Proteomic analysis of swine hepatitis E virus (sHEV)-infected livers reveals upregulation of apolipoprotein and downregulation of ferritin heavy chain.

Swine hepatitis E virus (sHEV) has been discovered to be almost ubiquitous in pigs, and is antigenically and genetically related to human HEV. Proteomic analysis was used to identify altered protein expression in swine liver, using two-dimensional electrophoresis and peptide mass fingerprinting. A total of 10 protein spots exhibited significant alterations in the sHEV-infected organ. The upregulation of apolipoprotein E (Apo E) and downregulation of ferritin heavy chain were confirmed by Western analysis and by semi-quantitative reverse transcription-PCR. The elevated expression of Apo E may provide a novel insight into molecular responses to HEV infection in swine.

Neutralization of infectivity of porcine circovirus type 2 (PCV2) by capsid-binding 2'F-RNA aptamers.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated diseases (PCVD), which is responsible for economic losses in the swine industry. The capsid protein of PCV2 has important role for virus neutralization that blocks viral infection. To develop the therapeutic agents, two 2'F-RNA aptamers that bound to the PCV2 capsid protein with nanomole affinity were isolated from a 2'F-RNA library by the Systematic Evolution of Ligands by EXponential enrichment (SELEX). The binding affinity of aptamers was analyzed by Electrophoretic Mobility shift assay (EMSA) and surface plasmon resonance (SPR) analysis. The RNA aptamers have been shown to exhibit high affinity and specificity to PCV2 capsid protein and to neutralize PCV2 infectivity in PK-15 cells in dose dependent manner. Neutralizing aptamers such as this could be promising candidates in developing efficacious anti-PCV2 drugs as well as therapeutic delivery reagent.

Genomic expression profiling in lymph nodes with lymphoid depletion from porcine circovirus 2-infected pigs.

Porcine circovirus type 2 (PCV2) is the main causative agent of porcine circovirus-associated disease, such as post-weaning multisystemic wasting syndrome, which involves lymphocyte depletion. However, little is known about the molecular mechanisms of lymphoid depletion. To gain insight into the interaction between virus and host cells, microarrays were used to analyse changes in genomic expression in lymph nodes following PCV2 infection of pigs, together with negative controls. Total RNA was subjected to microarray analysis with an Affymetrix Porcine Genome Array GeneChip. Of the 23,256 pig genes arrayed on a chip, 160 genes showed altered expression after infection (upregulated, 64; downregulated, 96). The altered genomic expression of 18 selected genes was confirmed by quantitative real-time PCR. The expression changes of numerous genes involved in innate immune defence (TLR1, CD14 and CD180), immunosuppressed responses (FGL2 and GPNMB), pro-inflammatory signals (galectin-3) and fasting processes (ANGPTL-4) indicate that PCV2 has developed an intricate mechanism to cause immunosuppression, inflammatory cell infiltration and weight loss in pigs. The results of this study provide a basis for understanding the molecular pathogenesis of PCV2 infection.

CD4 T-cell help programs a change in CD8 T-cell function enabling effective long-term control of murine gammaherpesvirus 68: role of PD-1-PD-L1 interactions.

We previously showed that agonistic antibodies to CD40 could substitute for CD4 T-cell help and prevent reactivation of murine gammaherpesvirus 68 (MHV-68) in the lungs of major histocompatibility complex (MHC) class II(-/-) (CII(-/-)) mice, which are CD4 T cell deficient. Although CD8 T cells were required for this effect, no change in their activity was detected in vitro. A key question was whether anti-CD40 treatment (or CD4 T-cell help) changed the function of CD8 T cells or another cell type in vivo. To address this question, in the present study, we showed that adoptive transfer of CD8 T cells from virus-infected wild-type mice or anti-CD40-treated CII(-/-) mice caused a significant reduction in lung viral titers, in contrast to those from control CII(-/-) mice. Anti-CD40 treatment also greatly prolonged survival of infected CII(-/-) mice. This confirms that costimulatory signals cause a change in CD8 T cells enabling them to maintain effective long-term control of MHV-68. We investigated the nature of this change and found that expression of the inhibitory receptor PD-1 was significantly increased on CD8 T cells in the lungs of MHV-68-infected CII(-/-), CD40(-/-), or CD80/86(-/-) mice, compared with that in wild-type or CD28/CTLA4(-/-) mice, correlating with the level of viral reactivation. Furthermore, blocking PD-1-PD-L1 interactions significantly reduced viral reactivation in CD4 T-cell-deficient mice. In contrast, the absence of another inhibitory receptor, NKG2A, had no effect. These data suggest that CD4 T-cell help programs a change in CD8 T-cell function mediated by altered PD-1 expression, which enables effective long-term control of MHV-68.

Immune-enhancing effect of fermented Maesil (Prunus mume Siebold & Zucc.) with probiotics against Bordetella bronchiseptica in mice.

