bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative Fluorogenic PCR Assay for Measuring Ovine Herpesvirus 2 Replication in Sheep
Authors: Hüssy D. , Stäuber N. , Leutenegger C. M. , Rieder S. , Ackermann M.
Published: July 2002

A fluorogenic PCR specific for ovine herpesvirus 2 (OvHV-2) DNA was developed and compared to a previously established conventional seminested PCR. Testing of a total of 152 blood samples from both positive and negative animals revealed that the results of both assays corresponded to each other in 100% of the cases. A second fluorogenic PCR for genomic sheep DNA was required to normalize the quantity of viral DNA in the sample. Separate standard curves had to be constructed for each PCR. The analytical sensitivity of the new PCRs ranged between at least 10 copies and sometimes even 1 copy of target DNA per reaction mixture. In dilution series of the target DNAs, linear decreases of the signals were observed over 7 orders of magnitude. Thus, it was possible to calculate the amounts of viral DNA in relation to the amounts of cellular DNA by normalizing the absolute quantity of OvHV-2 DNA with the amount of genomic sheep DNA. By this technique, it was possible for the first time to quantitatively characterize the course of OvHV-2 replication in naturally infected sheep.
Changes in CD4
+
, CD8
+
, CD4
+
CD8
+
, and Immunoglobulin M-Positive Peripheral Blood Mononuclear Cells of Postweaning Multisystemic Wasting Syndrome-Affected Pigs and Age-Matched Uninfected Wasted and Healthy Pigs Correlate with Lesions and Porcine Circovirus Type 2 Load in Lymphoid Tissues
Authors: Darwich Laila , Segalés Joaquim , Domingo Mariano , Mateu Enric
Published: July 2002

Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4
+
, CD8
+
, CD4
+
CD8
+
(double-positive [DP]), and immunoglobulin M-positive (IgM
+
) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8
+
and DP cell subsets compared to the other groups (
P
< 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (
P
< 0.05) as well as to the relative decrease in IgM
+
and CD8
+
cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.
Cell-Associated Interleukin-8 in Cord Blood of Term and Preterm Infants
Authors: Dembinski J. , Behrendt D. , Heep A. , Dorn C. , Reinsberg J. , Bartmann P.
Published: July 2002

To assess the effect of gestational age and labor on the interleukin-8 (IL-8) concentration in whole cord blood and serum, IL-8 levels were determined simultaneously in cord blood serum and lysate in 134 infants. Following the elimination of some of the samples due to exclusion criteria, the data for 99 uninfected infants (71 term and 28 preterm) and 9 infants with neonatal bacterial infection delivered either vaginally or by elective or emergency cesarean section were analyzed. The effects of labor and gestational age were tested by analysis of variance. IL-8 was not detectable in the serum of 25 infants, whereas IL-8 levels in whole blood were measurable in all of the samples. The median IL-8 conncentrations in whole cord blood lysate were 106 pg/ml (range, 20 to 415 pg/ml) in preterm infants and 176 pg/ml (range, 34 to 1,667 pg/ml) in term infants. In contrast to the IL-8 levels in serum, IL-8 levels in whole blood were reduced after ECS. Gestational age had no independent effect on the IL-8 concentrations in either serum or whole blood; these concentrations increased in infected infants after labor. We conclude that the neonatal proinflammatory response to labor stress was more evident in the concentrations of IL-8 in whole blood than in serum. The levels of IL-8 in whole-blood lysate reflect proinflammatory stimulation in neonates and may be a useful diagnostic tool for the early diagnosis of neonatal infection.
Pathogenesis of Cerebral Malaria: Recent Experimental Data and Possible Applications for Humans
Authors: Lou Jinning , Lucas Ralf , Grau Georges E.
Published: July 2002

Malaria still is a major public health problem, partly because the pathogenesis of its major complication, cerebral malaria, remains incompletely understood. Experimental models represent useful tools to better understand the mechanisms of this syndrome. Here, data generated by several models are reviewed both in vivo and in vitro; we propose that some pathogenic mechanisms, drawn from data obtained from experiments in a mouse model, may be instrumental in humans. In particular, tumor necrosis factor (TNF) receptor 2 is involved in this syndrome, implying that the transmembrane form of TNF may be more important than the soluble form of the cytokine. It has also been shown that in addition to differences in immune responsiveness between genetically resistant and susceptible mice, there are marked differences at the level of the target cell of the lesion, namely, the brain endothelial cell. In murine cerebral malaria, a paradoxical role of platelets has been proposed. Indeed, platelets appear to be pathogenic rather than protective in inflammatory conditions because they can potentiate the deleterious effects of TNF. More recently, it has been shown that interactions among platelets, leukocytes, and endothelial cells have phenotypic and functional consequences for the endothelial cells. A better understanding of these complex interactions leading to vascular injury will help improve the outcome of cerebral malaria.
Toxins, Butyric Acid, and Other Short-Chain Fatty Acids Are Coordinately Expressed and Down-Regulated by Cysteine in
Clostridium difficile
Authors: Karlsson Sture , Lindberg Anette , Norin Elisabeth , Burman Lars G. , Åkerlund Thomas
Published: July 2002

