Potentiation of the antiviral activity of human interferons by virazole in in vitro experiments

Combination of interferon and virazole results in potentiation of their antiviral activity. This was demonstrated with all three types of natural interferons and the recombinant RL-2-interferon. This effect is retained at a temperature of 37 degrees C and increases when the chemical drug is combined with a mixture of alpha- and gamma-interferon.     

Type I and type II interferons: differential antiviral actions in transformed cells

In transformed mouse embryo cells, type II interferon had much less antiviral activity than type I interferon. In non-transformed cells, the two interferons had similar high activity, Reversal of the phenotype of Moloney sarcoma virus (MSV) transformed cells by sodium butyrate restored their sensitivity to the antiviral action of type II interferon. Additional evidence for a role of the cytoskeletal network in the action of type II interferon is that its antiviral effect is reduced by cytochalasin B, colchicine or vinblastine. MSV-transformed cells, selected for their resistance to the antiviral action of type I interferon, were sensitive to type II interferon. These differences in the effects of type I and II interferon on transformed cells are at present unexplained, but suggest that they have at least partially separate mechanisms of action.     

Interferons induce an antiviral state in human pancreatic islet cells

Enterovirus infections, in particular those with Coxsackieviruses, have been linked to the development of type 1 diabetes (T1D). Although animal models have demonstrated that interferons (IFNs) regulate virus-induced T1D by acting directly on the beta cell, little is known on the human pancreatic islet response to IFNs. Here we show that human islet cells respond to IFNs by expressing signature genes of antiviral defense. We also demonstrate that they express three intracellular sensors for viral RNA, the toll like receptor 3 (TLR3) gene, the retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene-5 (MDA-5), which induce type I IFN production in infected cells. Finally, we show for the first time that the IFN-induced antiviral state provides human islets with a powerful protection from the replication of Coxsackievirus. This may be critical for beta cell survival and protection from virus-induced T1D in humans.     

二氧化锆梯度复合羟基磷灰石体外激活末梢血单个核细胞的实验研究

目的：体外检测二氧化锆（ZrO2）梯度复合羟基磷灰石(HAP)材料浸提液对健康青年人外周血单个核细胞（PBMC）的激活，来评价其免疫原性。 方法： 以二氧化锆（ZrO2）单纯复合羟基磷灰石(HAP)为对照材料，制备两种材料浸提液并分离健康年轻人PBMC。MTT法检测两种材料浸提液刺激PBMC转化的作用；ELISA法检测两种材料浸提液刺激PBMC分泌TNFα和IL-6的作用；流式细胞术检测两种材料浸提液刺激PBMC CD69、CD25的作用。 结果： 培养5 d，单纯复合组对PBMC的增殖作用弱于对照组（P<0.05），而梯度复合组与阴性对照组比较无差异；LPS共培养24 h，两材料组TNFα和IL-6水平差异显著（P<0.05）；PHA作用24 h，两材料组PBMC中CD69和CD25阳性细胞率差异显著（P<0.01）。 结论： ZrO2梯度复合HAP生物陶瓷材料免疫原性弱，对PBMC的激活程度较轻。     

人树突状细胞体外经HBsAg刺激后的抗病毒作用

目的观察人树突状细胞(DCs)经表面抗原(HBsAg)刺激、体外诱导自身特异性T淋巴细胞增殖后,对2.2.15细胞中HBeAg和HBsAg的特异性免疫抑制作用。方法 用粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白介素-4(IL-4)和肿瘤坏死因子(TNF-a)分化、诱导人外周血PBMC中的DCs,在DCs成熟前加入纯的HBsAg刺激,将成熟后的DCs体外与自身T淋巴细胞共培养,同时不加HBsAg刺激的DCs与T细胞共培养、T细胞加纯的HBsAg共培养以及单纯T细胞作对照,5 d后收集T细胞,分组加入2.2.15细胞培养液中,分别收集第1天、3天、5天和7天的培养上清液,检测其HBeAg和HBsAg的分泌情况。结果经抗原刺激后的DCs可以有效提呈病毒抗原,正常人与慢性乙肝患者负载抗原后的DCs刺激T淋巴细胞增殖的能力[cpm分别为(46 700±7 850)和(38 628±5 427)]明显高于未负载抗原的DCs[cpm分别为(40 450±4 645和33 924±4 498)]及对照组PBMC[cpm分别为(5 947±476)和(5 089±233)],P<0.01。负载抗原的DCs有强烈的免疫应答活性,并且其免疫刺激能力似乎与负载的抗原量成正比;经抗原刺激激活的T细胞可以有效地抑制HBeAg的表达,但对HBsAg未发现有明显的抑制作用。结论体外经HBsAg刺激后的DCs可有效地提呈病毒抗原,并可进一步激活T细胞产生,同时能显著地抑制2.2.15细胞上清     

