bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanical Fractionation Reveals Structural Requirements for Enteropathogenic
Escherichia coli
Tir Insertion into Host Membranes
Authors: Gauthier Annick , de Grado Myriam , Finlay B. Brett
Published: July 2002

Enteropathogenic
Escherichia coli
(EPEC) inserts its receptor for intimate adherence (Tir) into host cell membranes by using a type III secretion system. Detergents are frequently used to fractionate infected host cells to investigate bacterial protein delivery into mammalian cells. In this study, we found that the Triton X-100-soluble membrane fraction from EPEC-infected HeLa cells was contaminated with bacterial proteins. We therefore applied a mechanical method of cell lysis and ultracentrifugation to fractionate infected HeLa cells to investigate the biology and biochemistry of Tir delivery and translocation. This method demonstrates that the translocation of Tir into the host cell membrane requires its transmembrane domains, but not tyrosine phosphorylation or binding to Tir's ligand, intimin.
Differential Expression of Caveolin-1 in Lipopolysaccharide-Activated Murine Macrophages
Authors: Lei Mei G. , Morrison David C.
Published: July 2002

Five reciprocal cycles of subtractive hybridization using cDNA generated from fibroblasts with normal lipopolysaccharide (LPS) responsiveness (lpsn) and from hyporesponsive (lpsd) fibroblasts have led to the finding that caveolin-1 is expressed at markedly higher levels of mRNA inlpsdthan inlpsnfibroblasts. Caveolin-1 message can also be readily detected via reverse transcription-PCR in the RAW264.7 and J774.1 macrophage-like cell lines as well as in primary thioglycolate (TG)-elicited mouse peritoneal macrophages. In RAW264.7 cells, both caveolin-1 mRNA and protein levels are down-regulated by LPS. In TG-elicited C3HeB/FeJ peritoneal macrophages, in contrast, expression of both caveolin-1 protein and mRNA is up-regulated in vitro in response to LPS stimulation. The up-regulation of caveolin-1 protein expression in C3HeB/FeJ peritoneal macrophages can be demonstrated at concentrations as low as 1.0 pg of LPS/ml. However, LPS concentrations approximately 4 orders of magnitude higher (104pg/ml) were required to stimulate the LPS-hyporesponsive C3H/HeJ mice peritoneal macrophages such that significant caveolin-1 protein up-regulation was detected. Caveolin-1, a principal component of plasmalemmal caveolae, has been reported as a potentially important regulator for signal transduction during cellular stimulation. The results described in this report suggest that caveolin-1 expression may be associated with LPS signaling/internalization.
Characterization of Lymphocyte Response in the Female Genital Tract during Ascending Chlamydial Genital Infection in the Guinea Pig Model
Authors: Rank Roger G. , Bowlin Anne K. , Kelly Kathleen A.
Published: July 2002

It is well known that pathology caused by chlamydial infection is associated closely with the host response to the organism and that both innate and adaptive host responses contribute to tissue damage. While it is likely that the organism itself initiates the acute inflammatory response by eliciting cytokine and chemokine production from the host cell, the adaptive response is the result of activation of the cell-mediated immune response. While there are several studies describing the nature of the pathologic response in primate, guinea pig, and murine models, there is less information on the kinetics of the CD4 and CD8 response following primary and challenge infections. In this study, we have quantified by flow cytometry the mononuclear cell response to genital infection with the agent of guinea pig inclusion conjunctivitis in the cervix, endometrium, and oviducts at various times following a primary intravaginal infection and after a challenge infection. Tissues from individual animals were assessed for cells expressing CD4, CD8, or Mac-1 and for B cells. Peak responses of each subset occurred 10 to 14 days after a primary infection. The number of Mac-1-expressing cells in each tissue site was found to be dependent on the size of the inoculating dose of chlamydiae. The responses of each cell type were generally stronger in the cervix than in the upper genital tract. In contrast to the murine model but consistent with the primate models, there were equal numbers of CD4 and CD8 cells present in the infiltrates. Twenty-one days after challenge infection, which was performed 50 days after the primary infection, there was a significant increase in the number of CD4, CD8, and B cells in the oviduct compared to the number of these cells at the same time after a primary infection, providing clear cellular evidence for a cell-mediated immune pathologic response.
Homologous and Heterologous
Borrelia burgdorferi
Challenge of Infection-Derived Immune Rabbits Using Host-Adapted Organisms
Authors: Shang Ellen S. , Wu Xiao-yang , Lovett Michael A. , Miller James N. , Blanco David R.
Published: July 2002

We have recently found that strain B31 infection-immune rabbits are completely protected against homologous challenge with large numbers (>10
6
) of host-adapted
Borrelia burgdorferi
(HAB) (E. S. Shang, C. I. Champion, X. Wu, J. T. Skare, D. B. Blanco, J. N. Miller, and M. A. Lovett, Infect. Immun. 68:4189–4199, 2000). In this study, we have extended these findings to determine whether B31 strain infection-immune rabbits are also protected against heterologous HAB challenge. Infection-immune rabbits challenged with large numbers (>10
6
) of homologous HAB strain B31 were completely protected from erythema migrans (EM) and skin and disseminated infection. In contrast, infection-immune rabbits challenged with heterologous HAB strains N40 and Sh-2-82 were completely susceptible to EM and skin and disseminated infection; challenge with strain 297 also resulted in EM and infection of the skin and viscera, but clearance of infection occurred 3 weeks postchallenge. These findings confirm that immunity elicited in rabbits by B31 strain infection confers complete protection against large-dose homologous HAB challenge but not against a heterologous strain.
Th1 AdjuvantN-Acetyl-d-Glucosamine Polymer Up-Regulates Th1 Immunity but Down-Regulates Th2 Immunity against a Mycobacterial Protein (MPB-59) in Interleukin-10-Knockout and Wild-Type Mice
Authors: Shibata Yoshimi , Honda Ikuro , Justice J. Paul , Van Scott Michael R. , Nakamura Reiko M. , Myrvik Quentin N.
Published: July 2002

