Murine Macrophage Cell Line J774A Research Articles

Quantification of Macrophage Cellular Ferrous Iron (Fe2+) Content Using a Highly Specific Fluorescent Probe in a Plate Reader.

Macrophages are at the center of innate immunity and iron metabolism. In the case of an infection, macrophages adapt their cellular iron metabolism to deprive iron from invading bacteria to combat intracellular bacterial proliferation. A concise evaluation of the cellular iron content upon an infection with bacterial pathogens and diverse cellular stimuli is necessary to identify underlying mechanisms concerning iron homeostasis in macrophages. For the characterization of cellular iron levels during infection, we established an in vitro infection model where the murine macrophage cell line J774A.1 is infected with Salmonella enterica serovar Typhimurium (S.tm), the mouse counterpart to S. enterica serovar Typhi, under normal and iron-overload conditions using ferric chloride (FeCl3) treatment. To evaluate the effect of infection and iron stimulation on cellular iron levels, the macrophages are stained with FerroOrange. This fluorescent probe specifically detects Fe2+ ions and its fluorescence can be quantified photometrically in a plate reader. Importantly, FerroOrange fluorescence does not increase with chelated iron or other bivalent metal ions. In this protocol, we present a simple and reliable method to quantify cellular Fe2+ levels in cultured macrophages by applying a highly specific fluorescence probe (FerroOrange) in a TECAN Spark microplate reader. Compared to already established techniques, our protocol allows assessing cellular iron levels in innate immune cells without the use of radioactive iron isotopes or extensive sample preparation, exposing the cells to stress. Key features • Easy quantification of Fe2+ in cultured macrophages with a fluorescent probe. • Analysis of iron in living cells without the need for fixation. • Performed on a plate reader capable of 540 nm excitation and 585 nm emission by trained employees for handling biosafety level 2 bacteria.

Repeated Exposure of Macrophages to Synthetic Amorphous Silica Induces Adaptive Proteome Changes and a Moderate Cell Activation.

Synthetic amorphous silica (SAS) is a nanomaterial used in a wide variety of applications, including the use as a food additive. Two types of SAS are commonly employed as a powder additive, precipitated silica and fumed silica. Numerous studies have investigated the effects of synthetic amorphous silica on mammalian cells. However, most of them have used an exposure scheme based on a single dose of SAS. In this study, we have used instead a repeated 10-day exposure scheme in an effort to better simulate the occupational exposure encountered in daily life by consumers and workers. As a biological model, we have used the murine macrophage cell line J774A.1, as macrophages are very important innate immune cells in the response to particulate materials. In order to obtain a better appraisal of the macrophage responses to this repeated exposure to SAS, we have used proteomics as a wide-scale approach. Furthermore, some of the biological pathways detected as modulated by the exposure to SAS by the proteomic experiments have been validated through targeted experiments. Overall, proteomics showed that precipitated SAS induced a more important macrophage response than fumed SAS at equal dose. Nevertheless, validation experiments showed that most of the responses detected by proteomics are indeed adaptive, as the cellular homeostasis appeared to be maintained at the end of the exposure. For example, the intracellular glutathione levels or the mitochondrial transmembrane potential at the end of the 10 days exposure were similar for SAS-exposed cells and for unexposed cells. Similarly, no gross lysosomal damage was observed after repeated exposure to SAS. Nevertheless, important functions of macrophages such as phagocytosis, TNFα, and interleukin-6 secretion were up-modulated after exposure, as was the expression of important membrane proteins such as the scavenger receptors, MHC-II, or the MAC-1 receptor. These results suggest that repeated exposure to low doses of SAS slightly modulates the immune functions of macrophages, which may alter the homeostasis of the immune system.

A Low-Serum Culture System for Prolonged in Vitro Toxicology Experiments on a Macrophage System.

