Interferon-α Genes from Bos and Bubalus bubalus

Interferon-α genes were cloned from six breeds of three species of two genera (three Chinese native cattle breeds of yellow cattle, wild yak and HuanHu domestic yak, one European breed of Holstein cow, and two water buffalo breeds of FuAn water buffalo and FuZhong water buffalo) by direct PCR. The PCR products were directly inserted into the expression vector to be sequenced and expressed. Sequence analysis showed that IFN-α genes of six clones were composed of 498 nucleotides, encoding a mature polypeptide with 166 amino acids. Compared with the published BoIFN-α subtypes, the IFN-α gene of Holstein cow had only one point mutation with the BoIFN-αA subtype. The IFN-α gene of yellow cattle was similar to the BoIFN-αD subtype with amino acid identity of 97.0% and may be considered as a new subtype, namely, BoIFN-αD1. The other four IFN-α genes, cloned from wild yak and HuanHu domestic yak, FuAn water buffalo, and FuZhong water buffalo, represented four new subtypes, namely, BoIFN-αI, BoIFN-αJ, BuIFN-α1, and BuIFN-α2, respectively. Each of the six clones was expressed in E. coli with molecular weight of ～ 20kDa by SDS-PAGE and Western blot analyses. Antiviral activity assays showed that the six recombinant IFN-α (rIFN-α) all exhibited 1000 times higher antiviral activity in the MDBK/VSV cell line than in the CEF/VSV one. Moreover, the rIFN-αs could inhibit infectious bovine rhinotracheitis virus replication in the MDBK cell line using CPE inhibition method. The results suggested that rIFN-αs a potential agent for clinical application against virus diseases in cattle industry.     

Site-Specific Lipophilic Modification of Interferon-α

Interferon-α (IFNα),⁴ a cytokine with modulatory activities on many cell types, is useful for treating many types of cancer and infectious diseases. This study investigates whether modification of a protein, using IFNα as an example, with a lipophilic group can alter its distribution and kinetic properties in the body. Ser163 of IFNα2a was mutated to Cys to generate a free sulfhydryl group for site-specific chemical modification. IFNα2a(S163C) was conjugated by iodoacetamide derivatives of varying lengths, and the modified IFNα2a was purified by gel filtration chromatography. The biological activities of IFNα2a(S163C) and lipophilized IFNα2a(S163C) were similar to that of IFNα2a, as evidenced by their inhibitory effects on the growth of Daudi cells and on the replication of vesicular stomatitis virus in Madin-Darby bovine kidney cells. Lipophilized IFNα2a(S163C) bound to human serum albumin and cell membranes more readily than did IFNα2a. Future experiments will investigate whether lipophilized IFNα2a(S163C) has improved pharmacokinetic properties.     

干扰素(IFN)与AIDS疫苗

阐述了干扰素（IFN）在重组AIDS疫苗中的作用与意义。为AIDS的基因免疫治疗提出了新的研究方向。     

Synergistic production of TNFα and IFNα by human pDCs incubated with IFNλ3 and IL-3

In this study, we investigated whether IFNλ3 and IL-3 reciprocally influence their capacity to activate various functions of human plasmacytoid dendritic cells (pDCs). In fact, we preliminarily observed that IFNλ3 upregulates the expression of the IL-3Rα (CD123), while IL-3 augments the expression of IFNλR1 in pDCs. As a result, we found that combination of IFNλ3 and IL-3 induces a strong potentiation in the production of TNFα, IFNα, as well as in the expression of Interferon-Stimulated Gene (ISG) mRNAs by pDCs, as compared to either IFNλ3 or IL-3 alone. In such regard, we found that endogenous IFNα autocrinally promotes the expression of ISG mRNAs in IL-3-, but not in IFNλ3 plus IL-3-, treated pDCs. Moreover, we uncovered that the production of IFNα by IFNλ3 plus IL-3-treated pDCs is mostly dependent on endogenously produced TNFα. Altogether, our data demonstrate that IFNλ3 and IL-3 collaborate to promote, at maximal levels, discrete functional responses of human pDCs.     

