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Dry eye disease (DED) is a growing public health concern affecting quality of life and visual function, with a significant socio-economic impact. It is characterised by the loss of homoeostasis, resulting in tear film instability, hyperosmolarity and inflammation of the ocular surface. If the innate immune response is unable to cope with internal bodily or environmental adverse conditions, the persistent, self-maintaining vicious circle of inflammation leads to the chronic form of the disease. Treatment of DED should be aimed at the restoration of the homoeostasis of the ocular surface system. A proper diagnostic approach is fundamental to define the relevance and importance of each of the DED main pathogenic factors, namely tear film instability, epithelial damage and inflammation. Consideration also needs to be given concerning two other pathogenic elements: lid margin changes and nerve damage. All the factors that maintain the vicious circle of DED in the patient’s clinical presentation have to be considered and possibly treated simultaneously. The treatment should be long-lasting and personalised since it has to be adapted to the different clinical conditions observed along the course of the disease. Since DED treatment is frequently unable to provide fast and complete relief from symptoms, empathy with patients and willingness to explain to them the natural history of the disease are mandatory to improve patients’ compliance. Furthermore, patients should be instructed about the possible need to increase the frequency and/or change the type of treatment according to the fluctuation of symptoms, following a preplanned rescue regimen.

Corneal stromal fibroblasts are connected to each other via gap junctions, which contribute to maintenance of corneal homeostasis. Viral infection of the corneal stroma can result in inflammation and scarring. The effects of polyinosinic-polycytidylic acid [poly(I:C)], an analog of viral double-stranded RNA, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs) were examined.Cultured HCFs were exposed to poly(I:C) in the absence or presence of inhibitors of mitogen-activated protein kinase (MAPK) signaling or the antioxidantExposure of HCFs to poly(I:C) resulted in down-regulation of Cx43 expression and GJIC activity as well as in up-regulation of microRNA-21 expression. Poly(I:C) increased the amount of malondialdehyde and reduced the activity of SOD in the cells, and these effects were prevented by NAC. The inhibitory effects of poly(I:C) on both Cx43 expression and GJIC activity were attenuated by NAC and by c-Jun NHPoly(I:C) inhibited Cx43 expression and GJIC in cultured HCFs, possibly as a result of the associated up-regulation of microRNA-21. Poly(I:C) also increased oxidative stress in these cells, and such stress together with signaling by the MAPK JNK was implicated in the effects of poly(I:C) on Cx43 expression and GJIC activity. Down-regulation of GJIC activity among corneal fibroblasts by double-stranded RNA may thus contribute to the disruption of stromal homeostasis during viral infection of the cornea.

Viral infections induce exosomes containing viral material and inflammatory factors. During respiratory tract infection, exosomes can easily cross the blood-brain barrier and transmit the inflammatory signal to the brain; however, such a hypothesis has no experimental evidence. The study investigated whether exosomes from virus mimetic poly (I:C)-primed airway cells enter the brain and interact with brain immune cells microglia. Airway cells were isolated from Wistar rats and BALB/c mice; microglial cell cultures - from Wistar rats. Exosomes from poly (I:C)-stimulated airway cell culture medium were isolated by precipitation, visualised by transmission electron microscopy, and evaluated by nanoparticle analyser; exosomal markers CD81 and CD9 were determined by ELISA. For in vitro and in vivo tracking, exosomes were loaded with Alexa Fluor 555-labelled RNA. Intracellular reactive oxygen species (ROS) were evaluated by DCFDA fluo-rescence and mitochondrial superoxide - by MitoSOX. The exosomes from poly (I:C)-primed airway cells entered the brain within an hour after intranasal introduction, were internalised by microglia, and induced intracellular and intramitochondrial ROS production. There was no ROS increase in microglial cells was after treatment with exosomes from airway cells untreated with poly (I:C). The data indicate that virus-primed airway cell exosomes might enter the brain and induce the activation of microglial cells.

While there is a strong focus on the negative consequences of maternal immune activation (MIA) on the developing brain, very little attention is directed towards potential advantages of early life challenges. In this study we utilized a polyinosine-polycytidylic acid (poly(I:C)) MIA model to test visual discrimination (VD) and reversal learning (RL) in mice using touchscreen technology. Significant sex differences emerged in that MIA improved the latency for males to make a correct choice in the VD task while females reached criterion sooner, made fewer errors and utilized fewer correction trials in RL compared to saline-treated controls. These surprising improvements were accompanied by the sex-specific upregulation of several neural markers critical to cognitive functioning (e.g., Gabrg2, Grin1, Grin2b, Htr2a, Chrm1, Prkca, and Camk2a mRNA in prefrontal cortex (PFC)), indicative of compensatory plasticity in response to the MIA challenge. In contrast, when exposed to a "two-hit" stress model (MIA combined with loss of the social component of environmental enrichment (EE)), mice showed no evidence of anhedonia but required an increased number of PD and RL correction trials. These animals also had significant reductions of CamK2a mRNA in the PFC. Appropriate functioning of synaptic plasticity, via mediators such as this protein kinase and others, are critical for behavioral flexibility. Although EE has been implicated in delaying the appearance of symptoms associated with certain brain disorders, these findings are in line with evidence that it also makes individuals more vulnerable to its loss. Overall, with the right "dose", early life stress exposure can confer at least some functional advantages, which are lost when the number or magnitude of these exposures become too great.