Immunotoxicology sensu lato comprises not only toxicity toward immune cells, but also biological reactions from immune cells exposed to toxicants, reactions that may have deleterious effects at the organismal level. Within this wide frame, a specific case of interest is represented by the response of macrophages to particulate materials, with the epitome examples of asbestos and crystalline silica. For such toxicants that are both persistent and often encountered in an occupational setting, i.e. at low but repeated doses, there is a need for in vitro systems that can take into account these two parameters. Currently, most in vitro systems are used in an acute exposure mode, i.e., with a single dose and a readout made shortly if not immediately after exposure. We describe here how adequate changes of the culture methods applied to the murine macrophage cell line J774A.1 enable longer periods of culture (several days), which represents a first opportunity to address the persistence and dose-rate issues. To respond to this, the protocol uses a reduction in the concentration of the animal serum used for cell culture, as well as a switch from fetal to adult serum, which is less rich in proliferation factors. By doing so, we have considerably reduced cell proliferation, which is a problem with cell lines when they are supposed to represent slowly-dividing cells such as resident macrophages. We also succeeded in maintaining the differentiated functions of macrophages, such as phagocytosis or inflammatory responses, over the whole culture period. Furthermore, the presence of serum, even at low concentrations, provides excellent cell viability and keeps the presence of a protein corona on particulate materials, a feature that is known to strongly modulate their effects on cells and is lost in serum-free culture. Besides data showing the impact of these conditions on macrophages cell line cultures, illustrative examples are shown on silica- and cobalt-based pigments.

Mucoadhesive silver nanoparticle-based local drug delivery system for peri-implantitis management in COVID-19 era. Part 1: antimicrobial and safety in-vitro analysis

BackgroundPseudomonas aeruginosa, a major respiratory pathogen, has been isolated from peri-implant sites and is associated with dental implant failure. This in-vitro study (part 1) aimed to fabricate a novel mucoadhesive silver nanoparticle-based local drug delivery chip, evaluate its antimicrobial efficacy against P. aeruginosa, and its safety for the treatment of peri-implantitis. Materials and methodsSilver nanoparticles were synthesized and characterized using a transmission electron microscope (TEM). The local drug delivery chip was fabricated using gelatin, glycerol, silver nanoparticle solution (2.5 μg/ml, 5 μg/ml, 7.5 μg/ml, and 10 μg/ml), glutaraldehyde, and sodium alginate solution. These chips were evaluated for physical parameters, effect on viability of murine macrophage cell line J774A.1, and antimicrobial activity (using Kirby-Bauer disc diffusion method with 18 h incubation period) against P. aeruginosa ATCC 27853. ResultsSilver nanoparticle antimicrobial chip exhibited dimensions of 4 mm × 5 mm x 0.4 mm, 5.8 mg weight, pH 5–6, folding endurance 1.04, and one-year stability. P. aeruginosa was susceptible to ≥ 7.5 μg/ml concentration of silver nanoparticles (spherical shape with particle size ranging from 10 to 100 nm). Murine macrophage cells exhibited 93% viability after 24 h incubation with silver nanoparticle chips. ConclusionThe novel silver nanoparticle chip showed dimensional stability, minimal effect on murine macrophage cell viability, and significant antimicrobial activity against P. aeruginosa. With the further establishment of its effective dosage and safety, this chip could be used as an adjunct to mechanical debridement (as a non-aerosol generating procedure) in treating peri-implantitis, especially during the ongoing coronavirus disease 2019 (COVID-19) pandemic.

Extract of Solidago chilensis Meyen inflorescences: cytotoxicity and inhibitory activity on nitric oxide synthesis in activated macrophage cell line J774A.1

Solidago chilensis Meyen (= Solidago microglossa) popularly known as “Brazilian arnica” is used to treat of inflammatory disorders. S. chilensis is constant in the Therapeutic Memento of the Rio de Janeiro city and belongs to the medicinal species of Brazilian National List of Medicinal Plants of Interest of the Unified National Health System (SUS). There are no studies in the literature showing the direct activity of this plant species on immune system cells. The present study evaluated the chemical composition as well as the cytotoxic and pharmacological activity of the ether-ethanol extract from S. chilensis inflorescences (SCIE) in murine macrophage cell line J774A.1. The results showed that higher concentrations (50 to 200 µg/mL) of SCIE had significant cytotoxicity on J774A.1 cells, however, lower concentrations (from 10 to 0.1 µg/mL) did not produce significant cytotoxic effects and exhibited an inhibitory effect on nitric oxide production in LPS-stimulated J774A.1 cell line. The chemical analysis by HPLC-UV-PDA indicated that the SCIE contains flavonoid derived from quercetin and kaempferol; and diterpenes, probably labdanes. These findings complement data in the literature regarding the activity of this plant species on an important cell from the immune system involved in the innate and acquired immune response, the macrophages.

