Joong-Su Lee

Effect of Lactobacillus plantarum CJLP243 on the growth performance and cytokine response of weaning pigs challenged with enterotoxigenic Escherichia coli1

The purpose of the present study was to evaluate the effect of diets containing Lactobacillus plantarum CJLP243 on the growth and cytokine response of weaning pigs (Sus scrofa) challenged with enterotoxigenic Escherichia coli (ETEC). In a 28-d experiment (14 d before and 14 d after challenge), a total of 108 pigs at 20 ± 1 d of age were allotted to 1 of 6 diets. These were a control diet without ETEC challenge (CON) and 5 treatment diets with ETEC challenge, including a control diet with ETEC challenge (negative control, NC); a positive control diet containing antibiotics (PC); control diet plus (10(8), 10(9), or 10(10)) cfu/kg L. plantarum CJLP243 (T1, T2, and T3, respectively). After challenge, NC showed the least ADFI, whereas PC and T3 had the greatest ADFI (P = 0.002). The ADG of PC, T2, and T3 were greater (P = 0.001) than that of CON, NC, and T1 during wk 1 to wk 2. During wk 3 to wk 4, a marked decline was seen in NC (P = 0.001) compared with CON, whereas PC and T3 showed increased ADG (P = 0.001). The overall ADG of PC and T3 were greater (P < 0.001) than the remaining groups. The PC and T3 had the greatest G:F during the second 2 wk (P = 0.002), and the overall 4-wk experimental period (P = 0.003). At 3 h after challenge, all groups except CON had greater rectal temperatures (RT; P < 0.05). The RT decreased to prechallenge temperatures at 9 h (PC and T3), 24 h (T1 and T2), and remained increased until d 7 in NC. At 7 and 14 d postinfection, the number of animals detected positive for ETEC by PCR assay was the greatest in NC; however, the PC group had the fewest ETEC-positive animals (P < 0.05), which was similar to T3. All challenged pigs, except T2, had greater concentrations of serum haptoglobin compared with CON, with the greatest concentration observed in NC (P < 0.001). Challenged pigs had increased serum concentrations of tumor necrosis factor alpha (TNF-α) 3 to 48 h postinfection, with the greatest concentration of TNF-α at 48 h observed in NC (P < 0.05). Similarly, greater (P < 0.05) serum concentrations of interferon-γ were observed for 9 h (T1 and T3), 24 h (T2 and PC), and 48 h (NC) postinfection. The serum concentration of IL-6 increased (P < 0.05) for 3 h in T3 and 24 h in NC. In conclusion, our findings suggest that L. plantarum CJLP243, at a concentration of 10(10) cfu/kg, may serve as a potential alternative to antibiotic supplementation to improve the growth and health performance of weaning pigs, especially during acute inflammation of the gut after bacterial infections.

Mechanism of macrophage activation induced by β-glucan produced from Paenibacillus polymyxa JB115

Beta-glucans are heterogeneous groups of glucose polymers found in the cell walls of fungi, plants and some bacteria. Our previous report showed that a novel beta-1,3/1,6-glucan produced from Paenibacillus (P.) polymyxa JB115 can induce nitric oxide (NO) production in RAW264.7 cells. In the present study, the beta-glucan significantly increased luciferase activity in cells transfected with NFkappaB or AP1, but not STAT1, reporter vector DNA, which contain their binding promoter site. All specific NFkappaB and MAPKs pathway inhibitors (pyrrolidine dithiocarbamate, AG490, U0126, SB203580 and SP600125) remarkably attenuated NO production induced by the beta-glucan. Furthermore, Western blot analysis revealed that the stimulation of Raw264.7 cells by beta-glucan induced phosphorylation of IkappaB and the consequent translocation of NFkappaB into the nucleus. Meanwhile, phosphorylation of ERK1/2, JNK/SAPK and p38 MAPKs in cytoplasm were also confirmed. All these results indicated that beta-glucan from P. polymyxa JB115 activates macrophages through MAPKs and NFkappaB signaling pathway.

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.

