bluuebunny/crossref_metadata_2025 huggingface dataset:
Simultaneous Blocking of Human Toll-Like Receptors 2 and 4 Suppresses Myeloid Dendritic Cell Activation Induced byMycobacterium bovisBacillus Calmette-Guérin Peptidoglycan
Authors: Uehori Junji , Matsumoto Misako , Tsuji Shoutaro , Akazawa Takashi , Takeuchi Osamu , Akira Shizuo , Kawata Tsutomu , Azuma Ichiro , Toyoshima Kumao , Seya Tsukasa
Published: July 2003

TheMycobacterium bovisbacillus Calmette-Guérin (BCG) cell wall skeleton (CWS) consists of mycolic acids, arabinogalactan, and peptidoglycan (PGN) and activates Toll-like receptor 2 (TLR2) and TLR4. Here we investigated the ability of the essential portion of highly purified BCG CWS to support the TLR agonist function by using the following criteria: myeloid dendritic cell (DC) maturation, i.e., tumor necrosis factor alpha (TNF-α) production and CD83/CD86 up-regulation. The purified PGN region was sufficient to activate TLR2 and TLR4 in mouse DCs and macrophages; in TLR2 and TLR4 double-knockout cells the BCG PGN-mediated TNF-α production ability was completely impaired. Likewise, stimulation with BCG CWS of HEK293 cells expressing either human TLR2 or TLR4, MD-2, and CD14 resulted in NF-κB activation as determined by a reporter assay. Notably, specific blockers of extracellular human TLR2 (an original cocktail of monoclonal antibodies TLR2.45 and TH2.1) and TLR4 (E5531) inhibited BCG CWS-mediated NF-κB activation by 80%. Using this human TLR blocking system, we tested whether human myeloid DC maturation was TLR2 and TLR4 dependent. BCG PGN-mediated DC maturation was blocked by 70% by suppression of both TLR2 and TLR4 and by 30 to 40% by suppression of either of these TLRs. Similar but less profound suppression of BCG CWS-mediated DC maturation was observed. Hence, the presence of BCG PGN is a minimal requirement for activation of both TLR2 and TLR4 in human DCs, unlike the presence of PGNs of gram-positive bacteria, which activate only TLR2. Unexpectedly, however, BCG PGN, unlike BCG CWS, barely activated NF-κB in HEK293 cells coexpressing TLR2 plus TLR1, TLR2 plus TLR4, TLR2 plus TLR6, or TLR2 plus TLR10, suggesting that PGN receptors other than TLR2 and TLR4 present on human DCs but not on HEK293 cells are involved in TLR signaling for DC activation.
Critical Role of Multidrug Efflux Pump CmeABC in Bile Resistance and In Vivo Colonization ofCampylobacter jejuni
Authors: Lin Jun , Sahin Orhan , Michel Linda Overbye , Zhang Qijing
Published: July 2003

CmeABC functions as a multidrug efflux pump contributing to the resistance ofCampylobacterto a broad range of antimicrobials. In this study, we examined the role of CmeABC in bile resistance and its contribution to the adaptation ofCampylobacter jejuniin the intestinal tract of the chicken, a natural host and a major reservoir forCampylobacter. Inactivation ofcmeABCdrastically decreased the resistance ofCampylobacterto various bile salts. Addition of choleate (2 mM) in culture medium impaired the in vitro growth of thecmeABCmutants but had no effect on the growth of the wild-type strain. Bile concentration varied in the duodenum, jejunum, and cecum of chicken intestine, and the inhibitory effect of the intestinal extracts on the in vitro growth ofCampylobacterwas well correlated with the total bile concentration in the individual sections of chicken intestine. When inoculated into chickens, the wild-type strain colonized the birds as early as day 2 postinoculation with a density as high as 107CFU/g of feces. In contrast, thecmeABCmutants failed to colonize any of the inoculated chickens throughout the study. The minimum infective dose for thecmeABCmutant was at least 2.6 × 104-fold higher than that of the wild-type strain. Complementation of thecmeABCmutants with a wild-typecmeABCallele intransfully restored the in vitro growth in bile-containing media and the in vivo colonization to the levels of the wild-type strain. Immunoblotting analysis indicated that CmeABC is expressed and immunogenic in chickens experimentally infected withC. jejuni. Together, these findings provide compelling evidence that CmeABC, by mediating resistance to bile salts in the intestinal tract, is required for successful colonization ofC. jejuniin chickens. Inhibition of CmeABC function may not only control antibiotic resistance but also prevent the in vivo colonization of pathogenicCampylobacter.
Death Pathways Activated in CaCo-2 Cells by
Clostridium perfringens
Enterotoxin
Authors: Chakrabarti Ganes , Zhou Xin , McClane Bruce A.
Published: July 2003

Clostridium perfringens
enterotoxin (CPE), a 35-kDa polypeptide, induces cytotoxic effects in the enterocyte-like CaCo-2 cell culture model. To identify the mammalian cell death pathway(s) mediating CPE-induced cell death, CaCo-2 cultures were treated with either 1 or 10 μg of CPE per ml. Both CPE doses were found to induce morphological damage and DNA cleavage in CaCo-2 cells. The oncosis inhibitor glycine, but not a broad-spectrum caspase inhibitor, was able to transiently block both of those pathological effects in CaCo-2 cells treated with the higher, but not the lower, CPE dose. Conversely, a caspase 3/7 inhibitor (but not glycine or a caspase 1 inhibitor) blocked morphological damage and DNA cleavage in CaCo-2 cells treated with the lower, but not the higher, CPE dose. Collectively, these results indicate that lower CPE doses cause caspase 3/7-dependent apoptosis, while higher CPE doses induce oncosis. Apoptosis caused by the lower CPE dose was shown to proceed via a classical pathway involving mitochondrial membrane depolarization and cytochrome
c
release. As the CPE concentrations used in this study for demonstrating apoptosis and oncosis have pathophysiologic relevance, these results suggest that both oncosis and apoptosis may occur in the intestines during CPE-associated gastrointestinal disease.
Coordinate Cytokine Gene Expression In Vivo following Induction of Tuberculous Pleurisy in Guinea Pigs
Authors: Allen Shannon Sedberry , McMurray David N.
Published: July 2003

