Lipopolysaccharides

Detection of the Mycobacterium tuberculosis cell wall antigen lipoarabinomannan (LAM) in urine permits diagnoses of tuberculosis (TB) to be made in HIV-infected patients with advanced immunodeficiency. This can be achieved at the point-of-care within just 30 minutes using the Determine TB-LAM, which is a commercially available, lateral-flow urine 'strip test' assay. The assay has been shown to have useful diagnostic accuracy in patients enrolling in antiretroviral treatment services or in HIV-infected patients requiring admission to hospital medical wards in sub-Saharan Africa. Such patients have high mortality risk and have most to gain from rapid diagnosis of TB and immediate initiation of treatment. However, few studies using this assay have yet been reported and many questions remain concerning the correct use of the assay, interpretation of results, the role of the assay as an add-on test within existing diagnostic algorithms and the types of further studies needed. In this paper we address a series of questions with the aim of informing the design, conduct and interpretation of future studies. Specifically, we clarify which clinical populations are most likely to derive benefit from use of this assay and how patients enrolled in such studies might best be characterised. We describe the importance of employing a rigorous microbiological diagnostic reference standard in studies of diagnostic accuracy and discuss issues surrounding the specificity of the assay in different geographical areas and potential cross-reactivity with non-tuberculous mycobacteria and other organisms. We highlight the importance of careful procedures for urine collection and storage and the critical issue of how to read and interpret the test strips. Finally, we consider how the assay could be used in combination with other assays and outline the types of studies that are required to build the evidence base concerning its use.

The structures of the lipopolysaccharide (LPS) core and O antigen of Bordetella bronchiseptica and Bordetella parapertussis are known, but how these two regions are linked to each other had not been determined. We have studied LPS from several strains of these ...

Neutrophil infiltration into inflammatory sites is one of the hallmarks of acute inflammation. Locally produced chemotactic factors are presumed to mediate the sequence of events leading to the infiltration at inflammatory sites. Interleukin-8 (IL-8), a novel leukocyte chemotactic activating cytokine (chemokine), is produced by various types of cells upon stimulation with inflammatory stimuli and exerts a variety of functions on leukocytes, particularly, neutrophils in vitro. However, no definitive evidence has been presented on its role in recruiting and activating neutrophils in the lesions of various types of inflammatory reactions. We administered a highly specific neutralizing antibody against IL-8 in several types of acute inflammatory reactions, including lipopolysaccharide (LPS)-induced dermatitis, LPS/IL-1-induced arthritis, lung reperfusion injury, and acute immune complex-type glomerulonephritis. Anti-IL-8 treatment prevented neutrophil-dependent tissue damage as well as ...

EPR spectroscopic techniques have been developed for the measurement of oxygen and nitric oxide in vivo. Specifically, the methods for in vivo measurement of these molecules has been applied to the study of septic shock, utilising an experimental murine model developed in our laboratory. Oxygen was measured as pO 2 by the particlulate probes Gloxy and LiPc, which were surgically implanted at specific sites in tissues, and the soluble probe Trityl, which was administered intravenously. Nitric oxide was measured as the NO-Fe-(DETC) 2 complex after administration of Fe 2+ and DETC. LPS was seen to significantly decrease liver oxygen measured across the lobule and at the sinusoids by the Gloxy probe; there was a corresponding increase in nitric oxide both in the liver and systemically. The nitric oxide most likely originated from increased iNOS enzyme in the liver as demonstrated by Western blotting and the localisation of nitric oxide to the liver was confirmed with EPR imaging. LPS also caused a decrease in cerebral blood and tissue oxygenation, the rate of which was found to be dependent on the blood oxygenation. The development and applications of these in vivo EPR techniques for biomedical research and diagnostics is discussed.

Resveratrol is a natural molecule with antioxidant action. Moreover, resveratrol is also considered to be a molecule with anti-inflammatory action, an effect attributed to suppression of prostaglandin (PG) biosynthesis. The aim of the present study was to investigate the effects of resveratrol, a polyphenol present in most red wines, on reactive oxygen species formation as well as on arachidonic acid (AA) release, cyclooxygenase expression, and PG synthesis in murine resident peritoneal macrophages. Results show that resveratrol exerted a strong inhibitory effect on superoxide radical (O2−) and hydrogen peroxide (H2O2) produced by macrophages stimulated by lipopolysaccharides (LPS) or phorbol esters (PMA). Resveratrol also significantly decreased [3H]AA release induced by LPS and PMA or by exposure to O2− or H2O2. Resveratrol treatment caused a significant impairment of cyclooxygenase-2 (COX-2) induction stimulated by LPS and PMA or by O2− or H2O2 exposure. These effects of resveratrol on [3H]AA release and COX-2 overexpression were correlated with a marked reduction of PG synthesis. Our results indicate that the antioxidant action of resveratrol affects AA mobilization and COX-2 induction.

