Numerical Simulation of an Improved Updraft Biomass Gasifier Based on Aspen Plus.

In this paper, numerical investigation and optimization is conducted upon an improved updraft gasifier which is expected to overcome the weakness of conventional updraft gasifier. The comprehensive Aspen Plus model of the improved updraft gasifier is based on the RYield and RCSTR reactor. The tar prediction model is constructed, and the yield of tar is determined by the volatile of biomass and gasification temperature. The Aspen Plus simulation results agree very well with experiment results for the product yields and gasification efficiency, which shows the accuracy of the Aspen Plus model. The tar content in syngas of the improved gasifier is proved to be much lower than that of the conventional one by this model. The inflection point of the gasification efficiency occurs when air ratio is 0.25, and the optimum steam proportion in the air is 7.5%. Such a comprehensive investigation could provide necessary information for the optimal design and operation of the improved updraft gasifier.

Modulation of the tumor microenvironment by intratumoral administration of IMO-2125, a novel TLR9 agonist, for cancer immunotherapy.

The objective of cancer immunotherapy is to prime the host's immune system to recognize and attack malignant tumor cells. IMO­2125, a Toll­like receptor 9 (TLR9) agonist, exhibited potent antitumor effects in the murine syngeneic A20 lymphoma and the CT26 colon carcinoma models. IMO­2125 exhibited superior A20 antitumor activity when injected intratumorally (i.t.) compared with equivalent subcutaneous doses. In mice bearing dual CT26 grafts, the i.t. injection of right flank tumors elicited infiltration of cluster of differentiation (CD)3+ T lymphocytes into tumors, resulting in the regression of injected and uninjected left flank tumors. Depletion of CD8+, but not CD4+, T­cells abrogated the IMO­2125­mediated antitumor response, suggesting that CD8+ lymphocytes are required for the antitumor activity. In mice harboring right flank CT26 and left flank ß­galactosidase (ß­gal)­expressing CT26.CL25 grafts, the i.t. administration of IMO­2125 to the CT26 graft resulted in potent and dose­dependent antitumor activity against the two grafts. Splenic T­cells isolated from these mice responded to AH1 antigen (present in the two tumors) and ß­gal antigen (present only in CT26.CL25) in an interferon γ enzyme­linked immunospot assay, suggesting the clonal expansion of T­cells directed against antigens from the two tumors. Mice with ablated CT26 tumors by previous IMO­2125 treatment rejected re­implanted CT26 tumor cells, but not A20 tumor cells, demonstrating that the initial IMO­2125 treatment created a long­lived tumor­specific immune memory of CT26 antigens. A quantitative increase in CD3+ T lymphocytes in injected A20 tumors and an upregulation of selected checkpoint genes, including indoleamine 2,3­dioxygenase (IDO)­1, IDO­2, programmed cell death protein-1 (PD-1); programmed cell death protein ligand 1 (PD-L1), carcinoembryonic antigen­related cell adhesion molecule 1, tumor necrosis factor receptor superfamily member 4 (OX40), OX40 ligand, T­cell immunoglobulin and mucin­domain­containing 3 protein, lymphocyte­activation gene 3, cytotoxic T­lymphocyte­associated protein 4, were observed following IMO­2125 treatment. IMO­2125 also increased immune checkpoint gene expression in injected and uninjected contralateral CT26 tumors, suggesting that the co­administration of anti­CTLA­4, anti­PD­1 or anti­PD­L1 therapies with IMO­2125 may provide additional therapeutic efficacy.

A novel antagonist of Toll-like receptors 7, 8 and 9 suppresses lupus disease-associated parameters in NZBW/F1 mice.

