[Show abstract][Hide abstract] ABSTRACT: Patients with Sjögren's syndrome or head and neck cancer patients who have undergone radiation therapy suffer from severe dry mouth (xerostomia) due to salivary exocrine cell death. Regeneration of the salivary glands requires a better understanding of regulatory mechanisms by which stem cells differentiate into exocrine cells. In our study, bone marrow-derived mesenchymal stem cells were co-cultured with primary salivary epithelial cells from C57BL/6 mice. Co-cultured bone marrow-derived mesenchymal stem cells clearly resembled salivary epithelial cells, as confirmed by strong expression of salivary gland epithelial cell-specific markers, such as alpha-amylase, muscarinic type 3 receptor, aquaporin-5, and cytokeratin 19. To identify regulatory factors involved in this differentiation, transdifferentiated mesenchymal stem cells were analyzed temporarily by two-dimensional-gel-electrophoresis, which detected 58 protein spots (>1.5 fold change, p<0.05) that were further categorized into 12 temporal expression patterns. Of those proteins only induced in differentiated mesenchymal stem cells, ankryin-repeat-domain-containing-protein 56, high-mobility-group-protein 20B, and transcription factor E2a were selected as putative regulatory factors for mesenchymal stem cell transdifferentiation based on putative roles in salivary gland development. Induction of these molecules was confirmed by RT-PCR and western blotting on separate sets of co-cultured mesenchymal stem cells. In conclusion, our study is the first to identify differentially expressed proteins that are implicated in mesenchymal stem cell differentiation into salivary gland epithelial cells. Further investigation to elucidate regulatory roles of these three transcription factors in mesenchymal stem cell reprogramming will provide a critical foundation for a novel cell-based regenerative therapy for patients with xerostomia.
PLoS ONE 11/2014; 9(11):e112158. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evaluation of: Vergadi E, Vaporidi K, Theodorakis EE et al. Akt2 deficiency protects from acute lung injury via alternative macrophage activation and miR-146a induction in mice. J. Immunol. 192, 394-406 (2013). Acute respiratory distress syndrome currently has limited effective treatments; however, recent evidence suggests that modulation of alveolar macrophage responses may be an effective method for protection or repair of lung injury. Vergadi et al. are the first to demonstrate that depletion of Akt2 kinase and microRNA-146a induction in mice resulted in polarization of alveolar macrophages towards an M2 activation phenotype and resulted in less severe injury following acid-induced lung injury. However, this M2 polarization also resulted in increased lung bacterial load following infection with Pseudomonas aeruginosa.
[Show abstract][Hide abstract] ABSTRACT: Background: Xylitol is a well-known anti-caries agent and has been used for the prevention and treatment of dental caries. In this study, we evaluated the anti-inflammatory effects of xylitol for possible usage in the prevention and treatment of periodontal infections. Methods: Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis (P. gingivalis), and ELISA and a MILLIPLEX MAP kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining and co-stimulatory molecule expression was examined by flow cytometry. Results: Live P. gingivalis infection increased the production of representative proinflammatory cytokine, TNFα (Tumor necrosis factor) and IL-1β (Interleukin-1) in a MOI- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines such as IL-12-p40 (Interleukin 12), Eotaxin, IP-10 (Interferon gamma-induced protein 10), MCP-1 (Monocyte chemotactic protein-1), and MIP-1α (Macrophage inflammatory protein-1). The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted anti-phagocytic activity against both Escherichia coli and P. gingivalis. Conclusions: These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.
Journal of Periodontology 03/2014; · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: Although elevated IL-7 levels were reported in patients with primary Sjӧgren’s syndrome (pSjS), the role of IL-7 in this disease remains unclear. In this study, we characterized the previously unexplored in vivo role of IL-7 in the development and onset of pSjS using C57BL/6.NOD-Aec1Aec2 (B6.NOD-Aec)mouse model that replicates human pSjS.
Methods: For gain-of-function studies, recombinant human IL-7 (5 µg) or control PBS was injected intraperitoneally (i.p.) into female B6.NOD-Aec mice, 3 times per week for 8 weeks, starting from 12 weeks of age. For loss-of-function studies, 100 µg neutralizing anti-IL-7 receptor antibody (A7R34) or its isotype control IgG was administered i.p. into B6.NOD-Aec mice, 3 times per week for 8 weeks, starting from 16 weeks of age. Mice were then analyzed for various disease parameters by salivary flow measurement, salivary gland histological examination and serum antinuclear antibody assay.
