Highly pathogenic porcine reproductive and respiratory syndrome virus-induced inflammatory response in porcine pulmonary microvascular endothelial cells and effects of herbal ingredients on main inflammatory molecules.

The role of microvascular endothelial cells (MVECs) in viral infection has received increasing attention. Our previous study demonstrated the susceptibility of porcine pulmonary MVECs to highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV), while their responses to the viral infection remain unclear. This study aimed to understand effects of the HP-PRRSV infection on functions of porcine pulmonary MVECs and the intervention effects of Chinese herbal ingredients on them. Highly purified porcine pulmonary MVECs were separated using CD31-immunomagnetic beads and infected with HP-PRRSV JXA1 and HN strain. The virus particles in cells and the ultrastructural pathological changes of cells were revealed by transmission electron microscopy. High-throughput transcriptome sequencing indicated that 104 and 228 genes were differentially expressed at 36 h post-infection, respectively, including many inflammatory molecules such as interleukins, chemokines, and adhesion molecules. The expression kinetics of HP-PRRSV-induced IL-1α, IL-6, IL-8, and VCAM-1 were characterized at the mRNA and protein levels. Luteolin significantly down-regulated HP-PRRSV-induced increase of the four molecules at both levels, and glycyrrhetinic acid and baicalin reduced that of IL-6 and VCAM-1. Our results suggest that porcine pulmonary MVECs play important roles in the inflammatory lung injury caused by HP-PRRSV infection and that herbal ingredients have potential regulatory effects on the HP-PRRSV-induced dysfunction of MVECs.

Tylvalosin demonstrates anti-parasitic activity and protects mice from acute toxoplasmosis.

AIMS: Toxoplasmosis, caused by Toxoplasma gondii (Tg), is one of the most prevalent zoonotic diseases worldwide. Currently, safe and efficient therapeutic options for this disease are still being developed, and are urgently needed. Tylvalosin (Tyl), a broad-spectrum third-generation macrolide, exhibits anti-bacterial, anti-viral, and anti-inflammatory properties. The present study aims to explore the anti-parasitic and immunomodulation activities of Tyl against Tg, and the underlying mechanism. MAIN METHODS: Adhesion, invasion, replication, proliferation, plaque, reversibility, immunofluorescence assays and transmission electron microscopy were utilized to determine the anti-Toxoplasma effect of Tyl. With acute toxoplasmosis model and rabies virus-induced brain inflammation model, the anti-toxoplasmosis and immunomodulation activities of Tyl were assessed by colorimetric assay, histopathological and Oil red O staining, and real-time quantitative PCR. The involved molecular mechanisms were investigated by western blotting and immunohistochemical staining. KEY FINDINGS: Tyl (5 and 10 µg/ml) can inhibit Tg propagation, and damage its ultrastructure irreversibly. The combination of Tyl and Pyrimethamine (Pyr) exhibits a better synergistic effect. Tyl (50 and 100 mg/kg) treatment intraperitoneally can delay mice death and improve survival rate, accompanying the reduced histopathological score and parasite load in the indicated tissues, espically for ileum, liver, spleen, lung and brain. Furthermore, Tg can modulate host phospho-p38 MAPK (pp38), subtilisin/kexin-isozyme-1 (SKI-1)-sterol regulatory element binding protein-1 (SREBP-1) (SKI-1-SREBP-1) pathway and peroxisome proliferators-activated receptor δ (PPARδ), while Tyl is able to reverse these signal pathways close to normal levels. SIGNIFICANCE: Our findings indicate that Tyl exhibits anti-Toxoplasma activity and protects mice from acute toxoplasmosis.

Lipid metabolism is a novel and practical source of potential targets for antiviral discovery against porcine parvovirus.

