[Research Advance of CXCR4 Inhibitors in the Treatment of Acute Myeloid Leukemia--Review].

CXCL12/CXCR4 axis composed of chemokine CXCL12 and its specific ligand CXCR4 can regulate and control the adhesion of leukemia cells to protective bone marrow niche, promote cell survival, and resist apoptosis induced by signal transduction inhibitors and chemotherapeutic drugs. Therefore, CXCL12 /CXCR4 axis has become a new target for the treatment of acute myeloid leukemia. At present, CXCR4 inhibitors that have been developed are in different clinical trials, showing good anti-leukemia effect. In this review, the research advance of CXCR4 inhibitors in the treatment of acute myeloid leukemia is summarized briefly.

[The effect of Streptococcus mutans luxS gene on mixed-species biofilm communities].

PURPOSE: To evaluate the effect of S.mutans luxS gene on mixed-species biofilms communities. METHODS: Biofilms were formed by S. mutans (wild type strain, its luxS overexpression strain and luxS knockout strain) and Lactobacillus acidophilus (ATCC4356) with a ratio of 1:1 at 37℃ for 4 h, 14 h and 24 h. MTT assay was used to detect the quantification of the biofilms formed. The structures of biofilms were observed under confocal laser scanning microscopy after 24 h, and expression of biofilm-related genes (ftf, smu630, brpA, gbpB, gtfB, vicR, comDE and relA) was investigated by real-time PCR. Statistical analysis was performed with SPSS17.0 software package. RESULTS: The results showed that biofilm formed by S. mutans(wild type strain, its luxS overexpression strain and luxS knockout strain) and L.acidophilus after 14 h were 0.481±0.024, 0.591±0.023 and 0.279±0.019, respectively. The same findings were present after 24 h, the biofilm formed by S.mutans overexpression strain with L.acidophilus was higher than wild type strain, and the biofilm formed by knockout strain significantly decreased; but there was no significant difference at 4 h time points. CLSM images revealed that both S.mutans overexpression strain and its wild type strain tended to aggregate into distinct clusters and dense structures, whereas the luxS knockout strain appeared relatively sparse. Compared with wild type strain, all of the genes examined were upregulated in the biofilms formed by the overexpression strain, and were downregulated in the biofilms formed by the luxS mutant strain in mixed-species biofilm. CONCLUSIONS: S.mutans luxS gene can affect mixed-species biofilm formation with L.acidophilus, which provides evidences for further study.

[In vitro study of antimicrobial efficacy of different irrigations on Enterococcus faecalis biofilm formation in root canal].

PURPOSE: To compare the antimicrobial effect of different irrigations on Enterococcus faecalis biofilms in extracted teeth and evaluate the antimicrobial activity of irrigating solutions residual against E. faecalis biofilms formation, in order to provide a better strategy for clinician. METHODS: Extracted human premolar teeth with single root canal were clearly autoclaved. These teeth were contaminated with E. faecalis（ATCC33186） and incubated for 60 days. The samples were randomly assigned to 4 experimental groups. During biomechanical instrumentation, the root canal was irrigated with different irrigating agents. The bacteria samples were collected with sterile paper points before and after instrumentation to F2. Then, samples that had been instrumented and autoclaved again were randomly divided into 2 groups treated with normal saline and 1%NaOCl for 30 min. E. faecalis was used to contaminate these root canals. The bacteria samples were collected with sterile paper points after 2, 6, 24, 48 h. SPSS19.0 software package was used for statistical analysis. RESULTS: Group using 1% NaOCI with ultrasound devices was significantly more effective than NS alone groups. 1% NaOCI groups showed a better residual activity than NS group. CONCLUSIONS: NaOCl is still the most important irrigating solutions, and it could be a better choice after biomechanical instrumentation, because of its long time substantivity achieves residual antimicrobial activity. Ultrasound devices is recommended to coordinate with irrigation.

Porphyromonas gingivalis infection accelerates intimal thickening in iliac arteries in a balloon-injured rabbit model.

