A Role for Salivary Peptides in the Innate Defense Against Enterotoxigenic Escherichia coli.

Background: Diarrheal disease from enterotoxigenic Escherichia coli (ETEC) causes significant worldwide morbidity and mortality in young children residing in endemic countries and is the leading cause of traveler's diarrhea. As ETEC enters the body through the oral cavity and cotransits the digestive tract with salivary components, we hypothesized that the antimicrobial activity of salivary proteins might extend beyond the oropharynx into the proximal digestive tract. Results: Here, we show that the salivary peptide histatin-5 binds colonization factor antigen I pili, thereby blocking adhesion of ETEC to intestinal epithelial cells. Mechanistically, we demonstrate that histatin-5 stiffens the typically dynamic pili, abolishing their ability to function as spring-like shock absorbers, thereby inhibiting colonization within the turbulent vortices of chyme in the gastrointestinal tract. Conclusions: Our data represent the first report of a salivary component exerting specific antimicrobial activity against an enteric pathogen and suggest that histatin-5 and related peptides might be exploited for prophylactic and/or therapeutic uses. Numerous viruses, bacteria, and fungi traverse the oropharynx to cause disease, so there is considerable opportunity for various salivary components to neutralize these pathogens prior to arrival at their target organ. Identification of additional salivary components with unexpectedly broad antimicrobial spectra should be a priority.

The association between coeliac disease and periodontitis: Results from NHANES 2009-2012.

AIM: To investigate whether coeliac disease (CD) was associated with periodontitis among a nationally representative sample of US adults. MATERIALS AND METHODS: The National Health and Nutrition Examination Survey (NHANES) 2009-2012 enrolled 6,661 subjects with full-mouth periodontal examination and serological testing for antitissue transglutaminase (tTg) and antiendomysial (EMA) antibodies. CD was defined as (i) self-reported physician diagnosis while on a gluten-free diet; or (ii) tTg levels >10.0 U/ml and positive EMA results. Positive serology without self-reported diagnosis was defined as undiagnosed CD (UdxCD). Periodontitis was defined according to the CDC/AAP definition. Multivariable linear and logistic models were used to regress the mean probing depth (PD) or attachment loss (AL) outcomes across CD categories (none, diagnosed and undiagnosed). RESULTS: The prevalence of moderate/severe periodontitis and diagnosed/undiagnosed CD was 40% and 0.74%, respectively. Mean AL was lower among those with CD although results were not statistically significant (p = .67). The odds of periodontitis among individuals with diagnosed and undiagnosed CD were: 0.5(0.22, 1.16) and 0.62(0.1, 3.75), respectively. Mean PD levels among those without CD or with diagnosed or undiagnosed CD were 1.49 ± 0.02, 1.36 ± 0.11 and 1.31 ± 0.11 (p = .03). CONCLUSION: CD is associated with modestly lower levels of mean PD but was not associated with mean AL or periodontitis. Larger studies are necessary to enhance precision and strengthen conclusions.

Salivary Gluten Degradation and Oral Microbial Profiles in Healthy Individuals and Celiac Disease Patients.

