Late Antigen Expression Research Articles

A Luciferase Gene Driven by an Alphaherpesviral Promoter Also Responds to Immediate Early Antigens of the Betaherpesvirus HCMV, Allowing Comparative Analyses of Different Human Herpesviruses in One Reporter Cell Line.

Widely used methods for quantification of human cytomegalovirus (HCMV) infection in cell culture such as immunoblotting or plaque reduction assays are generally restricted to low throughput and require time-consuming evaluation. Up to now, only few HCMV reporter cell lines have been generated to overcome these restrictions and they are afflicted with other limitations because permanently expandable cell lines are normally not fully permissive to HCMV. In this work, a previously existing epithelial cell line hosting a luciferase gene under control of a Varicella-zoster virus promoter was adopted to investigate HCMV infection. The cells were susceptible to different HCMV strains at infection efficiencies that corresponded to their respective degree of epithelial cell tropism. Expression of early and late viral antigens, formation of nuclear inclusions, release of infectious virus progeny, and focal growth indicated productive viral replication. However, viral release and spread occurred at lower levels than in primary cell lines which appears to be due to a malfunction of virion morphogenesis during the nuclear stage. Expression of the luciferase reporter gene was specifically induced in HCMV infected cultures as a function of the virus dose and dependent on viral immediate early gene expression. The level of reporter activity accurately reflected infection efficiencies as determined by viral antigen immunostaining, and hence could discriminate the cell tropism of the tested virus strains. As proof-of-principle, we demonstrate that this cell line is applicable to evaluate drug resistance of clinical HCMV isolates and the neutralization capacity of human sera, and that it allows comparative and simultaneous analysis of HCMV and human herpes simplex virus type 1. In summary, the permanent epithelial reporter cell line allows robust, rapid and objective quantitation of HCMV infection and it will be particularly useful in higher throughput analyses as well as in comparative analyses of different human herpesviruses.

Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication.

Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. Latent EBV can periodically reactivate to produce infectious particles that allow the virus to spread and can lead to the death of the infected cell. In this study, we analyzed the relationship between autophagy and EBV reactivation in Burkitt's lymphoma cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation, suggesting that early after activation, the virus is able to suppress autophagy. We report here for the first time that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of Beclin1 gene, highly incremented EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that EBV activation induces the autophagic response, which is soon inhibited by the expression of EBV early lytic products. Moreover, our findings open the possibility that pharmacological inhibitors of autophagy may be used to enhance oncolytic viral therapy of EBV-related lymphomas.

Abstract 4568: Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication

Abstract Autophagy, an important degradation system involved in maintaining cellular homeostasis, serves also to eliminate pathogens and process their fragments for presentation to the immune system. Several viruses have been shown to interact with the host autophagic machinery to suppress or make use of this cellular catabolic pathway to enhance their survival and replication. Epstein Barr virus (EBV) is a γ-herpes virus associated with a number of malignancies of epithelial and lymphoid origin in which establishes a predominantly latent infection. EBV lytic cycle characterized by the sequential expression of immediate early (IE), early and late antigens results in the production of infectious particles which allow the virus to spread. In this study we analyzed the relationship between EBV and autophagy after inducing the virus productive cycle in Burkitt's lymphoma (BL) cells. By monitoring autophagy markers and EBV lytic genes expression, we demonstrate that autophagy is enhanced in the early phases of EBV lytic activation but decreases thereafter concomitantly with increased levels of EBV lytic proteins. In a cell line defective for late antigens expression, we found an inverse correlation between EBV early antigens expression and autophagosomes formation indicating that early after activation, the virus is able to suppress autophagy. We report that inhibition of autophagy by Bafilomycin A1 or shRNA knockdown of beclin1 gene, enhance EBV lytic genes expression as well as intracellular viral DNA and viral progeny yield. Taken together, these findings indicate that viral replication induces an autophagic response which can inhibit the further expression of EBV early lytic products. Moreover, our findings open the possibility to utilize pharmacological modulators of autophagy to control EBV infection and treat EBV-related lymphomas. Citation Format: Alessandra De Leo, Francesca Colavita, Fabiola Ciccosanti, Gian Maria Fimia, Paul Lieberman, Elena Mattia. Inhibition of autophagy in EBV-positive Burkitt's lymphoma cells enhances EBV lytic genes expression and replication. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4568. doi:10.1158/1538-7445.AM2015-4568

Statins demonstrate a broad anti-cytomegalovirus activity in vitro in ganciclovir-susceptible and resistant strains.

