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Genital bacterial infection is one of the most important causes of infertility, however, bacteria frequently exist in seminal fluid. Sperm express Toll-like receptors (TLRs) on their cell surfaces and bacterial recognition by TLRs induces sperm apoptosis. In this study, we examined the lactoferrin (LF) potentiality on sperm apoptosis induced by bacterial lipopolysaccharide (LPS). The TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay indicated that TUNEL-positive sperm cells were scarce in the group treated with LF and LPS (LF/LPS group) compared to the group treated with LPS only (LPS group). In addition, real-time RT-PCR detected lower mRNA expression levels of apoptosis-associated genes in the LF/LPS group compared to the LPS group. These results indicate that LF treatment of semen might decrease LPS-induced apoptosis of sperm.

The effect of lactoferrin (LF) on embryo development was investigated by using lipopolysaccharide (LPS)-treated mouse sperm. For the development rate of the 2-cell stage embryo, the embryo derived from LPS- and LF-treated sperm showed similar survival rate to the control embryo. On day 12 after the embryo transfer into the recipient, the frequent abnormality was observed in the embryo derived from LPS-treated sperm, and the abnormality was tended to be inhibited in the embryo derived from LPS- and LF-treated sperm. These results imply that LF treatment on sperm contaminated with bacteria may facilitate the embryo development, which contribute to the improvement of infertility.

Bordetella bronchiseptica (B. bronchiseptica) is associated with respiratory tract infections in laboratory animals. In our laboratory animal facility, B. bronchiseptica was isolated from 21 of 27 apparently healthy rabbits obtained from a breeding farm contaminated with B. bronchiseptica. Restriction fragment length polymorphism (RFLP) analysis showed that the flagellin genotype of isolates from the laboratory animal facility and breeding farm was type A, which is seen relatively frequently in rabbits in Europe. To examine its pathogenicity, guinea pigs, rats, and mice were inoculated intranasally with a representative strain isolated in the laboratory animal facility. Following inoculation of 10

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection., Author summary We show here that stimulation of PrPC with anti-PrP mAbs protected mice from lethal infection with IAVs by stimulating macrophage polarization to an anti-inflammatory M2 phenotype through activation of SFKs in infected lungs. IAVs are causative agents for seasonal epidemic outbreaks of influenza, and often cause high morbidity and mortality in infected people, particularly in the young and elderly and those with underlying chronic diseases. It has also been reported that highly pathogenic avian IAVs have the potential to infect humans and cause high morbidity and mortality in infected individuals. The other problem with IAV infections is that new IAV strains, which are resistant to currently available anti-influenza agents such as neuraminidase inhibitors, have been emerging in human populations. Our current results showing that targeting PrPC with anti-PrP mAbs protected against lethal infection with IAVs in mice give rise to the possibility that PrPC-targeting therapeutics might be beneficial in influenza infection. Moreover, since PrPC is a host molecule, PrPC-targeting drugs might not induce drug-resistant IAVs. Thus, PrPC might be a novel target molecule for therapeutic development for IAV infection, although further studies are needed.

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), possessing terminal sialic acid residues that suppress host immune response and provide a survival advantage to the pathogen. CPS binds to Siglec-9 expressed on neutrophils, which is expected to down-regulate the immune responsiveness of neutrophils. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of CPS to Siglec-9 on immune cells, leading to provide antibacterial benefit against GBS infection in the transgenic mouse line expressing sSiglec-9 (sSiglec-9 Tg). The sSiglec-9 in the sera of sSiglec-9 Tg bound to the sialylated-GBS strains belonging to serotypes Ia, Ib, II, III, IV and V in whole GBS cell ELISA. When GBS cells of serotype III that is a common serotype in late-onset GBS disease (LOD) were intraperitoneally inoculated into sSiglec-9 Tg, sSiglec-9 Tg showed a significant resistance as compared with non-transgenic littermates. Furthermore, GBS serotype III organisms were not detected in cultures of the blood from surviving mice (