The colony-stimulating factor 1 receptor (CSF1R) is a key regulator of myeloid lineage cells. Genetic loss of the CSF1R blocks the normal population of resident microglia in the brain that originates from the yolk sac during early development. However, the role of CSF1R signaling in microglial homeostasis in the adult brain is largely unknown. To this end, we tested the effects of selective CSF1R inhibitors on microglia in adult mice. Surprisingly, extensive treatment results in elimination of $99% of all microglia brainwide, showing that microglia in the adult brain are physiologically dependent upon CSF1R signaling. Mice depleted of microglia show no behavioral or cognitive abnormalities, revealing that microglia are not necessary for these tasks. Finally, we discovered that the microglia-depleted brain completely repopulates with new microglia within 1 week of inhibitor cessation. Microglial repopulation throughout the CNS occurs through proliferation of nestin-positive cells that then differentiate into microglia. Neuron CSF1R Regulates Resident Microglia Neuron 82, 380-397, April 16, 2014 ª2014 Elsevier Inc. 381

Phyllanthus amarus is a medicinal herb used in traditional Indian medicine for liver disorders. Several researches also show that it acts primarily in the liver, but still it remains unknown that which of these ingredients has a hepatoprotective effect. This study was to determine whether the lignans isolated from P. amarus attenuates the D-galactosamine (GalN) / Lipopolysaccharide (LPS)-induced acute hepatitis in mice. Standardize mixture of lignans (slPA) isolated from leaves of P. amarus using automated chromatographic technique was used for experiments. Experimental mice were orally pre-treated with slPA (10, 30 and100mg/kg) for 7 days before intra-peritoneal injection of GalN/LPS. Acute hepatitis was manifested by a significant increase of pro-inflammatory cytokines and the level of hepatotoxic markers (aspartate transaminase, alanine transaminase, alkaline phosphatase and bilirubin in serum and triglycerides, MDA level in liver homogenate). In-silico molecular docking studies ...

The Escherichia coli regulatory protein RfaH contributes to efficient colonization of the mouse gut. Extraintestinal pathogenic (ExPEC) as well as non-pathogenic probiotic E. coli strains rapidly outcompeted their isogenic rfaH mutants following oral mixed infections. LPS-core and O-...

Preterm birth (PTB) is a major public health problem, with a global prevalence of 9.6% and over a million annual neonatal deaths. In a mouse model of preterm labor (PTL) induced by intravaginal administration of a subclinical dose of lipopolysaccharide (LPS), we previously demonstrated that LPS ascends to the cervix, inducing complement activation, cervical remodeling and PTL. Here we show that complement activation also plays a role in myometrial contractions during PTL in this model. Increased levels of C5a were detected in the myometrium of LPS-treated mice but not in age-matched control or term myometrium. Human and mouse myometrium incubated with C5a showed increased frequency of contractions and expression of connexin 43, suggesting that C5a is an uterotonic molecule. Statins, which showed beneficial effects in preventing complement-mediated pregnancy complications, prevented cervical remodeling, myometrial contractions and PTL in the LPS model. The protective effects of stati...

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 b was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40fold respectively for IL-1b and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.

Ethnopharmacological relevance: The fruits of Theobroma cacao L. (Sterculiaceae) have been used as food and a remedy for more than 4000 years. Today, about 100 therapeutic applications of cacao are described involving the gastrointestinal, nervous, cardiovascular and immune systems. Pro-inflammatory cytokine interferon-␥ and related biochemical pathways like tryptophan degradation by indoleamine 2,3-dioxygenase and neopterin formation are closely associated with the pathogenesis of such disorders. Aim of the study: To determine the anti-inflammatory effect of cacao extracts on interferon-␥ and biochemical consequences in immunocompetent cells. Materials and methods: Effects of aqueous or ethanolic extracts of cacao were examined on mitogen-induced human peripheral blood mononuclear cells (PBMC) of healthy donors and on lipopolysaccharide-stimulated myelomonocytic THP-1 cells. Antioxidant activity of extracts was determined by oxygen radical absorption capacity (ORAC) assay. Results: In mitogen-stimulated PBMC, enhanced degradation of tryptophan, formation of neopterin and interferon-␥ were almost completely suppressed by the cacao extracts at doses of ≥5 g/mL. Cacao extracts had no effect on tryptophan degradation in lipopolysaccharide-stimulated THP-1 cells. Conclusions: There is a significant suppressive effect of cacao extracts on pro-inflammatory pathways in activated T-cells. Particularly the influence on indoleamine 2,3-dioxygenase could relate to some of the beneficial health effects ascribed to cacao.

Meriones unguiculatus commonly known as gerbils are widely used as animal models for a variety of parasitic infections such as Brugia malayi, Entamoeba histolytica, Giardia duodenalis, Toxoplasma gondi, Helicobacter pylori, Strongyloides stercoralis and Echinococcus multilocularis. Groups of BALB/c mice, gerbils and XID mice were studied for antibody responses to T-independent antigens. Gerbils were found to be signi®cantly de®cient in eliciting antibodies to both dextran and phosphorylcholine (PC) in comparison to BALB/c mice. The antibody response of gerbils to T-independent antigens was found to be similar to the response observed in Bruton's tyrosine kinase (Btk) de®cient XID mice, which are known to be poor responders to T-independent antigens. Similar to XID mice, normal gerbil sera were found to be de®cient in naturally occurring antibodies to single stranded DNA (SS-DNA), lipopolysaccharide (LPS) and phospholipids. This raises the possibility of a de®ciency of CD5 1 B-lymphocytes (also known as B-1 cells) in gerbils, since de®ciency of this sub-population of Blymphocytes has been attributed to the absence of such naturally occurring antibodies in XID mice. These results indicate the need to study immunogenicity of parasite T-independent antigens and their relationship to protective immunity in parasitic infections in gerbils.

