The current animal models of osteoarthritis (OA) can be divided into spontaneous models and induced models, both of which aim to simulate the pathophysiological changes of human OA. However, as the main symptom in the late stage of OA, pain affects the patients' daily life, and there are not many available models. The mono-iodoacetate (MIA)-induced model is the most widely used OA pain model, mainly used in rodents. MIA is an inhibitor of glyceraldehyde-3-phosphate dehydrogenase, which causes chondrocyte death, cartilage degeneration, osteophyte, and measurable changes in animal behavior. Besides, expression changes of matrix metalloproteinase (MMP) and pro-inflammatory cytokines (IL1 β and TNF α) can be detected in the MIA-induced model. Those changes are consistent with OA pathophysiological conditions in humans, indicating that MIA can induce a measurable and successful OA pain model. This study aims to describe the methodology of intra-articular injection of MIA in rats and discuss the resulting pain-related behaviors and histopathological changes.

Purpose: The effect of Actovegin® was investigated on PMA- and LPS-induced human peripheral blood mononuclear cells (PBMCs).

Methods: PBMCs (1 × 10⁶ cells/ml) from five blood donors (2 f, 3 m; 45-55 years) were grown in medium and exposed to Actovegin® in the presence or absence of PMA or LPS. Supernatants were collected to assess the concentration of cytokines (TNF-α, IL-1beta, IL-6 and IL-10). The reactive oxygen species (ROS) were assessed by a ROS-Glo^TM H₂O₂ assay.

Results: Stimulation of cells by PMA or LPS (without Actovegin®) significantly increased the secretion of IL-1beta, IL-6, IL-10 and TNF-α from PBMCs, compared to controls. Pre-treatment of cells with Actovegin® (1, 5, 25, 125 µg/ml) plus PMA significantly decreased the secretion of IL-1beta from PBMCs, compared to controls (PMA without Actovegin®). In contrast, addition of Actovegin® (1, 5, 25, 125 and 250 µg/ml) plus LPS did not alter the IL-1beta production, compared to controls (LPS without Actovegin®). TNF-α, IL-6 and IL-10 do not contribute to the reduction of inflammatory reactions with Actovegin®.

Conclusions: Actovegin® can reduce the PMA-induced IL-1beta release and the ROS production from PBMCs. These findings may help to explain the clinically known positive effects of Actovegin® on athletic injuries with inflammatory responses (e.g., muscle injuries, tendinopathies).

Keywords: Human PBMCs; IL1-beta; LPS; PMA; ROS; Sports.

<A<em>b</em>stractText>Endothelial dysfunction (ED) predisposes to venous throm<em>b</em>osis (VT) and post-throm<em>b</em>otic syndrome (PTS), a long-term VT-related complication. Sulodexide (SDX) is a highly purified glycosaminoglycan with antithrom<em>b</em>otic, pro-fi<em>b</em>rinolytic and anti-inflammatory activity used in the treatment of chronic venous disease (CVD), including patients with PTS. SDX has recently o<em>b</em>tained clinical evidence in the "extension therapy" after initial-standard anticoagulant treatment for the secondary prevention of recurrent deep vein throm<em>b</em>osis (DVT). Herein, we investigated how SDX counteracts ED.</A<em>b</em>stractText><A<em>b</em>stractText>Human um<em>b</em>ilical vein endothelial cells (HUVEC) were used. Meta<em>b</em>olic and non meta<em>b</em>olic-induced ED was induced <em>b</em>y treating with methylglyoxal (MGO) or irradiation (IR), respectively. Bafilomycin A1 was used to inhi<em>b</em>it autophagy. The production of reactive oxygen species (ROS), tetrazolium <em>b</em>romide (MTT) assay for cell via<em>b</em>ility, terminal deoxynucleotidyl transferase-mediated dUTP nick end la<em>b</em>eling (TUNEL) assay for cell apoptosis, Real-time PCR and Western <em>b</em>lot analysis for gene and protein expression were used.</A<em>b</em>stractText><A<em>b</em>stractText>SDX protected HUVEC from MGO- or IR-induced apoptosis <em>b</em>y counteracting the activation of the intrinsic and extrinsic caspase cascades. The cytoprotective effects of SDX resulted from a reduction in a) ROS production, <em>b</em>) neo-synthesis and release of pro-inflammatory cytokines (TNFα, <em>IL1</em>, IL6, IL8), c) DNA damage induced <em>b</em>y MGO or IR. These effects were reduced when autophagy was inhi<em>b</em>ited.</A<em>b</em>stractText><A<em>b</em>stractText>Data herein collected indicate the a<em>b</em>ility of SDX to counteract ED induced <em>b</em>y meta<em>b</em>olic or non-meta<em>b</em>olic stresses <em>b</em>y involving the intracellular autophagy pathway. Our experience significantly increases the knowledge of the mechanisms of action of SDX against ED and supports the use of SDX in the treatment of CVD, PTS and in the secondary prevention of recurrent DVT.</A<em>b</em>stractText>