How parvovirus manipulates host lipid metabolism to facilitate its propagation, pathogenicity and consequences for disease, is poorly characterized. Here, we addressed this question using porcine parvovirus (PPV) to understand the complex interactions of parvovirus with lipid metabolism networks contributing to the identification of novel and practical antiviral candidates. PPV significantly alters host lipid composition, characteristic of subclasses of phospholipids and sphingolipids, and induces lipid droplets (LDs) formation via regulating calcium-independent PLA2ß (iPLA2ß), phospholipase Cγ2 (PLCγ2), diacylglycerol kinase α (DKGα), phosphoinositide 3-kinase (PI3K), lysophosphatidic acid acyltransferase θ (LPAATθ), and sphingosine kinases (SphK1 and SphK2). PPV utilizes and exploits these enzymes as well as their metabolites and host factors including MAPKs (p38 and ERK1/2), protein kinase C (PKC) and Ca2+ to induce S phase arrest, apoptosis and incomplete autophagy, all benefit to PPV propagation. PPV also suppresses prostaglandin E2 (PGE2) synthesis via downregulating cyclooxygenase-1 (COX-1), indicating PPV hijacks COX-1-PGE2 axis to evade immune surveillance. Our data support a model where PPV to establishes an optimal environment for its propagation and pathogenicity via co-opting host lipid metabolism, being positioned as a source of potential targets.

Isolation of a Divergent Strain of Bovine Parainfluenza Virus Type 3 (BPIV3) Infecting Cattle in China.

Bovine parainfluenza virus type 3 (BPIV3) is one of the most important known viral respiratory pathogens of both young and adult cattle. It is also named "heat stress in transport", causing morbidity and mass death. New variants of BPIV3 have been detected or isolated in China since 2008. Here, we isolate one BPIV3 strain (named BPIV3 BJ) in Madin-Darby bovine kidney (MDBK) cells from nasal samples collected in China. Phylogenetic analysis showed that our isolate is related to BPIV3 of the genotype A. The comparison of BPIV3-BJ and the reference Chinese isolate NM09 showed that these strains are highly divergent. We found many differences in the amino acid composition in the nucleocapsid (NP) protein among these genotype A strains. Since the NP protein has been implicated in immunization studies, our BPIV3 isolate will be useful for the development of immune assays and vaccine studies. The diversity of BPIV3 lineages that we found in China indicated ongoing evolution for immune escape. Our study highlights the importance of genetic surveillance for determining the effect of BPIV3 variability on pathogen evolution and population-scale immunity.

Ketotifen fumarate attenuates feline gingivitis related with gingival microenvironment modulation.

Gingivitis is evidenced by inflammation of the free gingiva, and still reversible. If left untreated, it may then progress to periodontitis. In the present study, the therapeutical effect of ketotifen fumarate on gingivitis was explored. Domestic cats with varying degrees of gingivitis naturally were enrolled in this study. Subgroups of animals were treated twice daily for one week with or without ketotifen fumarate (5 mg/kg). Effects of ketotifen fumarate were measured on gingival index, cells accumulation, mediators release, receptor-ligand interaction, oxidative stress, MAPK and NF-κB pathways, epithelial barrier and apoptosis. Ketotifen fumarate attenuated the initiation and progression of gingivitis, inhibited the infiltrations of mast cells, B lymphocytes, T lymphocytes, macrophages, neutrophils and eosinophils as well as the release of IgE, ß-hexosaminidase, tryptase, chymase, TNF-α, IL-4, and IL-13, influenced endothelial cells, fibroblasts and epithelial cells proliferation and apoptosis, and induced Th2 cells polarization, where ketotifen fumarate also might affect their interactions. Ketotifen fumarate reduced the oxidative stress, and inhibited NF-κB and p38 MAPK related with mast cells and macrophages accumulation. Ketotifen fumarate improved the aberrant expression of ZO-1 and inhibits the following apoptosis. On the other hand, these cells and mediators augmented functional attributes of them involving SCF/c-Kit, α4ß7/VCAM-1 and IL-8/IL-8RB interactions, thus creating a positive feedback loop to perpetuate gingivitis, where an inflammation microenvironment was modeled. Our results showed a previously unexplored therapeutic potential of ketotifen fumarate for gingivitis and further suggest that, in addition to biofilms, targeting inflammation microenvironment could be new strategy for the treatment of gingivitis/periodontitis.