Phosphorodiamidate morpholino oligomers (PMO) are synthetic antisense molecules that interfere with translation, pre-mRNA splicing and RNA synthesis. Like other gene-silencing technologies, PMO are poorly taken up by primary leukocytes without the use of physical or chemical delivery techniques. We sought an alternative delivery mechanism of PMO into immune cells that eliminates the need for such manipulations. Here we demonstrate the first use of arginine-rich cell-penetrating peptides (CPPs) to deliver PMO (P-PMO) directly into primary murine leukocytes for inhibition of gene expression and promotion of altered pre-mRNA splicing. We compared the P-PMO delivery efficacy of four arginine-rich CPPs including HIV Tat and penetratin, and one histidine rich CPP, and found that the (RXR) 4 peptide was the most efficacious for PMO delivery and targeted antisense effect. The delivery and antisense effects of P-PMO are time-and dose-dependent and influenced by the activation and maturation states of T cells and dendritic cells, respectively. Targeted expression of several genes using P-PMO is shown including surface signaling proteins (CD45 and OX-40), a cytokine (interleukin-2), and a nuclear transcription factor (Foxp3). Considering the abundance of naturally occurring alternatively spliced gene products involved in immune regulation, P-PMO offer an effective method for modulating gene activity for immunological research and applications beyond traditional antisense approaches.

Background: 1E10 monoclonal antibody is a murine anti-idiotypic antibody that mimics N-glycolyl-GM3 gangliosides. This antibody has been tested as an anti-idiotypic cancer vaccine, adjuvated in Al(OH) 3 , in several clinical trials for melanoma, breast, and lung cancer. During early clinical development this mAb was obtained in vivo from mice ascites fluid. Currently, the production process of 1E10 is being transferred from the in vivo to a bioreactor-based method.

Central nervous system (CNS) has a completely separate immune system that communicates with the neurons by small molecules called cytokines. Cytokines are involved in many crucial processes in neuron including cell metabolism and neurotransmitter synthesis. It has been reported that cytokine imbalance is involved in the progression of many CNS diseases such as neuropsychiatric disorders (depression, schizophrenia, autism, and bipolar disorder) and neurodegenerative disorders (Parkinson's and Alzheimer's disease). Here, the effects of diclofenac, different antidepressants (sertraline, venlafaxine, and fluvoxamine), and vitamin B 6 (pyridoxine) on IL-10 and tumor necrosis factor-a (TNF-a) change with and without immune challenges with lipopolysaccharide (LPS) were investigated in in vitro culture of astrocytes from 2-day-old Swiss-Albino mice. Diclofenac and Sertraline significantly (p \ 0.05) improves anti-inflammatory cytokine (IL-10) while suppress (p \ 0.05) LPS-induced elevated level of pro-inflammatory mediators (TNF-a) in astrocyte culture.

Background: Alcohol, a substance that is most frequently abused, suppresses innate immune responses to microbial pathogens. The host senses pathogens via Toll-like receptors (TLRs). Recent studies indicate that alcohol affects TLR signaling.

GTPases of the p47 family involved in intracellular pathogen control. We tested TNF-α α α α and p47-GTPase induction in macrophages using classical LPSs [lipid As with glucosamine backbones, ester-and amidelinked C14:0(3-OH) and C12 to C16 in acyloxyacyl groups] of wild type and mutant Escherichia coli and Yersinia species and non-classical LPSs [lipid As with diaminoglucose, ester-linked 3-OH-fatty acids and C28:0(27-OH) and C23:0(29-OH) in acyloxyacyl groups] of plant endosymbionts ( Rhizobium ), intracellular pathogens ( Brucella and Legionella ) and phylogenetically related opportunistic bacteria ( Ochrobactrum ). Classical but not non-classical LPSs efficiently induced TNF-α α α α , IIGP and IGTP p47-GTPase expression. Remarkably, the acyloxyacyl groups in classical LPSs necessary to efficiently induce TNF-α α α α

Five new meroterpenoids, purpurogenolides A-E (1-5), and four known metabolites (6-9) were isolated from the solid substrate fermentation cultures of the fungus Penicillium purpurogenum MHz 111. The structures of the new meroterpenoids were elucidated by analysis of spectroscopic and spectrometric data (1D and 2D NMR, IR, and HRESIMS). The absolute configurations of 1 and 5 were determined by single-crystal X-ray crystallographic analysis, and those of 2-4 were elucidated on the basis of experimental and calculated electronic circular dichroism spectra. Compounds 2-4 and 6 showed inhibition of nitric oxide production in lipopolysaccharide-activated BV-2 microglial cells with IC50 values of 0.8-30.0 μM.

Use of probiotics, alone or as adjunct to other established therapies, has been reported to have potential benefits. Recently, we have reported protective potential of probiotic against Salmonella-induced liver injury. However, co-supplementation with prebiotics did not result in meaningful synergism at systemic level. Owing to the action of probiotics at the mucosal level and of arginine at systemic level, the present study was designed to evaluate the effect of Lactobacillus plantarum alone or in conjunction with arginine to combat endotoxin-mediated liver injury in rats. Bacterial endotoxin/lipopolysaccharide (LPS) was injected intraperitoneally and animals were sacrificed 8h post-challenge. Efficacy of L. plantarum alone or in conjunction with l-arginine was determined on the basis of enzyme markers, histology, levels of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-α) in addition to identification of amino acids by paper chromatography. Prior supplementation of LPS-cha...

Our earlier studies demonstrated that representative microbial substances--lipopolysaccharide, peptidoglycan, and poly-inosine: poly-cytosine (poly(I):(C))--increased the spontaneous discharge rates and sensory-evoked responses of isolated locus coeruleus (LC) neurons in a dose- and time-related manner after i.p. injection into rats. We then turned our attention to the mechanism by which microbial substances administered into the peritoneal cavity affect the LC neurons. The involvement of the subdiaphragmatic vagus nerves was examined in this regard since several brain responses to peripherally administered lipopolysaccharide have been found to depend upon the integrity of these nerves. The experiments reported here show that lipopolysaccharide, peptidoglycan, and poly(I):(C) all failed to excite LC neurons after i.p. injection into rats that had previously been subjected to complete transection of the subdiaphragmatic vagus nerves. Furthermore, selective transection of the subdiaph...

