The Human Cytomegalovirus Strain DB Activates Oncogenic Pathways in Mammary Epithelial Cells.

BACKGROUND: Human cytomegalovirus (HCMV) establishes a persistent life-long infection and increasing evidence indicates HCMV infection can modulate signaling pathways associated with oncogenesis. Breast milk is an important route of HCMV transmission in humans and we hypothesized that mammary epithelial cells could be one of the main cellular targets of HCMV infection. METHODS: The infectivity of primary human mammary epithelial cells (HMECs) was assessed following infection with the HCMV-DB strain, a clinical isolate with a marked macrophage-tropism. The impact of HCMV-DB infection on expression of p53 and retinoblastoma proteins, telomerase activity and oncogenic pathways (c-Myc, Akt, Ras, STAT3) was studied. Finally the transformation of HCMV-DB infected HMECs was evaluated using soft agar assay. CTH cells (CMV Transformed HMECs) were detected in prolonged cultures of infected HMECs. Tumor formation was observed in NOD/SCID Gamma (NSG) mice injected with CTH cells. Detection of long non coding RNA4.9 (lncRNA4.9) gene was assessed in CTH cells, tumors isolated from xenografted NSG mice and biopsies of patients with breast cancer using qualitative and quantitative PCR. RESULTS: We found that HCMV, especially a clinical strain named HCMV-DB, infects HMECs in vitro. The clinical strain HCMV-DB replicates productively in HMECs as evidenced by detection of early and late viral transcripts and proteins. Following infection of HMECs with HCMV-DB, we observed the inactivation of retinoblastoma and p53 proteins, the activation of telomerase activity, the activation of the proto-oncogenes c-Myc and Ras, the activation of Akt and STAT3, and the upregulation of cyclin D1 and Ki67 antigen. Colony formation was observed in soft agar seeded with HCMV-DB-infected HMECs. Prolonged culture of infected HMECs resulted in the development of clusters of spheroid cells that we called CTH cells (CMV Transformed HMECs). CTH cells when injected in NOD/SCID Gamma (NSG) mice resulted in the development of tumors. We detected in CTH cells the presence of a HCMV signature corresponding to a sequence of the long noncoding RNA4.9 (lncRNA4.9) gene. We also found the presence of the HCMV lncRNA4.9 sequence in tumors isolated from xenografted NSG mice injected with CTH cells and in biopsies of patients with breast cancer using qualitative and quantitative PCR. CONCLUSIONS: Our data indicate that key molecular pathways involved in oncogenesis are activated in HCMV-DB-infected HMECs that ultimately results in the transformation of HMECs in vitro with the appearance of CMV-transformed HMECs (CTH cells) in culture. CTH cells display a HCMV signature corresponding to a lncRNA4.9 genomic sequence and give rise to fast growing triple-negative tumors in NSG mice. A similar lncRNA4.9 genomic sequence was detected in tumor biopsies of patients with breast cancer.

Tumor control by human cytomegalovirus in a murine model of hepatocellular carcinoma.

Although viruses can cause cancer, other studies reported the regression of human tumors upon viral infections. We investigated the cytoreductive potential of human cytomegalovirus (HCMV) in a murine model of human hepatocellular carcinoma (HCC) in severe-immunodeficient mice. Infection of HepG2 cells with HCMV resulted in the absence of tumor or in a limited tumor growth following injection of cells subcutaneously. By contrast all mice injected with uninfected HepG2 cells and with HepG2 cells infected with UV-treated HCMV did develop tumors without any significant restriction. Analysis of tumors indicated that in mice injected with HCMV-infected-HepG2 cells, but not in controls, a restricted cellular proliferation was observed parallel to a limited activation of the STAT3-cyclin D1 axis, decreased formation of colonies in soft agar, and activation of the intrinsic apoptotic pathway. We conclude that HCMV can provide antitumoral effects in a murine model of HCC which requires replicative virus at some stages that results in limitation of tumor cell proliferation and enhanced apoptosis mediated through the intrinsic caspase pathway.

Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line.

Akt signaling plays a central role in many biological processes, which are key players in human immunodeficiency virus 1 (HIV-1) pathogenesis. We found that Akt interacts with HIV-1 Nef protein. In primary T cells treated with exogenous Nef or acutely infected with Nef-expressing HIV-1 in vitro, Akt became phosphorylated on serine(473) and threonine(308). In vitro, Akt activation mediated by Nef in T-cells was blocked by HIV protease inhibitors (PI), but not by reverse transcriptase inhibitors (RTI). Ex vivo, we found that the Akt pathway is hyperactivated in peripheral blood lymphocytes (PBLs) from cART naïve HIV-1-infected patients. PBLs isolated from PI-treated patients, but not from RTI-treated patients, exhibited decreased Akt activation, T-cell proliferation and IL-2 production. We found that PI but not RTI can block HIV-1 reactivation in latently infected J-Lat lymphoid cells stimulated with various stimuli. Using luciferase measurement, we further confirmed that Nef-mediated reactivation of HIV-1 from latency in 1G5 cells was blocked by PI parallel to decreased Akt activation. Our results indicate that PI-mediated blockade of Akt activation could impact the HIV-1 reservoir and support the need to further assess the therapeutic use of HIV-1 PI in order to curtail latently infected cells in HIV-1-infected patients.

