Immune responses to highly conserved influenza A virus matrix 1 peptides.

Influenza vaccine development is considered to be complicated and challenging. Constantly evolving influenza viruses require continuous global monitoring and reformulation of the vaccine strains. Peptides that are conserved among different strains and subtypes of influenza A virus are strongly considered to be attractive targets for development of cross protective influenza vaccines that stimulate cellular responses. In this study, three highly conserved (>90%) matrix 1 peptides that contain multiple T cell epitopes, ILGFVFTLTVPSERGLQRRRF (PM 1), LIRHENRMVLASTTAKA (PM 2) and LQAYQKRMGVQMQR (PM 3), were assessed for their immunogenic potential in vitro by subjecting peripheral blood mononuclear cells from healthy volunteers to repetitive stimulation with these chemically synthesised peptides and measuring their IFN-γ concentrations, proliferation by ELISA, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Seven samples were screened for immunogenicity of PM 1 and PM 2, and six for that of PM 3. All six samples had positive responses (IFN-γ secretion) to PM 3 stimulation, as did five and three for PM 2 and PM 1 respectively. In contrast, seven (PM 1 and PM 2) and four (PM 3) samples showed proliferative response as compared with unstimulated cells. The encouraging immunogenic response generated by these highly conserved matrix 1 peptides indicates they are prospective candidates for development of broadly reactive influenza vaccines.

S100A4 hypomethylation affects epithelial-mesenchymal transition partially induced by LMP2A in nasopharyngeal carcinoma.

To identify cellular target genes involved in NPC cell invasion and metastasis, gene expression profiles of CNE-1 cells with or without ectopic LMP2A expression were compared by using the metastatic gene array. S100 calcium binding protein A4 (S100A4) was the highest increased one among these genes both in mRNA and protein levels of NPC cells. Moreover, S100A4 was upregulated in LMP2A-positive NPC tissues. We found that CNE-1-S100A4 showed significantly increased invasion ability as compared to the controls both in vitro and in vivo, which indicated that S100A4 induced EMT occurrence and promoted metastasis. Notably, the DNA hypomethylation of S100A4 was found in LMP2A-positive NPC tissues. Besides, inhibition of DNA methyltransferases via 5-Aza-dC stimulated the expression of S100A4 in the cells without ectopic LMP2A expression. The methylation changes were confirmed by methylation specific PCR (MSP), suggesting that LMP2A ectopic expression led to the demethylation of S100A4 promoter. These results demonstrated that LMP2A-induced hypomethylation participated in regulating S100A4 expression in NPC. Our findings provide an evidence for the emerging notion that hypomethylation and activation of correlated genes are crucial for metastasis progression in cancer. © 2015 Wiley Periodicals, Inc.

Immunotherapy with Donor T Cells Sensitized with Overlapping Pentadecapeptides for Treatment of Persistent Cytomegalovirus Infection or Viremia.

We conducted a phase I trial of allogeneic T cells sensitized in vitro against a pool of pentadecapeptides (15-mer peptides) spanning the sequence of CMVpp65 for adoptive therapy of 17 allogeneic hematopoietic cell transplant recipients with cytomegalovirus (CMV) viremia or clinical infection persisting despite prolonged treatment with antiviral drugs. All but 3 of the patients had received T cell-depleted transplants without graft-versus-host disease (GVHD) prophylaxis with immunosuppressive drugs after transplantation. The CMVpp65-specific T cells (CMVpp65CTLs) generated were oligoclonal and specific for only 1 to 3 epitopes, presented by a limited set of HLA class I or II alleles. T cell infusions were well tolerated without toxicity or GVHD. Of 17 patients treated with transplant donor (n = 16) or third-party (n = 1) CMVpp65CTLs, 15 cleared viremia, including 3 of 5 with overt disease. In responding patients, the CMVpp65CTLs infused consistently proliferated and could be detected by T cell receptor Vß usage in CMVpp65/HLA tetramer + populations for period of 120 days to up to 2 years after infusion. Thus, CMVpp65CTLs generated in response to synthetic 15-mer peptides of CMVpp65 are safe and can clear persistent CMV infections in the post-transplantation period.

Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with rabies virus based bivalent vaccine.