Mounting evidence exists for the operation of a functional serotonin (5-HT) system in osteoclasts and osteoblasts, which involves both receptor activation and 5-HT reuptake. In previous work we showed that the serotonin transporter (5-HTT) is expressed in osteoclasts and that its activity is required by for osteoclast differentiation in vitro. The purpose of the current study was to determine the effect of treatment with fluoxetine, a specific serotonin reuptake inhibitor, on bone metabolism in vivo. Systemic administration of fluoxetine to Swiss-Webster mice for 6 weeks resulted in increased trabecular BV and BV/TV in femurs and vertebrae as determined by micro-computed tomography (mCT). This correlated with an increase in trabecular number, connectivity, and decreased trabecular spacing. Fluoxetine treatment also resulted in increased volume in vertebral trabecular bone. However, fluoxetine-treated mice were not protected against bone loss after ovariectomy, suggesting that its anabolic effect requires the presence of estrogen. The effect of blocking the 5-HTT on bone loss following an LPS-mediated inflammatory challenge was also investigated. Subcutaneous injections of LPS over the calvariae of Swiss-Webster mice for 5 days resulted in increased numbers of osteoclasts and net bone loss, whereas new bone formation and a net gain in bone mass was seen when LPS was given together with fluoxetine. We conclude that fluoxetine treatment in vivo leads to increased bone mass under normal physiologic or inflammatory conditions, but does not prevent bone loss associated with estrogen deficiency. These data suggest that commonly used anti-depressive agents may affect bone mass.

Animals must manage interactions with beneficial as well as detrimental microbes. Immunity therefore includes strategies for both resistance to and tolerance of microbial invaders. Transforming growth factor beta (TGFb) cytokines have many functions in animals including a tolerance-promoting (tolerogenic) role in immunity in vertebrates. TGFb pathways are present in basal metazoans such as cnidarians but their potential role in immunity has never been explored. This study takes a two-part approach to examining an immune function for TGFb in cnidarians. First bioinformatic analyses of the model anemone Aiptasia pallida were used to identify TGFb pathway components and explore the hypothesis that an immune function for TGFbs existed prior to the evolution of vertebrates. A TGFb ligand from A. pallida was identified as one that groups closely with vertebrate TGFbs that have an immune function. Second, cellular analyses of A. pallida were used to examine a role for a TGFb pathway in the regulation of cnidarian-dinoflagellate mutualisms. These interactions are stable under ambient conditions but collapse under elevated temperature, a phenomenon called cnidarian bleaching. Addition of exogenous human TGFb suppressed an immune response measured as LPS-induced nitric oxide (NO) production by the host. Addition of anti-TGFb to block a putative TGFb pathway resulted in immune stimulation and a failure of the symbionts to successfully colonize the host. Finally, addition of exogenous TGFb suppressed immune stimulation in heat-stressed animals and partially abolished a bleaching response. These findings suggest that the dinoflagellate symbionts somehow promote host tolerance through activation of tolerogenic host immune pathways, a strategy employed by some intracellular protozoan parasites during their invasion of vertebrates. Insight into the ancient, conserved nature of host-microbe interactions gained from this cnidarian-dinoflagellate model is valuable to understanding the evolution of immunity and its role in the regulation of both beneficial and detrimental associations.

Objective: Nutritional compounds that potentially limit inflammation and tissue factor expression may decrease the progression of chronic kidney disease (CKD) and associated cardiovascular disease. This project aimed to determine the effect of curcumin, bovine colostrum, and fish oil on inflammatory cytokine and tissue factor procoagulant activity of peripheral blood mononuclear cells (PBMCs) from patients with CKD before dialysis. Methods: Peripheral blood mononuclear cells from patients with CKD before dialysis (n ¼ 13) and age-and sex-matched healthy controls (n ¼ 12) were cultured alone and with low and high doses of the nutritional compounds for 24 h. Cells were cultured with and without lipopolysaccharide. Supernatants were analyzed for tumor necrosis factor-a, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, IL-1b, C-reactive protein, and tissue factor procoagulant activity. Results: The production of C-reactive protein, monocyte chemoattractant protein-1, IL-6, and IL-1b by PBMCs was inhibited by low-and high-dose fish oil in the CKD group (P < 0.05). Curcumin decreased secretion of IL-6 (P ¼ 0.015) and IL-1 b (P ¼ 0.016). Curcumin was more effective than colostrum at decreasing the procoagulant activity of PBMCs in the CKD and control groups (P < 0.019). Conclusion: Fish oil decreased inflammatory cytokine secretion from CKD PBMCs. In addition, the beneficial effects of curcumin were demonstrated in decreasing inflammation in vitro, often to a similar magnitude as fish oil.

