bluuebunny/crossref_metadata_2025 huggingface dataset:
C-Terminal Invariable Domain of VlsE Is Immunodominant but Its Antigenicity Is Scarcely Conserved among Strains of Lyme Disease Spirochetes
Authors: Liang Fang Ting , Bowers Lisa C. , Philipp Mario T.
Published: July 2002

VlsE, the variable surface antigen ofBorrelia burgdorferi, contains two invariable domains located at the amino and carboxyl terminal ends, respectively, and a central variable domain. In this study, both immunogenicity and antigenic conservation of the C-terminal invariable domain were assessed. Mouse antiserum to a 51-mer synthetic peptide (Ct) which reproduced the entire sequence of the C-terminal invariable domain of VlsE fromB. burgdorferistrain B31 was reacted on immunoblots with whole-cell lysates extracted from spirochetes of 12 strains from theB. burgdorferisensu lato species complex. The antiserum recognized only VlsE from strain B31, indicating that epitopes of this domain differed among these strains. When Ct was used as enzyme-linked immunosorbent assay (ELISA) antigen, all of the seven monkeys and six mice that were infected with B31 spirochetes produced a strong antibody response to this peptide, indicating that the C-terminal invariable domain is immunodominant. None of 12 monkeys and only 11 of 26 mice that were infected with strains other than B31 produced a detectable anti-Ct response, indicating a limited antigenic conservation of this domain among these strains. Twenty-six of 33 dogs that were experimentally infected by tick inoculation were positive by the Ct ELISA, while only 5 of 18 serum samples from dogs clinically diagnosed with Lyme disease contained detectable anti-Ct antibody. Fifty-seven of 64 serum specimens that were collected from American patients with Lyme disease were positive by the Ct ELISA, while only 12 of 21 European samples contained detectable anti-Ct antibody. In contrast, antibody to the more conserved invariable region IR6of VlsE was present in all of these dog and human serum samples.
Specificity of the Protective Antibody Response to Apical Membrane Antigen 1
Authors: Hodder Anthony N. , Crewther Pauline E. , Anders Robin F.
Published: July 2002

Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages of
Plasmodium falciparum
. Although the
ama1
gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in
P. falciparum
AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by
P. falciparum
merozoites. For these studies, we used antibodies to recombinant
P. falciparum
3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of
P. falciparum
. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.
Amebic Infection in the Human Colon Induces Cyclooxygenase-2
Authors: Stenson William F. , Zhang Zhi , Riehl Terrence , Stanley Samuel L.
Published: July 2002

We sought to determine if infection of the colon withEntamoeba histolyticainduces the expression of cyclooxygenase-2 and, if it does, to determine the contribution of prostaglandins produced through cyclooxygenase-2 to the host response to amebic infection. Human fetal intestinal xenografts were implanted subcutaneously in mice with severe combined immunodeficiency and allowed to grow; the xenografts were then infected withE. histolyticatrophozoites. Infection withE. histolyticaresulted in the expression of cyclooxygenase-2 in epithelial cells and lamina propria macrophages. Infection withE. histolyticaincreased prostaglandin E2(PGE2) levels 10-fold in the xenografts and resulted in neutrophil infiltration, as manifested by an 18-fold increase in myeloperoxidase activity. Amebic infection also induced an 18-fold increase in interleukin 8 (IL-8) production and a >100-fold increase in epithelial permeability. Treatment of the host mouse with indomethacin, an inhibitor of cyclooxygenase-1 and cyclooxygenase-2, or with NS-398, a selective inhibitor of cyclooxygenase-2, resulted in (i) decreased PGE2levels, (ii) a decrease in neutrophil infiltration, (iii) a decrease in IL-8 production, and (iv) a decrease in the enhanced epithelial permeability seen with amebic infection. These results indicate that amebic infection in the colon induces the expression of cyclooxygenase-2 in epithelial cells and macrophages. Moreover, prostaglandins produced through cyclooxygenase-2 participate in the mediation of the neutrophil response to infection and enhance epithelial permeability.
Tumor Necrosis Factor-Dependent Adhesions as a Major Protective Mechanism Early in Septic Peritonitis in Mice
Authors: Echtenacher Bernd , Weigl Karin , Lehn Norbert , Männel Daniela N.
Published: July 2002

The occurrence of peritoneal adhesions in surgical patients is positively correlated with tumor necrosis factor (TNF) levels. In a model of septic peritonitis—cecal ligation and puncture—TNF neutralization prevented formation of peritoneal adhesions and increased mortality, most likely because localization of the septic focus was prevented. To discriminate between the coagulation-independent protective TNF effect and a potential protective procoagulant TNF effect, formation of peritoneal adhesions after CLP was inhibited with heparin, hirudin, or urokinase. Each treatment increased mortality and increased the number of bacteria in the peritoneal lavage fluid, kidney, and liver to various degrees. Under these experimental conditions, antibiotics prevented death. In coagulation-compromised mice, lethality was further enhanced by additional TNF neutralization. These findings demonstrate that peritoneal adhesions early in septic peritonitis are an important mechanism of innate immunity that prevents increased spread of bacteria and reduces mortality.
Differential Induction of Gamma Interferon inLegionella pneumophila- Infected Macrophages from BALB/c and A/J Mice
Authors: Salins Sheldon , Newton Catherine , Widen Ray , Klein Thomas W. , Friedman Herman
Published: July 2002

