Influence of periodontal disease on Th1/Th2-type cytokines in saliva of HIV-positive individuals

Cytokines present during immune responses have a tremendous influence on resistance/susceptibility to oral diseases including periodontal disease and oral opportunistic infections in the immunocompromised individual, as seen by altered Th cytokines in saliva with human immunodeficiency virus (HIV) disease progression and oropharyngeal candidiasis. This study was designed to evaluate whether the presence of severe periodontal disease has any influence on Th cytokines in saliva of HIV-positive persons. For this, saliva from a cohort of HIV-positive persons with mild or severe periodontitis was evaluated for Th cytokines. A dominant Th2-type cytokine profile in saliva was validated in HIV-positive subjects with considerable immune suppression, irrespective of periodontal disease status. However, no significant differences in concentrations of Th1- or Th2-type cytokines in saliva were observed when stratified by periodontal status. Thus, the lack of salivary influences by periodontitis eliminates periodontal disease as a variable in interpretations regarding correlates of local cytokines during oral manifestations of HIV.     

Nerve growth factor concentration in human saliva

OBJECTIVE: The aims of this study were to measure the normal concentration of nerve growth factor (NGF) in healthy human saliva and to investigate the effects of age and gender differences on saliva NGF level. MATERIALS AND METHODS: Resting whole, stimulated parotid, and stimulated submandibular/sublingual saliva were collected from 127 healthy volunteers with ages ranging from 20 to 81 years. The saliva NGF concentration was measured by enzyme immunoassay. RESULTS AND CONCLUSIONS: The mean concentrations of NGF were 901.4 +/- 75.6 pg ml(-1) in resting whole saliva, 885.9 +/- 79.9 pg ml(-1) in stimulated parotid saliva, and 1066.1 +/- 88.1 pg ml(-1) in stimulated submandibular/sublingual saliva. The stimulated submandibular saliva showed lower NGF concentrations with increasing age (rho = -0.296, P = 0.001). The NGF concentrations of resting whole saliva (P = 0.025) and stimulated parotid saliva (P = 0.005) were significantly higher in women than men. The NGF concentration of stimulated submandibular saliva was significantly higher than stimulated parotid saliva (P = 0.005) and significantly correlated with stimulated parotid saliva NGF level (rho = -0.244, P = 0.008). We found measurable concentrations of NGF in all three sources of saliva; the concentration was affected by the source for the stimulated parotid and submandibular saliva, age for stimulated submandibular saliva, and gender difference for resting whole saliva and stimulated parotid saliva.     

Salivary secretory leukocyte protease inhibitor increases in HIV infection.

BACKGROUND: Secretory leukocyte protease inhibitor (SLPI) is an antimicrobial protein found in saliva and having anti-HIV activity. The concentrations of SLPI in parotid and submandibular/sublingual (SMSL) saliva were determined in an HIV(+) population and compared with uninfected controls. The effect of highly active antiretroviral therapy (HAART) on the concentrations in saliva was determined. METHODS: Stimulated parotid and SMSL saliva was collected from 65 HIV(+) patients and 19 healthy controls. Flow rates, total protein and SLPI concentrations were determined as well as the effect of HAART on these measurements. RESULTS: Mean flow rates were reduced for parotid (64%) and SMSL (44%) saliva of HIV(+) patients. Flow rate reductions were unaffected by HAART. Total protein concentration in HIV(+) parotid saliva was increased 56%; patients on HAART had higher concentrations than control. For both groups, SLPI concentrations of SMSL saliva were twice that of parotid saliva. For HIV(+) patients SLPI concentrations of both saliva types were 70% greater than control; the increase in parotid saliva was greater for those taking HAART. For each saliva type, the secretory rate and specific SLPI protein concentration were not different between the groups. Patients with low CD4(+) counts had greater SLPI concentrations in parotid saliva than control. There was a negative correlation between CD4(+) counts and the SLPI concentration of parotid saliva. CONCLUSIONS: Salivary flow rate is decreased and the concentration of SLPI is increased in the presence of HIV infection. SLPI concentration in parotid and SMSL saliva is greater with HAART.     

