Mi-Hyun Hwang

Mycoplasma hyopneumoniae induces pro-inflammatory cytokine and nitric oxide production through NFκB and MAPK pathways in RAW264.7 cells

In the present study, we confirmed the ability of M. hyopneumoniae to induce the secretion of large amount of proinflammatory cytokine and nitric oxide (NO) in murine macrophage RAW 264.7 cells. Moreover, M. hyopneumoniae-induced activation of the MAPK and NF-кB pathways by phosphorylation of ERK1/2, p38 and JNK/SAPK and by dissociation of IκB from NF-κB. Translocation of transcription factor NF-κB and its binding was confirmed through western blot and electromobility shift assay. From these results, we further hypothesized that these signal proteins were involved in M. hyopneumoniae-induced proinflammatory cytokines and NO productions in macrophages. Hence, we utilized specific blockers of MAPK and NF-κB to investigate the signaling pathway involvement in cytokine and NO production through pharmacological approaches. The results demonstrated significant inhibition of TNF-α, IL-1β, IL-6 and NO by MAPK inhibitors. NF-κB inhibitor PDTC significantly inhibited IL-1β and NO production. These findings contribute to the understanding of the mechanisms of immune reactivity and may ultimately prove useful in the development of new therapeutic strategies. In summary, we found critical evidence for the involvement of NF-κB and MAPK signaling pathways in the upregulation of proinflammatory cytokine and NO induced by M. hyopneumoniae.

Integration of Pharmacokinetic and Pharmacodynamic Indices of Orbifloxacin in Beagle Dogs after a Single Intravenous and Intramuscular Administration

ABSTRACT The pharmacokinetics (PK) and pharmacodynamics (PD) of orbifloxacin were studied in beagle dogs after intravenous (i.v.) and intramuscular (i.m.) administration at a dose of 2.5 mg/kg body weight. An absolute bioavailability of 100.1% ± 4.76%, a terminal half-life of 4.23 ± 0.2 h and 3.95 ± 0.15 h after i.v. and i.m. administration, a steady-state volume of distribution of 1.61 ± 0.13 liters/kg, and clearance of 0.31 ± 0.03 liters/h/kg were observed. Orbifloxacin showed rapid, concentration-dependent killing against the Escherichia coli, Staphylococcus aureus, Staphylococcus intermedius, and Proteus mirabilis clinical isolates. Computations based on PK-PD analysis indicated that the recommended dose is unlikely to be clinically effective against some strains like S. intermedius. Therefore, a higher dose of orbifloxacin would be worthy of consideration for treatment of certain bacterial infections in dogs.

Pharmacokinetics and pharmacokinetic/pharmacodynamic integration of orbifloxacin in Korean Hanwoo cattle.

The pharmacokinetics and pharmacodynamics of orbifloxacin were studied in six clinically healthy Hanwoo cows after intravenous (i.v.) and intramuscular (i.m.) administration at a dose of 3 mg/kg. Orbifloxacin concentrations were determined by high performance liquid chromatography with fluorescence detection. Steady-state volume of distribution and clearance of orbifloxacin after i.v. administration were 0.92 L/kg and 0.24 L/h x kg, respectively. Following i.m. administration, a slow and complete absorption with absolute bioavailability of 101.4%, and a maximum concentration (C(max)) of 1.17 microg/mL at 1.04 h were observed. The in vitro serum protein binding was 14.76%. The in vitro antibacterial activity of orbifloxacin against a pathogenic strain of Mannheimia haemolytica (M. haemolytica), Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) was determined. The ex vivo activity of orbifloxacin against M. haemolytica strain was also determined, and these data were integrated with the ex vivo bacterial counts to establish AUC(24h)/MIC values producing bacteriostatic action, bactericidal action and elimination of bacteria. Mean values were 32.7, 51.6 and 102.6 h, respectively. From these data, we predict that orbifloxacin, when administered i.m. at a dosage of 2.5-5 mg/kg once a day, would be effective against bovine pathogens, such as M. haemolytica. Additional studies may be needed to confirm its efficacy in a clinical setting, and to evaluate the penetration of the drug in diseased tissues.

Inflammatory responses to Mycoplasma hyopneumoniae in murine alveolar macrophage cell lines

