Murine Peritoneal Macrophages Research Articles

Immunomodulatory activity of Tityus serrulatus scorpion venom on human T lymphocytes.

BackgroundTityus serrulatus scorpion venom (TsV) contains toxins that act on K+ and Na+ channels and account for the venom’s toxic effects. TsV can activate murine peritoneal macrophages, but its effects on human lymphocytes have been poorly investigated. Considering that lymphocytes may play an important role in envenomation, we assessed whether TsV affects the expression of phenotypic (CD3, CD4, and CD8) and activation (CD69, CD25, and HLA-DR) markers, cell proliferation, and cytokine production in peripheral blood mononuclear cells.MethodsCytotoxicity of TsV was evaluated via the MTT assay. Cell proliferation, expression of phenotypic and activation markers, and release of cytokines were assessed using flow cytometry, after treatment with non-cytotoxic concentrations of TsV. The combined use of carboxyfluorescein diacetate succinimidyl ester and monoclonal antibodies against phenotypic and activation markers enabled us to simultaneously assess cell proliferation extent and cell activation status, and to discriminate among cell subpopulations.ResultsTsV at concentrations of 25 to 100 μg/mL were not cytotoxic towards peripheral blood mononuclear cells. TsV did not induce significant changes in lymphocyte subpopulations or in the expression of activation markers on CD4+ and CD8+ T cells. TsV inhibited the phytohemagglutinin-stimulated lymphocyte proliferation, particularly in the CD8+ CD25+ T lymphocyte subset. TsV alone, at 50 and 100 μg/mL, did not induce peripheral blood mononuclear cell proliferation, but elicited the production and release of IL-6, a proinflammatory cytokine that plays an important role in innate and adaptive immune responses.ConclusionsTsV is a potential source of molecules with immunomodulatory action on human T lymphocytes.Electronic supplementary materialThe online version of this article (doi:10.1186/s40409-015-0046-3) contains supplementary material, which is available to authorized users.

In vitro dose and duration dependent approaches for the assessment of ameliorative effects of nanoconjugated vancomycin against Staphylococcus aureus infection induced oxidative stress in murine peritoneal macrophages

AimsThe present study was aimed to evaluate the in vitro ameliorative effect of nanoconjugated vancomycin (NV) against vancomycin sensitive and resistant strains of Staphylococcus aureus infection-induced oxidative stress in murine peritoneal macrophage. MethodsPeritoneal macrophages from mice were treated with VSSA and VRSA (5 × 106 CFU/mL), VSSA + NV (5–250 μg/ml) and VRSA + NV (5–250 μg/ml) for 18 h, having 3 h interval in culture media; and the superoxide anion generation, lipid peroxidation, protein oxidation, antioxidant enzymes status and glutathione enzymes activity were monitored. ResultsThe significantly increased free radical generation, lipid peroxidation, protein carbonyls and oxidized glutathione levels were observed in VSSA and VRSA treated group as compared to control group; where as reduced glutathione level, antioxidant enzymes status and glutathione dependent enzymes were decreased significantly. All these changes come near to control in NV treated group in a dose and duration dependent fashion. Among the different doses and duration intervals of NV, maximum ameliorative effect was observed by 100 μg/ml for 12 h treatment which does not produce any damage to the cell. ConclusionsThese findings suggest the potential use and beneficial role of nanoconjugated vancomycin as a modulator of S. aureus infection-induced cellular damage in murine peritoneal macrophage.

A112: Vascular endothelial growth factor (VEGF) modulates functional activity of murine peritoneal macrophages in vitro