We have previously described the generation of a novel Ebola virus (EBOV) vaccine platform based on (a) replication-competent rabies virus (RABV), (b) replication-deficient RABV, or (c) chemically inactivated RABV expressing EBOV glycoprotein (GP). Mouse studies demonstrated safety, immunogenicity, and protective efficacy of these live or inactivated RABV/EBOV vaccines. Here, we evaluated these vaccines in nonhuman primates. Our results indicate that all three vaccines do induce potent immune responses against both RABV and EBOV, while the protection of immunized animals against EBOV was largely dependent on the quality of humoral immune response against EBOV GP. We also determined if the induced antibodies against EBOV GP differ in their target, affinity, or the isotype. Our results show that IgG1-biased humoral responses as well as high levels of GP-specific antibodies were beneficial for the control of EBOV infection after immunization. These results further support the concept that a successful EBOV vaccine needs to induce strong antibodies against EBOV. We also showed that a dual vaccine against RABV and filoviruses is achievable; therefore addressing concerns for the marketability of this urgently needed vaccine.

Targeting ß2 adrenergic receptors regulate human T cell function directly and indirectly.

It is well-established that central nervous system activation affects peripheral blood mononuclear cell (PBMCs) function through the release of the catecholamines (Epi) and norepinephrine (NE), which act on ß2-adrenergic receptors (ß2AR). However, most studies have used non-specific stimulation of cells rather than antigen-specific responses. Likewise, few studies have parsed out the direct effects of ß2AR stimulation on T cells versus indirect effects via adrenergic stimulation of antigen presenting cells (APC). Here we report the effect of salmeterol (Sal), a selective ß2AR agonist, on IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells following stimulation with Cytomegalovirus lysate (CMVL-strain AD169) or individual peptides spanning the entire region of the HCMV pp65 protein (pp65). Cells were also stimulated with Staphylococcal enterotoxin B. Additionally, we investigated the effect of Epi and Sal on cytotoxic cell killing of transfected target cells at the single cell level using the CD107a assay. The results show that Sal reduced the percentage of IFN-γ(+) CD4 and IFN-γ(+) CD8 T cells both when applied directly to isolated T cells, and indirectly via treatment of APC. These inhibitory effects were mediated via a ß2 adrenergic-dependent pathway and were stronger for CD8 as compared to CD4 T cells. Similarly, the results show that Sal suppressed cytotoxicity of both CD8 T and NK cells in vitro following stimulation with Chinese hamster ovary cell line transfected with MICA(*009) (T-CHO) and the human erythromyeloblastoid leukemic (K562) cell line. The inhibitory effect on cytotoxicity following stimulation with T-CHO was stronger in NK cells compared with CD8 T cells. Thus, targeting the ß2AR on lymphocytes and on APC leads to inhibition of inflammatory cytokine production and target cell killing. Moreover, there is a hierarchy of responses, with CD8 T cells and NK cells inhibited more effectively than CD4 T cells.

NetFCM: a semi-automated web-based method for flow cytometry data analysis.

Multi-parametric flow cytometry (FCM) represents an invaluable instrument to conduct single cell analysis and has significantly increased our understanding of the immune system. However, due to new techniques allowing us to measure an increased number of phenotypes within the immune system, FCM data analysis has become more complex and labor-intensive than previously. We have therefore developed a semi-automatic gating strategy (NetFCM) that uses clustering and principal component analysis (PCA) together with other statistical methods to mimic manual gating approaches. NetFCM is an online tool both for subset identification as well as for quantification of differences between samples. Additionally, NetFCM can classify and cluster samples based on multidimensional data. We tested the method using a data set of peripheral blood mononuclear cells collected from 23 HIV-infected individuals, which were stimulated with overlapping HIV Gag-p55 and CMV-pp65 peptides or medium alone (negative control). NetFCM clustered the virus-specific CD8+ T cells based on IFNγ and TNF responses into distinct compartments. Additionally, NetFCM was capable of identifying HIV- and CMV-specific responses corresponding to those obtained by manual gating strategies. These data demonstrate that NetFCM has the potential to identify relevant T cell populations by mimicking classical FCM data analysis and reduce the subjectivity and amount of time associated with such analysis. © 2014 International Society for Advancement of Cytometry.

