Distinct Pathways for Tumor Necrosis Factor Alpha and Ceramides in Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection can be fatal to immunocompromised individuals. We have previously reported that gamma interferon and tumor necrosis factor alpha (TNF-α) synergistically inhibit HCMV replication in vitro. Ceramides have been described as second messengers induced by TNF-α. To investigate the mechanisms involved in the inhibition of HCMV by TNF-α, in the present study we have analyzed ceramide production by U373 MG astrocytoma cells and the effects of TNF-α versus ceramides on HCMV replication. Our results show that U373 MG cells did not produce ceramides upon incubation with TNF-α. Moreover, long-chain ceramides induced by treatment with exogenous bacterial sphingomyelinase inhibited HCMV replication in synergy with TNF-α. Surprisingly, short-chain permeant C6-ceramide increased viral replication. Our results show that the anti-HCMV activity of TNF-α is independent of ceramides. In addition, our results suggest that TNF-α and endogenous long-chain ceramides use separate pathways of cell signalling to inhibit HCMV replication, while permeant C6-ceramide appears to activate a third pathway leading to an opposite effect.Human cytomegalovirus (HCMV) infections are well controlled in the immunocompetent host. Cellular immune responses (CD4⁺ and CD8⁺ T cells and NK cells) which accompany both acute and latent infections (for a review, see reference 4) are thought to be the main components of this control. HCMV infection during immunosuppression such as in cancer, transplantations, or AIDS results in severe pathology (4). We have previously shown that tumor necrosis factor alpha (TNF-α), in synergy with gamma interferon (IFN-γ), inhibits the replication of HCMV (7). In mice, TNF-α is involved in the clearance of CMV infection (25). TNF-α is a cytokine with multiple effects which is produced by many cell types, including macrophages and CD8⁺ and CD4⁺ T lymphocytes (for a review, see reference 40), and is known to possess antiviral effects (20, 47). The molecular mechanisms involved in the signalling by TNF-α depend on the type of receptor, p55 (TNF-R1) or p75 (TNF-R2) (5), to which it binds. Some cells express only one type of TNF-α receptor; however, expression of these receptors is not always mutually exclusive (5). The cytotoxicity of TNF-α has been reported to be mediated by TNF-R1 (38), whose intracellular region carries a death domain which signals for programmed cell death (39). Signalling through TNF-R1 with specific antibodies can also protect Hep-G2 cells from vesicular stomatitis virus-mediated cytopathic effects (48). Ceramide production after TNF-α treatment has been widely reported (16, 19, 31) and has also been shown to depend on signalling through the TNF-R1 receptor (45). In these experiments concerning myeloid cells, TNF-α induced the activation of a sphingomyelinase, which cleaved sphingomyelin to release ceramide and phosphocholine. The production of ceramides can lead to cell apoptosis (11, 14, 23) or cell cycle arrest (13). Induction of apoptosis by TNF-α has been mimicked by exogenous sphingomyelinase and by synthetic, short-chain, permeant ceramides, which suggests that ceramides, as second messengers, are sufficient to induce the cytotoxic effects of TNF-α (11, 23). Acidic and neutral sphingomyelinases activated in different cell compartments may be responsible for the diverse effects of TNF-α (46), with the former being involved in signalling through NF-κB (34) and the latter being involved in signalling through a ceramide-activated protein kinase and phospholipase A₂ (46).One of the characteristics of HCMV infection is the increase in the content of intracellular DNA, reported to be of viral (3, 8, 18) or cellular (12, 37) origin. Since TNF-α has been known to display antiproliferative properties and to block cells in the G₁ phase (29), we initially tested its effects on the cell cycle of infected cells. Then, based on studies reporting that TNF-α induces ceramides in cells (16, 19, 31) and on a study showing the role of ceramide in cell cycle blockade (13), we originally postulated that ceramide was responsible for the antiviral effect of TNF-α. In the present study, we used astrocytoma cells (U373 MG) as a model for brain cells, which are important targets of HCMV in vivo (22). In contrast to fibroblasts, infected U373 MG cells release smaller quantities of virus particles even though all the cells were infected in our experiments. We believe that the U373 MG model is closer to HCMV infection in vivo. We show that ceramides are not produced by U373 MG cells upon incubation with even high concentrations of TNF-α. In addition, we demonstrate that exogenously added sphingomyelinase induces anti-HCMV effects whereas permeant C6-ceramide increases HCMV proliferation in U373 MG cells. This suggests that lipid second messengers can modulate HCMV infection and that TNF-α and ceramides use distinct signalling pathways in the control of HCMV infection. This is supported by our observation that the protein kinase JNK1 is activated exclusively by TNF-α in U373 MG cells and that TNF-α and exogenous sphingomyelinase act in synergy on HCMV infection.     

