IFN-α mRNA Expression Research Articles

Duck Tembusu virus infection activates the MKK3/6-p38 MAPK signaling pathway to promote virus replication

Duck Tembusu virus (DTMUV) infection poses a serious threat to ducks, chickens, and geese, causing a range of detrimental effects, including reduced egg production, growth retardation, and even death. These consequences lead to substantial economic losses for the Chinese poultry industry. Although it is established that various viral infections can trigger activation of the p38 mitogen-activated protein kinase (MAPK) signaling pathway, the precise role and mechanisms underlying p38 MAPK activation in DTMUV infection remain poorly understood. To address this knowledge gap, we conducted a study to investigate whether the replication of DTMUV necessitates the activation of p38 MAPK. We found that DTMUV infection stimulates activation of the MKK3/6-p38 MAPK pathway, and the activation of p38 MAPK increases with viral titer. Subsequently, the use of the small molecule inhibitor SB203580 significantly reduced DTMUV replication by inhibiting p38 MAPK activity. Furthermore, downregulation of p38 MAPK protein expression by siRNA also inhibited DTMUV replication, whereas transient transfection of p38 MAPK protein promoted DTMUV replication. Interestingly, we found that the DTMUV capsid protein activates p38 MAPK, and there is interaction between DTMUV capsid and p38 MAPK. Finally, we found that DTMUV infection induces elevated mRNA expression of IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, and IL-12, which is associated with p38 MAPK activity. These results indicated that virus hijacking of p38 activation is a crucial event for DTMUV replication, and that pharmacological blockade of p38 activation represents a potential anti-DTMUV strategy.

The critical role of interleukin-6 in protection against neurotropic flavivirus infection.

West Nile virus (WNV) and Japanese encephalitis virus (JEV) are emerging mosquito-borne flaviviruses causing encephalitis globally. No specific drug or therapy exists to treat flavivirus-induced neurological diseases. The lack of specific therapeutics underscores an urgent need to determine the function of important host factors involved in flavivirus replication and disease progression. Interleukin-6 (IL-6) upregulation has been observed during viral infections in both mice and humans, implying that it may influence the disease outcome significantly. Herein, we investigated the function of IL-6 in the pathogenesis of neurotropic flavivirus infections. First, we examined the role of IL-6 in flavivirus-infected human neuroblastoma cells, SK-N-SH, and found that IL-6 neutralization increased the WNV or JEV replication and inhibited the expression of key cytokines. We further evaluated the role of IL-6 by infecting primary mouse cells derived from IL-6 knockout (IL-6-/-) mice and wild-type (WT) mice with WNV or JEV. The results exhibited increased virus yields in the cells lacking the IL-6 gene. Next, our in vivo approach revealed that IL-6-/- mice had significantly higher morbidity and mortality after subcutaneous infection with the pathogenic WNV NY99 or JEV Nakayama strain compared to WT mice. The non-pathogenic WNV Eg101 strain did not cause mortality in WT mice but resulted in 60% mortality in IL-6-/- mice, indicating that IL-6 is required for the survival of mice after the peripheral inoculation of WNV or JEV. We also observed significantly higher viremia and brain viral load in IL-6-/- mice than in WT mice. Subsequently, we explored innate immune responses in WT and IL-6-/- mice after WNV NY99 infection. Our data demonstrated that the IL-6-/- mice had reduced levels of key cytokines in the serum during early infection but elevated levels of proinflammatory cytokines in the brain later, along with suppressed anti-inflammatory cytokines. In addition, mRNA expression of IFN-α and IFN-β was significantly lower in the infected IL-6-/- mice. In conclusion, these data suggest that the lack of IL-6 exacerbates WNV or JEV infection in vitro and in vivo by causing an increase in virus replication and dysregulating host immune response.

Abstract 2123: Glycine decarboxylase regulates renal carcinoma progression via interferon stimulated gene factor 3-mediated pathway

