IFN-α/β and autophagy

Hepatitis C virus (HCV) infects approximately 130 million people worldwide. The clinical sequelae of this chronic disease include cirrhosis, functional failure and carcinoma of the liver. HCV induces autophagy, a fundamental cellular process for maintaining homeostasis and mediating innate immune response, and also inhibits autophagic protein degradation and suppresses antiviral immunity. In addition to this ploy, the HCV serine protease composed of the viral non-structural proteins 3/4A (NS3/4A) can enzymatically digest two cellular proteins, mitochondria-associated anti-viral signaling protein (MAVS) and Toll/interleukin-1 receptor domain containing adaptor inducing IFN-β (TRIF). Since these two proteins are the adaptor molecules in the retinoic acid-inducible gene I (RIG-I) and TLR3 pathways, respectively, their cleavage has been suggested as a pivotal mechanism by which HCV blunts the IFN-α/β signaling and antiviral responses. Thus far, how HCV perturbs autophagy and copes with IFN-α/β in the liver remains unclear.     

食用IFN-α薄脆饼卷土重来

号称能治疗艾滋病的α-干扰素(IFN-α)薄脆饼回来了。不过,这一次不再喳喳呼呼,而是带来一些可靠的科学试验数据。两年前,肯尼亚曾报道,吃过IFN-α薄脆饼的艾滋病患者减轻了许多艾滋病基本症状,包括厌食、疲倦、发烧、口腔溃疡,真菌感染、腹泻、淋巴结肿大等。肯尼亚医学界权威人士说,薄脆治疗使CD_4淋巴细胞水平大大降低,并且,在99个受试患者中,11名已查不出艾滋病抗体。平均每人增重10磅。     

Apoptosis-inducing factor (AIF) is targeted in IFN-α2a-induced Bid-mediated apoptosis through Bak activation in ovarian cancer cells

Previously we have shown that interferon (IFN)-α induced apoptosis is predominantly mediated by the upregulation of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) via the caspase-8 pathway. It was also shown that recruitment of mitochondria in IFN-α induced apoptosis involves the cleavage of BH3 interacting domain death agonist (Bid) to truncated Bid (tBid). In the present study, we demonstrate that tBid induced by IFN-α2a activates mitochondrial Bak to trigger the loss of mitochondrial membrane integrity, consequently causing release of apoptosis-inducing factor (AIF) in ovarian cancer cells, OVCAR3. AIF translocates from the mitochondria to the nucleus and induces nuclear fragmentation and cell death. Both a small molecule Bid inhibitor (BI-6C9) or Bid-RNA interference (RNAi) preserved mitochondrial membrane potential, prevented nuclear translocation of AIF, and abrogated IFN-α2a-induced cell death. Cell death induced by tBid was inhibited by AIF-RNAi, indicating that caspase-independent AIF signaling is the main pathway through which Bid mediates cell death. This was further supported by experiments showing that BI-6C9 did not prevent the release of cytochrome c from mitochondria to cytosol, while the release of AIF was prevented. In conclusion, IFN-α2a-induced apoptosis is mediated via the mitochondria-associated pathway involving the cleavage of Bid followed by AIF release that involves Bak activation and translocation of AIF from the mitochondria to the nucleus in OVCAR3 cells.     

Aberrant expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice

The Th2 locus control region (LCR) has been shown to be a crucial cis-acting element for Th2 cytokine expression and Th2 cell differentiation. To study the role of Th2 LCR in ifng locus regulation, we examined the expression of IFN-γ in Th2 cells from Th2 LCR-deficient mice. We found IFN-γ to be aberrantly up-regulated. In addition, histone 3(H3)-acetylation and histone 3 lysine 4 (H3-K4)-methylation greatly increased at the ifng locus of the Th2 cells. GATA-3 and STAT6 bound to the ifng promoter in Th2 cells from the wild type but not from the Th2 LCR-deficient mice, and they directly repressed ifng expression in transient reporter assay. Moreover, ectopic expression of GATA-3 and STAT6-VT repressed the aberrant expression of the ifng gene and restored repressive chromatin state at the ifng locus in Th2 cells from Th2 LCR-deficient mice. These results suggest that expression of the ifng gene and chromatin remodeling of the ifng locus are under the control of a Th2 LCR-mediated Th2 differentiation program.     

