Macrophage Cell Cultures Research Articles

Study on effects and mechanism of traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis in rats

To study the effects and immunoregulation mechanism of the traditional Mongolian medicine Wuweifengshi capsule on adjuvant arthritis (AA). Wister rats were divided into several groups: normal group, AA model group, Wuweifengshi capsule groups (with low, moderate, high dose of 0.2, 0.4, 0.8 g x kg(-1) x d(-1) respectively), and Zhonglun-5 group (original dose of 1.68 g x kg(-1) x d(-1)). The edema degree, the level of IL-1beta, TNF-alpha, PGE2, NO and MDA and the activity of SOD in serum were detected. Through cell culture, the effects of the medicine on AA rat's splenic cell's multiplication capacity were studied. The influence of celiac macrophage cell culture fluid of AA rats' on C57BL/6J mice thymic cell multiplication capacity under the medicine was evaluated. Wuweifengshi capsule showed an inhibiting function on the level of IL-1beta, TNF-alpha, PGE2, NO and increased the activity of SOD in serum, but showed no significant influence on MDA. It also inhibited the AA rat's splenic cell's multiplication capacity and the influence of celiac macrophage cell culture fluid of AA rat's on C57BL/6J mice thymic cell multiplication capacity. The anti-AA effect of Wuweifengshi capsule is possibly due to its inhibition of relevant cytokines and its adjustment of corresponding enzyme's activity and immunization organ's cell multiplication capacity.

Aromatic glycosyl disulfide derivatives: Evaluation of their inhibitory activities against Trypanosoma cruzi

Aromatic oligovalent glycosyl disulfides and some diglycosyl disulfides were tested against three different Trypanosoma cruzi strains. Di-(β-d-galactopyranosyl-dithiomethylene) benzenes 2b and 4b proved to be the most active derivatives against all three strains of cell culture-derived trypomastigotes with IC50 values ranging from 4 to 11μM at 37°C. The inhibitory activities were maintained, although somewhat lowered, at a temperature of 4°C as well. Three further derivatives displayed similar activities against at least one of the three strains. Low cytotoxicities of the active compounds, tested on confluent HeLa, Vero and peritoneal macrophage cell cultures, resulted in significantly higher selectivity indices (SI) than that of the reference drug benznidazole. Remarkably, several molecules of the tested panel strongly inhibited the parasite release from T. cruzi infected HeLa cell cultures suggesting an effect against the intracellular development of T. cruzi amastigotes as well.

Differential in Gel Electrophoresis (DIGE) Comparative Proteomic Analysis of Macrophages Cell Cultures in Response to Perthamide C Treatment

Secondary metabolites contained in marine organisms disclose diverse pharmacological activities, due to their intrinsic ability to recognize bio-macromolecules, which alter their expression and modulate their function. Thus, the identification of the cellular pathways affected by marine natural products is crucial to provide important functional information concerning their mechanism of action at the molecular level. Perthamide C, a marine sponge metabolite isolated from the polar extracts of Theonella swinhoei and endowed with a broad and interesting anti-inflammatory profile, was found in a previous study to specifically interact with heat shock protein-90 and glucose regulated protein-94, also disclosing the ability to reduce cisplatin-mediated apoptosis. In this paper, we evaluated the effect of this compound on the whole proteome of murine macrophages cells by two-dimensional DIGE proteomics. Thirty-three spots were found to be altered in expression by at least 1.6-fold and 29 proteins were identified by LC ESI-Q/TOF-MS. These proteins are involved in different processes, such as metabolism, structural stability, protein folding assistance and gene expression. Among them, perthamide C modulates the expression of several chaperones implicated in the folding of proteins correlated to apoptosis, such as Hsp90 and T-complexes, and in this context our data shed more light on the cellular effects and pathways altered by this marine cyclo-peptide.