Vasculoprotective and cholesterol-lowering properties are hallmarks of statins. Recently, statins have been found to exhibit antiviral activity. Little is known about the potential of statins against human cytomegalovirus (HCMV), a risk factor in the pathogenesis of atherosclerosis. In this study, the in vitro anti-CMV activity of four statins (atorva-, fluva-, prava-, and simvastatin) was explored in human aortic endothelial cells (HAEC) and fibroblasts. All statins dose-dependently reduced HCMV titers in both cell types. Whereas atorva-, fluva-, and simvastatin showed comparable EC50 and EC90 within a low micromolar range in HAEC, pravastatin exhibited only limited effects. In metabolite rescue experiments, mevalonate almost completely abrogated the anti-CMV activity of all statins, whereas cholesterol failed to counteract the effects. Geranylgeranyl-pyrophosphate partially reversed the anti-CMV activity of most statins, suggesting an involvement of the non-sterol isoprenoid arm of the mevalonate pathway as the mode-of-action. The accumulation of immediate early viral antigens was blocked after 1 dpi onwards, and early and late antigen expression was completely abolished in HAEC. The antiviral activity of statins was comparable to ganciclovir and was retained in a ganciclovir-resistant HCMV strain. These findings provide new insight into the beneficial effects of statins, adding antiviral activity against HCMV to their list of pleïotropic properties, and support further clinical investigations on combined therapy for the management of active HCMV disease.

Signal transducer and activator of transcription 3 (STAT3) controls susceptibility to Epstein-Barr virus reactivation in B cells

Epstein-Barr Virus (EBV) is known to cause malignancies in immunocompromised individuals, including lymphomas and lymphoproliferative disorders. Reactivation of EBV from latency is important in the pathogenesis of such malignancies. Therapeutically, oncolysis of tumors harboring lytic EBV holds promise. However, exposure of latently infected cultured B cells to lytic cycle inducing stimuli results in virus reactivation within only 20% to 50% of cells. Host cell determinants, that govern this susceptibility to lytic reactivat ion within latently infected B cells, are not well understood. Our research has previously identified that higher levels of signal transducer and activator of transcription 3 (STAT3) within cells correlate with resistance to EBV lytic reactivation. We now investigate whether inhibiting function of STAT3 promotes susceptibility of latently infected B cells to known lytic cycle inducing stimuli or results in induction of EBV lytic cycle. We found that pharmacological inhibition of STAT3 phosphorylation by Janus Kinase 2 (JAK2) inhibitors (AG490 or WP1066) in latently infected cells (HH514-16 Burkitt lymphoma and B95-8 lymphoblastoid cells) increased lytic reactivation by chemical stimuli that function by distinct mechanisms. Moreover, functional inhibition of STAT3 alone resulted in lytic reactivation in these cells. EBV-Lymphoblastoid cell lines (LCL) newly generated from healthy individuals also demonstrated increased susceptibility to lytic cycle inducing stimuli in the presence of AG490 but variable susceptibility to lytic reactivation when exposed to AG490 alone. Since this lack of uniform response to AG490 alone could be due to inadequate functional suppression of STAT3, we examined EBV-LCLs derived from patients with Autosomal Dominant Hyperimmunoglobulin-E syndrome (AD-HIES or Job syndrome). Patients with AD-HIES carry a dominant negative mutation in their Stat3 gene resulting in lower basal levels of functional STAT3. When LCLs from AD-HIES patients were exposed to a JAK2 inhibitor alone, we observed a strong increase in lytic reactivation by expression of early and late lytic antigens over LCLs derived from healthy individuals. Lytic reactivation in the presence of AG490 occurred in these cells despite lack of discernable increase from basal levels of expression of ZEBRA, the viral lytic switch protein, when compared to cells not exposed to AG490. Thus, STAT3 is important in determining susceptibility to EBV reactivation. Fully understanding how STAT3 governs such susceptibility can lead to novel therapeutic strategies for EBV-related diseases.