BACKGROUND: Current epidemiologic data suggest that a localized infection (periodontitis) can disseminate into the distant tissues, and subgingival bacteria can migrate in the bloodstream, thereby contributing to independent systemic disease processes. To test this hypothesis, we investigated the effect of repeated systemic inoculations with Porphyromonas gingivalis (Pg) on intimal hyperplasia in iliac arteries in a rabbit model of balloon injury. METHODS: One week after single balloon injury to the iliac artery, 30 male New Zealand rabbits were randomly assigned to intravenous inoculation with 100 microl live Pg (10(7) colony-forming units; n = 15) or vehicle (n = 15) once weekly for 4, 8, or 12 consecutive weeks. Arteries were fixed by perfusion and removed for analysis of neointimal lesion formation. We measured intimal and medial lesion areas in iliac artery cross-sections as well as the intimal/medial ratio (I/M). We also analyzed Pg 16S ribosomal DNA amplification with polymerase chain reaction, systemic proinflammatory mediators with enzyme-linked immunosorbent assay, and immunolocalization of macrophages in the balloon-injured arteries. RESULTS: At 12 weeks, iliac intimal hyperplasia was accelerated, and I/M was significantly increased in Pg-inoculated animals (I/M 3.961 +/- 0.536 in the Pg group versus 3.585 +/- 0.353 in the control animals; P <0.01). Pg-inoculated animals also had significant increases in macrophage infiltration at 12 weeks, C-reactive protein levels at all time points, and interleukin-6 levels at 12 weeks. Moreover, Pg ribosomal DNA was found in the injured arteries of Pg-inoculated animals, but only after 12 weeks. CONCLUSION: Long-term systemic challenge with Pg, an oral pathogen, may accelerate intimal hyperplasia in balloon-injured iliac arteries.

Melatonin Receptor Agonists as the "Perioceutics" Agents for Periodontal Disease through Modulation of Porphyromonas gingivalis Virulence and Inflammatory Response.

AIM: "Perioceutics" including antimicrobial therapy and host modulatory therapy has emerged as a vital adjunctive treatment of periodontal disease. Melatonin level was significantly reduced in patients with periodontal diseases suggesting melatonin could be applied as a potential "perioceutics" treatment of periodontal diseases. This study aims to investigate the effects of melatonin receptor agonists (melatonin and ramelteon) on Porphyromonas gingivalis virulence and Porphyromonas gingivalis-derived lipopolysaccharide (Pg-LPS)-induced inflammation. METHODS: Effects of melatonin receptor agonists on Porphyromonas gingivalis planktonic cultures were determined by microplate dilution assays. Formation, reduction, and viability of Porphyromonas gingivalis biofilms were detected by crystal violet staining and MTT assays, respectively. Meanwhile, biofilms formation was also observed by confocal laser scanning microscopy (CLSM). The effects on gingipains and hemolytic activities of Porphyromonas gingivalis were evaluated using chromogenic peptides and sheep erythrocytes. The mRNA expression of virulence and iron/heme utilization was assessed using RT-PCR. In addition, cell viability of melatonin receptor agonists on human gingival fibroblasts (HGFs) was evaluated by MTT assays. After pretreatment of melatonin receptor agonists, HGFs were stimulated with Pg-LPS and then release of cytokines (IL-6 and lL-8) was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Melatonin and ramelteon did exhibit antimicrobial effects against planktonic culture. Importantly, they inhibited biofilm formation, reduced the established biofilms, and decreased biofilm viability of Porphyromonas gingivalis. Furthermore, they at sub-minimum inhibitory concentration (sub-MIC) concentrations markedly inhibited the proteinase activities of gingipains and hemolysis in a dose-dependent manner. They at sub-MIC concentrations significantly inhibited the mRNA expression of virulence factors (kgp, rgpA, rgpB, hagA, and ragA), while increasing the mRNA expression of ferritin (ftn) or hemolysin (hem). They did not show obvious cytotoxicity toward HGFs. They inhibited Pg-LPS-induced IL-6 and IL-8 secretion, which was reversed by luzindole, the melatonin receptor antagonist. CONCLUSION: Melatonin receptor agonists can inhibit planktonic and biofilm growth of Porphyromonas gingivalis by affecting the virulent properties, as well as Pg-LPS-induced inflammatory response. Our study provides new evidence that melatonin receptor agonists might be useful as novel "perioceutics" agents to prevent and treat Porphyromonas gingivalis-associated periodontal diseases.