The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPΔOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp0/0 and Tg(PrPΔOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics., Author summary Influenza A virus (IAV) is an enveloped, negative sense, single-stranded RNA virus, causing seasonal epidemic outbreaks of influenza. Anti-influenza agents targeting viral molecules, such as neuraminidase inhibitors, are currently available. However, these agents have accelerated emergence of mutant IAVs that are resistant to these agents among human populations. Development of new types of anti-influenza agents is awaited. We show that the cellular prion protein PrPC has a protective role against lethal infection with IAVs through the octapeptide repeat (OR) region by abrogating lung epithelial cell apoptosis induced by reactive oxygen species (ROS) in infected lungs. We also show that PrPC could reduce ROS in IAV-infected lungs through the OR region by maintaining Cu ion homeostasis and thereby activating Cu/Zn-dependent superoxide dismutase, SOD1. These results highlight the protective role of PrPC in IAV infection. Elucidation of the exact mechanism underlying the PrPC-mediated protection against IAV infection would be important for further understanding the pathogenesis of IAV infection and could be useful for development of new types of anti-influenza therapeutics.

The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml−1 showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic., Preventive Veterinary Medicine, 103(2-3), pp.192-200; 2012

Pseudorabies virus is an alphaherpesvirus causing fatal neurological diseases in animals. Pseudorabies virus carries a gene encoding immediate-early (IE) protein IE180, which controls the transcription of other viral and host cell genes. Previously, we reported that transgenic expression of IE180 in mice causes severe ataxia and cerebellar deformity. Here we identified profound abnormalities in adult IE180 transgenic mice, including malpositioning of Purkinje cells (PCs), granule cells (GCs) and Bergmann glia (BG), impaired dendritogenesis and synaptogenesis in PCs, disoriented BG fibers, absence of molecular layer interneurons, and increased apoptosis of neurons and glia. In accordance with the cellular defects, we found the expression of IE180 in PCs, GCs and astrocytes during cerebellar development. We next examined transgenic mice expressing truncated IE180 mutants: dlN132 lacking the acidic transcriptional active domain, dlC629 lacking the nuclear localization signal and dlC1081 having all known domains but lacking the carboxyl-terminal sequence. Despite similar expression levels of the transgenes, ataxia and cerebellar defects were only manifested in the dlC1081 transgenic mice but their phenotypes were milder compared with the IE180 transgenic mice. In the dlC1081 transgenic mice, cerebellar neurons and glia were normally positioned but cerebellar size was severely reduced due to GC deficits. Interestingly, dlC1081 was mainly expressed in the GCs with low expression in a few BG. Taken together, the present findings clarified a causal relationship between cerebellar pathology and cellular expression of IE180, and further afforded an experimental insight into different symptomatic severity as a consequence of different cellular defects caused by such cytotoxic viral agents.

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

Neospora caninum was isolated from the brain of a 2-year-old dairy cow that had aborted confirmed N. caninum-infected fetuses on two occasions. The cow had an indirect fluorescent antibody titer of 1:1600 to N. caninum. The cow was killed 24 days after its second abortion and the brain was bioassayed for N. caninum in nude mice. Multifocal areas of perivascular cuffing and glial nodules were observed in the cerebrum and mesencephalon of the cow, but N. caninum was not identified in histological sections of the brain. All three nude mice inoculated with brain homogenate of the cow, developed emaciation and paralysis. Microscopical examination of the nude mice revealed systemic N. caninum infection with demonstrable tachyzoites in various organs. The parasites isolated from fresh mouse brain were transferred successfully into Vero cell cultures. PCR procedure on the purified tachyzoites obtained from the Vero cell cultures amplified the specific DNA sequence for N. caninum.

Insulin stimulates glucose transporter 4 (GLUT4) vesicle recruitment from its intracellular storage site to the plasma membrane. Cysteine string protein 1 (CSP1) is a SNARE-binding protein involved in the vesicular trafficking of neurotransmitters and other exocytic processes. In this study, we investigated the involvement of CSP1 in insulin-dependent GLUT4 recruitment in 3T3-L1 adipocytes. Over-expression of wild-type CSP1 led to attenuated insulin-stimulated glucose uptake without any change in GLUT4 content in the plasma membrane, rather it inhibits docking by blocking the association of VAMP2 with syntaxin 4. In contrast, knockdown of CSP1 enhanced insulin-stimulated glucose uptake. The mRNA and protein expression of CSP1 was elevated in 3T3-L1 adipocytes in insulin resistant states caused by high levels of palmitate and chronic insulin exposure. Taken together, the results of this study suggest that CSP1 is involved in insulin resistance by interrupting GLUT4 vesicle docking with the plasma membrane.

