bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Detection of
Helicobacter pylori
Gene Expression In Vivo and Relationship to Gastric Pathology
Authors: Peek Richard M. , van Doorn Leen-Jan , Donahue John P. , Tham Kyi T. , Figueiredo Ceu , Blaser Martin J. , Miller Geraldine G.
Published: July 2002

The
iceA
locus of
Helicobacter pylori
includes one of two mutually exclusive gene families,
iceA1
and
iceA2
. Colonization with
iceA1
strains is associated with enhanced acute mucosal inflammation, and adherence to gastric epithelial cells in vitro induces expression of
iceA1
but not
iceA2
mRNA; however, both transcripts can be detected in vivo. The aim of this study was to determine whether differing levels of
iceA
transcription in vivo may contribute to disease pathogenesis. RNA from 41
H. pylori-positive gastric biopsy specimens was reverse transcribed to cDNA. Quantitative PCR was performed using biotinylated iceA1
,
iceA2
, and 16S rRNA primers, and binding of biotinylated products to streptavidin-coated plates was detected by hybridization with a fluorescein-labeled probe.
iceA
genotypes were determined by PCR and sequence analysis. All 41 samples contained detectable
H. pylori
16S rRNA, with similar levels in
iceA1
- (
n
= 10) and
iceA2
(
n
= 31)-colonized patients (
P
= 0.34). Biopsy specimens from four (40%) and 19 (61%) persons colonized with
iceA1
or
iceA2
strains, respectively, had detectable
iceA
RNA. Acute inflammatory scores were significantly higher in
iceA1
RNA-positive patients than in
iceA1
RNA-negative,
iceA2
RNA-positive, or
iceA2
RNA-negative subjects (
P
≤ 0.05 for each). Within the
iceA2
RNA-positive group,
H. pylori
strains with a single 35-amino-acid cassette were associated with significantly higher mucosal
iceA2
transcript levels (
P
= 0.014 versus strains with two cassettes). These results indicate that the levels of transcription of
H. pylori iceA1
and
iceA2
and of 16S rRNA are independent and that particular
iceA2
gene structures are associated with enhanced transcription. The finding that
iceA1
transcription levels are significantly associated with the intensity of neutrophilic infiltration suggests that heterogeneity in inflammatory scores among persons colonized with
H. pylori iceA1
strains reflects levels of
iceA1
transcription in vivo.
New Method To Generate Enzymatically Deficient
Clostridium difficile
Toxin B as an Antigen for Immunization
Authors: Genth Harald , Selzer Jörg , Busch Christian , Dumbach Jürgen , Hofmann Fred , Aktories Klaus , Just Ingo
Published: July 2002

The family of the large clostridial cytotoxins, encompassing
Clostridium difficile
toxins A and B as well as the lethal and hemorrhagic toxins from
Clostridium sordellii
, monoglucosylate the Rho GTPases by transferring a glucose moiety from the cosubstrate UDP-glucose. Here we present a new detoxification procedure to block the enzyme activity by treatment with the reactive UDP-2′,3′-dialdehyde to result in alkylation of toxin A and B. Alkylation is likely to occur in the catalytic domain, because the native cosubstrate UDP-glucose completely protected the toxins from inactivation and the alkylated toxin competes with the native toxin at the cell receptor. Alkylated toxins are good antigens resulting in antibodies recognizing only the C-terminally located receptor binding domain, whereas formaldehyde treatment resulted in antibodies recognizing both the receptor binding domain and the catalytic domain, indicating that the catalytic domain is concealed under native conditions. Antibodies against the native catalytic domain (amino acids 1 through 546) and those holotoxin antibodies recognizing the catalytic domain inhibited enzyme activity. However, only antibodies against the receptor binding domain protected intact cells from the cytotoxic activity of toxin B, whereas antibodies against the catalytic domain were protective only when inside the cell.
Endotoxin-Induced Lung Inflammation Is Independent of the Complement Membrane Attack Complex
Authors: Brauer R. B. , Gegenfurtner C. , Neumann B. , Stadler M. , Heidecke C. D. , Holzmann B.
Published: July 2002

Several products of the activated complement system are known to modulate endothelial cell function in vitro. It has been shown that the membrane attack complex (MAC) (C5b-C9) can enhance tumor necrosis factor alpha (TNF-α)-induced expression of P- and E-selectin and intercellular adhesion molecule type 1 in cell cultures of human umbilical vein endothelial cells. In the present study the potential role of this synegism for lung injury during endotoxin-mediated septic shock in vivo was examined using a model of C6-deficient PVG (C−) (RT1C) rats and the congenic PVG (C+) (RT1C) strain. Following administration of a high (5 mg/kg) or low (0.5 mg/kg) dose of lipopolysaccharide (LPS) (Escherichia coliO55:B5), we determined the expression of cytokines, chemokines, and adhesion molecules as well as the recruitment of leukocytes in the lung. Challenge with intraperitoneal i.p. injections of LPS resulted in a strong induction of TNF-α, interleukin-1α/β, cytokine-induced neutrophil chemoattractant, interferon-inducible protein 10, macrophage inflammatory proteins 1α and 2, macrophage chemotactic protein 1, and P-selectin. However, there were no significant differences between PVG (C−) and PVG (C+) rats. Immunoperoxidase staining showed a similar increase of lung infiltration by CD11b/c+leukocytes in both rat strains. We therefore conclude that the described synergism between TNF-α and the MAC of the complement system on the induction of endothelial adhesion molecules is dispensable for inflammatory processes during endotoxin-mediated septic shock in vivo.
Cloning, Sequencing, and Role in Serum Susceptibility of Porin II from MesophilicAeromonas hydrophila
Authors: Nogueras Maria Mercé , Merino Susana , Aguilar Alicia , Benedi Vicente Javier , Tomás Juan M.
Published: July 2002