Salmonella Enteritidis (SE) is a facultative intracellular pathogen that colonizes the chicken gut leading to contamination of carcasses during processing. A reduction in intestinal colonization by SE could result in reduced carcass contamination thereby reducing the risk of illnesses in humans. Short chain fatty acids such as butyrate are microbial metabolites produced in the gut that exert various beneficial effects. However, its effect on SE colonization is not well known. The present study investigated the effect of sub-inhibitory concentrations (SICs) of sodium butyrate on the adhesion and invasion of SE in primary chicken enterocytes and chicken macrophages. In addition, the effect of sodium butyrate on the expression of SE virulence genes and selected inflammatory genes in chicken macrophages challenged with SE were investigated. Based on the growth curve analysis, the two SICs of sodium butyrate that did not reduce SE growth were 22 and 45 mM, respectively. The SICs of sodium butyrate did not affect the viability and proliferation of chicken enterocytes and macrophage cells. The SICs of sodium butyrate reduced SE adhesion by ∼1.7 and 1.8 Log CFU/mL, respectively. The SE invasion was reduced by ∼2 and 2.93 Log CFU/mL, respectively in chicken enterocytes (P < 0.05). Sodium butyrate did not significantly affect the adhesion of SE to chicken macrophages. However, 45 mM sodium butyrate reduced invasion by ∼1.7 Log CFU/mL as compared to control (P < 0.05). Exposure to sodium butyrate did not change the expression of SE genes associated with motility (flgG, prot6E), invasion (invH), type 3 secretion system (sipB, pipB), survival in macrophages (spvB, mgtC), cell wall and membrane integrity (tatA), efflux pump regulator (mrr1) and global virulence regulation (lrp) (P > 0.05). However, a few genes contributing to type-3 secretion system (ssaV, sipA), adherence (sopB), macrophage survival (sodC) and oxidative stress (rpoS) were upregulated by at least twofold. The expression of inflammatory genes (Il1β, Il8, and Mmp9) that are triggered by SE for host colonization was significantly downregulated (at least 25-fold) by sodium butyrate as compared to SE (P < 0.05). The results suggest that sodium butyrate has an anti-inflammatory potential to reduce SE colonization in chickens.

Keywords: Salmonella; anti-inflammatory; chicken macrophages; gene expression; primary chicken enterocytes; sodium butyrate.

Interleukin-1β (<em>IL1</em>) is a sleep regulatory su<em>b</em>stance. The <em>IL1</em>/<em>IL1</em> type 1 receptor complex requires a receptor accessory protein (AcP) to signal. There are three isoforms of AcP. In the current experiments, mice lacking a neuron-specific isoform, called AcP<em>b</em> knockout (AcP<em>b</em> KO), or mice lacking AcP + AcP<em>b</em> isoforms (AcP KO) or wild-type (WT) mice were used. Spontaneous sleep and sleep responses to sleep deprivation (SD) <em>b</em>etween zeitge<em>b</em>er time (ZT) 20-ZT4 and ZT8-ZT16 were characterized. Furthermore, somatosensory cortical protein extracts were examined for phosphorylated (p) proto-oncogene tyrosine-protein kinase sarcoma (Src) and p38MAPK levels at ZT4 and ZT16 and after SD. Spontaneous sleep was similar in the three strains, except rapid eye movement sleep (REMS) duration <em>b</em>etween ZT12-ZT16 was greater in AcP KO than WT mice. After SD at ZT4, only WT mice had non-REMS (NREMS) re<em>b</em>ounds. All mouse strains lacked an NREMS re<em>b</em>ound after SD at ZT16. All strains after <em>b</em>oth SD periods had REMS re<em>b</em>ounds. AcP<em>b</em> KO mice, <em>b</em>ut not AcP KO mice, had greater EEG delta wave (0.5-4 Hz) power during NREMS than WT mice. p-Src was very low at ZT16 <em>b</em>ut high at ZT4, whereas p-p38MAPK was low at ZT4 and high at ZT16. p-p38MAPK levels were not sensitive to SD. In contrast, p-Src levels were less after SD at the <i>P</i> = 0.08 level of significance in the strains lacking AcP<em>b</em>. We conclude that AcP<em>b</em> is required for NREMS responses to sleep loss, <em>b</em>ut not for SD-induced EEG delta wave or REMS responses.(<em>b</em>)NEW & NOTEWORTHY</<em>b</em>) Interleukin-1β (<em>IL1</em>), a well-characterized sleep regulatory su<em>b</em>stance, requires an <em>IL1</em> receptor accessory protein (AcP); one of its isoforms is neuron-specific (called AcP<em>b</em>). We showed that in mice, AcP<em>b</em> is required for nonrapid eye movement sleep responses following 8 h of sleep loss ending 4 h after day<em>b</em>reak <em>b</em>ut did not affect rapid eye movement sleep re<em>b</em>ound. Sleep loss reduced phosphorylation of proto-oncogene tyrosine-protein kinase sarcoma <em>b</em>ut not of the less sensitive p38MAPK, downstream <em>IL1</em> signaling molecules.

BACKGROUND

In inflammatory joint disease, such as osteoarthritis or arthritis, there is an increased level of pro-inflammatory cytokines, such as interleukin-1β. These cytokines stimulate the expression and release of matrix metalloproteases (MMP), leading to the degradation of cartilage extracellular matrix and subsequently mobility difficulty and suffering for patients. The aim of this study was to examine the therapeutic potential of a fatty acid copolymer in in vitro and in vivo models of cartilage inflammation.

METHODS

Inflammation was mimicked in vitro by treatment of human articular chondrocytes with interleukin-1β. Effects of a co-treatment with a copolymer of fatty acids (Ara 3000 beta®) were determined by evaluating MMP production by RT-PCR and ELISA, NO release by Griess assay, and PGE2 expression by ELISA. In addition, in vivo analysis (evolution of weight and edema) were also performed after injection of Freund adjuvant in rats treated or not with the copolymer of fatty acids.