Tuberculous pleurisy is a severe inflammatory response induced byMycobacterium tuberculosisorganisms that have escaped from lung granulomata into the pleural space during pulmonary infection. We have used the guinea pig model of tuberculous pleurisy to examine several aspects of the immune response to this antigen-specific inflammatory event. Pleurisy was induced by injection of heat-killedM. tuberculosisH37Rv directly into the pleural space of guinea pigs previously vaccinated withM. bovisBCG. Four animals were euthanized each day over a period of 9 days. Fluid in the pleural cavity was analyzed for transforming growth factor β1 (TGF-β1) and total interferon (IFN) protein levels. In addition, RNA was obtained from pleural cells and examined for TGF-β1, tumor necrosis factor alpha (TNF-α), IFN-γ, and interleukin-8 (IL-8) expression by real-time PCR. Finally, pleural cells were examined for the ability to proliferate in response to concanavalin A and purified protein derivative (PPD) in vitro. In the pleural fluid, TGF-β1 protein concentrations increased over the course of the inflammatory response while IFN protein levels were not significantly altered. Expression of TGF-β1 mRNA peaked on days 3 and 4, and IFN-γ mRNA expression peaked on day 3 and then returned to background levels. TNF-α mRNA expression was highest on days 2 to 4, and IL-8 mRNA levels remained elevated between days 2 and 5, peaking on day 3 before returning to background levels. PPD-induced proliferative responses were evident by day 3 and remained present throughout the study. Analysis of cytokine expression during tuberculous pleurisy may lead to a better understanding of the self-healing nature of this manifestation of tuberculosis.
Impaired Expression of Inflammatory Cytokines and Chemokines at Early Stages of Infection withLeishmania amazonensis
Authors: Ji Jiaxiang , Sun Jiaren , Soong Lynn
Published: July 2003

Infection of mice withLeishmania majorresults in disease progression or resolution, largely depending on the genetic backgrounds of the mouse strains. Infection withLeishmania amazonensis, on the other hand, causes progressive cutaneous lesions in most inbred strains of mice. We hypothesized that deficient activation of early immune responses contributes to the pathogenesis inL. amazonensis-infected mice. To distinguish early molecular events that determine the outcome ofLeishmaniainfections, we examined cytokine gene expression in C57BL/6 mice infected with eitherL. amazonensisorL. major(a healing model). After 2 to 4 weeks,L. amazonensis-infected mice had significantly delayed and depressed expression of inflammatory cytokines (interleukin-12 [IL-12], gamma interferon, IL-1α, IL-1β), CC chemokines (CC chemokine ligand 3 [CCL3]/macrophage inflammatory protein 1α [MIP-1α], CCL4/MIP-1β, CCL5/RANTES, MIP-2), and chemokine receptors (CCR1, CCR2, CCR5) in foot tissues and draining lymph nodes compared to the expression inL. major-infected controls. These findings correlated with defective T-cell responsiveness to parasite stimulation in vivo and in vitro. Adoptive transfer ofL. amazonensis-specific Th1 cells prior to infection overcame the immune defects of the animals, leading to complete control of the disease. Studies with gene knockout mice suggested that IL-10, but not IL-4, contributed partially to compromised immunity inL. amazonensis-infected hosts. The data suggest that there is impairment in multiple immune functions at early stages of infection withL. amazonensisparasites and provide a compelling rationale to explore immune augmentation as an intervention in American cutaneous leishmaniasis.
Expression of Cytokine and Chemokine Genes by Human Middle Ear Epithelial Cells Induced by Influenza A Virus andStreptococcus pneumoniaeOpacity Variants
Authors: Tong H. H. , Long J. P. , Shannon P. A. , DeMaria T. F.
Published: July 2003

Real-time PCR and enzyme-linked immunosorbent assay were used to evaluate the ability of influenza A virus andStreptococcus pneumoniaeopacity variants, either alone or in combination, to induce cytokine and chemokine genes in primary cultures of human middle ear epithelial (HMEE) cells. Following treatment with influenza A virus, the induction of gene expression, which occurred in a dose- and time-dependent manner, was strong for macrophage inflammatory protein 1α (MIP-1α) and MIP-1β; moderate for tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-8; and weak for IL-1β and monocyte chemotactic peptide 1 (MCP-1). Except for TNF-α, all the gene products were detected in the cell culture supernatants. In contrast, infection of HMEE cells withS. pneumoniaealone induced low levels of mRNA expression of MIP-1α and MIP-1β and did not significantly induce the transcription of the other cytokines and chemokines examined. However, bothS. pneumoniaeopacity variants increased mRNA expression of MIP-1α, MIP-1β, IL-6, and MCP-1 in HMEE cells activated by a prior influenza A virus infection compared to levels in cells treated with either agent alone. Up-regulation of IL-6, IL-8, and MCP-1 mRNA expression and production by the virus in combination with opaqueS. pneumoniaewas two- to threefold higher than that induced by the virus combined with the transparentS. pneumoniaevariant. These data indicate that the activation of HMEE cells by influenza A virus enhances the induction of cytokine and chemokine gene transcripts byS. pneumoniaeand that this effect appears to be most pronounced whenS. pneumoniaeis in the opaque phase.
Investigation of the Role of CD8+T Cells in Bovine Tuberculosis In Vivo
Authors: Villarreal-Ramos B. , McAulay M. , Chance V. , Martin M. , Morgan J. , Howard C. J.
Published: July 2003