Prior exposure to a stressor can potentiate CNS pro-inflammatory immune responses to a peripheral immune challenge. However, the neuroimmune substrate(s) mediating this effect has not been determined. The present investigation examined whether microglia serve as this neuroimmune substrate given that microglia are the primary immune effector cell in the CNS. The effect of inescapable shock (IS) on glial activation (MHC

We present a complete DNA sequence and metabolic analysis of the dominant oral bacterium Fusobacterium nucleatum. Although not considered a major dental pathogen on its own, this anaerobe facilitates the aggregation and establishment of several other species including the dental pathogens Porphyromonas gingivalis and Bacteroides forsythus. The F. nucleatum strain ATCC 25586 genome was assembled from shotgun sequences and analyzed using the ERGO bioinformatics suite (http://www.integratedgenomics.com). The genome contains 2.17 Mb encoding 2,067 open reading frames, organized on a single circular chromosome with 27% GC content. Despite its taxonomic position among the gram-negative bacteria, several features of its core metabolism are similar to that of gram-positive Clostridium spp., Enterococcus spp., and Lactococcus spp. The genome analysis has revealed several key aspects of the pathways of organic acid, amino acid, carbohydrate, and lipid metabolism. Nine very-high-molecular-weight outer membrane proteins are predicted from the sequence, none of which has been reported in the literature. More than 137 transporters for the uptake of a variety of substrates such as peptides, sugars, metal ions, and cofactors have been identified. Biosynthetic pathways exist for only three amino acids: glutamate, aspartate, and asparagine. The remaining amino acids are imported as such or as di-or oligopeptides that are subsequently degraded in the cytoplasm. A principal source of energy appears to be the fermentation of glutamate to butyrate. Additionally, desulfuration of cysteine and methionine yields ammonia, H 2 S, methyl mercaptan, and butyrate, which are capable of arresting fibroblast growth, thus preventing wound healing and aiding penetration of the gingival epithelium. The metabolic capabilities of F. nucleatum revealed by its genome are therefore consistent with its specialized niche in the mouth. Fusobacterium nucleatum is a gram-negative anaerobe, which belongs to the Bacteroidaceae family and is found naturally in the microflora of the mouth in healthy or diseased humans. F. nucleatum is a very long rod with tapering ends and is one of the dominant species of the 500 or more organisms that coexist in the oral cavity (21). Many of the oral flora are commensals but a few are opportunistic pathogens. F. nucleatum can be isolated not only from the mouth but also from infections such as skin ulcers, peritonsillar abscesses, septic arthritis, and endocarditis (5). Several species of Fusobacterium have been isolated and studied, including F. necrophorum (the causative agent of Lemierre's syndrome), F. ulcercans (skin ulcers), F. russi (animal bite infections), and F. varium (eye infections), with F. nucleatum and F. necrophorum considered to be the most pathogenic. In the initial stages of the periodontal disease process, saccharolytic, aerobic Streptococcus spp. and other bacteria adhere to and colonize the tooth enamel and root surface. This sets the stage for F. nucleatum to coaggregate with these early colonizers and to permit late colonizers, including dental pathogens, to eventually form a biofilm. These complex interactions result in the release of factors that lead to tooth decay. Physical interaction is very specific among various genera in this complex microbial community. Due to the unusual length, adhesive nature, and other cell surface properties of F. nucleatum, periodontal disease-causing bacteria such as Porphyromonas gingivalis, Bacteroides forsythus, Actinobacillus actinomycetemcomitans, Treponema denticola, and Streptococcus spp. aggregate and thrive; hence, F. nucleatum is referred to as a "bridge bacterium" (5). The interaction between F. nucleatum and P. gingivalis has been reported to be very specific (19), mediated by a lactose-binding adhesin (20). The same adhesin protein mediates the binding of F. nucleatum to a variety of eukaryotic cell types including HeLa cells, buccal epithelial cells, macrophages, polymorphonuclear leukocytes, and gingival and periodontal ligaments (46). Helicobacter pylori was also shown to adhere selectively to F. nucleatum (1). Research on Fusobacterium spp. has focused primarily on species identification, oral ecology, cell-cell communication, extracellular surfaces, amino acid degradation, carbohydrate metabolism, organic acid fermentation, and antibiotic resistance (5, 21). However, many metabolic pathways and their roles in survival in such specialized niches are not known. Genetic experiments have been hampered by the lack of molecular tools, although recently three plasmids, pFN1, pFN2, and pFN3, have been isolated and sequenced from the strain ATCC 10953. A transformation system has been developed for gene manipulation using the pFN1 ori sequence (14). Although many potential virulence candidates have been

The CCL2 chemokine mediates monocyte egress from bone marrow and recruitment into inflamed tissues through interaction with the CCR2 chemokine receptor, and its expression is upregulated by proinflammatory cytokines. Analysis of the gene expression profile in GM-CSF-and M-CSF-polarized macrophages revealed that a high CCL2 expression characterizes macrophages generated under the influence of M-CSF, whereas CCR2 is expressed only by GM-CSF-polarized macrophages. Analysis of the factors responsible for this differential expression identified activin A as a critical factor controlling the expression of the CCL2/CCR2 pair in macrophages, as activin A increased CCR2 expression but inhibited the acquisition of CCL2 expression by M-CSF-polarized macrophages. CCL2 and CCR2 were found to determine the extent of macrophage polarization because CCL2 enhances the LPS-induced production of IL-10, whereas CCL2 blockade leads to enhanced expression of M1 polarizationassociated genes and cytokines, and diminished expression of M2-associated markers in human macrophages. Along the same line, Ccr2-deficient bone marrow-derived murine macrophages displayed an M1-skewed polarization profile at the transcriptomic level and exhibited a significantly higher expression of proinflammatory cytokines (TNF-a, IL-6) in response to LPS. Therefore, the CCL2-CCR2 axis regulates macrophage polarization by influencing the expression of functionally relevant and polarizationassociated genes and downmodulating proinflammatory cytokine production.

