Toll-like receptor activation regulates the paracrine effect of adipose-derived mesenchymal stem cells on reversing osteoarthritic phenotype of chondrocytes.

BACKGROUND: The therapeutic efficacy of intra-articular mesenchymal stem cells (MSCs) injection for patients with osteoarthritis (OA) currently exhibits inconsistency, and the underlying mechanism remains elusive. It has been postulated that the immunomodulatory properties and paracrine activity of MSCs might be influenced by the inflammatory micro-environment within osteoarthritic joints, potentially contributing to this observed inconsistency. METHODS: Adipose-derived MSCs (ADSCs) were isolated from SD rats and pre-treated with Toll-like receptor 3 (TLR3) agonist Poly I:C or Toll-like receptor 4 (TLR4) agonist LPS. The pre-treated ADSCs were then co-cultured with IL-1ß-induced osteoarthritic chondrocytes using a Transwell system to analyze the paracrine effect of ADSCs on reversing the osteoarthritic phenotype of chondrocytes. RESULTS: RT-PCR and Western blot analysis revealed that Poly I:C and LPS pre-treatments up-regulated the expression of IL-10 and IL-6 in ADSCs, respectively. Furthermore, only Poly I:C-preconditioned ADSCs significantly promoted proliferation while inhibiting apoptosis in IL-1ß-treated chondrocytes. Additionally, Poly I:C-preconditioned ADSCs downregulated MMP13 expression while upregulating aggrecan and collagen II expression levels in IL-1ß-treated chondrocytes. CONCLUSIONS: TLR3 activation polarizes ADSCs into an immunomodulatory phenotype distinct from TLR4 activation, exerting differential effects on reversing the osteoarthritic phenotype of chondrocytes; thus indicating that MSCs' paracrine effect regulated by TLRs signaling impacts the efficacy of intra-articular MSCs injection.

Synergistic Inhibitory Effect of Lactobacillus Cell Lysates and Butyrate on Poly I:C-Induced IL-8 Production in Human Intestinal Epithelial Cells.

Postbiotics include cell lysates (CLs), enzymes, cell wall fragments, and heat-killed bacteria derived from probiotics. Although postbiotics are increasingly being considered for their potential health-promoting properties, the effects of postbiotics on virus-mediated inflammatory responses in the intestine have not been elucidated. Hence, the present study aimed to examine whether CLs of Lactipantibacillus plantarum (LP CL) and Lacticaseibacillus rhamnosus GG (LR CL) could inhibit virus-mediated inflammatory responses in the human intestinal epithelial cell line HT-29 in vitro. Pretreatment with LP CL and LR CL significantly inhibited interleukin (IL)-8 production, which was induced by poly I:C, a synthetic analog of double-stranded RNA (dsRNA) viruses, at the mRNA and protein levels in HT-29 cells. However, peptidoglycans and heat-killed L. plantarum and L. rhamnosus GG did not effectively inhibit IL-8 production. LP CL and LR CL attenuated the poly I:C-induced phosphorylation of ERK and JNK and the activation of NF-κB, suggesting that these CLs could inhibit poly I:C-induced IL-8 production by regulating intracellular signaling pathways in HT-29 cells. Furthermore, among the short-chain fatty acids, butyrate enhanced the inhibitory effect of CLs on poly I:C-induced IL-8 production at the mRNA and protein levels in HT-29 cells, while acetate and propionate did not. Taken together, these results suggest that both LP CL and LR CL could act as potent effector molecules that can inhibit virus-mediated inflammatory responses and confer synergistic inhibitory effects with butyrate in human intestinal epithelial cells.

Extracellular adenosine triphosphate regulates inflammatory responses of periodontal ligament cells.