It was recently found that a mixture of nine amino acids down-regulate
Clostridium difficile
toxin production when added to peptone yeast extract (PY) cultures of strain VPI 10463 (S. Karlsson, L. G. Burman, and T. Åkerlund, Microbiology 145:1683–1693, 1999). In the present study, seven of these amino acids were found to exhibit a moderate suppression of toxin production, whereas proline and particularly cysteine had the greatest impact, on both reference strains (
n
= 6) and clinical isolates (
n
= 28) of
C. difficile
(>99% suppression by cysteine in the highest toxin-producing strain). Also, cysteine derivatives such as acetylcysteine, glutathione, and cystine effectively down-regulated toxin expression. An impact of both cysteine and cystine but not of thioglycolate on toxin yield indicated that toxin expression was not regulated by the oxidation-reduction potential. Several metabolic pathways, including butyric acid and butanol production, were coinduced with the toxins in PY and down-regulated by cysteine. The enzyme 3-hydroxybutyryl coenzyme A dehydrogenase, a key enzyme in solventogenesis in
Clostridium acetobutylicum
, was among the most up-regulated proteins during high toxin production. The addition of butyric acid to various growth media induced toxin production, whereas the addition of butanol had the opposite effect. The results indicate a coupling between specific metabolic processes and toxin expression in
C. difficile
and that certain amino acids can alter these pathways coordinately. We speculate that down-regulation of toxin production by the administration of such amino acids to the colon may become a novel approach to prophylaxis and therapy for
C. difficile
-associated diarrhea.
Modulation of Neutrophil Chemokine Receptors byStaphylococcus aureusSupernate
Authors: Veldkamp K. E. , Heezius H. C. J. M. , Verhoef J. , van Strijp J. A. G. , van Kessel K. P. M.
Published: July 2002

In a previous study, we showed thatStaphylococcus aureussupernate (SaS) is a potent agonist for both neutrophils and mononuclear cells. To further investigate the immunomodulating effects of SaS, the effect on different neutrophil receptors was studied. Expression of various neutrophil receptors, before and after treatment with SaS, was quantified by flow cytometry. We found that SaS treatment of neutrophils resulted in a specific and total downregulation of the C5a and the fMLP receptor, both serpentine receptors, while other receptors were totally unaffected. Since these two receptors are both involved in chemotaxis, we tested the effect of SaS in calcium flux and chemotaxis assays. We showed that preincubation with SaS abrogated the rise in intracellular calcium concentration upon triggering with fMLP and C5a. We also showed that SaS is a potent inhibitor of neutrophil chemotaxis towards fMLP and C5a, but does not interfere with chemotaxis towards interleukin-8. These findings indicate thatS. aureusproduces a virulence factor extracellularly, which impairs chemotaxis towards the infected site.
Helicobacter pylori
Possesses Two CheY Response Regulators and a Histidine Kinase Sensor, CheA, Which Are Essential for Chemotaxis and Colonization of the Gastric Mucosa
Authors: Foynes Susan , Dorrell Nick , Ward Stephen J. , Stabler Richard A. , McColm Andy A. , Rycroft Andrew N. , Wren Brendan W.
Published: July 2002

Infection of the mucous layer of the human stomach by
Helicobacter pylori
requires the bacterium to be motile and presumably chemotactic. Previous studies have shown that fully functional flagella are essential for motility and colonization, but the role of chemotaxis remains unclear. The two-component regulatory system CheA/CheY has been shown to play a major role in chemotaxis in other enteric bacteria. Scrutiny of the 26695 genome sequence suggests that
H. pylori
has two CheY response regulators: one a separate protein (CheY1) and the other (CheY2) fused to the histidine kinase sensor CheA. Defined deletion mutations were introduced into
cheY1
,
cheY2
, and
cheA
in
H. pylori
strains N6 and SS1. Video tracking revealed that the wild-type
H. pylori
strain moves in short runs with frequent direction changes, in contrast to movement of
cheY2
,
cheAY2
, and
cheAY2 cheY1
mutants, whose motion was more linear. The
cheY1
mutant demonstrated a different motility phenotype of rapid tumbling. All mutants had impaired swarming and greatly reduced chemotactic responses to hog gastric mucin. Neither
cheY1
nor
cheAY2
mutants were able to colonize mice, but they generated a significant antibody response, suggesting that despite impaired chemotaxis, these mutants were able to survive in the stomach long enough to induce an immune response before being removed by gastric flow. Additionally, we demonstrated that
cheY1
failed to colonize gnotobiotic piglets. This study demonstrates the importance of the roles of
cheY1
,
cheY2
, and
cheA
in motility and virulence of
H. pylori
.
Characterization of Conserved T- and B-Cell Epitopes inPlasmodium falciparumMajor Merozoite Surface Protein 1
Authors: Parra Marcela , Hui George , Johnson Armead H. , Berzofsky Jay A. , Roberts Theodore , Quakyi Isabella A. , Taylor Diane W.
Published: July 2002

