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Psychosomatic Medicine 62:40-49 (2000)
© 2000 American Psychosomatic Society

ORIGINAL ARTICLES

Salivary MUC5B-Mediated Adherence (Ex Vivo) of Helicobacter pylori During Acute Stress

Jos A. Bosch, MSc, Eco J. C. de Geus, PhD, Toon J. M. Ligtenberg, PhD, Kamran Nazmi, BSc, Enno C. I. Veerman, PhD, Johan Hoogstraten, PhD and Arie V. Nieuw Amerongen, PhD

From the Departments of Oral Biology (J.A.B., T.J.M.L, K.N., E.C.I.V., A.V.N.A.), Community Dentistry and Dental Health Education (J.H.), Academic Centre for Dentistry Amsterdam; and Faculty of Psychology (E.J.C.D.G.), Department of Biological Psychology, Vrije Universiteit (E.J.C.D.G), Amsterdam, The Netherlands.

Address reprint requests to: Jos A. Bosch, Academic Centre for Dentistry Amsterdam, Department of Oral Biology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Email: ja.bosch.obc.acta{at}med.vu.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
OBJECTIVE: Biochemical host defenses at mucosal sites, such as the oral cavity, play a key role in the regulation of microbial ecology and the prevention of infectious disease. These biochemical factors have distinct features, some of which benefit the host and some that benefit bacteria. We investigated the effects of acute stress on the salivary levels of the carbohydrate structure sulfo-Lewisa (sulfo-Lea), which is linked to the mucosal glycoprotein MUC5B. Sulfo-Lea was recently identified as an adhesion molecule for Helicobacter pylori; therefore, we also measured saliva-mediated adherence (ex vivo) of H. pylori. The oral cavity is suspected to be involved in the transmission of H. pylori.

METHODS: Saliva was collected from 17 undergraduates before (baseline), during (stress), and after (recovery) exposure to a video showing surgical procedures. In addition, blood pressure, an impedance cardiogram, and an electrocardiogram were recorded.

RESULTS: During stressor exposure, participants reported increased state anxiety. In addition, stroke volume increased and heart rate decreased. The stressor induced a strong increase in salivary sulfo-Lea concentration (U/ml), sulfo-Lea output (U/min), sulfo-Lea/total protein ratio (U/mg protein), and saliva-mediated adherence (ex vivo) of H. pylori. As expected, sulfo-Lea concentration correlated with the adherence of H. pylori (r = 0.72, p < .05). It was demonstrated that the observed adherence was induced by MUC5B and that the carbohydrate structure sulfo-Lea contributed to this process.

CONCLUSIONS: Our study demonstrated a direct link between stress-mediated biochemical changes and altered host-microbe interactions in humans. Increased bacterial adherence may be a contributing factor in the observed relationship between stress and susceptibility to infectious disease.

Key Words: sulfo-mucin • MG1 • psychoneuroimmunology • innate immunity • laboratorystress • microbiology

Abbreviations: DBP = diastolic blood pressure; ECG = electrocardiogram; ELISA = enzyme-linked immunosorbent assay; HR = heart rate; ICG = impedance cardiogram; MANOVA = multivariate analysis ofvariance; PBS = phosphate-buffered saline; PEP = preinjectionperiod; RSA = respiratory sinus arrhythmia; SBP = systolicblood pressure; S-IgA = secretory immunoglobulin A; sulfo-Lea = sulfo-Lewisa; SV = strokevolume; U = ELISA units.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
For many years, investigators have documented the impact of behavioral factors on immune functioning as a possible determinant of the observed relationship between stress and susceptibility to infectious disease (1, 2). Infections are often initiated at mucosal surfaces, which form a boundary with the outside world. Host defense at mucosal surfaces is mediated by both nonspecific and immunologically specific mechanisms (3–5). Several studies in psychoimmunology have utilized S-IgA as an index of specific mucosal immune function (6). However, the effects of behavioral factors on nonspecific mucosal defenses remain, with a few exceptions (7–9), unexplored.

Nonspecific mucosal defense is established by a multitude of proteins, most of which are not produced by cells of the immune system but by secretory gland cells (4, 10). These secretory proteins protect the body against invading bacteria, viruses, yeasts, and toxins, by means of many biochemically different mechanisms. Examples of such antimicrobial mechanisms are killing, growth inhibition, inactivation of proteolytic enzymes, and prevention of adherence to host tissue (11–16). In this regard, the adjective "nonspecific" is a misnomer, considering the enormous functional diversity and specificity of this heterogeneous population of proteins.

The study described here addressed "nonspecific" host defense in saliva. Saliva plays a crucial role in the maintenance of oral health, which becomes dramatically clear in patients with xerostomia, or dry mouth syndrome. One of the symptoms of this syndrome is a rapid increase in dental caries and other oral infections, accompanied by an increase in pathogenic bacteria (17). However, the functions of saliva are not limited to the protection of the oral cavity because the mouth also serves as a reservoir and portal of entry for microorganisms that are active elsewhere in the body. These include several Streptococcus species (18), Hemophilus influenzae (19), Epstein-Barr virus, and herpes simplex virus (20). Here, the role of saliva is not necessarily a protective one. For example, whereas HIV is inactivated by saliva (21), pathogens such as Epstein-Barr virus and herpes simplex virus are, in fact, transmitted by saliva (20). It is for this reason that the pathological condition that results from Epstein-Barr virus infection is nicknamed "the kissing disease."

Because the mouth functions as the portal of entry to the gastrointestinal system, the possible role of the oral cavity in the transmission of Helicobacter pylori has been debated in recent years (22–24). H. pylori plays a causative role in the pathogenesis of gastritis, gastric atrophy, and peptic and duodenal ulcers (25, 26). Infection with this bacterium is also associated with an increased risk of gastric adenocarcinoma and the occurrence of mucosa-associated lymphoid tissue lymphoma (27, 28). Consequently, H. pylori is classified as a type I human carcinogen (29). Although the human stomach is the only well-established "reservoir" of H. pylori, in patients suffering from gastric infections, the microorganism has been detected at various sites of the oral cavity, such as the palate, cheeks, gingival pockets, dental plaque, and saliva (30–36). Therefore, the oral-oral route has been suggested as a modality of transmission for this infection. Although the debate on the oral-oral transmission of H. pylori is as yet undecided, alternative modalities of transmission (eg, fecal-oral and gastric-oral) also involve the oral cavity (22, 25, 37, 38). In addition, it has been suggested that the oral cavity is a possible source of reinfection in cases where H. pylori has been eradicated from the stomach (32, 39).

