Comparison of vancomycin area under the curve calculated based on Bayesian approach versus equation-based approach.

OBJECTIVE: Area under the curve (AUC)-based vancomycin dose adjustment is recommended to treat methicillin-resistant Staphylococcus aureus (MRSA) infections. AUC estimation methods include Bayesian software programs and simple analytical equations. This study compared the AUC obtained using the Bayesian approach with that obtained using an equation-based approach. MATERIALS AND METHODS: Patients receiving intravenous vancomycin for MRSA infection were included. Peak and trough levels were measured for each patient on days 3, 7, and 10 post vancomycin dosing (day 1). AUC was calculated using software based on the Bayesian method (MwPharm Online) and an equation-based calculator, Stanford Health Care (SHC) calculator. RESULTS: The AUC estimated using MwPharm Online was similar to that estimated using the SHC calculator. The geometric mean ratio (GMR) and their 90% confidence intervals (90% CI) were 1.08 (1.05 - 1.11), 1.03 (0.99 - 1.07), and 0.99 (0.94 - 1.05) at days 3, 7, and 10, respectively. Furthermore, according to the software used, there were no significant differences in the proportions of patients in the categories "within" and "below or above" the AUC target range. Additionally, trough levels predicted by both software programs were lower than the observed ones. Still, there was no significant difference between the predicted and observed peak levels for both software programs on day 10. CONCLUSION: AUC calculated using the Bayesian software allows for calculation with samples at a non-steady state, can integrate covariates, and is interconvertible with that estimated using an equation-based calculator, which is simpler and relies on fewer assumptions. Therefore, either method can be used, considering each method's strengths and limitations.

Exosomes: Nomenclature, Isolation, and Biological Roles in Liver Diseases.

The biogenesis and biological roles of extracellular vesicles (EVs) in the progression of liver diseases have attracted considerable attention in recent years. EVs are membrane-bound nanosized vesicles found in different types of body fluids and contain various bioactive materials, including proteins, lipids, nucleic acids, and mitochondrial DNA. Based on their origin and biogenesis, EVs can be classified as apoptotic bodies, microvesicles, and exosomes. Among these, exosomes are the smallest EVs (30-150 nm in diameter), which play a significant role in cell-to-cell communication and epigenetic regulation. Moreover, exosomal content analysis can reveal the functional state of the parental cell. Therefore, exosomes can be applied to various purposes, including disease diagnosis and treatment, drug delivery, cell-free vaccines, and regenerative medicine. However, exosome-related research faces two major limitations: isolation of exosomes with high yield and purity and distinction of exosomes from other EVs (especially microvesicles). No standardized exosome isolation method has been established to date; however, various exosome isolation strategies have been proposed to investigate their biological roles. Exosome-mediated intercellular communications are known to be involved in alcoholic liver disease and nonalcoholic fatty liver disease development. Damaged hepatocytes or nonparenchymal cells release large numbers of exosomes that promote the progression of inflammation and fibrogenesis through interactions with neighboring cells. Exosomes are expected to provide insight on the progression of liver disease. Here, we review the biogenesis of exosomes, exosome isolation techniques, and biological roles of exosomes in alcoholic liver disease and nonalcoholic fatty liver disease.

Study on chemotaxis and chemokinesis of bone marrow-derived mesenchymal stem cells in hydrogel-based 3D microfluidic devices.

BACKGROUND: Controlling the fate of mesenchymal stems cells (MSCs) including proliferation, migration and differentiation has recently been studied by many researchers in the tissue engineering field. Especially, recruitment of stem cells to injury sites is the first and crucial step in tissue regeneration. Although significant progress has been made in the chemotactic migration of MSCs, MSC migration in three dimensional environments remains largely unknown. We developed a 3D hydrogel-based microfluidic-device to study the migration behavior of human MSCs in the presence of stromal-cell derived factor-1α (SDF-1α), interleukin 8 (IL-8) and Substance P (SP) which have been utilized as chemoattractant candidates of human mesenchymal stem cells (hMSCs). RESULTS: We systematically investigated the chemotactic migration behaviors of hMSCs and their responses to SDF-1α, IL-8, and SP. SDF-1α was shown to be the most fascinating chemoattractant candidate among those factors at a certain time point. We also found that each chemokine showed different chemoattractant abilities according to their concentration. In the case of SP, this factor showed chemokinesis not chemotaxis. Especially at a 7-8 × 10(-8) M concentration range, the chemokinesis ability driven by SP was further increased. The data suggest that some factors at the optimal concentration exhibit chemokinesis or chemotaxis in a 3D hydrogel-based microfluidic device. CONCLUSION: In this study on chemotaxis and chemokinesis of hMSCs, the system parameters such as chemokine concentration, system stability, and 2D or 3D microenvironment are critically important to obtain meaningful results.

