Enhanced immune response to a dual-promoter anti-caries DNA vaccine orally delivered by attenuated Salmonella typhimurium.

The strength of immune responses induced by DNA vaccine is closely associated with the expression level of cloned antigens available to the antigen presenting cells (APCs). To acquire a larger and more persistent amount of antigen, a dual-promoter, which could double the target antigen output through its expression both in prokaryotic and eukaryotic cells, was employed in the constructed anti-caries DNA vaccine with attenuated Salmonella as mucosal delivery vector in this study. Here, both CMV and nirB promoters were included in the plasmid that harbors the genes encoding the functional epitopes of two virulence factors of S. mutans, i.e. the saliva-binding region (SBR) of PAc and the glucan-binding region (GBR) of glucosyltransferase-I (GTF-I). Delivered by attenuated Salmonella Typhimurium strain SL3261, the anti-caries vaccine was administered intragastrointestinally to BALB/c mice for evaluation of the effectiveness of this immune regime. Specific anti-SBR and anti-GBR antibodies were detected in the serum and saliva of experimental animals by week 3 after immunization. These immune responses were further enhanced after a booster vaccination at week 16. However, in mice receiving Salmonella expressing SBR and GBR under the control of nirB alone these antibody responses were significantly (P<0.01) lower. The serum IgG subclass profiles suggested a Th1/Th2-mixed but Th2 biased immune response to the cloned antigens, which was further confirmed by a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10) cytokines in splenocytes of immunized mice upon stimulation with SBR or GBR. To further determine the protective efficacy of these responses, a challenge test with S. mutans strain UA159 was performed in mice after the second immunization. Following challenge, mice immunized with Salmonella expressing SBR and GBR under the control of the CMV-nirB promoter showed a significant (P<0.01) reduction in the number of S. mutans in the dental plaque compared to the empty vector-immunized or unimmunized mice, and the reduction was also significant at weeks 3-8 (P<0.05) post-challenge when compared with those receiving Salmonella clones with nirB promoter alone. These results provide evidence for the effectiveness of a dual-promoter strategy in the anti-caries DNA vaccine when employing attenuated Salmonella as delivering vehicle for mucosal immunization.

Candida albicans Amphotericin B-Tolerant Persister Formation is Closely Related to Surface Adhesion.

Candida albicans persisters have so far been observed only in biofilm environment; the biofilm element(s) that trigger(s) persister formation are still unknown. In this study, we tried to further elucidate the possible relationship between C. albicans persisters and the early phases of biofilm formation, especially the surface adhesion phase. Three C. albicans strains were surveyed for the formation of persisters. We tested C. albicans persister formation dynamically at different time points during the process of adhesion and biofilm formation. The number of persister cells was determined based on an assessment of cell viability after amphotericin B treatment and colony-forming unit assay. None of the planktonic cultures contained persisters. Immediately following adhesion of C. albicans cells to the surface, persister cells emerged and the proportion of persisters reached a peak of 0.2-0.69 % in approximately 2-h biofilm. As the biofilm matured, the proportion of persisters decreased and was only 0.01-0.02 % by 24 h, while the number of persisters remained stable with no significant change. Persisters were not detected in the absence of an attachment surface which was pre-coated. Persisters were also absent in biofilms that were scraped to disrupt surface adhesion prior to amphotericin B treatment. These results indicate that C. albicans antifungal-tolerant persisters are produced mainly in surface adhesion phase and surface adhesion is required for the emergence and maintenance of C. albicans persisters.

Inhibition of nucleic acid biosynthesis makes little difference to formation of amphotericin B-tolerant persisters in Candida albicans biofilm.