The carbohydrate structures that are linked to glycoproteins often function as attachment sites for bacteria and viruses (40, 41). Recently, the carbohydrate structure sulfo-Lea was identified as an adhesion molecule for H. pylori (42, 43). In saliva, sulfo-Lea is uniquely present on the glycoprotein MUC5B1, terminating the carbohydrate chains linked to this glycoprotein (10). MUC5B, secreted by the sublingual, submandibular, and palatine salivary glands, is a very large mucosal glycoprotein, or mucin, present in a mucous protein layer that covers and protects the tissues of the oral cavity (44, 45). As a main constituent of this mucous layer, MUC5B is a potential site of attachment for organisms that colonize the oral cavity. MUC5B is also present in the secretions of the respiratory tract, eyes, and gastrointestinal tract (10). Although the exact physiological significance of the attachment of H. pylori to salivary MUC5B is still unclear, possible functions may be facilitation of transport to the stomach or colonization of the oral epithelium.


   AIM OF STUDY
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
There exists convincing evidence of links between stress and susceptibility to infectious disease in humans, and much research in behavioral medicine is dedicated to specifying the exact physiological pathways responsible for this link. However, most research in this field is concentrated on humoral and cellular immunity and almost completely ignores the possible role of innate biochemical defenses. In this study, we investigated the effects of acute stress on the secretion in saliva of the carbohydrate structure sulfo-Lea, which is linked to the glycoprotein MUC5B. In addition, we quantified the saliva-mediated adherence (ex vivo) of H. pylori. Adhesion is a process by which microbes gain a stable foothold at the portal of entry. Microbial adhesion forms a first and essential step in the course of an infection (46). The primary aim of this study was to establish whether stress-mediated biochemical alterations in mucosal secretions (salivary levels of sulfo-Lea) could be linked directly to an altered host-microbe interaction (saliva-mediated adherence of H. pylori).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
Participants and Procedure
Study participants were 18 male undergraduate volunteers ranging in age from 18 to 29 years (mean, 23 years). Participants received 15 Dutch guilders for their participation. None of the students were on medication, and all stated that they were in good health. Participants were instructed to abstain from smoking, drinking caffeine-containing beverages, and engaging in physical exercise for 1 hour before the experiment. All participants gave written informed consent on the aim and procedure of the study.

Measurements were recorded between 1:30 and 4:00 PM. On arrival, the experimental procedure was explained to the subject, electrodes for electrocardiography and impedance cardiography were attached, and the method of saliva collection was rehearsed. Participants then filled out a self-report questionnaire and were allowed to read self-selected magazines with neutral content for 20 minutes. The experiment started with a baseline measurement lasting 8 minutes. The experimental manipulation in this study was an 11-minute video showing bloody dental procedures, such as the extraction of front teeth and removal of a molar with a pick. This video was selected from among several videos on the basis of results of a separate pilot study (N = 20). Pilot study participants typically characterized the selected video as "horrible" or "gruesome" and reported increased anxiety during viewing.

Saliva was collected before the video was shown (baseline), during the final part of the video (stressor), and 10 minutes after the end of the video (recovery). Immediately after each saliva collection, participants also filled out a state anxiety questionnaire. The ECG and ICG were recorded continuously.

Saliva Collection
The method of saliva collection was practiced before the start of the experiment to ensure standardization. Saliva was collected by means of the spitting method according to the directions given by Navazesh (47). This method was shown to have adequate retest reliability (r = 0.79), which was confirmed by our data. The collection trial starts with the instruction to void the mouth of saliva by swallowing. Subsequently, saliva is allowed to accumulate in the floor of the mouth without stimulation of saliva secretion by means of orofacial movements (unstimulated saliva). The participant spits into a preweighed, ice-chilled test tube every 60 seconds. Each saliva collection period was 4 minutes. After collection, saliva was homogenized by vigorous shaking using a vortex and clarified by centrifugation (10,000g, 5 minutes) to eliminate buccal cells and oral microorganisms. The clear supernatant was divided into 500-µl aliquots and stored at -20°C until use.

Materials
Monoclonal antibody against sulfo-Lea was prepared in our laboratory as described by Veerman et al. (48, 49). A panel of polyclonal sera against H. pylori was kindly provided by Ben J. Appelmelk and Theo Verboom (Department of Medical Microbiology, Vrije Universiteit, Amsterdam). Details on the production of these antisera are described by Appelmelk et al. (50). The polyclonal antiserum used in this study was selected after testing a panel of anti-H. pylori polyclonal antisera for cross-reactivity with saliva. H. pylori strain NCTC 11637 was obtained from the National Collection of Type Cultures (Public Health Laboratory Service, London, UK). Bicinchoninic acid (BCA) protein assay reagent was obtained from Pierce (Rockford, UK), and microtiter plates were from Greiner (Recklinghausen, Germany) and Dynex (Chantilly, VA). Polymeric neoglycoconjugates for the blocking assay were obtained from Synstone (Munich, Germany). All other chemicals were obtained from Sigma Chemical Company (St. Louis, MO). Blood pressure was measured with a Dinamap Vital Signs Monitor (model 845 XT, Critikon, Tampa, FL). ICG and ECG signals were recorded using six Ag/AgCl spot electrodes (AMI type 1650–005, Medtronic, Haverhill, MA) and the Vrije Universiteit Ambulatory Monitoring Device (Amsterdam, The Netherlands) (51).

Cardiovascular Assessment
Assessment of cardiovascular response focused on blood pressure and cardiac autonomic balance. SBP and DBP were measured every 2 minutes during each of the three separate conditions. Indices of sympathetic and parasympathetic drive were obtained by analysis of the ECG and thoracic impedance (ICG) signals (52, 53). The ECG and ICG complexes were ensemble-averaged with reference to the ECG R wave across 1-minute periods (54). From these ensembles, condition averages were extracted for interbeat interval, PEP, SV, and left ventricular ejection time. Spectral power of HR was derived by frequency domain analysis of the continuously recorded interbeat interval time series using the CARSPAN program (55). Two frequency bands were deemed of interest in the integrated power density spectra: the frequency band around the intrinsic blood pressure oscillations (0.07–0.14, BLOOD) and the high-frequency band (0.15–0.40) that reflects RSA. In the time domain, the root mean square of successive differences was used as an alternative index of RSA. Changes in PEP and BLOOD have been used to index changes in cardiac sympathetic drive (56, 57), whereas changes in RSA and root mean square of successive differences are used to index changes in cardiac vagal tone.

Determination of MUC5B-Linked Sulfo-Lea and Total Salivary Protein
For the quantification of sulfo-Lea, an ELISA was used as described in detail by Veerman et al. (48). Protein was measured by the bicinchoninic acid method (58). This assay is described in detail by Bosch et al. (7). All assays have a split-half reliability of >0.98.

Bacteria
A strain of H. pylori NCTC 11637 (also designated ATCC 34504, American Type Culture Collection, Manassas, VA) was grown for 4 days under microaerophilic conditions (10% CO2, 5% O2, 85% N2) at 37°C on Dent agar plates (59) supplemented with 2,3,5-triphenyltetrazolium chloride (40 mg/ml). Cultures were harvested by centrifugation (4000g, 4°C), washed in sodium acetate buffer (100 mM, pH 5.0), and resuspended in sodium acetate buffer (with 0.5% Tween-20 added) to an optical density of 0.1 at 700 nm.