Abstract Glycine decarboxylase (GLDC) is a mitochondrial enzyme that mediates the degradation of glycine as part of the glycine cleavage system; it is implicated as a tumor suppressor in hepatocellular carcinoma but as an oncogene in the lung cancer. Although GLDC expression in the kidney is second highest next to the liver, very little is known as to the role of GLDC in the kidney. Thus, this study was designed to determine the role of GLDC in the renal carcinoma. We found markedly increased expressions of GLDC in patients with renal cell carcinoma (RCC) classified as poor-risk group based on the Memorial Sloan Kettering Cancer Center prognostic model. In vitro studies revealed that cellular proliferation, colony formation, and migration were decreased in GLDC-deficient renal carcinoma cells while those were increased in GLDC-overexpressing renal carcinoma cells. In vivo xenograft studies with GLDC-deficient and -overexpressing cells also revealed that tumor sizes were noticeably decreased in GLDC-deficient cell-injected mice while those were increased in GLDC-overexpressing cell-injected mice. Mechanistically, we found GLDC regulates renal carcinoma progression via interferon (IFN) stimulated gene factor 3 (ISGF3)-mediated pathway. Poly-IC induced mRNA expression of IFN-α more strongly in GLDC-deficient cells. Expressions of IRF9 and STAT2 were increased in GLDC-deficient cells while those were decreased in GLDC-overexpressing cells. Interferon-responsive genes (IFIT1, IFI44, IFI44L, and IFI27) were also regulated by GLDC as evidenced by increased mRNA expressions in GLDC-deficient cells and increased mRNA expressions in GLDC-overexpressing cells. Knock-down of IRF9 and STAT2 in GLDC-deficient cells reversed decreased cellular proliferation, colony formation, and migration. In summary, the data in this study indicates that GLDC regulates renal carcinoma progression via ISFG3-medated pathway and lay a scientific foundation that could support a therapeutic strategy that targets GLDC for the treatment of renal carcinoma. Citation Format: Jin Young Kim, Mai Thi Tuyet Pham, Byung Hoon Kim, Ji Hae Seo, Sojin Shin, Eunyoung Ha. Glycine decarboxylase regulates renal carcinoma progression via interferon stimulated gene factor 3-mediated pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2123.

Comparison of Cytokines Expression from Human Monocyte-Derived Macrophages Infected with Different Species of Mycobacteria.

Cytokines have an important role in mounting effective host immune response against mycobacteria. Latent tuberculosis infection (LTBI) is an indication of containment of mycobacteria by the host immune response, whereas active TB is an indication of a failure of the immune response to contain Mycobacterium tuberculosis. The dynamics of this host-immune response during in vitro infection experiment is believed to be indicative of behavior in the LTBI and active-TB cases. This relationship is, however, not fully elucidated. We investigated the cytokines expression at mRNA and protein level across 2 different protocols, that is, an in vitro protocol comparing human monocyte-derived macrophages (hMDMs; n = 12) infected with different species of mycobacteria, and a clinical protocol comparing TB-Antigen-Nil specimens from LTBI (n = 12) and active-TB (n = 12) cases. We found that in vitro infection of hMDMs with Mycobacterium bovis Bacillus Calmette-Guérin (BCG) and M. tuberculosis R179 showed increased colony-forming units at all time points postinfection. M. bovis BCG-infected hMDMs demonstrated higher levels of 5 cytokines [interferon (IFN)-γ, interleukin (IL)-6, IL-1β, IL-12p40, and IL-12p70] at different intervals compared to M. tuberculosis R179. Compared to LTBI, active-TB cases had higher mRNA expression of IFN-α, IL-6, and IL-8, and lower expression of IFN-γ, IL-1α, IL-1β, IL-4, and tumor necrosis factor-α. Overall, we observed differential host responses at mRNA and protein levels during experimentally controlled in vitro infection, but no prominent differences were observed in the clinical protocol. Therefore, the result of the in vitro experiment model of cytokine response against mycobacteria should be interpreted cautiously when relating to LTBI and active-TB.

Host immune response to infectious bronchitis virus Q1 in two commercial broiler chicken lines

This study investigated the pathogenesis of infectious bronchitis virus (Gammacoronavirus) strain Q1 in two commercial broiler chicken lines, and the host immune response to infection. Chicks from each line were grouped into either infected or control. Following Q1 infection at day-old, fast (Line-A) and slow (Line-B) growing chicks were monitored for clinical signs and body weights. At 3, 7, 9, 14, 21 and 28 days post infection (dpi), five birds were humanely euthanised, and trachea, kidney and proventriculus tissues were collected for quantitative RT-PCR and histopathology. Blood was collected weekly to determine IBV-specific ELISA antibody titres. Q1 infection significantly reduced the body weights of Line-A chicks at 14 and 21 dpi, but there were no significant differences in Line-B. Through qRT-PCR, significantly higher viral loads were found in the trachea, proventriculus and kidney tissues of Line-A chicks at 7–9 dpi. At day-old and at 28 dpi, the mean antibody titre in Line-B was notably higher than Line-A. Significant IFN-α mRNA expression was noted in the trachea and kidneys of Line-A, whereas no change occurred in Line-B. Chicks in Line-B, compared to those in Line-A, demonstrated a tissue-dependent increase of IFN-β, TLR3, IL-1β and IL-6 and LITAF gene transcription responses to IBV Q1. It appears that the level of maternal antibodies, growth rates, and other inherent host genetic factors could have influenced the differences in viral loads and immune responses.

