Concentrations Of Antagonists Research Articles

Equine autologous blood-based products contain variable quantities of transforming growth factor-β1, interleukin-1 receptor agonist, and α2-macroglobulin.

Quantify the concentration of α2-macroglobulin (A2M), immunomodulatory cytokines, and TGF-β1 factors in 4 commercially available autologous blood-based products including conditioned A2M (CA2M; Alpha2EQ; Astaria Global), autologous protein solution (APS; Pro-Stride; Zoetis), platelet-rich plasma (PRP; Restigen; Zoetis), and autologous conditioned plasma (ACP; Arthrex ACP). We hypothesized that CA2M would have higher concentrations of A2M and lower concentrations of cytokines and growth factors compared to APS, PRP, and ACP. Blood was obtained from 6 healthy, adult horses and processed into CA2M, APS, PRP, and ACP. The concentration of immunomodulatory cytokines, including IL-1β, IL-4, IL-6, IL-10, IL-17a, and TNF-α, and the concentration of the growth factor TGF-β1 were quantified using immunoassays. The concentration of the IL-1 receptor antagonist was quantified using ELISA. The concentration of A2M was quantified using mass spectrometry. No differences in the concentrations of IL-1β, IL-4, IL-6, IL-10, and IL-17a were found. Median TGF-β1 was significantly higher in APS (10,801 pg/mL; P < .05), PRP (6,219 pg/mL; P < .05), and ACP (5,263 pg/mL; P < .05) compared to CA2M (2,090 pg/mL). The IL-1 receptor antagonist was significantly higher in APS (58.78 ng/mL) and PRP (40.45 ng/mL). Median A2M concentration was significantly higher in APS (4.08 mg/mL; P < .001) compared to CA2M (1.99 mg/mL). Autologous blood-based products have notably different immunomodulatory and growth factor profiles. These differences likely reflect variable concentrations of platelets and WBCs, as well as processing methods. Equine veterinarians should be aware of the constituents of the different orthobiologics available before use.

Gonadotropin-releasing hormone II and its receptor regulate motility, morphology, and kinematics of porcine spermatozoa in vitro.

The second form of gonadotropin-releasing hormone (GnRH-II) and its receptor (GnRHR-II) are abundantly produced within the porcine testis and immunolocalize within the seminiferous tubules, suggesting a role in spermatogenesis and/or sperm function. The objective of this study was to quantify GnRH-II and GnRHR-II abundance within boar reproductive tract tissues and examine their role in porcine sperm function. Immunoblotting revealed GnRHR-II abundance was 12-fold greater (P < 0.0001) within the testis compared with other reproductive organs. Within seminiferous tubules, GnRHR-II prominently immunolocalized to elongating spermatids. In ejaculated spermatozoa, GnRHR-II immunolocalized to the connecting piece. GnRH-II was also detected in seminal plasma, likely originating from the testis as GnRH-II concentrations were greatest in testicular homogenates (P < 0.0001) compared with other reproductive tissues. To assess the effects of GnRH-II/GnRHR-II on sperm function, extended semen samples were treated with GnRHR-II analogues and evaluated via computer-assisted sperm analysis (CASA). In Experiment 1, semen treatment with increasing concentrations of GnRHR-II agonist (D-ala6 GnRH-II) revealed that two concentrations (0.1 and 100µM) tended to decrease the percentage of bent sperm tails versus vehicle-treated semen (P < 0.10). In Experiment 2, semen treatment with increasing concentrations of GnRHR antagonist (SB-75/Cetrorelix) indicated that only 10µM SB-75 impaired CASA metrics compared with vehicle-treated samples (P < 0.05). In Experiment 3, semen treatment with both 100µM D-ala6 GnRH-II and 10µM SB-75 partially rescued sperm motility and morphology measures. These data suggest that GnRH-II and its receptor regulate porcine sperm function in an autocrine/paracrine manner.

Assessment of the Monocyte Subpopulations and M1/M2 Macrophage Ratio in Concentrated Bone Marrow Aspirate.