人源性抗TNFα小分子抗体的改构和分析

在获得人源性抗人TNF α单链抗体 (ScFv)基因序列的基础上 ,对ScFv的连接肽部分进行基因改造 ,并构建了Fab抗体基因。改构前后的ScFv分别重组入表达载体pBV2 2 0 ,经 42℃热诱导 ,在E .coliDH5α中表达了ScFv蛋白 ,得到分子量约为 30kD的重组蛋白质 ,改构前后ScFv的表达量分别占菌体总蛋白质的 6 .5 %和13 .8%。同时构建Fab可溶性表达载体并转化非抑制型菌株HB2 15 1,经IPTG诱导 ,在约 5 0kD分子量处呈现一条新生蛋白质条带。从大肠杆菌裂解液中对ScFv进行了复性和层析纯化 ,对Fab基因的表达产物进行了亲和层析纯化 ,并证实 :(1)改构后ScFv在大肠杆菌中的表达量有所提高 ;(2 )改构前后的ScFv与Fab均具有与hTNF α相结合的活性 ,具有GGGGS连接肽的ScFv与hTNF α的亲和常数为 6 .70× 10 4 (mol/L) -1,而改构后具有(GGGGS) 3 连接肽的ScFv的亲和常数提高为 7.2 7× 10 5(mol/L) -1,Fab与hTNF α的亲和常数为 7.6 1× 10 5(mol/L) -1,Fab与改构后ScFv的亲和力无明显差异 ;(3)ScFv与Fab均有中和hTNF α细胞毒的作用 ,具有(GGGGS) 3 连接肽的ScFv与Fab的中和活性基本相同 ,但均明显低于一株鼠源性单抗     

人α-型肿瘤坏死因子cDNA在大肠杆菌中的高效表达

人α-型肿瘤坏死因子(Tumor Necrosis Factor,TNF-α)可以在体内或体外特异地杀伤多种肿瘤细胞,并同时具有抗病毒感染及免疫调节活性,是一种非常有前途的抗肿瘤及抗病毒生物制剂。最近,国外用基因工程技术将TNF-α基因克隆并在大肠杆菌及酵母细胞中表达成功。目前,人们正致力于提高其在大肠杆菌中表达产量的研究。据此,我们构建了P_L启动子控制的TNF-α cDNA表达载体,使其在大肠杆菌中获得了高水平表达。     

TLR-Mediated Production of Reactive Oxygen Species and Tumor Necrosis Factor Alpha by Human Peripheral Blood Neutrophils

This paper presents the study on TLR-mediated production of reactive oxygen species and tumor necrosis factor alpha by peripheral blood neutrophils in healthy donors stimulated with zymosan (TLR2/6 ligand), peptidoglycan (TLR2/1 ligand), and lipopolysaccharide (TLR4 ligand). Luminol- and lucigen-independent chemiluminescence was used to detect the production of reactive oxygen species. The concentration of tumor necrosis factor alpha was measured by enzyme immunoassay. The plots of dependence of the light sums of luminol- and lucigenin-dependent chemiluminescence on the concentration of each ligand were shaped as saturation curves. The comparison of the light sums of lucigenin-dependent chemiluminescence (the production of superoxide anion radical) and luminol-dependent chemiluminescence (the total production of reactive oxygen species) showed that the contribution of NADPH oxidase to the total TLR-mediated production of oxidants can reach 40–50%. Stimulation indices were calculated to compare the ability of TLR ligands to stimulate the production of reactive oxygen species and tumor necrosis factor alpha by neutrophils. It has been established that the activation of neutrophils with zymosan leads to higher (more than 8-fold) production of reactive oxygen species rather than production of tumor necrosis factor alpha. Unlike zymosan, lipopolysaccharide stimulated the production of tumor necrosis factor alpha to a greater extent (by more than 2 times) than the production of reactive oxygen species. Peptidoglycan takes an intermediate position between these ligands. Thus, the production of effector molecules (reactive oxygen species and tumor necrosis factor alpha) by human peripheral blood neutrophils depends on the nature of the TRL ligand.     