Recruitment of mononuclear phagocytes from the blood into tissues is considered to be a crucial process during inflammatory reactions, wound healing and tumor growth. Macrophages are known to reveal high plasticity and may change under the influence of microenvironment. The aim of the study was to evaluate the changes of macrophage functional activity under the influence of Vascular endothelial growth factor (VEGF) in vitro. This factor is known to be the main angiogenic factor but also possesses several immunomodulatory properties. Here we report that VEGF revealed a dose-dependent effects on cultured freshly isolated murine resident peritoneal macrophages: modulated iNOS mRNA expression, nitroxide and superoxide anion production, decreased 5′-nucleotidase (5′-N) activity, but had no influence on fluid-phase pinocytosis. Moreover, VEGF increased expression of its own mRNA via autocrine pathway as well as of VEGF protein expression. VEGF also induced up-regulation of extracellular matrix protein thrombospondin-1 (TSP-1) mRNA, which is considered as a part of macrophage activation phenotype. Production of cytokines and chemokines by macrophages was screened with the help of Multi- analyte ELISArray kits. It was found that incubation of macrophages in the presence of VEGF increased the production of angiogenic cytokines – TNF-α and IL-6 as well as several monocyte and leukocyte chemoattractants such as MCP-1, RANTES and MIP-1β. Therefore, we suggest that locally established VEGF gradient may influence inflammatory phenotype of tissue macrophages as well as potentiate their pro-angiogenic properties. This work was supported by Russian Foundation for Basic Research – Russia, Grant No. 15-04-06150 .

Activation effect of Ganoderma lucidum polysaccharides liposomes on murine peritoneal macrophages

The activation of murine peritoneal macrophages by Ganoderma lucidum polysaccharides liposomes (GLPL) was investigated in vitro. After treatment with GLPL, the changes of the nitric oxide (NO) secretion and iNOS (inducible nitric oxide synthase) activity were evaluated. The results showed that NO production and iNOS activity of macrophages were enhanced compared to GLP and BL group. In addition, both the phagocytic activity and levels of cytokines IL-1β, TNF-α and IFN-γ were enhanced in the peritoneal macrophages of mice by stimulation of GLPL. The expression of the major histocompatibility complex class II molecule (MHC II) on the surface of peritoneal macrophages significantly increased. These indicated that GLPL could enhance the activation of peritoneal macrophages and their potential for use as a delivery system of GLP.

OATP2A1/SLCO2A1-mediated prostaglandin E2 loading into intracellular acidic compartments of macrophages contributes to exocytotic secretion

There is significant evidence that the inducible cyclooxygenase isoform (COX-2) regulates the pericellular concentration of PGE2; however, the mechanism of the secretory process remains unclear. The present study, therefore, aimed to evaluate the role of prostaglandin transporter (OATP2A1) in PGE2 secretion from macrophages. Immunofluorescence staining for Oatp2a1 (Slco2a1) was primarily detected in cytoplasmic domains, and was partially co-localized with anti-PGE2 antibody, LysoTracker®, and anti-lysosome-associated membrane protein (Lamp) 1 antibody in murine macrophage-derived RAW264 cells and peritoneal macrophages (PMs). PGE2 uptake by subcellular fraction containing light lysosomes was reduced significantly in the presence of an OATP inhibitor and in Slco2a1+/− PMs. Secretion of PGE2 and lysosome-specific N-acetyl-β-d-glucosaminidase was enhanced in activated macrophagic cells, and diminished significantly under the Ca2+-depleted condition. The amount of PGE2 secreted from lipopolysaccharide-activated Slco2a1−/− PMs was significantly lower than that from PMs from wild type (WT) mice. Expression of Cox-2 and 15-hydroxyprostaglandin dehydrogenase (15-Pgdh) was unchanged between PMs from Slco2a1−/− and WT mice. These results suggest that OATP2A1 is involved in PGE2-loading into intracellular acidic compartments, including light lysosomes. Thus, OATP2A1 contributes to PGE2 secretion by macrophages via exocytosis induced by Ca2+ influx, independently of PGE2 synthesis and metabolism.

Synthesis of Unsymmetrical Sulfides and Their Oxidation to Sulfones to Discover Potent Antileishmanial Agents.

Unsymmetrical sulfides were first synthesized using combinations of a 1,3-dicarbonyl, an aromatic aldehyde and a thiol in the presence of 10 mol % ethanolic piperidine. These sulfides derivatives were subsequently converted into corresponding sulfones via oxidation in the presence of m-chloroperoxybenzoic acid (m-CPBA) at ice-bath to room temperature. The former reaction was achieved at room temperature through one-pot three-component. The later was obtained in good yields using mild reaction conditions with flexibility in choice from a range of substrates. The antimicrobial properties of the newly synthesized sulfone derivatives were investigated against the protozoan parasite, Leishmania donovani, a causative agent of visceral leishmaniasis (VL). Nine sulfone derivatives were found to be efficacious and exhibited significant antimicrobial activity. Further, these compounds were nontoxic on murine peritoneal macrophages thus eliminating potential cytoxicity in the host cells. These compounds may be indicated as potential leads in the treatment of visceral leishmaniasis.

