Macrocyclic lactones inhibit nasopharyngeal carcinoma cells proliferation through PAK1 inhibition and reduce in vivo tumor growth.

PURPOSE: The Epstein-Barr virus (EBV)-associated cancer nasopharyngeal carcinoma (NPC) is rare in Europe and North America but is a real public health problem in some regions of the world, such as southern Asia, North Africa, and for Inuit populations. Due to the anatomy and location of the nasopharynx, surgery is rarely used to treat primary NPC cancers. Treatment by radiotherapy, combined or not with chemotherapy, are efficient for primary tumors but often do not protect against fatal relapses or metastases. METHODS: Search for new therapeutic molecules through high content screening lead to the identification of Ivermectin (IVM) as a promising drug. IVM is a US Food and Drug Administration-approved macrocyclic lactone widely used as anthelmintic and insecticidal agent that has also shown protective effects against cancers. RESULTS: We show here that IVM has cytotoxic activity in vitro against NPC cells, in which it reduces MAPKs pathway activation through the inhibition PAK-1 activity. Moreover, all macrocyclic lactones tested and a PAK1 inhibitor are cytotoxic in vitro for EBV-positive and EBV-negative NPC tumor cells. We have also shown that IVM intraperitoneal repeated injections, at US Food and Drug Administration-approved doses, have no significant toxicity and decrease NPC subcutaneous tumors development in nude mice. CONCLUSION: Macrocyclic lactones appear as promising molecules against NPC targeting PAK-1 with no detectable adverse effect.

Real-Time Visualization and Quantification of Human Cytomegalovirus Replication in Living Cells Using the ANCHOR DNA Labeling Technology.

Human cytomegalovirus (HCMV) induces latent lifelong infections in all human populations. Between 30% and nearly 100% of individuals are affected depending on the geographic area and socioeconomic conditions. The biology of the virus is difficult to explore due to its extreme sophistication and the lack of a pertinent animal model. Here, we present the first application of the ANCHOR DNA labeling system to a herpesvirus, enabling real-time imaging and direct monitoring of HCMV infection and replication in living human cells. The ANCHOR system is composed of a protein (OR) that specifically binds to a short, nonrepetitive DNA target sequence (ANCH) and spreads onto neighboring sequences by protein oligomerization. When the OR protein is fused to green fluorescent protein (GFP), its accumulation results in a site-specific fluorescent focus. We created a recombinant ANCHOR-HCMV harboring an ANCH target sequence and the gene encoding the cognate OR-GFP fusion protein. Infection of permissive cells with ANCHOR-HCMV enables visualization of nearly the complete viral cycle until cell fragmentation and death. Quantitative analysis of infection kinetics and of viral DNA replication revealed cell-type-specific HCMV behavior and sensitivity to inhibitors. Our results show that the ANCHOR technology provides an efficient tool for the study of complex DNA viruses and a new, highly promising system for the development of innovative biotechnology applications.IMPORTANCE The ANCHOR technology is currently the most powerful tool to follow and quantify the replication of HCMV in living cells and to gain new insights into its biology. The technology is applicable to virtually any DNA virus or viruses presenting a double-stranded DNA (dsDNA) phase, paving the way to imaging infection in various cell lines, or even in animal models, and opening fascinating fundamental and applied prospects. Associated with high-content automated microscopy, the technology permitted rapid, robust, and precise determination of ganciclovir 50% and 90% inhibitory concentrations (IC50 and IC90) on HCMV replication, with minimal hands-on time investment. To search for new antiviral activities, the experiment is easy to upgrade toward efficient and cost-effective screening of large chemical libraries. Simple infection of permissive cells with ANCHOR viruses in the presence of a compound of interest even provides a first estimation of the stage of the viral cycle the molecule is acting upon.

Melanoma-expressed CD70 is involved in invasion and metastasis.

