Pleiotropic roles of autophagy in stem cell-based therapies.

Stem cells (SCs) have been proven to possess regenerative and immunomodulatory properties and can be used to treat diseases that involve loss of cells due to tissue damage or inflammation. For this approach to succeed, SCs or their derivatives should be able to engraft in the target tissue at least for a short period of time. Unfortunately, once injected, therapeutic SCs will encounter a hostile environment, including hypoxia, lack of nutrients and stromal support, and cells may also be targeted and rejected by the immune system. Therefore, SC's stress-response mechanisms likely play a significant role in survival of injected cells and possibly contribute to their therapeutic efficacy. Autphagy, a stress-response pathway, is involved in many different cellular processes, such as survival during hypoxia and nutrient deprivation, cellular differentiation and de-differentiation, and it can also contribute to their immunovisibility by regulating antigen presentation and cytokine secretion. Autophagy machinery interacts with many proteins and signaling pathways that regulate SC properties, including PI3K/Akt, mammalian target of rapamycin (mTOR), Wnt, Hedgehog and Notch, and it is also involved in regulating intracellular reactive oxygen species (ROS) levels. In this review, we contend that autophagy is an important therapeutic target that can be used to improve the outcome of SC-based tissue repair and regeneration. Further research should reveal whether inhibition or stimulation of autophagy increases the therapeutic utility of SCs and it should also identify appropriate therapeutic regimens that can be applied in the clinic.

Activation of Nrf2 Signaling Augments Vesicular Stomatitis Virus Oncolysis via Autophagy-Driven Suppression of Antiviral Immunity.

Oncolytic viruses (OVs) offer a promising therapeutic approach to treat multiple types of cancer. In this study, we show that the manipulation of the antioxidant network via transcription factor Nrf2 augments vesicular stomatitis virus Δ51 (VSVΔ51) replication and sensitizes cancer cells to viral oncolysis. Activation of Nrf2 signaling by the antioxidant compound sulforaphane (SFN) leads to enhanced VSVΔ51 spread in OV-resistant cancer cells and improves the therapeutic outcome in different murine syngeneic and xenograft tumor models. Chemoresistant A549 lung cancer cells that display constitutive dominant hyperactivation of Nrf2 signaling are particularly vulnerable to VSVΔ51 oncolysis. Mechanistically, enhanced Nrf2 signaling stimulated viral replication in cancer cells and disrupted the type I IFN response via increased autophagy. This study reveals a previously unappreciated role for Nrf2 in the regulation of autophagy and the innate antiviral response that complements the therapeutic potential of VSV-directed oncolysis against multiple types of OV-resistant or chemoresistant cancer.

Enhanced Influenza Virus-Like Particle Vaccination with a Structurally Optimized RIG-I Agonist as Adjuvant.

UNLABELLED: The molecular interaction between viral RNA and the cytosolic sensor RIG-I represents the initial trigger in the development of an effective immune response against infection with RNA viruses, resulting in innate immune activation and subsequent induction of adaptive responses. In the present study, the adjuvant properties of a sequence-optimized 5'-triphosphate-containing RNA (5'pppRNA) RIG-I agonist (termed M8) were examined in combination with influenza virus-like particles (VLP) (M8-VLP) expressing H5N1 influenza virus hemagglutinin (HA) and neuraminidase (NA) as immunogens. In combination with VLP, M8 increased the antibody response to VLP immunization, provided VLP antigen sparing, and protected mice from a lethal challenge with H5N1 influenza virus. M8-VLP immunization also led to long-term protective responses against influenza virus infection in mice. M8 adjuvantation of VLP increased endpoint and antibody titers and inhibited influenza virus replication in lungs compared with approved or experimental adjuvants alum, AddaVax, and poly(I·C). Uniquely, immunization with M8-VLP stimulated a TH1-biased CD4 T cell response, as determined by increased TH1 cytokine levels in CD4 T cells and increased IgG2 levels in sera. Collectively, these data demonstrate that a sequence-optimized, RIG-I-specific agonist is a potent adjuvant that can be utilized to increase the efficacy of influenza VLP vaccination and dramatically improve humoral and cellular mediated protective responses against influenza virus challenge. IMPORTANCE: The development of novel adjuvants to increase vaccine immunogenicity is an important goal that seeks to improve vaccine efficacy and ultimately prevent infections that endanger human health. This proof-of-principle study investigated the adjuvant properties of a sequence-optimized 5'pppRNA agonist (M8) with enhanced capacity to stimulate antiviral and inflammatory gene networks using influenza virus-like particles (VLP) expressing HA and NA as immunogens. Vaccination with VLP in combination with M8 increased anti-influenza virus antibody titers and protected animals from lethal influenza virus challenge, highlighting the potential clinical use of M8 as an adjuvant in vaccine development. Altogether, the results describe a novel immunostimulatory agonist targeted to the cytosolic RIG-I sensor as an attractive vaccine adjuvant candidate that can be used to increase vaccine efficacy, a pressing issue in children and the elderly population.

