bluuebunny/crossref_metadata_2025 huggingface dataset:
Inflammatory Mediators in Dengue Virus Infection in Children: Interleukin-8 and Its Relationship to Neutrophil Degranulation
Authors: Juffrie M. , van der Meer G. M. , Hack C. E. , Haasnoot K. , Sutaryo  , Veerman A. J. P. , Thijs L. G.
Published: July 2002

The chemokine interleukin-8 (IL-8) has chemoattractant activity for neutrophils and is able to activate and degranulate these cells. We investigated whether IL-8 may exert these effects in children with dengue virus infection. Circulating levels of IL-8, neutrophilic elastase (a constituent of the azurophilic granula of neutrophils), and lactoferrin, released from specific granula, were measured in 186 children with dengue virus infection, 33 healthy children as negative controls and 11 children with bacterial infections as positive controls. Levels of IL-8 on admission were elevated in 71% of the dengue patients, while the elastase and lactoferrin levels were increased in 68 and 17% of patients, respectively. These levels were significantly higher than in healthy children (P< 0.05) for IL-8 and elastase but not for lactoferrin (by the Wilcoxon-Mann-Whitney [WMW] U test). Similar levels of IL-8 were found in patients with bacterial infections. Levels of IL-8 and elastase in patients with shock were significantly higher than in patients without shock (P= 0.02; WMW), but those of lactoferrin were not. IL-8 correlated with elastase and lactoferrin (r= 0.19 andP= 0.009 versusr= 0.24 andP= 0.001, respectively; two-tailed Spearman rank correlation). Thus, IL-8 levels are increased in most patients with dengue virus infection and correlate with degranulation of neutrophils as well as with some clinical and hemodynamic variables. These findings suggest a role for IL-8 in the pathogenesis of dengue virus infection.
EnterohemorrhagicEscherichia coliInduces Apoptosis Which Augments Bacterial Binding and Phosphatidylethanolamine Exposure on the Plasma Membrane Outer Leaflet
Authors: Barnett Foster Debora , Abul-Milh Maan , Huesca Mario , Lingwood Clifford A.
Published: July 2002

EnterohemorrhagicEscherichia coli(EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenicE. coli(EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.
Antibodies against Ribosomal Phosphoprotein P0 ofPlasmodium falciparumProtect Mice against Challenge withPlasmodium yoelii
Authors: Chatterjee Sanchita , Singh Subhash , Sohoni Rashmi , Singh Nevil J. , Vaidya Akhil , Long Carole , Sharma Shobhona
Published: July 2002

Antibodies against thePlasmodium falciparumP0 ribosomal phosphoprotein (PfP0) have been detected exclusively but extensively in malaria-immune persons. Polyclonal rabbit and mice sera were raised against two recombinant polypeptides ofP. falciparumP0 protein, PfP0N and PfP0C, covering amino acids 17 to 61 and the remaining amino acids 61 to 316, respectively. Sera against both these domains detected a 35-kDa protein fromPlasmodium yoeliisubsp.yoelii, a rodent malarial parasite, and stained the surface of merozoites in immunofluorescence assays. Total immunoglobulin G (IgG) purified from rabbit and mouse anti-PfP0 sera by ammonium sulfate and DEAE-cellulose chromatography was used for passive transfer experiments in mice. Mice passively immunized with both anti-PfP0N and anti-PfP0C showed distinctly lower levels of parasitemia than control mice. With immunizations on days −1, 0, 1, 3, and 5, about 50% of both sets of mice receiving anti-PfP0N and anti-PfP0C cleared the lethal 17XL strain ofP. yoeliiand revived by day 25. All the control mice died by day 10. By extending the immunization schedule, the survival period of the mice could be extended for every mouse that received anti-PfP0 IgG. These data demonstrate the cross-protection of the anti-PfP0 IgG and establish parasite P0 protein as a target for invasion-blocking antibodies.
Regulation of Cytokine Expression in Mice Immunized with Cryptococcal Polysaccharide, a Glucuronoxylomannan (GXM), Associated with Peritoneal Antigen-Presenting Cells (APC): Requirements for GXM, APC Activation, and Interleukin-12
Authors: Blackstock Rebecca , McElwee Norma , Neller Eric , Shaddix-White Jeremy
Published: July 2002

Mice immunized with peritoneal exudate cells (PEC; used as antigen-presenting cells [APC]) that are pulsed ex vivo with cryptococcal capsular polysaccharide, a glucuronoxylomannan (GXM), exhibit increased survival times and delayed-type hypersensitivity reactions when they are infected withCryptococcus neoformans. These responses are GXM specific. The present study revealed that GXM-APC immunization enhanced development of anticryptococcal type-1 cytokine responses (interleukin-2 [IL-2] and gamma interferon) in mice infected withC. neoformans. The enhancement was not GXM specific, because immunization with GXM-APC and immunization with APC alone had similar effects. GXM-APC (or APC) immunization caused small increases in the expression of type-2 cytokines (IL-4 and IL-5), but the increases were not always statistically significant. IL-10 levels were not regulated by immunization with GXM-APC or APC. GXM-APC prepared with PEC harvested from mice injected with complete Freund's adjuvant (CFA) enhanced type-1 cytokine responses, while GXM-APC prepared with PEC induced with incomplete Freund's adjuvant were ineffective. The CFA-induced PEC had an activated phenotype characterized by increased numbers of F4/80+cells that expressed CD40, B7-1, and B7-2 on their membranes. The immunomodulatory activity of the CFA-induced APC population was not attributed to their production of IL-12 because GXM-APC prepared with peritoneal cells harvested from IL-12 knockout mice or their wild-type counterparts were equally effective in augmenting the type-1 response. Blocking of IL-12 in the recipients of GXM-APC early after APC infusion revealed that early induction of IL-12 secretion was not responsible for the immunomodulatory response elicited by GXM-APC. These data, considered together with previously reported data, reveal that the protective activity of GXM-APC immunization involves both antigen-specific and nonspecific activities of GXM-APC.
Presence of Phosphorylcholine on a Filarial Nematode Protein Influences Immunoglobulin G Subclass Response to the Molecule by an Interleukin-10-Dependent Mechanism
Authors: Houston Katrina M. , Wilson Emma H. , Eyres Linda , Brombacher Frank , Harnett Margaret M. , Alexander James , Harnett William
Published: July 2002