Saliva mediated adherence, aggregation and prevalence in dental plaque of Streptococcus mutans, streptococcus sanguis and actinomyces spp. in young and elderly humans

Salivary components in the pellicle mediate bacterial adherence to the tooth. Such components may also aggregate bacteria in saliva and prevent them becoming established in dental plaque. In the present study, the adherence and aggregation of Streptococcus mutans strain Ingbritt, S. sanguis strain 10556 and Actinomyces viscosus strain 19246 mediated by parotid and whole saliva from groups of young and elderly people were examined. Significant differences were found between test strains, salivary secretions and age groups. S. sanguis 10556 and A. viscosus 19246 generally adhered more strongly than S. mutans Ingbritt, which adhered better to pellicles from parotid saliva than from whole saliva. Strain 19246 bound in higher numbers to parotid saliva pellicles from elderly compared to young individuals. Strain 10556 adhered better to whole saliva than parotid saliva pellicles, and the difference was significant among the young individuals, indicating reduced adherence ability in elderly whole saliva. The streptococci were aggregated by parotid and whole saliva, and S. sanguis aggregation was less with whole saliva from the elderly than from the young participants. Besides a correlation between whole saliva aggregation of S. mutans and proportions of bacteria in plaque, no correlations were found for the individual binding properties of saliva and prevalence of bacteria in vivo. However, the level of saliva-mediated adherence in vitro was in the following order: S. mutans < Actinomyces S. sanguis, which corresponded to their isolation frequency in plaque. These findings emphasize the importance of initial adherence to salivary receptors in bacterial colonization on teeth. Further studies are needed to reveal if individual patterns in the in vitro binding characteristics of saliva lead to variation of colonization in vivo.     

Salivary calprotectin levels are raised in patients with oral candidiasis or Sjögren's syndrome but decreased by HIV infection

Calprotectin levels were determined in whole saliva from patients predisposed to oral candidiasis due to HIV infection or Sj?gren's syndrome and from patients with candidiasis associated with various oral disorders (e.g. lichen planus, oral ulceration). Mean calprotectin levels were higher in whole saliva (2 microgram/ml) than in parotid saliva (0.3 microgram/ml). Oral candidiasis was associated with raised whole saliva calprotectin levels in all groups studied. HIV infection was associated with lower levels of salivary calprotectin, in the presence of high or low salivary Candida counts, although CD4+ lymphocyte counts did not significantly correlate with calprotectin concentrations. Calprotectin levels were elevated in saliva from Sj?gren's syndrome patients with oral candidiasis, consistent with mucosal transudation of calprotectin from inflamed mucosa and limited dilution due to decreased salivary flow rates. This study indicates that oral candidiasis is associated with raised calprotectin levels secondary to mucosal inflammation, but that diminution of this candidacidal factor due to HIV infection may be a predisposing factor in the aetiology of oral candidiasis.     

Preliminary proton nuclear magnetic resonance studies of human saliva

Summary-Proton NMR spectra of gustatory stimulated healthy human whole, parotid and submandibular and sublingual saliva were measured. The typical patterns of their spectra were obtained. Among these three kinds of fluids the parotid saliva which contains preferentially serous saliva presented a relatively well resolved spectrum with satisfactory signal to noise ratio in a given short time(30min.). For eight subjects of parotid saliva collected in the afternoon the marked increase in signal intensity in the methyl and methylene proton region of proteins(peptides)was observed as compared with those collected in the morning, reflecting the circadian rhythms in the protein concentration in saliva. On the other hand the methyl peak of lactic acid presented the opposite tendency, which might be also correlated with the circadian rhythms of the glucose metabolism of the parotid gland. The proton NMR spectra of three patients suspected of salivary gland disease were different from those of healthy subjects.     