Results: Administration of exogenous IL-7 accelerated the development and onset of pSjS, whereas blockade of IL-7 receptor signaling almost completely abolished the development of this disease, based on parameters including salivary gland inflammation and apoptosis, autoantibody production and salivary gland secretory dysfunction. IL-7 positively regulated IFN-g-producing Th1 and CD8 T cells and overall IFN-g levels in the salivary glands, without affecting IL-17. IL-7 enhanced the expression of CXCR3 ligands in salivary glands in a T cell- and IFN-g-dependent fashion, suggesting a chemotactic mechanism that accelerates the infiltration of lymphocytes. All data were obtained from 6-9 mice from 2-3 independent experiments. Student’s t-test was performed for statistical analysis with P values smaller than 0.05 being considered statistically significant.
Conclusions: IL-7 plays a pivotal pathogenic role in SjS, which is underpinned by an enhanced Th1 response and IFN-g-CXCR3 ligand-mediated lymphocyte infiltration of target organs. These results suggest that targeting IL-7 pathway may be a potential future strategy to prevent and treat SjS.
IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
[Show abstract][Hide abstract] ABSTRACT: Since the discovery of RNA interference (RNAi), excitement has grown over its potential therapeutic uses. Targeting RNAi pathways provides a powerful tool to change biological processes post-transcriptionally in various health conditions such as cancer or autoimmune diseases. Optimum design of shRNA, siRNA, and miRNA enhances stability and specificity of RNAi-based approaches whereas it has to reduce or prevent undesirable immune responses or off-target effects. Recent advances in understanding pathogenesis of autoimmune diseases have allowed application of these tools in vitro as well as in vivo with some degree of success. Further research on the design and delivery of effectors of RNAi pathway and underlying molecular basis of RNAi would warrant practical use of RNAi-based therapeutics in human applications. This review will focus on the approaches used for current therapeutics and their applications in autoimmune diseases, including rheumatoid arthritis and Sjögren's syndrome.
[Show abstract][Hide abstract] ABSTRACT: Sjögren's syndrome (SjS) is a chronic autoimmune disease that mainly targets the salivary and lacrimal glands. It has been controversial whether anti-muscarinic type 3 receptor (α-M3R) autoantibodies in patients with SjS inhibit intracellular trafficking of aquaporin-5 (AQP5), water transport protein, leading to secretory dysfunction. To address this issue, GFP-tagged human AQP5 was overexpressed in human salivary gland cells (HSG-hAQP5) and monitored AQP5 trafficking to the plasma membrane following carbachol (CCh, M3R agonist) stimulation. AQP5 trafficking was indeed mediated by M3R stimulation, shown in partial blockage of trafficking by M3R-antagonist 4-DAMP. HSG-hAQP5 pre-incubated with SjS plasma for 24 hours significantly reduced AQP5 trafficking with CCh, compared with HSG-hAQP5 pre-incubated with healthy control (HC) plasma. This inhibition was confirmed by monoclonal α-M3R antibody and pre-absorbed plasma. Interestingly, HSG-hAQP5 pre-incubated with SjS plasma showed no change in cell volume, compared to the cells incubated with HC plasma showing shrinkage by twenty percent after CCh-stimulation. Our findings clearly indicate that binding of anti-M3R autoantibodies to the receptor, which was verified by immunoprecipitation, suppresses AQP5 trafficking to the membrane and contribute to impaired fluid secretion in SjS. Our current study urges further investigations of clinical associations between SjS symptoms, such as degree of secretory dysfunction, cognitive impairment, and/or bladder irritation, and different profiles (titers, isotypes, and/or specificity) of anti-M3R autoantibodies in individuals with SjS.
PLoS ONE 01/2013; 8(1):e53113. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objective: Sjögren’s syndrome (SjS) causes dry mouth and dry eyes by attacking exocrine glands. We reported that microRNA(miR)-146a is upregulated in the target tissue and PBMC of the SjS-mouse model and in human PBMC. In addition, elevated miR-146a altered innate immune functions such as phagocytic activity in vitro. To further identify the roles of miR-146a in innate immunity of SjS, co-stimulatory molecule CD80, which was identified as a potential target molecule by in silico analyses, was evaluated in this study.
Method: CD80 and CD86 were screened by western blot, immunohistochemistry, and flow cytometry in the salivary glands (SMX) and the spleen of SjS-prone C57BL/6.NOD-Aec1Aec2 (B6DC) and C57BL/6 (B6) at 8 (pre-disease) and 20 weeks (disease). MiR-146a in those tissues and CD80/CD86 in PBMC was measured by qRT-PCR. To determine if miR-146a is a direct target of CD80, HEK293 cells were transfected with a luciferase reporter vector containing human CD80 3’UTR along with pre-miR-146a-mimics (2.5-40nM). Data were analyzed by two-tailed-unpaired t-test or one-way ANOVA where appropriate.