Fcγ receptor antigen targeting potentiates cross-presentation by human blood and lymphoid tissue BDCA-3+ dendritic cells.

The reactivation of human cytomegalovirus (HCMV) poses a serious health threat to immune compromised individuals. As a treatment strategy, dendritic cell (DC) vaccination trials are ongoing. Recent work suggests that BDCA-3(+) (CD141(+)) subset DCs may be particularly effective in DC vaccination trials. BDCA-3(+) DCs had however been mostly characterized for their ability to cross-present antigen from necrotic cells. We here describe our study of human BDCA-3(+) DCs in elicitation of HCMV-specific CD8(+) T-cell clones. We show that Fcgamma-receptor (FcγR) antigen targeting facilitates antigen cross-presentation in several DC subsets, including BDCA-3(+) DCs. FcγR antigen targeting stimulates antigen uptake by BDCA-1(+) rather than BDCA-3(+) DCs. Conversely, BDCA-3(+) DCs and not BDCA-1(+) DCs show improved cross-presentation by FcγR targeting, as measured by induced release of IFNγ and TNF by antigen-specific CD8(+) T cells. FcγR-facilitated cross-presentation requires antigen processing in both an acidic endosomal compartment and by the proteasome, and did not induce substantial DC maturation. FcγRII is the most abundantly expressed FcγR on both BDCA-1(+) and BDCA-3(+) DCs. Furthermore we show that BDCA-3(+) DCs express relatively more stimulatory FcγRIIa than inhibitory FcγRIIb in comparison with BDCA-1(+) DCs. These studies support the exploration of FcγR antigen targeting to BDCA-3(+) DCs for human vaccination purposes.

Molecular determinants of susceptibility to oncolytic vesicular stomatitis virus in pancreatic adenocarcinoma.

BACKGROUND: M protein mutant vesicular stomatitis virus (M51R-VSV) has oncolytic properties against many cancers. However, some cancer cells are resistant to M51R-VSV. Herein, we evaluate the molecular determinants of vesicular stomatitis virus (VSV) resistance in pancreatic adenocarcinoma cells. METHODS: Cell viability and the effect of ß-interferon (IFN) were analyzed using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. Gene expression was evaluated via microarray analysis. Cell infectability was measured by flow cytometry. Xenografts were established in athymic nude mice and treated with intratumoral M51R-VSV. RESULTS: Four of five pancreatic cancer cell lines were sensitive to M51R-VSV, whereas Panc 03.27 cells remained resistant (81 ± 3% viability 72 h after single-cycle infection). Comparing sensitive MiaPaCa2 cells with resistant Panc 03.27 cells, significant differences in gene expression were found relating to IFN signaling (P = 2 × 10(-5)), viral entry (P = 3 × 10(-4)), and endocytosis (P = 7 × 10(-4)). MiaPaCa2 cells permitted high levels of VSV infection, whereas Panc 03.27 cells were capable of resisting VSV cell entry even at high multiplicities of infection. Extrinsic ß-IFN overcame apparent defects in IFN-mediated pathways in MiaPaCa2 cells conferring VSV resistance. In contrast, ß-IFN decreased cell viability in Panc 3.27 cells, suggesting intact antiviral mechanisms. VSV-treated xenografts exhibited reduced tumor growth relative to controls in both MiaPaCa2 (1423 ± 345% versus 164 ± 136%; P < 0.001) and Panc 3.27 (979 ± 153% versus 50 ± 56%; P = 0.002) tumors. Significant lymphocytic infiltration was seen in M51R-VSV-treated Panc 03.27 xenografts. CONCLUSIONS: Inhibition of VSV endocytosis and intact IFN-mediated defenses are responsible for M51R-VSV resistance in pancreatic adenocarcinoma cells. M51R-VSV treatment appears to induce antitumor cellular immunity in vivo, which may expand its clinical efficacy.

Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity.

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl(-)) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H(+)) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o-) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H(+), did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

Successful isolation and expansion of CMV-reactive T cells from G-CSF mobilized donors that retain a strong cytotoxic effector function.