Mycobacterium bovisis the causative agent of bovine tuberculosis (TB), and it has the potential to induce disease in humans. CD8+T cells (CD8 cells) have been shown to respond to mycobacterial antigens in humans, cattle, and mice. In mice, CD8 cells have been shown to play a role in protection against mycobacterial infection. To determine the role of CD8 cells in bovine TB in vivo, two groups of calves were infected with the virulentM. bovisstrain AF2122/97. After infection, one group was injected with a CD8 cell-depleting monoclonal antibody (MAb), and the other group was injected with an isotype control MAb. Immune responses to mycobacterial antigens were measured weekly in vitro. After 8 weeks, the animals were killed, and postmortem examinations were carried out. In vitro proliferation responses were similar in both calf groups, but in vitro gamma interferon (IFN-γ) production in 24-h whole-blood cultures was significantly higher in control cattle than in CD8 cell-depleted calves. Postmortem examination showed that calves in both groups had developed comparable TB lesions in the lower respiratory tract and associated lymph nodes. Head lymph node lesion scores, on the other hand, were higher in control calves than in CD8 cell-depleted calves. Furthermore, there was significant correlation between the level of IFN-γ and the head lymph node lesion score. These experiments indicate that CD8 cells play a role in the immune response toM. bovisin cattle by contributing to the IFN-γ response. However, CD8 cells may also play a deleterious role by contributing to the immunopathology of bovine TB.
The Major Cold Shock Gene,
cspA
, Is Involved in the Susceptibility of
Staphylococcus aureus
to an Antimicrobial Peptide of Human Cathepsin G
Authors: Katzif Samuel , Danavall Damien , Bowers Samera , Balthazar Jacqueline T. , Shafer William M.
Published: July 2003

A Tn
551
insertional library of
Staphylococcus aureus
strain ISP479 was challenged with an antimicrobial peptide (CG 117-136) derived from human neutrophil cathepsin G (CG). After repeated selection and screening of surviving colonies, a mutant was identified that expressed increased resistance to CG 117-136. Southern hybridization analysis revealed that the Tn
551
insert in this mutant (SK1) was carried on a 10.6-kb
Eco
RI chromosomal DNA fragment. Subsequent physical mapping of this Tn
551
insert revealed that it was positioned between a putative promoter sequence and the translational start codon of the
cspA
gene, which encodes a protein (CspA) highly similar to the major cold shock proteins CspA and CspB of
Escherichia coli
and
Bacillus subtilis
, respectively. This Tn
551
insertion as well as a separate deletion-insertion mutation in
cspA
decreased the capacity of
S. aureus
to respond to the stress of cold shock and increased resistance to CG 117-136. The results indicate for the first time that a physiologic link exists between bacterial susceptibility to an antimicrobial peptide and a stress response system.
VH3 Gene Usage in Neutralizing Human Antibodies Specific for theEntamoeba histolyticaGal/GalNAc Lectin Heavy Subunit
Authors: Tachibana Hiroshi , Watanabe Katsuomi , Cheng Xun-Jia , Tsukamoto Hideo , Kaneda Yoshimasa , Takeuchi Tsutomu , Ihara Seiji , Petri, William A.
Published: July 2003

A combinatorial human immunoglobulin gene library was constructed from peripheral lymphocytes of an asymptomaticEntamoeba histolyticacyst passer and screened for the production of Fab antibody to the parasite. One of the Fab clones, CP33, recognized the 260-kDa galactose- andN-acetyl-d-galactosamine (Gal/GalNAc)-specific lectin ofE. histolytica. By shuffling the heavy and light chains of CP33 with the heavy and light chains of two libraries derived from the cyst passer and a liver abscess patient, 18 additional clones were obtained. Sequence analysis of the heavy-chain genes, including CP33-H, revealed that all the nearest V-segment germ lines belonged to the VH3 family (VH3-21, VH3-30, VH3-48, and VH3-53), but the levels of homology were only 85 to 95%. The closest D-segment germ line was D2-2 or D6-6, and for the J-segment the closest germ line was JH4b or JH6b. On the other hand, all the light-chain genes, including CP33-L, belonged to the Vκ1 family, in which the closest Vκ germ line gene was 02/012 or L5, with the Jκ1, Jκ2, Jκ4, or Jκ5 segment. CP33 and three other Fabs obtained by light-chain shuffling were purified and analyzed further. All of these Fabs recognized the cysteine-rich domain of the 170-kDa heavy subunit of the Gal/GalNAc lectin. Preincubation ofE. histolyticatrophozoites with these Fabs significantly inhibited amebic adherence to Chinese hamster ovary cells and also inhibited erythrophagocytosis. The ability of the neutralizing antibodies to block erythrophagocytosis for the first time implicates the lectin in phagocytosis and VH3 antibodies in defense against parasitic infections. These results demonstrate the utility of a combinatorial human immunoglobulin gene library for identifying and characterizing neutralizing antibodies from humans with amebiasis.
Antibodies to thePlasmodium falciparumAntigens Circumsporozoite Protein, Thrombospondin-Related Adhesive Protein, and Liver-Stage Antigen 1 Vary by Ages of Subjects and by Season in a Highland Area of Kenya
Authors: John Chandy C. , Zickafoose Joseph S. , Sumba P. Odada , King Christopher L. , Kazura James W.
Published: July 2003

