Relationship between serum antibody titres to Porphyromonas gingivalis and hs-CRP levels as inflammatory markers of periodontitis.

OBJECTIVE: The present study was designed to investigate whether titres of antibody to two strains of Porphyromonas gingivalis, FDC381 and SU63, are associated with serum high-sensitivity C-reactive protein (hs-CRP) levels in Japanese periodontitis patients. DESIGN: Forty-nine patients with moderate to advanced periodontitis and 40 periodontally healthy control subjects were included in this study. hs-CRP levels and antibody titres to P. gingivalis were measured at baseline and reassessment 3-4 months after periodontal treatment in periodontitis patients as well as at the time of examination in the periodontally healthy subjects. Further, the effect of periodontal therapy, including surgical treatment and use of antibacterials on both markers, was analysed in patients. RESULTS: hs-CRP levels and antibody titres to P. gingivalis were higher in periodontitis patients than in control subjects, and they significantly decreased following periodontal treatment (p < 0.005). Also, a significant decrease in hs-CRP levels as a result of periodontal treatment was found in patients with hs-CRP levels >1 mgl(-1) at baseline (p < 0.005). Probing depth, clinical attachment level, and alveolar bone loss in patients were significantly associated with a higher antibody titre to both strains of P. gingivalis (p < 0.05), but were not related to hs-CRP levels. No relationship was observed between hs-CRP levels and tertiles as defined by titres of antibody to P. gingivalis strains FDC381 and SU63. CONCLUSIONS: Our data indicate that hs-CRP levels were independent of antibody titres to P. gingivalis in Japanese periodontitis patients.

Chronic oral infection with Porphyromonas gingivalis accelerates atheroma formation by shifting the lipid profile.

BACKGROUND: Recent studies have suggested that periodontal disease increases the risk of atherothrombotic disease. Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in the arteries. Although several studies have suggested that certain periodontopathic bacteria accelerate atherogenesis in apolipoprotein E-deficient mice, the mechanistic link between cholesterol accumulation and periodontal infection-induced inflammation is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: We orally infected C57BL/6 and C57BL/6.KOR-Apoe(shl) (B6.Apoeshl) mice with Porphyromonas gingivalis, which is a representative periodontopathic bacterium, and evaluated atherogenesis, gene expression in the aorta and liver and systemic inflammatory and lipid profiles in the blood. Furthermore, the effect of lipopolysaccharide (LPS) from P. gingivalis on cholesterol transport and the related gene expression was examined in peritoneal macrophages. Alveolar bone resorption and elevation of systemic inflammatory responses were induced in both strains. Despite early changes in the expression of key genes involved in cholesterol turnover, such as liver X receptor and ATP-binding cassette A1, serum lipid profiles did not change with short-term infection. Long-term infection was associated with a reduction in serum high-density lipoprotein (HDL) cholesterol but not with the development of atherosclerotic lesions in wild-type mice. In B6.Apoeshl mice, long-term infection resulted in the elevation of very low-density lipoprotein (VLDL), LDL and total cholesterols in addition to the reduction of HDL cholesterol. This shift in the lipid profile was concomitant with a significant increase in atherosclerotic lesions. Stimulation with P. gingivalis LPS induced the change of cholesterol transport via targeting the expression of LDL receptor-related genes and resulted in the disturbance of regulatory mechanisms of the cholesterol level in macrophages. CONCLUSIONS/SIGNIFICANCE: Periodontal infection itself does not cause atherosclerosis, but it accelerates it by inducing systemic inflammation and deteriorating lipid metabolism, particularly when underlying hyperlidemia or susceptibility to hyperlipidemia exists, and it may contribute to the development of coronary heart disease.

Analysis of immunostimulatory activity of Porphyromonas gingivalis fimbriae conferred by Toll-like receptor 2.

