Polyinosinic-cytidylic Acid Research Articles

Maternal Immune Activation in Mice Only Partially Recapitulates the Autism Spectrum Disorders Symptomatology

Prenatal viral/bacterial infections are considered risk factors for autism spectrum disorders (ASD) and rodent models of maternal immune activation (MIA) have been developed and extensively used in preclinical studies. Poly inosinic-cytidylic acid (Poly I:C) was injected in C57BL6/J dams to mimic a viral infection on gestational day 12.5; the experimental design includes 10/12 litters in each treatment group and data were analysed always considering the litter-effect; neonatal (spontaneous motor behaviour and ultrasonic vocalizations) and adult [open field, marble burying, social approach, fear conditioning, prepulse inhibition (PPI)] offspring of both sexes were tested. In vivo magnetic resonance imaging/spectroscopy (MRI-MRS) and high-performance liquid chromatography (HPLC) to quantify both aminoacid and/or neurotransmitter concentration in cortical and striatal regions were also carried out. In both sexes high levels of repetitive motor responses and sensory gating deficits in PPI were the more striking effects of Poly I:C, whereas no alteration of social responses were evidenced. Poly I:C treatment did not affect mean values, but, intriguingly, increased variability in the levels of four aminoacids (aspartate glycine and GABA) selectively in males. As a whole prenatal Poly I:C induced relevant long-term alterations in explorative-stereotyped motor responses and in sensory gating, sparing cognitive and social competences. When systematically assessing differences between male and female siblings within each litter, no significant sex differences were evident except for increased variability of four aminoacid levels in male brains. As a whole, prenatal Poly I:C paradigms appear to be a useful tool to investigate the profound and translationally-relevant effects of developmental immune activation on brain and behavioural development, not necessarily recapitulating the full ASD symptomatology.

Parental exposure to bisphenol A and its analogs influences zebrafish offspring immunity

Transgenerational effects of environmental pollutants on humans and animals are complex. Thus, we used zebrafish to evaluate the effects of parental whole-life cycle exposure to bisphenol A and its analogs (bisphenol S and F) on offspring innate immunity. At adulthood, offspring were examined with/without continued chemicals treatment until 72h post-fertilization (hpf). To measure offspring immune function, larvae at 72 hpf were expose for 24h with/without the viral mimic polyinosinic-cytidylic acid (Poly I:C) or the bacterial mimic Pam3Cys-Ser-Lys4 (PAM3CSK4). Data show modified immunity in offspring. Specifically, lysozyme activity was significantly induced in F1 larvae and respiratory burst response and oxidative defense genes were inhibited. Genes of the innate immune system including Toll-like receptors and their downstream molecules and inflammatory cytokines were significantly down-regulated, whereas matrix metalloproteinases were up-regulated in larvae. In addition, recombination-activating genes in the immature adaptive immune system were significantly reduced. Thus, immune defense is diminished by exposing parental generations of zebrafish to environmentally relevant concentration of bisphenols and this suggests that fish chronically exposed to bisphenols in the wild may be vulnerable to pathogens.

Molecular Characterization of MyD88 in Pinctada fucata and its Response to Lipopolysaccharides and Polyinosinic-cytidylic Acid

Myeloid differentiation factor 88 (MyD88) is a key and essential adapter involved in the interleukin-1 receptor (IL-1R) and toll-like receptor (TLR)-mediated activation signaling pathway. To investigate molecular characterization of MyD88 and its gene expression profile in response to stimulation by lipopolysaccharide (LPS) and polyinosinic-cytidylic acid (poly (I: C)), we isolated the MyD88 cDNA sequence in Pinctada fucata and analyzed expression patterns using quantitative real-time PCR. Sequence analysis indicated that Pf-MyD88 cDNA is 1463bp in length and contains a1050bp open reading frame that encodes a 349 α peptide. Pf-MyD88 has the highest similarity with homologues of Crassostrea gigas and highly conserved death and toll/IL-1R domains. Furthermore, during LPS and poly (I:C)-stimulated experiments in the gill, peak expression levels of Pf-MyD88 were detected at 2h and 8h with a 1.58-fold and 3.58-fold increase, respectively. The results revealed the existence of a MyD88-dependent signaling pathway in P. fucata and contributed to understanding the potential role of Pf-MyD88 in the TLR/IL-1R-mediated signaling pathway.

B16 cell lysates plus polyinosinic-cytidylic acid effectively eradicate melanoma in a mouse model by acting as a prophylactic vaccine.