The role of tumor cells in the modification of T lymphocytes activity — the expression of the early CD69<sup>+</sup>, CD71<sup>+</sup> and the late CD25<sup>+</sup>, CD26<sup>+</sup>, HLA/DR<sup>+</sup> activation markers on T CD4<sup>+</sup> and CD8<sup>+</sup> cells in squamous cell laryngeal carcinoma. Part I

The role of interactions between tumor cells and autologous immunocompetent cells, the impact on the modulation of the activity of T CD4(+) and CD8(+) lymphocytes, as well as the influence on the regulation and determination of antitumor cellular immune response in patients with head and neck squamous cell carcinomas (HNSCC) is not completely clear. The aim of this study was to analyze early and late activation antigens expression on T cells subpopulations modified under the influence of the presence of cancer cells to investigate the regulatory mechanisms of the local cellular immune response in carcinoma of the larynx. Cytofluorymetric analysis of the early (CD69(+), CD71(+)) and late activation markers (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD3(+)CD4(+) and CD3(+)CD8(+) cells subpopulations in mixed cellular cultures of freshly isolated tumor cells (MLTMC) and non-cancerous normal epithelial cells (MLNCC) with immunocompetent cells was performed in 55 cases of squamous cell laryngeal carcinoma. The whole peripheral blood concentrations of IL-10 and IFN-γ in 21 h and 72 h of experiments were also measured by ELISA. The relationships between the activation markers expression depending on the type of cells used in co-cultures, as well as the level of secreted cytokines, were investigated. Our work has revealed a statistically significant dependence of cytofluorymetric results on the presence of TMC or NCC in mixed cellular cultures. Increased expression of CD69(+), CD71(+) and CD25(+) (high), CD26(+), HLA/DR(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells was higher in MLTMC cultures, in comparison with MLNCC. We demonstrated negative significant relationships of IFN-γ and IL-10 secretion with regard to CD4(+)CD69(+), CD8(+)CD69(+), CD4(+)CD71(+), CD8(+)CD71(+) antigens expression in 21 h of experiments without mitogenic stimulation. Furthermore, this study revealed negative significant relationships of IFN-g secretion with regard to CD4(+)HLA/DR(+) and CD8(+)HLA/DR(+) as well as between IL-10 concentration and CD4(+)HLA/DR(+) in trials without PHA stimulation. Our findings have confirmed a key role for tumor cells in determining the function of T cells involved in the immunological processes and impact of neoplastic cells on modulating the activity of T CD4(+) and CD8(+) lymphocytes in laryngeal carcinoma.

Prognostic value of the immunological phenomena and relationship with clinicopathological characteristics of the tumor — the expression of the early CD69<sup>+</sup>, CD71<sup>+</sup>and the late CD25<sup>+</sup>, CD26<sup>+</sup>, HLA/DR <sup>+</sup> activation markers on T CD4<sup>+</sup> and CD8<sup>+</sup> lymphocytes in squamous cell laryngeal carcinoma. Part II

