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Objective:To investigate the feasibility of periodontal mechanical therapy for chronic periodontitis and coronary heart disease patients with low dose of aspirin.Methods:Sixty nine chronic periodontitis patients with coronary heart disease were randomly selected as the experimental group (medication group,group A),the control group (withdrawal group,group B) including 20 chronic periodontitis patients with coronary artery disease,stopping the drug for one week and another control group with 50 chronic periodontitis patients (group C).The three groups were examined with pocket probing,and received supragingival scaling,subgingival scaling,and root planning.Local bleeding after operation was observed.In 30 minutes after periodontal mechanical treatment,there was still a need to take some hemostatic measures (containing the oxidized cellulose putting in the periodontal pocket,gauze oppressing,and suturing).Nd:YAG laser was used to stop bleeding 60 minutes after operation.Results:At baseline,there was no significant difference in the three groups,as to the plaque index (PLI),the probing depth (PD),and the attachment loss (AL).The bleeding index (BI)in group A was significantly higher than that in group C (P =0.024),higher than that in group B (P =0.088).The platelet maximum aggregation rate (Aggmax) was detected in some subjects.The average Aggmax value group A was 15.2％,which was much greater than that in group B (60.7％) and group C (62.5 ％).The three groups were all safe in the treatment of periodontal therapy.There were five cases of active bleeding in group A,one case in group B and one case in group C in 30 minutes after operation.In 60 minutes after operation,there was one case of bleeding actively in group A.Nd:YAG laser was used to stop bleeding successfully.Conclusion:The chronic periodontitis and coronary heart disease patients with long-term oral administration of low dose of aspirin can be safely treated with periodontal mechanical treatment,and the effect of local hemostasis is positive without stopping the drug.
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Objective To evaluate the effect ofT-cell immunoglobulin and mucin domain-3 (TIM-3) on TRP-2180-188 peptide-stimulated murine spleen lymphocytes co-cultured with B16F10 murine melanoma cells.Methods A recombinant plasmid pFUSE-TIM-3-mIgG2Aae1-Fc2 encoding TIM-3 was constructed.Then,the recombinant plasmid and an empty plasmid pFUSE-mIgG2Aae1-Fc2 were transfected into human 293T epithelial cells followed by 48-hour culture for the preparation of supernatants containing TIM-3 and Ig-tail respectively.C57BL/6 mice were immunized with the TRP-2180-188 peptide vaccine for 4 sessions.One week after the last vaccination,C57BL/6 mice were sacrificed,and spleen lymphocytes were collected and then cultured with the TRP-21180-188 peptide and interleukin-2 (IL-2) for 5 days,with lymphocytes untreated with the TRP-2180-188 peptide or IL-2 serving as the control group.Mitomycin-treated B16F10 murine melanoma cells and TRP-2180-188 peptide-stimulated lymphocytes were co-cultured with the presence of supernatants of 293T cells that had been cultured for 48 hours (blank control group),TIM-3-containing supernatants (TIM-3 group) and Ig-tail-containing supernatants (negative control group) separately.After 24 and 48 hours of co-culture,cell counting kit-8 (CCK-8) assay was performed to estimate the proliferative activity of lymphocytes,enzyme-linked immunosorbent assay (ELISA) to determine the supernatant levels of interferon (INF)-γ and tumor necrosis factor (TNF)-α,flow cytometry to determine the percentage of CD8 + T cells in the co-culture system.Results Enzyme digestion and sequence analysis showed that the TIM-3 gene was successfully inserted into the eukaryotic expression plasmid.After 48-hour culture,TIM-3 and Ig-tail expressions were detected in the supernatants of 293T cells transfected with the recombinant plasmid and empty plasmid respectively.As CCK-8 assay showed,the proliferative activity of lymphocytes was significantly lower in the TIM-3 group than in the blank control group and negative control group after 24-and 48-hour culture (78.06％ ± 6.37％ vs.100.00％ ± 10.42％ and 108.70％ ± 9.90％ at 24 hours,42.93％ ± 5.93％ vs.100.00％ ± 6.24％ and 168.00％ ± 2.98％at 48 hours,all P ＜ 0.05),so was the ratio of cellular proliferative activity at 48 hours to that at 24 hours (all P ＜ 0.05).Compared with the blank control group and negative control group,the TIM-3 group showed significantly decreased supernatant levels of IFN-γ and TNF-α after 24-hour (IFN-γ:192.96 γ 5.05 ng/L vs.216.44 ± 7.85 ng/L and 223.67 ±7.79 ng/L,both P＜ 0.05;TNF-α:58.43 ± 0.26 ng/L vs.26.43 ± 0.01 ng/L and 86.85 ± 1.12 ng/L,both P＜ 0.05) and 48-hour culture (IFN-γ:54.95 ± 0.57 ng/L vs.230.06 ± 4.23 ng/L and 167.24 ± 3.33 ng/L,both P ＜ 0.05;TNF-α:30.23 ±0.26 ng/L vs.26.84 ± 0.20 ng/L and 45.34 ± 0.22 ng/L,both P ＜ 0.05).In addition,the median percentage of CD8+ T cells was significantly increased in the TIM-3 group compared with the blank control group and negative control group after 24-and 48-hour culture (3.30％ vs.0.421％ and 2.22％ at 24 hours,4.06％ vs.0.577％ and 0.691％ at 48 hours,all P＜ 0.05).Conclusion TIM-3 in vitro can suppress the proliferative activity of and secretion of IFN-γand TNF-α by lymphocytes,but increase the percentage of CD8 + T cells in the co-culture system of TRP-2180-188 peptide-stimulated lymphocytes and B16F10 cells.