Antiprotozoal activities of marine polyether triterpenoids

Chagas disease and leishmaniasis are tropical neglected diseases caused by kinetoplastids protozoan parasites of Trypanosoma and Leishmania genera, and a public health burden with high morbidity and mortality rates in developing countries. Among difficulties with their epidemiological control, a major problem is their limited and toxic treatments to attend the affected populations; therefore, new therapies are needed in order to find new active molecules. In this work, sixteen Laurencia oxasqualenoid metabolites, natural compounds 1–11 and semisynthetic derivatives 12–16, were tested against Leishmania amazonensis, Leishmania donovani and Trypanosoma cruzi. The results obtained point out that eight substances possess potent activities, with IC50 values in the range of 5.40–46.45 µM. The antikinetoplastid action mode of the main metabolite dehydrothyrsiferol (1) was developed, also supported by AFM images.The semi-synthetic active compound 28-iodosaiyacenol B (15) showed an IC50 5.40 µM against Leishmania amazonensis, turned to be non-toxic against the murine macrophage cell line J774A.1 (CC50 > 100). These values are comparable with the reference compound miltefosine IC50 6.48 ± 0.24 and CC50 72.19 ± 3.06 μM, suggesting that this substance could be scaffold for development of new antikinetoplastid drugs.

Bavachin attenuates LPS-induced inflammatory response and inhibits the activation of NLRP3 inflammasome in macrophages

BackgroundBavachin is a natural product isolated from Psoralea corylifolia L. that has been applied as a traditional medicine in Asian countries. However, the anti-inflammatory effects of bavachin on LPS-induced inflammation and NLRP3 inflammasome activation by macrophages remain unclear. PurposeWe investigated the anti-inflammatory effects of bavachin on LPS-activated murine macrophage cell line J774A.1 cells and murine peritoneal macrophages. MethodsJ774A.1 cells and murine peritoneal macrophages were pre-treated with bavachin following LPS treatment. The concentrations of NO, PGE2, IL-6 and IL-12p40 in cell culture supernatant were analyzed. The expressions of iNOS, COX-2, mPGES-1 and MAPKs were analyzed using Western blotting, while NF-κB activity was detected using promoter reporter assay. To examine the activation of NLRP3 inflammasome, J774A.1 cells were incubated with LPS, and then treated with bavachin following treatment with ATP. The concentration of IL-1β in the cell culture supernatant was measured. The expressions of NLRP3, ASC, caspase-1 and IL-1β were analyzed using Western blotting. The formation of inflammasome complex was observed by immunofluorescence microscopy. ResultsBavachin suppressed LPS-induced NO and PGE2 production, and decreased iNOS and mPGES-1 expression. Bavachin also reduced LPS-induced IL-6 and IL-12p40 production and decreased the activation of MAPKs and NF-κB. Additionally, bavachin suppressed NLRP3 inflammasome-derived IL-1β secretion, decreased caspase-1 activation, repressed mature IL-1β expression, and inhibited inflammasome complex formation. Furthermore, bavachin also suppressed the production of NO, IL-6 and IL-12p40 by LPS-stimulated murine peritoneal macrophages. ConclusionOur experimental results indicated anti-inflammatory effects of bavachin exhibit attenuation of LPS-induced inflammation and inhibit activation of NLRP3 inflammasome in macrophages. These results suggest that bavachin might have potential in treating inflammatory and autoimmune diseases.

Structural and functional characterization of the transcriptional regulator Rv3488 of Mycobacterium tuberculosis H37Rv.

Rv3488 of Mycobacterium tuberculosis H37Rv has been assigned to the phenolic acid decarboxylase repressor (PadR) family of transcriptional regulators that play key roles in multidrug resistance and virulence of prokaryotes. The binding of cadmium, zinc, and several other metals to Rv3488 was discovered and characterized by isothermal titration calorimetery to be an exothermic process. Crystal structures of apo-Rv3488 and Rv3488 in complex with cadmium or zinc ions were determined by X-ray crystallography. The structure of Rv3488 revealed a dimeric protein with N-terminal winged-helix-turn-helix DNA-binding domains composed of helices α1, α2, α3, and strands β1 and β2, with the dimerization interface being formed of helices α4 and α1. The overall fold of Rv3488 was similar to PadR-s2 and metal sensor transcriptional regulators. In the crystal structure of Rv3488-Cd complex, two octahedrally coordinated Cd2+ ions were present, one for each subunit. The same sites were occupied by zinc ions in the structure of Rv3488-Zn, with two additional zinc ions complexed in one monomer. EMSA studies showed specific binding of Rv3488 with its own 30-bp promoter DNA. The functional role of Rv3488 was characterized by expressing the rv3488 gene under the control of hsp60 promoter in Mycobacterium smegmatis Expression of Rv3488 increased the intracellular survival of recombinant M. smegmatis in murine macrophage cell line J774A.1 and also augmented its tolerance to Cd2+ ions. Overall, the studies show that Rv3488 may have transcription regulation and metal-detoxifying functions and its expression in M. smegmatis increases intracellular survival, perhaps by counteracting toxic metal stress.