One of the most important challenges in contemporary oncology is to find objective biomarkers of tumor aggressiveness, which help to identify more invasive phenotypes of the carcinoma. The purpose of this study was to investigate the relationships between the early and the late activation markers expression on T CD4(+) and CD8(+) cells subpopulations and certain clinicopathological characteristics of the neoplastic infiltration in order to determine their role as biomarkers for tumor behavior in squamous cell laryngeal carcinoma. Analysis of the early (CD69(+), CD71(+)) and the late activation antigens (CD25(+) (high), CD26(+), HLA/DR(+)) expression on T CD4+ and CD8(+) lymphocytes by cytofluorymetry in 55 patients treated for squamous cell laryngeal carcinoma was performed. Clinicomorphological analysis on the basis of TNM criteria and tumor front grading, which included tumor-related features and adjacent stroma-related characteristics of the peripheral edge of infiltration was carried out. The relationships between the activation markers expression and parameters of tumor aggressiveness were investigated. Our work revealed statistically significant differences in the expression of the studied activation markers on T cells with regard to certain clinicomorphological features. The expressions of CD69(+) and CD71(+) antigens on T CD3(+)CD4(+) and CD3(+)CD8(+) cells as well as CD4(+)HLA/DR(+) markers were higher for pT3 and pT4 tumors, in comparison with pT2 carcinomas. Moreover, tumors with the smallest number of TFG points were characterized by significantly lower values of the average expression of CD3(+)CD69(+) and CD3(+)CD71(+) as well as CD4(+)HLA/DR(+) markers on T lymphocytes. In addition, more aggressive and deeply infiltrating laryngeal carcinomas were most often characterized by significantly higher values of the average expression of CD69(+) and CD71(+) antigens on CD8(+) as well as HLA/DR(+) markers on CD4(+). Our study confirmed the implication of the early and the late activation antigens expression on CD4(+) and CD8(+) T lymphocytes in clinicomorphological parameters of the tumor, especially TFG total score and depth of invasion, and their importance as indicators of the invasive phenotype of laryngeal carcinoma.

Efficiency of porcine endothelial cell infection with human cytomegalovirus depends on both virus tropism and endothelial cell vascular origin

Human cytomegalovirus (HCMV) infection or reactivation has been linked to allograft rejection resulting from endothelial injury and immune activation. In pig-to-human xenotransplantation, currently investigated to circumvent the shortage of human organs in transplantation medicine, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV. We investigated the susceptibility of porcine endothelial cells (pEC) to HCMV infection. Immortalized porcine aortic (PEDSV15) and porcine microvascular bone-marrow derived EC (2A2) as well as a panel of primary pEC originated from different vascular beds were inoculated with the endotheliotropic (TB40/E) and the fibroblast propagated (TB40/F) HCMV strains at multiplicity of infection (MOI) ranging from 0.1 to 5. Viral replication kinetics, development of cytopathology and release of viral progeny were analyzed. All viral strains infected pEC with differences in both infection efficiency and kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV-infected pEC released viral particles but with a lower efficiency than infected HAEC. Our data demonstrate that HCMV productively infects pEC, therefore finding strategies to render pEC resistant to HCMV infection will be of interest to reduce the potential risk carried by HCMV reactivation in xenotransplantation.

Immunological consequences of cross‐species infection of porcine endothelial cells with human cytomegalovirus

