Using adjuvants to drive features of T cell responses to vaccine antigens is an important technol... more Using adjuvants to drive features of T cell responses to vaccine antigens is an important technological challenge in the design of new and improved vaccines against infections. Properties such as T helper cell function, T cell memory, and CD8+ T cell cytotoxicity may play critical roles in optimal and long-lived immunity through vaccination. Directly manipulating specific immune activation or antigen delivery pathways with adjuvants may selectively augment desired T cell responses in vaccination and may improve the effectiveness and durability of vaccine responses in humans. In this review we outline recently studied adjuvants in their potential for antigen presenting cell and T cell programming during vaccination, with an emphasis on what has been observed in studies in humans as available.
We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialid... more We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated ...
Serum obtained after immunization with an O18 polysaccharide-toxin A conjugate vaccine was evalua... more Serum obtained after immunization with an O18 polysaccharide-toxin A conjugate vaccine was evaluated for the estimation of protective levels of anti-O-specific lipopolysaccharide (LPS) immunoglobulin G (IgG) antibody against bacteremia and death caused by a homologous serotype of Escherichia coli K1 strains. Passive transfer of rabbit serum conferred significant protection from a lethal E. coli infection in a neonatal rat model. The overall incidence of bacteremia and mortality was 4% in rat pups receiving undiluted postvaccination serum, while that in control animals was 100% (P < 0.001). The overall incidences of bacteremia were 5 and 72% for animals with serum anti-O18 LPS IgG concentrations of > 1.0 and < 1.0 microgram/ml, respectively, while the overall incidences of mortality for animals with serum anti-O18 LPS IgG levels of > 1.0 and < 1.0 microgram/ml were 0 and 72%, respectively (P < 0.001). Protection against E. coli infection was also demonstrated with h...
The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers... more The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers resistance to phagocytosis. The treatment of encapsulated bacteria with salicylate to inhibit capsule expression was found to enhance the phagocytosis of encapsulated bacteria by human neutrophils only in the presence of cell surface-specific antibodies. Both type-specific rabbit antisera and anticapsular human hyperimmune globulin were employed as opsonins. Salicylate significantly enhanced phagocytosis with homologous, but not heterologous, whole-cell antisera. Antisera, diluted 1:40, no longer opsonized fully encapsulated bacteria but promoted the uptake of multiple salicylate-treated bacteria in > 90% of neutrophils. Salicylate (0.25 to 1.0 mM) also enhanced opsonization with globulin against homologous bacteria. Higher salicylate levels (1 to 2.5 mM) enhanced the opsonization of heterologous serotypes with human globulin. The nature of antibody attachment to encapsulated bacteri...
Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highl... more Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA "superfamily" has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN) sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s), presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously desc...
Despite decades of research, it has been remarkably difficult to demonstrate the role of endotoxi... more Despite decades of research, it has been remarkably difficult to demonstrate the role of endotoxin in human disease. The dilemma in part stems from our inability to accurately measure endotoxin in the circulation and body fluids and make meaningful clinical correlations. The most commonly used method for detecting endotoxin is the Limulus amebocyte lysate (LAL) assay. Routinely used in the pharmaceutical industry to detect endotoxin contamination, this assay measures endotoxin's activity, not endotoxin itself. Endotoxins activate a cascade of procoagulant proteins found in the blood amebocytes of the horseshoe crab, Limulus polyphemus, resulting in coagulation that may be detected by clot formation, turbidity or a chromogenic readout. Several factors may interfere with the sensitivity of the assay, including plasma proteins and lipids that bind the endotoxin. Consequently, heating and dilution of samples are required for more accurate measurement. The Limulus lysate also reacts with β-glucans found in fungi, but most commercial assay kits now include inhibitors of this reaction. In the current issue, Charbonney et al used a different assay, the Endotoxin Activity (EA) Assay, to evaluate the prevalence and kinetics of systemic endotoxemia in a cohort of 48 patients who were admitted to an ICU within 24 hr of sustaining severe trauma (1). While 46/48 of patients had no endotoxemia on admission, endotoxemia developed in 75% of them, particularly after shock or early surgery, and endotoxemia predicted organ dysfunction. Since few patients had Gram-negative bacterial infections, the authors concluded that a loss of gastrointestinal barrier integrity was the most likely source of the circulating endotoxin. The EA assay measures neither endotoxin nor its activity, but rather the ability of a putative LPS/Mab complex to prime the generation of reactive oxygen species (ROS) by the PMN in the patient's blood. The blood sample is mixed with an IgM Mab, E5, that targets a broad range of LPS species. If LPS is present in the blood and binds to E5, the LPS/E5 immune complex activates complement, which interacts with neutrophil complement receptors to prime the cells; when opsonized zymosan is added, greater amounts of ROS are produced. Copyright form disclosures: Dr. Cross disclosed past and present funding by the National Institutes of Health to develop vaccines for Gram-negative bacteria. In this editorial he states that vaccines and monoclonal antibodies directed at endotoxin are under development (by him and many others), but does not endorse any one of the endotoxin-directed therapies discussed.
Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung ... more Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC 50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to
Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the envir... more Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the environment, as quiescent endospores. These endospores must germinate inside host cells, forming vegetative bacilli, before they can express the virulence factors that enable them to evade host defenses and disseminate throughout the body. While the role of macrophages and dendritic cells in this initial interaction has been established, the role of polymorphonuclear leukocytes (PMNs) has not been adequately defined. We discovered that while B. anthracis 34F2 Sterne endospores germinate poorly within non-activated human PMNs, these phagocytes exhibit rapid microbicidal activity toward the outgrown vegetative bacilli, independent of superoxide and nitric oxide. These findings suggest that a non-free radical pathway kills B. anthracis bacilli. We also find in PMNs an autophagic mechanism of bacterial killing based on the rapid induction of LC-3 conversion, beclin-1 expression, sequestosome 1 (S...
The continued threat of worldwide influenza pandemics, together with the yearly emergence of anti... more The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence...
ABSTRACI'. Murine hybridoma antibodies directed against the capsule and 0-side chain determinants... more ABSTRACI'. Murine hybridoma antibodies directed against the capsule and 0-side chain determinants of the Escherichia coli strain Bort (018ac:Kl:H7) were evaluated for their ability to enhance bactericidal activity of cord blood against Kl E. coli strains possessing 0 antigens common in neonatal E. coli infection, i.e. 018, 07, and 01. The antibodies to the Kl capsule and 0-side chain efficiently enhanced cord polymorphonuclear leukocyte-mediated killing of K1 encapsulated E. coli strain possessing a homologous 0 antigen, but the IgM antibody to the K1 capsule exhibited approximately 10-fold greater activity than did the IgG3 antibody to 0-side chain (weight basis). Both antibodies required complement for their opsonic activities. Our findings indicate that antibodies directed against the capsule and 0-lipopolysaccharides are able to restore the opsonic activity of cord blood against K1 E. coli, suggesting that these antibodies may be useful in the prevention and therapy of neonatal E. coli infection. (Pediatr Res 28: 667-670, 1990) Abbreviations CFU, colony forming units LPS, lipopolysaccharide PMN, polymorphonuclear leukocyte In neonates, infections due to gram-negative bacilli are associated with significant morbidity and mortality despite appropriate antimicrobial therapy (1). The most common gram-negative organism causing sepsis and meningitis during the neonatal period is Escherichia coli (I). Given the plethora of E. coli serotypes (2), it is striking that E. coli strains possessing the K1 capsular polysaccharide are the predominant capsular serotype responsible for neonatal E. coli sepsis and meningitis (3-5) and that most of these K1 E. coli isolates are associated with a limited number of 0-LPS antigens (i.e. 0 18, 07, 0 1, 0 16) (6). We have developed a series of murine hybridomas producing MAb against different cell wall components of K1-encapsulated E. coli. MAb prepared against group B meningococcus and reactive with E. coli K1 polysaccharides and antibodies directed against 0-side chain determinants of a K1 E. coli strain were
To provide a database for the development of an O-antigen-polysaccharide-containing vaccine again... more To provide a database for the development of an O-antigen-polysaccharide-containing vaccine against Klebsiella spp., we examined the O-antigen seroepidemiology of 378 Klebsiella clinical isolates collected prospectively in two university centers. Strains were typed by competitive enzyme-linked immunosorbent assay with rabbit antisera specific for serogroups O1 to O12 and monoclonal antibodies (MAbs) specific for serogroups O1, O2ab, O2ac, and the genus-specific core antigen. The numbers of isolates (percentages) of individual O serogroups were as follows: 148 (39.2) for serogroup O1, 40 (10.6) for serogroup O2ab, 4 (1.1) for serogroup O2ac, 89 (23.6) for serogroup O3, 2 (0.5) for serogroup O4, 32 (8.5) for serogroup O5, none for serogroups O7, O9, and O12, and 21 (5.6) for serogroup O11. Forty-two (11.1) of the strains were non-O-typeable. O-serogroup distributions were virtually identical between isolates from invasive infections and those from noninvasive infections or colonizations. A vaccine containing the O-specific polysaccharides of serogroups O1, O2ab, O3, and O5 would cover 82% of clinically occurring O-antigen specificities. Three hundred thirty-eight of 378 isolates (89.4%) reacted with the genus-specific MAb V/9-5, which recognizes an epitope of the outer core region of Klebsiella lipopolysaccharide. Antibodies directed against this epitope may represent a further alternative for O-antigen-targeted immunoprophylaxis of Klebsiella infections. These data support further experimental investigations on the protective potential of O-antigen-based vaccines and/or hyperimmune globulins in Klebsiella infection.
Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell sur... more Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell surface glycoconjugates by one or both sialidase promotes cytokine production. Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibit...
MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseud... more MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-in...
Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia e... more Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia express catalytically active NEU1 and NEU3 sialidases, and NEU1 restrains the endothelial migratory response to wounding. Conclusion: NEU1 regulates endothelial remodeling in response to injury. Significance: Learning how NEU1 and NEU3 regulate sialylated molecules on the endothelial surface is key to understanding endothelial receptor-ligand, cell-cell, and host-pathogen interactions.
Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initia... more Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initiate repair programs. Results: NEU1 sialidase desialylates EGFR and MUC1 in airway epithelia to regulate their responsiveness to ligands and adhesiveness to P. aeruginosa. Conclusion: NEU1 provides an additional level of regulation over airway epithelial responsiveness to ligands and pathogens. Significance: The downstream effects of EGFR desialylation require further investigation. Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6-1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7-1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38-56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR-and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.
The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in ac... more The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an ·NO-dependent bactericidal response. Since NOS 2 also generates O 2 · − , experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO − ) from the reaction of ·NO with O 2 · − and if so, was ONOO − microbicidal toward B. anthracis . Our findings suggest that ONOO − was formed upon macrophage infection by B. anthracis endospores; however, ONOO − does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis , which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing ·NO levels in the macrophage. Thus, the ability of B. anthracis to subvert ·NO production has importa...
Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on t... more Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis . We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protect...
The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to intera... more The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dosedependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor±activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.
Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest ... more Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (• NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing Lornithine at the expense of • NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.
Using adjuvants to drive features of T cell responses to vaccine antigens is an important technol... more Using adjuvants to drive features of T cell responses to vaccine antigens is an important technological challenge in the design of new and improved vaccines against infections. Properties such as T helper cell function, T cell memory, and CD8+ T cell cytotoxicity may play critical roles in optimal and long-lived immunity through vaccination. Directly manipulating specific immune activation or antigen delivery pathways with adjuvants may selectively augment desired T cell responses in vaccination and may improve the effectiveness and durability of vaccine responses in humans. In this review we outline recently studied adjuvants in their potential for antigen presenting cell and T cell programming during vaccination, with an emphasis on what has been observed in studies in humans as available.
We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialid... more We previously reported that flagellin-expressing Pseudomonas aeruginosa (Pa) provokes NEU1 sialidase-mediated MUC1 ectodomain (MUC1-ED) desialylation and MUC1-ED shedding from murine lungs in vivo. Here, we asked whether Pa in the lungs of patients with ventilator-associated pneumonia might also increase MUC1-ED shedding. The levels of MUC1-ED and Pa-expressed flagellin were dramatically elevated in bronchoalveolar lavage fluid (BALF) harvested from Pa-infected patients, and each flagellin level, in turn, predicted MUC1-ED shedding in the same patient. Desialylated MUC1-ED was only detected in BALF of Pa-infected patients. Clinical Pa strains increased MUC1-ED shedding from cultured human alveolar epithelia, and FlaA and FlaB flagellin-expressing strains provoked comparable levels of MUC1-ED shedding. A flagellin-deficient isogenic mutant generated dramatically reduced MUC1-ED shedding compared with the flagellin-expressing wild-type strain, and purified FlaA and FlaB recapitulated ...
