Washing- and Separation-Free Electrochemical Detection of Porphyromonas gingivalis in Saliva for Initial Diagnosis of Periodontitis.

Indirect detection of Porphyromonas gingivalis in saliva, based on proteolytic cleavage by an Arg-specific gingipain (Arg-gingipain), has traditionally been used for simple, initial diagnosis of periodontitis. To accurately detect P. gingivalis using a point-of-care format, development of a simple biosensor that can measure the exact concentration of P. gingivalis is required. However, electrochemical detection in saliva is challenging due to the presence of various interfering electroactive species in different concentrations. Here, we report a washing- and separation-free electrochemical biosensor for sensitive detection of P. gingivalis in saliva. Glycine-proline-arginine conjugated with 4-aminophenol (AP) was used as an electrochemical substrate for a trypsin-like Arg-gingipain, and glycylglycine was used to increase the Arg-gingipain activity. The electrochemical signal of AP was increased using electrochemical-chemical (EC) redox cycling involving an electrode, AP, and tris(2-carboxyethyl)phosphine, and the electrochemical charge signal was corrected using the initial charge obtained before a 15 min incubation period. The EC redox cycling combined with the matrix-corrected signal facilitated a high and reproducible signal without requiring washing and separation steps. The proteolytic cleavage of the electrochemical substrate was specific to P. gingivalis. The calculated detection limit for P. gingivalis in artificial saliva was 5 × 105 colony-forming units/mL, and the concentration of P. gingivalis in human saliva could be measured. The developed biosensor can be used as an initial diagnosis method to distinguish between healthy people and patients with periodontal diseases.

Prognostic role of high cathepsin D expression in breast cancer: a systematic review and meta-analysis.

BACKGROUND: High cathepsin D has been associated with poor prognosis in breast cancer; however, the results of many studies are controversial. Here, we assessed the association between high cathepsin D levels and worse breast cancer prognosis by conducting a meta-analysis. METHODS: A comprehensive search strategy was used to search relevant literature in PUBMED and EMBASE by September 2018. The meta-analysis was performed in Review Manager 5.3 using hazard ratios (HRs) with 95% confidence intervals (CIs). RESULTS: A total of 15,355 breast cancer patients from 26 eligible studies were included in this meta-analysis. Significant associations between elevated high cathepsin D and poor overall survival (OS) (HR = 1.61, 95% CI: 1.35-1.92, p < 0.0001) and disease-free survival (DFS) (HR = 1.52, 95% CI: 1.31-2.18, p < 0.001) were observed. In the subgroup analysis for DFS, high cathepsin D was significantly associated with poor prognosis in node-positive patients (HR = 1.38, 95% CI: 1.25-1.71, p < 0.00001), node-negative patients (HR = 1.78, 95% CI: 1.39-2.27, p < 0.0001), early stage patients (HR = 1.73, 95% CI: 1.34-2.23, p < 0.0001), and treated with chemotherapy patients (HR = 1.60, 95% CI: 1.21-2.12, p < 0.001). Interestingly, patients treated with tamoxifen had a low risk of relapse when their cathepsin D levels were high (HR = 0.71, 95% CI: 0.52-0.98, p = 0.04) and a high risk of relapse when their cathepsin D levels were low (HR = 1.50, 95% CI: 1.22-1.85, p = 0.0001). CONCLUSIONS: Our meta-analysis suggests that high expression levels of cathepsin D are associated with a poor prognosis in breast cancer. Based on our subgroup analysis, we believe that cathepsin D can act as a marker for poor breast cancer prognosis and also as a therapeutic target for breast cancer.

Role of Aggregatibacter actinomycetemcomitans-induced autophagy in inflammatory response.