Bacterial fimbriae are an important pathogenic factor. It has been demonstrated that fimbrial protein encoded by fimA gene (FimA fimbriae) of Porphyromonas gingivalis not only contributes to the abilities of bacterial adhesion and invasion to host cells, but also strongly stimulates host innate immune responses. However, FimA fimbriae separated from P. gingivalis ATCC 33277 using a gentle procedure showed very weak proinflammatory activity compared with previous reports. Therefore, in the present study, biological characteristics of FimA fimbriae were further analyzed in terms of proinflammatory activity in macrophages. Macrophages differentiated from THP-1 cells were stimulated with native, heat-denatured, or either proteinase- or lipoprotein lipase-treated FimA fimbriae of P. gingivalis ATCC 33277. Stimulating activities of these FimA fimbriae were evaluated by TNF-alpha-inducing activity in the macrophages. To clarify the mode of action of FimA fimbriae, anti-Toll-like receptor (TLR) 2 blocking antibody was added prior to stimulation. Weak stimulatory activity of native FimA fimbriae was enhanced by heat treatment and low-dose proteinase K treatment. Higher dose of proteinase K treatment abrogated this up-regulation. The activity of treated FimA fimbriae was suppressed by anti-TLR2 antibody, and more substantially by lipoprotein lipase treatment. These results suggest that lipoproteins or lipopeptides associated with FimA fimbriae could at least in part account for signaling via TLR2 and subsequent TNF-alpha production in macrophages.

Elevated expression of IL-17 and IL-12 genes in chronic inflammatory periodontal disease.

BACKGROUND: A number of different theories have been postulated to explain the progression of gingivitis to periodontitis in the context of the Th1/Th2 paradigm. However, no consistent results have been obtained. Th17, a new T-cell subset producing IL-17, which is implicated in many aspect of inflammatory tissue destruction, overcomes many of the discrepant findings in the studies related to the Th1/Th2 hypothesis. We compared the gene expression profile of Th17-related molecules in gingivitis and periodontitis lesions showing distinct clinical entities. METHODS: Gingival tissue samples were obtained from 23 gingivitis and 24 periodontitis tissues. The gene expression was measured by using quantitative real-time PCR for IL-17A, IL-17F, CCR4, CCR6, IL-12 p35 and IL-23 p19. The difference of gene expressions between gingivitis and periodontitis was analyzed by Mann-Whitney U-test. Correlations between each gene expression were also analyzed. RESULTS: The expression level of IL-17A was higher than that of IL-17F and a significant difference in expression between gingivitis and periodontitis was observed only for IL-17A. CCR4 and CCR6 tended to be higher in periodontitis compared with gingivitis, although the differences were not statistically significant. Whereas the gene expression of IL-12 p35 was significantly higher in periodontitis compared with gingivitis, that of IL-23 p19 was not different between the two diseases. CONCLUSION: This study demonstrates the elevated expression of IL-17 and IL-12 in periodontitis, i.e., the tissue destruction form of periodontal diseases, as compared with gingivitis, and provides new insight into the T-cell mediated immunopathogenesis of periodontal disease.

[A case of interstitial pneumonia induced by S-1/irinotecan combination therapy].

A 67-year-old man with multiple liver metastases of colonic cancer was treated with combination therapy of S-1 and irinotecan (CPT-11): S-1 (120 mg/day) administered orally for 14 consecutive days followed by 14 days rest. CPT-11 (100 mg/m(2)) was given as a 2-hour infusion on day 1 and 15. The patient complained of high fever and subsequent exertional dyspnea in the middle of the second course of S-1/CPT-11 therapy. He was hospitalized with severe hypoxemia. CT scan showed extensive ground glass and consolidative changes in bilateral lungs. Steroid pulse therapy with oxygen therapy remarkably improved his symptoms, and abnormal findings on CT scan also resolved. Drug-induced pneumonia needs to be considered in the differential diagnosis when patients treated with S-1/CPT-11 combination therapy present high fever and dyspnea.

