Lawrence E. Wolinsky

The Proteomes of Human Parotid and Submandibular/Sublingual Gland Salivas Collected as the Ductal Secretions

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

The Inhibiting Effect of Aqueous Azadirachta indica (Neem) Extract Upon Bacterial Properties Influencing in vitro Plaque Formation

The purpose of this investigation was to examine the inhibitory effects of aqueous extracts derived from the bark-containing sticks (Neem stick) of Azadirachta indica upon bacterial aggregation, growth, adhesion to hydroxyapatite, and production of insoluble glucan, which may affect in vitro plaque formation. Neem stick extracts were screened for minimal bacterial growth inhibition (MIC) against a panel of streptococci by means of a broth dilution assay. Initial bacterial attachment was quantified by the measurement of the adhesion of 3H-labeled Streptococcus sanguis to saliva-conditioned synthetic hydroxyapatite. The effect of the Neem stick extract upon insoluble glucan synthesis was measured by the uptake of radiolabeled glucose from 14C-sucrose. Aggregating activity of the Neem stick extracts upon a panel of streptococci was also examined. No inhibition of bacterial growth was observed among the streptococcal strains tested in the presence of ≤ 320 pg/mL of the Neem stick extract. The pre-treatment of S. sanguis with the Neem stick extract or the gallotannin-enriched extract from Melaphis chinensis at 250 ug/mL resulted in a significant inhibition of the bacterial adhesion to saliva-conditioned hydroxyapatite. Pre-treatment of saliva-conditioned hydroxyapatite with the Neem stick or gallotannin-rich extract prior to exposure to bacteria yielded significant reductions in bacterial adhesion. The Neem stick extract and the gallotannin-enriched extract from Melaphis chinensis inhibited insoluble glucan synthesis. Incubation of oral streptococci with the Neem stick extract resulted in a microscopically observable bacterial aggregation. These data suggest that Neem stick extract can reduce the ability of some streptococci to colonize tooth surfaces.

Human Saliva Proteome and Transcriptome

This paper tests the hypothesis that salivary proteins and their counterpart mRNAs co-exist in human whole saliva. Global profiling of human saliva proteomes and transcriptomes by mass spectrometry (MS) and expression microarray technologies, respectively, revealed many similarities between saliva proteins and mRNAs. Of the function-known proteins identified in saliva, from 61 to 70% were also found present as mRNA transcripts. For genes not detected at both protein and mRNA levels, we made further efforts to determine if the counterpart is present. Of 19 selected genes detected only at the protein level, the mRNAs of 13 (68%) genes were found in saliva by RT-PCR. In contrast, of many mRNAs detected only by microarrays, their protein products were found in saliva, as reported previously by other investigators. The saliva transcriptome may provide preliminary insights into the boundary of the saliva proteome.

Oral mucositis in patients undergoing bone marrow transplantation

Thirty patients who received bone marrow transplantation treatment from HLA identical sibling donors for immunologic and malignant diseases were studied. In essentially all of the patients oral changes developed during the first 30 days following transplant. Oral symptoms frequently constituted the major complaints of the patients during the follow-up period. The oral changes included mucositis, xerostomia, pain, and bleeding. Mucositis was more severe and of longer duration when associated with herpes simplex infections and when optimal oral hygiene was not maintained. Xerostomia which accompanies engraftment was an early sign of acute graft-versus-host disease. A nonbrushing method of oral hygiene was effective in reducing the severity and duration of mucositis. This technique offers a short-term alternative to brushing in pancytopenic patients who are susceptible to bleeding or trauma.

Systemic Disease-Induced Salivary Biomarker Profiles in Mouse Models of Melanoma and Non-Small Cell Lung Cancer

Background Saliva (oral fluids) is an emerging biofluid poised for detection of clinical diseases. Although the rationale for oral diseases applications (e.g. oral cancer) is intuitive, the rationale and relationship between systemic diseases and saliva biomarkers are unclear. Methodology/Principal Findings In this study, we used mouse models of melanoma and non-small cell lung cancer and compared the transcriptome biomarker profiles of tumor-bearing mice to those of control mice. Microarray analysis showed that salivary transcriptomes were significantly altered in tumor-bearing mice vs. controls. Significant overlapping among transcriptomes of mouse tumors, serum, salivary glands and saliva suggests that salivary biomarkers have multiple origins. Furthermore, we identified that the expression of two groups of significantly altered transcription factors (TFs) Runx1, Mlxipl, Trim30 and Egr1, Tbx1, Nr1d1 in salivary gland tissue of melanoma-bearing mice can potentially be responsible for 82.6% of the up-regulated gene expression and 62.5% of the down-regulated gene expression, respectively, in the saliva of melanoma-bearing mice. We also showed that the ectopic production of nerve growth factor (NGF) in the melanoma tumor tissue as a tumor-released mediator can induce expression of the TF Egr-1 in the salivary gland. Conclusions Taken together, our data support the conclusion that upon systemic disease development, significant changes can occur in the salivary biomarker profile. Although the origins of the disease-induced salivary biomarkers may be both systemic and local, stimulation of salivary gland by mediators released from remote tumors plays an important role in regulating the salivary surrogate biomarker profiles.