RESULTS

The copolymer of fatty acids clearly reduces inflammation in joint. In vitro, it impairs IL1 stimulated-MMP production and release, as well as the release of NO and PGE2 and the activation of NFκB. Furthermore, in vivo experiments using adjuvant induced-arthritis corroborates the anti-inflammatory effects of the copolymer of fatty acids, with a reduction of edemas, erythemas and ankylosis in arthritic rats.

CONCLUSIONS

The results support the hypothesis that a copolymer of fatty acids, such as Ara 3000 beta®, is a powerful anti-inflammatory compounds, suggesting that it has a potential for preventing cartilage degradation associated with chronic inflammatory joint disease.

OBJECTIVE

To investigate in situ the expression of the integrin receptor subunits alpha 6 and beta 1 and the distribution of the ligand laminin in the synovia from osteoarthritis (OA) and rheumatoid arthritis (RA) patients and to study the effect of cytokines and antirheumatic drugs on the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of fibroblast-like synoviocytes (FBS) derived from OA and RA.

METHODS

The expression of the alpha 6 and beta 1 integrin subunits and the distribution of laminin were examined immunohistochemically in normal synovia and in synovia from patients with OA and RA. The effect of proinflammatory cytokines (IL1 beta and TNF alpha), and of antirheumatic drugs (salicylic acid, dexamethasone, and methotrexate) on the alpha 6 and beta 1 expression of cultured normal FBS and FBS from patients with OA and RA was determined by flow cytometry.

RESULTS

In normal synovia and in OA synovia samples with a low grade of inflammation, synovial lining cells (SLC) showed a parallel expression and distribution of alpha 6 and laminin. In synovia samples of OA with a higher grade of inflammation and in the majority of RA synovia samples laminin was pericellularly distributed in a low number of SLC, whereas alpha 6 was expressed on the surface of a high number of SLC. In RA synovia samples with severe inflammatory changes the gradual loss of laminin generally corresponded to a decrease of the alpha 6 integrin subunit. beta 1 was always strongly expressed in all synovia samples detected. Proinflammatory cytokines up regulated the expression of alpha 6 and beta 1 on OA-FBS, whereas these effectors decreases alpha 6 and beta 1 on RA-FBS. In contrast, antirheumatic drugs, in particular methotrexate and dexamethasone, reduced the expression of alpha 6 and beta 1 on OA-FBS, whereas the same treatment on RA-FBS stimulated the expression of these integrin subunits.

CONCLUSIONS

The gradual loss of laminin in chronic synovitis may contribute to the altered expression of alpha 6 in SLC. IL1 beta and TNF alpha down regulated the expression of the alpha 6 and beta 1 integrin subunits on long term cultures of FBS derived from RA. Therefore, these cytokines may be among the effectors regulating the expression of the alpha 6 integrin subunit in SLC in vivo. As antirheumatic drugs increase the expression of alpha 6 on RA-FBS, the presence of the laminin receptor may confer a protective effect on the synovia in vivo.

OBJECTIVE

This study investigated the effects of ascorbic acid and N-acetyl cysteine (NAC) antioxidants on the development of myringosclerosis (MS) in an experimental model.

METHODS

Myringotomies were performed in the ears of 15 guinea pigs, and Spongostan pieces were placed on the perforated regions of the tympanic membrane. The subjects were divided randomly into three groups and treated with three different solutions on the Spongostan-group 1: (control, 0.9% saline), group 2 (ascorbic acid), and group 3 (NAC). On day 15 after treatment, specimens from the tympanic membranes were obtained and examined via light microscopy. Sclerosis and inflammation scores and the tympanic membrane thicknesses were evaluated. Immunohistochemical methods were used to evaluate the expression of VEGF, TGF-β, iNOS, and IL1-β in all groups.

RESULTS

Lower sclerosis and inflammation scores and reduced tympanic membrane thicknesses were observed in groups treated with NAC or ascorbic acid compared with the control group. Immunohistochemical studies revealed significantly less expression of VEGF, TGF-β, and iNOS in groups 2 and 3 compared with group 1. Additionally, IL1-β expression was significantly less in group 3 than in group 1. Compared with group 1, group 2 animals exhibited reduced inflammation in the lamina propria, fewer active fibroblasts, less leukocyte infiltration, and decreased thickness of the vessels; group 3 animals exhibited decreased numbers of active fibroblasts and collagen fibers in the lamina propria.

CONCLUSIONS

Inflammation scores, cellular infiltration, and expression of VEGF, TGF-β, and iNOS were reduced by ascorbic acid and/or NAC treatments, thereby decreasing MS development. Decreased expression of IL1-β was observed only in animals treated with NAC.