It is generally considered that stress causes decreased immune function in fish. In this study we examined in Atlantic salmon (Salmo salar Linnaeus) the effects of both short-(a single 15 s out of water) and long-term (4 weeks of daily handling 15 s out of water) stress on plasma cortisol (free and total) and glucose levels, expression of interleukin-1b (IL-1b) and survival of head kidney (HK) macrophages under culture with Aeromonas salmonicida. In the short-term study, samples were collected prior to the application of the stressor, and at 1, 3, 6, 12 and 24 h post stress. Free and total plasma cortisol levels and the percentage of free cortisol increased significantly in the stressed group at 1 and 3 h post stress. Plasma glucose levels were significantly higher than those of control fish at 1, 3 and 6 h post stress. Constitutive expression of IL-1b in macrophages isolated from head kidneys in stressed fish was significantly higher at 1 and 3 h post stress. However, lipopolysaccharide (LPS) stimulated expression of IL-1b in HK macrophages, exhibited significantly higher fold increases in unstressed fish compared to stressed fish. In the long-term study, with the exception of an increase in plasma glucose levels at 1 week, there were no significant differences in stress parameters between groups. There was a significantly higher constitutive IL-1b expression in macrophages isolated from stressed fish over the first 2 weeks. At weeks 1, 2 and 3 the magnitude of IL-1b response of isolated HK macrophages to LPS stimulation was reduced in >90% of the stressed fish. At 4 weeks there was no significant difference in inducible IL-1b expression between the groups. Macrophages isolated from stressed fish also showed significantly decreased survival when exposed to A. salmonicida. This study shows a clear pattern from repeated handling stress, whereby effects on immune cells begin with increased constitutive expression of IL-1b, followed by decreased stimulation of leucocytes by extracellular antigen, and finally decreased

Aim of the study: This study is to elucidate the involvement of anti-inflammatory heme oxygenase-1 (HO-1) in the inhibitory activity of a Chrysanthemum boreale Makino (CB) extract on nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Materials and methods: Cell viability and NO assay were performed. In addition, iNOS expression was detected by Western blotting and real-time PCR. HO-1 expression was also evaluated by Western blotting, and blocking HO-1 activity on NO production was performed. Results: The CB extract at the highest concentration (100 g/ml) significantly inhibited NO production by approximately 90% and suppressed iNOS protein expression by approximately 84.8% compared to LPS-stimulated cells. Furthermore, the CB extract (100 g/ml) inhibited iNOS mRNA expression in a concentration-dependant manner and suppressed iNOS mRNA expression by 94.8%. The CB extract induced the expression of HO-1 in a dose-dependent manner, and blocking HO-1 activity abolished the inhibitory effects of the CB extract. Moreover, the addition of carbon monoxide such as tricarbonyl dichlororuthenium (II) dimmer (RuCO), a byproduct derived from heme degradation, mimicked the inhibitory action of low concentrations of CB extract. Conclusion: These results suggest that a CB extract has potent anti-inflammatory activity in RAW264.7 macrophages involving the induction of HO-1.

Rapid translocation of P-selectin from WPB to the surface of endothelial cells is crucial for early neutrophil recruitment to acute inflammatory lesions. Likewise, the chemokine CXCL8/IL-8 is sorted to WPB in human endothelial cells, but little is known about its functional importance in lack of a suitable animal model. Here, we explored the distribution of the functional IL-8 homologues CXCL1/KC, CXCL2/MIP-2, and CXCL5-6/LIX in resting and inflamed murine vessels by confocal microscopy and paired immunostaining with markers of WPB, discovering that these chemokines did not localize to WPB but displayed a granular pattern in a subset in healthy skin compatible with sorting to the type 2 endothelial compartment for regulated secretion. Moreover, all chemokines colocalized with VWF and P-selectin in platelets, suggesting that their storage in platelet ␣-granules might represent an alternative source of rapidly available, neutrophil-recruiting chemokines. In conclusion, WPB appear not to be involved in regulated secretion of chemokines in the mouse, and instead, the possible existence of type 2 granules and the role of platelets in rapid leukocyte adhesion deserve further attention. J. Leukoc. Biol. 87: 000 -000; 2010.

Lipopolysaccharide (LPS) is a highly proinflammatory molecule isolated from bacteria. This study demonstrated the existence of LPS in a medicinal fungus, Antrodia camphorata. Because no LPS had been identified in any fungus organism, the purification of LPS from A. camphorata was attempted. LPSs from six strains of A. camphorata (35396, 35398, 35716, B71, B85, and B86) were isolated. Chemical and functional properties were investigated on the fungus LPS. Compositional analysis revealed that sorbitol, fucose, galactose, and glucose were the neutral sugars in LPS of A. camphorata. Galactosamine, glucosamine, galactose, and glucose were the predominant monosaccharide species in E. coli O129 LPS molecules, whereas galactosamine and glucosamine were absent in A. camphorata LPS. Because these properties are different from those of bacterial LPS, the functions between fungus and bacterial LPS are also discussed. The vascular endothelial lining of blood vessels, which controls leucocyte traffic and activation, may be one of the primary targets of LPS action during sepsis. Assays for biological activity were performed on endothelial cells with anti-inflammatory effects associated with sepsis. A. camphorata LPS apparently showed a lesser extent of cytotoxicity than bacterial LPS. In contrary to the proinflammatory property of bacterial LPS, LPS from A. camphorata differentially reversed bacterial LPS-induced intercellular adhersion molecule-1 and monocyte adhesion; both were indicators during inflammatory process. In conclusion, basic chemical properties categorized A. camphorata extracts into lipopolysaccharide. However, the detailed functional structures and bioactivities of A. camphorata LPS were totally different from those of bacterial LPS. The investigation of the existence and anti-inflammatory effect of fungus LPS is at present a truly novel and important finding. These results show that LPS isolated from A. camphorata offers a novel therapeutic target for anti-inflammation against E. coli infection.