BACKGROUND: Various stimuli, that is, mechanical stresses or inflammation, induce the release of adenosine triphosphate (ATP) by human periodontal ligament cells (HPDLCs). Extracellular adenosine triphosphate (eATP) affects HPDLCs' functions such as immunosuppressive action and inflammatory responses. Lipopolysaccharide (LPS) is the key factor involved in periodontal inflammation. However, the possible correlation and detailed mechanism of inflammation-mediated eATP by LPS and inflammatory cascade formation in HPDLCs is unclarified. This study aims to examine the role of eATP on the HPDLCs' responses concerning inflammatory actions after LPS treatment. METHODS: HPDLCs were stimulated with Porphyromonas gingivalis LPS and polyinosinic:polycytidylic acid (poly I:C). The amount of ATP release was measured at different time points using a bioluminescence assay. HPDLCs were treated with eATP. The expression of pro-inflammatory and anti-inflammatory genes was determined. Specific P2X purinoreceptor 7 (P2X7) inhibitors (brilliant blue G [BBG] and KN62), a specific P2Y purinoreceptor 1 (P2Y1) inhibitors (MRS2179), calcium chelator (EGTA), protein kinase C (PKC) inhibitors, nuclear factor kappa-light-chain-enhancer of activated B cells (NF𝜅B) activation inhibitors, and cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) inhibitors (H89 dihydrochloride) and activators (forskolin) were used to dissect the mechanism of eATP-induced HPDLCs' inflammatory responses. RESULTS: LPS and poly I:C induced ATP release. A low concentration of eATP (50 µM) increased pro-inflammatory genes (COX2, IL1B, IL6, IL8, IL12, and TNFA), while a high concentration (500 µM) enhanced anti-inflammatory genes (IL4 and IL10). BBG, KN62, and NF𝜅B activation inhibitors impeded eATP-induced pro-inflammatory genes. MRS2179 and H89 markedly suppressed eATP-induced anti-inflammatory genes. Forskolin induced IL4 and IL10. CONCLUSION: HPDLCs respond to LPS by releasing ATP. eATP has dose-dependent dual functions on HPDLCs' inflammatory responses via different pathways. As regulation of inflammation is important in regeneration, eATP may help to limit inflammation and trigger periodontal regeneration.

Dexmedetomidine Alters the Inflammatory Profile of Rat Microglia In Vitro.

BACKGROUND: Microglia are a primary mediator of the neuroinflammatory response to neurologic injury, such as that in traumatic brain injury. Their response includes changes to their cytokine expression, metabolic profile, and immunophenotype. Dexmedetomidine (DEX) is an α2 adrenergic agonist used as a sedative in critically ill patients, such as those with traumatic brain injury. Given its pharmacologic properties, DEX may alter the phenotype of inflammatory microglia. METHODS: Primary microglia were isolated from Sprague-Dawley rats and cultured. Microglia were activated using multiple mediators: lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly I:C), and traumatic brain injury damage-associated molecular patterns (DAMP) from a rat that sustained a prior controlled cortical impact injury. After activation, cultures were treated with DEX. At the 24-h interval, the cell supernatant and cells were collected for the following studies: cytokine expression (tumor necrosis factor-α [TNFα], interleukin-10 [IL-10]) via enzyme-linked immunosorbent assay, 6-phosphofructokinase enzyme activity assay, and immunophenotype profiling with flow cytometry. Cytokine expression and metabolic enzyme activity data were analyzed using two-way analysis of variance. Cell surface marker expression was analyzed using FlowJo software. RESULTS: In LPS-treated cultures, DEX treatment decreased the expression of TNFα from microglia (mean difference = 121.5 ± 15.96 pg/mL; p < 0.0001). Overall, DEX-treated cultures had a lower expression of IL-10 than nontreated cultures (mean difference = 39.33 ± 14.50 pg/mL, p < 0.0001). DEX decreased IL-10 expression in LPS-stimulated microglia (mean difference = 74.93 ± 12.50 pg/mL, p = 0.0039) and Poly I:C-stimulated microglia (mean difference = 23.27 ± 6.405 pg/mL, p = 0.0221). In DAMP-stimulated microglia, DEX decreased the activity of 6-phosphofructokinase (mean difference = 18.79 ± 6.508 units/mL; p = 0.0421). The microglial immunophenotype was altered to varying degrees with different inflammatory stimuli and DEX treatment. CONCLUSIONS: DEX may alter the neuroinflammatory response of microglia. By altering the microglial profile, DEX may affect the progression of neurologic injury.