p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via the activation of NF-κB binding in the IL-7-induced migration and invasion of 5637 cells.

Interleukin-7 (IL-7) plays a pivotal role in the development and survival of lymphocytes, but its role in cancer cell responses remains unexplained. In this study, IL-7 treatment resulted in a significant induction in the wound-healing migration and Matrigel invasion of the 5637 bladder cancer cells, but it did not result in cell proliferation. In addition, IL-7 treatment strongly induced MMP-9 expression, and increased the binding activation of NF-κB and AP-1 motifs, the important transcription factors that regulate MMP-9 expression. Moreover, the treatment of 5637 cells with IL-7 stimulated the phosphorylation of ERK1/2. U0126, an ERK1/2-specific inhibitor, blocked IL-7-induced cell migration and invasion, and also suppressed the expression of MMP-9 in the presence of IL-7. Inhibition of the ERK1/2 function consistently reversed the binding activity of NF-κB without altering AP-1 activation in IL-7-stimulated cells. Among the cell cycle regulators examined, only the expression of the cell cycle inhibitor p27KIP1 was induced by IL-7. Moreover, the inhibition of p27KIP1 by small interfering RNA (siRNA) abolished the migration, invasion and phosphorylation of ERK1/2, the expression of MMP-9, and the binding activity of the NF-κB motif in IL-7-stimulated 5637 cells. These results demonstrated that the cell cycle inhibitor p27KIP1 is involved in ERK1/2-mediated MMP-9 expression via activation of the NF-κB binding motif, which leads to the migration and invasion of bladder cancer cells induced by IL-7. These novel results could help explain the migration and invasion of bladder tumor cells.

Interleukin-5 enhances the migration and invasion of bladder cancer cells via ERK1/2-mediated MMP-9/NF-κB/AP-1 pathway: involvement of the p21WAF1 expression.

Inflammatory cytokines may be a critical component of epithelial cancer progression. We examined the role of interleukin (IL)-5 in the migration of bladder cancer cells. The expression of IL-5 and its receptor IL-5Rα was enhanced in patients with muscle invasive bladder cancers (MIBC), and then it was detected in bladder cancer cell lines 5637 and T-24. IL-5 increased migration and MMP-9 expression via activation of transcription factors NF-κB and AP-1, and induced activation of ERK1/2 and Jak-Stat signaling in both cells. Treatment with ERK1/2 inhibitor U0126 significantly inhibited induction of migration, MMP-9 expression, and activation of NF-κB and AP-1 in IL-5-treated cells. However, none of the Jak inhibitors affected the IL-5-induced migration of bladder cancer cells. Moreover, gene knockdown for IL-5Rα, using siRNA transfection, suppressed migration, ERK1/2 activation, MMP-9 expression, as well as the binding activation of NF-κB and AP-1 in IL-5-treated bladder cancer cells. Similar results were observed in ßc siRNA (si-ßc) transfected cells. Unexpectedly, IL-5 treatment resulted in significant induction of p21WAF1 in both cell lines. The p21WAF1-specific small interfering RNA inhibited IL-5-induced cell migration, ERK activity, MMP-9 expression, and activation of NF-κB and AP-1 in bladder cancer cells. The effects of IL-5-induced cell responses were confirmed by transfection of IL-5 gene, which demonstrated that p21WAF1 participates in the induction of cell migration, leading to an increase in ERK1/2-mediated MMP-9 expression through activation of NF-κB and AP-1 in IL-5-treated bladder cancer cells. These unexpected results provide a theoretical basis for the therapeutic targeting of IL-5 in bladder cancer.