Th1 antigen-specific T cells secrete interferon-γ, which is able to kill antigen-specific cancer cells and is helpful for cancer vaccines. The aim of the present study was to explore whether B16 cell lysates plus polyinosinic-cytidylic acid (polyI:C) can effectively inhibit the progression of melanoma in an animal model. In the present study, C57BL/6 mice were divided into three groups, with each group containing more than six mice. The groups of mice were immunized twice with B16 cell lysates plus polyI:C, B16 cell lysates, or phosphate-buffered saline only, respectively. The in vivo results demonstrated that splenocytes from the mice immunized with B16 cell lysates plus poly I:C contained higher percentages of CD3+CD8+ Tlymphocytes and CD3+CD4+ Tlymphocytes, which were detected by a fluorescence-activated cell sorter, and produced higher levels of antigen-specific splenocyte proliferation activity, as detected by MTT assay. The splenocytes from the mice immunized with B16 cell lysates in combination with polyI:C produced higher levels of interferon‑γ, as detected by quantitativepolymerasechainreaction and ELISA, as well as cytotoxic T lymphocyte activity when stimulated in vitro with B16 lysates. Additionally, subcutaneous immunization of the C57BL/6 mice with B16 cell lysates plus polyI:C conferred greater protection against tumor-forming B16 melanoma cells than that of the mice immunized with injection of B16 cell lysate alone. In conclusion, the cancer vaccine of B16 cell lysates plus polyI:C exerts potently protective effects that polarize responses toward Th1 and elicit antitumor immunity.

Polyinosinic-cytidylic acid as an adjuvant on natural killer- and dendritic cell-mediated antitumor activities

Previously, we demonstrated that treatment with E7(44-62) and the adjuvant polyinosinic-cytidylic acid (poly(I:C)) in a rodent model generates antitumor immune responses, but the effect of E7(44-62) with poly(I:C) on natural killer (NK)- and dendritic cell (DC)-mediated antitumor activities is still unclear. Our goal was to examine the antitumor effects of E7(44-62) with poly(I:C). We examined the ability of E7(44-62) with poly(I:C) to induce toll-like receptor 3 (TLR3) expression, tumor necrosis factor-alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA expression, and tumor cell-killing activity in human NK cells as well as its ability to induce CD11c and CD86 expression and proliferation in human DCs. We found that E7(44-62) with poly(I:C) treatment markedly increased TLR3 expression and cytotoxicity against HeLa cells in human NK92 cells. Moreover, treatment with E7(44-62) and poly(I:C) markedly up-regulated IFN-γ and TNF-α mRNA expression in NK92 cells. Human patients with cervical cancer exhibited a marked decrease in the frequency of DCs; however, ex vivo treatment with E7(44-62) and poly(I:C) restored DC frequency. Stimulation of human DCs in patients with E7(44-62) and poly(I:C) led to high levels of CD11c and CD86 expression. Our data reveal the involvement of E7(44-62) combined with poly(I:C) in potentiating antitumor cytotoxicity and cytokine-producing activities in human NK92 cells and DCs.

Poly(I:C) combined with multi-epitope protein vaccine completely protects against virulent foot-and-mouth disease virus challenge in pigs

We designed a series of epitope proteins containing the G–H loops of three topotypes of foot-and-mouth disease virus (FMDV) serotype O and promiscuous artificial Th sites and selected one epitope protein (designated as B4) with optimal immunogenicity and cross-reactivity. Three out of five pigs immunized intramuscularly with this B4 were protected against virulent FMDV challenge after a single inoculation, while all pigs co-immunized with B4 and polyinosinic–cytidylic acid [poly(I:C)] conferred complete protection following FMDV challenge. Additionally, we demonstrated that all pigs co-immunized with B4 and poly(I:C) elicited FMDV-specific neutralizing antibodies, total IgG antibodies, type I interferon (IFN-α/β) and cytokines IFN-γ. In contrast, some pigs immunized with B4 alone produced parameters mentioned above, while some not, suggesting that poly(I:C) reduced animal-to-animal variations in both cellular and humoral responses often observed in association with epitope-based vaccines and up-regulated T-cell immunity often poorly observed in protein-based vaccines. We propose that poly(I:C) is an effective adjuvant for this epitope-based vaccine of FMDV. This combination could yield an effective and safe candidate vaccine for the control and eradication of FMD in pigs.

The antitumor effect of the toll-like receptor 3 ligand polyinosinic-cytidylic acid as an adjuvant.

