Systemic toll-like receptor 9 agonist CpG oligodeoxynucleotides exacerbates aminoglycoside ototoxicity.

The Toll-like receptor (TLR) signaling pathway is the key regulator of the innate immune system in response to systemic infection. Several studies have reported that the systemic TLR4 agonist lipopolysaccharide exacerbates aminoglycoside ototoxicity, but the influence of virus-associated TLR7 and TLR9 signaling cascades on the cochlea is unclear. The present study aimed to investigate the auditory effects of systemic TLR7 and TLR9 agonists during chronic kanamycin treatment. CBA/CaJ mice received the TLR7 agonist gardiquimod or TLR9 agonist CpG oligodeoxynucleotides (ODN) one day before kanamycin injection and on the 5th and 10th days during a 14-day course of kanamycin treatment. We observed that systemic gardiquimod or CpG ODN alone did not affect the baseline auditory brainstem response (ABR) threshold. Three weeks after kanamycin treatment, gardiquimod did not significantly change ABR threshold shifts, whereas CpG ODN significantly increased kanamycin-induced ABR threshold shifts. Furthermore, outer hair cell (OHC) evaluation revealed that CpG ODN reduced distortion product otoacoustic emission amplitudes and increased kanamycin-induced OHC loss. CpG ODN significantly elevated cochlear Irf-7, Tnf-α, Il-1, and Il-6 transcript levels. In addition, an increased number of Iba-1+ cells, which represented activated macrophages, was observed in the cochlea treated with CpG ODN. Our results indicated that systemic CpG ODN exacerbated kanamycin-induced ototoxicity and increased cochlear inflammation. This study implies that patients with underlying virus infection may experience more severe aminoglycoside-induced hearing loss if it occurs.

Aptamer-PROTAC Conjugates (APCs) for Tumor-Specific Targeting in Breast Cancer.

Development of proteolysis targeting chimeras (PROTACs) is emerging as a promising strategy for targeted protein degradation. However, the drug development using the heterobifunctional PROTAC molecules is generally limited by poor membrane permeability, low in vivo efficacy and indiscriminate distribution. Herein an aptamer-PROTAC conjugation approach was developed as a novel strategy to improve the tumor-specific targeting ability and in vivo antitumor potency of conventional PROTACs. As proof of concept, the first aptamer-PROTAC conjugate (APC) was designed by conjugating a BET-targeting PROTAC to the nucleic acid aptamer AS1411 (AS) via a cleavable linker. Compared with the unmodified BET PROTAC, the designed molecule (APR) showed improved tumor targeting ability in a MCF-7 xenograft model, leading to enhanced in vivo BET degradation and antitumor potency and decreased toxicity. Thus, the APC strategy may pave the way for the design of tumor-specific targeting PROTACs and have broad applications in the development of PROTAC-based drugs.

Evaluation of the AV7909 Anthrax Vaccine Toxicity in Sprague Dawley Rats Following Three Intramuscular Administrations.

AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.

Targeted delivery system using silica nanoparticles coated with chitosan and AS1411 for combination therapy of doxorubicin and antimiR-21.

Herein, a novel targeted delivery system was developed for intracellular co-delivery of doxorubicin (DOX) as a chemotherapeutic drug, antimiR-21 as an oncogenic antagomiR. In this system, DOX was loaded into mesoporous silica nanoparticles (MSNs) and chitosan was applied to cover the surface of MSNs. AS1411 aptamer as targeting nucleolin and antimiR-21 were electrostatically attached onto the surface of the chitosan-coated MSNs and formed the final nanocomplex (AACS nanocomplex). The study of drug release was based on DOX release under pH 7.4 and 5.5. Cellular toxicity and cellular uptake assessments of AACS nanocomplex were carried out in nucleolin positive (C26, MCF-7, and 4T1) and nucleolin negative (CHO) cell lines using MTT assay and flow cytometry analysis, respectively. Also, Anti-tumor efficacy of AACS nanocomplex was evaluated in C26 tumor-bearing mice. Overall, the results show that the combination therapy of DOX and antimiR-21, using AACS nanocomplex, could combat the cancer cell growth rate.

A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites.

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH2 -AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC50 value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 µg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.