The relationship between salivary histatin levels and oral yeast carriage

Candida species are common commensal inhabitants of the oral cavity. Human saliva contains antifungal proteins called histatins. We tested the hypothesis that oral yeast status is related to salivary histatin levels. Thirty subjects were divided into two groups based on the presence (n = 15) or absence (n = 15) of yeast on oral mucosa surfaces. Unstimulated and stimulated submandibular and sublingual and parotid saliva was collected from each subject. Salivary flow rates were measured and histatin concentrations were determined in the stimulated saliva samples. The yeast colony positive group showed lower median unstimulated parotid saliva flow rates as well as lower median concentrations of total histatins in submandibular and sublingual saliva. There was a negative correlation between yeast colony-forming units and unstimulated parotid saliva flow rates and between yeast colony-forming units and submandibular and sublingual saliva histatin concentration and secretion. The results suggest that oral yeast status may be influenced by unstimulated parotid saliva flow rates and by submandibular and sublingual histatin concentration and secretion.     

Age-related changes in salivary antibodies to commensal oral and gut biota

The prevalence of mucosally derived infections appears to increase with age, suggesting dysfunction at the mucosal surfaces. The present investigation was undertaken to examine any age-related changes in secretion rates and concentrations of secretory antibodies in whole and parotid saliva in a healthy adult population. A total of 116 subjects were subdivided into the following age groups: 20–39 years, 40–59 years, 60–79 years and 80 years and over. Specific immunoglobulin A (IgA), IgG and IgM antibodies in whole and parotid saliva to Streptococcus mutans (serotype c), Actinomyces viscosus NCTC 10951, and Escherichia coli NCTC 10418 were quantified by enzyme-linked immunosorbent assay. IgA antibodies to all three organisms examined increased with age in both whole and parotid saliva, whereas IgG antibody levels to S. mutans in whole saliva were significantly decreased with age. IgG antibodies to E. coli in parotid saliva were reduced in older age groups. IgM antibody levels to S. mutans were reduced with age in both secretions, whereas IgM antibodies to A. viscosus were greatest in the oldest age groups. No significant changes with age were observed in salivary IgM antibody levels to E. coli. No significant reduction in the secretion rates of IgA antibodies were observed in parotid or whole saliva, whereas IgG and IgM antibody secretion rates to all three microorganisms were reduced in most age groups in both whole and parotid saliva. The results of this investigation have demonstrated age-related changes with salivary antibodies, but, whereas salivary IgG and IgM antibodies showed decreases, salivary IgA levels generally increased with age. This suggests that the ability to form IgA antibody responses is not impaired with increased age, and that secretion rates and functional properties of antibodies may be as important as concentrations in protection against mucosal infective diseases.     

Influence of periodontal disease on Th1/Th2‐type cytokines in saliva of HIV‐positive individuals

Cytokines present during immune responses have a tremendous influence on resistance/susceptibility to oral diseases including periodontal disease and oral opportunistic infections in the immunocompromised individual, as seen by altered Th cytokines in saliva with human immunodeficiency virus (HIV) disease progression and oropharyngeal candidiasis. This study was designed to evaluate whether the presence of severe periodontal disease has any influence on Th cytokines in saliva of HIV‐positive persons. For this, saliva from a cohort of HIV‐positive persons with mild or severe periodontitis was evaluated for Th cytokines. A dominant Th2‐type cytokine profile in saliva was validated in HIV‐positive subjects with considerable immune suppression, irrespective of periodontal disease status. However, no significant differences in concentrations of Th1‐ or Th2‐type cytokines in saliva were observed when stratified by periodontal status. Thus, the lack of salivary influences by periodontitis eliminates periodontal disease as a variable in interpretations regarding correlates of local cytokines during oral manifestations of HIV.     

Salivary anti-candidal assays

Inhibition of Candida albicans blastospore viability by parotid, submandibular-sublingual and whole salivas could not be determined by direct assay of yeast cells in each respective saliva. Determination of antifungal activity could, however, be carried out if saliva was first preincubated with Candida cells and this was immediately followed by removal of saliva and resuspension of yeast cells in nonenriched buffers of pH 5-7 for appropriate incubation periods. To attain accurate reproducible quantitative data, parotid, submandibular-sublingual and whole salivas each required different preincubation times with C. albicans as well as prior acidification and boiling. Acidification was also necessary for optimizing the germ tube assay although, in contrast to blastospore viability, inhibition of blastospore-germ tube conversion could be determined directly in saliva. Salivary antifungal effects on blastospore division were negligible at yeast cell concentrations greater than 10(6) colony-forming units per ml and were found to be independent of pH, whereas salivary inhibition of germ tube formation was significant only at pH 5 in the assay systems employed. The requirement for acidification and an observed enhancement of antifungal activity on aqueous dilution of the saliva suggested that only a fraction of the salivary antifungal components present in saliva were available in the free form to exert their biological activity. These results open up the possibility of investigating salivary antifungal activity in human health and disease.     