Systemic Lupus Erythematosus is an autoimmune disease characterized by production of autoantibodies against nucleic acid-associated antigens. Endogenous DNA and RNA associated with these antigens stimulate inflammatory responses through Toll-like receptors (TLRs) and exacerbate lupus disease pathology. We have evaluated an antagonist of TLR7, 8 and 9 as a therapeutic agent in lupus-prone NZBW/F1 mice. NZBW/F1 mice treated with the antagonist had lower serum levels of autoantibodies targeting DNA, RNP, Smith antigen, SSA and SSB than did untreated mice. Reduction in blood urea nitrogen and proteinuria and improvements in kidney histopathology were observed in antagonist-treated mice. The antagonist treatment also reduced serum IL-12 and IL-1ß and increased IL-10 levels. Levels of mRNA for IL-6, iNOS and IL-1ß were lower in the kidneys and spleen of antagonist-treated mice than in those of untreated mice. Levels of mRNA for IP-10, TNFRSF9 and FASL were lower and IL-4 mRNA were higher in spleens of antagonist-treated mice than in spleens of untreated mice. mRNA for the inflammasome component NLRP3 was lower and mRNA for the antioxidant enzymes, catalase and glutathione peroxidase 1 was higher in the kidneys of antagonist-treated mice than in those of untreated mice. These results show that the antagonist of TLR7, 8 and 9 effectively inhibits inflammatory pathways involved in the development of lupus in NZBW/F1 mice and constitutes a potential therapeutic approach for the treatment of lupus and other autoimmune diseases.

A Toll-like receptor 7, 8, and 9 antagonist inhibits Th1 and Th17 responses and inflammasome activation in a model of IL-23-induced psoriasis.

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1ß. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases.

Immune-Stimulatory Dinucleotide at the 5'-End of Oligodeoxynucleotides Is Critical for TLR9-Mediated Immune Responses.

Oligodeoxynucleotides (ODNs) containing a CpG or certain synthetic dinucleotides, referred to as immune-stimulatory dinucleotides, induce Toll-like receptor 9 (TLR9)-mediated immune responses. Chemical modifications such as 2'-O-methylribonucleotides incorporated adjacent to the immune-stimulatory dinucleotide on the 5'-side abrogate TLR9-mediated immune responses. In this study, we evaluated the effect of the location of immune-stimulatory dinucleotides in ODNs on TLR9-mediated immune responses. We designed and synthesized ODNs with two immune-stimulatory dinucleotides, one placed toward the 5'-end region and the other toward the 3'-end region, incorporated 2'-O-methylribonucleotides selectively preceding the 5'- or 3'-immune-stimulatory dinucleotide or both, and studied TLR9-mediated immune responses of these compounds in cell-based assays and in vivo in mice. These studies showed that an immune-stimulatory dinucleotide located closer to the 5'-end is critical for and dictates TLR9-mediated immune responses. These studies provide insights for the use of ODNs when employed as TLR9 agonists and antagonists or antisense agents.

Impact of nature and length of linker incorporated in agonists on toll-like receptor 9-mediated immune responses.

Oligodeoxynucleotides containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). We previously reported a novel class of TLR9 agonists, referred to as immune-modulatory oligonucleotides (IMOs), in which two 11-mers of the same sequence are attached via their 3'-ends through a 1,2,3-propanetriol linker and contain a synthetic immune-stimulatory motif, Cp7-deaza-dG. In the present study, we have examined the impact of length, nature, and stereochemistry of the linker incorporated in agonists for TLR9 activation. The new linkers studied include (S)-(-)-1,2,4-butanetriol, 1,3,5-pentanetriol, cis,cis-1,3,5-cyclohexanetriol, cis,trans-1,3,5-cyclohexanetriol, 1,3,5-tris(2-hydroxyethyl)isocyanurate, tetraethyleneglycol, and hexaethyleneglycol in place of 1,2,3-propanetriol linker. Agonists with various linkers are studied for TLR9-mediated immune responses in HEK293 cells, human cell-based assays, and in vivo in mice. Results of these studies suggest that C3-C5 linkers, 1,2,3-propanetriol, (S)-(-)-1,2,4-butanetriol, or 1,3,5-pentanetriol, are optimal for stimulation of TLR9-mediated immune responses. Rigid C3 linkers with different stereochemistry have little effect on immune stimulation, while linkers longer than C5 reduced TLR9-mediated immune stimulation.