Gamma interferon (IFN-γ), a pleiotropic cytokine, is now known to be produced by macrophages as well as by NK cells, γδ cells, and activated T cells. The autocrine biological functions of IFN-γ on the macrophage include the upregulation of major histocompatibility complex MHC class II and the activation to an antiviral state. In this study, the production of IFN-γ by macrophages was demonstrated to correspond to antibacterial activity.Legionella pneumophilareplicates intracellularly in thioglycolate (TG)-elicited macrophages (TG-macrophages) from A/J mice, while TG-macrophages from BALB/c mice restrict bacterial growth after an initial period of growth. BALB/c TG-macrophages were shown to express IFN-γ mRNA at 24 and 28 h, which corresponded to the initiation of anti-L. pneumophilaactivity. Moreover, IFN-γneutralization by antibody treatment of the cultures resulted in increasedL. pneumophilagrowth in the macrophages. In contrast, no IFN-γ mRNA was expressed in TG-macrophages from A/J mice, whereL. pneumophilagrew unrestricted. As would be expected, IFN-γ treatment decreased bacterial growth. An IFN-γ-mediated antibacterial activity was, however, inducible in A/J macrophages by the addition of interleukin-12 followingL. pneumophilainfection. Thus, autocrine IFN-γ is involved in anti-L. pneumophilaactivity associated with different growth patterns and appears to be important during intracellular infection.
Identification of
Legionella pneumophila rcp
, a
pagP
-Like Gene That Confers Resistance to Cationic Antimicrobial Peptides and Promotes Intracellular Infection
Authors: Robey Marianne , O'Connell William , Cianciotto Nicholas P.
Published: July 2002

In the course of characterizing a locus involved in heme utilization, we identified a
Legionella pneumophila
gene predicted to encode a protein with homology to the product of the
Salmonella enterica
serovar Typhimurium
pagP
gene. In
Salmonella, pagP
increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the
L. pneumophila pagP
-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated
rcp
for resistance to cationic antimicrobial peptides. Furthermore,
Legionella
CAMP resistance was induced by growth in low-magnesium medium. To determine whether
rcp
had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a
Hartmannella vermiformis
coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact
rcp
gene. Mutations in
rcp
consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The
rcp
mutant was also more defective for lung colonization of A/J mice. Growth of
rcp
mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg
2+
medium, the
rcp
mutant was impaired in stationary-phase survival. This is the first demonstration of a
pagP
-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.
Expression of aMycobacterium tuberculosisArabinomannan Antigen In Vitro and In Vivo
Authors: Schwebach J. Reid , Casadevall Arturo , Schneerson Rachel , Dai Zhongdong , Wang Xiaojuan , Robbins John B. , Glatman-Freedman Aharona
Published: July 2002

The outermost layer ofMycobacterium tuberculosiscontains two major polysaccharides, arabinomannan (AM) and glucan (GC). We studied the in vitro and in vivo expression of anM. tuberculosisAM antigen using monoclonal antibody (MAb) 9d8 (2a), an isotype-switched variant of the immunoglobulin G3 (IgG3) MAb 9d8. MAb 9d8 had been previously shown to bindM. tuberculosisAM and theM. tuberculosissurface. Our in vitro experiments showed that MAb 9d8(2a) bound strongly to whole-cellM. tuberculosisErdman but not to the CDC 1551 strain grown in medium for an extended period. However, AM antigen was detected in the culture supernatant of both strains, and its concentration increased in a time-dependent manner. The detection of AM antigen from both strains was decreased in the presence of Tween 80. In mice infected withM. tuberculosisErdman, AM antigen accumulated in organ homogenates concomitant to an increase in bacterial organ burden and an increase in IgG and IgM titer to AM. These results (i) indicate that the surface expression of AM during in vitro growth changes with culture age, is strain dependent, and is affected by the presence of Tween 80 in the culture media; (ii) show that AM is produced by bacteria growth in vivo; and (iii) demonstrate that the amount of in vivo-detected AM can be dependent on the number of bacteria in the infected organ.
Comparison of Inflammation, Organ Damage, and Oxidant Stress Induced by
Salmonella enterica
Serovar Muenchen Flagellin and Serovar Enteritidis Lipopolysaccharide
Authors: Liaudet Lucas , Murthy Kanneganti G. K. , Mabley Jon G. , Pacher Pál , Soriano Francisco G. , Salzman Andrew L. , Szabó Csaba
Published: July 2002