Impact and prognosis of the expression of IFN-α among tuberculosis patients.

Mycobacterium tuberculosis (M.tb) infection stimulates the release of cytokines, including interferons (IFNs). IFNs are initiators, regulators, and effectors of innate and adaptive immunity. Accordingly, the expression levels of Type I (α, β) and II (γ) IFNs, among untreated tuberculosis (TB) patients and household contacts (HHC) clinically free of TB was assessed. A total of 264 individuals (TB patients-123; HHC-86; laboratory volunteers-55; Treated TB patients-36) were enrolled for this study. IFN-α mRNA expression levels predominated compared to IFN-γ and IFN-β among untreated TB patients. IFN-α transcripts were ~3.5 folds higher in TB patients compared to HHC, (p<0.0001). High expression of IFN-α was seen among 46% (56/ 123) of the TB patients and 26%, (22/86) of HHCs. The expression levels of IFN-α correlated with that of IFN transcriptional release factor 7 (IRF) (p<0.0001). In contrast, an inverse relationship exists between PGE2 and IFN-α expression levels; high IFN-α expressers were associated with low levels of PGE2 and vice-versa (Spearman's rho = -0.563; p<0.0001). In-vitro, IFN-α failed to restrict the replication of intracellular M.tb. The anti-mycobacterial activity of IFN-γ was compromised in the presence of IFN-α, but not by IFN-β. The expression of IFN-α and β diminished or is absent, among successfully treated TB patients. These observations suggest the utility of assessment of Type I IFNs expression levels as a prognostic marker to monitor tuberculosis patient response to chemotherapy because changes in Type I IFNs expression are expected to precede the clearance and /reduction in bacterial load.

Porcine enteric alphacoronavirus Inhibits IFN-α, IFN-β, OAS, Mx1, and PKR mRNA Expression in Infected Peyer's Patches in vivo.

Porcine enteric alphacoronavirus (PEAV) is a newly identified swine enteropathogenic coronavirus that causes watery diarrhea in neonatal piglets. The pathogenesis and host immune responses of PEAV infection are not fully characterized. The reason lies in the stomach environment, which would degrade cell-cultured live viruses via oral infection, making it difficult to establish an effective infection model to study the pathogenesis and host immune responses in pigs with a mature immune system. To solve this problem, in this study, coated PEAV-loaded microspheres were developed by centrifugal granulation-fluidized bed coating and demonstrated as an effective oral delivery system/animal infection model to protect PEAV virion against the complex gastrointestinal environment in vitro and to cause infection in weaned piglets in vivo. Weaned piglets orally inoculated with coated PEAV-loaded microspheres developed diarrhea and virus RNA was detected in rectal swabs from one to seven days post inoculation. In addition, microscopic lesions in the small intestine were observed, and viral antigens were also detected in the small intestines with PEAV immunohistochemical staining. Importantly, PEAV significantly inhibited mRNA expression of IFN-α, IFN-β, OAS, Mx1, and PKR, the genes involved in modulation of the host immune responses, in infected Peyer's patches, indicating that PEAV can overcome the antiviral response to cause damage when infection occurs. Collectively, our research successfully established a PEAV animal infection model in weaned piglets and suggested that the observed gene expression profile might help explain immunological changes associated with PEAV infection.

Niche Specific Microbiota-Dependent and Independent Bone Loss around Dental Implants and Teeth

Oral mucosal homeostasis is achieved by complex immunologic mechanisms, orchestrating host immunity to adapt to the physiologic functions of the various specialized niches in the oral cavity. Dental implants introduce a novel mucosal niche to the immune system to deal with. Nevertheless, the immune mechanisms engaged toward implants and whether they have broader effects are not well defined. Using a murine model, we found an accumulation of neutrophils and RANKL-expressing T and B lymphocytes in the implant-surrounding mucosa, accompanied by local bone loss. Surprisingly, the presence of implants had an impact on remote periodontal sites, as elevated inflammation and accelerated bone loss were detected in intact distant teeth. This was due to microbial dysbiosis induced by the implants, since antibiotic treatment prevented bone loss around teeth. However, antibiotic treatment failed to prevent the loss of implant-supporting bone, highlighting the distinct mechanisms mediating bone loss at each site. Further analysis revealed that implants induced chronic lymphocyte activation and increased mRNA expression of IFN-α and accumulation of IFN-α–producing plasmacytoid dendritic cells, which we previously reported as bone-destructive immune responses. Collectively, this study demonstrates that implants have a strong and broad impact on oral mucosal homeostasis, inducing periodontal bone loss in a niche-specific manner that is both microbiota dependent and independent.

