Ifit2 deficiency restricts microglial activation and leukocyte migration following murine coronavirus (m-CoV) CNS infection.

The interferon-induced tetratricopeptide repeat protein (Ifit2) protects mice from lethal neurotropic viruses. Neurotropic coronavirus MHV-RSA59 infection of Ifit2-/- mice caused pronounced morbidity and mortality accompanied by rampant virus replication and spread throughout the brain. In spite of the higher virus load, induction of many cytokines and chemokines in the brains of infected Ifit2-/- mice were similar to that in wild-type mice. In contrast, infected Ifit2-/- mice revealed significantly impaired microglial activation as well as reduced recruitment of NK1.1 T cells and CD4 T cells to the brain, possibly contributing to the lack of viral clearance. These two deficiencies were associated with a lower level of microglial expression of CX3CR1, the receptor of the CX3CL1 (Fractalkine) chemokine, which plays a critical role in both microglial activation and leukocyte recruitment. The above results uncovered a new potential role of an interferon-induced protein in immune protection.

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-1ß, IL-6 and NO by MAPK inhibitors. NF-κB inhibitor PDTC significantly inhibited IL-1ß 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.

Optimization of culture media of pathogenic Mycoplasma hyopneumoniae by a response surface methodology.

Composition of culture medium for mass production of Mycoplasma hyopneumoniae was optimized using a response surface methodology (RSM). Initially, the influence of glucose, thallium acetate, fresh yeast extract, horse serum, and porcine serum on the production of mycoplasmal protein was assessed using a 'one factor at a time' technique. Next, factors with a significant effect, including fresh yeast extract, and horse and porcine sera, were selected for further optimization using a central composite design (CCD) of RSM. The experimental results were fitted into a second order polynomial model equation. Estimated optimal condition of the factors for maximum production of mycoplasmal protein (i.e., triple-fold increase from 0.8 mg/L produced by basal mycoplasma media to 2.5 mg/L) was 10.9% fresh yeast extract, 15% horse serum, and 31.5% porcine serum (v/v). For the optimized conditions, a 2.96 mg/L experimental result was observed, similar to the estimated optimal conditions result of the CCD.

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

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

Integration of pharmacokinetic and pharmacodynamic indices of orbifloxacin in beagle dogs after a single intravenous and intramuscular administration.

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.

Mycoplasma hyopneumoniae Induces Grap, Gadd45ß, 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 45ß, and secreted phosphoprotein 1 in RAW264.7 cells. Subsequently, these genes may be targets for screening novel inhibitors of the mycoplasmal inflammatory response.

Comparative pharmacokinetics of tylosin or florfenicol after a single intramuscular administration at two different doses of tylosin-florfenicol combination in pigs.

Clinical pharmacokinetic profiles were investigated following intramuscular (i.m.) administration to pigs with a commercial tylosin-florfenicol combination product at a dose of 2.5 mg/kg tylosin and 5 mg/kg florfenicol or 10 mg/kg tylosin and 20 mg/kg florfenicol. The quantitation limit (QL) of florfenicol was 0.1 microg/ml, the inter-day and intra-day precision (CV%) were both beow 10%. The quantitation limit (QL) of tylosin was 0.05 microg/mL. The pharmacokinetic characteristics after i.m. doses were fitted by a one compartment open model. A fourfold decrease in the normal dose of each drug (20 mg/kg to 5 mg/kg for florfenicol, and 10 mg/kg to 2.5 mg/kg for tylosin) resulted in a corresponding two fold decrease in each drug of the maximum plasma concentration (C(max)) and the area under curve (AUC) values.

Surfactin C inhibits Mycoplasma hyopneumoniae-induced transcription of proinflammatory cytokines and nitric oxide production in murine RAW 264.7 cells.

Intact pathogenic Mycoplasma hyopneumoniae at 100 microg protein ml(-1) induced transcription of proinflammatory cytokines such as cyclooxygenase (COX)-2, tumor necrosis factor (TNF)-alpha, interleukin(IL)-1, IL-6 and inducible nitric oxide synthase (iNOS) in RAW 264.7 cells. After pretreatment with 50 microg surfactin C/ml, purified from Bacillus subtilis, transcription of the COX-2, IL-1beta, IL-6 and iNOS genes induced by M. hyopneumoniae was inhibited by 43%, 82%, 72% and 59%, respectively.