Treatment of mice with heat-killed (HK)Mycobacterium bovisBCG or 1- to 10-μm chitin particles (nonantigenicN-acetyl-d-glucosamine polymers) is known to induce innate immune responses, including gamma interferon (IFN-γ) production, which plays a Th1 adjuvant role. However, HK BCG further induces prostaglandin E2-releasing spleen macrophages (Mφ) (PGE2-Mφ), which potentially inhibit Th1 adjuvant activities. We found that chitin particles did not induce PGE2-Mφ formation. To further assess whether chitin has Th1 adjuvant effects, interleukin-10 (IL-10)-knockout (KO) mice and their wild-type (WT, C57BL/6) controls were immunized with a 30-kDa MPB-59 mycobacterial protein mixed with chitin. Immunization with MPB-59 alone induced Th2 responses, characterized by increases in total serum immunoglobulin E (IgE) and specific serum IgG1 levels and spleen Th2 cells producing IL-4, IL-5, and IL-10. No IFN-γ-producing spleen Th1 cells, specific serum IgG2a, or delayed-type hypersentivity (DTH) footpad reactions were detected. On the other hand, chitin–MPB-59 immunization significantly increased spleen Th1 responses, DTH reaction, and serum IgG2a levels along with decreases of Th2 responses. The magnitude of these Th1 adjuvant effects was greater in IL-10-KO mice than in WT mice. In contrast, immunization with HK BCG–MPB-59 showed little or no Th1 adjuvant effect. These data indicate that chitin has a unique Th1 adjuvant effect on the development of Th1 immunity against a mycobacterial antigen. IL-10 down-regulates the adjuvant effect of chitin.
Effect of AttenuatedSalmonella entericaSerovar Typhimurium Expressing aStreptococcus mutansAntigen on Secondary Responses to the Cloned Protein
Authors: Jespersgaard Christina , Zhang Ping , Hajishengallis George , Russell Michael W. , Michalek Suzanne M.
Published: July 2002

AttenuatedSalmonella entericaserovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route withSalmonellaexpressing an insoluble protein and to study the ability to augment recall responses by boosting with eitherSalmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor ofStreptococcus mutans, was recombinantly expressed in the insoluble phase inS. entericaserovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered inSalmonellaat week 1 (groups 1 and 2) and recall responses after a week 15 boost with eitherSalmonellaexpressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P< 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who receivedSalmonellafollowed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations withSalmonellaexpressing GLU. On the other hand, group 2 mice, which receivedSalmonellaexpressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate thatS. entericaserovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.
Survival ofChlamydia pneumoniae-Infected Mono Mac 6 Cells Is Dependent on NF-κB Binding Activity
Authors: Wahl Christian , Oswald Franz , Simnacher Ulrike , Weiss Sonja , Marre Reinhard , Essig Andreas
Published: July 2002

The respiratory tract pathogenChlamydia pneumoniaehas been associated with atherosclerosis. Monocytes are supposed to serve as a vehicle for systemic dissemination of intracellularC. pneumoniaefrom the lung to the artery vessel wall. We were therefore interested in pathogen-induced cellular events associated with NF-κB, a crucial transcription factor for both inflammatory cytokines and antiapoptotic molecules. In this study we demonstrate by electrophoretic mobility shift assay thatC. pneumoniaeinfection of the human monocytic cell line Mono Mac 6 induces activation of NF-κB over 48 h, with a maximum level at 1 h postinfection. As shown by supershift assay, the activated NF-κB complex consists of the subunits RelA (p65) and NF-κB1 (p50). Apoptotic host cells were not detected during the early stages of the infection when maximal activation of NF-κB was detected. Pretreatment of Mono Mac 6 with the antioxidant and NF-κB inhibitor PDTC (pyrrolidine dithiocarbamate) induced activation of caspase-3 and led to apoptotic cell death. TheC. pneumoniae-induced activation of the NF-κB complex was reduced by PDTC, which in parallel resulted in an increased apoptosis, as quantified by annexin V labeling and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction. In the complete absence of activated NF-κB, when Mono Mac 6 cells were pretreated with the more potent NF-κB inhibitors MG-132 and parthenolide aC. pneumoniae-mediated rescue of cells from induced apoptosis could not be achieved. Our results indicate that activation of NF-κB inC. pneumoniae-infected Mono Mac 6 cells is associated with protection of Mono Mac 6 cells against apoptosis and might thereby contribute to systemic spread of the pathogen.
Group A Streptococci Bind to Mucin and Human Pharyngeal Cells through Sialic Acid-Containing Receptors
Authors: Ryan Patricia A. , Pancholi Vijaykumar , Fischetti Vincent A.
Published: July 2002

