Comparison of anticancer activity between lactoferrin nanoliposome and lactoferrin in Caco-2 cells in vitro

The anticancer activities of Lactoferrin (Lf) and Lf nanoliposomes in Caco-2 cells were observed in this study, and mitochondrial function (MTT assay), count kit-8(CCK-8), detection of intracellular reactive oxygen species (ROS) and apoptosis induction (AO/EB staining) assays were used to evaluate the anticancer activity. MTT results demonstrated that Lf nanoliposomes and Lf reduced the mitochondrial activity of cells in a manner of dose and time effect, and the viabilities of Caco-2 cell were significantly decreased in vitro following exposure to Lf nanoliposomes at the concentrations of 5 and 10 mg/mL. LDH leakage and ROS significantly increased in cells exposed to Lf nanoliposomes (⩾5 mg/mL), while Lf induced ROS only at higher doses (10 mg/mL). CCK-8 evaluation of cell proliferation and AO/EB double staining supported the anti-proliferative effects of Lf liposomes. Our findings demonstrated that the presence of Lf nanoliposome is more significant than Lf in inhibiting human tumor cells proliferation. Therefore, it can be concluded that Lf nanoliposomes are a potential therapeutic modality in the management of tumors.     

Design and fungicidal activity of mucoadhesive lactoferrin tablets for the treatment of oropharyngeal candidosis

Lactoferrin (Lf) is a potential drug candidate for the treatment of oropharyngeal Candida infections. However, for an effective therapeutic treatment an appropriate dosage form is required. Therefore a mucoadhesive tablet for buccal application was developed. Tablets of sufficient strength could be produced on high speed tabletting machines, but they could only be obtained when the protein contained at least 7% moisture. The tablet contained sodium alginate both for its release-controlling properties as well as for its mucoadhesive properties. Furthermore, phosphate buffer was added to keep the pH of the saliva in the mouth within the range of 6.5 to 7.5. In this pH range, Lf has shown to have its highest activity against Candida growth inhibition. The tablet formulation containing Lf had the same antifungal properties as compared with Lf alone, because in most cases identical inhibitory concentrations were observed against several clinical isolates of Candida albicans and Candida glabrata . In human volunteers the tablets, containing 250 mg Lf and placed in each pouch, were able to keep the Lf concentration in the saliva at effective levels for at least 2 hr, while the pH of the saliva remained within the desired range. We concluded that the developed mucoadhesive tablet can improve the therapeutic efficacy of Lf and that it is suitable for further clinical research.     

Evaluation of the antiviral activity of (1'S,2'R)-9-[[1',2'-bis(hydroxymethyl)cycloprop-1'-yl]methyl]guanine (A-5021) against equine herpesvirus type 1 in cell monolayers and equine nasal mucosal explants

Equine herpesvirus 1 (EHV1) is a ubiquitous equine alphaherpesvirus that causes respiratory disease, neurological symptoms and abortions. Current vaccines are not fully protective and effective therapeutics are lacking. A-5021 [(1′S,2′R)-9-[[1′,2′-bis(hydroxymethyl)cycloprop-1′-yl]methyl]guanine], previously shown to possess potent anti-herpetic activity against most human herpesviruses, was evaluated for its potential to inhibit EHV1 replication. In equine embryonic lung (EEL) cells, infected with either a non-neurovirulent (97P70) or a neurovirulent (03P37) EHV1 isolate, A-5021 proved to be about 15-fold more potent than acyclovir in inhibiting viral replication. Moreover, in equine nasal mucosal explants, A-5021 (at 8 and 32 μM) was able to completely inhibit viral plaque formation whereas acyclovir did not exert an antiviral effect at these concentrations. Our data demonstrate that A-5021 is a potent inhibitor of EHV1 replication and may have potential for the treatment and/or prophylaxis of infections with this virus.     

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats

BackgroundAbnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells.MethodsThis study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA?HCC cell culture).ResultsUp-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA?HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation.ConclusionThese findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.     