Granulocyte-Macrophage Colony-Stimulating Factor and Tumor Necrosis Factor Alpha in Patients with Human Immunodeficiency Virus (HIV) Type 1 Infection

Variations in cytokine production in patients with human immunodeficiency virus (HIV) infection could be involved in the physiopathology and in the progression of the disease. Therefore we studied the level of granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor α (TNFα) produced in patients with HIV infection at stage II (asymptomatic seropositives) and stage IV (AIDS) of the CDC classification, by using an enzyme amplified sensitivity immunoassay. We measured the level of GM-CSF and TNFα in supernatant of phytohemagglutinin-activated peripheral blood mononuclear cells from patients and healthy individuals. In one out of 10 stage II patients and 4 out of 14 stage IV patients, we obtained higher levels of GM-CSF than the mean + 2 S.D. of controls, but in 3 stage IV patients with very low CD4+ T lymphocyte counts (< 50/mm^–3) compared to other patients, the GM-CSF values were very low. High levels of TNFα were detected in 3 out of 10 stage II and 6 out of 11 stage IV patients. The high values of TNFα were associated with high values of GM-CSF in stage II and in most of AIDS patients except those with very low CD4⁺ T cell counts, who produced low levels of GM-CSF. Plasma levels of cytokines were evaluated in 10 stage II, 22 stage IV patients and 20 controls. Increased levels of GM-CSF (more than 9 pg/ml) were observed in the plasma from 8 out of 10 stage II patients and 17 out of 22 stage IV patients. The tendency that increased levels of GM-CSF were associated with increased levels of TNFα was observed in plasma from stage IV patients. We report a disarray of GM-CSF production in patients with HIV infection that could be involved in clinical manifestations and progression of the disease.     

高温和红光诱导的鱼腥藻7120短藻丝体中TNF-α基因表达效率的提高

目前解决外源基因在蓝藻中低效表达的主要策略是改进供体DNA元件。这项工作尝试改变受体系统生理状态,研究对外源基因表达效率的影响。以前已经报道用高温(45℃)和红光处理鱼腥藻7120(Anabaena sp．PCC7120)可以诱导其形成短藻丝体,这里报道以此作为受体细胞,将构建的含重组人肿瘤坏死因子α(TNF—α)基因的穿梭表达载体pKT-TNF通过三亲接合转移法进行转化。红光和高温诱导形成的短藻丝体中,TNF—α基因接合转移效率为在正常营养藻丝中的5～6倍,Southern杂交结果证明pKT-FNF已在受体细胞中复制。Western印迹表明TNF—α基因已在受体系统中表达,放射免疫测定结果显示在短藻丝体中的表达效率提高到在正常营养藻丝中的4～5倍。这可能为提高外源基因在丝状蓝藻中的表达效率提供了一条新途径。此外,研究还分析了人TNF-α基因密码子使用偏向性对在鱼腥藻7120中表达效率的影响。     