An immunoenzyme quantitative assay for the antiviral effect of interferons

A technique is described for measurement of the antiviral activity of interferon by an immunoenzymatic assay for viral proteins. Cells treated by tested samples of interferon (IFN) are infected with vesicular stomatitis virus (VSV) and following the development of viral cytopathy are lysed by the addition of deoxycholate and then transferred into ELISA microplates. The viral proteins bind effectively to the microplates proportionally to their level in the culture and may be measured by incubating the plates sequentially with (1) rabbit antiserum against VSV, (2) a conjugate of alkaline phosphatase either to protein A or to an antibody against rabbit IgG and (3) p-nitrophenylphosphate. This procedure may be further simplified by using antibodies against VSV to which alkaline phosphatase has been directly conjugated. We found this immunoenzyme assay to be superior to the 'cytopathic effect inhibition' assay in precision and sensitivity and in being independent of the effectiveness of viral cytopathy.     

Myosin Mg-ATPase of molluscan muscles is slightly activated by F-actin under catch state in vitro

Molluscan muscle twitchin, a titin/connectin-related giant protein, regulates interactions between actin and myosin filaments at low Ca²⁺ concentrations. When it is dephosphorylated, actin filaments tightly bind to myosin filaments, resulting in the catch state known as the state of high passive tension with very low energy consumption. Yet when twitchin is phosphorylated actin filaments detach from the myosin filaments, resulting in relaxation of the catch. Here, steady-state Mg-ATPase activities of purified myosin were measured under various conditions: without twitchin, with dephosphorylated twitchin, or with phosphorylated twitchin; with or without phalloidin-stabilized F-actin; and at various Ca²⁺ concentrations. At low Ca²⁺ concentration, Mg-ATPase was activated by F-actin only in the presence of dephosphorylated twitchin (catch state). The activation was about two orders lower than that fully activated by Ca²⁺ and F-actin. In the absence of F-actin, twitchin and its phosphorylation state did not affect Mg-ATPase activities in any of the conditions we tested. Based on these results, we propose a molecular mechanism for the catch, where twitchin alone does not interact with the myosin catalytic motor domain but its complex with F-actin does, forming the bridge between actin and myosin filaments and the myosin slowly hydrolyzes Mg-ATP in the catch state.     

In vitro antiviral properties of endotoxin-activated rat Kupffer cells

Kupffer cells isolated from the liver of normal rats were checked for their antiviral activities. The intrinsic antiviral effect against vaccinia virus was high whether the cells were activated in vitro with endotoxin or not. The expression of the extrinsic antiviral activity measured by mixing isolated Kupffer cells with vaccinia-virus-infected target cells was remarkably enhanced by prior treatment of the Kupffer cells with LPS. Interferon was shown to be not responsible for that inhibitory activity. Different experimental data suggest that the virus replication in the target cells is blocked at a late stage in the replication cycle.     

Stimulation of activated rat T cells in vitro by Listeria monocytogenes antigens.

A soluble extract of Listeria monocytogenes bound firmly and in similar amounts to a variety of rat cells. Cells that bound this material differed in their capacity to stimulate the in vitro proliferation of lymphocytes obtained from the thoracic duct of Listeria-immune donors. The capacity of cells to serve as antigen-presenting cells in this system coincided or closely overlapped the expression on these cells of an Ia antigen-like structure. Three lines of evidence indicate that T cells respond to L. monocytogenes antigen: the responder cells are members of a nylon-wool nonadherent population that lacks readily detectable surface immunoglobulin; they express determinants recognized by the W3/25 monoclonal antibody (a surface marker of rat peripheral T cells); and they are stimulated optimally by L. monocytogenes antigen when the latter is displayed on cells that share a haplotype with the responder lymphocytes.     

Reversible inhibition of interferon-induced antiviral state by deoxyadenosine.

Deoxyadenosine reversibly inhibited the development of antiviral state (AVS) in chick embryo fibroblasts stimulated by interferon. Studies on cellular macromolecular syntheses in deoxyadenosine-treated cells suggested that the inhibition was related to nucleolar RNA synthesis. Reference experiments with actinomycin D and cordycepin (3'deoxyadenosine) also implied causal- or interrelation between AVS development and nucleolar RNA synthesis.     