Immunoglobulin G (IgG) antibodies to three vaccine candidate preerythrocyticPlasmodium falciparumantigens were evaluated in children and adults in an epidemic-prone highland area of Kenya during rainy (high-transmission) and dry (low-transmission) seasons. The frequencies and median levels of IgG antibodies to circumsporozoite protein (CSP) and thrombospondin-related adhesive protein (TRAP) were compared to the frequencies and median levels of IgG antibodies to liver-stage antigen 1 (LSA-1) reported previously. The frequencies and median levels of IgG antibodies to CSP and TRAP were similar in children and adults in the rainy season, but they were lower in children than in adults in the dry season. The frequencies and median levels of antibodies to LSA-1 were lower in children than in adults in both the rainy and dry seasons. Antibodies to CSP and LSA-1 were primarily members of the IgG1 and IgG3 subclasses, while antibodies to TRAP were primarily members of the IgG3 and IgG4 subclasses. In a treatment-reinfection study following dry season testing, antibodies to TRAP were associated with a trend toward protection from infection in children (P= 0.051) but not in adults. Antibodies to LSA-1 and CSP did not correlate with protection in children or adults. In this highland area of Kenya with unstable transmission, IgG antibodies to preerythrocyticP. falciparumantigens vary in subjects by age and season, and the protective effects of these antibodies against infection may be different in adults and children.
Characterization of New Members of the Group 3 Outer Membrane Protein Family of
Brucella
spp
Authors: Salhi Imed , Boigegrain Rose-Anne , Machold Jan , Weise Christoph , Cloeckaert Axel , Rouot Bruno
Published: July 2003

Impairment of the
omp25
gene in
Brucella
spp. leads to attenuated strains and confers protection to the host. Omp25 and Omp31, whose functions remain unknown, were the first characterized members of group 3 outer membrane proteins (Omps) (25 to 34 kDa). Recently, genomic and proteomic approaches identified five new putative members of this family, some of which are produced in
B. melitensis
or
B. abortus
. In the present study, using protein microsequencing, we identified new members of group 3 Omps proteins produced in
B. suis
. Since several monoclonal antibodies (MAbs) against Omp25 cross-reacted with other members of group 3 Omps, we also performed Western immunoblotting to compare wild-type
B. suis
with mutants systematically having
B. suis omp25
-related genes knocked out. We demonstrate the production of three paralogs of Omp31 and/or Omp25 in
B. suis
, and the existence of a common site of signal peptide cleavage (AXAAD), which is very similar to that present in the five homologous Omps of
Bartonella quintana
. The seven group 3 Omps were classified in four-subgroups on the basis of percentage amino acid sequence identities: Omp25 alone, the Omp25b-Omp25c-Omp25d cluster, the Omp31/31b subgroup, and the less related Omp22 protein (also called Omp3b). Together with previous data, our results demonstrate that all new members of group 3 Omps are produced in
B. suis
or in other
Brucella
species and we propose a nomenclature that integrates all of these proteins to facilitate the understanding of future
Brucella
interspecies study results.
Mechanism of Fluconazole Resistance in
Candida albicans
Biofilms: Phase-Specific Role of Efflux Pumps and Membrane Sterols
Authors: Mukherjee Pranab K. , Chandra Jyotsna , Kuhn Duncan M. , Ghannoum Mahmoud A.
Published: July 2003

Candida albicans
biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic
Candida
strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 μg/ml, respectively) than the wild-type strain (MIC > 256 μg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC ≥ 256 μg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that
Candida
biofilms expressed
CDR
and
MDR1
genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (
P
= 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (
P
< 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.
Identification of aMoraxella catarrhalisOuter Membrane Protein Exhibiting Both Adhesin and Lipolytic Activities
Authors: Timpe Jennifer M. , Holm Melissa M. , Vanlerberg Serena L. , Basrur Venkatesha , Lafontaine Eric R.
Published: July 2003

The UspA1 and Hag proteins have previously been shown to be involved in the ability of theMoraxella catarrhaliswild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library ofM. catarrhalisDNA fragments, which was introduced into a nonadherentEscherichia colistrain. RecombinantE. colibacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. Transposon mutagenesis of this plasmid identified the potential adhesin genemcaP(M. catarrhalisadherence protein). Sequence analysis revealed that McaP is related to autotransporter proteins and has substantial similarity with the GDSL family of lipolytic enzymes, particularly theMoraxella bovisphospholipase B. Expression of themcaPgene product byE. coliincreased adherence to Chang, A549, and 16HBE14o−polarized human bronchial cells 50- to 100-fold. Spectrophotometric assays withp-nitrophenol derivatives also demonstrated that McaP is an esterase. Furthermore, thin-layer chromatography revealed that McaP cleaves both phosphatidylcholine and lysophosphatidylcholine. McaP releases fatty acids and glycerophosphorylcholine upon cleavage of phosphatidylcholine, thus exhibiting phospholipase B activity. The construction and characterization of isogenicM. catarrhalisO35E mutants demonstrated that the lack of McaP expression abolishes esterase activity and considerably decreases adherence to several human cell lines.
Effect of an Orphan Response Regulator on
Streptococcus mutans
Sucrose-Dependent Adherence and Cariogenesis
Authors: Idone Vincent , Brendtro Stacy , Gillespie Robert , Kocaj Steve , Peterson Erica , Rendi Mara , Warren Wayne , Michalek Suzanne , Krastel Kirsten , Cvitkovitch Dennis , Spatafora Grace
Published: July 2003