BACKGROUND: CD70 is a costimulatory molecule of the tumour necrosis factor family expressed in activated immune cells and some solid tumours. In lymphocytes CD70 triggers T cell-mediated cytotoxicity and mitogen-activated protein kinase phosphorylation. METHODS: We evaluated the expression of CD70 in biopsies and melanoma cell lines. Using melanoma cell lines positive or not for CD70, we analysed CD70 function on melanoma progression. RESULTS: We report CD70 expression in human melanoma cell lines and tumour cells from melanoma biopsies. This expression was observed in 95% of primary melanomas but only 37% of metastases. Both monomeric and trimeric forms of CD70 were detected in tumour cell membrane fractions, whereas cytoplasmic fractions contained almost exclusively monomeric CD70. In vitro and in vivo experiments demonstrated that CD70 expression inhibited melanoma cell migration, invasion and pulmonary metastasis implantation independently of the tumour immune microenvironment. Increasing the levels of the trimeric form of CD70 through monoclonal antibody binding led to an increase in CD70+ melanoma cell invasiveness through MAPK pathway activation, RhoE overexpression, ROCK1 and MYPT1 phosphorylation decrease, and stress fibres and focal adhesions disappearance. CONCLUSIONS: Our results describe a new non-immunological function of melanoma-expressed CD70, which involves melanoma invasiveness through MAPK pathway, RhoE and cytoskeletal modulation.

Synthesis and Antiviral Activity Evaluation of Nitroporphyrins and Nitrocorroles as Potential Agents against Human Cytomegalovirus Infection.

Different nitroporphyrinoid derivatives were synthesized and studied as potential agents against human Cytomegalovirus. Interestingly, two nitrocorroles display strong activity against human Cytomegalovirus with IC 50 < 0.5 µM. These compounds also possess antiproliferative activities without detected in vivo toxicity. Therefore, nitrocorroles appear for the first time as potential active compounds that can be applied in human health.

Epstein-Barr virus late gene transcription depends on the assembly of a virus-specific preinitiation complex.

UNLABELLED: During their productive cycle, herpesviruses exhibit a strictly regulated temporal cascade of gene expression that has three general stages: immediate early (IE), early (E), and late (L). Promoter complexity differs strikingly between IE/E genes and L genes. IE and E promoters contain cis-regulating sequences upstream of a TATA box, whereas L promoters comprise a unique cis element. In the case of the gammaherpesviruses, this element is usually a TATT motif found in the position where the consensus TATA box of eukaryotic promoters is typically found. Epstein-Barr virus (EBV) encodes a protein, called BcRF1, which has structural homology with the TATA-binding protein and interacts specifically with the TATT box. However, although necessary for the expression of the L genes, BcRF1 is not sufficient, suggesting that other viral proteins are also required. Here, we present the identification and characterization of a viral protein complex necessary and sufficient for the expression of the late viral genes. This viral complex is composed of five different proteins in addition to BcRF1 and interacts with cellular RNA polymerase II. During the viral productive cycle, this complex, which we call the vPIC (for viral preinitiation complex), works in concert with the viral DNA replication machinery to activate expression of the late viral genes. The EBV vPIC components have homologs in beta- and gammaherpesviruses but not in alphaherpesviruses. Our results not only reveal that beta- and gammaherpesviruses encode their own transcription preinitiation complex responsible for the expression of the late viral genes but also indicate the close evolutionary history of these viruses. IMPORTANCE: Control of late gene transcription in DNA viruses is a major unsolved question in virology. In eukaryotes, the first step in transcriptional activation is the formation of a permissive chromatin, which allows assembly of the preinitiation complex (PIC) at the core promoter. Fixation of the TATA box-binding protein (TBP) is a key rate-limiting step in this process. This study provides evidence that EBV encodes a complex composed of six proteins necessary for the expression of the late viral genes. This complex is formed around a viral TBP-like protein and interacts with cellular RNA polymerase II, suggesting that it is directly involved in the assembly of a virus-specific PIC (vPIC).