Candida albicans persisters constitute a small subpopulation of biofilm cells and play a major role in recalcitrant chronic candidiasis; however, the mechanism underlying persister formation remains unclear. Persisters are often described as dormant, multidrug-tolerant, nongrowing cells. Persister cells are difficult to isolate and study not only due to their low levels in C. albicans biofilms but also due to their transient, reversible phenotype. In this study, we tried to induce persister formation by inducing C. albicans cells into a dormant state. C. albicans cells were pretreated with 5-fluorocytosine (planktonic cells, 0.8 µg ml(-1); biofilm cells, 1 µg ml(-1)) for 6 h at 37°C, which inhibits nucleic acid and protein synthesis. Biofilms and planktonic cultures of eight C. albicans strains were surveyed for persisters after amphotericin B treatment (100 µg ml(-1) for 24 h) and CFU assay. None of the planktonic cultures, with or without 5-fluorocytosine pretreatment, contained persisters. Persister cells were found in biofilms of all tested C. albicans strains, representing approximately 0.01 to 1.93% of the total population. However, the persister levels were not significantly increased in C. albicans biofilms pretreated with 5-fluorocytosine. These results suggest that inhibition of nucleic acid synthesis did not seem to increase the formation of amphotericin B-tolerant persisters in C. albicans biofilms.

Cerium promotes bone marrow stromal cells migration and osteogenic differentiation via Smad1/5/8 signaling pathway.

Cerium (Ce), one of the lanthanides (Ln), displays a variety of biochemical and physiological effects. However, the potential effect and mechanism of Ce on bone metabolism are not well understood. In this study, we investigated the putative role of Ce in regulating the migration and osteogenic differentiation of bone marrow stromal cells (BMSCs) and the underlying mechanism. The results indicated that Ce promoted BMSCs viability and ALP activity at lower concentrations (0.001 µM), and decreased the viability and ALP activity of BMSCs at higher concentrations (10 µM). Ce could also affect the expression of osteogenic transcription factors (Runx2, Satb2 and OCN) in BMSCs. Our results also showed that Ce promoted migration of BMSCs by increasing SDF-1 mRNA expression. As the Smad-dependent BMP signaling pathway plays an important role in migration and osteogenic differentiation of BMSCs, our results are in agreement with Ce promoting the phosphorylation of Smad1/5/8 and translocating to the nucleus by increase BMP2 expression. The activity of p-Smad1/5/8 increased SDF-1 and Runx2 expression level in BMSCs. In conclusion, our results support the notion that Ce promoted migration and osteogenic differentiation of BMSCs by Smad1/5/8 signaling pathway.

Expression of the chemokine CCL28 in pleomorphic adenoma and adenolymphoma of the human salivary glands.

Recent studies have proposed that the chemokine CCL28 is constitutively expressed by epithelial cells in salivary glands and play an important role in lymphocyte trafficking in oral immunity. To date, there is little information on the expression pattern of CCL28 in salivary gland tumors. The purpose of this study was to determine the expression of CCL28 in pleomorphic adenoma and adenolymphoma and to evaluate its potential function in regulating oral carcinogenesis. Immunohistochemical reactivity revealed CCL28 protein expression in the cytoplasm of acinar epithelial cells, both in tumorous tissues and normal adjacent tissues. The level of CCL28 mRNA was markedly reduced in 70% (28/40) of pleomorphic adenomas, and in 81% (26/32) of adenolymphomas, compared to the normal adjacent tissue. CCL28 protein expression was significantly lower in pleomorphic adenomas (P=0.0027, n=40) and in adenolymphomas (P=0.0003, n=32) compared to their normal adjacent tissues. Additionally, the CCL28 protein levels in saliva in the aforementioned patients were lower than those in healthy volunteers. Our study indicated that the reduced expression of CCL28 could possibly be a strategy by recruiting fewer antitumor immunocompetent cells to salivary glands. The expression and secretion of CCL28 may be associated with the pathogenesis of pleomorphic adenoma and adenolymphoma.

Local suppression of IL-21 in submandibular glands retards the development of Sjögren's syndrome in non-obese diabetic mice.

BACKGROUND: The aim of this study was to verify the validity of IL-21 local suppression in submandibular glands of preventing the development of Sjögren's syndrome in non-obese diabetic (NOD) mice and figure out the mechanism. METHODS: IL-21 levels in submandibular glands were suppressed by ductal cannulation of IL-21 shRNA lentivirus. Then, saliva flow rates (SFR) and histopathologic changes of submandibular glands were measured to assess the severity of disease development. Real-time PCR, flow cytometry, and immunohistochemistry were used to detect the changes of T helper cells and related cytokines. RESULTS: The reduction in SFRs in NOD mice was significantly alleviated from 9 to 17 weeks of age along with the suppression of IL-21 in submandibular glands. Lymphocytic infiltration was also milder than control NOD mice. Moreover, the lower level of IL-21 led to the down-regulation of follicular helper T (Tfh) cells. CONCLUSIONS: Local suppression of IL-21 in submandibular glands could retard the development of Sjögren's syndrome in NOD mice. IL-21 might contribute to the development of B-cell disorder in Sjögren's syndrome via Tfh cells pathway.