Although polyinosinic-polycytidylic acid (poly(I:C)) has been applied in tumor immunity as a Toll-like receptor 3 (TLR3) ligand, the interaction between poly(I:C) and TLR3 is still unclear, as are the mechanisms underlying the antitumor effect of poly(I:C). Our aim was to investigate the interaction between poly(I:C) and TLR3, as well as the mechanisms underlying the antitumor effect of poly(I:C). NK92 cells were maintained in medium (untreated group), or medium containing E7(44-62) (E7 group) or E7(44-62)+poly(I:C) (poly(I:C)/E7 group), and we measured the expression of TLR3 mRNA, p-p65, and IκB-α protein. The cells were first incubated in medium alone or medium containing TLR3 monoclonal antibody, and then in medium containing poly(I:C)/E7. Finally, we measured the level of interferon-beta (INF-β) in the supernatant and determined the tumor cell-killing effect of the NK92 cells. At 1 h, the expression of TLR3 mRNA in the poly(I:C)/E7 group was markedly higher than that in the untreated and E7 groups (P < 0.05). When compared with the poly(I:C)/E7 group, the expression of IκB-α was dramatically increased in the E7 and untreated groups, and the expression of p-p65 was dramatically decreased in the E7 and untreated groups (all P < 0.05). At 24 h, INF-β content and tumor cell-killing activity in the poly(I:C)/E7 group were markedly higher than those in the untreated group (P < 0.001, <0.05, respectively). Treatment with TLR3 monoclonal antibody significantly inhibited poly(I:C)/E7-induced INF-β secretion and tumor cell-killing activity in NK92 cells (P < 0.001, <0.05, respectively). The interaction between poly(I:C) and TLR3 plays an important role in the antitumor immunity of NK92 cells. In addition, the interaction between poly(I:C) and TLR3 increases INF-β expression, which may be attributed to the activation of NFκB.

The TAK1-JNK cascade is required for IRF3 function in the innate immune response

Interferon regulatory factor (IRF)3 is critical for the transcriptional induction of chemokines and cytokines during viral or bacterial invasion. The kinases Tank binding kinase (TBK)1 and Ikappa B kinase (IKK)epsilon can phosphorylate the C-terminal part of IRF3 and play important roles in IRF3 activation. In this study, we show that another kinase, c-Jun-NH(2)-terminal kinase (JNK), phosphorylates IRF3 on its N-terminal serine 173 residue, and TAK1 can stimulate IRF3 phosphorylation via JNK. JNK specific inhibitor SP600125 inhibits the N-terminal phosphorylation without affecting the C-terminal phosphorylation. In addition, IRF3-mediated gene expressions on lipopolysaccharide (LPS) or polyinosinic-cytidylic acid (polyI:C) treatment are severely impaired by SP600125, as well as for reporter gene assay of IRF3 activation. Knockdown of TAK1 further confirmed these observations. Interestingly, constitutive active IRF3(5D) can be inhibited by SP600125; JNK1 can synergize the action of IRF3(5D), but not the S173A-IRF3(5D) mutant. More importantly, polyI:C failed to induce the phosphorylation of mutant S173A and SP600125 dramatically abrogated IRF3 phosphorylation and dimerization that was stimulated by polyI:C. Thus, this study demonstrates that the TAK1-JNK cascade is required for IRF3 function, in addition to TBK1/IKKvarepsilon, uncovering a new mechanism for mitogen-activated protein (MAP) kinase to regulate the innate immunity.

Molecular cloning and expression analysis of interferon-γ inducible lysosomal thiol reductase (GILT)-like cDNA from disk abalone ( Haliotis discus discus)

Interferon Gamma (IFN-γ) Inducible Lysosomal Thiol reductase (GILT) has been described as a key enzyme in processing and presentation of major histocompatibility complex (MHC) class II restricted antigen (Ag) by catalyzing disulfide bond (S–S) reduction in mammals. Abalone GILT-like (AbGILT) full-length cDNA was isolated from the normalized disk abalone cDNA library. The 807-bp AbGILT cDNA consists of an open reading frame of 684-bp, encoding 228 amino acid residues. The predicted AbGILT protein has a molecular weight of 25 kDa and an isoelectric point of 7.8. The N-terminus of the AbGILT was found to have a putative signal peptide with a cleavage site amino acid position at 19–20. AbGILT contains two active site C-XX-C motifs, ( 23CLDC 26 and 46CPYC 49) which motif is highly conserved in GILT protein family. AbGILT exhibited a characteristic GILT signature sequence 92CQHGX 2ECX 2NX 4C 107 and 12 cysteine residues representing 5% in the mature peptide. Phylogenetic analysis showed that AbGILT has been derived from a common ancestor with other GILT proteins. RT-PCR results showed that AbGILT expression was up-regulated in the gill, mantle and digestive tract 24 h post injection of phytohemagglutinin (PHA) while Vibrio alginolyticus up-regulation appeared in the gill and digestive tract after 48 h. In contrast, AbGILT expression was not up-regulated by poly inosinic–cytidylic acid (poly I:C) during the 48 h induction. However, AbGILT was constitutively expressed in gill, mantle, and digestive tract tissues suggesting that it may maintain first line of innate immune defense at basal level in disk abalone.