Ibrutinib Prevents Acute Lung Injury via Multi-Targeting BTK, FLT3 and EGFR in Mice.

Ibrutinib has potential therapeutic or protective effects against viral- and bacterial-induced acute lung injury (ALI), likely by modulating the Bruton tyrosine kinase (BTK) signaling pathway. However, ibrutinib has multi-target effects. Moreover, immunity and inflammation targets in ALI treatment are poorly defined. We investigated whether the BTK-, FLT3-, and EGFR-related signaling pathways mediated the protective effects of ibrutinib on ALI. The intratracheal administration of poly I:C or LPS after ibrutinib administration in mice was performed by gavage. The pathological conditions of the lungs were assessed by micro-CT and HE staining. The levels of neutrophils, lymphocytes, and related inflammatory factors in the lungs were evaluated by ELISA, flow cytometry, immunohistochemistry, and immunofluorescence. Finally, the expression of proteins associated with the BTK-, FLT3-, and EGFR-related signaling pathways were evaluated by Western blotting. Ibrutinib (10 mg/kg) protected against poly I:C-induced (5 mg/kg) and LPS-induced (5 mg/kg) lung inflammation. The wet/dry weight ratio (W/D) and total proteins in the bronchoalveolar lavage fluid (BALF) were markedly reduced after ibrutinib (10 mg/kg) treatment, relative to the poly I:C- and LPS-treated groups. The levels of ALI indicators (NFκB, IL-1ß, IL-6, TNF-α, IFN-γ, neutrophils, and lymphocytes) were significantly reduced after treatment. Accordingly, ibrutinib inhibited the poly I:C- and LPS-induced BTK-, FLT3-, and EGFR-related pathway activations. Ibrutinib inhibited poly I:C- and LPS-induced acute lung injury, and this may be due to its ability to suppress the BTK-, FLT3-, and EGFR-related signaling pathways. Therefore, ibrutinib is a potential protective agent for regulating immunity and inflammation in poly I:C- and LPS-induced ALI.

Poly I:C Exacerbates Airway Inflammation and Remodeling in Cigarette Smoke-Exposed Mice.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is a chronic respiratory disorder characterized by chronic inflammation and airway remodeling. Cigarette smoke (CS) and respiratory viruses are major causes of COPD development and exacerbation, but the mechanisms of these compounding factors on inflammation and pathological changes in airway structure still need further investigation. PURPOSE: This work aimed to investigate the effects and mechanisms of Poly I:C on pathological changes in CS-induced COPD mice, such as airway inflammation and remodeling. METHODS: From 1 to 8 weeks, the mice were exposed to CS, Poly I:C, or a combination of both. To compare the pathological changes among different groups over time, the mice were sacrificed at week 4, 8, 16, and 24, then the lungs were harvested to measure pulmonary pathology, inflammatory cytokines, and airway remodeling. RESULTS: Our data revealed that the fundamental characteristics of COPD, such as pulmonary pathological damage, the release of inflammatory mediators, and the remodeling of airway walls, were observed at week 8 in CS-exposed mice and these pathological changes persisted to week 16. Compared with the CS group, the pathological changes, including decreased lung function, inflammatory cell infiltration, alveolar destruction, and airway wall thickening, were weaker in the Poly I:C group. These pathological changes were observed at week 8 and persisted to week 16 in Poly I:C-induced mice. Furthermore, Poly I:C exacerbated lung tissue damage in CS-induced COPD mice. The decreased lung function, airway inflammation and remodeling were observed in the combined group at week 4, and these pathological changes persisted to week 24. Our research indicated that Poly I:C enhanced the expression of p-P38, p-JNK and p-NF-κB in CS-exposed mice. CONCLUSION: Poly I:C could promote airway inflammation and remodeling in CS-induced COPD mice probably by NF-κB and MAPK signaling.