Streptococcus mutans
is the principal acidogenic component of dental plaque that demineralizes tooth enamel, leading to dental decay. Cell-associated glucosyltransferases catalyze the sucrose-dependent synthesis of sticky glucan polymers that, together with glucan binding proteins, promote
S. mutans
adherence to teeth and cell aggregation. We generated an
S. mutans
Tn
916
transposon mutant, GMS315, which is defective in sucrose-dependent adherence and significantly less cariogenic than the UA130 wild-type progenitor in germfree rats. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, and N-terminal sequence analysis confirmed the absence of a 155-kDa glucosyltransferase S (Gtf-S) from GMS315 protein profiles. Mapping of the unique transposon insertion in GMS315 revealed disruption of a putative regulatory region located upstream of
gcrR
, a gene previously described by Sato et al. that shares significant amino acid identity with other bacterial response regulators (Y. Sato, Y. Yamamoto, and H. Kizaki, FEMS Microbiol. Lett. 186: 187-191, 2000). The
gcrR
regulator, which we call “
tarC
,” does not align with any of the 13 proposed two-component signal transduction systems derived from in silico analysis of the
S. mutans
genome, but rather represents one of several orphan response regulators in the genome. The results of Northern hybridization and/or real-time reverse transcription-PCR experiments reveal increased expression of both Gtf-S and glucan binding protein C (GbpC) in a
tarC
knockout mutant (GMS900), thereby supporting the notion that TarC acts as a negative transcriptional regulator. In addition, we noted that GMS900 has altered biofilm architecture relative to the wild type and is hypocariogenic in germfree rats. Taken collectively, these findings support a role for signal transduction in
S. mutans
sucrose-dependent adherence and aggregation and implicate TarC as a potential target for controlling
S. mutans
-induced cariogenesis.
Kinetics of Cellular Responses to IntraperitonealBrugia pahangiInfections in Normal and Immunodeficient Mice
Authors: Ramalingam Thirumalai , Rajan Bhargavi , Lee James , Rajan T. V.
Published: July 2003

Filarial infections evoke exuberant inflammatory responses in the peritoneal cavities of immunocompetent mice. Clearance of infection appears to be dependent on complex interactions between B1 and B2 B lymphocytes, T cells, eosinophils, macrophages, and the products of these cells. In an earlier communication, we described the course of infection in normal immunocompetent mice. In this study, we utilize mice with well-characterized mutations that disable one or more effector components of adaptive immunity in order to determine their roles in host protection. We characterize peritoneal exudate cells by flow cytometry and determine the kinetics of accumulation of each of the different cell types following infection withBrugia pahangi. We find that (i) four-color flow-cytometric analysis of peritoneal exudate cells using anti-CD3, -CD11b, -CD19, and -Gr1 can distinguish up to six different populations of cells; (ii) an initial influx of neutrophils occurs within 24 h of infection, independent of the adaptive immune status of mice, and these cells disappear by day 3; (iii) an early influx of eosinophils is seen at the site of infection in all strains studied, but a larger, second wave occurs only in mice with T cells; (iv) the presence of T cells and eosinophils is important in causing an increase in macrophage size during the course of infection; and (v) most unexpectedly, T-cell recruitment appears to be optimal only if B cells are present, since JHD mice recruit significantly fewer T cells to the site of infection.
The
Arcanobacterium pyogenes
Collagen-Binding Protein, CbpA, Promotes Adhesion to Host Cells
Authors: Esmay Paula A. , Billington Stephen J. , Link Malen A. , Songer J. Glenn , Jost B. Helen
Published: July 2003

Arcanobacterium pyogenes
is an opportunistic pathogen associated with suppurative diseases in economically important food animals such as cattle, pigs, and turkeys.
A. pyogenes
adheres to host epithelial cells, and adhesion is promoted by the action of neuraminidase, which is expressed by this organism. However, a neuraminidase-deficient mutant of
A. pyogenes
only had a reduced ability to adhere to host epithelial cells, indicating that other factors are involved in adhesion. Far Western blotting revealed the presence of an approximately 120-kDa
A. pyogenes
cell wall protein that binds collagen type I. The 3.5-kb gene that encodes the 124.7-kDa CbpA protein was cloned, and sequence analysis indicated that CbpA contains a typical MSCRAMM protein domain structure. Recombinant, six-His-tagged CbpA (HIS-CbpA) was capable of binding collagen types I, II, and IV but not fibronectin. In addition, CbpA was involved in the ability of
A. pyogenes
to adhere to HeLa and 3T6 cells, as a
cbpA
knockout strain had 38.2 and 57.0% of wild-type adhesion, respectively. This defect could be complemented by providing
cbpA
on a multicopy plasmid. Furthermore, HIS-CbpA blocked
A. pyogenes
adhesion to HeLa or 3T6 cells in a dose-dependent manner.
cbpA
was only present in 48% of the
A. pyogenes
strains tested (
n
= 75), and introduction of plasmid-encoded
cbpA
into a naturally
cbpA
-deficient strain increased the ability of this strain to bind to HeLa and 3T6 cells 2.9- and 5.7-fold, respectively. These data indicate that CbpA, a collagen-binding protein of
A. pyogenes
, plays a role in the adhesion of this organism to host cells.
TransfectedPlasmodium knowlesiProduces Bioactive Host Gamma Interferon: a New Perspective for Modulating Immune Responses to Malaria Parasites
Authors: Ozwara Hastings , Langermans Jan A. M. , Kocken Clemens H. M. , van der Wel Annemarie , van der Meide Peter H. , Vervenne Richard A. W. , Mwenda Jason M. , Thomas Alan W.
Published: July 2003