Interferon Science的IFN-α产品被批准

Interferon Sciences Inc.公司(New Brunswick,NJ)新近获得人白血细胞α干扰素(IFN-α)产品的市场允许,该产品是用于治疗外阴疣的新药物。尽管另一家公司Schering-Plough Corp(Madison,NJ)已将Biogen公司(Cambridge,MA)开发的rDNAα干扰素打入市场,Interferon Sciences的产品仍是在美国获准的首例白细胞型α干扰素。从临床数据的比较来看,Interferon Sciences的产品可能优于Schering的重组DNA产品。白细胞型α干扰素治疗每个疣的基础平均剂量为225000单位。每周注射2次,共注     

Gamma Interferon (IFN-γ) Receptor Restricts Systemic Dengue Virus Replication and Prevents Paralysis in IFN-α/β Receptor-Deficient Mice

We previously reported that mice lacking alpha/beta and gamma interferon receptors (IFN-α/βR and -γR) uniformly exhibit paralysis following infection with the dengue virus (DENV) clinical isolate PL046, while only a subset of mice lacking the IFN-γR alone and virtually no mice lacking the IFN-α/βR alone develop paralysis. Here, using a mouse-passaged variant of PL046, strain S221, we show that in the absence of the IFN-α/βR, signaling through the IFN-γR confers approximately 140-fold greater resistance against systemic vascular leakage-associated dengue disease and virtually complete protection from dengue-induced paralysis. Viral replication in the spleen was assessed by immunohistochemistry and flow cytometry, which revealed a reduction in the number of infected cells due to IFN-γR signaling by 2 days after infection, coincident with elevated levels of IFN-γ in the spleen and serum. By 4 days after infection, IFN-γR signaling was found to restrict DENV replication systemically. Clearance of DENV, on the other hand, occurred in the absence of IFN-γR, except in the central nervous system (CNS) (brain and spinal cord), where clearance relied on IFN-γ from CD8⁺ T cells. These results demonstrate the roles of IFN-γR signaling in protection from initial systemic and subsequent CNS disease following DENV infection and demonstrate the importance of CD8⁺ T cells in preventing DENV-induced CNS disease.     

L-type Ca2+ channels’ involvement in IFN-γ-induced signaling in rat ventricular cardiomyocytes

Journal of Physiology and Biochemistry - The purpose of this study was to examine the effects of interferon-γ (IFN-γ) on calcium movement in rat ventricular myocytes. L-type Ca2+ currents...     

SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells

Background

We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.

Methodology/Principal Findings

In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.

Conclusions

These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.     

可诱导肿瘤靶向性IFN-α2a重组腺病毒的构建及其表达

腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗的研究,但这些肿瘤靶向载体缺乏可控性,其疗效和安全性受到很大的影响,因此开发新型可诱导的生物肿瘤靶向载体是当今抗肿瘤靶向药物研究的当务之急。该研究构建了可诱导肿瘤靶向性IFN-α2a重组腺病毒。重组腺病毒能够有效感染人肝癌细胞株HepG2等多种肿瘤细胞株,RT-PCR和Western blot结果表明肿瘤细胞能高效表达IFN-α2a,而其非肿瘤细胞株L02几乎没有表达。诱导试验表明重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a能被ZnSO4诱导表达。可诱导肿瘤靶向性重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a成功构建为下一步体内外抑癌实验研究打下了基础。     

绿头鸭IFN-α的可溶性表达及其活性分析

为了利用原核表达系统研制有生物活性的鸭IFN-α。以pMD18T-Ma IFN-α为模板扩增绿头鸭IFN-α成熟肽基因,将其克隆至原核表达载体pET-32 a中,在大肠杆菌BL21中进行变温诱导表达。SDS-PAGE分析表达结果,并用Western blotting进行验证。Ni2+树脂柱纯化目的蛋白后,测定其生物活性。结果表明,重组pET-32a(+)-Ma IFN-α在大肠杆菌BL21成功表达,主要以可溶性形式存在,Western blotting分析显示目的蛋白具有良好的抗原性。细胞病变抑制法测定重组鸭IFN-α的抗病毒活性约为8×104U/m L;荧光定量PCR方法检测显示表达的重组鸭IFN-α使NDV在DEF上的复制受到了明显的抑制,48 h时间点相对抑制率高达91%。这些都表明表达的重组鸭IFN-α具有良好抗病毒活性。     