Biocompatible and multi-responsive poly(N-vinylcaprolactam)-based microgels: The role of acidic comonomers in the colloidal properties and phase transition as a function of temperature and pH

Stimuli-responsive microgels have great potential for biomedical applications owing to their well-defined structure and tunable swelling-collapse behavior in response to environmental conditions changes. Herein, thermo- and pH-responsive biocompatible microgels have been developed via surfactant-free precipitation polymerization of N-vinylcaprolactam (NVCL) in aqueous media using acrylic or itaconic acid (AA or IA) as acidic comonomers. A comparative study was performed regarding the incorporation of acidic comonomers and their effect on the stimuli-responsive behavior and nanoparticles stability in aqueous dispersion. The particles size, polydispersity as well as temperature- and pH-responsiveness were characterized by Dynamic Light Scattering experiments. The results indicated that the acidic segments in the microgel networks not only rendered pH-responsiveness behavior but were also capable to stabilize the nanoparticles due to the negative charges from carboxyl groups present in AA and IA. Smaller particles diameters were obtained when IA was used as the ionizable comonomer. Moreover, IA proved to be more efficient in the stability of the dispersions against time. The microgels morphology was accessed by Electron Transmission Microscopy, which revealed their spherical shape and low polydispersity, although a tendency for particles aggregation. All microgels were well tolerated by murine peritoneal macrophages, as revealed by cytotoxic tests. These results provide important information for further studies on the incorporation of active ingredients in such microgels for controlled drug release systems.

Outer Surface Protein OspC Is an Antiphagocytic Factor That Protects Borrelia burgdorferi from Phagocytosis by Macrophages

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses. However, the exact biological function of OspC remains elusive. In this study, we showed that the ospC-deficient spirochete could not establish infection in NOD-scid IL2rγ(null) mice that lack B cells, T cells, NK cells, and lytic complement. The ospC mutant also could not establish infection in anti-Ly6G-treated SCID and C3H/HeN mice (depletion of neutrophils). However, depletion of mononuclear phagocytes at the skin site of inoculation in SCID and C3H/HeN mice allowed the ospC mutant to establish infection in vivo. In phagocyte-depleted mice, the ospC mutant was able to colonize the joints and triggered neutrophilia during dissemination. Furthermore, we found that phagocytosis of green fluorescent protein (GFP)-expressing ospC mutant spirochetes by murine peritoneal macrophages and human THP-1 macrophage-like cells, but not in PMN-HL60, was significantly higher than parental wild-type B. burgdorferi strains, suggesting that OspC has an antiphagocytic property. In addition, overproduction of OspC in spirochetes also decreased the uptake of spirochetes by murine peritoneal macrophages. Together, our findings provide evidence that mononuclear phagocytes play a key role in clearance of the ospC mutant and that OspC promotes spirochetes' evasion of macrophages during early Lyme borreliosis.

Angiopoietin-like protein 2 induces proinflammatory responses in peritoneal cells

Monocytes and macrophages are important effectors and regulators of inflammation, and both their differentiation and activation are regulated strictly in response to environmental cues. Angiopoietin-like protein 2 (Angptl2) is a multifaceted protein, displaying many physiological and pathological functions in inflammation, angiogenesis, hematopoiesis, and tumor development. Although recent studies implicate Angptl2 in chronic inflammation, the mechanisms of inflammation caused by Angptl2 remain unclear. The purpose of the present study was to elucidate the role of Angptl2 in inflammation by understanding the effects of Angptl2 on monocytes/macrophages. We showed that Angptl2 directly activates resident murine peritoneal monocytes and macrophages and induces a drastic upregulation of the transcription of several inflammatory genes including nitric oxide synthase 2 and prostaglandin-endoperoxide synthase 2, and several proinflammatory cytokine genes such as interleukin (IL)-1β, IL-6, TNFα, and CSF2, along with activation of ERK, JNK, p38, and nuclear factor kappa B signaling pathways. Concordantly, proinflammatory cytokines IL-1β, IL-6, TNFα, and GM-CSF, were rapidly elevated from murine peritoneal monocytes and macrophages. These results demonstrate a novel role for Angptl2 in inflammation via the direct activation of peritoneal monocytes and macrophages.