We cloned and sequenced the structural gene forAeromonas hydrophilaporin II from strain AH-3 (serogroup O:34). The genetic position of this gene, like that ofompFinEscherichia coli, is adjacent toaspCand transcribed in the same direction. However, upstream of the porin II gene no similarities withE. coliwere found. We obtained defined insertion mutants in porin II gene either inA. hydrophila(O:34) orA. veronii sobria(serogroup O:11) serum-resistant or -sensitive strains. Furthermore, we complemented these mutants with a plasmid harboring only the porin II gene, which allowed us to define the role of porin II as an important surface molecule involved in serum susceptibility and C1q binding in these strains.
Molecular and Biological Analysis of Eight Genetic Islands That Distinguish
Neisseria meningitidis
from the Closely Related Pathogen
Neisseria gonorrhoeae
Authors: Klee Silke R. , Nassif Xavier , Kusecek Barica , Merker Petra , Beretti Jean-Luc , Achtman Mark , Tinsley Colin R.
Published: July 2002

The pathogenic species
Neisseria meningitidis
and
Neisseria gonorrhoeae
cause dramatically different diseases despite strong relatedness at the genetic and biochemical levels.
N. meningitidis
can cross the blood-brain barrier to cause meningitis and has a propensity for toxic septicemia unlike
N. gonorrhoeae
. We previously used subtractive hybridization to identify DNA sequences which might encode functions specific to bacteremia and invasion of the meninges because they are specific to
N. meningitidis
and absent from
N. gonorrhoeae
. In this report we show that these sequences mark eight genetic islands that range in size from 1.8 to 40 kb and whose chromosomal location is constant. Five of these genetic islands were conserved within a representative set of strains and/or carried genes with homologies to known virulence factors in other species. These were deleted, and the mutants were tested for correlates of virulence in vitro and in vivo. This strategy identified one island, region 8, which is needed to induce bacteremia in an infant rat model of meningococcal infection. Region 8 encodes a putative siderophore receptor and a disulfide oxidoreductase. None of the deleted mutants was modified in its resistance to the bactericidal effect of serum. Neither were the mutant strains altered in their ability to interact with endothelial cells, suggesting that such interactions are not encoded by large genetic islands in
N. meningitidis
.
Clostridium difficile
Recombinant Toxin A Repeating Units as a Carrier Protein for Conjugate Vaccines: Studies of Pneumococcal Type 14,
Escherichia coli
K1, and
Shigella flexneri
Type 2a Polysaccharides in Mice
Authors: Pavliakova Danka , Moncrief J. Scott , Lyerly David M. , Schiffman Gerald , Bryla Dolores A. , Robbins John B. , Schneerson Rachel
Published: July 2002

Unlike the native protein, a nontoxic peptide (repeating unit of the native toxin designated rARU) from
Clostridium difficile
toxin A (CDTA) afforded an antigen that could be bound covalently to the surface polysaccharides of pneumococcus type 14,
Shigella flexneri
type 2a, and
Escherichia coli
K1. The yields of these polysaccharide-protein conjugates were significantly increased by prior treatment of rARU with succinic anhydride. Conjugates, prepared with rARU or succinylated (rARUsucc), were administered to mice by a clinically relevant dosage and immunization scheme. All conjugates elicited high levels of serum immunoglobulin G both to the polysaccharides and to CDTA. Conjugate-induced anti-CDTA had neutralizing activity in vitro and protected mice challenged with CDTA, similar to the rARU alone. Conjugates prepared with succinylated rARU, therefore, have potential for serving both as effective carrier proteins for polysaccharides and for preventing enteric disease caused by
C. difficile
.
Binding to and Opsonophagocytic Activity of O-Antigen-Specific Monoclonal Antibodies against Encapsulated and NonencapsulatedKlebsiella pneumoniaeSerotype O1 Strains
Authors: Held Thomas K. , Jendrike Nina R. M. , Rukavina Tomislav , Podschun Rainer , Trautmann Matthias
Published: July 2002

The high mortality of nosocomial infections caused byKlebsiellaspp. has acted as a stimulus to develop immunotherapeutic approaches targeted against surface molecules of these bacteria. Since O-antigen-specific antibodies may add to the protective effect of K antisera, we tested the functional and binding capacity of O-antigen-specific monoclonal antibodies (MAbs) raised against differentKlebsiellaO antigens. The MAbs tested were specific for the O-polysaccharide partial antigensd-galactan II (MAb Ru-O1),d-galactan I (MAb IV/4-5), or core oligosaccharide (MAb V/9-5) of theKlebsiellaserogroup O1 antigen. In enzyme-linked immunosorbent assay binding experiments, we found that all MAbs recognized their epitopes on intact capsule-free bacteria; however, binding to encapsulated wild-type strains belonging to different K-antigen serotypes was significantly reduced. The K2 antigen acted as the strongest penetration barrier, while the K7 and K21 antigens allowed some, though diminished, antibody binding. In vitro phagocytic killing experiments showed that MAb Ru-O1 possessed significant opsonizing activity for nonencapsulated O1 serogroup strains and also, to a much lesser extent, for encapsulated strains belonging to the O1:K7 and O1:K21 serotypes. MAbs or antisera specific for thed-galactan II antigen may thus be the most promising agents for further efforts to develop a second-generationKlebsiellahyperimmune globulin comprising both K- and O-antigen specificities.
l-Arginine Availability Modulates Local Nitric Oxide Production and Parasite Killing in Experimental Trypanosomiasis
Authors: Gobert Alain P. , Daulouede Sylvie , Lepoivre Michel , Boucher Jean Luc , Bouteille Bernard , Buguet Alain , Cespuglio Raymond , Veyret Bernard , Vincendeau Philippe
Published: July 2002

