J774 Macrophage Cell Research Articles

Understanding internalization of rotavirus VP6 nanotubes by cells: towards a recombinant vaccine

Rotavirus VP6 nanotubes are an attractive option for a recombinant vaccine against rotavirus disease. Protection against rotavirus infection and an adjuvant effect have been observed upon immunization with VP6 nanotubes. However, little information exists on how VP6 nanotubes interact with cells and trigger an immune response. In this work, the interaction between VP6 nanotubes and different cell lines was characterized. VP6 nanotubes were not cytotoxic to any of the animal or human cell lines tested. Uptake of nanotubes into cells was cell-line-dependent, as only THP1 and J774 macrophage cells internalized them. Moreover, the size and spatial arrangement of VP6 assembled into nanotubes allowed their uptake by macrophages, as double-layered rotavirus-like particles also displaying VP6 in their surface were not taken up. The internalization of VP6 nanotubes was inhibited by methyl-β-cyclodextrin, but not by genistein, indicating that nanotube entry is specific, depends on the presence of cholesterol in the plasma membrane, and does not require the activity of tyrosine kinases. The information generated here expands our understanding of the interaction of protein nanotubes with cells, which is useful for the application of VP6 nanotubes as a vaccine.

Proteome profiling of the dimorphic fungusPenicillium marneffeiextracellular proteins and identification of glyceraldehyde-3-phosphate dehydrogenase as an important adhesion factor for conidial attachment

Despite being the most important thermal dimorphic fungus causing systemic mycosis in Southeast Asia, the pathogenic mechanisms of Penicilliummarneffei remain largely unknown. By comparing the extracellular proteomes of P.marneffei in mycelial and yeast phases, we identified 12 differentially expressed proteins among which glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 60 (HSP60) were found to be upregulated in mycelial and yeast phases respectively. Based on previous findings in other pathogens, we hypothesized that these two extracellular proteins may be involved in adherence during P.marneffei-host interaction. Using inhibition assays with recombinant GAPDH (rGAPDH) proteins and anti-rGAPDH sera, we demonstrated that adhesion of P.marneffei conidia to fibronectin and laminin was inhibited by rGAPDH or rabbit anti-rGAPDH serum in a dose-dependent manner. Similarly, a dose-dependent inhibition of conidial adherence to A549 pneumocytes by rGAPDH or rabbit anti-rGAPDH serum was observed, suggesting that P.marneffei GAPDH can mediate binding of conidia to human extracellular matrix proteins and pneumocytes. However, HSP60 did not exhibit similar inhibition on conidia adherence, and neither GAPDH norHSP60 exhibited inhibition on adherence to J774 or THP-1 macrophage cell lines. This report demonstrates GAPDH as an adherence factor in P.marneffei by mediating conidia adherence to host bronchoalveolar epithelium during the early establishment phase of infection.

Development and characterization of sulfasalazine loaded fucosylated PPI dendrimer for the treatment of cytokine-induced liver damage

The present investigation was aimed at exploring the targeting potential of sulfasalazine (NF-κB inhibitor drug) loaded fucose tethered poly (propylene imine) (PPI) dendritic nanoarchitecture (SSZ–FUCO-PPID) to Kupffer cells for effective management of cytokine-induced liver damage. The SSZ–FUCO-PPID formulation was characterized for entrapment efficiency, in vitro release, stability, toxicological investigations, macrophage uptake, NF-κB inhibition, and in vivo studies. In cell uptake assay the uptake of SSZ–FUCO-PPID was found to be higher and preferentially by J774 macrophage cell line. Cytokine assay suggested that the SSZ–FUCO-PPID potentially inhibited the IL-12 p40 production in LPS activated macrophages. Western blot analysis clearly suggested that SSZ–FUCO-PPID inhibited the activation of NF-κB as indicated by the absence of p-IκB band. Pharmacokinetic study revealed improved bioavailability, half-life and mean residence time of SSZ upon fucosylation of dendrimers. The biodistribution pattern clearly established the higher amount of SSZ–FUCO-PPID in liver. Hematological data suggest that the fucosylated formulations are less immunogenic as compared to unconjugated formulations. The results suggest that the SSZ–FUCO-PPID formulation holds targeting potential to Kupffer cells for the treatment of cytokine-induced liver damage.