人类疱疹病毒7型的实验室检查初探

目的 :建立一种敏感、特异、快速诊断人类疱疹病毒 7型 (HHV- 7)的实验室检查方法。方法 :应用聚合酶链反应(PCR)和巢式聚合酶链反应 (n- PCR)对年龄 0～ 2岁正常儿童和患病儿童的吐出唾液和用无菌棉签放进口腔湿润唾液进行检测。结果 :用无菌棉签采集的口腔湿润唾液分别用 PCR和 n- PCR方法测得 HHV- 7总阳性率分别为 8.8%、6 1.6 % ,两者比较有非常显著性差异 (P <0 .0 1)。用 n- PCR方法测得吐出的唾液的总阳性率为 73% ,与同方法测得用无菌棉签采集的口腔湿润唾液标本比较 ,两者无显著性差异 (P >0 .0 5 )。结论 :用 n- PCR方法是一种临床检测 HHV- 7的快速有效手段 ,标本用无菌棉签采集的口腔湿润唾液和吐出唾液均可     

Antiviral activity of enterocin CRL35 against herpesviruses

Enterocin CRL35 is an antibacterial polypeptide of 3.5×10³ Da produced by Enterococcus faecium CRL35. A series of experiments are described that show the enterocin also had antiviral activity against thymidine-kinase positive (tk⁺) and deficient (tk⁻) strains of herpes simplex (HSV) type 1 and 2 in Vero and BHK-21 cells. This activity was observed at 100 μg/ml, 15-fold lower than the cytotoxic concentration. In both cell lines there was a 2 log inhibition of infectivity. The compound inhibited viral multiplication in a dose-dependent manner and had no virucidal effect. Enterocin CRL35 also inhibited the virion-associated host shutoff in infected Vero cells showing that intracellular viral multiplication was affected.     

Antiviral Activity of Ribosome-Inactivating Proteins

Ribosome-inactivating proteins (RIPs) are rRNA N-glycosylases from plants (EC 3.2.2.22) that inactivate ribosomes thus inhibiting protein synthesis. The antiviral properties of RIPs have been investigated for more than four decades. However, interest in these proteins is rising due to the emergence of infectious diseases caused by new viruses and the difficulty in treating viral infections. On the other hand, there is a growing need to control crop diseases without resorting to the use of phytosanitary products which are very harmful to the environment and in this respect, RIPs have been shown as a promising tool that can be used to obtain transgenic plants resistant to viruses. The way in which RIPs exert their antiviral effect continues to be the subject of intense research and several mechanisms of action have been proposed. The purpose of this review is to examine the research studies that deal with this matter, placing special emphasis on the most recent findings.     

Inhibition of HSV cell-to-cell spread by lactoferrin and lactoferricin

The milk protein lactoferrin (Lf) has multiple functions, including immune stimulation and antiviral activity towards herpes simplex virus 1 and 2 (HSV-1 and HSV-2); antiviral activity has also been reported for the N-terminal pepsin-derived fragment lactoferricin (Lfcin). The anti-HSV mode of action of Lf and Lfcin is assumed to involve, in part, their interaction with the cell surface glycosaminoglycan heparan sulfate, thereby blocking of viral entry. In this study we investigated the ability of human and bovine Lf and Lfcin to inhibit viral cell-to-cell spread as well as the involvement of cell surface glycosaminoglycans during viral cell-to-cell spread. Lf and Lfcin from both human and bovine origin, inhibited cell-to-cell spread of both HSV-1 and HSV-2. Inhibition of cell-to-cell spread by bovine Lfcin involved cell surface chondroitin sulfate. Based on transmission electron microscopy studies, human Lfcin, like bovine Lfcin, was randomly distributed intracellularly, thus differences in their antiviral activity could not be explained by differences in their distribution. In contrast, the cellular localization of iron-saturated (holo)-Lf appeared to differ from that of apo-Lf, indicating that holo- and apo-Lf may exhibit different antiviral mechanisms.     

乳铁蛋白多肽嵌合体对泛耐药铜绿假单胞菌生物被膜形成的作用

目的研究乳铁蛋白多肽嵌合体对泛耐药铜绿假单胞菌生物被膜形成的作用。方法在泛耐药铜绿假单胞菌培养基（OD600=0.05）中分别加入乳铁蛋白多肽（LFcin、LFampin）以及乳铁蛋白多肽嵌合体（LFchimera）溶液干预;结晶紫法定量检测生物被膜;以结晶紫染色法,在光镜下观察生物被膜形态。结果乳铁蛋白多肽以及乳铁蛋白多肽嵌合体均能抑制泛耐药铜绿假单胞菌生物被膜形成（P〈0.05）,生物被膜量与多肽浓度呈负相关（P〈0.05）;乳铁蛋白多肽嵌合体的作用强于乳铁蛋白多肽及其混合物（P〈0.05）。结论乳铁蛋白多肽和乳铁蛋白多肽嵌合体,尤其是LFchimera对泛耐药铜绿假单胞菌生物被膜形成具有显著的抑制作用,有望用于泛耐药铜绿假单胞菌感染的治疗。     