Effects of aqueous fractions of Uncaria tomentosa (Willd.) D.C. on macrophage modulatory activities

The D10a fraction obtained from the decoction of Uncaria tomentosa was chemically analyzed. An additional aqueous extract (1AERT) from commercial capsules containing powdered cat's claw stem bark was used for comparison purposes. D10a is composed mainly of polyphenols (86%), including the caffeoylquinic derivatives and flavonoid content such as chlorogenic acid, caffeic acid and rutin, quantified by HPLC-PDA.The polyphenolic composition of the 1AERT fraction was 42%. The arabinogalactan-protein contents of the D10a and 1AERT fractions were 0.3% and 24%, respectively. Both fractions were investigated using macrophage cell culture as an in vitro model for immunostimulant screening, which may be a useful approach to evaluate promising candidates for in vivo studies. In vitro macrophage activation by the D10a and 1AERT fractions was evidenced by the typical cell morphology of activated macrophages and by NO production in the case of 1AERT, although D10a did not affect NO production. Both the D10a and 1AERT fractions increased TNF-α and IL-6 levels, in different proportions, and reduced IL-1β levels. Because of the importance of the polyphenolic contents of aqueous extracts of this species, we suggest that the analysis of the composition of these components could be used as a biomarker for their standardization, as well as, for the design of future in vivo studies in the field of pharmacokinetics or nutrikinetic.

Evaluation of Antimicrobial Effects of Euphorbia cyparissias Extracts on Intramacrophages Salmonella typhi

Typhoid fever is a disease caused by Salmonella typhi (S. typhi) and one of the most important enteric infectious diseases in the world. Euphorbia cyparissias extracts showed antidiarrhoeic activity and a flavonoid, quercitrin, was isolated from this plant.This study evaluated on antimicrobial effects of Euphorbia cyparissias extracts against intramarophges S. typhi. Euphorbia cyparissias extracts were prepared in ethanol, aceton and distilled water. Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) for S. typhi were evaluated by broth macrodilution and agar well diffusion methods. Effect of extracts on intramacrophage surviving of S. typhi studied on cell culture of mouse peritoneal macrophages. MIC values respectively 2.96 to 24.5 mg/ml and MBC respectively 5.93 to 49 mg/ml (in this study acetone, methanol, water). In cell culture results indicated that all extracts were not inhibitory effect on macrophages. The results of intramacrophages study showed a significant decrease in the number of colony forming on the test plates comparing with control group. (p value≤0.05). Overall this study indicated that extracts of Eucalyptus cyparissias have antimicrobial effect against intramacrophage S. typhi and acetonic extract is the most effective antimicrobial activity on intramacrophage S. typhi, therefore these extracts can be useful in treatment of typhoid fever.

A comparison of Pattern Recognition Receptor requirement for human and horse strains of Streptococcus pneumoniae

Event Abstract Back to Event A comparison of Pattern Recognition Receptor requirement for human and horse strains of Streptococcus pneumoniae Hannah Williams1*, Clare Bryant1 and Gavin K. Paterson1 1 University of Cambridge, United Kingdom Streptococcus pneumoniae is an important respiratory pathogen of humans and horses. Pneumolysin and autolysin are major virulence factors of S. pneumoniae and damage host tissue through direct effects and/or the ability to induce activation of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs). In the horse functional pneumolysin and autolysin are absent due to a chromosome deletion, yet respiratory disease still occurs in these animals. How then do the horse strains of S. pneumonia cause disease? The response of horse or human TLR4 to pneumolysin was characterised using an in vitro transient transfection model. Pneumolysin is an agonist at human, mouse and horse TLR4. Disruption of pneumolysin binding using TLR4 antagonists revealed that the pneumolysin interaction occurs at the same binding pocket as the TLR4 agonist lipopolysaccharide (LPS). Molecular techniques, including fluorescent single molecule imaging, will be used to further investigate the nature of the interaction. Pneumolysin interaction with the TLR4/LPS binding pocket suggests that it may activate TLR4 in a similar manner to LPS. This study will be expanded to compare how horse, human and mutant pneumolysin- and autolysin strains interaction with PRRs using in vitro macrophage cell culture studies and mouse infection models. Acknowledgements This work is funded by the Wellcome Trust through a fellowship awarded to HE Williams. GK Paterson is funded by a Medical Research Council grant. CE Bryant is a BBSRC Research Development Fellow (BB/H021930/1). Keywords: Inflammation, Toll-Like Receptors, S. pneumoniae, pneumolysin, horse Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Innate immunity Citation: Williams H, Bryant C and Paterson GK (2013). A comparison of Pattern Recognition Receptor requirement for human and horse strains of Streptococcus pneumoniae. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00100 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Miss. Hannah Williams, University of Cambridge, Cambridge, United Kingdom, hew28@cam.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hannah Williams Clare Bryant Gavin K Paterson Google Hannah Williams Clare Bryant Gavin K Paterson Google Scholar Hannah Williams Clare Bryant Gavin K Paterson PubMed Hannah Williams Clare Bryant Gavin K Paterson Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Block of Death-Receptor Apoptosis Protects Mouse Cytomegalovirus from Macrophages and Is a Determinant of Virulence in Immunodeficient Hosts