Prevalence and quantification of the uncommon Archaea phylotype Thermoplasmata in chronic periodontitis.

OBJECTIVE: Chronic periodontitis is a chronic inflammatory disease of the periodontal tissues and is caused by invasion of certain types of bacteria and Archaea, with Methanobrevibacter oralis as the predominant archaeon. In this study, we investigated the prevalence and quantity of the newly discovered Archaea phylotype Thermoplasmata in patients with chronic periodontitis. METHODS: Subgingival plaque samples were obtained from 49 patients with chronic periodontitis and 45 periodontally healthy subjects. Qualitative analyses of Archaea and class Thermoplasmata were carried out by amplification of 16S rRNA genes in DNA extracts from plaque samples, and all the samples were quantitatively analyzed by real-time polymerase chain reaction (PCR). RESULTS: The prevalence of Archaea in patients with chronic periodontitis was 69.4% according to the conventional PCR results, but was 87.8% according to real-time PCR. In the control group, three samples were detected as positive, but none of these were confirmed in qualitative analyses. The prevalence of class Thermoplasmata was 18.4% by nested PCR and 24.5% by quantitative PCR in the chronic periodontitis group. The prevalence of Thermoplasmata was significantly lower than that of total Archaea. The relative abundances of Archaea and Thermoplasmata varied among samples. Thermoplasmata were not the predominant archaeons in the subgingival dental plaque. Among the clinical parameters of patients with periodontitis, probing depth was positively associated with Archaea detection. CONCLUSIONS: The existence of Archaea was correlated closely with the presence of chronic periodontitis. Thermoplasmata represented a minor archaeon in periodontal infection.

[Effect of different stress conditions on growth and biofilm formation capability of Enterococcus faecalis].

OBJECTIVE: To study the changes of growth and biofilm formation capability of Enterococcus faecalis (Ef) in different stress conditions. METHODS: The changes of growth of Ef in stress conditions were observed by measuring the A600 value with ultraviolet spectrophotometer. Ef was incubated on glass slide in stress conditions, biofilm formation capability of cells was investigated by colony-forming unit (CFU) counting of the culturable bacteria and fluorescence confocal laser scanning microscopy. RESULTS: Ef couldn't growth under the conditions of 2%, 5%NaClO, pH = 11 and 12, the A600 value was unchanged in 96 hours. But the growth curve changed at different levels in other stress conditions: under 1%NaClO, the A600 value peaked at 1.461 at 16 hour (the peaked level was 1.238 at 6 hours in control group) ; under 0,0.05%,0.15% glucose, it peaked at 0.645,0.890, 1.173, respectively, at 6 hour (it was maximized to 1.195 at 6 hours in control group); the A600 value peaked at 1.704 at 6 hours at pH = 9 and 1.225 at 10 hours at pH = 10 (the peak level was 1.732 at 6 hours at pH = 7) . Biofilm assay showed that Ef were able to form biofilm in these stress conditions except 5%NaClO and pH = 12. CONCLUSIONS: Ef could growth and form biofilms in energy starvation, low concentrations of sodium hypochlorite and weak alkaline stress.

Exploring the dynamic core microbiome of plaque microbiota during head-and-neck radiotherapy using pyrosequencing.