Rapid Differentiation of Host and Parasitic Exosome Vesicles Using Microfluidic Photonic Crystal Biosensor.

Parasite extracellular vesicles (EVs) are potential biomarkers that could be exploited for the diagnosis of infectious disease. This paper reports a rapid bioassay to discriminate parasite and host EVs. The EV detection assay utilizes a label-free photonic crystal (PC) biosensor to detect the EVs using a host-specific transmembrane protein (CD63), which is present on EV secreted by host cells (modeled by murine macrophage cell line J774A.1) but is not expressed on EV secreted by parasitic nematodes such as the gastrointestinal nematode Ascaris suum. The surface of PC is functionalized to recognize CD63, and is sensitive to the changes in refractive index caused by the immobilization of EVs. The biosensor demonstrates a detection limit of 2.18 × 109 EVs/mL and a capability to characterize the affinity constants of antibody-host EV bindings. The discrimination of murine host EVs from parasite EVs indicates the capability of the sensor to differentiate EVs from different origins. The label-free, rapid EV assay could be used to detection parasite infection and facilitate the exosome-based clinic diagnosis and exosome research.

Potential of surface-eroding poly(ethylene carbonate) for drug delivery to macrophages

Films composed of poly(ethylene carbonate) (PEC), a biodegradable polymer, were compared with poly(lactide-co-glycolide) (PLGA) films loaded with and without the tuberculosis drug rifampicin to study the characteristics and performance of PEC as a potential carrier for controlled drug delivery to macrophages.All drug-loaded PLGA and PEC films were amorphous indicating good miscibility of the drug in the polymers, even at high drug loading (up to 50wt.%). Polymer degradation studies showed that PLGA degraded slowly via bulk erosion while PEC degraded more rapidly and near-linearly via enzyme mediated surface erosion (by cholesterol esterase). Drug release studies performed with polymer films indicated a diffusion/erosion dependent delivery behavior for PLGA while an almost zero-order drug release profile was observed from PEC due to the controlled polymer degradation process. When exposed to polymer degradation products the murine macrophage cell line J774A.1 showed less susceptibility to PEC than to PLGA. However, when seeding the macrophages on PLGA and PEC films no relevant difference in cell proliferation/growth kinetics was observed.Overall, this study emphasizes that PEC is an attractive polymer for controlled drug release and could provide superior performance to PLGA for some drug delivery applications including the treatment of macrophage infections.

Intracellular translocation and localization of Edwardsiella tarda type III secretion system effector EseG in host cells

Edwardsiella tarda, an important fish pathogenic bacterium, could utilize type III secretion system (T3SS) to transfer multiple effector proteins into host cells during infection. EseG was identified to be an E. tarda T3SS effector, which could be injected by T3SS into non-phagocytic cells. Since E. tarda is a facultative intracellular pathogen that resides and replicates in macrophage, it is interesting to expand our knowledge about EseG translocation and localization within phagocytic cells. Here utilizing murine macrophage cell line J774A.1 as the cell model, we demonstrated that EseG could be transported into J774A.1 via T3SS only after E. tarda was internalized into macrophage cells, indicating that extracellular E. tarda could not inject EseG into host cells. Subcellular fractionation analysis gave the evidence that EseG was specifically localized in the membrane fraction of infected host cells. Furthermore, immunofluorescence detection indicated that EseG specifically targeted the E. tarda-containing vacuoles (ECVs) within macrophage cells. Finally the unique features for EseG were also confirmed in non-phagocytic cells. In summarize, this work illuminates internalization-depending translocation and ECV-targeting localization of E. tarda T3SS effector in both non-phagocytic and phagocytic cells, which might be important to interpret the interaction of EseG with host cells upon infection.

Mycobacterium marinum mmar_2318 and mmar_2319 are Responsible for Lipooligosaccharide Biosynthesis and Virulence Toward Dictyostelium.