Statins Reduce Melanoma Development and Metastasis through MICA Overexpression.

Survival of melanoma patients after metastases detection remains short. Several clinical trials have shown moderate efficiency in improving patient survival, and the search for pharmacological agents to enhance the immune response and reduce melanoma metastases is still necessary. Statins block the mevalonate pathway, which leads to decreases in GTPase isoprenylation and activity, particularly those of the Ras superfamily. They are widely used as hypocholesterolemic agents in cardiovascular diseases and several studies have shown that they also have protective effects against cancers. Furthermore, we have previously demonstrated that treatment of melanoma cells with inhibitors of the mevalonate pathway, such as statins, favor the development of specific adaptive immune responses against these tumors. In the present study, we tested statin impact on the innate immune response against human metastatic melanoma cells. Our data shows that treatment of two human melanoma cell lines with statins induced a weak but significant increase of MHC class I Chain-related protein A (MICA) membrane expression. Peroxisome Proliferator-Activated Receptor gamma is involved in this statin-induced MICA overexpression, which is independent of Ras and Rho GTPase signaling pathways. Interestingly, this MICA overexpression makes melanoma cells more sensitive to in vitro lysis by NK cells. The impact of statin treatment on in vivo development of melanoma tumors and metastases was investigated in nude mice, because murine NK cells, which express NKG2D receptors, are able to recognize and kill human tumor cells expressing MICA. The results demonstrated that both local tumor growth and pulmonary metastases were strongly inhibited in nude mice injected with statin-treated melanoma cells. These results suggest that statins could be effective in melanoma immunotherapy treatments.

The Epstein-Barr virus BcRF1 gene product is a TBP-like protein with an essential role in late gene expression.

That the expression of late genes is coupled to viral genome replication is well established for all herpesviruses, but the exact mechanisms of their regulation, especially by viral proteins, are poorly understood. Here, we report the identification of the Epstein-Barr virus (EBV) early protein BcRF1 as a viral factor crucial for the activation of late gene transcription following viral DNA replication during the productive cycle. In order to study the function of the BcRF1 protein, we constructed a recombinant EBV lacking this gene. In HEK293 cells, this recombinant virus underwent normal DNA replication during the productive cycle but failed to express high levels of late gene transcripts or proteins, resulting in a nonproductive infection. Interestingly, a TATT motif is present in the promoter of most EBV late genes, at the position of the TATA box. We show here that BcRF1 forms a complex with the TATT motif and that this interaction is required for activation of late viral gene expression. Moreover, our results suggest that BcRF1 acts via interaction with other viral proteins.

Paracrine inhibition of GM-CSF signaling by human cytomegalovirus in monocytes differentiating to dendritic cells.

A primary HCMV infection or virus reactivation may cause severe disease in hosts with a deficient immune system. The virus can disturb both innate and adaptive immunity by targeting dendritic cell (DC) functions. Monocytes, the precursors of DCs in vivo (MoDCs), are the primary targets of HCMV; they can also harbor latent virus. The DCs generated from infected monocytes (CMV-MoDCs) have an altered phenotype and functional defects. We have shown that CMV-MoDCs do not secrete IL-12 in response to lipopolysaccharide stimulation, cannot ingest dead cells, induce T(H)1 differentiation, or the proliferation of naive allogeneic CD4(+) T cells. We found that the GM-CSF signaling in an entire population of CMV-MoDCs was impaired, although only half of the cells were productively infected, and that IL-6 secretion and suppressors of cytokine signaling 3 induction contributed to this bystander effect. We also showed that MoDCs derived ex vivo from monocytes of viremic patients had the same altered phenotype as CMV-MoDCs, including decreased STAT5 phosphorylation, indicating defective GM-CSF signaling. We have thus described a new mechanism of HCMV-induced immunosupression, indicated how infection may disturb both GM-CSF-dependent physiologic processes and proposed GM-CSF-based therapeutic approaches.