番鸭IFN-α基因的克隆与序列分析

广东华南农业大学兽医学院王春霞、王林川和黄爱芳等6位先生从经免疫的番鸭外周血分离淋巴细胞,经PHA刺激培养后提取总RNA,采用RT—PCR法,按照GenBank中序列号为X84764鸭Ⅰ型IFN（DIFN1）基因设计引物,扩增出了番鸭IFN基因,命名为MDIFN-α,包含编码MDIFN-α成熟蛋白全部核苷酸序列。DIFN1开放阅读框架（ORF）有576个核苷酸,编码191个氨基酸,其中除去信号肽的IFN-α为成熟DIFN1,共有486个核苷酸组成,编码161个氨基酸,含有不完整信号肽的MDIFN-α与X84764的核苷酸同源性为97．7％。故他们6位先生从研究结果和大量参考资料中推导氨基酸同源性为95．7％,后来研究的结果显示,那种MDIFN-α可能为鸭Ⅰ型IFN一个新的亚型。     

High-level production of recombinant human IFN-α2a with co-expression of tRNAArg(AGG/AGA) in high-cell-density cultures ofEscherichia coli

The co-expression of theargU gene in a double-vector expression system of recombinantEscherichia coli BL21(DE3)[pET-IFN2a+pAC-argU] significantly enhanced the production level of recombinant human interferon-α2a (rhIFN-α2a) in high cell density cultures compared to a recombinantE coli culture containing only the single expression vector, pET-IFN2a. The dry cell mass concentration increased to almost 100 g/L, and more than 4 g/L of rhIFN-α2a was accumulated in the culture broth. Evidently, the synthesis of rhIFN-α2a was strongly dependent on the pre-induction growth rate and more efficient at a higher specific growth rate. The additional supply of tRNA^Arg(AGG/AGA) enhanced the expression level of the rhIFN-α2a gene in the early stage of the post-induction phase, yet thereafter the specific production rate of rhIFN-α2a rapidly decreased due to severe segregational instability of plasmid vector pET-IFN2a. It would appear that the plasmid instability, which only occurred to pET-IFN2a in the double vector system, was related to the effect of translational stress due to the overexpression of rhIFN-α2a.     

Metabolism of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane in the rabbit

The neutral urinary excretion products of 17β-hydroxy-2α,3α-cyclopropano-5α-androstane from the rabbit, dosed orally, were investigated. Column chromatography yielded five crystalline metabolites which were identified by GLC and spectroscopic measurements. Three of these substances were hydroxylated in the 4α-position and one in the 6a-position with the cyclopropane ring intact. The fifth substance, 17β-hydroxy-3β-methyl-5α-androstan-2-one, can be derived from initial hydroxylation of the cyclopropane ring at C-2 followed by ring opening. The dosed substance and triol material was shown to be present by GLC and m.s. measurements. GLC determinations show that hydroxylation has occurred at C-4?C-6>C-2.     

B Cell and BAFF dependence of IFN-α-exaggerated disease in systemic lupus erythematosus-prone NZM 2328 mice

IFN-α is a potent activator of innate and adaptive immunity, and its administration to preautoimmune (NZB×NZW)F1 mice promotes virulent systemic lupus erythematosus (SLE) disease. Given the known contributions of B cells and BAFF to SLE, we evaluated the ability of IFN-α administration to induce disease in wild-type (WT), B cell-deficient, and BAFF-deficient NZM 2328 mice. Whereas WT mice rapidly developed proliferative glomerulonephritis, marked proteinuria, and increased mortality in response to IFN-α administration, B cell-deficient mice developed neither renal pathology nor clinical disease. Moreover, BAFF-deficient mice, despite developing limited glomerular IgG and C3 deposition, also remained free of histological glomerulonephritis and clinical disease. Strikingly, similar T cell expansion and serum IgG responses were observed in adenovirus (Adv)-IFN-treated WT and BAFF-deficient mice despite their disparate pathological and clinical responses, whereas numbers of activated B cells increased in WT mice but not in BAFF-deficient mice. Nonetheless, B cell, plasma cell, and T cell infiltration of the kidneys in Adv-IFN-treated WT mice was similar to that in WT mice treated with Adv-control. Its ability to promote SLE disease in WT mice notwithstanding, IFN-α administration failed to drive the preferential expansion of CD4(+) memory T cells that occurs during the natural course of disease, and glomerular infiltration of macrophages failed to associate with development of disease. These results collectively suggest that therapeutic targeting in SLE of BAFF and/or B cells in SLE could be successful even in states of IFN-α overexpression. Moreover, our results document important biological differences between IFN-α-driven and spontaneous natural SLE disease.     