A dual sensor for real-time monitoring of glucose and oxygen

A dual glucose and oxygen sensor in a polymer format was developed. The dual sensor composed of a blue emitter as the glucose probe, a red emitter as an oxygen probe, and a yellow emitter as a built-in reference probe which does not respond to either glucose or oxygen. All the three probes were chemically immobilized in a polyacrylamide-based matrix. Therefore, the dual sensor possesses three well separated emission colors and ratiometric approach is applicable for analysis of the glucose and oxygen concentration at biological conditions. The sensor was applied for real-time monitoring of glucose and oxygen consumption of bacterial cells, Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis), and mammalian cells of mouse macrophage J774 and human cervical cancer HeLa cell lines. On the other hand, in order to achieve satisfactory sensing performance for glucose, compositions of the matrices among poly(2-hydroxyethyl methacrylate), polyacrylamide, and poly(6-aminohexyl methacrylamide) which is a linker polymer for grafting the glucose probe, were optimized.

IL-18 provided in dying bacterial-infected macrophages induces IFN-γ production in functional T-cell hybridoma B6HO3 through cell conjugates

We have previously reported that the co-culture of functional T-cell hybridoma B6HO3 with dying J774 macrophage cells infected with Listeria monocytogenes (LM) results in the production of IFN-γ by B6HO3 cells. Here, we explore the mechanism underlying this phenomenon. We found that IFN-γ production was dependent on IL-18, but that the dying LM-infected macrophages produced no more than 100 pg/ml of IL-18, much less than the amount of IL-18 required for stimulating B6HO3 cells to produce IFN-γ. Furthermore, IL-18 binding protein added to the co-culture was unable to easily gain access to IL-18 for neutralisation. B6HO3 cells formed cell conjugates with J774 macrophages, and IFN-γ-producing B6HO3 cells were spatially and temporally associated with LM-infected macrophage cell death that exhibited neither pyroptosis nor pyronecrosis. These results suggest that the IL-18 produced by dying LM-infected macrophages is released to the interface of the cell conjugates, thereby inducing B6HO3 cells to produce IFN-γ. Based on the present and also previous findings, we propose that IL-18 released from macrophages because of cell death caused by bacteria may be the primary cytokine that triggers the innate IFN-γ production that is required for activating the bactericidal functions of macrophages at early stages of bacterial infection.

Abstract 111: A Novel Assay that Incorporates Measurement of Ex Vivo LCAT Activity with Measurement of Cholesterol Efflux Capacity - Assessment of Two Biomarkers of Serum HDL Function

Atheroprotective properties of high-density lipoproteins (HDL) may be due to the ability to remove lipids from peripheral cells to the liver for excretion during reverse cholesterol transport (RCT). Assays that measure functionality of serum HDL are needed to evaluate the therapeutic potential of treatments targeting HDL metabolism. Recent work shows an inverse correlation between cholesterol efflux capacity of serum HDL and coronary artery disease, demonstrating the value of measuring HDL functionality. Another function of HDL that promotes RCT is the capacity for esterification of cholesterol by lecithin:cholesterol acyltransferase (LCAT). A novel assay was developed that in addition to measurement of serum HDL efflux capacity incorporates measuring the capacity for esterification of HDL-cholesterol by LCAT. Assay methodology includes measurement of global cholesterol efflux via all known pathways (ABCA1, SR-BI, ABCG1 and passive diffusion) from J774 macrophage cells to serum HDL, and measurement of the proportion of cholesterol transferred to serum HDL that is esterified by LCAT over 4h. Assay validation included assessment of intra-assay repeatability, inter-assay precision, linearity, LOQ and specificity. Results showed the intra-assay variability (%CV) from six independent preparations of serum HDL is ≤10% for both the % efflux and the % esterification of cholesterol. Inter-assay variability between four separate assays is ≤13% for both the % efflux and the % esterification of cholesterol. Cholesterol efflux is linear up to concentrations of 2.8% serum HDL and cholesterol esterification is linear up to concentrations of 4% serum. The LOQ for the assay is 0.15% cholesterol efflux and 0.40% cholesterol esterification. Global cholesterol efflux was determined to be specific for known cholesterol acceptors such as serum HDL (p≤0.0001 versus media blank). The cholesterol esterification measured was determined to be specific for the action of LCAT because the presence of LCAT inhibitor decreased the % ester by 86-97% (p≤0.0009). This novel assay allows the measurement of two functional aspects of an individual subject’s serum HDL, the capacities for both cholesterol efflux and for esterification of cholesterol by LCAT.

Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0–75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5kgmol−1 methoxy-terminated PEG corona. While a 5kgmol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8kgmol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associated with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This characterization of nanocarrier uptake and targeting provides promise for optimizing drug delivery to macrophages for TB treatment and establishes a general route for optimizing targeted formulations of nanocarriers for specific delivery at targeted sites.

The role of flagella and chemotaxis genes in host pathogen interaction of the host adapted Salmonella enterica serovar Dublin compared to the broad host range serovar S. Typhimurium

BackgroundThe importance of flagella and chemotaxis genes in host pathogen interaction in Salmonella enterica is mainly based on studies of the broad host range serovar, S. Typhimurium, while little is known on the importance in host specific and host adapted serovars, such as S. Dublin. In the current study we have used previously characterized insertion mutants in flagella and chemotaxis genes to investigate this and possible differences in the importance between the two serovars.ResultsfliC (encoding the structural protein of the flagella) was essential for adhesion and fliC and cheB (CheB restores the chemotaxis system to pre-stimulus conformation) were essential for invasion of S. Dublin into epithelial Int407 cells. In S. Typhimurium, both lack of flagella (fliC/fljB double mutant) and cheB influenced adhesion, and invasion was influenced by lack of both cheA (the histidine-kinase of the chemotaxis system), fliC/fljB and cheB mutation. Uptake in J774A.1 macrophage cells was significantly reduced in cheA, cheB and fliC mutants of S. Dublin, while cheA was dispensable in S. Typhimurium. Removal of flagella in both serotypes caused an increased ability to propagate intracellular in J774 macrophage cells and decreased cytotoxicity toward these cells. Flagella and chemotaxis genes were found not to influence the oxidative response. The induction of IL-6 from J774A-1 cells depended on the presence of flagella in S. Typhimurium, whilst this was not the case following challenge with S. Dublin. Addition of fliC from S. Typhimurium in trans to a fliC mutant of S. Dublin increased cytotoxicity but it did not increase the IL-6 production. Flagella were demonstrated to contribute to the outcome of infection following oral challenge of mice in S. Dublin, while an S. Typhimurium fliC/fljB mutant showed increased virulence following intra peritoneal challenge.ConclusionsThe results showed that flagella and chemotaxis genes differed in their role in host pathogen interaction between S. Dublin and S. Typhimurium. Notably, lack of flagella conferred a more virulent phenotype in S. Typhimurium at systemic sites, while this was not the case in S. Dublin. In vitro assays suggested that this could be related to flagella-induced induction of the IL-6 pro-inflammatory response, but further in vivo studies are needed to confirm this.

P2X7 Receptor Activation Induces Reactive Oxygen Species Formation and Cell Death in Murine EOC13 Microglia

The P2X7 purinergic receptor is a ligand-gated cation channel expressed on leukocytes including microglia. This study aimed to determine if P2X7 activation induces the uptake of organic cations, reactive oxygen species (ROS) formation, and death in the murine microglial EOC13 cell line. Using the murine macrophage J774 cell line as a positive control, RT-PCR, immunoblotting, and immunolabelling established the presence of P2X7 in EOC13 cells. A cytofluorometric assay demonstrated that the P2X7 agonists adenosine-5′-triphosphate (ATP) and 2′(3′)-O-(4-benzoylbenzoyl) ATP induced ethidium+ or YO-PRO-12+ uptake into both cell lines. ATP induced ethidium+ uptake into EOC13 cells in a concentration-dependent manner, with an EC50 of ~130 μM. The P2X7 antagonists Brilliant Blue G, A438079, AZ10606120, and AZ11645373 inhibited ATP-induced cation uptake into EOC13 cells by 75–100%. A cytofluorometric assay demonstrated that P2X7 activation induced ROS formation in EOC13 cells, via a mechanism independent of Ca2+ influx and K+ efflux. Cytofluorometric measurements of Annexin-V binding and 7AAD uptake demonstrated that P2X7 activation induced EOC13 cell death. The ROS scavenger N-acetyl-L-cysteine impaired both P2X7-induced EOC13 ROS formation and cell death, suggesting that ROS mediate P2X7-induced EOC13 death. In conclusion, P2X7 activation induces the uptake of organic cations, ROS formation, and death in EOC13 microglia.

Lipopolysaccharide induces CD38 expression and solubilization in J774 macrophage cells.