Radiotherapy is the primary treatment modality used for patients with head-and-neck cancers, but inevitably causes microorganism-related oral complications. This study aims to explore the dynamic core microbiome of oral microbiota in supragingival plaque during the course of head-and-neck radiotherapy. Eight subjects aged 26 to 70 were recruited. Dental plaque samples were collected (over seven sampling time points for each patient) before and during radiotherapy. The V1-V3 hypervariable regions of bacterial 16S rRNA genes were amplified, and the high-throughput pyrosequencing was performed. A total of 140 genera belonging to 13 phyla were found. Four phyla (Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria) and 11 genera (Streptococcus, Actinomyces, Veillonella, Capnocytophaga, Derxia, Neisseria, Rothia, Prevotella, Granulicatella, Luteococcus, and Gemella) were found in all subjects, supporting the concept of a core microbiome. Temporal variation of these major cores in relative abundance were observed, as well as a negative correlation between the number of OTUs and radiation dose. Moreover, an optimized conceptual framework was proposed for defining a dynamic core microbiome in extreme conditions such as radiotherapy. This study presents a theoretical foundation for exploring a core microbiome of communities from time series data, and may help predict community responses to perturbation as caused by exposure to ionizing radiation.

Characterization of oral bacterial diversity of irradiated patients by high-throughput sequencing.

The objective of this study was to investigate the compositional profiles and microbial shifts of oral microbiota during head-and-neck radiotherapy. Bioinformatic analysis based on 16S rRNA gene pyrosequencing was performed to assess the diversity and variation of oral microbiota of irradiated patients. Eight patients with head and neck cancers were involved in this study. For each patient, supragingival plaque samples were collected at seven time points before and during radiotherapy. A total of 147,232 qualified sequences were obtained through pyrosequencing and bioinformatic analysis, representing 3,460 species level operational taxonomic units (OTUs) and 140 genus level taxa. Temporal variations were observed across different time points and supported by cluster analysis based on weighted UniFrac metrics. Moreover, the low evenness of oral microbial communities in relative abundance was revealed by Lorenz curves. This study contributed to a better understanding of the detailed characterization of oral bacterial diversity of irradiated patients.

[The influence of different alkaline pH conditions on Enterococcus faecalis in planktonic and biofilm mode].

PURPOSE: To study the influence of different pH conditions on Enterococcus faecalis(E. faecalis) in planktonic and biofilm mode. METHODS: E. faecalis were prepared in planktonic and biofilm mode and cultured in TSB mediums 2 hours at pH 7,8,9,10,11 and 12. MTT assays were applied to evaluate the survival rate of bacterial cells in different pH value. SAS 6.12 software package was used for statistical analysis. RESULTS: Mild alkaline mediums (pH7-9) had no effect on cell vitality of E. faecalis and high alkaline condition (pH>10) led to significant declines of survival rate of cells. The biofilm cells of E. faecalis were more alkaline tolerant than corresponding planktonic cells. CONCLUSION: Biofilm formation is an important step in the development of alkaline tolerance of E. faecalis.

[Observation of genetic diversity in dental plaque of elder people with root caries].

PURPOSE: Bacterial community in dental plaque of elder people was analyzed to learn about the microhabitat composition and diversity. METHODS: Dental plaque samples were collected from 25 elders. PCR-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to evaluate the microbial diversity by displaying PCR-generated 16SrDNA fragments that migrate at different distances, reflecting the different sequence of fragment. SPSS12.0 software was used to analyze the variance of genotypes between different groups of bacteria. RESULTS: Genotypes of bacteria in dental plaques in the root caries group was significantly more than the other two groups. Crown caries group and caries-free group had no significant difference. CONCLUSIONS: The genetic diversity of the dental plaque microflora in the root caries group is significantly higher than coronal caries group and caries-free group.

[Analysis of community composition in dental plaque of elder people with root caries].

