Fecal proteolytic profiling of pediatric inflammatory bowel disease: A pilot study.

Colonoscopy is the gold standard for diagnosing inflammatory bowel disease (IBD). However, this invasive procedure has a high burden for pediatric patients. Previous research has shown elevated fecal amino acid concentrations in children with IBD versus controls. We hypothesized that this finding could result from increased proteolytic activity. Therefore, the aim of this study was to investigate whether fecal protease-based profiling was able to discriminate between IBD and controls. Protease activity was measured in fecal samples from patients with IBD (Crohn's disease (CD) n = 19; ulcerative colitis (UC) n = 19) and non-IBD controls (n = 19) using a fluorescence resonance energy transfer (FRET)-peptide library. Receiver operating characteristic (ROC) curve analysis was used to determine the diagnostic value of each FRET-peptide substrate. Screening the FRET-peptide library revealed an increased total proteolytic activity (TPA), as well as degradation of specific FRET-peptides specifically in fecal samples from IBD patients. Based on level of significance (p < .001) and ROC curve analysis (AUC > 0.85), the fluorogenic substrates W-W, A-A, a-a, F-h, and H-y showed diagnostic potential for CD. The substrates W-W, a-a, T-t, G-v, and H-y showed diagnostic potential for UC based on significance (p < .001) and ROC analysis (AUC > 0.90). None of the FRET-peptide substrates used was able to differentiate between protease activity in fecal samples from CD versus UC. This study showed an increased fecal proteolytic activity in children with newly diagnosed, treatment-naïve, IBD. This could lead to the development of novel, noninvasive biomarkers for screening and diagnostic purposes.

Natural and Synthetic Sortase A Substrates Are Processed by Staphylococcus aureus via Different Pathways.

Endogenous Staphylococcus aureus sortase A (SrtA) covalently incorporates cell wall anchored proteins equipped with a SrtA recognition motif (LPXTG) via a lipid II-dependent pathway into the staphylococcal peptidoglycan layer. Previously, we found that the endogenous S. aureus SrtA is able to recognize and process a variety of exogenously added synthetic SrtA substrates, including K(FITC)LPMTG-amide and K(FITC)-K-vancomycin-LPMTG-amide. These synthetic substrates are covalently incorporated into the bacterial peptidoglycan (PG) of S. aureus with varying efficiencies. In this study, we examined if native and synthetic substrates are processed by SrtA via the same pathway. Therefore, the effect of the lipid II inhibiting antibiotic bacitracin on the incorporation of native and synthetic SrtA substrates was assessed. Treatment of S. aureus with bacitracin resulted in a decreased incorporation of protein A in the bacterial cell wall, whereas incorporation of exogenous synthetic substrates was increased. These results suggest that natural and exogenous synthetic substrates are processed by S. aureus via different pathways.

Is TIMP-1 a biomarker for periodontal disease? A systematic review and meta-analysis.

OBJECTIVE: One of the most important families of proteases associated with periodontal disease is the family of the matrix metalloproteinases (MMPs). Their activity is regulated by tissue inhibitors of metalloproteinases (TIMPs), and an imbalance between MMP activity and regulation by TIMPs has been associated with the progression of periodontal disease. This strong interaction between TIMPs and MMPs might be an indication that TIMPs can be used as a biomarker to monitor periodontal disease progression in oral fluids. In particular, TIMP-1 is a frequently studied biomarker for periodontal diseases. Therefore, the aim of this systematic review was to evaluate the scientific literature regarding TIMP-1 concentrations in oral fluids of patients suffering from periodontitis or gingivitis in comparison to healthy individuals. MATERIAL AND METHODS: PubMed/ MedLine and Web of Science databases were searched electronically. Studies that met the inclusion criteria were systematically evaluated and assessed for eligibility and risk of bias. Meta-analysis was performed through the random effects model to assess the association between periodontitis/gingivitis and TIMP-1 concentration in stimulated saliva, unstimulated saliva, and gingival crevicular fluid (GCF). RESULTS: The search strategy provided a total of 322 studies of which 10 studies met all inclusion criteria. Two studies investigated TIMP-1 concentrations in GCF, three studies in unstimulated saliva, and five studies investigated TIMP-1 concentrations in stimulated saliva. Three studies revealed that TIMP-1 levels in oral fluids were significantly decreased in periodontal disease. Meta-analysis revealed that there is no statistically significant difference between TIMP-1 concentration in oral fluids of periodontitis/gingivitis patients in comparison to healthy individuals. CONCLUSIONS: This systematic review with meta-analysis shows that periodontal diseases are not associated with a statistically significant change in TIMP-1 concentration in oral fluids.