Peptide conjugation at the 5'-end of oligodeoxynucleotides abrogates toll-like receptor 9-mediated immune stimulatory activity.

Bacterial and synthetic DNA containing unmethylated CpG motifs act as ligands of Toll-like receptor 9 (TLR9). Our earlier studies showed that 5'-accessibility of synthetic oligodeoxynucleotides containing CpG motif (ODN) is required for TLR9-mediated immune stimulatory activity. Blocking the 5'-end of ODN through conjugation to a variety of moieties reduces immune stimulatory activity (Bioconjugate Chem. 2002, 13, 966-974). In the present study, we conjugated a model peptide, a 28-amino-acid-long beta-amyloid peptide, to either the 5'- or the 3'-end of an ODN via C3 and C6 alkyl linkers. We compared the immune stimulatory activity of the resulting conjugates with that of a parent ODN without conjugation in TLR9-transfected cells, mouse spleen cell cultures, and in vivo in mice. ODN with the peptide conjugated at the 3'-end via C3 and C6 linkers had immune stimulatory activity similar to that of the parent ODN in both in vitro and in vivo in mice. On the contrary, conjugation of peptide at the 5'-end of the ODN significantly abrogated immune stimulatory activity. In conclusion, the results presented here demonstrate that peptide/protein conjugation to ODN is optimal at the 3'-end with either C3 or C6 linker and conjugation at the 5'-end leads to significant loss of TLR9-mediated immune stimulation.

Toll-like receptor 7 selective synthetic oligoribonucleotide agonists: synthesis and structure-activity relationship studies.

We previously reported a novel class of stabilized immune-modulatory RNA (SIMRA) compounds that activates TLR8 or both TLR7 and TLR8 depending on the nucleotide composition and chemical modifications incorporated. In the present study, to identify TLR7-selective agonists, we designed and synthesized novel SIMRA compounds with varying sequence compositions substituting 7-deaza-G for natural guanosine and studied immune-stimulatory activity in cell-based assays and in vivo in mice. SIMRA compounds activated NF-kappaB in HEK293 cells expressing TLR7 and induced cytokine production in mouse spleen cells and human PBMCs and higher levels of IFN-alpha in human pDCs, which correlated with TLR7 activation. Subcutaneous administration of SIMRA compounds to mice increased serum cytokine levels. TLR knockout mouse studies showed that both TLR7 and MyD88 are required for activity of SIMRA compounds. The presence of a 5'-AA/CN (A > C and N = U/C/7-deaza-G) and/or C/AUU-3' (C > A) trinucleotide at the 5'- and 3'-ends of SIMRA compound along with a 5'-AN(1)N(2)UG1A-3' (N(1) = A/C; N(2) = U/C/7-deaza-G) or UG1AZ(1)G1Z(2)UU (Z(1) = A < C; Z(2) = C < A) motif confers TLR7 selectivity over other sequence compositions. In conclusion, we have designed and synthesized novel SIMRA compounds that selectively act as agonists of TLR7.

Modifications incorporated in CpG motifs of oligodeoxynucleotides lead to antagonist activity of toll-like receptors 7 and 9.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG dinucleotides act as agonists of TLR9 and induce Th1-type immune responses. In the present study, we synthesized CpG containing ODNs in which C or G was substituted with 2'-O-methylribonucleotides, 5-methyl-dC, or 2'-O-methyl-5-methyl-C and studied their immune stimulatory activity alone and in combination with TLR agonists. In mouse and human primary cell-based assays, modified ODNs did not stimulate immune responses but inhibited TLR9 agonist-induced immune stimulatory activity. In mice, modified ODNs did not induce cytokines but inhibited immune responses induced by agonists of TLR7 and TLR9. Modified ODNs did not inhibit endosomal TLR3- or cell-surface TLR4-agonist-induced cytokines. This study demonstrates that ODNs incorporated with chemical modifications in CpG dinucleotides do not induce immune stimulatory activity but act as antagonists of TLR7 and TLR9 in vitro and in vivo. These types of modifications are commonly employed in antisense sequences and thereby may affect the intended mechanism of action.