The inhibition of death-receptor apoptosis is a conserved viral function. The murine cytomegalovirus (MCMV) gene M36 is a sequence and functional homologue of the human cytomegalovirus gene UL36, and it encodes an inhibitor of apoptosis that binds to caspase-8, blocks downstream signaling and thus contributes to viral fitness in macrophages and in vivo. Here we show a direct link between the inability of mutants lacking the M36 gene (ΔM36) to inhibit apoptosis, poor viral growth in macrophage cell cultures and viral in vivo fitness and virulence. ΔM36 grew poorly in RAG1 knockout mice and in RAG/IL-2-receptor common gamma chain double knockout mice (RAGγC−/−), but the depletion of macrophages in either mouse strain rescued the growth of ΔM36 to almost wild-type levels. This was consistent with the observation that activated macrophages were sufficient to impair ΔM36 growth in vitro. Namely, spiking fibroblast cell cultures with activated macrophages had a suppressive effect on ΔM36 growth, which could be reverted by z-VAD-fmk, a chemical apoptosis inhibitor. TNFα from activated macrophages synergized with IFNγ in target cells to inhibit ΔM36 growth. Hence, our data show that poor ΔM36 growth in macrophages does not reflect a defect in tropism, but rather a defect in the suppression of antiviral mediators secreted by macrophages. To the best of our knowledge, this shows for the first time an immune evasion mechanism that protects MCMV selectively from the antiviral activity of macrophages, and thus critically contributes to viral pathogenicity in the immunocompromised host devoid of the adaptive immune system.

Inhibition of In Vitro TNF-α Production by Anisomeles malabarica R.Br. Reinforces Its Anti-Rheumatic and Immunomodulatory Properties

In-vivo studies have suggested the anti-rheumatic and immunomodulatory properties of the methanolic extract of the plant Anisomeles malabarica R.Br. However, the mechanism of action of this plant in the modulation of inflammation using the various in vitro models has not been explored earlier. Hence, the current investigation was undertaken to study the anti-rheumatic and immunomodulatory role of aerial parts, leaves and roots in lipopolysaccharide (LPS) mediated signaling in macrophage and mouse connective tissue cell cultures. It was observed from the present study that by employing tumor necrosis factor-α (TNF-α) bioassay, all the three extracts viz., aerial parts, leaves and roots inhibited TNF-α production in LPS (1 μg/mL) stimulated RAW-32 cells. 38.75 % inhibition of TNF-α was observed at 200 μg extracts of the aerial parts of the plant followed by 17.64 and 14.94 % by the roots and leaves respectively. Taken together, these findings from the present in vitro studies suggest the anti-rheumatic and immunomodulatory properties of the methanolic extracts of A. malabarica.

Effects of nanoparticles in cells infected by Toxoplasma gondii

AbstractCore-shell model drug carriers on 2 nanoscale size levels have been applied in cell culture studies, focused on Toxoplasmosis therapy. In synthesis, a seed of Rhodamin B-labelled polystyrene latex particles was coated by polybutyl cyanoacrylate under physical inclusion of 2 different, new drugs against Toxoplasmosis. Drug-loaded as well as drug-free core-shell model drug carriers were added to a cell culture of human macrophages, infected by Toxoplasma gondii, following an infection plan. Drug release from the carriers had been studied before by enzymatic degradation by means of pork liver esterase. Particle size decrease by degradation was investigated in a UV/VIS spectrometer via transmission measurements. Drug release profiles were obtained by HPLC studies. The dynamics in the population of infected human macrophages, Toxoplasma gondii as well as model drug carrier numbers were registered by a FACS (fluorescence-activated cell sorter). As main result, the drug-free references in the 2 series of core-shell model drug carriers achieved ca.85% of the observed maximum in Toxoplasmosis therapy efficiency. These data were correlated with an immune stimulant effect on the side of the human macrophages, caused by the cell uptake of colloidal substrate, foreign to the body.