Inflammation plays a major role in many diseases, for instance in arteriosclerosis, rheumatoid arthritis, autoimmune disorders and cancer. Since many plants contain compounds with anti-inflammatory activity, their consumption may be able to prevent the development of inflammatory-based diseases. Edible ferns are some of the most important wild vegetables in China and have traditionally been used both for dietary and therapeutic purposes. In this study we investigated the anti-inflammatory and antioxidant potential of fern extracts from Matteuccia struthiopteris, Osmundajaponica, Matteuccia orientalis and Pteridium aquilinum intended for use as nutraceuticals. Two modes of action were investigated: the inhibition of the pro-inflammatory gene expression of interleukin-1 beta (IL1-β) and interleukin-6 (IL6), and the gene expression of iNOS by LPS-elicited macrophages. The results showed a decrease of IL1-β gene expression for the five fern extracts. This effect was more pronounced for the extracts prepared from the roots of O. japonica (IC50 of 17.8 µg/mL) and the young fronds of M orientalis (50.0 µg/mL). Regarding the indirect measurement of NO, via iNOS gene expression, an interesting decrease of 50% was obtained with the extract of M. orientalis fronds at a low concentration (20 µg/mL) compared with P. aquilinum fronds (160 µg/mL) and leaves of O. japonica. The latter showed a higher decrease but at a high concentration of extract (160 µg/mL). The five fern extracts were also evaluated for their ability to scavenge 2,2-diphenyl-l-picrylhydrazyl (DPPH) radicals and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). All fern extracts exhibited antioxidant effects but the roots of O. japonica and the fronds of M orientalis were most efficient. The HPLC-MS analysis of the constituents of the fern extracts confirmed the presence of chlorogenic acid, caffeic acid, p-coumaric acid, ferulic acid, kaempferol and apigenin, molecules known to exhibit antiinflammatory and/or antioxidant properties.

OBJECTIVE

Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP) drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants.

METHODS

Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM) with or without IL1-β (10ng/ml) for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9), matrix metalloproteinases (MMPs), aggrecanases (ADAMTS5) and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG) synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence.

RESULTS

Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β.

CONCLUSIONS

Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b/β-catenin pathway.

In this work, for the first time, snail slime from garden snails "Helix Aspersa Müller", has been used to induce the formation of eco-friendly gold nanoparticles (AuNPs-SS) suitable for biomedical applications. An AuNPs-SS comprehensive investigation was performed and AuNPs with an average particle size of 14 ± 6 nm were observed, stabilized by a slime snail-based organic layer. Indeed, as recognized in high-resolution MALDI-MS analyses, and corroborated by FESEM, UV-Vis, ATR-FTIR, and XPS results, it was possible to assess the main presence of peptides and amino acids as the main components of the slime, that, combined with the AuNPs confers on them interesting properties. More specifically, we tested, in vitro, the AuNPs-SS safety in human keratinocytes and their potential effect on wound healing as well as their anti-inflammatory properties in murine macrophages. Moreover, the AuNPs-SS treatment resulted in a significant increase of the urokinase-type plasminogen activator receptor (uPAR), essential for keratinocyte adhesion, spreading, and migration, together with the reduction of LPS-induced IL1-β and IL-6 cytokine levels, and completely abrogated the synthesis of inducible nitric oxide synthase (iNOS).

Salinity changes on renal osmoregulation have often been investigated while the immune response of the kidney under osmotic stress is poorly understood in teleosts. Acute stress is generally associated with enhancement of circulating cortisol. The effects of osmotic stress on renal immune response and its regulation by cortisol deserve more attention. In the present study, the effects of exogenous cortisol treatment on the lipopolysaccharide (LPS)-induced immune response were analyzed in renal masses of Scatophagus argus under different osmotic stresses in vitro. mRNA expression of pro-inflammatory cytokines (TNF-α, IL1-β and IL-6) and immune-regulatory related genes (GR and SOCS1) was measured over a short course (15 h). Comprehensive analysis reveals that transcript abundances of pro-inflammatory cytokine genes such as TNF-α, IL-1β, and IL-6 induced by LPS, alone or in the combination of cortisol, are tightly associated with osmoregulation under acute osmotic stress. Our results showed that osmotic challenge could significantly enhance mRNA expression levels of pro-inflammatory cytokines in renal masses in vitro. Based on our analysis, it can be inferred that cortisol suppresses the magnitude of renal inflammatory response and attenuates LPS-induced immune response through GR signaling in the face of challenging environmental conditions.

Keywords: Cortisol; Inflammatory response; Kidney; Lipopolysaccharide; Osmotic stress; Scatophagus argus.

We recently reported that HSV-1 infection suppresses CD80 <em>b</em>ut not CD86 expression <i>in vitro</i> and <i>in vivo</i> This suppression required the HSV-1 ICP22 gene. We also reported that overexpression of CD80 <em>b</em>y HSV-1 exacer<em>b</em>ated corneal scarring in BALB/c mice. We now show that this recom<em>b</em>inant virus (HSV-CD80) expressed high levels of CD80 <em>b</em>oth <i>in vitro</i> in cultured ra<em>b</em><em>b</em>it skin cells and <i>in vivo</i> in infected mouse corneas. CD80 protein was detected on the surface of infected cells. Virulence of the recom<em>b</em>inant HSV-CD80 virus was similar to that of the parental strain and replication of HSV-CD80 was similar to that of control virus <i>in vitro</i> and <i>in vivo</i> Transcriptome analysis detected 75 known HSV-1 genes in the cornea of mice infected with HSV-CD80 or parental virus on day 4 post infection. Except for significantly higher CD80 expression in HSV-CD80 infected mice, HSV-1 gene expression was similar in corneas from HSV-CD80 infected and parental virus infected mice. The num<em>b</em>er of CD8⁺ T cells was higher, and CD4⁺ T cells lower, in corneas of HSV-CD80 infected mice than mice infected with parental virus. HSV-CD80 infected mice displayed a transient increase in DCs. Transcriptome analysis revealed mild differences in dendritic cell maturation, <em>IL1</em>-signaling pathways, and increased expression of Interferon Induced Protein with Tetratricopeptide Repeats 2 (Ifit2). Together, these results suggest that increased CD80 levels promote increased CD8⁺ T cells leading to exacer<em>b</em>ated eye disease in HSV-1 infected mice.(<em>b</em>)Importance</<em>b</em>) HSV-1 ocular infections are the leading cause of corneal <em>b</em>lindness. Eye disease is the result of prolonged immune response to the replicating virus. HSV-1, on the other hand, has evolved several mechanisms to evade clearance <em>b</em>y the host immune system. We descri<em>b</em>ed a novel mechanism of HSV-1 immune evasion via ICP22 dependent downregulation of the host T cell co-stimulatory molecule CD80. However, the exact role of CD80 in HSV-1 immune pathology is not clear. In this study, we show that eye disease is independent of HSV-1 replication, and that viral expression of CD80 has a detrimental role in corneal scarring, likely <em>b</em>y increasing CD8⁺ T cell recruitment and activation.

