microRNA Response to Listeria monocytogenes Infection in Epithelial Cells

microRNAs represent a family of very small non-coding RNAs that control several physiologic and pathologic processes, including host immune response and cancer by antagonizing a number of target mRNAs. There is limited knowledge about cell expression and the regulatory role of microRNAs following bacterial infections. We investigated whether infection with a Gram-positive bacterium leads to altered expression of microRNAs involved in the host cell response in epithelial cells. Caco-2 cells were infected with Listeria monocytogenes EGD-e, a mutant strain (ΔinlAB or Δhly) or incubated with purified listeriolysin (LLO). Total RNA was isolated and microRNA and target gene expression was compared to the expression in non-infected cells using microRNA microarrays and qRT-PCR. We identified and validated five microRNAs (miR- 146b, miR-16, let-7a1, miR-145 and miR-155) that were significantly deregulated following listerial infection. We show that expression patterns of particular microRNAs strongly depend on pathogen localization and the presence of bacterial effector proteins. Strikingly, miR-155 which was shown to have an important role in inflammatory responses during infection was induced by wild-type bacteria, by LLO-deficient bacteria and following incubation with purified LLO. It was downregulated following ΔinlAB infection indicating a new potent role for internalins in listerial pathogenicity and miRNA regulation. Concurrently, we observed differences in target transcript expression of the investigated miRNAs. We provide first evidence that L. monocytogenes infection leads to deregulation of a set of microRNAs with important roles in host response. Distinct microRNA expression depends on both LLO and pathogen localization

Isolation, Transfection, and Culture of Primary Human Monocytes

Human immunodeficiency virus (HIV) remains a major health concern despite the introduction of combined antiretroviral therapy (cART) in the mid-1990s. While antiretroviral therapy efficiently lowers systemic viral load and restores normal CD4 T cell counts, it does not reconstitute a completely functional immune system. A dysfunctional immune system in HIV-infected individuals undergoing cART may be characterized by immune activation, early aging of immune cells, or persistent inflammation. These conditions, along with comorbid factors associated with HIV infection, add complexity to the disease, which cannot be easily reproduced in cellular and animal models. To investigate the molecular events underlying immune dysfunction in these patients, a system to culture and manipulate human primary monocytes in vitro is presented here. Specifically, the protocol allows for the culture and transfection of primary CD14 monocytes obtained from HIV-infected individuals undergoing cART as well as from HIV-negative controls. The method involves isolation, culture, and transfection of monocytes and monocyte-derived macrophages. While commercially available kits and reagents are employed, the protocol provides important tips and optimized conditions for successful adherence and transfection of monocytes with miRNA mimics and inhibitors as well as with siRNAs

Role of Histamine and Related Signaling in Kaposi’s Sarcoma-Associated Herpesvirus Pathogenesis and Oncogenesis

Although Kaposi’s sarcoma-associated herpesvirus (KSHV) has been reported to cause several human cancers including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), the mechanisms of KSHV-induced tumorigenesis, especially virus–host interaction network, are still not completely understood, which therefore hinders the development of effective therapies. Histamine, together with its receptors, plays an important role in various allergic diseases by regulating different inflammation and immune responses. Our previous data showed that antagonists targeting histamine receptors effectively repressed KSHV lytic replication. In the current study, we determined that histamine treatment increased cell proliferation and anchorage-independent growth abilities of KSHV-infected cells. Furthermore, histamine treatment affected the expression of some inflammatory factors from KSHV-infected cells. For clinical relevance, several histamine receptors were highly expressed in AIDS-KS tissues when compared to normal skin tissues. We determined that histamine treatment promoted KSHV-infected lymphoma progression in immunocompromised mice models. Therefore, besides viral replication, our data indicate that the histamine and related signaling are also involved in other functions of KSHV pathogenesis and oncogenesis

Anti-glioblastoma effects of phenolic variants of benzoylphenoxyacetamide (BPA) with high potential for blood brain barrier penetration