Nitric oxide (NO) is an important effector molecule of the immune system in eliminating numerous pathogens. Peritoneal macrophages fromTrypanosoma brucei brucei-infected mice express type II NO synthase (NOS-II), produce NO, and kill parasites in the presence ofl-arginine in vitro. Nevertheless, parasites proliferate in the vicinity of these macrophages in vivo. The present study shows thatl-arginine availability modulates NO production. Trypanosomes usel-arginine for polyamine synthesis, required for DNA and trypanothione synthesis. Moreover, arginase activity is up-regulated in macrophages from infected mice from the first days of infection. Arginase competes with NOS-II for their common substrate,l-arginine. In vitro, arginase inhibitors decreased urea production, increased macrophage nitrite production, and restored trypanosome killing. In vivo, a dramatic decrease inl-arginine concentration was observed in plasma from infected mice. In situ restoration of NO production and trypanosome killing were observed when excessl-arginine, but notd-arginine orl-arginine plusNω-nitro-l-arginine (a NOS inhibitor), was injected into the peritoneum of infected mice. These data indicate the role ofl-arginine depletion, induced by arginase and parasites, in modulating thel-arginine–NO pathway under pathophysiological conditions.
Chlamydia pneumoniae
Induces Inflammatory Changes in the Heart and Aorta of Normocholesterolemic C57BL/6J Mice
Authors: Blessing Erwin , Lin Tsun-Mei , Campbell Lee Ann , Rosenfeld Michael E. , Lloyd Deborah , Kuo Cho-chou
Published: July 2002

Chlamydia pneumoniae
infection induces inflammatory changes in blood vessels in normocholesterolemic rabbits, but it is not known whether the same phenomenon occurs in other animal models. Thus, in this study, C57BL/6J mice were inoculated with
C. pneumoniae
. Inflammatory changes in the heart or aorta were observed in a small number of chronically infected mice. No evidence of atherosclerotic lesions was found in any of the mice. These findings suggest that chronic
C. pneumoniae
infection can induce inflammatory changes in the heart and aorta of C57BL/6J mice, but does not initiate definitive atherosclerosis.
Critical Role of Neutrophils in EliminatingListeria monocytogenesfrom the Central Nervous System during Experimental Murine Listeriosis
Authors: López Santiago , Marco Alberto Jesús , Prats Neus , Czuprynski Charles J.
Published: July 2002

Neutrophils are the main inflammatory cell present in lesions involving the central nervous system (CNS) during human and murine listeriosis. In this study, administration of the neutrophil-depleting monoclonal antibody RB6-8C5 during experimental murine listeriosis facilitated the multiplication ofListeria monocytogenesin the CNS. These data suggest that neutrophils play a key role in eliminating bacteria that gain access to the CNS compartment. In addition, we provide evidence that their migration into the CNS may be necessary for the subsequent recruitment of macrophages and activated lymphocytes.
CP30, a Cysteine Proteinase Involved in
Trichomonas vaginalis
Cytoadherence
Authors: Mendoza-López M. Remedios , Becerril-Garcia Cecilia , Fattel-Facenda Loriz V. , Avila-Gonzalez Leticia , Ruíz-Tachiquín Martha E. , Ortega-Lopez Jaime , Arroyo Rossana
Published: July 2002

We describe here the participation of a
Trichomonas vaginalis
30-kDa proteinase (CP30) with affinity to the HeLa cell surface in attachment of this parasite to host epithelial cells. The CP30 band is a cysteine proteinase because its activity was inhibited by E-64, a thiol proteinase inhibitor. In two-dimensional substrate gel electrophoresis of total extracts of the trichomonad isolate CNCD 147, three spots with proteolytic activity were detected in the 30-kDa region, in the pI range from 4.5 to 5.5. Two of the spots (pI 4.5 and 5.0) bound to the surfaces of fixed HeLa cells corresponding to the CP30 band. The immunoglobulin G fraction of the rabbit anti-CP30 antiserum that recognized a 30-kDa band by Western blotting and immunoprecipitated CP30 specifically inhibited trichomonal cytoadherence to HeLa cell monolayers in a concentration-dependent manner and reacted with CP30 at the parasite surface. CP30 degraded proteins found on the female urogenital tract, including fibronectin, collagen IV, and hemoglobin. Interestingly, CP30 digested fibronectin and collagen IV only at pH levels between 4.5 and 5.0. Moreover, trichomonosis patients whose diagnosis was confirmed by in vitro culture possessed antibody to CP30 in both sera and vaginal washes, and CP30 activity was found in vaginal washes. Our results suggest that surface CP30 is a cysteine proteinase necessary for trichomonal adherence to human epithelial cells.
Serum Amyloid P Component Prevents High-Density Lipoprotein-Mediated Neutralization of Lipopolysaccharide
Authors: de Haas Carla J. C. , Poppelier Miriam J. J. G. , van Kessel Kok P. M. , van Strijp Jos A. G.
Published: July 2002