Gram-negative sepsis is related to the activation of interconnected inflammatory cascades in response to bacteria and their products. Recent work showed that flagellin, the monomeric subunit of bacterial flagella, triggers innate immune responses mediated by Toll-like receptor 5. Here, we compared the effects of
Salmonella enterica
serovar Enteritidis lipopolysaccharide (LPS) and recombinant
Salmonella enterica
serovar Muenchen flagellin administered intravenously (100 μg) to mice. Flagellin and LPS both elicited a prototypical systemic inflammatory response, with increased levels of tumor necrosis factor alpha, gamma interferon, interleukin 6 and 10, and nitrate in plasma. Flagellin induced a widespread oxidative stress, evidenced by an increase in malondialdehyde and a decrease in reduced glutathione in most organs, as well as liver (increased plasma aminotransferases), but not renal, injury. Alternatively, LPS resulted in a less severe oxidative stress and triggered renal, but not liver, damage. Sequestration of polymorphonuclear neutrophils (increased myeloperoxidase activity) in the lungs was observed with both toxins, while only LPS recruited neutrophils in the gut. In additional experiments, the simultaneous administration of small doses of LPS and flagellin (10 μg) induced a synergistic enhancement of the production of proinflammatory cytokines. Our data support a novel concept implicating flagellin as a mediator of systemic inflammation, oxidant stress, and organ damage induced by gram-negative bacteria.
icmTIs Essential for Pore Formation-Mediated Egress ofLegionella pneumophilafrom Mammalian and Protozoan Cells
Authors: Molmeret Maelle , Alli O. A. Terry , Zink Steven , Flieger Antje , Cianciotto Nicholas P. , Kwaik Yousef Abu
Published: July 2002

The final step of the intracellular life cycle ofLegionella pneumophilaand other intracellular pathogens is their egress from the host cell after termination of intracellular replication. We have previously isolated five spontaneous mutants ofL. pneumophilathat replicate intracellularly similar to the wild-type strain but are defective in pore formation-mediated cytolysis and egress from mammalian and protozoan cells, and the mutants have been designatedrib(release of intracellular bacteria). Here, we show that theribmutants are not defective in the activity of enzymes secreted through the type II secretion system, including phospholipase A, lysophospholipase A, and monoacylglycerol lipase, although they are potential candidates for factors that lyse host cell membranes. In addition, thepilDandlspGmutants, which are defective in the type II secretion system, are not defective in the pore-forming toxin. We show that all fiveribmutants have an identical point mutation (deletion) following a stretch of poly(T) in theicmTgene. Spontaneous revertants of theribmutants, due to an insertion of a nucleotide following the poly(T) stretch inicmT,have been isolated and shown to have regained the wild-type phenotype. We constructed anicmTinsertion mutant (AA100kmT) in the chromosome of the wild-type strain by allelic exchange. The AA100kmT mutant was as defective as theribmutant in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. Both theribmutant and the AA100kmT mutant were complemented by theicmTgene for their phenotypic defect.rtxA, a gene that is thought to have a minor role in pore formation, was not involved in pore formation-mediated cytolysis and egress from mammalian and protozoan cells. We conclude that theicmTgene is essential for pore formation-mediated lysis of mammalian and protozoan cells and the subsequent bacterial egress.
Differential Binding of Host Complement Inhibitor Factor H byBorrelia burgdorferiErp Surface Proteins: a Possible Mechanism Underlying the Expansive Host Range of Lyme Disease Spirochetes
Authors: Stevenson Brian , El-Hage Nazira , Hines Melissa A. , Miller Jennifer C. , Babb Kelly
Published: July 2002

The Lyme disease spirochete,Borrelia burgdorferi, is capable of infecting a wide variety of vertebrates. This broad host range implies thatB. burgdorferipossesses the ability to contravene the immune defenses of many potential hosts.B. burgdorferiproduces multiple different Erp proteins on its outer membrane during mammalian infection. It was reported previously that one Erp protein can bind human factor H (J. Hellwage, T. Meri, T. Heikkilä, A. Alitalo, J. Panelius, P. Lahdenne, I. J. T. Seppälä, and S. Meri, J. Biol. Chem. 276:8427–8435, 2001). In this paper we report that the ability to bind the complement inhibitor factor H is a general characteristic of Erp proteins. Furthermore, each Erp protein exhibits different relative affinities for the complement inhibitors of various potential animal hosts. The data suggest that the presence of multiple Erp proteins on the surface can allow a singleB. burgdorferibacterium to resist complement-mediated killing in any of the wide range of potential hosts that it might infect. Thus, Erp proteins likely contribute to the persistence ofB. burgdorferiin nature and to the ability of this bacterium to cause Lyme disease in humans and other animals.
Fas Ligand-Expressing B-1a Lymphocytes Mediate CD4+-T-Cell Apoptosis during Schistosomal Infection: Induction by Interleukin 4 (IL-4) and IL-10
Authors: Lundy Steven K. , Boros Dov L.
Published: July 2002

A previous study of the murine model ofSchistosoma mansoniinfection has implicated splenic CD19+B lymphocytes as Fas ligand (FasL)-bearing mediators of CD4+T-lymphocyte apoptosis. The present study shows that B-cell deficiency leads to decreased CD4+T-cell apoptosis during infection and compares FasL expression and killer function of B-1a- and CD5−B-lymphocyte subsets. B-1a cells from uninfected mice displayed constitutive expression of FasL compared with that of CD5−B cells. FasL expression was enhanced following worm egg deposition and antigenic stimulation on both subsets of B cells. Purified B-1a cells from uninfected mice were potent effectors of CD4+T-cell apoptosis, and the killing effect was enhanced during schistosome infection. FasL expression by splenic B cells required CD4+-T-cell help that was replaced by addition of culture supernatants from antigen-stimulated splenocytes of infected mice. The culture-supernatant-stimulated FasL expression was inhibited by anti-interleukin 10 (IL-10) and anti-IL-4 antibodies. Culture of purified B cells with recombinant IL-4 (rIL-4), rIL-10, and soluble egg antigens (SEA) led to increased expression of FasL on B-1a cells. These results suggest that FasL-expressing, splenic B-1a cells are important mediators of SEA-stimulated CD4+-T-cell apoptosis and that maximal FasL expression on B-1a cells is dependent on antigenic stimulation and the presence of IL-4 and IL-10.
Transcriptional Regulators Cph1p and Efg1p Mediate Activation of the
Candida albicans
Virulence Gene
SAP5
during Infection
Authors: Staib Peter , Kretschmar Marianne , Nichterlein Thomas , Hof Herbert , Morschhäuser Joachim
Published: July 2002