The adjuvant effects of the toll-like receptor 3 ligand polyinosinic-cytidylic acid poly (I:C) on antigen-specific CD8+ T cell responses are partially dependent on NK cells with the induction of a beneficial cytokine milieu

Poly (I:C), a TLR3 ligand, has shown promise as a vaccine adjuvant to CD8 + T cell responses. The underlying mechanisms involved in creating this adjuvant response in vivo, however, have not been well defined. In this study, we explored the contribution of NK cells and inflammatory cytokines in mediation the poly (I:C) adjuvant effects. Enhanced antigen-specific CD8 + T cell responses were observed only when poly (I:C) was administered within 4 h of peptide vaccination. Poly (I:C) treatment was associated with a rapid induction of inflammatory cytokines in the serum, including IL-6, IL-10, MCP-1, TNF-α, IFN-α, and IFN-γ, and selective increases in the numbers of NK (NK1.1 +CD11b +) cells and Mϕ (NK1.1 −CD11b +), but not NK T (CD3 +NK1.1 +) cells. NK cells were required for the adjuvant effects of poly (I:C). Poly (I:C) treatment in TNF-α, type I IFNR, IFN-γ, IL-6, IL-12Rβ2, or IL-15 gene-deficient mice revealed a reciprocal interaction and interdependence in the induction of these cytokines, where the absence of one cytokine impacted on the production of others. Further, the adjuvant effects of poly (I:C) were dependent on the endogenous levels of type I IFNs, TNF-α, IFN-γ, IL-12, and IL-15. IFN-α and IFN-β, but not TNF-α or IL-6, were able to mimic the adjuvant effects of poly (I:C). We conclude that the adjuvant effects of poly (I:C) on antigen-specific CD8 + T cells appeared to be exquisitely dependent on the rapid induction of certain beneficial cytokines produced in part by NK cells.

Viral Double-Stranded RNA Aggravates Lupus Nephritis through Toll-Like Receptor 3 on Glomerular Mesangial Cells and Antigen-Presenting Cells

How viral infections trigger autoimmunity is poorly understood. A role for Toll-like receptor 3 (TLR3) was hypothesized in this context as viral double-stranded RNA (dsRNA) activates dendritic cells to secrete type I interferons and cytokines that are known to be associated with the disease activity in systemic lupus erythematosus (SLE). Immunostaining of nephritic kidney sections of autoimmune MRL(lpr/lpr) mice revealed TLR3 expression in infiltrating antigen-presenting cells as well as in glomerular mesangial cells. TLR3-positive cultured mesangial cells that were exposed to synthetic polyinosinic-cytidylic acid (pI:C) RNA in vitro produced CCL2 and IL-6. pI:C RNA activated macrophages and dendritic cells, both isolated from MRL(lpr/lpr) mice, to secrete multiple proinflammatory factors. In vivo, a single injection of pI:C RNA increased serum IL-12p70, IL-6, and IFN-alpha levels. A course of 50 microg of pI:C RNA given every other day from weeks 16 to 18 of age aggravated lupus nephritis in pI:C-treated MRL(lpr/lpr) mice. Serum DNA autoantibody levels were unaltered upon systemic exposure to pI:C RNA in MRL(lpr/lpr) mice, as pI:C RNA, in contrast to CpG-DNA, failed to induce B cell activation. It therefore was concluded that viral dsRNA triggers disease activity of lupus nephritis by mechanisms that are different from those of bacterial DNA. In contrast to CpG-DNA/TLR9 interaction, pI:C RNA/TLR3-mediated disease activity is B cell independent, but activated intrinsic renal cells, e.g., glomerular mesangial cells, to produce cytokines and chemokines, factors that can aggravate autoimmune tissue injury, e.g., lupus nephritis.

