Local Induction of B Cell Interleukin-10 Competency Alleviates Inflammation and Bone Loss in Ligature-Induced Experimental Periodontitis in Mice.

Interleukin-10 (IL-10)-producing B cells (B10 cells) play a critical role in the immune system balance by negatively regulating inflammatory responses. This study was conducted to determine the effect of local B10 cell induction on periodontal inflammation and bone loss in ligature-induced experimental periodontitis in vivo Purified spleen B cells from C57BL/6J mice (8 to 10 weeks old) were cultured with CD40 ligand (CD40L) and the Toll-like receptor 9 (TLR9) agonist cytidine-phosphate-guanosine oligodeoxynucleotide (CpG) to determine effective IL-10 induction in vitro Silk ligatures (size 7-0) were tied around the mouse maxillary second molars on day 0, followed by the injection of CD40L and CpG into the palatal gingiva on days 3, 6, and 9. All the mice were sacrificed, and samples were collected on day 14. CD40L and CpG significantly increased the level of IL-10 production by B cells in vitro, although the frequencies of CD1dhi CD5+ and IL-10-producing (IL-10+) CD45+ cells were decreased. IL-10 was predominantly produced by the CD1dhi CD5+ subpopulation of B cells. In vivo, both IL-10 mRNA expression and the number of IL-10+ CD45+ cells were significantly increased after gingival injection of CD40L and CpG. Periodontal bone loss was significantly decreased and the gingival expression of IL-1ß, tumor necrosis factor alpha, and RANKL was significantly reduced. The number of multinucleated tartrate-resistant acid phosphatase-positive cells along the alveolar bone surface was significantly decreased after gingival injection of CD40L and CpG. This study indicates for the first time that the local induction of B10 cell activity could inhibit periodontal inflammation and bone loss.

Pathogen-Mimicking Polymeric Nanoparticles based on Dopamine Polymerization as Vaccines Adjuvants Induce Robust Humoral and Cellular Immune Responses.

Aiming to enhance the immunogenicity of subunit vaccines, a novel antigen delivery and adjuvant system based on dopamine polymerization on the surface of poly(D,L-lactic-glycolic-acid) nanoparticles (NPs) with multiple mechanisms of immunity enhancement is developed. The mussel-inspired biomimetic polydopamine (pD) not only serves as a coating to NPs but also functionalizes NP surfaces. The method is facile and mild including simple incubation of the preformed NPs in the weak alkaline dopamine solution, and incorporation of hepatitis B surface antigen and TLR9 agonist unmethylated cytosine-guanine (CpG) motif with the pD surface. The as-constructed NPs possess pathogen-mimicking manners owing to their size, shape, and surface molecular immune-activating properties given by CpG. The biocompatibility and biosafety of these pathogen-mimicking NPs are confirmed using bone marrow-derived dendritic cells. Pathogen-mimicking NPs hold great potential as vaccine delivery and adjuvant system due to their ability to: 1) enhance cytokine secretion and immune cell recruitment at the injection site; 2) significantly activate and maturate dendritic cells; 3) induce stronger humoral and cellular immune responses in vivo. Furthermore, this simple and versatile dopamine polymerization method can be applicable to endow NPs with characteristics to mimic pathogen structure and function, and manipulate NPs for the generation of efficacious vaccine adjuvants.

Automated solid-phase synthesis of high capacity oligo-dT cellulose for affinity purification of poly-A tagged biomolecules.

Affinity purification of poly-adenylated biomolecules using solid supports that are derivatized with poly-thymidine oligonucleotides provides a powerful method for isolating cellular mRNA. These systems have also been used to purify mRNA-peptide fusions generated by RNA-display. However, the commercial source for high capacity oligo-dT cellulose was recently discontinued. To overcome this problem, we have developed a low cost solid-phase synthesis protocol to generate oligo-dT cellulose. Comparative binding studies indicate that chemically synthesized oligo-dT cellulose functions with superior loading capacity when compared to the discontinued product. We suggest that this method could be used to synthesize oligo-dT resin for routine purification of poly-adenylated biomolecules.