Background The neuroimmune system is required for normal neural processes, including modulation of cognition, emotion, and adaptive behaviors. Aberrant neuroimmune activation is associated with dysregulation of memory and emotion, though the precise mechanisms at play are complex and highly context dependent. Sex differences in neuroimmune activation and function further complicate our understanding of its roles in cognitive and affective regulation. Methods Here, we characterized the physiological sickness and inflammatory response of the hippocampus following intracerebroventricular (ICV) administration of a synthetic viral mimic, polyinosinic:polycytidylic acid (poly I:C), in both male and female C57Bl/6N mice. Results We observed that poly I:C induced weight loss, fever, and elevations of cytokine and chemokines in the hippocampus of both sexes. Specifically, we found transient increases in gene expression and protein levels of IL-1α, IL-1β, IL-4, IL-6, TNFα, CCL2, and CXCL10, where males showed a greater magnitude of response compared with females. Only males showed increased IFNα and IFNγ in response to poly I:C, whereas both males and females exhibited elevations of IFNβ, demonstrating a specific sex difference in the anti-viral response in the hippocampus. Conclusion Our data suggest that type I interferons are one potential node mediating sex-specific cytokine responses and neuroimmune effects on cognition. Together, these findings highlight the importance of using both males and females and analyzing a broad set of inflammatory markers in order to identify the precise, sex-specific roles for neuroimmune dysregulation in neurological diseases and disorders.

The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1β, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment., Highlights • Long-term effects of maternal poly(I:C) on immune mediators in the offspring appear limited. • Prenatal measurements and mid gestation poly(I:C) injection are associated with increases in IL-6 concentrations. • Variety in methodological conduct hampers identification of key elements that affect cytokine concentrations. • The quality of reporting of potential measures to minimize bias is poor.

Maternal immune activation (MIA) in response to infection during pregnancy has been linked through various epidemiological and preclinical studies to an increased risk of neurodevelopmental disorders such as autism spectrum disorder (ASD) and schizophrenia in exposed offspring. Sensory filtering disruptions occur in both of these disorders and are typically measured using the acoustic startle response in both humans and rodents. Our study focuses on characterizing the baseline reactivity, habituation and prepulse inhibition (PPI) of the acoustic startle response following exposure to MIA. We induced MIA using polyinosinic: polycytidylic acid (poly I:C) at gestational day (GD) 9.5 or 14.5, and we tested sensory filtering phenotypes in adolescent and adult offspring. Our results show that startle reactivity was robustly increased in adult GD9.5 but not GD14.5 poly I:C offspring. In contrast to some previous studies, we found no consistent changes in short-term habituation, long-term habituation or prepulse inhibition of startle. Our study highlights the importance of MIA exposure timing and discusses sensory filtering phenotypes as they relate to ASD, schizophrenia and the poly I:C MIA model. Moreover, we analyze and discuss the potential impact of between- and within-litter variability on behavioural findings in poly I:C studies., Highlights • Maternal immune activation (MIA) in rats by poly I:C is a model of neurodevelopmental disorder. • MIA at gestation day 9.5, but not 14.5, leads to robust startle hyperreactivity in response to sound. • No consistent changes in habituation or prepulse inhibition of startle are seen. • Analysis of within- and between-litter variability shows importance of experimental design to avoid random effects.

After the arrival of SARS COV2 (COVID 19) many biomedical devices have been innovated to mitigate the impact of the pandemic. Despite this, none have been developed to monitor patients with mild symptoms or the asymptomatic from their homes, forcing many hospitals worldwide to send these patients home with poor surveillance measures. The main objective of this project is to monitor from the HOMIL the vital signs of COVID 19 patients, who are currently isolated in their homes. For this purpose, a biomedical device in the form of a lowcost smartband has been developed. It is linked to a mobile application connected to a database where heart rate, oxygen saturation, and temperature parameters are sent in real time. The development was proposed in phases, which involve physical design, data acquisition and processing, and subsequent trial at the HOMIL. The smartband design and the mobile application has been developed, wireless communication between sensors has been achieved. Furthermore, the plan is to provide the HOMIL with the capability of initiating emergency protocols. The use of the smartband will be encouraged in patients who have tested positive for COVID 19 through PCR and will be taken off once they test negative.

Despite the potential of rodent models of maternal immune activation (MIA) to identify new biomarkers and therapeutic interventions for a range of psychiatric disorders, current approaches using these models ignore two of the most important aspects of this risk factor for human disease: (i) most pregnancies are resilient to maternal viral infection and (ii) susceptible pregnancies can lead to different combinations of phenotypes in offspring. Here, we report two new sources of variability—the baseline immunoreactivity (BIR) of isogenic females prior to pregnancy and differences in immune responses in C57BL/6 dams across vendors—that contribute to resilience and susceptibility to distinct combinations of behavioral and biological outcomes in offspring. Similar to the variable effects of human maternal infection, MIA in mice does not cause disease-related phenotypes in all pregnancies and a combination of poly(I:C) dose and BIR predicts susceptibility and resilience of pregnancies to aberrant repetitive behaviors and alterations in striatal protein levels in offspring. Even more surprising is that the intermediate levels of BIR and poly(I:C) dose are most detrimental to offspring, with higher BIR and poly(I:C) doses conferring resilience to measured phenotypes in offspring. Importantly, we identify the BIR of female mice as a biomarker before pregnancy that predicts which dams will be most at risk as well as biomarkers in the brains of newborn offspring that correlate with changes in repetitive behaviors. Together, our results highlight considerations for optimizing MIA protocols to enhance rigor and reproducibility and reveal new factors that drive susceptibility of some pregnancies and resilience of others to MIA-induced abnormalities in offspring.

Pneumonia is a common illness that continues to be the major killer of remaining to be a significant source of morbidity and mortality in the patient population. Many microorganisms cause pneumonia, and now concern is turning to the importance of the cause the new therapies for viral pneumonia. In the current study, we report the effect of andrographolide sulfonate, a water-soluble form of andrographolide (trade name: Xi-Yan-Ping Injection), on poly I: C-induced pneumonia. Andrographolide sulfonate was administrated through intraperitoneal injection to mice with poly I: C-induced pneumonia. Recruitment of airway inflammatory cells, alteration of lung histological induced by Poly I: C were significantly ameliorated by andrographolide sulfonate. The protein levels of pro-inflammatory cytokines in bronchoalveolar fluid (BALF) and serum were reduced by andrographolide sulfonate treatment. The levels of MUC5AC and MUC5B in lung tissue were also suppressed. These results reveal that andrographolide sulfate remarkably alleviated pneumonia induced by poly I:C in mice. Moreover, andrographolide sulfonate markedly inhibited the activation of nuclear factor-κB (NF-κB). Taken together, we demonstrated that andrographolide sulfonate ameliorated poly I: C-induced pneumonia in mice, suggesting the possible use of andrographolide sulfonate for virus-induced pneumonia in clinical.

