Convenient and reproducible in vivo gene transfer to mouse parotid glands

Oral Diseases (2010) 17 , 77–82 Objectives: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands. Methods: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen’s ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10⁷ vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme‐linked immunosorbent assay (hEpo). Vector biodistribution was measured by real‐time quantitative (Q) PCR. Results: The chosen volume for mouse parotid vector delivery was 20 μL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva. Conclusion: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre‐clinical studies with many murine disease models.     

水通道基因治疗小型猪腮腺放射损伤的研究

目的研究腺病毒介导水通道基因对小型猪腮腺放射损伤的治疗效果及其相应表达关系.方法19只实验用小型猪一侧腮腺给予20Gy放射剂量照射,照射后17周,转导不同浓度的水通道蛋白基因、荧光素酶基因和病毒缓冲液,分析唾液流率变化和目的基因表达.结果109pfu AdCMVhAQP1转导后3天和7天腮腺唾液流率明显增加,分别恢复到放射前腮腺唾液流率81±18%(P=0.024)和69±20%(P=0.058),14天下降到转导前水平,对照组均未见腮腺唾液流率明显变化.转导109pfu AdCMVhAQP1组免疫组织化学显示,AQP1主要表达在导管上皮,Western blotting分析有外源性水通道蛋白表达,而对照组未见AQP1表达.结论109pfu AdCMVhAQP1明显增加小型猪腮腺放射损伤唾液流率,转导的水通道基因具有潜在治疗涎腺放射损伤的临床应用价值.     

Gene transfer mediated by different viral vectors following direct cannulation of mouse submandibular salivary glands

The salivary gland has been suggested as an accessible organ for gene transfer to express recombinant proteins locally in the saliva, as well as for secretion to the blood circulation. The aim of this study was to evaluate the efficiency of gene transfer to salivary glands using different viral vectors: adenovirus, vaccinia, herpes simplex type 1 (HSV), and two retroviral vectors (murine leukemia virus (MuLV) and lentivirus). We show, by in situ staining and beta-galactosidase reporter activity assay, that the adenoviral and vaccinia vectors were able to deliver the reporter gene efficiently to acinar and duct cells. The HSV vector was less efficient and infected only the acinar cells. The lentiviral vector infected acinar and duct cells, but at a relatively low efficiency. The MuLV vector did not infect the salivary gland unless cell proliferation was induced. Host immune responses to viral infection, inflammation, apoptosis and lymphocyte infiltration, in the transduced glands, were assessed. The DNA viral vectors induced local lymphocyte infiltration and apoptosis. In contrast, the retroviral vectors did not induce an immune response. Our results describe the outcome of salivary gland infection with each of the five different viral vectors and indicate their advantages and limitations for transferring genes to the salivary glands.     

Effects of adenoviral‐mediated coexpression of bone morphogenetic protein‐7 and insulin‐like growth factor‐1 on human periodontal ligament cells

Yang L, Zhang Y, Dong R, Peng L, Liu X, Wang Y, Cheng X. Effects of adenoviral‐mediated coexpression of bone morphogenetic protein‐7 and insulin‐like growth factor‐1 on human periodontal ligament cells. J Periodont Res 2010; 45: 532–540. © 2010 John Wiley & Sons A/S Background and Objective: Bone morphogenetic protein‐7 (BMP‐7) and insulin‐like growth factor‐1 (IGF‐1) are important in periodontal reconstruction. However, their synergistic effect in periodontal regeneration by gene delivery has not been reported. In this study, gene delivery of these two growth factors to human periodontal ligament cells (hPDLCs) was examined for its effects on cell proliferation and differentiation. Material and Methods: Recombinant adenoviruses containing both human BMP‐7 and IGF‐1 cDNA created by introducing the internal ribosome entry site (IRES) sequence were used to transfer the genes into hPDLCs. 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and cell cycle analysis were used to observe their effects on cell proliferation, while alkaline phosphatase activity measurement, RT‐PCR and in vivo tests were conducted to investigate their effects on cell differentiation. Results: The proliferation of hPDLCs transduced by adenoviruses coexpressing BMP‐7 and IGF‐1 was suppressed while their differentiation ability was enhanced. There was a synergism of BMP‐7 and IGF‐1 in up‐regulating alkaline phosphatase activity and mRNA levels of collagen type I and Runx2. Implantation in vivo with scaffolds illustrated that the transduced cells exhibited osteogenic differentiation and formed bone‐like structures. Conclusion: The combined delivery of BMP‐7 and IGF‐1 genes using an IRES‐based strategy synergistically enhanced differentiation of hPDLCs. It is suggested that this could be a new potential method in gene therapy for periodontal reconstruction.     