707 Responsiveness of the Adenosine A2A receptor (A2AR) regulates immune and keratinocyte responses in psoriasis

Psoriasis is a chronic inflammatory disease of the skin and joints associated with both innate and adaptive immunity. An imbalance between the activators and the modulators of immunity and their interactions with keratinocytes leads to the chronic inflammatory hallmarks of the disease. The A2AR is a key modulator of both immunity and inflammation. We have described a means to enhance A2AR function through a small molecule that acts as a positive allosteric modulator (PAM; AEA061). The PAM has no intrinsic biologic activity at the A2AR but enhances adenosine-mediated A2AR function. In a mouse model of TLR 7 agonist imiquimod (IMQ)-induced psoriasis-like dermatitis, the oral administration of the PAM reduced ear thickness, skin erythema, scale formation and inflammatory cytokine expression in the skin, ear tissue and in plasma. Transcriptome analysis of the skin indicated differential expression of 111 genes upon PAM treatment. Ex-vivo and in culture, the PAM decreased IMQ-induced expression of IFN-α in pDCs and IL-23, IL-36α and IL-36β in cDCs. Moreover, the PAM inhibited TCR-mediated IL-17 expression in γδT cells and IFN-γ expression in CD4+ T cells. In human epidermal keratinocytes, the PAM attenuated IFN-α-mediated IL-22R expression, reducing their proliferative response to IL-22. We have extended our investigation into the effects of the PAM on human immune cell subsets that are central to the initiation and maintenance of psoriasis in man. We observed attenuation of mRNA expression of IFN-α, IFN-β, and IL-23 in human pDCs and IL-17F in human γδT cells. The PAM also inhibited the production of cytokines by Th cells that are implicated in psoriasis, including IL-2, IFN-γ, TNF-α, sCD40, GM-CSF, CXCL10, MIP-1α, MIP-1β, RANTES and eotaxin. Thus, the enhancement of A2AR responsiveness to endogenous adenosine, targeted through positive allosteric modulation, alters innate and adaptive immune responses towards a homeostatic state and reduces disease expression.

The relationship of serum vitamins A, D, E and LL-37 levels with allergic status, tonsillar virus detection and immune response.

BackgroundTonsils have an active role in immune defence and inducing and maintaining tolerance to allergens. Vitamins A, D, and E, and antimicrobial peptide LL-37 may have immunomodulatory effects. We studied how their serum levels were associated with allergy status, intratonsillar/nasopharyngeal virus detection and intratonsillar expression of T cell- and innate immune response-specific cytokines, transcription factors and type I/II/III interferons in patients undergoing tonsillectomy.Methods110 elective tonsillectomy patients participated. Serum levels of vitamins A, 25(OH)D, and E, LL-37 and allergen-specific IgE as well as nasopharyngeal/intratonsillar respiratory viruses were analyzed. The mRNA expression of IFN-α, IFN-β, IFN-γ, IL-10, IL-13, IL-17, IL-28, IL-29, IL-37, TGF-β, FOXP3, GATA3, RORC2 and Tbet in tonsils were analyzed by quantitative RT-PCR.ResultsThe median age of the patients was 16 years (range 3–60), 28% of subjects had atopy, and 57% carried ≥1 respiratory virus in nasopharynx. Detection of viruses decreased by age. Higher vitamin A levels showed borderline significance with less viral detection (P = 0.056). Higher 25(OH)D was associated with less allergic rhinitis and atopy (P < 0.05) and higher vitamin E with less self-reported allergy (P < 0.05). In gene expression analyses, 25(OH)D was associated with higher IL-37, vitamin A with higher IFN-γ and vitamin E with less IL-28 (P < 0.05). LL-37 was associated with less FOXP3, RORC2 and IL-17 in tonsils (P < 0.05).ConclusionsVitamin D and E levels were associated with less allergic disorders. Vitamin A was linked to antiviral and vitamin D with anti-inflammatory activity. LL-37 and was linked to T regulatory cell effects.

