Vitellogenin is a cidal factor capable of killing bacteria via interaction with lipopolysaccharide and lipoteichoic acid

Vitellogenin (Vg) has been shown to be involved in host immune defense. However, the underlying mechanism by which Vg functions is largely unknown, and which component in Vg is essential for the execution of its immune role remains lacking. Here, we demonstrate clearly that fish Vg is capable of killing the whole cells of Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus rather than their protoplasts; and that Vg has distinct binding sites specific for lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN), respectively. Of note, the interaction between Vg and bacterial cells via the different binding sites results in distinct effects: the binding of Vg to E. coli via LPS and to S. aureus via LTA is lethal, whereas the binding of Vg to S. aureus via PGN is not. Moreover, Vg exhibits a lectin-like activity because its antibacterial activities can be suppressed by the carbohydrates like d-mannose, N-acetyl-d-glucosamine and d-fucose. Finally, the polypeptide chain integrity and carbohydrate residues of Vg are indispensable for its antibacterial activity, but the lipidation and phosphorylation are not necessary. Taken together, Vg is a bacteriocidal factor capable of killing E. coli and S. aureus whole cells via interaction with LPS and LTA existing in the bacterial cell walls rather than attacking their plasma membranes.     

Expression and structural diversity of the lipopolysaccharide of Haemophilus influenzae: Implication in virulence

Lipopolysaccharide (LPS) is a major virulence determinant of the human bacterial pathogen Haemophilus influenzae. A characteristic feature of H. influenzae LPS is the extensive intra- and inter-strain heterogeneity of glycoform structure which is key to the role of the molecule in both commensal and disease-causing behaviour of the bacterium. Through the combination of genetics and detailed structural analyses, H. influenzae is an exemplar Gram-negative bacterium for which now the most extensive and detailed LPS structural data and functional correlates are available. LPS from H. influenzae consists of a conserved glucose-substituted triheptosyl inner-core moiety l-α-d-Hepp-(1→2)-[PEtn→6]-l-α-d-Hepp-(1→3)-[β-d-Glcp-(1→4)]-l-α-d-Hepp linked to lipid A via Kdo 4-phosphate. The inner-core unit provides the template for attachment of oligosaccharide- and non-carbohydrate substituents. Here, the structure, genetics and expression of LPS glycoforms in the outer core are reviewed as well as their implication on virulence.     

Characterization of Plasmodium falciparum serine hydroxymethyltransferase—A potential antimalarial target

Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme required for folate recycling and dTMP synthesis. A cDNA encoding Plasmodium falciparum (Pf) SHMT was expressed as a hexa-histidine tagged protein in Escherichia coli BL21-CodonPlus^® (DE3)-RIL. The protein was purified and the process yielded 3.6 mg protein/l cell culture. Recombinant His₆-tagged PfSHMT exhibits a visible spectrum characteristic of pyridoxal-5′-phosphate enzyme and catalyzes the reversible conversion of l-serine and tetrahydrofolate (H₄folate) to glycine and 5,10-methylenetetrahydrofolate (CH₂-H₄folate). Steady-state kinetics study indicates that His₆-tagged PfSHMT catalyzes the reaction by a ternary-complex mechanism. The sequence of substrate binding to the enzyme was also examined by glycine product inhibition. A striking property that is unique for His₆-tagged PfSHMT is the ability to use d-serine as a substrate in the folate-dependent serine–glycine conversion. Kinetic data in combination with expression result support the proposal of SHMT reaction being a regulatory step for dTMP cycle. This finding suggests that PfSHMT can be a potential target for antimalarial chemotherapy.     

