Activated Mouse Peritoneal Macrophages Research Articles

Isolation, Virtual Screening, and Evaluation of Hazelnut-Derived Immunoactive Peptides for the Inhibition of SARS-CoV-2 Main Protease.

The aim of this study is to validate the activity of hazelnut (Corylus avellana L.)-derived immunoactive peptides inhibiting the main protease (Mpro) of SARS-CoV-2 and further unveil their interaction mechanism using in vitro assays, molecular dynamics (MD) simulations, and binding free energy calculations. In general, the enzymatic hydrolysis components, especially molecular weight < 3 kDa, possess good immune activity as measured by the proliferation ability of mouse splenic lymphocytes and phagocytic activity of mouse peritoneal macrophages. Over 866 unique peptide sequences were isolated, purified, and then identified by nanohigh-performance liquid chromatography/tandem mass spectrometry (NANO-HPLC-MS/MS) from hazelnut protein hydrolysates, but Trp-Trp-Asn-Leu-Asn (WWNLN) and Trp-Ala-Val-Leu-Lys (WAVLK) in particular are found to increase the cell viability and phagocytic capacity of RAW264.7 macrophages as well as promote the secretion of the cytokines nitric oxide (NO), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β). Fluorescence resonance energy transfer assay elucidated that WWNLN and WAVLK exhibit excellent inhibitory potency against Mpro, with IC50 values of 6.695 and 16.750 μM, respectively. Classical all-atom MD simulations show that hydrogen bonds play a pivotal role in stabilizing the complex conformation and protein-peptide interaction. Molecular Mechanics/Generalized Born Surface Area (MM/GBSA) calculation indicates that WWNLN has a lower binding free energy with Mpro than WAVLK. Furthermore, adsorption, distribution, metabolism, excretion, and toxicity (ADMET) predictions illustrate favorable drug-likeness and pharmacokinetic properties of WWNLN compared to WAVLK. This study provides a new understanding of the immunomodulatory activity of hazelnut hydrolysates and sheds light on peptide inhibitors targeting Mpro.

Structural characterization and immune-enhancing activity of a novel acid proteoglycan from Black soybean

Black soybean ( Glycine max (L.) merr), as an edible seed of the leguminous plant, possesses a high amount of proteins and carbohydrates. However, few studies have been conducted on the protein-polysaccharide polymer (proteoglycan) in Black soybean. Here, we aim to characterize the proteoglycan in Black soybean. Firstly, a crude extract from Black soybean was obtained by water extraction and alcohol precipitation. Then, DEAE-52 cellulose and Sephadex G-100 column chromatography were used to isolate proteoglycans in the crude extract, and a novel proteoglycan named BBWPS2 was obtained. Moreover, the chemical analysis showed that BBWPS2 was a proteoglycan of 2.02 × 10 5 Da, and composed of 7 kinds of monosaccharides and 16 kinds of amino acids. β -elimination reaction identified the carbohydrate chain was connected to peptide chain with O- glycosidic bonds. Finally, the immunomodulatory tests indicated that BBWPS2 can significantly promote the primary splenocyte proliferation and activate mouse peritoneal macrophages to produce TNF-α. Subsequent pathway studies revealed that BBWPS2 activated macrophages through the mannose receptor-mediated NF-κB/MAPK signaling pathway. Our work indicated that BBWPS2 from Black soybean could be explored as a promising natural immunopotentiator in food and pharmaceutical industries.

Konjac Glucomannan from Amorphophallus konjac enhances immunocompetence of the cyclophosphamide-induced immunosuppressed mice.