Acute-phase proteins (APPs) have always had valued diagnostic potentialities in response to infection. This study aimed to evaluate the diagnostic accuracy of selected APPs and proinflammatory cytokines (PIC) in goats with contagious caprine pleuropneumonia (CCPP) under field conditions. Moreover, to highlight the role of tested biomarkers in CCPP pathogenesis. Fifty-eight goats (38 confirmed cases with CCPP and 20 healthy controls) were involved in this investigation. C-reactive protein (CRP), procalcitonin (PCT), haptoglobin (HP), fibrinogen (Fb), serum amyloid A (SAA), selected PIC (IL1-α, IL1-β, IL-6, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α)) levels were investigated in serum samples from all goats under investigation. Latex agglutination test was used for diagnosis of goats with CCPP. For microbiological investigations, nasopharyngeal swabs (from all goats), lung tissues and pleural fluids (from only necropsied goats) were collected. This study revealed that all tested parameters have a high to moderate degree of diagnostic performance for CCPP. Magnitudes of increase in levels of APPs (CRP, HP and SAA) were stronger than PIC, IFN-γ, Fb and PCT. All tested parameters showed high diagnostic accuracy (AUROC >90%), except HP (AUROC = 87.3%) and IFN-γ (AUROC = 78.8%) showed moderate accuracy in differentiation of goats with and without CCPP infection. For detecting goats with and without CCPP infection, HP had the lowest sensitivity (Se = 81.6%) and Fb had the lowest specificity (Sp = 85.0%) among the APPs parameters tested. However, PCT showed the highest Se (100%) and Sp (95.0%) to detect goats with and without CCPP infection among tested parameters. Conclusively, this study endorses the significance of selected APPs and PIC as additional screening diagnostic parameters for naturally occurring CCPP in goats. However, it does not replace traditional methods for diagnosis of CCPP in goats. Furthermore, APPs and PIC have an important role in disease pathogenesis in goats.

Keywords: Cytokines; C-reactive protein; Haptoglobin; Procalcitonin; Serum amyloid a.

Plantaginis Semen (PS) has been used to promote diuresis and clear away dampness. Recent reports have shown that PS can be used to treat gouty nephropathy (GN). However, the action and mechanism of PS have not been well defined in treating GN. The present study aimed to define the molecular mechanisms of PS as a potential therapeutic approach to treat GN. A combination of network pharmacology and validation experiments in GN is used to understand the potential mechanism. Information on pharmaceutically active compounds in PS and gene information related to GN was obtained from public databases. The compound target network and protein-protein interaction network were constructed to study the mechanism of action of PS in the treatment of GN. The mechanism of action of PS in the treatment of GN was analyzed via Gene Ontology (GO) biological process annotation and Kyoto Gene and Genomics Encyclopedia (KEGG) pathway enrichment. Validation experiments were performed to verify the core targets. The GN rat model was prepared by the method of combining yeast and adenine. Hematoxylin-eosin (HE) staining was used to observe the morphology of renal tissue in rats. ELISA was applied to detect TGF-βα, and IL-1β levels in renal tissue. The expressions of TGF-βα, and IL-1β were determined using immunohistochemistry. Through the results of network pharmacology, we obtained 9 active components, 118 predicted targets, and 149 GN targets from the public database. Based on the protein-protein interaction (PPI), 26 hub genes for interaction with PS treating for GN were screened, including MMP9, TNF, IL1β, and IL6. The enrichment analysis results showed that the treatment of GN with PS was mainly involved in the TGF-βκB signaling pathway, and PI3K Akt signaling pathway. Validation experiment results showed that PS could reduce the content of urinary protein and UA and deregulate the expression of TGF-βα, and IL-1β in the treatment of GN. The molecular mechanism of PS in the treatment of GN indicated the synergistic features of multicomponent, multitarget, and multipathway of traditional Chinese medicine, which provided an essential scientific basis for further elucidating the mechanism of PS in the treatment of GN.