Adherence Assay
Adherence of H. pylori was measured by means of an ELISA. In 96-well microtiter plates (Immulon 1B, medium affinity), 100 µl of saliva was threefold serially diluted in 0.1 M NaHCO3 buffer (pH 9.6) with a starting solution of 1:400. Microtiter plates were subsequently incubated for 3 hours at 37°C. After incubation, the wells were rinsed three times with PBS (50 mM potassium phosphate, 0.15 M sodium chloride, pH 7.4) containing 0.1% (v/v) Tween-20, followed by two rinses with demineralized water. This rinsing was done between each incubation step. Then, 100 µl of a freshly prepared bacterial suspension was added. After incubation (1 hour, 37°C) and subsequent washing, 100 µl of rabbit-anti-H. pylori polyclonal antibody (diluted 1:500 in PBS with 0.1% Tween-20 plus 3% bovine serum albumin) was added. After 1 hour of incubation and subsequent washing, horseradish peroxidase–conjugated goat anti-rabbit immunoglobulin (100 µl, diluted 1:6000 in PBS with 0.1% Tween-20 plus 3% bovine serum albumin) was added. After incubation (1 hour, 37°C), the microtiter wells were again rinsed, and 90 µl of the substrate-containing solution was added (1,1' orthophenylene diamine, 0.03% H2O2 in 50 mM citrate, 0.1 M disodium phosphate, pH 5.0). Color was allowed to develop until sufficient intensity was reached. The reaction was stopped by adding 50 µl of a 4M H2SO4 solution, and absorbance was measured at 492 nm using a microtiter plate reader (Dynatech MR 7000). Adherence of H. pylori was quantified by calibration against the dose-response curve obtained in the same assay of a pooled saliva sample. The level of adherence of this pooled sample was indexed on 100. The assay was performed in triplicate, and the average was used. The intraassay variability of the assay is 8.1%.

Purification of MUC5B
MUC5B was isolated from saliva by ultracentrifugation (100,000g, 24 hours) under dissociative conditions, followed by gel filtration (Sephacryl HR-500, Pharmacia, Uppsala, Sweden) in 50 mM Tris/HCl/4 M guanidine chloride, pH 7.4 (60). The mucin-containing fractions were pooled, dialyzed against double-distilled water, and stored as aliquots at -20°C.

Inhibition Assays
To test whether the adherence in our assay was mediated by MUC5B, purified MUC5B was added to the H. pylori suspension and incubated for 1 hour at 37°C. The suspensions contained three concentrations of MUC5B: 100, 400, and 1600 µg/ml. A bacterial suspension without MUC5B was used as a control. Subsequently, adherence assays were performed.

In a second experiment, the bacterial suspension was incubated with an excess (30 µg/ml) of synthetic sulfo-3-galactose, the terminal part of sulfo-Lea, bound to a polyacrylamide carrier (61). This synthetic oligosaccharide is known to compete with MUC5B for binding to H. pylori by occupying an adhesion molecule (designated neutrophil-activating protein) that specifically binds to sulfated carbohydrate (43). The bacterial suspension was incubated for 1 hour at 37°C. Subsequently, adherence assays were performed. Controls involved adherence of H. pylori after preincubation with buffer and with a nonrelevant carbohydrate-polyacrylamide conjugate (ß-galactose-polyacrylamide).

Questionnaires
Participants were administered the Dutch translation of the Spielberger State/Trait-Anxiety Inventory (62). The state anxiety part (20 items) of this questionnaire was administered immediately after each saliva collection. The instruction after the second saliva collection was adapted to report on the anxiety the subject felt while viewing the video.

Participants also filled out a self-report questionnaire on health (perceived health and use of medication or other medical treatment), life habits, and behavior on the day of the experiment (smoking, alcohol, tea and coffee consumption, physical exercise, and sleep duration and quality).

Data Analysis
Data were analyzed with MANOVA (SPSSWIN7.5) using the three conditions (baseline, film, recovery) as within-subject factors. Because of their skewed distribution, salivary parameters and spectral data were logarithmically transformed before statistical analyses. For readability, the original (untransformed) values are used in the figures.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
One participant terminated his participation in the experiment because he feared fainting. The analyses are confined to the remaining 17 subjects.

State Anxiety
Mean state anxiety increased from 11.8 (SEM = 2.4) at baseline to 19.3 (SEM = 2.6) during the stressor (t(16) = 2.7, p = .01). After recovery, mean state anxiety returned to just below the baseline value (mean = 9.9, SEM = 2.1; t(16) = 3.6, p = .002).

MUC5B-Linked Sulfo-Lea and Adherence of H.
pylori

Figure 1 shows the median, mean, and standard error of salivary concentration (U/ml), output (U/min), and total protein ratio (U/mg protein) of sulfo-Lea measured at baseline, during the stressor, and during recovery. Sulfo-Lea was quantified as ELISA units (U) using a pooled saliva sample as a standard. A measure of sulfo-Lea output (U/min) was computed to control for salivary flow rate and is obtained by multiplying concentration (U/ml) by flow rate (ml/min). The sulfo-Lea/protein ratio was computed to obtain a measure of specificity: An increased ratio indicates that sulfo-Lea output occurs at a higher rate than the output of other secretory proteins. Figure 2 shows the median, mean, and standard error of saliva-mediated adherence of H. pylori (ex vivo).



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  		Fig. 1. Values of sulfo-Lea concentration (U/ml), sulfo-Lea output (U/min), and sulfo-Lea/total protein ratio (U/mg protein). Values are expressed in ELISA units (U). Bars and error bars indicate mean values and standard errors, respectively. Horizontal lines indicate median values. For statistical analysis, log-transformed values were used.

 


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  		Fig. 2. Saliva-mediated adherence (ex vivo) of H. pylori. Bars and error bars indicate mean values and standard errors, respectively. Horizontal lines indicate median values. Adherence is quantified as an index value based on a pooled saliva sample as a standard, indexed on 100.

 
MANOVA indicated a significant effect of the manipulation on sulfo-Lea levels and adherence (F(8,9) = 4.59, p = .018). Subsequent multivariate analyses showed that baseline and stressor values differed significantly (F(4,13) = 9.68, p = .001) and that recovery and stressor values also differed significantly (F(4,13) = 4.61, p = .015), whereas baseline and recovery values did not differ significantly(F(4,13) = 1.44, p = .275).