Serum obtained after immunization with an O18 polysaccharide-toxin A conjugate vaccine was evalua... more Serum obtained after immunization with an O18 polysaccharide-toxin A conjugate vaccine was evaluated for the estimation of protective levels of anti-O-specific lipopolysaccharide (LPS) immunoglobulin G (IgG) antibody against bacteremia and death caused by a homologous serotype of Escherichia coli K1 strains. Passive transfer of rabbit serum conferred significant protection from a lethal E. coli infection in a neonatal rat model. The overall incidence of bacteremia and mortality was 4% in rat pups receiving undiluted postvaccination serum, while that in control animals was 100% (P < 0.001). The overall incidences of bacteremia were 5 and 72% for animals with serum anti-O18 LPS IgG concentrations of > 1.0 and < 1.0 microgram/ml, respectively, while the overall incidences of mortality for animals with serum anti-O18 LPS IgG levels of > 1.0 and < 1.0 microgram/ml were 0 and 72%, respectively (P < 0.001). Protection against E. coli infection was also demonstrated with h...
The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers... more The polysaccharide capsule of Klebsiella pneumoniae is an important virulence factor that confers resistance to phagocytosis. The treatment of encapsulated bacteria with salicylate to inhibit capsule expression was found to enhance the phagocytosis of encapsulated bacteria by human neutrophils only in the presence of cell surface-specific antibodies. Both type-specific rabbit antisera and anticapsular human hyperimmune globulin were employed as opsonins. Salicylate significantly enhanced phagocytosis with homologous, but not heterologous, whole-cell antisera. Antisera, diluted 1:40, no longer opsonized fully encapsulated bacteria but promoted the uptake of multiple salicylate-treated bacteria in > 90% of neutrophils. Salicylate (0.25 to 1.0 mM) also enhanced opsonization with globulin against homologous bacteria. Higher salicylate levels (1 to 2.5 mM) enhanced the opsonization of heterologous serotypes with human globulin. The nature of antibody attachment to encapsulated bacteri...
Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highl... more Neuraminidases (NAs) are critical virulence factors for several microbial pathogens. With a highly conserved catalytic domain, a microbial NA "superfamily" has been proposed. We previously reported that murine polymorphonuclear leukocyte (PMN) sialidase activity was important in leukocyte trafficking to inflamed sites and that antibodies to Clostridium perfringens NA recognized a cell surface molecule(s), presumed to be a sialidase of eukaryotic origin on interleukin-8-stimulated human and murine PMNs. These antibodies also inhibited cell sialidase activity both in vitro and, in the latter instance, in vivo We therefore hypothesized that mammalian sialidases share structural homology and epitopes with microbial NAs. We now report that antibodies to one of the isoforms of C. perfringens NA, as well as anti-influenza virus NA serum, recognize human NEU3 but not NEU1 and that antibodies to C. perfringens NA inhibit NEU3 enzymatic activity. We conclude that the previously desc...
Despite decades of research, it has been remarkably difficult to demonstrate the role of endotoxi... more Despite decades of research, it has been remarkably difficult to demonstrate the role of endotoxin in human disease. The dilemma in part stems from our inability to accurately measure endotoxin in the circulation and body fluids and make meaningful clinical correlations. The most commonly used method for detecting endotoxin is the Limulus amebocyte lysate (LAL) assay. Routinely used in the pharmaceutical industry to detect endotoxin contamination, this assay measures endotoxin's activity, not endotoxin itself. Endotoxins activate a cascade of procoagulant proteins found in the blood amebocytes of the horseshoe crab, Limulus polyphemus, resulting in coagulation that may be detected by clot formation, turbidity or a chromogenic readout. Several factors may interfere with the sensitivity of the assay, including plasma proteins and lipids that bind the endotoxin. Consequently, heating and dilution of samples are required for more accurate measurement. The Limulus lysate also reacts with β-glucans found in fungi, but most commercial assay kits now include inhibitors of this reaction. In the current issue, Charbonney et al used a different assay, the Endotoxin Activity (EA) Assay, to evaluate the prevalence and kinetics of systemic endotoxemia in a cohort of 48 patients who were admitted to an ICU within 24 hr of sustaining severe trauma (1). While 46/48 of patients had no endotoxemia on admission, endotoxemia developed in 75% of them, particularly after shock or early surgery, and endotoxemia predicted organ dysfunction. Since few patients had Gram-negative bacterial infections, the authors concluded that a loss of gastrointestinal barrier integrity was the most likely source of the circulating endotoxin. The EA assay measures neither endotoxin nor its activity, but rather the ability of a putative LPS/Mab complex to prime the generation of reactive oxygen species (ROS) by the PMN in the patient's blood. The blood sample is mixed with an IgM Mab, E5, that targets a broad range of LPS species. If LPS is present in the blood and binds to E5, the LPS/E5 immune complex activates complement, which interacts with neutrophil complement receptors to prime the cells; when opsonized zymosan is added, greater amounts of ROS are produced. Copyright form disclosures: Dr. Cross disclosed past and present funding by the National Institutes of Health to develop vaccines for Gram-negative bacteria. In this editorial he states that vaccines and monoclonal antibodies directed at endotoxin are under development (by him and many others), but does not endorse any one of the endotoxin-directed therapies discussed.
Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung ... more Neuraminidase-1 (NEU1) is the predominant sialidase expressed in human airway epithelia and lung microvascular endothelia where it mediates multiple biological processes. We tested whether the NEU1-selective sialidase inhibitor, C9-butyl-amide-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (C9-BA-DANA), inhibits one or more established NEU1-mediated bioactivities in human lung cells. We established the IC 50 values of C9-BA-DANA for total sialidase activity in human airway epithelia, lung microvascular endothelia and lung fibroblasts to be 3.74 µM, 13.0 µM and 4.82 µM, respectively. In human airway epithelia, C9-BA-DANA dose-dependently inhibited flagellin-induced, NEU1-mediated mucin-1 ectodomain desialylation, adhesiveness for Pseudomonas aeruginosa and shedding. In lung microvascular endothelia, C9-BA-DANA reversed NEU1-driven restraint of cell migration into a wound and disruption of capillary-like tube formation. NEU1 and its chaperone/transport protein, protective protein/cathepsin A (PPCA), were differentially expressed in these same cells. Normalized NEU1 protein expression correlated with total sialidase activity whereas PPCA expression did not. In contrast to
Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the envir... more Bacillus anthracis, the causative agent of anthrax, is acquired by mammalian hosts from the environment, as quiescent endospores. These endospores must germinate inside host cells, forming vegetative bacilli, before they can express the virulence factors that enable them to evade host defenses and disseminate throughout the body. While the role of macrophages and dendritic cells in this initial interaction has been established, the role of polymorphonuclear leukocytes (PMNs) has not been adequately defined. We discovered that while B. anthracis 34F2 Sterne endospores germinate poorly within non-activated human PMNs, these phagocytes exhibit rapid microbicidal activity toward the outgrown vegetative bacilli, independent of superoxide and nitric oxide. These findings suggest that a non-free radical pathway kills B. anthracis bacilli. We also find in PMNs an autophagic mechanism of bacterial killing based on the rapid induction of LC-3 conversion, beclin-1 expression, sequestosome 1 (S...
The continued threat of worldwide influenza pandemics, together with the yearly emergence of anti... more The continued threat of worldwide influenza pandemics, together with the yearly emergence of antigenically drifted influenza A virus (IAV) strains, underscore the urgent need to elucidate not only the mechanisms of influenza virulence, but also those mechanisms that predispose influenza patients to increased susceptibility to subsequent infection with Streptococcus pneumoniae. Glycans displayed on the surface of epithelia that are exposed to the external environment play important roles in microbial recognition, adhesion, and invasion. It is well established that the IAV hemagglutinin and pneumococcal adhesins enable their attachment to the host epithelia. Reciprocally, the recognition of microbial glycans by host carbohydrate-binding proteins (lectins) can initiate innate immune responses, but their relevance in influenza or pneumococcal infections is poorly understood. Galectins are evolutionarily conserved lectins characterized by affinity for β-galactosides and a unique sequence...