Functional analysis of domain of unknown function (DUF) 1943, DUF1944 and von Willebrand factor type D domain (VWD) in vitellogenin2 in zebrafish

Vitellogenin (Vg), the precursor of egg-yolk proteins in all oviparous organisms, shares a similar domain structure combination. In most cases, Vg contains the Vitellogenin_N domain, the domain of unknown function (DUF) 1943, and the von Willebrand factor type D domain (VWD), which are present in different forms of Vg from both vertebrates and invertebrates. Occasionally, a DUF1944 domain is also present in between DUF1943 and VWD in some Vg proteins of vertebrates. Recent studies have shown that Vg participates in immune defense of host with multiple functions. However, whether all Vg proteins encoded by different vg genes play an immune role is unknown. In addition, the correlation of different domains in Vg with the multiple immune functions remains completely unclear. Here we demonstrated clearly that recombinant proteins, rDUF1943, rDUF1944 and rVWD from zebrafish Vg2 interacted with both the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus and the Gram-negative bacteria Escherichia coli and Vibrio anguillarum, as well as their signature components LTA and LPS. Moreover, both rDUF1943 and rDUF1944 promoted the phagocytosis of E. coli and S. aureus by carp macrophages. These suggest that both DUF1943 and DUF1944 as well as VWD may contribute to the function of Vg as a pattern recognition receptor, and DUF1943 and DUF1944 also contribute to the function of Vg as an opsonin. This study also opens a new angle for identification of function of genes of unknown function, which have the domains DUF1943 and DUF1944.     

Comparison of effects of green tea catechins on apicomplexan hexose transporters and mammalian orthologues

Here we have investigated the inhibitory properties of green tea catechins on the Plasmodium falciparum hexose transporter (PfHT), the Babesia bovis hexose transporter 1 (BboHT1) and the mammalian facilitative glucose transporters, GLUT1 and GLUT5, expressed in Xenopus laevis oocytes. (−)-Epicatechin-gallate (ECG) and (−)-epigallocatechin-gallate (EGCG) inhibited d-glucose transport by GLUT1 and PfHT, and d-fructose transport by GLUT5, with apparent K_i values between 45 and 117 μM. BboHT1 was more potently inhibited by the ungallated catechins (−)-epicatechin (EC) and (−)-epigallocatechin (EGC), with apparent K_i values of 108 and 168 μM, respectively. Site-directed mutagenesis experiments provided little further support for previously reported models of catechin binding to hexose transporters. Furthermore, P. falciparum growth inhibition by catechins was not affected by the external d-glucose concentration. Our results provide new data on the inhibitory action of catechins against sugar transporters but were unable to elucidate the antimalarial mechanism of action of these agents.     

Oral contraceptive administration aggravates nitric oxide synthesis inhibition-induced high blood pressure in female rats

The use of estrogen–progestogen oral contraceptive (OC) is associated with high blood pressure, although mechanisms responsible are still unclear. This study sought to investigate the effects of administration of OC on high blood pressure resulting from nitric oxide (NO) synthesis inhibition in female Sprague–Dawley rats. Rats were given ethinyl estradiol in combination with norgestrel and were treated with NO synthase inhibitor, N^G-nitro-l-arginine methyl ester (l-NAME) in the drinking water or drinking water alone for 6 weeks. OC treatment alone led to a significant increase in blood pressure and positive water balance. Treatment with l-NAME alone resulted in a significant elevation of blood pressure without significant positive water balance. Concomitant treatment with OC and l-NAME produced significant increases in blood pressure and water balance. These magnitudes of increases were significantly greater than those observed in rats treated with OC or l-NAME alone. Treatment with OC did not affect NO biosynthesis with or without concurrent l-NAME treatment. Treatment with OC and/or l-NAME did not significantly affect body weight, food intake, heart rate, cardiac weight/body weight ratio, plasma sodium, glomerular filtration rate and urinary sodium output. Conclusion: These data demonstrate that OC administration resulted in a modest increased blood pressure via enhanced water retention that was not associated with impaired NO synthesis. On the other hand, these results showed that increased blood pressure induced by inhibition of NO synthesis was not associated with water retention. The study also indicated that OC administration aggravated increase in blood pressure during NO synthesis inhibition, via enhanced water retention.     