BACKGROUND: Aggressive periodontitis is characterized by the early-onset and rapid progression of periodontal destruction and is closely associated with Aggregatibacter actinomycetemcomitans. Autophagy is a conserved process that is critical for removing damaged proteins, organelles, and even intracellular pathogens. Therefore, this study examined whether A. actinomycetemcomitans induces autophagy. In addition, the relationship among autophagy, bacterial internalization, and inflammatory molecules in periodontal aggressive inflammation was analyzed. METHODS: The expression of autophagy-related proteins in human gingival tissue and THP-1 cells was assessed by Western blot analysis. The formation of light chain 3 (LC3) puncta was examined by confocal microscopy. The degree of bacterial internalization into the cells was determined by the viable cell count. Phagocytosis and reactive oxygen species (ROS) production were measured using confocal microscopy and flow cytometry. RESULTS: When macrophages were infected with live A. actinomycetemcomitans, the autophagy influx was activated by the increase in LC3-II, autophagy-related gene 5/12, and Beclin-1 expression through the Toll-like receptors and extracellular signal-regulated kinase signaling pathways. The inhibition of A. actinomycetemcomitans-induced autophagy suppressed bacterial internalization via phagocytosis into the macrophages and increased interleukin (IL)-1ß production. Moreover, treatment with an ROS inhibitor inhibited these enhanced inflammatory responses. CONCLUSIONS: A. actinomycetemcomitans-induced autophagy promotes bacterial internalization by phagocytosis, which restricts the excessive inflammatory response by downregulating IL-1ß and ROS production in macrophages. Thus, A. actinomycetemcomitans-induced autophagy and its role in regulating the inflammatory response may play an important role in the aggressive periodontal inflammatory process, and be a target for the development of new periodontal therapies.

Catechin ameliorates Porphyromonas gingivalis-induced inflammation via the regulation of TLR2/4 and inflammasome signaling.

BACKGROUND: Porphyromonas gingivalis is a major periodontopathogen found in patients with chronic periodontitis that can lead to alveolar bone or tooth loss. Interleukin-1ß (IL-1ß), a proinflammatory cytokine, is most relevant to the pathogenesis of periodontitis. Catechin is one of the main polyphenol compounds found in green tea and possesses a range of health benefits. This study examined the anti-inflammatory effects of catechin in THP-1-derived macrophages infected with P. gingivalis as well as its effects on P. gingivalis-induced periodontitis in a mouse model. METHODS: The cytokine levels and relevant protein expression in THP-1 cells were measured using an enzyme-linked immunosorbent assay and Western blot analysis, respectively. An apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) pyroptosome formation was measured by confocal laser scanning microscopy. Micro-computed tomography was used to determine the level of bone loss induced by a P. gingivalis oral infection. RESULTS: Catechin attenuated the production of IL-1ß by inhibiting pro-IL-1ß expression via the downregulation of nuclear factor-κB, p38 mitogen-activated protein kinase, and Toll-like receptor signaling. In addition, catechin inhibited the activation of inflammasomes induced by P. gingivalis, but did not affect the growth of P. gingivalis. Catechin reduced the level of alveolar bone loss in a P. gingivalis-induced periodontitis mouse model. CONCLUSION: Catechin possesses anti-inflammatory properties by reducing the level of IL-1ß production, suggesting that it can potentially be used for the prevention and treatment of periodontal inflammation caused by P. gingivalis.

Streptococcus mutans activates the AIM2, NLRP3 and NLRC4 inflammasomes in human THP-1 macrophages.

Streptococcus mutans (S. mutans), a major aetiologic agent of dental caries, is involved in systemic diseases, such as bacterial endocarditis, if it enters the bloodstream through temporary bacteraemia. Interleukin (IL)-1ß, a proinflammatory cytokine, is related to the host defences against pathogens, and its synthesis, maturation, and secretion are tightly regulated by the activation of the inflammasome, an inflammatory signalling complex. This study examined the signalling mechanism of IL-1ß secretion and the inflammasome pathway induced by S. mutans to explain the molecular mechanism through which systemic infection by oral streptococci can occur. After infection of THP-1 cells with S. mutans, the expression of inflammasome components was detected using various methods. S. mutans induced IL-1ß secretion via caspase-1 activation, and S. mutans-induced IL-1ß secretion required absent in melanoma (AIM2), NLR family pyrin domain-containing 3 (NLRP3) and NLR family CARD domain-containing 4 (NLRC4) inflammasome activation. In particular, the S. mutans-induced NLRP3 inflammasome was mediated by adenosine triphosphate (ATP) release, potassium depletion and lysosomal damage. Our study provides novel insight into the innate immune response against S. mutans infection.

Trans-cinnamic aldehyde inhibits Aggregatibacter actinomycetemcomitans-induced inflammation in THP-1-derived macrophages via autophagy activation.