Comparison of fertility between intracytoplasmic sperm injection and in vitro fertilization with a partial zona pellucida incision by using a piezo-micromanipulator in cryopreserved inbred mouse spermatozoa.

Cryopreservation of mouse spermatozoa has been widely applied for maintenance of transgenic and knockout lines. However, the fertility of cryopreserved spermatozoa from some inbred strains such as C57BL/6 and BALB/c is extremely poor. We have recently reported that a partial zona-pellucida incision by piezo-micromanipulator (ZIP) significantly improves the fertilization rate and subsequent embryonic development after in vitro fertilization (IVF) using cryopreserved C57BL/6 transgenic mouse spermatozoa and that inbred C57BL/6 mice could be produced by intracytoplasmic sperm injection (ICSI). These findings prompted us to compare the efficiency of fertilization and subsequent embryonic development between ICSI and IVF with ZIP (ZIP/IVF) using cryopreserved spermatozoa. In conventional IVF, BALB/cA, C57BL/6J, and B6C3F1 cryopreserved spermatozoa fertilized 19%, 0%, and 51% of oocytes, respectively. The fertilization rates of manipulated oocytes by ICSI versus ZIP/IVF using cryopreserved BALB/cA spermatozoa were 52% versus 68%, cryopreserved C57BL/6J spermatozoa were 43% versus 63%, and cryopreserved B6C3F1 spermatozoa were 58% versus 82%, respectively. In these strains, fertilization rates for ZIP/IVF were significantly higher (P < 0.05) than for other techniques. However, embryonic development to term for oocytes fertilized by cryopreserved spermatozoa was not significantly different between ZIP/IVF and ICSI in C57BL/6J and B6C3F1. The overall efficiency of mouse production in ZIP/IVF was higher than for ICSI and conventional IVF in C57BL/6J and B6C3F1. Furthermore, ZIP/IVF required approximately 3.3 times less manipulation time than did ICSI. Our results indicate that ZIP is a useful assisted reproductive technique for IVF of ova by cryopreserved spermatozoa and improves production in some mouse strains.

Effect of partial incision of the zona pellucida by piezo-micromanipulator for in vitro fertilization using frozen-thawed mouse spermatozoa on the developmental rate of embryos transferred at the 2-cell stage.

Cryopreservation of mouse spermatozoa is widely used, although considerable strain differences in fertilization rates using frozen-thawed mouse spermatozoa have been described. The C57BL/6 mouse strain is a very widely used for establishment of transgenic mice, but the fertilization rate associated with the use of cryopreserved C57BL/6 spermatozoa is very low compared with rates for other inbred strains. We have recently solved this difficulty by in vitro fertilization (IVF) in combination with partial zona pellucida dissection (PZD). However, this technique requires culture of fertilized eggs with PZD in vitro up to morula or blastocyst stage before transfer into the uterus because blastomeres are lost after transfer into the oviduct because of the relatively large artificial slit in the zona pellucida. To overcome this problem, we performed a partial zona pellucida incision by using a piezo-micromanipulator (ZIP) for IVF with frozen-thawed mouse spermatozoa. The blunt end of the micropipette touched the surface of the zona pellucida of the oocytes, and piezo pulses were used to incise the zona pellucida while the pipette was moved along by the surface of zona pellucida. The length of the incision was pir/6 microm. When cumulus-free ZIP and PZD oocytes were inseminated with frozen-thawed genetically modified C57BL/6J spermatozoa, the fertilization rates of ZIP and PZD oocytes were 52% and 48%, respectively. After embryo transfer at the 2-cell stage, 18% and 2% of the transferred embryos with ZIP and PZD developed to term, respectively. This difference was significant (P < 0.05). When ZIP and PZD zygotes were cultured to blastocyst stage and subsequently transferred to uterine horns of recipient animals, the difference between ZIP and PZD zygotes for development rate to full term was not significant. Our results indicate that ZIP is an effective alternative technique for IVF using cryopreserved mouse spermatozoa and subsequent embryo transfer.