The opportunistic fungal pathogen
Candida albicans
can cause superficial as well as systemic infections. Successful adaptation to the different host niches encountered during infection requires coordinated expression of various virulence traits, including the switch between yeast and hyphal growth forms and secretion of aspartic proteinases. Using an in vivo expression technology that is based on genetic recombination as a reporter of gene activation during experimental candidiasis in mice, we investigated whether two signal transduction pathways controlling hyphal growth, a mitogen-activated protein kinase cascade ending in the transcriptional activator Cph1p and a cyclic AMP-dependent regulatory pathway that involves the transcription factor Efg1p, also control expression of the
SAP5
gene, which encodes one of the secreted aspartic proteinases and is induced by host signals soon after infection. Our results show that both transcriptional regulators are important for
SAP5
activation in vivo.
SAP5
expression was reduced in a
cph1
mutant, although filamentous growth in infected tissue was not detectably impaired.
SAP5
expression was also reduced, but not eliminated, in an
efg1
null mutant, although this strain grew exclusively in the yeast form in infected tissue, demonstrating that in contrast to in vitro conditions,
SAP5
activation during infection does not depend on growth of
C. albicans
in the hyphal form. In a
cph1 efg1
double mutant, however,
SAP5
expression in infected mice was almost completely eliminated, suggesting that the two signal transduction pathways are important for
SAP5
expression in vivo. The avirulence of the
cph1 efg1
mutant seemed to be caused not only by the inability to form hyphae but also by a loss of expression of additional virulence genes in the host.
Transcript Heterogeneity of the
p44
Multigene Family in a Human Granulocytic Ehrlichiosis Agent Transmitted by Ticks
Authors: Zhi Ning , Ohashi Norio , Tajima Tomoko , Mott Jason , Stich Roger W. , Grover Debra , Telford Sam R. , Lin Quan , Rikihisa Yasuko
Published: July 2002

Human granulocytic ehrlichiosis (HGE) is an emerging tick-borne zoonosis caused by a strain of
Anaplasma phagocytophila
called the HGE agent, an obligatory intracellular bacterium. The agent expresses immunodominant 44-kDa outer membrane proteins (P44s) encoded by a multigene family. The present study established an experimental process for transmission of the HGE agent from infected mice (a reservoir model) to nymphal
Ixodes scapularis
ticks (a biological vector) and subsequently to horses (a patient model) by the adult infected ticks. Overall, a total of 20 different
p44
transcripts were detected in the mammals, ticks, and cell cultures. Among them, a transcript from a
p44
-
18
gene was major at acute stage in mice and horses but minor in ticks. Both mRNA and protein produced from the
p44
-
18
gene were detected in the HGE agent cultivated in HL-60 cells at 37°C, but their expression levels decreased in the organisms cultivated at 24°C, suggesting that temperature is one of the factors that influence the expression of members of the
p44
multigene family. Several additional
p44
transcripts that were not detected in the mammals at the acute stage of infection were detected in ticks. Phylogenetic analysis of the 20 different
p44
transcripts revealed that the major transcripts found in mammals and ticks were distinct, suggesting a difference in surface properties between populations of the HGE agent in different host environments. The present study provides new information for understanding the role of the
p44
multigene family in transmission of the HGE agent between mammals and ticks.
Lack of Fusion of Azurophil Granules with Phagosomes during Phagocytosis ofMycobacterium smegmatisby Human Neutrophils Is Not Actively Controlled by the Bacterium
Authors: Cougoule Céline , Constant Patricia , Etienne Gilles , Daffé Mamadou , Maridonneau-Parini Isabelle
Published: July 2002

Biogenesis of phagolysosomes is a very rapid event in neutrophils which takes place with nascent unclosed phagosomes, leading to the release of lysosomal enzymes such as β-glucuronidase in the extracellular medium. We have previously shown that, under nonopsonic conditions, both pathogenic and nonpathogenic mycobacteria uncouple phagocytosis from fusion of azurophil granules (specialized secretory lysosomes) with phagosomes. In the present study we questioned whether they actively act on neutrophils to block this process or use phagocytic receptors that negatively control the biogenesis of phagolysosomes. As for live unicellularMycobacterium smegmatis, we observed that nonopsonic phagocytosis of heat-killed mycobacteria did not induce the release of β-glucuronidase, indicating thatM. smegmatisdoes not actively act on the fusion process in neutrophils. In contrast, phagocytosis of unicellularM. smegmatisopsonized in immune serum or that of small nonopsonized mycobacterial aggregates restored the biogenesis of phagolysosomes. Aggregates were internalized in a CR3- and cholesterol-dependent manner as unicellular mycobacteria. However, aggregates but not unicellular bacteria triggered F-actin and Hck recruitment at the phagosomes, events that have been associated with lysosome fusion. Thus, we propose thatM. smegmatisdoes not actively control the fusion of azurophil granules at early time points postinfection and that mycobacterial aggregates recruit large clusters of receptors at the neutrophil surface which could trap proteins implicated in the biogenesis of phagolysosomes.
Pseudomonas aeruginosa
Quorum-Sensing Systems May Control Virulence Factor Expression in the Lungs of Patients with Cystic Fibrosis
Authors: Erickson David L. , Endersby Ryan , Kirkham Amanda , Stuber Kent , Vollman Dolina D. , Rabin Harvey R. , Mitchell Ian , Storey Douglas G.
Published: July 2002