The purpose of this study was to determine the M1/M2 macrophage ratio in concentrated bone marrow aspirate (cBMA) in patients undergoing surgical intervention augmented with cBMA for osteochondral lesions of the talus (OLTs). Samples of peripheral blood (PB), bone marrow aspirate (BMA), and cBMA were collected during the procedure. The samples were analyzed by automated cell counting and multicolor fluorescence-activated cell sorting with specific antibodies recognizing monocytes (CD14+ CD16+) and the M1 (CD86+) and M2 (CD163+CD206+) populations within that monocyte population. Cytokine concentrations within the samples were evaluated with enzyme-linked immunosorbent assay (ELISA). The composition of cBMA was compared between 2 commercially available BMA concentration systems. Thirty-eight patients with a mean age of 43.2 ± 10.1 years old undergoing a surgical procedure for the treatment of OLTs involving the use of cBMA were included. cBMA had a mean fold increase of 4.7 for all white blood cells, 6.1 for monocytes, 7.9 for lymphocytes, 2.4 for neutrophils, and 9.6 for platelets when compared to BMA. The mean M1/M2 ratio for PB, BMA, and cBMA was 15.2 ± 12.0, 20.8 ± 13.3, and 22.1 ± 16.0, respectively. There was a statistically significant higher concentration of interleukin-1 receptor antagonist (IL-1Ra) in the cBMA sample (8243.3 ± 14,837.4 pg/mL) compared to both BMA (3143.0 ± 2218.5 pg/mL) and PB (1847.5 ± 1520.4 pg/mL) samples. The IL-1Ra/IL-1β ratio for PB, BMA, and cBMA was 790.6 ± 581.9, 764.7 ± 675.2, and 235.7 ± 192.1, respectively. There was no difference in the cBMA M1/M2 ratio (19.0 ± 11.1 vs 24.0 ± 18.3) between the Magellan (Isto Biologics, Hopkinton, Massachusetts) and Angel systems (Arthrex Inc, Naples, Florida). This prospective study found that the M1/M2 ratio in cBMA was 22.1 ± 16.0, with significant patient to patient variation observed. Overall, there was no statistically significant difference in the M1/M2 ratio across PB, BMA, and cBMA samples. This is the first study to characterize the macrophage subpopulation within cBMA, which may have significant clinical implications in future studies.

Time Course of Reversal of Fentanyl-Induced Respiratory Depression in Healthy Subjects by Intramuscular Nalmefene and Intramuscular and Intranasal Naloxone.

The increase in opioid overdose deaths, particularly involving potent, long-acting synthetic opioids, has led to calls for stronger, longer-acting opioid-overdose-reversal agents. Using an opioid-induced respiratory depression model, we investigated the onset and time course of action of naloxone and a long-acting opioid antagonist, nalmefene, in reversing the effects of an ongoing intravenous fentanyl infusion over a period of up to 100 min. Healthy, moderately experienced opioid users received intramuscular (IM) nalmefene 1 mg, IM naloxone 2 mg, or intranasal (IN) naloxone 4 mg after fentanyl-induced respiratory depression was established based on reduction in respiratory minute volume (MV). Each participant received each opioid antagonist twice per a randomized crossover schedule. Reversal of respiratory depression, pharmacokinetics, and safety were investigated. Participants showed rapid increases in plasma opioid antagonist concentrations, and meaningful reversal of depressed MV tended to occur earlier with IM nalmefene and IM naloxone than with IN naloxone. Compared to naloxone, nalmefene provided extended exposure, and mean MV was maintained at a higher level. All participants experienced treatment-related adverse events, but none were severe, serious, or led to study drug discontinuation. This study provides evidence that IM nalmefene 1 mg achieves reversal of fentanyl-induced respiratory depression similar to or better than that achieved with standard-of-care naloxone treatments. No new safety concerns were raised for IM nalmefene at the tested dose. The pharmacokinetic and pharmacodynamic properties of IM nalmefene position it as an important treatment option in opioid overdose reversal, particularly given the increasing prevalence of overdoses involving potent, long-acting synthetic opioids.

Chondrocyte Response to Fresh Autologous Conditioned Serum Versus Freeze-Dried Allogenic Conditioned Serum.

To investigate the cytokine release profile and histological response of human cartilage after exposure to autologous conditioned serum (ACS) and freeze-dried allogenic conditioned serum (FD-CS). Cartilage explants were collected from 6 patients undergoing total knee arthroplasty. ACS and FD-CS were created from patient serum samples. Cartilage samples were divided into 6 groups: (1) untreated control, (2) ACS, (3) FD-CS, (4) untreated interleukin (IL)-1β (5 ng/ml), (5) IL-1β + ACS, and (6) IL-1β + FD-CS. After 12 days, cartilage samples were analyzed with glycosaminoglycan (GAG) concentration normalized to wet weight while comparing cytokine concentrations, and histological scoring. There was a significant decrease in pathology scoring for ACS (P = 0.0368) and FD-CS (P = 0.0368) in the IL-1β injury groups compared with the untreated IL-1β insult group. ACS and FD-CS significantly mitigate the IL-1β induced increase in basic fibroblast growth factor (bFGF) (P = 0.0009 and P = 0.0002, respectively). FD-CS showed a significant decrease in IL-1β concentration in the presence of IL-1β insult compared with the untreated IL-1β group (P < 0.0001). ACS-treated samples had significantly higher concentration of tumor necrosis factor (TNF)-α independent of IL-1β when compared with samples not treated with biologics (P = 0.0053). Explanted osteoarthritic cartilage responds favorably and equivalently to treatment with ACS and FD-CS from a histological perspective. Both ACS and FD-CS were able to mitigate the IL-1β-induced increases in bFGF and FD-CS lowered IL-1β concentration while increasing interleukin-1 receptor antagonist (IL-1Ra) concentration. Although the cytokine profile of cartilage tissue explants treated with FD-CS appears to be different than that of ACS, this difference does not seem to affect biologic activity of FD-CS.

