Carol M. Stewart

Salivary gland tissue expression of interleukin-23 and interleukin-17 in Sjogren's syndrome: findings in humans and mice.

OBJECTIVE Recently, the Th1/Th2 paradigm has been expanded by the discovery of Th17 cells, a subset of CD4+ memory T cells characterized by their unique ability to secrete interleukin-17 (IL-17) family cytokines. Importantly, Th17 cells appear to be intimately involved in autoimmunity. We undertook the present study to investigate whether the Th17/IL-23 system is up-regulated in Sjögrens syndrome (SS). METHODS Sera, saliva, and salivary glands from C57BL/6.NOD-Aec1Aec2 mice (a model for primary SS), as well as sera, saliva, and salivary gland biopsy specimens obtained from patients with primary SS, were evaluated for IL-17 and IL-23 expression by immunohistochemistry, real-time polymerase chain reaction, and the Luminex system. RESULTS Immunohistochemical stainings of submandibular glands from C57BL/6.NOD-Aec1Aec2 mice and of salivary gland biopsy specimens from SS patients revealed strong positive staining for both IL-17 and IL-23 within lymphocytic foci and diffuse staining on epithelial tissues. Temporal expression of IL-17 and IL-23 in submandibular glands of C57BL/6.NOD-Aec1Aec2 mice correlated with expression of retinoic acid-related orphan receptor gammat, the Th17 cell master control gene. While IL-17 could not be detected in saliva from 4-20-week-old C57BL/6.NOD-Aec1Aec2 mice, this cytokine was present in the blood of mice up to age 16 weeks. This contrasted with sera and saliva from SS patients, in which IL-17 and IL-6 were present at varying levels. CONCLUSION These results suggest that the Th17/IL-23 system is up-regulated in C57BL/6.NOD-Aec1Aec2 mice and SS patients at the time of disease. A correlation between up-regulated IL-17/IL-23 expression and specific clinical manifestations of SS has yet to be identified.

Altered miR-146a expression in Sjögren's syndrome and its functional role in innate immunity.

MicroRNAs (miRNAs), small non‐coding RNA molecules that post‐transcriptionally regulate gene expression, are known to play key roles in regulating immune responses and autoimmunity. We investigated miR‐146a expression in Sjögrens syndrome (SjS) patients as well as in the SjS‐prone C57BL/6.NOD‐Aec1Aec2 mouse model, to elucidate its involvement in SjS pathogenesis. Expression of miR‐146a was examined in the PBMCs of 25 SjS patients and ten healthy donors, as well as in PBMCs, salivary and lacrimal glands of SjS‐prone mice and WT C57BL/6J mice. Functional assays using THP‐1 human monocytes were conducted to determine the biological roles of miR‐146a in innate immunity. Expression of miR‐146a was significantly increased in SjS patients compared with healthy controls, and was upregulated in the salivary glands and PBMCs of the SjS‐prone mouse at both 8 wk (prior to disease onset) and 20 wk (full‐blown disease) of age. More importantly, functional analysis revealed roles for miR‐146a in increasing phagocytic activity and suppressing inflammatory cytokine production while migration, nitric oxide production and expression of antigen‐presenting/costimulatory molecules are not affected in human monocytic THP‐1 cells. Taken together, our data suggest that abnormal expression/regulation of microRNAs in innate immunity may contribute to, or be indicative of, the initiation and progression of SjS.

Overexpression of Dicer as a Result of Reduced let-7 microRNA Levels Contributes to Increased Cell Proliferation of Oral Cancer Cells