This present study was designed to evaluate the immunomodulatory activity of Konjac glucomannan (KGM) on immunosuppressed mice induced by cyclophosphamide (CTX) treatment. The mice immunodeficiency model was established by CTX. KGM was used to modulate the activities of immunosuppressive mice. It was proved that KGM could promote the proliferation of lymphocyte, thymus, and spleen indices, and alleviate the atrophy of immune organs and weight loss. Besides, in mice serum, the levels of cytokines including TNF‐α, IgG, IL‐2, and the contents of hemolysin were also increased after treatment with KGM. Furtherly, in nonspecific immunity, KGM could enhance natural killer (NK) cell lethality and pinocytic activity of mouse peritoneal macrophages. Therefore, all of these results revealed that KGM could improve the reduced immunity of CTX‐induced mice via modulation innate immunity and adaptive immunity.

Helminth-derived peptide GK-1 induces Myd88-dependent pro-inflammatory signaling events in bone marrow-derived antigen-presenting cells

GK-1 is an immunomodulatory, 18-aa-long peptide that has been proved to promote the activation of mouse peritoneal macrophages and LPS-pulsed mouse bone marrow-derived dendritic cells (BM-DCs). This study is aimed to explore the mechanisms underlying the activation of these antigen-presenting cells (APCs) by GK-1. In our study, GK-1 up-regulated in vitro the expression of CD86 and CD40, and it increased the secretion of NO in bone marrow-derived macrophages (BMDMs). In BM-DCs, GK-1 upregulated the expression of MHC class II and CD86. Additionally, GK-1 was found to be involved in the phosphorylation of MAPK p38, JNK and ERK 1/2 and in Myd88-dependent activation of NF-κB in both antigen-presenting cell types. In vivo, GK-1 increased the secretion of IL-15, CCL2, and IL-6 through a Myd88-dependent mechanism. This study demonstrated that GK-1 promotes the activation and effector activity of APCs through a mechanism dependent on Myd88, probably involving a Toll-like receptor as a target.

In vitro immunomodulatory effect of Bifidobacterium bifidum and Lactobacillus reuteri cell free extracts

Recent studies have shown that alterations of the immune response in the gastrointestinal mucosa are key components of the mechanism of the probiotic action of beneficial bacteria. Most of the beneficial effects of probiotics are due to the action of their structural components and metabolites. Macrophages are first-line defense cells of the immune system, which not only participate in the detection, phagocytosis and destruction of harmful microorganisms, but also determine the nature of the subsequent immune response by presenting antigens to T-cells and initiating inflammation by releasing cytokines. We researched the effect of two types of cell-free extracts (CFEs) containing probiotic derivatives (structural components and metabolites of bacteria) Bifidobacterium bifidum 1 (BbCFE) and Lactobacillus reuteri DSM 17938 (LrCFE) on the activity of mouse peritoneal macrophages and on the ability of peripheral human blood mononuclear cells to produce cytokines. CFEs were obtained by culturing probiotics in their own disintegrates and then removing cells and cell debris by centrifugation and filtration. Peritoneal macrophages were isolated from mice. Some of them were infected in vitro by Salmonella thyphimurium. Uninfected and infected macrophages were incubated in culture medium containing (30% vol) or not containing CFEs at 37 °С in a microaerobic atmosphere (5% СО2) for 18 hours. After incubation, peritoneal macrophages were lysed. The obtained suspensions were centrifuged and supernatants were carefully collected. Macrophages activity was assessed by the nitrites level, superoxide dismutase (SOD), lactate dehydrogenase (LDH) activity and antiinflammatory cytokines levels in supernatants using spectrophotometric method. Peripheral mononuclear cells were isolated from the blood of healthy volunteers. The ability of peripheral mononuclear blood cells to produce antiinflammatory cytokines was evaluated after cell stimulation with lipopolysaccharide (LPS) and incubation with or without CFEs. Cytokine levels in supernatants were determined using enzyme-linked immunosorbent assay (ELISA). After infection with S. thyphimurium in macrophages, nitrite levels increased 5.5-fold, SOD activity 4.8-fold, and LDH 2-fold. Both studied CFEs exerted a similar effect on the macrophages’ activity. Addition of BbCFE to the incubation medium of infected macrophages resulted in a 4-fold decrease in nitrite levels, and the addition of LrCFE was accompanied by a decrease in nitrite levels to levels in intact cells. Under the influence of both CFEs, the activity of SOD and LDH was significantly reduced and did not differ significantly from the activity of these enzymes in intact cells. BbCFE and LrCFE did not have a significant effect on nitrite levels, SOD and LDH activity in intact macrophages. Under the influence of BbCFE, there was a 2-fold decrease in the production of TNF, a 2-fold increase in IL10 production, and a 30% increase in IL6 production by mononuclear cells. LrCFE caused a decrease in TNF production by 26.7% and IL6 by 36%, and IL10 by 1.9 times. Thus, the studied CFEs normalized the nitrite levels in peritoneal macrophages infected with S. thyphymurium and infection-induced activation of SOD and LDH enzymes. This demonstrates their ability to modulate oxidative processes in macrophages. In addition, under the influence of the investigated CFEs, there was a decrease in the production of pro-inflammatory cytokines (TNFα and IL-6) and increased production of anti-inflammatory cytokine (IL-10) by human peripheral mononuclear cells. The results of the study indicate the ability of CFEs by influencing the functions of innate immunity cells to restrict the inflammatory response and oxidative stress. Based on this, CFEs can be considered as promising agents for the treatment of inflammatory diseases.