Enhancing CCL28 expression through the gene transfer to salivary glands for controlling cariogenic microbe.

The chemokine CCL28 participates in direct antimicrobial activities as well as homing of certain types of lymphocytes. The present study was conducted to harness these properties of the chemokine for the prevention of dental caries. The gene encoding CCL28 was transferred to salivary glands to enhance the production of this chemokine locally. First, a recombinant eukaryotic plasmid expressing CCL28 was constructed. Then, the CCL28 protein from 293 cells transfected with the recombinant plasmid was verified to inhibit the caries pathogen Streptococcus mutans (S. mutans) in a biofilm. Finally, the recombinant plasmid was retrogradely administered to the parotid glands of rats through the secretory ducts. The successful transfer of the gene encoding CCL28 to rat parotid acinar cells was confirmed by immunofluorescence and real-time PCR. Increases in both CCL28 and secretory IgA (SIgA) in the rat saliva were tested by ELISA. It was revealed that the CCL28 protein obtained from the study was able to strongly inhibit S. mutans living in biofilm in vitro. The delivery of the recombinant plasmid to the rat parotid glands was able to induce high levels of CCL28 and SIgA in saliva, and the increased levels of CCL28 and SIgA in saliva were maintained for 2 weeks. Notably, the dental plaque from the rats treated with the delivery of the recombinant plasmid in the study harbored significantly less S. mutans. These data indicated that the present strategy may hold hope for the effective prevention of dental caries.

Effect of dental plaque control on infection of Helicobacter pylori in gastric mucosa.

BACKGROUND: Data on the role of dental plaque in the transmission of Helicobactor pylori have varied. Furthermore, there has been few reports on the relationship between dental plaque control and H. pylori infection of gastric mucosa. The purpose of this study was to elucidate this potential relationship. METHODS: The 13C urea breath test was conducted on 56 subjects who received dental plaque control and 51 subjects who did not. RESULTS: The prevalence of H. pylori in the gastric mucosa was 19.64% in patients who received dental plaque control, which was significantly lower than in those without dental plaque control (84.31%). CONCLUSION: Long-term professional dental plaque control was associated with less gastric reinfection by H. pylori, suggesting that dental plaque control may help to prevent H. pylori-induced gastric disease or reinfection.

[Three-dimension finite element analyses of interior stress of two kinds of Replace implant].

OBJECTIVE: To establish three-dimension finite element model of mandible with two kinds of Replace implant, and to study stress of implant and abutment. METHODS: The data of components of the dental implant was measured, cross section of the mandible was scanned by spiral CT and image reconstruction technique was conducted. Three-dimension finite element analysis software UG and MSC. Marc/Mentat were used to built the conjunction model and bone model of two implant systems. Axial loading (200N) and 30 degrees oblique loading (100N) were applied on the models respectively, the stress distribution patterns of the implant and abutment of two implant systems were analyzed. RESULTS: The peak stress district was concentrated on the structure of the implant cervix, which was more obviously displayed on the Replace Select implant. The peak stress of oblique loading was higher than that of axial loading. The peak stress on the implant cervix of Replace Select implant was higher than that of Replace External Hex implant in all loadings. CONCLUSION: To Replace Select especially, oblique force should be avoided on clinical practice in case of the implant fracture.

Relationship between dental erosion and respiratory symptoms in patients with gastro-oesophageal reflux disease.