Age-related changes in arginine and its metabolites in memory-associated brain structures

l-arginine is metabolised by nitric oxide synthase (NOS) and arginase to form l-citrulline and nitric oxide, and l-ornithine and urea, respectively. The present study investigated NOS and arginase activities, and the levels of l-arginine, l-citrulline and l-ornithine, as well as glutamate and γ-aminobutyric acid (GABA), in memory-related brain structures in 4, 12 and 24 months old rats. Significantly increased NOS and arginase activities with age were found across the CA1, CA2/3 and dentate gyrus (DG) sub-regions of the hippocampus and the prefrontal, entorhinal, perirhinal, postrhinal and temporal cortices in a region-specific manner. For l-arginine, there were age-related increases in CA1 and the perirhinal and temporal cortices, and decreases in the entorhinal and postrhinal cortices. l-citrulline levels were decreased with age in the prefrontal, postrhinal and temporal cortices. There were age-related decreases in l-citrulline/l-arginine molar ratio in CA1 and CA2/3 and the prefrontal and temporal cortices, but an increase in the entorhinal cortex (EC). Increased l-ornithine levels and l-ornithine/l-arginine molar ratios with age were found in most of the brain regions examined. Glutamate levels were significantly decreased with age in the prefrontal, entorhinal, perirhinal and temporal cortices, whereas GABA level was largely unchanged except for age-related increase in CA1. There were significantly decreased glutamate/GABA molar ratios with age in six brain regions. Correlational analyses revealed no inverse relationship between NOS and arginase activities, and no positive correlations between the activities of the two enzymes and the tissue concentrations of their products. Interestingly, there were significant positive correlations between glutamate and GABA, and l-arginine and its metabolites in many brain regions. These results demonstrate that the aging process has dramatic effects on the NOS and arginase metabolic pathways of l-arginine and the glutamatergic neurotransmitter system. Since l-arginine metabolism is complex, there is a need to determine its metabolomic profile in vivo in the future.     

Characterization of L-aminocarnitine, an inhibitor of fatty acid oxidation

The pathogenesis of hypoketotic hypoglycemia and cardiomyopathy in patients with fatty acid oxidation (FAO) disorders is still poorly understood. In vitro studies are hampered by the lack of natural mutants to asses the effect of FAO inhibition. In addition, only a few inhibitors of FAO are known. Furthermore, most inhibitors of FAO are activating ligands of peroxisome proliferator-activated receptors (PPARs). We show that l-aminocarnitine (l-AC), a carnitine analog, inhibits FAO efficiently, but does not activate PPAR. l-AC inhibits carnitine palmitoyltransferase (CPT) with different sensitivities towards CPT1 and CPT2, as well as carnitine acylcarnitine translocase (CACT). We further characterized l-AC using fibroblasts cell lines from controls and patients with different FAO defects. In these cell lines acylcarnitine profiles were determined in culture medium after loading with [U-¹³C]palmitic acid. In control fibroblasts, l-AC inhibits FAO leading to a reduction of C2-acylcarnitine and elevation of C16-acylcarnitine. In very long-chain acyl-CoA dehydrogenase (VLCAD)-deficient fibroblasts, l-AC decreased the elevated C14-acylcarnitine and increased C16-acylcarnitine. In CACT and CPT2-deficient cell lines, l-AC did not change the already elevated C16-acylcarnitine level, showing that CPT1 is not inhibited. Oxidation of pristanic acid was only partly inhibited at high l-AC concentrations, indicating minimal CACT inhibition. Therefore, we conclude that in intact cells l-AC inhibits CPT2. Combined with our observation that l-AC does not activate PPAR, we suggest that l-AC is useful to simulate a FAO defect in cells from different origin.     

A combination of a SEM technique and X-ray microanalysis for studying the spore germination process of Clostridium tyrobutyricum

Clostridium tyrobutyricum is an anaerobic bacterium responsible for late blowing defects during cheese ripening and it is of scientific interest for biological hydrogen production. A scanning electron microscopy (SEM) coating technique and X-ray microanalysis were developed to analyze the architecture and chemical composition of spores upon germination in response to environmental changes. In addition, we investigated the effects of different compounds on this process. Agents and environmental conditions inducing germination were characterized monitoring changes in optical density (OD). Among all tested conditions, the greatest drop in OD₆₂₅ (57.4%) was obtained when spores were incubated in l-alanine/l-lactate buffer, pH 4.6. In addition, a carbon-coating SEM technique and X-ray microanalysis were used to observe the architecture of spores and to examine calcium dipicolinate release.Conditions inducing C. tyrobutyricum spore germination were identified and SEM X-ray microanalysis clearly distinguished germinating from dormant spores. We confirmed that calcium dipicolinate release is one of the first events occurring. These microscopy methods could be considered sensitive tools for evaluating morphological and chemical changes in spores of C. tyrobutyricum during the initial phase of germination. Information gathered from this work may provide new data for further research on germination.     

