Proteomic analysis of a noninvasive human model of acute inflammation and its resolution:the twenty-one day gingivitis model

The 21-day experimental gingivitis model, an established noninvasive model of inflammation in response to increasing bacterial accumulation in humans, is designed to enable the study of both the induction and resolution of inflammation. Here, we have analyzed gingival crevicular fluid, an oral fluid comprising a serum transudate and tissue exudates, by LC-MS/MS using Fourier transform ion cyclotron resonance mass spectrometry and iTRAQ isobaric mass tags, to establish meta-proteomic profiles of inflammation-induced changes in proteins in healthy young volunteers. Across the course of experimentally induced gingivitis, we identified 16 bacterial and 186 human proteins. Although abundances of the bacterial proteins identified did not vary temporally, Fusobacterium outer membrane proteins were detected. Fusobacterium species have previously been associated with periodontal health or disease. The human proteins identified spanned a wide range of compartments (both extracellular and intracellular) and functions, including serum proteins, proteins displaying antibacterial properties, and proteins with functions associated with cellular transcription, DNA binding, the cytoskeleton, cell adhesion, and cilia. PolySNAP3 clustering software was used in a multilayered analytical approach. Clusters of proteins that associated with changes to the clinical parameters included neuronal and synapse associated proteins.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 μM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

Avaliação comparativa da remodelação óssea periimplantar em implantes com junção cone-morse e hexágono externo: estudo clínico randomizado controlado, duplo cego, boca dividida

Background: Several theories, such as the biological width formation, the inflammatory reactions due to the implant-abutment microgap contamination, and the periimplant stress/strain concentration causing bone microdamage accumulation, have been suggested to explain early periimplant bone loss. However, it is yet not well understood to which extent the implant-abutment connection type may influence the remodeling process around dental implants. Aim: to evaluate clinical, bacteriological, and biomechanical parameters related to periimplant bone loss at the crestal region, comparing external hexagon (EH) and Morse-taper (MT) connections. Materials and methods: Twelve patients with totally edentulous mandibles received four custom made Ø 3.8 x 13 mm implants in the interforaminal region of the mandible, with the same design, but different prosthetic connections (two of them EH or MT, randomly placed based on a split-mouth design), and a immediate implant- supported prosthesis. Clinical parameters (periimplant probing pocket depth, modified gingival index and mucosal thickness) were evaluated at 6 sites around the implants, at a 12 month follow-up. The distance from the top of the implant to the first bone-to-implant contact – IT-FBIC was evaluated on standardized digital peri-apical radiographs acquired at 1, 3, 6 and 12 months follow-up. Samples of the subgingival microbiota were collected 1, 3 and 6 months after implant loading. DNA were extracted and used for the quantification of Tanerella forsythia, Porphyromonas gingivalis, Aggragatibacter actinomycetemcomitans, Prevotella intermedia and Fusobacterium nucleatum. Comparison among multiple periods of observation were performed using repeated-measures Analysis of Variance (ANOVA), followed by a Tukey post-hoc test, while two-period based comparisons were made using paired t- test. Further, 36 computer-tomographic based finite element (FE) models were accomplished, simulating each patient in 3 loading conditions. The results for the peak EQV strain in periimplant bone were interpreted by means of a general linear model (ANOVA). Results: The variation in periimplant bone loss assessed by means of radiographs was significantly different between the connection types (P<0.001). Mean IT-FBIC was 1.17±0.44 mm for EH, and 0.17±0.54 mm for MT, considering all evaluated time periods. All clinical parameters presented not significant differences. No significant microbiological differences could be observed between both connection types. Most of the collected samples had very few pathogens, meaning that these regions were healthy from a microbiological point of view. In FE analysis, a significantly higher peak of EQV strain (P=0.005) was found for EH (mean 3438.65 µ∑) compared to MT (mean 840.98 µ∑) connection. Conclusions: Varying implant-abutment connection type will result in diverse periimplant bone remodeling, regardless of clinical and microbiological conditions. This fact is more likely attributed to the singular loading transmission through different implant-abutment connections to the periimplant bone. The present findings suggest that Morse-taper connection is more efficient to prevent periimplant bone loss, compared to an external hexagon connection.