The first step in the colonization of group A streptococci (
Streptococcus pyogenes
) is adherence to pharyngeal epithelial cells. Prior to adherence to their target tissue, the first barrier that the streptococci encounter is the mucous layer of the respiratory tract. The present study was undertaken to characterize the interaction between mucin, the major glycoprotein component of mucus, and streptococci. We report here that
S. pyogenes
is able to bind to bovine submaxillary mucin in solid-phase microtiter plate assays. Western blots probed with
125
I-labeled mucin and a panel of monoclonal antibodies revealed that the streptococcal M protein is one of two cell wall-associated proteins responsible for this binding. The binding was further localized to the N-terminal portion of the M molecule. Further analysis revealed that the M protein binds to the sialic acid moieties on mucin, and this interaction seems to be based on M-protein conformation rather than specific amino acid sequences. We found that sialic acid also plays a critical role in the adherence of an M6 streptococcal strain to the Detroit 562 human pharyngeal cell line and have identified α2-6-linked sialic acid as an important sialylated linkage for M-protein recognition. Western blot analysis of extracted pharyngeal cell membrane proteins identified three potential sialic acid-containing receptors for the M protein. The results are the first to show that sialic acid not only is involved in the binding of the streptococci to mucin but also plays an important role in adherence of group A streptococci to the pharyngeal cell surface.
Genetics of Surface Antigen Expression inPneumocystis carinii
Authors: Stringer James R. , Keely Scott P.
Published: July 2002

This article reviews the molecular genetic data pertaining to the major surface glycoprotein (MSG) gene family ofPneumocystis cariniiand its role in surface variation and compares this fungal system to antigenic variation systems in the protozoanTrypanosoma bruceiand the bacteriaBorreliaspp.
Identification of Rgg-Regulated Exoproteins of
Streptococcus pyogenes
Authors: Chaussee Michael S. , Watson Robert O. , Smoot James C. , Musser James M.
Published: July 2002

Streptococcus pyogenes
secretes many proteins that influence host-pathogen interactions. Despite their importance, relatively little is known about the regulation of these proteins. The
rgg
gene (also known as
ropB
) is required for the expression of streptococcal erythrogenic toxin B (SPE B), an extracellular cysteine protease that contributes to virulence. Proteomics was used to determine if
rgg
regulates the expression of additional exoproteins. Exponential- and stationary-phase culture supernatant proteins made by
S. pyogenes
NZ131
rgg
and NZ131
speB
were separated by two-dimensional electrophoresis. Differences were identified in supernatant proteins from both exponential- and stationary-phase cultures, although considerably more differences were detected among stationary-phase supernatant proteins. Forty-two proteins were identified by peptide fingerprinting with matrix-assisted laser desorption mass spectrometry. Mitogenic factor, DNA entry nuclease (open reading frame [ORF 226]), and ORF 953, which has no known function, were more abundant in the culture supernatants of the
rgg
mutant compared to the
speB
mutant. ClpB, lysozyme, and autolysin were detected in the culture supernatant of the
speB
mutant but not the
rgg
mutant. To determine if Rgg affected protein expression at the transcriptional level, real-time (TaqMan) reverse transcription (RT)-PCR was used to quantitate Rgg-regulated transcripts from NZ131 wild-type and
speB
and
rgg
mutant strains. The results obtained with RT-PCR correlated with the proteomic data. We conclude that Rgg regulates the transcription of several genes expressed primarily during the stationary phase of growth.
Fas (CD95)-Fas Ligand Interactions Are Responsible for Monocyte Apoptosis Occurring as a Result of Phagocytosis and Killing ofStaphylococcus aureus
Authors: Baran J. , Weglarczyk K. , Mysiak M. , Guzik K. , Ernst M. , Flad H.-D. , Pryjma J.
Published: July 2002

Human peripheral blood monocytes become apoptotic following phagocytosis ofStaphylococcus aureus. In this study, we investigated the mechanisms involved in this phenomenon. Cells exposed to bacteria were examined for the surface expression of Fas and Fas ligand (FasL). The level of soluble form of FasL was also measured in the culture supernatants. As Fas-mediated apoptosis involves the activation of caspases, the activities of caspase-8 and caspase-3 were determined. Finally, the involvement of oxidative stress in apoptosis of infected monocytes was investigated. The data indicated that as a consequence of phagocytosis ofS. aureus, FasL is released from the monocyte surface and induces apoptosis of phagocytic monocytes and to some extent the bystander cells. The importance of this mechanism was confirmed by demonstrating that blockage of CD95 preventsS. aureus-induced apoptosis of monocytes. Cell death occurring after phagocytosis ofS. aureusinvolves the activation of caspase-3-like proteases, as the specific caspase-3 inhibitor suppressed apoptosis of infected cells. The generation of reactive oxygen intermediates by phagocytic monocytes by itself is not sufficient as a death signal but rather acts in up-regulating FasL shedding and possibly in modulating caspase activity.
Pheromone Cross-Inhibition between
Staphylococcus aureus
and
Staphylococcus epidermidis
Authors: Otto Michael , Echner Hartmut , Voelter Wolfgang , Götz Friedrich
Published: July 2002