Fibrinogen-related protein from amphioxus Branchiostoma belcheri is a multivalent pattern recognition receptor with a bacteriolytic activity

Fibrinogen-related proteins (FREPs) containing fibrinogen-like (FBG) domain have been shown to be involved in immune responses in both invertebrates and vertebrates, but the underlying mechanisms remain ill-defined. In this study we isolated a cDNA encoding amphioxus (Branchiostoma belcheri) FREP homolog, BbFREP. BbFREP encoded a protein of 286 amino acids, which included a C-terminal FBG domain and clustered together with human fibrinogen beta and gamma chains. Quantitative real time PCR revealed that the expression of BbFREP was significantly up-regulated following challenge with lipopolysaccharides (LPS) or lipoteichoic acid (LTA). The recombinant BbFREP expressed in Pichia pastoris was able to specifically recognize the pathogen-associated molecular patterns (PAMPs) on the bacterial surfaces including LPS, peptidoglycan (PGN) and LTA, and displayed strong bacteriolytic activities against both Gram-negative bacterium Escherichia coli and Gram-positive bacterium Staphylococcus aureus. BbFREP was also able to bind to both E. coli and S. aureus. In situ hybridization indicated that BbFREP was mainly expressed in the hepatic caecum and hind-gut, agreeing basically with the primary expression of vertebrate FREP genes in the liver. All these suggest that BbFREP can function as a pattern recognition receptor with a bacteriolytic activity via interaction with LPS, LTA and PGN. It also bolsters the notion that the hepatic caecum of amphioxus is equivalent to the vertebrate liver, acting as a major tissue in acute phase response.     

Heavy chain (LvH) and light chain (LvL) of lipovitellin (Lv) of zebrafish can both bind to bacteria and enhance phagocytosis

Lipovitellin (Lv) is an apoprotein in oviparous animals. Lv consists of a heavy chain (LvH) and a light chain (LvL) which are traditionally regarded as energy reserves for developing embryos. Recently, Lv has been shown to be involved in immune defense of developing embryos in fish. However, it remains unknown if each of LvH and LvL possesses immune activity; and if so, whether or not they function similarly. Here we clearly demonstrated that recombinant LvH (rLvH) and LvL (rLvL) from zebrafish vg1 gene bound to both the Gram-negative bacteria Escherichia coli and Vibrio anguillarum and the Gram-positive bacteria Staphylococcus aureus and Micrococcus luteus as well as the pathogen-associated molecular patterns LPS, LTA and PGN. In addition, both rLvH and rLvL were able to enhance the phagocytosis of bacteria E. coli and S. aureus by macrophages. All these data suggest that both LvH and LvL, in addition to being energy reserves, are also maternal immune-relevant factors capable of interacting with invading bacteria in zebrafish embryos/larvae.     

Inflammatory pain-induced signaling events following a conditional deletion of the N-methyl-D-aspartate receptor in spinal cord dorsal horn

The N-methyl-d-aspartate (NMDA) receptor in the spinal cord dorsal horn (SCDH) is one of the mechanisms involved in central sensitization during chronic pain. Previously, this laboratory created a spatio-temporal knockout (KO) of the N-methyl-d-aspartate receptor I (NR1) subunit in the mouse SCDH. The NR1 KO completely blocks NR1 gene and subsequent NMDA receptor expression and function in SCDH neurons. In the NR1 KO mice, the mechanical and cold allodynia induced at 24 h after complete Freund's adjuvant (CFA) was reduced. However, the protective effects of KO were transient and were not seen at 48 h after CFA. These observations suggest the presence of NMDA-independent pathways that contribute to CFA-induced pain. CFA induces the activation of several signaling cascades in the SCDH, including protein kinase C (PKC)γ and extracellular signal-regulated kinases (ERK1/2). The phosphorylation of PKCγ and ERK1/2 was inhibited in the SCDH of NR1 KO mice up to 48 h after CFA treatment, suggesting that these pathways are NMDA receptor-dependent. Interestingly, neuronal cyclooxygenase (COX) -2 expression and microglial p38 phosphorylation were induced in the SCDH of the NR1 KO at 48 h after CFA. Our findings provide evidence that inflammatory reactions are responsible for the recurrence of pain after NR1 KO in the SCDH.     