CHIP mediates down-regulation of nucleobindin-1 in preosteoblast cell line models.

Nucleobindin-1 (NUCB1), also known as Calnuc, is a highly conserved, multifunctional protein widely expressed in tissues and cells. It contains two EF-hand motifs which have been shown to play a crucial role in binding Ca(2+) ions. In this study, we applied comparative two-dimensional gel electrophoresis to characterize differentially expressed proteins in HA-CHIP over-expressed and endogenous CHIP depleted MC3T3-E1 stable cell lines, identifying NUCB1 as a novel CHIP/Stub1 targeted protein. NUCB1 interacts with and is down-regulated by CHIP by both proteasomal dependent and independent pathways, suggesting that CHIP-mediated down-regulation of nucleobindin-1 might play a role in osteoblast differentiation. The chaperone protein Hsp70 was found to be important for CHIP and NUCB1 interaction as well as CHIP-mediated NUCB1 down-regulation. Our findings provide new insights into understanding the stability regulation of NUCB1.

Self-assembly and release of peste des petits ruminants virus-like particles in an insect cell-baculovirus system and their immunogenicity in mice and goats.

Peste des petits ruminants (PPR) is an acute, febrile, viral disease of small ruminants that has a significant economic impact. For many viral diseases, vaccination with virus-like particles (VLPs) has shown considerable promise as a prophylactic approach; however, the processes of assembly and release of peste des petits ruminants virus (PPRV) VLPs are not well characterized, and their immunogenicity in the host is unknown. In this study, VLPs of PPRV were generated in a baculovirus system through simultaneous expression of PPRV matrix (M) protein and hemaglutin in (H) or fusion (F) protein. The released VLPs showed morphology similar to that of the native virus particles. Subcutaneous injection of these VLPs (PPRV-H, PPRV-F) into mice and goats elicited PPRV-specific IgG production, increased the levels of virus neutralizing antibodies, and promoted lymphocyte proliferation. Without adjuvants, the immune response induced by the PPRV-H VLPs was comparable to that obtained using equivalent amounts of PPRV vaccine. Thus, our results demonstrated that VLPs containing PPRV M protein and H or F protein are potential "differentiating infected from vaccinated animals" (DIVA) vaccine candidates for the surveillance and eradication of PPR.

Tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of NF-κB activation.

Tylvalosin, a new broad-spectrum, third-generation macrolides, may exert a variety of pharmacological activities. Here, we report on its anti-oxidative and anti-inflammatory activity in RAW 264.7 macrophages and mouse treated with lipopolysaccharide (LPS) as well as piglet challenged with porcine reproductive and respiratory syndrome virus (PRRSV). Tylvalosin treatment markedly decreased IL-8, IL-6, IL-1ß, PGE2, TNF-α and NO levels in vitro and in vivo. LPS and PRRSV-induced reactive oxygen species (ROS) production, and the lipid peroxidation in mice lung tissues reduced after tylvalosin treatments. In mouse acute lung injury model induced by LPS, tylvalosin administration significantly attenuated tissues injury, and reduced the inflammatory cells recruitment and activation. The evaluated phospholipase A2 (PLA2) activity and the increased expressions of cPLA2-IVA, p-cPLA2-IVA and sPLA2-IVE were lowered by tylvalosin. Consistent with the mouse results, tylvalosin pretreatment attenuated piglet lung scores with improved growth performance and normal rectal temperature in piglet model induced by PRRSV. Furthermore, tylvalosin attenuated the IκBα phosphorylation and degradation, and blocked the NF-κB p65 translocation. These results indicate that in addition to its direct antimicrobial effect, tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury through suppression of NF-κB activation.