Analyses of Streptococcus mutans in Saliva with Species-Specific Monoclonal Antibodies

Three species-specific monoclonal antibodies (MAbs) against Streptococcus mutans were used to detect and quantify S. mutans levels in saliva. This study shows that MAb-based salivary S. mutans tests exhibit significantly higher specificity and sensitivity than the commonly used selective culture method. Examination of nearly 2,000 human saliva samples shows that S. mutans counts in human saliva vary from less than 10,000 to a high 36 million cells/mL. Over 15% of the saliva samples examined have salivary S. mutans counts over 500,000 cells/mL. When saliva samples were collected at different time points during a day, the number of salivary S. mutans in the same human subject varied, especially before and after sugar uptake. Additionally, data obtained from stimulated versus unstimulated saliva in the same human subjects differed greatly and appear to be completely uncorrelated. This study provides useful information and tools for analyzing the role of S. mutans in human dental caries.

Osteoporosis and mandibular bone resorption: A prosthodontic perspective

Previous studies have examined the effects of osteoporosis on the vertebra, femur, and tibia. However, few studies have examined the effects on the mandible by using an animal model to quantify bone resorption. Osteoporosis was induced through pair feedings of a high protein, low calcium diet. Before this induction, experimental and control animals were injected subcutaneously with radioactive tritiated tetracycline. Bone resorption was quantified by measuring the amount of radioactivity present after the test diets were given for 90 days. Standard scintillation techniques were used for extracting the radioactivity from each half mandible. The following conclusions can be made from the results of this investigation: A significant difference in mandibular bone resorption was associated with an osteoporotic inducing diet high in protein and low in calcium. Bone resorption in the experimental group of animals was 17% greater than in the control group. Alkaline phosphatase may be an important indicator of osteoporosis in the Sprague-Dawley rat. Elevated levels were found in those with the osteoporotic diet. The animals in the control and experimental groups consumed similar amounts of their respective diets. No significant difference was found in the weight gains of either group. The histologic picture, although not pathognomonic for osteoporosis, was consistent with many findings in the literature describing osteoporosis. This study has shown that osteoporotic diets may increase the amount of bone resorption in the mandibles of Sprague-Dawley rats.

Induction of Activated Lymphocyte Killing by Bacteria Associated with Periodontal Disease

Complex interactions occur among host defense cells during bacterial infection. Bacteria and bacterial products may enhance or inhibit the effector and regulatory activity of human lymphocytes. Accordingly, we tested the ability of human periodontal pathogens to activate peripheral blood lymphocytes using standard 51chromium-release assays to measure lymphocyte-mediated cytolysis. Human adherent-cell depleted peripheral blood lymphocytes (PBL) with the addition of glutaraldehyde-fixed bacteria at a 5:1 bacteria:lymphocyte ratio were incubated at 37°C for 24 hr in RPMI 1640 medium. Six of eight bacteria tested significantly augmented lymphocyte killing of the natural killer (NK) cell-sensitive human erythroleukemia cell line K562. E. corrodens, representing activating bacteria, was also able to induce the killing of NK-resistant targets (M14, Raji), comparable with induction by interleukin-2. Lipopolysaccharides extracted from A. actinomycetemcomitans strains, when incubated with PBL, were able to enhance cytotoxicity without the presence of whole bacteria. A majority of cytotoxicity was mediated by NK cells bearing Leu-11 and NKH-1 markers.

Differential Modulation of Adherence of Oral Streptococci by Human Neutrophil Myeloperoxidase

In this study, the modulation of adherence of hydrogen peroxide (H2O2)-producing and non-H2O2-producing strains of oral streptococci by the host leukocyte enzyme myeloperoxidase (MPO) was examined. It was found that exposure to MPO decreased adherence of many strains of oral streptococci to saliva-coated hydroxyapatite beads in the presence of exogenous H2O 2 and chloride. The MPO-H2O2-Cl- system increased the adherence of one strain. In the absence of exogenous H 2O2, the MPO-H2O2-Cl- system decreased the adherence of H2O2producing strains only. Glucose increased streptococcal H2O2 production and also increased the anti-adhesive activity of MPO in the absence of exogenous H2O2. We conclude that: (1) host leukocytes can modulate the adherence of oral streptococci via MPO; (2) endogenous production of H 2O2 by the oral streptococci can provide sufficient substrate H2O2 to drive this system; and (3) MPO will exert differential modulatory effects on the adherence of oral streptococci, based in part upon the level of endogenous H2O2 production and in part upon the particular characteristics of the adhesins of the bacteria.

The Antimicrobial Activity of Pomegranate Polyphenol Extract (POMx) Lozenges in a Saliva-Derived Biofilm Model System

The ellagitannin type polyphenols present in Pomegranate extracts (POMx) have been associated with numerous health benefits including antibacterial activities. Despite their antibacterial potency, however, these purified pomegranate polyphenol extracts need to be incorporated into a delivery system for convenient human treatments. In this study, we performed a detailed investigation of the antimicrobial activity of pomegranate polyphenol fortified lozenges against oral bacteria including the major cariogenic species Streptococcus mutans. Since oral bacteria exert their cariogenic effects when they are attached as biofilms on the tooth surface, we developed a stationary saliva-derived biofilm model for high throughput parallel screening to assess effects on bacteria in their biologically relevant mode. We found strong antibacterial activities (up to >99% killing of biofilm cells) for the POMx lozenges at exposure times (15-20 min) relevant for lozenge consumption. Most interestingly, S. mutans appeared to be even more sensitive to these products than the general biofilm population. Furthermore, consistent with data derived for polyphenols extracted from other plants, the POMx lozenges completely prevented bacterial surface adherence as determined by live imaging. In summary, our data show that the strong antibacterial and anti-adherence activities of pomegranate extracts are maintained after processing in products such as the POMx lozenges that can be easily consumed and are therefore excellent candidates for prevention and possibly treatment of oral disease.