重组人肿瘤坏死因子(TNF)和干扰素αA(IFNαA)融合蛋白的构建及表达

本文采用定位诱导缺失突变技术,经137个单核苷酸缺失,将串联的重组人肿瘤坏死因子(rhTNF)和重组干扰素αA(rhIFNaA)基因融合成编码单一蛋白的基因。融合基因在大肠杆菌表达后,活性检测证实,存在一旣具有TNF抗肿瘤、又具有IFN抗病毒双重活性的蛋白质。融合蛋白的活性较TNF和IFNαA分别低24倍和15倍。分子筛分析证实,融合蛋白分子量大于25kD。     

Interferon-α abrogates tolerance induction by human tolerogenic dendritic cells

Background

Administration of interferon-α (IFN-α) represents an approved adjuvant therapy as reported for malignancies like melanoma and several viral infections. In malignant diseases, tolerance processes are critically involved in tumor progression. In this study, the effect of IFN-α on tolerance induction by human tolerogenic dendritic cells (DC) was analyzed. We focussed on tolerogenic IL-10-modulated DC (IL-10 DC) that are known to induce anergic regulatory T cells (iTregs).

Methodology/Principal Findings

IFN-α promoted an enhanced maturation of IL-10 DC as demonstrated by upregulation of the differentiation marker CD83 as well as costimulatory molecules. IFN-α treatment resulted in an increased capacity of DC to stimulate T cell activation compared to control tolerogenic DC. We observed a strengthened T cell proliferation and increased IFN-γ production of CD4⁺ and CD8⁺ T cells stimulated by IFN-α-DC, demonstrating a restoration of the immunogenic capacity of tolerogenic DC in the presence of IFN-α. Notably, restimulation experiments revealed that IFN-α treatment of tolerogenic DC abolished the induction of T cell anergy and suppressor function of iTregs. In contrast, IFN-α neither affected the priming of iTregs nor converted iTregs into effector T cells.

Conclusions/Significance

IFN-α inhibits the induction of T cell tolerance by reversing the tolerogenic function of human DC.     

Hypoxia-inducible factor-1α (HIF-1α) and autophagy in polycystic kidney disease (PKD)

Cyst expansion in polycystic kidney disease (PKD) results in localized hypoxia in the kidney that may activate hypoxia-inducible factor-1α (HIF-1α). HIF-1α and autophagy, a form of programmed cell repair, are induced by hypoxia. The purposes were to determine HIF-1α expression and autophagy in rat and mouse models of PKD. HIF-1α was detected by electrochemiluminescence. Autophagy was visualized by electron microscopy (EM). LC3 and beclin-1, markers of autophagy, were detected by immunoblotting. Eight-week-old male heterozygous (Cy/+) and 4-wk-old homozygous (Cy/Cy) Han:SPRD rats, 4-wk-old cpk mice, and 112-day-old Pkd2WS25/- mice with a mutation in the Pkd2 gene were studied. HIF-1α was significantly increased in massive Cy/Cy and cpk kidneys and not smaller Cy/+ and Pkd2WS25/- kidneys. On EM, features of autophagy were seen in wild-type (+/+), Cy/+, and cpk kidneys: autophagosomes, mitophagy, and autolysosomes. Specifically, autophagosomes were found on EM in the tubular cells lining the cysts in cpk mice. The increase in LC3-II, a marker of autophagosome production and beclin, a regulator of autophagy, in Cy/Cy and cpk kidneys, followed the same pattern of increase as HIF-1α. To determine the role of HIF-1α in cyst formation and/or growth, Cy/+ rats, Cy/Cy rats, and cpk mice were treated with the HIF-1α inhibitor 2-methoxyestradiol (2ME2). 2ME2 had no significant effect on kidney volume or cyst volume density. In summary, HIF-1α is highly expressed in the late stages of PKD and is associated with an increase in LC3-II and beclin-1. The first demonstration of autophagosomes in PKD kidneys is reported. Inhibition of HIF-1α did not have a therapeutic effect.     