Cross-inhibition by quorum-sensing pheromones between
Staphylococcus aureus
and
Staphylococcus epidermidis
was investigated using all known
S. aureus agr
pheromone subgroups. All
S. aureus
subgroups were sensitive towards the
S. epidermidis
pheromone, with the exception of the recently identified subgroup 4. The subgroup 4 pheromone was also the only
S. aureus
pheromone able to inhibit the
S. epidermidis agr
response. The close relation of subgroup 4 to subgroup 1 suggests that subgroup 4 might have evolved from subgroup 1 by mutation under the selective pressure of competition with
S. epidermidis
. The competition between
S. aureus
and
S. epidermidis
by means of quorum-sensing cross talk seems to be generally in favor of
S. epidermidis
, which might explain the predominance of
S. epidermidis
on the skin and in infections on indwelling medical devices.
Virulence of a Phosphoribosylaminoimidazole Carboxylase-Deficient
Candida
albicans
Strain in an Immunosuppressed Murine Model of Systemic Candidiasis
Authors: Donovan Matthew , Schumuke Jon J. , Fonzi William A. , Bonar Sheri L. , Gheesling-Mullis Karen , Jacob Gary S. , Davisson Vincent Jo , Dotson Stanton B.
Published: July 2002

The relative pathogenicities of three
Candida albicans
strains differing in the function of
ADE2
(the gene encoding phosphoribosylaminoimidazole carboxylase) were evaluated in a murine candidiasis model.
C. albicans
strain CAI7 (
ade2/ade2
), previously constructed by site-specific recombination, was avirulent in immunosuppressed mice compared to the parent strain, CAF2-1, and a heterozygous
ADE2
/
ade2
strain obtained by transforming CAI7 with a wild-type allele. The reduced virulence of CAI7 was correlated with the inability to proliferate in either synthetic medium or serum without the exogenous addition of >10 μg of adenine/ml. The loss of virulence upon site-specific disruption of the
ade2
locus, and the restoration of wild-type virulence with the repair of just one
ade2
allele, confirmed that the
ADE2
gene and de novo purine biosynthesis were required for
Candida
pathogenicity. The potential of the phosphoribosylaminoimidazole carboxylase enzyme as a novel target for antifungal drug discovery is discussed.
Intracellular Crystal Formation as a Mechanism of Cytotoxicity in Murine PulmonaryCryptococcus neoformansInfection
Authors: Feldmesser Marta , Kress Yvonne , Casadevall Arturo
Published: July 2002

Rod-like crystalline structures formed during eosinophilicCryptococcus neoformanspneumonia in C57BL/6 mice. Crystals were found associated with yeast cells and free in host cell cytoplasm. The crystals apparently formed because of the interaction of a host protein with the cryptococcal polysaccharide. Crystal formation likely contributes to pathogenesis by causing cellular damage.
Gamma Interferon and Lipopolysaccharide Interact at the Level of Transcription To Induce Tumor Necrosis Factor Alpha Expression
Authors: Lee Julia Y. , Sullivan Kathleen E.
Published: July 2002

Lipopolysaccharide (LPS) is a very potent inducer of tumor necrosis factor alpha (TNF-α) expression from monocytes and macrophages. Another inflammatory cytokine, gamma interferon (IFN-γ), can potentiate the effects of LPS, but the mechanism is not thoroughly understood. Previous reports emphasized the ability of IFN-γ to upregulate CD14 expression (the receptor for LPS), and nearly all studies have utilized sequential stimulation with IFN-γ followed by LPS to exploit this phenomenon. This study demonstrates that IFN-γ can upregulate the effect of LPS at the level of transcription. Human monoblastic Mono-Mac-6 cells produced up to threefold-greater levels of TNF-α when simultaneously stimulated with LPS and IFN-γ compared to treatment with LPS alone. RNase protection studies showed a similar increase in RNA beginning as early as within 30 min. The synthesis of TNF-α mRNA in IFN-γ- and LPS-treated Mono-Mac-6 cells was also temporally prolonged even though the message turnover rate was identical to that seen in LPS stimulated cells. The modulatory effect of IFN-γ may be mediated by Jak2.
Spa Contributes to the Virulence of Type 18 Group A Streptococci
Authors: McLellan Duncan G. J. , Chiang Edna Y. , Courtney Harry S. , Hasty David L. , Wei Shirley C. , Hu Mary C. , Walls Michael A. , Bloom Jackie J. , Dale James B.
Published: July 2002

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261–1268, 1999). In this study, we have determined the complete sequence of thespagene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.
Staphylococcus aureus
Induces Release of Bradykinin in Human Plasma
Authors: Mattsson Eva , Herwald Heiko , Cramer Henning , Persson Kristin , Sjöbring Ulf , Björck Lars
Published: July 2002

Staphylococcus aureus
is a prominent human pathogen. Here we report that intact
S. aureus
bacteria activate the contact system in human plasma in vitro, resulting in a massive release of the potent proinflammatory and vasoactive peptide bradykinin. In contrast, no such effect was recorded with
Streptococcus pneumoniae.
In the activation of the contact system, blood coagulation factor XII and plasma kallikrein play central roles, and a specific inhibitor of these serine proteinases inhibited the release of bradykinin by
S. aureus
in human plasma. Furthermore, fragments of the cofactor H-kininogen of the contact system efficiently blocked bradykinin release. The results suggest that activation of the contact system at the surface of
S. aureus
and the subsequent release of bradykinin could contribute to the hypovolemic hypotension seen in patients with severe
S. aureus
sepsis. The data also suggest that the contact system could be used as a target in the treatment of
S. aureus
infections.
Differential Gene Expression from Two Transcriptional Units in the
cag
Pathogenicity Island of
Helicobacter pylori
Authors: Joyce Elizabeth A. , Gilbert Joanne V. , Eaton Kathryn A. , Plaut Andrew , Wright Andrew
Published: July 2002