Individuals with cystic fibrosis (CF) are commonly colonized with
Pseudomonas aeruginosa
. The chronic infections caused by
P. aeruginosa
are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants,
P. aeruginosa
quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of
N
-(3-oxododecanoyl)-
l
-homoserine (3-oxo-C12-HSL) and
N
-butyryl-
l
-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene
lasI
and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of
lasI
and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.
Phase I Evaluation of Δ
virG Shigella sonnei
Live, Attenuated, Oral Vaccine Strain WRSS1 in Healthy Adults
Authors: Kotloff Karen L. , Taylor David N. , Sztein Marcelo B. , Wasserman Steven S. , Losonsky Genevieve A. , Nataro James P. , Venkatesan Malabi , Hartman Antoinette , Picking William D. , Katz David E. , Campbell James D. , Levine Myron M. , Hale Thomas L.
Published: July 2002

We conducted a phase I trial with healthy adults to evaluate WRSS1, a live, oral Δ
virG Shigella sonnei
vaccine candidate. In a double-blind, randomized, dose-escalating fashion, inpatient volunteers received a single dose of either placebo (
n
= 7) or vaccine (
n
= 27) at 3 × 10
3
CFU (group 1), 3 × 10
4
CFU (group 2), 3 × 10
5
CFU (group 3), or 3 × 10
6
CFU (group 4). The vaccine was generally well tolerated, although a low-grade fever or mild diarrhea occurred in six (22%) of the vaccine recipients. WRSS1 was recovered from the stools of 50 to 100% of the vaccinees in each group. The geometric mean peak anti-lipopolysaccharide responses in groups 1 to 4, respectively, were 99, 39, 278, and 233 for immunoglobulin (IgA) antibody-secreting cell counts; 401, 201, 533, and 284 for serum reciprocal IgG titers; and 25, 3, 489, and 1,092 for fecal IgA reciprocal titers. Postvaccination increases in gamma interferon production in response to
Shigella
antigens occurred in some volunteers. We conclude that WRSS1 vaccine is remarkably immunogenic in doses ranging from 10
3
to 10
6
CFU but elicits clinical reactions that must be assessed in further volunteer trials.
Construction of atetR-IntegratedSalmonella entericaSerovar Typhi CVD908 Strain That Tightly Controls Expression of the Major Merozoite Surface Protein ofPlasmodium falciparumfor Applications in Human Vaccine Production
Authors: Qian Feng , Pan Weiqing
Published: July 2002

AttenuatedSalmonellastrains are an attractive live vector for delivery of a foreign antigen to the human immune system. However, the problem with this vector lies with plasmid segregation and the low level of expression of the foreign gene in vivo when constitutive expression is employed, leading to a diminished immune response. We have established inducible expressions of foreign genes in theSalmonella entericaserovar Typhi CVD908 vaccine strain using the tetracycline response regulatory promoter. To set up this system, a tetracycline repressor (tetR) was integrated into a defined ΔaroClocus of the chromosome via suicide plasmid pJG12/tetR-neo. To remove theneogene conferring kanamycin resistance from the locus, acreexpression vector under the control of the tetracycline response promoter was transformed into the clone; expression of the Cre recombinase excised theneogene and generated the end strain CVD908-tetR. Expression of the luciferase reporter gene in this strain is dependent on the presence of tetracycline in the medium and can be regulated up to 4,773-fold. Moreover, the tightly controlled expression of major merozoite surface protein 1 (MSP1) and parts ofPlasmodium falciparumwas achieved, and the product yield was increased when the inducible expression system was employed. Inoculation of bacteria harboring plasmid pZE11/MSP142in mice produced the protein in liver and spleen controlled by the inducer. The persistence of the plasmid-carrying bacteria in mice was determined. Peak colonization of both liver and spleen was detected on the third day postinoculation and was followed by a decline in growth curves. After 14 days postinfection, the majority of the bacteria (>90%) recovered from the liver and spleen of the mice retained the plasmid when expression was induced; this clearly indicated that stability of the expression vector in vivo was improved by inducible expression. Establishment of the regulatory system in the vaccine strain may broaden the range of its use by enhancing plasmid stability and expression levels in vivo. Moreover, the availability of the vaccine strain inducibly expressing the entire MSP1 provides possibilities for examining its immunogenicity, particularly the cellular response in animal models.
Campylobacter Protein Glycosylation Affects Host Cell Interactions
Authors: Szymanski Christine M. , Burr Donald H. , Guerry Patricia
Published: July 2002