The dsRNA-mimetic poly (I:C) and IL-18 synergize for IFNγ and TNFα expression

Interleukin (IL)-18 bioactivity and dsRNA sensing by receptors of innate immunity are key components of anti-viral host defense. Despite extensive data on signal transduction activated by both pathways knowledge on cross-communication is incomplete. By using human PBMC and predendritic KG1 cells, as prototypic IL-18-responsive cellular models, we sought to assess cytokine production under the influence of IL-18 and the dsRNA-mimetic poly (I:C). Here, we report on potent synergy between both mediators concerning pro-inflammatory IFNγ and TNFα production. KG1 data revealed that synergistic induction likely relied on TLR3 and was associated with prolonged/increased activation of NF-κB, as detected by IκB analysis and luciferase reporter assays, respectively. Moreover, extended activation of JNK was mediated by IL-18/poly (I:C). Although vital for innate immunity, overwhelming induction of inflammatory cytokines during viral infections poses the threat of serious collateral tissue damage. The stunning synergism inherent to IL-18/dsRNA-induced TNFα/IFNγ detected herein may contribute to this pathological phenomenon.     

Protein Kinase Cα (PKCα) Regulates Bone Architecture and Osteoblast Activity

Bones'' strength is achieved and maintained through adaptation to load bearing. The role of the protein kinase PKCα in this process has not been previously reported. However, we observed a phenotype in the long bones of Prkca^−/− female but not male mice, in which bone tissue progressively invades the medullary cavity in the mid-diaphysis. This bone deposition progresses with age and is prevented by disuse but unaffected by ovariectomy. Castration of male Prkca^−/− but not WT mice results in the formation of small amounts of intramedullary bone. Osteoblast differentiation markers and Wnt target gene expression were up-regulated in osteoblast-like cells derived from cortical bone of female Prkca^−/− mice compared with WT. Additionally, although osteoblastic cells derived from WT proliferate following exposure to estradiol or mechanical strain, those from Prkca^−/− mice do not. Female Prkca^−/− mice develop splenomegaly and reduced marrow GBA1 expression reminiscent of Gaucher disease, in which PKC involvement has been suggested previously. From these data, we infer that in female mice, PKCα normally serves to prevent endosteal bone formation stimulated by load bearing. This phenotype appears to be suppressed by testicular hormones in male Prkca^−/− mice. Within osteoblastic cells, PKCα enhances proliferation and suppresses differentiation, and this regulation involves the Wnt pathway. These findings implicate PKCα as a target gene for therapeutic approaches in low bone mass conditions.     

生产高纯度干扰素(IFN)的新苗头

IFN的作用愈来愈引起人们的高度注视,一是它的抗病毒作用;二是它的抗癌作用,有所谓“妙药”之称,有掀起“工程菌合成IFN”之热。目的是生产大量人体IFN供临床之用。为获取IFN之应用,除保证量的供应外,还有两个重要之点:一是IFN的专异性,即人体IFN仅能用之于人,这样,使IFN应用就受到一定限制;二是IFN高度的     

Activation of Peroxisome Proliferator-activated Receptor α (PPARα) Suppresses Hypoxia-inducible Factor-1α (HIF-1α) Signaling in Cancer Cells