Lipopolysaccharide (LPS) is an amphipathic macromolecule that is highly aggregated in aqueous preparations. LPS-binding protein (LBP) catalyzes the transfer of single LPS molecules, segregated from an LPS aggregate, to high-density lipoproteins (HDL), which results in the neutralization of LPS. When fluorescein isothiocyanate-labeled LPS (FITC-LPS) is used, this transfer of LPS monomers to HDL can be measured as an increase in fluorescence due to dequenching of FITC-LPS. Recently, serum amyloid P component (SAP) was shown to neutralize LPS in vitro, although only in the presence of low concentrations of LBP. In this study, we show that SAP prevented HDL-mediated dequenching of FITC-LPS, even in the presence of high concentrations of LBP. Human bactericidal/permeability-increasing protein (BPI), a very potent LPS-binding and -neutralizing protein, also prevented HDL-mediated dequenching of FITC-LPS. Furthermore, SAP inhibited HDL-mediated neutralization of both rough and smooth LPS in a chemiluminescence assay quantifying the LPS-induced priming of neutrophils in human blood. SAP bound both isolated HDL and HDL in serum. Using HDL-coated magnetic beads prebound with SAP, we demonstrated that HDL-bound SAP prevented the binding of LPS to HDL. We suggest that SAP, by preventing LPS binding to HDL, plays a regulatory role, balancing the amount of LPS that, via HDL, is directed to the adrenal glands.
Application of Signature-Tagged Mutagenesis for Identification of
Escherichia coli
K1 Genes That Contribute to Invasion of Human Brain Microvascular Endothelial Cells
Authors: Badger Julie L. , Wass Carol A. , Weissman Scott J. , Kim Kwang Sik
Published: July 2002

Escherichia coli
K1 is the leading cause of gram-negative bacterial meningitis in neonates. It is principally due to our limited understanding of the pathogenesis of this disease that the morbidity and mortality rates remain unacceptably high. To identify genes required for
E. coli
K1 penetration of the blood-brain barrier (BBB), we used the negative selection strategy of signature-tagged transposon mutagenesis (STM) to screen mutants for loss or decreased invasion of human brain microvascular endothelial cells (HBMEC) which comprise the BBB. A total of 3,360 insertion mutants of
E. coli
K1 were screened, and potential HBMEC invasion mutants were subjected to a secondary invasion screen. Those mutants that failed to pass the serial invasion screens were then tested individually. Seven prototrophic mutants were found to exhibit significantly decreased invasive ability in HBMEC. We identified
traJ
and five previously uncharacterized loci whose gene products are necessary for HBMEC invasion by
E. coli
K1. In addition,
cnf1
, a gene previously shown to play a role in bacterial invasion, was identified. More importantly, a
traJ
mutant was attenuated in penetration of the BBB in the neonatal rat model of experimental hematogenous meningitis. This is the first in vivo demonstration that
traJ
is involved in the pathogenesis of
E. coli
K1 meningitis.
Phenotypic Analysis and Virulence of
Candida albicans LIG4
Mutants
Authors: Andaluz Encarnación , Calderone Richard , Reyes Guadalupe , Larriba Germán
Published: July 2002

In previous studies, we reported the isolation and preliminary characterization of a DNA ligase-encoding gene of
Candida albicans
. This gene (
LIG4
) is the structural and functional homologue of both yeast and human ligase IV, which is involved in nonhomologous end joining (NHEJ) of DNA double-strand breaks. In the present study, we have shown that there are no other
LIG4
homologues in
C. albicans
. In order to study the function of
LIG4
in morphogenesis and virulence, we constructed gene deletions.
LIG4
transcript levels were reduced in the heterozygote and were completely absent in null strains. Concomitantly, the heterozygote showed a pronounced defect in myceliation, which was slightly greater in the null strain. This was true with several solid and liquid media, such as Spider medium, medium 199, and 2% glucose–1% yeast extract–2% Bacto Peptone, at several pHs. Reintroduction of the wild-type allele into the null mutant partially restored the ability of cells to form hyphae. In agreement with the positive role of
LIG4
in morphogenesis, we detected a significant rise in mRNA levels during the morphological transition.
LIG4
is not essential for DNA replication or for the repair of DNA damage induced by ionizing radiation or UV light, indicating that these lesions are repaired primarily by homologous recombination. However, our data show that the NHEJ apparatus of
C. albicans
may control morphogenesis in this diploid organism. In addition, deletion of one or both copies of
LIG4
resulted in attenuation of virulence in a murine model of candidiasis.
Targeted Reduction in Expression ofTrypanosoma cruziSurface Glycoprotein gp90 Increases Parasite Infectivity
Authors: Málaga Sergio , Yoshida Nobuko
Published: July 2002

A previous study had shown that the expression of gp90, a stage-specific surface glycoprotein ofTrypanosoma cruzimetacyclic trypomastigotes, is inversely correlated with the parasite's ability to invade mammalian cells. By using antisense oligonucleotides complementary to a region of the gp90 gene implicated in host cell adhesion, we investigated whether the selective inhibition of gp90 synthesis affected the capacity of metacyclic forms to enter target cells. Parasites were incubated for 24 h with 20 μM PS1, a phosphorothioate oligonucleotide based on a sequence of the gp90 coding strand; PS2, the antisense counterpart of PS1; or PO2, the unmodified version of PS2 containing phosphodiester linkages, and the expression of surface molecules was analyzed by flow cytometry and immunoblotting using specific monoclonal antibodies. PS2 but not PS1 or PO2 inhibited the expression of gp90. Inhibition by PS2 was dose dependent. Northern blot analysis revealed that steady-state gp90 mRNA levels were diminished in PS2-treated parasites compared to untreated controls. Treatment with PS2 did not affect the expression of other metacyclic stage surface glycoproteins involved in parasite-host cell interaction, such as gp82 and the mucin-like gp35/50. Expression of gp90 was also inhibited by other phosphorothioate oligonucleotides targeted to the gp90 gene (PS4, PS5, PS6, and PS7) but not by PS3, with the same base composition as PS2 but a mismatched sequence. Parasites treated with PS2, PS4, or PS5 entered HeLa cells in significantly higher numbers than untreated controls, whereas the invasive capacity of PS1- and PS3-treated parasites was unchanged, confirming the inverse association between infectivity and gp90 expression.
Bordetella pertussis
Autoregulates Pertussis Toxin Production through the Metabolism of Cysteine
Authors: Bogdan John A. , Nazario-Larrieu Javier , Sarwar Jawad , Alexander Peter , Blake M. S.
Published: July 2002