Effects of dietary supplementation of Lactobacillus pentosus PL11 on the growth performance, immune and antioxidant systems of Japanese eel Anguilla japonica challenged with Edwardsiella tarda

The aim of this study was to determine the efficacy of dietary administration of Lactobacillus pentosus PL11 on growth performance and the immune and antioxidant systems in Japanese eel Anguilla japonica challenged with Edwardsiella tarda. A total of 75 Japanese eels (24.63±0.83 g) were grouped into 5 treatment diets which were a control diet (C) without E. tarda and 4 treatment diets with E. tarda challenge, including C for E. tarda challenge (NC), C plus L. pentosus PL11 supplemented diet (10⁸ cfu g⁻¹) (T-PL11), C plus L. pentosus KCCM 40997 supplemented diet (10⁸ cfu g⁻¹) (T-Lp) and C plus Weissella hellenica DS-12 supplemented diet (10⁸ cfu g⁻¹) (T-Wh) for 5 weeks (4 week before and 1 week after challenge). The results showed enhanced growth performance in fish fed the diet containing L. pentosus PL11 compared to others. The growth performance parameters including specific growth rate (SGR) and weight gain (WG), feed intake (FI), feed conversion ratio (FCR) and survival were significantly (P<0.05) higher in fish maintained on L. pentosus PL11 supplemented diet compared to C and NC. T-PL11 group also shows a significant increase in the levels of plasma immunoglobulin M, CAT and SOD activities compared to NC. Hematological parameters and mieloperoxidase were significantly better in fish fed the L. pentosus PL11 supplemented diet than in the control. L. pentosus PL11 supplementation recover the reduced expression of SOD, CAT and heat shock protein 70 genes in liver and intestine in pathogen challenged fishes. In conclusion the result of the current study demonstrated L. pentosus PL11 potential as an alternative to antibiotic supplementation to improve the growth and health performance of Japanese eel (A. japonica).

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.

Comparative mutant prevention concentration and mechanism of resistance to veterinary fluoroquinolones in Staphylococcus pseudintermedius

BACKGROUND The problem of antibacterial drug resistance is increasing worldwide, in part due to the therapeutic concentrations currently used based on the minimal inhibitory concentration (MIC) as a measure of potency are often the very concentrations required to selectively enrich the resistant mutant portion of the population. A mutant prevention concentration (MPC)-based dosing strategy is suggested to improve the therapeutic outcome based on the MIC. OBJECTIVE Our aim was to investigate the MPC and mechanism of resistance to various fluoroquinolones using recent Staphylococcus pseudintermedius isolates from canine pyoderma. METHODS The broth microdilution method for MIC and a series of agar plates containing different concentrations of fluoroquinolones were inoculated with ∼10(10) colony-forming units of the bacterial culture for MPC were used. PCR was used to identify mutation in the resistant isolates. RESULTS The rank order of potency based on MIC and MPC was ciprofloxacin = enrofloxacin ≥ marbofloxacin > difloxacin ≥ orbifloxacin. Integrating our data with reported pharmacokinetic data at the recommended dose ranges revealed that only high doses of ciprofloxacin, enrofloxacin and marbofloxacin could achieve a maximal plasma concentration (C(max)) greater than the MPC of 90% of isolates (C(max)/MPC(90)). The overall rank of potency against S. pseudintermedius, based on C(max)/MIC, C(max)/MPC, the area under concentration-time curve (AUC)/MIC and AUC/MPC values, was in decreasing order: enrofloxacin > ciprofloxacin ≥ marbofloxacin ≥ orbifloxacin = difloxacin. Sequencing of the quinolone resistant determining region of gyrA, gyrB, grlA and grlB of resistant strains showed a base-pair substitution in both gyrA and gyrB that resulted in Ser-84 to Leu and Ser-80 to Arg amino acid changes, respectively. CONCLUSIONS AND CLINICAL IMPORTANCE High doses of ciprofloxacin, enrofloxacin and marbofloxacin could minimize the selection of resistant mutants, whereas the possibility of selecting mutants with the conventional doses of difloxacin and orbifloxacin, and low clinical doses of all fluoroquinolones, seems high.

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.

Comparative Antitumor Activity of Different Solvent Fractions from an Auricularia auricula-judae Ethanol Extract in P388D1 and Sarcoma 180 Cells

The objective of this study was to evaluate and compare the antitumor activity of different solvent fractions (ethanol, dichloromethane, ethyl acetate, butanol and water) of the Auricularia auricula-judae 70% ethanol extract on the P388D1 macrophage and sarcoma 180 cells. A dose-dependent antitumor activity of each solvent fraction (from 0.01 mg/ml to 0.3 mg/ml) was shown against both cell types. These cytotoxic effects of all the tested fractions were confirmed on the MTT and SRB assays, without statistical differences each other. IC50 value of dichloromethane fraction was 94.2 μg/ml against sarcoma 180 cells lower than any other solvent fractions. The potent antitumor effect of the dichloromethane (DCM) fraction was also found against solid tumor in BALB/c mice. The splenomegaly and higher splenic index were found in tumor-bearing mice, with the DCM fraction returning to the negative control values. Thus, the results indicated the dichloromethane fraction may have potential ingredients as antitumor candidates.