immunostimulatory-agent-evaluation-lymphoid-tissue-extraction

Immunostimulatory Agent Evaluation: Lymphoid Tissue Extraction and Injection Route-Dependent Dendritic Cell Activation

For evaluation of a new therapeutic agent for immunotherapy or vaccination, analysis of immune cell activation in lymphatic tissues is essential. Here, we ...

pukyong national university

Pukyong National University

Breast cancer resistance protein (ABCG2), a member of the ATP-binding cassette transporters has been identified as a major determinant of multidrug resistance (MDR) in cancer cells, but ABC transporter inhibition has limited therapeutic value in vivo. In this research, we demonstrated that inhibition of efflux transporters ABCG2 induced the generation of tolerogenic DCs from human peripheral blood myeloid DCs (mDCs). ABCG2 expression was present in mDCs and was further increased by LPS stimulation. Treatment of CD1c+ mDCs with an ABCG2 inhibitor, Ko143, during LPS stimulation caused increased production of IL-10 and decreased production of pro-inflammatory cytokines and decreased expression of CD83 and CD86. Moreover, inhibition of ABCG2 in monocyte-derived DCs (MDDCs) abrogated the up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines in these cells in response to LPS. Furthermore, CD1c+ mDCs stimulated with LPS plus Ko143 inhibited the proliferation of allogeneic and superantigen-specific syngenic CD4+ T cells and promoted expansion of CD25+FOXP3+ regulatory T (Treg) cells in an IL-10-dependent fashion. These tolerogenic effects of ABCG2 inhibition could be abolished by ERK inhibition. Thus, we demonstrated that inhibition of ABCG2 in LPS-stimulated mDCs can potently induce tolerogenic potentials in these cells, providing crucial new information that could lead to development of better strategies to combat MDR cancer. 

Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation.

The Uba6-Use1 ubiquitin enzyme cascade is a poorly understood arm of the ubiquitin-proteasome system required for mouse development. Recently, we reported that Uba6 brain-specific knockout (termed NKO) mice display abnormal social behavior and neuronal development due to a decreased spine density and accumulation of Ube3a and Shank3. To better characterize a potential role for NKO mice in autism spectrum disorders (ASDs), we performed a comprehensive behavioral characterization of the social behavior and communication of NKO mice. Our behavioral results confirmed that NKO mice display social impairments, as indicated by fewer vocalizations and decreased social interaction. We conclude that UBA6 NKO mice represent a novel ASD mouse model of anti-social and less verbal behavioral symptoms. 

Impairment of social behavior and communication in mice lacking the Uba6-dependent ubiquitin activation system.

Advancement of biological process for the synthesis of bionanoparticles is evolving into a key area of research in nanotechnology. The present study deals with the biosynthesis, characterization of gold bionanoparticles by Nocardiopsis sp. MBRC-48 and evaluation of their antimicrobial, antioxidant and cytotoxic activities. The gold bionanoparticles obtained were characterized by UV-visible spectroscopy, X-ray diffraction analysis, Fourier transform infrared spectroscopy, field emission scanning electron microscopy, energy dispersive X-ray analysis and transmission electron microscopy (TEM). The synthesized gold bionanoparticles were spherical in shape with an average of 11.57 ± 1.24 nm as determined by TEM and dynamic light scattering (DLS) particle size analyzer, respectively. The biosynthesized gold nanoparticles exhibited good antimicrobial activity against pathogenic microorganisms. It showed strong antioxidant activity as well as cytotoxicity against HeLa cervical cancer cell line. The present study demonstrated the potential use of the marine actinobacterial strain of Nocardiopsis sp. MBRC-48 as an important source for gold nanoparticles with improved biomedical applications including antimicrobial, antioxidant as well as cytotoxic agent.

Extracellular synthesis of gold bionanoparticles by Nocardiopsis sp. and evaluation of its antimicrobial, antioxidant and cytotoxic activities.

Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems. 

Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

Our previous study demonstrated that ascophyllan, a sulfated polysaccharide purified from brown alga, has immune-activating effects. In this study, we evaluated ascophyllan as an adjuvant for its therapeutic and preventive effect on tumor in a mouse melanoma model. Ascophyllan induced migration of DCs to spleen and tumor-draining lymph node (drLN) in a mouse B16 melanoma model. Moreover, ascophyllan induced activation of dendritic cells (DCs), and promoted IFN-γ- and TNF-α-producing Th1 immune responses in tumor-bearing mice. In addition, treatment with a combination of ascophyllan and ovalbumin (OVA) in the tumor-bearing mice promoted proliferation of OVA-specific CD4 and CD8 T cells and migration of those cells into the tumor, consequently inhibiting the tumor growth. Immunization with the combination of ascophyllan and OVA caused enhanced OVA-specific antibody production and memory T cell responses compared to OVA immunization alone, and almost completely prevented B16-OVA tumor growth upon subsequent tumor challenge. Finally, the combination of ascophyllan and OVA prevented B16-OVA tumor invasion and metastasis into the liver. Thus, these results demonstrate that ascophyllan can function as an adjuvant to induce DC activation, antigen specific CTL activation, Th1 immune response and antibody production, and hence may be useful as a therapeutic and preventive tumor vaccine. 

