Browsing by Subject "Immunity, Natural--Physiology."

Now showing 1 - 5 of 5

Open Access
Enhanced immunomodulatory applications of nucleic acid encapsulating liposomes
(2009) Erikçi, Erdem
Recent studies have demonstrated that innate immune system has great ability to discriminate self from non-self through the action of innate immune receptors. The most extensively studied innate immune receptor family is the Toll-like receptors (TLRs). Endosomal/intracellular TLR3, TLR7/8 and TLR9 recognize dsRNA, ssRNA and unmethylated CpG DNA respectively. Upon activation following recognition of nucleic acids by endosomal TLRs, B cells secrete IL6, dendritic cells and macrophages secrete type I IFNs, IL12 and NK cells secrete IFNγ which yields Th1 type immune response. Modulating the immune response to mount such an immune response by TLR ligands are harnessed in medical applications such as anticancer, antiviral, antibacterial therapies, anti-allergen, as vaccine adjuvant and as immunoprotective agents. Promising clinical applications of TLR ligand nucleic acids are hampered due to their premature in vivo digestion by endonucleases and rapid clearance via serum protein absorption leading to limited stability and bioavailability. A powerful tool to overcome this problem can be achieved by encapsulating TLR ligands within liposomes, which increase in vivo stability as well as augment targeting and internalization to relevant innate immune cells. In this study we aimed to establish the most immunostimulatory liposome type encapsulating or coencapsulating CpG ODN and pIC. Five different liposomes possessing different physicochemical properties were prepared and their immunostimulatory potential when nucleic acid TLRs are loaded, were assessed. Following stimulation of splenocytes with combinations of these liposome types we have observed that neutral, anionic and stealth liposome encapsulating D-ODN, led to a dose dependent significantly higher IFNγ production over free counterpart. Stealth liposome encapsulating pIC induced both IL6 and IFNγ 10 and 250 fold respectively over free pIC. Neutral and anionic liposome coencapsulating D-ODN with pIC were very strong type 1 IFN as well as Th1 cytokine inducers both in vitro and ex vivo. Then, we immunized B6 mice with anionic liposome coencapsulating D-ODN and OVA to establish the immuno-adjuvant properties of liposome formulations in vivo. We assessed primary and secondary anti-OVA IgG subclass responses of mice. Results strongly implicated that even after primary immunization, we could obtain significantly higher anti-OVA IgG and IgG2a response over OVA mixed D-ODN group. After booster injection, 22 fold more IgG, 26 fold more IgG1 and 13 fold more IgG2a were obtained compared to free group. Our findings demonstrated that when simultaneous delivery of adjuvant (D-ODN) and antigen (OVA) within a proper depot system is given to a host, very potent antigen specific immunity is achieved. This knowledge will pave the way to design of novel effective vaccine adjuvants.
Open Access
Immunomodulatory effects of TLR ligands and polysaccharide combinations : strategies to augment innate immune response
(2007) Tincer, Gizem
Microbial infection initiates multiple TLR ligand mediated signaling cascade on innate immune cells. While some TLRs trigger a Th1 biased immune activation, others may lead to a Th2 dominant immune response. Extracellular (TLR1, 2, 4, 5, 6, 10, and 11) vs endosome-associated TLRs (TLR3, 7/8, and 9) display differential immune activation and cytokine milieu. Understanding contrasting and synergistic behaviors of these TLR subclasses when mixed together may lead to more potent formulations for immunotherapy. Delivery and retaining the stability of nucleic acid based labile TLR ligands to the site of immunologically relevant cells is also a challenge. In the first part of the thesis, optimum TLR combinations with differential immune effects will be brought into light. Next, immunomodulatory effect of a natural polysaccharide (PS) will be characterized. Finally the ability of a PS carrier to form complex with ligands of nucleic acid sensing TLRs and its potential as a controlled delivery vehicle to stimulate the immune cells will be documented. In brief, our results suggest that different PS types extracted from various mushroom sources are immunostimulatory and are targeted to TLR2/6 for delivery of other relevant stimulants. Moreover, certain TLR ligand combinations can be harnessed to induce more robust immune activation compared to their stand alone counterparts. This knowledge will pave the way for establishing an effective PS based carrier of DNA/RNA ligands thus, more effective immunotherapeutic strategies for treating infectious and other local or systemic diseases be possible.