Hormones like testosterone and progesterone in the humans play significant role in the regulation of various <em>b</em>iological processes like the <em>b</em>ody growth, reproduction, and others. In last two decades, researchers are using ionic liquids (ILs) extensively in different areas of sciences, and they are a novel class of compounds as well as their polarity can <em>b</em>e tuned. ILs are multidisciplinary in nature and can <em>b</em>e used in chemistry, materials science, chemical engineering, and environmental science. Further, ILs are <em>b</em>eing explored to increase the solu<em>b</em>ility of drugs or <em>b</em>iological potential molecules. Testosterone and progesterone are found to <em>b</em>e not very polar in nature; therefore, the authors attempt to increase the solu<em>b</em>ility of testosterone and progesterone via interaction with ILs. It was studied with density functional theory calculations using Gaussian, and an increase in the value of dipole moment is o<em>b</em>served for the complex of testosterone/progesterone with the ILs in comparison of individual one. The optimization energy and other thermodynamic energies of the ILs (<em>IL1</em>-IL3), testosterone (T), testosterone-IL (T-<em>IL1</em> to T-IL3), progesterone (P), and progesterone-ILs (P-<em>IL1</em> to P-IL3) are found to <em>b</em>e negative. Further, the change in free energy for the formation of complexes at room temperature is calculated. Further, the authors have investigated the synergistic effect of testosterone and progesterone against the main protease of new coronavirus using molecular docking. It is o<em>b</em>served that the testosterone-(<em>b</em>)<em>IL1</em></<em>b</em>) {<em>IL1</em>-3-(2-hydroxyethyl)-1-methyl-1H-imidazol-3-ium 2,4,6-trinitrophenolate} is found to <em>b</em>e prominent against the main protease of SARS-CoV-2.

Keywords: DFT calculations; ionic liquids; molecular docking; progesterone; testosterone.

<A<em>b</em>stractText>There is increased type I interferon signature in psoriasis patients. Interferon-kappa (IFN-κ) is a mem<em>b</em>er of type I interferon family that is constitutively expressed <em>b</em>y keratinocytes. In this study, we investigate whether IFN-κ is involved in psoriasis etiology.</A<em>b</em>stractText><p><div>(<em>b</em>)Patients and methods</<em>b</em>)</div>Twenty healthy individuals, 20 psoriasis vulgaris patients and 10 atopic dermatitis (AD) were included for this study. Immunohistochemistry staining, normal human epidermal keratinocytes (NHEK) culture, Ca<su<em>b</em>)2</su<em>b</em>)Cl-induced differentiation, quantitative reverse transcription (qRT-PCR), ELISA and murine experiments were performed.</p><p><div>(<em>b</em>)Results</<em>b</em>)</div>We found IFN-κ protein expression was extremely low in the epidermis of normal skin, <em>b</em>ut it was significantly increased in the supra<em>b</em>asal layers of epidermal keratinocytes in psoriatic skin lesions. However, its expression in the skin lesions of AD was similar to normal skin. Additionally, IFN-κ protein was detected in sera from psoriasis patients, <em>b</em>ut not in sera from normal su<em>b</em>jects and AD. We further investigated the regulation of <i>IFNk</i> gene expression in NHEK. We found that <i>IFNk</i> was significantly induced <em>b</em>y types of nucleic acid pathogen recognition receptor (PRR) agonists in NHEK. While its expression was significantly induced <em>b</em>y itself and IFN-γ, it was inhi<em>b</em>ited <em>b</em>y type 2 immunity cytokines IL4 and <em>IL1</em>3; other inflammatory cytokines including <em>IL1</em> super-family mem<em>b</em>ers and <em>IL1</em>7A did not alter its expression. Addition of recom<em>b</em>inant IFN-κ did not affect keratinocytes differentiation. Using the murine experimental model, we demonstrated that su<em>b</em>cutaneous administration of recom<em>b</em>inant IFN-κ did not increase skin thickness, <em>b</em>ut significantly increased the transcription of <i>TNFA</i> and <i><em>IL1</em>7A</i> in mice skin.</p><p><div>(<em>b</em>)Conclusion</<em>b</em>)</div>Increased IFN-κ in psoriasis may <em>b</em>e caused <em>b</em>y injured cells-released nucleic acids, increased IFN-γ and self-activation. Its enhancement may contri<em>b</em>ute to the etiology of the disease <em>b</em>y enhancing <i>TNFA</i> and <i><em>IL1</em>7A</i> gene expression.</p>

Iridovirus of Taiwan (TGIV) has been threatening the grouper farming since 1997, effective prophylaxis method is urgently needed. Subunit vaccine was proved to be useful to against the virus. Bath is the simplest method of vaccination and easy to be administrated without any stress to fish. In this research, we constructed a prokaryotic expression vector of TGIV's major capsid protein (MCP) to acquire the vaccine. Single-walled carbon nanotubes (SWCNTs) were used as the carrier to enhance the protective effect of bath vaccination for juvenile pearl gentian grouper (bath with concentrations of 5, 10, 20 mg/L for 6 h). Virus challenge was done after 28 days. Survival rates were calculated after 14 days. The level of antibody, activities of related enzymes in serums and expression of immune-related genes in kidneys and spleens were test. The results showed that vaccine with SWCNTs as carrier induced a higher level of antibody than that without. In addition, the activities of related enzymes (acid phosphatase, alkaline phosphatase, superoxide dismutase) and the expression of immune-related genes (Mx1, IgM, TNFαF, Lysozyme, CC chemokine 1, IL1-β, IL-8) had a significantly increase. What's more, higher survival rates (42.10%, 77.77%, 89.47%) were provided by vaccine with SWCNTs than vaccine without SWCNTs (29.41%, 38.09%, 43.75%). This study suggests that the protective effect of vaccine that against TGIV with the method of bath vaccination could be enhanced by SWCNTs and SWCNTs could be a potential carrier for other subunit vaccines.

Keywords: Bath vaccination; Iridovirus of Taiwan; Pearl gentian grouper; Single-walled carbon nanotubes; Vaccine.