Ascophyllan functions as an adjuvant to promote anti-cancer effect by dendritic cell activation.

Previous studies have demonstrated that pullulan, a polysaccharide purified from Aureobasidium pullulans, has immune-stimulatory effects on T and B cells. Moreover, pullulan has been used as a carrier in the delivery of the antigen (Ag) peptide to lymphoid tissues. However, the in vivo effect of pullulan on dendritic cells (DC) has not been well characterized. In this study, we assessed the effect of pullulan on DC activation and anti-cancer immunity. The results showed that the pullulan treatment up-regulated co-stimulatory molecule expression and enhanced pro-inflammatory cytokine production in bone marrow-derived DCs (BMDC) in vitro and in spleen DCs in vivo. Moreover, the combination of ovalbumin (OVA) and pullulan induced OVA antigen-specific T cell activations in vivo. In tumor-bearing mice, pullulan induced the maturation of DCs in spleen and tumor draining lymph node (drLN), and promoted the OVA-specific T cell activation and migration of the T cells into the tumor. In addition, the combination of OVA and pullulan inhibited B16-OVA tumor growth and liver metastasis. The combination of tyrosinase-related protein 2 (TRP2) peptide and pullulan treatment also suppressed B16 melanoma growth. Thus, the results demonstrated that pullulan enhanced DC maturation and function, and it acted as an adjuvant in promoting Ag-specific immune responses in mice. Thus, pullulan could be a new and useful adjuvant for use in therapeutic cancer vaccines.

Maturation of dendritic cells by pullulan promotes anti-cancer effect.

We introduce a versatile carrier system for in vitro and in vivo immune stimulation based on soft matter DNA nanoparticles (NPs). The incorporation of lipid-modified nucleotides into DNA strands enables the formation of micelles of uniform size. In a single self-assembly step, the micelles can be equipped with immune adjuvant (CpG) motifs and fluorescent probes. The immunological effects of CpG confined at the NP surface were studied in a comprehensive manner in animal experiments. Dose-dependent activation of spleen dendritic cells (DCs) by CpG-conjugated NP was observed, which was accompanied by the pronounced up-regulation of co-stimulatory molecule and cytokine production.

Modular delivery of CpG-incorporated lipid-DNA nanoparticles for spleen DC activation.

Lipopolysaccharide (LPS) is a well-known stimuli of dendritic cells (DCs). However, in vivo spleen DC maturation by Escherichia coli (E.coli) LPS has not been fully investigated. In this study, we examined the effect of LPS on the activation of spleen DCs and its subsets in a time-dependent manner on mice in vivo. The frequency, number and migration of spleen conventional DCs (cDCs) were increased 6 and 12h after completion of LPS treatment. Those increased DC numbers in spleen were then gradually decreased with apoptosis of the DCs. The highest levels of co-stimulatory molecule expression in the spleen cDCs and their subsets occurred 18h after LPS treatment, while the pro-inflammatory cytokines reached their maximum in the intracellular levels of the spleen cDCs and their subsets 3h after LPS treatment. The antigen presentation of the spleen cDCs and their subsets increased gradually from 3 to 12h after LPS treatment, but those levels decreased rapidly after 18h post-LPS treatment. Thus, by highlighting the importance of time in the stimulation of spleen DCs by LPS in mice in vivo, our data provided a model that could be used by immunologists when considering the manipulation of DC functions in vivo for experimental and clinical applications.

Time-dependent effect of E. coli LPS in spleen DC activation in vivo: Alteration of numbers, expression of co-stimulatory molecules, production of pro-inflammatory cytokines, and presentation of antigens.

