Identification of Sjögren's syndrome oral fluid biomarker candidates following high-abundance protein depletion.

OBJECTIVE: SS is an autoimmune exocrinopathy affecting ∼1 million patients in the USA that is diagnosed mostly in middle-aged women. Oral fluids (OFs) serving as the mirror of the body were suggested as an ideal non-invasive diagnostic tool. Previously we developed depletion techniques for OF high-abundance proteins to increase visualization of low-abundance proteins. Therefore the aim of this study was to examine the effect of depletion pretreatments on the identification potential of SS OF biomarker candidates. METHODS: Unstimulated OFs were collected from 18 female SS patients and 18 healthy age- and gender-matched controls. High-abundance proteins were depleted using affinity and immunodepletion methodologies followed by semi-quantitative two-dimensional gel electrophoresis and quantitative dimethylation liquid chromatography tandem mass spectrometry (LC-MS/MS). To initially validate the MS results, western blotting was performed. RESULTS: The use of depletion strategy before proteomics analysis increased identification ability by 3-fold. Overall, 79 biomarker candidates were identified. Proteins with the most pronounced fold changes were related to SS serum or tissue factors. In addition, bioinformatics analysis of proteins with a >3-fold increase in SS patients showed calcium-binding proteins, defence-response proteins, proteins involved in apoptotic regulation, stress-response proteins and cell motion-related proteins. Preliminary validation by western blotting of profilin and CA-I indicated similar expression profile trends to those identified by quantitative MS. CONCLUSION: The significance of OF novel depletion methodologies is clearly demonstrated for increased visibility of biomarker candidates as well as for unveiling possible mechanisms involved in this syndrome. This represents a major contribution to our ability to use OF as a future diagnostic fluid.

Effect of oxazaborolidines on immobilized fructosyltransferase analyzed by surface plasmon resonance.

Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harboring pathogenic bacteria. Fructosyltransferases (FTF) are extra cellular enzymes of several oral bacteria. FTF are associated with the formation of extracellular polysaccharide matrix (fructans) which play a role in biofilm formation and oral bacteria physiology. Oxazaborolidines have been shown to inhibit biofilm formation. The purpose of this study was to examine if the anti-biofilm effect is, in part, an effect on the immobilized enzymes synthesizing the extra cellular polysaccharide participating in biofilm formation. Eight different oxazaborolidines (BNO1-BNO8) were synthesized and evaluated for their affect on the synthesis of fructans by FTF using the biomolecular interaction analysis (BIAcore) system which involves the use of real-time surface plasmon resonance (SPR) technique. The tested oxazaborolidines demonstrated a significant and immediate inhibitory effect on immobilized FTF activity. This effect was reversible. Our results show that oxazaborolidines can act as enzymatic inhibitors of FTF immobilized on the surface, also at levels lower than their MIC. Part of the anti-biofilm effect of BNOs may be accounted for this enzymatic inhibition.

Identification of salivary protein biomarkers for orthodontically induced inflammatory root resorption.

PURPOSE: Orthodontically induced inflammatory root resorption (OIIRR) is one of the most prevalent and unavoidable consequence of orthodontic tooth movement. The aim of this study was to discover potential diagnostic protein biomarkers for detection of OIIRR in whole saliva (WS). MATERIAL AND METHODS: Unstimulated WS was collected from 72 subjects: 48 OIIRR patients and 24 untreated, generally healthy, age and gender matched controls. Radiographic assessment of periapical x-rays of four upper incisors taken before and 9 months after bonding was done. High-abundance proteins were depleted followed by two-dimensional-gel-electrophoresis and quantitative mass spectrometry (qMS). Finally, to initially validate qMS results, Western blotting was performed. RESULTS: qMS revealed differentially expressed proteins in the moderate-to-severe OIIRR group, which have never been found in WS before. Additionally, in the moderate-to-severe young OIIRR group, the pathogenetic mechanisms related to actin cytoskeleton regulation and Fc gamma R- mediated phagocytosis were detected, while in adults- to focal adhesion. Preliminary validation by Western blotting of fetuin-A and p21-ARC indicated expression profile trends similar to those identified by qMS. CONCLUSION: The significance of WS novel proteomic methodologies is clearly demonstrated for detecting new OIIRR biomarkers as well as for unveiling possible novel pathogenetic mechanisms in both young and adult patients.