Abstract AIM: To investigate the mechanism by which Mycoplasma hyopneumoniae induces inflammatory responses in murine alveolar macrophage (MH-S) cells. METHODS: A pathogenic strain of M. hyopneumoniae cultured in modified Friis medium was used to investigate the inflammatory response in MH-S cell lines. The effect of stimulation by M. hyopneumoniae on the production of nitric oxide (NO) and cytokines in MH-S cells and inhibition of their production, using specific inhibitors of signalling pathways, was investigated using the Griess reaction and ELISA respectively. A Western blot assay was used to confirm activation of the nuclear factor kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. Nuclear translocation of NF-κB was further confirmed using transient transfection and luciferase gene reporter assay. RESULTS: The results revealed dose-dependent production of NO in MH-S cells stimulated by M. hyopneumoniae. Increased concentrations of the cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β and IL-6 were also observed (p<0.05). Using immunoblot analysis, involvement of three MAPK pathways, extracellular signal-regulated kinase I/II (ERK1/2), p38 and Jun N-terminal kinases/stress-activated protein kinases (JNK/SAPK) was confirmed. Specific inhibitors of signal pathways also demonstrated their effect on the NO and cytokine responses of MH-S cells. Degradation and phosphorylation of inhibitory kappa B (IκB)-alpha was observed, while the luciferase gene reporter assays revealed activation of NF-κB after stimulation by M. hyopneumoniae. Inhibition of NF-κB by pyrrolidine dithiocarbamate decreased M. hyopneumoniae-induced production of NO and IL-1β (p<0.05), whereas no inhibitory effect was observed on concentrations of TNF-α, and IL-6. CONCLUSION: These findings indicate that M. hyopneumoniae induces NO and pro-inflammatory cytokines, and NF-κB and the three MAPK pathways are involved in the process.

A novel β-glucan produced by Paenibacillus polymyxa JB115 induces nitric oxide production in RAW264.7 macrophages

The effect of extracellular β-(1→3), (1→6)-glucan, produced by Paenibacillus polymyxa JB115, on nitric oxide (NO) production in RAW264.7 macrophages was investigated. β-glucan induced the production of NO by RAW264.7 macrophages in a concentration- and time-dependent manner. Moreover, β-glucan stimulation increased the mRNA expression of iNOS, COX-2 and IL-6 in RAW264.7 macrophages in a concentration-dependent manner.

Clinical pharmacokinetics of norfloxacin-glycine acetate after intravenous and oral administration in pigs.

The pharmacokinetics and dosage regimen of norfloxacin-glycine acetate (NFLXGA) was investigated in pigs after a single intravenous (i.v.) or oral (p.o.) administration at a dosage of 7.2 mg/kg body weight. After both i.v. and p.o. administration, plasma drug concentrations were best fitted to an open two-compartment model with a rapid distribution phase. After i.v. administration of NFLXGA, the distribution (t1/2α) and elimination half-life (t1/2β) were 0.36 ± 0.07 h and 7.42 ± 3.55 h, respectively. The volume of distribution of NFLXGA at steady state (Vdss) was 4.66 ± 1.39 l/kg. After p.o. administration of NFLXGA, the maximal absorption concentration (Cmax) was 0.43 ± 0.06 µg/ml at 1.36 ± 0.39 h (Tmax). The mean absorption (t1/2ka) and elimination half-life (t1/2β) of NFLXGA were 0.78 ± 0.27 h and 7.13 ± 1.41 h, respectively. The mean systemic bioavailability (F) after p.o. administration was 31.10 ± 15.16%. We suggest that the optimal dosage calculated from the pharmacokinetic parameters is 5.01 mg/kg per day i.v. or 16.12 mg/kg per day p.o.

Effect of Various Sawdusts and Logs Media on the Fruiting Body Formation of Phellinus gilvus.

Present experiments were conducted to determine the possibility of artificial culture with various sawdust of P. gilvus. The pH value was 6.0 of oak sawdust, 6.5 of mulberry sawdust, 6.6 of elm sawdust, 6.3 of acacia sawdust and 6.1 of apple tree sawdust. Mycelial density on elm sawdust and acacia sawdust were lower than those of oak sawdust, and apple sawdust. Weight of fresh fruiting body showed that 179 g on oak tree, 227 g on oak sawdust, 21 g on elm tree, 76 g on elm sawdust, 106 g on apple tree, and 170 g on apple sawdust. Among them, the yield of oak substrates was the highest whereas acacia sawdust was the lowest, and it is concluded that the yields of sawdust substrates were higher than log substrates. P. gilvus grown on various sawdusts and logs used in this study have shown similar in anti-tumor activity against P388.

Mycoplasma hyopneumoniae Induces Grap, Gadd45β, and secreted phosphoprotein 1 Gene Expression as Part of the Inflammatory Response in RAW264.7 Cells

Genes related to Mycoplasma hyopneumoniae-induced inflammation were identified using the gene-fishing technology, an improved method for identifying differentially expressed genes (DEGs) using an annealing control primer (ACP) system in RAW264.7 cells. After treatment with M. hyopneumo-niae, 16 DEGs were expressed in RAW264.7 cells using a pre-screening system. Among these 16 DEGs, 11 DEGs (DEGs 1, 4, 5–10, 12-15) were selected and sequenced directly, revealing that DEG12 (Grap), DEG14 (Gadd45), and DEG15 (secreted phosphoprotein 1) were related to inflammatory cytokines. This is the first report that intact M. hyopneumoniae induces the expression of Grap, Gadd 45β, and secreted phosphoprotein 1 in RAW264.7 cells. Subsequently, these genes may be targets for screening novel inhibitors of the mycoplasmal inflammatory response.