Knocking down Cav1 calcium channels implicated in Th2 cell activation prevents experimental asthma.

RATIONALE: Th2 cells orchestrate allergic asthma and the cytokines they produce (IL-4, IL-5, and IL-13) are deleterious in allergy. Therefore, it is important to identify key signaling molecules expressed by Th2 cells that are essential for their function. We have previously shown that dihydropyridines selectively modulate Th2 cell functions. OBJECTIVES: Because dihydropyridines bind to and modulate voltage-dependent calcium (Ca(v)1) channel in excitable cells, we aimed at showing that Th2 cells selectively express functional Ca(v)1-related channels, the inhibition of which may prevent asthma. METHODS: We looked for Ca(v)1 channel expression in Th2 and Th1 cells by real-time polymerase chain reaction and Western blotting. We sequenced the isoforms expressed by Th2 cells and tested whether Ca(v)1 antisense oligodeoxynucleotides (Ca(v)1AS) affected Ca(2+) signaling and cytokine production. Finally, we tested the effect of Ca(v)1AS in the passive asthma model by injection of ovalbumin-specific Th2 cells transfected with Ca(v)1AS into BALB/c mice challenged with intranasal ovalbumin and in the active model of asthma by intranasal delivery of Ca(v)1AS together with soluble ovalbumin in BALB/c mice previously immunized with ovalbumin in alum. MEASUREMENTS AND MAIN RESULTS: We show that mouse Th2 but not Th1 cells expressed Ca(v)1.2 and Ca(v)1.3 channels. Th2 cells transfected with Ca(v)1AS had impaired Ca(2+) signaling and cytokine production, and lost their ability to induce airway inflammation on adoptive transfer. Furthermore, intranasal administration of Ca(v)1AS suppressed airway inflammation and hyperreactivity in an active model of asthma. CONCLUSIONS: These results indicate that Th2 cells selectively express Ca(v)1 channels that may be efficiently targeted in T lymphocytes to prevent experimental asthma.

Activation of peroxisome proliferator-activated receptor gamma by human cytomegalovirus for de novo replication impairs migration and invasiveness of cytotrophoblasts from early placentas.

Human cytomegalovirus (HCMV) contributes to pathogenic processes in immunosuppressed individuals, in fetuses, and in neonates. In the present report, by using reporter gene activation assays and confocal microscopy in the presence of a specific antagonist, we show for the first time that HCMV infection induces peroxisome proliferator-activated receptor gamma (PPARgamma) transcriptional activity in infected cells. We demonstrate that the PPARgamma antagonist dramatically impairs virus production and that the major immediate-early promoter contains PPAR response elements able to bind PPARgamma, as assessed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Due to the key role of PPARgamma in placentation and its specific trophoblast expression within the human placenta, we then provided evidence that by activating PPARgamma human cytomegalovirus dramatically impaired early human trophoblast migration and invasiveness, as assessed by using well-established in vitro models of invasive trophoblast, i.e., primary cultures of extravillous cytotrophoblasts (EVCT) isolated from first-trimester placentas and the EVCT-derived cell line HIPEC. Our data provide new clues to explain how early infection during pregnancy could impair implantation and placentation and therefore embryonic development.

No evidence of occult hepatitis C virus (HCV) infection in serum of HCV antibody-positive HCV RNA-negative kidney-transplant patients.

