Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection

Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.     

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-α treatment

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG₂) under IFN-α treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG₂ cells as the most stable reference genes under IFN-α treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG₂ group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG₂ cells would greatly improve the normalization of the RT-qPCR data under IFN-α. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway.     

IL-6 and IFN-α from dsRNA-stimulated dendritic cells control expansion of regulatory T cells

Foxp3⁺CD4⁺ regulatory T cells (Treg) control not only autoimmunity but also the effective immune response against RNA virus infections, which produces virus-derived double-stranded RNA (dsRNA). To induce effective anti-viral immunity, it is a key issue to learn how Treg respond to dsRNA in vitro and in vivo. We here showed that synthetic dsRNA, polyI:C, caused peripheral expansion of functional Treg in a TICAM-1- and IL-6-dependent manner in vivo. PolyI:C did not expand Treg directly, but promoted the expansion of naturally occurring Treg indirectly through IL-6 produced from dendritic cells (DCs). In addition, the expansion of Treg by IL-6 was inhibited by IFN-α from polyI:C-stimulated DCs. These data suggest that the balance of IL-6 and IFN-α in the region of RNA virus infection may determine the number of peripheral Treg, which affects the effective immune responses against viruses.     

Clinical reagents of GM-CSF and IFN-α induce the generation of functional chronic myeloid leukemia dendritic cells in vitro

Dendritic cells (DCs) have been successfully induced in vitro from chronic myeloid leukemia (CML) cells, which may provide a promising immunotherapeutic protocol for CML. To facilitate the optimization of DCs-based vaccination protocols, we investigated the efficiency of in vitro generation of DCs from bone marrow mononuclear cells of CML patients by clinical reagents of GM-CSF and IFN-α. Bone marrow mononuclear cells were isolated from eight CML patients and CML-DCs were generated in the presence of different cytokines (Group A: GM-CSF for research and IL-4 for research; Group B: GM-CSF for injection and IFN-α for injection) in RMPI-1640 medium containing 10% human AB serum. After 8 days, the morphologic features of CML-DCs were observed and their immunophenotypes were analyzed by flow cytometry. The activity of CML-DCs was determined by evaluating their ability to stimulate allogeneic mixed lymphocyte reaction (allo-MLR) and anti-leukemic cytotoxic T lymphocytes (CTLs). The culture protocols were successful in generating functional CML-DCs from all the CML patients as evidenced by the significant upregulation of CD80, CD86, CD83 HLA-DR and CD1a compared to pre-cultured (p < 0.05), and increased allogeneic T cell stimulating proliferation capacity (p < 0.05). CML-DCs could stimulate a specific anti-leukemia response. In summary, we demonstrate that the combination of clinical reagents GM-CSF and IFN-α induced the generation of DCs that have the ability to stimulate a specific anti-leukemia CTLs response in vitro, indicating their feasibility for clinical vaccination protocols for CML patients.     

AAV2-mediated combined subretinal delivery of IFN-α and IL-4 reduces the severity of experimental autoimmune uveoretinitis

We previously showed that adeno-associated virus 2 (AAV2) mediated subretinal delivery of human interferon-alpha (IFN-α) could effectively inhibit experimental autoimmune uveoretinitis (EAU). In this study we investigated whether subretinal injection of both AVV2.IFN-α and AAV2.IL-4 had a stronger inhibition on EAU activity. B10RIII mice were subretinally injected with AAV2.IFN-α alone (1.5×10(7) vg), AAV2.IL-4 alone (3.55×10(7) vg), and AAV2.IFN-α combined with AAV2.IL-4. PBS, AAV2 vector encoding green fluorescent protein (AAV2.GFP) (5×10(7) vg) was subretinally injected as a control. IFN-α and IL-4 were effectively expressed in the eyes from three weeks to three months following subretinal injection of AAV2 vectors either alone or following combined administration and significantly attenuated EAU activity clinically and histopathologically. AAV2.IL-4 showed a better therapeutic effect as compared to AAV2.IFN-α. The combination of AAV2.IL-4 and AAV2.IFN-α was not significantly different as compared to AAV2.IL-4 alone. There was no difference concerning DTH (delayed-type hypersensitivity) reaction, lymphocyte proliferation and IL-17 production among the investigated treatment groups, suggesting that local retinal gene delivery did not affect the systemic immune response.     