Design of a new fluorescent probe: pyrrole/imidazole hairpin polyamides with pyrene conjugation at their γ-turn.

Fluorophores that are conjugated with N-methylpyrrole-N-methylimidazole (Py-Im) polyamides postulates versatile applications in biological and physicochemical studies. Here, we show the design and synthesis of new types of pyrene-conjugated hairpin Py-Im polyamides (1-5). We evaluated the steady state fluorescence of the synthesized conjugates (1-5) in the presence and absence of oligodeoxynucleotides 5'-CGTATGGACTCGG-3' (ODN 1) and 5'-CCGAGTCCATACG-3' (ODN 2) and observed a distinct increase in emission at 386nm with conjugates 4 and 5. Notably, conjugate 5 that contains a ß-alanine linker had a stronger binding affinity (K(D)=1.73×10(-8)M) than that of conjugate 4 (K(D)=1.74×10(-6)M). Our data suggests that Py-Im polyamides containing pyrene fluorophore with a ß-alanine linker at the γ-turn NH(2) position can be developed as the competent fluorescent DNA-binding probes.

In vivo delivery of nucleic acids via glycopolymer vehicles affords therapeutic infarct size reduction in vivo.

Using a new class of nontoxic and degradable glycopolymer-based vehicles termed poly(glycoamidoamine)s, we demonstrate virus-like delivery efficacy of oligodeoxynucleotide (ODN) decoys to cardiomyoblasts (H9c2), primary cardiomyocytes, and the mouse heart. These glycopolymers bind and compact ODN decoys into nanoparticle complexes that are internalized by the cell membrane and mediate nuclear uptake of DNA in 90+% of cultured primary cardiomyocytes and 87% of the mouse myocardium. Experimental results reveal that decoys delivered via these glycopolymers block the activation of the transcription factor NF-κB, a major contributor to ischemia/reperfusion injury. Decoy complexes formed with glycopolymer T4 significantly blocked downstream gene expression of Cox-2 and limited myocardial infarction in vivo, phenocopying a transgenic mouse model. These promising delivery vehicles may facilitate high-throughput genetic approaches in animal models. Additionally, the low toxicity, biodegradation, and outstanding delivery efficacy suggest that these nanomedicines may be clinically applicable for gene regulatory therapy.

The immunostimulatory activity of unmethylated and methylated CpG oligodeoxynucleotide is dependent on their ability to colocalize with TLR9 in late endosomes.

TLR9 recognizes CpG motifs present in pathogenic DNA and triggers potent immune responses. It is generally accepted that TLR9 distinguishes pathogenic DNA based, in part, on methylation status, where TLR9 binds unmethylated but not methylated CpG. However, we showed that methylated CpG induces potent TLR9-mediated responses when delivered in lipid nanoparticles. In this article, we report that methylation dictates the ability of free CpG DNA to colocalize with TLR9 in late endosomes. However, when delivered in lipid nanoparticles, CpG DNA and TLR9 colocalize, regardless of methylation status. Therefore, it is proposed that the ability of immune cells to distinguish unmethylated pathogenic from methylated mammalian DNA is controlled by a mechanism that regulates TLR9 mobilization and colocalization rather than a differential binding affinity.

Sublingual immunization with recombinant GroEL plus CpG-ODN inhibits Porphyromonas gingivalis-induced inflammation and alveolar bone loss.