Simple and rapid peptide nanoprobe biosensor for the detection of Legionellaceae.

This study demonstrates the development of a sensitive, specific, and quantitative peptide-based nanoprobe prototype assay for the detection of Legionellaceae in a simple way and in a short time. In this work, proteases present in the culture supernatants of Legionella spp. were used as a biomarker. Fluorogenic peptide substrates, specific to Legionella strains culture supernatant proteases, were identified. Peptidases produced a significant increase in the fluorescence intensity following the cleavage of the dipeptide fluorogenic substrates. The specific substrates were identified and coupled with carboxyl-terminated nano-magnetic particles (NMPs). On the other hand, the C-terminal was conjugated with the cysteine residue to covalently integrate with a gold sensing platform via the Au-S linkage. Four different sensors were fabricated from the four specific substrates, which were treated with the protesase of six different species of Legionella. In the presence of specific protease, the peptide sequence is digested and the magnetic nanobeads moved out of the gold surface, resulting in the apparence of gold color. One of the nanoprobes sensitivity detects as low as 60 CFU mL-1 of Legionella anisa, Legionella micdadei, and Fluoribacter dumoffii. The cross-reactivity of the sensors was tested using other closely associated bacterial species and no significant cross-reactivity of the sensors was found. It is envisaged that this assay could be useful for screening purposes or might be supportive for the fast and easy detection of Legionella protease activity for water monitoring purposes.

On site visual detection of Porphyromonas gingivalis related periodontitis by using a magnetic-nanobead based assay for gingipains protease biomarkers.

Porphyromonas gingivalis (P. gingivalis) is a pathogen causing periodontitis. A rapid assay is described for the diagnosis of periodontal infections related to P. gingivalis. The method is making use of gingipains, a group of P. gingivalis specific proteases as a detection biomarker. Magnetic-nanobeads were labeled with gingipain-specific peptide substrates and immobilized on a gold biosensing platform via gold-thiol linkage. As a result of this, the color of the gold layer turns black. Upon cleavage of the immobilized substrates by gingipains, the magnetic-nanobeads-peptide fragments were attracted by a magnet so that the golden surface color becomes visible again. This assay is highly sensitive and specific. It is capable of detecting as little as 49 CFU·mL-1 of P. gingivalis within 30 s. Examination of periodontitis patients and healthy control saliva samples showed the potential of the assay. The simplicity and rapidity of the assay makes it an effective point-of-care device. Graphical abstract Schematic of the assay for the detection of P. gingivalis proteases as one of the promising biomarkers associated with periodontal diseases.

Development of magnetic nanoparticle based calorimetric assay for the detection of bovine mastitis in cow milk.