The filarial nematode product ES-62 contains phosphorylcholine (PC) covalently attached to N-linked glycans. ES-62 induced high levels of immunoglobulin G1 (IgG1) antibodies, but no IgG2a, to non-PC epitopes of the molecule following subcutaneous injection into BALB/c mice. Conversely, mice given ES-62 lacking PC demonstrated significant production of both IgG subclasses. Thus, PC appears to block production of IgG2a antibodies to other epitopes on the parasite molecule. A role for interleukin-10 (IL-10) in this effect was shown by the ability of IL-10−/−mice to make an IgG2a antibody response to non-PC epitopes of ES-62.
Enhancement ofMycobacterium tuberculosis-Induced Tumor Necrosis Factor Alpha Production from Primary Human Monocytes by an Activated T-Cell Membrane-Mediated Mechanism
Authors: Warwick-Davies Jan , Watson Amanda J. , Griffin George E. , Krishna Sanjeev , Shattock Robin J.
Published: July 2002

Mycobacterium tuberculosisalone induces small, donor-variable amounts of tumor necrosis factor alpha (TNF-α) from primary human monocytes in vitro. However, TNF-α release is increased 5- to 500-fold when fixed activated T cells (FAT) or their isolated, unfixed membranes are added to this system. This FAT-induced synergy was at least as potent as that induced by gamma interferon (IFN-γ) at 100 U/ml. FAT-enhanced TNF-α production is at least in part transcriptionally mediated, as reflected by quantitative changes in TNF-α mRNA between 2 and 6 h poststimulation. Unlike IFN-γ-cocultured cells, FAT-treated monocytes appeared not to have enhanced TNF-α message stability, suggesting that de novo transcription may be involved in this effect. Furthermore,M. tuberculosisalone induced only minimal DNA binding of monocyte NF-κB, but cells treated withM. tuberculosisand FAT potentiated NF-κB activity more effectively. It is therefore possible that one mechanism by which FAT synergize withM. tuberculosisto stimulate TNF-α production is via NF-κB-enhanced transcription. These data strongly suggest that in the interaction of cells involved in the immune response toM. tuberculosis, T-cell stimulation of monocyte TNF-α production involves a surface membrane interaction(s) as well as soluble mediators.
Identification and Characterization of a Novel Genomic Island Integrated at
selC
in Locus of Enterocyte Effacement-Negative, Shiga Toxin-Producing
Escherichia coli
Authors: Schmidt H. , Zhang W.-L. , Hemmrich U. , Jelacic S. , Brunder W. , Tarr P. I. , Dobrindt U. , Hacker J. , Karch H.
Published: July 2002

The
selC
tRNA gene is a common site for the insertion of pathogenicity islands in a variety of bacterial enteric pathogens. We demonstrate here that
Escherichia coli
that produces Shiga toxin 2d and does not harbor the locus of enterocyte effacement (LEE) contains, instead, a novel genomic island. In one representative strain (
E
.
coli
O91:H
−
strain 4797/97), this island is 33,014 bp long and, like LEE in
E
.
coli
O157:H7, is integrated 15 bp downstream of
selC
. This
E
.
coli
O91:H
−
island contains genes encoding a novel serine protease, termed EspI; an adherence-associated locus, similar to
iha
of
E
.
coli
O157:H7; an
E
.
coli
vitamin B
12
receptor (BtuB); an AraC-type regulatory module; and four homologues of
E
.
coli
phosphotransferase proteins. The remaining sequence consists largely of complete and incomplete insertion sequences, prophage sequences, and an intact phage integrase gene that is located directly downstream of the chromosomal
selC
. Recombinant EspI demonstrates serine protease activity using pepsin A and human apolipoprotein A-I as substrates. We also detected Iha-reactive protein in outer membranes of a recombinant clone and 10 LEE-negative, Shiga toxin-producing
E
.
coli
(STEC) strains by immunoblot analysis. Using PCR analysis of various STEC, enteropathogenic
E
.
coli
, enterotoxigenic
E
.
coli
, enteroaggregative
E
.
coli
, uropathogenic
E
.
coli
, and enteroinvasive
E
.
coli
strains, we detected the
iha
homologue in 59 (62%) of 95 strains tested. In contrast,
espI
and
btuB
were present in only two (2%) and none of these strains, respectively. We conclude that the newly described island occurs exclusively in a subgroup of STEC strains that are
eae
negative and contain the variant
stx
2d
gene.
Evaluation of Cholera Vaccines Formulated with Toxin-Coregulated Pilin Peptide Plus Polymer Adjuvant in Mice
Authors: Wu Jia-Yan , Wade William F. , Taylor Ronald K.
Published: July 2002