Post hoc analysis, using paired t tests, indicated that sulfo-Lea concentration differed both between baseline and stressor (t(16) = 3.73, p = .002) and between stressor and recovery (t(16) = 3.14, p = .006) (Figure 1). Sulfo-Lea output (U/ml) also showed an increase during stressor exposure, as compared with both baseline (t(16) = 4.53, p < .001) and recovery (t(16) = 3.69, p = .002) (Figure 1), indicating that the observed increase in sulfo-Lea concentration is not due to alterations in flow rate. The sulfo-Lea/protein ratio showed the same pattern as the two previous parameters: a significant increase during the stressor, as compared with both baseline (t(16) = 2.90, p = .01) and recovery (t(16) = 2.47, p = .03) (Figure 1), indicating that the observed increase in sulfo-Lea output is specific and cannot be attributed to a nonspecifically increased protein secretion during stress. Finally, saliva-mediated adherence of H. pylori was significantly increased during the stressor, as compared with both baseline (t(16) = 4.31, p = .001) and recovery (t(16) = 4.05, p = .001) (Figure 2). Because of the relatively small sample size and skewness of our data, analyses were repeated using nonparametric tests (Wilcoxon and sign tests), yielding virtually the same results, with most significant p values well below the .01 criterion.

Salivary flow rate showed a nonsignificant increase during stressor exposure (F(2,15) = 1.50, p = .24). Variations in sulfo-Lea at baseline and during the stressor and recovery were consistently and significantly correlated with variations in adherence of H. pylori (with Spearman rank correlation coefficients between 0.62 and 0.84, .05 > p > .001). Variations in salivary flow rate did not correlate with adherence of H. pylori or sulfo-Lea (p > .2), indicating that the increased adherence of H. pylori was not a concentration artifact due to an altered salivary flow rate.

Inhibition Studies
To test whether the observed correlation between sulfo-Lea and adherence of H. pylori underlies a causal relationship, two additional assays were performed as described in Materials and Methods. Preincubation of H. pylori with purified MUC5B reduced adherence in a dose-dependent manner, completely blocking adherence at a level of 1600 µg MUC5B/ml suspension (Figure 3). This result indicates that MUC5B is responsible for the observed adherence of H. pylori to the saliva-coated microtiter plate.



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  		Fig. 3. Effects of preincubation of H. pylori suspension with 0, 100, 400, and 1600 µg/ml MUC5B. These results show that preincubation of H. pylori reduces adherence in a dose-dependent manner, indicating that the observed adherence is MUC5B dependent. Adherence was quantified as an index value based on a pooled saliva sample as a standard.

 
Preincubation of the H. pylori suspension with galactose-3-sulfate resulted in an approximately 4-fold decrease in adherence, as compared with the controls, preincubation with buffer, or preincubation with galactose-polyacrylamide (not shown). However, adherence was not blocked completely, indicating that besides sulfated glycans, other domains on the MUC5B molecule are involved in the binding to H. pylori.

Cardiovascular Response
Significant effects of condition emerged only for HR (F(2,15) = 4.40, p = .031) and SV (F(2,15) = 6.38, p = .019). Post hoc analysis indicated that HR decreased during the film in comparison with the baseline value, whereas baseline and recovery HR did not differ. The decrease in HR during the film was paralleled by an increase in SV. No other effects of condition emerged. Values of selected cardiovascular parameters are presented in Table 1.


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  		Table 1. Means and Standard Deviations of Selected Cardiovascular Parameters
 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
To the best of our knowledge, this is the first study that has succeeded in demonstrating a direct link between stress-mediated biochemical changes and altered host-microbe interactions in humans. Our results show enhanced saliva-mediated adherence (ex vivo) of H. pylori during an acute stressor. This increase was shown to correlate with an increase in sulfo-Lea, a carbohydrate structure linked to the mucous glycoprotein MUC5B. Subsequent experiments, aimed at identifying the biochemical factors involved, demonstrated that MUC5B is responsible for the observed saliva-mediated adherence and that the oligosaccharide galactose-3-sulfate (the terminal part of the carbohydrate moiety of sulfo-Lea) largely contributes to this process. These results are in agreement with those of recent studies showing that sulfated oligosaccharides on MUC5B function as adhesion molecules for H. pylori (42, 43).

The findings of our study resemble the results of animal studies investigating the effects of acute stressors (ie, water immersion and restraint stress) on the activity of secretory cells in the colon, stomach, and nasopharynx (63–71). These studies demonstrated robust effects of acute stress on mucin release, synthesis, and glycosylation (a process in which carbohydrate structures on proteins are formed). The relatively acute increase in the secretion of sulfo-Lea observed in our study is most likely due to an increased release of MUC5B, not to an altered synthesis or glycosylation of MUC5B, because the time necessary for glycosylation and intracellular transport far exceeds 10 minutes (72).

The release of salivary proteins is mainly governed by the synergistic actions of both the sympathetic and parasympathetic branches of the autonomic nervous system (73, 74) and is also modulated by several neuropeptides that are released in conjunction with the conventional transmitters acetylcholine and noradrenaline (75, 76). Therefore, stress-induced autonomic activation is the most likely explanation for the observed release of MUC5B. Surprisingly, no consistent association was observed between our cardiovascular indices and salivary parameters. Possibly, cardiac vagal and sympathetic drive do not properly reflect activity in parasympathetic and sympathetic branches that innervate the MUC5B-secreting salivary glands. Future pharmacological blockade studies may elucidate the neural mechanisms involved.

Most laboratory studies in psychoneuroimmunology make use of stressors that require mental effort and that are characterized by their potential to evoke a classic fight-flight cardiovascular response pattern (eg, mental arithmetic, Stroop task, and reaction tasks) (77–84). This clearly being the dominant paradigm, it may become overlooked that there is a body of literature showing that many stressful situations do not necessarily elicit a substantial cardiovascular response and may even be associated with a reduced HR (85–89). What becomes clear from our study is that such stressors are nevertheless capable of inducing robust biochemical changes with a clear effect on immunological functioning.

The binding of H. pylori to salivary mucins may play a role in the colonization of this pathogen through the oral route. Moreover, several pathogenic bacteria other than H. pylori bind to MUC5B and/or sulfo-Lea, and our results may therefore be extrapolated to these microorganisms. Examples are Escherichia coli (90), Staphylococcus aureus (91), Candida albicans (92), Bordetella pertussis (93), Hemophilus influenzae (94), and Fusobacterium nucleatum (Groenink J, Veerman ECI, Nieuw Amerongen AV. 1999, unpublished data). The latter two pathogens, which are involved in opportunistic infections of the respiratory tract and in periodontal disease (chronic infection of the tooth-supporting tissues), respectively, are especially of interest within the context of this study because there is a growing body of research showing an association of both pathologies with stress (2, 95).