Improved Treatment Options for Glaucoma with Brimonidine-Loaded Lipid DNA Nanoparticles.

Glaucoma is the second leading cause of irreversible blindness worldwide. Among others, elevated intraocular pressure (IOP) is one of the hallmarks of the disease. Antiglaucoma drugs such as brimonidine can lower the IOP but their adherence to the ocular surface is low, leading to a low drug uptake. This results in a frequent dropping regime causing low compliance by the patients. Lipid DNA nanoparticles (NPs) have the intrinsic ability to bind to the ocular surface and can be loaded with different drugs. Here, we report DNA NPs functionalized for loading of brimonidine through specific aptamers and via hydrophobic interactions with double stranded micelles. Both NP systems exhibited improved affinity toward the cornea and retained release of the drug as compared to controls both in vitro and in vivo. Both NP types were able to lower the IOP in living animals significantly more than pristine brimonidine. Importantly, the brimonidine-loaded NPs showed no toxicity and improved efficacy and hence should improve compliance. In conclusion, this drug-delivery system offers high chances of an improved treatment for glaucoma and thus preserving vision in the aging population.

Curcumin Can Decrease Tissue Inflammation and the Severity of HSV-2 Infection in the Female Reproductive Mucosa.

Herpes Simplex Virus Type 2 (HSV-2) is one of the most prevalent sexually transmitted viruses and is a known risk factor for HIV acquisition in the Female Genital Tract (FGT). Previously, we found that curcumin can block HSV-2 infection and abrogate the production of inflammatory cytokines and chemokines by genital epithelial cells in vitro. In this study, we investigated whether curcumin, encapsulated in nanoparticles and delivered by various in vivo routes, could minimize inflammation and prevent or reduce HSV-2 infection in the FGT. Female mice were pre-treated with curcumin nanoparticles through oral, intraperitoneal and intravaginal routes, and then exposed intravaginally to the tissue inflammation stimulant CpG-oligodeoxynucleotide (ODN). Local intravaginal delivery of curcumin nanoparticles, but not intraperitoneal or oral delivery, reduced CpG-mediated inflammatory histopathology and decreased production of pro-inflammatory cytokines Interleukin (IL)-6, Tumor Necrosis Factor Alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP-1) in the FGT. However, curcumin nanoparticles did not demonstrate anti-viral activity nor reduce tissue pathology when administered prior to intravaginal HSV-2 infection. In an alternative approach, intravaginal pre-treatment with crude curcumin or solid dispersion formulations of curcumin demonstrated increased survival and delayed pathology following HSV-2 infection. Our results suggest that curcumin nanoparticle delivery in the vaginal tract could reduce local tissue inflammation. The anti-inflammatory properties of curcumin delivered to the vaginal tract could potentially reduce the severity of HSV-2 infection and decrease the risk of HIV acquisition in the FGT of women.

Enhanced cancer therapy with pH-dependent and aptamer functionalized doxorubicin loaded polymeric (poly D, L-lactic-co-glycolic acid) nanoparticles.

Aptamer based drug delivery systems are gaining the importance in anticancer therapy due to their targeted drug delivery efficiency without harming the normal cells. The present work formulated the pH-dependent aptamer functionalized polymer-based drug delivery system against human lung cancer. The prepared aptamer functionalized doxorubicin (DOX) loaded poly (D, L-lactic-co-glycolic acid) (PLGA), poly (N-vinylpyrrolidone) (PVP) nanoparticles (APT-DOX-PLGA-PVP NPs) were spherical in shape with an average size of 87.168 nm. The crystallography and presence of the PLGA (poly (D, L-lactic-co-glycolic acid)) and DOX (doxorubicin) in APT-DOX-PLGA-PVP NPs were indicated by the X-ray diffraction (XRD), Fourier transforms infrared spectroscopy (FTIR), and 1H and 13C nuclear magnetic resonance spectrometer (NMR). The pH-dependent aptamer AS1411 based drug release triggered the cancer cell death was evidenced by cytotoxicity assay, flow cytometry, and fluorescent microscopic imaging. In addition, the cellular uptake of the DOX was determined and the apoptosis-related signaling pathway in the A549 cells was studied by Western blot analysis. Further, the in vivo study revealed that mice treated with APT-DOX-PLGA-PVP NPs were significantly recovered from cancer as evident by mice weight and tumor size followed by the histopathological study. It was reported that the APT-DOX-PLGA-PVP NPs induced the apoptosis through the activation of the apoptosis-related proteins. Hence, the present study revealed that the APT-DOX-PLGA-PVP NPs improved the therapeutic efficiency through the nucleolin receptor endocytosis targeted drug release.