肿瘤坏死因子-α在乳腺肿瘤中的表达及其与影像学特征的关系

目的:检测乳腺肿瘤中肿瘤坏死因子α(Tumor necrosis factor alpha,TNF-α)表达及其与影像学特征的关系。方法:利用免疫荧光和流式细胞术检测MDA-MB-231及MCF-7乳腺癌细胞中TNF-α的表达;收集82例经病理证实的乳腺疾病患者组织及其病理资料、影像学资料,通过免疫组化检测乳腺组织TNF-α的表达,并分析其表达量与病理特征及影像学特征之间的关系。结果:TNF-α在MDA-MB-231细胞中呈高表达,恶性乳腺肿瘤组织中TNF-α的表达显著高于乳腺良性肿瘤,其表达量与淋巴结转移、TNM分期相关、核磁共振(MRI)强化是否均匀、钼靶X射线的边界的平滑度形状是否规则以及B超中彩色血流信号强弱均显著相关(P0.05)。结论:乳腺肿瘤组织中TNF-α呈异常高表达,且与乳腺肿瘤的某些影像学特征密切相关。     

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。     

犬α干扰素在家蚕杆状病毒表达系统中的表达及其抗病毒活性的测定

犬α干扰素(Ca IFNα)在犬病防治过程中应用广泛。旨在开发一种高效的Ca IFNα表达方法。首先按家蚕密码子的偏好性对Gen Bank中的一个Ca IFNα基因进行了优化与合成,将其克隆到杆状病毒转移载体p VL1393中,并与亲本病毒Bm Bacmid共转染家蚕细胞进行细胞内重组。得到的重组病毒用于感染家蚕幼虫,收集发病幼虫的蚕血淋巴用于Ca IFNα检测。采用细胞病变抑制法在MDCK-VSV*GFP系统中测定其抗病毒活性。结果显示家蚕表达的Ca IFNα能有效抑制VSV*GFP在细胞内的复制,其抗病毒活性不低于1.78×106U/m L。     

Respiratory Mechanics and Plasma Levels of Tumor Necrosis Factor Alpha and Interleukin 6 Are Affected by Gas Humidification during Mechanical Ventilation in Dogs

The use of dry gases during mechanical ventilation has been associated with the risk of serious airway complications. The goal of the present study was to quantify the plasma levels of TNF-alpha and IL-6 and to determine the radiological, hemodynamic, gasometric, and microscopic changes in lung mechanics in dogs subjected to short-term mechanical ventilation with and without humidification of the inhaled gas. The experiment was conducted for 24 hours in 10 dogs divided into two groups: Group I (n = 5), mechanical ventilation with dry oxygen dispensation, and Group II (n = 5), mechanical ventilation with oxygen dispensation using a moisture chamber. Variance analysis was used. No changes in physiological, hemodynamic, or gasometric, and radiographic constants were observed. Plasma TNF-alpha levels increased in group I, reaching a maximum 24 hours after mechanical ventilation was initiated (ANOVA p = 0.77). This increase was correlated to changes in mechanical ventilation. Plasma IL-6 levels decreased at 12 hours and increased again towards the end of the study (ANOVA p>0.05). Both groups exhibited a decrease in lung compliance and functional residual capacity values, but this was more pronounced in group I. Pplat increased in group I (ANOVA p = 0.02). Inhalation of dry gas caused histological lesions in the entire respiratory tract, including pulmonary parenchyma, to a greater extent than humidified gas. Humidification of inspired gases can attenuate damage associated with mechanical ventilation.     

Class III Phosphatidylinositol 4-Kinase Alpha and Beta Are Novel Host Factor Regulators of Hepatitis C Virus Replication