Characterization of antiviral activity of spotted souslik (Spermophilus suslicus) interferons

The antiviral activity of three types of spotted souslik interferons So IFN alpha, So IFN beta and So IFN gamma was studied. Fibroblasts of lungs (SL), kidneys (SK) of adult sousliks and skin fibroblasts of foetuses (SE) were equally sensitive to three types of souslik interferons. In contrast to So IFN beta and So IFN gamma, So IFN alpha, exhibited a high (100% of activity in homologous cells) cross species antiviral activity in mouse embryo fibroblasts (MEF) and a low (3% to 10% of the activity) in bovine embryo fibroblasts (BEF) and human embryo fibroblasts (HEF). The development kinetics of antiviral activity not only depended on the interferon type but also on the temperature of incubation. In comparison with So IFN gamma, So IFN alpha and So IFN beta activated earlier the maximal antiviral state. Low incubation temperature (26 degrees C) did not decrease but only delayed the antiviral activity of spotted souslik interferons. Mixed preparations of So IFN gamma with So IFN alpha or So IFN beta exhibited synergistic antiviral activity at physiological (37 degrees C) and low (26 degrees C) temperatures. The development of antiviral activity of So IFN beta as well that of Mu IFN alpha/beta was inhibited by plant lectins which reacted with the cell membrane compounds. All three types of souslik interferons were completely destroyed by trypsin and boiling at 100 degrees C for 1 min. and partially by SDS. Their sensitivity to shaking, beta-mercaptoethanol and mouse antisera against So IFN beta was different in relation to the interferon type.     

Tumor-cell co-culture induced alternative activation of macrophages is modulated by interferons in vitro

Tumor-associated macrophages infiltrate tumors and facilitate tumor growth. Here, we analyzed M1 and M2 marker expression in the course of co-culture-driven macrophage differentiation and investigated the influence of interferons (IFNs) on this differentiation. To generate monocyte-derived macrophages (MDMs) 1×10? monocytes of healthy volunteers were cultivated either with 25×103 adherent A549/mL or in medium containing 50% A549 conditioned medium (CM) for 72?h in the presence or absence of IFN-α, β or γ, respectively. Supernatants were tested for CCL18 (M2 marker) and CXCL10 (M1 marker) by enzyme-linked immunosorbent assay. CCL18 and CXCL10 release by MDM is increased by the presence of A549 cells, but also when cultured in A549 CM. On stimulation with IFN-γ, we observe an increased release of the M1 marker CXCL10 and a decreased release of CCL18. Type I IFNs also increases CXCL10 release. Thus, A549 releases a soluble factor which enhances CCL18 production and M2 polarization, indicating that a localized specific cytokine milieu, as found in the environment of a tumor or in fibrotic lung tissue, favors alternative activation of macrophages. In the presence of IFN-γ, M2 differentiation is attenuated as shown by the decrease of the M2 chemokine CCL18 and by the increase of the M1 chemokine CXCL10. However, CXCL10 levels were also increased by the co-culture, which indicates a simultaneous classical activation (M1) or the formation of a M1/M2 hybrid.     

Production of virus-induced and mitogen-induced interferons by canine cells]

Cells of monocytic-phagocytic series and immunocompetent cells derived from dogs: bone marrow, leukocytes, spleen, thymus, lymph nodes (with the exception of the liver) were found to be capable of producing interferon (IFn) in vitro in response to inoculation of viral and non-viral inducers. This capacity is manifested in different ways: thymus and lymph node cells were less active interferon producers. Studies of physicochemical properties of the resulting interferons confirmed the belonging of the virus-induced IFn to alpha-interferons, PHA-induced IFn to immune alpha-IFn. The above-mentioned refers both to bone marrow and leukocyte interferons.     

Transforming growth factor-beta regulates in vitro heart valve repair by activated valve interstitial cells