Mastitis in dairy cattle is an inflammatory reaction of the udder tissue. Mastitis increases plasmin levels, leading to an increased proteolysis of milk proteins such as casein, resulting in a significant decrease in milk quality and related dairy products. Due to its key-role in mastitis, we used plasmin proteolytic activity as a biomarker for the detection of mastitis in bovine mastitic milk. Inspired by earlier studies on protease activity using mastitic milk samples, we developed a simple colorimetric assay to distinguish mastitic milk from milk derived from healthy animals. The plasmin substrate coupled to magnetic nanoparticles form a black self-assembled monolayer on a gold sensor surface. In the presence of increased levels of plasmin, the substrate is cleaved and the peptide fragment attached to the magnetic beads, will be attracted by the magnet which is present under the sensor strips revealing the golden surface. We found the area of the golden color surface proportional to plasmin activity. The sensitivity of this method was determined to be 1 ng/ml of plasmin in vitro. Next, we tested the biosensor using mastitis positive milk of which infection is confirmed by bacterial cultures. This newly developed colorimetric biosensor has high potential in applications for the diagnosis of mastitis with potential spin offs to health, food and environmental sectors.

Incorporation of a Valine-Leucine-Lysine-Containing Substrate in the Bacterial Cell Wall.

The emergence of antibiotic-resistant bacteria is a major public health threat, and therefore novel antimicrobial targets and strategies are urgently needed. In this regard, cell-wall-associated proteases are envisaged as interesting antimicrobial targets due to their role in cell wall remodeling. Here, we describe the discovery and characteristics of a protease substrate that is processed by a bacterial cell-wall-associated protease. Stationary-phase grown Gram-positive bacteria were incubated with fluorogenic protease substrates, and their cleavage and covalent incorporation into the cell wall was analyzed. Of all of the substrates used, only one substrate, containing a valine-leucine-lysine (VLK) motif, was covalently incorporated into the bacterial cell wall. Linkage of the VLK-peptide substrate appeared unrelated to sortase A and B activity, as both wild-type and sortase A and B knock out Staphylococcus aureus strains incorporated this substrate into their cell wall with comparable efficiency. Additionally, the VLK-peptide substrate showed significantly higher incorporation in the cell wall of VanA-positive Enterococcus faecium strains than in VanB- and vancomycin-susceptible isolates. In conclusion, the VLK-peptide substrate identified in this study shows promise as a vehicle for targeting antimicrobial compounds and diagnostic contrast agents to the bacterial cell wall.

Colorimetric Assay for the Detection of Typical Biomarkers for Periodontitis Using a Magnetic Nanoparticle Biosensor.

Periodontitis is a chronic disease which affects at least 10% of the population. If untreated, periodontitis can lead to teeth loss. Unfortunately, current diagnostic tests are limited in their sensitivity and specificity. In this study, a novel multiplex hand-held colorimetric diagnostic biosensor, using two typical inflammatory salivary biomarkers, Human Neutrophil Elastase (HNE) and Cathepsin-G, was constructed as proof of concept to potentially detect periodontitis. The biosensing method was based on the measurement of proteolytic activity using specific proteases probes. These probes consist of specific proteases substrates covalently bound to a magnetic bead from one end and to the gold sensor surface by the other end. When intact, this renders the golden sensor black. Upon proteolysis, the cleaved magnetic beads will be attracted by an external magnet revealing the golden color of the sensor surface observable by the naked eye. The biosensor was capable of specific and quantitative detection of HNE and Cathepsin-G in solution and in spiked saliva samples with a lower detection limit of 1 pg/mL and 100 fg/mL for HNE and Cathepsin-G, respectively. Examination of periodontitis patients' sample and a healthy control showed the potential of the multiplex biosensor to detect the presence of HNE and Cathepsin-G activity in situ. This approach is anticipated to be a useful biochip array amenable to low-cost point-of-care devices.

The papain inhibitor (SPI) of Streptomyces mobaraensis inhibits bacterial cysteine proteases and is an antagonist of bacterial growth.