BACKGROUND: Inflammation is an essential response against bacterial infection as a host defense mechanism, which can lead to tissue damage. Aggregatibacter actinomycetemcomitans (Aa) is major pathogen for aggressive periodontitis characterized by rapid destruction of periodontal tissue surrounding teeth. Trans-cinnamic aldehyde is a key bioactive compound of the cinnamon extracts, which has anti-inflammatory, antioxidant, antipyretic, antimicrobial, and anti-cancer properties. The objective of the present study was to investigate the anti-inflammatory effect of trans-cinnamic aldehyde against Aa infection in human THP-1 derived macrophages and on Aa-induced periodontitis in mice. METHODS: THP-1 cells were differentiated with phorbol 12-mystristate 13-acetate and were infected with live Aa. Trans-cinnamic aldehyde was pretreated 30 minutes before the bacterial infection. Cytokine production was determined by enzyme-linked immunosorbent assay (ELISA) and protein expressions were detected by Western blot analysis. Autophagosome formation was detected by Cyto-ID. Viable cell count was carried out to determine bacterial adhesion, internalization, and intracellular survival. Experimental periodontitis was induced by inoculating Aa orally to mice, and microcomputed tomography was used to evaluate bone loss. RESULTS: Pretreatment of trans-cinnamic aldehyde significantly inhibited Aa-stimulated release of tumor necrosis factor-α and interleukin (IL)-1ß. Pretreatment of trans-cinnamic aldehyde inhibited Aa-induced expression of TLR signaling pathway as well as the phosphorylation of JNK, p38, and nuclear factor (NF)-κB. Also, trans-cinnamic aldehyde treatment downregulated the expression of pro-IL-1ß, caspase-1, and inflammasome components. Trans-cinnamic aldehyde treatment significantly decreased intracellular survival of Aa. Moreover, the autophagosome formation and the expressions of autophagy markers including Beclin-1, ATG5, and LC3 were increased. Finally, trans-cinnamic aldehyde significantly inhibited bone loss in Aa-induced mouse periodontitis. CONCLUSIONS: Trans-cinnamic aldehyde inhibited Aa-stimulated expression of inflammatory responses and inhibited intracellular bacterial survival via autophagy activation. These results suggest that trans-cinnamic aldehyde may serve as an anti-inflammatory agent for aggressive periodontitis.

Elevated MicroRNA-128 in Periodontitis Mitigates Tumor Necrosis Factor-α Response via p38 Signaling Pathway in Macrophages.

BACKGROUND: Periodontitis is a chronic inflammatory disease resulting from an inflammatory response to subgingival plaque bacteria, including Porphyromonas gingivalis. MicroRNA (miRNA) is a current focus in regulating the inflammatory processes. In this study, the inflammatory miRNA expression in gingival tissues of patients with periodontitis and of healthy individuals is compared, and its role in regulating the inflammatory response is examined. METHODS: Gingival tissues from patients with periodontitis and healthy individuals were collected for miRNA microarray. THP-1 and CA9-22 cells were challenged with P. gingivalis, and miRNA expression was determined by real-time polymerase chain reaction. Target genes for miRNA were predicted using TargetScanHuman database, and miRNA gene expressions were reviewed using public databases. For the functional study, THP-1 cells were transfected with a miRNA-128 mimic, and target gene expression was compared with THP-1 cells challenged with P. gingivalis. For the tolerance test, THP-1 cells transfected with miRNA-128 mimic were treated with phorbol 12-myristate 13-acetate (PMA) or paraformaldehyde (PFA)-fixed Escherichia coli. Tumor necrosis factor (TNF)-α production was determined by enzyme-linked immunosorbent assay, and mitogen-activated protein kinase (MAPK) protein phosphorylation was determined by Western blot. RESULTS: Gingival tissues from patients with periodontitis showed increased expression of miRNA-128, miRNA-34a, and miRNA-381 and decreased expression of miRNA-15b, miRNA-211, miRNA-372, and miRNA-656. THP-1 cells and CA9-22 cells challenged with P. gingivalis showed increased miRNA-128 expression. Among the predicted miRNA-128 target genes, several genes that are involved in MAPK signaling pathway showed similar gene expression pattern between P. gingivalis challenge and miRNA-128 mimic transfection. In THP-1 cells transfected with miRNA-128 mimic, TNF-α production was lower, and phosphorylation of p38 was inhibited when challenged with PMA or PFA-fixed E. coli. CONCLUSION: miRNA-128 may be involved in mitigating the inflammatory response induced by P. gingivalis in periodontitis.

Aloin Inhibits Interleukin (IL)-1ß-Stimulated IL-8 Production in KB Cells.