Celiac disease (CD) is a chronic immune-mediated enteropathy induced by dietary gluten in genetically predisposed individuals. Saliva harbors the second highest bacterial load of the gastrointestinal (GI) tract after the colon. We hypothesized that enzymes produced by oral bacteria may be involved in gluten processing in the intestine and susceptibility to celiac disease. The aim of this study was to investigate salivary enzymatic activities and oral microbial profiles in healthy subjects versus patients with classical and refractory CD. Stimulated whole saliva was collected from patients with CD in remission (n = 21) and refractory CD (RCD; n = 8) and was compared to healthy controls (HC; n = 20) and subjects with functional GI complaints (n = 12). Salivary gluten-degrading activities were monitored with the tripeptide substrate Z-Tyr-Pro-Gln-pNA and the α-gliadin-derived immunogenic 33-mer peptide. The oral microbiome was profiled by 16S rRNA-based MiSeq analysis. Salivary glutenase activities were higher in CD patients compared to controls, both before and after normalization for protein concentration or bacterial load. The oral microbiomes of CD and RCD patients showed significant differences from that of healthy subjects, e.g., higher salivary levels of lactobacilli (P < 0.05), which may partly explain the observed higher gluten-degrading activities. While the pathophysiological link between the oral and gut microbiomes in CD needs further exploration, the presented data suggest that oral microbe-derived enzyme activities are elevated in subjects with CD, which may impact gluten processing and the presentation of immunogenic gluten epitopes to the immune system in the small intestine.IMPORTANCE Ingested gluten proteins are the triggers of intestinal inflammation in celiac disease (CD). Certain immunogenic gluten domains are resistant to intestinal proteases but can be hydrolyzed by oral microbial enzymes. Very little is known about the endogenous proteolytic processing of gluten proteins in the oral cavity. Given that this occurs prior to gluten reaching the small intestine, such enzymes are likely to contribute to the composition of the gluten digest that ultimately reaches the small intestine and causes CD. We demonstrated that endogenous salivary protease activities are incomplete, likely liberating peptides from larger gluten proteins. The potentially responsible microbes were identified. The study included refractory CD patients, who have been studied less with regard to CD pathogenesis.

Identification of food-grade subtilisins as gluten-degrading enzymes to treat celiac disease.

Gluten are proline- and glutamine-rich proteins present in wheat, barley, and rye and contain the immunogenic sequences that drive celiac disease (CD). Rothia mucilaginosa, an oral microbial colonizer, can cleave these gluten epitopes. The aim was to isolate and identify the enzymes and evaluate their potential as novel enzyme therapeutics for CD. The membrane-associated R. mucilaginosa proteins were extracted and separated by DEAE chromatography. Enzyme activities were monitored with paranitroanilide-derivatized and fluorescence resonance energy transfer (FRET) peptide substrates, and by gliadin zymography. Epitope elimination was determined in R5 and G12 ELISAs. The gliadin-degrading Rothia enzymes were identified by LC-ESI-MS/MS as hypothetical proteins ROTMU0001_0241 (C6R5V9_9MICC), ROTMU0001_0243 (C6R5W1_9MICC), and ROTMU0001_240 (C6R5V8_9MICC). A search with the Basic Local Alignment Search Tool revealed that these are subtilisin-like serine proteases belonging to the peptidase S8 family. Alignment of the major Rothia subtilisins indicated that all contain the catalytic triad with Asp (D), His (H), and Ser (S) in the D-H-S order. They cleaved succinyl-Ala-Ala-Pro-Phe-paranitroanilide, a substrate for subtilisin with Pro in the P2 position, as in Tyr-Pro-Gln and Leu-Pro-Tyr in gluten, which are also cleaved. Consistently, FRET substrates of gliadin immunogenic epitopes comprising Xaa-Pro-Xaa motives were rapidly hydrolyzed. The Rothia subtilisins and two subtilisins from Bacillus licheniformis, subtilisin A and the food-grade Nattokinase, efficiently degraded the immunogenic gliadin-derived 33-mer peptide and the immunodominant epitopes recognized by the R5 and G12 antibodies. This study identified Rothia and food-grade Bacillus subtilisins as promising new candidates for enzyme therapeutics in CD.

Despite sequence homologies to gluten, salivary proline-rich proteins do not elicit immune responses central to the pathogenesis of celiac disease.

Celiac disease (CD) is an inflammatory disorder triggered by ingested gluten, causing immune-mediated damage to the small-intestinal mucosa. Gluten proteins are strikingly similar in amino acid composition and sequence to proline-rich proteins (PRPs) in human saliva. On the basis of this feature and their shared destination in the gastrointestinal tract, we hypothesized that salivary PRPs may modulate gluten-mediated immune responses in CD. Parotid salivary secretions were collected from CD patients, refractory CD patients, non-CD patients with functional gastrointestinal complaints, and healthy controls. Structural similarities of PRPs with gluten were probed with anti-gliadin antibodies. Immune responses to PRPs were investigated toward CD patient-derived peripheral blood mononuclear cells and in a humanized transgenic HLA-DQ2/DQ8 mouse model for CD. Anti-gliadin antibodies weakly cross-reacted with the abundant salivary amylase but not with PRPs. Likewise, the R5 antibody, recognizing potential antigenic gluten epitopes, showed negligible reactivity to salivary proteins from all groups. Inflammatory responses in peripheral blood mononuclear cells were provoked by gliadins whereas responses to PRPs were similar to control levels, and PRPs did not compete with gliadins in immune stimulation. In vivo, PRP peptides were well tolerated and nonimmunogenic in the transgenic HLA-DQ2/DQ8 mouse model. Collectively, although structurally similar to dietary gluten, salivary PRPs were nonimmunogenic in CD patients and in a transgenic HLA-DQ2/DQ8 mouse model for CD. It is possible that salivary PRPs play a role in tolerance induction to gluten early in life. Deciphering the structural basis for the lack of immunogenicity of salivary PRPs may further our understanding of the toxicity of gluten.