Maternal immune activation (MIA) in response to a viral infection during early and mid-gestation has been linked through various epidemiological studies to a higher risk for the child to develop autism or schizophrenia-related symptoms.. This has led to the establishment of the pathogen-free poly I:C-induced MIA animal model for neurodevelopmental disorders, which shows relatively high construct and face validity. Depending on the experimental variables, particularly the timing of poly I:C administration, different behavioural and molecular phenotypes have been described that relate to specific symptoms of neurodevelopmental disorders such as autism spectrum disorder and/or schizophrenia. We here review and summarize epidemiological evidence for the effects of maternal infection and immune activation, as well as major findings in different poly I:C MIA models with a focus on poly I:C exposure timing, behavioural and molecular changes in the offspring, and characteristics of the model that relate it to autism spectrum disorder and schizophrenia.

Chemokine receptors play an important role in coordination of cell trafficking in many biological processes. In this study, a CC chemokine receptor 9 of cobia Rachycentron canadum (RcCCR9) was identified. Analysis of the ORF (1119 bp) of RcCCR9 revealed a predicted protein of 41.87 kDa with typical seven transmembrane domain architecture. RcCCR9 shared several conserved structural features with homologs from mammals and other fish, and had a consistent relationship with phylogenetic trees and sequence identities. Reverse transcription quantitative PCR analysis showed ubiquitous RcCCR9 transcripts in healthy cobia, mainly in immune-related organs, with the highest levels in blood and lower levels in intestines and brain. After challenge with inactivated Vibrio harveyi or viral mimic poly I:C, RcCCR9 expression was up-regulated in head kidney and down-regulated in spleen. Compared with poly I:C, V. harveyi induced a stronger up/down-regulation of CCR9 mRNA levels in the central immune organs. RcCCR9 seems to be strongly involved in host defense against bacterial infection.

Objective: Toll-like receptor (TLR) activation plays an important role in cerebral ischemia-reperfusion injury. In addition, increasing evidence suggests that TLRs may affect cognitive behavior through TLR-mediated signaling. Here, we explored the protective effects of TLR3 on cognitive dysfunction after ischemia in the context of poly(I:C) preconditioning.Materials and Methods : Mice (n=84) were randomly divided into the sham group, AAV (vector) group, middle cerebral artery occlusion (MCAO) model group, poly(I:C) (pre) + MCAO model group, and AAV (TRAF6) + poly(I:C) (pre) + MCAO model group. The mice were injected i.p. with poly(I:C) (1.25 mg/g) 24 h prior to cerebral ischemia. Then, neurological scores were assessed, and the infarct volume was measured after cerebral ischemia-reperfusion. We evaluated the poly(I:C) preconditioning-induced attenuation of neuronal damage using Nissl and TUNEL staining. We assessed the poly(I:C) preconditioning-mediated inhibition of I/R-induced glial activation, inflammatory factor levels and TRAF6 expression. We also assessed whether TRAF6 affects poly(I:C) preconditioning to improve cognitive dysfunction and neuroprotection.Results: The results showed that compared with those of the sham group and AAV (vector) group, the functional neurological scores and focal infarct volume of the MCAO group and poly(I:C) preconditioning group were significantly increased. The results also showed that compared with those of the MCAO group, the functional neurological scores and focal infarct volume of the poly(I:C) preconditioning group were significantly reduced. Our results indicated that poly(I:C) preconditioning significantly attenuated neuronal apoptosis and cell loss. Poly(I:C) preconditioning also inhibited I/R-induced glial cell activation and reduced NF-κB, TNF-α and IL-β levels. Our findings showed that poly(I:C) preconditioning affected cognitive dysfunction following cerebral I/R. Here, we observed that poly(I:C) preconditioning affected the expression and distribution of TRAF6 following cerebral I/R. TRAF6 overexpression abolished poly(I:C)-induced neuroprotection and worsened cognitive dysfunction in cerebral I/R injury.Significance: Our findings suggested that poly(I:C) preconditioning ameliorates cognitive dysfunction after cerebral I/R injury by inhibiting TRAF6 signaling, which is a potential therapeutic target for the treatment of cognitive dysfunction after stroke.

Maternal immune activation has been identified as a significant risk factor for schizophrenia. Using rodent models, past work has demonstrated various behavioral and brain impairments in offspring after immune-activating events. We applied 5 mg/kg of poly(I:C) on gestation day 9 to pregnant mouse dams, whose offspring were then stressed during puberty. We show impairments in attentional set-shifting in a T-maze, and a decreased number of parvalbumin-positive interneurons in the hippocampus as a result of peripubertal stress specifically in females.

Background and Purpose The potential for therapeutic antibody treatment of neurological diseases is limited by poor penetration across the blood–brain barrier. I.c.v. delivery is a promising route to the brain; however, it is unclear how efficiently antibodies delivered i.c.v. penetrate the cerebrospinal spinal fluid (CSF)-brain barrier and distribute throughout the brain parenchyma. Experimental Approach We evaluated the pharmacokinetics and pharmacodynamics of an inhibitory monoclonal antibody against β-secretase 1 (anti-BACE1) following continuous infusion into the left lateral ventricle of healthy adult cynomolgus monkeys. Key Results Animals infused with anti-BACE1 i.c.v. showed a robust and sustained reduction (~70%) of CSF amyloid-β (Aβ) peptides. Antibody distribution was near uniform across the brain parenchyma, ranging from 20 to 40 nM, resulting in a ~50% reduction of Aβ in the cortical parenchyma. In contrast, animals administered anti-BACE1 i.v. showed no significant change in CSF or cortical Aβ levels and had a low (~0.6 nM) antibody concentration in the brain. Conclusion and Implications I.c.v. administration of anti-BACE1 resulted in enhanced BACE1 target engagement and inhibition, with a corresponding dramatic reduction in CNS Aβ concentrations, due to enhanced brain exposure to antibody.