Host factor pathways are known to be essential for hepatitis C virus (HCV) infection and replication in human liver cells. To search for novel host factor proteins required for HCV replication, we screened a subgenomic genotype 1b replicon cell line (Luc-1b) with a kinome and druggable collection of 20,779 siRNAs. We identified and validated several enzymes required for HCV replication, including class III phosphatidylinositol 4-kinases (PI4KA and PI4KB), carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), and mevalonate (diphospho) decarboxylase. Knockdown of PI4KA could inhibit the replication and/or HCV RNA levels of the two subgenomic genotype 1b clones (SG-1b and Luc-1b), two subgenomic genotype 1a clones (SG-1a and Luc-1a), JFH-1 genotype 2a infectious virus (JFH1-2a), and the genomic genotype 1a (FL-1a) replicon. In contrast, PI4KB knockdown inhibited replication and/or HCV RNA levels of Luc-1b, SG-1b, and Luc-1a replicons. The small molecule inhibitor, PIK93, was found to block subgenomic genotype 1b (Luc-1b), subgenomic genotype 1a (Luc-1a), and genomic genotype 2a (JFH1-2a) infectious virus replication in the nanomolar range. PIK93 was characterized by using quantitative chemical proteomics and in vitro biochemical assays to demonstrate PIK93 is a bone fide PI4KA and PI4KB inhibitor. Our data demonstrate that genetic or pharmacological modulation of PI4KA and PI4KB inhibits multiple genotypes of HCV and represents a novel druggable class of therapeutic targets for HCV infection.Hepatitis C virus (HCV) causes liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (52). The HCV genome is a single-stranded RNA molecule where both the 5′ and the 3′ untranslated region (UTR) contain highly conserved RNA structures necessary for polyprotein translation and genome replication (43). The processed polyprotein yields at least three structural proteins and six nonstructural proteins. The structural proteins include the core, which forms the viral nucleocapsid, and the envelope glycoproteins E1 and E2. The viral proteins processed by signal peptidases form viral particles that assemble at the endoplasmic reticulum (ER) and/or Golgi bodies and are released from the host cell by viral budding. The structural protein coding regions are separated from nonstructural proteins by the short membrane peptide p7, thought to function as an ion channel (43, 53). The nonstructural proteins NS2, NS3/4A, NS5A, and NS5B are involved in coordinating the intracellular processes of the virus life cycle, including polyprotein processing and viral RNA replication (34).The Luc-1b cell is a human hepatoma cell line (Huh7) that contains a genotype 1b HCV subgenomic replicon, a luciferase reporter, and a neomycin selection marker, allowing HCV replication to be studied both in vitro and in vivo (8, 36). This subgenomic replicon lacks the coding regions for NS2 and the structural proteins but contains the nonstructural proteins in cis, which are required for replication of the viral RNA. Expression of the luciferase gene acts as a surrogate marker for levels of HCV RNA produced in the cell. The goal of the present study was to use this subgenomic HCV replicon to screen siRNA libraries and identify novel host proteins that are involved in HCV replication.A number of cellular pathways and proteins that play critical roles in HCV replication have recently been described (41, 42, 46). In particular, replication of HCV is tied closely to its localization and transport to various internal membranes and to lipid metabolism (2). Most of the HCV proteins appear to be targeted to the surface of the ER and replication complexes appear to be transported to lipid rafts, where RNA replication can occur (2). Infectious virus particle formation occurs in association with lipid droplets, and this process requires the core and NS5A proteins. In addition, cholesterol pathway production of geranylgeranyl-PP is important to geranylate the FBL2 protein, which serves as a membrane anchor for NS5A (62). The hVAP proteins involved in the localization and trafficking between internal membranous structures are known to be associated with the HCV proteins NS5A and NS5B (59). Thus, host factor lipid metabolism and intracellular protein transport are necessary for HCV replication in cells.Targeting host factors that are required for viral replication offers a strategy to overcome viral resistance and may allow treatment for more than one genotype of HCV and/or a related Flaviviridae virus such as Dengue, West Nile, or yellow fever virus. The current standard-of-care treatment for the genotype 1 strain of HCV infection is pegylated interferon alpha plus ribavirin over a 6-month time course with more than half of infected patients being refractory to this treatment (57). In addition to genotype 1, there are at least five naturally occurring genotype variants of HCV that can complicate a patient''s response to therapy when infected with more than one genotype. As well as the development of mutations, the presence of multiple variants coexisting in patients is thought to contribute to the rapid development of resistance (40). A variety of antiviral therapeutic strategies aim to inhibit viral proteins directly with small molecules or siRNAs (13, 31, 33). Although some small molecule approaches have been successful in preclinical studies, small-molecule strategies directed against the viral targets can still be rendered ineffective due to the development of mutant, treatment-resistant viral strains (13, 40). Thus, combination therapies are a necessary approach to treat the many variants of HCV that exist in the patient population.In the present study, a set of 779 SMARTpool small interfering RNAs (siRNAs) targeting the kinome and 4 siRNAs targeting 5,000 druggable genes (20,000 siRNAs) were tested for their ability to block replication of the Luc-1b HCV subgenomic replicon. siRNAs targeting CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase), a tripartite enzyme that catalyzes the first three steps of pyrimidine biosynthesis, inhibited both the Luc-1b replicon and JFH1-2a virus expression. This activity is consistent with the known inhibitor of this enzyme, leflunomide, which has been shown previously to inhibit both respiratory syncytial virus and HCV (12, 54). siRNAs targeting the mevalonate (diphospho) decarboxylase (MVD) enzyme, which catalyzes the formation of mevalonate, were found to inhibit Luc-1b replication (19). Inhibition of the cholesterol biosynthesis pathway and host cell geranylation has been previously reported to inhibit HCV subgenomic replication (3, 24, 51, 62, 67). siRNA-mediated knockdown of the class III phosphatidylinositol 4-kinases PI4KA and PI4KB inhibited luciferase expression not only for the genotype 1b subgenomic replicons (Luc-1a and Luc-1b) but also for the viral RNA levels of SG-1b, Luc-1b, and Luc-1a. PI4KA knockdown also inhibited Renilla expression in the JFH-1 genotype 2a infectious virus (JFH1-2a), genotype 2a subgenomic replicon (SG-1a), and a genomic and subgenomic genotype 1a replicon (FL-1a and SG-1a). Using the small-molecule inhibitor PIK93 in compound affinity competition experiments and in vitro biochemical assays, we demonstrated PIK93 could bind and inhibit both PI4KA and PI4KB enzymatic activity (58). PIK93 could inhibit luciferase expression in the Luc-1b, Luc-1a, and JFH1-2a infectious virus assays in the submicromolar range. Together, our data suggest that PI4KA and PI4KB regulate HCV replication and that pharmacological inhibition of these enzymes represents a new class of antiviral agents for multiple genotypes of HCV. Finally, since PI4KA and PI4KB are known to regulate protein and lipid transport to and from the ER and Golgi bodies, their function may hold clues as to how movement of HCV replication complexes throughout different organelles is regulated.     