Because the mouth is the habitat of many microorganisms and is involved in the transmission of many microorganisms, an increased adherence of MUC5B- and/or sulfo-Lea-binding organisms could affect susceptibility in three different ways. First, an increased opportunity for microorganisms to colonize the oral epithelium may imply an increased risk of being infected through the oral route. Second, the increased adherence to the tissues of the oral cavity might, in case of oral-oral contact with a carrier of the pathogen, cause exposure of another host to a relatively larger inoculating dose (the number of microorganisms transmitted). Because the inoculation dose is a crucial factor determining whether an infection will proceed (46), increased adherence may therefore imply a greater risk of infecting others. Third, a specifically increased release of MUC5B, as seen in our study, might favor the colonization of bacteria that preferentially bind to MUC5B and/or the carbohydrates linked to this glycoprotein. Likewise, microorganisms that do not bind MUC5B (among which are many streptococci and staphylococci) are likely to be relatively unaffected by an increase in MUC5B (94, 96–98). Consequently, stress-mediated release of MUC5B might affect the microbial homeostasis of the mucosa; that is, when stress induces biochemical changes that are favorable for some species but not necessarily for others, these changes may lead to a microbial population shift (99–101).


   CONCLUDING REMARKS
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 
Research on the effects of psychosocial factors on mucosal defense has concentrated almost exclusively on S-IgA, the predominant immunoglobulin in mucosal secretions (6). However, the fact that S-IgA deficiency is the most common form of immunodeficiency and that individuals deficient in S-IgA show only a marginally increased susceptibility to infectious disease (5) indicates that S-IgA is not the only factor. Instead, mucosal defense is brought about by the combined activity of many proteins, which for the most part are produced not by cells of the immune system but by the secretory glandular cells. In our opinion, the relevance of the current study lies in the fact that it exemplifies how psychological factors, by altering the secretion of such proteins, can affect host-microbe interactions.

The mucosa, especially that of the oral cavity, is an extremely complex environment in which an enormous diversity in microorganisms and host factors interact. Therefore, some caution is needed in extrapolating our results to the in vivo situation. Future studies may extend the approach presented in our current study by examining different aspects of host-microbe interactions simultaneously. The present study and other studies carried out by our group (7, 8) indicate that the secretion of nonimmunological proteins is altered in subjects under stress. Moreover, our results show that these alterations directly affect the way in which microorganisms are dealt with. We therefore conclude that the inclusion of innate defense-system parameters in studies in the field of behavioral medicine may contribute to our understanding of the relationship between stress and susceptibility to infectious disease.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
REFERENCES
 
We are very grateful to Ben J. Appelmelk, PhD, and Theo Verboom, PhD, for providing antisera against H. pylori. J. J. M. Bijlsma, MSc, advised on culturing H. pylori. M. Sparrius, BSc, advised on the adherence assay. P. M. de Groot, MSc, gave technical support on the AMS-46. Marcia Meerum-Terwogt and Willem G.J. van der Sluijs, DDS, assisted during the pilot phase of this study. We thank Bob Bermond, PhD, for his creative and supportive comments. The work in this study was financially supported by the Netherlands Institute for Dental Sciences and the Academic Center for Dentistry Amsterdam.


   NOTES
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
ACKNOWLEDGMENTS
 REFERENCES
 
Note added at proofs: In this paper, the term MUC5B denotes the whole high-molecular weight salivary mucin fraction, for which the term MG1 is in use also. This high-molecular weight mucin fraction may contain some other mucin gene products besides MUC5B. Back

Received for publication August 12, 1998.

Revision received July 1, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 AIM OF STUDY
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUDING REMARKS
 NOTES
 ACKNOWLEDGMENTS
 REFERENCES
 