Identification of Pseudolysin (lasB) as an Aciduric Gluten-Degrading Enzyme with High Therapeutic Potential for Celiac Disease.

OBJECTIVES: Immunogenic gluten proteins implicated in celiac disease (CD) largely resist degradation by human digestive enzymes. Here we pursued the isolation of gluten-degrading organisms from human feces, aiming at bacteria that would digest gluten under acidic conditions, as prevails in the stomach. METHODS: Bacteria with gluten-degrading activities were isolated using selective gluten agar plates at pH 4.0 and 7.0. Proteins in concentrated bacterial cell sonicates were separated by diethylaminoethanol chromatography. Enzyme activity was monitored with chromogenic substrates and gliadin zymography. Elimination of major immunogenic gluten epitopes was studied with R5 and G12 enzyme-linked immunosorbent assays. RESULTS: Gliadin-degrading enzyme activities were observed for 43 fecal isolates, displaying activities in the ~150-200 and <50 kDa regions. The active strains were identified as Pseudomonas aeruginosa. Gliadin degradation in gel was observed from pH 2.0 to 7.0. Liquid chromatography-electrospray ionization-tandem mass spectrometry analysis identified the enzyme as pseudolysin (lasB), a metalloprotease belonging to the thermolysin (M4) family proteases. Its electrophoretic mobility in SDS-polyacrylamide gel electrophoresis and gliadin zymogram gels was similar to that of a commercial lasB preparation, with tendency of oligomerization. Pseudolysin eliminated epitopes recognized by the R5 antibody, while those detected by the G12 antibody remained intact, despite destruction of the nearby major T-cell epitope QPQLPY. CONCLUSIONS: Pseudolysin was identified as an enzyme cleaving gluten effectively at extremely low as well as near-neutral pH values. The potential to degrade gluten during gastric transport opens possibilities for its application as a novel therapeutic agent for the treatment of CD.

Effect of Rothia mucilaginosa enzymes on gliadin (gluten) structure, deamidation, and immunogenic epitopes relevant to celiac disease.

Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzyme-treated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease.

High-resolution high-performance liquid chromatography with electrospray ionization mass spectrometry and tandem mass spectrometry characterization of a new isoform of human salivary acidic proline-rich proteins named Roma-Boston Ser22 (Phos) → Phe variant.

During a survey of human saliva by a top-down reversed-phase high-performance liquid chromatography with electrospray ionization mass spectrometry approach, two proteins eluting at 27.4 and 28.4 min, with average masses of 15 494 ± 1 and 11 142 ± 1 Da, were detected in a subject from Boston. The Δmass value (4352 Da) of the two proteins was similar to the difference in mass values between intact (150 amino acids, [a.a.]) and truncated acidic proline-rich proteins (aPRPs; 106 a.a.) suggesting an a.a. substitution in the first 106 residues resulting in a strong reduction in polarity, since under the same experimental conditions aPRPs eluted at ∼22.5 min (intact) and 23.5 min (truncated forms). Manual inspection of the high-resolution high-performance liquid chromatography with electrospray ionization tandem mass spectra of the truncated isoform showed the replacement of the phosphorylated Ser-22 in PRP-3 with a Phe residue. Inspection of the tandem mass spectra of the intact isoform confirmed the substitution, which is allowed by the code transition TCT→TTT and is in agreement with the dramatic increase in elution time. The isoform was also detected in two other subjects, one from Boston (unrelated to the previous) and one from Rome. For this reason we propose to name this variant PRP-1 (PRP-3) RB (Roma-Boston) Ser22 (phos)→Phe.