Change from mixed diet to lactovegetarian diet: influence on IgA levels in blood and saliva

IgA concentrations in human plasma, and whole and parotid saliva were measured before and 3 months after a shift to a lactovegetarian diet in 20 volunteers (four men and 16 women, mean age 44 yr, range 27–61). The major dietary trends observed were an increased intake of berries and other fruits, vegetables, and dairy products, and a decreased intake of fish, eggs, and meat; biscuits and buns; sweets; alcoholic beverages; coffee; and tea. The consumption of meat, fish, and eggs decreased to zero, showing that the participants had adopted a lactovegetarian diet. There was a decrease in fat, protein, sucrose, and alcohol intake and an increase in total carbohydrate and fiber intake. There was no significant change in energy, retinol equivalent, or zinc intake. Despite this change in diet, no significant changes were observed between the mixed diet period and the vegetarian diet period in IgA in plasma, 253±52 and 264±55; whole saliva, 2.5±0.4 and 2.4±0.4; or parotid saliva, 0.88±0.22 and 0.90±0.20 (mg/100 ml, mean values, 95% confidence interval). Moreover, the diet change did not after the secretion rate in whole and parotid saliva, the secretion rate of IgA in whole and parotid saliva, or the protein content of whole saliva. However, the protein content of parotid saliva increased significantly. Thus, this major diet change was apparently not drastic enough or sustained long enough to cause a change in IgA levels.     

The expression of the c-erbB-2 receptor protein in glandular salivary secretions

BACKGROUND: As the maintenance medium of the oral cavity, saliva is secreted from exocrine glands that include the parotid, submandibular, sublingual, and minor salivary glands. Considering that saliva is a fluid suffused with protein, it is possible that the solubilized by-products of oncogenic expression may be present in saliva. Recent studies suggest the presence of solubilized extracellular domain portion of the c-erbB-2 protein in serum, nipple aspirates, and saliva. As a consequence, the purpose of this study was to determine the presence and concentration of c-erbB-2 in major salivary gland secretions. METHODS: Fifteen healthy women had serum, stimulated whole (SWS), parotid (SP), and submandibular/sublingual (SS) salivary secretions collected. The specimens were analyzed for c-erbB-2 using enzyme linked immunosorbent assays (ELISAs). Western blots using c-erbB-2 were also performed on these specimens. RESULTS: The ELISAs revealed the presence of c-erbB-2 in SWS (24.50 Units/ml), SP (19.66 Units/ml), SS (15.59 Units/ml) and serum (1472.15 Units/ml). Western blots confirmed the presence of these 185 kDa proteins. CONCLUSIONS: These results suggest that the protein, c-erbB-2, is present in relatively equal amounts in both SP and SS glandular secretions. Elevated glandular salivary c-erbB-2 concentrations could be useful as a preliminary, non-invasive test in clinical decision making when diagnosing salivary gland carcinomas. Additionally, this marker may have utility in distinguishing between oral lesions that are benign, pre-malignant and malignant in the oral cavity. Further research is required to determine if these findings have clinical utility.     

Salivary viscosity and lubrication: influence of pH and calcium

Abstract – The viscosity of human whole saliva and six salivary protein fractions was studied at different pH levels in the presence and absence of calcium. Changes in pH significantly influenced the viscosity of whole saliva, and centrifugation and filtration removed one of the viscous components. Calcium depletion made the viscosity particularly susceptible to acidification. The protein fractions were obtained from pooled parotid saliva by ion exchange and gel permeation chromatography procedures. Except for a glycoprotein, these fractions generally exhibited the lowest viscosity in the pH region around their isoelectric points. Calcium generally increased the viscosity of these proteins.     

Humoral immunity in root caries in an elderly population. II.