Functional characterization of Vitellogenin_N domain,domain of unknown function 1943, and von Willebrand factor type D domain in vitellogenin of the non-bilaterian coral Euphyllia ancora: Implications for emergence of immune activity of vitellogenin in basal metazoan

Our understanding of the function of vitellogenin (Vg) in reproduction has undergone a transformation over the past decade in parallel with new insights into the role of Vg in immunity. However, the time when Vg was endowed with immunological activities during animal evolution remains elusive. Here we demonstrate for the first time that the recombinant proteins rVitellogenin_N, rDUF1943, and rVWD from Vg of the basal metazoan coral Euphyllia ancora not only interact with Gram-positive and negative bacteria as well as their conserved surface components LTA and LPS but also enhance phagocytosis of bacteria by macrophages. Moreover, challenge with LPS results in a marked up-regulation of vg in the coral E. ancora. These data suggest that E. ancora Vg, like that described in the bilaterian oviparous animals fish and amphioxus, is a molecule related to antibacterial defense, indicating that the timing of the emergence of immune role of Vg predates the divergence of the cnidarian (non-bilaterian) and bilaterian lineages.     

Muramyl Dipeptide Synergizes with Staphylococcus aureus Lipoteichoic Acid To Recruit Neutrophils in the Mammary Gland and To Stimulate Mammary Epithelial Cells