Infection with
Helicobacter pylori
strains containing the
cag
Pathogenicity Island (
cag
PAI) is strongly correlated with the development of severe gastric disease, including gastric and duodenal ulceration, mucosa-associated lymphoid tissue lymphoma, and gastric carcinoma. Although in vitro studies have demonstrated that the expression of genes within the
cag
PAI leads to the activation of a strong host inflammatory response, the functions of most
cag
gene products and how they work in concert to promote an immunological response are unknown. We developed a transcriptional reporter that utilizes urease activity and in which nine putative regulatory sequences from the
cag
PAI were fused to the
H. pylori ureB
gene. These fusions were introduced in single copies onto the
H. pylori
chromosome without disruption of the
cag
PAI. Our analysis indicated that while each regulatory region confers a reproducible amount of promoter activity under laboratory conditions, they differ widely in levels of expression. Transcription initiating upstream of
cag15
and upstream of
cag21
is induced when the respective fusion strains are cocultured with an epithelial cell monolayer. Results of mouse colonization experiments with an
H. pylori
strain carrying the
cag15-ureB
fusion suggested that this putative regulatory region appears to be induced in vivo, demonstrating the importance of the urease reporter as a significant development toward identifying in vivo-induced gene expression in
H. pylori
.
Tapeworm Infection Reduces Epithelial Ion Transport Abnormalities in Murine Dextran Sulfate Sodium-Induced Colitis
Authors: Reardon Colin , Sanchez Ana , Hogaboam Cory M. , McKay Derek M.
Published: July 2002

The rat tapewormHymenolepis diminutawas used to test the hypothesis that helminth infection could modulate murine colitis. Mice were infected with fiveH. diminutacysticercoids, and colitis was evoked via free access to 4% (wt/vol) dextran sulfate sodium (DSS)-containing drinking water for 5 days. BALB/c mice were either infected withH. diminutaand 7 days later exposed to DSS (prophylactic strategy) or started on DSS and infected withH. diminuta48 h later (treatment strategy). Naive andH. diminuta-only-infected mice served as controls. On autopsy, colonic segments were processed for histological examination and myeloperoxidase (MPO) measurement or mounted in Ussing chambers for assessment of epithelial ion transport. Cytokines (gamma interferon [IFN-γ], interleukin 12 [IL-12], and IL-10) were measured in serum and colonic tissue homogenates. DSS treatment resulted in reduced ion responses (indicated by short-circuit current [Isc]) to electrical nerve stimulation, the cholinergic agonist carbachol, and the adenylate cyclase activator forskolin compared to controls.H. diminutainfection, either prophylactic or therapeutic, caused a significant (P< 0.05) amelioration of these DSS-induced irregularities in stimulated ion transport. In contrast, the histopathology (i.e., mixed immune cell infiltrate, edema, and ulcerative damage) and elevated MPO levels that accompany DSS colitis were unaffected by concomitantH. diminutainfection. Similarly, there were no significant differences in levels of IFN-γ, IL-12, or IL-10 in serum or tissue from any of the treatment groups at the time of autopsy. We suggest that abolishment of colitis-induced epithelial ion transport abnormalities byH. diminutainfection provides proof-of-principle data and speculate that helminth therapy may provide relief of disease symptoms in colitis.
Interleukin-6 Deficiency Influences Cytokine Expression in Susceptible BALB Mice Infected withLeishmania majorbut Does Not Alter the Outcome of Disease
Authors: Titus Richard G. , DeKrey Gregory K. , Morris Robin V. , Soares Milena B. P.
Published: July 2002

Since interleukin-6 (IL-6) may promote Th2 responses, we infected BALB IL-6-deficient (IL-6−/−) mice withLeishmania major. There was not a significant difference between the courses of infection (lesion size and parasite burden) in IL-6−/−and wild-type mice, but IL-6−/−mice expressed lower levels of Th2- and Th1-associated cytokines.
Involvement of Mitogen-Activated Protein Kinase Pathways inStaphylococcus aureusInvasion of Normal Osteoblasts
Authors: Ellington John K. , Elhofy Adam , Bost Kenneth L. , Hudson Michael C.
Published: July 2002

Staphylococcus aureusinvades osteoblasts and can persist in the intracellular environment. The present study examined the role of osteoblast mitogen-activated protein kinase (MAPK) pathways in bacterial invasion.S. aureusinfection of normal human and mouse osteoblasts resulted in an increase in the phosphorylation of the extracellular signal-regulated protein kinases (ERK 1 and 2). This stimulation of ERK 1 and 2 correlated with the time course ofS. aureusinvasion, and bacterial adherence induced the MAPK pathway. ERK 1 and 2 phosphorylation was time and dose dependent and required activeS. aureusgene expression for maximal induction. The nonpathogenicStaphylococcus carnosuswas also able to induce ERK 1 and 2 phosphorylation, albeit at lower levels thanS. aureus. Phosphorylation of the stress-activated protein kinases was increased in both infected human and mouse osteoblasts; however, the p38 MAPK pathway was not activated in response toS. aureus. Finally, the transcription factor c-Jun, but not Elk-1 or ATF-2, was phosphorylated in response toS. aureusinfection.
DNA Sequences Encoding CD4+and CD8+T-Cell Epitopes Are Important for Efficient Protective Immunity Induced by DNA Vaccination with aTrypanosoma cruziGene
Authors: Fujimura Adriana E. , Kinoshita Sheila S. , Pereira-Chioccola Vera L. , Rodrigues Mauricio M.
Published: July 2002