Intracellular delivery of artificial transcription factors fused to the protein transduction domain of HIV-1 Tat.

Protein transduction domains (PTDs), such as the TAT peptide derived from HIV Tat protein, may transduce macromolecules into cells. In the present study, the TAT peptide-fused artificial transcription factors (ATFs) were generated by fusion of the N-terminal TAT peptide with SV40 promoter-targeted three-fingered C2H2 zinc finger proteins and the KRAB transcriptional repression domain. The fusion proteins were then expressed in an E .coli system and purified by Ni-NTA affinity chromatography. The purified fusion proteins were tested on mammalian cell lines CHO DG44 and L929. TAT-ATF-S, which contains the zinc fingers that bind to the SV40 promoter with high specificity, exhibited the desired transcriptional repression activity to the reported genes, indicating the successful cellular delivery and desired conformation of TAT-ATF-S. Our study has provided a new strategy for intracellular ATF delivery.

Creation of a six-fingered artificial transcription factor that represses the hepatitis B virus HBx gene integrated into a human hepatocellular carcinoma cell line.

Chronic hepatitis B virus (HBV) infection is an independent risk factor for the development of hepatocellular carcinoma (HCC). The HBV HBx gene is frequently identified as an integrant in the chromosomal DNA of patients with HCC. HBx encodes the X protein (HBx), a putative viral oncoprotein that affects transcriptional regulation of several cellular genes. Therefore, HBx may be an ideal target to impede the progression of HBV infection-related HCC. In this study, integrated HBx was transcriptionally downregulated using an artificial transcription factor (ATF). Two three-fingered Cys2-His2 zinc finger (ZF) motifs that specifically recognized two 9-bp DNA sequences regulating HBx expression were identified from a phage-display library. The ZF domains were linked into a six-fingered protein that specified an 18-bp DNA target in the Enhancer I region upstream of HBx. This DNA-binding domain was fused with a Krüppel-associated box (KRAB) transcriptional repression domain to produce an ATF designed to downregulate HBx integrated into the Hep3B HCC cell line. The ATF significantly repressed HBx in a luciferase reporter assay. Stably expressing the ATF in Hep3B cells resulted in significant growth arrest, whereas stably expressing the ATF in an HCC cell line lacking integrated HBx (HepG2) had virtually no effect. The targeted downregulation of integrated HBx is a promising novel approach to inhibiting the progression of HBV infection-related HCC.

Establishment of tetracycline-inducible, survivin-expressing CHO cell lines by an optimized screening method.

An optimized method based on tetracycline-inducible gene expression system T-REx was developed to screen and evaluate Tet repressor (TetR)-expressing cell lines using enhanced green fluorescence protein (EGFP) as reporter gene. To verify the effectiveness of the method, two TetR-expressing Chinese hamster ovary (CHO) cell lines, CHO-TR B2 (stringent) and B5 (less stringent), in which the EGFP genes were variantly controlled by tetracycline, were used to construct cell lines expressing the anti-apoptosis gene survivin upon induction with tetracycline. The resulting stable clones were analyzed for survivin expression. The analysis showed that all four B5-derived clones exhibited leaky survivin expression in the absence of tetracycline, while the B2-derived clones did not. DNA laddering and annexin V/PI staining assays further indicated that although tetracycline-inducible expression of survivin conferred resistance to NH4Cl- and staurosporine-induced apoptosis in both the B2- and the B5-derived stable cell lines, the B2-derived cell lines showed more stringent regulation in the absence of tetracycline. This represents successful utilization of the present screening method.

Cytotoxicity and genotoxicity of 1,4-bisdesoxyquinocetone, 3-methylquinoxaline-2-carboxylic acid (MQCA) in human hepatocytes.