C-terminus of heat shock protein 60 can activate macrophages by lectin-like oxidized low-density lipoprotein receptor 1

Immune responses against antigens generally require an efficient activation of antigen-presenting cells (APCs). Currently, the targeting of vaccine antigens to APCs has emerged as a promising strategy for boosting vaccine immunogenicity. Here, we reported that the C-terminus of heat shock protein 60 (HSP60C) can activate mouse peritoneal macrophages to secret a series of cytokines, and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and NF-κB p65 was involved in the pathway. We showed that the activation effect of HSP60C on macrophages was independent of toll-like receptor (TLR) 4 and the TLR-associated myeloide differentiation factor 88 (MyD88). Knockdown of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) reduced the activation of HSP60C-induced macrophage p38 MAPK, NF-κB p65 and cytokine secretion to some extent. Finally, we found that HSP60C up-regulated the expression of LOX-1 on macrophages and ovalbumin (OVA) model antigen fused with HSP60C markedly enhanced OVA-specific IgG responses. Thus, our results unravel a novel LOX-1-dependent pathway by which HSP60C can effectively activate macrophages and APCs targeting based on LOX-1 interaction is a promising approach to improve vaccines.

Immunization with pneumococcal elongation factor Tu enhances serotype-independent protection against Streptococcus pneumoniae infection

Vaccination is an effective strategy to prevent pneumococcal diseases. Currently, licensed vaccines include the pneumococcal polysaccharide vaccine (PPSV) and the pneumococcal conjugate vaccine (PCV), which target some of the most common of the 94 serotypes of S. pneumoniae based on their capsular composition. However, it has been reported that PPSV is not effective in children aged less than 2 years old and PCV induces serotype replacement, which means that the pneumococcal population has changed following widespread introduction of these vaccines, and the non-vaccine serotypes have increased in being the cause of invasive pneumococcal disease. Therefore, it is important that there is development of novel pneumococcal vaccines to either replace or complement current polysaccharide-based vaccines. Our previous study suggested that S. pneumoniae releases elongation factor Tu (EF-Tu) through autolysis followed by the induction of proinflammatory cytokines in macrophages via toll-like receptor 4, that may contribute to the development of pneumococcal diseases. In this study, we investigated the expression of EF-Tu in various S. pneumoniae strains and whether EF-Tu could be an antigen candidate for serotype-independent vaccine against pneumococcal infection. Western blotting and flow cytometry analysis revealed that EF-Tu is a common factor expressed on the surface of all pneumococcal strains tested, as well as intracellularly. In addition, we demonstrate that immunization with recombinant (r) EF-Tu induced the production of inflammatory cytokines and the IgG1 and IgG2a antibodies in mice, and increased the CD4+ T-cells proportion in splenocytes. We also reveal that anti-EF-Tu serum increased the phagocytic activity of mouse peritoneal macrophages against S. pneumoniae infection, independent of their serotypes. Finally, our results indicate that mice immunized with rEF-Tu were significantly and non-specifically protected against lethal challenges with S. pneumoniae serotypes (2 and 15A). Therefore, pneumococcal EF-Tu could be an antigen candidate for the serotype-independent vaccine against pneumococcal infection.