Repeat-Dose Toxicity Study of a Lyophilized Recombinant Protective Antigen-Based Anthrax Vaccine Adjuvanted With CpG 7909.

A recombinant protective antigen (rPA) anthrax vaccine candidate (rPA7909) was developed as a next-generation vaccine indicated for postexposure prophylaxis of disease resulting from suspected or confirmed Bacillus anthracis exposure. The lyophilized form of rPA7909-vaccinated candidate contains 75 µg purified rPA, 750 µg aluminum (as Alhydrogel adjuvant), and 250 µg of an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide CpG 7909 in a 0.5 mL phosphate-buffered suspension. General toxicity and local reactogenicity were evaluated in Sprague Dawley rats vaccinated with the full human dose of rPA7909 by intramuscular injection. Animals were immunized on study days 1, 15, and 29. Control groups were administered diluent only or adjuvant control (excipients, CpG 7909, and Alhydrogel adjuvant in diluent) intramuscularly at the same dose volume and according to the same schedule used for rPA7909. Toxicity was assessed based on the results of clinical observations, physical examinations, body weights, injection site reactogenicity, ophthalmology, clinical pathology (hematology, coagulation, and serum chemistry), organ weights, and macroscopic and microscopic pathology evaluation. The immune response to rPA7909 vaccination was confirmed by measuring serum anti-PA immunoglobulin G levels. The rPA7909 vaccine produced no apparent systemic toxicity and only transient reactogenicity at the injection site. The injection site reaction from animals receiving the adjuvant control was very similar to those receiving rPA7909 with respect to the inflammation. The inflammatory response observed in the injection site and the draining lymph nodes was consistent with expected immune stimulation. The overall results indicated a favorable safety profile for rPA7909.

Encapsulation of Poly I:C and the natural phosphodiester CpG ODN enhanced the efficacy of a hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticle vaccine in TC-1-grafted tumors.

FDA approval of CpG oligodeoxynucleotide (CpG ODN) adjuvants for a human hepatitis B virus vaccine has been delayed until late 2017 because of concerns regarding the severe side effects, which may be attributed to the high dosage and systemic diffusion of this proinflammatory material. Considering that PLGA could provide shelter to resist nucleases in tissue and that cationic lipids could confine anionic oligonucleotides in the nanoparticles via electrostatic attraction to avoid systemic diffusion, we encapsulated a natural phosphodiester or the expensive phosphorothioate CpG ODNs in our previously reported hyaluronic acid-modified cationic lipid-PLGA hybrid nanoparticles and evaluated vaccine efficacy in a TC-1-grafted mouse model. Our results showed that together with Poly I:C, CpG ODN could promote the maturation of bone marrow-derived dendritic cells and the cross-presentation of exogenous antigens in vitro. For the coencapsulation with Poly I:C, in vivo studies showed that adjuvant effects on the vaccine efficacy of tumor depression, immune cell activation, and memory T-cell elevation of phosphodiester CpG ODNs were comparable to those of the phosphorothioate CpG ODNs at a low concentration (5 µg/dose). In conclusion, the combination of oligonucleotide adjuvants and synthetic particulate systems not only potentiated the immunogenicity of these nanoparticles but also made these adjuvants safer and more economical, which may be helpful for their wide application.

A novel multivalent DNA helix-based inhibitor showed enhanced anti-HIV-1 fusion activity.