BACKGROUND: Interleukin (IL)-1ß, which is elevated in oral diseases including gingivitis, stimulates epithelial cells to produce IL-8 and perpetuate inflammatory responses. This study investigates stimulatory effects of salivary IL-1ß in IL-8 production and determines if aloin inhibits IL-1ß-stimulated IL-8 production in epithelial cells. METHODS: Saliva was collected from volunteers to determine IL-1ß and IL-8 levels. Samples from volunteers were divided into two groups: those with low and those with high IL-1ß levels. KB cells were stimulated with IL-1ß or saliva with or without IL-1 receptor agonist or specific mitogen-activated protein kinase (MAPK) inhibitors. IL-8 production was measured by enzyme-linked immunosorbent assay (ELISA). MAPK protein expression involved in IL-1ß-induced IL-8 secretion was detected by Western blot. KB cells were pretreated with aloin, and its effect on IL-1ß-induced IL-8 production was examined by ELISA and Western blot analysis. RESULTS: Saliva with high IL-1ß strongly stimulated IL-8 production in KB cells, and IL-1 receptor agonist significantly inhibited IL-8 production. Low IL-1ß-containing saliva did not increase IL-8 production. IL-1ß treatment of KB cells induced activation of MAPK signaling molecules as well as nuclear factor-kappa B. IL-1ß-induced IL-8 production was decreased by p38 and extracellular signal-regulated kinase (ERK) inhibitor treatment. Aloin pretreatment inhibited IL-1ß-induced IL-8 production in a dose-dependent manner and inhibited activation of the p38 and ERK signaling pathway. Finally, aloin pretreatment also inhibited saliva-induced IL-8 production. CONCLUSIONS: Results indicated that IL-1ß in saliva stimulates epithelial cells to produce IL-8 and that aloin effectively inhibits salivary IL-1ß-induced IL-8 production by mitigating the p38 and ERK pathway. Therefore, aloin may be a good candidate for modulating oral inflammatory diseases.

Aggregatibacter actinomycetemcomitans-Induced AIM2 Inflammasome Activation Is Suppressed by Xylitol in Differentiated THP-1 Macrophages.

BACKGROUND: Aggressive periodontitis is characterized by rapid destruction of periodontal tissue caused by Aggregatibacter actinomycetemcomitans. Interleukin (IL)-1ß is a proinflammatory cytokine, and its production is tightly regulated by inflammasome activation. Xylitol, an anticaries agent, is anti-inflammatory, but its effect on inflammasome activation has not been researched. This study investigates the effect of xylitol on inflammasome activation induced by A. actinomycetemcomitans. METHODS: The differentiated THP-1 macrophages were stimulated by A. actinomycetemcomitans with or without xylitol and the expressions of IL-1ß and inflammasome components were detected by real time PCR, ELISA, confocal microscopy and Immunoblot analysis. The effects of xylitol on the adhesion and invasion of A. actinomycetemcomitans to cells were measured by viable cell count. RESULTS: A. actinomycetemcomitans increased pro IL-1ß synthesis and IL-1ß secretion in a multiplicity of infection- and time-dependent manner. A. actinomycetemcomitans also stimulated caspase-1 activation. Among inflammasome components, apoptosis-associated speck-like protein containing a CARD (ASC) and absent in melanoma 2 (AIM2) proteins were upregulated by A. actinomycetemcomitans infection. When cells were pretreated with xylitol, proIL-1ß and IL-1ß production by A. actinomycetemcomitans infection was significantly decreased. Xylitol also inhibited ASC and AIM2 proteins and formation of ASC puncta. Furthermore, xylitol suppressed internalization of A. actinomycetemcomitans into differentiated THP-1 macrophages without affecting viability of A. actinomycetemcomitans within cells. CONCLUSIONS: A. actinomycetemcomitans induced IL-1ß production and AIM2 inflammasome activation. Xylitol inhibited these effects, possibly by suppressing internalization of A. actinomycetemcomitans into cells. Thus, this study proposes a mechanism for IL-1ß production via inflammasome activation and discusses a possible use for xylitol in periodontal inflammation caused by A. actinomycetemcomitans.

Xylitol induces cell death in lung cancer A549 cells by autophagy.