Sequence-Specific Modifications Enhance the Broad-Spectrum Antiviral Response Activated by RIG-I Agonists.

UNLABELLED: The cytosolic RIG-I (retinoic acid-inducible gene I) receptor plays a pivotal role in the initiation of the immune response against RNA virus infection by recognizing short 5'-triphosphate (5'ppp)-containing viral RNA and activating the host antiviral innate response. In the present study, we generated novel 5'ppp RIG-I agonists of varieous lengths, structures, and sequences and evaluated the generation of the antiviral and inflammatory responses in human epithelial A549 cells, human innate immune primary cells, and murine models of influenza and chikungunya viral pathogenesis. A 99-nucleotide, uridine-rich hairpin 5'pppRNA termed M8 stimulated an extensive and robust interferon response compared to other modified 5'pppRNA structures, RIG-I aptamers, or poly(I·C). Interestingly, manipulation of the primary RNA sequence alone was sufficient to modulate antiviral activity and inflammatory response, in a manner dependent exclusively on RIG-I and independent of MDA5 and TLR3. Both prophylactic and therapeutic administration of M8 effectively inhibited influenza virus and dengue virus replication in vitro. Furthermore, multiple strains of influenza virus that were resistant to oseltamivir, an FDA-approved therapeutic treatment for influenza, were highly sensitive to inhibition by M8. Finally, prophylactic M8 treatment in vivo prolonged survival and reduced lung viral titers of mice challenged with influenza virus, as well as reducing chikungunya virus-associated foot swelling and viral load. Altogether, these results demonstrate that 5'pppRNA can be rationally designed to achieve a maximal RIG-I-mediated protective antiviral response against human-pathogenic RNA viruses. IMPORTANCE: The development of novel therapeutics to treat human-pathogenic RNA viral infections is an important goal to reduce spread of infection and to improve human health and safety. This study investigated the design of an RNA agonist with enhanced antiviral and inflammatory properties against influenza, dengue, and chikungunya viruses. A novel, sequence-dependent, uridine-rich RIG-I agonist generated a protective antiviral response in vitro and in vivo and was effective at concentrations 100-fold lower than prototype sequences or other RNA agonists, highlighting the robust activity and potential clinical use of the 5'pppRNA against RNA virus infection. Altogether, the results identify a novel, sequence-specific RIG-I agonist as an attractive therapeutic candidate for the treatment of a broad range of RNA viruses, a pressing issue in which a need for new and more effective options persists.

Cellular oxidative stress response controls the antiviral and apoptotic programs in dengue virus-infected dendritic cells.