Acute pericardial postischemic inflammatory responses: Characterization using a preclinical porcine model

BackgroundPericardial fluid (PF) contains cells, proteins, and inflammatory mediators, such as cytokines, chemokines, growth factors, and matrix metalloproteinases. To date, we lack an adequate understanding of the inflammatory response that acute injury elicits in the pericardial space. ObjectiveTo characterize the inflammatory profile in the pericardial space acutely after ischemia/reperfusion. MethodsPigs were used to establish a percutaneous ischemia/reperfusion injury model. PF was removed from pigs at different time points postanesthesia or postischemia/reperfusion. Flow cytometry was used to characterize the immune cell composition of PF, while multiplex analysis was performed on the acellular portion of PF to determine the concentration of inflammatory mediators. There was a minimum of 3 pigs per group. ResultsWhile native PF mainly comprises macrophages, we show that neutrophils are the predominant inflammatory cell type in the pericardial space after injury. The combination of acute ischemia/reperfusion (IR) and repeatedly accessing the pericardial space significantly increases the concentration of interleukin-1 beta (IL-1β) and interleukin-1 receptor antagonist (IL-1ra). IR significantly increases the pericardial concentration of TGFβ1 but not TGFβ2. We observed that repeated manipulation of the pericardial space can also drive a robust pro-inflammatory response, resulting in a significant increase in immune cells and the accumulation of potent inflammatory mediators in the pericardial space. ConclusionIn the present study, we show that both IR and surgical manipulation can drive robust inflammatory processes in the pericardial space, consisting of an increase in inflammatory cytokines and alteration in the number and composition of immune cells.

Screening of Endophytic Antagonistic Bacteria in Wheat and Evaluation of Biocontrol Potential against Wheat Stripe Rust.

Wheat stripe rust is globally one of the most important diseases affecting wheat. There is an urgent need to develop environmentally safe and durable biological control options to supplement the control that is achieved with breeding and fungicides. In this study, endophytic bacteria were isolated from healthy wheat through the tissue separation method. Antagonistic endophytic bacteria were screened based on the control effect of urediniospore germination and wheat stripe rust (WSR). The taxonomic status of antagonistic strains was determined based on morphological, physiological, and biochemical characteristics and molecular biological identification (16S rDNA and gyrB gene sequence analysis). Finally, the potential growth-promoting effect of different concentrations of antagonists on wheat seedlings and the biological control effect of WSR were studied. A total of 136 strains of endophytic bacteria belonging to 38 genera were isolated. Pseudomonas was the most common bacterial genus, with 29 isolates (21%). The biological control effect of different isolates was assessed using an urediniospore germination assay. The isolate XD29-G1 of Paenibacillus polymyxa had the best performance, with 85% inhibition of spore germination during primary screening. In the deep screening, the control effect of XD29-G1 on wheat stripe rust was 60%. The antagonist XD29-G1 promoted the germination of wheat seeds and the growth of wheat seedlings at a solution dilution of 10-7 cfu/mL. The pot experiment results showed that different dilution concentrations of the strain had different levels of antibacterial activity against WSR, with the concentration of 10-1 cfu/mL having the best control effect and a control efficiency of 61.19%. XD29-G1 has better biological control potential against wheat stripe rust.