<A<em>b</em>stractText>To investigate the effects of systemically administered melatonin on inflammation and alveolar <em>b</em>one resorption in rats with experimentally induced periapical lesions.</A<em>b</em>stractText><p><div>(<em>b</em>)METHODOLOGY</<em>b</em>)</div>Thirty adult Sprague Dawley rats were divided equally into negative, positive control and melatonin groups. The pulp cham<em>b</em>ers of their mandi<em>b</em>ular first molars were exposed to the oral environment to induce experimental periapical lesions in the positive control and melatonin groups. The melatonin group received daily intraperitoneal injections of melatonin at a dose of 10 mg kg^-1 . After 21 days, the animals were euthanized; the hemi-mandi<em>b</em>le parts were prepared for radiological, histopathological, immunohistochemical (IL-1<em>β</em>, RANK, RANKL, OPG and tartrate-resistant acid phosphatase (TRAP) and Brown-Brenn (<em>b</em>acteria) evaluations. Data were analysed <em>b</em>y Kruskal-Wallis (for non-parametric data) and one-way anova tests (for parametric data) (P < 0.05).</p><A<em>b</em>stractText>The area of radiographic periapical <em>b</em>one loss was significantly smaller in rats that were given daily intraperitoneal injections of melatonin (P < 0.01). The histopathological scores of the melatonin group were significantly lower than those of positive control group (P < 0.01). Histomorphometrically, the area of periapical <em>b</em>one loss in the melatonin group was significantly smaller than the positive control group (P < 0.01). The expression of <em>IL1</em>-<em>β</em>, RANK and RANKL was significantly higher in the positive control group, whereas OPG was significantly higher in the melatonin group (P < 0.01). The num<em>b</em>er of osteoclasts was significantly greater in the positive control group <em>b</em>y TRAP staining analyses (P < 0.01). The scores for <em>b</em>acteria localization using Brown-Brenn staining in the melatonin group was significantly lower than that of the positive control group (P < 0.01).</A<em>b</em>stractText><A<em>b</em>stractText>Melatonin demonstrated antiresorptive effects on <em>b</em>one associated with experimentally induced periapical lesions in rats via its anti-inflammatory activity. Further studies are necessary to evaluate its possi<em>b</em>le effects on the healing of periapical lesions.</A<em>b</em>stractText>

Marine microalgae are known to be a source of bioactive molecules of interest to human health, such as n-3 polyunsaturated fatty acids (n-3 PUFAs) and carotenoids. The fact that some of these natural compounds are known to exhibit anti-inflammatory, antioxidant, anti-proliferative, and apoptosis-inducing effects, demonstrates their potential use in preventing cancers and cardiovascular diseases (CVDs). Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon (PAH), is an ubiquitous environmental pollutant known to contribute to the development or aggravation of human diseases, such as cancer, CVDs, and immune dysfunction. Most of these deleterious effects are related to the activation of the polycyclic aromatic hydrocarbon receptor (AhR). In this context, two ethanolic microalgal extracts with concentrations of 0.1 to 5 µg/mL are tested, Ostreoccoccus tauri (OT) and Phaeodactylum tricornutum (PT), in order to evaluate and compare their potential effects towards B[a]P-induced toxicity in endothelial HMEC-1 cells. Our results indicate that the OT extract can influence the toxicity of B[a]P. Indeed, apoptosis and the production of extracellular vesicles were decreased, likely through the reduction of the expression of CYP1A1, a B[a]P bioactivation enzyme. Furthermore, the B[a]P-induced expression of the inflammatory cytokines IL-8 and IL1-β was reduced. The PT extract only inhibited the expression of the B[a]P-induced cytokine IL-8 expression. The OT extract therefore seems to be a good candidate for counteracting the B[a]P toxicity.

We investigated the effects of two common dietary supplements on bone healing in dental extraction sockets in humans. In this randomized pilot trial, male subjects took Grape Seed Extract [GSE] or Grapefruit Extract [GFE] starting two weeks prior to dental extraction and maintained this regimen for sixty days after surgery. Extraction sockets were filled with a collagen plug. After 24 h, a socket sample was collected and processed for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) and an 84-gene wound healing assay. Sixty days after tooth extraction, a core of newly formed bone was obtained prior to dental implant placement and processed for histology. qRT-PCR revealed that GFE led to a significant decrease in platelet-derived growth factor and interleukin (IL)1-β compared to GSE, and a significant decrease in IL-6 and CXCL2 compared to control. GSE led to a significant increase in coagulation factor Von Willebrand and inflammatory marker IL1-β compared to GFE. WISP1 and CXCL5 were upregulated in both groups. Overall, GFE showed a downregulation of inflammation and GSE led to a decrease in collagen density and increased osteoclasts. This pilot trial highlights the need for further investigation on the mechanism of action of such supplements on bone healing and oral health.

Keywords: collagen; dietary supplements; flavonoid; osteoclast; phytochemicals; tooth socket.