Recent studies have demonstrated that hemichannels, which form gap junctions when paired from apposing cells, may serve additional roles when unpaired including cell adhesion and paracrine communication. Hemichannels in mammals are formed by connexins or pannexins, while in insects they are formed by pannexin homologues termed innexins. The formation of functional gap junctions by insect innexins has been established, although their ability to form functional nonjunctional hemichannels has not been reported. Here the characteristics of nonjunctional hemichannels were examined in three lepidopteran cell types, two cell lines (High Five and Sf9) and explanted hemocytes from Heliothis virescens (Fabricius) (Lepidoptera: Noctuidae). Selective fluorescent dye uptake by hemichannels was observed in a significant minority of cells, using fluorescence microscopy and flow cytometry. Carbenoxelone, an inhibitor of mammalian junctions, disrupted dye uptake, while flufenamic acid and mefloquine did not. The presence of Ca 2+ and Mg 2+ in the media increased hemichannel activity. Additionally, lipopolysaccharide, a stimulator of immune activity in lepidopterans, decreased dye uptake. These results demonstrate for the first time the activity of nonjunctional hemichannels in insect cells, as well as pharmacological tools to manipulate them. These results will facilitate the further examination of the role of innexins and nonjunctional hemichannels in insect cell biology, including paracrine signaling, and comparative studies of mammalian pannexins and insect innexins.

can be regulated by various mediators, including those which emanate from, and mimic, the sympathetic nervous system. Whereas Padrenergic agonists suppress, cY,-adrenergic agonists augment lipopolysaccharide (LPS)-stimulated tumor necrosis factor (TNF) production and gene expression. The susceptibility of M+s to regulation of LPS-induced TNF production and mRNA accumulation was examined following /3-adrenergic and cr,-adrenergic receptor activation at specific time points post-LPS challenge. Complete Freund's adjuvant-elicited murine M+s were incubated with LPS (30 ng/ml) in the presence or absence of adrenergic agonists or antagonists. We assessed the susceptibility of immunologically-activated M 4s to adrenergic receptor regulation: a) during the 1 h delay in the production of TNF after LPS-stimulation, and b) during the rapid increase in TNF production which follows. Disparate responsiveness of M 4s to adrenergic drugs was observed during this time course of TNF production and TNP mRNA accumulation. In particular, while the concomitant addition of an cr,-adrenergic antagonist and LPS resulted in 45% suppression of TNF production, this selective blockade of a,-adrenergic receptors on M& was equally effective throughout the first 45 min post-LPS challenge. After this initial period, the cu,-adrenergic receptor became progressively less responsive as demonstrated by the delayed addition of yohimbine (10e5 M) post-LPS challenge. The addition of the selective a,-adrenergic agonist UK-14304 (10m7 M) to LPS-activated M& augmented TNP mRNA accumulation. However, this augmentation was even greater when the addition of the a,-adrenergic agonist was delayed post-LPS challenge. It was also shown that the /3-adrenergic agonist isoproterenol (10e6 M) produced maximum suppression of TNF production within the first 1.5 h post-LPS challenge. Suppression by isoproterenol (lop6 M) of TNF mRNA accumulation occurred throughout the 2-h period assessed post-LPS stimulation of M&. The decline in isoproterenol-induced regulation was accompanied by an elevation in &-adrenergic receptor mRNA accumulation. Furthermore, suppression of TNF production induced by a maximum concentration of isoproterenol was observed at various LPS concentrations (0.001-1000 ng/ml), although this was not as pronounced a suppression as demonstrated for dibutyryl CAMP. These results demonstrate that the susceptibility of M#G to adrenergic receptor regulation changes throughout the time period necessary for gene activation and ultimate release of TNF. Thus, the production of TNF during LPS-dependent disease states may be regulated by adrenergic mediators throughout different temporal windows, better explaining the role played by the nervous system. . production by M 4s and, importantly, in M 4 sensitivity to these and other mediators (Spengler et al., 1989a, Spengler 0165-5728/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved PII SOl65-5728(96>00004-5

Previous studies have shown that IL-10 can induce the expression of the suppressor of cytokine signaling 3 (SOCS-3) mRNA in human monocytes and neutrophils, suggesting that the capacity of IL-10 to inhibit the expression of LPS-inducible proinflammatory genes may depend on SOCS-3 induction. However, no direct experimental evidence has been provided to support such hypothesis. Herein, we show that stable transfection of SOCS-3 into the mouse macrophage cell line J774 resulted in an inhibition of NO, TNF-␣, IL-6, and GM-CSF secretion in response to LPS at levels similar to those exerted by IL-10 in LPS-stimulated wild-type J774. Constitutive SOCS-3 expression also down-regulated the mRNA expression of inducible NO synthase and IL-6 and impaired the production of TNF-␣, mainly at a post-transcriptional level. In addition, SOCS-3-transfected cells displayed a constitutive expression of the IL-1R antagonist gene, consistent with the observation that IL-10 enhances IL-1R antagonist mRNA in LPS-stimulated wild-type cells. Furthermore, in peritoneal macrophages harvested from mice carrying heterozygous disruption of the SOCS-3 gene, IL-10 was less effective in repressing LPS-stimulated TNF-␣ and NO production. Taken together, our data show that SOCS-3 inhibits LPS-induced macrophage activation, strongly supporting the idea that it plays a role in the molecular mechanism by which IL-10 down-modulates the effector functions of LPS-activated macrophages. Finally, we show that forced expression of SOCS-3 significantly suppresses the ability of IL-10 to trigger tyrosine phosphorylation of STAT3. Therefore, SOCS-3 functions both as an LPS signal inhibitor and as a negative feedback regulator of IL-10/STAT3 signaling.