Glioblastomas are the most aggressive brain tumors for which therapeutic options are limited. Current therapies against glioblastoma include surgical resection, followed by radiotherapy plus concomitant treatment and maintenance with temozolomide (TMZ), however, these standard therapies are often ineffective, and average survival time for glioblastoma patients is between 12 and 18 months. We have previously reported a strong anti-glioblastoma activity of several metabolic compounds, which were synthetized based compounds, which were synthetized based on the chemical structure of a common lipid-lowering drug, fenofibrate, and share a general molecular skeleton of benzoylphenoxyacetamide (BPA). Extensive computational analyses of phenol and naphthol moieties added to the BPA skeleton were performed in this study with the objective of selecting new BPA variants for subsequent compound preparation and anti-glioblastoma testing. Initially, 81 structural variations were considered and their physical properties such as solubility (logS), blood–brain partitioning (logBB), and probability of entering the CNS calculated by the Central Nervous System—Multiparameter Optimization (MPO-CNS) algorithm were evaluated. From this initial list, 18 compounds were further evaluated for anti-glioblastoma activity in vitro. Nine compounds demonstrated desirable glioblastoma cell toxicity in cell culture, and two of them, HR51, and HR59 demonstrated significantly improved capability of crossing the model blood–brain-barrier (BBB) composed of endothelial cells, astrocytes and pericytes

Echinomycin as a promising therapeutic agent against KSHV-related malignancies

Abstract Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of several human cancers, including Kaposi’s sarcoma (KS) and primary effusion lymphoma (PEL), which preferentially arise in immunocompromised patients while lack of effective therapeutic options. Oncoproteins Myc and hypoxia-inducible factor-1α (HIF1α) have been found closely related to KSHV infection, replication and oncogenesis. However, the strategies of dual targeting these two oncoproteins have never been developed and tested for treatments of KSHV-related malignancies. In the current study, we report that treatment of echinomycin dramatically regresses cell growth both in vitro-cultured KSHV + tumor cells and in vivo KS or PEL xenograft mice models, through simultaneously inhibiting Myc and HIF1α expression. Echinomycin treatment also induces viral lytic gene expression whereas not increasing infectious virions production from KSHV + tumor cells. Our comparative transcriptomic analysis has identified a bunch of new Echinomycin-regulated, Myc- and HIF1α-related genes contributed to KSHV pathogenesis, including KDM4B and Tau, which are required for the survival of KSHV + tumor cells with functional validation. These data together reveal that dual targeting Myc and HIF1α such as using Echinomycin may represent a new and promising option for treatments of these virus-associated malignancies

KSHV miRNAs Decrease Expression of Lytic Genes in Latently Infected PEL and Endothelial Cells by Targeting Host Transcription Factors

Kaposi’s sarcoma-associated herpesvirus (KSHV) microRNAs are encoded in the latency-associated region. Knockdown of KSHV miR-K12-3 and miR-K12-11 increased expression of lytic genes in BC-3 cells, and increased virus production from latently infected BCBL-1 cells. Furthermore, iSLK cells infected with miR-K12-3 and miR-K12-11 deletion mutant viruses displayed increased spontaneous reactivation and were more sensitive to inducers of reactivation than cells infected with wild type KSHV. Predicted binding sites for miR-K12-3 and miR-K12-11 were found in the 3’UTRs of the cellular transcription factors MYB, Ets-1, and C/EBPα, which activate RTA, the KSHV replication and transcription activator. Targeting of MYB by miR-K12-11 was confirmed by cloning the MYB 3’UTR downstream from the luciferase reporter. Knockdown of miR‑K12-11 resulted in increased levels of MYB transcript, and knockdown of miR-K12-3 increased both C/EBPα and Ets-1 transcripts. Thus, miR-K12-11 and miR-K12-3 contribute to maintenance of latency by decreasing RTA expression indirectly, presumably via down‑regulation of MYB, C/EBPα and Ets-1, and possibly other host transcription factors

A Toolbox for Herpesvirus miRNA Research: Construction of a Complete Set of KSHV miRNA Deletion Mutants

Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes 12 viral microRNAs (miRNAs) that are expressed during latency. Research into KSHV miRNA function has suffered from a lack of genetic systems to study viral miRNA mutations in the context of the viral genome. We used the Escherichia coli Red recombination system together with a new bacmid background, BAC16, to create mutants for all known KSHV miRNAs. The specific miRNA deletions or mutations and the integrity of the bacmids have been strictly quality controlled using PCR, restriction digestion, and sequencing. In addition, stable viral producer cell lines based on iSLK cells have been created for wildtype KSHV, for 12 individual miRNA knock-out mutants (ΔmiR-K12-1 through -12), and for mutants deleted for 10 of 12 (ΔmiR-cluster) or all 12 miRNAs (ΔmiR-all). NGS, in combination with SureSelect technology, was employed to sequence the entire latent genome within all producer cell lines. qPCR assays were used to verify the expression of the remaining viral miRNAs in a subset of mutants. Induction of the lytic cycle leads to efficient production of progeny viruses that have been used to infect endothelial cells. Wt BAC16 and miR mutant iSLK producer cell lines are now available to the research community