Effects of sanitary conditions with lipopolysaccharide injection and dietary valine supplementation on growth performance, immune response, bacterial profile, and microbial metabolites in weaned pigs

ABSTRACT This study investigated the effects of dietary L-valine (Val) supplementation and sanitary conditions with lipopolysaccharide injection on growth performance, immune response, and intestinal bacterial profiles and metabolites in weaned pigs. Thirty-two weaned pigs (6.98 ± 0.47 kg) were randomly assigned to treatments in a 2 × 2 factorial arrangement based on dietary Val levels and sanitary conditions (low or high). The pigs were fed either a basal diet containing the standard levels of Val suggested by (NRC), (2012) or a basal diet supplemented with 0.1% L-Val. A room designated as a high sanitary room was washed weekly, whereas the designated low sanitary room was not washed throughout the experiment and 5 kg of manure from the nursery pig barn was spread on the pen floors on day 1. All data were analysed using a mixed procedure of SAS, with the individual pen as the experimental unit. The pigs raised in low sanitary conditions exhibited a lower (p < 0.05) average daily gain, average daily feed intake, and gain-to-feed ratio and a higher (p < 0.05) incidence of diarrhoea than those raised in high sanitary conditions during the 14-d experimental period. The pigs in the low sanitary group also had a lower (p < 0.05) concentration of butyrate in the jejunum and a higher (p < 0.05) concentration of NH3-N in the colon than those in the high sanitary group. Dietary Val supplementation was reduced (p < 0.05) plasma interleukin (IL)-1β and IL-1 receptor antagonist concentrations as well as isovalerate and NH3-N concentrations in the colon, regardless of sanitary conditions. Interactions between dietary Val supplementation and sanitary conditions were observed in the abundances of mRNA-encoding β-defensins 113, 125 and 129 (p < 0.05). In conclusion, dietary Val supplementation beneficially modulates inflammatory responses and microbial metabolites regardless of sanitary conditions while transcriptional levels of β-defensins are regulated by dietary Val supplementation in a manner dependent on housing hygiene conditions.

Revisiting pancreatic islet isolation in murine models: A practical and effective technical protocol.

The endocrine pancreas is composed of clusters of cell groups called pancreatic islets. These cells are responsible for the synthesis and secretion of hormones crucial for glycemic homeostasis, such as insulin and glucagon. Therefore, these cells were the targets of many studies. One method to study and/or understand endocrine pancreatic physiology is the isolation of these islets and stimulation of hormone production using different concentrations of glucose, agonists, and/or antagonists of specific secretagogues and mimicking the stimulation of hormonal synthesis and secretion. Many researchers studied pancreatic physiology in murine models due to their ease of maintenance and rapid development. However, the isolation of pancreatic islets involves meticulous processes that may vary between rodent species. The present study describes a simple and effective technical protocol for isolating intact islets from mice and rats for use as a practical guide for researchers. The method involves digestion of the acinar parenchyma by intraductal collagenase. Isolated islets are suitable for invitro endocrine secretion analyses, microscopy techniques, and biochemical analyses.

Changes in Induced-Antipredation Defense Traits and Transcriptome Regulations of Daphnia magna in Response to 5-HT1A Receptor Antagonist.

The serotonin signaling system plays a crucial role in regulating the ontogeny of crustaceans. Here, we describe the effects of different concentrations of the 5-hydroxytryptamine 1A receptor antagonist (WAY-100635) on the induced antipredation (Rhodeus ocellatus as the predator), morphological, behavioral, and life-history defenses of Daphnia magna and use transcriptomics to analyze the underlying molecular mechanisms. Our results indicate that exposure to WAY-100635 leads to changes in the expression of different defensive traits in D. magna when faced with fish predation risks. Specifically, as the length of exposure to WAY-100635 increases, high concentrations of WAY-100635 inhibit defensive responses associated with morphological and reproductive activities but promote the immediate negative phototactic behavioral defense of D. magna. This change is related to the underlying mechanism through which WAY-100635 interferes with gene expression of G-protein-coupled GABA receptors by affecting GABBR1 but promotes serotonin receptor signaling and ecdysteroid signaling pathways. In addition, we also find for the first time that fish kairomone can significantly activate the HIF-1α signaling pathway, which may lead to an increase in the rate of immediate movement. These results can help assess the potential impacts of serotonin-disrupting psychotropic drugs on zooplankton in aquatic ecosystems.

Leptin promotes proliferation of human undifferentiated spermatogonia by activating the PI3K/AKT/mTOR pathway.