Campylobacter jejuni
81-176
pgl
mutants impaired in general protein glycosylation showed reduced ability to adhere to and invade INT407 cells and to colonize intestinal tracts of mice.
NovelAeromonas hydrophilaPPD134/91 Genes Involved in O-Antigen and Capsule Biosynthesis
Authors: Zhang Y. L. , Arakawa E. , Leung K. Y.
Published: July 2002

The sequences of the O-antigen and capsule gene clusters of the virulentAeromonas hydrophilastrain PPD134/91 were determined. The O-antigen gene cluster is 17,296 bp long and comprises 17 genes. Seven pathway genes for the synthesis of rhamnose and mannose, six transferase genes, one O unit flippase gene, and one O-antigen chain length determinant gene were identified by amino acid sequence similarity. PCR and Southern blot analysis were performed to survey the distribution of these 17 genes among 11A. hydrophilastrains of different serotypes.A. hydrophilaPPD134/91 might belong to serotype O:18, as represented by JCM3980; it contained all the same O-antigen genes as JCM3980 (97 to 100% similarity at the DNA and amino acid levels). The capsule gene cluster ofA. hydrophilaPPD134/91 is 17,562 bp long and includes 13 genes, which were assembled into three distinct regions similar to those of the group II capsule gene cluster ofEscherichia coliand other bacteria. Regions I and III contained four and two capsule transport genes, respectively. Region II had five genes which were highly similar to capsule synthesis pathway genes found in other bacteria. Both the purified O-antigen and capsular polysaccharides increased the ability of the avirulentA. hydrophilastrain PPD35/85 to survive in naïve tilapia serum. However, the purified surface polysaccharides had no inhibitory effect on the adhesion ofA. hydrophilaPPD134/91 to carp epithelial cells.
Chlamydia pneumoniaeInfection Induces Differentiation of Monocytes into Macrophages
Authors: Yamaguchi Hiroyuki , Haranaga Shusaku , Widen Ray , Friedman Herman , Yamamoto Yoshimasa
Published: July 2002

Migration and differentiation of monocytes to the intima of blood vessels may be a crucial first step in the development of atherosclerosis associated withChlamydia(Chlamydophila)pneumoniae. However, the involvement ofC. pneumoniaeinfection in such steps is not clear. In the present study, therefore, the differentiation-inducing activity ofC. pneumoniaeto monocytes was examined. Human THP-1 monocytic cell line cells were infected withC. pneumoniae, and the differentiation of monocytes to macrophages was assessed by cell morphology, phagocytic activity, and expression of a cell surface adhesion molecule. The monocytic cells infected with viable bacteria markedly differentiated into macrophages associated with diffused cell morphology, increased uptake of polystyrene beads and increased ICAM-1 (intercellular adhesion molecule 1) expression on the cell surfaces. Heat-killed bacteria did not induce any morphological changes or increase of phagocytosis, but they did induce an increase of cell surface ICAM-1 expressions in THP-1 monocytic cells. The antibiotic minocycline treatment of infected cells resulted in marked inhibition of the cell differentiation as well asC. pneumoniaegrowth in the cells, but not ICAM-1 expression. In addition, the experiments with human peripheral blood monocytes infected withC. pneumoniaealso showed the differentiation of macrophages assessed by morphological change and phagocytic activity. These results indicate thatC. pneumoniaeinfection may directly induce the differentiation of monocytes to macrophages. However, antigenic stimulation of monocytes with bacteria may not be sufficient for a full macrophage differentiation.
Antigenic Diversity inEimeria maximaand the Influence of Host Genetics and Immunization Schedule on Cross-Protective Immunity
Authors: Smith Adrian L. , Hesketh Patricia , Archer Andrew , Shirley Martin W.
Published: July 2002

Eimeriaspp. are a group of highly successful intracellular protozoan parasites that develop within enterocytes.Eimeria maximafrom the chicken is characterized by high immunogenicity (a small priming infection gives complete immunity to subsequent homologous challenge) and naturally occurring antigenically variant populations that do not completely cross-protect. In this study we examined the expression of antigenic diversity inE. maxima, as manifested by cross-strain protection in a series of inbred chicken lines. The IAH line of Light Sussex chickens and all lines of inbred White Leghorns were susceptible to primary infections with either of two strains (H and W) ofE. maximaand were protected completely against challenge with the homologous strain of parasite. The extent of cross-protection against the heterologous parasite strain varied from 0 to almost 100% depending on host genetics. Interestingly, in one inbred line of chickens (line 15I) the cross-protective phenotype was directional and intensely influenced by the infection history of the host. The basis for the observed variation in cross-protection is not known, but our results suggest that the major histocompatibility complex is not a major genetic component of the phenotype. These results are discussed in relation to the number of protective antigens presented by complex pathogens and the development of immunoprotective responses in hosts of different genetic backgrounds.
Yersinia pseudotuberculosis
Produces a Cytotoxic Necrotizing Factor
Authors: Lockman Hank A. , Gillespie Rebecca A. , Baker Beth D. , Shakhnovich Elizabeth
Published: July 2002