Western blotting was performed to analyze Neospora caninum tachyzoite antigens recognized by mouse IgG at different stages of infection including recrudescence. At the early stage of infection, a 36-38 kDa antigen was clearly recognized by the mouse antisera. After day 48 postinoculation, the signal of the 36-38 kDa antigen gradually weakened. Meanwhile, a 43 kDa antigen was intensely and continuously recognized from 48 to 125 days postinoculation. This 43 kDa antigen was clearly detectable with the antisera from the mice under immunosuppression. Sera from naturally infected cattle strongly reacted with the 43 kDa antigen. Therefore, the 43 kDa antigen may be useful for immunological reactions to detect infected animals except in the early stage of the infection.

Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D (gD). Porcine nectin-1 mediates entry of pseudorabies virus (PRV), herpes simplex virus types 1 and 2 (HSV-1 and HSV-2), and bovine herpesvirus type 1 (BHV-1). The gD-binding domain of nectin-1 is the first or N-terminal immunoglobulin (Ig)-like domain of the entire ectodomain. Here, we generated three transgenic mouse lines expressing a fusion protein consisting of the first Ig-like domain of porcine nectin-1 and the Fc portion of porcine IgG1 to assess the antiviral potential of the first Ig-like domain of nectin-1 in vivo. All of the transgenic mouse lines showed significant resistance to PRV infection via intraperitoneal inoculation (survival rates of 67% to 100%). In the intranasal challenge, a lower but still significant protection was observed; 21% to 55% of the animals from the three transgenic mouse lines survived. The present results demonstrate that a soluble form of the first domain of porcine nectin-1 is able to exert a significant antiviral effect against pseudorabies virus infection.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a transgene encoding a soluble form of nectin-1, also known as herpesvirus entry mediator C. Nectin-1 is an α-herpesvirus receptor that binds to virion glycoprotein D. Nectin-1 mediates entry of PRV, herpes simplex virus types 1 and 2, and bovine herpesvirus type 1. To assess the antiviral potential of an ectopic expression of the nectin-1 ectodomain in vivo , six transgenic mouse lines expressing a soluble form of nectin-1, consisting of an extracellular domain of porcine nectin-1 and the Fc portion of human IgG1, were generated. All of the transgenic mouse lines showed nearly complete resistance to PRV infection by means of both i.p. and intranasal routes. These results suggest that the introduction into farm animals of a transgene encoding a soluble form of nectin-1 would offer a potent biological approach to generating α-herpesvirus-resistant livestock.

The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.

Purpose Pseudorabies virus (PRV), a representative member of the alpha-herpesvirus family, causes nervous symptoms and ocular lesions, such as keratoconjunctivitis and retinal degeneration in piglets. The immediate-early protein IE180 of the PRV is known to be essential, not only in viral gene expression, but also in the cellular gene expression in host cells. The purpose of this study was to examine the effect of IE180 on the development of the mouse eye, by using transgenic technology. Methods Transgenic mice expressing IE180 were generated and their eyes analyzed by histology, immunocytochemistry, and the bromodeoxyuridine cell proliferation assay. Results A fibrovascular retrolental tissue analogous to persistent hyperplastic primary vitreous (PHPV) in humans was observed in a transgenic mouse line expressing IE180. The gross anatomy of the eye showed white pupils. Analysis of hematoxylin and eosin-stained sections revealed that the retrolental tissue adhered to the neuroretina, the inner nuclear and ganglion cell layers were disorganized, and rosettelike arrangements of dysplastic photoreceptor cells were present. Bromodeoxyuridine-positive cells were detected in the retrolental tissues of postnatal day (P)1, P7, and P14 mice. The retrolental mass in the P7 transgenic mouse was composed of melanocytes and endothelial cells, which were detected by a cocktail of antibodies against endoglin, CD31, and VEGF receptor-2. Conclusions The observation that the eye disease in transgenic mice is similar to that in PHPV in humans raises the possibility that expression of the immediate-early gene of alpha-herpesviruses may contribute to PHPV.