It has been reported that GroEL, a heat shock protein (HSP) produced by the representative periodontopathogenic bacterium, Porphyromonas gingivalis, induces inflammation-induced osteoclastogenesis and promotes alveolar bone resorption. In this study, we demonstrated the efficacy of a mucosal vaccine targeting GroEL against bone resorption induced by P. gingivalis. Female BALB/c mice received sublingual CpG oligodeoxynucleotide as an adjuvant with recombinant GroEL (rGroEL) prior to P. gingivalis exposure. Animals were euthanized 30 days after P. gingivalis inoculation. Sublingual immunization (SLI) with rGroEL elicited significant rGroEL-specific serum immunoglobulin (Ig)G and salivary IgA antibody (Ab) responses, and these responses were sustained for approximately 1 year. Interestingly, 10-fold more GroEL-specific IgA Ab-producing cells were detected in the submandibular glands (SMGs) than in the spleen. Antigen (Ag)-specific cells isolated from the spleen and SMGs induced significantly higher levels of IFN-γ expression after Ag restimulation in vitro. Flow cytometry illustrated that the frequency of CD11b+ dendritic cells with enhanced expression of CD80, CD86, CD40, and major histocompatibility complex II molecules was significantly increased in the SMGs. Furthermore, SLI with rGroEL significantly suppressed P. gingivalis-induced alveolar bone resorption and P. gingivalis-stimulated tumor necrosis factor-α, interleukin-6, and HSP60 expression in the gingiva. These findings suggest that SLI with rGroEL and CpG oligodeoxynucleotide is a beneficial strategy for preventing periodontal disease, mainly by presenting Ags in the oral region and inducing antibody production in the mucosal and systemic systems.

Production of antibodies with peptide-CpG-DNA-liposome complex without carriers.

BACKGROUND: The screening of peptide-based epitopes has been studied extensively for the purpose of developing therapeutic antibodies and prophylactic vaccines that can be potentially useful for treating cancer and infectious diseases such as influenza virus, malaria, hepatitis B, and HIV. To improve the efficacy of antibody production by epitope-based immunization, researchers evaluated liposomes as a means of delivering vaccines; they also formulated adjuvants such as flagella and CpG-DNA to enhance the magnitude of immune responses. Here, we provide a potent method for peptide-based epitope screening and antibody production without conventional carriers. RESULTS: We present that a particular form of natural phosphodiester bond CpG-DNA encapsulated in a specific liposome complex (Lipoplex(O)) induces potent immunomodulatory activity in humans as well as in mice. Additionally, Lipoplex(O) enhances the production of IgG2a specific to antigenic protein in mice. Most importantly, immunization of mice with several peptides co-encapsulated with Lipoplex(O) without carriers significantly induces each peptide-specific IgG2a production in a TLR9-dependent manner. A peptide-specific monoclonal antibody produced against hepatocellular carcinoma-associated antigen has functional effects on the cancer cells. CONCLUSIONS: Our overall results show that Lipoplex(O) is a potent adjuvant and that complexes of peptide and Lipoplex(O) are extremely useful for B cell epitope screening and antibody production without carriers. Therefore, our strategy may be promptly used for the development of therapeutic antibodies by rapid screening of potent B cell epitopes.

Islet surface modification with urokinase through DNA hybridization.

Transplantation of islets of Langerhans (islets) has been proposed as a safe, effective approach to treating patients with insulin-dependent diabetes mellitus (type I diabetes). It has been reported, however, that many islets are lost in the early phase after intraportal transplantation by instant blood coagulation-mediated inflammatory reactions. In this study, DNA hybridization was applied to conjugate the fibrinolytic enzyme urokinase on the islet surface. We synthesized amphiphilic polymers, PEG-lipids carrying oligo(dT)(20) (oligo(dT)(20)-PEG-lipid; PEG MW = 5000) and urokinase (UK) carrying oligo(dA)(20). The oligo(dT)(20)-PEG-lipid was spontaneously incorporated into the cell membrane through interactions between the hydrophobic parts of the PEG-lipids and the lipid bilayer, and UK was conjugated on the cell surface through DNA hybridization between oligo(dT)(20) on the cell and complementary oligo(dA)(20) on the UK. The activity of UK was maintained on the islet surface. The surface modification with UK did not influence islet morphology or islet ability to secrete insulin in response to changes in glucose concentration. No practical volume increase was observed with our method, indicating that islet graft loss could be suppressed at the early stage of intraportal islet transplantation.

Self-catalyzed degradable cationic polymer for release of DNA.

The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen/phosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.

Controlling the Biological Fate of Liposomal Spherical Nucleic Acids Using Tunable Polyethylene Glycol Shells.