Oligodeoxyribonucleotide-based antagonists for Toll-like receptors 7 and 9.

Oligodeoxyribonucleotides containing unmethylated CpG motifs act as TLR9 agonists. In this study, we evaluated oligonucleotides containing an unmethylated CpG motif in which two nucleotides adjacent to the CpG dinucleotide were substituted with 2'-O-methylribonucleotides, resulting in TLR7 and TLR9 antagonists. In mouse and human cell cultures, antagonists did not stimulate immune activation but inhibited TLR7 and TLR9 agonist-induced activity. In mice, antagonists inhibited immune responses induced by TLR9 agonists for up to several days, and the inhibition was dose-dependent. Antagonists also inhibited immune responses induced by an RNA-based TLR7/8 agonist but not TLRs 2, 3, 4, or 5 agonists in mice. Additionally, antagonist inhibited TLR9 agonist-induced IL-6 in lupus-prone MRL/lpr mouse spleen cell cultures. These results indicate that antagonists described herein can suppress immune responses induced by TLR7 and TLR9 agonists. Antagonists may be suitable candidates for treating inflammatory and autoimmune diseases where inappropriate or uncontrolled TLR activation has been implicated.

Agonists of Toll-like receptor 9 containing synthetic dinucleotide motifs.

Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs activate Toll-like receptor 9 (TLR9). Our previous studies have shown that ODNs containing two 5'-ends are more immunostimulatory than those with one 5'-end. In the present study, to understand the role of functional groups in TLR9 recognition and subsequent immune response, we substituted C or G of a CpG dinucleotide with 5-OH-dC, 5-propyne-dC, furano-dT, 1-(2'-deoxy-beta- d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine, dF, 4-thio-dU, N(3)-Me-dC, N (4)-Et-dC, Psi-iso-dC, and arabinoC or 7-deaza-dG, 7-deaza-8-aza-dG, 9-deaza-dG, N(1)-Me-dG, N(2)-Me-dG, 6-Thio-dG, dI, 8-OMe-dG, 8-O-allyl-dG, and arabinoG in ODN containing two 5'-ends. Agonists of TLR9 containing cytosine or guanine modification showed activity in HEK293 cells expressing TLR9, mouse spleen, and human cell-based assays and in vivo in mice. The results presented here provide insight into which specific chemical modifications at C or G of the CpG motif are recognized by TLR9 and the ability to modulate immune responses substituting natural C or G in immune modulatory oligonucleotides.

Stabilized immune modulatory RNA compounds as agonists of Toll-like receptors 7 and 8.

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptor (TLR)7 and TLR8. However, single-stranded RNA is rapidly degraded by ubiquitous RNases, and the studies reported to date have used RNA with lipid carriers. To overcome nuclease susceptibility of RNA, we have synthesized several RNAs incorporating a range of chemical modifications. The present study describes one pool of RNA compounds, referred to as stabilized immune modulatory RNA (SIMRA) compounds, in which two RNA segments are attached through their 3' ends. SIMRA compounds showed greater stability in human serum compared with linear RNA and activated human TLR8, but not TLR7, in HEK293 cells without using lipid carriers. Interestingly, another set of SIMRA compounds containing 7-deazaguanosine substituted for natural guanosine activated human TLR7 and TLR8. Additionally, TLR7- and TLR8-activating compounds, but not the compounds that activated only TLR8, stimulated mouse immune cells in vitro and in vivo and produced dose-dependent T helper 1-type cytokines. Both types of compounds activated human peripheral blood mononuclear cells, but only TLR7- and TLR8-activating compounds activated plasmacytoid dendritic cells and produced high levels of IFN-alpha. In monkeys, s.c. administration of both types of SIMRA compounds induced transient changes in peripheral blood monocytes and neutrophils, and activated T lymphocytes, monocytes, and NK cells. Both types of compounds induced IFN-gamma-inducible protein 10, but only the 7-deazaguanosine-containing compound that activated both TLR7 and TLR8 induced IFN-alpha in monkeys. This is a comprehensive study of RNA-based compounds containing structures and synthetic stimulatory motifs in mouse, monkey, and human systems without using lipid carriers.