Result: Total protein expression in SMX of B6DC showed reduced CD80 expression compared to B6 at 8 and 20 weeks (n=6/group). In contrast, no substantial difference of CD80 or miR-146a was observed between the strains in the spleen. Luciferase activity was significantly reduced (p<0.001) in the presence of miR-146a mimic at the concentrations tested compared to a negative control, indicating that CD80 is a direct target of miR-146a. Expression of CD80 and CD86 in human PBMC indicates a trend of decreased expression in SjS patients compared to healthy controls, which requires further confirmation.
Conclusion: Our current study identified CD80 as a target molecule of miR-146a and altered CD80 expression by miR-146a was detected in the target tissue of SjS. Impact of altered expression of CD80 on antigen presentation in SjS is currently under active investigation.
[Show abstract][Hide abstract] ABSTRACT: Muscarinic type 3 receptor (M3R) plays a pivotal role in the induction of glandular fluid secretions. Although M3R is often the target of autoantibodies in Sjögren's syndrome (SjS), chemical agonists for M3R are clinically used to stimulate saliva secretion in patients with SjS. Aside from its activity in promoting glandular fluid secretion, however, it is unclear whether activation of M3R is related to other biological events in SjS. This study aimed to investigate the cytoprotective effect of chemical agonist-mediated M3R activation on apoptosis induced in human salivary gland (HSG) cells. Carbachol (CCh), a muscarinic receptor-specific agonist, abrogated tumor necrosis factor α/interferon γ-induced apoptosis through pathways involving caspase 3/7, but its cytoprotective effect was decreased by a M3R antagonist, a mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) inhibitor, a phosphatidylinositol 3-kinase/Akt inhibitor, or an epidermal growth factor receptor (EGFR) inhibitor. Ligation of M3R with CCh transactivated EGFR and phosphorylated ERK and Akt, the downstream targets of EGFR. Inhibition of intracellular calcium release or protein kinase C δ, both of which are involved in the cell signaling of M3R-mediated fluid secretion, did not affect CCh-induced ERK or Akt phosphorylation. CCh stimulated Src phosphorylation and binding to EGFR. A Src inhibitor attenuated the CCh/M3R-induced cytoprotective effect and EGFR transactivation cascades. Overall, these results indicated that CCh/M3R induced transactivation of EGFR through Src activation leading to ERK and Akt phosphorylation, which in turn suppressed caspase 3/7-mediated apoptotic signals in HSG cells. This study, for the first time, proposes that CCh-mediated M3R activation can promote not only fluid secretion but also survival of salivary gland cells in the inflammatory context of SjS.
[Show abstract][Hide abstract] ABSTRACT: The mouse model is the one of the most frequently used and well-established animal models, and is currently used in many research areas. To date, various mouse models have been utilized to elucidate underlying causes of multifactorial autoimmune conditions, including pathological immune components and specific signaling pathways. This review summarizes the more recent mouse models for Sjögren's syndrome, a systemic autoimmune disease characterized by lymphocytic infiltration in the exocrine glands, such as the salivary and lacrimal glands, and loss of secretory function, resulting in dry mouth and dry eyes in patients. Although every Sjögren's syndrome mouse model resembles the major symptoms or phenotypes of Sjögren's syndrome conditions in humans, the characteristics of each model are variable. Moreover, to date, there is no single mouse model that can completely replicate the human conditions. However, unique features of each mouse model provide insights into the roles of potential etiological and immunological factors in the development and progression of Sjögren's syndrome. Here, we will overview the Sjögren's syndrome mouse models. Lessons from these mouse models will aid us to understand underlying immune dysregulation in autoimmune diseases in general, and will guide us to direct future research towards appropriate diagnostic and therapeutic strategies.
[Show abstract][Hide abstract] ABSTRACT: Periodontal disease has been recently linked to a variety of systemic conditions such as diabetes, cardiovascular disease, preterm delivery, and oral cancer. The most common bacteria associated with periodontal disease, Porphyromonas gingivalis (P. gingivalis) has not yet been studied in the malignant gingival tissues. The objective of this study was to investigate the presence of P. gingivalis in specimens from squamous cell carcinoma patients. We have performed immunohistochemical staining to investigate the presence of P. gingivalis and Streptococcus gordonii (S. gordonii), a non invasive oral bacteria, in paraffin embedded samples of gingival squamous cell carcinoma (n = 10) and normal gingiva (n = 5). Staining for P. gingivalis revealed the presence of the bacteria in normal gingival tissues and gingival carcinoma, with higher levels (more than 33%, P < 0.05) detected in the carcinoma samples. The staining intensity was also significantly enhanced in the malignant tissue by 2 folds (P < 0.023) compared to specimens stained for the non-invasive S. gordonii. P. gingivalis is abundantly present in malignant oral epithelium suggesting a potential association of the bacteria with gingival squamous cell carcinoma.