Carapanosins D-F from the Seeds of Andiroba (Carapa guianensis, Meliaceae) and Their Effects on LPS-Activated NO Production.

A novel nor-phragmalin-type limonoid, named carapanosin D (1), and two novel mexicanolide-type limonoids, carapanosins E (2) and F (3), were isolated from the seed oil of andiroba (Carapa guianensis Aublet), a traditional medicine in Brazil and Latin American countries. Their structures were unambiguously determined on the basis of spectroscopic analyses using one-dimensional (1D) and two-dimensional (2D) NMR techniques and High resolution Fast Atom Bombardment Mass Spectrometry (HRFABMS). Compounds 1–3 were evaluated for their effects on the production of nitric oxide (NO) in Lipopolysaccharide (LPS)-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that compounds 2 and 3 have high potency as inhibitors of macrophage activation.

Role of the sseK1 gene in the pathogenicity of Salmonella enterica serovar enteritidis in vitro and in vivo

Salmonella enteritidis is a common food-borne pathogen associated with consumption of contaminated poultry meat and eggs, which frequently causes gastroenteritis in humans. Salmonella secreted effector K1 (SseK1), as a translocated and secreted protein has been identified to be essential for the virulence of Salmonella typhimurium in host cells. However, the role of the sseK1 gene in the pathogenicity of S. enteritidis remain unclear. In this study, a sseK1 deletion mutant of S. enteritidis was constructed and its biological characteristics were examined. It was found that the sseK1 deletion mutant did not affect the growth, adherence and invasion of Salmonella enteritidis when compared to the wild-type S. enteritidis. However, the mutant showed decreased formation of biofilm and significantly reduced intracellular survival of bacteria in activated mouse peritoneal macrophages, as well as showed reduced pathogenicity to a murine model by increasing the lethal dose 50% (LD50) value and decreasing the proliferation ratio of bacteria in vivo. Taken together, this study determined an important role for SseK1 in the pathogenicity of S. enteritidis in vitro and in vivo.

Prevention of Cyclophosphamide-Induced Immunosuppression in Mice with the Antimicrobial Peptide Sublancin.

Sublancin is a glycosylated antimicrobial peptide produced by Bacillus subtilis 168 with combined antibacterial and immunomodulatory activities. The purpose of this study was to evaluate the protective effects of sublancin on immunosuppression in cyclophosphamide-treated mice. In normal mice, the phagocytic activity of mouse peritoneal macrophages was significantly enhanced by oral administration of sublancin (1.0 mg/kg body weight) to BALB/c mice for 7 days (P < 0.01). In addition, the mRNA expression of IL-1β, IL-6, and TNF-α in peritoneal macrophages from sublancin- (1.0 mg/kg body weight) administered mice was significantly increased (P < 0.05). In cyclophosphamide-treated mice, oral sublancin administration accelerated the recovery of peripheral white blood cells, red blood cells, hemoglobins, and platelets and enhanced the macrophage phagocytic activity. Furthermore, sublancin restored the mRNA levels of IL-2, IL-4, and IL-6 in the spleen. Finally, the intestinal absorption of sublancin was poor as detected in the Caco-2 transwell system. Taken together, these findings suggest that sublancin plays a crucial role in the protection against immunosuppression in cyclophosphamide-treated mice and could be a potential candidate for use in immune therapy regimens.