Phenotypic and Genotypic Characteristics of Members of the Genus Streptobacillus

The genus Streptobacillus (S.) remained monotypic for almost 90 years until two new species were recently described. The type species, S. moniliformis, is one of the two etiological agents of rat bite fever, an under-diagnosed, worldwide occurring zoonosis. In a polyphasic approach field isolates and reference strains of S. moniliformis, S. hongkongensis, S. felis as well as divergent isolates were characterized by comparison of molecular data (n = 29) and from the majority also by their physiological as well as proteomic properties (n = 22). Based on growth-independent physiological profiling using VITEK2-compact, API ZYM and the Micronaut system fastidious growth-related difficulties could be overcome and streptobacilli could definitively be typed despite generally few differences. While differing in their isolation sites and dates, S. moniliformis isolates were found to possess almost identical spectra in matrix-assisted laser desorption ionization-time of flight mass spectrometry and Fourier transform infrared spectroscopy. Spectroscopic methods facilitated differentiation of S. moniliformis, S. hongkongensis and S. felis as well as one divergent isolate. Sequencing of 16S rRNA gene as well as functional genes groEL, recA and gyrB revealed only little intraspecific variability, but generally proved suitable for interspecies discrimination between all three taxa and two groups of divergent isolates.

The microbiological signature of human cutaneous leishmaniasis lesions exhibits restricted bacterial diversity compared to healthy skin

Localised cutaneous leishmaniasis (LCL) is the most common form of cutaneous leishmaniasis characterised by single or multiple painless chronic ulcers, which commonly presents with secondary bacterial infection. Previous culture- based studies have found staphylococci, streptococci, and opportunistic pathogenic bacteria in LCL lesions, but there have been no comparisons to normal skin. In addition, this approach has strong bias for determining bacterial composition. The present study tested the hypothesis that bacterial communities in LCL lesions differ from those found on healthy skin (HS). Using a high throughput amplicon sequencing approach, which allows for better populational evaluation due to greater depth coverage and the Quantitative Insights Into Microbial Ecology pipeline, we compared the microbiological signature of LCL lesions with that of contralateral HS from the same individuals. Streptococcus , Staphylococcus , Fusobacterium and other strict or facultative anaerobic bacteria composed the LCL microbiome. Aerobic and facultative anaerobic bacteria found in HS, including environmental bacteria, were significantly decreased in LCL lesions (p < 0.01). This paper presents the first comprehensive microbiome identification from LCL lesions with next generation sequence methodology and shows a marked reduction of bacterial diversity in the lesions.

New insights into vaginal environment during pregnancy: a multi-omics approach

The present thesis aims to provide a thorough comprehension of the vaginal ecosystem of pregnant women and enhance the knowledge of pregnancy pathophysiology. The first study emphasized the importance of limiting protein intake from animal sources, consuming carbohydrates, and avoiding starting pregnancy overweight to maintain a healthy vaginal environment characterized by lactobacilli and related metabolites. In the second paper, a reduction in bacterial diversity, an increase in Lactobacillus abundance, and a decrease in bacterial vaginosis-related genera were observed during pregnancy. Lactobacillus abundance correlated with higher levels of lactate, sarcosine, and amino acids, while bacterial vaginosis-related genera were associated with amines, formate, acetate, alcohols, and short-chain fatty acids. An association between intrapartum antibiotic prophylaxis for Group B Streptococcus and higher vaginal abundance of Prevotella was found. Moreover, women experiencing a first-trimester miscarriage displayed a higher abundance of Fusobacterium. The third study explored the presence of macrolides and tetracyclines resistance genes in the vaginal environment, highlighting that different vaginal microbiota types were associated with distinct resistance profiles. Lactobacilli-dominated ecosystems showed fewer or no resistance genes, while women with increased bacterial vaginosis-related genera were positive for resistance genes. The last two papers aimed to identify potential biomarkers of vaginal health or disease status. The fourth paper showed that positivity for Torquetenovirus decreased from the first to the third trimester, being more prevalent in women with higher vaginal leukocyte counts. Torquetenovirus-positive samples showed higher levels of cytokines, propionate, and cadaverine. Lactobacillus species decreased in Torquetenovirus-positive samples, while Sneathia and Shuttleworthia increased. The last work pointed out the association between clade 2 of Gardnerella vaginalis and bacterial vaginosis. Moreover, as the number of simultaneously detected G. vaginalis clades increased, bacterial vaginosis-associated bacteria also tended to increase. Additionally, sialidase gene levels negatively correlated with Lactobacillus and positively correlated with Gardnerella, Atopobium, Prevotella, Megasphaera, and Sneathia.