Trained Immunity in Primary Sjögren's Syndrome: Linking Type I Interferons to a Pro-Atherogenic Phenotype.

Background: Trained immunity - or innate immune memory - can be described as the long-term reprogramming of innate immune cells towards a hyperresponsive state which involves intracellular metabolic changes. Trained immunity has been linked to atherosclerosis. A subgroup of patients with primary Sjögren's syndrome (pSS) exhibits systemic type I interferon (IFN) pathway activation, indicating innate immune hyperactivation. Here, we studied the link between type I IFNs and trained immunity in an in vitro monocytic cell model and peripheral blood mononuclear cells (PBMCs) from pSS patients. Methods: The training stimuli heat killed Candida albicans, muramyl dipeptide, IFNß, and patient serum were added to THP-1 cells for 24 hours, after which the cells were washed, rested for 48 hours and subsequently re-stimulated with LPS, Pam3Cys, poly I:C, IFNß or oxLDL for 4-24 hours. PBMCs from pSS patients and healthy controls were stimulated with LPS, Pam3Cys, poly I:C or IFNß for 0.5-24 hours. Results: Training with IFNß induced elevated production of pro-atherogenic cytokines IL-6, TNFα and CCL2, differential cholesterol- and glycolysis-related gene expression, and increased glucose consumption and oxLDL uptake upon re-stimulation. Type I IFN production was increased in Candida albicans- and IFNß-trained cells after LPS re-stimulation, but was reduced after poly I:C re-stimulation. Training with muramyl dipeptide and IFNß, but not Candida albicans, affected the IFN-stimulated gene expression response to IFNß re-stimulation. PBMCs from pSS patients consumed more glucose compared with healthy control PBMCs and tended to produce more TNFα and type I IFNs upon LPS stimulation, but less type I IFNs upon poly I:C stimulation. Conclusions: Type I IFN is a trainer inducing a trained immunity phenotype with pro-atherogenic properties in monocytes. Conversely, trained immunity also affects the production of type I IFNs and transcriptional response to type I IFN receptor re-stimulation. The phenotype of pSS PBMCs is consistent with trained immunity. This connection between type I IFN, trained immunity and cholesterol metabolism may have important implications for pSS and the pathogenesis of (subclinical) atherosclerosis in these patients.

In vitro 2D and 3D cancer models to evaluate compounds that modulate macrophage polarization.

In vitro cancer models that can identify novel immunomodulating compounds are essential. Using a 3D multicellular tumor spheroid (MCTS) model comprising cancer cells, fibroblasts, and macrophages, we tested tumor-associated macrophage (TAM)-inhibiting compounds (CCL2 Ab, CSF1R inhibitor, CSF1R Ab) and TAM-reprograming compounds (poly I:C, CD40 Ab, CD40 ligand) for their effects on monocyte infiltration and polarization in tumor spheroids. For characterization of macrophage polarization, we measured the expression of CD206, CD163, CD86, MHC II, CD40, and CD14 and measured 43 soluble factors in the 3D MCTS cultures. 2D macrophage models were evaluated for comparison. A CSF1R inhibitor prevented infiltration of monocytes into pancreatic cancer spheroids, and macrophages treated with the inhibitor showed decreased expression of M2 markers. Treatment with a CD40 ligand and poly I:C induced M1 macrophage polarization in our models. We propose that these models can be used to improve the drug screening process of anti-cancer immunotherapies targeting macrophages.

Influence of exposure to microbial ligands, immunosuppressive drugs and chronic kidney disease on endogenous immunomodulatory gene expression in feline adipose-derived mesenchymal stem cells.