DNA helix-based HIV-1 fusion inhibitors have been discovered as potent drug candidates, but further research is required to enhance their efficiency. The trimeric structure of the HIV-1 envelope glycoprotein provides a structural basis for multivalent drug design. In this work, a "multi-domain" strategy was adopted for design of an oligodeoxynucleotide with assembly, linkage, and activity domains. Built on the self-assembly of higher-order nucleic acid structure, a novel category of multivalent DNA helix-based HIV-1 fusion inhibitor could be easily obtained by a simple annealing course in solution buffer, with no other chemical synthesis for multivalent connection. An optimized multivalent molecule, M4, showed significantly higher anti-HIV-1 fusion activity than did corresponding monovalent inhibitors. Examination of the underlying mechanism indicated that M4 could interact with HIV-1 glycoproteins gp120 and gp41, thereby inhibiting 6HB formation in the fusion course. M4 also showed anti-RDDP and anti-RNase H activity of reverse transcriptase. Besides, these assembled molecules showed improved in vitro metabolic stability in liver homogenate, kidney homogenate, and rat plasma. Moreover, little acute toxicity was observed. Our findings aid in the structural design and understanding of the mechanisms of DNA helix-based HIV-1 inhibitors. This study also provides a general strategy based on a new structural paradigm for the design of other multivalent nucleic acid drugs.

Signal management in pharmacovigilance and human risk assessment of CpG 7909, integrating embryo-fetal and post-natal developmental toxicity studies in rats and rabbits.

The potential reproductive and developmental toxicity of the synthetic oligodeoxynucleotide (ODN) CpG 7909, a component of GSK's AS15 immunostimulant, was examined in rat and rabbit studies following intermittent intramuscular injections. Previous studies using subcutaneous and intraperitoneal injections in mice, rats and rabbits revealed that CpG ODNs induced developmental effects. To analyze the safety signal, GSK conducted additional animal studies using the intended clinical route of administration. CpG 7909 injections were administered intramuscularly to rats or rabbits 28 and 14days before pairing, on 4 or 5 occasions during gestation, and on lactation day 7. The No Observed Adverse Effect Level for female fertility, embryo-fetal and pre- and post-natal development was 4.2mg/kg in both species, approximately 500-fold higher than the anticipated human dose. In conclusion, the anticipated risk to humans is considered low for sporadic intramuscular exposure to CpG 7909.

Self-assembled DNA nanocentipedes as multivalent vehicles for enhanced delivery of CpG oligonucleotides.

We report self-assembled nanocentipedes as multivalent vehicles for the delivery of immunostimulatory CpG ODNs. The multivalent vehicles could be internalized by RAW264.7 cells and stimulate the secretion of large amounts of cytokines, successively inducing effective apoptosis of cancer cells.

The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell.

AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent.

Exposure to stimulatory CpG oligonucleotides during gestation induces maternal hypertension and excess vasoconstriction in pregnant rats.

Bacterial infections increase risk for pregnancy complications, such as preeclampsia and preterm birth. Unmethylated CpG DNA sequences are present in bacterial DNA and have immunostimulatory effects. Maternal exposure to CpG DNA induces fetal demise and craniofacial malformations; however, the effects of CpG DNA on maternal cardiovascular health have not been examined. We tested the hypothesis that exposure to synthetic CpG oligonucleotides (ODNs) during gestation would increase blood pressure and cause vascular dysfunction in pregnant rats. Pregnant and nonpregnant female rats were treated with CpG ODN (ODN 2395) or saline (Veh) starting on gestational day 14or corresponding day for the nonpregnant groups. Exposure to CpG ODN increased systolic blood pressure in pregnant (Veh: 121 ± 2 mmHg vs. ODN 2395: 134 ± 2 mmHg,P< 0.05) but not in nonpregnant rats (Veh: 111 ± 2 mmHg vs. ODN 2395: 108 ± 5 mmHg,P> 0.05). Mesenteric resistance arteries from pregnant CpG ODN-treated rats had increased contractile responses to U46619 [thromboxane A2(TxA2) mimetic] compared with arteries from vehicle-treated rats [Emax(%KCl), Veh: 87 ± 4 vs. ODN 2395: 104 ± 4,P< 0.05]. Nitric oxide synthase (NOS) inhibition increased contractile responses to U46619, and CpG ODN treatment abolished this effect in arteries from pregnant ODN 2395-treated rats. CpG ODN potentiated the involvement of cyclooxygenase (COX) to U46619-induced contractions. In conclusion, exposure to CpG ODN during gestation induces maternal hypertension, augments resistance artery contraction, increases the involvement of COX-dependent mechanisms and reduces the contribution of NOS-dependent mechanisms to TxA2-induced contractions in mesenteric resistance arteries.