Targeting TNF-Alpha in HIV-1 Infection.

Highly active antiretroviral therapy (HAART) has dramatically extended the lifespan and quality of life of individuals infected with human immunodeficiency virus type 1 (HIV-1). HAART comprises of a cocktail of various pharmacological inhibitors which interfere with almost every stages of HIV-1 life cycle. However, constant application of drugs often results in the evolution of hostpathogen relationship resulting in the emergence of drug resistant viral strains. Drug resistant HIV-1 is a potent threat for the humankind. Therefore, there is a constant need to search for novel therapeutic molecules. HIV-1 infection results in the depletion of CD4+/CD8+T cells and alters the cytokine network in the infected individuals. Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, plays a critical role in HIV-1 pathogenesis. HIV-1 utilizes the TNF-alpha signaling pathway for expanding its reservoir. Several HIV-1 proteins mimic and regulate the TNF-alpha signaling pathway. TNF-alpha inhibitors have been used in several inflammatory pathologies with success to some extent. In the present mini review we will discuss the role of TNF-alpha in HIV-1 pathogenesis. Furthermore we will evaluate the TNF-alpha inhibitors as an additional therapeutic option for HIV-1 infection.

Dysregulated serum IL-23 and SIRT1 activity in peripheral blood mononuclear cells of patients with rheumatoid arthritis.

Sirtuin 1 (Sirt1) is a class III histone deacetylase (HDAC) that modulates gene expression and is involved in the regulation of proinflammatory cytokines. Interleukin-23 (IL-23) is produced by activated macrophages and dendritic cells and could fuel the progression of rheumatoid arthritis (RA). The goal of our study was to evaluate serum IL-23 levels and both Sirt1 activity and expression in peripheral blood mononuclear cells (PBMCs) in patients with RA compared to healthy controls (HC) and to determine the relationship between Sirt1 activity/expression and IL-23 levels. We assessed apoptosis in PBMCs of RA patients and its association with Sirt1 expression and serum IL-23. Serum IL-23 levels were increased in RA patients in comparison with controls. We found a positive correlation between the levels of serum IL-23 and serum IL-6 in RA patients. Decreased cytoplasmic Sirt1 activity was observed in RA patients with severe disease compared to HC. The expression of Sirt1 protein was significantly decreased in PBMCs of RA patients compared to HC using western blotting. Serum IL-23 levels correlated positively with the cytoplasmic Sirt1 activity in RA patients. Apoptosis rate of PBMCs isolated from RA patients was increased compared to HC and correlated negatively with the expression of Sirt1 protein and serum IL-23 levels. Levels of serum IL-23 and Sirt1 activity and expression were disturbed in RA parallel to increased PBMC apoptosis. Our findings might provide the rationale for the development of new therapeutic approaches in RA.

Type I interferons contribute to experimental cerebral malaria development in response to sporozoite or blood-stage Plasmodium berghei ANKA.

Cerebral malaria is a severe complication of Plasmodium falciparum infection. Although T-cell activation and type II IFN-γ are required for Plasmodium berghei ANKA (PbA)-induced murine experimental cerebral malaria (ECM), the role of type I IFN-α/ß in ECM development remains unclear. Here, we address the role of the IFN-α/ß pathway in ECM devel-opment in response to hepatic or blood-stage PbA infection, using mice deficient for types I or II IFN receptors. While IFN-γR1⁻/⁻ mice were fully resistant, IFNAR1⁻/⁻ mice showed delayed and partial protection to ECM after PbA infection. ECM resistance in IFN-γR1⁻/⁻ mice correlated with unaltered cerebral microcirculation and absence of ischemia, while WT and IFNAR1⁻/⁻ mice developed distinct microvascular pathologies. ECM resistance appeared to be independent of parasitemia. Instead, key mediators of ECM were attenuated in the absence of IFNAR1, including PbA-induced brain sequestration of CXCR3⁺-activated CD8⁺ T cells. This was associated with reduced expression of Granzyme B, IFN-γ, IL-12Rß2, and T-cell-attracting chemokines CXCL9 and CXCL10 in IFNAR1⁻/⁻ mice, more so in the absence of IFN-γR1. Therefore, the type I IFN-α/ß receptor pathway contributes to brain T-cell responses and microvascular pathology, although it is not as essential as IFN-γ for the development of cerebral malaria upon hepatic or blood-stage PbA infection.