Cytomegalovirus (CMV) infections post-haematopoietic stem cell transplantation (HSCT) can be effectively controlled through the adoptive transfer of donor-derived CMV-specific T cells (CMV-T). Current strategies involve a second leukapheresis collection from the original donor to manufacture CMV-T, which is often not possible in the unrelated donor setting. To overcome these limitations we have investigated the use of a small aliquot of the original granulocyte-colony stimulating factor (G-CSF) mobilized HSCT graft to manufacture CMV-T. We explored the T cell response to CMVpp65 peptide stimulation in G-CSF mobilized peripheral blood mononuclear cells (PBMC) and subsequently examined isolation of CMV-T based on the activation markers CD154 and CD25. CD25(+) enriched CMV-T from G-CSF mobilized PBMC contained a higher proportion of FoxP3 expression than non-mobilized PBMC and showed superior suppression of T cell proliferation. Expanded CMV-T enriched through CD154 were CD4(+) and CD8(+) , demonstrated a high specificity for CMV, secreted cytotoxic effector molecules and lysed CMVpp65 peptide-loaded phytohaemagglutinin-stimulated blasts. These data provide the first known evidence that CMV-T can be effectively manufactured from G-CSF mobilized PBMC and that they share the same characteristics as CMV-T isolated in an identical manner from conventional non-mobilized PBMC. This provides a novel strategy for adoptive immunotherapy that abrogates the need for successive donation.

Requirement for LMP1-induced RON receptor tyrosine kinase in Epstein-Barr virus-mediated B-cell proliferation.

EBV, an oncogenic human herpesvirus, can transform primary B lymphocytes into immortalized lymphoblastoid cell lines (LCLs) through multiple regulatory mechanisms. However, the involvement of protein tyrosine kinases in the infinite proliferation of B cells is not clear. In this study, we performed kinase display assays to investigate this subject and identified a specific cellular target, Recepteur d'Origine Nantais (RON) tyrosine kinase, expressed in LCLs but not in primary B cells. Furthermore, we found that latent membrane protein 1 (LMP1), an important EBV oncogenic protein, enhanced RON expression through its C-terminal activation region-1 (CTAR1) by promoting NF-κB binding to the RON promoter. RON knockdown decreased the proliferation of LCLs, and transfection with RON compensated for the growth inhibition caused by knockdown of LMP1. Immunohistochemical analysis revealed a correlation between LMP1 and RON expression in biopsies from posttransplantation lymphoproliferative disorder (PTLD), suggesting that LMP1-induced RON expression not only is essential for the growth of LCLs but also may contribute to the pathogenesis of EBV-associated PTLD. Our study is the first to reveal the impact of RON on the proliferation of transformed B cells and to suggest that RON may be a novel therapeutic target for EBV-associated lymphoproliferative diseases.

IL-21 imposes a type II EBV gene expression on type III and type I B cells by the repression of C- and activation of LMP-1-promoter.

Epstein-Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4(+) T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

Dominant inhibition of Akt/protein kinase B signaling by the matrix protein of a negative-strand RNA virus.

Vesicular stomatitis virus (VSV) is a rhabdovirus that alters host nuclear and cytoplasmic function upon infection. We have investigated the effect of VSV infection on cellular signaling through the phosphatidylinositol-3 kinase (PI3k)/Akt signaling pathway. Akt phosphorylation at both threonine 308 (Thr308) and serine 473 (Ser473) was inhibited in cells infected with VSV. This inhibition was rapid (beginning within the first 2 to 3 h postinfection) and correlated with the dephosphorylation of downstream effectors of Akt, such as glycogen synthase kinase 3ß (GSK3ß) and mammalian target of rapamycin (mTOR). The dephosphorylation of Akt occurred in the presence of growth factor stimulation and was not overcome through constitutive membrane targeting of Akt or high levels of phosphatidylinositol-3,4,5-triphosphate (PIP3) accumulation in the membrane. Akt dephosphorylation was not a result of alterations in PDK1 phosphorylation or activity, changes in phosphatase and tensin homologue deleted on chromosome 10 (PTEN) levels, or the downregulation of PI3k signaling. Inactivation of Akt was caused by the expression of the viral M protein in the absence of other viral components, and an M protein mutant that does not inhibit RNA polymerase II (Pol II) transcription and nuclear/cytoplasmic transport was also defective in inhibiting Akt phosphorylation. These data illustrate that VSV utilizes a novel mechanism to alter this central player in cell signaling and oncogenesis. It also suggests an inside-out model of signal transduction where VSV interruption of nuclear events has a rapid and significant effect on membrane signaling events.