CD38, an ADP ribosyl cyclase, is a 45 kDa type II transmembrane protein having a short N-terminal cytoplasmic domain and a long C-terminal extracellular domain, expressed on the surface of various cells including macrophages, lymphocytes, and pancreatic β cells. It is known to be involved in cell adhesion, signal transduction and calcium signaling. In addition to its transmembrane form, CD38 is detectable in biological fluids in soluble forms. The mechanism by which CD38 is solubilized from the plasma membrane is not yet clarified. In this study, we found that lipopolysaccharide (LPS) induced CD38 upregulation and its extracellular release in J774 macrophage cells. Furthermore, it also increased CD38 expression at the mRNA level by activating the Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway. However, LPS decreased the levels of CD38 in the plasma membrane by releasing CD38 into the culture supernatant. LPS-induced CD38 release was blocked by the metalloproteinase-9 inhibitor indicating that MMP-9 solubilizes CD38. In conclusion, the present findings demonstrate a potential mechanism by which C38 is solubilized from the plasma membrane.

Abstract 19253: Acute Inflammation Following Dental Treatment Alters Endothelial Effects, but not Cholesterol Efflux Capacity of HDL

Background: HDL elevating therapies have thus far failed to improve CV outcome and the loss of anti-atherosclerotic properties of HDL has recently been reported in CAD. This suggests specific biological activities of HDL, rather than its cholesterol content, may be crucial in mediating the anti-atherogenic effects of HDL. Aim: To determine the impact of acute systemic inflammation on various measures of HDL function. Methods: 50 subjects with periodontitis (PD) (Age 49.8 [6.5]) underwent dental treatment. In vivo changes in endothelial function were determined using Flow Mediated Dilatation (FMD). HDL function was assayed at baseline, at the peak of the inflammatory response (24 hours after PD treatment) and following resolution (6 months post PD treatment). HDL-mediated cholesterol efflux was measured in J774 macrophage cells, pre-treated with cAMP. Impact of HDL on cultured endothelial aortic cells was assayed by measuring HDL-dependent Nitric Oxide bioavailibility (NO) and superoxide (SO) production using ESR Spectroscopy. HDL-associated Paraoxonasae-1 (PON1) activity was measured by spectrophotometry. Results and Discussion: 24 hours after dental treatment HDL became dysfunctional, as shown by reduced NO and increased SO production by cultured endothelial cells (P<0.01 for both). In addition, PON1 activity was reduced (P=<0.05), but no difference was found in cholesterol efflux capacity. At 6 months, these changes in HDL function returned to baseline levels, whereas cholesterol efflux capacity remained unchanged. Endothelial effects of HDL tracked with changes in inflammatory markers and FMD. Conclusion: HDL becomes dysfunctional during acute inflammation resulting in abnormal endothelial responses, in the absence of any change in efflux capacity. Furthermore, the acute impairment of HDL function is reversible upon resolution of inflammation and tracks changes in FMD. These results suggest HDL function may contribute to the vascular alterations observed during an inflammatory response and may represent a novel mechanism whereby inflammation may promote atherosclerosis, i.e. by altering the endothelial effects of HDL.

Hepcidin Regulation by BMP Signaling in Macrophages Is Lipopolysaccharide Dependent

Hepcidin is an antimicrobial peptide, which also negatively regulates iron in circulation by controlling iron absorption from dietary sources and iron release from macrophages. Hepcidin is synthesized mainly in the liver, where hepcidin is regulated by iron loading, inflammation and hypoxia. Recently, we have demonstrated that bone morphogenetic protein (BMP)-hemojuvelin (HJV)-SMAD signaling is central for hepcidin regulation in hepatocytes. Hepcidin is also expressed by macrophages. Studies have shown that hepcidin expression by macrophages increases following bacterial infection, and that hepcidin decreases iron release from macrophages in an autocrine and/or paracrine manner. Although previous studies have shown that lipopolysaccharide (LPS) can induce hepcidin expression in macrophages, whether hepcidin is also regulated by BMPs in macrophages is still unknown. Therefore, we examined the effects of BMP signaling on hepcidin expression in RAW 264.7 and J774 macrophage cell lines, and in primary peritoneal macrophages. We found that BMP4 or BMP6 alone did not have any effect on hepcidin expression in macrophages although they stimulated Smad1/5/8 phosphorylation and Id1 expression. In the presence of LPS, however, BMP4 and BMP6 were able to stimulate hepcidin expression in macrophages, and this stimulation was abolished by the NF-κB inhibitor Ro1069920. These results suggest that hepcidin expression is regulated differently in macrophages than in hepatocytes, and that BMPs regulate hepcidin expression in macrophages in a LPS-NF-κB dependent manner.