Effects of Cold Stress on the Functional Activity of Mouse Peritoneal Macrophages in Conditions of Opiate Receptor Blockade

We report here studies of the effects of 10- and 60-min cold stress on the formation of reactive oxygen species (ROS) and cytokine production by peritoneal macrophages and corticosterone levels in conditions of opiate receptor blockade. Exposure of mice to a temperature of –20°C for 10 min led to suppression of ROS production by macrophages, and this effect was prevented by administration of naloxone. In animals exposed to 60-min stress, conversely, naloxone-dependent stimulation of ROS production was seen. Neither variant of cold stress had any significant effect on interleukin-1β production. TNF-α production on the background of 60-min cooling was stimulated in a naloxone-dependent manner in both unstimulated and zymosan-stimulated cultures. Production of IL-10 by macrophages increased regardless of the duration of cold exposure, with and without stimulation, and stopped on the background of opiate receptor blockade. Thus, the direction of action of acute cold stress on macrophage function depended on the parameter measured and the duration of exposure; opiate receptor blockade significantly modified the immunoregulatory effects of cold.

Influence of carrageenan on cytokine production and cellular activity of mouse peritoneal macrophages and its effect on experimental endotoxemia.

The in vivo effect of κ/β-carrageenan isolated from the red alga Tichocarpus crinitus on cytokine synthesis and cellular activity of murine peritoneal macrophages and also the protective effect of polysaccharides in LPS-induced endotoxemia in mice was studied. It was established that κ/β-carrageenan given orally at a dose of 100 mg/kg stimulates the induction of anti-inflammatory cytokines (IL-10) in mouse blood cells by more than 2.5-fold compared with control, with no effect on pro-inflammatory cytokine (TNF-α) production. Pretreating mice with carrageenan once a day before injecting LPS increased the levels of IL-10 by 2.5-fold and reduced TNF-α production by 2-fold compared with control. So, κ/β-carrageenan alone and in combination with LPS enhanced the cellular activity and mobility of peritoneal macrophages by increasing cell adhesion and migration compared with control. LPS activated cells intensively, sometimes resulting in their destruction by necrosis; carrageenan pretreatment reduced the excessive inflammatory cell activation caused by LPS. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1549-1557, 2017.

Three Novel Triterpenoids from Taraxacum officinale Roots.

Three novel lupane-, bauerane-, and euphane-type triterpenoids (1–3), in addition to seven known triterpenoids (4–10)—18β,19β-epoxy-21β-hydroxylupan-3β-yl acetate (4), 21-oxolup-18-en-3β-yl acetate (5), betulin (6), officinatrione (7), 11α-methoxyolean-12-en-3-one (8), eupha-7,24-dien-3-one (9), and 24-oxoeupha-7,24-dien-3β-yl acetate (10)—were isolated from the roots of Taraxacum officinale. Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D-NMR spectra and electron ionization mass spectrometry (EIMS). The effects of compounds 1–10 on the production of nitric oxide (NO) in lipopolysaccharide (LPS)-activated mouse peritoneal macrophages were evaluated. Compounds 4, 6, and 10 exhibited similar NO inhibitory activities to NG-monomethyl-l-arginine acetate (l-NMMA). These compounds did not exhibit cytotoxicity at an effective concentration. The results of present study suggest that compounds 4, 6, and 10 have potential as anti-inflammatory disease agents.

