p-Cymene Modulates In Vitro and In Vivo Cytokine Production by Inhibiting MAPK and NF-κB Activation

The present study was designed to investigate the effects of p-cymene on lipopolysaccharide (LPS)-induced inflammatory cytokine production both in vitro and in vivo. The production of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) in LPS-stimulated RAW 264.7 cells and C57BL/6 mice was evaluated by sandwich ELISA. Meanwhile, the mRNA levels of cytokine genes were examined in vitro by semiquantitative RT-PCR. In a further study, we analyzed the activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by western blotting. We found that p-cymene significantly regulated TNF-α, IL-1β, and IL-6 production in LPS-stimulated RAW 264.7 cells. Furthermore, the levels of relative mRNAs were also found to be downregulated. In in vivo trail, p-cymene markedly suppressed the production of TNF-α and IL-1β and increased IL-10 secretion. We also found that p-cymene inhibited LPS-induced activation of extracellular signal receptor-activated kinase 1/2, p38, c-Jun N-terminal kinase, and IκBα. These results suggest that p-cymene may have a potential anti-inflammatory action on cytokine production by blocking NF-κB and MAPK signaling pathways.     

Antibody repertoire development in fetal and neonatal piglets. XV. Porcine circovirus type 2 infection differentially affects serum IgG levels and antibodies to ORF2 in piglets free from other environmental factors

Porcine circovirus type 2 (PCV2) is an important pathogen in the porcine respiratory disease complex (PRDC) and its persistence may be due to dysregulation of systemic immunity. We examined this contention using isolator piglets. We present data on Ig levels in serum and bronchio-alveolar lavage (BAL), on antibody response to PCV2 and to TNP conjugates used as model antigens in 48 PCV2-infected isolator piglets. We compared these to data from TNP-immunized isolator piglets colonized with a probiotic flora, those infected with swine influenza (S-FLU) and those infected with porcine respiratory and reproductive syndrome virus (PRRSV). We found that PCV2 infection does not cause generalized hypergammaglobulinemia that characterizes PRRSV infections, but causes an unexplained increase in serum IgA. All animals had serum IgG to the ORF2 gene product of PCR2, but neither IgA nor IgG anti-ORF2 responses in BAL. PCV2 infection is a poor adjuvant since only natural anti-TNP antibodies were found. Unexpectedly, immunization appeared to result in lower Ig levels and lower anti-ORF2 responses. There was extreme variation in serum Ig levels in response to infection that could in part be traced to genetic and gender differences. These data suggest that non-replicating vaccines are unlikely to result in a significant primary antibody response but may prime the system for a secondary antibody and cytotoxic response following actual infection. In any case, developers may have to contend with significant genetic differences in the response of piglets to PCV2.     

Histamine-4 receptor antagonist ameliorates Parkinson-like pathology in the striatum

Growing evidence indicates that microglia activation and a neuroinflammatory trigger contribute to dopaminergic cell loss in Parkinson’s disease (PD). Furthermore, increased density of histaminergic fibers and enhanced histamine levels have been observed in the substantia nigra of PD-postmortem brains. Histamine-induced microglial activation is mediated by the histamine-4 receptor (H₄R). In the current study, gene set enrichment and pathway analyses of a PD basal ganglia RNA-sequencing dataset revealed that upregulation of H₄R was in the top functional category for PD treatment targets. Interestingly, the H₄R antagonist JNJ7777120 normalized the number of nigrostriatal dopaminergic fibers and striatal dopamine levels in a rotenone-induced PD rat model. These improvements were accompanied by a reduction of α-synuclein-positive inclusions in the striatum. In addition, intracerebroventricular infusion of JNJ7777120 alleviated the morphological changes in Iba-1-positive microglia and resulted in a lower tumor necrosis factor-α release from this brain region, as well as in ameliorated apomorphine-induced rotation behaviour. Finally, JNJ7777120 also restored basal ganglia function by decreasing the levels of γ-aminobutyric acid (GABA) and the 5-hydroxyindoleactic acid to serotonin (5-HIAA/5-HT) concentration ratios in the striatum of the PD model. Our results highlight H₄R inhibition in microglia as a promising and specific therapeutic target to reduce or prevent neuroinflammation, and as such the development of PD pathology.     