Role of QuantiFERON-TB Gold,Interferon Gamma Inducible Protein-10 and Tuberculin Skin Test in Active Tuberculosis Diagnosis

Background

The measurement of Interferon gamma or Interferon gamma inducible protein (IP)-10 in antigen stimulated blood samples is suggested as an alternative method for latent tuberculosis (TB) diagnosis. Nonetheless, their role in active TB diagnosis, particularly in TB endemic settings is yet to be defined. In this study, the sensitivities and specificities of Interferon gamma release assay (IGRA), IP-10 assay and tuberculin skin test (TST) in detecting active TB cases were assessed in human immunodeficiency virus (HIV) sero-negative TB patients and healthy controls respectively.

Methods/Principal Findings

A total of 177 adult TB patients and 100 healthy controls were included for this study. QuantiFERON-TB Gold In-tube (QFT-IT) method was used to analyze the sensitivity and specificity of IGRA. QFT-IT, IP-10 and TST yielded the diagnostic sensitivities of 90.6% (95%CI: 86.3%–94.9%), 92.5% (95%CI: 88.6%–96.4%) and 68.9% (95%CI: 60.6%–77.2%) and specificities of 55% (95% CI: 35.2%–54.8%), 48% (95% CI: 38.2%–57.8%) and 75.5% (95% CI: 66.8%–84.2%), respectively. The extent of pulmonary involvement or presence of diabetes mellitus did not appear to influence the sensitivities of any of these tests. The combination of any of the two tests among QFT-IT, IP-10 and TST showed >98% sensitivity among smear negative cases and particularly the combination of IP-10, TST and smear microscopy showed 100% sensitivity, however, the specificity was decreased to 44.8%.

Conclusions/Significance

QFT-IT and IP-10 were highly sensitive in detecting active TB cases. The combination with TST improved the sensitivity of QFT-IT and IP-10 significantly. Although the higher sensitivity of combination of QFT-IT/IP-10 and TST may be useful in active TB diagnosis, they are limited by their poor specificity due to the high prevalence of latent TB in our settings.     