Cholera is an acute diarrheal disease that is caused by the gram-negative bacteriumVibrio cholerae. The low efficacy of currently available killed-whole-cell vaccines and the reactinogenicity coupled with potential reversion of live vaccines have thus far precluded widespread vaccination for the control of cholera. Recent studies on the molecular nature of the virulence components that contribute toV. choleraepathogenesis have provided insights into possible approaches for the development of a defined subunit cholera vaccine. Genetic analysis has demonstrated that the toxin-coregulated pilus (TCP) is the major factor that contributes to colonization of the human intestine byV. cholerae. In addition, polyclonal and several monoclonal antibodies directed against TCP have been shown to provide passive immunity to disease in the infant mouse cholera model. In the present study, synthetic peptides corresponding to portions of the C-terminal disulfide region of TcpA pilin were formulated with polymer adjuvants currently in clinical trials and used to actively immunize adult female CD-1 mice. The experimental vaccine formulations elicited high levels of antigen-specific immunoglobulin G (IgG), including a broad spectrum of subclasses (IgG1, IgG2a, IgG2b, and IgG3), and lower levels of IgA. Infant mice born to the immunized mothers showed 100% protection against a 50% lethal dose (1 LD50) challenge and 50% protection against a 10-LD50challenge with virulent strain O395. These results indicate that specific regions of TcpA, including those delineated by the peptides used in this study, have the potential to be incorporated into an effective defined subunit vaccine for cholera.
Multiple Overlapping Epitopes in the Repetitive Unit of the Shed Acute-Phase Antigen from
Trypanosoma cruzi
Enhance Its Immunogenic Properties
Authors: Alvarez Paula , Leguizamón M. Susana , Buscaglia Carlos A. , Pitcovsky Tamara A. , Campetella Oscar
Published: July 2002

The repetitive shed acute-phase antigen (SAPA) from
Trypanosoma cruzi
was thoroughly mapped by SPOT peptides and phage display strategies, showing that a single SAPA repeat is composed of multiple overlapping B-cell epitopes. We propose that this intricate antigenic structure constitutes an alternative device to repetitiveness in order to improve its immunogenicity.
Initial Characterization of the Hemolysin Stachylysin fromStachybotrys chartarum
Authors: Vesper Stephen J. , Magnuson Matthew L. , Dearborn Dorr G. , Yike Iwona , Haugland Richard A.
Published: July 2002

Stachybotrys chartarumis a toxigenic fungus that has been associated with human health concerns, including pulmonary hemorrhage and hemosiderosis. This fungus produces a hemolysin, stachylysin, which in its apparent monomeric form has a molecular mass of 11,920 Da as determined by matrix-assisted laser desorption ionization–time of flight mass spectrometry. However, it appears to form polydispersed aggregates, which confounds understanding of the actual hemolytically active form. Exhaustive dialysis or heat treatment at 60°C for 30 min inactivated stachylysin. Stachylysin is composed of about 40% nonpolar amino acids and contains two cysteine residues. Purified stachylysin required more than 6 h to begin lysing sheep erythrocytes, but by 48 h, lysis was complete. Stachylysin also formed pores in sheep erythrocyte membranes.
Antibody Responses to Infections with Strains ofPlasmodium falciparumExpressing Diverse Forms of Merozoite Surface Protein 2
Authors: Weisman Simon , Wang Lina , Billman-Jacobe Helen , Nhan Doan Hanh , Richie Thomas L. , Coppel Ross L.
Published: July 2002

Individuals living in areas wherePlasmodium falciparumis endemic experience numerous episodes of infection. These episodes may or may not be symptomatic, with the outcome depending on a combination of parasite and host factors, several of which are poorly understood. One factor is believed to be the particular alleles of several parasite proteins to which the host is capable of mounting protective immune responses. We report a study examining antibody responses to MSP2 in 15 semi-immune teenagers and adults living in the Khanh-Hoa area of southern-central Vietnam, whereP. falciparumis highly endemic; subjects were serially infected with multiple strains ofP. falciparum. The MSP2 alleles infecting these subjects were determined by nucleotide sequencing. A total of 62 MSP2 genes belonging to both dimorphic families were identified, of which 33 contained distinct alleles, with 61% of the alleles being detected once. Clear changes in the repertoire occurred between infections. Most infections contained a mixture of parasites expressing MSP2 alleles from both dimorphic families. Two examples of reinfection with a strain expressing a previously encountered allele were detected. Significant changes in antibody levels to various regions of MSP2 were detected over the course of the experiment. There was no clear relation between the infecting form of MSP2 and the ensuing antibody response. This study highlights the complexity of host-parasite relationship for this important human pathogen.
Serological Basis for Use of Meningococcal Serogroup C Conjugate Vaccines in the United Kingdom: Reevaluation of Correlates of Protection
Authors: Borrow Ray , Andrews Nick , Goldblatt David , Miller Elizabeth
Published: July 2002