Male infertility is a common disease affecting male reproductive health. Leptin is an important hormone that regulates various physiological processes, including reproductive function. However, few experimental studies have been carried out to elucidate the mechanism of leptin's effects on male reproductive function. The purpose of this study was to investigate the effects of leptin on testicular spermatogenesis and its mechanism, so as to provide potential targets for the treatment of patients with spermatogenic dysfunction. Testicular tissues were collected from eight prostate cancer patients undergoing surgical castration. GPR125-positive spermatogonia were isolated by two consecutive magnetic activated cell sorting (MACS), followed by incubation with conditioned medium. To identify the signaling pathway(s) involved in the effects of leptin, undifferentiated spermatogonia were treated with different concentrations of leptin and antagonists of leptin-related pathways. The proliferative effect of leptin was evaluated by cell counting using a hemocytometer. Expressions of p-AKT, p-ERK, p-STAT, and p-S6K were determined by western blotting analysis. Leptin promoted the growth of human GPR125-positive spermatogonia in a concentration-dependent manner. The most significant proliferative effect was observed using 100ng/mL leptin after 6 days of culture. Leptin significantly increased the phosphorylation of STAT3, AKT, and ERK in undifferentiated spermatogonia. Phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 inhibited the leptin-induced activation of AKT, ERK, and downstream S6K. Treatment with the mammalian target of rapamycin (mTOR) inhibitor rapamycin also inhibited S6K phosphorylation. Moreover, both LY294002 and rapamycin were found to inhibit the leptin-induced proliferation of undifferentiated spermatogonia. These results suggested that the leptin-induced proliferation of GPR125-positive spermatogonia was dependent on the PI3K/AKT/mTOR pathway. Further exploration of proliferation and apoptotic markers suggested that leptin may alleviate cell apoptosis by regulating the expression of Bax and FasL. A certain concentration of leptin (25∼100ng/mL) could promote proliferation of undifferentiated spermatogonia, which was mediated by PI3K/AKT/mTOR pathway.

Functional characterization of three G protein-coupled acetylcholine receptors in parasitic nematode Trichinella spiralis

The physiological significance of metabotropic acetylcholine receptors in parasitic nematodes remains largely unexplored. Here, three different Trichinella spiralis G protein-coupled acetylcholine receptors (TsGAR-1, -2, and -3) were identified in the genome of T. spiralis. The phylogenetic analyses showed that TsGAR-1 and -2 receptors belong to a distinct clade specific to invertebrates, while TsGAR-3 is closest to the cluster of mammalian-type muscarinic acetylcholine receptors (mAChR). The mRNA of TsGAR-1, -2, and -3 was detected in muscle larvae, newborn larvae, and adults. The functional aequorin-based assay in Chinese hamster ovary cells revealed that all three types of T. spiralis GARs trigger the Gq/11 pathway upon activation of the receptor with the acetylcholine ligand. TsGAR-1 and TsGAR-2 showed atypical affinity with classical muscarinic agonists, while TsGAR-3 was sensitive to all muscarinic agonists tested. High concentrations of propiverine antagonist blocked the activities of all three TsGARs, while atropine and scopolamine antagonists effectively inhibited only TsGAR-3. Our data indicate that the distinct pharmacological profile of TsGAR-1 and -2 receptors, as well as the phylogenetic distance between them and their mammalian orthologs, place them as attractive targets for the development of selective anthelmintic drugs interfering with nematodes’ cholinergic system.

TRPA1 Antagonist LY3526318 Inhibits the Cinnamaldehyde-Evoked Dermal Blood Flow Increase: Translational Proof of Pharmacology.

Transient receptor potential Ankyrin 1 (TRPA1) is an ion channel expressed by sensory neurons, where it mediates pain signaling. Consequently, it has emerged as a promising target for novel analgesics, yet, to date, no TRPA1 antagonists have been approved for clinical use. In the present translational study, we utilized dermal blood flow changes evoked by TRPA1 agonist cinnamaldehyde as a target engagement biomarker to investigate the in vivo pharmacology of LY3526318, a novel TRPA1 antagonist. In rats, LY3526318 (1, 3, and 10 mg/kg, p.o.) dose-dependently reduced the cutaneous vasodilation typically observed following topical application of 10% v/v cinnamaldehyde. The inhibition was significant at the site of cinnamaldehyde application and also when including an adjacent area of skin. Similarly, in a cohort of 16 healthy human volunteers, LY3526318 administration (10, 30, and 100 mg, p.o.) dose-dependently reduced the elevated blood flow surrounding the site of 10% v/v cinnamaldehyde application, with a trend toward inhibition at the site of application. Comparisons between both species reveal that the effects of LY3526318 on the cinnamaldehyde-induced dermal blood flow are greater in rats relative to humans, even when adjusting for cross-species differences in potency of the compound at TRPA1. Exposure-response relationships suggest that a greater magnitude response may be observed in humans if higher antagonist concentrations could be achieved. Taken together, these results demonstrate that cinnamaldehyde-evoked changes in dermal blood flow can be utilized as a target engagement biomarker for TRPA1 activity and that LY3526318 antagonizes the ion channel both in rats and humans.