Persistence of hepatitis C virus (HCV) in patients who cleared HCV is still debated. Occult HCV infection is described as the presence of detectable HCV RNA in liver or peripheral blood mononuclear cells (PBMCs) of patients with undetectable plasma HCV-RNA by conventional PCR assays. We have assessed the persistence of HCV in 26 kidney-transplant patients, followed up for 10.5 years (range 2-16), after HCV elimination while on hemodialysis. If HCV really did persist, arising out of the loss of immune control caused by institution of the regimen of immunosuppressive drugs after kidney transplantation, HCV reactivation would have taken place. Their immunosuppression relied on calcineurin inhibitors (100%), and/or steroids (62%), and/or antimetabolites (94%). An induction therapy, given to 22 patients, relied on rabbit antithymocyte globulin (59%) or anti-IL2-receptor blockers (32%). All patients had undetectable HCV RNA as ascertained by several conventional tests. At the last follow-up, no residual HCV RNA was detected in the five liver biopsies, the 26 plasma, and in the 37 nonstimulated and 24 stimulated PBMCs tested with an ultrasensitive RT-PCR assay (detection limit, 2 IU/ml). No biochemical or virologic relapse was seen during follow-up. The absence of HCV relapse in formerly HCV-infected immunocompromised patients suggests the complete eradication of HCV after its elimination while on dialysis.

Calcium channel blocker prevents T helper type 2 cell-mediated airway inflammation.

RATIONALE: Ca(2+) signaling controls the production of T helper (Th) type 2 cytokines known to be deleterious in asthma. Recently, we showed that Ca(2+) signaling was dihydropyridine (DHP)-sensitive in Th2 lymphocytes and that the DHP derivate, nicardipine, used in the treatment of cardiovascular pathologies, prevents Th2-dependent B cell polyclonal activation. OBJECTIVES: We tested the effect of nicardipine in experimental allergic asthma. METHODS: BALB/c mice immunized with ovalbumin (OVA) in alum and challenged with intranasal OVA were treated with nicardipine once the Th2 response, or even airway inflammation, was induced. We also tested the effect of nicardipine in asthma induced by transferring OVA-specific Th2 cells in BALB/c mice exposed to intranasal OVA. We checked the impact of nicardipine on T-cell responses and airway inflammation. MEASUREMENTS AND MAIN RESULTS: Nicardipine inhibited in vitro Ca(2+) response in Th2 cells. In vivo, it impeded the development of Th2-mediated airway inflammation and reduced the capacity of lymphocytes from lung-draining lymph nodes to secrete Th2, but not Th1, cytokines. Nicardipine did not affect antigen presentation to CD4(+) T lymphocytes, nor the initial localization of Th2 cells into the lungs of mice exposed to intranasal OVA; however, it reduced the production of type 2 cytokines and the amplification of the Th2 response in mice with asthma. Conversely, nicardipine had no effect on Th1-mediated airway inflammation. CONCLUSIONS: Nicardipine improves experimental asthma by impairing Th2-dependent inflammation. This study could provide a rationale for developing drugs selectively targeting DHP receptors of Th2 lymphocytes, potentially beneficial in the treatment of asthma.

The Epstein-Barr virus oncoprotein LMP1 inhibits the activity of viral or cellular promoters without inducing cytostasis.

The Latent Membrane Protein 1 of the Epstein-Barr virus is required for human B lymphocyte immortalization and functions as a constitutively activated member of the TNF-receptor family, through recruitment of TRAFs and TRADD molecules on its Carboxy-terminal domain, leading to the activation of NF-kappaB and AP1 transcription factors. The formation of the signaling complexes requires LMP1 oligomerization, a role assigned to the membrane-spanning domains of the molecule. There is, however, increasing evidence that these membrane-spanning domains are not only confined to oligomerization but play a direct role in downregulation of promoter activity and cytostasis. Here, we describe a new inhibitory activity which is effective on viral or cellular promoters (even the endogenous ones), requires only membrane-spanning domains 3-4 or 5-6 and is neither associated with cytostasis nor with apoptosis.

Coexpression of CD40L and CD70 by semiallogenic tumor cells induces anti-tumor immunity.