The antibody data supporting the use of meningococcal serogroup C conjugate (MCC) vaccines in the United Kingdom were generated by serum bactericidal assay (SBA) using rabbit complement (rSBA). This may give higher titers than those obtained with human complement (hSBA), for which the “gold standard” correlate of protection for meningococcal C disease is a titer of ≥4. Comparison of rSBA and hSBA titers in sera from unvaccinated adults with an rSBA titer of ≥8 showed that for 93% (27 of 29) the titer was ≥4 by hSBA, confirming natural protection. Furthermore, sera from MCC vaccinees showed that an rSBA titer of <8 or ≥128 discriminated susceptibility and protection well (85% with rSBA titers of <8 had hSBA titers of <4, and 99% with rSBA titers of ≥128 had hSBA titers of ≥4). However, discrimination was poor in the rSBA titer range 8 to 64, with only 60% having hSBA titers of ≥4. In such cases we propose that protection can be assumed if there is a fourfold rise in titer between pre- and postvaccination sera or if there is a characteristic booster response to a polysaccharide challenge dose with, if available, evidence of antibody avidity maturation or an hSBA titer of result ≥4. Applying these criteria to toddlers, 10 to 40% of whom had titers in the range 8 to 64 after a single dose of MCC vaccine, showed that 94% had a fourfold rise in titer, including 98% of those in the titer range 8 to 64. In addition, of those with titers of <128 post-MCC vaccination, 90% had titers of ≥128 after a 10-μg polysaccharide booster dose, compared with only 7% of unprimed age-matched toddlers given a full 50-μg dose. Furthermore, the increase in geometric mean avidity index pre- and postbooster was independent of post-primary MCC titer. These results indicated that the majority of toddlers with an rSBA titer between 8 and 64, and some of those with an hSBA result of <4, have mounted a protective immune response with the induction of immunological memory.
Expression of Cytokine and Chemokine Genes by Human Middle Ear Epithelial Cells Induced by Formalin-KilledHaemophilus influenzaeor Its LipooligosaccharidehtrBandrfaDMutants
Authors: Tong H. H. , Chen Y. , James M. , Van Deusen J. , Welling D. B. , DeMaria T. F.
Published: July 2002

To define the role of nontypeableHaemophilus influenzae(NTHI) lipooligosaccharide (LOS) in the induction of proinflammatory cytokine gene expression during otitis media, we compared the abilities of formalin-killed NTHI strain 2019 and its LOShtrBandrfaDmutants to stimulate human middle ear epithelial (HMEE) cell cytokine and chemokine gene expression and production in vitro. Strain DK-1, anrfaDgene mutant, expresses a truncated LOS consisting of only three deoxy-d-manno-octulosonic acid residues, a single heptose, and lipid A. Strain B29, an isogenichtrBmutant, possesses an altered oligosaccharide core and an altered lipid A. HMEE cells were incubated with formalin-killed NTHI 2019, B29, or DK-1. The supernatants and the cells were collected at 2, 4, 8, and 24 h after stimulation. Expression of genes for the cytokines tumor necrosis factor alpha (TNF-α), interleukin lβ (IL-1β), and IL-6 and for the chemokines macrophage inflammatory protein 1β (MIP-1β), monocyte chemotactic peptide 1 (MCP-1), and IL-8 was quantitated by real-time PCR. NTHI B29 did not significantly stimulate any cytokine or chemokine mRNA expression in HMEE cells. In striking contrast, NTHI 2019 induced up to 105-, 139-, and 187-fold increases in HMEE cell expression of IL-1β, TNF-α, and MIP-1β, respectively (P< 0.01 [2019 versus B29]). NTHI 2019 also induced upregulation of IL-8, IL-6, and MCP-1 mRNA expression (by 26-, 44-, and 14-fold, respectively [P< 0.05 {2019 versus B29}]). The significant induction of cytokine genes was confirmed by quantitating the secretion of cytokines in culture supernatants with an enzyme-linked immunosorbent assay. There were no significant differences in mRNA expression of IL-8, IL-6, and MCP-1 between the 2019- and DK-1-treated groups. The low levels of gene transcripts observed after incubation of HMEE cells with B29 indicate that products of the disrupted NTHIhtrBLOS gene may play a major role in induction of these particular inflammatory mediators.
Vaccination withBordetella pertussis-Pulsed Autologous or Heterologous Dendritic Cells Induces a Mucosal Antibody Response In Vivo and Protects against Infection
Authors: George-Chandy Annie , Mielcarek Nathalie , Nordström Inger , Holmgren Jan , Eriksson Kristina
Published: July 2002

This study demonstrates for the first time that vaccination with either autologous or heterologous dendritic cells (DC) pulsed with specific antigen induces protective immune responses against noninvasive bacteria, namelyBordetella pertussis. The DC-mediated protection is associated with strongB. pertussis-specific immunoglobulin G (IgG) and IgA responses in the lung.
Establishment of a Persistent
Escherichia coli
Reservoir during the Acute Phase of a Bladder Infection
Authors: Mulvey Matthew A. , Schilling Joel D. , Hultgren Scott J.
Published: July 2002