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 (t(1/2alpha)) and elimination half-life (t(1/2beta)) were 0.36 +/- 0.07 h and 7.42 +/- 3.55 h, respectively. The volume of distribution of NFLXGA at steady state (Vd(ss)) was 4.66 +/- 1.39 l/kg. After p.o. administration of NFLXGA, the maximal absorption concentration (C(max)) was 0.43 +/- 0.06 microg/ ml at 1.36 +/- 0.39 h (T(max)). The mean absorption (t(1/2ka)) and elimination half-life (t(1/2beta)) 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.

Lipopolysaccharide-binding and neutralizing activities of surfactin C in experimental models of septic shock.

To evaluate the anti-endotoxin activity of surfactin C, we studied its lipopolysaccharide-binding activity in vitro and therapeutic efficacy in experimental models of gram-negative septic shock. The ability of surfactin C to bind LPS from Escherichia coli O111:B4 was determined using a limulus chromogenic assay. Male ICR mice and Sprague-Dawley rats were given intraperitoneal administration of 1x10(9) colony forming units of E. coli ATCC 25922. After bacterial challenge, all animals were randomized to receive intraperitoneally saline, polymyxin B or surfactin C. Surfactin C not only completely bound to the LPS (its median effective concentration being 13.75 microM) but also improved the survival and reduced of the number of inoculated bacteria in the mouse model of septic shock. Surfactin C reduced the plasma endotoxin, tumor necrosis factor-alpha and nitric oxide levels in response to septic shock in rats.

The effect of polyclonal antibody on intracellular calcium increase induced by Mycoplasma hyopneumoniae in porcine tracheal cells.

An investigation was undertaken to assess whether polyclonal convalescent and hyperimmune sera obtained from pigs inhibit Mycoplasma hyopneumoniae induced increases in intracellular calcium [Ca2+](i) in ciliated porcine tracheal cells. Basal [Ca2+](i) in the tracheal cells was 97+/-13 nM (n=22 cells in four experiments) and after exposure to M. hyopneumoniae (300 micro g/mL or 10(11) CCU/mL), [Ca2+](i) increased by 246+/-56 nM within 100 s. After pre-treatment with hyperimmune or convalescent serum, M. hyopneumoniae increased [Ca2+](i) by 196+/-43 and 223+/-65 nM, respectively. It was found that neither hyperimmune nor convalescent serum significantly prevented the increase in [Ca2+](i) compared with M. hyopneumoniae alone. It was concluded that polyclonal antibodies produced by mycoplasma vaccination or exposure to the pathogen do not prevent M. hyopneumoniae-induced increase in [Ca2+](i).

The anti-thrombotic activity of surfactins.

Platelet aggregation was inhibited and the density of platelet-rich plasma (PRP) clots was decreased by the preincubation of PRP with surfactins, an acidic lipopeptide of Bacillus subtilis complex BC1212 isolated from soybean paste, in dose-dependent manner. Our findings suggest that surfactins are able to prevent a platelet aggregation leading to an inhibition of additional fibrin clot formation, and to enhance fibrinolysis with facilitated diffusion of fibrinolytic agents.

Surfactin C inhibits the lipopolysaccharide-induced transcription of interleukin-1beta and inducible nitric oxide synthase and nitric oxide production in murine RAW 264.7 cells.

The anti-inflammatory activity of the surfactin C derived from Bacillus subtilis isolate was investigated in lipopolysaccharide (LPS, 1 microg ml(-1))-treated mouse RAW 264.7 cells. LPS increased mRNA transcription of cyclooxygenase (COX)-2, interleukin (IL)-1beta and inducible nitric oxide synthase (iNOS). However, surfactin C at 50 microg ml(-1 )inhibited the LPS-induced increase in the transcription of IL-1beta and iNOS and nitric oxide (NO) production in a dose-dependent manner.