bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACTNitric 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

ABSTRACT

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

ABSTRACTNeutrophils 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

ABSTRACT

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

ABSTRACTLipopolysaccharide (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

ABSTRACT

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

ABSTRACT

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

ABSTRACTA 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTLoss, 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

ABSTRACTDifferential 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.