The vast majority of urinary tract infections are caused by strains of uropathogenic
Escherichia coli
that encode filamentous adhesive organelles called type 1 pili. These structures mediate both bacterial attachment to and invasion of bladder epithelial cells. However, the mechanism by which type 1 pilus-mediated bacterial invasion contributes to the pathogenesis of a urinary tract infection is unknown. Here we show that type 1-piliated uropathogens can invade the superficial epithelial cells that line the lumenal surface of the bladder and subsequently replicate, forming massive foci of intracellular
E. coli
termed bacterial factories. In response to infection, superficial bladder cells exfoliate and are removed with the flow of urine. To avoid clearance by exfoliation, intracellular uropathogens can reemerge and eventually establish a persistent, quiescent bacterial reservoir within the bladder mucosa that may serve as a source for recurrent acute infections. These observations suggest that urinary tract infections are more chronic and invasive than generally assumed.
Phagocytosis of Wild-TypeLegionella pneumophilaOccurs through a Wortmannin-Insensitive Pathway
Authors: Khelef Nadia , Shuman Howard A. , Maxfield Frederick R.
Published: July 2002

Wild-typeLegionella pneumophilagrows in human macrophages within a replicative phagosome, avoiding lysosomal fusion, while nonreplicative mutants are killed in lysosomes. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) inhibitor, blocks phagocytosis of an avirulent mutant, but not of wild-typeL. pneumophila, without affecting membrane ruffling and actin polymerization. These results show that wild-type and mutantLegionellastrains use different entry pathways. They suggest that PI3Ks are involved in phagocytosis of an avirulentL. pneumophilamutant and regulate the ability of microorganisms to generate a replicative phagosome.
Role of Endogenous Interleukin-12 in Immune Response to Staphylococcal Enterotoxin B in Mice
Authors: Lauw Fanny N. , Florquin Sandrine , Speelman Peter , van Deventer Sander J. H. , van der Poll Tom
Published: July 2002

In the present study, the roles of interleukin 12 (IL-12) and IL-18 and their possible interaction during superantigen-induced responses were studied by injection of staphylococcal enterotoxin B (SEB) into mice. These data suggest that the role of IL-12 in SEB-induced responses is limited to sustaining gamma interferon release by an IL-18-independent mechanism.
STK Receptor Tyrosine Kinase Regulates Susceptibility to Infection withListeria monocytogenes
Authors: Lutz Michael A. , Gervais Francine , Bernstein Alan , Hattel Arthur L. , Correll Pamela H.
Published: July 2002

We have previously identified the STK receptor tyrosine kinase as a key regulator of macrophage activation and cell-mediated immune responses. Here we demonstrate that, although MSP activation of STK inhibits NO production by macrophages in response to heat-killedListeria monocytogenes,STK-deficient mice exhibit increased susceptibility to infection withListeria.
Modulation of Gamma Interferon-Induced Major Histocompatibility Complex Class II Gene Expression byPorphyromonas gingivalisMembrane Vesicles
Authors: Srisatjaluk Ratchapin , Kotwal Girish J. , Hunt Lawrence A. , Justus David E.
Published: July 2002

Gamma interferon (IFN-γ)-induced endothelial cells actively participate in initiating immune responses by interacting with CD4+T cells via class II major histocompatibility complex (MHC) surface glycoproteins. Previously,Porphyromonas gingivalismembrane vesicles were shown to selectively inhibit IFN-γ-induced surface expression of HLA-DR molecules by human umbilical cord vascular endothelial cells. In this study, we demonstrated an absence of HLA-DRα mRNA from IFN-γ-induced cells in the presence ofP. gingivalismembrane vesicles by using reverse transcriptase-PCR and Southern blotting. Vesicles also prevented transcription of the gene encoding class II transactivator, a transactivator protein required for IFN-γ-induced expression of MHC class II genes. In addition, the effects of vesicles on IFN-γ signal transduction involving Jak and Stat proteins were characterized by using immunoprecipitation and Western blot analyses. Jak1 and Jak2 proteins could not be detected in endothelial cells treated with membrane vesicles. Consequently, IFN-γ-induced phosphorylation of Jak1, Jak2, and Stat1α proteins was prevented. The class II-inhibitory effect of the membrane vesicles could be eliminated by heating vesicles at 100°C for 30 min or by treating them with a cysteine proteinase inhibitor. This indicates that the cysteine proteinases were most likely responsible for the absence of Jak proteins observed in vesicle-treated cells. The observed increased binding of radiolabeled IFN-γ to vesicle-treated cells suggests that vesicles may also modulate the IFN-γ interactions with the cell surface. However, no evidence was obtained demonstrating that vesicles affected the expression of IFN-γ receptors. Thus,P. gingivalismembrane vesicles apparently inhibited IFN-γ-induced MHC class II by disrupting the IFN-γ signaling transduction pathway. Vesicle-inhibited class II expression also occurred in other IFN-γ-inducible cells. This suggested that the ability ofP. gingivalismembrane vesicles to modulate antigen presentation by key cells may be an important mechanism used by this particular bacterium to escape immunosurveillance, thereby favoring its colonization and invasion of host tissues.
Balance of Pro- and Anti-Inflammatory Cytokines Correlates with Outcome of Acute ExperimentalPseudomonas aeruginosaKeratitis
Authors: Thakur A. , Xue M. , Stapleton F. , Lloyd A. R. , Wakefield D. , Willcox M. D. P.
Published: July 2002

