Polyriboinosinic-polyribocytidylic Acid Research Articles

MKK7 confers different activities to viral infection of Singapore grouper iridovirus (SGIV) and nervous necrosis virus (NNV) in grouper

Mitogen-activated protein kinase 7 (MKK7) is one of the major stress-activated protein kinase (SAPK)-activating kinases in response to environmental or physiological stimuli. Here a MKK7 named as Ec-MKK7 was identified from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-MKK7 was 1853 bp, with an open reading frame (ORF) of 1272 bp encoding a putative protein of 423 amino acids. A characteristic S-K-A-K-T motif was contained in the domain of dual-specificity protein kinase, mitogen-activated protein kinase kinase 7 (PKc_MKK7). Intracellular localization showed that Ec-MKK7 was localized in both the cytoplasm and the nucleus of grouper spleen (GS) and/or grouper brain (EAGB) cells. Moreover, Ec-MKK7 was universally expressed in all examined tissues and showed expression modulation to challenges of lipopolysacchride (LPS), Singapore grouper iridovirus (SGIV) and polyriboinosinic polyribocytidylic acid (poly I:C) in vivo. A gene targeting strategy over-expressing Ec-MKK7 was performed to examine the activities of MKK7 to viral infection in vitro. Our data showed that Ec-MKK7 was involved in the evasion and replication of SGIV but played an antiviral role to the infection of nervous necrosis virus (NNV). All results demonstrated that Ec-MKK7 could play important roles in grouper innate immunity and show distinct functions on virus infection.

Genome-wide DNA Methylation Changes in a Mouse Model of Infection-Mediated Neurodevelopmental Disorders

BackgroundPrenatal exposure to infectious or inflammatory insults increases the risk of neurodevelopmental disorders. Using a well-established mouse model of prenatal viral-like immune activation, we examined whether this pathological association involves genome-wide DNA methylation differences at single nucleotide resolution. MethodsPrenatal immune activation was induced by maternal treatment with the viral mimetic polyriboinosinic-polyribocytidylic acid in middle or late gestation. Following behavioral and cognitive characterization of the adult offspring (n = 12 per group), unbiased capture array bisulfite sequencing was combined with subsequent matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and quantitative real-time polymerase chain reaction analyses to quantify DNA methylation changes and transcriptional abnormalities in the medial prefrontal cortex of immune-challenged and control offspring. Gene ontology term enrichment analysis was used to explore shared functional pathways of genes with differential DNA methylation. ResultsAdult offspring of immune-challenged mothers displayed hyper- and hypomethylated CpGs at numerous loci and at distinct genomic regions, including genes relevant for gamma-aminobutyric acidergic differentiation and signaling (e.g., Dlx1, Lhx5, Lhx8), Wnt signaling (Wnt3, Wnt8a, Wnt7b), and neural development (e.g., Efnb3, Mid1, Nlgn1, Nrxn2). Altered DNA methylation was associated with transcriptional changes of the corresponding genes. The epigenetic and transcriptional effects were dependent on the offspring’s age and were markedly influenced by the precise timing of prenatal immune activation. ConclusionsPrenatal viral-like immune activation is capable of inducing stable DNA methylation changes in the medial prefrontal cortex. These long-term epigenetic modifications are a plausible mechanism underlying the disruption of prefrontal gene transcription and behavioral functions in subjects with prenatal infectious histories.

EP 18. Deep Brain Stimulation improves behavior and modulates neural circuits in a rodent model of schizophrenia