Dengue virus (DENV) is a re-emerging arthropod borne flavivirus that infects more than 300 million people worldwide, leading to 50,000 deaths annually. Because dendritic cells (DC) in the skin and blood are the first target cells for DENV, we sought to investigate the early molecular events involved in the host response to the virus in primary human monocyte-derived dendritic cells (Mo-DC). Using a genome-wide transcriptome analysis of DENV2-infected human Mo-DC, three major responses were identified within hours of infection - the activation of IRF3/7/STAT1 and NF-κB-driven antiviral and inflammatory networks, as well as the stimulation of an oxidative stress response that included the stimulation of an Nrf2-dependent antioxidant gene transcriptional program. DENV2 infection resulted in the intracellular accumulation of reactive oxygen species (ROS) that was dependent on NADPH-oxidase (NOX). A decrease in ROS levels through chemical or genetic inhibition of the NOX-complex dampened the innate immune responses to DENV infection and facilitated DENV replication; ROS were also essential in driving mitochondrial apoptosis in infected Mo-DC. In addition to stimulating innate immune responses to DENV, increased ROS led to the activation of bystander Mo-DC which up-regulated maturation/activation markers and were less susceptible to viral replication. We have identified a critical role for the transcription factor Nrf2 in limiting both antiviral and cell death responses to the virus by feedback modulation of oxidative stress. Silencing of Nrf2 by RNA interference increased DENV-associated immune and apoptotic responses. Taken together, these data demonstrate that the level of oxidative stress is critical to the control of both antiviral and apoptotic programs in DENV-infected human Mo-DC and highlight the importance of redox homeostasis in the outcome of DENV infection.

The intersection between viral oncolysis, drug resistance, and autophagy.

Resistance to both cytotoxic and targeted therapies is a major problem facing cancer treatment. The mechanisms of resistance to unrelated drugs share many common features, including up-regulation of detoxifying pathways, activation of pro-survival mechanisms, and ineffective induction of cell death. Oncolytic viruses (OVs) are promising biotherapeutics for cancer treatment that specifically replicate in and lyse cancer cells. In addition to direct viral lysis, the anti-tumor effects of OVs are mediated via innate and adaptive immune responses, and several adaptation mechanisms such as autophagy appear to contribute to their anti-tumor properties. Autophagy is a versatile pathway that plays a key role in cancer survival during stressful conditions such as starvation or cytotoxic drug challenges. Autophagy also plays a role in mediating innate and adaptive immune responses by contributing to antigen presentation and cytokine secretion. This role of autophagy in regulation of immune responses can be utilized to design therapeutic combinations using approaches that either stimulate or block autophagy to potentiate therapeutic efficacy of OVs. Additional studies are needed to determine optimal multimodal combination approaches that will facilitate future successful clinical implementation of OV-based therapies.

Histone deacetylase inhibitors potentiate vesicular stomatitis virus oncolysis in prostate cancer cells by modulating NF-κB-dependent autophagy.

Vesicular stomatitis virus (VSV) is an oncolytic virus that induces cancer cell death through activation of the apoptotic pathway. Intrinsic resistance to oncolysis is found in some cell lines and many primary tumors as a consequence of residual innate immunity to VSV. In resistant-tumor models, VSV oncolytic potential can be reversibly stimulated by combination with epigenetic modulators, such as the histone deacetylase inhibitor vorinostat. Based on this reversible effect of vorinostat, we reasoned that critical host genes involved in oncolysis may likewise be reversibly regulated by vorinostat. A transcriptome analysis in prostate cancer PC3 cells identified a subset of NF-κB target genes reversibly regulated by vorinostat, as well as a group of interferon (IFN)-stimulated genes (ISGs). Consistent with the induction of NF-κB target genes, vorinostat-mediated enhancement of VSV oncolysis increased hyperacetylation of NF-κB RELA/p65. Additional bioinformatics analysis revealed that NF-κB signaling also increased the expression of several autophagy-related genes. Kinetically, autophagy preceded apoptosis, and apoptosis was observed only when cells were treated with both VSV and vorinostat. VSV replication and cell killing were suppressed when NF-κB signaling was inhibited using pharmacological or genetic approaches. Inhibition of autophagy by 3-methyladenine (3-MA) enhanced expression of ISGs, and either 3-MA treatment or genetic ablation of the autophagic marker Atg5 decreased VSV replication and oncolysis. Together, these data demonstrate that vorinostat stimulates NF-κB activity in a reversible manner via modulation of RELA/p65 signaling, leading to induction of autophagy, suppression of the IFN-mediated response, and subsequent enhancement of VSV replication and apoptosis.

BCL-2 inhibitors sensitize therapy-resistant chronic lymphocytic leukemia cells to VSV oncolysis.