盐酸阿比朵尔抗呼吸道合胞病毒作用的实验研究

目的　研究盐酸阿比朵尔、盐酸金刚烷胺及利巴韦林在细胞Hep - 2培养中抑制呼吸道合胞病毒的作用。方法　采用细胞病变抑制法 ,测定 3种药物对呼吸道合胞病毒所致Hep - 2细胞病变效应的抑制作用。结果　 3种药物抗呼吸道合胞病毒的IC50 依次为盐酸阿比朵尔 (38 2 μg·ml-1)、盐酸金刚烷胺 (6 8 0 μg·ml-1)和利巴韦林 (80 3μg·ml-1) ,TI分别为 5 2、2 7、4 7,其中盐酸阿比朵尔表现出较强的抑制呼吸道合胞病毒的效果。结论　在体外抗呼吸道合胞病毒的 3种实验药物中 ,盐酸阿比朵尔应为首选。     

乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶表达的影响

目的通过观察鞘内注射乳铁蛋白对福尔马林致痛大鼠行为学及一氧化氮合酶（NOS）表达的影响，探讨乳铁蛋白可能的抗伤害机制。方法雄性SD大鼠80只，随机分为生理盐水组（NS组）和福尔马林组（F组）；实验组分为乳铁蛋白1ug-福尔马林组（F-R1组），乳铁蛋白10ug-福尔马林组（F—R10组）和乳铁蛋白100ug-福尔马林组（F—R100组）。五组鞘内分别给予NS 20ul，NS 20ul，乳铁蛋白1ug，乳铁蛋白10ug，乳铁蛋白100ug；10min后，除NS组大鼠组底注射NS 100ul外，其余各组均给予5％福尔马林100ul。经以上处理8只动物在福尔马林处理后30min取脊髓观察NOS的表达，另8只动物在福尔马林处理后0～1h观察行为学。结果F组的缩腿舔爪的时间和脊髓NOS表达显著长于，强于NS组；预先给予乳铁蛋白能抑制以上的作用，且呈剂量依赖性。结论乳铁蛋白明显抑制福尔马林致痛大鼠痛行为及NOS表达，可能是其产生抗伤害作用机制之一。     

Antiviral activity of glycyrrhizin against varicella-zoster virus in vitro

One of the plant extracts, glycyrrhizin (GL) was investigated for its antiviral action on varicella-zoster virus (VZV) in vitro. When human embryonic fibroblast (HEF) cells were treated with GL after inoculation of virus (post-treatment), the average 50%-inhibitory dose (ID50) for five VZV strains was 0.71 mM, and the selectivity index (ratio of ID50 for host-cell DNA synthesis to ID50 for VZV replication) was 30. GL was also effective against VZV replication when HEF cells were treated 24 h before the inoculation (pretreatment). Furthermore, at a concentration of 2.4 mM GL inactivated more than 99% of virus particles within 30 min at 37 degrees C. In combination with other anti-herpes drugs (acyclovir, adenine arabinoside, bromovinyldeoxyuridine, and phosphonoformate) or human native beta-interferon, GL had an additive or slightly synergistic effect on VZV replication. The mechanism of anti-VZV action is still unclear. We postulate that GL inhibits the penetration, uncoating or release of virus particles.     