Heat stress–associated changes in the intestinal barrier, inflammatory signals, and microbiome communities in dairy calves

Recent studies indicate that heat stress pathophysiology is associated with intestinal barrier dysfunction, local and systemic inflammation, and gut dysbiosis. However, inconclusive results and a poor description of tissue-specific changes must be addressed to identify potential intervention targets against heat stress illness in growing calves. Therefore, the objective of this study was to evaluate components of the intestinal barrier, pro- and anti-inflammatory signals, and microbiota community composition in Holstein bull calves exposed to heat stress. Animals (mean age = 12 wk old; mean body weight = 122 kg) penned individually in temperature-controlled rooms were assigned to (1) thermoneutral conditions (constant room temperature at 19.5°C) and restricted offer of feed (TNR, n = 8), or (2) heat stress conditions (cycles of room temperatures ranging from 20 to 37.8°C) along with ad libitum offer of feed (HS, n = 8) for 7 d. Upon treatment completion, sections of the jejunum, ileum, and colon were collected and snap-frozen immediately to evaluate gene and protein expression, cytokine concentrations, and myeloperoxidase activity. Digesta aliquots of the ileum, colon, and rectum were collected to assess bacterial communities. Plasma was harvested on d 2, 5, and 7 to determine cytokine concentrations. Overall, results showed a section-specific effect of HS on intestinal integrity. Jejunal mRNA expression of TJP1 was decreased by 70.9% in HS relative to TNR calves. In agreement, jejunal expression of heat shock transcription factor-1 protein, a known tight junction protein expression regulator, decreased by 48% in HS calves. Jejunal analyses showed that HS decreased concentrations of IL-1α by 36.6% and tended to decrease the concentration of IL-17A. Conversely, HS elicited a 3.5-fold increase in jejunal concentration of anti-inflammatory IL-36 receptor antagonist. Plasma analysis of pro-inflammatory cytokines showed that IL-6 decreased by 51% in HS relative to TNR calves. Heat stress alteration of the large intestine bacterial communities was characterized by increased genus Butyrivibrio_3, a known butyrate-producing organism, and changes in bacteria metabolism of energy and AA. A strong positive correlation between the rectal temperature and pro-inflammatory Eggerthii spp. was detected in HS calves. In conclusion, this work indicates that HS impairs the intestinal barrier function of jejunum. The pro- and anti-inflammatory signal changes may be part of a broader response to restore intestinal homeostasis in jejunum. The changes in large intestine bacterial communities favoring butyrate-producing organisms (e.g., Butyrivibrio spp.) may be part of a successful response to maintain the integrity of the colonic mucosa of HS calves. The alteration of intestinal homeostasis should be the target for heat stress therapies to restore biological functions, and, thus highlights the relevance of this work.

N-retinylidene-N-retinylethanolamine degradation in human retinal pigment epithelial cells via memantine- and ifenprodil-mediated autophagy.

N-methyl-D-aspartate (NMDA) receptors are ionic glutamine receptors involved in brain development and functions such as learning and memory formation. NMDA receptor inhibition is associated with autophagy activation. In this study, we investigated whether the NMDA receptor antagonists, memantine and ifenprodil, induce autophagy in human retinal pigment epithelial cells (ARPE-19) to remove Nretinylidene- N-retinylethanolamine (A2E), an intracellular lipofuscin component. Fluorometric analysis using labeled A2E (A2E-BDP) and confocal microscopic examination revealed that low concentrations of NMDA receptor antagonists, which did not induce cytotoxicity, significantly reduced A2E accumulation in ARPE-19 cells. In addition, memantine and ifenprodil activated autophagy in ARPE-19 cells as measured by microtubule-associated protein 1A/1B-light chain3-II formation and phosphorylated p62 protein levels. Further, to understand the correlation between memantine- and ifenprodil-mediated A2E degradation and autophagy, autophagy-related 5 (ATG5) was depleted using RNA interference. Memantine and ifenprodil failed to degrade A2E in ARPE-19 cells lacking ATG5. Taken together, our study indicates that the NMDA receptor antagonists, memantine and ifenprodil, can remove A2E accumulated in cells via autophagy activation in ARPE-19 cells.