Pharmacokinetic Profile and Acute Toxicological Properties of a Novel Radiosensitizer Cytosine-Phosphate-Guanosine Oligodeoxynucleotide 107 in Mice Following Intravenous and Orthotopic Administration.

The synthetic cytosine-phosphate-guanosine oligodeoxynucleotide 107 (CpG ODN107) is a novel radiosensitizer for glioma treatment. However, the information related to its pharmacokinetics and toxicity remains unclear. Therefore, the plasma pharmacokinetics, distribution, elimination, and acute toxicity of CpG ODN107 in mice were investigated in the present experiments. The results from the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay showed that the plasma elimination half-life (t1/2ß) of CpG ODN107 in BALB/c mice varied slightly with the dose, and it was 0.65, 0.49, and 0.50 h at the intravenous doses of 2.5, 5, and 10 mg/kg, respectively. CpG ODN107 rapidly and widely distributed in organs/tissues, except the brain and testes. The highest concentrations were found in the liver (28.6% of the administered dose after 0.5 h) and the kidneys (5.7% of the administered dose after 1 h). CpG ODN107 (0.3, 3, and 30 µg/mL) could highly bind to human and mouse plasma proteins in vitro. CpG ODN107 in the forms of prototype was excreted in urine (1.79%) and feces (0.91%), and its shortened metabolites were excreted in urine (2.1%) and feces (2.2%) within the first 24 h. The mice in vivo optical image showed CpG ODN107 labeled with Alexa Fluor 680 fluorochrome (AF680) accumulated in the brain after orthotopic injection, eliminated very slowly, and excreted in urine compared with poly T labeled with AF680. The median lethal dose (LD50) of CpG ODN107 was 75.7 mg/kg for mice; this dose only could produce apparent spleen and liver damage, in line with the distribution features of CpG ODN. In conclusion, our present pharmacokinetic and toxicity investigation will provide helpful information to further pharmacodynamic and pharmacokinetic research of CpG ODN107 and other oligodeoxynucleotide drugs in the future.

NF-κB decoy oligodeoxynucleotide enhanced osteogenesis in mesenchymal stem cells exposed to polyethylene particle.

Excessive generation of wear particles after total joint replacement may lead to local inflammation and periprosthetic osteolysis. Modulation of the key transcription factor NF-κB in immune cells could potentially mitigate the osteolytic process. We previously showed that local delivery of ultrahigh-molecular-weight polyethylene (UHMWPE) particles recruited osteoprogenitor cells and reduced osteolysis. However, the biological effects of modulating the NF-κB signaling pathway on osteoprogenitor/mesenchymal stem cells (MSCs) remain unclear. Here we showed that decoy oligodeoxynucleotide (ODN) increased cell viability when primary murine MSCs were exposed to UHMWPE particles, but had no effects on cellular apoptosis. Decoy ODN increased transforming growth factor-beta 1 (TGF-ß1) and osteoprotegerin (OPG) in MSCs exposed to UHMWPE particles. Mechanistic studies showed that decoy ODN upregulated OPG expression through a TGF-ß1-dependent pathway. By measuring the alkaline phosphatase activity, osteocalcin levels, Runx2 and osteopontin expression, and performing a bone mineralization assay, we found that decoy ODN increased MSC osteogenic ability when the cells were exposed to UHMWPE particles. Furthermore, the cellular response to decoy ODN and UHMWPE particles with regard to cell phenotype, cell viability, and osteogenic ability was confirmed using primary human MSCs. Our results suggest that modulation of wear particle-induced inflammation by NF-κB decoy ODN had no adverse effects on MSCs and may potentially further mitigate periprosthetic osteolysis by protecting MSC viability and osteogenic ability.

Intraperitoneal and subcutaneous injections of the TLR9 agonist ODN 1668 in rats: brain inflammatory responses are related to peripheral IL-6 rather than interferons.

Subcutaneous or intraperitoneal administration of Toll-like receptor (TLR)-9 agonist, ODN 1668 caused moderate fever and anorexia. In comparison to stimulation of other intracellular TLRs, activation of TLR9 did not result in pronounced peripheral induction of interferons, but rather induced interleukin-6. Expression of cytokines (TNFα, IL-1ß) and inducible forms of enzymes for prostaglandin E2 synthesis occurred in the brain, in conjunction with a moderate activation of the transcription factors STAT3 and NF-IL6 in brain endothelial cells. The lack of a septic-like state in ODN 1668-treated rats reinforces the therapeutic value of this drug.