Background A growing body of evidence suggests that schizophrenia (scz) is a neurodevelopmental disorder in which some brain alterations already occur during early brain development and others progress along the developmental course. This progressive nature of scz raises interest in the development of preventive avenues, i.e. therapeutic interventions prior to the full symptoms manifestation. For that however, a profounder understanding of the exact nature of altered brain development in scz is needed. Given methodological and ethical limitations of human studies, the use of appropriate animal models of schizophrenia is an invaluable tool. Methods This study used a maternal immune stimulation (MIS) rodent model of schizophrenia together with a neuromodulation approach, namely deep brain stimulation (DBS), to investigate the neurobiological and behavioral alterations during the course of disease. In this model, the exposure of pregnant rodents on their gestational day 15 to the viral mimic polyriboinosinic-polyribocytidylic acid (poly I:C) results in schizophrenia-relevant behavioral abnormalities which first emerge in adulthood, complying with the postnatal delay of this disorder. We applied DBS-interventions to the medial prefrontal cortex (mPFC) or nucleus accumbens (Nacc) to either poly I:C progeny or controls, at two different time points of scz development: a continuous DBS during a pre-symptomatic adolescence period and an acute DBS at the full symptomatic period of adulthood. In adulthood, we tested the effects of both interventions on two behavioral processes known to be disrupted in scz and in this model: sensorimotor gating and attentional selectivity. In addition, we studied the effects of DBS on several neurobiological parameters at post mortem . Results When applied at adulthood, DBS to both regions was found to be successful in alleviating sensorimotor gating and attentional selectivity deficits in the MIS model. However, when applied in adolescence only mPFC stimulation prevented both behavioral deficits, whereas Nacc stimulation only normalized sensorimotor deficits. Moreover, stimulation in those two brain sites induced different brain metabolic effects and neurochemical alterations, even if the effect on behavior was identical. Conclusions This study adds to the growing literature suggesting that electrical stimulation of specific brain regions improves behavioral deficits in neuro-psychiatric disorders and also scz. Moreover, this study indicates that early neuro-modulation might halt the progression of scz in reliance on stimulation site.

Two proprotein convertase subtilisin/kexin type 9 (PCSK9) paralogs from the tropical sea cucumber (Stichopus monotuberculatus): Molecular characterization and inducible expression with immune challenge.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a multifunctional protein that widely exists in eukaryotic species. In this study, two PCSK9 paralogs, named StmPCSK9-1 and StmPCSK9-2, were identified from the tropical sea cucumber (Stichopus monotuberculatus). The cDNAs of StmPCSK9-1 and StmPCSK9-2 are 1330kb and 1508kb in size, respectively. The open reading frames (ORF) for StmPCSK9-1 and StmPCSK9-2 cDNAs are 1128 and 1167bp in length, encoding the proteins of 375 and 388 amino acids with the deduced molecular weights of 38.76 and 41.07kDa, respectively. In accord with other members in PCSK9 family, the two StmPCSK9 paralogs possessed the inhibitor_I9 and peptidase_S8 functional domains, seven active sites, a catalytic triad and two calcium binding sites. For the gene structure, the splicing of the two StmPCSK9 paralogs was relatively conserved. In addition, the mRNA expression of StmPCSK9-1 and StmPCSK9-2 was only detected in the sea cucumber intestine and coelomocytes, and the expression levels of both the two StmPCSK9 paralogs were higher in intestine. Moreover, StmPCSK9-2 was found to be a cytoplasm protein without signal peptide, and show no response to the immune challenge. On the contrary, StmPCSK9-1 was a secreted protein and the transcriptional expression of StmPCSK9-1 was significantly up-regulated by lipopolysaccharides (LPS) treatment and slightly down-regulated by polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge in invitro experiments performed in the cultural primary coelomocytes, suggesting that the StmPCSK9-1 may play critical roles in the innate immune defense of sea cucumber, S.monotuberculatus, against bacterial and/or viral infections.