Vaccines forP. falciparumwill need to contain both T- and B-cell epitopes. Conserved epitopes are the most desirable, but they are often poorly immunogenic. The major merozoite surface protein 1 (MSP-1) is currently a leading vaccine candidate antigen. In this study, six peptides from conserved or partly conserved regions of MSP-1 were evaluated for immunogenicity in B10 congenic mice. Following immunization with the peptides, murine T cells were tested for the ability to proliferate in vitro and antibody responses to MSP-1 were evaluated in vivo. The results showed that one highly conserved sequence (MSP-1#1, VTHESYQELVKKLEALEDAV; located at amino acid positions 20 to 39) and one partly conserved sequence (MSP-1#23, GLFHKEKMILNEEEITTKGA; located at positions 44 to 63) contained both T- and B-cell epitopes. Immunization of mice with these peptides resulted in T-cell proliferation and enhanced production of antibody to MSP-1 upon exposure to merozoites. MSP-1#1 stimulated T-cell responses in three of the six strains of mice evaluated, whereas MSP-1#23 was immunogenic in only one strain. Immunization with the other four peptides resulted in T-cell responses to the peptides, but none of the resulting peptide-specific T cells recognized native MSP-1. These results demonstrate that two sequences located in the N terminus of MSP-1 can induce T- and B-cell responses following immunization in a murine model. Clearly, these sequences merit further consideration for inclusion in a vaccine for malaria.
Role of EspB in Experimental Human Enteropathogenic
Escherichia coli
Infection
Authors: Tacket Carol O. , Sztein Marcelo B. , Losonsky Genevieve , Abe Akio , Finlay B. Brett , McNamara Barry P. , Fantry George T. , James Stephen P. , Nataro James P. , Levine Myron M. , Donnenberg Michael S.
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC), a leading cause of diarrhea among infants in developing countries, induces dramatic alterations in host cell architecture that depend on a type III secretion system. EspB, one of the proteins secreted and translocated to the host cytoplasm via this system, is required for numerous alterations in host cell structure and function. To determine the role of EspB in virulence, we conducted a randomized, double-blind trial comparing the ability of wild-type EPEC and an isogenic Δ
espB
mutant strain to cause diarrhea in adult volunteers. Diarrhea developed in 9 of 10 volunteers who ingested the wild-type strain but in only 1 of 10 volunteers who ingested the Δ
espB
mutant strain. Marked destruction of the microvillous brush border adjacent to adherent organisms was observed in a jejunal biopsy from a volunteer who ingested the wild-type strain but not from two volunteers who ingested the Δ
espB
mutant strain. Humoral and cell-mediated immune responses to EPEC antigens were stronger among recipients of the wild-type strain. In addition, four of the volunteers who ingested the wild-type strain had lymphoproliferative responses to EspB. These results demonstrate that EspB is a critical virulence determinant of EPEC infections and suggest that EspB contributes to an immune response.
Salmonella enterica
Serovars Typhimurium and Dublin Can Lyse Macrophages by a Mechanism Distinct from Apoptosis
Authors: Watson Patricia R. , Gautier Anne V. , Paulin Sue M. , Bland A. Patricia , Jones Philip W. , Wallis Timothy S.
Published: July 2002

Salmonella enterica
serovars Typhimurium and Dublin lysed primary bovine alveolar macrophages and immortalized J774.2 macrophage-like cells in the absence of either the morphological changes or DNA fragmentation characteristic of apoptosis. Macrophage lysis was dependent on a subset of caspases and an intact
sipB
gene.
Identification of Three Essential Regulatory Gene Loci Governing Expression of
Staphylococcus epidermidis
Polysaccharide Intercellular Adhesin and Biofilm Formation
Authors: Mack Dietrich , Rohde Holger , Dobinsky Sabine , Riedewald Joachim , Nedelmann Max , Knobloch Johannes K.-M. , Elsner Holger-A. , Feucht Hubert H.
Published: July 2002

The formation of adherent multilayered biofilms embedded into a glycocalyx represents an essential factor in the pathogenesis of
Staphylococcus epidermidis
biomaterial-related infections. Using biofilm-producing
S. epidermidis
1457 and transposon Tn
917
carried on plasmid pTV1ts, we isolated nine isogenic biofilm-negative transposon mutants. Transduction by
S. epidermidis
phage 71 was used to prove the genetic linkage of transposon insertions and altered phenotypes. Mapping of the different transposon insertions by Southern hybridization and pulsed-field gel electrophoresis indicated that these were inserted in four unlinked genetic loci. According to their phenotypes, including quantitative differences in biofilm production in different growth media, in the amount of the polysaccharide intercellular adhesin (PIA) produced, in the hemagglutination titers, and in the altered colony morphology, the mutants could be separated into four phenotypic classes corresponding with the genetic classes. Synthesis of PIA was not detectable with class I and II mutants, whereas the amount of PIA produced reflected the residual degree of biofilm production of class III and IV mutants in different growth media. Chromosomal DNA flanking the transposon insertions of five class I mutants was cloned and sequenced, and the insertions were mapped to different locations of
icaADBC
, representing the synthetic genes for PIA. Expression of
icaADBC
from a xylose-dependent promoter in the different isogenic mutant classes reconstituted biofilm production in all mutants. In a Northern blot analysis no
icaADBC
-specific transcripts were observed in RNA isolated from mutants of classes II, III, and IV. Apparently, in addition to
icaADBC
, three other gene loci have a direct or indirect regulatory influence on expression of the synthetic genes for PIA on the level of transcription.
A Novel Factor Isolated fromActinobacillus actinomycetemcomitansStimulates Mouse B Cells and Human Peripheral Blood Mononuclear Cells
Authors: Jeong Soo-Jin , Yee Sung-Tae , Jo Wol-Soon , Yu Seong-Ho , Lee Sang-Hwa , Lim Young-Jin , Yoo Young-Hyun , Kim Jung-Man , Lee Jae-Dong , Jeong Min-Ho
Published: July 2002