International Journal of Oral Science 10/2011; 3(4):209-15. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Evaluation of: Nakasa T, Shibuya H, Nagata Y, Niimoto T, Ochi M: The inhibitory effect of microRNA-146a expression on bone destruction in collagen-induced arthritis. Arthritis Rheum. 63(6), 1582-1590 (2011). miRNA-146a (miR-146a) has been shown to play an important role in the negative regulation of inflammatory innate immune responses, and is differentially expressed in a number of human diseases including rheumatoid arthritis. However, evidence for the potential therapeutic use of miR-146a in human disease has been lacking. The current paper demonstrates the potential therapeutic application of miR-146a for rheumatoid arthritis by demonstrating the inhibitory effect of miR-146a on osteoclastogenesis in vitro. Moreover, using a collagen-induced arthritis mouse model, they were able to demonstrate that intravenous administration of double-stranded miR-146a resulted in the suppression of cartilage and bone destruction, despite relatively unaffected immune cell infiltration of the synovium and inflammatory cytokine expression.
[Show abstract][Hide abstract] ABSTRACT: Chronic infiltration of lymphocytes into the salivary and lacrimal glands of patients with Sjögren's syndrome (SS) leads to destruction of acinar cells and loss of exocrine function. Protein kinase C-delta (PKCδ) is known to play a critical role in B-cell maintenance. Mice in which the PKCδ gene has been disrupted have a loss of B-cell tolerance, multiple organ lymphocytic infiltration, and altered apoptosis. To determine whether PKCδ contributes to the pathogenesis of SS, we quantified changes in indicators of SS in PKCδ-/- mice as a function of age. Salivary gland histology, function, the presence of autoantibodies, and cytokine expression were examined.
Submandibular glands were examined for the presence of lymphocytic infiltrates, and the type of infiltrating lymphocyte and cytokine deposition was evaluated by immunohistochemistry. Serum samples were tested by autoantibody screening, which was graded by its staining pattern and intensity. Salivary gland function was determined by saliva collection at various ages.
PKCδ-/- mice have reduced salivary gland function, B220+ B-cell infiltration, anti-nuclear antibody production, and elevated IFN-γ in the salivary glands as compared to PKCδ+/+ littermates.
PKCδ-/- mice have exocrine gland tissue damage indicative of a SS-like phenotype.
[Show abstract][Hide abstract] ABSTRACT: Abstract A 66-year-old female presented with gum bleeding and soreness. Her medical history was significant for delayed wound healing, which appeared to cause blindness in her right eye. A gingival incisional biopsy revealed replacement of fibrous connective tissue by an amorphous and eosinophilic material. Direct immunofluorescent staining for antibodies was negative. After the differential diagnosis of ligneous gingivitis (LG) was rendered, the activity and antigenic level of plasminogen was tested and found to be significantly decreased. Partial improvement was observed within 1 year following a regimen of scaling, gingival curettage, topical steroids, and improved oral hygiene. LG is a rare condition, occurring as a result of plasminogen deficiency and subsequent fibrin accumulation. It presents as pseudomembranous gingiva and might affect the eyes and other organs. Dentists should be familiar with this condition, since they might assist in a diagnosis of this disease, with significant morbidity often missed by medical professionals.
Journal of Investigative and Clinical Dentistry. 05/2011; 2(3):207 - 211.
[Show abstract][Hide abstract] ABSTRACT: Sjögren's syndrome (SS) is characterized by xerophthalmia and xerostomia resulting from loss of secretory function due to immune cell infiltration in lacrimal and salivary glands. Current therapeutic strategies for SS use secretagogues to induce secretion via muscarinic receptor stimulation. The purpose of this study was to create a secretagogue-small interfering RNA (siRNA) conjugate to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues, such as proinflammatory or death signals, while simultaneously stimulating secretion.
Based on our expertise with type 3 muscarinic receptor (M3), we used carbachol, a ligand specific for muscarinic receptor, as the secretagogue. Carbachol was synthesized with an active choline group and was conjugated with an siRNA that targets caspase 3. A human salivary gland (HSG) cell line was used to test the efficacy of this secretagogue-siRNA conjugate.