Human cytomegalovirus (HCMV) infection or reactivationrepresents one of the most important infectious disease problems in allotransplantation. Transmission and activation of porcine cytomegalovirus in pig‐to‐baboon xenotransplantation is associated with xenograft injury and possibly an increased incidence of consumptive coagulopathy. However, stringent protocols used in the rearing of pig donors practically exclude a risk of porcine CMV. In contrast, in pig‐to‐human xenotransplantation, the porcine endothelium will inevitably be exposed to human pathogens such as HCMV, and the consequences of potential HCMV cross‐species infection of porcine xenografts, in particular endothelial injury and immune activation, remain to be elucidated. Since it is of outmost importance to anticipate any detrimental consequence of this contact, the susceptibility of porcine endothelial cells (pEC) to HCMV infection and its effect on cell surface molecule expression and human leukocyte recruitment was evaluated.Clinical and laboratory HCMV strains, including an EC propagated (TB40/E) and two fibroblast‐adapted (AD169 and TB40/F) strains, as well as UV‐inactivated, or heparin‐treated controls were inoculated to a panel of pEC stemming from different vascular beds.Infection at various MOI was analyzed by PCR, immediate early (IE), early and late antigen expression. To determine HCMV cytopathogenicity, morphological changes were monitored using live cell imaging and apoptosis was analyzed by flow cytometry. The capability of pEC to produce infectious virus was quantified by focus expansion assays. After inoculation, cell surface receptor expression was studied by flow cytometry on pEC bulk cultures and also differentially on IE‐positive (infected) and IE‐negative (bystander) pEC. Adhesion of human PBMC was tested on HCMV infected pEC monolayers and cell‐free pEC supernatants were analyzed for their cytokine content, chemotactic activity, and stimulatory effect on resting secondary pEC cultures.All viral strains infected pEC with differences in the infection efficiency (percentage of IE‐positive cells) and the kinetics of cytopathology. Moreover, differences in susceptibility of pEC derived from distinct vascular beds were observed. HCMV underwent a complete replication cycle in about 5% of the infected pEC. Comparing the permissiveness of pEC to human aortic EC (HAEC) revealed differences in strain susceptibility and lower rates of late antigen expression in pEC. Finally, HCMV‐infected pEC were demonstrated to release viral progeny with a lower efficiency than infected HAEC.Cell‐surface expression of E‐selectin and VCAM‐1 was upregulated not only in the IE‐positive fractions (infected) of HCMV‐inoculated cultures but also in the IE‐negative fractions, presumably by bystander effects. In contrast, porcine MHC class I expression was downregulated in IE‐positive, and upregulated in IE‐negative cells. The receptor alterations in the IE‐negative fraction were mediated by pEC‐derived soluble factors and consequently both porcine IL‐6 and IL‐8 were detected in the supernatants. The increased adhesion receptor expression was paralleled by enhanced human leukocyte chemotaxis and adhesion to infected pEC cultures. Pretreatment of HCMV with heparin blocking viral entry, but not UV‐inactivation blocking viral replication, prevented adhesion‐receptor modulation, and reversed the increased adhesion and chemotaxis.In summary, HCMV may productively infect pECfollowing pig‐to‐human solid organ transplantation and may alter and activate the porcine endothelium, rendering the xenograft more susceptible to human leukocyte recruitment and rejection. Therefore, understanding the mechanisms of cross‐species HCMV infection and finding strategies to render pEC resistant to HCMV infection willbe essential for xenotransplantation to become a clinical reality.

Inhibition of p38 MAP kinase pathway induces apoptosis and prevents Epstein Barr virus reactivation in Raji cells exposed to lytic cycle inducing compounds

BackgroundEBV lytic cycle activators, such as phorbol esters, anti-immunoglobulin, transforming growth factor β (TGFβ), sodium butyrate, induce apoptosis in EBV-negative but not in EBV-positive Burkitt's lymphoma (BL) cells. To investigate the molecular mechanisms allowing EBV-infected cells to be protected, we examined the expression of viral and cellular antiapoptotic proteins as well as the activation of signal transduction pathways in BL-derived Raji cells exposed to lytic cycle inducing agents.ResultsOur data show that, following EBV activation, the latent membrane protein 1 (LMP1) and the cellular anti-apoptotic proteins MCL-1 and BCL-2 were quickly up-regulated and that Raji cells remained viable even when exposed simultaneously to P(BU)2, sodium butyrate and TGFβ. We report here that inhibition of p38 pathway, during EBV activation, led to a three fold increment of apoptosis and largely prevented lytic gene expression.ConclusionThese findings indicate that, during the switch from the latent to the lytic phase of EBV infection, p38 MAPK phosphorylation plays a key role both for protecting the host cells from apoptosis as well as for inducing viral reactivation. Because Raji cells are defective for late antigens expression, we hypothesize that the increment of LMP1 gene expression in the early phases of EBV lytic cycle might contribute to the survival of the EBV-positive cells.

Increased human cytomegalovirus replication in fibroblasts after treatment with therapeutical plasma concentrations of valproic acid

Valproic acid (2-propylpentanoic acid, VPA), an effective inhibitor of histone deacetylases (HDAC) is used for the treatment of epilepsia. In this study, structure–activity relationships for the action of structurally modified VPA derivatives on human cytomegalovirus (HCMV) replication and HDAC inhibition were defined. Pretreatment of human foreskin fibroblasts with VPA (0.125–1 mM) caused a concentration-dependent increase of HCMV immediate early and antigen late antigen expression. Structure–activity relationships of VPA derivatives for HCMV stimulation were compared to those for teratogenic action and those for HDAC inhibition. Side chain elongation and introduction of a triple bond in 4-position of the other chain caused teratogenicity, stimulated HCMV replication, and increased HDAC inhibition, as demonstrated by enhanced levels of acetylated histones. Teratogenic VPA derivatives with a branched chain in 3-position as well as a non-teratogenic anticonvulsive active VPA derivative did not stimulate HCMV or accumulation of acetylated histones. This demonstrates a strict correlation between inhibition of HDAC and increased HCMV replication.