Pertussis toxin (Ptx) expression and secretion in
Bordetella pertussis
are regulated by a two-component signal transduction system encoded by the
bvg
regulatory locus. However, it is not known whether the metabolic pathways and growth state of the bacterium influence synthesis and secretion of Ptx and other virulence factors. We have observed a reduction in the concentration of Ptx per optical density unit midway in fermentation. Studies were conducted to identify possible factors causing this reduction and to develop culture conditions that optimize Ptx expression. Medium reconstitution experiments demonstrated that spent medium and a fraction of this medium containing components with a molecular weight of <3,000 inhibited the production of Ptx. A complete flux analysis of the intermediate metabolism of
B. pertussis
revealed that the sulfur-containing amino acids methionine and cysteine and the organic acid pyruvate accumulated in the media. In fermentation, a large amount of internal sulfate (SO
4
2−
) was observed in early stage growth, followed by a rapid decrease as the cells entered into logarithmic growth. This loss was later followed by the accumulation of large quantities of SO
4
2−
into the media in late-stage fermentation. Release of SO
4
2−
into the media by the cells signaled the decoupling of cell growth and Ptx production. Under conditions that limited cysteine, a fivefold increase in Ptx production was observed. Addition of barium chloride (BaCl
2
) to the culture further increased Ptx yield. Our results suggest that
B. pertussis
is capable of autoregulating the activity of the
bvg
regulon through its metabolism of cysteine. Reduction of the amount of cysteine in the media results in prolonged
vir
expression due to the absence of the negative inhibitor SO
4
2−
. Therefore, the combined presence and metabolism of cysteine may be an important mechanism in the pathogenesis of
B. pertussis
.
Inhibition of Attaching and Effacing Lesion Formation following Enteropathogenic
Escherichia coli
and Shiga Toxin-Producing
E. coli
Infection
Authors: Johnson-Henry Kathene , Wallace John L. , Basappa Naveen S. , Soni Rohini , Wu Gilbert K. P. , Sherman Philip M.
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) and Shiga toxin-producing
E. coli
(STEC) induce cytoskeletal changes in infected epithelial cells. To further characterize host cytosolic responses to infection, a series of specific cell-signaling inhibitors were employed. Initial bacterial adhesion to HEp-2 epithelial cells was not reduced, whereas α-actinin accumulation in infected cells was blocked by a phosphoinositide-specific phospholipase C inhibitor (ET-18-OCH
3
), phosphoinositide 3-kinase inhibitors (wortmannin and LY294002), and a 5-lipoxygenase inhibitor, nordihydroguaretic acid. A cyclooxygenase-2 inhibitor (NS-398), however, did not block α-actinin reorganization in response to EPEC and STEC infections. Understanding signal transduction responses to enteric pathogens could provide the basis for the development of novel therapeutic strategies.
Naturally Occurring
Clostridium perfringens
Nontoxic Alpha-Toxin Variant as a Potential Vaccine Candidate against Alpha-Toxin-Associated Diseases
Authors: Schoepe Heike , Pache Christian , Neubauer Axel , Potschka Heidrun , Schlapp Tobias , Wieler Lothar H. , Baljer Georg
Published: July 2002

Clostridium perfringens
mutant strain 121A/91 shows neither enzymatic (phospholipase C) nor hemolytic activity. Nevertheless, the
cpa
gene and the corresponding alpha-toxin variant are detectable. Vaccination with this genetically constructed alpha-toxin variant, rAT121/91, induces antibodies capable of significantly reducing activities induced by wild-type toxin. Thus, rAT121/91 could be a useful vaccine candidate.
Differences in Innate Defense Mechanisms in Endotoxemia and Polymicrobial Septic Peritonitis
Authors: Echtenacher Bernd , Freudenberg Marina A. , Jack Robert S. , Männel Daniela N.
Published: July 2002

Loss, reduction, or enhancement of the ability to respond to bacterial lipopolysaccharide (LPS) has no influence on survival of mice in a model of postoperative polymicrobial septic peritonitis induced by cecal ligation and puncture (CLP). This was demonstrated by using either mice with a defectiveTlr4gene, which encodes the critical receptor molecule for LPS responses, or mice deficient for LPS binding protein (LBP) or mice sensitized to LPS byPropionibacterium acnes. Though interleukin-12 (IL-12) and gamma interferon (IFN-γ) play an important role in the sensitivity to LPS as well as in the resistance to several infections, loss of these cytokine pathways does not affect survival after CLP. Thus, neutralization of neither endogenous IL-12 nor IFN-γ altered mortality. In addition, IFN-γ receptor-deficient mice demonstrated the same sensitivity to CLP as mice with a functional IFN-γ receptor. However, administration of IFN-γ at the time of operation or pretreatment of both IFN-γ-sensitive and IFN-γ-resistant mice with IL-12 significantly enhanced mortality. This indicates that in the present infection model activation of innate defense mechanisms is not dependent on LPS recognition and does not require endogenous IL-12 or IFN-γ function. Indeed, exogenous application of these two mediators had deleterious effects.
Streptococcus pneumoniaePstS Production Is Phosphate Responsive and Enhanced during Growth in the Murine Peritoneal Cavity
Authors: Orihuela Carlos J. , Mills Jeremy , Robb Christopher W. , Wilson Corey J. , Watson David A. , Niesel David W.
Published: July 2002