Transgenic pathogenic microorganisms expressing host cytokines such as gamma interferon (IFN-γ) have been shown to manipulate host-pathogen interaction, leading to immunomodulation and enhanced protection. Expression of host cytokines in malaria parasites offers the opportunity to investigate the potential of an immunomodulatory approach by generating immunopotentiated parasites. Using the primate malaria parasitePlasmodium knowlesi, we explored the conditions for expressing host cytokines in malaria parasites.P. knowlesiparasites transfected with DNA constructs for expressing rhesus monkey (Macaca mulatta) IFN-γ under the control of the heterologousP. bergheiapical membrane antigen 1 promoter, produced bioactive IFN-γ in a developmentally regulated manner. IFN-γ expression had no marked effect on in vitro parasite development. Bioactivity of the parasite-produced IFN-γ was shown through inhibition of virus cytopathic effect and confirmed by usingM. mulattaperipheral blood cells in vitro. These data indicate for the first time that it is feasible to generate malaria parasites expressing bioactive host immunomodulatory cytokines. Furthermore, cytokine-expressing malaria parasites offer the opportunity to analyze cytokine-mediated modulation of malaria during the blood and liver stages of the infection.
Human Alveolar Macrophages Infected by Virulent Bacteria Expressing SipB Are a Major Source of Active Interleukin-18
Authors: Obregon Carolina , Dreher Donatus , Kok Menno , Cochand Laurence , Kiama Gitahi S. , Nicod Laurent P.
Published: July 2003

Recent publications have demonstrated that the protease caspase-1 is responsible for the processing of pro-interleukin 18 (IL-18) into the active form. Studies on cell lines and murine macrophages have shown that the bacterial invasion factor SipB activates caspase-1, triggering cell death. Thus, we investigated the role of SipB in the activation and release of IL-18 in human alveolar macrophages (AM), which are the first line of defense against inhaled pathogens. Under steady-state conditions, AM are a more important source of IL-18 than are dendritic cells (DC) and monocytes. Cytokine production by AM and DC was compared after both types of cells had been infected with a virulent strain ofSalmonella entericaserovar Typhimurium and an isogenicsipBmutant, which were used as an infection model. Infection with virulentSalmonellaled to marked cell death with features of apoptosis while both intracellular activation and release of IL-18 were demonstrated. In contrast, thesipBmutant did not induce such cell death or the release of active IL-18. The specific caspase-1 inhibitor Ac-YVAD-CMK blocked the early IL-18 release in AM infected with the virulent strain. However, the type ofSalmonellainfection did not differentially regulate IL-18 gene expression. We concluded that the bacterial virulence factor SipB plays an essential posttranslational role in the intracellular activation of IL-18 and the release of the cytokine in human AM.
Biochemical and Functional Analyses of the Mip Protein: Influence of the N-Terminal Half and of Peptidylprolyl Isomerase Activity on the Virulence of
Legionella pneumophila
Authors: Köhler Rolf , Fanghänel Jörg , König Bettina , Lüneberg Edeltraud , Frosch Matthias , Rahfeld Jens-Ulrich , Hilgenfeld Rolf , Fischer Gunter , Hacker Jörg , Steinert Michael
Published: July 2003

The virulence factor Mip (macrophage infectivity potentiator) contributes to the intracellular survival of
Legionella pneumophila
, the causative agent of Legionnaires' disease. The protein consists of two domains that are connected via a very long α-helix (A. Riboldi-Tunnicliffe et al., Nat. Struct. Biol.
8:
779-783, 2001). The fold of the C-terminal domain (residues 100 to 213) is closely related to human FK506-binding protein (FKBP12), and like FKBP12, Mip exhibits peptidylprolyl
cis
/
trans
isomerase (PPIase) activity. The α-helical N-terminal domain is responsible for the formation of very stable Mip homodimers. In order to determine the importance of the homodimeric state of Mip for its biochemical activities and for infectivity of
Legionella
, a truncated, monomeric Mip variant [Mip
(77-213)
] was overexpressed in
Escherichia coli
and characterized biochemically. In vitro isomerase activity assays revealed that the altered protein exhibits full isomerase activity towards peptide substrates. However, the deletion resulted in a dramatic loss in the efficiency of refolding of reduced and carboxy-methylated RNase T
1
. By
cis
complementation of the Mip-negative mutant strain
L. pneumophila
JR32-2, we constructed the strain
L. pneumophila
JR32-2.4, which expresses an N-terminally truncated variant of Mip. Infection studies with these strains revealed that the N-terminal part and the dimerization of Mip but not its PPIase activity are necessary for full virulence in
Acanthamoeba castellanii
. Infection of guinea pigs showed that strains with dimerization-deficient Mip (JR32-2.4) or a very low PPIase activity (JR32-2.2) were significantly attenuated in the animal model. These results suggest a different role of the PPIase activity and the N-terminally mediated dimeric state of Mip in monocellular systems and during the infection of guinea pigs.
Oral Pretreatment of Mice with CpG DNA Reduces Susceptibility to Oral or Intraperitoneal Challenge with VirulentListeria monocytogenes
Authors: Ray Nancy B. , Krieg Arthur M.
Published: July 2003