Many primary cancers including chronic lymphocytic leukemia (CLL) are resistant to vesicular stomatitis virus (VSV)-induced oncolysis due to overexpression of the antiapoptotic and antiautophagic members of the B-cell lymphoma-2 (BCL-2) family. In the present study, we investigated the mechanisms of CLL cell death induced as a consequence of VSV infection in the presence of BCL-2 inhibitors, obatoclax, and ABT-737 in primary ex vivo CLL patient samples. Microarray analysis of primary CD19⁺ CD5⁺ CLL cells treated with obatoclax and VSV revealed changes in expression of genes regulating apoptosis, the mechanistic target of rapamycin (mTOR) pathway, and cellular metabolism. A combined therapeutic effect was observed for VSV and BCL-2 inhibitors in cells from untreated patients and from patients unresponsive to standard of care therapy. In addition, combination treatment induced several markers of autophagy--LC3-II accumulation, p62 degradation, and staining of autophagic vacuoles. Inhibition of early stage autophagy using 3-methyladenine (3-MA) led to increased apoptosis in CLL samples. Mechanistically, a combination of BCL-2 inhibitors and VSV disrupted inhibitory interactions of Beclin-1 with BCL-2 and myeloid cell leukemia-1 (MCL-1), thus biasing cells toward autophagy. We propose a mechanism in which changes in cellular metabolism, coupled with pharmacologic disruption of the BCL-2-Beclin-1 interactions, facilitate induction of apoptosis and autophagy to mediate the cytolytic effect of VSV.

Exosome RNA Sequencing as a Tool in the Search for Cancer Biomarkers.

Numerous noninvasive methods are currently being used to determine biomarkers for diseases such as cancer. However, these methods are not always precise and reliable. Thus, there is an unmet need for better diagnostic and prognostic biomarkers that will be used to diagnose cancer in early, more treatable stages of the disease. Exosomes are extracellular vesicles of endocytic origin released by the majority of cells. Exosomes contain and transport nucleic acids, proteins, growth factors, and cytokines from their parent cells to surrounding or even distant cells via circulation in biofluids. Exosomes have attracted the interest of researchers, as recent data indicate that exosome content may be indicative of disease stages and may contribute to disease progression via exosome-mediated extracellular communication. Therefore, the contents of these vesicles are being investigated as possible biomarkers for disease diagnosis and prognosis. The functions of exosomes and their contents in disease development are becoming clearer as isolation and analytical methods, such as RNA sequencing, advance. In this review, we discuss current advances and challenges in exosomal content analyses with emphasis on information that can be generated using RNA sequencing. We also discuss how the RNA sequencing of exosomes may be used to discover novel biomarkers for the detection of different stages for various cancers using specific microRNAs that were found to be differentially expressed between healthy controls and cancer-diagnosed subjects.

Exosomal RNAs in diagnosis and therapies.

The field of extracellular vesicles has been rapidly developing after it became evident that a defined subset of vesicles, called exosomes, can modulate several biological functions in distant cells and tissues. Exosomes range in a size from 40 to 160 nm in diameter, are released by majority of cells in our body, and carry molecules which reflect the cell of origin. The types of biomolecules packed, their respective purpose, and their impact on the physiological state of distinct cells and tissues should be understood to advance the using of exosomes as biomarkers of health and disease. Many of such physiological effects can be linked to exosomal RNA molecules which include both coding and non-coding RNAs. The biological role(s) of various exosomal RNAs have started being recognized after RNA sequencing methods became widely available which led to discovery of a variety of RNA molecules in exosomes and their roles in regulating of many biological processes are beginning to be unraveled. In present review, we outline and discuss recent progress in the elucidation of the various biological processes driven by exosomal RNA and their relevance for several major conditions including disorders of central nervous system, cardiovascular system, metabolism, cancer, and immune system. Furthermore, we also discuss potential use of exosomes as valuable therapeutics for tissue regeneration and for conditions resulting from excessive inflammation. While exosome research is still in its infancy, in-depth understanding of exosome formation, their biological effects, and specific cell-targeting will uncover how they can be used as disease biomarkers and therapeutics.

Effects of autophagy modulators tamoxifen and chloroquine on the expression profiles of long non-coding RNAs in MIAMI cells exposed to IFNγ.