Differential display-PCR (DDPCR) was used to identify aStreptococcus pneumoniaegene with enhanced transcription during growth in the murine peritoneal cavity. Northern dot blot analysis and comparative densitometry confirmed a 1.8-fold increase in expression of the encoded sequence following murine peritoneal culture (MPC) versus laboratory culture or control culture (CC). Sequencing and basic local alignment search tool analysis identified the DDPCR fragment aspstS, the phosphate-binding protein of a high-affinity phosphate uptake system. PCR amplification of the completepstSgene followed by restriction analysis and sequencing suggests a high level of conservation between strains and serotypes. Quantitative immunodot blotting using antiserum to recombinant PstS (rPstS) demonstrated an approximately twofold increase in PstS production during MPC from that during CCs, a finding consistent with the low levels of phosphate observed in the peritoneum. Moreover, immunodot blot and Northern analysis demonstrated phosphate-dependent production of PstS in six of seven strains examined. These results identifypstSexpression as responsive to the MPC environment and extracellular phosphate concentrations. Presently, it remains unclear if phosphate concentrations in vivo contribute to the regulation ofpstS. Finally, polyclonal antiserum to rPstS did not inhibit growth of the pneumococcus in vitro, suggesting that antibodies do not block phosphate uptake; moreover, vaccination of mice with rPstS did not protect against intraperitoneal challenge as assessed by the 50% lethal dose.
Role of Complement inMycobacterium aviumPathogenesis: In Vivo and In Vitro Analyses of the Host Response to Infection in the Absence of Complement Component C3
Authors: Bohlson Suzanne S. , Strasser Jennifer A. , Bower Jacquelyn J. , Schorey Jeffrey S.
Published: July 2002

We investigated the importance of the host complement system in the pathogenesis of disease mediated by the intramacrophage pathogenMycobacterium avium. Mycobacteria opsonized with complement are efficiently ingested by macrophages through various complement receptors. Furthermore, unlike other bacteria, mycobacteria can activate both the alternative and classical complement pathways in the absence of specific antibodies. Therefore, to examine the role of complement in the mycobacterial infection process in vivo, mice deficient in complement component C3 were infected withM. avium.Surprisingly, C3-deficient mice infected intravenously withM. aviumdisplayed no difference in bacterial burden or granulomatous response compared to wild-type control mice. C3-sufficient mice and C3-deficient mice were equally susceptible to infection byM. aviumregardless of the genotype at thebcglocus, a locus known to confer susceptibility to infection with intracellular pathogens. In vitro studies using mouse bone marrow-derived macrophages resulted in significantM. aviuminvasion of macrophages in the absence of C3; however, the kinetics of infection were delayed compared to complement-mediated invasion. The data indicate that complement does not play an essential role in mediatingM. aviuminfections in the mouse and suggest either that other invasion mechanisms can compensate for the absence of complement-mediated entry or that complement is not a major mycobacterial opsonin in vivo.
Toxoplasma gondiiInfection of Neurons Induces Neuronal Cytokine and Chemokine Production, but Gamma Interferon- and Tumor Necrosis Factor-Stimulated Neurons Fail To Inhibit the Invasion and Growth ofT. gondii
Authors: Schlüter D. , Deckert M. , Hof H. , Frei K.
Published: July 2002

The intracellular parasiteToxoplasmagondiihas the capacity to persist in the brain within neurons. In this study we demonstrated thatT. gondiiinfected murine cerebellar neurons in vitro and replicated within these cells. Stimulation with gamma interferon (IFN-γ) and/or tumor necrosis factor (TNF) did not enable neurons to inhibit parasite invasion and replication. Cultured neurons constitutively produced interleukin 1 (IL-1), IL-6, macrophage inflammatory protein 1α (MIP-1α), and MIP-1β but not transforming growth factor β1 (TGF-β1), IL-10, and granulocyte-macrophage colony-stimulating factor. Neuronal expression of some cytokines (IL-6, TGF-β1) and chemokines (MIP-1β) was regulated by infection and/or by IFN-γ and TNF.
Induction of Neutralizing Antibodies against Diphtheria Toxin by Priming with Recombinant
Mycobacterium bovis
BCG Expressing CRM
197
, a Mutant Diphtheria Toxin
Authors: Miyaji Eliane N. , Mazzantini Rogerio P. , Dias Waldely O. , Nascimento Ana L. T. O. , Marcovistz Rugimar , Matos Denise S. , Raw Isaias , Winter Nathalie , Gicquel Brigitte , Rappuoli Rino , Leite Luciana C. C.
Published: July 2002

BCG, the attenuated strain of
Mycobacterium bovis
, has been widely used as a vaccine against tuberculosis and is thus an important candidate as a live carrier for multiple antigens. With the aim of developing a recombinant BCG (rBCG) vaccine against diphtheria, pertussis, and tetanus (DPT), we analyzed the potential of CRM
197
, a mutated nontoxic derivative of diphtheria toxin, as the recombinant antigen for a BCG-based vaccine against diphtheria. Expression of CRM
197
in rBCG was achieved using
Escherichia coli
-mycobacterium shuttle vectors under the control of
p
BlaF*, an upregulated β-lactamase promoter from
Mycobacterium fortuitum
. Immunization of mice with rBCG-CRM
197
elicited an anti-diphtheria toxoid antibody response, but the sera of immunized mice were not able to neutralize diphtheria toxin (DTx) activity. On the other hand, a subimmunizing dose of the conventional diphtheria-tetanus vaccine, administered in order to mimic an infection, showed that rBCG-CRM
197
was able to prime the induction of a humoral response within shorter periods. Interestingly, the antibodies produced showed neutralizing activity only when the vaccines had been given as a mixture in combination with rBCG expressing tetanus toxin fragment C (FC), suggesting an adjuvant effect of rBCG-FC on the immune response induced by rBCG-CRM
197
. Isotype analysis of the anti-diphtheria toxoid antibodies induced by the combined vaccines, but not rBCG-CRM
197
alone, showed an immunoglobulin G1-dominant profile, as did the conventional vaccine. Our results show that rBCG expressing CRM
197
can elicit a neutralizing humoral response and encourage further studies on the development of a DPT vaccine with rBCG.
Interleukin-12 Promotes Pathologic Liver Changes and Death in Mice Coinfected withSchistosoma mansoniandToxoplasma gondii
Authors: Araujo Maria Ilma , Bliss Susan K. , Suzuki Yasuhiro , Alcaraz Ana , Denkers Eric Y. , Pearce Edward J.
Published: July 2002

