Tamer Kahraman

Immunostimulatory activity of polysaccharide-poly(I:C) nanoparticles.

Immunostimulatory properties of mushroom derived polysaccharides (PS) as stand-alone agents were tested. Next, PS were nanocomplexed with polyI:C (pIC) to yield stable nanoparticles around 200 nm in size evidenced by atomic force microscopy and dynamic light scattering analyses. PSs were selectively engaged by cells expressing TLR2 and initiated NFκB dependent signaling cascade leading to a Th1-biased cytokine/chemokine secretion in addition to bactericidal nitric oxide (NO) production from macrophages. Moreover, cells treated with nanoparticles led to synergistic IL6, production and upregulation of TNFα, MIP3α, IFNγ and IP10 transcript expression. In mice, PS-Ovalbumin-pIC formulation surpassed anti-OVA IgG responses when compared to either PS-OVA or pIC-OVA mediated immunity. Our results revealed that signal transduction initiated both by TLR2 and TLR3 via co-delivery of pIC by PS in nanoparticle depot delivery system is an effective immunization strategy. The present work implicate that the PS and nucleic acid based nanoparticle approach along with protein antigens can be harnessed to prevent infectious diseases.

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.

Differential alteration of IL-8 in liver cancer stem cell enrichment in response to PI3K/Akt/mTOR inhibitors and sorafenib

Liver cancer stem cells (LCSCs) are derived from damaged and transformed Hepatic progenitor cells (HPCs) during precancerous cirrhosis stage. Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are significantly deregulated in liver cancer. The activation of PI3K/AKT/mTOR pathway in LCSC population is one of the reasons for acquired resistance to Sorafenib in advanced Hepatocellular carcinoma (HCC) patients. Therefore, identifying novel inhibitors targeting this pathway acting on LCSCs is highly essential. We therefore elucidated the bioactivities of small molecule kinase inhibitors on LCSCs acting through PI3K/Akt/mTOR pathway in comparison with DAPT (CSC inhibitor), DNA intercalators and Sorafenib. For this purpose, CD133+/EpCAM+ cells originated from HCC cells were analyzed by flow cytometry and effective inhibitors on LCSCs were further tested for their potential combinatorial effects. Treatment of cells with Sorafenib, and DNA intercalators resulted in enrichment of CD133+/EpCAM+ cells. Yet, mTOR inhibitor Rapamycin, and Notch pathway inhibitor DAPT significantly reduced CD133/EpCAM positivity. Combination studies revealed that sequential treatment strategy, which involves treatment of cells with Rapamycin prior to Sorafenib treatment, decreased the ratio of LCSCs as opposed to Sorafenib treatment alone or Sorafenib treatment prior to Rapamycin. The effect of the inhibitors were also demonstrated with LCSC sphere formation. Additionaly, a large panel of genes involved in cancer pathways were analyzed using Nanostring® nCounter® Technology to identify the differentially expressed genes in Rapamycin, Sorafenib or DAPT treated cells. Pathways involved in stemness (Wnt and Notch pathways) were differentially regulated between Rapamycin or DAPT treated cells and Sorafenib treated cells. Interleukin 8 (IL-8), FLNC, FLNA expressions were down-regulated upon treatment with DAPT or Rapamycin, yet up-regulated upon Sorafenib treatment. Following IL-8 inhibition CD133/EpCAM positivity of cells decreased significantly, indicating that IL-8 signaling is crucial for the conservation of stemness features of cancer cells. Conclusion PI3K/Akt/mTOR pathway inhibitors alter hepatic CSC composition and gene expression in favor or to the detriment of cancer stem cell survival. Blockade of IL-8 signaling provides a promising therapeutic approach for prevention of LCSC enrichment.

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behçet's disease severity

ABSTRACT Behçet’s disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1β, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. Abbreviations: BD: Behçet’s disease; EV: extracellular vesicle; BB: binding buffer; AnV: annexin V; autologEV: autologous extracellular vesicles; alloEV: allogeneic extracellular vesicles