OBJECTIVES: Feline autologous mesenchymal stem cells (MSCs) show promise for immunomodulatory activity, but the functional impact of chronic kidney disease (CKD), concurrent immunosuppressive drug administration or infection is unknown. The study objectives compare endogenous cytokine gene expression (interleukin [IL]-6, IL-10, IL-12p40, IL-18 and transforming growth factor beta [TGF-ß]) in adipose-derived MSCs (aMSCs) from cats with and without CKD, following in vitro exposure to microbial ligands and treatment with common immunosuppressive drugs. METHODS: Previously obtained aMSCs, phenotype CD44+, CD90+, CD105+ and MHCII-, from cats with (n = 6) and without (n = 6) CKD were compared via real-time PCR (RT-PCR) for immunomodulatory gene expression. aMSCs were exposed in vitro to lipopolysaccharide (LPS), peptidoglycan or polyinosinic:polycytidylic acid (Poly I:C), simulating bacterial or viral exposure, respectively. aMSCs were also exposed to ciclosporin, dexamethasone or methotrexate. Gene expression was measured using RT-PCR, and Cq was utilized after each run to calculate the delta cycle threshold. RESULTS: aMSCs isolated from healthy and CKD cats showed no significant differences in gene expression in the five measured cytokines. No significant changes in measured gene expression after drug treatment or microbial ligand stimulation were observed between normal or CKD affected cats. Proinflammatory genes (IL-6, IL-12p40 and IL-18) showed altered expression in aMSCs from both groups when compared with the same cells in standard culture after exposure to methotrexate. Poly I:C altered IL-6 and TGF-ß gene expression in aMSCs from both healthy and CKD cats when compared with the same cells in standard culture. CONCLUSIONS AND RELEVANCE: The five genes tested showed no statistical differences between aMSCs from healthy or CKD cats. There was altered cytokine gene expression between the control and treatment groups of both healthy and CKD cats suggesting feline aMSCs have altered function with immunosuppressive treatment or microbial ligand exposure. Although the current clinical relevance of this pilot study comparing brief exposure to select agents in vitro in aMSCs from a small number of cats is unknown, the study highlights a need for continued investigation into the effects of disease and concurrent therapies on use of cell-based therapies in feline patients.

Pan-Caspase Inhibitor zVAD Induces Necroptotic and Autophagic Cell Death in TLR3/4-Stimulated Macrophages.

In addition to inducing apoptosis, caspase inhibition contributes to necroptosis and/or autophagy depending on the cell type and cellular context. In macrophages, necroptosis can be induced by co-treatment with Toll-like receptor (TLR) ligands (lipopolysaccharide [LPS] for TLR4 and polyinosinic-polycytidylic acid [poly I:C] for TLR3) and a cell-permeable pan-caspase inhibitor zVAD. Here, we elucidated the signaling pathways and molecular mechanisms of cell death. We showed that LPS/zVAD- and poly I:C/zVAD-induced cell death in bone marrow-derived macrophages (BMDMs) was inhibited by receptor-interacting protein kinase 1 (RIP1) inhibitor necrostatin-1 and autophagy inhibitor 3-methyladenine. Electron microscopic images displayed autophagosome/autolysosomes, and immunoblotting data revealed increased LC3II expression. Although zVAD did not affect LPS- or poly I:C-induced activation of IKK, JNK, and p38, it enhanced IRF3 and STAT1 activation as well as type I interferon (IFN) expression. In addition, zVAD inhibited ERK and Akt phosphorylation induced by LPS and poly I:C. Of note, zVAD-induced enhancement of the IRF3/IFN/STAT1 axis was abolished by necrostatin-1, while zVAD-induced inhibition of ERK and Akt was not. Our data further support the involvement of autocrine IFNs action in reactive oxygen species (ROS)-dependent necroptosis, LPS/zVAD-elicited ROS production was inhibited by necrostatin-1, neutralizing antibody of IFN receptor (IFNR) and JAK inhibitor AZD1480. Accordingly, both cell death and ROS production induced by TLR ligands plus zVAD were abrogated in STAT1 knockout macrophages. We conclude that enhanced TRIF-RIP1-dependent autocrine action of IFNß, rather than inhibition of ERK or Akt, is involved in TLRs/zVAD-induced autophagic and necroptotic cell death via the JAK/STAT1/ROS pathway.