Cell extracts from
Yersinia pseudotuberculosis
induced multinucleation in HEp-2 cells in a manner similar to the effect caused by
Escherichia coli
cytotoxic necrotizing factor (CNF). The activity was not dependent on the
Yersinia
70-kb virulence plasmid, and the activity was not inhibited by antibodies capable of neutralizing
E. coli
CNF type 1. The nucleotide sequence of the
Yersinia cnf
gene was 65.1% identical to the
E. coli cnf
gene.
Regulation of Antigen-Specific Immunoglobulin G Subclasses in Response to Conserved and PolymorphicPlasmodium falciparumAntigens in an In Vitro Model
Authors: Garraud Olivier , Perraut Ronald , Diouf Ababacar , Nambei Wilfrid S. , Tall Adama , Spiegel André , Longacre Shirley , Kaslow David C. , Jouin Hélène , Mattei Denise , Engler Gina M. , Nutman Thomas B. , Riley Eleanor M. , Mercereau-Puijalon Odile
Published: July 2002

Cytophilic antibodies (Abs) play a critical role in protection againstPlasmodium falciparumblood stages, yet little is known about the parameters regulating production of these Abs. We used an in vitro culture system to study the subclass distribution of antigen (Ag)-specific immunoglobulin G (IgG) produced by peripheral blood mononuclear cells (PBMCs) from individuals exposed toP. falciparumor unexposed individuals. PBMCs, cultivated with or without cytokines and exogenous CD40/CD40L signals, were stimulated with a crude parasite extract, recombinant vaccine candidates derived from conserved Ags (19-kDa C terminus of merozoite surface protein 1 [MSP119], R23, and PfEB200), or recombinant Ags derived from the polymorphic Ags MSP1 block 2 and MSP2. NoP. falciparum-specific Ab production was detected in PBMCs from unexposed individuals. PBMCs from donors exposed frequently toP. falciparuminfections produced multiple IgG subclasses when they were stimulated with the parasite extract but usually only one IgG subclass when they were stimulated with a recombinant Ag. Optimal Ab production required addition of interleukin-2 (IL-2) and IL-10 for all antigenic preparations. The IgG subclass distribution was both donor and Ag dependent and was only minimally influenced by the exogenous cytokine environment. In vitro IgG production and subclass distribution correlated with plasma Abs to some Ags (MSP119, R23, and MSP2) but not others (PfEB200 and the three MSP1 block 2-derived Ags). Data presented here suggest that intrinsic properties of the protein Ag itself play a major role in determining the subclass of the Ab response, which has important implications for rational design of vaccine delivery.
Expression and Characterization of StreptococcalrgpGenes Required for Rhamnan Synthesis inEscherichia coli
Authors: Shibata Yukie , Yamashita Yoshihisa , Ozaki Kazuhisa , Nakano Yoshio , Koga Toshihiko
Published: July 2002

Six genes (rgpAthroughrgpF) that were involved in assembling the rhamnose-glucose polysaccharide (RGP) inStreptococcus mutanswere previously identified (Y. Yamashita, Y. Tsukioka, K. Tomihisa, Y. Nakano, and T. Koga, J. Bacteriol.180:5803-5807, 1998). The group-specific antigens of Lancefield group A, C, and E streptococci and the polysaccharide antigen ofStreptococcus sobrinushave the same rhamnan backbone as the RGP ofS. mutans. Escherichia coliharboring plasmid pRGP1 containing all sixrgpgenes did not synthesize complete RGP. However,E. colicarrying a plasmid with all of thergpgenes except forrgpEsynthesized the rhamnan backbone of RGP without glucose side chains, suggesting that in addition torgpE, another gene is required for glucose side-chain formation. Synthesis of the rhamnan backbone inE. colirequired the initiation of transfer ofN-acetylglucosamine to a lipid carrier and the expression of thergpCandrgpDgenes encoding the putative ABC transporter specific for RGP. The similarities in RGP synthesis betweenE. coliandS. mutanssuggest common pathways for rhamnan synthesis. Therefore, we evaluated the rhamnosyl polymerization process inE. coliby high-resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the lipooligosaccharide (LOS). AnE. colitransformant harboringrgpAproduced the LOS modified by the addition of a single rhamnose residue. Furthermore, thergpA,rgpB, andrgpFgenes of pRGP1 were independently mutated by an internal deletion, and the LOS chemotypes of their transformants were examined. The transformant with anrgpAdeletion showed the same LOS profile asE. coliwithout a plasmid. The transformant with anrgpBdeletion showed the same LOS profile asE. coliharboringrgpAalone. The transformant with anrgpFdeletion showed the LOS band with the most retarded migration. On the basis of these results, we speculated that RgpA, RgpB, and RgpF, in that order, function in rhamnan polymerization.
Oral Vaccination with Subunit Vaccines Protects Animals against Aerosol Infection withMycobacterium tuberculosis
Authors: Doherty T. Mark , Olsen Anja Weinrich , van Pinxteren Laurens , Andersen Peter
Published: July 2002