A novel immunostimulating factor (ISTF) ofActinobacillus actinomycetemcomitansATCC 29522 was isolated and characterized as inducing proliferation of mouse B cells and human peripheral blood mononuclear cells. This factor was isolated from the bacterial culture medium and purified by size exclusion chromatography, dye-ligand affinity chromatography, immunoaffinity chromatography using monoclonal antibodies, and preparative electrophoresis. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified ISTF migrated as a single band corresponding to a molecular mass of 13 kDa. ISTF was a proteinaceous material distinct from lipopolysaccharide; it directly induced the proliferation of B lymphocytes but had no effect on the proliferation of T lymphocytes, even in the presence of antigen-presenting cells. A B-lymphocyte-mitogenic activity of ISTF was also shown by flow cytometric analysis of responding cell subpopulations. Immunoblot analysis revealed that ISTF was a component of the outer membranes of bacteria, could exist as a soluble form, and was released by growing and/or lysed bacteria. These results suggest that ISTF produced byA. actinomycetemcomitansmay play an important role in immunopathologic changes associated withA. actinomycetemcomitansinfections.
Single-Copy
IMH3
Allele Is Sufficient To Confer Resistance to Mycophenolic Acid in
Candida albicans
and To Mediate Transformation of Clinical
Candida
Species
Authors: Beckerman Janna , Chibana Hiroji , Turner Joshua , Magee P. T.
Published: July 2002

Parasexual genetic analysis of
Candida albicans
utilized the dominant selectable marker that conferred resistance to mycophenolic acid. We cloned and sequenced the
IMH3
r
gene from
C. albicans
strain 1006, which was previously identified as resistant to mycophenolic acid (MPA) (A. K. Goshorn and S. Scherer, Genetics 123:213–218, 1989). MPA is an inhibitor of IMP dehydrogenase, an enzyme necessary for the de novo biosynthesis of GMP. G. A. Kohler et al. (J. Bacteriol. 179:2331–2338, 1997) have shown that the wild-type
IMH3
gene, when expressed in high copy number, will confer resistance to this antibiotic. We demonstrate that the
IMH3
r
gene from strain 1006 has three amino acid changes, two of which are nonconservative, and demonstrate that at least two of the three mutations are required to confer resistance to MPA. We used this gene as a dominant selectable marker in clinical isolates of
C. albicans
and
Candida tropicalis
. We also identified the presence of autonously replicating sequence elements that permit autonomous replication in the promoter region of this gene. Finally, we found the excision of a φ-type long terminal repeat element outside the
IMH3
open reading frame of the gene in some strains. We used the
IMH3
r
allele to disrupt one allele of
ARG4
in two clinical isolates, WO-1 and FC18, thus demonstrating that a single ectopic integration of this dominant selectable marker is sufficient to confer resistance to MPA.
Release of Toll-Like Receptor-2-Activating Bacterial Lipoproteins inShigella flexneriCulture Supernatants
Authors: Aliprantis Antonios O. , Weiss David S. , Radolf Justin D. , Zychlinsky Arturo
Published: July 2002

Shigellaspp. cause dysentery, a severe form of bloody diarrhea. Apoptosis, or programmed cell death, is induced duringShigellainfections and has been proposed to be a key event in the pathogenesis of dysentery. Here, we describe a novel cytotoxic activity in the sterile-culture supernatants ofShigella flexneri. An identical activity was identified in purifiedS. flexneriendotoxin, defined here as a mixture of lipopolysaccharide (LPS) and endotoxin-associated proteins (EP). Separation of endotoxin into EP and LPS revealed the activity to partition exclusively to the EP fraction. Biochemical characterization ofS. flexneriEP and culture supernatants, including enzymatic deactivation, reverse-phase high-pressure liquid chromatography analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a Toll-like receptor-2 (TLR2) activation assay, indicates that the cytotoxic component is a mixture of bacterial lipoproteins (BLP). We show that biologically active BLP are liberated into culture supernatants of actively growingS. flexneri. In addition, our data indicate that BLP, and not LPS, are the component of endotoxin of gram-negative organisms responsible for activating TLR2. The activation of apoptosis by BLP shed fromS. flexneriis discussed as a novel aspect of the interaction of bacteria with the host.
Plasmodium falciparumGenotypes, Low Complexity of Infection, and Resistance to Subsequent Malaria in Participants in the Asembo Bay Cohort Project
Authors: Branch Oralee H. , Takala Shannon , Kariuki Simon , Nahlen Bernard L. , Kolczak Margaret , Hawley William , Lal Altaf A.
Published: July 2002

To assess the relationship between the within-host diversity of malaria infections and the susceptibility of the host to subsequent infection, we genotyped 60 children's successive infections from birth through 3 years of life. MSP-1 Block2 genotypes were used to estimate the complexity of infection (COI). Malaria transmission and age were positively associated with the number of K1 and Mad20 alleles detected (COIKM) (P< 0.003). Controlling for previous parasitemia, transmission, drug treatment, parasite density, sickle cell, and age, COIKMwas negatively correlated with resistance to parasitemia of >500/μl (P< 0.0001). Parasitemias with the RO-genotype were more resistant than those without this genotype (P< 0.0000). The resistance in low COIKMinfections was not genotype specific. We discuss the impact of genotype-transcending immunity to conserved antigenic determinants. We also propose a diversity-driven immunomodulation hypothesis that may explain the delayed development of natural immunity in the first few years of life and suggest that interventions that decrease the COIKMcould facilitate the development of protective immunity.
Gene Discovery through Genomic Sequencing of
Brucella abortus
Authors: Sánchez Daniel O. , Zandomeni Ruben O. , Cravero Silvio , Verdún Ramiro E. , Pierrou Ester , Faccio Paula , Diaz Gabriela , Lanzavecchia Silvia , Agüero Fernán , Frasch Alberto C. C. , Andersson Siv G. E. , Rossetti Osvaldo L. , Grau Oscar , Ugalde Rodolfo A.
Published: July 2002