Quinoxaline-1,4-dioxides, widely used as medicinal feed additives as antibacterial growth promoters, have been shown to exert diverse toxicities. Their toxicities are hypothesized to be closely related to the formation of N-oxide reductive metabolites. 1,4-Bisdesoxyquinocetone and MQCA are important N-oxide reductive metabolites of quinocetone or olaquindox. In this study, we evaluated the cytotoxicity and genotoxicity of the metabolites, 1,4-bisdesoxyquinocetone and MQCA, as well as their parental drugs (quinocetone and olaquindox) in two human hepatocyte cell lines, L-02 and Chang liver cells. All these compounds inhibited the growth of cells in a dose-dependent and time-dependent manner by the MTT assay. Hormesis effects were found in L-02 cells treated with quinocetone at low doses. In the comet assay, although the two metabolites induced dose-related DNA damage in both cell lines, the levels of damage were less than that demonstrated for the parent drugs. The flow cytometric analysis showed that only the two metabolites induced cell cycle arrest at the S phase, and a decrease in the G0/G1, G2/M phase of Chang liver cells, which was not found for the L-02 cells treated with any compounds. The results indicate that 1,4-bisdesoxyquinocetone and MQCA are toxic to L-02 and Chang liver cells, and provide important new information towards understanding the olaquindox and quinocetone toxic mechanisms.

Immunolocalization of estrogen receptor α in Neomysis japonica oocytes and follicle cells during ovarian development.

Estrogen induces oocytes development and vitellogenesis in crustacean by interacting with estrogen receptor (ER) subtypes. In the present study, we detect for the first time the ERα in oocytes and follicle cells and hepatopancreas cells of mysis by immunohistochemistry using a specific ERα antibody. ERα was mainly localized in the nuclei of oocytes and follicle cells, while mainly detected in nuclei of oogonia (OG), previtellogenic oocyte (PR) and endogenous vitellogenic oocyte (EN) at previtellogenic and early vitellogenic stage (I-early III). Follicle cells in all stages of ovary (all vitellogenic stages) showed strong ERα positive reaction, and they were able to gradually move to oocytes during the development of oocytes. In addition, ERα was also localized in the nuclei and cytoplasm of four hepatopancreas cells (including E-, R-, F- and B-cell) in all ovary stages. These findings suggest, for the first time to our knowledge, that there could be a close link between oogenesis, follicle cells, hepatopancreas cells and endocrine regulation, and estrogens might be involved in the regulation of oocytes at early ovarian stage in mysis.

Microbial ecology of swine farms and PRRS vaccine vaccination strategies.

The present study investigated the microbial ecology and vaccination strategies against porcine reproductive and respiratory syndrome (PRRS) in field condition. Four representative farms with a history of PRRS were included in this study. Over the almost 3-year period, the average detection rate was 68.9%, making PRRSV the first most frequently detected virus, followed by porcine circovirus type 2 (PCV2) (64.2%), pseudorabies virus (PRV) (11.03%) and classical swine fever virus (CSFV) (4.41%). Streptococcus suis (77.92%), Haemophilus parasuis (51.25%) and Escherichia coli (52.39%), Pasteurella multocida (26.77%) were isolated most frequently in association with PRRSV. Under the present microbial ecology, production performances of sows their offspring after mass vaccination with a PRRS attenuated vaccine were evaluated. In addition, large scale PRRS vaccines usage and efficacy were further performed. The results indicated that mass vaccination following our immunization program can improve health status and production performances of both sows (2ml/i.m. booster after 4 weeks, and then immunized quarterly) and their offsprings (1ml/i.m. on 14-18 days of age).

Combination therapy of an inhibitor of group VIA phospholipase A2 with paclitaxel is highly effective in blocking ovarian cancer development.