We previously demonstrated that mice concurrently infected withSchistosoma mansoniandToxoplasma gondiiundergo accelerated mortality which is preceded by severe liver damage. Abnormally high levels of serum tumor necrosis factor alpha (TNF-α) in the dually infected mice suggested a role for this and related proinflammatory mediators in the pathologic alterations. In order to evaluate the factors involved in increased inflammatory-mediator production and mortality, interleukin-12−/−(IL-12−/−) mice were coinfected withS. mansoniandT. gondii, and survival and immune responses were monitored. These IL-12−/−mice displayed decreased liver damage and prolonged time to death relative to wild-type animals also coinfected with these parasites. Relative to the response of cells from the coinfected wild-type animals, levels of TNF-α, gamma interferon, and NO produced by splenocytes from coinfected IL-12−/−mice were reduced, and levels of IL-5 and IL-10 were increased, with the net result that the immune response of the dually infected IL-12−/−mice was similar to that of the wild-type mice infected withS. mansonialone. While dually infected wild-type animals succumb in the absence of overt parasitemia, the delayed death in the absence of IL-12 is associated with relatively uncontrolledT. gondiireplication. These data support the view thatS. mansoni-infected mice are acutely sensitive to infection withT. gondiias a result of their increased hepatic sensitivity to high levels of proinflammatory cytokines; IL-12 and TNF-α are implicated in this process.
Implication of the Structure of theHelicobacter pylori cagPathogenicity Island in Induction of Interleukin-8 Secretion
Authors: Audibert C. , Burucoa C. , Janvier B. , Fauchère J. L.
Published: July 2002

Helicobacter pylorivirulence is associated with the presence of thecagpathogenicity island (PAI). ThecagPAI is involved in the ability to induce interleukin-8 (IL-8) secretion by human cells, which is implicated in the inflammatory response of the gastric mucosa toH. pyloriinfection. The aim of this study was to determine whether the genetic structure of thecagPAI is conserved and whether it is linked to IL-8 induction ability. Detection of specific markers (cagA, picB, cag13-cag14, virD4, and IS605) by PCR and dot blot hybridization and long-distance PCR determination of the presence of cagI, cagII, and the middle region of thecagPAI were performed on 153 strains isolated from adults suffering from ulcers (n= 79) or gastritis (n= 74). IL-8 induction ability was evaluated by coculture of the strains with HEp-2 cells. Eighty-three strains (54.3%) had an entirecagPAI, 12 strains (7.8%) had thecagPAI split in two, 49 strains (32%) had nocagPAI, and 9 strains exhibited other structural combinations. The presence of an entirecagPAI was statistically correlated with the presence of IS605(P= 0.006) and the ability to induce IL-8 secretion but not with clinical presentation of the infection. The structure of thecagPAI appears to be rather conserved and is related to the proinflammatory power of a strain. The existence of strains inducing IL-8 secretion regardless of thecagPAI structure suggests that this region is not the only requirement for IL-8 secretion.
Evaluation of De-O-Acetylated Meningococcal C Polysaccharide-Tetanus Toxoid Conjugate Vaccine in Infancy: Reactogenicity, Immunogenicity, Immunologic Priming, and Bactericidal Activity against O-Acetylated and De-O-Acetylated Serogroup C Strains
Authors: Richmond Peter , Borrow Ray , Findlow Jamie , Martin Sarah , Thornton Carol , Cartwright Keith , Miller Elizabeth
Published: July 2002

The polysaccharide capsule of serogroup C
Neisseria meningitidis
(MenC) has been integral to vaccine development. Licensed MenC vaccines contain the O-acetylated (OAc+) form of polysaccharide. Some MenC strains have de-O-acetylated (OAc−) polysaccharide, which may affect antibody specificity and functional activity when used in a vaccine. We evaluated an OAc-MenC conjugate-tetanus toxoid conjugate (MCC-TT) vaccine given concomitantly with whole-cell diphtheria-tetanus-pertussis,
Haemophilus influenzae
type b, and oral polio immunization in 83 infants at 2, 3, and 4 months of age. Serum bactericidal activities (SBA) against OAc+ and OAc− MenC strains and OAc+ and OAc− polysaccharide-specific immunoglobulin G (IgG) levels were evaluated. MCC-TT vaccine was well tolerated. All infants produced SBA titers of ≥8 after a single dose at 2 months of age. The SBA geometric mean titer for OAc+ strain C11 increased from 2.7 (95% confidence interval [CI] 2.2 to 3.2) to 320 (95% CI, 237 to 432), 773 (95% CI, 609 to 982), and 1,063 (95% CI, 856 to 1319) after one, two, and three doses of MCC-TT, respectively. OAc− IgG levels were twice as high as OAc+ IgG levels after the primary series of MCC-TT vaccine, and the SBA was significantly higher against the OAc− MenC strain. Antibody responses to booster vaccination with either OAc+ MenC polysaccharide vaccine (MACP) or a fourth dose of MCC-TT at 14 months of age provided evidence of immunologic memory. The acetylation status of the booster vaccine influenced the specificity of the response, with significantly higher OAc− IgG levels and SBA after MCC-TT vaccine compared to MACP vaccine but similar OAc+ antibody levels. MCC-TT vaccine is highly immunogenic and primes for immunologic memory against OAc+ and OAc− MenC strains in infancy.
Differential Gene Expression during Meningeal-Meningococcal Interaction: Evidence for Self-Defense and Early Release of Cytokines and Chemokines
Authors: Wells Damien B. , Tighe Patrick J. , Wooldridge Karl G. , Robinson Karen , Ala' Aldeen Dlawer A. A.
Published: July 2002