Liposomal spherical nucleic acids (LSNAs) modified with polyethylene glycol (PEG) units are studied in an attempt to understand how the circulation time and biodistribution of the constructs can be manipulated. Specifically, the effect of (1) PEG molecular weight, (2) PEG shell stability, and (3) PEG modification method (PEG in both the core and shell versus PEG in the shell only) on LSNA blood circulation, biodistribution, and in vivo cell internalization in a syngeneic, orthotopic triple-negative breast cancer mouse model is studied. Generally, high PEG molecular weight extends blood circulation lifetime, and a more lipophilic anchor stabilizes the PEG shell and improves circulation and tumor accumulation but at the cost of cell uptake efficiency. The PEGylation strategy has a minor effect on in vitro properties of LSNAs but significantly alters in vivo cell uptake. For example, surface-only PEG in one design contributed to higher in vivo cell internalization than its counterpart with PEG both in the shell and core. Taken together, this work provides guidelines for designing LSNAs that exhibit maximal in vivo cancer cell uptake characteristics in the context of a breast cancer model.

pH-sensitive PEG-coated hyper-branched ß-d-glucan derivative as carrier for CpG oligodeoxynucleotide delivery.

ß-d-glucan is a natural non-digestible polysaccharide that can be selectively recognized by recognition receptors such as Dectin-1 receptors, resulting in an emerging interest on exploring its capacity for carrying biological information to desired organs or cells. CpG oligodeoxynucleotide (ODN) has the potentiality to initiate an immune-stimulatory cascade via activating B cells inducing proinflammatory cytokines, which is conducive to immunotherapy and nucleic acid vaccine. Herein, we developed a pH-sensitive delivery system loading with CpG ODN by introducing poly-ethylenimine (PEI) to a hyperbranched ß-d-glucan (HBB) and coating with poly-ethylene glycol (PEG) shell via acidic liable Schiff bond. This delivery system exhibited a favorable biocompatibility and facilitated the cellular uptake of CpG ODN at pH 6.8 with the possibility of having higher accumulation in acidic cancer microenvironment. Furthermore, this carrier together with class B CpG ODN could enhance the secretion of cytokines including interleukin-6 and interferon-α as well as capable of interferon-α induction.

G-protein coupled receptor array technologies: site directed immobilisation of liposomes containing the H1-histamine or M2-muscarinic receptors.

This paper describes a novel strategy to create a microarray of G-protein coupled receptors (GPCRs), an important group of membrane proteins both physiologically and pharmacologically. The H(1)-histamine receptor and the M(2)-muscarinic receptor were both used as model GPCRs in this study. The receptor proteins were embedded in liposomes created from the cellular membrane extracts of Spodoptera frugiperda (Sf9) insect cell culture line with its accompanying baculovirus protein insert used for overexpression of the receptors. Once captured onto a surface these liposomes provide a favourable lipidic environment for the integral membrane proteins. Site directed immobilisation of these liposomes was achieved by introduction of cholesterol-modified oligonucleotides (oligos). These oligo/cholesterol conjugates incorporate within the lipid bilayer and were captured by the complementary oligo strand exposed on the surface. Sequence specific immobilisation was demonstrated using a quartz crystal microbalance with dissipation (QCM-D). Confirmatory results were also obtained by monitoring fluorescent ligand binding to GPCRs captured on a spotted oligo microarray using Confocal Laser Scanning Microscopy and the Zepto-READER microarray imaging system. Sequence specific immobilisation of such biologically important membrane proteins could lead to the development of a heterogeneous self-sorting liposome array of GPCRs which would underpin a variety of future novel applications.

Use of quantitative (1)H NMR chemical shift changes for ligand docking into barnase.