Chemosynthesis pathway and bioactivities comparison of saponins in radix and flower of Panax notoginseng (Burk.) F.H. Chen

Ethnopharmacological relevancePanax notoginseng (Burk.) F.H. Chen is a well known medicinal plant. Its radix is used in the history while its flower is recently used for health care. In this study we compared chemical ingredients and bioactivities in cell culture for radix and flower of Panax notoginseng (Burk.) F.H. Chen. Materials and methodsThe liquid chromatography-mass spectrometry system was applied to determine the contents of saponins in flower and radix of Panax notoginseng (Burk.) F.H. Chen. Transcription specific luciferase reporter assay and qPCR method for selected RNA were carried out to assess the impacts of flower and radix extract on the transcription signal pathways. ResultsThe results of chemical analysis showed that the contents of saponins in flower and radix are very different: the contents of Rg1, Rb1, Re, R1, Rg3-20R, Rh1 and Rf in radix are abundant; in contrast, the contents of Rb3, Fc, Ft1, Rb2 and Rh2-20s in flowers are plentiful. There are substantial variations of those saponin contents from one batch vs another. Based on relative content of saponins, the chemosynthesis pathway of ingredients in radix and flower are proposed: for radix, both PPT (Protopanaxatriol) and PPD (Protopanaxadiol) type triterpenoids are involved, the main pathway is PPT→Rb1→Rg1→R1 or PPD→Rh2 20s→Rg3(20s)→Rd→Rb1; for flowers, only PPD is main passage with PPD→Rh2 (20s)→Rg3(20s)→Rd→Rb2→Fc.The results of signal transcription assays demonstrated that herb water extract of radix and flower had no significant influences on most of transcription activities. However, total saponins of radix and flower which have highly content of saponins were able to inhibit the inflammatory related transcriptional activities and their related mRNA expression of IFNα, TNFα, il-6 and TGFβ as well as induce anti-oxygen NrF2 activities.In summary, although chemical ingredients and chemosynthesis pathway of flower and radix for Panax notoginseng (Burk.) F.H. Chen were different, these differences might not result in their differences of pharmacological activities.

Role of the suppressor of cytokine signaling 2 (SOCS2) during meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5)

The role of suppressors of cytokine signaling (SOCS) in meningoencephalitis caused by Bovine herpesvirus 5 (BoHV-5) was evaluated by intracranial infection in C57BL/6 wild-type mice (WT) and SOCS2 deficient mice (SOCS2(-/-)). Both infected groups presented weight loss, ruffled fur and hunched posture. Additionally, infected SOCS2(-/-) mice showed swollen chamfer and progressive depression. Infected WT animals developed mild meningitis, characterized by infiltration of mononuclear cells. Moreover, viral DNA was detected in liver and lung from infected WT group. This group also showed elevated brain levels of IFN-γ, IL-10, CXCL1 and CCL5, when compared with non-infected WT animals. Brain inflammation was exacerbated in infected SOCS2(-/-) mice with widespread distribution of the virus and increased brain levels of TNF-α, IFN-γ, IL-10, IL-12, CXCL1 and CCL5, when compared with WT infected mice. Moreover, infected SOCS2 deficient mice exhibited reduced brain mRNA expression of IFNα and IFNβ and increased expression of mRNA of SOCS1, compared with infected WT mice. Taken together, our study provides an insight into the role of SOCS2 in modulating the immune response to BoHV-5 infection.

Antiviral effect of emodin from Rheum palmatum against coxsakievirus B5 and human respiratory syncytial virus in vitro.

Viral infections are the major causes of morbidity and mortality in elderly people and young children throughout the world. The most common pathogens include coxsackie virus (CV) and respiratory syncytial virus (RSV). However, no antiviral agents with low toxicity and drug resistance are currently available in clinic therapy. The present study aimed to examine the antiviral activities of emodin (an ingredient of Rheum palmatum) against CVB5 and RSV infections, in an attempt to discover new antiviral agents for virus infection. The monomer emodin was extracted and isolated from Rheum palmatum. The antiviral activities of emodin on HEp-2 cells were evaluated, including virus replication inhibition, virucidal and anti-absorption effects, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-razolium bromide (MTT) assay and plaque reduction assay (PRA). The kinetics of virus inhibition by emodin in a period of 14 h was further determined by plaque assay and quantitative real time PCR (qPCR). Cytokine (IFN-γ, TNF-α) mRNA expressions after emodin treatment (7.5, 15, 30 μmol/L) were also assessed by qPCR post-infection. The results showed that emodin had potent inhibitory activities against CVB5 and RSV, with the 50% effective concentration (EC50) ranging from 13.06 to 14.27 μmol/L and selectivity index (SI) being 5.38–6.41 μmol/L. However, emodin couldn’t directly inactivate the viruses or block their absorption to cells. It acted as a biological synthesis inhibitor against CVB4 and RSV in a concentration- and time-dependent manner, especially during the first 0–4 h post-infection. Moreover, emodin could decrease the mRNA expression of IFN-α but enhance TNF-γ expression significantly compared to the viral controls in vitro. Our results provide a molecular basis for development of emodin as a novel and safe antiviral agent for human enterovirus and respiratory virus infection in the clinical therapy.