Influence of histatin 5 on Candida albicans mitochondrial protein expression assessed by quantitative mass spectrometry.

Individual aspects of the mode of action of histatin 5, a human salivary antifungal protein, have been partially elucidated, but the mechanism likely involves a complex set of events that have not been characterized. Previous evidence points toward histatin-induced alterations in mitochondrial function. The purpose of the present study was to verify and quantify changes in the mitochondrial proteome of Candida albicans treated with histatin 5. Cell killing was determined by plating and differential protein expression levels in the mitochondrial samples were determined by quantitative proteomics approaches employing mTRAQ and ICAT labeling and Western blotting. Relative quantitation ratios were established for 144 different proteins. Up-regulated mitochondrial proteins were predominantly involved in genome maintenance and gene expression, whereas proteins that constitute the respiratory enzyme complexes were mostly down-regulated. The differential expression of ATP synthase gamma chain and elongation factor 1-alpha were confirmed by Western blotting by comparison to levels of cytochrome c which were unchanged upon histatin treatment. The mTRAQ and ICAT proteomics results suggest that key steps in the histatin 5 antifungal mechanism involve a bioenergetic collapse of C. albicans, caused essentially by a decrease in mitochondrial ATP synthesis.

The antifungal activity of human parotid secretion is species-specific.

Candida albicans is the major fungal colonizer of the oral cavity and causes oral candidiasis in immunocompromised patient populations. While antifungal proteins in saliva have been identified and the virulence factors of C. albicans have been well studied, little is known about the role saliva plays in the preferential colonization of the oral cavity by C. albicans. We report that the fungistatic activity of human parotid secretion toward six C. albicans strains is considerably lower than towards nine non-C. albicans fungal species (average IC50 values >1000 mg/l and <70 mg/l, respectively). The species-specific activity of parotid secretion suggests that saliva may play a determining role in oral fungal colonization patterns.

Mass spectrometric identification of key proteolytic cleavage sites in statherin affecting mineral homeostasis and bacterial binding domains.

Human salivary statherin inhibits both primary and secondary calcium phosphate precipitation and, upon binding to hydroxyapatite, associates with a variety of oral bacteria. These functions, crucial in the maintenance of tooth enamel integrity, are located in defined regions within the statherin molecule. Proteases associated with saliva, however, cleave statherin effectively, and it is of importance to determine how statherin functional domains are affected by these events. Statherin was isolated from human parotid secretion by zinc precipitation and purified by reversed-phase high performance liquid chromatography (RP-HPLC). To characterize the proteolytic process provoked by oral proteases, statherin was incubated with whole saliva and fragmentation was monitored by RP-HPLC. The early formed peptides were structurally characterized by reversed phase liquid chromatography electrospray-ionization tandem mass spectrometry. Statherin was degraded 3.6× faster in whole saliva than in whole saliva supernatant. The main and primary cleavage sites were located in the N-terminal half of statherin, specifically after Arg(9), Arg(10), and Arg(13); after Phe(14) and Tyr(18); and after Gly(12), Gly(15), Gly(17) and Gly(19) while the C-terminal half of statherin remained intact. Whole saliva protease activities separated the charged N-terminus from the hydrophobic C-terminus, negatively impacting on full length statherin functions comprising enamel lubrication and inhibition of primary calcium phosphate precipitation. Cryptic epitopes for bacterial binding residing in the C-terminal domain were likewise affected. The full characterization of the statherin peptides generated facilitates the elucidation of their novel functional roles in the oral and gastro-intestinal environment.

Large-scale phosphoproteome of human whole saliva using disulfide-thiol interchange covalent chromatography and mass spectrometry.