Cytokine profiles in parotid saliva from HIV‐1‐infected individuals: changes associated with opportunistic infections in the oral cavity

The purpose of this study was to quantitate levels of cytokines in parotid saliva of subjects infected with human immunodeficiency virus‐1 (HIV‐1) and to determine if the cytokine profiles differ in subjects with an oral opportunistic infection, i.e., candidiasis or oral hairy leukoplakia. Parotid saliva samples were obtained from HIV‐infected individuals with or without candidiasis or oral hairy leukoplakia and from healthy controls and were assessed by ELISA for levels of interleukin (IL)‐1, IL‐2, IL‐4, IL‐5, IL‐10, transforming growth factor‐β, tumor necrosis factor‐α and interferon (IFN)‐γ. Saliva from HIV‐infected subjects with oral candidiasis had significantly higher levels of IFN‐γ than that seen in HIV‐infected individuals with no oral disease and significantly higher levels of IL‐2, IL‐5 and IFN‐γ than saliva of healthy controls. No significant difference was seen in cytokine levels in saliva from HIV‐infected subjects with no oral infections and healthy controls. The HIV‐infected subjects with oral hairy leukoplakia displayed significantly higher levels of both IL‐1α and IFN‐γ compared with the HIV and no oral disease group and a higher level of IFN‐γ than seen in saliva from the healthy control group. In comparing cytokine levels from both HIV and oral disease groups, significant differences were detected in levels of IL‐5 and IL‐10. These results indicate that the profile of salivary cytokines is altered as a result of the oral opportunistic infection candidiasis or oral hairy leukoplakia and also by concurrent HIV infection.     

Lentivirus‐mediated gene silencing of beta‐catenin inhibits growth of human tongue cancer cells

J Oral Pathol Med (2011) 40 : 643–650 Background: Beta‐catenin is one of the key components of Wnt signaling pathway. Increased level of this protein has been proved to be associated with enhanced cellular proliferation and the development of many kinds of cancers. But its role in the carcinogenesis in human tongue squamous cell carcinoma, one of the most common carcinomas of the human oral cavity, remains poorly characterized. Methods: In this study, we used lentivirus‐mediated RNA interference (RNAi) targeted against beta‐catenin to determine the effects of decreasing the high constitutive level of this protein in human tongue carcinoma cell line Tca8113. Results: Our studies demonstrated that RNAi directly against beta‐catenin markedly decreased beta‐catenin gene expression and inhibited cellular proliferation as reflected in the reduced growth of tongue cancer cells both in vitro and in nude mice. Conclusions: RNA interference (RNAi) targeting against beta‐catenin can induce cell growth suppression of tongue cancer and may have the potential as a therapeutic modality to treat human tongue cancer.     

The host defense peptide LL‐37 is detected in human parotid and submandibular/sublingual saliva and expressed in glandular neutrophils

The human host defense peptide, LL‐37, is an important player in the first line of defense against invading microorganisms. LL‐37 and its precursor, hCAP18, have been detected in unstimulated whole saliva but no reports showing hCAP18/LL‐37 in isolated, parotid, and/or submandibular/sublingual saliva have been presented. Here, we measured the levels of hCAP18/LL‐37 in human parotid and submandibular/sublingual saliva and investigated the expression of hCAP18/LL‐37 in parotid and submandibular gland tissue. Parotid and submandibular/sublingual saliva was collected from healthy volunteers, and the levels of hCAP18/LL‐37 in saliva were analyzed by dot blot, ELISA, and western blotting. Cellular expression of hCAP18/LL‐37 in human parotid and submandibular glands was investigated by immunohistochemistry. Immunoreactivity for hCAP18/LL‐37 was detected in both parotid and submandibular/sublingual saliva of all individuals. The concentration of hCAP18/LL‐37 was similar in parotid and submandibular/sublingual saliva, and was determined by densitometric scanning of each dot and normalization to the total protein concentration of each sample, and by ELISA. Double immunohistochemistry revealed that intravascular neutrophils of both parotid and submandibular glands express hCAP18/LL‐37. For the first time, we demonstrate hCAP18/LL‐37 in isolated human parotid and submandibular/sublingual saliva and expression of hCAP18/LL‐37 in glandular intravascular neutrophils, indicating that neutrophils of the major salivary glands contribute to the LL‐37 content of whole saliva.     