Saliva specimens stored for 18 months at -20 degrees C with or without glycerol and the anti-protease benzamidine-HCl, lost all antibody activity for S. mutans. IgA activity in processed whole saliva decreased significantly after one week when stored either at 4 degrees C or -20 degrees C with or without glycerol, although it was stable in parotid saliva for at least 40 days. Loss of activity prior to processing was significant in the first 24 h, and the addition of 50% glycerol and storage at -70 degrees after processing, prevented loss of antibody activity in both whole and parotid saliva. Diurnal variations in IgA, lactoferrin and the IgA secretion rate were insignificant in parotid saliva but showed some fluctuations in whole saliva. Albumin and lactoferrin levels exhibited the greatest fluctuation in whole saliva specimens although IgA and IgA antibody levels were still more characteristic of the patient than the time of sampling. Monthly variations in IgA, IgA antibody activity and other parameters were least in parotid saliva and e.g., values for parameters that were high in patients samples on the first month, remained high during the 4-month study period. Statistical analyses showed a high correlation between values obtained for most of the 15 parameters that were measured in parotid and whole saliva specimens collected from greater than 20 patients during 2 successive visits. Whole saliva values for albumin, lactoferrin and albumin levels in parotid saliva, were most variable but differences were not significant. Hence, patients with very low or very high values, even in whole saliva, can be identified within the population on the basis of specimens collected at a single time.     

Saliva composition and caries development in asthmatic patients treated with β2-adrenoceptor agonists: a 4-year follow-up study

Abstract – In an earlier study, we found that chronic treatment with β₂-adrenoceptor agonists in asthmatic subjects gave an impaired saliva secretion and a higher caries prevalence than in healthy controls. Twenty-one of the asthmatics and their matched controls were examined 4 yr later in a follow-up study. Samples of whole saliva stimulated by chewing and parotid saliva stimulated by citric acid were collected and dental caries was scored. In the asthmatic group the secretory rates of stimulated whole and parotid saliva decreased by 20% and 35%, respectively, compared to the control group. The number of lactobacilli increased. The asthmatic subjects had a decreased output per minute of total protein, amylase, hexosamine, salivary peroxidase, lysozymc, secretory IgA, a bacteria-aggregating glycoprotein, potassium, and calcium in stimulated parotid saliva. Initial and manifest caries lesions as well as the number of DFS were significantly increased in the asthma group. We conclude that asthmatic patients treated with β₂-adrenoccptor agonists have an increased caries susceptibility due to an impaired saliva secretion caused by the use of β-adrenergic agonists.     

Humoral immunity in root caries in an elderly population. I.

IgA, IgG and IgM antibody activity (ELISA Units/ml) to Streptococcus mutans, Actinomyces viscous and Escherichia coli CF8 in serum, parotid saliva and whole saliva was measured using the amplified ELISA (a-ELISA) while the concentration (microgram/ml) of each isotype of immunoglobulin as well as albumin and lactoferrin, was determined using sandwich ELISAs. Selection of suitable reagents from those commercially available was based on specificity tests using purified human immunoglobulin; most polyclonal reagents required further absorption to attain class specificity. Cross-absorption studies indicated the absence of patient antibodies that were cross-reactive among the bacteria studied, except for IgM in some cases. Expression of response in ELISA Units (E.U.) per microgram of immunoglobulin, i.e. specific activity, revealed that IgG specific activity was significantly higher in parotid saliva than in either whole saliva or serum for all bacteria studied; serum and whole saliva did not differ except for the higher specific activity in whole saliva to E. coli. The value of one E.U. was determined using the Comparative Antibody-immunoglobulin Capture Assay (CACA). Using this novel method, we estimated that about 0.05 percent of serum IgA was specific for Streptococcus mutans, 0.008 for Actinomyces viscosus and 0.004 for Escherichia coli CF8. The percentage of specific IgM antibodies was higher than for IgA and IgG. The concentration of IgA anti-Streptococcus mutans, Actinomyces viscosus and Escherichia coli levels are approximately 92 ng/ml, 25 ng/ml and 16 ng/ml in whole saliva and 46 ng/ml, 9.4 ng/ml and 6.3 ng/ml in parotid saliva.(ABSTRACT TRUNCATED AT 250 WORDS)     