Immunization of BALB/c mice with a plasmid containing the gene forTrypanosoma cruzi trans-sialidase (TS) induced antibodies that inhibited TS enzymatic activity, CD4+Th1 and CD8+Tc1 cells, and protective immunity against infection. We used this model to obtain basic information on the requirement of CD4 or CD8 or B-cell epitopes for an effective DNA-induced immunity againstT. cruziinfection. For that purpose, mice were immunized with plasmids containing DNA sequences encoding (i) the entire TS protein, (ii) the TS enzymatic domain, (iii) the TS CD4+T-cell epitopes, (iv) the TS CD8+T-cell epitope, or (v) TS CD4+and CD8+T-cell epitopes. Plasmids expressing the entire TS or its enzymatic domain elicited similar levels of TS-inhibitory antibodies, γ interferon (IFN-γ)-producing T cells, and protective immunity against infection. Although the plasmid expressing TS CD4 epitopes was immunogenic, its protective efficacy against experimental infection was limited. The plasmid expressing the CD8 epitope was poorly immunogenic and provided little protective immunity. The reason for the limited priming of CD8+T cells was due to a requirement for CD4+T cells. To circumvent this problem, a plasmid expressing both CD4+and CD8+T-cell epitopes was produced. This plasmid generated levels of IFN-γ-producing T cells and protective immunity comparable to that of the plasmid expressing the entire catalytic domain of TS. Our observations suggest that plasmids expressing epitopes recognized by CD4+and CD8+T cells may have a better protective potential against infection withT. cruzi.
Cardiac Myosin Autoimmunity in Acute Chagas' Heart Disease
Authors: Leon Juan S. , Godsel Lisa M. , Wang Kegiang , Engman David M.
Published: July 2002

Infection withTrypanosoma cruzi, the agent of Chagas' disease, may induce antibodies and T cells reactive with self antigens (autoimmunity). Because autoimmunity is generally thought to develop during the chronic phase of infection, one hypothesis is that autoimmunity develops only after long-term, low-level stimulation of self-reactive cells. However, preliminary reports suggest that autoimmunity may begin during acuteT. cruziinfection. The goal of the present study was to investigate whether cardiac autoimmunity could be observed during acuteT. cruziinfection. A/J mice infected with the Brazil strain ofT. cruzifor 21 days developed severe myocarditis, accompanied by humoral and cellular autoimmunity. Specifically,T. cruziinfection induced immunoglobulin G (IgG) autoantibodies and delayed type hypersensitivity (DTH) to cardiac myosin. This autoimmunity resembles that which develops in A/J mice immunized with myosin in complete Freund's adjuvant in that myosin-specific antibodies and DTH responses both develop by 21 days postinfection or postimmunization. While the levels of myosin IgG inT. cruzi-infected mice were slightly lower than those in myosin-immunized mice, the magnitude of myosin DTH in the two groups was statistically equivalent. In contrast, C57BL/6 mice, which are resistant to myosin-induced myocarditis and its associated autoimmunity, developed undetectable or low levels of myosin IgG and did not exhibit myosin DTH or myocarditis uponT. cruziinfection. Therefore, humoral and cellular cardiac autoimmunity can develop during acuteT. cruziinfection in the genetically susceptible host.
Surface Accessibility of the 70-KilodaltonChlamydia trachomatisHeat Shock Protein following Reduction of Outer Membrane Protein Disulfide Bonds
Authors: Raulston Jane E. , Davis Carolyn H. , Paul Terry R. , Hobbs J. Dave , Wyrick Priscilla B.
Published: July 2002

Numerous investigations have shown that 70-kDa heat shock protein (Hsp70) homologs interact tightly with hydrophobic proteins and functionally assist proteins in membranous organelles and environments. One such protein is theChlamydia trachomatisHsp70 that is associated with isolated outer membrane complexes of infectious elementary bodies (EB). Previous observations have indicated that chlamydial Hsp70 plays a role in EB attachment to, or entry into, endometrial epithelial cells. In this study, immunofluorescence microscopy and transmission electron microscopy observations showed that chlamydial Hsp70 is not a surface-displayed ligand on purified EB. However, brief exposure of EB to the thiol reducing agent dithiothreitol (DTT) led to surface accessibility of the Hsp70 substrate-binding domain. Reduction of the highly disulfide-cross-linked EB outer membrane proteins with DTT resulted in a decrease in EB attachment and infectivity. Interestingly, exposure of EB to the membrane-impermeable thiol-alkylating reagent 5,5′-dithiobis(2-nitrobenzoic acid) enhanced attachment but compromised infectivity, suggesting that EB outer membrane proteins must be reduced for entry and productive infection. Together, our data suggest that (i) the structural integrity of the EB outer membrane, maintained by protein disulfide bonds, is important during the initial stages of attachment; (ii) reduction occurs within the localized microenvironment of host cell surfaces once intimate contact is established between EB and host cells; and (iii) subsequent conformational changes in EB ultrastructure allow productive infection in host cells. The accessibility of the Hsp70 substrate-binding domain may support the hypothesis that this protein plays a role in events following the initial stage of attachment instead of serving as a primary, surface-displayed adhesin.
Mycoplasma pneumoniae
Induces Chronic Respiratory Infection, Airway Hyperreactivity, and Pulmonary Inflammation: a Murine Model of Infection-Associated Chronic Reactive Airway Disease
Authors: Hardy Robert D. , Jafri Hasan S. , Olsen Kurt , Hatfield Jeanine , Iglehart Janie , Rogers Beverly B. , Patel Padma , Cassell Gail , McCracken George H. , Ramilo Octavio
Published: July 2002