OBJECTIVES: Both dental erosion and respiratory symptoms are extra-oesophageal manifestations of gastro-oesophageal reflux disease (GERD). The aim of this study was to determine whether dental erosion was correlated with respiratory symptoms in GERD patients. METHODS: 88 GERD patients were recruited and assigned to three groups mainly according to the frequency of respiratory symptoms: Group I: never; Group II: occasional (1-2 days a week or less); Group III: frequent (3-5 days a week or more). All patients underwent medical evaluations, including medical history, questionnaire answering and alimentary tract examinations. Dental examinations were carried out on these patients and 36 healthy controls. Dental erosions were measured by modified method of Smith and Knight Tooth Wear Index (TWI). Location and severity of dental erosion were recorded. RESULTS: The prevalence of dental erosion in Group III (64.52%) was higher (p<0.05) than that in Groups I (36.67%) and II (44.44%). GERD patients were presented with dental erosion with TWI scores ranging from 1 to 4. Though proportion of dental erosion with Score 2 (7/20) in Group III was higher than that in Group I (2/11) and Group II (3/12), there was no statistical significance in the proportions of erosion scores among three patient groups. Correlation coefficient between airway symptoms and scores of dental erosion was 0.231 (p<0.05). Palatal erosion of upper incisor was seen in 8 persons (72.7%) in Group I, 9 persons (75%) in Group II and 16 persons (80%) in Group III (p>0.05). Labial erosion of upper incisors was found in 1 person in Groups I and II respectively and 4 persons in Group III. All patients with labial erosion on upper incisors had palatal erosion, except 1 patient in Group III. CONCLUSIONS: In GERD patients, dental erosions are more prevalent in patients with frequent respiratory symptoms than those in patients with occasional and without respiratory symptoms. Palatal erosion of upper incisor is the main manifestation in patients. Acid reflux is the main causative factor of dental erosion in GERD patients with airway symptoms.

The expression of tumstatin is down-regulated in renal carcinoma.

Tumstatin is the 28 kDa NC1 domain of the alpha3 chain of type IV collagen that inhibits pathological angiogenesis and suppresses endothelial cell proliferation and tumor growth. In the present paper, we expressed and purified recombinant human tumstatin protein and then prepared the anti-tumstatin polyclonal antibody. To investigate the expression of tumstatin in renal carcinoma, tumstatin protein was detected by western blotting using the prepared anti-tumstatin antibody and tumstatin mRNA levels were assayed by RT-PCR. The results showed that the expression of tumstatin gene was down-regulated in renal carcinoma tissues and cells. Our study suggests that as a novel endogenous angiogenesis inhibitor, tumstatin gene expression may be a marker for diagnosis, therapy and prognosis of renal carcinoma.

[Comparison of CCL28 in human labial glands and parotids].

OBJECTIVE: To compare the expression of CCL28 in minor and major salivary glands and clarify the role it plays in IgA secreting by minor salivary glands in oral cavity. METHODS: Labial gland and parotid samples were analyzed with real-time fluorescent quantitative PCR assay for CCL28 mRNA. Rank-sum test was used for data analysis using SPSS 10.0 software package. RESULTS: CCL28 mRNA was abundantly expressed in labial glands of healthy adults. Its expression was higher than that in parotids (P<0.01). CONCLUSION: The results of this article suggest that the expression level of CCL28 in labial glands is remarkably higher than that in parotids, which reminds us that the high concentration of IgA in minor salivary glands may be associated with their high expression of CCL28.

Effect of dental plaque control on infection of Helicobacter pylori in gastric mucosa.

BACKGROUND: Data on the role of dental plaque in the transmission of Helicobacter pylori have varied. Furthermore, there has been few reports on the relationship between dental plaque control and H. pylori infection of gastric mucosa. The purpose of this study was to elucidate this potential relationship. METHODS: The (13)C urea breath test was conducted on 56 subjects who received dental plaque control and 51 subjects who did not. RESULTS: The prevalence of H. pylori in the gastric mucosa was 19.64% in patients who received dental plaque control, which was significantly lower than in those without dental plaque control (84.31%). CONCLUSION: Long-term professional dental plaque control was associated with less gastric reinfection by H. pylori, suggesting that dental plaque control may help to prevent H. pylori-induced gastric disease or reinfection.

[Effects on Helicobacter pylori reinfection in gastric mucosa by two oral plaque control methods].