Staphylococcus aureus, a major pathogen for the mammary gland of dairy ruminants, elicits the recruitment of neutrophils into milk during mastitis, but the mechanisms are incompletely understood. We investigated the response of the bovine mammary gland to muramyl dipeptide (MDP), an elementary constituent of the bacterial peptidoglycan, alone or in combination with lipoteichoic acid (LTA), another staphylococcal microbial-associated molecular pattern (MAMP). MDP induced a prompt and marked influx of neutrophils in milk, and its combination with LTA elicited a more intense and prolonged influx than the responses to either stimulus alone. The concentrations of several chemoattractants for neutrophils (CXCL1, CXCL2, CXCL3, CXCL8, and C5a) increased in milk after challenge, and the highest increases followed challenge with the combination of MDP and LTA. MDP and LTA were also synergistic in inducing in vitro chemokine production by bovine mammary epithelial cells (bMEpC). Nucleotide-binding oligomerization domain 2 (NOD2), a major sensor of MDP, was expressed (mRNA) in bovine mammary tissue and by bMEpC in culture. The production of interleukin-8 (IL-8) following the stimulation of bMEpC by LTA and MDP was dependent on the activation of NF-κB. LTA-induced IL-8 production did not depend on platelet-activating factor receptor (PAFR), as the PAFR antagonist WEB2086 was without effect. In contrast, bMEpC and mammary tissue are known to express Toll-like receptor 2 (TLR2) and to respond to TLR2 agonists. Although the levels of expression of the inflammatory cytokines tumor necrosis factor alpha (TNF-α) and IL-1β were increased by LTA and MDP at the mRNA level, no protein could be detected in the bMEpC culture supernatant. The level of induction of IL-6 was low at both the mRNA and protein levels. These results indicate that MDP and LTA exert synergistic effects to induce neutrophilic inflammation in the mammary gland. These results also show that bMEpC could contribute to the inflammatory response by recognizing LTA and MDP and secreting chemokines but not proinflammatory cytokines. Overall, this study indicates that the TLR2 and NOD2 pathways could cooperate to trigger an innate immune response to S. aureus mastitis.Staphylococcus aureus is a pathogen frequently isolated from mastitis milk or mammary abscesses in the lapine, ovine, bovine, and human species (4, 5, 23, 41, 53). The recognition of S. aureus by the mammary gland is not as well known as the recognition of Escherichia coli, another major pathogen for the mammary gland. The E. coli outer membrane lipopolysaccharide (LPS) has been shown to be the major pathogen-associated molecular pattern (MAMP) recognized by the mammary gland through interactions with Toll-like receptor 4 (TLR4), which is expressed by bovine mammary epithelial cells (MEpC) (bMEpC) (18, 24). LPS has been extensively used to mimic mastitis due to E. coli, and it was shown that the responses that it provokes are in many respects representative of those induced by live bacteria (7). The counterpart of LPS, as a proinflammatory bacterial agonist of the mammary gland innate immune system, has not yet been established for S. aureus. This pathogen releases various MAMPs during infection, and two of these have been the subject of many studies. Lipoteichoic acid (LTA) was shown to be an important pattern for immune recognition of S aureus (66). LTA signals through TLR2 (56), a receptor which is expressed in the mammary gland, in particular by bMEpC (18, 24). We have recently established the ability of LTA to elicit an intense inflammatory response in the bovine mammary gland (49). Another major staphylococcal MAMP is the minimal bioactive constituent of the bacterial peptidoglycan MurNac-l-Ala-d-iso-Gln (MDP). A major sensor of MDP is the nucleotide-binding oligomerization domain 2 (NOD2) protein, which is encoded by the caspase recruitment domain 15 gene (CARD15) (17). NOD2 is expressed mainly by two cell types that are exposed to microorganisms that produce peptidoglycan, i.e., antigen-presenting cells and epithelial cells (25, 63).During infection, host-pathogen interactions are very complex and difficult to unravel. The use of isolated MAMPs makes it possible to decipher the components of the innate immune response at play in the mammary gland. Information gained from in vitro studies of the responses of relevant cells to the same bacterial agonists would increase our understanding of udder-pathogen interactions. The in vitro data can be compared with the immune response to experimentally induced infections. Similarities and differences are expected to point to important bacterial agonists of the host response and to important facets of this response. In turn, the new knowledge acquired can be used to devise new approaches with a view to modulating the inflammatory and the immune responses in the mammary gland. As information on the immune response of the mammary gland and mammary epithelial cells to staphylococcal MAMPs is relatively scarce, we decided to use two major staphylococcal MAMPs to induce mastitis and to stimulate MEpC. Epithelial cells, which line the lumen of the mammary gland, are the first and most abundant cells to be in contact with invading bacteria and their secreted products. Bovine MEpC have been shown to react to MAMPs such as LPS and LTA (62, 72).We investigated the ability of MDP to trigger an inflammatory response in the bovine mammary gland. As it was reported previously that MDP and LTA exert synergistic effects on a variety of cells (30, 63), we sought to determine whether these NOD2 and TLR2 agonists could induce additive or synergistic effects in vivo in the mammary gland and in vitro on bMEpC. Our results indicate that MDP induces neutrophilic inflammation and synergizes with LTA in the mammary gland to recruit neutrophils. Moreover, MDP and LTA synergized to induce the secretion of neutrophil-oriented chemokines by bMEpC but did not induce the secretion of the major proinflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). These new findings support the hypothesis that MDP and LTA are likely to play an important part in the initiation of inflammation during S. aureus mastitis. They contribute to an improved understanding of the detection of S. aureus by the mammary gland.     