Selenium is recognized as essential in animal and human nutrition. Several hypotheses have been advanced for its biological activity. The aim of this study was to investigate the in vivo effect of selenium on rat liver manganese superoxide dismutase (MnSOD), a key antioxidant enzyme, under naïve and inflammatory conditions. Rats received sodium selenite supplementation and LPS injection. Wholeliver samples, isolated hepatocytes, Kupffer cells and blood samples were subjected to protein, RNA and biochemical analysis. Liver enrichment with selenium increased whole-liver MnSOD levels due to an increase in MnSOD transcription in hepatocytes. This was due to an increase in the ratio of specificity protein 1 to activating enhancer binding protein 2 DNA-binding activity. The inflammatory stimulus further elevated MnSOD levels in the whole-liver that was abrogated in sodium selenite supplementation due to reduced transcription of MnSOD in Kupffer cells. Moreover, selenium enrichment decreased Kupffer cells IL-6 transcription in LPS-injected animals. Anti-inflammatory activity of selenium was demonstrated by normalized blood levels of ALT and IL-6 in LPS-injected animals. In conclusion, selenium up-regulates hepatocytes MnSOD expression, probably improving their anti-oxidant defense, while decreasing MnSOD and IL-6 transcription in Kupffer cells in the presence of inflammatory stimuli, attenuating their inflammatory response. This selective mechanism may explain the anti-inflammatory and hepato-protective effect of selenium.

We herein report the synthesis of diosgenin analogues from commercially available diosgenin as the starting material. The structures of newly synthesised compounds were confirmed by 1 H NMR, 13 C NMR and mass spectrometry. All analogues were evaluated for in-vitro anti-inflammatory profile against LPS-induced inflammation in primary peritoneal macrophages isolated from mice by quantification of pro-inflammatory (TNF-␣, IL-6 and IL-1␤) cytokines in cell culture supernatant using the ELISA technique followed by in-vitro cytotoxicity study. Among the synthesised analogues, analogue 15 [(E) 26-(3 ,4 ,5 -trimethoxybenzylidene)-furost-5en-3␤-acetate)] showed significant anti-inflammatory activity by inhibiting LPS-induced pro-inflammatory cytokines in a dose-dependent manner without any cytotoxicity. Efficacy and safety of analogue 15 were further validated in an in-vivo system using LPS-induced sepsis model and acute oral toxicity in mice. Oral administration of analogue 15 inhibited the proinflammatory cytokines in serum, attenuated the liver and lung injury and reduced the mortality rate in sepsis mice. Acute oral toxicity study showed that analogue 15 is non-toxic at higher dose in BALB/c mice. Molecular docking study revealed the strong binding affinity of diosgenin analogues to the active site of the pro-inflammatory proteins. These findings suggested that analogue 15 may be a useful therapeutic candidate for the treatment of inflammatory diseases.

. a EACTIVE OXYGEN species (ROS) have been impli-R cated in the pathogenesis of endotoxemia, sepsis, ischemia-reperfusion injury, and alcohol int~xication.'-~ The liver is highly susceptible to the deleterious effects of ROS because this organ is mainly responsible for clearance of bacterial endotoxin and the metabolism of ethanol. The liver is composed of >85% hepatocytes. However, the other 15% that include the sinusoidal endothelial cells, Kupffer cells, fat-storing cells NK cells, and a small number of neutrophils also contribute significantly to the overall homeostasis of the liver. During endotoxemia, chronic alcohol intoxication and hepatic-ischemia followed by reperfusion, hepatic sequestration of neutrophils, O C C U~S .~-~ Because phagocytic cells are potent producers of ROS, the most likely sources of these radicals in the liver are Kupffer cells and the sequestered neutrophils. Sinusoidal endothelial cells contribute to ROS formation in the liver to a lesser extent. Although hepatocytes are ideal sources of ROS, these cells do not produce these radicals extracellularly in significant amount. Thus, the production of ROS by inflammatory leukocytes and activated resident macrophages in liver is likely to contribute to hepatotoxicity.

Improved methods of cell culture from eye stalk, hepatopancreas, muscle, ovary, and hemocytes of shrimp (Penaeus vannamei) were established using synthetic media and shrimp muscle extract (SME). For hemocytes and ovarian cell cultures, Grace’s insect medium supplemented with 10% (v/v) fetal bovine serum and 10% SME (v/v) showed enhanced attachment and proliferation of the cells. The hemocyte and ovarian cell cultures could be maintained for 48 and 66 days, respectively, and have been sub-cultured four and six times, respectively. Both ovary and hemocyte cell cultures contained primarily epithelial-like cells. Cells derived from ovary tissue grew preferably between 26°C and 28°C with 5% CO2. Although the temperature preference of hemocyte cells was the same as ovarian cells, CO2 supplementation did not show any difference in the growth of hemocyte cells. When the shrimp were injected with lipopolysaccharide (8 μg/g of shrimp) and hemolymph was drawn 24 h post-injection, the in vitro multiplicity of hemocytes dramatically improved. The growth of eye stalk, hepatopancreas, and muscle-derived cells was much less compared to ovarian cells and hemocytes under the conditions described above. The optimal culture conditions for ovarian cells and hemocytes were also different from that for eye stalk, hepatopancreas, and muscle cell culture. The proliferation efficiencies of primary cultures of hepatopancreas, eyestalk, and muscle cells were about 30, 12, and <7 d, respectively. The improved culture conditions described here, particularly for hemocytes and ovary, will be very useful for in vitro studies involving viruses infecting shrimp and in shrimp genomic studies.