Combined anti-tumor effects of IFN-α and sorafenib on hepatocellular carcinoma in vitro and in vivo

Hepatocellular carcinoma (HCC) is among the most common and aggressive cancers worldwide, and novel therapeutic strategies are urgently required to improve clinical outcome. Interferon-alpha (IFN-α) and sorafenib are widely used as anti-tumor agents against various malignancies. In this study, we investigated the combined effects of IFN-α and sorafenib against HCC. We demonstrated that the combination therapy synergistically suppressed HCC cellular viability, arrested cell cycle propagation and induced apoptosis in HCC cells. Further research revealed that IFN-α and sorafenib collaboratively regulated the expression levels of cell cycle-related proteins Cyclin A and Cyclin B as well as the pro-survival Bcl-2 family proteins Mcl-1, Bcl-2 and Bcl-X(L). Moreover, sorafenib inhibited IFN-α induced oncogenic signaling of STAT3, AKT and ERK but not the activation of the tumor suppressor STAT1. Xenograft experiments also confirmed the combined effects of IFN-α and sorafenib on tumor growth inhibition and apoptosis induction in vivo. In conclusion, these results provide rationale for the clinical application of IFN-α and sorafenib combination therapy in HCC treatment.     

The Effect of Bovine IFN-α on the Immune Response in Guinea Pigs Vaccinatedwith DNA Vaccine of Foot-and-Mouth Disease Virus

Foot-and-mouth disease virus (FMDV) belongs to thegenus Aphthovirus of the family Picornavidae. The FMDVgenome is a copy of positive-sense, single-stranded RNA,which contains one large open reading frame (ORF). TheORF is translated into a polypeptide, which undergoesautoproteolytic cleavage to produce the structural and non-structural proteins and ultimately forms mature viral pro-teins [1,2]. FMD is caused by the FMDV, which is a highly conta-gious vesicular disease of cloven-hoofe…     

Activation of ERα Signaling Differentially Modulates IFN-γ Induced HLA-Class II Expression in Breast Cancer Cells

The coordinate regulation of HLA class II (HLA-II) is controlled by the class II transactivator, CIITA, and is crucial for the development of anti-tumor immunity. HLA-II in breast carcinoma is associated with increased IFN-γ levels, reduced expression of the estrogen receptor (ER) and reduced age at diagnosis. Here, we tested the hypothesis that estradiol (E₂) and ERα signaling contribute to the regulation of IFN-γ inducible HLA-II in breast cancer cells. Using a panel of established ER⁻ and ER⁺ breast cancer cell lines, we showed that E₂ attenuated HLA-DR in two ER⁺ lines (MCF-7 and BT-474), but not in T47D, while it augmented expression in ER⁻ lines, SK-BR-3 and MDA-MB-231. To further study the mechanism(s), we used paired transfectants: ERα⁺ MC2 (MDA-MB-231 c10A transfected with the wild type ERα gene) and ERα⁻ VC5 (MDA-MB-231 c10A transfected with the empty vector), treated or not with E₂ and IFN-γ. HLA-II and CIITA were severely reduced in MC2 compared to VC5 and were further exacerbated by E₂ treatment. Reduced expression occurred at the level of the IFN-γ inducible CIITA promoter IV. The anti-estrogen ICI 182,780 and gene silencing with ESR1 siRNA reversed the E₂ inhibitory effects, signifying an antagonistic role for activated ERα on CIITA pIV activity. Moreover, STAT1 signaling, necessary for CIITA pIV activation, and selected STAT1 regulated genes were variably downregulated by E₂ in transfected and endogenous ERα positive breast cancer cells, whereas STAT1 signaling was noticeably augmented in ERα⁻ breast cancer cells. Collectively, these results imply immune escape mechanisms in ERα⁺ breast cancer may be facilitated through an ERα suppressive mechanism on IFN-γ signaling.     

鹌鹑和三黄鸡IFN-α cDNA的克隆及序列分析

根据GenBank已登录的鸡和鹌鹑IFN-α基因序列,用Oligo6.0设计一对能同时扩增鸡和鹌鹑INF-α基因的共用引物,利用RT-PCR方法从经ConA诱导培养的三黄鸡、鹌鹑外周血淋巴细胞中分别扩增到IFN-αORF全长cDNA,并克隆到pET-28a( )载体上。将测序结果分别与GenBank中已收录的家禽类IFN-α核苷酸序列进行同源性比较及遗传进化分析。结果发现鸡、鹌鹑IFN-αcDNA遗传进化稳定,与GenBank中收录的鸡、鹌鹑IFN-α核苷酸序列同源性为98.6%~99.8%、99.8%。鸡与其它家禽类的IFN-α核苷酸序列比较分析发现,与鹌鹑亲缘性较高,同源性达86.9%~87.1%,但与家水禽(鸭、鹅)差距较大,同源性最高也只有67.6%;鹌鹑IFN-α核苷酸序列与家水禽同源性在也只在62.7%~62.9%之间;基因遗传进化树分析可见家禽类IFN-α被分成两个大分枝,分别是由鸡和鹌鹑两个小分支组成的一个大分支与家水禽独立分支组成。     

The possible role of zinc and metallothionein in the liver on the therapeutic effect of IFN-α to hepatitis C patients

