Mayda Gursel

Biological properties of extracellular vesicles and their physiological functions.

In the past decade, extracellular vesicles (EVs) have been recognized as potent vehicles of intercellular communication, both in prokaryotes and eukaryotes. This is due to their capacity to transfer proteins, lipids and nucleic acids, thereby influencing various physiological and pathological functions of both recipient and parent cells. While intensive investigation has targeted the role of EVs in different pathological processes, for example, in cancer and autoimmune diseases, the EV-mediated maintenance of homeostasis and the regulation of physiological functions have remained less explored. Here, we provide a comprehensive overview of the current understanding of the physiological roles of EVs, which has been written by crowd-sourcing, drawing on the unique EV expertise of academia-based scientists, clinicians and industry based in 27 European countries, the United States and Australia. This review is intended to be of relevance to both researchers already working on EV biology and to newcomers who will encounter this universal cell biological system. Therefore, here we address the molecular contents and functions of EVs in various tissues and body fluids from cell systems to organs. We also review the physiological mechanisms of EVs in bacteria, lower eukaryotes and plants to highlight the functional uniformity of this emerging communication system.

Potential role of phosphatidylinositol 3 kinase, rather than DNA-dependent protein kinase, in CpG DNA-induced immune activation.

Unmethylated CpG motifs present in bacterial DNA stimulate a strong innate immune response. There is evidence that DNA-dependent protein kinase (DNA-PK) mediates CpG signaling. Specifically, wortmannin (an inhibitor of phosphatidylinositol 3 kinase [PI3]-kinases including DNA-PK) interferes with CpG-dependent cell activation, and DNA-PK knockout (KO) mice fail to respond to CpG stimulation. Current studies establish that wortmannin actually inhibits the uptake and colocalization of CpG DNA with toll-like receptor (TLR)-9 in endocytic vesicles, thereby preventing CpG-induced activation of the NF-κB signaling cascade. We find that DNA-PK is not involved in this process, since three strains of DNA-PK KO mice responded normally to CpG DNA. These results support a model in which CpG signaling is mediated through TLR-9 but not DNA-PK, and suggest that wortmannin-sensitive member(s) of the PI3-kinase family play a critical role in shuttling CpG DNA to TLR-9.

CpG Oligodeoxynucleotides Protect Normal and SIV-Infected Macaques from Leishmania Infection

Oligodeoxynucleotides containing CpG motifs (CpG ODNs) mimic microbial DNA and activate effectors of the innate immune response, which limits the spread of pathogens and promotes an adaptive immune response. CpG ODNs have been shown to protect mice from infection with intracellular pathogens. Unfortunately, CpG motifs that optimally stimulate humans are only weakly active in mice, mandating the use of nonhuman primates to monitor the activity and safety of “human” CpG ODNs in vivo. This study demonstrates that CpG ODN treatment of rhesus macaques significantly reduces the severity of the lesions caused by a challenge with Leishmania. Leishmania superinfection is common in immunocompromised hosts, particularly those infected with HIV. This study shows that PBMCs from HIV-infected subjects respond to stimulation with CpG ODNs. To determine whether CpG ODNs can protect retrovirus-infected primates, SIV-infected macaques were treated with CpG ODNs and then challenged with Leishmania. Both lesion size and parasite load were significantly reduced in the CpG-treated animals. These findings support the clinical development of CpG ODNs as immunoprotective agents in normal and HIV-infected patients.

Immunoadjuvant action of plasmid DNA in liposomes

Bacterial DNA and oligodeoxynucleotides containing immunostimulatory sequences with a CpG motif stimulated a Th1 type response in vivo. The adjuvant action of a non-coding plasmid DNA derived from pRc/CMV HBS (encoding the S region of hepatitis B surface antigen, HBsAg) in mice was investigated. The role of methylation on the adjuvanticity of the plasmid as well as the effect of vaccine formulation employed on the outcome of antigen-specific humoral and cellular responses were also studied. The results demonstrated that plasmid DNA acted as a Th1 promoting adjuvant when mixed as such or co-entrapped in liposomes with a very low dose of antigen. However, the adjuvant activity was lost when separate liposome entrapped formulations of both the antigen and the plasmid DNA were mixed, indicating a necessity for the antigen and the plasmid DNA to contact the same APC for optimal immune activation. A decreased adjuvanticity of plasmid DNA upon methylation with HpaII methyltransferase was also demonstrated. A mechanism that may help partially explain the reduction in adjuvanticity after modification of C residues is also discussed.