Apoptosis induction and release of inflammatory cytokines in the oviduct of egg-laying hens experimentally infected with H9N2 avian influenza virus

The H9N2 subtype avian influenza virus (AIV) can cause serious damage to the reproductive tract of egg-laying hens, leading to severe egg-drop and poor egg shell quality. However, previous studies in relation to the oviductal-dysfunction resulted from this agent have not clearly been elucidated. In this study, apoptosis and pathologic changes in the oviducts of egg-laying hens caused by H9N2 AIV were evaluated. To understand the immune response in the pathogenic processes, 30-week old specific pathogen free (SPF) egg-laying hens inoculated with H9N2 subtype of AIV through combined intra-ocular and intra-nasal routes. H9N2 AIV infection resulted in oviductal lesions, triggered apoptosis and expression of immune related genes accompanied with infiltration of CD3+CD4+ and CD3+CD8α+ cells. Significant tissue damage and apoptosis were observed in the five oviductal parts (infundibulum, magnum, isthmus, uterus and vagina) at 5 days post-inoculation (dpi). Furthermore, immune-related genes, including chicken TLR3 (7, 21), MDA5, IL-2, IFN-β, CXCLi1, CXCLi2, XCL1, XCR1 and CCR5 showed variation in the egg-laying hens infected with H9N2 AIV. Notably, mRNA expression of IFN-α was suppressed during the infection. These results show distinct expression patterns of inflammatory cytokines and chemokines amongst segments of the oviduct. Differential gene expression of inflammatory cytokines and lymphocytes aggregation occurring in oviducts may initiate the infected tissue in response to virus replication which may eventually lead to excessive cellular apoptosis and tissue damage.

Expression of antiviral cytokines in Crandell-Reese feline kidney cells pretreated with Korean red ginseng extract or ginsenosides

The antiviral activity and protective mechanism of Korean red ginseng (KRG) is not well understood. The aim of this study was to investigate the protective mechanism of KRG extract and ginsenosides against feline calicivirus (FCV), a human norovirus surrogate. CRFK cells that were pretreated for 48h with 10μg/mL of KRG extract or purified ginsenoside Rb1 or Rg1, were inoculated with FCV. RNA extracted from each treated group was examined for the expression of antiviral cytokines, including interferon-α (IFN-α), interferon-β (IFN-β), interferon-ω (IFN-ω), Mx, and zinc finger antiviral protein shorter isoform (ZAPS), by relative real-time reverse transcription-polymerase chain reaction. mRNA expression of IFN-α, IFN-β, IFN-ω, Mx, and ZAPS was significantly induced in the FCV-challenged group pretreated with the KRG extract or ginsenosides, and it was higher than the group treated with FCV alone. Mx protein expression was confirmed by western blotting of CRFK cells pretreated with the ginsenoside Rb1 or with Rg1. Induction of antiviral cytokines contributes to the reduction of the viral titer in CRFK cells pretreated with the KRG extract and purified ginsenosides. In future studies, the antiviral protective mechanism of KRG should be demonstrated using other viruses such as human norovirus.

Npro of classical swine fever virus contributes to pathogenicity in pigs by preventing type I interferon induction at local replication sites

Classical swine fever (CSF) caused by CSF virus (CSFV) is a highly contagious disease of pigs. The viral protein Npro of CSFV interferes with alpha- and beta-interferon (IFN-α/β) induction by promoting the degradation of interferon regulatory factor 3 (IRF3). During the establishment of the live attenuated CSF vaccine strain GPE-, Npro acquired a mutation that abolished its capacity to bind and degrade IRF3, rendering it unable to prevent IFN-α/β induction. In a previous study, we showed that the GPE- vaccine virus became pathogenic after forced serial passages in pigs, which was attributed to the amino acid substitutions T830A in the viral proteins E2 and V2475A and A2563V in NS4B. Interestingly, during the re-adaptation of the GPE- vaccine virus in pigs, the IRF3-degrading function of Npro was not recovered. Therefore, we examined whether restoring the ability of Npro to block IFN-α/β induction of both the avirulent and moderately virulent GPE--derived virus would enhance pathogenicity in pigs. Viruses carrying the N136D substitution in Npro regained the ability to degrade IRF3 and suppress IFN-α/β induction in vitro. In pigs, functional Npro significantly reduced the local IFN-α mRNA expression in lymphoid organs while it increased quantities of IFN-α/β in the circulation, and enhanced pathogenicity of the moderately virulent virus. In conclusion, the present study demonstrates that functional Npro influences the innate immune response at local sites of virus replication in pigs and contributes to pathogenicity of CSFV in synergy with viral replication.

