Neurotropic murine coronavirus mediated demyelination: Factors dampening pathogenesis.

Virus infections and autoimmune responses are implicated as primary triggers of demyelinating diseases. Specifically, the association of Epstein-Barr virus (EBV) infection with development of multiple sclerosis (MS) has re-ignited an interest in virus induced autoimmune responses to CNS antigens. Nevertheless, demyelination may also be caused by immune mediated bystander pathology in an attempt to control direct infection in the CNS. Tissue damage as a result of anti-viral responses or low level viral persistence may lead to immune activation manifesting in demyelinating lesions, axonal damage and clinical symptoms. This review focuses on the neurotropic mouse coronavirus induced demyelination model to highlight how immune responses activated during the acute phase pave the way to dampen pathology and promote repair. We specifically discuss the role of immune dampening factors programmed cell death ligand 1 (PD-L1) and interleukin (IL)-10, as well as microglia and triggering receptor expressed on myeloid cells 2 (Trem2), in limiting demyelination independent of viral persistence.

The Impact of Innate Components on Viral Pathogenesis in the Neurotropic Coronavirus Encephalomyelitis Mouse Model.

Recognition of viruses invading the central nervous system (CNS) by pattern recognition receptors (PRRs) is crucial to elicit early innate responses that stem dissemination. These innate responses comprise both type I interferon (IFN-I)-mediated defenses as well as signals recruiting leukocytes to control the infection. Focusing on insights from the neurotropic mouse CoV model, this review discusses how early IFN-I, fibroblast, and myeloid signals can influence protective anti-viral adaptive responses. Emphasis is placed on three main areas: the importance of coordinating the distinct capacities of resident CNS cells to induce and respond to IFN-I, the effects of select IFN-stimulated genes (ISGs) on host immune responses versus viral control, and the contribution of fibroblast activation and myeloid cells in aiding the access of T cells to the parenchyma. By unraveling how the dysregulation of early innate components influences adaptive immunity and viral control, this review illustrates the combined effort of resident CNS cells to achieve viral control.

Trem2 deficiency impairs recovery and phagocytosis and dysregulates myeloid gene expression during virus-induced demyelination.

BACKGROUND: Triggering receptor expressed on myeloid cells 2 (Trem2) plays a protective role in neurodegenerative diseases. By contrast, Trem2 functions can exacerbate tissue damage during respiratory viral or liver infections. We, therefore, investigated the role of Trem2 in a viral encephalomyelitis model associated with prominent Th1 mediated antiviral immunity leading to demyelination. METHODS: Wild-type (WT) and Trem2 deficient (Trem2-/-) mice were infected with a sublethal glia tropic murine coronavirus (MHV-JHM) intracranially. Disease progression and survival were monitored daily. Leukocyte accumulation and pathological features including demyelination and axonal damage in spinal cords (SC) were determined by flow cytometry and tissue section immunofluorescence analysis. Expression of select inflammatory cytokines and chemokines was measured by RT-PCR and global myeloid cell gene expression in SC-derived microglia and infiltrated bone-marrow-derived macrophages (BMDM) were determined using the Nanostring nCounter platform. RESULTS: BMDM recruited to SCs in response to infection highly upregulated Trem2 mRNA compared to microglia coincident with viral control. Trem2 deficiency did not alter disease onset or severity, but impaired clinical recovery after onset of demyelination. Disease progression in Trem2-/- mice could not be attributed to altered virus control or an elevated proinflammatory response. A prominent difference was increased degenerated myelin not associated with the myeloid cell markers IBA1 and/or CD68. Gene expression profiles of SC-derived microglia and BMDM further revealed that Trem2 deficiency resulted in impaired upregulation of phagocytosis associated genes Lpl and Cd36 in microglia, but a more complex pattern in BMDM. CONCLUSIONS: Trem2 deficiency during viral-induced demyelination dysregulates expression of other select genes regulating phagocytic pathways and lipid metabolism, with distinct effects on microglia and BMDM. The ultimate failure to remove damaged myelin is reminiscent of toxin or autoimmune cell-induced demyelination models and supports that Trem2 function is regulated by sensing tissue damage including a dysregulated lipid environment in very distinct inflammatory environments.

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.

Neuronal Ablation of Alpha/Beta Interferon (IFN-α/ß) Signaling Exacerbates Central Nervous System Viral Dissemination and Impairs IFN-γ Responsiveness in Microglia/Macrophages.