OBJECTIVE: To investigate the reinfection rate of Helicobacter pylori (H. pylori) in gastric mucosa by two measures of oral plaque control on patients, and to demonstrate the necessity and better method of plaque control on those patients. METHODS: 148 patients suffered gastritis or gastroduodenal ulcer were assigned into test group 1 (54 patients), test group 2 (55 patients) and control group (39 patients). 13C-urea breath test proved that there were no H. pylori in their gastric mucosa. Daily plaque control was used in test group 1, oral professorial interventions were added into test group 2, neither daily plaque control nor oral professorial interventions was conducted in control group. All patients were conducted 13C-urea breath test again after half a year to determine the reinfection rate of H. pylori in gastric mucosa. RESULTS: 5 patients were eliminated because of stopping mouthwash in the test group 1, 8 patients failed to control dental plaque in the test group 2. The infection rates of H. pylori in gastric mucosa of test group 1, test group 2 and control group were 67.3%, 19.1%, 82.1%, respectively. The infection rate of H. pylori of test group 2 was lower significantly than that in control group and test group 1 (chi2=33, P<0.05; chi2=31.06, P<0.05). There were no significant difference between test group 1 and control group (chi2=2.43, 0.1

Adenovirus-mediated expression of spermidine/spermine N1-acetyltransferase gene induces S-phase arrest in human colorectal cancer cells.

Spermidine/spermine N1-acetyltransferase (SSAT) is a key enzyme of polyamine catabolism. In a previous study, we constructed a recombinant adenovirus, Ad-SSAT, which can express human SSAT. In the present study, we investigated the effect of Ad-SSAT on the growth and cell cycle of colorectal cancer cells. We found that Ad-SSAT increased the expression of SSAT and inhibited the growth of HT-29 and Lovo cells. The growth inhibition was caused by cell cycle arrest in the S phase. Furthermore, Ad-SSAT was shown to suppress the expression of cyclin A and nuclear factor E2F-1 in HT-29 and Lovo cells. The inhibitory effect of Ad-SSAT on cyclin A promoter activity was also observed in a reporter gene assay. Our results suggest that the expression of SSAT mediated by Ad-SSAT infection inhibits the growth of colorectal cancer cells and induces cell cycle arrest at the S phase, through a mechanism involving the suppression of cyclin A and E2F-1 expression.

[Three-dimensional finite element analyses of bone surface stress of two kinds of conjunction implant].

OBJECTIVE: To establish a three-dimension finite element model of mandible with two kinds of dental implant and to study the stress of implant-bone interface. METHODS: Measuring the data of the components of the dental implant and using spiral CT image reconstruction technique to scan the cross section of the mandible. Three-dimension finite element analysis software Unigraphics and MSC. Marc/Mentat were used to build the conjunction model and bone model of two implant systems. Loading 200 N axially and 100 N 30 degrees obliquely on the models respectively, the stress distribution patterns of the bone interface of two implant systems were analyzed. RESULTS: The stress distribution on the bone interface of two implant systems was similar. The peak stress of oblique loading was higher than that of axial loading. The peak stress district of the bone was concentrated on the stricture of the implant cervix, which was more obviously displayed on the Replace Select implant. The peak stresses on the bone interface of Replace Select implant were higher than that of Replace implant in all loadings. CONCLUSION: To Replace Select especially, oblique force should be avoided in clinical practice in case of the bone absorption.

[Nasal immunization with co-expression plasmid harboring genes encoding porphyromonas gingivalis FimA and human interleukin-15 in mice].

OBJECTIVE: To observe the antibody responses induced by recombinant plasmid plRES-fimA:IL15 via nasal immunization to BABL/c mice and the regulation of IL-15 to sIgA. METHODS: BABL/c mice were immunized with recombinant plasmids pIRES-fimA:IL15 and pIRES-fimA via nasal or intramuscular route. Serum IgG and salivary sIgA levels after immunization were analyzed by ELISA. RESULTS: Nasal immunization with plasmids pIRESfimA:IL15 or pIRES-fimA elicited significant higher level of salivary FimA-specific sIgA responses compared with intramuscular immunization. There was no significant difference of the serum IgG responses between nasal immunization mice and intramuscular immunization mice. Nasal immunization with plasmid pIRES -fimA:IL15 elicited significant higher level of salivary sIgA response than with pIRES-fimA (P<0.05). CONCLUSION: Nasal dropping may be an effective mucosal immunization route of anti-Porphyromonas gingivalis DNA vaccine to elicit specific antibody responses in serum and oral region. IL-15 has a positive regulation effect to sIgA response.