Nucleic acid binding and other biomedical properties of artificial oligolysines.

In the present study, we report the interaction of an artificial oligolysine (referred to as AOL) realized in our laboratory with targets of biomedical importance. These included polyinosinic acid (poly rI) and its complex with polycytidylic acid (poly I:C), RNAs with well-known interferon-inducing ability, and double-stranded (ds) DNA. The ability of the peptide to bind both single-stranded poly rI and ds poly I:C RNAs emerged from our circular dichroism (CD) and ultraviolet (UV) studies. In addition, we found that AOL forms complexes with dsDNA, as shown by spectroscopic binding assays and UV thermal denaturation experiments. These findings are encouraging for the possible use of AOL in biomedicine for nucleic acid targeting and oligonucleotide condensation, with the latter being a key step preceding their clinical application. Moreover, we tested the ability of AOL to bind to proteins, using serum albumin as a model protein. We demonstrated the oligolysine-protein binding by CD experiments which suggested that AOL, positively charged under physiological conditions, binds to the protein regions rich in anionic residues. Finally, the morphology characterization of the solid oligolysine, performed by scanning electron microscopy, showed different crystal forms including cubic-shaped crystals confirming the high purity of AOL.

Polyinosinic acid blocks adeno-associated virus macrophage endocytosis in vitro and enhances adeno-associated virus liver-directed gene therapy in vivo.

Adeno-associated virus serotype 8 (AAV8) has been demonstrated to be effective for liver-directed gene therapy in humans. Although hepatocytes are the main target cell for AAV8, there is a loss of the viral vector because of uptake by macrophages and Kupffer cells. Reducing this loss would increase the efficacy of viral gene therapy and allow a dose reduction. The receptor mediating this uptake has not been identified; a potential candidate seems the macrophage scavenger receptor A (SR-A) that is involved in the endocytosis of, for instance, adenovirus. In this study we show that SR-A can mediate scAAV8 endocytosis and that blocking it with polyinosinic acid (poly[i]) reduces endocytosis significantly in vitro. Subsequently, we demonstrate that blocking this receptor improves scAAV-mediated liver-directed gene therapy in a model for inherited hyperbilirubinemia, the uridine diphospho-glucuronyl transferase 1A1-deficient Gunn rat. In male rats, preadministration of poly[i] increases the efficacy of a low dose (1×10¹¹ gc/kg) but not of a higher dose (3×10¹¹ gc/kg) scAAV8-LP1-UT1A1. Administration of poly[i] just before the vector significantly increases the correction of serum bilirubin in female rats. In these, the effect of poly[i] is seen by both doses but is more pronounced in the females receiving the low vector, where it also results in a significant increase of bilirubin glucuronides in bile. In conclusion, this study shows that SR-A mediates the endocytosis of AAV8 in vitro and in vivo and that blocking this receptor can improve the efficacy of AAV-mediated liver-directed gene therapy.

Structure and functional studies of the ribonuclease binase Glu43Ala/Phe81Ala mutant.

Ribonuclease from Bacillus intermedius (binase) is a small basic protein with antitumour activity. The three-dimensional structure of the binase mutant form Glu43Ala/Phe81Ala was determined at 1.98 Å resolution and its functional properties, such as the kinetic parameters characterizing the hydrolysis of polyinosinic acid and cytotoxicity towards Kasumi-1 cells, were investigated. In all crystal structures of binase studied previously the characteristic dimer is present, with the active site of one subunit being blocked owing to interactions within the dimer. In contrast to this, the new mutant form is not dimeric in the crystal. The catalytic efficiency of the mutant form is increased 1.7-fold and its cytotoxic properties are enhanced compared with the wild-type enzyme.