Environmental agents, including viral and bacterial infectious agents, are involved in the alteration of physicochemical and biological parameters in the nasal epithelium. Hyaluronan (HA) has an important role in the regulation of tissue healing properties. High molecular weight HA (HMW-HA) shows greater anti-inflammatory responses than medium molecular weight HA (MMW-HA) and low molecular weight HA (LMW-HA). We investigated the effect of HMW-HA, MMW-HA and LMW-HA on the regulation of physicochemical and biological parameters in an ``in vitro'' model that might mimic viral infections of the nasal epithelium. Human nasal epithelial cell line RPMI2650 was stimulated with double-stranded RNA (dsRNA) Poly(I:C) for 5 days in air-liquid-interface (ALI) culture (3D model of airway tissue). dsRNA Poly(I:C) treatment significantly decreased transepithelial electrical resistance (TEER) in the stratified nasal epithelium of RPMI2650 and increased pH values, rheological parameters (elastic G' and viscous G `'), and Muc5AC and Muc5B production in the apical wash of ALI culture of RPMI2650 in comparison to untreated cells. RPMI2650 treated with dsRNA Poly(I:C) in the presence of HMW-HA showed lower pH values, Muc5AC and Muc5B production, and rheological parameters, as well as increased TEER values in ALI culture, compared to cells treated with Poly(I:C) alone or pretreated with LMW-HA and MMW-HA. Our 3D ``in vitro'' model of epithelium suggests that HMW-HA might be a coadjuvant in the pharmacological treatment of viral infections, allowing for the control of some physicochemical and biological properties affecting the epithelial barrier of the nose during infection.

The viral mimetic polyinosinic:polycytidylic acid (poly(I:C)) is increasingly used to induce maternal immune activation (mIA) to model neurodevelopmental disorders (NDDs). Robust and reproducible phenotypes across studies are essential for the generation of models that will enhance our understanding of NDDs and enable the development of improved therapeutic strategies. However, differences in mIA-induced phenotypes using poly(I:C) have been widely observed, and this has prompted the reporting of useful and much needed methodological guidelines. Here, we perform a detailed investigation of molecular weight and endotoxin variations in poly(I:C) procured from two of the most commonly used suppliers, Sigma and InvivoGen. We demonstrate that endotoxin contamination and molecular weight differences in poly(I:C) composition lead to considerable variability in maternal IL-6 response in rats treated on gestational day (GD)15 and impact on fetal outcomes. Specifically, both endotoxin contamination and molecular weight predicted reductions in litter size on GD21. Further, molecular weight predicted a reduction in placental weight at GD21. While fetal body weight at GD21 was not affected by poly(I:C) treatment, male fetal brain weight was significantly reduced by poly(I:C), dependent on supplier. Our data are in agreement with recent reports of the importance of poly(I:C) molecular weight, and extend this work to demonstrate a key role of endotoxin on relevant phenotypic outcomes. We recommend that the source and batch numbers of poly(I:C) used should always be stated and that molecular weight variability and endotoxin contamination should be minimised for more robust mIA modelling.

BACKGROUND: Rodent models of maternal immune activation (MIA) are used as an experimental approach for neurodevelopmental disorders such as schizophrenia to study neuronal and behavioral dysfunctions of offspring. In particular, injection of the viral mimic poly(I:C) in pregnant mice causes disruption of neuronal development of the offspring leading to deficits in social behavior, cognition, sensorimotor processing and/or dopaminergic system in adulthood [1]. BI 409306 is a selective phosphodiesterase-9 (PDE9) inhibitor [2] currently under development for the prevention of relapse in patients with schizophrenia taking antipsychotic medications (NCT03351244). PDE9 inhibition is hypothesized to improve the NMDA-receptor signaling cascade by increasing cGMP levels which subsequently leads to strengthening of synaptic plasticity. Abnormalities in glutamatergic neurotransmission related to NMDA-receptor hypofunction, along with dysfunction of the dopaminergic circuitry, are believed to play a crucial role in the pathophysiology of schizophrenia. In this study, we have investigated the effect of BI 409306 on the behavioral deficits of adult offspring from the poly(I:C)-based MIA mouse model. METHODS: Pregnant C57BL6/N mice were treated with poly(I:C) (5 mg/kg, i.v.) or control (saline, i.v.) solution on gestation day 12. All male offspring (n=9/group) were administered orally once daily with BI 409306 (0.2, 0.5 or 1 mg/kg) or vehicle starting at post-natal day (PND) 70 until subjected to a battery of consecutive behavioral tests two weeks later which lasts until PND120. The order of these tests were: Y-maze spontaneous alternation for working memory performance, social interaction to assess social behavior, pre-pulse inhibition (PPI) for sensorimotor processing and amphetamine-induced hyperlocomotion to assess the dopaminergic system. Statistical analyses were done by one-way ANOVA followed by Fisher’s LSD post-hoc. RESULTS: Maternal poly(I:C)-induced immune challenge led to a significant decrease in social interaction (p

Central fatigue is a condition associated with impairment of the central nervous system often leading to the manifestation of a range of debilitating symptoms. Fatigue can be a consequence of systemic inflammation following an infection. Administration of lipopolysaccharide (LPS) and polyriboinosinic:polyribocytidlic (poly I:C) to animals can induce systemic inflammation by mimicking a bacterial or viral infection respectively and therefore have been used as models of fatigue. We evaluated a range of phenotypic behaviors exhibited in the LPS and poly I:C animal models to assess whether they adequately replicate fatigue symptomology in humans. In addition to standard observation- and intervention-based behavioral assessments, we used powerful in-cage monitoring technology to quantify rodent behavior without external interference. LPS and poly I:C treated Sprague Dawley rats displayed ‘sickness behaviors’ of elevated temperature, weight loss and reduced activity in the open field test and with in-cage monitoring within 24 h post-treatment, but only LPS-treated rats displayed these behaviors beyond these acute timepoints. Once sickness behavior diminished, LPS-treated rats exhibited an increase in reward-seeking and motivation behaviors. Overall, these results suggest that the LPS animal model produces an extensive and sustained fatigue-like phenotype, whereas the poly I:C model only produced acute effects. Our results suggest that the LPS animal model is a more suitable candidate for further studies on central fatigue-like behavior.