Natural compounds as lead therapeutic agents against diabetic eye disease

AbstractDiabetes is a leading cause of new cases of blindness throughout the world. Given the rapid increase in diabetes incidence in recent years, diabetic eye disease will likely become an even bigger problem in the future. There are currently no medical therapies for prevention of early stage ophthalmic complications of diabetes, and treatment for late stage disease is complicated and prohibitively expensive for deployment on a large scale. We have centered our efforts on identifying therapeutic compounds identified as natural products, with a particular focus on plant materials identified over time to be associated with efficacy against diabetes and its complications. The Indian gooseberry (Emblica officinalis), commonly known as Amla, is used in the practice of Indian traditional medicine (Ayurveda) to minimize the effects of diabetes and its complications. We previously showed that treatment of experimentally diabetic rats with crude extracts from Amla fruit delayed the onset and progression of cataracts and prevented the accumulation of sorbitol and diabetes‐induced markers of lipid peroxidation and protein oxidation products in the eye. Because these results were consistent with the effects of aldose reductase (AR) inhibition, we embarked on a bioassay‐guided scheme to search for putative AR inhibitors (ARI), using human AR (AKR1B1) activity as an assay read out. Fractionation of materials in an Amla extract resolved several compounds with ARI activity. Structure elucidation by NMR identified the major inhibitor as 1‐O‐galloyl‐beta‐D‐glucose, also known as beta‐glucogallin (bGG). We and others have shown that AR inhibition by sorbinil and a variety of other validated ARIs suppresses inflammatory markers associated with exposure to lipopolysaccharide (LPS) endotoxins. Our studies show that treatment of LPS‐exposed mice with bGG dose‐dependently suppresses infiltration of inflammatory cells in the anterior and posterior chambers and prevents morphological disruption of retinal layers. Using macrophage cell cultures, we demonstrated that bGG down‐regulates endotoxin‐induced expression of a variety of pro‐inflammatory cytokines including TNFa and IL1b. Similar studies are being carried out with a novel drug lead recently identified in a family of flowering plants commonly cultivated in India and Malaysia (Piperaceae). Overall, our studies suggest that natural products may be a rich source of lead compounds useful for development as therapeutic agents against diabetic complications.

Effect of exogenous erythritol on growth and survival of Brucella

Erythritol has been considered as an important factor for the pathogenesis of Brucella abortus 2308 and its ability to cause abortion in ruminants. There is a lack of laboratory models to study the Brucella–erythritol relationship, as commonly used murine models do not have erythritol. We tested the effect of exogenous erythritol on the growth of Brucella in iron minimal medium (IMM), in infected macrophage culture and in infected mice to determine if these models can be used to study the relationship between Brucella and erythritol. An effect of erythritol on Brucella growth was only seen in IMM. There appear to be no effect of erythritol on Brucella growth in macrophage cell cultures or in mice. This shows that administration of erythritol to the mice or macrophages cannot mimic the environment in ruminants during pregnancy and thus cannot be used as models to understand the effect of erythritol on Brucella pathogenesis.

Chemical and biological properties of a highly branched β-glucan from edible mushroom Pleurotus sajor-caju

Hot aqueous extraction of the basidiocarps of the mushroom Pleurotus sajor-caju provided a cold water-soluble, gel-like glucan, which was characterized chemically, and its effects on RAW 264.7 cell line (mouse leukaemic monocyte macrophage) activation were determined. NMR spectroscopy, HPSEC, methylation analysis, and a controlled Smith degradation showed it to have a branched structure with a (1→3)-linked β-Glcp main-chain, substituted at O-6 by single-unit β-Glcp side-chains, on the average of two to every third residues of the backbone, with a molar mass of 9.75×105gmol−1. In macrophage cell culture, the β-glucan induced production of NO and the cytokines TNF-α, IL-1β, these effects being very similar as those of Escherichia coli serotype 0111:B4 Sigma–Aldrich lipopolysaccharide (LPS), although not modifying the response of LPS-activated macrophages. The results suggest that the (1→3), (1→6)-linked β-glucan from P. sajor-caju may have potential for immunological activities, although additional experiments are necessary for a better understanding of the mechanisms involved.