We and others have shown that calcium-independent phospholipase A(2) (iPLA(2)) is involved in epithelial ovarian cancer (EOC). Hence, we propose that iPLA(2) is a potential effective and novel target for EOC. We tested this concept and found that bromoenol lactone (BEL), a selective inhibitor of iPLA(2), significantly inhibited EOC metastatic tumor growth in mouse xenograft models using human SKOV3 and HEY ovarian cancer cells. Moreover, the combination of BEL with paclitaxel (PTX), one of the most commonly used therapeutic agents in EOC, almost completely blocked tumor development in the xenograft mouse model. BEL showed no detectable cytotoxic effects in mice. Another iPLA(2) inhibitor, FKGK11, also inhibited tumor development in the xenograft mouse model, supporting that the major target of action was iPLA(2). The additional effects of BEL with PTX in vivo likely stem from their distinct cellular effects. BEL and FKGK11 reduced adhesion, migration, and invasion of EOC cells in vitro; the reduced ability to adhere, migrate, and invade seems to increase the vulnerability of tumor cells to PTX. These results provide an important basis for the development of new treatment modalities for EOC.

Immunogenicity of a DNA vaccine expressing the Neospora caninum surface protein NcSRS2 in mice.

The immunogenicity of a DNA vaccine expressing the surface protein NcSRS2 of Neospora caninum was studied in BALB/c mice. The NcSRS2-encoding DNA was obtained by PCR amplification of the NcSRS2 ORF gene from the p43 plasmid encoding the N. caninum surface protein NcSRS2, ligated to the mammalian expression vector pcDNA3.1/Zeo(+) and propagated in E. coli DH5alpha to produce the N. caninum NcSRS2 DNA vaccine. BALB/c mice were immunised by two intramuscular injections of the DNA vaccine with or without complete Freund's adjuvant (CFA). Serum antibody titres and nitric oxide (NO) concentrations, and splenocyte proliferation and cytokine expression were measured after immunisation. The DNA vaccine induced T-cell-mediated immunity as shown by significantly increased NO concentrations, cytokine gene (IL-2 and IFN-gamma) expression, and NcSRS2 protein-stimulated lymphocyte proliferation in mice immunised with the DNA vaccine. The vaccine also induced weak humoral immunity. The immunogenicity of the DNA vaccine was slightly enhanced by CFA. The immune response was specific to NcSRS2. No immune response was observed in mice immunised with the pcDNA3.1/Zeo(+) vector alone.

Cultivation, LD(50) determination and experimental model of Streptococcus suis serotype 2 strain HA9801.

The effects of nutritional components and submerged culture conditions on colony-forming unit (CFU) counts by Streptococcus suis serotype 2 strain HA9801 in flask culture was investigated, and the optimal medium and cultivation conditions was confirmed by using a 50l bioreactor. The LD(50) values of HA9801 in pigs before and after fermentation were 1.8 x 10(7)CFU, which indicated that the virulence of HA9801 was very stable in the fermentation process. In addition, an experimental model that closely mimics naturally occurring disease in conventional pigs was established.

Liquid chromatography-tandem mass spectrometry analysis of nitazoxanide and its major metabolites in goat.

A rapid, sensitive and specific liquid chromatography-electrospray ionization (ESI) tandem mass spectrometry (LC-MS-MS) method has been developed for the identification of nitazoxanide metabolites in goat plasma and urine. The purified samples was separated using an XTerra MS C8 column with the mobile phase consisted of acetonitrile and 10-mM ammonium acetate buffer (pH 2.5) followed a linear gradient elution, and detected by MS-MS. Identification and structural elucidation of the metabolites were performed by comparing their retention-times, full scan, product ion scan, precursor ion scan and neutral loss scan MS-MS spectra with those of the parent drug or other available standard. Four metabolites (tizoxanide, tizoxanide glucuronide, tizoxanide sulfate and hydroxylated tizoxanide sulfate) were found and identified in goat after single oral administration of 200mg/kg dose of nitazoxanide. In addition, the possible metabolic pathway was proposed for the first time. The results proved that the established method was simple, reliable and sensitive, revealing that it could be used to rapid screen and identify the structures of active metabolites responsible for pharmacological effects of nitazoxanide and to better understand its in vivo metabolism.