OBJECTIVE: To analyze the community in dental plaque of elder people with root caries. METHODS: Total DNAs were extracted from the root caries dental plaques of nine elders over 60 years of age. Polymerase chaid reaction-based denaturing gradient gel electrophoresis (PCR-DGGE) was used to analyze the microbial composition, DGGE bands were excised from the gels for sequencing and identification. RESULTS: The dominant genus in root caries dental plaque of elder people were: Acinetobacte [0.9% (1/114)], Actinobaculum [1.8% (2/114)], Actinomyces [15.8% (18/114)], Aggregatibacter [0.9% (1/114)], Capnocytophaga [14.0% (16/114)], Corynebacterium [0.9% (1/114)], Haemophilus [0.9% (1/114)], Mobiluncus [0.9% (1/114)], Naxibacter [0.9% (1/114)], Neisseriaceae [10.5% (12/114)], Porphyromonas [0.9% (1/114)], Prevotella [12.3% (14/114)], Selenomonas [6.1% (7/114)], Staphylococcus [1.8% (2/114)], Oralis streptococcus [6.1% (7/114)], Mutans streptococcu [7.9% (9/114)], Tannerella [0.9% (1/114)], Treponema [1.8% (2/114)], Veillonella [10.5% (12/114)] and two uncultured unknown genus [1.8% (2/114)]. Uncultred genotypes accounted for 19.30% of the total. Gram-positive bacteria genotype accounted for 31.6% (36/114), and Gram-negative bacteria genotype accounted for 66.7% (76/114). CONCLUSIONS: There were many bacteria genotypes in root caries dental plaque in the elderly, which were widely distributed. Gram-negative bacteria accounted for the majority. Genotype-specific pathogenic bacteria were not found.

[Quantitative evaluation of residual endodontic microorganisms after mechanical root canal preparation different chemical preparations by real-time PCR].

PURPOSE: To establish a quick, sensitive method for quantifying root canal flora and investigate the effects of different root canal preparations on the pathogenic bacteria at RNA level. METHODS: A total of 24 single-rooted teeth with chronic apical periodontitis were selected and prepared using 3% H2O2 combined with 1% NaClO, EDTA combined with 3% H(2)O(2),1% NaClO, respectively,the samples were taken before and after root canal preparation. After isolation of total RNA from the root canal samples, cDNA was synthesized by reverse transcription, and detected by real-time PCR. The data were analyzed with SAS 6.12 software package. RESULTS: The number of bacteria in the root canal reduced dramatically after mechanical preparation and irrigated using 3% H(2)O(2) and 1% NaClO(P<0.01). Further combined with EDTA, its effect was better than that of simply irrigated using 3% H(2)O(2) and 1% NaClO(P<0.05). CONCLUSIONS: Real-time PCR can be employed in the identification of bacteria flora in the root canal, both methods of root canal preparation can effectively reduce the number of bacteria flora.

[Identification and quantitative analysis of Archaea involved in periodontal disease].

OBJECTIVE: To make qualitative and quantitative analysis of Archaea in subgingival plaque sample and to investigate the relationship between periodontal disease and Archaea. METHODS: Subgingival plaque was collected from 23 patients with aggressive periodontitis, 29 with chronic periodontitis, 35 with plaque-induced gingivitis and 38 healthy controls. Qualitative and quantitative analysis of methanogenic archaea was performed by amplification of the 16S rRNA genes in the DNA extracted from the plaque samples. RESULTS: Archaea were found in 65% of aggressive periodontitis patients, 72% of chronic periodontitis, 26% of gingivitis and zero of healthy subjects. Quantitative analysis showed the average abundance of archaeal 16S rRNA gene in Archaea-positive patients was different among the three groups. The average 16S rRNA gene copy number from per microg wet plaque was 6.66 x 10(6) in aggressive periodontitis sufferers, 4.47 x 10(6) in chronic periodontitis and 1.78 x 10(6) in gingivitis groups. The prevalence of Archaea and the average Archaea 16S rRNA gene numbers in periodontitis groups were higher than those in gingivitis group (P < 0.05). CONCLUSIONS: This suggests that Archaea may be implicated as causative agents for periodontitis.

[Quantitive study of interleukin 1beta in periapical exudates of chronic periapical periodontitis in the course of root canal therapy].

