-like appendages. Based on phylogenetic data and phenotypic and chemotaxonomic characteristics, it is proposed that the isolates represent a novel species, Paenibacillus stellifer sp. nov., with IS 1(T) (=DSM 14472(T)=CCUG 45566(T)) as the type strain.

Entacapone does not induce conformational changes in liver mitochondria or skeletal muscle in vivo.

Entacapone and tolcapone, novel catechol-O-methyl-transferase (COMT) inhibitors, have been developed for the treatment of Parkinson's disease in combination with levodopa. Three fatal cases of drug-induced hepatitis, one with hepatic necrosis and mitochondrial changes have been reported in clinical use of tolcapone. In vitro tolcapone has been shown to induce uncoupling of oxidative phosphorylation. Liver and skeletal muscle tissues from an oral rat toxicity study were used to investigate the influence of entacapone, tolcapone (300 and 500 mg/kg/day) or a known uncoupling agent, 2,4-dinitrophenol (DNP), (20 mg/kg/ day) on the cell morphology. Centrolobular hypertrophy was revealed in the histopathology of the liver in tolcapone-treated rats. Transmission electron microscopy (TEM) of the liver and skeletal muscle tissue, revealed mitochondrial swelling and reduced matrix density with deformation of cristae in the tolcapone and DNP groups. Intermyofibrillar edema was characteristic of the skeletal muscle tissue of DNP- and tolcapone-exposed animals. In the tolcapone group, also the sarcomeres were prominent. Treatment-related light microscopic or TEM findings were not observed either in entacapone-treated or control animals. The similarity of structural damages induced by both tolcapone- and DNP suggests that uncoupling of oxidative phosphorylation may contribute to the toxicity of tolcapone in the rat.

Permeabilizing action of polyethyleneimine on Salmonella typhimurium involves disruption of the outer membrane and interactions with lipopolysaccharide.

Polyethyleneimine (PEI), a polycationic polymer substance used in various bioprocesses as a flocculating agent and to immobilize enzymes, was recently shown to make Gram-negative bacteria permeable to hydrophobic antibiotics and to detergents. Because this suggests impairment of the protective function of the outer membrane (OM), the effect of PEI on the ultrastructure of Salmonella typhimurium was investigated. Massive alterations in the OM of PEI-treated and thin-sectioned bacteria were observed by electron microscopy. Vesicular structures were seen on the surface of the OM, but no liberation of the membrane or its fragments was evident. Since a potential mechanism for the action of PEI could be its binding to anionic LPSs on the OM surface, the interaction of PEI with isolated LPSs was assayed in vitro. The solubility of smooth-type LPSs of Salmonella, regardless of the sugar composition of their O-specific chains, was not affected by PEI, nor was that of Ra-LPS (lacking O-specific chains but having a complete core oligosaccharide). PEI strongly decreased the solubility of rough-type LPSs of the chemotypes Rb2 and Re, whereas it had only a weak effect on the abnormally cationic Rb2-type pmrA mutant LPS, suggesting that the negative charge to mass ratio of LPS plays a critical role in the interaction.

Crystalline surface protein of Peptostreptococcus anaerobius.

The surface ultrastructure of three anaerobic Gram-positive cocci frequently encountered in oral infections, Peptostreptococcus micros, P. magnus and P. anaerobius, was studied. The type strains of P. micros (DSM 20468) and P. anaerobius (ATCC 27337), several clinical isolates of both species and the type strain of P. magnus (DSM 20470) were included. Thin-sectioned cells studied by electron microscopy revealed a homogeneous layer outside the peptidoglycan layer in P. anaerobius. In P. micros and P. magnus a more amorphous layer was present. No periodic structures were seen in negatively stained whole cells of these three species. However, in freeze-etched cells of P. anaerobius a crystalline surface protein layer (S-layer) was detected. No periodicity was seen in any of the P. micros strains or the P. magnus type strain by the methods used, but a periodic pattern was observed in negatively stained specimens of cell wall fragments of sonicated P. anaerobius cells. No capsular material was visible outside the S-layer in P. anaerobius. The cells of the Peptostreptococcus spp. were extracted for 30 min with detergents and urea. One per cent SDS and M urea both extracted a major 78 kDa protein from all strains of P anaerobius. Extraction of P. micros and P. magnus cells did not reveal any major protein bands comparable to that of P. anaerobius. Surface biotinylation of cells followed by Western blotting and detection by alkaline-phosphatase-conjugated extravidin showed strong staining of the 78 kDa band in P. anaerobius, further indicating that this molecule is located on the surface of the cell and is the S-protein.(ABSTRACT TRUNCATED AT 250 WORDS)