Mesenchymal stromal cells (MSCs) can be utilized clinically for treatment of conditions that result from excessive inflammation. In a pro-inflammatory environment, MSCs adopt an anti-inflammatory phenotype resulting in immunomodulation. A sub-type of MSCs referred to as "marrow-isolated adult multilineage inducible" (MIAMI) cells, which were isolated from bone marrow, were utilized to show that the addition of autophagy modulators, tamoxifen (TX) or chloroquine (CQ), can alter how MIAMI cells respond to IFNγ exposure in vitro resulting in an increased immunoregulatory capacity of the MIAMI cells. Molecularly, it was also shown that TX and CQ each alter both the levels of immunomodulatory genes and microRNAs which target such genes. However, the role of other non-coding RNAs (ncRNAs) such as long non-coding RNAs (lncRNAs) in regulating the response of MSCs to inflammation has been poorly studied. Here, we utilized transcriptomics and data mining to analyze the putative roles of various differentially regulated lncRNAs in MIAMI cells exposed to IFNγ with (or without) TX or CQ. The aim of this study was to investigate how the addition of TX and CQ alters lncRNA levels and evaluate how such changes could alter previously observed TX- and CQ-driven changes to the immunomodulatory properties of MIAMI cells. Data analysis revealed 693 long intergenic non-coding RNAS (lincRNAs), 480 pseudogenes, and 642 antisense RNAs that were differentially regulated with IFNγ, IFNγ+TX and IFNγ+CQ treatments. Further analysis of these RNA species based on the existing literature data revealed 6 antisense RNAs, 2 pseudogenes, and 5 lincRNAs that have the potential to modulate MIAMI cell's response to IFNγ treatment. Functional analysis of these genomic species based on current literature linking inflammatory response and ncRNAs indicated their potential for regulation of several key pro- and anti-inflammatory responses, including NFκB signaling, cytokine secretion and auto-immune responses. Overall, this work found potential involvement of multiple pro-and anti-inflammatory pathways and molecules in modulating MIAMI cells' response to inflammation.

Combined anticancer effects of sphingosine kinase inhibitors and sorafenib.

The pro-apoptotic lipid sphingosine is phosphorylated by sphingosine kinases 1 and 2 (SK1 and SK2) to generate the mitogenic lipid sphingosine-1-phosphate (S1P). We previously reported that inhibition of SK activity delays tumor growth in a mouse mammary adenocarcinoma model. Because SK inhibitors and the multikinase inhibitor sorafenib both suppress the MAP kinase pathway, we hypothesized that their combination may provide enhanced inhibition of tumor growth. Therefore, we evaluated the effects of two SK inhibitors, ABC294640 (a SK2-specific inhibitor) and ABC294735 (a dual SK1/SK2 inhibitor), alone and in combination with sorafenib on human pancreatic adenocarcinoma (Bxpc-3) and kidney carcinoma (A-498) cells in vitro and in vivo. Exposure of either Bxpc-3 or A-498 cells to combinations of ABC294640 and sorafenib or ABC294735 and sorafenib resulted in synergistic cytotoxicity, associated with activation of caspases 3/7 and DNA fragmentation. Additionally, strong decreases in ERK phosphorylation were observed in Bxpc-3 and A-498 cells exposed to either the sorafenib/ABC294640 or the sorafenib/ABC294735 combination. Oral administration of either ABC294640 or ABC294735 to mice led to a delay in tumor growth in both xenograft models without overt toxicity to the animals. Tumor growth delay was potentiated by co-administration of sorafenib. These studies show that combination of an SK inhibitor with sorafenib causes synergistic inhibition of cell growth in vitro, and potentiates antitumor activity in vivo. Thus, a foundation is established for clinical trials evaluating the efficacy of combining these signaling inhibitors.

Correction: Characterization of zika virus infection of human fetal cardiac mesenchymal stromal cells.

[This corrects the article DOI: 10.1371/journal.pone.0239238.].

A novel sphingosine kinase inhibitor induces autophagy in tumor cells.