Listeria monocytogenesis an enteroinvasive intracellular bacterial pathogen that infects humans and other animals, including mice, sometimes resulting in severe systemic infections. Previous studies showed that intraperitoneal (i.p.) pretreatment of susceptible BALB/c mice with immune-stimulatory CpG DNA 48 to 96 h prior to i.p. challenge with virulentL. monocytogenesreduces bacterial numbers in livers by greater than 100-fold, correlating with recovery from infection. Here we show that oral pretreatment of BALB/c mice with CpG DNA results in decreased susceptibility to either oral or i.p. challenge withL. monocytogenes. A single dose of 200 μg of CpG DNA administered to BALB/c mice orally by gavage 48 h or 7 days before oral challenge with virulentL. monocytogenesreduces bacterial numbers approximately 10- to 100-fold in livers and spleens. Lymphotoxin alpha knockout mice lacking Peyer's patches (PPs) and pretreated orally with CpG DNA 48 h prior to oral challenge withL. monocytogenesalso have reduced susceptibility to infection, suggesting that PPs are required neither for oral infection nor for CpG-induced resistance against oral infection withL. monocytogenes. Surprisingly, 48-h oral pretreatment of BALB/c mice with 100 to 200 μg of CpG DNA results in approximately 100-fold-decreased bacterial numbers in livers following i.p. challenge withL. monocytogenes, suggesting, along with other data in this report, that orally delivered CpG DNA induces systemic resistance to infection. These results indicate that oral administration of CpG DNA induces systemic innate immune defenses against either oral or systemic infection with virulentL. monocytogenes.
Contribution of a Response Regulator to the Virulence of
Streptococcus pneumoniae
Is Strain Dependent
Authors: Blue Clare E. , Mitchell Tim. J.
Published: July 2003

Bacterial two-component signal transduction systems (TCS) enable bacteria to respond to environmental changes and regulate a range of genes accordingly. They have a crucial role in regulating many cellular responses and have excellent potential as antibacterial-drug targets. We have constructed mutations in a TCS response regulator gene for two different strains of the human pathogen
Streptococcus pneumoniae
. These mutants have been analyzed in our murine model of infection. Data suggest that in a D39 background the response regulator gene is essential for virulence; an isogenic mutant is avirulent via intraperitoneal, intranasal, and intravenous routes of infection. This mutant, which does not show impaired growth in vitro, is unable to grow in the lung tissue or in blood. Mutation of the response regulator in a 0100993 background results in a strain that is fully virulent intraperitoneally and intravenously but shows decreased levels of bacteremia and increased murine survival following intranasal infection. The ability to grow in the lung tissue is not impaired in this mutant, suggesting that it has an impaired ability to disseminate from the lungs to the systemic circulation. Our data highlight the importance of assessing the contribution of putative virulence factors to the infection process at different sites of infection and provide evidence that virulence determinants can behave very differently based on the genetic background of the bacterial strain. These important findings may be relevant to other bacterial pathogens.
Phosphoinositide 3 Kinase Mediates Toll-Like Receptor 4-Induced Activation of NF-κB in Endothelial Cells
Authors: Li Xianwu , Tupper Joan C. , Bannerman Douglas D. , Winn Robert K. , Rhodes Christopher J. , Harlan John M.
Published: July 2003

Many of the proinflammatory effects of gram-negative bacteria are elicited by the interaction of bacterial lipopolysaccharide (LPS) with Toll-like receptor 4 (TLR4) expressed on host cells. TLR4 signaling leads to activation of NF-κB and transcription of many genes involved in the inflammatory response. In this study, we examined the signaling pathways involved in NF-κB activation by TLR4 signaling in human microvascular endothelial cells. Akt is a major downstream target of phosphoinositide 3 kinase (PI3-kinase), and PI3-kinase activation is necessary and sufficient for Akt phosphorylation. Consequently, Akt kinase activation was used as a measure of PI3-kinase activity. In a stable transfection system, dominant-negative mutants of myeloid differentiation factor 88 (MyD88) and interleukin-1 (IL-1) receptor-associated kinase 1 (IRAK-1) (MyD88-TIR and IRAK-DD, respectively) blocked Akt kinase activity in response to LPS and IL-1β. A dominant-negative mutant (Mal-P/H) of MyD88 adapter-like protein (Mal), a protein with homology to MyD88, failed to inhibit LPS- or IL-1β-induced Akt activity. Moreover, a dominant-negative mutant of p85 (p85-DN) inhibited the NF-κB luciferase activity, IL-6 production, and IκBα degradation elicited by LPS and IL-1β but not that stimulated by tumor necrosis factor alpha. The dominant-negative mutant of Akt partially inhibited the NF-κB luciferase activity evoked by LPS and IL-1β. However, expression of a constitutively activated Akt failed to induce NF-κB luciferase activity. These findings indicate that TLR4- and IL-1R-induced PI3-kinase activity is mediated by the adapter proteins MyD88 and IRAK-1 but not Mal. Further, these studies suggest that PI3-kinase is an important mediator of LPS and IL-1β signaling leading to NF-κB activation in endothelial cells and that Akt is necessary but not sufficient for NF-κB activation by TLR4.
Modification of In Vivo and In Vitro T- and B-Cell-Mediated Immune Responses by thePseudomonas aeruginosaQuorum-Sensing MoleculeN-(3-Oxododecanoyl)-l-Homoserine Lactone
Authors: Ritchie Adam J. , Yam Andrew O. W. , Tanabe Kara M. , Rice Scott A. , Cooley Margaret A.
Published: July 2003