Latent Membrane Protein 1 as a molecular adjuvant for single-cycle lentiviral vaccines.

BACKGROUND: Molecular adjuvants are a promising method to enhance virus-specific immune responses and protect against HIV-1 infection. Immune activation by ligands for receptors such as CD40 can induce dendritic cell activation and maturation. Here we explore the incorporation of two CD40 mimics, Epstein Barr Virus gene LMP1 or an LMP1-CD40 chimera, into a strain of SIV that was engineered to be limited to a single cycle of infection. RESULTS: Full length LMP1 or the chimeric protein LMP1-CD40 was cloned into the nef-locus of single-cycle SIV. Human and Macaque monocyte derived macrophages and DC were infected with these viruses. Infected cells were analyzed for activation surface markers by flow cytometry. Cells were also analyzed for secretion of pro-inflammatory cytokines IL-1ß, IL-6, IL-8, IL-12p70 and TNF by cytometric bead array. CONCLUSIONS: Overall, single-cycle SIV expressing LMP1 and LMP1-CD40 produced a broad and potent T(H)1-biased immune response in human as well as rhesus macaque macrophages and DC when compared with control virus. Single-cycle SIV-LMP1 also enhanced antigen presentation by lentiviral vector vaccines, suggesting that LMP1-mediated immune activation may enhance lentiviral vector vaccines against HIV-1.

Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies.

BACKGROUND: Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8(+) and CD4(+) T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65(495-503)-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for ex vivo expansion of CMV-specific CTLs. METHODS: CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65(495-503) peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65(495-503) complex. RESULTS: About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65(495-503) peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8(+) T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC. CONCLUSION: This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.

Characterization of the antiviral activity for influenza viruses M1 zinc finger peptides.

We sought to investigate the cellular uptake and antiviral activity for the M1 zinc finger peptides derived from influenza A and influenza B viruses in vitro. No cellular uptake was detected by fluorescent microscopy for the synthetic zinc finger peptides. When flanked to a cell permeable peptide Tp10, the zinc finger recombinant proteins were efficiently internalized by MDCK cells. However, no antiviral activity was detected against homologous or heterologous virus infections for the synthetic peptides or the Tp10-flanked recombinant proteins, regardless treated with or without Zn(2+). Nevertheless, MDCK cell constitutively expressing the M1 zinc finger peptides in cell nuclei potently inhibited replication of homologous, but not heterologous influenza viruses. Adenoviral vector delivered M1 zinc finger peptides also exhibited potent antiviral activity against homologous viruses challenge. Transduction at 100 PFU dose of recombinant adenovirus efficiently protected 99% of the cells from 100 TCID(50) of different virus infections for both peptides. These results brought new insight to the antiviral researches against influenza virus infections.

[Antitumor effects of matrix protein of vesicular stomatic virus on EL4 lymphoma mice].

OBJECTIVE: To explore antitumor effects of plasmid pcDNA3. 1-MP encoding matrix protein of vesicular stomatitis virus (VSV) complexed with cationic liposome (DOTAP:CHOL) in mice with EL4 lymphoma. METHODS: C57BL/6 mouse model with EL4 lymphoma was established. Sixty mice bearing EL4 lymphoma were divided randomly into five groups including Lip-MP, Lip-pVAX, Lip, ADM and NS groups, which were intravenously injected with liposome-pcDNA 3. 1-MP complex, liposome-pVAX complex, empty liposome, Adriamycin and normal saline respectively every three days. Tumor volumes and survival time were monitored. Microvessel density and tumor proliferative index in tumor tissues were determined by CD31, Ki-67 immunohistochemistry staining, meanwhile the tumor apoptosis index was measured by TUNEL method. RESULTS: From 6 days after treatments on, the tumor volume in Lip-MP group was much smaller than that in Lip-pVAX, Lip and NS group (P < 0.05). The median survival time of mice in Lip-MP group, 44 days after inoculation of tumor cells, was significantly higher than that in other groups (P < 0.05), which was 39 days, 38.5 days and 34 days in Lip-pVAX, Lip and NS groups respectively. The MVD value in tumor tissues in Lip-MP group was less than that in Lip-pVAX, Lip and NS groups (P < 0.05). Ki67 staining revealed that Lip-MP complex apparently suppressed the proliferation of EL4 tumor cells in vivo (P < 0.05). TUNEL assays showed that apoptosis index of tumor cells in Lip-MP group, 10.60 +/- 1.71, was much higher than that in other three groups (P < 0.05). CONCLUSIONS: Lip-MP complex, the plasmid encoding matrix protein of VSV (VSV-MP) encapsulated in cationic liposome, significantly inhibited the growth of tumor and prolonged the survival of mice bearing EL4 lymphoma, which may be related to the induction of tumor cell apoptosis, inhibition of tumor angiogenesis, and suppression of tumor cell proliferation.