Hepatocyte apoptotic bodies encasing nonstructural HCV proteins amplify hepatic stellate cell activation: implications for chronic hepatitis C.

Chronic hepatitis C infection leads to increased hepatocyte apoptosis. Because engulfment of apoptotic bodies (ABs) by hepatic stellate cells (HSC) is profibrogenic, we compared the effects of ABs derived from hepatitis C virus (HCV)-negative vs HCV-infected (Con1+) Huh7 hepatoblastoma cells on fibrogenic and activation-related mRNA expression by a human HSC line (LX2). Uptake of Huh7(Con1+) ABs by LX2 cells dose dependently upregulated profibrotic genes (COL1A1, TGFB1; TIMP1; TIMP2). When normalized to the apoptotic cytokeratin-18 M30 neoepitope, HCV(+) ABs exhibited a more pronounced effect than HCV(-) ABs. In contrast, neither noningested ABs nor nucleic acids obtained from Huh7, Huh7(Con1+) or HepG2 cells triggered those AB-dependent effects. Both the engulfment of Huh7(Con1+) ABs and their effects were partially blocked by masking of phosphatidylserine with annexin V and completely inhibited by the class-A scavenger receptor ligand, polyinosinic acid. Our findings demonstrate that AB uptake stimulates HSCs and indicate that HCV infection leads to amplified fibrogenic mRNA expression and enhanced HSC activation.

Polymers for improving the in vivo transduction efficiency of AAV2 vectors.

BACKGROUND: Adeno-associated virus has attracted great attention as vehicle for body-wide gene delivery. However, for the successful treatment of a disease such as Duchenne muscular dystrophy infusion of very large amounts of vectors is required. This not only raises questions about the technical feasibility of the large scale production but also about the overall safety of the approach. One way to overcome these problems would be to find strategies able to increase the in vivo efficiency. METHODOLOGY: Here, we investigated whether polymers can act as adjuvants to increase the in vivo efficiency of AAV2. Our strategy consisted in the pre-injection of polymers before intravenous administration of mice with AAV2 encoding a murine secreted alkaline phosphatase (mSeAP). The transgene expression, vector biodistribution and tissue transduction were studied by quantification of the mSeAP protein and real time PCR. The injection of polyinosinic acid and polylysine resulted in an increase of plasmatic mSeAP of 2- and 12-fold, respectively. Interestingly, polyinosinic acid pre-injection significantly reduced the neutralizing antibody titer raised against AAV2. CONCLUSIONS: Our results show that the pre-injection of polymers can improve the overall transduction efficiency of systemically administered AAV2 and reduce the humoral response against the capsid proteins.

Non-hybridization saturable mechanisms play a role in the uptake of (68)Ga-Labeled LNA-DNA mixmer antisense oligonucleotides in rats.

Oligonucleotides (ODN) are key molecules for the aim of preventing translation of a gene product or monitoring gene expression in tissues. However, multiple methodological and biological hurdles need to be solved before in vivo application in humans will be possible. For positron emission tomography (PET) investigations, a 20-mer DNA-locked nucleic acid (LNA) mixmer ODN specific for rat chromogranin-A mRNA was labeled with (68)Ga and its uptake was examined in vivo in rats with and without blocking of scavenger receptors by polyribonucleotides. In addition, uptake studies of (68)Ga-LNA were performed with respect to time and concentration in human and rat cell lines. The human cell lines did not express the target mRNA. Both polyinosinic acid (poly-I) and polyadenylic acid (poly-A) reduced the uptake in rat tissues and in human cell lines. Poly-I was found to be more effective in the liver whereas poly-A was more effective in the kidney. In addition, the blockade by poly-I was statistically significant in the pancreas, adrenal gland, bone marrow, intestine, testis, urinary bladder, muscle, parotid gland, and heart, whereas poly-A also caused significant reduction in pancreas, adrenal gland, and bone marrow but not as much as in kidney. Cell culture study showed a 2-phase dose-dependent uptake characteristic with a saturable and a passive diffusion-like phase; however, these 2 phases were not so well expressed in the rat cell line. The results suggest that scavenger receptors or other saturable processes unrelated to hybridization may be involved in the tissue uptake of (68)Ga-LNA and in the clearance of antisense ODN through the liver, kidney, spleen, and bone marrow. The fact that these processes may be sequence-dependent suggests that proof of in vivo hybridization through imaging may not be obtained by only comparing sense and antisense sequences and proving dose-dependency.