The role of orexin-1 receptors within the ventral tegmental area in the extinction and reinstatement of methamphetamine place preference

Targeting the orexin system has recently been identified as one of the promising options for treating drug addiction. It may be more feasible and achievable if we investigate the accurate function of the orexin system in brain areas implicated in reward and addiction, such as the ventral tegmental area (VTA) by animal reward models. This study investigated the contribution of the orexin system, mainly the orexin-1 receptors (OX1R) in the VTA, in the extinction and reinstatement of methamphetamine (METH) related memories in the conditioned place preference (CPP) model. Animals after the acquisition of METH place preference were subjected to two separate sets of extinction and reinstatement experiments to receive various concentrations of selective OX1R antagonist, SB334867 into the bilateral VTA before extinction sessions (1, 3, and 10 nmol/0.3 μl DMSO per side) or only on the reinstatement phase (3, 10, and 30 nmol/0.3 μl DMSO per side), respectively. Intra-VTA infusion of SB334867 throughout the extinction phase could remarkably facilitate the extinction process and decrease the maintenance of reinforcing effects of METH at the highest dosage (10 nmol; p < 0.0001). Data also indicated a single microinfusion of SB334867 into the VTA before reinstatement of the METH-seeking behavior could considerably prevent the relapse of previously formed reward-context memories (10 nmol; p < 0.01 and 30 nmol; p < 0.001). The present study provided evidence supporting the potential therapeutic effects of the orexin system modulation, specifically in the VTA, on different stages of METH-induced place preference.

Cardiac surgery elicits pericardial inflammatory responses that are distinct compared with postcardiopulmonary bypass systemic inflammation

ObjectivesCardiac surgery using cardiopulmonary bypass contributes to a robust systemic inflammatory process. Local intrapericardial postsurgical inflammation is believed to trigger important clinical implications, such as postoperative atrial fibrillation and postsurgical intrathoracic adhesions. Immune mediators in the pericardial space may underlie such complications. MethodsIn this prospective pilot clinical study, 12 patients undergoing isolated coronary artery bypass graft surgery were enrolled. Native pericardial fluid and venous blood samples (baseline) were collected immediately after pericardiotomy. Postoperative pericardial fluid and venous blood samples were collected 48-hours after cardiopulmonary bypass and compared with baseline. Flow cytometry determined proportions of specific immune cells, whereas multiplex analysis probed for inflammatory mediators. ResultsNeutrophils are the predominant cells in both the pericardial space and peripheral blood postoperatively. There are significantly more CD163lo macrophages in blood compared with pericardial effluent after surgery. Although there are significantly more CD163hi macrophages in native pericardial fluid compared with baseline blood, after surgery there are significantly fewer of these cells present in the pericardial space compared with blood. Postoperatively, concentration of interleukin receptor antagonist 6, and interleukin 8 were significantly higher in the pericardial space compared with blood. After surgery, compared with blood, the pericardial space has a significantly higher concentration of matrix metalloproteinase 3, matrix metalloproteinase 8, and matrix metalloproteinase 9. The same trend was observed with transformational growth factor β. ConclusionsCardiac surgery elicits an inflammatory response in the pericardial space, which differs from systemic inflammatory responses. Future work should determine whether or not this distinct local inflammatory response contributes to postsurgical complications and could be modified to influence clinical outcomes.

Exposure to ambient air pollutants and acute respiratory distress syndrome risk in sepsis.

Exposures to ambient air pollutants may prime the lung enhancing risk of acute respiratory distress syndrome (ARDS) in sepsis. Our objective was to determine the association of short-, medium-, and long-term pollutant exposures and ARDS risk in critically ill sepsis patients. We analyzed a prospective cohort of 1858 critically ill patients with sepsis, and estimated short- (3 days), medium- (6 weeks), and long- (5 years) term exposures to ozone, nitrogen dioxide (NO2), sulfur dioxide (SO2), carbon monoxide (CO), particulate matter < 2.5μm (PM2.5), and PM < 10μm (PM10) using weighted averages of daily levels from monitors within 50km of subjects' residences. Subjects were followed for 6days for ARDS by the Berlin Criteria. The association between each pollutant and ARDS was determined using multivariable logistic regression adjusting for preselected confounders. In 764 subjects, we measured plasma concentrations of inflammatory proteins at presentation and tested for an association between pollutant exposure and protein concentration via linear regression. ARDS developed in 754 (41%) subjects. Short- and long-term exposures to SO2, NO2, and PM2.5 were associated with ARDS risk (SO2: odds ratio (OR) for the comparison of the 75-25th long-term exposure percentile 1.43 (95% confidence interval (CI) 1.16, 1.77); p < 0.01; NO2: 1.36 (1.06, 1.74); p = 0.04, PM2.5: 1.21 (1.04, 1.41); p = 0.03). Long-term exposures to these three pollutants were also associated with plasma interleukin-1 receptor antagonist and soluble tumor necrosis factor receptor-1 concentrations. Short and long-term exposures to ambient SO2, PM2.5, and NO2 are associated with increased ARDS risk in sepsis, representing potentially modifiable environmental risk factors for sepsis-associated ARDS.