Activation of peroxisome proliferator-activated receptor α (PPARα) has been demonstrated to inhibit tumor growth and angiogenesis, yet the mechanisms behind these actions remain to be characterized. In this study, we examined the effects of PPARα activation on the hypoxia-inducible factor-1α (HIF-1α) signaling pathway in human breast (MCF-7) and ovarian (A2780) cancer cells under hypoxia. Incubation of cancer cells under 1% oxygen for 16 h significantly induced HIF-1α expression and activity as assayed by Western blotting and reporter gene analysis. Treatment of the cells with PPARα agonists, but not a PPARγ agonist, prior to hypoxia diminished hypoxia-induced HIF-1α expression and activity, and addition of a PPARα antagonist attenuated the suppression of HIF-1α signaling. Activation of PPARα attenuated hypoxia-induced HA-tagged HIF-1α protein expression without affecting the HA-tagged HIF-1α mutant protein level, indicating that PPARα activation promotes HIF-1α degradation in these cells. This was further confirmed using proteasome inhibitors, which reversed PPARα-mediated suppression of HIF-1α expression under hypoxia. Using the co-immunoprecipitation technique, we found that activation of PPARα enhances the binding of HIF-1α to von Hippel-Lindau tumor suppressor (pVHL), a protein known to mediate HIF-1α degradation through the ubiquitin-proteasome pathway. Following PPARα-mediated suppression of HIF-1α signaling, VEGF secretion from the cancer cells was significantly reduced, and tube formation by endothelial cells was dramatically impaired. Taken together, these findings demonstrate for the first time that activation of PPARα suppresses hypoxia-induced HIF-1α signaling in cancer cells, providing novel insight into the anticancer properties of PPARα agonists.     

Differential regulation of IFNα, IFNβ and IFNε gene expression in human cervical epithelial cells

Interferonε (IFNε) is a unique type I IFN that has distinct functions from IFNα/β. IFNε is constitutively expressed at mucosal tissues, including the female genital mucosa, and is reported to be modulated by estrogen and seminal plasma. However, its regulation by cytokines, including TNFα, IL-1β, IL-6, IL-8, IL-17, IL-22 and IFNα, which are commonly present in the female genital mucosa, is not well documented in freshly isolated primary cervical cells from tissues. We determined the effect of these cytokines on gene expression of type I IFNs in an immortalized endocervical epithelial cell line (A2EN) and in primary cervical epithelial cells. Several pro-inflammatory cytokines were found to induce IFNε, and TNFα induced the strongest response in both cell types. Pretreatment of cells with the IκB inhibitor, which blocks the NF-κB pathway, suppressed TNFα-mediated IFNε gene induction and promoter activation. Expression of IFNα, IFNβ, and IFNε was differentially regulated in response to various cytokines. Taken together, our results show that regulation of these IFNs depends on cell type, cytokine concentration, and incubation time, highlighting the complexity of the cytokine network in the cervical epithelium.     

生产高纯度干扰素(IFN)的新苗头

IFN的作用愈来愈引起人们的高度注视,一是它的抗病毒作用;二是它的抗癌作用,有所谓“妙药”之称,有掀起“工程菌合成IFN”之热。目的是生产大量人体IFN供临床之用。为获取IFN之应用,除保证量的供应外,还有两个重要之点:一是IFN的专异性,即人体IFN仅能用之于人,这样,使IFN应用就受到一定限制;二是IFN高度的     

干扰素(IFN)研究在我国的发展

干扰素(IFN)是由诱生剂在高等生物细胞中诱导生成的一种糖蛋白,具有抗病毒、抗肿瘤和调节免疫系统的基本作用,在临床上得到实际应用,主要有三种类型即IFN-d、IFN-β、IFN-T。直接从高等生物提取它有一定的局限性,产量有限,价格比黄金还要贵的多。为此通过基因工程办法能使微生物具有合成这三种IFN的能力,这已在国内外取得成功,并有新发展,有的已商品化生产,其产品应用于临床取得较好效果,有的还在继续探究之中,如IFN的分子改造、提高IFN的活性以及扩大新的应用范围等等。我国科研人员经过十来年的努力为微生物合成IFN打下了扎实的基础,使研究工作取得突破性进展,应该说,这是我国基因工程研究最早地取得“工程干扰素”这一重要成果。现在,通过基因工程构建的“工程微生物”能合成不同类型的[FN,有的已进入中试生产阶段。这里着重把近年来IFN基因工程研究所取得的成果包括IFN-r和IL-2、IFN-α、IFN-β与α及其作用以及IFN蛋白工程等方面作些简介。 IFN-r(免疫干扰素)具有较强的免疫调节和细胞抑制活性。     