Immunomodulatory and toxicological evaluation of the fruit seeds from Platonia insignis, a native species from Brazilian Amazon Rainforest

The “bacuri” (Platonia insignis Mart., Clusiaceae) is a native tropical fruit from the Brazilian Amazon and Northeast Regions. Its seeds are used to treat inflammatory diseases, diarrhea and skin problems in traditional medical practices. Regarding its widespread medicinal uses, it is important to evaluate the biological and toxicological potential of this species. This way, the aim of this study was to investigate the in vitro cytotoxic and immunomodulatory effects of the hexanic extract of P. insignis seeds, as well as its in vivo acute oral toxicity. The biological evaluation was performed by the determination of cytotoxic (MTT and hemolysis assay) and immunomodulatory (phagocytic capacity, lysosomal volume and nitrite production) activities of EHSB in murine peritoneal macrophages. In addition, the oral acute toxicity was evaluated using female Wistar rats treated with EHSB (2.0g/kg), in accordance with the OECD 423 Guideline. The EHSB showed low toxicity for macrophages in the MTT test (CC50 value: 90.03μg/ml), as well as for erythrocytes, which caused only 2.5% hemolysis at the highest concentration. A strong immunomodulatory activity was observed by a markedly increase of the NO production, phagocytic ability and lysosomal volume. On the other hand, it was not observed deaths or changes in the clinical and behavioral parameters in the toxicological evaluation. This manner, the present study contributes to the knowledge about the immunomodulatory and toxicological properties of the P. insignis. This may provide perspectives for the evaluation and development of effective and safe phytomedicines created from the Brazilian local biodiversity.

Benzophenone derivatives as cysteine protease inhibitors and biological activity against Leishmania(L.) amazonensis amastigotes.

The leishmanicidal potential of benzophenones has been described, some of them highlighting their potential as cysteine protease inhibitors. Therefore, this work described leishmanicidal activity of nine benzophenone derivatives (1a-c;2a-c;3a-c) against intramacrophage amastigote forms of Leishmania(L.)amazonensis (IC50) and the cytotoxic effect on murine peritoneal macrophages (CC50). The derivative 1c exhibited a selectivity index SI (CC50/IC50) of 6.7, besides cytotoxicity lower than Amphotericin B (p< 0.05). Moreover it showed inhibitory activity against papain (42.8±0.3, p<0.05), and when tested on trypanosomatids cysteine proteases 1c also proved to be a potent inhibitor of rCPB2.8, rCPB3.0 and cruzain, showing non-competitive inhibition mechanism by enzymatic assays in vitro.So, benzophenone 1c is interesting drug candidate prototype, with a multi-target directed mode of action, inhibiting rCPB2.8, rCPB3.0 and cruzain.

Immunomodulatory activity of essential oil from Tetrania riparia (Hochstetter) Codd in murine macrophages

AbstractTetradenia riparia is a plant that is used as a traditional medicine in South Africa to treat inflammatory and infectious diseases. The extracts and essential oil of this plant have shown antioxidant, anticarcinogenic, and antimicrobial properties. Some infections that resolve depend on the specific pathogens and host immune response balance, but only a few studies have screened the immunomodulatory effects of T.riparia. Thus, we studied the immunomodulatory effects of T. riparia essential oil (TrEO) on macrophages. Murine peritoneal macrophages were incubated with 30 ng/ml TrEO for 3, 6, and 24 h. Cytokine expression and production were determined by semiquantitative reverse‐transcriptase polymerase chain reaction and flow cytometry, respectively. TrEO affected cytokine synthesis at all time points, whereas mRNA expression was altered only at 3 and 6 h. Interleukin (IL)‐1β, IL‐12, IL‐17, and interferon‐γ expression were highly induced by TrEO at 3 h. Only IL‐1β expression was elevated at 6 h, and its production gradually increased until 24 h. IL‐2 was produced at high levels in the initial response, and granulocyte monocyte colony stimulating factor and IL‐17 were produced at high levels at 24 h. TrEO inhibited IL‐10. These results indicate that TrEO can modulate proinflammatory cytokines and downregulate IL‐10 and IL‐6. This profile is associated with stimulation of the innate cellular immune response and TH2 cell suppression. The essential oil from T. riparia may be an alternative therapy for carcinogenic, autoimmune, and infectious diseases in which cellular responses are critical for their resolution. Copyright © 2015 John Wiley &amp; Sons, Ltd.