We have studied zinc deficiency in hepatitis C patients (complete responder [C,R] 22, nonresponder [NR] 25) with relation to the therapeutic effect of interferon-α (IFN-α). Circadian variations in serum zinc levels were high in the morning (basal level) and then gradually decreased during the day in both chronic hepatitis C patients and healthy controls. Basal zinc levels in serum were significantly lower in chronic hepatitis C patients (73±3 μg/dL,n=12) than in controls (93±5 μg/dL). An injection of 10 MU of IFN-α to hepatitis C patients augmented the serum zinc reductions, up to 40% in 8 h. Serum cortisol levels were significantly elevated 8 h (25.6±2.3 μg/dL) after IFN-α dose. Forty-seven chronic hepatitis C patients were treated with IFN-α for 24 wk, and serum zinc and copper levels were determined 12 and 24 wk during and after the completion of IFN-α therapy. Serum zinc levels and zinc/copper ratio were higher in CRs than in NRs to IFN therapy at each time-point. Hepatic metallothionein staining became prominent after IFN therapy in most of CRs, whereas it diminished NRs. These data suggest that nutritional status of zinc influences the effect of IFN on hepatitis C patients.     

Cutting edge: the role of IFN-α receptor and MyD88 signaling in induction of IL-15 expression in vivo

IL-15 plays a multifaceted role in immune homeostasis, but the unreliability of IL-15 detection has stymied exploration of IL-15 regulation in vivo. To visualize IL-15 expression, we created a transgenic mouse expressing emerald-GFP (EmGFP) under IL-15 promoter control. EmGFP/IL-15 was prevalent in innate cells including dendritic cells (DCs), macrophages, and monocytes. However, DC subsets expressed varying levels of EmGFP/IL-15 with CD8(+) DCs constitutively expressing EmGFP/IL-15 and CD8(-) DCs expressing low EmGFP/IL-15 levels. Virus infection resulted in IL-15 upregulation in both subsets. By crossing the transgenic mice to mice deficient in specific elements of innate signaling, we found a cell-intrinsic dependency of DCs and Ly6C(+) monocytes on IFN-α receptor expression for EmGFP/IL-15 upregulation after vesicular stomatitis virus infection. In contrast, myeloid cells did not require the expression of MyD88 to upregulate EmGFP/IL-15 expression. These findings provide evidence of previously unappreciated regulation of IL-15 expression in myeloid lineages during homeostasis and following infection.     

PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2

Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3'Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3'UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.     

Ribavirin enhances IFN-α signalling and MxA expression: a novel immune modulation mechanism during treatment of HCV

The nucleoside analogue Ribavirin significantly increases patient response to IFN-α treatment of HCV, by directly inhibiting viral replication. Recent studies indicate that Ribavirin also regulates immunity and we propose that Ribavirin enhances specific interferon sensitive gene (ISG) expression by amplifying the IFN-α-JAK/STAT pathway. We found that IFN-α-induced STAT1 and STAT3 phosphorylation was increased in hepatocytes co-treated with Ribavirin and IFN-α, compared to IFN-α alone. Ribavirin specifically enhanced IFN-α induced mRNA and protein of the anti-viral mediator MxA, which co-localised with HCV core protein. These novel findings indicate for the first time that Ribavirin, in addition to its viral incorporation, also enhances IFN-α-JAK/STAT signalling, leading to a novel MxA-mediated immuno-modulatory mechanism that may enhance IFN-α anti-viral activity against HCV.     

Effect of Lactobacillus pentosus S-PT84 Ingestion on IFN-α Production from Plasmacytoid Dendritic Cells by Virus Stimulation

We investigated the effect of ingesting Lactobacillus pentosus S-PT84 on the interferon-α (IFN-α) production from splenocytes and plasmacytoid dendritic cells by virus stimulation. IFN-α production by the Lactobacillus pentosus S-PT84 ingestion group was significantly greater under the virus-infected condition than that by the control group. Lactobacillus pentosus S-PT84 could enhance the production of IFN-α which is known as an important cytokine for preventing virus infection. It may therefore become a prophylactic tool against such virus infection.     

Effect of Lactobacillus pentosus S-PT84 ingestion on IFN-α production from plasmacytoid dendritic cells by virus stimulation

We investigated the effect of ingesting Lactobacillus pentosus S-PT84 on the interferon-α (IFN-α) production from splenocytes and plasmacytoid dendritic cells by virus stimulation. IFN-α production by the Lactobacillus pentosus S-PT84 ingestion group was significantly greater under the virus-infected condition than that by the control group. Lactobacillus pentosus S-PT84 could enhance the production of IFN-α which is known as an important cytokine for preventing virus infection. It may therefore become a prophylactic tool against such virus infection.