Lipofectamine transfection of the conjugate into HSG cells resulted in a 78% reduction in the expression of the caspase 3 gene, while external conjugate treatment of HSG cells resulted in intracellular calcium release and induction of endocytosis at levels similar to those of carbachol stimulation, indicating that the siRNA and carbachol portions of the conjugate retained their function after conjugation. HSG cells treated with conjugate (without Lipofectamine transfection) exhibited a 50% reduction in caspase 3 gene and protein expression, indicating that our conjugate was effective in delivering functional siRNA into cells via receptor-mediated endocytosis. Furthermore, tumor necrosis factor α-induced apoptosis was significantly reduced in conjugate-treated cells.
Our secretagogue-siRNA conjugate prevented cytokine-induced apoptosis in salivary gland epithelial cells, which is critical to maintaining fluid secretion and potentially reversing the clinical hallmark of SS.
[Show abstract][Hide abstract] ABSTRACT: 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 Sjögren's 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.
European Journal of Immunology 04/2011; 41(7):2029-39. · 4.52 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
CIP2A may play a significant role in oral malignant transformation and therefore, it may be a potential target for chemotherapy of OSCC.
Cancer biology & therapy 11/2010; 10(7):694-9. · 3.29 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The receptor for advanced glycation end products (RAGE) is a multiligand receptor expressed in a number of cell types, including gingival epithelia. RAGE mediates inflammation and induces cellular oxidative stress. Upregulation of RAGE is associated with various diseases, such as periodontal and cardiovascular diseases. This study examines the hypothesis that the gingiva of rats fed a calorie-restriction (CR) diet expresses lower levels of RAGE than the gingiva of rats fed an ad libitum (AL) diet.
Male F344BN rats (n = 16) from the National Institute on Aging (NIA) were fed a CR (n = 8) or AL (n = 8) diet according to NIA recommendations. Rats were sacrificed by guillotine at 8 (n = 5), 18 (n = 3), 29 (n = 4), and 38 (n = 4) months of age. The gingiva from around the molars was dissected and submitted for histologic and molecular analyses.
Immunohistochemistry revealed that RAGE was expressed in the plasma membrane and cytoplasm of gingival epithelial cells and endothelial cells from both groups. RAGE mRNA levels were quantified relative to levels of GAPDH mRNA by real-time reverse-transcriptase polymerase chain reaction. The mean relative RAGE mRNA level in the CR group (7.26 ± 0.54) was lower than in the AL group (10 ± 1.4) (P <0.05). There were no detectable differences in RAGE expression according to animal age.
Gingival RAGE expression in rats is reduced by calorie restriction.
Journal of Periodontology 10/2010; 81(10):1481-7. · 2.57 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Objectives: Sjgren's syndrome (SjS) is characterized by xerophthalmia and xerostomia resulting from immune cell infiltration in lacrimal and salivary glands. Current SjS therapeutic strategies employ secretagogues to induce secretion via muscarinic receptor (MR) stimulation. Based on our expertise on MR in SjS, we are utilizing ligands specific for MR to deliver siRNA into cells via receptor-mediated endocytosis, thereby altering epithelial cell responses to external cues such as pro-inflammatory or death signals while simultaneously stimulating secretion. Methods: Carbachol was synthesized with an active choline group and directly conjugated with siRNA targeting caspase-3. HSG cells were externally treated or transfected with conjugate and qRT-PCR and immunofluorescence were used to detect caspase-3 gene and protein expression, respectively. Carbachol functionality was assessed using intracellular calcium release assays. A FAM-labeled DNA probe was designed to target the antisense strand of caspase-3 siRNA and in situ hybridization was utilized to detect conjugate entry into cells. Results: Conjugate transfection into cells resulted in 80% reduction in caspase-3 gene expression, confirming retained function of siRNA after conjugation. External conjugate treatment of HSG cells resulted in similar intracellular calcium release and induction of endocytosis as carbachol stimulation indicating that carbachol portion of conjugate is functional. Using the FAM-labeled probe, conjugate was visualized in approximately 30% of conjugate-treated HSG cells, and in those cells caspase-3 protein expression was reduced 69%. Conclusion: Both siRNA and carbachol portions of the conjugate were shown to retain function, and external treatment of HSG cells with conjugate shows promising results. Further studies are needed to optimize conjugate entry and efficacy in the restoration of abnormal glandular homeostasis or cytokine-induced apoptosis. This therapeutic strategy can easily be manipulated to target different genes of interest while maintaining cell-type specificity, paving a way for potential clinical applications in the treatment of SjS. Supported by Sjgren's Syndrome Foundation/NIDCR-DE016705