CpG ODN Nanorings Induce IFNa from Plasmacytoid Dendritic Cells and Demonstrate Potent Vaccine Adjuvant Activity

Nanorings act as CpG oligodeoxynucleotide surrogates that may function as vaccine adjuvants and anticancer and antiviral agents. An Adjuvant That Has a Ring to It Adjuvants are vaccines’ little helpers—they modify the immune response to the vaccine antigen. CpG oligodeoxynucleotides (ODN) are short single-stranded synthetic DNA molecules that activate the immune system. Hence, ODN are considered prime candidates for use as adjuvants. Indeed, they’ve been shown to be effective for both preventing and treating infectious disease and cancers in animal models and early clinical trials. Yet, there are limitations to widespread use of ODN as an adjuvant, one of which is structurally optimizing the ODN to induce the desired immune response. Now, Gungor et al. use the HIV-derived peptide Tat(47–57) to multimerize K-type ODN, forming a nanoring. The authors found that these nanorings targeted the ODN to early endosomes and induced a type I interferon response in human plasmacytoid dendritic cells. The ODN nanorings were then tested in animal models. They induced TH1 immune responses in mice vaccinated with inactivated foot and mouth disease virus, and improved antitumor immunity in a mouse cancer model. These data suggest that these nanorings may be valuable as either antiviral or anticancer agents. CpG oligodeoxynucleotides (ODN) are short single-stranded synthetic DNA molecules that activate the immune system and have been found to be effective for preventing and treating infectious diseases, allergies, and cancers. Structurally distinct classes of synthetic ODN expressing CpG motifs differentially activate human immune cells. K-type ODN (K-ODN), which have progressed into human clinical trials as vaccine adjuvants and immunotherapeutic agents, are strong activators of B cells and trigger plasmacytoid dendritic cells (pDCs) to differentiate and produce tumor necrosis factor–α (TNFα). In contrast, D-type ODN (D-ODN) stimulate large amounts of interferon-α (IFNα) secretion from pDCs. This activity depends on the ability of D-ODN to adopt nanometer-sized G quadruplex–based structures, complicating their manufacturing and hampering their progress into the clinic. In search of a D-ODN substitute, we attempted to multimerize K-ODN into stable nanostructures using cationic peptides. We show that short ODN with a rigid secondary structure form nuclease-resistant nanorings after condensation with the HIV-derived peptide Tat(47–57). The nanorings enhanced cellular internalization, targeted the ODN to early endosomes, and induced a robust IFNα response from human pDCs. Compared to the conventional K-ODN, nanorings boosted T helper 1–mediated immune responses in mice immunized with the inactivated foot and mouth disease virus vaccine and generated superior antitumor immunity when used as a therapeutic tumor vaccine adjuvant in C57BL/6 mice bearing ovalbumin-expressing EG.7 thymoma tumors. These results suggest that the nanorings can act as D-ODN surrogates and may find a niche for further clinical applications.

Differential immune activation following encapsulation of immunostimulatory CpG oligodeoxynucleotide in nanoliposomes

The immunogenicity of a vaccine formulation is closely related to the effective internalization by the innate immune cells that provide prolonged and simultaneous delivery of antigen and adjuvant to relevant antigen presenting cells. Endosome associated TLR9 recognizes microbial unmethylated CpG DNA. Clinical applications of TLR9 ligands are significantly hampered due to their pre-mature in vivo digestion and rapid clearance. Liposome encapsulation is a powerful tool to increase in vivo stability as well as enhancing internalization of its cargo to relevant immune cells. The present study established that encapsulating CpG motifs in different liposomes having different physicochemical properties altered not only encapsulation efficiency, but also the release and delivery rates that ultimately impacted in vitro and ex-vivo cytokine production rates and types. Moreover, different liposomes encapsulating CpG ODN significantly increased Th1-biased cytokines and chemokines gene transcripts Additional studies demonstrated that co-stimulatory and surface marker molecules significantly upregulated upon liposome/CpG injection. Finally, co-encapsulating model antigen ovalbumin with CpG ODN adjuvant in nanoliposomes profoundly augmented Th1 and cell mediated anti-Ova specific immune response. Collectively, this work established an unappreciated immunoregulatory property of nanoliposomes mediating immunity against protein antigen and could be harnessed to design more effective therapeutic vaccines or stand alone immunoprotective agents targeting infectious diseases, as well as cancer or allergy.

Activation of type I interferon‐dependent genes characterizes the “core response” induced by CpG DNA

Synthetic ODNs expressing CpG motifs trigger an innate immune response via TLR9. pDCs are major effectors of this response. Two structurally distinct classes of CpG ODNs have been identified that differentially activate pDCs. “K” ODNs trigger the production of TNF‐α and IL‐6, whereas “D” ODNs preferentially induce the secretion of IFN‐α. As K and D ODNs have distinct therapeutic effects, knowledge of their shared and sequence‐specific activity is of considerable importance. This work uses the CAL‐1 human pDC line to analyze the effect of CpG stimulation on gene expression. Genes up‐regulated by both K and D ODNs (n=92) were largely dependent on type I IFN signaling and characterized functionally by antiviral activity. K ODNs induced a short‐term increase in IFN‐α/β production and uniquely up‐regulated genes that supported antibacterial responses. In contrast, D ODNs triggered a persistent increase in IFN‐α/β production and uniquely up‐regulated genes associated with metabolic functions. Thus, the core functionality of human pDCs mediated by TLR9 ligation rests on a type I IFN response that differs from the response induced by the structural elements unique to specific classes of ODNs.