A novel papain inhibitory protein (SPI) from Streptomyces mobaraensis was studied to measure its inhibitory effect on bacterial cysteine protease activity (Staphylococcus aureus SspB) and culture supernatants (Porphyromonas gingivalis, Bacillus anthracis). Further, growth of Bacillus anthracis, Staphylococcus aureus, Pseudomonas aeruginosa, and Vibrio cholerae was completely inhibited by 10 µM SPI. At this concentration of SPI, no cytotoxicity was observed. We conclude that SPI inhibits bacterial virulence factors and has the potential to become a novel therapeutic treatment against a range of unrelated pathogenic bacteria.

Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases.

Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these potential functions, the authors designed and used a protease substrate fluorescence resonance energy transfer (FRET) library comprising 115 short d- and l-amino-acid-containing fluorogenic substrates as a tool to generate proteolytic profiles for a wide range of bacteria. Bacterial specificity of the d-amino acid substrates was confirmed using enzymes isolated from both eukaryotic and prokaryotic organisms. Interestingly, bacterial proteases that are known to be involved in housekeeping and nutrition, but not in virulence, were able to degrade substrates in which a d-amino acid was present. Using our FRET peptide library and culture supernatants from a total of 60 different bacterial species revealed novel, bacteria-specific, proteolytic profiles, although in-species variation was observed for Pseudomonas aeruginosa, Porphyromonas gingivalis, and Staphylococcus aureus. Overall, the specific characteristic of our substrate peptide library makes it a rapid tool to high-throughput screen for novel substrates to detect bacterial proteolytic activity.

Differential Modulation of Saliva-Derived Microcosm Biofilms by Antimicrobial Peptide LL-31 and D-LL-31.

Microbiome modulation, aiming to restore a health-compatible microbiota, is a novel strategy to treat periodontitis. This study evaluated the modulation effects of antimicrobial peptide LL-31 and its D-enantiomer (D-LL-31) on saliva-derived microcosm biofilms, spiked with or without Porphyromonas gingivalis. To this end, one-day-old biofilms were incubated for 24 h with biofilm medium alone, or medium containing 40 µM LL-31 or D-LL-31, after which biofilms were grown for 5 days. Biofilms were assessed at 1 day and 5 days after intervention for the total viable cell counts, dipeptidyl peptidase IV (DPP4) activity, P. gingivalis amount (by qPCR) and microbial composition (by sequencing). The results showed that D-LL-31, not LL-31, significantly reduced the total viable cell counts, the P. gingivalis amount, and the DPP4 activity of the biofilms spiked with P. gingivalis, but only at 1 day after intervention. In the biofilms spiked with P. gingivalis, D-LL-31 tended to reduce the α-diversity and the compositional shift of the biofilms in time as compared to the control and LL-31 groups. In conclusion, D-LL-31 showed a better performance than LL-31 in biofilm modulation. The biofilm modulation function of the peptides could be impaired when the biofilms were in a severely dysbiotic state.

Highly specific protease-based approach for detection of porphyromonas gingivalis in diagnosis of periodontitis.

Porphyromonas gingivalis is associated with the development of periodontitis. Here we describe the development of a highly specific protease-based diagnostic method for the detection of P. gingivalis in gingival crevicular fluid. Screening of a proteolytic peptide substrate library, including fluorogenic dipeptides that contain d-amino acids, led to the discovery of five P. gingivalis-specific substrates. Due to the presence of lysine and arginine residues in these substrates, it was hypothesized that the cleavage was mediated by the gingipains, a group of P. gingivalis-specific proteases. This hypothesis was confirmed by the observation that P. gingivalis gingipain knockout strains demonstrated clearly impaired substrate cleavage efficacy. Further, proteolytic activity on the substrates was increased by the addition of the gingipain stimulators dithiothreitol and l-cysteine and decreased by the inhibitors leupeptin and N-ethylmaleimide. Screening of saliva and gingival crevicular fluid of periodontitis patients and healthy controls showed the potential of the substrates to diagnose the presence of P. gingivalis proteases. By using paper points, a sensitivity of approximately 10(5) CFU/ml was achieved. P. gingivalis-reactive substrates fully composed of l-amino acids and Bz-l-Arg-NHPhNO(2) showed a relatively low specificity (44 to 85%). However, the five P. gingivalis-specific substrates that each contained a single d-amino acid showed high specificity (96 to 100%). This observation underlines the importance of the presence of d-amino acids in substrates used for the detection of bacterial proteases. We envisage that these substrates may improve the specificity of the current enzyme-based diagnosis of periodontitis associated with P. gingivalis.