Anti-inflammatory effect of desoxo-narchinol-A isolated from Nardostachys jatamansi against lipopolysaccharide

We previously reported that Nardostachys jatamansi (NJ) exhibits anti-inflammatory activity against lipopolysaccharide (LPS). However, the active compound in NJ is unknown. Therefore, here, we examined the effects of desoxo-narchinol-A (DN) isolated from NJ against LPS-induced inflammation. To demonstrate the anti-inflammatory effect of DN against LPS, we used two models; murine endotoxin shock model for in vivo model, and peritoneal macrophage responses for in vitro. In endotoxin shock model, DN was administrated intraperitoneally 1h before LPS challenge, then we evaluated mice survival rates and organ damages. Pretreatment with DN (0.05mg/kg, 0.1mg/kg, or 0.5mg/kg) dramatically reduced mortality in a murine LPS-induced endotoxin shock model. Furthermore, DN inhibited tissue injury and production of pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α), in the liver and lung. In in vitro macrophage model, we examined the inflammatory mediators and regulatory mechanisms such as mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB). DN inhibited the production of inflammatory mediators, such as inducible nitric oxide synthase (iNOS) and its derivative nitric oxide (NO), cyclooxygenase-2 (COX-2), prostaglandin E2 (PGE2), IL-1β, IL-6 and TNF-α and H3 protein acetylation in murine peritoneal macrophages. DN also inhibited p38 activation, but not extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and NF-κB. These results suggest that DN from NJ exhibits protective effects against LPS-induced endotoxin shock and inflammation through p38 deactivation.

Octarphin--Nonopioid Peptide of the Opioid Origin

The data on the properties and mechanism of action of the peptide octarphin (TPLVTLFK, the fragment 12-19 of β-endorphin)--a selective agonist of nonopioid (insensitive to the action of the opioid antagonist naloxone) β-endorphin receptor found on n immune cells (peritoneal macrophages, T and B lymphocytes of spleen and blood), endocrine (adrenal cortex, hypothalamus), cardiovascular (cardiomyocytes) systems are analyzed and systematized. Binding to the receptor octarphin increases increases the mitogen-induced pro- liferation of human and mouse T and B lymphocytes in vitro, activates murine peritoneal macrophages in vitro and in vivo, stimulates growth of human T-lymphoblast cell lines Jurkat and MT-4, inhibits adenylate cyclase activity of rat adrenal cortex membranes and suppresses the secretion of glucocorticoids from the adrenal gland into the blood. It was shown that in a concentration range of 1-1000 nM the peptide increases the activity of inducible NO-synthase (iNOS), and the content of NO and cGMP in lipopolysaccharide-activated murine peritoneal macrophages. Taking into account that NO acts as a primary activator of soluble guanylate cyclase (sGC), it can be assumed that the activating effect of octarphin on macrophages is realized in the following way: increase in th iNOS expression --> increase in the NO production --> increase in the sGC activity --> increase in intracellular levels of cGMP.

α7 Nicotinic Acetylcholine Receptor is a Novel Mediator of Sinomenine Anti-Inflammation Effect in Macrophages Stimulated by Lipopolysaccharide.

Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effects and has been used for rheumatoid arthritis treatment in China. This study aims to verify the hypothesis that SIN acts on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit the activation of macrophages stimulated by lipopolysaccharide. The prototypical α7nAChR antagonist α-bungarotoxin and mecamylamine attenuated the effect of SIN on tumor necrosis factor-α and interleukin-6 in RAW264.7 murine macrophage-like cells and primary peritoneal macrophages of mouse induced by lipopolysaccharide. With the knockdown of α7nAChR expression in RAW264.7 cells by small interfering RNA, the inhibitory effect of SIN on tumor necrosis factor-α and interleukin-6 was reversed. Sinomenine decreased p65 expression in nuclear and increased IκBα expression in cytoplasm, and these effects were reversed by the α7nAChR small interfering RNA as well. These results indicate that the anti-inflammatory effects of SIN on macrophages in vitro depend on α7nAChR.