Resistance to phagocyte killing is an important virulence factor in mycobacteria. Dictyostelium has been used to study the interaction between phagocytes and bacteria, given its similarity to the mammalian macrophage. Here, we investigated the genes responsible for virulence to Dictyostelium by screening 1728 transposon mutants of the Mycobacterium marinum NTUH-M6094 strain. A total of 30 mutants that permissive for Dictyostelium growth were identified. These mutants revealed interruptions in 20 distinct loci. Of the 20 loci, six genes (losA, mmar_2318, mmar_2319, wecE, mmar_2323 and mmar_2353) were located in the lipooligosaccharide (LOS) synthesis cluster. LOS are antigenic glycolipids and the core LOS structure from LOS-I to LOS-IV have been reported to exist in M. marinum. Two-dimensional thin-layer chromatography (2D-TLC) glycolipid profiles revealed that deletion of mmar_2318 or mmar_2319 resulted in the accumulation of LOS-III and deficiency of LOS-IV. Deletion and complementation of mmar_2318 or mmar_2319 confirmed that these genes both contributed to virulence toward Dictyostelium but not entry and replication inside Dictyostelium. Co-incubation with a murine macrophage cell line J774a.1 or PMA-induced human monocytic cell line THP-1 demonstrated that mmar_2318 or mmar_2319 deletion mutant could grow in macrophages, and their initial entry rate was not affected in J774a.1 but significantly increased in THP-1. In conclusion, although mmar_2319 has been reported to involve LOS biosynthesis in a previous study, we identified a new gene, mmar_2318 that is also involved in the biosynthesis of LOS. Deletion of mmar_2318 or mmar_2319 both exhibits reduction of virulence toward Dictyostelium and increased entry into THP-1 cells.

Morphology and its underlying genetic regulation impact the interaction between Cryptococcus neoformans and its hosts.

Cryptococcus neoformans is a fungus that causes the majority of fatal cryptococcal meningitis cases worldwide. This pathogen is capable of assuming different morphotypes: yeast, pseudohypha, and hypha. The yeast form is the most common cell type observed clinically. The hyphal and pseudohyphal forms are rarely observed in the clinical setting and are considered attenuated in virulence. However, as a ubiquitous environmental pathogen, Cryptococcus interacts with various organisms, and it is known to be parasitic to different hosts. Capitalizing on recent discoveries, morphogenesis regulators were manipulated to examine the impact of cell shape on the cryptococcal interaction with three different host systems: the soil amoeba Acanthamoeba castellanii (a protist), the greater wax moth Galleria mellonella (an insect), and the murine macrophage cell line J774A.1 (mammalian cells). The regulation of Ace2 and morphogenesis (RAM) pathway is a highly conserved pathway among eukaryotes that regulates cytokinesis. Disruption of any of five RAM components in Cryptococcus renders cells constitutively in the pseudohyphal form. The transcription factor Znf2 is the master activator of the yeast to hyphal transition. Deletion of ZNF2 locks cells in the yeast form, while overexpression of this regulator drives hyphal growth. Genetic epistasis analyses indicate that the RAM and the Znf2 pathways regulate distinct aspects of cryptococcal morphogenesis and independently of each other. These investigations using the Cryptococcus RAM and ZNF2 mutants indicate that cell shape, cell size, and likely cell surface properties weigh differently on the outcome of cryptococcal interactions with different hosts. Thus, certain traits evolved in Cryptococcus that are beneficial within one host might be detrimental when a different host is encountered.

Purification and immunomodulating activity of C-phycocyanin from Spirulina platensis cultured using power plant flue gas

In this study, flue gas from a power plant smokestack was applied to culture Spirulina platensis microalgae. Our results will not only achieve the fixation of carbon from the emissions, products can also be produced from the algal biomass that possess physiological activities which could be beneficial to human health. An improved one-step process of chromatography was used to produce high-purity C-phycocyanin with a PC ratios >3.5. Adding different concentrations of ammonium sulfate produced different amounts of C-phycocyanin, with 40% generating the highest yield, followed by 35% and 30% concentrations. Immunomodulating activities were evaluated in the murine macrophage cell line J774A.1. We found that C-phycocyanin had the capability to induce secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and that these results were not due to contamination with LPS. Treatment with C-phycocyanin also increased proIL-1β and COX-2 protein expression dose-dependently. Furthermore, C-phycocyanin rapidly stimulated phosphorylation of inflammatory-related signaling molecules, including ERK, JNK, p38 and IκB. In addition, although C-phycocyanin decreased production of LPS-induced ROS, it did not inhibit LPS-induced inflammatory cytokines in J774A.1 cells. This is the first report to show that C-phycocyanin exhibited a detailed molecular mechanism of bioactivity by boosting immunomodulation performance.