In Vitro Anti‐Cytomegalovirus Activity of Kampo (Japanese Herbal) Medicine

We examined the effect of three types of Kampo medicines on human cytomegalovirus (CMV) replication in the human embryonic fibroblast cell line, MRC-5. Treatment of cells with at least 0.01 μg/ml of Kampo medicines inhibited the cytopathic effects of CMV-infected MRC-5 cells. Moreover, Kampo medicine decreased the replication of CMV without affecting the inhibition of host cells, with a concomitant decrease in CMV DNA levels. However, Kampo medicine demonstrated no virocidal effect on cell-free CMV. Furthermore, western blotting analysis demonstrated that the Kampo medicine decreased the amount of 65 kDa late antigen expression in the infected cells. These results suggest that Kampo medicine may be sufficient to inhibit viral DNA replication and late protein synthesis, resulting in anti-CMV effects. Therefore, these three Kampo medicines have the potential of being a source of new powerful anti-CMV compounds.

Disruption of gammaherpesvirus 68 gene 50 demonstrates that Rta is essential for virus replication.

Gammaherpesvirus pathogenesis is dependent on the ability of these viruses to establish a lifelong latent infection and the ability to reactivate from latency. Immediate-early genes of theses viruses are thought to be critical regulators of lytic replication and reactivation from latency. The gene 50-encoded Rta is the only immediate-early gene product that appears to be conserved among all characterized gammaherpesviruses. Previous studies have demonstrated that, in Epstein-Barr virus (EBV), Kaposi's sarcoma-associated virus, and gammaherpesvirus 68 (gamma HV68, also referred to as murine gammaherpesvirus 68), ectopic expression of Rta in latently infected cell lines can lead to induction of the viral cycle. Recently, studies employing null mutants of EBV have provided a formal demonstration that both Rta and the BZLF1 gene product, Zta, the two EBV immediate-early gene products, are essential for EBV replication. Here we generate and characterize a gene 50-null mutant gamma HV68 and demonstrate that the gene 50 product Rta is essential for virus replication. Providing gamma HV68 Rta in trans was sufficient to restore replication of the gene 50-null virus. Notably, Rta expressed from the spliced form of the gene 50 transcript was sufficient to complement growth of the gene 50-null virus. In addition, we provide evidence that loss of Rta expression leads to a complete defect in viral DNA replication and a significant defect in late antigen expression. This work lays the foundation for characterizing the role of Rta in gamma HV68 chronic infection of mice.

Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts

Lactoferrin is mainly produced by polymorphonuclear leukocytes and has been demonstrated in mammalian milk and external secretions. Lactoferrin is an iron-binding, multifunctional protein and may play an important role in immune regulation and in defense mechanisms against bacteria, fungi and viruses. Lactoferricin is a potent antimicrobial peptide generated from the N-terminal part of lactoferrin by pepsin cleavage. We demonstrate that lactoferrins from different species and its N-terminal peptide lactoferricin (particularly the cyclic form) inhibit expression of early and late antigens, as well as production of infectious viral progeny during human cytomegalovirus (HCMV) infection in vitro. Iron-saturated lactoferrin did not affect HCMV antigen expression. Heparin had the same effects as iron-depleted lactoferrin. Yet, mixtures of lactoferrin and heparin did not inhibit HCMV multiplication i.e. lactoferrin and heparin seemed to mutually block each other's antiviral activities. HCMV-infected cells exposed to lactoferrin and cyclic lactoferricin contained less intracellular virus than unexposed cells. The antiviral activity of cyclic lactoferricin was more than seven-fold weaker than that of the maternal molecule. Lactoferrin and cyclic lactoferricin prevented HCMV entrance into the host cell.