The sphingolipids ceramide, sphingosine, and sphingosine 1-phosphate (S1P) regulate cell signaling, proliferation, apoptosis, and autophagy. Sphingosine kinase-1 and -2 (SK1 and SK2) phosphorylate sphingosine to form S1P, shifting the balanced activity of these lipids toward cell proliferation. We have previously reported that pharmacological inhibition of SK activity delays tumor growth in vivo. The present studies demonstrate that the SK2-selective inhibitor 3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide (ABC294640) induces nonapoptotic cell death that is preceded by microtubule-associated protein light chain 3 cleavage, morphological changes in lysosomes, formation of autophagosomes, and increases in acidic vesicles in A-498 kidney carcinoma cells. ABC294640 caused similar autophagic responses in PC-3 prostate and MDA-MB-231 breast adenocarcinoma cells. Simultaneous exposure of A-498 cells to ABC294640 and 3-methyladenine, an inhibitor of autophagy, switched the mechanism of toxicity to apoptosis, but decreased the potency of the SK2 inhibitor, indicating that autophagy is a major mechanism for tumor cell killing by this compound. Induction of the unfolded protein response by the proteasome inhibitor N-(benzyloxycarbonyl)leucinylleucinylleucinal Z-Leu-Leu-Leu-al (MG-132) or the heat shock protein 90 inhibitor geldanamycin synergistically increased the cytotoxicity of ABC294640 in vitro. In severe combined immunodeficient mice bearing A-498 xenografts, daily administration of ABC294640 delayed tumor growth and elevated autophagy markers, but did not increase terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling-positive cells in the tumors. These data suggest that ABC294640 promotes tumor cell autophagy, which ultimately results in nonapoptotic cell death and a delay of tumor growth in vivo. Consequently, ABC294640 may effectively complement anticancer drugs that induce tumor cell apoptosis.

Pharmacology and antitumor activity of ABC294640, a selective inhibitor of sphingosine kinase-2.

Sphingolipid-metabolizing enzymes control the dynamic balance of the cellular levels of important bioactive lipids, including the apoptotic compound ceramide and the proliferative compound sphingosine 1-phosphate (S1P). Many growth factors and inflammatory cytokines promote the cleavage of sphingomyelin and ceramide leading to rapid elevation of S1P levels through the action of sphingosine kinases (SK1 and SK2). SK1 and SK2 are overexpressed in a variety of human cancers, making these enzymes potential molecular targets for cancer therapy. We have identified an aryladamantane compound, termed ABC294640 [3-(4-chlorophenyl)-adamantane-1-carboxylic acid (pyridin-4-ylmethyl)amide], that selectively inhibits SK2 activity in vitro, acting as a competitive inhibitor with respect to sphingosine with a K(i) of 9.8 muM, and attenuates S1P formation in intact cells. In tissue culture, ABC294640 suppresses the proliferation of a broad panel of tumor cell lines, and inhibits tumor cell migration concomitant with loss of microfilaments. In vivo, ABC294640 has excellent oral bioavailability, and demonstrates a plasma clearance half-time of 4.5 h in mice. Acute and chronic toxicology studies indicate that ABC294640 induces a transient minor decrease in the hematocrit of rats and mice; however, this normalizes by 28 days of treatment. No other changes in hematology parameters, or gross or microscopic tissue pathology, result from treatment with ABC294640. Oral administration of ABC294640 to mice bearing mammary adenocarcinoma xenografts results in dose-dependent antitumor activity associated with depletion of S1P levels in the tumors and progressive tumor cell apoptosis. Therefore, this newly developed SK2 inhibitor provides an orally available drug candidate for the treatment of cancer and other diseases.

"Skittish" Abca2 knockout mice display tremor, hyperactivity, and abnormal myelin ultrastructure in the central nervous system.