Recent reports have demonstrated that Dicer, an RNase III endonuclease required for microRNA (miRNA) maturation, is aberrantly expressed in different types of cancer. Furthermore, Dicer has been reported to be regulated by the let‐7 family of miRNA genes. We hypothesize that Dicer is aberrantly expressed in oral cancer cells due to altered expressions of let‐7 and that Dicer contributes to the development and progression of the disease. Western blot examination of Dicer protein levels in four head and neck squamous cell carcinoma (HNSCC) cell lines, including two oral cancer cell lines, demonstrated that Dicer had between 4‐ and 24‐fold higher expression levels when compared to normal human primary gingival epithelial cells. Furthermore, five of six oral cancer tissues analyzed by indirect immunofluorescence had increased Dicer protein expression, compared to normal gingival epithelial tissue. The Dicer mRNA levels were not found to correlate well with protein expression in the HNSCC cell lines, suggesting that Dicer protein expression was post‐transcriptionally regulated. Analysis of let‐7a and let‐7b levels in HNSCC cell lines by real‐time PCR demonstrated that let‐7b, but not let‐7a, was significantly reduced in the HNSCC cell lines compared to control cells. Lastly, transfection of oral cancer cells with chemically synthesized let‐7b and small interfering RNAs targeting Dicer significantly inhibited cell proliferation up to 83% and >100%, respectively, as early as 3 days post‐transfection. Together, these data demonstrate that elevated expression levels of Dicer in oral cancer cells correlate with downregulation of let‐7b and increased cell proliferation.

Oral granular cell tumors: A clinicopathologic and immunocytochemical study

To investigate the histogenesis of the granular cell, a large series of granular cell tumors was studied for clinical and histopathologic features with emphasis on immunocytochemical markers. The nongingival granular cell tumors (NGGCT) were found to be more prevalent among females than males by a ratio of 2:1 and arose on the tongue (67%), the buccal mucosa (13%), the lips (8%), the soft palate (6%), and other sites (6%). With the use of the avidin-biotin-peroxidase method, polyclonal rabbit antisera were employed. The antisera were directed to the following antigens: S-100 protein, myoglobin, myosin, actin, desmin, alpha-1-antitrypsin, and muramidase. Results indicated that granular cell tumors are not homogenous for immunocytochemical markers. Nongingival granular cell tumors were universally positive for S-100 protein and failed to exhibit immunoreactivity for myogenous or histiocytic markers. Alternatively, the gingival granular cell tumor of infancy was negative for all markers, whereas rhabdomyoma was reactive with myogenous markers and a subpopulation of tumor cells displayed S-100 protein immunoreactivity. The granular cell ameloblastoma was reactive only with antiserum to alpha-1-antitrypsin. Ultrastructurally, granular cells from one of two NGGCT showed a direct evolution from skeletal muscle fibers. It is concluded that the oral NGGCT is a tumor positive for S-100 protein that may arise from muscle or nerve sheath.

The Cytobrush Plus cell collector in oral cytology

The Cytobrush Plus cell collector (cytobrush) was compared with the wooden tongue depressor during oral exfoliative cytology. The degree of patient discomfort, the convenience to the clinician, and the quantity and distribution of epithelial cells collected were evaluated. Two-factor analysis of variance and parametric and nonparametric analyses were performed. Because of the favorable findings revealed in this study, we recommend that the cytobrush be considered for use when obtaining diagnostic cytologic smears from the oral mucosa.

CIP2A expression and localization in oral carcinoma and dysplasia

Aims: Oral squamous cell carcinoma (OSCC) is the most prevalent malignancy of the oral cavity resulting in severe morbidity and mortality. To date only few proteins have been suggested as potential biomarkers or targets for this type of cancer. Cancerous inhibitor of PP2A (CIP2A) is a protein expressed in epithelial tissues that stabilizes the oncogene c-Myc and causes cell transformation. This study was designed to investigate the expression of CIP2A in OSCC cell lines and tissues representing human normal, dysplasia and OSCC. Methods: Using quantitative real time PCR, mRNA quantification for CIP2A was performed in a primary gingival cell line and OSCCs CAL 27 and SCC-25. Paraffin embedded human specimen classified as normal, dysplastic or OSCC were immunohistochemically stained for CIP2A expression. EGFR and CIP2A were also stained by immunofluorescence for co-localization. Samples of human normal oral tissue and OSCC were studied by PCR for mRNA expression of CIP2A. Results: CIP2A was significantly increased in the human carcinoma cell lines compared to the primary gingival cell line. CIP2A was overexpressed in the human oral dysplasia and OSCC tissues compared to normal oral tissues. CIP2A was also preferentially localized in the dysplastic and OSCC epithelial areas compared to EGFR that was expressed mainly in areas of relatively normal epithelium and in dysplastic tissues above the basal layers. Conclusions: CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.