Evaluation of oxazaborolidine activity on Streptococcus mutans biofilm formation.

Dental diseases are among the most prevalent afflictions of humankind. These diseases are associated with the formation of biofilms harbouring pathogenic bacteria. Eight different derivatives of oxazaborolidines were synthesised and evaluated for their affect on Streptococcus mutans adhesion and biofilm formation. Structure-activity relationship was observed. The B-butyl moiety of the oxazaborolidines contributed an anti-adhesion effect for all derivatives, whilst its effect diminished when the boron atom was incorporated in a fused heterocyclic ring. The B-phenyl group induced bacterial adhesion in all tested compounds Oxazaborolidines may serve as novel agents for affecting oral biofilm formation. Moreover, the ability to alter the oxazaborolidine molecule and thus affect biofilms offers an excellent opportunity to study biofilm formation.

Regulation of fructosyltransferase activity by carbohydrates, in solution and immobilized on hydroxyapatite surfaces.

We tested the effect of several carbohydrates on the activity of cell-free fructosyltransferases (FTF) in solution and immobilized onto hydroxyapatite (HA) and found an inhibitory dose-dependent effect of glucose on FTF activity, both on the surface and in solution. Glucose at 160 mM inhibits FTF activity by 75% both on HA and in solution. Fructose at 160 mM inhibited FTF activity by 25% in solution and by 15% on HA. Levan inhibited FTF activity by 30% in solution, while dextrans and inulin had a limited effect on FTF activity. Circular dichroism and infrared analysis demonstrated no major changes in the chemical structure of fructans synthesized by cell-free FTF on HA and in solution, in the presence or absence of glucose. However, as verified by size-exclusion chromatography, glucose inhibited the synthesis of high molecular-weight fructans. The results indicate that glucose, a byproduct of the FTF enzymatic reaction, is the main carbohydrate affecting FTF activity. Selective inhibition of high molecular-weight fructan production by glucose, may indicate that two mechanisms are involved in the synthesis of fructans, both in solution and on the surface.

Prevention of initial biofilm formation on ureteral stents using a sustained releasing varnish containing chlorhexidine: in vitro study.

BACKGROUND AND PURPOSE: Ureteral stents are being used exceedingly in the field of urology, and with advancements in endourology, this trend is increasing. Bacterial colonization and proliferation on the stent surface may result in urinary tract infections (UTIs) necessitating the administration of antibiotics that, in turn, may lead to the development of antibiotic-resistant bacterial strains. Several studies have shown that sustained release varnish (SRV) combined with antibiotics or antiseptics can prevent the proliferation of bacteria on urethral catheters. This is the first study that evaluates this technique implemented on ureteral stents. MATERIALS AND METHODS: We evaluated growth inhibition on ureteral stent segments coated with chlorhexidine (CHX) 1% SRV. The tests were conducted using common urinary pathogens: Enterococci, Pseudomonas, and Escherichia coli. Coated stent segments were inserted into bacterial suspensions. Controls included uncoated stent segments and stents coated with placebo SRV (without CHX). RESULTS: Bacterial growth measured as turbidity and as colony-forming units showed a significant inhibition effect of initial bacteria adhesion to the CHX-SRV coated stent segments compared with the controls (P<0.001). This inhibitory effect was apparent in each of the bacteria tested and was confirmed by inspection of the stent segments under an electron microscope. In a kinetic experiment using CHX 2% SRV, we were able to prolong the growth inhibition effect from 1 week to nearly 2 weeks. CONCLUSIONS: We believe this technique may play a significant role in reducing ureteral stent-associated UTIs. Further studies are needed before this approach can be implemented in clinical practice.