(1)H NMR complexation-induced changes in chemical shift (CIS) of HN protons have been used to characterize the complexes of barnase with the deoxyoligonucleotides d(GC) and d(CGAC). Quantitative shift changes are used not only to locate the most probable binding site (using ring-current shifts), but also to determine the orientation of the ligand within the binding site, based on a more complete shift calculation including bond magnetic anisotropies and electric field effects. For both ligands, the guanine is in the same binding site cleft, in the same position as identified in the crystal structure of the d(CGAC) complex. By contrast, a previous X-ray crystal structure of the d(GC) complex showed the ligand in the mouth of the active site, rather than at the guanyl-specific site, implying that the location may be an artifact of the crystallisation process.

HTLV-I Tax protein stimulation of DNA binding of bZIP proteins by enhancing dimerization.

The Tax protein of human T cell leukemia virus type-1 (HTLV-I) transcriptionally activates the HTLV-I promoter. This activation requires binding sites for activating transcription factor (ATF) proteins, a family of cellular proteins that contain basic region-leucine zipper (bZIP) DNA binding domains. Data are presented showing that Tax increases the in vitro DNA binding activity of multiple ATF proteins. Tax also stimulated DNA binding by other bZIP proteins, but did not affect DNA binding proteins that lack a bZIP domain. The increase in DNA binding occurred because Tax promotes dimerization of the bZIP domain in the absence of DNA, and the elevated concentration of the bZIP homodimer then facilitates the DNA binding reaction. These results help explain how Tax activates viral transcription and transforms cells.

Flow cytometry and live confocal analysis for the evaluation of the uptake and intracellular distribution of FITC-ODN into HaCaT cells.

In this study, the mechanism of the internalization and the cellular distribution of 59 fluorescein conjugated PS-ODN (FITC-ODN) after transfection with different mixed lipidic vesicles/oligo complexes (lipoplexes) have been investigated. Mixed lipidic vesicles were prepared with one of the most used cationic lipid (DOTAP) and different amounts of a cholic acid (UDCA) to release the oligo into HaCaT cells. Using flow cytometry, the cellular uptake of the oligo was studied with and without different inhibitors able to block selectively the different pathways involved in the internalization mechanism. The intracellular distribution of the oligo was analyzed by confocal laser scanning microscopy (CLSM), treating the cells with the lipoplexes and directly observing without any fixing procedure. To better carry out the colocalization studies, fluorescent-labeled markers, specific for the different cellular compartments, were coincubated with 59 fluorescein-conjugated 29-mer phosphorotioate oligonucleotide (FITC-ODN). The different lipidic vesicles affect the internalization mechanism of FITC-ODN. After using the inhibitors, the uptake of complexes involved a different internalization mechanism. The live CLSM analysis demonstrated that, after 1 hour from the complex incubation, the oligo was transferred into cells and localized into the endosomes; after 24 hours, the oligo was intracellularly localized close to the nuclear structure in a punctuate pattern. However, the results from fusion experiments showed also a binding of a quite low amount of oligo with the cell membranes.

New Insights into the Interactions of a DNA Oligonucleotide with mPEGylated-PAMAM by Circular Dichroism and Solution NMR.

Dendrimers are well-defined, highly branched, synthetic three-dimensional molecules with a large number of reactive end groups. PAMAM dendrimers form stable complexes with DNA chemistries and constitute an important class of nonviral, cationic vectors in gene delivery. The aim of this study is to examine the interactions of a 12 bp DNA oligonucletide with PAMAM-G2 and mPEG- b-PAMAM-G3 having eight surface amine groups under physiological conditions, using constant DNA concentration but varying dendrimer concentration. 1D 31P NMR, 2D NOESY, and CD spectroscopic methods were employed to investigate the interactions between the dendrimer and the DNA. The CD experiments carried out with a constant DNA concentration of 25 µM and dendrimer concentrations from 0 to 100 µM indicated minimal change to the chirality of the DNA for both types of dendrimers. While the PAMAM-G2 dendrimer caused aggregation of the majority of the DNA, the 2D NMR data of the DNA with an mPEG- b-PAMAM-G3 dendrimer indicated general broadening of the 1D 31P peaks from the DNA phosphates, a small number of 1H chemical shift perturbations (CSPs), and reduction of specific 1H-1H NOE intensities. These data suggest there is minimal structural alteration of the DNA in the complex and indicate preferential binding of the dendrimer to the central AATT region of the DNA sequence. The results herein are the first such results demonstrating a soluble DNA complex with the mPEG- b-PAMAM-G3 dendrimer analyzed by multidimensional NMR.