Comparative Characterization of Sequence Structure, Gene Expression, and Enzyme Activity of Alpha‐2‐macroglobulin between Stichopus monotuberculatus (Stichopodidae) and Holothuria atra (Holothuriidae)

AbstractAlpha‐2‐macroglobulin (A2M) is essential for hosts defending against pathogen infections. In this study, two distinct A2Ms were identified from sea cucumbers Stichopus monotuberculatus and Holothuria atra, which belong to different genera in the Phylum Echinodermata, and named StmA2M and HaA2M, respectively. Structural analysis of StmA2M and HaA2M revealed that all contain the bait region domain, thioester domain, and receptor‐binding domain. Phylogenetic analysis indicated that StmA2M and HaA2M all belong to the echinoderm A2Ms. The cleavage site RXXR and the catalytic histidine residue for complement component C3 were found in StmA2M and HaA2M. Compared with human A2M, the three‐dimensional structures of StmA2M and HaA2M were more similar to that of common carp A2M. Both StmA2M and HaA2M showed the strongest response at transcriptional levels to lipopolysaccharide (LPS) at 12 h and polyriboinosinic polyribocytidylic acid [Poly (I: C)] at 24 h, respectively. Activity of StmA2M showed the strongest response to Poly (I: C) at 6 h, while the strongest change of HaA2M activity was induced by LPS at 24 h. The differential mRNA expression and enzyme activity levels suggested that A2M in sea cucumbers might be involved in different immune responses to LPS or Poly (I: C) challenge.

Identification of differentially expressed genes in the spleens of polyriboinosinic polyribocytidylic acid (poly I:C)-stimulated yellow catfish Pelteobagrus fulvidraco.

The yellow catfish, Pelteobagrus fulvidraco (Siluriformes: Bagridae) is an economically important fish in China. However, genomic research and resources on this species are largely unavailable and still in infancy. In the present study, we constructed a cDNA library following poly I:C injection to screen for immune response genes in the spleens of P. fulvidraco using suppression subtractive hybridization (SSH). A total of 420 putative expressed sequence tag (EST) clones were identified at 24h post-injection, which contain 103 genes consisting of 25 immune response genes, 12 cytoskeleton genes, 7cell cycle and apoptosis genes, 7 respiration and energy metabolism genes, 7 transport genes, 26 metabolism genes, 10 stress response genes, 9 translational regulation genes, and 71 unknown genes. Real-time quantitative reverse transcription-PCR (qRT-PCR) results revealed that a set of randomly selected immune response genes were identified to be up-regulated after 24h of poly I:C stimulation compared to controls. Our study provides an annotation of immune genes in detail and insight into fish immunity.

Maternal Inflammation Disrupts Fetal Neurodevelopment via Increased Placental Output of Serotonin to the Fetal Brain.

Maternal inflammation during pregnancy affects placental function and is associated with increased risk of neurodevelopmental disorders in the offspring. The molecular mechanisms linking placental dysfunction to abnormal fetal neurodevelopment remain unclear. During typical development, serotonin (5-HT) synthesized in the placenta from maternal l-tryptophan (TRP) reaches the fetal brain. There, 5-HT modulates critical neurodevelopmental processes. We investigated the effects of maternal inflammation triggered in midpregnancy in mice by the immunostimulant polyriboinosinic-polyribocytidylic acid [poly(I:C)] on TRP metabolism in the placenta and its impact on fetal neurodevelopment. We show that a moderate maternal immune challenge upregulates placental TRP conversion rapidly to 5-HT through successively transient increases in substrate availability and TRP hydroxylase (TPH) enzymatic activity, leading to accumulation of exogenous 5-HT and blunting of endogenous 5-HT axonal outgrowth specifically within the fetal forebrain. The pharmacological inhibition of TPH activity blocked these effects. These results establish altered placental TRP conversion to 5-HT as a new mechanism by which maternal inflammation disrupts 5-HT-dependent neurogenic processes during fetal neurodevelopment. The mechanisms linking maternal inflammation during pregnancy with increased risk of neurodevelopmental disorders in the offspring are poorly understood. In this study, we show that maternal inflammation in midpregnancy results in an upregulation of tryptophan conversion to serotonin (5-HT) within the placenta. Remarkably, this leads to exposure of the fetal forebrain to increased concentrations of this biogenic amine and to specific alterations of crucially important 5-HT-dependent neurogenic processes. More specifically, we found altered serotonergic axon growth resulting from increased 5-HT in the fetal forebrain. The data provide a new understanding of placental function playing a key role in fetal brain development and how this process is altered by adverse prenatal events such as maternal inflammation. The results uncover important future directions for understanding the early developmental origins of mental disorders.