Scavenger Receptor Mediated Endocytosis of Silver Nanoparticles into J774A.1 Macrophages Is Heterogeneous

We investigated the scavenger receptor mediated uptake and subsequent intracellular spatial distribution and clustering of 57.7 ± 6.9 nm diameter silver nanoparticles (zeta-potential = -28.4 mV) in the murine macrophage cell line J774A.1 through colorimetric imaging. The NPs exhibited an overall red-shift of the plasmon resonance wavelength in the cell ensemble as function of time and concentration, indicative of intracellular NP agglomeration. A detailed analysis of the NP clustering in individual cells revealed a strong phenotypic variability in the intracellular NP organization on the single cell level. Throughout the observation time of 24h cells containing non- or low-agglomerated NPs with a characteristic blue color coexisted with cells containing NPs with varying degrees of agglomeration, as evinced by distinct spectral shifts of their resonance wavelengths. Pharmacological inhibition studies indicated that the observed differences in intracellular NP organization resulted from coexisting actin- and clathrin-dependent endocytosis mechanisms in the macrophage population. Correlation of intracellular NP clustering with macrophage maturity marker (F4/80, CD14) expression revealed that differentiated J774A.1 cells preferentially contained compact NP agglomerates, whereas monocyte-like macrophages contained non-agglomerated NPs.

Effect of entF deletion on iron acquisition and erythritol metabolism by Brucella abortus 2308

Brucella abortus has been shown to produce two siderophores: 2,3-dihydroxybenzoic acid (2,3-DHBA) and brucebactin. Previous studies on Brucella have shown that 2,3-DHBA is associated with erythritol utilization and virulence in pregnant ruminants. The biosynthetic pathway and role of brucebactin are not known and the only gene shown to be involved so far is entF. Using cre-lox methodology, an entF mutant was created in wild-type B. abortus 2308. Compared with the wild-type strain, the ΔentF strain showed significant growth inhibition in iron minimal media that became exacerbated in the presence of an iron chelator. For the first time, we have demonstrated the death of the ΔentF strain under iron-limiting conditions in the presence of erythritol. Addition of FeCl(3) restored the growth of the ΔentF strain, suggesting a significant role in iron acquisition. Further, complementation of the ΔentF strain using a plasmid containing an entF gene suggested the absence of any polar effects. In contrast, there was no significant difference in survival and growth between the ΔentF and wild-type strains grown in the murine macrophage cell line J774A.1, suggesting that an alternate iron acquisition pathway is present in Brucella when grown intracellulary.

Effect of Heparin, Fucoidan and Other Polysaccharides on Adhesion of Enterohepatic Helicobacter Species to Murine Macrophages

Helicobacter species have been isolated and cultured from both the gastric and enterohepatic niches of the gastrointestinal tract and are associated with a wide spectrum of diseases. Some members of the enterohepatic Helicobacter species (EHS), which include Helicobacter bilis, Helicobacter hepaticus and Helicobacter pullorum, are associated with chronic inflammatory and proliferative bowel inflammation, hepatitis and in experimental murine studies with hepatic cancer. The present study aimed to explore if polysulphated polysaccharides can prevent adhesion of EHS to the murine macrophage cell line J774A.1. A competitive binding assay showed that heparin and heparan sulphate at a concentration of 1.25mg/ml reduced binding of H. hepaticus and H. pullorum to the host cells, but not H. bilis. Of the tested Helicobacter spp, the highest inhibition by heparin was demonstrated for H. pullorum (P < 0.01), the most hydrophilic strain. Partially or completely de-sulphated heparin derivatives lost the ability to inhibit adherence of EHS, indicating the importance of sulphated groups of heparin. The most efficient inhibitor of EHS binding to macrophages was fucoidan, which reduced bacterial adhesion of the three enterohepatic Helicobacter species to a greater extent than heparin, 60-90% inhibition vs 30-70% inhibition by heparin. Identification of receptors that EHS ligands bind to is important for understanding the development of infection and may provide a rational target to prevent infection and therapy.