Antiviral activity of ganciclovir elaidic acid ester against herpesviruses

A fatty acid derivative of ganciclovir (GCV), elaidic acid ganciclovir (E-GCV), has been evaluated for its inhibitory activity against laboratory and clinical strains of herpes simplex type 1 (HSV-1) and type 2 (HSV-2), varicella-zoster virus (VZV) and human cytomegalovirus (HCMV) in human embryonic lung fibroblasts. GCV, cidofovir, acyclovir (ACV), brivudin (BVDU) and foscarnet (PFA) were included as reference compounds. The viruses studied were wild-type, thymidine kinase-deficient (TK(-)) and PFA-resistant (PFA(r)) HSV strains. The IC(50) values obtained for E-GCV were 5- to 30-fold lower than those observed for GCV, the IC(50) value of E-GCV for HSV-1 strain KOS being 0.07 nM. A similarly increased activity of E-GCV (as compared to GCV) was noted for TK(-) and PFA(r) HSV-1 or HSV-2 strains. However, E-GCV did not exhibit superior activity over GCV to VZV or HCMV in vitro. The antiviral efficacy of E-GCV was also evaluated in vivo against intracerebral HSV-2 infection in NMRI mice. Animals were treated intraperitoneally or perorally with E-GCV, GCV or placebo once daily for 10 days, starting the day of infection. E-GCV compared to GCV at equimolar doses, proved markedly more efficacious than GCV in terms of reduction of mortality rate and delay of mean time of death. The elaidic acid ester of GCV should therefore be considered as a novel approach towards the treatment of HSV infections.     

展枝马尾藻硫酸多糖对呼吸道合胞病毒的抗病毒活性及其机理研究

目的研究从褐藻展枝马尾藻Sargassumpatens中分离到的一种硫酸多糖SP2的抗病毒活性及抗病毒作用机理。方法通过细胞致病效应实验、MTT测定和空斑形成抑制实验对该多糖的抗病毒活性、细胞毒性和可能的抗病毒机理进行测定。结果通过空斑形成抑制实验测定结果显示,该多糖对呼吸道合胞病毒有好的抗病毒活性,其EC50值为1.1但没有抗流感病毒A和B、副流感病毒的活性。多糖的CC50高达3000μg.mL-1。其对呼吸道合胞病毒的抗病毒选择性指数被估计大于2727。用SP2预处理宿主细胞,不显示任何对病毒感染的保护作用。SP2对呼吸道合胞病毒有一定的杀病毒活性,其EC50值超过100μg.mL-1。时间加入实验证明,只有在37℃呼吸道合胞病毒吸附时期加入SP2才能完全抑制病毒的复制。结论SP2可能是一种有潜力的抗呼吸道合胞病毒感染的多糖。其对呼吸道合胞病毒的抗病毒作用机制与抑制病毒的吸附有关。     

Antiviral activity of type I and type III interferons against porcine reproductive and respiratory syndrome virus (PRRSV)

The newly identified type III interferons (IFNs), also known as IFN-λ1/IL-29, IFN-λ2/IL-28A and IFN-λ3/IL-28B, like type I IFNs, have antiviral activity against a broad spectrum of viruses. We therefore examined whether type III IFNs, as well as type I IFNs, has the ability to inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in MARC-145 cells. We found that replication of PRRSV in MARC-145 cells was significantly reduced following treatment with IFN-λ1, IFN-λ2 and IFN-λ3, respectively, and such inhibition was dose-dependent. However, type III IFNs (IFN-λ1, IFN-λ2 and IFN-λ3) was less effective than type I IFNs (IFN-α and IFN-β) in antiviral activity against PRRSV. Mixture of two types of IFNs could not improve the antiviral activity of each type alone. In addition, all types of IFNs in our study were able to induce the expression of ISG56, 2′,5′-OAS and MxA in MARC-145 cells. These data demonstrate that type III IFNs had antiviral activity against PRRSV and may serve as useful antiviral agents against infectious swine diseases.     

Antiviral activity of tenofovir against Cauliflower mosaic virus and its metabolism in Brassica pekinensis plants

The antiviral effect of the acyclic nucleoside phosphonate tenofovir (R)-PMPA on double-stranded DNA Cauliflower mosaic virus (CaMV) in Brassica pekinensis plants grown in vitro on liquid medium was evaluated. Double antibody sandwich ELISA and PCR were used for relative quantification of viral protein and detecting nucleic acid in plants. (R)-PMPA at concentrations of 25 and 50 mg/l significantly reduced CaMV titers in plants within 6-9 weeks to levels detectable neither by ELISA nor by PCR. Virus-free plants were obtained after 3-month cultivation of meristem tips on semisolid medium containing 50 mg/l (R)-PMPA and their regeneration to whole plants in the greenhouse. Studying the metabolism of (R)-PMPA in B. pekinensis revealed that mono- and diphosphate, structural analogs of NDP and/or NTP, are the only metabolites formed. The data indicate very low substrate activity of the enzymes toward (R)-PMPA as substrate. The extent of phosphorylation in the plant’s leaves represents only 4.5% of applied labeled (R)-PMPA. In roots, we detected no radioactive peaks of phosphorylated metabolites of (R)-PMPAp or (R)-PMPApp.     