Antibacterial and antimycotic activity of a cross-linked electrospun poly(vinyl pyrrolidone)–iodine complex and a poly(ethylene oxide)/poly(vinyl pyrrolidone)–iodine complex

Photo-cross-linked poly(vinyl pyrrolidone) (PVP) and poly(ethylene oxide)(PEO)/PVP electrospun nanofibrous mats containing complex-bound iodine have been studied. FT-IR spectroscopy analyses have proved that coordination of molecular iodine with carbonyl group and nitrogen atom of pyrrolidone rings of the PVP chains has taken place. The distribution of iodine along the fibers is uniform as revealed by X-ray mapping. The microbiological tests have demonstrated that the iodine complex-containing electrospun mats are highly effective against the Gram-positive bacterium Staphylococcus aureus, the Gram-negative bacterium Escherichia coli and the fungus Candida albicans. Comparison with iodine complex-containing films has shown that the iodine complex-containing nanofibers exhibit a higher killing rate than the films against bacteria E. coli. SEM observations showed that PVP–iodine nanofibrous mats inhibit the adhesion of bacteria S. aureus. These characteristic features make the electrospun iodine-containing nanofibers good candidates for wound-dressing materials.     

Cefdinir-induced modification of the susceptibility of bacteria to the antibacterial activity of human serum and polymorphonuclear neutrophils

The effect of serum on the bactericidal activity of cefdinir, and the ability of the antibiotic to modify the interaction of bacteria with human polymorphonuclear neutrophils were assessed. In the presence of antibiotic, serum-resistantEscherichia coli were sensitised to the bactericidal activity of normal human serum. Cefdinir enhanced opsonophagocytic killing ofEscherichia coli andStaphylococcus aureus at suprainhibitory concentrations. Significant potentiation of killing occurred with the combination of inhibitory concentrations of cefdinir, neutrophils and sub-optimal levels of serum opsonins. Pre-exposure ofEscherichia coli, but notStaphylococcus aureus, to cefdinir enhanced phagocytic uptake and killing of the antibiotic-damaged bacteria. These results indicate that cefdinir-mediated phenotypic modification ofEscherichia coli renders the bacteria susceptible to serum antibacterial activity and phagocytic uptake and intracellular killing.     

Apolipoprotein B100 is a suppressor of Staphylococcus aureus‐induced innate immune responses in humans and mice

Plasma lipoproteins such as LDL (low‐density lipoprotein) are important therapeutic targets as they play a crucial role in macrophage biology and metabolic disorders. The impact of lipoprotein profiles on host defense pathways against Gram‐positive bacteria is poorly understood. In this report, we discovered that human serum lipoproteins bind to lipoteichoic acid (LTA) from Staphylococcus aureus and thereby alter the immune response to these bacteria. Size‐exclusion chromatography and solid‐phase‐binding analysis of serum revealed the direct interaction of LTA with apolipoproteins (Apo) B100, ApoA1, and ApoA2. Only ApoB100 and the corresponding LDL exerted biological effects as this binding significantly inhibited LTA‐induced cytokine releases from human and murine immune cells. Serum from hypercholesterolemic mice or humans significantly diminished cytokine induction in response to S. aureus or its LTA. Sera taken from the patients with familial hypercholesterolemia before and after ApoB100‐directed immuno‐apheresis confirmed that ApoB100 inhibited LTA‐induced inflammation in humans. In addition, mice in which LDL secretion was pharmacologically inhibited, displayed significantly increased serum cytokine levels upon infection with S. aureus in vivo. The present study identifies ApoB100 as an important suppressor of innate immune activation in response to S. aureus and its LTA.     

Macrophage-released neutrophil chemotactic factor (MNCF) induces PMN-neutrophil migration through lectin-like activity

We have previously reported that rat peritoneal macrophages stimulated with LPS release a factor (MNCF) which induces neutrophil migration that is not blocked by glucocorticoids. The supernatant of macrophage monolayers stimulated with LPS was submitted to affinity chromatography on immobilized sugar columns. We observed that thed-gal binding fraction retained MNCF activity. This fraction, consisting of four protein components, was submitted to chromatography on Superdex 75, yielding a homogeneous preparation of the active component. MNCF has a MW of 54 KDa (gel filtration and SDS-PAGE) and pI<4.0 (isoelectrofocusing and chromatofocusing).d-gal did not interfere with the behaviour of known interleukins (IL-1β, IL-6, IL-8 TNF-α), but blocked MNCF activity in anin vitro migration assay. The present results reinforce our previous suggestion that MNCF may correspond to a novel monokine which induces neutrophil migration through a direct mechanism involving thed-gal binding site of the molecule.