Gramicidin A is hydrolyzed by a d-stereospecific peptidase produced by Bacillus anthracis.

Previously we described the discovery of a Bacillus spp. specific peptidase activity related to d-stereospecific peptidases (DSPs). The peptidase showed a strong preference for d-leucine and d-valine amino acids. These amino acids are present in the structure of the non-ribosomal peptide (NRP) antibiotics gramicidin A, B and C and polymyxin E. To examine if the Bacillus spp. DSP-related peptidase can hydrolyze these NRPs, the effect of gramicidin A and C and polymyxin E on peptidase activity in Bacillus anthracis culture supernatant was monitored. It was found that both gramicidins inhibited the DSP-related activity in a competitive manner. MALDI-TOF analysis revealed that upon incubation with B. anthracis culture supernatant gramicidin A hydrolyzation products appeared. This study shows that the Bacillus spp. specific DSP-like peptidase was potentially produced by the bacteria to gain intrinsic resistance against NRP antibiotics. These results are of utmost importance in research towards antimicrobial resistance, whereas transfer of DSP-related activity to other clinically relevant pathogens can be a serious threat to human health.

Novel Antiamoebic Tyrocidine-Derived Peptide against Brain-Eating Amoebae.

Acanthamoeba castellanii (A. castellanii) can cause Acanthamoeba keratitis, a sight-threatening infection, as well as a fatal brain infection termed granulomatous amoebic encephalitis, mostly in immunocompromised individuals. In contrast, Naegleria fowleri (N. fowleri) causes a deadly infection involving the central nervous system, recognized as primary amoebic encephalitis, mainly in individuals partaking in recreational water activities or those with nasal exposure to contaminated water. Worryingly, mortality rates due to these infections are more than 90%, suggesting the need to find alternative therapies. In this study, antiamoebic activity of a peptide based on the structure of the antibiotic tyrocidine was evaluated against A. castellanii and N. fowleri. The tyrocidine-derived peptide displayed significant amoebicidal efficacy against A. castellanii and N. fowleri. At 250 µg/mL, the peptide drastically reduced amoebae viability up to 13% and 21% after 2 h of incubation against N. fowleri and A. castellanii., whereas, after 24 h of incubation, the peptide showed 86% and 94% amoebicidal activity against A. castellanii and N. fowleri. Furthermore, amoebae pretreated with 100 µg/mL peptide inhibited 35% and 53% A. castellanii and N. fowleri, while, at 250 µg/mL, 84% and 94% A. castellanii and N. fowleri failed to adhere to human cells. Amoeba-mediated cell cytopathogenicity assays revealed 31% and 42% inhibition at 100 µg/mL, while at 250 µg/mL 75% and 86% A. castellanii and N. fowleri were inhibited. Assays revealed inhibition of encystation in both A. castellanii (58% and 93%) and N. fowleri (73% and 97%) at concentrations of 100 and 250 µg/mL respectively. Importantly, tyrocidine-derived peptide depicted minimal cytotoxicity to human cells and, thus, may be a potential candidate in the rational development of a treatment regimen against free-living amoebae infections. Future studies are necessary to elucidate the in vivo effects of tyrocidine-derived peptide against these and other pathogenic amoebae of importance.

Peptide-based fluorescence resonance energy transfer protease substrates for the detection and diagnosis of Bacillus species.