Cell cycling status of human cord blood CD34+ cells during ex vivo expansion is related to the level of very late antigen expression.

Very late antigen-4 (VLA-4), which binds to the extracellular matrix protein fibronectin, is an integrin molecule known to be modulated during mobilization of CD34+ cells, and to be involved in signaling the mobilization stimuli. On the hypothesis that cell cycling status might be different depending on the level of VLA-4 expression, we investigated the DNA contents of human cord blood CD34+ cells during ex vivo expansion by recombinant human thrombopoietin and flt3-ligand with simultaneous measurement of surface VLA-4 at the 1st and 4th week. During this ex vivo expansion, expression of VLA-4 increased and almost all cells became VLA-4+ until the 4th day of culture. Expression of VLA-4 was maintained in the major population of the cultured cells until the 4th week. The cells in S/G2/M phase were greater in number in VLA-4 high fraction than in VLA-4 low fraction (n=4, p<.001). Furthermore, the fraction of cells in S/G2/M phase increased as the expression of VLA-4 became higher. These results suggest that cord blood CD34+ cells expressing high levels of VLA-4 have more proliferative activities.

Cytomegalovirus infection decreases expression of thrombospondin-1 and -2 in cultured human retinal glial cells: effects of antiviral agents.

In fibroblasts, infection with human cytomegalovirus (HCMV) inhibits expression of the extracellular matrix proteins thrombospondin-1 and -2 (TSP-1 and TSP-2). These effects may depend on expression of HCMV immediate-early (IE) genes, which are activated by cellular transcription factor NF-kappaB. The influence of HCMV infection on TSP-1 and TSP-2 expression and the ability of different antiviral drugs to prevent these cellular changes in permissive cultures of human retinal glial cells were observed. Ganciclovir inhibited only HCMV late antigen (LA) expression, whereas antisense oligonucleotide ISIS 2922 and peptide SN50, inhibitors of HCMV IE expression and NF-kappaB activity, respectively, inhibited both IE and LA expression. ISIS 2922 and SN50, but not ganciclovir, prevented down-modulation of TSP-1 and TSP-2. The results showed that HCMV-induced down-modulation of TSP-1 and TSP-2 in retinal glial cells is prevented by inhibition of HCMV IE expression. These findings may be relevant to pathogenesis and treatment of HCMV retinitis.

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts.

Human cytomegalovirus (HCMV) infection is associated with excessive proinflammatory immune responses such as cytokine/chemokine production or upregulation of adhesion molecules on the host cells. It is assumed that these features of HCMV-related immunopathology can not be treated effectively with currently available anti HCMV drugs. In the present study the efficacy of ganciclovir (GCV), foscarnet (PFA), cidofovir (HPMPC), and ISIS 2922, an antisense oligonucleotide complementary to HCMV immediate-early (IE) mRNA, was investigated on HCMV-induced secretion and functional activity of the C-X-C chemokine IL-8 and the expression of the intercellular adhesion molecule-1 (ICAM-1). As compared with mock-infected cells IL-8 production was increased up to 9-fold and ICAM-1 expression up to 4-fold in HCMV-infected fibroblasts. Treatment of infected cells with GCV (40 microM), PFA (200 microM) or HPMPC (2 microM) suppressed completely virus replication as demonstrated by quantification of late (L) antigen expression and infectious virus production. These drugs, however, failed to inhibit IE antigen expression and did not prevent HCMV-induced upregulation of IL-8 and ICAM-1. In contrast, ISIS 2922 (1 microM) suppressed both IE and L antigen expression by 99% and inhibited infectious virus production by 10(4)-fold. Moreover, ISIS 2922 significantly suppressed HCMV-induced upregulation of both IL-8 and ICAM-1 expression on the transcriptional and on the protein level. Our results indicate that ISIS 2922 but not inhibitors of HCMV DNA prevents HCMV-induced upregulation of IL-8 and ICAM-1, both hallmarks of inflammatory processes. Thus, inhibition of HCMV IE expression with ISIS 2922 may be an important strategy for the treatment of HCMV-related immunopathogenesis.