Using microarray technology, we studied the early differential expression of 3,528 genes in human meningothelial cells in response to meningococcal challenge. Thirty-two genes were up-regulated, and four were down-regulated. Those up-regulated included the tumor necrosis factor alpha, interleukin-6 (IL-6), and IL-8 (but not IL-1β) genes, suggesting that meningeal cells may be a local and early source of these cytokines. Also, a trend in up-regulation of anti-apoptotic genes and down-regulation of pro-apoptotic genes was observed. This is the first evidence that meningothelial cells may mount cytoprotective responses to pathogenic bacteria.
Role ofHelicobacter pylori cagRegion Genes in Colonization and Gastritis in Two Animal Models
Authors: Eaton Kathryn A. , Kersulyte Dange , Mefford Megan , Danon Stephen J. , Krakowka Steven , Berg Douglas E.
Published: July 2002

TheHelicobacter pylorichromosomal region known as the cytotoxin-gene associated pathogenicity island (cagPAI) is associated with severe disease and encodes proteins that are believed to induce interleukin (IL-8) secretion by cultured epithelial cells. The objective of this study was to evaluate the relationship between thecagPAI, induction of IL-8, and induction of neutrophilic gastric inflammation. Germ-free neonatal piglets and conventional C57BL/6 mice were given wild-type orcagdeficient mutant derivatives ofH. pyloristrain 26695 or SS1. Bacterial colonization was determined by plate count, gastritis and neutrophilic inflammation were quantified, and IL-8 induction in AGS cells was determined by enzyme-linked immunosorbent assay. Deletion of the entirecagregion or interruption of thevirB10orvirB11homolog had no effect on bacterial colonization, gastritis, or neutrophilic inflammation. In contrast, these mutations had variable effects on IL-8 induction, depending on theH. pyloristrain. In the piglet-adapated strain 26695, which induced IL-8 secretion by AGS cells, deletion of thecagPAI decreased induction. In the mouse-adapted strain SS1, which did not induce IL-8 secretion, deletion of thecagII region or interruption of any of threecagregion genes increased IL-8 induction. These results indicate that in mice and piglets (i) neither thecagPAI nor the ability to induce IL-8 in vitro is essential for colonization or neutrophilic inflammation and (ii) there is no direct relationship between the presence of thecagPAI, IL-8 induction, and neutrophilic gastritis.
Heat-Inducible Surface Stress Protein (Hsp70) Mediates Sulfatide Recognition of the Respiratory PathogenHaemophilus influenzae
Authors: Hartmann Evamarie , Lingwood Clifford A. , Reidl Joachim
Published: July 2002

The in vitro glycolipid binding specificity of clinical strains of nontypeableHaemophilus influenzaeis altered to include sulfated glycolipids following a brief heat shock. We have constructed, expressed, and purified a recombinant protein ofH. influenzaeHsp70, which showed significant specific binding to sulfated galactolipids in vitro. Furthermore, indirect immunofluorescence demonstrates that Hsp70 proteins are surface exposed inH. influenzaeonly after heat shock and are contained in the outer membrane protein fractions.
Escherichia coliHeat-Labile Enterotoxin B Subunit Is a More Potent Mucosal Adjuvant than Its Closely Related Homologue, the B Subunit of Cholera Toxin
Authors: Millar Douglas G. , Hirst Timothy R. , Snider Denis P.
Published: July 2002

Although cholera toxin (Ctx) andEscherichia coliheat-labile enterotoxin (Etx) are known to be potent mucosal adjuvants, it remains controversial whether the adjuvanticity of the holotoxins extends to their nontoxic, receptor-binding B subunits. Here, we have systematically evaluated the comparative adjuvant properties of highly purified recombinant EtxB and CtxB. EtxB was found to be a more potent adjuvant than CtxB, stimulating responses to hen egg lysozyme when the two were coadministered to mice intranasally, as assessed by enhanced serum and secretory antibody titers as well as by stimulation of lymphocyte proliferation in spleen and draining lymph nodes. These results indicate that, although structurally very similar, EtxB and CtxB have strikingly different immunostimulatory properties and should not be considered equivalent as prospective vaccine adjuvants.
Helicobacter pyloriLipopolysaccharides Preferentially Induce CXC Chemokine Production in Human Monocytes
Authors: Innocenti M. , Svennerholm A.-M. , Quiding-Järbrink M.
Published: July 2002

Helicobacter pyloriinfection can cause duodenal ulcers and may also induce gastric adenocarcinoma. The bacteria colonize the gastric mucosa and areas of gastric metaplasia in the duodenum for decades, resulting in active chronic inflammation in the infected areas. A characteristic feature of the infection is the ongoing recruitment of neutrophils to the infected sites. To evaluate the role ofH. pylorilipopolysaccharides (LPS) in the recruitment of leukocytes to the gastric mucosa, we have examined the cytokine and chemokine production from human monocytes stimulated with LPS isolated from differentH. pyloristrains, as well as from several other gram-negative bacteria. Our results show thatH. pyloriLPS induce a large production of neutrophil-recruiting CXC chemokines (interleukin-8 and growth-related oncogene alpha) from purified human monocytes, to almost the same extent asEscherichia coliLPS. However, and in agreement with previous studies,H. pyloriLPS was much less potent in inducing production of proinflammatory cytokines by purified human monocytes and was also a weak inducer of the CC chemokine RANTES. There was no difference between LPS preparations from differentH. pyloristrains in their ability to induce cytokines and chemokines. The preferential production of CXC chemokines after stimulation withH. pyloriLPS indicates an important contribution of this molecule in maintaining neutrophil recruitment during the infection, irrespective of the infecting strain.