Oral administration of a synthetic agonist of Toll-like receptor 9 potently modulates peanut-induced allergy in mice.

BACKGROUND: Agonists of Toll-like receptor 9 have been shown to induce potent T(H)1-type immune responses and prevent and reverse ovalbumin-induced T(H)2-dominant allergic asthma in mice. OBJECTIVE: We examined oral administration of a synthetic agonist of Toll-like receptor 9 (immune modulatory oligonucleotide [IMO]) to modulate peanut-induced allergy in mice. METHODS: In the prevention model mice were sensitized 3 times by means of oral administration of peanut in the presence or absence of IMO. In a treatment protocol mice were sensitized orally with peanut on days 0 and 14 and treated 4 times with oral administration of IMO starting on day 21. RESULTS: In the prevention study mice that received the combination of IMO/peanut showed decreased IgE and increased IgG2a levels in the serum and intestinal tissue compared with mice sensitized with peanut only. In spleen cell recall experiments, production of IL-5 and IL-13 was inhibited and production of IFN-gamma was increased in mice immunized with the peanut/IMO combination compared with those sensitized with peanut only. In the treatment model IMO-treated mice showed decreased IgE, IL-5, and IL-13 levels and increased IgG2a and IFN-gamma levels in the serum, intestines, and spleen cells compared with PBS-treated mice. A reduction in local inflammation and restoration of normal structure in the intestines was found in the mice that received IMO in both models. CONCLUSION: These results indicate that IMOs can switch peanut-induced T(H)2 immune responses toward T(H)1 responses accompanied by reduced inflammation in the gastrointestinal tract and anaphylaxis in both the prevention and treatment models. CLINICAL IMPLICATIONS: IMOs might be suitable candidates for the management of peanut-induced allergy.

Novel oligodeoxynucleotide agonists of TLR9 containing N3-Me-dC or N1-Me-dG modifications.

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation.

Modulation of ovalbumin-induced Th2 responses by second-generation immunomodulatory oligonucleotides in mice.

Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG DNAs) prevent development of T-helper type 2 (Th2) immune responses and reverse established allergic responses in mouse models. We recently reported that second-generation immunomodulatory oligonucleotides (IMOs) containing novel structures (immunomers) and a synthetic immunostimulatory CpR (R=2'-deoxy-7-deazguanosine) motif induce the production of distinct cytokine secretion profiles in vitro and in vivo. In the present study, we evaluated IMOs containing CpG and CpR motifs to modulate allergen-induced Th2 immune responses in prevention and treatment models. Mice sensitized and challenged with ovalbumin (OVA) were treated with a CpG DNA or an IMO by administration either at the time of OVA sensitization (co-administration; prevention) or after establishment of an allergic response (treatment). Spleens, blood, and lungs were collected and analyzed for immune responses. Spleen-cell cultures harvested from OVA-sensitized mice showed a significant decrease in Th2 cytokine levels with a concomitant increase in Th1 cytokine levels only when CpG DNA or IMOs were co-administered with OVA. The co-administration of CpG DNA or IMOs during OVA sensitization significantly reduced serum OVA-specific and total IgE levels in mice. The mice who received CpG DNA or IMOs co-administered with OVA showed a small reduction in serum OVA-specific and total IgG1 levels and a significant increase in serum OVA-specific and total IgG2a levels. Similar results were found in mice with established allergic responses who received IMO treatment. IMO treatment also resulted in strong inhibition of inflammatory cell infiltration and goblet cell hyperplasia in the lungs compared with untreated mice lungs. These data demonstrate that IMOs prevent antigen-induced Th2 immune responses when co-administered to mice during OVA sensitization and that IMOs reverse established allergic responses induced by OVA.