Xylitol is a widely used anti-caries agent that has anti-inflammatory effects. We have evaluated the potential of xylitol in cancer treatment. It's effects on cell proliferation and cytotoxicity were measured by MTT assay and LDH assay. Cell morphology and autophagy were examined by immunostaining and immunoblotting. Xylitol inhibited cell proliferation in a dose-dependent manner in these cancer cells: A549, Caki, NCI-H23, HCT-15, HL-60, K562, and SK MEL-2. The IC50 of xylitol in human gingival fibroblast cells was higher than in cancer cells, indicating that it is more specific for cancer cells. Moreover, xylitol induced autophagy in A549 cells that was inhibited by 3-methyladenine, an autophagy inhibitor. These results indicate that xylitol has potential in therapy against lung cancer by inhibiting cell proliferation and inducing autophagy of A549 cells.

Xylitol, an anticaries agent, exhibits potent inhibition of inflammatory responses in human THP-1-derived macrophages infected with Porphyromonas gingivalis.

BACKGROUND: Xylitol is a well-known anticaries agent and has been used for the prevention and treatment of dental caries. In this study, the anti-inflammatory effects of xylitol are evaluated for possible use in the prevention and treatment of periodontal infections. METHODS: Cytokine expression was stimulated in THP-1 (human monocyte cell line)-derived macrophages by live Porphyromonas gingivalis, and enzyme-linked immunosorbent assay and a commercial multiplex assay kit were used to determine the effects of xylitol on live P. gingivalis-induced production of cytokine. The effects of xylitol on phagocytosis and the production of nitric oxide were determined using phagocytosis assay, viable cell count, and Griess reagent. The effects of xylitol on P. gingivalis adhesion were determined by immunostaining, and costimulatory molecule expression was examined by flow cytometry. RESULTS: Live P. gingivalis infection increased the production of representative proinflammatory cytokines, such as tumor necrosis factor-α and interleukin (IL)-1ß, in a multiplicity of infection- and time-dependent manner. Live P. gingivalis also enhanced the release of cytokines and chemokines, such as IL-12 p40, eotaxin, interferon γ-induced protein 10, monocyte chemotactic protein-1, and macrophage inflammatory protein-1. The pretreatment of xylitol significantly inhibited the P. gingivalis-induced cytokines production and nitric oxide production. In addition, xylitol inhibited the attachment of live P. gingivalis on THP-1-derived macrophages. Furthermore, xylitol exerted antiphagocytic activity against both Escherichia coli and P. gingivalis. CONCLUSION: These findings suggest that xylitol acts as an anti-inflammatory agent in THP-1-derived macrophages infected with live P. gingivalis, which supports its use in periodontitis.

Activation of NLRP3 and AIM2 inflammasomes by Porphyromonas gingivalis infection.

Porphyromonas gingivalis, a major periodontopathogen, is involved in the pathogenesis of periodontitis. Interleukin-1ß (IL-1ß), a proinflammatory cytokine, regulates innate immune responses and is critical for the host defense against bacterial infection. However, excessive IL-1ß is linked to periodontal destruction. IL-1ß synthesis, maturation, and secretion are tightly regulated by Toll-like receptor (TLR) signaling and inflammasome activation. We found much higher levels of inflammasome components in the gingival tissues from patients with chronic periodontitis than in those from healthy controls. To investigate the molecular mechanisms by which P. gingivalis infection causes IL-1ß secretion, we examined the characteristics of P. gingivalis-induced signaling in differentiated THP-1 cells. We found that P. gingivalis induces IL-1ß secretion and inflammatory cell death via caspase-1 activation. We also found that P. gingivalis-induced IL-1ß secretion and pyroptic cell death required both NLRP3 and AIM2 inflammasome activation. The activation of the NLRP3 inflammasome was mediated by ATP release, the P2X7 receptor, and lysosomal damage. In addition, we found that the priming signal via TLR2 and TLR4 activation precedes P. gingivalis-induced IL-1ß release. Our study provides novel insight into the innate immune response against P. gingivalis infection which could potentially be used for the prevention and therapy of periodontitis.

Caffeic acid-conjugated chitosan derivatives and their anti-tumor activity.

In this study, we synthesized caffeic acid (CFA)-conjugated chitosan (ChitoCFA) as an anti-cancer compound. CFA was conjugated to the amine groups of chitosan (ChitoCFA) and its chemical composition was confirmed using (1)H nuclear magnetic resonance spectra, which indicates that specific peaks of CFA was observed at ChitoCFA conjugates. The anti-cancer effects of CFA and ChitoCFA were studied using CT26 colorectal carcinoma cells. In this cytotoxicity study, CFA and ChitoCFA revealed a dose-dependent decrease of cell viability while chitosan had lower cytotoxicity against tumor cells. CFA and ChitoCFA also proved an anti-proliferative effect against tumor cells. In comparison with CFA, ChitoCFA may accelerate an apoptosis of tumor cells. Furthermore, ChitoCFA demonstrated good anti-invasive efficacy at Matrigel(®) invasion assay against tumor cells. We suggest that ChitoCFA is a promising candidate as an anti-cancer compound.