Maesil (Prunus mume) has long been used as a traditional drug and healthy food in East Asian countries. It possesses a number of beneficial biological activities including potential antimicrobial effects against pathogens. Probiotics also have antibacterial effects. Moreover, some probiotics have an important role in regulating the immune system. The present study evaluated the immune enhancing effects of fermented Maesil with probiotics (Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus acidophilus) in mice, especially against Bordetella bronchiseptica, as an initial step towards the development of feed supplements for the promotion of immune activity and prevention of disease, especially in pigs. Continuous ingestion of fermented Maesil with probiotics markedly increased the macrophage ratio in peripheral blood and the T lymphocyte ratio in the spleen. In addition, antibody production against formalin-killed B. bronchiseptica significantly increased in the mice fed fermented Maesil compared with the control group. The number of leukocytes was significantly higher in the bronchio-alveolar lavage obtained from the fermented Maesil-fed animals compared to it in the control group at day 3 (maximal peak time) after experimental B. bronchiseptica infection. Moreover, at 7 day post-infection, relative messenger RNA expression levels of tumor necrosis factor- α and interferon-γ were significantly increased in splenocytes of mice fed fermented Maesil compared with those in the control group. Taken together, these findings suggest that feed containing fermented Maesil with probiotics enhances immune activity in mice, especially against B. bronchiseptica, via the potent stimulation of non-specific immune responses.

Fermented Maesil (Prunus mume) with probiotics inhibits development of atopic dermatitis-like skin lesions in NC/Nga mice.

Maesil (Prunus mume Siebold & Zucc.), a potential source of free radical scavengers and inhibitor of pro-inflammatory mediators, is used in traditional Korean medical preparations as a remedy for skin disorders as have probiotics. The action of a probiotic fermented Maesil preparation on the development of atopic dermatitis (AD)-like skin lesions was determined in a NC/Nga mouse model as an initial step towards the development of a therapeutic feed supplement for use in dogs. Continuous ingestion of the experimental feed markedly inhibited the development of the AD-like skin lesions, as evidenced by a marked decrease in skin signs and reduced inflammation within the skin lesions. Efficacy was confirmed by significant decreases in eosinophil ratio and serum IgE concentration, and a reduction in the number of Staphylococcus aureus recovered from the ear. Relative mRNA expression levels of IL-4, interferon-gamma and tumour necrosis factor-alpha in the spleens of the experimental animals were also decreased and there was an increased serum concentration of IL-10 with a concurrent decreased IL-4 concentration in comparison to a control group. Taken together, the results indicate that some component(s) of fermented Maesil have the ability to suppress the development of AD-like skin lesions, possibly by stimulation of IL-10. Beneficial effects of fermented Maesil may thus be expected in dogs with AD, although this and the nature of the active pathway remain to be explored.

A mouse model of lethal synergism between influenza virus and Haemophilus influenzae.

Secondary bacterial infections that follow infection with influenza virus result in considerable morbidity and mortality in young children, the elderly, and immunocompromised individuals and may also significantly increase mortality in normal healthy adults during influenza pandemics. We herein describe a mouse model for investigating the interaction between influenza virus and the bacterium Haemophilus influenzae. Sequential infection with sublethal doses of influenza and H. influenzae resulted in synergy between the two pathogens and caused mortality in immunocompetent adult wild-type mice. Lethality was dependent on the interval between administration of the bacteria and virus, and bacterial growth was prolonged in the lungs of dual-infected mice, although influenza virus titers were unaffected. Dual infection induced severe damage to the airway epithelium and confluent pneumonia, similar to that observed in victims of the 1918 global influenza pandemic. Increased bronchial epithelial cell death was observed as early as 1 day after bacterial inoculation in the dual-infected mice. Studies using knockout mice indicated that lethality occurs via a mechanism that is not dependent on Fas, CCR2, CXCR3, interleukin-6, tumor necrosis factor, or Toll-like receptor-4 and does not require T or B cells. This model suggests that infection with virulent strains of influenza may predispose even immunocompetent individuals to severe illness on secondary infection with H. influenzae by a mechanism that involves innate immunity, but does not require tumor necrosis factor, interleukin-6, or signaling via Toll-like receptor-4.

Toxicological effects of acrylamide on rat testicular gene expression profile.

Toxicological effects of acrylamide on differential gene expression profile of rat testis were evaluated. Acrylamide induced morphological sperm defects, and decreased sperm concentration in cauda epididymis. Serum testosterone level and Leydig cell viability were also decreased dose-dependently, which resulted in decreased spermatogenesis. Acrylamide-induced histopathological lesions, such as formation of multinucleated giant cells and vacuolation, and numerous apoptotic cells were observed in seminiferous tubules. cDNA microarray analysis revealed that genes related to testicular-functions, apoptosis, cellular redox, cell growth, cell cycle, and nucleic acid-binding were up/down-regulated in testes isolated from acrylamide-treated group (60 mg/kg/day). Acrylamide toxicity appears to increase Leydig cell death and perturb gene expression levels, contributing to sperm defects and various abnormal histopathological lesions including apoptosis in rat testis.