Expression of Human Herpesvirus-6 Antigens in Benign and Malignant Lymphoproliferative Diseases

Immunohistochemistry was used to look for the expression of human herpesvirus-6 (HHV-6) antigens in a well characterized series of benign, atypical, and malignant lymphoid lesions, which tested positive for the presence of HHV-6 DNA. A panel of specific antibodies against HHV-6 antigens, characteristic either of the early (p41) or late (p101K, gp106, and gp116) phases of the viral cycle, was applied to the lymphoid tissues from 15 non-Hodgkin's lymphomas, 14 Hodgkin's disease cases, 5 angioimmunoblastic lymphadenopathies with dysproteinemia, 14 reactive lymphadenopathies, and 2 cases of sinus histiocytosis with massive lymphadenopathy (Rosai-Dorfman disease). In lymphomatous tissues, the expression of late antigens was documented only in reactive cells, and mainly in plasma cells. Of interest, the expression of the early p41 antigen was detected in the so-called "mummified" Reed-Sternberg cells, in two Hodgkin's disease cases. In reactive lymphadenopathies, the HHV-6 late antigen-expressing cells were plasma cells, histiocytes, and rare granulocytes distributed in interfollicular areas. In both cases of Rosai-Dorfman disease, the p101K showed an intense staining in follicular dendritic cells of germinal centers, whereas the gp106 exhibited an intense cytoplasmic reaction in the abnormal histiocytes, which represent the histological hallmark of the disease. The expression of HHV-6 antigens is tightly controlled in lymphoid tissues. The lack of HHV-6 antigen expression in neoplastic cells and the limited expression in degenerating Reed-Sternberg cells argue against a major pathogenetic role of the virus in human lymphomagenesis. The detection of a rather unique pattern of viral late antigen expression in Rosai-Dorfman disease suggests a possible pathogenetic involvement of HHV-6 in some cases of this rare lymphoproliferative disorder.

Generation of a nude mouse tumor model for in vivo replication of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a common opportunistic infection resulting in retinitis in 15%-40% of AIDS patients. Several anti-HCMV therapies are currently available, and new treatments are in various stages of development. An HCMV animal model involving in vivo infection of human cells without the dependence on human fetuses or multiple surgical procedures has been developed. A human glioblastoma cell line that is permissive for HCMV replication (U373MG) was adapted to grow as a subcutaneous tumor in nude mice. These tumors arise in approximately 7 days and grow progressively. An evaluation of HCMV DNA replication demonstrated an increase in the accumulation of HCMV DNA within infected tumors from 48 to 168 h after infection. Immunohistochemical analysis showed focal areas of HCMV infection in which expression of immediate-early and late antigens was detected. In addition, it was demonstrated that ganciclovir inhibited HCMV DNA replication in vivo in a dose-dependent manner.

Inhibition of cytomegalovirus late antigen expression and cytomegalovirus replication in human fibroblasts and differentiated monocytic cells by liposome-encapsulated foscarnet

Foscarnet is one of the drugs of choice for the treatment of cytomegalovirus (CMV) infections, but its efficacy is limited by systemic toxicity, poor penetration into cells and a short plasma half-life. In this study, two types of sized, foscarnet-containing liposomes were evaluated with respect to their in vitro efficacy against human CMV in differentiated monocytic (THP-1) cells and embryonic lung fibroblasts. Both preparations of liposomal foscarnet inhibited CMV replication and CMV late antigen expression in either cell type. In contrast to free foscarnet, the extent of inhibition was dependent on the cell type, with superior activity towards CMV in THP-1 cells. The antiviral effect was most likely mediated by direct association of liposome-encapsulated foscarnet with THP-1 cells. Free foscarnet, however, showed an equal or greater antiviral activity than liposome-encapsulated foscarnet. At the IC 50 concentrations of free and liposome-encapsulated foscarnet, toxicity towards the permissive cells was not observed. Together with our previous findings of the prolonged circulation time of one of the liposome preparations used in this study, these results indicate that liposome-encapsulated foscarnet warrants further exploration to assess its potential for the treatment of CMV-infections.