The regulation of valve interstitial cell (VIC) function in response to tissue injury and valve disease is not well understood. Because transforming growth factor-β (TGF-β) has been implicated in tissue repair, we tested the hypothesis that TGF-β is a regulator of VIC activation and associated cell responses that occur during early repair processes. We used a well-characterized wound model that was created by mechanical denudation of a confluent VIC monolayer to study activation and repair 24 hours after wounding. VIC activation was demonstrated by immunofluorescent localization of α-smooth muscle actin (α-SMA), and α-SMA mRNA levels were quantified by real-time polymerase chain reaction. Proliferation and apoptosis were quantified by bromodeoxyuridine staining and terminal deoxynucleotidyl transferase dUTP nick end labeling, respectively. Repair was quantified by measuring VIC extension into the wound, and TGF-β expression was shown by immunofluorescent localization of intracellular TGF-β. Compared with nonwounded monolayers, VICs at the wound edge showed α-SMA staining, increased α-SMA mRNA content, elongation into the wound with stress fibers, proliferation, and apoptosis. VICs at the wound edge also showed increased TGF-β and pSmad2/3 staining with co-expression of α-SMA. Addition of TGF-β neutralizing antibody to the wound decreased VIC activation, α-SMA mRNA content, proliferation, apoptosis, wound closure rate, and stress fibers. Conversely, exogenous addition of TGF-β to the wound increased VIC activation, proliferation, wound closure rate, and stress fibers. Thus, wounding activates VICs, and TGF-β signaling modulates VIC response to injury.     

Effect of ammonium salts on the interferon-induced antiviral state in mouse L cells.

The addition of ammonium salts to cells treated with interferon prevents the development of the antiviral state and destroys it when already established. This treatment does not seem to act on the binding of interferon to the cells but blocks a further step of activation on the cell membrane. The anti-interferon effect of ammonium salts is reversible with a complete recovery of the antiviral state. It is postulated that these salts may stabilize the interferon-receptor complex and thus prevent the changes in configuration necessary for the establishment and maintenance of its biological functions.     

Inhibition of Epstein-Barr virus infection in vitro by recombinant human interferons alpha and gamma

The inhibitory effects of pure recombinant human interferons alpha A and gamma (reIFN-alpha A and -gamma) on Epstein-Barr virus (EBV) infection of a human EBV-negative B cell line, BJAB, and of normal adult B lymphocytes were studied. With pretreatment for 24 h, both types of reIFNs were effective in suppressing the production of EBV specific nuclear antigen (EBNA-1) in BJAB cells 24 h after EBV-infection, as determined by the immunoblotting technique. ReIFN-alpha A was, however, a much more potent inhibitor than reIFN-gamma. With treatment starting 1 h after EBV infection, both types of reIFNs were less effective in the suppression of EBNA production. Neither of the reIFNs showed any inhibitory effect on EBNA production in the latently EBV-infected cell lines, Raji and Daudi. These results suggest that reIFNs act in the early phase of EBV infection. Both types of reIFNs were also effective in inhibiting EBV infection of normal adult B lymphocytes as demonstrated by a reduction both in [3H]thymidine incorporation 6 days after EBV infection and in the total number of proliferating cells 21 days after EBV infection. Again, reIFN-alpha A showed a greater inhibitory effect than reIFN-gamma. We also showed that in BJAB cells, reIFN-alpha A strongly induced (2'-5')oligoadenylate synthetase activity, whereas reIFN-gamma increased the surface expression of HLA class I antigens.     

Non-cytotoxic antiviral activities of granzymes in the context of the immune antiviral state

Summary: Viruses are obligatory intracellular parasites, whose replication depends on components encoded by the virus and pathways and functions of the host cell. In addition to the pathways required for viral synthesis, viruses activate multiple mechanisms to evade immune attack, promoting viral propagation while avoiding or slowing the host immune response. To achieve efficient control of viral infections, the immune system in vertebrates relies on diverse and synergistic antiviral pathways (both at the innate and adaptive immune response), which target and inactivate viral and host components involved both in viral replication and in viral defenses that block host antiviral activities. During this process, the immune system uses mechanisms to slow down viral propagation, while apoptotic pathways are triggered to kill (when possible) the infected cell. Granzymes (granule enzymes) are key components of the immune response that play important roles in eliminating host cells infected by intracellular pathogens. Although the induction of target cell death has been considered the central function for these proteases, recent evidence supports that granzymes can achieve direct antiviral activities through the cleavage of viral and host factors required for viral replication and viral defense. In addition, granzyme A can stimulate the production of pro-inflammatory cytokines. The focus of this review is to discuss recent views on antiviral mechanisms involved in controlling viral infections, with special interest in novel and potential non-death-related antiviral functions of the granzymes, and how these unique functions complement and synergize with the ‘antiviral state’ created by interferons and cytotoxic lymphocytes in response to virus.     

Aleutian disease of mink: production of 14C-labeled antiviral antibodies by mink lymphoid cells in vitro.

Lymphoid cells from mink infected with Aleutian disease virus produced labeled, specific antiviral antibody when incubated in medium containing [14C]tyrosine.