Transcriptome profiling of the antiviral immune response in Atlantic cod macrophages

A study was conducted to determine the transcriptome response of Atlantic cod (Gadus morhua) macrophages to the viral mimic, polyriboinosinic polyribocytidylic acid (pIC), using a 20K Atlantic cod microarray platform and qPCR. We identified 285 significantly up-regulated and 161 significantly down-regulated probes in cod macrophages 24 h after pIC stimulation. A subset of 26 microarray-identified transcripts was subjected to qPCR validation using samples treated with pIC or phosphate-buffered saline (control) over time (3, 6, 12, 24, 48 h), and 77% of them showed a significant response to pIC. The microarray and qPCR analyses in this study showed that pIC induced the expression of cod macrophage transcripts involved in RLR- and TLR-dependent pathogen recognition (e.g. tlr3, tlr7, mda5 and lgp2), as well as signal transducers (e.g. stat1 and nfkbia) and transcription activators (e.g. irf7 and irf10) in the MyD88-independent and dependent signalling pathways. Several immune effectors (e.g. isg15s, viperin, herc4, mip2 and ccl13) were significantly up-regulated in pIC-stimulated cod macrophages. The expression of some transcripts (e.g. irf7, irf10, viperin) was significantly up-regulated by pIC as early as 12 h. All pIC-induced transcripts had peak expression at either 24 h (e.g. tlr7, irf7, mip2) or 48 h (e.g. tlr3, lgp2, stat1). This study suggests possible roles of both vertebrate-conserved (e.g. tlr3 as an up-regulated gene) and fish-specific (tlr22g as a down-regulated gene) receptors in dsRNA recognition, and the importance of conserved and potentially fish-specific interferon stimulated genes in cod macrophages.

Discovery, structural characterization and functional analysis of alpha-2-macroglobulin, a novel immune-related molecule from Holothuria atra

The non-specific protease inhibitor alpha-2-macroglobulin (A2M) is a key macromolecular glycoprotein that involved in host immune defense against pathogens in vertebrates and invertebrates. However, no research regarding A2M has been developed in echinoderms to date. In this study, the full-length cDNA of A2M was cloned from the sea cucumber (Holothuria atra), which is a tropical species widely distributed along the coasts of the South China Sea and designated HaA2M. HaA2M possesses all three conserved functional domains of known A2M proteins, including the bait region domain, thioester domain and receptor-binding domain. Compared to fish and shrimp A2Ms, the histidine residue from the catalytical regions is well conserved in HaA2M. HaA2M mRNA was predominantly expressed in coelomocytes and, to a lesser extent, in the body wall, intestine and respiratory tree. A2M activity was detected in the coelomic fluids of H. atra. The mRNA expression and activity levels were investigated in the major immune tissues and coelomic fluids of H. atra after challenge with inactivated Vibrio alginolyticus or polyriboinosinic polyribocytidylic acid [Poly (I: C)]. RNA interference (RNAi)-mediated knockdown of HaA2M resulted in a significant reduction of HaA2M gene transcript level (86%). RNAi-mediated silencing of HaA2M gene significantly decreased the A2M activity (38%) and increased the number of viable bacteria (2.8-fold) in the coelomic fluids of H. atra infected by V. alginolyticus. Our study, as a whole, supplied the evidences for HaA2M as an immune-relevant molecule and it might have multiple functions in the innate immune system of H. atra.