To date, only a handful of phosphoproteins with important biological functions have been identified and characterized in oral fluids, and these include some of the abundant protein constituents of saliva. Whole saliva (WS) samples were trypsin digested, followed by chemical derivatization using dithiothreitol (DTT) of the phospho-serine/threonine-containing peptides. The DTT-phosphopeptides were enriched by covalent disulfide-thiol interchange chromatography and analysis by nanoflow liquid chromatography and electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The specificity of DTT chemical derivatization was evaluated separately under different base-catalyzed conditions with NaOH and Ba(OH)(2), blocking cysteine residues by iodoacetamide and enzymatic O-deglycosylation prior to DTT reaction. Further analysis of WS samples that were subjected to either of these conditions provided supporting evidence for phosphoprotein identifications. The combined chemical strategies and mass spectrometric analyses identified 65 phosphoproteins in WS; of these, 28 were based on two or more peptide identification criteria with high confidence and 37 were based on a single phosphopeptide identification. Most of the identified proteins (∼80%) were previously unknown phosphoprotein components. This study represents the first large-scale documentation of phosphoproteins of WS. The origins and identity of WS phosphoproteome suggest significant implications for both basic science and the development of novel biomarkers/diagnostic tools for systemic and oral disease states.

Kinetics of histatin proteolysis in whole saliva and the effect on bioactive domains with metal-binding, antifungal, and wound-healing properties.

The present study was undertaken to investigate the rate and mode of degradation of individual histatin proteins in whole saliva to establish the impact on its functional domains. Pure synthetic histatins 1, 3, and 5 were incubated with whole saliva supernatant as the enzyme source, and peptides in the resultant digests were separated by reverse-phase-HPLC and structurally characterized by electrospray ionization-tandem mass spectrometry. The overall V(max)/K(m) ratios, a measure of proteolytic efficiency, were on the order of histatin-5 > histatin-3 > histatin-1. Mathematical models predict that histatins 1, 3, and 5 levels in whole saliva stabilize at 5.1, 1.9, and 1.2 microM, representing 59, 27, and 11% of glandular histatins 1, 3, and 5 levels, respectively. Monitoring of the appearance and disappearance of histatin fragments yielded the identification of the first targeted enzymatic cleavage sites as K(13) and K(17) in histatin 1, R(22), Y(24), and R(25) in histatin 3, and Y(10), K(11), R(12), K(13), H(15), E(16), K(17), and H(18) in histatin 5. The data indicate that metal-binding, antifungal, and wound-healing domains are largely unaffected by the primary cleavage events in whole saliva, suggesting a sustained functional activity of these proteins in the proteolytic environment of the oral cavity.

Gluten Degrading Enzymes for Treatment of Celiac Disease.

Celiac disease (CeD) affects about 1% of most world populations. It presents a wide spectrum of clinical manifestations, ranging from minor symptoms to mild or severe malabsorption, and it may be associated with a wide variety of autoimmune diseases. CeD is triggered and maintained by the ingestion of gluten proteins from wheat and related grains. Gluten peptides that resist gastrointestinal digestion are antigenically presented to gluten specific T cells in the intestinal mucosa via HLA-DQ2 or HLA-DQ8, the necessary genetic predisposition for CeD. To date, there is no effective or approved treatment for CeD other than a strict adherence to a gluten-free diet, which is difficult to maintain in professional or social environments. Moreover, many patients with CeD have active disease despite diet adherence due to a high sensitivity to traces of gluten. Therefore, safe pharmacological treatments that complement the gluten-free diet are urgently needed. Oral enzyme therapy, employing gluten-degrading enzymes, is a promising therapeutic approach. A prerequisite is that such enzymes are active under gastro-duodenal conditions, quickly neutralize the T cell activating gluten peptides and are safe for human consumption. Several enzymes including prolyl endopeptidases, cysteine proteases and subtilisins can cleave the human digestion-resistant gluten peptides in vitro and in vivo. Examples are several prolyl endopeptidases from bacterial sources, subtilisins from Rothia bacteria that are natural oral colonizers and synthetic enzymes with optimized gluten-degrading activities. Without exception, these enzymes must cleave the otherwise unusual glutamine and proline-rich domains characteristic of antigenic gluten peptides. Moreover, they should be stable and active in both the acidic environment of the stomach and under near neutral pH in the duodenum. This review focuses on those enzymes that have been characterized and evaluated for the treatment of CeD, discussing their origin and activities, their clinical evaluation and challenges for therapeutic application. Novel developments include strategies like enteric coating and genetic modification to increase enzyme stability in the digestive tract.