We describe the development of a highly specific enzyme-based fluorescence resonance energy transfer (FRET) assay for easy and rapid detection both in vitro and in vivo of Bacillus spp., among which are the members of the B. cereus group. Synthetic substrates for B. anthracis proteases were designed and exposed to secreted enzymes of a broad spectrum of bacterial species. The rational design of the substrates was based on the fact that the presence of D-amino acids in the target is highly specific for bacterial proteases. The designed D-amino acids containing substrates appeared to be specific for B. anthracis but also for several other Bacillus spp. and for both vegetative cells and spores. With the use of mass spectrometry (MS), cleavage products of the substrates could be detected in sera of B. anthracis infected mice but not in healthy mice. Due to the presence of mirrored amino acids present in the substrate, the substrates showed high species specificity, and enzyme isolation and purification was redundant. The substrate wherein the D-amino acid was replaced by its L-isomer showed a loss of specificity. In conclusion, with the use of these substrates a rapid tool for detection of B. anthracis spores and diagnosis of anthrax infection is at hand. We are the first who present fluorogenic substrates for detection of bacterial proteolytic enzymes that can be directly applied in situ by the use of D-oriented amino acids.

Salivary Biomarkers for Dental Caries Detection and Personalized Monitoring.

This study investigated the potential of salivary bacterial and protein markers for evaluating the disease status in healthy individuals or patients with gingivitis or caries. Saliva samples from caries- and gingivitis-free individuals (n = 18), patients with gingivitis (n = 17), or patients with deep caries lesions (n = 38) were collected and analyzed for 44 candidate biomarkers (cytokines, chemokines, growth factors, matrix metalloproteinases, a metallopeptidase inhibitor, proteolytic enzymes, and selected oral bacteria). The resulting data were subjected to principal component analysis and used as a training set for random forest (RF) modeling. This computational analysis revealed four biomarkers (IL-4, IL-13, IL-2-RA, and eotaxin/CCL11) to be of high importance for the correct depiction of caries in 37 of 38 patients. The RF model was then used to classify 10 subjects (five caries-/gingivitis-free and five with caries), who were followed over a period of six months. The results were compared to the clinical assessments of dental specialists, revealing a high correlation between the RF prediction and the clinical classification. Due to the superior sensitivity of the RF model, there was a divergence in the prediction of two caries and four caries-/gingivitis-free subjects. These findings suggest IL-4, IL-13, IL-2-RA, and eotaxin/CCL11 as potential salivary biomarkers for identifying noninvasive caries. Furthermore, we suggest a potential association between JAK/STAT signaling and dental caries onset and progression.

OralDisk: A Chair-Side Compatible Molecular Platform Using Whole Saliva for Monitoring Oral Health at the Dental Practice.

Periodontitis and dental caries are two major bacterially induced, non-communicable diseases that cause the deterioration of oral health, with implications in patients' general health. Early, precise diagnosis and personalized monitoring are essential for the efficient prevention and management of these diseases. Here, we present a disk-shaped microfluidic platform (OralDisk) compatible with chair-side use that enables analysis of non-invasively collected whole saliva samples and molecular-based detection of ten bacteria: seven periodontitis-associated (Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia, Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola) and three caries-associated (oral Lactobacilli, Streptococcus mutans, Streptococcus sobrinus). Each OralDisk test required 400 µL of homogenized whole saliva. The automated workflow included bacterial DNA extraction, purification and hydrolysis probe real-time PCR detection of the target pathogens. All reagents were pre-stored within the disk and sample-to-answer processing took < 3 h using a compact, customized processing device. A technical feasibility study (25 OralDisks) was conducted using samples from healthy, periodontitis and caries patients. The comparison of the OralDisk with a lab-based reference method revealed a ~90% agreement amongst targets detected as positive and negative. This shows the OralDisk's potential and suitability for inclusion in larger prospective implementation studies in dental care settings.

Selection of antimicrobial frog peptides and temporin-1DRa analogues for treatment of bacterial infections based on their cytotoxicity and differential activity against pathogens.