Therapeutic effect of neonatal mouse Cytomegalovirus hepatitis by CpG Oligodeoxynucleotide 2395

Objective To study the treatment effects of newborn mice murine Cytomegalovirus (MCMV)hepatitis by using CpG Oligodeoxynucleotide 2395(CpG ODN2395). Methods Specific pathogen-free BALB/c newborn mice were divided into 3 groups according to the broods randomly: control group, virus group and treatment group.In control group 9 g/L sodium chloride 20×10-6 L was intraperitoneally injected every other day.In virus group MCMV(TCID50=108.31/L) 20×10-6 L was intraperitoneally injected once and 9 g/L sodium chloride solution 20×10-6 L was intraperitoneally injected every other day.In treatment group murine MCMV(TCID50=108.31/L) 20×10-6 L was intraperitoneally injected once and from the first day CpG ODN2395 20 mg/kg was intraperitoneally injected every other day.Growths and development of mice were observed.Murine body weights were measured.Pathology of livers was observed by means of hematoxylin and eosin stain.The levels of serum ALT and IFN-α were measured by adopting enzyme linked immunosorbent assay.MCMV loads in liver were measured by way of polymerase chain reaction.The expression levels of IFN-α mRNA in liver were detected by using reverse transcription polymerase chain reaction.The expression levels of Toll-like receptor 9(TLR9) and myeloid cell differentiation factor 88(MyD88) in liver were detected by adopting Western blot.The results were analyzed by using SPSS 16.0 statistics software. Results 1.Compared with control group and treatment group, growth and development of virus group mice fell behind and on day 7, 14 body weights were lowest(F=18.919, 25.543, all P<0.05). Growth and development of treatment group mice were between control group and virus group.Body weights of treatment group mice were lower than those of control group, and there was statistical difference on day 7(t=3.187, P<0.05).2.Compared with control group and treatment group, the levels of ALT in virus group was highest.ALT levels of treatment group were higher than those of control group and treatment group(F=11.407, 11.791, 154.656, all P<0.05).3.The pathologic change of liver tissue: the HAI of virus group reached the peak on day 3, then decreased gradually.The HAI of treatment group also reached the peak on day 3, but liver damage was obviously less than those of virus group.The liver damage also relieved gradually and the mean of HAI was obviously lower than that of virus group on day 7 and 14.4.MCMV DNA in liver was negative at control group.The magnitude of viral loading in livers of virus group was higher than that of treatment group.5.The levels of IFN-α in treatment group and virus group reached a peak on day 3 and declined gradually on day 7, 14.The levels of IFN-α on treatment group were higher than that of virus group and control group.The levels of IFN-α virus group were higher than those of control group, but had no statistical difference.6.The mRNA expression of IFN-α in livers of treatment group and virus group began to increase on day 3 and reached a peak on day 7, and declined on day 14.The mRNA expression of IFN-α was higher than that of virus group and control group.The mRNA expression of virus group was higher than that of control group.7.The expressions of TLR9 and MyD88 in livers of treatment group were higher than that of virus group and control group.The expressions of TLR9 and MyD88 of virus group were higher than those of control group. Conclusions CpG ODN2395 can obviously improved liver function and histopathological lesions and reduce MCMV DNA load within liver tissues as well.CpG ODN2395 can improve the expression levels of TLR9 in liver and activate secretion interferon alpha by MyD88-dependent pathway, which were likely to play an important role in its treatment of murine CMV infection. Key words: Cytomegalovirus; CpG Oligodeoxynucleotide 2395; Hepatitis; Mouse

Protein-bound polysaccharide-K augments the anticancer effect of fluoropyrimidine derivatives possibly by lowering dihydropyrimidine dehydrogenase expression in gastrointestinal cancers