Inflammatory and bone regulators expression in murine macrophages under exposure of commercial and experimental mineral trioxide aggregate

Mineral trioxide aggregate (MTA) has been used in a variety of surgical and non-surgical endodontic applications. The aim of this study was to evaluate the gene expression and protein production of TNF-α, IL-1β and IL-6, as well as the gene expression of RANKL and OPG using both commercial and experimental MTA in macrophage cell cultures. Peritoneal macrophage cell culture was performed. Viability, gene expression of cytokines, RANKL and OPG, and protein levels in experimental- and commercial-grey MTA co-cultured with peritoneal macrophages was determined by tryptan blue, real time PCR and ELISA. The expression of TNF-α for both commercial and experimental MTA was higher, while the expression of IL-1β and IL-6 was similar when compared to the negative control. At protein expression level, no differences were observed between the negative control and cements. RANKL did not show a significant improvement in gene expression when compared with the negative control, but OPG expression in cement samples was higher when compared to the negative control. This study suggests that commercial and experimental MTA promotes anti-inflammatory processes, as well as bone healing capacity.

Macrophage Infection by Dimorphic Fungi

Mammalian infection by dimorphic fungi occurs through the inhalation of asexual spores (conidia), which are phagocytosed by host pulmonary alveolar macrophages of the innate immune system. Once phagocytosed, fungal conidia germinate into the pathogenic cell type; unicellular yeast cells which divide by fission (Vanittanakom et al., 2006; Boyce et al., 2011). To investigate if mutation of a particular fungal gene affects macrophage phagocytosis or the production of yeast cells, a murine macrophage cell culture assay can be utilized. This protocol was developed for Penicillium marneffei but is applicable to most dimorphic fungi., 两形态真菌的哺乳动物感染通过吸入无性孢子（分生孢子）发生，所述无性孢子被先天免疫系统的宿主肺泡巨噬细胞吞噬。 一旦被吞噬，真菌分生孢子萌发成致病细胞类型; 通过裂变分裂的单细胞酵母细胞（Vanittanakom et al。，2006; Boyce et al。，2011）。 为了研究特定真菌基因的突变是否影响巨噬细胞吞噬作用或酵母细胞的产生，可以利用鼠巨噬细胞培养测定法。 该方案是为马尼埃青霉开发的，但适用于大多数二形真菌。

Use of near-infrared luminescent gold nanoclusters for detection of macrophages

We determined the effect of aggregation and coating thickness of gold on the luminescence of nanoparticles engulfed by macrophages and in gelatin phantoms. Thin gold-coated iron oxide nanoclusters (nanoroses) have been developed to target macrophages to provide contrast enhancement for near-infrared optical imaging applications. We compare the brightness of nanoroses luminescent emissions in response to 635 nm laser excitation to other nanoparticles including nanoshells, nanorods, and Cy5 conjugated iron oxide nanoparticles. Luminescent properties of all these nanoparticles were investigated in monomeric and aggregated form in gelatin phantoms and primary macrophage cell cultures using confocal microscopy. Aggregation of the gold nanoparticles increased luminescence emission and correlated with increased surface mass of gold per nanoparticle (nanoshells 37 ± 14.30 × 10(-3) brightness with 1.23 × 10(-4) wt of gold (g)/nanoparticle versus original nanorose 1.45 ± 0.37 × 10(-3) with 2.10 × 10(-16) wt of gold/nanoparticle, p<0.05). Nanoshells showed greater luminescent intensity than original nanoroses or Cy5 conjugated iron oxide nanoparticles when compared as nanoparticles per macrophage (38 ± 10 versus 11 ± 2.8 versus 17 ± 6.5, p<0.05, respectively, ANOVA), but showed relatively poor macrophage uptake (1025 ± 128 versus 7549 ± 236 versus 96,000 nanoparticles/cell, p<0.05, student t-test nanoshells versus nanoroses). Enhancement of gold fluorescent emissions by nanoparticles can be achieved by reducing the thickness of the gold coating, by clustering the gold on the surface of the nanoparticles (nanoshells), and by clustering the gold nanoparticles themselves.