Salivary anti-candidal assays

Inhibition of Candida albicans blastospore viability by parotid, submandibular-sublingual and whole salivas could not be determined by direct assay of yeast cells in each respective saliva. Determination of antifungal activity could, however, be carried out if saliva was first preincubated with Candida cells and this was immediately followed by removal of saliva and resuspension of yeast cells in nonenriched buffers of pH 5-7 for appropriate incubation periods. To attain accurate reproducible quantitative data, parotid, submandibular-sublingual and whole salivas each required different preincubation times with C. albicans as well as prior acidification and boiling. Acidification was also necessary for optimizing the germ tube assay although, in contrast to blastospore viability, inhibition of blastospore-germ tube conversion could be determined directly in saliva. Salivary antifungal effects on blastospore division were negligible at yeast cell concentrations greater than 10⁶ colony-forming units per ml and were found to be independent of pH, whereas salivary inhibition of germ tube formation was significant only at pH 5 in the assay systems employed. The requirement for acidification and an observed enhancement of antifungal activity on aqueous dilution of the saliva suggested that only a fraction of the salivary antifungal components present in saliva were available in the free form to exert their biological activity. These results open up the possibility of investigating salivary antifungal activity in human health and disease.     

Salivary gland function in persons with ectodermal dysplasias

Ectodermal dysplasias (EDs) constitute a group of conditions comprising developmental defects in two or more of the following tissues: hair, teeth, nails, and sweat glands. The aim of the present study was to contribute to a better understanding of salivary gland involvement in EDs. An ED group (n = 39, median age 12 yr; 24 males, 15 females) and a healthy age- and sex-matched control group were studied. Citric acid stimulated submandibular and parotid salivary flow rates and salivary concentrations, and output of total protein, acidic proline-rich proteins and histatins were analysed. The associations between quantitative and qualitative salivary parameters were also studied. In the ED group, 13 persons (33%) demonstrated a significantly reduced secretion of submandibular and/or parotid saliva, in addition to a low unstimulated and/or chewing-stimulated whole salivary flow. In the ED group as a whole, a reduced median secretory rate of submandibular saliva was found, whereas the median concentrations of some protein parameters were increased. However, the overall output of proteins was normal or reduced. Submandibular glands seemed to be more affected than parotid glands in EDs. In conclusion, salivary secretory tests are recommended in persons with known or suspected EDs.     

Salivary calprotectin levels are raised in patients with oral candidiasis or Sjögren’s syndrome but decreased by HIV infection

Calprotectin levels were determined in whole saliva from patients predisposed to oral candidiasis due to HIV infection or Sjögren’s syndrome and from patients with candidiasis associated with various oral disorders (e.g. lichen planus, oral ulceration). Mean calprotectin levels were higher in whole saliva (2 μg/ml) than in parotid saliva (0.3 μg/ml). Oral candidiasis was associated with raised whole saliva calprotectin levels in all groups studied. HIV infection was associated with lower levels of salivary calprotectin, in the presence of high or low salivary Candida counts, although CD4⁺ lymphocyte counts did not significantly correlate with calprotectin concentrations. Calprotectin levels were elevated in saliva from Sjögren’s syndrome patients with oral candidiasis, consistent with mucosal transudation of calprotectin from inflamed mucosa and limited dilution due to decreased salivary flow rates. This study indicates that oral candidiasis is associated with raised calprotectin levels secondary to mucosal inflammation, but that diminution of this candidacidal factor due to HIV infection may be a predisposing factor in the aetiology of oral candidiasis.     

Evidence for the presence of carbamino compounds in human saliva.

The bicarbonate concentrations of whole saliva and of protein-free filtrates of saliva were measured to test for the presence of carbamino compounds. If no acid-evolved CO2 were derived from the salivary proteins, then the two measured bicarbonate concentrations should be identical. Instead, the bicarbonate concentrations of the whole saliva samples were on the average 30% higher than that of their filtrates. The results are consistent with the conclusion that carbamino compounds account for the differences. Similar findings were obtained with samples of resting whole saliva and parotid saliva.