VARIABLE EFFECTS OF IN VIVO STIMULATORS OR INHIBITORS OF HEPATIC CYTOCHROME P450 (HC-P450) ON BILIRUBIN (B) OXIDATION BY BRAIN MITOCHONDRIAL MEMBRANES (MM). 322

Background: Brain MM oxidize B at a rate that appears to be physiologically significant. We investigated the effects of in vivo HC-P450 simulators (phenobarbital [PB], β-naphthoflavone [BN]) or inhibitors (spironolactone [SP], 8-methoxypsoralen [MP], polyinosinic-cytidylic acid [pIC] on B oxidation by MM. Methods: Adult male Sprague-Dawley rats were injected ip with PB (60mg/kg/dx10d,n=4), BN (40mg/kg/dx4d, n=3), SP (100mg/kg BIDx4d, n=4), MP (55mg/kg, 1hr, n=4), pIC(10mg/kg, 24hr, n=5) All rats were killed by pentobarbital ip and brain vasculature was flushed in situ. Crude MM fractions (“P2”) were produced by homogenization and differential centrifugation in 0.32M sucrose. The P2 were resuspended in distilled H2O and sonicated to rupture the mitochondria. The protein concentrations were standardized to 2.5mg/ml of which 200μL MM was added to 2.5mL of a 0.1M barbital buffer pH 8.2 containing 10nM B, 5mM EDTA, and 500U/ml catalase. Optical density at 440nM was measured pre and post 60 min incubation at 37.5°C after centrifugation of a 1mL aliquot. Results: The hepatic inhibitors MP and pIC increased B oxidation by brain MM, while the hepatic inducer PB decreased B oxidation (ANOVA F=33.94, p<0.0001). BN and SP had no significant effects(results not shown). Data is presented as percent of control activity + SD(see figure). Conclusion: In vivo HC-P450 inducers and inhibitors have different effects on B oxidation by brain MM. B oxidation by brain MM is altered by P450 inducers and inhibitors, but the effect appears to be opposite that on HC-P450 activity. HC-P450 activity is primarily found in microsomes, while brain P450 enzymes appear to be mainly localized to mitochondria. It is at present not known whether this difference in intracellular localization is related to the apparently inverse response to the agents tested here.

Adjuvant actions of polyclonal lymphocyte activators: I. Comparison and characterization of their actions in antibody response to deaggregated bovine serum albumin

Various polyclonal lymphocyte activators (PLA) were compared in their effects to trigger the initiation of the immune response and the amplification of the once-initiated immune response. When PLA were injected into mice subcutaneously together with deaggregated bovine serum albumin (DBSA), all of the nine kinds of PLA tested, such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide, dextran sulfate, concanavalin A, pokeweed mitogen, phytohemagglutinin, polyadenylic-uridylic acid, and polyinosinic-cytidylic acid, exhibited more or less the adjuvant action to trigger the antibody response and to increase immunological memory to DBSA. Among the PLA tested the action of CPS-K was the strongest. On the other hand, none of these PLA, including CPS-K, acted to augment the secondary antibody response to the optimal dose of DBSA in mice primed with DBSA together with CPS-K. It was concluded that PLA act generally to trigger the initiation of the antibody response, although the intensity of their actions distributes in a wide range of diversity, but that they do not stimulate the amplification of the response.

Hyporeactivity of infection: potential limitation to therapeutic use of interferon-inducing agents.

Interferon inducers are generally most effective as antiviral agents when used prophylactically. One possible explanation for this is that animals develop a state of hyporeactivity during the course of a virus infection. Such a progressive loss of capacity to produce interferon was observed with a representative group of interferon-inducing agents (polyinosinic-cytidylic acid, Tilorone hydrochloride, New-castle disease virus, or a strain of encephalomyocarditis virus) during the course of a model picornavirus infection in mice.

Polyinosinic-cytidylic acid complex (poly I:C) and viral infections in mice.

Poly I:C, the double-stranded complex of polyinosinic and polycytidylic acids, was tested for activity in mice against infections produced by Columbia SK virus and viruses of clinical importance. Poly I:C had high activity against the SK virus. It had marginal activity, and only when given intranasally, against strains of influenza virus belonging to types A, A1, A2 and B. It was active against infections produced by parainfluenza 1 virus, vaccinia virus, and herpes simplex virus. It was inactive against Coxsackie A21 and B1 virus.

Inhibition of tumor growth by polyinosinic-polycytidylic acid.

The synthetic double-stranded RNA, polyinosinic-cytidylic acid, inhibits the growth of some tumors in mice. Two days after implantation of a reticulum cell sarcoma, a lymphatic lymphoma, a fibrosarcoma, two leukemias, and a human adenovirus 12-induced tumor, treatment of groups of mice resulted in decreased growth rates of the tumors and increased survival times of the animals. In the two tumors tested (the reticulum cell sarcoma and the adenovirus 12-induced tumor) initiation of treatment after the tumor was grown to moderate size caused a regression of the tumor. In the case of the reticulum cell sarcoma, the tumor had not reappeared in some of the animals two months after cessation of treatment.