  1. Herbert TB, Cohen S. Stress and immunity in humans: a meta-analytic review. Psychosom Med 1993; 55: 364–79.[Abstract/Free Full Text]
  2. Cohen S, Herbert TB. Psychological factors and physical disease from the perspective of human psychoneuroimmunology. Annu Rev Psychol 1996; 47: 113–42.[Medline]
  3. Kraehenbuhl JP, Neutra MR. Molecular and cellular basis of immune protection of mucosal surfaces. Physiol Rev 1992; 72: 853–79.[Free Full Text]
  4. Schenkels LCPM, Veerman ECI, Nieuw Amerongen AV. Biochemical composition of human saliva in relation to other mucosal fluids. Crit Rev Oral Biol Med 1995; 6: 161–75.[Abstract/Free Full Text]
  5. Lamm ME. Interaction of antigens and antibodies at mucosal surfaces. Annu Rev Microbiol 1997; 51: 311–40.[Medline]
  6. Valdimarsdottir HB, Stone AA. Psychosocial factors and secretory immunoglobulin A. Crit Rev Oral Biol Med 1997; 8: 461–74.[Abstract/Free Full Text]
  7. Bosch JA, Brand HS, Ligtenberg AJM, Bermond B, Hoogstraten J, Nieuw Amerongen AV. Psychological stress as a determinant of protein levels and salivary-induced aggregation of Streptococcus gordonii in human whole saliva. Psychosom Med 1996; 58: 374–82.[Abstract/Free Full Text]
  8. Bosch JA, Brand HS, Ligtenberg AJM, Bermond B, Hoogstraten J, Nieuw Amerongen AV. The response of salivary protein levels and S-IgA to an academic examination are associated with daily stress. J Psychophysiol 1998; 12: 384–91.
  9. Perera S, Uddin M, Hayes JA. Salivary lysozyme—a noninvasive marker for the study of the effects of stress on natural immunity. Int J Behav Med 1997; 4: 170–8.
  10. Nieuw Amerongen AV, Bolscher JGM, Bloemena E, Veerman ECI. Sulfomucins in the human body. Biol Chem Hoppe-Seyler 1998; 379: 1–18.
  11. Bevins CL. Antimicrobial peptides as agents of mucosal immunity. Ciba Found Symp 1994; 186: 250–69.[Medline]
  12. Ganz T, Lehrer RI. Defensins. Pharmacol Ther 1995; 66: 191–205.[Medline]
  13. Nieuw Amerongen AV, Bolscher JGM, Veerman ECI. Salivary mucins—protective functions in relation to their diversity. Glycobiology 1995; 5: 733–40.[Free Full Text]
  14. Lonnerdal B, Iyer S. Lactoferrin: molecular structure and biological function. Annu Rev Nutr 1995; 15: 93–110.[Medline]
  15. Henskens YMC, Veerman ECI, Nieuw Amerongen AV. Cystatins in health and disease. Biol Chem Hoppe-Seyler 1996; 1377: 71–86.
  16. McClellan KA. Mucosal defense of the outer eye. Surv Ophthalmol 1997; 42: 233–46.[Medline]
  17. Atkinson JC, Wu AJ. Salivary gland dysfunction: causes, symptoms, treatment. J Am Dent Assoc 1994; 125: 409–16.[Abstract]
  18. Jenkinson HF, Lamont RJ. Streptococcal adhesion and colonization. Crit Rev Oral Biol Med 1997; 8: 175–200.[Abstract/Free Full Text]
  19. Liljemark WF, Fenner LJ, Bloomquist CG. In vivo colonization of salivary pellicle by Haemophilus, Actinomyces and Streptococcus species. Caries Res 1986; 20: 481–97.[Medline]
  20. Merchant VA. Herpes viruses and other microorganisms of concern in dentistry. Dent Clin North Am 1991; 35: 283–98.[Medline]
  21. Shine N, Konopka K, Duzgunes N. The anti-HIV-1 activity associated with saliva. J Dent Res 1997; 76: 634–40.[Abstract/Free Full Text]
  22. Cave DR. How is Helicobacter pylori transmitted? Gastroenterology 1997; 113: 9–14.
  23. Thomas E, Jiang C, Chi DS, Li C, Ferguson DA Jr. The role of the oral cavity in Helicobacter pylori infection. Am J Gastroenterol 1997; 92: 2148–54.[Medline]
  24. Madinier IM, Fosse TM, Monteil RA. Oral carriage of Helicobacter pylori: a review. J Periodontol 1997; 68: 2–6.[Medline]
  25. Dunn BE, Cohen H, Blaser MJ. Helicobacter pylori. Clin Microbiol Rev 1997; 10: 720–41.[Abstract/Free Full Text]
  26. Goodwin CS, Mendall MM, Northfield TC. Helicobacter pylori infection. Lancet 1997; 349: 265–9.[Medline]
  27. Parsonnet J, Friedman GD, Vandersteen D, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991; 325: 1127–31.[Abstract]
  28. Parsonnet J, Hansen S, Rodriguez L, Gelb AB, Warnke RA, Jellum E, Orentreich N, Vogelman JH, Friedman GD. Helicobacter pylori infection and gastric lymphoma. N Engl J Med 1994; 330: 1267–71.[Abstract/Free Full Text]
  29. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Schisostomes, liver flukes and Helicobacter pylori. In: IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. Vol 61. Lyon,France: International Agency for Research on Cancer; 1994.
  30. Banatvala N, Lopez CR, Owen R, Abdi Y, Davies G. Helicobacter pylori in dental plaque [letter]. Lancet 1993; 341: 380.[Medline]
  31. Ferguson DA, Li C, Patel NR, Mayberry WR, Chi DS, Thomas E. Isolation of Helicobacter pylori from saliva. J Clin Microbiol 1993; 31: 2802–4.[Abstract/Free Full Text]
  32. Mapstone NP, Lynch DA, Lewis FA, Axon AT, Tompkins DS, Dixon MF, Quirke P. Identification of Helicobacter pylori-DNA in the mouths and stomachs of patients with gastritis, using PCR. J Clin Pathol 1993; 46: 540–3.[Abstract/Free Full Text]
  33. Nguyen AM, Engstrand L, Genta RM, Graham DY, El-Zaatari FA. Detection of Helicobacter pylori in dental plaque by reverse transcription-polymerase chain reaction. J Clin Microbiol 1993; 31: 783–7.[Abstract/Free Full Text]
  34. Namavar F, Roosendaal R, Kuipers EJ, De Groot P, Van der Bijl MW, Pena AS, De Graaff J. Presence of Helicobacter pylori in the oral cavity, oesophagus, stomach and faeces of patients with gastritis. Eur J Clin Microbiol Infect Dis 1995; 14: 234–7.[Medline]
  35. Li CF, Musich PR, Ha TZ, Ferguson DA, Patel NR, Chi DS, Thomas EA. High prevalence of Helicobacter pylori in saliva demonstrated by a novel PCR assay. J Clin Pathol 1995; 48: 662–6.[Abstract/Free Full Text]
  36. Li CF, Ha TZ, Ferguson DA, Chi DS, Zhao RG, Patel NR, Krishnaswamy G, Thomas EA. A newly developed PCR assay of H. pylori in gastric biopsy, saliva, and feces—evidence of high prevalence of H. pylori in saliva supports oral transmission. Dig Dis Sci 1996; 41: 2142–9.[Medline]
  37. Axon ATR. Transmission of Helicobacter pylori. Yale J Biol Med 1997; 70: 1–6.
  38. Xia HH, Talley NJ. Natural acquisition and spontaneous elimination of Helicobacter pylori infection: clinical implications. Am J Gastroenterol 1997; 92: 1780–7.[Medline]
  39. Pytko-Polonczyk J, Konturek SJ, Karczewska E, Bielanski W, Kacmarczyk-Stachowska A. Oral cavity as permanent reservoir of Helicobacter pylori and potential source of reinfection. J Physiol Pharmacol 1996; 47: 121–9.[Medline]
  40. Karlsson KA. Microbial recognition of target-cell glycoconjugates. Curr Opin Struct Biol 1995; 5: 622–35.[Medline]