The effects on immune response of levamisole treatment following infection of U-937 macrophages with Candida albicans

The aim of this study was to investigate the effects on the immune response of levamisole alone and in conjunction with Candida albicans stimulation in human macrophage cell culture by determining the alterations in the levels of cytokine release. Levamisole treatment was performed before, during and after infecting U-937 human macrophage cells with C. albicans. In cell supernatants, interleukin (IL)-1b, IL-12, IL-18, tumour necrosis factor alpha (TNF-α) levels were measured by ELISA. In vitro levamisole treatment accompanied by C. albicans stimulation significantly increased IL-12, IL-1β and IL-18 production in macrophage cells (p < 0.05). It was observed that when administered before C. albicans infection, levamisole significantly increased IL-12 and IL-1β production in macrophage cells (p < 0.05). Another finding was that when applied to macrophage cells simultaneously with C. albicans infection, or before infection with C. albicans, levamisole suppressed the TNF-β production stimulating effect of C. albicans (p < 0.05). These results indicated that levamisole could be useful in treating patients infected with C. albicans or in protecting individuals under the risk of being infected with this pathogen. There is a need for further experimental and clinical studies on this hypothesis.

Abstract PR-7: Antitumor activity of the immunomodulatory peptide SCV-07 correlates with changes in macrophage STAT3 signal transduction and cytokine secretion.

Abstract The synthetic dipeptide gamma-D-glutamyl-L-tryptophan (SCV-07) is able to enhance immune responses in preclinical anti-viral and vaccine models and is currently under clinical evaluation for attenuation of radiation-induced oral mucositis in HNSCC patients. We recently demonstrated that SCV-07 also has anti-tumor efficacy in various syngeneic and nude mouse xenograft models despite lack of direct inhibition of the tumor cell growth in standard proliferation assays. Based on these data, along with observations that SCV-07 enhances a Th1-type immune response in vivo and binds with high affinity to cells and membranes from mouse thymocytes and macrophages, we sought to explore the hypothesis that SCV-07 has anti-tumor activity through modulation of immune cells in the tumor microenvironment. Towards this end we evaluated effects of SCV-07 on signal transduction and biological properties of T cells and macrophages in cell culture and in mouse tumor models. Using phosphoprotein detection arrays broad changes in global phosphorylation patterns were observed upon SCV-07 treatment of Jurkat T cells including STATs 3, 5 and 6, SHP2, several Src family kinases and various cytoplasmic and membrane proteins. We subsequently showed that SCV-07 leads to inhibition of cytokine-induced but not basal STAT3 tyrosine phosphorylation (PY) in Jurkat T cells and this required tyrosine phosphatase activity. New data using phosphospecific antibodies in western blots of Jurkat cells demonstrate that SCV-07 mediates an increase in basal and cytokine-induced phosphorylation of SHP2, a tyrosine phosphatase known to dephosphorylate STAT3. The phosphorylated residue was identified as Tyr542, known to activate SHP2. New findings also demonstrate that SCV-07 inhibits IL-6 stimulated STAT3 PY and induces SHP2 Tyr542 phosphorylation in primary mouse peritoneal macrophages treated in culture, similar to the findings with cell lines. Further evaluation of human PBMC showed that SCV-07 inhibited IL-6, IL-10 and IFNα induced STAT3 PY in monocytes from 2 of 3 PBMC donors. In a syngeneic B16 melanoma model we initially determined that SCV-07 had anti-tumor efficacy and this was accompanied by a decrease in serum CCL2, a cytokine produced by and chemotactic for monocytes/macrophages and which has its expression regulated by STAT3. New results show that SCV-07 can directly inhibit basal and cytokine induced CCL2 secretion from purified primary mouse peritoneal macrophages. Experiments are underway to evaluate the activation state and distribution of tumor infiltrated macrophages and T cells in B16 melanoma xenografts undergoing efficacy response to SCV-07 treatment and these findings will be presented. Taken together our data support a model whereby SCV-07 could mediate an anti-tumor effect by reducing both chemotaxis of tumor-growth promoting macrophages and secretion of tumor growth promoting cytokines in the tumor microenvironment, and also promote a shift from Th2 to Th1 profile of local T cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr PR-7.