The purpose of this study was to elucidate the expression of pro- and anti-inflammatory cytokines in mouse corneas infected withPseudomonas aeruginosa. Three bacterial strains (invasive, cytotoxic, or CLARE [contact lens-induced acute red eye]) which have recently been shown to produce distinct patterns of corneal disease in the mouse were used. The left mouse (BALB/c) corneas were scarified and infected with 2 × 106CFU of one of the threeP. aeruginosastrains, while right eyes served as controls. Animals were examined at 1, 4, 8, 16, and 24 h with a slit lamp biomicroscope to grade the severity of infection. Following examination, eyes were collected and processed for histopathology, multiprobe RNase protection assay for cytokine mRNA, enzyme-linked immunosorbent assay to quantitate cytokine proteins, and myeloperoxidase activity to quantitate polymorphonuclear leukocytes. The kinetics of appearance and magnitude of expression of key cytokines varied significantly in the three different phenotypes ofP. aeruginosainfection. The predominant cytokines expressed in response to all three phenotypes were interleukin-1β (IL-1β), IL-1Ra, and IL-6. In response to the invasive strain, which induced severe corneal inflammation, significantly lower ratios of IL-1Ra to IL-1β were present at all time points, whereas corneas challenged with the CLARE strain, which induced very mild inflammation, showed a high ratio of IL-1Ra to IL-1β. The outcome of infection in bacterial keratitis correlated with the relative induction of these pro- and anti-inflammatory cytokines, and exogenous administration of recombinant rIL-1Ra (rIL-1Ra) was able to reduce the disease severity significantly. These findings point to the therapeutic potential of rIL-1Ra protein in possible treatment strategies for bacterial keratitis.
Cloning of a Mucin-Desulfating Sulfatase Gene from
Prevotella
Strain RS2 and Its Expression Using a
Bacteroides
Recombinant System
Authors: Wright Damian P. , Knight Catriona G. , Parkar Shanthi G. , Christie David L. , Roberton Anthony M.
Published: July 2002

A gene encoding the mucin-desulfating sulfatase in
Prevotella
strain RS2 has been cloned, sequenced, and expressed in an active form. A 600-bp PCR product generated using primers designed from amino acid sequence data was used to isolate a 5,058-bp genomic DNA fragment containing the mucin-desulfating sulfatase gene. A 1,551-bp open reading frame encoding the sulfatase proprotein was identified, and the deduced 517-amino-acid protein minus its signal sequence corresponded well with the published mass of 58 kDa estimated by denaturing gel electrophoresis. The sulfatase sequence showed homology to aryl- and nonarylsulfatases with different substrate specificities from the sulfatases of other organisms. No sulfatase activity could be detected when the sulfatase gene was cloned into
Escherichia coli
expression vectors. However, cloning the gene into a
Bacteroides
expression vector did produce active sulfatase. This is the first mucin-desulfating sulfatase to be sequenced and expressed. A second open reading frame (1,257 bp) was identified immediately upstream from the sulfatase gene, coding in the opposite direction. Its sequence has close homology to iron-sulfur proteins that posttranslationally modify other sulfatases. By analogy, this protein is predicted to catalyze the modification of a serine group to a formylglycine group at the active center of the mucin-desulfating sulfatase, which is necessary for enzymatic activity.
Synechocystis
Strain PCC 6803
cya
2, a Prokaryotic Gene That Encodes a Guanylyl Cyclase
Authors: Ochoa de Alda Jesús A. G. , Ajlani Ghada , Houmard Jean
Published: July 2002

Synechocystis
strain PCC 6803 exhibits similar levels of cyclic AMP (cAMP) and cyclic GMP (cGMP). A thorough analysis of its genome showed that Cya2 (Sll0646) has all the sequence determinants required in terms of activity and purine specificity for being a guanylyl cyclase. Insertional mutagenesis of
cya2
caused a marked reduction in cGMP content without altering the cAMP content. Thus, Cya2 represents the first example of a prokaryotic guanylyl cyclase.
Transcriptional Control of the Citrate-Inducible
citMCDEFGRP
Operon, Encoding Genes Involved in Citrate Fermentation in
Leuconostoc paramesenteroides
Authors: Martín Mauricio , Magni Christian , López Paloma , de Mendoza Diego
Published: July 2002

In this study we describe the expression pattern of the
Leuconostoc paramesenteroides citMCDEFGRP
operon in response to the addition of citrate to the growth medium. An 8.8-kb polycistronic transcript, which includes the
citMCDEFGRP
genes, was identified; its synthesis was dramatically induced upon addition of citrate to the growth medium. We also found that expression of the
cit
operon is subjected to posttranscriptional regulation, since processing sites included in four complex secondary structures (I, II, III, and IV) were identified by Northern blot analysis and mapped by primer extension. Upstream of the
citMCDEFGRP
operon a divergent open reading frame, whose expression was also increased by citrate, was identified by DNA sequencing and designated
citI
. The start and end sites of transcription of the
cit
operon and
citI
gene were mapped. The start sites are separated by a stretch of 188 bp with a very high A+T content of 77% and are preceded by transcriptional promoters. The end sites of the transcripts are located next to the 3′ end of two secondary structures characteristic of ρ-independent transcriptional terminators. The effect of the
citI
gene on expression of the
cit
operon was studied in
Escherichia coli
. The presence of the
citI
gene in
cis
and in
trans
resulted in increased activity of the
cit
promoter. These data provide the first evidence that citrate fermentation in
Leuconostoc
is regulated at the transcriptional level by a transcriptional activator rather than by a repressor.
Characterization of Spores of
Bacillus subtilis
Which Lack Dipicolinic Acid
Authors: Paidhungat Madan , Setlow Barbara , Driks Adam , Setlow Peter
Published: July 2002