TNF受体（P55）胞外区基因的克隆及其在大肠杆菌中的表达

TNF与多种疾病密切相关。为了获得大量具有生物学活性的可溶性TNF受体用以拮抗TNF的毒性作用,在原核表达系统中表达了TNFR（P55）的胞外区与TrxA的融合蛋白。将TNFR（P55）胞外区去信号肽的前三个结构域基因克隆入融合蛋白表达载体pET-32a,在大肠杆菌BL21（DE3）中高效表达了TrxA-TNFR融合蛋白。表达产物以包涵体形式存在,经过变性和复性,并经镍金属鳌和柱亲和层析纯化,得到了纯度较高的可溶性受体蛋白的初纯品。免疫学实验及L929细胞体外实验均表明：该蛋白具有TNFR（P55）特异的抗原性、与TNF结合的活性以及良好的抑制TNF的TNF生物学活性。     

Effects of Interferon and PKC Modulators on Human Glioma Protein Kinase C,Cell Proliferation,and Cell Cycle

The in-vitro effects of human interferon -2b (HuIFN -2b), protein kinase C (PKC) agonist [TPA (12-0-tetra-decanoyl-phorbol-13-acetate)] and PKC inhibitor (calphostin C) on human glioma (U-373MG) PKC activity, cell proliferation and cell cycle were compared. HuIFN -2b and TPA increased PKC activity, elevated the number of cells in DNA synthesis (S) phase and decreased cell proliferation by similar magnitudes. Calphostin C inhibited PKC activity, increased the number of cells in S phase and produced strong cytotoxic effects (IC₅₀ 150 nM). Higher concentrations of calphostin C with or without serum induced an additional block in gap2 and mitosis. We conclude that HuIFN -2b's mode of action may be directly or indirectly affecting PKC. The response produced by HuIFN -2b is similar to TPA (potent PKC activation and S phase arrest).     

Antioxidants Inhibit the Human Cortical Neuron Apoptosis Induced by Hydrogen Peroxide,Tumor Necrosis Factor Alpha,Dopamine and Beta-amyloid Peptide 1-42

Several substances related to the neurodegenerative diseases of Alzheimer and Parkinson, such as hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, have been shown to induce apoptosis in tumoral cell lines and rat neurons but not in human neurons. Moreover, the role of mitochondria (membrane potential) during neuronal apoptosis is still a matter of debate. We present here, for the first time, in cultured human cortical neurons that the DNA fragmentation induced by these substances was preceded by a decrease of the mitochondrial membrane potential. We have also examined the antiapoptotic effect of the antioxidants glutathione, N -acetyl-cysteine and ascorbic acid. All these antioxidants inhibited the apoptosis induced by hydrogen peroxide, tumor necrosis factor alpha, dopamine and beta-amyloid peptide 1-42, since they were able to inhibit completely the mitochondrial membrane potential depolarization and the DNA fragmentation.     

Concomitant Interferon Alpha Stimulation and TLR3 Activation Induces Neuronal Expression of Depression-Related Genes That Are Elevated in the Brain of Suicidal Persons

We have previously identified 15 genes that are associated with the development of severe depressive side effects during the standard therapy with interferon alpha and ribavirin in the peripheral blood of hepatitis C virus infected patients. An enhanced expression of these genes was also found in the blood of psychiatric patients suffering severe depressive episode. Herein, we demonstrate that the same depression-related interferon-inducible genes (DRIIs) are also upregulated in post-mortem brains of suicidal individuals. Using cultured mouse hippocampal and prefrontal neurons we show that costimulation with murine IFN (mIFN) and the TLR3 agonist poly(I:C) promotes the expression of the described DRIIs, at the same time inducing pro-inflammatory cytokine expression through Stat1 and Stat3 activation, promoting neuronal apoptosis. Consequently, the upregulation of selective DRIIs, production of inflammatory cytokines and inhibition of neuronal plasticity may be involved in the pathogenesis of IFN-associated depression.