Different responses are elicited in cytotoxic T lymphocytes by different levels of T cell receptor occupancy.

We have investigated the level of TCR occupancy required to elicit different biological responses in human CTL clones specific for an influenza matrix peptide. Specific cytotoxicity could be detected at extremely low peptide concentrations (10(-12) to 10(-15) M). However, IFN-gamma production, responsiveness to IL-2 and Ca++ fluxes were observed only at peptide concentrations > 10(-9) M, while autonomous proliferation required even higher peptide concentrations. In parallel experiments we measured TCR downregulation to estimate the number of TCRs triggered. We observed that at low peptide concentrations, where only cytotoxicity is triggered, TCR downregulation was hardly detectable. Conversely, induction of IFN-gamma production and proliferation required triggering of at least 20-50% of TCRs. Taken together these results indicate that a single CTL can graduate different biological responses as a function of antigen concentration and that killing of the specific target does not necessarily result in full activation.

[Research progress of anti-influenza virus agents].

Influenza is a major threat to millions of people worldwide. Vaccines and antiviral agents are two main options available to reduce the impact of the influenza virus, while anti-influenza agents are the most effective means to prevent the transmission of the highly contagious virus and to treat the epidemics of disease. At present, four anti-influenza agents have been approved by the FDA for the treatment of influenza, including two M2 protein ion channel inhibitors-amantadine and rimantadine and two neuraminidase inhibitors-zanamivir and oseltamivir. Arbidol hydrochloride, launched in Russia, is a potent inhibitor of influenza virus, too. Neuraminidase inhibitors could be classified generally by structure into six different kinds: sialic acid derivatives, benzoic acid derivatives, cyclohexene derivatives, cyclopentane derivatives, pyrrolidine derivatives and natural products. In this paper, recent progress in the research of the action mechanisms and structure-activity relationships of these anti-influenza virus agents were reviewed.

Effect of Cytomegalovirus Recombinant Phosphoprotein 150 (pp150) on Maturation and Function of Murine Dendritic Cells: an In-Vitro Study.

BACKGROUND: Tegument protein pp150 of cytomegaloviruses (CMVs) plays a vital role in all stages of viral life cycle, representing the most important tegument protein candidate for HCMV treatment. However, the exact role of pp150 in immune regulation is yet to be elucidated. OBJECTIVE: To examine the effects of pp150 on the maturity and function of murine dendritic cells (DCs). METHODS: Maturity status (CD40, CD86, and MHC-II expression) and phagocytic capacity of DCs (dextran uptake assay) were characterized. Gene expression profiles of ROR-γ, GATA-3, T-bet, and FOXP-3 as well as the protein expression of INF-γ (Th1), IL-4 (Th2), IL-35 (Treg), IL-17A (Th17), IL-22, TNF-α, IL-6, and IL-2 were evaluated in T cells co-cultured with DCs. RESULTS: A significant increase in CD40, CD86, and CCR7 expression and a reduction in the phagocytosis rate were observed in pp150-stimulated DCs compared with unstimulated DCs. T cells co-cultured with stimulated DCs showed higher expressions of ROR-γ, IL-6, IL-2, IL-17A, IL-22, and TNF-α. CONCLUSION: Despite improvements in maturity status, pp150-stimulated DCs did not seem to be able to induce Th1 or Th2 immunity. In fact, Th17 and its mediators, IL-17A and IL-22, might be the main inflammatory factors involved in pp150-stimulated DC's mechanism of action. However, it is necessary to conduct further investigations to corroborate these observations.