Alzheimer's disease is marked by the accumulation of the amyloid-beta (Aβ) peptide, and increases in phosphorylation of the microtubule associated protein, tau. Changes in these proteins are considered responsible, in part, for the progressive neuronal degeneration and cognitive deficits seen in AD. We examined the effect of repeated consecutive peripheral poly I:C injections on cognitive deficits, central Aβ, and phosphorylated tau accumulation, following three treatment durations: 7, 14, and 21 days. Forty-eight hours after the final injection, animals were trained in a contextual fear-conditioning paradigm, and tested 24h later. Immediately after testing, the hippocampus was collected to quantify Aβ and phosphorylated tau accumulation. Results showed that, although poly I:C-induced Aβ was significantly elevated at all time points examined, poly I:C only disrupted cognition after 14 and 21 days of administration. Moreover, elevations in phosphorylated tau were not seen until the 14-day time point. Interestingly, phosphorylated tau expression then declined at the 21-day time point. Finally, we demonstrated that Aβ levels are a stronger predictor of cognitive dysfunction, explaining 37% of the variance, whereas phosphorylated tau levels only accounted for 0.2%. Taken together, these results support the hypothesis that inflammation-induced elevation in Aβ disrupts cognition, independently of phosphorylated tau, and suggest that long-term administration of poly I:C may provide a model to investigate the contribution of long-term inflammation toward the development of Alzheimer's-like pathology.

Sociocommunicative deficits commonly observed in autism spectrum disorder (ASD) can be experimentally modeled using rodents' ultrasonic vocalizations (USVs). For example, USVs emitted by pups, separated from their mothers and nest, serve as a useful tool to identify autistic-like behaviors during the early period of development. Being sensitive to social context, these neonatal calls may help to reveal reduced social attachment or abnormal processing of social information. The aim of the present study was to characterize quantitative and structural changes in USVs emitted during isolation by male and female rat pups prenatally exposed to either valproic acid (VPA) or poly(I:C). To determine whether those pups differed from controls in sensitivity to social stimuli, isolation-induced USVs were recorded under two bedding conditions, i.e., novel bedding and soiled bedding from their home cages. Our results demonstrated early communication deficits in both models of autism. We reported a reduced number of USVs emitted by both VPA- and poly(I:C)-exposed males and females. Moreover, compared to the controls, VPA (but not poly(I:C)) pups emitted shorter calls with a higher peak frequency. While VPA offspring demonstrated fewer USVs on the "safe" bedding imbued with maternal/nest odors, this calming effect was not observed in poly(I:C) males, suggesting a more specific deficit in social communication. The present results demonstrate that qualitative along with quantitative analyses of neonatal vocalizations are a useful tool for assessing early sociocommunicative deficits in ASD models. Notably, more specific changes in USV emission may be detected when introducing social context.

Matrix metalloproteinase (MMP)-9 is thought to be involved in the etiopathogenesis of chronic rhinosinusitis (CRS) with nasal polyps and cleaves collagen IV, causing hyperpermeability of the basement membrane within mucosal tissue. It is known that MMP-9 expression is negatively affected by sirtuin (SIRT)-1 in human monocytotic cells, retinal endothelial cells, and epithelial carcinoma cells. However, it is unknown which factors affect MMP-9 expression and activity in human nasal epithelial cells (HNECs). To examine factors affecting MMP-9 expression and activity in HNECs, HNECs were stimulated with Toll-like receptor (TLR) agonists, followed by quantitative PCR, immunofluorescence, and zymography to examine MMP-9 expression and activity. MMP-9 expression was evaluated in sinonasal tissue of control subjects without CRS, and patients with CRS without nasal polyps and those with CRS with nasal polyps, in relation to the expression of SIRT1 using a tissue microarray. The effect of SIRT1 stimulation/inhibition on MMP-9 expression in HNECs was also tested. TLR3 agonists increased MMP-9 mRNA expression (473 fold, P = 0.0198) and activity (20.4-fold, P 0.05). SIRT1 activation or inhibition reciprocally affected MMP-9 expression in the presence of TLR3 agonists. MMP-9 and SIRT1 expression within the epithelial layer of sinonasal tissue was inversely correlated only in patients with CRS but not in control subjects. TLR3 agonists increased MMP-9 expression and activity in HNECs, and the effect was abolished in the presence of SIRT1 activation. SIRT1 and MMP-9 expression was inversely correlated in CRS tissue, supporting SIRT1 as a possible therapeutic target for nasal polyp formation.