PURPOSE: To evaluate the relationship between IL-1beta and clinical findings of chronic apical periodontitis and to explore the function of IL-1beta during the endodontic interappointment flare-ups. METHODS: Periapical exudates samples were obtained from 19 teeth suffering from endodontic flare-ups after root canal preparation and 20 teeth without any symptoms and signs at the second visit after root canal preparation. The levels of IL-1beta were determined by ELISA and the data was analyzed by SAS6.12 software package. RESULTS: Significantly higher levels of IL-1beta were found in periapical exudates from teeth suffering from endodontic flare-ups than that before canal preparation(P<0.001). The mean IL-1beta levels significantly decreased following the endodontic therapy if there were no symptom at the second visit (P<0.001). CONCLUSION: The level of IL-1beta in the exudates of root canals were related with the exist of infection which might take an active part in the occurrence of endodontic interappointment flare-up.

[Comparison of the sucrose dependent cell adherence of Streptococcus mutans isolates from caries-active and caries-free children].

PURPOSE: To study the sucrose dependent cell adhesive ability of Streptococcus mutans islolated from the caries-active and caries-free children. METHODS: 60 isolated Streptococcus mutans strains were selected and identified from the dental plaque of 10 caries-active children and 10 caries-free children (3 to 5 years), in which 39 strains were from caries-active group(dmfs>or=6) and 21 strains from caries-free group(dmfs=0). With the use of ultraviolet spectrophotometer, the sucrose dependent cell adherence to glass wall of the sucrose-containing testing tubes was analyzed. One-way ANOVA was used by SPSS12.0 software package to determine the statistical difference of the adhesive ability between the two groups. RESULTS: The average adhesive ratio of the Streptococcus mutans strains isolated from caries-active group was 55.49%+26.16% in the 1% sucrose-containing culture medium, while the average adhesive ratio of the Streptococcus mutans strains isolated from caries-free group was 27.01%+18.39%. The difference between the two groups was statistically significant (P<0.01). CONCLUSION: In the sucrose-containing circumstance, the sucrose dependent cell adhesive ability of the Streptococcus mutans isolated from the caries-active children was significantly higher than that from the caries-free children. This indicated that the adhesive ability may be related to the caries-causing tendency.

[Identification of P. gingivalis P. intermedia P. nigrescens by 16S rDNA and membrane microarray chip].

PURPOSE: The aim of this study is to identify three kinds of black-pigmented periodontal pathogens P. gingivalis Pg, P. intermedia Pi, P. nigrescens Pn by 16S rDNA and microarray. METHODS: A pair of universal primers which can amplify a section of conservative domain of bacterial 16S rDNA based on the sequences of 16S rDNA in Genebank were designed. Then the specific oligonucleotide probes for Pg Pi Pn based on the sequences of the conservative domain were constructed. Standard bacterial genomic DNAs were amplified using the designed universal primers by PCR, and labeled by digoxigenin at the same time, the products of PCR were hybridized with the microarray in which the specific probes were added. The results of hybridization were analysed. RESULTS: The results of hybridization showed that the specific probes of Pg Pi Pn on microarray reacted only with corresponding PCR products of Pg Pi Pn, not reacted with others. CONCLUSION: The method of 16S rDNA and membrane microarray could be useful to identify Pg Pi Pn, and had high specificity. It will be developed into a kind of clinical bacterial detective system.

[Preliminary study on adult odontoblast culture in situ].

PURPOSE: To establish an in situ culture model of adult odontoblasts. METHODS: Thirty intact and healthy third molars freshly prepared from 20-30 year old individuals were randomly divided into three groups. Each group had 10 molars. Group 1 was pulp tissue extraction group. Group 2 was serum-containing culture group. Group 3 was serum-free culture group. The root was dissected from the crown and the pulp was pulled out to make odontoblasts remaining in the crown. The odontoblasts were cultured in situ either in medium containing serum or serum-free medium for up to 7 days. The growth status of the cells was examined by light microscopy and cell morphology and distribution was analyzed by scanning electron microscopy. Cell viability was determined by trypan blue staining. RESULTS: After pulp removal at room temperature, odontoblasts remained in the wall of the pulp chamber, and kept viable and good morphology during the 7-day culture. CONCLUSION: We have successfully established an in situ culture model of adult primary odontoblasts in either serum-containing or serum-free medium.