Insights into the structural dynamics of Liver kinase B1 (LKB1) by the binding of STe20 Related Adapterα (STRADα) and Mouse protein 25α (MO25α) co-activators

LKB1, the tumour suppressor, is found mutated in Peutz-Jeghers syndrome (PJS). The LKB1 is a serine-threonine kinase protein that is allosterically activated by the binding of STRADα and MO25α without phosphorylating the Thr212 present at activation loop. The present study aims to highlight the structural dynamics and complexation mechanism during the allosteric activation of LKB1 by these co-activators using molecular dynamics simulations. The all atom simulations performed on the complexes of LKB1 with ATP, STRADα, and MO25α for a period of 30 ns reveal that binding of STRADα and MO25α significantly stabilizes the highly flexible regions of LKB1 such as ATP binding region (β1-β2 loop), catalytic & activation loop segments and αG helix. Also, binding of STRADα and MO25α to LKB1 promotes coordinated motion between N- and C-lobes along with the catalytic & activation loops by forming H-bonds between LKB1 and co-activators, which further facilitate to establish the conserved attributes of active LKB1 such as (i) formation of salt bridge between Lys78 and Glu98, (ii) formation of stable hydrophobic R- and C-spines, and (iii) interaction between both catalytic and activation loops. Especially, the residues of LKB1 interacting with STRADα (Arg74, Glu342) and MO25α (Glu165, Pro203 and Phe204) are observed to play a significant role in stabilizing the (LKB1-ATP)-(STRADα-ATP)-MO25α complex. Overall, the present work highlighting the structural dynamics of LKB1 by the binding of allosteric co-activators is expected to provide a basic understanding on drug design specific to PJS syndrome.     

A Human Cell Line Model for Interferon-α Driven Dendritic Cell Differentiation

The CD34⁺ MUTZ-3 acute myeloid leukemia cell line has been used as a dendritic cell (DC) differentiation model. This cell line can be cultured into Langerhans cell (LC) or interstitial DC-like cells using the same cytokine cocktails used for the differentiation of their primary counterparts. Currently, there is an increasing interest in the study and clinical application of DC generated in the presence of IFNα, as these IFNα-DC produce high levels of inflammatory cytokines and have been suggested to be more potent in their ability to cross-present protein antigens, as compared to the more commonly used IL-4-DC. Here, we report on the generation of IFNα-induced MUTZ-DC. We show that IFNα MUTZ-DC morphologically and phenotypically display characteristic DC features and are functionally equivalent to “classic” IL-4 MUTZ-DC. IFNα MUTZ-DC ingest exogenous antigens and can subsequently cross-present HLA class-I restricted epitopes to specific CD8⁺ T cells. Importantly, mature IFNα MUTZ-DC express CCR7, migrate in response to CCL21, and are capable of priming naïve antigen-specific CD8⁺ T cells. In conclusion, we show that the MUTZ-3 cell line offers a viable and sustainable model system to study IFNα driven DC development and functionality.     

Inhibiting autophagy potentiates the anticancer activity of IFN1@/IFNα in chronic myeloid leukemia cells

IFN1@ (interferon, type 1, cluster, also called IFNα) has been extensively studied as a treatment for patients with chronic myeloid leukemia (CML). The mechanism of anticancer activity of IFN1@ is complex and not well understood. Here, we demonstrate that autophagy, a mechanism of cellular homeostasis for the removal of dysfunctional organelles and proteins, regulates IFN1@-mediated cell death. IFN1@ activated the cellular autophagic machinery in immortalized or primary CML cells. Activation of JAK1-STAT1 and RELA signaling were required for IFN1@-induced expression of BECN1, a key regulator of autophagy. Moreover, pharmacological and genetic inhibition of autophagy enhanced IFN1@-induced apoptosis by activation of the CASP8-BID pathway. Taken together, these findings provide evidence for an important mechanism that links autophagy to immunotherapy in leukemia.