Cationic, amphipathic, α-helical host-defense peptides (HDPs) that are naturally secreted by certain species of frogs (Anura) possess potent broad-spectrum antimicrobial activity and show therapeutic potential as alternatives to treat infections by multidrug-resistant pathogens. Fourteen amphibian skin peptides and twelve analogues of temporin-1DRa were studied for their antimicrobial activities against clinically relevant human or animal skin infection-associated pathogens. For comparison, antimicrobial potencies of frog skin peptides against a range of probiotic lactobacilli were determined. We used the VITEK 2 system to define a profile of antibiotic susceptibility for the bacterial panel. The minimal inhibitory concentration (MIC) values of the naturally occurring temporin-1DRa, CPF-AM1, alyteserin-1c, hymenochirin-2B, and hymenochirin-4B for pathogenic bacteria were threefold to ninefold lower than the values for the tested probiotic strains. Similarly, temporin-1DRa and its [Lys4 ], [Lys5 ], and [Aib8 ] analogues showed fivefold to 6.5-fold greater potency against the pathogens. In the case of PGLa-AM1, XT-7, temporin-1DRa and its [D-Lys8 ] and [Aib13 ] analogues, no apoptosis or necrosis was detected in human peripheral blood mononuclear cells at concentrations below or above the MIC. Given the differential activity against commensal bacteria and pathogens, some of these peptides are promising candidates for further development into therapeutics for topical treatment of skin infections.

Activation of Toll-like receptors and dendritic cells by a broad range of bacterial molecules.

Activation of pattern recognition receptors such as Toll-like receptors (TLRs) by pathogens leads to activation and maturation of dendritic cells (DC), which orchestrate the development of the adaptive immune response. To create an overview of the effects of a broad range of pathogenic bacteria, their capacity to activate TLRs and to affect DC maturation, cytokine production and T cell polarizing capacity were determined. Different bacterial species differed in their potency to affect these parameters. In general, on the DC level differences were found in the maturation-inducing capacity of gram-negative and gram-positive bacteria. Remarkably, these differences did not result in differential polarization of the T cell response. With respect to TLRs, TLR4 activation by pathogens correlated with their ability to induce DC maturation, while for TLR2 and TLR5 such a correlation was absent. Taken together, this study provides insight into qualitative differences and general effects of pathogen-derived molecules on dendritic cells.

Salivary Total Protease Activity Based on a Broad-Spectrum Fluorescence Resonance Energy Transfer Approach to Monitor Induction and Resolution of Gingival Inflammation.

OBJECTIVE: Salivary total protease and chitinase activities were measured by a broad-spectrum fluorescence resonance energy transfer approach as predictors of induction and resolution of gingival inflammation in healthy individuals by applying an experimental human gingivitis model. METHODS: Dental biofilm accumulated (21 days, Induction Phase) by omitting oral hygiene practices followed by a 2-week Resolution Phase to restore gingival health in an experimental gingivitis study. Plaque accumulation, as assessed by the Turesky Modification of the Quigley-Hein Plaque Index (TQHPI), and gingival inflammation, assessed using the Modified Gingival Index (MGI), scores were recorded and unstimulated saliva was collected weekly. Saliva was analysed for total protein, albumin, total protease activity and chitinase activity (n = 18). RESULTS: The TQHPI and MGI scores, as well as total protease activity, increased until day 21. After re-establishment of oral hygiene, gingival inflammation levels returned to values similar to baseline (day 0). Levels of protease activity decreased significantly, but not to baseline values. Furthermore, 'fast' responders, who responded immediately to plaque, exhibited significantly higher proteolytic activity throughout the experimental course than 'slow' responders, who showed a lagged inflammatory response. CONCLUSION: The results indicate that differential inflammatory responses encompass inherent variations in total salivary proteolytic activities, which could be further utilised in contemporary diagnostic, prognostic and treatment modalities for periodontal diseases.