Open Access
Immunoregulatory activities of nanoparticle-forming oligodeoxynucleotides
(2009) Karatepe, Kutay
Innate immune system is activated by a wide range of microbial by products leading to an immediate immune activation primarily designed to neutralize and control the invading insult. The cells of the innate immune system also instruct the development of antigen-specific adaptive immunity. While TLR9 is triggered by bacterial DNA, extended and over-exuberant immune response poses a threat since it may exacerbate cell and tissue destruction leading to organ failure. Telomeric TTAGGG conserved motifs are previously reported to antagonize TLR mediated events. The down-regulatory effect of these motifs may help to restore the desired homeostatic balance of the immune system. While CpG ODN patterned after bacterial DNA can be harnessed in different clinical settings to provide an advantage to host to resist infectious diseases, control tumor growth or alleviate allergic symptoms, the immunosuppressive telomeric motifs could be effectively applied in controlling systemic anti-inflammatory or autoimmune related disorders. Several challenges exist in the utilization of synthetic ODNs in the clinic. The first challenge is that conventional classes of synthetic ODNs exhibit different properties. K-type ODNs are more effective in proliferation and activation of B cells and DC. D-type ODNs are in nanoparticle forms, lead to anti-viral type I IFN production and mature monocytes into DCs. Of note, the efficacy of these synthetic ODNs is reduced under physiological conditions due to premature clearance and low levels of internalization. Moreover, D-ODNs as one of the most potent IFNα inducing TLR9 ligands possess a large-scale production problem due to 3’polyGruns, which hamper their entry into the clinic. We have designed a novel class of ODN, designated as ODN420, devoid of polyGs that can undergo nanoparticle formation necessary for its IFNα induction. Ex vivo stimulation of mouse splenocytes and in vivo administration of ODN420 have revealed that this ODN exhibits higher immunostimulatory potential and is more stable than most commonly used ODNs due to its nanoparticle-forming ability. Another interesting finding is that ODN420 with the natural phosphodiester (PO) backbone is at least as potent as its more stable counterpart with the modified phosphorothioate backbone. Furthermore, it combines superior properties of conventional classes of K and D-ODNs. These results have been reproduced in human peripheral blood mononuclear cells by various assays. Next, we have analyzed whether this ODN could be utilized as a vaccine adjuvant and an anti-cancer agent with two independent experiments. Our immunization results demonstrate that ODN420 induces a higher level of Th1-mediated response than conventional ODNs and is a promising candidate as a vaccine adjuvant. This response is hampered when ODN420 is used in combination with ODN-A151. In the tumor xenograft model, ODN420 has promoted partial remission of the tumors or delayed the tumor growth. This knowledge will pave the way for more effective immunotherapeutic approaches.
Open Access
Immunostimulatory mechanisms and immunotherapeutic applications of polysaccharide nanocarrier complexed with nucleic acid based TLR ligands
(2013) König, Gizem Tincer
Open Access
Investigation of improved immunostimulatory activity of D and K type CpG ODNs in liposomes
(2013) Dereli, İhsan
CpG ODNs are potent immunotherapeutic agents. In human, two major classes of CpG ODNs were shown to induce differential immune activation. D ODNs are strong IFNα inducers, thus promising antiviral agents, whereas K ODNs are effective against bacterial infections. However, their effects cannot be combined. When K and D type ODNs are used simultaneously, K ODN cancels D specific effect, a phenomenon known as K and D ODN dichotomy. The prime reason for this counter acting K ODN action was subcellular compartmentalization of K type CpG ODNs upon internalization. Besides, CpG ODNs have labile nature. When investigated in clinical trials, these nucleic acid based ligands are eliminated upon administration and displayed limited bio-availability due to nuclease digestion. Hence, efforts to protect in vivo performance, and increase stability and accumulation near target cells became a crucial task. Liposome technology offers a simple and mild approach to harbor these ODNs within membrane bilayers and protect them. We also reasoned that, if we use liposomes that alter subcellular fate of K and D ODNs, we can retain both K and D effect when liposomal ODNs are co-administered and the breadth of immunotherapeutic spectrum could be improved. This thesis was designed to understand and characterize different types of CpG ODNs loaded into different liposomes and aimed to determine their activities in different in vitro and in vivo settings. Our results revealed that when two different classes of clinically important CpG ODNs were encapsulated within proper liposome types, it is possible to recapitulate both K and D type ODN effect in PBMCs. Furthermore, in a vaccine model against H. felis, although initially did not induce significantly higher anti H.felis immunity, liposomal CpG ODNs improved persisting antibody levels for extended periods compared to free counterparts. Collectively, our results demonstrate that this platform allows more effective in vivo utilization of CpG ODNs and can be formulated to develop more efficient means to combat several health problems, ranging from cancer to allergy.