Interleukin-20 promotes migration of bladder cancer cells through extracellular signal-regulated kinase (ERK)-mediated MMP-9 protein expression leading to nuclear factor (NF-κB) activation by inducing the up-regulation of p21(WAF1) protein expression.

The role of inflammatory cytokine interleukin-20 (IL-20) has not yet been studied in cancer biology. Here, we demonstrated up-regulation of both IL-20 and IL-20R1 in muscle-invasive bladder cancer patients. The expressions of IL-20 and IL-20R1 were observed in bladder cancer 5637 and T-24 cells. We found that IL-20 significantly increased the expression of matrix metalloproteinase (MMP)-9 via binding activity of NF-κB and AP-1 in bladder cancer cells and stimulated the activation of ERK1/2, JNK, p38 MAPK, and JAK-STAT signaling. Among the pathways examined, only ERK1/2 inhibitor U0126 significantly inhibited IL-20-induced migration and invasion. Moreover, siRNA knockdown of IL-20R1 suppressed migration, invasion, ERK1/2 activation, and NF-κB-mediated MMP-9 expression induced by IL-20. Unexpectedly, the cell cycle inhibitor p21(WAF1) was induced by IL-20 treatment without altering cell cycle progression. Blockade of p21(WAF1) function by siRNA reversed migration, invasion, activation of ERK signaling, MMP-9 expression, and activation of NF-κB in IL-20-treated cells. In addition, IL-20 induced the activation of IκB kinase, the degradation and phosphorylation of IκBα, and NF-κB p65 nuclear translocation, which was regulated by ERK1/2. IL-20 stimulated the recruitment of p65 to the MMP-9 promoter region. Finally, the IL-20-induced migration and invasion of cells was confirmed by IL-20 gene transfection and by addition of anti-IL-20 antibody. This is the first report that p21(WAF1) is involved in ERK1/2-mediated MMP-9 expression via increased binding activity of NF-κB, which resulted in the induction of migration in IL-20/IL-20R1 dyad-induced bladder cancer cells. These unexpected results might provide a critical new target for the treatment of bladder cancer.

Identification of pro-inflammatory cytokines associated with muscle invasive bladder cancer; the roles of IL-5, IL-20, and IL-28A.

We used gene expression profiling to identify inflammatory cytokines that correlate with bladder cancer development. Gene expression profiles of the tissue samples were investigated using cDNA microarrays that contained 103 non-muscle invasive bladder cancers (NMIBC), 62 muscle invasive bladder cancers (MIBC), 58 samples of histologically normal-looking surrounding tissues, and 10 normal, healthy subjects who served as the control cohort for comparison. We grouped the data-sets according to biological characterizations and focused on immune response genes with at least 2-fold differential expression in MIBC vs. controls. The experimental data-set identified 36 immune-related genes that were significantly altered in MIBC samples. In addition, 10 genes were up-regulated and 26 genes were down-regulated in MIBC samples compared with the normal tissues. Among the 10 up-regulated molecules examined, the capacity for both wound-healing migration and invasion was enhanced in response to IL-5, IL-20, and IL-28A in bladder cancer cell lines (253J and EJ cells), compared with untreated cells. The expression levels of IL-5, IL-20, and IL-28A were increased in patients with MIBC. All 3 cytokines and their receptors were produced in bladder cancer cell lines, as determined by real-time PCR, immunoblot analysis and confocal immunofluorescence. Up-regulation of MMP-2 and MMP-9 was found after IL-5, IL-20, and IL-28A stimulation in both cell types. Moreover, an EMSA assay showed that treatment with IL-5, IL-20, and IL-28A induced activation of the transcription factors NF-κB and AP-1 that regulate the MMP-9 promoter. Finally, activation of MAPK and Jak-Stat signaling was observed after the addition of IL-5, IL-20, and IL-28A to bladder cancer cells. This study suggests the presence of specific inflammatory cytokine (IL-5, IL-20, and IL-28A)-mediated association in bladder cancer development. All 3 cytokines may be important new molecular targets for the modulation of migration and invasion in bladder cancer.

IL-5-induced migration via ERK1/2-mediated MMP-9 expression by inducing activation of NF-κB in HT1376 cells.