A New Piece of the Shigella Pathogenicity Puzzle: Spermidine Accumulationby Silencing of the speG Gene

The genome of Shigella, a gram negative bacterium which is the causative agent of bacillary dysentery, shares strong homologies with that of its commensal ancestor, Escherichia coli. The acquisition, by lateral gene transfer, of a large plasmid carrying virulence determinants has been a crucial event in the evolution towards the pathogenic lifestyle and has been paralleled by the occurrence of mutations affecting genes, which negatively interfere with the expression of virulence factors. In this context, we have analysed to what extent the presence of the plasmid-encoded virF gene, the major activator of the Shigella regulon for invasive phenotype, has modified the transcriptional profile of E. coli. Combining results from transcriptome assays and comparative genome analyses we show that in E. coli VirF, besides being able to up-regulate several chromosomal genes, which potentially influence bacterial fitness within the host, also activates genes which have been lost by Shigella. We have focused our attention on the speG gene, which encodes spermidine acetyltransferase, an enzyme catalysing the conversion of spermidine into the physiologically inert acetylspermidine, since recent evidence stresses the involvement of polyamines in microbial pathogenesis. Through identification of diverse mutations, which prevent expression of a functional SpeG protein, we show that the speG gene has been silenced by convergent evolution and that its inactivation causes the marked increase of intracellular spermidine in all Shigella spp. This enhances the survival of Shigella under oxidative stress and allows it to better face the adverse conditions it encounters inside macrophage. This is supported by the outcome of infection assays performed in mouse peritoneal macrophages and of a competitive-infection assay on J774 macrophage cell culture. Our observations fully support the pathoadaptive nature of speG inactivation in Shigella and reveal that the accumulation of spermidine is a key determinant in the pathogenicity strategy adopted by this microrganism.

In vitro and in vivo evaluation of the inflammatory response to nanoscale grooved substrates

The immune response to an implanted biomaterial is orchestrated by macrophages. In this study various nanogrooved patterns were created by using laser interference lithography and reactive ion etching. The created nanogrooves mimic the natural extracellular matrix environment. Macrophage cell culture demonstrated that interleukin 1β and TNF-α cytokine production were upregulated on nanogrooved substrates. In vivo subcutaneous implantation in a validated mouse cage model for 14 days demonstrated that nanogrooves enhanced and guided cell adhesion, and few multinucleated cells were formed. In agreement with the in vitro results, cytokine production was found to be nanogroove dependent, as interleukin 1β, TNF-α, TGF-β and osteopontin became upregulated. The results indicate that biomaterial surface texturing, especially at the nanometric scale, can be used to control macrophage activation to induce a wound healing response, rather than a profound inflammatory response. From the Clinical EditorThe authors investigate various nano-grooved patterns that mimic the natural extracellular matrix environment and demonstrate (both in macrophage cultures and in vivo) that interleukin 1β and TNF-α cytokine production is dependent upon surface texturing at the nanometric scale. They propose that modified surfaces may trigger macrophage activation to promote a wound healing response.

Adipokine resistin predicts anti-inflammatory effect of glucocorticoids in asthma

BackgroundAdipokines are protein mediators secreted by adipose tissue. Recently, adipokines have also been involved in the regulation of inflammation and allergic responses, and suggested to affect the risk of asthma especially in obese female patients. We assessed if adipokines predict responsiveness to glucocorticoids and if plasma adipokine levels are associated with lung function or inflammatory activity also in non-obese (body mass index (BMI) ≤ 30 kg/m2) women with newly-diagnosed steroid-naïve asthma.MethodsLung function, exhaled NO, plasma levels of adipokines leptin, resistin, adiponectin and adipsin, and inflammatory markers were measured in 35 steroid-naïve female asthmatics and in healthy controls. The measurements were repeated in a subgroup of asthmatics after 8 weeks of treatment with inhaled fluticasone. Adipokine concentrations in plasma were adjusted for BMI.ResultsHigh baseline resistin concentrations were associated with a more pronounced decrease in serum levels of eosinophil cationic protein (ECP) (r = -0.745, p = 0.013), eosinophil protein X (EPX) (r = -0.733, p = 0.016) and myeloperoxidase (MPO) (r = -0.721, p = 0.019) during fluticasone treatment. In asthmatics, leptin correlated positively with asthma symptom score and negatively with lung function. However, no significant differences in plasma adipokine levels between non-obese asthmatics and healthy controls were found. The effects of resistin were also investigated in human macrophages in cell culture. Interestingly, resistin increased the production of proinflammatory factors IL-6 and TNF-α and that was inhibited by fluticasone.ConclusionsHigh resistin levels predicted favourable anti-inflammatory effect of inhaled glucocorticoids suggesting that resistin may be a marker of steroid-sensitive phenotype in asthma. High leptin levels were associated with a more severe disease suggesting that the link between leptin and asthma is not restricted to obesity.