Protective mechanism of curcumin against Vibrio vulnificus infection.

Curcumin, a natural polyphenolic flavonoid extracted from the rhizome of Curcuma longa L., has many beneficial biological activities. However, there are relatively few reports of the effects of curcumin on pathogen infections. This study examined the effect of curcumin on a Vibrio vulnificus infection. The cytotoxicity of V. vulnificus to HeLa cells was significantly inhibited by curcumin (at 10 or 30 µM). To further examine the inhibitory mechanism of curcumin against V. vulnificus-mediated cytotoxicity, the level of bacterial growth, bacterial motility, cell adhesion, RTX toxin expression and host cell reactions were evaluated. Curcumin inhibited V. vulnificus growth in HI broth. Curcumin inhibited both bacterial adhesion and RTX toxin binding to the host cells, which can be considered the major protective mechanisms for the decrease in V. vulnificus cytotoxicity. Curcumin also inhibited host cell rounding and actin aggregation, which are the early features of cell death caused by V. vulnificus. In addition, curcumin decreased the V. vulnificus-induced NF-κB translocation in HeLa cells. Finally, curcumin protected mice from V. vulnificus-induced septicemia. In conclusion, curcumin may be an alternative antimicrobial agent against fatal bacterial infections.

Relationship between a hopeful attitude and cellular immunity in patients with breast cancer.

OBJECTIVES: To evaluate the relationship between hopefulness and immune function in patients with breast cancer. METHODS: A total of 196 patients with breast cancer were enrolled. The subjects were divided into two groups using the abbreviated version of the seven-item Beck Hopelessness Scale (BHS-7). Subsets of circulating lymphocytes were assessed using flow cytometry: CD3(+), CD4(+), CD8(+), CD19(+) and CD56(+). The Beck Depression Inventory (BDI), Montgomery-Asberg Depression Rating Scale (MADRS) and EORTC QLQ-C30 were administered. RESULTS: A total of 104 patients (53.6%) showed a hopeful attitude, with a score of 0 on the BHS-7. Scores on the MADRS and BDI were significantly higher in the nonhopeful group, whereas global and total functioning scores on the EORTC QLQ-C-30 were significantly higher in the hopeful group. The hopeful group showed significantly higher CD8(+) T-cell percentage and counts and significantly lower CD4(+) T-cell percentage and CD19(+) B-cell percentage and counts compared with the nonhopeful group. All statistically significant differences between the two groups were maintained after adjusting for age and scores on the BDI and EORTC QLQ-C-30 as covariates, except for CD 19(+) cell counts. CONCLUSION: The results suggest that hopefulness may be associated with immunity in patients with breast cancer, independent of depression and quality of life.

Bone-marrow-derived mesenchymal stem cell transplantation enhances closing pressure and leak point pressure in a female urinary incontinence rat model.

PURPOSE: The purpose of this study was to determine whether periurethral injection of allogenic mesenchymal stem cells (MSCs) could increase the leak point pressure (LPP) in a rat model of stress urinary incontinence. MATERIALS AND METHODS: Female Sprague-Dawley rats (230-240 g, n = 30) were divided into 3 groups: sham operation (group C), saline-treated (group S) and MSC-treated (group M). Bilateral pudendal nerve dissection followed by normal saline or MSC injection on both sides of the urethra was done. LPP and closing pressure (CP) testing was performed after the treatment. The specific markers for smooth muscle cells in the transplantation sites of the urethra were determined. RESULTS: Both the LPP and CP were significantly lower in group S than controls. However, these were restored to the control values in group M (p < 0.05). The LPPs of groups C, S and M were 29.1 ± 2.1, 22.0 ± 2.2 and 43.1 ± 3.2 cm H(2)O, respectively. The CPs of groups C, S and M were 27.1 ± 3.1, 21.1 ± 3.2, and 32.1 ± 2.1 cm H(2)O, respectively. The injected MSCs stained positive for muscle-specific markers. CONCLUSION: This study suggests that MSCs might differentiate into muscle lineage cells and may contribute to the repair of damaged muscle tissue.