Interleukin-5 (IL-5) plays an important role in the growth and differentiation of human B cells and eosinophils. However, little is known about the effect of IL-5 on cancer cells. In this study, we investigated the molecular mechanisms involved in the IL-5-induced migration of HT1376 bladder cancer cells. Our results indicated that IL-5 significantly enhanced migration and MMP-9 expression in HT1376 cells. We also found that IL-5 induces transcriptional activation of the binding of NF-κB and AP-1, which are two important nuclear transcription factors that are linked to MMP-9 expression in HT1376 cells. In subsequent experiments, we found activation of ERK1/2 in IL-5-treated HT1376 cells. To examine the involvement of the ERK1/2 signaling pathway on IL-5-induced cell responses, we pretreated HT1376 cells with the ERK1/2 inhibitor U0126 followed by IL-5 treatment. The results showed that U0126 treatment inhibited migration of IL-5-treated HT1376 cells. Moreover, IL-5-stimulated MMP-9 expression was suppressed by the addition of U0126. Inhibition of ERK1/2 function consistently rescued transcriptional activity of NF-κB, without altering AP-1 activation, in IL-5-treated cells. Finally, inhibition of the IL-5-specific receptor IL-5Rα by small interfering RNA (siRNA) suppressed migration, ERK1/2 activation, MMP-9 expression and binding activation of NF-κB in IL-5-treated HT1376 cells. The results of the present study indicate that the IL-28A/IL-28AR1 dyad induces cell migration through ERK1/2-mediated expression of MMP-9 by binding activation of NF-κB in bladder cancer cells. In conclusion, these novel findings indicate that binding of IL-5 to IL-5Rα plays a critical role in MMP-9 expression, which may be involved in the migration of bladder cancer.

Cordycepin suppresses TNF-alpha-induced invasion, migration and matrix metalloproteinase-9 expression in human bladder cancer cells.

Cordycepin (3'-deoxyadenosine), a nucleoside derivative isolated from Cordyceps militaris, reportedly has antitumor effects, but its effect on the regulation of matrix metalloproteinases-9 (MMP-9), which regulates invasion and migration by cancer cells, has not been clearly elucidated. Cancer cell invasion and migration was investigated using a Matrigel invasion assay and wound healing analysis in two different bladder cancer cell lines: 5637 and T-24. The results of the present study show that TNF-α-induced invasion and migration of cancer cells were inhibited by cordycepin. In addition, cordycepin inhibited TNF-α-induced proliferation in cancer cells, independent of the apoptosis pathway. Furthermore, the TNF-α-induced MMP-9 expression was suppressed by cordycepin, but MMP-2 expression was not. The inhibited MMP-9 expression by cordycepin was associated with a decreased promoter activity of the MMP-9 gene. Finally, cordycepin reduced the transcriptional activity of the transcription factors, nuclear factor kappaB (NF-KB) and activator protein-1 (AP-1), which were identified by gel-shift assay as cis-elements for TNF-α activation of the MMP-9 promoter in both bladder cancer cell lines. These results suggest that cordycepin maybe an effective therapeutic approach to treat bladder cancer.

Naringin inhibits matrix metalloproteinase-9 expression and AKT phosphorylation in tumor necrosis factor-alpha-induced vascular smooth muscle cells.

Citrus fruits are high in naringin, which has a beneficial effect on cardiovascular diseases. However, the matrix metalloproteinase-9 (MMP-9) regulation involved in cell migration and invasion remains to be identified. Naringin inhibited tumor necrosis factor-alpha (TNF-alpha)-induced expression of MMP-9, under 10-25 microM concentration conditions in vascular smooth muscle cells (VSMC). The TNF-alpha-induced invasion and migration of VSMC were inhibited by naringin. Furthermore, naringin suppressed TNF-alpha-mediated release of interleukin-6 and -8 (IL-6 and IL-8). However, naringin (10-25 microM) treatment of VSMC in the presence of TNF-alpha did not affect cell growth and apoptosis. In additional experiments, naringin reduced the transcriptional activity of activator protein-1 and nuclear factor kappaB (NF-kappaB), which are two important nuclear transcription factors that are involved in MMP-9 expression. Also, naringin treatment blocked PI3K/AKT/mTOR/p70S6K pathway in TNF-alpha-induced VSMC. Treatment of aglycone naringenin (10-25 microM) had same effect on the levels of MMP-9 expression, invasion, migration, and AKT phosphorylation in TNF-alpha-induced VSMC, compared with naringin treatment. These results suggest that naringin represses PI3K/AKT/mTOR/p70S6K pathway, invasion and migration, and subsequently suppresses MMP-9 expression through the transcription factors NF-kappaB and activator protein-1 in TNF-alpha-induced VSMC. These novel findings provide a theoretical basis for the preventive use of naringin for atherosclerosis disease.