Comparison of diverse affinity based high-abundance protein depletion strategies for improved bio-marker discovery in oral fluids.

Oral fluids (OF) have been suggested as a source of biomarkers for oral and systemic health, but as with other bio-fluids, the presence of high-abundance proteins interferes with the detection of lower-abundance biomarkers. Here, we compared the performance of four depletion treatments: triple depletion (TD) of amylases, albumins and immunoglobulin G; multiple depletion (MD) of amylases and a panel of 20 proteins, a combination of the two (EMD) and combinatorial peptide ligand library based depletion termed CPLL. TD, MD, EMD and CPLL removed 76%, 83%, 85% and 94% of total proteins, respectively, coupled with increased low abundance protein detection and narrowed dynamic range. 2-DE revealed that all depletion pretreatments successfully clarified areas hampered by high-abundance proteins; however, EMD and CPLL exposed the highest number of proteins. Quantitative MS of EMD samples relative to none treated samples indicated that most of downregulated proteins (>90%) were EMD target proteins. In conclusion, a multiple step EMD and CPLL depletion approaches bring about the highest number of protein detection ability and the best hampered-area clearance. As CPLL requires at least 10 fold more protein starting material, we suggest EMD pretreatment as a new detection tool in instances of low protein starting material.

An approach to remove alpha amylase for proteomic analysis of low abundance biomarkers in human saliva.

Proteomic characterization of human whole saliva for the identification of disease-specific biomarkers is guaranteed to be an easy-to-use and powerful diagnostic tool for defining the onset, progression and prognosis of human systemic diseases and, in particular, oral diseases. The high abundance of proteins, mainly alpha amylase, hampers the detection of low abundant proteins appearing in the disease state and therefore should be removed. In the present study a 2-DE was used to analyze human whole saliva following the removal of alpha amylase by affinity adsorption to potato starch. After alpha amylase removal whole saliva was analyzed by SDS-PAGE showing at least sixfold removal efficiency and by an alpha amylase activity assay showing 97% reduced activity. MS identification of the captured alpha amylase after elution demonstrated specific removal; 2-DE analysis showed the selective removal of alpha amylase and consequently increased gel resolution. MS identification of protein spots in the 60 kDa area revealed 15 proteins, which were masked before alpha amylase removal. In conclusion, treatment of human whole saliva with an alpha amylase removal device increases gel resolution and enables a higher protein sample for analysis.

Reduced expression of gamma interferon in serum and marked lymphoid depletion induced by Porphyromonas gingivalis increase murine morbidity and mortality due to cytomegalovirus infection.

Porphyromonas gingivalis, a gram-negative anaerobe, is a major etiological agent of severe forms of periodontal disease. Although periodontal disease is considered a localized disease, accumulating evidence indicates that it may lead to a predisposition to a decline in immunocompetence. Human cytomegalovirus (CMV) commonly infects all human populations without producing significant clinical symptoms. Immunocompromised patients usually develop a primary or reactivated CMV infection, which is associated with high rates of morbidity and mortality. The aim of this study was to determine whether P. gingivalis increases animal susceptibility to CMV infection. Mice were inoculated with CMV and infected locally with P. gingivalis 3 days after the virus inoculation. Mortality rates were monitored, and traces of viral DNA and bacterial infection were detected systemically by using real-time PCR. Local and systemic cytokine secretion was measured, and histological sections were used to assess the pathological state of infected organs. P. gingivalis- and CMV-coinfected mice showed dramatically higher mortality rates than mice infected with P. gingivalis or CMV only. Although the organs of coinfected mice exhibited decreased viral titers, distinct necrosis and tissue damage were more evident in the livers and spleens of these mice than in those of mice infected with CMV only. Furthermore, systemic gamma interferon levels were decreased in coinfected mice, and marked lymphoid depletion was observed in their necrotic organs. In parallel control Escherichia coli-CMV coinfection experiments, the mortality and pathological results were the same as those found in mice infected with CMV only. Our results suggest a specific influence of P. gingivalis on the mouse immune response, causing increased susceptibility to CMV infection.