Protein-bound polysaccharide-K (PSK) enhances the antitumor effect of anticancer drug when used clinically in combination with such drugs. PSK is known to act by immune-mediated mechanisms; however, the relationship between PSK and metabolic enzymes of anticancer drugs is unknown. We used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST) clinically to evaluate the sensitivity of anticancer drugs. In the present study, we modified the CD-DST by adding peripheral blood mononuclear cells (PBMCs) (immuno-CD-DST) and examined the antitumor effect of PSK in combination with anticancer drugs. First, HCT116 human colon cancer cells were cultured with PSK and 5-fluorouracil (5-FU) or 5'-deoxy-5-fluorouridine (5'-DFUR) in the presence or absence of PBMCs, and the antiproliferative effects were compared. In the presence of PBMCs, PSK augmented the inhibitory effects of 5-FU and 5'-DFUR on HCT116 cell proliferation. Next, using human gastric cancer and colon cancer cell lines, the effects of PSK on mRNA expression of various metabolic enzymes of fluoropyrimidines: dihydropyrimidine dehydrogenase (DPD), thymidylate synthase, thymidine phosphorylase and orotate phosphoribosyl transferase, were examined by real-time PCR. PSK significantly enhanced DPD mRNA expression in all of the cancer cell lines tested, but not those of the other enzymes. Addition of IFN-α and TRAIL, cytokines known to inhibit DPD expression, to the cultures reduced DPD mRNA expression in the cancer cells. When PBMC samples collected from healthy volunteers were cultured with PSK, IFN-α mRNA expression increased in 3 of the 5 PBMC samples, while TRAIL mRNA expression was unchanged. The present results propose the possibility that PSK induces PBMCs to express IFN-α which inhibits DPD expression, and consequently augments the antitumor effect of 5-FU or 5'-DFUR. Immuno-CD-DST is useful for evaluating drugs with immunological mechanisms of action.

Mucosal application of cationic poly(d,l-lactide-co-glycolide) microparticles as carriers of DNA vaccine and adjuvants to protect chickens against infectious bursal disease

Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens. The virus protein (VP) 2 induces neutralizing antibodies, which protect chickens against the disease. The aim of this study was to develop a cationic poly(d,l-lactide-co-glycolide) (PLGA) microparticle (MP) based IBDV-VP2 DNA vaccine (MP-IBDV-DNA) for chickens to be delivered orally and by eye drop route. The tested IBDV-VP2 DNA vaccines were immunogenic for specific-pathogen-free chickens and induced an antibody response after intramuscular application. Co-inoculation with a plasmid encoding chicken IL-2 (chIL-2) or CpG-ODN did not significantly improve protection against IBDV challenge. However, the application of a MP-IBDV-DNA vaccine alone or in combination with a delayed oral and eye drop application of cationic MP loaded with CpG-ODN or chIL-2 improved protection against challenge. The MP-IBDV-DNA-vaccinated chickens showed less pathological and histopathological bursal lesions, a reduced IBDV antigen load as well as T-cell influx into the bursa of Fabricius (BF) compared to the other groups (p<0.05). The addition of chIL-2 loaded MP improved challenge virus clearance from the BF as demonstrated by lower neutralizing antibody titers and reduced IL-4 and IFN-α mRNA expression in the bursa at 7 days postchallenge compared to the other challenged groups. Overall, the efficacy of the IBDV-DNA vaccine was improved by adsorption of the DNA vaccine onto cationic PLGA-MP, which also allowed mucosal application of the DNA vaccine.

Interferon-α Is the Primary Plasma Type-I IFN in HIV-1 Infection and Correlates with Immune Activation and Disease Markers

Type-I interferon (IFN-I) has been increasingly implicated in HIV-1 pathogenesis. Various studies have shown elevated IFN-I and an IFN-I-induced gene and protein expression signature in HIV-1 infection, yet the elevated IFN-I species has not been conclusively identified, its source remains obscure and its role in driving HIV-1 pathogenesis is controversial. We assessed IFN-I species in plasma by ELISAs and bioassay, and we investigated potential sources of IFN-I in blood and lymph node tissue by qRT-PCR. Furthermore, we measured the effect of therapeutic administration of IFNα in HCV-infected subjects to model the effect of IFNα on chronic immune activation. IFN-I bioactivity was significantly increased in plasma of untreated HIV-1-infected subjects relative to uninfected subjects (p = 0.012), and IFNα was the predominant IFN-I subtype correlating with IFN-I bioactivity (r = 0.658, p<0.001). IFNα was not detectable in plasma of subjects receiving anti-retroviral therapy. Elevated expression of IFNα mRNA was limited to lymph node tissue cells, suggesting that peripheral blood leukocytes are not a major source of IFNα in untreated chronic HIV-1 infection. Plasma IFN-I levels correlated inversely with CD4 T cell count (p = 0.003) and positively with levels of plasma HIV-1 RNA and CD38 expression on CD8 T cells (p = 0.009). In hepatitis C virus-infected subjects, treatment with IFN-I and ribavirin increased expression of CD38 on CD8 T cells (p = 0.003). These studies identify IFNα derived from lymph nodes, rather than blood leukocytes, as a possible source of the IFN-I signature that contributes to immune activation in HIV-1 infection.