重组酵母产人IFN-α2a与LL-37融合蛋白的工艺优化

通过单因素试验探索重组酵母P.pastoris GS115LI产人LL-37基因与IFN-α2a融合蛋白的最佳条件。结果显示,重组酵母菌产蛋白酶的最佳表达条件是利用BMMY为诱导培养基,以接种量OD600=5.5、温度26℃、p H6.0、诱导剂(甲醇)为每隔24 h添加1.0%、振荡速度为200 r/min条件下连续诱导144 h。在最佳培养条件下,发酵液中的LL-37最高抗菌活性达27.9 mm。与初始的发酵条件相比,人LL-37基因与IFN-α2a融合蛋白的产量提高了32.29%。     

IFN-γ promoter gene polymorphism in psoriasis vulgaris

This study was performed to investigate the association between interferon (IFN)-γ single nucleotide polymorphism (SNP) and susceptibility for psoriasis vulgaris. DNA from 78 patients with psoriasis vulgaris (54 patients with type I psoriasis, 24 with type II psoriasis) and 74 healthy volunteers was investigated. IFN-γ promoter gene SNP in position 874 was evaluated by polymerase chain reaction with sequence-specific primers (PCR-SSP) and the results were compared between a group of psoriatic patients, divided into early onset of psoriasis (type I) and late onset of psoriasis (type II) subgroups, and healthy control subjects. A significant difference in the genotype frequencies between psoriasis patients and healthy controls was found (p <0.02) and no significant differences were observed analyzing subsets of psoriatic patients (gender, type of disease) also in carriage and allele frequencies. The results suggest that IFN-γ polymorphism is associated with susceptibility to psoriasis vulgaris.     

Fixed-time deep uterine insemination in PGF2α-synchronized goats

The aim of this investigation was to optimize fixed-time insemination in goats by clustering ovulations in prostaglandin F_2α-synchronized goats either with gonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (hCG). The underlying intention was to reduce the incidence of short cycles by providing a more sustained stimulation of the corpus luteum by substituting the commonly used GnRH with longer-acting hCG. It was conjectured that this might render the corpus luteum less prone to premature regression. Sixty pluriparous does were administered 5 mg of the prostaglandin F_2α preparation dinoprost (Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) during the luteal phase of the estrous cycle. Twenty of these does were administered 0.004 mg of the GnRH analog buserelin (Receptal; Intervet, Unterschleissheim, Germany) 48 hours later; another 20 does received 500 IU hCG (Chorulon; Intervet, Unterschleissheim, Germany) instead. Sixteen hours later the does were inseminated with frozen-thawed semen. The remaining 20 does served as controls and were inseminated 16-18 h after the onset of detected estrus. All 60 treated goats displayed estrous symptoms, the time of onset being similar for all groups (42.6, 37.6, and 40.5 hours after treatment for GnRH-treated, hCG-treated, and control does, respectively). The duration of estrus in the GnRH-treated group was 10 h less than in the other groups (45.1 vs. 56.4 and 54.4 h, P < 0.05). The number of ovulations (assessed by ultrasound monitoring) did not differ among groups (2.4, 2.1, and 2.5, P > 0.05). Monitoring of serum progesterone revealed that the incidence of corpus luteum insufficiency was significantly higher in GnRH- and hCG-treated does than in the control group (40% and 35% vs. 5%, P < 0.05). The pregnancy rate was 50% in the GnRH and 35% in the hCG group as compared with 60% in the controls. Corresponding kidding rates were 40%, 35%, and 60% (P > 0.05). When disregarding does with corpus luteum insufficiency, pregnancy rates would have been 83%, 54%, and 63%, and kidding rates 67%, 54%, and 63%, respectively. The average number of kids born was 1.88, 1.71, and 1.83, respectively (P > 0.05). It may be concluded that fixed time insemination of cycling does treated with prostaglandin F_2α during the luteal phase, followed by ovulation induction with GnRH or hCG, would be an effective management tool if it were possible to control the high incidence of corpus luteum insufficiency. The attempt to achieve this by substituting GnRH with hCG, was not met with success. Until a solution for the problem has been found, it is advisable to inseminate prostaglandin-synchronized does 16-18 hours after the onset of detected estrus.     