Expression of Interleukin‐22 in Sjögren’s Syndrome: Significant Correlation with Disease Parameters

Sjögren’s syndrome (SS) is an autoimmune disease targeting the exocrine glands resulting in xerostomia/keratoconjunctivitis sicca. Presently, we examined the levels and clinical correlations of IL‐22 in SS. Patients with SS together with normal controls were randomly selected. IL‐22 was detected at significantly higher levels in sera of patients with SS. The levels of IL‐22 present in sera showed statistically significant direct correlations with hyposalivation, anti‐SSB, anti‐SSA/SSB combined, hypergammaglobulinemia and rheumatoid factor. IL‐22 showed a direct correlation with major clinical parameters. The data suggest that IL‐22 plays a critical role in the development of SS, and further study is needed to examine its function in human SS.

Atypical odontalgia: Phantom tooth pain

The findings in 30 cases diagnosed as atypical odontalgia are presented. The clinical characteristics of these cases are compared with other cases reported in the literature. Three cases are described in detail. Patient understanding and treatment with tricyclic antidepressants are discussed together with medication side effects and interactions. The importance of deferring invasive procedures is emphasized.

Experimental oral foreign body reactions: Commonly employed dental materials

Foreign bodies and tissue reactions to foreign materials are commonly encountered in the oral cavity. The more common lesions include apical deposition of endodontic materials, mucosal amalgam and graphite tattoos, myospherulosis, oil granulomas, and traumatically introduced dental materials and instruments. Since many foreign materials are unidentifiable histologically, commonly used dental materials were experimentally implanted subcutaneously in rats to assess local host responses and characterize the nature of these materials microscopically. The histologic characteristics of these foreign body reactions are detailed herein. The implanted materials corresponded to reactions seen in human subjects.

Salivary Mucin 19 Glycoproteins: Innate Immune Functions in Streptococcus mutans-Induced Caries in Mice and Evidence for Expression in Human Saliva

Background: We investigated murine salivary Muc19, in vivo and in vitro, to protect against dental infections. Results: Muc19 attenuates dental infections by Streptococcus mutans and promotes its aggregation. Evidence for human salivary MUC19 is presented. Conclusion: Murine Muc19 helps limit colonization via clearance of S. mutans. Significance: A role for Muc19 in clearance expands upon acknowledged innate immune functions of salivary gel-forming mucins. Saliva functions in innate immunity of the oral cavity, protecting against demineralization of teeth (i.e. dental caries), a highly prevalent infectious disease associated with Streptococcus mutans, a pathogen also linked to endocarditis and atheromatous plaques. Gel-forming mucins are a major constituent of saliva. Because Muc19 is the dominant salivary gel-forming mucin in mice, we studied Muc19−/− mice for changes in innate immune functions of saliva in interactions with S. mutans. When challenged with S. mutans and a cariogenic diet, total smooth and sulcal surface lesions are more than 2- and 1.6-fold higher in Muc19−/− mice compared with wild type, whereas the severity of lesions are up to 6- and 10-fold higher, respectively. Furthermore, the oral microbiota of Muc19−/− mice display higher levels of indigenous streptococci. Results emphasize the importance of a single salivary constituent in the innate immune functions of saliva. In vitro studies of S. mutans and Muc19 interactions (i.e. adherence, aggregation, and biofilm formation) demonstrate Muc19 poorly aggregates S. mutans. Nonetheless, aggregation is enhanced upon adding Muc19 to saliva from Muc19−/− mice, indicating Muc19 assists in bacterial clearance through formation of heterotypic complexes with salivary constituents that bind S. mutans, thus representing a novel innate immune function for salivary gel-forming mucins. In humans, expression of salivary MUC19 is unclear. We find MUC19 transcripts in salivary glands of seven subjects and demonstrate MUC19 glycoproteins in glandular mucous cells and saliva. Similarities and differences between mice and humans in the expression and functions of salivary gel-forming mucins are discussed.