Polyinosinic acid is a ligand for toll-like receptor 3.

Innate immune responses are critical in controlling viral infections. Viral proteins and nucleic acids have been shown to be recognized by pattern recognition receptors of the Toll-like receptor (TLR) family, triggering downstream signaling cascades that lead to cellular activation and cytokine production. Viral DNA is sensed by TLR9, and TLRs 3, 7, and 8 have been implicated in innate responses to RNA viruses by virtue of their ability to sense double-stranded (ds) RNA (TLR3) or single-stranded RNA (murine TLR7 and human TLR8). Viral and synthetic dsRNAs have also been shown to be a potent adjuvant, promoting enhanced adaptive immune responses, and this property is also dependent on their recognition by TLR3. It has recently been shown that mRNA that is largely single-stranded is a ligand for TLR3. Here we have investigated the ability of single-stranded homopolymeric nucleic acids to induce innate responses by murine immune cells. We show for the first time that polyinosinic acid (poly(I)) activates B lymphocytes, dendritic cells, and macrophages and that these responses are dependent on the expression of both TLR3 and the adaptor molecule, Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF). We therefore conclude that TLR3 is able to sense both single-stranded RNA and dsRNA.

Low molecular weight chitosan is an efficient inhibitor of ribonucleases.

RNase inhibitors are commonly used to block the RNase activity in manipulations with RNA-containing preparations. Recently RNase inhibitors, either synthetic or natural, have been intensively sought because they appeared to be promising for therapy of cancer and allergy. However, there is only a limited number of efficient RNase inhibitors. We have shown that a low molecular weight chitosan (M(r) approximately 6 kDa) inhibits activity of pancreatic RNase A and some bacterial RNases with inhibition constants in the range of 30-220 nM at pH 7.0 and ionic strength 0.14 M. The preferential contribution to the chitosan complex formation with RNases is due to establishment of 5-6 ion pairs. The results of this work show that polycations may efficiently inhibit ribonuclease activities.

Na+ and Mg2+ ion effect on the stability of a poly I . poly A . poly I triple helix.

Differential UV spectroscopy was used to study the temperature dependence of the conformational equilibrium in aqueous poly I . poly A . poly I (A2I) solutions containing Na+ (0.1-2 M) and Mg2+ (10(-5)-0.005 M) ions. Over the whole range of the studied Na+ and Mg2+ concentrations, the heating-induced destruction of the triple A2I helix is actually the A2I --> A + I + I (3 --> 1) transition. The rise of the transition temperature with increasing Na+ and Mg2+ contents is well described by Manning's and the "ligand" theories, which makes it possible to estimate the linear charge density on the single-stranded poly I (xi = 1.9 +/- 0.1) and the Mg2+-A2I binding constant (K = 1,250 M(-1) for the zero degree of binding). An analytical expression has been obtained, which correlates the constants of Mg2+ binding to three- and single-stranded polynucleotides (K3 and K1, respectively) and the linear charge density on them. There are only minor distinctions between the K3 values for A2U and A2I because these polynucleotides have similar structures. The difference in the K1 values is also slight as single-stranded poly U, poly I, and poly A have similar conformations. Dependence of the conformational transition temperatures of two triple helices with changing Mg2+ concentration.

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa.

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.