Spores of
Bacillus subtilis
with a mutation in
spoVF
cannot synthesize dipicolinic acid (DPA) and are too unstable to be purified and studied in detail. However, the spores of a strain lacking the three major germinant receptors (termed Δ
ger3
), as well as
spoVF
, can be isolated, although they spontaneously germinate much more readily than Δ
ger3
spores. The Δ
ger3 spoVF
spores lack DPA and have higher levels of core water than Δ
ger3
spores, although sporulation with DPA restores close to normal levels of DPA and core water to Δ
ger3 spoVF
spores. The DPA-less spores have normal cortical and coat layers, as observed with an electron microscope, but their core region appears to be more hydrated than that of spores with DPA. The Δ
ger3 spoVF
spores also contain minimal levels of the processed active form (termed P
41
) of the germination protease, GPR, a finding consistent with the known requirement for DPA and dehydration for GPR autoprocessing. However, any P
41
formed in Δ
ger3 spoVF
spores may be at least transiently active on one of this protease's small acid-soluble spore protein (SASP) substrates, SASP-γ. Analysis of the resistance of wild-type, Δ
ger3
, and Δ
ger3 spoVF
spores to various agents led to the following conclusions: (i) DPA and core water content play no role in spore resistance to dry heat, dessication, or glutaraldehyde; (ii) an elevated core water content is associated with decreased spore resistance to wet heat, hydrogen peroxide, formaldehyde, and the iodine-based disinfectant Betadine; (iii) the absence of DPA increases spore resistance to UV radiation; and (iv) wild-type spores are more resistant than Δ
ger3
spores to Betadine and glutaraldehyde. These results are discussed in view of current models of spore resistance and spore germination.
Identification of
Brucella abortus
OxyR and Its Role in Control of Catalase Expression
Authors: Kim Jeong-a , Mayfield John
Published: July 2002

We report the cloning and sequencing of the
Brucella abortus oxyR
homolog and provide evidence that the transcription product of this gene binds to the
B. abortus
catalase promoter region. A gene replacement/deletion
Brucella oxyR
mutant exhibits increased sensitivity to prolonged exposure to H
2
O
2
and is unable to adapt to H
2
O
2
in the environment.
YsxC, a Putative GTP-Binding Protein Essential for Growth of
Bacillus subtilis
168
Authors: Prágai Zoltán , Harwood Colin R.
Published: July 2002

YsxC is a member of a family of GTP-binding proteins carried by a diverse range of organisms from bacteria to yeasts, plants, and humans. To resolve the issue of whether
ysxC
of
Bacillus subtilis
is essential for growth, we attempted to construct mutants in which
ysxC
was either inactivated or placed under the control of an inducible promoter. Viable mutants were obtained only in the latter case, and these were inducer dependent, demonstrating unambiguously that
ysxC
is an essential gene.
Extracellular Oxidoreduction Potential Modifies Carbon and Electron Flow in
Escherichia coli
Authors: Riondet Christophe , Cachon Rémy , Waché Yves , Alcaraz Gérard , Diviès Charles
Published: July 2002

Wild-type
Escherichia coli
K-12 ferments glucose to a mixture of ethanol and acetic, lactic, formic, and succinic acids. In anoxic chemostat culture at four dilution rates and two different oxidoreduction potentials (ORP), this strain generated a spectrum of products which depended on ORP. Whatever the dilution rate tested, in low reducing conditions (−100 mV), the production of formate, acetate, ethanol, and lactate was in molar proportions of approximately 2.5:1:1:0.3, and in high reducing conditions (−320 mV), the production was in molar proportions of 2:0.6:1:2. The modification of metabolic fluxes was due to an ORP effect on the synthesis or stability of some fermentation enzymes; thus, in high reducing conditions, lactate dehydrogenase-specific activity increased by a factor of 3 to 6. Those modifications were concomitant with a threefold decrease in acetyl-coenzyme A (CoA) needed for biomass synthesis and a 0.5- to 5-fold decrease in formate flux. Calculations of carbon and cofactor balances have shown that fermentation was balanced and that extracellular ORP did not modify the oxidoreduction state of cofactors. From this, it was concluded that extracellular ORP could regulate both some specific enzyme activities and the acetyl-CoA needed for biomass synthesis, which modifies metabolic fluxes and ATP yield, leading to variation in biomass synthesis.
prpR
,
ntrA
, and
ihf
Functions Are Required for Expression of the
prpBCDE
Operon, Encoding Enzymes That Catabolize Propionate in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Palacios Sergio , Escalante-Semerena Jorge C.
Published: July 2002