Brucella abortus
is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of
B. abortus
strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10
−5
) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the
Brucella
genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to
Agrobacterium tumefaciens
and
Rhizobium meliloti
proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several
B. abortus
homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from
Salmonella enterica
serovar Typhimurium and the AidA-1 gene from
Escherichia coli
. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that
Brucella
might also have an active type III secretion machinery.
Lipopolysaccharide ofBurkholderia cepaciaand Its Unique Character To Stimulate Murine Macrophages with Relative Lack of Interleukin-1β-Inducing Ability
Authors: Shimomura Hirofumi , Matsuura Motohiro , Saito Shinji , Hirai Yoshikazu , Isshiki Yasunori , Kawahara Kazuyoshi
Published: July 2002

Lipopolysaccharide (LPS) ofBurkholderia cepaciawas purified by the conventional phenol-water extraction method (preparation BcLPS-1), followed by enzymatic treatments with DNase, RNase, trypsin, and proteinase K (preparation BcLPS-2), and finally by deoxycholate-phenol-water extraction (preparation BcLPS-3). Cells of LPS-hyporesponsive C3H/HeJ mice were activated by both the BcLPS-1 and the BcLPS-2 preparations but barely activated by BcLPS-3. When LPS-responsive C3H/HeN mice were used as targets, endotoxic activities such as lethal toxicity to galactosamine-sensitized mice, mitogenicity to spleen cells, and activation of macrophages to induce tumor necrosis factor alpha and interleukin-6 (IL-6) were strongly exhibited even by highly purified BcLPS-3 at levels comparable to those of the highly active enterobacterial LPS ofSalmonella entericaserovar Abortus-equi (SaeLPS), used as the control. The ability of BcLPS-3 to activate murine macrophages for induction of IL-1β was, however, much weaker than that of SaeLPS. Both accumulation of pro-IL-1β protein and expression of IL-1β mRNA in macrophages by stimulation with BcLPS-3 were much weaker than by stimulation with SaeLPS. These results indicate that LPS ofB. cepaciahas the potential to play a role as a pathogenic factor with strong activity comparable to that of usual enterobacterial LPS, but unlike the latter, this LPS has a relative lack of ability in the activation of murine macrophages to induce IL-1β. The lack of IL-1β-inducing ability appears to be caused by incomplete signal transduction somewhere in the upstream step(s) of IL-1β gene transcription.
Pyrogenicity and Cytokine-Inducing Properties ofStreptococcus pyogenesSuperantigens: Comparative Study of Streptococcal Mitogenic Exotoxin Z and Pyrogenic Exotoxin A
Authors: Müller-Alouf Heide , Proft Thomas , Zollner Thomas M. , Gerlach Dieter , Champagne Eric , Desreumaux Pierre , Fitting Catherine , Geoffroy-Fauvet Christiane , Alouf Joseph E. , Cavaillon Jean-Marc
Published: July 2002

Streptococcal mitogenic exotoxin Z (SMEZ), a superantigen derived fromStreptococcus pyogenes, provoked expansion of human lymphocytes expressing the Vβ 2, 4, 7 and 8 motifs of T-cell receptor. SMEZ was pyrogenic in rabbits and stimulated the expression of the T-cell activation markers CD69 and cutaneous lymphocyte-associated antigen. A variety of cytokines was released by human mononuclear leukocytes stimulated with SMEZ, which was 10-fold more active than streptococcal pyrogenic exotoxin A. Th2-derived cytokines were elicited only by superantigens and not by streptococcal cells.
Down-Modulation of L-Selectin by Lipopolysaccharide Is Not Required for Lipopolysaccharide-Induced Expression of CD14 in Mouse Bone Marrow Granulocytes
Authors: Pédron Thierry , Girard Robert , Chaby Richard
Published: July 2002

We established in previous studies that a constitutive lipopolysaccharide (LPS) receptor of low affinity is present on mouse bone marrow granulocytes (BMG). This yet-unidentified receptor is involved in the LPS-induced expression of a second LPS receptor, CD14. Because it has been claimed that L-selectin (CD62L) is a low-affinity LPS receptor in mature granulocytes (polymorphonuclear leukocytes), it may be asked whether this molecule could be the constitutive LPS receptor in BMG. We show in this study thatl-selectin is constitutively present on BMG and is down-regulated after exposure of the cells to LPS. A phorbol ester induced a down-regulation of CD62L and blocked the LPS-induced expression of CD14. However, a metalloproteinase inhibitor (BB-3103) blocked the former but not the latter effect of PMA. We also observed an absence of cross-reactivity between LPS and a CD62L ligand (fucoidan) in binding studies with radiolabeled derivatives of the two agents. Furthermore, BMG froml-selectin-deficient mice expressed normal levels of CD14 in response to LPS. Taken together, these results demonstrate that in BMG,l-selectin is not the constitutive LPS receptor required for the LPS-induced expression of CD14.
In Situ Activation of Helper T Cells in the Lung
Authors: Raju Bindu , Tung Chung F. , Cheng Debbie , Yousefzadeh Nora , Condos Rany , Rom William N. , Tse Doris B.
Published: July 2002