[Establishment of HLH-like mouse model with CPG-ODN and IFN-γ].

OBJECTIVE: To establish a hemophagocytic lymphohistiocytosis (HLH)-like mouse model induced by CpG oligodeoxynucleotide (CpG-ODN1826) and interferon (IFN)-γ for further study on therapy. METHODS: Wild type adult C57BL/6 mice were administered with PBS or CpG-ODN1826 (50 µg) by intraperitoneal injection every two day and IFN-γ subcutaneous injection every day. Parameters of HLH were evaluated on day 10. RESULTS: As compared to control, HLH-like symptoms in CpG group were characterized with pancytopenia accompanied by increased ratios of monocytes, alanine aminotransferase [(198.7±54.2)IU/L], triglyceride level [(12.1±0.6)g/L], and serum ferritin [(708.4±11.8)pmol/L]; decreased albumin [(217.7±4.3)g/L], fibrinogen [(17.1±1.9)g/L] (all P<0.05). Hepatosplenomegaly was obvious in CpG group. The liver in CpG group had multifocal hepatocytes necrosis and perivascular inflammations. Spleen had expanding red pulp and hyperplastic nucleated cells. Furthermore, macrophages in the liver and spleen were largely activated. Hemophagocytosis were observed in liver, spleen and bone marrow smear. The CpG group was alive during experiment, other than significant decreased activity after the first injection of CpG-ODN. CONCLUSION: These data demonstrate that repeated administration of CpG-ODN1826 and IFN-γ could induce HLH-like symptoms without fatal condition in wild type C57B/L mice. This protocol could establish a mild HLH-like mouse model, which could be useful for further study on HLH.

The impact of single and pairwise Toll-like receptor activation on neuroinflammation and neurodegeneration.

BACKGROUND: Toll-like receptors (TLRs) enable innate immune cells to respond to pathogen- and host-derived molecules. The central nervous system (CNS) exhibits most of the TLRs identified with predominant expression in microglia, the major immune cells of the brain. Although individual TLRs have been shown to contribute to CNS disorders, the consequences of multiple activated TLRs on the brain are unclear. We therefore systematically investigated and compared the impact of sole and pairwise TLR activation on CNS inflammation and injury. METHODS: Selected TLRs expressed in microglia and neurons were stimulated with their specific TLR ligands in varying combinations. Cell cultures were then analyzed by immunocytochemistry, FlowCytomix, and ELISA. To determine neuronal injury and neuroinflammation in vivo, C57BL/6J mice were injected intrathecally with TLR agonists. Subsequently, brain sections were analyzed by quantitative real-time PCR and immunohistochemistry. RESULTS: Simultaneous stimulation of TLR4 plus TLR2, TLR4 plus TLR9, and TLR2 plus TLR9 in microglia by their respective specific ligands results in an increased inflammatory response compared to activation of the respective single TLR in vitro. In contrast, additional activation of TLR7 suppresses the inflammatory response mediated by the respective ligands for TLR2, TLR4, or TLR9 up to 24 h, indicating that specific combinations of activated TLRs individually modulate the inflammatory response. Accordingly, the composition of the inflammatory response pattern generated by microglia varies depending on the identity and combination of the activated TLRs engaged. Likewise, neuronal injury occurs in response to activation of only selected TLRs and TLR combinations in vitro. Activation of TLR2, TLR4, TLR7, and TLR9 in the brain by intrathecal injection of the respective TLR ligand into C57BL/6J mice leads to specific expression patterns of distinct TLR mRNAs in the brain and causes influx of leukocytes and inflammatory mediators into the cerebrospinal fluid to a variable extent. Also, the intensity of the inflammatory response and neurodegenerative effects differs according to the respective activated TLR and TLR combinations used in vivo. CONCLUSIONS: Sole and pairwise activation of TLRs modifies the pattern and extent of inflammation and neurodegeneration in the CNS, thereby enabling innate immunity to take account of the CNS diseases' diversity.