Pharmacological Inhibition of β3 Integrin Reduces the Inflammatory Toxicities Caused by Oncolytic Adenovirus without Compromising Anticancer Activity

Adenoviruses have been clinically tested as anticancer therapies but their utility has been severely limited by rapid, systemic cytokine release and consequent inflammatory toxicity. Here, we describe a new approach to tackling these dangerous side effects. Using human ovarian cancer cell lines as well as malignant epithelial cells harvested from the ascites of women with ovarian cancer, we show that tumor cells do not produce cytokines in the first 24 hours following in vitro infection with the oncolytic adenovirus dl922-947. In contrast, dl922-947 does induce inflammatory cytokines at early time points following intraperitoneal delivery in mice with human ovarian cancer intraperitoneal xenografts. In these animals, cytokines originate predominantly in murine tissues, especially in macrophage-rich organs such as the spleen. We use a nonreplicating adenovirus to confirm that early cytokine production is independent of adenoviral replication. Using β3 integrin knockout mice injected intraperitoneally with dl922-947 and β3 null murine peritoneal macrophages, we confirm a role for macrophage cell surface β3 integrin in this dl922-947-induced inflammation. We present new evidence that co-administration of a cyclic RGD-mimetic-specific inhibitor of β3 integrin significantly attenuates the cytokine release and inflammatory hepatic toxicity induced by dl922-947 in an intraperitoneal murine model of ovarian cancer. Importantly, we find no evidence that β3 inhibition compromises viral infectivity and oncolysis in vitro or anticancer efficacy in vivo. By enabling safe, systemic delivery of replicating adenoviruses, this novel approach could have a major impact on the future development of these effective anticancer agents.

Corticotropin-Releasing Hormone (CRH) Promotes Macrophage Foam Cell Formation via Reduced Expression of ATP Binding Cassette Transporter-1 (ABCA1).

Atherosclerosis, the major pathology of cardiovascular disease, is caused by multiple factors involving psychological stress. Corticotropin-releasing hormone (CRH), which is released by neurosecretory cells in the hypothalamus, peripheral nerve terminals and epithelial cells, regulates various stress-related responses. Our current study aimed to verify the role of CRH in macrophage foam cell formation, the initial critical stage of atherosclerosis. Our quantitative real-time reverse transcriptase PCR (qRT-PCR), semi-quantitative reverse transcriptase PCR, and Western blot results indicate that CRH down-regulates ATP-binding cassette transporter-1 (ABCA1) and liver X receptor (LXR)-α, a transcription factor for ABCA1, in murine peritoneal macrophages and human monocyte-derived macrophages. Oil-red O (ORO) staining and intracellular cholesterol measurement of macrophages treated with or without oxidized LDL (oxLDL) and with or without CRH (10 nM) in the presence of apolipoprotein A1 (apoA1) revealed that CRH treatment promotes macrophage foam cell formation. The boron-dipyrromethene (BODIPY)-conjugated cholesterol efflux assay showed that CRH treatment reduces macrophage cholesterol efflux. Western blot analysis showed that CRH-induced down-regulation of ABCA1 is dependent on phosphorylation of Akt (Ser473) induced by interaction between CRH and CRH receptor 1(CRHR1). We conclude that activation of this pathway by CRH accelerates macrophage foam cell formation and may promote stress-related atherosclerosis.

A lupane-triterpene isolated from Combretum leprosum Mart. fruit extracts that interferes with the intracellular development of Leishmania (L.) amazonensis in vitro.

