Lysosome-associated membrane protein 3 misexpression in salivary glands induces a Sjögren's syndrome-like phenotype in mice.

OBJECTIVES: Sjögren's syndrome (SS) is an autoimmune sialadenitis with unknown aetiology. Although extensive research implicated an abnormal immune response associated with lymphocytes, an initiating event mediated by salivary gland epithelial cell (SGEC) abnormalities causing activation is poorly characterised. Transcriptome studies have suggested alternations in lysosomal function are associated with SS, but a cause and effect linkage has not been established. In this study, we demonstrated that altered lysosome activity in SGECs by expression of lysosome-associated membrane protein 3 (LAMP3) can initiate an autoimmune response with autoantibody production and salivary dysfunction similar to SS. METHODS: Retroductal cannulation of the submandibular salivary glands with an adeno-associated virus serotype 2 vector encoding LAMP3 was used to establish a model system. Pilocarpine-stimulated salivary flow and the presence of autoantibodies were assessed at several time points post-cannulation. Salivary glands from the mice were evaluated using RNAseq and histologically. RESULTS: Following LAMP3 expression, saliva flow was significantly decreased and serum anti-Ro/SSA and La/SSB antibodies could be detected in the treated mice. Mechanistically, LAMP3 expression increased apoptosis in SGECs and decreased protein expression related to saliva secretion. Analysis of RNAseq data suggested altered lysosomal function in the transduced SGECs, and that the cellular changes can chemoattract immune cells into the salivary glands. Immune cells were activated via toll-like receptors by damage-associated molecular patterns released from LAMP3-expressing SGECs. CONCLUSIONS: These results show a critical role for lysosomal trafficking in the development of SS and establish a causal relationship between LAMP3 misexpression and the development of SS.

Effects of human bone morphogenetic protein 2 (hBMP2) on tertiary dentin formation.

Formation of tertiary dentin to maintain pulp vitality is a major odontoblastic response to dental pulp injury. Human bone morphogenetic protein 2 (hBMP2) can promote proliferation and differentiation of odontoblasts. Current study is interested in evaluating if the hBMP2 can promote the regeneration of tertiary dentin and cure dental pulp injury using the adenoviral vector to deliver hBMP2 cDNA into the pulp. Primary culture of dental pulp cells of exfoliated deciduous teeth (hDPCs) was established. Human serotype 5 adenoviral vector, AdCMV-hBMP2, was created. AdCMV-hBMP2 was used to transduce hDPCs in vitro and dental pulp cells in animal model in vivo. Data clearly demonstrated that hBMP2 increased ALP and mineralization. Reverse transcription-real time quantitative PCR (RT-QPCR) data showed that hBMP2 dramatically increased gene expressions of Runx2 (Runt-related transcription factor 2), ALP, Col Iα (Collagen 1a1), SP7 (Osterix), DMP1 (dentin matrix acidic phosphoprotein 1), DSPP (dentin sialophosphoprotein), and BSP (bonesialoprotein), which are normally involved in osteogenesis/odontogenesis. Data from in vivo assays demonstrated that hBMP2 promoted pulp cell proliferation and increased formation of tertiary dentin in dental pulp. Our in vitro and in vivo data suggest that hBMP2 gene can efficiently be delivered into the dental pulp cells by adenovirus, and show potential clinical application for the treatment of dental pulp damage.

Effect of same-dose single or dual field irradiation on damage to miniature pig parotid glands.

AIM: To evaluate the effect of single or dual field irradiation (IR) with the same dose on damage to miniature pig parotid glands. METHODOLOGY: Sixteen miniature pigs were divided into two IR groups (n=6) and a control group (n=4). The irradiation groups were subjected to 20 Gy X-radiation to one parotid gland using single-field or dual-field modality by linear accelerator. The dose-volume distributions between two IR groups were compared. Saliva from parotid glands and blood were collected at 0, 4, 8 and 16 weeks after irradiation. Parotid glands were removed at 16 weeks to evaluate tissue morphology. RESULTS: The irradiation dose volume distributions were significantly different between single and dual field irradiation groups (t=4.177, P=0.002), although dose volume histogramin (DVH) indicated the equal maximal dose in parotid glands. Saliva flow rates from IR side decreased dramatically at all time points in IR groups, especially in dual field irradiation group. The radiation caused changes of white blood cell count in blood, lactate dehydrogenase and amylase in serum, calcium, potassium and amylase in saliva. Morphologically, more severe radiation damage was found in irradiated parotid glands from dual field irradiation group than that from single field irradiation group. CONCLUSION: Data from this large animal model demonstrated that the radiation damage from the dual field irradiation was more severe than that of the single field irradiation at the same dose, suggesting that dose-volume distribution is an important factor in evaluation of the radiobiology of parotid glands.

[Targeting transgene effect of adTERT-TRAIL on salivary adenoid cystic carcinoma cell line SACC-83].

OBJECTIVE: To investigate the targeting expression of TRAIL gene driven by human telomerase reverse transcriptase (hTERT) promoter in SACC-83 cell with telomerase activity. METHODS: Adenovirus vector AdTERT-TRAIL was constructed by homologus recombination. After transfecting AdTERT-TRAIL into SACC-83 cell and HEL cell, its effect on these cells in vitro was investigated using RT-PCR technique, MTT method and flow cytometry. RESULTS: After transfection of AdTERT-TRAIL, expression of extrinsic TRAIL gene driven was detected in SACC-83, the proliferation of SACC-83 cell showed significant inhibitory effect (the relative cell viability was 49.70%) and its apoptotic rate was promoted (30.49%), whereas no TRAIL gene was detected in HEL cell, also no inhibitory effect was observed in HEL cell and its apoptotic rate showed little change. CONCLUSION: Adenovirus vector AdTERT-TRAIL was successfully constructed, which can be used to induce expression of TRAIL gene in SACC-83 cell with targeting effect.