Fixed-time deep uterine insemination in PGF2α-synchronized goats

The aim of this investigation was to optimize fixed-time insemination in goats by clustering ovulations in prostaglandin F_2α-synchronized goats either with gonadotropin releasing hormone (GnRH) or human chorionic gonadotropin (hCG). The underlying intention was to reduce the incidence of short cycles by providing a more sustained stimulation of the corpus luteum by substituting the commonly used GnRH with longer-acting hCG. It was conjectured that this might render the corpus luteum less prone to premature regression. Sixty pluriparous does were administered 5 mg of the prostaglandin F_2α preparation dinoprost (Dinolytic; Pharmacia and Upjohn, Erlangen, Germany) during the luteal phase of the estrous cycle. Twenty of these does were administered 0.004 mg of the GnRH analog buserelin (Receptal; Intervet, Unterschleissheim, Germany) 48 hours later; another 20 does received 500 IU hCG (Chorulon; Intervet, Unterschleissheim, Germany) instead. Sixteen hours later the does were inseminated with frozen-thawed semen. The remaining 20 does served as controls and were inseminated 16–18 h after the onset of detected estrus. All 60 treated goats displayed estrous symptoms, the time of onset being similar for all groups (42.6, 37.6, and 40.5 hours after treatment for GnRH-treated, hCG-treated, and control does, respectively). The duration of estrus in the GnRH-treated group was 10 h less than in the other groups (45.1 vs. 56.4 and 54.4 h, P < 0.05). The number of ovulations (assessed by ultrasound monitoring) did not differ among groups (2.4, 2.1, and 2.5, P > 0.05). Monitoring of serum progesterone revealed that the incidence of corpus luteum insufficiency was significantly higher in GnRH- and hCG-treated does than in the control group (40% and 35% vs. 5%, P < 0.05). The pregnancy rate was 50% in the GnRH and 35% in the hCG group as compared with 60% in the controls. Corresponding kidding rates were 40%, 35%, and 60% (P > 0.05). When disregarding does with corpus luteum insufficiency, pregnancy rates would have been 83%, 54%, and 63%, and kidding rates 67%, 54%, and 63%, respectively. The average number of kids born was 1.88, 1.71, and 1.83, respectively (P > 0.05). It may be concluded that fixed time insemination of cycling does treated with prostaglandin F_2α during the luteal phase, followed by ovulation induction with GnRH or hCG, would be an effective management tool if it were possible to control the high incidence of corpus luteum insufficiency. The attempt to achieve this by substituting GnRH with hCG, was not met with success. Until a solution for the problem has been found, it is advisable to inseminate prostaglandin-synchronized does 16–18 hours after the onset of detected estrus.     

Caspase-3 is Involved in IFN-γ- and TNF-α-Mediated MIN6 Cells Apoptosis via NF-κB/Bcl-2 Pathway

TNF-α and IFN-γ are the major pro-inflammatory cytokines in the β-cell destruction. However, the underlying mechanism remains unclear. The present study used a murine insulinoma cell line MIN6 for further investigation of the effect of Caspase-3 on the cytokines-induced pancreatic β-cell apoptosis and analyzed the mechanisms involved in the activation of Caspase-3. It was showed that the combination of IFN-γ and TNF-α significantly reduced the viability of MIN6 cells and the observed cells growth inhibition was due to cell apoptosis as judged by the morphological changes under a confocal laser scanning microscopy and FACS assay of Annexin-V/7-AAD double staining. Accompanying with NF-κB activation and Bcl-2 downregulation, both the cleaved Caspase-3 and PARP, a known substrate of Caspase-3 in vivo, were observed at 24 and 12 h, respectively, after cells exposure to IFN-γ and TNF-α treatment. Pretreatment of Caspase-3 inhibitors remarkably attenuated IFN-γ- and TNF-α-induced cells apoptosis. Inhibition of NF-κB activation led to the increase in Bcl-2 expression, a significant attenuation in Caspase-3 activity, and an obvious amelioration in cells viability in IFN-γ- and TNF-α-treated MIN6 cells. Taken together, our results indicate that Caspase-3 is critical for the induction of MIN6 cells apoptosis and it’s activation is further confirmed to be related to the NF-κB-mediated Bcl-2 downregulation, which may be the underlying mechanism of IFN-γ- and TNF-α-mediated MIN6 cells apoptosis.