[Construction and verification of the dual-promoter plasmid pCN-SSISG as bivalent anti-caries DNA vaccine].

PURPOSE: The aim of the study is to construct an anti-caries DNA vaccine with high immunogenicity due to its unique properties of being able to express target antigens both in eukaryotic and prokaryotic host cells and harboring two main virulence genes from caries pathogen Streptoccocus mutans (S. mutans). METHODS: The gene encoding glucan binding region (GBR) of glucosyltransferase-I (GTF-I) was amplified from the plasmid harboring the gtfB gene from S. mutans by PCR. Then the GBR gene that was upstream linked with tissue-type plasminogen activator signal peptide (tPA-SP) was directly cloned into the plasmid pCN-SSIE containing sSBR gene encoding tPA-SP and saliva binding region (SBR) of antigen protein I/II (AgI/II) from S. mutans. And finally the recombinant plasmid pCN-SSISG was analysed by DNA sequencing and endonuclearase digestion mapping. RESULTS: DNA sequencing and endonuclearase digestion mapping showed that the open reading frame (ORF) and the tPA-SP sequence of the recombinant plasmid pCN-SSISG were identical with the expectant ones. CONCLUSION: We have successfully constructed the dual-promoter bivalent recombinant plasmid pCN-SSISG.

Construction of a sIgA-enhancing anti-Porphyromonas gingivalis FimA vaccine and nasal immunization in mice.

Porphyromonas gingivalis is implicated in the etiology of chronic periodontitis. Fimbriae are one of several critical surface virulence factors of P. gingivalis. Interleukin 15 (IL-15) is a critical important cytokine for the differentiation of B-1 cells into IgA-inducing cells in mucosal tissues and the proliferation of B cells. The present study constructed a co-expression plasmid pIRES-fimA:IL-15 encoding fimbrinllin (FimA), a subunit of fimbriae and IL-15 as a sIgA-enhancing anti-P. gingivalis FimA vaccine. The plasmid pIRES-fimA:IL-15 was transfected to CHO cells. The expressions of FimA and IL-15 in CHO cells were verified by Western blot and ELISA. Mice were immunized with pIRES-fimA:IL-15 via nasal or intramucusal route. The results showed that nasal immunization was capable of promoting Ag-specific immune responses in the oral region as well as systemic immunity. When immunized via nasal route, IL-15 expressed by the plasmid enhanced FimA-specific sIgA antibody response. In conclusion, a co-expression plasmid pIRES-fimA:IL-15 has been constructed, and when immunized via nasal route, antigen-specific sIgA antibody response could be modulated positively in immunized mice.

[Construction of expression vector containing S. mutans GBR gene and confirmation of the immunogenicity of the recombinant GBR protein].

PURPOSE: The purpose of this study is to construct the expressing plasmid vector containing the GBR gene of Streptococcus mutans and study the immunogenicity of recombinant GBR protein. METHODS: The GBR gene was cloned into the expression vector pTriEx-4 through gene cloning techniques by PCR,T-A clone and etc,the recombinant plasmid pTriEx-4-GBR was analyzed by DNA sequencing and endonuclearase digestion mapping. The recombinant GBR(rGBR) protein expression was induced with IPTG in E. coli JM109(DE3) which was transformed with plasmid pTriEx-4-GBR and then the rGBR protein was purified by affinity chromatography. Balb/c mice were randomly divided into two groups:mice of experimental group were inoculated subcutaneously with the rGBR protein and the rGBR protein was replaced for NS in the control group, then the anti-serum was evaluated by ELISA.The data were analysed by statistical software PEMS3.0. RESULTS: The DNA sequence and the reading frame of GBR gene in the reconstructed vector pTriEx-4-GBR was in corresponding with the initial design and the rGBR protein was purified. The level of specific anti-rGBR serum IgG in the experimental group was significantly higher than that in the control group(P<0.005). CONCLUSION: The results showed that the expressing plasmid carrying the GBR gene was constructed successfully and the purified recombinant GBR protein can elicit specific murine system immune response, which is necessary for further experiments including construction of bivalent anti-caries DNA vaccine and studies both in vitro and in vivo.