To better understand the lung and systemic responses of helper T cells mediating memory immunity toMycobacterium tuberculosis, we used three- and four-color flow cytometry to study the surface phenotype of CD4+lymphocytes. Bronchoalveolar lavage (BAL) fluid and peripheral blood (PB) samples were obtained from a total of 25 subjects, including 10 tuberculosis (TB)-infected subjects, 8 purified-protein-derivative-negative subjects, and 7 purified-protein-derivative-positive subjects. In marked contrast to CD4+lymphocytes from PB (9% ± 5% expressing CD45RA and CD29), the majority (55% ± 16%) of CD4+lymphocytes in BAL (ALs) simultaneously expressed CD45RA, a naı̈ve T-cell marker, and CD29, members of the very late activation family. Further evaluation revealed that CD4+ALs expressed both CD45RA and CD45RO, a memory T-cell marker. In addition, the proportion of CD4+lymphocytes expressing CD69, an early activation marker, was drastically increased in BAL fluid (83% ± 9%) compared to PB (1% ± 1%), whereas no significant difference was seen in the expression of CD25, the low-affinity interleukin 2 receptor (34% ± 15% versus 40% ± 16%). More importantly, we identified a minor population of CD69brightCD25brightCD4+lymphocytes in BAL (10% ± 6%) that were consistently absent from PB (1% ± 1%). Thus, CD4+lymphocytes in the lung paradoxically coexpress surface molecules characteristic of naı̈ve and memory helper T cells as well as surface molecules commonly associated with early and late stages of activation. No difference was observed for ALs obtained from TB-infected and uninfected lung segments in this regard. It remains to be determined if these surface molecules are induced by the alveolar environment or if CD4+lymphocytes coexpressing this unusual combination of surface molecules are selectively recruited from the circulation. Our data suggest that ex vivo experiments on helper T-cell subsets that display distinctive phenotypes may be pivotal to studies on the human immune response to potential TB vaccines.
Production ofNeisseria meningitidisTransferrin-Binding Protein B by RecombinantBordetella pertussis
Authors: Coppens Isabelle , Alonso Sylvie , Antoine Rudy , Jacob-Dubuisson Françoise , Renauld-Mongénie Geneviève , Jacobs Eric , Locht Camille
Published: July 2002

Neisseria meningitidisserogroup B infections are among the major causes of fulminant septicemia and meningitis, especially severe in young children, and no broad vaccine is available yet. Because of poor immunogenicity of the serogroup B capsule, many efforts are now devoted to the identification of protective protein antigens. Among those are PorA and, more recently, transferrin-binding protein B (TbpB). In this study, TbpB ofN. meningitidiswas genetically fused to the N-terminal domain of theBordetella pertussisfilamentous hemagglutinin (FHA), and thefha-tbpBhybrid gene was expressed inB. pertussiseither as a plasmid-borne gene or as a single copy inserted into the chromosome. The hybrid protein was efficiently secreted by the recombinant strains, despite its large size, and was recognized by both anti-FHA and anti-TbpB antibodies. A single intranasal administration of recombinant virulent or pertussis-toxin-deficient, attenuatedB. pertussisto mice resulted in the production of antigen-specific systemic immunoglobulin G (IgG), as well as local IgG and IgA. The anti-TbpB serum antibodies were of the IgG1, IgG2a, and IgG2b isotypes and were found to express complement-mediated bactericidal activity againstN. meningitidis. These observations indicate that recombinantB. pertussismay be a promising vector for the development of a mucosal vaccine against serogroup B meningococci.
Intermediate Subunit of the Gal/GalNAc Lectin of
Entamoeba histolytica
Is a Member of a Gene Family Containing Multiple CXXC Sequence Motifs
Authors: Cheng Xun-Jia , Hughes Molly A. , Huston Christopher D. , Loftus Brendan , Gilchrist Carol A. , Lockhart Lauren A. , Ghosh Salil , Miller-Sims Vanessa , Mann Barbara J. , Petri William A. , Tachibana Hiroshi
Published: July 2002

Killing by
Entamoeba histolytica
requires parasite adherence to host galactose- and
N
-acetyl-
d
-galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric
E. histolytica
Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no.
AF337950
and
AF337951
). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The
igl
genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.
The
Helicobacter pylori
Homologue of the Ferric Uptake Regulator Is Involved in Acid Resistance
Authors: Bijlsma Jetta J. E. , Waidner Barbara , Vliet Arnoud H. M. van , Hughes Nicky J. , Häg Stephanie , Bereswill Stefan , Kelly David J. , Vandenbroucke-Grauls Christina M. J. E. , Kist Manfred , Kusters Johannes G.
Published: July 2002