Regulation of LPS-induced mRNA expression of pro-inflammatory cytokines via alteration of NF-κB activity in mouse peritoneal macrophages exposed to fluoride

F toxicity to immune system, especially to macrophage, has been studied a lot recently. Nuclear factor-kappa B (NF-κB), as a transcription factor, plays a central role in immune and inflammatory responses via the regulation of downstream gene expression. Recent studies indicated that fluoride effect on inflammatory cytokine secretion, however, the molecular mechanism was less understood. In our study, peritoneal macrophages (PMs) were divided several groups and were administrated sodium fluoride (NaF, 50, 100, 200, 400, 800 μM) and/or lipopolysaccharide (LPS, 30 ng/mg). The mRNA expression of p65, inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF-α) and interleukin-1 beta (IL-1β) in macrophages exposed to fluoride was determined by quantitative real-time RT-PCR respectively. The translocation of NF-κB from cytoplasm to nucleus, which in a way reflects NF-κB activity, was demonstrated by Immunofluorescence and ELISA. Our results showed that fluoride had a dose-dependent effect on NF-κB activity, which coincided with LPS-induced mRNA expression of its downstream genes, iNOS and IL-1β. Fluoride alone causes no effect on gene expression. However, the mRNA expression of TNF-α showed non-NF-κB-dependent manner. Therefore, we come to the conclusion that fluoride can regulate LPS-induced mRNA expression of iNOS and IL-1β via NF-κB pathway in mouse peritoneal macrophages.

Telocytes in Inflammatory Gynaecologic Diseases and Infertility.

Women suffered with inflammatory gynecologic diseases, such as endometriosis (EMs) and acute salpingitis (AS) often complained of sub- or infertility, even in those women without obvious macroscopic anatomical pelvic abnormalities also have unexplained infertility. Generally, besides the well-known impairment of classically described oviduct cells caused by inflammatory diseases, such as the ciliated cells, fibroblasts and myofibroblasts, the involvement of the newly identified telocytes (TCs) in disease-affected oviduct tissues and potential pathophysiological roles in fertility problems remain unknown. In this chapter, TCs was investigated in rat model of EMs- and AS-affected oviduct tissues. Results showed inflammation and ischaemia-induced extensive ultrastructural damages of TCs both in cellular body and prolongations, with obvious TCs loss and interstitial fibrotic remodelling. Such in vivo pathological alterations might contribute to structural and functional abnormalities of oviduct tissue and potentially engaged in sub- or infertility. And especially, TCs connected to various activated immunocytes in both normal and diseased tissues, thus might participate in local immunoregulation (either repression or activation) and serve a possible explanation for immune-mediated pregnancy failure. Then, in vitro cell co-culture study showed that uterine TC conditioned media (TCM) can activate mouse peritoneal macrophages and subsequently trigger its cytokine secretion, thus providepreliminary evidence that, TCs are not simply innocent bystanders, but are instead potential functional players in local immunoregulatory and immunosurveillance.

Effects of alkaloids from Coptidis Rhizoma on mouse peritoneal macrophages in vitro

This work was mainly studied the effects of the four alkaloids from Coptidis Rhizoma on the mouse peritoneal macrophages in vitro and preliminarily discussed the regulating mechanisms. The effect of alkaloids from Coptidis Rhizoma on the vitality of macrophages was measured by the MTT assay. The effect of alkaloids on the phagocytosis of macrophages was determined by neutral red trial and respiratory burst activity was tested by NBT. The expressions of respiratory-burst-associated genes influenced by alkaloids were detected by qRT-PCR. The conformation change of membrane protein in macrophages by the impact of alkaloids was studied by fluorospectro-photometer. Results showed that the four alkaloids from Coptidis Rhizoma could increase the phagocytosis of macrophages in different level and berberine had the best effect. Berberine, coptisine and palmatine had up-regulation effects on respiratory burst activity of mouse peritoneal macrophages stimulated by PMA and regulatory activity on the mRNA expression of PKC, p40phox or p47phox, whereas the epiberberine had no significant influence on respiratory burst. Moreover, alkaloids from Coptidis Rhizoma could change the conformation of membrane protein and the berberine showed the strongest activity. The results suggested that the four alkaloids from Coptidis Rhizoma might activate macrophages through changing the conformation of membrane protein of macrophages and then enhanced the phagocytosis and respiratory burst activity of macrophages. Furthermore, the regulatory mechanism of alkaloids on the respiratory burst activity of macrophages may be also related to the expression level of PKC, p40phox and p47phox.