Anti-leishmanial and toxicity activities of some selected Iranian medicinal plants

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania. Cutaneous leishmaniasis is the most common form of leishmaniasis in Iran. As there is not any vaccine for leishmaniasis, treatment is important to prevent the spreading of parasites. There is, therefore, a need to develop newer drugs from different sources. The aim of this study was to assess anti-leishmanial activity of the ethanolic extracts of 17 different medicinal plants against Leishmania major promastigotes and macrophage cell line J774. The selection of the hereby studied 17 plants was based on the existing information on their local ethnobotanic history. Plants were dried, powdered, and macerated in a hydroalcoholic solution. Resulting extracts have been assessed for in vitro anti-leishmanial and brine shrimp toxicity activities. Four plants, Caesalpinia gilliesii, Satureia hortensis, Carum copticum heirm, and Thymus migricus, displayed high anti-leishmanial activity (IC50, 9.76 ± 1.27, 15.625 ± 3.76, 15.625 ± 5.46, and 31.25 ± 15.44 μM, respectively) and were toxic against the J774 macrophage cell line at higher concentrations than those needed to inhibit the parasite cell growth (IC50, 45.13 ± 3.17, 100.44 ± 17.48, 43.76 ± 0.78, and 39.67 ± 3.29 μM, respectively). Glucantime as positive control inhibited the growth of L. major promastigotes with IC50 = 254 μg/ml on promastigotes (1 × 10(6)/100 μ/well) of a log phase culture, without affecting the growth of J774 macrophages. These data revealed that C. gilliesii, S. hortensis, C. copticum heirm, and T. migricus extracts contain active compounds, which could serve as alternative agents in the control of cutaneous leishmaniasis. The activity of these herbs against L. major promastigotes and macrophage cell line J774 was reported for the first time in our study.

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus

To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

Comparative analysis of the roles of catalases KatB and KatG in the physiological fitness and pathogenesis of fish pathogen Edwardsiella tarda

The aim of this study was to reveal functional redundancy and variation of the two catalases KatB and KatG in Edwardsiella tarda. Genome sequencing of fish pathogen Edw. tarda EIB202 reveals that it contains two genes putatively encoding catalases, katB (ETAE_1368) and katG (ETAE_0889). Under free-living conditions, single disruption in katB or katG resulted in no growth impairment, whereas double mutation of the two genes led to moderate decrease in growth, indicating that these two catalases were together essential for the physiological fitness by dissipating the endogenous H(2) O(2) . katG mutant exhibited much more elevated sensitivity to exogenous H(2) O(2) than katB mutant did, indicating that KatG was quasi-essential in detoxifying external reactive oxygen species (ROS) in Edw. tarda EIB202. Further comparative analysis indicated that katB or katG disruption showed different effects on the virulence-related processes of Edw. tarda such as haemolysin production, bile and serum resistance, as well as the internalization within fish epithelial cells. Moreover, both of the katB and katG mutants exhibited incapacity to replicate in murine macrophage J774 cell model, although the deficiency was seen much severe for ΔkatB/katG mutant. With regard to in vivo virulence, katB and katG mutants displayed delayed lethality and increased LD(50) values for zebrafish. KatB and KatG in Edw. tarda serve for the physiological fitness, and pathogenesis related the bacterial survival in macrophage and in vivo of fish. Counteracting ROS for systemic infection, Edw. tarda catalase KatG and KatB merits as potential targets for attenuated live vaccine construction.

Abstract P353: Acute Consumption of Walnuts Increases Ex Vivo Cholesterol Efflux and Postprandial Lipid Response in Overweight and Obese Adults