Sequence diversity in the major fimbrial subunit gene (flp‐1) of Actinobacillus actinomycetemcomitans

Cells of the periodontal pathogen Actinobacillus actinomycetemcomitans exhibit tight adherence to surfaces such as glass, plastic and hydroxyapatite, a property that probably plays an important role in the ability of this bacterium to colonize teeth and other surfaces. Tight adherence is mediated by long fibrils of bundled pili (fimbriae) that form on the surface of the cell. The flp‐1 gene encodes the major pilin protein component of A. actinomycetemcomitans fimbriae. In this study we compared flp‐1 DNA sequences from 43 strains of A. actinomycetemcomitans isolated in Europe, Japan and the United States and identified seven distinct flp‐1 allelic classes. DNA and predicted protein sequences were almost completely conserved within each flp‐1 class but were highly divergent between classes. Most amino acid substitutions occurred in the C‐terminus of the pilin protein, a region that has been shown to be important for the bundling and adhesive properties of the pili. flp‐1 classes correlated with serotypes and 16S rRNA genotypes in most strains. At least five strains showed evidence of horizontal transfer of flp‐1 between strains of different serotypes and 16S rRNA genotypes. Four of the seven flp‐1 classes were present in geographically diverse isolates. Strains representing all seven flp‐1 classes, but not a strain carrying a transposon insertion in flp‐1, bound avidly to polystyrene in an in vitro adherence assay. Strains representing six of the seven flp‐1 classes were isolated from localized juvenile periodontitis patients, suggesting that phylogenetically diverse strains carry pathogenic potential. Our findings provide a framework for future biochemical, immunological and genetic studies of A. actinomycetemcomitans fimbriae.     

Streptococcus mutans binding to solid phase dextran mediated by the glucan‐binding protein C

Streptococcus mutans GbpC is a wall‐anchored surface protein which is involved in dextran‐dependent aggregation. The GbpC phenotype is observed only in cells grown under stress conditions. In order to detect the GbpC protein of S. mutans, we isolated the wall fraction following digestion of the cell wall of this organism by N‐acetylmuramidase, and detected the GbpC protein from S. mutans cells by western analysis with anti‐GbpC serum. Interestingly, S. mutans cells exhibiting the negative dextran(α‐1,6 glucan)‐dependent aggregation (ddag) phenotype expressed the protein and could bind to immobilized dextran.     

The high expression level of programmed death‐1 ligand 2 in oral lichen planus and the possible costimulatory effect on human T cells

J Oral Pathol Med (2011) 40 : 525–532 Background: Oral lichen planus (OLP) is a T‐cell‐mediated chronic autoimmune disease whose precise etiology is unknown. The recently identified costimulatory programmed death‐1 (PD‐1) molecule and its ligands, PD‐L1 and PD‐L2, have been identified as CD28‐B7 family molecules and constitute a regulatory pathway of potential therapeutic use in immune‐mediated diseases. Methods: We examined the expression of two ligands of PD‐1 at both the protein and gene level in the focal mucosa and peripheral blood of OLP patients using immunohistochemistry and real‐time PCR. Next, we used the PD‐L2.Ig fusion protein and observed its effects on T cells, which were co‐cultured with IFN‐γ‐treated keratinocytes (KCs) in the presence of PHA. Results: We found that the expression of PD‐L2 at both the gene and protein level was statistically different in peripheral blood and local lesion tissue of patients with OLP compared to the normal controls. The proliferation ability of T cells and the expression level of IFN‐γ in the supernatant of the above co‐culture model were significantly augmented (P < 0.05). PD‐L2.Ig fusion protein significantly aggravated the apoptosis of T cells, inhibited the proliferation of T cells and decreased the release levels of IL‐2 and IFN‐γ in the model (P < 0.05). Conclusion: These data show that the increased expression of PD‐L2, as a costimulatory molecule, may have an important modulatory function on the local immune responses of OLP in vivo.     

Activation of the TREM‐1 pathway in human monocytes by periodontal pathogens and oral commensal bacteria

Periodontitis is a highly prevalent disease caused in part by an aberrant host response to the oral multi‐species biofilm. A balance between the oral bacteria and host immunity is essential for oral health. Imbalances in the oral microbiome lead to an uncontrolled host inflammatory response and subsequent periodontal disease (i.e. gingivitis and periodontitis). TREM‐1 is a signaling receptor present on myeloid cells capable of acting synergistically with other pattern recognition receptors leading to amplification of inflammatory responses. The aim of this study was to investigate the activation of the TREM‐1 pathway in the human monocyte‐like cell line THP‐1 exposed to both oral pathogens and commensals. The relative expression of the genes encoding TREM‐1 and its adapter protein DAP12 were determined by quantitative real‐time polymerase chain reaction. The surface expression of TREM‐1 was determined by flow cytometry. Soluble TREM‐1 and cytokines were measured by enzyme‐linked immunosorbent assay. The results demonstrate that both commensal and pathogenic oral bacteria activate the TREM‐1 pathway, resulting in a proinflammatory TREM‐1 activity‐dependent increase in proinflammatory cytokine production.