Alteration of transcriptional networks in the entorhinal cortex after maternal immune activation and adolescent cannabinoid exposure

Maternal immune activation (MIA) and adolescent cannabinoid exposure (ACE) have both been identified as major environmental risk factors for schizophrenia. We examined the effects of these two risk factors alone, and in combination, on gene expression during late adolescence. Pregnant rats were exposed to the viral infection mimic polyriboinosinic-polyribocytidylic acid (poly I:C) on gestational day (GD) 15. Adolescent offspring received daily injections of the cannabinoid HU210 for 14days starting on postnatal day (PND) 35. Gene expression was examined in the left entorhinal cortex (EC) using mRNA microarrays. We found prenatal treatment with poly I:C alone, or HU210 alone, produced relatively minor changes in gene expression. However, following combined treatments, offspring displayed significant changes in transcription. This dramatic and persistent alteration of transcriptional networks enriched with genes involved in neurotransmission, cellular signalling and schizophrenia, was associated with a corresponding perturbation in the expression of small non-coding microRNA (miRNA). These results suggest that a combination of environmental exposures during development leads to significant genomic remodeling that disrupts maturation of the EC and its associated circuitry with important implications as the potential antecedents of memory and learning deficits in schizophrenia and other neuropsychiatric disorders.

Application of Magnesium Pyrophosphate–Based Sponge-Like Microparticles to Enhance the Delivery Efficiency and Adjuvant Effects of Polyriboinosinic-Polyribocytidylic Acid in Immune Cells

The magnesium pyrophosphate particle (MgPP) is a unique and safe carrier that is prepared by simply mixing magnesium chloride and sodium pyrophosphate. In this study, we investigated whether MgPP can be used to deliver nucleic acid–based adjuvants to immune cells. Polyriboinosinic-polyribocytidylic acid (polyI:C), a ligand for toll-like receptor 3, was selected as a model nucleic acid–based adjuvant. PolyI:C-loaded MgPP (polyI:C-MgPP) was prepared by adding polyI:C during the MgPP preparation process. Efficient loading of polyI:C into MgPP was confirmed by measuring the absorbance at 260 nm after disruption of polyI:C-MgPP by ethylenediaminetetraacetic acid. Scanning electron microscopy revealed that both MgPP and polyI:C-MgPP had a unique sponge-like shape with a diameter of approximately 1 μm. PolyI:C-MgPP was more efficiently taken up by toll-like receptor 3–positive RAW264.7 cells than naked polyI:C, and its uptake stimulated increased tumor necrosis factor–α production. When the presentation of ovalbumin (OVA), a model antigen, was evaluated after the addition of OVA along with naked polyI:C or polyI:C-MgPP to mouse dendritic DC2.4 cells, polyI:C-MgPP substantially increased OVA presentation. These results indicate that MgPP is a useful delivery vehicle for polyI:C and that polyI:C-MgPP is an effective immune cell adjuvant.

Cloning and expression analysis of interferon regulatory factor 7 in the Pacific cod, Gadus macrocephalus.

Interferon regulatory factor 7 (IRF7) plays an important role in regulating the response of type I interferon (IFN) to viral infection. To understand the mechanisms underlying immune reactions in the Pacific cod, Gadus macrocephalus, the gene encoding G. macrocephalus IRF7 was cloned and characterized. The cDNA of G. macrocephalus IRF7 was also cloned and sequenced. A cDNA sequence of 2032bp was assembled using polymerase chain reaction (PCR) products. It contains an open reading frame of 1323bp in length, which encoded a 440-amino acid polypeptide that comprised a DNA-binding domain (DBD), an IRF association domain (IAD), and a serine-rich domain (SRD). In the DBD, the tryptophan cluster consisted of only four tryptophans, which is a unique characteristic in fish IRF7. The mRNA of IRF7 was detected in various tissues, including in the spleen, thymus, kidney, intestine, and gills, using relative quantification PCR (R-qPCR). Dynamic expression of IRF7 was observed in larvae throughout post-hatching (ph) development, with the highest level detected at day of ph (dph) 25. Response to immune stimulation was examined by challenging larvae with polyriboinosinic polyribocytidylic acid (pIC) to mimic viral infection and elicit an immune reaction. R-qPCR revealed that the expression of IRF7 significantly increased in pIC-treated groups relative to that in the control groups, in a time-dependent manner, with peak responses at 48 and 72h after pIC-treatment. These results show that IRF7 is expressed in various tissues of adult fish and larvae and is sensitive to viral infection, suggesting that it plays a role in antiviral immune defense in G. macrocephalus.