BACKGROUND: In the last two decades, the concept of early intervention in schizophrenia has gained much attention. Although several studies showed that treatment with atypical antipsychotic drugs (APDs) in the early clinical stages of the disorder may reduce the conversion rate to first-episode psychosis, it remains unknown if intervention can halt the progression of structural brain aberrations, and whether structural abnormalities and symptom emergence are related. Given the diagnostic, ethical and methodological limitations of human studies, the efficacy and utility of early intervention are bound to remain debatable and controversial. Here we used the maternal activation model to show that prevention of behavioral abnormalities requires normalization of structural deficits and must be implemented prior to the emergence of behavioral abnormalities. METHODS: On gestation day 15, pregnant dams were injected intravenously with Poly-I:C (4 mg/kg/) or saline. Eight different cohorts of poly-I:C or saline female offspring were injected with risperidone (RIS, 0.045mg/kg) or clozapine (CLOZ, 7.5mg/kg) and respective vehicle at different developmental windows. Four cohorts were injected with RIS or vehicle at 4 time windows (TW): PNDs 34–47 (TW1), PNDs 48–61 (TW2), or PNDs 106–119 (TW3), and three cohorts were injected with CLOZ at the first three windows (third window was ineffective). All animals underwent behavioral testing (latent inhibition and amphetamine-induced locomotion) and imaging about six weeks after drug cessation. RESULTS: Adult females exposed prenatally to poly-I:C had smaller hippocampal, striatal, and pre-fronto-cortical volumes and larger lateral ventricular volumes, as well as disrupted latent inhibition and higher amphetamine-induced activity, compared to their controls. The efficacy of a two-week ultra-low dose RIS or CLOZ treatment in preventing these behavioral and structural abnormalities in poly-I:C offspring was a function of the drug and their time of administration. RIS was effective in preventing both structural and behavioral abnormalities when administered in the first three windows, whereas at the PND 106–120 window, RIS failed to prevent both structural and behavioral abnormalities. CLOZ was effective in preventing structural and behavioral abnormalities in the first two windows but failed to prevent both already in the third window. CONCLUSIONS: The present results provide strong support for structure-function relationship, since behavioral deficits were prevented only if structural deficits were prevented. Furthermore, they demonstrate that intervention must be given prior to the emergence of behavioral abnormalities in order to be effective. We previously showed that whereas volumetric reductions are present from early adolescence, disrupted latent inhibition and increased amphetamine-induced activity emerges in the female offspring on PND90. We therefore expected that in the present study, RIS and CLOZ will be effective only if given before PND 90, and this was indeed found. The difference in the preventive efficacies between RIS and CLOZ most likely stem from the fact that at the doses used, RIS acts as a 5HT2A/C antagonist without dopaminergic blockade, whereas CLOZ exerts substantial DA blockade. Importantly, the prevention trajectories in females differed from those we found previously in males and will be discussed.

Yang, X.-q., Wang, L., Li, H.-t. and Liu, D. 2015. Immune responses of porcine airway epithelial cells to poly(I:C), a synthetic analogue of viral double-stranded RNA. Can. J. Anim. Sci. 95: 13–20. Swine respiratory disease (SRD) is one of the most economically important diseases affecting the pig industry. The main infectious agents that cause SRD are viruses, but the molecular pathogenesis of viral SRD has not been extensively studied. Here, using digital gene expression tag profiling, the global transcriptional responses to poly(I:C), a synthetic analogue of viral double-stranded RNA, was analyzed in porcine airway epithelial cells (PAECs). The profiling analysis revealed numerous differentially expressed genes (DEGs), including unknown sequences in the porcine nucleotide databases. Gene ontology enrichment analysis showed that DEGs were mainly enriched in response to stress (GO: 0006950), of which, defense response is one sub-process. Poly(I:C) challenge induced a general inflammation response as indicated by marked upregulation of a variety of pathogen recognition receptors, interferon-stimulated genes, proinflammatory cytokines, and chemokines, together with the significant downregulation of anti-inflammatory molecules. Furthermore, the antiapoptotic pathway was triggered, as demonstrated by the significant suppression of molecules involved in the induction of apoptosis, together with the significant stimulation of putative inhibitor of apoptosis. The results indicate that PAECs initiated defense against poly(I:C) challenge through the inflammation responses, whereas poly(I:C) can utilize antiapoptotic pathway to evade host defense.

Subunit vaccines are typically poorly immunogenic when administered alone, and require adjuvants for robust responses. Triggering TLRs to boost antigen-specific adaptive immunity is an attractive approach to increase the potency and quality of vaccines. However, recent reports suggest that alterations in TLR expression are associated with the pathogenesis of inflammatory and autoimmune diseases. To compare genetic studies with adjuvant studies, we examined whether stimulation through a TLR agonist induces or increases the autoimmune phenotype of healthy or autoimmune mice. C57BL/6, MRL/lpr, and Fcγr2b-deficient mice were dosed i.p. with Poly I:C every other day for 3 weeks, and monitored for signs of autoimmunity over 3 months. A separate group of mice was vaccinated three times i.m. with rPA anthrax antigen with or without Poly I:C with 2 weeks between doses. Immunized groups exhibited robust responses to vaccine and C57BL/6 and MRL/lpr mice showed a statistically significant increase in anti-rPA IgG responses in the presence of Poly I:C. Interestingly, Fcγr2b-/- mice showed increases with the base rPA vaccine, which was not significantly increased when Poly I:C was used as an adjuvant. In the chronically dosed groups, we also observed subtle alterations in levels of total antibody and some autoantibodies. However, there were no statistically significant differences in autoimmune syndrome, as measured by proteinurea, kidney pathology, weight loss, and mortality, with Poly I:C administration in chronic or vaccination mode. Taken together, these results suggest that administration of TLR3 agonists in chronic or vaccination mode does not induce or exacerbate models of systemic lupus erythematosus.

Alzheimer's disease (AD) is a progressive disorder characterized by neuronal and behavioral deterioration. Two hallmark pathologies of AD are amyloid-beta (Aβ) plaques and neurofibrillary tangles, and the presence of such pathology can limit cell-to-cell communication, leading to cognitive deficits, and neuronal cell death. Although Aβ plaques were originally thought to cause the cognitive deficits, more simple forms of Aβ, such as monomers, dimers, tetramers and oligomers, have also been shown to be neurotoxic. Moreover, chronic inflammation has also been implicated in the onset and progression of these AD-related pathologies. The current study was designed to further our understanding of peripheral inflammation-induced AD-like pathology, by administering polyinosinic:polycytidylic acid (poly I:C), a viral mimetic. Mice were administered intraperitoneal injections of poly I:C or saline once daily for 7 consecutive days. Hippocampal tissue from animals receiving poly I:C contained significantly higher levels of the Aβ₁₋₄₂ peptide. Even after ensuring that potential sickness behavior could not confound cognitive testing, we found that animals administered poly I:C displayed significant cognitive deficits in the hippocampus-dependent contextual fear conditioning paradigm. These results confirm our hypothesis that peripheral inflammation can lead to increased levels of hippocampal-Aβ and associated cognitive deficits.