The immune system is potentially qualified to detect and eliminate tumor cells, but various mechanisms developed by tumor cells allow tumor escape. Strategies selected to promote antitumor responses have included genetic modifications of tumor cells to induce expression of costimulatory molecules. Moreover, alloantigens can also act as strong enhancers of the immune response. In this work, we have associated the expression of two costimulatory members of the TNF superfamily, CD40L and CD70 along with an allogenic MHC Class I (H-2K(d)) molecule expression on melanoma cells (B16F10, H-2(b)) to favor the antitumor immune response. B16F10 tumor growth slows significantly when CD40L and CD70 are coexpressed by tumor cells and the association with the allogenic molecule (H-2K(d)) enhances this effect. Growth kinetics of mock and CD40L-CD70-H-2K(d)-expressing B16F10 tumors in immunocompetent versus nu/nu and beige mice suggested that CD8(+) T lymphocytes and NK cells were involved in this antitumor immunity. A delay in mock tumor growth was observed when CD40L-CD70-H-2K(d)-expressing B16F10 cells and mock tumor cells were injected simultaneously and contralaterally. It was also shown that in vivo immunization of immunocompetent mice with CD40L-CD70-H-2K(d) B16F10 tumor cells improved the generation of cytotoxic lymphocytes against the wild-type melanoma cells expressing the syngenic MHC Class I molecule H-2K(b) (B16K1). These observations lay a path for new immunotherapeutic trials using semiallogenic fibroblasts expressing costimulatory molecules and tumor-associated antigens.

Dihydropyridine receptors are selective markers of Th2 cells and can be targeted to prevent Th2-dependent immunopathological disorders.

Th1 cells that produce IFN-gamma are essential in the elimination of intracellular pathogens, and Th2 cells that synthetize IL-4 control the eradication of helminths. However, highly polarized Th1 or Th2 responses may be harmful and even lethal. Thus, the development of strategies to selectively down-modulate Th1 or Th2 responses is of therapeutic importance. Herein, we demonstrate that dihydropyridine receptors (DHPR) are expressed on Th2 and not on Th1 murine cells. By using selective agonists and antagonists of DHPR, we show that DHPR are involved in TCR-dependent calcium response in Th2 cells as well as in IL-4, IL-5, and IL-10 synthesis. Nicardipine, an inhibitor of DHPR, is beneficial in experimental models of Th2-dependent pathologies in rats. It strongly inhibits the Th2-mediated autoimmune glomerulonephritis induced by injecting Brown Norway (BN) rats with heavy metals. This drug also prevents the chronic graft vs host reaction induced by injecting CD4(+) T cells from BN rats into (LEW x BN)F(1) hybrids. By contrast, treatment with nicardipine has no effect on the Th1-dependent experimental autoimmune encephalomyelitis triggered in LEW rats immunized with myelin. These data indicate that 1) DHPR are a selective marker of Th2 cells, 2) these calcium channels contribute to calcium signaling in Th2 cells, and 3) blockers of these channels are beneficial in the treatment of Th2-mediated pathologies.

[Role of L-type calcium channels in the calcium response and interleukin 4 (IL-4) synthesis by Th2 lymphocytes]]. / Rôle de canaux de type L dans la réponse calcique et la production d'interleukine (IL-)4 par les lymphocytes Th2.

CD4+ T lymphocytes are divided in Th1 cells that produce interferon (IFN) gamma and Th2 cells that synthesize IL-4. These subsets may arise from a common precursor: a combination of IL-12 plus anti-IL-4 monoclonal antibody (mAb) drives Th1 cell differentiation while IL-4 plus anti-IFN gamma mAb favor Th2 cell development. TCR stimulation activates protein kinase C that controls a calcium entry through L type calcium channels in Th2 cells. L type calcium channels are induced during Th2 but not Th1 cell differentiation. In addition, L type calcium channel inhibitors may be successfully used in the treatment of an experimental model of Th2 cell-mediated immunopathology. Thus, this signaling pathway that characterizes Th2 cells can be a target for the treatment of Th2 diseases.