The ATP-binding cassette transporter 2 (ABCA2) is an endolysosomal protein most highly expressed in the central and peripheral nervous system tissues and macrophages. Previous studies indicated its role in cholesterol/steroid (estramustine, estradiol, and progesterone) trafficking/sequestration, oxidative stress response, and Alzheimer's disease. Developmental studies have shown its expression during macrophage and oligodendrocyte differentiation, processes requiring membrane growth. To determine the in vivo function(s) of this transporter, we generated a knockout mouse from a gene-targeted disruption of the murine ABCA2 gene. Knockout males and females are viable and fertile. However, a non-Mendelian inheritance pattern was shown among male progeny of heterozygous crosses. Compared to wild-type and heterozygous littermates, knockout mice displayed a tremor without ataxia, hyperactivity, and reduced body weight; the latter two phenotypes were more marked in females than in males. This sexual disparity suggests a role for ABCA2 in hormone-dependent neurological and/or developmental pathways. Myelin sheath thickness in the spinal cords of knockout mice was greatly increased compared to that in wild-type mice, while a significant reduction in myelin membrane periodicity (compaction) was observed in both spinal cords and cerebra of knockout mice. Loss of ABCA2 function in vivo resulted in abnormal myelin compaction in spinal cord and cerebrum, an ultrastructural defect that we propose to be the cause of the phenotypic tremor.

Differences and similarities between cancer and somatic stem cells: therapeutic implications.

Over the last decades, the cancer survival rate has increased due to personalized therapies, the discovery of targeted therapeutics and novel biological agents, and the application of palliative treatments. Despite these advances, tumor resistance to chemotherapy and radiation and rapid progression to metastatic disease are still seen in many patients. Evidence has shown that cancer stem cells (CSCs), a sub-population of cells that share many common characteristics with somatic stem cells (SSCs), contribute to this therapeutic failure. The most critical properties of CSCs are their self-renewal ability and their capacity for differentiation into heterogeneous populations of cancer cells. Although CSCs only constitute a low percentage of the total tumor mass, these cells can regrow the tumor mass on their own. Initially identified in leukemia, CSCs have subsequently been found in cancers of the breast, the colon, the pancreas, and the brain. Common genetic and phenotypic features found in both SSCs and CSCs, including upregulated signaling pathways such as Notch, Wnt, Hedgehog, and TGF-ß. These pathways play fundamental roles in the development as well as in the control of cell survival and cell fate and are relevant to therapeutic targeting of CSCs. The differences in the expression of membrane proteins and exosome-delivered microRNAs between SSCs and CSCs are also important to specifically target the stem cells of the cancer. Further research efforts should be directed toward elucidation of the fundamental differences between SSCs and CSCs to improve existing therapies and generate new clinically relevant cancer treatments.

Characterization of zika virus infection of human fetal cardiac mesenchymal stromal cells.

Zika virus (ZIKV) is a single-stranded RNA virus belonging to the family Flaviviridae. ZIKV predominantly enters cells using the TAM-family protein tyrosine kinase receptor AXL, which is expressed on a range of cell types, including neural progenitor cells, keratinocytes, dendritic cells, and osteoblasts. ZIKV infections have been associated with fetal brain damage, which prompted the World Health Organization to declare a public health emergency in 2016. ZIKV infection has also been linked to birth defects in other organs. Several studies have reported congenital heart defects (CHD) in ZIKV infected infants and cardiovascular complications in adults infected with ZIKV. To develop a better understanding of potential causes for these pathologies at a cellular level, we characterized ZIKV infection of human fetal cardiac mesenchymal stromal cells (fcMSCs), a cell type that is known to contribute to both embryological development as well as adult cardiac physiology. Total RNA, supernatants, and/or cells were collected at various time points post-infection to evaluate ZIKV replication, cell death, and antiviral responses. We found that ZIKV productively infected fcMSCs with peak (~70%) viral mRNA detected at 48 h. Use of an antibody blocking the AXL receptor decreased ZIKV infection (by ~50%), indicating that the receptor is responsible to a large extent for viral entry into the cell. ZIKV also altered protein expression of several mesenchymal cell markers, which suggests that ZIKV could affect fcMSCs' differentiation process. Gene expression analysis of fcMSCs exposed to ZIKV at 6, 12, and 24 h post-infection revealed up-regulation of genes/pathways associated with interferon-stimulated antiviral responses. Stimulation of TLR3 (using poly I:C) or TLR7 (using Imiquimod) prior to ZIKV infection suppressed viral replication in a dose-dependent manner. Overall, fcMSCs can be a target for ZIKV infection, potentially resulting in CHD during embryological development and/or cardiovascular issues in ZIKV infected adults.