The only known niche of the human pathogen
Helicobacter pylori
is the gastric mucosa, where large fluctuations of pH occur, indicating that the bacterial response and resistance to acid are important for successful colonization. One of the few regulatory proteins in the
H. pylori
genome is a homologue of the ferric uptake regulator (Fur). In most bacteria, the main function of Fur is the regulation of iron homeostasis. However, in
Salmonella enterica
serovar Typhimurium, Fur also plays an important role in acid resistance. In this study, we determined the role of the
H. pylori
Fur homologue in acid resistance. Isogenic
fur
mutants were generated in three
H. pylori
strains (1061, 26695, and NCTC 11638). At pH 7 there was no difference between the growth rates of mutants and the parent strains. Under acidic conditions, growth of the
fur
mutants was severely impaired. No differences were observed between the survival of the
fur
mutant and parent strain 1061 after acid shock. Addition of extra iron or removal of iron from the growth medium did not improve the growth of the
fur
mutant at acidic pH. This indicates that the phenotype of the
fur
mutant at low pH was not due to increased iron sensitivity. Transcription of
fur
was repressed in response to low pH. From this we conclude that Fur is involved in the growth at acidic pH of
H. pylori
; as such, it is the first regulatory protein implicated in the acid resistance of this important human pathogen.
Identification and Testing of
Porphyromonas gingivalis
Virulence Genes with a pPGIVET System
Authors: Wu Yi , Lee Seok-Woo , Hillman Jeffrey D. , Progulske-Fox Ann
Published: July 2002

An in vivo expression technology (IVET) system was designed to identify previously unknown virulence genes of
Porphyromonas gingivalis.
Fourteen
ivi
(for in vivo induced) genes that are induced during infection in a mouse abscess model were identified in our study. Of these, seven had homology to genes in the NCBI database, and the rest had no homology to reported DNA sequences. In order to determine virulence-related properties of these genes, three mutant strains, deleted of
ivi
8 (no homology to genes in the database),
ivi
10 (homologous to a putative TonB-dependent outer membrane receptor protein), and
ivi
11 (an immunoreactive 33-kDa antigen PG125 in
P. gingivalis
), were created. The mutants were tested in a mouse abscess model for alterations in virulence relative to the wild type by a competition assay in BALB/c mice. After 5 days we observed the enrichment of the wild-type strain over mutant strains Δ
ivi
10 and Δ
ivi
11, which indicated that mutant strains Δ
ivi
10 and Δ
ivi
11 are less able to survive in this model than the wild-type strain, while Δ
ivi
8 survives as well as the wild-type strain. We propose that knockout of these
ivi
genes reduced the ability of the mutated
P. gingivalis
to survive and cause infection compared to the wild-type strain at the site of injection. Also, in separate experiments, groups of mice were challenged with subcutaneous injections of each individual mutant strain (Δ
ivi
8, Δ
ivi
10, and Δ
ivi
11) or with the wild-type strain alone and were then examined to assess their general health status. The results showed that knockout of these
ivi
genes conferred a reduction in virulence. The ability of the mutants to invade KB cells compared to the wild type was also determined. Interestingly, the CFU counts of the mutant strain Δ
ivi
10 recovered from KB cells were eight times lower than those of the wild type, indicating that this mutant has a lower capacity for invasion. These results demonstrate that IVET is a powerful tool in discovering virulence genes and the significant role that
ivi
genes play in the pathogenesis of this species.
Intracellular Induction of
Listeria monocytogenes actA
Expression
Authors: Shetron-Rama Lynne M. , Marquis Hélène , Bouwer H. G. Archie , Freitag Nancy E.
Published: July 2002

Following entry into the host cytosol, the bacterial pathogen
Listeria monocytogenes
dramatically increases the expression of several key virulence factors. The expression of
actA
, whose protein product is required for
L
.
monocytogenes
actin-based intracellular motility, is increased by more than 200-fold in cytosolic bacteria in comparison to broth-grown cultures. Two distinct promoter elements have been reported to regulate
actA
expression. One promoter is located immediately upstream of
actA
coding sequences, while the second promoter is contributed by the upstream
mpl
gene via the generation of an
mpl-actA-plcB
transcript. A series of
L
.
monocytogenes
mutants were constructed to define the contributions of individual promoter elements to
actA
expression. The intracellular induction of
actA
expression was found to be dependent upon the
actA
proximal promoter; the
mpl
promoter appeared to contribute to the extracellular induction of
actA
but did not affect intracellular levels of expression. The
actA
promoter is dependent upon a regulatory factor known as PrfA for transcriptional activation; however, no increase in
actA
expression was detected following the introduction of a high-affinity PrfA binding site within the
actA
promoter. The presence of a mutationally activated form of PrfA, known as PrfA*, increased overall
actA
expression in broth-grown cultures of both wild-type and
actA
promoter mutant strains, but the levels of induction observed were still approximately 50-fold lower than those observed for intracellularly grown
L
.
monocytogenes
. Collectively, these results indicate that the dramatic induction of
actA
expression that occurs in the host cell cytosol is mediated through a single promoter element. Furthermore, intracellular induction of
actA
appears to require additional steps or factors beyond those necessary for the activation and binding of PrfA to the
actA
promoter.
Dendritic Cells Pulsed with a Recombinant Chlamydial Major Outer Membrane Protein Antigen Elicit a CD4+Type 2 Rather than Type 1 Immune Response That Is Not Protective
Authors: Shaw Jennifer , Grund Vernon , Durling Luke , Crane Debbie , Caldwell Harlan D.
Published: July 2002