保元湯의 免疫調節 作用에 관한 硏究

Objectives : The water extract of Bowon-tang composited with thePanax, AstragalusandGlycyrrhiza Radixhas been traditionally used for treatment of a sickly child and smallpox in oriental medicine. However, little is known about the regulatory effects of Bowon-tang on the production, expression and activity of immune mediators [nitric oxide, prostaglandin E2, inducible nitric oxide synthetase, cyclooxygenase-2], the macrophage activation factor production, the proliferation, subset expression, the killing activity, and the capping in immune cells.Methods : In this study, we investigated the effects of water extracts from Bowon-tang,Panax, AstragalusorGRin mouse immune cells or human Jurkat T cells. Each extract (25-200 ㎍/㎖)perse had no cytotoxic effect in unstimulated macrophages, but concentration-dependently regulated NO and PGE₂production, iNOS expression, and COX-2 activity in mouse peritoneal macrophages with MAF stimulation. These regulatory effects were synergistically increased by their combination (Bowon-tang).Results : The extract of Bowon-tang concentration-dependently regulated T cell proliferation, CD4⁺and CD8⁺expression, and NK killing activity in mouse splenocytes and capping in Jurkat T cells.Conclusions : These results suggest that the water extract of Bowon-tang composited with thePanax, AstragalusandGRmay be useful for therapeutic drugs against a sickly constitution and immune diseases, probably by regulating the production of immune mediators.

Pleurotus nebrodensis polysaccharide (PN-S) enhances the immunity of immunosuppressed mice

In the present study, the effects of Pleurotus nebrodensis polysaccharide (PN-S) on the immune functions of immunosuppressed mice were determined. The immunosuppressed mouse model was established by treating the mice with cyclophosphamide (40 mg/kg/2d, CY) through intraperitoneal injection. The results showed that PN-S administration significantly reversed the CY-induced weight loss, increased the thymic and splenic indices, and promoted proliferation of T lymphocyte, B lymphocyte, and macrophages. PN-S also enhanced the activity of natural killer cells and increased the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in the serum. In addition, PN-S treatment significantly increased the phagocytic activity of mouse peritoneal macrophages. PN-S also increased the levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), and nitric oxide (NOS) in splenocytes. qRT-PCR results also indicated that PN-S increased the mRNA expression of IL-6, TNF-α, INF-γ, and nitric oxide synthase (iNOS) in the splenocytes. These results suggest that PN-S treatment enhances the immune function of immunosuppressed mice. This study may provide a basis for the application of this fungus in adjacent immunopotentiating therapy against cancer and in the treatment of chemotherapy-induced immunosuppression.

Abstract 216: The Effect of Fisetin on Lipopolysccharide-induced Inflammation and MMP Activity in Mouse Peritoneal Macrophages

Objective: Fisetin is a flavonoid that is prominent in strawberries. Fisetin has been shown to have several favorable effects, including anti-aging, anti-inflammatory and anti-carcinogenic. Fisetin is also known to have anti-oxidant properties as with other flavonoids. The purpose of this study was to investigate the influence of fisetin on lipopolysccharide (LPS) -induced inflammation and matrix metalloproteinase (MMP) activity in mouse peritoneal macrophages (MPMs) in vivo. Methods and Results: MPMs were harvested from BALB/c mice and cultured. After 30 minutes incubation with fisetin (10, 30 or 100 uM), MPMs were incubated with LPS (10 ng/ml) for 15 minutes, 60 minutes or 24 hours. LPS incubation increased concentrations of nitric oxide in supernatants that was attenuated by fisetin in a concentration-dependent manner. Similarly, mRNA abundance of inflammatory genes, such as MCP-1, IL-1b and iNOS, were enhanced by LPS, but were decreased by fisetin incubation in a concentration-dependent manner. Gelatin zymography demonstrated that LPS increased MMP-9 activity in supernatants that were decreased by the highest fisetin concentration. LPS-enhanced mRNA abundance of MMP-2 and MMP-9 were also attenuated by the highest concentration of festin. Fisetin also decreased LPS increased protein abundance uPA, as determined by Western blotting, and uPAR gene expression. Furthermore, fisetin also attenuated LPS enhanced ERK and JNK phosphorylation. Conclusion: Fisetin attenuated LPS-induced oxidative stress, inflammation, and MMPs activity in MPMs.