Molecular clone and characterization of c-Jun N-terminal kinases 2 from orange-spotted grouper, Epinephelus coioides.

c-Jun N-terminal kinase 2 (JNK2) is a multifunctional mitogen-activated protein kinases involving in cell differentiation and proliferation, apoptosis, immune response and inflammatory conditions. In this study, we reported a new JNK2 (Ec-JNK2) derived from orange-spotted grouper, Epinephelus coioides. The full-length cDNA of Ec-JNK2 was 1920bp in size, containing a 174bp 5'-untranslated region (UTR), 483bp 3'-UTR, and a 1263bp open reading frame (ORF), which encoded a putative protein of 420 amino acids. The deduced protein sequence of Ec-JNK2 contained a conserved Thr-Pro-Tyr (TPY) motif in the domain of serine/threonine protein kinase (S-TKc). Ec-JNK2 has been found to involve in the immune response to pathogen challenges in vivo, and the infection of Singapore grouper iridovirus (SGIV) in vitro. Immunofluorescence staining showed that Ec-JNK2 was localized in the cytoplasm of grouper spleen (GS) cells, and moved to the nucleus after infecting with SGIV. Ec-JNK2 distributed in all immune-related tissues examined. After challenging with lipopolysaccharide (LPS), SGIV and polyriboinosinic polyribocytidylic acid (poly I:C), the mRNA expression of Ec-JNK2 was significantly (P<0.01) up-regulated in juvenile orange-spotted grouper. Over-expressing Ec-JNK2 in fathead minnow (FHM) cells increased the SGIV infection and replication, while over-expressing the dominant-negative Ec-JNK2Δ181-183 mutant decreased it. These results indicated that Ec-JNK2 could be an important molecule in the successful infection and evasion of SGIV.

Therapeutic Targets for Neurodevelopmental Disorders Emerging from Animal Models with Perinatal Immune Activation.

Increasing epidemiological evidence indicates that perinatal infection with various viral pathogens enhances the risk for several psychiatric disorders. The pathophysiological significance of astrocyte interactions with neurons and/or gut microbiomes has been reported in neurodevelopmental disorders triggered by pre- and postnatal immune insults. Recent studies with the maternal immune activation or neonatal polyriboinosinic polyribocytidylic acid models of neurodevelopmental disorders have identified various candidate molecules that could be responsible for brain dysfunction. Here, we review the functions of several candidate molecules in neurodevelopment and brain function and discuss their potential as therapeutic targets for psychiatric disorders.

Maternal intranasal immunization with HTLV-1 Env induces Env specific antibody responses in breast milk

Human T cell lymphotropic virus type-1 (HTLV-1) is the etiologic agent of Adult T cell Leukemia and Lymphoma (ATLL) and the neurological disorder HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The virus prevalent worldwide with foci of high prevalence. However, effective vaccines or drugs against HTLV-1 infection are not available. Because of the severity of the diseases and the absence of a satisfactory treatment, there is indeed an urgent need for an efficient vaccine against HTLV-1 in endemic regions. HTLV-1 is associated with persistent and lifelong infections and a low incidence of lymphomas within its hosts, and can be spread through the contact with bodily fluids containing infected cells, most often from mother to offspring through breast milk. These previous observations suggest that HTLV-1 has common features in infection and protective immune mechanisms with Bovine Leukemia virus (BLV). Imm une protection of newborns by vaccination is difficult to achieve since there is not enough time to mount an immune response before exposure to the virus. Interestingly, previous study revealed that antibodies from the maternal colostrum protect from BLV infection. On the basis of the above reports, we have designed a vaccination strategy mediating transfer of neutralizing antibodies from the mother to the offspring during pregnancy and / or lactation. In the present study, we investigated the effectiveness of intranasal vaccine using HTLV-1 Env protein as an antigen in combination with a synthetic double-stranded RNA analog for the induction of specific antibody in serum and breast milk in mother mice. Full-length Env protein was synthesized using the wheat germ cell-free protein production system. Intranasal administration for mother mice with the Env protein and the synthetic double-stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid [poly(I:C)] generated significant Env-specific systemic and mucosal immune responses as evidenced by the prominent induction of serum and milk antibodies. Our results provide the first evidence that intranasal co-administration of HTLV-1 Env protein with the poly(I:C) adjuvant augments the viral-specific immunity against HTLV-1.