Inhibition of proliferation and migration by piceatannol in vascular smooth muscle cells.

Piceatannol (3,5,3',4'-tetrahydroxy- trans-stilbene), a resveratrol analogue, is a polyphenol present in the skins of grapes and in wine and other foods. The present study aimed to investigate for the first time the cardioprotective effects of piceatannol on vascular smooth muscle cells (VSMC). The treatment of cells with piceatannol inhibited cell proliferation by reducing extracellular signal-regulated kinase (ERK) 1/2 and JNK activity in cultured VSMC in the presence of tumor necrosis factor-alpha (TNF-alpha). These inhibitory effects were also associated with G1 cell cycle arrest, and resulted in a decrease in cyclin-dependent kinases (CDKs) and cyclins. Piceatannol treatment strongly induced the expression of p21WAF1 via independence of p27KIP and p53 expression. The effect of piceatannol was not restricted to cell proliferation, as TNF-alpha-induced invasion and migration was also suppressed in VSMC. Moreover, piceatannol treatment strongly decreased matrix metalloproteinase-9 (MMP-9) expression and promoter activity in a dose-dependent manner in response to TNF-alpha. It was further demonstrated that piceatannol abrogated the transcriptional activity of nuclear factor kappa B (NF-kappaB), an important nuclear transcription factor involved in MMP-9 expression. Overall, these results demonstrate that piceatannol inhibits proliferation and migration of VSMC treated with TNF-alpha. Therefore, piceatannol may be an effective therapeutic approach to treat atherosclerosis.

Naringin-induced p21WAF1-mediated G(1)-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells.

The flavonoid naringin has been shown to play a role in preventing the development of cardiovascular disease. However, the exact molecular mechanisms underlying the roles of integrated cell cycle regulation and MAPK signaling pathways in the regulation of naringin-induced inhibition of cell proliferation in vascular smooth muscle cells (VSMCs) remain to be identified. Naringin treatment resulted in significant growth inhibition and G(1)-phase cell cycle arrest mediated by induction of p53-independent p21WAF1 expression; expression of cyclins and CDKs in VSMCs was also down-regulated. In addition, among the pathways examined, blockade of ERK function inhibited naringin-dependent p21WAF1 expression, reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Moreover, naringin treatment increased both Ras and Raf activations. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, naringin-induced reduction in cell proliferation and cell cycle protein was abolished in the presence of RasN17 and RafS621A mutant genes. The Ras/Raf/ERK pathway participates in p21WAF1 induction, leading to a decrease in cyclin D1/CDK4 and cyclin E/CDK2 complexes and in naringin-dependent inhibition of cell growth. These novel and unexpected findings provide a theoretical basis for preventive use of flavonoids to the atherosclerosis disease.

Cloning, expression, and characterization of a glycoside hydrolase family 50 beta-agarase from a marine Agarivorans isolate.

The gene for a thermostable beta-agarase from Agarivorans sp. JA-1 was cloned and sequenced. It comprised an open reading frame of 2,988 base pairs, which encode a protein of 109,450 daltons consisting of 995 amino acid residues. A comparison of the entire sequence showed that the enzyme has 98.8% sequence similarities to beta-agarase from Vibrio sp. JT1070, indicating that it belongs to the family glycoside hydrolase (GH)-50. The gene corresponding to a mature protein of 976 amino acids was inserted and expressed in Escherichia coli. The recombinant beta-agarase was purified to homogeneity. It had maximal activity at 40 degrees C and pH 8.0 in the presence of 1 mM NaCl and 1 mM CaCl(2). The enzyme hydrolyzed agarose as well as neoagarohexaose and neoagarotetraose to yield neoagarobiose as the main product. Thus, the enzyme would be useful for the industrial production of neoagarobiose.