  41. Sharon N, Lis H. Microbial lectins and their glycoprotein receptors. In: Montreuil J, Vliegenthart JFG, Schachter H, editors. Glycoproteins. Vol 2. London: Elsevier Science BV; 1997. p. 475–506.
  42. Veerman ECI, Bank CM, Namavar F, Appelmelk BJ, Bolscher JGM, Nieuw Amerongen AV. Sulfated glycans on oral mucin as receptors for Helicobacter pylori. Glycobiology 1997; 7: 737–43.[Abstract/Free Full Text]
  43. Namavar F, Sparrius M, Veerman ECI, Appelmelk BJ, Vandenbroucke-Grauls CM. Neutrophil-activating protein mediates adhesion of Helicobacter pylori to sulfated carbohydrates on high-molecular-weight salivary mucin. Infect Immun 1998; 66: 444–7.[Abstract/Free Full Text]
  44. Prakobphol A, Levine MJ, Tabak LA, Reddy MS. Purification of a low-molecular-weight, mucin-type glycoprotein from human submandibular-sublingual saliva. Carbohydr Res 1982; 108: 111–22.[Medline]
  45. Loomis RE, Prakobphol A, Levine MJ, Reddy MS, Jones PC. Biochemical and biophysical comparison of two mucins from human submandibular-sublingual saliva. Arch Biochem Biophys 1987; 258: 452–64.[Medline]
  46. Talaro K, Talaro A. Foundations in microbiology. 2nd ed. Dubuque (IA): William C Brown Publishers; 1996.
  47. Navazesh J. Methods for collecting saliva. Ann NY Acad Sci 1993; 694: 73–7.
  48. Veerman ECI, Valentijn-Benz M, Van den Keijbus PAM, Rathman WM, Sheehan JK, Nieuw Amerongen AV. Immunochemical analysis of high-molecular-weight human salivary mucins (MG1) using monoclonal antibodies. Arch Oral Biol 1991; 36: 923–32.[Medline]
  49. Veerman ECI, Bolscher JGM, Appelmelk BJ, Bloemena E, Van den Berg TK, Nieuw Amerongen AV. A monoclonal antibody directed against high mr salivary mucins recognizes the SO3-3gal ß1–3glcNac moiety of sulfo-Lewisa: a histochemical survey of human and rat tissue. Glycobiology 1997; 7: 37–43.[Abstract/Free Full Text]
  50. Appelmelk BJ, Simoons-Smit I, Negrini R, Moran AP, Aspinall GO, Forte JG, De Vries T, Quan H, Verboom T, Maaskant JJ, Ghiara P, Kuipers EJ, Bloemena E, Tadema TM, Townsend RR, Tyagarajan K, Crothers JM Jr, Monteiro MA, Savio A, De Graaff J. Potential role of molecular mimicry between Helicobacter pylori lipopolysaccharide and host Lewis blood group antigens in autoimmunity. Infect Immun 1996; 64: 2031–40.[Abstract/Free Full Text]
  51. De Geus EJC, van Doornen LJP. Ambulatory assessment of parasympathetic/sympathetic balance by impedance cardiography. In: Fahrenberg J, Myrtek M, editors. Ambulatory assessment: computer-assisted psychological and psychophysiological methods in monitoring and filed studies. Seattle (WA): Hogrefe and Huber; 1996. p. 141–63.
  52. Willemsen GH, De Geus EJC, Klaver CH, Van Doornen LJP, Carroll D. Ambulatory monitoring of the impedance cardiogram. Psychophysiology 1996; 33: 184–93.[Medline]
  53. Berntson GG, Bigger T, Eckberg DL, Grossman P, Kaufmann PG, Malik M, Nagajara HN, Porges S, Saul JP, Stone PH, van der Molen M. Heart rate variability: origins, methods, and interpretive caveats. Psychophysiology 1997; 34: 623–48.[Medline]
  54. Muzi M, Ebert TJ, Tristani FE, Jeutter DC, Barney JA, Smith JJ. Determination of cardiac output using ensemble-averaged impedance cardiograms. J Appl Physiol 1985; 58: 200–5.[Abstract/Free Full Text]
  55. Mulder LJM. Assessment of cardiovascular reactivity by means of spectral analysis [dissertation]. Groningen, The Netherlands: Universiteitsdrukkerij Rijksuniversiteit Groningen; 1988.
  56. Sherwood A, Allen MT, Fahrenberg J, Kelsey RM, Lovallo WR, van Doornen LJP. Methodological guidelines to impedance cardiography. Psychophysiology 1990; 27: 1–23.[Medline]
  57. Pagani M, Mazzuero G, Ferrari A, Liberati D, Cerutti S, Vaitl D, Tavazzi L, Malliani A. Sympathovagal interaction during mental stress: a study using spectral analysis of heart rate variability in healthy control subjects and patients with a prior myocardial infarction. Circulation 1991; 83 (Suppl 2): II43–51.
  58. Smith PK, Krohn RI, Hermanson GT. Measurement of protein using bicinchoninic acid. Ann Biochem 1985; 150: 76–85.
  59. Veerman ECI, Valentijn-Benz M, Bank RA, Nieuw Amerongen AV. Isolation of high molecular weight mucins from human whole saliva by ultracentrifugation. J Biol Buccale 1989; 17: 307–12.[Medline]
  60. Bovin NV, Korchagina EY, Zemlyanukhina TV, Byramova NE, Galanina ME, Zemlyakov AE, Ivanov AE, Zubov VP, Mochalova LV. Synthesis of polymeric neoglycoconjugates based on N-substituted polyacrylamides. Glycoconj J 1993; 10: 142–51.[Medline]
  61. Dent JC, McNulty CA. Evaluation of a new selective medium for Campylobacter pylori. Eur J Clin Microbiol Infect Dis 1988; 7: 555–8.[Medline]
  62. Van der Ploeg HM. Handleiding bij de Zelfbeoordelings Vragenlijst ZBV: een nederlandstalige bewerking van de Spielberger State/Trait Anxiety Inventory. Lisse, The Netherlands: Zwets and Zeitlinger; 1988.
  63. Murakami S, Mori Y. Effect of water immersion stress on the biosynthesis of rat gastric glycoproteins with or without sulfate. J Pharmacobiodynamics 1985; 8: 235–45.[Medline]
  64. Somasundaram K, Ganguly AK. Gastric mucosal protection during restraint stress in rats: alteration in gastric adherent mucus and dissolved mucus in gastric secretion. Hepatogasteroenterology 1985; 32: 24–6.
  65. Tsukada H, Seino Y, Ueda S, Uchino H, Sakai M. Influence of water-immersion stress on synthesis of mucus glycoprotein in the rat gastric mucosa. Scand J Gastroenterol 1989; 162: 19–22.
  66. Tachibana M, Senuma H, Ebara T, Kumamoto K. Stress increases the secretory product of rat nasal mucosa goblet cells. Res Commun Chem Pathol Pharmacol 1991; 73: 153–8.[Medline]
  67. Rubio CA, Huang CB. Quantification of the sulphomucin-producing cell population of the colonic mucosa during protracted stress in rats. In Vivo 1992; 6: 81–4.
  68. Castagliuolo I, Leeman SE, Bartolak-Suki E, Nikulasson S, Qiu B, Carraway RE, Pothoulakis C. A neurotensin antagonist, SR 48692, inhibits colonic responses to immobilization stress in rats. Proc Natl Acad Sci USA 1996; 93: 12611–5.[Abstract/Free Full Text]
  69. Castagliuolo I, LaMont JT, Qiu B, Fleming SM, Bhaskar KR, Nikulasson S, Kornetsky C, Pothoulakis C. Acute stress causes mucin release from rat colon: role of corticotropin releasing factor and mast cells. Am J Physiol 1996; 271: G884–92.[Abstract/Free Full Text]