Intestinal Microbiota in Patients with Spinal Cord Injury

Human intestinal flora comprises thousands of bacterial species. Growth and composition of intestinal microbiota is dependent on various parameters, including immune mechanisms, dietary factors and intestinal motility. Patients with spinal cord injury (SCI) frequently display neurogenic bowel dysfunction due to the absence of central nervous system control over the gastrointestinal system. Considering the bowel dysfunction and altered colonic transit time in patients with SCI, we hypothesized the presence of a significant change in the composition of their gut microbiome. The objective of this study was to characterize the gut microbiota in adult SCI patients with different types of bowel dysfunction. We tested our hypothesis on 30 SCI patients (15 upper motor neuron [UMN] bowel syndrome, 15 lower motor neuron [LMN] bowel syndrome) and 10 healthy controls using the 16S rRNA sequencing. Gut microbial patterns were sampled from feces. Independent of study groups, gut microbiota of the participants were dominated by Blautia, Bifidobacterium, Faecalibacterium and Ruminococcus. When we compared all study groups, Roseburia, Pseudobutyrivibrio, Dialister, Marvinbryantia and Megamonas appeared as the genera that were statistically different between groups. In comparison to the healthy group, total bacterial counts of Pseudobutyrivibrio, Dialister and Megamonas genera were significantly lower in UMN bowel dysfunction group. The total bacterial count of Marvinbryantia genus was significantly lower in UMN bowel dysfunction group when compared to the LMN group. Total bacterial counts of Roseburia, Pseudobutyrivibrio and Megamonas genera were significantly lower in LMN bowel dysfunction group when compared to healthy groups. Our results demonstrate for the first time that butyrate-producing members are specifically reduced in SCI patients when compared to healthy subjects. The results of this study would be of interest since to our knowledge, microbiome-associated studies targeting SCI patients are non-existent and the results might help explain possible implications of gut microbiome in SCI.

Enhanced immunostimulatory activity of cyclic dinucleotides on mouse cells when complexed with a cell-penetrating peptide or combined with CpG.

Recognition of pathogen‐derived nucleic acids by immune cells is critical for the activation of protective innate immune responses. Bacterial cyclic dinucleotides (CDNs) are small nucleic acids that are directly recognized by the cytosolic DNA sensor STING (stimulator of IFN genes), initiating a response characterized by proinflammatory cytokine and type I IFN production. Strategies to improve the immune stimulatory activities of CDNs can further their potential for clinical development. Here, we demonstrate that a simple complex of cylic‐di‐GMP with a cell‐penetrating peptide enhances both cellular delivery and biological activity of the cyclic‐di‐GMP in murine splenocytes. Furthermore, our findings establish that activation of the TLR‐dependent and TLR‐independent DNA recognition pathways through combined use of CpG oligonucleotide (ODN) and CDN results in synergistic activity, augmenting cytokine production (IFN‐α/β, IL‐6, TNF‐α, IP‐10), costimulatory molecule upregulation (MHC class II, CD86), and antigen‐specific humoral and cellular immunity. Results presented herein indicate that 3′3′‐cGAMP, a recently identified bacterial CDN, is a superior stimulator of IFN genes ligand than cyclic‐di‐GMP in human PBMCs. Collectively, these findings suggest that the immune‐stimulatory properties of CDNs can be augmented through peptide complexation or synergistic use with CpG oligonucleotide and may be of interest for the development of CDN‐based immunotherapeutic agents.

Anticarcinogenic effects of the ethanolic extract of Salix aegyptiaca in colon cancer cells: involvement of Akt/PKB and MAPK pathways.

The bark from Salix species of plants has been traditionally consumed for its antiinflammatory properties. Because inflammation frequently accompanies the progress of colorectal cancer (CRC), we have evaluated the anticancer properties of the ethanolic extract from the bark (EEB) of S. aegyptiaca, a Salix species endogenous to the Middle East, using HCT-116 and HT29 CRC cell lines. Fresh bark from S. aegyptiaca was extracted with ethanol, fractionated by solvent-solvent partitioning and the fractions were analyzed by tandem mass spectrometry. Catechin, catechol, and salicin were the most abundant constituents of the extract. Interestingly, EEB showed the highest anticancer effect in the colon cancer cells followed by its fractions in ethyl acetate and water, with catechin, catechol, and salicin showing the least efficacy. EEB could strongly reduce the proliferation of the cancer cells, but not of CCD-18Co, normal colon fibroblast cell line. Accompanying this was cell cycle arrest at G1/S independent of DNA damage in the cancer cells, induction of apoptosis through a p53 dependent pathway and an inhibition of PI3K/Akt and MAP Kinase pathways at levels comparable to known commercial inhibitors. We propose that the combination of the polyphenols and flavonoids in EEB contributes toward its potent anticarcinogenic effects. [Supplementary materials are available for this article. Go to the publishers online edition of Nutrition and Cancer for the following free supplemental resource(s): Supplementary Figure 1 and Supplementary Figure 2.]