Anti-inflammatory interleukin 1 receptor antagonist concentration in plasma correlates with blood-brain barrier integrity in the primary lesion area in traumatic brain injury patients

PurposeBlood-brain barrier (BBB) dysregulation and pro-inflammatory signalling molecules are secondary factors that have been associated with injury severity and long-term clinical outcome following traumatic brain injury (TBI). However, the association between BBB permeability and inflammation is unknown in human TBI patients. In this study, we investigated whether BBI integrity as measured by Dynamic Contrast-Enhanced (DCE) Magnetic Resonance Imaging (MRI) correlates with plasma levels of immunological markers following TBI. MethodsThirty-two TBI patients recruited from a neurosurgical unit were included in the study. Structural three-dimensional T1-weighted and DCE-MRI images were acquired on a 3T MRI at the earliest opportunity once the participant was sufficiently stable after patient admission to hospital. Blood sampling was performed on the same day as the MRI. The location and extents of the haemorrhagic and contusional lesions were identified. Immunological biomarkers were quantified from the participants’ plasma using a multiplex immunoassay. Demographic and clinical information, including age and Glasgow Coma Scale (GCS) were also collected and the immunological biomarker profiles were compared across controls and the TBI severity sub-groups. Contrast agent leakiness through blood-brain barriers (BBB) in the contusional lesions were assessed by fitting DCE-MRI using Patlak model and BBB leakiness characteristics of the participants were correlated with the immunological biomarker profiles. ResultsTBI patients showed reduced plasma levels of interleukin (IL)-1β, IFN-γ, IL-13, and chemokine (C–C motif) ligands (CCL)2 compared to controls and significantly higher levels of platelet-derived growth factor (PDGF-BB), IL-6, and IL-8. BBB leakiness of the contusional lesions did not significantly differ across different TBI severity sub-groups. IL-1ra levels significantly and positively correlated with the contusional lesion's BBB integrity as measured with DCE-MRI via an exponential curve relationship. DiscussionThis is the first study to combine DCE-MRI with plasma markers of inflammation in acute TBI patients. Our finding that plasma levels of the anti-inflammatory cytokine IL-1ra correlated negatively with increased leakiness of the BBB.

E069 Antagonism of IL-27 bioactivity

Abstract Background/Aims Rheumatoid arthritis (RA) is a chronic autoimmune disease that results in joint damage and bony destruction. The inflammatory process is central in the pathology of RA, with cytokines mediating the inflammatory response. Cytokines, in particular IL-6, have been targeted as treatments for RA. The IL-27 cytokine is part of the IL-6 family, and acts via the same signalling pathway. IL-27 can have the same function as IL-6, but can also antagonise IL-6. The presence of ectopic lymphoid structures (ELSs) is associated with more severe disease and worse joint damage, and is also associated with low IL-27 levels. Hence, targeting IL-27 may aid in severe RA and offer a novel treatment option. Aims: 1) To generate a recombinant IL-27 antagonist, 2) to determine the concentration and bioactivity of IL-27 antagonist, 3) to test the bioactivity of IL-27 antagonist as an inhibitor of IL-27 activity. Methods Chinese hamster ovarian (CHO) cells were transduced with adenovirus to express the IL-27 antagonist. The concentration of these IL-27 antagonists was determined using ELISA. To determine the bioactivity of the IL-27 antagonist, both acute leukaemia cell lines (THP1 cells) and cells from C57BL/6J mice were treated with human IL-27 and IL-27 antagonists and STAT3 activation determined using flow cytometry and ELISA. Results THP1 cells treated with both 5ng/ml and 15ng/ml of human recombinant IL-27 showed STAT3 activation, with 29.4% and 30.7% of cells being STAT3 positive respectively. Cells that remained untreated only showed STAT3 activation in 2.54% of cells, indicating IL-27 has a role in the activation of the STAT3 pathway. STAT3 activation was not witnessed in CD4 cells from C57BL/6J mice treated with human recombinant IL-27. Furthermore, the recombinant IL-27 antagonist was not seen to be able to antagonise the actions IL-27 and alter STAT3 activation. Conclusion In conclusion, this study has demonstrated that IL-27 treatment of cells can cause increased STAT3 activation, however we were unable to demonstrate that the IL-27 antagonist was able to inhibit the actions of IL-27. IL-27 shows great potential for use as a novel therapy, since both its pro and anti-inflammatory functions can be targeted, however there needs to be much more information about the action of IL-27 as this is still largely unknown. Future studies could aim to optimise the protocol in order to understand the bioactivity of the IL-27 antagonist, thus understanding its role in altering STAT3 activation. Disclosure L. Green: None.