Opioid use disorder (OUD) affects over two million in the United States and is an increasing public health crisis. The abuse of fentanyl and the emergence of potent fentanyl derivatives increases the risk for the user to succumb to overdose, but also to develop OUD. While intense attention is currently focused on understanding the complexity of behaviors and neural functions that contribute to OUD, much remains to be discovered concerning the interactions of opioid intake with the immune response in the central nervous system (CNS). In the present studies, we tested the hypothesis that short-term abstinence from fentanyl self-administration associates with altered expression of innate immune markers. Male Sprague-Dawley rats were trained to self-administer fentanyl (0.0032 mg/kg/infusion) to stability followed by 24 h of abstinence. Several innate immune markers, as well as opioid receptors (ORs) and intracellular pattern recognition receptors (PRRs), were interrogated within nodes of the neurocircuitry involved in OUD processes, including the prefrontal cortex (PFC), nucleus accumbens (NAc), caudate putamen (CPu), hippocampus (HIP) and midbrain (MB). In the present study, few immune targets were impacted in the PFC and MB during short-term abstinence from fentanyl (relative to saline) self-administration. However, increased expression of cytokines [e.g., interleukin (IL)1<em>β</em>, IL5], chemokines [e.g., C-C motif chemokine 20 (MIP3α)], tumor necrosis factor α (TNF α) and interferon (IFN) proteins (e.g., IFN <em>β</em> and IFNγ)] was seen in the NAc, while decreased expression of cytokines (e.g., several ILs), chemokines [e.g., granulocyte-macrophage colony-stimulating factor (GMCSF), monocyte chemoattractant protein (MCP) MCP1, MIP3α], the chemokine ligand 5 (RANTES) and interferons (e.g., IFN <em>β</em> and IFNγ) in the HIP. Positive correlations were observed between cumulative fentanyl intake and expression of <em>IL1</em> <em>β</em> and IL6 in the NAc, and significant negative correlations with fentanyl intake and IFN <em>β</em>, IL2, IL5, <em>IL1</em>2p70 and <em>IL1</em>7 in the HIP. Few changes in OR expression was observed during early abstinence from fentanyl self-administration. Excitingly, the expression of the PRR, stimulator of interferon genes (STING) negatively correlated with cumulative fentanyl intake and significantly correlated to specific cytokines, chemokines and interferon proteins in the HIP. Although the CPu appears relatively invulnerable to changes in innate immune markers, the highest correlations between cumulative fentanyl intake with MAVS and/or STING was measured in the CPu. Our findings provide the first evidence of CNS innate immune responses and implicate STING as novel mechanistic targets of immunomodulation during short-term abstinence from fentanyl self-administration.

Background: The endothelium is the first line of defence against harmful microenvironment risks, and microRNAs (miRNAs) involved in vascular inflammation may be promising therapeutic targets to modulate atherosclerosis progression. In this study, we aimed to investigate the mechanism by which microRNA-216a (miR-216a) modulated inflammation activation of endothelial cells. Methods. A replicative senescence model of human umbilical vein endothelial cells (HUVECs) was established, and population-doubling levels (PDLs) were defined during passages. PDL8 HUVECs were transfected with miR-216a mimics/inhibitor or small interfering RNA (siRNA) of SMAD family member 7 (Smad7). Real-time PCR and Western blot assays were performed to detect the regulatory role of miR-216a on Smad7 and NF-κB inhibitor alpha (IκBα) expression. The effect of miR-216a on adhesive capability of HUVECs to THP-1 cells was examined. MiR-216a and Smad7 expression in vivo were measured using human carotid atherosclerotic plaques of the patients who underwent carotid endarterectomy (n = 41).

Results: Luciferase assays showed that Smad7 was a direct target of miR-216a. Smad7 mRNA expression, negatively correlated with miR-216a during endothelial aging, was downregulated in senescent PDL44 cells, compared with young PDL8 HUVECs. MiR-216a markedly increased endothelial inflammation and adhesive capability to monocytes in PDL8 cells by promoting the phosphorylation and degradation of IκBα and then activating NF-κB signalling pathway. The effect of miR-216a on endothelial cells was consistent with that blocked Smad7 by siRNAs. When inhibiting endogenous miR-216a, the Smad7/IκBα expression was rescued, which led to decreased endothelial inflammation and monocytes recruitment. In human carotid atherosclerotic plaques, Smad7 level was remarkably decreased in high miR-216a group compared with low miR-216a group. Moreover, miR-216a was negatively correlated with Smad7 and IκBα levels and positively correlated with interleukin 1 beta (IL1β) expression in vivo.

Conclusion: In summary, our findings suggest a new mechanism of vascular endothelial inflammation involving Smad7/IκBα signalling pathway in atherosclerosis.

BACKGROUND

We report the use of an ex vivo precision cut liver slice (PCLS) mouse model for studying hepatic schistosomiasis. In this system, liver tissue is unfixed, unfrozen, and alive for maintenance in culture and subsequent molecular analysis.

RESULTS

Using thick naive mouse liver tissue and sterile culture conditions, the addition of soluble egg antigen (SEA) derived from Schistosoma japonicum eggs, followed 4, 24 and 48 hrs time points. Tissue was collected for transcriptional analysis and supernatants collected to quantitate liver enzymes, cytokines and chemokines. No significant hepatotoxicity was demonstrated by supernatant liver enzymes due to the presence of SEA. A proinflammatory response was observed both at the transcriptional level and at the protein level by cytokine and chemokine bead assay. Key genes observed elevated transcription in response to the addition of SEA included: IL1-α and IL1-β, IL6, all associated with inflammation. The recruitment of antigen presenting cells was reflected in increases in transcription of CD40, CCL4 and CSF1. Indications of tissue remodeling were seen in elevated gene expression of various Matrix MetalloProteinases (MMP3, 9, 10, 13) and delayed increases in TIMP1. Collagen deposition was significantly reduced in the presence of SEA as shown in COL1A1 expression by qPCR after 24 hrs culture. Cytokine and chemokine analysis of the culture supernatants confirmed the elevation of proteins including IL6, CCL3, CCL4 and CXCL5.

CONCLUSIONS

This ex vivo model system for the synchronised delivery of parasite antigen to liver tissue provides an insight into the early phase of hepatic schistosomiasis, corresponding with the release of soluble proteins from dying schistosome eggs.