Multi-Stage Tuberculosis Subunit Vaccine Candidate LT69 Provides High Protection against Mycobacterium tuberculosis Infection in Mice.

Effective tuberculosis (TB) vaccine should target tubercle bacilli with various metabolic states and confer long-term protective immunity. In this study, we constructed a novel multi-stage TB subunit vaccine based on fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-HspX (LT69 for short) which combined early expressed antigens and latency-associated antigen. The fusion protein was mixed with an adjuvant being composed of N, N’-dimethyl-N, N’-dioctadecylammonium bromide (DDA) and polyriboinosinic polyribocytidylic acid (PolyI:C) to construct subunit vaccine, whose immunogenicity and protective ability were evaluated in C57BL/6 mice. The results showed that LT69 had strong immunogenicity and high protective effect against Mycobacterium tuberculosis (M. tuberculosis) H37Rv aerosol challenge. Low-dose (2 μg) of LT69 generated long-term immune memory responses and provided effective protection, which was even higher than traditional vaccine BCG did at 30 weeks post the last vaccination. In conclusion, multistage subunit vaccine LT69 showed high and long-term protection against M. tuberculosis infection in mice, whose effect could be enhanced by using a relative low dosage of antigen.

The suppression of maternal-fetal leukemia inhibitory factor signal relay pathway by maternal immune activation impairs brain development in mice.

Recent studies in rodents suggest that maternal immune activation (MIA) by viral infection is associated with schizophrenia and autism in offspring. Although maternal IL-6 is though t to be a possible mediator relating MIA induced these neuropsychiatric disorders, the mechanism remains to be elucidated. Previously, we reported that the maternal leukemia inhibitory factor (LIF)–placental ACTH–fetal LIF signaling relay pathway (maternal–fetal LIF signal relay) promotes neurogenesis of fetal cerebrum in rats. Here we report that the maternal–fetal LIF signal relay in mice is suppressed by injection of polyriboinosinic-polyribocytidylic acid into dams, which induces MIA at 12.5 days post-coitum. Maternal IL-6 levels and gene expression of placental suppressor of cytokine signaling 3 (Socs3) increased according to the severity of MIA and gene expression of placental Socs3 correlated with maternal IL-6 levels. Furthermore, we show that MIA causes reduction of LIF level in the fetal cerebrospinal fluid, resulting in the decreased neurogenesis in the cerebrum. These findings suggest that maternal IL-6 interferes the maternal–fetal LIF signal relay by inducing SOCS3 in the placenta and leads to decreased neurogenesis.

Identification and expression analysis of the interferon-induced protein with tetratricopeptide repeats 5 (IFIT5) gene in duck (Anas platyrhynchos domesticus).

The interferon-induced proteins with tetratricopeptide repeats (IFITs) protein family mediates antiviral effects by inhibiting translation initiation, cell proliferation, and migration in the interferon (IFN) dependent innate immune system. Several members of this family, including IFIT1, IFIT2, IFIT3 and IFIT5, have been heavily studied in mammals. Avian species contain only one family member, IFIT5, and little is known about the role of this protein in birds. In this study, duck IFIT5 (duIFIT5) full-length mRNA was cloned by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of the cDNA ends (RACE). Based on the sequence obtained, we performed a series of bioinformatics analyses, and found that duIFIT5 was most similar to homologs in other avian species. Also, duIFIT5 contained eight conserved TPR motifs and two conserved multi-domains (TPR_11 and TPR_12). Finally, we used duck hepatitis virus type 1 (DHV-1) and polyriboinosinicpolyribocytidylic acid (poly (I:C)) as a pathogen or a pathogen-associated molecular pattern induction to infect three-day-old domestic ducklings. The liver and spleen were collected to detect the change in duIFIT5 transcript level upon infection by quantitative real-time PCR (qRT-PCR). DuIFIT5 expression rapidly increased after DHV-1 infection and maintained a high level, while the transcripts of duIFIT5 peaked at 8h after poly (I:C) infection and then returned to normal. Taken together, these results provide a greater understanding of avian IFIT5.