Antiviral properties of new arylsulfone derivatives with activity against human betaherpesviruses

Based on our previous experience with arylsulfone derivatives displaying antiherpetic activity, we synthesized several analogues in which the sulfonyl group is part of a bicyclic structure. The benzene-fused derivative 2H-3-(4-chlorophenyl)-3,4-dihydro-1,4-benzo-thiazine-2-carbonitrile 1,1-dioxide and its thiophene-fused analogue were shown to have favorable activity and selectivity against the betaherpesviruses human cytomegalovirus (HCMV) and human herpesvirus 6 (HHV-6) and 7 (HHV-7). The benzene-fused derivative retained its anti-HCMV activity when evaluated against virus strains resistant to foscarnet, ganciclovir, and/or cidofovir. The compound conferred >or=95% inhibition of viral DNA synthesis in HHV-6-infected cells. RT-PCR analysis of immediate-early, early and late gene products revealed that this arylsulfone compound acts at a step preceding late gene expression, and coinciding with the inhibition exerted by foscarnet. No inhibitory effect was seen in an enzyme assay for DNA elongation catalyzed by the HCMV or HHV-6 DNA polymerase catalytic subunit. The arylsulfone derivatives had no effect on the functional interaction between the catalytic subunit of HCMV DNA polymerase and its accessory protein, nor did they disrupt the physical interaction between the two proteins. We conclude that these arylsulfone derivatives represent new betaherpesvirus inhibitors with a novel mode of action that results in indirect inhibition of viral DNA synthesis.     

Antiviral activity of sulfated polysaccharides against African swine fever virus.

The polyanionic substances lambda and kappa carrageenan, pentosan polysulfate, fucoidan, dextran sulfate and heparin were investigated for their inhibitory effect on the replication of African swine fever virus (ASFV) in vitro. Lambda carrageenan was the most efficacious with a selectivity index, as based on the ratio of the 50% cytotoxic concentration to the 50% antiviral effective concentration, of 120, followed by pentosan polysulfate with 30, kappa carrageenan 13.3 and fucoidan 10. Dextran sulfate and heparin were almost inactive. In general, the substances had low toxicity for Vero cells. The studies with radiolabeled ASF virions suggest that the sulfated polysaccharides inhibit virus adsorption. Inhibition of virus replication was found for all the polysaccharides only when the substances were present during virus adsorption, with the exception of lambda and kappa carrageenan, which were also inhibitory when added immediately after the adsorption period.     

Inhibitory activity of bovine lactoferrin against echovirus induced programmed cell death in vitro

Lactoferrin is a glycoprotein and plays an important role in defence against pathogens. Although the antiviral activity of lactoferrin is one of the major biological functions of such protein, the mechanism of action is still under debate. The effect of lactoferrin on echovirus 6 infection in vitro was analysed and results showed that (i) cells infected with echovirus 6, died as a result of apoptosis and that (ii) programmed cell death was inhibited by lactoferrin treatment. In this report, we demonstrate that lactoferrin can exert its anti-enteroviral activity by preventing viral-induced apoptosis.     

天然(口山)酮化合物抗柯萨奇病毒B_5的作用

目的 研究天然咄酮类化合物抗柯萨奇病毒B_5(CVB_5)的作用及其机理.方法 通过改变给药途径,观察病毒引起的细胞病变效应(CPE),MTT法检测细胞活性,评价酮化合物在Hep-2细胞培养中的抗CVB_5作用.结果 酮具有抗CVB_5的活性,对Hep-2细胞的TC_(50)为54.44 μg·mL~(-1),其EC_(50)分别为26.378、25.813、25.405 μg·mL~(-1),治疗指数(TI)分别为2.064、2.109、2.143;在32 μg·mL~(-1)时,对CVB_5的抑制率达50%.结论 酮化合物通过直接杀灭病毒、阻止病毒的吸附、抑制病毒的生物合成而发挥其抗CVB_5的作用;但需对结构做进一步的修饰,以降低其毒性.