Alpha/beta interferon (IFN-α/ß) signaling through the IFN-α/ß receptor (IFNAR) is essential to limit virus dissemination throughout the central nervous system (CNS) following many neurotropic virus infections. However, the distinct expression patterns of factors associated with the IFN-α/ß pathway in different CNS resident cell populations implicate complex cooperative pathways in IFN-α/ß induction and responsiveness. Here we show that mice devoid of IFNAR1 signaling in calcium/calmodulin-dependent protein kinase II alpha (CaMKIIα) expressing neurons (CaMKIIcre:IFNARfl/fl mice) infected with a mildly pathogenic neurotropic coronavirus (mouse hepatitis virus A59 strain [MHV-A59]) developed severe encephalomyelitis with hind-limb paralysis and succumbed within 7 days. Increased virus spread in CaMKIIcre:IFNARfl/fl mice compared to IFNARfl/fl mice affected neurons not only in the forebrain but also in the mid-hind brain and spinal cords but excluded the cerebellum. Infection was also increased in glia. The lack of viral control in CaMKIIcre:IFNARfl/fl relative to control mice coincided with sustained Cxcl1 and Ccl2 mRNAs but a decrease in mRNA levels of IFNα/ß pathway genes as well as Il6, Tnf, and Il1ß between days 4 and 6 postinfection (p.i.). T cell accumulation and IFN-γ production, an essential component of virus control, were not altered. However, IFN-γ responsiveness was impaired in microglia/macrophages irrespective of similar pSTAT1 nuclear translocation as in infected controls. The results reveal how perturbation of IFN-α/ß signaling in neurons can worsen disease course and disrupt complex interactions between the IFN-α/ß and IFN-γ pathways in achieving optimal antiviral responses.IMPORTANCE IFN-α/ß induction limits CNS viral spread by establishing an antiviral state, but also promotes blood brain barrier integrity, adaptive immunity, and activation of microglia/macrophages. However, the extent to which glial or neuronal signaling contributes to these diverse IFN-α/ß functions is poorly understood. Using a neurotropic mouse hepatitis virus encephalomyelitis model, this study demonstrated an essential role of IFN-α/ß receptor 1 (IFNAR1) specifically in neurons to control virus spread, regulate IFN-γ signaling, and prevent acute mortality. The results support the notion that effective neuronal IFNAR1 signaling compensates for their low basal expression of genes in the IFN-α/ß pathway compared to glia. The data further highlight the importance of tightly regulated communication between the IFN-α/ß and IFN-γ signaling pathways to optimize antiviral IFN-γ activity.

Intercellular Communication Is Key for Protective IFNα/ß Signaling During Viral Central Nervous System Infection.

A variety of viruses can induce central nervous system (CNS) infections and neurological diseases, although the incidence is rare. Similar to peripheral infections, IFNα/ß induction and signaling constitutes a first line of defense to limit virus dissemination. However, CNS-resident cells differ widely in their repertoire and magnitude of both basal and inducible components in the IFNα/ß pathway. While microglia as resident myeloid cells have been implicated as prominent sentinels of CNS invading pathogens or insults, astrocytes are emerging as key responders to many neurotropic RNA virus infections. Focusing on RNA viruses, this review discusses the role of astrocytes as IFNα/ß inducers and responders and touches on the role of IFNα/ß receptor signaling in regulating myeloid cell activation and IFNγ responsiveness. A summary picture emerges implicating IFNα/ß not only as key in establishing the classical "antiviral" state, but also orchestrating cell mobility and IFNγ-mediated effector functions.

Dynamics of Virus-Specific Memory B Cells and Plasmablasts following Viral Infection of the Central Nervous System.

Humoral responses within the central nervous system (CNS) are common to many neurotropic viral infections, with antibody (Ab)-secreting cells (ASC) contributing to local protection. However, a role for virus-specific memory B cells (Bmem) within the CNS is poorly explored due to lack of robust phenotypic or functional identification in mice. This study takes advantage of the progeny of mice expressing tamoxifen-inducible Cre recombinase (Cre-ERT2) under the Aicda promoter crossed with Rosa26-loxP-tdTomato reporter mice (AIDCre-Rosa26tdTomato) to monitor B cells having undergone activation-induced cytidine deaminase (AID)-mediated somatic hypermutation (SHM) following neurotropic coronavirus infection. AID detection via tdTomato expression allowed tracking of virus-specific ASC and Bmem in priming and effector sites throughout infection. In draining lymph nodes, tdTomato-positive (tdTomato+) ASC were most prevalent prior to germinal center (GC) formation, but total tdTomato+ B cells only peaked with robust GC formation at day 14 p.i. Moreover, their proportion of Bmem dominated over the proportion of ASC throughout infection. In the CNS, tdTomato+ cells started emerging at day 14 p.i. While they initially comprised mainly Bmem, the proportions of ASC and Bmem became similar as tdTomato+ B cells increased throughout viral persistence. Delayed tamoxifen treatment demonstrated ongoing CNS recruitment of tdTomato+ B cells, mainly ASC, primed late during GC reactions. Overall, the data support the idea that virus-induced B cells exhibiting SHM require peripheral GC formation to emerge in the CNS. Ongoing GC reactions and regional signals further regulate dynamics within the CNS, with preferential maintenance of tdTomato+ B cells in spinal cords relative to that in brains during viral persistence.IMPORTANCE The prevalence and role of antigen-specific Bmem in the CNS during viral encephalomyelitis is largely undefined. A lack of reliable markers identifying murine Bmem has made it difficult to assess their contribution to local antiviral protection via antigen presentation or conversion to ASC. Using reporter mice infected with neurotropic coronavirus to track virus-specific Bmem and ASC, this report demonstrates that both subsets only emerge in the CNS following peripheral GC formation and subsequently prevail. While early GC reactions supported preferential Bmem accumulation in the CNS, late GC reactions favored ASC accumulation, although Bmem outnumbered ASC in draining lymph nodes throughout infection. Importantly, virus-specific B cells undergoing sustained GC selection were continually recruited to the persistently infected CNS. Elucidating the factors governing temporal events within GCs, as well as regional CNS cues during viral persistence, will aid intervention to modulate CNS humoral responses in the context of infection and associated autoimmune pathologies.

