bluuebunny/crossref_metadata_2025_split huggingface dataset:
Survival, Replication, and Antibody Susceptibility ofEhrlichia chaffeensisoutside of Host Cells
Authors: Li Julia Shu-yi , Winslow Gary M. 
Published: July 2003

ABSTRACTEhrlichia chaffeensis, an obligate intracellular, tick-transmitted bacterium, is susceptible to antibody-mediated host defense, but the mechanism by which this occurs is not understood. One possible explanation is that antibodies directly access the bacteria in the extracellular environment of the host, perhaps during bacterial intercellular transfer. Accordingly, we investigated whether bacteria could be found outside of host cells during infection. Host cell-free plasma obtained from infected mice was found to contain ehrlichiae, and the host cell-free ehrlichiae readily transferred disease to susceptible SCID recipients. The host cell-free ehrlichiae were found during infection of both immunocompetent BALB/c and immunocompromised BALB/c-scidmice and reached levels as high as 108/ml in plasma during persistent infection in SCID mice. Approximately 10% of the blood-borne bacteria were found outside of host cells. Although it is generally accepted that replication of ehrlichiae occurs only within host cells, the cell-free bacteria were shown to undergo DNA replication and cell division in vitro for 3 to 5 days when incubated at 37°C in plasma. Paradoxically, both infectivity and virulence were lost after 24 h of ex vivo culture. The data indicate thatE. chaffeensisis exposed to the extracellular milieu during infection, presumably during intercellular transfer, and reveal that these intracellular bacteria do not require the environment of the host cell for replication. Our findings reveal a possible mechanism by which antibodies can access the intracellular bacteria upon their release into the extracellular milieu and mediate host defense and also have implications for understanding the replication and transmission of this vector-borne pathogen.
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

ABSTRACTTheMycobacterium 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

ABSTRACTCmeABC 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

ABSTRACT

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

ABSTRACTTuberculous 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

ABSTRACTInfection 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

ABSTRACTReal-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

ABSTRACTMycobacterium 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

ABSTRACT

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

ABSTRACTA 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

ABSTRACTImmunoglobulin 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

ABSTRACT

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

ABSTRACT

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.