N-3-(oxododecanoyl)-l-homoserine lactone (OdDHL), a quorum-sensing molecule ofPseudomonas aeruginosa, plays an important role in the pathogenesis of the organism through its control of virulence factor expression. Several reports have suggested that OdDHL can also directly modulate host immune responses. However, the nature of the modulation is controversial, with different reports suggesting promotion of either humoral (Th2-mediated) or inflammatory (Th1-mediated) responses. This report describes a series of studies which demonstrate for the first time that in vivo administration of OdDHL can modulate the course of an antibody response, with an increase in ovalbumin (OVA)-specific immunogloblulin G1 (IgG1) but not IgG2a in OdDHL-treated OVA-immunized BALB/c mice compared to levels for controls. In vitro stimulation of lymphocytes from both Th1-biased C57Bl/6 and T-cell receptor transgenic mice and Th2-biased BALB/c mice in the presence of OdDHL demonstrated that OdDHL inhibits in vitro cytokine production in response to both mitogen and antigen, with gamma interferon (IFN-γ) tending to be more inhibited than interleukin-4 (IL-4). In vitro mitogen or antigen restimulation of cells from mice treated with OdDHL in vivo shows effects on cytokine production which depend on the underlying immune bias of the mouse strain used, with a relative increase of IFN-γ in Th1-biased C57Bl/6 mice and a relative increase of IL-4 in Th2-biased BALB/c mice. Thus, the mode of action of OdDHL on T-cell cytokine production is likely to be a relatively nonspecific one which accentuates an underlying immune response bias rather than one which specifically targets either Th1 or Th2 responses.
Effect of Complement Component C3 Deficiency on Experimental Lyme Borreliosis in Mice
Authors: Lawrenz Matthew B. , Wooten R. Mark , Zachary James F. , Drouin Scott M. , Weis Janis J. , Wetsel Rick A. , Norris Steven J.
Published: July 2003

Mice deficient in complement component C3 (C3−/−) and syngeneic C57BL/6 control mice were challenged withBorrelia burgdorferito determine the role of complement in immune clearance and joint histopathology during experimental Lyme borreliosis. Tibiotarsal joint, ear, and heart tissues were monitored for spirochete numbers at 2, 4, 8, and 12 weeks postinoculation with 105B. burgdorferiB31 clone 5A4 by using quantitative real-time PCR. The spirochete load in joint and ear tissue remained higher in the C3−/−mice than in the wild-type counterparts throughout the 12-week study, whereas the numbers in heart tissue of both groups of mice decreased substantially at 8 to 12 weeks postinfection. Histopathology scores for joint tissue were generally higher in the C3−/−mice compared to C57BL/6 controls at 2 and 4 weeks postinfection, which may reflect the presence of higher numbers of bacteria in the joints at these early time points. Levels of anti-B. burgdorferiimmunoglobulin G tended to be reduced in the C3−/−mice compared to control mice. Furthermore, a 5.5-fold-lower number of the complement-sensitiveBorrelia gariniiwas needed to infect C3−/−mice compared to C57BL/6 mice, indicating that its sensitivity to complement is one barrier to infection of the mouse model byB. garinii. These results indicate that the complement system may be important in controlling the early dissemination and progression ofB. burgdorferiinfection.
Role for Interleukin-1β inTrypanosoma cruzi-Induced Cardiomyocyte Hypertrophy
Authors: Petersen Christine A. , Burleigh Barbara A.
Published: July 2003

Chagas' disease, the leading cause of heart failure in Latin America, results from infection with the intracellular protozoan parasiteTrypanosoma cruzi. Host cell responses elicited in the myocardium early in the infective process are thought to be critical for establishment of infection by this pathogen; however, these changes have not been well characterized. We report here that primary cardiomyocytes undergo hypertrophy as an early response toT. cruziinfection. TheT. cruzi-elicited hypertrophic response is characterized by increased expression of genes encoding the contractile proteins MyHCβ and MyHCα, followed by an approximately twofold increase in cell size. Hypertrophy was observed in both parasite-containing and noninfected cell populations represented inT. cruzi-infected cultures, indicating the involvement of a soluble mediator in this process. Conditioned medium harvested fromT. cruzi-infected cultures, which contained significant levels of interleukin-1β (IL-1β) but not endothelin-1 or tumor necrosis factor alpha, was sufficient to induce hypertrophy in isolated cardiomyocytes. Addition of a high-affinity receptor chimera, IL-1 trap, to cardiomyocyte cultures blocked the overall increase in cell size elicited byT. cruzi. These novel findings indicate that IL-1β, which is rapidly induced in response toT. cruzi, promotes cardiomyocyte hypertrophy early in the infective process and may contribute to maintenance of cardiomyocyte function during establishment ofT. cruziinfection in the heart.
Caspase 1 Involvement in Human Monocyte Lysis Induced by
Actinobacillus actinomycetemcomitans
Leukotoxin
Authors: Kelk P. , Johansson A. , Claesson R. , Hänström L. , Kalfas S.
Published: July 2003

Actinobacillus actinomycetemcomitans
, an oral bacterium implicated in the etiology of periodontal diseases, produces a leukotoxin that selectively lyses primate neutrophils and monocytes, the major populations of defense cells in the periodontium. Though lysis requires expression of the receptor lymphocyte function-associated molecule 1 (LFA-1) on the cell surface, not all LFA-1-expressing leukocyte populations are equally susceptible to the toxin. In this study, the susceptibility of human leukocytes to leukotoxin-induced lysis is compared to their expression of LFA-1 and the activity of caspase 1. Cytolysis was determined by the activity of lactate dehydrogenase released from peripheral human leukocytes after 1-h exposure to leukotoxin. Monocytes were lysed at leukotoxin concentrations of ≥5 ng/ml, while the corresponding values for neutrophils and lymphocytes were approximately 10 times greater. Similar LFA-1 expression was found in all susceptible cell populations irrespective of their degree of sensitivity to the toxin. Exposure of monocytes to leukotoxin increased their caspase 1 activity about fivefold within 10 to 20 min. Presence of the caspase 1 inhibitor Ac-YVAD-CMK significantly blocked the leukotoxin-induced lysis of monocytes only. At sublytic concentrations, leukotoxin induced no apoptotic activity in monocytes, as revealed by the lack of caspase 3 activation and DNA fragmentation. Monocytes are the most lysis-sensitive leukocytes for
A. actinomycetemcomitans
leukotoxin. Their lysis by this toxin depends on caspase 1 activation and proceeds through a process that differs from classical apoptosis.