Outer membrane vesicles of Vibrio vulnificus deliver cytolysin-hemolysin VvhA into epithelial cells to induce cytotoxicity.

The Gram-negative bacterium Vibrio vulnificus produces cytotoxins that induce the acute death of host cells. However, the secretory mechanisms of such cytotoxins have not been extensively studied. Previously, we reported that substantial amounts of V. vulnificus cytolysin-hemolysin (VvhA) are produced in vivo during the bacterial infection in mice and that this cytotoxin, in conjunction with RtxA1, mediates cytotoxicity. In this study, we investigated whether V. vulnificus cells release outer membrane vesicles (OMVs), which are used by some Gram-negative bacteria to deliver virulence factors into host cells. We found that V. vulnificus produce OMVs and that these vesicles can induce host cell death. This process appears to be mediated by VvhA, as evidenced by the finding that OMVs isolated from VvhA-null mutants do not induce cytotoxicity. In addition, cholesterol sequestration in the host cells prevents OMV-mediated VvhA delivery, indicating that VvhA-bearing OMVs interact with cholesterol on the host cell surface. Furthermore, intracellular expression experiments revealed that VvhA-mediated cytotoxicity is driven by its N-terminal leukocidin domain.

Combination antitumor effects of micelle-loaded anticancer drugs in a CT-26 murine colorectal carcinoma model.

Experiments were designed to evaluate the in vitro cytotoxic interactions of anticancer drugs in combination, evaluate synergistic activity in vivo and utilize micelle-forming polymeric drugs as drug carriers in a murine cancer model. Antitumor effects of 5-fluorouracil, cisplatin, CPT-11, oxaliplatin, etoposide, mitomycin-C, doxorubicin and paclitaxel were evaluated by determination of in vitro cytotoxicity to CT-26 colorectal tumor cells or in vivo following a subcutaneous transplant in BALB/c mice. Single agent and combination in vivo studies were also performed using drug-loaded polymeric micelles composed of poly(gamma-benzyl L-glutamate) and poly(ethylene oxide) (GEG) or poly(L-lactide)/poly(ethylene glycol) (LE) diblock copolymer. After 3 days exposure, the mean IC(50) (microg/mL) for 5-fluorouracil, cisplatin, CPT-11, oxaliplatin, etoposide, mitomycin-C, doxorubicin and paclitaxel were 0.95, 2.01, 4.47, 3.34, 3.5, 1.96, 1.8 and 2.1, respectively. When tumor cells were exposed to doxorubicin concurrently with etoposide or paclitaxel, evidence of synergy was observed in CT-26 cells in vitro. Doxorubicin and paclitaxel loaded into GEG or LE copolymers at a high concentration (19.5 and 16.7 wt%, respectively) were almost completely released (83.2% and 93.7%, respectively) by day 3. When tumor-bearing mice were treated in combination with doxorubicin-paclitaxel or doxorubicin-etoposide, substantial antitumor activity was evident compared with single therapy. These data suggest that in vitro cytotoxicity of anticancer drugs is related to in vivo results, and chemotherapy using micelle-loaded anticancer drugs represents a promising potential as a carrier system in modulating drug delivery.

Effect of iron on cytolysin A expression in Salmonella enterica serovar Typhi.

Previously, a novel protein ClyA (Cytolysin A) has been identified in Escherichia coli K-12, Salmonella enterica serovars Typhi and Paratyphi A and Shigella. Salmonella spp. synthesize substantial amounts of ClyA upon infection of the human host, although the mechanism by which ClyA is induced in vivo is unclear. Since environmental signals could control the expression of virulence determinants, ClyA expression in S. Typhi Ty2 was tested by Western blotting in the presence of normal pooled human serum (NPS). The level of ClyA expression increased in the presence of NPS in a concentration-dependent manner. RPMI 1640 medium similarly induced ClyA expression. ClyA expression was inversely proportional to the concentration of iron in RPMI medium. Therefore, we speculated that iron inhibited the expression of ClyA in S. Typhi Ty2, and free iron depletion may be one of the causes of S. Typhi-mediated induction of ClyA in vivo. Transcription from a clyA-lacZ fusion gene decreased as iron concentration increased, but not as significantly as the ClyA protein expression. It is concluded that the regulatory effect of iron on clyA expression is mainly at translational level.