  70. Delalastra CA, Martin MJ, Lacasa MC, Lopez A, Motilva V. Effects of cisapride on ulcer formation and gastric secretion in rats—comparison with ranitidine and omeprazole. Gen Pharmacol 1996; 27: 1415–20.[Medline]
  71. Kiuchi Y, Isobe Y, Kijima H, Muramatsu M, Higuchi S. Effect of cold-stress and indomethacin on the biosynthesis of gastric sulfated mucin in rats. Res Commun Mol Pathol Pharmacol 1997; 98: 179–89.[Medline]
  72. Nieuw Amerongen AV, Aarsman MEG, Bos-Vreugdenhil AP, Roukema PA. Influence of isoproterenol on the incorporation in vitro of [3H]-leucine and N-acetyl-[14C]-mannosamine into proteins and glycoproteins of the parotid glands of the mouse. Arch Oral Biol 1982; 27: 659–65.[Medline]
  73. Garrett JR. The proper role of nerves in salivary secretion: a review. J Dent Res 1987; 66: 387–97.[Abstract/Free Full Text]
  74. Baum BJ. Neurotransmitter control of secretion. J Dent Res 1987; 66: 626–32.
  75. Sabbadini E, Berczi I. The submandibular gland: a key organ in the neuro-immuno-regulatory network? Neuroimmunomodulation 1995; 2: 184–202.[Medline]
  76. Kusakabe T, Matsuda H, Kawakami T, Syoui N, Kurihara K, Tsukuda M, Takenaka T. Distribution of neuropeptide-containing nerve fibers in the human submandibular gland, with special reference to the difference between serous and mucous acini. Cell Tissue Res 1997; 288: 25–31.[Medline]
  77. Naliboff BD, Benton D, Solomon GF, Morley JE, Fahey JL, Bloom ET, Makinodam T, Gilmore SL. Immunological changes in young and old adults during brief laboratory stress. Psychosom Med 1991; 53: 121–32.[Abstract]
  78. Herbert TB, Cohen S, Marsland AL, Bachen EA, Rabin BS, Muldoon MF, Manuck SB. Cardiovascular reactivity and the course of immune response to an acute psychological stressor. Psychosom Med 1994; 56: 337–44.[Abstract/Free Full Text]
  79. Benschop RJ, Jacobs R, Sommer B, Schurmeyer TH, Raab JR, Schmidt RE, Schedlowski M. Modulation of the immunologic response to acute stress in humans by beta-blockade or benzodiazepines. FASEB J 1996; 10: 517–24.[Abstract]
  80. Benschop RJ, Nieuwenhuis EE, Tromp EA, Godaert GL, Ballieux RE, Van Doornen LJP. Effects of beta-adrenergic blockade on immunologic and cardiovascular changes induced by mental stress. Circulation 1994; 89: 762–9.[Abstract/Free Full Text]
  81. Bachen EA, Manuck SB, Cohen S, Muldoon MF, Raible R, Herbert TB, Rabin BS. Adrenergic blockade ameliorates cellular immune responses to mental stress. Psychosom Med 1995; 57: 366–72.[Abstract/Free Full Text]
  82. Cacioppo JT, Malarkey WB, Kiecolt-Glaser JK, Uchino BN, Sgoutas-Emch SA, Sheridan JF, Berntson GG, Glaser R. Heterogeneity in neuroendocrine and immune responses to brief psychological stressors as a function of autonomic cardiac activation. Psychosom Med 1995; 57: 154–64.[Abstract/Free Full Text]
  83. Carroll D, Ring C, Shrimpton J, Evans P, Willemsen G, Hucklebridge F. Secretory immunoglobulin A and cardiovascular responses to acute psychological challenge. Int J Behav Med 1996; 3: 266–79.
  84. Willemsen G, Ring C, Carroll D, Evans P, Clow A, Hucklebridge F. Secretory immunoglobulin A and cardiovascular reactions to mental arithmetic and cold pressor. Psychophysiology 1998; 35: 252–9.[Medline]
  85. Carruthers M, Taggart P. Vagotonicity of violence: biochemical and cardiovascular responses to violent films and television programmes. BMJ 1973; 3: 384–9.
  86. Taggart P, Hedworth-Whitty R, Carruthers M, Gordon PD. Observations on electrocardiogram and plasma catecholamines during dental procedures: the forgotten vagus. BMJ 1976; 2: 787–9.
  87. Vingerhoets AJJM. The role of the parasympathetic division of the autonomic nervous system in stress and the emotions. Int J Psychosom 1985; 32: 28–32.
  88. Hubert W, de Jong-Meyer R. Autonomic, neuroendocrine, and subjective responses to emotion-inducing film stimuli. Int J Psychophysiol 1991; 11: 131–40.[Medline]
  89. Vingerhoets AJJM, Ratcliff-Crain J, Jabaaij L, Menges LJ, Baum A. Self-reported stressors, symptom complaints and psychobiological functioning. I. Cardiovascular stress reactivity. J Psychosom Res 1996; 40: 177–90.[Medline]
  90. Moshier A, Reddy MS, Scannapieco FA. Role of type 1 fimbriae in the adhesion of Escherichia coli to salivary mucin and secretory immunoglobulin A. Curr Microbiol 1996; 33: 200–8.[Medline]
  91. Thomas LV, Wimpenny JW, Davis JG. Effect of three preservatives on the growth of Bacillus cereus, vero cytotoxigenic Escherichia coli and Staphylococcus aureus on plates with gradients of pH and sodium chloride concentration. Int J Food Microbiol 1993; 17: 289–301.[Medline]
  92. Edgerton M, Scannapieco FA, Reddy MS, Levine MJ. Human submandibular-sublingual saliva promotes adhesion of Candida albicans to polymethylmethacrylate. Infect Immun 1993; 61: 2644–52.[Abstract/Free Full Text]
  93. Geuijen CA, Willems RJ, Mooi FR. The major fimbrial subunit of Bordetella pertussis binds to sulfated sugars. Infect Immun 1996; 64: 2657–65.[Abstract/Free Full Text]
  94. Veerman ECI, Ligtenberg AJM, Schenkels LCPM, Walgreen-Weterings E, Nieuw Amerongen AV. Binding of human high-molecular-weight salivary mucins (MG1) to Haemophilus (para)influenzae. J Dent Res 1995; 74: 351–7.[Abstract/Free Full Text]
  95. Monteiro da Silva AM, Newman HN, Oakley DA. Psychosocial factors in inflammatory periodontal diseases. J Clin Periodontol 1995; 22: 516–26.[Medline]
  96. Murray PA, Prakobphol A, Lee T, Hoover CI, Fisher SJ. Adherence of oral streptococci to salivary glycoproteins. Infect Immun 1992; 60: 31–8.[Abstract/Free Full Text]
  97. Ligtenberg AJM, Walgreen-Weterings E, Veerman ECI, de Soet JJ, de Graaff J, Nieuw Amerongen AV. Influence of saliva on aggregation and adherence of Streptococcus gordonii HG222. Infect Immun 1992; 60: 3878–84.[Abstract/Free Full Text]
  98. Schenkels LCPM, Ligtenberg AJM, Veerman ECI, Nieuw Amerongen AV. Interaction of the salivary glycoprotein EP-GP with the bacterium Streptococcus salivarius HB. J Dent Res 1993; 72: 1559–65.[Abstract/Free Full Text]
  99. Marsh PD. Microbial ecology of dental plaque and its significance in health and disease. Adv Dent Res 1994; 8: 263–71.[Abstract/Free Full Text]
  100. Scannapieco FA. Saliva-bacterium interactions in oral microbial ecology. Crit Rev Oral Biol Med 1994; 5: 203–48.[Abstract/Free Full Text]
  101. Rudney JD. Does variability in salivary protein concentrations influence oral microbial ecology and oral health? Crit Rev Oral Biol Med 1995; 6: 343–67.[Abstract/Free Full Text]



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