 polysaccharide (RGP) has shown an activation of immune cells . However, the immune stimulatory effect of RGP in a mouse  is not well studied. In this study, we examined the effect of RGP on dendritic cell (DC) activation and anticancer immunity . Treatments of RGP in C56BL/6 mice induced increased levels of co-stimulatory molecule expression and pro-inflammatory cytokine production in spleen DCs dependent on toll-like receptor 4 (TLR4), and those DCs promoted interferon-gamma (IFNγ) production in CD4 and CD8 T cells. RGP also enhanced ovalbumin (OVA) antigen (Ag)-specific immune activation in tumor-bearing mice, including Ag presentation in DCs, OT-I and OT-II T-cell proliferation, migration of OT-I and OT-II T cells into the B16-OVA tumor, OVA-specific IFNγ production, and the specific killing of OVA-coated splenocytes, which consequently inhibited B16-OVA tumor growth dependent on TLR4 and CD8 T cells. Finally, the combination of RGP and self-Ag treatment efficiently inhibited CT26 carcinoma and B16 melanoma tumor growth in BLAB/c and C57BL/6 mice, respectively. These data demonstrate that RGP could be a useful adjuvant molecule for immunotherapy against cancer.

 polysaccharide induces toll-like receptor 4 dependent spleen dendritic cell maturation and anti-cancer immunity.

Nucleic acids represent very appealing building blocks for the construction of nano-scaled objects with great potential applications in the field of drug delivery where multifunctional nanoparticles (NPs) play a pivotal role. One opportunity for DNA nanotechnology lies in the treatment of ophthalmic diseases as the efficacy of eye drops is impaired by the short survival time of the drug on the eye surface. As a consequence, topical administration of ocular therapeutics requires high drug doses and frequent administration, still rarely providing high bioavailability. To overcome these shortcomings we introduce a novel and general carrier system that is based on DNA nanotechnology. Non-toxic, lipid-modified DNA strands (12mers with 4 lipid modified thymines at the 5' end) form uniform NPs (micelles), which adhere to the corneal surface for extended periods of time. In a single self-assembly step they can be equipped with different drugs by hybridization with an aptamer. The long survival times of DNA NPs can be translated into improved efficacy. Their functionality was demonstrated in several ex-vivo experiments and in an in-vivo animal model. Finally, the NPs were confirmed to be applicable even for human tissue.

DNA nanoparticles for ophthalmic drug delivery.

To meet the ultimate goal of cancer therapy, which is treating not only the primary tumor but also preventing metastatic cancer, the concept of combining immunotherapy with photothermal therapy (PTT) is gaining great interest. Here, we studied the new material, lipopolysaccharide (LPS) coated copper sulfide nanoparticles (LPS-CuS), for the immuno-photothermal therapy. We evaluated the effect of LPS-CuS for induction of apoptosis of CT26 cells and activation of dendritic cells. Moreover, the LPS-CuS and laser irradiation was examined anti-metastasis effect by liver metastasis model mouse . Through PTT, LPS-CuS induced elimination of CT26 tumor in BALB/c mice, which produced cancer antigens. In addition, released LPS and cancer antigen by PTT promoted dendritic cell activation in tumor draining lymph node (drLN), and consequently, enhanced the tumor antigen-specific immune responses. Finally, the primary tumor cured mice by LPS-CuS-mediated PTT completely resisted secondary tumor injection in the spleen and also prevented liver metastasis. Our results demonstrated the potential usage of LPS-CuS for the immuno-photothermal therapy against various types of cancer by showing the clear elimination of primary colon carcinoma with complete prevention of spleen and liver metastasis.

Lipopolysaccharide-coated CuS nanoparticles promoted anti-cancer and anti-metastatic effect by immuno-photothermal therapy.

In advanced cancer therapy, the combinational therapeutic effect of photothermal therapy (PTT) using near-infrared (NIR) light-responsive nanoparticles (NPs) and anti-cancer drug delivery-mediated chemotherapy has been widely applied. In the present study, using a facile, low-cost, and solution-based method, we developed and synthesized fucoidan, a natural polymer isolated from seaweed that has demonstrated anti-cancer effect, and coated NPs with it as an ideal candidate in chemo-photothermal therapy against cancer cells. Fucoidan-coated copper sulfide nanoparticles (F-CuS) act not only as a nanocarrier to enhance the intracellular delivery of fucoidan but also as a photothermal agent to effectively ablate different cancer cells (e.g., HeLa, A549, and K562), both  and , with the induction of apoptosis under 808 nm diode laser irradiation. These results point to the potential usage of F-CuS in treating human cancer.

Fucoidan-coated CuS nanoparticles for chemo-and photothermal therapy against cancer.