Because chronic
Mycoplasma pneumoniae
respiratory infection is hypothesized to play a role in asthma, the potential of
M. pneumoniae
to establish chronic respiratory infection with associated pulmonary disease was investigated in a murine model. BALB/c mice were intranasally inoculated once with
M. pneumoniae
and examined at 109, 150, 245, 368, and 530 days postinoculation.
M. pneumoniae
was detected in bronchoalveolar lavage fluid by culture or PCR in 70 and 22% of mice at 109 and 530 days postinoculation, respectively. Lung histopathology was normal up to 368 days postinoculation. At 530 days, however, 78% of the mice inoculated with
M. pneumoniae
demonstrated abnormal histopathology characterized by peribronchial and perivascular mononuclear infiltrates. A mean histopathologic score (HPS) at 530 days of 5.1 was significantly greater (
P
< 0.01) than that for controls (HPS score of 0). Serum anti-
M. pneumoniae
immunoglobulin G was detectable in all of the mice inoculated with
M. pneumoniae
and was inversely correlated with HPS (
r
= −0.95,
P
= 0.01) at 530 days postinoculation. Unrestrained whole-body plethysmography measurement of enhanced pause revealed significantly elevated airway methacholine reactivity in
M. pneumoniae
-inoculated mice compared with that in controls at 245 days (
P
= 0.03) and increased airway obstruction at 530 days (
P
= 0.01). Murine
M. pneumoniae
respiratory infection can lead to chronic pulmonary disease characterized by airway hyperreactivity, airway obstruction, and histologic inflammation.
Use of Bacteriophage Ba1 To Identify Properties Associated withBordetella aviumVirulence
Authors: Shelton Celia B. , Temple Louise M. , Orndorff Paul E.
Published: July 2002

Bordetella aviumcauses bordetellosis, an upper respiratory disease of birds. Commercially raised turkeys are particularly susceptible. We report here on the use of a recently describedB. aviumbacteriophage, Ba1, as a tool for investigating the effects of lysogeny and phage resistance on virulence. We found that lysogeny had no effect on any of the in vivo or in vitro measurements of virulence we employed. However, two-thirds (six of nine) spontaneous phage-resistant mutants of our virulent laboratory strain, 197N, were attenuated. Phage resistance was associated, in all cases, with an inability of the mutants to bind phage. Further tests of the mutants revealed that all had increased sensitivities to surfactants, and increased amounts of incomplete (O-antigen-deficient) lipopolysaccharide (LPS) compared to 197N. Hot phenol-water-extracted 197N LPS inactivated phage in a specific and dose-dependent manner. Acid hydrolysis and removal of lipid A had little effect upon the ability of isolated LPS to inactivate Ba1, suggesting that the core region and possibly the O antigen were required for phage binding. All of the mutants, with one exception, were significantly more sensitive to naive turkey serum and, without exception, significantly less able to bind to tracheal rings in vitro than 197N. Interestingly, the three phage-resistant mutants that remained virulent appeared to be O antigen deficient and were among the mutants that were the most serum sensitive and least able to bind turkey tracheal rings in vitro. This observation allowed us to conclude that even severe defects in tracheal ring binding and serum resistance manifested in vitro were not necessarily indicative of attenuation and that complete LPS may not be required for virulence.
Identification of a
Mycobacterium tuberculosis
Putative Classical Nitroreductase Gene Whose Expression Is Coregulated with That of the
acr
Gene within Macrophages, in Standing versus Shaking Cultures, and under Low Oxygen Conditions
Authors: Purkayastha Anjan , McCue Lee Ann , McDonough Kathleen A.
Published: July 2002

Tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. This effort will benefit greatly from a better understanding of gene regulation in
Mycobacterium tuberculosis,
particularly with respect to this pathogen's response to its host environment. We examined the behavior of two promoters from the divergently transcribed
M. tuberculosis
genes
acr
/
hsp
X/Rv2031c (α-crystallin homolog) and Rv2032/
acg
(
acr
-coregulated gene) by using a promoter-GFP fusion assay in
Mycobacterium bovis
BCG. We found that Rv2032 is a novel macrophage-induced gene whose expression is coregulated with that of
acr
. Relative levels of intracellular induction for both promoters were significantly affected by shallow standing versus shaking bacterial culture conditions prior to macrophage infection, and both promoters were strongly induced under low oxygen conditions. Deletion analyses showed that DNA sequences within a 43-bp region were required for expression of these promoters under all conditions. Multiple sequence alignment and database searches performed with PROBE indicated that Rv2032 is one of eight
M. tuberculosis
genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment. These findings show that mycobacterial culture conditions can greatly influence the results and interpretation of subsequent gene regulation experiments. We propose that these differences might be exploited for dissection of the regulatory factors that affect mycobacterial gene expression within the host.
Antibody-Mediated Protection in MurineCryptococcus neoformansInfection Is Associated with Pleotrophic Effects on Cytokine and Leukocyte Responses
Authors: Feldmesser Marta , Mednick Aron , Casadevall Arturo
Published: July 2002