Objective. To explore the correlation between Thymic Stromal Lymphopoietin (TSLP) and the Nuclear Factor- (NF-) κB signaling pathways in bronchial epithelial cells and to clarify whether the traditional Chinese medicine formula Yi-Qi-Ping-Chuan-Fang (YQPC) reduces inflammation by inhibiting TSLP/NF-κB signaling pathways. Methods. Cells were stimulated with LPS + Poly(I:C) and treated with YQPC. The expressions of TSLP and NF-κB signaling pathways related proteins P65, IκK, IκBa, P-P65, P-IκK, P-IκBa were detected. The effects of NF-κB upstream molecules, Toll-like receptors 3 and 4, myeloid differentiation primary response gene 88 (Myd88), TIR-domain-containing adapter-inducing interferon-β (TRIF), and downstream inflammatory cytokines, TNF-α, IL-1β, IL-6, and IL-8, were assessed. Results. The mRNA and protein expressions of TSLP were significantly increased after LPS + Poly(I:C) stimulation, the total protein IκBa and IκK decreased (P < 0.05), and the phosphorylated protein P-P65, P-IκK, and P-IκBα increased. After YQPC treatment, the expression of TSLP, P-P65, P-IκBa, and P-IκK was significantly inhibited (P < 0.05). The activation of TLR4 and MyD88 decreased, and release of IL-1β, IL-6, IL-8, and TNF-α reduced (P < 0.05). Conclusion. In summary, the expression of TSLP is activated by the NF-κB signaling pathway. YQPC alleviated inflammation by inhibiting TSLP through regulating the NF-κB activation and translocation.

Increasing evidence has suggested that natural killer (NK) cells contribute to the pathogenesis of human immunological liver injury. Previous studies have demonstrated that Sophocarpine exerts activity in immune modulation. It also has a therapeutic effect on liver protection in that it can alleviate liver fibrosis by suppressing both the activation of hepatic stellate cells and the proliferation of the activated hepatic stellate cells. However, whether Sophocarpine protects the liver by regulating NK cell activity remains unclear. In this study, the modulating effect of Sophocarpine on NK cells in the liver was investigated. The results showed that Sophocarpine dramatically decreased the production of pro-inflammatory cytokines and attenuated the liver injury induced by PolyI: C/D-GalN in C57BL/6- mice. More importantly, Sophocarpine pre-treatment significantly suppressed NK cell activation and downregulated the expression of NKG2D, a receptor responsible for NK cell activation. Moreover, the protein levels of DAP12, ZAP76 and Syk decreased, as did their corresponding mRNA levels. Overall, our study demonstrates that Sophocarpine inhibits NK cell activity, thus making it a promising therapy for immunological liver injury.

Glycolytic metabolism plays a critical role in ischemia/reperfusion (I/R) injury. The Yes associated protein (YAP) is a core effector of the Hippo pathway that regulates cell proliferation and apoptosis. We observed that poly (I:C) enhanced glycolysis and Yap activation in neonatal cardiomyocytes. This study investigated whether poly (I:C) will attenuate myocardial I/R injury via a glycolytic dependent mechanism. Mice (n=6/group) were treated with poly (I:C) (10 μg/25g body weight) one h before the hearts were subjected to ischemia (45 min) followed by reperfusion (24 h). Sham surgery served as sham control. Poly (I:C) treatment significantly reduced infarct size by 35% and enhanced EF% by 20.5% and FS% by 24.9% compared with I/R group. The expression of miR-143 was markedly reduced and Yap levels were significantly increased in poly (I:C) treated hearts. In vitro data show that poly (I:C) treatment enhanced extracellular acidification rate (ECAR) and lactate production in HL-1 cardiomyocytes. In vivo inhibition of hexokinase 2 abolished poly (I:C)-induced cardioprotection. To determine the role of miR-143 in regulation of glycolysis, we transfected HL-1 cardiomyocytes with anti-miR-143 mimics before the cells were subjected to hypoxia/reoxygenation. We observed that anti-miR-143 significantly enhanced cell viability, reduced LDH release, and increased hexokinase 2 levels and extracellular acidification rate (ECAR). To determine whether suppression of miR-143 will induce protection against myocardial I/R injury, we loaded anti-miR-143 on exosomes (Exo-antimiR-143) by transection of bone marrow stromal cells with anti-miR-143 mimics. MiR-control mimics served as control (Exo-miR-control). Exo-miR-143 was delivered into the myocardium through the right carotid artery immediately before the hearts (n=6/group) were subjected to I/R. We observed that delivery of Exo-antimiR-143 significantly enhanced EF% by 20.5% and FS% by 26.4% and decreased infarct size by 42.2%, when compared with untreated I/R group. Delivery of Exo-miR-control did not alter I/R-induced cardiac dysfunction and infarct size. We conclude that poly (I:C) attenuates myocardial I/R injury via glycolytic dependent YAP mechanism and suppression of miR-143 expression.

Maternal immune activation (MIA) model is increasingly well appreciated as a rodent model for the environmental risk factor of various psychiatric disorders. Numerous studies have demonstrated that MIA model is able to show face, construct, and predictive validity that are relevant to autism and schizophrenia. To model MIA, investigators often use viral mimic polyinosinic:polycytidylic acid (poly(I:C)) to activate the immune system in pregnant rodents. Generally, the offspring from immune activated dam exhibit behavioral abnormalities and physiological alterations that are associated with autism and schizophrenia. However, poly(I:C) injection with different dosages and at different time points could lead to different outcomes by perturbing brain development at different stages. Here we provide a detailed method of inducing MIA by intraperitoneal (i.p.) injection of 20 mg/kg poly(I:C) at mid-gestational embryonic 12.5 days (E12.5). This method has been shown to induce acute inflammatory response in the maternal-placental-fetal axis, which ultimately results in the brain perturbations and behavioral phenotypes that are associated with autism and schizophrenia.