Isolation of Poly(A)+ Messenger RNA Using Magnetic Oligo(dT) Beads.

This is a general protocol for the isolation of mRNA from total RNA using oligo(dT) coupled to magnetic beads. First, total RNA is dissolved in a high-salt buffer and heated briefly to 65°C-70°C, followed by immediate cooling on ice to disrupt secondary structures. The RNA is subsequently annealed to the oligo(dT)-magnetic beads at room temperature; the high-salt binding buffer stabilizes the poly(A)-oligo(dT) complexes. A high-salt washing buffer is then used to wash away unbound RNAs while retaining oligo(dT)-bound poly(A)+ mRNAs. To elute the poly(A)+ mRNAs from the beads, a low-salt buffer (or water) is used to destabilize the poly(A)-oligo(dT) complexes. Alternatively, poly(A)+ mRNAs can be retained on the beads for downstream applications (e.g., solid-phase cDNA synthesis).

Engineering release kinetics with polyelectrolyte multilayers to modulate TLR signaling and promote immune tolerance.

Autoimmune disorders, such as multiple sclerosis and type 1 diabetes, occur when immune cells fail to recognize "self" molecules. Recently, studies have revealed aberrant inflammatory signaling through pathogen sensing pathways, such as toll-like receptors (TLRs), during autoimmune disease. Therapeutic inhibition of these pathways might attenuate disease development, skewing disease-causing inflammatory cells towards cell types that promote tolerance. Delivering antagonistic ligands to a TLR upstream of an inflammatory signaling cascade, TLR9, has demonstrated exciting potential in a mouse model of MS; however, strategies that enable sustained delivery could reduce the need for repeated administration or enhance therapeutic efficacy. We hypothesized that GpG - an oligonucleotide TLR9 antagonist - which is inherently anionic, could be self-assembled into polyelectrolyte multilayers (PEMs) with a cationic, degradable poly(ß-amino ester) (Poly1). We hypothesized that degradable Poly1/GpG PEMs could promote sustained release of GpG and modulate inflammatory immune cell functions. Here we demonstrate layer-by-layer assembly of degradable PEMs, as well as subsequent degradation and release of GpG. Following assembly and release, GpG maintains the ability to reduce dendritic cell activation and inflammatory cytokine secretion, restrain TLR9 signaling, and polarize myelin specific T cells towards regulatory phenotypes and functions in primarily immune cells. These results indicate that degradable PEMs may be able to promote tolerogenic immune function in the context of autoimmunity.

Pegylation of liposomes favours the endosomal degradation of the delivered phosphodiester oligonucleotides.

Liposomal vesicles have been widely investigated as carriers for the intracellular delivery of oligonucleotides (ONs). To avoid unspecific uptake by the reticulo endothelial system, 'pegylation' of the liposomes, by incorporating polyethyleneglycol (PEG) at the surface, has been an attractive strategy. While pegylation has a clear benefit on the systemic level, one could wonder if pegylation also benefits the delivery efficacy of liposomes at the intracellular level. We compared the intracellular distribution of non-pegylated and pegylated liposomes, with special attention to the integrity of the oligonucleotides they are carrying. After uptake in the cells, the non-pegylated liposomes efficiently escaped from the endosomes thereby releasing phosphodiester oligonucleotides (PO-ONs) in the cytoplasm of the cells. The PO-ONs were however rapidly degraded in the intracellular environment. In contrast to non-pegylated liposomes, pegylated liposomes failed in protecting the PO-ONs they were carrying, leading to rapid degradation of the PO-ONs in the endosomal compartment. Furthermore, the PEG chains inhibited the endosomal escape of the degraded ONs. These intracellular findings explain why pegylated liposomes failed in establishing an antisense effect.