Valproic acid exposure sequentially activates Wnt and mTOR pathways in rats

BackgroundAutism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impaired social interaction, limited verbal communication and repetitive behaviors. Recent studies have demonstrated that Wnt signaling and mTOR signaling play important roles in the pathogenesis of ASD. However, the relationship of these two signaling pathways in ASD remains unclear.ResultsWe assessed this question using the valproic acid (VPA) rat model of autism. Our results demonstrated that VPA exposure activated mTOR signaling and suppressed autophagy in the prefrontal cortex, hippocampus and cerebellum of autistic model rats, characterized by enhanced phospho-mTOR and phospho-S6 and decreased Beclin1, Atg5, Atg10, LC3-II and autophagosome formation. Rapamycin treatment suppressed the effect of VPA on mTOR signaling and ameliorated the autistic-like behaviors of rats in our autism model. The administration of VPA also activated Wnt signaling through up-regulating beta-catenin and phospho-GSK3beta. Suppression of the Wnt pathway by sulindac relieved autistic-like behaviors and attenuated VPA-induced mTOR signaling activation in autistic model rats.ConclusionsOur results demonstrate that VPA exposure sequentially activates Wnt signaling and mTOR signaling in rats. Suppression of the Wnt signaling pathway relieves autistic-like behaviors partially by deactivating the mTOR signaling pathway in VPA-exposed rats.     

Whole genome alignment based one-step real-time RT-PCR for universal detection of avian orthoreoviruses of chicken,pheasant and turkey origins

Newly emerging avian orthoreovirus (ARV) variants have been continuously detected in Pennsylvania poultry since 2011. In this paper, we report our recent diagnostic assay development of one-step real-time RT-PCR (rRT-PCR) for the rapid and universal detection of all ARVs or reference strains of chicken, pheasant and turkey origins and six σC genotypes of the newly emerging field ARV variants in Pennsylvania (PA) poultry. Primers and probes for the rRT-PCR were designed from the conserved region of the M1 genome segment 5′ end based on the whole-genome alignment of various ARV strains, including six field variants or novel strains obtained in PA poultry. The detection limit of the newly developed rRT-PCR for ARV was as low as 10 copies/reaction of viral RNA, and 10^0.50–10^0.88 tissue culture infectious dose (TCID₅₀)/100 μL of viruses. This new rRT-PCR detected all six σC genotypes from the 66 ARV field variant strains and reference strains tested in this study. There were no cross-reactions with other avian viruses. Reproducibility of the assay was confirmed by intra- and inter-assay tests with variability from 0.12% to 2.19%. Sensitivity and specificity of this new rRT-PCR for ARV were achieved at 100% and 88%, respectively, in comparison with virus isolation as the “gold standard” in testing poultry tissue specimen.     

Development of a lyophilized soybean paste certified reference material for the analysis of ochratoxin A

A certified reference material (CRM) [²KRISS CRM # 108-10-018] for the analysis of ochratoxin A (OTA) in doenjang (fermented soybean paste and popular food in Korea) was produced to ensure the reliability of analytical results in testing laboratories. A home-made doenjang was chosen as a raw material after testing its OTA level. The raw material was freeze-dried, pulverized, sieved and homogenized. An isotope-dilution-liquid chromatography/tandem mass spectrometric method (ID-LC/MS/MS) which was previously developed and validated in this laboratory was used as a higher-order reference method for characterization, homogeneity studies, and short-term stability studies. The CRM had good between-bottle homogeneity with 0.56% relative standard deviation among 10 selected units. The stability of the CRM at −70 °C (the storage condition in our laboratory) and at −20 °C (the possible storage temperature at user sites) were tested for up to 8 months. No change in the OTA content was observed within the measurement uncertainty. The stability of the CRM at room temperature (for regular use and transportation) was also tested and confirmed. The certified value was (49.50 ± 1.17) μg/kg, where the expanded uncertainty was in the confidence level of 95%.     

HEPATOPROTECTIVE EFFECT OF AMOORA ROHITUKA

Abstract

Antihepatotoxic activity of a resuspended residue of the alcohol extract of Amoora rohituka W & A. (Meliaceae) was studied in rats with hepatic injury induced by carbon tetrachloride. Carbon tetrachloride (1 ml/kg, i.p.) was administered twice a week for 3 weeks and an extract of A. rohituka (50 mg/kg/day) was given orally for the same period. The rats were sacrificed 24 h after the last CC1₄ challenge. Carbon tetrachloride induced elevations of alkaline phosphatase (ALP), glutamate pyruvate transaminase (CPT), alanine aminotransferase (ALAT), glutamate oxaloacetate transaminase (GOT), aspartate aminotransferase (ASAT) and lactate dehydrogenase (LDH) and total plasma bilirubin concentration as well as depression of total plasma cholesterol concentration were reduced significantly by the concurrent treatment of rats with A. rohituka suspension. Changes in the histological architecture of the liver produced by CC1₄ where also protected by the administration of A. rohituka suspension. These results indicate that A. rohituka suspension possesses hepatoprotective action.