A dinucleotide motif in oligonucleotides shows potent immunomodulatory activity and overrides species-specific recognition observed with CpG motif.

Bacterial and synthetic DNAs containing CpG dinucleotides in specific sequence contexts activate the vertebrate immune system through Toll-like receptor 9 (TLR9). In the present study, we used a synthetic nucleoside with a bicyclic heterobase [1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine; R] to replace the C in CpG, resulting in an RpG dinucleotide. The RpG dinucleotide was incorporated in mouse- and human-specific motifs in oligodeoxynucleotides (oligos) and 3'-3-linked oligos, referred to as immunomers. Oligos containing the RpG motif induced cytokine secretion in mouse spleen-cell cultures. Immunomers containing RpG dinucleotides showed activity in transfected-HEK293 cells stably expressing mouse TLR9, suggesting direct involvement of TLR9 in the recognition of RpG motif. In J774 macrophages, RpG motifs activated NF-kappa B and mitogen-activated protein kinase pathways. Immunomers containing the RpG dinucleotide induced high levels of IL-12 and IFN-gamma, but lower IL-6 in time- and concentration-dependent fashion in mouse spleen-cell cultures costimulated with IL-2. Importantly, immunomers containing GTRGTT and GARGTT motifs were recognized to a similar extent by both mouse and human immune systems. Additionally, both mouse- and human-specific RpG immunomers potently stimulated proliferation of peripheral blood mononuclear cells obtained from diverse vertebrate species, including monkey, pig, horse, sheep, goat, rat, and chicken. An immunomer containing GTRGTT motif prevented conalbumin-induced and ragweed allergen-induced allergic inflammation in mice. We show that a synthetic bicyclic nucleotide is recognized in the C position of a CpG dinucleotide by immune cells from diverse vertebrate species without bias for flanking sequences, suggesting a divergent nucleotide motif recognition pattern of TLR9.

Effect of cytofectins on the immune response of murine macrophages to mammalian DNA.

DNA, depending on base sequence, can induce a wide range of immune responses. While bacterial DNA is stimulatory, mammalian DNA is inactive alone and can, moreover, inhibit the response to bacterial DNA. To determine whether the mode of cell entry affects the immune properties of mammalian DNA, we have investigated the effects of the cytofectin agents Fugene 6 (Roche Diagnostics Corp., Indianapolis, IN), Lipofectin and Lipofectamine (Life Technologies, Grand Island, NY) on the responses of murine macrophages to DNA from calf thymus and human placenta. Whereas calf thymus and human placenta DNA alone failed to stimulate J774 or RAW264.7 cell lines or bone marrow-derived macrophages, these DNAs in complexes with cytofectin agents stimulated macrophages to produce nitric oxide but not interleukin 12. Both single-stranded and double-stranded DNAs were active in the presence of cytofectins. Macrophage activation by the DNA-cytofectin complexes was reduced by chloroquine, suggesting a role of endosomal acidification in activation. As shown by flow cytometry and confocal microscopy, the cytofectins caused an increase in the uptake of DNA into cells. Our findings indicate that macrophages vary in their response to DNA depending on uptake pathway, suggesting that activation by DNA reflects not only sequence but also context or intracellular location.

Divergent synthetic nucleotide motif recognition pattern: design and development of potent immunomodulatory oligodeoxyribonucleotide agents with distinct cytokine induction profiles.