Immunity againstMycobacterium tuberculosisdepends largely on activation of cell-mediated responses, and gamma interferon has been shown to play a crucial role in this process in both humans and animal models. Since the lung is normally the organ in which infection is initiated and is the major site of pathology, immune responses in the lung play a significant role in restricting initial infection withM. tuberculosis. The aim of the present study was to stimulate efficient immunity in the lung by targeting the gut mucosa. Detoxified monophosphoryl lipid A (MPL) has been shown to be a relatively nontoxic adjuvant which efficiently promotes the induction of type 1 responses when it is given by the traditional subcutaneous route. We have therefore compared subcutaneous immunization of mice to oral immunization by using a model subunit vaccine carrying two immunodominant proteins fromM. tuberculosis, in combination with MPL-based adjuvants. While less effective when used to prime a response, a heterologous priming and boosting vaccination strategy employing oral boosting induced significant systemic type 1 responses which equaled and surpassed those attained by subcutaneous immunization protocols. Moreover, the increased immune responses observed correlated with the induction of substantial protection against subsequent aerosol infection with virulentM. tuberculosisat levels comparable to, or better than, those obtained by multiple subcutaneous vaccinations. These results demonstrate that booster vaccinations via mucosal surfaces, by combining efficient subunit vaccines with the potent adjuvant MPL, may be an effective method of addressing some of the shortcomings of current vaccination strategies.
DNA Immunization in a Mouse Model of Latent Tuberculosis: Effect of DNA Vaccination on Reactivation of Disease and on Reinfection with a Secondary Challenge
Authors: Repique Charlene J. , Li Amy , Collins Frank M. , Morris Sheldon L.
Published: July 2002

Individuals who are latently infected with
Mycobacterium tuberculosis
can develop active disease via either endogenous reactivation of the latent bacilli or exogenous reinfection with a second mycobacterial strain. In this study, we investigated whether immunization with a tuberculosis DNA vaccine cocktail that induces significant protective responses in mice could prevent reactivation of disease in a murine latent-tuberculosis model. In addition, we assessed whether DNA vaccination could retard the growth of a secondary aerogenic infection with
M. tuberculosis
(exogenous reinfection) in latently infected mice. In the reactivation studies, administration of the DNA vaccine combination did not prevent recrudescence of the latent infection after injection of dexamethasone. Moreover, for the reinfection experiments, only a modest decrease in the growth of a secondary
M. tuberculosis
challenge in DNA-vaccinated animals, compared to controls, was observed 14 and 28 days after the reinfection of previously exposed mice. Interestingly, although proliferation of the secondary challenge was reduced significantly in a nonvaccinated chronic-infection group relative to the naïve controls, the number of bacilli still increased by 500-fold 1 month after the secondary challenge in mice with active tuberculosis. These results indicate that novel immunotherapeutic approaches will be required to prevent reactivation of infection or reinfection of individuals with latent tuberculosis.
Multiple-Locus Variable-Number Tandem Repeat Analysis Reveals Genetic Relationships within
Bacillus anthracis
Authors: Keim P. , Price L. B. , Klevytska A. M. , Smith K. L. , Schupp J. M. , Okinaka R. , Jackson P. J. , Hugh-Jones M. E.
Published: July 2002

Bacillus anthracis
is one of the most genetically homogeneous pathogens described, making strain discrimination particularly difficult. In this paper, we present a novel molecular typing system based on rapidly evolving variable-number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) uses the combined power of multiple alleles at several marker loci. In our system, fluorescently labeled PCR primers are used to produce PCR amplification products from eight VNTR regions in the
B. anthracis
genome. These are detected and their sizes are determined using an ABI377 automated DNA sequencer. Five of these eight loci were discovered by sequence characterization of molecular markers (
vrrC
1
,
vrrC
2
,
vrrB
1
,
vrrB
2
, and CG3), two were discovered by searching complete plasmid nucleotide sequences (pXO1-aat and pXO2-at), and one was known previously (
vrrA
). MLVA characterization of 426
B. anthracis
isolates identified 89 distinct genotypes. VNTR markers frequently identified multiple alleles (from two to nine), with Nei's diversity values between 0.3 and 0.8. Unweighted pair-group method arithmetic average cluster analysis identified six genetically distinct groups that appear to be derived from clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. Human commerce doubtlessly has contributed to the dispersal of particular clones in ancient and modern times.
Hyperrecombination in
Streptococcus pneumoniae
Depends on an Atypical
mutY
Homologue
Authors: Samrakandi Moulay Mustapha , Pasta Franck
Published: July 2002

The unusual behavior of the mutation
ami36
, which generates hyperrecombination in two point crosses, was previously attributed to a localized conversion process changing A/G mispairs into CG pairs. Although the mechanism was found to be dependent on the DNA polymerase I, the specific function responsible for this correction was still unknown. Analysis of the pneumococcal genome sequence has revealed the presence of an open reading frame homologous to the gene
mutY
of
Escherichia coli
. The gene
mutY
encodes an adenine glycosylase active on A/G and A/7,8-dihydro-8-oxoguanine (8-OxoG) mismatches, inducing their repair to CG and C/8-OxoG, respectively. Here we report that disrupting the pneumococcal
mutY
homologue abolishes the hyperrecombination induced by
ami36
and leads to a mutator phenotype specifically enhancing AT-to-CG transversions. The deduced amino acid sequence of the pneumococcal MutY protein reveals the absence of four cysteines, highly conserved in the endonuclease III/MutY glycosylase family, which ligate a [4Fe-4S]
2+
cluster. The actual function of this cluster is still intriguing, inasmuch as we show that the pneumococcal gene complements a
mutY
strain of
E. coli.