Background3beta,6beta,16beta-trihydroxylup-20(29)-ene is a lupane triterpene isolated from Combretum leprosum fruit. The lupane group has been extensively used in studies on anticancer effects; however, its possible activity against protozoa parasites is yet poorly known. The high toxicity of the compounds currently used in leishmaniasis chemotherapy stimulates the investigation of new molecules and drug targets for antileishmanial therapy.MethodsThe activity of 3beta,6beta,16beta-trihydroxylup-20(29)-ene was evaluated against Leishmania (L.) amazonensis by determining the cytotoxicity of the compound on murine peritoneal macrophages, as well as its effects on parasite survival inside host cells. To evaluate the effect of this compound on intracellular amastigotes, cultures of infected macrophages were treated for 24, 48 and 96 h and the percentage of infected macrophages and the number of intracellular parasites was scored using light microscopy.ResultsLupane showed significant activity against the intracellular amastigotes of L. (L.) amazonensis. The treatment with 109 μM for 96 h reduced in 80 % the survival index of parasites in BALB/c peritoneal macrophages. At this concentration, the triterpene caused no cytotoxic effects against mouse peritoneal macrophages. Ultrastructural analyses of L. (L.) amazonensis intracellular amastigotes showed that lupane induced some morphological changes in parasites, such as cytosolic vacuolization, lipid body formation and mitochondrial swelling. Bioinformatic analyses through molecular docking suggest that this lupane has high-affinity binding with DNA topoisomerase.ConclusionTaken together, our results have showed that the lupane triterpene from C. leprosum interferes with L. (L.) amazonensis amastigote replication and survival inside vertebrate host cells and bioinformatics analyses strongly indicate that this molecule may be a potential inhibitor of topoisomerase IB. Moreover, this study opens major prospects for the development of novel chemotherapeutic agents with leishmanicidal activity.

녹차 효소 처리 다당의 화학적 특성 및 면역증진 활성

In order to develop new immuno-stimulating ingredients from mature leaves of green tea, crude polysaccharides were isolated from pectinase digests of tea leaves (green tea enzyme digestion, GTE-0), after which their immuno-stimulating activities and chemical properties were examined. GTE-0 mainly contained neutral sugars (54.9%) such as glucose (14.2%), arabinose (12.2%), rhamnose (11.1%), and galacturonic acid (45.1%), which are characteristic of pectic polysaccharides. The anti-complementary activity of GTE-0 was similar to that of polysaccharide K (used as positive control). Number of morphologically activated macrophages was significantly increased in the GTE-0-treated group. GTE-0 significantly augmented H₂O₂ and reactive oxygen species production by murine peritoneal macrophage cells in a dose-dependent manner, whereas production of nitric oxide showed the highest activity at a dose of 100 μg/mL among all tested concentrations. Murine peritoneal macrophages stimulated with GTE-0 showed enhanced production of various cytokines such as interleukin (IL)-6, IL-12, and tumor necrosis factors-α in a dose-dependent manner. Further, GTE-0 induced higher phagocytic activity in a dose-dependent manner. In ex vivo assay for cytolytic activity of murine peritoneal macrophages, GTE-0-treated group showed significantly higher activity compared to the untreated group at an effector-to-target cell ratio of 20. The above results lead us to conclude that polysaccharides from leaves of green tea have a potent immuno-stimulating effect on murine peritoneal macrophage cells.

Heparin inhibits lipopolysaccharide-induced inflammation via inducing caveolin-1 and activating the p38/mitogen-activated protein kinase pathway in murine peritoneal macrophages.

Heparin is a soluble glycosaminoglycan largely used as an anti-coagulant drug and with well known anti‑inflammatory effects. However, heparin is currently not used as an anti‑inflammatory agent in the clinic due to a risk of bleeding as well as its complex mechanism of action. The underlying mechanism of the anti‑inflammatory action of heparin and its effector targets have remained to be fully elucidated. The present study confirmed the anti‑inflammatory effects of heparin in lipopolysaccharide (LPS)‑induced murine peritoneal macrophages through decreasing the levels of the inflammatory cytokines tumor necrosis factor alpha (TNF‑α), interleukin 6 (IL‑6), IL‑8 and IL‑1β. Caveolin‑1 participated in the anti‑inflammatory process and it was able to be induced by heparin. Transfection of small interfering RNA of caveolin‑1 into murine peritoneal macrophages attenuated the anti‑inflammatory effects of heparin. Furthermore, following caveolin‑1 silencing, the p38/mitogen‑activated protein kinase (MAPK) pathway was still able to be activated by heparin, while the extracellular signal‑regulated kinase and c‑Jun N‑terminal kinase pathways were inhibited. In conclusion, these results suggested that heparin inhibits LPS‑induced inflammation via inducing caveolin‑1 and activating the p38/MAPK pathway in murine peritoneal macrophages. Revealing the anti‑inflammatory mechanisms of heparin will aid in its development for clinical treatment in the future.