Chlamydophila pneumoniae induces production of the defensin-like MIG/CXCL9, which has in vitro antichlamydial activity

CXC chemokines that lack the ELR motif, including the monokine induced by IFN-γ (MIG/CXCL9), the IFN-induced protein of 10 kDa (IP-10/CXCL10), and the IFN-inducible T-cell α-chemoattractant (I-TAC/CXCL11), have been shown to mediate the generation of type 1 immune responses and to possess defensin-like bactericidal effects. This study revealed that the infection of mice with Chlamydophila pneumoniae via the intranasal route resulted in the local expression of MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. The expression of IP-10/CXCL10 and I-TAC/CXCL11 mRNA peaked on day 4. On day 7, the expression of MIG/CXCL9 mRNA in the infected lungs was increased 156-fold relative to that in the uninfected mouse lungs. MIG/CXCL9 was also detected at a protein level from day 1, with the highest concentration in the supernatants of the infected lungs on day 7. The expression of IFN-γ displayed similar kinetics. C. pneumoniae and its inactivated form also induced the production of MIG/CXCL9 in mouse fibroblasts and in the murine macrophage cell line J774A in vitro. Cotreatment of the tissue cultures with C. pneumoniae and different quantities of IFN-γ resulted in strong increases in MIG/CXCL9 production. Recombinant MIG/CXCL9 exerted dose-dependent antibacterial activity against C. pneumoniae. Significant antichlamydial activity of MIG/CXCL9 was observed after a 15-min incubation period. Chlamydial proteins at a molecular weight of 60 kDa were identified by Far-Western blot assay and liquid chromatography-tandem mass spectrometry as binding molecules of MIG/CXCL9. The results of these experiments suggest that MIG/CXCL9 might play an important role in the innate and acquired defense mechanisms against C. pneumoniae.

In-vitro anti-inflammatory effect of Eucalyptus globulus and Thymus vulgaris: nitric oxide inhibition in J774A.1 murine macrophages.

It is well known that nitric oxide (NO) plays an important role in the pathogenesis of inflammatory diseases. Eucalyptus globulus Labill. and Thymus vulgaris L. have been used in traditional medicine in the treatment of bronchitis, asthma and other respiratory diseases. The present study focuses on the effects of these two extracts on NO production induced by lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) in the murine macrophage cell line J774A.1. In addition, cell viability, scavenging activity and inducible nitric oxide synthase (iNOS) mRNA expression were evaluated. E. globulus and T. vulgaris extracts significantly inhibited the enhanced production of NO induced by LPS and IFN-gamma in a dose-dependent manner. Treatment with these two extracts did not reduce cell viability at any dose used. Both plant extracts showed significant scavenging of NO radicals released by an NO donor, PAPA-NONOate. Results also show that pre-treatment with E. globulus and T. vulgaris extracts significantly inhibits iNOS mRNA expression. This study thus suggests that the inhibition of net NO production by these two extracts may be due to their NO scavenging activity and/or their inhibitory effects on iNOS gene expression.

In-vitro anti-inflammatory activity of Pinus sylvestris and Plantago lanceolata extracts: effect on inducible NOS, COX-1, COX-2 and their products in J774A.1 murine macrophages

Extracts of the plant species Pinus sylvestris L. and Plantago lanceolata L. have been used in traditional medicine for the treatment of certain respiratory diseases, but little is known about their precise effects and mechanisms of action. In this study, we investigated the effect of these plant extracts on the production of nitric oxide (NO) and prostaglandin E(2), NO synthase (NOS) type II, cyclooxygenase-1 (COX-1) and COX-2 mRNA expression in the murine macrophage cell line J774A.1. We found that Pinus sylvestris and Plantago lanceolata extracts inhibited NO production in a concentration-dependent manner in this cell line, without obvious cytotoxic effects as tested by MTT assay. The Plantago lanceolata extract at all doses used, and the Pinus sylvestris extract at high doses, showed significant scavenging of NO radicals released by the NO donor PAPA-NONOate. Our data also show that pre-treatment with these extracts significantly inhibits inducible NOS (iNOS) mRNA production in this cell line, without affecting COX-1 mRNA expression. COX-2 mRNA levels and PGE(2) levels induced by lipopolysaccharide/interferon-gamma were not modified upon pre-treatment with the extracts. Thus, our results suggest that the anti-inflammatory properties of Pinus sylvestris and Plantago lanceolata extracts may reflect decreased NO production, possibly due to inhibitory effects on iNOS gene expression or to NO-scavenging activity.