Alpha/Beta Interferon (IFN-α/ß) Signaling in Astrocytes Mediates Protection against Viral Encephalomyelitis and Regulates IFN-γ-Dependent Responses.

The contribution of distinct central nervous system (CNS) resident cells to protective alpha/beta interferon (IFN-α/ß) function following viral infections is poorly understood. Based on numerous immune regulatory functions of astrocytes, we evaluated the contribution of astrocyte IFN-α/ß signaling toward protection against the nonlethal glia- and neuronotropic mouse hepatitis virus (MHV) strain A59. Analysis of gene expression associated with IFN-α/ß function, e.g., pattern recognition receptors (PRRs) and interferon-stimulated genes (ISGs), revealed lower basal mRNA levels in brain-derived astrocytes than in microglia. Although astrocytes poorly induced Ifnß mRNA following infection, they upregulated various mRNAs in the IFN-α/ß pathway to a higher extent than microglia, supporting effective IFN-α/ß responsiveness. Ablation of the IFN-α/ß receptor (IFNAR) in astrocytes using mGFAPcre IFNARfl/fl mice resulted in severe encephalomyelitis and mortality, coincident with uncontrolled virus replication. Further, virus spread was not restricted to astrocytes but also affected microglia and neurons, despite increased and sustained Ifnα/ß and ISG mRNA levels within the CNS. IFN-γ, a crucial mediator for MHV control, was not impaired in infected mGFAPcre IFNARfl/fl mice despite reduced T cell CNS infiltration. Unexpectedly however, poor induction of IFN-γ-dependent major histocompatibility complex (MHC) class II expression on microglia supported that defective IFN-γ signaling contributes to uncontrolled virus replication. A link between sustained elevated IFN-α/ß and impaired responsiveness to IFN-γ supports the novel concept that temporally limited early IFN-α/ß responses are critical for effective antiviral IFN-γ function. Overall, our results imply that IFN-α/ß signaling in astrocytes is not only critical in limiting early CNS viral spread but also promotes protective antiviral IFN-γ function.IMPORTANCE An antiviral state established by IFN-α/ß contains initial viral spread as adaptive immunity develops. While it is apparent that the CNS lacks professional IFN-α/ß producers and that resident cells have distinct abilities to elicit innate IFN-α/ß responses, protective interactions between inducer and responder cells require further investigation. Infection with a glia- and neuronotropic coronavirus demonstrates that astrocytes mount a delayed but more robust response to infection than microglia, despite their lower basal mRNA levels of IFN-α/ß-inducing components. Lethal, uncontrolled viral dissemination following ablation of astrocyte IFN-α/ß signaling revealed the importance of IFN-α/ß responses in a single cell type for protection. Sustained global IFN-α/ß expression associated with uncontrolled virus did not suffice to protect neurons and further impaired responsiveness to protective IFN-γ. The results support astrocytes as critical contributors to innate immunity and the concept that limited IFN-α/ß responses are critical for effective subsequent antiviral IFN-γ function.

Early endonuclease-mediated evasion of RNA sensing ensures efficient coronavirus replication.