ABSTRACI'. Murine hybridoma antibodies directed against the capsule and 0-side chain determinants... more ABSTRACI'. Murine hybridoma antibodies directed against the capsule and 0-side chain determinants of the Escherichia coli strain Bort (018ac:Kl:H7) were evaluated for their ability to enhance bactericidal activity of cord blood against Kl E. coli strains possessing 0 antigens common in neonatal E. coli infection, i.e. 018, 07, and 01. The antibodies to the Kl capsule and 0-side chain efficiently enhanced cord polymorphonuclear leukocyte-mediated killing of K1 encapsulated E. coli strain possessing a homologous 0 antigen, but the IgM antibody to the K1 capsule exhibited approximately 10-fold greater activity than did the IgG3 antibody to 0-side chain (weight basis). Both antibodies required complement for their opsonic activities. Our findings indicate that antibodies directed against the capsule and 0-lipopolysaccharides are able to restore the opsonic activity of cord blood against K1 E. coli, suggesting that these antibodies may be useful in the prevention and therapy of neonatal E. coli infection. (Pediatr Res 28: 667-670, 1990) Abbreviations CFU, colony forming units LPS, lipopolysaccharide PMN, polymorphonuclear leukocyte In neonates, infections due to gram-negative bacilli are associated with significant morbidity and mortality despite appropriate antimicrobial therapy (1). The most common gram-negative organism causing sepsis and meningitis during the neonatal period is Escherichia coli (I). Given the plethora of E. coli serotypes (2), it is striking that E. coli strains possessing the K1 capsular polysaccharide are the predominant capsular serotype responsible for neonatal E. coli sepsis and meningitis (3-5) and that most of these K1 E. coli isolates are associated with a limited number of 0-LPS antigens (i.e. 0 18, 07, 0 1, 0 16) (6). We have developed a series of murine hybridomas producing MAb against different cell wall components of K1-encapsulated E. coli. MAb prepared against group B meningococcus and reactive with E. coli K1 polysaccharides and antibodies directed against 0-side chain determinants of a K1 E. coli strain were
To provide a database for the development of an O-antigen-polysaccharide-containing vaccine again... more To provide a database for the development of an O-antigen-polysaccharide-containing vaccine against Klebsiella spp., we examined the O-antigen seroepidemiology of 378 Klebsiella clinical isolates collected prospectively in two university centers. Strains were typed by competitive enzyme-linked immunosorbent assay with rabbit antisera specific for serogroups O1 to O12 and monoclonal antibodies (MAbs) specific for serogroups O1, O2ab, O2ac, and the genus-specific core antigen. The numbers of isolates (percentages) of individual O serogroups were as follows: 148 (39.2) for serogroup O1, 40 (10.6) for serogroup O2ab, 4 (1.1) for serogroup O2ac, 89 (23.6) for serogroup O3, 2 (0.5) for serogroup O4, 32 (8.5) for serogroup O5, none for serogroups O7, O9, and O12, and 21 (5.6) for serogroup O11. Forty-two (11.1) of the strains were non-O-typeable. O-serogroup distributions were virtually identical between isolates from invasive infections and those from noninvasive infections or colonizations. A vaccine containing the O-specific polysaccharides of serogroups O1, O2ab, O3, and O5 would cover 82% of clinically occurring O-antigen specificities. Three hundred thirty-eight of 378 isolates (89.4%) reacted with the genus-specific MAb V/9-5, which recognizes an epitope of the outer core region of Klebsiella lipopolysaccharide. Antibodies directed against this epitope may represent a further alternative for O-antigen-targeted immunoprophylaxis of Klebsiella infections. These data support further experimental investigations on the protective potential of O-antigen-based vaccines and/or hyperimmune globulins in Klebsiella infection.
Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell sur... more Neu1 and Neu3 are up-regulated as monocytes differentiate into DCs; and desialylation of cell surface glycoconjugates by one or both sialidase promotes cytokine production. Removal of sialic acid from glycoconjugates on the surface of monocytes enhances their response to bacterial LPS. We tested the hypothesis that endogenous sialidase activity creates a permissive state for LPS-induced cytokine production in human monocyte-derived DCs. Of the four genetically distinct sialidases (Neu1–4), Neu1, Neu3, and Neu4 are expressed in human monocytes, but only Neu1 and Neu3 are up-regulated as cells differentiate into DCs. Neu1 and Neu3 are present on the surface of monocytes and DCs and are also present intracellularly. DCs contain a greater amount of sialic acid than monocytes, but the amount of sialic acid/mg total protein declines during differentiation to DCs. This relative hyposialylation of cells does not occur in mature DCs grown in the presence of zanamivir, a pharmacologic inhibit...
MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseud... more MUC1 (MUC1 in human and Muc1 in nonhumans) is a membrane-tethered mucin that interacts with Pseudomonas aeruginosa (PA) through flagellin. In this study, we compared PA pulmonary clearance and proinflammatory responses by Muc1−/− mice with Muc1+/+ littermates following intranasal instillation of PA or flagellin. Compared with Muc1+/+ mice, Muc1−/− mice showed increased PA clearance, greater airway recruitment of neutrophils, higher levels of TNF-α and KC in bronchoalveolar lavage fluid, higher levels of TNF-α in media of flagellin-stimulated alveolar macrophages, and higher levels of KC in media of tracheal epithelial cells. Knockdown of MUC1 enhanced flagellin-induced IL-8 production by primary human bronchial epithelial cells. Expression of MUC1 in HEK293T cells attenuated TLR5-dependent IL-8 release in response to flagellin, which was completely ablated when its cytoplasmic tail was deleted. We conclude that MUC1/Muc1 suppresses pulmonary innate immunity and speculate its anti-in...
Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia e... more Background: The vascular endothelial surface is highly sialylated. Results: Vascular endothelia express catalytically active NEU1 and NEU3 sialidases, and NEU1 restrains the endothelial migratory response to wounding. Conclusion: NEU1 regulates endothelial remodeling in response to injury. Significance: Learning how NEU1 and NEU3 regulate sialylated molecules on the endothelial surface is key to understanding endothelial receptor-ligand, cell-cell, and host-pathogen interactions.
Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initia... more Background: Airway epithelia express sialoglycoproteins that respond to danger signals and initiate repair programs. Results: NEU1 sialidase desialylates EGFR and MUC1 in airway epithelia to regulate their responsiveness to ligands and adhesiveness to P. aeruginosa. Conclusion: NEU1 provides an additional level of regulation over airway epithelial responsiveness to ligands and pathogens. Significance: The downstream effects of EGFR desialylation require further investigation. Epithelial cells (ECs) lining the airways provide a protective barrier between the external environment and the internal host milieu. These same airway epithelia express receptors that respond to danger signals and initiate repair programs. Because the sialylation state of a receptor can influence its function and is dictated in part by sialidase activity, we asked whether airway epithelia express catalytically active sialidase(s). Human primary small airway and A549 ECs expressed NEU1 sialidase at the mRNA and protein levels, and NEU1 accounted for >70% of EC sialidase activity. Blotting with Maackia amurensis and peanut agglutinin lectins established epidermal growth factor receptor (EGFR) and MUC1 as in vivo substrates for NEU1. NEU1 associated with EGFR and MUC1, and NEU1-EGFR association was regulated by EGF stimulation. NEU1 overexpression diminished EGF-stimulated EGFR Tyr-1068 autophosphorylation by up to 44% but enhanced MUC1-dependent Pseudomonas aeruginosa adhesion by 1.6-1.7-fold and flagellin-stimulated ERK1/2 activation by 1.7-1.9-fold. In contrast, NEU1 depletion increased EGFR activation (1.5-fold) and diminished MUC1-mediated bacterial adhesion (38-56%) and signaling (73%). These data indicate for the first time that human airway epithelia express catalytically active NEU1 sialidase that regulates EGFR-and MUC1-dependent signaling and bacterial adhesion. NEU1 catalytic activity may offer an additional level of regulation over the airway epithelial response to ligands, pathogens, and injurious stimuli.
The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in ac... more The ability of the endospore-forming, gram-positive bacterium Bacillus anthracis to survive in activated macrophages is key to its germination and survival. In a previous publication, we discovered that exposure of primary murine macrophages to B. anthracis endospores upregulated NOS 2 concomitant with an ·NO-dependent bactericidal response. Since NOS 2 also generates O 2 · − , experiments were designed to determine whether NOS 2 formed peroxynitrite (ONOO − ) from the reaction of ·NO with O 2 · − and if so, was ONOO − microbicidal toward B. anthracis . Our findings suggest that ONOO − was formed upon macrophage infection by B. anthracis endospores; however, ONOO − does not appear to exhibit microbicidal activity toward this bacterium. In contrast, the exosporium of B. anthracis , which exhibits arginase activity, protected B. anthracis from macrophage-mediated killing by decreasing ·NO levels in the macrophage. Thus, the ability of B. anthracis to subvert ·NO production has importa...
Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on t... more Bacillus collagen-like protein of anthracis (BclA) is an immunodominant glycoprotein located on the exosporium of Bacillus anthracis . We hypothesized that antibodies to this spore surface antigen are largely responsible for the augmented immunity to anthrax that has been reported for animals vaccinated with inactivated spores and protective antigen (PA) compared to vaccination with PA alone. To test this theory, we first evaluated the capacity of recombinant, histidine-tagged, nonglycosylated BclA (rBclA) given with adjuvant to protect A/J mice against 10 times the 50% lethal dose of Sterne strain spores introduced subcutaneously. Although the animals elicited anti-rBclA antibodies and showed a slight but statistically significant prolongation in the mean time to death (MTD), none of the mice survived. Similarly, rabbit anti-rBclA immunoglobulin G (IgG) administered intraperitoneally to mice before spore inoculation increased the MTD statistically significantly but afforded protect...
The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to intera... more The amount of sialic acid on the surface of the neutrophil (PMN) influences its ability to interact with other cells. PMN activation with various stimuli mobilizes intracellular sialidase to the plasma membrane, where it cleaves sialic acid from cell surfaces. Because enhanced PMN adherence, spreading, deformability, and motility each are associated with surface desialylation and are critical to PMN diapedesis, we studied the role of sialic acid on PMN adhesion to and migration across pulmonary vascular endothelial cell (EC) monolayers in vitro. Neuraminidase treatment of either PMN or EC increased adhesion and migration in a dosedependent manner. Neuraminidase treatment of both PMNs and ECs increased PMN adhesion to EC more than treatment of either PMNs or ECs alone. Moreover, neuraminidase treatment of ECs did not change surface expression of adhesion molecules or release of IL-8 and IL-6. Inhibition of endogenous sialidase by either cross-protective antineuraminidase antibodies (45.5% inhibition) or competitive inhibition with pseudo-substrate (41.2% inhibition) decreased PMN adhesion to ECs; the inhibitable sialidase activity appeared to be associated with activated PMNs. Finally, EC monolayers preincubated with activated PMNs became hyperadhesive for subsequently added resting PMNs, and this hyperadhesive state was mediated through endogenous PMN sialidase activity. Blocking anti-E-selectin, anti-CD54 and anti-CD18 antibodies decreased PMN adhesion to tumor necrosis factor±activated ECs but not to PMN-treated ECs. These data implicate desialylation as a novel mechanism through which PMN-EC adhesion can be regulated independent of de novo protein synthesis or altered adhesion molecule expression. The ability of activated PMNs, through endogenous sialidase activity, to render the EC surface hyperadherent for unstimulated PMNs may provide for rapid amplification of the PMN-mediated host response.
Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest ... more Interactions between Bacillus anthracis (B. anthracis) and host cells are of particular interest given the implications of anthrax as a biological weapon. Inhaled B. anthracis endospores encounter alveolar macrophages as the first line of defense in the innate immune response. Yet, the consequences of this interaction remain unclear. We have demonstrated that B. anthracis uses arginase, inherent in the endospores, to reduce the ability of macrophages to produce nitric oxide (• NO) from inducible nitric oxide synthase (NOS2) by competing for L-arginine, producing Lornithine at the expense of • NO. In the current study, we used genetically engineered B. anthracis endospores to evaluate the contribution of germination and the lethal toxin (LT) in mediating signaling pathways responsible for the induction of NOS2 and ornithine decarboxylase (ODC), which is the rate-limiting enzyme in the conversion of L-ornithine into polyamines. We found that induction of NOS2 and ODC expression in macrophages exposed to B. anthracis occurs through the activation of p38 and ERK1/2 MAP kinases, respectively. Optimal induction of NOS2 was observed following exposure to germination-competent endospores, whereas ODC induction occurred irrespective of the endospores' germination capabilities and was more prominent in macrophages exposed to endospores lacking LT. Our findings suggest that activation of kinase signaling cascades that determine macrophage defense responses against B. anthracis infection occurs through distinct mechanisms.
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