Evaluation Of Targeting CD47-SIRPα Using Primary Effusion Lymphoma Xenograft Mouse Model

IntroductionRecently, the critical roles of CD47 on the surface of resistant cancer cells and its ligand, signal regulatory protein alpha (SIRPα), have been proposed in their evasion of immunosurveillance. Primary effusion lymphoma (PEL) is a subtype of aggressive non-Hodgkin lymphoma that shows serous lymphomatous effusion without tumor masses in body cavities especially in advanced advanced acquired immunodeficiency syndrome (AIDS). The lack of optimal therapy combined with the aggressive nature of PEL results in a short median survival of less than 6 months. There is therefore a need for the development of new therapies. In this study, we transplanted PEL cells intraperitoneally into immunodeficient mice and evaluated the effect of targeting CD47-SIRPα signal with anti-CD47 antibody (Ab) and NOD-specific SIRPα on PEL. MethodsSurface CD47 expressions of six PEL cell lines and peripheral blood mononuclear cells (PBMC) from six different donors were examined by flow cytometry. The antiproliferative activities of anti-CD47Ab against PEL cell lines were measured by the methylthiotetrazole (MTT) method. Efficacy of anti-CD47 Ab-mediated phagocytosis against PEL was evaluated using mouse peritoneal macrophages and human macrophages in vitro. In a direct xenograft mouse model, primary PEL cells were injected intraperitoneally into NOD/Rag-2/Jak3 double-deficient mice to assess the in vivo efficacy of anti-CD47 Ab. To clarify the effect of NOD-specific SIRPα on PEL, we compared the phagocytic activities of peritoneal macrophages against PEL and the in vivo engraftment of PEL among Rag-2/Jak3 double-deficient mice with NOD and non-NOD (Balb/c and C57/BL6) genetic backgrounds. Organ invasion by PEL cells was evaluated by immunohistochemistry and flow cytometry. ResultsSurface CD47 of PEL cell lines was highly expressed compared with that of PBMC. Anti-CD47 Ab did not have a direct antitumor effect against PEL cell lines. Treatment with anti-CD47 Ab promoted phagocytic activities of mouse peritoneal macrophages and human macrophages in vitro. In addition, the expression of CD47 was significantly correlated with the fold increase of phagocytic activity. Treatment with anti-CD47 Ab inhibited the ascites formation and organ invasion completely in vivo compared with control IgG-treated mice. Anti-CD47 Ab-treated mice seemed to stay in healthy and show no apparent change. Although the numbers of peritoneal macrophages were not different among Rag-2/Jak3 double-deficient mice with NOD and non-NOD genetic backgrounds, the phagocytic activity in NOD mice was significantly decreased compared with non-NOD mice. Consistent with the data of phagocytosis assay, the development of PEL cells in NOD mice was superior to those in non-NOD mice. ConclusionsCD47 and SIRPα play the pivotal role in the immune evasion of PEL. NOD-specific SIRPα polymorphism has been reported to produce signals for macrophages not to engulf human cells. Thus, NOD-specific SIRPα is considered to contribute to the attenuation of macrophage phagocytosis and enable the development of PEL. CD47-SIRPα signal could be a therapeutic target for the immunotherapy of PEL. Disclosures:No relevant conflicts of interest to declare.