Chlamydia trachomatisis an obligate intracellular bacterium that infects the oculogenital mucosae.C. trachomatisinfection of the eye causes trachoma, the leading cause of preventable blindness. Infections of the genital mucosae are a leading cause of sexually transmitted diseases. A vaccine to prevent chlamydial infection is needed but has proven difficult to produce by using conventional vaccination approaches. Potent immunity to vaginal rechallenge in a murine model of chlamydial genital infection has been achieved only by infection or by immunization with dendritic cells (DC) pulsed ex vivo with whole inactivated organisms. Immunity generated by infection or ex vivo antigen-pulsed DC correlates with a chlamydia-specific interleukin 12 (IL-12)-dependent CD4+Th1 immune response. Because of the potent antichlamydial immunizing properties of DC, we hypothesized that DC could be a powerful vehicle for the delivery of individual chlamydial antigens that are thought to be targets for more conventional vaccine approaches. Here, we investigated the recombinant chlamydial major outer membrane protein (rMOMP) as a target antigen. The results demonstrate that DC pulsed with rMOMP secrete IL-12 and stimulate infection-sensitized CD4+T cells to proliferate and secrete gamma interferon. These immunological properties implied that rMOMP-pulsed DC would be potent inducers of MOMP-specific CD4+Th1 immunity in vivo; however, we observed the opposite result. DC pulsed ex vivo with rMOMP and adoptively transferred to naive mice generated a Th2 rather than a Th1 anti-MOMP immune response, and immunized mice were not protected following infectious challenge. We conclude from these studies that the immunological properties of ex vivo pulsed DC are not necessarily predictive of the immune response generated in vivo following adoptive transfer. These findings suggest that the nature of the antigen used to pulse DC ex vivo influences the Th1-Th2 balance of the immune response in vivo.
Clonal Diversity of
Escherichia coli
Colonizing Stools and Urinary Tracts of Young Girls
Authors: Schlager Theresa A. , Hendley J. Owen , Bell Alison L. , Whittam Thomas S.
Published: July 2002

Intestinal carriage of
Escherichia coli
in prepubertal girls without a history of urinary tract infection was examined by collecting weekly stools and periurethral and urine samples over 3 to 4 weeks of study. Dominant and minor clones were defined by grouping 28
E. coli
isolates into clonal types. Multiple enteric clones of
E. coli
, which changed week to week, were found in the 13 girls during the study (median, 3 clones/girl; range, 1 to 16 clones/girl). Dominance of an enteric clone did not predict persistence in the stool. In only 10 (34%) of the 29 episodes in which a dominant clone present in one weekly sample could have been detected the following week did it persist as the dominant clone in the next weekly sample. In 5 (17%) of the 29 episodes, a dominant clone found in one weekly sample was classified as a minor clone the next week. Both dominant and minor clones were observed to colonize the urinary tract. However, when colonization of the periurethra or bladder urine occurred, it was brief and often did not reflect the dominant stool flora from the same week. In fact, in only 40% of episodes was a clone that was detected either on the periurethra or in the urine also recovered from the stool the same week. Our findings suggest that the intestinal flora of healthy girls is multiclonal with frequent fluctuations in composition.
Selective T-Cell Recognition of the N-Terminal Peptide of GroES in Tuberculosis
Authors: Chua-Intra Boosbun , Wilkinson Robert J. , Ivanyi Juraj
Published: July 2002

Peptides derived from the whole sequence of mycobacterial GroES heat shock proteins were tested for the ability to induce the proliferation of blood mononuclear cells from tuberculosis patients and sensitized healthy subjects. The response to the N-terminal peptide (residues 1 to 16) was found to be more frequent and stronger in tuberculosis patients. This finding is exceptional, considering that recognition of all other GroES peptides by patients was either diminished or not different from that of controls.
Discordance between T-Cell Receptor Expression and Effector Function in Mice Infected withHistoplasma capsulatum
Authors: Deepe George S. , Gibbons Reta S. , Ward Susan R.
Published: July 2002

Vβ10+and Vβ14+T cells were selectively increased 7 to 14 days following infection in the lungs of naive mice infected withHistoplasma capsulatum. Following secondary challenge of immune mice, Vβ1+and Vβ8.1+cells were sporadically increased. Elimination of Vβ10+and Vβ14+cells from naive mice did not alter the course of infection over a period of 21 days. Thus, overexpression of Vβ families does not necessarily signify a key role in host defense.
Involvement of PhoP-PhoS Homologs in
Enterococcus faecalis
Virulence
Authors: Teng Fang , Wang Ling , Singh Kavindra V. , Murray Barbara E. , Weinstock George M.
Published: July 2002

Eleven PhoP-PhoS homolog pairs were identified by searching the
Enterococcus faecalis
V583 genome sequence database at The Institute for Genomic Research with the
Bacillus subtilis
PhoP-PhoS sequences. Each pair appears to be a potential two-component system composed of a response regulator and a sensor kinase. Seven of the homologs were disrupted in
E. faecalis
strain OG1RF. TX10293, a mutant disrupted in one of these genes (
etaR
, the first gene of the gene pair designated
etaRS
), showed delayed killing and a higher 50% lethal dose in a mouse peritonitis model. The predicted EtaR protein sequence showed greatest similarity to LisR of
Listeria monocytogenes
(77%) and CsrR of
Streptococcus pyogenes
(70%); EtaS is 53% similar to LisK and 54% similar to CsrS. When grown in vitro, the TX10293 mutant was more sensitive to low pH (pH 3.4) and more resistant to high temperature (55°C) than wild-type OG1RF. In conclusion, many potential two-component systems are identified for
E. faecalis
, one of which, EtaRS, was shown to be involved in stress response and virulence.