Unmethylated CpG dinucleotides present within certain specific sequence contexts in bacterial and synthetic DNA stimulate innate immune responses and induce cytokine secretion. Recently, we showed that CpG DNAs containing two 5'-ends, immunomers, are more potent in both regards. In this study, we show that an immunomer containing a synthetic CpR motif (R = 2'-deoxy-7-deazaguanosine) is a potent immunostimulatory agent. However, the profile of cytokine induction is different from that with immunomers containing a natural CpG motif. In general, a CpR immunomer induced higher interleukin (IL)-12 and lower IL-6 secretion. Compared with conventional CpG DNAs, both types of immunomers showed a rapid and enhanced activation of the transcription factor NF-kappaB in J774 cells. NF-kappaB activation by CpG DNA corresponded to degradation of IkappaBalpha in J774 cells. All three immunostimulatory oligonucleotides activated the p38 mitogen-activated protein kinase pathway as expected. Immunomers containing CpG and CpR motifs showed potent reversal of the antigen-induced Th2 immune response towards a Th1 type in antigen-sensitized mouse spleen cell cultures. Immunomers containing a CpR motif showed significant antitumor activity in nude mice bearing MCF-7 human breast cancer and U87MG glioblastoma xenografts. These studies suggest the ability for a divergent synthetic nucleotide motif recognition pattern of the receptor involved in the immunostimulatory pathway and the possibility of using synthetic nucleotides to elicit different cytokine response patterns.

Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides.

Depending on sequence and backbone structure, DNA can inhibit as well as stimulate immune responses. As previously shown, single-base phosphorothioate (Ps) oligodeoxynucleotides (ODN) can inhibit murine macrophage activation. To determine whether these compounds can also affect dendritic cells (DC), the effects of 30-mer Ps ODN (SdA, SdT, SdG, and SdC) on DC activation were assessed in an in vitro system. With DC preparations obtained from murine bone marrow cultured in granulocyte macrophage-colony stimulating factor, the Ps ODN blocked the production of interleukin-12 and nitric oxide induced by bacterial DNA, an immunostimulatory cytosine phosphate guanosine dinucleotide (CpG) ODN and lipopolysaccharide (LPS). Furthermore, these compounds inhibited up-regulation of costimulatory molecules CD40 and CD86 as well as major histocompatibility complex-II molecules, indicating an effect on DC maturation. Although the Ps ODN limited uptake of CpG ODN as assessed by flow cytometry, the Ps ODN did not affect LPS uptake, suggesting that these compounds inhibit DC responses by effects on downstream signaling pathways. Together, these observations extend the range of action of inhibitory ODN to DC and suggest a role of these compounds as immunomodulatory agents.

Potent CpG oligonucleotides containing phosphodiester linkages: in vitro and in vivo immunostimulatory properties.

Bacterial and synthetic DNAs, containing CpG dinucleotides in specific sequence contexts, activate the vertebrate immune system. Unlike phosphorothioate (PS) CpG DNAs, phosphodiester (PO) CpG DNAs require either palindromic sequences and/or poly(dG) sequences at the 3(')-end for activity. Here, we report 'PO-immunomers' having two PO-CpG DNA molecules joined through their 3(')-ends. These PO-imunomers permitted us, for the first time, to assess immunostimulatory properties of PO-CpG DNAs in vitro and in vivo without the need for palindromic and/or poly(dG) sequences. In medium containing 10% fetal bovine serum, PO-immunomers were more resistant than PO-CpG DNAs to nucleases. Compared to PS-CpG DNA in BALB/c and C3H/HeJ mice spleen cell culture assays, PO-immunomers showed increased IL-12 secretion and minimal amounts of IL-6 secretion. PO-immunomers activated NF-kappa B and induced cytokine secretion in J774 cell cultures. In addition, PO-immunomers showed antitumor activity in nude mice bearing human breast (MCF-7) and prostate (DU145) cancer xenografts.