Combination therapies enhance immunoregulatory properties of MIAMI cells.

BACKGROUND: Mesenchymal stromal cells (MSCs), adult stromal cells most commonly isolated from bone marrow (BM), are being increasingly utilized in various therapeutic applications including tissue repair via immunomodulation, which is recognized as one of their most relevant mechanism of action. The promise of MSC-based therapies is somewhat hindered by their apparent modest clinical benefits, highlighting the need for approaches that would increase the efficacy of such therapies. Manipulation of cellular stress-response mechanism(s) such as autophagy, a catabolic stress-response mechanism, with small molecules prior to or during MSC injection could improve MSCs' therapeutic efficacy. Unfortunately, limited information exists on how manipulation of autophagy affects MSCs' response to inflammation and subsequent immunoregulatory properties. METHODS: In this study, we exposed BM-MSC precursor cells, "marrow-isolated adult multilineage inducible" (MIAMI) cells, to autophagy modulators tamoxifen (TX) or chloroquine (CQ), together with IFN-γ. Exposed cells then underwent RNA sequencing (RNAseq) to determine the effects of TX or CQ co-treatments on cellular response to IFN-γ at a molecular level. Furthermore, we evaluated their immunoregulatory capacity using activated CD4+ T cells by analyzing T cell activation marker CD25 and the percentage of proliferating T cells after co-culturing the cells with MIAMI cells treated or not with TX or CQ. RESULTS: RNAseq data indicate that the co-treatments alter both mRNA and protein levels of key genes responsible for MSCs' immune-regulatory properties. Interestingly, TX and CQ also altered some of the microRNAs targeting such key genes. In addition, while IFN-γ treatment alone increased the surface expression of PD-L1 and secretion of IDO, this increase was further enhanced with TX. An improvement in MIAMI cells' ability to decrease the activation and proliferation of T cells was also observed with TX, and to a lesser extent, CQ co-treatments. CONCLUSION: Altogether, this work suggests that both TX and CQ have a potential to enhance MIAMI cells' immunoregulatory properties. However, this enhancement is more pronounced with TX co-treatment.

Immunomodulatory effects of interferon-γ on human fetal cardiac mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs), due to their regenerative and immunomodulatory properties, are therapeutically used for diseases, including heart failure. As early gestational-phase embryonic tissues exhibit extraordinary regenerative potential, fetal MSCs exposed to inflammation offer a unique opportunity to evaluate molecular mechanisms underlying preferential healing, and investigate their inherent abilities to communicate with the immune system during development. The principal aim of this study was to evaluate the effects of interferon-γ (IFNγ) on the immunomodulatory effects of first-trimester human fetal cardiac (hfc)-MSCs. METHODS: hfcMSCs (gestational week 8) were exposed to IFNγ, with subsequent analysis of the whole transcriptome, based on RNA sequencing. Exploration of surface-expressed immunoregulatory mediators and modulation of T cell responses were performed by flow cytometry. Presence and activity of soluble mediators were assessed by ELISA or high-performance liquid chromatography. RESULTS: Stimulation of hfcMSCs with IFNγ revealed significant transcriptional changes, particularly in respect to the expression of genes belonging to antigen presentation pathways, cell cycle control, and interferon signaling. Expression of immunomodulatory genes and associated functional changes, including indoleamine 2,3-dioxygenase activity, and regulation of T cell activation and proliferation via programmed cell death protein (PD)-1 and its ligands PD-L1 and PD-L2, were significantly upregulated. These immunoregulatory molecules diminished rapidly upon withdrawal of inflammatory stimulus, indicating a high degree of plasticity by hfcMSCs. CONCLUSIONS: To our knowledge, this is the first study performing a systematic evaluation of inflammatory responses and immunoregulatory properties of first-trimester cardiac tissue. In summary, our study demonstrates the dynamic responsiveness of hfcMSCs to inflammatory stimuli. Further understanding as to the immunoregulatory properties of hfcMSCs may be of benefit in the development of novel stromal cell therapeutics for cardiovascular disease.