Introduction: Epidemiological and clinical evidence demonstrate benefits of walnuts on cardiometabolic risk factors. Hypothesis: Previously, we conducted a study that evaluated the acute, postprandial effects of walnut components [separated nut skins (5.6 g), de-fatted nutmeat (34 g), and nut oil (51 g)] versus whole walnuts (85 g) on lipid/lipoprotein responses and other measures of CVD risk. The results reported herein are from an ancillary study that compared postprandial whole walnut values to fasting values, to test the hypothesis that the whole walnut treatment would improve reverse cholesterol transport. Methods: A randomized 4-period crossover design was conducted with healthy overweight and obese adults (n=15; 9 women and 6 men) with moderate hypercholesterolemia. Plasma lipids were measured at 0 (fasting), 30, 60, 120, 240, and 360 min post meal consumption; serum for ex vivo cholesterol efflux analysis was collected at 0 and 240 min. The mean fractional efflux of radiolabeled cholesterol was conducted in J774 macrophage cells cultured with 2.5% human serum. Results: A mixed linear model demonstrated an increase in TG postprandially (P < 0.01). TG levels were significantly increased at 120, 240, and 360 min compared to baseline (30.1 ± 5.1 mg/dl, 42.7 ± 5.2 mg/dl, 21.1 ± 5.1 mg/dl, respectively; P < 0.01 for all). HDL-C and the total cholesterol:HDL-C (TC:HDL) ratio also increased postprandially (P = 0.04 and P < 0.01, respectively). HDL-C increased at 60 min (1.8 ± 0.7 mg/dl; P = 0.01) and TC:HDL increased at 120, 240, and 360 min (0.2 ± 0.1, 0.3 ± 0.1, 0.3 ± 0.1, respectively; P < 0.01 for all) compared to baseline. Cholesterol efflux was increased by 3.3 % in cells cultured with postprandial serum relative to fasting baseline (baseline: 18.6 ± 0.3 %, 240 min: 19.2 ± 0.3 %; P = 0.02). Cholesterol efflux correlated positively with plasma HDL-C concentrations (r = 0.43; P = 0.02). Conclusion: Acute consumption of walnuts increased ex vivo cholesterol efflux, suggesting a novel mechanism by which walnut consumption may reduce cardiovascular risk.

The viability of the genetically diverse C. jejuni and C. coli strains in the macrophage J774 cell line

The intracellular survival of Campylobacter has been described within epithelial cells as well as in macrophages in vitro. The goal of this study was to estimate the viability of the genetically diverse C. jejuni and C. coli strains in the macrophage J774 cell line. Strains selected for analysis differed with regard to the occurrence of genes encoding specific virulence factors. The present work indicates that was no correlation between the source of isolates and relative intracellular survival.

Temperature-Dependent Production of Various PlcR-Controlled Virulence Factors in Bacillus weihenstephanensis Strain KBAB4

The Bacillus cereus sensu lato complex has recently been divided into several phylogenetic groups with clear differences in growth temperature range. However, only a few studies have investigated the actual pathogenic potential of the psychrotolerant strains of the B. cereus group at low temperature, and little information is available concerning gene expression at low temperature. We found that vegetative cells of the psychrotolerant B. weihenstephanensis strain KBAB4 were pathogenic against the model insect Galleria mellonella at 15°C but not at 30°C. A similar temperature-dependent difference also was observed for the supernatant, which was cytotoxic to Vero epithelial cell lines and to murine macrophage J774 cells at 15°C but not at 30°C. We therefore determined the effect of low temperature on the production of various proteins putatively involved in virulence using two-dimensional protein gel electrophoresis, and we showed that the production of the Hbl enterotoxin and of two proteases, NprB and NprP2, was greater at a growth temperature of 15°C than at 30°C. The quantification of the mRNA levels for these virulence genes by real-time quantitative PCR at both temperatures showed that there was also more mRNA present at 15°C than at 30°C. We also found that at 15°C, hbl mRNA levels were maximal in the mid- to late exponential growth phase. In conclusion, we found that the higher virulence of the B. cereus KBAB4 strain at low temperature was accompanied by higher levels of the production of various known PlcR-controlled virulence factors and by a higher transcriptional activity of the corresponding genes.

Anti-Inflammatory Effects of Shea Butter through Inhibition of Inos, Cox-2, and Cytokines via the Nf-Kb Pathway in Lps-Activated J774 Macrophage Cells

Shea butter is traditionally used in Africa for its anti-inflammatory and analgesic effects. In this study we explored the anti-inflammatory activities of the methanolic extract of shea butter (SBE) using lipopolysaccharide (LPS)-induced murine macrophage cell line J774. It was observed that SBE significantly reduced the levels of LPS-induced nitric oxide, Tumor necrosis factor-α (TNF-α), interleukins, 1β (IL-1β), and -12 (IL-12) in the culture supernatants in a dose dependent manner. Expression of pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) were also inhibited by SBE. These anti-inflammatory effects were due to an inhibitory action of SBE on LPS-induced iNOS, COX-2, TNF-α, IL-1β, and IL-12 mRNA expressions. Moreover, SBE efficiently suppressed IκB phosphorylation and NF-κB nuclear translocation induced by LPS. These findings explain the molecular bases of shea butter's bioactivity against various inflammatory conditions and substantiate it as a latent source of novel therapeutic agents.

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.