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Objective To evaluate in vitro effects of specific small interfering RNA (siRNA)-silencing of the casitas B-lineage lymphoma b (Cbl-b)gene on immunocompetence of primary murine lymphocytes. Methods Spleens were resected from C57BL/6 mice, and splenic lymphocytes were sterily isolated and cultured in vitro. These lymphocytes were divided into 3 groups: silence group transfected with a Cbl-b-specific siRNA using the EntransterTM-R 4000 reagent, negative control group transfected with a negative control siRNA using the EntransterTM-R4000 reagent, blank control group receiving no treatment. After additional culture for 72 hours, ELISA was performed to measure levels of interferon γ(IFN-γ)and tumor necrosis factor α (TNF-α)in culture supernatants of lymphocytes. In addition, the Cbl-b gene-silenced lymphocytes were co-cultured with B16F10 melanoma cells to evaluate their immunocytotoxic effects on melanoma cells. Results Splenic lymphocytes were successfully isolated from C57BL/6 mice and cultured in vitro, and the Cbl-b-specific siRNA was also successfully transfected into the primary murine lymphocytes and effectively down-regulated the expression of Cbl-b gene in them. Compared with the negative control group and blank control group, the silence group showed significantly increased supernatant levels of IFN-γ and TNF-α(all P < 0.05). The immunocytotoxic effect of lymphocytes on melanoma cells was significantly stronger in the silence group than in the negative control group. Conclusion Cbl-b gene silencing can promote secretion of IFN-γ and TNF-α by murine lymphocytes, and enhance their immunocytotoxic effects on B16F10 melanoma cells in vitro.
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Objective To construct a eukaryotic expression plasmid vector encoding Cbl-b gene-specific short hairpin RNAs (shRNAs),and to evaluate its interference effect,so as to lay a foundation for further study on the role of Cbl-b in the immunotherapy of malignant melanoma.Methods According to the sequence of Cbl-b cDNA,4 pairs of shRNAs targeting the Cbl-b gene were designed and synthesized,and then inserted into the plasmid PGPU6/GFP/Neo to construct recombinant plasmids.After identification by DNA sequencing,the 4 shRNA expression vectors were cotransfected into 293T cells with the Cbl-b gene eukarytic expresson plasmid,respectively.The knockdown efficiency of these shRNA expression plasmids on Cbl-b expression was evaluated by real-time (RT) fluorescence-based quantitative PCR and Western blot at 48 hours aftert transfection.Results Sequencing analysis revealed that all the 4 pairs of shRNAs were successfully inserted into the eukarytic expression vector PGPU6/GFP/Neo.As RT-PCR and Western blot showed,all the 4 shRNA-expressing vectors could downregulate Cbl-b expession,and the NO.1 shRNA-expressing vector displayed the strongest interference effect(P ＜ 0.05).Conclusions A eukaryotic expression plasmid vector was successfully constructed for Cbl-b gene-specific shRNAs,and the most effective shRNA was selected in this study.
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<p><b>BACKGROUND</b>The occurrence and development of aortic aneurysm (AA) are associated with infection. Some researchers have detected the DNA of periodontal pathogens in AA samples in certain populations. However, it has not been done in Chinese population. The objective of this study was to evaluate the prevalence of periodontal pathogens in oral tissue samples and aneurysm samples of AA patients.</p><p><b>METHODS</b>Eighty-nine subjects with AA and 59 subjects without AA were examined. Periodontal clinical parameters were evaluated. Unstimulated saliva and subgingival plaque samples were collected from all subjects. Twenty-six dissected AA samples were obtained. Evidence of eight periodontal pathogens including Porphyromonas gingivalis (Pg), Actinobacillus actinomycetemcomitans (Aa), Prevotella intermedia (Pi), Tannerella forsythensis (Tf), Treponema denticola (Td), Campylobacter rectus (Cr), Fusobacterium nucleatum (Fn), and Prevotella nigrescens (Pn) was ascertained in all samples by 16S rRNA-based polymerase chain reaction (PCR) assay.</p><p><b>RESULTS</b>The periodontal indexes including plaque index (PLI), probing depth (PD), bleeding index (BI), and clinical attachment loss (CAL), of the six Ramfjord index teeth were significantly higher in the AA group than those in the control group (P < 0.01). Eight periodontal pathogens in subgingival plaque samples were more frequently detected in the AA group than in control group. The difference in prevalence between the groups was significant for six (out of eight) periodontal pathogens assayed (Pg, Pi, Fn, Pn, Tf, and Td, P < 0.01). Additionally, all eight periodontal pathogens were more frequently detected in saliva samples of the AA group than in those of the control group, again with six (out of eight) (Pg, Pi, Fn, Cr, Tf, and Td) displaying significant differences in prevalence between the two groups (P < 0.01). Out of 26 aneurysm samples examined, Pg, Pi, Fn, Cr and Tf were detected in 6 (23.1%), 2 (7.7%), 3 (11.5%), 1 (3.8%), 2 (7.7%), respectively, and Aa, Pn, and Td were not detected in dissected aneurysm samples.</p><p><b>CONCLUSION</b>Results of this study suggested that periodontal infection is associated with the occurrence of AA.</p>