Commensal Bacterium Rothia aeria Degrades and Detoxifies Gluten via a Highly Effective Subtilisin Enzyme.

Celiac disease is characterized by a chronic immune-mediated inflammation of the small intestine, triggered by gluten contained in wheat, barley, and rye. Rothia aeria, a gram-positive natural colonizer of the oral cavity and the upper digestive tract is able to degrade and detoxify gluten in vitro. The objective of this study was to assess gluten-degrading activity of live and dead R. aeria bacteria in vitro, and to isolate the R. aeria gluten-degrading enzyme. METHODS: After an overnight fast, Balb/c mouse were fed a 1 g pellet of standard chow containing 50% wheat (and 4% gliadin) with or without 1.6 × 107 live R. aeria bacteria. After 2 h, in vivo gluten degradation was assessed in gastric contents by SDS-PAGE and immunoblotting, and immunogenic epitope neutralization was assessed with the R5 gliadin ELISA assay. R. aeria enzyme isolation and identification was accomplished by separating proteins in the bacterial cell homogenate by C18 chromatography followed by gliadin zymography and mass spectrometric analysis of excised bands. RESULTS: In mice fed with R. aeria, gliadins and immunogenic epitopes were reduced by 20% and 33%, respectively, as compared to gluten digested in control mice. Killing of R. aeria bacteria in ethanol did not abolish enzyme activity associated with the bacteria. The gluten degrading enzyme was identified as BAV86562.1, here identified as a member of the subtilisin family. CONCLUSION: This study shows the potential of R. aeria to be used as a first probiotic for gluten digestion in vivo, either as live or dead bacteria, or, alternatively, for using the purified R. aeria enzyme, to benefit the gluten-intolerant patient population.

Fiber-optic microsphere-based antibody array for the analysis of inflammatory cytokines in saliva.

Antibody microarrays have emerged as useful tools for high-throughput protein analysis and candidate biomarker screening. We describe here the development of a multiplexed microsphere-based antibody array capable of simultaneously measuring 10 inflammatory protein mediators. Cytokine-capture microspheres were fabricated by covalently coupling monoclonal antibodies specific for cytokines of interest to fluorescently encoded 3.1 microm polymer microspheres. An optical fiber bundle containing approximately 50,000 individual 3.1 microm diameter fibers was chemically etched to create microwells in which cytokine-capture microspheres could be deposited. Microspheres were randomly distributed in the wells to produce an antibody array for performing a multiplexed sandwich immunoassay. The array responded specifically to recombinant cytokine solutions in a concentration-dependent fashion. The array was also used to examine endogenous mediator patterns in saliva supernatants from patients with pulmonary inflammatory diseases such as asthma and chronic obstructive pulmonary disease (COPD). This array technology may prove useful as a laboratory-based platform for inflammatory disease research and diagnostics, and its small footprint could also enable integration into a microfluidic cassette for use in point-of-care testing.

A general enhancement of autonomic and cortisol responses during social evaluative threat.

OBJECTIVE: To examine the Social Self Preservation Theory, which predicts that stressors involving social evaluative threat (SET) characteristically activate the hypothalamic-pituitary-adrenal (HPA) axis. The idea that distinct psychosocial factors may underlie specific patterns of neuroendocrine stress responses has been a topic of recurrent debate. METHODS: Sixty-one healthy university students (n = 31 females) performed a challenging speech task in one of three conditions that aimed to impose increasing levels of SET: performing the task alone (no social evaluation), with one evaluating observer, or with four evaluating observers. Indices of sympathetic (preejection period) and parasympathetic (heart rate variability) cardiac drive were obtained by impedance- and electrocardiography. Salivary cortisol was used to index HPA activity. Questionnaires assessed affective responses. RESULTS: Affective responses (shame/embarrassment, anxiety, negative affect, and self-esteem), cortisol, heart rate, sympathetic and parasympathetic activation all differentiated evaluative from nonevaluative task conditions (p < .001). The largest effect sizes were observed for cardiac autonomic responses. Physiological reactivity increased in parallel with increasing audience size (p < .001). An increase in cortisol was predicted by sympathetic activation during the task (p < .001), but not by affective responses. CONCLUSION: It would seem that SET determines the magnitude, rather than the pattern, of physiological activation. This potential to perturb broadly multiple physiological systems may help explain why social stress has been associated with a range of health outcomes. We propose a threshold-activation model as a physiological explanation for why engaging stressors, such as those involving social evaluation or uncontrollability, may seem to induce selectively cortisol release.