In our previous study, we showed that Rehmannia glutinosa polysaccharide (RGP) treatment induced maturation of dendritic cells (DCs) and that it had an anticancer effect in mice. The effect of RGP has not been studied in human DCs, including monocyte-derived DCs (MDDCs) and peripheral blood DCs (PBDCs). In this study, we examined DC activation by RGP in human cells. The dendritic morphology of RGP-treated MDDCs was substantially altered as compared with that of phosphate-buffered saline (PBS)-treated control cells. Moreover, RGP treatment markedly decreased phagocytic activity and increased expression levels of co-stimulatory molecules in MDDCs. In addition, RGP treatment elevated the production of proinflammatory cytokines. Furthermore, RGP-induced activation of MDDCs was dependent on the phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). RGP-treated MDDCs promoted upregulation of T-cell activation, including proliferation and interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) production. Analysis of the effect of RGP in PBDC subsets revealed that it induced upregulation of co-stimulatory molecule expression and proinflammatory cytokine production. These data suggest that RGP may function as an immune stimulatory molecule in humans.

Rehmannia glutinosa polysaccharide promoted activation of human dendritic cells.

Europium ion (Eu ) doped Sr SiO phosphors with greenish-yellow emission were synthesized using microwave-assisted sintering. The phase structure and photoluminescence (PL) properties of the obtained phosphor samples were investigated. The PL excitation spectra of the Sr SiO :Eu phosphors exhibited a broad band in the range of 260 nm to 485 nm with a maximum at 361 nm attributed to the 5f-4d allowed transition of the Eu ions. Under an excitation at 361 nm, the Sr SiO :Eu phosphor exhibited a greenish-yellow emission peak at 541 nm with an International-Commission-on-Illumination (CIE) chromaticity of (0.3064, 0.4772). The results suggest that the microwave-assisted sintering method is promising for the synthesis of phosphors owing to the decreased sintering time without the use of additional reductive agents.

Microwave-assisted sintering synthesis of greenish-yellow emitting Sr SiO :Eu phosphors.

The generation of virus-mimetic nanoparticles has received much attention in developing a new vaccine for overcoming the limitations of current vaccines. Thus, a method, encompassing most viral features for their size, hydrophobic domain and antigen display, would represent a meaningful direction for the vaccine development. In the present study, a polymer-templated protein nanoball with direction oriented hemagglutinin1 on its surface (H1-NB) was prepared as a new influenza vaccine, exhibiting most of the viral features. Moreover, the concentrations of antigen on the particle surface were controlled, and its effect on immunogenicity was estimated by in vivo studies. Finally, H1-NB efficiently promoted H1-specific immune activation and cross-protective activities, which consequently prevented H1N1 infections in mice.

Article	Year
Inhibition of breast cancer resistance protein (ABCG2) in human myeloid dendritic cells induces potent tolerogenic functions during LPS stimulation. PloS one\| PubMed ID: 25111504	2014
Impairment of social behavior and communication in mice lacking the Uba6-dependent ubiquitin activation system. Behavioural brain research\| PubMed ID: 25523030	2015
Extracellular synthesis of gold bionanoparticles by Nocardiopsis sp. and evaluation of its antimicrobial, antioxidant and cytotoxic activities. Bioprocess and biosystems engineering\| PubMed ID: 25645365	2015
Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations. Electrophoresis\| PubMed ID: 26095642	2015
Ascophyllan functions as an adjuvant to promote anti-cancer effect by dendritic cell activation. Oncotarget\| PubMed ID: 27008707	2016
Maturation of dendritic cells by pullulan promotes anti-cancer effect. Oncotarget\| PubMed ID: 27341129	2016
Modular delivery of CpG-incorporated lipid-DNA nanoparticles for spleen DC activation. Biomaterials\| PubMed ID: 27886556	2017
Time-dependent effect of E. coli LPS in spleen DC activation in vivo: Alteration of numbers, expression of co-stimulatory molecules, production of pro-inflammatory cytokines, and presentatio... Molecular immunology\| PubMed ID: 28285188	2017
polysaccharide induces toll-like receptor 4 dependent spleen dendritic cell maturation and anti-cancer immunity. Oncoimmunology\| PubMed ID: 28811960	2017
DNA nanoparticles for ophthalmic drug delivery. Biomaterials\| PubMed ID: 29258013	2018
Lipopolysaccharide-coated CuS nanoparticles promoted anti-cancer and anti-metastatic effect by immuno-photothermal therapy. Oncotarget\| PubMed ID: 29285274	2017
Fucoidan-coated CuS nanoparticles for chemo-and photothermal therapy against cancer. Oncotarget\| PubMed ID: 29560098	2018
Rehmannia glutinosa polysaccharide promoted activation of human dendritic cells. International journal of biological macromolecules\| PubMed ID: 29715554	2018
Microwave-assisted sintering synthesis of greenish-yellow emitting Sr SiO :Eu phosphors. Luminescence : the journal of biological and chemical luminescence\| PubMed ID: 29927536	2018
Virus-mimetic polymer nanoparticles displaying hemagglutinin as an adjuvant-free influenza vaccine. Biomaterials\| PubMed ID: 30176403	2018

Minseok Kwak

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