Background The length of time for cigarette smoke (CS) exposure to cause emphysema in mice is drastically reduced when CS exposure is combined with viral infection. However, the extent of inflammatory responses and lung pathologies of mice exposed to CS and infected with influenza A virus (IAV), respiratory syncytial virus (RSV), or treated with the viral derivative dsRNA (polyinosine-polycytidylic acid [poly (I:C)] have not been compared. Methods Mice were exposed to CS or filtered air for 4 weeks and received a single dose of vehicle, AV, or RSV infection and extent of inflammation and emphysema was evaluated 14 d later. In another set of experiments, mice were instilled with poly (I:C) twice a week during the third and fourth weeks of CS exposure and immediately analyzed for extent of inflammation and lung pathologies. Results In CS-exposed mice, inflammation was characterized mainly by macrophages, lymphocytes, and neutrophils after IAV infection, mainly by lymphocytes, and neutrophils after RSV infection, and mainly by lymphocytes and neutrophils after poly (I:C) instillations. Despite increased inflammation, extent of emphysema by poly (I:C) was very mild; but was robust and similar for both IAV and RSV infections with enhanced MMP-12 mRNA expression and TUNEL positivity. Both IAV and RSV infections increased the levels of IL-17, IL-1β, IL-12b, IL-18, IL-23a, Ccl-2, Ccl-7 mRNAs in the lungs of CS-exposed mice with IAV causing more increases than RSV. Conclusion CS-induced inflammatory responses and extent of emphysematous changes differ depending on the type of viral infection. These animal models may be useful to study the mechanisms by which different viruses exacerbate CS-induced inflammation and emphysema.

Neuroinflammation induced during immune responses to viral infections in the brain affect behavior. Unexpected evidence that oral gavage of an algal virus in its host algal cells could alter cognition was further examined by directly injecting purified algal virus ATCV-1 intracranially into C57BL/6 mice. After 4weeks, the ATCV-1 infection impaired delayed location recognition memory, and also reduced and anxiety. Corresponding to these effects, heightened ATCV-1, IL-6, iNOS, IFN-γ, and CD11b expression in brains was observed 3-days and/or 8-weeks post infection compared with control mice. These results imply that ATCV-1 infection damages the hippocampus via induction of inflammatory factors.

Nervous necrosis virus (NNV), the causative agent of viral nervous necrosis (VNN), is an indigenous virus in Korean coastal areas and a threat to the farming industry. In the present study, we investigated the curative and preventive effects against VNN in sevenband grouper ( Epinephelus septemfasciatus ) using polyinosinic polycytidylic acid [Poly(I:C)]. Fish challenged with brain-filtrates from VNN-affected fish at 0, 2, and 4 d after Poly(I:C) injection showed

It remains unclear whether immune response to viral infection is inhibited by severe exercise. We determined whether exhaustive exercise inhibits interferon (IFN)-β and tumor necrosis factor (TNF)-α production after injection of synthetic double-stranded (ds) RNAs, a polyriboinosinic polyribocytidylic acid (poly I:C), as viral infection model. Male C3H/HeN mice, which were divided into exhaustive-exercised and non-exercised groups, were injected with poly I:C (5 mg/kg). Although TNF-α in response to poly I:C was significantly inhibited by exhaustive exercise, IFN-β was no different in both groups. In in-vitro experiments, catecholamines inhibited poly I:C-induced TNF-α, but not IFN-β, production in macrophages. These results suggest that anti-virus cytokine IFN-β in response to poly I:C might be maintained despite severe stressful exercise.

It was recently reported that Poly(I:C) immunization of fish confers protection against viral infection. In this process, fish are immunized with a live pathogenic virus followed by administration of Poly(I:C), which induces a transient, non-specific antiviral state. As a result, fish survive the initial immunization with live virus, which would otherwise be lethal. In the present study, we tried to determine the exact dosage of red-spotted grouper nervous necrosis virus (RGNNV), the causative agent of viral nervous necrosis, required for Poly(I:C) immunization of sevenband grouper Epinephelus septemfasciatus . In Poly(I:C) immunization with more than 90% relative percentage survival (RPS) against RGNNV challenge with 10 4.3 TCID 50 /fish, approximately 10 4.0 TCID 50 /fish or greater of RGNNV was administered by intramuscular (IM) injection, whereas 10 5.3 TCID 50 /fish or greater of RGNNV was required for immersion methods. It was concluded that the degree of RGNNV infection must be similar to a fatal dose in order for the fish to become immune to RGNNV. Interestingly, antibody against RGNNV was not detected in the sera of fish immunized by immersion. Thus, Poly(I:C) immunization by immersion may have some interesting effects on the epidermal immune response in fish.

Maternal immune activation (MIA) is a principal environmental risk factor contributing to autism spectrum disorder (ASD), which compromises fetal brain development at critical periods of pregnancy and might be causally linked to ASD symptoms. We report that endogenous activation of the purinergic ion channel P2X7 (P2rx7) is necessary and sufficient to transduce MIA to autistic phenotype in male offspring. MIA induced by poly(I:C) injections to P2rx7 WT mouse dams elicited an autism-like phenotype in their offspring, and these alterations were not observed in P2rx7-deficient mice, or following maternal treatment with a specific P2rx7 antagonist, JNJ47965567. Genetic deletion and pharmacological inhibition of maternal P2rx7s also counteracted the induction of IL-6 in the maternal plasma and fetal brain, and disrupted brain development, whereas postnatal P2rx7 inhibition alleviated behavioral and morphological alterations in the offspring. Administration of ATP to P2rx7 WT dams also evoked autistic phenotype, but not in KO dams, implying that P2rx7 activation by ATP is sufficient to induce autism-like features in offspring. Our results point to maternal and offspring P2rx7s as potential therapeutic targets for the early prevention and treatment of ASD. SIGNIFICANCE STATEMENT Autism spectrum disorder (ASD) is a neurodevelopmental psychiatric disorder caused by genetic and environmental factors. Recent studies highlighted the importance of perinatal risks, in particular, maternal immune activation (MIA), showing strong association with the later emergence of ASD in the affected children. MIA could be mimicked in animal models via injection of a nonpathogenic agent poly(I:C) during pregnancy. This is the first report showing the key role of a ligand gated ion channel, the purinergic P2X7 receptor in MIA-induced autism-like behavioral and biochemical features. We show that genetic or pharmacological inhibition of both maternal and offspring P2X7 receptors could reverse the compromised brain development and autistic phenotype pointing to new possibilities for prevention and treatment of ASD.