Cryptococcus neoformans, an encapsulated yeast, is a common cause of life-threatening meningoencephalitis in immunosuppressed patients. We previously observed that administration of a monoclonal antibody (MAb) to the capsular polysaccharide to mice with pulmonary infection prolonged survival and enhanced granulomatous inflammation without reducing lung CFU. To understand the mechanism of MAb action, we studied leukocyte recruitment and cytokine profiles in lungs of A/JCr mice. B lymphocytes were the predominant cell type in lung infiltrates, comprising 15 to 30% of the leukocytes. Despite alterations in histological appearance, fluorescence-activated cell sorter analysis revealed no significant difference in total numbers of lung leukocytes in MAb-treated mice and controls. Differences in the immune response toC. neoformansbetween MAb-treated mice and controls included (i) an increase in the percentage of granulocytes among lung leukocytes on day 14, (ii) higher macrophage surface expression of CD86 on day 28, (iii) larger amounts of IL-10 in lung homogenates at day 7, (iv) a trend toward smaller amounts of gamma interferon mRNA and protein on day 7, and (v) a smaller increase in the levels of interleukin-4 mRNA and protein on day 7. Hence, the immune responses toC. neoformansinfection in the presence and absence of specific antibody were qualitatively similar, and antibody administration was associated with several subtle quantitative differences in immune response parameters that could translate into enhanced survival. MAb may function partly by down-regulating the inflammatory response and reducing host damage. Our findings demonstrate unexpected complexity in the interaction between specific MAb and other components of the host immune response.
Lipopolysaccharide-Specific but Not Anti-Flagellar Immunoglobulin A Monoclonal Antibodies PreventSalmonella entericaSerotype Enteritidis Invasion and Replication within HEp-2 Cell Monolayers
Authors: Iankov Ianko D. , Petrov Dragomir P. , Mladenov Ivan V. , Haralambieva Iana H. , Mitov Ivan G.
Published: July 2002

The protective potential of immunoglobulin A (IgA) monoclonal antibodies (MAbs) directed against O and H antigens ofSalmonella entericaserotype Enteritidis to prevent bacterial adhesion to and invasion of HEp-2 cells was evaluated. Although anti-flagellar IgA MAbs showed strong agglutinating capacities, they did not protect cell monolayers. In contrast, IgA MAbs specific for the O:9 epitope ofSalmonellalipopolysaccharide antigen alone preventedS. entericaserotype Enteritidis entry and replication within HEp-2 cells, and the protection was not mediated by direct binding of antibodies to bacterial adhesins or by agglutination of microorganisms.
Host Immune Response toSalmonella entericaSerovar Typhimurium Infection in Mice Derived from Wild Strains
Authors: Sebastiani Giovanna , Blais Véronique , Sancho Vanessa , Vogel Stefanie N. , Stevenson Mary M. , Gros Philippe , Lapointe Jean-Martin , Rivest Serge , Malo Danielle
Published: July 2002

Studies of mouse models of endotoxemia and sepsis with gram-negative bacteria have shown that the host response is genetically controlled. Mice infected with the gram-negative bacteriumSalmonella entericaserovar Typhimurium exhibit marked genetic differences in disease manifestation, and the wild-derived strainMus musculus molossinusMOLF/Ei is extremely susceptible toS. entericaserovar Typhimurium. The kinetics of bacterial proliferation within the liver and the spleen and histological examination of tissue sections have suggested that MOLF/Ei mice do not succumb to infection because of overwhelming bacterial growth in the reticuloendothelial organs or massive tissue necrosis, as observed in otherSalmonella-susceptible strains. MOLF/Ei mice respond normally to lipopolysaccharide (LPS) in vivo and in vitro, as determined by the production of tumor necrosis factor alpha and spleen cell mitogenesis. However, they have a unique cytokine profile in response to infection compared to that observed for otherSalmonella-susceptible mice. There was increased expression of mRNA of the interleukin-1α (IL-1α) and IL-1β genes as the infection in the spleens and livers of MOLF/Ei mice progressed. Despite the fact that MOLF/Ei mice have the ability to respond to LPS and the fact that there are significant increases in IL-1α and IL-1β mRNA,Nos2in the spleen is not upregulated and nitrite production by spleen cells is reduced. At the central level, the inflammatory response is characterized by strong upregulation of the inhibitory factor kappa B alpha and Toll-like receptor 2 genes, two genes known to be regulated by LPS and IL-1 in the brain. The high levels of IL-1 expression in the spleens and livers of MOLF/Ei mice may have important implications for the activation of peripheral and central innate immune mechanisms.
Intravaginal and Intranasal Immunizations Are Equally Effective in Inducing Vaginal Antibodies and Conferring Protection against Vaginal Candidiasis
Authors: De Bernardis Flavia , Boccanera Maria , Adriani Daniela , Girolamo Antonietta , Cassone Antonio
Published: July 2002

Oophorectomized, estrogen-treated rats were immunized by the intravaginal or intranasal route with a mannoprotein extract (MP) or secreted aspartyl proteinases (Sap) of
Candida albicans
, with or without cholera toxin as a mucosal adjuvant. Both routes of immunization were equally effective in (i) inducing anti-MP and anti-Sap vaginal antibodies and (ii) conferring a high degree of protection against the vaginal infection by the fungus. These data suggest that appropriate fungal antigens and adjuvant can be used to protect against candidal vaginitis, by either route.