The genes required for the catabolism of propionate in
Salmonella enterica
serovar Typhimurium are organized as two transcriptional units (
prpR
and
prpBCDE
) that are divergently transcribed from one another. Sequence homology to genes encoding members of the sigma-54 family of transcriptional activators and the identification of a consensus sigma-54 promoter 5′ to the
prpBCDE
operon suggested that PrpR was required to activate expression of this operon. We isolated insertions in
prpR
and showed that
prpR
function was needed for growth on propionate as a carbon and energy source. A medium-copy-number plasmid carrying the
lacZ
gene under the control of the native sigma-54 promoter of
prpBCDE
was used to study
prpBCDE
operon expression. Transcription of the
lacZ
reporter in
prpR
,
ntrA
, and
ihfB
mutants was 85-, 83-, and 15-fold lower than the level of transcription measured in strains carrying the wild-type allele of the gene tested. These data indicated that PrpR, IHF, and transcription sigma factor RpoN were required for the expression of the
prpBCDE
operon. Further analysis of the involvement of the integration host factor (IHF) protein in the expression of this operon is required due to the well-documented pleiotropic effect the lack of this global regulator has on gene expression. Deletion of the 5′ 615-bp portion of the
prpR
gene resulted in a PrpR
c
mutant protein that activated
prpBCDE
transcription regardless of the ability of the strain to synthesize 2-methylcitrate, the putative coactivator of PrpR. These results indicate that the N terminus of PrpR is the coactivator-sensing domain of the protein. When placed under the control of the arabinose-inducible promoter P

araBAD

, expression of

prpR
c

allele by arabinose had a strong negative effect on growth of the cell. It is proposed that this deleterious effect of PrpR
c
may be due to an uncontrolled ATPase activity of PrpR or to cross-activation of genes whose functions negatively affect cell growth under the conditions tested.
The Detergent-Soluble Maltose Transporter Is Activated by Maltose Binding Protein and Verapamil
Authors: Reich-Slotky Ronit , Panagiotidis Cynthia , Reyes Moraima , Shuman Howard A.
Published: July 2002

The maltose transporter FGK2 complex of
Escherichia coli
was purified with the aid of a glutathione
S
-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the
V
max
of the ATPase 2.7-fold but had no effect on the

K
m

of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN
3
, as well as a mercurial,
p
-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.
Calcofluor Antifungal Action Depends on Chitin and a Functional High-Osmolarity Glycerol Response (HOG) Pathway: Evidence for a Physiological Role of the
Saccharomyces cerevisiae
HOG Pathway under Noninducing Conditions
Authors: García-Rodriguez L. J. , Durán A. , Roncero C.
Published: July 2002

We have isolated several
Saccharomyces cerevisiae
mutants resistant to calcofluor that contain mutations in the
PBS2
or
HOG1
genes, which encode the mitogen-activated protein kinase (MAPK) and MAP kinases, respectively, of the high-osmolarity glycerol response (HOG) pathway. We report that blockage of either of the two activation branches of the pathway, namely,
SHO1
and
SLN1
, leads to partial resistance to calcofluor, while simultaneous disruption significantly increases resistance. However, chitin biosynthesis is independent of the HOG pathway. Calcofluor treatment also induces an increase in salt tolerance and glycerol accumulation, although no activation of the HOG pathway is detected. Our results indicate that the antifungal effect of calcofluor depends on its binding to cell wall chitin but also on the presence of a functional HOG pathway. Characterization of one of the mutants isolated,
pbs2-14
, revealed that resistance to calcofluor and HOG-dependent osmoadaptation are two different physiological processes. Sensitivity to calcofluor depends on the constitutive functionality of the HOG pathway; when this is altered, the cells become calcofluor resistant but also show very low levels of basal salt tolerance. Characterization of some multicopy suppressors of the calcofluor resistance phenotype indicated that constitutive HOG functionality participates in the maintenance of cell wall architecture, a conclusion supported by the antagonism observed between the protein kinase and HOG signal transduction pathways.
Phosphorylation of HPr by the Bifunctional HPr Kinase/P-Ser-HPr Phosphatase from
Lactobacillus casei
Controls Catabolite Repression and Inducer Exclusion but Not Inducer Expulsion
Authors: Dossonnet Valérie , Monedero Vicente , Zagorec Monique , Galinier Anne , Pérez-Martínez Gaspar , Deutscher Josef
Published: July 2002

We have cloned and sequenced the
Lactobacillus casei hprK
gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant
L. casei
HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When
hprK
was inactivated, carbon catabolite repression of
N
-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the
hprK
mutant. However, inducer expulsion of
methyl
β-
d
-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in
ptsH
mutants altered at Ser-46 of HPr and the
hprK
mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.
H-NS Is a Repressor of the
Proteus mirabilis
Urease Transcriptional Activator Gene
ureR
Authors: Coker Christopher , Bakare Olubunmi O. , Mobley Harry L. T.
Published: July 2002

Expression of
Proteus mirabilis
urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A
6
TA
2
CA
2
TGGTA
5
GA
6
TGA
5
, is located 16 bp upstream of the ς
70
-like
ureR
promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured β-galactosidase activity of wild-type
Escherichia coli
MC4100 (H-NS
+
) and its isogenic derivative ATM121 (
hns
::Tn
10
) (H-NS
−
) harboring a
ureR-lacZ
operon fusion plasmid (pLC9801). β-Galactosidase activity in the H-NS
−
host strain was constitutive and sevenfold greater (
P
< 0.0001) than that in the H-NS
+
host. A recombinant plasmid containing cloned
P. mirabilis hns
was able to complement and restore repression of the
ureR
promoter in the H-NS
−
host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in β-galactosidase activity in the H-NS
+
host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS
−
host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of
P. mirabilis
) was equivalent in both the H-NS
−
background and the H-NS
+
background in the presence of urea but was eightfold greater (
P
= 0.0001) in the H-NS
−
background in the absence of urea. We conclude that H-NS represses
ureR
expression in the absence of urea induction.