Isobavachalcone attenuates lipopolysaccharide-induced ICAM-1 expression in brain endothelial cells through blockade of toll-like receptor 4 signaling pathways

Inflammation has been implicated in the pathogenesis of various cerebral diseases. Thus, control of brain inflammation is regarded as one of the important therapeutic strategies for the treatment of neurodegenerative diseases such as Alzheimer׳s disease and stroke. Isobavachalcone, a flavonoid from Psoralea corylifolia, is known to possess a wide spectrum of biological activities and is expected to be useful in preventing or treating neurodegenerative diseases. However, very little is known regarding its effects on cerebral inflammation. In this study, we examined the effect of isobavachalcone on leukocyte adhesion and intercellular adhesion molecule-1 (ICAM-1) expression in brain endothelial cells activated with lipopolysaccharide (LPS) and explored the possible mechanisms involved. Isobavachalcone significantly down-regulated LPS-induced ICAM-1 expression and leukocyte–endothelial cell adhesion and suppressed NF-κB activity which is implicated in the expression of ICAM-1. It attenuated ICAM-1 expression as well as NF-κB transcriptional activity induced by macrophage-activating lipopeptide 2-kDa (MALP-2) or polyriboinosinic polyribocytidylic acid (poly[I:C]). Isobavachalcone also down-regulated LPS or poly[I:C]-induced expression of IFN-β, which can indirectly activate NF-κB. These data imply that isobavachalcone can modulate both MyD88-dependent and TRIF-dependent signaling of toll-like receptor 4 (TLR4). Taken together, our data suggest that isobavachalcone inhibits LPS-induced ICAM-1 expression and leukocyte adhesion to brain endothelial cell by blocking TLR4 signaling and thus, has the potential to ameliorate neuronal injury in brain diseases associated with inflammation.

The first echinoderm gamma-interferon-inducible lysosomal thiol reductase (GILT) identified from sea cucumber (Stichopus monotuberculatus)

Gamma-interferon-inducible lysosomal thiol reductase (GILT) has been described as a key enzyme that facilitating the processing and presentation of major histocompatibility complex class II-restricted antigen in mammals. In this study, the first echinoderm GILT named StmGILT was identified from sea cucumber (Stichopus monotuberculatus). The StmGILT cDNA is 1529 bp in length, containing a 5′-untranslated region (UTR) of 87 bp, a 3′-UTR of 674 bp and an open reading frame (ORF) of 768 bp that encoding a protein of 255 amino acids with a deduced molecular weight of 27.82 kDa and a predicted isoelectric point of 4.73. The putative StmGILT protein possesses all the main characteristics of known GILT proteins, including a signature sequence, a reductase active site CXXC, twelve conserved cysteines, and two potential N-linked glycosylation sites. For the gene structure, StmGILT contains four exons separated by three introns. In the promoter region of StmGILT gene, an NF-κB binding site and an IFN-γ activation site were found. The thiol reductase activity of recombinant StmGILT protein was also demonstrated in this study. In addition, the highest level of mRNA expression was noticed in coelomocytes of S. monotuberculatus. In in vitro experiments performed in coelomocytes, the expression of StmGILT mRNA was significantly up-regulated by lipopolysaccharides (LPS), inactivated bacteria or polyriboinosinic polyribocytidylic acid [poly (I:C)] challenge, suggested that the sea cucumber GILT might play critical roles in the innate immune defending against bacterial and viral infections.