Pharmaceutically modified subtilisins withstand acidic conditions and effectively degrade gluten in vivo.

Detoxification of gluten immunogenic epitopes is a promising strategy for the treatment of celiac disease. Our previous studies have shown that these epitopes can be degraded in vitro by subtilisin enzymes derived from Rothia mucilaginosa, a natural microbial colonizer of the oral cavity. The challenge is that the enzyme is not optimally active under acidic conditions as encountered in the stomach. We therefore aimed to protect and maintain subtilisin-A enzyme activity by exploring two pharmaceutical modification techniques: PEGylation and Polylactic glycolic acid (PLGA) microencapsulation. PEGylation of subtilisin-A (Sub-A) was performed by attaching methoxypolyethylene glycol (mPEG, 5 kDa). The PEGylation protected subtilisin-A from autolysis at neutral pH. The PEGylated Sub-A (Sub-A-mPEG) was further encapsulated by PLGA. The microencapsulated Sub-A-mPEG-PLGA showed significantly increased protection against acid exposure in vitro. In vivo, gluten immunogenic epitopes were decreased by 60% in the stomach of mice fed with chow containing Sub-A-mPEG-PLGA (0.2 mg Sub-A/g chow) (n = 9) compared to 31.9% in mice fed with chow containing unmodified Sub-A (n = 9). These results show that the developed pharmaceutical modification can protect Sub-A from auto-digestion as well as from acid inactivation, thus rendering the enzyme more effective for applications in vivo.

Use of colorimetric test strips for monitoring the effect of hemodialysis on salivary nitrite and uric acid in patients with end-stage renal disease: a proof of principle.

BACKGROUND: Initial screening of potential biomarkers for monitoring dialysis was performed with saliva samples collected from patients with end-stage renal disease (ESRD). A more thorough analysis of the most promising markers identified in the initial screening was conducted with saliva samples acquired at hourly intervals throughout dialysis to monitor analyte concentrations as dialysis progressed. We observed that salivary nitrite (NO(2)(-)) and uric acid (UA) concentrations consistently decreased as dialysis proceeded. METHODS: Solution-based colorimetric-detection chemistries for NO(2)(-) and UA were converted to a test strip format to produce a simple method for semiquantitatively measuring NO(2)(-) and UA concentrations in the clinic or at the patient's home. We assessed the test strips with saliva samples collected from both ESRD patients undergoing dialysis and healthy control volunteers to qualitatively monitor the effect of dialysis on salivary NO(2)(-) and UA. We used computer software to analyze digital images of the resulting test strip color intensities. RESULTS: Test strip measurements showed that mean salivary concentrations of NO(2)(-) and UA were decreased in ESRD patients by 86% and 39%, respectively, compared with 15% and 9% for time-matched controls. Comparison of test strip results with calibrated solution-based assays suggests that the test strips can semiquantitatively measure salivary concentrations of NO(2)(-) and UA. CONCLUSIONS: The colorimetric test strips monitored changes in salivary NO(2)(-) and UA concentrations that occurred in ESRD patients during dialysis. The test strips may prove useful for noninvasively evaluating dialysis progress and may also be useful for monitoring renal disease status.

Whole saliva proteolysis: wealth of information for diagnostic exploitation.

Whole saliva (WS) protein profiles differ significantly from those of glandular salivary secretions. Rapid proteolysis of the prominent members of the salivary protein families by WS resident proteases appears to be a major cause for the observed differences. We propose that the rate and mode of glandular salivary protein degradation in the oral cavity contains information that could be of unique value in the diagnosis of oral disease.