Coronaviruses are of veterinary and medical importance and include highly pathogenic zoonotic viruses, such as SARS-CoV and MERS-CoV. They are known to efficiently evade early innate immune responses, manifesting in almost negligible expression of type-I interferons (IFN-I). This evasion strategy suggests an evolutionary conserved viral function that has evolved to prevent RNA-based sensing of infection in vertebrate hosts. Here we show that the coronavirus endonuclease (EndoU) activity is key to prevent early induction of double-stranded RNA (dsRNA) host cell responses. Replication of EndoU-deficient coronaviruses is greatly attenuated in vivo and severely restricted in primary cells even during the early phase of the infection. In macrophages we found immediate induction of IFN-I expression and RNase L-mediated breakdown of ribosomal RNA. Accordingly, EndoU-deficient viruses can retain replication only in cells that are deficient in IFN-I expression or sensing, and in cells lacking both RNase L and PKR. Collectively our results demonstrate that the coronavirus EndoU efficiently prevents simultaneous activation of host cell dsRNA sensors, such as Mda5, OAS and PKR. The localization of the EndoU activity at the site of viral RNA synthesis-within the replicase complex-suggests that coronaviruses have evolved a viral RNA decay pathway to evade early innate and intrinsic antiviral host cell responses.

Distinct CD4 T-cell effects on primary versus recall CD8 T-cell responses during viral encephalomyelitis.

CD4 T-cell help is not a universal requirement for effective primary CD8 T cells but is essential to generate memory CD8 T cells capable of recall responses. This study examined how CD4 T cells affect primary and secondary anti-viral CD8 T-cell responses within the central nervous system (CNS) during encephalomyelitis induced by sublethal gliatropic coronavirus. CD4 T-cell depletion before infection did not impair peripheral expansion, interferon-γ production, CNS recruitment or initial CNS effector capacity of virus-specific CD8 T cells ex vivo. Nevertheless, impaired virus control in the absence of CD4 T cells was associated with gradually diminished CNS CD8 T-cell interferon-γ production. Furthermore, within the CD8 T-cell population short-lived effector cells were increased and memory precursor effector cells were significantly decreased, consistent with higher T-cell turnover. Transfer of memory CD8 T cells to reduce viral load in CD4-depleted mice reverted the recipient CNS CD8 T-cell phenotype to that in wild-type control mice. However, memory CD8 T cells primed without CD4 T cells and transferred into infected CD4-sufficient recipients expanded less efficiently and were not sustained in the CNS, contrasting with their helped counterparts. These data suggest that CD4 T cells are dispensable for initial expansion, CNS recruitment and differentiation of primary resident memory CD8 T cells as long as the duration of antigen exposure is limited. By contrast, CD4 T cells are essential to prolong primary CD8 T-cell function in the CNS and imprint memory CD8 T cells for recall responses.

IL-21 optimizes T cell and humoral responses in the central nervous system during viral encephalitis.

Acute coronavirus encephalomyelitis is controlled by T cells while humoral responses suppress virus persistence. This study defines the contribution of interleukin (IL)-21, a regulator of T and B cell function, to central nervous system (CNS) immunity. IL-21 receptor deficiency did not affect peripheral T cell activation or trafficking, but dampened granzyme B, gamma interferon and IL-10 expression by CNS T cells and reduced serum and intrathecal humoral responses. Viral control was already lost prior to humoral CNS responses, but demyelination remained comparable. These data demonstrate a critical role of IL-21 in regulating CNS immunity, sustaining viral persistence and preventing mortality.

CD4 T cells promote CD8 T cell immunity at the priming and effector site during viral encephalitis.

CD4 T cell activation during peripheral infections not only is essential in inducing protective CD8 T cell memory but also promotes CD8 T cell function and survival. However, the contributions of CD4 T cell help to antiviral CD8 T cell immunity during central nervous system (CNS) infection are not well established. Encephalitis induced by the sublethal coronavirus JHMV was used to identify when CD4 T cells regulate CD8 T cell responses following CNS infection. Peripheral expansion of virus-specific CD8 T cells was impaired when CD4 T cells were ablated prior to infection but not at 4 days postinfection. Delayed CD4 T cell depletion abrogated CD4 T cell recruitment to the CNS but only slightly diminished CD8 T cell recruitment. Nevertheless, the absence of CNS CD4 T cells was associated with reduced gamma interferon (IFN-γ) and granzyme B expression by infiltrating CD8 T cells, increased CD8 T cell apoptosis, and impaired control of infectious virus. CD4 T cell depletion subsequent to CD4 T cell CNS migration restored CD8 T cell activity and virus control. Analysis of γc-dependent cytokine expression indicated interleukin-21 (IL-21) as a primary candidate optimizing CD8 T cell activity within the CNS. These results demonstrate that CD4 T cells play critical roles in both enhancing peripheral activation of CD8 T cells and prolonging their antiviral function within the CNS. The data highlight the necessity for temporally and spatially distinct CD4 T cell helper functions in sustaining CD8 T cell activity during CNS infection.

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.

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.